William E. Cullinan

Pattern and time course of immediate early gene expression in rat brain following acute stress

The pattern and time course of brain activation in response to acute swim and restraint stress were examined in the rat by in situ hybridization using complementary RNA probes specific for transcripts encoding the products of the immediate early genes c-fos, c-jun and zif/268. A widespread pattern of c-fos messenger RNA expression was detected in response to these stressors; surprisingly, the expression patterns were substantially similar following both swim and restraint stress. A dramatic induction of c-fos messenger RNA was observed in numerous neo- and allocortical regions, the lateral septal nucleus, the hypothalamic paraventricular and dorsomedial nuclei, the anterior hypothalamic area, the lateral portion of the retrochiasmatic area, the medial and cortical amygdaloid nuclei, the periaqueductal gray, and the locus coeruleus; however, a prominent induction of c-fos was also seen in numerous additional subcortical and brainstem regions. Although not as widely expressed in response to stress as c-fos, induction of zif/268 messenger RNA was also detected throughout many brain areas; these regions were largely similar to those in which c-fos was induced, although in a number of regions zif/268 was expressed in regions devoid of c-fos messenger RNA. Few brain areas showed increased expression of c-jun following stress; these regions also showed induction of c-fos and/or zif/268. The time courses of expression of all three immediate early genes were similar, with peak levels observed at the 30 or 60 min time point, and a markedly reduced signal evident at 120 min post-stress. However, in a number of cases a delayed and/or prolonged induction was noted that may be indicative of secondary neuronal activation. A number of recent studies have attempted to define neural pathways which convey stress-related information to the hypothalamic-pituitary-adrenal axis. The present results reveal a widespread pattern of neuronal activation in response to acute swim or restraint stress. These findings may aid in the identification of stress-specific neural circuits and are thus likely to have important implications for our understanding of neuronal regulation of the stress response.

Elicitation and reduction of fear: behavioural and neuroendocrine indices and brain induction of the immediate-early gene c-fos

The elicitation and reduction of fear were indexed with fear-potentiated startle and corticosterone release and induction of the immediate-early gene c-fos as a marker of neural activity in male Sprague-Dawley rats. Conditioning consisted of pairing one stimulus with footshock, which was withheld when the conditioned stimulus was preceded by a different modality stimulus, the conditioned inhibitor. On the test day, approximately 60% of the rats were used for c-fos in situ hybridization, and were presented with either the conditioned stimulus alone, the conditioned inhibitor alone, a compound of the two stimuli, or no stimuli, and killed 30 min following the presentation of 10 such stimuli. The remaining rats were tested with the fear-potentiated startle paradigm. Rats displayed reliable fear-potentiated startle and corticosterone release to the conditioned stimulus, and both measures were reduced when the conditioned stimulus was preceded by the conditioned inhibitor. The ventral bed nucleus of the stria terminalis, septohypothalamic nucleus, some tegmental nuclei, and the locus coeruleus had particularly high c-fos induction in rats that received the conditioned inhibitor, providing one of the first functional indication that these nuclei might be important in behavioural or endocrine inhibition. Conditioning specific c-fos induction in the three groups that received a stimulus on the test day was observed in many hypothalamic areas, the medial geniculate body and the central gray, structures previously involved in fear and anxiety. The cingulate, infralimbic and perirhinal cortex, nucleus accumbens, lateral septum, dorsal endopiriform nucleus, and ventral tegmental area had higher c-fos induction in rats presented with the fearful conditioned stimulus, confirming previous studies. The amygdala and hippocampus of conditioned rats did not show higher c-fos induction than in rats repeatedly exposed to the context. Many regions displayed c-fos messenger RNA induction in the control condition, suggesting that processes other than fear and anxiety participate in c-fos induction.

Gene expression of prohormone and proprotein convertases in the rat CNS: a comparative in situ hybridization analysis

Posttranslational processing of proproteins and prohormones is an essential step in the formation of bioactive peptides, which is of particular importance in the nervous system. Following a long search for the enzymes responsible for protein precursor cleavage, a family of Kexin/subtilisin-like convertases known as PC1, PC2, and furin have recently been characterized in mammalian species. Their presence in endocrine and neuroendocrine tissues has been demonstrated. This study examines the mRNA distribution of these convertases in the rat CNS and compares their expression with the previously characterized processing enzymes carboxypeptidase E (CPE) and peptidylglycine alpha-amidating monooxygenase (PAM) using in situ hybridization histochemistry. Furin mRNA was ubiquitously distributed and detected both in neurons and non- neuronal tissue throughout the brain with a higher abundance in ependyma, the circumventricular organs, the islands of Calleja, hippocampus, and allocortex. The cellular localization of PC1 and PC2 was exclusively neuronal with highest concentrations in known neuropeptide-rich brain regions. In general, PC2 was more widely expressed than PC1 in the CNS, although many regional variations were detected. The identification of specific combinations of convertase expression together with CPE and PAM expression in neuropeptide-rich brain regions suggests that specific enzymatic pathways are involved in neuropeptide precursor processing, and that these specific combinations are responsible for region-specific differences of posttranslational processing.

