Withdrawal from chronic alcohol impairs the serotonin-mediated modulation of GABAergic transmission in the infralimbic cortex in male rats.

The infralimbic cortex (IL) is part of the medial prefrontal cortex (mPFC), exerting top-down control over structures that are critically involved in the development of alcohol use disorder (AUD). Activity of the IL is tightly controlled by γ-aminobutyric acid (GABA) transmission, which is susceptible to chronic alcohol exposure and withdrawal. This inhibitory control is regulated by various neuromodulators, including 5-hydroxytryptamine (5-HT; serotonin). We used chronic intermittent ethanol vapor inhalation exposure, a model of AUD, in male Sprague-Dawley rats to induce alcohol dependence (Dep) followed by protracted withdrawal (WD; 2 weeks) and performed ex vivo electrophysiology using whole-cell patch clamp to study GABAergic transmission in layer V of IL pyramidal neurons. We found that WD increased frequencies of spontaneous inhibitory postsynaptic currents (sIPSCs), whereas miniature IPSCs (mIPSCs; recorded in the presence of tetrodotoxin) were unaffected by either Dep or WD. The application of 5-HT (50 µM) increased sIPSC frequencies and amplitudes in naive and Dep rats but reduced sIPSC frequencies in WD rats. Additionally, 5-HT2A receptor antagonist M100907 and 5-HT2C receptor antagonist SB242084 reduced basal GABA release in all groups to a similar extent. The blockage of either 5-HT2A or 5-HT2C receptors in WD rats restored the impaired response to 5-HT, which then resembled responses in naive rats. Our findings expand our understanding of synaptic inhibition in the IL in AUD, indicating that antagonism of 5-HT2A and 5-HT2C receptors may restore GABAergic control over IL pyramidal neurons. SIGNIFICANCE STATEMENT: Impairment in the serotonergic modulation of GABAergic inhibition in the medial prefrontal cortex contributes to alcohol use disorder (AUD). We used a well-established rat model of AUD and ex vivo whole-cell patch-clamp electrophysiology to characterize the serotonin modulation of GABAergic transmission in layer V infralimbic (IL) pyramidal neurons in ethanol-naive, ethanol-dependent (Dep), and ethanol-withdrawn (WD) male rats. We found increased basal inhibition following WD from chronic alcohol and altered serotonin modulation. Exogenous serotonin enhanced GABAergic transmission in naive and Dep rats but reduced it in WD rats. 5-HT2A and 5-HT2C receptor blockage in WD rats restored the typical serotonin-mediated enhancement of GABAergic inhibition. Our findings expand our understanding of synaptic inhibition in the infralimbic neurons in AUD.

Distinct engrams control fear and extinction memory.

Memories are stored in engram cells, which are necessary and sufficient for memory recall. Recalling a memory might undergo reconsolidation or extinction. It has been suggested that the original memory engram is reactivated during reconsolidation so that memory can be updated. Conversely, during extinction training, a new memory is formed that suppresses the original engram. Nonetheless, it is unknown whether extinction creates a new engram or modifies the original fear engram. In this study, we utilized the Daun02 procedure, which uses c-Fos-lacZ rats to induce apoptosis of strongly activated neurons and examine whether a new memory trace emerges as a result of a short or long reactivation, or if these processes rely on modifications within the original engram located in the basolateral amygdala (BLA) and infralimbic (IL) cortex. By eliminating neurons activated during consolidation and reactivation, we observed significant impacts on fear memory, highlighting the importance of the BLA engram in these processes. Although we were unable to show any impact when removing the neurons activated after the test of a previously extinguished memory in the BLA, disrupting the IL extinction engram reactivated the aversive memory that was suppressed by the extinction memory. Thus, we demonstrated that the IL cortex plays a crucial role in the network involved in extinction, and disrupting this specific node alone is sufficient to impair extinction behavior. Additionally, our findings indicate that extinction memories rely on the formation of a new memory, supporting the theory that extinction memories rely on the formation of a new memory, whereas the reconsolidation process reactivates the same original memory trace.

Infralimbic cortex plays a similar role in the punishment and extinction of instrumental behavior.

