Colocalization of peptide- and tyrosine hydroxylase-like immunoreactivities with Fos-immunoreactive neurons in rat central amygdaloid nucleus after immobilization stress.

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.     

The organization of projections from the central nucleus of the amygdala to brainstem sites involved in central autonomic regulation: A combined retrograde transport-immunohistochemical study

The central nucleus of the amygdala (ACe) in the rat sends a considerable projection to, and receives projections from, the parabrachial nucleus (PB) and the dorsal vagal complex (DVC; the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve). In each part of this 'triangle', immunohistochemical staining for the following peptides has been observed in perikarya and fibers: neurotensin, somatostatin, substance-P, Leu-enkephalin and corticotropin-releasing factor. The aim of the present study was to investigate whether any of these peptides are involved in projections to the brainstem from the ACe, and to characterize the distribution of each cell type in the ACe. The results of double retrograde tracing studies indicate that most of the ACe neurons projecting to the PB and DVC are present in the medial part of ACe (ACem), and that many of them project to both the 1 B and the DVC. The combined use of immunohistochemistry with a retrograde fluorescent tracer, True Blue, indicated that the peptide-containing perikarya are found predominantly in the lateral part of ACe (ACe1), and that only a small proportion of neurotensin, somatostatin and corticotropin-releasing factor-stained neurons contained True Blue after injections into the PB or the DVC. The results suggest that most of the fibers in the descending projection from the ACe to the brainstem do not contain the peptides examined here.     

Effects of neurotensin on neurons in the rat central amygdaloid nucleus in vitro

The effects of neurotensin (NT) on neurons in the central amygdaloid nucleus (ACe) were investigated in rat brain slice preparations by adding the peptide to the perfusing medium. Of 115 ACe neurons, 69 cells (60%) showed excitatory responses and 10 cells (9%) showed inhibitory responses to application of NT. The excitatory response to NT was observed in a dose-dependent manner and the threshold concentration was approximately 3 × 10⁻⁹ M. The excitatory effects of NT persisted under blockade of synaptic transmission. The NT fragment neurotensin 8–13 and the NT analogue neuromedin N showed effects similar to those of NT, whereas the NT fragment neurotensin 1–8 had no effect on ACe neurons. Of 43 neurons in the septal nucleus, 8 cells (19%) and 3 cells (7%) showed excitatory and inhibitory responses, respectively, to NT. The results suggest that NT exerts a potent excitatory effect on ACe neurons through a direct action on specific receptors, in which NT may play a role in amygdala-relevant functions.     

Collateral axonal projections to limbic structures from ventrolateral medullary A1 noradrenergic neurons

Experiments were done to investigate whether catecholaminergic neurons within the ventrolateral medulla (VLM) send collateral axonal projections to the central nucleus of the amygdala (ACe) and the bed nucleus of the stria terminalis (BST). Unilateral microinjections of the fluorescent retrograde tracers fluorogold (FG) or rhodamine labelled latex micro-beads (Rd) were made into either ACe or BST in the rat. Brainstem sections were then processed immunohistochemically for the identification of cell bodies containing the catecholamine biosynthetic enzymes tyrosine hydroxylase, dopamine β-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT). Retrogradely labelled cell bodies projecting to either ACe or BST were found throughout the rostrocaudal extent of VLM, bilaterally. Approximately 44% Of these retrogradely labelled neurons were found to contain both eetrograde tracers. In addition, approximately 91% of the VLM neurons that send collateral axonal projections to ACe and BST were also immunoreactive to DBH. None were found to contain PNMT immunoreactivity. These results demonstrate that noradrenergic neurons of the A1 cell group in VLM innervate ACe and BST via collateral axonal projections and suggest that these VLM neurons may be directly involved in relaying cardiovascular afferent and/or visceral afferent information directly to these limbic structures.     

