Peptidergic transmission: From morphological correlates to functional implications

Micron, Vol. 27, No. 1, pp. 35-91, 1996

Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0968--4328/96 $32.00

Pergamon 096&-4328(95)00028-3 REVIEW PAPER

Peptidergic Transmission: From Morphological Correlates to Functional Implications GUNTHER K. H. ZUPANC* Max-Planck-Institut ffir Entwicklungsbiologie, Abteilung Physikalische Biologie, Postfach 21 09, D-72011 Tfibingen, Germany

Abstract--Like non-peptidcrgic transmitters, neuropcptides and their receptors display a wide distribution in specific cell types of the nervous system. The p¢ptides are synthesized, typically as part o f a larger precursor molecule, on the rough endoplasmic reticulum in the cell body. In the trans-Golgi network, they arc sorted to the regulated secretory pathway, packaged into so-called large dense-core vesicles, and concentrated. Large dense-core vesicles are preferentially located at sites distant from active zones of synapses. Exocytosis may occur not only at synaptic specializations in axonai terminals but frequently also at nonsynaptic release sites throughout the neuron. Large dense-core vesicles are distinguished from small, clear synaptic vesicles, which contain 'classical' transmitters, by their morphological appearance and, partially, their biochemical composition, the mode o f stimulation required for release, the type o f calcium channels involved in the ¢xocytotie process, and the time course o f recovery after stimulation. The frequently observed 'diffuse' release o f neuropcptides and their occurrence also in areas distant to release sites is paralleled by the existence of pronounced pcptidc--peptid¢ receptor mismatches found at the light microscopic and ultrastructural level. Coexistence of neuropcptides with other pcptidergie and nonpeptidergic substances within the same neuron or even within the same vesicle has been established for numerous neuronal systems. In addition to exerting excitatory and inhibitory transmitter-like effects and modulating the release o f other neuroactive substances in the nervous system, several neuropeptides are involved in the regulation of neuronal development. Copyright © 1996 Elsevier Science Ltd. Key words: Synapse, ncuropeptides, transmitters, somatostatin, vasoactivc intestinal polypeptidc, protein sorting, synaptotagmin, synaptophysin, synaptobrcvin, colocalization, coexistence, ncuromodulation, large dense-core vesicles, secretion, parasynaptic release, neurotrophic factors.

CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. From molecule to vesicle: Synthesis, sorting, and packaging o f neuropeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Two secretory pathways ...................................................................................................................... C. Cellular localization o f the machinery for sorting and packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Universality o f sorting and packaging information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Possible mechanisms o f sorting and packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. The role o f clathrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Concentration .................................................................................................................................. H. Segregation and mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Release, receptor interaction, and recycling o f vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Spatial distribution o f large dense-core vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Exocytosis at nonsynaptic release sites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Differential release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The role o f calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Range o f action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Peptide--peptide receptor mismatches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Recovery o f large dense-core vesicles after stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Molecular constituents o f large dense-core vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Vesicle fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Synaptotagmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Synaptophysin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Synaptobrevin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Coexistence o f neuropeptides with other neuroactive substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Techniques used in colocalization research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Advances and limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Principles o f interaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Future approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Functional implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . * Tel.: +49°(0) 70 71-601-338 and -339. Fax: +49-(0) 70 71-369498. E-mail [email protected] 35

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VII.

G.K.H.

Zupanc

B. Neuropeptides as transmitters a n d modulators: The case o f somatostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Neuropeptides as regulators o f neuronal development: The cases of vasoactive intestinal polypeptide a n d somatostatin . . . . . . . . . Synaptic versus nonsynaptic action: A n evolutionary perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. INTRODUCTION

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the degradation mixtures with dansyl chloride, the dansyl derivatives of the amides are isolated from Whereas electrical signalling between neurons via other products and analysed by thin-layer chromatosmall non-peptidergic molecules such as acetylcholine, L- graphy. As a result of this approach, neuropeptides glutamate, or 7-aminobutyric acid is one of the best- YY and HI (Tatemoto and Mutt, 1980), neuropeptide examined phenomena in biology, the role of peptides in Y (Tatemoto et al., 1982), and galanin (Tatemoto et this process is far less well understood. Based on al., 1983) were isolated. In the 1980s, molecular morphological observations, Ernst and Berta Scharrer cloning was introduced to endocrinology. The first proposed that some nerve cells display, in addition to peptide discovered by applying this technique was characteristics of neurons, features of gland cells (for calcitonin gene-related peptide (Amara et al., 1982). review, Scharrer and Scharrer, 1954). In the supraoptic By now, the primary structure of more than one and paraventricular nuclei of the hypothalamus, such hundred neuropeptides within the animal kingdom has 'neurosecretory cells' synthesize the posterior pituitary been described (Zimmermann, 1993). Soon after the initial discovery of the hypothalamic hormones oxytocin and vasopressin. Following transport along the axons of the hypothalamic-hypophysial hormones regulating pituitary function, many peptides tract, these peptides are stored in nerve endings in the were localized in extrahypothalamic areas of the central posterior lobe of the pituitary gland, where they are nervous system as well, thus suggesting functions in finally exocytosed. Electron microscopy revealed that addition to the neurosecretory role that the peptides fibers of neurosecretory cells do not always end blindly play in the hypothalamus. This progress was sparked by in close spatial relationship to the blood system; rather, the advances made in the refinement of techniques such cells may also form synaptic contacts with other employed for radioimmunoassay and immunohistocells (Knowles and Vollrath, 1965). For peptide- chemistry (cf. Yalow, 1978; Polak and Van Noorden, synthesizing neurons exhibiting synaptic specializations, 1986). While examination of tissue by radioimmunothe terms 'peptidergic neuron' and 'peptidergic synapse' assay provided evidence for the existence of a given peptide in various parts of the central nervous system were suggested (Bargmann et al., 1967). The modern era in neuroendocrinology began with and enabled quantification of the amount of a peptithe pioneering work performed in the laboratories of dergic substance present, application of immunohistoRoger Guillemin and Andrew V. Schally in the late chemistry led to detailed mapping studies revealing the 1960s and early 1970s when the structure of the distribution of numerous neuropeptides. In addition, hypothalamic factors controlling secretion of pituitary many peptides originally characterized as gastrointesthormones was determined and found to be peptidergic inal hormones (such as vasoactive intestinal polypep(for reviews, Schally et al., 1973; Gulllemin, 1978). tide, insulin, glucagon, and gastrin) were also identified Milestones during this initial stage of molecular in the central nervous system. Other peptides, initially neuroendocrinology were the determination of the described to occur in neural tissue, were also found in structure of porcine thyrotropin-releasing hormone secreting elements of peripheral organs. Representatives (Nair et al., 1970), the isolation and characterization of the latter category are substance P, neurotensin, of porcine luteinizing hormone-releasing hormone enkephalin, and somatostatin. (Matsuo et aL, 1971), and the isolation, characterizaThe investigations carried out on the distribution of tion, and synthesis of growth-hormone release-in- the various neuropeptides were supplemented by studies hibiting hormone, also called "somatostatin" (Brazeau employing in-situ hybridization, thus providing direct et al., 1973). These hypothalamic peptides were identi- evidence for the synthesis of these peptides in the fied by extracting huge amounts of hypothalamic tissue nervous system and revealing their sites of origin (for (in the case of thyrotropin-releasing hormone, 50 tons of examples, see Uhl, 1986; Valentino et al., 1987). New tissue to isolate 1 mg of the peptide) and monitoring vistas in neuropeptide research were opened in 1977 each purification step by a specific bioassay. when Tomas Hfkfelt and associates (Hfkfelt et al., An alternative strategy for the detection of new 1977) discovered that a peptide, somatostatin, and a peptides was developed in the laboratory of Victor "classical" molecule, noradrenaline, were colocalized in Mutt (Tatemoto and Mutt, 1978). This technique is the same neuron. A few years later, Pelletier and based on the observation that a COOH-terminal ~- colleagues (Pelletier et al., 1981b) found, by immunoamide group is a characteristic feature of many electron microscopy, that substance P and serotonin biologically active peptides. Concentrates of these may be present not only in the same neuron but even in peptides are exposed to conditions of enzymatic the same vesicle. By now, coexistence of peptides with degradation so that the characteristic COOH-terminal classical transmitters has been found in numerous amides are released. Following fluorescent labeling of systems (for review, Hfkfelt et al., 1987).

Peptidergic Transmission The question arises whether interaction takes place between classical transmitters and neuropeptides, and if so, what the function of this interaction might be. In some systems, an effect of the peptide(s) on the action of the classical transmitter(s) could be demonstrated. This interaction has quite vaguely been termed "neuromodulation" (for review, Kaczmarek and Levitan, 1987). In other systems, peptides appear to exert their effect more in a conventional transmitter-like fashion than in a neuromodulatory way. In recent years, some neuropeptides have also been suspected to play a functional role for the development or maintenance of neurons, thus acting as a trophic-like substance (for review, Strand et al., 1991). In general, however, there is still an enormous gap between molecular and neurohistochemical data that have been collected over the past twenty years and the information available on the functional significance of these results. The action of neuropeptides in the nervous system is mediated by specific receptors. Although autoradiographic studies employing radiolabeled ligands for various neuropeptides (e.g. opioid peptides: Young and Kuhar, 1979; neurotensin: Young and Kuhar, 1981; substance P: Quirion et al., 1983; somatostatin: Uhl et aL, 1985) have shown distinct patterns of binding, the existence of specific peptide receptors is not evident a priori. Theoretically, binding sites for these receptors could also be located on receptors for classical transmitter molecules. However, in the late 1980s cloning of receptors for various neuropeptides began, resulting in the elucidation of the structure and the pharmacological properties of specific receptors for various neuropeptides such as substance K (Masu et al., 1987), substance P (Yokota et al., 1989; Hershey and Krause, 1990), neurotensin (Tanaka et al., 1990), and somatostatin (Yamada et al., 1992a) (for review, Burbach and Meijer, 1992). Following the availability of specific probes, the distribution of these receptors has been mapped by in-situ hybridization (e.g. Elde et al., 1990; Breder et al., 1992). Information on the structure of these receptors is also likely to facilitate the design of specific antagonists for the usage in pharmacological investigations and for the development of specific antibodies enabling the localization of these receptors by immunohistochemical means. Neuropeptides are distinct from classical transmitters not only by their chemical nature and the specificity of their receptors, but also by many other features. Like proteins, they are synthesized in the cell body, packaged into so-called large dense-core vesicles which are morphologically (and, partially, also biochemically) different from other vesicle types, and transported to their release site. In contrast to acetylcholine, biogenic amines, and amino acids, neuropeptides are not recycled at the level of the synapse; replenishment is possible only through new synthesis in the soma. Since, depending on the distance between cell body and release site, transport may take a considerable amount of time, effects leading to a change in the synthesis will become apparent at the synapse after time periods of order of magnitude longer

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than we are accustomed to in the case of classical transmitters. This dualism between neuropeptides and classical-transmitter molecules has also become evident when studying mechanisms of release. Unfortunately, the concentration of neuropeptides in the central nervous system is, with typically 10-12-10 -15 moles per milligram protein, extremely low. In comparison, concentrations of amino acids range between 10 -6 and 10 - s moles per milligram protein, and for acetylcholine and monoamines concentrations between 10 -9 and 10 -1° moles per milligram protein are characteristic (Krieger, 1983). Consequently, when studying mechanisms of synthesis, packaging, and secretion, often nonneuronal model systems exhibiting secreting elements such as exocrine pancreas are used instead of neurons. Although a neuron can be regarded as a specialized secretory cell, and given that neurons and secretory cells of peripheral organs have many features in common, questions regarding neuron-specificity remain open. Generalizations have, therefore, to be formulated with caution. Today, more than twenty years after the breakthroughs achieved in the isolation and characterization of hypothalamic peptides, neuropeptide research is still a field expanding at a tremendous pace. Currently, more than five hundred papers are published every year on somatostatin alone. In order to manage this wealth of information, a rigorous selection process is required. This review will, therefore, focus mainly on peptides present in vertebrate species (for reviews on neuropeptides in invertebrates, Penzlin, 1989; N/issel, 1994). I will try to draw general principles illustrated by the presentation of a few well-studied examples.

II. F R O M MOLECULE TO VESICLE: SYNTHESIS, SORTING, AND PACKAGING OF NEUROPEPTIDES A. Overview

Neuropeptides, like the vast majority of secretory proteins in eukaryotic cells, are synthesized in the rough endoplasmic reticulum. A common mechanism for biosynthesis is the generation of a larger precursor molecule carrying a signal peptide. Interaction of this signal peptide with a signal recognition particle assumes that the precursor is synthesized on the rough endoplasmic reticulum and translocated into the lumen of this organelle. Upon synthesis, the signal peptide is rapidly cleaved by a signal peptidase, and the propeptide takes the route to the cis-Golgi network. From there, the propeptide is, together with other proteins, transported through the Golgi stacks until it finally reaches the trans-Golgi network. It is in this compartment that the various proteins are sorted and packaged into secretory vesicles. The initial steps of processing and transfer, which are common to almost all proteins, are not the subject of this article (for reviews, Palade, 1975; Farquhar and

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G.K.H. Zupanc

Palade, 1981; Pfeffer and Rothman, 1987; Rothman and Orci, 1992; Gilmore, 1993; Rothman, 1994). In this section, I will rather focus on mechanisms thought to be effective in the sorting and packaging of secretory proteins. Special emphasis will be placed on these processes in neuropeptides.

B. Two secretory pathways In the cell, two secretory pathways can be distinguished: the constitutive pathway and the regulated secretory pathway (Griffiths and Simons, 1986). Constitutive proteins, which are common to all ceils, leave the Golgi apparatus in short-lived membrane vesicles that fuse with the plasma membrane continuously. Proteins transported in these vesicles are not concentrated, and stimulation is not required to trigger exocytosis. In contrast, proteins and peptides destined for the regulated secretory pathway are characterized by transportation in large dense-core vesicles (LDCVs) where they are highly concentrated. This vesicle type has received its name from the electron-dense appearance of a core structure situated in the rather large vesicle. Proteins destined for the regulated pathway can be stored for considerable periods of time, which results in a large intracellular pool of mature secretory product. Furthermore, their release is dependent upon stimulation with an appropriate secretagogue. This mode is, therefore, characteristic for exocrine cells, endocrine cells, and neurons. Although neuropeptides appear to be directed exclusively to the regulated pathway, it remains elusive whether a minor amount of a given neuropeptide can also be released via the constitutive pathway. Both constitutive and regulated secretion may coexist in the same cell. It is thought that the constitutive pathway operates by a bulk flow mechanism, thus representing the default pathway. Proteins designated ~'or regulated secretion must be directed to this pathway by 'retention signals' (for review, Burgess and Kelly, 1987).

do not colocalize outside the Golgi area. Insulin is concentrated in LDCVs, whereas hemagglutinin is found predominantly in clear 100-300 um vesicles. The site at which the two proteins diverge is the tram-most cisterua of the Golgi complex (Figs 1 and 2). This corresponds to the localization where, in most cell types, concentration and packaging of secretory products occurs (Palade, 1975; Farquhar and Palade, 1981). Similarly as observed in vivo, sorting of a constitutive and a regulated secretory protein upon exit from the trans-Golgi network has been found in a cell-free system (Tooze and Huttner, 1990).

D. Universality of sorting and packaging information Gene transfer experiments have revealed that secreted proteins of different endocrine, exocrine, neuroendocrine, and endothelial cells share common sorting and packaging information. Comb et al. (1985) have used this approach to introduce a plasmid containing the human proenkephalin gene into the mouse anterior pituitary tumor cell line AtT-20. These cells express the protein precursor pro-opiomelanocortin (POMC) but not the endogenous mouse proenkephalin gene which encodes sequences of the opioid peptides Met-enkephalin and Leu-enkephalin. AtT-20 transformants that express the human proenkephalin mRNA also express proenkephalin protein and cleave the protein correctly to form free Metenkephalin. The release of both ACTH, a cleavage product of POMC, and Met-enkephalin from these cells is stimulated by corticotropin releasing factor, a natural secretagogue for ACTH. Similarly, human growth hormone transfected into AtT-20 cells is transported to the regulated pathway with an efficiency comparable to the one for ACTH, while exogenous vesicular stomatitis virus G protein (a membrane protein) exits the cells via the constitutive pathway (Moore and Kelly, 1985).

E. Possible mechanisms of sorting and packaging C. Cellular localization of the machinery for sorting and packaging The existence of two separate pathways for constitutive secretion and regulated secretion requires an effective sorting mechanism in the cell. On the basis of results obtained by combined cell fractionation and autoradiographic studies on the pancreas, it has been suggested that the intracellular site where this sorting event takes place are the trans elements of the Golgi apparatus (Tartakoff et al., 1978). This has been confirmed directly by the application of immunocytochemical techniques (Orci et al., 1987). For this purpose, the routes taken by a regulated hormone--insulin--and a constitutive protein--the viral membrane protein hemagglutinin--have been followed by immunolabeling. Both proteins have been identified in individual Golgi stacks where they appear randomly distributed throughout the cisternae. In contrast, the two proteins

How is sorting achieved in molecular terms? Blobel (1980) has suggested that the information for targeting proteins could reside in discrete segments of the polypeptide chain. These 'sorting domains' are expected to be similar for proteins that are assigned similar routes of intracellular trafficking. Although no consensus domain has yet been identified as a sorting signal within proteins, evidence that regulated secretory proteins and neuropeptides can contain sorting information has been accumulated by examining several systems. Normal endogenous mouse proinsulin, for example, is almost exclusively handled via a regulated secretory pathway. Transgenic mice that contain copies of a coding mutation in the human insulin gene (His-B10---~Asp) express high levels of the mutant prohormone in islets of Langerhans. In contrast to the normal proinsulin, up to 15% of the human mutant proinsulin is rapidly secreted after

Peptidergic Transmission

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Fig. 1. Intracellular routes of sorting and packaging taken by a regulated hormone and a constitutive protein. Consecutive serial sections of the Golgi region (A and B) and of post-Golgi compartments at intermediate (C and D) and peripheral areas (E and F) of pituitary AtT-20 cells. The cells were infected with influenza virus and immunostained with antisera directed against insulin (insulin; left side) and the viral membrane protein hemagglutinin (HA; right side). For visualization, a protein A-gold technique has been utilized. The pairs of consecutive sections show that both proteins are present in individual Golgi stacks (G), where they appear randomly distributed throughout the cisternae (A and B). In contrast, in post-Golgi compartments (C through F) their localization differs. Insulin is concentrated in large dense-core vesicles (open arrowheads), as shown by the accumulation of gold particles on these organelles (C and E). Hemagglutinin immunolabeling, on the other hand, is predominantly associated with clear vesicles (D and F). Several individual gold particles are encircled on the plasma membrane. (A) and (B) 32,000 x ; (C) and (D) 27,000 x; (E) and (F) 31,000 x. (Reproduced with permission of Cell Press, from Fig. 5 in Orci et al., 1987.)

