Calcineurin dephosphorylates topoisomerase IIß and regulates the formation of neuronal-activity-induced DNA breaks.

Neuronal activity induces topoisomerase IIß (Top2B) to generate DNA double-strand breaks (DSBs) within the promoters of neuronal early response genes (ERGs) and facilitate their transcription, and yet, the mechanisms that control Top2B-mediated DSB formation are unknown. Here, we report that stimulus-dependent calcium influx through NMDA receptors activates the phosphatase calcineurin to dephosphorylate Top2B at residues S1509 and S1511, which stimulates its DNA cleavage activity and induces it to form DSBs. Exposing mice to a fear conditioning paradigm also triggers Top2B dephosphorylation at S1509 and S1511 in the hippocampus, indicating that calcineurin also regulates Top2B-mediated DSB formation following physiological neuronal activity. Furthermore, calcineurin-Top2B interactions following neuronal activity and sites that incur activity-induced DSBs are preferentially localized at the nuclear periphery in neurons. Together, these results reveal how radial gene positioning and the compartmentalization of activity-dependent signaling govern the position and timing of activity-induced DSBs and regulate gene expression patterns in neurons.

Cocaine engages a non-canonical, dopamine-independent, mechanism that controls neuronal excitability in the nucleus accumbens.

Drug-induced enhanced dopamine (DA) signaling in the brain is a canonical mechanism that initiates addiction processes. However, indirect evidence suggests that cocaine also triggers non-canonical, DA-independent, mechanisms that contribute to behavioral responses to cocaine, including psychomotor sensitization and cocaine self-administration. Identifying these mechanisms and determining how they are initiated is fundamental to further our understanding of addiction processes. Using physiologically relevant in vitro tractable models, we found that cocaine-induced hypoactivity of nucleus accumbens shell (NAcSh) medium spiny neurons (MSNs), one hallmark of cocaine addiction, is independent of DA signaling. Combining brain slice studies and site-directed mutagenesis in HEK293T cells, we found that cocaine binding to intracellular sigma-1 receptor (σ1) initiates this mechanism. Subsequently, σ1 binds to Kv1.2 potassium channels, followed by accumulation of Kv1.2 in the plasma membrane, thereby depressing NAcSh MSNs firing. This mechanism is specific to D1 receptor-expressing MSNs. Our study uncovers a mechanism for cocaine that bypasses DA signaling and leads to addiction-relevant neuroadaptations, thereby providing combinatorial strategies for treating stimulant abuse.

Reduced GluN1 in mouse dentate gyrus is associated with CA3 hyperactivity and psychosis-like behaviors.

Recent findings from in vivo-imaging and human post-mortem tissue studies in schizophrenic psychosis (SzP), have demonstrated functional and molecular changes in hippocampal subfields that can be associated with hippocampal hyperexcitability. In this study, we used a subfield-specific GluN1 knockout mouse with a disease-like molecular perturbation expressed only in hippocampal dentate gyrus (DG) and assessed its association with hippocampal physiology and psychosis-like behaviors. First, we used whole-cell patch-clamp recordings to measure the physiological changes in hippocampal subfields and cFos immunohistochemistry to examine cellular excitability. DG-GluN1 KO mice show CA3 cellular hyperactivity, detected using two approaches: (1) increased excitatory glutamate transmission at mossy fibers (MF)-CA3 synapses, and (2) an increased number of cFos-activated pyramidal neurons in CA3, an outcome that appears to project downstream to CA1 and basolateral amygdala (BLA). Furthermore, we examined psychosis-like behaviors and pathological memory processing; these show an increase in fear conditioning (FC), a reduction in prepulse inhibition (PPI) in the KO animal, along with a deterioration in memory accuracy with Morris Water Maze (MWM) and reduced social memory (SM). Moreover, with DREADD vectors, we demonstrate a remarkably similar behavioral profile when we induce CA3 hyperactivity. These hippocampal subfield changes could provide the basis for the observed increase in human hippocampal activity in SzP, based on the shared DG-specific GluN1 reduction. With further characterization, these animal model systems may serve as targets to test psychosis mechanisms related to hippocampus and assess potential hippocampus-directed treatments.

