Rats respond to aversive emotional arousal of human handlers with the activation of the basolateral and central amygdala.

Reading danger signals may save an animal's life, and learning about threats from others allows avoiding first-hand aversive and often fatal experiences. Fear expressed by other individuals, including those belonging to other species, may indicate the presence of a threat in the environment and is an important social cue. Humans and other animals respond to conspecifics' fear with increased activity of the amygdala, the brain structure crucial for detecting threats and mounting an appropriate response to them. It is unclear, however, whether the cross-species transmission of threat information involves similar mechanisms, e.g., whether animals respond to the aversively induced emotional arousal of humans with activation of fear-processing circuits in the brain. Here, we report that when rats interact with a human caregiver who had recently undergone fear conditioning, they show risk assessment behavior and enhanced amygdala activation. The amygdala response involves its two major parts, the basolateral and central, which detect a threat and orchestrate defensive responses. Further, we show that humans who learn about a threat by observing another aversively aroused human, similar to rats, activate the basolateral and centromedial parts of the amygdala. Our results demonstrate that rats detect the emotional arousal of recently aversively stimulated caregivers and suggest that cross-species social transmission of threat information may involve similar neural circuits in the amygdala as the within-species transmission.

Optogenetic and chemogenetic approaches reveal differences in neuronal circuits that mediate initiation and maintenance of social interaction.

For social interaction to be successful, two conditions must be met: the motivation to initiate it and the ability to maintain it. This study uses both optogenetic and chemogenetic approaches to reveal the specific neural pathways that selectively influence those two social interaction components.

Prolonged Consumption of Sweetened Beverages Lastingly Deteriorates Cognitive Functions and Reward Processing in Mice.

We investigated the detrimental effects of chronic consumption of sweet or sweetened beverages in mice. We report that consumption of beverages containing small amounts of sucrose during several weeks impaired reward systems. This is evidenced by robust changes in the activation pattern of prefrontal brain regions associated with abnormal risk-taking and delayed establishment of decision-making strategy. Supporting these findings, we find that chronic consumption of low doses of artificial sweeteners such as saccharin disrupts brain regions' activity engaged in decision-making and reward processes. Consequently, this leads to the rapid development of inflexible decisions, particularly in a subset of vulnerable individuals. Our data also reveal that regular consumption, even at low doses, of sweet or sweeteners dramatically alters brain neurochemistry, i.e., dopamine content and turnover, and high cognitive functions, while sparing metabolic regulations. Our findings suggest that it would be relevant to focus on long-term consequences on the brain of sweet or sweetened beverages in humans, especially as they may go metabolically unnoticed.

Relaying Aversive Ultrasonic Alarm Calls Depends on Previous Experience. Empathy, Social Buffering, or Panic?

Ultrasonic vocalizations are among the oldest evolutionarily forms of animal communication. In order to study the communication patterns in an aversive social situation, we used a behavioral model in which one animal, the observer, is witnessing as his cagemate, the demonstrator, is experiencing a series of mild electrical foot shocks. We studied the effect of the foot shock experience on the observer and the influence of a warning sound (emitted shortly before the shock) on USV communication. These experiments revealed that such a warning seems to increase the arousal level, which differentiates the responses depending on previous experience. This can be identified by the emission of characteristic, short 22 kHz calls of a duration below 100 ms. Two rats emitted calls that overlapped in time. Analysis of these overlaps revealed that in 'warned' pairs with a naive observer, 22 kHz calls were mixed with 50 kHz calls. This fact, combined with a high fraction of very high-pitched 50 kHz calls (over 75 kHz), suggests the presence of the phenomenon of social buffering. Pure 22 kHz overlaps were mostly found in 'warned' pairs with an experienced observer, suggesting a possible fear contagion with distress sharing. The results show the importance of dividing 22 kHz calls into long and short categories.

Distinct circuits in rat central amygdala for defensive behaviors evoked by socially signaled imminent versus remote danger.

