Charlotte Deleuze

Agonist action of taurine on glycine receptors in rat supraoptic magnocellular neurones: possible role in osmoregulation

1 To evaluate the implication of taurine in the physiology of supraoptic neurones, we (i) investigated the agonist properties of taurine on glycine and GABAA receptors of supraoptic magnocellular neurones acutely dissociated from adult rats, using whole‐cell voltage clamp, (ii) studied the effects of taurine and strychnine in vivo by extracellular recordings of supraoptic vasopressin neurones in anaesthetized rats, and (iii) measured the osmolarity‐dependent release of endogenous taurine from isolated supraoptic nuclei by HPLC. 2 GABA, glycine and taurine evoked rapidly activating currents that all reversed close to the equilibrium potential for Cl−, indicating activation of Cl− selective channels. Glycine‐activated currents were reversibly blocked by strychnine (IC50 of 35 nM with 100μm glycine), but were unaffected by the GABAA antagonist gabazine (1–3 μm). GABA‐activated currents were reversibly antagonized by 3 μm gabazine, but not by strychnine (up to 1 μm). 3 Responses to 1 mm taurine were blocked by strychnine but not by gabazine and showed no additivity with glycine‐induced currents, indicating selective activation of glycine receptors. Responses to 10 mm taurine were partially antagonized by gabazine, the residual current being blocked by strychnine. Thus, taurine is also a weak agonist of GABAA receptors. 4 In the presence of gabazine, taurine activated glycine receptors with an EC50 of 406 μm. Taurine activated at most 70% of maximal glycine currents, suggesting that it is a partial agonist of glycine receptors. 5 In vivo, locally applied strychnine (300 mm) increased and taurine (1 mm) decreased the basal electrical activity of vasopressin neurones in normally hydrated rats. The effect of strychnine was markedly more pronounced in water‐loaded rats. 6 Taurine, which is concentrated in supraoptic glial cells, could be released from isolated supraoptic nuclei upon hyposmotic stimulation. Decreases in osmolarity of 15 and 30% specifically enhanced basal release of taurine by 42 and 124%, respectively. 7 We conclude that supraoptic neurones express high amounts of glycine receptors, of which taurine may be regarded as a major natural agonist. We postulate that taurine, which can be released in hyposmotic situations, acts on glycine receptors to exert an inhibitory control on magnocellular neurones during alterations of body fluid homeostasis, implicating an active participation of glial cells in this neuroendocrine regulatory loop.

Osmotic regulation of neuronal activity: a new role for taurine and glial cells in a hypothalamic neuroendocrine structure

Maintenance of osmotic pressure is a primary regulatory process essential for normal cell function. The osmolarity of extracellular fluids is regulated by modifying the intake and excretion of salts and water. A major component of this regulatory process is the neuroendocrine hypothalamo-neurohypophysial system, which consists of neurons located in the paraventricular and supraoptic nuclei. These neurons synthesize the neurohormones vasopressin and oxytocin and release them in the blood circulation. We here review the mechanisms responsible for the osmoregulation of the activity of these neurons. Notably, the osmosensitivity of the supraoptic nucleus is described including the recent data that suggests an important participation of taurine in the transmission of the osmotic information. Taurine is an amino acid mainly known for its involvement in cell volume regulation, as it is one of the major inorganic osmolytes used by cells to compensate for changes in extracellular osmolarity. In the supraoptic nucleus, taurine is highly concentrated in astrocytes, and released in an osmodependent manner through volume-sensitive anion channels. Via its agonist action on neuronal glycine receptors, taurine is likely to contribute to the inhibition of neuronal activity induced by hypotonic stimuli. This inhibitory influence would complement the intrinsic osmosensitivity of supraoptic neurons, mediated by excitatory mechanoreceptors activated under hypertonic conditions. These observations extend the role of taurine from the regulation of cell volume to that of the whole body fluid balance. They also point to a new role of supraoptic glial cells as active components in a neuroendocrine regulatory loop.

Properties and glial origin of osmotic-dependent release of taurine from the rat supraoptic nucleus

1 Taurine, prominently concentrated in glial cells in the supraoptic nucleus (SON), is probably involved in the inhibition of SON vasopressin neurones by peripheral hypotonic stimulus, via activation of neuronal glycine receptors. We report here the properties and origin of the osmolarity‐dependent release of preloaded [3H]taurine from isolated whole SO nuclei. 2 Hyposmotic medium induced a rapid, reversible and dose‐dependent increase in taurine release. Release showed a high sensitivity to osmotic change, with a significant enhancement with less than a 5 % decrease in osmolarity. Hyperosmotic stimulus decreased basal release. 3 Evoked release was independent of extracellular Ca2+ and Na+, and was blocked by the Cl− channel blockers DIDS (4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid) and DPC (N‐phenylanthranilic acid), suggesting a diffusion process through volume‐sensitive Cl− channels. 4 Evoked release was transient for large osmotic reductions (≥ 15 %), probably reflecting regulatory volume decrease (RVD). However, it was sustained for smaller changes, suggesting that taurine release induced by physiological variations in osmolarity is not linked to RVD. 5 Basal and evoked release were strongly inhibited by an incubation of the tissue with the glia‐specific toxin fluorocitrate, but were unaffected by a neurotoxic treatment with NMDA, demonstrating the glial origin of the release of taurine in the SON. 6 The high osmosensitivity of taurine release suggests an important role in the osmoregulation of the SON function. These results strengthen the notion of an implication of taurine and glial cells in the regulation of the whole‐body fluid balance through the modulation of vasopressin release.

