Chemogenetic Excitation of Accumbens-Projecting Infralimbic Cortical Neurons Blocks Toluene-Induced Conditioned Place Preference

Abstract

Abuse rates for inhalants among adolescents continue to be high, yet preclinical models for studying mechanisms underlying inhalant abuse remain limited. Our laboratory has previously shown that, in male rats, an acute binge-like exposure to toluene vapor that mimics human solvent abuse modifies the intrinsic excitability of mPFC pyramidal neurons projecting to the NAc. These changes showed region (infralimbic; IL vs prelimbic; PRL), layer (shallow; 2/3 vs deep; 5/6), target (core vs shell), and age (adolescent vs adult) dependent differences (Wayman and Woodward, 2017). To expand these findings using reward-based models that may better mimic human drug abuse, we used whole-cell electrophysiology and drug receptors exclusively activated by designer drugs to examine changes in neuronal function and behavior in rats showing a conditioned place preference (CPP) to toluene. Repeated pairings of adolescent rats to binge concentrations of toluene vapor previously shown to enhance dopamine release in reward-sensitive areas of the brain produced CPP that persisted for 7 but not 30 d. Toluene-induced CPP was associated with increased excitability of IL5/6 mPFC neurons projecting to the core of the NAc and reduced excitability of those projecting to the NAc shell. No changes in PRL-NAc-projecting neurons were found in toluene-CPP rats. Chemogenetic reversal of the toluene-induced decrease in IL5/6-NAc shell neurons blocked the expression of toluene-induced CPP while manipulating IL5/6-NAc core neuron activity had no effect. These data reveal that alterations in selective mPFC-NAc pathways are required for expression of toluene-induced CPP.SIGNIFICANCE STATEMENT Disturbed physiology of pyramidal neurons projecting from the mPFC to the NAc has been shown to have different roles in drug-seeking behaviors for a number of drugs (e.g., methamphetamine, cocaine, ecstasy, alcohol, heroin). Here, we report that rats repeatedly exposed to the volatile organic solvent toluene, a member of the class of abused inhalants often used for intoxicating purposes by adolescents, induces a preference for the drug-paired environment that is accompanied by altered physiology of a specific population of NAc-projecting mPFC neurons. Chemogenetic correction of this deficit before testing prevented expression of drug preference. Overall, these findings highlight the importance of corticolimbic circuitry in mediating the rewarding properties of abused inhalants.

Keywords: DREADDs; core; electrophysiology; infralimbic; shell; volatile organic solvent.

Figure 1.

Toluene-induced CPP is associated with alterations in mPFC-NAc neuron excitability. A, Experimental timeline showing alternating exposures to air or toluene vapor. B, Toluene-exposed rats exhibit a significant preference for the drug-paired chamber (previously identified as the nonpreferred chamber) compared with rats that received air in both chambers during the conditioning paradigm. ****p < 0.0001 (paired t test). C, Representative image depicting the injection sites of the NAcc and NAcs as well as the recording sites of the PRL and IL prefrontal cortex (redrawn from Paxinos and Watson (2005)). D, Toluene-induced CPP does not alter evoked firing of PRL5/6-NAcc mPFC neurons (n = 12). E, Toluene-induced CPP was associated with enhanced firing of IL5/6-NAcc mPFC neurons (n = 12). **p < 0.01 (two-way mixed ANOVA). F, Toluene-induced CPP did not affect the firing of PRL5/6-NAcs mPFC neurons (n = 12). G, Toluene-induced CPP was associated with reduced firing of IL5/6-NAcs mPFC neurons (n = 12). **p < 0.01 (two-way mixed ANOVA). Insets, All example traces obtained at the 250 pA current injection step.

Figure 2.

Toluene-induced CPP and underlying changes in mPFC-NAc neuronal excitability persist for at least 7 d. A, Experimental timeline showing alternating exposures to air or toluene vapor. B, Preference scores were significantly different between air- and toluene-exposed rats on WD1 (***p < 0.001), and this preference persisted to at least WD7 (**p < 0.01, two-way mixed ANOVA Sidak's post hoc). C, Toluene-induced CPP did not alter the firing of PRL5/6-NAcc mPFC neurons (n = 12). D, Toluene-induced CPP was associated with enhanced firing of IL5/6-NAcc mPFC neurons that persist for 7 d after the last exposure (n = 12; **p < 0.01, two-way mixed ANOVA). E, Toluene-induced CPP did not affect the firing of PRL5/6-NAcs mPFC neurons (n = 12). F, Toluene-induced CPP was associated with reduced firing of IL5/6-NAcs mPFC neurons that persist for 7 d after the last exposure (n = 12; **p < 0.01, two-way mixed ANOVA). Insets, All representative traces obtained at the 250 pA current injection step.

