Effect of intracerebroventricular administration of the octadecaneuropeptide on the expression of pro-opiomelanocortin,neuropeptide Y and corticotropin-releasing hormone mRNAs in rat hypothalamus

Intracerebroventricular (i.c.v.) administration of the octadecaneuropeptide (diazepam-binding inhibitor [33-50]; ODN) exerts a potent anorexigenic effect in the rat. We studied the effect of ODN on three neuropeptides involved in feeding behaviour: the orexigenic peptide neuropeptide Y (NPY) and two anorexigenic peptides, corticotropin-releasing hormone (CRH) and the pro-opiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone. The effect of i.c.v. administration of ODN (0.1 microg/kg and 1 microg/kg) on mRNA expression of the peptides in male rat hypothalamus was evaluated by semiquantitative in situ hybridization. In the arcuate nucleus, NPY-expressing neurones were mostly found in the inner zone in close proximity of the third ventricle. ODN at the dose of 0.1 microg/kg induced a significant decrease of 17.4% in NPY mRNA expression, while the depressing effect was more marked (31.4%) with the highest dose of ODN (1 microg/kg). POMC-expressing neurones were more laterally located in the arcuate nucleus. Administration of ODN at 0.1 microg/kg and 1 microg/kg doses induced increases of 33.5% and 27.4% in POMC mRNA expression, respectively. Labelling obtained with the CRH cRNA probe was essentially distributed throughout the medial parvocellular area of the hypothalamic paraventricular nucleus. ODN, at doses of 0.1 and 1 microg/kg, resulted in 17.8% and 32.8% decreases in CRH mRNA expression, respectively. The present data suggest that ODN might exert its anorexigenic effect by increasing mRNA expression of POMC and decreasing mRNA expression of NPY in the arcuate nucleus.     

Glutamate-stimulated neuropeptide Y mRNA expression in the rat dentate gyrus: A prominent role of metabotropic glutamate receptors

The influence of intrahippocampal injections of glutamate receptor agonists on neuropeptide Y (NPY) mRNA expression was investigated in granule cells and interneurons of the rat dentate gyrus. One day after local injection of non-neurodegenerative doses (20 and 70 nmol) of the metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)ACPD], NPY mRNA levels were more than doubled in ipsilateral granule cells and interneurons. Doses of 200 and 400 nmol caused up to 15.9- and 4.6-fold mRNA increases in granule cells and interneurons, respectively. The group I metabotropic glutamate receptor agonist (RS)-3,5-dihydroxyphenylglycine (DHPG; 50 nmol), but not the group III receptor agonist L(+)-2-amino-4-phosphonobutyrate (L-AP4; 20 and 200 nmol) exerted a similar action. The general metabotropic glutamate receptor antagonist (+)-α-methyl-4-carboxyphenylglycine (MCPG; 200 nmol), the group I receptor antagonist (S)-4-carboxyphenylglycine (4-CPG; 200 nmol) and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (1 mg/kg; i.p.) partially blocked the (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate-induced increase in NPY mRNA in granule cells, but not in interneurons. (S)-4-carboxyphenylglycine (200 nmol) by itself increased NPY mRNA levels in ipsilateral interneurons threefold, indicating the activation of phospholipase D coupled receptors. Non-neurodegenerative doses of (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA, 0.3 nmol) caused modest increases in NPY mRNA levels in ipsilateral interneurons, whereas neurodegenerative doses (1–10 nmol) induced markedly increased NPY mRNA levels in granule cells (up to 11-fold) and interneurons (up to threefold). It is suggested that activation of metabotropic glutamate receptors stimulates NPY mRNA expression in granule cells and interneurons in the rat dentate gyrus. Whereas in granule cells NPY mRNA upregulation is preferentially mediated by group I metabotropic glutamate receptors, it may involve ionotropic and metabotropic glutamate receptors in interneurons. Hippocampus 1998;8:274–288. © 1998 Wiley-Liss, Inc.     

