Distinct Thalamo-Subcortical Circuits Underlie Painful Behavior and Depression-Like Behavior Following Nerve Injury

Abstract

Clinically, chronic pain and depression often coexist in multiple diseases and reciprocally reinforce each other, which greatly escalates the difficulty of treatment. The neural circuit mechanism underlying the chronic pain/depression comorbidity remains unclear. The present study reports that two distinct subregions in the paraventricular thalamus (PVT) play different roles in this pathological process. In the first subregion PVT posterior (PVP), glutamatergic neurons (PVP^Glu) send signals to GABAergic neurons (VLPAG^GABA) in the ventrolateral periaqueductal gray (VLPAG), which mediates painful behavior in comorbidity. Meanwhile, in another subregion PVT anterior (PVA), glutamatergic neurons (PVA^Glu) send signals to the nucleus accumbens D1-positive neurons and D2-positive neurons (NAc^D1→D2), which is involved in depression-like behavior in comorbidity. This study demonstrates that the distinct thalamo-subcortical circuits PVP^Glu→VLPAG^GABA and PVA^Glu→NAc^D1→D2 mediated painful behavior and depression-like behavior following spared nerve injury (SNI), respectively, which provides the circuit-based potential targets for preventing and treating comorbidity.

Keywords: chronic pain; comorbidity; depression; nucleus accumbens; paraventricular thalamus anterior; paraventricular thalamus posterior; ventrolateral periaqueductal gray.

Figure 1

The hyper‐excitability of PVPGlu and hypo‐excitability of PVAGlu were observed in the SNI‐induced comorbidity mice. a) SNI induced a persistent mechanical allodynia (Sham: n = 6 mice; SNI: n = 6 mice; F(1,10) = 44.68, p < 0.0001). b) Depression‐like behaviors including OFT, EPM, SPT, TST and FST were performed in week 6 in mice (OFT: Sham, n = 13 mice; SNI, n = 10 mice; U = 18, p = 0.0025; EPM: Sham, n = 13 mice; SNI, n = 13 mice; U = 3.5, p < 0.0001; SPT: Sham, n = 6 mice; SNI, n = 9 mice; t(13) = 5.092, p = 0.0002; TST: Sham, n = 11 mice; SNI, n = 11 mice; t(20) = 3.061, p = 0.0062; FST: Sham, n = 13 mice; SNI, n = 11 mice; t(22) = 8.871, p < 0.0001). c) Left: from sagittal slice, cFos expression was increased in PVP, but not PVA, in comorbidity mice. Scale bar, 1000 um. Right: the magnified images. Scale bar, 200 um (PVA: Sham, n = 13 mice; SNI, n = 10 mice; U = 55, p = 0.5629; PVP: Sham, n = 13 mice; SNI, n = 10 mice; t(21) = 2.381, p = 0.0268). d) Left: the immunostaining results for the coronal section showed that the expression of cFos in PVA and PVP was colocalized with CaMKIIa‐positive neurons. Scale bar, 100 um. Right: quantitative analysis of the colocalization (n = 12 images from 3 mice). e) Schema of PVP with AAV‐DIO‐mCherry in CaMKIIa‐Cre mice. f) Typical images and quantitative analysis of immunofluorescence showed the colocalization of mCherry‐positive cells with CaMKIIa‐positive neurons following intra‐PVP injection of AAV‐DIO‐mCherry in CaMKIIa‐Cre mice. Scale bar, 100 um (n = 10 images from 3 mice). g) Representative trace (upper) and statistical data (lower) for the action potential firing recorded from PVPGlu neurons in week 6 following SNI or sham (Sham, n = 25 cells from 5 mice; SNI, n = 20 cells from 4 mice; F(1, 43) = 4.968, p = 0.0311). h) Scheme for the location of AAV‐CaMKIIa‐Gcamp6s injection (upper), typical image of viral expression and optical fiber in PVP of mice (lower). Scale bar, 500 um. i) Example of fiber photometry traces (upper) and quantitative analysis for histogram of area under the curve (AUC) of fiber photometry traces (lower) from PVPGlu neurons in mice treated with sham or SNI (Sham, n = 8 mice; SNI, n = 8 mice; U = 12, p = 0.0379). j) Schema of PVA injection with AAV‐DIO‐mCherry in CaMKIIa‐Cre mice (upper). Typical immunofluorescence images of the injection site and viral expression was co‐located with CaMKIIa‐positive neurons (lower). Scale bar, 100 um. k) Representative traces (upper) and statistical data (lower) for the action potential firing recorded from PVAGlu neurons on week 6 in mice treated with sham or SNI (Sham, n = 30 cells from 5 mice; SNI, n = 24 cells from 4 mice; F(1, 52) = 7.361, p = 0.009). l) Schema of the location of AAV‐CaMKIIa‐GCamp6s injection (upper), typical image of viral expression and optical fiber in PVA of mice (lower). Scale bar, 1000 um. m) Example (upper) and histogram of area under the curve (AUC) (lower) of fiber photometry traces from PVAGlu neurons in mice treated with sham or SNI (Sham, n = 7 mice; SNI, n = 11 mice; t(16) = 2.833, p = 0.012). All data were presented as the mean ± s.e.m. *P < 0.05, **P < 0.01. For detailed statistical information, see Supporting Table.

