The distribution of somatostatin binding sites in the brain of gymnotiform fish, Apteronotus leptorhynchus

I~umanatom~t_ E L ~

Journal of Chemical Neuroanatomy 7 (1994) 49-63

The distribution of somatostatin binding sites in the brain of gymnotiform fish, Apteronotus leptorhynchus Giinther K.H. Zupanc *a'c, Danielle C6cyre b, Leonard Maler c, Marianne M. Zupanc c'l, R6mi Quirion b aAbteilung Physikalische Biologie, Max-Planck-lnstitut j~r Entwicklungsbiologie, Postfach 21 09, D-72011 Tiibingen, Germany bDouglas Hospital Research Centre and Department of Psychiatry, Faculty of Medicine, McGill University, 6875 Boul. LaSalle, Verdun, Quebec, Canada H4H IR3 CDepartment of Anatomy and Neurobiology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario Canada KIH 8M5 Accepted 19 December 1993

Abstract

The neuropeptide somatostatin (SS) and its binding sites display a wide distribution in the central nervous system of vertebrates. By employing .semi-quantitative autoradiography, we identified such binding sites in the brain of the weakly electric fish Apteronotus leptorhynchus(Gymnotiformes, Teleostei). Whereas (SSj) binding sites for the octapeptide analogue TyrLSMS-201995 appear to be absent in the gymnotiform brain, (SS2) binding sites for the analogue [Tyr°-t>TrpS]-somatostatin-14 were found in many brain regions and Showed a similar distribution to that observed by other authors in the amphibian and mammalian central nervous system. TelencephalonWhile binding in the ventral telencephalon was typically low, all cell groups of the dorsal portion displayed a high degree of binding. The highest density of binding sites was found in the dorsal and caudal subdivision 2 of the dorsomedial telencephalon. DiencephalonMany cell groups of the diencephalon showed a medium to high degree of binding density. The highest level was seen in the habenula. MesencephalonAll layers of the optic tectum contained a medium number of binding sites, except the stratum marginale. In the torus semicircularis, the different layers displayed distinct binding density. While laminae 7-8 showed the highest degree of binding, the lowest density was found in lamina 6. RhombencephalonBinding was generally low or absent in the tegmentum. Low levels of binding density were observed in the electrosensory lateral line lobe. CerebellumExtremely high levels of binding were found in the eminentia granularis medialis and the eminentia granularis posterior. Throughout most regions of the brain, the relative density of binding sites and the relative amount of somatostatin immunoreactivity in fibres, as determined in previous studies, were in good agreement.

Keywords: Somatostatin receptor; Neuropeptide; Neuromodulation; Electric fish

1. Introduction

Somatostatin (SS) was originally isolated from ovine hypothalamus and chemically characterized as a tetradecapeptide inhibiting the release o f growth hormone from cells of the anterior pituitary (Brazeau et al., 1973). Besides their existence in the hypothalamus, SS and its messenger RNA display a wide distribution in specific neuronal cell types of the central nervous system * Corresponding author, Tfibingen address. I Present address: Abteilung Mikrobiologie, Max-Pianck-Institut ffir Biologie, Corrensstr. 38, D-72076 Ttibingen, Germany. 0891-0618/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0891-0618(94)00005-4

in mammals including man (Johansson et al., 1984; Vincent et al., 1985; Fitzpatrick-McElligott et al., 1988; Priestley et al., 1991; Mengod et al., 1992) and fish (Sas and Maler, 1991; Zupanc et al., 1991b). The existence of SS in neuronal tissue as well as the synthesis of its precursor in the brain (Zingg and Patel, 1982; Gluschankof et al., 1985), its presence in synaptosomal fractions (Epelbaum et al., 1977), its calcium-dependent release upon depolarization (Berelowitz et al., 1978; Iversen et al., 1978), as well as the existence of SS binding sites and SS receptors in neuronal tissue (Reubi et al., 1981; Srikant and Patel, 1981; Tapia-Arancibia et al., 1981; for review, see Epelbaum, 1992; Krantic et al.,

50

G.K.H. Zupanc et al./J. Chem. Neuroanat. 7 (1994) 49-63

1992a; Bell and Reisine, 1993; Epelbaum et al., 1994) suggest that this peptide may act in a neurotransmitterlike fashion. In the mammalian central nervous system, SS exhibits a wide range of effects when applied to neurons and other electrically excitable cells (Renaud et al., 1975; Dodd and Kelly, 1978; Dunlap and Fischbach, 1978; Ioffe et al., 1978; Randi6 and Mileti6, 1978; Pittman and Siggins, 1981; Delfs and Dichter, 1983; Mueller et al., 1986; Ikeda and Schofield, 1989). A remarkable feature is its modulatory action on the release of other neurotransmitters such as acetylcholine (Nemeth and Cooper, 1979; Mancillas et al., 1986), noradrenaline (Gfthert, 1980), dopamine (Chesselet and Reisine, 1983; Beal and Martin, 1984), and ~,-aminobutyric acid (GABA) (Meyer et al., 1989; Xie and Sastry, 1992). This effect is paralleled by the coexistence of SS with other classical transmitters or neuropeptides in many brain regions (for review, see Lundberg and H6kfelt, 1983; H6kfelt, 1991). In the present investigation, we examined the distribution of SS binding sites in the brain of the weakly electric fish Apteronotus leptorhynchus (Gymnotiformes, Teleostei) by autoradiography. Since by application of this technique the existence of at least two binding sites for SS in the central nervous system of rats has been suggested (Reubi, 1984; Tran et al., 1985; Krantic et al., 1990), two iodinated ligands were used: the octapeptide analogue Tyr3-SMS-201-995 (Bauer et al., 1982), which in mammals specifically recognizes the high-affinity (SS0 binding site, and the analogue Tyr°-D-Trp8-SSi4, which acts both on the high- and low-affinity (SS2) binding sites. The results of this mapping were compared with the distribution of SS immunoreactivity (Sas and Maler, 1991; Zupanc et al., 1991a; Stroh and Zupanc, 1993a) and the localization of SS mRNA (Zupanc et al., 1991b) in the gymnotiform brain. These neuroanatomical studies will provide the basis for future functional investigations. Apteronotus leptorhynchus may prove to be a good model system for a functional analysis since peptides such as SS have been implicated to participate in the control of electromotor behaviour (Sas and Maler, 1991; Zupanc et al., 1991a; Maler, 1992; Zupanc and Maler, 1993). Furthermore, in some of the SS-immunoreactive brain regions, other neurotransmitters and neuropeptides (Johnston et al., 1990; Weld and Maler, 1992; Yamamoto et al., 1992; Stroh and Zupanc, 1993b) as well as their respective receptors (Maler and Monaghan, 1991; Weld et al., 1994) have been identified, thus providing the perspective to investigate the coexistence and pre- and postsynaptic interaction of SS with these molecules. Preliminary results of this investigation were reported at the annual meeting of the study group 'Neurochemistry' of the 'Gesellschaft fiir Biologische Chemie'

held in Mainz (Germany) on 5th and 6th October 1992 (Zupanc et al., 1992). 2. Materials and methods

2.1. Animals Fifteen juvenile and adult fish, purchased from tropical fish importers, were used for this investigation. Their total lengths ranged from 98 to 265 mm and their body weights from 1.2 to 46.5 g. Immediately after killing the fish, the sex was determined by gonadal inspection. Seven were males, five females, and three could not be sexed. The relative gonadal weight (= fresh weight of gonads divided by body weight) ranged from 0.0007 to 0.0050 in males and 0.008 to 0.091 in females, thus covering the whole range between immature and mature individuals.

2.2. Preparation of brain tissue Fish were killed in ice-water, their brains rapidly removed from the skull and snap-frozen in 2-methylbutane at -40 to -50°C. Twenty-micrometer-thick transverse sections were cut on a cryostat at -17 to -18°C, thaw-mounted onto chrome alum-/gelatincoated slides, and dried in a desiccator at 5°C overnight. They were stored at -80°C until assayed. In some experiments, the sections of one rat brain were processed in parallel with the sections obtained from the brains of Apteronotus leptorhynchus.

2.3. Chemicals The tetradecapeptide somatostatin (SS-14) and its analogue [Tyr°-D-TrpS]-somatostatin-14 (T°-DS-SS-14) were synthesized and kindly given by Dr P. Gaudreau (H6pital Notre-Dame, Montr6al, Qu6bec), while the octapeptide analogues SMS 201-995 and [Tyr3]-SMS 201995 (code name SMS 204-090) were generously provided by Dr J.C. Reubi (Sandoz Co., Switzerland). [12SI]Na salt (2100 Ci/mmol) was obtained from ICN Biomedicals (Irvine, California). Hyperfilm-3H and iodine Micro-Scales were purchased from Amersham Canada (Oakville, Ontario). Sep-Pak Cl8 cartridges and #-Bondapak Cl8 columns were obtained from Waters Associates (Montr6al, Qu6bec). Bovine serum albumin (BSA) fraction V was purchased from Boehringer Mannheim (Montr6al, Qu6bec). Bacitracin and chloramine-T were obtained from Sigma Chemicals (StLouis, Missouri). All other chemicals were purchased from standard commercial sources.