Figure 3.

Chemogentic modification of IL5/6-NAcs activity prevents expression of toluene-induced CPP. A, Experimental timeline showing alternating exposures to air or toluene vapor and CPP testing. B, IL5/6-NAcc-hM4Di rats exhibited a clear preference for the toluene-paired chamber following injection of either the saline vehicle (**p < 0.01, two-way mixed ANOVA Sidak's post hoc) or CNO (**p < 0.01, two-way mixed ANOVA Sidak's post hoc). C, Toluene-exposed IL5/6-NAcs-hM3Dq rats exhibited a preference for the toluene-paired chamber following injection of saline (*p < 0.05, two-way mixed ANOVA Sidak's post hoc) but not CNO (^n.s.p > 0.05, two-way mixed ANOVA Sidak's post hoc). CPP for the drug-paired chamber in toluene-exposed IL5/6-NAcs-hM3Dq during saline injection was blocked when they were given CNO. ^#p < 0.05 (two-way mixed ANOVA Sidak's post hoc).

Figure 4.

Toluene-induced CPP and altered excitability of IL-NAc-projecting neurons is no longer present 30 d after the last conditioning day. No differences in preference scores were observed between rats paired with toluene or air when tested after 30 d of withdrawal. A, IL5/6-NAcc hM4Di rats. B, IL5/6-NAcs hM3Dq rats. C, No differences in evoked firing of IL5/6-NAcc mPFC neurons (n = 8) between air- and toluene-exposed animals 30 d after the last pairing session. D, No differences in evoked firing of IL5/6-NAcs mPFC neurons (n = 8) between air- and toluene-exposed animals 30 d after the last pairing session. Insets, All representative traces obtained at the 250 pA current injection step.

Figure 5.

Validation of expression and function of Gi- and Gq-DREADDs. A, Representative images and atlas drawings showing location and expression of hM4Di Gi-DREADDs. Atlas diagrams of cortical rat brain slices (redrawn from Paxinos and Watson, 2005) show extent of hM4Di Gi-DREADD labeling in IL5/6-NAcc pyramidal neurons used for recording. Images show IL5/6-NAcc pyramidal neurons expressing hM4Di Gi-DREADD labeling at 2× (right), 10× (middle), and 40× (left). B, Firing of IL5/6-NAcc neurons (n = 8) in mPFC slices from air-exposed hM4Di-infected rats is inhibited by bath application of 5 μm CNO. ^##p < 0.01 (two-way repeated-measures ANOVA). C, Firing of IL5/6-NAcc neurons (n = 8) in mPFC slices from toluene-exposed hM4Di-infected rats is inhibited by bath application of 5 μm CNO. *p < 0.05 (two-way repeated-measures ANOVA). D, Representative images and atlas drawings showing location and expression of hM3Dq Gq-DREADDs. Atlas diagrams of cortical rat brain slices (redrawn from Paxinos and Watson, 2005) show extent of hM3Dq Gq-DREADD labeling in IL5/6-NAcs pyramidal neurons used for recording. Images show IL5/6-NAcs pyramidal neurons expressing hM3Dq Gq-DREADD labeling at 2× (right), 10× (middle), and 40× (left). E, Firing of IL5/6-NAcs neurons (n = 8) in mPFC slices from air-exposed hM3Dq-infected rats exhibit enhanced firing following application of 10 μm CNO. ^#p < 0.05 (two-way repeated-measures ANOVA). F, Firing of IL5/6-NAcs neurons (n = 8) in mPFC slices from toluene-exposed hM3Dq-infected rats exhibit enhanced firing following application of 10 μm CNO. **p < 0.01 (two-way repeated-measures ANOVA). Insets, All representative traces obtained at the 250 pA current injection step.