Figure 2

PVP^Glu hyperexcitability contributed to the painful behavior in the comorbidity mice. a) Schedule of experimental process (left) and schematic diagram (upper right) of the injection of AAV‐flex‐taCasp3 in PVP in CaMKIIa‐Cre mice. AAV‐flex‐taCasp3 application ablated the glutamatergic neurons of PVP in CaMKIIa‐Cre mice (lower right). Scale bar, 100 um. b) Ablation of PVP^Glu significantly attenuated the mechanical allodynia induced by SNI in CaMKIIa‐Cre mice (mCherry, n = 5 mice; taCasp3, n = 5 mice. t(8) = 3.191, p = 0.0128). c) Schedule of experimental process (left) and schematic diagram and typical image (right) for the injection location of the GABA_A receptor agonist muscimol (Mus). Scale bar, 1000 um. d) Intra‐PVP injection Mus relieved the mechanical allodynia in the comorbidity mice (Saline, n = 6 mice, Mus, n = 6 mice; t(10) = 2.321, p = 0.0427). e) Schedule and image for the injection of AAV‐DIO‐hM4Di‐mCherry into PVP and intraperitoneal injection CNO, and whole‐cell recording showed the inhibition of CNO on PVP^Glu neurons activity following intra‐PVP injection of AAV‐DIO‐hM4Di‐mCherry in CaMKIIa‐Cre mice. Scale bar, 100 um. f) The mechanical allodynia induced by SNI surgery was attenuated by intraperitoneal injection CNO following injection AAV‐DIO‐hM4Di‐mCherry into PVP in CaMKIIa‐Cre mice (mCherry, n = 4 mice; t(3) = 0.6441, p = 0.5654. hM4Di, n = 7 mice; t(6) = 3.383, p = 0.0148). g) Schedule and image for the injection of AAV‐DIO‐eNpHR‐mCherry into PVP, and yellow light inhibited the PVP^Glu neurons activity following intra‐PVP injection of AAV‐DIO‐eNpHR‐mCherry in CaMKIIa‐Cre mice. Scale bar, 500 um. h) The paw withdrawal threshold significantly increased by yellow light stimulation (594 nm, 10 mW, constant) in comorbidity mice after SNI surgery (eNpHR, n = 8 mice; t(7) = 4.373, p = 0.0033). i) Schedule and schematic diagram for the injection of virus, whole‐cell recording showed the activation of CNO on PVP^Glu neurons following intra‐PVP injection of AAV‐DIO‐hM3Dq‐mCherry in CaMKIIa‐Cre mice. j) Application with CNO (i.p.) decreased the paw withdrawal threshold in naïve CaMKIIa‐Cre mice with injection of AAV‐DIO‐hM3Dq‐mCherry (mCherry, n = 5 mice; t(4) = 0.0311, p = 0.9767. hM3Dq, n = 8 mice; t(7) = 4.258, p = 0.0038). k) Schedule and schematic diagram for the injection of optogenetic virus, and the traces to show that blue light activated the PVP^Glu neurons. l) The blue light stimulation (473 nm, 10 mW, 20 Hz, 5 ms) significantly induced painful behavior in naive mice (ChR2, n = 7 mice; t(6) = 3.35, p = 0.0154). All data were presented as the mean ± s.e.m. *P < 0.05, **P < 0.01. For detailed statistical information, see Supporting Table.