2.4. Iodination of ligands The SS analogues T°-DS-SS-14 and SMS 204-090 were iodinated by a modification of procedures describ-

G.K.H. Zupanc et al. / J. Chem. Neuroanat. 7 (1994) 49-63

ed by Hunter and Greenwood (1962), Rorstad et al. (1979) and Enjalbert et al. (1982). Briefly, 5/~g peptide dissolved in 10/~1 water was mixed with 20/~1 0.5 M phosphate buffer, pH 7.5, 1 mCi [125I]Na salt, and 10 #1 fresh chlorarnine-T (1 mg/ml) as the oxidizing agent. After a reaction time of 40 s at room temperature, 20 #1 sodium metabisulfite (1 mg/ml) dissolved in water and 100 /~1 0.1% trifluoroacetic acid were added. The radioiodinated peptides were purified by means of a Sep-Pak Ci8 cartridge and high performance liquid chromatography on a/~-Bondapak C~s column with a linear gradient of 0-80% acetonitrile in 0.05% trifluoroacetic acid as previously reported (Krantic et al., 1990, 1992b). Monoiodinated peptides were eluted between 30 and 50% acetonitrile. Corresponding fractions were pooled, diluted in 0.1 M citrate buffer (pH 6.0) containing 1% BSA and kept at -20°C until use.

2.5. Somatostatin binding autoradiography Four different batches of brain sections were processed with four independent batches of iodinated ligands for SS binding autoradiography. In order to dissociate bound endogenous SS, slides were pre-incubated at room temperature in 150 mM Tris-HCl buffer, pH 7.5, for 1 h. Incubation with the radioligand was carried out in a fresh batch of the same buffer, supplemented by 5 mM MgCI2, 0.1% BSA and 0.05% bacitracin. Four sets of slides were incubated in this buffer for 2 h at room temperature with the following ligands present: (1) 2 × 105 countsdmin [t25I]T°-D8-SS-14 per ml (50 pM); (2) 2 × 105 counts/min [125IIT°-DS-SS-14 per ml (50 pM) and 1.0 t~M SS-14; (3) 6 × 105 counts/min [125IISMS-204-090 per ml (125 pM); (4) 6 × 105 counts/rain [125I]SMS-204-090 per ml (125 pM) and 1.0 /~M SMS-201-995. The conditions (2) and (4) represented the non-specific binding when assaying both the SSI and SS2 binding types (1) or the SS1 (3) binding sites, respectively (Krantic et al., 1990). The incubation was terminated by sequentially rinsing the slides four times for 5 min each in 150 mM Tris-HCl buffer, pH 7.5, at 4°C, followed by a dip in distilled water. They were rapidly dried with a stream of cold air and juxtaposed to Hyperfilm-3H. After 2 days (SSI binding subtype) or 4 days (SS l and SS2 binding subtypes) films were developed in Kodak D-19 developer as described before (Krantic et al., 1990). After exposure of the slides to autoradiographic film, the sections were fixed in 2% glutaraldehyde in 0.1 M phosphate buffer, dehydrated in a graded ethanol series, air-dried, and coated by dipping in Kodak NTB-2 emulsion diluted 1:1 with distilled water. After an exposure time of 10 days, the autoradiograms were developed in Kodak D-19, stained with cresyl violet, dehydrated in graded concentrations of ethanol, defatted in xylene,

51

embedded in Permount and coverslipped. These emulsion-coated sections allowed precise localization of SS binding sites even in small nuclear groups when the resolution of the film autoradiograms was not satisfactory.

2.6. Image analysis and quantification Images of the film autoradiograms were acquired by using a Sony CCD camera, model XC-77 (Sony Corp.), with a Micro-Nikkor 55 mm, 1:2.8 lens, a Northern Light Illuminator Model 890 (Imaging Research, Inc.) and a QuickCapture frame grabber card (Data Translation) installed on a Macintosh llci computer (Apple Computer, Inc.). The autoradiograms were quantified by densitometry using the image analysis program 'Image', versions 1.33 and 1.47. Relative optical densities were converted into levels of radioactivity by reference to a series of plastic-embedded standard [125I]micro scales which had been co-exposed with radiolabelled sections. The level of specific binding was determined by subtracting the non-specific binding (binding in the presence of unlabelled ligands) from the total binding. Non-specific binding amounted to approximately 10% in the rhombencephalic eminentia granularis medialis, which showed the highest level of radioactivity in the brain. Radioactivity equal to or less than 120% of the radioactivity measured for nonspecific binding was considered background. The radioactivity values between this threshold level and the maximum value found in the brain were divided into ten categories of equal size. In order to standardize the use of terms, we will employ the following convention: categories 1 and 2 indicate very low densities of binding sites, 3 and 4 low densities, 5 and 6 moderate densities, 7 and 8 high densities, and 9 and 10 very high densities. A total of five fish was quantitatively analysed. These cases were chosen on the basis of the completeness of the sections taken from their brains at all levels of the neuraxis. The five individuals included both sexes and displayed a similar range of total length, body weight, and relative gonadal weight as the entire population of fish. The localization of binding sites was confirmed in an additional ten individuals. 3. Results

3.1. Binding characteristics In contrast to previous experiments, in which similar conditions as in the present study were employed for the processing of rat brain sections (Krantic et al., 1990), application of the radioligand SMS 204-090 to the gymnotiform brain did not reveal specific binding. While the binding pattern in the rat brain sections processed in parallel was in agreement with the results published

52

G.K.H. Zupanc et al. / J. Chem. Neuroanat. 7 (1994) 49-63

Fig. 1. Bindingof [ 125I]T°-DS-ss-14 in the absence(left) or presence(right) of a saturating concentrationof the unlabelled ligand, thus representing total binding or control sections, respectively.Increase in binding density is represented by increase in darkness. In the total binding section moderate to dense binding is observedin the torus semicircularis(TSd), tectum opticum(TeO) and inferiorlobe (IL), Backgroundlevelsof binding are seenin the white matter tracts (lateral lemniscus, LL; commissuraansulata, cANS; medial longitudinal fasciculus,MLF). In the control section, by contrast, there is a lower levelof binding observed;this binding is homogenouslydistributed over gray and white matter. CCb, corpus cerebelli; Hc, hypothalamuscaudalis; nRP, nucleus recessus posterioris; VCbl, valvula cerebelli pars lateralis; VCbm, valvula cerebellipars medialis. Scale bars, 1 mm. (Krantic et al., 1990), the binding density in fish brain sections processed for total binding and in adjacent sections reacted for non-specific binding was indistinguishable. Thus, assuming that the fish brain tissue requires similar conditions as the rat brain, this positive control excludes a technical failure and suggests the absence of high affinity (SS0 binding sites in fish brain. Incubation of the fish brain sections with [125I]T°-D8SS-14, on the other hand, resulted in binding densities far above background (Fig. la). When this type of assay was performed in the presence of saturating concentrations of the corresponding unlabelled ligand, the levels of non-specific binding were low and just above film background (Fig. lb). Specific binding was highly heterogeneously distributed (see below) and was absent in regions of white matter such as the anterior commissure, postoptic commissure, posterior commissure, and lateral lemniscus. Regression analysis of the standard curve obtained for [125I]T°-D8-SS-14 binding showed a linear relationship between the amount of radioactivity and the optical density of the standard in the range which was typical for our experiments (r = 0.93). Thus, the ten categories into which the binding densities were grouped reflect a linear scale of radioactivity measured.

3.2. Distribution of binding sites in the brain For the following description of the sites for [125I]T°D8-SS-14 binding in the brain of Apteronotus leptorhynchus, the nomenclature used in the atlas of Maler et al.

(1991) was employed. The results are summarized in Table 1 and Figs. 2-5. The data obtained from the analysis of the autoradiograms of the five fish examined quantitatively were in good agreement with the observations made in the remaining ten fish, which were studied qualitatively, and with the results obtained from the emulsion-coated slides. No obvious differences could be found between males and females as well as between juvenile and adult fish.

Telencephalon (Fig. 2) The olfactory bulb, which is small and attached to the rostroventral part of the telencephalon, was essentially devoid of [125I]T°-D8-SS-14 binding. While binding in the ventral telencephalon was typically low, all cell groups of the dorsal portion of the telencephalon displayed a moderate to very high degree of binding (for morphological classification of the telencephalon, see Nieuwenhuys, 1963; Northcutt and Davis, 1983). The highest density of binding sites was found in the dorsal and caudal subdivision 2 of the dorsomedial telencephalon. Moderate densities were found in two olfactorecipient portions of the dorsal telencephalon, namely the ventral subdivision of the dorsolateral telencephaion and the lateral subdivision of the caudal dorsal posterior telencephalon. A high level of binding was present in the central division of the dorsal forebrain.

