somatostatin receptors: distribution in rat central nervous system and human frontal cortex

THE JOURNAL OF COMPARATIVE NEUROLOGY 240:288-304 (1985)

Somatostatin Receptors: Distribution in Rat Central Nervous System and Human

Frontal Cortex

GEORGE R. UHL, VINH TRAN, SOLOMON H. SNYDER AND JOSEPH B. MARTIN Departments of Neuroscience (G.R.U., S.H.S.) and Neurology (G.R.U.), Johns Hopkins

Hospital, Baltimore, Maryland 21205; Department of Neurology, Massachusetts General Hospital (G.R.U., V.T., J.B.M.) and Neuroscience Group, Howard Hughes Medical Institute, Massachusetts General Hospital and Harvard Medical School

(G.R.U.), Boston, Massachusetts 02114

ABSTRACT Somatostatins are a brain peptide family centered on a 14-amino acid

cyclic peptide (SS-14) and a 28-amino acid N-terminally extended form (SS- 28). Using radioiodinated analogs of SS-14 and SS-28, we have identified specific binding sites in rat and human brain sections that display pharma- cological properties anticipated for somatostatin receptors and discrete pat- terns of anatomical localization. High binding densities are found in many forebrain regions, with special densities in infragranular cerebral cortical laminae in rat and human brain. In the rat, other densities lie in olfactory zones, lateral and triangular septa1 nuclei, the CA-1 hippocampal region, and claustrum with moderate densities in the striatum. Discrete hypotha- lamic areas, especially the median preoptic, paraventricular, and periventri- cular nuclei, display elevated binding levels, while the thalamus shows only scattered areas of modest binding. Midbrain receptor concentrations are found in portions of the periaqueductal gray, interpeduncular nucleus, and the substantia nigra. Notable pontine and medullary densities lie in the locus coeruleus, fourth ventricular floor, parabrachial, solitary, prepositus hypoglossal, dorsal column, and caudal trigeminal zones. Although the cer- ebellar cortex shows unimpressive densities, each of the deep cerebellar nuclei is heavily labeled. Modest spinal cord receptor densities are concen- trated in the substantia gelatinosa and central cord regions.

These localizations show many paraIIeIs with the distributions of SS- immunoreactive neurons, fibers, and terminals determined previously by immunohistochemistry. They provide plausible loci for several reported physiological or pharmacological activities of the SS-peptides, and may im- prove understanding of the role of the SS alterations described in several human neurodegenerative disorders.

Key words: cerebral cortex, receptor autoradiography, hypothalamus, neuropeptides, Alzheimer's disease

Somatostatin peptides (SS) were originally isolated from hypothalamus on the basis of inhibition of growth hormone release (Brazeau et al., '73). These peptides are now linked to diverse functions in many brain areas and implicated in several human neurodegenerative diseases (Reichlin, '83). Selective reductions in cerebral cortical SS levels in the dementias accompanying Alzheimer's and Parkinson's dis- eases, for example, are consistent with a role for this cere-

bral cortical peptide in cognitive function (Davies et al., '80; Rossor et al., '80; Epelbaum et al., '83). Similarly, the selectively increased striatal SS levels found in Hunting- ton's disease hint a t a role for the peptide in the genesis of the choreoathetotic movements found in this disorder (Cooper et al., '81; Nemeroff et al., '82).

Accepted July 2, 1985.

0 1985 ALAN R. LISS, INC.

CNS SOMATOSTATIN RECEPTORS 289

The somatostatin originally isolated from hypothalamic extracts was a 14-amino acid cyclic peptide (SS-14). Subse- quent studies have revealed additional peptides displaying sequences and bioactivities partially homologous to SS-14, especially SS-28 (Pradayral et al., '80). SS-28 contains the 33-14 sequence plus an additional 14-amino acids at its N terminus. This longer form is localized in brain synapto- somes and released under physiological conditions (Reich- lin, '83).

Somatostatin receptors have been identified in brain, pi- tuitary, and peripheral tissue homogenates by binding tech- niques (Reubi et al., '81; Srikrant et al., '81; Epelbaum et al., '82; Perry et al., '83; Tran et al., '84a). Recent studies have revealed binding of high affinity and fairly high den- sity in homogenates of rat and human brain, with mem- branes from human cinguIate cortex displaying a 1 nM KD and 2.96 fmoVmg B,, while rat cerebral cortical mem- branes show a KD = 0.17 nM and B,, of 5.2 fmol/mg (Beale et al. '85). Receptor binding studies have been aided by synthesis of less lipophilic, degradation-resistant SS an- alogs that display good bioactivity, less nonspecific binding, and accessibility to radioiodination (Reubi et al., '82). These studies have demonstrated physiological relevance since peptide analog potencies in displacing radiolabeled SS from binding sites have paralleled their bioactivities in physio- logic test systems (Rorstadt et al., '83; Schonbrunn et al., '83). Though several binding site subtypes have been re- ported (Taborsky et al., '79; Reubi et al., '82; Tran et al., '84a), clear-cut evidence for strongly SS-14 or SS-28 prefer- ring receptors has been more difficult to obtain in brain.

