Remifentanil self-administration in mice promotes sex-specific prefrontal cortex dysfunction underlying deficits in cognitive flexibility

Abstract

Opioid-based drugs are frequently used for pain management in both males and females despite the known risk of prefrontal cortex dysfunction and cognitive impairments. Although poorly understood, loss of cognitive control following chronic drug use has been linked to decreased activation of frontal cortex regions. Here, we show that self-administration of the potent opioid, remifentanil, causes a long-lasting hypoactive basal state evidenced by a decrease in ex vivo excitability that is paralleled by an increase in firing capacity of layer 5/6 pyramidal neurons in the prelimbic, but not infralimbic region of the medial prefrontal cortex. This phenomenon was observed in females after as few as 5 days and up to 25-30 days of self-administration. In contrast, pyramidal neurons in males showed increased excitability following 10-16 days of self-administration, with hypoactive states arising only following 25-30 days of self-administration. The emergence of a hypoactive, but not hyperactive basal state following remifentanil self-administration aligned with deficits in cognitive flexibility as assessed using an operant-based attentional set-shifting task. In females, the hypoactive basal state is driven by a reduction in excitatory synaptic transmission mediated by AMPA-type glutamate receptors. Alternatively, hyper- and hypoactive states in males align selectively with decreased and increased GABA_B signaling, respectively. Chemogenetic compensation for this hypoactive state prior to testing restored cognitive flexibility, basal hypoactive state, and remifentanil-induced plasticity. These data define cellular and synaptic mechanisms by which opioids impair prefrontal function and cognitive control; indicating that interventions aimed at targeting opioid-induced adaptations should be tailored based on biological sex.

Fig. 1. Bidirectional effect of remifentanil self-administration on PrLC L5/6 pyramidal neurons in males and females.

a Self-administration, abstinence timeline, and location of whole-cell recordings in the PrLC. b For mice that received 10–16 days of self-administration, no difference was observed for active presses during maintenance between remifentanil males (blue, unfilled) and females (blue, filled) whereas females had greater inactive responses (gray, filled) compared to males (gray, unfilled). c Cumulative remifentanil infusions during the entire 10–16-day self-administration procedure did not differ in males (M) vs. females (F). d Comparison of mean action potential (AP) threshold (rheobase) in PrLC L5/6 pyramidal neurons following 14–21-day abstinence from 10- to 16-day self-administration in males and females showed that rheobase was similar in females compared to males under saline conditions (SAL). Alternatively, remifentanil (REM) increased rheobase in females but reduced it in males compared to respective controls. e For 25–30-day self-administration groups, active lever responding did not differ between remifentanil males (orange, unfilled) and females (orange, filled) or across days during maintenance. Alternatively, males exhibited significantly lower inactive lever responding compared to females on days 9, 13, and 19–25. f Cumulative remifentanil infusions were elevated in females (F) compared to males (M). g Comparison of rheobase following 14–21-day abstinence from 25–30 days of self-administration showed a significantly higher firing threshold in remifentanil males and females compared to respective saline controls. h–i Current-spike analysis in 10–16-day self-administering mice showed a trend toward increased firing frequency in remifentanil vs. saline males (h) whereas i remifentanil females exhibited similar firing at lower currents and increased firing at higher potentials (380 pA) compared to controls. j Following 25–30 days of self-administration, current-spike analysis showed firing frequency was reduced at lower currents (140–220 pA) but increased at more depolarized potentials (340–380 pA) in remifentanil vs. saline males. k Remifentanil females showed reduced firing frequency at lower currents (100–180 pA) but similar firing frequency at more depolarized potentials vs. saline females. Representative scale bars: 20 pA/200 ms. Representative spike firing following 200 pA current injection. All data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001; ^^^p < 0.001 main effect of treatment.

Fig. 2. Remifentanil effects on PrLC L5/6 pyramidal neuron GABA_B-GIRK signaling.

a Self-administration and abstinence timepoints. b Baclofen-evoked currents (I_Baclofen) from male (unfilled) and female (filled) PrLC L5/6 pyramidal neurons 14–21 days after saline (gray), 10–16-day remifentanil self-administration (blue), or 25–30-day remifentanil self-administration (orange). I_Baclofen was no different in 10–16-day vs. 25–30-day saline males or females and thus were combined. I_Baclofen was reduced in 10–16-day remifentanil males vs. saline males and 25–30-day remifentanil males, whereas 25–30-day remifentanil males showed an increase in I_Baclofen vs. saline males. I_Baclofen did not differ when comparing saline females with 10–16- or 25–30-day remifentanil females. A difference was observed between 10- and 16-day remifentanil males and females. c Representative I_Baclofen traces in males (top) and females (bottom) across treatments. Representative scale bars: 50 pA/200 s. *p < 0.05, ***p < 0.001.

