Biologically active pituitary hormones in the rat brain amygdaloid nucleus.

While an attempt was being made to identify the source of the growth hormone releasing factor present in cerebral spinal fluid of man, it was discovered that cells of the rat amygdaloid nucleus, grown in tissue culture, produce a material that is immunologically and chromatographically identical to growth hormone found in the pituitary. Immunoperoxidase staining revealed dense accumulation of the peroxidase-antibody to growth hormone complex in amygdala cells. Significant amounts of growth hormone and adrenocorticotropin could be extracted from this limbic structure. Extracts containing immunoequivalent amounts of growth hormone were measured by bioassay in hypophysectomized rats. Stimulation of the growth of epiphyseal cartilage by extracts of the amygdala was comparable to the stimulation by extracts of anterior pituitary glands. The stimulatory effect of amygdala extracts on adrenal and gonadal size and weight and on growth of thyroid follicular epithelium was also comparable to that of pituitary extracts.

Autoradiographic analysis of tritiated imipramine binding in the human brain post mortem: effects of suicide.

In vitro quantitative autoradiography of high-affinity tritiated imipramine binding sites was performed on brains of 12 suicide victims and 12 matched controls. Region-specific differences in imipramine binding were found between the two groups. Thus, the pyramidal and molecular layers of the cornu ammoni hippocampal fields and the hilus of the dentate gyrus exhibited 80%, 60%, and 90% increases in binding in the suicide group, respectively. The postcentral cortical gyrus, insular cortex, and claustrum had 45%, 28%, and 75% decreases in binding in the suicide group, respectively. No difference in imipramine binding was observed in prefrontal cortical regions, in the basal ganglia, and in mesencephalic nuclei. No sex and postmortem delay effects on imipramine binding were found. Imipramine binding was positively correlated with age, the effect of age being most pronounced in portions of the basal ganglia and temporal cortex.

Levels of corticotropin releasing factor-like immunoreactivity in mammalian hypothalamic and extrahypothalamic brain tissue as determined with a monoclonal antibody to the ovine material.

A monoclonal antibody to ovine corticotropin releasing factor (CRF) has been produced by fusion of a non-producing plasmacytoma cell line P3U1 with spleen cells of Balb/c mice immunized with the synthetic 41 amino acid peptide coupled covalently with rabbit myosin by a heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. A total immunizing dose of 500 micrograms resulted in a highly specific, high-affinity antibody with a Ka of 0.15 x 10(12) M-1, which was used to establish a specific RIA with a sensitivity of 10 pg/tube. Levels of corticotropin releasing factor-like immunoreactivity (CRF-LI) in a pg/mg of hypothalamic tissue ranged from 4-10 in ovine, 2.5-8 in bovine, 47.5-67.5 in mouse and 2.3-20 in human tissue. Moreover, CRF-LI was widely distributed in extrahypothalamic mouse brain at concentrations approximately one half those seen in hypothalamus.

Immunohistochemical localization of adrenocorticotropin in the rat brain.

ACTH and some of its fragments have been shown to play a role in a variety of adaptative mechanisms. To clearly identify the nervous structures containing ACTH in the rat brain, an immunohistochemical localization of this peptide was conducted at both light and electron microscopic levels. Nervous fibers staining for ACTH were found to be largely distributed throughout regions of hypothalamus, thalamus, and midbrain. Positive fibers could also be occasionally observed in the spinal cord. Immunostained neuronal cell bodies were only detected in the arcuate nucleus. Essentially, the same results were obtained 2 and 8 weeks after hypophysectomy. In animals pretreated with colchicine, the intensity in the staining of cell bodies was markedly increased, making possible the detection of a larger number of cell bodies. At the electron microscopic level, it was demonstrated that ACTH is contained in dense core vesicles present in nervous fibers and endings. These results indicate that ACTH of nonpituitary origin is synthesized in the central nervous system and could probably be considered as a neurotransmitter of still undefined function.

Morphology of peptide-immunoreactive neurons in the rat central nucleus of the amygdala.

