CRF potentiates GABA A receptor-mediated IPSCs via presynaptic CRF-R1

We recorded stimulus-evoked GABA A -mediated inhibitory postsynaptic currents (eIPSCs) from VTA dopamine neurons in acute brain slices from C57BL-6J mice. The concentration dependence of two non-selective agonists (CRF and UCN) and one CRF-R2 agonist (UCN-3) were evaluated after 10 min of bath application, over a range of 10–300 nM (Fig. 1a). Both CRF and UCN potentiated the amplitude of eIPSCs with near maximal effects at 200 nM (Fig. 1a; CRF: t (12) = 20.19, p < 0.0001 relative to baseline; UCN: t (10) = 26.01, p < 0.0001 relative to baseline). UCN-3 had no measurable effect on eIPSCs (Fig. 1a, 200 nM UCN-3: t (8) = 1.148, p = 0.1884 relative to baseline). The half-maximal effective concentration (EC 50 ) for CRF and UCN was 34 nM and 41 nM, respectively (Fig. 1a). At 300 nM, CRF also caused a persistent inward current (8.1 ± 3.1 pA), consistent with previous reports [44]. At the end of the experiments, the GABA A receptor antagonist picrotoxin (picro, 100 µM) was applied to confirm the specificity of GABA A receptor-mediated currents (Fig. 1a).

Fig. 1 Presynaptic CRF-1 receptors potentiate GABA A IPSCs in VTA DA neurons. a Traces of stimulus-evoked, picrotoxin-sensitive IPSCs showing potentiation by CRF (200 nM) and urocortin (UCN; 200 nM), but not the CRF-R2 agonist UCN-3 (300 nM). For the timeline, the amplitude of the evoked current was normalized for each cell using the mean amplitude recorded during the first 5 min and plotted as function of time (% of baseline, mean ± SEM). Although CRF (200 nM) and UCN (200 nM) potentiated IPSCs, UCN-3 did not. The smooth curves in the agonist concentration–response plot are the best fit to the data by the logistic equation. b CRF (300 nM) increased the amplitude of the first eIPSC (S1), with little measurable change in the second eIPSC (S2). Inset shows the first eIPSCs (S1) normalized to the second eIPSC (S2). CRF significantly decreased the paired pulse ratio (PPR; IPSC2/IPSC1, ***p < 0.001. c CRF (300 nM) enhances the frequency of spontaneous IPSCs (sIPSCs). Inset shows magnification of sIPSCs. Both UCN and CRF increased the frequency of sIPCS relative to their baseline (***p < 0.001). d, e The CRF (300 nM) potentiation is blocked by co-application of (e) CRF-R1 antagonists (CP154156 (CP154) or antalarmin (AA), but not (d) CRF-R2 antagonists (K41498 (K414) or astressin-2B (A2B) (*p < 0.05). f The CRF (300 nM) shift in the PPR is reduced by CRF-R1 blockers (CP154 or AA), but not CRF-R2 blockers (K414 or A2B). One-way ANOVA, *p < 0.05 vs CRF alone (Dunnett’s post test). g The CRF (200 nM) enhancement of sIPSC frequency is prevented by co-application of CRF-R1 blockers (CP154 or AA), but not CRF-R2 blockers (K414 or A2B). One-way ANOVA, ***p < 0.001 vs CRF alone (Dunnett’s post test). a–g Number of cells/mice per group: a CRF 13/7; UCN 11/6, UCN-3 9/4 for both timelines and concentration–response curves; b 7/4; c 7/5, d, e K414 9/5, A2B 9/5, CP154 8/4, AA 7/3; f 7/3-4 per group; g CRF+K414 7/4, and the following groups contained 8-9/4-5: UCN+A2B, CRF+A2B, CRF+CP154, and CRF+AA Full size image

To assess whether the CRF acted at a pre- or postsynaptic site, we recorded paired pulse ratios (PPRs, IPSC2/IPSC1, Fig. 1b). PPRs correlate inversely with release probability [45]. CRF (300 nM) increased the amplitude of the first eIPSC (Fig. 1b, S1, paired t-test t (6) = 9.625, p < 0.0001), with little measurable change in the second eIPSC (Fig. 1b, S2, paired t-test t (6) = 0.7661, p = 0.2663). This resulted in a significant decrease in the PPR in the presence of CRF (Fig. 1b inset, paired t-test, t (6) = 8.2601, p = 0.0002), thus suggesting a presynaptic locus for CRF actions on GABA A eIPSCs.

