Intact catecholamine inputs to the forebrain are required for appropriate regulation of corticotrophin-releasing hormone and vasopressin gene expression by corticosterone in the rat paraventricular nucleus.

Corticotrophin-releasing hormone (CRH) neuroendocrine neurones in the paraventricular nucleus of the hypothalamus (PVH) drive adrenocorticotrophic hormone (ACTH) and thereby glucocorticoid release from pituitary corticotrophs and the adrenal cortex, respectively. Glucocorticoids suppress the ability of neuroendocrine corticotrophin-releasing hormone (CRH) neurones to synthesise and release ACTH secretogogues. Despite the importance of glucocorticoids as regulatory signals to CRH neurones in the extended time domain, how and where they act in this capacity is still not fully understood. Ascending catecholamine projections encode important cardiovascular, metabolic and other visceral information to the rat PVH and surrounding hypothalamus. These afferents have previously been implicated as targets for glucocorticoid action, including a role in the feedback regulation of PVH neuroendocrine neurones. To determine the contribution of these neurones to the long-term actions of corticosterone on CRH and vasopressin (AVP) gene expression in the PVH, we used an immunocytotoxin (a conjugate of the cytotoxin saporin and an antibody against dopamine-ß-hydroxylase) that specifically ablates adrenergic and noradrenergic neurones. Lesions were administered to intact animals and to adrenalectomised animals with either no corticosterone or corticosterone replacement that provided levels above those required to normalise Crh expression. The ability of elevated levels of corticosterone to suppress Crh expression was abolished in animals lacking catecholaminergic innervation of the PVH. No effect was seen in the absence of corticosterone or in animals with intact adrenals. Furthermore, Avp expression, which is increased in CRH neurones following adrenalectomy, was suppressed in adrenalectomised catecholaminergic-lesioned animals. Interactions between corticosterone and catecholaminergic projections to the hypothalamus therefore make significant contributions to the regulation of Crh and Avp expression. However, the importance of catecholamine inputs is only apparent when circulating corticosterone concentrations are maintained either below or above those required to maintain the activity of the hypothalamic-pituitary-adrenal axis that is seen in intact animals.

Basic organization of projections from the oval and fusiform nuclei of the bed nuclei of the stria terminalis in adult rat brain.

The organization of axonal projections from the oval and fusiform nuclei of the bed nuclei of the stria terminalis (BST) was characterized with the Phaseolus vulgaris-leucoagglutinin (PHAL) anterograde tracing method in adult male rats. Within the BST, the oval nucleus (BSTov) projects very densely to the fusiform nucleus (BSTfu) and also innervates the caudal anterolateral area, anterodorsal area, rhomboid nucleus, and subcommissural zone. Outside the BST, its heaviest inputs are to the caudal substantia innominata and adjacent central amygdalar nucleus, retrorubral area, and lateral parabrachial nucleus. It generates moderate inputs to the caudal nucleus accumbens, parasubthalamic nucleus, and medial and ventrolateral divisions of the periaqueductal gray, and it sends a light input to the anterior parvicellular part of the hypothalamic paraventricular nucleus and nucleus of the solitary tract. The BSTfu displays a much more complex projection pattern. Within the BST, it densely innervates the anterodorsal area, dorsomedial nucleus, and caudal anterolateral area, and it moderately innervates the BSTov, subcommissural zone, and rhomboid nucleus. Outside the BST, the BSTfu provides dense inputs to the nucleus accumbens, caudal substantia innominata and central amygdalar nucleus, thalamic paraventricular nucleus, hypothalamic paraventricular and periventricular nuclei, hypothalamic dorsomedial nucleus, perifornical lateral hypothalamic area, and lateral tegmental nucleus. Moderately dense inputs are found in the parastrial, tuberal, dorsal raphé, and parabrachial nuclei and in the retrorubral area, ventrolateral division of the periaqueductal gray, and pontine central gray. Light projections end in the olfactory tubercle, lateral septal nucleus, posterior basolateral amygdalar nucleus, supramammillary nucleus, and nucleus of the solitary tract. These and other results suggest that the BSTov and BSTfu are basal telencephalic parts of a circuit that coordinates autonomic, neuroendocrine, and ingestive behavioral responses during stress.

Corticosterone modulation of ACTH secretogogue gene expression in the paraventricular nucleus.

