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Putative neuroendocrine cells in the Aplysia cerebral ganglion
liEUROSCIENCE RESERRCH ELSEVIER
Neuroscience Research 25 (1996) 209 216
Putative neuroendocrine cells in the
Aplysia cerebral ganglion
Seppo Soinila a'b'*, George J. Mpitsos a aMark O. Hatfield Marine Science Center, Oregon State University, Newport, OR 97365, USA bDepartments of Anatomy and Neurology, University of Helsinki, Haartmaninkatu 4, 00290 Helsinki. Finland Received 5 January 1996; accepted 1 April 1996
Abstract
We report on a special population of cells in the Aplysia cerebral ganglion that are characterized by several features compatible with neuroendocrine function. These cells can be recognized in living ganglia by their small size, white color and their typical distribution as a compact cluster in the central medial region of the dorsal ganglion surface. Upon intracellular recording, these cells generate action potentials of relatively long duration (about 25 ms), as compared with the faster action potentials of larger white cells or of non-white cells (about 4 ms). Intracellular injection of the small white cells with Lucifer yellow after recording revealed a dual projection area: single cells have one process which branches extensively into m~ny varicose terminals as it courses through the neuropil, and then sends varicose terminals to the vascular sheath at the periphery of the ganglion. In cryostat sections, these cells were specifically characterized by their content of large granules, the staining characteristics of which distinguish them from lipochondria or lysosomes. Their ability to bind fluorochromes nonspecifically is of particular importance for the interpretation of histochemical localization studies based on immunofluorescence techniques.
Keywords: Mollusk; Fluorescein isothiocyanin; Identified neuron; Histochemistry; Electrophysiology
1. Introduction
The use of the nervous systems of marine mollusks as neurobiological models is based on several advantages of these systems (Kandel, 1979): many neurons have relatively constant morphological features, such as cell size, pigmentation or location, which allow their repeatable identification in living whole-ganglion preparations. Gross-anatomical identification can be supplemented by intracellular labeling of individual neurons to reveal their projections, by intracellular recordings to characterize their electrophysiological activity and connections, and by immunohistochemical stainings to define their neurotransmitters. As a result, several model systems are available in which the synaptic connections of the individual neurons, their transmitter content and firing pattern are known. Such * Corresponding author. Tel.: +358 0 191 8463; fax: + 358 0 4714009.
systems of identified neurons have opened up possibilities to study how hierarchically different neurons work as a group to produce various forms of behavior and how each individual neuron affects the output response produced by the group (Mpitsos, 1989; Mpitsos and Cohan, 1986b,a; Mpitsos et al., 1988). The present work was initiated by our observation that certain cells in sections cut through the cerebral ganglion of Aplysia californica contain large granules which give these cells characteristic staining properties and make them anatomically identifiable. Our original observation of these cells came from studies of histamine immunofluorescence (Soinila et al., 1990), in which we found that certain cells exhibit nonspecific histamine immunofluorescence. Using extensive controls, we have now analyzed the nature of the nonspecific fluorescence and discuss problems that might arise generally from nonspecific binding in immunohistochemical studies. In addition, we have identified these cells under phase contrast optics in whole ganglia,
0168-0102/96/$15.00 © 1996 Elsevier Science Ireland Ltd. All rights reserved
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S. Soinila, G.J. Mpitsos / Neuroscience Research 24 (1996) 209-216
described their projections using intraceUular injections of fluorescent dyes, and characterized their electric activity. The evidence indicates that these cells form a specific population of neuroendocrine cells, which, due to their convenient location and identifiability, may be a useful model to study neurohormonal modification of nervous functions. Historically, our observations were preceded almost 60 years ago by those of Scharrer (Scharrer, 1935) who suggested the presence of neuroendocrine cells in the cerebral ganglion of Aplysia limacina and Pleurobranchaea meckeli. The present study provides clarification and functional extension of the observations made at that time.
2. Experimental procedures The marine mollusks Aplysia californica and Pleurobranchaea californica, both ranging between 50-200 g in weight, were used in this study. The animals were obtained from Dr. Rim Fay of Pacific Biomarine Laboratories (Venice, Ca) and maintained in an open seawater system until used. The animals were anaesthetized with 0.3 M MgC12 (1/3 body weight), the nervous system was dissected out into sea water and pinned on a Sylgard-coated dish.
2.1. Gross-anatomical identification and electrophysiology The cerebral ganglion was bathed in artificial sea-water (Instant Ocean R from Aquarium Systems, Inc.) containing 0.3 M sucrose and its dorsal surface was desheathed mechanically. Each hemiganglion contained on its dorsal surface a compact cluster of white cells located close to the medial margin of the ganglion. The small size of these cells (30-50 mm in diameter) distinguished them from other, larger white cells found on this surface. Intracellular recording was performed using standard electrophysiological methods and micropipettes filled with potassium chloride (0.8 M) or Lucifer Yellow (Sigma; at 50% saturated solutions at 40°C). Physiological recordings were made in artificial sea water solutions maintained at 12°C. Following intracellular recording, some cells were iontophoretically injected with Lucifer Yellow by passing hyperpolarizing current from the recording micropipette for at least 2 h before fixation.
2.2. Whole-mount preparations The ganglia were fixed overnight at 4°C with Bouin's solution containing 30 parts saturated picric acid, 10 parts 37% formaldehyde and 2 parts glacial acetic acid. The ganglia were then rinsed extensively with phosphate-buffered saline (PBS), pH 7.3, until the yellow
color of the picric acid had disappeared, and transferred into PBS containing 20% sucrose. Some ganglia were incubated with 10 - 6 M solution of tetramethylrhodamine isothiocyanate (TRITC) for 2-24 h. The ganglia were dehydrated in alcohol-xylene series.