Diencephalon (Fig. 3) Many cell groups in the diencephalon showed a low to moderate degree of binding density. The habenula

53

G.K.H. Zupanc et al./d. Chem. Neuroanat. 7 (1994) 49-63

Table I Distribution of relative densities of [ 1251]T°-DS-SS-14 b i n d i n g sites in the brain of g y m n o t i f o r m fish, Apteronotus leptorhynchus Brain area

Relative density o f SS 2 b i n d i n g sites Mean

Telencephalon DC DDi DDmg DDs DLc DLd DLp DLv DM 1 DM2c DM2d DM2r DM2v DP1 DPm Er MOTF nOr OB Vd VI Vlr VI Vn Vs/Vp Vv Diencephalon A/B CP/PPn DLTh/DMTIVAV ! G H Ha Hd HI/Hc Hv IL MFB/PPp nE PGI/PGr PGm PPa PT PTh PTI SE TA tOVL TPP

Relative a m o u n t of SS-IR in fibres

Relative a m o u n t o f SS-IR in s o m a t a

Relative a m o u n t o f SSm R N A in s o m a t a

0 0 +

Standard deviation

7 6 6 7 7 6 6 6 6 8 9 7 7 5 0 0 3 6

1.2 0.9 0.8 1.0 1.2 0.8 0.8 0.8 1.6 1.8 1.7 1.5 0.8 0.9 0.6 1.3

+ ++ ++ ++ ++ ++ +++ ++ ++ + ++ +++ + + ++ + + ++

0 0 + 0 0 0 0 + 0 0 0 0 0 0 + +++ 0 0

1

0

+

+

5 4 4 0 0 5 4

1,4 1.6 1.4 ! .7 1.0

+ + ++ + ++ + +

0 + ++ +++ 0 + 0

5 4 5 4 7 4 0 5 4 6 4 4 5 4

1.6 1.0 1.8 1.8 2.8 0.8 1.3 0.8 1.2 1.3 0.8 2.9 2.2

+ +++ ++ + ++ + + +++ + ++ + + + +

0 +++ 0 + 0 + 0 ++ ++ 0 +++ 0 0 0

4

l. 1

+

0

2 4 4

0.6 1.2 0.4

+ + +

0 0 0 ++ 0 0 ++

0

-

+

6 0 3

0.6 0.8

+ 0 +++

Mesencephalon cTSd eTS IPn

I 1 4

0.5 0 0.8

0 + ++

MRF nl

1 I

0.4 0.4

+ +

0 0 + 0 0

0 0 0 0 + 0 0 ++ ++ 0 0 + +++ 0 0 0 0 0 +++ +++ ++ ++ 0

0 +/++ 0 + 0 ++ + +/++ + 0 ++ 0 0 0 0 0 0 0 ++ 0 + ++

0 0 + 0 0

54

G.K.H. Zupanc et al./J. Chem. Neuroanat. 7 (1994) 49-63

Table 1 (Continued) Brain area

Relative density of SS2 binding sites Mean

PL1 PLm Rc TeO TL TSd, TSd, TSd, TSd, TSd, TSd, TSv

layers 2-5 layer 6 layers 7-8 layer 8d layer 9 total

Rhombencephalon CC CG/LCe/VPn ELL (dml) ELL, CMS (g) ELL, LS/CLS (g) ELL, MS (g) ELL, CMS (p) ELL, LS/CLS (p) ELL, MS (p) nM nXs Pdl Pdm PMRF RF STr Cerebellum CCb CCb (c) EGa EGm EGp/ZT (g) EGp/ZT (m) VCbl (g) VCbl (m) VCbm (c) VCbm (g) VCbm (m)

Relative amount of SS-IR in fibres

Relative amount of SS-IR in somata

Relative amount of SS-mRNA in somata

Standard deviation

2 I 2 4 2 3 2 6 4 5 4

0.5 0.4 0.5 0.5 1.0 0.5 0.5 0.8 1.0 1.0 0.4

+ + + + 0 + 0 + + + + +

+ 0 0 + 0 0 0 + + + + 0

0 0 0 0 0 + 0

1 4 7 3 1 2 3 1 1 2 4 3 4

0.4 0.8 1.2 0.5 0 0 1 0 0 0.5 0.7 0.8 0.6

0 ++ 0 + + + 0 0 0 0 ++ 0 0 + + +

0 + 0 0 0 0 0 0 0 0 ++ 0 0 ++ 0 0

+ + + 0 + + 0 + + 0 +

0 0 0 0 0 0 0 0 0 0 0

0

-

1 0

0 -

0 ++ 0 + + + 0 0 0 0 ++ + 0 + + +++

0.5 0 3.1 0.4 1.2 1.5 0.4 1.1 1.0 0.4 0.7

+ + + 0 + + 0 + + 0 +

1 7 4 10 7 8 2 3 6 1 3

The radioactivity associated with the different binding sites was measured and averaged in individual fish, then grouped into ten categories, and finally averaged over five individuals. While '0' indicates lack of binding, the ten categories represent the range between minimum (' 1') and maximum ('10') level of binding density found. The variability among individuals is depicted by the standard deviation of the mean. For comparison, the relative amount of somatostatin immunoreactivity (SS-IR) found in fibre plexuses and cell bodies as well as the relative amount of somatostatin messenger RNA (SS-mRNA) contained in somatostatin-producing somata are included (0 no, + weak, ++ intermediate, +++ intense labelling). The intensities for SS-IR were estimated from the results of the immunohistochemical studies by Sas and Maler (1991), Zupanc et al. (1991a) and Stroh and Zupanc (1993a). The data for the relative amount of SS-mRNA were obtained by in situ hybridization and taken from Zupanc et al. The nomenclature employed for the different brain regions is based on the atlas published by Maler et al. (1991). For abbreviations, see Appendix.

displayed a high density of binding. In the pretectum,

i n g d e n s i t y in t h e p r e t e c t a l n u c l e u s w a s v e r y l o w . I n t h e

n u c l e i A a n d B a s w e l l a s n u c l e u s e l e c t r o s e n s o r i u s (a high-order electrosensory processing station) exhibited a

complex of the central posterior/prepacemaker nucleus of the thalamus, the capability for [125I]T°-DS-ss-14

low to moderate number of binding sites, whereas bind-

binding

(at l o w level) w a s n o t e w o r t h y

s i n c e t h i s cell

G.K.H. Zupanc et al./J. Chem. Neuroanat. 7 (1994) 49-63

55

Fig. 2. Distribution of [125I]T°-DS-SS-14 binding sites in the telencephalon of gymnotiform fish, Apteronotus leptorhynchus, as revealed by autoradiography. The transverse sections have roughly been taken through levels 32 (left) and 27 (right) of the atlas of the gymnotiform brain (Maler et al., 1991). For abbreviations, see Appendix. Scale bars, I ram.

group has been shown to be crucial for the control of eleetrocommunicatory behaviour (Kawasaki et al., 1988), and the neurons in this region receive a massive input from SS-immunopositive fibres (Sas and Maler, 1991; Zupanc et al., 1991a). More rostrally, a region corresponding to the dorsolateral thalamus, the dor-

somedial thalamus, and the area ventrolateralis of the thalamus showed moderate levels of binding density. In the hypothalamus, all nuclei (except hypothalamus dorsalis that lacked binding sites) displayed a low to moderate degree of binding; the highest level was seen in the central nucleus and the nucleus diffusus lateralis of the inferior lobe (average binding density: category 6). In the glomerular and tuberal regions (e.g., the periventricular nucleus of the posterior tuberculum), the density of binding sites was similar to that in most of the other cell groups of the dicencephalon.

Mesencephalon (Fig. 4)

Fig. 3. Distribution of [125I]T°-Ds-SS-14 binding sites in the rostral dienc~phalon of gymnotiform fish, Apteronotus leptorhynchus. The transverse section has roughly been taken through level 25 of the atlas of the gymnotiform brain (Maler et al., 1991). For abbreviations, see Appendix. Scale bar, i ram.

All layers of the optic tectum (for classification, see Sas and Maler, 1986) contained a low level of [125I]T°D8-SS-14 binding sites. The only exception was the stratum marginale, which was devoid of labelling in the autoradiograms. Among the labelled portion of the optic tectum, the pattern of binding was uniform, and no division into individual layers was visible. The torus semicircularis is a midbrain structure that is enlarged and laminated in gymnotiforms (for review, see Carr and Maler, 1986). While the 12 laminae of the dorsal part are devoted to the processing of electrosensory input, the two divisions of the ventral part receive input from auditory and mechanoreceptive systems. The different laminae are characterized by distinct cell types, afferent and efferent connections, and physiological responses. The laminar structure was also maintained in the autoradiograms, although typically only laminae 2-5, 6, 7-8, 8d and 9 could be resolved. Of them, laminae 7-8 displayed the highest (average: category 6)

56

G.K.H. Zupanc et al./ J. Chem. Neuroanat. 7 (1994) 49-63

and lamina 6 the lowest density of binding sites (average: category 2).

Rhombencephalon (Fig. 5)

Fig. 4. Distribution of [t25I]T°-DS-SS-14 binding sites near the boundary of dienc~phalon and mesencephalon of gymnotiform fish, Apteronotus leptorhynchus. The transverse sections have roughly been taken through levels 18 (top), 14 (middle) and I 1 (bottom) of the atlas of the gymnotiform brain (Maler et al., 1991). For abbreviations, see Appendix. Scale bars, I mm.