Animals display a rich repertoire of defensive responses adequate to the threat proximity. In social species, these reactions can be additionally influenced by the behavior of fearful conspecifics. However, the majority of neuroscientific studies on socially triggered defensive responses focuses on one type of behavior, freezing. To study a broader range of socially triggered reactions and underlying mechanisms, we directly compared two experimental paradigms, mimicking occurrence of the imminent versus remote threat. Observation of a partner currently experiencing aversive stimulation evokes passive defensive responses in the observer rats. Similar interaction with a partner that has just undergone the aversive stimulation prompts animals to increase active exploration. Although the observers display behaviors similar to those of the aversively stimulated demonstrators, their reactions are not synchronized in time, suggesting that observers' responses are caused by the change in their affective state rather than mimicry. Using opsins targeted to behaviorally activated neurons, we tagged central amygdala (CeA) cells implicated in observers' responses to either imminent or remote threat and reactivated them during the exploration of a novel environment. The manipulation revealed that the two populations of CeA cells promote passive or active defensive responses, respectively. Further experiments confirmed that the two populations of cells at least partially differ in expression of molecular markers (protein kinase C-δ [PKC-δ] and corticotropin-releasing factor [CRF]) and connectivity patterns (receiving input from the basolateral amygdala or from the anterior insula). The results are consistent with the literature on single subjects' fear conditioning, suggesting that similar neuronal circuits control defensive responses in social and non-social contexts.

Social deprivation substantially changes multi-structural neurotransmitter signature of social interaction: Glutamate concentration in amygdala and VTA as a key factor in social encounter-induced 50-kHz ultrasonic vocalization.

Ultrasonic vocalizations are important for coordinating social behavior in rats. Examination of the neurochemical mechanisms that govern social behavior and ultrasonic vocalization emission is crucial for understanding the social impairments that occur in many neuropsychiatric disorders. To elucidate neurochemical changes in the brain structures related to social behavior and their mutual relationships, we conducted three-phase experiment. Neurochemicals were measured in the following behavioral situations: without social encounter, with short social encounter, with long social encounter in isolated and non-isolated rats. The aims of this study were to: (1) extract the most important neurotransmitters and their metabolites that are involved in social encounter-induced emission of 50 kHz calls; (2) to elucidate mutual relationships among the neurochemical changes in the selected, six brain structures, and analyze compound relationships by step analysis; (3) create a model of all-to-all neurotransmitter correlations; (4) find the neurochemical basis of 50-kHz USVs emission during social encounter. Our behavioral and neurochemical analysis indicated that social encounter was a triggering factor of the glutamatergic neurotransmission in the ventral tegmental area (VTA), hippocampus, and amygdala; serotonergic neurotransmission in the NAcc, CPu, and amygdala; the dopaminergic neurotransmission in the caudate putamen (CPu) and hippocampus; GABAergic neurotransmission in the hippocampus and VTA. Social encounter-induced 50-kHz USVs were bound up with changes in glutamate in amygdala and VTA, glycine in the amygdala, VTA, hippocampus, nucleus accumbens and CPu, and dopamine metabolites in VTA and CPu.

Inter-individual differences in serotonin and glutamate co-transmission reflect differentiation in context-induced conditioned 50-kHz USVs response after morphine withdrawal.

