Pain modulates dopamine neurons via a spinal-parabrachial-mesencephalic circuit

Abstract

Pain decreases the activity of many ventral tegmental area (VTA) dopamine (DA) neurons, yet the underlying neural circuitry connecting nociception and the DA system is not understood. Here we show that a subpopulation of lateral parabrachial (LPB) neurons is critical for relaying nociceptive signals from the spinal cord to the substantia nigra pars reticulata (SNR). SNR-projecting LPB neurons are activated by noxious stimuli and silencing them blocks pain responses in two different models of pain. LPB-targeted and nociception-recipient SNR neurons regulate VTA DA activity directly through feed-forward inhibition and indirectly by inhibiting a distinct subpopulation of VTA-projecting LPB neurons thereby reducing excitatory drive onto VTA DA neurons. Correspondingly, ablation of SNR-projecting LPB neurons is sufficient to reduce pain-mediated inhibition of DA release in vivo. The identification of a neural circuit conveying nociceptive input to DA neurons is critical to our understanding of how pain influences learning and behavior.

Extended Data Fig. 1.. Neuroanatomical characterization of LPB→SNR and LPB→VTA neurons.

(a) eYFP-expressing LPB_VGLUT2 neurons. (b-g) LPB_VGLUT2 terminals expressing eYFP in different brain regions. Bregma: 0.14 mm (b), −1.22 mm (c), −1.46 mm (d), −1.46 mm (e), 3.40 mm (f), −3.40 mm (g). Note fibers of passage in VTA (red arrow) (scale bar 50 μm). (h) Mean eYFP fluorescence intensity in different brain areas (n = 5 mice; Data represent mean ± SEM). (i) Synaptophysin-mCherry expression in LPB (left) and ventral midbrain (right) from VGLUT2-Cre mice (scale bars 50 μm). (j) High resolution images of synaptophysin-expressing LPB_VGLUT2 terminals close to lateral VTA (lVTA) TH-immunopositive and SNR GABA-immunopositive neurons (scale bar 20 μm). (k) Mean synaptophysin intensity (quantified as number of particles in a defined region) for different ventral midbrain subregions (n = 3 mice). (l) Experimental design. (m) Sample of a recorded and neurobiotin (NB)-filled, mCherry-positive SNR cell (scale bar 50 μm). (n) Light-evoked EPSC recorded in mCherry-positive SNR cell (black) in response to stimulation of LPB inputs (red trace: 20 μM CNQX). (o) Mean EPSC amplitude before (ACSF) and after CNQX application. (p) eYFP-expressing LPB_GAD2 neurons at bregma −5.30 mm. (q-s) LPB_GAD2 terminals expressing eYFP in (q) DLPAG (Bregma: −4.60 mm), but not in (r) ventral midbrain (Bregma: −3.30 mm) or (s) amygdala (Bregma: −1.50 mm; scale bar 200 μm). (t) Left: Retrobead injection site in NAcLat of a VGLUT2-Cre mouse for experiment in Fig. 1g (scale bar 200 μm). Middle/Right: lVTA cell that was recorded for experiment in Fig. 1g and filled with neurobiotin (NB). It is retrogradely labeled (i.e., projects to NAcLat) and TH-immunopositive (scale bar 50 μm). (u) Same as in (t) but cell is in SNR and TH-immunonegative (scale bar 50 μm). (v) Recorded VTA cell from a GAD2tdTomato mouse and filled with NB (Refers to Fig. 1k; scale bar 50 μm). Data represent mean ± SEM. Significance was calculated by means of one-way RM ANOVA with Tukey’s post-hoc test (k) or paired t-test (o). * p < 0.05, ** p < 0.01, *** p < 0.001.

Extended Data Fig. 2.. Whole brain mapping of monosynaptic inputs to LPB→SNR and LPB→VTA neurons.

