title: Excitatory interneurons in the mouse spinal cord with emphasis on those expressing gastrin-releasing peptide
Creators: name: Bell, Andrew McKenzie
Publisher: name: University of Glasgow
Year: 2020
Subjects: subject: Pain, itch, dorsal horn, interneuron, gastrin-releasing peptide.

description: The neurons of the dorsal horn of the spinal cord are critically important components of neural circuits for pain and itch. The vast majority of cells in this area are interneurons and these have axons that remain in the cord and do not project to supra-spinal targets. It is likely that specific classes of interneurons are functionally highly specialised to process specific somatosensory modalities and mediate cross-modality interactions. However, we have a poor understanding of the dorsal horn circuitry that underlies pain and itch sensation and defining functional populations of interneurons remains a challenge. Dorsal horn excitatory interneurons display a marked heterogeneity in terms of their lineage, morphology, physiology, neurochemistry, transcriptomic identity and connectivity. A proposed class of lamina II excitatory interneurons includes cells that express gastrin-releasing peptide (GRP), and these can be identified by the expression of enhanced green fluorescent protein (EGFP) in a GRP-EGFP transgenic mouse line. These cells are distinct from other defined neurochemical classes of interneuron in the dorsal horn and have been proposed to act as secondary pruritoceptors in a labelled-line for itch. The underlying hypothesis of the work presented here is that these cells are not simply neurochemically distinct but rather represent a distinct functional population of excitatory interneurons. We present a series of studies that aims to provide evidence for this by further categorising these neurons in terms of their morphology, responses to stimuli, synaptic inputs and molecular identity. In order to investigate the morphology of GRP-EGFP cells, neurobiotin-filled neurons were reconstructed following patch-clamp recording. Many of the GRP-EGFP cells were classed as having central type morphology. As the majority of these cells also displayed transient firing patterns, they are likely to represent ‘transient central cells’, which are a component of a previously proposed circuit for tactile allodynia. We also compared the morphology of GRP-EGFP cells to cells of another neurochemical class defined by the expression of substance P. Substance P-expressing cells were revealed using virally mediated Brainbow labelling in the Tac1Cre mouse. Many of these cells had radial morphology. Additionally, the two populations of neurons were shown to have distinct somatodendritic morphology using hierarchical cluster analysis. Furthermore, we aimed to determine the responses of GRP-EGFP cells to noxious and pruritic stimuli using activity-dependent markers as surrogate measures of neuronal activation. Despite observing a robust expression of Fos and phosphorylation of ERK following intradermal injection of the pruritogen chloroquine, we found that GRP-EGFP cells were significantly less likely to show these markers than other neurons. We also seldom observed phosphorylation of ERK in GRP-EGFP cells in response to the pruritogen histamine or to a variety of noxious stimuli. The patterns of synaptic input to a cell have implications for function. Here the proportions of synapses arising from specific classes of primary afferent neurons to the GRP-EGFP cells were determined using an anatomical quantification technique. Excitatory synapses were identified using the post-synaptic density protein Homer. We show that these cells receive much of their excitatory synaptic input from MrgprA3/MrgprD‐expressing pruritoceptive/nociceptive afferents or from C‐low threshold mechanoreceptors. Although the cells were not innervated by a class of pruritoceptors that express brain natriuretic peptide (BNP) most of them contained mRNA for NPR1, the receptor for BNP. These cells received only around 10% of their excitatory input from other interneurons, and this is in stark contrast to the equivalent proportion in SP-expressing cells, for which around 50% of excitatory synapses arise from other local neurons. Finally, recent transcriptomic studies have shown that mRNA for GRP is widely distributed among excitatory interneurons in the superficial dorsal horn. This finding is at odds with observations based on the GRP-EGFP mouse, which reveals a homogenous and distinct group of neurons. We show using multiplex in situ hybridisation that Grp mRNA is present in several transcriptomically‐defined populations including those expressing Tac1, Tac2 and a newly recognised class expressing Npff. However, EGFP is restricted to a discrete subset of Grp-expressing cells in the GRP-EGFP mouse. These are different from those that express Tac1, Tac2 and Npff, although some of them express Nmur2 and likely belong to a cluster defined as Glut8. Altogether, these findings demonstrate that the GRP‐EGFP cells constitute a discrete population of excitatory interneurons. In support of this we provide evidence of distinctive somatodendritic morphology, and a characteristic pattern of synaptic inputs. Many of these findings are consistent with the proposed role of these cells in itch circuits. However, the functional implications of the mechano-nociceptive and low threshold inputs identified here are still to be established