Figure 4. Temporally Coordinated Presynaptic Inhibition of Primary Afferents by GABA/Glycine and Enkephalins from Penk+ Neurons.
(A) Experimental design used to assess the effect of ChR2-mediated activation of Penk+ neurons on synaptic transmission between primary afferent and spinal neurons based on the amplitude of EPSCs evoked by dorsal root stimulation (Penk-negative neurons were recorded).
(B) Activation of Penk+ neurons reduced synaptic transmission between primary afferent and spinal neurons for up to 2 s after light stimulation. Results are expressed as mean ± SEM.
(C) Example traces of EPSCs evoked by dorsal root stimulation and modulated by light during the early phase of inhibition of synaptic transmission (50 ms after light stimulation). Bicuculline and strychnine, but not the DOR and MOR antagonists Tipp-psi and CTOP, blocked the reduction in EPSC amplitude during the early phase of synaptic transmission inhibition. “S” indicates dorsal root stimulation artifacts.
(D) Quantification of (C).
(E) Light-evoked increase in the paired-pulse ratio (PPR), which indicates presynaptic inhibition, was also blocked by bicuculline and strychnine during the early phase of synaptic transmission inhibition.
(F) Example traces of EPSCs evoked by dorsal root stimulation and regulated by light during the late phase of presynaptic inhibition (1,000 ms after light). DOR and MOR antagonists Tipp-psi and CTOP, but not bicuculline and strychnine, prevented the reduction in EPSC amplitude during the early phase of presynaptic inhibition. “S” indicates dorsal root stimulation artifacts.
(G) Quantification of (H).
(H) The increase in the PPR during the late phase of light-induced presynaptic inhibition was also blocked by Tipp-psi and CTOP.
(I) Immunostaining in spinal cord sections from PenkCre mice injected with AAV-FLEx-YFP (green) showed that enkephalins detected in the processes of Penk+ neurons do not co-localize with the somato-dendritic marker MAP2 (red), suggesting enkephalin presence in axons.
(J) Enkephalins (red) co-localized with the presynaptic marker synaptotagmin (blue) and were present in close proximity to primary afferent axon terminals containing CGRP (gold) and synaptotagmin. Arrow heads indicate a process from YFP+ Penk+ neuron (green) forming an enkephalinergic en passant synapse with a CGRP+ primary afferent axon terminal.
Kruskal-Wallis test, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars represent 10 μm. All bar graphs represent mean ± SEM.
See also Figure S6.