The melanin-concentrating hormone (MCH) system in an animal model of depression-like behavior.

Selective breeding for divergence in locomotion in a novel environment (bHR, bred High-Responder; bLR, bred Low-Responder) correlates with stress-reactivity, spontaneous anxiety-like behaviors and predicts vulnerability in a rodent model of depression. Identifying genetic factors that may account for such vulnerability are key determinants not only for the illness outcome but also for the development of better-tailored treatment options. Melanin-concentrating hormone (MCH) is a neuropeptide that exhibits some of the hallmarks of a regulator of affective states. The aim of this study was to ascertain the role of the MCH system in depression-like behaviors in bHR vs. bLR rats. bLR rats showed a 44% increase in hypothalamic pMCH mRNA and a 14% decrease in hippocampal CA1 MCH1R mRNA when compared to bHR rats. Interestingly, the amount of time that rats spent immobile in the FST (depressive-like behavior) correlated positively with the amount of hypothalamic pMCH mRNA and negatively with that of hippocampal CA1 MCH1R. The results indicate that the bLR-bHR is a useful rat model to investigate individual basal genetic differences that participate in the monitoring of emotional responsiveness (i.e., depression- and anxiety-like behaviors). They also point to the MCH system (i.e., chronically higher pMCH expression and consequently receptor down-regulation) as a candidate biomarker for the severity of depressive-like behavior. The data indicate that MCH1R participates in the modulation of depression-like behavior through a process that involves the CA1 region of the hippocampus, supporting the possible use of MCH1R antagonists in the treatment of depression.

A food predictive cue must be attributed with incentive salience for it to induce c-fos mRNA expression in cortico-striatal-thalamic brain regions.

Cues associated with rewards acquire the ability to engage the same brain systems as rewards themselves. However, reward cues have multiple properties. For example, they not only act as predictors of reward capable of evoking conditional responses (CRs), but they may also acquire incentive motivational properties. As incentive stimuli they can evoke complex emotional and motivational states. Here we sought to determine whether the predictive value of a reward cue is sufficient to engage brain reward systems, or whether the cue must also be attributed with incentive salience. We took advantage of the fact that there are large individual differences in the extent to which reward cues are attributed with incentive salience. When a cue (conditional stimulus, CS) is paired with delivery of food (unconditional stimulus, US), the cue acquires the ability to evoke a CR in all rats; that is, it is equally predictive and supports learning the CS-US association in all. However, only in a subset of rats is the cue attributed with incentive salience, becoming an attractive and desirable incentive stimulus. We used in situ hybridization histochemistry to quantify the ability of a food cue to induce c-fos mRNA expression in rats that varied in the extent to which they attributed incentive salience to the cue. We found that a food cue induced c-fos mRNA in the orbitofrontal cortex, striatum (caudate and nucleus accumbens), thalamus (paraventricular, intermediodorsal and central medial nuclei), and lateral habenula, only in rats that attributed incentive salience to the cue. Furthermore, patterns of "connectivity" between these brain regions differed markedly between rats that did or did not attribute incentive salience to the food cue. These data suggest that the predictive value of a reward cue is not sufficient to engage brain reward systems-the cue must also be attributed with incentive salience.

Analysis of 5-HT6 and 5-HT7 receptor gene expression in rats showing differences in novelty-seeking behavior.

Sensation-seeking is a human personality trait associated with a greater propensity to use psychoactive substances. A rat model showing face validity of this human trait has been developed. The model is based on the variety of behavioral responses that rats exhibit in a novel and inescapable environment, with some animals (high-responders, HR) being highly active, and others (low-responders, LR) showing less exploration. More active rats (HR) also show increased drug-taking and decreased anxiety-like behavior. There is evidence that response to novelty may rely on differential 5-HT-mediated neurotransmission. This research focuses on the recently discovered 5-HT6 and 5-HT7 receptors which share affinity for neuroleptic drugs and hallucinogens. To date, emerging evidence suggests that 5-HT6 and 5-HT7 may be involved in cognition and mood regulation, respectively. To further our knowledge of their behavioral attributes, we compared patterns of gene expression for these receptors in the brains of HR and LR rats. As a control, gene expression for the 5-HT3 receptor was investigated because its contribution to anxiety and addiction is only weakly demonstrated. Transcript levels for 5-HT6 in the olfactory tubercle inversely correlated with the level of locomotion in a novel environment. Phenotype differences in mRNA signal for 5-HT6 showed a complex pattern in the dentate gyrus. LR rats were statistically higher in the most anterior region of the dentate gyrus, while HR rats were higher in median areas of the dentate gyrus. Levels of 5-HT7 transcript in HR rats were significantly lower than LR rats in pivotal areas for information trafficking, such as thalamo-cortical projection areas and dorsal hippocampus. By contrast, phenotype differences in 5-HT3 expression were not found in areas of the limbic cortex and mesolimbic system. Taken together, these results provide new insight into the potential contribution of 5-HT to novelty-seeking behavior and associated behaviors such as substance abuse.

