In Vivo Interrogation of Spinal Mechanosensory Circuits

Abstract

Spinal dorsal horn circuits receive, process, and transmit somatosensory information. To understand how specific components of these circuits contribute to behavior, it is critical to be able to directly modulate their activity in unanesthetized in vivo conditions. Here, we develop experimental tools that enable optogenetic control of spinal circuitry in freely moving mice using commonly available materials. We use these tools to examine mechanosensory processing in the spinal cord and observe that optogenetic activation of somatostatin-positive interneurons facilitates both mechanosensory and itch-related behavior, while reversible chemogenetic inhibition of these neurons suppresses mechanosensation. These results extend recent findings regarding the processing of mechanosensory information in the spinal cord and indicate the potential for activity-induced release of the somatostatin neuropeptide to affect processing of itch. The spinal implant approach we describe here is likely to enable a wide range of studies to elucidate spinal circuits underlying pain, touch, itch, and movement.

Keywords: itch; nociception; optogenetics; somatostatin; spinal cord; touch.

Figure 1. Implantation of fiber optic ferrules for light delivery to the spinal cord

(a) Schematic showing relevant surgical landmarks. (b)–(e) Schematics showing process of implantation of fiber optic cannula. (f) Representative longitudinal spinal cord sections from control and implanted mice stained for astrocyte (GFAP) or microglia activations (Iba1). (2 sections each from n = 3 experimental and n = 3 control were analyzed) Scale bar: 1 mm. (g) Close up of implantation region for Iba1 and GFAP stained longitudinal sections. Scale bar: 250 μm (h) Schematic showing catwalk and representative gait trace of cannulated mouse. (i) Stride length of ipsilateral vs. contralateral paws of implanted mice (n = 5. P = 0.397). (j) Mechanical withdrawal thresholds of cannulated and uncannulated mice, as measured on the von Frey test (n = 10 implanted, 10 wild type. P = 0.528). Thermal withdrawal latencies of cannulated and uncannulated mice, as measured on the Hargreaves test (n = 10 implanted, 10 wild type. P = 0.47). All group data is shown as mean ± s.e.m.

Figure 2. Optogenetic and chemogenetic modulation of somatostatin interneurons

(a) Diagram of primary afferent to brain circuit containing somatostatin interneurons. Hypothesized and/or polysynaptic connection shown in dotted lines. (b) Histology showing somatostatin expression (sections from n = 3 mice were examined for quantification). Scale bar: 250 μm, inset: 100 μm. (c) Spontaneous response score of YFP mice compared to ChR2 mice in somatostatin interneurons (n = 5 ChR2, 5 YFP, P = 4×10⁻⁴). (d) Latency to lick response of somatostatin mice vs. threshold light power (n = 7 mice, each normalized to their individual threshold, binned in 5 second intervals, and then averaged across mice). (e) Thermal withdrawal latency during ‘subthreshold’ blue light illumination of YFP mice and ChR2 mice (n = 5 ChR2, 5 YFP. P = 0.1 YFP, P = 0.21 control). (f) Mechanical withdrawal thresholds of YFP and ChR2 mice at baseline and during subthreshold blue light illumination (n = 5 ChR2, 5 YFP. P = 0.421 YFP, P = 0.0173 ChR2). (g) Conditioned place aversion (CPA) ratios, calculated as the ratio of the percentage of time spent in the stimulation chamber on initial (pre-test) day, and after three days of conditioning (n = 6 ChR2, 6 YFP. ChR2: P = 0.006, control: P = 0.208). (h) Histology indicating robust expression of hM4D in the spinal cord dorsal horn following intraspinal injection of AAV5::hM4D, indicating expressing in lamina II that is non-overlapping with PKCγ. Top row, transverse spinal cord section: hM4D-mCherry (red), PKCγ (cyan), overlap. Scale bar: 100 μm. Bottom row, dorsal root ganglion section: hM4D-mCherry (red), DAPI (cyan). Scale bar: 250 μm. (i) Mechanical withdrawal thresholds following injection of CNO or saline in SOM-hM4D+ mice (n = 8 post-CNO, n = 8 post-saline, P (post-CNO) = 0.013, P (post-saline) = 0.52). (j) Thermal withdrawal latency following injection of CNO or saline in SOM-hM4D+ mice (n = 7 post-CNO, n = 7 post-saline, P (post-CNO) = 0.047, P (post-saline) = 0.66). (k) Cotton swab sensitivity following injection of CNO or saline in SOM-hM4D+ mice before and after intraplantar CFA. (Pre-CFA: n = 8 post-CNO, n = 8 post-saline. Post-CFA: n = 8 post-CNO, n = 8 post-saline. Pre-CFA: P (post-CNO) = 1, P (post-saline) = 0.84. Post-CFA: P (post-CNO) = 0.015, P (post-saline) = 0.60). All group data is shown as mean ± s.e.m.

Figure 3. Temporally sparse optogenetic stimulation of somatostatin interneurons modulates pruritoception

(a) Histology indicating significant spatial proximity between ChR2+ neurons and SST2R immunoreactivity in SOM-ChR2+ mice. Clockwise from top-left: green: ChR2-eYFP, blue: DAPI, red: SST2R immunoreactivity, overlay. Scale bar: 100 μm. (b) Time spent itching during sparse optogenetic stimulation in SOM-ChR2 and SOM-mCherry mice, with concurrent intrathecal CYN-154806 or intrathecal saline. (n = 6 SOM-ChR2+ mice, n = 5 SOM-mCherry+ mice, P (SOM-ChR2) = 0.028, P (SOM-mCherry) = 0.47). (c) Thermal withdrawal latency during sparse optogenetic stimulation in SOM-ChR2 and SOM-mCherry mice, with concurrent intrathecal CYN-154806 or intrathecal saline (n = 5 mice in all conditions, P (SOM-ChR2 + CYN-154806) = 0.042, P (SOM-ChR2 + saline) = 0.77, P (SOM-mCherry + CYN-154806) = 0.54, P (SOM-mCherry + saline) = 0.78)). (d) Mechanical withdrawal thresholds during sparse optogenetic stimulation in SOM-ChR2 and SOM-mCherry mice, with concurrent intrathecal CYN-154806 or intrathecal saline (n = 5 mice in all conditions, P (SOM-ChR2 + CYN-154806) = 0.37, P (SOM-ChR2 + saline) = 0.10, P (SOM-mCherry + CYN-154806) = 0.22, P (SOM-mCherry + saline) = 0.25). All group data is shown as mean ± s.e.m.

Figure 4. c-Fos activation in the dorsal horn after stimulation of somatostatin interneurons

(a) ChR2-eYFP fluorescence in the dorsal horn after intraspinal injection of AAVDJ:ef1a:DIO:ChR2-eYFP in SOM-IRES-Cre mice. (b) Immunostaining for NK1R in the dorsal horn of the spinal cord. (c) c-Fos expression in the dorsal horn after expression of somatostatin in the spinal cord. (d) Merge of c-Fos, ChR2-eYFP, and NK1R channels. Scale bar (a–d): 250 μm. (e) Examples of overlap of c-Fos and YFP (closed arrows), or c-Fos and NK1R (open arrows). Scale bar: 250 μm. (f) Quantification of overlap of c-Fos, YFP and NK1R expression. Note: 56 ± 8 % of ChR2+ cells are also c-Fos+, The ChR2+/c-Fos+ to ChR2−/c-Fos+ ratio is 2.55 ± 0.44. (g) Visualization of overlap. (h) Quantification of depth of c-Fos expressing neurons in the dorsal horn. Lamina depths are denoted with dotted lines and roman numerals.