Rolipram Attenuated Cocaine-Induced Locomotor Sensitization

We examined the effects of the PDE4 inhibitor rolipram on cocaine-induced locomotor sensitization in male (n=20) and female (n=19) mice. Mice were first allowed to habituate to the open-field chamber for 2 days. Female mice exhibited significantly higher basal locomotor activity than male mice (t 37 =5.4, p<0.001; Supplementary Figure 1a). Starting from day 3, the male and female mice were randomly divided into four groups, and the mice received daily injections of saline or cocaine (15 mg/kg, i.p.) for 5 days. Rolipram (1 mg/kg, i.p.) or vehicle was injected 30 min before cocaine or saline injection. Three-way ANOVA of locomotor activity from day 3 to day 7 indicated that cocaine produced significant increases in locomotor activity in both sexes, a significantly greater increase in locomotor activity in female mice than in male mice, and that rolipram pretreatments significantly attenuated the cocaine-induced increase in locomotor activity in both male and female mice; there was no significant interaction between rolipram and sex (Supplementary Figure 1b,c,d). The detailed statistics summary is shown in Supplementary Table 1.

Rolipram Pretreatments Blocked the Reduction of GABAergic Inhibition Induced by Repeated Cocaine Exposure In Vivo

Repeated cocaine exposure in vivo leads to a decrease in IPSCs in VTA dopamine neurons, and this reduced GABAergic inhibition may contribute to the increase in the excitability of these neurons (Bocklisch et al, 2013; Liu et al, 2005). Having shown that rolipram pretreatments attenuated cocaine-induced locomotor sensitization (Supplementary Figure 1), we examined whether spontaneous IPSCs (sIPSCs) were altered in VTA dopamine neurons in slices prepared from these mice. One day after the locomotor test, mice shown in Supplementary Figure 1 were killed and midbrain slices were prepared. sIPSCs were recorded from VTA dopamine neurons in slices from these eight groups of mice. Male and female mice did not exhibit significant differences of the mean frequency and amplitude of sIPSCs among corresponding groups (Supplementary Figure 2); the data from both sexes were pooled in each group for subsequent analysis. Two-way ANOVA showed that cocaine and rolipram treatments had significant effects on the mean frequency of sIPSCs (cocaine: F (1,57) =5.7, p=0.020; rolipram: F (1,57) =20.7, p<0.001; cocaine × rolipram interaction: F (1,57) =8.6, p=0.005; Figures 1a and b), and the mean amplitude of sIPSCs (cocaine: F (1,57) =6.9, p=0.011; rolipram: F (1,57) =6.4, p=0.014; cocaine x rolipram interaction: F (1,57) =4.7, p=0.035; Figures 1a and c). Tukey’s post hoc tests indicated that cocaine injections led to significant decreases in the mean frequency (p<0.001; Figure 1b) and amplitude of sIPSCs (p<0.001; Figure 1c) in vehicle-pretreated mice. The cocaine-induced decreases in the frequency and amplitude of sIPSCs were blocked in slices from mice that received rolipram pretreatment (p<0.001; Figures 1b and c). The cumulative distribution for inter-event intervals of sIPSCs was right-shifted in the vehicle/cocaine group compared to that in the vehicle/saline group, and this shift was blocked by rolipram pretreatment (Figure 1d). The cumulative distribution for the amplitude of sIPSCs was left-shifted in the vehicle/cocaine group, and this shift was blocked by rolipram pretreatments (Figure 1e). Together, these results indicate that repeated cocaine exposure in vivo led to decreases in sIPSC frequency and amplitude, and these decreases were blocked by rolipram pretreatments.

Figure 1 Rolipram pretreatments blocked the reduction of GABAergic inhibition to dopamine neurons induced by repeated cocaine exposure in vivo. (a), Representative sIPSCs recorded from VTA dopamine neurons in slices prepared from saline- or cocaine-injected mice pre-treated with vehicle or rolipram. (b, c) The averaged frequency (b) and amplitude (c) of sIPSCs in VTA dopamine neurons in these four groups of mice. The mean frequency and amplitude of sIPSCs were significantly decreased in cocaine-injected vehicle-treated mice (***p<0.001, n=15–16), and this decrease was blocked by rolipram pretreatments (***p<0.001, n=14–16). (d, e), The cumulative probability plots indicated that cocaine exposure led to shifts in the distribution of the inter-event intervals (d) and amplitude (e) in vehicle-treated mice; these shifts were blocked by rolipram pretreatments (p<0.001, n=13–16). PowerPoint slide Full size image