Relation between the hypothalamic-pituitary-thyroid (HPT) axis and the hypothalamic-pituitary-adrenal (HPA) axis during repeated stress.

Previous work has indicated that acute and repeated stress can alter thyroid hormone secretion. Corticosterone, the end product of hypothalamic-pituitary-adrenal (HPA) axis activation and strongly regulated by stress, has been suggested to play a role in hypothalamic-pituitary-thyroid (HPT) axis regulation. In the current study, we sought to further characterize HPT axis activity after repeated exposure to inescapable foot-shock stress (FS), and to examine changes in proposed regulators of the HPT axis, including plasma corticosterone and hypothalamic arcuate nucleus agouti-related protein (AGRP) mRNA levels. Adult male Sprague-Dawley rats were subjected to one daily session of inescapable FS for 14 days. Plasma corticosterone levels were determined during and after the stress on days 1 and 14. Animals were killed on day 15, and trunk blood and brains were collected for measurement of hormone and mRNA levels. Repeated exposure to FS led to a significant decrease in serum levels of 3,5,3'-triiodothyronine (T3) and 3,5,3',5'-tetraiodothyronine (T4). Stress-induced plasma corticosterone levels were not altered by repeated exposure to the stress. Despite the decrease in peripheral hormone levels, thyrotropin-releasing hormone (TRH) mRNA levels within the paraventricular nucleus of the hypothalamus were not altered by the stress paradigm. Arcuate nucleus AGRP mRNA levels were significantly increased in the animals exposed to repeated FS. Additionally, we noted significant correlations between stress-induced plasma corticosterone levels and components of the HPT axis, including TRH mRNA levels and free T4 levels. Additionally, there was a significant correlation between AGRP mRNA levels and total T3 levels. Changes in body weight were also correlated with peripheral corticosterone and TRH mRNA levels. These results suggest that repeated exposure to mild-electric foot-shock causes a decrease in peripheral thyroid hormone levels, and that components of the HPA axis and hypothalamic AGRP may be involved in stress regulation of the HPT.

Differential distribution and regulation of OX1 and OX2 orexin/hypocretin receptor messenger RNA in the brain upon fasting.

To further understand the functions of the orexin/hypocretin system, we examined the expression and regulation of the orexin/hypocretin receptor (OX1R and OX2R) mRNA in the brain by using quantitative in situ hybridization. Expression of OX1R and OX2R mRNA exhibited distinct distribution patterns. Within the hypothalamus, expression for the OX1R mRNA was largely restricted in the ventromedial (VMH) and dorsomedial hypothalamic nuclei, while high levels of OX2R mRNA were contained in the paraventricular nucleus, VMH, and arcuate nucleus as well as in mammilary nuclei. In the amygdala, OX1R mRNA was expressed throughout the amygdaloid complex with robust labeling in the medial nucleus, while OX2R mRNA was only present in the posterior cortical nucleus of amygdala. High levels of OX2R mRNA were also observed in the ventral tegmental area. Moreover, both OX1R and OX2R mRNA were observed in the hippocampus, some thalamic nuclei, and subthalamic nuclei. Furthermore, we analyzed the effect of fasting on levels of OX1R and OX2R mRNA in the hypothalamic and amygdaloid subregions. After 20 h of fasting, levels of OX1R mRNA were significantly increased in the VMH and the medial division of amygdala. An initial decrease (14 h) and a subsequent increase (20 h) in OX1R mRNA levels after fasting were observed in the dorsomedial hypothalamic nucleus and lateral division of amygdala. Levels of OX2R mRNA were augmented in the arcuate nucleus, but remained unchanged in the dorsomedial hypothalamic nucleus, paraventricular hypothalamic nucleus, and amygdala following fasting. The time-dependent and region-specific regulatory patterns of OX1R and OX2R suggest that they may participate in distinct neural circuits under the condition of food deprivation.

