Home Cage Control Research Articles

Effects of Immunization With the Soil-Derived Bacterium Mycobacterium vaccae on Stress Coping Behaviors and Cognitive Performance in a "Two Hit" Stressor Model.

Previous studies demonstrate that Mycobacterium vaccae NCTC 11659 (M. vaccae), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with M. vaccae is an effective countermeasure in a “two hit” stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day –28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of M. vaccae (0.1 mg, administered subcutaneously on days –21, –14, –7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0–56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days –1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with M. vaccae induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with M. vaccae. Immunization with M. vaccae stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with M. vaccae also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with M. vaccae stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience.

Expression of a heroin contextually conditioned immune effect in male rats requires CaMKIIα-expressing neurons in dorsal, but not ventral, subiculum and hippocampal CA1

The physiological and motivational effects of heroin and other abused drugs become associated with environmental (contextual) stimuli during repeated drug use. As a result, these contextual stimuli gain the ability to elicit drug-like conditioned effects. For example, after context-heroin pairings, exposure to the heroin-paired context alone produces similar effects on peripheral immune function as heroin itself. Conditioned immune effects can significantly exacerbate the adverse health consequences of heroin use. Our laboratory has shown that exposure to a heroin-paired context suppresses lipopolysaccharide (LPS)-induced splenic nitric oxide (NO) production in male rats, and this effect is mediated in part by the dorsal hippocampus (dHpc). However, specific dHpc output regions, whose efferents might mediate conditioned immune effects, have not been identified, nor has the contribution of ventral hippocampus (vHpc) been investigated. Here, we evaluated the role of CaMKIIα-expressing neurons in the dHpc and vHpc main output regions by expressing Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) under a CaMKIIα promoter in the dorsal subiculum and CA1 (dSub, dCA1) or ventral subiculum and CA1 (vSub, vCA1). After context-heroin conditioning, clozapine-N-oxide (CNO, DREADD agonist) or vehicle was administered systemically prior to heroin-paired context (or home-cage control) exposure and LPS immune challenge. Chemogenetic inhibition of CaMKIIα-expressing neurons in dHpc, but not vHpc, output regions attenuated the expression of conditioned splenic NO suppression. These results establish that the main dHpc output regions, the dSub and dCA1, are critical for this context-heroin conditioned immune effect.

Lack of change in CA1 dendritic spine density or clustering in rats following training on a radial-arm maze task.

Background:Neuronal plasticity is thought to underlie learning and memory formation. The density of dendritic spines in the CA1 region of the hippocampus has been repeatedly linked to mnemonic processes. Both the number and spatial location of the spines, in terms of proximity to nearest neighbour, have been implicated in memory formation. To examine how spatial training impacts synaptic structure in the hippocampus, Lister-Hooded rats were trained on a hippocampal-dependent spatial task in the radial-arm maze. Methods:One group of rats were trained on a hippocampal-dependent spatial task in the radial arm maze. Two further control groups were included: a yoked group which received the same sensorimotor stimulation in the radial-maze but without a memory load, and home-cage controls. At the end of behavioural training, the brains underwent Golgi staining. Spines on CA1 pyramidal neuron dendrites were imaged and quantitatively assessed to provide measures of density and distance from nearest neighbour. Results: There was no difference across behavioural groups either in terms of spine density or in the clustering of dendritic spines. Conclusions:Spatial learning is not always accompanied by changes in either the density or clustering of dendritic spines on the basal arbour of CA1 pyramidal neurons when assessed using Golgi imaging.

Effects of chronic sleep deprivation on upper respiratory tract mucosal histology and mucociliary clearance on rats.

In this study, we aimed to investigate the effects of chronic sleep deprivation on mucociliary clearance, which is the primary defence mechanism of the upper airway tract and nasal mucosal histology. Forty-two Wistar Albino rats (250-300g), 8 or 12 weeks old, were randomly assigned into three groups as follows. The first sleep-deprivation group consisted of 14 rats (A), another 14 of them were assigned to platform group (B), and the remaining 14 were included to the home cage control group (C). For the two deprivation groups (A and B), the modified multiple platform method (MMPM) was used to induce sleep deprivation for 21days. Tc-99m MAA rhinoscintigraphy was performed to assess mucociliary clearance and the nasal histopathological changes of the sacrificed rats were also examined. Mucociliary clearance was significantly higher in sleep deprivation (A) and deprivation control (B) groups than the control group (C) (p=.037). The ratio of columnar ciliary was significantly higher in group A and B than in the control group (p=.003). The transitional epithelial ratio in groups A and B was also significantly increased compared with group C (p=.04). The control group's squamous epithelial ratio was increased compared to the sleep-deprived groups (p=.003). There was a significantly increased inflammatory response in the ciliated columnar epithelium in groups A and B compared to group C (p=.02). For the first time in the literature, we demonstrated that chronic sleep deprivation has caused a significant increase in mucociliary clearance speed and in the number of ciliary cells.

