Traumatic Brain Injury-Induced Fear Generalization in Mice Involves Hippocampal Memory Trace Dysfunction and Is Alleviated by (R,S)-Ketamine.

BACKGROUND: Traumatic brain injury (TBI) is a debilitating neurological disorder caused by an impact to the head by an outside force. TBI results in persistent cognitive impairments, including fear generalization and the inability to distinguish between aversive and neutral stimuli. The mechanisms underlying fear generalization have not been fully elucidated, and there are no targeted therapeutics to alleviate this symptom of TBI. METHODS: To identify the neural ensembles mediating fear generalization, we utilized ArcCreERT2 × enhanced yellow fluorescent protein (EYFP) mice, which allow for activity-dependent labeling and quantification of memory traces. Mice were administered a sham surgery or the controlled cortical impact model of TBI. Mice were then administered a contextual fear discrimination paradigm and memory traces were quantified in numerous brain regions. In a separate group of mice, we tested if (R,S)-ketamine could decrease fear generalization and alter the corresponding memory traces in TBI mice. RESULTS: TBI mice exhibited increased fear generalization when compared with sham mice. This behavioral phenotype was paralleled by altered memory traces in the dentate gyrus, CA3, and amygdala, but not by alterations in inflammation or sleep. In TBI mice, (R,S)-ketamine facilitated fear discrimination, and this behavioral improvement was reflected in dentate gyrus memory trace activity. CONCLUSIONS: These data show that TBI induces fear generalization by altering fear memory traces and that this deficit can be improved with a single injection of (R,S)-ketamine. This work enhances our understanding of the neural basis of TBI-induced fear generalization and reveals potential therapeutic avenues for alleviating this symptom.

( R,S )-ketamine's rapid-acting antidepressant effects are modulated by NR2B-containing NMDA receptors on adult-born hippocampal neurons.

Standard antidepressant treatments often take weeks to reach efficacy and are ineffective for many patients. ( R,S )-ketamine, an N -methyl-D-aspartate (NMDA) antagonist, has been shown to be a rapid-acting antidepressant and to decrease depressive symptoms within hours of administration. While previous studies have shown the importance of the NR2B subunit of the NMDA receptor (NMDAR) on interneurons in the medial prefrontal cortex (mPFC), no study has investigated the influence of NR2B-expressing adult-born granule cells (abGCs). In this study, we examined whether ( R,S )-ketamine's efficacy depends upon these adult-born hippocampal neurons using a genetic strategy to selectively ablate the NR2B subunit of the NMDAR from Nestin + cells. To validate our findings, we also used several other transgenic lines including one in which NR2B was deleted from an interneuron (Parvalbumin (PV) + ) population. We report that in male mice, NR2B expression on 6-week-old adult-born neurons is necessary for ( R,S )-ketamine's effects on behavioral despair in the forced swim test (FST) and on hyponeophagia in the novelty suppressed feeding (NSF) paradigm, as well on fear behavior following contextual fear conditioning (CFC). In female mice, NR2B expression is necessary for effects on hyponeophagia in the NSF. We also find that ablating neurogenesis increases fear expression in CFC, which is buffered by ( R,S )-ketamine administration. In line with previous studies, these results suggest that 6-week-old adult-born hippocampal neurons expressing NR2B partially modulate ( R,S )-ketamine's rapid-acting effects. Future work targeting these 6-week-old adult-born neurons may prove beneficial for increasing the efficacy of ( R , S )-ketamine's antidepressant actions.

Dissociable control of motivation and reinforcement by distinct ventral striatal dopamine receptors.

Dopamine release in striatal circuits, including the nucleus accumbens (NAc), tracks separable features of reward such as motivation and reinforcement. However, the cellular and circuit mechanisms by which dopamine receptors transform dopamine release into distinct constructs of reward remain unclear. Here, we show that dopamine D3 receptor (D3R) signaling in the NAc drives motivated behavior by regulating local NAc microcircuits. Furthermore, D3Rs co-express with dopamine D1 receptors (D1Rs), which regulate reinforcement, but not motivation. Paralleling dissociable roles in reward function, we report non-overlapping physiological actions of D3R and D1R signaling in NAc neurons. Our results establish a novel cellular framework wherein dopamine signaling within the same NAc cell type is physiologically compartmentalized via actions on distinct dopamine receptors. This structural and functional organization provides neurons in a limbic circuit with the unique ability to orchestrate dissociable aspects of reward-related behaviors that are relevant to the etiology of neuropsychiatric disorders.