2.3. Serial sections The ganglia were fixed as described above. Some ganglia were frozen unfixed in liquid nitrogen. Complete serial sections (18 mm thickness) were cut with a cryostat and collected on gelatin-coated glass slides and subjected to one of the following staining procedures: 1% Eosin B (Fisher) in distilled water for 5 min; 1% Toluidine Blue O (Allied Chemical Corp.) in distilled water for 2 min; saturated solution of Sudan Black B (Allied Chemical Corp.) in 70% ethanol according to Ackerman's method (Pearse, 1980); saturated solution of Sudan III (Matheson, Coleman and Bell) in alcoholacetone according to Kay's and Whitehead's method (Pearse, 1980). Some series of sections were shortly rehydrated in PBS containing 0.25% Triton X-100 (Triton-PBS), and subjected to a routine indirect immunofluorescence procedure (Coons, 1958) which we used for molluscan nervous tissue in previous studies (Soinila et al., 1990; Soinila and Mpitsos, 1988). Briefly, the sections were incubated with 10% nonimmune swine serum for 10 min at room temperature, and with nonimmune rabbit serum, a substitute for the primary antiserum, diluted 1:500 in PBS-Triton overnight at 4°C. After rinsing with PBS-Triton, the sections were incubated with swine anti-rabbit IgG conjugated with either tetramethylrhodamine isothiocyanate (TRITC) (DAKO R156) or fluorescein isothiocyanate (FITC) (DAKO F205), both diluted 1:50 in PBS-Triton. The same batch of each conjugated fluorochrome was used throughout the study to eliminate variation in the protein-fluorochrome ratio as a source of non-specific staining (Hebert et al., 1967). After incubation for 60 min at room temperature, the slides were rinsed with PBS, embedded in glycerol-PBS (50/50). Peroxidase-antiperoxidase (PAP) procedure was performed on some sections as described by Sternberger (Sternberger, 1994). FITC and TRITC concentrations of the secondary antibody solutions used in the routine immunohistochemical procedure were determined using a Beckman DU-6 spectrophotometer at 500 and 550 nm, respectively, and a series of known concentrations of the corresponding unconjugated FITC and TRITC (Sigma). Accordingly, sections through the cerebral ganglion were then incubated with 1.8 x 10 - 6 M unconjugated TRITC or with 2.5 x 10-6 M unconjugated FITC. For control experiments, PBS-Triton was supplemented with 0.5 M NaC1.
Fig. 1. Granular cells in the Aplysia cerebral ganglion. (A) Low-power photomicrograph of the dorsal surface of a fresh ganglion showing a cluster of small white cells (arrow head), larger white cells (arrow) and large pigmented cells (asterisk). (B) Micrograph of a section containing small granular cells as shown by Nomarski optics (arrow head). Regular neurons shown with these optics are nongranular and barely visible (asterisk). (C) The same section as in (B) viewed through a catecholamine filter. The small granular cell shown by the arrow head in (B) has been iontophoretically injected with Lucifer Yellow. (D) A section through the neuropil region of the ganglion shown in (B) and (C) showing the axonal ramification of the cell marked in (B) and (C). (E) Three semiconsecutive sections showing the projection of the cell shown in (B) and (C) in the wall of an invagination of the haemolymph compartment (large arrow). Small arrows indicate the anterior contour of the interganglionic connective. Bar = 1 mm (A), or 50 /~m (B-E).
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S. Soinila, G.J. Mpitsos / Neuroscience Research 24 (1996) 209-216
For fluorescence microscopy, the specimens were examined with a Zeiss Universal microscope equipped with an HBO mercury lamp for epiillumination and Zeiss filter blocks 487712 for TRITC (excitation maximum 546 nm, emission barrier > 590 nm), filter block 487709 for FITC (excitation maximum 450-490 nm, emission barrier > 520 nm) and filter block 487702 for catecholamines (excitation maximum 365 nm, emission barrier > 420 nm). The same microscope was used for examination with phase contrast and differential interference contrast optics.
3. Results
3. I. Whole-mount preparations and electrophysiology Each Aplysia cerebral hemiganglion contained on its dorsal surface a compact cluster of about 20 small white cells. These cells were distinguished from other, larger white cells by their size (approximately 50 mm versus 100 mm in diameter) and their location in the central medial region (Fig. 1A). Larger white cells were found routinely just posterior to these cells on the midline, and white cells having intermediate sizes were found laterally and somewhat anteriorly. Examination of whole, fixed ganglia under phase contrast optics revealed that the small white cells have granular cytoplasm, while at magnifications ranging from 250 x to 400 x all other cells showed nongranular cytoplasm. Intracellular recording from the small granular cells showed that they generate action potentials in response to depolarizing electrical currents passed through the recording microelectrode. Their action potentials were of relatively long duration (25 ms), as compared with those of the larger white cells whose action potentials were approximately 4 ms in duration (Fig. 2). Examination of whole ganglia after iontophoretic application of Lucifer yellow revealed that both the small and the larger white cells have a single axon projecting towards the neuropil region.