Cannabinoids prevent the differential long-term effects of exposure to severe stress on hippocampal- and amygdala-dependent memory and plasticity.

Exposure to excessive or uncontrolled stress is a major factor associated with various diseases including posttraumatic stress disorder (PTSD). The consequences of exposure to trauma are affected not only by aspects of the event itself, but also by the frequency and severity of trauma reminders. It was suggested that in PTSD, hippocampal-dependent memory is compromised while amygdala-dependent memory is strengthened. Several lines of evidence support the role of the endocannabinoid (eCB) system as a modulator of the stress response. In this study we aimed to examine cannabinoids modulation of the long-term effects (i.e., 1 month) of exposure to a traumatic event on memory and plasticity in the hippocampus and amygdala. Following exposure to the shock and reminders model of PTSD in an inhibitory avoidance light-dark apparatus rats demonstrated: (i) enhanced fear retrieval and impaired inhibitory extinction (Ext), (ii) no long-term potentiation (LTP) in the CA1, (iii) impaired hippocampal-dependent short-term memory in the object location task, (iv) enhanced LTP in the amygdala, and (v) enhanced amygdala-dependent conditioned taste aversion memory. The cannabinoid CB1/2 receptor agonist WIN55-212,2 (0.5mg/kg, i.p.) and the fatty acid amide hydrolase (FAAH) inhibitor URB597 (0.3mg/kg, i.p.), administered 2 hr after shock exposure prevented these opposing effects on hippocampal- and amygdala-dependent processes. Moreover, the effects of WIN55-212,2 and URB597 on Ext and acoustic startle were prevented by co-administration of a low dose of the CB1 receptor antagonist AM251 (0.5mg/kg, i.p.), suggesting that the preventing effects of both drugs are mediated by CB1 receptors. Exposure to shock and reminders increased CB1 receptor levels in the CA1 and basolateral amygdala 1 month after shock exposure and this increase was also prevented by administering WIN55-212,2 or URB597. Taken together, these findings suggest the involvement of the eCB system, and specifically CB1 receptors, in the opposite effects of severe stress on memory and plasticity in the hippocampus and amygdala.

Cannabinoids and post-traumatic stress disorder: clinical and preclinical evidence for treatment and prevention.

There is substantial evidence from studies in humans and animal models for a role of the endocannabinoid system in the control of emotional states. Several studies have shown an association between exposure to trauma and substance use. Specifically, it has been shown that there is increased prevalence of cannabis use in post-traumatic stress disorder (PTSD) patients and vice versa. Clinical studies suggest that PTSD patients may cope with their symptoms by using cannabis. This treatment-seeking strategy may explain the high prevalence of cannabis use among individuals with PTSD. Preliminary studies in humans also suggest that treatment with cannabinoids may decrease PTSD symptoms including sleep quality, frequency of nightmares, and hyperarousal. However, there are no large-scale, randomized, controlled studies investigating this specifically. Studies in animal models have shown that cannabinoids can prevent the effects of stress on emotional function and memory processes, facilitate fear extinction, and have an anti-anxiety-like effect in a variety of tasks. Moreover, cannabinoids administered shortly after exposure to a traumatic event were found to prevent the development of PTSD-like phenotype. In this article, we review the existing literature on the use of cannabinoids for treating and preventing PTSD in humans and animal models. There is a need for large-scale clinical trials examining the potential decrease in PTSD symptomatology with the use of cannabis. In animal models, there is a need for a better understanding of the mechanism of action and efficacy of cannabis. Nevertheless, the end result of the current clinical and preclinical data is that cannabinoid agents may offer therapeutic benefits for PTSD.