(a) Schematic showing AAV helper virus (DIO-TVA and DIO-RVG) injections into LPB and EvA-RV-GFP into SNR or VTA of VGLUT2-Cre mice. (b) Sample coronal brain section showing VTA-projecting starter cells in LPB (green: EnvA-ΔG-GFP; red: TVA-mCherry, blue: DAPI; scale bar 100 μm). (c) Bar graph showing quantification of TVA-expressing cells (red) in LPB. (d) Bar graph showing number of starter cells in LPB. (e) Total number of EnvA-ΔG-GFP labeled cells across all brain regions analyzed. (f) Sample images showing GFP-expressing cells (green) that make monosynaptic inputs onto LPB→SNR or LPB→VTA neurons for different brain areas (scale bar 100 μm). (g) Quantification of inputs to LPB→SNR and LPB→VTA neurons. Data are presented as a percentage of total input neurons counted in each individual brain region (BNST: bed nucleus of the stria terminalis, CeA: central nucleus of the amygdala, BLA: basolateral amygdala, LH: lateral hypothalamic area, ZI: zona incerta, VTA: ventral tegmental area, SNR: substantia nigra, reticular part, MG: medial geniculate nucleus, PAG: periaqueductal grey, vlPAG: ventrolateral periaqueductal gray, SC: superior colliculus, LL: lateral lemniscus, DR: dorsal raphe nucleus, CnF: cuneiform nucleus, PPT: pedunculopontine tegmental nucleus, LDT: laterodorsal tegmental nucleus, MPB: medial parabrachial nucleus, LPBi: ipsilateral LPB, PACRt:parvicellular reticular nucleus, Gi: gigantocellular reticular nucleus; n = 4–5 mice). Data represent mean ± SEM. Significance was calculated by means of unpaired t-test. * p < 0.05. Comparisons with no asterisk had p > 0.05 and were not considered significant.

Extended Data Fig. 3.. Heterogenous responses to noxious stimuli in LPB neurons.

(a) Schematic of experimental design showing in vivo electrophysiological recordings of non-projection defined LPB neurons. Animals were exposed to tail pinch, heat, and electrical shock. (b) Pie graphs represent the proportion of LPB cells that are excited (green), inhibited (red) and do not respond (grey) to tail pinch, heat, or electrical tail shock (n = 63–97 cells from n = 5 mice). (c) Samples of spike raster plots and firing patterns for two different LPB cells that either excited (top) or inhibited (bottom) in response to noxious stimuli. Note that the top and bottom graphs are from the same LPB cell (i.e., an LPB cell that was excited by all three noxious stimuli (top) and another LPB cell that was inhibited by all three noxious stimuli (bottom)). (d,e) Analysis for LPB cells that were recorded in response to all three noxious stimuli (n = 60 cells). Overlap between the proportion of LPB cells that were excited (top, green, n = 30 cells) or inhibited (bottom, red, n = 16 cells) in response to individual noxious stimuli (d) and LPB cells that show both excitatory and inhibitory responses for individual noxious stimuli (left, n = 9 cells) or did not respond at all (right, n = 5 cells) (e).

Extended Data Fig. 4.. Noxious stimuli activate LPB→SNR neurons and alter excitatory and inhibitory transmission.

(a) Mice were subjected to a hot plate test (c,e,f) or received unilateral intraplantar injection of 1% formalin or saline (d,g,h). (b) LPB subregions are highlighted in different colors to demonstrate location of LPB subpopulations projecting to SNR (blue), VTA (red) or CeA (yellow). (c) Mean number of c-Fos-immunopositive cells in animals exposed to 37°C (white triangle) or 55°C (red triangle) heat and mean number of FG labeled cells for each temperature level (white and red circle, respectively) for different LPB subregions described in (b) (37°C: n = 3 mice; 55°C: n = 4 mice). (d) Same as in (c), but for animals that received intraplantar injections of 1% formalin or saline (n = 4 mice). (e) Retrogradely-labeled SNR-projecting LPB cells (green, FG) and c-Fos immunoreactivity (red) in response to 37°C (left) or 55°C (right) heat (scale bar 50 μm). (f) Animals that have been exposed to 55°C heat display significantly increased c-Fos immunoreactivity in LPB cells (left) and in SNR-projecting LPB neurons (right, FG-positive cells) when compared to 37°C heat. No difference in mean number of FG-labeled LPB cells between 37°C and 55°C heat (middle). (g,h) Same as in (e,f), but for animals that received intraplantar injections of 1% formalin or saline. (i-k) Sample traces of miniature inhibitory postsynaptic currents (mIPSCs) from SNR-projecting LPB neurons recorded from animals that received intraplantar injections of 1% formalin (red) or saline (grey; cells recorded in 1 μM TTX, 20 μM CNQX, 50 μM D-AP5) (i). Cumulative probability plots and bar graphs of the means from the frequencies (j) and amplitudes (k) of mIPSCs recorded from SNR-projecting LPB neurons (saline: n = 13 cells; formalin: n = 14 cells). (l-n) Same as in (i-k), but for recordings of miniature excitatory postsynaptic currents (mEPSCs; cells recorded in 1 μM TTX, 100 μM picrotoxin; saline: n = 11 cells; formalin: n = 12 cells). Data represent mean ± SEM. Significance was calculated by means of unpaired t-test for across group comparison (f,h,j,k,m,n). * p < 0.05, ** p < 0.01.

