5-HT_1A and benzodiazepine receptors in the basolateral amygdala modulate anxiety in the social interaction test, but not in the elevated plus-maze

Abstract

In order to investigate the role of the 5-HT_1A receptors of the amygdala in modulating anxiety, rats were implanted with bilateral cannulae aimed at the basolateral nucleus of the amygdala complex and infused with either artificial cerebrospinal fluid (aCSF) or the selective 5-HT_1A receptor agonist 8-OH-DPAT (50–200 ng) and tested in two animal models of anxiety. In the elevated plus-maze test, no significant effects were detected in this dose range. In contrast, 8-OH-DPAT caused an overall reduction in levels of social investigation, thus indicating anxiogenic actions in the social interaction test. At 50 ng, 8-OH-DPAT had a selective action on anxiety, while at 200 ng there was a concomitant reduction in locomotor activity and, in some animals, signs of the 5-HT_1A syndrome. Evidence that the anxiogenic effect of 8-OH-DPAT (50 ng) was due to activation of 5-HT_1A receptors came from the finding that (−)-tertatolol, a 5-HT_1A receptor antagonist, reversed this effect at a dose (1.5 μg) which was silent when given alone. The benzodiazepine receptor agonist, midazolam (1 and 2 μg) was bilaterally administered into the basolateral nucleus of the amygdala and evoked clear-cut anxiolytic effects in the social interaction test. These data indicate that the agonist activation of post-synaptic 5-HT_1A receptors in the basolateral nucleus of the amygdala may produce anxiogenic effects, while agonist activation of BDZ receptors in the same areas evokes anxiolytic effects. Our results from the social interaction test are similar to those previously reported from tests of anxiety using punished paradigms, but contrast with those found in the elevated plus-maze. Thus, it is concluded that either the two tests have different sensitivities to midazolam and 8-OH-DPAT or more intriguingly, the tests are evoking fundamentally different states of anxiety, with that evoked by the plus-maze being mediated via brain areas or receptors different from those studied here.

Introduction

The amygdala has been critically implicated in the modulation of fear and anxiety [8]. For example, electrolytic or N-methyl-D-aspartate lesions of central or lateral nuclei of the amygdala have been reported to block or reduce anxiety expression 22, 27, while electrical stimulation of neurons within the amygdaloid complex elicit clear-cut signs of fear in many animals including humans 26, 7. Furthermore, electrophysiological studies have shown that both unconditioned and conditioned aversive stimuli can evoke neural activity in the amygdala 3, 15, 32 and mRNA levels of c-fos proto-oncogene were increased in the amygdala during conditioned and unconditioned fear situations [5].

Serotonin (5-hydroxytryptamine; 5-HT) release has been shown to be increased in the amygdaloid complex in the anxiogenic situation of diazepam withdrawal [9], and increases in extracellular 5-HT have been detected in the basolateral nucleus of the amygdala as measured by microdialysis techniques during stressful situations [20]. Administration of 5-HT directly into the amygdala has been shown to evoke anxiogenic effects as measured in the social interaction test [16] and in a punished paradigm [17]. The amygdala is a main projection site from the dorsal raphé nucleus 4, 21, and it is now widely held that neuronal inhibition by different manipulations of the serotonergic neurones from the dorsal raphé nucleus leads to consistent anxiolytic effects across several animal models (for review see [14]). All these findings indicate that post-synaptic serotonergic receptors in the amygdala may modulate anxiety.

Although there is a relatively high density of 5-HT_1A receptors within the amygdaloid complex 6, 33, the role of these receptors in modulating anxiety has been rather scarcely investigated. Hodges et al. [17] reported that infusions of the 5-HT_1A receptor agonist 8-OH-DPAT (125 or 500 ng/0.5 μl) into the lateral and basolateral nuclei of the amygdala decreased punished responding, thus indicating anxiogenic actions. Conversely, Takao et al. [30], examined the effects of two 5-HT_1A receptor agonists, buspirone and d-AP159 (N-cyclohexyl-1,2,3,4-tetrahydrobenzo(b)thieno-(2,3c)pyridine)-3-carboamide HCl) when injected into the central nucleus of the amygdala; d-AP159 but not buspirone (a partial agonist) had anticonflict activity. This suggests that the activation of 5-HT_1A receptors in basolateral and central nuclei may have opposite consequences for anxiety. However, there is not enough evidence to support the specificity of such effects. Thus, the actions of 5-HT_1A ligands have not been replicated in those or other animal tests of anxiety and it has not yet been shown whether the effects of direct infusions of 5-HT_1A receptor agonists are blocked by 5-HT_1A receptor antagonists.

In the following experiments we investigated the effects of 8-OH-DPAT when injected into the basolateral nucleus of the amygdala in the social interaction and the elevated plus-maze, two ethologically derived tests of anxiety [11]. The dose range of 8-OH-DPAT was selected on the basis of those doses found to be active in previous studies [1]; the highest dose (200 ng) was determined by its solubility in aCSF. The low light test condition of the social interaction test was used since this is the most sensitive to anxiogenic effects [10].

We used (−)-tertatolol as a selective 5-HT_1A receptor antagonist [18], since it is soluble in artificial cerebrospinal fluid (aCSF) at pH 7.4 and, at the time of study, WAY 100635 was not available. The dose of (−)-tertatolol (1.5 μg) was selected based on the findings of a previous study [2].

Drugs acting at the GABA-benzodiazepine receptor complex have consistent anxiolytic effects in conflict paradigms when administered into the amygdaloid complex 17, 28, 31, and such effects have also been found in the social interaction test [16]. However, there is no clear agreement regarding the relative importance of the central and basolateral nuclei of the amygdala. For example, while Shibata et al. [29], using the Geller-Seifter conflict paradigm, found anticonflict effects of benzodiazepines when injected into the central but not the basolateral nucleus, Hodges et al. [17] found significant anticonflict effects of benzodiazepines injected into the basolateral areas using the same paradigm. The lack of specific histological procedures to identify nuclei boundaries may account for these discrepancies. We therefore examined whether bilateral administration of midazolam to the basolateral nucleus of the amygdala was able to increase social investigation, indicating an anxiolytic effect, and have examined the injection sites using an acetylcholinesterase staining procedure which delineates the basolateral nucleus from the central nucleus of the amygdala. Although Higgins et al. [16] used high light, unfamiliar test conditions, the high light familiar condition was selected for this study to determine whether high light was a sufficient aversive factor in making the test sensitive to benzodiazepines.