Lamina II contains a large number of interneurons involved in modulation and transmission of somatosensory (including nociceptive) information. However, its neuronal circuitry is poorly understood due to the difficulty of identifying functional populations of interneurons. This information is important for understanding nociceptive processing and for identifying changes that underlie chronic pain. In this study, we compared morphology, neurotransmitter content, electrophysiological and pharmacological properties for 61 lamina II neurons recorded in slices from adult rat spinal cord. Morphology was related to transmitter content, since islet cells were GABAergic, while radial and most vertical cells were glutamatergic. However, there was considerable diversity among the remaining cells, some of which could not be classified morphologically. Transmitter phenotype was related to firing pattern, since most (18/22) excitatory cells, but few (2/23) inhibitory cells had delayed, gap or reluctant patterns, which are associated with A-type potassium (IA) currents. Somatostatin was identified in axons of 14/24 excitatory neurons. These had variable morphology, but most of those tested showed delayed-firing. Excitatory interneurons are therefore likely to contribute to pain states associated with synaptic plasticity involving IA currents. Although noradrenaline and serotonin evoked outward currents in both inhibitory and excitatory cells, somatostatin produced these currents only in inhibitory neurons, suggesting that its pro-nociceptive effects are mediated by disinhibition. Our results demonstrate that certain distinctive populations of inhibitory and excitatory interneuron can be recognised in lamina II. Combining this approach with identification of other neurochemical markers should allow further clarification of neuronal circuitry in the superficial dorsal horn.

Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, in particular the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb neurons, and we demonstrate that Nppb/somatostatin-cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch, and characterize a contrasting anti-nociceptive role for the peptide.

GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABA(A) and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA-immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA- and/or glycine-immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham-operated and nai;ve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I-III of the ipsilateral dorsal horn that showed GABA- or glycine-immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham-operated or nai;ve animals. In addition, we did not see any evidence for alterations of GABA- or glycine-immunostaining in the neuropil of laminae I-III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.

Excitatory interneurons account for the majority of neurons in the superficial dorsal horn, but despite their presumed contribution to pain and itch, there is still limited information about their organisation and function. We recently identified two populations of excitatory interneuron defined by expression of gastrin-releasing peptide (GRP) or substance P (SP). Here we demonstrate that these cells show major differences in their morphological, electrophysiological and pharmacological properties. Based on their somatodendritic morphology and firing patterns, we propose that the SP cells correspond to radial cells, which generally show delayed firing. In contrast, most GRP cells show transient or single-spike firing, and many are likely to correspond to the so-called transient central cells. Unlike the SP cells, few of the GRP cells had long propriospinal projections, suggesting that they are involved primarily in local processing. The two populations also differed in responses to neuromodulators, with most SP cells, but few GRP cells, responding to noradrenaline and 5-HT; the converse was true for responses to the μ-opioid agonist DAMGO. Although a recent study suggested that GRP cells are innervated by nociceptors and are strongly activated by noxious stimuli [60], we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signal-regulated kinases in response to noxious stimuli. These findings indicate that the SP and GRP cells differentially process somatosensory information.

Peripheral nerve injury leads to structural and functional changes in the spinal dorsal horn, and these are thought to be involved in the development of neuropathic pain. In the chronic constriction injury (CCI) model, abnormal 'dark' neurons and apoptotic nuclei have been observed in laminae I-III of the dorsal horn in the territory innervated by the injured sciatic nerve. These findings have been taken as evidence that there is significant neuronal death in this model, and it has been suggested that loss of inhibition resulting from death of GABAergic inhibitory interneurons contributes to the neuropathic pain. However, loss of neurons from the dorsal horn has not been directly demonstrated in neuropathic models, even though this issue is of considerable importance for our understanding of the mechanisms that underlie neuropathic pain. In this study, we have looked for evidence of neuronal death by using a stereological method (the optical disector) with NeuN-immunostaining, and examining spinal cords of naïve rats, and of rats that had undergone CCI or sham operations. All of the CCI animals showed clear signs of thermal hyperalgesia. However, the numbers of neurons in laminae I-III of the ipsilateral dorsal horn in these animals did not differ significantly from those on the contralateral side, nor from those of sham-operated or naïve animals. These results do not, therefore, support the suggestion that there is significant neuronal death in the dorsal horn in this model.

