Dorsal raphe 5-HT(2C) receptor and GABA networks regulate anxiety produced by cocaine withdrawal

Abstract

The serotonin system is intimately linked to both the mediation of anxiety and long-term effects of cocaine, potentially through interaction of inhibitory 5-HT2C receptor and gamma-aminobutyric acid (GABA) networks. This study characterized the function of the dorsal raphe (DR) 5-HT2C receptor and GABA network in anxiety produced by chronic cocaine withdrawal. C57BL/6 mice were injected with saline or cocaine (15 mg/kg) 3 times daily for 10 days, and tested on the elevated plus maze 30 min, 25 h, or 7 days after the last injection. Cocaine-withdrawn mice showed heightened anxiety-like behavior at 25 h of withdrawal, as compared to saline controls. Anxiety-like behavior was not different when mice were tested 30 min or 7 days after the last cocaine injection. Electrophysiology data revealed that serotonin cells from cocaine-withdrawn mice exhibited increased GABA inhibitory postsynaptic currents (IPSCs) in specific DR subregions dependent on withdrawal time (25 h or 7 d), an effect that was absent in cells from non-withdrawn mice (30 min after the last cocaine injection). Increased IPSC activity was restored to baseline levels following bath application of the 5-HT2C receptor antagonist, SB 242084. In a separate cohort of cocaine-injected mice at 25 h of withdrawal, both global and intra-DR blockade of 5-HT2C receptors prior to elevated plus maze testing attenuated anxiety-like behavior. This study demonstrates that DR 5-HT2C receptor blockade prevents anxiety-like behavior produced by cocaine withdrawal, potentially through attenuation of heightened GABA activity, supporting a role for the 5-HT2C receptor in mediating anxiety produced by cocaine withdrawal.

Keywords: Addiction; Anxiety; Cocaine; Electrophysiology; GABA; Serotonin.

Figure 1

A-B. Anxiety-like behaviors as measured by the elevated plus maze and open field test. (A) Elevated plus maze testing was conducted 30 minutes, 25 hours, or 7 days after the last injection of saline or cocaine. Cocaine-treated mice after 25 hours of withdrawal spent significantly less time on the open arms of the elevated plus maze, as compared to saline controls (***p<0.001), demonstrating an anxiogenic phenotype. Mice that were subjected to elevated plus maze testing 30 minutes or 7 days after the last cocaine injection did not demonstrate anxiety-like behavior on the elevated plus maze as compared to saline controls (n=10-23 mice/group). (B) Open field testing was performed 25 hours after the last injection of saline or cocaine. Cocaine-treated mice after 25 hours of withdrawal spent significantly less time in the center of the open field apparatus (*p<0.05), demonstrating an anxiogenic phenotype (n=8 mice/group).

Figure 2

A-I. Spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in dorsal raphe serotonin neurons using whole cell patch-clamp electrophysiology techniques in vitro. (A) Serotonin neurons in the dmDR region from cocaine-treated mice demonstrated significantly heightened GABA_A sIPSC frequency after 25 hours of withdrawal (n= 6-17 cells/group, ***p<0.001), and no significant differences were observed at 30 minutes or 7 days of withdrawal (n=6-8 cells/group). (B) No significant differences in dmDR sIPSC amplitudes at 30 minutes, 25 hours or 7 days after the last injection were observed. (C) Serotonin cells from the vmDR aspect of the dorsal raphe exhibited heightened GABA_A sIPSC frequency at 25 hours of withdrawal (n=6-15, p<0.001), and no differences were observed in sIPSC frequency of vmDR cells at 30 minutes or 7 days after the last injection (n=6-8 cells/group). (D) No significant differences in sIPSC amplitude were observed in the vmDR subregion at 30 minutes, 25 hours or 7 days after the last saline or cocaine injection (n=6-17 cells/group). (E) Within the lateral wing subregion, serotonin neurons from cocaine withdrawn mice at 7 days of withdrawal exhibited significantly heightened sIPSC frequency as compared to cells from saline controls (n=6-13, p<0.01), while no differences were observed at 30 minutes or 25 hours after the last injection. (F) No significant differences were observed in sIPSC amplitudes of lwDR cells at 30 minutes, 25 hours or 7 days after the last injection. (G) Average amplitude traces for vmDR 25 hr cell data are shown. (H) Representative traces providing 1 minute bins of data are shown for vmDR 25 hr cell data. (I) Recorded cells were filled with biocytin during the whole cell electrophysiology recording and post-hoc immunohistochemical analysis was performed to confirm serotonin expression in recorded cells (TPH = tryptophan hydroxylase).