As other modifications of short-term plasticity can alter the PPR independent of changes in release probability [46], we confirmed a presynaptic locus of CRF action measuring the frequency and amplitude of sIPSCs and mIPSCs. CRF or UCN increased the sIPSC frequency in all cells tested (Fig. 1c; CRF: t (7) = 4.567, p = 0.0003; UCN: t (7) = 4.324, p = 0.0003). Similar increases were observed with CRF on mIPSC recordings (medium containing 200 nM TTX; CRF: 68.8% ± 4.26% increase relative to baseline; t (7) = 5.885, p = 0.0023). CRF did not alter sIPSC or mIPSC amplitudes (sIPSCs: 5.56% ± 4.6%, t (14) = 0.03597, p = 0.4860; mIPSCs: 2.94% ± 11.13%, t (7) = 0.5172, p = 0.3105). Taken together, these results indicate a presynaptic action of CRF on GABA synapses on to VTA DA neurons.

CRF potentiation of GABA A receptor-mediated IPSCs requires CRF-R1

To assess whether the CRF facilitation of GABA A currents was mediated by CRF-R1 or -R2, CRF was applied in the presence of blockers for CRF-R1 (300 nM CP154156, CP154; 200 nM antalarmin (AA)) or CRF-R2 (300 nM K41498, K414; 200 nM astressin-2B (A2B)). Slices were incubated with antagonists 10 min prior to and during CRF challenges. The resulting changes in evoked currents, the PPR, and the frequency of sIPSCs was examined.

Neither of the CRF-R2 blockers, K414 or A2B, prevented the CRF potentiation of eIPSCs (Fig. 1d; CRF + K414: t (6) = 3.564, p = 0.0121; CRF + A2B: t (7) = 4.234, p = 0.0223). However, co-application of the CRF-R1 antagonists CP154 or AA blocked the CRF increase of eIPSCs (Fig. 1e; CRF + CP154 t (5) = 0.03185, p = 0.5241; CRF+AA: t (7) = 0.02354, p = 0.7123). To verify that these effects were mediated by presynaptic CRF-R1, we evaluated the PPR (Fig. 1f) and sIPSC frequency (Fig. 1g) in the presence of selective CRF receptor antagonists. A one-way between-subject ANOVA evaluating the effects of the four antagonists on the CRF-mediated changes in the PPR (Fig. 1f) or in the frequency of sIPSC (Fig. 1g) indicated significant differences at the p < 0.05 level (PPR F (4, 22) = 3.884, p = 0.0156; frequency F (4, 64) = 13.16, p < 0.0001). Separate post hoc tests (multiple comparisons Dunnett’s) revealed that the CRF changes in the PPR (Fig. 1f) were absent when blocking CRF-R1 with CP154 (p = 0.9997) or AA (p = 0.9745), but were observed during co-application of the CRF-R2 blockers K414 (p = 0.0137) or A2B (p = 0.0413). Post hoc tests revealed that the CRF changes in frequency (Fig. 1g) followed the same pattern: CRF changes were absent when blocking CRF-R1 with CP154 (p = 0.9831) or AA (p = 0.7991), but observed during co-application of the CRF-R2 blockers K414 (p < 0.0001) or A2B (p < 0.001). Thus, presynaptic CRF-R1 receptors regulate GABA release.

The possibility that endogenous release of CRF tonically activates CRF receptors was also tested using selective antagonists. Application of either of the CRF-R1 or CRF-R2 antagonists alone, without CRF, produced no measurable change in eIPSCs (one-way ANOVA, F (4, 41) = 0.3584, p = 0.8367), nor in the frequency of sIPSCs (one-way ANOVA F (4, 30) = 0.0194, p = 0.9618), suggesting the absence of a measurable tone on CRF receptors in slices prepared from naive mice. Together, the results from the PPR studies and sIPSC/mIPSC recordings are consistent with a presynaptic action of CRF via CRF-R1 on GABA synapses on to VTA DA neurons.

