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Immunodetection of catecholamine biosynthetic enzymes is frequently used for the visualization of central nervous catecholaminergic systems. Because of the method's limited specificity for the different catecholamines, interpretation of the results often presents difficulties. To determine criteria for the identification of dopaminergic, noradrenergic, and adrenergic afferents to the rat amygdaloid complex, comparative immunolabelling for tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyl-transferase (PNMT) was carried out using single- and double-labelling for fluorescence, light- and electron microscopy. The observations were complemented by findings in brainstem and hypothalamic areas. The results indicated that TH-labelling detected preferentially dopaminergic afferents in the lateral central and intercalated amygdaloid nuclei. DBH-labelling detected noradrenergic axons in nuclei lacking PNMT-immunoreactive fibres, and PNMT was a marker for adrenergic axons in the entire complex. For nuclei with combined dense dopaminergic, noradrenergic and/or adrenergic innervation, morphological and immunolabelling characteristics were described which, to a certain extent, enabled identification of the different afferents in anti-TH or anti-DBH-preparations. Using a monoclonal TH-antiserum, noradrenergic and adrenergic axons displayed weaker immunoreactivity than dopaminergic ones, and possessed characteristic morphological features. TH-immunoreactivity in noradrenergic axons differed depending on their origin, and showed intra-axonal compartmentalization. The present study provides a basis for the use of the detection of biosynthetic enzymes in future investigations into the ultrastructure and connectivity of the catecholaminergic amygdala innervation.

The medial nucleus of the amygdala, bed nucleus of the stria terminalis and medial preoptic area play critical roles in the regulation of mating behavior in the male hamster. Destruction of these nuclei or the pathways that connect them severely disrupt copulation. We have begun identifying the neuropeptides contained in these neurons as a prelude to determining the role of peptide neurotransmitters in the regulation of male copulatory behavior. We have found that substance P is localized within these neurons and is regulated by gonadal steroids. In this study we report 1) that a closely related peptide, NKA (substance K), is also present in the medial nucleus of the amygdala, the bed nucleus of the stria terminalis and the medial preoptic area; 2) that all those neurons which contain SP also contain NKA and 3) testosterone also regulates the production of NKA. Thus, NKA may also play a role in the regulation of male copulatory behavior.

The nuclear protein product (FOS) of the immediate-early gene, c-fos, was visualized immunocytochemically in the brains of male rats after they either achieved 8 intromissions with an estrous female or were left alone in a test arena. Mating induced equivalent increments in the number of FOS immunoreactive (IR) neurons present in the medial preoptic area (mPOA), the bed nucleus of the stria terminalis (BNST), and the medial amygdala in groups of males that were gonadally intact or had been castrated and treated for 7 days with either testosterone propionate, dihydrotestosterone propionate, estradiol benzoate (EB), or oil vehicle. Equivalent, low numbers of FOS-IR neurons were seen in these brain regions in additional groups of castrated males that received either EB or oil vehicle but were not paired with a female before being killed. Circulating sex steroids apparently contribute little to the mating-induced stimulation of c-fos gene expression, even in brain regions known to contain high levels of androgen and estrogen receptor.

Chemosensory and hormonal signals, both of which are essential for mating in the male Syrian hamster, are relayed through a distinct forebrain circuit. Immunocytochemistry for tyrosine hydroxylase, a catecholamine biosynthetic enzyme, previously revealed immunoreactive neurons in the anterior and posterior medial amygdaloid nucleus, one of the nuclei within this pathway. In addition, dopamine-immunoreactive neurons were located in the posterior, but not the anterior, medial amygdala. In the present study, tyrosine hydroxylase-immunostained neurons were also observed in other areas of the chemosensory pathway, including the posteromedial bed nucleus of the stria terminalis and the posterior, lateral part of the medial preoptic area, while dopamine immunostaining was only seen in the posteromedial bed nucleus of the stria terminalis. The colocalization of tyrosine hydroxylase and androgen receptors was examined in these four tyrosine hydroxylase cell groups by a double immunoperoxidase technique. The percentage of tyrosine hydroxylase-immunolabeled neurons that were also androgen receptor-immunoreactive was highest in the posterior medial amygdaloid nucleus (74%) and the bed nucleus of the stria terminalis (79%). Fewer tyrosine hydroxylase-immunostained neurons in the anterior medial amygdala (33%) and the medial preoptic area (4%) contained androgen receptors. Surprisingly, castration resulted in a significant decrease in the number of tyrosine hydroxylase-immunoreactive neurons only in the anterior medial amygdaloid nucleus, and this effect was transient. Six weeks after castration, the anterior medial amygdala contained 61% fewer tyrosine hydroxylase-immunolabeled neurons, but 12 weeks after gonadectomy, immunostaining returned to intact values. The number of immunostained neurons in testosterone-replaced, castrated hamsters was not significantly different from that of intact or castrated animals at any time. The results of this study indicate that a substantial number of tyrosine hydroxylase-immunostained neurons in the chemosensory pathway are influenced by androgens; the majority of these neurons in the posterior medial amygdala and the posteromedial bed nucleus of the stria terminalis produce androgen receptors, and tyrosine hydroxylase immunoreactivity is altered by castration in the anterior medial amygdala.

