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Somatostatin-like immunoreactivity in the forebrain of Pseudemys turtles
h’eurosckncr Vol. 9, No. 2, pp. 297-307, Printed in Great Britain
0306-4522/83
1983
$3.00+0.00
Pergamon Press Ltd cc’ 1983 IBRO
SOMATOSTATIN-LIKE TMMUNOREACTIVITY IN THE FOREBRAIN OF PSEUDEMYS TURTLES M. F. BEAR and F. F. EBNER Section
of Neuroscience,
Division
of Biology and Medicine, RI 02912, U.S.A.
Brown
University,
Providence,
Abstract-Previous investigations of cortical organization in the brain of the turtle have revealed many features typical of mammalian neocortex. Recent evidence suggests that many neocortical neurons contain neuroactive peptides. The possibility that one such peptide, somatostatin, is found in the turtle brain was tested using immunocytochemical techniques. Intense somatostatin-like immunoreactivity was observed in many neurons and fibers in turtle cortex, as well as in several forebrain nuclei. Cortical neurons with several different dendritic configurations showed immunoreactive labelling, including bipolar, stellate and pyramidal cell types. In addition, stained cells and processes were observed in close association with the
ependyma of the lateral ventricle. Other forebrain regions containing immunoreactive neurons included the dorsal ventricular ridge, the basal telencephalic nuclei and the hypothalamus. These data support the idea that peptidergic neurons existed in the pallium of an ancestor common to modern mammals and reptiles. We speculate that somatostatin plays function of all types of cortex and suggest that turtle cortex may provide of this cortical neuropeptide.
The ICamino acid peptide somatostatin was first identified as the hypothalamic growth hormone release inhibiting factor by Brazeau, Vale, Burgus, Ling, Butcher, Rivier and Guillemin“ in 1973. It has since been shown by radioimmunoassay’ and by immunocytochemistry*,” that this peptide has a widespread distribution in neurons of the mammalian central nervous system. Several lines of evidence suggest a transmitter role for somatostatin. For example, somatostatin has been localized in synaptic terminals”’ and is released from synaptosomal preparations by high [K +].3 In addition, it is released from brain slices in a calcium-dependent fashion” and has a potent physiological effect when applied by iontophoresis onto cortical neurons.33 The presence of numerous somatostatin neurons in mammalian neocortex* raises the question of whether equivalent peptidergic neurons exist in reptiles. To investigate this possibility, we undertook the following immunocytochemical study in Pseudemys turtles, a species whose cortex we have studied in some detail. A partial report of these data has been published in abstract form.’ EXPERIMENTAL
2448 h at 4°C. After rinsing briefly in distilled water, the brain was cut on a freezing microtome at either 50 or 100pm. Tissue sections were then washed in phosphate buffered saline (PBS) and incubated overnight in normal turtle serum diluted I : 5 with PBS at 4°C. After a PBS rinse, sections were reacted for immunocytochemistry either mounted on gelatin-coated slides or free floating, according to the unlabeled antibody enzyme method of Sternberger.35 Rabbit anti-somatostatin antibody was generated against a carbodiamide conjugate with keyhole limpet hemocyanin and synthetic somatostatin-14 (Immuno Nuclear lot No. 29189). All antibody dilutions were made in PBS that contained 0.27; Triton X-100 (Sigma Chemicals). Tissue sections were first incubated overnight at 4°C in rabbit anti-somatostatin diluted I: 100-500. After a I h wash in PBS, the sections were incubated in goat anti-rabbit IgG (Miles-Yeda) diluted 1: 50 for 1h at room temperature. The tissue was again washed in PBS and incubated in rabbit peroxidase antiperoxidase (Miles-Yeda) diluted 1: 100 for I h at room temperature. After a final PBS wash, sections were reacted for 1&I 5 min in an incubation medium consisting of 2.0 ml PBS, pH. 7.3, 1.2 mg. 3,3’-diaminobenzidine and 0.03% H,O,. This reaction was stopped by transferring the sections to fresh PBS. Finally, sections were dehydrated through a series of alcohols, cleared in xylene and coverslipped. Sections were examined with a Leitz Orthoplan microscope using both normal bright-field and interference contrast optics. In some cases, adjacent sections were stained with cresyl violet acetate to enhance the localization of nuclear borders.
PROCEDURES
Adults turtles (Pseudemy.7 scripta) of both sexes were anesthetized with sodium pentobarbital, cooled to 6°C and perfused though the ascending aorta with 6°C saline followed by I I. of cold 4% phosphate buffered paraformaldehyde, pH 7.4. The brain was removed and immersed in the same fixative containing 30% sucrose for
Three strategies were employed to address the question of specificity. To test the possibility that the observed staining resulted from non-immunologic reactions of the histochemical reagents, the rabbit anti-somatostatin antiserum was omitted from the immunocytochemical protocol. To test the specificity of the primary antiserum, 0.5ml of diluted rabbit anti-somatostatin was adsorbed with 100 pg of synthetic somatostatin-14 (Sigma) for 24 h prior to the immunocytochemical reaction. Although a diffuse background of HRP reaction product frequently persisted after the second test, both procedures eliminated all perikaryal and fiber staining. Finally. as a biological control’R
Correspondence: F. F. Ebner Biology and Medicine, Box G Brown University Providence, RI 02912, U.S.A. Abbreaiofions: CSF, cerebrospinal fluid; GABA y-aminobutyric acid; HRP, horseradish peroxidase; PBS, phosphate buffered saline. YS(9/2
297 <
a similar role in the normal a useful model for the study
298
M. F. Bear and F. F. Ebner
2.0mm
Fig. I. Coronal sections through the turtle forebrain to show the distribution of somatostatin-containing neurons. Immunoreactive neurons are charted on the left; cytoarchitectural subdivisions on the right. Inset: lateral view of the turtle brain showing the approximate level of each frontal section. Abbreviations are: h, hippocampus; sb, subiculum; gc, general cortex; pa th, pallial thickening; dvr, dorsal ventricular ridge; pyr, pyriform cortex; BTN, basal telencephalic nuclei; ot, optic tract; pv, periventricular nucleus. Both scale bars = 2 mm.