39

40

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Fig. 2. Model of the Golgi complex and its role in sorting. Peptides and proteins synthesized at the rough endoplasmic reticulum are transferred from the c/s-Golgi network to the trans-Golgi network via the Golgi stacks in non-clathrin-eoated vesicles. In the latter compartment, lysosomal proteins and secretory peptides or proteins are actively sorted and packaged into lysosomes and large dense-core vesicles (LDCVs), respectively. Immature lysosomes and LDCVs carry a clathrin coat. LDCVs are destined for the regulated pathway. It is presumed that delivery to constitutive vesicles, which lack a clathrin coat, represents the default pathway. 0Vlodified after Halban and Irminger, 1994.)

synthesis via an unregulated or constitutive pathway (Carroll et al., 1988). Similarly, reduction by dithiothreitol of the single disulfide bond of chromogranin B causes missorting of this normally regulated secretory protein to the constitutive secretory pathway in the trans-Golgi network of PC12 cells (Chanat et al., 1993). Thus, correct sorting of chromogranin B to secretory granules is dependent upon the integrity of its disulfide bond and the resulting formation of a 20 amino acid loop in this polypeptide. Attachment of putative sorting sequences to regions encoding a constitutively secreted protein and expression of these genes in appropriate cell lines can direct such products to the regulated pathway. However, a prerequisite for accurate processing appears to be that the cells transfected contain the machinery necessary for processing and packaging secretory products. Recipient cells lacking secretory vesicles have been found to synthesize and release only unprocessed pro-hormone (Laub and Rutter, 1983; Robins et al., 1982). In contrast, transfection into secretory cells has proven to be successful in several instances. For instance, a constitutively secreted viral protein can be directed to LDCVs by attachment to the sequence of human growth hormone, a regulatory secreted peptide hormone. Cells expressing the hybrid protein have been found to target it to LDCVs with an efficiency close to that observed for the parental human growth hormone (Moore and Kelly, 1986).

In anglerfish, two different forms of somatostatin (SS14 and SS-28) are derived from two different precursors, anglerfish preprosomatostatin-1 (aPPSS-1) and anglerfish preprosomatostatin-2 (aPPSS-2). aPPSS-1 is, in its sequence, closely related to the mammalian form of preprosomatostatin (Argos et al., 1983; cf. Section VI.B). When expression vectors of the two anglerfish preprosomatostatins and of rat preprosomatostatin are introduced into mammalian cell lines, aPPSS-2 is targeted to the regulated secretory pathway much less efficiently than is the rat precursor and the aPPSS-1. This deficiency in targeting of the aPPSS-2 is likely to reflect the absence of prohormonal sorting sequences. A fusion gene containing the leader sequence and the amino-terminal 54 amino acids of the pro-region of rat preprosomatostatin linked to the carboxy-terminal 48 residues of aPPSS-2 is sufficient to redirect aPPSS-2 to the regulated pathway (Sevarino et al., 1989). Stoller and Shields (1989) have obtained similar results when using a fusion protein which contains the pro-region of the somatostatin-precursor and ~-globin. Unlike the normally expressed ~-globin, the fusion protein facilitates packaging into vesicles whose secretion is dependent upon stimulation. Thus, a possible function of the proregion of at least some peptides may be to provide sorting information. This hypothesis is especially attractive since in the past it has proven to be rather difficult to attribute a function to peptide fragments originating from this region after protein conversion. However, the universality of this notion appears questionable since deletion of the entire proregion of insulin, the C-peptide chain, does not prevent targeting to secretory granules at high efficiency (Powell et al., 1988). In addition to providing sorting information, the proregion of peptides also appears to be important for proper folding and for regulating biological activity of the final product. An alternative model that is in agreement with the specificity of packaging proposes that proteins destined to enter the regulated pathway selectively assemble with each other, and then the aggregate binds specifically to membrane protein of the LDCVs (Kelly, 1985, 1991; Burgess and Kelly, 1987). Sorting would thus be achieved by excluding 'non-aggregating' molecules from the forming secretory vesicles. In this model it would be sufficient that one element of the core associates with the membrane; the other elements may be sorted by binding to that element. In addition to the specificity in sorting, the proposed mechanism of protein condensation is compatible with the following two observations: first, secretory product is concentrated into a dense core; second, the process of concentration is not dependent upon continuous expenditure of energy (Jamieson and Palade, 1971). Further, if more than one secretory product occurs, the segregation of these materials into domains within the same LDCV (cf. Section II.H) has been used to favor the concentrationsorting model (Burgess and Kelly, 1987). Experimental data on granins have shown that low pH and high calcium milieu may indeed be sufficient to

PeptidergicTransmission induce aggregation of these regulated secretory proteins in the trans-Golgi network, thus segregating them from constitutive secretory proteins (Chanat and Huttner, 1991). Conditions such as those used in these experiments are believed to be comparable to the ones present in the lumenal milieu of the trans-Golgi network. Although sorting is highly effective, it is important to stress that exclusion of constitutively secreted proteins from the regulated pathway is not necessarily absolute. Thyroid cells, for example, can direct a small amount of a constitutive protein, p500, to the regulated pathway (Aryan and Lee, 1991). Similarly, transgenic mice overexpressing fl2-microglobulin via an insulin promotor have detectable amounts of fl2-microglobulin in their insulin-containing secretory granules (Allison et al., 1991).

41

process in the anterior pituitary by electron microscopic autoradiography employing pulse labeling with [3H]leueine has indicated that the specific activity of immature LDCVs may be as high as 200 times the activity observed in the nearby Golgi complex cisternae (Salpeter and Farquhar, 1981). As a result of this concentration process, the electron-dense core of LDCVs results. In the core, secretory products are thought to be kept in an osmotically inert state (for review, Farquhar and Palade, 1981). Experiments quantifying the content of LDCVs in the posterior pituitary have indicated the presence of as many as 85,000 molecules of peptide hormone within one secretory vesicle, corresponding to a concentration of roughly 60 mM (Nordmann and Morris, 1984). H. Segregation and mixing

F. The role of clathrin

As shown directly by immunocytochemistry (Orci et al., 1987), the site where sorting and packaging of secretory products into granules takes place is the region of the trans-Golgi network (cf. Section II.C). Frequently, structures containing condensing secretory protein in the cisternae of the Golgi apparatus, as well as the detached immature granules in the peri-Golgi region, have surface coats (Fig. 2). These coats have been identified as clathrin by immunocytochemistry (Orci et al., 1985a; Tooze and Tooze, 1986). In contrast, the mature granules below the plasma membrane do not display such surface coats. The unperturbed passage through the clathrin-coated membrane compartments of the Golgi appears to be necessary for processing of secretory material. When the intracellular transit of 3H-labeled proinsulin polypeptides has been perturbed by the carboxylic ionophore monesin in the pancreatic B-cells, proinsulin conversion is impaired and the radioactive peptides accumulate in a clathrin-coated membrane compartment related to the Golgi apparatus (Orci et al., 1984). Under these experimental conditions, non-coated secretory granules do not become significantly labeled. These observations have led to the assumption that clathrin and its adaptor molecules, which link the clathrin cage to the enclosed membrane, might be involved in sorting, packaging, and/or maturation of secretory proteins (Pearse and Bretscher, 1981). How this is achieved, remains elusive. G. Concentration

In the rims of the transmost cisternae of the Golgi complex, not only packaging but also concentration of secretory product(s) takes place. This has been corroborated by autoradiography, immunohistochemistry, and cell fractionation. The results of these experiments have demonstrated greatly increased specific activity in the content of forming and mature LDCVs as compared to the activity found in the rough endoplasmic reticulum and the Golgi cisternae (for review, Farquhar and Palade, 1981). A quantitative analysis of the secretory

In cells that produce more than one secretory product destined for the regulated pathway, the various materials may occur mixed within the same LDCV or be packaged into different vesicles. Ultrastructural localization of growth hormone and prolactin in cow anterior pituitary employing specific antibodies and protein-A gold particles of different sizes has revealed three modes of packaging of these two hormones in mixed (somatomammotropic) cells (Fumagalli and Zanini, 1985; Hashimoto et al., 1987). Growth hormone and prolactin are either stored within distinct types of LDCVs in the same cell or in the same LDCV where they are segregated in different portions, or in the same LDCV but evenly intermixed. Differential storage of secretory products in topologically segregated domains has also been found in ~ granules of human pancreatic A cells containing glucagon and glicentin-like material (Ravazzola and Orci, 1980). While the glucagon antigenic sites are mostly restricted to the dense core of the ~ granule, glicentin antigenic sites are preferentially located on the mantle layer. The ratio of two or more secretory products contained in the same LDCVs does not appear to be fixed among the vesicle population. This problem of relative packaging of secretory proteins has been addressed in zymogen granules of rat exocrine pancreas (Mroz and Lechene, 1986). The absolute amounts per granule of both chymotrypsin and amylase enzyme activities, studied by means of micromanipulation and microfluorometric methods, vary over wide ranges, and no significant correlation between chymotrypsin and amylase content among granules is detectable. I. Processing

An important aspect of maturation comprises cleavage of the protein precursors to the final secretory products by limited proteolysis (for review, Halban and Irminger, 1994). In most systems examined, at least part of the cleavage process takes place in the secretory vesicles. By using a proinsulin-specific monoclonal antibody, Orci et al. (1985b) have provided evidence

42

G.K.H. Zupanc

that in pancreatic B-cells coated secretory granules are the major, if not the only, cellular site of proinsulin to insulin conversion. In these cells, the Golgi stack appears not to be involved in the conversion process. During maturation, the interior of the secretory vesicles has to reach a critical pH before conversion of proinsulin occurs (Orci et al., 1986). Similarly, in AtT20 cells at least some POMC is packaged into secretory granules before its proteolytic cleavage to ACTH and fllipotropic hormone takes place. Anti cleavage sitespecific antibodies still label one quarter to one third of peripheral secretory granules, while anti-ACTH antiserum labels all these granules (Tooze et al., 1987). A modification of the above described scheme for packaging and cleavage of a precursor molecule has been found in the marine snail Aplysia californica by Scheller and his associates (Fisher et al., 1988; Jung and Scheller, 1991; Sossin et al., 1989, 1990). In this mollusc, egg-laying behavior is controlled by bag cells in the abdominal ganglion. Each cell is filled with many neuropeptide-containing LDCVs. When exocytosed, these peptides elicit egg-laying behavior. The egg-laying hormone (ELH) of the bag cells gives rise to different peptides. Products that are located on the COOHterminal side of the first endoproteolytic cleavage are packaged into one set of vesicles, whereas products that originate from the NH2-terminal side of the first cleavage are localized in distinct sets of vesicles which are directed to different parts of the neuron. This process takes place when the ELH prohormone enters the trans-Golgi network. When cDNA encoding ELH precursor is transfected into AtT-20 cells, ELH is packaged, processed, and stored via a regulated secretory pathway in LDCVs, while NH2-terminal products are degraded or constitutively released (Jung and Scheller, 1991). Deletion of the site where the first cleavage occurs results in re-routing of the NH2terminal intermediate products from the constitutive pathway to the regulated secretory pathway (Jung et al., 1993).

HI. RELEASE, RECEPTOR INTERACTION, AND RECYCLING OF VESICLES A. Spatial distribution o f large dense-core vesicles Sections through terminal areas of neurons display an abundance of small clear vesicles (SCVs). In addition to this vesicle type, LDCVs are also frequently found, although typically they comprise only a minor fraction of the total vesicle population (Fig. 3). When examining electron micrographs of such areas of unstimulated neurons, it appears as if LDCVs, compared to SCVs, are not randomly distributed within the profile area but instead are concentrated more distantly from the active zone. A quantitative analysis of terminals making synaptic contact with unambiguously identified neurons of the diencephalic prepacemaker nucleus in weakly electric gymnotiform fish has confirmed this impression

(Zupanc, 1991). In these boutons, the number of LDCVs found in an area stretching along the active zone is much lower than the number expected if these vesicles were randomly distributed within the whole profile (Fig. 4). This distinct spatial distribution could not only provide a basis for release of LDCVs at unspecialized sites of the synaptic bouton rather than at the active zone (see Section III.B). Mobilization of LDCVs from this distant pool, if they are destined for release at the active zone, is also likely to require more intensive stimulation than the mobilization of SCVs (cf. Sections III.C and III.D). B. Exocytosis at nonsynaptic release sites Small clear vesicles typically release their contents at membrane specializations ('active zones') in the terminal region of the axon. At the ultrastructural level, active zones are characterized by electron-dense material associated with the presynaptic membrane. At this site, the presynaptic membrane is apposed to a postsynaptic element. Upon release, the transmitters are thought to interact with the corresponding receptors located at the postsynaptic element (for review, Jahn and Siidhof, 1994; cf. Section III.F). However, notable exceptions to this rule have been reported: in the mammalian substantia nigra, dopamine is released from dendrites (Geffen et al., 1976; Korf et al., 1976; Nieoullon et al., 1977); in the cerebral cortex, only very few nerve terminals prelabeled with [3H]norepinephrine show synaptic specializations (Descarries et al., 1977; Beaudet and Descarries, 1978); in the rat dorsal horn, the majority of serotonergic and noradrenergic varicosities are characterized by nonsynaptic contacts (Ridet et al., 1993); LDCVs containing aminergic substances may be released at morphologically unspecialized regions of the central and peripheral nervous system (Zhu et al., 1986; for review, Thureson-Klein and Klein, 1990); in preganglionic axons of sympathetic nerve trunk, release of acetylcholine does not appear to be confined to axon terminals but may also take place in a nonsynaptic fashion from other parts of the axon (Viziet al., 1983); and GABA may be released at nonsynaptic sites (Cuello, 1982). Release of neurotransmitters at nonsynaptic dendritic sites may be directly related to backpropagating of action potentials into dendrites after being triggered in the axon (Stuart and Sakmann, 1994; Spruston et al., 1995; Magee and Johnston, 1995). Although much less is known about the topography, time course, and mechanism of release of LDCVs, they appear to exocytose their contents typically at morphologically undifferentiated sites distant to active zones, a process which has been called "nonsynaptic release." If discharge occurs at areas of the terminal plasmalemma situated adjacent to the synaptic specialization, often the more restricted term "parasynaptic release" is applied (for reviews, Bach-y-Rita, 1993; Golding, 1994). Nonsynaptic release has been suggested by several observations: (1) Peptides may not only be located in axons and cell bodies but also in dendrites (Pelletier et

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Fig. 3. Nerve endings of different types containing large dense-core vesicles (arrows). The boutons shown in (a) and (b) make synaptic contact with dendrites originating from neurons in a subnucleus of the diencephalic prepacemaker nucleus, the so-called PPn-C. The neurons of this cell cluster were identified by retrograde tracing with horseradish l~roxidase, which, after histochemical processing, results in a black reaction product. The terminal in (a) is, in addition, in synaptic contact with a second, unlabeled dendrite (*). The small clear vesicles displayed by the bouton in (a) are predominantly of round shape; they are believed to contain excitatory transmitter. In contrast, many of the small clear vesicles in Co) are flat or pleiomorphic, thus probably mediating an inhibitory action, m, mitochondrion. Scale bars, 0.5 ~tm. (Reproduced with permission of Chapman & Hall, from Fig. 2 in Zupanc, 1991.)

44

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Peptidergic Transmission

45

al., 1981a; Sofroniew and Glasmann, 1981; Guy et aL, LDCVs are immobilized as the granule cores are 1982; Lehman et al., 1986; Castel and Morris, 1988). (2) exposed to the extracellular space. As a result, cores Axonal terminals of peptidergic neurons, although caught in the act of discharge progressively accumulate displaying immunoreactive LDCVs, may not form until the tissue is eventually fixed. Therefore, after typical synaptic contacts (Pelletier et al., 1981a; Guy et appropriate incubation time with tannic acid the al., 1982; Oka and Ichikawa, 1991; van Heumen and number of exocytotic events visible at the ultrastructural Roubos, 1991; Beckers et al., 1993). (3) Despite their level is drastically increased compared to the application presence in cell bodies, peptides may be absent in axons of conventional methods. or axonal terminals. We have obtained evidence for this By fixation with tannic acid, nonsynaptic release of phenomenon in the prepacemaker nucleus of gymnoti- LDCVs could be demonstrated in a variety of systems: form fish by combining in-vitro tracing techniques with the central nervous system of the snail Lymnaea immunohistochemistry for the detection of somatostatin stagnalis, the cockroach Periplaneta americana, and the (Stroh and Zupanc, 1995). Although a large portion rat (Buma and Roubos, 1986); in the enteric nervous of retrogradely labeled neurons of this diencephalic system of the snail Helix pomatia (Benedeczky and nucleus exhibits somatostatin-like immunoreactivity, Halasy, 1988); in the cerebral ganglia of the platyneither their proximal axons nor their anterogradely helminth Dendrocoelum lacteum (Golding and labeled terminals display significant amounts of im- Bayraktaroglu, 1984); in terminals innervating the munoreactivity (Fig. 5). (4) Mismatches between the adrenal chromaffin tissue of goldfish (Golding and projection sites of peptidergic neurons and the locations Pow, 1987; Golding, 1992) as well as of frog (Rana of corresponding receptors may occur (see Section pipiens), hamster, and rat (Golding and Pow, 1987); and III.F). in Light Green Cells expressing insulin-related peptides Direct ultrastructural evidence for exocytosis of (van Heumen and Roubos, 1990) and in caudodorsal LDCVs at nonsynaptic-release sites stems from the cells producing ovulation hormone (van Heumen and observation of omega-shaped membrane invaginations Roubos, 1991) in Lymnaea stagnalis. In the latter two on the plasma membrane associated with a dense core in studies, the nature of the peptide released from LDCVs their lumen. In general, it has been extremely difficult to has been identified by immunolabeling at the ultravisualize such exocytotic processes by routine electron structural level. microscopic procedures, presumably because the neuroNonsynaptic release of LDCVs has also been found active substances dissolve almost instantaneously upon after surgical manipulation by conventional electronrelease. This problem can be overcome by incubation microscopic methods in the trigeminal subnucleus of the tissue in (or perfusion of the whole animal with) caudalis of the cat (Zhu et al., 1986). In the latter Ringer containing tannic acid, followed by conventional study, of 370 exocytotic profiles involving LDCVs, only gluteraldehyde/osmium fixation (Buma et aL, 1984). In four occurred at active zones of well-defined synapses. addition, the tissue may be stimulated, e.g. with high Pow and Golding (1987) report only a solitary case concentrations of K +, during the incubation with tannic "among the many hundreds encountered" where disacid to achieve a high release rate. The use of tannic charge immediately adjacent to a synaptic density was acid, a complex polymer of gallic acid, is based on its observed. A similar observation has been made by ability to stain specifically extracellular substances but Golding and Bayraktaroglu (1984) when examining the not to penetrate plasma membranes of living or neuropil of annelids. As the authors state, "not one of gluteraldehyde-fixed cells. In addition, tannic acid the hundreds of exocytotic figures we have encountered shows high-binding affinity for heavy metal ions, e.g. has involved discharge of the contents of a secretory osmium and lead, thus leading to an electron-dense granule into a synaptic cleft." In a quantitative precipitate. During tissue incubation with tannic acid, investigation on the release of peptidergic LDCVs in exocytosis proceeds but the exteriorized contents of the magnocellular supraopticoneurohypophyseal system

Legend for page 46 Fig. 5. Evidence for a nonsynaptic function of a neuropeptide revealed by immunohistochemistry combined with in vitro-tract tracing. The figures demonstrate that neurons of the prepacemaker nucleus (PPn), although the majority of them express somatostatin in their cell bodies, lack detectable amounts of this neuropeptide in their axons and in their terminals innervating the pacemaker nucleus (Pn). The transverse section through the PPn (A-C) shows retrogradely labeled neurons (A, green) and somatostatin-immunopositive somata (C, red). Double-labeled somata display a yellow color in the double-exposed micrograph (B). A cell that is retrogradely labeled but does not exhibit somatostatin-like immunoreactivity is indicated by an arrowhead in A and in B. The retrogradely traced PPn axons (D, green), approximately 100 pan caudally to the PPn, do not display somatostatinlike immunoreactivity (F). They appear green in the double-exposed photomicrograph and are located among red-labeled somatostatin-llke immunoreactive terminals (E). One somatostatin-positive bouton is marked by an arrowhead in E and in F. The transverse section through the Pn (G-I) also demonstrates a lack of somatostatin-immunoreactivity. Anterogradely labeled fibers and boutons are seen throughout the nucleus (G, H, arrowheads), they are especially dense around the profiles of the somata of one cell class in the Pn, the relay cells (marked by * in H). The traced structures are devoid of somatostatin-like immunoreactivity as indicated by their green color in the double-exposed micrograph (H) and by the lack of specific somatostatin-immunolluorescence in the Pn (I). Note that relay cell somata show unspecific fluorescence with all excitation filters employed. (Modified after Stroh and Zupanc, 1995.)

