Complex plastic changes in the neuropeptide Y system during ethanol intoxication and withdrawal in the rat brain

Previous studies show that chronic ethanol treatment induces prominent changes in brain neuropeptide Y (NPY). The purpose of the present study was to explore ethanol effects at a deeper NPY‐system level, measuring expression of NPY and its receptors (Y1, Y2, Y5) as well as NPY receptor binding and NPY‐stimulated [³⁵S]GTPγS functional binding. Rats received intragastric ethanol repeatedly for 4 days, and the NPY system was studied in the hippocampal dentate gyrus (DG), CA3, CA1, and piriform cortex (PirCx) and neocortex (NeoCx) during intoxication, peak withdrawal (16 hr), late withdrawal (3 days), and 1 week after last ethanol administration. NPY mRNA levels decreased during intoxication and at 16 hr in hippocampal regions but increased in the PirCx and NeoCx at 16 hr. NPY mRNA levels were increased at 3 days and returned to control levels in most regions at 1 week. Substantial changes also occurred at the receptor level. Thus Y1, Y2, and Y5 mRNA labelling decreased at 16 hr in most regions, returning to control levels at 3 days, except for PirCx Y2 mRNA, which increased at 3 days and 1 week. Conversely, increases in NPY receptor binding occurred in hippocampal regions during intoxication and in functional binding in the DG and NeoCx during intoxication and at 16 hr and in PirCx during intoxication and at 1 week. Thus this study shows that ethanol intoxication and withdrawal induce complex plastic changes in the NPY system, with decreased/increased gene expression or binding occurring in a time‐ and region‐specific manner. These changes may play an important role in mediating ethanol‐induced changes in neuronal excitability. © 2009 Wiley‐Liss, Inc.     

The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y and not Y receptors

Neuropeptide Y (NPY) potently inhibits glutamate release and seizure activity in rodent hippocampus in vitro and in vivo, but the nature of the receptor(s) mediating this action is controversial. In hippocampal slices from rats and several wild-type mice, a Y2-preferring agonist mimicked, and the Y2-specific antagonist BIIE0246 blocked, the NPY-mediated inhibition both of glutamatergic transmission and of epileptiform discharges in two different slice models of temporal lobe epilepsy, stimulus train-induced bursting (STIB) and 0-Mg2+ bursting. Whereas Y5 receptor-preferring agonists had small but significant effects in vitro, they were blocked by BIIE0246, and a Y5 receptor-specific antagonist did not affect responses to any agonist tested in any preparation. In slices from mice, NPY was without effect on evoked potentials or in either of the two slice seizure models. In vivo, intrahippocampal injections of Y2- or Y5-preferring agonists inhibited seizures caused by intrahippocampal kainate, but again the Y5 agonist effects were insensitive to a Y5 antagonist. Neither Y2- nor Y5-preferring agonists affected kainate seizures in mice. A Y5-specific antagonist did not displace the binding of two different NPY ligands in WT or mice, whereas all NPY binding was eliminated in the mouse. Thus, we show that Y2 receptors alone mediate all the anti-excitatory actions of NPY seen in the hippocampus, whereas our findings do not support a role for Y5 receptors either in vitro or in vivo. The results suggest that agonists targeting the Y2 receptor may be useful anticonvulsants.     

Differential suppression of seizures via Y2 and Y5 neuropeptide Y receptors

Neuropeptide Y (NPY) prominently inhibits epileptic seizures in different animal models. The NPY receptors mediating this effect remain controversial partially due to lack of highly selective agonists and antagonists. To circumvent this problem, we used various NPY receptor knockout mice with the same genetic background and explored anti-epileptic action of NPY in vitro and in vivo. In Y2 (Y2-/-) and Y5 (Y5-/-) receptor knockouts, NPY partially inhibited 0 Mg2+-induced epileptiform activity in hippocampal slices. In contrast, in double knockouts (Y2Y5-/-), NPY had no effect, suggesting that in the hippocampus in vitro both receptors mediate anti-epileptiform action of NPY in an additive manner. Systemic kainate induced more severe seizures in Y5-/- and Y2Y5-/-, but not in Y2-/- mice, as compared to wild-type mice. Moreover, kainate seizures were aggravated by administration of the Y5 antagonist L-152,804 in wild-type mice. In Y5-/- mice, hippocampal kindling progressed faster, and afterdischarge durations were longer in amygdala, but not in hippocampus, as compared to wild-type controls. Taken together, these data suggest that, in mice, both Y2 and Y5 receptors regulate hippocampal seizures in vitro, while activation of Y5 receptors in extra-hippocampal regions reduces generalized seizures in vivo.     

