Controllable stress elicits circuit-specific patterns of prefrontal plasticity in males, but not females.

Actual or perceived behavioral control during a traumatic event can promote resilience against future adversity, but the long-term cellular and circuit mechanisms by which this protection is conferred have not been identified. Clinical outcomes following trauma exposure differ in men and women, and, therefore, it is especially important in preclinical research to dissect these processes in both males and females. In male adult rats, an experience with behavioral control over tail shock ("escapable stress", ES) has been shown to block the neurochemical and behavioral outcomes produced by later uncontrollable tail shock ("inescapable stress", IS), a phenomenon termed "behavioral immunization". Here, we determined whether behavioral immunization is present in females. Unlike males, the stress-buffering effects of behavioral control were absent in female rats. We next examined the effects of ES and IS on spine morphology of dorsal raphe nucleus (DRN)-projecting prelimbic (PL) neurons, a circuit critical to the immunizing effects of ES in males. In males, IS elicited broad, non-specific alterations in PL spine size, while ES elicited PL-DRN circuit-specific spine changes. In contrast, females exhibited broad, non-specific spine enlargement after ES but only minor alterations after IS. These data provide evidence for a circuit-specific mechanism of structural plasticity that could underlie sexual divergence in the protective effects of behavioral control.

Immunization with Mycobacterium vaccae induces an anti-inflammatory milieu in the CNS: Attenuation of stress-induced microglial priming, alarmins and anxiety-like behavior.

Exposure to stressors induces anxiety- and depressive-like behaviors, which are mediated, in part, by neuroinflammatory processes. Recent findings demonstrate that treatment with the immunoregulatory and anti-inflammatory bacterium, Mycobacterium vaccae (M. vaccae), attenuates stress-induced exaggeration of peripheral inflammation and stress-induced anxiety-like behavioral responses. However, the effects of M. vaccae on neuroimmune processes have largely been unexplored. In the present study, we examined the effect of M. vaccae NCTC11659 on neuroimmune regulation, stress-induced neuroinflammatory processes and anxiety-like behavior. Adult male rats were immunized 3× with a heat-killed preparation of M. vaccae (0.1 mg, s.c.) or vehicle. M. vaccae induced an anti-inflammatory immunophenotype in hippocampus (increased interleukin (Il)4, Cd200r1, and Mrc1 mRNA expression) and increased IL4 protein 8 d after the last immunization. Central administration of recombinant IL4 recapitulated the effects of M. vaccae on Cd200r1 and Mrc1 mRNA expression. M. vaccae reduced basal levels of genes (Nlrp3 and Nfkbia) involved in microglial priming; thus, we explored the effects of M. vaccae on stress-induced hippocampal microglial priming and HMGB1, which mediates priming. We found that M. vaccae blocked stress-induced decreases in Cd200r1, increases in the alarmin HMGB1, and priming of the microglial response to immune challenge. Furthermore, M. vaccae prevented stress-induced increases in anxiety-like behavior. The present findings suggest that M. vaccae enhances immunomodulation in the CNS and mitigates the neuroinflammatory and behavioral effects of stress, which may underpin its capacity to impart a stress resilient phenotype.

Behavioural and neural sequelae of stressor exposure are not modulated by controllability in females.

The degree of behavioural control that an organism has over a stressor is a potent modulator of the stressor's impact; controllable stressors produce none of the neurochemical and behavioural sequelae that occur if the stressor is uncontrollable. Research demonstrating the importance of control and the neural mechanisms responsible has been conducted almost entirely with male rats. It is unknown if behavioural control is stress blunting in females, and whether or not a similar resilience circuitry is engaged. Female rats were exposed to controllable, yoked uncontrollable or no tailshock. In separate experiments, behavioural (juvenile social exploration, fear and shuttle box escape) and neurochemical (activation of dorsal raphe serotonin and dorsal raphe-projecting prelimbic neurons) outcomes, which are sensitive to the dimension of control in males, were assessed. Despite successful acquisition of the controlling response, behavioural control did not mitigate dorsal raphe serotonergic activation and behavioural outcomes induced by tailshock, as it does in males. Moreover, behavioural control failed to selectively engage prelimbic cells that project to the dorsal raphe as in males. Pharmacological activation of the prelimbic cortex restored the stress-buffering effects of control. Collectively, the data demonstrate stressor controllability phenomena are absent in females and that the protective prelimbic circuitry is present but not engaged. Reduced benefit from coping responses may represent a novel approach for understanding differential sex prevalence in stress-related psychiatric disorders.

