Morphine Differentially Alters the Synaptic and Intrinsic Properties of D1R- and D2R-Expressing Medium Spiny Neurons in the Nucleus Accumbens

Abstract

Exposure to opioids reshapes future reward and motivated behaviors partially by altering the functional output of medium spiny neurons (MSNs) in the nucleus accumbens shell. Here, we investigated how morphine, a highly addictive opioid, alters synaptic transmission and intrinsic excitability on dopamine D1-receptor (D1R) expressing and dopamine D2-receptor (D2R) expressing MSNs, the two main output neurons in the nucleus accumbens shell. Using whole-cell electrophysiology recordings, we show, that 24 h abstinence following repeated non-contingent administration of morphine (10 mg/kg, i.p.) in mice reduces the miniature excitatory postsynaptic current (mEPSC) frequency and miniature inhibitory postsynaptic current (mIPSC) frequency on D2R-MSNs, with concomitant increases in D2R-MSN intrinsic membrane excitability. We did not observe any changes in synaptic or intrinsic changes on D1R-MSNs. Last, in an attempt to determine the integrated effect of the synaptic and intrinsic alterations on the overall functional output of D2R-MSNs, we measured the input-output efficacy by measuring synaptically-driven action potential firing. We found that both D1R-MSN and D2R-MSN output was unchanged following morphine treatment.

Keywords: intrinsic excitability; morphine; neuronal activity; nucleus accumbens; opioid use disorder; synaptic transmission.

Figure 1

Repeated morphine administration reduces miniature excitatory postsynaptic current (mEPSC) frequency on dopamine D2-receptor medium spiny neurons (D2R-MSNs) on abstinence day 1. (A) Representative traces showing mEPSCs recorded from D1R- or D2R-MSNs from animals treated with saline or morphine (10 mg/kg, i.p.). Scale bar: 20 pA, 1 s. (B,C) Cumulative plot of a representative neuron showing the distribution of mEPSC amplitudes recorded from D1R-MSNs (B) or D2R-MSNs (C) in animals treated with saline or morphine. (D) Summary graph showing the average mEPSC amplitude recorded D1R- or D2R-MSNs following saline or morphine treatment (F_(3,42) = 2.92, p = 0.045; One-way ANOVA. Bonferroni post-test, D1R-MSN: saline vs. morphine, p > 0.999; D2R-MSN: saline vs. morphine, p > 0.999). (E,F) Cumulative plot of a representative neuron showing the distribution of mEPSC inter-event intervals (I-I) recorded from D1R-MSNs (E) or D2R-MSNs (F) in animals treated with saline or morphine. (G) Summary graph showing the average mEPSC frequency recorded from D1R- or D2R-MSNs following saline or morphine treatment (F_(3,42) = 6.73, p = 0.0008; One-way ANOVA with Bonferroni post-test; *p < 0.05). (H) Summary graph showing the average rise time of mEPSC recorded from D1R- or D2R-MSNs following saline or morphine treatment. (I) Summary graph showing the average decay tau of mEPSC recorded from D1R- or D2R-MSNs following saline or morphine treatment. Circle = neuron.

Figure 2

Short-term abstinence from in vivo morphine treatment has no effect on the evoked excitatory/inhibitory (E/I) ratio and does not alter the temporal relationship between spontaneous EPSCs (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) on D1R- or D2R-MSNs in the nucleus accumbens shell. (A) Representative traces showing evoked AMPA receptor (AMPAR)- and GABA receptor (GABAR)-mediated currents on D1R- or D2R-MSNs 24 h following repeated saline or morphine treatments. Neurons were held at −70 mV. (B) Summary graph showing the E/I ratio of evoked currents on D1R- or D2R-MSNs 24 h following repeated saline (sal) or morphine (mor; 10 mg/kg, i.p.) treatments. There were no significant differences between groups in male or female mice. (C) Representative traces showing spontaneous EPSCs (inward current) and IPSCs (outward current) when D1R- or D2R MSNs were held at −30 mV 24 h following in vivo morphine treatment. Scale bars: 20 pA, 0.5 s (Lower). Electrophysiological recordings in whole-cell patch-clamp configuration showing the inter-event intervals (inter. interv.) of sEPSCs to sIPSCs (left) or sIPSCs to sEPSCs (right) in a MSN held at −30 mV. Scale bars: 20 pA, 0.125 s. (D) Summary graph showing no significant changes in the inter-event interval (I-I) between sEPSCs and sIPSCs on D1R- or D2R-MSNs 24 following repeated saline or morphine administration in male mice. (E) Summary graph showing no significant changes in the inter-event interval (I-I) between sIPSCs and sEPSCs on D1R- or D2R-MSNs 24 following repeated saline or morphine administration in male mice. (F) Summary graph showing that morphine exposure had no effect on the frequency ratio of sEPSC to sIPSC events within D1R- or D2R-MSNs in male mice.