Distinct neurochemical populations in the rat central nucleus of the amygdala and bed nucleus of the stria terminalis: evidence for their selective activation by interleukin-1beta.

The lateral division of the central nucleus of the amygdala (CEAl) and the oval nucleus of the bed nucleus of the stria terminalis (BSTov) have been linked closely anatomically and functionally. To determine whether these regions may be subdivided further on a neurochemical basis, dual in situ hybridization was used to determine the colocalization of corticotropin-releasing hormone (CRH), enkephalin (ENK), or neurotensin (NT) with glutamic acid decarboxylase isoforms 65 and 67 [used concurrently as a marker for gamma-aminobutyric acid GABA] in these nuclei. It was found that, for both regions, each peptide invariably was localized in a GABAergic cell. Although there was a similar overlap in the distribution of NT with ENK in the BSTov and CEAl, it was observed that CRH and ENK rarely were colocalized in either nucleus. To determine whether these distinct neuronal populations could be activated differentially, male rats were given a systemic injection of interleukin-1beta (IL-1beta; 5 microg/kg, i.p.), a stimulus that results in a robust increase in c-fos mRNA expression in the BSTov and CEAl. The neurochemical identity of these activated neurons showed striking similarities between the BSTov and the CEAl; All IL-1beta-responsive cells were GABAergic, the majority of c-fos- positive cells expressed ENK mRNA (BSTov, 81%; CEAl, 94%), and some expressed NT mRNA (BSTov, 23%; CEAl, 22%), whereas very few expressed CRH mRNA (BSTov, 4%; CEAl, 1%). These data provide evidence for the existence of discrete neural circuits within the BSTov and CEAl, and the similarities in the patterns of neurochemical colocalization in these nuclei are consistent with the concept of an extended amygdala. Furthermore, these data indicate that intraperitoneal IL-1beta recruits neurochemically distinct pathways within the BSTov and CEAl, and it is suggested that this differential activation may mediate specific aspects of immune, limbic, and/or autonomic processes.

Alpha, beta, and gamma mineralocorticoid receptor messenger ribonucleic acid splice variants: differential expression and rapid regulation in the developing hippocampus.

Two different types of corticoid receptor molecules bind circulating corticosterone in brain: mineralocorticoid receptors (MR) and glucocorticoid receptors. MR exhibit the highest affinity for the endogenous glucocorticoid in the rat, corticosterone. During development, low corticosterone levels influence neurogenesis, and these effects are probably MR mediated. Three MR complementary DNA clones, alpha, beta, and gamma, have been identified in the rodent. All of these MR complementary DNA clones have identical coding regions, but differ significantly at the 5'-untranslated end. Although the functional significance of these three messenger RNA (mRNA) species remains unknown, one hypothesis is that they reflect the ability of the brain to regulate the expression of MR, allowing multiple factors to differentially control transcription in a tissue- and time-specific manner. To investigate this possibility, we examined the presence of these distinct mRNA forms in the developing rat hippocampus (HC). In situ hybridization with specific alpha, beta, and gamma complementary RNA probes was performed in the HC of 3-, 5-, 7-, 12-, 14-, 28-, 35-, and 65-day-old animals. We found that there is differential expression of these forms in each of the HC subfields from infancy to adulthood. y expression appears to be associated with periods of cell birth and increased axonal sprouting. beta expression, on the other hand, may be best linked to periods of synaptogenesis, growth of commissural and associative terminal fields, and possibly active pruning. To explore the possibility that the differential gene expression may be related to corticosterone environment, adrenalectomy was performed. A rapid modulation of the MR mRNA variants (14 h) in an age- and site-specific fashion was seen. These findings suggest that the variation in expression and regulation during development of the multiple MR transcripts could reflect a complex pattern of developmental regulation that may involve a multitude of factors unique to each postnatal age and to the different neuronal populations within the hippocampal formation.