Learning to stop responding is a fundamental process in instrumental learning. Animals may learn to stop responding under a variety of conditions that include punishment-where the response earns an aversive stimulus in addition to a reinforcer-and extinction-where a reinforced response now earns nothing at all. Recent research suggests that punishment and extinction may be related manifestations of a common retroactive interference process. In both paradigms, animals learn to stop performing a specific response in a specific context, suggesting direct inhibition of the response by the context. This process may depend on the infralimbic cortex (IL), which has been implicated in a variety of interference-based learning paradigms including extinction and habit learning. Despite the behavioral parallels between extinction and punishment, a corresponding role for IL in punishment has not been identified. Here we report that, in a simple arrangement where either punishment or extinction was conducted in a context that differed from the context in which the behavior was first acquired, IL inactivation reduced response suppression in the inhibitory context, but not responding when it "renewed" in the original context. In a more complex arrangement in which two responses were first trained in different contexts and then extinguished or punished in the opposite one, IL inactivation had no effect. The results advance our understanding of the effects of IL in retroactive interference and the behavioral mechanisms that can produce suppression of a response.

Effects of social dominance and acute social stress on morphology of microglia and structural integrity of the medial prefrontal cortex.

Chronic stress increases activity of the brain's innate immune system and impairs function of the medial prefrontal cortex (mPFC). However, whether acute stress triggers similar neuroimmune mechanisms is poorly understood. Across four studies, we used a Syrian hamster model to investigate whether acute stress drives changes in mPFC microglia in a time-, subregion-, and social status-dependent manner. We found that acute social defeat increased expression of ionized calcium binding adapter molecule 1 (Iba1) in the infralimbic (IL) and prelimbic (PL) and altered the morphology Iba1+ cells 1, 2, and 7 days after social defeat. We also investigated whether acute defeat induced tissue degeneration and reductions of synaptic plasticity 2 days post-defeat. We found that while social defeat increased deposition of cellular debris and reduced synaptophysin immunoreactivity in the PL and IL, treatment with minocycline protected against these cellular changes. Finally, we tested whether a reduced conditioned defeat response in dominant compared to subordinate hamsters was associated with changes in microglia reactivity in the IL and PL. We found that while subordinate hamsters and those without an established dominance relationships showed defeat-induced changes in morphology of Iba1+ cells and cellular degeneration, dominant hamsters showed resistance to these effects of social defeat. Taken together, these findings indicate that acute social defeat alters microglial morphology, increases markers of tissue degradation, and impairs structural integrity in the IL and PL, and that experience winning competitive interactions can specifically protect the IL and reduce stress vulnerability.

Sex bias in social deficits, neural circuits and nutrient demand in Cttnbp2 autism models.

Autism spectrum disorders caused by both genetic and environmental factors are strongly male-biased neuropsychiatric conditions. However, the mechanism underlying the sex bias of autism spectrum disorders remains elusive. Here, we use a mouse model in which the autism-linked gene Cttnbp2 is mutated to explore the potential mechanism underlying the autism sex bias. Autism-like features of Cttnbp2 mutant mice were assessed via behavioural assays. C-FOS staining identified sex-biased brain regions critical to social interaction, with their roles and connectivity then validated by chemogenetic manipulation. Proteomic and bioinformatic analyses established sex-biased molecular deficits at synapses, prompting our hypothesis that male-biased nutrient demand magnifies Cttnbp2 deficiency. Accordingly, intakes of branched-chain amino acids (BCAA) and zinc were experimentally altered to assess their effect on autism-like behaviours. Both deletion and autism-linked mutation of Cttnbp2 result in male-biased social deficits. Seven brain regions, including the infralimbic area of the medial prefrontal cortex (ILA), exhibit reduced neural activity in male mutant mice but not in females upon social stimulation. ILA activation by chemogenetic manipulation is sufficient to activate four of those brain regions susceptible to Cttnbp2 deficiency and consequently to ameliorate social deficits in male mice, implying an ILA-regulated neural circuit is critical to male-biased social deficits. Proteomics analysis reveals male-specific downregulated proteins (including SHANK2 and PSD-95, two synaptic zinc-binding proteins) and female-specific upregulated proteins (including RRAGC) linked to neuropsychiatric disorders, which are likely relevant to male-biased deficits and a female protective effect observed in Cttnbp2 mutant mice. Notably, RRAGC is an upstream regulator of mTOR that senses BCAA, suggesting that mTOR exerts a beneficial effect on females. Indeed, increased BCAA intake activates the mTOR pathway and rescues neuronal responses and social behaviours of male Cttnbp2 mutant mice. Moreover, mutant males exhibit greatly increased zinc demand to display normal social behaviours. Mice carrying an autism-linked Cttnbp2 mutation exhibit male-biased social deficits linked to specific brain regions, differential synaptic proteomes and higher demand for BCAA and zinc. We postulate that lower demand for zinc and BCAA are relevant to the female protective effect. Our study reveals a mechanism underlying sex-biased social defects and also suggests a potential therapeutic approach for autism spectrum disorders.

