A role for δ subunit-containing GABA A receptors on parvalbumin positive neurons in maintaining electrocortical signatures of sleep states.

GABA A receptors containing δ subunits have been shown to mediate tonic/slow inhibition in the CNS. These receptors are typically found extrasynaptically and are activated by relatively low levels of ambient GABA in the extracellular space. In the mouse neocortex, δ subunits are expressed on the surface of some pyramidal cells as well as on parvalbumin positive (PV+) interneurons. An important function of PV+ interneurons is the organization of coordinated network activity that can be measured by EEG; however, it remains unclear what role tonic/slow inhibitory control of PV+ neurons may play in shaping oscillatory activity. After confirming a loss of functional δ mediated tonic currents in PV cells in cortical slices from mice lacking Gabrd in PV+ neurons (PV δcKO), we performed EEG recordings to survey network activity across wake and sleep states. PV δcKO mice showed altered spectral content of EEG during NREM and REM sleep that was a result of increased oscillatory activity in NREM and the emergence of transient high amplitude bursts of theta frequency activity during REM. Viral reintroduction of Gabrd to PV+ interneurons in PV δcKO mice rescued REM EEG phenotypes, supporting an important role for δ subunit mediated inhibition of PV+ interneurons for maintaining normal REM cortical oscillations. Significance statement: The impact on cortical EEG of inhibition on PV+ neurons was studied by deleting a GABA A receptor subunit selectively from these neurons. We discovered unexpected changes at low frequencies during sleep that were rescued by viral reintroduction.

Endoplasmic reticulum stress, autophagy, neuroinflammation, and sigma 1 receptors as contributors to depression and its treatment.

The etiological factors contributing to depression and other neuropsychiatric disorders are largely undefined. Endoplasmic reticulum stress pathways and autophagy are well-defined mechanisms that play critical functions in recognizing and resolving cellular stress and are possible targets for the pathophysiology and treatment of psychiatric and neurologic illnesses. An increasing number of studies indicate the involvement of endoplasmic reticulum stress and autophagy in the control of neuroinflammation, a contributing factor to multiple neuropsychiatric illnesses. Initial inflammatory triggers induce endoplasmic reticulum stress, leading to neuroinflammatory responses. Subsequently, induction of autophagy by neurosteroids and other signaling pathways that converge on autophagy induction are thought to participate in resolving neuroinflammation. The aim of this review is to summarize our current understanding of the molecular mechanisms governing the induction of endoplasmic reticulum stress, autophagy, and neuroinflammation in the central nervous system. Studies focused on innate immune factors, including neurosteroids with anti-inflammatory roles will be reviewed. In the context of depression, animal models that led to our current understanding of molecular mechanisms underlying depression will be highlighted, including the roles of sigma 1 receptors and pharmacological agents that dampen endoplasmic reticulum stress and associated neuroinflammation.

Glyphosate as a direct or indirect activator of pro-inflammatory signaling and cognitive impairment.

Glyphosate-based herbicides are widely used around the world, making it likely that most humans have significant exposure. Because of habitual exposure, there are concerns about toxicity including neurotoxicity that could result in neurological, psychiatric, or cognitive impairment. We recently found that a single injection of glyphosate inhibits long-term potentiation, a cellular model of learning and memory, in rat hippocampal slices dissected 1 day after injection, indicating that glyphosate-based herbicides can alter cognitive function. Glyphosate-based herbicides could adversely affect cognitive function either indirectly and/or directly. Indirectly, glyphosate could affect gut microbiota, and if dysbiosis results in endotoxemia (leaky gut), infiltrated bacterial by-products such as lipopolysaccharides could activate pro-inflammatory cascades. Glyphosate can also directly trigger pro-inflammatory cascades. Indeed, we observed that acute glyphosate exposure inhibits long-term potentiation in rat hippocampal slices. Interestingly, direct inhibition of long-term potentiation by glyphosate appears to be similar to that of lipopolysaccharides. There are several possible measures to control dysbiosis and neuroinflammation caused by glyphosate. Dietary intake of polyphenols, such as quercetin, which overcome the inhibitory effect of glyphosate on long-term potentiation, could be one effective strategy. The aim of this narrative review is to discuss possible mechanisms underlying neurotoxicity following glyphosate exposure as a means to identify potential treatments.