Comparative distribution of neuropeptide Y Y1 and Y5 receptors in the rat brain by using immunohistochemistry

Neuropeptide Y (NPY) Y1 and Y5 receptor subtypes mediate many of NPY's diverse actions in the central nervous system. The present studies use polyclonal antibodies directed against the Y1 and Y5 receptors to map and compare the relative distribution of these NPY receptor subtypes within the rat brain. Antibody specificity was assessed by using Western analysis, preadsorption of the antibody with peptide, and preimmune serum controls. Immunostaining for the Y1 and Y5 receptor subtypes was present throughout the rostral-caudal aspect of the brain with many regions expressing both subtypes: cerebral cortex, hippocampus, hypothalamus, thalamus, amygdala, and brainstem. Further studies using double-label immunocytochemistry indicate that Y1R immunoreactivity (-ir) and Y5R-ir are colocalized in the cerebral cortex and caudate putamen. Y1 receptor ir was evident in the central amygdala, whereas both Y1- and Y5-immunoreactive cells and fibers were present in the basolateral amygdala. Corresponding with the physiology of NPY in the hypothalamus, both Y1R- and Y5R-ir was present within the paraventricular (PVN), supraoptic, arcuate nuclei, and lateral hypothalamus. In the PVN, Y5R-ir and Y1R-ir were detected in cells and fibers of the parvo- and magnocellular divisions. Intense immunostaining for these receptors was observed within the locus coeruleus, A1-5 and C1-3 nuclei, subnuclei of the trigeminal nerve and nucleus tractus solitarius. These data provide a detailed and comparative mapping of Y1 and Y5 receptor subtypes within cell bodies and nerve fibers in the brain which, together with physiological and electrophysiological studies, provide a better understanding of NPY neural circuitries.     

Distribution of NPY Y5-like immunoreactivity in the rat brain

The pharmacology and brain mRNA distribution of the neuropeptide Y (NPY) rat Y5 (rY5) receptor has led to the hypothesis that this receptor might mediate the hypothalamic feeding response to NPY in addition to many other physiologic functions. However, through the use of autoradiographic techniques, only very low levels of Y5-like immunoreactive (Y5-ir) binding are detected in the rat brain. To localize the Y5 protein in the rat brain, polyclonal antibodies were raised to the carboxyl terminus of the rY5 receptor. The resulting antisera were affinity purified and characterized by specific binding to HEK293 cells that had been stably transfected with the rY5 receptor. Utilizing immunohistochemical techniques, we found a discrete pattern of Y5-ir in the rat brain. In initial studies, very low levels of Y5-ir were detected, and TSA amplification was required to visualize the staining. Areas with the highest levels of expression in clude the piriform cortex, supraoptic nucleus, and hippocampus. Areas with moderate levels of expression include the lateral septum, amygdala, arcuate nucleus, paraventricular hypothalamic nucleus, locus coeruleus, and cerebellum. With several exceptions, this pattern of distribution is consistent with earlier reports of rY5 mRNA and receptor protein expression.     

Multiple receptors for neuropeptide Y in the hippocampus: putative roles in seizures and cognition

Neuropeptide Y (NPY) is widely distributed throughout the central nervous system (CNS) and is one of the most conserved peptides in evolution, suggesting an important role in the regulation of basic physiological functions, including learning and memory. In addition, experimental studies have suggested that NPY, together with its receptors, may have a direct implication in several pathological disorders, including epilepsy/seizure. NPY-like immunoreactivity and NPY receptors have been shown to be present throughout the brain, but is concentrated in the hippocampus. The hippocampal formation has been repeatedly implicated in the modulation of cognition, as well as the pathogenesis of seizure. This review will concentrate on the hippocampal distribution of NPY, its receptors and the putative role played by this peptide in seizure, together with the regulation of cognitive function associated with learning and memory.     

Quantitative autoradiographic localization of neuropeptide Y receptors in the rat lower brainstem

Using quantitative autoradiography, we have characterized the binding of ¹²⁵I-Bolton-Hunter coupled neuropeptide Y ([¹²⁵I]NPY) and observed the localization of ¹²⁵I-NPY receptors in the rat lower brainstem. [¹²⁵I]NPY bound to the receptors in a specific, saturable and reversible manner with high affinity. The binding was blocked only by unlabeled NPY but not by NPY-related peptides i.e. peptide YY, pancreatic polypeptide (avian and human), nor by neurotensin. [¹²⁵I]NPY receptors were revealed to be coupled to the guanosine triphosphate (GTP)-binding protein. Regional distribution study showed that [¹²⁵I]NPY has a distinctive pattern of distribution in the rat lower brainstem, being particularly concentrated in the area postrema and the medial subnucleus of the nucleus tractus solitarii. These results suggest that such NPY receptors have an important role in cardiovascular regulation.     

