Expression of green fluorescent protein defines a specific population of lamina II excitatory interneurons in the GRP::eGFP mouse

Abstract

Dorsal horn excitatory interneurons that express gastrin-releasing peptide (GRP) are part of the circuit for pruritogen-evoked itch. They have been extensively studied in a transgenic line in which enhanced green fluorescent protein (eGFP) is expressed under control of the Grp gene. The GRP-eGFP cells are separate from several other neurochemically-defined excitatory interneuron populations, and correspond to a class previously defined as transient central cells. However, mRNA for GRP is widely distributed among excitatory interneurons in superficial dorsal horn. Here we show that although Grp mRNA is present in several transcriptomically-defined populations, eGFP is restricted to a discrete subset of cells in the GRP::eGFP mouse, some of which express the neuromedin receptor 2 and likely belong to a cluster defined as Glut8. We show that these cells receive much of their excitatory synaptic input from MrgA3/MrgD-expressing nociceptive/pruritoceptive afferents and C-low threshold mechanoreceptors. Although the cells were not innervated by pruritoceptors expressing brain natriuretic peptide (BNP) most of them contained mRNA for NPR1, the receptor for BNP. In contrast, these cells received only ~ 10% of their excitatory input from other interneurons. These findings demonstrate that the GRP-eGFP cells constitute a discrete population of excitatory interneurons with a characteristic pattern of synaptic input. Neuronal circuits in the spinal dorsal horn process somatosensory information, which is then transmitted to the brain by projection cells. The vast majority of dorsal horn neurons are interneurons, and these can be assigned to two main classes: excitatory and inhibitory cells 1,2. Nociceptive and pruritoceptive primary afferents terminate in the superficial dorsal horn (SDH; laminae I-II), and ~ 75% of neurons in this region are excitatory interneurons 2. Recent studies have shown that these cells can be assigned to several largely non-overlapping populations, based on the expression of different neuropeptides 3-6 , and these are thought to have distinct roles in pain and itch. There is compelling evidence that gastrin-releasing peptide (GRP) and its receptor (GRPR) play a key role in itch. Intrathecal administration of GRP evokes scratching, while GRPR antagonists reduce scratching in response to pruritogens 7-11. In addition, mice lacking GRPR, or those in which GRPR-expressing dorsal horn neurons are ablated, show reduced responses in several itch models 7,8,12,13. Excitatory interneurons within the SDH provide a source of GRP 7,14-16 , and several studies have investigated the role of these cells by using BAC transgenic mouse lines from the GENSAT project 17 in which either enhanced green fluorescent protein (eGFP) 7,14,15,18-20 or Cre recombinase 18-21 is expressed under control of the Grp promoter

Neuron-specific spinal cord translatomes reveal a neuropeptide code for mouse dorsal horn excitatory neurons

“…In total, we find that the 11 different neuropeptides analysed in this study span dorsal horn laminae I–V, thus potentially setting up a map for a neuropeptide sensory signalling code. This is in good agreement with recent literature, as a number of these genes have either already been suggested to define specific subsets of spinal neurons 11 , 30 – 32 or have already been used as driver genes for functional manipulation of spinal neuron subtypes 14 , 33 – 39 . These studies demonstrate that neuronal subpopulations marked by the expression of specific neuropeptides exert modality specific functions in sensory processing.…”

Characterisation of lamina I anterolateral system neurons that express Cre in a Phox2a-Cre mouse line

“…Spinal cord tissue was cut into 60 μm thick section in either the transverse or horizontal plane with a vibrating blade microtome (Leica VT1200 or VT1000). Sections were processed for multiple-labelling immunofluorescence, as described previously [52][53][54] . They were incubated at 4 °C in mixtures of primary antibodies (see Table S1) for 3 days and then in species-specific secondary antibodies for 1 day.…”

How Do Neurons Signal Itch?

“…Implications for clinical approaches: The “phenotypic switch” of nociceptors that start to express GRP after axotomy ( 29 ) not only adds to the controversy about the role of this peptide in primary afferents ( 62 , 63 ), but may have major implications for clinical pruritus. Beyond the direct involvement of GRP in the generation of pruritus it may render the spinal cord more susceptible for evoked pruritus: in particular acute electrical stimulation may provoke mainly pain in healthy volunteers.…”