Homogenate binding studies provide only general infor- mation about receptor distributions. Receptor autoradiog- raphy can yield much more precise anatomic detail. Preliminary studies have demonstrated the feasibility of this approach for SS (Tran et al., '84b; Leroux and Pelletier, '84). Because of the wide anatomic distribution of SS im- munoreactivity, its broad range of possible important roles in normal brain function, and its implication in human neurodegenerative disorders, we have extended these pre- liminary studies. We now report validation of the SS-recep- tor autoradiographic technique in rat and human brain, and detailed mapping of receptor distributions throughout the rat neuraxis and in human cerebral cortex.

METHODS Peptide iodination and purification

Leu8-D-Trp22,Tyr25-SS-28, "(LTT)," and Tyrll SS-14 were synthesized and generously supplied by Drs. Jean Rivier and Wylie Vale (Laboratory of Peptide Biology, The Salk Institute, La Jolla, California). Five micrograms of peptide dissolved in 0.1 N acetic acid was added to 10 pl of freshly-prepared Chloramine T solution (1 mg/ml) (Kodak) and 1 mCi Na lZ5I (Amersham) (2,200 CUmmol) in 25 p1 NaP04 buffer, 0.6 M, pH 7.4. Reaction for 90 seconds at 22°C was terminated by addition of 100 pl 10 mM tyrosine (Sigma). Reaction products were separated from unreacted Na lZ5I by elution with 0.1 N HAC through a Sep-Pack (Waters Assoc.) preequilibrated with 0.1 N HAC. The first 1-ml fraction was subjected to isocratic elution high-perfor- mance liquid chromatography (HCLC) on a Waters HPLC a t 1 nil/minute with the aid of a C18 microbondapack col- umn in 0.25 M triethylaminelformic acid (pH 3.5) with 17% n-propanol. Active fractions were identified by monitoring radioactivity peaks; iodinated material elutes at 30 min- utes in this protocol. Since uniodinated peptide elutes at 15

minutes, this provides consistent separation of iodinated from uniodinated peptide. We calculate specific activities of our peptide products to be 2,200 Ciimmol based on incorpo- ration of one lZ5I for each tyrosine residue.

Tissue preparation Male Sprague-Dawley rats (225-250 g) and frozen speci-

mens from autopsy-derived human cerebral cortex were used for these studies. Rats were anesthetized with pento- barbital and perfused intracardially with 0.32 M sucrose in 0.1 M sodium phosphate buffer, pH 7.4. Brains were re- moved, cut into 5.0-mm coronal slabs, and frozen onto cryo- stat chucks with powdered dry ice. Frontal pole specimens from six neurologically normal individuals (ages 50-74 years, postmortem intervals 5.5-29 hours) were frozen on dry ice, maintained at -7O"C, and mounted on cryostat chucks. Twelve-micron cryostat sections were thaw- mounted onto slides pretreated with gelatin and chrome alum, stored at -7O"C, then thawed and dried at room temperature prior to autoradiographic incubations.

Incubations Approximately 0.16 nM '251-Tyrll SS-14 or 0.04 nM 1251-

LIT SS-28 was incubated with sections in 150 nM Tris buffer, pH 7.4, containing 0.1% bacitracin (Sigma), 0.1 %, bovine serum albumin (Sigma, fraction V), and 5 mM MgS04. Ligand concentrations used were based on prelim- inary studies on slide-mounted sections, as well as exten- sive saturation analyses of rat and human cerebral cortical membranes in tissue homogenate preparations (Beale et al., '85). Standard incubations were 60 minutes at 25°C. In parallel experiments, various peptides or other agents were added to separate incubations with adjacent sections and with identical ligand content. Control "blank" conditions were obtained using 2 x M SS-14 displacer. Sections were washed in 150 mM Tris, pH 7.4. Routine washing consisted of three 15-minute washes a t 4°C.

Biochemical assay Sections were wiped from slides while still wet by means

of glass fiber filters (Young and Kuhar, '79) and residual radioactivity was counted in an LKB gamma counter a t 90% efficiency. Values presented are derived from at least two experiments, in which three slides were assayed for each "total" and "blank" condition described.

Autoradiography and analysis For autoradiographic experiments, sections were rapidly

dried under cool, dry air, stored overnight with dessicant at 4"C, and then apposed to LKB ultrofilm or to flexible, emulsion-coated coverslips (Kodak NTB3) as described (Young and Kuhar, '79). For each level of the neuraxis of each individual rat at least five adjacent sections were examined; two by film and three by coverslip autoradiog- raphy. Studies reported here represent examination of eight rat brains; each level described in detail has been observed in at least two animals. Following 10-day exposures, films and coverslip emulsions were developed. For coverslipped autoradiograms, the emulsion was developed, underlying tissue was stained with cresyl violet, and the slide and coverslip were then reapposed with Permount medium. An- atomic localizations were derived from examination of both types of images, consultation with anatomic atlases, and reference to descriptive papers in particular cases (Krieg, '46; Berman and Bowers, '67; Nauta and Haymaker, '69;

290 G.R. UHL ET AL.

Chavis, '69; Steiner and Turner, '72; Smaha and Kaelben, '73; Palkovits and Jacobowitz, '74; Herkenham and Nauta, '77; Swanson et al., '78; Krettek and Price, '78; Konig and Klippel, '63).