Mapping catalytically engaged TOP2B in neurons reveals the principles of topoisomerase action within the genome.

We trapped catalytically engaged topoisomerase IIß (TOP2B) in covalent DNA cleavage complexes (TOP2Bccs) and mapped their positions genome-wide in cultured mouse cortical neurons. We report that TOP2Bcc distribution varies with both nucleosome and compartmental chromosome organization. While TOP2Bccs in gene bodies correlate with their level of transcription, highly expressed genes that lack the usually associated chromatin marks, such as H3K36me3, show reduced TOP2Bccs, suggesting that histone posttranslational modifications regulate TOP2B activity. Promoters with high RNA polymerase II occupancy show elevated TOP2B chromatin immunoprecipitation sequencing signals but low TOP2Bccs, indicating that TOP2B catalytic engagement is curtailed at active promoters. Surprisingly, either poisoning or inhibiting TOP2B increases nascent transcription at most genes and enhancers but reduces transcription within long genes. These effects are independent of transcript length and instead correlate with the presence of intragenic enhancers. Together, these results clarify how cells modulate the catalytic engagement of topoisomerases to affect transcription.

Atypical Modes of CTCF Binding Facilitate Tissue-Specific and Neuronal Activity-Dependent Gene Expression States.

Multivalent binding of CTCF to variable DNA sequences is thought to underlie its ability to mediate diverse cellular functions. CTCF typically binds a 20 base-pair consensus DNA sequence, but the full diversity of CTCF binding sites (CBS) within the genome has not been interrogated. We assessed CTCF occupancy in cultured cortical neurons and observed surprisingly that ~ 22% of CBS lack the consensus CTCF motif. We report here that sequence diversity at most of these atypical CBS involves degeneracy at specific nucleotide positions within the consensus CTCF motif, which likely affect the binding of CTCF zinc fingers 6 and 7. This mode of atypical CTCF binding defines most CBS at gene promoters, as well as CBS that are dynamically altered during neural differentiation and following neuronal stimulation, revealing how atypical CTCF binding could influence gene activity. Dynamic CBS are distributed both within and outside loop anchors and TAD boundaries, suggesting both looping-dependent and independent roles for CTCF. Finally, we describe a second mode of atypical CTCF binding to DNA sequences that are completely unrelated to the consensus CTCF motif, which are enriched within the bodies of tissue-specific genes. These tissue-specific atypical CBS are also enriched in H3K27ac, which marks cis-regulatory elements within chromatin, including enhancers. Overall, these results indicate how atypical CBS could dynamically regulate gene activity patterns during differentiation, development, and in response to environmental cues.

Differential effects of cannabinoid receptor agonist on social discrimination and contextual fear in amygdala and hippocampus.

We examined whether the cannabinoid receptor agonist WIN55,212-2 (WIN; 5 µg/side) microinjected into the hippocampus or the amygdala would differentially affect memory processes in a neutral vs. an aversive task. In the aversive contextual fear task, WIN into the basolateral amygdala impaired fear acquisition/consolidation, but not retrieval. In the ventral subiculum (vSub), WIN impaired fear retrieval. In the neutral social discrimination task, WIN into the vSub impaired both acquisition/consolidation and retrieval, whereas in the medial amygdala WIN impaired acquisition. The results suggest that cannabinoid signaling differentially affects memory in a task-, region-, and memory stage-dependent manner.

Gut colonization by Proteobacteria alters host metabolism and modulates cocaine neurobehavioral responses.