3.2. Neuroanatomical experiments To obtain better resolution of the projection areas of Lucifer-injected cells, the tissue was fixed and a complete series of sections was cut through the ganglion. Examination of serial sections using phase contrast, differential interference or Nomarski optics revealed in each cerebral hemiganglion a cluster of about 20-30 granule-containing cells measuring 40-70 mm in diameter (Fig. 1B). Fig. 3 shows a schematic reconstruction obtained from the serial sections, indicating that these cells were located in the central medial region of the dorsal surface and showed approximately symmetrical distribution in the two hemiganglia. The cytoplasm
of these cells was densely packed with large granules, the size of which reached up to few micrometers in diameter (Fig. 4A). Such granular cells were not found in the Pleurobranchaea cerebral ganglion, or in any other ganglia of either species, although these other ganglia contained white cells having sizes equivalent to the intermediate and larger white cells found in Aplysia. The granular cells in Aplysia stained intensely with eosin, while the nongranular cells remained unstained. On the other hand, Toluidine blue stained the cytoplasm of the nongranular cells intensely blue and the cytoplasm of the granular cells pale blue, but left the granules themselves unstained. The granular cells did not stain with Sudan III or Sudan black B, while regions of cytoplasm in nongranular neurons stained intensely brown with the former and completely black with the latter stain (data not shown). The Lucifer Yellow-filled cells and their processes were readily visible in sections when they were epiilluminated with UV-light and viewed through the catecholamine filter. The same section could be viewed alternatively or simultaneously using transillumination with bright light or differential interference optics (Fig. 1B, C). This revealed that all small white cells injected with Lucifer yellow had a coarsely granular cytoplasm, while the larger white cells had a nongranular cytoplasm. Furthermore, the small white cells had a single axon which projected into the neuropil region and often could be traced to the connective tissue sheath of the vascular compartment (Fig. 1D). The close proximity of the processes with the haemocele was particularly evident in sections showing the anterior edge. of the haemocele compartment just in front of the interganglionic connective (Fig. 1E). The larger white cells also had a single axon which projected to a large area of the neuropil. Neither the small nor the large white cells extended processes into the nerve roots. Separate series of sections were stained with several antisera specific for classical or putative neurotransmitters, such as histamine, dopamine, acetylcholine, vasoactive intestinal polypeptide, cholecystokinin, somatostatin, substance P, methionine-enkephalin, FM-
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Fig. 2. lntracellular recording from a large white cell (A) and small granular white cells (B). Calibration = 100 ms.
S. Soinila, G.J. Mpitsos /Neuroscience Research 25 (1996) 209-216
213
confined to the cytoplasmic granules and was clearly more intense than the slight autofluorescence showed by these cells (Fig. 4B-D). Also the granular cells of unfixed ganglia stained with unconjugated fluorochromes, although less intensely.
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4. Discussion
4. I. Neuroendocrme characteristics of granular cells
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CEREBRAL GANGLION Fig. 3. A schematic presentation of the Aplysia cerebral ganglion. The set of four drawings summarizes the information obtained from the examination of a complete series of sections through the ganglion. The top drawing represents the most dorsal zone, and the bottom drawing the most ventral zone. The black circles represent the fluorochrome-binding granular cells on the dorsal surface or subsurface zones, atn, anterior tentacular nerve; cbc, buccal-cerebral connective; iln, inferior labial nerve; on, optic nerve; pdc, cerebral-pedal connective; plc, cerebral pleural connective; ptn, posterior tentacular nerve; sin, superior labial nerve; stcn, statocyst nerve.
RFamide and small cardioactive peptide B. None of these antisera stained the granular cells, when the PAP method was used. The granular cells, however, showed distinct staining when immunofluorescence method with either TRITC- or FITC-labeled secondary antiserum was used. To further analyze this obviously nonspecific staining, solutions of unconjugated TRITC and FITC were prepared that contained the same concentrations of fluorochromes as the secondary antibody solution of the actual immunohistochemical procedure. Both solutions resulted in intense staining that was
We have characterized a specific population of cells in the Aplysia cerebral ganglion which show morphological and electrophysiological features of neuroendocrine function. The cytoplasm of these cells is packed with granules, which are considerably larger than those in previously described Aplysia neuroendocrine cells, such as the neurons R3 to RI5 or the bag cells in the abdominal ganglion (Coggeshall, 1967; Coggeshall et al., 1966; Dorsett, 1986; Frazier et al., 1967; Kupfermann and Kandel, 1970; Smock and Arch, 1977). These granules exhibit several characteristic staining properties: they are strongly eosinophilic and fail to stain with basophilic dyes, indicating that the granular membrane or its contents have a net positive charge. This, and the observation that the cytoplasm of the granular cells exhibits only weak basophilic staining, suggest that the product(s) of these cells is not a peptide or protein. In this respect, the granule-containing cells differ from previously reported molluscan neurosecretory cells, which are strongly basophilic (Rittenhouse and Price, 1986; Smock and Arch, 1977). The granule-containing cells did not stain with the lipid stains, Sudan III or Sudan black, indicating that the granules are not lipochondria or .lysosomes (Baur, 1977). Granular cells have been previously described, in the Aplysia cerebral ganglion, which show similar distributions to the cells described here (Simpson et at., 1963). These cells stained intensely with paraldehyde-fuchsin, which also stains several vertebrate (Pearse, 1980) and other molluscan neuroendocrine cells (Chiu and Strumwasser, 1981; Coggeshall et al., 1966). The observation that the granular cells reported in our study were strongly eosinophilic while the previously described Aplysia neuroendocrine cells are basophilic suggests that the former cells are a distinct class of neurons, although due to their location they may be part of so-called Hanstrom's X-organ described in opistobranch species by Scharrer (Scharrer, 1935). Intracellular injection of the fluorescent dye Lucifer Yellow revealed that each granular cell has one axon which projects into the ganglion neuropil. This indicates that these cells are neurons, rather than pure endocrine cells. Moreover, the ramification of the granular cells could be followed in serial sections into the connective tissue sheath between the ganglion and the
214
S. Soinila, G.J. Mpitsos / Neuroscience Research 24 (1996) 209-216
Fig. 4. Four photomicrographs of the same horizontal section through the Aplysia cerebral ganglion showing a cluster of granular cells (black arrowheads in A). (A) and (B) are controls, i.e. subjected to incubation with buffer only and taken with differential interference contrast optics and FITC-specific fluorescence optics, respectively. White arrowheads in (B) point out autoftuorescence in regular neurons and small granular cells. (C) and (D) are fluorescence micrographs taken after incubation with unconjugated FITC and TRITC, respectively. Bar = 50 mm.
