Soon-Wook Choi

Role of the α1G T-Type Calcium Channel in Spontaneous Absence Seizures in Mutant Mice

Alterations in thalamic T-type Ca2+ channels are thought to contribute to the pathogenesis of absence seizures. Here, we found that mice with a null mutation for the pore-forming α1A subunits of P/Q-type channels (α1A–/– mice) were prone to absence seizures characterized by typical spike-and-wave discharges (SWDs) and behavioral arrests. Isolated thalamocortical relay (TC) neurons from these mice showed increased T-type Ca2+ currents in vitro. To examine the role of increased T-currents in α1A–/– TC neurons, we cross-bred α1A–/– mice with mice harboring a null mutation for the gene encoding α1G, a major isotype of T-type Ca2+ channels in TC neurons. α1A–/–/α1G–/– mice showed a complete loss of T-type Ca2+ currents in TC neurons and displayed no SWDs. Interestingly, α1A–/–/α1G+/– mice had 75% of the T-type Ca2+ currents in TC neurons observed in α1A+/+/α1G+/+ mice and showed SWD activity that was quantitatively similar to that in α1A–/–/α1G+/+ mice. Similar results were obtained using double-mutant mice harboring the α1G mutation plus another mutation also used as a model for absence seizures, i.e., lethargic (β4lh/lh), tottering (α1Atg/tg), or stargazer (γ2stg/stg). The present results reveal that α1G T-type Ca2+ channels play a critical role in the genesis of spontaneous absence seizures resulting from hypofunctioning P/Q-type channels, but that the augmentation of thalamic T-type Ca2+ currents is not an essential step in the genesis of absence seizures.

γ-Band deficiency and abnormal thalamocortical activity in P/Q-type channel mutant mice

Thalamocortical in vivo and in vitro function was studied in mice lacking P/Q-type calcium channels (CaV2.1), in which N-type calcium channels (CaV2.2) supported central synaptic transmission. Unexpectedly, in vitro patch recordings from thalamic neurons demonstrated no γ-band subthreshold oscillation, and voltage-sensitive dye imaging demonstrated an absence of cortical γ-band-dependent columnar activation involving cortical inhibitory interneuron activity. In vivo electroencephalogram recordings showed persistent absence status and a dramatic reduction of γ-band activity. Pharmacological block of T-type calcium channels (CaV3), although not noticeably affecting normal control animals, left the knockout mice in a coma-like state. Hence, although N-type calcium channels can rescue P/Q-dependent synaptic transmission, P/Q calcium channels are essential in the generation of γ-band activity and resultant cognitive function.

CaV3.1 is a tremor rhythm pacemaker in the inferior olive

The rhythmic motor pathway activation by pacemaker neurons or circuits in the brain has been proposed as the mechanism for the timing of motor coordination, and the abnormal potentiation of this mechanism may lead to a pathological tremor. Here, we show that the potentiation of CaV3.1 T-type Ca2+ channels in the inferior olive contributes to the onset of the tremor in a pharmacological model of essential tremor. After administration of harmaline, 4- to 10-Hz synchronous neuronal activities arose from the IO and then propagated to cerebellar motor circuits in wild-type mice, but those rhythmic activities were absent in mice lacking CaV3.1 gene. Intracellular recordings in brain-stem slices revealed that the CaV3.1-deficient inferior olive neurons lacked the subthreshold oscillation of membrane potentials and failed to trigger 4- to 10-Hz rhythmic burst discharges in the presence of harmaline. In addition, the selective knockdown of CaV3.1 gene in the inferior olive by shRNA efficiently suppressed the harmaline-induced tremor in wild-type mice. A mathematical model constructed based on data obtained from patch-clamping experiments indicated that harmaline could efficiently potentiate CaV3.1 channels by changing voltage-dependent responsiveness in the hyperpolarizing direction. Thus, CaV3.1 is a molecular pacemaker substrate for intrinsic neuronal oscillations of inferior olive neurons, and the potentiation of this mechanism can be considered as a pathological cause of essential tremor.

Subthreshold membrane potential oscillations in inferior olive neurons are dynamically regulated by P/Q- and T-type calcium channels: a study in mutant mice.

