Effects of acidic-astressin and ovine-CRF microinfusions into the ventral hippocampus on defensive behaviors in rats

This study investigated a possible role for ventral hippocampal corticotropin-releasing factor (CRF) in modulating both unconditioned and conditioned defensive behaviors by examining the effects of pre-training ventral hippocampal ovine-CRF (oCRF) or acidic-astressin ([Glu^11,16]Ast) microinfusions in male Long-Evans hooded rats exposed to various threat stimuli including the elevated plus-maze (EPM) (oCRF), cat odor (oCRF and [Glu^11,16]Ast) and a live cat ([Glu^11,16]Ast). Unconditioned defensive behaviors were assessed during threat exposure, while conditioned defensive behaviors were assessed in each predator context 24 h after the initial threat encounter. Pre-training infusions of the CRF₁ and CRF₂ receptor agonist oCRF significantly increased defensive behaviors during both the unconditioned and conditioned components of the cat odor test, as well as exposure to the EPM. In contrast to the behavioral effects of oCRF microinfusions, the CRF₁ and CRF₂ receptor antagonist [Glu^11,16]Ast significantly decreased defensive behaviors during exposure to cat odor, while producing no discernible effects following a second injection in the cat exposure test. During conditioned test trials, pre-training infusions of [Glu^11,16]Ast also significantly reduced defensive behaviors during re-exposure to both predator contexts. These results suggest a specific role for ventral hippocampal CRF receptors in modulating anxiety-like behaviors in several ethologically relevant animal models of defense.     

CRF type 1 receptors in the dorsal periaqueductal gray modulate anxiety-induced defensive behaviors

The dorsal periaqueductal gray (dPAG) is involved in defensive coping reactions to threatening stimuli. Corticotropin releasing factor (CRF) is substantially implicated as a direct modulator of physiological, endocrine and behavioral responses to a stressor. Previous findings demonstrate a direct role of the central CRF system in dPAG-mediated defensive reactions toward a threatening stimulus. These include anxiogenic behaviors in the elevated plus maze (EPM) in rats and defensive reactions in both the mouse defense test battery (MDTB) and rat exposure test (RET) paradigms in mice. Furthermore, CRF was shown to directly and dose-dependently excite PAG neurons in vitro. The aim of the present series of experiments was to directly evaluate the role of the CRF1 receptor (CRF1) in dPAG-induced defensive behaviors in the MDTB and the RET paradigms. For this purpose, cortagine, a novel CRF1-selective agonist, was directly infused into the dPAG. In the RET the high dose of cortagine (100 ng) significantly affected spatial avoidance measures and robustly increased burying behavior, an established avoidance activity, while having no effects on behaviors in the MDTB. Collectively, these results implicate CRF1 in the dPAG as a mediator of temporally and spatially dependent avoidance in response to controllable and constant stimuli.     

Increased corticosterone levels in mice subjected to the rat exposure test

In recent years, there has been a notable interest in studying prey-predator relationships to develop rodent-based models for the neurobehavioral aspects of stress and emotion. However, despite the growing use of transgenic mice and results showing important differences in the behavioral responses of rats and mice, little research has been conducted regarding the responses of mice to predators. The rat exposure test (RET), a recently developed and behaviorally validated prey-predator (mouse-rat)-based model, has proven to be a useful tool in evaluating the defensive responses of mice facing rats. To further validate the RET, we investigated the endocrine and behavioral responses of mice exposed to this apparatus. We first constructed a plasma corticosterone secretion curve in mice exposed to a rat or to an empty cage (control). Rat-exposed mice showed a pronounced rise in corticosterone levels that peaked 15 min from the beginning of the predator exposure. The corticosterone levels and behavioral responses of mice exposed to a rat or to a toy in the RET apparatus were then measured. We observed high plasma corticosterone levels along with clear avoidance behaviors represented by decreases in tunnel and surface area exploration and increases in risk assessment behaviors and freezing. This strongly suggests that the test elicits a repertoire of behavioral responses compatible with an aversion state and indicates that it is a promising model for the evaluation of prey-predator interactions. However, more physiological, neurochemical, and pharmacological studies are needed to further validate the test.     

