Altered neuronal activity in the ventromedial prefrontal cortex drives nicotine intake escalation

Abstract

Nicotine addiction develops after prolonged drug use and escalation of drug intake. However, because of difficulties in demonstrating escalation of nicotine use in rats, its underlying neuroadaptations still remain poorly understood. Here we report that access to unusually high doses of nicotine (i.e., from 30 µg to 240 µg/kg/injection) for self-administration precipitated a rapid and robust escalation of nicotine intake and increased the motivation for the drug in rats. This nicotine intake escalation also induced long-lasting changes in vmPFC neuronal activity both before and during nicotine self-administration. Specifically, after escalation of nicotine intake, basal vmPFC neuronal activity increased above pre-escalation and control activity levels, while ongoing nicotine self-administration restored these neuronal changes. Finally, simulation of the restoring effects of nicotine with in vivo optogenetic inhibition of vmPFC neurons caused a selective de-escalation of nicotine self-administration.

Fig. 1. Nicotine intake escaladed with increasing doses of nicotine.

a Schematic diagram of the three stages nicotine escalation procedure (top). Mean (±SEM) nicotine intake during the 3 h session is plotted as a function of days for rats in the no-escalation group (No ES, light blue, n = 12) that has access to the unit dose of 30 µg/kg/injection, and rats in the escalation group (ES, blue gradient, n = 27) that had access to increasing doses of nicotine (30 µg, 120 µg and 240 µg/kg/injection) (bottom). b Average nicotine intake (mean ± SEM) during the last 3 days at each nicotine dose for the No ES and ES groups. c Mean (±SEM) breaking point reached at the end of the procedure for the No ES and ES groups (d, e) Mean (±SEM) number of lever presses (d) and nicotine injections (e) before and after escalation during a test session with the unit dose of nicotine 30 µg/kg/injection. f Mean number (±SEM) of nicotine injections per 10 min in the No ES and ES groups during the test session with the unit dose of nicotine 30 µg/kg/injection. *p < 0.001, different from the lowest dose of 30 µg/kg/injection. ⁺p < 0.001^, different from the No ES group.

Fig. 2. Nicotine escalation induced convulsive-like behavior.

a Incidence and (b) intensity (as revealed by the behavioral score) of the convulsive responses induced after the 1st injection of nicotine at 240 µg/kg/injection in the escalation group are plotted as a function of days. *p < 0.01, different from the 1st day. c Mean (±SEM) latency to initiate nicotine self-administration as a function of days and (d) averaged during the last 3 days at each nicotine dose for the No ES and ES groups. *p < 0.01, different from the lowest dose of 30 µg/kg/injection.

Fig. 3. Changes in vmPFC neuronal activity during nicotine intake escalation.

a Schematic of the location of individual wire tips within the vmPFC. The numbers indicate millimeters anterior to bregma. b Mean (±SEM) tonic firing rates (log Hz) of vmPFC neurons measured 30 min period before (Before SA) and during the 3 h nicotine self-administration (During SA) across recording days in the no-escalation (No ES, n = 5) and the escalation (ES, n = 5) groups. Hab = habituation day; Initial SA = initial phase after 15 days of nicotine self-administration; and Final SA = final phase after 45 days of nicotine self-administration. *p < 0.05, different from Hab. ^$p < 0.01, different from Initial SA. ⁺p < 0.01, difference between Before SA vs During SA. c Average firing rate (Hz) of vmPFC neurons measured 30 min period before and during the 3 h nicotine self-administration during final SA is plotted as a function of time in the No ES and the ES groups. Time 0 on the abscissa corresponds to the start of the self-administration session. d Percentage of vmPFC neurons exhibiting a sustained increase (white bars) or decrease (hatched bars) in firing rate during the self-administration session relative to the pre-drug baseline period in the No ES and the ES groups. ***p < 0.001, different from No ES. Mean (±SEM) (e) nicotine intake and (f) difference in firing activity before vs during SA (∆ firing activity) in the No ES (light blue) and the ES (dark blue) groups during final SA. *p < 0.05 and **p < 0.01 different from No ES. g Examples of long-duration change in firing showing an increase (light blue) or a decrease (dark blue) in firing. Each perievent histogram shows the average firing rate of a single neuron 5 min before and 10 min after nicotine injection. Time 0 on the abscissa corresponds to the nicotine injection. h Percentage of vmPFC neurons exhibiting a long-duration increase (white bars) or decrease (hatched bars) in firing rate after nicotine injection in the No ES (light blue) and the ES (dark blue) groups. **p < 0.01, different from No ES.

Fig. 4. In vivo optogenetic inhibition of vmPFC decreased nicotine self-administration.

a (Left) Schematic of the location of individual optic fiber tips within the vmPFC. The numbers indicate millimeters anterior to bregma. (Right) Representative images of coronal sections showing the virus expression and fibers implants in the vmPFC (top) and immunostaining of eYFP (green) expression in vmPFC pyramidal neurons (red, CAMKII) (bottom). b (Left) Average firing frequency (Hz) of light-evoked responses in vmPFC neurons expressing ArchT3.0. (Right) Mean (±SEM) firing rate prior (Pre), during (Light) and after (Post) stimulation application in vmPFC neurons expressing ArchT3.0. *p < 0.01, as compared to pre- and post-light. c, d Mean (±SEM) total number of (c) lever presses and (d) nicotine injections during the 1 h nicotine self-administration session at 30 µg/kg/injection in the no-escalation (No ES) and the escalation groups (ES) injected either with eYFP (n = 5 for each group) or ArChT3.0 (n = 8 for each group) under light or no-light conditions. ***p < 0.001 and **p < 0.01, different from the No ES group. ⁺⁺p < 0.01 and ⁺p < 0.05, different from No Light. e, f (e) Response time course and (f) and injection time course are plotted as a function of time in the no-escalation (No ES) and the escalation group (ES) injected with ArChT3.0 (n = 8 for each group). **p < 0.01, different from No Light.

Fig. 5. In vivo optogenetic inhibition of vmPFC had no effect on sucrose self-administration.

a Schematic of the location of individual optic fiber tips within the vmPFC. The numbers indicate millimeters anterior to bregma. b, c Mean (±SEM) total number of lever presses during (b) FR1 and (c) FR3 sucrose self-administration in rats injected with ArChT3.0 (n = 8).