Augmented hypothalamic corticotrophin-releasing hormone mRNA and corticosterone responses to stress in adult rats exposed to perinatal hypoxia.

Stressful events before or just after parturition alter the subsequent phenotypical response to stress in a general process termed programming. Hypoxia during the period before and during parturition, and in the postnatal period, is one of the most common causes of perinatal distress, morbidity, and mortality. We have found that perinatal hypoxia (prenatal day 19 to postnatal day 14) augmented the corticosterone response to stress and increased basal corticotrophin-releasing hormone (CRH) mRNA levels in the parvocellular portion of the paraventricular nucleus (PVN) in 6-month-old rats. There was no effect on the levels of hypothalamic parvocellular PVN vasopressin mRNA, anterior pituitary pro-opiomelanocortin or CRH receptor-1 mRNA, or hippocampus glucocorticoid receptor mRNA. We conclude that hypoxia spanning the period just before and for several weeks after parturition programmes the hypothalamic-pituitary-adrenal axis to hyper-respond to acute stress in adulthood, probably as a result of drive from the parvocellular CRH neurones.

Abused drugs modulate RGS4 mRNA levels in rat brain: comparison between acute drug treatment and a drug challenge after chronic treatment.

While different classes of abused drugs interact with distinct signaling substrates, it appears that all utilize receptors in the mesolimbic dopamine system to mediate their reinforcing effects. The regulator of G-protein signaling (RGS) proteins modulate G-protein coupled receptor (GPCR) signaling by increasing the rate of GTP hydrolysis of G proteins. This study was undertaken to determine whether morphine, cocaine, or amphetamine would modulate RGS4 mRNA levels in relevant brain regions. Acute administration of morphine and cocaine decreased levels of RGS4 mRNA in the reticulotegmental pontine nucleus (RtTg) and locus coeruleus (LC). Increases in RGS 4 mRNA levels were observed in the nucleus accumbens (NAc) and dorsal central gray (CGD). Acute drug challenge after chronic drug administration increased RGS4 mRNA in the CGD and decreased RGS4 levels in the red nucleus and RtTg. Interestingly, the LC exhibited biphasic modulation, with decreased RGS4 mRNA levels after acute administration and increased levels after chronic administration. These findings indicate that RGS4 mRNA levels are modulated in a similar manner by different drugs of abuse and imply that a common substrate could mediate some effects of abused drugs.

Increased mRNA expression for the alpha(1) subunit of the GABA(A) receptor following nitrous oxide exposure in mice.

The mechanisms by which nitrous oxide (N(2)O) produces physical dependence and withdrawal seizures are not well understood, but both N(2)O and ethanol exert some of their effects via the GABA(A) receptor and several lines of evidence indicate that withdrawal from N(2)O and ethanol may be produced through similar mechanisms. Expression levels of mRNA transcripts encoding several GABA(A) receptor subunits change with chronic ethanol exposure and, therefore, we hypothesized that N(2)O exposure would produce changes in mRNA expression for the alpha(1) subunit. Male, Swiss--Webster mice, 10--12 weeks of age, were exposed for 48 h to either room air or a 75%:25% N(2)O:O(2) environment. Brains were sectioned and mRNA for the alpha(1) subunit was detected by in situ hybridization using an 35S-labelled cRNA probe. N(2)O exposure produced a significant increase in expression levels of the alpha(1) subunit mRNA in the cingulate cortex, the CA1/2 region of the hippocampus, the dentate gyrus, the subiculum, the medial septum, and the ventral tegmental area. These results lend support to the hypothesis that N(2)O effects are produced, at least in part, through the GABA(A) receptor and that N(2)O produces these effects through actions in the cingulate cortex, hippocampus, ventral tegmental area and medial septum. These results are also further evidence that ethanol and N(2)O produce dependence and withdrawal through common mechanisms.

GABA(A) receptor subunit expression within hypophysiotropic CRH neurons: a dual hybridization histochemical study.

