Conditioned Freezing Response Research Articles

Bidirectional fear modulation by discrete anterior insular circuits in male mice.

The brain's ability to appraise threats and execute appropriate defensive responses is essential for survival in a dynamic environment. Humans studies have implicated the anterior insular cortex (aIC) in subjective fear regulation and its abnormal activity in fear/anxiety disorders. However, the complex aIC connectivity patterns involved in regulating fear remain under investigated. To address this, we recorded single units in the aIC of freely moving male mice that had previously undergone auditory fear conditioning, assessed the effect of optogenetically activating specific aIC output structures in fear, and examined the organization of aIC neurons projecting to the specific structures with retrograde tracing. Single-unit recordings revealed that a balanced number of aIC pyramidal neurons' activity either positively or negatively correlated with a conditioned tone-induced freezing (fear) response. Optogenetic manipulations of aIC pyramidal neuronal activity during conditioned tone presentation altered the expression of conditioned freezing. Neural tracing showed that non-overlapping populations of aIC neurons project to the amygdala or the medial thalamus, and the pathway bidirectionally modulated conditioned fear. Specifically, optogenetic stimulation of the aIC-amygdala pathway increased conditioned freezing, while optogenetic stimulation of the aIC-medial thalamus pathway decreased it. Our findings suggest that the balance of freezing-excited and freezing-inhibited neuronal activity in the aIC and the distinct efferent circuits interact collectively to modulate fear behavior.

A novel automated software and video analysis system for quantifying physiological responses of conditioned fear in mice

BACKGROUND: Conditioned fear is an aversive learning process that is highly sensitive to sensory conditions such as environmental context, threat intensity and circadian rhythms. To better understand these external variables that impact conditioned fear learning, we developed a novel software application that allows for programmable remote conditioned stimulus (CS, ie., auditory tone) delivery and recording of group-housed mice in a homecage, or familiar environment. The goal of our study was therefore to leverage this technology to examine conditioned fear in the homecage across light-dark (LD) circadian cycles. METHODS: Male C57bl/6J mice underwent Pavlovian auditory fear conditioning (20 CS/US pairings), with a CS-only group as a control (n=4/group). Mice were then returned to the homecage, equipped with speaker and camera. Using a customized open-source software to remotely evoke an enhanced physiological state of fear in the homecage, mice received a select number of conditioned auditory stimuli (30sec. intermittent tones) throughout the LD cycle over 14 days. The software was configured to deliver six tones (6kHz) over a 24hr period (3 light, 3 dark). The first CS during each LD cycle was recorded and percent freezing behavior was manually scored. On day 15, to evaluate whether the HC-specific evoked fear response transfers to a new environment, mice were placed into a novel context and CS-dependent freezing was measured. RESULTS: Over 14 days, compared to controls, fear-conditioned pair-housed mice exhibited strong CS-evoked freezing in the homecage ( total freezing: FC 70.46%, CS-only 3.69%, F(2.29, 11.14)=10.78, p<0.01). Interestingly, when we evaluated CS-specific circadian responses, fear-conditioned mice exhibited enhanced freezing in the light versus the dark phase ( total freezing: light 84.56%, dark 56.36%, F(2.15, 6.46)=11.04, p<0.01). In fear-conditioned mice, there was a 36% reduction in total CS-dependent homecage freezing over 14 days. However, this was a context-dependent effect, as the reduction in freezing on day 14 was not observed in the novel context on day 15 ( t(3)=3.16, p=0.05). CONCLUSIONS: Here we demonstrate for the first time, using an open-source software developed in our laboratory, environment and circadian-dependent conditioned freezing responses in the homecage of group-housed mice. This study provides a proof-of-principle innovative approach for examining physiological outcomes of conditioned fear across ethologically relevant environments, which may help further the understanding for heightened physiological responses in neuropsychiatric diseases such as posttraumatic stress disorder (PTSD). To further elucidate impaired physiological mechanisms of disordered fear, future studies will integrate multimodal behavioral (DeepLabCut TM ) and cardiovascular telemetry-based approaches. Congressionally Directed Medical Research Programs (CDMRP) PR210574 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Involvement of D2-like dopaminergic receptors in contextual fear conditioning in female rats: influence of estrous cycle.

