Calretinin positive neurons form an excitatory amplifier network in the spinal cord dorsal horn

Abstract

Nociceptive information is relayed through the spinal cord dorsal horn, a critical area in sensory processing. The neuronal circuits in this region that underpin sensory perception must be clarified to better understand how dysfunction can lead to pathological pain. This study used an optogenetic approach to selectively activate spinal interneurons that express the calcium-binding protein calretinin (CR). We show that these interneurons form an interconnected network that can initiate and sustain enhanced excitatory signaling, and directly relay signals to lamina I projection neurons. Photoactivation of CR interneurons in vivo resulted in a significant nocifensive behavior that was morphine sensitive, caused a conditioned place aversion, and was enhanced by spared nerve injury. Furthermore, halorhodopsin-mediated inhibition of these interneurons elevated sensory thresholds. Our results suggest that dorsal horn circuits that involve excitatory CR neurons are important for the generation and amplification of pain and identify these interneurons as a future analgesic target.

Three neurotransmitters regulate diverse inhibitory and excitatory Parvalbumin interneuron circuits in the dorsal horn

“…Our in vivo phototstimulation experiments show that when fully intact and strongly activated, ePVINs can recruit signalling in lamina I, observed as Fos activation in this region. In addition, several excitatory interneuron populations in lamina II are known to synapse with lamina I projection neurons including vertical cells (Lu and Perl., 2005;Cordero-Erausquin et al, 2009), calretinin-expressing interneurons (Smith et al, 2019;Petitjean et al, 2019) and CCK-expressing interneurons (Liu et al, 2018). Given that we now show that most ePVINs co-express CCK, and that ePVINs provide direct input to identified lamina I projection neurons, as well as presumptive excitatory interneurons in this region, our findings position these ePNINs as an element of this nociceptive circuitry.…”

“…We assessed the pattern of Fos labelling following in vivo photostimulation PVINs in anaesthetised PV Cre ;Ai32 mice (n = 3) to determine the pattern of spinal activation following selective recruitment of these cells. The dorsal spinal cord (L4 segment) of these animals was exposed by laminectomy and an optic fibre (400μm diameter) applied unilateral photostimulation (10 ms pulses @ 10 Hz for 10 minutes) as described previously (Smith et al, 2019). Animals were maintained under anaesthesia for a further 2 hours before overdose and transcardial perfusion fixation.…”

“…The postsynaptic circuits targeted by PVINs were assessed by delivering spinal photostimulation to anaesthetised PV Cre ;Ai32 animals and then processing spinal cords for Fos-protein as described previously (Smith et al, 2019). Animals (n = 3) were anaesthetised with isoflurane (5% initial, 1.5-2% maintenance) and secured in a stereotaxic frame.…”

Dorsal horn CGRP-expressing interneurons contribute to nerve injury-induced mechanical hypersensitivity

“…For example, the transiently expressing VGLUT3 population is located ventral to the CGRP interneurons, receives low-threshold mechanoreceptive input and their chemogenetic activation also enhances mechanical sensitivity (Cheng et al, 2017; Peirs et al, 2015). Dorsal to the CGRP interneuron are PKCγ and calretinin excitatory interneurons that contribute to nerve injury induced mechanical allodynia (Malmberg et al, 1997; Neumann et al, 2008; Peirs et al, 2015; Petitjean et al, 2019; Smith et al, 2019).…”

Projection Neuron Axon Collaterals in the Dorsal Horn: Placing a New Player in Spinal Cord Pain Processing

“…The anatomy and function of PNs have been studied extensively for over 60 years (Wercberger and Basbaum, 2019) as they play such a key role in channeling highly processed information from the dorsal horn to supraspinal targets. This view is supported by several studies that show a dorsal flow of sensory signals through anatomically and functionally connected dorsal horn IN populations to PNs that can then relay this information to the brain (Cordero-Erausquin et al, 2009;Hachisuka et al, 2018;Smith et al, 2019). The fact that PNs project to readily accessed brainstem centers such as the PBN and periaqueductal gray has facilitated their identification and study in spinal cord slices through retrograde labeling approaches (Ruscheweyh et al, 2004;Dahlhaus et al, 2005).…”