Identification of Early RET+ Deep Dorsal Spinal Cord Interneurons in Gating Pain

Abstract

The gate control theory (GCT) of pain proposes that pain- and touch-sensing neurons antagonize each other through spinal cord dorsal horn (DH) gating neurons. However, the exact neural circuits underlying the GCT remain largely elusive. Here, we identified a new population of deep layer DH (dDH) inhibitory interneurons that express the receptor tyrosine kinase Ret neonatally. These early RET+ dDH neurons receive excitatory as well as polysynaptic inhibitory inputs from touch- and/or pain-sensing afferents. In addition, they negatively regulate DH pain and touch pathways through both pre- and postsynaptic inhibition. Finally, specific ablation of early RET+ dDH neurons increases basal and chronic pain, whereas their acute activation reduces basal pain perception and relieves inflammatory and neuropathic pain. Taken together, our findings uncover a novel spinal circuit that mediates crosstalk between touch and pain pathways and suggest that some early RET+ dDH neurons could function as pain “gating” neurons.

Semi-intact ex vivo approach to investigate spinal somatosensory circuits

“…Recently, several Cre and/or Flp alleles have been used to target genetically defined populations of spinal interneurons and investigate their role through loss- or gain-of-function approaches in vivo (Bourane et al, 2015a, 2015b; Cui et al, 2016; Duan et al, 2014; Peirs et al, 2015; Petitjean et al, 2015). Less is known, however, about the specific functions of these cells within the context of a spinal microcircuit.…”

“…However, to a large extent, these tools have been vetted and optimized in the brain, rather than the spinal cord. There is little precedent for the use of these tools in dorsal horn interneurons (Cui et al, 2016; Foster et al, 2015), and the degree to which they will work is unclear. We therefore undertook a basic characterization of optogenetic tools in dorsal horn interneurons using several distinct Cre lines and opsins.…”

“…In particular, the typical criteria used to establish mono-synaptic connectivity using electrical simulation (absence of failure upon stimulation at 20 Hz for Aδ fibers and 2 Hz for C fibers [Nakatsuka et al, 1999]) is not applicable to optogenetic stimulation, since light-evoked action potentials have longer latency and show Cre latency jitter than those evoked by electric stimulation (Figure 5—figure supplement 1F). For this reason, we and others used a less stringent criterion (e.g., 0.1 Hz optogenetic stimulation) to differentiate between mono- and polysynaptic input in the dorsal horn (Honsek et al, 2015; Wang and Zylka, 2009; Cui et al, 2016). Verification of monosynaptic connections will likely require additional tests involving, for instance, the use of tetrodotoxin together with the potassium channel antagonist 4-aminopyridine (Nelson et al, 2014; Petreanu et al, 2009).…”

“…However, a considerable limitation to this approach is that it is difficult to target known cell types in the dorsal horn. With the advent of a new generation of molecular genetic tools, it has been possible to overcome this constraint by recording from genetically defined subsets of neurons (Bourane et al, 2015a, 2015b; Cui et al, 2016; Duan et al, 2014; Hantman et al, 2004; Heinke et al, 2004; Kardon et al, 2014; Peirs et al, 2015; Petitjean et al, 2015; Zeilhofer et al, 2005). Generally, though, these experiments have been performed within the context of a spinal slice, which suffers from the shortcoming that key components of the nociceptive circuit, such as the input, are severed.…”

Lamina‐specific population encoding of cutaneous signals in the spinal dorsal horn using multi‐electrode arrays

“…Recently, intra-vital imaging has been used to quantify population data from the DRG (Emery et al 2016;Kim et al 2016;Yarmolinsky et al 2016) and the DH superficial laminae (Nishida et al 2014;Matsumura et al 2015). The predominant and segregated study of superficial and deep laminae persists despite many studies suggesting that communication between laminae, especially between the intermediate and superficial laminae, is crucial to the sensory experience and plays a major role in the development of sensory disorders (Cui et al 2016;Gutierrez-Mecinas et al 2016;Yarmolinsky et al 2016). Unfortunately, our understanding of the processing, modulation and relaying of information, something that is crucial to truly understand the manner in which sensation arises, is lacking due to an absence of assessment of DH function at a network or population level.…”