The morphological characteristics of peptide-immunoreactive neurons in the rat central nucleus of the amygdala were examined. Observations were compared with details of neuron morphology available from Golgi-stained tissue to determine whether peptide immunoreactivity was associated with specific cell types in the central nucleus. The lateral subdivision (CL) of the central nucleus contained mainly medium-sized, densely spiny neurons. Larger, pyramiform spiny neurons; medium-sized, sparsely spinous neurons; and small, aspinous cells were also present in CL. Somatostatin-, neurotensin-, corticotropin-releasing factor (CRF)-, and enkephalin-immunoreactive neurons in CL were characterized as the medium spiny and larger, pyramiform types. No obvious morphological differences were evident among medium spiny neurons containing different peptides. In the medial subdivision, substance P, neurotensin, somatostatin, and CRF were present within pyramiform, sparsely spinous neurons with long dendrites. Galanin immunoreactivity in the medial subdivision was associated with moderately spiny, pyramiform neurons and a larger, aspinous, polygonal neuron. The ventral subdivision of the central nucleus contained neurons similar to those found in the adjacent medial and lateral subdivisions. In addition, this subdivision contained a characteristic ovoid neuron with long, sparsely spinous dendrites. Vasoactive intestinal polypeptide (VIP) and neurotensin appeared to be present within this cell type. In the lateral capsular subdivision, neurotensin and enkephalin were present in cells resembling the medium spiny neurons characteristic of this part of the central nucleus. Numbers of spindle-shaped, biopolar somatostatin, and VIP neurons were identified in the medial, lateral, and ventral subdivisions. The present results provide evidence for a heterogeneous morphology of peptide-immunoreactive neurons in the rat central nucleus that are distributed across cytoarchitectonic boundaries. Except for substance P, neuropeptides in the central nucleus appear to be expressed by a variety of neurons rather than morphologically characteristic types of cell.

Distribution of cholecystokinin-immunoreactive cell bodies in the male and female rat: II. Bed nucleus of the stria terminalis and amygdala.

The distribution of cholecystokinin-immunoreactive (CCK-I) cell bodies was studied in the bed nucleus of the stria terminalis (BST) and amygdaloid complex of colchicine-treated male and female rats. Immunoreactive cells were visualized in the BST medial amygdaloid (MeA), central lateral, basolateral, basolateral ventral, medial, intercalated, anterior cortical, and posterior cortical nuclei and the amygdalohippocampal zone. Several significant sex differences were observed. In the male, a dense aggregation of CCK-I cell bodies was visualized in the MeA, especially in the dorsocaudal part and in the encapsulated part of the BST. In comparison, female rats had relatively fewer immunoreactive cells in both of these regions. In the lateral and basolateral amygdaloid nuclei, however, more CCK-I cells were visualized in the female than in the male, but the difference was not statistically significant. These data provide characterization of a sexually differentiated CCK system. In addition, we observed that the number of CCK-I cells in the BST and posterodorsal part of the MeA was substantially reduced after castration. The number of CCK-I cells in female rats, however, was not significantly reduced after ovariectomy in any of the regions studied. These findings imply that the steroid regulation of CCK is sexually differentiated. The sexually dimorphic distribution of CCK-I cells in areas that are targets of steroid hormones and regulate reproductive processes is consistent with the possibility that CCK participates in central integration of sensory and steroidal input that modulates reproductive behavior.

Neuronal architecture in the rat central nucleus of the amygdala: a cytological, hodological, and immunocytochemical study.

