Parvalbumin and Somatostatin Interneurons Contribute to the Generation of Hippocampal Gamma Oscillations.

γ-frequency oscillations (30-120 Hz) in cortical networks influence neuronal encoding and information transfer, and are disrupted in multiple brain disorders. While synaptic inhibition is important for synchronization across the γ-frequency range, the role of distinct interneuronal subtypes in slow (<60 Hz) and fast γ states remains unclear. Here, we used optogenetics to examine the involvement of parvalbumin-expressing (PV+) and somatostatin-expressing (SST+) interneurons in γ oscillations in the mouse hippocampal CA3 ex vivo, using animals of either sex. Disrupting either PV+ or SST+ interneuron activity, via either photoinhibition or photoexcitation, led to a decrease in the power of cholinergically induced slow γ oscillations. Furthermore, photoexcitation of SST+ interneurons induced fast γ oscillations, which depended on both synaptic excitation and inhibition. Our findings support a critical role for both PV+ and SST+ interneurons in slow hippocampal γ oscillations, and further suggest that intense activation of SST+ interneurons can enable the CA3 circuit to generate fast γ oscillations.SIGNIFICANCE STATEMENT The generation of hippocampal γ oscillations depends on synchronized inhibition provided by GABAergic interneurons. Parvalbumin-expressing (PV+) interneurons are thought to play the key role in coordinating the spike timing of excitatory pyramidal neurons, but the role distinct inhibitory circuits in network synchronization remains unresolved. Here, we show, for the first time, that causal disruption of either PV+ or somatostatin-expressing (SST+) interneuron activity impairs the generation of slow γ oscillations in the ventral hippocampus ex vivo We further show that SST+ interneuron activation along with general network excitation is sufficient to generate high-frequency γ oscillations in the same preparation. These results affirm a crucial role for both PV+ and SST+ interneurons in hippocampal γ oscillation generation.

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains.

Advances in membrane cell biology are hampered by the relatively high proportion of proteins with no known function. Such proteins are largely or entirely devoid of structurally significant domain annotations. Structural bioinformaticians have developed profile-profile tools such as HHsearch (online version called HHpred), which can detect remote homologies that are missed by tools used to annotate databases. Here we have applied HHsearch to study a single structural fold in a single model organism as proof of principle. In the entire clan of protein domains sharing the pleckstrin homology domain fold in yeast, systematic application of HHsearch accurately identified known PH-like domains. It also predicted 16 new domains in 13 yeast proteins many of which are implicated in intracellular traffic. One of these was Vps13p, where we confirmed the functional importance of the predicted PH-like domain. Even though such predictions require considerable work to be corroborated, they are useful first steps. HHsearch should be applied more widely, particularly across entire proteomes of model organisms, to significantly improve database annotations.

Semi-automated seizure detection using interpretable machine learning models.

Despite the vast number of seizure detection publications there are no validated open-source tools for automating seizure detection based on electrographic recordings. Researchers instead rely on manual curation of seizure detection that is highly laborious, inefficient, error prone, and heavily biased. Here we developed an open-source software called SeizyML that uses sensitive machine learning models coupled with manual validation of detected events reducing bias and promoting efficient and accurate detection of electrographic seizures. We compared the validity of four interpretable machine learning models (decision tree, gaussian naïve bayes, passive aggressive classifier, and stochastic gradient descent classifier) on an extensive electrographic seizure dataset that we collected from chronically epileptic mice. We find that the gaussian naïve bayes and stochastic gradient descent models achieved the highest precision and f1 scores, while also detecting all seizures in our mouse dataset and only require a small amount of data to train the model and achieve good performance. Further, we demonstrate the utility of this approach to detect electrographic seizures in a human EEG dataset. This approach has the potential to be a transformative research tool overcoming the analysis bottleneck that slows research progress.

Sex-dependent effects of early life stress on network and behavioral states.

