Histological analyses

Histological analysis revealed injector placements localized within the anatomical boundaries of the shell subdivision of the NASh, localized to the anterior vs. posterior anatomical divisions (see methods). In Fig. 1a, a representative microphotograph showing a typical intra-aNASh injector tip location is shown. In Fig. 1b, a representative microphotograph showing bilateral intra-aNASh injector locations is shown. In Fig. 1c, a schematic summary showing representative aNASh experimental group bilateral infusion locations is presented. In Fig. 1d, a representative microphotograph showing a typical intra-pNASh injector tip location is shown. In Fig. 1e, a representative microphotograph showing bilateral intra-pNASh injector locations is shown. In Fig. 1f, a schematic summary showing representative pNASh experimental group bilateral infusion locations is presented.

Figure 1 Histological analysis of intra-NASh microinjection sites. (a) Microphotograph of representative injector placement within the anterior portion of the nucleus accumbens shell. (b) Schematic representation of select intra-anterior-NASh injector locations; ● = 100 ng THC group, ◆ = 100 ng THC + 1 µg CYP. (c) Microphotograph of representative intra-anterior-NASh bilateral cannulae placements. (d) Microphotograph of representative injector placement within the posterior portion of the nucleus accumbens shell. (e) Schematic representation of select intra-posterior-NASh injector locations; ● = 100 ng THC group, ◆ = 100 ng THC + 1 µg CYP. (f) Microphotograph of representative intra-posterior-NASh bilateral cannulae placements. Full size image

Intra-NASh THC produces dose-dependent, anatomically dissociable reward or aversion effects via separate opioid receptor substrates

Given previous evidence demonstrating functional differences in anterior vs. posterior NASh region in reward vs. aversion processing29 and evidence demonstrating that cannabinoid signaling can modulate reward or aversion signals via MOR vs KOR receptor substrates22, we hypothesized that aNASh THC would produce rewarding effects through a MOR-dependent mechanism, whereas pNASh THC would produce aversive behavioral effects through a KOR-dependent. We first examined the effects of THC (10 ng or 100 ng/0.5 µl), directly in the anterior NASh, or in combination with the selective MOR antagonist [CYP (0.5 µg–1 µg/0.5 µl)] or KOR [nor-BNI (1 µg/0.5 µl)] antagonist using an unbiased CPP procedure (see methods). Two-way ANOVA comparing times spent in the THC-paired vs. VEH- paired environments revealed a main effect of environment (F (1,110) = 46.094, p < 0.001) and a significant treatment x environment interaction (F (5,110) = 9.802, p < 0.001; Fig. 2a). Post-hoc analyses showed that intra-aNASh THC produced dose-dependent CPP for THC-paired environments as rats receiving a higher THC dose showed significant CPP (100 ng; n = 10; p = 0.002) vs. rats receiving the lower dose (10 ng; n = 10; p > 0.05; Fig. 2a). Co-administration with the selective MOR antagonist, cyprodime, dose-dependently blocked the rewarding effects of aNASh THC as rats receiving a lower dose of CYP (0.5 μg) + THC (100 ng) still showed significant CPP for THC-paired environments (0.5 μg; n = 10; p = 0.01) while a higher dose of CYP (1 μg; n = 11) blocked THC CPP (p > 0.05; Fig. 2a). In contrast, rats receiving THC co-administration with a selective KOR antagonist, nor-BNI, still displayed robust CPP for THC-paired environments (1 μg; n = 10; p = 0.002). VEH control rats (n = 10) displayed neither preference nor aversion for either environment (p > 0.05; Fig. 2a). The same data are presented in the form of difference scores to highlight the relative change in time spent in each environment (Fig. 2b). No significant difference was observed for locomotor activity between groups when comparing activity in the different drug vs. VEH conditioning environments (data not shown). Thus, THC in the anterior NASh produces dose-dependent rewarding effects which are dependent upon local MOR transmission but independent of KOR transmission.