46

G . K . H . Zupanc

Peptidergic Transmission

and in the hypothalamus immediately around the supraoptic nucleus of the rat, Morris and Pow (1991) observed exocytosis in more than half of the LDCVs at sites distant from the synaptic cleft. Furthermore, at least in the latter systems, nonsynaptic release of LDCVs is not restricted to certain portions of axons, e.g. to terminal parts adjacent to synaptic thickenings. Morris and Pow (1991) found in their study on the magnocellular neurosecretory system and the hypothalamus that release of peptidergic LDCVs may occur over all parts of the neuronal membrane, even on dendrites.

47

acetylcholine, but they also contain a minor complement of LDCVs, which represent about 1% of the total vesicle population. At least a subpopulation of these LDCVs store the regulatory peptide calcitonin gene-related peptide, as has been shown by electron microscopy with colloidal-gold immunolabeling of ultrathin frozen sections (Matteoli et al., 1988). Treatment of nervemuscle preparations with ~-latrotoxin causes a complete depletion of SCVs without significantly affecting the number of LDCVs (Matteoli et al., 1988) (Fig. 6). This effect is independent of the presence of extracellular Ca 2 ÷ and occurs both at room temperature and at low temperature (1-3°C).

C. Differential release

Not only the sites for storage and release of neuropeptides differ significantly from the corresponding sites of non-peptidergic substances but also the process and mechanism of release. In general, release of peptides from LDCVs requires more prolonged periods of stimulation and/or higher stimulation frequencies than is necessary for exocytosis of classical transmitters from SCVs. Wiley et al. (1987) have shown that electrical stimulation of the mammalian superior cervical sympathetic ganglion at 10 Hz for 5 min results in a reduction of the vesicle packing density ( = number of vesicles per area of the boutonic profile) of SCVs to 58%, whereas the LDCV-packing density is reduced only to 83% of control levels when applying the same stimulation regime. Stimulation at 10 Hz for 30 min, however, leads to a further depletion of LDCVs to 41% of the control ganglion values. In the frog neuromuscular junction, stimulation at 30 Hz for 0.3 hr significantly reduces the number of LDCVs compared to preparations that have been stimulated over the same period of time but with only 2 Hz (Lynch, 1980). The differences in the time course of release suggest a different mechanism of exocytosis for SCVs and LDCYs. This notion is supported by physiological and pharmacological evidence. In the cat, nerve terminals in the submaxillary gland display immunoreactivity to vasoactive intestinal peptide. This peptide is released within the gland in response to preganglionic stimulation of the chorda tympani and causes vasodilatation. Andersson et al. (1982) have examined the effect of different modes of stimulus delivery. The mean output of vasoactive intestinal peptide from the gland is significantly increased when the same total number of impulses is delivered in the form of bursts at higher frequency than when a continuous preganglionic stimulation regime at low frequency is applied. Similarly, in the marine snail Aplysia californica significant depletion of small cardioactive peptides synthesized in identified motor neurons requires either high-frequency stimulation or prolonged bursts at lower frequency (Whim and Lloyd, 1989). A sophisticated demonstration of differential release has been accomplished by the application of a specific toxin to the frog motor nerve endings. These terminals are not only densely populated by SCVs, which store

D. The role of calcium

Calcium ions play a pivotal role for triggering of transmitter release (for reviews, Smith and Augustine, 1988; Augustine et al., 1991; Cheek and Barry, 1993; Burgoyne and Morgan, 1995; Ghosh and Greenberg, 1995). Calcium channels of various types are located in the plasma membrane of the presynaptic terminal, often forming clusters at the active zones. Presynaptic depolarization opens these voltage-gated channels so that Ca 2+ enters the presynaptic cytoplasm and diffuses

Fig. 6. Differential effect of ct-latrotoxin on exocytosis f r o m small clear synaptic vesicles and from large dense-core vesicles at the frog neuromuscular junction. An electron micrograph o f a control nerve terminal densely populated by small clear vesicles is shown in A. After treatment with ct-latrotoxin, n e r v e endings are swollen and completely depleted of small clear vesicles, as shown in B. However, large dense-core vesicles are not affected by this toxin. Arrowheads in A and B indicate large dense-core vesicles. Scale bars, 500 nm in A and 750 nm in B. (Modified after Matteoli et al., 1988.)

48

G.K.H. Zupanc

from the channel mouth away into the interior of the terminal. This Ca 2+ influx may be followed by a secondary rise in Ca 2+, as has been observed on continued depolarization of chromattin cells (O'Sullivan et aL, 1989). It was hypothesized that this elevation in the concentration of Ca 2+ is due to the release of Ca 2+ from internal stores (Hua et al., 1993). In rat pituitary melanotrophs and in bovine chromarlin cells, different phases of exocytotic secretion can be distinguished in response to a step rise in cytosolic Ca 2+ concentration (Neher and Zucker, 1993; Thomas et aL, 1993a,b). A small cohort of vesicles is exocytosed within less than 40 ms. These vesicles appear to form a pool of LDCVs docked to the presynaptic membrane. Release of LDCVs in subsequent, slower phases, which last for up to several seconds, might involve the mobilization of vesicles from pools distant to presynaptic release sites. Since the concentration of Ca 2+ rapidly decreases with distance from the channel, it is likely that the requirements for Ca 2+ concentration are different for vesicles of the readily releasable pool and for vesicles undergoing a rather long translocalization process. Different types of calcium channels appear to be involved in the exocytotic process of SCVs and LDCVs. In neurons, multiple types of calcium channels have been identified (for review, Tsien et al., 1988; Llin~s et al., 1992; Olivera et al., 1994; Dunlap et al., 1995). N-type channels are implicated in mediating the release of SCVs, whereas secretion of LDCVs may be initiated by influx of Ca 2+ through L-type channels. N-type channels display a relatively low conductance and a moderate inactivation rate. In contrast, the conductance of L-type channels is high and the inactivation rate very slow. Extensive stimulation at high frequencies favors the activation of L-type channels. Pharmacologically, N-type channels and Ltype channels can be distinguished on the basis of their sensitivity to dihydropyridines. While N-type Ca 2+ channels are resistant against treatment by dihydropyridines, L-type Ca 2+ channels react in a sensitive manner. In cultured sensory dorsal root ganglia of neonatal rats, for example, substance-P release is extremely sensitive to modulation by dihydropyridine drugs, thus pointing to an involvement of L-type Ca 2+ channels. Release of norepinephrine from sympathetic neurons taken from the same animal, on the other hand, is generally resistant to these drugs (Perney et al., 1986). Similarly, secretion of the neuropeptide vasopressin (AVP) from rat neurohypophysis is modulated by dihydropyridine drugs (Cazalis et al., 1987), while potassium-evoked release of norepinephrine from cultured superior cervical ganglia is little affected by the dihydropyridine drug nitrendipine (Hirning et al., 1988). This differential behavior does not appear to depend so much upon the type of transmitter or neuropeptide involved but more on the type of vesicle released. Release of catecholamines from secretory granules in chromaffin cells of the adrenal medulla is mediated by dihydropyridine-sensitive calcium channels (Garcia et al., 1984; Artalejo et al., 1994), thus

resembling the control of LDCVs containing peptiderglc substances. E. Range o f action

The range of action of a neuroactive substance depends on the distance it crosses by diffusion or active transport, the rate of inactivation through enzymes, and the distribution of the corresponding receptors. Unfortunately, only very little information is available on the range over which neuropeptides spread. Anatomical and physiological investigations on frog sympathetic ganglia indicate that a peptide resembling luteinizing-hormone-releasing hormone (LHRH) probably diffuses for tens of micrometers before it reaches some target neurons (Jan et aL, 1980; Jan and Jan, 1982; for review, Jan and Jan, 1983). These ganglia are composed of two cell types, B cells and C cells. LHRHpositive preganglionic C fibers make synaptic contact with C cells only. However, C fibers are able to generate late slow excitatory postsynaptic potentials via a nonsynaptic mechanism in B cells. The activation lasts for many seconds, probably due to a slow degradation process of the peptide. This case is of special interest: it demonstrates that, although a peptide transmitter is localized to nerve terminals making classical synaptic contacts with other neurons, it may diffuse and act upon neurons which are not in synaptic contact with the peptidergic terminals. A direct approach to measure the range of diffusion has been developed by Duggan et al. (1988a). Antibodycoated microelectrodes are placed into the central nervous system at regions of interest. Then, the electrodes are incubated with a solution of a radioactively labeled form of the peptide under investigation. The existence of endogenous peptide is detected on autoradiographs by the presence of zones on the microprobes in which binding of the radioactively labeled peptide is inhibited. Experiments employing such probes in the spinal cord of cats have shown that neurokinin A is not restricted to the sites of release, but the level of immunoreactive neurokinin A increases diffusely throughout the dorsal horn following stimulation. Furthermore, the release of neurokinin A persists for at least 30 min beyond the period of stimulation (Duggan et al., 1990). This pattern of persistence and the wide distribution of immunoreactive neurokinin A may underlie long-term changes in the spinal cord induced by noxious stimuli. In contrast to this diffuse mode of action, stimulusevoked release of substance P in the same system produces discrete focal increases in the level of immunoreactivity. The bands of immunoreactive substance P approximate to the sites of substance Pcontaining terminals in the upper dorsal horn (Duggan and Hendry, 1986; Duggan et al., 1988b). Thus, both diffuse spread and focal release appear to dictate the action of neuropeptides in the central nervous system. Spread of neuropeptides over long distances is clearly not restricted to the neuropil. Peptidergic neurons are

Peptidergic Transmission frequently located in areas near ventricles. In such areas, released neuropeptides readily reach the ventricular cavities. Substances using the cerebrospinal fluid as a vehicle can potentially reach numerous, even very distant, locations within the central nervous system. This hypothesis is supported by experiments in which a protein tracer, horseradish peroxidase, was infused into the lateral cerebral ventricle. After histochemical processing, the resulting reaction product could be detected in perivascular spaces as well as in extracellular spaces of the adjacent parenchyma throughout the entire brain after just a few minutes (Rennels et al., 1985). F. Peptide-peptide receptor mismatches

The notion of a predominant parasynaptic or nonsynaptic release of neuropeptides, followed by diffusion away from the site of release, is in agreement with the distribution of potential receptors. Initially, models of the action of peptidergic and non-peptidergic transmitters in the central nervous system were dominated by theories derived from observations at the neuromuscular junction. At the endplate, receptor sites for binding of acetylcholine are restricted to the post-junctional membrane. This has been revealed by employing radioactively labeled ct-bungarotoxin and autoradiography at the electron microscopic level (Porter and Baruard, 1975; Matthews-Bellinger and Salpeter, 1978) or by utilization of ~-bungarotoxin directly labeled with horseradish peroxidase and subsequent histochemical reaction for peroxidase (Lentz et al., 1977). Results of investigations conducted on the distribution of classical transmitters and their receptor sites in the central nervous system are controversial. While several authors have found no evidence for a nonsynaptic localization of the respective receptors (e.g. glutamate: Fagg and Matus, 1984; Petralia and Wenthold, 1992; glycine: Triller et al., 1985; van den Pol and Gorcs, 1988), other groups have discovered obvious mismatches between the ultrastructural localization of synapses and receptor sites. Such extrasynaptic localization has been found for nicotinic acetylcholine receptors (Jacob et al., 1986; Sargent et al., 1989) as well as for kainic acid receptors (Dechesne et al., 1990). In peptidergic systems, numerous cases of mismatches have been reported (for review, Herkenham, 1987), although in many instances a high degree of gross correspondence between the localization of peptides and their receptor sites exists at the light microscopic level. A striking mismatch occurs between the distribution of substance P-immunoreactive fibers and the density of substance-P binding sites in the substantia nigra. Consistently among groups and independent of the method used, an extremely high concentration of substance P-immunoreactive material has been localized to the substantia nigra (Brownstein et aL, 1976, 1977; CueUo and Kanazawa, 1978; Ljungdahl et al., 1978). These findings are in sharp contrast to the lack of substance P-binding sites (Quirion et al., 1983; Mantyh et al., 1984; Shults et al., 1984) and the absence of

49

mRNA encoding the NK-1 tachykinin receptor, for which substance P acts as the endogenous ligand, in this brain region (Maeno et al., 1993). A drastic quantitative mismatch between the concentration of somatostatin-producing cells and the density of somatostatin-binding sites occurs in the cerebellum of gymnotiform fish. In the caudal part of the cerebellum, immunoreactive cell bodies and fibers could be detected only by highly sensitive methods, and immunostaining occurs only in restricted areas (Stroh and Zupanc, 1993; cf. Sas and Maler, 1991). In the areas of existence, the concentration of immunoreactive material is clearly low. This corresponds to the absence of detectable levels of somatostatin mRNA-expressing cells in this brain region (Zupanc et al., 1991). In contrast, the density of somatostatin binding sites in the caudal cerebellum is the highest of the whole brain, and binding sites are abundant even in regions where no somatostatin immunoreactivity has been found at all (Zupanc et al., 1994) (Fig. 7). A significant amount of information on the mismatch problem has been added by two ultrastructural studies. By using the Met-enkephalin analogue [12SI]FK 33-824, which labels both # and ~ opioid receptors, Hamel and Beaudet (1984) have investigated enkephalin binding in rat neostriatum. Although the vast majority of binding sites are localized at axo-dendritic, axo-axonic, and axosomatic appositions of neuronal membrane, only 7% of the total number of binding sites are associated with synaptic junctions. A similar degree of mismatch has been observed by Liu et al. (1994), who used anti-serum directed against the NK-1 subtype of tachykinin receptors in rat brain. Labeling for this receptor subtype is concentrated on membranes, thus effectively outlining the surface of neuronal profiles. In cortical neurons, 68% of the surface membrane is immunoreactive for the substance P receptor, but only 9% of the substance P receptor-laden membrane apposes synaptic profiles. In the striatum and in lamina I of the spinal cord, the corresponding figures are 72% vs. 4% and 65% vs. 16% (Fig. 8). These data demonstrate that a large portion of the surface of neurons may be acted upon by the respective peptide, but only a minor fraction of these receptor sites receives focal input from directly opposed synapses.

G. Recovery of large dense-core vesicles after stimulation

The time course and mechanism of recovery of LDCVs after stimulation are different from that of SCVs. Studies in the mammalian superior cervical ganglion have revealed a rapid recovery of SCVs (Wiley et al., 1987). After electrical stimulation of the preganglionic trunk at 10 Hz for 5 rain, the vesicle packing density is, on the average, reduced to 58% compared to the unstimulated contralateral control ganglion. Already 5 min following stimulation, the vesicle packing density has returned to 78% of the controls. When terminals are allowed to recover for

50

G.K.H. Zupanc boutons. This idea is consistent with experimental data: the total turnover time for the re-supply of splenic nerves with neuropeptide Y by axonal transport has been calculated to be around 11 days (Lundberg et al., 1989). Similarly, the turnover time of vesicles containing vasoactive intestinal polypeptide in sympathetic terminals of limbs has been estimated to be approximately 5 days (Lundberg et al., 1981).

~t

IV. M O L E C U L A R CONSTITUENTS OF LARGE DENSE-CORE VESICLES A. Vesicle fusion

Pn

1 mm I

!

I

ii

Fig. 7. Distributionof somatostatin-bindingsitesin a transverse section taken through the rhombencephalon of the gymnotiform fish, Apteronotus leptorhynchus. For autoradiography,the 0 8 iodinated somatostatin analogue [125I]Tyr-D-Trp-somatostatin-14 was employed.While a large portion of the caudal cerebellum, namely the granule cell layers of the eminentia granularis medialis (EGm), the eminentia granularis pars posterior (EGp) and the transitional zone (ZT) as well as the corresponding molecular layers (EGp/ZT (tool)), display an extremely high density of binding sites, the medullary pacemaker nucleus (Pn) lacks detectable levels of binding. The absence of somatostatinbinding sites in the Pn is in agreement with the lack of somatostatin as revealed by immunohistochemistry (of. Fig. 5). On the other hand, the abundance of binding sites in the cerebellum is in sharp contrast to the absence (EGm) or the low levels of somatostatin immunoreactivity (EGp/ZT and the molecular layers) in this region (Stroh and Zupane, 1993). CC, crista cerebellaris;CCb, corpus cerebelli; ELL, electrosensorylateral line lobe with pyramidal cell layer (p) and granule cell layer (g) and four segments(LS, lateral segment; CLS, centrolateral segment; CMS, centromedial segment; MS, medial segment); MLF, medial longitudinal fasciculus; nM, nucleus medialis; nXs, vagal sensory nucleus; RF, reticular formation. (Modifiedafter Zupanc et al., 1994.)

