A quantitative study of neurochemically defined excitatory interneuron populations in laminae I–III of the mouse spinal cord

Abstract

BackgroundExcitatory interneurons account for the majority of neurons in laminae I–III, but their functions are poorly understood. Several neurochemical markers are largely restricted to excitatory interneuron populations, but we have limited knowledge about the size of these populations or their overlap. The present study was designed to investigate this issue by quantifying the neuronal populations that express somatostatin (SST), neurokinin B (NKB), neurotensin, gastrin-releasing peptide (GRP) and the γ isoform of protein kinase C (PKCγ), and assessing the extent to which they overlapped. Since it has been reported that calretinin- and SST-expressing cells have different functions, we also looked for co-localisation of calretinin and SST.ResultsSST, preprotachykinin B (PPTB, the precursor of NKB), neurotensin, PKCγ or calretinin were detected with antibodies, while cells expressing GRP were identified in a mouse line (GRP-EGFP) in which enhanced green fluorescent protein (EGFP) was expressed under control of the GRP promoter. We found that SST-, neurotensin-, PPTB- and PKCγ- expressing cells accounted for 44%, 7%, 12% and 21% of the neurons in laminae I–II, and 16%, 8%, 4% and 14% of those in lamina III, respectively. GRP-EGFP cells made up 11% of the neuronal population in laminae I–II. The neurotensin, PPTB and GRP-EGFP populations showed very limited overlap, and we estimate that between them they account for ~40% of the excitatory interneurons in laminae I–II. SST which is expressed by ~60% of excitatory interneurons in this region, was found in each of these populations, as well as in cells that did not express any of the other peptides. Neurotensin and PPTB were often found in cells with PKCγ, and between them, constituted around 60% of the PKCγ cells. Surprisingly, we found extensive co-localisation of SST and calretinin.ConclusionsThese results suggest that cells expressing neurotensin, NKB or GRP form largely non-overlapping sets that are likely to correspond to functional populations. In contrast, SST is widely expressed by excitatory interneurons that are likely to be functionally heterogeneous.

Expression of cholecystokinin by neurons in mouse spinal dorsal horn

“…Because we had previously found that many neurotensin‐expressing cells also showed moderate‐strong labeling for PKCγ (Gutierrez‐Mecinas et al, ), we examined the relationship between neurotensin and CCK among PKCγ‐expressing cells. For this analysis, we excluded cells that showed weak PKCγ‐immunoreactivity, because we have reported that many of these are PPTB‐immunoreactive (Gutierrez‐Mecinas et al, ), and PPTB shows minimal coexpression with either CCK or neurotensin. We identified a total of 838 cells with moderate‐strong PKCγ‐immunoreactivity in sections from three mice, and found that 44% of these were pro‐CCK+ only, 40% were neurotensin+ only, while 2% had both types of peptide immunoreactivity, and 15% had neither (Figure a–d,h).…”

“…We had previously shown that many of the cells with moderate‐strong PKCγ immunoreactivity contained neurotensin (Gutierrez‐Mecinas et al, ). Although PPTB cells were often PKCγ‐positive, the level of PKCγ in these cells was generally much lower, and they may therefore not contribute significantly to the allodynia attributed to PKCγ, or to PKCγ‐expressing neurons.…”

“…The GRP cells were identified by the presence of enhanced green fluorescent protein (eGFP) in a BAC transgenic mouse line (GRP::eGFP) in which eGFP is expressed under control of the GRP promoter (Gutierrez-Mecinas, Watanabe, & Todd, 2014;Mishra & Hoon, 2013;Solorzano et al, 2015). Neurotensin and NKB neurons, which are concentrated in inner lamina II (lamina IIi) and lamina III, partly correspond to cells that express protein kinase Cγ (PKCγ; Gutierrez-Mecinas et al, 2016). PKCγ-expressing cells are of particular interest, because they are thought to form part of a circuit that is responsible for mechanical allodynia (Lu et al, 2013;Malmberg, Chen, Tonegawa, & Basbaum, 1997;Miraucourt, Dallel, & Voisin, 2007).…”

Making sense out of spinal cord somatosensory development

“…The origin of the excitatory calretinin population is mixed because most, but not all cells are derived from the Lbx1 lineage (Duan et al, 2014;Peirs et al, 2015). The SOM + population makes up a large proportion (∼59%) of the excitatory interneurons in lamina II (Gutierrez-Mecinas et al, 2016). Those residing at the lamina II/III border overlap with PKCγ neurons, a population also implicated in mechanical allodynia (Malmberg et al, 1997;Petitjean et al, 2015).…”

Neuron Type-Specific Translatomes in Spinal Cords of Naïve and Neuropathic Mice

“…This is especially reflected for example by the lamina specific innervation pattern by the different types of peripheral sensory neurons: unmyelinated C fibers, which mainly carry noxious and thermal information, terminate in the superficial dorsal horn (laminae I-II), while thickly myelinated Ab fibers, which convey innocuous signals including touch and proprioceptive information, terminate in the deep dorsal horn (laminae III-V) [3,6,7]. A laminar organization of neuronal function is also supported by gene expression patterns that follow laminar patterns [8][9][10][11][12]. Furthermore, optogenetic and chemogenetic experiments support a modality-specific processing by distinct genetically defined neuron populations [7,[13][14][15].…”