Introduction: Dopamine has been increasingly recognized as a key neurotransmitter regulating fear/anxiety states. Nevertheless, the influence of sex and estrous cycle differences on the role of dopamine in fear responses needs further investigation. We aimed to evaluate the effects of sulpiride (a dopaminergic D2-like receptor antagonist) on contextual fear conditioning in females while exploring the influence of the estrous cycle. Methods: First, using a contextual fear conditioning paradigm, we assessed potential differences in acquisition, expression, and extinction of the conditioned freezing response in male and female (split in proestrus/estrus and metestrus/diestrus) Wistar rats. In a second cohort, we evaluated the effects of sulpiride (20 and 40 mg/kg) on contextual conditioned fear in females during proestrus/estrus and metestrus/diestrus. Potential nonspecific effects were assessed in motor activity assays (catalepsy and open-field tests). Results: No sex differences nor estrous cycle effects on freezing behavior were observed during the fear conditioning phases. Sulpiride reduced freezing expression in female rats. Moreover, females during the proestrus/estrus phases of the estrous cycle were more sensitive to the effects of sulpiride than females in metestrus/diestrus. Sulpiride did not cause motor impairments. Discussion: Although no sex or estrous cycle differences were observed in basal conditioned fear expression and extinction, the estrous cycle seems to influence the effects of D2-like antagonists on contextual fear conditioning.

Caffeine facilitates extinction of auditory fear conditioning inrats.

Caffeine is the most widely consumed psychostimulant drug which could affect learning and memory acting through central adenosine receptors. Although caffeine has been suggested to impair the acquisition and the expression of auditory fear conditioning, its effect on the extinction has not been elucidated. To address this issue, in the present study, we investigated whether caffeine affects the extinction of conditioned fear in an auditory fear conditioning paradigm. During conditioning, rats received pairings of auditory cues (conditioned stimulus, CS) and foot shocks (unconditioned stimulus). On the next day, the rats were intraperitoneally administrated saline or caffeine (5 or 10mg/kg) and then subjected to the extinction training, in which CSs were repeatedly presented without the foot shocks. Twenty-four hours later, the rats were re-exposed to the presentations of CSs (retrieval test). We found an acute caffeine administration dose-dependently decreased freezing rates during the presentations of CS in the extinction training. Furthermore, caffeine-treated animals showed lower conditioned freezing responses in the retrieval test. These findings suggest that caffeine facilitates the extinction of conditioned fear.

Adolescent Stress Confers Resilience to Traumatic Stress Later in Life: Role of the Prefrontal Cortex

BackgroundAdolescent brains are sensitive to stressors. However, under certain circumstances, developmental stress can promote an adaptive phenotype, allowing individuals to cope better with adverse situations in adulthood, thereby contributing to resilience. MethodsSprague Dawley rats (50 males, 48 females) were subjected to adolescent chronic variable stress (adol CVS) for 2 weeks at postnatal day 45. At postnatal day 85, a group was subjected to single prolonged stress (SPS). After a week, animals were evaluated in an auditory-cued fear conditioning paradigm, and neuronal recruitment during reinstatement was assessed by Fos expression. Patch clamp electrophysiology (17–35 cells/group) was performed in male rats to examine physiological changes associated with resilience. ResultsAdol CVS blocked fear potentiation evoked by SPS. We observed that SPS impaired extinction (males) and enhanced reinstatement (both sexes) of the conditioned freezing response. Prior adol CVS prevented both effects. SPS effects were associated with a reduction of infralimbic (IL) cortex neuronal recruitment after reinstatement in males and increased engagement of the central amygdala in females, both also prevented by adol CVS, suggesting different neurocircuits involved in generating resilience between sexes. We explored the mechanism behind reduced IL recruitment in males by studying the intrinsic excitability of IL pyramidal neurons. SPS reduced excitability of IL neurons, and prior adol CVS prevented this effect. ConclusionsOur data indicate that adolescent stress can impart resilience to the effects of traumatic stress on neuroplasticity and behavior. Our data provide a mechanistic link behind developmental stress-induced behavioral resilience and prefrontal (IL) cortical excitability in males.

Divergent projections of the paraventricular nucleus of the thalamus mediate the selection of passive and active defensive behaviors.