This review will describe effects of corticosterone on the temporal dynamics of components within the hypothalamo-pituitary-adrenal (HPA) axis in response to sustained hypovolemia. The characterization of the synthetic and secretory profiles of HPA elements in these rat models reveals the complexities of steroid-mediated regulation of neuroendocrine and corticotrope function during a sustained stress event. Collectively, our data suggest activation of gene transcription and secretion are independently controlled, and that corticosterone affects adrenocorticotropin hormone (ACTH) gene expression in the parvicellular neuroendocrine part of the hypothalamic paraventricular nucleus using two mechanisms: first, an inhibition which contributes to classic negative feedback, and second, a facilitation, which is seen at low plasma concentrations.

Neuropeptides and the integration of motor responses to dehydration.

Drinking and eating are critically important motivated behaviors whose expression is usually tightly linked; under conditions of spontaneous intake, disruption of one usually disturbs the other. This characteristic is exemplified by dehydration-induced anorexia in which increasing plasma osmolality leads to a centrally generated reduction in food intake, which is then rapidly reversed as water is again made available. This review discusses, at a systems level, how the brain is organized to generate these behaviors and how dehydration affects the expression of neuropeptides in sets of anatomically defined forebrain circuits that contribute to the integration of motor outputs. These findings are then used to consider how altered neuropeptidergic signaling operates within motor drive networks and how these changes may impact the way neuroendocrine, autonomic, and behavioral motor systems respond to this fundamental homeostatic challenge.

Adrenalectomy dramatically modifies the dynamics of neuropeptide and c-fos gene responses to stress in the hypothalamic paraventricular nucleus.

We have used in situ hybridization and radio-immunoassay to compare temporal dynamics of components in the hypothalamo-pituitary limb of the hypothalamo-pituitary-adrenal axis during sustained hypovolemic stress in adrenalectomized (ADX) rats to those previously reported in intact animals. We asked three questions: first, does corticotropin-releasing hormone (CRH) gene transcription occur in neuroendocrine neurones of the hypothalamic paraventricular nucleus (PVH) of ADX rats, and if so, how is it temporally organized; second, what is the expression pattern of the vasopressin and other genes known to be colocalized in these neuroendocrine neurones; third, if adrenocorticotropin hormone (ACTH) secretion occurs, what is its temporal profile? We found that sustained hypovolemia evoked a brief episode of CRH gene transcription in ADX rats that occurred earlier than in intact rats. However, in contrast to saline-injected controls, this activation was not maintained because declines in CRH hnRNA and mRNA were seen as the stress continued. Although increased vasopressin gene transcription was not seen in intact hypovolemic rats, robust increases were measured throughout in ADX rats, suggesting that in the absence of corticosterone the vasopressin gene is transcribed preferentially to the CRH gene during sustained hypovolemia. c-fos and preproenkephalin mRNA profiles also exhibited earlier onsets compared to intact rats. Finally, the onset and duration of ACTH secretion was the same in ADX rats as previously reported in intact rats. Collectively, these data support two hypotheses regarding the actions of corticosterone. First, that it provides some form of facilitatory signal allowing neuroendocrine CRH transcriptional mechanisms to remain active during sustained hypovolemia. Second, that it strongly inhibits the response of the vasopressin gene to hypovolemic stress.

Understanding the neural control of ingestive behaviors: helping to separate cause from effect with dehydration-associated anorexia.

Eating and drinking are motivated behaviors that are made up of coordinated sets of neuroendocrine, autonomic, and behavioral motor events. Although the spinal cord, hindbrain, and hypothalamus contain the motor neurons and circuitry sufficient to maintain the reflex parts of these motor events, inputs from the telencephalon are required to furnish the behavioral components with a motivated (goal-directed) character. Each of these motor events derives from the complex interaction of a variety of sensory inputs with groups of neural networks whose components are distributed throughout the brain and collectively support motor expression and coordination. At a first approximation based on a variety of data, these networks can be divided into three groups: networks that stimulate, those that inhibit, and those that disinhibit motor functions. A fourth contributor is the circadian timing signal that originates in the hypothalamic suprachiasmatic nucleus and provides the temporal anchor for the expression of all behaviors. This article discusses the nature of these networks using neuroanatomical (tract-tracing and neuropeptide in situ hybridization), endocrine, and behavioral evidence from a variety of experimental models. A persistent problem when studying the control of food intake from a neural systems perspective has been the difficulty in separating those neuronal changes that result in hunger from those that are as a consequence of eating. To address this problem, dehydration-associated anorexia is presented as a particularly useful experimental model because it can be used to distinguish between neural mechanisms underlying anorexia and those changes that occur as a consequence of anorexia. The article concludes by highlighting the potential role of neuropeptidergic action in the operation of these networks, using forebrain neuropeptidergic innervation of the parabrachial nucleus as an example.