The distribution of somatostatin receptors (SRIF-R) was analyzed in the limbic system of the adult rat by in vitro autoradiography with [125I-Tyr0,DTrp 8]S14 as a radioligand. Precise quantification of the density of binding sites, at 0.2 mm intervals throughout the different areas revealed a marked heterogeneity of labeling in most structures. In particular, SRIF-R were concentrated in the basal (104.4 +/- 3.3 fmol/mg proteins) and basolateral amygdaloid nuclei (94.8 +/- 4.3 fmol/mg proteins), and in the nucleus of the lateral olfactory tract (121.6 +/- 2.4 fmol/mg proteins), whereas moderate densities were detected in the amygdalo-hippocampal nucleus (76.4 +/- 2.8 fmol/mg proteins). The medial (41.3 +/- 1.9 fmol/mg proteins) and the central (24.0 +/- 1.4 fmol/mg proteins) amygdaloid nuclei contained lower SRIF-R concentrations. It appears from these observations, in the light of the anatomical pathways of the amygdala, that intra-amygdalian SRIF-containing neurons project to the amygdalo-hippocampal nucleus, and that SRIF-R in the basolateral complex are the target of afferents from limbic cortical areas. SRIF-R were detected at different levels of the hippocampal formation but their distribution was more restricted than that of SRIF-containing fibers. The maximal density of sites was detected in the ventral and dorsal parts of the subiculum (115.0 +/- 3.4 and 87.0 +/- 2.8 fmol/mg proteins, respectively) and in the parasubiculum (100.1 +/- 5.4 fmol/mg proteins). In Ammon's horn, the stratum oriens and stratum radiatum of the CA1 field were the only sites enriched in SRIF-R (74.1 +/- 2.0 and 74.6 +/- 1.9 fmol/mg proteins, respectively). The apparent lack of receptors in the pyramidal cell layer indicated that, in Ammon's horn, SRIF is involved in intra-hippocampal communication. Low levels of receptors were found in the hippocampal CA2 and CA3 fields. SRIF-R in the dentate gyrus were mainly concentrated in the molecular layer (57.3 +/- 1.2 fmol/mg proteins). A very high density of sites was also observed in the entorhinal cortex (up to 123.1 +/- 1.5 fmol/mg proteins). A clear mismatch between SRIF and SRIF-R was detected in the septum and the habenula. In the profound layers of the cingulum and retrosplenial cortex, a heterogeneous distribution of SRIF-R was observed. High concentrations of sites were detected in the rostral zone of the cingulate cortex (93.4 +/- 2.0 fmol/mg proteins) while the posterior cingulate only exhibited moderate concentrations of sites (66.5 +/- 0.7 fmol/mg proteins).(ABSTRACT TRUNCATED AT 400 WORDS)

Calbindin-D28k is a calcium-binding protein located in a variety of neuronal cell types in many regions of the central nervous system. In the present study, we describe the distribution of calbindin-D28k-immunoreactive cells, fibers, and terminals in the monkey amygdaloid complex. Calbindin-D28k-immunoreactive neurons could be divided into four major cell types. Neurons of the first three cell types demonstrated clearly stained dendrites that were either aspiny or had a few spines on their distal portions. Type 1 cells were small, stellate, or multipolar and found throughout the amygdala. Type 2 cells were large, multipolar and were most commonly found in the deep nuclei, particularly in the lateral nucleus, intermediate division of the basal nucleus, accessory basal nucleus and in the periamygdaloid cortex. Type 3 cells were fusiform, of various sizes, and were found throughout the amygdala. Type 4 cells were quite large and lightly stained; the dendrites of these cells were usually unstained. The size, shape, and location of Type 4 labeled cell bodies suggested that they might be the large, modified pyramidal cells that constitute the projection neurons of the amygdala. Type 4 cells were observed primarily in the lateral, basal, and accessory basal nuclei and in the periamygdaloid cortex. Calbindin-D28k-immunoreactive fibers and terminals were difficult to observe in the amygdala partly because of a diffuse, finely granular neuropil labeling that was particularly dense in the anterior cortical and medial nuclei, in the central nucleus, and in the periamygdaloid cortex. The neuropil labeling was substantially lighter in the lateral, basal, and accessory basal nuclei. Conspicuous linear profiles resembling the "calbindin bundles" of the neocortex were evident in large numbers in the accessory basal nucleus, the medial portion of the parvicellular division of the basal nucleus, in the amygdalohippocampal area, and in the periamygdaloid cortex. There were calbindin-D28k-positive fibers in the stria terminalis and in the ventral amygdalofugal pathway. When the distributions of calbindin-D28k and parvalbumin immunoreactivity in the monkey amygdaloid complex were compared, it appeared that the overall distribution of these two calcium-binding proteins was generally complementary rather than overlapping.