Tachykinin-induced stimulation of neuropeptide Y gene expression in the rat arcuate nucleus

Previous neurocytochemical data indicate the presence of synaptic contacts between tachykinergic terminals and neuropeptide Y (NPY) neurons in the arcuate nucleus of the rat suggesting that tachykinins may regulate NPY neuronal activity. To examine the functional signification of such regulation, the effect of intracerebroventricular administration of neurokinin A on NPY mRNA levels was studied using in situ hybridization. Repeated treatment with NKA (40 microg/day for 3 days) induced a 44% increase in NPPY mRNA expression compared with saline-injected control animals. These results demonstrate a positive effect of tachykinins on NPY gene expression and suggest either a direct or indirect control of arcuate NPY neurons by endogenous tachykinins.     

Oestrogenic regulation of pro-opiomelanocortin, neuropeptide Y and corticotrophin-releasing hormone mRNAs in mouse hypothalamus

It is well documented that oestrogen suppresses food intake by an action at the hypothalamic level. Using in situ hybridisation, we studied the effect of castration (CX) and short-term administration of oestradiol (E2) in CX female mice for three neuropeptides involved in feeding behaviour: two anorexigenic peptides, (i) the pro-opiomelanocortin (POMC)-derived peptide alpha-melanocyte-stimulating hormone and (ii) corticotrophin-releasing hormone (CRH), and the orexigenic peptide, (iii) neuropeptide Y (NPY). POMC-expressing neurones were mostly laterally located in the arcuate nucleus. POMC mRNA expression was decreased following CX and a single injection of E2 induced an increase in mRNA levels at 12- and 24-h time intervals. In the parvocellular area of the paraventricular nucleus, CRH mRNA levels were similarly decreased after CX and completely restored to normal levels at 12 and 24 h following E2 injection. On the other hand, the levels of NPY mRNA expressed in neurones located in the inner zone of the arcuate nucleus were increased by CX and decreased to the levels observed in intact animals by E2 injection (3-24 h). The present data suggest that oestrogen might exert an anorexigenic action by stimulating POMC and CRH mRNA expression and decreasing NPY mRNA expression in the hypothalamus.     

The Anorexigenic Action of the Octadecaneuropeptide (ODN) in Goldfish is Mediated Through the MC4R- and Subsequently the CRH Receptor-Signaling Pathways

Intracerebroventricular (ICV) administration of the octadecaneuropeptide (ODN), a peptide derived from diazepam-binding inhibitor, reduces food intake in goldfish as in rodents. However, the neurochemical pathways involved in the anorexigenic action of ODN have not yet been identified in goldfish. Alpha-melanocyte-stimulating hormone (α-MSH), corticotropin-releasing hormone (CRH), and CRH-related peptides play a major role in the control of food consumption in goldfish. In this species, the anorexigenic action of α-MSH is mediated via the CRH/CRH receptor neuronal system. Therefore, in the present study, we examined whether the anorexigenic effect of ODN in goldfish could be mediated through α-MSH and/or CRH neuronal pathways. ICV injection of ODN (10 pmol/g body weight (BW)) significantly reduced food intake, and the anorexigenic effect of ODN was suppressed by ICV preinjection of the melanocortin 4 receptor (MC4R) antagonist HS024 (40 pmol/g BW) or the CRH receptor 1/receptor 2 antagonist α-helical CRH_(9–41) (100 pmol/g BW). ICV injection of ODN (10 pmol/g BW) induced a significant increase of proopiomelanocortin mRNA level but had no effect on CRH mRNA level, while ICV injection of the MC4R agonist, melanotan II (100 pmol/g BW), significantly enhanced CRH mRNA expression. These results suggest that, in goldfish, the anorexigenic action of ODN is mediated by the MC4R- and subsequently through the CRH receptor-signaling pathways.     

