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.     

Fos expression in forebrain afferents to the hypothalamic paraventricular nucleus following swim stress

The paraventricular nucleus of the hypothalamus (PVN) serves as the origin of the final common pathway in the secretion of glucocorticoid hormones in response to stress. Various stress-related inputs converge upon the cells of the medial parvocellular division of the PVN. These neurons, which synthesize and release corticotropin-releasing hormone, arginine vasopressin, and other secretagogues, are responsible for a cascade of events which culminates in the adrenocorticotropin-induced release of corticosteroids from the adrenal cortex. Previous data have suggested complex afferent regulation of PVN neurons, although the neuronal pathways by which the effects of stress are mediated remain to be fully disclosed. The present experiment sought to identify forebrain areas potentially involved in afferent regulation of the PVN in response to an acute stressor. Discrete injections of the retrograde tracer Fluoro-gold were delivered to the PVN, and rats were subsequently subjected to an acute swim stress. Brains were processed immunocytochemically for the simultaneous detection of the tracer and Fos, the protein product of the immediate early gene c-fos, utilized as a marker for neuronal activation. The majority of Fluoro-gold/Fos labeled neurons were detected in the parastrial nucleus, the medial preoptic area, the anterior hypothalamic area, the dorsomedial hypothalamic nucleus and adjacent posterior hypothalamic area, and, to a lesser extent, the supramammillary nucleus. These findings are discussed in relation to neural pathways mediating activation and inhibition of the hypothalamic-pituitary-adrenocortical axis. © 1996 Wiley-Liss, Inc.     

Antinociception after microinjection of neurotensin into the central amygdaloid nucleus of the rat

Neurotensin (NT) is an endogenous peptide which has been hypothesized to function in the central nervous systems as a neurotransmitter. Injection of NT into the cerebral ventricular system of rodents produces antinociception in a variety of analgesia tests. In the hot plate test, direct microinjection of NT into the central nucleus of the amygdala (AC) produced a significant increase in the nociceptive threshold of the rat, while injections into tissue adjacent to the AC were generally ineffective. Antinociception following intra-AC injection of NT occurred at an ED₅₀ dose of 2.4 μg NT, and was significantly lower than the ED₅₀ dose observed when NT was given into the lateral ventricles (93.2 μg NT). Lesions of the stria terminalis totally abolished the antinociceptive effect of intra-AC administration of NT, indicating that AC efferent or afferent fibers within the stria terminalis are necessary for the observed increase in nociceptive threshold.     

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.     

Implication of the serotoninergic system in the decreased ACTH response to stress after lesion of the amygdaloid central nucleus

1. The present study was designed to ascertain the possible implication of the serotoninergic system and the central amygdaloid nucleus in the control of ACTH secretion in response to immobilization stress.

2. The response to immobilization stress of intact and lesioned animals was studied by monitoring the plasma and pituitary ACTH concentration and the activity of the serotoninergic system within specific hypothalamic and amygdaloid nuclei.

3. Bilateral lesions of the central nucleus of the amygdala significantly decreased the secretion of ACTH in response to immobilization. Moreover, the serotoninergic activity in most of the hypothalamic and in all the amygdaloid nuclei studied was greatly increased. A 60-min immobilization stress prevented this increase in the hypothalamic nuclei but not in the amygdala.

4. These results indicate that the central nucleus of the amygdala participates in the regulation of ACTH secretion in response to immobilization stress. Furthermore, they substantiate the hypothesis of a participation of the serotoninergic system in limbic areas, particularly in nuclei which contain neurons possessing glucocorticoid receptors such as the medial, basomedial and cortical amygdaloid nuclei.     

Psychological stress increased corticotropin-releasing hormone mRNA and content in the central nucleus of the amygdala but not in the hypothalamic paraventricular nucleus in the rat

The central administration of corticotropin-releasing hormone (CRH) to experimental animals sets into motion a coordinated series of physiological and behavioral events that promote survival during threatening situation. A large body of evidence suggest that CRH in the central nucleus of the amygdala (CEA) induces fear-related behaviors and is essential to fear conditioning; however, evidence of CRH-mediated activation of the amygdala under physiological situation is still limited. We report here a study of the impact of a psychological stressor on hypothalamic and amygdala CRH systems in the rat. Non-footshocked rats placed in a floored compartment surrounded by footshocked rats were defined as the psychological stress group. Rats were exposed to psychological stress for 15 min, and then sacrificed 1.5 and 3 h after cessation of stress. We found that our psychological stressor induced an increase in both CRH mRNA levels, as assessed by in situ hybridization histochemistry, and CRH content, as assessed by micropunch RIA, in the CEA. Exposure to the psychological stressor also caused a significant increase in CRH mRNA levels with a trend for an increase in CRH content in the dorsolateral subdivision of the bed nucleus of the stria terminalis (BNST) which is anatomically associated with the CEA. In contrast, psychological stress induced a small, but significant increase in type-1 CRH receptor (CRHR-1) mRNA in the hypothalamic paraventricular nucleus (PVN), while it failed to elevate either PVN CRH mRNA levels or content, CRH content in the median eminence (ME), or levels of plasma ACTH or corticosterone (CORT). Thus, in the context of a psychological stressor, the activation of the amygdala CRH system can occur without robust activation of the hypothalamic CRH system. In the light of previous data that the psychological stress-induced loss of sleep was reversed by the central administration of a CRH antagonist, these data suggest that CRH in the CEA may contribute to the psychological stress-evoked fear-related behavior such as hyperarousal. These data also indicate that in response to a psychological stressor, the amygdala CRH system is much more sensitive than is the CRH system emanating from the PVN.     