Figure 2 Effects of intra-NASh THC and selective MOR antagonists on place conditioning behaviors. (a) Anterior NASh (+2.5 mm from bregma) microinfusions of THC dose dependently increases preference for the drug paired side. Co-administration of cyprodime, but not Nor-BNI, dose-dependently blocks this effect. (b) Difference scores of the data presented in (a) showing the effect of THC in the anterior NASh. (c) In contrast, posterior NASh (+1.5 mm from bregma) THC dose dependently produces a conditioned place aversion. Co-administration of Nor-BNI, but not cyprodime, dose-dependently blocks this effect. (d) Difference scores of the data presented in (c) showing the effect of THC in the posterior NASh. (e) Infusions of the effective doses of cyprodime (1 μg) or Nor-BNI (1 μg) vs. VEH produce neither reward nor aversion effects in the CPP paradigm. (f) THC in the anterior (but not posterior) NASh (100 ng) selectively potentiates the rewarding effects of a sub-reward threshold CPP conditioning dose of morphine. Full size image

Next, we examined the potential motivational effects of THC in the posterior NASh. Again, using two different doses of THC (10 ng and 100 ng/0.5 µl), two doses of the selective KOR antagonist, nor-BNI (0.5 µg and 1 µg/0.5 µl) in combination with the higher dose of THC, nor-BNI alone (1 µg/0.5 µl) or the higher dose of THC in combination with the MOR antagonist, CYP (1 µg/0.5 µl). Two-way ANOVA comparing times spent in THC vs. VEH-paired environments revealed a significant main effect of environment (F (1,116) = 26.584, p < 0.001) and a significant treatment x environment interaction (F (5,116) = 7.950, p < 0.001; Fig. 2c). Post-hoc analyses showed that intra-pNASh THC produced dose-dependent conditioned place aversions (CPA) for THC-paired environments as rats receiving a higher THC dose showed significant CPA (100 ng; n = 11; p = 0.004) vs. rats receiving the lower THC dose, who showed neither preference nor aversion behaviors (10 ng; n = 10; p > 0.05; Fig. 2c). THC co-administration with the selective KOR antagonist, nor-BNI, dose-dependently blocked the aversive effects of pNASh THC as rats receiving a lower dose of nor-BNI (0.5 μg) + THC (100 ng) still showed significant CPA for THC-paired environments (0.5 μg; n = 11; p = 0.009) while the higher dose of nor-BNI blocked THC CPA(1 μg; n = 11; p > 0.05; Fig. 2c). In contrast, rats receiving THC co-administration with the selective MOR antagonist, cyprodime, still displayed robust CPA for THC-paired environments (1 μg; n = 11; p < 0.001). Rats receiving vehicle (n = 10) displayed neither preference nor aversion for either environment. The same data are presented in the form of difference scores to highlight the relative change in time spent in each environment (Fig. 2d). No significant difference was observed for locomotor activity between groups. Thus, THC in the posterior NASh produces dose-dependent aversion effects which are dependent upon local KOR transmission but independent of MOR transmission.

Finally, to control for the potential motivational effects of either nor-BNI or CYP in and of themselves, separate control groups received either the effective dose of intra-aNASh CYP (1 µg; n = 9) vs. VEH or intra-pNASh nor-BNI (1 μg; n = 10) vs. VEH. Neither group displayed a preference or aversion for drug vs. VEH-paired environments (p’s > 0.05; Fig. 2e). Together, this data demonstrates a double-dissociation between the rewarding and aversive motivational effects of THC in the anterior vs. posterior NASh, mediated through separate MOR vs. KOR signaling mechanisms, while blockade of aNASh MOR or pNASh KOR transmission has no motivational effects in and of itself.

Intra-aNASh THC potentiates sub-threshold morphine reward salience

Given our findings that aNASh THC produced robust rewarding effects through a MOR-dependent substrate and previous evidence showing that stimulation of µORs within the NASh can potentiate drug reward salience30, we next examined how intra-NASh THC may modulate the motivational effects of an exogenous opioid, morphine, using a sub-reward threshold conditioning dose of morphine (0.05 mg/kg; i.p.; see methods). To control for the previously characterized rewarding or aversive effects of THC (in the anterior vs. posterior NAc, respectively; Fig. 2a,c), 4 separate groups received either intra-aNASh or pNASh THC (100 ng) prior to both morphine and saline conditioning sessions or aNASh or pNASh VEH. Thus, the motivational properties of THC were counterbalanced across both saline and morphine conditioning environments, meaning that place preferences/aversions would be associated with the potential effects of morphine vs. saline, rather than the effects of intra-NASh THC in and of itself. Two-way ANOVA comparing times spent in morphine vs. saline-paired environments revealed a significant main effect of treatment on times spent in morphine vs. saline-paired environments (F (3,24) = 3.702, p = 0.026). Post-hoc analyses revealed that there was no significant difference between intra-pNASh THC (100 ng; n = 7) vs. vehicle CPP times, as both groups displayed neither preference nor aversions to either environment (n = 7; p’s > 0.05; Fig. 2e). In contrast, rats receiving intra-aNASh THC (100 ng; n = 7), displayed a significant CPP for morphine-paired environments relative to VEH controls (n = 7; p = 0.030; Fig. 2e). Thus, consistent with the ability of aNASh THC to produce rewarding effects through a MOR-dependent substrate, aNASh THC potentiated the reward salience of normally sub-reward threshold morphine conditioning cues. In contrast, THC in the pNASh had no effect on sub-threshold morphine CPP behaviors.

