Introduction

Chronic pain is a thorny medical problem, with the current treatment measures failing to achieve satisfactory results [1]. Chronic pain is often accompanied by several mental diseases or abnormalities, of which anxiety is prominent [2,

Fig. 5: Map** and chemogenetic activation of axonal projections from LS GABAergic neurons.
figure 5

A, B The distribution pattern of the LS GABAergic projection. A Schematics and timeline of antegrade virus (AAV2/9-DIO-EGFP) tracing. B Schematic summary of mapped recipient brain regions of LS projection fibers with the distribution pattern from whole-brain tracing. C–O By using chemogenetic manipulation, LS GABAergic projections to HDB, LH, PAG were indirectly activated in behavioral experiments. C Experimental scheme of virus injections and behavior tests. Schematics of the stereotaxic surgery targeting the (D) LS-HDB, (H) LS-LH, and (L) LS-PAG circuits. Coronal sections illustrating hM4Di mCherry-positive HDB, LH and PAG neurons at the injection site. Scale bar, 100 μm. E–G Behavioral results for LS-HDB activation. E Withdrawal threshold of the hind paw measured with the von Frey test. F OFT (Left) total distance and (Right) percentage of time in center. G EPM (Left) total entries in arms and (Right) time in open arms. I–K Same as (E–G) but for LS-LH activation. M–O Same as (E–G) but for LS-PAG activation. *P < 0.05, **P < 0.01. ns, no significant difference (P > 0.05). Data are presented as the means ± SEM. For further details of statistical data analysis see Table S1. DM, dorsomedial hypothalamus; HDB, horizontal limb of the diagonal band; HPC, hippocampus; LH, lateral hypothalamus; LPO and MPO, lateral and medial preoptic area; NAc, nucleus accumbens; PAG, periaqueductal gray; VDB, vertical limb of the diagonal band; VMH, ventromedial hypothalamus; VTA, ventral tegmental area.

Full size image

Activation of LS projection induces comorbid pain and anxiety behavior in naïve mice

Different subregions of LS, such as the dorsal and ventral parts, are known to play different roles in the regulation of emotion and other related behaviors [20]. We speculated that these differences might also be reflected in the differences in the anatomical projection targets (i.e., downstream brain regions) associated with the distinct LS subpopulations. To examine the differences in the downstream projection of different LS subregions, we injected anterograde vectors expressing EGFP and mCherry into the dorsal and ventral LS, respectively (Fig. S8A, B). These experiments revealed obvious differences in the brain-wide distribution of the downstream projections between the two subregions of LS. The projection of dorsal LS tends to HDB, LH, VTA et al. while the projection of ventral LS tends to DM, VMH and other brain regions (Fig. S8C, D).

Next, we investigated the role of the different LS projection pathways in pain and anxiety regulation. In selected known target regions of LS projections, we bilaterally injected a retrograde virus vector of a Cre recombinant enzyme (Retro-AAV-Cre) into HDB, LH, PAG, medial preoptic area (MPOA), ventral HPC (vHPC) and VTA, and we also injected a Cre-dependent chemogenetic vector (AAV2/9-DIO-hM3Dq-mCherry) into the bilateral LS (Fig. S9A, E, I; Fig. S10A, E, I) for the selective chemogenetic activation of LS projection neurons. Three weeks after full expression of the virus in the LS, intraperitoneal injection of CNO (2 mg/kg) activates LS projection. We then assessed OFT and EPM testing behaviors. We observed activation of LS projections to HDB and LH reduced time spent in OFT center, EPM open arms and entries in open arms; The projection of LS to HDB decreased the total distance in OFT, but the projection of LS to LH did not affect the total distance (Fig. S9C, D, G, H). Interestingly, the activation of the LS to LH projection had an effect on the pain threshold, the projection of LS to HDB did not change the pain threshold (Fig. S9B, F). The activation of LS projections to PAG also reduced pain thresholds but did not induce anxiety-like behavior (Fig. S9J–L). Chemogenetic activation of the MPOA-projecting, vHPC-projecting, and VTA-projecting LS neurons had no effect on the pain threshold or anxiety behaviors (Fig. S10A–L).

