Role of GABAA receptors in dorsal raphe nucleus in stress-induced reinstatement of morphine-conditioned place preference in rats.

RATIONALE: The serotonin (5-hydroxytryptamine, 5-HT) system plays an important role in stress-related psychiatric disorders and substance abuse. Our data indicate that stress inhibits the dorsal raphe nucleus (DRN)-5-HT system via stimulation of GABA synaptic activity by the stress neurohormone corticotropin-releasing factor and, more recently, that morphine history sensitizes DRN-5-HT neurons to GABAergic inhibitory effects of stress. OBJECTIVES: We tested the hypothesis that DRN GABAA receptors contribute to stress-induced reinstatement of morphine-conditioned place preference (CPP). METHODS: First, we tested if activation of GABAA receptors in the DRN would reinstate morphine CPP. Second, we tested if blockade of GABAA receptors in the DRN would attenuate swim stress-induced reinstatement of morphine CPP. CPP was induced by morphine (5 mg/kg) in a 4-day conditioning phase followed by a conditioning test. Upon acquiring conditioning criteria, subjects underwent 4 days of extinction training followed by an extinction test. Upon acquiring extinction criteria, animals underwent a reinstatement test. For the first experiment, the GABAA receptor agonist muscimol (50 ng) or vehicle was injected into the DRN prior to the reinstatement test. For the second experiment, the GABAA receptor antagonist bicuculline (75 ng) or vehicle was injected into the DRN prior to a forced swim stress, and then, animals were tested for reinstatement of CPP. RESULTS: Intraraphe injection of muscimol reinstated morphine CPP, while intraraphe injection of bicuculline attenuated swim stress-induced reinstatement. CONCLUSIONS: These data provide evidence that GABAA receptor-mediated inhibition of the serotonergic DRN contributes to stress-induced reinstatement of morphine CPP.

Urocortin 2 increases c-Fos expression in serotonergic neurons projecting to the ventricular/periventricular system.

Serotonin plays an important role in the regulation of anxiety states and physiological responses to aversive stimuli. Intracerebroventricular (i.c.v.) injection of the stress- and anxiety-related neuropeptide urocortin 2 (Ucn 2) increases c-Fos expression in serotonergic neurons in the dorsal (DRD) and caudal (DRC) parts of the dorsal raphe nucleus. These regions contain a subset of serotonergic neurons that projects via the dorsal raphe periventricular tract to periventricular structures, including the subfornical organ and ependymal layer, and to the ventricular system. To determine if Ucn 2 activates ventricle/periventricular-projecting serotonergic neurons in the midbrain raphe complex, we made i.c.v. injections of the retrograde tracer Fluoro-Gold into the lateral ventricle, followed 7 days later by i.c.v. injection of Ucn 2. The DRD at -8.18 mm and the DRC at -8.54 mm and -9.16 mm bregma were analyzed using a combined bright field and immunofluorescence technique. Approximately 40% of the ventricle/periventricular-projecting neurons in the subdivisions sampled were serotonergic. Urocortin 2 increased c-Fos expression in ventricle/periventricular-projecting serotonergic neurons in the DRC and in non-ventricle/periventricular-projecting serotonergic neurons in the DRD and DRC. Of the total population of ventricle/periventricular-projecting serotonergic neurons in the DRC at -8.54 and -9.16 mm bregma, 35% expressed c-Fos following Ucn 2 injections. These data are consistent with previous studies showing that i.c.v. injection of Ucn 2 activates subpopulations of serotonergic neurons restricted to the mid-rostrocaudal DRD and DRC and further demonstrate that these include both subsets of serotonergic neurons that do and do not project to the ventricle/periventricular system.

Serotonergic systems, anxiety, and affective disorder: focus on the dorsomedial part of the dorsal raphe nucleus.

Depressed suicide patients have elevated expression of neuronal tryptophan hydroxylase 2 (TPH2) mRNA and protein in midbrain serotonergic neurons, as well as increases in brain serotonin turnover. The mechanisms underlying these changes are uncertain, but increased TPH2 expression and serotonin turnover could result from genetic influences, adverse early life experiences, or acute stressful life events, all of which can alter serotonergic neurotransmission and have been implicated in determining vulnerability to major depression. Emerging evidence suggests that there are several different stress-related subsets of serotonergic neurons, each with a unique role in the integrated stress response. Here we review our current understanding of how genetic and environmental factors may influence TPH2 mRNA expression and serotonergic neurotransmission, focusing in particular on the dorsomedial part of the dorsal raphe nucleus. This subdivision of the dorsal raphe nucleus is selectively innervated by key forebrain structures implicated in regulation of anxiety states, it gives rise to projections to a distributed neural system mediating anxiety states, and serotonergic neurons within this subdivision are selectively activated by a number of stress- and anxiety-related stimuli. A better understanding of the anatomical and functional properties of specific stress- or anxiety-related serotonergic systems should aid our understanding of the neural mechanisms underlying the etiology of anxiety and affective disorders.

Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors: relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus.

Serotonergic systems arising from the mid-rostrocaudal and caudal dorsal raphe nucleus (DR) have been implicated in the facilitation of anxiety-related behavioral responses to anxiogenic drugs or aversive stimuli. In this study we attempted to determine a threshold to engage serotonergic neurons in the DR following exposure to aversive conditions in an anxiety-related behavioral test. We manipulated the intensity of anxiogenic stimuli in studies of male Wistar rats by leaving them undisturbed (CO), briefly handling them (HA), or exposing them to an open-field arena for 15-min under low-light (LL: 8-13 lx) or high-light (HL: 400-500 lx) conditions. Rats exposed to HL conditions responded with reduced locomotor activity, reduced time spent exploring the center of the arena, a lower frequency of rearing and grooming, and an increased frequency of facing the corner of the arena compared to LL rats. Rats exposed to HL conditions had small but significant increases in c-Fos expression within serotonergic neurons in subdivisions of the rostral DR. Exposure to HL conditions did not alter c-Fos responses in serotonergic neurons in any other DR subdivision. In contrast, rats exposed to the open-field arena had increased c-Fos expression in non-serotonergic cells throughout the DR compared to CO rats, and this effect was particularly apparent in the dorsolateral part of the DR. We conclude that exposure to HL conditions, compared to LL conditions, increased anxiety-related behavioral responses in an open-field arena but this stimulus was at or below the threshold required to increase c-Fos expression in serotonergic neurons.

Evidence supporting a role for corticotropin-releasing factor type 2 (CRF2) receptors in the regulation of subpopulations of serotonergic neurons.

Corticotropin-releasing factor (CRF)-related peptides can modulate stress-related physiology and behavior. Some of these effects may be mediated via the CRF type 2 (CRF2) receptor on serotonergic neurons in the dorsal raphe nucleus (DR). To determine if the CRF2 receptor agonist urocortin 2 (Ucn 2) increases c-Fos expression in rat DR serotonergic neurons via actions on CRF2 receptors, we gave intracerebroventricular (icv) injections of mouse Ucn 2 after icv injections of either saline or the CRF2 receptor antagonist antisauvagine-30 (ASV-30). Double immunostaining methods for c-Fos and tryptophan hydroxylase revealed that, consistent with previous studies, mouse Ucn 2 increased c-Fos expression in tryptophan hydroxylase immunostained neurons in the middle and caudal parts (-8.18, -8.54, and -9.16 mm bregma) of the dorsal subdivision of the dorsal raphe nucleus 2 h after drug treatment. Pre-treatment with ASV-30 blocked these effects. Mouse Ucn 2 had no effect on c-Fos expression within the median raphe nucleus, consistent with the hypothesis that Ucn 2 has specific actions on an anatomically and functionally distinct subset of serotonergic neurons via activation of CRF2 receptors. These findings are also consistent with the hypothesis that Ucn 2, or another CRF-related neuropeptide acting at CRF2 receptors, modulates physiological and behavioral responses to stress-related stimuli via actions on a specific subset of serotonergic neurons within the dorsal raphe nucleus.

Urocortin 2 increases c-Fos expression in topographically organized subpopulations of serotonergic neurons in the rat dorsal raphe nucleus.

Corticotropin-releasing factor (CRF)-related peptides modulate stress-related physiology and behavior. Some of the physiological and behavioral effects of CRF-related peptides may be due to actions on CRF type 2 (CRF2) receptors modulating serotonergic systems in the dorsal raphe nucleus (DR). To determine if CRF2 receptor activation has effects on serotonergic neurons in the DR in conscious behaving rats, we gave intracerebroventricular (icv) injections of the selective CRF2 receptor agonist urocortin 2 (0, 0.01, 0.1, or 1.0 mug in 2 microl saline) to adult male Wistar rats and quantified c-Fos expression in topographically organized subpopulations of serotonergic neurons within the DR. In addition, home cage behaviors were recorded for 30 min prior to drug treatment and for 2 h following drug treatment. Two hours following drug treatment, rats were anesthetized, transcardially perfused with fixative, and brain tissues were processed for immunohistochemistry. Urocortin 2, in the absence of any effects on most behavioral endpoints studied, consistently increased c-Fos expression in subpopulations of serotonergic neurons identified by either tryptophan hydroxylase or serotonin immunostaining within specific subdivisions of the DR, particularly the dorsal region of the mid-rostrocaudal and caudal DR (-7.64, -8.18, -8.54, and -9.16 mm bregma). These studies demonstrate that urocortin 2 has selective actions on a subset of DR serotonergic neurons. Urocortin 2 actions on serotonergic systems described here may contribute to delayed behavioral effects of urocortin 2 described previously, including orexigenic, locomotor, and anxiety-related effects in a variety of behavioral tests as well as potentiation of conditioned fear and induction of escape deficits in a model of learned helplessness.