CRF-R1 acts through PKC and calcium-induced calcium release

CRF receptors couple to the Gs alpha subunit to activate adenylyl cyclase, as well as the Gq alpha subunit to activate PLC–PKC [47]. To better understand the intracellular pathway regulating the CRF potentiation of GABA onto dopamine (DA) cells, we evaluated eIPSCs during co-manipulation of CRF-R1 and the PKC enzyme.

As previously shown, CRF increased the amplitude of eIPSCs (Fig. 2a; t (9) = 4.6327, p = 0.0143) and these responses were further potentiated by phorbol-ester-dybutyrate (PDBU), an activator of PKC (Fig. 2a; t (7) = 4.1032, p = 0.0178). However, if PDBU (1 µM) was applied first, application of CRF (300 nM) produced no additional increase in eIPSC amplitude (Fig. 2b). The lack of a CRF action in the presence of PDBU was not due to a ceiling effect of maximal chloride conductance, because at the end of the experiment a more intense synaptic stimulation (~35%) further increased the inward current (158.4% ± 51.6% relative to baseline, t (2) = 16.99, p = 0.0017). The PDBU potentiation of eIPSC amplitude was not reduced by the CRF-R1 antagonist K41498 (PDBU vs PDBu + K414: 0.56% ± 11.7%, t (5) = 0.3577, p < 0.3676), suggesting an activation of PKC activity downstream of CRF-R1.

Fig. 2 The CRF potentiation of GABA A receptor IPSCs requires PKC and intracellular calcium stores. a The CRF (200 nM) potentiation of eIPSCs is further increased by the PKC activator Phorbol 12,13-dibutyrate (PDBU; 1 µM). Bath incubation of slices with the PKC inhibitor chelerythrine (Chele, 1 µM), blocked the actions of CRF (200 nM) and PDBU (1 µM), whereas inclusion of the cell impermeable PKC inhibitor PKC 19-36 (300 nM) in the internal recording solution did not. The timeline shows the eIPSC amplitude as a percentage of pre-drug baseline ± SEM, normalized for each cell using the mean amplitude recorded during the first 5 min of pre-drug application and plotted as a function of time. b Application of PDBU (1 µM) prior to CRF (200 nM) reduces the CRF potentiation of eIPSCs. c Incubation of brain slices with the PKA inhibitor (H-89: 10 µM; incubation > 30 min) did not prevent the CRF potentiation of eIPSCs. d Summary bars showing pharmacological manipulation of calcium stores reduces the actions of CRF. Drugs include antagonists/inhibitors of: Ca2+-ATPases [cyclopiazonic acid (CPA 10 µM), thapsigargin (Thaps, 5 µM)], ryanodine receptors (ryanodine, 50 µM), IP3 receptors (2-aminoethoxydiphenyl-borate (2-APB, 10 µM)); voltage-dependent calcium channels (cadmium, Cad 300 µM). Also tested is caffeine (caff, 10 mM), an agonist of ryanodine receptors used to deplete intracellular calcium stores. CRF (300 nM) data in bar graph are duplicate from Fig. 1c, shown again for comparison. One-way ANOVA, ***p < 0.001 vs CRF alone (Dunnett’s post test). a–d Number of cells/mice per group: a control 10/5, Chele 15/7, PKC 19-36 8/4; b 6/3; c 10/5; d CRF 15/7, CRF+CPA 8/4, CRF+Thaps 5/3, CRF+ryanodine 6/4, CRF+2-APB 5/3, CRF+caff, 6/3, Cad 8/4 Full size image