Involvement of the bed nucleus of the stria terminalis in tonic regulation of paraventricular hypothalamic CRH and AVP mRNA expression.

The bed nucleus of the stria terminalis (BNST) occupies a central position in pathways regulating hypothalamo‐pituitary‐adrenocortical (HPA) stress regulation. The potential role of the BNST in tonic neural control of HPA function was assessed by examining effects of selective BNST lesions on expression of ACTH secretagogues in HPA‐integrative neurons of the medial parvocellular paraventricular nucleus. Anterior BNST lesions (ABN) involved major portions of the anteromedial, anterolateral, ventromedial, ventrolateral, dorsolateral and juxtacapsular subnuclei. These lesions resulted in significant (30%) decreases in corticotropin‐releasing hormone (CRH) mRNA expression across the rostrocaudal extent of the medial parvocellular PVN, with no accompanying changes in basal arginine vasopressin (AVP) mRNA levels. Posterior BNST (PBN) lesions involved large but subtotal damage to the posterior intermediate, posterior medial, posterior lateral and preoptic subnuclei; these lesions resulted in small but significant changes in CRH mRNA and slight increases in number of AVP mRNA‐producing parvocellular neurons. PBN effects on CRH mRNA expression were most pronounced at the caudal extent of the medial parvocellular zone, suggesting a topographic input from the posterior BNST to the PVN that is only partially compromised by PBN lesions. Analysis of individual cases revealed a correlation between damage of the anterolateral BNST and decreased CRH mRNA levels, and damage of the posterior intermediate and/or posterior medial BNST and increased CRH mRNA levels. The results suggest differential BNST input into HPA regulation, perhaps reflecting the diversity of limbic input into the BNST region.

Local circuit regulation of paraventricular nucleus stress integration Glutamate: GABA connections

Limbic neurocircuits play a central role in regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Limbic influences on adrenocortical hormone secretion are mediated by transynaptic activation or inhibition of hypophysiotrophic neurons in the medial parvocellular paraventricular nucleus (PVN). Projections from the ventral subiculum, prefrontal cortex, medial amygdala, lateral septum, paraventricular thalamus and suprachiasmatic nucleus (SN) terminate in the immediate surround of the PVN, an area heavily populated by GABAergic interneurons. As such, these regions are positioned to modulate paraventricular output via excitation or inhibition of interneuronal projections into the PVN. In addition, the same limbic and diencephalic regions have projections to local PVN-projecting hypothalamic and basal telencephalic nuclei, including the dorsomedial and medial preoptic nuclei and the bed nucleus of the stria terminalis. These regions are involved in both inhibitory and excitatory regulation of the stress axis, indicating that they contain heterogeneous neuronal populations whose relative impact on the PVN is determined by the nature of afferent stimuli. Thus, limbic modulation of the pituitary-adrenocortical system appears to be a multisynaptic process integrated at the level of local PVN-projecting neurocircuits. Local circuits are likely the primary integrators of anticipatory stress responses, and may indeed be the focus of HPA dysfunction seen with aging or affective disease.

Distribution of vesicular glutamate transporter mRNA in rat hypothalamus

Two isoforms of the vesicular glutamate transporter, VGLUT1 and VGLUT2, were recently cloned and biophysically characterized. Both VGLUT1 and VGLUT2 specifically transport glutamate into synaptic vesicles, making them definitive markers for neurons using glutamate as a neurotransmitter. The present study takes advantage of the specificity of the vesicular transporters to afford the first detailed map of putative glutamatergic neurons in the rat hypothalamus. In situ hybridization analysis was used to map hypothalamic distributions of VGLUT1 and VGLUT2 mRNAs. VGLUT2 is clearly the predominant vesicular transporter mRNA found in the hypothalamus; rich expression can be documented in regions regulating energy balance (ventromedial hypothalamus), neuroendocrine function (preoptic nuclei), autonomic tone (posterior hypothalamus), and behavioral/homeostatic integration (lateral hypothalamus, mammillary nuclei). Expression of VGLUT1 is decidedly more circumspect and is confined to relatively weak labeling in lateral hypothalamic regions, neuroendocrine nuclei, and the suprachiasmatic nucleus. Importantly, dual‐label analysis revealed no incidence of colocalization of VGLUT1 or VGLUT2 mRNAs in glutamic acid decarboxylase (GAD) 65‐positive neurons, indicating that GABA neurons do not express either transporter. Our data support a major role for hypothalamic glutamatergic neurons in regulation of all aspects of hypothalamic function. J. Comp. Neurol. 448:217–229, 2002.