Distinct patterns of neuropeptide gene expression in the lateral hypothalamic area and arcuate nucleus are associated with dehydration-induced anorexia.

We have investigated the hormonal and hypothalamic neuropeptidergic substrates of dehydration-associated anorexia. In situ hybridization and hormone analyses of anorexic and paired food-restricted rats revealed two distinct profiles. First, both groups had the characteristic gene expression and endocrine signatures usually associated with starvation: increased neuropeptide Y and decreased proopiomelanocortin and neurotensin mRNAs in the arcuate nucleus (ARH); increased circulating glucocorticoid but reduced leptin and insulin. Dehydrated animals are strongly anorexic despite these attributes, showing that the output of leptin- and insulin-sensitive ARH neurons that ordinarily stimulate eating must be inhibited. The second pattern occurred only in anorexic animals and had two components: (1) reduced corticotropin-releasing hormone (CRH) mRNA in the neuroendocrine paraventricular nucleus (PVH) and (2) increased CRH and neurotensin mRNAs in the lateral hypothalamic (LHA) and retrochiasmatic areas. However, neither corticosterone nor suppressed PVH CRH gene expression is required for anorexia after dehydration because PVH CRH mRNA in dehydrated adrenalectomized animals is unchanged from euhydrated adrenalectomized controls. We also showed that LHA CRH mRNA was strongly correlated with the intensity of anorexia, increased LHA CRH gene expression preceded the onset of anorexia, and dehydrated adrenalectomized animals (which also develop anorexia) had elevated LHA CRH gene expression with a distribution pattern similar to intact animals. Finally, we identified specific efferents from the CRH-containing region of the LHA to the PVH, thereby providing a neuroanatomical framework for the integration by the PVH of neuropeptidergic signals from the ARH and the LHA. Together, these observations suggest that CRH and neurotensin neurons in the LHA constitute a novel anatomical substrate for their well known anorexic effects.

Dehydration-associated anorexia: development and rapid reversal.

Dehydration in rats results in anorexia that is proportional to the degree of dehydration. The aims of this study were first, to determine when anorexia develops in response to drinking hypertonic (2.5%) saline for 4 days; and second, to determine the organization of ingestive behaviors after access to water is resumed. Body weights, food, and fluid intake were measured morning and evening before, during, and after a 4-day period of dehydration caused by drinking hypertonic saline. A profile of the behaviors expressed immediately after rehydration was determined. The data make three points. First, dehydration-associated anorexia does not emerge until the second night of dehydration when the composition of the fluid compartments can no longer be homeostatically buffered. Second, dehydration reduces the amount food eaten nocturnally, but leaves diurnal food consumption largely unaffected. Animals very rapidly return to predehydration nocturnal ingestion patterns, whereas the amounts of food and water ingested during the day are significantly increased. Increased diurnal food intake may play a significant role in normalizing metabolism after dehydration. Finally, anorexia is reversed within minutes of rehydration. The data suggest a model where dehydration simultaneously activates two sets of circuits within the brain that will independently stimulate or inhibit feeding. Eating is inhibited during dehydration through the action of a set of inhibitory circuits, which masks the output of circuits that stimulate eating. However, when drinking water resumes, sensory inputs to these circuits rapidly release the inhibition and allow eating to proceed freely.

Dehydration modifies somal CRH immunoreactivity in the rat hypothalamus: an immunocytochemical study in the absence of colchicine.

Corticotropin-releasing hormone immunoreactive (CRH-ir) neurons were examined in the hypothalamus of euhydrated and dehydrated rats without using colchicine. CRH-ir cells were observed in the lateral hypothalamus, retrochiasmatic, and in magnocellular parts of the supraoptic and paraventricular hypothalamic nucleus (PVH) in dehydrated but not euhydrated animals. However, CRH-ir neurons were decreased in the medial parvicellular part of the PVH. These results indicate that altered CRH mRNA levels previously reported in dehydrated animals translate into changes in peptide immunoreactivity.