The organization of neurons in the rat central nucleus of the amygdala (CNA) has been examined by using Nissl stain and immunocytochemical and retrograde tracing techniques. Four main subdivisions were identified on the basis of quantitative analyses of Nissl-stained material: medial (CM), lateral (CL), lateral capsular (CLC), and ventral (CV). An intermediate subdivision (CI), previously described by McDonald ('82), was apparent only in animals that had HRP-WGA injected into the bed nucleus of the stria terminalis. Large populations of neurotensin-, corticotropin-releasing factor (CRF)-, and enkephalin-immunoreactive neurons were present within the lateral divisions (mainly CL), although they were also seen within CM. Somatostatin-immunoreactive neurons were distributed mainly within CL and CM. Within CL, neurotensin- and enkephalin-immunoreactive neurons were more numerous laterally whereas CRF- and somatostatin-immunoreactive neurons were more numerous medially. Substance P-immunoreactive neurons were almost exclusively confined to CM. Only a few cholecystokinin- and vasoactive-polypeptide-immunoreactive neurons were seen in the CNA, and they were observed within CL, CV, and CM. The majority of neurons projecting to the dorsal medulla, hypothalamus, and ventral tegmental area were located within CM, although a significant number of cells were also seen within CL. Efferent projections to the bed nucleus of the stria terminalis were found to arise from neurons located within all subdivisions of the CNA. Thus, the distributional patterns of peptidergic and efferent neurons were not confined to individual cytoarchitectonically- defined subdivisions of the CNA. Rather, the results suggest overlapping medial to the lateral trends. Comparisons with the results of previous studies indicate that peptidergic and afferent terminal distribution patterns are more restricted to individual cytoarchitectonically defined subregions of the CNA. These observations suggest that the detailed cytoarchitecture of the CNA more likely reflects the functional integration of afferents rather than the organization of the CNA output neurons.

Evidence for corticotropin-releasing factor, neurotensin, and somatostatin in the neural pathway from the central nucleus of the amygdala to the parabrachial nucleus.

The central nucleus of the amygdala (CNA) and the parabrachial nucleus of the pons (PBN) are included within a group of brain nuclei involved in autonomic responses. Previous studies have shown that the CNA sends a considerable projection to the PBN and that both nuclei contain neurons immunoreactive to many different peptides. In the present study, we used the combined retrograde fluorescence-immunofluorescence method to determine whether the CNA projection to the PBN contains any of the following neuropeptides: corticotropin-releasing factor (CRF), neurotensin (NT), somatostatin (SS), and enkephalin (ENK). Following injections of fluorescent dye into the PBN, neurons within both lateral and medial subdivisions of the CNA were retrogradely labeled. A significant percentage of CRF (54-66%)-, NT (40-53%)-, and SS (31-50%)-immunoreactive neurons were retrogradely labeled, predominantly within the lateral CNA. Enkephalin-immunoreactive neurons were never retrogradely labeled, although they were often found adjacent to retrogradely labeled neurons. Our results show that the lateral CNA is a major source of CRF, NT, and SS terminals within the PBN. Neurons in the medial CNA also provide a significant contribution to the CNA-PBN pathway, but their chemical nature remains to be determined. We conclude that CRF, NT, and SS are important putative neurotransmitters in the CNA's regulation of PBN function. This CNA-PBN peptidergic pathway may participate in stress-related cardiovascular and respiratory responses.

Zinc distribution in mouse brain subsequent to hippocampal lesions.

The role of zinc in central nervous system metabolism remains obscure but it has been shown in animals that diphenylthiocarbazone (Dithizone) selectively colors intravitally the hippocampus and adnexa, parts affected in human temporal lobe epilepsy. To examine the effect of experimental cerebral lesions on zinc storage in that region, 80 mice were divided into two operative and two control groups. The operative groups had unilateral lesions placed in the hippocampus or frontal lobe. One control group and both lesion groups were injected subcutaneously with zinc lactate daily for ten days before intravenous injection of diphenylthiocarbazone. The other control group was used to determine if zinc storage occurs as a result of its increased systemic availability. Hippocampal lesion mice showed more extensive staining of lethal amygdala and associated cortex on the side of the lesion. Frontal lesions remained unstained and frontal lesions did not alter the staining of hippocampus and related parts. Lack of difference between controls shows that greater availability of systemic zinc does not increase its content in the hippocampal-lateral amygdalar region. Increased zinc uptake in this instance appears to be a local phenomenon.

Vasoactive intestinal polypeptide afferents to the bed nucleus of the stria terminalis in the rat: an immunohistochemical and biochemical study.