60 min after stimulation at 10 Hz for 30 min, they have reached 94% of the vesicle packing density of controls. In contrast, recovery of LDCVs after stimulation appears to occur at a much slower time course. Electrical stimulation of the superior cervical ganglion of cats at 40 Hz for 2 hr leads to a marked decrease of the number of LDCVs within synaptic boutons (Weldon et al., 1990). Boutons of ganglia excised 2 hr after the end of the stimulation period show only 14% of the number of LDCVs of controls. Even after a recovery period of I0 days the number of LDCVs per bouton has, with 79% of the number observed in contralateral control ganglia, still not reached normal values (Fig. 9). This long time-course argues against a local retrieval of vesicle membrane of LDCVs during the process of recovery as has been proposed for SCVs (Kadota and Kadota, 1982). Rather, re-supply by anterograde axoplasmic transport is likely to be responsible for the gradual re-appearance of LDCVs within the ganglionic

Secretion via vesicular exocytosis occurs in eukaryotic organisms as divergent as yeast and man. Despite fundamental differences between organisms (e.g. secretion in yeast is a constitutive process, whereas synaptic transmission in neural tissue follows the regulated pathway), recent studies have revealed common mechanisms in intracellular events involving vesicle fusion (DeBeUo et al., 1995; for reviews, Bennett and Scheller, 1993; Stidhof et al., 1993; Ferro-Novick and Jahn, 1994; Rothman, 1994). This implies that similar molecular constituents are likely to be present in different types of vesicles. In LDCVs, several vesicle proteins previously found in SCVs have been identified. On the other hand, certain differences in the composition and/or mode of action of the molecular fusion machinery are expected to be found between SCVs and LDCVs, since pronounced differences exist in the release process between these two vesicle types (cf. Section III). Three important vesicle proteins--synaptotagmin, synaptophysin, and synaptobrevin--will be discussed in detail below. B. Synaptotagmin

Synaptotagmin, the first integral membrane protein of synaptic vesicles to be identified (Matthew et al., 1981), comprises 7-8% of the total vesicle protein (Chapman and Jahn, 1994). Because of its molecular mass of 65 kD it is also referred to as p65. Cloning and sequencing of the synaptotagmin gene (Perin et al., 1990; Wendland et al., 1991) has provided information about the structure of this protein. It consists of a small intravesicular amino-terminal domain that is glycosylated, a single transmembrane region, and a large cytoplasmic tail containing two identical repeats that are homologous to the regulatory calcium- and phospholipid-binding (C2) region of protein kinase C. Synaptotagmin is highly conserved throughout phylogeny (Matthew et al., 1981; Perin et aL, 1991b) and exists in multiple isoforms. The various genes comprising the synaptotagmin family may be expressed in different but overlapping patterns in the central nervous system (Geppert et al., 1991; Wendland et al., 1991). Besides its existence in SCVs, synaptotagmin is common to chromaffin granules (Fournier and Trifar6, 1988; Perin et al., 1991a) and LDCVs (Floor

Peptidergic Transmission

51

Fig. 8. Electron micrographs of a neuronal cell body in rat striatum, illustrating mismatches between substance-P receptors and corresponding synapses at the ultrastructural level. Substance-P receptors were localized by employing an antiserum directed against the NKI taehykiuin receptor subtype. Sites with substance-P receptors are distinguishedby the dark reaction product. A. Although substance P-receptor immunoreactivityis concentrated on the neuronal surface, thus effectivelyoutlining the soma, no synaptie boutons are visible that contact the cell body in this section. B. The micrograph of a cell body taken at higher magnification shows that the reaction product is not continuously deposited but exhibits alternating patches of labeled and unlabeled (arrows) portions on the surface membrane. N, nucleus; n, nucleolus. (Micrographs courtesy Dr Hantao Liu.) and Leeman, 1985; Fournier and Trifar6, 1988; WalchSolimena et al., 1993; Egger et al., 1994) (Fig. 10). The cytoplasmic domain of synaptotagmin binds calcium at physiological concentrations in a complex with negatively charged phospholipids (Brose et al., 1992; Chapman and Jahn, 1994). In addition, synaptotagmin displays a high affinity to calmodulin (Bader et al., 1985; for review, Trifar6 et al., 1989). It is also associated with syntaxins (Bennett et al., 1992) and the ~-latrotoxin receptor 0aetrenko et al., 1991; Hata et al., 1993; Perin, 1994), a member o f the neurexin family which mediates the releasing effect of ~t-latrotoxin observed in SCVs. Furthermore, synaptotagrnin appears to interact with N-

type calcium channels, presumably via syntaxin (Leveque et al., 1992; Bennett et al., 1992; L6v~que et al., 1994). These observations have led to the suggestion that synaptotagmin, syntaxin, neurexin, and calcium channels form a complex ("synaptosecretosome"; O ' C o n n o r et al., 1993). This complex was postulated to participate in the docking and/or fusion process of secretory vesicles, with synaptotagmin possibly acting as a calcium sensor (for reviews, DeBello et al., 1993; Greengard et al., 1993; Popoli, 1993; Popov and Poo, 1993; Kelly, 1995; Littleton and Bellen, 1995). Results of investigations employing mutants with disrupted synaptotagrnin-gene expression or in-vitro

52

G . K . H . Zupanc

(Shoji-Kasai et al., 1992). However, recent evidence suggests that not only synaptotagmin-I (which was altered in this study) is present in endocrine cells, but also, at high levels of expression, synaptotagmin-III as an additional isoform (Mizuta et al., 1994). It is, therefore, possible that synaptotagmin-III compensates for the lack of function of synaptotagmin-I in these cells.

a1

t--

6-

~t

--I

2~t

C. Synaptophysin O-

Control

2 hrs

1 day

10 days

Fig. 9. Time course of recovery of large dome-core vesicles (LDCVs) in the superior cervical ganglion of cats after electrical stimulation. Stimulation of the ganglion at 40 Hz for 2 hr results in a marked decrease of the number of LDCVs within synaptie boutons, compared to the values found in the unstimulated contralateral ganglion (control). This number is lowest 2 hr following the end of the stimulation, but even after a recovery period of 10 days the number of LDCVs per bouton has not yet reached normal values. The vertical lines indicate the standard error of the mean. *, number of LDCVs per bouton sitmiflcanfly different at P<0.05 from control values. (Modified after Weldon et al., 1990.)

preparations in which the level of this vesicle protein has been altered are consistent with this notion. Microinjection of peptides from the C2 domains of synaptotagmin into the giant presynaptic terminals of the squid Loligo pealei rapidly and reversibly inhibits neurotransmitter release (Bommert et al., 1993). This inhibition is associated with an accumulation of docked synaptic vesicles. Presumably, the C2-domain peptides block exocytosis by preventing the binding of synaptotagmin to an "accepter" protein. In line with these results are the observations of Elferink and associates that microinjection of antibodies raised against synaptotagmin and of bacterially expressed forms of this vesicle protein both perturb the function of catecholamine-containing LDCVs in PC12 cells (Elferink et al., 1993). Hippoeampal neurons cultured from homozygous mice carrying a mutation in the synaptotagmin-I gene have the synchronous, fast component of Ca 2+dependent neurotransmitter release decreased, while spontaneous synaptic activity is unaffected (Geppert et al., 1994). Mutations in the synaptotagmin gene of Drosophila show a drastic reduction in evoked synaptic transmission (Littleton et al., 1993, 1994; Broadie et al., 1994). Moreover, some mutations decrease the dependence of release on Ca 2÷ by approximately half (Littleton et al., 1994). Concomitantly with these deficits, an increase in the frequency of spontaneous miniature excitatory junctional potentials is observed in the mutant fruit flies. The latter result, together with the findings of Geppert et al. (1994) that mutations in the synaptobrevin gene do not affect spontaneous synaptic activity, provides evidence for a mechanistic distinction between evoked and spontaneous secretion. In contrast to the above results, synaptotagmindeficient clonal variants of rat PC12 cells still secrete catecholamines and adenosine triphosphate in response to elevated intracellular concentrations of calcium

Synaptophysin, a polypeptide with a molecular weight of 38,000 (hence also known as p38), is probably the most abundant integral membrane protein of SCVs (Jahn et al., 1985; Wiedenmann and Franke, 1985). Consequently, its expression is widely distributed in the brain, as has been demonstrated by in-situ hybridization (Mahata et al., 1993). Immunochemically, synaptophysin is identical to synaptin (Gaardsvoll et al., 1988). The latter protein, originally termed "membrane antigen CI" (Beck et al., 1974; Beck and Jorgensen, 1975), was discovered in synaptic-vesicle fractions where it is extremely enriched. Because of its potential to serve as a marker protein for synaptic vesicles, the name "synaptin" has been suggested (Beck et al., 1975). Whereas electron microscopic studies employing immunogold labeling initially suggested that synaptophysin is selectively associated with SCVs but not with LDCVs or chromaffin granules (Navone et al., 1986), biochemical investigations demonstrated low levels of this protein in the latter two organelles (Lowe et al., 1988; Obendorf et al., 1988; Schilling and Gratzl, 1988; Wiedenmann et al., 1988; Agoston and Whittaker, 1989; Agoston et al., 1989; Fournier et al., 1989; Schmidle et al., 1991; Morin et al., 1991) (Fig. 11). Furthermore, subcellular-fractionation experiments have indicated its association with vesicles carrying peptides such as vasoactive intestinal polypeptide (Agoston and Whittaker, 1989; Agoston et al., 1989) and tachykinins (Morin et al., 1991). It is unknown whether the different concentrations of synaptophysin in SCVs and LDCVs are causally linked to differences in the release properties of these vesicle types. The difference in the results obtained by the immunogold labeling and the biochemical experiments can be explained by the low amount of synaptophysin present in chromaffin granules and LDCVs. Schilling and Gratzl (1988) have calculated that the number of synaptophysin molecules present in a chromatfin granule is 20 and thus similar to the number estimated for SCVs. However, due to the differences in the size of these two organelles, the surface area of chromaffin granules is approximately 30 times larger than that of SCVs. This leads to a drastically reduced density of synaptophysin in LDCVs. Similar results are obtained when estimating the amount of synaptophysin relative to the amount of membrane protein (3.75 ~tg vs. 75 ~tg of synaptophysin/mg protein). The reduced density of synaptophysin in the membrane of chromaffin granules

t,

m

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ib gl)

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O

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D ~¸

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¢

I %,,

q) %

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Fig. 10. Isolation of vesicles from rat hypothalamus using immunobeads. The immunobeads were prepared by coupling monoclonai antibodies directed against either synaptophysin ('synaptophysin beads'), synaptotagmin-I Csynaptotagmin beads'), or bovine IgG ('control beads') to the surface of beads. These beads were then incubated in homogenates from rat hypothaiamus and processed for electron microscopy. The surface of the synaptophysin beads (A) and the synaptotagmin beads (]3) is mainly covered with small vesicular profiles corresponding to sm~ll clear vesicles. The synaptotagmin beads contain, in addition, the profile of one large densecore vesicle (arrowhead). The control beads (C) arc essentially devoid of vesicular profiles. Scale bars, 200 nm. (Reproduced with permission of the Society for Neuroscience, from Fig. 2 in Walch-Solimena et al., 1993.)

54

G . K . H . Zupanc

i :if(

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~

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•

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•~ ~

~ ~~

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Fig. 11. Immunoisolation of vesicles containing synaptophysin. Chromatfin granules purified from bovine adrenal medulla (A) and rat pheochromocytoma (PC12) cells (B) were incubated with anti-synaptophysin antibody bound to beads and then processed for electron microscopy. In both micrographs, dense-core granules (*) are attached to sectioned beads Cod). In addition to one granule, a small clear vesicle (arrow) is bound to the bead in B. In contrast, immunoprecipitation of chromaflin granules with beads prepared with a control antibody does not reveal adherent vesicles. (Modified after Lowe et al., 1988.)

and LDCVs causes a decrease in the number of gold particles associated with the synaptophysin antigen per cross-section of a vesicle to background level. In contrast, 20 molecules of synaptophysin per vesicle are likely to be sufficient to bind chromaffin granules as well as clear vesicles to beads, as has been demonstrated by Lowe et al. (1988). Synaptophysin contains four transmembrane regions and a cytoplasmic amino- and carboxyl-terminal domain (Siidhof et al., 1987; Johnston et al., 1989). Incorporation of purified synaptophysin into planar lipid bilayers results in the formation of voltage-sensitive

channel activity (Thomas et al., 1988). Based on this observation and on the similarity between the structure of synaptophysin and gap-junction proteins it has been suggested that synaptophysin may be involved in the formation of a fusion pore that precedes exocytosis (for review, Betz, 1990). Since several properties of this putative ion channel are difficult to reconcile with the function of a fusion pore (Siidhof and Jahn, 1991), an alternative model has been proposed in which synaptophysin plays a role for the structural organization of the synaptic vesicle (Johnston and Siidhof, 1990; for review, Jahn and Siidhof, 1994).

Peptidergic Transmission D. Synaptobrevin

Synaptobrevin (also called vesicle-associated membrane protein, V A M P ) is an integral membrane protein (Trimble et al., 1988; Baumert et al., 1989). It occurs in two isoforms termed synaptobrevin-1 and synaptobrevin-2. The predicted protein of synaptobrevin-1 from rat brain contains 118 amino acid residues (Mr = 12,760), while synaptobrevin-2 is composed of 116 amino acids (Mr = 12,671) (Elferink et al., 1989). The deduced amino acid sequences of human synaptobrevin1 and synaptobrevin-2 are 77°/'o identical (Archer et al., 1990). Each isoform is highly conserved across species, and both transcripts are expressed differentially in the central nervous system. The two isoforms are cleaved by tetanus toxin (which is produced by the bacterium Clostridium tetanO and botulinum toxin (which, in seven types designated A, B, C, D, E, F, and G, is generated by Clostridium botulinum). Both exotoxins are responsible for the clinical manifestation of tetanus and botulism by causing a long-lasting inhibition of transmitter release from presynaptic nerve endings. The clostridial neurotoxins are zinc endopeptidases consisting of a heavy chain (100 kD) and a light chain (50 kD). While the heavy chain mediates cell entry by binding selectively to certain neurons, the protease activity has been localized at the light chain (for reviews, Niemann, 1991; Niemann et al., 1994). Synaptobrevin is cleaved by tetanus toxin as well as the botulinum neurotoxins types B, D, F, and G (Link et al., 1992; Sehiavo et al., 1992; Poulain et al., 1993; Hunt et al., 1994; Llin~s et al., 1994). The same inhibitory effect has been observed in the exocytotic process of secretory granules from adrenal chromaffin cells (H6hne-Zell et aL, 1994) and in the release of Metenkephalin (in addition to glutamate, aspartate, and GABA) from guinea pig cerebrocortical synaptosomes (McMahon et al., 1992). These observations suggest that synaptobrevin might be involved in a step of the exocytotic process common to both SCVs and LDCVs. Synaptobrevin has been found in nerve terminals where it is associated with SCVs (Trimble et al., 1988; Baumert et al., 1989; Chin and Goldman, 1992), secretory granules from adrenal chromaffin cells (Hrhne-Zell et al., 1994), and LDCVs containing catecholamines in PC12 neuroendocrine cells (Papini et al., 1995). In the latter cell line, the light chain of tetanus neurotoxin, as well as the botulinum neurotoxins F and G, effectively cleave LDCV-associated synaptobrevin-2 (Papini et al., 1995). Synaptobrevin forms complexes with other proteins thought to be involved in the docking and]or fusion of synaptic vesicles. These proteins include synaptophysin, syntaxin, SNAP-25, and synaptotagmin (Calakos et al., 1994; Chapman et al., 1994; Edelmann et al., 1995; E1 Far et al., 1995; McMahon and Siidhof, 1995; Washbourne et al., 1995). It has been hypothesized that synaptobrevin acts as the vesicle receptor responsible for vesicle docking to the active zone and/or fusion with the presynaptic membrane, as suggested by the

55

SNARE hypothesis (S611ner et al., 1993a,b; Washbourne et al., 1995). Cleavage of synaptobrevin by tetanus toxin increases the number of docked vesicles instead of decreasing it as proposed, if this protein were involved in the docking process (Hunt et al., 1994). Thus, synaptobrevin appears to participate in vesicle exocytosis at a step upstream of vesicle docking.

V. COEXISTENCE OF NEUROPEPTIDES WITH OTHER NEUROACTIVE SUBSTANCES A. Terminology

Until the 1970s, different transmitters were thought to exist in distinct populations of neurons. Consequently, nerve cells were characterized based upon their content, e.g. as "noradrenergic" (containing noradrenaline = norepinephrine) or "GABAergic" [producing y-aminobutyric acid (GABA)]. This rather simple picture drastically changed in 1977 when Hrkfelt and associates demonstrated the coexistence of a peptide (somatostatin) and a non-peptidergic transmitter (norepinephrine) in some peripheral neurons. In numerous subsequent studies, various 'classical transmitters' (such as dopamine, epinephrine, norepinephrine, serotonin, histamine, acetylcholine, GABA, glutamate, and glycine) have been detected to coexist with neuropeptides. The term "coexistence" is used to describe the existence of two neuroactive substances within the same neuron, regardless of whether these substances are colocalized within the same synaptic bouton or even the same synaptic vesicle of this neuron. On the other hand, if the two molecules are present in two different, though neighboring cells, this relationship will not be referred to as coexistence. B. Techniques used in colocalization research

The progress made in studying the colocalization of neuropeptides with other neuroactive substances has been catalyzed tremendously by advances made in the development of techniques to localize more than one antigen in a given cell. Initially, the method of choice at the light microscopic level was to stain adjacent, thin (<10 ~tm) sections with different antisera. Based on landmarks, bisected cells were localized and compared for immunoreactivity. Obvious drawbacks of this method are difficulties in identifying respective landmarks in both sections and the necessity to obtain very thin sections, since otherwise the same cell may not be present in both sections. Due to these limitations, labeling of two antigens in the same section is desirable. One of the methods widely used in the first decade of colocalization research was the elution-restaining technique. A section is immunolabeled for the first antigen, and structures of interest are photographed. Then the antibody is removed from the tissue by immersing the section in solutions of high ionic strength or low pH (Nakane, 1968) or by treatment with

56

G.K.H. Zupanc

acid potassium permanganate (Tramu et al., 1978), (Scherer-Singler et al., 1983) can be visualized by followed by incubation with the new primary antibody histochemical techniques as well. and localization of bound antibodies by an appropriate Today, the most widely used techniques for localizavisualization system. The completeness of removal of tion of two antigens in the same section at the light the first primary antibody can be checked by processing microscopic level are methods employing immunothe section after the elution step with the same labeling with two different fluorophores or chromogens. visualization system as was used for the initial labeling. This is particularly useful (although not necessary) if In cases where one of the molecules of interest can be primary antisera from different host animals (e.g. rat identified by histochemical means, a combination of and rabbit) are available. Then the appropriate immunohistochemistry and histochemistry may prove secondary antibodies conjugated to different fluorouseful. This approach has often been utilized when phores (e.g. fluorescein isothiocyanate (FITC)-conjuexamining neurons that contain catecholamines. gated goat anti-rat IgG and Texas Red-conjugated goat Norepinephrine-containing cells, for example, can be anti-rabbit IgG) can be applied sequentially or simulinduced to exhibit a yellow fluorescence when exposed taneously. Visualization is performed by means of a to formaldehyde vapor ("Falck-Hillarp's fluorescence fluorescence microscope equipped with the appropriate method"; of. Falck et al., 1962) or when reacted with filter sets. Triple immunofluorescence, allowing the glyoxylic acid (Furness and Costa, 1975; Imai et al., localization of three different antigens in the same 1982). Similarly, structures that contain acetylcholines- section, is possible by using three different fluorophores terase (e.g. Newman et al., 1980) or NADPH diaphorase [e.g. fluorescein, rhodamine, and 7-amino-4-methyl-

C

i I\! i

Fig. 12. Amacrine cells in the retina of chicken are immunopositive for enkephalin, somatostatin, and neurotensin, as revealed by triple immunolabeling. The set of three microphotographs shows the same retinal section processed for enkephalin (visualization with AMCA; A), somatostatin (visualization with Texas Red; B), and neurotensin (visualization with fluorescein isothiocyanate; C). The thin arrows point to two triple-labeled somata in the inner nuclear layer (INL) that are immunopositive for the three peptides. The open arrow in B points to labeled processes ramifying in the inner plexiform layer (IPL) along the border of INL. Within the inner one-half of IPL, a broad band of labeled processes is present (denoted by an asterisk in A). The arrowheads indicate a few out of many triple-labeled segments of processes that are observed throughout IPL. GCL, ganglion cell layer. Scale bar, 20 pan. (Reproduced with permission of Elsevier Science, from Fig. 2 in Watt and Florack, 1994.)

Peptidergic Transmission

coumarin-3-acetic acid (AMCA)] thus emitting light in the green, red and blue range, respectively. An example for triple immunolabeling is shown in Fig. 12. For examination of sections under transmitted fight, the two different primary antibodies are visualized by employing two different enzyme systems or a two-color immunoperoxidase procedure. Possible choices for two independent enzyme systems are, for example, 8galactosidase and peroxidase. For the two-color immunoperoxidase procedure, e.g. the avidin-biotin immunoperoxidase technique using 3,3'-diaminobenzidine (DAB) as one chromogen to generate a brown reaction product and benzidine dihydrochloride (BDHC) as the second chromogen to achieve a dark blue reaction product, have proven reliable. Correspondingly, at the electron microscopic level, combination of pre-embedding peroxidase-antiperoxidase immunostaining for one antigen and post-embedding immunocolloidal gold staining for another antigen or double-immunogold labeling with gold particles of different sizes has found wide application (Fig. 13). Combination of immunohistochemistry and radioimmunohistochemistry, although suitable at both the light microscopic and ultrastructural level, has been favored in relatively few colocalization studies. The various modifications of the radioimmunohistochemical technique involve the use of internally-radiolabeled monoclonal antibodies (CueUo et al., 1982), [1251]secondary IgG (Pickel et al., 1986), complexes formed

57

by binding of [1251]- or [3H]-labeled neuropeptides to one of the two binding sites of their specific antibodies (Alonso and Siaud, 1989), or [125I]-labeled protein A (Kachidian and Bosler, 1991) to detect antigenic sites. These sites are then visualized by autoradiography. C. Advances and limitations

In Table 1, various neuropeptides and neuroactive substances found to coexist with each other in individual neurons of the peripheral and central nervous system of vertebrates are listed. From this list it becomes clear that there are no simple rules governing the coexistence of a given neuropeptide with other peptides or non-peptidergic neuroactive substances. In fact, the combination of messengers found might reflect the state of technical development rather than the actual mixture of substances present in a given neuron. A peptide that coexists with a second substance in one system does not necessarily coexist with the same substance in other systems or animals (or may do so at levels too low to be detected by the methods employed). Okado et aL (1991), for example, found great species differences in the coexistence ratios of substance P and serotonin in cat, rat, chicken, and monkey. Such differences may not only represent differences in intraneuronal concentrations but could also be due to structural dissimilarities of a peptide in various species. Although such dissimilarities may be

Fig. 13. Colocafization of substance P and calcitonin gene-related peptide within the same large dense-core vesicles. A terminal of a primary sensory neuron from rat dorsal horn is labeled with a rat monoclonal antibody against substance P (10 nm gold particles) and a rabbit polyelonal antibody against calcitonin gene-related peptide (20 nm gold particles). Several large dense-core vesicles contain each of the two peptides. One example of such a double-labeled vesicle is indicated by an arrowhead (10 nm gold particle) and an arrow (20 nm gold particle). (Micrograph courtesy Dr Adalberto Merighi.)