Identification of neurons containing orexin-B (hypocretin-2) immunoreactivity in limbic structures

Orexins (hypocretins) are neuropeptides which have recently been identified exclusively within lateral hypothalamic and perifornical neurons, and these orexin (ox) containing neurons appear to have extensive projections to all levels of the neuraxis. In this study, we report the identification of two distinct clusters of neurons containing ox-B-like immunoreactivity within the amygdaloid complex of the rat. A cluster of small to medium size ovoid shaped neurons containing ox-B-like immunoreactivity was found predominantly within the lateral division of the central nucleus of the amygdala (ACe). A second distinct, but smaller group of ox-B labelled neurons with similar shapes and sizes to those in ACe was also identified in the anterior lateral subnucleus of the bed nucleus of the stria terminalis (BST) immediately adjacent the internal capsule, and in an area just ventral to the lateral ventricle. Neurons containing ox-A-like immunoreactivity were not observed in either structure. However, both structures contained ox-A- and ox-B labelled varicose fibers. Unilateral electrolytic lesions of the lateral hypothalamic area that contained ox-A and ox-B neurons did not alter the labelling of either ACe or BST ox-B pericarya. As both the ACe and BST are known to be involved in integrating complex homeostatic mechanisms associated with behaviours, these data suggest that a specific subset of ox-B neurons within the amygdaloid complex may serve as a component of neuronal circuits coordinating these responses.     

Effect of noradrenergic inputs on the cardiovascular depressor responses to stimulation of central nucleus of the amygdala

Experiments were done in chloralose anesthetized, paralyzed and artificially ventilated male Wistar rats to investigate the effects of microinjections of either norepinephrine (NE) or tyramine into the central nucleus of the amygdala (ACe) on the arterial pressure (AP) and heart rate (HR) responses elicited by glutamate (Glu) stimulation of the ACe. Microinjections of Glu into the ACe elicited decreases in mean AP (-23+/-3 mmHg) and HR (-11+/-3 bpm). Microinjections of NE or tyramine into these sites did not elicit cardiovascular responses. However, Glu into the ACe in the presence of NE or tyramine elicited depressor or bradycardic response that were significantly smaller (70-100%) in magnitude than to Glu alone. These data suggest that noradrenergic mechanisms in the ACe alter the excitability of ACe neurons involved in mediating changes in systemic AP and HR.     

Bed nucleus of the stria terminalis: cytoarchitecture, immunohistochemistry, and projection to the parabrachial nucleus in the rat

The bed nucleus of the stria terminalis (BST) sends a dense projection to the parabrachial nucleus (PB) in the pons. The BST contains many different types of neuropeptidelike immunoreactive cells and fibers, each of which exhibits its own characteristic distribution within cytoarchitecturally distinct BST subnuclei. Corticotropin releasing factor (CRF)-, neurotensin (NT)-, somatostatin (SS)-, and enkephalin (ENK)-like immunoreactive (ir) neurons are particularly numerous within areas of the BST that project to the PB. In this study, we use the combined retrograde fluorescence-immunofluorescence method to determine whether neurons in the BST that project to the PB contain these immunoreactivities. After Fast Blue injections into PB, retrogradely labeled neurons were numerous throughout the lateral part of the BST, particularly in the dorsal lateral (DL) and posterior lateral subnuclei. Retrogradely labeled neurons were also present in the preoptic, ventral lateral, and supracapsular BST subnuclei and in the parastrial nucleus. Many of the CRF-ir, NT-ir, and SS-ir neurons in DL were retrogradely labeled. A few double-labeled cells of each type were also found in the posterior lateral, ventral lateral and supracapsular BST subnuclei ENK-ir neurons were never retrogradely labeled. Our results show that BST neurons that project to the PB stain for the same neuropeptides as those in the central nucleus of the amygdala (CeA) that project to the PB, demonstrating further the close anatomical relations between these two structures.     

Effects of vasopressin and involvement of receptor subtypes in the rat central amygdaloid nucleus in vitro

Effects of arginine-vasopressin (AVP) on neurons in the central amygdaloid nucleus (ACe) were investigated with rat brain slice preparations using extracellular recording methods. Of 160 ACe neurons tested, 70 cells (44%) were excited and 9 cells (6%) were inhibited by bath application of AVP at 3×10⁻⁷ M. The excitatory effects of AVP were dose-dependent and the threshold concentration was approximately 10⁻¹⁰ to 10⁻⁹ M. The excitatory effects of AVP persisted under blockade of synaptic transmission by perfusing with Ca²⁺-free and high-Mg²⁺ medium, whereas the inhibitory effects were abolished by synaptic blockade. AVP-induced effects were mimicked by a V₁-receptor agonist and completely blocked by a selective V₁-antagonist. V₂-agonist produced no effects on ACe neurons and V₂-antagonist had no effect on AVP-induced excitation. These results showed that the excitatory effect of AVP on ACe neurons was produced by a direct action through the V₁-receptors, whereas the inhibitory response of ACe neurons to AVP seemed to be produced by an indirect action. The results of this study suggest that AVP is involved in the amygdala-relevant functions as a neurotransmitter or a neuromodulator.     