Acute Rolipram Perfusion Increased GABAergic Inhibition of VTA Dopamine Neurons

VTA dopamine neurons receive inhibitory synaptic inputs from local GABAergic neurons in the VTA as well as GABAergic axonal projections from the nucleus accumbens (NAc) (Bocklisch et al, 2013). We examined whether acute rolipram perfusion altered the strength of inhibitory inputs from VTA GABAergic neurons to VTA dopamine neurons by selectively expressing channelrhodopsin2 (hChR2) in GABAergic neurons. AAV DJ-EF1-DIO-hChR2-(H134R)-eYFP (denoted hChR2-eYFP for simplicity) was bilaterally microinjected into the VTA of Gad2-IRES-Cre mice. After recovery for 1 week, immunohistochemical staining indicated that the hChR2-eYFP was co-labeled with NeuN, a neuronal marker, but not with tyrosine hydroxylase (TH), a marker for dopamine neurons. The hChR2-eYFP was expressed in 95.1±4.6% of TH- neurons and was largely limited to the VTA (Supplementary Figure 3), indicating robust targeting of non-dopaminergic, putative GABAergic neurons in the VTA (Figures 2a, n=3 mice/group). hChR2-eYFP was expressed in axonal terminals in the NAc, which is consistent with long-range GABA projections from the VTA GABAergic neurons to NAc (Brown et al, 2012). Importantly, we did not observe any hChR2-eYFP expression in the somata of NAc neurons as shown by Nissl co-staining (Supplementary Figure 4). The lack of retrograde hChR2-eYFP expression suggests that optogenetic stimulation activates hChR2 expressed on local VTA GABAergic neurons.

Figure 2 Optogenetic interrogation of rolipram mechanisms on inhibitory synaptic plasticity in VTA dopamine neurons. (a), Expression of AAV DJ-EF1-DIO-hChR2(H134R)-eYFP in VTA GABAergic neurons in Gad2-IRES-Cre mice following intra-VTA viral injection. The majority of tyrosine hydroxylase negative (TH−) neurons in the VTA were infected with the AAVs. (b), Laser stimulation (473 nm, 1 ms pulse) induced an inward current in VTA dopamine neurons, which was abolished by the GABA A receptor blocker picrotoxin (p<0.001, n=3). Representative recordings of the laser-evoked currents at the indicated membrane potentials. Linear regression fit of I–V curve indicates the reversal potential of laser-evoked IPSCs is −21 mV (n = 7). (c), Bath perfusion of rolipram induced a significantly greater increase in the amplitude of laser-evoked IPSCs in slices from cocaine-injected mice than in saline-injected mice (p=0.031, n=6–7). PowerPoint slide Full size image

We first recorded IPSCs in VTA dopamine neurons evoked by optogenetic stimulation of axonal terminals from local GABAergic neurons. Dopamine neurons were identified by electrophysiological properties as well as the lack of eYFP fluorescence. Single laser pulses (473 nm, 1 ms duration) induced an inward current in VTA dopamine neurons at −60 mV, which was abolished by the GABA A receptor blocker picrotoxin (50 μM, n=3; Figure 2b). The currents were voltage-dependent and had a reversal potential of -20.5±3.6 mV (n=7; Figure 2b), consistent with the calculated E Cl − (−21.0 mV) for the high-Cl− (54 mM) internal solution we used (Supplementary Materials and Methods). Thus, laser stimulation induced GABA A receptor-mediated IPSCs in VTA dopamine neurons. IPSCs evoked by electrical stimulation had a similar reversal potential (−20.9±3.8 mV; n=8), and were blocked by picrotoxin (n=2; Supplementary Figure 5a). We also made recordings from eYFP+ GABAergic neurons in the VTA. The same single laser pulses induced currents that showed strong inward rectification, reversed at ~0 mV and were not altered by picrotoxin (n=2, Supplementary Figure 5b). Thus, these currents are mediated by direct activation of hChR2 expressed on GABAergic neurons.

We examined whether acute application of rolipram altered laser-evoked IPSCs in slices prepared from mice that received five daily saline or cocaine injections in vivo. Bath perfusion of rolipram (10 μM) produced significant increases in the amplitude of laser-evoked IPSCs in slices prepared from saline-injected mice (t 10 =2.8, p=0.020) and cocaine-injected mice (t 12 =5.8, p<0.001; Figure 2c; Supplementary Figure 6). The IPSC amplitude from cocaine-exposed animals was significantly greater than that of saline-exposed animals after the application of rolipram (t 11 =2.5, p=0.031; Figure 2c). Thus, application of rolipram produces greater potentiation of IPSCs in slices prepared from cocaine-injected mice than that in saline-injected mice. These results suggest that in vivo cocaine exposure, which suppresses inhibitory inputs onto VTA dopamine neurons, permits greater PDE4 inhibition-induced potentiation compared to saline exposure.