The role of P/Q‐ and T‐type calcium channels in the rhythmic oscillatory behaviour of inferior olive (IO) neurons was investigated in mutant mice. Mice lacking either the CaV2.1 gene of the pore‐forming α1A subunit for P/Q‐type calcium channel, or the CaV3.1 gene of the pore‐forming α1G subunit for T‐type calcium channel were used. In vitro intracellular recording from IO neurons reveals that the amplitude and frequency of sinusoidal subthreshold oscillations (SSTOs) were reduced in the CaV2.1−/− mice. In the CaV3.1−/− mice, IO neurons also showed altered patterns of SSTOs and the probability of SSTO generation was significantly lower (15%, 5 of 34 neurons) than that of wild‐type (78%, 31 of 40 neurons) or CaV2.1−/− mice (73%, 22 of 30 neurons). In addition, the low‐threshold calcium spike and the sustained endogenous oscillation following rebound potentials were absent in IO neurons from CaV3.1−/− mice. Moreover, the phase‐reset dynamics of oscillatory properties of single neurons and neuronal clusters in IO were remarkably altered in both CaV2.1−/− and CaV3.1−/− mice. These results suggest that both α1A P/Q‐ and α1G T‐type calcium channels are required for the dynamic control of neuronal oscillations in the IO. These findings were supported by results from a mathematical IO neuronal model that incorporated T and P/Q channel kinetics.

Blocking effects of human tau on squid giant synapse transmission and its prevention by T-817 MA

Filamentous tau inclusions are hallmarks of Alzheimers disease and related neurodegenerative tauopathies, but the molecular mechanisms involved in tau-mediated changes in neuronal function and their possible effects on synaptic transmission are unknown. We have evaluated the effects of human tau protein injected directly into the presynaptic terminal axon of the squid giant synapse, which affords functional, structural, and biochemical analysis of its action on the synaptic release process. Indeed, we have found that at physiological concentration recombinant human tau (h-tau42) becomes phosphorylated, produces a rapid synaptic transmission block, and induces the formation of clusters of aggregated synaptic vesicles in the vicinity of the active zone. Presynaptic voltage clamp recordings demonstrate that h-tau42 does not modify the presynaptic calcium current amplitude or kinetics. Analysis of synaptic noise at the post-synaptic axon following presynaptic h-tau42 microinjection revealed an initial phase of increase spontaneous transmitter release followed by a marked reduction in noise. Finally, systemic administration of T-817MA, a proposed neuro-protective agent, rescued tau-induced synaptic abnormalities. Our results show novel mechanisms of h-tau42 mediated synaptic transmission failure and identify a potential therapeutic agent to treat tau-related neurotoxicity.

Role of Rab27 in synaptic transmission at the squid giant synapse

Small GTPase Rab is a member of a large family of Ras-related proteins, highly conserved in eukaryotic cells, and thought to regulate specific type(s) and/or specific step(s) in intracellular membrane trafficking. Given our interest in synaptic transmission, we addressed the possibility that Rab27 (a close isoform of Rab3) could be involved in cytosolic synaptic vesicle mobilization. Indeed, preterminal injection of a specific antibody against squid Rab27 (anti-sqRab27 antibody) combined with confocal microscopy demonstrated that Rab27 is present on squid synaptic vesicles. Electrophysiological study of injected synapses showed that the anti-sqRab27 antibody inhibited synaptic release in a stimulation-dependent manner without affecting presynaptic action potentials or inward Ca2+ current. This result was confirmed in in vitro synaptosomes by using total internal reflection fluorescence microscopy. Thus, synaptosomal Ca2+-stimulated release of FM1-43 dye was greatly impaired by intraterminal anti-sqRab27 antibody. Ultrastructural analysis of the injected giant preterminal further showed a reduced number of docked synaptic vesicles and an increase in nondocked vesicular profiles distant from the active zone. These results, taken together, indicate that Rab27 is primarily involved in the maturation of recycled vesicles and/or their transport to the presynaptic active zone in the squid giant synapse.

Tau pathology-mediated presynaptic dysfunction.