Activation of a Habenulo-Raphe Circuit Is Critical for the Behavioral and Neurochemical Consequences of Uncontrollable Stress in the Male Rat.

Exposure to uncontrollable stress [inescapable tailshock (IS)] produces behavioral changes that do not occur if the stressor is controllable [escapable tailshock (ES)] an outcome that is mediated by greater IS-induced dorsal raphe nucleus (DRN) serotonin [5-hydroxytryptamine (5-HT)] activation. It has been proposed that this differential activation occurs because the presence of control leads to top-down inhibition of the DRN from medial prefrontal cortex (mPFC), not because uncontrollability produces greater excitatory input. Although mPFC inhibitory regulation over DRN 5-HT activation has received considerable attention, the relevant excitatory inputs that drive DRN 5-HT during stress have not. The lateral habenula (LHb) provides a major excitatory input to the DRN, but very little is known about the role of the LHb in regulating DRN-dependent behaviors. Here, optogenetic silencing of the LHb during IS blocked the typical anxiety-like behaviors produced by IS in male rats. Moreover, LHb silencing blocked the increase in extracellular basolateral amygdala 5-HT during IS and, surprisingly, during behavioral testing the following day. We also provide evidence that LHb-DRN pathway activation is not sensitive to the dimension of behavioral control. Overall, these experiments highlight a critical role for LHb in driving DRN activation and 5-HT release into downstream circuits that mediate anxiety-like behavioral outcomes of IS and further support the idea that behavioral control does not modulate excitatory inputs to the DRN.

Previous Ketamine Produces an Enduring Blockade of Neurochemical and Behavioral Effects of Uncontrollable Stress.

Recent interest in the antidepressant and anti-stress effects of subanesthetic doses of ketamine, an NMDA receptor antagonist, has identified mechanisms whereby ketamine reverses the effect of stress, but little is known regarding the prophylactic effect ketamine might have on future stressors. Here we investigate the prophylactic effect of ketamine against neurochemical and behavioral changes that follow inescapable, uncontrollable tail shocks (ISs) in Sprague Dawley rats. IS induces increased anxiety, which is dependent on activation of serotonergic (5-HT) dorsal raphe nucleus (DRN) neurons that project to the basolateral amygdala (BLA). Ketamine (10 mg/kg, i.p.) administered 2 h, 1 week, or 2 weeks before IS prevented the increased extracellular levels of 5-HT in the BLA typically produced by IS. In addition, ketamine administered at these time points blocked the decreased juvenile social investigation produced by IS. Microinjection of ketamine into the prelimbic (PL) region of the medial prefrontal cortex duplicated the effects of systemic ketamine, and, conversely, systemic ketamine effects were prevented by pharmacological inhibition of the PL. Although IS does not activate DRN-projecting neurons from the PL, IS did so after ketamine, suggesting that the prophylactic effect of ketamine is a result of altered functioning of this projection. SIGNIFICANCE STATEMENT: The reported data show that systemic ketamine, given up to 2 weeks before a stressor, blunts behavioral and neurochemical effects of the stressor. The study also advances understanding of the mechanisms involved and suggests that ketamine acts at the prelimbic cortex to sensitize neurons that project to and inhibit the DRN.

