In the rat lumbar spinal cord the major supraspinal targets for lamina I projection neurons are the caudal ventrolateral medulla (CVLM), lateral parabrachial area (LPb) and periaqueductal grey matter (PAG). In this study we have estimated the number of lamina I neurons retrogradely labelled from each of these sites in the L4 segment, as well as the proportion that can be labelled by injecting different tracers into two separate sites. Our results suggest that this segment contains approximately 400 lamina I projection neurons on each side, and that approximately 85% of these can be labelled from either the CVLM or the LPb on the contralateral side. Around 120 lamina I cells in L4 project to the PAG, and over 90% of these cells can also be labelled from the CVLM or LPb. Most lamina I neurons projecting to CVLM or LPb are located in the contralateral dorsal horn, but in each case some cells were found to have bilateral projections. We also examined horizontal sections to investigate morphology and the expression of the neurokinin 1 (NK1) receptor in cells labelled from CVLM, LPb or PAG. There were no consistent morphological differences between these groups, however, while cells with strong or moderate NK1 receptor-immunostaining were labelled from LPb or CVLM, they seldom projected to the PAG. These results suggest that many lamina I cells project to more than one site in the brain and that those projecting to PAG may represent a distinct subclass of lamina I projection neuron.

Lamina I of the spinal cord is densely innervated by nociceptive primary afferents, many of which contain substance P. It contains numerous projection neurons: the majority of these respond to noxious stimuli, however some are activated by cooling. In the rat, approximately 80% of the projection neurons express the neurokinin 1 (NK1) receptor, on which substance P acts, and most cells with this receptor are activated by noxious stimuli. Lamina I neurons can be classified morphologically into pyramidal, multipolar, and fusiform types. It has been reported in the cat that pyramidal neurons are activated only by cooling and that in monkey relatively few pyramidal cells are NK1 receptor-immunoreactive. We have used immunocytochemistry to examine the innervation of lamina I projection neurons in the rat by substance P-containing primary afferents and their responses to a noxious stimulus (subcutaneous formalin injection). NK1 receptor-immunoreactive projection cells received a significantly higher density of contacts from substance P-containing afferents than neurons that lacked the receptor. Most contacts on NK1 receptor-immunoreactive cells were associated with synapses. Formalin injection induced c-Fos in approximately 80% of projection neurons with the NK1 receptor and in 25-45% of those without it. More than 80% of pyramidal neurons expressed the receptor, and for both substance P innervation and c-Fos expression there were no significant differences among different morphological types of NK1 receptor-immunoreactive neuron. We conclude that presence or absence of the NK1 receptor is a better indicator of function than morphology for lamina I projection neurons in the rat.

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.

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