Abstract
Interneurons of the spinal dorsal horn are central to somatosensory and nociceptive processing. A mechanistic understanding of their function depends on profound knowledge of their intrinsic properties and their integration into dorsal horn circuits. Here, we have used BAC transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of the vesicular glutamate transporter (vGluT2) gene (vGluT2::eGFP mice) to perform a detailed electrophysiological and morphological characterisation of excitatory dorsal horn neurons, and to compare their properties to those of GABAergic (Gad67::eGFP tagged) and glycinergic (GlyT2::eGFP tagged) neurons. vGluT2::eGFP was detected in about one-third of all excitatory dorsal horn neurons and, as demonstrated by the co-expression of vGluT2::eGFP with different markers of subtypes of glutamatergic neurons, probably labelled a representative fraction of these neurons. Three types of dendritic tree morphologies (vertical, central, and radial), but no islet cell-type morphology, were identified in vGluT2::eGFP neurons. vGluT2::eGFP neurons had more depolarised action potential thresholds and longer action potential durations than inhibitory neurons, while no significant differences were found for the resting membrane potential, input resistance, cell capacitance and after-hyperpolarisation. Delayed firing and single action potential firing were the single most prevalent firing patterns in vGluT2::eGFP neurons of the superficial and deep dorsal horn, respectively. By contrast, tonic firing prevailed in inhibitory interneurons of the dorsal horn. Capsaicin-induced synaptic inputs were detected in about half of the excitatory and inhibitory neurons, and occurred more frequently in superficial than in deep dorsal horn neurons. Primary afferent-evoked (polysynaptic) inhibitory inputs were found in the majority of glutamatergic and glycinergic neurons, but only in less than half of the GABAergic population. Excitatory dorsal horn neurons thus differ from their inhibitory counterparts in several biophysical properties and possibly also in their integration into the local neuronal circuitry.
Highlights
The spinal dorsal horn serves as the first relay station for sensory and nociceptive signals reaching the CNS from the periphery
Transverse sections of PFA-fixed spinal cord tissue were prepared from three mice and endogenous enhanced green fluorescent protein (eGFP) fluorescence was enhanced through staining with anti-GFP antisera. eGFP-positive somata were rather densely packed in the superficial dorsal horn and around the central canal (Fig. 1Aa,b)
Within the spinal grey matter, the gross distribution of vGluT2-eGFP-positive neuropil largely paralleled that of eGFP-positive somata, but intense neuropil staining was present in dorsolateral funiculus (Fig. 1A and Ba), which contains the axons of contralateral lamina I projection neurons (McMahon & Wall, 1985)
Summary
The spinal dorsal horn serves as the first relay station for sensory and nociceptive signals reaching the CNS from the periphery. Nociceptive (high-threshold) afferent fibres terminate mainly in its superficial layers (laminae I and II), while low-threshold mechanosensitive afferent fibres preferentially innervate the deep dorsal horn (laminae III-V) In both the superficial and the deep dorsal horn more than 90% of the neurons are local interneurons. Mice expressing enhanced green fluorescent protein (eGFP) in GABAergic neurons under the transcriptional control of the Gad or Gad gene, or in glycinergic neurons under the control of the GlyT2 (Slc6a5) gene have been successfully used to characterize dorsal horn inhibitory interneurons (Heinke et al, 2004; Zeilhofer et al, 2005; Gassner et al, 2009; Labrakakis et al, 2009; Cui et al, 2011). We used a bacterial artificial chromosome (BAC) transgenic mouse line, which expresses eGFP under the transcriptional control of the vGluT2 gene, to perform targeted recordings from this interneuron population and to compare their intrinsic biophysical properties and their synaptic connections with those of GABAergic and glycinergic interneurons in Gad67eGFP and GlyT2-eGFP transgenic mice
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