Milena de Barros Viana

Trial 2 in the elevated plus-maze: a different form of fear?

A factor analysis of the scores from rats given two trials in the elevated plus-maze showed that four independent factors emerged. Measures of anxiolytic activity on trial 1 (number of open arm entries and time spent on open arms) loaded on factor 1, measures of anxiolytic activity on trial 2 loaded on factor 2, the measure of general activity (number of closed arm entries) on both trials loaded on factor 3, and a measure of decision time (time spent in central square) for both trials loaded on factor 4. The independence of trials 1 and 2 anxiety measures raises the possibility that the state of anxiety/fear on the second trial in the plus-maze is qualitatively different from that on trial 1. This difference is reflected in the loss of anxiolytic action of diazepam (2 mg/kg) on trial 2. However, this occurs only when the trials are short (5 min); when they are longer (10 min) diazepam retains anxiolytic efficacy. It is concluded that during a brief (5 min) trial in the plus-maze rats acquire a specific phobic anxiety, which is relatively resistant to benzodiazepines. With a longer exposure to the plus-maze this form of fear extinguishes.

The elevated T-maze: A new animal model of anxiety and memory

In an attempt to analyze different types of anxiety, and at the same time assess memory, a new experimental model was developed. The apparatus, named the elevated T-maze, consisted of three arms of equal dimensions (50 x 10 cm) elevated 50 cm from the ground. One arm, enclosed by 40-cm high walls, was perpendicular to two open arms. The first experimental session was conducted 25 min after IP injection of either drug or saline. To assess inhibitory (passive) avoidance, the rat was placed at the end of the enclosed arm and the time taken to withdraw from this arm was recorded three times in succession. Soon afterwards, the rat was placed at the end of one of the open arms and the time taken to withdraw from this arm was measured, thus estimating one-way escape. To assess memory, inhibitory avoidance and escape were measured again 3 days later, without drug. Dose-response curves were determined for the benzodiazepine anxiolytic and amnestic agent diazepam (DZP, 0.5-4 mg/kg), as well as for ipsapirone (IPS, 0.25-2 mg/kg), an azapirone anxiolytic that is devoid of clinically significant amnestic effects. The doses of 1, 2, and 4 mg/kg DZP and of 1 and 2 mg/kg IPS impaired inhibitory avoidance, an effect that may be viewed as anxiolytic. Inhibitory avoidance remained impaired 3 days later in the rats treated with 1-4 mg/kg DZP, indicating anterograde amnesia. This effect was not due to state-dependent learning, because rats injected both at pretraining and pretesting with 2 mg/kg DZP still showed complete amnesia.(ABSTRACT TRUNCATED AT 250 WORDS)

Dual role of 5-HT in defense and anxiety

Pharmacological results obtained in animals tested in approach/avoidance conflict situations have led to the suggestion that 5-HT enhances anxiety by acting on forebrain structures. In contrast, results with intracerebral drug injection associated with aversive electrical brain stimulation indicate that 5-HT inhibits aversion in the dorsal periaqueductal gray (DPAG). To reconcile this evidence, it has been suggested that 5-HT may enhance conditioned fear in the amygdala while inhibiting innate fear in the DPAG. To test this hypothesis, we used three drug treatments known to increase the release of 5-HT from terminals of the dorsal raphe nucleus (DR): (1) intra-DR microinjection of the benzodiazepine inverse agonist FG 7142, (2) intra-DR microinjection of the excitatory amino acid kainic acid and (3) intraperitoneal injection of the 5-HT releaser and uptake blocker D-fenfluramine. All drug treatments enhanced inhibitory avoidance (learned fear) in the elevated T-maze, a new animal model of anxiety. Intra-raphe kainate and D-fenfluramine also decreased one-way escape (innate fear) in the T-maze. In contrast, reduction of 5-HT release by intra-DR injection of 8-OH-DPAT impaired inhibitory avoidance without affecting one-way escape. Overall, these results agree with the above hypothesis. Clinical implications are discussed, especially with regard to panic disorder.

Antipanic-like effect of serotonin reuptake inhibitors in the elevated T-maze

There has been an increasing interest in the development of animal models that address different types of anxiety disorders. The elevated T-maze (ETM) was developed based on the assumption that inhibitory avoidance and one-way escape are respectively related to generalized anxiety (GAD) and panic disorder (PD). Although anxiolytic effects on the inhibitory avoidance task have been shown for drugs used in clinical settings to treat GAD, positive effects of antipanic drugs in one-way escape have been described only for imipramine. The study extends the pharmacological validation of the ETM to other serotonin reuptake inhibitors that have antipanic properties. To this end, we investigated the effects of acute and chronic (21 days) IP administration of clomipramine (3, 10 and 30 mg/kg) and fluoxetine (5, 10 and 15 mg/kg). Buspirone (0.3, 1 and 3 mg/kg) was used as a negative control since the drug is effective in GAD but not PD. Chronically, both clomipramine and fluoxetine impaired one-escape, an antipanic-like effect, not altering avoidance latencies. Clomipramine, but not fluoxetine, also affected locomotion. Acute injection of buspirone impaired avoidance, an anxiolytic-like effect. Neither acute nor chronic buspirone altered one-escape. These results corroborate the suggestion that one-way escape is related to PD.