Somatostatin
in turtle
immunocytochemical staining was performed in the rat brain yielding results consistent with those reported in the literature.2.‘7
The possibility of antibody cross reactivity cannot be excluded, and the “somatostatin-like” immunoreactivity reported here is referred to as “somatostatin” reservation clearly in mind.
with
this
RESULTS Cells and fibers displaying somatostatin-like immunoreactivity were observed in several regions of the turtle forebrain. Figure 1 illustrates the overall distribution of immunoreactive cells in a series of coronal sections taken from one representative case. In the telencephalon, labeled cells were observed in cortex, the dorsal ventricular ridge, and among the nuclei of the basal telencephalon. Labeled neurons were distributed in all types of cortex: lateral or olfactory, dorsal or general, and medial or hippocampal. In dorsal and hippocampal cortex, these cells were concentrated in the main cell layer, but were also commonly observed in the molecular layer and subcellular zone. The frequency of immunoreactive cortical cells appeared very similar in sections over a wide anterior-posterior extent of the hemisphere. In the dorsal ventricular ridge, stained cells were observed both in the anterior part of the core nucleus and the corticoid region. In the basal telencephalon, occasional labeled neurons were observed in the “globus pallidus”3’ and “area d”.34 In the diencephalon, immunoreactive cells were clustered in the nucleus periventricularis hypothalami. Stained fibers arising from these neurons were traced posteriorly to where they form a dense plexus over the median eminence. The dorsal thalamus and the other nuclei of the hypothalamus were largely free of immunoreactive neurons. Immunoreactive neurons in cerebral cortex Many cortical neurons displaying somatostatinlike immunoreactivity fell into morphologically distinct categories. Bipolar or fusiform neurons were the most frequently identified. Such cells with dendrites oriented parallel to the ventricular surface were particularly common in the subcellular zone of dorsal cortex. This zone consists of a narrow strip of neuropil that separates the main layer of cortical neurons from the underlying ependymal cells. Bipolar cells with radially-oriented dendrites (Fig. 2a) were found most frequently in the molecular and the main cell layers of dorsal cortex and hippocampus. The majority of the immunoreactive cortical cell bodies were located in the main cell layer as shown in Fig. 1. Unfortunately, the processes of these cells rarely contained enough reaction product to characterize the dendritic morphology. Occasionally, a cell would be stained adequately to identify it as a pyramidal neuron. Figure 2(b) shows an example of such a cell from the subiculum. Interestingly, den-
forebrain
299
dritic spines were not labeled on any immunoreactive neurons. When the brains were cut into 100pm sections, some cortical cells presented a striking Golgi-like image (Figs 2c and d). Figure 2(c) is a photomicrograph of an immunoreactive cell situated in the molecular layer of general cortex. A camera lucida drawing of this cell (Fig. 2d) demonstrates the extent of the dendritic field that could be visualized in this single section. This molecular-layer cell belongs to the broad category of stellate neurons and is representative of a third morphological type of immunoreactive cortical cell. Neurons displaying somatostatin immunoreactivity were also consistently observed in the pyriform cortex. These cells also ranged in dendritic morphology from stellate to pyramidal. Staining of periependymal telencephalon
cells and axons in the
A surprisingly high proportion of the immunoreactive cells in the dorsal and medial cortices were found in close association with the ependymal cell bodies that line the lateral ventricle. Figure 3(a) shows a neural element in close apposition to the ependymal cell layer. The ependymal perikarya are viewed as a flat sheet of cells in this fortuitous sagittal section that cut the ependymal layer of the medial wall tangentially. A labeled process, presumably an axon, may be seen coursing along mosaic image formed by the ependymal cell bodies. This axon gives rise to a number of branches, several of which appear to end in bulbous swellings. Figure 3(b) is a photomicrograph of an immunoreactive neuron from the same case. Again, it appears to lie in close contact with the ependyma. In fact, in almost every section regardless of the plane, stained cortical neurons were observed to give rise to processes projecting among ependyma and ending near the ventricle-ependymal interface. Occasionally periependymal cells emitted immunoreactive processes that projected radially and appeared to traverse the entire thickness of cortex to terminate at the pial surface. Examples of these fibers from hippocampal cortex appear in Figs 3(c) and 3(d). Immunoreactive neurons in the dorsal ventricular ridge basal telencephalon Other cell groups in the telencephalon consistently contained immunoreactive cells. Labeled neurons were occasionally observed in the dorsal ventricular ridge, particularly in the dorsolateral region that blends with the pallial thicknening. These cells were often among the corticoid cell clusters, but immunoreactive neurons were also observed in the core nucleus, and as in cortex, in close association with the ependyma. Dendritic morphology of immunoreactive dorsal ventricular ridge neurons was also quite varied, including both pyramidal and stellate cell types.
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M. F. Bear and F. F. Ebner
Fig. 2. Immunoreactive cell types in turtle cortex. Labeled cells fall into three broad morphological categories: bipolar a, pyramidal b, and stellate c. Figure 2(d) is a camera lucida reconstruction of the neuron photographed in 2(c) showing the extent of visible dendrites in this single 100 nm-thick section. Scale bars: a = 20 pm, b = 10 pm, c = 20 pm. Fig. 3. Somatostatin-like immunoreactivity in periependymal cells and fibers. a. Labeled axon coursing among the ependyma cell bodies. A neuron adjacent to this ependymal lining is illustrated in b. Figures 3(c) and (d) are low power photomicrographs of frontal sections through hippocampal cortex. The pial surface is at the top and in c, the lateral ventricle lies just below the lower margin of the picture. Immunoreactive fibers which arise from periependymal cells may be seen traversing the entire thickness of cortex and ramifying close to the pial surface. The dark band at the bottom of 3(c) was not eliminated by routine antibody controls and thus reflects non-specific staining. Scales bars: a = IS pm, b = 10 pm. c=20pm, d= 15pm.
2d 301
i-,
3c 1 II
’
.
. .
Fig. 4. Immunoreactive neurons in the basal telencephalon. These cells, from two different cases, lie in area d. Both emit long stained fibers that course toward the ventrolateral surface of the brain. Scale bars = 15 pm.
303
immunoreactivity in the turtle hypothalamus. Laheled cells in the periw mtric ular Fig. 5. somatostati’n-like to terminate in a dense plexus over the em give rise to axons that sweep ventrally and posteriorly median eminence. The third ventricle is indicated by a star. Scale bar = 100 pm.
