Chronic amphetamine treatment enhances corticotropin-releasing factor-induced serotonin release in the amygdala

Abstract

Amphetamine use is associated with dysphoric states, including heightened anxiety, that emerge within 24h of withdrawal from the drug. Corticotropin-releasing factor increases serotonin release in the central nucleus of the amygdala, and this neurochemical circuitry may play a role in mediating fear and anxiety states. We have previously shown that chronic amphetamine treatment increases corticotropin-releasing factor receptor type-2 levels in the serotonergic dorsal raphe nucleus of the rat. Therefore, we hypothesized that chronic amphetamine treatment would enhance the amygdalar serotonergic response to corticotropin-releasing factor infused into the dorsal raphe nucleus. Male rats were injected once-daily with d-amphetamine (2.5mg/kg i.p., or saline) for two weeks. Serotonin release within the central nucleus of the amygdala in response to intra-raphe infusion of corticotropin-releasing factor (100 ng) was measured 24h after the last treatment in urethane-anesthetized (1.8 mg/kg, i.p.) rats using in vivo microdialysis. Rats pretreated with amphetamine showed significantly enhanced serotonin release in the central nucleus of the amygdala in response to corticotropin-releasing factor infusion when compared to saline pretreated rats. Furthermore, this enhanced response was blocked by the corticotropin-releasing factor type-2 receptor antagonist antisauvagine-30 (2 microg) infused into the dorsal raphe nucleus. These results suggest increased sensitivity to corticotropin-releasing factor as mediated by type-2 receptors following chronic amphetamine treatment, which may underlie dysphoric states observed during amphetamine withdrawal.

Fig. 1. Representative coronal diagrams for microdialysis probe and drug infusion cannula placements

Microdialysis probe membrane placement (black bars) in the central nucleus of the amygdala (CeA) and location of drug infusion cannula tips in the dorsal raphe nucleus (dRN; black circles) and cannula tip placements outside the dRN (black stars) of (A) untreated rats, (B) saline pretreated rats and (C) amphetamine pretreated rats. (D) Representative stained sections of CeA microdialysis probe placement and dRN cannula tip placement. Figs were adapted from Paxinos and Watson (1998).

Fig. 2. Effects of corticotropin-releasing factor in the dorsal raphe nucleus on serotonin release in the central nucleus of the amygdala in rats with no previous amphetamine/saline exposure

Corticotropin-releasing factor (CRF; 100 ng/0.5 µl) infused into the dorsal raphe nucleus resulted in an immediate and transient increase in serotonin release in the central nucleus of the amygdala (CeA). Vehicle (aCSF, 0.5µl) infused into the dRN had no effect on serotonin in the CeA. Data represent mean ± S.E.M. # = significantly different from pre-infusion levels, * = significant differences between treatment groups, p < 0.05. Arrow represents time of dRN infusion.

Fig. 3. Effects of corticotropin-releasing factor in the dorsal raphe nucleus on serotonin release in the central nucleus of the amygdala in amphetamine and saline pretreated rats

(A) Corticotropin-releasing factor (CRF; 100 ng/0.5µl) infused into the dorsal raphe nucleus of rats previously treated with amphetamine (2.5 mg/kg, i.p.) caused an immediate and transient increase in serotonin release in the central nucleus of the amygdala (CeA), but had no effect on rats previously treated with saline. (B) Vehicle (aCSF, 0.5 µl) infused into the dRN had no effect on serotonin release in the CeA in either pretreatment group. Data represent mean ± S.E.M. # = significantly different from pre-infusion levels, * = significant differences between treatment groups, p < 0.05. Arrows indicate time of infusion, 1 = vehicle for ASV-30 and 2 = CRF (A) or aCSF (B).

Fig. 4. Effects of corticotropin-releasing factor outside the dorsal raphe nucleus on serotonin release in the central nucleus of the amygdala in amphetamine and saline pretreated rats

Corticotropin-releasing factor (CRF 100 ng/0.5µl) infused outside but adjacent to the dorsal raphe nucleus of rats previously treated with amphetamine (2.5 mg/kg, i.p.) or saline had no effect on the release of serotonin in central nucleus of the amygdala (CeA). Data represent mean ± S.E.M. # = significantly different from pre-infusion levels. Arrows indicate time of infusion, 1 = vehicle for ASV-30 and 2 = CRF.

Fig. 5. Effects of corticotropin-releasing factor receptor 2 antagonism in the dorsal raphe nucleus on corticotropin-releasing factor elicited serotonin release in the central nucleus of the amygdala in amphetamine pretreated rats

Pre-infusion of the dorsal raphe nucleus (dRN) with the corticotropin-releasing factor (CRF) receptor 2 antagonist antisauvagine-30 (ASV-30, 2 µg/0.5 µl) blocked the stimulatory effect of intra-dRN CRF (100 ng/0.5 µl) on central nucleus of the amygdala (CeA) serotonin levels in rats previously exposed to amphetamine (2.5 mg/kg, i.p.). Data represent mean ± S.E.M. Arrows indicate time of infusion, 1 = ASV-30 and 2 = CRF or aCSF (vehicle). Dashed line represents the positive control for this study (vehicle followed by CRF infusion into the dRN of amphetamine pretreated rats) previously shown in Fig. 3A and plotted here to illustrate the impact of ASV-30 on this CRF-induced response.