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The amplitude of the whole-body acoustic startle response is reliably enhanced when elicited in the presence of foreground signals, such as light, previously paired with footshocks. It has been shown that this enhancement is evident by moderate fear levels, but is less affected by high fear levels. Potentiation of the acoustic startle reflex has also been reported in the presence of background cues previously associated with footshocks. However, the effects of anxiolytic drugs on different levels of fear elicited by moderate and intense contextual fear conditioning associated with startle reflex have not been examined yet. To approach this issue, we examined the effects of the anxiolytic, midazolam, on two intensities of contextual fear; freezing behavior and the startle response to loud noise. First, we compared the magnitude of the freezing behavior and the startle amplitude during the testing sessions in groups of rats submitted to fear conditioning using 0.3 and 0.6 mA as unconditioned stimuli (10 stimuli of 1 s each, intertrial interval from 60 to 180 s). Afterwards, the effects of midazolam (0.5 and 1.0 mg/kg) were assessed in these two conditions. Rats showed a potentiated startle reflex and a significant freezing behavior to moderate fear conditioning, which were both attenuated by midazolam. Higher levels of fear conditioning caused more intense freezing behavior without enhancing the startle reflex. Whereas midazolam reduced this freezing response, the startle response was unaffected. These results are indicative that anxiolytic-sensitive freezing and fear-potentiated startle are triggered by moderate contextual fear conditioning, while contextual conditioning with the use of high footshocks causes a distinct pattern of behavioral responses, which is only partially affected by midazolam. Due to the differential sensitivity to midazolam of these two patterns of startle responses generated as a function of the intensity of contextual fear conditioning, it is proposed that they represent moderate and intense aversive states that may be related to anxiety or panic/phobic conditions, respectively.

Consistent evidence has shown that learning may be produced in paradigms using electrical stimulation of the inferior colliculus as unconditioned stimulus (US). Much evidence has also been provided for the involvement of dopamine in the setting up of adaptive responses to aversive states generated at this mesencephalic level. The aim of the present study was to determine whether dopaminergic mechanisms are involved in the conditioned freezing behavior and latent inhibition (LI) using either foot shocks (Experiment 1) or inferior colliculus stimulation (Experiment 2) as US and light as conditioned stimulus (CS). To this end, the authors examined the effects of the dopaminergic agonist apomorphine (AP; 0, 0.5, 1.0, and 2.0 mg/kg) and the atypical antipsychotic clozapine (CLZ 0, 1.0, 2.5, and 5.0 mg/kg) on the acquisition of conditioned freezing and LI. Rats were either simply placed in the experimental chamber non-preexposed (NPE) or preexposed (PE) to 50 light presentations. Next, they were submitted to 10 light plus foot shock (or inferior colliculus stimulation) pairings. In the testing session, conditioned freezing under the CS was measured. In both experiments, conditioned freezing was highly significant in the NPE group while previous exposure to nonreinforced light (PE) weakened the strength of the conditioning. Although weaker than that caused by foot shock/light pairings, the occurrence of freezing and LI with the use of inferior colliculus stimulation as US gives additional support to the participation of this structure in the filtering processes of relevant information to higher brain regions. The drug effects depended on whether foot shock or inferior colliculus stimulation was used as US. In Experiment 1, AP produced a dose dependent increase in conditioned freezing without changing LI. On the other hand, CLZ did not change freezing and LI. In Experiment 2 while AP did not change freezing and LI, CLZ clearly increased freezing without changing LI with the use of inferior colliculus as US. The selective effect of AP on conditioned freezing due to light/foot shocks association is consistent with the heightened attentional and cognitive functions of dopaminergic mechanisms of the mesocorticolimbic systems in the setting up of adaptive responses aimed at coping with or signaling the presence of stimuli of aversive nature. The selective effect of CLZ on conditioned freezing is due to light/IC stimulation and may also be due to the known antagonism of 5-HT2 receptors produced by low doses of this atypical antipsychotic.