IlUCl
304
Somatostatin
in turtle forebrain
A few immunoreactive neurons were observed in the basal telencephalon. Specifically, labeled cells were found in the ventrolateral, large celled region termed “globus pallidus” by Powers and Reined’ and in area d of Riss et ~1.~~which is ventroiateral to the floor of the lateral ventricle. Examples of labeled neurons from areas are presented in the photomicrographs of Fig. 4. These cells give rise to long processes that course ventrolaterally (down and to the left in the micrographs). In Fig. 4(a), the labeled fibers project across the lateral forebrain bundle, and one of the beaded processes in Fig. 4(b) could be followed almost to the pial surface. Somutostatin-like cephalon
immunoreactiuity
in
the
dien -
Somatostatin immunoreactivity in the turtle diencephalon was largely confined to the hypothalamus. Specifically, labeled neurons were observed in the nucleus periventricularis hypothalami (Fig. 5). Stained processes from these cells often could be traced through the ependyma to the surface of the third ventricle. However, most observable fibers arising from the periventricular cells swept ventrally and posteriorly to form a rich terminal plexus in the median eminence.
DISCUSSION Turtle dorsal cortex receives thalamic inputs, some of which are known to relay sensory information.” The same area of turtle cortex receives noradrenergic input from the Iocus coeruleus,27~Z serotoninergic input from the raphe nuclei,26 and an input, possibly cholinergic, from the basal telencephalon’s~26. In addition, a population of non-pyramidal cells with aspinous dendrites is present in turtle cortex that contains glutamate decarboxylase, the synthesizing enzyme for y-aminobutyrate (GABA) (L. M. Smith, C. Houser, R. L. Patrick and F. F. Ebner, unpublished observations). Each of these chemical and connectional features of turtle cortex is shared by mammalian neocortex. The results from the present study reveal yet another striking similarity to neocortex; namely, the presence of somatostatin-containing neurons and processes. Somatostatin-containing neurons in mammalian neocortex are located primarily in layers II, III and VI. These cells have a dendritic morphology that characterizes them as stellate, bipolar or pyramidal neurons.* Though turtle cortex consists of only a single prominent cell layer, the immunoreactive neurons still fall into these three morphological categories. Interestingly, somatostatin-positive neurons in the mammalian hippocampus, another mono-layer cortex, fall in the same three classes’ suggesting that morphological diversity is a characteristic of somatostatin-positive neurons wherever they are found in the forebrain. Furthermore, the recent local-
30.5
ization of somatosatin-like immunoreactivity to neurons of the dorsal pallium in the frog, Rana temporuria,37 and the lizard, CtenQsuur~s pectinata,” supports the idea that peptidergic neurons may have existed in the pallium of an ancestor common to modern terrestrial vertebrates, A previous immunocytochemical study indicated that many, if not all, of the stellate cells in turtle cortex contain glutamate decarboxylase and release GABA at their synapses (Unpublished observations). In this light, the finding of somatostatin-like immunoreactivity in this cell type is particularly interesting. The two obvious inte~retations are that either two (or more) chemically distinct populations of stellate cell exist in turtle cortex or that GABA and somatostatin are contained within the same neurons. The latter possibility is not without precedent; Graybiel, Ahmad-Mirza and Elde” have provided a logical argument that these two molecules coexist in the cells of the cat nucleus reticularis thalami. The resolution of this question in turtle cortex awaits direct evidence from double labelling procedures. The function of somatostatin in cortex remains obscure. Renaud et a1.33 have reported a potent inhibition of spontaneous unit activity after iontophoresis of somatostatin in neocortex, whereas, in the hippocampus Dodd and Kelly’ report neuronal depolarization after application of this peptide. Havliceki4 suggests that somatostatin may interact with central noradrenergic mechanisms. Turtle cortex has eight times the norepinephrine content of mammalian neocortex per unit wet weight.” Radioimmunoassay in the tortoise brain indicates that the concentration of extrahypothalamic brain somatostatin is about twice that in the rat,” and our immunocytochemical results indicate that the relative concentration of somatostatin-positive neurons is considerably higher in cortex than in the subcortical regions. Taken together, these data suggest that turtle cortex might be a valuable preparation to investigate further the interactions of norepinephrine, GABA, and somatostatin. Per~ependym~~ cells In this study we have identified many cells and processes with somatostatin-like immunoreactivity that lie within or in close apposition to the ependymal lining of the lateral and third ventricles. In some cases, axons from nearby neurons are observed to contact the ependyma and occasionally appear to terminate at the ventricular surface. Such a relationship has been described for cells in the third ventricle of several vertebrate species. These cells have been termed “liquor-contacting” neurons by Knowles.23 In other cases, stained fibers are seen projecting radially from the zone of periependymal cells to the surface of cerebral cortex. The characteristics of these elements are strikingly similar to the neurosecretory neurons described by Vigh et a1.39in the spinal cord of turtles. Our data suggest that many
M. F. Bear and F. F. Ebner
306
cells in the turtle cerebral cortex and hypothalamus contain somatostatin. lmmunoreactive somatostatin has been found in the cerebrospinal fluid (CSF)29 and in the intercellular clefts of several brain regions, including cerebral cortex.24 Since it is likely that the extracellular space of the brain is exposed to CSF,6 the proposal has been made that the CSF might provide one route for this peptide to reach its target cells and thus produce periphysiological effects.22 The immunoreactive ependymal cells we have observed in the turtle forebrain must be considered as one likely source of somatostatin in both ventricular and subarachnoid CSF, and free somatostatin in the subarachnoid CSF may play an important role in the functioning of cerebral cortex. It is interesting to note that somatostatin-positive fibers have not been observed in close contact with the lateral ventricles of mammals, in the manner we have described in turtles. However, cells in neocortical layer VI apparently do contain this peptide’ and layer VI cells are known to give rise to corticothalamic projections.” Thus, corticofugal feedback to the thalamus from cortical somatostatin neurons may employ direct synaptic connections as well as a neurohumoral mechanism for transmission of the same chemical signals. CSF-contacting