Tyrosine phosphorylation modulates the osmosensitivity of volume‐dependent taurine efflux from glial cells in the rat supraoptic nucleus

1 In the supraoptic nucleus, taurine, selectively released in an osmodependent manner by glial cells through volume‐sensitive anion channels, is likely to inhibit neuronal activity as part of the osmoregulation of vasopressin release. We investigated the involvement of various kinases in the activation of taurine efflux by measuring [3H]taurine release from rat acutely isolated supraoptic nuclei. 2 The protein tyrosine kinase inhibitors genistein and tyrphostin B44 specifically reduced, but did not suppress, both the basal release of taurine and that evoked by a hypotonic stimulus. Inhibition of tyrosine phosphatase by orthovanadate had the opposite effect. 3 The tyrosine kinase and phosphatase inhibitors shifted the relationship between taurine release and medium osmolarity in opposite directions, suggesting that tyrosine phosphorylation modulates the osmosensitivity of taurine release, but is not necessary for its activation. 4 Genistein also increased the amplitude of the decay of the release observed during prolonged hypotonic stimulation. Potentiation of taurine release by tyrosine kinases could serve to maintain a high level of taurine release in spite of cell volume regulation. 5 Taurine release was unaffected by inhibitors and/or activators of PKA, PKC, MEK and Rho kinase. 6 Our results demonstrate a unique regulation by protein tyrosine kinase of the osmosensitivity of taurine efflux in supraoptic astrocytes. This points to the presence of specific volume‐dependent anion channels in these cells, or to a specific activation mechanism or regulatory properties. This may relate to the particular role of the osmodependent release of taurine in this structure in the osmoregulation of neuronal activity.

Molecular determinants of emerging excitability in rat embryonic motoneurons

Molecular determinants of excitability were studied in pure cultures of rat embryonic motoneurons. Using RT‐PCR, we have shown here that the spike‐generating Na+ current is supported by Nav1.2 and/or Nav1.3 α‐subunits. Nav1.1 and Nav1.6 transcripts were also identified. We have demonstrated that alternatively spliced isoforms of Nav1.1 and Nav1.6, resulting in truncated proteins, were predominant during the first week in culture. However, Nav1.6 protein could be detected after 12 days in vitro. The Navβ2.1 transcript was not detected, whereas the Nav β1.1 transcript was present. Even in the absence of Navβ2.1, α‐subunits were correctly inserted into the initial segment. RT‐PCR (at semi‐quantitative and single‐cell levels) and immunocytochemistry showed that transient K+ currents result from the expression of Kv4.2 and Kv4.3 subunits. This is the first identification of subunits responsible for a transient K+ current in spinal motoneurons. The blockage of Kv4.2/Kv4.3 using a specific toxin modified the shape of the action potential demonstrating the involvement of these conductance channels in regulating spike repolarization and the discharge frequency. Among the other Kv α‐subunits (Kv1.3, 1.4, 1.6, 2.1, 3.1 and 3.3), we showed that the Kv1.6 subunit was partly responsible for the sustained K+ current. In conclusion, this study has established the first correlation between the molecular nature of voltage‐dependent Na+ and K+ channels expressed in embryonic rat motoneurons in culture and their electrophysiological characteristics in the period when excitability appears.

Minimal alterations in T-type calcium channel gating markedly modify physiological firing dynamics

Non‐technical summary  Voltage‐dependant calcium channels constitute a heterogeneous group playing ubiquitous roles in excitable cells. Among them the low‐voltage activated T‐type channels generate a family of currents that differ in their biophysical properties reflecting structural or neuromodulatory diversity. These T‐type calcium channels are highly expressed in neurons located in the thalamus, a brain structure considered as the gateway to the cortex. Thalamic T‐type calcium channels are critically involved in oscillatory neuronal activities associated with sleep or epilepsy and may contribute to sensory processing. Using injections of computer‐simulated T‐type conductances (a real time mimicry of ionic currents) in biological thalamic neurons, we dissect how the diversity in T‐type currents impact on the output of thalamic neurons. We show that very subtle modifications in the properties of the T current that were overlooked so far affect drastically the physiological output of the thalamic neurons and therefore condition the dynamics of thalamo‐cortical information integration.