The appropriate selection of passive and active defensive behaviors in threatening situations is essential for survival. Previous studies have shown that passive defensive responses depend on activity of the central nucleus of the amygdala (CeA), whereas active ones primarily rely on the nucleus accumbens (NAc). However, the mechanisms underlying flexible switching between these two types of responses remain unknown. Here, we show in mice that the paraventricular thalamus (PVT) mediates the selection of defensive behaviors through its interaction with the CeA and the NAc. We show that the PVT–CeA pathway drives conditioned freezing responses, whereas the PVT–NAc pathway is inhibited during freezing and instead signals active avoidance events. Optogenetic manipulations revealed that activity in the PVT–CeA or PVT–NAc pathway biases behavior toward the selection of passive or active defensive responses, respectively. These findings provide evidence that the PVT mediates flexible switching between opposing defensive behaviors.

Behavioural and cardiovascular effects of orexin-A infused into the central amygdala under basal and fear conditions in rats

The neuropeptide orexin-A (OX-A) has diverse functions, including maintaining arousal, autonomic control, motor activity and stress responses. These functions are regulated at different terminal regions where OX-A is released. The current study examined the physiological and behavioural effects of OX-A microinjections into the central amygdala (CeA) under basal and stressed conditions in rats. When OX-A was microinjected into the CeA and the animals returned to the home-cage, heart rate and mean arterial pressure were increased compared to vehicle-injected controls. General activity of the animal was also increased, indicating that OX-A activity in CeA contributes to increased arousal. This outcome is similar to the effects of central intracerebroventricular infusions of OX-A, as well as the cardiovascular effects previously demonstrated at many of OX’s efferent hypothalamic and brainstem structures. In a second study, animals were fear-conditioned to a context by delivery of electric footshocks and then animals were re-exposed to the conditioned context at test. When OX-A was microinjected at test, freezing behaviour was reduced and there was a corresponding increase in the animal’s activity but no impact on the pressor and cardiac responses (i.e, blood pressure and heart rate were unchanged). This reduction in freezing suggests that OX-A activates amygdala neurons that inhibit freezing, which is similar to the actions of other neuropeptides in the CeA that modulate the appropriate defence response to fearful stimuli. Overall, these data indicate that the CeA is an important site of OX-A modulation of cardiovascular and motor activity, as well as conditioned freezing responses.

Male, but not female, Sprague Dawley rats display enhanced fear learning following acute ethanol withdrawal (hangover)

The present studies investigated the effects of withdrawal from a single binge-like dose of ethanol (hangover) on fear conditioning in male and female Sprague Dawley rats. In Experiment 1, males and females were given 0 or 3.5 g/kg ethanol intraperitoneally (i.p.) and then conditioned to contextual fear 24 h post injection. Withdrawal from acute ethanol enhanced expression of the conditioned freezing response in males, but not in females. Experiment 2 demonstrated that in males, withdrawal from acute ethanol administered 24 h prior to conditioning enhanced contextual fear conditioning, but not auditory-cued fear conditioning. In Experiment 3, male and female rats were given 3.5 g/kg ethanol, and blood ethanol concentrations (BECs) were assessed at various time points for determination of ethanol clearance. Female rats cleared ethanol at a higher rate than males, with 10 h required for females and 14 for males to eliminate ethanol from their systems. Because females cleared ethanol faster than males, in Experiment 4, females were conditioned 18 h after ethanol administration to keep the interval between ethanol clearance and fear conditioning similar to that of males. Withdrawal from acute ethanol given 18 h prior to conditioning did not affect both contextual and auditory-cued fear conditioning in females. In summary, these results highlight sex differences in the impact of withdrawal from acute ethanol (hangover) on fear learning; suggesting that males are more sensitive to hangover-associated enhancement of negative affect than females.

Acquisition and extinction of contextual fear conditioning in Carioca high- and low-conditioned freezing rats

Carioca High- and Low-conditioned Freezing (CHF and CLF, respectively) rats are two bidirectional breeding lines of animals with high and low levels of defensive conditioned freezing response to contextual cues that are associated with an aversive stimulus. In the last decade, various studies have investigated their physiology and response to other external stimuli as a model of trait anxiety. However, the process of the extinction of their characteristic freezing response is not well understood. The present study investigated the acquisition and extinction of a contextual fear conditioning protocol among these animals. The results showed that CLF rats were unable to consolidate aversive memories and thus were unresponsive to extinction. In contrast, CHF rats still exhibited a large freezing response to contextual cues and were susceptible to extinction, reaching levels of freezing that were comparable to control, randomly bred animals. These findings contribute to validating CHF rats as a model of trait anxiety and their usefulness for evaluating exposure protocols to ameliorate excessive levels of context-conditioned fear.

