Testing Emotional Vulnerability to Threat in Adults Using a Virtual Reality Paradigm of Fear Associated With Autonomic Variables.

Fear and anxiety are generally assessed as responses of prey to high or low levels of threatening environments, fear-conditioned or unconditioned stimuli, or the intensity and distance between predator and prey. Depending on whether a threat is close to or distant from the individual, the individual exhibits specific behaviors, such as being quiet (freezing in animals) if the threat is distant or fleeing if the threat is close. In a seminal paper in 2007, Dean Mobbs developed an active prevention virtual reality paradigm (VRP) to study a threat's spatial imminence using finger shocks. In the present study, we used a modified VRP with a distinctive feature, namely a dynamic threat-of-loud noise paradigm. The results showed a significant reduction in the number of times the subjects were captured in the high predator phase (85 dB) vs. control phases, suggesting that the participants were motivated to avoid the high predator. Concomitant with avoidance behavior, a decrease in respiratory rate and an increase in heart rate characterized the defense reaction. These results demonstrate behavioral and autonomic effects of threat intensity in volunteers during a VRP, revealing a profile of defense reaction that reflects the individual emotional susceptibility to the development of anxiety.

Context-Specific Tolerance and Pharmacological Changes in the Infralimbic Cortex-Nucleus Accumbens Shell Pathway Evoked by Ketamine.

Like other drugs, ketamine is abused due to its ability to act as a positive reinforcer in the control of behavior, just as natural reinforcers do. Besides, through Pavlovian conditioning, tolerance to drug effects can become conditioned to specific contextual cues showing that environmental stimuli can act as powerful mediators of craving and relapse. In the present study, we shall investigate the effects of long-term ketamine administration and withdrawal on behavioral measures and emotionality, the drug-context-specific influence on the tolerance to the sedative effects of an anesthetic dose of ketamine, and the neuropharmacological events underlying this phenomenon, in rats conditioned with 10 mg/kg of ketamine and later challenged with a dose of ketamine of 80 mg/kg in a familiar and non-familiar environment. Variations in dopamine and serotonin efflux in the infralimbic cortex-nucleus accumbens shell circuitry (IL-NAcSh) was further recorded in the same conditions. Our results highlight that besides its well-known reinforcing properties, ketamine also shares the ability to induce behavioral and pharmacological conditioned tolerance, associated with increases in cortical (IL), and decreases in striatal (NAcSh) dopamine release. To our knowledge, we are presenting the first set of behavioral and neurochemical data showing that, like other drugs of abuse, ketamine can induce learned context-specific tolerance.

Differential effects of early exposure to alcohol on alcohol preference and blood alcohol levels in low- and high-anxious rats.

Emotional disturbances emerge following alcohol withdrawal. The anxiolytic effect of alcohol may be one important motivation for its consumption in conditions where alcohol intake is anxiety reducing. Besides, early alcohol experience will predict future alcohol-related problems at some point in their lives. Rats classified according to their anxiety-like behavior phenotype show a higher preference for alcohol. Yet, despite preclinical studies have identified the behavioral and physiological effects of alcohol withdrawal, little has been shown on relapse to alcohol after a period of abstinence following intermittent long-term alcohol consumption in low-(LA) and high-anxiety (HA) phenotype rats. This study attempts to assess the role of emotional reactivity of early-aged LA and HA rats on later alcohol preference, through an operant response procedure. For this, a sweetened liquid fading procedure associated with a long-term and intermittent voluntary alcohol drinking was used, with the animals being tested on different withdrawal periods. Alcohol withdrawal symptoms were recorded, and blood alcohol levels were assessed at two intervals to examine the influence of anxiety phenotype on blood alcohol clearance. Data from HA control and HA withdrawn rats show that emotionality selectively influences alcohol preference. Blood alcohol decay was faster in HA than in LA alcohol pretreated rats. HA rats showed an ascending curve of alcohol intake, exhibiting a high level of alcohol drinking on withdrawal and withdrawal symptoms. Moreover, HA alcohol experienced rats show a high frequency of lever-pressing response during extinction, associated with a facilitation of bar-pressing recovery trials, an indication of alcohol-seeking behavior.

