Neuronal circuitry for pain processing in the dorsal horn

Abstract

Neurons in the spinal dorsal horn process sensory information, which is then transmitted to several brain regions, including those responsible for pain perception. The dorsal horn provides numerous potential targets for the development of novel analgesics and is thought to undergo changes that contribute to the exaggerated pain felt after nerve injury and inflammation. Despite its obvious importance, we still know little about the neuronal circuits that process sensory information, mainly because of the heterogeneity of the various neuronal components that make up these circuits. Recent studies have begun to shed light on the neuronal organization and circuitry of this complex region.

Figure 1. Laminar organisation of the dorsal horn and primary afferent inputs

Rexed divided the grey matter of the cat dorsal horn into a series of parallel laminae based on variations in the size and packing density of neurons, and this scheme has since been applied to other species. a | A transverse section of rat mid-lumbar spinal cord that is immunostained using an antibody (NeuN) that specifically labels neurons. Laminar boundaries are indicated by the dashed lines. Laminae I and II (also known as the marginal zone and substantia gelatinosa, respectively) constitute the superficial dorsal horn, and are characterised by the presence of numerous small neurons. Lamina II can be divided into outer (IIo) and inner (IIi) parts, with the latter having a somewhat lower density of neurons. Image is modified, with permission, from REF. . b | Primary afferents arborise within the dorsal horn in an orderly way: a laminar termination pattern based on fibre diameter and function is superimposed on a somatotopic distribution that determines mediolateral and rostrocaudal location. The central terminations of the major primary afferent types (excluding proprioceptors) are shown. In the 1970s and 1980s a series of intra-axonal labelling studies revealed the projections of different types of myelinated afferents^-. These showed that Aβ tactile and hair afferents end mainly in laminae III-VI with some extension into lamina IIi, the precise arrangement depending on their function. Aδ hair-follicle afferents arborise on either side of the lamina II/III border, whereas Aδ nociceptors end mainly in lamina I, with some giving branches to laminae V and X. More recent studies have identified myelinated nociceptors with conduction velocities in the Aβ range that arborise throughout laminae I-V (not shown). Peptidergic primary afferents (which also include some Aδ nociceptors) arborise mainly in lamina I and IIo, with some fibres penetrating more deeply, whereas most non-peptidergic C fibres form a band that occupies the central part of lamina II.

Figure 2. Classification of lamina II interneurons

Various experimental approaches have been used to classify interneurons, including somatodendritic morphology, firing pattern in response to injected current, and neurochemistry. Examples of these are illustrated. a | Confocal images of 4 lamina II neurons labelled with Neurobiotin following whole cell patch-clamp recording. The cells are seen in sagittal sections and correspond to each of the main classes identified by Grudt and Perl. Islet cells have dendritic trees that are elongated (>400 μm) in the rostrocaudal axis, with little dorsoventral or mediolateral spread. Central cells are similar, but have much shorter dendritic trees (<400 μm long). Radial cells have compact dendritic trees with primary dendrites radiating in several directions. Vertical cells typically have a dorsally placed soma and dendrites that fan out ventrally, often occupying a conical shape. Immunocytochemical staining of axons with antibodies against the vesicular glutamate transporter VGLUT2 and the vesicular GABA (γ-aminobutyric acid) transporter VGAT revealed that the islet cell was GABAergic (VGAT⁺), whereas the other 3 cells in this figure were glutamatergic (VGLUT2⁺) (not shown). Scale bar = 100 μm. b | Firing patterns in response to 1 sec pulses of injected current in 3 different lamina II neurons. Values at the left of each trace indicate initial membrane voltage or current before application of the pulses, and the arrow shows the ‘gap’ seen after the initial spike in gap-firing neurons. The gap and delayed firing pattern, which are thought to result from the presence of I_A currents, were associated with most excitatory (18/22), but few inhibitory (2/23) cells. c | This image shows a confocal optical slice through part of lamina II in a section immunostained to reveal neuropeptide Y (NPY, green), the neuronal form of nitric oxide synthase (nNOS, red) and parvalbumin (PV, blue), compounds that are present in the dorsal horn and found in non-overlapping groups (A.J. Todd and E. Polgár, unpublished data). A single cell body containing each compound is visible. Scale bar = 10 μm. Parts a and b reproduced, with permission, from REF © 2010 Elsevier.

