Carola Eva

Physiology and gene regulation of the brain NPY Y1 receptor

Neuropeptide Y (NPY) is one of the most prominent and abundant neuropeptides in the mammalian brain where it interacts with a family of G-protein coupled receptors, including the Y(1) receptor subtype (Y(1)R). NPY-Y(1)R signalling plays a prominent role in the regulation of several behavioural and physiological functions including feeding behaviour and energy balance, sexual hormone secretion, stress response, emotional behaviour, neuronal excitability and ethanol drinking. Y(1)R expression is regulated by neuronal activity and peripheral hormones. The Y(1)R gene has been isolated from rodents and humans and it contains multiple regulatory elements that may participate in the regulation of its expression. Y(1)R expression in the hypothalamus is modulated by changes in energetic balance induced by a wide variety of conditions (fasting, pregnancy, hyperglycaemic challenge, hypophagia, diet induced obesity). Estrogens up-regulate responsiveness to NPY to stimulate preovulatory GnRH and gonadotropin surges by increasing Y(1)R gene expression both in the hypothalamus and the pituitary. Y(1)R expression is modulated by different kinds of brain insults, such as stress and seizure activity, and alteration in its expression may contribute to antidepressant action. Chronic modulation of GABA(A) receptor function by benzodiazepines or neuroactive steroids also affects Y(1)R expression in the amygdala, suggesting that a functional interaction between the GABA(A) receptor and Y(1)R mediated signalling may contribute to the regulation of emotional behaviour. In this paper, we review the state of the art concerning Y(1)R function and gene expression, including our personal contribution to many of the subjects mentioned above.

The murine NPY-1 receptor gene. Structure and delineation of tissue-specific expression.

The murine gene for the NPY‐1 receptor subtype for neuropeptide Y was characterized by DNA sequencing and expression studies. It comprises three exons with a 6,400 bp 5′‐untranslated and a 80 bp internal intronic sequence. The 5′‐flanking region of this gene lacks TATA or CCAAT consensus sequences in the proximity to the multiple transcription initiation sites. A 1,300 bp genomic fragment of the 5′‐flanking region drives the expression of the lacZ reporter gene in NG108‐15 cells and primary cultured neurons but not in glial and human embryonic kidney cells. In addition, it contains consensus sequences for various transcription factors including cAMP‐ and glucocorticoid‐responsive elements.

M1 muscarinic receptors increase calcium current and phosphoinositide turnover in guinea-pig ventricular cardiocytes.

1. Physiological and molecular evidence for the presence and functional role of M1 muscarinic cholinergic receptors (mAChRs) in adult guinea‐pig ventricular cells is presented. 2. Whole‐cell clamp measurements of the L‐type calcium current (ICa) in isolated myocytes were performed. Caesium was used to suppress potassium currents. ICa was increased by the muscarinic agonist carbachol in cells pretreated with pertussis toxin which blocked the M2 mAChR‐triggered cascade of intracellular signalling, while it was not changed in untreated cells. 3. If the M2‐mediated regulation of ICa was blocked by directly saturating the cell with cyclic adenosine monophosphate (cAMP) through the patch pipette, application of carbachol induced a further small increase of the current above the level reached after cAMP perfusion. This increase was more pronounced in cells pretreated with pertussis toxin. 4. The carbachol‐induced increase of ICa was blocked by the selective M1 mAChR antagonist pirenzepine. 5. The application of high concentrations of carbachol increased the accumulation of [3H]inositol monophosphate up to 240% above control levels. This increase was reduced by application of pirenzepine. 6. The expression of M1 receptor mRNA in ventricular cardiocytes was shown by reverse transcriptase‐polymerase chain reaction. 7. These results suggest that M1 mAChR regulation of ICa can be a component of the paradoxical positive inotropism induced by high concentrations of muscarinic agonists.