Effects of Cocaine Exposure and Rolipram Pretreatments on the AMPAR/NMDAR Ratio in VTA Dopamine Neurons

We determined whether the AMPAR/NMDAR ratio was affected in mice that received saline or cocaine injections with rolipram or vehicle pretreatments. A new cohort of mice received 5 daily injections of saline or cocaine (15 mg/kg, i.p.). Rolipram (1 mg/kg, i.p.) or vehicle was injected 30 min before cocaine or saline injection. One day after locomotor activity was assessed, the mice were euthanized and midbrain slices were prepared. The AMPAR/NMDAR ratio was measured in VTA dopamine neurons prepared from these eight groups of mice. There were no significant sex differences of the AMPAR/NMDAR ratio between corresponding groups (Supplementary Figure 7), so the data from both sexes were pooled in each group. We found that cocaine (F (1,43) =21.0, p=0.001) and rolipram (F (1,43) =18.2, p<0.001) had significant effects on the AMPAR/NMDAR ratio, and there was a significant interaction between cocaine and rolipram (F (1,43) =19.4, p<0.001; Figure 3a). Tukey’s post hoc tests indicated that repeated cocaine injections led to a significant increase in the AMPAR/NMDAR ratio compared with saline injections (p<0.001; Figure 3a). Interestingly, compared with the vehicle group, rolipram pretreatments alone increased the AMPAR/NMDAR ratio in saline-injected mice (p<0.001) but did not cause any further increase in the AMPAR/NMDAR ratio in cocaine-injected mice (p=0.871; Figure 3a). Together, these results indicate that rolipram pretreatments alone increase the AMPAR/NMDAR ratio and do not further increase the cocaine-induced potentiation in the AMPAR/NMDAR ratio in VTA dopamine neurons.

Figure 3 Effects of cocaine exposure and rolipram pretreatments on the AMPAR/NMDAR ratio in VTA dopamine neurons. (a) Representative AMPAR- and NMDAR-mediated evoked EPSCs recorded from VTA dopamine neurons. Compared with vehicle, rolipram pretreatments increased the AMPAR/NMDAR ratio in slices from saline-injected mice (***p<0.001, n=11) and did not further increase the AMPAR/NMDAR ratio in slices from cocaine-injected mice (p=0.871, n=11-12). (b) Acute rolipram perfusion caused a significantly greater increase in the amplitude of evoked EPSCs in slices from saline-injected mice than that in cocaine-injected mice (p=0.024, n=6–7). The effect of rolipram in slices from saline- or cocaine-injected mice was blocked by the PKA inhibitor H89 (saline, p<0.001, n=7; cocaine, p=0.011, n=6). PowerPoint slide Full size image

To investigate potential mechanisms involved, we investigated the effects of acute bath application of rolipram on evoked EPSCs. Mice received five daily saline or cocaine injections without vehicle or rolipram pretreatment. One day after the last saline or cocaine injection, mice were euthanized and midbrain slices were prepared. Whole-cell recordings of dopamine neurons voltage-clamped at −60 mV were performed. In slices prepared from saline-injected mice, bath application of rolipram significantly increased the amplitude of evoked EPSCs (t 12 =5.5, p<0.001; Figure 3b; Supplementary Figure 8). In slices prepared from cocaine-injected mice, rolipram application also induced a significant increase in the amplitude of evoked EPSCs (t 10 =2.9, p=0.016; Figure 3b; Supplementary Figure 8), with smaller EPSC potentiation relative to saline-treated animals (t 11 =2.6, p=0.024; Figure 3b). The effects of rolipram in both groups were blocked by the PKA inhibitor H89 (saline, 89.1±2.7%, t 12 =6.3, p<0.001; cocaine, 95.7±6.3%, t 10 =3.1, p=0.011; Figure 3b), suggesting that rolipram increased EPSCs via cAMP-PKA signaling. Thus, repeated cocaine injections in vivo induces synaptic potentiation of glutamatergic inputs onto VTA dopamine neurons, and rolipram pretreatments do not produce further potentiation of excitatory synaptic inputs.