Peptide gene activation, secretion, and steroid feedback during stimulation of rat neuroendocrine corticotropin-releasing hormone neurons.

We have used colloid-induced hypovolemia to investigate mechanisms operating in CRH neuroendocrine neurons of the hypothalamic paraventricular nucleus during a sustained stress. Specifically, three questions have been addressed using in situ hybridization and RIA. 1) Do neuropeptide secretion and gene activation share the same stimulus threshold? 2) Does corticosterone modulate mechanisms regulating CRH gene expression during sustained stress? 3) How are neuropeptides commonly colocalized with CRH affected? Our results show that the secretion of ACTH and activation of the CRH gene have distinct and separate stimulus thresholds. The threshold is higher for CRH gene activation than for ACTH secretion, suggesting some degree of mechanistic separation. In addition, corticosterone secreted during the first 3 h of sustained hypovolemia does not inhibit CRH gene expression. However, feedback inhibition may occur in the delayed time domain. Finally, neuropeptides colocalized with CRH are differentially regulated by sustained hypovolemia. Proenkephalin messenger RNA levels show a slower temporal response than those of CRH, while the vasopressin gene is not activated at any time in parvicellular neuroendocrine neurons. Our results emphasize that CRH neuroendocrine neurons respond to a stress event in a stimulus-specific manner in terms of both the profiles of secretion and gene expression, and the structure of glucocorticoid feedback.

Corticosterone can facilitate as well as inhibit corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus.

We have used in situ hybridization to investigate how basal levels of circulating corticosterone modulate CRH gene transcription in the neuroendocrine parvicellular part of the hypothalamic paraventricular nucleus (PVHmpd) during sustained hypovolemia. In the absence of the stressor, the accumulation rate of the CRH primary transcript exhibited a dose dependency on low maintained levels of plasma corticosterone similar to that previously reported for the mature messenger RNA (mRNA); levels declined as plasma corticosterone increased. In response to hypovolemia, the absence of corticosterone compromised CRH gene transcription mechanisms to mount the activated response seen in intact animals. However, adrenalectomized rats with low doses of corticosterone (insufficient to normalize thymus weights) showed an augmented mRNA response compared with that in intact animals. When replaced corticosterone normalized thymus weights, the magnitude of the mRNA response was reduced to that seen in intact animals. In contrast to CRH gene regulation, PVHmpd proenkephalin mRNA levels were unaffected by corticosterone concentrations. These results suggest that corticosterone affects CRH gene transcription in the PVHmpd using two mechanisms: first, inhibition, which probably uses type II glucocorticoid receptor-dependent mechanisms and contributes to classic negative feedback; and second, facilitation, which is seen at low plasma concentrations and maintains gene transcription in the presence of sustained stress, possibly using type I mechanisms. This suggests that one reason why adrenal insufficiency severely compromises survival of sustained stress is that CRH gene transcription cannot be maintained without previous exposure to low levels of plasma corticosterone.

The region of the pontine parabrachial nucleus is a major target of dehydration-sensitive CRH neurons in the rat lateral hypothalamic area.

Neurons in a restricted part of the lateral hypothalamic area (LHA) show increased expression of corticotropin-releasing hormone (CRH) mRNA as a consequence of cellular dehydration. In the present study, we have investigated the organization of their efferent projections by using anterograde and retrograde tracing techniques. Additionally, we have compared the distribution of CRH mRNA-containing neurons after cellular dehydration and intraventricular (i.c.v.) colchicine injections. Our results show that cellular dehydration activates a more restricted neuronal population than does i.c.v. colchicine. Iontophoretic injections of Phaseolus vulgaris leucoagglutinin (PHAL) were placed in the LHA of animals drinking hypertonic saline and their proximity to activated CRH neurons determined by in situ hybridization for CRH mRNA. Although labelled fibers from these injections were seen throughout the brain, the region of the parabrachial nucleus and nucleus of the solitary tract (NTS) were most conspicuous in also having CRH immunoreactive fibers. Injections of Fluoro-Gold placed in these two structures were used to confirm these findings in dehydrated animals. Significant numbers of neurons containing both Fluoro-Gold and CRH mRNA were seen in the lateral hypothalamus after injections in the lateral and medial parts of the parabrachial nucleus; far fewer were seen after injections in the NTS. These results strongly suggest that the CRH neurons in the LHA activated by cellular dehydration provide an input to the region of the parabrachial nucleus. The altered biochemical composition of this pathway may well be able to modify sensory and motor patterns both during and after dehydration.