Prelimbic NMDA receptors stimulation mimics the attenuating effects of clozapine on the auditory electrophysiological rebound induced by ketamine withdrawal.

Ketamine (KET) is a non-competitive N-Methyl-d-aspartate (NMDA) receptors antagonist that intensifies sensory experiences, prompts hallucinations and delusions, exacerbates previously installed psychosis and disrupts physiological evoked potentials (AEPs). Pharmacologically, KET stimulates glutamate efflux in the medial prefrontal cortex, mainly in the prelimbic (PrL) sub-region. Efferences from this region exert a top-down regulatory control of bottom-up sensory processes either directly or indirectly. In the midbrain, the central nucleus of the inferior colliculus (CIC) plays a fundamental role in the processing of auditory ascending information related to sound localization, sensorimotor gating, and preattentive event-related potentials. Auditory hallucinations elicited during a psychotic outbreak are accompanied by CIC neural activation. Thus, it is possible that NMDA-mediated glutamate neurotransmission in the PrL indirectly modulates CIC neuronal firing. The aim of the present study was to assess the effects of KET on the latency and amplitude of AEPs elicited in the CIC of rats tested during KET effects and following withdrawal from the chronic administration. Changes on emotionally induced by KET treatment were evaluated with the use of the elevated zero maze (EZM). Unlike typical neuroleptics, the atypical antipsychotic clozapine (CLZ) potently blocks the disruption of the sensorimotor gating induced by NMDA antagonists. Therefore, the effects of KET withdrawal on AEPs were challenged with a systemic injection of CLZ. In addition, we further investigated the role of NMDA receptors of the PrL on the AEPs expression recorded in the CIC through intra-PrL infusions of NMDA itself. Our results showed that the processing of sensory information in the CIC is under indirect control of PrL. These data suggest that the long-term KET treatment disrupts the collicular auditory field potentials, possibly through influencing PrL glutamate activity on intrinsic 5-HT mechanisms in the dorsal raphe and CIC.

Behavioral and Auditory Electrophysiological Rebound as a Compensatory Response to the Reinforcing Effects of Morphine.

Auditory-evoked potentials (AEPs) can be modified by associative learning, where the appearance of learned compensatory responses (CCRs) may result in the emergence of drug withdrawal symptoms and relapse. Although CCRs' influence on later attentive and cognitive domains has been extensively examined, contextual conditioned tolerance occurring in preattentive mechanisms operating at earlier stages of information processing has remained largely unexplored. To extend our knowledge on this subject, compensatory changes on the motor and emotional aspects of behavior evoked by contextual cues were investigated with an electronic open field in morphine-pretreated rats challenged with two morphine overdoses (40 and 80 mg/kg). CCRs influence on the AEPs recorded in the central nucleus of the inferior colliculus (CIC) was analyzed with the help of a field potential recording device and a two-chamber shuttle box placed inside a Faraday cage system. The emergence of electrophysiological CCRs was analyzed by recording AEP latency and amplitude elicited in the central nucleus of the IC (CIC) with the aid of a field potential recording device and a two-chamber shuttle box placed inside a Faraday cage system. Behavioral analysis indicated that CCRs ensue in non-familiar contexts. Electrophysiological data revealed increases in the amplitude of AEPs evoked in a non-familiar context. Our results indicate that behavioral learning responses emerge following Pavlovian conditioning even with the use of low and regular doses of morphine over a short-term treatment. Changes in the CIC electrophysiology may indicate that the development of drug dependence occurs covertly in the early stages of sensory information processing.

Environmental enrichment may protect against neural and behavioural damage caused by withdrawal from chronic alcohol intake.