Infralimbic YTHDF1 is necessary for the beneficial effects of acute mild exercise on auditory fear extinction retention.

Exposure therapy is the most effective approach of behavioral therapy for anxiety and post-traumatic stress disorder (PTSD). But fear is easy to reappear even after successful extinction. So, identifying novel strategies for augmenting exposure therapy is rather important. It was reported that exercise had beneficial effects on cognitive and memory deficits. However, whether exercise could affect fear memory, especially for fear extinction remained elusive. Here, our results showed that exposure to acute mild exercise 1 or 2 h before extinction training can augment recent fear extinction retention and 2 h for the remote fear extinction retention. These beneficial effects could be attributed to increased YTHDF1 expression in medial prefrontal cortex (mPFC). Furthermore, by using an AAV-shRNA-based approach to silence YTHDF1 expression via stereotactic injection in prelimbic cortex (PL) or infralimbic cortex (IL), respectively, we demonstrated that silence YTHDF1 in IL, but not in PL, blunted augmentation of exposure therapy induced by acute mild exercise and accompanied with decreased NR2B and GluR1 expression. Moreover, YTHDF1 modulated dendritic spines remodeling of pyramidal neuron in IL. Collectively, our findings suggested that acute mild exercise acted as an effective strategy in augmenting exposure therapy with possible implications for understanding new treatment underlying PTSD.

Second-order fear conditioning involves formation of competing stimulus-danger and stimulus-safety associations.

How do animals process experiences that provide contradictory information? The present study addressed this question using second-order fear conditioning in rats. In second-order conditioning, rats are conditioned to fear a stimulus, S1, through its pairings with foot-shock (stage 1); and some days later, a second stimulus, S2, through its pairings with the already-conditioned S1 (stage 2). However, as foot-shock is never presented during conditioning to S2, we hypothesized that S2 simultaneously encodes 2 contradictory associations: one that drives fear to S2 (S2-danger) and another that reflects the absence of the expected unconditioned stimulus and partially masks that fear (e.g. S2-safety). We tested this hypothesis by manipulating the substrates of danger and safety learning in the brain (using a chemogenetic approach) and assessing the consequences for second-order fear to S2. Critically, silencing activity in the basolateral amygdala (important for danger learning) reduced fear to S2, whereas silencing activity in the infralimbic cortex (important for safety learning) enhanced fear to S2. These bidirectional changes are consistent with our hypothesis that second-order fear conditioning involves the formation of competing S2-danger and S2-safety associations. More generally, they show that a single set of experiences can produce contradictory associations and that the brain resolves the contradiction by encoding these associations in distinct brain regions.

Social buffering diminishes fear response but does not equal improved fear extinction.

Social support during exposure-based psychotherapy is believed to diminish fear and improve therapy outcomes. However, some clinical trials challenge that notion. Underlying mechanisms remain unknown, hindering the understanding of benefits and pitfalls of such approach. To study social buffering during fear extinction, we developed a behavioral model in which partner's presence decreases response to fear-associated stimuli. To identify the neuronal background of this phenomenon, we combined behavioral testing with c-Fos mapping, optogenetics, and chemogenetics. We found that the presence of a partner during fear extinction training causes robust inhibition of freezing; the effect, however, disappears in subjects tested individually on the following day. It is accompanied by lowered activation of the prelimbic (PL) and anterior cingulate (ACC) but not infralimbic (IL) cortex. Accordingly, blocking of IL activity left social buffering intact. Similarly, inhibition of the ventral hippocampus-PL pathway, suppressing fear response after prolonged extinction training, did not diminish the effect. In contrast, inhibition of the ACC-central amygdala pathway, modulating social behavior, blocked social buffering. By reporting that social modulation of fear inhibition is transient and insensitive to manipulation of the fear extinction-related circuits, we show that the mechanisms underlying social buffering during extinction are different from those of individual extinction.