A growing body of research provides compelling evidence that in rats 50-kHz USVs are a form of expression of positive emotions. Context-induced 50-kHz USVs emission is variable among rats, indicating individual differences in contextual response bound up with pharmacological reward. The aims of this study were to: extract the most important neurotransmitters related to context-induced conditioned 50-kHz USVs response; find biological basis of existing inter-individual differences in context-induced conditioned 50-kHz USVs response; create a model of all-to-all neurotransmitters correlations. The data collected here confirms that re-exposure to the context of morphine administration after the withdrawal period increases the level of 50-kHz USVs and this contextual response is associated with elevated serotonin concentrations in amygdala, hippocampus and mPFC and with increased Glu/Gln ratio in nucleus accumbens. The concentration of serotonin increases simultaneously in amygdala, nucleus accumbens and hippocampus. Moreover, 5-HT concentration in amygdala is bound up with glutamate level in this structure as well as in hippocampus. Furthermore, Glu/Gln ratio in nucleus accumbens has strong associations with Glu/Gln ratio simultaneously in VTA, amygdala, striatum and hippocampus. All-to-all-analysis indicate that concentration of glutamate in hippocampus is proportional to glutamate in VTA and GABA concentration in the hippocampus. We have also demonstrated that Glu/GABA ratio in VTA and amygdala was elevated after post withdrawal re-exposure to the pharmacological reward paired context. Presented analysis indicates a strong correlation between serotonergic and glutamatergic systems in context-induced conditioned response. The strength of this co-transmission correlates with the number of 50-kHz USVs emitted in response to morphine-paired context.

Non-parametric analysis of neurochemical effects and Arc expression in amphetamine-induced 50-kHz ultrasonic vocalization.

A number of studies have identified the importance of dopaminergic, opioid, serotonergic, noradrenergic and glutamatergic neurotransmission in amphetamine-induced "50-kHz" ultrasonic vocalizations (USVs). Amphetamine became a topic of interest for many researchers interested in USVs due to its ability to induce 50-kHz USVs. To date, it has been difficult to identify the neurotransmitters responsible for this phenomenon. The aim of this study was to determine the following: (i) concentrations of neurotransmitters in selected structures of the rat brain after re-exposure of the rats to amphetamine administration; (ii) changes in Arc in the medial prefrontal cortex, striatum, nucleus accumbens core and shell, hippocampus, amygdala and ventral tegmental area; and (iii) a biological basis for differences in 50-kHz USV emissions in response to amphetamine administration. Re-exposure to amphetamine increased 50-kHz USVs. This parameter do not correlate with distance covered by the investigated animals. An increased concentration of noradrenaline in the nucleus accumbens (NAcc) strongly correlated with the number of 50-kHz USVs. We found that NAcc noradrenaline concentrations negatively correlated with the concentration of dopamine and dopamine metabolites and positively correlated with the concentration of GABA and 5-HIAA (serotonin metabolite) in this structure. We have also identified a positive correlation between striatal 3-MT (dopamine metabolite) concentrations and Arc expression in the hippocampal DG as well as a negative correlation between the concentration of GABA in the amygdala and Arc expression in the central amygdala. Thus, the relationship between the emission of 50-kHz USVs and the neurochemical changes that occur after re-exposure to amphetamine indicates cross-talk between NA, DA, 5-HT and GABA neurotransmission in the NAcc.

κ-opioid receptor as a key mediator in the regulation of appetitive 50-kHz ultrasonic vocalizations.

RATIONALE: Acute administration of high doses of morphine reduced 50-kHz ultrasonic vocalizations (USVs). Although morphine meets the classical criteria for inducing 50-kHz USVs (it causes place preference and induces dopamine release in nucleus accumbens), it also inhibits appetitive vocalizations. OBJECTIVE: The aims of this study were to (i) study the pharmacological impact of κ-opioid (KOR) and µ-opioid receptor (MOR) ligands on the emission of 50-kHz USVs triggered by social interaction after long-term isolation and (ii) analyze the concentrations of the main neurotransmitters in reward-related structures (ventral tegmental area (VTA), nucleus accumbens (NAcc), and medial prefrontal cortex (mPFC)). METHODS: In an attempt to define the effects of opioid-receptor activation on the reward system, we used a social interaction test (after 21 days isolation). HPLC analysis was used to determine the monoamine and amino acid concentrations in reward-related structures. RESULTS: U-50488 (10.0 mg/kg), morphine (5.0 and 1.0 mg/kg), and naltrexone (5.0 mg/kg) decreased, and nor-BNI (10.0 mg/kg) increased 50-kHz USVs. Acute pretreatment with nor-BNI or naltrexone reduced the 50-kHz suppression induced via morphine. The biochemical data showed several variations between groups regarding dopamine concentrations, serotonin, and their metabolites; these data may suggest that the levels of emitted ultrasound in the 50-kHz band are inversely proportional to the 5-hydroxyindoleacetic acid (5-HIAA)/3-methoxytyramine (3-MT) ratio in the VTA. CONCLUSIONS: These results indicate an important role for KOR in the regulation of 50-kHz USV emissions and suggest that KOR activation may be a key mediator in the regulation of reward responses. Changes in the balance between serotonin and dopamine concentrations in the VTA may be a key predictor for 50-kHz USV emission.

