Synaptic Dynamics of the Feed-forward Inhibitory Circuitry Gating Mechanical Allodynia in Mice

Abstract

Background The authors’ previous studies have found that spinal protein kinase C γ expressing neurons are involved in the feed-forward inhibitory circuit gating mechanical allodynia in the superficial dorsal horn. The authors hypothesize that nerve injury enhances the excitability of spinal protein kinase C γ expressing interneurons due to disinhibition of the feed-forward inhibitory circuit, and enables Aβ primary inputs to activate spinal protein kinase C γ expressing interneurons. Methods Prkcg-P2A-tdTomato mice were constructed using the clustered regularly interspaced short palindromic repeats and clustered regularly interspaced short palindromic repeats-associated nuclease 9 technology, and were used to analyze the electrophysiologic properties of spinal protein kinase C γ expressing neurons in both normal conditions and pathologic conditions induced by chronic constriction injury of the sciatic nerve. Patch-clamp whole cell recordings were used to identify the nature of the dynamic synaptic drive to protein kinase C γ expressing neurons. Results Aβ fiber stimulation evoked a biphasic synaptic response in 42% (31 of 73) of protein kinase C γ expressing neurons. The inhibitory components of the biphasic synaptic response were blocked by both strychnine and bicuculline in 57% (16 of 28) of neurons. Toll-like receptor 5 immunoreactive fibers made close contact with protein kinase C γ expressing neurons. After nerve injury, the percentage of neurons double-labeled for c-fos and Prkcg-P2A-tdTomato in animals walking on a rotarod was significantly higher than that in the nerve injury animals (4.1% vs. 9.9%, 22 of 539 vs. 54 of 548,P < 0.001). Aβ fiber stimulation evoked burst action potentials in 25.8% (8 of 31) of protein kinase C γ expressing neurons in control animals, while the proportion increased to 51.1% (23 of 45) in nerve injury animals (P = 0.027). Conclusions The Prkcg-P2A-tdTomato mice the authors constructed provide a useful tool for further analysis on how the spinal allodynia gate works. The current study indicated that nerve injury enhanced the excitability of spinal protein kinase C γ expressing interneurons due to disinhibition of the feed-forward inhibitory circuit, and enabled Aβ primary inputs to activate spinal protein kinase C γ expressing interneurons. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Molecular and Electrophysiological Characterization of Dorsal Horn Neurons in a GlyT2-iCre-tdTomato Mouse Line

“…Methods for in situ hybridization (ISH) which were related to vesicular glutamate transporter 2 (Vlgut2) have been described in previous articles. 10,18,19 Briefly, sections from three GlyT2-tdTomato male mice were applied in this procedure. For the acquisition of tdTomato fluorescent images, slices were captured under a confocal scanning microscope installed with laser (Olympus FV1200, Japan).…”

“…According to the protocol utilized in our previous study, 6,7,10,20 spinal cord slice was placed in the bath filled with circulated artificial cerebrospinal fluid. The GlyT2-tdTomato neurons were observed by the upright microscope which was installed with infrared differential interference contrast (IR-DIC), together with green luminescence.…”

“…The procedures for dorsal root stimulation have been reported previously. 6,7,10,20 0.1 to 0.5 ms graded pulses were applied by suction electrodes to stimulate the dorsal roots. The conduction velocities (CVs) of afferent fibers were calculated by the combination of latency with the conduction distance of the recorded response.…”

“…The classification criteria for morphological features of GlyT2 neurons were in accordance with our previous study. 10 Central cells belong to the neuron types which extended their dendrites mainly in the rostro-caudal direction with their dendrite length extended less than 200μm. Islet cells, similar to the central cells, elongated their dendrites longer than 450μm and even reach 600μm.…”

“…3 By using the Prkcg-P2A-tdTomato mice, we have recently documented that spinal PKCγ + neurons receive the feedforward inhibition mediated by Aβ inputs. 10,11 Mechanical allodynia was gated by the convergent inputs which regulate the function of PKCγ + neurons. We further confirmed that the feedforward control on PKCγ + neurons was predominantly glycinergic.…”