Thalamic CGRP neurons define a spinothalamic pathway for affective pain

Abstract

Pain is both a sensory and emotional experience caused by various harmful stimuli. While numerous studies have explored peripheral and central pain mechanisms, the specific neural circuits linking the spinal cord to the brain remain poorly defined. In this study, we demonstrate the involvement of calcitonin gene-related peptide (CGRP)-positive neurons in the parvicellular part of the subparafascicular nucleus (SPFp) in pain. Tracing revealed that CGRP neurons in the SPFp (CGRP^SPFp) receive projections from the dorsal horn. Increased calcium activity was observed in CGRP^SPFp neurons during mechanical, thermal, and inflammatory stimuli. Genetic silencing of these neurons resulted in reduced pain responses in animals. Furthermore, optogenetic activation of CGRP^SPFp neurons induced aversive memory but did not alter mechanical or thermal pain thresholds. This study reveals a distinct neural circuit involving CGRP^SPFp neurons that mediates pain, which differs from CGRP neurons in the parabrachial nucleus. Understanding these circuits could lead to better pain treatments with fewer side effects.

Keywords: CGRP; affective pain; pain; thalamus.

Fig. 1.

CGRP^SPFp neurons receive inputs from the spinal cord. (A) Schematic diagrams and images of Cre-dependent expression of TVA and G in SPFp neurons of Calca^Cre mice for the rabies tracing. (B) Representative images of the SPFp five days after EnvA-RVdG-mCherry injection. Yellow indicates the starter cells. (Scale bar, 200 µm.) (C) A schematic diagram of identifying presynaptic neurons by monosynaptic rabies tracing. (D) Representative images of superficial and deep layer dorsal horn neurons that project to CGRP^SPFp neurons. (Scale bar, 100 µm.) (E) The number of spinal dorsal horn neurons in the superficial (Sup) and deep layers project to CGRP^SPFp neurons. n = 5 mice. (F) The number of spinal cord neurons in different spinal segments that project to CGRP^SPFp neurons. n = 5 mice. Data in E and F are shown as mean ± SEM; two-tailed paired t test (E) or repeated measure one-way ANOVA with post hoc Tukey’s multiple comparison (F). See also SI Appendix, Table S1 for statistical details. *P < 0.05.

Fig. 2.

CGRP^SPFp neurons are activated by multimodal nociceptive stimuli. (A) A diagram of the fiber photometry calcium imaging experiment in an anesthetized mouse. (B) A schematic and representative image of the Cre-dependent expression of GCaMP6m in the Calca^Cre mice with an optical fiber implanted (red dashed line in the overlay image indicates fiber track) above the SPFp. (Scale bar, 200 µm.) (C–E) Heatmap (C), average traces of all four paws (D), and the area under curve (AUC, E) of CGRP^SPFp neuronal calcium activity in response to a pressure meter (0, 50, 100, 200, and 300 g). Intensity-dependent calcium activity increase was observed. AUC was quantified during 0 to 10 s. n = 6 (GCaMP6m), n = 4 mice (EYFP). (F–H) Intensity-dependent calcium increase in CGRP^SPFp neurons in response to a temperature-controlled rod (25, 35, 45, or 55 °C). n = 6 (GCaMP6m), n = 4 mice (EYFP). (I) Calcium signal increases in CGRP^SPFp neurons following subcutaneous injection of formalin (4%) into the paw. n = 6 (GCaMP6m), n = 4 mice (EYFP). (J) AUC quantification of CGRP^SPFp neuronal activity during the acute phase (5 to 10 min, pink box) of formalin response. (K) AUC quantification of CGRP^SPFp neuronal activity during the inflammatory phase (15 to 45 min, yellow box) of formalin injection. The red dotted box in C and F and blue box in D and G indicates the stimulation period. Data are shown as mean ± SEM.; repeated measure two-way ANOVA with post hoc Sidak’s multiple comparison (E and H) or two-tailed unpaired t test (J and K). See also SI Appendix, Table S1 for statistical details. *P < 0.05, ***P < 0.001.

Fig. 3.

Silencing CGRP^SPFp neurons attenuates responses to pain. (A) Schematic of Cre-dependent TetTox expression targeting CGRP^SPFp neurons. (Scale bar, 200 µm.) (B) Automatic von Frey assay with CGRP^SPFp silenced mice. n = 15 (EYFP), n = 16 mice (TetTox). (C) Hot plate assay at 48 and 55 °C with CGRP^SPFp silenced mice. 48 °C: n = 5 (EYFP), n = 6 mice (TetTox); 55 °C: n = 15 (EYFP), n = 16 mice (TetTox). (D) A schematic diagram of the Formalin assay to test inflammatory pain responses. (E) CGRP^SPFp silenced mice displayed significantly alleviated inflammatory pain responses. n = 10 (EYFP), n = 9 mice (TetTox). Data in B, C, and E are shown as mean ± SEM.; two-tailed unpaired t test (B and C) or repeated measure two-way ANOVA with post hoc Sidak’s multiple comparison (E). See also SI Appendix, Table S1 for statistical details. *P < 0.05, **P < 0.01.

Fig. 4.

Modulation of CGRP^SPFp neurons in anxiety and fear conditioning tests. (A) Schematic of Cre-dependent TetTox expression targeting CGRP^SPFp neurons. (Scale bar, 200 µm.) (B) Schematic diagram of the elevated plus maze (EPM) test (Left) and the results in CGRP^SPFp silenced mice (Right). n = 7 mice (EYFP), n = 9 mice (TetTox). (C) Schematic diagram of the context-dependent fear conditioning (Left), and the results in CGRP^SPFp silenced mice (Right). n = 9 mice (EYFP), n = 8 mice (TetTox). (D) Schematic and Cre-dependent ChR2 expression targeting CGRP^SPFp neurons. (Scale bar, 200 µm.) (E) Scheme of the cue-dependent optogenetic conditioning. (F) The result of the context test on Day 3. n = 8 (EYFP), n = 11 mice (ChR2). (G) The result of the cue test on Day 4. n = 8 (EYFP), n = 11 mice (ChR2). Data are shown as mean ± SEM.; two-tailed unpaired t test (B, C, and F) or repeated measure two-way ANOVA with post hoc Sidak’s multiple comparison (G). See also SI Appendix, Table S1 for statistical details. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5.

Active transcriptome profiling of CGRP^SPFp and CGRP^PBel neurons. (A) Correlation plot showing expression (transcript per million, TPM in log 10 scale) of genes enriched in CGRP^SPFp neurons compared with the total SPFp inputs using RiboTag transcriptome profiling. Up and down-regulated genes were separated based on 2.5 or −2.5-fold enrichment. (B) Correlation plot of the transcriptome profiles of CGRP^PBel neurons versus total PBel inputs. (C) Heatmaps showing fold enrichment of genes in the SPFp and PBel that encode membrane proteins. (D) Heatmaps showing fold enrichment of genes in the SPFp and PBel that encode neuropeptides, (E) Heatmaps showing fold enrichment of genes in the SPFp and PBel that encode markers of inhibitory neuron or glia. (F–H) Coexpression of NaV1.7 (F), CaV2.1 (G), or FAAH (H) with CGRP by double IHC. (Scale bar, 50 µm.)