Purpose: Spinal glycinergic neurons function as critical elements of a spinal gate for pain and itch. We have recently documented that spinal PKCγ⁺ neurons receive the feedforward inhibitory input driven by Aβ primary afferent. The glycinergic neurons control the excitability of PKCγ⁺ neurons and therefore gate mechanical allodynia. However, a dynamic or electrophysiological analysis of the synaptic drive on spinal glycinergic interneurons from primary afferent fibers is largely absent. The present study was aimed to analyze the synaptic dynamics between spinal glycinergic interneurons and primary afferents using a genetic labeled animal model.
Materials and Methods: The GlyT2-P2A-iCre mice were constructed by the CRISPR/Cas9 technology. The GlyT2-iCre-tdTomato mice were then generated by crossing the GlyT2-P2A-iCre mice with fluorescent reporter mice. Patch-clamp whole-cell recordings were used to analyze the dynamic synaptic inputs to glycinergic neurons in GlyT2-iCre-tdTomato mice. The distribution of GlyT2-tdTomato neurons in the spinal dorsal horn was examined by the immunohistochemistry method. The firing pattern and morphological features of GlyT2-tdTomato neurons were also examined by electrophysiological recordings and intracellular injection of biocitin.
Results: The GlyT2-P2A-iCre and GlyT2-tdTomato mice were successfully constructed. GlyT2-tdTomato fluorescence was colocalized extensively with immunoreactivity of glycine, GlyT2 and Pax2 in somata, confirming the selective expression of the transgene in glycinergic neurons. GlyT2-tdTomato neurons were mainly distributed in spinal lamina IIi through IV. The firing pattern and morphological properties of GlyT2-tdTomato neurons met the features of tonic central or islet type of spinal inhibitory interneurons. The majority (72.1%) of the recorded GlyT2-tdTomato neurons received primary inputs from Aβ fibers.
Conclusion: The present study indicated that spinal GlyT2-positive glycinergic neurons mainly received primary afferent Aβ fiber inputs; the GlyT2-P2A-iCre and GlyT2-tdTomato mice provided a useful animal model to further investigate the function of the GlyT2⁺-PKCγ⁺ feedforward inhibitory circuit in both physiological and pathological conditions.
Keywords: glycinergic neurons, spinal cord, GlyT2, Aβ fiber, feed-forward inhibitory circuitry