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Dorsal root ganglia control nociceptive input to the central nervous system [1]

['Han Hao', 'Department Of Pharmacology', 'Hebei Medical University', 'The Key Laboratory Of Neural', 'Vascular Biology', 'Ministry Of Education', 'China', 'The Key Laboratory Of New Drug Pharmacology', 'Toxicology', 'Hebei Province']

Date: 2023-01

Accumulating observations suggest that peripheral somatosensory ganglia may regulate nociceptive transmission, yet direct evidence is sparse. Here, in experiments on rats and mice, we show that the peripheral afferent nociceptive information in mice undergoes dynamic filtering within the dorsal root ganglion (DRG) and suggest that this filtering occurs at the axonal bifurcations (t-junctions). Using synchronous in vivo electrophysiological recordings from the peripheral and central processes of sensory neurons (in the spinal nerve and dorsal root), ganglionic transplantation of GABAergic progenitor cells, and optogenetics, we demonstrate existence of tonic and dynamic filtering of action potentials traveling through the DRG. Filtering induced by focal application of GABA or optogenetic GABA release from the DRG-transplanted GABAergic progenitor cells was specific to nociceptive fibers. Light-sheet imaging and computer modeling demonstrated that, compared to other somatosensory fiber types, nociceptors have shorter stem axons, making somatic control over t-junctional filtering more efficient. Optogenetically induced GABA release within DRG from the transplanted GABAergic cells enhanced filtering and alleviated hypersensitivity to noxious stimulation produced by chronic inflammation and neuropathic injury in vivo. These findings support “gating” of pain information by DRGs and suggest new therapeutic approaches for pain relief.

Funding: This work was supported by the National Natural Science Foundation of China grants (84870872 & 313400048) to X.D., Key Basic Research Project of Applied Basic Research Program of Hebei Province (16967712D) to X.D. and Science Fund for Creative Research Groups of Natural Science Foundation of Hebei Province (H2020206474) to X.D.; National Natural Science Foundation of China (91732108, 81871075) to H.Z. and S&T Program of Hebei Province (193977144D) grants to H.Z.; Innovation fund for graduate students of Hebei Province (CXZZBS2018077) to H.H.; the Wellcome Trust Investigator Award 212302/Z/18/Z and Medical Research Council project grant (MR/V012738/1) to N.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

An intrinsic GABAergic signaling system within DRGs was recently proposed as a modulator of ganglionic filtering [ 15 ]. Yet, our understanding of how information can be modified within DRGs still remains sparse. Here, we obtained direct in vivo evidence for ganglionic filtering mediated by the GABA signaling system and assessed if such filtering can be exploited to control pain. Using in vivo electrophysiological recordings from the peripheral and central processes of sensory neurons (in the spinal nerve (SN) and dorsal root (DR)), optogenetic manipulations, stem axon morphometry, and biophysical modeling, we demonstrate that the DRG is a bona fide processing device controlling and modifying nociceptive signaling into the central nervous system (CNS). These findings support the existence of a “peripheral gate” in somatosensory system and suggest new ways of how sensory ganglia can be targeted for pain control.

Current understanding of the somatosensory information processing largely assumes that peripheral nerves faithfully deliver peripherally born action potentials to the spinal cord. The first synapse in the dorsal horn of the spinal cord is assumed to be the first major integration point for action potentials generated at the periphery. Such a view is represented by the Gate Control Theory of pain [ 1 ] and its subsequent refinements and modifications [ 2 – 4 ]. While it has been proposed that information processing is more efficient the earlier it begins within the sensory pathway [ 5 – 7 ], the absence of true synaptic connections or interneurons within peripheral somatosensory nerves and ganglia reasonably led researchers to dismiss them as possible information processing sites. Despite this, growing evidence suggests that a degree of crosstalk between the peripheral fibers [ 8 , 9 ] or sensory neuron somata [ 10 – 12 ] might exist. Moreover, there is substantial experimental evidence that action potentials propagating from the peripheral nerve endings of nociceptive nerve terminals to the spinal cord can fail (or be “filtered”) at axonal bifurcation points (t-junctions) within the dorsal root ganglion (DRG) [ 13 – 19 ].