Cortical input to the basal forebrain

The arborization pattern and postsynaptic targets of corticofugal axons in basal forebrain areas have been studied by the combination of anatomical tract-tracing and pre- and postembedding immunocytochemistry. The anterograde neuronal tracer Phaseolus vulgaris leucoagglutinin was iontophoretically delivered into different neocortical (frontal, parietal, occipital), allocortical (piriform) and mesocortical (insular, prefrontal) areas in rats. To identify the transmitter phenotype in pre- or postsynaptic elements, the tracer staining was combined with immunolabeling for either glutamate or GABA, or with immunolabeling for choline acetyltransferase or parvalbumin. Tracer injections into medial and ventral prefrontal areas gave rise to dense terminal arborizations in extended basal forebrain areas, particularly in the horizontal limb of the diagonal band and the region ventral to it. Terminals were also found to a lesser extent in the ventral part of the substantia innominata and in ventral pallidal areas adjoining ventral striatal territories. Similarly, labeled fibers from the piriform and insular cortices were found to reach lateral and ventral parts of the substantia innominata, where terminal varicosities were evident. In contrast, descending fibers from neocortical areas were smooth, devoid of terminal varicosities, and restricted to the myelinated fascicles of the internal capsule en route to more caudal targets. Ultrastructural studies obtained indicated that corticofugal axon terminals in the basal forebrain areas form synaptic contact primarily with dendritic spines or small dendritic branches (89%); the remaining axon terminals established synapses with dendritic shafts. All tracer labeled axon terminals were immunonegative for GABA, and in the cases investigated, were found to contain glutamate immunoreactivity. In material stained for the anterograde tracer and choline acetyltransferase, a total of 63 Phaseolus vulgaris leucoagglutinin varicosities closely associated with cholinergic profiles were selected for electron microscopic analysis. From this material, 37 varicosities were identified as establishing asymmetric synaptic contacts with neurons that were immunonegative for choline acetyltransferase, including spines and small dendrites (87%) or dendritic shafts (13%). Unequivocal evidence for synaptic interactions between tracer labeled terminals and cholinergic profiles could not be obtained in the remaining cases. From material stained for the anterograde tracer and parvalbumin, 40% of the labeled terminals investigated were found to establish synapses with parvalbumin-positive elements; these contacts were on dendritic shafts and were of the asymmetrical type. The present data suggest that corticofugal axons innervate forebrain neurons that are primarily inhibitory and non-cholinergic; local forebrain axonal arborizations of these cells may represent a mechanism by which prefrontal cortical areas control basal forebrain cholinergic neurons outside the traditional boundaries of pallidal areas.

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.

CONTRIBUTION OF THE VENTRAL SUBICULUM TO INHIBITORY REGULATION OF THE HYPOTHALAMO-PITUITARY-ADRENOCORTICAL AXIS

Anatomical studies indicate that the ventral subiculum is in a prime position to mediate hippocampal inhibition of the hypothalamo‐pituitary‐adrenocortical (HPA) axis. The present study evaluated this hypothesis by assessing HPA function following ibotenic acid lesion of the ventral subiculum region. Rats with lesions of the ventral subiculum (vSUB) or ventral hippocampus (vHIPPO) did not show changes in basal corticosterone (CORT) secretion at either circadian peak or nadir time points when compared to sham‐lesion rats (SHAM) or unoperated controls. However, rats with vSUB lesions exhibited a prolonged glucocorticoid stress response relative to all other groups. Baseline CRH mRNA levels were significantly increased in the medial parvocellular paraventricular nucleus (PVN) of the vSUB group relative to controls. CRH mRNA differences were particularly pronounced at caudal levels of the nucleus, suggesting topographic organization of vSUB interactions with PVN neurons. Notably, the vHIPPO group, which received large lesions of ventral CA1, CA3 and dentate gyrus without significant subicular damage, showed no change in stress‐induced CORT secretion, suggesting that the ventral subiculum proper is principally responsible for ventral hippocampal actions on the HPA stress response. No differences in medial parvocellular PVN AVP mRNA expression were seen in either the vSUB or vHIPPO groups. The results indicate a specific inhibitory action of the ventral subiculum on HPA activation. The increase in CRH biosynthesis and stress‐induced CORT secretion in the absence of changes in baseline CORT secretion or AVP mRNA expression suggests that the inhibitory actions of ventral subicular neurons affect the response capacity of the HPA axis.

Functional role of local GABAergic influences on the HPA axis

Neuronatomical and pharmacological studies have established GABA-mediated inhibition of the HPA axis at the level of the PVN. The origin of this innervation is a series of local hypothalamic and adjacent forebrain regions that project to stress-integrative hypophysiotropic CRH neurons. While a role in tonic inhibition of the stress axis is likely, this system of inhibitory loci is also capable of producing a dynamic braking capacity in the context of the neuroendocrine stress response. The latter function is mediated in large part by glutamatergic forebrain afferents that increase GABA release at the level of the PVN. In addition, this local GABA system can be inhibited by upstream GABAergic projection neurons, producing activation of the HPA axis via removal of GABAergic tone. This PVN projecting GABA network interfaces with a wide range of homeostatic mechanisms, and is capable of biochemical plasticity in response to chronic stress. Collectively, the elements of this system provide for exquisite control of neuroendocrine activation in the face of stressful stimuli, and loss of this regulatory capacity may underlie many stress-related disorders.