Brainstem prolactin-releasing peptide neurons are sensitive to stress and lactation.

Prolactin-releasing peptide (PrRP) was originally thought to participate in the control of adenohypophyseal prolactin secretion, but its predominant expression in a subset of medullary noradrenergic neurons is more in line with roles in interoceptive and/or somatosensory information processing. To better define functional contexts for this peptide system, immuno- and hybridization histochemical methods were used to monitor the capacity of PrRP neurons to display activational responses to lactation, suckling, acute footshock or hypotensive hemorrhage. PrRP mRNA signal was reduced in the medulla of lactating dams, relative to both male and diestrus female controls, with cell counts revealing 42% and 43% reductions in the number of positively hybridized cells in the nucleus of the solitary tract (NTS) and ventrolateral medulla, respectively. Lactating mothers killed after a 90 min suckling episode (following 4 h pup removal) failed to show induced Fos expression in identified medullary PrRP neurons, despite the fact that responsive neurons were detected in other aspects of the caudal NTS. By contrast, acute exposure to hypotensive (25%) hemorrhage or footshock each activated substantial complements of medullary neurons expressing PrRP mRNA. A substantially greater fraction of the total medullary PrRP population exhibited sensitivity to footshock than hemorrhage (71 versus 39%, respectively). These results suggest that medullary PrRP neurons are negatively regulated by (presumably hormonal) changes in lactation, and are not recruited to activation by suckling stimuli. These populations exhibit differential sensitivity to distinct acute stressors, and may participate in the modulation of adaptive neuroendocrine and autonomic responses to each.

Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor.

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors.

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)(+) RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 microg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.

Paradoxical activational effects of a corticotropin-releasing factor-binding protein "ligand inhibitor" in rat brain.

The corticotropin-releasing factor-binding protein is distinct from known corticotropin-releasing factor receptors, but can bind the peptide and neutralize its biological actions. Recent interest has centered about the therapeutic potential of "ligand inhibitors" of binding protein action, synthetic corticotropin-releasing factor fragments which are inactive at corticotropin-releasing factor receptors, but can displace the peptide from the binding protein, thereby increasing levels of free corticotropin-releasing factor. To identify sites of action of such ligands, the distribution of Fos expression seen following intracerebroventricular administration of rat/human corticotropin-releasing factor(6-33) (5-50 microg) was charted in relation to corticotropin-releasing factor-binding protein and receptor expression. It was expected that Fos induction would mimic aspects of the distribution of the two known corticotropin-releasing factor receptors, but the far greater correspondence was seen with that of the binding protein itself. This included neurons in the isocortex, the olfactory system, amygdala and a number of discrete brainstem cell groups; many Fos-immunoreactive neurons in each were found to co-express corticotropin-releasing factor-binding protein messenger RNA. Subsets of activated neurons co-expressed Type 1 corticotropin-releasing factor receptor messenger RNA, though these were largely limited to cell groups that also express the corticotropin-releasing factor-binding protein, and where binding protein immunoreactivity and Type 1 receptor transcripts were found to co-exist. Responsive neurons displaying Type 2 corticotropin-releasing factor receptor message were seen reliably only in the lateral septal nucleus. These findings support only a limited capacity of the ligand inhibitor to activate neurons bearing corticotropin-releasing factor receptors. The more pervasive activation seen among neurons that express the corticotropin-releasing factor-binding protein may be indicative of an unexpected role for this protein in signaling by corticotropin-releasing factor-related peptides.

Effects of selective sinoaortic denervations on phenylephrine-induced activational responses in the nucleus of the solitary tract.