Effects of Complete and Partial Loss of the 24S-Hydroxycholesterol-Generating Enzyme Cyp46a1 on Behavior and Hippocampal Transcription in Mouse.

Brain cholesterol metabolic products include neurosteroids and oxysterols, which play important roles in cellular physiology. In neurons, the cholesterol oxidation product, 24S-hydroxycholesterol (24S-HC), is a regulator of signaling and transcription. Here, we examined the behavioral effects of 24S-HC loss, using global and cell-selective genetic deletion of the synthetic enzyme CYP46A1. Mice that are globally deficient in CYP46A1 exhibited hypoactivity at young ages and unexpected increases in conditioned fear memory. Despite strong reductions in hippocampal 24S-HC in mice with selective loss of CYP46A1 in VGLUT1-positive cells, behavioral effects were not recapitulated in these conditional knockout mice. Global knockout produced strong, developmentally dependent transcriptional effects on select cholesterol metabolism genes. These included paradoxical changes in Liver X Receptor targets. Again, conditional knockout was insufficient to recapitulate most changes. Overall, our results highlight the complex effects of 24S-HC in an in vivo setting that are not fully predicted by known mechanisms. The results also demonstrate that the complete inhibition of enzymatic activity may be needed for a detectable, therapeutically relevant impact on gene expression and behavior.

Periodic and aperiodic changes to cortical EEG in response to pharmacological manipulation.

Cortical electroencephalograms (EEGs) may help understanding of neuropsychiatric illness and new treatment mechanisms. The aperiodic component (1/f) of EEG power spectra is often treated as noise, but recent studies suggest that changes to the aperiodic exponent of power spectra may reflect changes in excitation/inhibition balance, a concept linked to antidepressant effects, epilepsy, autism, and other clinical conditions. One confound of previous studies is behavioral state, because factors associated with behavioral state other than excitation/inhibition ratio may alter EEG parameters. Thus, to test the robustness of the aperiodic exponent as a predictor of excitation/inhibition ratio, we analyzed video-EEG during active exploration in mice of both sexes during various pharmacological manipulations with the fitting oscillations and one over f (FOOOF) algorithm. We found that GABAA receptor (GABAAR)-positive allosteric modulators increased the aperiodic exponent, consistent with the hypothesis that an increased exponent signals enhanced cortical inhibition, but other drugs (ketamine and GABAAR antagonists at subconvulsive doses) did not follow the prediction. To tilt excitation/inhibition ratio more selectively toward excitation, we suppressed the activity of parvalbumin-positive interneurons with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Contrary to our expectations, circuit disinhibition with the DREADD increased the aperiodic exponent. We conclude that the aperiodic exponent of EEG power spectra does not yield a universally reliable marker of cortical excitation/inhibition ratio.NEW & NOTEWORTHY Neuropsychiatric illness may be associated with altered excitation/inhibition balance. A single electroencephalogram (EEG) parameter, the aperiodic exponent of power spectra, may predict the ratio between excitation and inhibition. Here, we use cortical EEGs in mice to evaluate this hypothesis, using pharmacological manipulations of known mechanism. We show that the aperiodic exponent of EEG power spectra is not a reliable marker of excitation/inhibition ratio. Thus, alternative markers of this ratio must be sought.

Sertraline modulates hippocampal plasticity and learning via sigma 1 receptors, cellular stress and neurosteroids.