5-HT1 receptors in migraine pathophysiology and treatment

Migraine is a frequent paroxysmal headache disorder of unknown aetiology. Genetic factors may control attack frequency and possibly attack severity. Serotonin_1D (5-HT_1Dβ) receptors have a prominent position within the final common pathway of the mechanisms involved in the headache and associated symptoms. Stimulation of these receptors by selective 5-HT_1Dβ receptor agonists such as sumatriptan and newer compounds including MK-462 and 311C90, rapidly and fully blocks the symptoms of the headache phase. The efficacy depends on factors such as timing of administration during or before the headache, speed of initial rise of drug plasma levels, and possibly degree of brain penetration. All agonists at S-HT_1Dβ receptors share a short duration of action resulting in recurrence of the headache symptoms within 24 h in about one-third of attacks in clinical trials. The risk for headache recurrence seems patient dependent: about 10% of patients treating multiple attacks experience headache recurrence in every treated attack, whereas 40% never experience recurrence. These differences are not related to simple pharmacokinetic differences between patients or drugs. Increasing plasma half-life of the drug will most likely not reduce the risk of recurrence. “Breakthrough of peripheral suppressive effect” with an ongoing “central migraine generator”, rather than the occurrence of a new attack, seems to be the most likely underlying mechanism for headache recurrence. In a minority of, possibly predisposed, patients, use of sumatriptan may induce increase of attack frequency. Four mechanisms have been suggested for the antimigraine action of 5-HT_1Dβ receptor agonists: (1) vasoconstriction of cranial, most likely meningeal and dural blood vessels; (2) inhibition of release of vasoactive neuropeptides from perivascular trigeminal nerve terminals within dura mater and meninges; (3) blockade of trigeminal nerve terminal depolarization; and (4) central inhibition within the trigeminal nucleus caudatus in the brainstem. Which of these mechanisms is the most important, and whether or not vasoconstrictor action is necessary for antimigraine efficacy, is currently under extensive investigation. At this point all drugs with proven antimigraine efficacy share the ability to contract blood vessels and thus all feature also the potential risk of causing vasoconstriction of coronary vessels. In relation herewith, major efforts are put into the search for “the antimigraine receptor” and which receptor subtype mediates which action of sumatriptan-like drugs. At this point, the 5-HT_1Dβ receptor subtype is thought to mediate vasoconstriction. Some investigators feel that the 5-HT_1Dα receptor subtype mediates the neuronal effects of sumatriptan, while others are much less convinced about the physiological role of this subtype of receptor. Further research into receptor subtype specificity and affinity of compounds may promote the development of even better antimigraine drugs.     

Characterization of the neuropeptide Y5 receptor in the human hypothalamus: a lack of correlation between Y5 mRNA levels and binding sites

Neuropeptide Y (NPY) is a 36-amino-acid peptide that appears to play a central role in the control of feeding behavior. Recently, a cDNA encoding a novel NPY receptor subtype (Y5) was cloned from the rat and human hypothalamus, and shown to have a pharmacology consistent with NPY-induced feeding. We have subsequently cloned this cDNA from human hypothalamus and stably expressed it in CHO cells. Consistent with earlier reports, hY5 has a high affinity for NPY, [Leu³¹,Pro³⁴]NPY, and NPY(3–36), but low affinity for larger C-terminal deletions of NPY and BIBP3226. High levels of hY5 mRNA were found in the human testis, brain, spleen and pancreas, with lower levels in several other tissues. In the human brain, hY5 mRNA levels were typically higher than hY2, but lower in comparison to hY1 receptor mRNA. To quantify the relative amounts of hY1, hY2 and hY5 mRNA in the human hypothalamus, we employed competitive RT-PCR. Interestingly, the relative amount of hY5 mRNA was substantially higher than either hY1 or hY2. However, pharmacological characterization of NPY binding sites in human hypothalamus membranes revealed predominantly the hY2 subtype. These data establish that while hY5 mRNA levels are very high in the human hypothalamus, conventional radioligand binding techniques do not detect hY5-like binding site. Whether hY5-like binding sites exist in the other human tissues that express hY5 mRNA (and what function hY5 has in those tissues) awaits future investigation.     