Exposure to stress and prolonged exposure to alcohol leads to neuronal damages in several brain regions, being the medial prefrontal cortex (mPFC) one of the most affected. These changes presumably reduce the ability of the organism to cope with these stimuli and may underlie a series of maladaptive behaviours among which include drug addiction and withdrawal. Drug-addicted individuals show a pattern of behavior similar to patients with lesions of the mPFC. This impairment in the decision-making could be one of the mechanisms responsible for the transition from the casual to compulsive drug use. The environmental enrichment (EE) has a protective effect on the neural and cognitive impairments induced by psychoactive drugs, including ethyl alcohol. The present study aims to determine the influence of withdrawal from intermittent long-term alcohol exposure on alcohol preference, emotional reactivity and neural aspects of early isolated or grouped reared rats kept under standard or complex environments and the influence of social isolation on these measures, as well. Our results point out new insights on this matter showing that the EE can attenuate the adverse effects of withdrawal and social isolation on rat's behavior. This effect is probably due to its protective action on the mPFC integrity, including the cingulate area 1 (Cg1), and the prelimbic (PrL) and infralimbic cortex (IL), what could account for the absence of changes in the emotional reactivity in EE alcohol withdrawal rats. We argue that morphological changes at these cortical levels can afford the emotional, cognitive and behavioural dysregulations verified following withdrawal from chronic alcohol intake.

5-HT1A receptors of the prelimbic cortex mediate the hormonal impact on learned fear expression in high-anxious female rats.

Hormones highly influence female behaviors. However, research on this topic has not usually considered the variable hormonal status. The prelimbic cortex (PrL) is commonly engaged in fear learning. Connections from and to this region are known to be critical in regulating anxiety, in which serotonin (5-HT) plays a fundamental role, particularly through changes in 5-HT1A receptors functioning. Also, hormone fluctuations can greatly influence anxiety in humans and anxiety-related behavior in rodents, and this influence involves the functioning of 5-HT brain systems. The present investigation sought to determine whether fluctuations in ovarian hormones relative to the estrous cycle would influence the expression of learned fear in female rats previously selected as low- (LA) or high-anxious (HA). Furthermore, we investigate the role of the 5-HT system of the PrL, particularly the 5-HT1A receptors, as a possible modulator of estrous cycle influence on the expression of learned fear through intra-PrL microinjections of 5-HT itself or the full 5-HT1A agonist 8-OH-DPAT (8-hydroxy-2-(di-n-propylamine)tetralin). Behavioral changes were assessed using the fear-potentiated startle (FPS) procedure. The results showed that fear intensity is associated with hormonal decay, being more accentuated during the estrus phase. This increase in fear levels was found to be negatively correlated with the expression of potentiated startle. In rats prone to anxiety and tested during the proestrus and estrus phases, 5-HT mechanisms of the PrL seem to play a regulatory role in the expression of learned fear. These results were not replicated in the LA rats. Similar but less intense results were found regarding the early and late diestrus. Our data indicate that future studies on this subject need to take into account the dissociation between low- and high-responsive females to understand how hormones affect emotional behavior.

Impaired fear inhibitory properties of GABAA and µ opioid receptors of the dorsal periaqueductal grey in alcohol-withdrawn rats.

The dorsal periaqueductal grey (DPAG) is a midbrain region that plays a fundamental role on the expression of the anxiety and fear-related responses. Increased fear and anxiety levels are the most frequent symptoms present in the alcohol withdrawal syndrome. This study aims to assess whether GABA and opioid receptors of the DPAG could be implicated in the expression of the conditioned fear state elicited in alcohol withdrawn-rats. For this purpose, we used the fear-potentiated startle (FPS) procedure. Drugs used were the selective GABAA agonist muscimol (1 nmol/0.2 µL) and the predominantly mu opiate receptors agonist morphine (10 nmol/0.2 µL). Exposure to aversive cues of the elevated-plus-maze (EPM) was used in order to validate the influence of alcohol withdrawal on emotionality. Data from FPS pointed out to an anxiogenic-like profile of withdrawal, a result sustained by the data collected from the EPM test. Muscimol and morphine showed their well-known anxiolytic-like profile, decreasing the fear-potentiated startle (FPS) amplitude in control subjects. However, the drugs had no effect on the levels of fear evoked in rats pre-treated with and withdrawn from alcohol. It is suggested that this lack of effect was possibly due to the desensitization of the GABAA receptors, as well as by the decrease on the responsiveness of the functions of µ opioid receptors, resulting from chronic administration of ethyl alcohol. These findings shed light on some aspects of the anxiety-like behavior elicited during alcohol withdrawal bringing new information on the influence of GABA and opioid receptors of the DPAG on the expression of unconditioned and conditioned fear responses.