malian neocortex and amygdala possess somatostatin-like immunoreactivity, thus the finding of labeled neurons in all divisions of the turtle dorsal ventricular ridge does not clarify the interpretation of forebrain relationships. In the basal telencephalon, immunoreactive cells were largely confined to two regions; the globus pallidus and area d. The turtle globus pallidus is a large-celled zone of the basolateral telencephalon that Johnston” included in the “nucleus lentiformis”. This cell group is characterized by a relative paucity of acetylcholinesterase, rich substance P immunoreactivity, and efferent connections to the tegmentum.32 It is generally considered homologous to the mammalian globus pallidus. In the turtle, we have observed only occasional somatostatin-like immunoreactivity associated with neurons in this nucleus, and it appears to be virtually absent in the rat globus pallidus. Area d of Riss er al.” is characterized by a high acetylcholinesterase content, rich substance P immunoreactivity and intense catecholamine fluorescence.32 We find occasional neurons in area d with strong somatostatin-like immunoreactivity. These data support the suggestion by Reiner” that this region is homologous to the mammalian nucleus accumbens. Hypothulamus
Dorsul
t’entriculur ridge und Basal telencephalon
In this study,
we have identified immunoreactive neurons in both the dorsal ventricular ridge and the basal telencephalic nuclei. The reptilian dorsal ventricular ridge has no established mammalian homology. The dorsal ventricular ridge was originally considered to be homologus to mammalian neostriatum (caudate) by Johnston, 20but the finding of a massive input to the anterior dorsal ventricular ridge from the tectal-recipient nucleus rotundus13 led to the suggestion that the dorsal ventricular ridge is related to extrastriate cortex. The posterior division of the ridge which does not receive a rotunda1 input has been likened to the amygdaloid complex.*’ Both the mam-
Somatostatin has been established to be a potent inhibitor of growth hormone release from the mammalian pituitary.36 Accordingly, many immunocytochemical studies in mammalian brain have confirmed the presence of somatostatin-containing neurons in the hypothalamic periventricular nucleus whose axons project caudally to terminate in the median eminence.‘~‘~” A similar arrangement has been described for lizards” and frogs.37 Our results confirm the existence of somatostatin-positive cells in the hypothalamus of Pseudemys turtles. Acknowledgemenrs-We Maria and E. Waymire
gratefully for typing
acknowledge the manuscript.
K.
De
REFERENCES I. Bear M. F. and Ebner
2. 3. 4. 5. 6. 7. 8. 9.
F. F. (1981) Somatostatin-like immunoreactivity in the cerebral cortex of the turtle. Pseudem~~ scriptu. Anat. Rec. 199, (3) 22A. Bennett-Clarke C., Romagnano M. A. and Joseph S. A. (1980) Distribution of somatostatin in the rat brain: telencephalon and diencephalon. Brain Res. 188, 473486. Bennett G. W., Edwardson J. A. Marcano de Cotte D., Berelowitz M., Dimstone B. L. and Kronheim S. (1979) Release of somatostatin from rat brain synaptosomes. .I. Neurochem. 32, 1127-I 130. Brazeau P., Vale W., Burgus R., Ling N., Butcher M., Rivier J. and Guillemin R. (1973) Hypothalamic polypeptide that inhibits the secretions of immunoreactive pituitary growth hormone. Science, N. Y. 179, 77-79. Brownstein M.. Arimura A., Sato H., Schally A. V. and Kizer J. S. (1975) The regional distribution of somatostatin in the rat brain. Endocrinology 96, 1456-1461. Cserr H. F., Fenstermacher J. D. and Fend F. (Eds) (1975) Fluid Environment of’the Brain. Academic Press, New York. Dodd J. and Kelly J. S. (1978) Is somatostatin an excitatory transmitter in the hippocampus? Nature. Land. 273, 674-675. Elde R. P. and Parsons J. A. (1975) Immunocytochemical localization of somatostatin in cell bodies of the rat hypothalamus. Am. J. Anat. 144, 541-548. Feldman S. C.. Drefus C. F. and Lichtenstein E. (1981) SRIF neurons in the rodent hippocampus. Anal. Rec. 199, (3). 81 A.
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10 Gilbert C. D. and Kelly J. P. (1975) The projections of cells in different layers of the cat visual cortex. J. camp. Neural. 163, 81-106. II. Goosens N., Dierickx K. and Vandesande F. (1980) Immunocytochemical localization of somatostatin in the brain of the lizard, Ctenosauria pectinata. Cell Tissue Res. 208, 499-505. 12. Graybiel A. M., Ahmad-Mirza R. and Elde R. P. (1981) Somatostatin-like immunoreactivity characterizes neurons of the nucleus reticularis thalami in the cat. Anat. Rec. 199, (3) 99A. 13. Hall W. C. and Ebner F. F. (1970) Thalamotelencephalic projections in the turile, Pseudemys scripta and Chrysemys picta. J. camp. Neurol. 140, 101-102. 14. Havlicek V. (1978) Somatostatin action on cyclic AMP in rat brain: Role of central adrenergic mechanisms. Fedn. Proc. Fed. Am. Sot. Exp. Biol. 35, 3175.
15. Hohmann C. F., Carroll P. T. and Ebner F. F. (1983) Acetylcholine levels and choline acetyltransferase activity in turtle cortex. Brain Res. 258, 12&123. 16. Hokfelt T., Efendic S., Johansson O., Luft R. and Arimura A. (1974) Immunocytochemical localization of somatostatin (growth hormone release-inhibiting factor) in the guinea-pig brain. Brain Res. 80, 165-169. 17. Hokfelt T., Elde R., Johansson O., Ljungdahl A., Schultzberg M., Fuxe K., Goldstein M., Nilsson G., Pemow B., Terenius L., Ganten D., Jeffcoate S. L., Rehfled J. and Said S. (1978) Distribution of peptide-containing neurons. In Psychopharmacology: A Generation of Progress (eds Lipton M. S., DiMascio A. and Killam K. F.) Raven Press, New York. 18. Iversen L. L., Iversen S. D., Bloom F. E., Douglas C., Brown M. and Vale W. (1978) Calcium-dependent release of somatostatin and neurotensin in vitro. Nature, Land. 273, 151-163. 19. Johnston J. B. (1915) The cell masses in the forebrain in the turtle (Cistudo Carolina) J. camp. Neurol. 25, 393-468. 20. Johnston J. B. (1918) The history of the nucleus candatus and the stria terminals in vertebrates. Anat. Rec. 19, 41. 21. King J. A. and Millar R. P. (1979) Phylogenetic and anatomical distribution of somatostatin in vertebrates. Endocrinology 105, (6) 1322-1329. 22. Knigge K. M., Bennett-Clarke C., Burchanowski B., Joseph S. A., Romagnano M. A. and Sternberger L. (1979) Relationships of some RF-producing neuron systems to the ventricles of the brain. In The Endocrine Functions of the Brain (eds Molta M. and Martini L.) Chap. 12. Raven Press, New York. 23. Knowles Sir F. (1972) Ependyma of the third ventricle in relation to pituitary function. In Topics in Neuroendocrinology. Progress in Brain Research (eds Ariens Kappers J. and Schade J. P.) Vol. 38, pp. 255-270. Elsevier, New York. 24. Krisch B. and Leonhardt H. (1980) Neurohormones in the intercellular clefts and in glia-like cells of the rat brain. Cell Tissue Res. 211, 251-268.