Extended Data Fig. 5.. Optogenetic silencing of the LPB→SNR pathway does not affect locomotor activity and general licking behavior.

(a) Serial reconstruction of viral injection sites in LPB. Right panels show representative examples of NpHR-eYFP (green) injection sites across the rostro-caudal extent of the LPB (scale bar 100 μm). Left panels show schematics of the corresponding brain regions in which NpHR-eYFP was detected. Each color represents the expression profile from a single mouse that was used for the experiments shown in Fig. 4a–f. (b) Corresponding serial reconstructions of optical fiber locations in the SNR (TH: red; scale bar 100 μm). (c) Schematic design of open field test for assessing the effects of optogenetic silencing of the LPB→SNR pathway on general locomotor activity. (d) Representative trajectories of animals expressing NpHR (top) or eYFP (bottom). (e,f) Optogenetic silencing of the LPB→SNR pathway does not have a significant effect on the (e) mean distance traveled (a measure for locomotor activity) or (f) time spent in the center of the box in NpHR- (n = 9 mice) and eYFP-expressing (n = 5 mice) mice. (g) Schematic showing control experiment for studying general licking behavior in response to optogenetic silencing of LPB→SNR neurons. (h) Bar graphs showing mean number (left) and duration (right) of licks for sucrose solution in NpHR- (n = 8 mice) and eYFP-expressing (n = 6 mice) mice. Data represent mean ± SEM. Significance was calculated by means of one-way RM ANOVA with Tukey’s post-hoc test (e,f) or unpaired t-test (h). Comparisons with no asterisk had p > 0.05 and were not considered significant.

Extended Data Fig. 6.. Genetic ablation of LPB→SNR neurons reduces behavioral responses to formalin-induced pain.

(a) Injection of retrogradely-transported pseudotyped equine infectious anemia virus expressing Cre-recombinase (RG-EIAVCre) into the right SNR and AAV carrying Cre-dependent Caspase 3 (CASP) or eYFP into the right LPB of C57BL/6 mice. 5 weeks later, mice received bilateral intraplantar injections of 1% formalin into the hind paws. (b) Comparison between animals in which SNR-projecting LPB neurons were genetically ablated using CASP (left) and animals that express a control vector (eYFP) in SNR-projecting LPB neurons (middle). Sections were stained using an eYFP antibody (green; scale bar 50 μm). CASP animals show significantly reduced number of eYFP-positive SNR-projecting LPB cells when compared to control animals (right; CASP: n = 5 mice, eYFP: n = 5 mice). (c) Mean total distance traveled was not significantly different between CASP and eYFP animals in the open field test (CASP: n = 10 mice, eYFP: n = 7 mice). (d) Number of licks in response to formalin injection in CASP mice (n = 10 mice) for the left (blue) and right (grey) hind paws (left). Mean number of licks during phase I and phase II of the formalin test for comparison of left and right hind paws (right). (e) Same as in (d) but for analysis of lick duration. (f,g) Same as in (d,e) but for eYFP control animals (n = 7 mice). (h) Comparison of mean total number of licks (left) and mean total lick duration (right) for CASP and eYFP mice for individual hind paws. Data represent mean ± SEM. Significance was calculated by means of unpaired t-test (a,b,d,e,f,g) and one-way RM ANOVA with Tukey’s post-hoc test (h). * p < 0.05, ** p < 0.01, *** p < 0.001.

Extended Data Fig. 7.. Comparison of transsynaptic distribution of AAV1-Cre versus CAV2-Cre.