Changes on auditory physiology in response to the inactivation of amygdala nuclei in high anxiety rats expressing learned fear.

The inferior colliculus (IC) is primarily involved in the processing of acoustic stimuli, including those emitted by prey and predators. The role of the central nucleus of the IC (CIC) in fear and anxiety has been suggested based on electrophysiological, behavioral and immunohistochemical studies. The reactivity of high-anxiety rats (HA) to diverse challenges is different from low-anxiety ones (LA). In humans and laboratory animals, pathological anxiety is often accompanied by heightened vigilance and alertness, hyperactivity of the amygdala (AM), and increased amplitude of the auditory evoked potentials (AEP) from the IC. This study aims to evaluate the influence of the inactivation of the central (CEA) and basolateral (BLA) nuclei of the amygdala, after local infusions of the full GABAA agonist muscimol (1nmol/0.2µl), on the AEP elicited in the CIC of rats tested under a learned fear state. Our results showed that both BLA and CEA inactivation change the expression of conditioned fear, in a paradigm using the context as the conditioned stimulus (CS). These changes are correlated to the innate anxiety levels of the animals. It is supposed that this shortcoming is in addition to the imbalance between the regulatory role of the top-down and bottom-up processes in the control of anxiety.

Modulation of auditory-evoked potentials recorded in the inferior colliculus by GABAergic mechanisms in the basolateral and central nuclei of the amygdala in high- and low-anxiety rats.

The central nucleus of the inferior colliculus (CIC) is an important relay station for acoustic information that has an aversive connotation. γ-Aminobutyric acid (GABA) mechanisms exert tonic inhibitory control over the neural substrates of aversion in the midbrain tectum. Recent evidence obtained in this laboratory showed that low- (LA) and high-anxiety (HA) rats selected in the elevated plus maze (EPM) exhibit low and high auditory-evoked potential (AEP) amplitudes, respectively, recorded in the CIC when subjected to loud click noises. The present study investigated the eventual top-down regulation exerted by GABA mechanisms in the basolateral (BLA) and central (CeA) nuclei of the amygdala on aversive information processing at the level of the CIC. The GABA inhibitors bicuculline (10 ng/0.2 µl) and semicarbazide (7 µg/0.2 µl) and GABA agonist muscimol (1 nmol/0.2 µl) were locally infused into the CeA and BLA in rats subjected to an AEP recording procedure that used electrodes implanted in the CIC. Freezing behavior induced by these intra-amygdala injections was also measured. The results confirmed that the processing of aversive acoustic information depends on anxiety levels in LA and HA rats. Freezing behavior and the increased AEPs induced by intra-CeA injections of bicuculline and semicarbazide were more pronounced in HA than in LA animals. Intra-CeA injections of muscimol did not change either the basal levels of freezing or AEP amplitudes in LA and HA rats. However, freezing and the enhanced AEPs were of small magnitude following intra-BLA injections of bicuculline and semicarbazide. An unexpected magnitude of freezing and enhanced AEPs was observed following muscimol infusions into the BLA in HA rats. GABA mechanisms in the CeA appear to play a regulatory role in aversive states, and inactivation of the BLA with muscimol produced pro-aversive effects, suggesting that inactivation of the BLA removed the descending inhibitory control of the neural substrates of aversion in the CIC exerted by the BLA. Because the latter effects were observed only in HA rats, a dysfunctional BLA may underlie the differences between trait and state anxiety.