Traumatic brain injury-induced fear generalization in mice involves hippocampal memory trace dysfunction and is alleviated by ( R,S )-ketamine.

INTRODUCTION: Traumatic brain injury (TBI) is a debilitating neurological disorder caused by an impact to the head by an outside force. TBI results in persistent cognitive impairments, including fear generalization, the inability to distinguish between aversive and neutral stimuli. The mechanisms underlying fear generalization have not been fully elucidated, and there are no targeted therapeutics to alleviate this symptom of TBI. METHODS: To identify the neural ensembles mediating fear generalization, we utilized the ArcCreER T2 x enhanced yellow fluorescent protein (EYFP) mice, which allow for activity-dependent labeling and quantification of memory traces. Mice were administered a sham surgery or the controlled cortical impact (CCI) model of TBI. Mice were then administered a contextual fear discrimination (CFD) paradigm and memory traces were quantified in numerous brain regions. In a separate group of mice, we tested if ( R,S )-ketamine could decrease fear generalization and alter the corresponding memory traces in TBI mice. RESULTS: TBI mice exhibited increased fear generalization when compared with sham mice. This behavioral phenotype was paralleled by altered memory traces in the DG, CA3, and amygdala, but not by alterations in inflammation or sleep. In TBI mice, ( R,S )-ketamine facilitated fear discrimination and this behavioral improvement was reflected in DG memory trace activity. CONCLUSIONS: These data show that TBI induces fear generalization by altering fear memory traces, and that this deficit can be improved with a single injection of ( R,S )-ketamine. This work enhances our understanding of the neural basis of TBI-induced fear generalization and reveals potential therapeutic avenues for alleviating this symptom.

Sex-specific neurobiological actions of prophylactic (R,S)-ketamine, (2R,6R)-hydroxynorketamine, and (2S,6S)-hydroxynorketamine.

Enhancing stress resilience in at-risk populations could significantly reduce the incidence of stress-related psychiatric disorders. We have previously reported that the administration of (R,S)-ketamine prevents stress-induced depressive-like behavior in male mice, perhaps by altering α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated transmission in hippocampal CA3. However, it is still unknown whether metabolites of (R,S)-ketamine can be prophylactic in both sexes. We administered (R,S)-ketamine or its metabolites (2R,6R)-hydroxynorketamine ((2R,6R)-HNK) and (2S,6S)-hydroxynorketamine ((2S,6S)-HNK) at various doses 1 week before one of a number of stressors in male and female 129S6/SvEv mice. Patch clamp electrophysiology was used to determine the effect of prophylactic drug administration on glutamatergic activity in CA3. To examine the interaction between ovarian hormones and stress resilience, female mice also underwent ovariectomy (OVX) surgery and a hormone replacement protocol prior to drug administration. (2S,6S)-HNK and (2R,6R)-HNK protected against distinct stress-induced behaviors in both sexes, with (2S,6S)-HNK attenuating learned fear in male mice, and (2R,6R)-HNK preventing stress-induced depressive-like behavior in both sexes. (R,S)-ketamine and (2R,6R)-HNK, but not (2S,6S)-HNK, attenuated large-amplitude AMPAR-mediated bursts in hippocampal CA3. All three compounds reduced N-methyl-D-aspartate receptor (NMDAR)-mediated currents 1 week after administration. Furthermore, ovarian-derived hormones were necessary for and sufficient to restore (R,S)-ketamine- and (2R,6R)-HNK-mediated prophylaxis in female mice. Our data provide further evidence that resilience-enhancing prophylactics may alter AMPAR-mediated glutamatergic transmission in CA3. Moreover, we show that prophylactics against stress-induced depressive-like behavior can be developed in a sex-specific manner and demonstrate that ovarian hormones are necessary for the prophylactic efficacy of (R,S)-ketamine and (2R,6R)-HNK in female mice.