Immunohistochemical localization of the cloned kappa 1 receptor in the rat CNS and pituitary.

Several lines of evidence have demonstrated the presence of three opioid receptor types in the CNS and periphery. These receptors are referred to as mu, delta and kappa, and have been implicated in a wide variety of functions. The present study examines the localization of the kappa 1 receptor, a region of the receptor that has little homology with mu and delta receptors. Immunohistochemical studies in Zamboni-fixed rat tissue demonstrate immunoreactive perikarya and/or fibers in such regions as the deep layers of the parietal, temporal and occipital cortex, parasubiculum, central and medial amygdala, bed nucleus stria terminalis, nucleus accumbens, olfactory tubercle, endopiriform nucleus, claustrum, hypothalamic nuclei, median eminence, midline thalamic nuclei, zona incerta, central gray, caudal linear and dorsal raphe, substantia nigra, pars reticulata, ventral tegmental area, parabrachial nucleus, spinal trigeminal nucleus, nucleus of the solitary tract, spinal cord and the dorsal root ganglia. Specific kappa 1 receptor-like immunohistochemical staining is also observed in the pituitary, where immunoreactive perikarya and fibers are localized in the neural and intermediate lobes. Transfection and preabsorption controls suggest that the antibody is selective for the cloned kappa 1 receptor, and does not recognize mu or delta. This immunohistochemical localization corresponds well to previously described kappa 1 receptor mRNA and binding distributions and provides new insights into the cellular localization and pre- and postsynaptic organization of the kappa 1 receptor-like proteins in the rat brain and pituitary. The functional implications of these results are discussed in light of the kappa 1 receptors play in hormonal regulation, antinociception and reward.

Differential pattern of c-fos mRNA in rat brain following central and systemic administration of interleukin-1-beta: implications for mechanism of action.

Interleukin-1-beta (IL-1 beta) is a potent activator of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in the release of corticosteroids from the adrenal glands. This effect is evident after both central and peripheral administration, and controversy surrounds the mechanism(s) by which systemic administration of this peptide, which should not cross the blood-brain barrier, may activate the HPA axis. In the present study, IL-1 beta was administered systemically (5 micrograms/kg i.p.) or centrally (100 ng i.c.v.) to male rats. Both routes of administration of IL-1 beta resulted in significant and comparable activation of the HPA axis, as assessed by analysis of plasma conrticosterone. In addition, both routes of administration of IL-1 beta resulted in c-fos mRNA induction in specific regions, as determined by in situ hybridization. These included the meninges, cerebral vasculature, choroid plexus and circumventricular organs. Semiquantitative analysis revealed that both routes of administration resulted in significant and comparable induction of c-fos mRNA in the paraventricular nucleus of the hypothalamus, as compared with control animals. In contrast, in the nucleus tractus solitarius (NTS) and central nucleus of the amygdala (CeA), levels of c-fos mRNA were 3-4 times higher in animals treated intraperitoneally compared with intracerebroventricularly. A similar differential activation of c-fos mRNA was observed in the lateral divisions of the parabrachial nucleus (PBN) and bed nucleus of the stria terminalis (BNST). These data indicate that following systemic administration, IL-1 beta may activate specific brain areas through mechanisms distinct from those involved following central administration. The differential magnitude of the c-fos mRNA response in the NTS, PBN, CeA and BNST is consistent with vagal activation. Physiologically, these results suggest that IL-1 beta may have differential central effects depending on its source or point of entry to the brain.

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.

A chimeric study of the molecular basis of affinity and selectivity of the kappa and the delta opioid receptors. Potential role of extracellular domains.