Background: Adverse childhood experiences (ACEs) are associated with numerous detriments in health, including increased vulnerability to psychiatric illnesses. Early life stress (ELS) in rodents has been shown to effectively model several of the behavioral and endocrine impacts of ACEs and has been utilized to investigate the underlying mechanisms contributing to disease. However, the precise neural mechanisms responsible for mediating the impact of ELS on vulnerability to psychiatric illnesses remain largely unknown. Methods: We use behavior, immunoassay, in vivo LFP recording, histology, and patch clamp to describe the effects of ELS on stress behaviors, endocrinology, network states, protein expression, and cellular physiology in male and female mice. Results: We demonstrate that a murine maternal separation (MS) ELS model causes sex-dependent alterations in behavioral and hormonal responses following an acute stressor. Local field potential (LFP) recordings in the basolateral amygdala (BLA) and frontal cortex (FC) reveal similar sex-dependent alterations at baseline, in response to acute ethological stress, and during fear memory extinction, supporting a large body of literature demonstrating that these network states contribute to stress reactivity and vulnerability to psychiatric illnesses. Sex differences were accompanied by altered physiology of BLA principal neurons in males and BLA PV interneurons in females. Conclusions: Collectively, these results implicate novel, sex-dependent mechanisms through which ACEs may impact psychiatric health, involving altered cellular physiology and network states involved in emotional processing.

Basolateral amygdala parvalbumin interneurons coordinate oscillations to drive reward behaviors.

The basolateral amygdala (BLA) mediates both fear and reward learning.1,2 Previous work has shown that parvalbumin (PV) interneurons in the BLA contribute to BLA oscillatory states integral to fear expression.3,4,5,6,7 However, despite it being critical to our understanding of reward behaviors, it is unknown whether BLA oscillatory states and PV interneurons similarly contribute to reward processing. Local field potentials in the BLA were collected as male and female mice consumed sucrose reward, where prominent changes in the beta band (15-30 Hz) emerged with reward experience. During consumption of one water bottle during a two-water-bottle choice test, rhythmic optogenetic stimulation of BLA PVs produced a robust bottle preference, showing that PVs can sufficiently drive reward seeking. Finally, to demonstrate that PV activity is necessary for reward value use, PVs were chemogenetically inhibited following outcome devaluation, rendering mice incapable of using updated reward representations to guide their behavior. Taken together, these experiments provide novel information about the physiological signatures of reward while highlighting BLA PV interneuron contributions to behaviors that are BLA dependent. This work builds upon established knowledge of PV involvement in fear expression and provides evidence that PV orchestration of unique BLA network states is involved in both learning types.

ALS-related p97 R155H mutation disrupts lysophagy in iPSC-derived motor neurons.

Mutations in the AAA+ ATPase p97 cause multisystem proteinopathy 1, which includes amyotrophic lateral sclerosis; however, the pathogenic mechanisms that contribute to motor neuron loss remain obscure. Here, we use two induced pluripotent stem cell models differentiated into spinal motor neurons to investigate how p97 mutations perturb the motor neuron proteome. Using quantitative proteomics, we find that motor neurons harboring the p97 R155H mutation have deficits in the selective autophagy of lysosomes (lysophagy). p97 R155H motor neurons are unable to clear damaged lysosomes and have reduced viability. Lysosomes in mutant motor neurons have increased pH compared with wild-type cells. The clearance of damaged lysosomes involves UBXD1-p97 interaction, which is disrupted in mutant motor neurons. Finally, inhibition of the ATPase activity of p97 using the inhibitor CB-5083 rescues lysophagy defects in mutant motor neurons. These results add to the evidence that endo-lysosomal dysfunction is a key aspect of disease pathogenesis in p97-related disorders.

Hypothalamic-pituitary-adrenal axis dysfunction elevates SUDEP risk in a sex-specific manner.