We have determined the distribution of angiotensin II receptor subtypes in rabbit brain using in vitro autoradiography. AT1 receptors were found in very high concentrations in the forebrain circumventricular organs--the subfornical organ, organum vasculosum of the lamina terminalis, and the median eminence as observed in other mammals. However, there was very little labeling in the area postrema. In the paraventricular nucleus, median preoptic nucleus, supraoptic nucleus there were high levels of predominantly AT1 receptors. High densities of AT1 receptors were also found in the nucleus of the solitary tract and the rostral and caudal ventrolateral medulla. All of these regions have putative roles in the regulation of blood pressure and fluid and electrolyte balance. In the rabbit brain there is less AT2 receptor binding than the rat, with most AT2 binding found in the molecular layer of the cerebellum and in the septohypothalamic nucleus. In the subthalamic nucleus, the mediodorsal and ventroposterior nuclei of the thalamus, locus coeruleus and inferior olivary nuclei, areas containing mostly AT2 receptors in the rat, no binding was detected in the rabbit except in the locus coeruleus which contains moderate levels of AT1 receptors. Taken in conjunction with our previous results in the rat and human brains, these results reveal that AT1 receptors predominate in rostral forebrain, hypothalamus and autonomic control centers of the medulla oblongata in all three species. However, the distribution and density of AT2 bearing sites in regions such as the septum, thalamus subthalamic nuclei, locus coeruleus, cerebellum and inferior olivary nuclei show marked species differences.

The central amygdaloid nucleus (ACe) is part of the amygdaloid body, and it has been shown to participate in several stress related reactions. The ACe is densely innervated by tyrosine hydroxylase- (TH), corticotropin releasing factor- (CRF), calcitonin gene-related peptide- (CGRP), neurotensin- (NT), somatostatin- (SOM), enkephalin- (ENK), substance P- (SP), vasoactive intestinal polypeptide- (VIP) and cholecystokinin- (CCK) immunoreactive (IR) nerve terminals. In addition, the ACe contains numerous CRF-, NT-, SOM-, ENK- and SP-IR perikarya. In previous studies it has been shown that stress stimulates the expression of the immediate early gene c-fos in the ACe. The aim of this study was to demonstrate the colocalization of the Fos-IR neurons with the peptide- and TH-IR structures using an immunocytochemical double staining technique. In intact animals the ACe contained only a few Fos-IR neurons. After immobilization stress about 100 Fos-IR neurons were seen per section. They were mainly located in the area, which was enriched by peptide- and TH-IR nerve terminals. The close contacts observed between the Fos-IR neurons and the peptide- and TH-IR nerve endings suggest that the Fos-IR neurons were innervated by these nerve terminals. Furthermore, several NT-, ENK-, SOM- and CRF-IR neurons were observed and the vast majority of these cells exhibited Fos-like immunoreactivity. These results suggest that stress enhances the synaptic activity of the ACe, which stimulates the expression of c-fos. Subsequently, Fos may regulate the expression of the NT, ENK, SOM and CRF genes and thus affect the peptidergic efferents from the ACe.

1. Concentrations of the neurotransmitter amines noradrenaline (NA), dopamine (DA), and 5-hydroxytryptamine (5-HT) and the acid metabolites homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were determined in four regions of postmortem brains of demented patients with or without Alzheimer's disease (AD). 2. NA was deficient in the temporal cortex (BA 21) of AD, but not of non-AD, patients. 3. Caudate, in particular, had an impaired dopaminergic system in AD patients, with low HVA levels. 4. In all regions investigated [amygdala, caudate, putamen, temporal cortex (BA 21)] 5-HT was significantly depleted in AD patients, and 5-HIAA was also depleted in amygdala and caudate. 5. These results indicate that neurotransmitter systems other than cholinergic systems are also widely affected in AD and suggest that these deficits may also play an important role in determining the symptomatology of AD.

This study was designed to assess the effects of the 5-hydroxytryptamine (5-HT) indirect agonist fenfluramine on the brain distribution of Fos- and corticotropin-releasing factor-like immunoreactivity (F-LI and CRF-LI). A single intraperitoneal injection of either DL-fenfluramine (25 mg/kg) or saline was given to resting Sprague-Dawley rats housed on a 12-12 h light-dark cycle and fed libitum. Sixty min following injections, rats were killed and brains removed and sliced (40 microns thick) in a coronal plane from the anterior olfactory bulb to the brainstem. Brain slices were then stored at -40 degrees C pending the tissue localization of F-LI and CRF-LI. F-LI and CRF-LI were determined by means of a double immunostaining procedure using the peroxidase-avidin:biotin complex (ABC) method. Fenfluramine injection led to a marked increase in F-LI in the caudate-putamen (CPu), the parvocellular division of the hypothalamic paraventricular nucleus (PVN), and the central amygdaloid nucleus (CeA). In the PVN, most of the F-LI was co-localized with CRF-LI. There was no attempt to identify which types of neurons displayed F-LI in CPu and CeA. While F-LI was readily observable in all rats treated with fenfluramine, it was not discernible in the control animals. This study provides evidence for an involvement of the immediate-early genes c-fos in the central action of fenfluramine.