haemolymph compartment, suggesting secretory function. Although secretion is not directly demonstrated by our observations, it is strongly suggested by projection of granular cell fibers to the wall of the haemocele, particularly evident on the anterior edge of this compartment (Scharrer, 1935; Dorsett, 1986; Willows, 1986). Interestingly, the two previously reported neuroendocrine cell types of the Aplysia abdominal ganglion, the white neurons R3-R 15 and the bag cells, also have similar
dual projection (Rittenhouse and Price, 1986; Chiu and Strumwasser, 1981; Coggeshall, 1967). Our intracellular recordings confirm the neuronal nature of the granular cells: each cell generated spontaneously action potentials. However, the duration of the action potentials was several times as long as that of neighboring neurons. This is also characteristic of previously described neuroendocrine cells of the Aplysia abdominal ganglion (Coggeshall et al., 1966; K u p f e r m a n n and Kandel, 1970).
s. Soinila, G.J. Mpitsos / Neuroscience.Research 25(1996)209-216 4.2. Implications of immunofluorescence staining Nonspecific staining of the granular cells in Aplysia in immunofluorescence procedures points to several important implications. First, lack of staining when the PAP procedure was applied excludes the possibility that staining with F I T C or T R I T C is due to nonimmunological binding of the secondary antibody, a phenomenon occurring in m a n y vertebrate neurons (Aarli et al., 1975) and endocrine cells (Grube, 1980). Rather, both fluorochromes T R I T C and F I T C were bound by these cells to the same extent regardless of whether they were conjugated to an immunoglobulin. Nonspecific fluorochrome binding m a y be based on hydrophobic mechanisms (Riggs et al., 1958), since both T R I T C and F I T C are polar molecules. In accordance with the c o m m o n experience that formaldehyde increases tissue hydrophobicity (Sternberger, 1994), we found similarly distributed but more intense fluorochrome binding in fixed, as compared to unfixed tissue. Nonspecific fluorochrome binding was not found in any other cells in any other Aplysia ganglia or in any cells of Pleurobranchaea. In addition, none of these other cells, some of which are known to be neurosecretory, appeared granular under phase contrast optics. Therefore, a second implication is that the small granular cells in Aplysia californica, and possibly those described by Scharrer (Scharrer, 1935) in Aplysia limacina, may be a functionally unique class of neurosecretory cells, or at least a class of neurosecretory cells that are physicochemically distinct from other types. It is important to note that no nonimmunological binding of fluorochromes has been reported in previous studies applying the indirect immunofluorescence technique on Aplysia nervous tissue. This may be due to differences in fixation and staining procedures, or in sampling of control sections. Nevertheless, the third implication of the present results points to the importance of controlling for specificity in complete serial sections through each ganglion in which staining has been observed. Lastly, the nonspecific binding of the granular cells may prove useful in studies of endocrine functions. Being restricted to the granular cells themselves, the nonspecific binding of T R I T C and F I T C provides a useful histological marker for the granular cell population. Since the granular material remains chemically unidentified and since its actual secretion has not yet been demonstrated, we still must consider the granular cells putatively neuroendocrine. However, their convenient location, distinct morphological identity and several unique staining properties make them a useful model for studies of the neurohumoral modulation of the nervous system. This is expected to be especially useful in testing our previous proposal that a set of
215
neurons having constant connections might produce qualitatively different activity patterns by slight changes in parameters that affect the dynamic state of the system (Mpitsos et al., 1988; Mpitsos, 1989; Mpitsos and Cohan, 1986b,a). Neuroendocrine cells, which act relatively nonselectively and produce relatively longlasting effects, m a y provide a way for the nervous system to perform such adjustments.
Acknowledgements This study was supported by the grant A F O S R 890262 to G J M and by grants from the Finnish Cultural Foundation and University of Helsinki to SS. We thank Dr. Lavern Weber for making facilities available to us at the Hatfield Marine Science Center.
References Aarli, J.A.., Aparicio, S.R., Lumsden, C.E. and Tonder, O. (1975) Binding of normal human IgG to myelin sheaths, glia and neurons. Immunology, 28: 171-185. Baur, P.S.J. (1977) Lipochondria and the light response of Aplysia giant neurons, J. Neurobiol., 8: 19-42. Chiu, A.Y. and Strumwasser, F. (1981) An immunohistochemical study of the neuropeptidergic bag cells of Aplysia. J. Neurosci., 1: 812-826. Coggeshall, R.E., Kandel, E.R., Kupfermann, I. and Waziri, R. (1966) A morphological and functional study on a cluster of identifiable neurosecretory cells in the abdominal ganglion of Aplysia. J. Cell Biol., 31: 363-368. Coggeshall, R.E. (1967) A light and electron microscope study of the abdominal ganglion of Aplysia californica. J. Neurophysiol., 30: 1263-1287. Coons, A.H. (1958) Fluorescent antibody methods. In: J.F. Danielli (Ed.), General Cytochemical Methods, Academic Press, New York, pp. 399-422. Dorsett, D.A. (1986) Brains to cells: The neuroanatomy of selected gastropod species. In: A.O.D. Willows (Ed.). The Mollusca, Academic Press, New York, pp. 101-187. Frazier, W.T., Kandel, E.R., Kupfermann, 1., Waziri, R. and Coggeshall, R.E. (1967) Morphological and functional properties of identified neurons in the abdominal ganglion of Aplysia californica. J. Neurophysiol., 30: 1288-1351. Grube, D. (1980) Immunoreactivities of gastrin (G-) ceils. II. Nonspecific binding of immunoglobulins to G-cells by ionic interactions. Histochemistry, 66:149 167. Hebert, G.A., Pittman, B. and Cherry, W.B. (1967) Factors affecting the degree of nonspecific staining given by fluorescein isothiocyanate labelled globulins. J. Immunol., 98:1204-1212. Kandel, E.R. (1979) Behavioral Biology of Aplysia, Freeman & Co., San Francisco. Kupfermann, I. and Kandel, E.R. (1970) Electrophysiologicalproperties and functional interconnections of two symmetrical neurosecretory clusters (bag cells) in abdominal ganglion of Aplysia. J. Neurophysiol., 33: 865-876. Mpitsos, G.J. and Cohan, C.S. (1986a) Comparison of differential Pavlovian conditioning in whole animals and physiologicalpreparations of Pleurobranchaea: implications of motor pattern variability. J. Neurobiol., 17: 499-516.