Mineralocorticoid receptors in the medial prefrontal cortex and hippocampus mediate rats' unconditioned fear behaviour

Corticosterone is released from the adrenal cortex in response to stress, and binds to glucocorticosteroid receptors (GRs) and mineralocorticosteroid receptors (MRs) in the brain. Areas such as the dorsal hippocampus (DH), ventral hippocampus (VH) and medial prefrontal cortex (mPFC) all contain MRs and have been previously implicated in fear and/or memory.The purpose of the following experiments was to examine the role of these distinct populations of MRs in rats’ unconditioned fear and fear memory.The MR antagonist (RU28318) was microinfused into the DH, VH, or mPFC of rats. Ten minutes later, their unconditioned fear was tested in the elevated plus-maze and the shock-probe tests, two behavioral models of rat “anxiety.” Twenty-four hours later, conditioned fear of a non-electrified probe was assessed in rats re-exposed the shock-probe apparatus.Microinfusions of RU28318 into each of the three brain areas reduced unconditioned fear in the shock-probe burying test, but only microinfusions into the VH reduced unconditioned fear in the plus-maze test. RU28318 did not affect conditioned fear of the shock-probe 24 hr later.MRs in all three areas of the brain mediated unconditioned fear to a punctate, painful stimulus (probe shock). However, only MRs in the ventral hippocampus seemed to mediate unconditioned fear of the more diffuse threat of open spaces (open arms of the plus maze). In spite of the known roles of the hippocampus in spatial memory and conditioned fear memory, MRs within these sites did not appear to mediate memory of the shock-probe.     

Anxiety and panic responses to a predator in male and female Ts65Dn mice, a model for Down syndrome

Hyperactivity is a feature frequently reported in behavioral studies on the Ts65Dn (TS) mouse, the most widely accepted model of Down syndrome, when tested in anxiety-provoking situations such as the plus-maze and the open-field tests. Although this behavior could be considered as an expression of reduced anxiety, it has been considered as a consequence of a lack of behavioral inhibition and/or reduced attention. This study addressed anxiety and panic behavior of male and female TS mice by evaluating serum biochemical parameters and behavioral responses to a predator in the Mouse Defense Test Battery. Flight, risk assessment, defensive threat/attack and escape attempts were measured during and after rat confrontation. When confronted to a rat, male TS mice showed similar biochemical and behavioral responses as control mice. However, female control and TS mice presented lower serum adrenocorticotropic hormone (ACTH) levels under basal conditions and higher corticosterone levels after predator exposure than male mice. Thus, there was a larger increase in ACTH and corticosterone levels after predator exposure with respect to the undisturbed condition in females than in males. In addition, TS females showed some alterations in defensive behaviors after predator exposure. The results emphasize the need to consider gender as a confounding factor in the behavioral assessment of TS mice.     

Tonic modulation of anxiety-like behavior by corticotropin-releasing factor (CRF) type 1 receptor (CRF1) within the medial prefrontal cortex (mPFC) in male mice: Role of protein kinase A (PKA)

The medial prefrontal cortex (mPFC) and the neuropeptide corticotropin-releasing factor (CRF) have recently been receiving more attention from those interested in the neurobiology of anxiety. Here, we investigated the CRF pathway in the modulation of anxiety-like behaviors in male mice exposed to the elevated plus-maze (EPM), through intra-mPFC injections of CRF, CP376395 [N-(1-ethylpropyl)-3,6-dimethyl-2-(2,4,6-trimethylphenoxy)-4-pyridinamine hydrochloride, a CRF type 1 receptor antagonist (CR F1)] or H-89 [N-[2-[[3-(4-bromophenyl)-2-propenyl]amino]ethyl]-5-isoquinolinesulfonamide dihydrochloride, a protein kinase (PKA) inhibitor]. We also investigated the effects of intra-mPFC injections of H-89 on the behavioral effects induced by CRF. Mice received bilateral intra-mPFC injections of CRF (0, 37.5, 75 or 150 pmol), CP376395 (0, 0.75, 1.5 or 3 nmol) or H-89 (0, 1.25, 2.5 or 5 nmol) and were exposed to the EPM, to record conventional and complementary measures of anxiety for 5 min. Results showed that while CRF (75 and 150 pmol) produced an anxiogenic-like effect, CP376395 (all doses) and H-89 (5 nmol) attenuated anxiety-like behavior. When injected before CRF (150 pmol), intra-mPFC H-89 (2.5 nmol, a dose devoid of intrinsic effects on anxiety) completely blocked the anxiogenic-like effects of CRF. These results suggest that (i) CRF plays a tonic anxiogenic-like role at CRF1 receptors within the mPFC, since their blockade per se attenuated anxiety indices and (ii) the anxiogenic-like effects following CRF1 receptor activation depend on cAMP/PKA cascade activation in this limbic forebrain area.     