The dorsal raphe nucleus exerts opposed control on generalized anxiety and panic-related defensive responses in rats

It has been proposed that the ascending dorsal raphe (DR)-serotonergic (5-HT) pathway facilitates conditioned avoidance responses to potential or distal threat, while the DR-periventricular 5-HT pathway inhibits unconditioned flight reactions to proximal danger. Dysfunction on these pathways would be, respectively, related to generalized anxiety (GAD) and panic disorder (PD). To investigate this hypothesis, we microinjected into the rat DR the benzodiazepine inverse receptor agonist FG 7142, the 5-HT(1A) receptor agonist 8-OH-DPAT or the GABA(A) receptor agonist muscimol. Animals were evaluated in the elevated T-maze (ETM) and light/dark transition test. These models generate defensive responses that have been related to GAD and PD. Experiments were also conducted in the ETM 14 days after the selective lesion of DR serotonergic neurons by 5,7-dihydroxytriptamine (DHT). In all cases, rats were pre-exposed to one of the open arms of the ETM 1 day before testing. The results showed that FG 7142 facilitated inhibitory avoidance, an anxiogenic effect, while impairing one-way escape, an anxiolytic effect. 8-OH-DPAT, muscimol, and 5,7-DHT-induced lesions acted in the opposite direction, impairing inhibitory avoidance while facilitating one-way escape from the open arm. In the light/dark transition, 8-OH-DPAT and muscimol increased the time spent in the lighted compartment, an anxiolytic effect. The data supports the view that distinct DR-5-HT pathways regulate neural mechanisms underlying GAD and PD.

Serotonergic regulation of inhibitory avoidance and one-way escape in the rat elevated T-maze.

It has been proposed that distinct 5-HT pathways modulate different types of anxiety. Activation of the ascending dorsal raphe (DR)-5-HT pathway, innervating the amygdala and frontal cortex, would facilitate learned defensive behaviors. On the other hand, activation of the DR-periventricular 5-HT pathway, which innervates the dorsal periaqueductal gray matter (DPAG), would inhibit innate flight or fight reactions. Dysfunction of these pathways has been suggested to relate to generalized anxiety disorder (GAD) and panic disorder (PD) in humans, respectively. The elevated T-maze has been developed to separate conditioned (inhibitory avoidance) from unconditioned (escape) defensive responses in the same rat. Pharmacological validation of this model has shown that the GAD-effective serotonergic anxiolytic buspirone or the putative anxiolytic ritanserin selectively impaired inhibitory avoidance while leaving one-way escape unchanged. Chronic injection of the 5-HT/noradrenaline reuptake inhibitor imipramine impaired inhibitory avoidance and prolonged escape, an effect that may be related to the therapeutic action of this drug on both GAD and PD. Like imipramine, intra-DPAG injection of the 5-HT(1A) agonist 8-OH-DPAT impaired both inhibitory avoidance and one-way escape. Intra-DPAG administration of the 5-HT(2A/2C) agonist DOI prolonged escape, without affecting inhibitory avoidance. The reversible inactivation of the DRN by muscimol impaired inhibitory avoidance, while facilitating escape from the open arm. Taken together, these results suggest that 5-HT exerts differential control on inhibitory avoidance and escape response in the elevated T-maze, mobilizing different types of 5-HT receptors in key structures implicated in fear/anxiety.

Opposed regulation by dorsal raphe nucleus 5-HT pathways of two types of fear in the elevated T-maze