Differential behavioural effects of chronic infusion of CRH 1 and CRH 2 receptor antisense oligonucleotides into the rat brain

The purpose of this study was to investigate the functional role the two corticotropin-releasing hormone (CRH) receptor subtypes play in regulating the behavioural performance of rats in various well-defined test situations. Antisense oligodeoxynucleotides (ODNs) corresponding to either the rat CRH1 or CRH2 receptor mRNA were infused chronically into the lateral ventricle of male rats via osmotic minipumps (5 microg/0.5 microl/h over 6 days). Control groups received infusions of either a scrambled sequence ODN or mixed bases ODN or vehicle. On day 4 after surgery, the rats were subjected to 10 min of social defeat and immediately afterwards tested on the elevated plus-maze. Compared to a scrambled sequence control ODN, CRH1 receptor antisense ODN infusion was found to exert an anxiolytic-like effect whereas CRH2 receptor antisense ODN infusion had no effect on defeat-induced anxiety-related behaviour. In contrast, the CRH2 receptor antisense ODN increased immobility in a forced swim test whereas CRH1 receptor ODN-treated rats did not differ from controls. No influence of either ODN was found on general locomotor activity in an open field or on short-term memory performance in a social discrimination test. Furthermore, the CRH2 receptor antisense ODN did not affect spatial learning in a Morris water maze task. An additional experiment comparing a mixture of both missense ODNs and a vehicle control group confirmed that the former failed to induce non-specific (toxic) side effects, further substantiating the specificity of the respective antisense effects measured in this study. The results support the hypothesis that the two CRH receptor subtypes selectively mediate differential effects of endogenous CRH or CRH-related peptides at the brain level with the CRHI receptor contributing predominantly to emotional behaviour and the CRH2 receptor being involved in the regulation of stress coping behaviour.     

Effects of Long-Term Treatment with Antidepressant Drugs on Proopiomelanocortin and Neuropeptide Y mRNA Expression in the Hypothalamic Arcuate Nucleus of Rats

Antidepressant drugs have in common a delayed onset of clinical efficacy. In rats, long-term, daily administration of four different types of clinically effective antidepressant drugs results in decreased corticotropin releasing hormone (CRH) mRNA expression levels in the hypothalamic paraventricular nucleus (PVN). Because a subpopulation of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (Arc) projects to the PVN, we measured NPY and POMC mRNA expression in the Arc using in situ hybridization histochemistry at several time points following daily administration of four different antidepressant drugs. After 14 and 56 days of imipramine treatment, Arc NPY mRNA levels are decreased to 85% and 75% of control levels, but are unchanged compared to control after one or five days of treatment. Arc POMC mRNA levels are unchanged compared to controls at 1, 5, 14, or 56 days following imipramine treatment. Unlike after imipramine, Arc NPY and POMC mRNA levels are increased significantly to 134–172% of control following 56-day treatment with the antidepressant drugs fluoxetine, phenelzine, or idazoxan. The divergent effects of imipramine vs the other 3 antidepressant drugs on Arc NPY mRNA expression are similar to the pattern of changes in tyrosine hydroxylase (TH) mRNA expression levels in the locus coeruleus (LC) using the same experimental paradigm, but are different from the unidirectional depressive effects of all four drugs on CRH mRNA expression in the PVN. Thus, the Arc NPY and LC noradrenergic systems may act coordinately in mediating antidepressant effects. The present data are consistent with the delayed onset of clinical efficacy for antidepressant drugs, and suggest that Arc NPY and POMC neurotransmitter systems play a role in the pathophysiology of depression.     

