Functional status of the serotonin 5-HT2C receptor (5-HT2CR) drives interlocked phenotypes that precipitate relapse-like behaviors in cocaine dependence

Abstract

Relapse vulnerability in cocaine dependence is rooted in genetic and environmental determinants, and propelled by both impulsivity and the responsivity to cocaine-linked cues ('cue reactivity'). The serotonin (5-hydroxytryptamine, 5-HT) 5-HT2C receptor (5-HT2CR) within the medial prefrontal cortex (mPFC) is uniquely poised to serve as a strategic nexus to mechanistically control these behaviors. The 5-HT2CR functional capacity is regulated by a number of factors including availability of active membrane receptor pools, the composition of the 5-HT2CR macromolecular protein complex, and editing of the 5-HT2CR pre-mRNA. The one-choice serial reaction time (1-CSRT) task was used to identify impulsive action phenotypes in an outbred rat population before cocaine self-administration and assessment of cue reactivity in the form of lever presses reinforced by the cocaine-associated discrete cue complex during forced abstinence. The 1-CSRT task reliably and reproducibly identified high impulsive (HI) and low impulsive (LI) action phenotypes; HI action predicted high cue reactivity. Lower cortical 5-HT2CR membrane protein levels concomitant with higher levels of 5-HT2CR:postsynaptic density 95 complex distinguished HI rats from LI rats. The frequency of edited 5-HT2CR mRNA variants was elevated with the prediction that the protein population in HI rats favors those isoforms linked to reduced signaling capacity. Genetic loss of the mPFC 5-HT2CR induced aggregate impulsive action/cue reactivity, suggesting that depressed cortical 5-HT2CR tone confers vulnerability to these interlocked behaviors. Thus, impulsive action and cue reactivity appear to neuromechanistically overlap in rodents, with the 5-HT2CR functional status acting as a neural rheostat to regulate, in part, the intersection between these vulnerability behaviors.

Figure 1

Impulsive action phenotypes are identifiable in an outbred rat population. Rats were required to meet an acquisition criteria of a minimum of 50 correct responses, >80% accuracy and <20% omissions on the final one-choice serial reaction time (1-CSRT) task training stage (0.5 s stimulus duration, 5 s limited hold and 5 s intertrial interval, ITI5) before being tested on an ITI8 challenge session to more easily detect phenotypic differences in inherent impulsive action. (a) The number of premature responses made during the ITI8 challenge session was used to stratify rats as high impulsive (HI) or low impulsive (LI) relative to rats in the interquartile range. (b) HI rats exhibited higher numbers of premature responses vs LI rats on two separate ITI8 challenge sessions conducted when the cohort initially met criteria (ITI8 challenge #1; day 25–30 on average) and then upon retest (ITI8 challenge #2; day 92 of 1-CSRT task maintenance), respectively (*p<0.001 vs LI). (c) The impulsive phenotype was identifiable and stable throughout >75 days of 1-CSRT sessions (ITI5) (HI vs LI at day 35 (*p<0.05 vs LI), day 50 (*p<0.05 vs LI) and day 75 (*p<0.05 vs LI)).

Figure 2

Inherent impulsive action predicts cocaine cue reactivity. Following phenotype identification, the one-choice serial reaction time (1-CSRT) task sessions were terminated, and HI rats and LI rats were retained in their home cages and allowed to free-feed for 7 days before surgical implantation of a jugular catheter. (a) In daily 180 min sessions, HI and LI rats readily acquired cocaine self-administration (0.75 mg/kg/0.1 ml infusion) to stability. (b) On forced abstinence (FA) day 14, HI rats exhibited a higher number of previously active (*p<0.05 vs LI), but not inactive lever presses (n.s.), during the cue reactivity test session vs LI rats. (c) There was a positive correlation between premature responses on the 1-CSRT task and cue-reinforced lever presses during the cue reactivity test session for individual subjects (r=0.502; p<0.05).