Dual hybridization histochemical studies were conducted to investigate the extent of colocalization of mRNA transcripts encoding the alpha1-2 and beta1-3 subunits of the gamma aminobutyric acid (GABA)(A) receptor with those for corticotropin-releasing hormone (CRH) within the rat hypothalamic paraventricular nucleus (PVN). A vast majority of CRH neurons (>94.5%) were found to express transcripts specific for the the alpha2, beta1 and beta3 subunits; mRNAs for the alpha1 and beta2 subunits of the GABA(A) receptor were detected within 53.3% and 65.7% of PVN CRH neurons, respectively. The results may have important implications for studies aimed at understanding GABAergic influences upon the hypothalamic-pituitary-adrenocortical (HPA) axis. Hypophysiotropic CRH neurons serve as the origin of the final common pathway for glucocorticoid secretion in response to stressful stimuli, and GABAergic afferents have been implicated in afferent control of these neurons. The subunit composition of GABA(A) receptors at this key regulatory locus may affect the efficacy of a major inhibitory input, and thus the magnitude and/or duration of stress-induced glucocorticoid secretion. The present findings reveal basal expression patterns of transcripts encoding several subunits of the GABA(A) receptor within stress-integrative CRH neurons, data which may be used to guide regulatory studies of GABAergic influences on the HPA axis under a variety of conditions.

Chronic stress regulates levels of mRNA transcripts encoding beta subunits of the GABA(A) receptor in the rat stress axis.

Semi-quantitative hybridization histochemical analyses were undertaken to determine expression levels of mRNA transcripts encoding the beta1-3 subunits of the GABA(A)receptor within the rat hypothalamic paraventricular nucleus (PVN) and hippocampal formation following exposure to a chronic non-habituating stress protocol. After delivery of a battery of stressors on a randomized schedule over a 3-week period, expression levels of the beta1 subunit of the GABA(A) receptor were found to be decreased in the medial parvocellular PVN (mpPVN) by 48.3% relative to control animals. Levels of beta2 mRNA following chronic stress were also found to be decreased in the mpPVN (29.8%), but increased in hippocampal subfields CA(1) and CA(3) (33.9 and 23.2%, respectively) and increased (24%) in the dentate gyrus. The results suggest that GABA(A) receptor subunit composition may be altered at a key regulatory site, and may have important implications for studies aimed at understanding GABAergic inhibitory influences upon the hypothalamic-pituitary-adrenocortical (HPA) axis. Hypophysiotropic CRH neurons serve as the origin of the final common pathway for glucocorticoid secretion in response to stressful stimuli, and GABAergic afferents have been implicated in afferent control of these neurons. Regulation of GABA(A) receptors at these sites may alter the efficacy of a major inhibitory influence upon the stress axis, and thereby modulate stress-induced glucocorticoid secretion.

Region-specific regulation of glutamic acid decarboxylase (GAD) mRNA expression in central stress circuits.

Neurocircuit inhibition of hypothalamic paraventricular nucleus (PVN) neurons controlling hypothalamo-pituitary-adrenocortical (HPA) activity prominently involves GABAergic cell groups of the hypothalamus and basal forebrain. In the present study, stress responsiveness of GABAergic regions implicated in HPA inhibition was assessed by in situ hybridization, using probes recognizing the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD65 and GAD67 isoforms). Acute restraint preferentially increased GAD67 mRNA expression in several stress-relevant brain regions, including the arcuate nucleus, dorsomedial hypothalamic nucleus, medial preoptic area, bed nucleus of the stria terminalis (BST) and hippocampus (CA1 and dentate gyrus). In all cases GAD67 mRNA peaked at 1 hr after stress and returned to unstimulated levels by 2 hr. GAD65 mRNA upregulation was only observed in the BST and dentate gyrus. In contrast, chronic intermittent stress increased GAD65 mRNA in the anterior hypothalamic area, dorsomedial nucleus, medial preoptic area, suprachiasmatic nucleus, anterior BST, perifornical nucleus, and periparaventricular nucleus region. GAD67 mRNA increases were only observed in the medial preoptic area, anterior BST, and hippocampus. Acute and chronic stress did not affect GAD65 or GAD67 mRNA expression in the caudate nucleus, reticular thalamus, or parietal cortex. Overall, the results indicate preferential upregulation of GAD in central circuitry responsible for direct (hypothalamus, BST) or multisynaptic (hippocampus) control of HPA activity. The distinct patterns of GAD65 and GAD67 by acute versus chronic stress suggest stimulus duration-dependent control of GAD biosynthesis. Chronic stress-induced increases in GAD65 mRNA expression predict enhanced availability of GAD65 apoenzyme after prolonged stimulation, whereas acute stress-specific GAD67 upregulation is consistent with de novo synthesis of active enzyme by discrete stressful stimuli.