Lack of change in CA1 dendritic spine density or clustering in rats following training on a radial-arm maze task

Background: Neuronal plasticity is thought to underlie learning and memory formation. The density of dendritic spines in the CA1 region of the hippocampus has been repeatedly linked to mnemonic processes. Both the number and spatial location of the spines, in terms of proximity to nearest neighbour, have been implicated in memory formation. To examine how spatial training impacts synaptic structure in the hippocampus, Lister-Hooded rats were trained on a hippocampal-dependent spatial task in the radial-arm maze. Methods: One group of rats were trained on a hippocampal-dependent spatial task in the radial arm maze. Two further control groups were included: a yoked group which received the same sensorimotor stimulation in the radial-maze but without a memory load, and home-cage controls. At the end of behavioural training, the brains underwent Golgi staining. Spines on CA1 pyramidal neuron dendrites were imaged and quantitatively assessed to provide measures of density and distance from nearest neighbour. Results: There was no difference across behavioural groups either in terms of spine density or in the clustering of dendritic spines. Conclusions: Spatial learning is not always accompanied by changes in either the density or clustering of dendritic spines on the basal arbour of CA1 pyramidal neurons when assessed using Golgi imaging.

Lack of change in CA1 dendritic spine density or clustering in rats following training on a radial-arm maze task.

Background:Neuronal plasticity is thought to underlie learning and memory formation. The density of dendritic spines in the CA1 region of the hippocampus has been repeatedly linked to mnemonic processes. Both the number and spatial location of the spines, in terms of proximity to nearest neighbour, have been implicated in memory formation. To examine how spatial training impacts synaptic structure in the hippocampus, Lister-Hooded rats were trained on a hippocampal-dependent spatial task in the radial-arm maze. Methods:One group of rats were trained on a hippocampal-dependent spatial task in the radial arm maze. Two further control groups were included: a yoked group which received the same sensorimotor stimulation in the radial-maze but without a memory load, and home-cage controls. At the end of behavioural training, the brains underwent Golgi staining. Spines on CA1 pyramidal neuron dendrites were imaged and quantitatively assessed to provide measures of density and distance from nearest neighbour. Results: There was no difference across behavioural groups either in terms of spine density or in the clustering of dendritic spines. Conclusions:Spatial learning is not always accompanied by changes in either the density or clustering of dendritic spines on the basal arbour of CA1 pyramidal neurons when assessed using Golgi imaging.

Retrotransposition Bursts in the Adult Brain: Stress, Running, and Instrumental Learning Increase the Number of L1 Retrotransposon DNA Copies in the Mouse Nervous System

Retrotransposons, the DNA elements which proliferate by reverse transcription of their RNA intermediates, comprise a major fraction of mammalian genomes. In the human genome a particularly abundant retrotransposon ‐ long interspersed nuclear element‐1 (LINE‐1 or L1) has 5x105 copies 80–100 of which are active and competent for retrotransposition. Recent findings indicate that LINE‐1 is active in neuronal precursor cells during differentiation, and its de novo insertions can change expression of neuron‐specific genes and cause somatic mosaicism in the adult brain. However, there has been limited understanding whether such retrotranspositions happen in the adult brain, when and how they occur, and what is their importance for the CNS function. In our study we tested whether prolonged stress, wheel running, or operant learning can lead to changes in L1 DNA copy number in the mouse brain. For this purpose, we subjected mice to immobilization stress (2 hours a day, 10 days), wheel running (unlimited access, 10 days) or nose‐poke operant training (1 session a day, 10 days). We collected the samples of hippocampus, frontal cortex, cerebellum, muscle and testicle tissues from experimental and home cage control mice. We performed qPCR for analysis of 3′‐UTR and ORF‐1 copy number. We observed significant increase in the 3′‐UTR and ORF‐1 copy number in all brain structures of the stress group compared to the home cage group. Nose‐poke training was associated with specific increase in 3′‐UTR copy number in the hippocampus. We also observed a small increase in 3′‐UTR and ORF‐1 copy number in all investigated brain structures in wheel running group. Altogether our results show that retrotranspositions is a common event in the adult brain under different conditions of novel experience.Support or Funding InformationRFBR # 19‐015‐00550

Medial prefrontal and ventral hippocampal contributions to incidental context learning and memory in adolescent rats.