(a,b) Sample fluorescent images showing tdTomato labeled neurons (red) in different brain regions in response to (a) CAV2-Cre or (b) AAV1-Cre injection into the LPB of Ai14 mice (n = 2 mice for each condition) (DAPI: blue; scale bars 100 μm). (c) Left: Schematic showing injection of AAV1-Cre into SNR and AAV-DIO-eYFP into LPB of C57BL/6 mice (n = 2 mice). Right: Sample fluorescent image showing that eYFP-expressing cells are predominantly located in the LPBc (DAPI: blue; scale bar 100 μm).

Extended Data Fig 8.. Whole-brain projections of LPB-targeted SNR neurons.

(a) Schematic showing unilateral targeting of anterogradely-transported AAV1-Cre to LPB and AAV carrying Cre-dependent eYFP to SNR of C57BL/6 mice. (b) Representative fluorescent images showing coronal brain sections of eYFP-expressing cells in the SNR (upper left image) and eYFP-expressing terminals across different brain regions (scale bar 100 μm). (c) Quantification of fluorescence intensity of eYFP-expressing terminals in different brain regions (n = 3 mice). (d) Schematic showing injection of fluorescent retrobeads and AAV-DIO-ChR2 into the LPB of VGLUT2-Cre mice. Whole-cell patch-clamp recordings were performed from retrogradely-labeled (i.e., beads-positive) cells in the lateral SNR. (e) Left: Sample trace showing light-evoked EPSC from LPB-projecting SNR neurons (black trace) in response to light stimulation of LPB inputs. Light-evoked EPSCs are blocked by bath application of 20 μM CNQX (red trace). Right: Bar graph showing mean EPSC amplitudes before (ACSF) and after bath application of CNQX (n = 5 cells). (f) Sample image of retrogradely-labeled (beads, red) cells in the lateral SNR that were filled with neurobiotin (NB, green) during whole-cell patch-clamp recordings (scale bar 50 μm). Significance was calculated by means of paired t-test (e). * p < 0.05. Data represent mean ± SEM.

Extended Data Fig. 9.. SNR→LPB neurons have very few collaterals to NAcLat-projecting VTA DA neurons.

(a) Schematic showing injection of fluorogold (FG) into the LPB and AAVs encoding the cellular receptor for subgroup A avian leukosis viruses (TVA) and rabies virus glycoprotein (RG) in the VTA of DAT-Cre mice. In the same animals, EnvA-pseudotyped, glycoprotein-deficient rabies virus expressing GFP (EnvA-RV-GFP) was targeted to the NAcLat. (b) Representative example of coronal section of the ventral midbrain showing retrogradely-labeled cells in the lateral SNR (i.e., LPB-projecting, FG-positive, red). GFP-positive cells (green) make monosynaptic connections onto VTA DA neurons projecting to NAcLat and are mainly located in the substantia nigra pars compacta (SNc), ventral SNR (vSNR) and lateral VTA, but do not overlap with the lateral SNR (lSNR; scale bar 100 μm). (c) Pie chart showing proportion of analyzed cells (n = 4035 cells from n = 3 mice) in the ventral midbrain that express GFP (green, 19.7%) or are labeled by FG (red, 79.4%) or contain both GFP and FG (yellow, 0.9%).

Extended Data Fig. 10.. Optogenetic stimulation of LPB→VTA DA neurons does not affect locomotor activity but promotes reward-related behavior.

