Changes in Oestrogen Receptor α Expression in the Nucleus of the Solitary Tract of the Rat Over the Oestrous Cycle and Following Ovariectomy

Oestrogen is capable of modulating autonomic outflow and baroreflex function via actions on groups of neurones in the brainstem. We investigated the presence of oestrogen receptor (ER) α in a part of the nucleus of the solitary tract (NTS) associated with central cardiovascular control, aiming to determine whether ERα mRNA and protein expression is correlated with levels of circulating oestrogen during the oestrous cycle. Polymerase chain reaction (PCR) detected ERα mRNA in the NTS at each stage of the oestrous cycle, from ovariectomised, sham‐operated and male rats. Real‐time PCR showed variations in ERα mRNA expression during the oestrous cycle, with the highest levels seen in oestrus, and lowest levels in metoestrus (P < 0.05 versus oestrus) and proestrus (P < 0.05 versus oestrus). Expression in males was lower than in dioestrus and oestrus females (P < 0.05). After ovariectomy, ERα mRNA levels were decreased compared to sham‐operated animals (P < 0.01). Confocal fluorescence immunohistochemistry with stereological analysis showed that numbers of ERα immunoreactive cell nuclei per mm³ of tissue in the caudal NTS were significantly greater in proestrus than in other groups of rats (P < 0.05). There were also differences among the groups in the extent of colocalisation of ERα in neurones immunoreactive for tyrosine hydroxylase and nitric oxide synthase. These results imply a complex pattern of region‐specific oestrogen signalling in the NTS and suggest that ERα expression in this important autonomic nucleus may be related to circulating oestrogen levels. This may have consequences for the regulation of autonomic tone and baroreflex sensitivity when oestrogen levels decline, for example following menopause.     

Roles of GABA<Subscript>B</Subscript> receptor subtypes in presynaptic auto- and heteroreceptor function regulating GABA and glutamate release

Γ-Aminobutyric acid B (GABA_B) receptors are heterodimers composed of two subunits GABA_B(1) and GABA_B(2), the former existing in two isoforms GABA_B(1a) and GABA_B(1b). The contributions of individual receptor subunits and isoforms to GABA_B auto- and heteroreceptor functions were investigated, using release experiments in cortical slice preparations from corresponding knockout mice. Presynaptic GABA_B autoreceptors are located on GABAergic terminals and inhibit GABA release, whereas presynaptic GABA_B heteroreceptors control the release of other neurotransmitters (e.g. glutamate). Neither baclofen nor the selective antagonist CGP55845 at maximally active concentrations affected [³H]GABA release in slices from GABA_B(1)−/− mice. The amount of [³H]GABA released per pulse was unaffected by the stimulation frequency in slices from GABA_B(1)−/− and GABA_B(2)−/− demonstrating a loss of GABA_B autoreceptor function in these knockout animals. The GABA_B receptor agonist baclofen was ineffective in modulating glutamate release in cortical slices from GABA_B(2)−/− mice, showing that heteroreceptor function was abolished as well. Next we investigated knockout mice for the two predominant GABA_B(1) isoforms expressed in brain, GABA_B(1a) and GABA_B(1b). In cortical, hippocampal and striatal slices from both GABA_B(1a)−/− and GABA_B(1b)−/− mice, the frequency dependence of [³H]GABA released per pulse was maintained, suggesting that both isoforms participate or can substitute for each other in GABA_B autoreceptor function. By contrast, the efficacy of baclofen to inhibit glutamate release was substantially reduced in GABA_B(1a)−/−, but essentially unaltered in GABA_B(1b)−/− mice. Our data suggest that functional GABA_B heteroreceptors regulating glutamate release are predominantly, but not exclusively composed of GABA_B(1a) and GABA_B(2) subunits. An erratum to this article can be found at     

Studies on the Origin and Release of Somatostatin-Immunoreactivity in the Nucleus of the Solitary Tract

The somatostatin content of the nucleus of the solitary tract (NTS) was regionally distributed within the nucleus and a calcium-dependent release of the neuropeptide was evoked by potassium-induced depolarization in vitro. The origin of the somatostatin in the NTS was studied with various denervation procedures. Unilateral NTS deafferentation decreased the concentration of somatostatin in the NTS. However, neither midbrain hemisection nor nodose ganglionectomy reduced the somatostatin content of the NTS. In addition, the nodose ganglion contained very low quantities of the peptide. These results suggest that (1) somatostatin may function as a neurotransmitter in the NTS and (2) the somatostatin-containing innervation of the NTS is at least in part from extrinsic neurons located in the lower brainstem and not from vagal afferent or forebrain neurons.     