Collateral axonal projections from ventrolateral medullary non-catecholaminergic neurons to central nucleus of the amygdala

Retrograde tract-tracing techniques were used to investigate whether catecholaminergic neurons in the ventrolateral medulla (VLM) send collateral axonal projections to both central nuclei of the amygdala (ACe) in the rat. Rhodamine-labelled latex microspheres or fluorogold (2%) were microinjected into the region of either the right or left ACe. After a survival period of 10–12 days, the rats were sacrificed and transverse sections of the brainstem were processed immunohistochemically for the identification of cell bodies containing the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) or phenylethanolamine-N-methyltransferase (PNMT). Neuronal perikarya containing the retrogradely transported tracers were observed throughout the rostrocaudal extent of VLM, bilaterally. Approximately 10% of the retrogradely labelled neurons were observed to contain both retrograde tracers. The majority (79 ± 6.8%) of these double labelled neurons were located within the caudal VLM and their number decreased rostrally. In addition, the proportion of double labelled neurons to single labelled neurons in VLM decreased rostrally; approximately 11% in the caudal VLM and 6% in the rostral VLM. Furthermore, approximately 21% of all VLM neurons that projected to ACe were found to be catecholaminergic: 75% of these were immunoreactive to TH and 25% to PNMT. However, no neurons were found in VLM that contained both retrograde tracers and immunoreactivity to TH or PNMT. These data demonstrate that axons originating from non-catecholaminergic neurons in VLM bifurcate to innervate ACe bilaterally. Although the function of these VLM neurons that project to both ACe is not known, they may be the anatomical substrate by which VLM neurons relay simultaneously autonomic and/or visceral sensory information to influence the activity of ACe.     

Effect of the NR3 subunit on ethanol inhibition of recombinant NMDA receptors

Central stress regulatory pathways utilize various neurotransmitters/neuropeptides, such as urocortin (Ucn) and catecholamines. Ucn is most abundantly expressed in the Edinger-Westphal nucleus (E-WN), co-distributed with catecholaminergic terminals. Acute stress recruits E-WN neurons, and ascending catecholaminergic pathways also contribute to the activation of various brain areas in response to stress. We hypothesized that catecholamine and Ucn interactions in the E-WN mediated the recruitment of these neurons in response to stress. Using double-labeling immunohistochemistry, we found close appositions between urocortin-immunoreactive nervous structures and dopaminergic terminals, however, depletion of them had no effect on the activation pattern of E-WN neurons upon acute immune challenge. From these results we conclude that dopaminergic terminals innervating E-WN Ucn neurons do not play a major role in mediating the responses of E-WN neurons upon acute immune challenge.     

Neuropeptides in the Cat Amygdala

The distribution of seven neuropeptides was studied in the cat amygdala using an indirect immunoperoxidase technique. No labeling was found for luteinizing hormone-releasing hormone or β-endorphin (1–27). Sparse α-melanocyte-stimulating hormone-immunoreactive fibers were found in the basomedial nucleus of the amygdala, whereas a low density of fibers containing α-neo-endorphin was observed in the anterior amygdaloid area. Neurotensin was observed in fibers of the anterior amygdaloid area (low density) and both the lateral (low density) and the medial part (moderate density) of the central nucleus. A low density of fibers containing neurokinin A was found in the anterior amygdaloid area, the basolateral nucleus, and the medial part of the central nucleus. A moderate density was observed in the basomedial nucleus and in the medial and cortical nuclei. Fibers containing somatostatin-28 (fragment 1–12) were observed in all the amygdaloid nuclei, whereas immunoreactive cell bodies were found in all the nuclei except in the medial part of the central nucleus and the medial nucleus. Perikarya containing neurokinin A were observed in the latter nucleus. The results point to a discrete distribution of peptidergic fibers in the cat amygdala, as well as the occurrence of neurons containing neurokinin A and somatostatin-28 (fragment 1–12). The distribution of the peptides studied in the cat is compared with the location of the same peptides in the amygdala of other species. The possible diencephalic origin of the peptidergic fibers is also discussed.     