25. Northcutt R. G. (1978) Forebrain and midbrain organization in lizards and its phylogenetic significance. In Behavior and Neurology of Lizards (eds Greenberg N. and MacLean P. D.) NIMH. U.S. Govt. Printing Off. 26. Ouimet C. C. and Ebner F. F. (1981) Extrathalamic inputs to the cerebral cortex of the turtle Pseudemys scripta. Anat. Rec. 199, (3) 189A. 27. Ouimet C. C., Patrick R. L. and Ebner F. F. (1981) An ultrastructural and biochemical analysis of norepinephrinecontaining varicosities in the cerebral cortex of the turtle Pseudemys. J. camp. Neurol. 195, 289-304. 28. Parent A. and Poitras D. (1974) The origin and distribution of catecholaminergic axon terminals in the cerebral cortex of the turtle (Chrysemys picta). Brain Res. 78, 345-358. 29. Pate1 Y. C., Yao K. and Reichlin S. (1977) Somatostatin in human cerebrospinal fluid. New Eng. J. Med. 2%,529-533. 30. Petrusz P., Sar M., Grossman G. H. and Kizer J. S. (1977) Synaptic terminals with somatostatin-like immunoreactivity in the rat brain. Brain Res. 137, 181-187. 31. Powers A. S. and Reiner A. (1980) A stereotaxic atlas of the forebrain and midbrain of the eastern painted turtle. (Chrysemys picta picta). J. Hirnforsch. 21, 125-159.
32. Reiner A. (1979) the paleostriatal complex in turtles. Sot. Neurosci. Abs. 5, 466. 33. Renaud L. P., Martin J. B. and Brazeau P. (1975) Depressant action of TRH, LH-RH, and somatostatin on activity of central neurons. Nature, Lond. 255, 233-235. 34. Riss W., Halpern M. and Scalia F. (1969) The quest for clues to forebrain evolution-the study of reptiles. Brain Behau. Evol. 2, I-50.
35. Sternberger L. A. (1979) Immunocytochemistry. Wiley, New York. 36. Vale W., Brazeau P., Rivier C., Brown M., Boss B., Rivier J., Burgus R., Ling N. and Guillemin R. V. (1975) Somatostatin. Recent Prog. Horm. Res. 31, 365-397. 37. Vandesande F. and Dierickx K. (1980) Immunocytochemical localization of somatostatin-containing neurons in the brain of Rana temporaria. Cell Tissue Res. 205, 45-53. 38. Vaughn J. E., Barber R. P., Ribak C. E. and Houser C. R. (1981) Methods for immunocytochemical localization of proteins and peptides involved in neurotransmission. In Current Trends in Morphological Techniques. (ed Johnson J. F., Jr.) CRC Press, West Palm Beach, Florida. 39. Vigh B., Vigh-Teichmann I. and Aros B. (1977) Special dendritic and axonal endings formed by the cerebrospinal fluid contacting neurons of the spinal cord. Cell Tissue Res. 183, 541-542. (Accepted 6 November 1982)
0306-4522/83
1983
$3.00+0.00
Pergamon Press Ltd cc’ 1983 IBRO
SOMATOSTATIN-LIKE TMMUNOREACTIVITY IN THE FOREBRAIN OF PSEUDEMYS TURTLES M. F. BEAR and F. F. EBNER Section
of Neuroscience,
Division
of Biology and Medicine, RI 02912, U.S.A.
Brown
University,
Providence,
Abstract-Previous investigations of cortical organization in the brain of the turtle have revealed many features typical of mammalian neocortex. Recent evidence suggests that many neocortical neurons contain neuroactive peptides. The possibility that one such peptide, somatostatin, is found in the turtle brain was tested using immunocytochemical techniques. Intense somatostatin-like immunoreactivity was observed in many neurons and fibers in turtle cortex, as well as in several forebrain nuclei. Cortical neurons with several different dendritic configurations showed immunoreactive labelling, including bipolar, stellate and pyramidal cell types. In addition, stained cells and processes were observed in close association with the
ependyma of the lateral ventricle. Other forebrain regions containing immunoreactive neurons included the dorsal ventricular ridge, the basal telencephalic nuclei and the hypothalamus. These data support the idea that peptidergic neurons existed in the pallium of an ancestor common to modern mammals and reptiles. We speculate that somatostatin plays function of all types of cortex and suggest that turtle cortex may provide of this cortical neuropeptide.