Intra-NASh THC has no effect on sucrose reward processing

To determine if the effects of intra-NASh THC on affective processing may modulate non-drug-related motivational effects, we next examined the processing of natural, sucrose-related reward (see methods). Percent sucrose consumed was calculated by dividing the amount of sucrose consumed by the amount of total liquid (water plus sucrose) consumed. Although there was a trend towards an increase in sucrose consumption by rats receiving intra-pNASh THC, statistical comparison showed no significant difference between rats receiving intra-aNASh vehicle (n = 7) and intra-aNASh THC (n = 8; t (13) = −0.051, p > 0.05) or intra-pNASh vehicle (n = 8) and intra-pNASh THC (n = 8, t (14) = 0.152, p > 0.05; Fig. 3a), indicating that while THC in the aNASh is capable of potentiating drug-related reward salience (morphine), this effect does not influence a natural reward cue (sucrose).

Figure 3 Effects of intra-NASh THC on sucrose preference and social recognition. (a) THC has no significant effect on the percent consumption of sucrose vs total liquid consumed. (b,c) Summary of apparatus and experimental procedure for the sociability and social recognition test phases. (d) Microinfusions of THC in the pNASh, but not the aNASh, significantly reduced sociability scores *p < 0.05. (e) Microinfusions of THC in the pNASh, but not the aNASh, significantly reduced social recognition scores *p < 0.05. Full size image

THC induces social interaction and cognition deficits selectively in the posterior NASh

Previous studies have demonstrated that cannabinoid signaling can strongly modulate social behavioral phenomena through actions in the NASh29. Social behaviors are also naturally rewarding for rats therefore social interaction can also act as another model of natural reward. Therefore, we examined the potential effects of intra-NASh THC on social motivation behaviors and cognition (social memory). A simplified diagram of the experimental procedure is presented in Fig. 3b,c. In phase 1, sociability scores (measuring motivation to interact with a novel rat) were calculated by measuring times spent interacting with a novel rat and subtracting times spent interacting with an empty box. ANOVA showed a main effect of treatment on Phase 1 sociability scores (F (3,26) = 3.156, p = 0.042). Post-hoc analyses revealed a significant difference between intra-pNASh THC (n = 8) and intra-pNASh vehicle (n = 8; p = 0.009), intra-aNASh vehicle (n = 7; p = 0.029) and intra-aNASh THC (n = 7; p = 0.038) (Fig. 3d). No significant differences were observed between any other groups. In phase 2, social memory scores were calculated by taking times spent with a new, novel rat and subtracting times spent with the previously encountered, familiar rat. ANOVA showed a main effect of treatment for social memory scores (F (3,26) = 3.516, p = 0.029). Post-hoc testing revealed a significant difference between intra-pNASh THC (n = 8) and intra-pNASh vehicle (n = 8; p = 0.013), intra-aNASh vehicle (n = 7; p = 0.009) and intra-aNASh THC (n = 7; p = 0.013) (Fig. 3e). Thus, intra-NASh THC selectively impairs natural social motivation and social memory cognition selectively in the posterior region of the NASh.

