Identifying functional populations among the interneurons in laminae I-III of the spinal dorsal horn

Abstract

The spinal dorsal horn receives input from primary afferent axons, which terminate in a modality-specific fashion in different laminae. The incoming somatosensory information is processed through complex synaptic circuits involving excitatory and inhibitory interneurons, before being transmitted to the brain via projection neurons for conscious perception. The dorsal horn is important, firstly because changes in this region contribute to chronic pain states, and secondly because it contains potential targets for the development of new treatments for pain. However, at present, we have only a limited understanding of the neuronal circuitry within this region, and this is largely because of the difficulty in defining functional populations among the excitatory and inhibitory interneurons. The recent discovery of specific neurochemically defined interneuron populations, together with the development of molecular genetic techniques for altering neuronal function in vivo, are resulting in a dramatic improvement in our understanding of somatosensory processing at the spinal level.

Keywords: Itch; neuropeptide; pain; spinal cord.

Figure 1.

Confocal images of four lamina II neurons recorded in parasagittal spinal cord slices from young adult rats in the study by Yasaka et al. Neurobiotin in the pipette allowed labelling with fluorescent avidin after whole-cell recording. The cells correspond to the four main classes recognised by Grudt and Perl. Reproduced with permission from Yasaka et al.

Figure 2.

Neurochemical populations among the inhibitory interneurons. Four largely non-overlapping populations can be identified among the inhibitory interneurons in lamina I-III, defined by expression of neuropeptide Y (NPY), galanin (Gal), neuronal nitric oxide synthase (nNOS) and parvalbumin (PV). This confocal image shows a single optical plane through a transverse section of rat lumbar spinal cord that had been immunostained to reveal each of these substances. A single cell of each type is visible, and these are indicated with asterisks. Approximate positions of laminae are shown. Scale bar = 20 µm. Reproduced with permission from Battaglia AA: An Introduction to Pain and its relation to Nervous System Disorders. John Wiley and Sons; 2016.

Figure 3.

Populations of excitatory neurons in laminae I-II defined by neuropeptide expression. The pie chart shows the proportions of all excitatory neurons in this region that express neurokinin B (NKB), neurotensin (NT), gastrin-releasing peptide (GRP) or substance P (SP). The GRP cells were detected by the presence of eGFP in mice in which eGFP is under control of the GRP promoter, while the other populations were revealed by immunocytochemistry for the neuropeptide or its precursor protein. Note that there is limited overlap between the neurotensin cells and those that express NKB or GRP. Between them, these four populations account for just over half of the excitatory neurons in laminae I-II. Unlike these four neuropeptides, somatostatin is widely expressed among excitatory neurons. We have estimated that it is present in between 60% and 90% of the cells that express each of the other neuropeptides, as well as in some of the remaining excitatory neurons. For further details, see text.

Figure 4.

The distribution of tdTom- and eGFP-positive cells in the dorsal horn following intraspinal injection of AAV.flex.eGFP into a Tac1^Cre;Ai9 mouse. The section has been scanned to reveal tdTom (red), eGFP (green) and PKCγ (blue). (a) TdTom⁺ neurons are concentrated in the superficial laminae and scattered through the deep dorsal horn. (b) The distribution of eGFP⁺ neurons is more restricted, as most of these lie dorsal to the band of PKCγ-immunoreactive neurons, which occupy lamina IIi. Note that none of the eGFP⁺ cells are PKCγ-immunoreactive. (c) In the merged image, it can be seen that there are many tdTom⁺ neurons that lack eGFP (and therefore appear red), and that these include PKCγ-immunoreactive cells (some indicated with arrowheads). The two large cells that are indicated with arrows are likely to be ALT projection neurons, some of which express substance P. The images are projected from 45 optical sections at 1 µm z-spacing. Scale bar = 50 µm. Reproduced with permission from Gutierrez-Mecinas et al.

Figure 5.

A schematic diagram showing some of the synaptic circuits discussed. Cells that express GFP under control of the prion promoter (PrP) correspond to a subset of inhibitory interneurons that express nNOS and/or galanin and dynorphin. These cells receive synaptic input from a variety of primary afferents, including C fibres that express TRPV1 or TRPM8, non-peptidergic C nociceptors that express MrgD and myelinated low-threshold myelinated afferents (A-LTMRs) that conduct in the Aδ or Aβ range. There is evidence that different classes of primary afferent converge on the same cell. They form reciprocal (inhibitory) synaptic connections with lamina II islet cells, and they are also presynaptic to lamina II vertical (ver) cells and to projection neurons in lamina I, which include giant cells. Lamina II vertical cells are thought to form part of a polysynaptic pathway that can transmit input from A-LTMRs to NK1r-expressing lamina I projection neurons. This pathway involves PKCγ-expressing excitatory interneurons in lamina IIi/III, together with transient central (TrC) cells in lamina II. A feedforward circuit involving inhibitory interneurons (including some that express parvalbumin, PV) normally limits the activation of PKCγ cells by A-LTMRs, and this could occur through both GABAergic presynaptic inhibition of the A-LTMR terminals and glycinergic postsynaptic inhibition of the PKCγ cells. A-LTMR afferents are also thought to innervate the ventral dendrites of vertical cells and this synaptic input may also be presynaptically inhibited by the PV cells. There is also evidence that both TrC and vertical cells receive nociceptive input from TRPV1-expressing primary afferents, indicating that the pathway involving these cells normally transmits nociceptive information. Note that dendrites are only illustrated on vertical cells to show that these enter lamina III, where they may receive A-LTMR input. Excitatory and inhibitory synapses are represented by open and closed circles, respectively. For further details, see text.