Increases in the numerical density of GAT-1 positive puncta in the barrel cortex of adult mice after fear conditioning.

Three days of fear conditioning that combines tactile stimulation of a row of facial vibrissae (conditioned stimulus, CS) with a tail shock (unconditioned stimulus, UCS) expands the representation of "trained" vibrissae, which can be demonstrated by labeling with 2-deoxyglucose in layer IV of the barrel cortex. We have also shown that functional reorganization of the primary somatosensory cortex (S1) increases GABAergic markers in the hollows of "trained" barrels of the adult mouse. This study investigated how whisker-shock conditioning (CS+UCS) affected the expression of puncta of a high-affinity GABA plasma membrane transporter GAT-1 in the barrel cortex of mice 24 h after associative learning paradigm. We found that whisker-shock conditioning (CS+UCS) led to increase expression of neuronal and astroglial GAT-1 puncta in the "trained" row compared to controls: Pseudoconditioned, CS-only, UCS-only and Naïve animals. These findings suggest that fear conditioning specifically induces activation of systems regulating cellular levels of the inhibitory neurotransmitter GABA.

Diverging frequency-modulated 50-kHz vocalization, locomotor activity and conditioned place preference effects in rats given repeated amphetamine treatment.

Behavioral sensitization and tolerance to repetitive exposure to addictive drugs are commonly used for the assessment of the early stages of the drug dependence progress in animals. The orchestra of tools for studying the progress of drug dependence in laboratory rodents has been considerably enriched in the 1980s by the introduction of ultrasonic vocalization (USV) detection and characterization. However, the relationship between the results of this technology and those of traditional behavioral tests is not clear. We attempted to elucidate some of the respective ambiguities by comparing the effects of an intermittent amphetamine treatment, which was aimed both at the induction of sensitization and tolerance to this drug and at testing the persistence of these effects, on the locomotor activity and 50-kHz USV responses to both the drug and the context of drug exposure in adult male rats showing diverging susceptibility for sensitization to amphetamine. Categorization of the rats into low and high responders/callers based on sensitization of their frequency-modulated 50-kHz USV responsiveness showed some correspondence with conditioned place preference effects, but not with responses to amphetamine. The study showed distinct changes in the rate and latency of the frequency-modulated 50-kHz USV responses to repetitive amphetamine treatment, which were reminiscent of classical behavioral signs of sensitization and tolerance. These results show the utility of the appetitive USV for monitoring of early phases of complex processes leading to drug dependence. However, USV, locomotor activity and conditioned place preference seem to reflect different aspects of these phenomena.

Inter-individual diversity and intra-individual stability of amphetamine-induced sensitization of frequency-modulated 50-kHz vocalization in Sprague-Dawley rats.