Epilepsy is often comorbid with psychiatric illnesses, including anxiety and depression. Despite the high incidence of psychiatric comorbidities in people with epilepsy, few studies address the underlying mechanisms. Stress can trigger epilepsy and depression. Evidence from human and animal studies support that hypothalamic-pituitary-adrenal (HPA) axis dysfunction may contribute to both disorders as well as their comorbidity (Kanner, 2003). Here, we investigate if HPA axis dysfunction may influence epilepsy outcomes and psychiatric comorbidities. We generated a novel mouse model (Kcc2/Crh KO mice) lacking the K+/Cl- co-transporter, KCC2, in corticotropin-releasing hormone (CRH) neurons, which exhibit stress- and seizure-induced HPA axis hyperactivation (Melon et al., 2018). We used the Kcc2/Crh KO mice to examine the impact on epilepsy outcomes, including seizure frequency/burden, comorbid behavioral deficits, and SUDEP risk. We found sex differences in HPA axis dysfunction's effect on chronically epileptic KCC2/Crh KO mice seizure burden, vulnerability to comorbid behavioral deficits, and SUDEP. Suppressing HPA axis hyperexcitability in this model using pharmacological or chemogenetic approaches decreased SUDEP incidence, suggesting that HPA axis dysfunction may contribute to SUDEP. Altered neuroendocrine markers were present in SUDEP cases compared to people with epilepsy or individuals without epilepsy. Together, these findings implicate HPA axis dysfunction in the pathophysiological mechanisms contributing to psychiatric comorbidities in epilepsy and SUDEP.Significance Statement Our work provides new insight into a potential novel pathophysiological mechanism contributing to psychiatric illnesses and SUDEP in epilepsy patients, implicating HPA axis dysfunction in negative outcomes associated with epilepsy. This study is the first to link HPA axis dysfunction to SUDEP risk. Chronically epileptic male mice with exaggerated HPA axis dysfunction had increased SUDEP incidence. The translational relevance of these findings is supported by neuroendocrine abnormalities observed in postmortem samples from individuals that died of SUDEP. These data suggest that neuroendocrine mechanisms should be further explored in psychiatric comorbidities in epilepsy and SUDEP risk. Further, neuroendocrine markers may be a biomarker for SUDEP risk.

Neurosteroid influence on affective tone.

Affective disorders such as depression and anxiety are among the most prevalent psychiatric illnesses and causes of disability worldwide. The recent FDA-approval of a novel antidepressant treatment, ZULRESSO® (Brexanolone), a synthetic neurosteroid has fueled interest into the role of neurosteroids in the pathophysiology of depression as well as the mechanisms mediating the antidepressant effects of these compounds. The majority of studies examining the impact of neurosteroids on affective states have relied on the administration of exogenous neurosteroids; however, neurosteroids can also be synthesized endogenously from cholesterol or steroid hormone precursors. Despite the well-established influence of exogenous neurosteroids on affective states, we still lack an understanding of the role of endogenous neurosteroids in modulating affective tone. This review aims to summarize the current literature supporting the influence of neurosteroids on affective states in clinical and preclinical studies, as well as recent evidence suggesting that endogenous neurosteroids may set a baseline affective tone.

Influence of chronic stress on network states governing valence processing: Potential relevance to the risk for psychiatric illnesses.

Stress is a major risk factor for psychiatric illnesses and understanding the mechanisms through which stress disrupts behavioral states is imperative to understanding the underlying pathophysiology of mood disorders. Both chronic stress and early life stress alter valence processing, the process of assigning value to sensory inputs and experiences (positive or negative), which determines subsequent behavior and is essential for emotional processing and ultimately survival. Stress disrupts valence processing in both humans and preclinical models, favoring negative valence processing and impairing positive valence processing. Valence assignment involves neural computations performed in emotional processing hubs, including the amygdala, prefrontal cortex, and ventral hippocampus, which can be influenced by neuroendocrine mediators. Oscillations within and between these regions are critical for the neural computations necessary to perform valence processing functions. Major advances in the field have demonstrated a role for oscillatory states in valence processing under physiological conditions and emerging studies are exploring how these network states are altered under pathophysiological conditions and impacted by neuroendocrine factors. The current review highlights what is currently known regarding the impact of stress and the role of neuroendocrine mediators on network states and valence processing. Further, we propose a model in which chronic stress alters information routing through emotional processing hubs, resulting in a facilitation of negative valence processing and a suppression of positive valence processing.