Kainate down-regulates a subset of GABA<Subscript>A</Subscript> receptor subunits expressed in cultured mouse cerebellar granule cells

The effect of kainate, an agonist selective for ionotropic AMPA/kainate type of glutamate receptors, on GABAA receptor subunit expression in cultured mouse cerebellar granule cells was studied using quantitative RT-PCR, ligand binding and electrophysiology. Chronic kainate treatment, without producing excitotoxicity, resulted in preferential, dose- and time-dependent down-regulation of alpha1, alpha6 and beta2 subunit mRNA expression, the expression of beta3, gamma2 and delta subunit mRNAs being less affected. The down-regulation was reversed by DNQX, an AMPA/kainate-selective glutamate receptor antagonist. A 14-day kainate treatment resulted in 46% decrease of total [3H]Ro 15-4513 binding to the benzodiazepine sites. Diazepam-insensitive [3H]Ro 15-4513 binding was decreased by 89% in accordance with very low amount of alpha6 subunit mRNA present. Diazepam-sensitive [3H]Ro 154513 binding was decreased only by 40%, contrasting >90% decrease in alpha1 subunit mRNA expression. However, this was consistent with lower potentiation of GABA-evoked currents in kainate-treated than control cells by the alpha1-selective benzodiazepine site ligand zolpidem, suggesting compensatory expression of alpha5 (and/or alpha2 or alpha3) subunits producing diazepam-sensitive but zolpidem-insensitive receptor subtypes. In conclusion, chronic kainate treatment of cerebellar granule cells selectively down-regulates oil, alpha6 and beta2 subunits resulting in altered GABAA receptor pharmacology.     

Functional analysis of GABAA receptors in nucleus tractus solitarius neurons from neonatal rats

To gain insight into specific GABA_A receptor configurations functionally expressed in the nucleus tractus solitarius (NTS), we conducted several physiological and pharmacological assessments. NTS neurons were characterized in thin brain slices from 1–14 day old rats using whole-cell patch clamp recordings. GABA_A− receptor-mediated currents were detected in all neurons tested, with an average EC₅₀ of 22.2 μM. GABA currents were consistently stimulated by diazepam (EC₅₀=63 nM), zolpidem (EC₅₀=85 nM), loreclezole (EC₅₀=10.1 μM) and the neurosteroid 5α-pregnan-3α-hydroxy-20-one (3α-OH-DHP). In contrast, GABA-gated currents of the NTS were inhibited by the divalent cation Zn²⁺ (IC₅₀=33.6 μM) picrotoxin (IC₅₀=2.4 μM) and blockade of endogenous protein tyrosine kinase. GABA-activated currents were insensitive to furosemide (10–1000 μM) in all NTS neurons tested. Collectively, the data suggest that in neonatal rats, the predominant α subunit isoform present in GABA_A receptors of the NTS appears to be the α1 and/or α2 subunit. β2 and/or β3 subunits are the major β isoform, while the predominant γ subunit is likely γ2. Our data suggest the contribution to NTS GABA currents by α3–α6, β1, γ1 and δ subunits, if present, is minor by comparison.     

Endogenous GABA in the commissural subnucleus of the NTS inhibits the carotid chemoreceptor reflex via GABA<Subscript>A</Subscript> receptors in rats

Summary. Using urethane-chloralose anesthetized rats, we investigated which GABA receptor is responsible for the action of endogenous GABA on the carotid chemoreceptor reflex in the commissural subnucleus of the nucleus tractus solitarius (commNTS). Microinjection of the selective GABA uptake inhibitor nipecotic acid (40 nmol) into the commNTS attenuated the increases in respiration (respiratory movement and rate) and the elevation in arterial blood pressure elicited by carotid chemoreceptor stimulation. These effects were completely antagonized by premicroinjection of the GABA_A antagonist bicuculline (20 pmol), but not of the GABA_B antagonist 2-OH-saclofen (400 pmol), into the same site. These findings suggest that endogenous GABA mainly acts on GABA_A receptors, and inhibits the chemoreceptor reflex in the commNTS in rats. Received May 16, 2002; accepted August 8, 2002 Published online December 9, 2002 Authors' address: M. Suzuki, Department of Pharmacology, Saitama Medical School, Moroyama, Iruma-gun, Saitama 350-0495, Japan, e-mail: mhsuzuki@saitama-med.ac.jp     

