Presynaptic N-type and P/Q-type Ca2+ channels mediating synaptic transmission at the calyx of Held of mice. (73/132)

At the nerve terminal, both N- and P/Q-type Ca2+ channels mediate synaptic transmission, with their relative contribution varying between synapses and with postnatal age. To clarify functional significance of different presynaptic Ca2+ channel subtypes, we recorded N-type and P/Q-type Ca2+ currents directly from calyces of Held nerve terminals in alpha1A-subunit-deficient mice and wild-type (WT) mice, respectively. The most prominent feature of P/Q-type Ca2+ currents was activity-dependent facilitation, which was absent for N-type Ca2+ currents. EPSCs mediated by P/Q-type Ca2+ currents showed less depression during high-frequency stimulation compared with those mediated by N-type Ca2+ currents. In addition, the maximal inhibition by the GABAB receptor agonist baclofen was greater for EPSCs mediated by N-type channels than for those mediated by P/Q-type channels. These results suggest that the developmental switch of presynaptic Ca2+ channels from N- to P/Q-type may serve to increase synaptic efficacy at high frequencies of activity, securing high-fidelity synaptic transmission.  (+info)

Kv1 channels selectively prevent dendritic hyperexcitability in rat Purkinje cells. (74/132)

Purkinje cells, the sole output of the cerebellar cortex, encode the timing signals required for motor coordination in their firing rate and activity pattern. Dendrites of Purkinje cells express a high density of P/Q-type voltage-gated calcium channels and fire dendritic calcium spikes. Here we show that dendritic subthreshold Kv1.2 subunit-containing Kv1 potassium channels prevent generation of random spontaneous calcium spikes. With Kv1 channels blocked, dendritic calcium spikes drive bursts of somatic sodium spikes and prevent the cell from faithfully encoding motor timing signals. The selective dendritic function of Kv1 channels in Purkinje cells allows them to effectively suppress dendritic hyperexcitability without hindering the generation of somatic action potentials. Further, we show that Kv1 channels also contribute to dendritic integration of parallel fibre synaptic input. Kv1 channels are often targeted to soma and axon and the data presented support a major dendritic function for these channels.  (+info)

Dominant-negative effects of human P/Q-type Ca2+ channel mutations associated with episodic ataxia type 2. (75/132)

Episodic ataxia type 2 (EA2) is an inherited autosomal dominant disorder related to cerebellar dysfunction and is associated with mutations in the pore-forming alpha(1A)-subunits of human P/Q-type Ca(2+) channels (Cav2.1 channels). The majority of EA2 mutations result in significant loss-of-function phenotypes. Whether EA2 mutants may display dominant-negative effects in human, however, remains controversial. To address this issue, five EA2 mutants in the long isoform of human alpha(1A)-subunits were expressed in Xenopus oocytes to explore their potential dominant-negative effects. Upon coexpressing the cRNA of alpha(1A)-WT with each alpha(1A)-mutant in molar ratios ranging from 1:1 to 1:10, the amplitude of Ba(2+) currents through wild-type (WT)-Cav2.1 channels decreased significantly as the relative molar ratio of alpha(1A)-mutants increased, suggesting the presence of an alpha(1A)-mutant-specific suppression effect. When we coexpressed alpha(1A)-WT with proteins not known to interact with Cav2.1 channels, we observed no significant suppression effects. Furthermore, increasing the amount of auxiliary subunits resulted in partial reversal of the suppression effects in nonsense but not missense EA2 mutants. On the other hand, when we repeated the same coinjection experiments of alpha(1A)-WT and mutant using a splice variant of alpha(1A)-subunit that contained a considerably shorter COOH terminus (i.e., the short isoform), no significant dominant-negative effects were noted until we enhanced the relative molar ratio to 1:10. Altogether, these results indicate that for human WT-Cav2.1 channels comprising the long-alpha(1A)-subunit isoform, both missense and nonsense EA2 mutants indeed display prominent dominant-negative effects.  (+info)

Eye movements of the murine P/Q calcium channel mutant tottering, and the impact of aging. (76/132)

Mice carrying mutations of the gene encoding the ion pore of the P/Q calcium channel (Cacna1a) are an instance in which cerebellar dysfunction may be attributable to altered electrophysiology and thus provide an opportunity to study how neuronal intrinsic properties dictate signal processing in the ocular motor system. P/Q channel mutations can engender multiple effects at the single neuron, circuit, and behavioral levels; correlating physiological and behavioral abnormalities in multiple allelic strains will ultimately facilitate determining which alterations of physiology are responsible for specific behavioral aberrations. We used videooculography to quantify ocular motor behavior in tottering mutants aged 3 mo to 2 yr and compared their performance to data previously obtained in the allelic mutant rocker and C57BL/6 controls. Tottering mutants shared numerous abnormalities with rocker, including upward deviation of the eyes at rest, increased vestibuloocular reflex (VOR) phase lead at low stimulus frequencies, reduced VOR gain at high stimulus frequencies, reduced gain of the horizontal and vertical optokinetic reflex, reduced time constants of the neural integrator, and reduced plasticity of the VOR as assessed in a cross-axis training paradigm. Unlike rocker, young tottering mutants exhibited normal peak velocities of nystagmus fast phases, arguing against a role for neuromuscular transmission defects in the attenuation of compensatory eye movements. Tottering also differed by exhibiting directional asymmetries of the gains of optokinetic reflexes. The data suggest at least four pathophysiological mechanisms (two congenital and two acquired) are required to explain the ocular motor deficits in the two Cacna1a mutant strains.  (+info)