The nucleus praeminentialis dorsalis, a large isthmic cell group, receives etectrosensory input and is devoted nearly exclusively to feedback projections in the electrosensory system (for review, see Carr and Maler, 1986). Both medial and lateral portions displayed low levels of [12SI]T°-D8-SS-14 binding, but the density of binding sites was slightly lower in the lateral part (average densities: category 4 vs. category 3). Binding was generally absent in the tegmentum of the rhombencephalon. The few exceptions, which showed very low or low levels of binding density, comprised the following cell groups: medial and lateral subdivision of the paralemniscal nucleus; interpeduncular nucleus; locus ceruleus, central gray, and nucleus of the valvular peduncle (the latter three nuclei could not be distinguished in the autoradiograms); nucleus raph6 centralis; and vagal sensory nucleus. In teleosts, the cerebellum consists of three subdivisions: corpus cerebelli; valvula cerebelli; and eminentia granularis (Finger, 1983). While the corpus cerebelli corresponds to that found in mammals, the valvula cerebelli and eminentia granularis are absent in amniotes. In many teleosts, the valvula can be divided into lateral and medial portions (cf. Bass, 1982; Finger, 1983). In gymnotiform fish, the eminentia granularis consists of three parts: the eminentia granularis posterior, a granule cell mass with its associated molecular layers, which is devoted to feedback in the elec. trosensory system; eminentia granularis anterior, which receives octavolateral input (its molecular layer is the crista cerebellaris of the nucleus medialis); and eminentia granularis medialis, a granule cell mass located at the midline just dorsally to the cerebello-medullary cistern and lacking an obvious molecula r layer. The granule cell layer as well as the molecular layer of t h e corpus cerebelli displayed only very loW levels o f SS binding sites. In the valvula cerebelli, the granule cell layer showed a very low density of [125I]T°-D8-SS-14 binding, whereas in the molecular layer the density was slightly higher. This is also the case at more caudal levels, where lateral (VCbl) and medial portions (VCbm) can be distinguished. At the most caudal level, where a large number of cells is found at the ventral extension of the valvula cerebelli (cf. Maler et al., 1991, atlas level 5), the autoradiograms showed a moderate degree of labelling in this region. In the eminentia granularis anterior, binding density was low. However, at levels where the medial eminentia granularis anterior becomes confluent with the caudal-most granule cells of the corpus cerebelli, the density was high. The highest level of [125I]T°-DS-SS-14 binding in the gymnotiform brain was found in the granule cells of the eminentia granularis medialis (average

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57

density: category 10). High levels of [125I]T°-DS-SS-14 binding were also found in the granule cell mass of the eminentia granularis posterior and the transitional zone as well as the molecular layer located between eminentia granularis posterior and eminentia granularis medialis. The electrosensory lateral line lobe, a first:order electrosensory region, is a laminar structure with two cellular layers: the granular and pyramidal cell layers. In addition, there are two input layers: the deep fibre layer and the molecular layer. The molecular layer can further be divided into ventral and dorsal portions; the dorsal portion is continuous with, and shares parallel fibre input with, the overlying EGp of the cerebellum (Carr and Maler, 1986). The electrosensory lateral line lobe is also divided into four functionally distinct segments (for review, see Cart and Maler, 1986): medial, centromedial, centrolateral and lateral segments; the medial segment receives ampullary electroreceptor input, while the three lateral segments receive tuberous electroreceptor input. Overall, the granular and the pyramidal cell layers displayed very low or low levels of [125I]T°-DS-SS-14binding density, and the density varied across the segments. Binding was higher in the centromedial segment than in the medial, lateral and centrolateral segments. The emulsion-coated sections demonstrated that SS binding was associated with the basilar dendrites of the pyramidal cells. In the dorsal molecular layer of the electrosensory lateral line lobe, binding density was high. The electrosensory lateral line lobe has been proposed to have evolved from the nucleus medialis in parallel with the evolution of electroreceptors from lateral line receptors (J6rgensen, 1982). It is, therefore, interesting that the nucleus medialis and the crista cerebellaris (molecular layer of the nucleus medialis with parallel fibres emanating from the granule cells of the eminentia granularis anterior) displayed very low levels of [125I]T°-DS-SS-14 binding. 4. Discussion 4.1. Subtypes o f S S binding sites

Fig. 5. Distribution of [125I]T°-DS-SS-14 binding sites in the rhombencephalonof gymnotiformfish, Apteronotus leptorhynchus. Thetransversesectionshaveroughlybeentakenthroughlevels7 (top), 2-3 (middle)and -7 (bottom)of the atlas of the gymnotiformbrain (Maler et al., 1991). For abbreviations, see Appendix. Scale bars, I ram.

The tetradecapeptide SS and its N-terminal-extended forms are widely distributed in neuronal as well as in non-neuronal tissue from fish to mammals. This neuropeptide produces its multiple effects through interaction with membrane-bound receptors (for review, see Epelbaum, 1992; Bell and Reisine, 1993; Epelbaum et al., 1994). By using shorter-ringed analogues, which are less prone to enzymatic degradation, pharmacologically distinct subtypes of SS binding sites can be distinguished. In cortex, hippocampus and striatum of mammals, for example, the cyclic octapeptide SMS 201-995 competes with [125I][Tyrll]-SS-14 binding in a biphasic manner, while the competition curves in pituitary and

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pancreas are monophasic (Reubi, 1984; Tran et al., 1985). Thus, in contrast to the periphery, in the mammalian brain SMS 201-995 appears to be able to differentiate between two subtypes of SS binding sites: one (termed SS 0 with nanomolar affinity to the peptide, the other (termed SS2) with micromolar affinity. In the brain of rats (Reubi, 1984; Maurer and Reubi, 1985; Tran et al., 1985; Krantic et al., 1989, 1990) and humans (Reubi et al., 1986, 1987; Krantic et al., 1992b) these two subtypes of binding sites display a differential distribution, thus suggesting functional differences between the high- and low-affinity SS binding sites. The notion of the existence of multiple types of SS binding sites in the mammalian brain is supported and extended by investigations in which additional SS analogues were employed (Leroux et al., 1985; Uhl et al., 1985; Raynor and Reisine, 1989; Martin et al., 1991; Raynor et al., 1992), by photocrosslinking techniques (Thermos et al., 1989), and by recent advances made in the cloning of several genes encoding different types of SS receptors (for review, see Bell and Reisine, 1993; Epelbaum et al., 1994). By using in situ hybridization, the distribution of the mRNA of two of these receptors has been mapped in the mouse brain (Breder et al., 1992). In contrast to mammals, application of the radioligand [125I]SMS 204-090 to brain tissue of gymnotiform fish did not reveal specific binding. Assuming that the experimental conditions employed in the present study were suitable for fish brain tissue, this result appears to suggest the absence of SS1 binding sites in fish brain. Unfortunately, nothing is known from other investigations about the existence of SSI binding sites in other vertebrates besides mammals. Thus, it would be of special interest to study the existence and possible distribution of SMS 201-995 binding sites in the brain of other fish as well as of amphibians, reptiles and birds.

4.2. Comparative and functional aspects of somatostatin binding in the gymnotiform brain Since application of the ligand [125I]SMS 204-090 did not result in specific binding in the gymnotiform brain, we will in the following compare the pattern of binding obtained by using the unspecific ligand [t2SI]T°-DS-SS14 with the distribution of SS binding in the brain of rat and frog as well as with the in situ-hybridization mapping of two cloned SS receptor types (SSTR 1 and SSTR2; see Breder et al., 1992) in mouse brain. In general, there appears to be a good correlation between the distribution of SS binding sites in the brain of Apteronotus leptorhynchus and that of both amphibians and mammals (see below), thus suggesting that these binding sites label SS receptors which have an important and conservative function in the vertebrate brain. Across the telencephalon of gymnotiform fish, amphi-

bians and mammals, the distribution of SS binding sites is remarkably consistent. High densities are found in the pallial portion of the forebrain and lower densities in the subpallium. It is not possible, at present, to compare in detail the pallium of teleosts with that of amniotes (Nieuwenhuys and Meek, 1990). Nevertheless, it is striking that in both gynmotiform fish and mammals the deeper portions of the pallium have a higher density of SS binding sites than the superficial portions. In mammals, the infragraiiular layers, which project to subcortical structures, have the highest density of SS receptors (Uhl et al., 1985; Breder et al., 1992). In .4pteronotus leptorhynchus, the dorsal subdivision of the dorsolateral telencephalon (DLd) receives ascending afferent input from the diencephalon (Striedter, 1992); DLd, in turn, innervates the central division of the dorsal forebrain (DC), which then projects to sub-telencephalic levels (Sas and Maler, 1989, unpublished observations). The efferent portion of the gymnotiform pallium, namely DC, which is at least analogous to the infragranular layers of the mammalian cortex, had a similar density of SS binding sites as DLd, despite the fact that it displayed few SS-immunoreactive fibres in comparison to DLd. The subpaUial portion of the gymnotiform telencephalon includes areas similar to the septal region (ventral subdivision of ventral telencephalon, Vv, and supracommissural subdivision of ventral telencephalon, Vs) and possibly the ventral striatum (intermediate rostral subdivision of ventral telencephalon, VIr) of other vertebrates (Sas et al., 1993). These areas contained low to moderate densities of [125I]T°-DS-SS-14 binding sites, similar to the receptor localizations reported for these subpallial regions in amphibians (Laquerriere et al., 1989) and mammals (Reubi and Maurer, 1985; Uhl et al., 1985). In the diencephalon, the habenula displayed the highest density of SS binding sites in Apteronotus leptorhynchus. For the mammalian habenula, results are contradictory: Leroux et al. (1985) found low densities of unspecific SS binding sites in the medial habenula, whereas Uhl et al. (1985) report moderate to high densities in this portion and low densities over the lateral habenula. Reubi and Maurer (1985), using SMS 204-090 as a ligand, observed heavy labelling in the medial habenula. The latter finding is in agreement with the in situ-hybridization study of Breder et al. (1992) who noted a high degree of expression of SSTR2 receptors (which bind SMS 204-090) in the medial habenula. Consistent with results in mammals (Leroux et al., 1985; Reubi and Maurer, 1985; Uhl et al., 1985; Martin et al., 1991; Breder et al., 1992), thalamus and hypothalamus of Apteronotus leptorhynchus displayed low to moderate densities of binding sites, although immunoreactive fibres and boutons are dense in some nuclei of the gymnotiform diencephalon (Sas and Maler, 1991; Zupanc et al., 1991a) and of the mammalian hypothal-