To corroborate that PKC may be involved in this response, we performed similar experiments in the presence of various PKC or PKA inhibitors. Experiments performed using an internal solution containing the selective, non-membrane permeable pseudo-substrate blocker of PKC, PKC 19-36 (5 µM internal solution; infusion >20 min) [48] showed that CRF still potentiated eIPSCs, suggesting an action independent of postsynaptic PKC (Fig. 2a; t (5) = 4.2340, p = 0.0117). However, treatment of slices with chelerythrine (chele: 1 μM, >30 min), which has been reported to inhibit the catalytic subunit of PKC [49, 50], did block the effects of both CRF and the PKC activator PDBU on eIPSCs (Fig. 2a; CRF+chele t (7) = 0.03016, p = 0.5781) [49]. Although this suggests that CRF may enhance GABA A IPSCs via a presynaptic PKC-dependent mechanism, chelerythrine is not universally accepted to be a PKC inhibitor and has other actions unrelated to PKC [51,52,53,54]. So, caution is warranted. However, we noted that incubation with the membrane permeant PKA inhibitor H89 (10 μM; >30 min incubation; present during recording) did not prevent the CRF potentiation of IPSCs (Fig. 2c; t (4) = 4.0124, p = 0.0223). If indeed the CRF-R1 action in mice is PKC dependent, then this would differ from the CRF-R2/PKA-dependent mechanism we previously observed in rats [34].

Neuropeptides can regulate transmitter release by a kinase-dependent mobilization of calcium release from intracellular stores [47]. Accordingly, we observed that depletion of stores by incubating slices (>15 min) with cyclopiazonic acid (CPA; 10 µM) reduced the CRF facilitation of mIPSC frequency (29.2% ± 2.84% decrease relative to pre-CPA; t (9) = 10.89, p < 0.0001), but not the amplitude (mIPSC amplitudes 9.26% ± 13.12% decrease relative to baseline, t (9) = 0.5246, p = 0.3063). As the frequency of the recordable mIPSCs decreased with CPA, we confirmed the results by also measuring sIPSCs. Slices were treated with CPA, thapsigargin (5 µM), ryanodine, caffeine, or 2-APB (Fig. 2d)—drugs that either block IP3/ryanodine receptors or deplete calcium stores [55,56,57]. Because the frequency of sIPSCs varied considerably between slices/cells even under control conditions without antagonist (range 4–8 Hz, mean 4.39 ± 0.43 Hz), we determined the CRF-related increase in the frequency within cells before CRF (in the presence of the antagonist) and during CRF. A one-way between-subject ANOVA comparing CRF responses in the presence of the antagonists indicated significant differences (Fig. 2d; F (6, 45) 16.21, p < 0.0001). Post hoc tests (multiple comparisons Dunnett’s) indicated the change was significant for all the compounds (Fig. 2d; p < 0.0001 all drugs, except cadmium; cadmium, p < 0.006). A summary of the frequency and amplitude of sIPSCs with different treatments is shown in Table 1. The observation that these different treatments all reduced the CRF action on sIPSCs (Fig. 2d), indicates a need for functional calcium stores. It was notable that treatment with the non-selective calcium channel blocker cadmium reduced, but did not block, the CRF action on sIPSCs (Fig. 2d). Thus, although CRF facilitation does not require extracellular calcium, calcium entering through voltage-gated calcium channels likely potentiates the calcium release from intracellular stores (calcium-induced calcium release) [55].

Table 1 Frequency (Hz) and amplitude (pA) of sIPSCs Full size table

Reduced CRF and EtOH effects on GABA A currents after long-term withdrawal from CIE exposure

Similar to other brain regions, ethanol can enhance GABA release onto VTA dopamine cells [58, 59]. Using VTA slices from naive mice, we measured the frequency of GABA A IPSCs during ethanol application (50 mM; 10 min) and confirmed that the potentiation also occurs under our recording conditions (Fig. 3a; t (5) = 5.733, p = 0.0023). The ethanol action may require CRF-R1 and the release of calcium from intracellular stores, as observed in other brain regions [37], and so we wondered if this also occurs in the VTA. To evaluate this, we measured the ethanol enhancement of sIPSC frequency in slices from naive mice during bath application of: (1) ethanol, (2) ethanol plus the CRF-R1 antagonist CP154, or (3) ethanol plus CRF. A one-way ANOVA comparing responses under these conditions indicated no significant differences (Fig. 3a; F (2, 32) = 0.1467, p = 0.8644). The absence of an additive effect of CRF on ethanol or any reduction in the ethanol action by the CRF-R1 antagonist suggests that the “stress-like” actions of ethanol may reflect intracellular alterations somewhere downstream of the CRF-R1 receptor.

