Neuroactive steroids are allosteric modulators of GABAA receptors and are implicated in the etiology and treatment of neuropsychiatric disorders. Existing treatments are helpful but have drawbacks. Neuroactive steroids recently gained attention as rapidly acting antidepressants in postpartum depression and other indications. Unlike other GABAA receptor modulators, neurosteroids may possess anti-inflammatory actions, potentially contributing to therapeutic benefit. Here we seek to understand neuroactive steroid structure-activity relationships relevant to these anti-inflammatory effects. We used murine microglial BV2 cells challenged with lipopolysaccharide (LPS) as an inflammation model. We investigated structure-activity profile of neuroactive steroids and oxysterol-like compounds on cytokine transcription. LPS increased transcripts for cytokines IL-1β, IL-6 and TNF-α. Both allopregnanolone and its enantiomer significantly suppressed these LPS-induced increases, with no effect in the absence of LPS. Conclusions Our results suggest that neuroactive steroids exhibit a distinct structure-activity profile compared with their GABAA receptor modulation effects. Certain neuroactive steroids may selectively target neuroinflammation. The enantiomer of AlloP could be a tool compound to differentiate anti-inflammatory effects of neuroactive steroids from GABAergic and other enantioselective effects.

Neuroactive steroids modulate GABAA and NMDA receptors allosterically, typically requiring specific structural features for their activity. In this study, we characterize YX84, a novel neuroactive steroid bearing a 3β sulfate and p-trifluoroacetylbenzyl alcohol attached in an ether linkage to a hydroxyl group at steroid carbon 17. This compound and similar analogues exhibit an atypical pharmacological profile, with three distinct actions at GABAA receptors. First, YX84 is a full agonist, with EC50 near 1 μM and comparable efficacy to GABA at GABAA receptors in native hippocampal neurons. It presents as a full agonist relative to GABA at α4/δ subunit-containing receptors. Second, YX84 acts as a slow-onset, potent positive allosteric modulator (PAM) of GABAA receptors at concentrations below those that gate a response. Finally, YX84 exhibits rapid desensitizing and/or blocking kinetics; voltage dependence is consistent with a contribution of channel block. Structure-activity relationship analyses reveal that both functional groups are essential for gating activity, while classical requirements such as carbon 3 hydroxyl stereoselectivity and carbon 5 reduction are dispensable. YX84 also modestly inhibits NMDA receptor currents, suggesting weak negative allosteric modulation. Behavioral assays show that intraperitoneal administration of YX84 (30 mg/kg) does not impair sensorimotor function, unlike allopregnanolone. These findings identify YX84 as a structurally distinct neuroactive steroid with dual receptor activity and favorable behavioral tolerability, offering a promising scaffold for therapeutic development targeting excitatory/inhibitory imbalance in neuropsychiatric disorders if pharmacokinetic considerations can be overcome.

Abstract Neurosteroids are powerful endogenous modulators of inhibition and emerging therapeutics for anxiety, epilepsy, and mood disorders, yet their actions at defined receptor subtypes and within specific neuronal populations remain poorly resolved. Here, we engineer a neurosteroid DART (Drug Acutely Restricted by Tethering) platform to deliver neuroactive steroid (NAS) activity with cellular precision and receptor-subunit selectivity. From a screen of seventeen NAS analogs, we identified seven scaffolds suitable for further engineering, and we discovered that linker attachment at the steroid C11 position uniquely preserves NAS positive allosteric modulation of GABA A receptors, whereas C2 and C17 attachment abolished activity. C11-linked NAS-DARTs slowed IPSC decay kinetics and showed variable off-target modulation of NMDA and AMPA EPSCs. The lead DART compound, YX85.1 DART.2 , enhanced GABA-evoked currents in neurons expressing engineered α4/δ-containing GABA A receptors but spared γ2-containing receptors. A complementary benzodiazepine DART BZP.1 DART.2 showed the opposite selectivity. Together, these tools enable cell-restricted, subunit-resolved interrogation of neurosteroid action on inhibitory microcircuits and provide a strategy to dissect how distinct GABA A receptor subclasses contribute to circuit function and therapeutic outcomes.