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Mpitsos, G.J. and Cohan, C.S. (1986b) Convergence in a distributed nervous system: parallel processing and self-organization. J. Neurobiol., 17:517 545. Mpitsos, G.J., Creech, H.C., Cohan, C.S. and Mendelson, M. (1988) Variability and chaos: Neurointegrative principles in self-organization of motor patterns. In: J.A.S. Kelso, A.J. Mandell and M.F. Schlesinger (Eds.), Dynamic Patterns in Complex Systems, World Scientific, Singapore, pp. 162 190. Mpitsos, G.J. (1989) Chaos in brain function and the problem of nonstationarity: A commentary. In: E. Basar and T.H. Bullock (Eds.), Dynamics of Sensory and Cognitive Processing by the Brain, Springer Verlag, New York, pp. 521-535. Pearse, A.G.E. (1980) Histochemistry, Theoretical and Applied, Churchill & Livingstone, Edinburgh. Riggs, J.L., Seiwald, R.J., Burckhalter, J.H., Downs, C.M. and Metcalf, T.G. (1958) Isothiocyanate compounds as fluorescent labeling agents for immune serum. Am. J. Pathol., 32:1081 1097. Rittenhouse, A.R. and Price, C.H. (1986) Electrophysiological and anatomical identification of the peripheral axons and target tis-
sues of Aplysia neurons R2-R14 and their status as multifunctional, multimessenger neurons. J. Neurosci., 6: 2071-2084. Scharrer, B. (1935) Uber das Hanstr6msche Organ X bei Opistobranchiern. Pubbl. Stat. Zool. Napoli, 15: 132-142. Simpson, L., Bern, H.A. and Nishioka, R.S. (1963) Inclusions in the neurons of Aplysia californica (Cooper, 1983) (Gastropoda Opistobranchiata). J. Comp. Neurol., 121: 237-257. Smock, T. and Arch, S. (1977) A cytochemical study of the bag cell organs of Aplysia californica. J. Histochem. Cytochem., 25: 1345-1350. Soinila, S. and Mpitsos, G.J. (1988) Immunohistochemical study on vasoactive intestinal polypeptide in the buccal ganglion of Aplysia californica and Pleurobranchaea californica. Soc. Neurosci. Abstr., 14: 986. Soinila, S., Mpitsos, G.J. and Panula, P. (1990) Comparative study of histamine immunoreactivity in nervous systems of Aplysia and Pleurobranchaea. J. Comp. Neurol., 298: 83-96. Sternberger, L.A. (1994) Immunocytochemistry, Wiley, New York. Willows, A.O.D. (1986) The Mollusca, Academic Press, New York.
Neuroscience Research 25 (1996) 209 216
Putative neuroendocrine cells in the
Aplysia cerebral ganglion
Seppo Soinila a'b'*, George J. Mpitsos a aMark O. Hatfield Marine Science Center, Oregon State University, Newport, OR 97365, USA bDepartments of Anatomy and Neurology, University of Helsinki, Haartmaninkatu 4, 00290 Helsinki. Finland Received 5 January 1996; accepted 1 April 1996
Abstract
We report on a special population of cells in the Aplysia cerebral ganglion that are characterized by several features compatible with neuroendocrine function. These cells can be recognized in living ganglia by their small size, white color and their typical distribution as a compact cluster in the central medial region of the dorsal ganglion surface. Upon intracellular recording, these cells generate action potentials of relatively long duration (about 25 ms), as compared with the faster action potentials of larger white cells or of non-white cells (about 4 ms). Intracellular injection of the small white cells with Lucifer yellow after recording revealed a dual projection area: single cells have one process which branches extensively into m~ny varicose terminals as it courses through the neuropil, and then sends varicose terminals to the vascular sheath at the periphery of the ganglion. In cryostat sections, these cells were specifically characterized by their content of large granules, the staining characteristics of which distinguish them from lipochondria or lysosomes. Their ability to bind fluorochromes nonspecifically is of particular importance for the interpretation of histochemical localization studies based on immunofluorescence techniques.
Keywords: Mollusk; Fluorescein isothiocyanin; Identified neuron; Histochemistry; Electrophysiology
1. Introduction
The use of the nervous systems of marine mollusks as neurobiological models is based on several advantages of these systems (Kandel, 1979): many neurons have relatively constant morphological features, such as cell size, pigmentation or location, which allow their repeatable identification in living whole-ganglion preparations. Gross-anatomical identification can be supplemented by intracellular labeling of individual neurons to reveal their projections, by intracellular recordings to characterize their electrophysiological activity and connections, and by immunohistochemical stainings to define their neurotransmitters. As a result, several model systems are available in which the synaptic connections of the individual neurons, their transmitter content and firing pattern are known. Such * Corresponding author. Tel.: +358 0 191 8463; fax: + 358 0 4714009.
systems of identified neurons have opened up possibilities to study how hierarchically different neurons work as a group to produce various forms of behavior and how each individual neuron affects the output response produced by the group (Mpitsos, 1989; Mpitsos and Cohan, 1986b,a; Mpitsos et al., 1988). The present work was initiated by our observation that certain cells in sections cut through the cerebral ganglion of Aplysia californica contain large granules which give these cells characteristic staining properties and make them anatomically identifiable. Our original observation of these cells came from studies of histamine immunofluorescence (Soinila et al., 1990), in which we found that certain cells exhibit nonspecific histamine immunofluorescence. Using extensive controls, we have now analyzed the nature of the nonspecific fluorescence and discuss problems that might arise generally from nonspecific binding in immunohistochemical studies. In addition, we have identified these cells under phase contrast optics in whole ganglia,
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described their projections using intraceUular injections of fluorescent dyes, and characterized their electric activity. The evidence indicates that these cells form a specific population of neuroendocrine cells, which, due to their convenient location and identifiability, may be a useful model to study neurohormonal modification of nervous functions. Historically, our observations were preceded almost 60 years ago by those of Scharrer (Scharrer, 1935) who suggested the presence of neuroendocrine cells in the cerebral ganglion of Aplysia limacina and Pleurobranchaea meckeli. The present study provides clarification and functional extension of the observations made at that time.
