Adaptive alterations in the mesoaccumbal network after peripheral nerve injury

Abstract

The nucleus accumbens (NAc) and the ventral tegmental area (VTA) are critical hubs in the brain circuitry controlling chronic pain. Yet, how these 2 regions interact to shape the chronic pain state is poorly understood. Our studies show that in mice, spared nerve injury (SNI) induced alterations in the functional connectome of D2-receptor expressing spiny projection neurons in the core region of the NAc-enhancing connections with prelimbic cortex and weakening them with basolateral amygdala. These changes, which were attributable in part to SNI-induced suppression of VTA dopaminergic signaling, were adaptive because mimicking them chemogenetically alleviated the anxiety and social withdrawal accompanying injury. By contrast, chemogenetic enhancement of activity in VTA dopaminergic neurons projecting to the medial shell of the NAc selectively suppressed tactile allodynia in SNI mice. These results suggest that SNI induces regionally specific alterations in VTA dopaminergic signaling in the NAc to promote environmental reengagement after injury. However, countervailing, homeostatic mechanisms limit these adaptive changes, potentially leading to the chronic pain state.

Figure 1.. The strength of BLA synapses on cNAc d2SPNs was reduced whereas the strength of PL synapses on cNAc d2SPNs was increased in SNI animals.

a, Sample slices displaying the expression of ChR2-mCherry (red) in BLA (larger image) and cNAc (smaller image) after virus injection in the BLA. Scale bars represent 0.5 mm. b-e, Optical stimulation-evoked d2SPN AMPA/NMDA receptor responses (A/N ratio) were inhibited in SNI (n = 7–8 neurons from 5 mice per group; U = 9, p = 0.0289), but no change was detected in d1SPNs (j, n = 6 neurons from 5 mice per group with U = 17, p = 0.8983). b and d AMPA and NMDA receptor-mediated currents elicited in the cNAc SPNs with optical stimulation of BLA inputs (calibration: 50 pA, 100 ms). f, Sample images displaying the expression of ChR2-mCherry (red) in prelimbic (PL)-PFC and cNAc after virus injection in BLA (scale bar: 0.5 mm). g-j, Optically evoked d2SPNs AMPA/NMDA receptor responses were enhanced in SNI, but no change was detected in d1SPNs (n = 6 neurons from 5 mice per group, Mann–Whitney U test with U = 2, p = 0.0087 for c and U = 16, p = 0.8182 for e). g and i showed the representative AMPA and NMDA receptor-mediated currents elicited in the cNAc SPNs with optical stimulation of PL inputs (calibration: 50 pA, 100 ms). Data are presented as whisker box plots displaying median, lower and upper quartiles, and whiskers representing minimum and maximum of the data.

Figure 2.. D2 receptor signaling modulated state-dependent LTD at PL-cNAc d2SPN synapses.

a-b, Representative images showing Chronos-GFP (green) expression in PL-PFC and BLA at 4-week post injection (scale bar: 0.5 mm). c-d, Bath application of CB1 receptor agonist WIN 55,212–2 induced LTD of AMPAR EPSC amplitude in PL-cNAc d2SPN synapses, but had no effect on BLA-d2SPN synapses (n = 5 neurons from 5 mice per group; Wilcoxon test with W = −4543, p < 0.0001; calibration for c: 50 pA, 10 ms). e, Diagram of D2R-dependence of the eCb-dependent LTD. f, The representative recording traces showing the EPSCs of PL-cNAc d2SPNs synapses before and after state-dependent LTD induction in the presence of D2 receptor agonist quinpirole (10 μM, calibration: 50 pA, 10 ms). g, State-dependent LTD induction protocol (calibration: 300 pA, 500 ms). h, At PL-cNAc d2SPNs synapses, the induction protocol elicited a robust LTD in the presence of quinpirole but not sulpiride (10 μM of quinpirole and sulpiride, n = 5 neurons from 5 mice per group; Wilcoxon test with W = −5130, p < 0.0001). Data are presented as median with shaded interquartile (quartile 1 to quartile 3).