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation's behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Small lesions of the dorsal or ventral hippocampus subregions are associated with distinct impairments in working memory and reference memory retrieval, and combining them attenuates the acquisition rate of spatial reference memory.

The importance of the hippocampus in spatial learning is well established, but the precise relative contributions by the dorsal (septal) and ventral (temporal) subregions remain unresolved. One debate revolves around the extent to which the ventral hippocampus contributes to spatial navigation and learning. Here, separate small subtotal lesions of dorsal hippocampus or ventral hippocampus alone (destroying 18.9 and 28.5% of total hippocampal volume, respectively) spared reference memory acquisition in the water maze. By contrast, combining the two subtotal lesions significantly reduced the rate of acquisition across days. This constitutes evidence for synergistic integration between dorsal and ventral hippocampus in mice. Evidence that ventral hippocampus contributes to spatial/navigation learning also emerged early on during the retention probe test as search preference was reduced in mice with ventral lesions alone or combined lesions. The small ventral lesions also led to anxiolysis in the elevated plus maze and over-generalization of the conditioned freezing response to a neutral context. Similar effects of comparable magnitudes were seen in mice with combined lesions, suggesting that they were largely due to the small ventral damage. By contrast, small dorsal lesions were uniquely associated with a severe spatial working memory deficit in the water maze. Taken together, both dorsal and ventral poles of the hippocampus contribute to efficient spatial navigation in mice: While the integrity of dorsal hippocampus is necessary for spatial working memory, the acquisition and retrieval of spatial reference memory are modulated by the ventral hippocampus. Although the impairments following ventral damage (alone or in combination with dorsal damage) were less substantial, a wider spectrum of spatial learning, including context conditioning, was implicated. Our results encourage the search for integrative mechanism between dorsal and ventral hippocampus in spatial learning. Candidate neural substrates may include dorsoventral longitudinal connections and reciprocal modulation via overlapping polysynaptic networks beyond hippocampus.

Differential Phosphorylation of the Glucocorticoid Receptor in Hippocampal Subregions Induced by Contextual Fear Conditioning Training.

Aversive events induce the release of glucocorticoid stress hormones that facilitate long-term memory consolidation, an effect that depends on the activation of glucocorticoid receptors (GRs). GRs are distributed widely in the hippocampus. The dorsal region of the hippocampus has been related to cognitive functions and the ventral region to stress and emotion. GR acts as a transcription factor which after hormone binding becomes phosphorylated, affecting its cellular distribution and transcriptional activity. Two functionally well-described GR phosphorylation sites are serine 232 (pSer232), which enhances gene expression, and serine 246 (pSer246), having the opposite effect. Since gene expression is one of the plastic mechanisms needed for memory consolidation, we investigated if an aversive learning task would induce GR phosphorylation in the dorsal (DH) and the ventral (VH) hippocampus. We trained rats in contextual fear conditioning (CFC) using different foot-shock intensities (0.0, 0.5, or 1.5 mA). One subgroup of animals trained with each intensity was sacrificed 15 min after training and blood was collected to quantify corticosterone (CORT) levels in serum. Another subgroup was sacrificed 1 h after training and brains were collected to evaluate the immunoreactivity (IR) to GR, pSer232 and pSer246 by SDS-PAGE/Western blot in DH and VH, and by immunohistochemistry in dorsal and ventral CA1, CA2, CA3, and dentate gyrus (DG) hippocampal regions. The conditioned freezing response increased in animals trained with 0.5 and 1.5 mA during training and extinction sessions. The degree of retention and CORT levels were directly related to the intensity of the foot-shock. Although total GR-IR remained unaffected after conditioning, we observed a significant increase of pSer246-IR in the dorsal region of CA1 and in both dorsal and ventral DG. The only region in which pSer232-IR was significantly elevated was ventral CA3. Our results indicate that fear conditioning training is related to GR phosphorylation in specific subregions of the hippocampus, suggesting that its transcriptional activity for gene expression is favored in ventral CA3, whereas its repressor activity for gene-silencing is increased in dorsal CA1 and in both dorsal and ventral DG.