Mating behavior in the male golden hamster is regulated by both gonadal steroids and photoperiod. Gonadal steroids may regulate mating behavior by actions on the medial nucleus of the amygdala, bed nucleus of the stria terminalis, and medial preoptic area. Neurons in these areas actively accumulate gonadal steroids and lesions of these nuclei disrupt mating behavior in male hamsters. Photoperiodic regulation of mating behavior is regulated, at least in part, by decreased responsiveness to gonadal steroids. Therefore, we sought to determine if the changes induced by changes in gonadal steroids would mimic those induced by changes in photoperiod. The number of substance P-containing neurons in these areas decrease following castration and are restored with testosterone treatment suggesting that this peptide may mediate steroidal regulation of male mating behavior. To determine the effect of photoperiod on substance P, peptide containing neurons were counted in (1) enucleates (n = 6), (2) enucleated castrates treated with testosterone (n = 6), (3) castrates treated with testosterone (n = 4), and (4) intact controls (n = 6). Bilateral enucleation caused a decrease in the number of substance P neurons in the medial nucleus, bed nucleus of the stria terminalis, and medial preoptic area (P less than 0.05). Testosterone treatment prevented this decrease (P less than 0.05). Thus, a decrease in daylength causes a decrease in substance P in the medial nucleus of the amygdala, the medial bed nucleus of the stria terminalis and the medial preoptic area that is mediated by changes in testosterone levels.

Populations of dopamine (DA) neurons in the rat brain are selectively activated by stress, and the response is attenuated by the administration of anxiolytics. Given the role of the component nuclei of the amygdaloid complex in conditioned associations, stress responses and the anxiolytic effects of benzodiazepines, we hypothesized that particular mesoamygdaloid DA projections might be especially sensitive to the effects of conditioned stress and to diazepam (DZ). We mapped the effect of a conditioned stressor on the concentration of the DA metabolite homovanillic acid (HVA) in distinct amygdaloid nuclei and other brain nuclei and areas and the effect of DZ (1 or 3 mg/kg) on the conditioned response in drug-experienced subjects. The conditioned stress paradigm resulted in significant elevations in classical indices of stress, including serum corticosterone and plasma epinephrine. Conditioned stress-induced increases in the estimated activity of DA neurons were specific for DA neurons projecting to the central, basolateral and lateral amygdaloid nuclei, and for DA projections to the dorsal septal nucleus. Conditioned stress-induced increases in the HVA concentration of responsive amygdaloid nuclei were antagonized by low, anxiolytic doses of DZ. These results indicate a role for a subset of mesoamygdaloid DA projections in transducing the impact of perceived stressors on the output of the amygdaloid complex. A role for particular amygdaloid DA projections in the formation of conditioned fear or anticipatory anxiety and its modulation by anxiolytics is also suggested.

The effects of bilateral intraamygdala microinjection of flumazenil, a benzodiazepine receptor (BZD-R) antagonist, on the exploratory activity in an elevated plus maze were examined in chronically implanted rats. This compound induced a significant decrease in the time spent in the open arms, which is consistent with an anxiogenic action. No effect was observed after intrastriatal injections of flumazenil. Naive rats exposed to the elevated plus maze showed a rapid and selective decline in the content of BZD-like molecules in amygdala (-68%) but not in striatum and hippocampus. These data suggest that the anxiogenic effects of the intraamygdala injection of flumazenil is probably due to the blockade of BZD-like molecules released during the performance.

The fine structure of nerve fibers with corticotropin-releasing factor (CRF)-like immunoreactivity in the central amygdaloid nucleus and CRF-containing afferents to the nucleus were investigated by pre-embedding immunoelectron microscopy and by the combination of fluoro-gold tracing and the indirect immunofluorescence method. Significant numbers of CRF nerve endings and dendrites formed synapses with non-immunoreactive dendrites and axon terminals, respectively. Axon terminals devoid of CRF frequently made synapses with the soma of immunoreactive and non-immunoreactive neurons; CRF nerve endings in contact with the soma were fewer in number. Occasionally, CRF was localized to both pre- and postsynaptic structures in the central amygdaloid nucleus. After fluoro-gold injection into the central amygdaloid nucleus and adjacent areas, double-labeled cells with the tracer and CRF were observed mainly in the lateral hypothalamic area and occasionally in the dorsal raphe nucleus, and they were less numerous than single-labeled cells. These findings suggest that part of the CRF axon terminals identified in the electron micrographs arises from neurons in the lateral hypothalamic area and the dorsal raphe nucleus and the others from intra-amygdaloid CRF neurons. The immunoreactive dendrites are likely to derive from neurons in the central amygdaloid nucleus, which are shown to send axons to the lower brainstem. Thus, this study demonstrates that CRF structures constitute a more complex neuronal network in the central amygdaloid nucleus than previously considered.

The central amygdaloid nucleus (ACe) is part of the amygdaloid complex that participates in adrenocorticotrophin secretion, stress-related reactions and behavioral functions. The ACe contains numerous glucocorticoid receptor (GR)-immunoreactive (IR) neurons, and in addition it has been shown to contain several neuropeptide-IR somata and nerve terminals. In order to study the relationship between the GR- and neuropeptide-IR structures we mapped the distribution of GR-like immunoreactivity (LI) in amygdaloid complex and colocalized the neuropeptide- and GR-LIs in the ACe. In the amygdaloid complex the central, medial and cortical nuclei contained a high number of GR-IR neurons, whereas a moderate number of GR-IR neurons were observed in the basolateral and basomedial nuclei. Only a few GR-IR neurons were seen in the lateral nucleus. In the ACe, the majority of corticotrophin-releasing factor (CRF)-, met-enkephalin (met-ENK)-, neurotensin (NT)- and somatostatin (SOM)-IR neurons contained also GR-IR. About half of the substance P (SP)-IR neurons were seen to contain GR-IR, whereas only some of the few vasoactive intestinal polypeptide and cholecystokinin-IR neurons showed GR-LI. Nerve terminals containing calcitonin gene-related peptide and the above mentioned peptides were seen in close contact with the GR-IR neurons. These results suggest that the glucocorticoids may modulate directly the neurotransmitter synthesis of the CRF-, met-ENK, NT-, SOM- and SP-IR cells in the ACe.