Fig. 3. Remifentanil effects on PrLC L5/6 pyramidal neuron excitatory and inhibitory transmission.

Representative miniature excitatory postsynaptic current (mEPSC) traces from PrLC L5/6 pyramidal neurons in a males and b females following 14–21 days abstinence from 10–16 and 25–30 days of self-administration. c mEPSC frequency was significantly greater in saline females compared to 10–16- and 25–30-day remifentanil females as well as saline males. mEPSC frequency did not differ when comparing saline to 10–16-day or 25–30-day remifentanil males. d mEPSC amplitude was elevated in 25–30-day remifentanil mice compared to 10–16-day and saline mice. Representative miniature inhibitory postsynaptic current (mIPSC) traces in males (e) and females (f) following 14–21 days abstinence from 10–16 and 25–30 days of self-administration. g mIPSC frequency was greater in 10–16-day remifentanil females vs. saline and 25–30-day remifentanil females, with no differences between 25- and 30-day remifentanil and saline groups. No differences were observed when comparing saline males to 10–16- or 25–30-day remifentanil males. h Mean mIPSC amplitude was elevated in females compared to males regardless of treatment. Scale bars: 10 pA/100 ms. *p < 0.05, **p < 0.01, ***p < 0.001; ⁺⁺⁺p < 0.001 main effect of sex, ^p < 0.05 vs. Rem10-16, ^##p < 0.01 vs Rem25-30.

Fig. 4. Remifentanil-induced deficits in cognitive flexibility.

a Timeline of behavioral assessments following 10–16 or 25–30-day self-administration and schematic of operant set-shifting procedure depicting levers (gray), dipper (white), cue location (yellow), and correct response (star). b, e Testing following 10–16 days of self-administration in males and females. b Number of days to pass lever training criterion (2 consecutive days ≤ 5 omissions) was similar acrosss treatment and sex. c Number of trials to criterion during a visual cue test also showed no significant effect of sex, treatment, or interaction. d Number of trials to criterion during the extradimensional (ED) shift test showed remifentanil females required more trials vs. saline females and remifentanil males, whereas performance in remifentanil vs. saline males was similar. e Number of trials to criterion during a reversal test showed no significant effect of sex, treatment, or interaction. f–h Testing following 25–30 days of self-administration in males. f, g No difference was observed in number of days to pass lever training criterion or trials to reach criterion during a visual cue test. h During the ED test, remifentanil males required more trials to reach criterion compared to saline. *p < 0.05, **p < 0.01.

Fig. 5. Increased excitation of PrLC pyramidal neurons in remifentanil females reverses deficits in cognitive flexibility.

a Timeline of DREADD experiments involving 10–16-day saline and remifentanil female mice expressing an excitatory CaMKII-hM3Dq(Gq)-DREADD or control GFP AAV in the PrLC. All mice received a saline or CNO (1.5–2.0 mg/kg, i.p.) injection 30 min prior to the visual cue and ED shift test, respectively. b Comparison of trials required to reach criterion during visual cue testing showed no effect of treatment, virus, or treatment by virus interaction. c For trials to criterion during the ED shift, female GFP-remifentanil mice required more trials vs. GFP-saline and Gq-remifentanil mice. Gq-remifentanil mice performed significantly better than Gq-saline mice. There was no significant difference between GFP-saline and Gq-saline. d–f Ex vivo comparison of rheobase and synaptic transmission following in vivo systemic CNO injection. d Comparison of rheobase in PrLC pyramidal neurons showed a treatment by virus interaction, with more current required to evoke an action potential in female GFP-remifentanil mice vs. GFP-saline and Gq-remifentanil mice. Rheobase in Gq-saline did not differ compared to GFP-saline or Gq-remifentanil. e Spontaneous EPSC (sEPSC) frequency (left) and amplitude (right) in GFP- and Gq-expressing PrLC pyramidal neurons. Pyramidal neurons in GFP-remifentanil mice showed a reduction in frequency compared to GFP-saline and Gq-remifentanil mice. sEPSC frequency in Gq-saline was not significantly different compared to GFP-saline or Gq-remifentanil. For sEPSC amplitude, Gq+ cells exhibited an overall greater amplitude compared to GFP. f Spontaneous IPSC (sIPSC) frequency and amplitude in GFP and Gq-expressing PrLC pyramidal neurons. sIPSC frequency was increased in remifentanil mice compared to saline mice regardless of virus. For sIPSC amplitude, no pairwise comparisons of interest were significant. Scale bars: 10 pA/100 ms. *p < 0.05, **p < 0.01, ***p < 0.001, ^^p < 0.01 main effect of virus, ⁺p < 0.05 main effect of treatment.