The influence of chemosensory input and gonadotropin releasing hormone on mating behavior circuits in male hamsters

Chemosensory input is important for mating behavior in male hamsters. Chemosignals found in female hamster vaginal fluid activate regions of the brain that receive input from the vomeronasal/accessory olfactory system and are important for mating behavior. Mating or exposure to these chemosignals produces increased Fos protein expression in the amygdala, bed nucleus of the stria terminalis, and medial preoptic area (MPOA). These brain regions contain cell bodies and/or fibers of gonadotropin releasing hormone (GnRH) neurons, suggesting potential relationships between chemosensory systems and GnRH. GnRH is released naturally when male rodents (mice and hamsters) encounter female chemosignals, and intracerebrally injected GnRH restores mating behavior in sexually naive male hamsters after removal of the vomeronasal organs. We report here that the combination of pheromone exposure and intracerebrally-injected GnRH increases Fos expression in the MPOA above the increase seen in pheromone-exposed males, or in males given only the exogenous GnRH. In males with vomeronasal organs removed (VNX), there was an also an increment in Fos expression in the MPOA when these pheromone exposed males were injected with GnRH, provided they had previous sexual experience. Males with vomeronasal organs removed and without sexual experience showed increased Fos expression in the medial amygdala when pheromone exposure and GnRH injection were combined, but not in the medial preoptic area.     

Selective depletion of somatostatin in rat brain by cysteamine

A single injection of cysteamine (300 mg/kg, subcutaneously) results in a 70–80% decrease in somatostatin levels in the periventricular nucleus where somatostatin-producing neurons are located and the median eminence where somatostatinergic nerve terminals are. The drug seems quite selective: no changes in levels of other neuropeptides — LH-RH, vasopressin, enkephalin, VIP, CCK — were observed in the same animals.     

Coexpression of prodynorphin and corticotrophin-releasing hormone in the rat central amygdala: evidence of two distinct endogenous opioid systems in the lateral division

The lateral subdivision of the central nucleus of the amygdala (CeA) comprises two groups of gamma-aminobutyric acid (GABA) neurons that express corticotrophin-releasing hormone (CRH) and enkephalin. Regulation of the expression and release of these neuropeptides by glucocorticoids and other factors has been suggested to have a regulatory function on the diverse somatic, autonomic, and neuroendocrine responses that are coordinated by the CeA. Because another opioid peptide, dynorphin, has been reported to be also expressed by neurons in the lateral CeA, this study examined the neuronal expression of this kappa-opioid (KOP) receptor-preferring ligand by using immunohistochemistry for the precursor peptide prodynorphin. Prodynorphin neurons in the extended amygdala were observed mostly in the medial and central regions of the lateral CeA and the oval of the bed nucleus of the stria terminalis (BST). About one-third of the prodynorphin neurons in the CeA coexpressed CRH, whereas no coexpression with CRH was detected in the BST. Prodynorphin was not expressed by calbindin neurons in the medial part of the lateral CeA, and indirect evidence suggested that it was not expressed by enkephalin neurons. Coexpression of prodynorphin in extrahypothalamic CRH neurons in the CeA could provide an anatomical basis for regulation of the stress responses and other CRH-related functions by the brain dynorphin/KOP receptor system.     

Projection Sites of Medial Preoptic Area and Ventral Bed Nucleus of the Stria Terminalis Neurons that Express Fos during Maternal Behavior in Female Rats

Medial preoptic area (MPOA) and ventral bed nucleus of the stria terminalis (VBST) neurons are involved in maternal behavior, but the neural sites to which the maternally relevant neurons project have not been determined. Since MPOA and VBST neurons express Fos during maternal behavior, we used a double-labeling immunocytochemical procedure to detect both Fos and a retrograde tracer, wheat germ agglutinin (WGA), in order to determine where these Fos neurons project. On Day 4 postpartum, fully maternal females were separated from their litters. On Day 5, WGA was iontophoretically injected into one of the following regions known to receive MPOA and/or VBST input: Lateral septum, medial hypothalamus at the level of the ventromedial nucleus, lateral habenula, ventral tegmental area, retrorubral field, or periaqueductal gray. On Day 7, females received a 2-h test with either pups or candy, after which they were perfused and their brains were processed for the detection of Fos and WGA. As expected, females tested with pups had more Fos-containing neurons in the MPOA and VBST than did females tested with candy. After WGA injections into several brain sites, the number of double-labeled cells observed in the MPOA and VBST was greater for the maternal females when compared to the non-maternal females. Therefore, these results pinpointed neural circuits that were activated during maternal behavior. For the maternal females, Fos-containing neurons in the MPOA projected most strongly to the medial hypothalamus at the level of the ventromedial nucleus and to the lateral septum, while Fos-containing neurons in the VBST projected most strongly to the retrorubral field, ventral tegmental area, and medial hypothalamus. Although relatively few MPOA and VBST neurons which expressed Fos during maternal behavior projected to the periaqueductal gray, these Fos-expressing neurons made up a relatively large proportion of the MPOA and VBST projection to the periaqueductal gray. This study suggests that MPOA and VBST efferents project to a variety of regions to promote full maternal responsiveness.     

