GABAB receptors suppress burst-firing in reticular thalamic neurons

Burst-firing in thalamic neurons is known to play a key role in mediating thalamocortical (TC) oscillations that are associated with non-REM sleep and some types of epileptic seizure. Within the TC system the primary output of GABAergic neurons in the reticular thalamic nucleus (RTN) is thought to induce the de-inactivation of T-type calcium channels in thalamic relay (TR) neurons, promoting burst-firing drive to the cortex and the propagation of TC network activity. However, RTN neurons also project back onto other neurons within the RTN. The role of this putative negative feedback upon the RTN itself is less well understood, although is hypothesized to induce de-synchronization of RTN neuron firing leading to the suppression of TC oscillations. Here we tested two hypotheses concerning possible mechanisms underlying TC oscillation modulation. Firstly, we assessed the burst-firing behavior of RTN neurons in response to GABAB receptor activation using acute brain slices. The selective GABAB receptor agonist baclofen was found to induce suppression of burst-firing concurrent with effects on membrane input resistance. Secondly, RTN neurons express CaV3.2 and CaV3.3 T-type calcium channel isoforms known contribute towards TC burst-firing and we examined the modulation of these channels by GABAB receptor activation. Utilizing exogenously expressed T-type channels we assessed whether GABAB receptor activation could directly alter T-type calcium channel properties. Overall, GABAB receptor activation had only modest effects on CaV3.2 and CaV3.3 isoforms. The only effect that could be predicted to suppress burst-firing was a hyperpolarized shift in the voltage-dependence of inactivation, potentially causing lower channel availability at membrane potentials critical for burst-firing. Conversely, other effects observed such as a hyperpolarized shift in the voltage-dependence of activation of both CaV3.2 and CaV3.3 as well as increased time constant of activation of the CaV3.3 isoform would be expected to enhance burst-firing. Together, we hypothesize that GABAB receptor activation mediates multiple downstream effectors that combined act to suppress burst-firing within the RTN. It appears unlikely that direct modulation of T-type calcium channels is major contributor to this suppression

Pattern of neurological recovery in persons with an acute cervical spinal cord injury over the first 14 days post injury

IntroductionFollowing a traumatic spinal cord injury (SCI) it is critical to document the level and severity of injury. Neurological recovery occurs dynamically after injury and a baseline neurological exam offers a snapshot of the patient's impairment at that time. Understanding when this exam occurs in the recovery process is crucial for discussing prognosis and acute clinical trial enrollment. The objectives of this study were to: (1) describe the trajectory of motor recovery in persons with acute cervical SCI in the first 14 days post-injury; and (2) evaluate if the timing of the baseline neurological assessment in the first 14 days impacts the amount of motor recovery observed.MethodsData were obtained from the Rick Hansen Spinal Cord Injury Registry (RHSCIR) site in Vancouver and additional neurological data was extracted from medical charts. Participants with a cervical injury (C1–T1) who had a minimum of three exams (including a baseline and discharge exam) were included. Data on the upper-extremity motor score (UEMS), total motor score (TMS) and American Spinal Injury Association (ASIA) Impairment Scale (AIS) were included. A linear mixed-effect model with additional variables (AIS, level of injury, UEMS, time, time2, and TMS) was used to explore the pattern and amount of motor recovery over time.ResultsTrajectories of motor recovery in the first 14 days post-injury showed significant improvements in both TMS and UEMS for participants with AIS B, C, and D injuries, but was not different for high (C1–4) vs. low (C5–T1) cervical injuries or AIS A injuries. The timing of the baseline neurological examination significantly impacted the amount of motor recovery in participants with AIS B, C, and D injuries.DiscussionTiming of baseline neurological exams was significantly associated with the amount of motor recovery in cervical AIS B, C, and D injuries. Studies examining changes in neurological recovery should consider stratifying by severity and timing of the baseline exam to reduce bias amongst study cohorts. Future studies should validate these estimates for cervical AIS B, C, and D injuries to see if they can serve as an “adjustment factor” to control for differences in the timing of the baseline neurological exam

Characterization of the +SSTR and ΔSSTR splice variants of the Cav2.1 P/Q-type voltage-gated calcium channel