The medial amygdaloid nucleus (Me) is part of a neural pathway that regulates sexual behavior in the male Syrian hamster. To characterize the neurochemical content of neurons in this nucleus, brains from colchicine-treated adult male and female hamsters were immunocytochemically labeled using antibodies that recognize the catecholamine-synthesizing enzymes, tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), as well as dopamine. A large population of TH-immunoreactive (TH-IR) neurons was observed throughout Me of male and female hamsters, primarily concentrated in the midrostral and caudal portions of the nucleus. The somata were generally small to medium in size and bipolar. Brains from animals that did not receive colchicine contained a limited number of TH-IR neurons in Me as reported previously. The DBH and PNMT antisera did not label any cells in Me of colchicine-treated animals, and the dopamine antiserum labeled neurons in the same location as the caudal group of TH-IR cells. Therefore, these caudal TH-IR neurons are interpreted to be dopaminergic. The rostral group of TH-IR neurons, on the other hand, may be producing only the immediate precursor of dopamine, L-3,4-dihydroxyphenylalanine (L-DOPA). The TH-synthesizing neurons in Me of the Syrian hamster appear to be a species-specific group of cells located outside of the previously described catecholaminergic cell groups.

Immunocytochemical methods were used to study the regional and cellular distribution of diazepam binding inhibitor (DBI)-, ODN (DBI 33-50)- and TTN (DBI 17-50)-like immunoreactivity (LI) in monkey brain. DBI-, ODN- and TTN-LI were found to have similar regional but different cellular distributions. The most intense staining was detected in limbic structures, especially the amygdala. The most notable staining discrepancy was the weak-to-moderate uniform DBI-LI and the very high patchy ODN-LI and TTN-LI in the ventral striatum. At the cellular level, DBI-LI was preferentially localized in glia-like cells, whereas ODN-LI and TTN-LI were mainly found in neurons.

Calcitonin gene-related peptide (CGRP) may be present in two forms in nervous tissue. Reversed-phase high-performance liquid chromatography has previously been found to be insufficient to clearly separate alpha-CGRP and beta-CGRP. A method for the separation of CGRPs by capillary zone electrophoresis has been developed. Separation of human or rat alpha-CGRP and beta-CGRP was achieved at pH values between 3.5 and 4.5 and a potential of 20 kV in a fused-silica capillary. Electrophoresis of an extract of rat amygdala in a micropreparative way, with subsequent radioimmunoassay, revealed for the first time the presence of alpha-CGRP and beta-CGRP in this brain area. The method may thus be used for separation of CGRPs, to reveal the distribution of alpha-CGRP and beta-CGRP, and for purity control.

GABA-immunoreactive (GABA-I) elements (neuronal somata and neuropile) are detected in turtle Emys orbicularis and lizard Ophysaurus spodus in all structures of ventral and dorsal parts of amygdaloid complex (AC) considered as phylogenetic more ancient and younger, respectively by means of the immunohistochemical method. Their maximal quantity in the ventral section of AC is found in the lateral region, lesser--in the ventral, central and medial regions. Besides in lizards a specialized laminar distribution of GABA-I elements in n. sphaericus is observed. GABA-I neurons are also detected in structures of dorsal part in turtles and lizards against the background of the immunopositive neuropile of a moderate density. It is supposed that GABA-ergic innervation of AC is liable to considerable variations in connection with taxonomic, ecological and other factors.

Glutamate immunoreactivity was found in 9%, in the case of Wistar Kyoto rats (WKY), and 14%, in the case of spontaneously hypertensive rats (SHR), of neurons located in the medial division of the central amygdaloid nucleus (CeM) projecting ipsilaterally to the subretrofacial nucleus (SRF) in the rostral ventrolateral medulla using a double-labeling technique in combination with glutamate immunocytochemistry. The results indicate that possibly-glutamatergic neurons located in the CeM project to the SRF, in which vasomotor neurons are present, suggesting involvement of the CeM in blood pressure regulation. No significant difference was found between the distribution of labeled CeM neurons in SHR and WKY rats.

The present study has employed the technique of fast cyclic voltammetry to measure electrically-evoked dopamine release within the central amygdaloid complex in a rat brain slice. Local electrical stimulation caused the release of an electroactive substance which was identified, biochemically and pharmacologically, as being neuronal dopamine. Dopamine release could be inhibited by the dopamine D2 receptor agonist, quinpirole, but not by the D1 receptor agonist, SKF38393. Quinpirole-induced inhibitions were antagonized by sulpiride, metoclopramide and clozapine but not by SCH23390. It is concluded that dopamine release in the amygdala can be modulated by presynaptic D2 receptors which appear to be the same type as those found in striatum and nucleus accumbens.