Abstract Peripheral nerve injury induces long-lasting changes in sensory neurons that contribute to the development of neuropathic pain. Although the histamine H₃ receptor (H₃R) is best known for regulating neurotransmitter release in the central nervous system, its expression and role in dorsal root ganglia (DRG) remain poorly understood. In this study, we examined the expression, cellular distribution, and functional role of H₃R in DRG and implications in neuropathic pain. Sciatic nerve injury increased membrane-associated H₃R protein expression in the DRG, suggesting enhanced receptor trafficking or stabilization at the neuronal membrane. Single-cell transcriptomic analysis revealed that H₃R mRNA expression in DRG is predominantly restricted to peptidergic sensory neurons and C-LTMRs rodents. Fluorescence imaging and transcriptomic analysis also suggest that the majority of H3R co-express with TrkA + (a NGF receptor) sensory neuron populations. These findings identify a subset of NGF-responsive nociceptors in which H₃R may directly influence injury-induced sensitization. Pain behavior assessment demonstrated a paradoxical role of an H3R inverse agonist (GSK334429), which reduces mechanical hypersensitivity caused by chronic constriction injury (CCI) with no change in thermal pain. Patch-clamp recordings show that the GSK334429 attenuates NGF-induced hyperexcitability in DRG neuronal cultures. Overall, our work suggests a contributing role of H3R in modality-specific effects on sensory processing through its enrichment in specific neuronal populations in DRG. Graphical Abstract

Background and purpose: Neurosteroids modulate neuronal function and are promising therapeutic agents for neuropsychiatric disorders. Neurosteroid analogues are approved for treating postpartum depression and are of interest in other disorders. GABA-A receptors are well characterized targets of natural neurosteroids, but other biological pathways are likely relevant to therapeutic mechanisms and/or to off-target effects. We performed hypothesis-generating in silico analyses and broad in vitro biological screens to assess the range of actions of neurosteroids analogues of varying structural attributes. Key Results: We employed in silico molecular similarity analysis and network pharmacology to elucidate likely targets. This analysis confirmed likely targets beyond GABA-A receptors. We then functionally screened 19 distinct neurosteroid structures across 78 targets representing interconnected signaling pathways, complemented with a limited screen of kinase activation. Results revealed unanticipated modulation of targets by neurosteroids with some structural selectivity. Many compounds-initiated androgen receptor translocation with little or no enantioselectivity. Modulation of multiple G-protein receptors was also unexpected. Conclusions and implications: Neurosteroids are ascendant treatments in neuropsychiatry, but their full spectrum of actions remains unclear. This virtual and biological screening discovery approach opens new vistas for exploring mechanism of neurosteroids analogues. The multifaceted approach provides an unbiased, holistic exploration of the potential effects of neurosteroids across various molecular targets and provides a platform for future validation studies to aid drug discovery.

GABAA 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 by 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 validating expected functional loss of δ-associated current in cortex of PV δcKO mice of both sexes, 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.