Figure 3

The reduced excitability of PVAGlu contributed to SNI‐induced depression‐like behaviors in mice. a) Schema of cannula implantation in PVA (left) and the representative image of cannula trace (right). Scale bar, 100 um. b) Microinjection of Gabazine (GBZ) into the PVA increased the time spent in center in OFT and in open arms in EPM, and also decreased the immobility time in TST and FST (OFT: Saline, n = 6 mice; GBZ, n = 6 mice; t(10) = 2.498, p = 0.0316. EPM: Saline, n = 6 mice; GBZ, n = 6 mice; U = 3, p = 0.0152. TST: Saline, n = 5 mice; GBZ, n = 5 mice; t(8) = 2.932, p = 0.0189. FST: Saline, n = 6 mice; GBZ, n = 6 mice; t(10) = 3.935, p = 0.0028). c) Scheme for the injection location of AAV‐DIO‐hM3Dq‐mCherry in PVA of CaMKIIa‐Cre mice. d) The depression‐like behaviors were explored before and after by intraperitoneal injection of CNO (OFT: hM3Dq, n = 8 mice; t(7) = 3.801, p = 0.007. EPM: hM3Dq, n = 8 mice; t(7) = 3.343, p = 0.012. SPT: hM3Dq, n = 5 mice; t(4) = 3.467, p = 0.026. TST: hM3Dq, n = 5 mice; t(4) = −4.117, p = 0.015. FST: hM3Dq, n = 6 mice; t(5) = −3.273, p = 0.022). e) Scheme of PVA injection with AAV‐DIO‐ChR2‐mCherry and the implantation of optical fiber for blue light stimulation (left) and the typical image of localized fluorescence and optical fiber trace (right). Scale bar, 100 um. f) Photostimulation attenuated the depression‐like behaviors in comorbidity mice after SNI treatment (OFT: ChR2, n = 7 mice; t(6) = 2.677, p = 0.037. EPM: ChR2, n = 7 mice; t(6) = 3.387, p = 0.015. TST: ChR2, n = 8 mice; t(7) = −4.567, p = 0.003. FST: ChR2, n = 6 mice; t(5) = −5.746, p = 0.002). g) Scheme for the injection location of AAV‐DIO‐hM4Di‐mCherry in PVA of CaMKIIa‐Cre mice. h) Intraperitoneal injection CNO significantly reduced the time spent in center of OFT, in open arms of EPM and the sucrose preference of SPT, and also increased the immobility time in TST and FST in naïve mice (OFT: hM4Di, n = 9 mice; t(8) = −4.361, p = 0.002. EPM: hM4Di, n = 9 mice; Z = −2.547, p = 0.011. SPT: hM4Di, n = 8 mice; t(7) = −2.796, p = 0.027. TST: hM4Di, n = 5 mice; t(4) = 2.953, p = 0.042. FST: hM4Di, n = 8 mice; t(7) = 4.367, p = 0.003). i) Scheme of PVA injection with AAV‐DIO‐eNpHR 3.0‐mCherry and the implantation of optical fiber for yellow light stimulation (left) and the typical image of localized fluorescence and optical fiber trace (right). Scale bar, 100 um. j) Stimulation with yellow light (594 nm) dramatically induced the depressive‐like behaviors in naïve mice (t‐test, *p < 0.05 versus the corresponding to before stimulation, eNpHR group (OFT: eNpHR, n = 7 mice; t(6) = −2.957, p = 0.025. EPM: eNpHR, n = 7 mice; t(6) = −2.816, p = 0.031. TST: eNpHR, n = 7 mice; t(6) = 3.321, p = 0.016. FST: eNpHR, n = 6 mice; t(5) = 3.336, p = 0.021). *P < 0.05, **P < 0.01. For detailed statistical information, see Supporting Table.

