Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system

Glycine receptors are ionotropic receptors formed by either the homomeric assembly of ligand-binding alpha subunits or the heteromeric combination of an alpha subunit and the auxiliary beta subunit. Glycine receptors in the brain are found at either pre- or post-synaptic sites. Rat supraoptic nucleus neurons express glycine receptors on the membrane of both their soma and dendrites within the supraoptic nucleus, and their axon terminals in the neurohypophysis. Taking advantage of the well-separated cellular compartments of this system, we correlated the structural properties of the receptors to their subcellular localization. Immunohistochemical study using the generic mAb4a antibody revealed that somatodendritic receptors were clustered, whereas axonal glycine receptors showed a more diffuse distribution. This was paralleled by the presence of clusters of the glycine receptor aggregating protein gephyrin in the supraoptic nucleus and its complete absence in the neurohypophysis. Moreover, another antibody recognizing the alpha1/alpha2 subunits similarly labeled the axonal glycine receptors, but did not recognize the somatodendritic receptor clusters of supraoptic nucleus neurons, indicative of structural differences between somatic and axonal glycine receptors. Furthermore, the subunits composing the somatic and axonal receptors have different molecular weight. Functional study further differentiated the two types of glycine receptors on the basis of their sensitivity to picrotoxin, identifying somatic receptors as alpha/beta heteromers, and axonal receptors as alpha homomers. These results indicate that targeting of glycine receptors to axonal or somatodendritic compartment is directly related to their subunit composition, and set the hypothalamo-neurohypophysial system as an excellent model to study the mechanisms of targeting of proteins to various neuronal cellular compartments

Pharmacological characterization of volume-sensitive, taurine permeable anion channels in rat supraoptic glial cells

International audience1 To characterize the volume-sensitive, osmolyte permeable anion channels responsible for the osmodependent release of taurine from supraoptic nucleus (SON) astrocytes, we investigated the pharmacological properties of the [ 3 H]-taurine eux from acutely isolated SON. 2 Taurine release induced by hypotonic stimulus (250 mosmol l 71) was not antagonized by the taurine transporter blocker guanidinoethyl sulphonate, con®rming the lack of implication of the transporter. 3 The osmodependent release of taurine was blocked by a variety of Cl 7 channel inhibitors with the order of potency: NPPB4ni¯umic acid4DPC4DIDS4ATP. On the other hand, release of taurine was only weakly aected by other compounds (dideoxyforskolin, 4-bromophenacyl bromide, mibefradil) known to block volume-activated anion channels in other cell preparations, and was completely insensitive to tamoxifen, a broad inhibitor of these channels. 4 Although the molecular identity of volume-sensitive anion channels is not ®rmly established, a few genes have been postulated as potential candidates to encode such channels. We checked the expression in the SON of three of them, ClC 3 , phospholemman and VDAC 1 , and found that the transcripts of these genes are found in SON neurons, but not in astrocytes. Similar observation was previously reported for ClC 2. 5 In conclusion, the osmodependent taurine permeable channels of SON astrocytes display a particular pharmacological pro®le, suggesting the expression of a particular type or subtype of volume-sensitive anion channel, which is likely to be formed by yet unidenti®ed proteins

Age-impaired fluid homeostasis depends on the balance of IL-6/IGF-I in the rat supraoptic nuclei

International audienceAdaptive metabolic changes associated with bacterial infections are likely to cause dehydration. Activation of hypothalamic neurons in the supraoptic nucleus that release anti-diuretic arginine-vasopressin in plasma provides water retention. Aging is characterized by arginine-vasopressin neuron hyper-activity and over-expression of pro-inflammatory cytokines like interleukin (IL)-6. Conversely, insulin-like growth factor (IGF)-I, known to exhibit anti-inflammatory properties, decreases with age. We compared activation of arginine-vasopressin neurons in adult (3 months) and aged (22 months) Wistar rats by measuring not only c-fos expression, plasma arginine-vasopressin and diuresis but also the expression of IL-6 and IGF-I in the supraoptic nuclei after intraperitoneal lipopolysaccharide injection. Aged rats displayed a heightened, shorter lasting activation of arginine-vasopressin neurons following lipopolysaccharide as compared to adults. IL-6 mRNA was 3-fold higher while IGF-I mRNA was 10-fold lower in aged than in adult rats. Brain pre-treatment with neutralizing anti-IL-6 antibodies or recombinant IGF-I in aged rats reversed lipopolysaccharide-induced anti-diuresis. These data extend the concept of neuroendocrine-immune interactions to the arginine-vasopressin neuronal system by establishing a relationship between brain IL-6/IGF-I balance and age-associated arginine-vasopressin neuronal dysfunction

Differential coupling of the vasopressin V1b receptor through compartmentalization within the plasma membrane.

International audienceWe show here that the rat vasopressin V(1b) receptor simultaneously activates both the G(q/11)-inositol phosphate (IP) and G(s)-cAMP pathways when transiently expressed in Chinese hamster ovary, human embryonic kidney (HEK) 293, and COS-7 cells and stimulated with arginine-vasopressin. Higher concentrations of the hormone, however, were needed to trigger the cAMP pathway. The nonmammalian analog arginine-vasotocin and the selective V(1b) agonist d[Cha(4)]vasopressin also activated the cAMP and IP pathways, although d[Cha(4)]-vasopressin elicited the two responses with equivalent potencies. We determined that the V(1b) receptor is present as a homodimer at the plasma membrane. Treatment of V(1b)-transfected HEK-293 cells with methyl-beta-cyclodextrin, a drug known to dissociate cholesterol-rich domains of the plasma membrane, shifted the EC(50) of the vasopressin-induced cAMP accumulation to lower concentrations and, remarkably, increased the hormone efficacy related to the activation of this second messenger system. In parallel, the vasopressin-mediated activation of the IP pathway was slightly reduced without modification of its EC(50). These results suggest that, as with many other G protein-coupled receptors, when transfected in heterologous cell systems, the V(1b) receptor forms dimers that signal differentially through the G(q/11) and G(s) proteins depending on the nature of the ligand as well as on its localization within specialized compartments of the plasma membrane. The present study thus illustrates how signal transduction associated with the activation of a G protein-coupled receptor can be versatile and highly dependent on both the cell context and the chemical nature of the extracellular signaling messenger

The V 1a and V 1b , But Not V 2 , Vasopressin Receptor Genes Are Expressed in the Supraoptic Nucleus of the Rat Hypothalamus, and the Transcripts Are Essentially Colocalized in the Vasopressinergic Magnocellular Neurons

International audienceWe have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraop-tic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V 1a , but also V 1b , subtypes of VP receptors are expressed from birth to adult-hood. No V 2 receptor messenger RNA (mRNA) was detected. Furthermore , the single-cell RT-nested PCR indicated that the V 1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V 1a and V 1b receptor mRNAs in supraoptic and para-ventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immu-nocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V 1a or V 1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V 1a and V 1b subtype receptors