Endogenous angiotensin II facilitates GABAergic neurotransmission afferent to the Na+-responsive neurons of the rat median preoptic nucleus.

The median preoptic nucleus (MnPO) is densely innervated by efferent projections from the subfornical organ (SFO) and, therefore, is an important relay for the peripheral chemosensory and humoral information (osmolality and serum levels ANG II). In this context, controlling the excitability of MnPO neuronal populations is a major determinant of body fluid homeostasis and cardiovascular regulation. Using a brain slice preparation and patch-clamp recordings, our study sought to determine whether endogenous ANG II modulates the strength of the SFO-derived GABAergic inputs to the MnPO. Our results showed that the amplitude of the inhibitory postsynaptic currents (IPSCs) were progressively reduced by 44 +/- 2.3% by (Sar(1), Ile(8))-ANG II, a competitive ANG type 1 receptor (AT(1)R) antagonist. Similarly, losartan, a nonpeptidergic AT(1)R antagonist decreased the IPSC amplitude by 40.4 +/- 5.6%. The facilitating effect of endogenous ANG II on the GABAergic input to the MnPO was not attributed to a change in GABA release probability and was mimicked by exogenous ANG II, which potentiated the amplitude of the muscimol-activated GABA(A)/Cl(-) current by 53.1 +/- 11.4%. These results demonstrate a postsynaptic locus of action of ANG II. Further analysis reveals that ANG II did not affect the reversal potential of the synaptic inhibitory response, thus privileging a cross talk between postsynaptic AT(1) and GABA(A) receptors. Interestingly, facilitation of GABAergic neurotransmission by endogenous ANG II was specific to neurons responding to changes in the ambient Na(+) level. This finding, combined with the ANG II-mediated depolarization of non-Na(+)-responsive neurons reveals the dual actions of ANG II to modulate the excitability of MnPO neurons.

[Role of Ca(2+) in the pacemaker-like property of spinal motoneurons]. / Rôle du Ca(2+) dans la propriété de type pacemaker des motoneurones spinaux.

It is generally accepted that locomotion in vertebrate species is produced by signals coded and integrated by neurons of the spinal cord. In fact, the basic features of locomotion, including patterns and rhythms, are generated by a network of neurons called the CPG (central pattern generator) essentially localized in the lumbar segments of the spinal cord. However, the detailed mechanisms underlying the rhythmic aspect of CPG-generated locomotion are not fully understood. Here, we report data of studies that focus on the role of Ca(2+)-related mechanisms involved in the expression of the pacemaker property of lumbar motoneurons that innervate the hindlimbs. In fact, it has become increasingly clear that Ca(2+) plays a determinant function in the expression of this active and conditional rhythmic property. In addition to NMDA-mediated currents (NMDA is an agonist of the calcium permeable ionotropic glutamatergic receptor) and to a Ca(2+)-dependent K(+) current that were found twenty years ago to contribute to intrinsic voltage oscillations in motoneurons, a pivotal role for voltage-gated channels (e.g., CaV1.3) and intracellular Ca(2+) concentrations ([Ca(2+)]i) have recently been shown. Increasing evidence of a role for metabotropic receptor subtypes, calmodulin (a calcium binding protein), ryanodine and IP3-sensitive intracellular stores of Ca(2+) suggests that additional mechanisms are yet to be identified. A detailed understanding of the complex role of Ca(2+) in mediating the auto-rhythmic property of spinal neurons may contribute to the development of novel therapeutic approaches to induce locomotion after spinal cord injury.

Heterogeneous chloride homeostasis and GABA responses in the median preoptic nucleus of the rat.

The median preoptic nucleus (MnPO) is an integrative structure of the hypothalamus receiving periphery-derived information pertinent to hydromineral and cardiovascular homeostasis. In this context, excitability of MnPO neurones is controlled by fast GABAergic, glutamatergic and angiotensinergic projection from the subfornical organ (SFO). Taking advantage of a brain slice preparation preserving synaptic connection between the SFO and the MnPO, and appropriate bicarbonate-free artificial cerebrospinal fluid (CSF), we investigated a possible implication of an active outward Cl- transport in regulating efficacy of the GABA(A) receptor-mediated inhibitory response at the SFO-MnPO synapse. When somata of the MnPO neurones was loaded with 18 mm chloride, stimulation of the SFO evoked outward inhibitory postsynaptic currents (IPSCs) in 81% of the MnPO neurones held at -60 mV. Accordingly, E(IPSC) was found 25 mV hyperpolarized from the theoretical value calculated from the Nernst equation, indicating that IPSC polarity and amplitude were driven by an active Cl- extrusion system in these neurones. E(IPSC) estimated with gramicidin-based perforated-patch recordings amounted -89.2 +/- 4.3 mV. Furosemide (100 microm), a pharmacological compound known to block the activity of the neurone-specific K(+)-Cl- cotransporter, KCC2, reversed IPSC polarity and shifted E(IPSC) towards its theoretical value. Presence of the KCC2 protein in the MnPO was further detected with immunohistochemistry, revealing a dense network of KCC2-positive intermingled fibres. In the presence of a GABA(B) receptor antagonist, high-frequency stimulation (5 Hz) of the SFO evoked a train of IPSCs or inhibitory postsynaptic potentials (IPSPs), whose amplitude was maintained throughout the sustained stimulation. Contrastingly, similar 5 Hz stimulation carried out in the presence of furosemide (50 microm) evoked IPSCs/IPSPs, whose amplitude collapsed during the high-frequency stimulation. Similar reduction in inhibitory neurotransmission was also observed in MnPO neurones lacking the functional Cl- extrusion mechanism. We conclude that a majority of MnPO neurones were characterized by a functional Cl- transporter that ensured an efficient activity-dependent Cl- transport rate, allowing sustained synaptic inhibition of these neurones. Pharmacological and anatomical data strongly suggested the involvement of KCC2, as an essential postsynaptic determinant of the inhibitory neurotransmission afferent to the MnPO, a key-structure in the physiology of the hydromineral and cardiovascular homeostasis.