THC inhibits spontaneous medium spiny neuron activity in the anterior NASh through a MOR-dependent mechanism

The activity states of NASh MSN neurons are strongly correlated with reward vs. aversive motivational states17 and we have previously demonstrated that cannabinoid CB1 transmission can produce rewarding effects by inhibiting NASh MSN neurons or aversive effects by activating these same neurons18. To determine if the anatomically localized effects of THC on reward or aversion were correlated with MSN activity state modulation, we next performed in vivo single-unit recordings in the posterior and anterior NASh, combined with ICV infusions of THC. We used a THC dose 10x our highest behaviorally effective dose (1 µg/µl) for these systemic electrophysiological recording studies to control for potential CSF diffusion effects (see methods). First, a total of n = 15 MSNs were isolated in the aNASh and we compared frequency rates pre vs. post THC administration (see Fig. 4a for representative aNASh recording location). Population analysis of aNASh MSNs revealed that 66.6% showed decreased activity, 0% increased, and 33.3% were unchanged, relative to baseline, following ICV THC administration (Fig. 4b). Thus, a plurality of aNASh MSNs showed a decrease in spontaneous firing frequency following ICV THC administration. An analysis of average firing frequency recorded 10 min pre vs. post ICV THC infusion revealed that THC significantly decreased firing rates in the aNASh (t (14) = 2.738, p = 0.016; Fig. 4c).

Figure 4 Effects of ICV THC and CYP on anterior NASh medium spiny neurons activity patterns. (a) Representative microphotograph showing typical intra-aNASh in vivo MSN recording location (b) Summary of experimental neuronal groups showing relative changes (no change, increase, or decrease) in firing frequencies following ICV pharmacological treatments (C) ICV THC significantly decreased spontaneous aNASh MSN neuronal firing frequency. (d) ICV THC alone (1 µg/μl) caused a significant decrease in spontaneous aNASh MSN neuronal firing frequency rates vs baseline. This inhibitory effect was reversed by co-administration of the behaviorally effective dose of CYP (10 µg/μl). (e) Sample rastergram showing typical aNASh MSN response pattern following ICV THC (1 µg/µl) infusion (arrows indicate intra-NAc infusion event). (f) Sample rastergram showing typical aNASh MSN response pattern following ICV THC (1 µg/µl) and CYP (10 µg/µl) infusion, demonstrating the block of THC’s neuronal effects with MOR blockade (arrows indicate intra-NAc infusion event). Full size image

Given our previous behavioral findings (Fig. 2a,c) showing THC-induced reward or aversion mediation through differential MOR vs. KOR transmission, respectively, we next performed in vivo single-unit recordings in the posterior and anterior NASh to determine if the anatomically dissociable effects of THC on MSN activity states may similarly depend upon differential opioid receptor signaling. Therefore, we performed co-administration studies using THC + CYP, or THC + nor-BNI, using 10x our behaviorally effective doses of THC (1 µg/µl), CYP (10 µg/µl), and nor-BNI (10 µg/µl).

In the aNASh, we sampled a total of n = 40 MSNs (Vehicle: n = 12, THC: n = 14, THC + CYP: n = 14). For rats receiving ICV vehicle, 58.3% of neurons showed no change, 8.3% increased and 43.4% showed decreased activity. For rats receiving ICV THC + CYP, 42.8% of neurons showed no change, 14.3% increased and 42.8% showed decreased activity (Fig. 4b, far right side). Analyses of pre vs. post infusion activity rates for aNASh MSNs revealed average changes from baseline of −4.9% for rats treated with vehicle, −34.5% for rats treated with THC, and −11% with THC + CYP. ANOVA comparing groups revealed a significant main effect of treatment (F (2,38) = 3.889, p = 0.029; Fig. 4d). Post-hoc analysis revealed that rats treated with ICV THC showed significantly decreased activity relative to VEH controls (p = 0.014; Fig. 4d) and from the THC + CYP group (p = 0.039; Fig. 4d). The vehicle group did not differ significantly from the THC + CYP group (p > 0.05; Fig. 4d). Co-treatment with CYP, therefore, reversed THC-induced inhibition of spontaneous MSN activity. A representative rastergram showing a typical inhibitory response to THC in the aNASh is shown in Fig. 4e. A sample neuronal rastergram from a THC + CYP treated rat are shown in Fig. 4f, showing the typical blockade of THC-induced neuronal inhibition.