Intravenous administration of phenylephrine provokes a pattern of cellular activation in the nucleus of the solitary tract that resembles the central distributions of primary baroreceptor afferents supplied by the carotid sinus and aortic depressor nerves. Transganglionic transport and denervation methods were used in an experimental setting to test the dependence of phenylephrine-induced Fos immunoreactivity on the integrity of buffer nerve afferents, and to identify the subregions of the nucleus of the solitary tract supplied by each. Cholera toxin B-horseradish peroxidase injections into either or both nerves revealed terminal labeling concentrated in, but not restricted to, the dorsal commissural part of the nucleus of the solitary tract at the level of the apex of calamus scriptorius, and extending into the dorsal subnucleus at the level of the area postrema. Preferential ramifications of carotid sinus and aortic depressor nerve afferents at the levels of the commissural part of the nucleus and the area postrema, respectively, were reflected in the extent to which labeled fibers comingled with neurons exhibiting phenylephrine-induced Fos in dual labeling experiments. Complete sinoaortic denervation reduced by 90% the number of neurons exhibiting drug-induced Fos expression. Selective carotid and aortic sinus denervations effected partial reductions manifest preferentially in the caudal and rostral foci of the distribution, respectively. Reduced activational responses at the level of the area postrema of aortic sinus-denervated rats were accompanied by a reduction in cellular nicotinamide adenine dinucleotide phosphate-diaphorase activity in this region. Animals killed 30 days after complete sinoaortic denervation displayed no evidence of recovery of phenylephrine-induced Fos, while the strength and distribution of the response in rats that received selective carotid sinus denervation were indistinguishable from those seen in controls. These findings (i) support the dependence of phenylephrine-induced Fos expression on the integrity of carotid sinus and aortic depressor nerve afferents, (ii) provide anatomical and functional evidence that the two buffer nerves distribute differentially within the nucleus of the solitary tract, and (iii) implicate central reorganization as a likely basis for functional recovery of baroreflex mechanisms following partial sinoaortic denervation.

Glucocorticoid negative feedback selectively targets vasopressin transcription in parvocellular neurosecretory neurons.

To identify molecular targets of corticosteroid negative feedback effects on neurosecretory neurons comprising the central limb of the hypothalamo-pituitary-adrenal (HPA) axis, we monitored ether stress effects on corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) heteronuclear RNA (hnRNA) expression in rats that were intact or adrenalectomized (ADX) and replaced with corticosterone (B) at constant levels ranging from nil to peak stress concentrations. Under basal conditions, relative levels of both primary transcripts varied inversely as a function of plasma B titers. In response to stress, the kinetics of CRF hnRNA responses of intact and ADX rats replaced with low B were similar, peaking at 5 min after stress. By contrast, intact rats showed a delayed AVP hnRNA response (peak at 2 hr), the timing of which was markedly advanced in ADX/low B-replaced animals (peak at 5-30 min). Transcription factors implicated in these responses responded similarly. Manipulation of B status did not affect the early (5-15 min) phosphorylation of transcription factor cAMP-response element-binding protein (CREB) but accelerated maximal Fos induction from 2 hr after stress (intact) to 1 hr (ADX). Assays of binding by proteins in hypothalamic extracts of similarly manipulated rats toward consensus CRE and AP-1 response elements supported a role for the stress-induced plasma B increment in antagonizing AP-1, but not CRE, binding. These findings suggest that glucocorticoid negative feedback at the transcriptional levels is exerted selectively on AVP gene expression through a mechanism that likely involves glucocorticoid receptor interactions with immediate-early gene products.

Do centrally administered neuropeptides access cognate receptors?: an analysis in the central corticotropin-releasing factor system.

