Developmental age and fatty acid amide hydrolase genetic variation converge to mediate fear regulation in female mice.

Anxiety disorders are more prevalent in females than in males, yet a majority of basic neuroscience studies are performed in males. Furthermore, anxiety disorders peak in prevalence during adolescence, yet little is known about neurodevelopmental trajectories of fear expression, particularly in females. To examine these factors, we fear conditioned juvenile, adolescent, and adult female mice and exposed them to fear extinction and a long-term recall test. For this, we used knock-in mice containing a common human mutation in the gene for fatty acid amide hydrolase (FAAH), the primary catabolic enzyme for the endocannabinoid anandamide (FAAH-IN). This mutation has been shown to impart a low-anxiety phenotype in humans, and in rodents relative to their wild-type littermates. We find an impact of the FAAH polymorphism on developmental changes in fear behavior. Specifically, the FAAH polymorphism appears to induce a state of hypervigilance (increased fear) during adolescence. We also used markerless pose estimation software to classify alternative behaviors outside of freezing. These analyses revealed age differences in vigilance to indicators of threat and in the propensity of mice to explore an aversive environment, though genotypic differences were minimal. These findings address a gap in the literature regarding developmental patterns of fear learning and memory as well as the mechanistic contributions of the endocannabinoid system in females.

Inhibitory regulation of calcium transients in prefrontal dendritic spines is compromised by a nonsense Shank3 mutation.

The SHANK3 gene encodes a postsynaptic scaffold protein in excitatory synapses, and its disruption is implicated in neurodevelopmental disorders such as Phelan-McDermid syndrome, autism spectrum disorder, and schizophrenia. Most studies of SHANK3 in the neocortex and hippocampus have focused on disturbances in pyramidal neurons. However, GABAergic interneurons likewise receive excitatory inputs and presumably would also be a target of constitutive SHANK3 perturbations. In this study, we characterize the prefrontal cortical microcircuit in awake mice using subcellular-resolution two-photon microscopy. We focused on a nonsense R1117X mutation, which leads to truncated SHANK3 and has been linked previously to cortical dysfunction. We find that R1117X mutants have abnormally elevated calcium transients in apical dendritic spines. The synaptic calcium dysregulation is due to a loss of dendritic inhibition via decreased NMDAR currents and reduced firing of dendrite-targeting somatostatin-expressing (SST) GABAergic interneurons. Notably, upregulation of the NMDAR subunit GluN2B in SST interneurons corrects the excessive synaptic calcium signals and ameliorates learning deficits in R1117X mutants. These findings reveal dendrite-targeting interneurons, and more broadly the inhibitory control of dendritic spines, as a key microcircuit mechanism compromised by the SHANK3 dysfunction.

Extinction trial spacing across days differentially impacts fear regulation in adult and adolescent male mice.

Fear regulation changes as a function of age and adolescence is a key developmental period for the continued maturation of fear neural circuitry. A consistent finding in the literature is diminished extinction retention in adolescents. However, these studies often directly compare adolescents to adults using a single protocol and therefore provide little insight into learning parameters that improve adolescent fear regulation. Studies in adults highlight the benefits of spaced learning over massed learning. These findings have been extended to fear regulation, with adult rodents exhibiting improved extinction learning and retention when cues are distributed over days versus a single session. However, similar studies have not been performed in adolescents. Here, we systematically examine the impact of trial spacing across days on fear regulation. Adolescent or adult male mice were exposed to one of three extinction paradigms that presented the same number of trials but differed in the temporal distribution of trials across days (one day, two days, or four days). We found that introducing consolidation events into the protocol improves adult extinction learning and short-term extinction retention but these effects disappear after two weeks. For adolescents, all three protocols were comparably effective in reducing freezing across extinction training and improved retention at both short-term and long-term fear recall time points relative to extinction-naive mice. These findings suggest that extinction protocols that incorporate consolidation events are optimal for adults but additional booster training may be required for enduring efficacy. In contrast, protocols incorporating either massed or spaced presentations show immediate and enduring benefits for adolescents.

Synaptic plasticity via receptor tyrosine kinase/G-protein-coupled receptor crosstalk.