Most photographs presented here were generated by printing the ultrofilm image of the brain; since data were obtained from different films developed separately, grain sizes may vary from image to image. Each anatomic local- ization has been confirmed by reference to cell-stained ma- terial. Photographs were cut from their surroundings to eliminate distracting images in mounting medium, and to separate multiple images cut on single chucks.

RESULTS Biochemical-pharmacological binding

site characterization In biochemical experiments, tissue sections were wiped

from slides and the radioactivity was counted. Preliminary studies with 1251-LTT-SS-28 in rat (coronal sections through mid-striatum) and human (frontal or temporal cortex) re- vealed specific:nonspecific binding ratios of 2:l to 3:l that were achieved after 60-90 minutes of incubation at 22°C. Sections through the brainstem revealed lower ratios. Characterization of these regions could conceivably yield somewhat different values, but the low signal-to-noise ratio in these sections renders this difficult. Slow apparent dis- sociation at 4°C allowed maximal specific binding ratios after 20-60 minutes total washing (data not shown). Per- haps most importantly, pharmacological characterization

of these sites was consistent with properties of SS-receptors described in homogenate-binding studies (Srikrant and Pa- tel, '81; Reubi et al., '81, '82; Perry et al., '83; Tran et al., '84a). Active SS-peptides, including SS-14, SS-28, Tyr-11-SS- 14, and LTT-SS-28, at concentrations of less than 10 nM, were all active in displacing this binding in both rat and human studies (Table 1). Conversely, closely related phys- iologically inactive peptides failed to displace binding sig- nificantly at 0.1 pM concentration (Taborsky et al., '79; Reubi et al., '81; Schonbrunn et al., '83; Brown et al., '77). These analogs include 8- and 9-"active-site" Ala substitu- tions, multiple amino acid deletions, and the 1-12 potential SS-28 cleavage product. Furthermore, none of a series of unrelated eptides displayed significant potency at this site.

Use of g51-Tyr-11-SS-14 in such studies yielded consis- tently lower specific:nonspecific binding ratios, approxi- mately 151.

Autoradiographic distribution Rut. Autoradiographic images reveal strikingly local-

ized binding with substantial heterogeneity among gray- matter regions, little binding in most white-matter regions, and predominance of binding in more rostra1 areas of the neuraxis. Control "blank" sections obtained by using 1251- L1T-SS-28 display very little nonspecific binding (Fig. lA,B), while blank sections generated with 1251-Tyr-ll-SS-14 show more prominent background (Fig. lC,D). Nevertheless, re- sults obtained with the two ligands reveal virtually identi- cal patterns of receptor distribution at several forebrain and midbrain levels (Figs lA,C, 3C, 4A). Results reported

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amygdala, basal nuclei anterior commissure amygdala, cortical nucleus amygdala, lateral nuclei amygdala, medial nucleus arcuate nucleus, hypothalamus bed nucleus of stria terminalis CA1 region of hippocampus CA3 region of hippocampus amygdala, central nucleus cerebellar cortex corpus callosum colliculus, inferior claustrum cochlear nuclei caudatelputamen colliculus, superior cuneate nucleus cerebral cortex denate gyrus fornix fimbria of hippocampus granular cerebral cortical layer (IV) globus pallidus gracile nuclei hypothalamus, anterior hypothalamus, dorsal hippocampus hypothalamus, lateral hypothalamus, posterior hypothalamus, ventral infragranular cortex intermediate deep cerebellar nucleus interpeduncular nucleus lateral deep cerebellar nucleus locus coeruleus lateral habenula mamillary bodies medial deep cerebellar nucleus

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medial habenula lateral premamillary nucleus medial thalamus dorsal tegmental nucleus, Gudden nucleus of the solitary tract lateral olfactory tract nucleus olfactory tract pyramid periaqueductal gray paraventricular hypothalamic nucleus periventricular hypothalamic nucleus parahypoglossal nucleus pontine nuclei lateral preoptic area medial preoptic area paratenial nucleus of thalamus dorsal raphe nucleus reuniens thalamic nucleus supragranular cortex subfornical organ "substantia gelatinosa." dorsal horn of spinal cord lateral septal nucleus medial septal nucleus substantia nigra supraoptic nucleus stria terminalis "substantia gelatinosa," caudal nucleus of trigeminal thalamus, anteromedial nucleus optic tract thalamus, reticular nucleus triangular septal nucleus thalamus, ventral nuclei spinal tract of the trigeminal vestibular nuclei seventh nerve white matter, cerebral cortical Zona incerta

CNS SOMATOSTATIN RECEPTORS 291

Fig. 1. Somatostatin receptor autoradiograms. Prints of ultrofilm images that 5 x M unlabeled SS-14 was added to incubations to establish a (increased whiteness corresponds to increased grain density). Magnification control. C. 1251-Tyr11-SS-14 autoradiogram, anterior forebrain. Arrow- x 7.9 before reduction. A. '251-Lm-SS-28 autoradiogram, anterior forebrain. heads, granular cortical layer. D. '"1-Tyr1'-SS-14 autoradiogram "blank" Arrowheads denote the granular cortical layer. B. '251-L'lT-SS-28 autoradio- adjacent section to C and treated identically except that 5 x M gram "blank" control, adjacent section to A and treated identically except unlabeled SS-14 was added to incubations to establish a control.