Fig. 3 Reduction in responses to CRF or ethanol following long-term withdrawal from chronic intermittent ethanol (CIE) exposure. a In neurons from naive mice, bath application of CRF (300 nM) or ethanol (EtOH; 50 mM) increases the sIPSC frequency relative to their baseline to a similar extent. Ethanol responses are not significantly altered by co-application of the CRF-R1 antagonist CP154156 (CP154; 300 nM; incubation > 12 min) or CRF (CRF+EtOH). The CRF traces are shown for comparison. See Fig. 1c for average CRF data; ***p < 0.001 compared to baseline. b Schematic showing experimental timeline for chronic intermittent ethanol (CIE) exposure. Habituation (5 days) was followed by vapor chamber exposure to ethanol (or air for controls) over 4 cycles (16 h per day; 4 days per week). Blood ethanol concentrations (BEC) were measured after each cycle and averaged ~210 mg/dl—a concentration sufficient to produce tolerance and dependence (see Materials and methods). After the final air/CIE treatment, mice were returned to the home cage. Brain slices were later prepared to compare physiological responses after a period of short-term withdrawal (ST; 3 days after treatment) or long-term withdrawal (LT; pooled responses from 20, 30 and 45 days after treatment and reported as a group). c Sample sIPSC traces from LT-air (top) and LT-CIE (bottom) cells before (baseline) and during bath application of CRF (300 nM) or ethanol (EtOH; 50 mM). d Summary graph showing differences in baseline frequency of sIPSC in ST and LT withdrawal groups without application of CRF or ethanol. One-way ANOVA, ***p < 0.001 ST-CIE vs LT-CIE (Dunnett’s post test). e During ST withdrawal, CRF (200 nM) and ethanol (EtOH; 50 mM) enhance sIPSC frequency similar to control levels. f During LT withdrawal, the CRF (200 nM) and EtOH (50 mM) action is reduced in CIE group relative to controls. Two-way ANOVA, p < 0.0001 for effect of treatment. a–f Number of cells/mice per group: a EtOH 6/4, CP154+EtOH 6/4, CRF+EtOH 7/5; d ST-air 5/4; ST-CIE 6/5, LT-air 5/4, LT-CIE 11/6; e ST-air-CRF 18/9; ST-CIE-CRF 13/6, ST-air-EtOH 10/5, ST-CIE- EtOH 17/8; f LT-air-CRF 18/9; LT-CIE-CRF 11/6, LT-air-EtOH 10/7, LT-CIE-EtOH 9/6 Full size image

CIE exposure produces dependence and robustly activates CRF stress systems [31, 39, 41]. So, we predicted that CIE treatment (Fig. 3b) would also alter the GABA A responses to CRF and ethanol. To test this, we prepared VTA slices from mice to evaluate the air or CIE treatments under two conditions: short-term (ST) withdrawal of 72 h or a long-term (LT) withdrawal of 20–45 days. We recorded basal frequency of sIPSCs (in Hz) in the ST and LT groups (Fig. 3c, d) and found a significant interaction between treatment and time (two-way ANOVA, F (1, 21) = 4.457, p = 0.0469; post hoc Dunnett’s multiple comparison test: ST- vs LT-CIE p = 0.0007, ST- vs LT-air p = 0.7219). A summary of the frequency and amplitude of sIPSCs with different treatments is shown in Table 1. This suggests that the CIE-induced change in basal release of GABA is temporary: During the ST withdrawal periods, the frequency of sIPSCs increase, whereas during LT withdrawal periods, the spontaneous release of GABA decreases toward control levels.