58

G. K. H. Zupanc Table 1. Coexistence of neuropeptides with other neuroactive substances in the nervous system of vertebrates

Colocalizad substance(s) Corticotropin-releasing factor + met-enkephalinArg6-GlyT-Leus Galanin Lcu-Enkephalin

Structure(s) (species) Arginm"e vasopressia LDCVs in median eminence (rat) Supraoptic, paraventricttlar, and suprachiasmatic nuclei of hypothalamus (man) Hypothalamus (rat)

Method(s)

Reference(s)

Immunohistochemistry, LM/EM

Hisano et al., 1987

Immunohistochemistry, LM

G~fi et al., 1990

Immunohistochemistry, LM

Sakanaka et al., 1990

Ar.~i'ninevasotociu (see corticotropin-releasing factor)

Acetylcholine+ substance P

Calcitonin gene-related peptide + somatostatin + substance P

Acetylcholine Acetylcholine+ met-enkephalin Cholecystokinin + galanin+ substance P Galanin Galanin+ neuropeptide Y Glutamate + substance P Neuropeptide K, somatostatin-14, somatostatin-28, substance P Neuropeptide Y + substance P + vasoactive intestinal polypeptide Neurotensin Serotonin

Atriopeplia Pedunculopontine and laterodorsal tegmental nuclei (rat) Bombesin Lumbar and sacral dorsal root ganglia (cat)

lmmtmohistochemistry, LM

Standaert et al., 1986

Immunohistochemistry, LM

Cameron et al., 1988

Caleitoniu geae-related peplide (see also bombesin, somatostatin, substance P) lmmunohistochemistry, LM Hypogiossal, facial, and ambiguus nuclei (rat) Vestibular efferent neurons (rat) lmmunohistochemistry, LM Lateral efferent neurons within lateral superior lmmunohistochemistry, LM

olive (rat) Dorsal horn of spinal cord (rat)

Takami eta/., 1985 O h n o et aL, 1991 Satieddine and Eybalin, 1992 Zhang et al., 1993

Terminals in dorsal hem (rat) LDCVs in dorsal root ganglia (rat)

Immunohistochvmistry, LM/EM lmmunohistochemistry, LM lmmunohistocbemistry, LM/EM Immunohistocbemistry, EM lmmunohistochemistry, EM

Nerve fibers of carotid labyrinth (bullfrog, Rana

lmmunohistochemistry, LM

Kusakabe et al., 1994

Immunohistochemistry, LM

Yamano et al., 1988a

lmmunohistochvmistry, LM

Arvidsson et al., 1990

Immunohistocbemistry, LM

Yamano et al., 1988b

lmmunohistochomistry, LM

K u s a k a b e et al., 1993a

lmmunohistochemistry, LM

K u s a k a b e et al., 1993b

lmmunohistochemistry, LM

Venesio et al., 1987

Dorsal root ganglia (cat) Dorsal hem of spinal cord (rat)

Arvidsson et al., 1991 Zhang et al., 1993 Merighi et al., 1991 Merighi et al., 1988

catesbeiana)

Projection from lateral parabrachial nucleus to central amygdaloid nucleus (rat) Bulbospinal pathway (monkey, Macacafascicu-

lar/s) Substance P

External subdivision of lateral parabrachial nucleus (rat) Nerve fibers of internal gills(larvae of bullfrog, Rana catesbeiana)

Nerve fibers of intervascular stroma of carotid labyrinth Coullfrog,Rana catesbeiana) Primary afferent fibers in spinal cord (frog, Rana esculenta)

Secretory vesiclesin peripheral nerves (guinea pig) Immunohistochemistry, EM

Gulbenkian et al., 1986

CholecystoHnin(see also calcitonin gene-related peptide, cholecystokinin mRNA, cholecystokinin/gastrin) Corticotropin-releasing Paraventricular nucleus of hypothalamus (rat) lmmunohistochemistry, LM Mezey et al., 1986 factor + vasopressin Cerebral cortex (cat, monkey) GABA lmmunohistochemistry, LM Hendry et al., 1984 Hippocampus; visual cortex (cat) lmmunohistocbemistry, LM Somogyi et al., 1984 Hippocampus; dentate gyms (rat) lmmunohistocbemistry, LM Kosaka et al., 1985 Non-pyramidal neurons of basolateral amygdala Immunohistochemistry, LM McDonald and Pearson, (rat) 1989 Ventral mesencephalon (rat) Neurotensin lmmunohist ochvmistry, LM Seroogy et al., 1987 Serotonin Medulla oblongata (rat) lmmunohistochvmistry, LM Mantyh and Hunt, 1984 Somatostatin + substance Sensory neurons (cat) Immunohistochemistry, LM Leah et al., 1985 P + vasoactive intestinal polypeptide Substance P Sensory and autonomic regions of spinal chord Immunohistocbemistry, LM Tuchschervr et al., 1987 segment L6 (rat) Tyrosine hydroxylase mRNA Ventral mesencephalon (rat) Immunohistochemistry//n situ Seroogy et al., 1989

hybridization,L M

Tyrosine hydroxylase mRNA

CholeeystoklninmRNA (see also cholecystokinin, cholecystokinin/gastrin) Ventral tegmental area; substantia nlgra pars In situ hybridization, LM compacta (rat)

Savasta et al., 1989

59

Peptidergic Transmission Table 1---continued Colocalized substance(s)

Structure(s) (species)

Method(s)

Tyrosine hydroxylase

Choleeystokinin/Gastrin (see also cholecystokinin, cholecystokinin mRNA) Meso-limbic neurons (rat) lmmunohistochemistry, LM

Reference(s) Htkfeit et al., 1980a,b

Corticotropin-rele~Lqlngfactor (see also arginine vasopressin, cholecystokinin, corticotropin-like intermediate lobe peptide) Paraventricolar nucleus (snake, Natrix maura) Immunohistochemistry, LM Mancera et al., 1991 Arginine vasotoein, mesotocin Parvicellular neurons of paraventricular nucleus of lmm tmohistochemistry/in situ Pretel and Piekut, 1990 Enkephalin hybridization, LM hypothalamus (rat) Zhang and Eldred, 1992 Amacrine ceils of retina (turtle, Pseudemys scripta lmm tmohistochemistry, LM GABA elegans)

Met-Enkephalin-Argt-Gly7-

Median eminence (rat)

Immunohistochemistry, EM

Hisano et al., 1986

Dorsal subdivision of lateral bed nucleus of the stria terminalis; lateral subdivision of central amygdaloid nucleus (rat) Oval nucleus of the bed nuclei of the stria

Immunohistochemistry, LM

Shimada et al., 1989

Immunohistochemistry, LM

Ju and Hart, 1989

Immunohistochemistry, LM

Papadopoulos etal., 1985

Len s

Neurotensin

terminalis (rat)

Paraventricular nucleus of hypothalamus (rat, sheep)

Oxytocin

Delta sleep-inducing peptide

Luteinizing hormonereleasing hormone

Corlicotropin-like intermedinte lobe pepfide (see also corticotropin-reieasing facto0 Hypophysis (man) lmmtmohistochemistry, LM

Vallet et al., 1988

Delta sleep-imlneing peptide (see also corticotropin-like intermediate lobe peptide) Basal forebrain; hypothalamus (rabbit) Immtmohistochemistry, LM

Charnay et al., 1989

Dynurphin (see also substance P)

Dopamine-fl-hydroxylase

Paracervieal ganglia (guinea pig)

Immunohistochemistry, LM

Enkephalin

Dorsal gray commissure of sixth lumbar and first sacral spinal cord segments (rat)

Immunohistochemistry, LM

Morris and Gibbins, 1987 Sasek and Elde, 1986

Enke#aalin (see also arginine vasopressin, caleitonin gene-related peptide, corticotropin-releasing factor, dynorphin, I"~t 7 Len-enkephalin, Met-enkephalin, Met-enkephalin-Arg6--Gly -Leu,8 Met-enkephalin-Arg6 -Phe7 ) Immunohistochemistry, LM Fried et al., 1986 Dopamine-fl-hydroxylase, Celiac ganglion (cow) tyrosine hydroxylase Immunohistocbemistry, LM Mize, 1989 Superior coilicttlns (cat) GABA Immunohistochemistry, LM Keast, 1991 Galanin, neuropeptide Y, Major pelvic ganglion (rat) vasoactive intestinal polypeptide lmmtmohistochemistry, LM Ltger et aL, 1986 Serotonin Raphe nuclei (cat) Immunohistochemistry, LM Li et al., 1990; Hamano Amacrine ceils of retina (chicken) Somatostatin et al., 1989 Perifornical region of hypothalamus (rat) Immunohistochemistry, LM Merchenthaler, 1991 Thyrotropin-releasing hormone Immunohistocbemistry, LM l.,tger et aL, 1983 Tyrosine hydroxylase Dorsolateral pens (eat) Immunohistoebemistry, LM Charnay et al., 1982 Locus coerulens (cat) FMRFmide

Neuropeptide Y

Telencephalon, thalamus, tegmentum, brainstem (Atlantic salmon, Saline salar)

lmmunohistochemistry, LM

Vecino and Ekstrtm, 1992

Galnnin (see also arginine vasopressin, ealeitonin gene-related peptide, enkephalin, substance P)

Septum-basal forebraln complex (rat) Septum-basal forebrain complex; hippoeampus (owl monkey, Aotus trivirgatus) GABA, tyrosine hydroxylase, Hypothalamns (rat) vasopressin, Preoptic area of hypothalamus (rat) Luteinizing hormonereleasing hormone Locus coeruleus (rat) Norepinephrine Supraoptic and paraventricular nuclei of Oxytocin hypothalamns (man) MagnoceUular neurons in supraoptic and Oxytocin mRNA paraventricular nuclei (rat) Mesencephalic and medullary raphe nuclei; Serotonin hippucampus (rat) Ventral horn of spinal cord (cat) Substance P Dorsal horn of spinal cord (cat) Acetylcholine

Immunohistuchemistry, LM Immunohistoehemistry/ Histoebemistry, LM Immunohistocbemistry, LM

Meiander et al., 1985 Melander and Stalnes, 1986 Meiander et al., 1986

Immunohistochemistry, LM lmmtmohistochemistry, LM lmmtmohistochemistry, LM

Merchenthaler et al., 1990 Melander et al., 1986 Gai et al., 1990

Immunohistochenfistry /in situ

Landry et al., 1991

hybridization, LM lmmunohistochemistry, LM

Melander et al., 1986

Immunohistochemistry, LM Immunohistochemistry, LM

Arvidsson et al., 1991 Arvidsson et al., 1991

60

G.K.H. Zupanc Table 1--continued

Colocalized substance(s)

Structure(s)(species)

Method(s)

Reference(s)

GABA

Ghlcagon Amacrine cells of retina (turtle, Pseudemys scripta

Immtmohistoehemistry, LM

Zhang and Eldred, 1992

elegans)

Len-Enkephalin(see also arginine vasopressin, enkephalin) GABA

Amacrine cellsof retina (chicken) Amacrine cells of retina (turtle, Pseudemys scripta

Immunohistochemistry/ Autoradiography, LM lmmunohistochemistry, LM

Watt et al., 1984

Sugimoto and Mizuno, 1987 Berk et aL, 1993 Watt and Florack, 1994 Armstrong et aL, 1984

Zhang and Eldred, 1992

elegans)

GABA + neurotensin

Caudate nucleus; nucleus aeeumbens (cat)

Immunohistochemistry, LM

Neurotensin Neurotensin + somatostatin Serotonin

Nucleus of the solitary tract (pigeon) Amacrine cells of retina (chicken) Area postrema of medulla oblongata (rat)

Immanohistochemistry, LM lmmunohistochemistry, LM Immtmohist ochemistry/ Autoradiograpby, EM Immtmohistochemistry/ Radioimmunohistochemistry, LM Immunohistochemistry, LM Immunohistochemistry, LM

Nuclei raphe magnus, raphe obscurus, and raphe pallidus (rat) Substance P

Hypothalamus (rat) Rostral subdivision of interpedancular nucleus (rat)

Kachidianet al., 1991 Slaimada et al., 1987 Shinoda et al., 1988

Luteinizlng hormone-releasing hormone (see delta sleep-inducing peptide, galanin) a-melanocyte-stimulating hormone

Melanin-concentrating hormone Dorsolateral hypothalamus (rat) Immunohistochemistry, LM

Naito et al., 1986

a-Melanocyte-slimalaliag hormone (see melanin-concentrating hormone) Mesotocin (see also corticotropin-releasing factor)

Met-Enkephalin

Neural lobe of pituitary (frog, Rana nigromaculata)

Immtmohistochemistry, EM

Takeda et al., 1990

Met-Enkephalin (see also calcitonin gene-related peptide, enkephalin, mesotocin, Met-enkephalin-Argt-GlyV-Leus, Met-enkephalin-Argt-Phe7) Acetylcholine Lateral superior olive (guinea pig) Immunohistochemistry/ Altschuler et aL, 1983 Histochemistry, LM Immtmohistochemistry, LM Zahm et al., 1985 Ventral pallidum (rat) GABA Density gradient Bastiaensen et al., 1988 LDCVs in ganglion stellatum (cow) Norepinephrine centrifugation Density gradient De Potter et al., 1987 Vas deferens (cow) centrifugation; immunohistochemistry, EM Immunohistochemistry, LM Hunt and Lovick, 1982 Nuclei raphe magnus and raphe paUidus (cat) Serotonin Kachidian et al., 1991 Nuclei raphe magnus, raphe obscurus, and raphe lmmunohistochemistry/ Radioimmunohistopallidns (rat) chemistry, LM Immtmohistochemistry, LM Todd and Spike, 1992 Somatostatin Spinal dorsal horn (rat) Met-Enkephalin-Argt-GlyV-Leus (see also arginine vasopressin, corticotropin-relcasing factor, enkephalin) Sympathetic preganglionic neurons (rat) Immunohistochemistry, LM Kondo et al., 1985 A I/C1 neurons of medulla oblongata (rat) Immunohistochemistry/ Okamura et al., 1989 Histochemistry, LM Catecholamines + AI/C1 neurons of medulla oblongata (rat) Immtmohistoehemistry/ Murakami et al., 1989b neuropeptide Y Histochemistry, LM Golgi cells of cerebellum (rat) Immunohistochemistry, LM GABA Ibuki et aL, 1988

Acetylcholine Catecholamines

GABA + substance P

Met-Enkephalln-Argt-Phe 7 (see also enkephalin) Tuberomammillary nucleus (rat) Immunohistochemistry, LM

K6hler et al., 1985

Neuropeptide K (see caleitonin gene-related peptide, somatostatin, somatostatin-28) Neuropeplide Y (see also calcitonin gene-related peptide, enkephalin, FMRFamide, Met-enkephalin-Argt-GlyT-Leu s) Cranial parasympathetic neurons (rat) Acetylcholine, vusoaetive Immtmohistoehemistry, LM Leblanc et al., 1987 intestinal polypeptide Acetylcholine + vasoaetive Ganglionic cells of larynx (rat) Immunohistochemistry/ Domeij et al., 1991 intestinal polypeptide Histoehemistry, LM Catvcholamines Paracervical ganglia (guinea pig) Immunohistochemistry/ Morris and Gibbins, Histochemistry, LM 1987 Dopamine~ff-hydroxylase Puracervical ganglia (guinea pig) Immunohistochemistry, LM Morris and Gibbins, 1987 Dopamine-p-hydroxylase, Celiac ganglion (cow) Immunohistochemistry, LM Fried et al., 1986 tyrosinehydroxylase Epinephrine C2 cell group of dorsal medulla oblongata (rat) lmmunohistochemistry, LM Everitt et aL, 1984 Hypothalamic and thalamic paraventricular nuclei; lmmunohistochemistry/ Kachidian and Bosler, arenate nucleus (rat) Radioimmunohisto1991 chemistry, LM/EM

61

Peptidergic Transmission Table 1--continued Colocalized substance(s)

GABA

GABA + somatostafin Norepinephrine

Somatostatin Somatostatin-28

Tyrosine hydroxylase Vasoaetive intestinal polypeptide

Structure(s) (species)

Method(s)

Reference(s)

Neurons of C1, C2, and C3 group of medulla oblongata (rat) Axon terminals innervating intermediate lobe (frog, Rana ridibtmda)

Immunohistocbemistry, LM

Sawchenko et al., 1985

Immunohistochemistry/ Radioimmunohistochemistry, EM Immunohistocbemistry, LM Immunohistochemistry, LM

Tonon et al., 1992

Cerebral cortex (cat, monkey) Non-pyramidal neurons of basolateral amygdala (rat) Cerebral cortex (lizards, Psammodromus algirus and Podarcis hispanica) A1/C1 cell group of ventrolateral medulla oblongata; A6 cell group of locus coeruleus (rat) LDCVs in ganglion steUatum (cow) Neurons of A1 cell group of medulla oblongata (rat) Ventrolateral medulla oblongata (rat) Amygdala (rat) Hippocampus; cortex (man) Medial septal area; nucleus accumbens and olfactory tubercle; laterodorsal septal nucleus; infralimbic cortex (layer VI) and subcortieal white matter (man) Major pelvic ganglion (rat) Medulla oblongata (man) Rostral medulla (rabbit) Cerebrovascular nerves (rat) Submucous nerves of jejunum (rat)

Immunohistochemistry, LM

Hendry et al., 1984 McDonald and Pearson, 1989 Dfivila et al., 1991

Immunohistochemistry, LM

Everitt et aL, 1984

Density gradient centrifugation Immunohistochemistry, LM

Bastiaensen et aL, 1988 Sawchenko et al., 1985

Immunohistochemistry, LM Immunohistochemistry, LM Immunohistocbemistry, LM Immunohistocbemistry, LM

Everitt et al., 1984 McDonald, 1989 Chan-Palay, 1987 Gaspar et al., 1987

Immunohistochemistry, LM Immunohistochemistry, LM Immunohistochemistry, LM Immunohistoebemistry, EM

Keast, 1991 Htkfelt et al., 1983 Blessing et al., 1986 Cavanagh et al., 1990

Immunohistochemistry, EM

Cox et al., 1994

Neurotensln (see also ealcitonin gene-related peptide, cholecystokinin, corticotropin-releasing factor, Leu-enkephalin) Arcuate nucleus of hypothalamus (rat) lmmunohistoebemistry/ Ibata et al., 1983 Histochemistry, LM Arcuate nucleus of hypothalamus; ventral Immunohistochemistry, LM Htkfelt et al., 1984 mesencephalon (rat) Immunohistocbemistry, LM Epinephrine H6kfelt et al., 1984 Medulla oblongata (rat) Glycine Amacrine cells of retina (turtle, Pseudemys scripta Immunohistochemistry/ Weiler and Ball, 1984 elegans) Autoradiography, LM Substance P Amacrine cells of retina (goldfish, Carassius Immunohistocbemistry, LM Li et al., 1986 Dopamine

auratus)

