Slobodan M. Todorovic

Although opioids are effective in treating pain, they cause serious side effects. The use of regional anesthesia, although effective in the perioperative period, may not be suitable if mobility and lack of numbness is desired. Hence, there is a clear need for novel pain therapies. Low-voltage activated (T-type) calcium channels (Ca_V3.2 isoform) could be a promising therapeutic target for the development of novel pain therapies. Indeed, our published findings suggest that novel neuroactive steroid (NAS) analogs that modulate the activity of Ca_V3.2 channels have unique anti-nociceptive properties. However, the concern with current NASs appears to be their hypnotic/sedative properties, thus potentially hindering the future development of NASs for novel pain therapies. Hence, we developed a new line of NASs that are effective blockers of neuronal Ca_V3.2 channels in pain pathways while having more favorable pharmacodynamic properties, i.e., lack of sedative/hypnotic side effects. We present two promising novel analogs of NASs—B372 ((3β,5α,17β)-3-Hydroxyandrostan-17-carbonitrile) and YX23 ((3β,5α,17β)-3-Methoxyestran-17-ol). Using an in vitro approach, we show that B372 and YX23 are effective in blocking Ca_V3.2 channels. Using an in vivo approach, we show that they are effective anti-nociceptives in wild-type but not Ca_V3.2 knock-out mice. Importantly, we show that they lack sedative/hypnotic effects.

Neuroactive steroids (NAS) have long been recognized for their hypnotic and anesthetic properties in both clinical and preclinical settings. While sex differences in NAS sensitivity are acknowledged, the underlying mechanisms remain poorly understood. Here, we examined sex-specific responses to an endogenous NAS epipregnanolone (EpiP) in wild-type mice using behavioral assessment of hypnosis (loss of righting reflex, LORR) and in vivo electrophysiological recordings. Specifically, local field potentials (LFPs) were recorded from the central medial thalamus (CMT) and electroencephalogram (EEG) signals were recorded from the barrel cortex. We found that EpiP-induced LORR exhibited clear sex differences, with females showing increased sensitivity. Spectral power analysis and thalamocortical (TC) and corticocortical (CC) phase synchronization further supported enhanced hypnotic susceptibility in female mice. Our findings reveal characteristic sex-dependent effects of EpiP on the synchronized electrical activity in both thalamus and cortex. These results support renewed exploration of endogenous NAS as clinically relevant anesthetic agents.

Although substantial progress has been made in the last three decades towards our understanding of how general anesthetics (GAs) act at the molecular level, much less is known about how GAs cause loss of consciousness at the level of neuronal networks. The role of thalamus as an important brain region in anesthetic-induced hypnosis is relatively well established, but the specific roles of voltage-gated ion channels in different functional regions of the thalamus in anesthetic mechanisms are not well studied. To address this gap in knowledge, we selectively silenced the Cacna1g gene that encodes the low-threshold-activated Ca_V3.1 T-type voltage-gated calcium channel subunit by injecting short-hairpin RNA (shRNA) into midline and intralaminar - nonspecific thalamus (MIT) and sensory - specific ventrobasal (VB) thalamic nuclei in wild-type (WT) mice. Control animals were injected with scrambled shRNA. To validate our silencing approach, we performed patch-clamp experiments in acute thalamic slices ex vivo. In injected animals we determined anesthetic endpoints such as hypnosis measured with loss of righting reflex (LORR) and immobilization measured with loss of withdrawal reflex (LOWR) in vivo after administration of a traditional volatile GA isoflurane. Effective Ca_V3.1 channel knock-down was documented by greatly diminished amplitudes of T-currents and absence of rebound burst firing in our patch-clamp recordings from thalamic slices. We found that knocking down Ca_V3.1 channels in MIT significantly decreased inhaled isoflurane concentration that is required to induce LORR, but it did not affect speed of anesthetic induction and the immobilizing effect of isoflurane. In contrast, knocking down the Ca_V3.1 channel in the VB thalamus did not affect any of the measured anesthetic endpoints. Hence, we concluded that Ca_V3.1 channels in nonspecific MIT thalamus have a preferential role in anesthetic hypnosis when compared to the sensory VB thalamus.