The amygdala has a crucial role in detecting motivationally significant inputs and in communicating relevant information to other limbic structures. Behavioural studies have shown that the central (CeA) and basolateral (BLA) nuclei of amygdala differentially regulate conditioned and unconditioned fear. Indeed, much evidence has accumulated suggesting that regulatory mechanisms in the BLA serve as a filter for unconditioned and conditioned aversive information that ascends to higher structures from the brainstem, whereas the CeA is the main output for the autonomic and somatic components of fear reaction through major projections to other limbic regions. It is still unclear, however, how amygdaloid nuclei function in high and open spaces so as to determine the characteristic exploratory behaviour of rats submitted to the elevated plus-maze test (EPM). In the present study, we carried out an ethopharmacological analysis of the behaviour of rats submitted to the elevated plus-maze test together with analysis of the tissue content of monoamine dopamine (DA) and serotonin (5-HT) and their metabolites in the dorsal hippocampus (DH), nucleus accumbens (NAC) and dorsal striatum (DS) of animals injected with saline or muscimol (1.0 nmol/0.2 microL) into the BLA or CeA. The data obtained show that injections of muscimol into the CeA, but not into the BLA, caused anxiolytic-like effects in the EPM. Such effects of muscimol into the CeA were accompanied by increases in 5-HT content of the DH, whereas corresponding injections into the BLA caused a reduction in the DA content of the NAC. There was no change in the turnover rates of these monoamines. These data suggest that the BLA and CeA have distinct roles in the exploratory behaviour of rodents in the EPM. While BLA appears to be related to the detection and validation of threatening stimuli, the CeA appears to be involved in the expression of fear behaviours in the EPM.

It has been demonstrated that exposure to a variety of stressful experiences enhances fearful reactions when behavior is tested in current animal models of anxiety. Until now, no study has examined the neurochemical changes during the test and retest sessions of rats submitted to the elevated plus maze (EPM). The present study uses a new approach (HPLC) by looking at the changes in dopamine and serotonin levels in the prefrontal cortex, amygdala, dorsal hippocampus, and nucleus accumbens in animals upon single or double exposure to the EPM (one-trial tolerance). The study involved two experiments: i) saline or midazolam (0.5 mg/kg) before the first trial, and ii) saline or midazolam before the second trial. For the biochemical analysis a control group injected with saline and not tested in the EPM was included. Stressful stimuli in the EPM were able to elicit one-trial tolerance to midazolam on re-exposure (61.01%). Significant decreases in serotonin contents occurred in the prefrontal cortex (38.74%), amygdala (78.96%), dorsal hippocampus (70.33%), and nucleus accumbens (73.58%) of the animals tested in the EPM (P < 0.05 in all cases in relation to controls not exposed to the EPM). A significant decrease in dopamine content was also observed in the amygdala (54.74%, P < 0.05). These changes were maintained across trials. There was no change in the turnover rates of these monoamines. We suggest that exposure to the EPM causes reduced monoaminergic neurotransmission activity in limbic structures, which appears to underlie the "one-trial tolerance" phenomenon.

The involvement of dopaminergic mechanisms in fear and anxiety is still unclear. Behavioral studies aimed to disclose the involvement of dopamine in anxiety have reported anxiolytic-like, anxiogenic-like and lack of effects with the use of dopaminergic agonists and antagonists in animal models of anxiety. This work was an attempt to contribute to this field by providing evidence that these discrepancies may be due to the kind of aversive situation the animals experience in these models. The present study examined the effects of a dopaminergic agonist apomorphine, a dopaminergic D(1) antagonist SCH 23390 and a D(2) receptor antagonist sulpiride on the two-way avoidance response test (CAR) and on the switch-off responses to light (SOR). In both tests, learning was assessed by the performance of the animals across four blocks of 10 trials in which light was paired to footshocks (CAR) or only light was presented to the animals (SOR). The obtained data show that rats learn to make a shuttling response to avoid the shock in the CAR test and maintain a regular pace of switch-off responses in the SOR. While sulpiride and SCH 23390 administrations prevented learning of the avoidance responses, apomorphine injections produced a dose-dependent enhancement in the conditioned learning in the CAR test. The number of escape responses was unchanged by these drugs. In the light-induced switch-off test, apomorphine reduced the number of switch-off responses whereas sulpiride increased these responses. These findings suggest that the involvement of dopaminergic mechanisms in threatening situations depends on the nature of the aversive stimulus. Activation of D(1) and D(2) receptors seems to be implicated in the heightened aversiveness to conditioned stressful situations, as assessed by the CAR test. Thus, blockade of D(1) and D(2) receptors may be necessary for attenuating the aversiveness triggered by these conditioned fear stimuli. In contrast, mechanisms mediated by D(2) receptors seem to be involved in the setting up of adaptive responses to innate fear reactions. Therefore, the signal of the modulatory dopaminergic mechanisms on defensive behavior will depend on the type of emotional stimuli triggering the coping reaction.