Behavioral effects of buspirone in a mouse model of posttraumatic stress disorder

Buspirone presents a unique profile of action, which involves activation of 5-HT1A receptors and complex effects on D2-like dopaminergic receptors. This medication is studied in terms of potential clinical repositioning to conditions that are associated with dopaminergic dysfunctions including schizophrenia and substance use disorder. Buspirone antagonizes D3 and D4 receptors, however, depending on the dose it differentially interacts with D2 receptors. Previously, we reported that some of D2/D3 dopaminergic agonists attenuate PTSD-like behavioral symptoms in mice. Here we investigated whether buspirone could also affect PTSD-like symptoms. We used the single prolonged stress (mSPS) protocol to induce PTSD-like behavior in adult male CD-1 mice. Buspirone (0.5, 2, or 10 mg/kg, i.p.) was injected for 15 consecutive days. The subjects were repeatedly examined in a variety of behavioral tests measuring conditioned freezing response, antidepressant-like effects, anxiety, and ultrasonic vocal response to the restraint stress. Mouse SPS resulted in prolonged immobility in the forced swim test and freezing in the fear-conditioning test, and produced symptoms of anxiety. Buspirone dose-dependently decreased the exaggerated freezing response in mice, but only at the dose of 2 mg/kg exhibited the anxiolytic-like effect in the elevated plus maze test. Buspirone reduced the number of ultrasonic calls in mSPS-exposed mice but revealed no antidepressant-like effect in the forced swim test. Present data suggest some positive effects of buspirone in the treatment of selected PTSD-like symptoms and prompt for its further clinical evaluation.

Novel behavioural characteristics of the superoxide dismutase 1 G93A (SOD1G93A ) mouse model of amyotrophic lateral sclerosis include sex-dependent phenotypes.

Amyotrophic lateral sclerosis (ALS) involves the rapid degeneration of upper and lower motor neurons leading to weakening and paralysis of voluntary movements. Mutations in copper-zinc superoxide dismutase 1 (SOD1) are a known genetic cause of ALS, and the SOD1 G93A mouse has been used extensively to investigate molecular mechanisms in ALS. In recent years, evidence suggests that ALS and frontotemporal dementia form a spectrum disorder ranging from motor to cognitive dysfunctions. Thus, we tested male and female SOD1 G93A mice for the first time before the onset of debilitating motor impairments in behavioural domains relevant to both ALS and frontotemporal dementia. SOD1 G93A males displayed reduced locomotion, exploration and increased anxiety-like behaviours compared with control males. Intermediate-term spatial memory was impaired in SOD1 G93A females, whereas long-term spatial memory deficits as well as lower acoustic startle response, and prepulse inhibition were identified in SOD1 G93A mice of both sexes compared with respective controls. Interestingly, SOD1 G93A males exhibited an increased conditioned cue freezing response. Nosing behaviours were also elevated in both male and female SOD1 G93A when assessed in social paradigms. In conclusion, SOD1 G93A mice exhibit a variety of sex-specific behavioural deficits beyond motor impairments supporting the notion of an ALS-frontotemporal spectrum disorder. Thus, SOD1 G93A mice may represent a useful model to test the efficacy of therapeutic interventions on clinical symptoms in addition to declining motor abilities.

Fear conditioning and extinction induce opposing changes in dendritic spine remodeling and somatic activity of layer 5 pyramidal neurons in the mouse motor cortex

Multiple brain regions including the amygdala and prefrontal cortex are crucial for modulating fear conditioning and extinction. The primary motor cortex is known to participate in the planning, control, and execution of voluntary movements. Whether and how the primary motor cortex is involved in modulating freezing responses related to fear conditioning and extinction remains unclear. Here we show that inactivation of the mouse primary motor cortex impairs both the acquisition and extinction of freezing responses induced by auditory-cued fear conditioning. Fear conditioning significantly increases the elimination of dendritic spines on apical dendrites of layer 5 pyramidal neurons in the motor cortex. These eliminated spines are further apart from each other than expected from random distribution along dendrites. On the other hand, fear extinction causes the formation of new spines that are located near the site of spines eliminated previously after fear conditioning. We further show that fear conditioning decreases and fear extinction increases somatic activities of layer 5 pyramidal neurons in the motor cortex respectively. Taken together, these findings indicate fear conditioning and extinction induce opposing changes in synaptic connections and somatic activities of layer 5 pyramidal neurons in the primary motor cortex, a cortical region important for the acquisition and extinction of auditory-cued conditioned freezing responses.