Executive Function Deficits and Social-Behavioral Abnormality in Mice Exposed to a Low Dose of Dioxin In Utero and via Lactation

An increasing prevalence of mental health problems has been partly ascribed to abnormal brain development that is induced upon exposure to environmental chemicals. However, it has been extremely difficult to detect and assess such causality particularly at low exposure levels. To address this question, we here investigated higher brain function in mice exposed to dioxin in utero and via lactation by using our recently developed automated behavioral flexibility test and immunohistochemistry of neuronal activation markers Arc, at the 14 brain areas. Pregnant C57BL/6 mice were given orally a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at a dose of either 0, 0.6 or 3.0 µg/kg on gestation day 12.5. When the pups reached adulthood, they were group-housed in IntelliCage to assess their behavior. As a result, the offspring born to dams exposed to 0.6 µg TCDD/kg were shown to have behavioral inflexibility, compulsive repetitive behavior, and dramatically lowered competitive dominance. In these mice, immunohistochemistry of Arc exhibited the signs of hypoactivation of the medial prefrontal cortex (mPFC) and hyperactivation of the amygdala. Intriguingly, mice exposed to 3.0 µg/kg were hardly affected in both the behavioral and neuronal activation indices, indicating that the robust, non-monotonic dose-response relationship. In conclusion, this study showed for the first time that perinatal exposure to a low dose of TCDD in mice develops executive function deficits and social behavioral abnormality accompanied with the signs of imbalanced mPFC-amygdala activation.     

Source Odor,Intensity, and Exposure Pattern Affect Antipredatory Responses in the Subterranean Rodent Ctenomys talarum

Predation is a strong selective force, and prey species may show specific adaptations that allow recognition, avoidance, and defense against predators. Facing a situation of predatory risk, anxiety constitutes a reaction of adaptive value, allowing to evaluate the potential risk of this encounter as well as to generate a physiological and behavioral response. Previous studies in the subterranean rodent Ctenomys talarum revealed that exposure to predator odors (urine or fur) generates an anxiety state and induces behavioral changes. However, no differences between the responses generated by both odor sources were observed, although fur odors may indicate a higher level of predatory immanence. Therefore, the aim of this study was to evaluate the behavioral and physiological responses of C. talarum to different intensities of predator odors (urine and fur) and to the repeated exposition to the same odorous stimulus. When comparing the highest behavioral effects elicited by both predatory odors on C. talarum, our study supports the assumption that fur odors are more anxiogenic than urine, while the former provoked significant changes in the distance traveled, the number of arm entries and time in transparent arms in the elevated plus maze; cat urine only caused slight changes on those behavioral parameters. Furthermore, we also found that the intensity of natural predator odor presented to tuco‐tucos has a role on the appearance of defensive behaviors, although an amount‐dependent relationship between predator odor and anxiety levels was not observed. Finally, while individuals exposed for 1 day to fur odor displayed an evident anxiety state, those exposed repeatedly for 5 consecutive days did not differ with the control group in their behavioral response, indicating a clear habituation to the predatory cue. In our intensity and habituation experiments, we did not find differences in the measured physiological parameters among control individuals, exposed to different cues intensity (urine and fur odor) and exposed only once or for 5 days to fur odor. These results provide valuable evidence that the types of predatory odor, along with the frequency of exposition, are important determinants of the appearance, strength, and extinction of defensive behaviors in the subterranean rodent C. talarum.     

