PROJECT SUMMARY Inflammatory injury can have substantial, lasting effects on nociceptive neurocircuits, and potentiates peripheral sensory neuron synapses onto spinal neurons projecting to the midbrain periaqueductal gray (PAG). Although these findings suggest that inflammation strengthens excitatory spinal input to the PAG, complex and heterogenous neurocircuitry in the PAG differentially affects pain transmission. Rodent studies suggest GABA- and glutamatergic PAG neurons facilitate nociception and analgesia, respectively. I propose to isolate spinal input to the PAG using optogenetics and determine if ascending inputs to GABAergic PAG neurons are strengthened, consequently reducing excitation of the glutamatergic PAG population. Furthermore, cannabinoids may promote analgesia via presynaptic inhibition in the PAG, which could be reduced by inflammation—a possibility tested in this proposal. Moreover, since inflammatory injury results in long-term potentiation (LTP) of peripheral synapses onto spinal projection neurons, this proposal will test for LTP in the spino-PAG circuit. Finally, I will use this optogenetic approach in vivo to determine if inhibiting and activating spinal afferents in the PAG after can alleviate or worsen pain after inflammatory injury, respectively. Aim 1: Ascertain if inflammatory injury strengthens spinal input to GABAergic PAG neurons, thereby reducing input to glutamatergic PAG neurons. I will address this hypothesis using in vitro patch-clamp recordings in PAG slices from formalin-injected (inflammatory injury) and control animals. Genetically modified mice expressing channelrhodopsin (Chr2) in ascending spinal projections will allow for selective and temporally precise stimulation of spinal afferents in the PAG. I will label GABA- and glutamatergic PAG neurons using promoter- specific viruses. A) I will determine if inflammation affects the presynaptic strength (number of release sites and probability of release), magnitude of feedforward inhibition, and cannabinoid sensitivity of spinal input to GABA- and glutamatergic PAG neurons. B) I will determine if inflammatory injury potentiates spinal input to the PAG using stimulation protocols and an adenylyl cyclase activator (forskolin) to generate LTP in control animals and assess if this effect is occluded by inflammatory injury. Aim 2: Determine if inhibiting spinal input to the PAG reduces inflammatory pain, and if activating these inputs can exacerbate and/or replicate inflammatory pain. This aim will use optical implants over the PAG region of genetically modified mice expressing opsins in ascending spinal afferents, while measuring pain behaviors. A) Mice expressing halorhodopsin will be used to determine if inhibiting spinal afferents reduces formalin-evoked inflammatory pain. B) Mice expressing the high-fidelity ChETA variant of Chr2 (to mimic the burst firing patterns of spino-PAG projection neurons) will be used to determine if activating ascending spinal te...