The impact of maternal separation and isolation stress during stress hyporesponsive period on fear retention and extinction recall memory from 5-week- to 1-year-old rats.

The purpose of the present study was to determine whether age would disrupt fear retention and extinction memory in rats pre-exposed to maternal separation and isolation stress; these rats are called MS rats. MS stress was induced by exposing rat pups into maternal separation followed by isolation stress from peer groups (MS) daily/6h during stress hyporesponsive period, while controls rats that were undisturbed during this period are called NMS rats. 5, 8, 15 and 52weeks later, these animals were exposed to classical fear conditioning test by pairing auditory stimulus (conditioned stimulus, CS+) with electric footshock. 24h later, conditioned freezing response to CS+ was measured during fear retention, extinction and extinction recall trials. The normal ageing per se did not affect the formation of fear memory, retention and fear extinction memory. MS stress, on the other hand, disrupted fear memory at young adulthood age exhibiting increased freezing response to CS+ during retention test and reduced during fear extinction memory test when compared to NMS groups. On the other hand, rats at adolescence age exhibited reduced freezing during fear retention and enhanced freezing response to CS+ during extinction recall test. However, MS-induced changes in freezing response during fear retention and extinction tests were not seen in adulthood and 1-year-old age groups. These data demonstrate the young adulthood age is highly vulnerable to fear memory and extinction processes. The differences in freezing response to CS+ during fear conditioning from adolescence to old age, thus, appear to be related to the maturation of the limbic circuit.

Inhibitory gain modulation of defense behaviors by zona incerta

Zona incerta (ZI) is a functionally mysterious subthalamic nucleus containing mostly inhibitory neurons. Here, we discover that GABAergic neurons in the rostral sector of ZI (ZIr) directly innervate excitatory but not inhibitory neurons in the dorsolateral and ventrolateral compartments of periaqueductal gray (PAG), which can drive flight and freezing behaviors respectively. Optogenetic activation of ZIr neurons or their projections to PAG reduces both sound-induced innate flight response and conditioned freezing response, while optogenetic suppression of these neurons enhances these defensive behaviors, likely through a mechanism of gain modulation. ZIr activity progressively increases during extinction of conditioned freezing response, and suppressing ZIr activity impairs the expression of fear extinction. Furthermore, ZIr is innervated by the medial prefrontal cortex (mPFC), and silencing mPFC prevents the increase of ZIr activity during extinction and the expression of fear extinction. Together, our results suggest that ZIr is engaged in modulating defense behaviors.

Characterization of Behavioral, Signaling and Cytokine Alterations in a Rat Neurodevelopmental Model for Schizophrenia, and Their Reversal by the 5-HT6 Receptor Antagonist SB-399885

Post-weaning social isolation of rats produces neuroanatomical, neurochemical and behavioral alterations resembling some core features of schizophrenia. This study examined the ability of the 5-HT6 receptor antagonist SB-399885 to reverse isolation-induced cognitive deficits, then investigated alterations in hippocampal cell proliferation and hippocampal and frontal cortical expression of selected intracellular signaling molecules and cytokines. Male Lister hooded rats (weaned on post-natal days 21–24 and housed individually or in groups of 3–4) received six i.p. injections of vehicle (1% Tween 80, 1 mL/kg) or SB-399885 (5 or 10 mg/kg) over a 2-week period starting 40 days post-weaning, on the days that locomotor activity, novel object discrimination (NOD), pre-pulse inhibition of acoustic startle and acquisition, retention and extinction of a conditioned freezing response (CFR) were assessed. Tissue was collected 24 h after the final injection for immunohistochemistry, reverse-phase protein microarray and western blotting. Isolation rearing impaired NOD and cue-mediated CFR, decreased cell proliferation within the dentate gyrus, and elevated hippocampal TNFα levels and Cdc42 expression. SB-399885 reversed the NOD deficit and partially normalized CFR and cell proliferation. These effects were accompanied by altered expression of several members of the c-Jun N-terminal Kinase (JNK) and p38 MAPK signaling pathways (including TAK1, MKK4 and STAT3). Although JNK and p38 themselves were unaltered at this time point hippocampal TAK1 expression and phosphorylation correlated with visual recognition memory in the NOD task. Continued use of this neurodevelopmental model could further elucidate the neurobiology of schizophrenia and aid assessment of novel therapies for drug-resistant cognitive symptoms.