G.K.H. Zupanc et al./ J. Chem. Neuroanat. 7 (1994) 49-63

arnus (Johansson et al., 1984; Vincent et al., 1985). For the role that SS may play in this region, the complex of the thalamic central posterior/pacemaker nucleus (cP/pPn) is of special interest. A portion of the cells comprising this nucleus is immunoreactive for SS, and neurons of the CP/PPn receive a massive input from SSpositive fibres and boutons (Sas and Maler, 1991; Zupanc et al., 1991a,b; Stroh and Zupanc, submitted). Interestingly, the density of this SS-positive innervation changes in Eigenmannia sp., a related gymnotiform species, with sexual maturity (Zupanc et al., 1991a). This immunological change may be causally linked to maturity-dependent alterations in chirping behaviour, brief frequency modulations of the electric organ discharge which are controlled by this nucleus. SS binding sites were present at low to moderate levels within the major hypophysiotrophic nuclei of Apteronotus ieptorhynchus (PPa, PPp, Ha, Hv, HI, Hc; see Johnston and Maler, 1992). This suggests, as already proposed for mammals (Uhl et al., 1985; Breder et al., 1992), that hypothalamic SS receptors may mediate the neuroendocrine effects of SS. In the mesencephalon of Apteronotus leptorhynchus, SS binding sites were found at low to moderate levels in the interpeduncular nucleus, torus semicircularis and optic tectum, and at very low levels elsewhere. In the mammalian interpeduncular nucleus, low levels of binding density were reported for the SS analogues [125I]T°DS-SS-14 (Leroux et al., 1985) and [125I]CGP 23996 (Martin et al., 1991), whereas Uhl et al. (1985), using the radioiodinated analogues Tyrll-SS-14 and LeuS-DTrp22-Tyr2LSS-28, found high densities in this region. The presence of moderate densities of SS binding sites in both the dorsal and ventral torus semicircularis as well as in the tectum opticum suggests a functional role in the processing of sensory signals, namely: lateral line/auditory input (ventral torus is equivalent to the inferior collieulus; Carr and Maler, 1986), electrosensory input (dorsal torus; see Carr and Maler, 1986) and visual input (tectum opticum is equivalent to the superior colliculus). It is thus interesting that all workers, having employed various SS analogues for autoradiography or having used in situ-hybridization techniques, consistently report the presence of SS binding sites and of SSTR 1 receptors in the inferior and superior colliculus of mammals (Leroux et al., 1985; Reubi and Maurer, 1985; Uhl et al., 1985; Martin et al., 1991; Breder et al., 1992) and in the tectum opticum of amphibians (Laquerriere et al., 1989). Whether this points to an involvement of SS and its receptors in the control of sensory functions, as has been suggested for mammals (Leroux et al., 1985; Reubi and Maurer, 1985), will have to be demonstrated by physiological investigations. The density of SS binding sites was very low or low throughout most of the gymnotiform cerebellum (corpus cerebelli, CCb, and valvula cerebelli, VCb), thus

59

being consistent with the absence of messenger RNA for SS receptors (Breder et al., 1992) and with reports of low levels of binding for SS-14 analogues (Uhl et al., 1985; Leroux et al., 1985) and for SMS 204-090 (Reubi and Maurer, 1985) in the adult mammalian cerebellar cortex. The cerebellar cortex of the frog shows a low to moderate level of binding, despite the apparent lack of SS immunoreactivity within it (Laquerriere et al., 1989). The caudal-most part of the cerebellum of Apteronotus leptorhynchus (eminentia granularis pars anterior, EOa, and caudal corpus cerebelli, CCb (c)), and especially that portion concerned with the electroreceptive system (eminentia granularis pars medialis, EGrn, and eminentia granularis pars posterior, EGp; see Sas and Maler, 1987) had relative to CCb and VCb high densities of binding sites. It would, therefore, be of interest to assess whether there are also regional differences in SS binding in the amphibian or mammalian cerebellum. Although they do not comment upon it, Leroux et al. (1985) show what appears to be moderate binding in the 10th cerebellar lobule of the rat cerebellum (Fig. 3 in Leroux et al., 1985; dense band in the ventral cerebeUar cortex, above the 'X' which points to the vagus nerve nucleus); this part of the cerebellum is concerned with vestibular input and is thus most similar to EGa, Zone T, and the caudal corpus cerebelli (Sas and Maler, 1987). The electrosensory lateral line lobe (ELL) of gymnotiform fish displays only a few SS-immunopositive cells and fibres (Sas and Maler, 1991), and these are confined to the deep neuropil and granular layer. It was, therefore, surprising to find a high density of SS binding sites in the dorsal molecular layer of the ELL. The ELL is in many respects similar to the dorsal cochlear nucleus (DCN) of mammals, e.g. both structures have a molecular layer in receipt of cerebellar granule cell axons (Sas and Maler, 1987; Mugnaini and Maler, 1993). It is, therefore, remarkable that the DCN of rats displays binding of radioactively labelled SS at moderate densities within its molecular layer (Fig. 3 in Leroux et al., 1985; Uhl et al., 1985), despite a paucity of SSimmunoreactive fibres (Vincent et al., 1985). The source of the endogenous ligand(s), if any, for the SS binding sites in the molecular layers of ELL and DCN, and the functional effects of such ligands on electrosensory and auditory physiology may provide clues to common modes of sensory processing in these octavolateral areas. SS binding sites were also found, albeit at a low density, in the cellular layers of the ELL. Ovoid cells, which are immunopositive for SS (Sas and Maler, 1991), have recently been shown to generate a bilateral GABAergic plexus in the deep layer of the ELL around the basilar dendrites of the pyramidal cells (Maler and Mugnaini, 1994). Use of the extremely bright fluorescent chromophore Cy3 for the detection of SS immunoreactivity has confirmed that the climbing fibres forming this plexus

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are SS positive. Thus, it is interesting that the emulsioncoated sections demonstrated a preferential association of the SS binding sites with the basilar dendrites. This suggests an interaction of SS and its receptors with inhibitory synapses.

4.3. Mismatches between the distribution of SSimmunopositive cells and of [ml]T°-DS-SS-14 binding sites In Table 1, in addition to the relative density of [125I]T°-DS-SS-I,1 binding sites, the relative amount of SS immunoreactivity in fibres and cell bodies, as well as the relative amount of SS messenger RNA is listed. A comparison of the relative density of these binding sites and the relative amount of SS-like immunoreactivity in fibres (which contain the presumptive release sites for SS) shows that, in general, these two parameters are in good agreement. Only in a few brain regions SS-positive fibres are absent, while [125I]T°-DS-SS-14 binding sites are present, or vice versa. In the following areas low to moderate levels of SS-like immunoreactivity in fibres has been found in a previous investigation (Sas and Maler, 1991), while no binding sites could be identified in the present study: nother subdivision of the ventral telencephalon (Vn), intermediate subdivision of the ventral telencephalon (VI), rostral entopeduncular nucleus (Er) and medial subdivision of the caudal dorsal posterior telencephalon (DPm); hypothalamus dorsalis (Hd) and subelectrosensorius nucleus (SE) of the diencephalon; and paramedian reticular formation (PMRF) and subtrigeminal nucleus (STr) of the rhombencephalon. Conversely, in the following regions low to moderate levels of relative density of [t25I]T°-D8SS-14 binding sites were found, but at the corresponding sites no immunoreactivity could be detected: torus longitudinalis (TL) and commissure of the torus semicircularis dorsalis (cTSd) of the mesencephalon; and nucleus praeminentialis dorsalis pars medialis (Pdm), crista cerebellaris (CC), all four segments of the electrosensory lateral line lobe (ELL) including its dorsal molecular layer (dml), and nucleus medialis (nM) of the rhombencephalon. Qualitative and quantitative mismatches between the localization of SS and its binding sites were found in different parts of the cerebellum. While SS immunoreactivity is absent in the eminentia granularis medialis, EGm, and low throughout the other portions of the cerebellum (Stroh and Zupanc, 1993a), all cerebeilar parts exhibited binding of [125I]T°-D8-SS14, and some (eminentia granularis medialis, EGm, and eminentia granularis posterior/transitional zone, EGp/ZT) even displayed high and very high binding levels. Several causes may account for these qualitative and quantitative mismatches (for an extensive review on possible reasons for mismatches between neurotransmitter