The Context Preexposure Facilitation Effect (CPFE) is a contextual fear conditioning (CFC) paradigm in which context learning, context-shock learning, and retrieval of contextual fear occur in three distinct phases. The medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC), and ventral hippocampus (vHPC) are required for the acquisition and/or consolidation of a context representation during incidental context exposure (Heroux et al., 2017; Robinson-Drummer et al., 2016; Rudy & Matus-Amat, 2006). This exposure also induces the expression of the immediate early genes (IEGs) c-Fos, Arc, Egr-1, and Npas4 in these regions (Heroux et al., 2018, 2019). Despite these studies, it is still unclear how mPFC and vHPC contribute to incidental context learning and memory. The current study examined whether prefrontal or ventral hippocampal inactivation during context preexposure interferes with long-term context memory and IEG activity in the mPFC, vHPC, dHPC and the ventral midline thalamus (VMT, a region connected to both the mPFC and HPC). Adolescent Long-Evans rats were given intra-mPFC (Experiment 1) or intra-vHPC (Experiment 2) infusions of the GABAA receptor agonist muscimol or PBS prior to context preexposure, and then were sacrificed 30 min later and whole mPFC, dHPC, vHPC, and VMT were collected and assayed for IEG mRNA expression via qPCR. Prefrontal or ventral hippocampal inactivation during context exposure abolished subsequent post-shock and retention test freezing in behaviorally-tested littermates of the sacrificed groups. In Experiment 1, prefrontal inactivation reduced expression of c-Fos, Arc, Egr-1, and Npas4 in the mPFC, c-Fos, Arc, and Npas4 in the vHPC, and c-Fos in the VMT, to the level of behaviorally-naïve home-cage controls. Prefrontal inactivation did not alter IEG expression in the dHPC during context exposure. In Experiment 2, ventral hippocampal inactivation impaired expression of all IEGs in the mPFC, dHPC, and vHPC, with no effect in the VMT. Taken together, these results suggest that context memory processes on the preexposure day of the CPFE may depend on mPFC-vHPC circuitry not typically emphasized in studies of incidental or configural learning and memory.

Local inhibition of uptake2 transporters augments stress-induced increases in serotonin in the rat central amygdala

Organic cation transporter 3 (OCT3) is a corticosterone-sensitive, low-affinity, high-capacity transporter. This transporter functions, in part, to clear monoamines, including serotonin (5-HT), from the extracellular space. The central nucleus of the amygdala (CeA) is an important structure controlling fear- and anxiety-related behaviors. The CeA has reciprocal connections with brainstem nuclei containing monoaminergic systems, including serotonergic systems arising from the dorsal raphe nucleus, which are thought to play an important role in modulation of CeA-mediated behavioral responses. Organic cation transporter 3 (OCT3) is expressed in the CeA, but little is known about the role of OCT3 within the CeA in modulating serotonergic signaling. We hypothesized that inhibition of OCT3-mediated transport in the CeA during restraint stress would increase extracellular 5-HT. In Experiment 1, rats received unilateral reverse dialysis of either corticosterone or normetanephrine, which interfere with OCT3-mediated transport, into the CeA under home cage control conditions. In Experiment 2, rats received unilateral reverse dialysis of corticosterone, normetanephrine, or vehicle into the CeA, while undergoing a 40-min period of restraint stress. Infusion of these drugs had no effect on extracellular concentrations of 5-HT during home cage control conditions, but, in contrast, markedly increased extracellular concentrations of 5-HT during restraint stress, relative to vehicle-treated controls. These findings suggest a role for OCT3 in the CeA in control of serotonergic signaling during stressful conditions.