Guanidino compounds are intrinsic chemoconvulsants. We investigated the regional and time-dependent changes of these compounds in the amygdala (AM) of kindled rats versus electroconvulsive shock (ECS) seizures using high-performance liquid chromatography (HPLC). Twenty-eight days after the last AM-kindled seizure, guanidinoacetic acid (GAA) and methylguanidine (MG) levels were significantly increased in the bilateral AM as compared with those of control rats, which had been implanted with electrodes but were not stimulated. Both compounds, however, tended to decrease in the bilateral AM after ECS seizure. In addition, we measured these compounds after induction of one afterdischarge (AD) in the AM. These compounds increased significantly 7 days after AD was induced in the ipsilateral AM. We suggest that the increase of these compounds is coincident with AD generation and specific to AM kindling and the kindled state.

Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are structurally similar, share the same high affinity site in same peripheral tissues and increase the intracellular content of adenylate cyclase. To establish which neural circuits are signaling with each of these two peptides, we systematically compared the immunohistochemical distribution of PACAP and VIP in selected rat forebrain regions using previously characterized antiserum. The PACAP antiserum recognized both PACAP27 and PACAP38, and PACAP immunoreactivity was unaffected by preincubation with various other peptides. PACAP-immunoreactive perikarya and fibers were observed in both hypothalamic and extrahypothalamic regions. In the hypothalamus PACAP perikarya were located in the supraoptic, paraventricular, anterior commissural, periventricular, and perifornical nuclei. In intact rats PACAP immunolabeled fibers were present in the internal zone of the median eminence and posterior pituitary. One week after hypophysectomy the intensity of staining in the internal zone was enhanced and immunoreactive fibers appeared in the external zone of the median eminence. Two or 3 weeks later a dense fiber network was observed around the portal capillaries in the external zone, and immunoreactive material further accumulated in the fibers of the internal zone. PACAP-immunoreactive perikarya and fibers were also observed in several extrahypothalamic regions including central thalamic nuclei, amygdaloid complex, bed nucleus of stria terminalis, septum, hippocampus and cingulate, and entorhinal cortices. In the lateral septum and entorhinal cortex PACAP fibers surrounded unstained neuronal cell bodies and small blood vessels. In intact rats, VIP-immunoreactive perikarya were present in all regions of the cerebral cortex, hippocampus, amygdaloid complexus and in the suprachiasmatic nucleus, but not in the paraventricular and supraoptic nuclei. In colchicine-treated rats the VIP perikarya appeared in the preoptic area and paraventricular nucleus. The fibers were organized in two main pathways: the stria terminalis and an ascending pathway from the suprachiasmatic nucleus to the paraventricular area. Hypophysectomy induced the appearance of VIP-immunoreactive fibers in the internal zone of the median eminence and perikarya in the supraoptic and paraventricular nuclei in addition to the suprachiasmatic nucleus. The dissimilar distributions of PACAP and VIP suggest that PACAP neural circuits are independent of that of VIP in the rat forebrain. These findings support possible multifunctional roles for PACAP as a posterior pituitary hormone, a hypophysiotrophic factor, and a neurotransmitter/neuromodulator.

Alterations in a variety of neurotransmitter systems have been identified in experimental models of epilepsy and in brain tissue from patients with intractable temporal lobe seizures. The availability of new high-affinity radioligands permits the study of some neuroreceptors in vivo with positron emission tomography (PET). We previously characterized the in vivo binding of 11C-carfentanil, a potent and selective mu opiate receptor agonist, and described increases in 11C-carfentanil binding in the temporal neocortex of patients with unilateral temporal lobe epilepsy. These studies have been extended to 11C-diprenorphine, which labels mu, kappa, and delta opiate receptor subtypes. Paired measurements of opiate receptor binding were performed with PET using 11C-carfentanil and 11C-diprenorphine in patients with unilateral temporal lobe seizures. Carfentanil binding, reflecting changes in mu opiate receptors, was increased in the temporal neocortex and decreased in the amygdala on the side of the epileptic focus. Diprenorphine binding, reflecting mu as well as non-mu opiate subtypes, was not significantly different among regions in the focus and nonfocus temporal lobes. Regional glucose metabolism, measured using 18F-2-fluoro-2-deoxyglucose, was decreased in the mesial and lateral aspects of the temporal lobe ipsilateral to the epileptogenic focus. The variation in pattern of carfentanil and diprenorphine binding supports a differential regulation of opiate subtypes in unilateral temporal lobe epilepsy.