The ICamino acid peptide somatostatin was first identified as the hypothalamic growth hormone release inhibiting factor by Brazeau, Vale, Burgus, Ling, Butcher, Rivier and Guillemin“ in 1973. It has since been shown by radioimmunoassay’ and by immunocytochemistry*,” that this peptide has a widespread distribution in neurons of the mammalian central nervous system. Several lines of evidence suggest a transmitter role for somatostatin. For example, somatostatin has been localized in synaptic terminals”’ and is released from synaptosomal preparations by high [K +].3 In addition, it is released from brain slices in a calcium-dependent fashion” and has a potent physiological effect when applied by iontophoresis onto cortical neurons.33 The presence of numerous somatostatin neurons in mammalian neocortex* raises the question of whether equivalent peptidergic neurons exist in reptiles. To investigate this possibility, we undertook the following immunocytochemical study in Pseudemys turtles, a species whose cortex we have studied in some detail. A partial report of these data has been published in abstract form.’ EXPERIMENTAL
2448 h at 4°C. After rinsing briefly in distilled water, the brain was cut on a freezing microtome at either 50 or 100pm. Tissue sections were then washed in phosphate buffered saline (PBS) and incubated overnight in normal turtle serum diluted I : 5 with PBS at 4°C. After a PBS rinse, sections were reacted for immunocytochemistry either mounted on gelatin-coated slides or free floating, according to the unlabeled antibody enzyme method of Sternberger.35 Rabbit anti-somatostatin antibody was generated against a carbodiamide conjugate with keyhole limpet hemocyanin and synthetic somatostatin-14 (Immuno Nuclear lot No. 29189). All antibody dilutions were made in PBS that contained 0.27; Triton X-100 (Sigma Chemicals). Tissue sections were first incubated overnight at 4°C in rabbit anti-somatostatin diluted I: 100-500. After a I h wash in PBS, the sections were incubated in goat anti-rabbit IgG (Miles-Yeda) diluted 1: 50 for 1h at room temperature. The tissue was again washed in PBS and incubated in rabbit peroxidase antiperoxidase (Miles-Yeda) diluted 1: 100 for I h at room temperature. After a final PBS wash, sections were reacted for 1&I 5 min in an incubation medium consisting of 2.0 ml PBS, pH. 7.3, 1.2 mg. 3,3’-diaminobenzidine and 0.03% H,O,. This reaction was stopped by transferring the sections to fresh PBS. Finally, sections were dehydrated through a series of alcohols, cleared in xylene and coverslipped. Sections were examined with a Leitz Orthoplan microscope using both normal bright-field and interference contrast optics. In some cases, adjacent sections were stained with cresyl violet acetate to enhance the localization of nuclear borders.
PROCEDURES
Adults turtles (Pseudemy.7 scripta) of both sexes were anesthetized with sodium pentobarbital, cooled to 6°C and perfused though the ascending aorta with 6°C saline followed by I I. of cold 4% phosphate buffered paraformaldehyde, pH 7.4. The brain was removed and immersed in the same fixative containing 30% sucrose for
Three strategies were employed to address the question of specificity. To test the possibility that the observed staining resulted from non-immunologic reactions of the histochemical reagents, the rabbit anti-somatostatin antiserum was omitted from the immunocytochemical protocol. To test the specificity of the primary antiserum, 0.5ml of diluted rabbit anti-somatostatin was adsorbed with 100 pg of synthetic somatostatin-14 (Sigma) for 24 h prior to the immunocytochemical reaction. Although a diffuse background of HRP reaction product frequently persisted after the second test, both procedures eliminated all perikaryal and fiber staining. Finally. as a biological control’R
Correspondence: F. F. Ebner Biology and Medicine, Box G Brown University Providence, RI 02912, U.S.A. Abbreaiofions: CSF, cerebrospinal fluid; GABA y-aminobutyric acid; HRP, horseradish peroxidase; PBS, phosphate buffered saline. YS(9/2
297 <
a similar role in the normal a useful model for the study
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2.0mm
Fig. I. Coronal sections through the turtle forebrain to show the distribution of somatostatin-containing neurons. Immunoreactive neurons are charted on the left; cytoarchitectural subdivisions on the right. Inset: lateral view of the turtle brain showing the approximate level of each frontal section. Abbreviations are: h, hippocampus; sb, subiculum; gc, general cortex; pa th, pallial thickening; dvr, dorsal ventricular ridge; pyr, pyriform cortex; BTN, basal telencephalic nuclei; ot, optic tract; pv, periventricular nucleus. Both scale bars = 2 mm.
Somatostatin
in turtle
immunocytochemical staining was performed in the rat brain yielding results consistent with those reported in the literature.2.‘7
The possibility of antibody cross reactivity cannot be excluded, and the “somatostatin-like” immunoreactivity reported here is referred to as “somatostatin” reservation clearly in mind.
with
this
RESULTS Cells and fibers displaying somatostatin-like immunoreactivity were observed in several regions of the turtle forebrain. Figure 1 illustrates the overall distribution of immunoreactive cells in a series of coronal sections taken from one representative case. In the telencephalon, labeled cells were observed in cortex, the dorsal ventricular ridge, and among the nuclei of the basal telencephalon. Labeled neurons were distributed in all types of cortex: lateral or olfactory, dorsal or general, and medial or hippocampal. In dorsal and hippocampal cortex, these cells were concentrated in the main cell layer, but were also commonly observed in the molecular layer and subcellular zone. The frequency of immunoreactive cortical cells appeared very similar in sections over a wide anterior-posterior extent of the hemisphere. In the dorsal ventricular ridge, stained cells were observed both in the anterior part of the core nucleus and the corticoid region. In the basal telencephalon, occasional labeled neurons were observed in the “globus pallidus”3’ and “area d”.34 In the diencephalon, immunoreactive cells were clustered in the nucleus periventricularis hypothalami. Stained fibers arising from these neurons were traced posteriorly to where they form a dense plexus over the median eminence. The dorsal thalamus and the other nuclei of the hypothalamus were largely free of immunoreactive neurons. Immunoreactive neurons in cerebral cortex Many cortical neurons displaying somatostatinlike immunoreactivity fell into morphologically distinct categories. Bipolar or fusiform neurons were the most frequently identified. Such cells with dendrites oriented parallel to the ventricular surface were particularly common in the subcellular zone of dorsal cortex. This zone consists of a narrow strip of neuropil that separates the main layer of cortical neurons from the underlying ependymal cells. Bipolar cells with radially-oriented dendrites (Fig. 2a) were found most frequently in the molecular and the main cell layers of dorsal cortex and hippocampus. The majority of the immunoreactive cortical cell bodies were located in the main cell layer as shown in Fig. 1. Unfortunately, the processes of these cells rarely contained enough reaction product to characterize the dendritic morphology. Occasionally, a cell would be stained adequately to identify it as a pyramidal neuron. Figure 2(b) shows an example of such a cell from the subiculum. Interestingly, den-
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dritic spines were not labeled on any immunoreactive neurons. When the brains were cut into 100pm sections, some cortical cells presented a striking Golgi-like image (Figs 2c and d). Figure 2(c) is a photomicrograph of an immunoreactive cell situated in the molecular layer of general cortex. A camera lucida drawing of this cell (Fig. 2d) demonstrates the extent of the dendritic field that could be visualized in this single section. This molecular-layer cell belongs to the broad category of stellate neurons and is representative of a third morphological type of immunoreactive cortical cell. Neurons displaying somatostatin immunoreactivity were also consistently observed in the pyriform cortex. These cells also ranged in dendritic morphology from stellate to pyramidal. Staining of periependymal telencephalon
cells and axons in the
A surprisingly high proportion of the immunoreactive cells in the dorsal and medial cortices were found in close association with the ependymal cell bodies that line the lateral ventricle. Figure 3(a) shows a neural element in close apposition to the ependymal cell layer. The ependymal perikarya are viewed as a flat sheet of cells in this fortuitous sagittal section that cut the ependymal layer of the medial wall tangentially. A labeled process, presumably an axon, may be seen coursing along mosaic image formed by the ependymal cell bodies. This axon gives rise to a number of branches, several of which appear to end in bulbous swellings. Figure 3(b) is a photomicrograph of an immunoreactive neuron from the same case. Again, it appears to lie in close contact with the ependyma. In fact, in almost every section regardless of the plane, stained cortical neurons were observed to give rise to processes projecting among ependyma and ending near the ventricle-ependymal interface. Occasionally periependymal cells emitted immunoreactive processes that projected radially and appeared to traverse the entire thickness of cortex to terminate at the pial surface. Examples of these fibers from hippocampal cortex appear in Figs 3(c) and 3(d). Immunoreactive neurons in the dorsal ventricular ridge basal telencephalon Other cell groups in the telencephalon consistently contained immunoreactive cells. Labeled neurons were occasionally observed in the dorsal ventricular ridge, particularly in the dorsolateral region that blends with the pallial thicknening. These cells were often among the corticoid cell clusters, but immunoreactive neurons were also observed in the core nucleus, and as in cortex, in close association with the ependyma. Dendritic morphology of immunoreactive dorsal ventricular ridge neurons was also quite varied, including both pyramidal and stellate cell types.