Corticotropin-releasing hormone (CRH) downregulates the function of its receptor (CRF1) and induces CRF1 expression in hippocampal and cortical regions of the immature rat brain

In addition to regulating the neuroendocrine stress response, corticotropin-releasing hormone (CRH) has been implicated in both normal and pathological behavioral and cognitive responses to stress. CRH-expressing cells and their target neurons possessing CRH receptors (CRF1 and CRF2) are distributed throughout the limbic system, but little is known about the regulation of limbic CRH receptor function and expression, including regulation by the peptide itself. Because CRH is released from limbic neuronal terminals during stress, this regulation might play a crucial role in the mechanisms by which stress contributes to human neuropsychiatric conditions such as depression or posttraumatic stress disorder. Therefore, these studies tested the hypothesis that CRH binding to CRF1 influenced the levels and mRNA expression of this receptor in stress-associated limbic regions of immature rat. Binding capacities and mRNA levels of both CRF1 and CRF2 were determined at several time points after central CRH administration. CRH downregulated CRF1 binding in frontal cortex significantly by 4 h. This transient reduction (no longer evident at 8 h) was associated with rapid increase of CRF1 mRNA expression, persisting for >8 h. Enhanced CRF1 expression-with a different time course-occurred also in hippocampal CA3, but not in CA1 or amygdala, CRF2 binding and mRNA levels were not altered by CRH administration. To address the mechanisms by which CRH regulated CRF1, the specific contributions of ligand-receptor interactions and of the CRH-induced neuronal stimulation were examined. Neuronal excitation without occupation of CRF1 induced by kainic acid, resulted in no change of CRF1 binding capacity, and in modest induction of CRF1 mRNA expression. Furthermore, blocking the neuroexcitant effects of CRH (using pentobarbital) abolished the alterations in CRF1 binding and expression. These results indicate that CRF1 regulation involves both occupancy of this receptor by its ligand, as well as "downstream" cellular activation and suggest that stress-induced perturbation of CRH-CRF1 signaling may contribute to abnormal neuronal communication after some stressful situations.     

Anorectic Effect of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in Rats: Lack of Evidence for Involvement of Hypothalamic Neuropeptide Gene Expression

We investigated the effect of centrally administered pituitary adenylate cyclase activating polypeptide (PACAP) on feeding in rats, and the involvement of hypothalamic neuropeptide gene expression using in situ hybridization. lntracerebroventricular injection of PACAP (1000  pmol/rat) significantly decreased food intake in a dose-dependent manner. In PACAP-treated rats, neuropeptide Y (NPY) mRNA levels in the arcuate nucleus and galanin mRNA levels in the paraventricular nucleus increased, and corticotropin-releasing hormone (CRH) mRNA levels in the paraventricular nucleus decreased. In rats fasted for 72  h, NPY mRNA levels increased, and CRH mRNA levels decreased, but galanin mRNA levels were unchanged. These results indicate that the anorectic function of PACAP is not mediated by NPY or CRH, and that PACAP increases galanin synthesis.     

Intrahippocampal infusion of antisense oligodeoxynucleotide to the GABA(A) receptor gamma2 subunit enhances neuropeptide Y gene expression

The effects of hippocampal treatment with a phosphorothioate oligodeoxynucleotide (ODN) antisense to the gamma-aminobutyric acid (GABA)A receptor gamma2 subunit on neuropeptide Y (NPY) were studied. Adult male Wistar rats were treated with unilateral intrahippocampal infusion of gamma2 subunit antisense ODN for 5 days. Rats infused with mismatch ODN and na?ve rats served as controls. Brain sections were analysed for levels of NPY mRNA by in situ hybridisation, NPY-immunoreactivity (NPY-ir) by means of immunocytochemistry, and specific NPY binding sites by in vitro receptor autoradiography. Following infusion of antisense ODN, a marked increase in cytoplasmic NPY-ir was observed in hilar neurones of the fascia dentata. Further, intense NPY-ir was visualised in the mossy fibres and in cell bodies of the entorhinal cortex and throughout the neocortex. High levels of NPY mRNA were detected in the same cortical areas of antisense treated rats. A very large increase was observed in the piriform and parietal areas. NPY gene expression also occurred in the granular cell layer, in which no NPY mRNA could be detected in normal animals. The level and distribution of cells displaying high levels of NPY mRNA differed among animals, perhaps as a result of the distinct anatomical location of ODN infusion. Finally, hippocampal levels of NPY specific binding increased, suggesting that NPY neurotransmission is markedly increased. These findings are reminiscent of reported changes in the expression of NPY mRNA and immunoreactivity in conditions of increased neuronal excitation and support the usefulness of the present animal model for the study of epileptic phenomena.     