Rolipram Pretreatments Blocked Cocaine-Induced Disruption of Excitatory/Inhibitory Balance

We determined whether repeated cocaine exposure and rolipram pretreatments in vivo altered the balance of excitation and inhibition in VTA dopamine neurons. Mice received 5 daily injections of saline or cocaine (15 mg/kg, i.p.). Rolipram (1 mg/kg, i.p.) or vehicle was injected 30 min before each cocaine or saline injection. One day after the behavioral test, the mice were euthanized and midbrain slices were prepared. We recorded evoked EPSCs and IPSCs sequentially from the same VTA dopamine neurons in slices prepared from these four groups of mice. The NMDAR antagonist CPP (5 μM) was present throughout the experiment. VTA dopamine neurons were voltage-clamped alternatively at the reversal potential for IPSCs (−60 mV) and EPSCs (0 mV) to isolate EPSCs and IPSCs, respectively. Indeed, IPSCs recorded at 0 mV were abolished by the GABA A receptor blocker picrotoxin (50 μM), while EPSCs recorded at −60 mV were abolished by the AMPAR antagonist CNQX (20 μM; Figure 4a). We next determined whether cocaine and rolipram treatments altered the E/I ratio in these four groups of mice. Two-way ANOVA indicated that cocaine and rolipram pretreatments had significant effects on the E/I ratio (cocaine: F (1,37) =12.7, p=0.001; rolipram: F (1,37) =8.9, p=0.005; cocaine × rolipram interaction: F (1,37) =10.0, p=0.004; Figures 4b and c). Tukey’s post hoc tests indicated that the E/I ratio was significantly increased in the vehicle/cocaine group compared to the vehicle/saline group (p<0.001; Figure 4c). The increase in E/I ratio induced by in vivo cocaine exposure was blocked by rolipram pretreatment (p<0.001; Figure 4c). Thus, rolipram pretreatment prevents the disruption of excitatory/inhibitory balance in VTA dopamine neurons induced by repeated cocaine exposure.

Figure 4 Rolipram pretreatments restored the cocaine-induced imbalance of excitation and inhibition in the VTA. (a) EPSCs and IPSCs were isolated by voltage-clamping VTA dopamine neurons at the reversal potentials of IPSCs (−60 mV) and EPSCs (0 mV), respectively. EPSCs were blocked by the AMPAR antagonist CNQX (n=3), while IPSCs were blocked by the GABA A receptor blocker picrotoxin (n=3). (b) Sample EPSCs and IPSCs recorded in slices from saline- or cocaine-injected mice that received vehicle or rolipram pretreatments. (c) Repeated cocaine injections led to an increase in the E/I ratio (***p<0.001, n=10–11). This increase was blocked by rolipram pretreatments (***p<0.001, n=9-11). PowerPoint slide Full size image

Rolipram Pretreatment Blocked Cocaine-Induced Increase in In Vivo Action Potential Firing in VTA Dopamine Neurons

Previous studies have shown that repeated cocaine exposure in vivo leads to increased excitability in VTA dopamine neurons (Bocklisch et al, 2013; Liu et al, 2005). We examined whether cocaine-induced behavioral sensitization was associated with changes in action potential firing in VTA dopamine neurons in vivo. One day after the locomotor activity tests, mice were anesthetized with urethane and in vivo single-unit recordings were performed. Dopamine neurons were identified by firing characteristics (see Materials and Methods) and were validated by juxtacellular labelling with neurobiotin and post hoc TH immunostaining (Chaudhury et al, 2013; Ungless et al, 2004) (Figures 5a and b). We found that cocaine exposure and rolipram pretreatments had significant effects on the frequency of action potential firing (cocaine: F (1,66) =27.4, p<0.001; rolipram: F (1,66) =5.4, p=0.024; cocaine x rolipram interaction: F (1,66) =21.2, p<0.001; Figure 5c), and percentage of spikes in bursts in VTA dopamine neurons (cocaine: F (1,43) =12.5, p=0.001; rolipram: F (1,43) =4.5, p=0.040; cocaine x rolipram interaction: F (1,43) =18.1, p<0.001; Figure 5d). Tukey’s post hoc tests indicated that cocaine exposure significantly increased the frequency of action potential firing and the percentage of spikes in bursts in vehicle-pretreated mice (both p<0.001). These increases were blocked by rolipram pretreatments (p>0.05). There was no significant difference in the firing frequency and the percentage of spikes in bursts between vehicle- and rolipram-pretreated mice that received saline injections (p>0.05). Cocaine exposure and rolipram pretreatments had no significant effects on the proportion of dopamine neurons showing burst firing (cocaine: F (1,15) =0.2, p=0.705; rolipram: F (1,15) =0.4, p=0.525; cocaine x rolipram interaction: F (1,15) =4.0, p=0.068; Figure 5e). Thus, cocaine locomotor sensitization was accompanied by an increase in the activity of VTA dopamine neurons, and this increase was blocked by rolipram pretreatments.