Prenatal Ethanol Exposure Persistently Alters Endocannabinoid Signaling and Endocannabinoid-Mediated Excitatory Synaptic Plasticity in Ventral Tegmental Area Dopamine Neurons

Abstract

Prenatal ethanol exposure (PE) leads to increased addiction risk which could be mediated by enhanced excitatory synaptic strength in ventral tegmental area (VTA) dopamine (DA) neurons. Previous studies have shown that PE enhances excitatory synaptic strength by facilitating an anti-Hebbian form of long-term potentiation (LTP). In this study, we investigated the effect of PE on endocannabinoid-mediated long-term depression (eCB-LTD) in VTA DA neurons. Rats were exposed to moderate (3 g/kg/d) or high (6 g/kg/d) levels of ethanol during gestation. Whole-cell recordings were conducted in male offspring between 4 and 10 weeks old.We found that PE led to increased amphetamine self-administration. Both moderate and high levels of PE persistently reduced low-frequency stimulation-induced eCB-LTD. Furthermore, action potential-independent glutamate release was regulated by tonic eCB signaling in PE animals. Mechanistic studies for impaired eCB-LTD revealed that PE downregulated CB1 receptor function. Interestingly, eCB-LTD in PE animals was rescued by metabotropic glutamate receptor I activation, suggesting that PE did not impair the synthesis/release of eCBs. In contrast, eCB-LTD in PE animals was not rescued by increasing presynaptic activity, which actually led to LTP in PE animals, whereas LTD was still observed in controls. This result shows that the regulation of excitatory synaptic plasticity is fundamentally altered in PE animals. Together, PE leads to impaired eCB-LTD at the excitatory synapses of VTA DA neurons primarily due to CB1 receptor downregulation. This effect could contribute to enhanced LTP and the maintenance of augmented excitatory synaptic strength in VTA DA neurons and increased addiction risk after PE.SIGNIFICANCE STATEMENT Prenatal ethanol exposure (PE) is among many adverse developmental factors known to increase drug addiction risk. Increased excitatory synaptic strength in VTA DA neurons is a critical cellular mechanism for addiction risk. Our results show that PE persistently alters eCB signaling and impairs eCB-LTD at the excitatory synapses, an important synaptic plasticity that weakens synaptic strength. These effects combined with PE-induced anti-Hebbian long-term potentiation reported in a previous study could result in the maintenance of enhanced excitatory synaptic strength in VTA DA neurons, which in turn contributes to PE-induced increase in addiction risk. Our findings also suggest that restoring normal eCB signaling in VTA DA neurons could be a useful strategy for treating behavioral symptoms caused by PE.

Keywords: CB1 receptors; addiction risk; endocannabinoid-mediated long-term depression; long-term potentiation; synaptic homeostasis; ventral tegmental area dopamine neurons.

Figure 1.

Number of amphetamine infusions and responses (lever presses) in control and PE animals during six progressive ratio schedule self-administration sessions. Animals were first pretrained for lever pressing and went through fixed ratio training for amphetamine self-administration at 0.02 mg/kg/infusion. Increased infusions (A) and lever presses (B) were observed in PE animals indicating increased work output to obtain more amphetamine. Left, The number of infusions and lever presses at each session. Right, The average across 6 d. *p < 0.05, ***p < 0.001 indicate differences between control and PE animals.

Figure 2.

Prenatal ethanol exposure leads to reduced eCB-LTD of AMPA receptor-mediated EPSCs in VTA DA neurons. A, The induction of eCB-LTD via pairing low-frequency stimulation (2 Hz) and moderate membrane depolarization (−30 mV) led to a prominent reduction in evoked EPSC amplitude in control (○), but significantly less reduction in either moderate PE (3 g/kg/d ethanol; ▴), or high PE animals (6 g/kg/d ethanol; ○). B, Summary bar graph of the magnitude of eCB-LTD as percentage of baseline EPSC amplitude at time points indicated in A. The magnitude of eCB-LTD was profoundly reduced in both PE groups as indicated by reduced inhibition of EPSC amplitude. There were no differences between the moderate and high PE groups. Upper traces depict representative EPSC recordings in control (left) and PE animals before and after LTD induction at time points indicated in A. ***p < 0.001 difference between control and PE animals; #p < 0.05 difference before and after LTD induction.

Figure 3.

Prenatal ethanol exposure leads to presynaptic CB1 receptor downregulation in VTA DA neurons. A, Left, PE profoundly reduced the inhibitory effect of CB1 receptor agonist WIN 55,212-2 at 10 μm on evoked EPSC amplitude. Right, The summary graph of WIN 55,212-2 effect as percentage of baseline at time points indicated on the left. Upper traces depict representative EPSCs recorded at time points indicated on the left. B, Left, At a higher WIN 55,212-2 concentration (30 μm), there were no differences between control and PE animals in WIN 55,212-2-induced inhibitory effect. Right, Summary graph of WIN 55,212-2 effect as percentage of baseline at time points indicated on the left. Upper traces depict representative EPSCs recorded at time points indicated on the left. **p < 0.01 difference between control and PE animals.

