Remodeling of glial coverage of glutamatergic synapses in the rat nucleus tractus solitarii after ozone inhalation.

Besides the well-described inflammatory and dysfunction effects on the respiratory tract, accumulating evidence indicates that ozone (O3 ) exposure also affects central nervous system functions. However, the mechanisms through which O3 exerts toxic effects on the brain remain poorly understood. We previously showed that O3 exposure caused a neuronal activation in regions of the rat nucleus tractus solitarii (NTS) overlapping terminal fields of vagal lung afferents. Knowing that O3 exposure can impact astrocytic protein expression, we decided to investigate whether it may induce astroglial cellular alterations in the NTS. Using electron microscopy and immunoblot techniques, we showed that in O3 -exposed animals, the astrocytic coverage of NTS glutamatergic synapses was 19% increased while the astrocyte volume fraction and membrane density were not modified. Moreover, the expression of glial fibrillary acidic protein and S100ß, which are known to be increased in reactive astroglia, did not change. These results indicate that O3 inhalation induces a glial plasticity that is restricted to the peri-synaptic coverage without overall astroglial activation. Taken together, these findings, along with our previous observations, support the conclusion that O3 -induced pulmonary inflammation results in a specific activation of vagal lung afferents rather than non-specific overall brain alterations mediated by blood-borne agents. Exposure to ozone, a major atmospheric pollutant, induces an increase in the glial coverage of neurons that is restricted to peri-synaptic compartments. This observation does not support the view that the ozone-induced neuronal disorders are related to non-specific overall brain alterations. It rather argues for a specific activation of the vagus nerve in response to pulmonary inflammation.

Kv4 channels underlie A-currents with highly variable inactivation time courses but homogeneous other gating properties in the nucleus tractus solitarii.

In the nucleus of the tractus solitarii (NTS), a large proportion of neurones express transient A-type potassium currents (I KA) having deep influence on the fidelity of the synaptic transmission of the visceral primary afferent inputs to second-order neurones. Up to now, the strong impact of I KA within the NTS was considered to result exclusively from its variation in amplitude, and its molecular correlate(s) remained unknown. In order to identify which Kv channels underlie I KA in NTS neurones, the gating properties and the pharmacology of this current were determined using whole cell patch clamp recordings in slices. Complementary information was brought by immunohistochemistry. Strikingly, two neurone subpopulations characterized by fast or slow inactivation time courses (respectively about 50 and 200 ms) were discriminated. Both characteristics matched those of the Kv4 channel subfamily. The other gating properties, also matching the Kv4 channel ones, were homogeneous through the NTS. The activation and inactivation occurred at membrane potentials around the threshold for generating action potentials, and the time course of recovery from inactivation was rapid. Pharmacologically, I KA in NTS neurones was found to be resistant to tetraethylammonium (TEA), sea anemone toxin blood-depressing substance (BDS) and dendrotoxin (DTX), whereas Androctonus mauretanicus mauretanicus toxin 3 (AmmTX3), a scorpion toxin of the α-KTX 15 family that has been shown to block all the members of the Kv4 family, inhibited 80 % of I KA irrespectively of its inactivation time course. Finally, immunohistochemistry data suggested that, among the Kv4 channel subfamily, Kv4.3 is the prevalent subunit expressed in the NTS.

Ozone inhalation activates stress-responsive regions of the CNS.

Ozone (O(3)), a major component of air pollution, has considerable impact on public health. Besides the well-described respiratory tract inflammation and dysfunctions, there is accumulating evidence indicating that O(3) exposure affects brain functions. However, the mechanisms through which O(3) exerts toxic effects on the brain remain poorly understood. This work aimed at precisely characterizing CNS neuronal activation after O(3) inhalation using Fos staining in adult rat. We showed that, together with lung inflammation, O(3) exposure caused a sustained time- and dose-dependent neuronal activation in the dorsolateral regions of the nucleus tractus solitarius overlapping terminal fields of lung afferents running in vagus nerves. Furthermore, we highlighted neuronal activation in interconnected central structures such as the caudal ventrolateral medulla, the parabrachial nucleus, the central nucleus of the amygdala, the bed nucleus of the stria terminalis and the paraventricular hypothalamic nucleus. In contrast, we did not detect any neuronal activation in the thoracic spinal cord where lung afferents running in spinal nerves terminate. Overall, our results demonstrate that O(3) challenge evokes a lung inflammation that induces the activation of nucleus tractus solitarius neurons through the vagus nerves and promotes neuronal activation in stress-responsive regions of the CNS.