Gut-microbiota membership is associated with diverse neuropsychological outcomes, including substance use disorders (SUDs). Here, we use mice colonized with Citrobacter rodentium or the human γ-Proteobacteria commensal Escherichia coli HS as a model to examine the mechanistic interactions between gut microbes and host responses to cocaine. We find that cocaine exposure increases intestinal norepinephrine levels that are sensed through the bacterial adrenergic receptor QseC to promote intestinal colonization of γ-Proteobacteria. Colonized mice show enhanced host cocaine-induced behaviors. The neuroactive metabolite glycine, a bacterial nitrogen source, is depleted in the gut and cerebrospinal fluid of colonized mice. Systemic glycine repletion reversed, and γ-Proteobacteria mutated for glycine uptake did not alter the host response to cocaine. γ-Proteobacteria modulated glycine levels are linked to cocaine-induced transcriptional plasticity in the nucleus accumbens through glutamatergic transmission. The mechanism outline here could potentially be exploited to modulate reward-related brain circuits that contribute to SUDs.

D-cycloserine into the BLA reverses the impairing effects of exposure to stress on the extinction of contextual fear, but not conditioned taste aversion.

We investigated whether the N-methyl-D-aspartate (NMDA) receptor partial agonist D-cycloserine (DCS, 20 microg/side) microinfused into the basolateral amygdala (BLA) would reverse stress-induced impairment of extinction in two aversive learning paradigms: contextual fear conditioning and conditioned taste aversion (CTA). We found that DCS in the BLA show differential involvement in the extinction of these two paradigms and in its modulation of stress-induced impairment of extinction. This may suggest that the dysfunctional extinction of fear and taste aversion following exposure to a stressful experience may be modulated by different mechanisms.

Cocaine-Induced Synaptic Redistribution of NMDARs in Striatal Neurons Alters NMDAR-Dependent Signal Transduction.

The consequence of repeated cocaine exposure and prolonged abstinence on glutamate receptor expression in the nucleus accumbens has been extensively studied. However, the early effects of cocaine on NMDAR signaling remain unknown. NMDAR signaling depends on the subunit composition, subcellular localization, and the interaction with proteins at the postsynaptic density (PSD), where NMDARs and other proteins form supercomplexes that are responsible for the signaling pathways activated by NMDAR-induced Ca2+ influx. Here, we investigated the effect of cocaine on NMDAR subunit composition and subcellular localization after both intraperitoneal non-contingent cocaine and response-contingent intravenous cocaine self-administration in mice. We found that repeated cocaine exposure, regardless of the route or contingency of drug administration, decreases NMDAR interactions with the PSD and synaptic lipid rafts in the accumbens shell and dorsal striatum. We provide evidence that cocaine triggers an early redistribution of NMDARs from synaptic to extrasynaptic sites, and that this adaptation has implications in the activation of downstream signaling pathways. Thus, consistent with a loss of NMDAR function, cocaine-induced ERK phosphorylation is attenuated. Because early NMDAR activity contributes to the initiation of lasting addiction-relevant neuroadaptations, these data may hold clues into cellular mechanisms responsible for the development of cocaine addiction.

Role of endocannabinoids in the hippocampus and amygdala in emotional memory and plasticity.

Posttraumatic stress disorder (PTSD) is characterized by the reexperiencing of a traumatic event and is associated with slower extinction of fear responses. Impaired extinction of fearful associations to trauma-related cues may interfere with treatment response, and extinction deficits may be premorbid risk factors for the development of PTSD. We examined the effects of exposure to a severe footshock followed by situational reminders (SRs) on extinction, plasticity, and endocannabinoid (eCB) content and activity in the hippocampal CA1 area and basolateral amygdala (BLA). We also examined whether enhancing eCB signaling before extinction, using the fatty acid amide hydrolase (FAAH) inhibitor URB597, could prevent the shock/SRs-induced effects on fear response and plasticity. URB597 administered systemically (0.3 mg/kg) or locally into the CA1 or BLA (0.1 µg/side) prior to extinction decreased fear retrieval and this effect persisted throughout extinction training and did not recuperate during spontaneous recovery. A low dose of the CB1 receptor antagonist AM251 (0.3 mg/kg i.p. or 0.01 µg/0.5 µl intra-CA1 or intra-BLA) blocked these effects suggesting that the effects of URB597 were CB1 receptor-dependent. Exposure to shock and reminders induced behavioral metaplasticity with opposite effects on long-term potentiation (LTP) in the hippocampus (impairment) and the BLA (enhancement). URB597 was found to prevent the opposite shock/SR-induced metaplasticity in hippocampal and BLA-LTP. Exposure to shock and reminders might cause variation in endogenous cannabinoid levels that could affect fear-circuit function. Indeed, exposure to shock and SRs affected eCB content: increased 2-arachidonoyl-glycerol (2-AG) and N-arachidonylethanolamine (AEA) levels in the CA1, decreased serum and BLA AEA levels while shock exposure increased FAAH activity in the CA1 and BLA. FAAH inhibition before extinction abolished fear and modulated LTP in the hippocampus and amygdala, brain regions pertinent to emotional memory. The findings suggest that targeting the eCB system before extinction may be beneficial in fear memory attenuation and these effects may involve metaplasticity in the CA1 and BLA.

