Role of a Lateral Orbital Frontal Cortex-Basolateral Amygdala Circuit in Cue-Induced Cocaine-Seeking Behavior

Abstract

Cocaine addiction is a disease characterized by chronic relapse despite long periods of abstinence. The lateral orbitofrontal cortex (lOFC) and basolateral amygdala (BLA) promote cocaine-seeking behavior in response to drug-associated conditioned stimuli (CS) and share dense reciprocal connections. Hence, we hypothesized that monosynaptic projections between these brain regions mediate CS-induced cocaine-seeking behavior. Male Sprague-Dawley rats received bilateral infusions of a Cre-dependent adeno-associated viral (AAV) vector expressing enhanced halorhodopsin 3.0 fused with a reporter protein (NpHR-mCherry) or a control AAV (mCherry) plus optic fiber implants into the lOFC (Experiment 1) or BLA (Experiment 2). The same rats also received bilateral infusions of a retrogradely transported AAV vector expressing Cre recombinase (Retro-Cre-GFP) into the BLA (Experiment 1) or lOFC (Experiment 2). Thus, NpHR-mCherry or mCherry expression was targeted to lOFC neurons that project to the BLA or to BLA neurons that project to the lOFC in different groups. Rats were trained to lever press for cocaine infusions paired with 5-s CS presentations. Responding was then extinguished. At test, response-contingent CS presentation was discretely coupled with optogenetic inhibition (5-s laser activation) or no optogenetic inhibition while lever responding was assessed without cocaine/food reinforcement. Optogenetic inhibition of lOFC to BLA, but not BLA to lOFC, projections in the NpHR-mCherry groups disrupted CS-induced reinstatement of cocaine-seeking behavior relative to (i) no optogenetic inhibition or (ii) manipulations in mCherry control or (iii) NpHR-mCherry food control groups. These findings suggest that the lOFC sends requisite input to the BLA, via monosynaptic connections, to promote CS-induced cocaine-seeking behavior.

Figure 1

Circuit specific, cre-dependent expression of NpHR-mCherry. (a) Diagrams of the AAV (serotype 5) expressing a cre-dependent, enhanced halorhodopsin 3.0 fused to mCherry (NpHR-mCherry) and AAV (serotype 6) expressing Cre-recombinase-IRES2-GFP (Retro-Cre-GFP) utilized in Experiments 1 and 2. Both AAVs contained transgenes under the control of an EF1α promoter. Representative images showing (b) expression of NpHR-mCherry in the lOFC and (c) in the BLA. Scale bar=200 μm, × 10 magnification. NpHR-mCherry expression was co-localized with immunoreactivity for (d) the excitatory marker, CaMKII, but not for (e) the GABAergic marker glutamic acid decarboxylase-67 (GAD-67). Scale bar=50 μm, × 20 magnification.

Figure 2

Virus spread and optic fiber placements. Schematics depicting mCherry and NpHR-mCherry viral spread (shaded areas) and the most ventral point of optic fiber tracts for rats that received mCherry (white circles) or NpHR-mCherry (black circles) into the (a) lOFC (mCherry, n=8; NpHR-mCherry, n=8) or (b) BLA (mCherry, n=6; NpHR-mCherry, n=6). Numbers denote distance from bregma in mm on the schematics modified from the rat brain atlas of Paxinos and Watson (1997).

Figure 3

Effect of optogenetic inhibition of lOFC afferents to the BLA on CS-induced reinstatement of cocaine-seeking behavior. (a) Schematic depicting the timeline of the behavioral experiments: cocaine self-administration training (SA), extinction training (EXT), and counterbalanced CS-induced reinstatement tests (1 h each) with or without laser stimulation. (b) Active lever responses (mean±SEM) during each phase of the experiment for the mCherry (n=8) and NpHR-mCherry (n=8) groups. (c) Time course of active lever responses (mean±SEM) during each 20-min bin of the Laser ON test. (d) Response rates (mean responses/second±SEM) on the active lever during the Laser ON test when active lever presses were reinforced with CS presentation (REIN) or were not reinforced during the 5-s CS presentation (CS) and the remaining 15-s (No CS) of the timeout period. (e) Total number of CS-reinforced active lever responses during the Laser ON test. Symbols represent difference between mCherry and NpHR-mCherry groups (*, b, c: Tukey's test, P<0.05; d, e: t-test, P<0.05), between the Laser OFF and Laser ON test sessions (^#, Tukey's test, P<0.05), and change from Bin 1 (^‡, Tukey's test, P<0.05).

Figure 4

Effect of optogenetic inhibition of BLA afferents to the lOFC on CS-induced reinstatement of cocaine-seeking behavior. (a) Schematic depicting the timeline of the behavioral experiments: cocaine self-administration training (SA), extinction training (EXT), and counterbalanced CS-induced reinstatement tests with or without laser stimulation (1 h each). (b) Active lever responses (mean±SEM) during each phase of the experiment for the mCherry (n=6) and NpHR-mCherry (n=6) groups. (c) Time course of active lever responses (mean±SEM) during each 20-min bin of the Laser ON test. (d) Response rates (mean responses/second±SEM) on the active lever during the Laser ON test when active lever presses were reinforced with CS presentation (REIN) or were not reinforced during the 5-s CS presentation (CS) and the remaining 15-s (No CS) of the timeout period. (e) Total number of CS-reinforced active lever responses during the Laser ON test. Symbols represent change from Bin 1 (^‡, Tukey's test, P<0.05).

Figure 5

Effects of optogenetic manipulation on neuronal membrane potential and evoked action potential firing in BLA afferents to the lOFC. (a) Overlay of current-clamp recording of BLA neuronal membrane potential in response to three consecutive light pulses (560 nm, 5 s, shaded area). (b) Mean change in membrane potential (±SEM) in NpHR-mCherrry-expressing neurons (n=5) at the peak and plateau of the light response. (c, d) Representative recordings of action potential firing evoked by current injection steps in the absence (c) and presence (d) of a light pulse (560 nm, 1 s, shaded area). (e) Current injection-evoked firing rates (mean±SEM, normalized to maximum rate in ‘Light OFF' condition) in NpHR-mCherrry-expressing BLA neurons in the presence and absence of a light pulse (560 nm, 1 s). Symbols represent difference relative to baseline membrane potential (^#, t-tests, P<0.05), relative to the Light OFF condition (*, ANOVA light main effect, P<0.05), and relative to current steps 0–75 pA (^, Tukey's tests, P<0.05).