Glutamatergic neurotransmission in the nucleus tractus solitarii: structural and functional characteristics.

Glutamate is the main excitatory transmitter in the central nervous system. As such, it plays a major role in transmitting and processing visceral sensory information within the nucleus tractus solitarii (NTS). Here, we review current knowledge on NTS glutamatergic transmission. We describe the main organizational features of NTS glutamatergic synapses as determined by work performed during the last decade using antibodies against glutamate receptors and transporters proteins. In light of these recent neuronatomical findings, we discuss some functional properties of developing and adult NTS glutamatergic synapses.

Absence of regulation of the T-type calcium current by Cav1.1, beta1a and gamma1 dihydropyridine receptor subunits in skeletal muscle cells.

The subunit structure of low voltage activated T-type Ca2+ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type alpha1 subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Cav3.2 (alpha1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca2+ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of alpha1S (Cav1.1), beta1 and gamma1 DHPR subunits was tested using dysgenic (alpha1S-null), beta1a and gamma1 knockout mice. The results show that the absence of alpha1S, beta1 or gamma1 DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca2+ currents in skeletal muscle, suggesting that (1) native Cav3.2 is not regulated by beta1 or gamma1 DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.

Glutamatergic synapses in the rat nucleus tractus solitarii develop by direct insertion of calcium-impermeable AMPA receptors and without activation of NMDA receptors.

Calcium influxes through ionotropic glutamate receptors (AMPA and NMDA receptors, AMPARs and NMDARs) are considered to be critical for the shaping and refinement of neural circuits during synaptogenesis. Using a combined morphological and electrophysiological approach, we evaluated this hypothesis at the level of the nucleus tractus solitarii (NTS), a brainstem structure that is a gateway for many visceral sensory afferent fibres. We confirmed that in the NTS, the first excitatory synapses appeared at embryonic day 18. We next characterized the biophysical properties of NTS AMPARs. Throughout perinatal development, both evoked and miniature EPSCs recorded in the presence of an NMDAR blocker were insensitive to polyamines and had linear current-voltage relationships. This demonstrated that AMPARs at NTS excitatory synapses were calcium-impermeable receptors composed of a majority of GluR2 subunits. We then investigated the influence of calcium influxes through NMDARs on the development of NTS synaptic transmission. We found that NMDAR expression at synaptic sites did not precede AMPAR expression. Moreover, NMDAR blockade in utero did not prevent the development of AMPAR synaptic currents and the synaptic clustering of GluR2 subunits. Thus, our data support an alternative model of synaptogenesis that does not depend on calcium influxes through either AMPARs or NMDARs. This model may be particularly relevant to the formation of neural networks devoted to basic behaviours required at birth for survival.

Early expression of AMPA receptors and lack of NMDA receptors in developing rat climbing fibre synapses.

Whether nascent glutamatergic synapses acquire their AMPA receptors constitutively or via a regulated pathway triggered by pre-existing NMDA receptor activation is still an open issue. Here, we provide evidence that some glutamatergic synapses develop without expressing NMDA receptors. Using immunocytochemistry, we showed that synapses between developing rat climbing fibres and Purkinje cells expressed GluR2-containing AMPA receptors as soon as they were formed (i.e. on embryonic day 19) but never carried detectable NMDA receptors. This was confirmed by electrophysiological recordings. Excitatory synaptic currents were recorded in Purkinje cells as early as P0. However, no NMDA receptor-mediated component was found in either spontaneous or evoked synaptic responses. In addition, we ruled out a possible role of extrasynaptic NMDA receptors by showing that AMPA receptor clustering at nascent climbing fibre synapses was not modified by chronic in utero NMDA receptor blockade.

Membrane cholesterol modulates dihydropyridine receptor function in mice fetal skeletal muscle cells.