Opioid peptides Immunohistochemistry, LM

GABA

Basal ganglia (rat)

Nitric oxide

Oxytodn (see also corticotropin-releasing factor, galanin) Hypothalamus (rat) Immunohistocbemistry/ Histochemistry, LM

Substance P

Pancreatic polypeptide Amacrine cells of retina (chicken)

Vasoactive intestinal polypeptide

Pepfide histidine isoleudae/peptide histidine methionine Amacrine and displaced amaerine cells of retina Immunohistochemistry, LM (rabbit)

Immunohistocbemistry, LM

Oertel and Mugnaini, 1984

Miyagawa et al., 1994; Sfinchez et al., 1994

Katayama-Kumoi et al., 1985

Pachter et al., 1989

Prodynorphin peplides (see substance P) Somatostatin (see also bombesin, calcitonin gene-related peptide, cholecystokinin, enkephalin, Leu-enkephalin, Met-enkephalin, neuropeptide Y, somatostatin-28) Spinal cord motoneurons (embryonic chicken) Immunohistochemistry, LM ViUaret al., 1988 Calcitonin gene-related peptide, vasoactive intestinal polypeptide Immunohistochemistry, LM Morris and Gibbins, Paracervieal ganglia (guinea pig) Dopamine-fl-hydroxylase 1987 Immunohistochemistry, LM Sur et al., 1994 Afferent terminals presynaptic to Mautlmer GABA cells(goldfish, Carassius auratus) lmmunohistochemistry, LM Li et al., 1990 Amacrine ceils of retina (chicken) lmmunohistochemistry/ Watt and Florack, 1993 Amacrine cells of retina (larval tiger salamander, Autoradiography, LM Ambystoma tigrinum) Hendry et al., 1984 Immunohistochemistry, LM Cerebral cortex (eat, monkey) Schmecbel et al., 1984 Immunohistochemistry, LM Cerebral cortex; hippoeampus (rat, cat, monkey)

62

G . K . H . Zupanc Table I--continued

Colocalized substance(s)

Glutamate Glyciue Neuropeptide K, substance P Nitric oxide Substance P

Structure(s) (species)

Method(s)

Reference(s)

Hippoeampus (rat) Hippocampus; visual cortex (cat) Non-pyramidal neurons of basolateral amygdala (rat) Nucleus reticularis thalami (cat) Ventral lamina III and lamina IV of spinal dorsal horn (rat) Afferent terminals presynaptic to Mauthner cells (goldfish, Carassius auratus) Ventral lamina III and lamina IV of spinal dorsal horn (rat) LDCVs in dorsal root ganglia (rat) Dentate hilus of hippocampus (rat) Periventricular parvicellular subdivision of pamventrieular nucleus of hypothalamus (rat) Ventral subdivision of lateral bed nucleus of the stria terminalis; medial subdivision of central amygdaloid nucleus (rat)

lmmunohistoehemistry, LM lmmunohistochemistry, LM lmmtmohistochemistry, LM lmmunohistochemistry, LM lmmtmohistochemistry, LM

Jirikowski et al., 1984 Somogyi et al., 1984 McDonald and Pearson, 1989 Oertel et aL, 1983 Proudloek et al., 1993

Immunohistochemistry, LM

S u r e t al., 1994

Immunohistochemistry, LM

Proudlock et al., 1993

lmmunohistochemistry, EM lmmunohistocbemistry, LM lmmunohistochemistry/ Histochemistry, LM Immunohistochemistry, LM

Merighi et al., 1988 Dun et al., 1994 Alonso et al., 1992 Shimada et al., 1989

Somatustafin-28 (see also calcitonin gene-related peptide, somatostatin, neuropeptide Y) Neuropeptide K, substance P LDCVs in dorsal root ganglia (rat) Immunohistocbemistry, EM Merighi et al., 1988 Substance P (see also atriopeptin, bombesin, calcitonin gene-related peptide, choleeystokinin, galanin, Leu-enkephalin, Met-enkephalin-Arg6-Phe 7, ueurotensin, pancreatic polypeptide, somatostatin, somatostatin-28) lmmunohistochemistry, LM Vincent et aL, 1983 Ascending reticular system (rat) Acetylcholine Immunohistochemistry, LM Tuchseherer and Varicosities within superficial laminae of dorsal Calcitonin gene-related Seybold, 1989 horn of spinal cord (rat) peptide, dynorphin, galanin Immtmohistochemistry, LM Pourcho and Goebel, Amacrine cells of retina (cat) GABA 1988 lmm unohistochemistry/ Watt et al., 1993 Amacrine cells of retina (larval tiger salamander, Autoradiography, LM Ambystoma tigrinum) lmm uimhistochemistry, LM Murakami et al., 1989a Entopeduneular nucleus (rat) Immanohistochemistry, LM Caruso et al., 1990 Ganglion cells of retina (rat) Immunohistochemistry, LM Kosaka et al., 1988 GABA, tyrosine hydroxylase Periglomerular region of main olfactory bulb (hamster) Neal et al., 1989 Immunohistochemistry, LM Medial nucleus of the amygdala; medial bed Prodynorphin peptides: nucleus of the stria terminalis; medial preoptic dynorphin A(1-17), area (Syrian hamster) dynorphin B( 1-13), C-peptide Marson, 1989 Bulbospinal neurons of raphe nuclei and of caudal Immunohistochemistry, LM Serotonin ventrolateral medulla (cat) Immunohistochemistry, LM Appel et aL, 1986 Fibers in iutermediolateral cell column of spinal cord (rat) Immunohistochemistry, LM Lovick and Hunt, 1983 Medulla oblongata (cat) Immunohistochemistry, LM H6kfelt et al., 1978 Medulla oblongata (rat) Kachidian et al., 1991 Nuclei raphe magnus, raphe obscurus, and raphe lmmunohistochemistry/ Radioimmunohistopallidus (rat) chemistry, LM lmmunohistochemistry/ Chan-Palay et al., 1978 Raphe nuclei and nucleus interfascicularis Histochemistry/ hypoglossi of medulla oblongata (rat) Autoradiography lmm tmohistochemistry, LM Okado et al., 1991 Raphe-spinal motor-neuron fibers (monkey, cat, rat, chicken) Immtmohistochemistry, LM Wessendorf and Elde, Spinal cord (rat) 1987 lmmanohistochemistry, LM Ozald et al., 1991 Ventral horn of lumbar spinal cord (rat) lmm unohistochemistry, Lid Johansson et al., 1981 Medulla oblongata (rat) Serotouln + thyrotropinlmm anohistochemistry, LM Sasek et al., 1990 Ventral medulla (rat) releasing hormone Serotonin

Thyrotrop'm-releasiag hormone (see also enkephalin, substance P) Nuclei raphe magnus, raphe obscurus, and raphe Immanohistochemistry/ pallidus (rat) Radioimmunohistochemistry, LM Var/cosities innervating spinothalamic-tract lmmunohistoehemistry, LM neurons (rat) Ventral medulla (rat) Lesions/Radioimmunoassay/ Fluorometry

Kachidian et al., 1991 Wu and Wessendorf, 1992 Helke et al., 1986

Vasoacfive intestinal polypeptide (see also calcitouln gene-related peptide, cholccystokinin, enkcphalin, neuropeptide Y, peptide h/stidine isoleucine/peptide h/stidine methioniue, somatostatin) Dopamine-~-hydroxylase Paracervical ganglia (guinea pig) Immunohistocbemistry, LM Morris and Gibbins, 1987 GABA Hippocampus; dentate gyrus (rat) Immunohistochemistry, LM Kosaka et al., 1985 Non-pyramidal neurons of basolateral amygdala Immunohistochemistry, LM McDonald and Pearson, (rat) 1989

Pepfidergic Transmission

63

Table l---continued Colocalized substance(s)

Structure(s)(species)

Method(s)

Nitric oxide

Vasopressin (see also arginine vasopressin,cholecystokinin,galanin) Hypothalamus (rat) Immunohistochemistry/ Histochemistry, LM

Reference(s) S~nchez et al., 1994

In several studies, colocalizationof a given neuropeptidein the neural systemexaminedhas been demonstrated to occur with more than one neuroactive substance. If these substances, listed in the first column, also colocalizewith each other, then they are linked together by a "+ ". If these substances have not been tested for a possible colocalization,or if they are expressedin separate populations of cells, then a "," has been placed between them in the listing. EM: Electron microscopy.LDCVs: Large dense-corevesicles. LM: Light microscopy. small, they could lead to marked differences in binding affinity if the epitope involved in antibody binding is affected. Furthermore, peptides are often present only in a subpopulation of neurons expressing a second neuroactive substance, and vice versa. This leads to an enormous degree o f variability in the expression pattern of neuroactive substances in neurons. Negative results in investigations examining the (co)existence of peptides have to be viewed with caution. Generally, the concentration o f a neuropeptide in a neuron is rather low compared to many other neuroactive substances, and the low number of antigenic sites may be even further reduced in the course of immunohistochemical processing. Wilson et al. (1980) found in bovine splenic nerve a molar ratio of opiate-like peptides to norepinephrine of 1:60. In the same system, Fried et al. (1986) measured molar ratios of neuropeptide Y, enkephalin, somatostatin, substance P, and vasoactive intestinal peptide to norepinephrine of 1:25, 1:350, 1:50,000, 1:30,000, and 1:20,000, respectively. Neuroactive substances that coexist within the same terminal do not necessarily also coexist within the same synaptic vesicle. The two or more substances may occur in distinct populations of vesicles. An important physiological consequence of this situation is that different stimulatory conditions may be required to trigger release. Corelease may occur especially in cases where the two substances are colocalized within the same vesicle. However, although colocalization per definitionem represents the morphological basis for cotransmission, the existence of two substances within the same terminal (in different vesicles or in the same vesicle) does not necessarily prove corelease. Evidence for cotransmission has been obtained under experimental conditions employing massive depolarizing stimulation triggered by exposure of the neuron to high concentrations of potassium or by electrical stimulation at high frequency for prolonged periods of time. It is not clear whether similar events of corelease occur under normal operation of a neuron as well. Indeed, to date in no case has cotransmission been demonstrated unequivocally under physiological conditions. D. Principles o f interaction Interaction of neuropeptides with other neuroactive substances does not require involvement of cotransmis-

sion a priori. The principles of interaction are similar, independent of whether the different substances are released from the same terminal, as in cotransmission, or from separate sources when, for example, two neurons converge on a single postsynaptic target (Fig. 14). In both cases, the interactions can be synergistic or antagonistic, and they may occur at the level of pre- and postsynaptic receptors as well as through altering the activity of degradative enzymes, e.g. endopeptidases (for reviews, Lundberg and H6kfelt, 1983; H6kfelt, 1991). Presynaptically, such interactions could lead to an altered proportion of the two substances released. At the postsynaptic site of action, one substance could alter the affinity of a second substance to its receptors, the rate of desensitization, or the rate of recovery from desensitization of a receptor for its agonists (for review, Kupfermann, 1991). Selected examples for interaction between somatostatin and other neuroactive substances will be presented below (see Section VI.B.3).

E. Future approaches Quantification of the relative (or absolute) amount of different neuroactive substances that are colocalized in tissue is highly desirable. To date, the only approach available is biochemical means applied to rather large tissue samples, thus tremendously limiting the spatial resolution. A quantitative electron microscopic immunohistochemical technique, which in a modified form, may prove useful for the analysis of neuropeptides, has been developed by Ottersen and colleagues (Somogyi et al., 1986; Ottersen, 1987, 1989a,b) for neuroactive amino acids. Model-antigen systems are prepared by conjugating amino acids to a crude extract of brain macromolecules by means of glutaraldehyde, thus mimicking the conjugates formed in tissue during fixation. These conjugates are embedded in resin as is done for tissue--cut, incubated with the respective antiserum, and processed for immunogold labeling. Test sections from tissue are placed next to "graded sections" which are cut from model systems containing a series of known antigen concentrations. This series allows to determine the relationship between antigen concentration and gold particle density. The approach is especially useful in determining relative concentrations of antigens; calculation of absolute concentrations is possible, although such values should be viewed with

64

G.K.H. Zupanc

~

~o ~

J

°ii ,° .~'~

~'~

Peptidergic Transmission caution due to inherent inaccuracies in the method employed. In principle, an approach similar to the one used for assessing concentrations of amino acids appears feasible for the determination of amounts of neuropeptides in tissue. A major obstacle in the development of a modified procedure is likely to be the fragile nature and the short half-life of many neuropeptides. Likewise, fixation of neuropeptides in model systems may affect the structure in a different way to that after fixation in situ, thus resulting in altered antigenicity. Furthermore, for optimal fixation, neuropeptides and classical transmitters often require different protocols, which hinders simultaneous optimal preservation of the different antigens. Nevertheless, aside from these technical difficulties, it is clear that future studies in colocalization research will also have to add the quantitative dimension to their strategy.

VI. FUNCTIONAL IMPLICATIONS A. General remarks

Despite the enormous advances made over the last 25 years in the structural characterization of neuropeptides and their receptors, as well as the progress achieved in the elucidation of their anatomical distribution, still very little is known about the function these molecules exert in the central nervous system. Their actions have often been characterized as "complex", reflecting the difficulty to formulate a unifying hypothesis. These difficulties are due to several factors: • The function of a given neuropeptide is likely not to be universal, but to vary from brain region to brain region and, within an area, from cell type to cell type. In most physiological studies carried out to date, differences in the responses of individual cells have been related only insufficiently to possibly different cell types, although anatomical tools, such as the filling of a neuron with an appropriate dye after intracellular recording, are available. • Within a brain region, the function of a neuropeptide may change in the course of ontogenic development. • A major feature of neuropeptides appears to be their interaction with other neuroactive substances; consequently, the effect of a neuropeptide often becomes apparent only after probing the influence of such systems as well. • Neuropeptides typically exist and act at low concentrations, requiring sometimes sensitive techniques for the detection in neural tissue. • The half-life of a peptide may be rather short; this may destructively interfere with the speed required by many physiological experiments. • Many effects elicited by neuropeptides are very prone to the experimental conditions employed. Such factors include the method of peptide application, the concentration used, the choice of anesthesia, and

65

the question whether an experiment has been preceded by stimulation in the same region. • For many neuropeptides, no good receptor-specific agonists or antagonists are available, thus impeding the effort to analyse the cellular mechanisms of a possible effect. Several of these difficulties will also be addressed in the following discussion. I will focus on reviewing the findings of functional studies on two well-examined representatives, somatostatin and vasoactive intestinal polypeptide. B. Neuropeptides as transmitters and modulators: The case o f somatostatin 1. Molecular f o r m s and distribution

Somatostatin (SS) is one of the most abundant neuropeptides in the mammalian brain, displaying concentrations between 1000 and more than 4000 pmol/g protein (Crawley, 1985). In mammals, the biologically active forms of SS, a tetradecapeptide (SS14) and an N-terminally extended form of SS-14 consisting of 28 amino acids (SS-28), are synthesized as part of a large precursor molecule (prepro-SS) comprising 116 amino acids (for review, Patel, 1992). This molecule, encoded by a single gene, is rapidly cleaved into the prohormone form (pro-SS; 92 amino acids), which is further processed into various smaller molecules including SS-14 and SS-28 (Fig. 15). SS-14 is totally conserved from fish to man (Vale et al., 1976; King and Millar, 1979), suggesting an important role of this peptide. Additional molecular forms of SS have been identified in several fish species. These fish-specific forms are derived from a second gene which apparently has been lost in the course of evolution leading to higher vertebrates (for review, Patel, 1992). Originally, SS-14 was isolated from ovine hypothalamus on the basis of the potent inhibitory activity it exerts on the release of growth hormone from anterior pituitary (Brazeau et al., 1973). Besides this endocrine function in adenohypophysial secretion (for reviews, Patel and Srikant, 1986; Tannenbaum et al., 1990), a role of SS as a transmitter or modulator in the process of synaptic transmission has been proposed. This notion is based on the wide distribution of SS and its messenger RNA in extrahypothalamic areas of the brain of various vertebrate species, as revealed by radioimmunoassay (e.g. Brownstein et al., 1975; Kobayashi et al., 1977; Palkovits et al., 1976), immunohistochemistry (e.g. Finley et al., 1981; Johansson et aL, 1984; Vincent et al., 1985; Sas and Maler, 1991; Y/tfiez et al., 1992), and in-situ hybridization (e.g. Fitzpatrick-McElligott et aL, 1988; Priestley et al., 1991; Zupanc et al., 1991; Mengod et al., 1992). Substantial support for this hypothesis has been obtained from the results of numerous physiological and pharmacological investigations discussed below, and by the following observations: SS is contained in synaptosomal fractions (Epelbaum et aL, 1977; Berelowitz et al., 1978a) and localized in LDCVs

66

G . K . H . Zupanc

Preprosomatostatin 1 - 116

1 - 24

Prosomatostatin 25 - 116

Somatostatin-28 89 - 116

Somatostatin-14 103 - 116

Fig. 15. Schematic representation of the major forms of mammalian somatostatin. The precursor molecule, preprosomatostatin, is cleaved into prosomatostatin. The two biologieaUy active forms, somatostatin-14 and somatostatin-28, are C-terminal cleavage products of this larger prohormone.

within synaptic terminals (Foster and Johansson, 1985; Ribeiro-da-Silva and Cuello, 1990; de Stefano et aL, 1993; Sur et al., 1994); its release from neural tissue upon stimulation is dependent on calcium (Berelowitz et al., 1978b; Lee and Iversen, 1981); and its effects are mediated by specific receptors expressed in the central nervous system (for reviews, Bell and Reisine, 1993; Epelbaum et al., 1994; Hoyer et al., 1994; Reisine and Bell, 1995). 2. Excitatory and inhibitory effects

When SS is applied to neurons, a great variety of different, and sometimes even contradictory, effects has been described. Inhibitory functions of SS, evident from a reduction of excitability or a hyperpolarization of neurons, have been found in cat and rat dorsal horn neurons (Randi6 and Miletir, 1978; Murase et al., 1982), neurons of submucous plexus of guinea-pig caecum and ileum (Mihara et al., 1987), cultured locus coeruleus neurons from neonatal rats (Inoue et al., 1988), spinal cord neurons of lampreys (Christenson et aL, 1991), neurons of the solitary tract complex (Jacquin et al., 1988), and CA1 neurons of rat and guinea-pig hippocampus (Pittman and Siggins, 1981; Xie and Sastry, 1992; Schweitzer et al., 1993). In contrast, excitatory effects have been observed in vagal motoneurons of rat (Wang et al., 1993), neurons of sensorimotor cortex of rabbits (Ioffe et al., 1978), neurons in the pyramidal layer of the CA1 and CA2 region of rat hippocampus (Dodd and Kelly, 1978), and pyramidal neurons in the CA1 area of guinea-pig and rabbit hippocampus (Mueller et al., 1986), although in the latter study hyperpolarization could be evoked in a minority of the ceils. Remarkably, several authors have reported inhibitory as well as excitatory effects in a given system. Twery and Gallagher (1989, 1990) examined the effect of SS in slice preparations of rat dorsolateral septal nucleus by intracellular recordings. SS was applied by superfusion or pressure ejection. In 16 of 27 neurons tested, SS inhibited the intracellularly recorded fast inhibitory

postsynaptic potential and the late hyperpolarizing potential elicited by focal electrical stimulation. In 11 of the 27 neurons, on the other hand, treatment with SS increased the amplitude of either the fast inhibitory postsynaptic potential or the late hyperpolarizing potential. In some systems, the respective effect----excitatory or inhibitory--appears to depend on the conditions applied. Katayama and North (1980) found depolarizing responses in ganglion neurons of myenteric plexus of guinea-pig ileum to be predominantly associated with iontophoretic application of SS, whereas hyperpolarizing responses were preferentially elicited through SS application by superfusion of tissue slices. Similarly, in rat cortical neurons grown in dispersed cell culture, SS causes predominantly excitatory effects; inhibitory actions, recorded in fewer cases, appear to be dependent upon the application of higher concentrations of SS. Furthermore, tachyphylaxis, a progressive diminution of responses to repeated application of SS, has been observed in this system as well (Delfs and Dichter, 1983). Consequently, the response of a given neuron will depend on the stimulation regime previously applied to this system. 3. Interaction with other neuroactive substances

