Chronic pain. Decreased motivation during chronic pain requires long-term depression in the nucleus accumbens

Abstract

Several symptoms associated with chronic pain, including fatigue and depression, are characterized by reduced motivation to initiate or complete goal-directed tasks. However, it is unknown whether maladaptive modifications in neural circuits that regulate motivation occur during chronic pain. Here, we demonstrate that the decreased motivation elicited in mice by two different models of chronic pain requires a galanin receptor 1-triggered depression of excitatory synaptic transmission in indirect pathway nucleus accumbens medium spiny neurons. These results demonstrate a previously unknown pathological adaption in a key node of motivational neural circuitry that is required for one of the major sequela of chronic pain states and syndromes.

Copyright © 2014, American Association for the Advancement of Science.

Figures

Fig. 1. Motivation is impaired in models of chronic pain

(A) Schematic of mouse in the operant chamber. (B) Sum of nose pokes required to earn rewards on PR schedule. (C) Kymograph illustrating distance from nose poke port (y axis) versus time (x axis). Green arrows indicate times at which rewards 4 to 6 were earned. (D) Time line of experiments. Results from 2 PR tests before induction of pain models were compared with results from 6 PR tests at three time points after induction. (E) Nose pokes per animal during 2 days of baseline testing. (F to H) Both CFA and SNI induction reduced number of nose pokes, resulting in a drop in the rewards earned at the respective time points (control, n = 12 mice, includes SNI sham surgery n = 4, CFA sham injections n = 5, untreated n = 3; CFA n = 10 mice; SNI n = 8 mice). (G) **P < 0.01 versus control. (H) CFA †P < 0.05, †† P < 0.01; SNI #P < 0.05, ##P < 0.01; post hoc t tests. (I and J) During PR tests, there was no difference in searches for rewards before or after induction nor differences in the sucrose preference test (control n = 6 mice, CFA n = 6 mice, SNI n = 5 mice). (K) Both pain models reduce mechanical threshold, and this is ameliorated by analgesic administration (diclofenac, subcutaneous, n = 8 mice, *P < 0.05; clonidine, intrathecal, n = 5 mice, **P < 0.01; Student’s t tests). (L) Neither acute analgesic affects the reduction in rewards earned after model induction. For all figures, error bars are SEM.

Fig. 2. Excitatory synapses are modified 7 to 12 days after induction of chronic pain

(A) Time course of experiments in (B) to (K). (B) Sample EPSCs recorded at +40 mV (top traces) and two EPSCs evoked with a 50-ms interval recorded at −70 mV (bottom traces) from NAc D2-MSNs and D1-MSNs at 7 to 12 days after sham procedures or after model induction. EPSC amplitudes are normalized to peaks at +40 mV. (C) Summary showing AMPAR/ NMDAR ratios in D2-MSNs and D1-MSNs in both pain models (D2, control n = 15 cells, CFA n = 13 cells, SNI n = 12 cells, *P < 0.05 post hoc t test; D1, control n = 12 cells, CFA n = 7 cells, SNI n = 11 cells). (D) Average traces of 100 mEPSCs from representative D2-MSNs. (E) mEPSC frequency was unaffected by CFA and SNI treatments. (F and G) Cumulative distribution of mEPSC amplitudes (F) and averages (G) in D2-MSNs and D1-MSNs in both pain models (D2, control n = 14 cells, CFA n = 10 cells, SNI n = 12 cells, *P < 0.05 Student’s t test; D1, control n = 13 cells, CFA n = 14 cells, SNI n = 13 cells). (H) Sample normalized average traces of pharmacologically isolated NMDAR EPSCs. (I) Summary data of NMDAR EPSC decay kinetics from D2-MSNs and D1-MSNs in both pain models (D2, control n = 12 cells, CFA n = 9 cells, SNI n = 6 cells,*P < 0.05 post hoc t tests; D1, control n = 9 cells, CFA n = 7 cells, SNI n = 12 cells). (J and K) In both models, ifenprodil (3 μM) caused a larger depression of NMDAR EPSCs in D2-MSNs versus D1-MSNs (D2, control n = 8 cells, CFA n = 8 cells, SNI n = 6 cells; D1, control n = 9 cells, CFA n = 7 cells, SNI n = 5 cells; *P < 0.05 post hoc t tests).

Fig. 3. NMDAR EPSCs are slower in D2-MSNs 12 hours after induction of chronic pain

(A) Experimental protocol. (B) Sample EPSCs from D2-MSNs recorded 12 hours after saline (control) or CFA injection (CFA12hrs). Traces are normalized to peak at +40 mV. (C) Summary of AMPAR/NMDAR ratios in D2-MSNs and D1-MSNs at this time point (D2, control, n = 15 cells, CFA12hrs n = 12 cells; D1, control n = 12 cells, CFA12hrs n = 9 cells). (D and E) Cumulative distribution and average of mEPSC amplitudes from D2-MSNs at this time point (control n = 14 cells, CFA12hrs n = 10 cells). (F) Sample normalized average NMDAR EPSCs from D2-MSN neurons. (G) Summary of NMDAR EPSC weighted decay time constants 12 hours after CFA (control n = 12 cells; CFA12hrs n = 16 cells; **P < 0.01 Student’s t test).

