Reduced GABAergic inhibition in the superficial dorsal horn of the spinal cord contributes to the development of persistent pain after peripheral nerve injury (neuropathic pain). We show that the decrease in spinal inhibition is caused by nerve injury-induced transsynaptic apoptosis in the dorsal horn, which leads to a substantial loss (~25%) of GABAergic interneurons. Our goal is to elucidate the mechanisms responsible for the transsynaptic induction of apoptosis and establish strategies to prevent the loss of spinal inhibitory interneurons. We hypothesize that following nerve injury, excitotoxic levels of glutamate released from abnormally active primary afferents trigger the degeneration of dorsal horn neurons via the mitochondrial apoptosis pathway. This hypothesis is based on the observations that (1) blocking conduction of afferent input from an injured nerve attenuates apoptosis in the dorsal horn, (2) apoptosis is decreased after intrathecal administration of an NMDA receptor antagonist, and (3) caspase inhibition reduces the loss of GABAergic interneurons. In order to explore the factors that mediate glutamate excitotoxicity in the dorsal horn, we propose: (1) To determine if NMDA receptor activation is required for glutamate excitotoxicity. We will produce a spatially restricted deletion of functional NMDA receptors by injecting a Cre recombinase-expressing virus into the ipsilateral dorsal horn of adult mice with a floxed NMDA receptor subunit 1 (NR1) gene. (2) To examine if increased glutamate release driven by ongoing afferent activity and a decrease in glutamate uptake are responsible for excitotoxic cell death. Using microdialysis we will measure extracellular glutamate concentrations in the dorsal horn after nerve injury. We will study the expression and activity of glutamate transporters and explore if inhibition of glutamate uptake produces apoptosis. (3) To test whether blockade of mitochondrial apoptosis inhibits neuronal loss. We will employ mice overexpressing Bcl-2 and Bcl-xL under neuron-specific promoters to prevent initiation of the mitochondrial apoptosis pathway. In addition, we will examine whether blocking mitochondrial apoptosis with minocycline or the selective caspase-9 inhibitor zLEHD reduces the loss of neurons. We will determine if rescuing dorsal horn interneurons preserves spinal inhibition and attenuates neuropathic pain-like behavior. ? ? ?
