![]() (B) Representative images showing immunoreactivities to dendritic and synaptic markers in DRG/spinal cord neuron co‐cultures in the presence or absence of TSP4 (20 nM) for 4 days in different chambers. (A) Diagram of Campenot chamber set‐up showing spinal cord neurons were cultured in the outer chambers and DRG neurons were in the inner chamber, where their axons could grow through the inner chamber wall to reach the outer chambers. TSP4 induces synaptogenesis by interacting with Ca vα 2δ 1 subunits at the presynaptic terminals but not DRG neuron somata. * P < 0.05, significantly different from control (PBS) treatment non‐parametric Kruskal–Wallis test with multiple comparisons. Means ± SEM from 20 neurons per group, five neurons per well randomly selected from multiple culture plates of independent experiments. (E) Quantification of synapse counts on individual spinal cord neurons showing that TSP4‐induced excitatory synaptogenesis can be blocked by early, but not delayed, gabapentin treatment. (D) Close‐up images of spinal cord neuron dendrite segments and associated excitatory synapses after early or delayed gabapentin (GBP) treatment. * P < 0.05 significantly different from control (no TSP4 treatment) non‐parametric Kruskal–Wallis test with multiple comparisons. (C) Quantification of synapse counts on individual spinal cord neurons showing that 8–50 nM TSP4 strongly induce synaptogenesis. (B) Close up images of spinal cord neuron dendrite segments and associated synapses after treatment with different concentrations of TSP4 for 4 days. The box denotes a sampling area taken for close‐up analysis of co‐localized dendritic (MAP2, blue) and synaptic (VGlut2, red PSD95, green) marker immunoreactivities shown in the top panels, which are also shown in the top row panels in (B). ![]() (A) Representative images of a mouse DRG (10×) showing RFP‐labelled neurons (left) and a stained spinal cord neuron in co‐culture (right). ![]() TSP4 induces excitatory synaptogenesis between sensory and spinal cord neurons that can be blocked by early, but not delayed, gabapentin treatment. © 2018 The British Pharmacological Society. To view the other articles in this section visit. This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. Early intervention with gabapentin may prevent the development of injury-induced chronic pain, resulting from Ca v α 2 δ 1 /TSP4-initiated abnormal synapse formation. This process was blocked by early, but was not reversed by delayed, treatment with gabapentin. In vivo, early, but not delayed, treatment with low-dose gabapentin blocked this pathway and the development of the pain state.Ĭa v α 2 δ 1 /TSP4 interactions were critical for the initiation, but not for the maintenance, of abnormal synapse formation between sensory and spinal cord neurons. Ca v α 2 δ 1 /TSP4 interactions were not required for maintenance of already formed synapses. Only presynaptic, but not postsynaptic, Ca v α 2 δ 1 subunits interacted with TSP4 to initiate excitatory synaptogenesis through a pathway modulated by T-type calcium channels. Anti-synaptogenic actions of gabapentin were studied in TSP4-injected mice. We investigated the contributions of Ca v α 2 δ 1 subunits and TSP4 to synaptogenesis, and the pathways involved in vitro, and whether treatment with gabapentin could block this process and pain development in vivo.Ī co-culture system of sensory and spinal cord neurons was used to study the contribution from each protein to synaptogenesis and the pathway(s) involved. ![]() Interactions of these proteins promote aberrant excitatory synaptogenesis that contributes to neuropathic pain state development through unknown mechanisms. Nerve injury induces concurrent up-regulation of the voltage-gated calcium channel subunit Ca v α 2 δ 1 and the extracellular matrix protein thrombospondin-4 (TSP4) in dorsal root ganglia and dorsal spinal cord, leading to the development of a neuropathic pain state. ![]()
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