Nucleus accumbens dopamine release modulation by mesolimbic GABAA receptors—an in vivo electrochemical study

The role of GABA receptors in regulating the mesolimbic dopamine (DA) system and drug reinforced behaviors has not been well characterized. Using fast-cyclic voltammetry, the effects of specific GABA receptor modulation on DA release in the nucleus accumbens (NAcc) and heroin self-administration (SA) behavior was investigated. The GABA_A agonist muscimol, administered either intravenously or directly into the ventral tegmental area (VTA), significantly increased DA release in the NAcc in 7 of the 10 rats tested. DA release decreased in the remaining three rats; both effects were blocked by pretreatment with the GABA_A receptor antagonist bicuculline. In contrast, the GABA_B agonist baclofen decreased, while 2-OH-saclofen (a GABA_B antagonist) increased DA release in the NAcc. However, when VTA GABA_B receptors were previously activated or inactivated by microinjections of baclofen or 2-OH-saclofen, systemic injections of muscimol caused an inhibition of NAcc DA release. These results suggest that GABA_A receptors may be co-localized on both DA neurons and non-DA (GABAergic) interneurons in the VTA, with the effects of GABA_A determined by the net effect of both direct inhibition and indirect disinhibition of DA neurons. Finally, although a DA releaser, muscimol was neither self-administered in drug naive rats, nor did it substitute for heroin in rats previously trained to self-administer heroin, suggesting that GABA_A receptors appear to play a complex role in mediating drug reinforcement, depending upon the dynamic functional state of GABA_A receptors on both tegmental DA and non-DA neurons.     

Benzodiazepine-sensitive GABA(A) receptors in the commissural subnucleus of the NTS are involved in the carotid chemoreceptor reflex in rats

We studied the role of benzodiazepine (BDZ) receptors in the commissural subnucleus of the nucleus tractus solitarius (commNTS) in chemoreceptor reflex in urethane-anesthetized, pancronium-immobilized, artificially ventilated and bilaterally vagotomized rats. A BDZ agonist, diazepam (1-4 micromol/kg), administered intravenously reduced resting phrenic nerve activity (PNA) and blood pressure (BP). Stimulation of carotid chemoreceptors induced an increase in PNA and an increase in BP. Diazepam inhibited this chemoreceptor reflex. The effects of intravenous injection of diazepam (4 micromol/kg) on the chemoreceptor reflex were antagonized by microinjection of the BDZ antagonist flumazenil (100 pmol) into the commNTS. Microinjection of flumazenil (100 pmol) alone had no effect on the basal PNA and BP, and the chemoreceptor reflex. These results suggest that BDZ receptors are present in the carotid chemoreceptor reflex pathway in the commNTS and potentiate GABA(A) transmission.     

Molecular diversity of GABA-gated chloride channels in the rat anterior pituitary

mRNA expression of GABA-gated Cl⁻-channels in rat antepituitary was evaluated by using an reverse-transcribed (RT)-polymerase chain reaction (RT-PCR) method with degenerate and specific oligonucleotides. The main result of our findings is that the antepituitary expresses mRNAs encoding α4 and π1 GABA receptor subunits. These two subunits are believed to be, respectively, constituents of benzodiazepine-insensitive GABA_A and GABA_C receptors in the CNS. This molecular analysis is consistent with the pharmacological diversity of GABA receptors in pituitary cells.     

Blood levels of homocysteine,cysteine, glutathione,folic acid,and vitamin B<Subscript>12</Subscript> in the acute phase of atherothrombotic stroke

Blood levels of total homocysteine (tHcy), cysteine (Cys), total and reduced glutathione (tGSH and rGSH), folic acid (FA), and vitamin B₁₂ (B₁₂) change during ischemic stroke as accompaniment of the tissue damage. The relationship between these changes remains scantly investigated. We evaluated the variation of these molecules in the 48 h after acute large artery atherothrombotic stroke (LAAS) and searched for the presence of matched variation of them. The study involved 50 subjects affected by acute LAAS and 49 healthy controls. Plasma levels of tHcy and Cys were significantly higher and serum levels of FA and B₁₂ and plasma levels of rGSH were significantly lower in the patients than in the control group. Acute LAAS was associated with increased Hcy—decreased tGSH and decreased FA/tGSH. Pathways involved in cellular stress and in tissue repair are activated during acute LAAS.     