Results

Transplantation of forebrain GABAergic neuron precursors into the adult mouse DRG in vivo delivers an analgesic mechanism To test how GABAergic filtering of nociceptive transmission at the DRG can be exploited in vivo, we adopted an approach developed by Basbaum’s group, who were able to transplant and functionally integrate into dorsal spinal cord, embryonic GABAergic progenitor cells from the medial ganglionic eminence (MGE). Transplanted MGE cells were able to compensate for the loss of spinal GABAergic inhibitory system observed in neuropathic pain models [26,27]. We transplanted embryonic MGE cells derived from VGAT-ChR2-eYFP into the L4 DRG of WT C57 mice. L4 DRG was chosen in this case as it is the major contributor to the sciatic nerve in mice [28]. At 4 weeks after the DRG injection, we observed numerous YFP-positive cells in the DRG sections (Fig 3A); fluorescent cells were entirely absent in vehicle-injected control animals. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Transplantation of forebrain GABAergic neuron progenitor cells into the adult mouse DRG in vivo delivers an analgesic mechanism. (A) Fluorescence micrographs of DRG sections of mice 4 weeks after the injection with a suspension of MGE cells derived from VGAT-ChR2-eYFP mice. Control mice (right images) received vehicle injections. (B) Bright-field and overlaid fluorescence images of “loosened” DRG preparation used for patch clamp recording. The whole L4 DRG transplanted with VGAT-ChR2-eYFP MGE cells mice was extracted 4 weeks after transplantation. Recording was made from a DRG neuron juxtaposed to fluorescent MGE cell (top image) using whole-cell voltage clamp. (C) Top: example trace of continuous current recording (−60 mV) from the cell shown in B; stimulation with 473 nm blue laser (3 mV) induced inward current, similar in amplitude and kinetics to the current induced by perfusion of GABA (200 μm). Bottom: similar recording from a DRG of a vehicle-injected mice. (D) Summary for panel C; one-way ANOVA: F(2,23) = 5.9; p < 0.01; Bonferroni post hoc test: ***significant difference between +MGE vs. -MGE (p < 0.001). Number of recorded/responsive cells is indicated within each bar. (E) Schematic of the in vivo optogenetic DRG stimulation. (F) Timeline of the in vivo behavioral testing after the MGE cell transplantation and hindpaw injection of CFA. (G, H) Hypersensitivity to mechanical (G) and thermal (H) stimulation caused by hindpaw injection of CFA 2 weeks after MGE cells transplantation into L4 DRG of mice. At a time of MGE transplantation, mice were also implanted with the fiberoptic light guide. Mechanical and thermal sensitivity was measured using the von Frey and Hargreaves methods, respectively. Starting at 1 week after the CFA injection, measurements were performed while stimulating the L4 DRG with 473 nm laser. Black and blue symbols denote control mice DRG-injected with vehicle without and with optogenetic stimulation, respectively. Red and green symbols denote MGE-transplanted mice without and with optogenetic stimulation, respectively. BL1: baseline before transplantation; BL2: baseline after transplantation; CFA: 1 day after the plantar injection of CFA. (G) Three-factor (MGE vs. vehicle, time after CFA, laser stimulation) ANOVA: main effects associated with MGE transplantation [F(1,24) = 50.9; p < 0.001], time after CFA [F(2,23) = 6.6; p < 0.01]; laser stimulation [F(1,24) = 8.9; p < 0.01]. Bonferroni post hoc test: red* indicate the difference between MGE group and vehicle group within the corresponding time point; green* indicate the difference between MGE with laser stimulation group and vehicle with laser stimulation group; *p < 0.05, **p < 0.01, ***p < 0.001. (H) Three-factor (MGE vs. vehicle, time after CFA, laser stimulation) repeated measures ANOVA: main effects associated with MGE transplantation [F(1,24) = 37.4; p < 0.001], time after CFA [F(2,23) = 6.1; p < 