Figure 4

The enhanced excitability of VLPAG^GABA contributed to the chronic pain in the comorbid. a) Schema of PVP injection of AAV‐CaMKIIa‐EGFP in mice. b) Representative image of EGFP+ fiber in PAG of mice with PVP injection of AAV‐CaMKIIa‐EGFP. Scale bar, 100 µm. c) Schema of VLPAG injection AAV‐DIO‐mCherry in GAD2‐Cre mice (left) and typical images of restricted fluorescence in VLPAG (right). Scale bar, 100 µm. d) Representative traces (left) and summary (right) of action potential firing responsive to depolarizing current injections in VLPAG^GABA neurons (Sham, n = 15 cells from 3 mice; SNI, n = 23 cells from 4 mice; F(1, 36) = 5.42, p = 0.0256). e) Schema of VLPAG injection AAV‐DIO‐hM4Di‐mCherry in GAD2‐Cre mice and typical images of restricted fluorescence in VLPAG. Scale bar, 100 µm. f) The mechanical allodynia induced by SNI treatment was significantly alleviated by intraperitoneal application CNO (hM4Di, n = 6 mice; t(5) = 2.864, p = 0.035). g) Schema of injection of AAV‐DIO‐eNpHR‐mCherry into VLPAG and implantation of optic fiber in GAD2‐Cre mice (left) and the typical images of injection site (right). Scale bar, 100 µm. h) The paw withdrawal threshold was increased by inhibition of VLPAG^GABA with yellow light stimulation (eNpHR, n = 6 mice; t(5) = 3.375, p = 0.0198). i) Schema of VLPAG injection with AAV‐DIO‐hM3Dq‐mCherry in GAD2‐Cre mice and typical fluorescence images of injection site in VLPAG. Scale bar, 100 µm. j) Activation of VLPAG^GABA by CNO‐induced mechanical allodynia in the naïve mice (hM3Dq, n = 5 mice; t(4) = −3.276, p = 0.0306). k) Schema of injection of AAV‐DIO‐ChR2‐mCherry into VLPAG and implantation of optic fiber in GAD2‐Cre mice (left) and the typical images of injection site (right). Scale bar, 100 µm. l) The paw withdrawal threshold was decreased by activation of VLPAG^GABA with blue light stimulation in naïve mice (ChR2, n = 4 mice; t(3) = −3.952, p = 0.0289). All data were presented as the mean ± s.e.m. *P < 0.05, **P < 0.01. For detailed statistical information, see Supplementary Table.