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.

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.

Neurocircuitry of stress: central control of the hypothalamo-pituitary-adrenocortical axis.

Integration of the hypothalamo-pituitary-adrenal stress response occurs by way of interactions between stress-sensitive brain circuitry and neuroendocrine neurons of the hypothalamic paraventricular nucleus (PVN). Stressors involving an immediate physiologic threat ('systemic' stressors) are relayed directly to the PVN, probably via brainstem catecholaminergic projections. By contrast, stressors requiring interpretation by higher brain structures ('processive' stressors) appear to be channeled through limbic forebrain circuits. Forebrain limbic sites connect with the PVN via interactions with GABA-containing neurons in the bed nucleus of the stria terminalis, preoptic area and hypothalamus. Thus, final elaboration of processive stress responses is likely to involve modulation of PVN GABAergic tone. The functional and neuroanatomical data obtained suggest that disease processes involving inappropriate stress control involve dysfunction of processive stress pathways.

Direct catecholaminergic-cholinergic interactions in the basal forebrain. I. Dopamine-beta-hydroxylase- and tyrosine hydroxylase input to cholinergic neurons.

Immunocytochemical double-labeling techniques were used at the light and electron microscopic levels to investigate whether dopamine-beta-hydroxylase and tyrosine hydroxylase-containing axons contact basal forebrain cholinergic neurons. Dopamine-beta-hydroxylase- and tyrosine hydroxylase-positive fibers and terminals were found in close proximity to cholinergic neurons throughout extensive basal forebrain areas, including the vertical and horizontal limb of the diagonal band nuclei, the sublenticular substantia innominata, bed nucleus of the stria terminalis, ventral pallidum, and ventrolateral globus pallidus. Cholinergic cells in some aspects of the globus pallidus appeared to be contacted by tyrosine hydroxylase-positive but not dopamine-beta-hydroxylase-positive fibers, suggesting dopaminergic input to cholinergic neurons in these regions. Direct evidence for the termination of dopamine-beta-hydroxylase and tyrosine hydroxylase-positive fibers on cholinergic neurons was obtained in electron microscopic double-immunolabeling studies. Using high magnification light microscopic screening, both qualitative and quantitative differences were noted in the catecholaminergic innervation of forebrain cholinergic neurons. For example, while many cholinergic neurons were in close proximity to single dopamine-beta-hydroxylase-positive varicosities, others, particularly those located in the substantia innominatabed nucleus of the stria terminalis continuum, were apparently contacted by labeled fibers in repetitive fashion. The findings of the present study, together with our preliminary biochemical experiments (Zaborszky et al. [1993] Prog. Brain Res. 98:31-49) suggest that catecholaminergic afferents can differentially modulate forebrain cholinergic neurons. Such interactions may be important in learning and memory processes, and their perturbations may contribute to the cognitive decline seen in aging and in disorders such as Alzheimer's and Parkinson's diseases.

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.

Neuronal circuit regulation of the hypothalamo-pituitary-adrenocortical stress axis.

The hypothalamo-pituitary-adrenocortical (HPA) axis is the primary modulator of the adrenal glucocorticoid stress response. Activation of this axis occurs by way of a discrete set of neurons in the hypothalamic paraventricular nucleus (PVN). The PVN neuron appears to be affected by multiple sources, including (1) brainstem aminergic/peptidergic afferents; (2) blood-borne information; (3) indirect input from limbic system-associated regions, including the prefrontal cortex, hippocampus, and amygdala; and (4) local-circuit interactions with the preoptic-hypothalamic continuum. Analysis of the literature suggests that different classes of stressor employ different stress circuits. Severe physiologic ("systemic") stress appears to trigger brainstem/circumventricular organ systems that project directly to the paraventricular nucleus. In contrast, stressors requiring interpretation with respect to previous experience ("processive" stressors) reach the PVN by way of multisynaptic limbic pathways. Limbic regions mediating processive stress responses appear to have bisynaptic connections with the PVN, forming intervening connections with preoptic/hypothalamic GABAergic neurons. Stressors of the latter category may thus require interaction with homeostatic information prior to promoting an HPA response. The HPA stress response thus appears to be a product of both the physiologic importance of the stimulus and the specific pathways a given stimulus excites.