Previous spatial learning is required for the development of place preference in rats following chronic administration of and withdrawal from morphine

The positive reinforcing properties of addictive drugs have a primary role in the development of drug dependence. In the field of opiates, great attention has been given to this phenomenon, as well as to the negative properties eliciting craving and inducing relapse during withdrawal. This study was designed to evaluate whether elevated plus-maze (EPM) experienced rats withdrawn from low doses of subcutaneous (s.c.) morphine (10 mg/kg), in which a high anxiety level is the most prominent withdrawal symptom, acquire place preference when submitted to a conflict paradigm in which drug effects are paired with an aversive context: the distal part of the open arms of an EPM. Both the anxiety test and place preference conditioning were measured in the same apparatus, a biased version of the plus-maze. In order to verify the influence of previous EPM spatial learning on the performance of morphine-withdrawn rats, half the animals in this study experienced the EPM prior to treatment. Additional groups were also tested under the influence of morphine effects. The effects of the treatments were quantified through the analysis of three types of measures: anxiety was inferred from the use of the conventional measures (percentage of entries and time spent in the open-arms) and risk-assessment behaviours (frequency of stretched-attending postures - SAP, and time spent at the centre of the maze). Place preference conditioning was evaluated through analysis of the number of entries, total time spent and distance run in the open-arm extremities, which is where the animals were conditioned. The number of closed-arm entries was taken as an index of locomotor activity. Our results showed that (i) EPM naïve rats pre-treated with morphine did not develop place preference, behaving like control rats; (ii) rats that had previous experience in the EPM showed no changes in open-arm avoidance on the second exposure, when compared with rats naïve for this condition; ...(AU)

Previous spatial learning is required for the development of place preference in rats following chronic administration of and withdrawal from morphine

The positive reinforcing properties of addictive drugs have a primary role in the development of drug dependence. In the field of opiates, great attention has been given to this phenomenon, as well as to the negative properties eliciting craving and inducing relapse during withdrawal. This study was designed to evaluate whether elevated plus-maze (EPM) experienced rats withdrawn from low doses of subcutaneous (s.c.) morphine (10 mg/kg), in which a high anxiety level is the most prominent withdrawal symptom, acquire place preference when submitted to a conflict paradigm in which drug effects are paired with an aversive context: the distal part of the open arms of an EPM. Both the anxiety test and place preference conditioning were measured in the same apparatus, a biased version of the plus-maze. In order to verify the influence of previous EPM spatial learning on the performance of morphine-withdrawn rats, half the animals in this study experienced the EPM prior to treatment. Additional groups were also tested under the influence of morphine effects. The effects of the treatments were quantified through the analysis of three types of measures: anxiety was inferred from the use of the conventional measures (percentage of entries and time spent in the open-arms) and risk-assessment behaviours (frequency of stretched-attending postures - SAP, and time spent at the centre of the maze). Place preference conditioning was evaluated through analysis of the number of entries, total time spent and distance run in the open-arm extremities, which is where the animals were conditioned. The number of closed-arm entries was taken as an index of locomotor activity. Our results showed that (i) EPM naïve rats pre-treated with morphine did not develop place preference, behaving like control rats; (ii) rats that had previous experience in the EPM showed no changes in open-arm avoidance on the second exposure, when compared with rats naïve for this condition; (iii) previous spatial learning of the EPM contextual cues was, in fact, a requirement for anxiety-inducing place preference for the open-arms in morphine-withdrawn rats and, (iiii) conditioned place preference was achieved both in rats under the effects of morphine and in withdrawal, probably through the influence of the positive or negative reinforcing effects promoted by the presence or absence of the drug in the central nervous system.

Brainstem areas activated by diazepam withdrawal as measured by Fos-protein immunoreactivity in rats.

