A calcium-activated, large conductance and non-selective cation channel in Paramecium cell

AbstractA non-selective cation channel was found in mutant Paramecium cells (K115). This cell had been selected as a resistant mutant in a high-K+ solution. In patch clamp studies of these cells in the inside-out configuration, this channel was activated by bath applications of elevated Ca2+ concentrations. The channels became very active when the Ca2+ concentration was above 3.2 μM. The channel was also activated by depolarization. The voltage dependency was steep upon depolarization, whereas upon hyperpolarization the channel activity barely changed. This channel had poor selectivity for monovalent alkali cations. Using the Goldman–Hodgkin–Katz equation for the reversal potential, the permeability ratios with respect to K+ for Na+, Rb+, Cs+ and Li+ were nearly 1. Although the permeability ratios were similar for each cation, the single channel conductances differed. The single channel conductances were 467 pS with K+ as the charge carrier, 406 pS with Na+, 397 pS with Rb+, 253 pS with Cs+ and 198 pS with Li+ upon depolarization in 100 mM cation solutions. A similar calcium-activated large conductance channel was observed in the wild-type (G3) Paramecium cells but was very rare

Disease-specific monoclonal antibodies targeting glutamate decarboxylase impair GABAergic neurotransmission and affect motor learning and behavioral functions

Autoantibodies to the smaller isoform of glutamate decarboxylase (GAD) can be found in patients with type 1 diabetes and a number of neurological disorders, including stiff-person syndrome, cerebellar ataxia and limbic encephalitis. The detection of disease-specific autoantibody epitopes led to the hypothesis that distinct GAD autoantibodies may elicit specific neurological phenotypes. We explored the in vitro/in vivo effects of well-characterized monoclonal GAD antibodies. We found that GAD autoantibodies present in patients with stiff person syndrome (n = 7) and cerebellar ataxia (n = 15) recognized an epitope distinct from that recognized by GAD autoantibodies present in patients with type 1 diabetes mellitus (n = 10) or limbic encephalitis (n = 4). We demonstrated that the administration of a monoclonal GAD antibody representing this epitope specificity; (1) disrupted in vitro the association of GAD with γ-Aminobutyric acid containing synaptic vesicles; (2) depressed the inhibitory synaptic transmission in cerebellar slices with a gradual time course and a lasting suppressive effect; (3) significantly decreased conditioned eyelid responses evoked in mice, with no modification of learning curves in the classical eyeblink-conditioning task; (4) markedly impaired the facilitatory effect exerted by the premotor cortex over the motor cortex in a paired-pulse stimulation paradigm; and (5) induced decreased exploratory behavior and impaired locomotor function in rats. These findings support the specific targeting of GAD by its autoantibodies in the pathogenesis of stiff-person syndrome and cerebellar ataxia. Therapies of these disorders based on selective removal of such GAD antibodies could be envisioned.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Cerebellar Globular Cells Receive Monoaminergic Excitation and Monosynaptic Inhibition from Purkinje Cells

Inhibitory interneurons in the cerebellar granular layer are more heterogeneous than traditionally depicted. In contrast to Golgi cells, which are ubiquitously distributed in the granular layer, small fusiform Lugaro cells and globular cells are located underneath the Purkinje cell layer and small in number. Globular cells have not been characterized physiologically. Here, using cerebellar slices obtained from a strain of gene-manipulated mice expressing GFP specifically in GABAergic neurons, we morphologically identified globular cells, and compared their synaptic activity and monoaminergic influence of their electrical activity with those of small Golgi cells and small fusiform Lugaro cells. Globular cells were characterized by prominent IPSCs together with monosynaptic inputs from the axon collaterals of Purkinje cells, whereas small Golgi cells or small fusiform Lugaro cells displayed fewer and smaller spontaneous IPSCs. Globular cells were silent at rest and fired spike discharges in response to application of either serotonin (5-HT) or noradrenaline. The two monoamines also facilitated small Golgi cell firing, but only 5-HT elicited firing in small fusiform Lugaro cells. Furthermore, globular cells likely received excitatory monosynaptic inputs through mossy fibers. Because globular cells project their axons long in the transversal direction, the neuronal circuit that includes interplay between Purkinje cells and globular cells could regulate Purkinje cell activity in different microzones under the influence of monoamines and mossy fiber inputs, suggesting that globular cells likely play a unique modulatory role in cerebellar motor control

Differential Modulation of GABAA Receptors Underlies Postsynaptic Depolarization- and Purinoceptor-Mediated Enhancement of Cerebellar Inhibitory Transmission: A Non-Stationary Fluctuation Analysis Study.