2. Experimental procedures The marine mollusks Aplysia californica and Pleurobranchaea californica, both ranging between 50-200 g in weight, were used in this study. The animals were obtained from Dr. Rim Fay of Pacific Biomarine Laboratories (Venice, Ca) and maintained in an open seawater system until used. The animals were anaesthetized with 0.3 M MgC12 (1/3 body weight), the nervous system was dissected out into sea water and pinned on a Sylgard-coated dish.
2.1. Gross-anatomical identification and electrophysiology The cerebral ganglion was bathed in artificial sea-water (Instant Ocean R from Aquarium Systems, Inc.) containing 0.3 M sucrose and its dorsal surface was desheathed mechanically. Each hemiganglion contained on its dorsal surface a compact cluster of white cells located close to the medial margin of the ganglion. The small size of these cells (30-50 mm in diameter) distinguished them from other, larger white cells found on this surface. Intracellular recording was performed using standard electrophysiological methods and micropipettes filled with potassium chloride (0.8 M) or Lucifer Yellow (Sigma; at 50% saturated solutions at 40°C). Physiological recordings were made in artificial sea water solutions maintained at 12°C. Following intracellular recording, some cells were iontophoretically injected with Lucifer Yellow by passing hyperpolarizing current from the recording micropipette for at least 2 h before fixation.
2.2. Whole-mount preparations The ganglia were fixed overnight at 4°C with Bouin's solution containing 30 parts saturated picric acid, 10 parts 37% formaldehyde and 2 parts glacial acetic acid. The ganglia were then rinsed extensively with phosphate-buffered saline (PBS), pH 7.3, until the yellow
color of the picric acid had disappeared, and transferred into PBS containing 20% sucrose. Some ganglia were incubated with 10 - 6 M solution of tetramethylrhodamine isothiocyanate (TRITC) for 2-24 h. The ganglia were dehydrated in alcohol-xylene series.
2.3. Serial sections The ganglia were fixed as described above. Some ganglia were frozen unfixed in liquid nitrogen. Complete serial sections (18 mm thickness) were cut with a cryostat and collected on gelatin-coated glass slides and subjected to one of the following staining procedures: 1% Eosin B (Fisher) in distilled water for 5 min; 1% Toluidine Blue O (Allied Chemical Corp.) in distilled water for 2 min; saturated solution of Sudan Black B (Allied Chemical Corp.) in 70% ethanol according to Ackerman's method (Pearse, 1980); saturated solution of Sudan III (Matheson, Coleman and Bell) in alcoholacetone according to Kay's and Whitehead's method (Pearse, 1980). Some series of sections were shortly rehydrated in PBS containing 0.25% Triton X-100 (Triton-PBS), and subjected to a routine indirect immunofluorescence procedure (Coons, 1958) which we used for molluscan nervous tissue in previous studies (Soinila et al., 1990; Soinila and Mpitsos, 1988). Briefly, the sections were incubated with 10% nonimmune swine serum for 10 min at room temperature, and with nonimmune rabbit serum, a substitute for the primary antiserum, diluted 1:500 in PBS-Triton overnight at 4°C. After rinsing with PBS-Triton, the sections were incubated with swine anti-rabbit IgG conjugated with either tetramethylrhodamine isothiocyanate (TRITC) (DAKO R156) or fluorescein isothiocyanate (FITC) (DAKO F205), both diluted 1:50 in PBS-Triton. The same batch of each conjugated fluorochrome was used throughout the study to eliminate variation in the protein-fluorochrome ratio as a source of non-specific staining (Hebert et al., 1967). After incubation for 60 min at room temperature, the slides were rinsed with PBS, embedded in glycerol-PBS (50/50). Peroxidase-antiperoxidase (PAP) procedure was performed on some sections as described by Sternberger (Sternberger, 1994). FITC and TRITC concentrations of the secondary antibody solutions used in the routine immunohistochemical procedure were determined using a Beckman DU-6 spectrophotometer at 500 and 550 nm, respectively, and a series of known concentrations of the corresponding unconjugated FITC and TRITC (Sigma). Accordingly, sections through the cerebral ganglion were then incubated with 1.8 x 10 - 6 M unconjugated TRITC or with 2.5 x 10-6 M unconjugated FITC. For control experiments, PBS-Triton was supplemented with 0.5 M NaC1.
Fig. 1. Granular cells in the Aplysia cerebral ganglion. (A) Low-power photomicrograph of the dorsal surface of a fresh ganglion showing a cluster of small white cells (arrow head), larger white cells (arrow) and large pigmented cells (asterisk). (B) Micrograph of a section containing small granular cells as shown by Nomarski optics (arrow head). Regular neurons shown with these optics are nongranular and barely visible (asterisk). (C) The same section as in (B) viewed through a catecholamine filter. The small granular cell shown by the arrow head in (B) has been iontophoretically injected with Lucifer Yellow. (D) A section through the neuropil region of the ganglion shown in (B) and (C) showing the axonal ramification of the cell marked in (B) and (C). (E) Three semiconsecutive sections showing the projection of the cell shown in (B) and (C) in the wall of an invagination of the haemolymph compartment (large arrow). Small arrows indicate the anterior contour of the interganglionic connective. Bar = 1 mm (A), or 50 /~m (B-E).