RATIONALE: Propensity for drug dependence shows great diversity that is related to intrinsic neurobiological factors. This diversity is important both for the understanding of these traits and for the development of therapies. OBJECTIVES: The goals of the study were (1) to define, using ultrasonic vocalization characteristics, inter-individual differences in rats' propensity for sensitization to amphetamine, (2) to test whether possible resistance to this effect could be overcome with repetitive treatment, and (3) to seek useful predictors of the propensity. METHODS: Rats were subject to tests meant to characterize their anxiety, pain sensitivity, and responses to novelty and natural rewards. Then they were subject to the so-called two-injection protocol of sensitization (using amphetamine) followed by 2 weeks of daily amphetamine treatment, 2-week withdrawal, and final amphetamine challenge. The development and outcome of sensitization were monitored by measuring 50-kHz vocalization. RESULTS: The two-injection protocol yielded three patterns of changes in the frequency-modulated 50-kHz vocalization response to amphetamine. These patterns persisted after completion of the extended drug treatment. Rats with lower sensitivity to pain or with longer latency of their vocalization response to the first drug exposure showed an increased propensity for ultrasonic vocalization sensitization. CONCLUSION: Vulnerability to sensitization of frequency-modulated 50-kHz vocalization response of Sprague-Dawley rats to amphetamine, which supposedly reflects rats' propensity for amphetamine dependence, shows large inter-individual diversity. Resistance to this effect, which is evident in a majority of the rats, cannot be overcome even with prolonged intermittent drug treatment under the conditions (novelty) that promote sensitization.

The effects of morphine and morphine conditioned context on 50 kHz ultrasonic vocalisation in rats.

The 50 kHz ultrasonic vocalisations (USVs) that are emitted by rats are dependent on activity of dopaminergic neurons projecting from the ventral tegmental area to the limbic and cortical structures. According to many experimental data, emission of the 50 kHz USV reflects a positive emotional state. The appetitive calls are also emitted in response to the administration of drugs of abuse, e.g., cocaine or amphetamine (AMPH), or in a reply to a positively conditioned context. However, there is no strong evidence in the literature that morphine can also modulate 50 kHz USVs. The aim of this paper is to study the effects of morphine and morphine-conditioned context on 50 kHz USVs, using spontaneously or drug-modulated 50 kHz USVs. Our results showed that acute administration of morphine to rats after withdrawal period inhibited the emission of 50 kHz USVs. The stimulating effect of morphine-conditioned context on 50 kHz USVs appeared on the post-withdrawal challenge day immediately before drug injection, 14 days after the last episode of morphine-induced context conditioning. The context-induced 50 kHz USVs can be used as a sensitive test for drug dependency. The current study also shows that 50 kHz USVs can be useful tool for studying the mechanisms of long lasting central effects of morphine.

The effects of midazolam and D-cycloserine on the release of glutamate and GABA in the basolateral amygdala of low and high anxiety rats during extinction trial of a conditioned fear test.

In this study, we investigated how midazolam and d-cycloserine regulate the tonic activity and/or phasic reactivity of brain neurotransmitter systems to fear-evoking stimuli in rats with varying intensities of a fear response. We used a new animal model composed of high (HR) and low (LR) anxiety rats, selected according to their behaviour in the contextual fear test (i.e., the duration of a freezing response was used as a discriminating variable). In these rats, we examined the effects of both drugs on the release of glutamate and GABA in the basolateral amygdala (BLA) during the first extinction trial of a conditioned fear test. The results showed that administration of d-cycloserine (15 mg/kg, i.p.) significantly enhanced the inhibition of an aversive context-induced freezing response observed during the extinction session in HR and LR rats. In contrast, midazolam (0.75 mg/kg, i.p.) accelerated the attenuation of fear responses only in HR rats. The less anxious behaviour of LR animals given saline was accompanied by elevated basal levels of glutamate in the BLA, in comparison with HR rats, and a stronger elevation of GABA in response to contextual fear. In HR animals, the pretreatment of rats with d-cycloserine and midazolam significantly increased the local concentration of GABA and inhibited the expression of contextual fear. These findings suggest that animals more vulnerable to stress have innate deficits in brain systems that control the activity of the BLA mediating the central effect of stress. These results contribute to our understanding of observed individual differences in the effects of anxiolytic drugs among patients with anxiety disorders.

The effects of buspirone and diazepam on aversive context- and social isolation-induced ultrasonic vocalisation.