Specific Na+ sensors are functionally expressed in a neuronal population of the median preoptic nucleus of the rat.

Whole-cell patch-clamp recordings were performed on acute brain slices of male rats to investigate the ability of the neurons of the median preoptic nucleus (MnPO) to detect fluctuation in extracellular osmolarity and sodium concentration ([Na+]out). Local application of hypotonic and hypertonic artificial CSF hyperpolarized and depolarized the neurons, respectively. Similar responses obtained under synaptic isolation (0.5 microM TTX) highlighted the intrinsic ability of the MnPO neurons to detect changes in extracellular osmolarity and [Na+]out. Manipulating extracellular osmolarity, [Na+]out, and [Cl-]out showed in an independent manner that the MnPO neurons responded to a change in [Na+]out exclusively. The specific Na+ response was voltage insensitive and depended on the driving force for Na+ ions, indicating that a sustained background Na+ permeability controlled the membrane potential of the MnPO neurons. This specific response was not reduced by Gd3+, amiloride, or benzamil, ruling out the participation of mechanosensitive cationic channels, specific epithelial Na+ channels, and Phe-Met-Arg-Phe-gated Na+ channels, respectively. Combination of in situ hybridization, using a riboprobe directed against the atypical Na+ channel (Na(X)), and immunohistochemistry, using an antibody against neuron-specific nuclei protein, revealed that a substantial population of MnPO neurons expressed the Na(X) channel, which was characterized recently as a concentration-sensitive Na+ channel. This study shows that a neuronal population of the MnPO acts as functional Na+ sensors and that the Na(X) channel might represent the molecular basis for the extracellular sodium level sensing in these neurons.

Heterogeneous co-localization of AT 1A receptor and Fos protein in forebrain neuronal populations responding to acute hydromineral deficit.

The present study investigates co-localization of AT(1A) receptor subtype and Fos protein in neuronal populations of the lamina terminalis (LT) that have been recruited during acute Na(+) and water depletion mediated by furosemide injections. For that purpose, we combined high cellular resolution of in situ hybridization technique to reveal neurons expressing AT(1A) receptor gene (AT(1A) mRNA) with the specificity of Fos protein immunoreactivity as a marker of neuronal activation (Fos-ir). As expected, furosemide treatment dramatically increased the density of Fos-immunoreactive neuronal population in all the regions of the LT compared to control (saline-injected animals). Distribution analysis of Fos-ir neurons and AT(1A) receptor-expressing neurons performed consecutively to furosemide-induced Na(+) and water depletion indicated that double-labeled neurons (AT(1A) mRNA+Fos-ir) represented the majority (67%) of the neuronal population that expressed AT(1A) receptor in the rim of the vascular organ of the lamina terminalis (OVLT). Double-labeled neurons amounted about 60% of the neurons that expressed AT(1A) receptor in the core of the subfornical organ (SFO) and 34% in the periphery of the SFO. In the median preoptic nucleus (MnPO), the density of the double-labeled neuronal population observed in the furosemide-treated animals remained weak compared to the control group of animals. Double-labeled neuronal population estimated in the MnPO of the furosemide-treated group of animals represented 17% of the neurons that express AT(1A) receptor gene. Our results report a heterogeneous distribution of the neuronal populations that co-localize AT(1A) receptor and Fos protein in the lamina terminalis after an acute Na(+) and water depletion. This study gives anatomical support to a direct action of endogenous AngII on c-fos transcription via binding on AT(1A) receptor in specific areas of the circumventricular organs (rim of the OVLT and core of the SFO). In the MnPO, our data indicate that intracellular signaling pathways unlikely couple AT(1A) receptor with c-fos transcription. The expression of Fos protein in this nucleus might be therefore secondary to the recruitment of excitatory inputs different from AngII. This observation underlines the complexity of molecules and neurocircuits in the preoptic region that are involved in the control of acute Na(+) and water deficit.