In addition to modulating serotonin transport, selective serotonin reuptake inhibitors (SSRIs) have multiple other effects that may contribute to clinical effects, and some of these latter actions prompt repurposing of SSRIs for non-psychiatric indications. We recently observed that the SSRIs fluvoxamine and fluoxetine prevent the acute adverse effects of pro-inflammatory stimulation on long-term potentiation (LTP) in the CA1 hippocampal region. Sertraline showed markedly different effects, acutely inhibiting LTP at a low micromolar concentration through inverse agonism of sigma 1 receptors (S1Rs). In the present studies, we pursued mechanisms contributing to sertraline modulation of LTP in rat hippocampal slices. We found that sertraline partially inhibits synaptic responses mediated by N-methyl-D-aspartate receptors (NMDARs) via effects on NMDARs that express GluN2B subunits. A selective S1R antagonist (NE-100), but not an S1R agonist (PRE-084) blocked effects on NMDARs, despite the fact that both S1R ligands were previously shown to prevent LTP inhibition. Both NE-100 and PRE-084, however, prevented adverse effects of sertraline on one-trial learning. Because of the important role that S1Rs play in modulating endoplasmic reticulum stress, we examined whether inhibitors of cellular stress alter effects of sertraline. We found that two stress inhibitors, ISRIB and quercetin, prevented LTP inhibition, as did inhibitors of the synthesis of endogenous neurosteroids, which are homeostatic regulators of cellular stress. These studies highlight complex effects of sertraline, S1Rs and neurosteroids on hippocampal function and have relevance for understanding therapeutic and adverse drug actions.

Neuroactive steroid effects on autophagy in a human embryonic kidney 293 (HEK) cell model.

Neuropsychiatric and neurodegenerative disorders are correlated with cellular stress. Macroautophagy (autophagy) may represent an important protective pathway to maintain cellular homeostasis and functionality, as it targets cytoplasmic components to lysosomes for degradation and recycling. Given recent evidence that some novel psychiatric treatments, such as the neuroactive steroid (NAS) allopregnanolone (AlloP, brexanolone), may induce autophagy, we stably transfected human embryonic kidney 293 (HEK) cells with a ratiometric fluorescent probe to assay NAS effects on autophagy. We hypothesized that NAS may modulate autophagy in part by the ability of uncharged NAS to readily permeate membranes. Microscopy revealed a weak effect of AlloP on autophagic flux compared with the positive control treatment of Torin1. In high-throughput microplate experiments, we found that autophagy induction was more robust in early passages of HEK cells. Despite limiting studies to early passages for maximum sensitivity, a range of NAS structures failed to reliably induce autophagy or interact with Torin1 or starvation effects. To probe NAS in a system where AlloP effects have been shown previously, we surveyed astrocytes and again saw minimal autophagy induction by AlloP. Combined with other published results, our results suggest that NAS may modulate autophagy in a cell-specific or context-specific manner. Although there is merit to cell lines as a screening tool, future studies may require assaying NAS in cells from brain regions involved in neuropsychiatric disorders.

Periodic and aperiodic changes to cortical EEG in response to pharmacological manipulation.

Cortical electroencephalograms (EEG) may help understanding of neuropsychiatric illness and new treatment mechanisms. The aperiodic component (1/ f ) of EEG power spectra is often treated as noise, but recent studies suggest that changes to the aperiodic exponent of power spectra may reflect changes in excitation/inhibition (E/I) balance, a concept linked to antidepressant effects, epilepsy, autism, and other clinical conditions. One confound of previous studies is behavioral state, because factors associated with behavioral state other than E/I ratio may alter EEG parameters. Thus, to test the robustness of the aperiodic exponent as a predictor of E/I ratio, we analyzed active exploration in mice using video EEG following various pharmacological manipulations with the Fitting Oscillations & One Over F (FOOOF) algorithm. We found that GABA A receptor (GABA A R) positive allosteric modulators increased the aperiodic exponent, consistent with the hypothesis that an increased exponent signals enhanced cortical inhibition, but other drugs (ketamine and GABA A R antagonists at sub-convulsive doses) did not follow the prediction. To tilt E/I ratio more selectively toward excitation, we suppressed the activity of parvalbumin (PV) interneurons with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). Contrary to our expectations and studies demonstrating increased cortical activity following PV suppression, circuit disinhibition with the DREADD increased the aperiodic exponent. We conclude that the aperiodic exponent of EEG power spectra does not yield a universally reliable marker of E/I ratio. Alternatively, the concept of E/I state may be sufficiently oversimplified that it cannot be mapped readily onto an EEG parameter. Significance StateBment: Neuropsychiatric illness is widely prevalent and debilitating. Causes are not well understood, but some hypotheses point toward altered excitation/inhibition (E/I) balance. Here, we use cortical electroencephalograms (EEG) in mice, given applicability of cortical EEG across species, and evaluate the impact of validated drugs, including anxiolytics (pentobarbital and diazepam), along with novel rapid-acting antidepressants (ketamine and allopregnanolone). We focus on analyzing the aperiodic component of EEG power spectra, which may be associated with changes in E/I ratio. We show that aperiodic exponent of EEG power spectra is not a reliable marker of E/I ratio. Moreover, the concept of E/I ratio may be too broad and complex to be defined by an EEG parameter.