Structural organization and chromosomal localization of the human Na,K-ATPase beta 3 subunit gene and pseudogene.

We have cloned and characterized the Na,K-ATPase beta 3 subunit gene (ATP1B3), and a beta 3 subunit pseudogene (ATP1B3P1), from a human PAC genomic library. The beta 3 subunit gene is > 50 kb in size and is split into 7 exons. The exon/intron organization of the beta 3 subunit gene is identical to that of the Na,K-ATPase beta 3 subunit gene, indicating that these two genes evolved from a common evolutionary ancestor. Comparison of the promoter region of the human and mouse beta 3 subunit gene reveals a high degree of homology within a 300-bp segment located immediately upstream of the translation start site, suggesting that control elements that serve to regulate the cell-specific expression of the beta 3 subunit gene are likely to be located within this conserved region. Dot blot analysis of beta 3 subunit transcripts revealed expression within virtually all human tissues, while in situ hybridization showed expression of beta 3 mRNA in both neurons and glia of rat brain. Fluorescence in situ hybridization with PAC DNA clones localized ATP1B3 to the q22-->23 region of Chromosome (Chr) 3, and the beta 3 pseudogene to the p13-->15 region of Chr 2.

The impact of physiological stimuli on the expression of corticotropin-releasing hormone (CRH) and other neuropeptide genes.

The article reviews some of the recent work showing how physiological stimuli act to alter neuropeptide gene expression. It describes how neural and humoral factors activated by physiological stimuli interact with the mechanisms regulating neuropeptide gene expression in neurons with either vascular (neurosecretory) or cellular (centrally directed) synapses. Although the focus will be on corticotropin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus, comparisons will be made between this neurosecretory cell group and others that express this gene. The regulation of neuropeptide genes colocalized in neurons that synthesize CRH is also considered. The review begins with a brief historical introduction, placing peptides in the overall functional perspective of neurosecretory and centrally directed neurons. It then describes studies using in vitro preparations that reveal details of the signal transduction mechanisms responsible for altering the expression of neuropeptide genes. For the CRH gene they are providing the foundations for future work on how physiological stimuli alter mRNA levels in the whole animal. Physiological stimuli provide a very broad range of signals to neuropeptide neurons commensurate with the wide variety of motor responses they initiate. One important humoral signal impacting neuropeptide neurons is plasma corticosterone, and many workers have addressed this aspect of its function. Corticosterone appears capable of interacting with at least two different neuronal mechanisms to regulate CRH mRNA levels: one is clearly seen in paraventricular neurosecretory neurons, where increasing plasma corticosteroid reduces CRH mRNA levels; the other, seen in neurons in the central nucleus of the amygdala, acts to increase them. Since physiological stimuli present a complex mixture of humoral and neural signals to the CNS, integration of these two signal types is a critical aspect of peptide metabolism that requires detailed attention. Studies that are beginning to address this important question are described. Circadian influences play an important role in organizing homeostatic processes, and their influence on CRH gene expression is considered. The viscerosensory-motor integration associated with dehydration offers a useful model for investigating the role of peptides in neuronal function and motor architecture. Much of our work has concentrated on how peptide genes are regulated by alterations to fluid homeostasis, and these studies, along with those of other investigators, are described in this integrative context. Finally, consideration is given to the many studies that have addressed the impact of nonviscerosensory stimulation on neuropeptide gene expression.

Mediation of dehydration-induced peptidergic gene expression in the rat lateral hypothalamic area by forebrain afferent projections.