Figure 5

The PVP^Glu→VLPAG^GABA circuit contributed to the mechanical allodynia in comorbidity mice. a) Schema of virus injection in mice. b) Representative image and quantitative analysis of the colocalization of mCherry‐positive neurons and GABA‐positive in VLPAG. Scale bar, 50 µm (n  =  25 images from 3 mice). c) Schema of the Cre‐dependent retrograde trans‐monosynaptic RV tracing strategy in GAD2‐Cre mice. d) Left: typical image of the injection site and viral expression within the VLPAG of GAD2‐Cre mice. Starter cells (yellow) co‐express AAV‐DIO‐TVA‐EGFP, AAV‐DIO‐RVG (green) and RV‐ENVA‐ΔG‐DsRed (red). Scale bar, 200 µm. The middle images depicted the area shown in the dotted line square of the VLPAG. Scale bar, 100 µm. Right: DsRed‐labeled neurons within the PVP. Scale bar, 200 µm. e) Schema of PVP injection of AAV‐CaMKIIa‐ChR2‐mCherry and VLPAG injection of AAV‐DIO‐EGFP in GAD2‐Cre mice and the recording configuration in acute slices (Left). Typical images of the injection site and viral expression (Right). Scale bar, 200 µm. f) Representative traces for the recording of light‐evoked current (473 nm, 20 ms) before and after DNQX (20 µM) treatment. g) One example of EPSCs recorded in VLPAG^GABA neuron in ACSF and after the sequential application of TTX (1 µM) and 4‐AP (100 µM). h) Schema of virus injection and optical fiber photometer recording configuration. i) The heatmaps (left) and the mean (right) showed that the Ca²⁺ signal rapidly increased by the persistent red‐light stimulation (635 nm) in CaMKIIa‐Cre mice with PVP injection of AAV‐DIO‐ChrimsionR‐mCherry and VLPAG injection of AAV‐CaMKIIa‐GCamp6s (n = 4). j) Schema of injection of AAV‐DIO‐hM4Di‐mCherry into PVP of CaMKIIa‐Cre mice and implantation of cannula into VLPAG (Left) and typical images of mCherry+ fiber and cannula tract (Right). Scale bar, 200 µm. k) Intra‐VLPAG injection of CNO relieved the SNI‐induced mechanical allodynia in the mice microinjected with AAV‐DIO‐hM4Di‐mCherry into PVP of CaMKIIa‐Cre mice (mCherry, n = 5 mice; Z = −0.365, p = 0.715. hM4Di, n = 6 mice; Z = −2.201, p = 0.028). l) Schema of injection of AAV1‐hSyn‐Cre into PVP and AAV‐DIO‐hM4Di‐mCherry into VLPAG. m) The mechanical allodynia was rescued by intraperitoneal injection of CNO in SNI‐treated comorbidity mice (hM4Di, n = 4 mice; t(3) = 3.481, p = 0.04). n) Schema of injection of AAV1‐hSyn‐Cre into PVP and AAV‐DIO‐eNpHR‐mCherry into VLPAG and implantation of cannula into VLPAG. o) The paw withdrawal threshold was increased through inhibition PVP‐projected VLPAG neurons with light stimulation (eNpHR, n = 6 mice; t(5) = 2.802, p = 0.038). p) Schema of PVP^Glu projections onto VLPAG^GABA neurons. The schematic diagram was created with BioRender.com. *P < 0.05. For detailed statistical information, see Supporting Table.