Early life stress impairs VTA coordination of BLA network and behavioral states.

Motivated behaviors, such as social interactions, are governed by the interplay between mesocorticolimbic structures, such as the ventral tegmental area (VTA), basolateral amygdala (BLA), and medial prefrontal cortex (mPFC). Adverse childhood experiences and early life stress (ELS) can impact these networks and behaviors, which is associated with increased risk for psychiatric illnesses. While it is known that the VTA projects to both the BLA and mPFC, the influence of these inputs on local network activity which govern behavioral states - and whether ELS impacts VTA-mediated network communication - remains unknown. Our study demonstrates that VTA inputs influence BLA oscillations and mPFC activity, and that ELS weakens the ability of the VTA to coordinate BLA network states, likely due to ELS-induced impairments in dopamine signaling between the VTA and BLA. Consequently, ELS mice exhibit increased social avoidance, which can be recapitulated in control mice by inhibiting VTA-BLA communication. These data suggest that ELS impacts social reward via the VTA-BLA dopamine network.

Loss of PV interneurons in the BLA contributes to altered network and behavioral states in chronically epileptic mice.

Psychiatric disorders, including anxiety and depression, are highly comorbid in people with epilepsy. However, the mechanisms mediating the shared pathophysiology are currently unknown. There is considerable evidence implicating the basolateral amygdala (BLA) in the network communication of anxiety and fear, a process demonstrated to involve parvalbumin-positive (PV) interneurons. The loss of PV interneurons has been well described in the hippocampus of chronically epileptic mice and in postmortem human tissue of patients with temporal lobe epilepsy (TLE). We hypothesize that a loss of PV interneurons in the BLA may contribute to comorbid mood disorders in epilepsy. To test this hypothesis, we employed a ventral intrahippocampal kainic acid (vIHKA) model of chronic epilepsy in mice, which exhibits profound behavioral deficits associated with chronic epilepsy. We demonstrate a loss of PV interneurons and dysfunction of remaining PV interneurons in the BLA of chronically epileptic mice. Further, we demonstrate altered principal neuron function and impaired coordination of BLA network and behavioral states in chronically epileptic mice. To determine whether these altered network and behavioral states were due to the loss of PV interneurons, we ablated a similar percentage of PV interneurons observed in chronically epileptic mice by stereotaxically injecting AAV-Flex-DTA into the BLA of PV-Cre mice. Loss of PV interneurons in the BLA is sufficient to alter behavioral states, inducing deficits in fear learning and recall of fear memories. These data suggest that compromised inhibition in the BLA in chronically epileptic mice contributes to behavioral deficits, suggesting a novel mechanism contributing to comorbid anxiety and epilepsy. Significance Statement: Psychiatric illnesses and epilepsy are highly comorbid and negatively impact the quality of life of people with epilepsy. The pathophysiological mechanisms mediating the bidirectional relationship between mood disorders and epilepsy remain unknown and, therefore, treatment options remain inadequate. Here we demonstrate a novel mechanism, involving the loss of PV interneurons in the BLA, leading to a corruption of network and behavioral states in mice. These findings pinpoint a critical node and demonstrate a novel cellular and circuit mechanism involved in the comorbidity of psychiatric illnesses and epilepsy.

Experience-dependent information routing through the basolateral amygdala.

The basolateral amygdala (BLA) is an emotional processing hub and is well-established to influence both positive and negative valence processing. Selective engagement of a heterogeneous cell population in the BLA is thought to contribute to this flexibility in valence processing. However, how this process is impacted by previous experiences which influence valence processing is unknown. Here we demonstrate that previous positive (EE) or negative (chronic unpredictable stress) experiences differentially influence the activity of specific populations of BLA principal neurons projecting to either the nucleus accumbens core or bed nucleus of the stria terminalis. Using chemogenetic manipulation of these projection-specific neurons we can mimic or occlude the effects of chronic unpredictable stress or enriched environment on valence processing to bidirectionally control avoidance behaviors and stress-induced helplessness. These data demonstrate that previous experiences influence the responsiveness of projection-specific BLA principal neurons, biasing information routing through the BLA, to govern valence processing.