Chronic alcohol exposure alters behavioral and synaptic plasticity of the rodent prefrontal cortex

In the present study, we used a mouse model of chronic intermittent ethanol (CIE) exposure to examine how CIE alters the plasticity of the medial prefrontal cortex (mPFC). In acute slices obtained either immediately or 1-week after the last episode of alcohol exposure, voltage-clamp recording of excitatory post-synaptic currents (EPSCs) in mPFC layer V pyramidal neurons revealed that CIE exposure resulted in an increase in the NMDA/AMPA current ratio. This increase appeared to result from a selective increase in the NMDA component of the EPSC. Consistent with this, Western blot analysis of the postsynaptic density fraction showed that while there was no change in expression of the AMPA GluR1 subunit, NMDA NR1 and NRB subunits were significantly increased in CIE exposed mice when examined immediately after the last episode of alcohol exposure. Unexpectedly, this increase in NR1 and NR2B was no longer observed after 1-week of withdrawal in spite of a persistent increase in synaptic NMDA currents. Analysis of spines on the basal dendrites of layer V neurons revealed that while the total density of spines was not altered, there was a selective increase in the density of mushroom-type spines following CIE exposure. Examination of NMDA-receptor mediated spike-timing-dependent plasticity (STDP) showed that CIE exposure was associated with altered expression of long-term potentiation (LTP). Lastly, behavioral studies using an attentional set-shifting task that depends upon the mPFC for optimal performance revealed deficits in cognitive flexibility in CIE exposed mice when tested up to 1-week after the last episode of alcohol exposure. Taken together, these observations are consistent with those in human alcoholics showing protracted deficits in executive function, and suggest these deficits may be associated with alterations in synaptic plasticity in the mPFC.     

Fear‐related behaviors in Lurcher mutant mice exposed to a predator

The Lurcher mutant mice are characterized by massive cerebellar cortex degeneration. Besides their motor and cognitive disturbances, they exhibit both exaggerated blood corticosterone (CORT) level surge and behavioral disinhibition when confronted to anxiogenic conditions (i.e. to a potential threat). In this study, we assessed if such physiological and behavioral hyperactivity was also detectable in a fear‐eliciting situation (actual threat). For this purpose, the behaviors and CORT level elevations in Lurcher mice were compared with those of littermate controls in the predator exposure test: mice were exposed either to a rat (exposure) or to a brief wave of the experimenter's hand (sham exposure). While the basal CORT concentrations (24 h before testing) were not significantly different between mice of both genotypes, the post‐exposure ones were higher in Lurcher than in control mice whatever the condition of the experimental design (exposure or sham exposure). Predator exposure did not provoke significant increase of CORT levels whatever the genotype. On the contrary, our data clearly showed that fear‐related behaviors of cerebellar mutants facing a real threat were exacerbated in comparison to those of control mice. These results suggest that the cerebellar cortex not only participates to fear conditioning and anxiety but also actively contributes to the modulation of the innate fear‐related behaviors.     

Effects of the stimulus and chamber size on unlearned fear across development

Predator odors have been found to induce unconditioned fear in adult animals and provide the opportunity to study the mechanisms underlying unlearned and learned fear. Predator threats change across an animal's lifetime, as do abilities that enable the animal to learn or engage in different defensive behaviors. Thus, the objective of this study was to determine the combination of factors that successfully induce unlearned fear to predator odor across development. Infant, juvenile, adolescent, and adult rats were exposed to one of the three odor stimuli (control odor, cat urine, or cat fur) in either a small or large chamber. Though all ages displayed fear-related behavior to cat odors, differences were reflected only in freezing behavior and not, as expected, risk-assessment. Infant and juvenile animals also increased freezing to cat urine compared to the control odor, possibly because these age groups possess limited defensive options to cope with threat and so may respond with freezing to all predator stimuli. Unexpectedly, chamber size had no effect on either freezing or risk-assessment in this study. Once the parameters of unconditioned fear are understood, they can be exploited to develop a learning paradigm to predator odors that could be used in early life.     