Abstract # 2044 Glucocorticoids mediate stress-induced upregulation of the alarmin HMGB1 and downregulation of the inhibitory receptor CD200R1 in hippocampus and amygdala: A mechanism of neuroinflammatory priming

Glucocorticoids (GCs) have universally been considered anti-inflammatory. However, mounting evidence now demonstrates that GCs can exert paradoxical effects on innate immune function. In the context of stress exposure, GCs have been found to mediate stress-induced priming of neuroinflammatory responses to subsequent immune challenges. Recent evidence from our laboratory suggests that the alarmin HMGB1 as well as the microglial inhibitory receptor CD200R1 play a causal role in stress-induced neuroinflammatory priming. Here, we explored whether GCs mediate the effects of stress on HMGB1 and CD200R1. Male Sprague-Dawley rats were injected (SC) with vehicle or the GC receptor antagonist RU486 (50 mg/kg). 24 h after injection, rats were exposed to inescapable tailshock (IS) or served as home cage controls (HCCs). Stress exposure increased HMGB1 levels, while decreasing CD200R1 levels in hippocampus and amygdala. Treatment with RU486 blocked these effects of stress on HMGB1 and CD200R1. To examine whether GCs are sufficient to induce these changes in HMGB1 and CD200R1 expression, rats were injected (SC) with vehicle or corticosterone (2.5 mg/kg), which recapitulates the pattern of the GC response to stress exposure. 2 h after injection, corticosterone treatment decreased CD200R1 expression and increased HMGB1 levels. The present results suggest that GCs mediate the effects of stress on HMGB1 and CD200R1, which provides insight into the mechanisms by which stress-induced GCs prime neuroinflammatory processes to subsequent immune challenges.

Dynamic Patterns of Threat-Associated Gene Expression in the Amygdala and Blood.

Stress and trauma profoundly influence psychiatric biobehavioral outcomes. The identification of treatment and biomarker targets would be accelerated by a broad understanding of the biological responses to these events. The goal of this study was to determine genes responsive to auditory fear conditioning (FC), a well-characterized amygdala-dependent rodent model of threat-exposure, in the presence or absence of prior stress history, providing insight into the physiological processes underlying response to trauma. RNA-sequencing was performed in blood and amygdala from mice that underwent fear conditioning with (Immo+FC) and without (FC) prior immobilization stress, a paradigm that induces HPA axis, and behavioral stress sensitization. In the amygdala, 607 genes were regulated by FC vs. home-cage (HC) controls, and 516 genes differed in stress-sensitized mice (Immo+FC vs. FC). In the former, we observed an enhancement of specific biological processes involved in learning and synaptic transmission, and in the latter processes associated with cell proliferation and the cellular response to drugs. In the blood of stress-sensitized animals, 468 genes were dynamically regulated when compared to FC, and were enriched for the biological pathways of inflammation and cytokine signaling. This study identified genes and pathways that respond to threat in the amygdala and blood of mice with and without a prior stress history and reveals the impact of stress history on subsequent inflammation. Future studies will be needed to examine the role of these dynamically regulated genes may play in human clinical stress and trauma-related disorders.

Circadian misalignment has differential effects on affective behavior following exposure to controllable or uncontrollable stress

In modern 24 h society, circadian disruption is pervasive, arising from night shift work, air travel across multiple time zones, irregular sleep schedules, and exposure to artificial light at night. Disruption of the circadian system is associated with many adverse health consequences, including mood disorders. Here we investigate whether inducing circadian misalignment using a phase advance protocol interferes with the ability to cope with a stressor, thereby increasing susceptibility to the negative consequences of stress. Male rats were maintained on a standard 12:12 light: dark (LD) cycle or subjected to a chronic phase advance (CPA) protocol involving 4 weekly 6 h phase shifts (earlier light onset) of the LD cycle. Rats were then exposed to escapable stress (ES), inescapable stress (IS), or no stress (home cage control; HC) and performance on juvenile social exploration and active escape learning in the two-way shuttlebox test was assessed 24 h and 48 h following stress, respectively. CPA alone had no effect on pre-stress juvenile social exploration, and it also did not interfere with the protective effect of ES on the stress-induced reduction in juvenile social exploration. In contrast, CPA impaired escape learning in the two-way shuttlebox to the same extent as IS in all subjects, regardless of stress history. Additionally, CPA produced somatic alterations that included increased body mass, increased epididymal adiposity, and decreased adrenal mass. These data indicate that CPA differentially modulated the stress-protective effects of behavioral control depending on the type of affective behavior examined.

