An in vitro assay system for studying synapse formation between nociceptive dorsal root ganglion and dorsal horn neurons

Abstract

Synapses between nociceptive dorsal root ganglion (DRG) neurons and spinal cord dorsal horn neurons represent the first loci for transmission of painful stimuli. Our knowledge of the molecular organization and development of these synapses is sparse due, partly, to a lack of a reliable model system that reconstitutes synaptogenesis between these two neuronal populations. To address this issue, we have established an in vitro assay system consisting of separately purified DRG neurons and dorsal horn neurons on astrocyte microislands. Using immunocytochemistry, we have found that 97%, 93%, 98%, 96%, and 94% of DRG neurons on these microislands express markers often associated with nociceptive neurons including Substance P, TRPV1, calcitonin-gene related peptide (CGRP), TrKA, and peripherin, respectively. Triple labeling with these nociceptive-like markers, synaptic vesicle marker Vglut2 and using MAP2 as a dendritic marker revealed the presence of nociceptive-like markers at synaptic terminals. Using this immunocytochemical approach, we counted contact points as overlapping MAP2/Vglut2 puncta and showed that they increased with time in culture. Single and dual patch-clamp recordings showed that overlapping Vglut2/MAP2 puncta observed after a few days in culture are likely to be functional synapses between DRG and dorsal horn neurons in our in vitro assay system. Taken together, these data suggest our co-culture microisland model system consists of mostly nociceptive-like DRG neurons that express presynaptic markers and form functional synapses with their dorsal horn partners. Thus, this model system may have direct application for studies on factors regulating development of nociceptive DRG/dorsal horn synapses.

Fig. 1

DRG/dorsal horn neuron microisland assay system. (A) Schematic of the homemade device for making microisland coverslips showing the perforations through which collagen is pushed out by air to generate a matrix of dots on a PDL-agarose pre-coated coverslip. (B) Example of a microisland with a DRG (Arrowhead) and two dorsal horn neurons (Arrows). (C, D) DRG (Arrowheads) and dorsal horn neurons (Arrows) at day 1 (C) and 5 (D) in vitro are positive for the neuronal marker Tuj1. Neurons avoid the nonpermissive agarose and are confined to the permissive substrate (collagen/cortical astrocytes). Note that a DRG neuron is easily identified by its size difference from the dorsal horn neuron. Scale bar is 50μm.

Fig. 2

DRG neurons on microislands are predominantly nociceptive. DRG/dorsal horn microisland cultures were fixed and immunostained with antibodies against markers associated with nociceptors. (A) Arrowheads point to DRG neurons positively stained for the nociceptive markers peripherin, TRPV1, and TrkA, whereas arrows in DIC images point to unstained dorsal horn neurons. (B) Shows that most DRG neurons in our microisland culture system are nociceptive with 96% (913/947) expressing substance P (SP), 97% (658/680) CGRP, 96.5% (860/890) TrkA, 94% (851/903) TRPV1, and 98.5% (957/972) peripherin. Cells were counted over 10 different cultures. Scale bar is 50 μm.

Fig. 3

Time-course for accumulation of nociceptive DRG and dorsal horn synaptic boutons. (A, B) representative immunostaining images at day 1 and 5 against TrkA, VGlut2, and MAP2. Arrowhead and broken arrow point to a DRG and dorsal horn neuron, respectively. Insets in lower left and right panels of A and B illustrate the triple-labeled boutons (Arrows) quantified in C. The number of synaptic boutons increased over time in culture (0.48 ± 0.1, 3 ± 0.30, 7.7 ± 0.64, 13.4 ± 1.1, 16.4 ± 1.02 for 1-5 DIV cultures, respectively; P<0.0001 by ANOVA test, n≥27 for each time point). All values are given in ± SEM and scale bars are 50μm.

Fig. 4

DRG and dorsal horn neurons on microisland assay system form functional synapses. (A) Representative compressed trace showing a view of all the events during the sampling period. The trace below is an expanded view of an mEPSC event. (B) Cumulative amplitude distribution of mEPSCs recorded from dorsal horn neurons after 5 days in culture (13.8 ± 1.8pA, n=6).

Fig. 5

Paired-recording assay. (A) Typically, a microisland with a DRG neuron and a dorsal horn neuron (Maximum numbers of cell is 3) is selected for patching. DRG neuron is patched in the current-clamp mode and the dorsal horn neuron in the voltage clamp mode. (B) Current steps are given to DRG neurons to assess threshold for action potential and synaptic connection. (C) Sub-threshold stimulation of DRG neurons generally fails to generate action potentials and eEPSCs in the synaptically coupled dorsal horn neuron. (D) Suprathreshold stimulation of DRG neurons generates action potentials, resulting in eEPSCs in the dorsal horn neuron (120 ± 10.02 pA, n=5).