Persistent Brain Connectivity Changes in Healthy Volunteers Following Nitrous Oxide Inhalation.

Background: Nitrous oxide holds promise in the treatment of major depressive disorder. Its psychotropic effects and NMDA receptor antagonism have led to comparisons with ketamine. Despite longstanding use, persistent effects of nitrous oxide on the brain have not been characterized. Methods: Sixteen healthy volunteers were recruited in a double-blind crossover study. In randomized order, individuals underwent a 1-hour inhalation of either 50% nitrous oxide/oxygen or air/oxygen mixtures. At least two 7.5-minute echo-planar resting-state functional magnetic resonance imaging scans were obtained before and at 2 and 24 hours after each inhalation (average 130 min/participant). Using the time series of preprocessed, motion artifact-scrubbed, and nuisance covariate-regressed imaging data, interregional signal correlations were measured and converted to T scores. Hierarchical clustering and linear mixed-effects models were employed. Results: Nitrous oxide inhalation produced changes in global brain connectivity that persisted in the occipital cortex at 2 and 24 hours postinhalation (p < .05, false discovery rate-corrected). Analysis of resting-state networks demonstrated robust strengthening of connectivity between regions of the visual network and those of the dorsal attention network, across 2 and 24 hours after inhalation (p < .05, false discovery rate-corrected). Weaker changes in connectivity were found between the visual cortex and regions of the frontoparietal and default mode networks. Parallel analyses following air/oxygen inhalation yielded no significant changes in functional connectivity. Conclusions: Nitrous oxide inhalation in healthy volunteers revealed persistent increases in global connectivity between regions of primary visual cortex and dorsal attention network. These findings suggest that nitrous oxide inhalation induces neurophysiological cortical changes that persist for at least 24 hours.

The herbicide glyphosate inhibits hippocampal long-term potentiation and learning through activation of pro-inflammatory signaling.

Glyphosate, a herbicide marketed as Roundup, is widely used but there are concerns this exposure could impair cognitive function. In the CA1 region of rat hippocampal slices, we investigated whether glyphosate alters synaptic transmission and long-term potentiation (LTP), a cellular model of learning and memory. Our hypothesis is that glyphosate alters neuronal function and impairs LTP induction via activation of pro-inflammatory processes. Roundup depressed excitatory synaptic potentials(EPSPs) in a dose-dependent manner with complete suppression at 2000 mg/L. At concentrations ≤ 20 mg/L Roundup did not affect basal transmission, but 4 mg/L Roundup administered for 30 min inhibited LTP induction. Acute administration of 10-100 µM glyphosate also inhibited LTP induction. Minocycline, an inhibitor of microglial activation, and TAK-242, an inhibitor of toll-like receptor 4 (TLR4), both overcame the inhibitory effects of 100 µM glyphosate. Similarly, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS), a different TLR4 antagonist, overcame the inhibitory effects. In addition, ISRIB (integrated stress response inhibitor) and quercetin, an inhibitor of endoplasmic reticulum stress, overcame the inhibitory effects. We also observed that in vivo glyphosate injection (16.9 mg/kg i.p.) impaired one-trial inhibitory avoidance learning. This learning deficit was overcome by TAK-242. These observations indicate that glyphosate can impair cognitive function through pro-inflammatory signaling in microglia.

The Taylor Family Institute at Washington University: Novel Treatments in Psychiatry.

Efforts to develop more effective treatments for psychiatric illnesses will require innovative approaches that address changes in brain networks underlying cognition, emotion and motivation, cardinal symptoms that cut across all psychiatric disorders. This effort will include new molecular entities that modulate neuronal excitability, synapses, cellular stress and inflammation. Other opportunities will come from repurposing existing treatments. This overview highlights current and future goals in treatment development in the Taylor Family Institute for Innovative Psychiatric Research at Washington University.