Compensatory contribution of Cav2.3 channels to acetylcholine release at the neuromuscular junction of tottering mice. (77/132)

Tottering (Tg) mice carry the mutation P601L in their Cacna1a encoded Cav2.1 channels. Transmitter release at the wild-type neuromuscular junction (NMJ) is almost exclusively mediated by Cav2.1 channels, and we used this model synapse to study synaptic consequences of the Tg mutation. With electrophysiology, and using subtype-specific Cav2 channel-blocking toxins, we assessed a possible compensatory contribution of non-Cav2.1 channels to evoked acetylcholine (ACh) release at Tg NMJs. Release was reduced by approximately 75% by the Cav2.1 channel blocker omega-agatoxin-IVA, which was less than the approximately 95% reduction observed in wild-type. Release at Tg NMJs, but not at wild-type synapses, was reduced by approximately 15% by SNX-482, a Cav2.3 channel blocker. No Cav2.2 channel involvement was found. Probably, there is a small reduction in functional presynaptic Cav2.1 channels at Tg NMJs, which is compensated for by Cav2.3 channels. The remaining Cav2.1 channels are likely to convey enlarged Ca2+ flux, because evoked ACh release at Tg NMJs, at low extracellular Ca2+ concentration, was approximately sixfold higher than at wild-type NMJs. This is the first report of compensatory expression of non-Cav2.1 channels at NMJs of mice with a single amino acid change in Cav2.1.  (+info)

Pharmacological characterization of inhibitory effects of postsynaptic opioid and cannabinoid receptors on calcium currents in neonatal rat nucleus tractus solitarius. (78/132)

1. The profile of opioid and cannabinoid receptors in neurons of the nucleus tractus solitarius (NTS) has been studied using the whole-cell configuration of the patch clamp technique. 2. Experiments with selective agonists and antagonists of opioid, ORL and cannabinoid receptors indicated that mu-opioid, kappa-opioid, ORL-1 and CB1, but not delta-opioid, receptors inhibit VDCCs in NTS. 3. Application of [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; mu-opioid receptor agonist), Orphanin FQ (ORL-1 receptor agonist) and WIN55,122 (CB1 receptor agonist) caused inhibition of I(Ba) in a concentration-dependent manner, with IC50's of 390 nM, 220 nM and 2.2 microM, respectively. 4. Intracellular dialysis of the G(i)-protein antibody attenuated DAMGO-, Orphanin FQ- and WIN55,122-induced inhibition of I(Ba). 5. Both pretreatment with adenylate cyclase inhibitor and intracellular dialysis of the protein kinase A (PKA) inhibitor attenuated WIN55,122-induced inhibition of I(Ba) but not DAMGO- and Orphanin FQ-induced inhibition. 6. Mainly N- and P/Q-type VDCCs were inhibited by both DAMGO and Orphanin FQ, while L-type VDCCs were inhibited by WIN55,122. 7. These results suggest that mu- and kappa-opioid receptors and ORL-1 receptor inhibit N- and P/Q-type VDCCs via G alpha(i)-protein betagamma subunits, whereas CB1 receptors inhibit L-type VDCCs via G alpha(i)-proteins involving PKA in NTS.  (+info)

Identification of the REST regulon reveals extensive transposable element-mediated binding site duplication. (79/132)

The genome-wide mapping of gene-regulatory motifs remains a major goal that will facilitate the modelling of gene-regulatory networks and their evolution. The repressor element 1 is a long, conserved transcription factor-binding site which recruits the transcriptional repressor REST to numerous neuron-specific target genes. REST plays important roles in multiple biological processes and disease states. To map RE1 sites and target genes, we created a position specific scoring matrix representing the RE1 and used it to search the human and mouse genomes. We identified 1301 and 997 RE1s inhuman and mouse genomes, respectively, of which >40% are novel. By employing an ontological analysis we show that REST target genes are significantly enriched in a number of functional classes. Taking the novel REST target gene CACNA1A as an experimental model, we show that it can be regulated by multiple RE1s of different binding affinities, which are only partially conserved between human and mouse. A novel BLAST methodology indicated that many RE1s belong to closely related families. Most of these sequences are associated with transposable elements, leading us to propose that transposon-mediated duplication and insertion of RE1s has led to the acquisition of novel target genes by REST during evolution.  (+info)

Motor deficits in homozygous and heterozygous p/q-type calcium channel mutants. (80/132)

P/Q-type voltage-dependent Ca(2+) channels (VDCCs) are highly expressed in the cerebellum, and mutations of these channels are associated with disrupted motor function. Several allelic variants of the alpha1A pore-forming subunit of P/Q-type VDCCs have been described, and mice homozygous for these mutations exhibit gait ataxia, as do alpha1A knockout mice. Here we report that heterozygous alpha1A mutants also have a motor phenotype. Mice heterozygous for the leaner and alpha1A knockout mutations exhibit impaired motor learning in the vestibulo-ocular reflex (VOR), suggesting that subtle disruption of P/Q Ca(2+) currents is sufficient to disrupt motor function. Basal VOR and optokinetic reflex performance were normal in the heterozygotes but severely impaired in the leaner and alpha1A knockout homozygotes.  (+info)