Potassium Ion Facilitation of Phosphoinositide Turnover Activation by Muscarinic Receptor Agonists in Rat Brain

Abstract: In rat hippocampal slices kept in Krebs‐Henseleit medium, an increase of K+ ions to 12 mMpotentiates the stimulation of phosphoinositide turnover elicited by carbachol and (±)‐cis‐methyldioxolane. Oxotremorine is inactive if tested in Krebs‐Henseleit medium but it stimulates by 220% the phosphoinositide turnover when K+ is increased to 12 mM. The K+ facilitation of the carbachol stimulation of phosphoinositide turnover was blocked by pirenzepine, a muscarinic antagonist. This drug was equally potent in inhibiting the carbachol stimulation of phosphoinositide turnover both in normal and 12 mM K+ Krebs medium. This facilitatory effect of K+ appears to be preferential for muscarinic receptors, since it failed to increase the activation of phosphoinositide breakdown induced by norepinephrine and histamine. The K+ potentiation of the muscarinic stimulation of phosphoinositide turnover is not mediated by a release of one of the endogenous neurotransmitters stored in these slices because such a facilitation occurs in Ca2+ ‐deprived Krebs‐Henseleit medium and failed to occur following a depolarizing dose of veratrine. Our experiments excluded that K+ facilitates carbachol stimulation of phosphoinositide turnover because it modifies the binding characteristics of muscarinic receptors; however, they cannot exclude that K+ acts at the receptor transducer coupling.

Decrease of the D4 dopamine receptor messenger RNA expression in lymphocytes from patients with major depression

OBJECTIVES The evaluation of the possible role of dopamine in psychiatric disorders has been limited by the relative inadequacy of tools. A tempting approach to examine alterations of dopaminergic system in major depression is to examine the expression of dopamine receptors in peripheral blood mononuclear cells (PBMC). METHODS D4 dopamine receptor (D4DR) messenger RNA (mRNA) expression in PBMC from 12 patients with major depressive disorder was examined before and after an 8-week treatment with paroxetine at 20-50 mg/day. Ten healthy subjects were analyzed in parallel. The relative content of D4DR mRNA was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). using beta-actin as internal standard. RESULTS D4DR mRNA levels were significantly decreased in untreated depressed patients as compared to controls. D4DR mRNA expression returned to control levels after paroxetine treatment, when patients achieved a significant improvement of depressive symptoms. CONCLUSIONS Results of our study suggest the role of PBMC D4DR mRNA expression as a peripheral marker of the central dopaminergic function in major depression.

Increased expression of the neuropeptide Y receptor Y(1) gene in the medial amygdala of transgenic mice induced by long-term treatment with progesterone or allopregnanolone.

The neurosteroid allopregnanolone, a reduced metabolite of progesterone, induces anxiolytic effects by enhancing GABAA receptor function. Neuropeptide Y (NPY) and GABA are thought to interact functionally in the amygdala, and this interaction may be important in the regulation of anxiety. By using Y1R/LacZ transgenic mice, which harbour a fusion construct comprising the promoter of the mouse gene for the Y1 receptor for NPY linked to the lacZ gene, we previously showed that long‐term treatment with benzodiazepine receptor ligands modulates Y1 receptor gene expression in the medial amygdala. We have now investigated the effects of prolonged treatment with progesterone or allopregnanolone on Y1R/LacZ transgene expression, as determined by quantitative histochemical analysis of β‐galactosidase activity. Progesterone increased both the cerebrocortical concentration of allopregnanolone and β‐galactosidase expression in the medial amygdala. Finasteride, a 5α‐reductase inhibitor, prevented both of these effects. Long‐term administration of allopregnanolone also increased both the cortical concentration of this neurosteroid and transgene expression in the medial amygdala. Treatment with neither progesterone nor allopregnanolone affected β‐galactosidase activity in the medial habenula. These data suggest that allopregnanolone regulates Y1 receptor gene expression through modulation of GABAA receptor function, and they provide further support for a functional interaction between GABA and neuropeptide Y in the amygdala.