The effect of adrenalectomy on stress-induced c-fos mRNA expression in the rat brain.

Previously, we determined the pattern of stress-induced c-fos mRNA expression throughout the brain in order to gain further insight into the identification of the neural circuits mediating stress-induced regulation of the hypothalamic-pituitary-adrenal axis. In the present study, we determined if rapid effects of increased glucocorticoid levels after stress contribute to changes in c-fos mRNA expression. To this end, stress-induced c-fos expression was characterized in adrenalectomized (ADX) or adrenalectomized and corticosterone replaced (ADX/B) male rats. Animals were sacrificed 30 min post-onset of a 10 min swim stress, and in situ hybridization histochemistry was used to detect c-fos mRNA throughout the brain. The pattern of c-fos induction in the ADX and ADX/B animals was similar to that observed in the sham operated animals. Additionally, densitometric measurements were made to quantify the c-fos response in the paraventricular nucleus of the hypothalamus and the CA1/2 region of the hippocampus. We found that ADX did not alter the magnitude of the c-fos response to stress in these areas, but there was a slight dampening of the response in ADX/B animals. In sum, these results suggest that the pattern of c-fos expression observed 30 min post-stress is independent of stress-induced increases in circulating glucocorticoid concentrations.

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.

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.

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.

Ventral subicular interaction with the hypothalamic paraventricular nucleus: evidence for a relay in the bed nucleus of the stria terminalis.

The axonal projections of the ventral subiculum to the bed nucleus of the stria terminalis (BST) were examined in the rat with the anterograde neuronal tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Axons originating in the ventral subiculum coursed to the BST through either the fimbria-fornix, or a pathway involving the stria terminalis via the amygdala. Ventral subicular axons gave rise to dense terminal networks that were preferentially distributed in medial and ventral subregions of the BST. The distribution of subicular fibers and terminals was examined in relation to BST neurons that project to the hypothalamic paraventricular nucleus (PVN). In these cases, discrete iontophoretic injections of the retrograde tracer Fluoro-gold were made in the PVN, with PHA-L delivered to the ipsilateral ventral subiculum. An immunocytochemical double-labeling protocol was then employed for the simultaneous detection of PHA-L and Fluoro-gold, and provided light microscopic evidence for subicular input to PVN-projecting cells located within the BST. In a second series of experiments, the gamma-amino butyric acid (GABA)ergic nature of the BST was examined by in situ hybridization histochemistry for detection of transcripts encoding GAD67 mRNA. The studies revealed that a high proportion of BST neurons express GAD67 transcripts. Also, experiments combining Fluoro-gold tracing with GAD67 in situ hybridization suggested that a proportion of PVN-projecting neurons in the BST are GABAergic. Taken together, the results of these sets of studies suggest that the inhibitory influences of the hippocampus on the PVN might be relayed through specific portions of the BST. These findings may have important implications for our understanding of the neural regulation of the hypothalamic-pituitary-adrenal axis.

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.

Region specific expression of furin mRNA in the rat brain.

The distribution of furin mRNA was examined in the rat central nervous system. Northern blot analysis reveals the presence of a 4.4 kb band in all brain tissues examined. In situ hybridization analysis of frozen rat brain sections using a radioactively labeled antisense cRNA probe to rat furin demonstrated moderate to low levels of expression in both neuronal and non-neuronal tissue in all areas examined. Interestingly, higher levels of furin were expressed in selective regions which include the ventricles (the choroid plexus and ependymal cells), the islands of Calleja, the hippocampus and the pineal gland. the ubiquitous localization of furin in the brain is consistent with its postulated role as a vital convertase important in the processing of proproteins negotiating the constitutive pathway of secretion. However, the higher expression of furin mRNA in distinct brain areas suggests a more active role in the processing of proproteins synthesized in these tissues.