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Fig. 2. Immunoreactive cell types in turtle cortex. Labeled cells fall into three broad morphological categories: bipolar a, pyramidal b, and stellate c. Figure 2(d) is a camera lucida reconstruction of the neuron photographed in 2(c) showing the extent of visible dendrites in this single 100 nm-thick section. Scale bars: a = 20 pm, b = 10 pm, c = 20 pm. Fig. 3. Somatostatin-like immunoreactivity in periependymal cells and fibers. a. Labeled axon coursing among the ependyma cell bodies. A neuron adjacent to this ependymal lining is illustrated in b. Figures 3(c) and (d) are low power photomicrographs of frontal sections through hippocampal cortex. The pial surface is at the top and in c, the lateral ventricle lies just below the lower margin of the picture. Immunoreactive fibers which arise from periependymal cells may be seen traversing the entire thickness of cortex and ramifying close to the pial surface. The dark band at the bottom of 3(c) was not eliminated by routine antibody controls and thus reflects non-specific staining. Scales bars: a = IS pm, b = 10 pm. c=20pm, d= 15pm.
2d 301
i-,
3c 1 II
’
.
. .
Fig. 4. Immunoreactive neurons in the basal telencephalon. These cells, from two different cases, lie in area d. Both emit long stained fibers that course toward the ventrolateral surface of the brain. Scale bars = 15 pm.
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immunoreactivity in the turtle hypothalamus. Laheled cells in the periw mtric ular Fig. 5. somatostati’n-like to terminate in a dense plexus over the em give rise to axons that sweep ventrally and posteriorly median eminence. The third ventricle is indicated by a star. Scale bar = 100 pm.
IlUCl
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Somatostatin
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A few immunoreactive neurons were observed in the basal telencephalon. Specifically, labeled cells were found in the ventrolateral, large celled region termed “globus pallidus” by Powers and Reined’ and in area d of Riss et ~1.~~which is ventroiateral to the floor of the lateral ventricle. Examples of labeled neurons from areas are presented in the photomicrographs of Fig. 4. These cells give rise to long processes that course ventrolaterally (down and to the left in the micrographs). In Fig. 4(a), the labeled fibers project across the lateral forebrain bundle, and one of the beaded processes in Fig. 4(b) could be followed almost to the pial surface. Somutostatin-like cephalon
immunoreactiuity
in
the
dien -
Somatostatin immunoreactivity in the turtle diencephalon was largely confined to the hypothalamus. Specifically, labeled neurons were observed in the nucleus periventricularis hypothalami (Fig. 5). Stained processes from these cells often could be traced through the ependyma to the surface of the third ventricle. However, most observable fibers arising from the periventricular cells swept ventrally and posteriorly to form a rich terminal plexus in the median eminence.
DISCUSSION Turtle dorsal cortex receives thalamic inputs, some of which are known to relay sensory information.” The same area of turtle cortex receives noradrenergic input from the Iocus coeruleus,27~Z serotoninergic input from the raphe nuclei,26 and an input, possibly cholinergic, from the basal telencephalon’s~26. In addition, a population of non-pyramidal cells with aspinous dendrites is present in turtle cortex that contains glutamate decarboxylase, the synthesizing enzyme for y-aminobutyrate (GABA) (L. M. Smith, C. Houser, R. L. Patrick and F. F. Ebner, unpublished observations). Each of these chemical and connectional features of turtle cortex is shared by mammalian neocortex. The results from the present study reveal yet another striking similarity to neocortex; namely, the presence of somatostatin-containing neurons and processes. Somatostatin-containing neurons in mammalian neocortex are located primarily in layers II, III and VI. These cells have a dendritic morphology that characterizes them as stellate, bipolar or pyramidal neurons.* Though turtle cortex consists of only a single prominent cell layer, the immunoreactive neurons still fall into these three morphological categories. Interestingly, somatostatin-positive neurons in the mammalian hippocampus, another mono-layer cortex, fall in the same three classes’ suggesting that morphological diversity is a characteristic of somatostatin-positive neurons wherever they are found in the forebrain. Furthermore, the recent local-
30.5
ization of somatosatin-like immunoreactivity to neurons of the dorsal pallium in the frog, Rana temporuria,37 and the lizard, CtenQsuur~s pectinata,” supports the idea that peptidergic neurons may have existed in the pallium of an ancestor common to modern terrestrial vertebrates, A previous immunocytochemical study indicated that many, if not all, of the stellate cells in turtle cortex contain glutamate decarboxylase and release GABA at their synapses (Unpublished observations). In this light, the finding of somatostatin-like immunoreactivity in this cell type is particularly interesting. The two obvious inte~retations are that either two (or more) chemically distinct populations of stellate cell exist in turtle cortex or that GABA and somatostatin are contained within the same neurons. The latter possibility is not without precedent; Graybiel, Ahmad-Mirza and Elde” have provided a logical argument that these two molecules coexist in the cells of the cat nucleus reticularis thalami. The resolution of this question in turtle cortex awaits direct evidence from double labelling procedures. The function of somatostatin in cortex remains obscure. Renaud et a1.33 have reported a potent inhibition of spontaneous unit activity after iontophoresis of somatostatin in neocortex, whereas, in the hippocampus Dodd and Kelly’ report neuronal depolarization after application of this peptide. Havliceki4 suggests that somatostatin may interact with central noradrenergic mechanisms. Turtle cortex has eight times the norepinephrine content of mammalian neocortex per unit wet weight.” Radioimmunoassay in the tortoise brain indicates that the concentration of extrahypothalamic