The dorsal horn of the spinal cord is the first node in the somatosensory pathway, and is an area essential for controlling the flow of sensory information sent to the brain. Interneurons constitute the vast majority of neurons in this area, and between 25-40% of those in laminae I-III are inhibitory. These inhibitory interneurons are critical for normal somatosensation, for example, by suppressing pain in the absence of noxious stimuli. Interneurons of the dorsal horn are poorly understood due to their morphological and functional diversity, and this is a major factor limiting our understanding of the neuronal circuitry of the dorsal horn. In order to better understand sensory processing in the dorsal horn it is first necessary to characterise the neurons in this area, and to determine the neuronal circuits in which they are integrated. To address this issue, two separate and non-overlapping populations of inhibitory interneurons in the dorsal horn were thoroughly characterised in terms of their morphological and physiological properties. To achieve this, whole-cell recordings were taken from neurons labelled with green fluorescent protein (GFP) under the control of the Prion promoter (PrP) and the neuropeptide Y (NPY) promoter in spinal cord slices from mice. The recording electrodes contained Neurobiotin, which filled the cells during recording and was revealed with fluorescent molecules, enabling three-dimensional reconstruction of cell bodies and dendrites and axons of neurons. Slices containing these labelled neurons were then resectioned for immunocytochemical reactions to determine their neurochemical content and their synaptic inputs and outputs.This study demonstrated that both PrP- and NPY-GFP cells were morphologically heterogeneous although neither group contained islet cells, which are a distinct morphological class of interneuron. PrP- and NPY-GFP cells in lamina II could not be distinguished from each other by using hierarchical cluster analysis with measures of somatodendritic morphology. This suggests that morphological properties may not be useful in distinguishing these populations of interneurons. The vast majority of PrP- and NPY-GFP cells either displayed tonic or initial burst firing of action potentials. However, these groups of cells showed significant differences in some of their active and passive membrane properties, such as membrane resistance, spike frequency adaptation and mV drop in action potential height. When hierarchical cluster analysis was used to group these cells in lamina II based on physiological parameters, PrP- and NPY-GFP cells could be distinguished with some accuracy. This suggests that some physiological differences may exist between these two groups.Within the PrP-GFP group there was a subset that included lamina I among its synaptic outputs, and these cells could provide inhibition to the projection neurons located in this lamina, since GFP boutons from this mouse line can form synapses with giant cells and neurokinin-1 receptor (NK1r)-expressing lamina I neurons. Some PrP-GFP cells showed immunoreactivity for neuronal nitric oxide synthase (nNOS) or galanin, and these two groups had slight morphological differences, which included their laminar location and the spread of their processes. Several experimental approaches, such as electrophysiological, pharmacological and anatomical techniques, indicated that PrP-GFP cells received input from many different types of primary afferent fibre, including peptidergic and non-peptidergic C-afferents, as well as low-threshold mechanosensory fibres. Taken together these findings establish the PrP-GFP cells as a much more functionally heterogeneous group than previously reported. NPY-GFP cells were located in laminae II and III, but were preferentially found in lamina III. The lamina III cells had dendrites with a greater dorsoventral extent than the lamina II cells, and this extent was seen be more dorsal from the soma than ventral. Many NPY-GFP cells received synaptic input from C-fibres, and a subset of those tested lacked TRPV1. Since the TRPV1-lacking C-fibres mostly correspond to the non-peptidergic C-fibres, including non-peptidergic nociceptors and C-low threshold mechanoreceptors, this suggests that NPY-GFP cells could receive input from these fibres. Dorsal root stimulation experiments showed that labelled NPY-GFP cells with somata located in lamina III often received synaptic input from unmyelinated C-fibres, and NPY-expressing neurons in lamina III could respond to noxious mechanical stimuli. A select group of NPY-GFP cells were seen to innervate putative anterolateral tract (ALT) neurons located in lamina III, which could be identified by their dense innervation by bundles of axons containing either NPY or calcitonin gene related peptide (CGRP).Taken together these data suggest that the PrP- and NPY-GFP neurons are distinct populations based on their primary afferent input and post-synaptic targets, and that more than one functional population exists within each of these groups. Despite their many differences, morphological parameters do not appear to be useful in distinguishing the PrP- and NPY-GFP cells, or detecting different functional populations within these groups. The PrP-GFP cells are more morphologically heterogeneous than previous reports suggested, and due to similar features with cells that require the transcription factor Bhlhb5 to develop, they may include a population that are involved in suppressing itch-related signals. NPY-GFP cells could play a role in limiting the spread and intensity of noxious stimuli due to their input from C-fibres, and a small subset of these could inhibit ALT neurons in lamina III. These results further support the view that different neurochemical populations of inhibitory neurons have distinct functional roles, and also highlight the complexity of the neuronal circuitry in the superficial dorsal horn.
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Characterisation of distinct inhibitory interneuron populations in the spinal dorsal horn
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