It was not clear whether the decrease in sIPSCs represented an active change or simply recovery from the acute effects of CIE. On this basis, we examined the actions of CRF and ethanol on sIPSCs starting with the ST withdrawal group. A two-way ANOVA revealed no significant differences in response to CRF or ethanol for sIPSCs (Fig. 3e: treatment (F (1, 20) = 3.435, p = 0.0786), drug (F (1, 20) = 0.0016, p = 0.9682); interaction of treatment × drug (F (1, 20) = 1.14, p = 0.2984)), nor for eIPSCs (treatment (F (1, 20) = 0.3054, p = 0.5866), drug (F (1,20) = 0.1761, p = 0.6792); interaction of treatment × drug (F (1, 20) = 0.0026, p = 0.9625)). This suggests that during ST withdrawal, the mechanisms that facilitate GABA release remains functional. However, a similar comparison after LT withdrawal did reveal differences in the ability of CRF and ethanol to alter the frequency of sIPSCs (Fig. 3c, f). For sIPSCs, a two-way ANOVA showed a significant effect of treatment (F (1, 17) = 142.2, p < 0.0001), but not drug (F (1, 17) = 0.8148, p = 0.3793) or interaction (F (1, 17) = 1.15, p = 0.2986). As a point of control, additional experiments were performed measuring eIPSCs and a two-way ANOVA of eIPSCs also revealed an effect of treatment (F (1, 17) = 10.64, p < 0.0046), but not drug (F (1, 17) = 0.03609, p = 0.8516 or interaction (F (1, 17) = 0.1804, p = 0.6764). Thus, the actions of CRF and alcohol on the release of GABA appear similar, but measurably change following LT withdrawal from CIE treatments.

CB1-mediated inhibition opposes the actions of CRF

To determine the signaling mechanism underlying the blunted CRF and ethanol modulation of GABA A currents after 20–45 days of withdrawal from CIE treatment, we retested the response to CRF and the PKC activator PDBU using eIPSCs, as they are easily discernable due to their large size. While CRF and PDBU responses in LT-air mice were robust (Fig. 4a; CRF: t (6) = 3.3810, p = 0.0221, PDBU: t (4) = 03.6542, p = 0.0323) and resembled those in naive tissue as noted previously, no increase was observed in response to PDBU in slices from LT-CIE mice (Fig. 4a; CRF: t (6) = 0.3415, p = 0.3524, PDBU: t (5) = 0.2153, p = 0.2972). Although acute somatic withdrawal from alcohol is known to increase the synthesis of CRF [60], no evidence of a ceiling effect, nor a CRF tone, was observed, as application of the CRF-R1 antagonist alone produced no significant change in eIPSCs during LT withdrawal (CP154156 (300 nM): 4.95 ± 1.98% increase relative to baseline; t (3) = 0.6470, p = 0.2819). Thus, during LT withdrawal, CRF-R1 did not seem to be tonically activated and some other functional adaptation must emerge in the weeks following chronic ethanol exposure that limits the ability of CRF, ethanol, and PKC-to facilitate GABAergic neurotransmission.

Fig. 4 The reduction in CRF-R1 activity during LT withdrawal from CIE is associated with enhanced CB1 receptor-mediated inhibition. a Sample eIPSCs traces and summary data showing reduced responding to CRF (300 nM) and PDBU (1 µM) in neurons from CIE treated mice. b,c Sample eIPSCs traces and summary data showing LT-CIE cells are (b) less sensitive to CRF (300 nM) and the CB1 agonist WIN55212 (WIN; 1 µM) but (c) more sensitive to the CB1 antagonist AM251 (1 µM). In (b), note the WIN-related reversal of the CRF potentiation in the air controls. d,e Sample eIPSCs traces and summary data illustrating that enhanced calcium buffering (1 mM BAPTA vs 0.1 mM EGTA in the recording solution) in postsynaptic LT-CIE cells rescues the (d) CRF (300 nM) and PDBU (1 mM) mediated increase in eIPSC amplitude (Two-way ANOVA, ***p < 0.0001 for effect of treatment (internal chelator)),as well as reduces the sensitivity to the (e) CB1 receptor antagonist AM251 (***p < 0.0001). f Schematic of proposed mechanism of CRF regulation of GABA release onto VTA dopamine neurons during LT withdrawal following CIE. Left, in controls, presynaptic CRF-R1 facilitates the release of GABA onto GABA A receptors via PKC mediated recruitment of intracellular calcium. During LT withdrawal from CIE, enhancement of endocannabinoid (eCB) inhibition opposes the CRF action. Chelation of intracellular calcium with BAPTA in the dopamine neuron reduces eCB synthesis, reducing the CB1-mediated suppression of the presynaptic CRF dependent mechanism. (a-d) Number of cells and mice per LT-group: (a) air 8/4, CIE 8/5; (b) air 8/4, CIE 8/5; (c) air 8/4, CIE 8/5;(d) for both EGTA and BAPTA: CRF 7/6, PDBU 6/4; (e) for both EGTA and BAPTA: AM251 6/4. All timelines show eIPSC amplitude % of baseline, mean ± SEM Full size image