To determine the extent to which centrally administered corticotropin-releasing factor (CRF) activates neurons that express CRF receptors (CRF-Rs), we followed the kinetics and distribution (relative to those of CRF-Rs) of Fos induction seen in response to intracerebroventricular (icv) injection of the peptide (1-10 microg). CRF provoked widespread Fos expression: its strength was dose-related, it peaked at 2 hr after injection, and it was antagonized in a dose-dependent manner by coinjection of CRF-R antagonists. The activation pattern closely mimicked the distribution of CRF-R1 mRNA, in including widespread Fos induction throughout the cortical mantle, in cell groups involved in sensory information processing, and in the cerebellum and several of its major afferents and targets. Dual labeling revealed extensive correspondence of CRF-stimulated Fos-immunoreactivity (Fos-ir) and CRF-R1 mRNA at these and other loci. Unique sites of CRF-R2 expression were relatively unresponsive to CRF but were more so after icv administration of urocortin (UCN), a new mammalian CRF-related peptide. Both CRF and UCN elicited activational responses in cell groups that are involved in central autonomic control but that express neither CRF-R, including the central amygdaloid and paraventricular hypothalamic nuclei, and brainstem catecholaminergic cell groups. The results support an ability of CRF-related peptides in the ventricular system to access receptor-expressing cells directly but leave open questions as to the basis for the recruitment of central autonomic structures, many of which have been identified as stress-related sites of CRF action.

Ultrastructural localization of the corticotropin-releasing factor-binding protein in rat brain and pituitary.

Preembedding immunoperoxidase staining methods were used to permit ultrastructural analyses of the distribution in rat brain and pituitary of the corticotropin-releasing factor-binding protein (CRF-BP), a moiety distinct from CRF receptors, but which is nonetheless capable of binding the peptide and reversibly neutralizing its biological actions. In anterior pituitary, CRF-BP immunoreactivity (ir) was detected in corticotropelike cells, with reaction product associated principally with secondary lysosomes and multivesicular bodies and not at all with secretory granules. In brain, marked regional differences in the subcellular pattern of CRF-BP staining were evident. In isocortex, where BP/peptide colocalization is rare, BP-ir was distributed in cells and processes in a manner similar to that of a prototypic neuropeptide, including in terminals commonly engaging in synaptic contacts with unlabeled dendritic profiles. In the bed nucleus of the stria terminalis, a site that contains overlapping accumulations of CRF-BP-ir projections and CRF-ir perikarya, BP staining was restricted to vesicle-laden varicosities that rarely engaged in synaptic contacts with somatic or dendritic elements but were frequently apposed to unlabeled axon varicosities and terminals. In the ventromedial medulla, a site of partial CRF/BP overlap, most cells displayed a subcellular localization CRF-BP-ir like that seen in cortex, whereas in others the distribution shared similarities with that observed in pituitary. The results suggest that the function of the CRF-BP may differ in different cellular contexts. In cellular targets of CRF or in neurons in which peptide and BP coexist, the CRF-BP may play a role in processing and degradation of CRF and/or ligand-receptor complexes. In other areas of the central nervous system, the BP seems positioned to serve as a transmitter/modulator at conventional synapses or as an autocrine or paracrine modulator of local CRF effects.

Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development.

Corticotropin releasing factor (CRF) is a major integrator of adaptive responses to stress. Two biochemically and pharmacologically distinct CRF receptor subtypes (CRFR1 and CRFR2) have been described. We have generated mice null for the CRFR1 gene to elucidate the specific developmental and physiological roles of CRF receptor mediated pathways. Behavioral analyses revealed that mice lacking CRFR1 displayed markedly reduced anxiety. Mutant mice also failed to exhibit the characteristic hormonal response to stress due to a disruption of the hypothalamic-pituitary-adrenal (HPA) axis. Homozygous mutant mice derived from crossing heterozygotes displayed low plasma corticosterone concentrations resulting from a marked agenesis of the zona fasciculata region of the adrenal gland. The offspring from homozygote crosses died within 48 hr after birth due to a pronounced lung dysplasia. The adrenal agenesis in mutant animals was attributed to insufficient adrenocorticotropic hormone (ACTH) production during the neonatal period and was rescued by ACTH replacement. These results suggest that CRFR1 plays an important role both in the development of a functional HPA axis and in mediating behavioral changes associated with anxiety.