A remarkable feature of SS is its modulatory action on the release or turnover rate of other neuroactive substances. In synaptosomal preparations from rat hippocampus, SS augments [3H]acetylcholine release (Nemeth and Cooper, 1979). In slices of rat hippocampus, exogenous SS-14 and SS-28 enhance K +evoked release of endogenous acetylcholine (Araujo et al., 1990). This increase is antagonized by calciumchannel antagonists, thus implicating voltage-sensitive calcium channels in this effect. In contrast, release of [3H]acetylcholine at the neuromuscular junction between ciliary ganglion neurons and the choroidal smooth muscle of the chick eye is reduced to 5% of the values obtained after control-evoked release (Gray et al., 1989). A similar effect has been observed in slices of

PeptidergicTransmission rat caudate nucleus, where potassium-induced release of [3H]acetylcholine is inhibited by SS (Arneri6 and Reis, 1986). This action of SS may be mediated by dopaminergic mechanisms since, as has been shown by the same authors, sulpiride, a dopamine receptor antagonist, prevents the effects of SS. Not only the release of acetylcholine but also the release of other transmitters and neuropeptides may be modulated by SS. In organ cultures of rat hypothalamus, SS inhibits basal release of thyrotropin-releasing hormone (Hirooka et al., 1978). A similar inhibitory effect is observed when release of the latter peptide is evoked by stimulation with norepinephrine. An inhibitory action of SS becomes also evident in superfused mediobasal hypothalami of rat, where the depolarization-induced release of immunoreactive thyrotropinreleasing hormone is reduced by SS (Tapia-Arancibia et al., 1984). In the same system, SS induces a dosedependent inhibition of luteinizing-hormone releasinghormone release in vitro (Rotsztejn et al., 1982). [3H]norepinephrine release is inhibited by SS in rat hypothalamic slices (G6thert, 1980) but stimulated in rat cerebral cortex slices (Tsujimoto and Tanaka, 1981). By using superfused slices of rat cerebral cortex, hippocampus, and hypothalamus, Tanaka and Tsujimoto (1981) have demonstrated a facilitatory, dose-dependent effect of SS on the electrically- or potassium-stimulated release of [3H]serotonin. From rat striatal slices, SS increases spontaneous [3H]dopamine release in a dosedependent manner (Chesselet and Reisine, 1983). A similar effect has been shown by the same authors in the caudate nucleus and the substantia nigra of cats in vivo using a push-pull cannula technique. Both facilitatory and inhibitory effects of SS have been found by Meyer and associates (Meyer et al., 1989) on the release of GABA in rat caudatoputamen. Slices of this brain region were loaded with [3H]glutamine and examined for the release of [3H]GABA. Low concentrations of SS (1 mmol/1) enhanced moderate release but had no effect on the more pronounced release. Higher concentrations (10 mmol/1), on the other hand, did not show any effect on moderate release but diminished the pronounced one. Somatostatin also affects the turnover-rate of several transmitters in various parts of the brain following intraventricular administration with or local application through injection of SS and SS analogues. So far, only stimulatory effects have been reported, namely on acetylcholine (Malthe-Sorenssen et al., 1978), monoamine (Garcia-Sevilla et al., 1978), and catecholamine turnover (Beal and Martin, 1984). It is not surprising that the actions of SS on the release or turnover-rate of other neuroactive substances are also reflected in the physiological responses of neurons elicited by these substances. Examination of such effects is important, since the distribution of SS-like immunoreactivity at terminal sites often overlaps with the distribution of other neuroactive molecules. Thus, physiological effects observed after application of SS alone may not primarily be caused by this neuropeptide

67

but may actually occur through interaction with other systems (cf. Section V). Again, these actions may be very complex. In rat parietal cortex and dorsal hippocampus, Mancillas et al. (1986) examined the effect of SS on acetylcholine-evoked responses. In these systems, SS inhibits spontaneous firing of nearly all cells tested. Acetylcholine, on the other hand, facilitates spontaneous firing. In contrast to its inhibitory effect on spontaneous firing, sustained iontophoretic application of SS enhances acetylcholine-induced excitation in the majority of the ceils tested. Thus, SS may improve the signal-to-noise ratio by depressing the basal firing in the absence of acetylcholine and increasing the responsiveness of the neurons during simultaneous excitation by acetylcholine. Interaction of SS with the GABAergic system has been suggested by Twery and Gallagher (1990), who observed depressant actions of SS on synaptic responses of neurons in the dorsolateral septal nucleus. Hyperpolarizing responses to GABAA stimulation (after application of the GABAA receptor agonist isoguvacine) and to GABAB stimulation (after application of the GABAB receptor agonist baclofen) are significantly decreased during superfusion of SS-14. 4. Differential effect o f SS-14 and SS-28

In most of the above studies, only the effect of SS-14 on the excitability of neurons or the release/turnover rate of other neuroactive substances has been examined. Jacquin et aL (1988) and Araujo et al. (1990), who investigated the effect of both SS-14 and SS-28, have found the two forms of SS to be equally active. In contrast, Wang et al. (1989), who used cultures of rat cerebral cortical neurons and whole-cell patch-clamp techniques, have observed SS-14 to increase a delayed rectifier K + current (II0 in neurons of the cerebral cortex, while SS-28 reduced IK. Both effects occur in a concentration-dependent manner. Thus, SS-14 and SS28 may induce opposite changes in IK in the same neuron. Pretreatment of the cells with pertussis toxin abolishes both SS-14 and SS-28 modulations of IK, suggesting that receptors for the two types of SS are coupled to IK via GTP-binding proteins. The results of this study are consistent with the notion suggesting the existence of separate binding sites (Leroux et al., 1985) and different cloned receptors (Meyerhof et al., 1992) for the two major forms of SS in mammals. 5. The role o f potassium channels

Hyperpolarizing actions of SS recorded in neurons of the solitary tract complex (Jacquin et al., 1988) and in CA1 pyramidal neurons of the hippocampus (Pittman and Siggins, 1981; Xie and Sastry, 1992) can, at least in part, be explained by the augmentation of a noninactivating, time- and voltage-dependent outward potassium current (M-current, IM) (Jacquin et al., 1988; Moore et al., 1988). Schweitzer et al. (1990, 1993) found evidence that a pathway which generates

68

G.K.H. Zupanc

arachidonic acid is involved in the mechanism leading to an opening of K + channels, since metabolites of arachidonic acid are able to mimic the effect of SS. Interestingly, the muscarinic cholinergic agonists carbachol and muscarine antagonize the action of SS on 1M, a result which implies a reciprocal regulation of the M-current by SS and acetylcholine (Jacquin et aL, 1988; Moore et al., 1988).

6. Effect on calcium concentration Somatostatin has been shown to modify the concentration of intracellular calcium ([Ca2+]i) in several systems. In isolated rat superior cervical ganglion neurons, application of the SS analogue [D-TrpS]-ss 14 results in a rapid, reversible and concentrationdependent reduction of Ca 2+ current (Ikeda et aL, 1987; Ikeda and Schofield, 1989) (Fig. 16). Miyoshi et al. (1989), who measured changes of [Ca2+]i in response to SS on a singie-cell basis by fura-2 fluorometry in cultured rat hippocampal neurons, have found a dosedependent increase in [Ca2+]i after SS application. Since this effect is completely blocked by o~-conotoxin GVIA, SS receptors are thought to couple with N-type voltagesensitive Ca 2+ channels in this system. Modulation of Ca 2+ channels by SS has also been found in various other systems, e.g. in rat hippocampus (Araujo et al., 1990), cholinergic terminals of chick choroid (Gray et al., 1990), rat spinal cord neurons (Sah, 1990), and chick sympathetic ganglia (Golard and Siegelbaum, 1993). Pertussis toxin pretreatment prevents modulation of the calcium current, suggesting involvement of G-proteins in this process (lkeda and Schofield, 1989; Golard and Siegelbaum, 1993; Shapiro and HiUe, 1993). This is in agreement with the identification of cloned mammalian SS receptors as members of the family of G proteincoupled receptors with seven putative membrane-span-

ning regions (for reviews, Bell and Reisine, 1993; Epelbaum et aL, 1994; Hoyer et al., 1994; Reisine and Bell, 1995). The mechanism by which G-protein activation is linked to Ca 2+ channel modulation is controversial. In addition to a membrane-delimited pathway, SS receptors may initiate a second cascade, which also acts through a G protein targeting the Ca 2 + channel. As has been demonstrated by the use of perforated-patch recordings in chick ciliary ganglion neurons, this second soluble pathway involves a cyclic GMP-dependent protein kinase (Meriney et al., 1994).

C. Neuropeptides as regulators of neuronal development." The cases of vasoactive intestinal polypeptide and somatostatin In addition to transmitting information in a fashion similar to 'classical' transmitters, neuropeptides also participate in the establishment of structural elements during ontogenesis of the nervous system and in the expression of neuronal plasticity during adult life. The latter effects show a rather long time-course compared to actions elicited by classical synaptic transmission; they may include genomic effects as well. Interestingly, a similar function has been proposed for several "classical" transmitters (for reviews, Lipton and Kater, 1989; Leslie, 1993). In the following, two peptidergic substances, vasoactive intestinal peptide and somatostatin, will be discussed in detail. 1. Vasoactive intestinal polypeptide (a) Overview. Vasoactive intestinal polypeptide (VIP) was originally isolated from the small intestine of the hog (Said and Mutt, 1970; Mutt and Said, 1974). Besides its existence in the gastrointestinal tract, VIP occurs in various regions of the peripheral and central

B

A

60

1.0 (

vo~ 0.8

o

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0.6

o

~6 20

0.4-

ss

0.2.

o

o

rn 0

0.0 0

100

1 0

~me (s)

2 0

250

-10

-9

-

-7

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F i g . 16. T i m e c o u r s e a n d c o n c e n t r a t i o n r e s p o n s e o f s o m a t o s t a t i n effects o n v o l t a g e - g a t e d C a 2+ c u r r e n t in n e u r o n s o f a d u l t rat

superior cervical ganglia. Voltage-clamp recordings, using the whole-cellpatch-clamp technique, were performed on acutely isolated neuronal somata. A. Time course. A Ca2 + currentwas elicited with a depolarizing step to + 10 mV from a holding potential of -80 mV every 10 s. One minute after the current had stabilized,0.1 Im [D-Trps ]-SS-14was applied to the cell via a micropipette(solidbar). The applicationresultedin a rapid decreasein the current amplitudefollowedby a progressiveincreasein the current amplitude during the continued presence of the SS analogue. B. Concentration-responsecurve. Using a similar paradigm as in A, the maximalSS-inducedreduction of the Ca2+ current was determinedfor various concentrationsof the SS analogue. Each point represents the mean for 2-4 cells;bars representstandard errors of the mean. The maximumattainableblock of Ca2+ current by [D-TrpS]-SS-14was 50%. (Modifiedafter Ikeda et al., 1987.)

Peptidergic Transmission

nervous system, as has been demonstrated by radioimmunoassay and immunocytochemistry (e.g. Larsson et al., 1976, 1977; Said and Rosenberg, 1976; Fuxe et al., 1977; Emson et al., 1978; Schultzberg et al., 1978; Besson et al., 1979; Lor6n et al., 1979; Morrison et al., 1984) (Fig. 17). At the ultrastructural level, this peptide has been localized in LDCVs of nerve endings (Johansson and Lundberg, 1981). Binding sites for VIP have been identified in brain tissue (Taylor and

69

Pert, 1979; Masuo et al., 1992), thus pointing to the existence of VIP receptors in the central nervous system. In membrane preparations from guinea pig brain, VIP stimulates adenylate cyclase activity (DeschodtLanckman et al., 1977). Its biological actions include systemic vasodilation, hypotension, increased cardiac output, respiratory stimulation, and hyperglycemia in the periphery. In the central nervous system, VIP also exhibits diverse effects such as regulation of cerebral

Pia

-

100 IJm

- 2 0 0 IJm

- 300 #m

- 4 0 0 iJm

- 500 ~m

- 600 #m

- 7 0 0 iJm

Fig. 17. Vasoactive intestinal polypeptide-positive neurons in the lateral neocortex of adult rat. Cortical depth, measured from the pia, is marked on the right side. Note that most cell bodies have a major ascending and descending process. These processes extend radially for several hundred micrometers within the plane of section. (Modified after Morrison et al., 1984.)

70

G.K.H. Zupanc

blood circulation, control of central energy metabolism, modification of specific enzymatic activities, modulation of serotoninergic systems, interaction with adrenal steroids, and stimulation of pituitary hormone release (for review, Rost6ne, 1984). Studies from recent years indicate that VIP may also play an important role in the generation and differentiation of neuronal and nonneuronal ceils during embryonic development (for review, Gozes and Brenneman, 1993).

(b) Experimentalstudies. To study the effect of VIP on neuronal development, most investigators have employed an in-vitro approach. One extensively studied system is dissociated spinal cord/dorsal root ganglion cultures. Initial investigations (Brenneman et al., 1985; Brenneman and Eiden, 1986) have shown that blockade of electrical activity in these cultures by application of tetrodotoxin (TTX) results in a significant loss of neurons during embryonic development. This decrease in neuronal cell number can be prevented by addition of VIP to the culture. A similar effect has been observed when the medium of neuron cell cultures treated with TTX is supplemented by conditioned medium from VIP-stimulated astrocyte-enriched cell cultures (Brenneman et al., 1987). These data indicate that VIP released during spontaneous electrical activity may interact in a paracrine fashion with non-neuronal cells to produce neurotrophic substances which promote neuronal survival. Among these gila-derived substances is an interleukin-l-like molecule (Brenneman et al., 1992). In the cerebral cortex of adult rats, interleukin-1 has been shown to stimulate astrogliosis and neovascularization (Giulian et al., 1988). The hypothesis of an indirect effect of VIP via glial cells is supported by the finding of functional receptors for VIP on astroglia (Magistretti et al., 1983). Two mechanisms contributing to the effects observed in the cell cultures have been proposed: In the short-term, VIP acts as a secretagogue for neuron survival-promoting factors. In the long-term, VIP increases astroglial mitosis (Brenneman et al., 1990). Whereas in central cultures VIP appears to act through glial intermediaries, a direct effect of this polypeptide has been suggested for dissociated cell cultures composed of an enriched population of sympathetic precursors derived from superior cervical ganglia (Pincus et al., 1990, 1994). In this system, VIP increases the rate of mitosis, promotes neurite outgrowth, and influences survival of neuroblasts (Figs 18 and 19). As VIP is present in presynaptic fibers that innervate neurons of the superior cervical ganglion, it is possible that the effects are mediated by trans-synaptic regulation. Alternatively, the peptide may exert its effects through autocrine or even intracellular routes, since VIP has been identified both in mature sympathetic neurons and in embryonic sympathetic neuroblasts. Similar stimulatory effects on cell division have been observed in neural and non-neural tissues of explanted embryonic mice (Gressens et al., 1993), and

in a human neuroblastoma cell line (Wollman et al., 1993). (c) Expression o f vasoactive intestinal polypeptide during development. The hypothesis of a mitogenic and/ or neurotrophic function of VIP, based on in-vitro investigations, is in agreement with in-vivo observations on the development of VIP systems in the mammalian central nervous system. While in the rat brain the peptide, as well as its messenger RNA, are undetectable at embryonic stages, they both reach their maxima a few weeks after birth and decrease then to adult levels (Emson et al., 1979; McGregor et aL, 1982; Nobou et al., 1987; Gozes et al., 1987). Similarly, in the cerebral cortex of the macaque monkey, VIP is detectable only at low levels at embryonic stage El20, while it displays very high levels at E165 (the time of birth). During adulthood, only between 9 and 50% of the values measured by radioimmunoassay at E165 are present in the various regions of the cerebral cortex (Hayashi and Oshima, 1986; Hayashi, 1992) (Fig. 20). The drastic rise in VIP immunoreactivity and in the expression of VIP mRNA during late embryogenesis or early postnatal development coincides with the period of rapid brain growth associated with axonal outgrowth and synaptogenesis.

(d) Vasoactive intestinal polypeptide and HIV. It is interesting that VIP reveals its survival-promoting activity not only during normal ontogenesis. It is also capable of counteracting neuronal deficits emerging after infection with human immunodeficiency virus (HIV) (Brenneman et al., 1988). Addition of purified envelope glycoprotein gpl20 of the HIV virus causes significant neural cell death in dissociated hippocampal cultures derived from fetal mice. However, the apparent toxic effect of gpl20 can be antagonized by exogenous VIP. Studies undertaken in vivo corroborate these results (Glowa et al., 1992). Administration of gpl20 into the cerebral ventricles of adult rats impairs the acquisition of spatial control in a learning assay. While similar results are obtained with a VIP antagonist, the impairment can be attenuated by concurrent administration of VIP. 2. Somatostatin

(a) Overview. For several years, SS has been suspected of playing a role in the structural organization of the nervous system during development and/or of the maintenance of neuronal structures during adult life. This hypothesis has been sparked by numerous reports that SS and SS analogues exert an anti-proliferative effect on non-neuronal cells, especially tumor cells, in a variety of tissues (e.g. Mascardo and Sherline, 1982; de Quijada et al., 1983; Redding and Schally, 1983; Mascardo et al., 1984; Morisset, 1984; Liebow et al., 1986, 1989, 1990; Chou et al., 1987; Conteas and Majumdar, 1987; Paz-Bouza et al., 1987; Setyono-Han et al., 1987; Viguerie et al., 1989; Bensa'id et al., 1992; Pan et al., 1992; Weckbecker et al., 1992; Pinski et al., 1993; Ain and Taylor, 1994; Buscail et al., 1994; for

Peptidergie Transmission

71

4

Fig. 18. Effectof vasoactiveintestinalpolypeptideon developmentof neuriticprocesses.Neuroblasts from superiorcervicalganglia of embryonicrats weregrownin controlmedium(A, C) or in mediumcontaining 1 Imvasoactiveintestinalpolypeptide(B, D). The effecton the developmentof neurites was evaluatedby phase contrast microscopyafter 10 hr (A, B) and 24 hr (C, D). Ten hours after plating, neurite initiation is enhanced in the presence of vasoaetive intestinal polypeptide. At 24 hr, the percentage of neuroblasts bearing long processes is significantlyincreasedby vasoaetiveintestinalpolypeptide(13, D). (Modifiedafter Pineus et al., 1990.) review, Schally, 1988). In the nervous system, strong experimental support for a neurotrophic function of SS comes from two studies on invertebrates and one study using PC12 pheochromocytoma cells (see below). The results of these investigations suggest a role of SS for neurite outgrowth. Whether the effect of SS is restricted to this function is unclear. One experimental study examining the effect of SS on the rate of survival in motoneurons of rat spinal cord has found rather minor effects (Weill, 1991). (b) Experimental studies. In the snail Helisoma trivolvis, subsequent to axotomy neurons of the buccal ganglia exhibit rapid regeneration from the cut axonal stump and from sites close to and upon the soma. This regenerative process can be observed both during incubation of isolated ganglia in saline, as well as during long-term cultures of the ganglia in appropriate media. Under both experimental conditions, neurite outgrowth is enhanced by SS (Fig. 21), while concurrent incubation of the preparations with SS and the SS

inhibitor cyclo (7-amino-heptanoyl-L-phenylalanyl-Dtryotophyl-L-lysyl-L-threonyl) acetate results in a sprouting pattern not different from controls (Bulloch, 1987). Similar results have been obtained for dissociated neuron cell cultures of the gastropod Physella heterostropha (Grimm-J~rgensen, 1987). When these cells are incubated in defined medium, only 2.2*/0 of them extend processes. Addition of SS significantly increases the number of cells observed with neurites in 48 hr old cultures. The increase in the number of neuriteextending cells is dose-dependent and is as large as fivefold above values obtained by culturing neurons in control media. SS may exert its neurite-growth-promoting effect by lowering the concentration of intracellular free calcium, thus contributing to the preservation of cytoskeletal components required for the initiation of axonal outgrowth. In PC12 cells, SS increases neurite outgrowth after 2 days in culture and enhances neurite outgrowth after exposure to nerve growth factor (Ferriero et al., 1994). This effect is inhibited by application of SS antiserum and pertussis toxin.