Voltage-dependent anion channels (VDACs) are the most abundant proteins in the outer mitochondrial membrane (OMM) and key regulators of mitochondrial function under physiological and pathological conditions. These channels are modulated by multiple agents and are known binding sites for neuroactive steroids (NAS) including allopregnanolone (AlloP). Using erastin, an agent that promotes VDAC activation by preventing inhibition by tubulin, we assessed the impact of VDAC activation on hippocampal function. Brief erastin administration had no effect on basal transmission but completely inhibited induction of long-term potentiation (LTP) in the Schaffer collateral pathway of rat hippocampal slices. This LTP inhibition was prevented by VBIT-4, an agent that inhibits VDAC oligomerization. VDAC-mediated LTP inhibition was also prevented by inhibitors of the NLRP3 inflammasome and caspase-1, downstream effectors of VDACs, but not by inhibition of cGAS-STING, narrowing the neuroinflammatory pathways involved. Similarly, effects of erastin were prevented by AlloP and at lower concentrations by its unnatural enantiomer ( ent -AlloP), an agent that unlike AlloP has little effect on GABAA receptors. Erastin also inhibited memory formation in a hippocampal-dependent form of one-trial learning and these effects were prevented by VBIT-4 and ent -AlloP, but not by a non-sedating dose of AlloP. These results have relevance for understanding the role of VDACs as mediators of neuronal stress and for the further development of NAS as neurotherapeutics and modulators of cellular stress. SIGNIFICANCE STATEMENT Voltage-dependent anion channels (VDACs) are important regulators of mitochondrial function, playing roles in cellular metabolism, stress responses and neuroinflammation, and contribute to the pathogenesis of neuropsychiatric illnesses. Here we show that erastin, an agent that activates VDACs initiates specific neuroinflammatory responses to acutely disrupt synaptic plasticity in the rodent hippocampus and abrogate learning. These adverse effects are prevented by the neuroactive steroids allopregnanolone, which binds VDACs and is used clinically for postpartum depression, and its unnatural enantiomer, suggesting that these agents could have therapeutic effects in a broad range of brain illnesses. ### Competing Interest Statement CFZ previously served as a member of the Scientific Advisory Board for Sage Therapeutics and held equity in the company. Sage Therapeutics had no role in the design or interpretation of these experiments. The remaining authors declare no competing financial interests. NIH Common Fund, https://ror.org/001d55x84, MH123748, MH122379 Taylor Family Institute for Innovative Psychiatric Research, N/A Bantly Foundation, N/A

Nitrous oxide (N2O) induces rapid and durable antidepressant effects. The cellular and circuit mechanisms mediating this process are not known. Here we find that a single dose of inhaled N2O induces rapid and specific activation of layer V (L5) pyramidal neurons in the cingulate cortex of rodents exposed to chronic stress conditions. N2O-induced L5 activation rescues a stress-associated hypoactivity state, persists following exposure, and is necessary for its antidepressant-like activity. Although NMDA-receptor antagonism is believed to be a primary mechanism of action for N2O, L5 neurons activate even when NMDA-receptor function is attenuated through both pharmacological and genetic approaches. By examining different molecular and circuit targets, we identify N2O-induced inhibition of calcium-sensitive potassium (SK2) channels as a key molecular interaction responsible for driving specific L5 activity along with ensuing antidepressant-like effects. These results suggest that N2O-induced L5 activation is crucial for its fast antidepressant action and this effect involves novel and specific molecular actions in distinct cortical cell types.

Background and purposeNeurosteroids (NS) modulate neuronal function and are promising therapeutic agents for neuropsychiatric disorders. NS analogues are approved for treating postpartum depression and are of interest in other disorders. Gamma-aminobutyric acid type A (GABAA) receptors are well characterised targets of natural NS, but other biological pathways are likely relevant to therapeutic mechanisms and/or to off-target effects.Experimental ApproachWe performed hypothesis-generating in silico analyses using machine learning and broad in vitro biological screens to assess the range of actions of NS analogues of varying structural attributes. We employed machine learning for molecular similarity analysis and network pharmacology to elucidate likely targets.Key ResultsThis analysis confirmed likely targets beyond GABAA receptors. We then functionally screened 19 distinct NS structures across 78 targets representing interconnected signalling pathways, complemented with a limited screen of kinase activation. Results revealed the unanticipated modulation of targets by natural and synthetic NS analogues. Many compounds initiated androgen receptor (AR) translocation, with little or no enantioselectivity. Modulation of multiple G-protein receptors also was unexpected.Conclusions and ImplicationsNS compounds are ascendant treatments in neuropsychiatry, but their full spectrum of actions remains unclear. This virtual and biological screening discovery approach opens new vistas for exploring the mechanism of NS analogues. The multifaceted approach provides an unbiased, holistic exploration of the potential effects of NS compounds across various molecular targets and provides a platform for future validation studies to aid drug discovery.