Effects of testosterone on neuronal nitric oxide synthase and tyrosine hydroxylase

Male rat copulatory ability decreases dramatically following castration. This may be due in part to the impairment of medial preoptic area (MPOA) dopamine (DA) release. Previous studies showed that extracellular DA levels in the MPOA of castrates were lower than in intact males, both during basal conditions and in the presence of a receptive female. However, tissue levels of DA in the MPOA were higher in castrates than in intact males, suggesting that DA synthesis may be normal or increased in castrates, but that release may be compromised. The current study found that neither long term (2 months) nor short term (2 weeks) castration had any effect on the number of neurons in the DA A₁₄ area that were immunoreactive (ir) for tyrosine hydroxylase (TH), the rate limiting enzyme for DA synthesis. Therefore, castration may not affect DA synthesis in the MPOA. Tissue levels of neurotransmitter reflect release, as well as synthesis. We previously reported that nitric oxide (NO) may increase DA release in the MPOA. The present study tested whether castration affected the number of NO producing cells in the MPOA. Long term, but not short term, castration significantly decreased the number of NADPH-d (nicotinamide adenine dinucleotide phosphate diaphorase) positive neurons and brain nitric oxide synthase immunoreactive (bNOS-ir) neurons in the medial preoptic nucleus (MPN). This suggests that in gonadally intact animals testosterone may activate NOS, which increases the production of NO. Long or short term castration had no effect on the numbers of bNOS-ir neurons in the paraventricular nucleus (PVN) or medial amygdala. However, short term castration decreased bNOS-ir neurons in the bed nucleus of stria terminalis (BNST). Thus, one means by which testosterone promotes male sexual behavior may be by increasing production of NO in the MPOA, which increases local DA release.     

NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like immunoreactivities

Certain neurons in the brain are specifically and intensely stained by a histochemical method which demonstrates nicotinamide adenine dinucleotide phosphate NADPH-diaphorase activity. The cell types containing this enzyme in certain areas of the rat forebrain were examined by combining NADPH-diaphorase histochemistry with the indirect immunofluorescence technique. Neurons containing somatostatin- or avian pancreatic polypeptide (APP)-like immunoreactivities were found throughout the forebrain including the striatum and neocortex. These two neuropeptides were also found to coexist in many telencephalic neurons. After photography, the sections processed for immunohistochemistry were stained for NADPH-diaphorase activity by a histochemical method. It was found that within the striatum all of the neurons that were selectively stained by this technique also contained both somatostatin- and APP-like immunoreactivities. Also in the neocortex NADPH-diaphorase was found only in those neurons displaying somatostatin- or APP-like immunoreactivity. In other brain regions such as the nucleus laterodorsalis tegmenti, NADPH-diaphorase-containing cells did not contain these neuropeptides. The results indicate that NADPH-diaphorase histochemistry provides a simple, reliable, histochemical method to demonstrate those striatal neurons in which somatostatin- and APP-like immunoreactivities coexist. The selective occurrence of this enzyme within these neurons may provide a useful target for pharmacological studies of these neuropeptide-containing cells.     

Fos and jun in rat central amygdaloid nucleus and paraventricular nucleus after stress.

The present paper describes the effect of capsaicin-induced stressful stimulus on the expression of immediate early genes (IEGs) c-fos, c-jun, junB and junD in the hypothalamic paraventricular nucleus (PVN) and the central amygdaloid nucleus (ACe) using in situ hybridization. Stress caused an intense expression of c-fos, c-jun and junB especially in the PVN and ACe and also a clear induction of junD was observed in the PVN. This suggests that the PVN and the ACe are two major targets of stress in the brain. The intense expression of the IEGs in the ACe and PVN suggests that stress may affect neurotransmitter gene expression through Fos and Jun proteins in both these nuclei.