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For fluorescence microscopy, the specimens were examined with a Zeiss Universal microscope equipped with an HBO mercury lamp for epiillumination and Zeiss filter blocks 487712 for TRITC (excitation maximum 546 nm, emission barrier > 590 nm), filter block 487709 for FITC (excitation maximum 450-490 nm, emission barrier > 520 nm) and filter block 487702 for catecholamines (excitation maximum 365 nm, emission barrier > 420 nm). The same microscope was used for examination with phase contrast and differential interference contrast optics.
3. Results
3. I. Whole-mount preparations and electrophysiology Each Aplysia cerebral hemiganglion contained on its dorsal surface a compact cluster of about 20 small white cells. These cells were distinguished from other, larger white cells by their size (approximately 50 mm versus 100 mm in diameter) and their location in the central medial region (Fig. 1A). Larger white cells were found routinely just posterior to these cells on the midline, and white cells having intermediate sizes were found laterally and somewhat anteriorly. Examination of whole, fixed ganglia under phase contrast optics revealed that the small white cells have granular cytoplasm, while at magnifications ranging from 250 x to 400 x all other cells showed nongranular cytoplasm. Intracellular recording from the small granular cells showed that they generate action potentials in response to depolarizing electrical currents passed through the recording microelectrode. Their action potentials were of relatively long duration (25 ms), as compared with those of the larger white cells whose action potentials were approximately 4 ms in duration (Fig. 2). Examination of whole ganglia after iontophoretic application of Lucifer yellow revealed that both the small and the larger white cells have a single axon projecting towards the neuropil region.
3.2. Neuroanatomical experiments To obtain better resolution of the projection areas of Lucifer-injected cells, the tissue was fixed and a complete series of sections was cut through the ganglion. Examination of serial sections using phase contrast, differential interference or Nomarski optics revealed in each cerebral hemiganglion a cluster of about 20-30 granule-containing cells measuring 40-70 mm in diameter (Fig. 1B). Fig. 3 shows a schematic reconstruction obtained from the serial sections, indicating that these cells were located in the central medial region of the dorsal surface and showed approximately symmetrical distribution in the two hemiganglia. The cytoplasm
of these cells was densely packed with large granules, the size of which reached up to few micrometers in diameter (Fig. 4A). Such granular cells were not found in the Pleurobranchaea cerebral ganglion, or in any other ganglia of either species, although these other ganglia contained white cells having sizes equivalent to the intermediate and larger white cells found in Aplysia. The granular cells in Aplysia stained intensely with eosin, while the nongranular cells remained unstained. On the other hand, Toluidine blue stained the cytoplasm of the nongranular cells intensely blue and the cytoplasm of the granular cells pale blue, but left the granules themselves unstained. The granular cells did not stain with Sudan III or Sudan black B, while regions of cytoplasm in nongranular neurons stained intensely brown with the former and completely black with the latter stain (data not shown). The Lucifer Yellow-filled cells and their processes were readily visible in sections when they were epiilluminated with UV-light and viewed through the catecholamine filter. The same section could be viewed alternatively or simultaneously using transillumination with bright light or differential interference optics (Fig. 1B, C). This revealed that all small white cells injected with Lucifer yellow had a coarsely granular cytoplasm, while the larger white cells had a nongranular cytoplasm. Furthermore, the small white cells had a single axon which projected into the neuropil region and often could be traced to the connective tissue sheath of the vascular compartment (Fig. 1D). The close proximity of the processes with the haemocele was particularly evident in sections showing the anterior edge. of the haemocele compartment just in front of the interganglionic connective (Fig. 1E). The larger white cells also had a single axon which projected to a large area of the neuropil. Neither the small nor the large white cells extended processes into the nerve roots. Separate series of sections were stained with several antisera specific for classical or putative neurotransmitters, such as histamine, dopamine, acetylcholine, vasoactive intestinal polypeptide, cholecystokinin, somatostatin, substance P, methionine-enkephalin, FM-
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S. Soinila, G.J. Mpitsos /Neuroscience Research 25 (1996) 209-216
213
confined to the cytoplasmic granules and was clearly more intense than the slight autofluorescence showed by these cells (Fig. 4B-D). Also the granular cells of unfixed ganglia stained with unconjugated fluorochromes, although less intensely.
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4. Discussion
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CEREBRAL GANGLION Fig. 3. A schematic presentation of the Aplysia cerebral ganglion. The set of four drawings summarizes the information obtained from the examination of a complete series of sections through the ganglion. The top drawing represents the most dorsal zone, and the bottom drawing the most ventral zone. The black circles represent the fluorochrome-binding granular cells on the dorsal surface or subsurface zones, atn, anterior tentacular nerve; cbc, buccal-cerebral connective; iln, inferior labial nerve; on, optic nerve; pdc, cerebral-pedal connective; plc, cerebral pleural connective; ptn, posterior tentacular nerve; sin, superior labial nerve; stcn, statocyst nerve.