Influence of Adrenal Glands on the Modulation of Prolactin Gene Expression by the Endogenous Benzodiazepine Ligand Octadecaneuropeptide in the Male Rat Pituitary Gland

Recently, an 86-amino acid polypeptide with high affinity for diazepam binding sites, termed diazepam-binding inhibitor (DBI), has been found in the rat brain. DBI, as well as a peptide derived from DBI, the octadecaneuropeptide DBI[33–50] (ODN), interacts with the GABA_A receptor complex. To investigate the role of these endogenous ligands for GABA_A receptors on prolactin gene expression, we studied the effects of acute intracerebroventricular administration (4  h before sacrifice) of ODN on prolactin mRNA levels in the male rat. Because, in some neuropeptidergic systems, glucocorticoids play a role in the response to ODN, we also studied the influence of adrenal glands and the effect of dexamethasone administration in the response of prolactin gene expression to ODN. ODN injection produced an increase in prolactin mRNA levels. Adrenalectomy performed 5 days before sacrifice resulted in an increase in prolactin gene expression and also potentiated the stimulating effect of ODN. Because castration has been shown to decrease prolactin gene expression in the male rat, we used castrated and adrenalectomized animals to study the role of dexamethasone in the response of lactotrophs to ODN. In these steroid-deprived animals, dexamethasone treatment (for 4 days) decreased prolactin mRNA levels but did not modify the response to ODN. These data indicate that an endogenous neuropeptide interacting with the GABA_A receptor complex can stimulate prolactin gene expression and suggest that the adrenal glands may produce factor(s) capable of decreasing prolactin mRNA. On the other hand, it does not appear that glucocorticoid hormones play a role in the effect of ODN on lactotroph activity.     

Intrahippocampal infusion of antisense oligodeoxynucleotide to the GABAA receptor γ2 subunit enhances neuropeptide Y gene expression

The effects of hippocampal treatment with a phosphorothioate oligodeoxynucleotide (ODN) antisense to the γ-aminobutyric acid (GABA)_A receptor γ2 subunit on neuropeptide Y (NPY) were studied. Adult male Wistar rats were treated with unilateral intrahippocampal infusion of γ2 subunit antisense ODN for 5 days. Rats infused with mismatch ODN and naïve rats served as controls. Brain sections were analysed for levels of NPY mRNA by in situ hybridisation, NPY-immunoreactivity (NPY-ir) by means of immunocytochemistry, and specific NPY binding sites by in vitro receptor autoradiography. Following infusion of antisense ODN, a marked increase in cytoplasmic NPY-ir was observed in hilar neurones of the fascia dentata. Further, intense NPY-ir was visualised in the mossy fibres and in cell bodies of the entorhinal cortex and throughout the neocortex. High levels of NPY mRNA were detected in the same cortical areas of antisense treated rats. A very large increase was observed in the piriform and parietal areas. NPY gene expression also occurred in the granular cell layer, in which no NPY mRNA could be detected in normal animals. The level and distribution of cells displaying high levels of NPY mRNA differed among animals, perhaps as a result of the distinct anatomical location of ODN infusion. Finally, hippocampal levels of NPY specific binding increased, suggesting that NPY neurotransmission is markedly increased. These findings are reminiscent of reported changes in the expression of NPY mRNA and immunoreactivity in conditions of increased neuronal excitation and support the usefulness of the present animal model for the study of epileptic phenomena.     