THC increases spontaneous medium spiny neuronal activity in the posterior NASh through a KOR-dependent mechanism

For MSN neurons recorded in the pNASh (n = 14; see Fig. 5a for representative pNASh recording location), population analysis revealed that 14.3% of MSNs showed decreased activity, 50% increased and 35.7% showed no change, relative to baseline, following ICV THC administration (Fig. 5b). Thus, a plurality of pNASh MSN neurons show an inhibitory response to ICV THC administration. Analysis of average firing frequencies recorded 10 min pre vs. post ICV THC revealed that THC significantly increased firing rates relative to baseline (t (13) = −2.288, p = 0.04; Fig. 5c). Given our previous findings showing that the effects of pNASh THC were dependent upon a KOR-transmission substrate, we next sampled a total of n = 42 pNASh MSNs (Vehicle: n = 13, THC: n = 14, THC + nor-BNI: n = 15). Population analyses revealed that for rats receiving ICV vehicle, 58.3% of neurons showed no change, 16.6% increased and 25% showed decreased activity. For rats receiving ICV THC + nor-BNI, 60% of neurons showed no change, 13.3% increased and 26.6% showed decreased activity (Fig. 5b, far right side). Analyses of pre vs. post infusion activity rates for pNASh MSNs revealed average changes from baseline of −1.7% for rats treated with vehicle, +31.3% for rats treated with THC, and −9.3% with THC + CYP. ANOVA comparing groups revealed a significant main effect of treatment (F (2,38) = 4.085, p = 0.025; Fig. 5d). Post-hoc analysis revealed that rats treated with ICV THC showed significantly decreased activity relative to VEH controls (p = 0.043; Fig. 5d) and from the THC + nor-BNI group (p = 0.01; Fig. 5d). The vehicle group did not differ significantly from the THC + nor-BNI group (p > 0.05; Fig. 5d). Thus, co-treatment with nor-BNI reversed THC-induced increases in spontaneous MSN activity in the posterior NASh. A representative rastergram showing a typical excitatory response pattern to THC administration in the pNASh is shown in Fig. 5e. A representative neuronal rastergram from a THC + nor-BNI treated rat is shown in Fig. 5f, showing a typical blockade of THC-induced neuronal excitation following in the presence of the KOR antagonist.

Figure 5 Effects of ICV THC and nor-BNI on MSN activity patterns in the pNASh. (a) Representative microphotograph showing typical intra-pNASh in vivo MSN recording location. (b) Summary of experimental neuronal groups showing relative changes (no change, increase, or decrease) in firing frequencies following ICV pharmacological treatments. (c) ICV THC significantly increased spontaneous pNASh MSN neuronal firing frequency. (d) ICV THC alone (1 µg/μl) caused a significant increase in spontaneous aNASh MSN neuronal firing frequency rates vs baseline activity. This excitatory effect was reversed by co-administration of the behaviorally effective dose of nor-BNI (10 µg/μl). (e) Sample rastergram showing typical pNASh MSN response pattern following ICV THC (1 µg/µl) infusion (arrows indicate intra-NAc infusion event). (f) Sample rastergram showing typical aNASh MSN response pattern following ICV THC (1 µg/µl) and nor-BNI (10 µg/µl) infusion (arrows indicate intra-NAc infusion event). Full size image

THC produces differential changes in the power of high-frequency γ-oscillations

In the above described studies, LFPs were recorded concurrently with single-unit activity. The signal was divided into bins of 2 seconds and 410 different frequency values. An analysis was performed to determine the power each frequency had on the signal. A sample spectrograph of an aNASh LFP recording is shown in Fig. 6a. ANOVA comparing the power of high-frequency γ-oscillations between treatment groups revealed a significant main effect of treatment (F (2,35) = 3.963, p = 0.028; Fig. 6b). Post-hoc analyses revealed that rats treated with ICV THC showed significantly increased power of high-frequency γ-oscillations relative to VEH controls (p = 0.010; Fig. 6b) or rats treated with ICV THC + CYP (p = 0.045; Fig. 6b). The VEH group also did not differ significantly from the ICV THC + CYP group (p > 0.05; Fig. 6b). THC, therefore, increased the power of high-frequency γ-oscillations in the aNASh and this effect was reversed by co-treatment with a MOR antagonist.

Figure 6 Effect of ICV THC on the power of high-frequency gamma oscillations in the local field potential signal in the NASh. (a) Sample spectrograph showing typical high-frequency γ-oscillations in the aNASh following ICV infusion of THC (b) ICV THC significantly decreases the power of high-frequency γ-oscillations in the aNASh. *p < 0.05 from the other two groups. (c) Sample spectrograph showing typical high-frequency γ-oscillations in the pNASh following ICV infusion of THC (d) ICV THC significantly increases the power of high-frequency γ-oscillations in the pNASh. Full size image