Hypothalamic effector neurons and extended circuitries activated in "neurogenic" stress: a comparison of footshock effects exerted acutely, chronically, and in animals with controlled glucocorticoid levels.

Immunolocalization of Fos protein was used to identify and characterize hypothalamic visceromotor populations responsive to acute and chronic intermittent footshock stress, and candidate afferent mediators of hypothalamic effects. Exposure to a single 30 minute footshock session induced maximal Fos expression in the paraventricular hypothalamic nucleus (PVH) 2 hours after the challenge; activated cells corresponded principally to hypophysiotropic neurons expressing corticotropin-releasing factor, with secondary involvement of magnocellular oxytocinergic and autonomic-related projection neurons. Extrahypothalamic cell groups activated in response to acute footshock included ones associated with the processing or modulation of somatosensory/nociceptive inputs, the limbic region of the telencephalon, and visceral sensory mechanisms. Rats with constant corticosterone levels displayed enhanced footshock-induced Fos expression in the parvicellular compartment of the PVH, as well as in certain limbic and somatosensory cell groups, the locus coeruleus, but not in medullary catecholaminergic cell groups. Animals subjected to chronic intermittent stress (2 sessions/day for 7 days) showed only modest evidence of habituation of cellular activation responses in the PVH and most extrahypothalamic regions. Rats bearing retrograde tracer deposits in the PVH and killed 2 hours after acute footshock displayed Fos-positive retrogradely labeled neurons principally in medullary catecholaminergic cell groups, with secondary foci in the hypothalamus, limbic region, and pontine tegmentum. This characterization of footshock-responsive systems identifies cell groups that are in a position to (1) mediate acute stress effects on hypothalamic visceromotor neurons, (2) comprise targets for corticosteroid negative feedback effects, and/or (3) underlie habituation of the neuroendocrine limb of the stress response.

Organization and transmitter specificity of medullary neurons activated by sustained hypertension: implications for understanding baroreceptor reflex circuitry.

In situ expression of c-fos observed in response to phenylephrine (PE)-induced hypertension provided a basis for characterizing the organization and neurotransmitter specificity of neurons at nodal points of medullary baroreflex circuitry. Sustained hypertension induced by a moderate dose of PE provoked patterns of c-fos mRNA and protein expression that conformed in the nucleus of the solitary tract (NTS) to the termination patterns of primary baroreceptor afferents and in the caudal ventrolateral medulla (CVLM) to a physiologically defined depressor region. A majority of barosensitive CVLM neurons concurrently displayed markers for the GABAergic phenotype; few were glycinergic. Phenylephrine-sensitive GABAergic neurons that were retrogradely labeled after tracer deposits in pressor sites of the rostral ventrolateral medulla (RVLM) occupied a zone extending approximately 1.4 mm rostrally from the level of the calamus scriptorius, intermingled partly with catecholaminergic neurons of the A1 and C1 cell groups. By contrast, barosensitive neurons of the NTS were found to be phenotypically complex, with very few projecting directly to the RVLM. Extensive colocalization of PE-induced Fos-IR and markers for the nitric oxide phenotype were seen in a circumscribed, rostral, portion of the baroreceptor afferent zone of the NTS, whereas only a small proportion of PE-sensitive neurons in the NTS were found to be GABAergic. PE treatment parameters have been identified that provide a basis for defining and characterizing populations of neurons at the first station in the central processing of primary baroreceptor input and at a key inhibitory relay in the CVLM.

Differential time- and dose-related effects of haemorrhage on tyrosine hydroxylase and neuropeptide Y mRNA expression in medullary catecholamine neurons.