However, we could not fully distinguish the specific roles of the HDB-, LH- and PAG-projecting LS neurons in the regulation of pain and anxiety using the neural circuit construction method described above. To overcome this limitation and to obtain more convincing evidence, C57BL/6 mice were infused with AAV2/1-Cre into the LS, which could anterograde transport from the neuronal cell body to the axon terminal and then across the monosynapse into its projecting neurons, and AAV2/9-DIO-hM4Di-mCherry into the HDB, LH and PAG (Fig. 5C, D, H, L). Through the above circuit construction and with the intraperitoneal injection of CNO (2 mg/kg), the HDB-, LH- and PAG-projecting LS neurons were indirectly activated. The results showed that activation of the neural projection from LS to HDB and to LH triggered anxiety-like behavior including decreased exploring time in the center and total distance in the OFT (Fig. 5F, J) and reduced the number of times and time into the open arm (Fig. 5G, K). The results of von Frey stimulation also showed that activation of neural projection from the LS to LH and PAG neurons induced hyperalgesia (Fig. 5I, M). Activation of LS projection to HDB did not affect the mechanical pain threshold in mice (Fig. 5E). We found that activation of neural projection from the LS to PAG neurons displayed no anxiety-like behaviors in the EPM and OFT tests (Fig. 5N, O). These findings are further confirmation of the remarkable functional differences of the HDB-, LH-, and PAG-projecting subpopulations of LS GABAergic neurons in the regulation of anxiety and pain.

To determine whether these divergent projections originate from different subsets of LS neurons or reflect collateral projections from the same LS population, we performed simultaneous retrograde tracing from the 3 target regions using fluorescent labels of different color. After injection of Retro-AAV-hSyn- expressing mCherry (red), EGFP (green) and mTagBFP-3XFlag (blue) into the HDB, LH and PAG, respectively (Fig. S11A, B), we found strong expression of mCherry, EGFP and mTagBFP-3XFlag in the LS. The expression of mCherry and mtagBFP-3xFlag partially overlapped with EGFP (46.19% and 20.65%), but little overlap between mCherry and mTagBFP-3XFlag (12.78%) (Fig. S11C, D). These experiments suggest that the HDB-, LH - and PAG -projecting LS neurons form largely distinct subpopulations.

Activity of HDB-, LH- and PAG-projecting LS neurons mediates anxiety-like behavior

Above we demonstrated how LS neuron activity affected the behavior of the animals. We next determined whether the activity of the HDB-, LH - and PAG -projecting LS neurons would change in response to noxious stimuli or anxiety behaviors. First, C57BL/6 mice were injected with AAV2/9-DIO-GCaMP6s into the HDB, LH or PAG, and AAV2/1-Cre into the LS. An optic-fiber cannula was implanted in the brain of the animal over the HDB, LH or PAG. We used mice of the CFA model to induce comorbid pain and anxiety behaviors. A 3-day CFA conditioning was conducted, followed by the von Frey, OFT and EPM tests combined with simultaneous monitoring of neuronal activity in the HDB, LH or PAG using position-synchronized in vivo fiber photometry recordings (Fig. S12A). The green fluorescence signals observed in the LS projection areas verified that the AAV2/1-Cre vector crossed the synapse at the LS axon terminals in HDB, LH, and PAG and helped the expression of GCaMP6s in the soma of HDB, LH or PAG neurons (Fig. S12B, F, J).

Mechanical stimulation inhibited calcium signaling in HDB, LH and PAG neurons (Fig. S12C, G, K). The position-synchronized in vivo calcium recording revealed that CFA mice exhibited significant inactivation of GCaMP6s activity following emotional stress exposure of HDB, LH neurons monosynaptically innervated by GABAergic LS projections (Fig. S12D, E, H, I). Calcium signal in PAG was responsive to EMP test, but not to OFT test (Fig. S12L, M). The above results indirectly reflect that mechanical pain stimulation, OFT and EPM test can activate the GABAergic neurons in LS.

Notably, above studies have shown that the LSGABAergic-LH circuit played an importance role in the regulation of comorbid pain and anxiety. To further establish a functional link between the LS and LH, ChR2-mCherry was virally expressed in LH neurons that were the monosynaptic targets of axon projections originated from LS GABAergic neurons (Fig. S13A). In vivo electrophysiological recordings showed that blue laser pulses inhibited LH neurons firing in a time-locked manner (Fig. S13B, C). In CFA model animals exhibiting comorbidity of pain and anxiety behaviors, we next used a genetically encoded GABA sensor (AAV2/9-DIO-iGABASnFR) to determine the dynamics of GABA release in and around the LH from LS projection axon terminals (Fig. S13, D). There were large increases in GABA release around the LH from LS during von Frey stimulation, center of OFT and open-arm exploration of EPM (Fig. S13E–G). These findings represent strong evidence that the LSGABAergic-LH circuit exhibits robust activation during exposure to nocifensive stimulation and it constitutes a key part of the neuronal substrate that functionally mediates anxiety-like behavior in CFA mice.