Neurons Specifically Activated by Fear Learning in Lateral Amygdala Display Increased Synaptic Strength.

The lateral amygdala (LA) plays a critical role in the formation of fear-conditioned associative memories. Previous studies have used c-fos regulated expression to identify a spatially restricted population of neurons within the LA that is specifically activated by fear learning. These neurons are likely to be a part of a memory engram, but, to date, functional evidence for this has been lacking. We show that neurons within a spatially restricted region of the LA had an increase in both the frequency and amplitude of spontaneous postsynaptic currents (sPSC) when compared to neurons recorded from home cage control mice. We then more specifically addressed if this increased synaptic activity was limited to learning-activated neurons. Using a fos-tau-LacZ (FTL) transgenic mouse line, we developed a fluorescence-based method of identifying and recording from neurons activated by fear learning (FTL+) in acute brain slices. An increase in frequency and amplitude of sPSCs was observed in FTL+ neurons when compared to nonactivated FTL− neurons in fear-conditioned mice. No learning-induced changes were observed in the action potential (AP) input-output relationships. These findings support the idea that a discrete LA neuron population forms part of a memory engram through changes in synaptic connectivity.

Attenuated cocaine-seeking after oxytocin administration in male and female rats.

Initial drug abstinence (modeled here as Extinction Day 1, ED1) is a critical time point in the progression of addiction that is strongly influenced by stress and sex. ED1 induces corticosterone release in both sexes, and cocaine-seeking during ED1 can be mitigated by corticotrophin-releasing factor (CRF) antagonists more effectively in female rats. Oxytocin (OXT) is a neuropeptide that has several biological functions, including regulation of stress pathways. To investigate a relationship between OXT, sex, and cocaine-seeking, we examined Fos on ED1 in OXT neurons of paraventricular (PVN) and supraoptic nuclei (SON) compared to homecage (cocaine experienced) or naïve male and female rats. We also administered OXT 30min prior to ED1 testing or cued reinstatement testing. OXT neurons had decreased activity (as reflected by Fos protein) in PVN and SON on withdrawal day 1 (homecage) compared to naïve rats. Fos in OXT neurons was further decreased on ED1, compared to homecage controls, in both males and females even though in SON, cocaine exposure increased the number of OXT-expressing neurons. In addition, systemically administered OXT reduced cocaine-seeking during ED1 and cue-induced reinstatement of cocaine-seeking but delayed extinction, similarly among male and female rats. These data indicate that OXT neurons in PVN and SON may be involved in cocaine-seeking during ED1 and support OXT as a possible therapeutic to decrease cocaine-seeking during initial abstinence and in response to cocaine-associated cues.

Metabolic profile associated with distinct behavioral coping strategies of 129Sv and Bl6 mice in repeated motility test

We investigated the metabolic outcome of different coping strategies in 129S6/SvEvTac (129Sv) and C57BL/6Ntac (Bl6) strains. Two different batches of male 129Sv and Bl6 mice were used. One batch was not subjected to any behavioral manipulations (home cage control; HCC), whereas the other batch was treated with saline for 11 days and exposed after every treatment to the motor activity measurement (repeated motility tested; RMT). Bl6 RMT mice displayed a robust increase in number of rearings during repeated testing. 129Sv RMT mice experienced significant loss of body weight, but showed enhanced weight gain in HCC batch compared to Bl6. Serum metabolites (acylcarnitines, amino acids, biogenic amines, hexoses, glycerophospholipids and sphingolipids) were determined with AbsoluteIDQ p180 kit. Results of the metabolomic study revealed prominent peculiarities between strains in two different conditions. Comparison of both batches of mice demonstrated that in Bl6 biogenic amines (acetyl-ornithine, alpha-amionadipic acid, carnosine) and lysophosphatidylcholine PC(16:1/0:0) dominated. However in 129Sv acylcarnitine C5 clearly dominated, indicating shift towards short-chain acylcarnitines. Stable strain-specific ratios also emerged for both lines, ratio of glycine/PC ae C38:2 for Bl6 and ratios of C5/C0 as well as PC(16:0/0:0)/PC(16:1/0:0) for 129Sv. The described metabolic changes probably reflect different behavioral coping strategies of 129Sv and Bl6 mice.