New Role of Taurine as an Osmomediator between Glial Cells and Neurons in the Rat Supraoptic Nucleus

Taurine has an established function as an osmolyte in the nervous system. Present inside cells at high concentration, it is released upon cell swelling induced by a decrease in extracellular tonicity. The loss of taurine is believed to contribute in a major way to the subsequent regulatory volume decrease (RVD) undergone by both neurons and astrocytes25. However, some observations indicate that the osmoregulatory role of taurine in the nervous system may be more complicated than sometimes thought. First, the distribution of taurine is not homogenous, as it is concentrated in selected sets of cells in various brain areas. For instance, it is found almost exclusively in Purkinje neurons in the cerebellum24, primarily in neurons in hippocampal and cortical formations, but prominently in astrocytes in the hypothalamus and brain stem18,32. While volume regulation of all brain cells should be critical for proper neuronal function, the uneven distribution of taurine suggests a differential use of this amino acid by the various cell populations. Second, taurine is not an inactive compound as it would be expected for an “ideal” osmolyte. Taurine is an agonist of the inhibitory ligand-gated channels glycine and GABAA receptors , binds to the metahtropic GABAB receptor14,17, and alters the electrical activity of neurons and other excitable cells to various extents9,31. Therefore, release of taurine from brain cells under hypotonic stress may well serve other function 4,9,11,36

Cortically-Controlled Population Stochastic Facilitation as a Plausible Substrate for Guiding Sensory Transfer across the Thalamic Gateway

The thalamus is the primary gateway that relays sensory information to the cerebral cortex. While a single recipient cortical cell receives the convergence of many principal relay cells of the thalamus, each thalamic cell in turn integrates a dense and distributed synaptic feedback from the cortex. During sensory processing, the influence of this functional loop remains largely ignored. Using dynamic-clamp techniques in thalamic slices in vitro, we combined theoretical and experimental approaches to implement a realistic hybrid retino-thalamo-cortical pathway mixing biological cells and simulated circuits. The synaptic bombardment of cortical origin was mimicked through the injection of a stochastic mixture of excitatory and inhibitory conductances, resulting in a gradable correlation level of afferent activity shared by thalamic cells. The study of the impact of the simulated cortical input on the global retinocortical signal transfer efficiency revealed a novel control mechanism resulting from the collective resonance of all thalamic relay neurons. We show here that the transfer efficiency of sensory input transmission depends on three key features: i) the number of thalamocortical cells involved in the many-to-one convergence from thalamus to cortex, ii) the statistics of the corticothalamic synaptic bombardment and iii) the level of correlation imposed between converging thalamic relay cells. In particular, our results demonstrate counterintuitively that the retinocortical signal transfer efficiency increases when the level of correlation across thalamic cells decreases. This suggests that the transfer efficiency of relay cells could be selectively amplified when they become simultaneously desynchronized by the cortical feedback. When applied to the intact brain, this network regulation mechanism could direct an attentional focus to specific thalamic subassemblies and select the appropriate input lines to the cortex according to the descending influence of cortically-defined “priors”.

Heterogeneous firing rate response of mouse layer V pyramidal neurons in the fluctuation‐driven regime

We recreated in vitro the fluctuation‐driven regime observed at the soma during asynchronous network activity in vivo and we studied the firing rate response as a function of the properties of the membrane potential fluctuations. We provide a simple analytical template that captures the firing response of both pyramidal neurons and various theoretical models. We found a strong heterogeneity in the firing rate response of layer V pyramidal neurons: in particular, individual neurons differ not only in their mean excitability level, but also in their sensitivity to fluctuations. Theoretical modelling suggest that this observed heterogeneity might arise from various expression levels of the following biophysical properties: sodium inactivation, density of sodium channels and spike frequency adaptation.

Firing rate response of neocortical neurons in the fluctuation-driven regime.

Characterizing the input-output properties of neocortical neurons is of crucial importance to understand the properties emerging at the network level. While deriving those input-output relations for artificial neuronal models has been the object of intense investigations, determining those properties for real neocortical cells remains both experimentally and theoretically challenging [1]. Here, we identify four somatic variables that characterize the dynamical state at the soma in the fluctuation-driven regime and we investigate the firing rate response of pyramidal neocortical neurons in this four dimensional space by means of perforated patch recordings in mice cortex in vitro. We compare our measurements with different single compartment models (Integrate and Fire, Exponential Integrate and Fire and Hodgkin-Huxley models) and we discuss the limitations imposed by those models in the input-output relationship. We also construct an analytical template for the firing rate response and we show that it is able to capture the behavior of both neuronal models and neocortical neurons. Because of the difficulty to reproduce the measured characteristics with single compartment models, our results argue in favor of phenomenological models for the cellular computation, where the complex details of the biophysical features are absorbed within a flexible higher-level description. Moreover, the transfer functions obtained from neurons in slices will enable building mean-field models based on realistic neuronal properties.