and receptor localizations, see Herkenham, 1987). Besides technical failures and limitations of the methods employed, the most likely reasons are that additional, yet unidentified, types of binding sites and other forms of SS exist in the gymnotiform brain. In anglerfish and catfish two forms of SS have been characterized, respectively (for review, see Patel, 1992). One form, existing in both fishes, is identical to the mammalian tetradecapeptide SS-14. In contrast, the anglerfish SS-28 (consisting of 28 amino acids) and the catfish SS-22 (comprising 22 amino acids), both identified in pancreas, are different from any known mammalian form of SS. Since gymnotiform fish are probably a sister group of catfish (Fink and Fink, 1981), it appears reasonable to assume that a form of SS similar to catfish SS-22 also occurs in gymnotiforms. Unfortunately, to our knowledge no specific antiserum is available against the catfish SS-22. Thus, it has not been possible yet to identify and localize SS-22 in other tissues besides pancreas in catfish and to examine the possible existence of catfish-like SS-22 immunohistochemically in gymnotiform fish. It is unlikely that the monoclonal antibody used in the previous immunohistochemical mapping studies (Sas and Maler, 1991; Zupanc et al., 1991a; Stroh and Zupanc, 1993a) is able to recognize SS-22, since this antibody is directed to the C-terminal part of SS-14 (Vincent et al., 1985), and this part shows only little similarity to the respective region of SS-22 (for a comparison of the amino acid sequence between SS-22 and SS-14, see Magazin et al., 1982). On the other hand, it is also reasonable to assume that the catfish-like SS-22 form may be bound by [125I]T°-D8-SS-14 binding sites, since the residues corresponding to amino acids 7 to l0 of the mammalian SS-14 are highly homologous in catfish SS-22, and this region has been shown to represent the active site of the molecule (for review, see Epelbaum, 1992). Thus, it is possible that the [t25I]T°-D8-SS-14 binding sites identified in the present study include potential binding sites for catfish SS-22, but a 'mismatch' may appear since the localizations of the corresponding peptide are not known.

Acknowledgements This work was supported by grant MT-8580 of the Medical Research Council of Canada to R.Q. and grant 10530 of the Medical Research Council of Canada to L.M.G.K.H.Z. is a 'Hermann von Helmholtz Fellow' of the Bundesministerium fiir Forschung und Technologie, R.Q. a 'Chercheur-Boursier' of the Fonds de la Recherche en Sant6 du Qu6bec. We thank Dr P. Gaudreau (H6pital Notre-Dame, Montr6al, Qu6bec) for synthesizing SS-14 and its analogue T°-DS-SS-14, Dr J. C. Reubi (Sandoz Co., Switzerland) for generously providing SMS 201-995 and SMS-204-090, Thomas Stroh (MaxPlanck-Institut fiir Entwicklungsbiologie, Tiibingen) for helpful comments on the manuscript as well as Ingrid

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Horschke and Jiirgen Jung (both Max-Planck-lnstitut fiir Entwicklungsbiologie, Tiibingen) for excellent technical assistance. Appendix: abbreviations A, pretectal nucleus A; AVI, area ventrolateralis (thalami); B, pretectal nucleus B; c, caudal; cANS, commissura ansulata; CC, crista cerebellaris; CCb, corpus cerebelli; CG, central gray; CLS, centrolateral segment; CMS, centromedial segment; CP, central-posterior nucleus (diencephalon); cT, tectal commissure; cTSd, commissure of the torus semicircularis dorsalis; DC, central division of dorsal forebrain; DD, dorsal division of the dorsal forebrain; DDi, inferior subdivision of DD; DDmg, magnocellular subdivision of DD; DDs, superficial subdivision of DD; DFI, nucleus diffusus lateralis of the inferior lobe; DLc, dorsolateral telencephalon, central subdivision; DLd, dorsolaterai telencephalon, dorsal subdivision; DLp, dorsolateral telencephalon, posterior subdivision; DLTh, dorsolateral thalamus; DLv, dorsolateral telencephalon, ventral subdivision; DMI, dorsomedial telencephalon, subdivision 1; DM2c, dorsomedial telencephalon, subdivision 2, caudal; DM2d, dorsomedial telencephalon, subdivision 2, dorsal; DM2r, dorsomedial telencephalon, subdivision 2, rostal; DM2v, dorsomedial telencephalon, subdivision 2, ventral; DMTh, dorsomedial thalamus; DP, dorsal posterior telencephalon; DPI, lateral subdivision of caudal DP; DPm, medial subdivision of caudal DP; EGa, eminentia granularis pars anterior; EGm, eminentia granularis par medialis; EGp, eminentia granularis pars posterior; ELL, electrosensory lateral line lobe; ELL (dml), dorsal molecular layer of ELL; Er, rostral entopeduncular nucleus; eTS, torus semicircularis efferents; FB, forebrain bundle; g, granule cell layer; G, glomerular nucleus; H, habenula; Ha, hypothalamus anterioris; Hc, hypothalamus caudalis; Hd, hypothalamus dorsalis; HI, hypothalamus lateralis; Hv, hypothalamus ventralis; IL, inferior lobe; IPn, interpeduncular nucleus; LCe, locus ceruleus; LL, lateral lemniscus; LS, lateral segment; MFB, medial forebrain bundle; MLF, medial longitudinal fasciculus; mol, molecular layer; MOTF, medial olfactory terminal field; MRF, mesencephalic reticular formation; MS, medial segment; nE, nucleus electrosensorius; hi, nucleus isthmi; nM, nucleus medialis; nOr, rostral olfactory nucleus; nRP, nucleus recessus posietioris; nXs, vagal sensory nucleus; OB, olfactory bulb; p, pyramidal cell layer; Pdl, nucleus praeminentialis dorsalis pars lateralis; Pdm, nucleus praeminentialis dorsalis pars medialis; PGI, preglomerular nucleus, lateral subdivision; PGm, preglomerular nucleus, medial subdivision; PGr, preglomerular nucleus, rostral subdivision; PL1, paralemniscal nucleus, lateral subdivision; PLm, paralemniscal nucleus, medial subdivision; PMRF, paramedian

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reticular formation; POR, preoptic region; PPa, nucleus preopticus periventricularis, anterior subdivision; PPn, prepacemaker nucleus; PPp, nucleus preopticus periventricularis, posterior subdivision; PT, pretectal nucleus; PTh, nucleus prethalamicus; PTI, lateral pretectal nucleus; Rc, nucleus raph6 centralis; Rd, nucleus raph6 dorsalis; RF, reticular formation (rhombencephalon); SE, subelectrosensorius nucleus; STr, subtrigeminal nucleus; TA, nucleus tuberis anterior; TeO, optic teetum; TL, torus longitudinalis; tOVL, tractus opticus ventrolateralis; TPP, periventricular nucleus of the posterior tuberculum; TSd, torus semicircularis, dorsal subdivision; TSv, torus semicircularis, ventral subdivision; Vc, ventral telencephalon, central subdivision; VCb, valvular cerebelli; VCbl, valvula cerebelli pars lateralis; VCbm, valvula cerebelli pars medialis; Vd, ventral telencephalon, dorsal subdivision; VI, ventral telencephalon, intermediate subdivision; VIr, ventral telencephalon, intermediate rostral subdivision; VLTh, ventrolateral thalamus; Vn, ventral telencephalon, nother subdivision; VP, ventral telencephalon, posterior subdivision; VPn, nucleus of the valvular peduncle; Vs, ventral telencephalon, supracommissural subdivision; Vv, ventral telencephalon, ventral subdivision; ZT, transitional zone. References Bass, A.H. (1982) Evolution of the vestibulolateral lobe of the cerebellum in electroreceptive and nonelectroreceptive teleosts. J. Morphol. 174, 335-348. Bauer, W., Briner, U., Doepfner, W., Hailer, R., Hugenin, R., Marbach, P., Peotcher, T.J. and Pless J. (1982) SMS 201-995: a very potent and selective octapeptide analogue of somatostatin with prolonged action. Life ScL 31, 1133-1139. Beal, M.F. and Martin, J.B. (1984) The effect of somatostatin on striatal catecholamines. Neurosci. Lett. 44, 271-276. Bell, G.I. and Reisine, T. 0993) Molecular biology of somatostatin receptors. Trends NeuroscL 16, 34-38. Berelowitz, M., Kronheim, S., Pimstone, B. and Sheppard, M. (1978) Potassium-stimulated calcium dependent release of immunoreactire somatostatin from incubated rat hypothalamus. J. Neurochem. 31, 1537-1539. Brazeau, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J. and Guillemin, R. (1973) Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. Science 179, 77-79. Breder, C.D., Yamada, Y., Yasuda, K., Seino, S., Saper, C.B. and Bell, G.I. (1992) Differential expression of somatostatin receptor subtypes in brain. J. Neurosci. 12, 3920-3934. Carr, C.E. and Maler, L. (1986) Electroreception in gymnotiform fish: central anatomy and physiology. In Electroreception (eds Bullock, T.H. and Heiligenberg, W.), pp. 319-373. John Wiley & Sons, New York. Chesselet, M.-F. and Reisine, T.D. (1983) Somatostatin regulates dopamine release in rat striatal slices and cat caudate nuclei. J. Neurosci. 3, 232-236. Dells, J.R. and Dichter, M.A. (1983) Effects of somatostatin on mammalian cortical neurons in culture: physiological actions and unusual dose response characteristics. J. Neurosci. 3, 1176-1188. Dodd, J. and Kelly, J.S. (1978) Is somatostatin an excitatory transmitter in the hippocampus? Nature 273, 674-675.