The expression of three beta subunit (beta 1, beta 2, and beta 3) mRNAs for gamma-aminobutyric acidA receptor in the postnatal rat forebrain was examined by in situ hybridization histochemistry with probes synthesized for the respective subunit mRNAs. The developmental expression of these subunit mRNAs conformed to one of three patterns. Pattern I was high expression of the mRNA at birth and a constant or increasing expression thereafter. In contrast, pattern II was no or very low expression of the mRNA at birth, with expression quickly increasing to reach the adult level in the early postnatal period. Pattern III was the transient expression of the subunit mRNA or else a marked decrease of its expression after a peak in the early postnatal period. On the basis of this classification, the expression of beta 3 subunit mRNA followed pattern I in most regions of the forebrain, such as the isocortex, the olfactory bulb and some of its related areas, the hippocampal formation, the amygdala, the septum, the bed nucleus of the stria terminalis, the caudate-putamen, the nucleus accumbens, the globus pallidus, the ventral pallidum, and the hypothalamus. In some areas, such as the magnocellular preoptic nucleus, the thalamus, and the subthalamic nucleus, pattern III was seen for this subunit. However, none of the regions of the brain showed pattern II expression of beta 3 subunit mRNA. In contrast, the expression of beta 1 and beta 2 subunit mRNAs followed pattern II in most regions of the forebrain. These included the expression of beta 1 subunit mRNA in the isocortex, the olfactory bulb, the hippocampal formation, the amygdala, the septum, the bed nucleus of the stria terminalis, the thalamus, and the hypothalamus, and the expression of beta 2 subunit mRNA in the isocortex, the olfactory bulb and some of its related areas, the amygdala, the nucleus of the diagonal band, the caudate-putamen, the thalamus, and the hypothalamus. Pattern I was not found for beta 1 subunit mRNA, although it was seen in some areas for beta 2 subunit mRNA, such as the ventral pallidum, the globus pallidus, and the magnocellular preoptic nucleus. On the other hand, pattern III was followed by beta 1 subunit mRNA in the anterior olfactory nucleus, the olfactory tubercle, and the piriform cortex, and the same pattern for the beta 2 subunit was also found in the olfactory tubercle, the hippocampal formation, the septum, the bed nucleus of the stria terminalis, and the nucleus accumbens.(ABSTRACT TRUNCATED AT 400 WORDS)

The organization of calcitonin gene-related peptide-like immunoreactive (CGRPir) innervation of the amygdala and caudate-putamen in the rat was examined by using immunohistochemistry for CGRP combined with retrograde transport of the fluorescent dye fluoro-gold, as well as anterograde transport of Phaseoleus vulgaris leucoagglutinin (PHA-L). The lateral part of the central nucleus of the amygdala and the amygdalostriatal transition zone was densely innervated by CGRPir terminals at all anterior-posterior levels. More caudally, the lateral part of the caudate-putamen also had large numbers of CGRPir terminals. Injections of fluoro-gold into the amygdala and amygdalostriatal transition area followed by immunohistochemistry for CGRP revealed double-labeled neurons in the subparafascicular, lateral subparafascicular, and posterior intralaminar nuclei of the thalamus and peripeduncular nucleus. Injections into the caudate-putamen demonstrated double-labeled neurons in the more lateral parts of this same nuclear complex. PHA-L injections into the posterior thalamic nuclei from which the CGRPir projections arise confirmed the medial-to-lateral organization of the projections to the amygdala and striatum. The subparafascicular nucleus and the rostral portion of the lateral subparafascicular nucleus primarily projected to the medial amygdala and the amygdalostriatal transition area, while the more lateral cell groups, including the caudal part of the lateral parafascicular, posterior intralaminar, and peripeduncular nuclei projected to the lateral amygdala and the caudate-putamen. These CGRPir projections may be involved in mediating conditioned autonomic and behavioral responses to acoustic stimuli or somatosensory stimuli.

Quantitative autoradiography was used to examine central binding sites for L-[3H]glutamate in amygdaloid-kindled rats since receptors for excitatory amino acids have been implicated in epileptiform activity and seizure behaviors. In tissue from rats killed five days after two kindled seizures, the ipsilateral hippocampus, entorhinal, perirhinal and parietal cortices had significantly (35-100%) greater densities of binding sites for L-[3H]glutamate than the opposite, contralateral side or operated, unstimulated controls. These regions receive excitatory inputs from the amygdala via the entorhinal cortex. Dissociation constants were not altered and significant differences were not observed in the binding parameters for L-[3H]glutamate between control and kindled rats or ipsilateral and contralateral sides of the amygdala, corpus striatum, nucleus accumbens or substantia nigra. The proportion and affinity of N-methyl-D-aspartate (NMDA)-sensitive binding sites for L-[3H]glutamate was unchanged after kindling, as were the relative proportions of kainate- and AMPA-(DL-alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid) sensitive sites. However, the density of NMDA and non-NMDA receptor subtypes was increased in the ipsilateral hippocampus, entorhinal, perirhinal and parietal cortices of kindled rats. These findings of specific, unilateral glutamate receptor up-regulation may indicate adaptive responses to the enhanced excitation found in kindling, and are consistent with other neuronal changes reported in early kindling.

We have measured the amount of Gi (the inhibitory G-protein) or Go (a similar G-protein of unknown function) in 5 areas of the medial temporal lobe of control and schizophrenic brains utilizing pertussis toxin-catalyzed ADP ribosylation. The material used has previously been shown to have asymmetrical structural abnormalities of the ventricular system. The amount of Gi or Go was reduced on the left side in the hippocampus, amygdala and parahippocampal gyrus, the difference reaching significance in the hippocampus. This data is the first report of a neurochemical correlate of the structural change in the brains of patients with schizophrenia. Decreased Gi or Go in hippocampus may relate to other reported neurochemical deficits or other transmembrane signalling abnormalities. Further investigations of these indices of secondary messenger function in relation to structural changes are indicated.