Prefrontal representation of affective stimuli: importance of stress, sex, and context.

Stress-related disorders such as depression and anxiety exhibit sex differences in prevalence and negatively impact both mental and physical health. Affective illness is also frequently accompanied by changes in ventromedial prefrontal cortical (vmPFC) function. However, the neurobiology that underlies sex-specific cortical processing of affective stimuli is poorly understood. Although rodent studies have investigated the prefrontal impact of chronic stress, postmortem studies have focused largely on males and yielded mixed results. Therefore, genetically defined population recordings in behaving animals of both sexes were used to test the hypothesis that chronic variable stress (CVS) impairs the neural processing of affective stimuli in the rodent infralimbic region. Here, we targeted expression of a calcium indicator, GCaMP6s, to infralimbic pyramidal cells. In males, CVS reduced infralimbic responses to social interaction and restraint stress but increased responses to novel objects and food reward. In contrast, females did not have CVS-induced changes in infralimbic activity, which was partially dependent on the ovarian status. These results indicate that both male and female vmPFC cells encode social, stress, and reward stimuli. However, chronic stress effects are sex-dependent and behavior-specific. Ultimately, these findings extend the understanding of chronic stress-induced prefrontal dysfunction and indicate that sex is a critical factor for cortical processing of affective stimuli.

Inhibition of Indoleamine 2,3-Dioxygenase Exerts Antidepressant-like Effects through Distinct Pathways in Prelimbic and Infralimbic Cortices in Rats under Intracerebroventricular Injection with Streptozotocin.

The application of intracerebroventricular injection of streptozotocin (ICV-STZ) is considered a useful animal model to mimic the onset and progression of sporadic Alzheimer's disease (sAD). In rodents, on day 7 of the experiment, the animals exhibit depression-like behaviors. Indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme catalyzing the conversion of tryptophan (Trp) to kynurenine (Kyn), is closely related to depression and AD. The present study aimed to investigate the pathophysiological mechanisms of preliminary depression-like behaviors in ICV-STZ rats in two distinct cerebral regions of the medial prefrontal cortex, the prelimbic cortex (PrL) and infralimbic cortex (IL), both presumably involved in AD progression in this model, with a focus on IDO-related Kyn pathways. The results showed an increased Kyn/Trp ratio in both the PrL and IL of ICV-STZ rats, but, intriguingly, abnormalities in downstream metabolic pathways were different, being associated with distinct biological effects. In the PrL, the neuroprotective branch of the Kyn pathway was attenuated, as evidenced by a decrease in the kynurenic acid (KA) level and Kyn aminotransferase II (KAT II) expression, accompanied by astrocyte alterations, such as the decrease in glial fibrillary acidic protein (GFAP)-positive cells and increase in morphological damage. In the IL, the neurotoxicogenic branch of the Kyn pathway was enhanced, as evidenced by an increase in the 3-hydroxy-kynurenine (3-HK) level and kynurenine 3-monooxygenase (KMO) expression paralleled by the overactivation of microglia, reflected by an increase in ionized calcium-binding adaptor molecule 1 (Iba1)-positive cells and cytokines with morphological alterations. Synaptic plasticity was attenuated in both subregions. Additionally, microinjection of the selective IDO inhibitor 1-Methyl-DL-tryptophan (1-MT) in the PrL or IL alleviated depression-like behaviors by reversing these different abnormalities in the PrL and IL. These results suggest that the antidepressant-like effects linked to Trp metabolism changes induced by 1-MT in the PrL and IL occur through different pathways, specifically by enhancing the neuroprotective branch in the PrL and attenuating the neurotoxicogenic branch in the IL, involving distinct glial cells.