Cellular signaling involves a large repertoire of membrane receptors operating in overlapping spatiotemporal regimes and targeting many common intracellular effectors. However, both the molecular mechanisms and the physiological roles of crosstalk between receptors, especially those from different superfamilies, are poorly understood. We find that the receptor tyrosine kinase (RTK) TrkB and the G-protein-coupled receptor (GPCR) metabotropic glutamate receptor 5 (mGluR5) together mediate hippocampal synaptic plasticity in response to brain-derived neurotrophic factor (BDNF). Activated TrkB enhances constitutive mGluR5 activity to initiate a mode switch that drives BDNF-dependent sustained, oscillatory Ca2+ signaling and enhanced MAP kinase activation. This crosstalk is mediated, in part, by synergy between Gßγ, released by TrkB, and Gαq-GTP, released by mGluR5, to enable physiologically relevant RTK/GPCR crosstalk.

Synaptic plasticity via receptor tyrosine kinase/G protein-coupled receptor crosstalk.

Cellular signaling involves a large repertoire of membrane receptors operating in overlapping spatiotemporal regimes and targeting many common intracellular effectors. However, both the molecular mechanisms and physiological roles of crosstalk between receptors, especially those from different superfamilies, are poorly understood. We find that the receptor tyrosine kinase (RTK), TrkB, and the G protein-coupled receptor (GPCR), metabotropic glutamate receptor 5 (mGluR5), together mediate a novel form of hippocampal synaptic plasticity in response to brain-derived neurotrophic factor (BDNF). Activated TrkB enhances constitutive mGluR5 activity to initiate a mode-switch that drives BDNF-dependent sustained, oscillatory Ca 2+ signaling and enhanced MAP kinase activation. This crosstalk is mediated, in part, by synergy between Gßγ, released by TrkB, and Gα q -GTP, released by mGluR5, to enable a previously unidentified form of physiologically relevant RTK/GPCR crosstalk.

The Added Value of Crosstalk Between Developmental Circuit Neuroscience and Clinical Practice to Inform the Treatment of Adolescent Anxiety.

Significant advances have been made in recent years regarding the developmental trajectories of brain circuits and networks, revealing links between brain structure and function. Emerging evidence highlights the importance of developmental trajectories in determining early psychiatric outcomes. However, efforts to encourage crosstalk between basic developmental neuroscience and clinical practice are limited. Here, we focus on the potential advantage of considering features of neural circuit development when optimizing treatments for adolescent patient populations. Drawing on characteristics of adolescent neurodevelopment, we highlight two examples, safety cues and incentives, that leverage insights from neural circuit development and may have great promise for augmenting existing behavioral treatments for anxiety disorders during adolescence. This commentary seeks to serve as a framework to maximize the translational potential of basic research in developmental populations for strengthening psychiatric treatments. In turn, input from clinical practice including the identification of age-specific clinically relevant phenotypes will continue to guide future basic research in the same neural circuits to better reflect clinical practices. Encouraging reciprocal communication to bridge the gap between basic developmental neuroscience research and clinical implementation is an important step toward advancing both research and practice in this domain.

Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor.

We show that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R)-hydroxynorketamine. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. A better understanding of the specific and quantitative contributions of the sex of human experimenters to study outcomes in rodents may improve replicability between studies and, as we have shown, reveal biological and pharmacological mechanisms.

An Adolescent Sensitive Period for Threat Responding: Impacts of Stress and Sex.

Anxiety and fear-related disorders peak in prevalence during adolescence, a window of rapid behavioral development and neural remodeling. However, understanding of the development of threat responding and the underlying neural circuits remains limited. Preclinical models of threat conditioning and extinction have provided an unparalleled glimpse into the developing brain. In this review we discuss mouse and rat studies on the development of threat response regulation, with a focus on the adolescent period. Evidence of nonlinear patterns of threat responding during adolescence and the continued development of the underlying circuitry is highly indicative of an adolescent sensitive period for threat response regulation. While we highlight literature in support of this unique developmental window, we also emphasize the need for causal studies to clarify the parameters defining such a sensitive period. In doing so, we explore how stress and biological sex affect the development and expression of threat response regulation during adolescence and beyond. Ultimately, a deeper understanding of how these factors interact with and affect developmental trajectories of learning and memory will inform treatment and prevention strategies for pediatric anxiety disorders.