brain somatostatin is about twice that in the rat,” and our immunocytochemical results indicate that the relative concentration of somatostatin-positive neurons is considerably higher in cortex than in the subcortical regions. Taken together, these data suggest that turtle cortex might be a valuable preparation to investigate further the interactions of norepinephrine, GABA, and somatostatin. Per~ependym~~ cells In this study we have identified many cells and processes with somatostatin-like immunoreactivity that lie within or in close apposition to the ependymal lining of the lateral and third ventricles. In some cases, axons from nearby neurons are observed to contact the ependyma and occasionally appear to terminate at the ventricular surface. Such a relationship has been described for cells in the third ventricle of several vertebrate species. These cells have been termed “liquor-contacting” neurons by Knowles.23 In other cases, stained fibers are seen projecting radially from the zone of periependymal cells to the surface of cerebral cortex. The characteristics of these elements are strikingly similar to the neurosecretory neurons described by Vigh et a1.39in the spinal cord of turtles. Our data suggest that many
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cells in the turtle cerebral cortex and hypothalamus contain somatostatin. lmmunoreactive somatostatin has been found in the cerebrospinal fluid (CSF)29 and in the intercellular clefts of several brain regions, including cerebral cortex.24 Since it is likely that the extracellular space of the brain is exposed to CSF,6 the proposal has been made that the CSF might provide one route for this peptide to reach its target cells and thus produce periphysiological effects.22 The immunoreactive ependymal cells we have observed in the turtle forebrain must be considered as one likely source of somatostatin in both ventricular and subarachnoid CSF, and free somatostatin in the subarachnoid CSF may play an important role in the functioning of cerebral cortex. It is interesting to note that somatostatin-positive fibers have not been observed in close contact with the lateral ventricles of mammals, in the manner we have described in turtles. However, cells in neocortical layer VI apparently do contain this peptide’ and layer VI cells are known to give rise to corticothalamic projections.” Thus, corticofugal feedback to the thalamus from cortical somatostatin neurons may employ direct synaptic connections as well as a neurohumoral mechanism for transmission of the same chemical signals. CSF-contacting
malian neocortex and amygdala possess somatostatin-like immunoreactivity, thus the finding of labeled neurons in all divisions of the turtle dorsal ventricular ridge does not clarify the interpretation of forebrain relationships. In the basal telencephalon, immunoreactive cells were largely confined to two regions; the globus pallidus and area d. The turtle globus pallidus is a large-celled zone of the basolateral telencephalon that Johnston” included in the “nucleus lentiformis”. This cell group is characterized by a relative paucity of acetylcholinesterase, rich substance P immunoreactivity, and efferent connections to the tegmentum.32 It is generally considered homologous to the mammalian globus pallidus. In the turtle, we have observed only occasional somatostatin-like immunoreactivity associated with neurons in this nucleus, and it appears to be virtually absent in the rat globus pallidus. Area d of Riss er al.” is characterized by a high acetylcholinesterase content, rich substance P immunoreactivity and intense catecholamine fluorescence.32 We find occasional neurons in area d with strong somatostatin-like immunoreactivity. These data support the suggestion by Reiner” that this region is homologous to the mammalian nucleus accumbens. Hypothulamus
Dorsul
t’entriculur ridge und Basal telencephalon
In this study,
we have identified immunoreactive neurons in both the dorsal ventricular ridge and the basal telencephalic nuclei. The reptilian dorsal ventricular ridge has no established mammalian homology. The dorsal ventricular ridge was originally considered to be homologus to mammalian neostriatum (caudate) by Johnston, 20but the finding of a massive input to the anterior dorsal ventricular ridge from the tectal-recipient nucleus rotundus13 led to the suggestion that the dorsal ventricular ridge is related to extrastriate cortex. The posterior division of the ridge which does not receive a rotunda1 input has been likened to the amygdaloid complex.*’ Both the mam-
Somatostatin has been established to be a potent inhibitor of growth hormone release from the mammalian pituitary.36 Accordingly, many immunocytochemical studies in mammalian brain have confirmed the presence of somatostatin-containing neurons in the hypothalamic periventricular nucleus whose axons project caudally to terminate in the median eminence.‘~‘~” A similar arrangement has been described for lizards” and frogs.37 Our results confirm the existence of somatostatin-positive cells in the hypothalamus of Pseudemys turtles. Acknowledgemenrs-We Maria and E. Waymire
gratefully for typing
acknowledge the manuscript.
K.
De
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2. 3. 4. 5. 6. 7. 8. 9.
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35. Sternberger L. A. (1979) Immunocytochemistry. Wiley, New York. 36. Vale W., Brazeau P., Rivier C., Brown M., Boss B., Rivier J., Burgus R., Ling N. and Guillemin R. V. (1975) Somatostatin. Recent Prog. Horm. Res. 31, 365-397. 37. Vandesande F. and Dierickx K. (1980) Immunocytochemical localization of somatostatin-containing neurons in the brain of Rana temporaria. Cell Tissue Res. 205, 45-53. 38. Vaughn J. E., Barber R. P., Ribak C. E. and Houser C. R. (1981) Methods for immunocytochemical localization of proteins and peptides involved in neurotransmission. In Current Trends in Morphological Techniques. (ed Johnson J. F., Jr.) CRC Press, West Palm Beach, Florida. 39. Vigh B., Vigh-Teichmann I. and Aros B. (1977) Special dendritic and axonal endings formed by the cerebrospinal fluid contacting neurons of the spinal cord. Cell Tissue Res. 183, 541-542. (Accepted 6 November 1982)