Post-weaning social isolation of rats leads to long-term disruption of the gut microbiota-immune-brain axis

Early-life stress is an established risk for the development of psychiatric disorders. Post-weaning isolation rearing of rats produces lasting developmental changes in behavior and brain function that may have translational pathophysiological relevance to alterations seen in schizophrenia, but the underlying mechanisms are unclear. Accumulating evidence supports the premise that gut microbiota influence brain development and function by affecting inflammatory mediators, the hypothalamic–pituitaryadrenal axis and neurotransmission, but there is little knowledge of whether the microbiota-gut-brain axis might contribute to the development of schizophrenia-related behaviors. To this end the effects of social isolation (SI; a well-validated animal model for schizophrenia)-induced changes in rat behavior were correlated with alterations in gut microbiota, hippocampal neurogenesis and brain cytokine levels. Twenty-four male Lister hooded rats were housed in social groups (group-housed, GH, 3 littermates per cage) or alone (SI) from weaning (post-natal day 24) for four weeks before recording open field exploration, locomotor activity/novel object discrimination (NOD), elevated plus maze, conditioned freezing response (CFR) and restraint stress at one week intervals. Post-mortem caecal microbiota composition, cortical and hippocampal cytokines and neurogenesis were correlated to indices of behavioral changes. SI rats were hyperactive in the open field and locomotor activity chambers traveling further than GH controls in the less aversive peripheral zone. While SI rats showed few alterations in plus maze or NOD they froze for significantly less time than GH following conditioning in the CFR paradigm, consistent with impaired associative learning and memory. SI rats had significantly fewer BrdU/NeuN positive cells in the dentate gyrus than GH controls. SI rats had altered microbiota composition with increases in Actinobacteria and decreases in the class Clostridia compared to GH controls. Differences were also noted at genus level. Positive correlations were seen between microbiota, hippocampal IL-6 and IL-10, conditioned freezing and open field exploration. Adverse early-life stress resulting from continuous SI increased several indices of ‘anxiety-like’ behavior and impaired associative learning and memory accompanied by changes to gut microbiota, reduced hippocampal IL-6, IL-10 and neurogenesis. This study suggests that early-life stress may produce long-lasting changes in gut microbiota contributing to development of abnormal neuronal and endocrine function and behavior which could play a pivotal role in the aetiology of psychiatric illness.

Dopamine D2-like receptors modulate freezing response, but not the activation of HPA axis, during the expression of conditioned fear.

Considering the complexity of aversive information processing and defensive response expression, a combined action of stress modulators may be required for an optimal performance during threatening situations. Dopamine is now recognized as one of the most active modulators underlying states of fear and anxiety. On the other hand, activation of hypothalamic-pituitary-adrenocortical (HPA) axis, which leads to the release of corticosterone in rodents, has been considered a key part of the stress response. The current study is an extension of prior work investigating modulatory effects of dopamine and corticosterone on conditioned fear expression. We have showed that corticosterone, acting through mineralocorticoid receptors in the ventral tegmental area (VTA), upregulates dopaminergic system in the basolateral amygdala (BLA), enabling the expression of conditioned freezing response. The novel question addressed here is whether VTA-BLA dopaminergic signaling is necessary for increases in corticosterone during conditioned fear expression. Using site-specific treatment with D2-like agonist quinpirole (VTA) and D2-like antagonist sulpiride (BLA), we evaluated freezing and plasma corticosterone in rats exposed to a light used as aversive conditioned stimulus (CS). Intra-VTA quinpirole and intra-BLA sulpiride significantly decreased freezing expression in the conditioned fear test, but this anxiolytic-like effect of the dopaminergic drugs was not associated with changes in plasma corticosterone concentrations. Altogether, data suggest that interferences with the ability of the CS to activate the dopaminergic VTA-BLA pathway reduce the expression of freezing, but activation of the HPA axis seems to occur upstream of the recruitment of dopaminergic mechanisms in conditioned fear states.