Regional and cell-type-specific effects of DAMGO on striatal D1 and D2 dopamine receptor-expressing medium-sized spiny neurons

Abstract

The striatum can be divided into the DLS (dorsolateral striatum) and the VMS (ventromedial striatum), which includes NAcC (nucleus accumbens core) and NAcS (nucleus accumbens shell). Here, we examined differences in electrophysiological properties of MSSNs (medium-sized spiny neurons) based on their location, expression of DA (dopamine) D1/D2 receptors and responses to the μ-opioid receptor agonist, DAMGO {[D-Ala(2)-MePhe(4)-Gly(ol)(5)]enkephalin}. The main differences in morphological and biophysical membrane properties occurred among striatal sub-regions. MSSNs in the DLS were larger, had higher membrane capacitances and lower Rin (input resistances) compared with cells in the VMS. RMPs (resting membrane potentials) were similar among regions except for D2 cells in the NAcC, which displayed a significantly more depolarized RMP. In contrast, differences in frequency of spontaneous excitatory synaptic inputs were more prominent between cell types, with D2 cells receiving significantly more excitatory inputs than D1 cells, particularly in the VMS. Inhibitory inputs were not different between D1 and D2 cells. However, MSSNs in the VMS received more inhibitory inputs than those in the DLS. Acute application of DAMGO reduced the frequency of spontaneous excitatory and inhibitory postsynaptic currents, but the effect was greater in the VMS, in particular in the NAcS, where excitatory currents from D2 cells and inhibitory currents from D1 cells were inhibited by the largest amount. DAMGO also increased cellular excitability in the VMS, as shown by reduced threshold for evoking APs (action potentials). Together the present findings help elucidate the regional and cell-type-specific substrate of opioid actions in the striatum and point to the VMS as a critical mediator of DAMGO effects.

Keywords: ACSF, artificial cerebrospinal fluid; AHP, after hyperpolarization; AP, action potential; AP-5, dl-2-amino-5-phosphonovaleric acid; BIC, bicuculline; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; CsMeth, Cs-methanesulfonate; D1/D2 receptors; DA, dopamine; DAMGO, [d-Ala2-MePhe4-Gly(ol)5]enkephalin; DLS, dorsolateral striatum; EGFP, enhanced green fluorescent protein; EPSC, excitatory postsynaptic current; IPSC, inhibitory postsynaptic current; KGluc, K-gluconate; MSSN, medium-sized spiny neuron; NAcC, nucleus accumbens core; NAcS, nucleus accumbens shell; RMP, resting membrane potential; Rin, input resistance; TBST, TBS containing 0.1% Tween 20; TTX, tetrodotoxin; UCLA, University of California at Los Angeles; VMS, ventromedial striatum; VTA, ventral tegmental area; electrophysiology; mEPSC, miniature EPSC; mIPSC, miniature IPSC; nucleus accumbens; opioid receptors; sEPSC, spontaneous EPSC; sIPSC, spontaneous IPSC; striatum.

Figure 1. Morphology of MSSNs

(A) Confocal images of D1/D2 receptor-expressing MSSNs from the three sub-regions of striatum. Scale bar: 25 μm and applies to all panels. (B–D) Graphs show the somatic area (B), the number of primary (C) and secondary dendrites (D) per D1/D2 MSSN in the striatum. n = 4–6 in each group. In this and other Figures, data are expressed as means±S.E.M. Data were analysed by two-way ANOVA followed by Bonferroni post hoc test. **P<0.01, ***P<0.001 respectively compared with DLS; #P<0.05, D1 versus D2 cells in the same sub-region.

Figure 2. Whole-cell current clamp recordings from striatal MSSNs

(A) Sample traces show a gradient of inward rectification evaluated by recording responses to hyperpolarizing current steps: there is an increase from DLS to VMS (note increased voltage deflections below dashed lines in VMS compared with DLS). (B) I–V plots from groups of D1 (left panel) or D2 (right panel) receptor-expressing MSSNs, n = 9–10 in each group. The data were analysed using ANOVA with repeated measures followed by Bonferroni post hoc test. *P<0.05, ***P<0.001 respectively.

Figure 3. Regional and cell-type-specific differences in frequency of sEPSCs in MSSNs

(A) Representative traces of sEPSCs in D1/D2 cells from each sub-region (holding potential −70 mV). (B) Histograms show the decreased frequency of sEPSCs as well as increased differences in frequency of sEPSCs between D1 versus D2 cells in the VMS. (C–E) Differences of cumulative inter-event interval probability between D1 versus D2 cells in the DLS, NAcC and NAcS. For (B–E), n = 10–13 in each group. The data were analysed using two-way ANOVA (B) or ANOVA with repeated measures (C–E) followed by Bonferroni post hoc tests. ***P<0.001, compared with the DLS; #P<0.05, D1 versus D2 cells.