We have previously shown in dehydrated rats that cellular levels of the mRNAs encoding the precursor peptides for corticotropin-releasing hormone and neurotensin/neuromedin N significantly increase in a restricted region of the lateral hypothalamic area (Watts, 1992, Brain Res. 581:208-216). The experiments reported here address the role that forebrain osmosensitive cells groups or regions associated with autonomic regulation play in developing this mRNA response. The first experiment showed that unilateral knife cuts placed between the rostral forebrain and the lateral hypothalamic area (LHA) will unilaterally attenuate the mRNA response in the LHA to dehydration. In a second experiment, small injections of the retrograde tracer Fluorogold into the region of the LHA containing these mRNAs revealed a direct input from the osmosensitive median preoptic nucleus and subfornical organ and from the fusiform nucleus of the bed nuclei of the stria terminalis, which is part of a complex of cell groups associated with autonomic regulation. We found that at least 30% of the neurons in the median preoptic nucleus and subfornical organ and 14% of the neurons in the fusiform nucleus of the bed nuclei of the stria terminalis that project to the LHA responded to a rapid increase in plasma osmolality with increased c-fos mRNA levels. In the final experiment, injections of Fluorogold into the LHA were made simultaneously with ipsilateral rostral knife cuts. Here the numbers of neurons accumulating Fluorogold in the median preoptic nucleus, subfornical organ, and the fusiform nucleus were all significantly decreased concomitantly with attenuated mRNA responses in the LHA to dehydration. We conclude that the LHA receives direct and functional projections from the median preoptic nucleus, subfornical organ, and the fusiform nucleus. These projections appear capable of mediating a substantial part of the response of peptidergic mRNAs in the LHA to dehydration.

Neuropeptides and thirst: the temporal response of corticotropin-releasing hormone and neurotensin/neuromedin N gene expression in rat limbic forebrain neurons to drinking hypertonic saline.

The authors have demonstrated in rats that the ingestion of hypertonic saline for 5 days provides an increasingly complex dehydrating stimulus to the rats. Initially, the stimulus leads to cellular dehydration, but extracellular dehydration develops as ingestion continues beyond 3 days. The initial cellular dehydration provokes modifications to corticotropin-releasing hormone and neurotensin/neuromedin N messenger RNAs (mRNAs) in some neurons of the limbic forebrain, changes that are either maintained or are modified as extracellular dehydration develops. These changes in mRNA content occur in neurosecretory neurons as well as in neurons in hypothalamic and telencephalic regions associated with behavior and autonomic regulation. The authors propose that alterations in peptide mRNAs are allied to altered neuronal signaling processes that direct the different components of the homeostatic response to dehydration.

A cell-specific role for the adrenal gland in regulating CRH mRNA levels in rat hypothalamic neurosecretory neurons after cellular dehydration.

In the rat, the cellular dehydration induced by water deprivation rapidly increases CRH mRNA in magnocellular neurosecretory neurons, but gradually reduces mRNA levels in hypothalamic paraventricular parvicellular neurosecretory neurons. Using in situ hybridization we investigated a possible role for corticosterone as a mediator of the effects of water deprivation on the levels of CRH mRNA in the paraventricular and supraoptic nuclei. Following adrenalectomy and water deprivation, the reduction of CRH mRNA in the medial parvicellular part of the paraventricular nucleus was inhibited. However, replacement of low-doses of corticosterone to dehydrated adrenalectomized animals was not sufficient to reduce parvicellular CRH mRNA levels to those seen in intact dehydrated animals. Neither adrenalectomy nor corticosterone replacement had any effect on the increased CRH mRNA levels in magnocellular neurosecretory neurons. We conclude that an intact adrenal gland is required for the decreased levels of CRH mRNA seen during water deprivation in parvicellular paraventricular neurosecretory neurons, but not magnocellular neurosecretory neurons. These effects may be mediated by the increased corticosterone secretion seen during water deprivation.

Region-specific regulation of neuropeptide mRNAs in rat limbic forebrain neurones by aldosterone and corticosterone.