Prophylactic (R,S)-ketamine selectively protects against inflammatory stressors.

Individuals with peripheral inflammation are a particularly vulnerable population for developing depression and are also more resistant towards traditional antidepressants. This signals the need for novel drugs that can effectively treat this patient population. Recently, we have demonstrated that (R,S)-ketamine is a prophylactic against a variety of stressors, but have yet to test if it is protective against inflammatory-induced vulnerability to a stressor. Here, male 129S6/SvEv mice were administered saline or (R,S)-ketamine (30 mg/kg) 6 days before an injection of vehicle (VEH) or lipopolysaccharide (LPS) (0.83 or 1.0 mg/kg, serotypes O111:B4 or O127:B8). Twenty-four hours after LPS administration, mice were administered a contextual fear conditioning (CFC) paradigm, followed by a context re-exposure and the forced swim test (FST). In a separate cohort, we tested if (R,S)-ketamine was effective as a prophylactic against polyinosinic-polycytidylic acid (PIC), a viral mimetic. (R,S)-ketamine was effective as a prophylactic for attenuating learned fear in the O111:B4 and O127:B8 strains of LPS. (R,S)-ketamine was also effective as a prophylactic for decreasing stress-induced depressive-like behavior in the O111:B4 and O127:B8 strains of LPS. Both of these effects were limited to administration of 1.0, but not 0.83 mg/kg of the O111:B4 and O127:B8 strains of LPS. (R,S)-ketamine was not effective against either stress phenotype following PIC administration. These data suggest that prophylactic (R,S)-ketamine may protect against selective inflammation-induced stress phenotypes following an inflammatory challenge. Future studies will be necessary to determine if (R,S)-ketamine can be useful in patient populations with peripheral inflammation.

Antidepressant but Not Prophylactic Ketamine Administration Alters Calretinin and Calbindin Expression in the Ventral Hippocampus.

Ketamine has been found to have rapid, long-lasting antidepressant effects in treatment-resistant (TR) patients with major depressive disorder (MDD). Recently, we have also shown that ketamine acts as a prophylactic to protect against the development of stress-induced depressive-like behavior in mice, indicating that a preventative treatment against mental illness using ketamine is possible. While there is significant investigation into ketamine's antidepressant mechanism of action, little is known about ketamine's underlying prophylactic mechanism. More specifically, whether ketamine's prophylactic action is molecularly similar to or divergent from its antidepressant action is entirely unknown. Here, we sought to characterize immunohistochemical signatures of cell populations governing ketamine's antidepressant and prophylactic effects. 129S6/SvEv mice were treated with saline (Sal) or ketamine (K) either before a social defeat (SD) stressor as a prophylactic, or after SD as an antidepressant, then subsequently assessed for depressive-like behavior. Post-fixed brains were processed for doublecortin (DCX), calretinin (CR) and calbindin (CB) expression. The number of DCX+ neurons in the dentate gyrus (DG) of the hippocampus (HPC) was not affected by prophylactic or antidepressant ketamine treatment, while the number of CR+ neurons in the ventral hilus increased with antidepressant ketamine under SD conditions. Moreover, antidepressant, but not prophylactic ketamine administration significantly altered CR and CB expression in the ventral HPC (vHPC). These data show that while antidepressant ketamine treatment mediates some of its effects via adult hippocampal markers, prophylactic ketamine administration does not, at least in 129S6/SvEv mice. These data suggest that long-lasting behavioral effects of prophylactic ketamine are independent of hippocampal DCX, CR and CB expression in stress-susceptible mice.

Ventral CA3 Activation Mediates Prophylactic Ketamine Efficacy Against Stress-Induced Depressive-like Behavior.