62

G.K.H. Zupanc et al. / J. Chem. Neuroanat. 7 (1994) 49-63

Dunlap, K. and Fischbach, G.D. (1978) Neurotransmitters decrease the calcium component of sensory neurone action potentials. Nature 276, 837-839. Enjalbert, A., Tapia-Arancibia, L., Rieutort, M., Brazeau, P., Kordon, C. and Epelbaum, J. (1982) Somatostatin receptors on rat anterior pituitary membranes. Endocrinology 110, 1634-1 640. Epelbaum, J. (1992) Somatostatin receptors in the central nervous system. In Somatostatin (eds Weil, C., Miiller, E.E. and Thorner, M.O.), pp. 17-28. Springer-Verlag, Berlin. Epelbaum, J., Brazeau, P., Tsang, D., Brawer, J. and Martin, J.B. (1977) Subcellular distribution of radioimmunoassayable somatostatin in rat brain. Brain Res. 126, 309-323. Epelbaum, J., Dournaud, P., Fodor, M. and Viollet, C. (1994) The neurobiology of somatostatin. Crit. Rev. Neurobiol. 8, 25-44. Finger, T. (1983) Organization of the teleost cerebellum. In Fish Neurobiology, Vol. 1 (eds Northcutt, R.G. and Davis, R.E.), pp. 261-284. The University of Michigan Press, Ann Arbor. Fink, S.V. and Fink, W.L. (1981) Interrelationships of the ostariophysan fishes (Teleostei). Zool. J. Linn. Soc. 72, 297-353. Fitzpatrick-McEIligott, S., Card, J.P. Baldino, Jr., F. and Lewis, M. E. (1988) Neuronal localization of prosomatostatin mRNA in the rat brain with in situ hybridization histochemistry. J. Comp. Neurol. 273, 558-572. Gluschankof, P., Morel, A., Benoit, R. and Cohen, P. (1985) The somatostatin-28 convertase of rat brain cortex generates both somatostatin-14 and somatostatin-28 (1-12). Biochem. Biophys. Res. Commun. 128, 1051-1057. G&hert, M. (1980) Somatostatin selectively inhibits noradrenaline release from hypothalamic neurones. Nature 288, 86-88. Herkenham, M. (1987) Mismatches between neurotransmitter and receptor localizations in brain: Observations and implications. Neuroscience 23, 1-38. H6kfelt, T. (1991) Neuropeptides in perspective: The last ten years. Neuron 7, 867-879. H6kfelt, T., Millhorn, D., Seroogy, K., Tsuruo, Y., Ceccatelli, S., Lindh, B., Meister, B., Melander, T., Schalling, M., Bartfai, T. and Terenius, L. (1987) Coexistence of peptides with classical neurotransmitters. Experientia 43, 768-780. Hunter, W.M. and Greenwood, F.C. (1962) Preparation of iodine- 131 labelled human growth hormone of high specific activity. Nature 194, 495-496. lkeda, S.R. and Schofield, G.G. (1989) Somatostatin blocks a calcium current in rat sympathetic ganglion neurones. J. Physiol. 409, 221-240. loffe, S., Havlicek, V., Friesen, H. and Chernick, V. (1978) Effect of somatostatin (SRIF) and L-glutamate on neurons of the sensorimotor cortex in awake habituated rabbits. Brain Res. 153, 414-418. Iversen, L.L., Iversen, S.D., Bloom, F., Douglas, C., Brown, M. and Vale, W. (1978) Calcium-dependent release of somatostatin and neurotensin from rat brain in vitro. Nature 273, 161-163. Johansson, O., H6kfelt, T. and Elde, R.P. (1984) lmmunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience 13, 265-339. Johnston, S.A. and Maler, L. (1992) Anatomical organization of the hypophysiotrophic systems in the electric fish, Apteronotus leptorhynchus. J. Comp. Neurol. 317, 421-437. Johnston, S.A., Maler, L. and Tinner, B. (1990) The distribution of serotonin in the brain of Apteronotus leptorhynchus: an immunohistochemical study. J. Chem. Neuroanat. 3, 429-465. J~rgensen, J.M. (1982) Fine structure of the ampullary organs of the bichir Polypterus senegalus Cuvier, 1829 (Pisces: Brachiopterygii) with some notes on the phylogenetic development of electroreceptors. Acta Zool. (Stockh.) 63, 211-217. Kawasaki, M., Maler, L., Rose, G.J. and Heiligenberg, W. (1988) Anatomical and functional organization of the prepacemaker

nucleus in gymnotiform electric fish: the accommodation of two behaviors in one nucleus. J. Comp. Neurol. 276, 113-131. Krantic, S., Martel, J.-C., Weissmann, D., Pujol, J.-F. and Quirion, R. (1990) Quantitative radioautographic study of somatostatin receptors heterogeneity in the rat extrahypothalamic brain. Neuroscience 39, 127-137. Krantic, S., Martel, J.C., Weismann, D. and Quirion, R. (1989) Radioautographic analysis of somatostatin receptor sub-type in rat hypothalamus. Brain Res. 498, 267-278. Krantic, S., Quirion, R. and Uhl, G. (1992a) Brain somatostatin receptors. In Handbook of Chemical Neuroanatomy, Vol. 11 (eds Bj6rklund, A., H6kfelt, T. and Kuhar, M.), pp. 321-346. Elsevier, Amsterdam. Krantic, S., Robitaille, Y. and Quirion, R. (1992b) Deficits in the somatostatin SS1 receptor sub-type in frontal and temporal cortices in Alzheimer's disease. Brain Res. 573, 299-304. Laquerriere, A., Leroux, P., Gonzalez, B.J., Bodenant, C., Benoit, R. and Vaudry, H. (1989) Distribution of somatostatin receptors in the brain of the frog Rana ridibunda: correlation with the localization of somatostatin-containing neurons. J. Comp. Neurol. 280, 451-467. Leroux, P., Quirion, R. and Pelletier, G. (1985) Localization and characterization of brain somatostatin receptors as studied with somatostatin-14 and somatostatin-28 receptor radioautography. Brain Res. 347, 74-84. Lundberg, J.M. and Hfkfelt, T. (1983) Coexistence of peptides and classical neurotransmitters. Trends Neurosci. 6, 325-333. Magazin, M., Minth, C.D., Funckes, C.L., Deschenes, R., Tavianini, M. A. and Dixon, J.E. (1982) Sequence of a cDNA encoding pancreatic preprosomatostatin-22. Proc. Natl. Acad. Sci. USA 79, 5152-5156. Maler, L. (1992) Regulation of electrocommunicatory behavior by sensory input and chemically coded hypothalamic input. Proc. Third Int. Congress Neuroethology, Montreal, Canada, 5-6. Maler, L. and Monaghan, D. (1991) The distribution of excitatory amino acid binding sites in the brain of an electric fish, Apteronotus leptorhynchus. J. Chem. Neuroanat. 4, 39-61. Maler, L. and Mugnaini, E. (1994) Correlating gamma-aminobutyric acidergic circuits and sensory function in the electrosensory lateral line lobe of a gymnotiform fish. J. Comp. Neurol. 345, 224-252. Maler, L., Sas, E., Carr, C.E. and Matsubara, J. (1982) Efferent projections of the posterior lateral line lobe in gymnotiform fish. J. Comp. Neurol. 211, 154-164. Maler, L., Sas, E., Johnston, S. and Ellis, W. (1991) An atlas of the brain of the electric fish Apteronotus leptorhynchus. J. Chem. Neuroanat. 4, 1-38. Mancillas, J.R., Siggins, G.R. and Bloom, F.E. (1986) Somatostatin selectively enhances acetylcholine-induced excitations in rat hippocampus and cortex. Proc. Natl. Acad. Sci. USA 83, 7518-7521. Martin, J.-L., Chesselet, M.-F., Raynor, K., Gonzales, C. and Reisine, T. (1991) Differential distribution of somatostatin receptor subtypes in rat brain revealed by newly developed somatostatin analogs. Neuroscience 41, 581-593. Maurer, R. and Reubi, J.C. (1985) Brain somatostatin receptor subpopulation visualized by autoradiography. Brain Res. 333, 178-181. Mengod, G., Rigo, M., Savasta, M., Probst, A. and Palacios, J.M. (1992) Regional distribution of neuropeptide somatostatin gene expression in the human brain. Synapse 12, 62-74. Meyer, D.K., Conzelmann, U. and Schultheiss, K. (1989) Effects of somatostatin-14 on the in vitro release of [3H]GABA from slices of rat caudatoputamen. Neuroscience 28, 61-68. Mueller, A.L., Kunkel, D.D. and Schwartzkroin, P.A. (1986). Electrophysiological actions of somatostatin (SRIF) in hippocampus: an in vitro study. Cell. Mol. Neurobiol. 6, 363-379. Mugnaini, E. and Maler, L. (1993) Comparison between the fish elec-