1. We have determined in adrenalectomized male rats the effects of clamping plasma corticosterone and aldosterone at various concentrations on corticotropin-releasing hormone (CRH), neurotensin/neuromedin N (NT/N) and proenkephalin (pENK) mRNAs in the hypothalamus and amygdala using semi-quantitative in situ hybridization. 2. Corticosterone differentially regulated the levels of CRH and NT/N but not pENK mRNA. These effects were cell specific. CRH mRNA was reduced in the hypothalamic paraventricular nucleus (PVH), but increased in the central nucleus of the amygdala and bed nuclei of the stria terminalis. NT/N mRNA was never seen in the PVH, whereas levels increased in the central nucleus of the amygdala, but were unaffected in the lateral hypothalamic area. In those regions expressing pENK mRNA, levels were unaffected in all treatment groups. 3. CRH mRNA in both the central nucleus of the amygdala and PVH, and NT/N mRNA in the central nucleus of the amygdala were most sensitive to plasma corticosterone concentrations of less than 120 ng ml-1, i.e. those seen away from the peak of the diurnal rhythm. In adrenalectomized animals CRH mRNA in both the central nucleus of the amygdala and PVH could be set at levels usually seen in intact animals by the same plasma concentration of corticosterone. 4. The levels of CRH mRNA in the PVH and the central nucleus of the amygdala were closely correlated, while CRH and NT/N mRNA levels were similarly correlated in the central nucleus of the amygdala suggesting the existence of a common regulatory mechanism. The ED50 of their responses to corticosterone and correlations with thymus weight suggested the operation of glucocorticoid (type II) receptor mechanisms. 5. In the absence of corticosterone, aldosterone increased CRH and NT/N mRNA accumulation in the central nucleus of the amygdala, and increased CRH but not NT/N mRNA accumulation in the PVH. Aldosterone also blunted the dose-response effects of corticosterone on CRH and NT/N mRNA levels in the central nucleus of the amygdala, but not in the PVH. 6. These results suggest that, in intact animals, adrenal steroids play a major role in maintaining the levels of neuropeptide mRNAs in the PVH, bed nuclei of the stria terminalis and central nucleus of the amygdala. The results underscore the importance of cell-specific mechanisms operating to regulate the expression of neuropeptide genes in different cell types in response to diverse physiological conditions.

Physiological regulation of peptide messenger RNA colocalization in rat hypothalamic paraventricular medial parvicellular neurons.

In the present study, we used subcutaneous polyethylene glycol injections to show that a physiologically relevant stimulus, hypovolemia, will selectively increase the expression of neuropeptide genes in a restricted population of parvicellular corticotropin-releasing hormone-containing neurons in the hypothalamic paraventricular nucleus. Our results show that a large reduction in extracellular fluid maintained over approximately 20 hours is associated with a significant increase in the level of corticotropin-releasing hormone mRNA in the medial parvicellular division of the paraventricular nucleus. Additionally, there are concomitant increases in cellular levels of both neurotensin/neuromedin N and proenkephalin mRNAs. Our colocalization results show that the increases in neurotensin/neuromedin N and proenkephalin mRNAs after polyethylene glycol injection occur to a significant degree in cells that also contain corticotropin-releasing hormone mRNA. Furthermore, significant numbers of cells containing proenkephalin mRNA also contain neurotensin/neuromedin N mRNA, raising the possibility that some neurons have increased levels of all three mRNAs. Finally, in the medial parvicellular division of the paraventricular nucleus, the number of identified corticotropin-releasing hormone neurons also containing vasopressin mRNA is very low in control animals and is not increased by polyethylene glycol injections, suggesting that, within this period, activation of the vasopressin gene may not be a critical event in the neuroendocrine response of corticotropin-releasing hormone neurosecretory neurons to extracellular dehydration. Considered together with the effects of adrenalectomy on peptide colocalization, our results suggest the existence of several phenotypically distinct sets of neurons within the medial parvicellular division of the paraventricular nucleus, each characterized by its ability to regulate the expression of neuropeptide genes in a stimulus-specific manner.

Autoradiographic visualization of 35S-labeled cRNA probes combined with immunoperoxidase detection of choleragenoid: a double-labeling light microscopic method for in situ hybridization and retrograde tract tracing.

We describe a protocol for simultaneous light microscopic visualization of a neuron's efferent projections and its expression of mRNA. We have combined immunohistochemical visualization of the retrograde marker cholera toxin subunit B (CTb) with autoradiographic visualization of 35S-labeled cRNA probes. Injections of CTb were made into rat brain. Immunoreactivity for CTb was demonstrated by modification of the peroxidase-anti-peroxidase immunohistochemical technique, with DAB and nickel ammonium sulfate or cobalt acetate as chromogen. On the same sections, in situ hybridization was performed with a 35S-labeled RNA probe complementary to preproenkephalin mRNA or tyrosine hydroxylase mRNA. Many double-labeled neurons were detected. These neurons contained peroxidase reaction product and were covered by an accumulation of silver grains in the overlaying emulsion layer. The present method has several advantages over double-labeling methods using the combination of fluorescent tracers and oligonucleotide probes. Both reaction products are permanent and can be visualized simultaneously by light microscopy. Furthermore, both CTb and cRNA probes are very sensitive markers. In addition, the sections can be counterstained.