Pre-adolescent stress disrupts adult, but not adolescent, safety learning.

Anxiety disorders are highly prevalent across the lifespan, although diagnoses peak early in adolescence. As a method for inhibiting fear, safety signals have the potential to augment conventional treatments for anxiety. However, the ability to acquire and use safety signals during adolescence remains unclear. Moreover, the impact of stress on safety learning has received surprisingly little attention given that stress is a major factor preceding anxiety onset. In this study, mice were trained in a discriminative conditioning protocol to facilitate safety learning and were tested for fear inhibition using a conditioned safety signal. Next, independent groups of mice were exposed to chronic unpredictable stress (CUS) conditions between postnatal day 22 and 28, followed by tests for anxiety-like phenotypes or fear inhibition using a safety signal, performed either 24 h or five weeks following CUS. Pre-adolescent CUS reduced weight in adolescence and this effect endured into adulthood. CUS also increased specific anxiety-like behaviors in adolescence that were unique from the increase in anxiety observed in adulthood. Despite increased anxiety-like behaviors, adolescents were able to learn about and effectively use safety signals to inhibit fear. In contrast, adults that experienced CUS showed a subtle increase in anxiety but had impaired safety signal learning and usage. Together, these findings indicate that pre-adolescent stress has immediate and enduring effects on anxiety-like behaviors but impairs the capacity for conditioned inhibition only following incubation.

Ketamine disinhibits dendrites and enhances calcium signals in prefrontal dendritic spines.

A subanesthetic dose of ketamine causes acute psychotomimetic symptoms and sustained antidepressant effects. In prefrontal cortex, the prevailing disinhibition hypothesis posits that N-methyl-d-aspartate receptor (NMDAR) antagonists such as ketamine act preferentially on GABAergic neurons. However, cortical interneurons are heterogeneous. In particular, somatostatin-expressing (SST) interneurons selectively inhibit dendrites and regulate synaptic inputs, yet their response to systemic NMDAR antagonism is unknown. Here, we report that ketamine acutely suppresses the activity of SST interneurons in the medial prefrontal cortex of the awake mouse. The deficient dendritic inhibition leads to greater synaptically evoked calcium transients in the apical dendritic spines of pyramidal neurons. By manipulating NMDAR signaling via GluN2B knockdown, we show that ketamine's actions on the dendritic inhibitory mechanism has ramifications for frontal cortex-dependent behaviors and cortico-cortical connectivity. Collectively, these results demonstrate dendritic disinhibition and elevated calcium levels in dendritic spines as important local-circuit alterations driven by the administration of subanesthetic ketamine.

GABA interneurons are the cellular trigger for ketamine's rapid antidepressant actions.

A single subanesthetic dose of ketamine, an NMDA receptor (NMDAR) antagonist, produces rapid and sustained antidepressant actions in depressed patients, addressing a major unmet need for the treatment of mood disorders. Ketamine produces a rapid increase in extracellular glutamate and synaptic formation in the prefrontal cortex, but the initial cellular trigger that initiates this increase and ketamine's behavioral actions has not been identified. To address this question, we used a combination of viral shRNA and conditional mutation to produce cell-specific knockdown or deletion of a key NMDAR subunit, GluN2B, implicated in the actions of ketamine. The results demonstrated that the antidepressant actions of ketamine were blocked by GluN2B-NMDAR knockdown on GABA (Gad1) interneurons, as well as subtypes expressing somatostatin (Sst) or parvalbumin (Pvalb), but not glutamate principle neurons in the medial prefrontal cortex (mPFC). Further analysis of GABA subtypes showed that cell-specific knockdown or deletion of GluN2B in Sst interneurons blocked or occluded the antidepressant actions of ketamine and revealed sex-specific differences that are associated with excitatory postsynaptic currents on mPFC principle neurons. These findings demonstrate that GluN2B-NMDARs on GABA interneurons are the initial cellular trigger for the rapid antidepressant actions of ketamine and show sex-specific adaptive mechanisms to GluN2B modulation.