Olfactory mucosal (OM) tissue, a potential source of stem cells, is currently being assessed in the clinic as a candidate tissue for transplant‐mediated repair of spinal cord injury. We examined the ability of embryonic rat OM tissue to generate stem cells using culture conditions known to promote neural stem cell proliferation. Primary spheres formed that proliferated and exhibited two main morphologies: (a) CNS neurosphere‐like (OM‐I) and (b) small, tight spheroid‐like (OM‐II). The OM‐I spheres expressed the neural stem cell marker nestin but also markers of peripheral glia, neurons, and connective tissue. Further studies demonstrated the presence of multipotential mesenchymal‐like stem cells within OM‐I spheres that differentiated into bone, adipose, and smooth muscle cells. In contrast, the OM‐II spheres contained mainly cytokeratin‐expressing cells. Immunolabeling of rat olfactory tissue with Stro‐1, CD90, and CD105 showed the presence of multipotent mesenchymal cells in the lamina propria, whereas cytokeratin was expressed by the epithelial cells of the olfactory epithelium. In addition, a comparable pattern of immunoreactivity was detected in human tissue using Stro‐1 and cytokeratin, suggesting the presence of similar cells in this tissue. The identification of a nonepithelial multipotent cell in the OM may explain the varied reports on olfactory stem cell differentiation capacity in vitro and in vivo and illustrates the cellular complexity of this tissue as a potential source of stem cells for transplantation and translation to the clinic. STEM CELLS 2009;27:2196–2208

One of the main research strategies to improve treatment for spinal cord injury involves the use of cell transplantation. This review looks at the advantages and possible caveats of using glial cells from the olfactory system in transplant-mediated repair. These glial cells, termed olfactory ensheathing cells (OECs), ensheath the axons of the olfactory receptor neurons. The primary olfactory system is an unusual tissue in that it can support neurogenesis throughout life. In addition, newly generated olfactory receptor neurons are able to grow into the CNS environment of the olfactory bulb tissue and reform synapses. It is thought that this unique regenerative property depends in part on the presence of OECs. OECs share some of the properties of both astrocytes and Schwann cells but appear to have advantages over these and other glial cells for CNS repair. In particular, OECs are less likely to induce hypertrophy of CNS astrocytes. As well as remyelinating demyelinated axons, OEC grafts appear to promote the restoration of functions lost following a spinal cord lesion. However, much of the evidence for this is based on behavioural tests, and the mechanisms that underlie their potential benefits in transplant-mediated repair remain to be clarified.

The central arborizations of large myelinated cutaneous afferents normally extend as far dorsally as the ventral part of lamina II in rat spinal cord. Woolf et al. (1992) reported that after nerve injury some of these afferents sprouted into lamina I and the dorsal part of lamina II, and it has been suggested that this could contribute to allodynia associated with neuropathic pain. Part of the evidence for sprouting was on the basis of the use of cholera toxin B subunit as a selective tracer for A-fibers, and the validity of this approach has recently been questioned; however, sprouting was also reported in experiments involving intra-axonal labeling of chronically axotomized afferents. We have used intra-axonal labeling in the rat to examine central terminals of 58 intact sciatic afferents of presumed cutaneous origin and 38 such afferents axotomized 7-10 weeks previously. Both normal and axotomized populations included axons with hair follicle afferent-like morphology and arbors that entered the ventral half of lamina II; however, none of these extended farther dorsally. We also performed bulk labeling of myelinated afferents by injecting biotinylated dextran into the lumbar dorsal columns bilaterally 8-11 weeks after unilateral sciatic nerve section. We observed that both ipsilateral and contralateral to the sectioned nerve, arbors of axons with hair follicle afferent-like morphology in the sciatic territory extended only as far as the ventral half of lamina II. Therefore these results do not support the hypothesis that Abeta afferents sprout into the superficial laminas after nerve section.