Hypotensive haemorrhage induces nuclear Fos expression and upregulates tyrosine hydroxylase (TH) mRNA in catecholamine-containing cell groups of the rat medulla oblongata. To shed light on the significance of the coexistence of neuropeptide Y (NPY) in aminergic neurons, the impact of graded levels of haemorrhage on temporal changes in the expression of TH and NPY mRNAs was compared; concurrent staining for Fos permitted comparisons between cells that ostensibly were and were not targeted by the stimulus. A 15% haemorrhage provoked increased NPY expression in all medullary catecholamine cell groups except the A2; these changes were detected predominantly in Fos-immunoreactive neurons (Fos-ir) at later (2-4 h) time points. Upregulation of TH and NPY mRNAs in Fos-ir neurons followed distinct time courses, with NPY responses peaking more rapidly, particularly in the C1 and C2 cell groups. Adrenergic cell groups displayed greater maximal increases in NPY expression than the A1 noradrenergic cell group while the converse was true of TH mRNA response. Increasing the severity of haemorrhage resulted in more pronounced increases in both mRNA responses in each aminergic region. These findings indicate that haemorrhage differentially affects TH and NPY expression in medullary catecholamine cell groups that participate in the maintenance of cardiovascular homeostasis. The differential nature of these responses suggests them not to be a simple consequence of metabolic alterations pursuant to increased synaptic activity. The prompt and robust NPY mRNA responses in adrenergic neurons suggests a mechanism by which peptide content of these cell groups' terminal projections is defended.

Evidence for an intramedullary prostaglandin-dependent mechanism in the activation of stress-related neuroendocrine circuitry by intravenous interleukin-1.

We have provided evidence that the stimulatory effects of intravenous interleukin-1 (IL-1) on neurosecretory neurons in the paraventricular nucleus (PVH) that express corticotropin-releasing factor (CRF) depend specifically on the integrity of catecholaminergic projections originating in caudal medulla. Here we report on experiments designed to test alternative means by which circulating IL-1 might access medullary aminergic neurons, including mechanisms involving sensory components of the vagus, the area postrema, or perivascular cells bearing IL-1 receptors. Neither abdominal vagotomy nor area postrema lesions reliably altered Fos expression induced in the medulla or PVH in response to a moderately suprathreshold dose of IL-1beta. Cytokine-stimulated increases in CRF mRNA in the PVH were also unaffected by either ablation. By contrast, systemic administration of the cyclooxygenase inhibitor indomethacin resulted in parallel dose-related attenuations of IL-1 effects in hypothalamus and medulla. Microinjections of prostaglandin E2 (PGE2; >/=10 ng) in rostral ventrolateral medulla, the principal seat of IL-1-sensitive neurons that project to the PVH, provoked discrete patterns of cellular activation in hypothalamus and medulla that mimicked those seen in response to intravenous IL-1. We interpret these findings as supporting the hypothesis that paracrine effects of PGE2 released from perivascular cells in the medulla as a consequence of IL-1 stimulation and, acting through prostanoid receptors on or near local aminergic neurons that project to the PVH, contribute to the stimulatory effects of increased circulating IL-1 on neurons constituting the central limb of the hypothalamo-pituitary-adrenal axis.

Hypophysiotropic CRF neurons display a sustained immediate-early gene response to chronic stress but not to adrenalectomy.