Figure 3

A-I. Miniature inhibitory postsynaptic currents (mIPSCs) using TTX (1 μM) were recorded in dorsal raphe serotonin neurons using whole cell patch-clamp in vitro electrophysiology techniques. (A and B) No significant differences were observed in mIPSC frequency (A) or amplitude (B) in dmDR cells at 30 minutes, 25 hours or 7 days after the last cocaine injection (n=5-12 cells/group). (C and D) Serotonin neurons from cocaine withdrawn mice demonstrated significantly heightened GABA_A mIPSC frequency (n= 5-10 cells/group, ***p<0.001) and amplitude (n=6-14 cells/group, p<0.05) in the ventromedial aspect of the dorsal raphe at 25 hours of withdrawal. (E and F) No differences were observed in mIPSC frequency or amplitude in lwDR cells from cocaine-treated mice at 30 minutes, 25 hours or 7 days following the last injection, as compared to cells from saline controls (n=7-12 cells/group). (G) Average amplitude traces are shown. (H) Representative traces providing 1 minute bins of data from vmDR cells are shown. (I) Recorded cells were filled with biocytin during the whole cell electrophysiology recording and post-hoc immunohistochemical analysis was performed to confirm serotonin expression in recorded cells (TPH = tryptophan hydroxylase).

Figure 4

Linear regression analyses were performed to establish a correlation between either sIPSC or mIPSC frequency and time spent on the open arms of the elevated plus maze. Averages of single cell data from each mouse (both saline- and cocaine-injected) were used in analyses. There was a significant negative correlation in A) sIPSC (n=37 mice, p<0.001) and B) mIPSC (n=31 mice, p<0.01) frequencies of vmDR serotonin cells and the time spent on the open arms of the elevated plus maze.

Figure 5

IPSC frequency of serotonin cells at 25 hours after repeated saline or cocaine: Effect of 5-HT_2C receptor blockade. (A) Serotonin cells from cocaine-withdrawn mice demonstrated significantly heightened sIPSC frequency as compared to saline controls (**p<0.01). Bath application of the 5-HT_2C receptor antagonist, SB 242084 (20 nM) significantly attenuated GABA_A sIPSC frequency in cells from anxious, cocaine-withdrawn mice (n=6 cells/group, #p<0.05). (B) An increase in mIPSC frequency was observed in cells from cocaine-withdrawn mice at 25 hours of withdrawal, as compared to saline controls, although no statistical significance was revealed. Bath application of SB 242084 had no significant effect on mIPSC frequency (n = 7-12 cells/group).

Figure 6

5-HT_2C receptor regulation of anxiety during cocaine withdrawal was evaluated using the elevated plus maze. (A) Mice exposed to a chronic binge cocaine paradigm and 24 hours of withdrawal with a saline injection (i.p.) 1 hour prior to elevated plus maze testing spent significantly less time in the open arms than the saline-administered controls (n=8-13 mice/group, ***p<0.001). Administration of 1 mg/kg SB 242084 (i.p.) 1 hour prior to elevated plus maze testing blocked the expression of anxiety-like behavior in cocaine-withdrawn mice (***p<0.001). (B) Mice exposed to a chronic binge cocaine paradigm and 24 hours of withdrawal with a saline microinjection into the dorsal raphe prior to elevated plus maze testing spent significantly less time on the open arms than the saline-administered controls (n=6-8 mice/group, ***p<0.001). Intra-dorsal raphe administration of 25nmol SB 242084 1 hour prior to elevated plus maze testing prevented the expression of anxiety-like behavior in cocaine-withdrawn mice (**p<0.01). (C) Microinjection locations along the rostrocaudal gradient of the dorsal raphe (images from Paxinos and Watson, 1996. (D) Representative coronal section (40 μ) of the dorsal raphe with blue dye injected for confirmation of accurate injection site.

Figure 7

Representative schematic illustrating the dorsal raphe 5-HT_2C receptor and GABA negative feedback mechanism. (A) Activation of 5-HT_2C receptors leads to stimulation of GABA release from GABA neurons. GABA then activates GABA_A receptors located on serotonin neurons, resulting in the influx of chloride ions and hyperpolarization of the cell, thus overall inhibition of serotonin release. (B) During cocaine withdrawal, this negative feedback mechanism is dysregulated and a heightened release of GABA through the 5-HT_2C receptor contributes to anxiety at this stage of addiction.