Figure 4.

Prenatal ethanol exposure decreases evoked glutamate release in VTA DA neurons. A, Prenatal ethanol exposure increased the PPR (left) and CV (right) in VTA DA neurons, indicating a decrease in evoked glutamate release. B, Neither PPR (left) nor CV (right) was influenced by CP-AMPAR activation. These parameters were evaluated in a subset of neurons before and after bath application of NASPM (20 μm). NASPM did not alter PPR or CV in control or PE animals, indicating that CP-AMPAR-dependent short-term plasticity did not influence the values of PPR or CV. Increased PPR and CV in PE animals should be attributed to a reduction in evoked glutamate release. Upper traces depict representative EPSC recordings in conditions described in the bar graph below. *p < 0.05; **p < 0.01 indicate difference between control and PE animals.

Figure 5.

Prenatal ethanol exposure decreases action potential-independent glutamate release in VTA DA neurons. A, Representative mEPSCs recorded in the presence of TTX in VTA DA neurons in control and PE animals. B, Left, The mEPSC frequency was significantly lower in PE animals, indicated by a significant right shift in the cumulative probability curve of inter-event intervals (K–S test). Right, Bar graph showing group difference in mean mEPSC frequency can also be detected by t test. These results indicate that action potential-independent glutamate release was reduced in PE animals. C, The mEPSC amplitude was greater in VTA DA neurons recorded from PE animals, indicated by a significant right shift in the cumulative probability curve of mEPSC amplitude (K–S test). This effect is consistent with increased expression of CP-AMPARs which have larger conductance in PE animals. Right, Summary bar graph showing no group differences in mean amplitude were detected using t test. **p < 0.01; ***p < 0.001 indicate differences between control and PE animals.

Figure 6.

Prenatal ethanol exposure induces tonic eCB signaling to regulate action potential-independent but not evoked glutamate release. A, A lack of tonic eCB signaling on evoked EPSCs. Bath applied AM251, a CB1 receptor antagonist, did not alter evoked EPSC amplitude in either control or PE animals, indicating a lack of tonic eCB signaling regulating evoked glutamate release. Left, Normalized evoked EPSC amplitude before and after AM251 administration. Middle, Summary bar graph of normalized evoked EPSC after AM251 application. Right, PPR as percentage baseline was not altered by AM251 in control or PE animals. Upper traces depict recordings of PPR at time points indicated on the left. B, Left, mEPSC frequency was not altered by AM251 in control animals but increased in PE animals, indicated by a significant left shift in the cumulative probability of inter-event interval of mEPSC (K–S test). Upper traces depict representative mEPSC recordings. Right, Summary bar graph showing increased mean mEPSC frequency by AM251 in PE animals could also be detected by t test. C, Left, mEPSC amplitude was not altered by AM251 in control or PE animals indicated by a lack of shift in the cumulative probability of inter-event interval of mEPSCs. Right, Summary bar graph showing average mEPSC amplitude before and after AM251. ***p < 0.001 indicate difference before and after AM251 administration.

Figure 7.

Prenatal ethanol exposure does not impair metabotropic glutamate receptor 1 (mGluR1) activation-induced eCB-LTD. A, Left, Bath administration of mGluR1 agonist DHPG led to LTD induction, indicated by reduced amplitude of evoked EPSCs in control and PE animals. Right, Summary graph shows the averaged magnitude of mGluR-LTD did not differ between control and PE animals. Upper traces depict the representative EPSCs recorded at time points indicated on the left. B, Left, eCB signaling is required for mGluR-LTD. LTD induced by DHPG was completely blocked in the presence of CB1 receptor antagonist AM251. Right, Summary graph showing the average EPSC amplitude did not change after DHPG administration in the presence of AM251. Top, The representative EPSCs recorded at time points indicated on the left.

Figure 8.

Prenatal ethanol exposure promotes LTP in VTA DA neurons. A, When presynaptic stimulation frequency was increased from 2 to 5 Hz during eCB-LTD induction, LTD indicated by reduced EPSC amplitude was still observed in control animals, whereas robust LTP was observed in PE animals. This observation shows increasing presynaptic activity cannot rescue eCB-LTD. In addition, the excitatory synapses were prone to further strengthening in PE animals. B, Summary bar graph depicting the average EPSC amplitude collected at time points indicated in A. Upper traces show representative EPSCs at time points indicated in A. #p < 0.05, difference between two time points indicated in A; **p < 0.01, difference between control and PE groups.