The potential contribution of the ion channels that control the excitability of the midline and intralaminar nuclei of the thalamus to the modulation of behaviors has not been well studied. In this study, we used both global genetic deletion (knock-out, KO) and thalamus-specific molecular knock-down (KD) approaches to investigate the role of thalamic Ca_V3.1 T-type calcium channels (T-channels) in fear learning and fear responses. Previously, we have shown that the dominant subtype of T-channels in the central medial nucleus of the thalamus (CMT) is the Ca_V3.1 isoform and that CMT neurons from Ca_V3.1 KO animals have decreased burst firing. By specifically knocking down Ca_V3.1 T-channels in the CMT using the shRNA approach, we also reduced burst firing without affecting the tonic firing mode of the transfected neurons. We report that global Ca_V3.1 KO animals showed stronger freezing behaviors during both the conditioning and testing phases of contextual fear conditioning, while CMT-specific Ca_V3.1 KD mice only had stronger fear responses during testing. In contrast, the cue-mediated fear responses were similar between Ca_V3.1 KO and Ca_V3.1 KD mice and the controls. Our findings validate thalamic Ca_V3.1 T-channels as a potential new target for the development or treatment of different psychiatric diseases, such as post-traumatic stress disorder, schizophrenia, anxiety, and substance abuse disorders.

BACKGROUND: Normal sleep architecture is important for brain development, and we previously demonstrated that a single exposure to isoflurane during the neonatal period did not induce changes in the sleep architecture and only minimally altered neuronal beta oscillations in adolescent rats. Here, we hypothesized that a more clinically relevant scenario of repeated shorter exposures to isoflurane during brain development may have more profound effects on sleep and wake behavior and associated delta and theta oscillations, respectively. METHODS: Male and female rat pups were exposed to sham anesthesia (30% oxygen) or repeated isoflurane delivery for 2 hours each on 3 consecutive days (total exposure of 6 hours). The rat pups were divided into 2 cohorts. In cohort 1, we evaluated the neurotoxic effects of exposure postanesthesia. In cohort 2, electroencephalogram electrodes were implanted into the rat cortex between postnatal days 21–23, and sleep architecture was classified as wake, nonrapid eye movement (NREM), and rapid-eye movement (REM) sleep. Electroencephalogram power spectra were also measured in adolescent rats over a 72-hour period. RESULTS: Isoflurane exposure (n = 11) increased neuroapoptosis to 27. 7 ± 6.5 per mm^-2 when compared to the sham group (9. 6 ± 3.0 per mm^-2, n = 12, P < .001) and disrupted sleep architecture in adolescent rats. Specifically, there was an increase in the total sleep time (light + dark period) from 89. 9 ± 14.2 minutes in sham group (n = 9) to 111. 2 ± 32.2 minutes in the experimental group (n = 11, P < .05). Furthermore, there were fewer transitions during the dark period from 157. 1 ± 43.3 in sham group (n = 9) to 110. 6 ± 52.5 in the experimental group (n = 11, P < .05). The absolute power of delta oscillations was significantly decreased during the light period of NREM from an average 2217 ± 2016 μV² in the sham group (n = 8) to 791 ± 659 μV² in the experimental group (n = 11, P < .05). Further, theta oscillations in the wake stage were significantly decreased in the light period from 1579 ± 885 μV² in sham group (n = 8) to 690 ± 413 μV² in the experimental group (n = 11, P < .05) and light + dark period from 1390 ± 808 μV² in sham group (n = 8) to 691 ± 421 μV² in the experimental group (n = 11, P < .05). CONCLUSIONS: Exposing neonatal rats to isoflurane repeatedly causes significant neurotoxicity, and alters delta and theta thalamocortical oscillations, as well as sleep architecture in adolescence. This contrasts with a single continuous exposure to isoflurane, in which we previously reported no significant effects on sleep-wake architecture and only minimal effect on beta oscillations despite similar acute neurotoxicity.