Within the large family of G-protein-coupled receptors, a picture is emerging which contrasts the binding of small ligands and the binding of peptides to the seven-helix configuration of the proteins. Because of its unique richness in both peptide and non-peptide ligands, the opioid receptor family offers several advantages for achieving a better understanding of similarities and differences in ligand/receptor interactions across different classes of agonists and antagonists. Since multiple, naturally occurring, ligands interact with the multiple receptors with varying degrees of selectivity, this family is also an excellent model for examining the structural basis of selectivity. Thus, the molecular basis of binding affinity and selectivity of the kappa and the delta opioid receptors was investigated by the construction of four kappa/delta chimeric receptors. The pharmacological profiles of these chimeras as well as those of the wild type kappa and delta receptors were determined by their binding with several different categories of opioid ligands. A linear model was used to deduce the relative contribution of each corresponding pairs of kappa-delta receptor segments to the binding of a given ligand. The results show that the kappa and delta receptors bind the same opioid core differently and achieve their selectivity through different mechanisms. In addition, the interaction of a peptide ligand with a receptor appears to be different from that of a small ligand. Furthermore, these results point to a particularly important role of the second extracellular loop and the top half of transmembrane domain 4 in the binding of prodynorphin products. Together, the results suggest that these peptide receptors can be bound and activated via multiple binding pockets as a function of their own topography and the nature of the interacting ligand.

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.

Primary structure and functional expression of a guinea pig kappa opioid (dynorphin) receptor.

A full-length cDNA encoding the guinea pig kappa opioid (dynorphin) receptor has been isolated. The deduced protein contains 380 aa and seven hydrophobic alpha-helices characteristic of the G protein-coupled receptors. This receptor is 90% identical to the mouse and rat kappa receptors, with the greatest level of divergence in the N-terminal region. When expressed in COS-7 cells, the receptor displays high affinity and stereospecificity toward dynorphin peptides and other kappa-selective opioid ligands such as U50, 488. It does not bind the mu- and delta-selective opioid ligands. The expressed receptor is functionally coupled to G protein(s) to inhibit adenylyl cyclase and Ca2+ channels. The guinea pig kappa receptor mRNA is expressed in many brain areas, including the cerebellum, a pattern that agrees well with autoradiographic maps of classical guinea pig kappa binding sites. Species differences in the pharmacology and mRNA distribution between the cloned guinea pig and rat kappa receptors may be worthy of further examination.

Cloning and pharmacological characterization of a rat kappa opioid receptor.

A full-length cDNA was isolated from a rat striatal library by using low-stringency screening with two PCR fragments, one spanning transmembrane domains 3-6 of the mouse delta opioid receptor and the other unidentified but homologous to the mouse delta receptor from rat brain. The novel cDNA had a long open reading frame encoding a protein of 380 residues with 59% identity to the mouse delta receptor and topography consistent with a seven-helix guanine nucleotide-binding protein-coupled receptor. COS-1 cells transfected with the coding region of this clone showed high-affinity binding to kappa opioid receptor-selective ligands such as dynorphin A and U-50,488 and also nonselective opioid ligands such as bremazocine, ethylketocyclazocine, and naloxone. Not bound at all (or bound with low affinity) were dynorphin A-(2-13), enantiomers of naloxone and levophanol [i.e., (+)-naloxone and dextrorphan], and selective mu and delta opioid receptor ligands. Activation of the expressed receptor by kappa receptor agonists led to inhibition of cAMP. Finally, in situ hybridization revealed a mRNA distribution in rat brain that corresponded well to the distribution of binding sites labeled with kappa-selective ligands. These observations indicate that we have cloned a cDNA encoding a rat kappa receptor of the kappa 1 subtype.

A bacterially expressed mineralocorticoid receptor is associated in vitro with the 90-kilodalton heat shock protein and shows typical hormone- and DNA-binding characteristics.

