Attenuation of cocaine seeking in rats via enhancement of infralimbic cortical activity using stable step-function opsins

Abstract

Rationale: The infralimbic cortex (IL) and its downstream projection target the nucleus accumbens shell (NAshell) mediate the active suppression of cocaine-seeking behavior. Although an optogenetic approach would be beneficial for stimulating the IL and its efferents to study their role during reinstatement of cocaine seeking, the use of channelrhodopsin introduces significant difficulties, as optimal stimulation parameters are not known.

Objectives: The present experiments utilized a stable step-function opsin (SSFO) to potentiate endogenous activity in the IL and in IL terminals in the NAshell during cocaine-seeking tests to determine how these manipulations affect cocaine-seeking behaviors.

Methods: Rats first underwent 6-h access cocaine self-administration followed by 21-27 days in the homecage. Rats then underwent cue-induced and cocaine-primed drug-seeking tests during which the optogenetic manipulation was given. The same rats then underwent extinction training, followed by cue-induced and cocaine-primed reinstatements.

Results: Potentiation of endogenous IL activity did not significantly alter cue-induced or cocaine-primed drug seeking following the homecage period. However, following extinction training, enhancement of endogenous IL activity attenuated cue-induced reinstatement by 35% and cocaine-primed reinstatement by 53%. Stimulation of IL terminals in the NAshell did not consistently alter cocaine-seeking behavior.

Conclusion: These results suggest the utility of an SSFO-based approach for enhancing activity in a structure without driving specific patterns of neuronal firing. However, the utility of an SSFO-based approach for axon terminal stimulation remains unclear. Moreover, these results suggest that the ability of the IL to reduce cocaine seeking depends, at least in part, on rats first having undergone extinction training.

Keywords: Channelrhodopsin; Cocaine seeking; Incubation of craving; Optogenetics; Reinstatement.

Figure 1.. Confirmation of Stable Step-function Opsin expression and function.

A. Schematic of fiber-optic placement targeted at the IL (first panel). Anti-eYFP immunohistochemical staining from an SSFO-transduced rat (second panel). Fluorescent image shows SSFO expression in the IL (third panel). Cresyl violet staining shows a fiber optic terminating in the IL (fourth panel). B. Schematic of fiber-optic placement targeted at the NAshell (first panel). Anti-eYFP immunohistochemical staining of axonal fibers from the IL in the NAshell from an SSFO-transduced rat (second panel). Cresyl violet staining shows a fiber optic terminating in the NAshell (third panel). ac, anterior commissure C. Whole-cell voltage-clamp recording from an IL neuron transduced with SSFO. The representative trace shows inward current recorded following blue laser illumination (473 nm, 10 ms, blue rectangle). Trace shows return to baseline following yellow laser illumination (561 nm, 10 ms, yellow rectangle). D. In vivo electrophysiological recording from an IL neuron transduced with SSFO. Blue laser illumination (473 nm, 2 s, blue rectangle) enhanced 40–60 Hz activity, which was attenuated following yellow laser illumination (561 nm, 10 s, yellow rectangle).

Figure 2.. SSFO-mediated enhancement of IL activity during cocaine-seeking tests following the drug-free homecage period and extinction training.

A. First part of the experimental timeline for Experiment 1 in which IL activity was enhanced with SSFOs during cocaine-seeking tests. B. Number of active and inactive lever presses and cocaine infusions for the last 10 d of cocaine self-administration for all rats included in the final analysis. C. Following the last d of self-administration (baseline craving) and the last d of the drug-free homecage period (incubation of craving), rats underwent 1-h cue-induced drug-seeking tests. Left, Rats made significantly more cocaine-seeking responses during the incubation-of-craving test than during the baseline-craving test, demonstrating incubation of cocaine craving. Right, Active lever presses from baseline- and incubation-of-craving tests, broken down into 15-min bins. *, p < 0.05 compared to baseline test. D. Schematic diagrams of fiber optic placement (left) and manipulations given during cocaine-seeking and open field tests (right). One min before the session, a 473 nm laser was used to activate the SSFOs and, following the session, a 561 nm laser was used to close SSFO channels. E. Active lever presses during the cue-induced cocaine-seeking test. Activation of SSFOs in the IL did not alter cue-induced cocaine seeking. F. Active lever presses during the cocaine-primed (5 mg/kg) drug-seeking test. Activation of SSFOs in the IL did not alter cocaine-primed drug seeking. G. Experimental timeline for subsequent behavioral procedures in Experiment 1. H. Left, Activation of SSFOs in the IL during cue-induced reinstatement significantly decreased cocaine seeking following extinction training. Inset, All rats decreased cocaine-seeking behavior following laser treatment compared to sham treatment. Right, Cue-induced reinstatement data divided into 15-min bins suggest that, following laser treatment, rats pressed the active lever less during the first and fourth bins compared to sham treatment. *, p < 0.05 compared to extinction baseline; #, p < 0.05 relative to sham treatment. EXT, extinction. I. Left, Enhancement of IL activity with an SSFO during cocaine-primed reinstatement significantly decreased cocaine seeking following extinction training. *, p < 0.05 relative to extinction baseline; #, p < 0.05 relative to sham treatment. Inset, All rats but one decreased cocaine-seeking behavior following laser treatment compared to sham treatment. Right, Cocaine-primed reinstatement data divided into 15-min bins suggest that rats pressed the active lever less during the first bin following laser treatment compared to sham treatment. J. Rats traveled a significantly greater distance in the open-field test following laser treatment compared to sham treatment. Note the decrease in subjects from 10 to 9 due to one death. *, p < 0.05 compared to sham treatment.