Brain tauopathies are characterized by abnormal processing of tau protein. While somatodendritic tau mislocalization has attracted considerable attention in tauopathies, the role of tau pathology in axonal transport, connectivity and related dysfunctions remains obscure. We have previously shown using the squid giant synapse that presynaptic microinjection of recombinant human tau protein (htau42) results in failure of synaptic transmission. Here, we evaluated molecular mechanisms mediating this effect. Thus, the initial event, observed after htau42 presynaptic injection, was an increase in transmitter release. This event was mediated by calcium release from intracellular stores and was followed by a reduction in evoked transmitter release. The effect of htau42 on synaptic transmission was recapitulated by a peptide comprising the phosphatase-activating domain of tau, suggesting activation of phosphotransferases. Accordingly, findings indicated that htau42-mediated toxicity involves the activities of both GSK3 and Cdk5 kinases.

Altered thalamocortical rhythmicity and connectivity in mice lacking CaV3.1 T-type Ca2+ channels in unconsciousness

Significance Defining the neuronal activity underlying unconscious states is a fundamental prerequisite to understanding cognitive brain function. The present findings indicate that the neuronal activity associated with unconsciousness is directly related to the voltage-gated ionic conductances supporting thalamic low-frequency electrical oscillations. In particular, voltage-gated calcium channels are important players in the generation of prolonged unconsciousness. Thus, mice with a deletion of voltage-gated CaV3.1 channels are incapable of maintaining the low-frequency oscillatory activity associated with unconsciousness. These results indicate that CaV3.1, though functionally very significant in the generation of physiological unconsciousness, is not the only channel involved in the generation of the low-frequency spontaneous brain activity that supports nonpathological unconsciousness. In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of CaV3.1 T-type Ca2+ channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking CaV3.1 channels (CaV3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that CaV3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (CaV3.1 WT). Intriguingly, we also found that CaV3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, CaV3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in CaV3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of CaV3.1 WT mice, during unconsciousness. These results suggest that the CaV3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states.

Pathophysiological implication of CaV3.1 T-type Ca2+ channels in trigeminal neuropathic pain

Significance Understanding the pathophysiological mechanism of central neuropathic pain has attracted much attention, especially because neuropathic pain is often unresponsive to existing medical treatments. In this study, we investigated the role of CaV3.1 T-type Ca2+ channels in the development of trigeminal neuropathic pain (TNP) in mice. Our results show that, intriguingly, CaV3.1 knockout mice had attenuated TNP. Specifically, we demonstrate that increased low-frequency rhythmicity and widely spread noncolumnar activity were present in wild-type TNP mice but not in knockout TNP mice. Moreover, abnormally pronounced coupling between low-frequency and high-frequency rhythms in the thalamocortical network of wild-type mice was absent in CaV3.1 knockout mice. Our results clearly imply that the presence of CaV3.1 channels is a crucial element in the pathophysiology of TNP. A crucial pathophysiological issue concerning central neuropathic pain is the modification of sensory processing by abnormally increased low-frequency brain rhythms. Here we explore the molecular mechanisms responsible for such abnormal rhythmicity and its relation to neuropathic pain syndrome. Toward this aim, we investigated the behavioral and electrophysiological consequences of trigeminal neuropathic pain following infraorbital nerve ligations in CaV3.1 T-type Ca2+ channel knockout and wild-type mice. CaV3.1 knockout mice had decreased mechanical hypersensitivity and reduced low-frequency rhythms in the primary somatosensory cortex and related thalamic nuclei than wild-type mice. Lateral inhibition of gamma rhythm in primary somatosensory cortex layer 4, reflecting intact sensory contrast, was present in knockout mice but severely impaired in wild-type mice. Moreover, cross-frequency coupling between low-frequency and gamma rhythms, which may serve in sensory processing, was pronounced in wild-type mice but not in CaV3.1 knockout mice. Our results suggest that the presence of CaV3.1 channels is a key element in the pathophysiology of trigeminal neuropathic pain.

RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production

We have examined the effects of RNS60, a 0.9% saline containing charge‐stabilized oxygen nanobubble‐based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringers solution, (2) RNS60‐based Ringers solution, (3) RNS10.3 (TCP‐modified saline without excess oxygen)‐based Ringers, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)‐based Ringers. RNS60‐based Ringers solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial‐based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60‐based Ringers solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.