Impaired Endogenous Neurosteroid Signaling Contributes to Behavioral Deficits Associated With Chronic Stress.

BACKGROUND: Chronic stress is a major risk factor for psychiatric illnesses, including depression. However, the pathophysiological mechanisms whereby stress leads to mood disorders remain unclear. Allopregnanolone acts as a positive allosteric modulator preferentially on δ subunit-containing GABAA (gamma-aminobutyric acid A) receptors. Accumulating clinical and preclinical evidence supports the antidepressant effects of exogenous administration of allopregnanolone analogs; yet, the role of endogenous allopregnanolone in the pathophysiology of depression remains unknown. METHODS: We utilized a chronic unpredictable stress (CUS) mouse model, followed by behavioral and biochemical assays, to examine whether altered neurosteroid signaling contributes to behavioral outcomes following CUS. We subsequently performed in vivo CRISPR (clustered regularly interspaced short palindromic repeats) knockdown of rate-limiting enzymes involved in allopregnanolone synthesis, 5α-reductase type 1 and 2 (5α1/2), in addition to lentiviral overexpression of 5α1/2 in the basolateral amygdala (BLA) of mice that underwent CUS to assess the impact of 5α1/2 on behavioral outcomes. RESULTS: The expression of δ subunit-containing GABAA receptors and endogenous levels of allopregnanolone were reduced in the BLA following CUS. Treatment with an exogenous allopregnanolone analog, SGE-516, was sufficient to increase allopregnanolone levels in the BLA following CUS. Knockdown of 5α1/2 in the BLA mimicked the behavioral outcomes associated with CUS. Conversely, overexpression of 5α1/2 in the BLA improved behavioral outcomes following CUS. CONCLUSIONS: Our findings demonstrate that chronic stress impairs endogenous neurosteroid signaling in the BLA, which is sufficient to induce behavioral deficits. Further, these studies suggest that allopregnanolone-based treatments may directly target the underlying pathophysiology of mood disorders suggesting that targeting endogenous neurosteroidogenesis may offer a novel therapeutic strategy.

Sex Differences in the Alcohol-Mediated Modulation of BLA Network States.

Alcohol use, reported by 85% of adults in the United States, is highly comorbid with mood disorders, like generalized anxiety disorder and major depression. The basolateral amygdala (BLA) is an area of the brain that is heavily implicated in both mood disorders and alcohol use disorder. Importantly, the modulation of BLA network/oscillatory states via parvalbumin (PV)-positive GABAergic interneurons has been shown to control the behavioral expression of fear and anxiety. Further, PV interneurons express a high density of δ subunit-containing GABAA receptors (GABAARs), which are sensitive to low concentrations of alcohol. Therefore, we hypothesized that the effects of alcohol may modulate BLA network states that have been associated with fear and anxiety behaviors via δ-GABAARs on PV interneurons in the BLA. Given the impact of ovarian hormones on the expression of δ-GABAARs, we also examined the ability of alcohol to modulate local field potentials in the BLA from male and female C57BL/6J and Gabrd-/- mice after acute and repeated exposure to alcohol. Here, we demonstrate that acute and repeated alcohol can differentially modulate oscillatory states in male and female C57BL/6J mice, a process that involves δ-GABAARs. This is the first study to demonstrate that alcohol is capable of altering network states implicated in both anxiety and alcohol use disorders.

Gq neuromodulation of BLA parvalbumin interneurons induces burst firing and mediates fear-associated network and behavioral state transition in mice.