Publisher Correction: Stimulation of entorhinal cortex-dentate gyrus circuitry is antidepressive.

In the version of this article originally published, a URL provided in the Methods section was incorrect. The URL had a solidus at the end but should have appeared as http://www.nature.com/authors/policies/image.html. The error has been corrected in the PDF and HTML versions of this article.

Stimulation of entorhinal cortex-dentate gyrus circuitry is antidepressive.

Major depressive disorder (MDD) is considered a 'circuitopathy', and brain stimulation therapies hold promise for ameliorating MDD symptoms, including hippocampal dysfunction. It is unknown whether stimulation of upstream hippocampal circuitry, such as the entorhinal cortex (Ent), is antidepressive, although Ent stimulation improves learning and memory in mice and humans. Here we show that molecular targeting (Ent-specific knockdown of a psychosocial stress-induced protein) and chemogenetic stimulation of Ent neurons induce antidepressive-like effects in mice. Mechanistically, we show that Ent-stimulation-induced antidepressive-like behavior relies on the generation of new hippocampal neurons. Thus, controlled stimulation of Ent hippocampal afferents is antidepressive via increased hippocampal neurogenesis. These findings emphasize the power and potential of Ent glutamatergic afferent stimulation-previously well-known for its ability to influence learning and memory-for MDD treatment.

Cannabinoids and Glucocorticoids in the Basolateral Amygdala Modulate Hippocampal-Accumbens Plasticity After Stress.

Acute stress results in release of glucocorticoids, which are potent modulators of learning and plasticity. This process is presumably mediated by the basolateral amygdala (BLA) where cannabinoids CB1 receptors have a key role in regulating the hypothalamic-pituitary-adrenal (HPA) axis. Growing attention has been focused on nucleus accumbens (NAc) plasticity, which regulates mood and motivation. The NAc integrates affective and context-dependent input from the BLA and ventral subiculum (vSub), respectively. As our previous data suggest that the CB1/2 receptor agonist WIN55,212-2 (WIN) and glucocorticoid receptor (GR) antagonist RU-38486 (RU) can prevent the effects of stress on emotional memory, we examined whether intra-BLA WIN and RU can reverse the effects of acute stress on NAc plasticity. Bilateral, ipsilateral, and contralateral BLA administration of RU or WIN reversed the stress-induced impairment in vSub-NAc long-term potentiation (LTP) and the decrease in cAMP response element-binding protein (CREB) activity in the NAc. BLA CB1 receptors were found to mediate the preventing effects of WIN on plasticity, but not the preventing effects of RU, after stress. Inactivating the ipsilateral BLA, but not the contralateral BLA, impaired LTP. The possible mechanisms underlying the effects of BLA on NAc plasticity are discussed; the data suggest that BLA-induced changes in the NAc may be mediated through neural pathways in the brain's stress circuit rather than peripheral pathways. The results suggest that glucocorticoid and cannabinoid systems in the BLA can restore normal function of the NAc and hence may have a central role in the treatment of a variety of stress-related disorders.