Neuroinflammation is an increasingly important target for therapeutics in neuropsychiatry and contributes to cognitive dysfunction, disability and death across a range of illnesses. We previously found that acute effects of pro-inflammatory stimulation with lipopolysaccharide (LPS) on hippocampal long-term potentiation (LTP), a form of synaptic plasticity involved in learning and memory, requires synthesis of the oxysterol, 25-hydroxycholesterol (25HC) and exogenous 25HC mimics effects of LPS. However, downstream mechanisms engaged by LPS and 25HC remain uncertain. Here we use rat hippocampal slices and in vivo behavioral studies to provide evidence that acute modulation of synaptic plasticity by both LPS and 25HC requires activation of the NLRP3 inflammasome, caspase-1 and interleukin-1 receptor. Furthermore, both LPS and 25HC engage cellular stress responses including synthesis of 5α-reduced neurosteroids and effects on plasticity are prevented by modulators of these responses. In studies of acute learning using a one-trial inhibitory avoidance task, inhibition of learning by LPS and 25HC are prevented by pre-treatment with an inhibitor of NLRP3. The present studies provide strong support for the role of 25HC as a mediator of pro-inflammatory stimulation on hippocampal synaptic plasticity and for the importance of NLRP3 inflammasome and caspase-1 activation in the deleterious effects of acute inflammation.

In medial prefrontal cortex (mPFC), fast-spiking parvalbumin (PV) interneurons regulate excitability and microcircuit oscillatory activity important for cognition. Although PV interneurons inhibit pyramidal neurons, they themselves express δ subunits of GABAA receptors important for slow inhibition. However, the specific contribution of δ-containing GABAA receptors to the function of PV interneurons in mPFC is unclear. We explored cellular, synaptic, and local-circuit activity in PV interneurons and pyramidal neurons in mouse mPFC after selectively deleting δ subunits in PV interneurons (cKO mice). In current-clamp recordings, cKO PV interneurons exhibited a higher frequency of action potentials and higher input resistance than wild type (WT) PV interneurons. Picrotoxin increased firing and GABA decreased firing in WT PV interneurons but not in cKO PV interneurons. The δ-preferring agonist THIP reduced spontaneous inhibitory postsynaptic currents disproportionately in WT pyramidal neurons compared with cKO pyramidal neurons. In WT slices, depolarizing the network with 400 nM kainate increased firing of pyramidal neurons but had little effect on PV interneuron firing. By contrast, in cKO slices kainate recruited PV interneurons at the expense of pyramidal neurons. At the population level, kainate induced broadband increases in local field potentials in WT but not cKO slices. These results on cells and network activity can be understood through increased excitability of cKO PV interneurons. In summary, our study demonstrates that δ-containing GABAA receptors in mPFC PV interneurons play a crucial role in regulating their excitability and the phasic inhibition of pyramidal neurons, elucidating intricate mechanisms governing cortical circuitry.

The success of ketamine, a dissociative anesthetic and non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist, as a rapidly acting antidepressant has ignited efforts to identify other novel depression treatments. In recent years, several clinical trials indicated that nitrous oxide (N2O), an inhalational dissociative anesthetic in clinical use for over 150 years, also has rapid and durable antidepressant effects in patients with major depressive disorder (MDD) and treatment resistant major depression (TRMD). N2O is a non-competitive NMDAR inhibitor but acts on NMDARs by mechanisms distinct from ketamine. Cellular and neuronal circuit studies of N2O-induced psychotropic and antidepressant effects are in their infancy and suggest that N2O shares at least some downstream mechanisms with ketamine, while also having unique effects on neurophysiology and signaling. Human neuroimaging and brain network connectivity studies of N2O have begun to identify acute and persisting effects of the drug on brain circuits likely relevant for antidepressant responses. In this review, we highlight the current state of clinical and preclinical research into the effects of N2O and emphasize major unanswered questions, some of which are currently being explored. We emphasize future directions and potential barriers to clinical use of N2O for treatment of patients with psychiatric illnesses.