TABLE 1. Peptide Efficacy in Displacing '251-L1T-SS 28 From Slide-Mounted Tissue Sections'

here are thus obtained with 1251-Lm-SS-28, unless other- wise indicated (Figs. 1-7).

Forebrain. In the cerebral cortex, very dense binding is found in infragranular layers (Figs. 1-5,8). A variable and relatively receptor-poor zone corresponding to the granular layer (lamina IV) separates this very dense deep binding from moderately dense receptors in supragranular layers. This pattern persists across all neocortical regions.

In the olfactory bulb and anterior olfactory area very dense binding is also apparent (Fig. 1). Elevated receptor concentrations are also found in the nucleus of the lateral olfactory tract, though the tract itself shows little binding

The septal nuclei and adjacent regions display heteroge- neous binding (Fig. 2A,B). The lateral septum shows mod- erate receptor densities while the medial septal nuclei possess more modest densities. High densities are also found in the triangular septal nucleus (Fig. 9A,B).

At more caudal levels, receptors are dense in the subforn- ical organ. Moderate to high concentrations are found in the bed nucleus ofthe stria terminalis, with higher concen-

Active Inactive ( I C ~ ~ < 1 0 - 7 ~ ) (rcs0 > 1 0 - 7 ~ )

SS 14 (1-12) SS-28 SS 28 d Ala 8-SS-14 Tyr-1 1 -SS- 14 (Leu 8-D-Trp 22,Tyr 25)-SS-28

d Ala 9-SS-14 des 1 ,2 ,4 ,5 , 12-d *pa, SS-14 dCys 14-SS-14 des ', 2, 4, 57 12, 13-d Trpa, "-14

Vasoactive intestinal peptide Neurotensin met-Enkephalin (Fig. 2C,D). Bombesin Substance P Angiotensin I1 Thyrotropin-releasing hormone Pro-leu-glycinamide Oxytocin Dynorphin Eledoisin

1-Cys 14-SS-14

'Pharmacological profile of '251-L1T-SS-28 binding to slide-mounted tissue sections

cortical gyrus) brain. Sections were incubated as described in Methods with 125 LTT- SS-28 and each of the above peptides. Sections were washed, scraped from slides, and residual radioactivity was assayed in a gamma counter. More than 506 inhibition of Regions imp1icated in extrapyramidal motor function binding to both rat and human tissue (at M) was noted by the "active" compounds, while none of the "inactive" substances reduced binding significantly at lo-' M. Individual peptide ICs0 concentrations are best determined by homogenate binding are heterogeneous, with modest to moderate densities in- studios (Reale et al.. '85).

from rat (coronal sections through mid-striaturn) and human (sections of a temporal trations more ventrally disposed.

display receptors (Fig. 2B,C). Striatal receptor densities

creasing somewhat in ventral and medial zones. Some

292 G.R. UHL ET AL.

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294 G.R. UHL ET AL.

Fig. 4. Somatostatin receptor utoradiograms (‘“I-~r”-SS-l4) . Prints of ultrofilm images, Magnification x 6.4 before reduction. A. Level through hippocampus. B. “Blank” adjacent section to A, with 5 x M unlabeled SS-14 added to primary incubations to establish a control.

patchiness is notable though without a fully developed “striosomal” pattern (Graybiel et al., ’81). The globus pal- lidus reveals modest to low levels of binding, while the zona incerta displays moderately dense binding.

The hippocampus shows a striking region- and lamina- dependent binding specificity (Figs. 3,4,5,10). In each of the hippocampal zones, low binding in the pyramidal cell layer contrasts with higher binding densities in the strata oriens and radiata. Superimposed on this lamina specicity is a marked variation from one hippocampal field to the next. Receptor densities are high in CA-1 and the subiculum, moderate in the dentate gyrus, and low in CA-213-4 (Swan- son et al., ’78).

The stria terminalis shows substantial receptor binding along its course; this is confirmed by comparison of grain densities with cell-stained material (Figs. 2,3). Other sub- cortical white matter tracts are virtually devoid of binding, including the corpus callosum, anterior commissure, inter- nal capsule, stria medullaris, fornix, mammilothalamic tract, optic tract, and olfactory tract.

The amygdala displays variability in the generally rich binding among its nuclei (Fig. 3). Most of the lateral group, especially the lateroposterior nucleus, displays very high receptor concentrations (Krettek et al., ’78). High receptor concentrations are also found in the cortical nucleus. Den-

Fig. 5 . Somatostatin receptor autoradiograms (‘”“ILTT-SS-28). Prints of ultrofilm images. Magnification ~ 5 . 5 before reduction. A. Level of mammil- lary bodies. B. Level of interpeduncular nucleus.

sities in the basolateral area, however, are more modest. Moderate receptor concentrations also occur in central and medial amygdaloid nuclei. As noted above, the associated nucleus of the lateral olfactory tract also displays binding densities.

