Anxiogenic Stimuli Research Articles

Conditioned fear-induced changes in behavior and in the expression of the immediate early gene c-fos: with and without diazepam pretreatment

The synthesis of Fos, the protein product of the immediate early gene c-fos, was used to map metabolically some of the neural substrates of conditioned fear in the rat. Analysis of the behaviors emitted by the rats during the test session provided strong evidence that the conditioning procedure was effective. Exposure to the environment in which they had previously received footshock significantly increased the number of Fos-like immunoreactive neurons in nearly 50 brain regions, both cortical and subcortical. Among the structures showing the most dramatic increases in fear-induced c-fos expression were the cingulate, piriform, infralimbic, and retrosplenial cortices, the anterior olfactory nucleus, claustrum, endopiriform nucleus, nucleus accumbens shell, lateral septal nucleus, various amygdalar nuclei, paraventricular thalamic nucleus, ventral lateral geniculate nucleus, the ventromedial, lateral, and dorsal hypothalamic nuclei, the ventral tegmental area, and the supramammillary area. These data demonstrate that a relatively simple classical conditioning procedure activates a large number of widely dispersed cortical and subcortical structures. Some of the structures showing increased c-fos expression have important autonomic functions and may therefore have reflected centrally mediated changes in blood pressure and respiration produced by the anxiogenic stimuli. In a second experiment, the effects of pretreatment with the anxiolytic drug diazepam (2.5, 5.0, or 10 mg/kg) were evaluated. The benzodiazepine produced dose-related decreases in the frequency of crouching (freezing) elicited by the aversively conditioned contextual cues. Diazepam also produced dose-related decreases in conditioned stress-induced c-fos expression in all but one structure, the effects being statistically significant in 38 of 60 sampled structures. Diazepam dose dependently increased fear-induced c-fos expression in the central nucleus of the amygdala. There was considerable regional variability with respect to sensitivity to diazepam, the retrosplenial cortex and the supramammillary area being the only two structures to show decreases after the lowest dose of diazepam. In contrast, the entorhinal cortex, nucleus accumbens core, ventromedial and posterior hypothalamic nuclei, median raphe, and locus coeruleus were particularly resistant to diazepam, all failing to show statistically significant decreases in conditioned fear-induced c-fos expression even at the highest dose. The extent to which diazepam decreased conditioned stress-induced c-fos expression was unrelated to previous estimates of benzodiazepine receptor density in the sampled structures.

Behavioural effects of methyl-parathion in adult rats

Environmental enrichment can modulate mild and chronic stress, responses to anxiogenic stimuli as well as drug vulnerability in a number of animal models. The current study was designed to examine the impact of postnatal environmental enrichment on selectively bred 4th generation high- (HAn) and low-anxiety (LAn) male rats. After weaning, animals were placed in isolated (IE), social (SE) and enriched environments (EE) (e.g., toys, wheels, ropes, changed weekly). We measured anxiety-like behavior (ALB) on the elevated plus maze (EPM; trial 1 at postnatal day (PND) 46, trial 2 at PND 63), amphetamine (AMPH) (0.5 mg/kg, IP)-induced locomotor behavior, basal and post anxiogenic stimuli changes in (1) plasma corticosterone, (2) blood pressure and (3) core body temperature. Initially, animals showed consistent trait differences on EPM with HAn showing more ALB but after 40 days in select housing, HAn rats reared in an EE showed less ALB and diminished AMPH-induced activity compared to HAn animals housed in IE and SE. In the physiological tests, animals housed in EE showed elevated adrenocortical responses to forced novel object exposure but decreased body temperature and blood pressure changes after an air puff stressor. All animals reared in EE and SE had elevated brain-derived neurotrophic factor (BDNF)-positive cells in the central amygdala (CeA), CA1 and CA2 hippocampal regions and the caudate putamen, but these differences were most pronounced in HAn rats for CeA, CA1 and CA2. Overall, these findings suggest that environmental enrichment offers benefits for trait anxiety rats including a reduction in behavioral and physiological responses to anxiogenic stimuli and AMPH sensitivity, and these responses correlate with changes in BDNF expression in the central amygdala, hippocampus and the caudate putamen.