OBJECTIVE:Mouse models of genetic dystonias have demonstrated abnormal striatal cholinergic interneuron excitability, but do not consistently demonstrate subjective dystonic features. To determine whether striatal cholinergic interneuron excitation can cause potentially dystonic motor behaviors, we first determined features correlated specifically with dystonia severity in people and then determined whether these features emerged in mice following striatal cholinergic interneuron excitation. METHODS:Eight movement disorders experts rated dystonia severity in 193 videos of people with cerebral palsy doing a seated task. Leg adduction variability metrics, which are known to correlate with leg dystonia severity during gait, were quantified in these videos of seated tasks. Metrics significantly associated with leg dystonia severity during seated tasks in people were then quantified in mice and compared between mice who underwent chemogenetic striatal cholinergic interneuron excitation (n = 17) and mice who did not (n = 17). RESULTS:Leg adduction variability correlated well with experts' leg dystonia severity scores in people. Leg adduction variability was also significantly increased in mice that underwent striatal cholinergic interneuron excitation compared to mice that did not (p < 0.05). This difference was not present with acute excitation and emerged only after 14 days of ongoing excitation. INTERPRETATION:We demonstrate that leg adduction variability correlates with leg dystonia severity in people with cerebral palsy and that chronic, but not acute, striatal cholinergic interneuron excitation can cause leg adduction variability in mice. These results support targeting striatal cholinergic interneurons for dystonia drug development and demonstrate the potential value of using quantifiable leg adduction metrics to study dystonia pathophysiology. ANN NEUROL 2025;98:726-740.

While nitrous oxide (N2O) has demonstrated antidepressant properties in treatment-resistant major depression (TRD), little is known about neural mechanisms mediating these effects. Employing serial resting-state functional magnetic resonance imaging (rs-fMRI), we compared spatiotemporal effects of inhaled N2O on brain functional connectivity in TRD patients (n=14) and non-depressed healthy controls (n=16, CNTL). Participants received sequential, one-hour inhalations of either 50% N2O/oxygen or air/oxygen (placebo), with sessions separated by at least one month in random cross-over order. BOLD-contrast rs-fMRI scans were acquired at three time points: pre-inhalation, 2 hours post-inhalation, and 24 hours post-inhalation. For the rs-fMRI functional connectivity analyses, five a priori seeds in medial limbic structures targeted cortical networks implicated in major depression - the salience, anterior and posterior default mode, reward, and cingulo-opercular networks - and a nexus in the dorsal paracingulate region previously identified in MDD ("dorsal nexus"). Depression, dissociation, and psychosis assessments were made before and after inhalations. In TRD patients, functional connectivity was reduced in all seeded networks and the voxel-wise global analysis after N2O exposure. N2O progressively decreased connectivity in patients with TRD but increased connectivity in healthy controls. In TRD patients, each seeded network demonstrated post-inhalation functional connectivity reductions in the dorsal paracingulate gyrus ("dorsal nexus"). This study further elucidates neural mechanisms underlying the antidepressant properties of N2O, supporting the notion that N2O specifically alters mood-associated brain regions in the depressed brain state by reducing functional connectivity within these brain networks. The trial was registered at ClinicalTrials.gov (NCT02994433).