Thalamic paraventricular nucleus lesions facilitate central amygdala neuronal responses to acute psychological stress

The thalamic paraventricular nucleus (PVT) is activated by stress and projects to forebrain structures directly implicated in processing stress-related information. Accordingly, it seems likely the PVT plays an important role in modulating stress responses. We examined effects of excitotoxic PVT lesions on forebrain Fos expression patterns normally elicited by an acute psychological stressor. PVT lesions significantly increased stress-induced Fos in a key stress-processing region, the central amygdala.     

Intrinsic connections of the rat amygdaloid complex: Projections originating in the central nucleus

Inputs from the amygdaloid and extraamygdaloid areas terminate in various divisions of the central nucleus. To elucidate the interconnections between the different regions of the central nucleus and its connectivity with the other amygdaloid areas, we injected the anterograde tracer, Phaseolus vulgaris-leucoagglutinin (PHA-L) into the capsular, lateral, intermediate, and medial divisions of the central nucleus in rat. There were a number of labeled terminals near the injection site within each division. The intrinsic connections between the various divisions of the central nucleus were organized topographically and originated primarily in the lateral division, which projected to the capsular and medial divisions. Most of the connections were unidirectional, except in the capsular division, which received a light reciprocal projection from its efferent target, the medial division. The intermediate division did not project to any of the other divisions of the central nucleus. Extrinsic projections from the central nucleus to the other amygdaloid nuclei were meager. Light projections were observed in the parvicellular division of the basal nucleus, the anterior cortical nucleus, the amygdalohippocampal area, and the anterior amygdaloid area. No projections to the contralateral amygdala were found. These data show that the central nucleus has a dense network of topographically organized intradivisional and interdivisional connections that may integrate the intraamygdaloid and extraamygdaloid information entering the different regions of the central nucleus. The sparse reciprocal connections to the other amygdaloid nuclei suggest that the central nucleus does not regulate the other amygdaloid regions but, rather, executes the responses evoked by the other amygdaloid nuclei that innervate the central nucleus. J. Comp. Neurol. 395:53–72, 1998. © 1998 Wiley-Liss, Inc.     

Chronic treatment with the antidepressant tianeptine attenuates lipopolysaccharide-induced Fos expression in the rat paraventricular nucleus and HPA axis activation

The antidepressant tianeptine has been shown to decrease the response of the hypothalamic-pituitary-adrenal (HPA) axis to stress and to attenuate the behavioral effects of the cytokine inducer, lipopolysaccharide (LPS). Since LPS also activates the HPA axis, the objective of this study was to assess the effects of tianeptine on the HPA axis activation and Fos expression induced by intraperitoneal (i.p.) administration of LPS (30 and 250 microg/kg respectively). Chronic, but not acute, tianeptine treatment (10 mg/kg twice a day for 15 days, i.p.) attenuated LPS-induced increase of plasma ACTH and corticosterone in rats bearing an indwelling catheter in the jugular vein and Fos immunoreactivity in the paraventricular nucleus (PVN). These results open new vistas on the pharmacological activity of tianeptine and provide further insights on the action mechanisms of antidepressants in clinics.     

Role of the central amygdaloid nucleus in shaping the discharge of gustatory neurons in the rat parabrachial nucleus

The central amygdaloid nucleus (CeA) receives projection from the parabrachial nucleus (PBN) gustatory neurons and descendingly projects to the PBN. To assess if the CeA is involved in modulating the activity of gustatory neurons in the PBN, the effects of electrical stimulation and electrolytic lesion of CeA on PBN gustatory neurons were observed. Of 60 neurons observed, 30 were classified as NaCl-best, 18 as HCl-best, 5 as Quinine HCl (QHCl)-best, and 7 as sucrose-best. During CeA stimulation, the responses to at least one effective stimulus were inhibited in most PBN neurons, with the response magnitudes to HCl and QHCl significantly decreased (P<0.01). In contrast, bilateral lesions of CeA facilitated the responses to HCl and QHCl (P<0.01). According to the best-stimulus category, the effects on the responses to HCl and QHCl were similarly subjected to these modulations either during electrical stimulation or after electrolytic lesions of CeA. Analyses of across-unit patterns indicated that the CeA stimulation increased the chemical selection of PBN taste neurons while the CeA lesions depressed the effect on the chemical selection between NaCl and QHCl. These findings suggest that the CeA may be involved in mediating feeding behavior via modulating the activity of gustatory neurons of PBN.     