Differential Effects of Forced Locomotion, Tail‐Pinch, Immobilization, and Methyl‐β‐Carboline Carboxylate on Extracellular 3,4‐Dihydroxyphenylacetic Acid Levels in the Rat Striatum, Nucleus Accumbens, and Prefrontal Cortex: An In Vivo Voltammetric Study

In vivo voltammetry with carbon fiber electrodes was used to assess extracellular 3,4-dihydroxyphenylacetic acid (DOPAC) levels in striatum, nucleus accumbens, and anteromedial prefrontal cortex of freely moving rats subjected to altered motor activity or anxiogenic stimuli. Forced locomotion on a rotarod for 40 min caused an increase in extracellular DOPAC levels in the striatum and to a lesser extent in the nucleus accumbens but not in the prefrontal cortex. Subcutaneous injection of the anxiogenic agent methyl-beta-carboline carboxylate (10 mg/kg) increased extracellular DOPAC levels to a similar extent in prefrontal cortex and nucleus accumbens. Immobilization for 4 min augmented dopamine (DA) metabolism preferentially in the nucleus accumbens and to a lesser extent in the prefrontal cortex. Tail-pinch caused a selective activation of DA metabolism in the nucleus accumbens. None of these stimuli altered extracellular striatal DOPAC levels. These results confirm the involvement of dopaminergic systems projecting to the striatum and nucleus accumbens in motor function and suggest that mesolimbic and mesocortical dopaminergic systems can be specifically activated by certain kinds of anxiogenic stimuli; the relative activation of either of these latter systems could depend primarily on the nature (sensory modality, intensity) of the acute stressor.

Mesocorticolimbic dopaminergic systems and emotional states

We have used the technique of in vivo voltammetry with carbon fibre electrodes to investigate further the involvement of ascending mesencephalic dopaminergic systems in emotional states in freely moving rats. In Sprague-Dawley rats, forced locomotion caused an increase in extracellular DOPAC levels in the striatum and nucleus accumbens but not in the prefrontal cortex. Immobilization (4 min) or systemic injection of the anxiogenic agent methyl-β-carboline carboxylate enhanced extracellular DOPAC in both prefrontal cortex and nucleus accumbens but not in striatum whereas tail-pinch provoked a selective increase in this parameter in the nucleus accumbens. These data suggest that mesolimbic and mesocortical dopaminergic systems can be specifically activated by certain kinds of anxiogenic stimuli. To evaluate the relationship between emotional status and the response of mesocortical dopaminergic neurons to stress, we investigated the effects of stressful conditions on dopamine metabolism in the prefrontal cortex of 2 genetically selected lines of rats which differ drastically in their level of emotionality. Introduction of the animals into an unfamiliar environment, application of a high-intensity loud noise or immobilization were associated with an increase in extracellular cortical DOPAC levels in the hypoemotional (RHA) but not in the hyperemotional (RLA) line. These results suggest that the increase in cortical dopamine metabolism induced by stress is not connected to the emotional reaction caused by the aversive nature of the stressor but may reflect activation of cognitive processes in an attempt to cope with the stressor.

Animal models of drug withdrawal symptoms

There have been few attempts to model subjective symptoms of drug withdrawal using animals as subjects. Two approaches for developing such models are reviewed. First, using drug discrimination methodology, it may be possible to train animals to detect the effects of withdrawal. This method has two difficulties: 1) the only discriminations trained to date involve precipitated withdrawal, and 2) the stimulus controlling behavior is difficult to specify. Second, withdrawal from many drugs of abuse produces the symptom of anxiety, and it seems likely that animal models of anxiety could be useful for studying drug withdrawal. This hypothesis has been explored most fully using subjects trained to detect the discriminative stimulus properties of the putative anxiogenic drug pentylenetetrazole (PTZ). Withdrawal from benzodiazepines or ethanol substitutes fully for PTZ, and withdrawal from cocaine, morphine, and nicotine substitutes partially for PTZ. Emerging data suggest that other animal models of anxiety may also be useful for detecting drug withdrawal. The final portion of this review examines a behavioral test that is very sensitive for detecting physical signs of withdrawal in animals. In subjects maintained on an operant baseline using food as a reinforcer, withdrawal from a drug of dependence frequently is associated with disruption of that operant behavior. For example, tetrahydrocannabinol and cocaine, drugs that are not traditionally seen as having significant withdrawal signs, produce disruption of operant responding when high-dose administration is terminated, and their readministration reverses this behavioral disruption. Based on the observation that withdrawal is associated with anxiogenic stimuli, we suggest a method to determine if disruption of operant behavior may be related to these stimuli.