We and others have shown that activation of CB1 receptors can suppress VTA GABA release [61, 62], and in amygdala neurons this reverses the facilitation produced by acute exposure to ethanol [16]. Consistent with these reports, we noted that application of the CB1 agonist WIN55212 (1 μM) alone in slices from LT-air mice significantly decreased eIPSCs and subsequent co-application of CRF (300 nM) reversed this action of WIN55212 (Supplemental Fig. 2; WIN: 52.8% ± 4.97% decrease relative to baseline; t (4) = 4.085, p = 0.0075; WIN + CRF, 43.6% ± 3.85% increase relative to WIN alone t (4) = 3.918, p = 0.0139). The same concentrations of WIN55212 reversed the CRF (300 nM) potentiation of eIPSCs in LT-air controls (Fig. 4b; pre-CRF vs CRF + WIN: t (4) = 0.25465, p = 0.4701). In contrast, in slices from LT-CIE mice, neither CRF nor co-application of WIN55212 with CRF measurably changed IPSC amplitudes (Fig. 4b; CRF, t (4) = 0.2314, p = 0.3138; WIN, t (5) = 0.2834, p = 0.3807).

To determine if the diminished actions of CRF and WIN55212 in LT-CIE mice reflected an enhancement of endocannabinoid CB1 receptor inhibition, we measured the effects of CB1R antagonist AM251 (1 μM) on eIPSCs. In control LT-air tissue, AM251 produced no change in eIPSC amplitudes when applied after CRF (Fig. 4c; t (6) = 0.2128, p = 0.3781), suggesting the CRF potentiation of eIPSC amplitude was not restricted by inhibitory tone on CB1 receptors under control conditions. Bath application of AM251 (1 μM) alone in the LT-air control group produced no measurable change in eIPSCs (AM251: 8.67% ± 6.13% increase relative to baseline; t (6) = 0.3408, p = 0.7449). This finding with VTA GABA A IPSCs is consistent with our previous published findings by us and others indicating the absence of CB1 receptor tone in drug-naive animals [61, 63]. In tissue from LT-CIE mice, as noted above, CRF applied alone produced no change in IPSC amplitude (Fig. 4c; t (6) = 0.2128, p = 0.3781). However, application of the CB1 antagonist AM251 in the presence of CRF produced a robust enhancement of eIPSC amplitude (Fig. 4c; t (6) = 32.01, p < 0.0001). This suggests that LT withdrawal from CIE treatment is associated with a functional enhancement of CB1 inhibition on GABA synapses of VTA DA neurons. This CB1-mediated tone blocks subsequent modulation of GABA release by CRF or ethanol and may explain the reduction in the basal frequency of sIPSCs observed in Fig. 3d.

As endocannabinoid synthesis requires increases in intracellular calcium in the postsynaptic neuron [64], we tested whether chelating intracellular calcium post-synaptically in the recorded DA neuron would restore CRF and PDBU sensitivity in LT-CIE tissue. Under normal recording conditions (0.1 mM EGTA) in LT-CIE tissue, CRF and PdBU produced minimal changes in eIPSC amplitude (Fig. 4d). In contrast, when recording with an internal solution containing the fast calcium chelator BAPTA (1 mM), both CRF and PDBU enhanced eIPSC amplitude (Fig. 4d). A two-way ANOVA that revealed a significant effect of treatment (internal chelator; F (1, 16) = 51.92, p < 0.0001), but not for drug (F (1, 16) = 3.892, p = 0.0660) or interaction (F (1, 16) = 2.126, p = 0.1641). Thus, chelation of intracellular calcium in the postsynaptic neurons of LT-CIE mice restored the ability of bath applied CRF, which acts on the presynaptic CRF-R1, to potentiate GABA A IPSCs.