Vasoactive intestinal polypeptide (VIP) has a markedly heterogeneous distribution in the rat bed nucleus of the stria terminalis. The dorsal bed nucleus contains the highest concentration of VIP in the rat brain, with the exception of the suprachiasmatic nucleus, 4-fold higher than the VIP concentration in the frontal cortex. These biochemical findings agree well with the immunohistochemical analysis of this area. The bed nucleus is also a heterogeneous nucleus with respect to the afferent VIP pathways which innervate it. A combination of immunohistochemical and biochemical techniques was used to examine VIP innervation of the bed nucleus after knife cuts designed to interrupt ascending brainstem, stria terminalis and ventral amygdalofugal inputs to the bed nucleus. The results obtained suggest that (1) ascending pathways arising in the mesencephalon at the level of the dorsal raphe nucleus send VIP fibers to the dorsal but not the ventral bed nucleus, (2) afferent VIP fibers which travel to the bed nucleus via the stria terminalis contribute a diffuse VIP innervation to both the dorsal and ventral bed nucleus and (3) a newly described ventral amygdalofugal VIP pathway to the bed nucleus contributes a major input to the dorsal, but not to the ventral bed nucleus. These three pathways probably account for the entire extrinsic VIP input to the bed nucleus. The finding that the bed nucleus is heterogeneous both with respect to VIP content and afferent VIP inputs serves to clarify previous, apparently discrepant, reports that both the stria terminalis and ascending pathways constitute the major VIP input to the bed nucleus of the stria terminalis.

Kainic acid induced seizures: changes in somatostatin, substance P and neurotensin.

The neuropeptides somatostatin, neurotensin and substance P were investigated in rats during and after limbic seizures induced by systemic injection of kainic acid (10 mg/kg, i.p.). Three hours after injection of the toxin, pronounced decreases (40-50%) in somatostatin-like immunoreactivity in frontal cortex, striatum, dorsal hippocampus and amygdala/pyriform cortex were observed. Concomitantly, neurotensin-like and substance P-like immunoreactivities were also reduced in the frontal cortex and the hippocampus. These early decreases in peptide levels may result from increased release and subsequent inactivation of the peptides during acute seizures. At later time intervals, 3, 10 and 30 days after injection of kainic acid, the initially decreased peptide levels were partially normalized. However, the reduction in somatostatin-like immunoreactivity in amygdala/pyriform cortex and striatum persisted up to 30 days. Neurotensin-like immunoreactivity remained decreased in the frontal cortex. On the other hand, neurotensin- and substance P-like immunoreactivities were increased in the striatum and substantia nigra 10-30 days after injection of kainic acid. These late changes in peptide levels may suggest destruction of peptidergic neurons or adaptive changes induced by the convulsions. Pretreatment of rats with cysteamine (100 mg/kg, i.p.), an agent which decreases brain somatostatin levels, had no effect on the intensity of kainic acid induced convulsions, although a slightly earlier onset of seizures was observed. The changes in peptide levels, especially the marked decreases in somatostatin content after systemic injection of kainic acid, suggest considerable acute and chronic alterations in peptidergic systems caused by limbic convulsions.

Corticotrophin-releasing factor in extra-hypothalamic brain of the mouse: demonstration by immunoassay and immunoneutralization of bioassayable activity.

Corticotrophin-releasing factor (CRF) bioactivity has been described in the extra-hypothalamic brain, but its relationship to hypothalamic CRF has remained questionable. Of the seven regions of the mouse brain examined, highest concentrations of CRF-like immunoreactivity (CRF-LI) and bioassayable CRF activity were present in the median eminence and hypothalamus. However, substantial CRF-LI and bioassayable CRF activity were also seen in brain extracts from the amygdala, thalamus, frontal cortex, pons medulla and cerebellum. Bioactivity was largely neutralized by prior incubation with heat-inactivated antiserum to ovine CRF. These findings, in conjunction with previous immunocytochemical evidence, strongly suggest that a substance closely resembling hypothalamic CRF is present in the extrahypothalamic brain of the mouse.

Multiple forms of somatostatin-like immunoreactivity in the hypothalamus and amygdala of the rat: selective localization of somatostatin-28 in the median eminence.