Operant behavior and reactivity to the anticonflict effect of diazepam in perinatally undernourished rats

Adult female rats perinatally submitted to a protein deprivation schedule and then recovered with balanced chow, were tested under two different operant schedules of reinforcement, variable ratio-20 (VR-20) and differential reinforcement of low rate-5 sec (DRLR-5″). Undernourished animals showed higher response rates in the VR-20 schedule and a lower efficiency in the DRLR-5 as compared to controls, effects that could be attributed to the hyperreactivity to aversive or stressful situations characteristic of these animals. As the behavioral responses to anxiogenic stimuli are sensitive to drugs acting on the GABAergic system, the functionalism of the central GABAergic system in the undernourished group was evaluated by studying the reactivity to the anticonflict effect of diazepam in two conflict tests. Undernourished animals had a lower response to the anticonflict effect of diazepam when compared to control rats. The altered reactivity to the anticonflict effect of diazepam between both groups suggest that some aspects of the GABAergic neurotransmission may be involved in the behavioral abnormalities present in adult rats that were undernourished at perinatal age.

The effects of compounds related to gamma-aminobutyrate and benzodiazepine receptors on behavioural responses to anxiogenic stimuli in the rat: extinction and successive discrimination.

In a first set of experiments rats were trained to run in a straight alley for food reward on a continuous reinforcement schedule and the running response was then extinguished. On the last 2 days of training and daily throughout extinction different groups of animals were injected IP with saline, 5 mg/kg chlordiazepoxide, 0.75 mg/kg picrotoxin, chlordiazepoxide + picrotoxin, chlordiazepoxide + 1.5 mg/kg bicuculline, 0.00125 or 0.25 mg/kg muscimol, 1 mg/kg baclofen, chlordiazepoxide + baclofen, or 0.00125 mg/kg muscimol + baclofen. Chlordiazepoxide increased resistance to extinction, a well-known anxiolytic effect. This effect was blocked by both picrotoxin and bicuculline. Picrotoxin on its own reduced resistance to extinction (an anxiogenic-like effect). Whether given alone or in combination with other drugs, muscimol and baclofen had no effect. In a second set of experiments rats were trained in a successive operant discrimination (signalled by a flashing or steady light) between components in which sucrose reward was available on a variable-interval schedule for barpressing and components in which no reward was given. Chlordiazepoxide at 10 mg/kg increased responding in both rewarded and nonrewarded components, but more in the latter than could be accounted for by change in the former. This effect is as expected with an anxiolytic drug. It was not altered by administration of bicuculline at 1.5 or 1.75 mg/kg; at 2 mg/kg bicuculline acted synergistically with chlordiazepoxide. Picrotoxin (1 and 1.5 mg/kg) also acted synergistically with chlordiazepoxide, enhancing the latter's rate-increasing effects, but only during rewarded components. Neither muscimol (0.00125 and 0.25 mg/kg) nor baclofen (0.01 mg/kg) affected response rates, whether given alone or in combination. However, baclofen in a dose of 1 mg/kg, provided it was given to rats also injected with muscimol (0.00125 or 0.25 mg/kg) at other times, significantly reduced responding during nonrewarded components (an apparently anxiogenic effect). The results of the two sets of experiments are discussed in relation to the hypothesis that anxiolytic drugs affect behaviour by increasing GABAergic inhibition.

The effects of compounds related to gamma-aminobutyrate and benzodiazepine receptors on behavioural responses to anxiogenic stimuli in the rat: punished barpressing.

Rats were trained to press a bar for sucrose reward on a random-interval (RI) schedule and footshock punishment was then introduced for 3-min intrusion periods (signalled by a tone) on an independent RI schedule. Shock intensity was individually adjusted to produce stable intermediate levels of response suppression during the tone for each animal. Groups of animals were then allocated to a number of separate experiments in which they were systemically injected with anxiolytics (chlordiazepoxide HCl or sodium amylobarbitone), GABA antagonists (picrotoxin or bicuculline), the GABA (A) agonist muscimol, the GABA(B) agonist baclofen, an antagonist (RO 15-1788) at the benzodiazepine receptor and, an inverse agonist (FG 7142) at this receptor. The results showed that the alleviation of punishment-induced suppression of barpressing produced by chlordiazepoxide was blocked or partially blocked by RO 15-1788, picrotoxin and bicuculline but not by FG 7142; that picrotoxin (but not FG 7142) increased the suppression of responding by punishment; that neither muscimol nor baclofen affected responding on their own, but their combination weakly but reliably released punished responding from suppression; and that the anti-punishment effect of amylobarbitone was unaffected by either picrotoxin or bicuculline, though the barbiturate reversed the punishment-enhancing effect of picrotoxin. These results are discussed in the light of the hypothesis that anxiolytic behavioural effects are due to increased GABAergic inhibition.