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.     

Rat central amygdaloid nucleus projections to the bed nucleus of the stria terminalis.

The projections from the central amygdaloid nucleus (Ce) to different subdivisions of the bed nucleus of the stria terminalis (BNST) were investigated using retrograde transport of fluorescent dyes. Iontophoretic injections of either Fast Blue (FB) or bisbenzimide (BB) were applied to the anterior medial, posterior medial, anterior lateral and posterior lateral parts of the bed nucleus of the stria terminalis. The anterior medial BNST receives projections from caudal part of medial Ce (CeM). The posterior medial BNST receives projections specifically from the intermediate subdivision of Ce, though in some cases projections from the ventral subdivision (CeV) of Ce were seen. The anterior lateral BNST receives projections primarily from the caudal lateral Ce (CeL) as well as middle and caudal part of CeM. The posterior lateral BNST receives projection from rostral CeL as well as the CeV and lateral capsular Ce. In general, the results indicate that the major subdivisions of the BNST receive projections from Ce subdivisions having similar connections with diencephalic or brainstem cell groups. Additional evidence is presented suggesting that Ce-BNST projections are part of an extensive system of intrinsic connections linking similar groups of neurons in both the Ce and BNST as well as within Ce.     

Projection from the ventrolateral medullary neurons containing tyrosine hydroxylase to the central amygdaloid nucleus in the rat.

By using a double-labeling produce of retrograde horseradish peroxidase (HRP) tracing and the immunocytochemical localization of tyrosine hydroxylase (TH), the present study ascertained that the axonal fibers of catecholaminergic neurons in the caudal ventrolateral medulla projected to the central amygdaloid nucleus in the rat. The majority of double-labeled cells were observed primarily around the level of the obex. 92% of HRP retrogradely labeled cells contained TH-like immunoreactive (TH-IR), but only 31% of TH-IR cells was labeled with HRP.     

Selective reduction of glucocorticoid receptor immunoreactivity in the hippocampal formation and central amygdaloid nucleus of the aged rat.

Hippocampal cell loss during aging has been related to the toxic effects of corticosterone on this cell population. It is not known which receptor mediates corticosterone cytotoxicity. At least two types of receptors for corticosterone have been recognized in the rat brain, type I (corticosterone preferring receptor, CR) and type II (glucocorticoid receptor, GR). In the present study the possible changes in GR immunoreactivity (IR) in various tel- and diencephalic regions of the aged rat have been investigated using immunocytochemistry coupled with computer-assisted image analysis. Male Sprague-Dawley rats of 3, 12 and 24 months of age were used (n = 5/group). A selective decrease of GR-IR was observed in the CA1 hippocampal field and central amygdaloid nucleus of the 24-month-old with respect to both 3- and 12-month-old rats. While in the former region GR-IR decrease was paralleled by a decrease of IR field area, no age-related decrease of GR-IR profile number was detected in central amygdaloid nucleus. A significant decrease of GR-IR and IR field area was also observed in the dentate gyrus of 24- vs 12-month-old rats but not vs 3-month-old rats. The analysis of adjacent sections stained with Cresyl violet showed a pattern of age-related changes (decrease of neuronal profiles in CA1 field pyramidal layer and dentate gyrus granular layer, and no change in the central amygdaloid nucleus of aged rats) which paralleled the observed changes in GR-IR in the same areas. This study provides evidence that GR are selectively decreased in the hippocampal formation and in the central amygdaloid nucleus of the aged rat.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrical self-stimulation in the central amygdaloid nucleus after ibotenic acid lesion of the lateral hypothalamus

This experiment was carried out in order to investigate the involvement of lateral hypothalamus (LH) in electrical self-stimulation of the central amygdaloid nucleus (CeA). Adult male Sprague-Dawley rats were bilaterally implanted with a guide cannula situated above each LH and with two electrodes in the CeA. Self-stimulation was subsequently obtained separately from both right and left electrodes. The LH was then lesioned unilaterally by ibotenic acid (IBO) injection. Eight days later, the effect of this unilateral lesion on self-stimulation of the ipsilateral and contralateral CeA was tested. Then the neurons of the remaining non-lesioned LH side were lesioned with IBO and self-stimulation was tested 15 days after the second lesion. Both unilateral as well as bilateral lesions of LH produced a significant decrease in CeA self-stimulation rates but had no significant effect on the reward effectiveness. The unilateral lesions did not produce any modification of the rate-intensity function in the contralateral CeA. This lesion-induced depression in performance was reversed by treatment with phenobarbital. These results provide clear evidence that the rewarding effects of CeA electrical stimulation do not result from the activation of the LH outputs and that the apparent decrease in CeA self-stimulation may result from the LH lesion-induced increase in the frequency of epileptiform manifestations that occur following amygdaloid stimulation.     