G. K. H. Zupanc

72 1200

Moreover, in a protein kinase A-deficient cell line, SS has not shown a significant effect on neurite outgrowth. These results indicate that SS may function as a neurite extension factor in vertebrates as well; its action appears to be mediated through inhibition o f c A M P synthesis.

tO •~ 1000 0 o

800

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600

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Fig. 19. Effect of vasoactive intestinal polypeptide on mitotic activity as determined by [3H]thymidiueincorporation. For the assay, a dissociatedcell culture systemcomposed of embryonic neuroblasts from superior cervicalganglia was used. Cells were obtained from 15.5-days old rats. Cultures were incubated 48 hrs in control medium (control) or in medium containing 101m vasoactive (VIP) or 10~tg/ml insulin (Insulin). [3H]thymidiue (1 ~tCi/ml) was added at 24 hrs of incubation. At 48 hrs, cells were collected and radioactivity determinedby scintillation spectroscopy. The bars represent the mean incorporation of four culture wells and are expressed as mean counts per minute. The vertical lines on top of the bars indicate the standard error of the mean. Values obtained after incubation with vasoactiveintestinal polypeptidediffer significantly from the control values at P<0.01 (*). Results obtained after insulin treatment differfrom control values at P<0.01 and from vasoactive intestinal polypeptide values at P<0.05 (**; one-wayANOVA and ScheffeF-test). (Modifiedafter Pincus et al., 1990.)

0.8

O

0.4

E D.

Fig. 20. Concentration of vasoactive intestinal polypeptide in the various subdivisions of the cerebral cortex of the macaque monkey at embryonic stages El20 (hatched bars), E165 (open bars), and adult stages (closed bars). The concentration of the peptide was measured by radioimmunoassayand related to the total amount of protein present. The vertical lines on top of the bars represent standard errors of the mean. At E165, which corresponds to the time of birth, the concentration of vasoactive intestinal polypeptide is drastically increased in all cortical areas, compared to the levels found at El20. In the adult monkey, the levels of vasoactiveintestinal polypeptidein the various cortical regions are reduced to 9-50% of the values found at E165, and are thus comparable to the concentrations present at El20. (Modifiedafter Hayashi and Oshima, 1986.)

(c) SomaWstatin and its receptors in the developing cerebellum. Additional evidence for the hypothesis of a developmental role o f SS in vertebrates, though being circumstantial, is derived from observations in the cerebellum. While in mammals the number o f SSpositive structures is low in the adult cerebellum, SSimmunoreactive structures appear in the cerebellar primordium o f fetal rats as early as gestational day 16 (Inagaki et al., 1982). F r o m that time on, the SS-positive structures increase in number and reach their maximum in the first week after birth; afterwards, these structures decrease remarkably in number (Inagaki et al., 1982; Villar et al., 1989). In the macaque monkey, SS immunoreactivity is observed at four months o f embryonic life (full term in this species is 5.5 months), and at that time the concentration measured by radioimmunoassay is up to 18 times (when expressed per g tissue), or up to 40 times (when expressed per g protein) higher than during adulthood (Hayashi, 1987, 1992). It is interesting that gymnotiform fish, whose cerebellum continues to grow beyond early ontogenic stages by the addition of new neurons (Zupanc et aL, in press), display an appreciable amount o f SS immunoreactivity in various cerebellar regions during adulthood (Stroh and Zupanc, 1993). The time course o f the development o f SS-positive structures is paralleled by temporal events observed in the expression o f SS binding sites and SS receptor genes. In the rat, SS binding sites have not been identified in the adult cerebellum (Reubi and Maurer, 1985; Uhl et al., it;, 1988; Gonzalez et al., 1988; Martin et al., 1991). During ontogenesis, autoradiographic labeling is first seen at El5. After birth (E22), the density o f SS binding sites increases drastically between postnatal days P4 and P13, while at P23 the labeling disappears in most lobes of the cerebellum (Gonzalez et al., 1988, 1989) (Fig. 22). The binding sites are present in close association with the external granule cell layer o f the cerebellum, a transient germinal matrix located at the surface o f the cerebellar cortex. The external granule cell layer contains essentially the stem cells of the granule cells. The decrease and disappearance of SS binding sites coincides with the involution of the external granule cell layer. In the course of the latter process, which starts at P10, the internal granule cell layer in the mature cerebellum is formed. A similar temporal pattern has been observed by immuneblotting using antiserum for a 60 kD brain SS receptor (Theveniau and Reisine, 1993), SS immunoreactivity is first detectable at El6, increases in levels at birth, is apparent from P3 to PS, and then disappears. Culture preparations have demonstrated that the binding sites associated with the external granule cell layer o f newborn rats represent functional SS receptors located on immature cerebellar granule cells (Gonzalez

Peptidergic Transmission

73

Fig. 21. Neurite outgrowth from regenerating Helisoma neurons induced by somatostatin. Buccal ganglia were incubated for 20 hrs in saline, supplemented with 0.15 gu bovine serum albumin (control condition) or 1 ~m somatostatin (experimental condition), and the extent of neurite outgrowth displayed after nerve crush by neuron 5 was determined. While a control neuron (A) sprouted 140 gm as measured from the distal axon stump (*), an experimental neuron (B) sprouted 210 Itm. Scale bar, 50 inn. (Reproduced with permission of Elsevier Science, from Fig. 1 in BuUoch, 1987.)

et al., 1992). In these neurons, SS induces a dosedependent inhibition of forskolin-evoked cAMP formation and causes a marked reduction of intracellular calcium concentration. The hypothesis of a functional role of SS and its receptors during the development of the cerebeUum is further supported by observations in Brattleboro rats, which exhibit a selective impairment of the granule cell layer of the cerebellum. Homozygous individuals display, at the age of 13 days, a marked deficiency of the number of SS binding sites compared to heterozygous Brattleboro rats or Long-Evans rat (Gonzalez et aL, 1990). Despite the intriguing correlation between the expression of SS binding sites and the development of the rat cerebeUum, it is important to emphasize that this phenomenon is not universal in vertebrates. The cerebellum of human fetuses, as well as that of adult humans, contains high concentrations of SS binding sites (Laquerri6re et al., 1992, 1994). In adult gymnotiform fish, three cerebellar structures---the eminentia granularis pars medialis, the eminentia granularis pars posterior, and the molecular layer located between these two granule cell layers---show an abundance of SS binding sites (Zupanc et al., 1994) (Fig. 7). While the function of SS binding sites in the adult human cerebellum is unknown, it can be speculated that in

the gymnotiform cerebellum SS may play a neuroregulatory role similar to the one proposed for the embryonic development of the cerebellum in rats. This notion is based on the observation that, during adulthood, the eminentia granularis pars medialis displays extremely high levels of mitotic activity (Zupanc and Horschke, 1995); the eminentia granularis pars posterior, on the other hand, represents the target region which the cells born in the eminentia granularis pars medialis reach after migration through the molecular layer (Zupanc et al., in press). How the results obtained by autoradiographic localization of SS binding sites correspond to the expression of the various molecular forms of SS receptors is unclear. Five subtypes of SS receptors have been cloned (Yamada et al., 1992a,b, 1993; Kluxen et al., 1992; Li et al., 1992; Yasuda et al., 1992; Meyerhof et al., 1992; Vanetti et al., 1992; Bruno et al., 1992; O'Carroll et al., 1992; Corness et al., 1993; Demchyshyn et al., 1993; Rohrer et al., 1993; Xu et al., 1993; Matsumoto et al., 1994; Panetta et al., 1994; for reviews, Bell and Reisine, 1993; Epelbaum et al., 1994; Hoyer et al., 1994; Reisine and Bell, 1995). Surprisingly, despite the very low number of binding sites in the cerebellum of adult rats (Reubi and Maurer, 1985; Uhl et al., 1985; Gonzalez et al., 1988; Martin et al., 1991), in-situ hybridization

74

G . K . H . Zupanc

4D

,.,

13D

23D

B

C

E

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i

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Fig. 22. Transient expression of somatostatin binding sites in the rat cerebellum during development. Sections through the cerebellum were stained with cresyl violet (A--C) or processed for autoradiographic lofa!jT~tion of [125I][Tyr°-D-TrpS]-SS-14 binding sites (D--F). At postnatal day 4, the external granule cell layer (large arrows) appears as a continuous cell layer at the cortex surface (A), and autoradiographic labefing is confined to this layer (D). Maximal labeling density is observed at postnatal day 13 (E), when the external granule ceil layer is maximally developed (13). At postnatal day 23, in the vermis, the external granule cell layer has completely disappeared (C). This is paralleled by a decline in the autoradiographic labeling. Small arrows indicate the internal granule cell layer; the arrowhead points to the paraflocculus. Scale bar, 3 mm. (Reproduced with permission of Elsevier Science, from Fig. 1 in Gonzalez et al., 1988.)

pattern (Chun et al., 1987; Naus et al., 1988; Chtm and Shatz, 1989; Cavanagh and Parnavelas, 1988). Specific binding of SS to crude membranes of cerebral cortex of rats also indicates a transient expression of SS receptors in the early phase of postnatal development (Kimura, 1989). Complementary to these results are observations that show a differential expression of the gene encoding somatostatin receptor SStl (previously termed SSTR-1) in rat cortex (Wulfsen et al., 1993). While during prenatal stages only the superficial cortical layers express this gene, at postnatal days 7 and 14 the pattern of expression is present in all layers. In contrast, around postnatal day 28 the expression pattern is (d) Somatostatin in extracerebellar regions o f the brain confined to the deep cortical layers. during development. During development, one characAside from the cerebellum and cerebral cortex, teristic feature appears to be the early onset of SS transient expression o f SS and/or SS receptors during immunoreactivity, a process that often starts well before ontogenesis has been found in numerous types of the formation of connections between neurons and the neuronal tissue including paravertebral sympathetic establishment of synapses. In the cerebral cortex of the ganglia of embryonic quail (Maxwell et al., 1984), macaque monkey, SS-immunoreactive cells are observed lumbosacral sympathetic ganglia of developing chicken from El20 on, increase in number at late embryonic (New and Mudge, 1986), dorsal root ganglia of the stages and the time period immediately after birth, and rat (Maubert et al., 1992), pyramidal paths of fetal then decrease to a much smaller number during and newborn human spinal cord (Charnay et al., adulthood (Yamashita et al., 1989; Hayashi, 1992) 1988), spinal cord and the sensory derivatives of the (Fig. 23). In cortical areas of other animals conflicting rat (Maubert et al., 1994), peripheral sensory and results exist as to whether SS immunoreactivity increases sympathetic neurons of the rat (Katz et al., 1992), rat during development and then plateaus (McDonald et auditory brainstem (Kungel and Friauf, 1995), nasal al., 1982; Hogan and Berman, 1993), or increases over a and forebrain regions of chick embryo (Murakami certain developmental time period and then decreases to and Arai, 1994), various forebraln areas of mice adult levels, thus displaying a transient-expression (Forloni et al., 1990), and visual system of the rat experiments indicate the expression of at least two subtypes of SS receptors (Meyerhof et al., 1992; Kaupmann et al., 1993; P6rez et al., 1994). The intensity of the signal found is moderate for sst2 (previously termed SSTR-2) mRNA and extremely high for sst3 (previously termed SSTR-3) mRNA. P6rez and colleagues (P6rez et al., 1994) have explained this discrepancy by differences between mRNA and protein turnover. A closer examination of this phenomenon, as well as investigations on the expression of the various SS receptor subtypes during ontogeny, will certainly be required.

Peptidergic Transmission

75

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3.0 ¢-

2.0 Q..

o 1.0 ¢-

O9

0

B E120

E140

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P60

Ad

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/:.: "1 •

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Fig. 23. A. Concentration of somatostatin in the cerebral subdivisions of the macaque monkey at embryonicstages E120 (hatched bars), E165 (open bars), and adult stages (closedbars). The concentration of the peptide was measured by radioimmunoassayand related to the total amount of protein present. The verticallines on top of the bars representstandard errors of the mean. Similaras for vasoactive intestinal polypeptide (el. Fig. 20), in all subdivisions of the cerebral cortex, the concentration of somatostatin is highest at E165. B. Distribution of somatostatin-immunoreactivecells in the posterior parietal cortex at various stages of development(El20, embryonicday 120;E140, embryonicday 140;Nb, newborn; P60, postnatal day 60; Ad, adult stage). Each dot represents a somatostatin-positivecell body. The dotted lines indicate the border between gray matter (GM) and white matter (WM). (Modifiedafter Yamashita et al., 1989.)

(Ferriero and Sagar, 1987; Ferriero et al., 1990; Bodenant et al., 1991). However, transient expression has not been found in the retina of chicken (Morgan et al., 1983) and guinea pig (Spira et al., 1984). Senba and colleagues (Senba et aL, 1982), who studied the ontogeny of SS in the spinal cord of rat, have observed a number of SS immunoreactive structures that are only transiently expressed while others maintain their immunoreactivity in adult life at a level comparable to the one found during embryonic development. Similar observations have been made in the forebrain and diencephalon of rats (Shiosaka et al., 1982). It is possible that these different time courses in the development of SS systems reflect different functions in which different subtypes of SS receptors are involved. Use o f probes specific for individual subtypes o f SS receptors are, therefore, likely to shed light on these aspects.

(e) Somatostatin in Alzheimer's disease. In senile dementia, as well as in Alzheimer's disease, low levels of SS have been found in the cerebrospinal fluid (Oram et al., 1981; W o o d et al., 1982; Francis et al., 1984; Serby et al., 1984; Soininen et al., 1984, 1988; Cramer et al., 1985; Beal et al., 1986; Bissette et al., 1986; Davis et al., 1988; Gomez et al., 1986; Sunderland et al., 1987). Decreased concentrations of SS (Davies et al., 1980; Rossor et al., 1980; Candy et al., 1985; Roberts et al., 1985; Beal et al., 1986; Chan-Palay, 1987; Tamminga et al., 1987) and reduced levels o f SS binding sites (Beal et al., 1985; Krantic et al., 1992) have been demonstrated in various cortical and subcortical regions o f human postmortem brains of Alzheimer's patients. Furthermore, SS immunoreactivity has been found in neuritic plaques of Alzheimer's patients (Morrison et al., 1985). The clinical relevance of these observations is unclear, but generally they have been regarded as

76

G . K . H . Zupane

epiphenomena (for reviews, Beal, 1990; Rubinow et al., 1992; Auchus et aL, 1994). However, in addition to a neurotransmitter-like function, SS may also exert neurotrophic effects in cortical tissue. Alterations in the SS system could, thus, be a primary cause for the cognitive impairments observed in Alzheimer's patients. Defining the exact role of SS in the pathology of this disease may be facilitated by the results obtained following recent attempts to produce transgenic mice displaying Alzheimer-like features (Games et aL, 1995; LaFerla et al., 1995).

VII. SYNAPTIC VERSUS NONSYNAPTIC ACTION: AN EVOLUTIONARY PERSPECTIVE Chemical synapses are means for intercellular communication via neuroactive substances. Morphologically, a neuro-neuronal chemical synapse consists of membrane specializations of two juxtaposed neurons, separated by a narrow synaptic cleft, and distinguished by the presence of synaptic vesicles in the presynaptic element and of receptors associated with the postsynaptic membrane. Following release of the contents of the synaptic vesicles, the neuroactive substance diffuses to the postsynaptic site and binds to the respective receptors. As a result, a cellular response is generated in the postsynaptic neuron. The main difference between this point-to-point transmission exerted by synapses and the nonsynaptic mechanism of interneuronal signalling is found in the spatial relationship between the presynaptic neuron and the postsynaptic neuron. Synaptic transmission is directed towards a small target region. Due to the short distance required for diffusion, a fast response in the postsynaptic neuron is generated. Parasynaptic or nonsynaptic release of peptides may delay the cellular response of the "postsynaptic" neuron considerably; the extent of this time lag mainly depends on the distance the neuroactive substance crosses. Since in nonsynaptic transmission typically many neurons scattered over a wide region are involved at the postsynaptic site, the action is nondirected or diffuse. However, the elements involved in nonsynaptic transmission are very similar to the elements used in synaptic transmission: A "presynaptic" element which mediates the release of the neuroactive substance by discharge of synaptic vesicles, and a "postsynaptie" element equipped with appropriate receptors and the machinery necessary to translate binding of the respective ligands into a cellular response. The structural correlate of nonsynaptic peptidergic transmission, therefore, resembles that of a synapse with a giant synaptic cleft. The most extreme realization of this idea is found in neuroendocrine cells. They release peptide hormones into the blood stream, thus mediating signals to distant targets. Evolution proceeds by modification of pre-existing structures. Even subtle structural changes can result in major functional alterations. In the case of neuropeptides, it is an attractive hypothesis to assume that

peptidergic transmission by point-to-point mechanisms evolved from processes in which a long-distance action of peptides was exerted. If this assumption is correct, the principal elements used for synaptic signalling were already existent prior to the "invention" of this mode of transmission; modifications primarily brought "presynaptic" and "postsynaptic" neurons closer to each other. It is likely that both mechanisms of peptidergic transmission have begun to coexist very early in the evolution of nervous systems. Coelenterates, the lowest animal group having a nervous system, release peptides not only nonsynaptically, but they also display synaptic structures (Jha and Mackie, 1967). These terminals are filled with SCVs as well as LDCVs. In higher invertebrates and in vertebrates, synaptic and nonsynaptic mechanisms of peptidergic transmission have continued to coexist. This clearly underlines the importance of each of the two processes. Unravelling the details of the various modes of peptidergic transmission and exploring the functional significance of this process is certain to remain a major challenge for neurobiologists beyond the twentieth century. Acknowledgements---I am grateful to Stephen J. Duguay, Heinz Schwarz, Thomas Stroh, Cecilia Uhilla, Lutz Vollrath, and Marianne M. Zupanc for their comments on the manuscript, and to Ingrid Horschke for her assistance in managing the reference database. Karl-Heinz Nill helped in the preparation of several figures. Ira B. Black, Floyd E. Bloom, Andrew G. M. BuUoch, Emanuel DiCiccoBloom, Pietro V. de Camilli, Regis B. Kelly, philippe Leroux, Hantao Liu, Michela Matteoli, Adalberto Merighi, John H. Morrison, Lelio Orci, Julia M. Polak, Christiane Waleh-Solimena, Thomas Stroh, and Carl B. Watt kindly contributed figures from their original works to this review. Financial support for the author's own investigations was provided by the Howard Hughes Medical Institute, the United States National Institutes of Health, the Canadian Medical Research Council, the Friedrich Ebert Foundation, the Max Planck Society, and the Bundesministerium fiir Forschung und Technologie. Part of this review has been written while the author was a Visiting Professor with the Department of Anatomy and Neurobiology of the University of Ottawa and a Visiting Scholar with the Howard Hughes Medical Institute of the University of Chicago and the Scripps Institution of Oceanography of the University of California, San Diego.

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