In the 1970s, chronic treatment with benzodiazepines was supposed not to cause dependence. However, by the end of the decade several reports showed that the interruption of a prolonged treatment with diazepam leads to a withdrawal syndrome characterized, among other symptoms, by an exaggerated level of anxiety. In laboratory animals, signs that oscillate from irritability to extreme fear-like behaviors and convulsions have also been reported. In recent years many studies have attempted to disclose the neural substrates responsible for the benzodiazepines withdrawal. However, they have focused on telencephalic structures such as the prefrontal cortex, nucleus accumbens and amygdala. In this study, we examined the Fos immunoreactivity in brain structures known to be implicated in the neural substrates of aversion in rats under spontaneous diazepam-withdrawal. We found that the same group of structures that originally modulate the defensive responses evoked by fear stimuli, including the dorso-medial hypothalamus, the superior and inferior colliculus and the dorsal periaqueductal gray, were most labeled following diazepam withdrawal. It is suggested that an enhanced neural activation of neural substrates of fear in the midbrain tectum may underlie the aversive state elicited in diazepam-withdrawn rats.

The blockade of AMPA-kainate and NMDA receptors in the dorsal periaqueductal gray reduces the effects of diazepam withdrawal in rats.

It is well established that the most persistent sign of withdrawal from chronic benzodiazepine use in humans is anxiety. In contrast to other types of drugs of abuse, the emergence of this anxiety does not seem to be linked directly to alterations in the levels of dopamine in the mesolimbic system. Some studies have proposed that fear-like behaviors elicited by benzodiazepine withdrawal could be the result either of alterations in the sensitivity of GABAA receptors or in the neuronal hyperexcitability that results from neuroadaptative responses to chronic treatment, probably mediated by glutamate. The increased fear-like behaviors induced by benzodiazepine withdrawal are similar to the defense reaction displayed by animals exposed to dangerous situations or submitted to electrical or chemical stimulation of the dorsal periaqueductal gray (dPAG), a key structure of the brain aversive system. However, the involvement of the dPAG in drug abuse has been investigated only in the context of the physical effects of drug dependence. Thus, in this study we investigated the effects of injections into the dPAG of the glutamic acid diethyl ester (GDEE) and 2-amino-7-phosphonoheptanoate (AP-7) (AMPA-kainate and NMDA receptors antagonists, respectively) on fear-like behaviors promoted by benzodiazepine withdrawal in rats submitted to aversive events (foot-shocks) immediately before chronic diazepam administration in a conditioning place-preference paradigm, using a light-dark box. Our results showed that inhibition of the glutamatergic neurotransmission in the dPAG reduces the consequence of the diazepam withdrawal in rats, implicating the excitatory amino acids of the dPAG in the modulation of the aversive state induced by benzodiazepine drugs withdrawal.

Substance P injected into the dorsal periaqueductal gray causes anxiogenic effects similar to the long-term isolation as assessed by ultrasound vocalizations measurements.

Housing conditions change the emotional state of the animals. Ultrasound vocalizations (USVs) termed as 22 kHz are the usual components of the defensive responses of rats exposed to threatening conditions such as isolation. The amount of emission of 22 kHz USVs depends on the intensity of the aversive stimuli. While short periods of isolation caused an anxiolytic-sensitive enhancement of the defensive responses, long-term isolation tended to reduce the defensive performance of the animals to aversive stimuli. The dorsal periaqueductal gray (dPAG) is an important vocal center and a crucial structure for the expression of defensive response. While it has been shown that Substance P (SP) at this midbrain level is involved in the modulation of the defensive response, its role in the emission of ultrasound vocalizations has not been evaluated. In this study we examined whether the resocialization and local injections of SP into the dPAG have an influence on the isolation-induced 22 kHz USVs recorded within the frequency range of 18-26 kHz. Rats isolated for 1 day showed a significant increase in the number and duration of USVs, which were reversed by resocialization. On the other hand, 2-week isolation reduced the number and duration of 22 kHz USVs, which could not be reversed by resocialization. SP injections into the dPAG (35 pmol/0.2 microL) caused a reduction in the 22 kHz USVs. Pretreatment with the NK-1 receptor antagonist spantide (100 pmol/0.2 microL) blocked these effects but exhibited no effect when given alone. These findings suggest that 1-day and 2-week isolation recruit distinct brain defensive systems. Also, in agreement with the notion that intense fear is associated with the neural substrates of fear of the dPAG, activation of NK-1 receptors of this midbrain structure reduces the 22 kHz USVs.