Differential Fos expression in the paraventricular nucleus of the hypothalamus, sacral parasympathetic nucleus and colonic motor response to water avoidance stress in Fischer and Lewis rats

The responsiveness of hypothalamic CRF to various stressors is reduced in the young female Lewis relative to the histocompatible Fischer rat. Whether such a difference impacts the brain-gut response to water avoidance stress was investigated by monitoring Fos immunoreactivity in the brain and sacral spinal cord and fecal pellet output. Exposure for 60 min to water avoidance stress increased the number of Fos positive cells in the paraventricular nucleus of the hypothalamus (PVN), nucleus tractus solitarius (NTS), and the parasympathetic nucleus of the lumbo-sacral spinal cord (L6-S1) in both Lewis and Fischer rats compared with non stress groups. The Fos response was lower by 32.0% in the PVN, and 63% in sacral parasympathetic nucleus in Lewis compared with Fischer rats while similar Fos expression was observed in the NTS. Stress-induced defecation was reduced by 52% in Lewis compared with Fischer rats while colonic motor response to CRF injected intracisternally resulted in a similar pattern and magnitude of defecation in both strains. The CRF receptor antagonist [ -Phe12,Nle^21,38C^aMeLeu³⁷]-CRF_12–41 injected intracisternally antagonized partly the defecation response in Lewis and Fischer rats. These data indicate that a lower activation of PVN and sacral parasympathetic nuclei in Lewis compared with Fisher rats may contribute to the differential colonic motor response and that the blunted CRF hypothalamic response to stress, unlike responsiveness to central CRF plays a role.     

Role of the paraventricular nucleus microenvironment in stress integration

The hypothalamic paraventricular nucleus is the primary controller of hypothalamo-pituitary-adrenocortical glucocorticoid release. In performing this function, the paraventricular nucleus summates a variety of information from both external and internal sources into a net secretory signal to the adrenal cortex. In this review, we will provide an overview of neuronal circuit mechanisms governing activation and inhibition of hypophysiotrophic neurons, highlight recent developments in our understanding of nonsynaptic mechanisms regulating paraventricular cellular activity, including dendritic neuropeptide release, direct steroid feedback, cytokine cascades and gaseous neurotransmission, and illustrate the capacity for hypophysiotrophic, neurohypophysial and preautonomic paraventricular effector pathways to work together in control of glucocorticoid release. The current state of knowledge reveals the paraventricular nucleus to be a dynamic entity, capable of integrating diverse classes of signals into control of adrenocortical activation.     

Sex-dependent and differential responses to acute restraint stress of corticotropin-releasing factor-producing neurons in the rat paraventricular nucleus, central amygdala, and bed nucleus of the stria terminalis

Male and female rodents respond differently to acute stress. We tested our hypothesis that this sex difference is based on differences in stress sensitivity of forebrain areas, by determining possible effects of a single acute psychogenic stressor (1-hr restraint stress) on neuronal gene expression (c-Fos and FosB immunoreactivities), storage of corticotropin-releasing factor (CRF) immunoreactivity, and CRF production (CRF mRNA in situ hybridization) as well as the expression of genes associated with epigenetic processes (quantitative RT-PCR) in the rat paraventricular nucleus (PVN), the oval and fusiform subdivisions of the bed nucleus of the stria terminalis (BSTov and BSTfu, respectively), and the central amygdala (CeA), in both males and females. Compared with females, male rats responded to the stressor with a stronger rise in corticosterone titer and a stronger increase in neuronal contents of c-Fos, CRF mRNA, and CREB-binding protein mRNA in the PVN. In the BSTov, females but not males showed an increase in c-Fos, whereas the CRF mRNA content was increased in males only. In the BSTfu, males and females showed similar stress-induced increases in c-Fos and FosB, whereas in the CeA, both sexes revealed similar increases in c-Fos and in CRF mRNA. We conclude that male and female rats differ in their reactivity to acute stress with respect to possibly epigenetically mediated (particularly in the PVN) neuronal gene expression and neuropeptide dynamics (PVN and BSTov) and that this difference may contribute to the sex dependence of the animal's physiological and behavioral responses to an acute stressor.