Fig. 4. Increase in NMDAR EPSC decay 12 hours after induction of chronic pain requires GalR1

(A) Images of neurons retrogradely labeled by rabies virus expressing eGFP (green) injected into NAc overlaid with Gal-antibody labeling (red) from basoateral amygdala (BLA), paraventricular nucleus of thalamus (PVN), and arcuate nucleus of hypothalamus (ARC). (B) Sample NMDAR EPSCs from D2-MSN taken at time points indicated on summary graph showing transient effects of Gal (1 μM). (C) Summary of change in NMDAR EPSC decay kinetics after wash-out of Gal (n = 8 cells; *P < 0.05 repeated measure t test). (D) Ifenprodil causes larger depression of NMDAR EPSCs in D2-MSNs versus D1-MSNs from slices preincubated in Gal (D2, nontreated from Fig. 2K, post-Gal+ n = 6 cells; D1, nontreated from Fig. 2K, post-Gal+ n = 6 cells; **P < 0.01 Student’s t test). (E) Schematic of adeno-associated virus vector expressing GalR1 shRNA and eGPF, with images showing eGFP expression and GalR1 staining 1 month after infection of NAc. Graph shows relative mRNA levels from sister hippocampal cultures infected with eGFP and GalR1 shRNA, respectively (n = 3 experiments; ++P = 0.006 Student’s t test). (F) Time line of experiments in (G) to (I). (G) Sample normalized NMDAR EPSCs from D2-MSNs. (H) Summary graph of NMDAR EPSC decay kinetics 12 hours after CFA. KD, knockdown. (D2, control taken from Fig. 3, vehicle+CFA12hrs n = 9 cells, M40 +CFA12hrs n = 12 cells, KD+CFA12hrs n = 8 cells; **P < 0.01, *P < 0.05 post hoc t tests). (I) Effects of ifenprodil on NMDAR EPSCs 12 hours after CFA (control n = 8 cells, Vehicle+CFA12hrs n = 5 cells, KD+CFA12hrs n = 5 cells; *P < 0.05 post hoc t test).

Fig. 5. GalR1 in NAc is required for pain-induced synaptic depression in D2-MSNs and reduction in motivation

(A) Time line of acute slice experiments in (B) to (E). (B) Decrease in AMPAR/NMDAR ratios in D2-MSNs in both pain models is prevented by GalR1 KD [control, CFA and SNI (left group) taken from Fig. 2; GalR1 KD, sham n = 5 cells, CFA n = 11 cells, SNI n = 8 cells). (C to E) Summary graphs of LTD in D2-MSNs in both pain models and effects of GalR1 KD (control, n = 11 cells, CFA n = 10 cells, GalR1 KD+CFA n = 7 cells, SNI n = 6 cells, GalR1 KD+SNI n = 6 cells). (F) Timeline of behavior experiments in (H) to (J). (G) Schematics of virus constructs. (H) Cumulative distribution of nose pokes per session and average nose pokes per day during before-induction baseline period (PRE). (sham n = 12 mice includes eGFP n = 5 mice, naive n = 7 mice; GalR1 KD n = 18 mice, Rep. GalR1 KD n = 6 mice). (I and J) Effects of GalR1 KD in NAc on nose pokes after model induction and rescue by coexpression of shRNA-resistant GalR1 (Rep.GalR1 KD) in CFA model. (I) CFA n = 6 mice includes eGFP n = 3 mice, naive n = 3 mice; GalR1 KD+CFA n = 12 mice, Rep. GalR1 KD+CFA n = 6 mice, **P < 0.01, ***P < 0.001 Tukey’s post hoc t test. (J) SNI n = 6 mice includes eGFP n = 2 mice, naïve n = 4 mice; GalR1 KD +SNI n = 6 mice, *P < 0.05 Student’s t test.

Fig. 6. NMDAR-dependent LTD in NAc is required for pain-induced decrease in motivation

(A) Timeline for virus injection and acute slice experiments in (B) to (D). (B and C) Schematics of AKAP replacement constructs and summary LTD graphs for D2-MSNs expressing these constructs (AKAPWT n = 4 cells; AKAPΔPP2B n = 8 cells). (D) Summary of AMPAR/NMDAR ratios in D2-MSNs after CFA treatment (control and CFA from Fig. 2; AKAPΔPP2B, sham n = 7 cells, CFA n = 9 cells). (E) Timeline for behavior experiments in (F) to (I). (F to H) Cumulative distribution of nose pokes per session and average nose pokes per day during before-induction baseline (PRE) and after model induction (POST). (F) AKAPWT/eGFP n = 13 mice includes eGFP n = 5 mice, AKAPWT n = 8 mice; AKAPΔPP2B n = 10 mice. (G) AKAPWT/eGFP CFA n = 7 mice includes eGFP n = 3 mice, AKAPWT, n = 4 mice; AKAPΔPP2B+CFA n = 4. (H) AKAPWT/eGFP SNI n = 6 mice includes eGFP n = 2 mice, AKAPWT n = 4 mice; AKAPΔPP2B+SNI n = 6 mice; *P < 0.05, ***P < 0.001 Student’s t test. (I) Summary of effects of GalR1 KD and AKAPΔPP2B expression on nose pokes per day after model induction (data pooled from Figs. 1, 5, and 6) (*P < 0.05,***P < 0.001 post hoc t test versus control; ++P < 0.01, +++P < 0.001 Tukey’s post hoc t test versus respective pain model). (J) Schematic summarizing temporal order of synaptic and behavioral changes after pain induction.