Voltage-gated calcium channels are important for thalamocortical (TC) oscillations related to spike-wave discharges (SWDs) during absence seizures. The role of Ca_V2.3 R-type channels expressed in the thalamic intralaminar complex in SWDs, however, is not well studied. We investigated pharmacologically induced SWDs from the central medial thalamus (CMT) and somatosensory cortex in a Ca_V2.3 knockout (KO) mouse model using local field potential (LFP), and electroencephalographic (EEG) recordings. The duration of cumulative SWDs was significantly decreased in Ca_V2.3 KO mice compared with wild-type (WT) mice. A characteristic increase in the delta and theta waves was observed in both the CMT and somatosensory cortex during SWDs with delta (1–4 Hz) band TC synchronization increasing only in WT animals. Specifically, in the KO mice, LFPs recorded from the CMT showed no significant changes in the delta band and a significant decrease in the theta (4–8 Hz) band, and cortical EEG recordings showed a significant increase in the delta band, but no changes in the theta band. The baseline TC phase synchronization in the delta band was also more pronounced in the Ca_V2.3 KO mice than in WT mice. These findings suggest that R-type calcium channels in the CMT play a crucial role in sustaining and promoting the oscillatory activity of the TC network during absence seizures.

The Ca _V 3.2 isoform of T-type voltage-gated calcium channels plays a crucial role in regulating the excitability of nociceptive neurons; the endogenous molecules that modulate its activity, however, remain poorly understood. Here, we used serum proteomics and patch-clamp physiology to discover a novel peptide albumin (1-26) that facilitates channel gating by chelating trace metals that tonically inhibit Ca _V 3.2 via H191 residue. Importantly, serum also potently modulated T-currents in human and rodent dorsal root ganglion (DRG) neurons. In vivo pain studies revealed that injections of serum and albumin (1-26) peptide resulted in robust mechanical and heat hypersensitivity. This hypersensitivity was abolished with a T-channel inhibitor, in Ca _V 3.2 null mice and in Ca _V 3.2 H191Q knock-in mice. The discovery of endogenous chelators of trace metals in the serum deepens our understanding of the role of Ca _V 3.2 channels in neuronal hyperexcitability and may facilitate the design of novel analgesics with unique mechanisms of action.

Published evidence over the past few decades suggests that general anesthetics could be neurotoxins especially when administered at the extremes of age. The reported pathology is not only at the morphological level when examined in very young and aged brains, given that, importantly, newly developing evidence suggests a variety of behavioral impairments. Since anesthesia is unavoidable in certain clinical settings, we should consider the development of new anesthetics. A promising and safe solution could be a new family of anesthetics referred to as neuroactive steroids. In this review, we summarize the currently available evidence regarding their anesthetic and analgesic properties.

Preclinical studies have established that neonatal exposure to contemporary sedative/hypnotic drugs causes neurotoxicity in the developing rodent and primate brains. Our group recently reported that novel neuroactive steroid (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH) induced effective hypnosis in both neonatal and adult rodents but did not cause significant neurotoxicity in vulnerable brain regions such as subiculum, an output region of hippocampal formation particularly sensitive to commonly used sedatives/hypnotics. Despite significant emphasis on patho-morphological changes, little is known about long-term effects on subicular neurophysiology after neonatal exposure to neuroactive steroids. Hence, we explored the lasting effects of neonatal exposure to 3β-OH on sleep macrostructure as well as subicular neuronal oscillations in vivo and synaptic plasticity ex vivo in adolescent rats. At postnatal day 7, we exposed rat pups to either 10 mg/kg of 3β-OH over a period of 12 h or to volume-matched cyclodextrin vehicle. At weaning age, a cohort of rats was implanted with a cortical electroencephalogram (EEG) and subicular depth electrodes. At postnatal day 30–33, we performed in vivo assessment of sleep macrostructure (divided into wake, non-rapid eye movement, and rapid eye movement sleep) and power spectra in cortex and subiculum. In a second cohort of 3β-OH exposed animals, we conducted ex vivo studies of long-term potentiation (LTP) in adolescent rats. Overall, we found that neonatal exposure to 3β-OH decreased subicular delta and sigma oscillations during non-rapid eye movement sleep without altering sleep macrostructure. Furthermore, we observed no significant changes in subicular synaptic plasticity. Interestingly, our previous study found that neonatal exposure to ketamine increased subicular gamma oscillations during non-rapid eye movement sleep and profoundly suppressed subicular LTP in adolescent rats. Together these results suggest that exposure to different sedative/hypnotic agents during a critical period of brain development may induce distinct functional changes in subiculum circuitry that may persist into adolescent age.