Diencephalon. The thalamus contains moderate to low receptor densities. At rostra1 levels, the reticular nucleus displays modest binding densities (Fig. ZC,D). Paratenial and periventricular nuclei reveal receptor densities that are somewhat lower than those found in the reticular nu- cleus. In more caudal areas, however, only modest to low receptor densities can be found (Fig. 3A-D). Modest densi- ties are found in reuniens, periventricular, suprafascicu- lar, and the middle of the reticular nucleus, with low levels in anteromedial, anteroventral, medial, lateral, ventral, parafascicular, and caudal aspects of the reticular nucleus. At the level of the posterior commissure (Fig. 5A), moder- ate to modest receptor contents lie in the lateral geniculate and pretectal nuclei. Lower concentrations surround the third ventricle. The lateral geniculate displays significant binding only a t its medial border.

Preoptic and hypothalamic areas reveal patterned distri- butions of moderate receptor densities (Figs. 2-4). Periven- tricular, preoptic, median preoptic, and adjacent portions of medial preoptic nuclei display moderately dense recep- tors, while lateral preoptic zones are labeled with only modest density (Fig. 2B). Anterior hypothalamus and peri- ventricular hypothalamus show modest to moderate recep- tor densities that are accentuated in the supraoptic nuclei

CNS SOMATOSTATIN RECEPTORS 295

Fig. 6. Somatostatin receptor autoradiograms ('"I-LTT-SS-28). Prints of ultrofilm images. Magnification x 7.9 before reduction. A. Level of the locus coeruleus. B. Level of deep cerebellar nuclei. C. Level of vestibular nuclei. D. Level of dorsal column nuclei.

(Fig. 2C). Lateral hypothalamus, conversely, displays only low receptor densities. Moderate to high receptor concen- trations occur in paraventricular zones (Fig. 2D). Moderate levels are found in periventricular nuclei, with modest to moderate labeling in dorsal, ventral, and lateral hypotha- lamic nuclei at this level. Somewhat more caudally, mod- est dorsal and ventral hypothalamic densities are accentuated in an arclike pattern just medial to the fornix (Fig. 3B,D), and in the arcuate nucleus. In the median eminence, modest to moderate receptor densities may be somewhat accentuated in external layers. At still more caudal levels, modest receptor densities extend through the dorsal-ventral extent of the hypothalamus from the third ventricle to the plane of the fornix and the mamil- lothalamic tract. These densities are accentuated in the arcuate and at the lateral border of the premamillary nuclei, where a high receptor density lies in the prelateral mamillary nucleus (Fig. 3C). Conversely, lower densities extend laterally from the plane of the fornix into the lat- eral hypothalamus. Low binding in the mamillary nuclei is accentuated slightly in the supramamillary region (Fig. 5).

Regions of the epithalamus and the zona incerta reveal substantial binding (Figs. 3,4). Moderate to high densities

are found in the medial habenula, with low densities over the lateral habenula. Moderate to high levels also occur in the zona incerta, as noted above.

Midbruin. Highest midbrain binding densities are found in dorsal and medial portions of the interpeduncular nu- cleus, which appear to correspond to the apical and central subnuclei described in cats (Berman and Bowers, '67) (Figs. 5B, 11). Moderately high binding is noted in the substantia nigra with higher densities in zona reticulata. Modest to moderate levels are noted in the adjacent ventral tegmen- tal area of Tsai. In the periaqueductal gray, moderate binding densities are accentuated in a limited distribution in dorsolateral quadrants, while receptors elsewhere are of lower density (Fig. 5B). The superior colliculus displays modest to moderate receptor densities. Low receptor den- sities in the mesencephalic reticular formation are accen- tuated in the cuneiform nucleus and aspects of the linear and raphe nuclei. The inferior colliculus displays low re- ceptor densities.

In the pons, moderate binding in the dorsolateral periaqueductal gray continues (Fig. 5A). Moderately to modestly dense receptors are found in the locus coeruleus, with a band of elevated density along the floor of the fourth ventricle, extending along the medial border of the dorsal

Pons.

296 G.R. UHL ET AL.

Fig. 7. Somatostatin receptor autoradiograms ('"SI-L?T-SS-28). Prints of ultrofilm images. Magnification ~ 5 . 5 before reduction. A. Caudal medulla. B. Cervical spinal cord. C. Lumbar spinal cord.

tegmental nucleus of Von Gudden. Dorsal and ventral para- brachial regions and the areas of the principal sensory and motor nuclei of the trigeminal show modest to moderate binding. Low receptor densities are noted in pontine retic- ular zones.

Cerebellum. The cerebellar cortex shows little binding. Deep cerebellar nuclei, however, are each heavily labeled (Fig. 6).