γ-Aminobutyric acid receptor subtype antagonists differentially alter opioid-induced feeding in the shell region of the nucleus accumbens in rats

Food intake is significantly increased by administration of μ-selective opioid agonists into the nucleus accumbens, particularly its shell region. Pretreatment with either opioid (μ, δ₁, δ₂ or κ₁) or dopaminergic (D₁) receptor antagonists in the nucleus accumbens shell reduce μ opioid agonist-induced feeding. Selective GABA_A (muscimol) and GABA_B (baclofen) agonists administered into the nucleus accumbens shell each stimulate feeding which is respectively and selectively blocked by GABA_A (bicuculline) and GABA_B (saclofen) antagonists. The present study investigated whether feeding elicited by the μ-selective opioid agonist, [ -Ala²,NMe⁴,Gly-ol⁵]-enkephalin in the nucleus accumbens shell was decreased by intra-accumbens pretreatment with an equimolar dose range of either GABA_A or GABA_B antagonists, and further, whether general opioid or selective GABA antagonists decreased feeding elicited by GABA_A or GABA_B agonists in the nucleus accumbens shell. Feeding elicited by the μ-selective opioid agonist was dose-dependently increased following intra-accumbens pretreatment with GABA_A (bicuculline) antagonism; this enhancement was significantly blocked by pretreatment with general or μ-selective opioid antagonists. In contrast, μ opioid agonist-induced feeding elicited from the nucleus accumbens shell was dose-dependently decreased by GABA_B (saclofen) antagonism. Neither bicuculline nor saclofen in the nucleus accumbens shell altered baseline food intake. Whereas muscimol-induced feeding elicited from the nucleus accumbens shell was reduced by bicuculline and naltrexone, but not saclofen pretreatment, baclofen-induced feeding elicited from the nucleus accumbens shell was reduced by saclofen, but not by bicuculline or naltrexone. These data indicate that GABA_A and GABA_B receptor subtype antagonists differentially affect feeding elicited by μ opioid receptor agonists within the nucleus accumbens shell in rats.     

c-Fos immunoreactivity induced by intraperitoneal LPS administration is reduced in the brain of mice lacking the microsomal prostaglandin E synthase-1 (mPGES-1)

The aim of the present study was to investigate the impact of the deletion of the microsomal prostaglandin E synthase-1 (mPGES-1) gene on lipopolysaccharide (LPS)-induced neuronal activation in central nervous structures. The mPGES-1 catalyses the conversion of COX-derived PGH(2) to PGE(2) and has been described as a regulated enzyme whose expression is stimulated by proinflammatory agents. Using the immediate-early gene c-fos as a marker of neuronal activation, we determined whether deletion of the mPGES-1 gene altered the neuronal activation induced by LPS in structures classically recognized as immunosensitive regions. No significant difference in the c-Fos immunostaining was observed in the brain of saline-treated mPGES-1+/+, mPGES-1+/- and mPGES-1-/- mice. However, we observed that LPS-induced neuronal activation was reduced in most of the centres known as immunosensitive nuclei in mPGES-1-/- mice compared with heterozygous and wild-type mice. The decrease in the number of c-Fos positive nuclei occurred particularly in the caudal ventrolateral medulla, the medial, intermediate and central parts of the nucleus tractus solitarius, area postrema, parabrachial nucleus, locus coeruleus, paraventricular nucleus of the hypothalamus, ventromedial preoptic area, central amygdala, bed nucleus of the stria terminalis and to a lesser extent in the ventrolateral part of the nucleus tractus solitarius and rostral ventrolateral medulla. These results suggest that the mPGES-1 enzyme is strongly needed to provide sufficient PGE(2) production required to stimulate immunosensitive brain regions and they are discussed with regard to the recent works reporting impaired sickness behavior in mPGES-1-/- mice.     

Expression of CB<Subscript>1</Subscript> cannabinoid receptor in the anterior cingulate cortex in schizophrenia,bipolar disorder,and major depression

Summary The human endogenous cannabinoid system is an appealing target in the investigation of psychiatric disorders. In schizophrenia, endocannabinoids and their receptors are involved in the pathology of the disease. Previous studies reported an increased radioligand binding to cannabinoid receptors 1 (CB₁) in schizophrenia, both in the dorsolateral prefrontal cortex and in the anterior cingulate cortex (ACC). We analyzed the expression of the CB₁ receptors in the ACC at the protein level using immunohistochemistry. In a quantitative postmortem study, 60 patients suffering from schizophrenia, bipolar disorder, major depression and controls were included. Numerical densities of neurons and glial cells immunopositive for CB₁ receptors were evaluated. No evidence of an increased or decreased density of CB₁ receptor immunopositive cells in schizophrenia or bipolar disorder was found. In major depression, CB₁ receptor immunopositive glial cells in the grey matter were decreased. Furthermore, our data show that different medications have an impact on the expression of CB₁ receptors in the ACC.     