Corticostriatal Suppression of Appetitive Pavlovian Conditioned Responding.

The capacity to suppress learned responses is essential for animals to adapt in dynamic environments. Extinction is a process by which animals learn to suppress conditioned responding when an expected outcome is omitted. The infralimbic (IL) cortex to nucleus accumbens shell (NAcS) neural circuit is implicated in suppressing conditioned responding after extinction, especially in the context of operant cocaine-seeking behavior. However, the role of the IL-to-NAcS neural circuit in the extinction of responding to appetitive Pavlovian cues is unknown, and the psychological mechanisms involved in response suppression following extinction are unclear. We trained male Long Evans rats to associate a 10 s auditory conditioned stimulus (CS; 14 trials per session) with a sucrose unconditioned stimulus (US; 0.2 ml per CS) in a specific context, and then following extinction in a different context, precipitated a renewal of CS responding by presenting the CS alone in the original Pavlovian conditioning context. Unilateral, optogenetic stimulation of the IL-to-NAcS circuit selectively during CS trials suppressed renewal. In a separate experiment, IL-to-NAcS stimulation suppressed CS responding regardless of prior extinction and impaired extinction retrieval. Finally, IL-to-NAcS stimulation during the CS did not suppress the acquisition of Pavlovian conditioning but was required for the subsequent expression of CS responding. These results are consistent with multiple studies showing that the IL-to-NAcS neural circuit is involved in the suppression of operant cocaine-seeking, extending these findings to appetitive Pavlovian cues. The suppression of appetitive Pavlovian responding following IL-to-NAcS circuit stimulation, however, does not appear to be an extinction-dependent process.SIGNIFICANCE STATEMENT Extinction is a form of inhibitory learning through which animals learn to suppress conditioned responding in the face of nonreinforcement. We investigated the role of the IL cortex inputs to the NAcS in the extinction of responding to appetitive Pavlovian cues and the psychological mechanisms involved in response suppression following extinction. Using in vivo optogenetics, we found that stimulating the IL-to-NAcS neural circuit suppressed context-induced renewal of conditioned responding after extinction. In a separate experiment, stimulating the IL-to-NAcS circuit suppressed conditioned responding in an extinction-independent manner. These findings can be used by future research aimed at understanding how corticostriatal circuits contribute to behavioral flexibility and mental disorders that involve the suppression of learned behaviors.

Ventral hippocampal projections to infralimbic cortex and basolateral amygdala are differentially activated by contextual fear and extinction recall.

Fear and extinction learning are thought to generate distinct and competing memory representations in the hippocampus. How these memory representations modulate the expression of appropriate behavioral responses remains unclear. To investigate this question, we used cholera toxin B subunit to retrolabel ventral hippocampal (vHPC) neurons projecting to the infralimbic cortex (IL) and basolateral amygdala (BLA) and then quantified c-Fos immediate early gene activity within these populations following expression of either contextual fear recall or contextual fear extinction recall. Fear recall was associated with increased c-Fos expression in vHPC projections to the BLA, whereas extinction recall was associated with increased activity in vHPC projections to IL. A control experiment was performed to confirm that the apparent shift in projection neuron activity was associated with extinction learning rather than mere context exposure. Overall, results indicate that hippocampal contextual fear and extinction memory representations differentially activate vHPC projections to IL and BLA. These findings suggest that hippocampal memory representations orchestrate appropriate behavioral responses through selective activation of projection pathways.

Ventral Hippocampal Input to Infralimbic Cortex Is Necessary for the Therapeutic-Like Effects of Extinction in Stressed Rats.