Immediate-early genes (IEGs) are now widely used to identify neurons acutely activated by extracellular stimuli. Though challenge-induced IEG expression is typically transient, examples of sustained elevation have been reported, including in hypothalamic magnocellular neurosecretory neurons of osmotically challenged rats. Another chronic stimulus, adrenalectomy (ADX), targets parvocellular neurosecretory neurons in the paraventricular nucleus of the hypothalamus (PVH), resulting in a persistent elevation in the synthesis and secretion of corticotropin-releasing factor (CRF). In the present study we used hybridization histochemical methods to compare the effects of ADX on the induction over time of two independent IEG markers, c-fos and NGFI-B, in hypophysiotropic CRF neurons. The induction of both c-fos and NGFI-B mRNA was greatest 3 to 6 after ADX, diminished by 12 h, and no longer detectable at 24, 48, 72 h, or 6 days after surgery. Immunohistochemical analyses using a Fos-specific N-terminally directed antiserum also revealed Fos immunoreactivity (-ir) in the PVH at early time points (3 h), but not later than 12 h after ADX. Combined immuno- and hybridization histochemistry on tissue from 3 h ADX rats localized Fos-ir and NGFI-B mRNA to parvocellular CRF-expressing neurons, the majority of which expressed both Fos and NGFI-B. These early IEG responses, however, were not paralleled by increases in CRF mRNA, which was not significantly elevated until 48 h after ADX. In the chronically ADX rat, CRF neurons are capable of c-fos expression since animals subjected to an acute injection of hypertonic saline 5 days after ADX displayed a robust induction of Fos-ir in the parvocellular PVH. Furthermore, a chronic challenge, insulin-induced hypoglycemia, provoked comparable c-fos mRNA and protein expression in CRF neurons in the PVH of ADX and sham-operated rats, which was observed both acutely (2 h) and chronically (5 days) after the onset of hypoglycemia. The maintenance of Fos expression in parvocellular CRF neurons following chronic hypoglycemia, but not ADX, suggests an involvement of distinct signaling pathways in the maintenance of hypophysiotropic neuron responses to chronic steroid withdrawal and stress.

Corticotropin-releasing factor-binding protein ligand inhibitor blunts excessive weight gain in genetically obese Zucker rats and rats during nicotine withdrawal.

Elevation of the neuropeptide corticotropin-releasing factor (CRF) in the brain is associated with a reduction of food intake and body weight gain in normal and obese animals. A protein that binds CRF and the related peptide, urocortin, with high affinity, CRF-binding protein (CRF-BP), may play a role in energy homeostasis by inactivating members of this peptide family in ingestive and metabolic regulatory brain regions. Intracerebroventricular administration in rats of the high-affinity CRF-BP ligand inhibitor, rat/human CRF (6-33), which dissociates CRF or urocortin from CRF-BP and increases endogenous brain levels of "free" CRF or urocortin significantly blunted exaggerated weight gain in Zucker obese subjects and in animals withdrawn from chronic nicotine. Chronic administration of CRF suppressed weight gain nonselectively by 60% in both Zucker obese and lean control rats, whereas CRF-BP ligand inhibitor treatment significantly reduced weight gain in obese subjects, without altering weight gain in lean control subjects. Nicotine abstinent subjects, but not nicotine-naive controls, experienced a 35% appetite suppression and a 25% weight gain reduction following acute and chronic administration, respectively, of CRF-BP ligand inhibitor. In marked contrast to the effects of a CRF-receptor agonist, the CRF-BP ligand inhibitor did not stimulate adrenocorticotropic hormone secretion or elevate heart rate and blood pressure. These results provide support for the hypothesis that the CRF-BP may function within the brain to limit selected actions of CRF and/or urocortin. Furthermore, CRF-BP may represent a novel and functionally selective target for the symptomatic treatment of excessive weight gain associated with obesity of multiple etiology.

Distinct mechanisms underlie activation of hypothalamic neurosecretory neurons and their medullary catecholaminergic afferents in categorically different stress paradigms.