Medulla Rostra1 medullary regions with modest to moderate receptor densities include the vestibular and cochlear nuclei (Figs. 6,7A). In the more caudal medulla, moderate to high receptor densities overlie the prepositus hypoglossal nuclei and solitary and vagal nuclei. Modest to moderate densities lie over the dorsal column nuclei. Low receptor densities in much of the medullary reticular formation are accentuated somewhat in the raphe magnus and in the "substantia gelatinosa" of the caudal trigem- inal nucleus.

Spinal cord. In cervical, thoracic, and lumbosacral spinal cord, modest to moderate binding is seen in the dorsal horn substantia gelatinosa region (Steiner and Turner, '71) (Fig. 7). Low to modest binding in other cord laminae is slightly accentuated around the central canal, and perhaps in the interomediolateral cell column in tho- racic segments.

Human. Human cerebral cortex reveals high receptor densities that are most concentrated in infragranular lam-

inae (Carpenter and Sutin, '83) (Fig. 12). Receptors in layers I-IV are of more moderate density and there is a hint of a relatively receptor-sparse layer corresponding to layer IV. The distribution is apparent in postmortem samples taken from each of six neurologically normal individuals. Com- parison of frontal and temporal cortical specimens suggests higher binding densities in temporal specimens; we have not examined variations across different frontal cortical fields.

DISCUSSION Our anatomic results should be considered in relation to

the pharmacologic specificity of observed binding, correla- tions of receptor distributions with peptide distribution, comparisons between rat and human data, and possible relationships of regional receptor concentrations to normal and pathological brain function.

Pharmacological characterization The SS binding sites labeled in our autoradiographic

studies of human and rat brain display close parallels with SS-receptors identified in physiological studies and in ho- mogenate binding experiments. The observed binding time- course resembles data from homogenate studies. However, the pharmacologic binding profile may provide the strong- est evidence for such an interaction. In our studies, as well as in homogenate binding and in physiological experi- ments, L1T-SS-28, SS-14, Tyr-11-SS-14, and SS-28 all showed potency at the SS site (Srikrant et al., '81; Reubi et al., '81, '82; Epelbaum et al., '82; Perry et al., '83, Tran et al., '84b). Closely related peptides with amino acid substitutions or deletions from critical regions of the molecule showed much less potency in autoradiographic analyses of binding, in accord with their weakness in homogenate binding and physiological test systems. In both rat and human tissue, therefore, pharmacological profiles support our labeling of a physiologically relevant receptor.

Recently, heterogeneities of SS-receptors have been de- scribed based on differing affinities of certain analogs (Sri- krant et al., '81; Perry et al., '83; Tran et al., '84a). In homogenate binding studies of rat cortex, however, compar- isons between the SS-14 and SS-28 analogs used here do not seem to distinguish between these receptor subclasses.

Our routine use of unlabeled SS-14 as a "blank" control displacer could have biased us toward observation of only a SS-14-preferring receptor subpopulation. However, bio- chemical experiments revealed similar binding displace- ment with either SS-14 or SS-28. In autoradiographic studies, furthermore, virtually all brain-associated radio- activity is displaced by unlabeled SS-14 (Fig. 1). This low background fails to support dis lacement by unlabeled SS- 14 of only a small fraction of 51-LTT-SS-28. These obser- vations, coupled with the parallels between anatomic dis- tributions of sites labeled with SS-14 and SS-28 analogs thus do not allow differential SS-receptor subtype localiza- tion in our studies.

Studies in autopsy-derived human tissue are also compli- cated by the potential for age-related, agonal, or postmor- tem receptor alterations. Binding to this tissue does retain a receptor like pharmacological profile identical to that observed in fresher rat tissue. The observed human cere- bral cortical receptor distribution pattern also parallels dis- tributions noted in rat studies. These features do support our valid labeling of reasonably stable cortical SS-receptor populations in sections of postmortem human tissue.

b?

CNS SOMATOSTATIN RECEPTORS

Fig. 8. Somatostatin receptor autoradiogram ('251-LTT-SS-28). Photomicrographs of coverslipped autoradio- gram. Magnification x 380 before reduction. A. Darkfield image, cerebral cortex, '251-L1T-SS-28. B. Brightfield image, same field as A, toluidine blue cell stain.

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298 G.R. UHL ET AL.

Fig. 9. Somatostatin receptor autoradiograms ('"I-L?"r-SS-28). Photomi- crographs of coverslipped autoradiograms. Magnification X200 before re- duction (A,B), x235 before reduction (C,D). A. Darkfield image, triangular

septal nucleus. B. Brightfield image, triangular septal nucleus. C. Darkfield image, subfornical organ. D. Brightfield image, subfornical organ.

Anatomic characterization The detailed distribution of SS-receptors revealed by these

studies shows considerable agreement with the distribution of SS immunoreactivity (Hokfelt et al., '74; Brownstein et al., '75; Elde and Parsons, '75; Elde et al., 78; Baker and Wu, '76; Koboyoshi et al., '77; Krisch, '78; Bennett-Clark et al., '80; Borden et al., '81; Finley et al., '81; Takatsuki et al., '81; Vincent et al., '82; Sorensen, '82; McDonald et al., '82; De Figlia and Aronin, '82; Kohler and Chan-Palay, '82; Morrison et al., '82; Graybiel and Elde, '83; Robert et al., '84; Johansson et al., '84) and correlates with known phys- iological SS functions. In addition, these results point to- ward loci where SS-receptors could play roles in the pathophysiology of specific neurologic diseases.