Investigating Tryptophan Hydroxylase Immunoreactivity in the Dorsal Raphe of Rats Exposed to Early Adversity

Prior studies link anxiety to alterations in serotonergic neurotransmission. Tao and Menard (unpublished data) found that female rats exposed to intermittent physical stress during early adolescence display greater levels of anxiety-like behaviours when tested as adults in social interaction tests. Interestingly, these behavioural changes were associated with changes in serotonin (5-HT) fiber density in the medial prefrontal cortex. Whether these changes in 5-HT fibre density are accompanied by changes in the number of 5-HT-producing cells in the dorsal raphe is not known. To examine this research question, tryptophan-hydroxylase (TPH) immunoreactivity (-IR) was used to label 5-HT cells in the dorsal raphe. Half the animals from the original adolescent stress study were exposed to IPS, and the remaining animals were handled only, serving as the control (CON) group. In adulthood, half of each of those groups were tested in the SI test. The remaining animals were not behaviourally tested, serving as home-cage controls (HCC). A two-factor analysis of variance will be performed with Treatment (IPS vs CON) and Testing (SI vs HCC) as the two factors. It is expected that, regardless of Testing, IPS rats will display higher levels of TPH-IR in the dorsal raphe than no-stress controls. The number of TPH-IR-positive cells will also be correlated with archival behavioural data. It is expected that that higher levels of TPH-IR will be associated with higher levels of anxiety-like behaviour. This study provides insights into the role that early adversity plays in shaping the structure and function of the brain’s serotonergenic system.

Two models of inescapable stress increase tph2 mRNA expression in the anxiety-related dorsomedial part of the dorsal raphe nucleus

Expression of TPH2, the rate-limiting enzyme for brain serotonin synthesis, is elevated in the dorsal raphe nucleus (DR) of depressed suicide victims. One hypothesis is that this increase in TPH2 expression is stress-induced. Here, we used an established animal model to address whether exposure to an acute stressor, inescapable tail shock (IS), increases tph2 mRNA and Tph2 protein expression, and if IS sensitizes the DR to a subsequent, heterotypic stressor. In Experiment 1, we measured tph2 mRNA expression 4 h after IS or home cage (HC) control conditions in male rats, using in situ hybridization histochemistry. In Experiment 2, we measured Tph2 protein expression 12 h or 24 h after IS using western blot. In Experiment 3, we measured tph2 mRNA expression following IS on Day 1, and cold swim stress (10 min, 15 °C) on Day 2. Inescapable tail shock was sufficient to increase tph2 mRNA expression 4 h and 28 h later, selectively in the dorsomedial DR (caudal aspect of the dorsal DR, cDRD; an area just rostral to the caudal DR, DRC) and increased Tph2 protein expression in the DRD (rostral and caudal aspects of the dorsal DR combined) 24 h later. Cold swim increased tph2 mRNA expression in the dorsomedial DR (cDRD) 4 h later. These effects were associated with increased immobility during cold swim, elevated plasma corticosterone, and a proinflammatory plasma cytokine milieu (increased interleukin (IL)-6, decreased IL-10). Our data demonstrate that two models of inescapable stress, IS and cold swim, increase tph2 mRNA expression selectively in the anxiety-related dorsomedial DR (cDRD).

Argan Oil Supplementation Reverses Anxiety and Depressive-Like Behaviors, Neurodegeneration and Oxidative Stress in Amygdala Induced by Chronic Mild Stress in Rats

Background: Argan Oil (AO) has been used as a natural remedy in traditional medicine, mainly in Morocco, for several centuries. In this study, we evaluated the beneficial effects of AO dietary on vulnerability of rats to the chronic unpredictable mild stress (UCMS) using behavioral tests, biochemical and histological markers of depression or anxiety. Method: Rats were handled daily (home cage control) or subjected to the UCMS procedure during 6 weeks (i.e., from 43th to 85 Post-natal Day (PND)) (Stress group, n=12). Animals were previously administered orally by NaCl 0.9% (Control group, n=11) or AO (10 ml/kg/day) (AO+Stress group, n=12) for 10 weeks (i.e., from weaning 21th to 93 PND). The efficacy of UCMS or AO dietary on behavioral performances of the animals in the open field, the forced swimming, the light/dark, the novelty suppression of feeding and sucrose preference tests, was measured. Following behavioral assays, oxidative stress in amygdala, histologic semiquantitative analysis of neurodegeneration in the hippocampus, frontal cortex and basolateral amygdala (BLA) subregions, and corticosterone level in plasma was also performed. Results: Our data supports pharmacological and biochemical evidences for the antidepressant and anxiolytic-like effects of AO. Prolonged supplementation with AO reverses all the behavioral changes that occurred due to UCMS and restored corticosterone level in the plasma, oxidative status of amygdala and the neurons level in the CA3 subregion of rats’ hippocampus. Conclusion: This study suggests that antidepressant and anxiolytic like effects of AO in adult rats can be the result of modulation of brain antioxidant enzyme activities, the activation of hippocampal neurogenesis and the modulation of HPA axis activity. However, more experiment and detailed analysis is required for definitive conclusion.