Figure 6

NAc^D2 participated in the depression‐like behavior in comorbidity mice. a) Schema of PVA injection of AAV‐CaMKIIa‐EGFP in mice (Left) and typical image of injection site (Right). Scale bar, 100 µm. b) Representative image of EGFP⁺ fiber in NAc of mice with PVA injection of AAV‐CaMKIIa‐EGFP. Scale bar, 100 µm. c) Schematic of virus injection. d) Images (left) and statistic pie chart (right) showing that EGFP‐labeled neurons were colocalized with D2‐positive neurons in NAc. Scale bar, 10 µm (n  =  16 images from 3 mice). e) Schema of the Cre‐dependent retrograde trans‐monosynaptic RV tracing strategy in D2‐Cre mice. f) Left: typical image of the injection site and viral expression in the NAc of D2‐Cre mice. Scale bar, 100 µm. Right: DsRed‐labeled neurons within the PVA. Scale bar, 100 µm. g) Schema of NAc injection of AAV‐DIO‐mCherry in D2‐Cre mice. h) Representative traces and summary (right) of action potential firing rates in response to depolarizing current injections in D2 neurons (Sham, n = 21 neurons from 4 mice; SNI, n = 26 neurons from 4 mice; F(1, 45) = 6.812, p = 0.0122). i) Schema of injection of AAV‐DIO‐hM3Dq‐mCherry into NAc. j) The depression‐like behaviors were explored before and after by intraperitoneal injection of CNO (OFT: hM3Dq, n = 5 mice; t(4) = 3.319, p = 0.029. EPM: hM3Dq, n = 5 mice; t(4) = 00617, p = 0.022. SPT: hM3Dq, n = 5 mice; t(4) = 3.167, p = 0.034. TST: hM3Dq, n = 5 mice; t(4) = −3.407, p = 0.027. FST: hM3Dq, n = 5 mice; t(4) = −3.169, p = 0.034). k) Schema of injection of AAV‐DIO‐ChR2‐mCherry and implantation of optical fiber into NAc (left), and typical image of virus expression and implantation site in NAc in D2‐Cre mice (Right). Scale bar, 100 µm. l) The depression‐like behaviors were examined before and after blue light stimulation (OFT: ChR2, n = 5 mice; t(4) = 3.913, p = 0.017. EPM: ChR2, n = 5 mice; t(4) = 2.939, p = 0.042. TST: ChR2, n = 5 mice; t(4) = −3.852, p = 0.018. FST: ChR2, n = 5 mice; t(4) = −5.177, p = 0.007). m) Schema of NAc injection of AAV‐DIO‐hM4Di‐mCherry in D2‐Cre mice. n. Continuous injection of CNO induced depression‐like behaviors. (OFT: hM3Dq, n = 6 mice; t(5) = −2.614, p = 0.047. EPM: hM3Dq, n = 6 mice; t(5) = −2.629, p = 0.047. SPT: hM3Dq, n = 6 mice; t(5) = −2.971, p = 0.031. TST: hM3Dq, n = 6 mice; t(5) = 4.242, p = 0.008. FST: hM3Dq, n = 6 mice; t(5) = 2.849, p = 0.036). *P < 0.05, **P < 0.01. For detailed statistical information, see Supporting Table.

Figure 7

The role of PVA^Glu→NAc^D2 in the depression‐like behavior in comorbidity mice. a) Schema of PVA injection of AAV‐DIO–ChR2–mCherry and NAc injection of AAV‐D2–EGFP in CaMKII‐Cre mice (Left) and typical images of injection site and viral expression in NAc (Right). Scale bar, 100 µm. b) Blue light stimulation (470 nm, 5 ms) of PVA^Glu fiber induced an EPSCs and an IPSCs in each responsive NAc^D2 neurons, and blocked by bath application of DNQX (20 µM). c) An example of EPSCs recorded in NAc^D2 neuron in ACSF and after the sequential application of TTX (1 µM) and 4‐AP (100 µM). d) Schematic of PVA injection of AAV‐CaMKIIa‐ChrimsonR‐mCherry and NAc injection of AAV‐DIO‐GCamp6s in D2‐Cre mice. e) The heatmaps (left) and the mean (right) show that Ca2+ signal rapidly increased with persistent red‐light stimulation (635 nm) in D2‐Cre mice with PVA injection of AAV‐CaMKIIa‐ChrimsionR‐mCherry and NAc injection of AAV‐DIO‐GCamp6s (n = 7). f) Schema of injection of AAV1‐hSyn‐Cre into PVA and AAV‐DIO‐hM3Dq‐mCherry into NAc (left), and typical image of injection site in NAc (Right). Scale bar, 100 µm. g) The depression‐like behaviors were explored before and after intraperitoneal injection of CNO (OFT: hM3Dq, n = 7 mice; t(6) = 4.89, p = 0.003. EPM: hM3Dq, n = 7 mice; t(6) = 3.195, p = 0.019. SPT: hM3Dq, n = 7 mice; t(6) = 4.152, p = 0.006. TST: hM3Dq, n = 7 mice; t(6) = −2.452, p = 0.0497. FST: hM3Dq, n = 6 mice; t(5) = −7.865, p = 0.001). h) Schema of injection of AAV1‐hSyn‐Cre into PVA and AAV‐DIO‐ChR2‐mCherry into NAc, and implantation of optical fiber into NAc. i) The depression‐like behaviors were tested before and after blue‐like stimulation (OFT: ChR2, n = 4 mice; t(3) = 5.744, p = 0.0105. EPM: ChR2, n = 4 mice; t(3) = 3.419, p = 0.042. TST: ChR2, n = 4 mice; t(3) = −5.665, p = 0.011. FST: ChR2, n = 4 mice; t(3) = −10.205, p = 0.002). j) Schema of PVA injection of AAV1‐Cre and NAc injection of AAV‐DIO‐hM4Di‐mCherry. k) Depression‐like behaviors were performed before and after injection of CNO (OFT: hM3Dq, n = 6 mice; t(5) = −3.512, p = 0.017. EPM: hM3Dq, n = 6 mice; t(5) = −3.233, p = 0.023. SPT: hM3Dq, n = 6 mice; t(5) = −4.036, p = 0.01. FST: hM3Dq, n = 6 mice; t(5) = 5.382, p = 0.003). *P < 0.05, **P < 0.01. For detailed statistical information, see Supporting Table.