Figure 3

Functional status of the 5-HT_2CR is a neuromolecular substrate for inherent impulsive action. Following phenotype identification in two separate cohorts of rats, the one-choice serial reaction time (1-CSRT) task sessions were terminated, and HI and LI rats were retained in their home cages (food restriction was maintained) and killed within 2–3 days for ex vivo neurochemical analyses of 5-HT_2CR protein and mRNA. (a) Qualitative (inset) and quantitative demonstration that HI rats expressed lower levels of membrane medial prefrontal cortex (mPFC) 5-HT_2CR vs LI rats (*p<0.05 vs LI). (b) There was an inverse correlation between mPFC 5-HT_2CR membrane protein levels and premature responding on the 1-CSRT task for individual subjects (r=−0.709; p<0.01). (c) Immunoprecipitation (IP) for postsynaptic density 95 (PSD95) followed by immunoblot (IB) for 5-HT_2CR yielded 5-HT_2CR immunoreactivity in both HI and LI rats (inset). Synaptosomal 5-HT_2CR associated with PSD95 in the mPFC to a greater extent in HI vs LI rats (*p<0.05 vs LI); comparable levels of PSD95 were immunoprecipitated between HI and LI rats (inset). (d) The 5-HT_2CR mRNA editing efficiency was significantly different in HI vs LI rats; editing at the E and D sites was higher in HI vs LI rats (*p<0.05). (e) The frequencies of ABD, AEC, ABED, AECD, and ABECD 5-HT_2CR mRNA variants were elevated in HI rats compared with LI rats. HI rats expressed higher levels of predicted protein isoforms VNV and VGV vs LI rats (*p<0.05 vs LI). (f) Total mPFC 5-HT_2CR mRNA levels as assessed by RT-PCR did not differ between HI and LI rats (n.s.).

Figure 4

Genetic loss of 5-HT_2CR in medial prefrontal cortex (mPFC) confers aggregate impulsive action/cue reactivity. (a) The shRNA expression plasmid targeting the 5-HT_2CR efficiently silenced 5-HT_2CR mRNA in a cell line transiently expressing 5-HT_2CR (*p<0.05 vs LI). (b) The 5-HT_2CR shRNA-eGFP AAV knocked down 5-HT_2CR protein levels in the mPFC (∼50% decrease) relative to the NSC-eGFP AAV. (c) Stereotaxic placement and NSC-eGFP AAV infection (green) in mPFC (left). The NSC-eGFP AAV (green) did not alter 5-HT_2CR protein expression (red) in infected neurons (yellow arrows) relative to non-AAV infected neurons (white arrows) (right). (d) Stereotaxic placement and 5-HT_2CR shRNA-eGFP AAV infection (green) in mPFC (left). The 5-HT_2CR shRNA-eGFP AAV (green) induced a significant knockdown of 5-HT_2CR protein (red) in infected neurons (yellow arrows) relative to non-infected neurons (white arrows) (right). (e) Following intra-mPFC transgene delivery and stable viral vector expression, control and 5-HT_2CR knockdown rats were subjected to the one-choice serial reaction time (1-CSRT) task. The 5-HT_2CR knockdown rats expressed significantly higher premature responses vs control rats on the final 1-CSRT task training stage (0.5 s stimulus duration, 5 s limited hold and 5 s intertrial interval (ITI5)) (*p<0.05 vs NSC-eGFP AAV). Upon completion of 1-CSRT task assessments, control and 5-HT_2CR knockdown rats were retained in their home cages and allowed to free-feed for 7 days before surgical implantation of a jugular catheter. Cocaine self-administration commenced 7 days post catheterization. (f) On forced abstinence (FA) day 14, 5-HT_2CR knockdown rats exhibited higher cue-reinforced lever presses (*p<0.05 vs NSC-eGFP AAV), but not inactive, lever presses (n.s.), during a cue reactivity test session vs control rats. (g) There was a positive correlation between premature responses and cue-reinforced lever presses during the cue reactivity test session for individual subjects (r=0.577; p<0.01).