Caveolae and transverse (T-) tubules are membrane structures enriched in cholesterol and glycosphingolipids. They play an important role in receptor signalling and myogenesis. The T-system is also highly enriched in dihydropyridine receptors (DHPRs), which control excitation-contraction (E-C) coupling. Recent results have shown that a depletion of membrane cholesterol alters caveolae and T-tubules, yet detailed functional studies of DHPR expression are lacking. Here we studied electrophysiological and morphological effects of methyl-beta-cyclodextrin (MbetaCD), a cholesterol-sequestering drug, on freshly isolated fetal skeletal muscle cells. Exposure of fetal myofibres to 1-3 mM MbetaCD for 1 h at 37 degrees C led to a significant reduction in caveolae and T-tubule areas and to a decrease in cell membrane electrical capacitance. In whole-cell voltage-clamp experiments, the L-type Ca(2+) current amplitude was significantly reduced, and its voltage dependence was shifted approximately 15 mV towards more positive potentials. Activation and inactivation kinetics were slower in treated cells than in control cells and stimulation by a saturating concentration of Bay K 8644 was enhanced. In addition, intramembrane charge movement and Ca(2+) transients evoked by a depolarization were reduced without a shift of the midpoint, indicating a weakening of E-C coupling. In contrast, T-type Ca(2+) current was not affected by MbetaCD treatment. Most of the L-type Ca(2+) conductance reduction and E-C coupling weakening could be explained by a decrease of the number of DHPRs due to the disruption of caveolae and T-tubules. However, the effects on L-type channel gating kinetics suggest that membrane cholesterol content modulates DHPR function. Moreover, the significant shift of the voltage dependence of L-type current without any change in the voltage dependence of charge movement and Ca(2+) transients suggests that cholesterol differentially regulates the two functions of the DHPR.

Alpha(1H) mRNA in single skeletal muscle fibres accounts for T-type calcium current transient expression during fetal development in mice.

Calcium channels are essential for excitation-contraction coupling and muscle development. At the end of fetal life, two types of Ca(2+) currents can be recorded in muscle cells. Whereas L-type Ca(2+) channels have been extensively studied, T-type channels have been poorly characterized in skeletal muscle. We describe here the functional and molecular properties of T-type calcium channels in developing mouse skeletal muscle. The T-type current density increased transiently during prenatal myogenesis with a maximum at embryonic day E16 followed by a drastic decrease until birth. This current showed similar electrophysiological and pharmacological properties at all examined stages. It displayed a wide window current centred at about -35 and -55 mV in 10 and 2 mM external Ca(2+), respectively. Activation and inactivation kinetics were fast (3 and 16 ms, respectively). The current was inhibited by nickel and amiloride with an IC(50) of 5.4 and 156 microM, respectively, values similar to those described for cloned T-type alpha(1H) channels. Whole muscle tissue RT-PCR analysis revealed mRNAs corresponding to alpha(1H) and alpha(1G) subunits in the fetus but not in the adult. However, single-fibre RT-PCR demonstrated that only alpha(1H) mRNA was present in prenatal fibres, suggesting that the alpha(1G) transcript present in muscle tissue must be expressed by non-skeletal muscle cells. Altogether, these results demonstrate that the alpha(1H) subunit generates functional T-type calcium channels in developing skeletal muscle fibres and suggest that these channels are involved in the early stages of muscle differentiation.

Effects of extracellular ATP on freshly isolated mouse skeletal muscle cells during pre-natal and post-natal development.

Extracellular adenosine 5'-triphosphate (ATP) has profound effects on membrane conductance and on the intracellular free [Ca(2+)] ([Ca(2+)](i)) in cultured skeletal muscle cells. The aim of the present study was to examine the occurrence and to characterize the properties of such responses during mammalian muscle development in vivo. The effect of ATP (0.2 mM) was tested on membrane current and [Ca(2+)](i) in freshly isolated pre- and post-natal mouse skeletal muscle cells. Pre-natal cells were from 14- to 19-day-old fetuses. In pre- and early post-natal cells, very small elevations of [Ca(2+)](i) (<50 nM) following ATP application could be detected with the fluorescent indicator fura-2. A clear subsarcolemmal rise in [Ca(2+)] was however associated to the presence of ATP, as demonstrated by increased activity of plasma membrane Ca(2+)-activated K(+) channels in cells bathed in a depolarizing, high-calcium-containing solution. In cells voltage-clamped at -80 mV in external Tyrode, ATP induced an inward current associated with an increased membrane conductance. The mean maximal amplitude of the ATP-induced current was -0.84 +/- 0.07 A/F ( n=39). The response to ATP was still present after birth, although its amplitude tended to decrease with post-natal development and was completely absent in muscle cells from 3- to 6-month-old mice. The ATP-induced current could be abolished reversibly by suramin. Our results suggest that, over the range of developmental stages examined, skeletal muscle cells display an ionotropic purinergic signalling pathway with functional properties qualitatively consistent with what is observed in cultured myotubes.