In both rat and human cerebral cortex, the strikingly high densities of SS-receptors in deeper, infragranular lay- ers correlate well with at least some reports describing relatively higher densities of SS-immunoreactive perikarya in these zones (Finley et al., '81; Sorensen et al., '82; Hendry et al., '84; but also see McDonald et al., '82). Such parallels between the distributions of cells and receptors could be

consistent with a role for cortical SS in interneuronal func- tion. These receptors could mediate the SS-induced excita- tion found in physiological studies of rat or rabbit cortical neurons (Ioffe et al., '78; Phillis et al., '80). Interestingly, human cortical SS levels are selectively decreased in the dementias of Alzheimer's and Parkinson's diseases (Epel- baum et al., '83; Davies et al., '80). Conceivably, these cerebral cortical receptors may thus play a role in circuits important for intact cognition.

Olfactory nucleus receptors may also be positioned to receive local influences, since the deeper laminae of this structure are enriched in SS-immunoreactive perikarya (Finley et al., '81; Bennet-Clark et al., '80). In this area, as in several primary afferent ways stations such as the dorsal horn of the spinal cord, substantia gelatinosa of the caudal trigeminal nucleus, and cochlear and vestibular nuclei, SS- receptors could allow modulation of sensory information.

Observations of SS-immunoreactive perikarya and pro- cesses in lateral but not medial septal nuclei correlate well with the selective receptor localization to the lateral sep- tum (Bennet-Clark et al., '80; Finley et al., '81; Shiosaka, '82). Terminal densities in the receptor-rich triangular sep-

CNS SOMATOSTATIN RECEPTORS

Fig. 10. Somatostatin receptor autoradiograms ('""IL1T-SS-28). Photomlcrographs of coverslipped autoradi- ograms. Magnification x207 before reduction. A. Darkfield image, hippocampus CA1-CA3 border. B. Brightfield image, same field as A.

299

Figure 11

CNS SOMATOSTATIN RECEPTORS 301

Fig. 12. Somatostatin receptor autoradiograms ('"-I-LTT-SS-28). Prints of ultrofilm images. Magnification M unlabeled x5.5 before reduction. A. Human frontal cerebral cortex. B. "Blank" adjacent to A, with 5 x 10

SS-14 added to primary incubation to establish a control.

tal nucleus are not reported, however. "he amygdala, bed nucleus of the stria terminalis, and interconnecting stria

Fig. 11. Somatostatin receptor autoradiograms ('"I-L1T-SS-28). Photo- micrograph of coverslipped autoradiograms. Magnification ~ 2 2 3 before re- duction. A. Darkfield image, interpeduncular nucleus. B. Brightfield image,

terminal is all display receptor densities. The two gray- matter structures contain SS~immunoreactive peri-

same as A. karya and terminals (Robert et al., '84).

302 G.R. UHL ET AL.

SS-containing cell bodies are distributed to each major amygdaloid region, and at least modest receptor densities are present in each nucleus despite the above-mentioned variability.

In hippocampus, receptor distributions seem well posi- tioned to interact with the two SS-immunoreactive systems recently delineated there (Bennett-Clark et al., '80; Finley et al., '81; Kohler and Chan-Palay, '82; Morrison et al., '82). Molecular-layer receptor densities could receive input from the fiberkerminal plexus described there, while locally ram- ifying processes of polymorphic-layer SS-immunoreactive cells could innervate SS-receptors in this layer. Although the receptors concentrated in CA-1 (and probably CA-2) (cf. Swanson et al., '78) could receive input from Ss-containing perikarya intrinsic to this hippocampal zone, subiculum, CA-1, and regio superior are also targets for both intrahip- pocampal and extrahippocampal projection systems possi- bly using SS as their transmitters. Electrophysiological studies also support the existence of physiological hippo- campal SS-receptors (Dodd and Kelly, '78; Olpe et al., '80; Pittman and Siggins, '81h

Morrison et al. ('82) have used multiple antisera to sug- gest preferential involvement of SS-28-like peptides in hip- pocampus with little SS-1Clike immunostaining. Our autoradiographic studies with SS-14- and SS-%-derived li- gands reveal similar receptor patterns; we cannot yet sup- port differential hippocampal receptivity for SS-14 and SS- 28 peptides. Conceivably, SS systems in the hippocampus and amygdala could play a role in the limbic/emotional functions thought to be centered here.