Cerebellar GABAergic inhibitory transmission between interneurons and Purkinje cells (PCs) undergoes a long-lasting enhancement following different stimulations, such as brief depolarization or activation of purinergic receptors of postsynaptic PCs. The underlying mechanisms, however, are not completely understood. Using a peak-scaled non-stationary fluctuation analysis, we therefore aimed at characterizing changes in the electrophysiological properties of GABAA receptors in PCs of rat cerebellar cortex during depolarization-induced "rebound potentiation (RP)" and purinoceptor-mediated long-term potentiation (PM-LTP), because both RP and PM-LTP likely depend on postsynaptic mechanisms. Stimulation-evoked inhibitory postsynaptic currents (eIPSCs) were recorded from PCs in neonatal rat cerebellar slices. Our analysis showed that postsynaptic membrane depolarization induced RP of eIPSCs in association with significant increase in the number of synaptic GABAA receptors without changing the channel conductance. By contrast, bath application of ATP induced PM-LTP of eIPSCs with a significant increase of the channel conductance of GABAA receptors without affecting the receptor number. Pretreatment with protein kinase A (PKA) inhibitors, H-89 and cAMPS-Rp, completely abolished the PM-LTP. The CaMKII inhibitor KN-62 reported to abolish RP did not alter PM-LTP. These results suggest that the signaling mechanism underlying PM-LTP could involve ATP-induced phosphorylation of synaptic GABAA receptors, thereby resulting in upregulation of the channel conductance by stimulating adenylyl cyclase-PKA signaling cascade, possibly via activation of P2Y11 purinoceptor. Thus, our findings reveal that postsynaptic GABAA receptors at the interneuron-PC inhibitory synapses are under the control of two distinct forms of long-term potentiation linked with different second messenger cascades

Rebound potentiation (RP) and purinoceptor-mediated long-term potentiation (PM-LTP) of eIPSCs at basket cell-PC synapses.

<p>(A) Time course changes of eIPSC amplitude in the course of RP (n = 8, left panel) and PM-LTP (n = 6, right panel). RP was induced by depolarization pulses given to individual PCs at the time point indicated by an arrow. PM-LTP was induced by bath-application of 100 μM ATP during the period of 5 min indicated by a horizontal bar. (B) Mean amplitude of eIPSCs during the period of 10 to 15 min after induction of RP or PM-LTP. Asterisk indicates a statistically significant difference compared to mean amplitude of eIPSCs before induction of RP or PM-LTP (p<0.01).</p

Effects of PKA and CaMKII inhibitors on PM-LTP.

<p>(A, B,C) Time course change of the eIPSC amplitudes in experiments where ATP (100 μM) was applied after treatments with PKA inhibitors, H-89 (A) and cAMPS-Rp (B), and a CaMKII inhibitor, KN-62 (C). Inset shows representative averaged traces of eIPSC recorded before (thin line) and after ATP application (thick line). (D) Effects of PKA and CaMKII inhibitors on the mean amplitudes of eIPSCs recorded during the periods10 to 15 min after ATP application. (E) Effects PKA and CaMKII inhibitors on the mean values of changes in the number of functioning GABA_A receptors (<i>N</i>) and the size of unitary current through the GABA_A receptor (<i>i</i>). Filled bars (ATP) indicate the values obtained after application of ATP alone (data taken from Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150636#pone.0150636.g002" target="_blank">2B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150636#pone.0150636.g003" target="_blank">3C</a>). Asterisk indicates a level of statistically significant difference (*: p<0.05, **: p<0.01, N.S.: no significant difference).</p

Validation of PS-NSFA applied to eIPSCs at GABAergic synapses between basket cells and PCs.

<p>(A) Effects of changing the driving force for GABA_A receptor activation on eIPSCs. Upper panel: averaged traces of eIPSCs recorded at the holding membrane potential of –40 mV (thin line) and -60 mV (thick line). Inset shows the averaged trace of IPSCs recorded at −40 mV was scaled to the same amplitude of those recorded at –60 mV (Normalized). Lower panel: mean-variance curves at the membrane potentials of –40 (open circles) and –60 mV (closed circles). <i>N</i> and <i>i</i> indicate the number of active GABA_A receptors and the size of unitary current through GABA_A receptors estimated from the mean-variance curves, respectively. (B) Effects of changing the GABA_A receptor availability by bicuculline on eIPSCs. Upper panel: averaged traces of eIPSCs before (control) and after bath-application of the GABA_A receptor antagonist bicuculline (100 nM). Inset shows averaged trace of IPSCs in the presence of bicuculline was scaled to the same amplitude of those recorded in control solution (Normalized). Note that the decay time was prolonged after partial blockade of GABA_A receptors by bicuculline. Lower panel: mean-variance curves before (open circles) and after (closed circles) application of bicuculline. <i>N</i> and <i>i</i> indicate the number of active GABA_A receptors and the size of unitary current through the GABA_A receptor estimated from the mean-variance curves, respectively.</p