Cav2.1 P/Q-type voltage-gated calcium channels are essential for neurotransmission in many regions of the mammalian central nervous system (CNS). Alternative splicing generates functional diversity between Cav2.1 splice isoforms and is thought to be a mechanism by which fine-tuning and complexity of Cav2.1-mediated activities occur. The Cav2.1 +SSTR splice variant, located in the S3-S4 linker of domain III, has been identified in rodent brain although its effects on the biophysical and pharmacological properties of Cav2.1 have not been previously studied. Here, by performing splice variant-specific quantitative real-time PCR on selected regions of the rat CNS I demonstrate that +SSTR variant channels are differentially expressed spatially with predominant expression in the brainstem, reticular thalamus and spinal cord. Using whole-cell patch-clamp electrophysiology performed on transfected HEK 293 cells I have shown that compared to ΔSSTR channels, +SSTR variants exhibit faster activation kinetics and a hyperpolarizing shift in the voltage-dependence of activation and inactivation. Additionally, the +SSTR and ΔSSTR variants respond differently to increasing durations of action potential waveforms (APWs) with the charge transference through +SSTR channels being significantly less sensitive to APW broadening than ΔSSTR channels. Together, these data suggest that the unique biophysical properties of the Cav2.1 splice variants contribute to distinct roles in CNS synaptic physiology by relaying different types of action potential-encoding synaptic information. Lastly, I examined whether the +SSTR variant affected the sensitivity of Cav2.1 to the gating modifier peptide toxin ω-Agatoxin-IVA. Using whole-cell patch-clamp electrophysiology I found that the effects of ω-Agatoxin-IVA on current block did not significantly differ between the +SSTR and ΔSSTR splice variants suggesting that SSTR insertion does not affect the binding of ω-Agatoxin-IVA to Cav2.1 channels. The differential expression of Cav2.1 splice variants and their unique channel properties provides insight into the mechanisms by which complexity of P/Q-type calcium channel-mediated signaling contributes to CNS physiology.Medicine, Faculty ofGraduat

Epigallocatechin-3-gallate elicits Ca2+ spike in MCF-7 breast cancer cells: Essential role of Cav3.2 channels

We used MCF-7 human breast cancer cells that endogenously express Cav3.1 and Cav3.2 T-type Ca2+ channels toward a mechanistic study on the effect of EGCG on [Ca2+]i. Confocal Ca2+ imaging showed that EGCG induces a [Ca2+]i spike which is due to extracellular Ca2+ entry and is sensitive to catalase and to low-specificity (mibefradil) and high-specificity (Z944) T-type Ca2+channel blockers. siRNA knockdown of T-type Ca2+ channels indicated the involvement of Cav3.2 but not Cav3.1. Application of EGCG to HEK cells expressing either Cav3.2 or Cav3.1 induced enhancement of Cav3.2 and inhibition of Cav3.1 channel activity. Measurements of K+ currents in MCF-7 cells showed a reversible, catalase-sensitive inhibitory effect of EGCG, while siRNA for the Kv1.1 K+ channel induced a reduction of the EGCG [Ca2+]i spike. siRNA for Cav3.2 reduced EGCG cytotoxicity to MCF-7 cells, as measured by calcein viability assay. Together, data suggest that EGCG promotes the activation of Cav3.2 channels through K+ current inhibition leading to membrane depolarization, and in addition increases Cav3.2 currents. Cav3.2 channels are in part responsible for EGCG inhibition of MCF-7 viability, suggesting that deregulation of [Ca2+]i by EGCG may be relevant in breast cancer treatment

Heantos-4, a natural plant extract used in the treatment of drug addiction, modulates T-type calcium channels and thalamocortical burst-firing

Heantos-4 is a refined combination of plant extracts currently approved to treat opiate addiction in Vietnam. In addition to its beneficial effects on withdrawal and prevention of relapse, reports of sedation during clinical treatment suggest that arousal networks in the brain may be recruited during Heantos administration. T-type calcium channels are implicated in the generation of sleep rhythms and in this study we examined whether a Heantos-4 extraction modulates T-type calcium channel currents generated by the Cav3.1, Cav3.2 and Ca3.3 subtypes. Utilizing whole-cell voltage clamp on exogenously expressed T-type calcium channels we find that Heantos inhibits Cav3.1 and Cav3.3 currents, while selectively potentiating Cav3.2 currents. We further examined the effects of Heantos-4 extract on low-threshold burst-firing in thalamic neurons which contribute to sleep oscillations. Using whole-cell current clamp in acute thalamic brain slices Heantos-4 suppressed rebound burst-firing in ventrobasal thalamocortical neurons, which express primarily Cav3.1 channels. Conversely, Heantos-4 had no significant effect on the burst-firing properties of thalamic reticular neurons, which express a mixed population of Cav3.2 and Cav3.3 channels. Examining Heantos-4 effects following oral administration in a model of absence epilepsy revealed the potential to exacerbate seizure activity. Together, the findings indicate that Heantos-4 has selective effects both on specific T-type calcium channel isoforms and distinct populations of thalamic neurons providing a putative mechanism underlying its effects on sedation and on the thalamocortical network.Medicine, Faculty ofOther UBCNon UBCPsychiatry, Department ofReviewedFacult