(a) Targeting of AAVs encoding the cellular receptor for subgroup A avian leukosis viruses (TVA) and rabies virus glycoprotein (RG) as well as EnvA-pseudotyped, glycoprotein-deficient rabies virus expressing ChR2 or GFP (EnvA-RV-ChR2/-GFP) to VTA of DAT-Cre mice. Patch-clamp recordings were performed from LPB neurons (d-f) or bilateral optical fibers were implanted above the LPB for assessment of locomotor activity in open field test (g-j). (b) Injection site in VTA (scale bar 100 μm). (c) ChR2-expressing cells in LPB, which make monosynaptic connections onto VTA DA neurons (scale bar 100 μm). (d) Sample patch-clamp recordings from LPB neurons showing light-evoked action potentials in response to 5 Hz, 10 Hz, 20 Hz or 40 Hz stimulation (scale bars 20 mV/0.5 sec). (e) Mean number of spikes in response to different stimulation frequencies (5 Hz: n = 5 cells, 10 Hz: n = 5 cells, 20 Hz: n = 5 cells, 40 Hz: n = 5 cells). (f) Neurobiotin (NB)-filled LPB cell expressing ChR2 (scale bar 20 μm). (g) Experimental design. (h) Representative trajectories of animals expressing ChR2 (top) or GFP (bottom) in LPB→VTA DA neurons. (i) 20 Hz stimulation of LPB→VTA DA neurons does not have significant effect on the mean distance traveled between ChR2- (left) and GFP-expressing (right) mice. (j) No significant difference in time spent in center area between ChR2 and GFP mice. (k) Schematic of real-time (RT) place preference assay. (l) Trajectories of sample ChR2- and GFP-expressing mice during RT place preference test. (m) ChR2- (left) but not GFP-expressing (right) mice spent significantly more time on the side of the chamber paired with light stimulation of LPB→VTA DA neurons (ChR2: n = 9 mice, GFP: n = 7 mice). Data represent mean ± SEM. Significance was calculated by means of one-way RM ANOVA with Tukey’s post-hoc test (i) and two-way RM ANOVA with Holm-Sidak’s post-hoc test (j) or paired t-tests (m). * p < 0.05.

Fig. 1.. Functional neuroanatomy of LPB projections to the ventral midbrain.

(a) Experimental design. (b) Whole brain fluorescent image showing LPB_VGLUT2 projections (eYFP = green; upper row: horizontal; lower row: sagittal). VTA, SNR and CeA are highlighted in different colors (scale bar 1 mm). (c) LPB_VGLUT2 terminals and fibers of passage (white arrows) in different ventral midbrain subregions (scale bar 50 μm). (d-f) Left: CAV2-Cre injection into (d) SNR, (e) VTA and (f) CeA of Ai14 mice (n = 3 mice for each projection target). Middle: Retrogradely labeled (tdTomato-positive, red) neurons in different LPB subregions (DAPI: blue; scale bars 100 μm). Right: Quantification of retrogradely labeled cells for different LPB subregions (LPBd: lateral parabrachial nucleus - dorsal part; LPBc: lateral parabrachial nucleus - central part; LPBe: lateral parabrachial nucleus – external part). (g) Experimental design. (h) EPSCs generated at −70 mV by light stimulation of LPB_VGLUT2 inputs to retrogradely-labeled VTA DA (i.e., TH-immunopositive; Extended Data Fig. 1t) neurons projecting to NAcLat (red trace) or non-DA (i.e., TH-immunonegative; Extended Data Fig. 1u) cells in the SNR (black trace). (i) Mean EPSC amplitudes produced by light stimulation of LPB_VGLUT2 inputs to different cell populations (DA→NAcLat: n = 21 cells; SNR: n = 25 cells; recorded in ACSF). (j) Application of 20 μM CNQX and 50 μM APV blocks EPSCs in SNR cells (n = 16 cells) (left) and NAcLat-projecting DA neurons (n = 7 cells) (right). (k) Experimental design (left) and EPSC amplitudes produced by light stimulation of excitatory LPB inputs onto GAD2-tdTomato-positive VTA neurons (right, VTA_GAD2+, n = 10 cells; recorded in ACSF; Extended Data Fig. 1v). (l,m) Spontaneous firing from (l) NAcLat-projecting VTA DA neurons and (m) SNR cells in response to 10 Hz light stimulation of LPB_VGLUT2 inputs. LPB_VGLUT2 stimulation significantly increases firing of both NAcLat-projecting DA neurons and SNR cells (NAcLat: n = 12 cells; SNR: n = 12 cells). Significance was calculated by means of paired t-test within group comparison (j), unpaired t-test (i) or one-way RM ANOVA test with Tukey’s post-hoc test (l,m). ** p < 0.01, *** p < 0.001. Data represent mean ± SEM.