Figure 4. Regional and cell-type-specific differences in frequency of sIPSCs in MSSNs

(A) Representative traces of sIPSCs in D1/D2 cells from the DLS, NAcC and NAcS (holding potential +10 mV). (B) Histograms show the increased frequency of sIPSCs in the VMS compared with the DLS. (C–E) Show inter-event interval cumulative probabilities between D1 versus D2 cells in the DLS, NAcC and NAcS. For (B–E), n = 9–11 in each group. The data were analysed using two-way ANOVA (B) or ANOVA with repeated measures (C–E) or followed by Bonferroni post hoc test. *P<0.05, **P<0.01, respectively, compared with the DLS.

Figure 5. Effects of DAMGO on sEPSCs in MSSNs

(A) Representative recordings of sEPSCs (holding potential −70 mV) in a D2 receptor-expressing MSSN from NAcS before, during, and after washout of DAMGO (1 μM). (B) Representative time-course of a typical recording from another D2 receptor-expressing MSSN from NAcS before, during and after DAMGO (1 μM). DAMGO decreased the frequency of sEPSCs, which was reversed after DAMGO washout. (C, D) Histograms show the effects of DAMGO on sEPSCs (C) and mEPSCs (D) in D1/D2 cells in the DLS, NAcC and NAcS. In (C, D), data were calculated as changes of average frequencies of EPSCs during versus before DAMGO application, divided by the corresponding values before DAMGO. n = 10–14 (C) or n = 5 (D) in each group. The data were analysed using two-way ANOVAs followed by Bonferroni post hoc tests. *P<0.05, **P<0.01, ***P<0.0001 compared with the DLS. Also, paired t tests were performed between the average frequencies of EPSCs before versus during DAMGO application. #P<0.05, ##P<0.01, ###P<0.001 respectively.

Figure 6. Effects of DAMGO on sIPSCs in MSSNs

(A) Representative traces of sIPSCs (holding potential +10 mV) in a D1 receptor-expressing MSSN from NAcS before, during and after washout of DAMGO (1 μM). (B) Typical time-course of a representative recording from another D1 receptor-expressing MSSN from NAcS before, during and after DAMGO (1 μM). DAMGO decreased the frequency of sIPSCs, which recovered after DAMGO washout. Histograms in (C, D) show the effects of DAMGO on sIPSCs (C) and mIPSCs (D) in D1/D2 cells in the striatum. In (C, D), data were calculated as described in Figure 5. n = 8–10 (C) or n = 5 (D) in each group. The data were analysed using two-way ANOVAs followed by Bonferroni post hoc tests. *P<0.05, **P<0.01 respectively compared with the DLS or NAcC. Also, paired t tests were performed between the average frequencies of IPSCs before versus after DAMGO. #P<0.05, ##P<0.01 respectively.

Figure 7. Effects of DAMGO on the intrinsic excitability of MSSNs in the NAcC/NAcS

(A) Representative traces (left panel) show that DAMGO decreased the AP threshold of D2 cells in NAcS. Effects of DAMGO on AP threshold of D1/D2 cells in the striatum are shown in the histograms (right panel). (B) Representative traces show that DAMGO decreased AHP amplitude of D1 cells in the NAcS. Effects of DAMGO on AHP amplitude of D1/D2 cells in the striatum are shown in the histograms. (C) Representative traces show that DAMGO decreased the rheobase of D1 cells in NAcS. Effects of DAMGO on rheobase of D1/D2 cells in the striatum are shown in the histograms. n = 5–7 cells in each group. Data were calculated by changes of the value before versus during DAMGO application, divided by the corresponding values before DAMGO; analysed by two-way ANOVAs followed by Bonferroni post hoc tests, *P<0.05, **P<0.01, compared with the DLS. Also, paired t tests were performed between the average frequencies of IPSCs before versus during DAMGO in (C, D); #P<0.05, ##P<0.01, respectively.

Figure 8. Effects of DAMGO on the intrinsic excitability of MSSNs in the DLS

(A) Representative traces and histograms show that DAMGO increased the membrane Rin of D1 cells in DLS, recorded in current clamp mode and calculated from the voltage response to a hyperpolarizing current pulse (−100 pA). (B) Representative traces show that DAMGO decreased firing of a D1 MSSNs in DLS; (C) DAMGO caused a rightward shift in the current–response curve of D1 and D2 MSSNs in DLS. n = 7 in each group. Data are expressed as means±S.E.M., and analysed using ANOVA with repeated measures (A) and paired t test (B); *P<0.05, **P<0.01, ***P<0.001 respectively compared with the control value before DAMGO application.