BACKGROUND: Posttraumatic stress disorder is characterized by deficits in cognitive flexibility related to dysfunction of the medial prefrontal cortex (mPFC). Exposure therapy can effectively reverse these deficits. Fear extinction in rodents bears similarity to exposure therapy. Extinction reverses chronic stress-induced deficits in cognitive flexibility on the attentional set-shifting test (AST), an mPFC-mediated process. This therapeutic effect requires activity of pyramidal neurons and brain derived neurotrophic factor (BDNF) signaling in infralimbic cortex (IL). However, the circuit mechanisms governing BDNF-mediated plasticity initiated by extinction in IL are unknown. The ventral hippocampus (vHipp) plays a role in regulating IL activity during extinction, and plasticity in vHipp is necessary for extinction memory consolidation. Therefore, we investigated the role of vHipp input to IL in the effects of extinction in reversing stress-induced cognitive deficits. METHODS: vHipp input to IL was silenced using a Gi-Designer Receptors Exclusively Activated by Designer Drugs (DREADD) via local infusion of clozapine-N-oxide (CNO) into IL before extinction. A day later, rats were tested on AST. In a separate experiment, we tested whether vHipp input to the IL induces BDNF signaling to exert therapeutic effects. We activated the vHipp using a Gq-DREADD, and injected an anti-BDNF neutralizing antibody into IL. Rats were tested on the AST 24 hours later. RESULTS: Silencing the vHipp input to IL prevented the beneficial effects of extinction in reversing stress-induced cognitive deficits. Activating vHipp input to IL in the absence of extinction was sufficient to reverse stress-induced deficits in set-shifting. The beneficial effects were blocked by local infusion of a neutralizing anti-BDNF antibody into IL. CONCLUSIONS: vHipp-driven BDNF signaling in IL is critical for extinction to counteract the deleterious cognitive effects of chronic stress.

Preferential In Vivo Inhibitory Action of Serotonin in Rat Infralimbic versus Prelimbic Cortex: Relevance for Antidepressant Treatments.

The infralimbic (IL) cortex is the rodent equivalent of human ventral anterior cingulate cortex (vACC), which plays a key role in the pathophysiology and treatment of major depressive disorder (MDD). The modulation of glutamatergic neurotransmission in IL [but not in the adjacent prelimbic (PrL) cortex] evokes antidepressant-like or depressive-like behaviors, associated with changes in serotonin (5-HT) function, highlighting the relevance of glutamate/serotonin interactions in IL for emotional control. 5-HT modulates neuronal activity in PrL and cingulate (Cg) cortex but its effects in IL are largely unknown. We therefore compared the in vivo effects of 5-HT on pyramidal neuron activity in IL (n = 61) and PrL (n = 50) of anesthetized rats. IL pyramidal neurons were more responsive to physiological dorsal raphe stimulation (0.9 Hz) than PrL neurons (84% vs. 64%, respectively) and were inhibited to a greater extent (64% vs. 36%, respectively). Orthodromic activations (8% in PrL) were absent in IL, whereas biphasic responses were similar (20%) in both areas. Excitations were mediated by 5-HT2A-R activation, whereas inhibitions involved 3 different components: 5-HT1A-R, 5-HT3-R and GABAA-R, respectively. The remarkable inhibitory action of 5-HT in IL suggests that 5-HT-enhancing drugs may exert their antidepressant action by normalizing a glutamatergic hyperactivity in the vACC of MDD patients.

Differential Modulation of Dorsal Raphe Serotonergic Activity in Rat Brain by the Infralimbic and Prelimbic Cortices.

The reciprocal connectivity between the medial prefrontal cortex (mPFC) and the dorsal raphe nucleus (DR) is involved in mood control and resilience to stress. The infralimbic subdivision (IL) of the mPFC is the rodent equivalent of the ventral anterior cingulate cortex, which is intimately related to the pathophysiology/treatment of major depressive disorder (MDD). Boosting excitatory neurotransmission in the IL-but not in the prelimbic cortex, PrL-evokes depressive-like or antidepressant-like behaviors in rodents, which are associated with changes in serotonergic (5-HT) neurotransmission. We therefore examined the control of 5-HT activity by both of the mPFC subdivisions in anesthetized rats. The electrical stimulation of IL and PrL at 0.9 Hz comparably inhibited 5-HT neurons (53% vs. 48%, respectively). However, stimulation at higher frequencies (10-20 Hz) revealed a greater proportion of 5-HT neurons sensitive to IL than to PrL stimulation (86% vs. 59%, at 20 Hz, respectively), together with a differential involvement of GABAA (but not 5-HT1A) receptors. Likewise, electrical and optogenetic stimulation of IL and PrL enhanced 5-HT release in DR in a frequency-dependent manner, with greater elevations after IL stimulation at 20 Hz. Hence, IL and PrL differentially control serotonergic activity, with an apparent superior role of IL, an observation that may help to clarify the brain circuits involved in MDD.