Differences in regional brain concentrations of neuropeptide Y in spontaneously hypertensive (SH) and Wistar-Kyoto (WKY) rats

Regional brain concentrations of neuropeptide Y immunoreactivity (NPY) were measured in age-matched Wistar-Kyoto (WKY) and spontaneously hypertensive (SH) rats using a sensitive and specific radioimmunoassay developed within our laboratory. In 5 of the 9 brain regions examined the SH rats had significantly lower NPY levels compared to the WKY strain. The largest differences occurred within the cortex (−43%), and cervical (−30%) and thoracic spinal cord (−30%), whilst smaller differences were observed in the midbrain (−11%) and medulla oblongata-pons (−18%). The concentration of NPY in the hypothalamus and hippocampus did not vary between the strains. The SH rats contained significantly greater (+18%) NPY levels in the striatum compared to the WKY rats.     

Antinociceptive effects induced by intra-periaqueductal grey administration of neuropeptide Y in rats

Hindpaw withdrawal latency (HWL) to thermal and mechanical stimulation increased dose-dependently after intra-periaqueductal grey (PAG) injection of neuropeptide Y (NPY). Furthermore, the NPY-induced increases in HWLs were attenuated by intra-PAG injection of the Y1 receptor antagonist NPY28-36. The results demonstrated that NPY plays an important role in antinociception in PAG, in which Y1 receptor is involved in.     

Central nervous system receptors involved in mediating the inhibitory action of neuropeptide Y on luteinizing hormone secretion in the male rhesus monkey (Macaca mulatta)

An earlier finding that gonadotropin-releasing hormone (GnRH) secretion may be triggered prematurely in the juvenile male monkey by central administration of 1229U91, a Y1 receptor antagonist, contributed to our current hypothesis that neuropeptide Y (NPY) is a major component of the brake that holds pulsatile GnRH release in check during prepubertal development in primates. However, 1229U91 is also a Y4 receptor agonist, and the present study was conducted to further examine the role of the Y1 receptor in mediating the putative inhibitory action of NPY on GnRH release. Agonadal juvenile and postpubertal male monkeys were implanted with i.v. and i.c.v. cannulae to gain continuous access to the venous and cerebroventricular circulations without sedation. Luteinizing hormone (LH) secretion was measured to provide an indirect index of GnRH release. The specific Y1 antagonists, VD-11 (476 microg; n = 4) and isopropyl 3-chloro-5-[1-((6-[2-(5-ethyl-4-methyl-1,3-thiazol-2-yl)ethyl]-4-morpholin-4-ylpyridin-2-yl)amino)ethyl]phenylcarbamate (Compound A, 300 microg; n = 4), did not mimic the stimulatory action of 1229U91 on GnRH secretion in the juvenile male monkey. Additionally, neither NPY (200 microg; n = 2), a general Y receptor agonist, nor rPP (100 microg; n = 4), a Y4 agonist, mimicked the action of 1229U91 in stimulating GnRH release. Moreover, previous exposure of the hypothalamus of juvenile monkeys (n = 5) to NPY (660 microg) failed to block 1229U91-induced (200 microg) GnRH release. However, the action of NPY (364 microg) in inhibiting GnRH release postpubertally was attenuated by 1229U91 (300 microg). We conclude that, although the action of exogenous NPY to suppress GnRH release from the postpubertal hypothalamus appears to be mediated, at least in part, by the Y1 receptor, the existence of a Y1 receptor pathway inhibitory to GnRH release in the prepubertal hypothalamus remains to be substantiated.     

Orexin-induced food intake involves neuropeptide Y pathway

Orexins (orexin-A and -B) are recently identified neuropeptides, which are thought to be implicated in the regulation of feeding behavior. We used a NPY-Y1 receptor specific antagonist, BIBO3304, to examine whether NPY is involved in orexin-induced feeding behavior. Intracerebroventricular administration of orexin-A (10 nmol) induced food intake in rats (food intake for 3 h; vehicle 0.3+/-0.2 g vs. orexin-A 10 nmol, 4.0+/-0.5 g, n=4). Orexin-induced feeding behavior was partially inhibited by prior administration of BIBO3304 (3 h food intake: orexin-A 10 nmol, 4.0+/-0.5 g vs. BIBO3304 (60 microgram) + orexin-A 10 nmol, 2.2+/-0.2 g, n=4). A low dose of BIBO3304 (30 microgram) did not show a significant inhibitory effect. BIBO3457, an inactive enantiomer, used as a negative control, did not show any inhibitory effect on orexin-A-induced feeding behavior. Fos expression was observed in NPY-containing neurons in the arcuate nucleus 1 h after orexin-A (10 nmol) was administered intracerebroventricularly (control 0.3+/-0.08%, orexin-A 10.2+/-0.8%, n=5 rats/group). These observations suggest that NPY is involved in orexin-induced feeding behavior. However, BIBO3304 did not completely abolish the effect of orexin-A. These results suggest that orexin-A elicits feeding behavior partially via the NPY pathway. The NPY system could be the one of downstream pathways by which orexin-A induces feeding behavior. Another pathway may also be involved in orexin-A-induced feeding behavior, because BIBO3304 did not completely abolish orexin-A-induced feeding behavior.     