Age-specific threats induce CRF expression in the paraventricular nucleus of the hypothalamus and hippocampus of young rats

Young animals respond to threatening stimuli in an age-specific way. Their endocrine and behavioral responses reflect the potential threat of the situation at a given age. The aim of the present study was to determine whether corticotropin-releasing factor (CRF) is involved in the endocrine and behavioral responses to threat and their developmental changes in young rats. Preweaning 14-day-old and postweaning 26-day-old rats were exposed to two age-specific threats, cat odor and an adult male rat. The acute behavioral response was determined during exposure. After exposure, the time courses of the corticosterone response and of CRF expression in the paraventricular nucleus of the hypothalamus (PVN) and in extrahypothalamic areas were assessed. Preweaning rats became immobile when exposed to cat odor or the male rat, whereas postweaning rats became immobile to cat odor only. Male exposure increased serum corticosterone levels in 14-day-old rats, but cat odor failed to increase levels at either age. Exposure induced elevation of CRF mRNA levels in the PVN that paralleled changes in corticosterone levels. CRF may thus play a role in endocrine regulation and its developmental changes during early life. Neither cat odor nor the adult male altered CRF mRNA levels in the bed nucleus of the stria terminalis (BNST) or the amygdala, but both stimuli increased levels in the hippocampus. Hippocampal CRF mRNA expression levels did not parallel cat odor or male-induced immobility, indicating that CRF is not involved in this response in young rats but may be involved in aspects of learning and memory.     

Single units in the medial prefrontal cortex with anxiety-related firing patterns are preferentially influenced by ventral hippocampal activity

The medial prefrontal cortex (mPFC) and ventral hippocampus (vHPC) functionally interact during innate anxiety tasks. To explore the consequences of this interaction, we examined task-related firing of single units from the mPFC of mice exploring standard and modified versions of the elevated plus maze (EPM), an innate anxiety paradigm. Hippocampal local field potentials (LFPs) were simultaneously monitored. The population of mPFC units distinguished between safe and aversive locations within the maze, regardless of the nature of the anxiogenic stimulus. Strikingly, mPFC units with stronger task-related activity were more strongly coupled to theta-frequency activity in the vHPC LFP. Lastly, task-related activity was inversely correlated with behavioral measures of anxiety. These results clarify the role of the vHPC-mPFC circuit in innate anxiety and underscore how specific inputs may be involved in the generation of behaviorally relevant neural activity within the mPFC.     

Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus

Zika virus (ZIKV) infection during pregnancy is linked to various developmental brain disorders. Infants who are asymptomatic at birth might have postnatal neurocognitive complications. However, animal models recapitulating these neurocognitive phenotypes are lacking, and the circuit mechanism underlying behavioral abnormalities is unknown. Here, we show that ZIKV infection during mouse pregnancy induces maternal immune activation (MIA) and leads to autistic‐like behaviors including repetitive self‐grooming and impaired social memory in offspring. In the medial prefrontal cortex (mPFC), ZIKV‐affected offspring mice exhibit excitation and inhibition imbalance and increased cortical activity. This could be explained by dysregulation of inhibitory neurons and synapses, and elevated neural activity input from mPFC‐projecting ventral hippocampus (vHIP) neurons. We find structure alterations in the synaptic connections and pattern of vHIP innervation of mPFC neurons, leading to hyperconnectivity of the vHIP‐mPFC pathway. Decreasing the activity of mPFC‐projecting vHIP neurons with a chemogenetic strategy rescues social memory deficits in ZIKV offspring mice. Our studies reveal a hyperconnectivity of vHIP to mPFC projection driving social memory deficits in mice exposed to maternal inflammation by ZIKV.     

Chronic Toxoplasma infection modifies the structure and the risk of host behavior

The intracellular parasite Toxoplasma has an indirect life cycle, in which felids are the definitive host. It has been suggested that this parasite developed mechanisms for enhancing its transmission rate to felids by inducing behavioral modifications in the intermediate rodent host. For example, Toxoplasma-infected rodents display a reduction in the innate fear of predator odor. However, animals with Toxoplasma infection acquired in the wild are more often caught in traps, suggesting that there are manipulations of intermediate host behavior beyond those that increase predation by felids. We investigated the behavioral modifications of Toxoplasma-infected mice in environments with exposed versus non-exposed areas, and found that chronically infected mice with brain cysts display a plethora of behavioral alterations. Using principal component analysis, we discovered that most of the behavioral differences observed in cyst-containing animals reflected changes in the microstructure of exploratory behavior and risk/unconditioned fear. We next examined whether these behavioral changes were related to the presence and distribution of parasitic cysts in the brain of chronically infected mice. We found no strong cyst tropism for any particular brain area but found that the distribution of Toxoplasma cysts in the brain of infected animals was not random, and that particular combinations of cyst localizations changed risk/unconditioned fear in the host. These results suggest that brain cysts in animals chronically infected with Toxoplasma alter the fine structure of exploratory behavior and risk/unconditioned fear, which may result in greater capture probability of infected rodents. These data also raise the possibility that selective pressures acted on Toxoplasma to broaden its transmission between intermediate predator hosts, in addition to felid definitive hosts.     