Figure 3.. Chemogenetically activating cNAc d2SPNs relieved anxiety and social recognition deficits in SNI mice.

a, In AAV-PSAM^L141F,Y115F-5HT3 HC-GFP transduced A2A::Cre mice, GFP fluorescence was exclusively expressed in cNAc. b-c, PSEM89S induced firing in PSAM-5HT3-GFP expressing cNAc d2SPNs from A2a-Cre mice (n = 7 neurons from 5 mice per group in c, Wilcoxon test with W = 28, p = 0.0156 for PRE versus SPEM and W = 28, p = 0.0156 for PSEM versus WASH). d, The timeline for behavioral tests in the PSAM-5HT3 infected mice (n = 7 in Sham, n = 5–6 in Saline-SNI, n = 8 in PSEM-SNI; Mann–Whitney U test for data statistics). e, Activating cNAc d2SPNs by i.p. injection of PSEM^89S had no effect on SNI-induced tactile allodynia (U = 1, p = 0.0051 for Sham versus Saline-SNI; U = 15.50, p = 0.5462 for Saline-SNI versus PSEM-SNI).f-g, In open field test, PSAM-5HT3 expressing SNI mice receiving PSEM89S treatment spent more time in the center zone of open field than those receiving saline treatment (U = 2, p = 0.0062 for Sham versus Saline-SNI; U = 2, p = 0.0062 for Saline-SNI versus PSEM-SNI). h-i, In social recognition task, PSEM^89S treatment abolished the deficits in social recognition associated with SNI (n = 7 in Sham, n = 6 in Saline-SNI, n = 8 in PSEM-SNI; U = 1, p = 0.0023 for Sham versus Saline-SNI; U = 6, p = 0.02 for Saline-SNI versus PSEM-SNI). Data are presented as whisker box plots displaying median, lower and upper quartiles, and whiskers representing minimum and maximum of the data.

Figure 4.. VTA neurons innervating cNAc and msNAc differentially affected pain-associated behaviors in SNI mice.

a, RV-Cre was stereotaxically injected into cNAc and AAV-DIO- PSAM-GlyR constructs were injected into the VTA respectively. Representative image demonstrating 4-week expression of PSAM-GlyR-GFP in lateral VTA (l-VTA)-cNAc neurons (GFP expression). Robust PSAM-GlyR-GFP expression was routinely detected in the VTA, but no attempt was made to rigorously estimate the percentage of the neurons labeled. b-c, PSEM^89S (The activator of PSAM) lowered spontaneous firing in these PSAM-GlyR expressing VTA neurons (n = 12 neurons from 6 mice, Wilcoxon test with W = 12, p = 0.0024). d-e, Diagram showing chemogenetic inhibition of VTA neurons innervating cNAc subsequently elevated the excitability of cNAc d2SPNs (d), and administration of PSEM89S in these virus-infected mice normalized the SNI-induced social recognition deficits (n = 6 in control group, n = 6 in PSEM group; U = 4, p = 0.026). f, By the similar virus strategy (msNAc with RV-Cre injection and VTA with PSAM-5HT3 injection), the PSAM-5HT3-GFP neurons are detected in medial VTA (m-VTA). g-h, In the slices from SPAM-5HT3 infected mice, PSEM increased the excitability of VTA-msNAc neurons (GFP positive neurons, n = 11 neurons from 6 mice, Wilcoxon test with W = 66, p = 0.00100). i-j, In SNI mice, chemogenetic activation of VTA neurons projecting to msNAc reversed tactile allodynia (n = 5 in control group, n = 6 in PSEM group; U = 0, p = 0.0043). Data are analyzed by Mann–Whitney U test and presented as whisker box plots displaying median, lower and upper quartiles, and whiskers representing minimum and maximum of the data.