Rats emit two types of high-frequency vocalisations (aversive and appetitive calls) in different behavioural situations. The aims of this paper were to examine an animal model of appetitive behaviour (as an element of social interaction) and to study the effects of selected psychotropic drugs on appetitively evoked ultrasonic vocalisation (USVs) and aversive context-evoked USVs. Specifically, we analysed the impact of the encounter of pairs of adult rats after long-term isolation on ultrasound vocalisation. It was found that isolation of the adult rats significantly enhanced the appetitive ultrasound vocalisations (50-kHz) during encounters between pairs of rats. In the pharmacological part of the study, we found that diazepam (1.0 mg kg(-1)) significantly increased isolation-induced appetitive USVs (50 kHz) and decreased aversive context-evoked USVs (22-kHz). Buspiron (3.0 mg kg(-1)) decreased the aversive context-evoked USVs and had no effect on isolation-induced appetitive USVs. These data indicate that long-term, isolation-induced 50-kHz USVs in adult rats represent a new behavioural parameter under control of the central GABAergic system, which can be used to study the effects of anxiolytic drugs.

Mapping of c-Fos expression in the rat brain during the evolution of pentylenetetrazol-kindled seizures.

c-Fos protein immunocytochemistry was used to map the brain structures recruited during the evolution of seizures that follows repeated administration of a subconvulsive dose (35mg/kg, ip) of pentylenetetrazol in rats. c-Fos appeared earliest in nucleus accumbens shell, piriform cortex, prefrontal cortex, and striatum (stages 1 and 2 of kindling in comparison to control, saline-treated animals). At the third stage of kindling, central amygdala nuclei, entorhinal cortex, and lateral septal nuclei had enhanced concentrations of c-Fos. At the fourth stage of kindling, c-Fos expression was increased in basolateral amygdala and CA1 area of the hippocampus. Finally, c-Fos labeling was enhanced in the dentate gyrus of the hippocampus only when tonic-clonic convulsions were fully developed. The most potent changes in c-Fos were observed in dentate gyrus, piriform cortex, CA1, lateral septal nuclei, basolateral amygdala, central amygdala nuclei, and prefrontal cortex. Piriform cortex, entorhinal cortex, prefrontal cortex, lateral septal nuclei, and CA3 area of the hippocampus appeared to be the brain structures selectively involved in the process of chemically induced kindling of seizures.

The expression of c-Fos and colocalisation of c-Fos and glucocorticoid receptors in brain structures of low and high anxiety rats subjected to extinction trials and re-learning of a conditioned fear response.

We designed an animal model to examine the mechanisms of differences in individual responses to aversive stimuli. We used the rat freezing response in the context fear test as a discriminating variable: low responders (LR) were defined as rats with a duration of freezing response one standard error or more below the mean value, and high responders (HR) were defined as rats with a duration of freezing response one standard error or more above the mean value. We sought to determine the colocalisation of c-Fos and glucocorticoid receptors-immunoreactivity (GR-ir) in HR and LR rats subjected to conditioned fear training, two extinction sessions and re-learning of a conditioned fear. We found that HR animals showed a marked decrease in conditioned fear in the course of two extinction sessions (16 days) in comparison with the control and LR groups. The LR group exhibited higher activity in the cortical M2 and prelimbic areas (c-Fos) and had an increased number of cells co-expressing c-Fos and GR-ir in the M2 and medial orbital cortex after re-learning a contextual fear. HR rats showed increased expression of c-Fos, GR-ir and c-Fos/GR-ir colocalised neurons in the basolateral amygdala and enhanced c-Fos and GR-ir in the dentate gyrus (DG) in comparison with LR animals. Our data indicate that recovery of a context-related behaviour upon re-learning of contextual fear is accompanied in HR animals by a selective increase in c-Fos expression and GRs-ir in the DG area of the hippocampus.

Stress-opioid interactions: a comparison of morphine and methadone.