Behavioral and neuroendocrine response of Brandt's voles,Lasiopodomys brandtii,to odors of different species

Predator odors are nonintrusive natural stressors of high ethological relevance. Behavioral and hormonal responses and changes in the expression of medial hypothalamic c-fos mRNA were examined in Brandt's voles (Lasiopodomys brandtii) after exposure to feces of a domestic cat (Felis catus), weasel (Mustela sibirica), snake (Xenopeltis hainanensis), goat (Capra aegagrus), and distilled water (control). One hundred voles were tested in the defensive withdrawal apparatus. Voles showed an aversion to the predator odors by showing significant high levels of flight-related behaviors, more freezing behavior, and less and higher grooming behavior when they exposed to weasel and cat feces and more vigilant rearing compared to goat and control groups. Adrenocorticotropic hormone and corticosterone serum levels significantly increased when voles were exposed to the predator odors compared to voles exposed to goat and control odors. A significant high c-fos mRNA level indicates a strong predator odor-induced activation of the medial hypothalamus of the tested voles. Our results display significant fear responses of Brandt's voles exposed to predator odors and that they can skillfully differentiate between odors from different species with odors from sympatric predators triggering the highest responses.     

Fear-like behavioral responses in mice in different odorant environments: Trigeminal versus olfactory mediation under low doses

Odors can have repulsive effects on rodents based on two complementary adaptive behaviors: the avoidance of predator odors (potentially dangerous) and the avoidance of trigeminal stimulants (potentially noxious). The present study aimed to compare the behavioral effects on mice of odors according to their trigeminal properties and ecological significance. We used three different odors: 2,4,5-trimethylthiazoline (TMT: a fox feces odor frequently used to elicit fear-induced behaviors), toluene (a strong stimulant of the trigeminal system) and phenyl ethyl alcohol (PEA: a selective stimulant of the olfactory system). First, we checked preference and avoidance behaviors in mice with and without anosmia towards these odors to ensure their olfactory/trigeminal properties. Secondly, we used a standard test (open-field and elevated plus-maze) to assess the behaviors of mice when exposed to these odors. The results show that the anosmic and control mice both avoided TMT and toluene odors. In the open-field and the elevated plus-maze, mice exhibited "anxious" behaviors when exposed to TMT. Conversely, exposure to PEA induced "anxiolytic" effects confirmed by low blood corticosterone levels resulting from completion of the elevated plus-maze. Compared with TMT exposure, toluene exposure induced moderate "anxious" effects.     

Selective modifications in the nucleus accumbens of dopamine synaptic transmission in rats exposed to chronic stress

Stressful events are accompanied by modifications in dopaminergic transmission in distinct brain regions. As the activity of the neuronal dopamine (DA) transporter (DAT) is considered to be a critical mechanism for determining the extent of DA receptor activation, we investigated whether a 3-week exposure to unavoidable stress, which produces a reduction in DA output in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC), would affect DAT density and DA D1 receptor complex activity in the NAcS, mPFC and caudate-putamen (CPu). Rats exposed to unavoidable stress showed a decreased DA output in the NAcS accompanied by a decrease in the number of DAT binding sites, and an increase in the number of DA D1 binding sites and Vmax of SKF 38393-stimulated adenylyl cyclase. In the mPFC, stress exposure produced a decrease in DA output with no modification in DAT binding or in DA D1 receptor complex activity. Moreover, in the CPu stress exposure induced no changes in DA output or in the other neurochemical variables examined. This study shows that exposure to a chronic unavoidable stress that produces a decrease in DA output in frontomesolimbic areas induced several adaptive neurochemical modifications selectively in the nucleus accumbens.