Comparison of prefrontal cortex sucrose seeking ensembles engaged in multiple seeking sessions: Context is key.

Encoding of memories, including those associated with prior drug or reward, is thought to take place within distinct populations of neurons, termed ensembles. Neuronal ensembles for drug- and reward-seeking have been identified in regions of the medial prefrontal cortex, but much of our understanding of these ensembles is based on experiments that take place in a single reward-associated environment and measure ensemble encoding over short durations of time. In contrast, reward seeking behavior is evident across different reward-associated environments and persists over time. Using TetTag mice and Fos immunohistochemistry, we examined the relationship between persistent sucrose-seeking and ensemble encoding in mice that undergo seeking sessions in the same or different sucrose self-administration contexts 2 weeks apart. We found that prelimbic (PrL) and anterior cingulate cortex ensembles tagged in the first seeking session were highly sensitive to the context in which a second seeking session took place: reactivation of these ensembles was reduced in the same context but elevated in a distinct sucrose self-administration context. Correlational analyses revealed that ensemble reactivation in the PrL was proportional to the persistence of sucrose seeking behavior across sessions in differing ways in female mice. In the same context, reactivation was proportional to the persistence of non-reinforced operant responses, whereas in a distinct context, reactivation was proportional to the persistence of non-reinforced head entries into the sucrose receptacle. This study underlines the importance of the medial prefrontal cortex importance in maintaining a reward-seeking ensemble over time and identifies context-dependent changes in behavioral correlates of ensemble reactivation.

Hippocampal neuropeptide Y2 receptor blockade improves spatial memory retrieval and modulates limbic brain metabolism.

INTRODUCTION: The neuropeptide Y (NPY) is broadly distributed in the central nervous system (CNS), and it has been related to neuroprotective functions. NPY seems to be an important component to counteract brain damage and cognitive impairment mediated by drugs of abuse and neurodegenerative diseases, and both NPY and its Y2 receptor (Y2R) are highly expressed in the hippocampus, critical for learning and memory. We have recently demonstrated its influence on cognitive functions; however, the specific mechanism and involved brain regions where NPY modulates spatial memory by acting on Y2R remain unclear. METHODS: Here, we examined the involvement of the hippocampal NPY Y2R in spatial memory and associated changes in brain metabolism by bilateral administration of the selective antagonist BIIE0246 into the rat dorsal hippocampus. To further evaluate the relationship between memory functions and neuronal activity, we analysed the regional expression of the mitochondrial enzyme cytochrome c oxidase (CCO) as an index of oxidative metabolic capacity in limbic and non-limbic brain regions. RESULTS: The acute blockade of NPY Y2R significantly improved spatial memory recall in rats trained in the Morris water maze that matched metabolic activity changes in spatial memory processing regions. Specifically, CCO activity changes were found in the dentate gyrus of the dorsal hippocampus and CA1 subfield of the ventral hippocampus, the infralimbic region of the PFC and the mammillary bodies. CONCLUSIONS: These findings suggest that the NPY hippocampal system, through its Y2R receptor, influences spatial memory recall (retrieval) and exerts control over patterns of brain activation that are relevant for associative learning, probably mediated by Y2R modulation of long-term potentiation and long-term depression.

Rodent mnemonic similarity task performance requires the prefrontal cortex.