Stimulation of nicotinic receptors in the lateral septal nucleus increases anxiety

The present study investigated the role of nicotinic receptors in the lateral septum in the modulation of anxiety. The effects of direct injections of nicotine into the lateral septum were first investigated in two tests of anxiety, social interaction and elevated plus-maze tests. Intra-septal injection of nicotine (1 and 4 microgram) induced consistent anxiogenic effects in both tests. The reversal of nicotinic effects with mecamylamine was then studied in the social interaction test. Intra-septal injection of mecamylamine at a low dose (15 ng) induced an anxiolytic effect, suggesting the presence of intrinsic cholinergic tone increasing anxiety. At higher doses (30-50 ng), mecamylamine was without effect in the social interaction test, but blocked the anxiogenic effects of nicotine (4 microgram). These findings provide further evidence for the role of the lateral septum in the modulation of anxiety and suggest that cholinergic projections to this brain area facilitate anxiety through nicotinic receptors.     

Activation of neuropeptide Y(2) receptors exerts an excitatory action on cardio-respiratory variables in anaesthetized rats

The respiratory effects of stimulation of NPYY₂ receptors were studied in spontaneously breathing rats that were either (i) neurally intact and subsequently bilaterally vagotomized in the neck, or (ii) neurally intact and subjected to supranodosal vagotomy or (iii) neurally intact treated with pharmacological blockade of NPY_1-2 receptors. Before neural interventions an intravenous (iv) bolus of the NPYY₂ receptor agonist NPY 13-36 (10 μg/kg) increased breathing rate, tidal volume and mean arterial blood pressure (MAP).Section of the midcervical vagi abrogated NPY 13-36-evoked increase in respiratory rate but had no effect on augmented tidal volume, minute ventilation and blood pressure. Supranodosal vagotomy prevented the increase in tidal volume and slightly reduced the pressor response. Blockade of NPYY₂ receptor with intravenous doses of BIIE 0246 eliminated cardio-respiratory effects of NPY 13-36 injection. BMS 193885 - an antagonist of NPYY₁ receptor-was not effective in abrogating cardio-respiratory response.The present study showed that (i) NPY 13-36 induced stimulation of breathing results from activation of NPYY₂ receptors associated with pulmonary vagal afferentation; (ii) the increase in the frequency of breathing is mediated by midcervical vagi and augmentation of tidal volume relies on the intact supranodosal trunks (iii) the pressor response results from the excitation of NPYY₂ receptors outside of the vagal pathway.     

Identification of neuropeptide Y receptors in cultured astrocytes from neonatal rat brain

Specific binding sites for neuropeptide Y could be demonstrated in primary cultures of astrocytes from neonatal rat brain. Neuropeptide Y binding was saturable, reversible, and temperature dependent as revealed by saturation studies and kinetic experiments. Scatchard analysis of equilibrium binding data indicated a single population of high-affinity binding sites with respective K_D and B_max values of 0.43 nM and 6.9 fmol/2.7 × 10⁵ cells. Physiological responses induced by neuropeptide Y could be detected in a distinct subpopulation of cultured astrocytes on the basis of two criteria: (1) electrophysiological responses and (2) single cell measurements of changes in [Ca²⁺]_i. In that fraction of cells responding (20–70%, varying among cultures from different preparations), brief application of neuropeptide Y led to a membrane potential depolarization, lasting several minutes. When the membrane was clamped close to the resting membrane potential using the whole-cell patch-clamp technique, neuropeptide Y induced an inward current with a similar time course as the neuropeptide Y-induced membrane depolarization. As detected by single cell microfluorimetric (fura-2) measurements neuropeptide Y induced an increase of [Ca²⁺]_i which was caused by the entry of extracellular Ca²⁺. Both the [Ca²⁺]_i increase and the electrophysiological responses were unaffected by pretreatment of the astrocytes with pertussis toxin. © 1993 Wiley-Liss, Inc.