Whole-cell Patch-clamp Recordings in Brain Slices.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices, a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular, when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

Cannabinoid receptor activation prevents the effects of chronic mild stress on emotional learning and LTP in a rat model of depression.

Most psychiatric disorders are characterized by emotional memory or learning disturbances. Chronic mild stress (CMS) is a common animal model for stress-induced depression. Here we examined whether 3 days of treatment using the CB1/2 receptor agonist WIN55,212-2 could ameliorate the effects of CMS on emotional learning (ie, conditioned avoidance and extinction), long-term potentiation (LTP) in the hippocampal-accumbens pathway, and depression-like symptoms (ie, coping with stress behavior, anhedonia, and weight changes). We also examined whether the ameliorating effects of WIN55,212-2 on behavior and physiology after CMS are mediated by CB1 and glucocorticoid receptors (GRs). Rats were exposed to CMS or handled on days 1-21. The agonist WIN55,212-2 or vehicle were administered on days 19-21 (IP; 0.5 mg/kg) and behavioral and electrophysiological measures were taken on days 23 and 28. The CB1 receptor antagonist AM251 (IP; 0.3 mg/kg) or the GR antagonist RU-38486 (IP; 10 mg/kg) were co-administered with WIN55,212-2. Our results show that CMS significantly modified physiological and behavioral reactions, as observed by the impairment in avoidance extinction and LTP in the hippocampal-accumbens pathway, and the alterations in depression-like symptoms, such as coping with stress behavior, weight gain, and sucrose consumption. The most significant effect observed in this study was that 3 days of WIN55,212-2 administration prevented the CMS-induced alterations in emotional memory (ie, extinction) and plasticity. This effect was mediated by CB1 receptors as the CB1 receptor antagonist AM251 prevented the ameliorating effects of WIN55,212-2 on extinction and LTP. The GR antagonist RU-38486 also prevented the CMS-induced alterations in extinction and plasticity, and when co-administered with WIN55,212-2, the preventive effects after CMS were maintained. The findings suggest that enhancing cannabinoid signaling could represent a novel approach to the treatment of cognitive deficits that accompany stress-related depression.

Stress hormones receptors in the amygdala mediate the effects of stress on the consolidation, but not the retrieval, of a non aversive spatial task.

This study examined the effects of the arousal level of the rat and exposure to a behavioral stressor on acquisition, consolidation and retrieval of a non-aversive hippocampal-dependent learning paradigm, the object location task. Learning was tested under two arousal conditions: no previous habituation to the experimental context (high novelty stress/arousal level) or extensive prior habituation (reduced novelty stress/arousal level). Results indicated that in the habituated rats, exposure to an out-of-context stressor (i.e, elevated platform stress) impaired consolidation and retrieval, but not acquisition, of the task. Non-habituated animals under both stressed and control conditions did not show retention of the task. In habituated rats, RU-486 (10 ng/side), a glucocorticoid receptor (GR) antagonist, or propranolol (0.75 µg/side), a beta-adrenergic antagonist, injected into the basolateral amygdala (BLA), prevented the impairing effects of the stressor on consolidation, but not on retrieval. The CB1/CB2 receptor agonist WIN55,212-2 (WIN, 5 µg/side) microinjected into the BLA did not prevent the effects of stress on either consolidation or retrieval. Taken together the results suggest that: (i) GR and ß-adrenergic receptors in the BLA mediate the impairing effects of stress on the consolidation, but not the retrieval, of a neutral, non-aversive hippocampal-dependent task, (ii) the impairing effects of stress on hippocampal consolidation and retrieval are mediated by different neural mechanisms (i.e., different neurotransmitters or different brain areas), and (iii) the effects of stress on memory depend on the interaction between several main factors such as the stage of memory processing under investigation, the animal's level of arousal and the nature of the task (neutral or aversive).