Vascular neuropeptide Y Y1-receptors in the rat kidney: vasoconstrictor effects and expression of Y1-receptor mRNA

Neuropeptide Y (NPY) -receptor subtypes were studied in the rat kidney in vivo by systemic administration of NPY, the two agonists [Leu(31), Pro(34)]NPY (Y1-receptor agonist) and NPY (13-36) (Y2-receptor agonist), or the Y1-receptor antagonist BIBP 3226. Effects on mean arterial blood pressure (MAP) and renal arterial blood flow were recorded. The Y1-receptor agonist evoked a dose-dependent increase in MAP concomitantly with a reduction in renal blood flow. At the largest dose administered (1.42 pmol/g), the Y1-agonist [Leu(31), Pro(34)] NPY increased MAP by 20 +/- 6 mmHg and reduced the renal vascular conductance by more than 50%. The same dose of the Y2-agonist NPY (13-36) did not evoke any clear-cut effects on the renal blood flow or MAP. Furthermore, administration of the Y1-receptor antagonist BIBP 3226 reduced the NPY-induced renal vasoconstriction, but did not affect the response to angiotensin II or phenylephrine. The effects evoked by 0.71 pmol/g NPY were almost abolished by 3 mg/kg BIBP 3226. In situ hybridization histochemistry was used to study the expression of Y1-receptor mRNA in the developing rat kidney. The levels of Y1-receptor mRNA expression in the vascular smooth muscle of the rat kidney varied at different ages, with low levels at postnatal day 10 and high levels at 20 days and again low levels at 40 days. In summary, the present study show a maturation-specific expression pattern of NPY Y1-receptor mRNA as well as functional effects of vascular NPY receptors of the Y1-subtype in the rat kidney.     

Expression of spinal cord Fos protein in response to intrathecal adrenomedullin and CGRP in conscious rats

Adrenomedullin (AM) immunoreactivity and mRNA, in addition to a large number of specific AM-binding sites, exist in the rat spinal cord. However, no phenotype has been reported for AM in the spinal cord. Here, expression of c-fos in response to intrathecal (i.t.) administration of AM, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) was examined in the thoracic, lumbar and sacral regions of spinal cord in conscious rats. Two hours after i.t. administration of either CGRP (2.5 and 10 microg) or AM (10 microg), the number of c-Fos immunoreactive nuclei was increased in all the spinal regions examined in this study, with the highest increase observed in the superficial dorsal horn. Few cells with c-fos immunoreactivity were found in the spinal cord of rats 2 h after i.t. injection of either saline or PAMP. Effects of AM (10 microg) and CGRP (2.5 microg) on c-fos expression were blocked when rats were pretreated with 40 microg of intrathecal CGRP8-37 (CGRP1 receptor antagonist). Fos-like immunoreactivity induced by i.t. CGRP and/or AM were also significantly abolished by i.t. administration of the nitric oxide (NO) inhibitor, l-NAME, indicating that endogenous NO is a necessary intermediary in CGRP and AM induced c-fos expression in the rat spinal cord. In conclusion, AM induces c-fos expression in rat spinal cord when administered intrathecally, with the pattern being similar to those produced by i.t. CGRP. Effects of the two peptides are sensitive to CGRP8-37 and l-NAME.     

Serotonergic stimulation of corticotropin-releasing hormone and pro-opiomelanocortin gene expression