The utility of methadone and morphine for analgesia and of methadone for substitution therapy for heroin addiction is a consequence of these drugs acting as opioid receptor agonists.We compared the cataleptogenic and antinociceptive effects of single subcutaneous doses of methadone hydrochloride (1-4 mg/kg) and morphine sulfate (2.5-10 mg/kg) using catalepsy and hot-plate tests, and examined the effects of the highest doses of the drugs on Fos protein expression in selected brain regions in male Sprague-Dawley rats. Methadone had greater cataleptogenic and analgesic potency than morphine. Fos immunohistochemistry revealed substantial effects on the Fos response of both the stress induced by the experimental procedures and of the drug exposure itself. There were three response patterns identified: 1) drug exposure, but not stress, significantly elevated Fos-positive cell counts in the caudate-putamen; 2) stress alone and stress combined with drug exposure similarly elevated Fos-positive cell counts in the nucleus accumbens and cingulate cortex; and 3) methadone and morphine (to a lesser extent) counteracted the stimulatory effect of nonpharmacological stressors on Fos protein expression in the somatosensory cortex barrel field, and Fos-positive cell counts in this region correlated negatively with both the duration of catalepsy and the latency time in the hot-plate test. The overlap between brain regions reacting to nonpharmacological stressors and those responding to exogenous opioids suggests that stress contributes to opioid-induced neuronal activation.

The effects of group III mGluR ligands on pentylenetetrazol-induced kindling of seizures and hippocampal amino acids concentration.

Considering the contribution of hippocampal formation and glutamate-mediated signalling to epileptogenesis, we investigated the effects of group III metabotropic glutamate receptor (mGluR)-selective ligands on the kindling of seizures. We also examined the concentration of the amino acid glutamate, GABA, alanine and taurine in the hippocampus of rats using a microdialysis technique. Pentylenetetrazol (PTZ), a non-competitive antagonist of the GABA(A) receptor, was administered intraperitoneally at 35 mg/kg body weight to induce seizures. It was determined that the kindling of PTZ-induced seizures could be attenuated by post intracerebroventricular administration of 100 nmol of the group III mGluR antagonist CPPG [(RS)-a-cyclopropyl-4-phosphonophenylglycine]. There were significant differences in tested parameters during the final stages of the kindling procedure. The group III mGluR agonist L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid at 100 nmol, i.c.v.] did not significantly affect the kindling of seizures in comparison to control rats, although there was acceleration of the process as compared to CPPG treated animals. We demonstrated that the baseline concentrations of glutamate, GABA, alanine, taurine, and the glutamine/GABA ratio were elevated in the hippocampus of fully kindled rats. Intracerebroventricular administration of a single dose of CPPG increased the concentrations of glutamate in the hippocampus of control, non-kindled rats. Intracerebroventricular administration of L-AP4 did not affect the hippocampal amino acid concentration in either animal group. Overall, these data suggest that there is a shift in the balance between neurotransmitters towards increased production of excitatory amino acids, and this may be mediated by group III mGluRs during seizure kindling.

The effect of CRF and alpha-helical CRF((9-41)) on rat fear responses and amino acids release in the central nucleus of the amygdala.

The effects of intracerebroventricular injections of CRF and a non-selective CRF receptor antagonist, alpha-helical CRF((9-41)), on the release of glutamate, aspartate, and GABA in the central nucleus of the amygdala (CeA), were examined in the course of testing rat anxiety-like behaviour in the conditioned fear test (a freezing response), using the microdialysis technique. It was found that CRF (1 microg/rat), given to animals exposed to the stress of novelty only, insignificantly increased the glutamate concentration in the CeA, up to 200% of the control level. In the fear-conditioned animals, the influence of CRF on the local concentration of aspartate, glutamate, and Glu/GABA ratio was much more pronounced (up to a 400% increase above the baseline level of aspartate concentration), preceded an increased expression of anxiety-like responses, and appeared as early as 15 min after the drug administration. The intracerebroventricular administration of alpha-helical CRF((9-41)) (10 microg/rat) significantly decreased the rat freezing responses and increased the local concentration of GABA during the first 30 min of observation. In sum, these are new findings, which show an important role of CRF in the CeA in the regulation of fear-controlled amino acids release and suggest an involvement of amino acids in the central nucleus of the amygdala in the effects of this neurohormone on the expression of conditioned fear.