Striatum and other regions implicated in "extrapyrami- dal" motor functions display modest to moderate binding. In striatum, the increasing receptor density in more ventral and more medial zones, and in the nucleus accumbens, parallels the distribution of SS immunoreactivity measured by micropunch techniques (Beale et al., '83). Although there is some heterogeneity of the striatal receptor pattern, "striosomal" or "island" (Graybiel et al., '81) patterns are not clear-cut (Graybiel and Elde '83). Conceivably, such patterns may be more evident in higher mammalian spe- cies, as has been the case for other striatal receptor types. Binding densities in the substantia nigra support the like- lihood of SS action on nigrostriatal dopaminergic systems. This interaction is supported by SS effects on dopamine release from superfused striatal slices (Chesselet and Re- isine, '83), and by our localization of human nigral SS receptors to dopaminergic cells, using the lesion of idio- pathic Parkinsonism (Uhl et al., '85 ). SS-containing neu- rons in striatum could thus act on other striatal neurons andor on nigrostriatal afferents to exert influences on movement. Recent findings of elevated SS immunoreactiv- ity in Huntington's disease striatum make further delinea- tion of these relationships of interest (Martin, '84). Receptor densites in claustrum and zona incerta provide other possi- ble foci for motor influences.

Modest thalamic receptor levels are consistent with un- impressive densities of SS immunoreactivity found there. Somewhat elevated receptor densities are noted in portions of the reticular nucleus, where Graybiel and Elde ('83) have recently reported SS-immunoreactive neurons in feline and monkey brains. Low receptor densities in nuclei of origin of thalamocortical projections accord well with reduced corti- cal receptors in zones of termination of many of these in- puts, especially layer IV.

Preoptic and hypothalamic SS neuronal clusters in peri- ventricular and Daraventricular zones found bv immuno-

histochemistry are accompanied by increased SS-receptor densities in each case (Finley et al., '81). Modest to moder- ate receptor densities in more medial hypothalamic areas are generally greater than those in lateral zones, a finding that parallels the distribution of hypothalamic immunore- activity (Brownstein et al., '75; Elde and Parsons, '75; Ko- boyoshi et al., '77; Krisch, '78; Bennett-Clark et al., '80; Borden et al., '81; Finley et al., '81). Median eminence receptors are only of modest to moderate density; SS re- leased here may instead interact with the densities of an- terior pituitary SS-receptors recently described by Perry et al. ('83). These hypothalamic and pituitary receptors could mediate endocrine effects of the hormone such as its classi- cal inhibition of growth hormone release, and the peptide's effects on thermoregulation k i n , '82; Reichlin, '83).

The interconnected habenula and interpenduncular nu- cleus each possesses a focal receptor density. Two features suggest that these receptor densities are not localized to habenula-interpeduncular projection neurons. First, the fasciculus retroflexus is virtually devoid of receptor grains. Second, the heavily labeled medial habenula projects to the lightly labeled paramedian nuclei of the interpenduncular nucleus, and not to the more receptor-rich apical and cen- tral zones (Smaha and Kaelben, '73). Interestingly, the tri- angular septa1 nucleus displaying a high receptor density is intimately connected with the receptor-dense medial ha- benula (Herkenham and Nauta, '77).

Elevated receptor densities in laterahentral quadrants of the periaqueductal gray correspond well with the re- ported ventral and lateral localization of SS-immunoreac- tive parikarya (Finley et al., '81).

SS can influence release of norepinephrine, serotonin, and dopamine in terminal fields of locus coeruleus, median raphe, and ventral tegmental area monoaminergic neu- rons, respectively (Garcia-Sevilla et al., '78; Tsujimoto and Taraka, '81; Tanko and Tsujimoto, '81). These physiological data fit with our observation of at least modest to moderate receptor-binding concentrations in these three regions.

Several brainstem receptor clusters suggest possible foci for SS interaction with visceral regulatory processes. Para- brachial region SS receptors could represent plausible sites for the peptide influences on respiratory control. Receptors in the nucleus of the solitary tract, fourth ventricular floor zones, and vagal nuclei could possibly function in cardiovas- cular and visceromotor regulation. In each case, these re- ceptor densities are accompanied by increased numbers of immunoreactive SS-containing elements in immunohisto- chemical studies (Finley et al., '81).

The striking receptor density in deep cerebellar nuclei, accompanied by SS-immunoreactive processes, could pro- vide another site for peptidergic motor alterations.

We have found biochemically and anatomically specific SS,receptors positioned discretely through the rat neuraxis and in human cerebral cortex, in a fashion consistent with peptide distribution and known SS physiological actions. Conceivably, these detailed localizations will provide the basis for further elucidation of the normal and pathophys- iological roles of this interesting peptide.

ACKNOWLEDGMENTS We gratefully acknowledge helpful discussions with D.

Manning, D. Perry, R. Grzanna, M. Kuhar, M. DiFiglia, and M. Molliver, generous peptide supplies from W. Vale and J. Rivier, excellent technical assistance by G. Hackney, thorough assistance with manuscriut DreDaration bv S. Cronin- and P. Douglas, and support frbm- the McKLight

CNS SOMATOSTATIN RECEPTORS 303

Foundation, the Sloan Foundation, the American Parkin- son’s Disease Association, the Julianne Dorn Fund for Neu- rological Research, and the NIH (# DA-00266, MH-18501, NS-16375, DA-00074, NS-16367, AM-26252).

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