The presence of multiple forms of somatostatin-like immunoreactivity (SSLI) in the rat hypothalamus was confirmed using a sensitive radioimmunoassay in conjunction with gel filtration chromatography and high performance liquid chromatography (HPLC). Gel filtration chromatography of hypothalamic extracts revealed the presence of four forms of SSLI with estimated molecular weights of 1500, 3000, 6000 and 10000. Analysis by HPLC indicated that the 1500 and 3000 mol. wt forms of SSLI corresponded respectively to somatostatin-14 (SS14) and somatostatin-28 (SS28) whereas the 6000 and 10000 mol. wt forms eluted together as a composite peak of high molecular weight somatostatin (HMW-SS). The proportions of SS14 (63%), SS28 (12%) and HMW-SS (25%) present in the hypothalamus were similar to those in the amygdala (59, 9 and 32% respectively). In contrast, the median eminence contained a greater proportion of SS28 than the other tissues: SS14, SS28 and HMW-SS were present in the proportions 40: 24: 26%. These results show that the rat median eminence differs from the hypothalamus as a whole in containing SS14 and SS28 in almost equimolar concentrations. The localized abundance of SS28 in the nerve terminals of the median eminence suggests a specific role for this peptide in the hypothalamic regulation of growth hormone secretion.

Muscarinic cholinergic receptors and the canine model of narcolepsy.

The role of the muscarinic cholinergic receptor in narcolepsy was examined using radioligand binding to various brain regions of normal and genetically narcoleptic Doberman pinschers. In this multi-litter study, a previous report of a proliferation of muscarinic cholinergic receptors in the brainstem was confirmed, and the concentration of the M2 receptor subtype, in particular, was elevated. This up-regulation of brainstem cholinergic receptors suggests a problem with release of acetylcholine, which, together with previous reports of an impairment of dopamine release, may be indicative of a fundamental membrane problem in narcolepsy.

The effect of repeated administration of antidepressant drugs on the thyrotropin-releasing hormone (TRH) content of rat brain structures.

The present study investigated the effect of repeated treatment with the antidepressant drugs imipramine, amitryptyline, citalopram and mianserin (10 mg/kg PO, twice daily for 14 days) on levels of thyrotropin-releasing hormone (TRH) in several brain structures (cerebral cortex, amygdala + pyriform cortex, hippocampus, nucleus accumbens, striatum and hypothalamus) of the rat. Amitriptyline caused a marked increase in the TRH content in the striatum and nucleus accumbens. Citalopram and mianserin produced a smaller but significant increase in the TRH content in the striatum only, while imipramine did not significantly affect the TRH concentrations in any of the brain structures. None of the antidepressant drugs administered acutely significantly affected the TRH concentrations in the nucleus accumbens or the striatum. These results indicate that changes in brain TRH induced by antidepressant drugs are not related to their therapeutic activity.

Detection of vasopressin mRNA in cells of the medial amygdala but not the locus coeruleus by in situ hybridization.

Vasopressin (AVP)-immunoreactive cells have been previously reported in the medial amygdala (AME) and in the locus coeruleus (LC). The present study was designed to verify the presence of AVP-synthesizing neurons in these areas using in situ hybridization histochemistry. A 35S-labelled oligonucleotide probe, complementary to the glycopeptide portion of the vasopressin-encoding mRNA, was used to label cells expressing the AVP gene in brain sections from male Wistar rats. AVP mRNA-positive cells were identified in the AME and were located throughout the anterodorsal and posterodorsal aspect of the nucleus. Cells in the LC, however, did not exhibit labelling for the glycopeptide portion of the AVP gene. The highest density of labelled cells in the medial amygdala occurred 2.30 to 2.80 mm caudal to bregma. The labelling intensity of the cells averaged 53.8 +/- 3.9 grains/cells and was constant throughout the rostro-caudal extent of the AME. These data demonstrate the presence of AVP-synthesizing cells in the AME and provide a method for quantifying their activity. In addition, these data suggest that the cells in the LC may not synthesize vasopressin.

Early postnatal handling alters glucocorticoid receptor concentrations in selected brain regions.