A recombinant system was developed for generation of steroid-receptor complexes in vitro. The DNA- and steroid-binding domains of the rat mineralocorticoid receptor were expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The identity of the expressed recombinant protein was confirmed by Western blot analysis. Protein preparations purified by affinity chromatography, avoiding the use of detergents or high ionic strength buffers, exhibited negligible steroid binding. However, after incubation of these preparations with rabbit reticulocyte lysate, known to promote the association of isolated steroid receptors with heat shock proteins, the [3H]aldosterone-binding activity gradually increased. This temperature-dependent effect reached a maximum after 1 h at 30 degrees C and was favored by ATP supplementation (Bmax = 22 +/- 3 pmol/mg of protein). The apparent Kd value for aldosterone (0.6 +/- 0.2 nM) and the steroid-binding specificity of the recombinant protein were in accordance with those reported for the native mineralocorticoid receptor. The sedimentation and DNA-cellulose-binding characteristics of the radioactive complexes were also in agreement with those reported for the native heteromeric receptor. Complexes sedimented at 8.9 +/- 0.2 or 4.2 +/- 0.2 S in sucrose gradients containing 20 mM sodium molybdate or 0.4 M KCl, respectively. Monoclonal antibody 8D3 against the 90-kDa heat shock protein (hsp90) was able to bind to the 8.9S complexes, increasing its sedimentation coefficient. Treatment of the complexes with 100 mM sodium thiocyanate, known to activate the native receptor to a DNA-binding state, caused a 79% increase in DNA-cellulose binding over the control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Diurnal CRH mRNA rhythm in the hypothalamus: decreased expression in the evening is not dependent on endogenous glucocorticoids.

Levels of hypothalamic corticotropin-releasing hormone (CRH) mRNA and plasma glucocorticoids vary diurnally as a result of circadian influences on the hypothalamopituitary-adrenal axis. CRH mRNA expression increases from morning to afternoon in rats but decreases rapidly near the onset of dark as glucocorticoids reach peak concentrations in plasma. Since glucocorticoids are normally inhibitory on hypothalamic CRN mRNA expression, we determined whether the glucocorticoid secretion at the diurnal peak reduced CRH mRNA concentration in the evening. We found that adrenalectomy did not prevent the decrease in CRH mRNA levels near the onset of dark. It appears that the drop in CRH mRNA expression occurs via a steroid-independent mechanism. While the mean CRH mRNA level increased after adrenalectomy, the shape of the CRH mRNA rhythm remained unchanged except in the morning. Interestingly, adrenalectomy increased CRH mRNA levels disproportionately in the morning, producing a sharp rise followed by a plateau during the light phase instead of the gradual rise observed in intact animals. We subsequently treated adrenalectomized animals with corticosterone pellets to determine whether a constant steroid signal was sufficient in restoring the normal shape of the mRNA rhythm during the light phase. Results indicate that the endogenous steroid rhythm is not necessary for generating the normal CRH mRNA rhythm during the light phase. Instead, a constant exposure to corticosterone at approximately 50% of the daily mean (2.4-3 micrograms/dl) appears to be sufficient for regulation of the mRNA rhythm.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of morphine treatment on pro-opiomelanocortin systems in rat brain.

In previous studies to determine whether chronic opiate administration might negatively feedback upon endogenous opioid systems in the CNS, investigators found no changes in steady-state concentrations of opioid peptides following morphine pelleting. However, since only steady-state levels were measured, it was still not clear whether morphine treatment altered the release and/or biosynthesis of opioid-containing neurons. The goal of the present study was to assess the effects of chronic morphine pelleting on the dynamics of beta-endorphin (beta E) biosynthesis in rats. Hence, at several times during a 7-day morphine treatment, concentrations of total beta E-immunoreactivity (-ir), as well as chromatographically sieved forms of beta E, were determined by RIA, and mRNA levels of pro-opiomelanocortin (POMC) were measured by a solution phase protection assay using a mouse or rat POMC 32P-labelled riboprobe. Concentrations of total beta E-ir or different forms of beta E-ir peptides (i.e. beta-lipotropin, beta E1-31, or beta E1-27/beta E1-26) in the hypothalamus or midbrain following either 1 or 7 days of treatment were similar in morphine- and placebo-pelleted animals. However, a significant increase in total hypothalamic beta E-ir was observed following 3 days of morphine pelleting; chromatographic analyses indicated that this was primarily due to a selective increase in the opiate inactive forms of beta E, i.e. beta E1-27/beta E1-26. After 7 days of pelleting, morphine-treated animals tended to have lower POMC mRNA levels than those of placebo controls (20 to 50% decrease in different studies). The accumulation of hypothalamic beta E-ir at 3 days, and the apparent decline in POMC mRNA levels at 7 days, lend support to the hypothesis that morphine negatively feeds back upon POMC neurons in the brain by inhibiting beta E release and biosynthesis.