Gabaergic regulation of the neural organization of fear in the midbrain tectum.

In midbrain tectum (MT) structures, such as the dorsal periaqueductal gray (dPAG), the superior colliculus (SC) and the inferior colliculus (IC) GABAergic neurons exert a tonic control on the neural substrates involved in the expression of defensive reactions. In this review, we summarize behavioral, immunohistochemical (brain Fos distribution) and electrophysiological (auditory evoked potentials) data obtained with the reduction of GABA transmission by local injections of a GABA receptor blocker (bicuculline, BIC) or a glutamic acid decarboxylase inhibitor (semicarbazide, SMC) into the MT. Distinct patterns of Fos distribution were obtained following the freezing and escape reactions induced by MT injections of SMC and BIC, respectively. While only the laterodorsal nucleus of the thalamus was labeled after SMC-induced freezing, a widespread increase in Fos expression in the brain occurred after BIC-induced escape. Also, injections of SMC into the IC increased the auditory evoked potentials recorded from this structure. It is suggested that GABAergic mechanisms of MT are also called into play when sensory gating of the MT is activated during different emotional states.

Isolation-induced changes in ultrasonic vocalization, fear-potentiated startle and prepulse inhibition in rats.

Isolation causes important changes in the behavioral reactivity of rats to environmental stimuli. These changes include deficit in sensorimotor gating and altered fear-like responses to aversive stimuli. Measures of ultrasound vocalizations at 20-22 kHz when rats are exposed to threatening conditions, such as novelty, have been taken as a good measure of fear. The fear-potentiated startle to loud sounds and the prepulse inhibition tests have been considered reliable indicators of anxiety and attention impairments, respectively. Rats reared under conditions of isolation from weaning display clear deficits in prepulse inhibition. Taking into account that housing condition changes the emotional state of the animals, we evaluated in this work the performance of rats in the fear-potentiated startle test, prepulse inhibition and emission of ultrasound vocalizations to novelty when isolated for 10 days and after resocialization for 1 week in comparison to grouped rats. Isolated rats showed greater reactivity to loud sounds in the fear-potentiated startle test than grouped animals. They also emitted less ultrasound vocalizations at 20-22 kHz than grouped animals when exposed to a novel environment. In contrast to the well-known deficit in prepulse inhibition displayed by isolation-reared animals, in the present study isolation for 10 days caused a significant increase in prepulse inhibition. Resocialization was not able to counteract the effects of isolation in all three tests. The results suggest that the emotional state of the animals is altered by 10 days of isolation; they do not vocalize characteristically as grouped rats when submitted to novelty; unconditioned responses to loud sounds are enhanced and increased prepulse inhibition is shown rather than a deficit as largely documented in studies with isolation-reared animals. It is suggested that the assessment of the emotional state of the animals is a prerequisite in the evaluation of prepulse inhibition. The level of defensive reactivity displayed by isolated animals is crucial for the functioning of sensory gating and, by extension, to the expression of prepulse inhibition.

Effects of acute and chronic fluoxetine and diazepam on freezing behavior induced by electrical stimulation of dorsolateral and lateral columns of the periaqueductal gray matter.