Changes in the Gene Expression of GABAA Receptor α1 and α2 Subunits and Metabotropic Glutamate Receptor 5 in the Basal Ganglia of the Rats with Unilateral 6-Hydroxydopamine Lesion and Embryonic Mesencephalic Grafts

By using an animal model of parkinsonism, we examined the expression of GABA_A receptor (R) and metabotropic glutamate receptor (mGluR) 5 in the basal ganglia after transplantation with dopamine-rich tissue. The adult rats were unilaterally lesioned by the injection of 6-hydroxydopamine to their left medial forebrain bundles. At 5–10 weeks following the dopaminergic denervation, the levels of GABA_AR in the left caudate-putamen and globus pallidus were about 20 and 16% lower than that of the right intact (control) sides, as shown by [³H]flunitrazepam binding autoradiography on the brain sections. However, the receptor density increased to around 132 and 130% of control levels in the entopeduncular nucleus and substantia nigra pars reticulata of the lesioned sides. Furthermore, in situ hybridization analysis exhibited parallel trends of changes in the levels of the GABA_AR α₁ and α₂ subunit and mGluR5 mRNAs in the neurons of the brain regions with that of the proteins detected by the binding assay. A number of the rats 5 weeks postlesion were transplanted with the ventral mesencephalon of the embryonic rat into their left striata. Five weeks later, the changes in the [³H]flunitrazepam binding seemed to be recovered by approximately 50–63% on the grafted sides of the areas. Moreover, the transplantation appeared to produce a nearly complete reversal of the lesion-induced alterations in the levels of the mRNAs. Thus, the data indicate the mechanism of gene regulation for the modified expression of the receptors and could implicate the participation of the receptors in the pathogenesis of Parkinson's disease.     

A Novel Class of Succinimide-Derived Negative Allosteric Modulators of Metabotropic Glutamate Receptor Subtype 1 Provides Insight into a Disconnect in Activity between the Rat and Human Receptors

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Expression of the α7, α4 and α3 nicotinic receptor subtype in the brain and adrenal medulla of transgenic mice carrying genes coding for human AChE and β-amyloid

Human AChE-enzyme (hAChE) enhances the over-expression of beta-amyloid (Abeta) containing plaques in the brain of transgenic mice (APP(SWE)/hAChE-Tg) carrying mutated genes for human amyloid precursor protein (APP(SWE)) and hAChE. In this study, we showed that interaction of hAChE with Abeta affects the plasticity of the alpha7 nicotinic acetylcholine receptors (nAChRs) both in the brain and adrenal medulla. An age-related increase in the (125)I-alphabungarotoxin ((125)I-alphaBTX) binding (specific to alpha7 nAChRs) was observed in the adrenal medulla of 3, 7 and 10 months old control mice. In contrast, a significant decrease in (125)I-alphaBTX binding was detected in the adrenal medulla of 10 months old APP(SWE)/hAChE-Tg. A significantly higher alpha7 nAChR mRNA level was observed in the brain of APP(SWE)/hAChE-Tg at 3 and 7 months of age and in the adrenal medulla at 3 and 10 months of age compared to those of the control mice. The alpha3 nAChR mRNA level was significantly higher in the brain of APP(SWE)/hAChE-Tg at 3 months of age and in the adrenal medulla at 10 months of age. The alpha4 nAChR mRNA level remained unchanged in the brain and adrenal medulla of APP(SWE)/hAChE-Tg for all age groups. Based on these observations, we conclude that a high load of Abeta and an over-expression of hAChE induce differences in the expression of the nAChR subtypes at various ages in the brain and in the adrenal medulla of hAChE/APP(SWE)Tg mice. The findings may have implications for a better understanding the underlying mechanism for AD-related pathogenesis.     

Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes

The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T^3.40 was selected based on sequence alignments both between subtypes and between species and G^2.68, L^4.60 and Q^6.55 also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T^3.40I and Q^6.55A. Increased affinity was also observed for PYY to Q^6.55A. PYY(3-36) displayed decreased affinity for G^2.68N and L^4.60A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T^3.40I, L^4.60A and Q^6.55A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.