The STOP COVID 2 Study: Fluvoxamine vs Placebo for Outpatients With Symptomatic COVID-19, a Fully Remote Randomized Controlled Trial.

Background: Prior randomized clinical trials have reported benefit of fluvoxamine ≥200 mg/d vs placebo for patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: This randomized, double-blind, placebo-controlled, fully remote multisite clinical trial evaluated whether fluvoxamine prevents clinical deterioration in higher-risk outpatients with acute coronavirus disease 2019 (COVID-19). Between December 2020 and May 2021, nonhospitalized US and Canadian participants with confirmed symptomatic infection received fluvoxamine (50 mg on day 1, 100 mg twice daily thereafter) or placebo for 15 days. The primary modified intent-to-treat (mITT) population included participants who started the intervention within 7 days of symptom onset with a baseline oxygen saturation ≥92%. The primary outcome was clinical deterioration within 15 days of randomization, defined as having both (1) shortness of breath (severity ≥4 on a 0-10 scale or requiring hospitalization) and (2) oxygen saturation <92% on room air or need for supplemental oxygen. Results: A total of 547 participants were randomized and met mITT criteria (n = 272 fluvoxamine, n = 275 placebo). The Data Safety Monitoring Board recommended stopping early for futility related to lower-than-predicted event rates and declining accrual concurrent with vaccine availability in the United States and Canada. Clinical deterioration occurred in 13 (4.8%) participants in the fluvoxamine group and 15 (5.5%) participants in the placebo group (absolute difference at day 15, 0.68%; 95% CI, -3.0% to 4.4%; log-rank P = .91). Conclusions: This trial did not find fluvoxamine efficacious in preventing clinical deterioration in unvaccinated outpatients with symptomatic COVID-19. It was stopped early and underpowered due to low primary outcome rates. Clinical Trials Registration: ClinicalTrials.gov Identifier: NCT04668950.

Precision Neuroimaging for Localization-Related Psychiatry.

This Viewpoint describes how precision functional mapping may be helpful for associating neuroanatomical regions with specific psychiatric disorders.

Striatal mGlu5-mediated synaptic plasticity is independently regulated by location-specific receptor pools and divergent signaling pathways.

Metabotropic glutamate receptor 5 (mGlu5) is widely expressed throughout the central nervous system and is involved in neuronal function, synaptic transmission, and a number of neuropsychiatric disorders such as depression, anxiety, and autism. Recent work from this lab showed that mGlu5 is one of a growing number of G protein-coupled receptors that can signal from intracellular membranes where it drives unique signaling pathways, including upregulation of extracellular signal-regulated kinase (ERK1/2), ETS transcription factor Elk-1, and activity-regulated cytoskeleton-associated protein (Arc). To determine the roles of cell surface mGlu5 as well as the intracellular receptor in a well-known mGlu5 synaptic plasticity model such as long-term depression, we used pharmacological isolation and genetic and physiological approaches to analyze spatially restricted pools of mGlu5 in striatal cultures and slice preparations. Here we show that both intracellular and cell surface receptors activate the phosphatidylinositol-3-kinase-protein kinase B-mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, whereas only intracellular mGlu5 activates protein phosphatase 2 and leads to fragile X mental retardation protein degradation and de novo protein synthesis followed by a protein synthesis-dependent increase in Arc and post-synaptic density protein 95. However, both cell surface and intracellular mGlu5 activation lead to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor GluA2 internalization and chemically induced long-term depression albeit via different signaling mechanisms. These data underscore the importance of intracellular mGlu5 in the cascade of events associated with sustained synaptic transmission in the striatum.

The herbicide glyphosate inhibits hippocampal long-term potentiation and learning through activation of pro-inflammatory signaling.