Mnemonic similarity task performance, in which a known target stimulus must be distinguished from similar lures, is supported by the hippocampus and perirhinal cortex. Impairments on this task are known to manifest with advancing age. Interestingly, disrupting hippocampal activity leads to mnemonic discrimination impairments when lures are novel, but not when they are familiar. This observation suggests that other brain structures support discrimination abilities as stimuli are learned. The prefrontal cortex (PFC) is critical for retrieval of remote events and executive functions, such as working memory, and is also particularly vulnerable to dysfunction in aging. Importantly, the medial PFC is reciprocally connected to the perirhinal cortex and neuron firing in this region coordinates communication between lateral entorhinal and perirhinal cortices to presumably modulate hippocampal activity. This anatomical organization and function of the medial PFC suggests that it contributes to mnemonic discrimination; however, this notion has not been empirically tested. In the current study, rats were trained on a LEGO object-based mnemonic similarity task adapted for rodents, and surgically implanted with guide cannulae targeting prelimbic and infralimbic regions of the medial PFC. Prior to mnemonic discrimination tests, rats received PFC infusions of the GABAA agonist muscimol. Analyses of expression of the neuronal activity-dependent immediate-early gene Arc in medial PFC and adjacent cortical regions confirmed muscimol infusions led to neuronal inactivation in the infralimbic and prelimbic cortices. Moreover, muscimol infusions in PFC impaired mnemonic discrimination performance relative to the vehicle control across all testing blocks when lures shared 50-90% feature overlap with the target. Thus, in contrast hippocampal infusions, PFC inactivation impaired target-lure discrimination regardless of the novelty or familiarity of the lures. These findings indicate the PFC plays a critical role in mnemonic similarity task performance, but the time course of PFC involvement is dissociable from that of the hippocampus.

The role of the serotonin transporter in prefrontal cortex glutamatergic signaling following short- and long-access cocaine self-administration.

Vulnerability to drug addiction relies on substantial individual differences. We previously demonstrated that serotonin transporter knockout (SERT-/- ) rats show increased cocaine intake and develop signs of compulsivity. However, the underlying neural mechanisms are not fully understood. Given the pivotal role of glutamate and prefrontal cortex in cocaine-seeking behavior, we sought to investigate the expression of proteins implicated in glutamate neurotransmission in the prefrontal cortex of naïve and cocaine-exposed rats lacking SERT. We focused on the infralimbic (ILc) and prelimbic (PLc) cortices, which are theorized to exert opposing effects on the control over subcortical brain areas. SERT-/- rats, which compared to wild-type (SERT+/+ ) rats show increased ShA and LgA intake short-access (ShA) and long-access (LgA) cocaine intake, were sacrificed 24 h into withdrawal for ex vivo molecular analyses. In the ILc homogenate of SERT-/- rats, we observed a sharp increase in glial glutamate transporter 1 (GLT-1) after ShA, but not LgA, cocaine intake. This was paralleled by ShA-induced increases in GluN1, GluN2A, and GluN2B NMDA receptor subunits and their scaffolding protein SAP102 in the ILc homogenate, but not postsynaptic density, of these knockout animals. In the PLc, we found no major changes in the homogenate; conversely, the expression of GluN1 and GluN2A NMDA receptor subunits was increased in the postsynaptic density under ShA conditions and reduced under LgA conditions. These results point to SERT as a critical regulator of glutamate homeostasis in a way that differs between the subregions investigated, the duration of cocaine exposure as well as the cellular compartment analyzed.

mTOR Knockdown in the Infralimbic Cortex Evokes A Depressive-like State in Mouse.

Fast and sustained antidepressant effects of ketamine identified the mammalian target of rapamycin (mTOR) signaling pathway as the main modulator of its antidepressive effects. Thus, mTOR signaling has become integral for the preclinical evaluation of novel compounds to treat depression. However, causality between mTOR and depression has yet to be determined. To address this, we knocked down mTOR expression in mice using an acute intracerebral infusion of small interfering RNAs (siRNA) in the infralimbic (IL) or prelimbic (PrL) cortices of the medial prefrontal cortex (mPFC), and evaluated depressive- and anxious-like behaviors. mTOR knockdown in IL, but not PrL, cortex produced a robust depressive-like phenotype in mice, as assessed in the forced swimming test (FST) and the tail suspension test (TST). This phenotype was associated with significant reductions of mTOR mRNA and protein levels 48 h post-infusion. In parallel, decreased brain-derived neurotrophic factor (BDNF) expression was found bilaterally in both IL and PrL cortices along with a dysregulation of serotonin (5-HT) and glutamate (Glu) release in the dorsal raphe nucleus (DRN). Overall, our results demonstrate causality between mTOR expression in the IL cortex and depressive-like behaviors, but not in anxiety.