Role of Neuronal VEGF Signaling in the Prefrontal Cortex in the Rapid Antidepressant Effects of Ketamine.

OBJECTIVE: The N-methyl-d-aspartate receptor antagonist ketamine produces rapid and sustained antidepressant actions even in patients with treatment-resistant depression. Vascular endothelial growth factor (VEGF) has been implicated in the effects of conventional monoamine-based antidepressants, but the role of VEGF in the rapid antidepressant actions of ketamine remains unclear. The authors examined whether neuronal VEGF signaling in the medial prefrontal cortex (mPFC) mediates the rapid antidepressant actions of ketamine. METHODS: The authors used a combination of approaches, including conditional, neuron-specific knockout of VEGF or its receptor, Flk-1; antibody neutralization; viral-mediated knockdown of Flk-1; and pharmacological inhibitors. Further in vitro and in vivo experiments were performed to examine whether neuronal VEGF signaling was required for the neurotrophic and synaptogenic actions of ketamine that underlie its behavioral actions. RESULTS: The behavioral actions of systemic ketamine are blocked by forebrain excitatory neuron-specific deletion of either VEGF or Flk-1 or by intra-mPFC infusion of a VEGF neutralizing antibody. Moreover, intra-mPFC infusions of VEGF are sufficient to produce rapid ketamine-like behavioral actions, and these effects are blocked by neuron-specific Flk-1 deletion. The results also show that local knockdown of Flk-1 in mPFC excitatory neurons in adulthood blocks the behavioral effects of systemic ketamine. Moreover, inhibition of neuronal VEGF signaling blocks the neurotrophic and synaptogenic effects of ketamine. CONCLUSIONS: Together, these findings indicate that neuronal VEGF-Flk-1 signaling in the mPFC plays an essential role in the antidepressant actions of ketamine.

Rapid-Acting Antidepressants: Mechanistic Insights and Future Directions.

PURPOSE OF REVIEW: Ketamine produces rapid (within hours) antidepressant actions, even in patients considered treatment resistant, and even shows promise for suicidal ideation. Here, we review current research on the molecular and cellular mechanisms of ketamine and other novel rapid-acting antidepressants, and briefly explore gender differences in the pathophysiology and treatment of MDD. RECENT FINDINGS: Ketamine, an NMDA receptor antagonist, increases BDNF release and synaptic connectivity, opposing the deficits caused by chronic stress and depression. Efforts are focused on the development of novel rapid agents that produce similar synaptic and rapid antidepressant actions, but without the side effects of ketamine. The impact of gender on the response to ketamine and other rapid-acting antidepressants is in early stages of investigation. SUMMARY: The discovery that ketamine produces rapid therapeutic actions for depression and suicidal ideation represents a major breakthrough and much needed alternative to currently available medications. However, novel fast acting agents with fewer side effects are needed, as well as elucidation of the efficacy of these rapid-acting antidepressants for depression in women.

Predicting Barriers to Treatment for Depression in a U.S. National Sample: A Cross-Sectional, Proof-of-Concept Study.

OBJECTIVE: Even though safe and effective treatments for depression are available, many individuals with a diagnosis of depression do not obtain treatment. This study aimed to develop a tool to identify persons who might not initiate treatment among those who acknowledge a need. METHODS: Data were aggregated from the 2008-2014 U.S. National Survey on Drug Use and Health (N=391,753), including 20,785 adults given a diagnosis of depression by a health care provider in the 12 months before the survey. Machine learning was applied to self-report survey items to develop strategies for identifying individuals who might not get needed treatment. RESULTS: A derivation cohort aggregated between 2008 and 2013 was used to develop a model that identified the 30.6% of individuals with depression who reported needing but not getting treatment. When applied to independent responses from the 2014 cohort, the model identified 72% of those who did not initiate treatment (p<.01), with a balanced accuracy that was also significantly above chance (71%, p<.01). For individuals who did not get treatment, the model predicted 10 (out of 15) reasons that they endorsed as barriers to treatment, with balanced accuracies between 53% and 65% (p<.05 for all). CONCLUSIONS: Considerable work is needed to improve follow-up and retention rates after the critical initial meeting in which a patient is given a diagnosis of depression. Routinely collected information about patients with depression could identify those at risk of not obtaining needed treatment, which may inform the development and implementation of interventions to reduce the prevalence of untreated depression.