0.01], laser stimulation [F(1,24) = 2.4; p = 0.12]; significant interaction between time and laser stimulation [F(2,23) = 2.5; p = 0.09] and between MGE and laser stimulation [F(2,23) = 3.2; p = 0.08]. Bonferroni post hoc test: red* indicate the difference between MGE group and vehicle group within the corresponding time point; green* indicate the difference between MGE with laser stimulation group and vehicle with laser stimulation group; *p < 0.05, **p < 0.01, ***p < 0.001. Metadata for quantifications presented in this figure can be found at https://archive.researchdata.leeds.ac.uk/1042/. Schematics are drawn with Canvas X 2019; vector diagram of laboratory mouse from Wikimedia was used in the panel E, https://commons.wikimedia.org/wiki/File:Vector_diagram_of_laboratory_mouse_(black_and_white).svg. CFA, complete Freund’s adjuvant; DRG, dorsal root ganglion; MGE, medial ganglionic eminence. https://doi.org/10.1371/journal.pbio.3001958.g003 In order to confirm that transplanted MGE cells can function as GABAergic neurons within DRG, we performed patch clamp recordings from the DRG neurons juxtaposed to the MGE cells (Fig 3B–3D) using “loosened” whole L4 DRGs from mice pre-injected (4 weeks) with the VGAT-ChR2-eYFP-expressing MGE (see Materials and methods). Stimulation of the ganglion with the 473 nm blue light induced inward currents in 9/9 DRG neurons, which were in close juxtaposition with MGE cells (Fig 3B–3D). These same neurons also responded to perfusion of 200 μm GABA with very similar inward currents. In contrast, DRG neurons from vehicle-injected mice never responded to blue light (0/8 neurons) but these did respond to GABA (Fig 3C and 3D). These results suggest that (i) implanted MGE progenitor cells can survive and maturate to produce GABA-releasing neurons in DRG; and (ii) stimulus-induced release of GABA by resident neurons can induce a response in neighboring neurons. Next, we tested if optogenetic release of GABA from the implanted MGE cells can alleviate hypersensitivity to noxious stimuli in chronic pain models. In these experiments, a fiber-optic light guide was implanted into the DRG immediately after the MGE cells transplantation (Fig 3E; Materials and methods). Chronic inflammation with hind paw injection of complete Freund’s adjuvant (CFA, 20 μl) induced significant hypersensitivity to mechanical and thermal stimuli (Fig 3F–3H). We then performed mechanical (Fig 3G) or thermal (Fig 3H) sensitivity tests while stimulating ipsilateral L4 DRG with blue light. Optical stimulation significantly reduced both types of hypersensitivity in MGE-injected mice. Interestingly, starting from the second week after the CFA injection, both mechanical and thermal hypersensitivity in the MGE-implanted mice started to recover even in the absence of optogenetic stimulation and by the third week after the CFA injection blue light stimulation no longer produced any additional analgesic effect (Fig 3G and 3H). We hypothesized that a buildup of tonic GABA release from the transplanted MGE cells in DRG could be responsible for the light-stimulation-independent recovery of the CFA-induced hypersensitivity. This hypothesis was corroborated in experiments, similar to the ones presented in Fig 3G and 3H, but in which no optogenetic stimulation was used, to avoid inducing any stimulus-induced GABA release (S8A and S8B Fig). We also utilized a chronic constriction injury (CCI) model of neuropathic pain in similar experiments (S8C and S8D Fig). In both models, hypersensitivity developed in control (vehicle-injected) and MGE-implanted mice. However, the latter group displayed significantly quicker and more complete recovery. Collectively, these data suggest that DRG-implanted MGE cells can be stimulated to release GABA locally in vivo and that such release produces analgesic effect.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001958

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