The defensive responses induced by electrical stimulation of the dorsal periaqueductal gray matter (dPAG) of the rat have been proposed as a model of panic attacks in humans. In the present study we investigated the acute and chronic effects of fluoxetine and diazepam on freezing and escape reactions elicited by electrical stimulation of the dorsolateral (dlPAG) and lateral (lPAG) columns of the periaqueductal gray matter (PAG). The frequencies of crossing, rearing, bouts of micturition and fecal boli were also recorded. Electrodes were unilaterally implanted in the brainstem aimed at the PAG. Drug treatments were given daily for 2 weeks with fluoxetine (5, 10 and 20 mg/kg ip), a selective inhibitor of serotonin reuptake, diazepam (1, 2 and 4 mg/kg ip), or saline. Drug effects were assessed acutely (15 min after the first injection) and chronically (15 min after the 14th injection). Chronic, but not acute, administration of fluoxetine caused a significant increase in the threshold of freezing without affecting the escape response elicited by dlPAG/lPAG stimulation. This characteristic pattern of effects could not be attributed to motor deficit, since this drug did not change the number of crossings and rearings. In contrast, no significant threshold changes were observed following acute and chronic treatment with diazepam. These data give further evidence for (a) an antiaversive effect of chronic treatment with fluoxetine, which caused a selective reduction in freezing behavior and neurovegetative responses associated with fearlike reaction elicited by dlPAG/lPAG electrical stimulation; (b) the involvement of the dlPAG and lPAG in the generation and organization of defensive responses and that freezing may probably be associated with panic attacks; and (c) the lack of effect of diazepam in this model is in line with its inefficacy as a panicolytic drug. The study of the unconditioned freezing behavior evoked by dlPAG/lPAG stimulation may constitute a new and interesting model for the study of panic disorder.

Analysis of freezing behavior and ultrasonic vocalization in response to foot-shocks, ultrasound signals and GABAergic inhibition in the inferior colliculus: effects of muscimol and midazolam.

Freezing behavior is a common response to distal threatening stimuli. It has been reported that experimenter-presented 20-kHz tones produce freezing in Wistar rats. The present studies were designed to determine the acoustic specificity for induction of the emission of ultrasound vocalizations (USV) and freezing of rats to either ultrasound tones or to a standard stressor, foot-shocks. We also examined whether GABA-benzodiazepine mechanisms, known for modulating anxiety-related processes, are involved in the regulation of defensive responses to these two aversive unconditioned stimuli. It was found that emission of USV was only observed with foot-shocks. Moreover, rats exhibited freezing when they were exposed to foot-shocks and 20-25-kHz ultrasound signals. Likewise, removal of the GABAergic inhibitory control on the neural substrates of aversion in the inferior colliculus by local microinjections of semicarbazide, a blocker of the glutamic acid decarboxylase, caused freezing behavior without emission of USV. All these responses were significantly reduced by midazolam and muscimol. It is suggested that the neural substrates of aversion in the inferior colliculus are under inhibitory control of GABAergic mechanisms and are different from those triggered by foot-shocks.

Enhancement of acoustic evoked potentials and impairment of startle reflex induced by reduction of GABAergic control of the neural substrates of aversion in the inferior colliculus.

The neural network of the inferior colliculus (IC), implicated in the generation of defensive behavior to aversive acoustic stimuli, is under tonic GABAergic control. Dopamine also seems to have a modulatory role in these neural circuits. It is still unclear how such changes in transmission of acoustic information influence the motor expression of the defensive behavior. Startle reaction to a sudden noise has been used as an effective way to measure the motor reactivity of rats to fearful acoustic stimuli. In this work we examined the processing of sensorial information--assessed by the recording of auditory evoked potentials (AEP)--and the behavioral effects--evaluated by the freezing and startle responses--during the reduction of GABA levels caused by microinjections of semicarbazide (SMC, 6 microg/0.2 microl), a glutamic acid decarboxylase inhibitor, into the IC. These data were compared to the effects of the overall arousal elicited by apomorphine (APO, 0.5 mg/kg, i.p.). The results obtained show that IC microinjections of SMC induced freezing behavior, enhanced the AEP and impaired the startle reaction to a loud sound. On the other hand, APO changed neither the AEP nor the startle in the same experimental conditions. These results suggest that the release of GABAergic control of the neural substrates of aversion in the IC results in an increased processing of auditory information along with an inhibitory influence on the motor pathways responsible for the startle response.