BACKGROUND: We previously reported that a single injection of ketamine prior to stress protects against the onset of depressive-like behavior and attenuates learned fear. However, the molecular pathways and brain circuits underlying ketamine-induced stress resilience are still largely unknown. METHODS: Here, we tested whether prophylactic ketamine administration altered neural activity in the prefrontal cortex and/or hippocampus. Mice were injected with saline or ketamine (30 mg/kg) 1 week before social defeat. Following behavioral tests assessing depressive-like behavior, mice were sacrificed and brains were processed to quantify ΔFosB expression. In a second set of experiments, mice were stereotaxically injected with viral vectors into ventral CA3 (vCA3) in order to silence or overexpress ΔFosB prior to prophylactic ketamine administration. In a third set of experiments, ArcCreERT2 mice, a line that allows for the indelible labeling of neural ensembles activated by a single experience, were used to quantify memory traces representing a contextual fear conditioning experience following prophylactic ketamine administration. RESULTS: Prophylactic ketamine administration increased ΔFosB expression in the ventral dentate gyrus and vCA3 of social defeat mice but not of control mice. Transcriptional silencing of ΔFosB activity in vCA3 inhibited prophylactic ketamine efficacy, while overexpression of ΔFosB mimicked and occluded ketamine's prophylactic effects. In ArcCreERT2 mice, ketamine administration altered memory traces representing the contextual fear conditioning experience in vCA3 but not in the ventral dentate gyrus. CONCLUSIONS: Our data indicate that prophylactic ketamine may be protective against a stressor by altering neural activity, specifically the neural ensembles representing an individual stressor in vCA3.

Prophylactic ketamine alters nucleotide and neurotransmitter metabolism in brain and plasma following stress.

Recently, we have shown that ketamine given prior to stress exposure protects against the development of depressive-like behavior in mice. These data suggest that it may be possible to prevent the induction of affective disorders before they develop by administering prophylactic pharmaceuticals, a relatively nascent and unexplored strategy for psychiatry. Here, we performed metabolomics analysis of brain and plasma following prophylactic ketamine treatment in order to identify markers of stress resilience enhancement. We administered prophylactic ketamine in mice to buffer against fear expression. Following behavioral analyses, untargeted metabolomic profiling was performed on both hemispheres of the prefrontal cortex (PFC) and the hippocampus (HPC), and plasma. We found that prophylactic ketamine attenuated learned fear. Eight metabolites were changed in the PFC and HPC upon ketamine treatment. Purine and pyrimidine metabolism were most significantly changed in the HPC, PFC, and, interestingly, plasma of mice two weeks after prophylactic administration. Moreover, most precursors to inhibitory neurotransmitters were increased whereas precursors to excitatory neurotransmitters were decreased. Strikingly, these long-term metabolomic changes were not observed when no stressor was administered. Our results suggest that prophylactic treatment differentially affects purine and pyrimidine metabolism and neurotransmission in brain and plasma following stress, which may underlie the long-lasting resilience to stress induced by a single injection of ketamine. These data may provide novel targets for prophylactic development, and indicate an interaction effect of prophylactic ketamine and stress. To our knowledge, this is the first study that identifies metabolomic alterations and biomarker candidates for prophylactic ketamine efficacy in mice.

Prophylactic Ketamine Attenuates Learned Fear.

Ketamine has been reported to be an efficacious antidepressant for major depressive disorder and posttraumatic stress disorder. Most recently, ketamine has also been shown to be prophylactic against stress-induced depressive-like behavior in mice. It remains unknown, however, when ketamine should be administered relative to a stressor in order to maximize its antidepressant and/or prophylactic effects. Moreover, it is unknown whether ketamine can be prophylactic against subsequent stressors. We systematically administered ketamine at different time points relative to a fear experience, in order to determine when ketamine is most effective at reducing fear expression or preventing fear reactivation. Using a contextual fear conditioning (CFC) paradigm, mice were administered a single dose of saline or ketamine (30 mg/kg) at varying time points before or after CFC. Mice administered prophylactic ketamine 1 week, but not 1 month or 1 h before CFC, exhibited reduced freezing behavior when compared with mice administered saline. In contrast, ketamine administration following CFC or during extinction did not alter subsequent fear expression. However, ketamine administered before reinstatement increased the number of rearing bouts in an open field, possibly suggesting an increase in attentiveness. These data indicate that ketamine can buffer a fear response when given a week before as prophylactic, but not when given immediately before or after a stress-inducing episode. Thus, ketamine may be most useful in the clinic if administered in a prophylactic manner 1 week before a stressor, in order to protect against heightened fear responses to aversive stimuli.