Chronic modulation of the GABAA receptor complex regulates Y1 receptor gene expression in the medial amygdala of transgenic mice

NPY exerts anxiolytic effects, which are mediated by activation of Y1 receptors in the amygdala. It has been shown that diazepam counteracts the anxiogenic effect of Y1 receptor antagonists, suggesting that NPYergic and GABAergic systems are coupled in the regulation of anxiety. We used a transgenic mouse model, expressing a mouse Y1 receptor-beta-galactosidase fusion gene (Y1R/LacZ), to study the effect of positive or negative modulators of GABA(A) receptors on Y1 receptor gene expression. Mice were treated for 14 days with diazepam (4 or 20 mg/kg), the anxiolytic beta-carboline-derivative abecarnil (0.3 or 6 mg/kg) and the anxiogenic beta-carboline FG7142 (20 mg/kg). Transgene expression was determined by quantitative analysis of beta-galactosidase histochemical staining in the medial amygdala and in the medial habenula as a control region. Chronic treatment with 20 mg/kg diazepam or 6 mg/kg abecarnil significantly increased, whereas FG 7142 decreased, transgene expression in the medial amygdala. A transient decrease in transgene expression was observed in the medial amygdala six hours after the acute treatment with 20 mg/kg FG 7142 but not with diazepam or abecarnil. No significant changes were observed in the medial habenula. These data suggest that modulation of GABA(A) receptor function may regulate Y1 receptor gene expression in medial amygdala.

GABAergic and NPY-Y1 network in the medial amygdala: a neuroanatomical basis for their functional interaction

We used Y(1)R/LacZ transgenic mice to investigate the interaction between NPY, GABA and Y(1) receptors in the amygdala. Immunolabeling of GABA and NPY positive neurons and histochemical staining of beta-galactosidase revealed NPY and GABA colocalization and close contacts of NPY-positive fibers with GABAergic neurons also expressing the Y(1)R/LacZ transgene.

Regulatory functions of limbic Y1 receptors in body weight and anxiety uncovered by conditional knockout and maternal care

Neuropeptide Y (NPY) plays an important role in stress, anxiety, obesity, and energy homeostasis via activation of NPY-Y1 receptors (Y1Rs) in the brain. However, global knockout of the Npy1r gene has low or no impact on anxiety and body weight. To uncover the role of limbic Y1Rs, we generated conditional knockout mice in which the inactivation of the Npy1r gene was restricted to excitatory neurons of the forebrain, starting from juvenile stages (Npy1rrfb). Npy1rrfb mice exhibited increased anxiety and reduced body weight, less adipose tissue, and lower serum leptin levels. Npy1rrfb mutants also had a hyperactive hypothalamic–pituitary–adrenocortical axis, as indicated by higher peripheral corticosterone and higher density of NPY immunoreactive fibers and corticotropin releasing hormone immunoreactive cell bodies in the paraventricular hypothalamic nucleus. Importantly, through fostering experiments, we determined that differences in phenotype between Npy1rrfb and Npy1r2lox mice became apparent when both genotypes were raised by FVB/J but not by C57BL/6J dams, suggesting that limbic Y1Rs are key targets of maternal care-induced programming of anxiety and energy homeostasis.

[3H]N-methylscopolamine binding to muscarinic receptors in human peripheral blood lymphocytes: Characterization, localization on T-lymphocyte subsets and age-dependent changes

The properties of the binding of the muscarinic receptor ligands, [3H]quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS) in human mononuclear cells were compared. The binding of [3H]QNB showed a high, non-specific component and lack of saturability in both intact mononuclear cells and preparations of lysed mononuclear cell membranes. Conversely the specific binding of [3H]NMS had a high affinity and was saturable at concentrations greater than 30 nM in both intact and broken cells. Classical muscarinic receptor antagonists displaced specific binding of [3H]NMS binding according to the law of mass action, while displacement curves for pirenzepine and muscarinic agonists were very shallow (nH less than 1), suggesting the presence of more than one subtype of muscarinic receptor on mononuclear cell membranes. Binding studies with [3H]NMS to purified mononuclear cell subpopulations demonstrated that muscarinic binding sites were mainly localized on thymus-derived (T) lymphocytes and large granule lymphocytes. Moreover evidence is presented of an age-dependent increase of the density of muscarinic binding sites on T-lymphocytes. The results are discussed in terms of the usefulness of the binding of [3H]NMS in studying the physiological function of muscarinic receptors on human T-lymphocytes and their possible changes in patients with neurological diseases.