The effect of imipramine, desipramine, ketanserin and lithium on Type II glucocorticoid receptor (GR) mRNA levels was studied in rat brain regions involved in the control of the hypothalamo-pituitary-adrenal (HPA) axis, the dysregulation of which has been implicated in the pathophysiology of major depression. Northern blot analysis of Type II GR mRNA showed that treatment of male rats with either desipramine or imipramine increased hypothalamic and hippocampal GR mRNA levels. Upregulation of GR mRNA following administration of imipramine was found in brain regions of female rats, while desipramine had no effect. Ketanserin increased levels of GR mRNA in hippocampus of male, but not female, rats. Lithium also was able to induce important increases rat brain GR mRNA; this effect was particularly marked in females. We conclude that desipramine, imipramine, ketanserin and lithium can modulate GR mRNA in regions of rat brain involved in the control of the HPA axis and may have a common mechanism of action at the level of the GR gene. Sexual dimorphism for drug regulation of brain GR mRNA content was shown and may be related to sex differences in the prevalence of certain affective disorders.

The distribution of cholecystokinin (CCK)-immunoreactive nerve fibers and cell bodies was studied in the forebrain of control and colchicine-treated guinea pigs by using an antiserum directed against the carboxyterminus of CCK octapeptide (CCK-8) in the indirect immunoperoxidase technique. Virtually all forebrain areas examined contained immunoreactive nerve fibers. A dense innervation was visualized in; neocortical layers II-III, piriform cortex, the medial amygdala, the medial preoptic area, a circumventricular organ-like structure located at the top of the third ventricle in the preoptic area, the subfornical organ, the posterior bed nucleus of the stria terminalis, the posterior globus pallidus (containing labeled woolly fiber-like profiles), the ventromedial hypothalamus, the median eminence, and the premammillary nucleus. A moderately dense innervation was visualized elsewhere excepted in the septum and thalamus where labeled axons were comparatively few. Immunoreactive perikarya were abundant in: neocortex (especially layers II-III), piriform cortex, amygdala, the median preoptic nucleus, the bed nucleus of the stria terminalis, the hypothalamic paraventricular (parvicellular part), arcuate, and dorsomedial (pars compacta) nuclei, the dorsal and perifornical hypothalamic areas, and throughout the thalamus. Areas also containing a moderate number of labeled cell bodies were the medial preoptic area, the globus pallidus, the caudate-putamen, and the periventromedial area in the hypothalamus. Immunostained perikarya were absent or only occasionally observed in the septum, the suprachiasmatic nucleus, the magnocellular hypothalamoneurohypophyseal nuclei, and the ventral mesencephalon. In the adenohypophysis, corticomelanotrophs were labeled in both males and females, and thyrotrophs were labeled in females only. This distribution pattern of CCK-8 immunoreactivity is compared to those previously recorded in other mammals. This shows that very few features are peculiar to the the guinea pig. It is discussed whether some interspecific differences in immunostaining are real rather than methodological.

DNA and RNA contents in 20 brain regions or nuclei of the rat were determined by a highly sensitive method using high-performance liquid chromatography with electrochemical detection. The high DNA and RNA contents were found in the hypothalamic nuclei, especially the median eminence-arcuate nucleus. These results may be available for the preparation of nucleic acids as the regional control.

The physicochemical characteristics of solubilized crosslinked CRF receptor-ligand complexes were studied in the anterior and intermediate pituitary lobes and brain of the rat. In all tissues studied, there was a major labeled band with a molecular weight of 72 +2- 3.5 kDa, (n = 15), 71 +/- 1.3 (n = 6), 73 +2- 2.5 (n = 7) and 75 +2- 3.5 (n = 7) kDa in the anterior and intermediate lobes of the pituitary, amygdala and cerebral cortex, respectively. The density of this band was inhibited by CRF analogs, but not by unrelated peptides. This is consistent with the comparable binding properties of CRF in membrane preparations of these tissues. These results suggest that differences in receptor regulation and the reported ability of CRF to stimulate cAMP is due to differences in ligand receptor processing and coupling to membrane transduction systems, rather than to major differences in the binding protein.

5-HT1 and 5-HT1A binding sites were measured in brain tissue obtained at postmortem from 19 suicides, with definite evidence of depression, and 19 sex and age-matched controls. Thirteen of the depressed suicides had not been prescribed psychoactive drugs recently (drug-free suicides); six had been receiving antidepressant drugs, alone or in combination with other drugs (antidepressant-treated suicides). No significant differences were found in the number or affinity of 5-HT1 and 5-HT1A binding sites in frontal or temporal cortex between drug-free suicides and controls. The number of 5-HT1 sites was significantly lower (by 20%), affinity unaltered, in hippocampus and the affinity significantly lower (by 33%), number unaltered, in amygdala of drug-free suicides than controls. The number of 5-HT1 binding sites tended to be higher and the affinity lower in the antidepressant-treated compared to drug-free suicides, and significantly so in hippocampus. The present results, together with our previous studies, provide no evidence of altered cortical 5-HT markers in depressed suicides, but further emphasise abnormalities in the hippocampus.

Levels of mu, delta and kappa opioid receptors in 4 subnuclei of the rat amygdala were determined by quantitative autoradiography following chronic treatment with naloxone or saline. A different distribution of each receptor subtype was observed, with mu binding greatest in the lateral nucleus (La), delta greatest in the basolateral (Bl), and kappa greatest in the medial (Me). Levels of all 3 receptors were very low in the central nucleus. Receptor upregulation following chronic naloxone treatment was also anatomically heterogeneous. Increases in mu receptors were statistically significant in the Me, Bl and La, while increases in delta and kappa receptors were significant only in the Bl.