The neurotransmitter serotonin (5-HT) stimulates adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary gland via activation of central 5-HT1 and 5-HT2 receptors. The effect of 5-HT is predominantly indirect and may be mediated via release of hypothalamic corticotropin-releasing hormone (CRH). We therefore investigated the possible involvement of CRH in the serotonergic stimulation of ACTH secretion in male rats. Increased neuronal 5-HT content induced by systemic administration of the precursor 5-hydroxytryptophan (5-HTP) in combination with the 5-HT reuptake inhibitor fluoxetine raised CRH mRNA expression in the paraventricular nucleus (PVN) by 64%, increased pro-opiomelanocortin (POMC) mRNA in the anterior pituitary lobe by 17% and stimulated ACTH secretion five-fold. Central administration of 5-HT agonists specific to 5-HT1A, 5-HT1B, 5-HT2A or 5-HT2C receptors increased CRH mRNA in the PVN by 15-50%, POMC mRNA in the anterior pituitary by 15-27% and ACTH secretion three- to five-fold, whereas a specific 5-HT3 agonist had no effect. Systemic administration of a specific anti-CRH antiserum inhibited the ACTH response to 5-HTP and fluoxetine and prevented the 5-HTP and fluoxetine-induced POMC mRNA response in the anterior pituitary lobe. Central or systemic infusion of 5-HT increased ACTH secretion seven- and eight-fold, respectively. Systemic pretreatment with the anti-CRH antiserum reduced the ACTH responses to 5-HT by 80% and 64%, respectively. It is concluded that 5-HT via activation of 5-HT1A, 5-HT2A, 5-HT2C and possibly also 5-HT1B receptors increases the synthesis of CRH in the PVN and POMC in the anterior pituitary lobe, which results in increased ACTH secretion. Furthermore, the results indicate that CRH is an important mediator of the ACTH response to 5-HT.     

Distribution of corticotropin releasing hormone receptor immunoreactivity in the rat hypothalamus: coexpression in neuropeptide Y and dopamine neurons in the arcuate nucleus

An abundance of physiological data suggests an interaction between neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH) in the regulation of endocrine and autonomic functions. Previously, studies in our laboratory have indicated that NPY neurons in the arcuate nucleus of the hypothalamus (ARH) project to and come in close contact with CRH neurons in the paraventricular nucleus of the hypothalamus (PVH). Conversely, it has been demonstrated that the ventromedial portion of the ARH, an area containing NPY neurons, displays CRH receptor binding and CRH receptor mRNA. These data suggest a possible reciprocal feedback regulation between NPY and CRH neurons. The ARH also contains several other populations of neurons that may be targets of the CRH system and express CRH receptors; most notable are tuberoinfundibular dopaminergic neurons (TIDA). The PVH is an important component in the regulation of prolactin secretion and may play a role in the suppression of TIDA activity, which is a critical step in the prolactin stress response. The purpose of the present study was to characterize the distribution and cellular localization of CRH R(1) receptor-like immunoreactivity (CRH R(1)-ir) in the rat hypothalamus and to determine the phenotype of neurons in the ARH that contain CRH R(1)-ir. CRH R(1)-ir was present throughout the rat brain. Hypothalamic regions with the highest levels of immunostaining were the supraoptic nucleus, magnocellular PVH, ARH, and suprachiasmatic nucleus. Double label immunofluorescence was used to demonstrate that CRH R(1)-ir in the ARH was localized to NPY cell bodies. Furthermore, TIDA neurons in the ARH also displayed CRH R(1)-ir. However, despite an abundance of CRH R(1)-ir cells in the ARH, CRH-ir fiber innervation to the ARH was extremely sparse. Therefore, although this study provides neuroanatomical evidence for direct CRH R(1) regulation of ARH NPY and TIDA neurons in the rat, it is not consistent with the idea of a reciprocal feedback loop and suggests the involvement of other CRH-like ligands, such as urocortin.     

Neuropeptide Y mRNA expression levels following chronic olanzapine, clozapine and haloperidol administration in rats