Intermittent electrical footshock induces c-fos expression in parvocellular neurosecretory neurons expressing corticotropin-releasing factor and in other visceromotor cell types of the paraventricular hypothalamic nucleus (PVH). Since catecholaminergic neurons of the nucleus of the solitary tract and ventrolateral medulla make up the dominant loci of footshock-responsive cells that project to the PVH, these were evaluated as candidate afferent mediators of hypothalamic neuroendocrine responses. Rats bearing discrete unilateral transections of this projection system were exposed to a single 30-min footshock session and sacrificed 2 hr later. Despite depletion of the aminergic innervation on the ipsilateral side, shock-induced up-regulation of Fos protein and corticotropin-releasing factor mRNA were comparable in strength and distribution in the PVH on both sides of the brain. This lesion did, however, result in a substantial reduction of Fos expression in medullary aminergic neurons on the ipsilateral side. These results contrast diametrically with those obtained in a systemic cytokine (interleukin 1) challenge paradigm, where similar cuts ablated the Fos response in the ipsilateral PVH but left intact the induction seen in the ipsilateral medulla. We conclude that (i) footshock-induced activation of medullary aminergic neurons is a secondary consequence of stress, mediated via a descending projection transected by our ablation, (ii) stress-induced activation of medullary aminergic neurons is not necessarily predictive of an involvement of these cell groups in driving hypothalamic visceromotor responses to a given stressor, and (iii) despite striking similarities in the complement of hypothalamic effector neurons and their afferents that may be activated by stresses of different types, distinct mechanisms may underlie adaptive hypothalamic responses in each.

Sequence of stress-induced alterations in indices of synaptic and transcriptional activation in parvocellular neurosecretory neurons.

Immediate-early genes (IEGs) are widely used to mark endocrine hypothalamic neurons that are activated in response to stress, yet their relationship to the transcriptional control of relevant effector molecule expression is unclear. Acute ether stress provokes increased adrenocorticotropic hormone (ACTH) and corticosterone secretion that peaks at 5 and 30 min, respectively, after the challenge. Using probes complementary to intronic sequences of genes encoding ACTH secretagogues in parvocellular neurosecretory neurons of the paraventricular nucleus, we found these events to be accompanied by rapid and transient increases in corticotropin-releasing factor heteronuclear RNA (CRF hnRNA; peak at 5 min) and by a delayed upregulation of arginine vasopressin (AVP) hnRNA (120 min). To identify candidate mechanisms regulating peptide expression, we followed the timing of ether effects on representatives of three transcription factor classes: IEGs [c-fos and nerve growth factor I-B (NGFI-B)], a POU-domain factor (Brn-2), and the cAMP response element-binding protein (CREB), using antisera specific to its transcriptionally active, phosphorylated form (pCREB). After ether exposure, c-fos and NGFI-B mRNA induction were maximal at 30--60 min, whereas Fos protein peaked at 60--120 min. Brn-2 mRNA was expressed constitutively in the PVH and was unresponsive to stress. By contrast, pCREB was induced in parvocellular neurons with a time course parallel to that of CRF hnRNA expression. Stress-induced transcriptional activation of the CRF and AVP genes in hypophysiotropic neurons follows distinct time courses that are compatible with control mechanisms involving phosphorylation events and de novo protein synthesis, respectively.

Regulation of stress-induced transcriptional changes in the hypothalamic neurosecretory neurons.

Transcriptional changes in corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) gene expression were studied by in situ hybridization histochemistry using cRNA probes directed against intronic sequences. Acute ether stress resulted in a rapid induction of CRF and a delayed activation of vasopressin heteronuclear (hn)RNA in the parvocellular neurosecretory neurons within the paraventricular nucleus (PVN) of the hypothalamus. To explore possible molecular mechanisms regulating stress-related neuropeptide expression in vivo, the time-courses of stress-induced activation of different transcription factor classes were compared to that of changes in neuropeptide transcription. The peak of CRF transcription was parallel to that of cAMP response-element binding protein (CREB) phosphorylation but preceded the induction of c-fos and NGFI-B mRNAs and Fos protein. In contrast, AVP expression occurred in step with immediate-early gene (IEG) responses, suggesting involvement of different mechanisms underlying stress-induced neuropeptide responses. The interference of glucocorticoid hormones with stress-induced neuropeptide and transcription-factor responses has also been revealed in rats acutely or chronically pretreated with glucocorticoids. Acute dexamethasone injection did not prevent neuropeptide and transcription factor responses to either inhalation, whereas chronic corticosterone administration completely blocked IEG and neuropeptide induction in the stress-related neurosecretory neurons.