Norway rat pups were either handled (H) or undisturbed (nonhandled, NH) in the period between birth and weaning on Day 21. Following weaning, half of the animals in each group were housed socially (Soc), and half were housed in isolation (Isol). At 120-150 days of age, all animals were sacrificed, and the following regions were dissected and frozen at -70 degrees C until the time of assay: frontal cortex, hippocampus, hypothalamus, amygdala, septum, and pituitary. [3H]Dexamethasone (3H Dex) binding in each region was examined by an in vitro, cytosol, receptor assay. 3H Dex binding was significantly higher in the hippocampus of both H-Soc and H-Isol than in NH groups. In the frontal cortex, 3H Dex binding was higher in the H-Soc animals than in the H-Isol and NH-Isol animals. There were no significant handling or housing effects found in the amygdala, hypothalamus, septum, or pituitary. Thus, early postnatal handling appears to influence the development of the glucocorticoid receptor system in the hippocampus and frontal cortex. These results are discussed as providing a possible mechanism for some of the previously reported effects of early handling on the development of the pituitary-adrenal response to stress.

Satiety, hunger and regional brain content of cholecystokinin/gastrin and met-enkephalin immunoreactivity in sheep.

Cholecystokinin (CCK) and met-enkephalin (MEK) related peptides have been shown to alter feeding behavior subsequent to their injection into the peripheral circulation or directly into the brains of several species. To evaluate the potential role of endogenous brain pools of these peptides in feeding, groups of sheep were sacrificed either immediately following a meal (satiated) or after various intervals of food deprivation (hungry). Content of CCK-gastrin immunoreactivity in the anterior hypothalami of satiated sheep was elevated compared to 2, 4, or 24 hours of food deprivation. Content of MEK increased progressively with longer intervals of fasting (4 and 24 hours) in the amygdala and basomedial hypothalamus, whereas olfactory bulb content decreased with a similar time course. The results support a potential role for anterior hypothalamic CCK/gastrin in behaviors of satiety, whereas MEK neurons of limbic/rhinencephalic regions appear to form part of a separate circuit gradually activated by increasing hunger. Results are discussed in terms of potential target regions of the peptides, as well as the regional levels and feeding response of sheep as compared to available data from other species.

Synaptology of three peptidergic neuron types in the central nucleus of the rat amygdala.

The synaptology of neurotensin (NT)-, somatostatin (SS)- and vasoactive intestinal polypeptide (VIP)-immunoreactive neurons was studied in the central nucleus of the rat amygdala (CNA). Three types of axon terminals formed synaptic contacts with peptide-immunoreactive neurons in the CNA: Type A terminals containing many round or oval vesicles; Type B terminals containing many pleomorphic vesicles; and Type C terminals containing fewer, pleomorphic vesicles. Peptide-immunoreactive terminals were type A. All three types of terminals formed symmetrical axosomatic and asymmetrical axodendritic contacts. However, type B and peptide-immunoreactive terminals frequently formed symmetrical axodendritic synaptic contacts. VIP-immunoreactive terminals also formed asymmetrical axodendritic contacts. SS- and NT-immunoreactive terminals commonly formed symmetrical contacts on SS- and NT-immunoreactive cell bodies, respectively. VIP-immunoreactive axon terminals were postsynaptic to nonreactive terminals. Type B terminals appeared more frequently on VIP neurons than on NT or SS neurons.

Organization and interrelationship of neuropeptides in the central amygdaloid nucleus of the rat.

The organization and interactions of neuropeptides in the central nucleus of the amygdala (Ce) were studied using single and double label immunocytochemical techniques. Immunocytochemical localization of substance P (SP), neurotensin (NT), met-enkephalin (m-ENK), somatostatin (SS) and vasoactive intestinal polypeptide (VIP) revealed all of these peptides within discrete regions of the Ce. The regions differed from the classical medial and lateral anatomical divisions reported for the Ce. Instead, three easily recognizable neuropeptidergic subdivisions were evident: a medial zone, a central zone and a lateral capsular zone. Two types of interrelationships between peptides were noted. The first involved a peptidergic fiber in apposition to a peptidergic perikarya. The most prevalent peptidergic interaction of this type occurred between SP and NT. The second interrelationship involved two different peptidergic fibers in apposition to an immunonegative cell. Two interactions of this type were commonly observed. The first involved NT and m-ENK fibers simultaneously apposed to an unstained cell. The second involved SP and m-ENK fibers adjacent to the same immunonegative cell. The interactions between peptidergic systems may suggest a role of these substances in the regulation of autonomic functions in the Ce.