The effects of compounds related to ?-aminobutyrate and benzodiazepine receptors on behavioural responses to anxiogenic stimuli in the rat: Choice behaviour in the T-maze

Two methods were used to test rats' responses to novelty in the T-maze: (1) a test of spontaneous alternation allowing separate measurement of place and body turn alternation; and (2) a test of entry into an arm of changed brightness ("response to stimulus change"). Chlordiazepoxide reduced spontaneous alternation by specifically weakening body turn alternation and eliminated the response to stimulus change. These findings are similar to those previously reported for the barbiturate sodium amylobarbitone. The same pattern of change in the two tests was seen after a low dose of the GABAA agonist muscimol (0.00125 mg/kg); when the dose of muscimol was raised (0.01 and 0.25 mg/kg), place alternation was also reduced. Picrotoxin but not bicuculline (both GABAA blockers) reversed the effects of muscimol and partially those of chlordiazepoxide on the response to stimulus change; in the spontaneous alternation test picrotoxin only marginally affected the response to 0.25 mg/kg muscimol and actually enhanced the effect of 0.000125 mg/kg. The GABAB agonist baclofen (1 mg/kg) acted in the test of response to stimulus change like chlordiazepoxide and muscimol; however, when baclofen was combined with muscimol, the two drugs tended to show mutual blocking. These results are generally consistent with the hypothesis that GABAergic mechanisms play a role in anxiolytic behavioural activity, but many details are difficult to explain.

Behavioral analogues of anxiety animal models

In the absence of fully characterized biological indexes, anxiety is at present measured as unpleasant effects reported verbally by patients. Because of the subjective nature of the syndrome, animal analogues have been difficult to design, but quests for new anxiolytics and a deeper understanding of the neurobiology of anxiety have fostered the development of several animal models. Usually, animals are exposed to exteroceptive or interoceptive stimuli which can be interpreted as capable of causing anxiety in humans. Then, the animals are observed for responses or behavioral deficits resulting from those stimuli in order to provide an index of anxiety. Behavioral responses that are reliably produced by those stimuli and that are also antagonized by anxiolytic drugs are accepted as analogues of anxiety. Exteroceptive stimuli, useful in this respect, consist of a variety of noxious treatments such as exposure to conflictsituations or unavoidable electric shock, whereas interoceptive stimuli consist of treatment with anxiogenic drugs or electrical stimulation of selected brain areas. Elicitation of unconditioned behavior or changes in the rate of conditioned (learned) responding have been employed as measures of anxiety responses following application of either exteroceptive or interoceptive stimuli. These measures, although useful in detecting anxiolytic drugs, possess several weaknesses. They suffer from difficulties in obtaining quantitative and objective data, they do not differentiate between anxiety and stress or fear, they are unable to measure further deterioration of behavior expected to occur when more potent anxiogenic stimuli are tested and they often present difficulty in differentiating direct motor effects of a number of stimuli not related to anxiety. More recently, interest in the development of other analogues of anxiety has led to the use of drug-discrimination paradigms. In this approach, interoceptive discriminative stimuli, produced by anxiogenic drugs, are used as analogues of anxiety in animals. As an example of this approach, data are reviewed showing that pentylenetetrazol, an anxiogenic drug in humans, produces interoceptive stimuli which can be readily discriminated by rats. Further, these stimuli can be easily quantified through dose-response analysis. All known anxiogenic drugs generalize to pentylenetetrazol-induced interoceptive discriminative stimuli. Similarly, other anxiety-provoking situations in humans, such as withdrawal from dependence on benzodiazepines, also generalize to the pentylenetetrazol-induced stimuli. Alternatively, all known anxiolytic drugs antagonize these stimuli with a relative potency similar to those reported clinically. The data obtained from the drug discrimination procedure are collected through automatic devices, are objective and are in a format easily amenable to quantitative statistical treatment. Moreover, the measurements are independent of response rates and are, therefore, not confounded by the motor effects of either drug or other anxiogenic treatments. The novel development in animal analogues of anxiety as indicated by the drug discrimination technique shows potential for facilitating research on the neurobiology of anxiety as well as on mechanisms underlying anxiety disorders. It also provides a novel approach for the discovery of new drugs to treat anxiety.

Attenuation of chemically induced anxiogenic stimuli as a novel method for evaluating anxiolytic drugs: A comparison of clobazam with other benzodiazepines

Male rats were trained to emit responses on one lever after they were injected with pentylenetetrazol and a different lever when injected with saline in order to obtain food reinforcement. It was shown that the pentylenetetrazol action, which served as a cue for appropriate response selection, was predictive of anxiogenic activity. This cue was blocked by anxiolytic drugs in a dose-dependent manner but not by anticonvulsant or depressant drugs. This procedure was shown to reliably predict elicitation of anxiogenicity or its blockade.