Pro-inflammatory changes contribute to multiple neuropsychiatric illnesses. Understanding how these changes are involved in illnesses and identifying strategies to alter inflammatory responses offer paths to potentially novel treatments. We previously found that acute pro-inflammatory stimulation with high (μg/ml) lipopolysaccharide (LPS) for 10-15 min dampens long-term potentiation (LTP) in the hippocampus and impairs learning. Effects of LPS involved non-canonical inflammasome signaling but were independent of toll-like receptor 4 (TLR4), a known LPS receptor. Low (ng/ml) LPS also inhibits LTP when administered for 2-4 h, and here we report that this LPS exposure requires TLR4. We also found that effects of low LPS on LTP involve the oxysterol, 25-hydroxycholesterol, akin to high LPS. Effects of high LPS on LTP are blocked by inhibiting synthesis of 5α-reduced neurosteroids, indicating that neurosteroids mediate LTP inhibition. 5α-Neurosteroids also have anti-inflammatory effects, and we found that exogenous allopregnanolone (AlloP), a key 5α-reduced steroid, prevented effects of low but not high LPS on LTP. We also found that activation of TLR2, TLR3 and TLR7 inhibited LTP and that AlloP prevented the effects of TLR2 and TLR7, but not TLR3. The enantiomer of AlloP, a steroid that has anti-inflammatory actions but low activity at GABAA receptors, prevented LTP inhibition by TLR2, TLR3 and TLR7. In vivo, both AlloP enantiomers prevented LPS-induced learning defects. These studies indicate that neurosteroids play complex roles in network effects of acute neuroinflammation and have potential importance for development of AlloP analogues as therapeutic agents.

Late-onset Alzheimer’s disease (LOAD) is the most common form of Alzheimer’s disease (AD). However, modeling sporadic LOAD that endogenously captures hallmark neuronal pathologies such as amyloid-β (Aβ) deposition, tau tangles, and neuronal loss remains an unmet need. We demonstrate that neurons generated by microRNA (miRNA)–based direct reprogramming of fibroblasts from individuals affected by autosomal dominant AD (ADAD) and LOAD in a three-dimensional environment effectively recapitulate key neuropathological features of AD. Reprogrammed LOAD neurons exhibit Aβ-dependent neurodegeneration, and treatment with β- or γ-secretase inhibitors before (but not subsequent to) Aβ deposit formation mitigated neuronal death. Moreover inhibiting age-associated retrotransposable elements in LOAD neurons reduced both Aβ deposition and neurodegeneration. Our study underscores the efficacy of modeling late-onset neuropathology of LOAD through high-efficiency miRNA-based neuronal reprogramming.

Abstract Brain tumors arise in close association with neurons, suggesting that these non-neoplastic cells may be critical stromal drivers of brain tumor initiation and growth. Previously, we have shown that murine low-grade optic glioma formation and progression in the setting of Neurofibromatosis type 1 (NF1) is dictated by neurons and neuronal activity. In these studies, these neuronal dependencies reflected neuronal activity-driven enzymatic cleavage of a growth factor (neuroligin-3) from oligodendrocyte precursors cells (tumor initiation) or neuronal production of a paracrine factor to stimulate T cell support of optic glioma growth (tumor progression). Since neurons typically communicate with other neurons through neurotransmitters, we sought to explore the possibility that neurotransmitters operate to modulate low-grade glioma growth using humanized mouse models of pilocytic astrocytoma (PA). Leveraging single cell RNA sequencing of three independent sets of pediatric PAs, we identified neurotransmitter pathway enrichment in the tumor cells. This neurotransmitter pathway enrichment reflected aberrant expression of specific neurotransmitter receptors, which we confirmed in three independently generated PA tissue microarrays and in five distinct primary PA cell lines grown in vitro. Moreover, this aberrant neurotransmitter receptor expression established differential neurotransmitter PA growth dependencies in vitro. Importantly, interruption of neurotransmitter signaling in human PA xenografts attenuated tumor growth and ERK activation in Rag1-/- mice in vivo. Finally, we discovered crosstalk between neurotransmitter receptor and receptor tyrosine kinase signaling that revealed another target for therapeutic inhibition. Taken together, these findings elucidate a previously unknown neurotransmitter PA growth dependency amenable to therapeutic targeting.

In addition to modulating serotonin transport, selective serotonin reuptake inhibitors (SSRIs) have multiple other mechanisms that may contribute to clinical effects, and some of these latter actions prompt repurposing of SSRIs for non-psychiatric indications. In a recent study of the SSRIs fluvoxamine, fluoxetine and sertraline we found that, unlike the other two SSRIs, sertraline acutely inhibited 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 contain GluN2B subunits. A selective S1R antagonist (NE-100), but not an S1R agonist (PRE-084) blocked effects on NMDARs, even though 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.

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.