Enhancing Endocannabinoid Neurotransmission Augments The Efficacy of Extinction Training and Ameliorates Traumatic Stress-Induced Behavioral Alterations in Rats.

Exposure to a traumatic event may result in the development of post-traumatic stress disorder (PTSD). Endocannabinoids are crucial modulators of the stress response, interfere with excessive retrieval and facilitate the extinction of traumatic memories. Exposure therapy, combined with pharmacotherapy, represents a promising tool for PTSD treatment. We investigated whether pharmacological manipulations of the endocannabinoid system during extinction learning ameliorates the behavioral changes induced by trauma exposure. Rats were exposed to inescapable footshocks paired with social isolation, a risk factor for PTSD. One week after trauma, rats were subjected to three spaced extinction sessions, mimicking human exposure therapy. The anandamide hydrolysis inhibitor URB597, the 2-arachidonoylglycerol hydrolysis inhibitor JZL184 or the cannabinoid agonist WIN55,212-2 were administered before or after the extinction sessions. Rats were tested for extinction retention 16 or 36 days after trauma and 24-h later for social interaction. Extinction training alone reduced fear of the trauma-associated context but did not restore normal social interaction. Traumatized animals not exposed to extinction sessions exhibited reductions in hippocampal anandamide content with respect to home-cage controls. Noteworthy, all drugs exerted beneficial effects, but URB597 (0.1 mg/kg) induced the best improvements by enhancing extinction consolidation and restoring normal social behavior in traumatized rats through indirect activation of CB1 receptors. The ameliorating effects remained stable long after treatment and trauma exposure. Our findings suggest that drugs potentiating endocannabinoid neurotransmission may represent promising tools when combined to exposure-based psychotherapies in the treatment of PTSD.

Differential expression of the immediate early genes c-Fos, Arc, Egr-1, and Npas4 during long-term memory formation in the context preexposure facilitation effect (CPFE).

The context preexposure facilitation effect (CPFE) is a contextual fear conditioning paradigm in which learning about the context, acquiring the context-shock association, and retrieving/expressing contextual fear are temporally dissociated into three distinct phases (context preexposure, immediate-shock training, and retention). The current study examined changes in the expression of plasticity-associated immediate early genes (IEGs) during context and contextual fear memory formation on the preexposure and training days of the CPFE, respectively. Using adolescent Long-Evans rats, preexposure and training day expression of the IEGs c-Fos, Arc, Egr-1, and Npas4 in the medial prefrontal cortex (mPFC), dorsal hippocampus (dHPC), and basolateral amygdala (BLA) was analyzed using qPCR as an extension of previous studies from our lab examining Egr-1 via in situ hybridization (Asok, Schreiber, Jablonski, Rosen, & Stanton, 2013; Schreiber, Asok, Jablonski, Rosen, & Stanton, 2014). In Expt. 1, context preexposure induced expression of c-Fos, Arc, Egr-1 and Npas4 significantly above that of home-cage (HC) controls in all three regions. In Expt. 2, immediate-shock was followed by a post-shock freezing test, resulting in increased mPFC c-Fos expression in a group preexposed to the training context but not a control group preexposed to an alternate context, indicating expression related to associative learning. This was not seen with other IEGs in mPFC or with any IEG in dHPC or BLA. Finally, when the post-shock freezing test was omitted in Expt. 3, training-related increases were observed in prefrontal c-Fos, Arc, Egr-1, and Npas4, hippocampal c-Fos, and amygdalar Egr-1 expression. These results indicate that context exposure in a post-shock freezing test re-engages IEG expression that may obscure associatively-induced expression during contextual fear conditioning. Additionally, these studies suggest a key role for long-term synaptic plasticity in the mPFC in supporting the CPFE.