Control Over Stress Accelerates Extinction of Drug Seeking Via Prefrontal Cortical Activation.

Extinction is a form of inhibitory learning viewed as an essential process in suppressing conditioned responses to drug cues, yet there is little information concerning experiential variables that modulate its formation. Coping factors play an instrumental role in determining how adverse life events impact the transition from casual drug use to addiction. Here we provide evidence in rat that prior exposure to controllable stress accelerates the extinction of cocaine-seeking behavior relative to uncontrollable or no stress exposure. Subsequent experimentation using high-speed optogenetic tools determined if the infralimbic region (IL) of the ventral medial prefrontal cortex mediates the impact of controllable stress on cocaine-seeking behavior. Photoinhibition of pyramidal neurons in the IL during coping behavior did not interfere with subject's ability to control the stressor, but prevented the later control-induced facilitation of extinction. These results provide strong evidence that the degree of behavioral control over adverse events, rather than adverse events per se, potently modulates the extinction of cocaine-seeking behavior, and that controllable stress engages prefrontal circuitry that primes future extinction learning.

A novel variable delay Go/No-Go task to study attention, motivation and working memory in the head-fixed rodent.

In order to parse the causal elements underlying complex behaviors and decision-making processes, appropriate behavioral methods must be developed and used in concurrence with molecular, pharmacological, and electrophysiological approaches. Presented is a protocol for a novel Go/No-Go behavioral paradigm to study the brain attention and motivation/reward circuitry in awake, head-restrained rodents. This experimental setup allows: (1) Pharmacological and viral manipulation of various brain regions via targeted guide cannula; (2) Optogenetic cell-type specific activation and silencing with simultaneous electrophysiological recording and; (3) Repeated electrophysiological single and multiple unit recordings during ongoing behavior. The task consists of three components. The subject first makes an observing response by initiating a trial by lever pressing in response to distinctive Go or No-Go tones.  Then, after a variable delay period, the subject is presented with a challenge period cued by white noise during which they must respond with a lever press for the Go condition or withhold from lever pressing for the duration of the cue in the No-Go condition. After correctly responding during the challenge period (Challenge) and a brief delay, a final reward tone of the same frequency as the initiation tone is presented and sucrose reward delivery is available and contingent upon lever pressing. Here, we provide a novel procedure and validating data set that allows researchers to study and manipulate components of behavior such as attention, motivation, impulsivity, and reward-related working memory during an ongoing operant behavioral task while limiting interference from non task-related behaviors.

High fidelity optogenetic control of individual prefrontal cortical pyramidal neurons in vivo.

Precise spatial and temporal manipulation of neural activity in specific genetically defined cell populations is now possible with the advent of optogenetics. The emerging field of optogenetics consists of a set of naturally-occurring and engineered light-sensitive membrane proteins that are able to activate (e.g. channelrhodopsin-2, ChR2) or silence (e.g. halorhodopsin, NpHR) neural activity. Here we demonstrate the technique and the feasibility of using novel adeno-associated viral (AAV) tools to activate (AAV-CaMKllα-ChR2-eYFP) or silence (AAV-CaMKllα-eNpHR3.0-eYFP) neural activity of rat prefrontal cortical prelimbic (PL) pyramidal neurons  in vivo.  In vivo single unit extracellular recording of ChR2-transduced pyramidal neurons showed that delivery of brief (10 ms) blue (473 nm) light-pulse trains up to 20 Hz via a custom fiber optic-coupled recording electrode (optrode) induced spiking with high fidelity at 20 Hz for the duration of recording (up to two hours in some cases). To silence spontaneously active neurons, we transduced them with the NpHR construct and administered continuous green (532 nm) light to completely inhibit action potential activity for up to 10 seconds with 100% fidelity in most cases. These versatile photosensitive tools, combined with optrode recording methods, provide experimental control over activity of genetically defined neurons and can be used to investigate the functional relationship between neural activity and complex cognitive behavior.