Blockade of local spinal cord inhibition mimics the behavioral hypersensitivity that manifests in chronic pain states. This suggests that there is a pathway capable of mediating allodynia/hyperalgesia that exists but is normally under strong inhibitory control. Lamina I and III neurokinin 1 (NK1) receptor expressing (NK1Rϩ) dorsal horn neurons, many of which are projection neurons, are required for the development of this hypersensitivity and are therefore likely to be a component of this proposed pathway. To investigate, whole-cell patch-clamp recordings were made from lamina I and III NK1Rϩ neurons in the spinal cord slice preparation with attached dorsal root. Excitatory postsynaptic currents were recorded in response to electrical stimulation of the dorsal root. Lamina I NK1Rϩ neurons were shown to receive high-threshold (A␦/C fiber) monosynaptic input, whereas lamina III NK1Rϩ neurons received low-threshold (A␤ fiber) monosynaptic input. In contrast, lamina I neurons lacking NK1 receptor (NK1RϪ) received polysynaptic A fiber input. Blockade of local GABAergic and glycinergic inhibition with bicuculline (10 M) and strychnine (300 nM), respectively, revealed significant A fiber input to lamina I NK1Rϩ neurons that was predominantly A␤ fiber mediated. This novel A fiber input was polysynaptic in nature and required NMDA receptor activity to be functional. In lamina I NK1RϪ and lamina III NK1Rϩ neurons, disinhibition enhanced control-evoked responses, and this was also NMDA receptor dependent. These disinhibition-induced changes, in particular the novel polysynaptic low-threshold input onto lamina I NK1Rϩ neurons, may be an underlying component of the hypersensitivity present in chronic pain states.

Translational control through the mammalian target of rapamycin (mTOR) is critical for synaptic plasticity, cell growth, and axon guidance. Recently, it was also shown that mTOR signaling was essential for the maintenance of the sensitivity of subsets of adult sensory neurons. Here, we show that persistent pain states, but not acute pain behavior, are substantially alleviated by centrally administered rapamycin, an inhibitor of the mTOR pathway. We demonstrate that rapamycin modulates nociception by acting on subsets of primary afferents and superficial dorsal horn neurons to reduce both primary afferent sensitivity and central plasticity. We found that the active form of mTOR is present in a subpopulation of myelinated dorsal root axons, but rarely in unmyelinated C-fibers, and heavily expressed in the dorsal horn by lamina I/III projection neurons that are known to mediate the induction and maintenance of pain states. Intrathecal injections of rapamycin inhibited the activation of downstream targets of mTOR in dorsal horn and dorsal roots and reduced the thermal sensitivity of A-fibers. Moreover, in vitro studies showed that rapamycin increased the electrical activation threshold of A␦-fibers in dorsal roots. Together, our results imply that central rapamycin reduces neuropathic pain by acting both on an mTOR-positive subset of A-nociceptors and lamina I projection neurons and suggest a new pharmacological route for therapeutic intervention in persistent pain states.

NMDA receptors have the potential to produce complex activity-dependent regulation of transmitter release when localized presynaptically. In the somatosensory system, NMDA receptors have been immunocytochemically detected on presynaptic terminals of primary afferents, and these have been proposed to drive release of substance P from central terminals of a subset of nociceptors in the spinal cord dorsal horn. Here we report that functional NMDA receptors are indeed present at or near the central terminals of primary afferent fibers. Furthermore, we show that activation of these presynaptic receptors results in an inhibition of glutamate release from the terminals. Some of these NMDA receptors may be expressed in the preterminal axon and regulate the extent to which action potentials invade the extensive central arborizations of primary sensory neurons.

The dorsal horn of the spinal cord in the newborn rat is characterized by large cutaneous mechanoreceptive fields, a predominance of A-fibre synaptic inputs and diffuse primary afferent A-fibre projections, all of which are gradually reduced and refined over the first postnatal weeks. This may be partly responsible for the reduction in cutaneous flexion reflex sensitivity of rats over the postnatal period. Here we show that chronic, local exposure of the dorsal horn of the lumbar spinal cord to the NMDA antagonist MK801 from birth prevents the normal functional and structural reorganization of A-fibre connections. Dorsal horn cells in spinal MK801-treated animals, investigated at eight weeks of age by in vivo electrophysiological recording, had significantly larger cutaneous mechanoreceptive fields and greater A-fibre evoked responses than vehicle controls. C-fibre evoked responses were unaffected. Chronic MK801 also prevented the normal structural reorganization of A-fibre terminals in the spinal cord. The postnatal withdrawal of superficially projecting A-fibre primary afferents to deeper laminae did not occur in treated animals although C-fibre afferent terminals and cell density in the dorsal horn were apparently unaffected. Spinal MK801-treated animals also had significantly reduced behavioural reflex thresholds to mechanical stimulation of the hindpaw compared to naïve and vehicle-treated animals, whereas noxious heat thresholds remained unaffected. The results indicate that the normal postnatal structural and functional development of A-fibre sensory connectivity within the spinal cord is an activity-dependent process requiring NMDA receptor activation.

Central sensitization in inflammatory pain conditions results in behavioral mechanical hypersensitivity. Specifically, C-fiber-driven spinal hyperexcitability enables A fibers to gain access to specific spinal circuitry, via heterosynaptic facilitatory mechanisms, to mediate mechanical hypersensitivity. However, the precise circuitry engaged is not known. Lamina I neurokinin 1 (NK1) receptor expressing (NK1R Control neurons predominantly received monosynaptic C-fiber input (69%) with a smaller proportion receiving monosynaptic A␦-fiber input (28%). In contrast, CFA inflammation significantly increased the incidence (by twofold) and magnitude (by 75% in a subset) of monosynaptic A␦-fiber but not monosynaptic C-fiber-evoked responses. A␤-fiber input to lamina I NK1Rϩ neurons was minimal, polysynaptic in nature, and unaltered by CFA inflammation. Additional examination of control neurons revealed that a proportion received silent monosynaptic A␦-fiber input, suggesting that these may provide the substrate for the novel A␦ inputs observed in CFA inflammation. This inflammation induced unmasking and strengthening of monosynaptic A␦ drive to lamina I NK1R ϩ neurons may contribute to the heterosynaptic facilitatory mechanisms underlying mechanical hyperalgesia in inflammatory pain.