To investigate the influence of dorsal raphe nucleus (DRN) 5-HT pathways on different types of fear, we microinjected into the rat DRN the benzodiazepine inverse agonist FG 7142 and the excitatory amino acid kainic a acid. In addition, we systemically administered the 5-HT releasing drug D-fenfluramine. The behavioral effects of these drugs were measured in an elevated T-maze, consisting of three arms of equal dimensions (50 x 10 cm), elevated 50 cm from the floor. One arm is enclosed by walls (40 cm) and stands perpendicular to the two open arms. Inhibitory (passive) avoidance--representing learned fear--was measured by placing a rat at the end of the enclosed arm and recording the time to withdraw from this arm with the four paws during three consecutive trials. Soon afterwards, the same animal was placed at the end of one of the open arms and the time to withdraw from this arm with the four paws was recorded. This one-way escape response represents unconditioned fear. Intra-DRN FG 7142 (40 pmol) facilitated inhibitory avoidance (anxiogenic effect), but did not affect one-way escape. Kainic acid (60 pmol) also facilitated inhibitory avoidance and, in addition, impaired one-way escape (anxiolytic effect). These effects are unlikely to be due to motor deficit, because intra-DRN kainate did not change locomotor activity and rearing behavior of rats placed inside a circular arena for 10 min. Finally, D-fenfluramine (0.03, 0.1, and 0.3 mg/kg, IP) tended to enhance inhibitory avoidance while depressing one-way escape in a dose-dependent way. Because the three drug treatments are believed to increase 5-HT release from DRN nerve terminals, these results support the hypothesis that ascending DRN 5-HT pathways facilitate learned fear while inhibiting unconditioned fear. The former may be related to generalized anxiety and the latter to panic disorder.

Anxiolytic effect of intra-amygdala injection of midazolam and 8-hydroxy-2-(di-n-propylamino)tetralin in the elevated T-maze.

The effects of intra-amygdala injection of midazolam (20 nmol) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 8 and 16 nmol) were investigated in rats submitted to the elevated T-maze, a new animal model of anxiety. This test allows the measurement, in the same rat, of conditioned and unconditioned fear/anxiety responses. Both drugs impaired inhibitory avoidance of the open arms of the T-maze (task representing conditioned fear), indicating an anxiolytic effect, but did not change escape performance from one of the open arms (representing unconditioned fear). The results further implicate gamma-aminobutyric acid (GABA)/benzodiazepine and serotonergic systems within the basolateral/lateral amygdala in the modulation of conditioned anxiety responses.

Kainate microinjection into the dorsal raphe nucleus induces 5-HT release in the amygdala and periaqueductal gray.

Earlier results obtained in one of our laboratories showed that microinjection into the dorsal raphe nucleus (DRN) of the excitatory amino acid kainic acid, the benzodiazepine (BZD) inverse agonist FG 7142, and the 5-HT1A receptor agonist 8-OHDPAT changed the behavior of rats in the elevated T-maze, an animal model of anxiety. The present study investigates biochemical correlates of these results in awake rats by measuring 5-HT release with in vivo microdialysis in two brain structures innervated by the DRN-the amygdala (Am) and the dorsal periaqueductal gray matter (DPAG)-that have been implicated in anxiety. Microinjection of kainic acid (60 pmol) into the DRN significantly increased 5-HT release in both the Am and the DPAG. In the DPAG, the increase was 14-fold higher with respect to the baseline and occurred only at the first sample, which was collected 30 min after the injection. In the Am, the increase was less pronounced (nearly fourfold) but persistent, lasting until the fourth sample, which was collected 120 min from the injection. FG 7142 (40 pmol) and 8-OH-DPAT (8 nmol) were ineffective. Because only intra-DRN kainate both increased inhibitory avoidance and decreased one-way escape in the elevated T-maze, the present behavioral results support the suggestion that 5-HT facilitates conditioned fear in the Am and inhibits unconditioned fear in the DPAG.

Localization in the amygdala of the amnestic action of diazepam on emotional memory

It is well known that systemically administered benzodiazepines (BZDs) induce anterograde amnesia in a variety of learning tasks. BZs effects are mediated through the GABAA complex by enhancing GABA-induced synaptic inhibition. As the GABAergic system in the amygdaloid complex (AC) is a site of action for the anxiolytic effects of BZs, such findings suggest that BZs may also influence memory through the amygdala. The present report summarizes a recent series of experiments designed to examine this implication. In a first experiment rats received either sham or bilateral AC lesion using N-methyl-D-aspartic acid (NMDA). One week later, animals were trained on an inhibitory avoidance task and tested 48 h later. Diazepam (DZP; 1.0 and 2.0 mg/kg, i.p.) or vehicle was injected 30 min prior to acquisition. The results demonstrate that DZP-induced retention deficits was blocked in rats with AC lesions. In a second experiment, in an attempt to localize the site of BZDs amnestic action in the AC, we tested the effects of DZP in rats with bilateral ibotenic acid-induced lesions of central (CE), lateral (LAT) or basolateral (BL) amygdala nuclei. The results shown that retention was impaired in animals with CE and LAT lesions but not in animals with BL lesions. In a third experiment we tested the effects of DZP microinjections in different nuclei of the AC on retention performance of rats trained in an avoidance task. The results demonstrate that DZP microinjection prior training in the BL/LAT, but not CE nuclei produce anterograde amnesia.(ABSTRACT TRUNCATED AT 250 WORDS)