Fig. 2.. Spinal cord, dorsal horn neurons target LPB→SNR neurons

(a) Overview showing viral injection of CAV2-Cre into the VTA or SNR and ChR2 (green) into the dorsal horn (lower image) of the spinal cord (L4-L5) of Ai14 mice (DAPI: blue; scale bar 100 μm). (b) ChR2-expressing terminals from the dorsal horn (green) are more frequently detected in the LPBd adjacent to retrogradely labeled (tdTomato-positive, red) cells projecting to SNR (right image) than in the LPBc, which contains mainly retrogradely labeled (tdTomato-positive, red) cells projecting to VTA (left image; DAPI: blue; scp: superior cerebellar peduncle; scale bar 50 μm). Insets show higher magnification images of LPB→VTA and LPB→SNR neurons in the LPBc and LPBd, respectively. (c) Schematic showing dorsal horn spinal cord projections to different LPB subregions, which are highlighted in different colors to demonstrate the locations of LPB subpopulations projecting to SNR (violet) or VTA (magenta). Note that CeA-projecting LPB neurons are predominantly located in the lateral parabrachial nucleus - external part (LPBe) (MPB: medial parabrachial nucleus). (d) EPSC generated at −70 mV by light stimulation of dorsal horn inputs to a retrogradely-labeled LPB neuron projecting to SNR (red trace: sample response after bath application of 20 μM CNQX; n = 10 cells; left) and mean EPSC amplitudes generated by light stimulation of dorsal horn inputs to LPB→SNR (n = 33 cells) and LPB→VTA neurons (n = 32 cells; right). Significance was calculated by means of unpaired t-test (q). Comparisons with no asterisk had p > 0.05 and were not considered significant. Data represent mean ± SEM.

Fig. 3.. LPB→SNR neurons represent diverse painful stimuli.

(a) Experimental design. (b) Response from LPB→SNR neurons to tail shock (red dashed lines; scale bars 0.5 mA/2 s). (c-e) Z score averages for LPB→SNR fluorescence in response to (c) tail shock, (d) tail brush, and (e) tail pinch for all animals (n = 8 trials per stimuli; n = 5 mice). (f) Sample response from LPB→SNR neurons to tail pinch (red dashed lines). (g) Z score averages for LPB→SNR fluorescence in response to heat exposure at 40°C (orange), 50°C (purple), or 55°C (magenta). Shading represents SEM (n = 3 trials per temperature level; n = 5 mice). (h) Significantly greater GCaMP7f response from LPB→SNR neurons in response to heat exposure at 50°C and 55°C compared to 40°C (n = 3 trials per stimuli; n = 5 mice). (i) Experimental design. Activity of opto-tagged LPB neurons was recorded in response to heat (50°C, 10 sec) and tail shock (0.5 mA, 50 or 200 ms), n = 6 mice. (j) Location of optrode, AAVretro-Cre-eGFP (green) and ChR2 (red) in LPB (scale bar 100 μm). (k) Left: Raster plot showing latency of light-evoked spikes relative to light pulses (5 ms, blue; top) and corresponding spike firing frequency (bottom). Right: Mean response latency to laser stimulation for ChR2-tagged LPB→SNR neurons (n = 17 cells). (l) Sample waveform for evoked and spontaneous firing. (m) Sample of spike raster plot (top) and spontaneous firing frequency before and after (shaded) heat exposure (50°C, 10 sec) from an opto-tagged LPB→SNR neuron. (n) Left: Proportion of LPB→SNR neurons with significant increase (green), significant decrease (red) or no significant change (grey) in firing in response to heat (n = 9 cells). Right: Mean firing frequency of LPB→SNR neurons pre and post heat (n = 9 cells). (o,p) Same as in (m,n) but for electrical shock (n = 14 cells). Significance was calculated by means of one-way RM ANOVA with Tukey’s post-hoc test (h) or paired t-test (n,p). * p < 0.05. Comparisons with no asterisk had p > 0.05 and were not considered significant. Data represent mean ± SEM.

Fig. 4.. Inhibition of LPB→SNR reduces behavioral responses to pain.