Figure 8

The local NAc^D1→D2 circuits. a) Schema of PVA injection of AAV‐DIO–ChR2–mCherry and NAc injection of AAV‐D2–EGFP in CaMKII‐Cre mice. b) The IPSCs were blocked by the incubation of bicuculline (BIC, 10 µM). c) Images (left) and statistic pie chart (right) showed that EGFP‐labeled neurons were colocalized with D2‐positive neurons in NAc. Scale bar, 10 µm (n  =  14 images from 3 mice). d) Schema of NAc injection of AAV‐DIO‐mCherry in D1‐Cre mice. e) Representative traces (left) and summary (right) of action potential firing rates in response to depolarizing current injections in D1 neurons (Sham, n = 18 cells from 4 mice; SNI, n = 21 cells from 4 mice; F(1, 37) = 7.155, p = 0.0111). f) Schema of PVA injection of AAV‐DIO–ChR2–mCherry and NAc injection of AAV‐D1–EGFP in CaMKII‐Cre mice and the recording configuration in the acute brain slices (left), and typical image of virus expression (right). Scale bar. 100 µm. g) Optostimulation (blue light, 470 nm, 5 ms) of PVA^Glu fiber terminal induced an EPSCs in NAc^D1 neurons, which were blocked by the bath application of DNQX (20 µM). h) Schema of PVA injection of AAV‐CaMKIIa‐ChrimsonR‐mCherry and NAc injection of AAV‐DIO‐GCamp6s in D1‐Cre mice (left), and typical image of viral expression of injection site (right). Scale bar, 50 µm. i) The heatmaps (left) and the mean (right) showed that Ca²⁺ signal rapidly increased following persistent red‐light stimulation (635 nm) in D1‐Cre mice (n = 4). j) Schema of injection of AAV‐D1‐ChR2‐mCherry and AAV‐DIO‐EGFP into NAc in D2‐Cre mice (left), and typical images of injection site and viral expression in NAc (right). Scale bar, 10 µm. k) For recording in the NAc^D2 neurons, optostimulation (blue light, 470 nm, 5 ms) induced an IPSC, which was blocked by incubation of BIC (10 µM). l) A model of long‐range and local PVA^Glu→ NAc^D1→D2 circuits. The schematic diagram was created with BioRender.com. *P < 0.05. For detailed statistical information, see Supporting Table.