In vitro release of hypothalamic beta-endorphin (beta E) by arginine vasopressin, corticotropin-releasing hormone and 5-hydroxytryptamine: evidence for release of opioid active and inactive beta E forms.

The aims of the present experiments were: 1) to test whether substances which modulate beta-endorphin-immunoreactive (beta E-ir) release from the pituitary gland might act similarly in hypothalamic tissue; and 2) to further characterize the beta E-ir forms which are released from hypothalamus. To address these questions, hypothalamic tissue was incubated in vitro for 10 min periods in either normal media (basal conditions) or in media containing 55 mM KCl or one of several other test substances (stimulation conditions) and release was estimated by measuring the beta E-ir concentrations in the media. Depolarizing concentrations of K+ increased beta E-ir release 2-3 fold over basal levels and this effect appeared to be Ca2(+)-dependent. Dose-dependent increases in beta E-ir release were elicited by nanonolar to micromolar concentrations of either corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), or 5-hydroxytryptamine (5-HT). Conversely, dopamine (1 microM) inhibited both the basal and K(+)-stimulated release of beta E-ir from hypothalamus. Gel filtration chromatography revealed that beta E1-31 and beta E1-27/beta E1-26 were the primary beta E-ir peptides released under either basal or CRH-stimulated conditions; the relative amounts of the beta E-ir peptides found in the media were nearly identical to those found in the hypothalamus itself. This result indicates that the release of different beta E-ir peptides into the media appears to be proportional to the relative amounts stored in tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Two precursors of melanin-concentrating hormone: DNA sequence analysis and in situ immunochemical localization.

Two precursors to Chinook salmon (Oncorhynchus tshawytscha) melanin-concentrating hormone, an important factor in teleosts involved in the control of skin pigmentation and stress responsiveness, have been identified from DNA sequence analysis. Both precursors encode proteins of 132 amino acids and they share 107/132 amino acid identities. The biologically active 17-residue peptide is located at the C terminus of both precursors and can be liberated by proteolytic cleavage following two adjacent arginine residues. Additional putative proteolytic processing sites are located within the two precursors. Northern analysis demonstrated an intense hybridization signal of 750 nucleotides in the hypothalamus. Immunocytochemical studies as well as in situ hybridization analyses identify intensely staining cell bodies in the hypothalamus in the area of the lateral tuberal nucleus.

The posttranslational processing of prodynorphin in the rat anterior pituitary.

The posttranslational processing of prodynorphin (Pro-Dyn) is not well understood. The rat anterior pituitary is an interesting tissue which merits examination to address this issue since it is known that Dyn immunoreactivity is stored as high mol wt (HMW) intermediates and not as free products such as dynorphin-A-(1-17) (Dyn-A17) or dynorphin-B-(1-13) (Dyn-B). The aim of our study is to characterize the Pro-Dyn products in the rat anterior pituitary quantitatively as well as qualitatively by keeping a close account of each of the possible domains that are known to compose the protein structure. This was achieved by a convergence of tools: designing RIA with antibodies to each of these domains, including antibodies to Dyn-A17, Dyn-B, alpha-neo-endorphin, bridge peptide, and Pro-Dyn carboxyl-terminal peptide (C-peptide), and using these antisera with gel filtration chromatography, reverse phase HPLC, immunoaffinity, and immunoprecipitation techniques. Our data indicate the presence of at least six distinct molecules which are classified as HMW intermediates (greater than 3.5K). By gel filtration chromatography they have apparent mol wt of 16,000, 10,000, 8,000, 6,000, 4,000, and 3,500, respectively. Each of these structures is characterized by multiple immunoreactivities to account for the observed mass. Based on the relative content of each structure we present a scheme for the posttranslational processing pathway of Pro-Dyn in the rat anterior pituitary. We also analyze other tissues, spinal cord and hypothalamus, for their content of Pro-Dyn HMW intermediates. Our results indicate that these tissues store Pro-Dyn HMW molecules of similar sizes and immunoreactive properties, suggesting that Pro-Dyn may be processed in a similar manner, at least in the initial phases, across tissues.