Ultrastructural localization of inhibin beta- and somatostatin-28-immunoreactivities in the paraventricular and supraoptic nuclei.

Recent studies have supported the existence of projections to the paraventricular and supraoptic nuclei of the hypothalamus that arise from non-catecholaminergic neurons in the nucleus of the solitary tract, whose terminal distribution is suggestive of interactions with both parvocellular and magnocellular neurosecretory neurons. Pre-embedding immunolabeling methods were used to compare and characterize the termination patterns of axons immunoreactive for two putative markers for this projection system, inhibin beta and somatostatin-28, at the ultrastructural level. Axon terminal profiles stained for either peptide were found to form symmetric or asymmetric junctions predominantly with the shafts of unlabeled dendrites of varying caliber. A small percentage of peptidergic terminals was found in both hypothalamic nuclei to engage in so-called 'shared synapses', where a single terminal profile contacted two postsynaptic elements. Axo-somatic terminations were relatively rarely seen in the supraoptic nucleus, but were somewhat more abundant in the paraventricular nucleus. These comprised principally symmetric junctions onto the somatic membranes of an ostensibly mixed population of cells, some of which bore apparent neurosecretory specializations. Combined immunoperoxidase and immuno-autoradiographic staining methods were used to estimate the extent to which either terminal type interacts with oxytocin neurons. Oxytocin stained elements comprised a minority of the postsynaptic targets of both peptidergic terminal types in the paraventricular nucleus, and a scant majority of those in the supraoptic nucleus. These results support the view that peptidergic neurons in the caudal nucleus of the solitary tract interact synaptically with multiple cell types in the parvocellular division of the paraventricular nucleus, and preferentially with oxytocinergic elements in the

Hemodynamic regulation of tyrosine hydroxylase messenger RNA in medullary catecholamine neurons: a c-fos-guided hybridization histochemical study.

Medullary catecholamine cell groups are involved in multiple modes of cardiovascular regulation and display indices of functional activation, including widespread c-fos expression, in response to hypotensive hemorrhage. Assessments of the impact of such challenges on transmitter-related gene expression are complicated by the biochemical and connectional heterogeneity that characterize these cell groups. Quantitative hybridization histochemical methods were used to follow the effects of 15% hemorrhage on levels of messenger RNA encoding tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, in medullary aminergic neurons; concurrent staining for nuclear Fos-immunoreactivity permitted comparisons between cells that ostensibly were and were not targeted by the challenge. Increased levels of tyrosine hydroxylase messenger RNA were detected in Fos-immunoreactive neurons in all cell groups examined. Mean maximal increases ranged between 133 and 192% of control values, and were attained within 0.5-1 h post-hemorrhage in noradrenergic (A1 and A2) cell groups, and at 2 h in adrenergic ones (C1, C2, and C2d or dorsal strip). By 4 h after the challenge, tyrosine hydroxylase messenger RNA levels in Fos-immunoreactive neurons in all cell groups had returned to control values. By contrast, tyrosine hydroxylase messenger RNA in non-Fos-immunoreactive cells either did not change significantly over the course of the experiment (C2 and C2d), or showed a rapid and transient increase, whose magnitude tended to be less than that seen in Fos-immunoreactive cells. c-fos messenger RNA was prominently induced in catecholaminergic neurons in each of the medullary cell groups examined at 0.5 h after hemorrhage, suggesting that the early tyrosine hydroxylase messenger RNA response to hemorrhage in non-Fos-immunoreactive cells preceded the capacity of responsive neurons to manifest detectable Fos protein expression. These findings indicate that hemorrhage up-regulates tyrosine hydroxylase messenger RNA levels in medullary catecholaminergic cell groups which have access to adaptive neuroendocrine and/or autonomic control systems. The approach employed here should prove of general utility in assessing the impact of environmental events on messenger RNA expression in connectionally heterogeneous cell groups that share a common biochemical phenotype.