The dangerous stimuli may be potentially dangerous, distal or proximal and the recognition by the animals of each one of these conditions is determinant for the nature of the fear responses. In the present article a parallel with this particular process is drawn taking into account that different fear responses are generated by light, tones and contexts used as conditioned stimuli and by unconditioned stimulation of the dorsal periaqueductal gray (dPAG). In this review we summarize the efforts that have been made to characterize the neural circuits recruited in the organization of defensive reactions to the conditioned and unconditioned aversive stimulations, particularly evidence linking the brain's defense response systems to the concept of fear-stress-anxiety. The dPAG constitute the main neural substrates for the integration of aversive states in response to proximal aversive stimuli. In fact, panic-like behaviors often result when this structure is electrically or chemically stimulated. On the other hand, successful preparatory processes of danger-orientation and preparedness to flee seem to be linked to anxiety. The pre-frontal and cingulate cortex, median raphe nucleus, septum and hippocampus seem to be implicated in the elaboration and organization of these responses. As a working hypothesis, it is advanced that increasing the intensity and proximity of the danger may lead to an emotional shift. When the animals are submitted to this gradual increase in aversiveness there is a switch from the neural circuits responsible for the production of the orientated and organized motor patterns of appropriate defensive response to a conditioned stimulus towards the incomplete and uncoordinated defense responses related to panic attacks. The circuits in the amygdala and the medial hypothalamus responsible for the organization of the defense reaction may well subserve to this switch process.

A natureza das respostas de medo em animais expostos a situações ameaçadoras depende da intensidade e da distância do estímulo aversivo. Esses estímulos podem ser potencialmente perigosos, distais ou proximais ao animal. Esforços têm sido feitos no sentido de identificar os circuitos neurais recrutados na organização das reações defensivas a estas condições aversivas. Neste artigo, sumarizamos evidências que associam os sistemas cerebrais de defesa ao conceito de medo-stress-ansiedade. Respostas de orientação ao estímulo de perigo, à esquiva e à preparação para o enfrentamento do perigo parecem estar associados à ansiedade. O giro do cíngulo e o córtex pré-frontal de um lado; o núcleo mediano da rafe, septo e o hipocampo de outro fazem parte dos circuitos cerebrais que integram essas respostas emocionais. No outro extremo, estímulos de medo que induzem formas ativas de defesa, mas pouco elaboradas, determinam estados emocionais de natureza diferente e parecem associadas a manifestações elementares de medo. A substância cinzenta periaquedutal dorsal constitui o principal substrato neural para a integração desses estados aversivos no cérebro. Comportamentos defensivos desse tipo são produzidos pela estimulação elétrica e química desta estrutura. À medida que os estímulos ameaçadores, potenciais e distais dão lugar a estímulos de perigo muito intensos ou são substituídos por estímulos proximais de medo, ocorre uma comutação (switch) dos circuitos neurais usualmente responsáveis pela produção de respostas condicionadas de medo para reações defensivas com baixo nível de regulação e organização que se assemelham aos ataques de pânico. Portanto, dependendo da natureza do evento estressor ou do estímulo incondicionado, o padrão de respostas defensivas orientadas e organizadas cede lugar a respostas motoras incoordenadas e incompletas. A amígdala e o hipotálamo medial podem funcionar como uma espécie de interface comutando os estímulos para os substratos neurais apropriados para elaboração das respostas defensivas condicionadas ou incondicionadas. The dangerous stimuli may be potentially dangerous, distal or proximal and the recognition by the animals of each one of these conditions is determinant for the nature of the fear responses. In the present article a parallel with this particular process is drawn taking into account that different fear responses are generated by light, tones and contexts used as conditioned stimuli and by unconditioned stimulation of the dorsal periaqueductal gray (dPAG). In this review we summarize the efforts that have been made to characterize the neural circuits recruited in the organization of defensive reactions to the conditioned and unconditioned aversive stimulations, particularly evidence linking the brain's defense response systems to the concept of fear-stress-anxiety. The dPAG constitute the main neural substrates for the integration of aversive states in response to proximal aversive stimuli. In fact, panic-like behaviors often result when this structure is electrically or chemically stimulated. On the other hand, successful preparatory processes of danger-orientation and preparedness to flee seem to be linked to anxiety. The pre-frontal and cingulate cortex, median raphe nucleus, septum and hippocampus seem to be implicated in the elaboration and organization of these responses. As a working hypothesis, it is advanced that increasing the intensity and proximity of the danger may lead to an emotional shift. When the animals are submitted to this gradual increase in aversiveness there is a switch from the neural circuits responsible for the production of the orientated and organized motor patterns of appropriate defensive response to a conditioned stimulus towards the incomplete and uncoordinated defense responses related to panic attacks. The circuits in the amygdala and the medial hypothalamus responsible for the organization of the defense reaction may well subserve to this switch process.