(a) Experimental design. (b) Implant locations (scale bar 200 μm). (c) Number of licks in response to formalin injection in NpHR (orange; n = 8) and eYFP (grey; n = 5) mice while exposing LPB_VGLUT2 terminals in SNR to 589 nm light (left). Mean number of licks during phase I and phase II of formalin test for NpHR (orange) and eYFP (grey) mice. (d) Same as in (c) but for analysis of lick duration. (e) von Frey test. (f) Mean 50% paw withdrawal threshold (PWT) before formalin (-form) and after formalin (+form) injection for with (on) or without (off) 589 nm light for NpHR (left, n = 8) and eYFP (right; n = 5) mice. (g) Experimental design. (h,i) Mean 50% PWT measured 24 hours before and 8 days after SNL (left) or sham (right) surgery for (h) NpHR- and (i) eYFP-expressing mice. ‘On’ shows mean 50% PWT during 589 nm light exposure (SNL: NpHR: n = 8 mice, eYFP: n = 7 mice; sham: NpHR: n = 8 mice, eYFP: n = 7 mice). (j) Mean withdrawal latency during hot plate test at 50°C, 8 days after sham or SNL surgeries with (on) or without (off) 589 nm light exposure for NpHR- (left) and eYFP-expressing (right) mice (SNL: NpHR: n = 8, eYFP: n = 7; sham: NpHR: n = 8, eYFP: n = 7). (k) Experimental design. (l) Trajectories of sample NpHR SNL NpHR (top), NpHR sham (middle) and eYFP SNL (bottom) animals. Pre-tests were performed 8 days after SNL or sham surgeries. (m) NpHR SNL mice show significant increase in preference time for the side of the chamber paired with light stimulation compared to NpHR sham and eYFP SNL mice (NpHR SNL: n = 8; NpHR sham: n = 7; eYFP SNL: n = 7). Significance was calculated by means of two-way ANOVA with Holm-Sidak’s post-hoc test (f,j,m), one-way RM ANOVA with Tukey’s post-hoc test (h,i) or unpaired t-test (c,d). * p < 0.05, ** p < 0.01, *** p < 0.001. Data represent mean ± SEM.

Fig. 5.. LPB targeted SNR neurons are excited by noxious stimuli.

(a) Schematic showing unilateral targeting of AAV-DIO-ChR2 to LPB and placement of an optrode in the SNR of VGLUT2-Cre mice. Activity from SNR neurons was recorded in response to optogenetic stimulation of LPB inputs, tail pinch, heat (50°C, 10 sec) and electrical tail shock (0.5 mA, 200 ms; n = 5 mice). (b) Coronal section showing LPB_VGLUT2 terminals expressing ChR2 (green) and location of optrode in lateral SNR (DAPI: blue; IPN: interpeduncular nucleus; VTA: ventral tegmental area; scale bar 100 μm). (c) Spike raster plot showing individual spikes in response to 5 ms laser pulse with each row representing individual trials (top) and the corresponding spike firing frequency (bottom). (d) Mean response latency to laser stimulation for LPB-targeted SNR neurons (n = 29 cells). (e) Proportion of SNR neurons that significantly increased firing in response to light stimulation of LPB inputs. (f) Samples of evoked and spontaneous action potential waveforms. (g) Proportion of LPB-targeted SNR neurons (i.e., SNR cells that showed significantly increased firing in response to light stimulation of LPB inputs) that were excited (green), inhibited (red) or did not respond (grey) following tail pinch, heat, or electrical shock (n = 15–29 cells from n = 5 mice). (h) Samples of spontaneous spike raster plots (top) and spontaneous firing frequencies (bottom) for LPB-targeted SNR neurons that were excited (left), inhibited (middle) or did not respond (right) to tail shock. (i) Responses of LPB-targeted SNR neurons that were exposed to all three noxious stimuli (n = 15 cells). Data represent mean ± SEM.

Fig. 6.. LPB-targeted SNR neurons innervate VTA DA neurons directly and indirectly