G.K.H. Zupanc et al. / J. Chem. Neuroanat. 7 (1994) 49-63 trosensory lateral line lobe and the mammalian dorsal cochlear nucleus. J. Comp. Physiol. A, 173, 683-685. Nemeth, E.F. and Cooper, J.R. (1979) Effect of somatostatin on acetylcholin¢ release from rat hippocampal synaptosomes. Brain Res. 165, 166-170. lqieuwenhuys, R. (1963) The comparative anatomy of the actinopterygian forebrain. J. Hirnforsch. 6, 171-192. Nieuwenhuys, R. and Meek, J. (1990) The telencephalon of Sarcopterygian fishes. In Cerebral Cortex (eds Jones, E.G. and Peters, A.), pp. 75-106. Plenum Press, New York. Northcutt, R.G. and Davis, R.E. (1983) Telencephalic organization in ray-finned fishes. In Fish Neurobiology, Vol. 2 (eds Northcutt, R.G. and Davis, R.E.), pp. 203-236. The University of Michigan Press, Ann Arbor. Patel, Y.C. (1992) General aspects of the biology and function of somatostatin. In Somatostatin (eds Weil, C., Miiller, E.E. and Thorner, M.O.), pp. i-16. Springer-Verlag, Berlin. Pittman, Q.J. and Siggins, G.R. (1981) Somatostatin hyperpolarizes hippocampai pyramidal cells in vitro. Brain Res. 221,402-408. Priestley, J.V., R6thelyi, M. and Lund, P.K. (1991). Semi-quantitative analysis of somatostatin mRNA distribution in the rat central nervous system using in situ hybridization. J. Chem. Neuroanat. 4, 131-153. Randi~, M. and Mileti~, V. (1978) Depressant actions of methionineenkephalin and somatostatin in cat dorsal horn neurons activated by noxious stimuli. Brain Res. 152, 196-202. Raynor, K. and Reisine, T. (1989) Analogs of somatostatin selectively label distinct subtypes of somatostatin receptors in rat brain. J. Pharmacol. Exp. Ther. 251,510-517. Raynor, K., Coy, D.C. and Reisine, T. (1992) Analogues of somatostatin bind selectively to brain somatostatin receptor subtypes. J. Neurochem. 59, 1241-1250. Renaud, L.P., Martin, J.B. and Brazeau, P. (1975) Depressant action of TRH, LH-RH and somatostatin on activity of central neurones. Nature 255, 233-235. Reubi, J.-C. (1984) Evidence for two somatostatin-14 receptor types in rat brain cortex. NeuroscL Lett. 49, 259-263. Reubi, J.C., Cort6s, R., Maurer, R., Probst, A. and Palacios, J.M. (1986) Distribution of somatostatin receptors in the human brain: an autoradiographic study. Neuroscience 18, 329-346. Reubi, J.C. and Maurer, R. (1985) Autoradiographic mapping of somatostatin receptors in the rat central nervous system and pituitary. Neuroscience 15, 1183-1193. Reubi, J.-C., Perrin, M., Rivier, J. and Vale, W. (1981) High affinity binding sites for a somatostatin-28 analog in rat brain. Life Sci. 28, 2191-2198. Reubi, J.C., Probst, A., Cort6s, R. and Palacios, J.M. (1987) Distinct topographical localisation of two somatostatin receptor subpopulations in the human cortex. Brain Res. 406, 391-396. Rorstad, O.P., Epelbaum, J., Brazeau, P. and Martin, J.B. (1979). Chromatographic and biological properties of immunoreactive somatostatin in hypothalamic and extrahypothalamic brain regions of the rat. Endocrinology 105, 1083-1092. Sas, E. and Maler, L. (1986) The optic tectum of gymnotiform teleosts Eigenmannia virescens and Apteronotus leptorhynchus: a Golgi study. Neuroscience 18, 215-246. Sas, E. and Maler, L. (1987) The organization of afferent input to the caudal lobe of the cerebellum of the gymnotid fish Apteronotus leptorhynchus. Anat. Embryol. 177, 55-79. Sas, E. and Maler, L. (1989) Olfactory bulb connectivity with special reference to telencephalic locus coeruleus and raphe inputs. Sac. Neurosci. Abstr. 15, 927. Sas, E. and Maler, L. (1991) Somatostatin-like immunoreactivity in the brain of an electric fish (Apteronotus leptorhynchus) identified with monoclonal antibodies. J. Chem. Neuroanat. 4, 155-186.

63

Sas, E., Maler, L. and Weld, M. (1993) Connections of the olfactory bulb in the gymnotiform fish, Apteronotus leptorhynchus. J. Comp. Neurol. 335, 486-507. Srikant, C.B. and Patel, Y.C. (1981) Somatostatin receptors: identification and characterization in rat brain membranes. Proc. Natl. dcad. Sci. USA 78, 3930-3934. Striedter, G.F. (1992) Phylogenetic changes in the connections of the lateral preglomerular nucleus in ostariophysan teleosts: a pluralistic view of brain evolution. Brain Behav. Evol. 39, 329-357. Stroh, T. and Zupanc, G.K.H. (1993a) Identification and localization of somatostatin-like immunoreactivity in the cerebellum of gymnotiform fish, Apteronotus leptorhynchus. Neurosci. Lett., 160, 145-148. Stroh, T. and Zupanc, G.K.H. (1993b) GABA and somatostatin in the region of the prepacemaker nucleus of .4pteronotus leptorhynchus: a double-labelling immunohistochemical study. In Gene - - Brain Behaviour (eds Eisner, N. and Heisenberg, M.), p. 613, Georg Thieme Verlag, Stuttgart. Tapia-Arancibia, L., Epelbaum, J., Enjalbert, A. and Kordon, C. (1981). Somatostatin binding sites in various structures of the rat brain. Eur. J. Pharmacol. 71,523-525. Thermos, K., He, H.-T., Wang, H.-L., Margolis, N. and Reisine, R. (1989) Biochemical properties of brain somatostatin receptors. Neuroseience 31, 131-141. Tran, V.T., Beal, M.F. and Martin, J.B. (1985) Two types of somatostatin receptors differentiated by cyclic somatostatin analogs. Science 228, 492-495. Uhl, G.R., Tran, V., Snyder, S.H. and Martin, J.B. (1985) Somatostatin receptors: distribution in rat central nervous system and human frontal cortex. J. Comp. Neurol. 240, 288-304. Vincent, S.R., Mclntosh, C.H.S., Buchan, A.M.J. and Brown, J.C. (1985) Central somatostatin systems revealed with monoclonal antibodies. J. Comp. Neurol. 238, 169-186. Weld, M.M, Kar, S., Maler, L. and Quirion, R. (1994) The distribution of tachykinin binding sites in the brain of an electric fish (Apteronotus leptorhynchus). J. Chem. Neuroanat., in press. Weld, M.M. and Maler, L. (1992) Substance P-like immunoreactivity in the brain of the gymnotiform fish Apteronotus leptorhynchus: presence of sex differences. J. Chem. Neuroanat. 5, 107-129. Xie, Z. and Sastry, B.R. (1992) Actions of somatostatin on GABAergic synaptic transmission in the CAI area of the hippocampus. Brain Res. 591,239-247. Yamamoto, T, Maler, L. and Nagy, J.I. (1992) Organization of galanin-like immunoreactive neuronal systems in weakly electric fish (Apteronotus leptorhynchus). J. Chem. Neuroanat. 5, 19-38. Zingg, H.H. and Patel, Y.C. (1982) Processing of synthetic somatostatin-28 and a related endogenous rat hypothalamic somatostatin-like molecule by hypothalamic enzymes. Life ScL 30, 525-533. Zupanc, G.K.H., C6cyre, D., Maler, L., Zupanc, M.M. and Quirion, R. (1992) Distribution of somatostatin binding sites in the brain of gymnotiform fish, Apteronotus leptorhynchus. Biol. Chem. HoppeSeyler 372, 967. Zupanc, G.K.H. and Maler, L. (1993) Evoked chirping in the weakly electric fish Apteronotus leptorhynchus: a quantitative biophysical analysis. Can. J. Zool. 71, 2301-2310. Zupanc, G.K.H., Maler, L. and Heiligenberg, W. (1991a). Somatostatin-like immunoreactivity in the region of the prepacemaker nucleus in weakly electric knifefish, Eigenmannia: a quantitative analysis. Brain Res. 559, 249-260. Zupanc, G.K.H., Okawara, Y., Zupanc, M.M., Fryer, J.N. and Maler, U (1991 b) In situ hybridization of putative somatostatin mRNA in the brain of electric gymnotiform fish. NeuroReport 2, 707-710. -

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