RFamide and small cardioactive peptide B. None of these antisera stained the granular cells, when the PAP method was used. The granular cells, however, showed distinct staining when immunofluorescence method with either TRITC- or FITC-labeled secondary antiserum was used. To further analyze this obviously nonspecific staining, solutions of unconjugated TRITC and FITC were prepared that contained the same concentrations of fluorochromes as the secondary antibody solution of the actual immunohistochemical procedure. Both solutions resulted in intense staining that was
We have characterized a specific population of cells in the Aplysia cerebral ganglion which show morphological and electrophysiological features of neuroendocrine function. The cytoplasm of these cells is packed with granules, which are considerably larger than those in previously described Aplysia neuroendocrine cells, such as the neurons R3 to RI5 or the bag cells in the abdominal ganglion (Coggeshall, 1967; Coggeshall et al., 1966; Dorsett, 1986; Frazier et al., 1967; Kupfermann and Kandel, 1970; Smock and Arch, 1977). These granules exhibit several characteristic staining properties: they are strongly eosinophilic and fail to stain with basophilic dyes, indicating that the granular membrane or its contents have a net positive charge. This, and the observation that the cytoplasm of the granular cells exhibits only weak basophilic staining, suggest that the product(s) of these cells is not a peptide or protein. In this respect, the granule-containing cells differ from previously reported molluscan neurosecretory cells, which are strongly basophilic (Rittenhouse and Price, 1986; Smock and Arch, 1977). The granule-containing cells did not stain with the lipid stains, Sudan III or Sudan black, indicating that the granules are not lipochondria or .lysosomes (Baur, 1977). Granular cells have been previously described, in the Aplysia cerebral ganglion, which show similar distributions to the cells described here (Simpson et at., 1963). These cells stained intensely with paraldehyde-fuchsin, which also stains several vertebrate (Pearse, 1980) and other molluscan neuroendocrine cells (Chiu and Strumwasser, 1981; Coggeshall et al., 1966). The observation that the granular cells reported in our study were strongly eosinophilic while the previously described Aplysia neuroendocrine cells are basophilic suggests that the former cells are a distinct class of neurons, although due to their location they may be part of so-called Hanstrom's X-organ described in opistobranch species by Scharrer (Scharrer, 1935). Intracellular injection of the fluorescent dye Lucifer Yellow revealed that each granular cell has one axon which projects into the ganglion neuropil. This indicates that these cells are neurons, rather than pure endocrine cells. Moreover, the ramification of the granular cells could be followed in serial sections into the connective tissue sheath between the ganglion and the
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S. Soinila, G.J. Mpitsos / Neuroscience Research 24 (1996) 209-216
Fig. 4. Four photomicrographs of the same horizontal section through the Aplysia cerebral ganglion showing a cluster of granular cells (black arrowheads in A). (A) and (B) are controls, i.e. subjected to incubation with buffer only and taken with differential interference contrast optics and FITC-specific fluorescence optics, respectively. White arrowheads in (B) point out autoftuorescence in regular neurons and small granular cells. (C) and (D) are fluorescence micrographs taken after incubation with unconjugated FITC and TRITC, respectively. Bar = 50 mm.
haemolymph compartment, suggesting secretory function. Although secretion is not directly demonstrated by our observations, it is strongly suggested by projection of granular cell fibers to the wall of the haemocele, particularly evident on the anterior edge of this compartment (Scharrer, 1935; Dorsett, 1986; Willows, 1986). Interestingly, the two previously reported neuroendocrine cell types of the Aplysia abdominal ganglion, the white neurons R3-R 15 and the bag cells, also have similar
dual projection (Rittenhouse and Price, 1986; Chiu and Strumwasser, 1981; Coggeshall, 1967). Our intracellular recordings confirm the neuronal nature of the granular cells: each cell generated spontaneously action potentials. However, the duration of the action potentials was several times as long as that of neighboring neurons. This is also characteristic of previously described neuroendocrine cells of the Aplysia abdominal ganglion (Coggeshall et al., 1966; K u p f e r m a n n and Kandel, 1970).
s. Soinila, G.J. Mpitsos / Neuroscience.Research 25(1996)209-216 4.2. Implications of immunofluorescence staining Nonspecific staining of the granular cells in Aplysia in immunofluorescence procedures points to several important implications. First, lack of staining when the PAP procedure was applied excludes the possibility that staining with F I T C or T R I T C is due to nonimmunological binding of the secondary antibody, a phenomenon occurring in m a n y vertebrate neurons (Aarli et al., 1975) and endocrine cells (Grube, 1980). Rather, both fluorochromes T R I T C and F I T C were bound by these cells to the same extent regardless of whether they were conjugated to an immunoglobulin. Nonspecific fluorochrome binding m a y be based on hydrophobic mechanisms (Riggs et al., 1958), since both T R I T C and F I T C are polar molecules. In accordance with the c o m m o n experience that formaldehyde increases tissue hydrophobicity (Sternberger, 1994), we found similarly distributed but more intense fluorochrome binding in fixed, as compared to unfixed tissue. Nonspecific fluorochrome binding was not found in any other cells in any other Aplysia ganglia or in any cells of Pleurobranchaea. In addition, none of these other cells, some of which are known to be neurosecretory, appeared granular under phase contrast optics. Therefore, a second implication is that the small granular cells in Aplysia californica, and possibly those described by Scharrer (Scharrer, 1935) in Aplysia limacina, may be a functionally unique class of neurosecretory cells, or at least a class of neurosecretory cells that are physicochemically distinct from other types. It is important to note that no nonimmunological binding of fluorochromes has been reported in previous studies applying the indirect immunofluorescence technique on Aplysia nervous tissue. This may be due to differences in fixation and staining procedures, or in sampling of control sections. Nevertheless, the third implication of the present results points to the importance of controlling for specificity in complete serial sections through each ganglion in which staining has been observed. Lastly, the nonspecific binding of the granular cells may prove useful in studies of endocrine functions. Being restricted to the granular cells themselves, the nonspecific binding of T R I T C and F I T C provides a useful histological marker for the granular cell population. Since the granular material remains chemically unidentified and since its actual secretion has not yet been demonstrated, we still must consider the granular cells putatively neuroendocrine. However, their convenient location, distinct morphological identity and several unique staining properties make them a useful model for studies of the neurohumoral modulation of the nervous system. This is expected to be especially useful in testing our previous proposal that a set of
215
neurons having constant connections might produce qualitatively different activity patterns by slight changes in parameters that affect the dynamic state of the system (Mpitsos et al., 1988; Mpitsos, 1989; Mpitsos and Cohan, 1986b,a). Neuroendocrine cells, which act relatively nonselectively and produce relatively longlasting effects, m a y provide a way for the nervous system to perform such adjustments.
Acknowledgements This study was supported by the grant A F O S R 890262 to G J M and by grants from the Finnish Cultural Foundation and University of Helsinki to SS. We thank Dr. Lavern Weber for making facilities available to us at the Hatfield Marine Science Center.
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