Satellite glial cells (SGCs), enveloping primary sensory neurons’ somas in the dorsal root ganglion (DRG), contribute to neuropathic pain upon nerve injury. Glial fibrillary acidic protein (GFAP) serves as an SGC activation marker, though its DRG satellite cell specificity is debated. We employed the hGFAP-CFP transgenic mouse line, designed for astrocyte studies, to explore its expression within the peripheral nervous system (PNS) after spared nerve injury (SNI). We used diverse immunostaining techniques, Western blot analysis, and electrophysiology to evaluate GFAP+ cell changes. Post-SNI, GFAP+ cell numbers increased without proliferation, and were found near injured ATF3+ neurons. GFAP+ FABP7+ SGCs increased, yet 75.5% of DRG GFAP+ cells lacked FABP7 expression. This suggests a significant subset of GFAP+ cells are non-myelinating Schwann cells (nmSC), indicated by their presence in the dorsal root but not in the ventral root which lacks unmyelinated fibres. Additionally, patch clamp recordings from GFAP+ FABP7−cells lacked SGC-specific K_ir4.1 currents, instead displaying outward K_v currents expressing K_v1.1 and K_v1.6 channels specific to nmSCs. In conclusion, this study demonstrates increased GFAP expression in two DRG glial cell subpopulations post-SNI: GFAP+ FABP7+ SGCs and GFAP+ FABP7− nmSCs, shedding light on GFAP’s specificity as an SGC marker after SNI.

The immune system is involved in the development of neuropathic pain. In particular, the infiltration of T-lymphocytes into the spinal cord following peripheral nerve injury has been described as a contributor to sensory hypersensitivity. We used the spared nerve injury (SNI) model of neuropathic pain in Sprague Dawley adult male rats to assess proliferation, and/or protein/gene expression levels for microglia (Iba1), T-lymphocytes (CD2) and cytotoxic T-lymphocytes (CD8). In the dorsal horn ipsilateral to SNI, Iba1 and BrdU stainings revealed microglial reactivity and proliferation, respectively, with different durations. Iba1 expression peaked at D4 and D7 at the mRNA and protein level, respectively, and was long-lasting. Proliferation occurred almost exclusively in Iba1 positive cells and peaked at D2. Gene expression observation by RT-qPCR array suggested that T-lymphocytes attracting chemokines were upregulated after SNI in rat spinal cord but only a few CD2/CD8 positive cells were found. A pronounced infiltration of CD2/CD8 positive T-cells was seen in the spinal cord injury (SCI) model used as a positive control for lymphocyte infiltration. Under these experimental conditions, we show early and long-lasting microglia reactivity in the spinal cord after SNI, but no lymphocyte infiltration was found.

Abnormal activity in injured nerve fibers triggers spinal microglial reactivity, a critical step in the development of chronic postoperative pain. It remains unclear whether spinal microglia respond to inputs from nociceptive fibers alone or require combined activity from nociceptors and nonnociceptors. Using electrical and optogenetic approaches, we selectively activated different fiber subtypes without causing neuronal injury. Our findings demonstrate that only simultaneous activation of both nociceptive and nonnociceptive fibers induces sustained hypersensitivity and a spinal microglia response. This response is characterized by increased cell proliferation, altered electrophysiological profile with hyperpolarized membrane potential, increased potassium currents, and a less ramified morphology. In contrast, activation of nociceptive or nonnociceptive fibers alone does not induce these changes. In addition, inhibiting spinal microglia with minocycline prevents those changes. Overall, our study suggests that both types of peripheral sensory input are necessary to elicit microglial reactivity in the spinal cord and the subsequent development of sustained pain-related behavior.

Development of self-regulatory competencies during adolescence is partially dependent on normative brain maturation. Here we report that juvenile rats as compared to adults exhibit impulsive and compulsive-like behavioral traits, the latter being associated with lower expression of mRNA levels of the immediate early gene zif268 in the anterior insula (AI). This observation suggests that deficits in AI function in juvenile rats could explain their immature pattern of interoceptive cue integration in rational decision-making and compulsive phenotype. In support of this, here we report hypoexcitability of juvenile layer-V pyramidal neurons in the AI, concomitant with reduced glutamatergic synaptic input to these cells. Chemogenetic activation of the AI attenuated the compulsive trait suggesting that delayed maturation of the AI results in suboptimal integration of sensory and cognitive information in adolescents and this contributes to inflexible behaviors in specific conditions of reward availability.

Macrophages and satellite glial cells are found between injured and uninjured neurons in the lumbar dorsal root ganglia (DRG). We explored the mechanism of neuro-immune and neuron–glia crosstalk leading to hyperexcitability of DRG neurons. After spared nerve injury (SNI), CX3CR1 ⁺ resident macrophages became activated, proliferated and increased inward-rectifying potassium channel K _ir 2.1 currents. Conditioned medium (CM) by macrophages, obtained from DRG of SNI mice, sensitised small DRG neurons from naïve mice. However, treatment with CM from GFAP ⁺ glial cells did not affect neuronal excitability. When subjected to this macrophage-derived CM, DRG neurons had increased spontaneous activity, current-evoked responses and voltage-gated Na _V 1.7 and Na _V 1.8 currents. Silencing K _ir 2.1 in macrophages after SNI prevented the induction of neuronal hyperexcitability from their CM. Blocking vesicular exocytosis or soluble tumour necrosis factor (TNF) in CM or interfering with the downstream intracellular p38 pathway in neurons, also prevented neuronal hyperexcitability. Blocking protein trafficking in neurons reduced the effect of CM, suggesting that the hyperexcitable state resulted from changes in Na _V channel trafficking. These results suggest that DRG macrophages, primed by peripheral nerve injury, contribute to neuron–glia crosstalk, Na _V channel dysregulation and neuronal hyperexcitability implicated in the development of neuropathic pain. Graphical