Patterned coordination of network activity in the basolateral amygdala (BLA) is important for fear expression. Neuromodulatory systems play an essential role in regulating changes between behavioral states, however the mechanisms underlying this neuromodulatory control of transitions between brain and behavioral states remain largely unknown. We show that chemogenetic Gq activation and α1 adrenoreceptor activation in mouse BLA parvalbumin (PV) interneurons induces a previously undescribed, stereotyped phasic bursting in PV neurons and time-locked synchronized bursts of inhibitory postsynaptic currents and phasic firing in BLA principal neurons. This Gq-coupled receptor activation in PV neurons suppresses gamma oscillations in vivo and in an ex vivo slice model, and facilitates fear memory recall, which is consistent with BLA gamma suppression during conditioned fear expression. Thus, here we identify a neuromodulatory mechanism in PV inhibitory interneurons of the BLA which regulates BLA network oscillations and fear memory recall.

Allopregnanolone Mediates Affective Switching Through Modulation of Oscillatory States in the Basolateral Amygdala.

BACKGROUND: Brexanolone (allopregnanolone) was recently approved by the Food and Drug Administration for the treatment of postpartum depression, demonstrating long-lasting antidepressant effects. Despite our understanding of the mechanism of action of neurosteroids as positive allosteric modulators of GABAA (gamma-aminobutyric acid A) receptors, we still do not fully understand how allopregnanolone exerts persistent antidepressant effects. METHODS: We used electroencephalogram recordings in rats and humans along with local field potential, functional magnetic resonance imaging, and behavioral tests in mice to assess the impact of neurosteroids on network states in brain regions implicated in mood and used optogenetic manipulations to directly examine their relationship to behavioral states. RESULTS: We demonstrated that allopregnanolone and synthetic neuroactive steroid analogs with molecular pharmacology similar to allopregnanolone (SGE-516 [tool compound] and zuranolone [SAGE-217, investigational compound]) modulate oscillations across species. We further demonstrated a critical role for interneurons in generating oscillations in the basolateral amygdala (BLA) and a role for δ-containing GABAA receptors in mediating the ability of neurosteroids to modulate network and behavioral states. Allopregnanolone in the BLA enhances BLA high theta oscillations (6-12 Hz) through δ-containing GABAA receptors, a mechanism distinct from other GABAA positive allosteric modulators, such as benzodiazepines, and alters behavioral states. Treatment with the allopregnanolone analog SGE-516 protects mice from chronic stress-induced disruption of network and behavioral states, which is correlated with the modulation of theta oscillations in the BLA. Optogenetic manipulation of the network state influences the behavioral state after chronic unpredictable stress. CONCLUSIONS: Our findings demonstrate a novel molecular and cellular mechanism mediating the well-established anxiolytic and antidepressant effects of neuroactive steroids.

Miro1-dependent mitochondrial dynamics in parvalbumin interneurons.

The spatiotemporal distribution of mitochondria is crucial for precise ATP provision and calcium buffering required to support neuronal signaling. Fast-spiking GABAergic interneurons expressing parvalbumin (PV+) have a high mitochondrial content reflecting their large energy utilization. The importance for correct trafficking and precise mitochondrial positioning remains poorly elucidated in inhibitory neurons. Miro1 is a Ca²+-sensing adaptor protein that links mitochondria to the trafficking apparatus, for their microtubule-dependent transport along axons and dendrites, in order to meet the metabolic and Ca2+-buffering requirements of the cell. Here, we explore the role of Miro1 in PV+ interneurons and how changes in mitochondrial trafficking could alter network activity in the mouse brain. By employing live and fixed imaging, we found that the impairments in Miro1-directed trafficking in PV+ interneurons altered their mitochondrial distribution and axonal arborization, while PV+ interneuron-mediated inhibition remained intact. These changes were accompanied by an increase in the ex vivo hippocampal γ-oscillation (30-80 Hz) frequency and promoted anxiolysis. Our findings show that precise regulation of mitochondrial dynamics in PV+ interneurons is crucial for proper neuronal signaling and network synchronization.

How Deep Learning Solved My Seizure Detection Problems.

[Box: see text].