(a) Experimental design. (b) Synaptophysin-expressing (red) terminals in lateral VTA (lVTA) and LPBc (scale bars 50 μm). (c) Experimental design. Inset: Neurobiotin (NB)-filled (green), TH-immunopositive (blue), retrogradely labeled (red) cell in lVTA (scale bar 10 μm). (d) Left: Light-evoked IPSC from NAcLat-projecting DA neuron (red trace: 100 μM picrotoxin (PCTX; scale bars 500 pA/100 ms). Right: Mean IPSC amplitudes recorded in NAcLat-projecting DA neurons before and after PCTX application (n = 8 cells). (e) Experimental design. Inset: Neurobiotin (NB)-filled (green), retrogradely labeled (red) LPBc cell (scale bar 10 μm). (f) Left: Light-evoked IPSC from LPB→VTA neuron (red trace: 100 μM PCTX; scale bars 500 pA/100 ms). Right: Mean IPSC amplitudes recorded in LPB→VTA neurons before and after PCTX application (n = 4 cells). (g) Left: Experimental design. Right: Neurobiotin (NB)-filled (green), retrogradely labeled (red) LPBc cell (scale bar 50 μm). (h) Left: Sample IPSC generated at −70 mV by light stimulation of SNR_VGAT inputs to LPB→VTA neuron (red trace: 100 μM PCTX; scale bars 500 pA/100 ms). Right: Mean IPSC amplitudes before and after PCTX application (n = 6 cells). (i) Left: Spontaneous firing from LPB→VTA neuron before (top) and after (bottom) PCTX application in response to 20 Hz light stimulation of SNR_VGAT inputs (scale bars 20 mV/1 sec). Right: SNR_VGAT stimulation significantly reduces firing before but not after PCTX application in LPB→VTA neurons (n = 4 cells). (j) Experimental design. (k) Left: Number of licks in response to formalin injection in ChR2- (blue; n = 8) and GFP-expressing (grey; n = 6) mice while stimulating LPB→VTA DA neurons with 20 Hz blue light. Right: Mean number of licks during phase I and phase II of the formalin test for ChR2- (blue) and eYFP-expressing (grey) mice. (l) Same as in (k) but for analysis of lick duration. Significance was calculated by means of paired t-test (d,f,h), unpaired t-test (k,l) and one-way RM ANOVA with Tukey’s post-hoc test (i). * p < 0.05, ** p < 0.01. Data represent mean ± SEM.

Fig. 7.. LPB contributes to pain-induced inhibition of DA release in vivo.

(a) Schematic of viral targeting of ChR2 to lateral SNR and GCaMP6m to the VTA of DAT-Cre mice. Optogenetic stimulation of SNR terminals in the LPB and fiber photometry recordings of VTA DA terminals in NAc medial shell (NAcMed) or lateral shell (NAcLat). (b) Left: Coronal brain slice image showing ChR2 (red) expression in lateral SNR and GCaMP6m (green) expression in VTA DA neurons (TH: blue, SNC: substantia nigra pars compacta, IPN: interpeduncular nucleus). Middle: Coronal brain slice image showing optical fiber for fiber photometry recordings in NAcMed (DAPI: blue). Right: Coronal brain slice image showing optical fiber for fiber photometry recordings in NAcLat (scale bar 100 μm). (c) Sample GCaMP6m responses from VTA DA terminals in NAcLat (top) and NAcMed (bottom) in response to 20 Hz optogenetic stimulation of SNR terminals in the LPB. (d) Comparison of mean GCaMP6m responses for recordings in NAcMed (red) and NAcLat (blue) in response to 20 Hz optogenetic stimulation of SNR terminals in the LPB. Shading represents SEM (n = 20–24 trials; NAcLat: n = 6 mice; NAcMed: n = 5 mice). (e) Comparison of mean AUC in NAcMed (orange) and NAcLat (blue) during optogenetic stimulation. (f) Schematic of experimental design showing bilateral targeting of AAVretro-Cre to SNR, unilateral targeting of AAV-DIO-Caspase 3 (CASP) to right LPB and control (AAV-DIO-mCherry) to left LPB of C57BL/6 mice. dLight1.2 and optical fibers were targeted bilaterally to NAcLat of the same animals (n = 9 mice). DA transients were recorded following electrical shock and tail pinch. (g) Top: Representative heat maps for NAcLat DA release from respective hemispheres as CASP and control vector expression in response to electrical shock. Bottom: Corresponding Z score averages (CASP: red, control: black). (h,i) Mean AUCs during shock (h) or tail pinch (i) for CASP and control sides. Significance was calculated by means of unpaired t-test (e) or paired t-test (h,i). * p < 0.05, ** p < 0.01. Comparisons with no asterisk had p > 0.05 and were not considered significant. Data represent mean ± SEM.

Fig. 8.. Circuit model.

Schematic illustrating a neural circuit for conveying nociceptive input from the dorsal horn spinal cord to midbrain dopamine neurons projecting to NAcLat.