Figure 3

Repeated morphine administration reduces miniature inhibitory postsynaptic current (mIPSC) frequency on D2R-MSNs on abstinence day 1. (A) Representative traces showing mIPSCs recorded from D1R- or D2R-MSNs from animals treated with saline or morphine (10 mg/kg, i.p.). Scale bar: 25 pA, 0.5 s. (B,C) Cumulative plot showing the distribution of mIPSC amplitudes recorded from D1R-MSNs (B) or D2R-MSNs (C) in animals treated with saline or morphine. (D) Summary graph showing the average mIPSC amplitude recorded D1R- or D2R-MSNs following saline or morphine treatment (F_(3,65) = 4.73, p = 0.005; one way ANOVA with Bonferroni post-test). *p < 0.05. (E,F) Cumulative plot of a representative neuron showing the distribution of mIPSC inter-event intervals (I-I) recorded from D1R-MSNs (E) or D2R-MSNs (F) in animals treated with saline or morphine. (G) Summary graph showing the average mIPSC frequency recorded from D1R- or D2R-MSNs following saline or morphine treatment (F_(3,65) = 8.94, p < 0.0001; one-way ANOVA with Bonferroni post-test; *p < 0.05, **p < 0.01). (H) Summary graph showing the average rise time of mIPSC recorded from D1R- or D2R-MSNs following saline or morphine treatment. (I) Summary graph showing the average decay tau of mIPSC recorded from D1R- or D2R-MSNs following saline or morphine treatment. Circle = neuron.

Figure 4

Repeated morphine administration increases membrane excitability on D2R-MSNs on abstinence day 1. (A) Representative traces, scale bar, 40 mV, 300 ms at 100 pA current injection. (B) Summary graph showing the average number spikes generated by injected current on D1R-MSNs following saline or morphine (10 mg/kg, i.p.) treatment (F_(7,238) = 1.05, p = 0.395; two-way repeated-measures ANOVA). (C) Summary graph showing the average number of spikes generated by the injected current on D2R-MSNs following saline or morphine treatment (F_(7,210) = 10.4, p = 0 < 0.0001; two-way repeated-measures ANOVA with Bonferroni post-test). *p < 0.05. (n/n = cells/animals).

Figure 5

Synaptically-driven action potential firing on D1R- or D2R-MSNs is unaffected by repeated morphine (10 mg/kg, i.p.) treatment. (A) Representative traces showing depolarizations or action potentials of a recorded MSN evoked by electrical current (in μA) of 20 (light gray), 60 (blue), 100 (black), or 100 in the presence of NBQX (red), an AMPA receptor antagonist. Scale bars, 12.5 mV, 50 ms. (B,C) Summary graphs showing the average spike number at each current injected for D1R- or D2R-MSNs following saline or morphine treatment (cells/animals).

Figure 6

(A) A strength-duration curve constructed from an MSN in the nucleus accumbens shell. Stimulus current was adjusted at each duration (from 0 to 2.0 ms with 0.2 ms increments) until an action potential was evoked using an electrical stimulus. The curve was fit with a two-phase exponential decay. The rheobase (gray dashed line) was calculated as the plateau of the curve and the chronaxie (black dashed line) was calculated as 2× the rheobase (Inset). Representative traces illustrating the stimulus (downward deflection) followed by the action potential at 2 ms duration). In the presence of NBQX (2 μM), action potentials are not elicited (2 ms duration, 35 μA of current). Scale bars, 20 mV, 12.5 ms. (B) Summary graph showing the average rheobase for D1R- or D2R-MSNs following saline (Sal) or morphine (Mor; 10 mg/kg, i.p.) treatment. (C) Summary graph showing the average chronaxie for D1R- or D2R-MSNs following saline or morphine treatment.