Figure 3.. Illumination of IL cells expressing eYFP during cocaine-seeking tests following the drug-free homecage period and extinction training.

A. First part of experimental timeline for Experiment 2 in which IL cell bodies expressing eYFP only (i.e., no SSFO expression) were illuminated during cocaine-seeking tests. B. Number of active and inactive lever presses and cocaine infusions for the last 10 d of cocaine self-administration for all rats included in the final analysis. C. Following the last d of self-administration (baseline craving) and the last d of the homecage period (incubation of craving), rats underwent 1-h cue-induced drug-seeking tests. Left, There was a trend toward increased active lever pressing during the incubation-of-craving test compared to the baseline-craving test. Right, Active lever presses from baseline- and incubation-of-craving tests, broken down into 15-min bins. &, p < 0.10 compared to baseline test. D. Schematic diagrams of fiber optic placement (left) and manipulations given during cocaine-seeking and open field tests (right). One min before the session, a 473 nm laser was used and, following the session, a 561 nm was used. E. Active lever presses during the cue-induced cocaine seeking test. IL illumination did not alter cue-induced drug seeking. F. Active lever presses during the cocaine-primed (5 mg/kg) drug-seeking test. IL illumination did not alter cocaine-primed drug seeking. G. Experimental timeline for subsequent behavioral procedures in Experiment 2. H. IL illumination during cue-induced reinstatement did not significantly alter cocaine seeking following extinction training. *, p < 0.05 compared to extinction baseline. EXT, extinction. Note decrease in subjects from 11 to 10 due to one rat’s death. I. IL illumination during cocaine-primed reinstatement did not significantly alter cocaine seeking following extinction training. J. Laser treatment did not significantly alter rats’ distance traveled in the open-field test compared to sham treatment. Note the decrease in subjects from 10 to 9 due to one rat’s death.

Figure 4.. Illumination of IL terminals in NAshell during cocaine-seeking tests following the drug-free homecage period and extinction training.

A. First part of experimental timeline for Experiment 3 in which IL terminals in the NAshell were illuminated during cocaine-seeking tests. B. Number of active and inactive lever presses and cocaine infusions for the last 10 d of cocaine self-administration for all rats included in the final analysis. C. Following the last d of self-administration (baseline craving) and the last d of the drug-free homecage period (incubation of craving), rats underwent 1-h cue-induced drug-seeking tests. Left, Rats pressed the active lever more during the incubation-of-craving test than during the baseline craving test, demonstrating incubation of cocaine craving. Right, Active lever presses from baseline- and incubation-of-craving tests, broken down into 15-min bins. *, p < 0.05 compared to baseline test. D. Schematic diagrams of fiber optic placement (left) and manipulations given during cocaine-seeking and open field tests (right). One min before the session, a 473 nm laser was used and, following the session, a 561 nm laser was used. E. Left, Active lever presses during the cue-induced cocaine seeking test. Laser treatment significantly attenuated cocaine seeking compared to sham treatment. Inset, Active lever presses for individual rats following either sham or laser treatment. *, p < 0.05 compared to sham treatment. F. Active lever presses during the cocaine-primed (5 mg/kg) drug-seeking test. Illumination of IL terminals in the NAshell did not alter cocaine-primed drug seeking. G. Experimental timeline for subsequent behavioral procedures in Experiment 3. H. Illumination of IL terminals in the NAshell during cue-induced reinstatement did not significantly alter cocaine seeking following extinction training. *, p < 0.05 compared to extinction baseline. EXT, extinction. Note the decrease in subjects from 16 to 14 due to 2 deaths. I. Illumination of IL terminals in the NAshell during cocaine-primed reinstatement did not significantly alter cocaine seeking following extinction training. *, p < 0.05 relative to extinction baseline. Note the decrease in subjects from 14 to 13 due to failure to re-extinguish following the cue-induced reinstatement. J. Laser treatment did not significantly alter rats’ distance traveled in the open field test compared to sham treatment. Note the decrease in subjects 13 to 12 due to one rat’s death prior to open field testing.