Figure 3. Projection neurons

a | This image shows a transverse section through the contralateral side of the L4 segment of a rat that received injections of two retrograde tracers: Fluorogold into the lateral parabrachial area (LPb) and cholera toxin B subunit (CTb) into the caudal ventrolateral medulla (CVLM). The section was immunostained for CTb (red), Fluorogold (green) and NeuN (blue). Injection of tracers into both of these regions has been previously shown to label virtually all lamina I projection neurons. Cells retrogradely labelled from the LPb appear green, those labelled from the CVLM are red, and those that received label from both sites are yellow. Note the high density of double-labelled cells in lamina I, but that even here they are outnumbered by other neurons (blue). Lamina II contains virtually no projection cells at lumbar levels, and there are scattered retrogradely labelled cells in deeper laminae and in the lateral spinal nucleus (LSN). b | A summary of quantitative data from a series of studies of projection neurons in the L4 segment of the rat spinal cord^,,-. These data suggest that there are approximately 400 projection neurons in lamina I (~5% of the total neuronal population in this lamina) (blue numbers). Most of these can be retrogradely labelled following injection of tracer into the LPb or CVLM, with smaller numbers projecting to the other sites. Studies involving paired injections indicate that virtually all lamina I cells that are retrogradely labelled following tracer injection into thalamus, periaqueductal grey matter (PAG) or nucleus of the solitary tract (NTS) also project to LPb. Note that as spinal projections to the CVLM terminate near the main bundle of ascending axons, retrograde labelling from this region may include cells labelled through uptake of tracer into fibres of passage, rather than from axon terminals. The numbers of neurokinin 1 receptor (NK1R)-expressing projection neurons in laminae III-IV that can be labelled from each site are also shown (red numbers). There are around 24 of these cells per side in the L4 segment (corresponding to ~0.1% of the neurons in this lamina). c | A horizontal section through lamina I scanned to reveal NK1R (green) and the glycine receptor-associated protein gephyrin (magenta). Two NK1R-positive neurons and a giant lamina I cell (outlined by gephyrin puncta) are visible. Although retrograde labelling was not performed, all lamina I neurons of this size are known to be projection cells^,. d | An example of a NK1R-expressing lamina III projection neuron in a sagittal section. The cell is retrogradely labelled with CTb (magenta) that had been injected into the LPb. Note the extensive dorsal dendrites that are labelled with the NK1R antibody (green), and can be followed into lamina I. Several retrogradely labelled lamina I cells are also visible in this field. Scale bars = 100 μm (a,d), 50 μm (c). Part a reproduced, with permission, from REF. . Part c reproduced, with permission, from REF^, © 2008 Society for Neuroscience. Part d reproduced, with permission, from REF © 2000 Wiley.

Fig. 4. Neuronal circuits involving projection neurons

a | A diagram showing some of the synaptic circuits identified in laminae I-III. Three types of projection neuron (PN) are shown: a neurokinin 1 receptor (NK1R)-expressing cell in lamina I (NK1R PN), an NK1R-expressing cell in lamina III, and a giant lamina I neuron^,. Both types of NK1R-expressing projection neuron are densely innervated by substance P-containing primary afferents (SP)^,, and the lamina III neurons also have an input from myelinated low threshold mechanoreceptive (LTM) afferents. The lamina III NK1R cells receive a substantial input from GABAergic interneurons that contain neuropeptide Y (GABA/NPY IN), whereas inhibitory interneurons that contain neuronal nitric oxide synthase (GABA/nNOS IN) innervate the giant lamina I cells. These cells receive a high density of synapses from vesicular glutamate transporter 2-containing boutons derived from unknown populations of glutamatergic interneuron (?GLU IN). NK1R-expressing lamina I projection neurons also receive an input from glutamatergic vertical cells (GLU Vertical cell), which are innervated by glutamatergic central cells (GLU Central cell). The primary afferents that synapse onto vertical cells include Aδ fibres, as well as C fibres that express both transient receptor potential A1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1). b and c show examples of synaptic inputs to projection neurons. b | A lamina III NK1R-positive projection neuron (blue), here seen in sagittal section, receives many contacts from NPY-containing boutons (red). The lower images show part of this region (boxed area) scanned to reveal gephyrin (green), NK1R and NPY. Gephyrin puncta (arrowheads) indicate the presence of synapses. c | The upper image (horizontal section) shows lamina I neurons labelled with Fluorogold from the lateral parabrachial area. The lower images show part of a dendrite of the cell marked with the asterisk (corresponding to the boxed area). The left image has been scanned to reveal GluA2 (blue) and GluA4 (green) subunits of the AMPA ((α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, together with Fluorogold (white). On the right, the same field has been scanned for GluA2, GluA4 and calcitonin gene-related peptide (CGRP, red). The high density of CGRP contacts on the dendrite indicate that this is likely to be a NK1R-expressing cell^,. The presence of puncta containing GluA2 and GluA4 at sites where CGRP boutons are in contact (arrowheads) indicates that these are forming synapses. Scale bars: main images = 25 μm, lower images = 5 μm. Part a modified, with permission, from REF . Part c reproduced, with permission, from REF © 2010 Elsevier.