Using quantitative in situ hybridization, this study examined regional changes in rat brain mRNA levels encoding neuropeptide Y (NPY) following olanzapine, clozapine and haloperidol administration (1.2, 1.5 and 2.0 mg/kg, oral) for 36 days. The NPY mRNA expression levels and patterns were examined after the last drug administration at both time points enabling the measurement of immediate effect at 2h and the effects after 48 h of drug administration. It was found that all these drugs had an immediate effect on NPY mRNA expression, while virtually all these changes normalized 48 h after the drug treatments. A similarity in altered NPY mRNA expression patterns was seen between the olanzapine and clozapine groups; however, haloperidol was very different. Olanzapine and clozapine administration decreased NPY mRNA levels in the nucleus accumbens, striatum and anterior cingulate cortex (from -60% to -77%, p<0.05). Haloperidol decreased NPY mRNA expression in the amygdala and hippocampus (-69%, -64%, p<0.05). In the lateral septal nucleus, NPY mRNA levels significantly decreased in the olanzapine group (-66%, p<0.05), a trend toward a decrease was observed in the clozapine group, and no change was found in the haloperidol treated group. These results suggest that the different effects of atypical and typical antipsychotics on NPY systems may reflect the neural chemical mechanisms responsible for the differences between these drugs in their effects in treating positive and negative symptoms of schizophrenia. The immediate decrease of NPY mRNA levels suggests an immediate reduction of NPY biosynthesis in response to these drugs.     

A single brain-derived neurotrophic factor injection modifies hypothalamo-pituitary-adrenocortical axis activity in adult male rats

Immobilization stress induces in adult male rats rapid activation of brain derived neurotrophic factor (BDNF) expression in the hypothalamic paraventricular nucleus (PVN) preceding the increases in corticotropin releasing hormone (CRH) and arginin-vasopressin (AVP) expression. The BDNF mRNA signal belatedly co-localizes with CRH and AVP mRNA signals in the PVN, as determined by in situ hybridization. Intracerebroventricular BDNF injections (5 microg/rat) in non-anesthetized adult male rats induce a gradual increase in the CRH mRNA signal whereas AVP mRNA signal progressively decreases in the parvocellular and magnocellular PVN portions. At the same time, the CRH hypothalamic content decreases while the AVP content increases. These variations are accompanied by increases in ACTH and corticosterone plasma concentrations. These results strongly suggest that BDNF could be a stress-responsive intercellular messenger since when it is exogenously administered acts as an important and early component in the activation and recruitment of hypothalamic CRH and AVP neurons.     

Neuropeptide Y‐stimulated [35S]GTPγs functional binding is reduced in the hippocampus after kainate‐induced seizures in mice

Kainate‐induced seizures constitute a model of temporal lobe epilepsy where prominent changes are observed in the hippocampal neuropeptide Y (NPY) system. However, little is known about the functional state and signal transduction of the NPY receptor population resulting from kainate exposure. Thus, in this study, we explored functional NPY receptor activity in the mouse hippocampus and neocortex after kainate‐induced seizures using NPY‐stimulated [³⁵S]GTPγS binding. Moreover, we also studied levels of [¹²⁵I]‐peptide YY (PYY) binding and NPY, Y1, Y2, and Y5 receptor mRNA in these kainate‐treated mice. Functional NPY binding was unchanged up to 12 h post‐kainate, but decreased significantly in all hippocampal regions after 24 h and 1 week. Similarly, a decrease in [¹²⁵I]‐PYY binding was found in the dentate gyrus (DG) 1 week post‐kainate. However, at 2 h, 6 h, and 12 h, [¹²⁵I]‐PYY binding was increased in all regions, and in the CA1 also at 24 h post‐kainate. NPY mRNA levels were prominently increased in hippocampal regions, reaching maximum at 12 and 24 h. Y1 and Y5 mRNA levels were lowered in the DG at 24 and 2 h, respectively, while Y2 mRNA levels were elevated at 24 h in the DG and CA3. This study confirms rat kainate studies by showing pronounced adaptive changes in the mouse hippocampus both with regard to NPY synthesis and NPY receptor synthesis and binding, which may contribute to regulating neuronal seizure susceptibility after kainate. However, the potential seizure‐suppressant effects of increased NPY gene expression at late time points post‐kainate could be attenuated by the novel finding of reduced NPY‐receptor G‐protein activation. Synapse 68:427–436, 2014 . © 2014 Wiley Periodicals, Inc.