Cacna1c in the Prefrontal Cortex Regulates Depression-Related Behaviors via REDD1.

The CACNA1C gene that encodes the L-type Ca2+ channel (LTCC) Cav1.2 subunit has emerged as a candidate risk gene for multiple neuropsychiatric disorders including bipolar disorder, major depressive disorder, and schizophrenia, all marked with depression-related symptoms. Although cacna1c heterozygous (HET) mice have been previously reported to exhibit an antidepressant-like phenotype, the molecular and circuit-level dysfunction remains unknown. Here we report that viral vector-mediated deletion of cacna1c in the adult prefrontal cortex (PFC) of mice recapitulates the antidepressant-like effect observed in cacna1c HET mice using the sucrose preference test (SPT), forced swim test (FST), and tail suspension test (TST). Molecular studies identified lower levels of REDD1, a protein previously linked to depression, in the PFC of HET mice, and viral-mediated REDD1 overexpression in the PFC of these HET mice reversed the antidepressant-like effect in SPT and TST. Examination of downstream REDD1 targets found lower levels of active/phosphorylated Akt (S473) with no change in mTORC1 phosphorylation. Examination of the transcription factor FoxO3a, previously linked to depression-related behavior and shown to be regulated in other systems by Akt, revealed higher nuclear levels in the PFC of cacna1c HET mice that was further increased following REDD1-mediated reversal of the antidepressant-like phenotype. Collectively, these findings suggest that REDD1 in cacna1c HET mice may influence depression-related behavior via regulation of the FoxO3a pathway. Cacna1c HET mice thus serve as a useful mouse model to further study cacna1c-associated molecular signaling and depression-related behaviors relevant to human CACNA1C genetic variants.

Emerging treatment mechanisms for depression: focus on glutamate and synaptic plasticity.

Major depression is a chronic and debilitating illness that effects approximately 1 in 5 people, but currently available treatments are limited by low rates of efficacy, therapeutic time lag, and undesirable side effects. Recent efforts have been directed towards investigating rapid-acting agents that reverse the behavioral and neuronal deficits of chronic stress and depression, notably the glutamate NMDA receptor antagonist ketamine. The cellular mechanisms underlying the rapid antidepressant actions of ketamine and related agents are discussed, as well as novel, selective glutamatergic receptor targets that are safer and have fewer side effects.

GABA interneurons mediate the rapid antidepressant-like effects of scopolamine.

Major depressive disorder (MDD) is a recurring psychiatric illness that causes substantial health and socioeconomic burdens. Clinical reports have revealed that scopolamine, a nonselective muscarinic acetylcholine receptor antagonist, produces rapid antidepressant effects in individuals with MDD. Preclinical models suggest that these rapid antidepressant effects can be recapitulated with blockade of M1-type muscarinic acetylcholine receptors (M1-AChR); however, the cellular mechanisms underlying activity-dependent synaptic and behavioral responses to scopolamine have not been determined. Here, we demonstrate that the antidepressant-like effects of scopolamine are mediated by GABA interneurons in the medial prefrontal cortex (mPFC). Both GABAergic (GAD67+) interneurons and glutamatergic (CaMKII+) interneurons in the mPFC expressed M1-AChR. In mice, viral-mediated knockdown of M1-AChR specifically in GABAergic neurons, but not glutamatergic neurons, in the mPFC attenuated the antidepressant-like effects of scopolamine. Immunohistology and electrophysiology showed that somatostatin (SST) interneurons in the mPFC express M1-AChR at higher levels than parvalbumin interneurons. Moreover, knockdown of M1-AChR in SST interneurons in the mPFC demonstrated that M1-AChR expression in these neurons is required for the rapid antidepressant-like effects of scopolamine. These data indicate that SST interneurons in the mPFC are a promising pharmacological target for developing rapid-acting antidepressant therapies.