TY - JOUR
T1 - Further Evidence that Inhibition of Neuronal Voltage-Gated Calcium Channels Contributes to the Hypnotic Effect of Neurosteroid Analogue, 3β-OH
AU - Timic Stamenic, Tamara
AU - Manzella, Francesca M.
AU - Maksimovic, Stefan
AU - Krishnan, Kathiresan
AU - Covey, Douglas F.
AU - Jevtovic-Todorovic, Vesna
AU - Todorovic, Slobodan M.
N1 - Funding Information:
We thank the University of Colorado Anschutz Medical Campus Rodent In Vivo Neurophysiology Core, which is partly supported by the NIH P30 NS048154 grant, for providing the facilities to acquire and review the video-EEG data. We thank Pavle Joksovic for the technical assistance with the experiment involving HEK cells, Toni Schneider for the kind donation of Ca2.3 knock-out animals and the stably transfected HEK cell line, Drs. Vasilije Tadic and Reza Ghodsi for their help with the qPCR, Chase Castro for help with the loss of righting reflex, and MacKenzie Walz for maintaining the mouse colonies. V
Funding Information:
This study was funded in part by grants from the National Institutes of Health (GRANT# R01GM123746 to ST and VJ-T, R35GM141802-01 to ST) and funds from the Department of Anesthesiology and School of Medicine at Anschutz Medical Campus.
Publisher Copyright:
Copyright © 2022 Timic Stamenic, Manzella, Maksimovic, Krishnan, Covey, Jevtovic-Todorovic and Todorovic.
PY - 2022/5/23
Y1 - 2022/5/23
N2 - We recently reported that a neurosteroid analogue with T-channel-blocking properties (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), induced hypnosis in rat pups without triggering neuronal apoptosis. Furthermore, we found that the inhibition of the CaV3.1 isoform of T-channels contributes to the hypnotic properties of 3β-OH in adult mice. However, the specific mechanisms underlying the role of other subtypes of voltage-gated calcium channels in thalamocortical excitability and oscillations in vivo during 3β-OH-induced hypnosis are largely unknown. Here, we used patch-clamp recordings from acute brain slices, in vivo electroencephalogram (EEG) recordings, and mouse genetics with wild-type (WT) and CaV2.3 knock-out (KO) mice to further investigate the molecular mechanisms of neurosteroid-induced hypnosis. Our voltage-clamp recordings showed that 3β-OH inhibited recombinant CaV2.3 currents. In subsequent current-clamp recordings in thalamic slices ex vivo, we found that selective CaV2.3 channel blocker (SNX-482) inhibited stimulated tonic firing and increased the threshold for rebound burst firing in WT animals. Additionally, in thalamic slices we found that 3β-OH inhibited spike-firing more profoundly in WT than in mutant mice. Furthermore, 3β-OH reduced bursting frequencies in WT but not mutant animals. In ensuing in vivo experiments, we found that intra-peritoneal injections of 3β-OH were less effective in inducing LORR in the mutant mice than in the WT mice, with expected sex differences. Furthermore, the reduction in total α, β, and low γ EEG power was more profound in WT than in CaV2.3 KO females over time, while at 60 min after injections of 3β-OH, the increase in relative β power was higher in mutant females. In addition, 3β-OH depressed EEG power more strongly in the male WT than in the mutant mice and significantly increased the relative δ power oscillations in WT male mice in comparison to the mutant male animals. Our results demonstrate for the first time the importance of the CaV2.3 subtype of voltage-gated calcium channels in thalamocortical excitability and the oscillations that underlie neurosteroid-induced hypnosis.
AB - We recently reported that a neurosteroid analogue with T-channel-blocking properties (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), induced hypnosis in rat pups without triggering neuronal apoptosis. Furthermore, we found that the inhibition of the CaV3.1 isoform of T-channels contributes to the hypnotic properties of 3β-OH in adult mice. However, the specific mechanisms underlying the role of other subtypes of voltage-gated calcium channels in thalamocortical excitability and oscillations in vivo during 3β-OH-induced hypnosis are largely unknown. Here, we used patch-clamp recordings from acute brain slices, in vivo electroencephalogram (EEG) recordings, and mouse genetics with wild-type (WT) and CaV2.3 knock-out (KO) mice to further investigate the molecular mechanisms of neurosteroid-induced hypnosis. Our voltage-clamp recordings showed that 3β-OH inhibited recombinant CaV2.3 currents. In subsequent current-clamp recordings in thalamic slices ex vivo, we found that selective CaV2.3 channel blocker (SNX-482) inhibited stimulated tonic firing and increased the threshold for rebound burst firing in WT animals. Additionally, in thalamic slices we found that 3β-OH inhibited spike-firing more profoundly in WT than in mutant mice. Furthermore, 3β-OH reduced bursting frequencies in WT but not mutant animals. In ensuing in vivo experiments, we found that intra-peritoneal injections of 3β-OH were less effective in inducing LORR in the mutant mice than in the WT mice, with expected sex differences. Furthermore, the reduction in total α, β, and low γ EEG power was more profound in WT than in CaV2.3 KO females over time, while at 60 min after injections of 3β-OH, the increase in relative β power was higher in mutant females. In addition, 3β-OH depressed EEG power more strongly in the male WT than in the mutant mice and significantly increased the relative δ power oscillations in WT male mice in comparison to the mutant male animals. Our results demonstrate for the first time the importance of the CaV2.3 subtype of voltage-gated calcium channels in thalamocortical excitability and the oscillations that underlie neurosteroid-induced hypnosis.
KW - EEG
KW - R-type calcium channels
KW - ex vivo in slice electrophysiology
KW - hypnosis
KW - neuroactive steroids
KW - thalamus
KW - voltage-gated calcium channels
UR - http://www.scopus.com/inward/record.url?scp=85131887519&partnerID=8YFLogxK
U2 - 10.3389/fphar.2022.850658
DO - 10.3389/fphar.2022.850658
M3 - Article
C2 - 35677453
AN - SCOPUS:85131887519
SN - 1663-9812
VL - 13
JO - Frontiers in Pharmacology
JF - Frontiers in Pharmacology
M1 - 850658
ER -