Figure 5.. SNI selectively decreased the intrinsic excitability of cNAc d2SPNs.

a, Sample recordings from Sham and SNI slices (calibration bars: 20 mV, 200 ms). 5 days after Sham or SNI surgery, acute slices of cNAc were collected and input/output responses of intrinsic d2SPN excitability were obtained with depolarizing current injections (current injections: −40, 60, 120 and 160 pA). b-d, In SNI slices, intrinsic d2SPN excitability was reduced (n = 10–11 neurons from 5 mice per group; b, U = 12717, p = 0.0008) and accompanied by a longer first spike latency (c, U = 139, p < 0.0001) and elevated rheobase current (d, U = 18, p = 0.0065) in cNAc d2SPNs from SNI. e-h, the excitability of cNAc d1SPNs (input/output curve, first-spike latency and rheobase current) was unchanged by SNI (each group n=10–11 neurons from 5 mice; current injection for e : −40, 100, 220 and 260 pA; U = 15534, p = 0.6657 for f, U = 484, p = 0.8831 for g, U = 44.5, p = 0.4714 for h). Data for b,c,f,g are shown as median with shaded interquartile (quartile 1 to quartile 3); data for d,h are presented as whisker box plots displaying median, lower and upper quartiles, and whiskers representing minimum and maximum of the data. Data are analyzed by Mann–Whitney U test.

Figure 6.. SNI had no effect on the morphological properties.

a: Representative reconstructed d2SPNs from sham and SNI groups. b, A three-dimensional Sholl analysis of reconstructed cNAc d2SPNs showed that the dendritic complexity of cNAc d2SPNs was unchanged by SNI (n = 8 neurons from 5 mice in each group; Mann–Whitney U test with U = 13647, p = 0.6000). c-e, SNI had no effect on the total dendritic length or average spine density of cNAc d2SPNs (n=8 neurons from 5 mice in each group with Mann–Whitney U test; U = 28, p = 0.7023 for c and U = 32, p > 0.9999 for e). Data for b are shown as median with shaded interquartile (quartile 1 to quartile 3); whisker box plots show median, lower and upper quartiles, and whiskers representing minimum and maximum of the data.

Figure 7.. SNI differentially modulated VTA dopaminergic innervation of cNAc and msNAc.

a-c, After the injection of RV-Cre into the cNAc of an Ai14-tdTomato-flox mouse, tdTomato signal was restricted to the cNAc (a) and retrogradely labeling was detected in the lateral VTA (lVTA, b-c). Robust tdTomato expression was routinely detected in the VTA, but no attempt was made to rigorously estimate the percentage of the neurons labeled. d-e, The intrinsic neuronal excitability of VTA-DA neurons innervating the cNAc was upregulated following SNI (n = 17 neurons from 6 mice per group, U = 76, p = 0.0173). d, Sample traces of VTA neurons innervating cNAc recorded from Sham and SNI slices. f-h, The VTA neurons projecting to NAc medial shell were dominantly located in medial VTA (mVTA) of the rostral midbrain. i-j, Medial VTA neurons projecting to msNAc from SNI mice had slower firing than that from Sham mice (n = 15 neurons from 6 mice per group, U = 61, p = 0.0319). ml: medial lemniscus; PBP: parabrachial pigmented nucleus of the VTA; SNr: substantia nigra, reticular part; VS: ventral subiculum; VTA: ventral tegmental area. Data are analyzed by Mann–Whitney U test and presented as whisker box plots displaying median, lower and upper quartiles, and whiskers representing minimum and maximum of the data.

Figure 8.. Schematic summarizing the adaptive synaptic and behavioral changes after pain induction.

Sustained aversive stimulation by SNI injury elevated the dopamine signaling in msNAc whereas decreased the dopamine in cNAc. This alterations of dopamine signaling in NAc appears to be adaptive – lessening the negative affect and enhancing the suppression of escape behavior, implying that cellular ‘homeostatic’ plasticity in NAc that comes with SNI is maladaptive at the network level, providing a novel therapeutic target for chronic pain.