Background: Glyphosate, a herbicide marketed under the trade name Roundup, is now widely used, in part because genetically modified organism plants that are resistant to this agent have been developed. Environmental or dietary exposure to glyphosate is omnipresent and there are concerns this exposure could impair cognitive function in addition to carcinogenicity. Methods: Using hippocampal slices from juvenile male rats, we investigated whether glyphosate alters synaptic transmission and induction of long-term potentiation (LTP), a cellular model of learning and memory. Our hypothesis is that glyphosate alters neuronal function and impairs LTP induction via activation of pro-inflammatory processes, because increases in pro-inflammatory cytokines and neuroinflammation have been reported following glyphosate exposure. LTP was induced by delivery of 100 Hz x 1 sec high frequency stimulation (HFS) of the Schaffer collateral pathway and excitatory synaptic potentials (EPSPs) were monitored 60 min after HFS. Resulsts: We first tested effects of Roundup on basal synaptic function and LTP induction. Roundup depressed EPSPs in a dose-dependent manner. Basal synaptic transmission was completely suppressed by 2000 ppm. At concentrations ≤ 20 ppm Roundup did not affect basal transmission, but 4 ppm Roundup administered 30 min before HFS inhibited LTP induction. We also observed that acute administration of 10-100 µM glyphosate inhibits LTP induction. Minocycline, an inhibitor of microglial activation, and TAK-242, an inhibitor of toll-like receptor 4 (TLR4), both overcame the inhibitory effects of 100M glyphosate. Similarly, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) overcame the inhibitory effects. In addition, ISRIB (integrated stress response inhibitor) and quercetin, an inhibitor of endoplasmic reticulum stress, allowed LTP induction in the presence of glyphosate. We also observed that in vivo glyphosate injection (16.9 mg/kg i.p.) impaired one-trial inhibitory avoidance learning. This learning deficit was overcome by TAK-242. Conclusion: While Roundup inhibits LTP induction, these observations indicate that glyphosate alone, the major ingredient of Roundup, can impair cognitive function through pro-inflammatory signaling in microglia. Manipulation of pro-inflammatory signaling could be a useful strategy to prevent cognitive impairment after exposure to a glyphosate-based herbicide (GBH).

Neurosteroid enantiomers as potentially novel neurotherapeutics.

Endogenous neurosteroids and synthetic neuroactive steroids (NAS) are important targets for therapeutic development in neuropsychiatric disorders. These steroids modulate major signaling systems in the brain and intracellular processes including inflammation, cellular stress and autophagy. In this review, we describe studies performed using unnatural enantiomers of key neurosteroids, which are physiochemically identical to their natural counterparts except for rotation of polarized light. These studies led to insights in how NAS interact with receptors, ion channels and intracellular sites of action. Certain effects of NAS show high enantioselectivity, consistent with actions in chiral environments and likely direct interactions with signaling proteins. Other effects show no enantioselectivity and even reverse enantioselectivity. The spectrum of effects of NAS enantiomers raises the possibility that these agents, once considered only as tools for preclinical studies, have therapeutic potential that complements and in some cases may exceed their natural counterparts. Here we review studies of NAS enantiomers from the perspective of their potential development as novel neurotherapeutics.

Beyond "Psychotropic": Repurposing Psychiatric Drugs for COVID-19, Alzheimer's Disease, and Cancer.

Importance: "Psychotropic" drugs have widespread reach and impact throughout the brain and body. Thus, many of these drugs could be repurposed for non-psychiatric indications of high public health impact.Observations: The selective serotonin reuptake inhibitor (SSRI) fluvoxamine was shown efficacious as a COVID-19 treatment based on randomized controlled trials (RCTs), and a benefit of other antidepressants has been posited based on observational and preclinical studies. In this review, we illuminate features of SSRIs and other psychiatric drugs that make them candidates to repurpose for non-psychiatric indications. We summarize research that led to fluvoxamine's use in COVID-19 and provide guidance on how to use it safely. We summarize studies suggestive of benefit of other antidepressants versus COVID-19 and long COVID. We also describe putative mechanisms of psychiatric drugs in treating long COVID, Alzheimer's disease, cancer, and other conditions.Conclusion and Relevance: There is a potentially great clinical and public health impact of psychotropic drug repurposing. Challenges exist to such repurposing efforts, but solutions exist for researchers, regulators, and funders that overcome these challenges.