The aim of this proposal is to provide a clearer understanding of the cellular and molecular mechanisms underlying the transmission of nociceptive information at synapses between small diameter primary afferent fibers and neurons in the dorsal horn of the mammalian spinal cord. Studies over the past decade have identified acidic amino acids and neuropeptides as two major classes of excitatory neurotransmitters that appear to be release from primary afferent fibers. However the detailed mechanism of action and interaction of these substances and the sensory modalities that they transmit are poorly understood. These problems will be addressed using two new in-vitro preparations; I/ a transverse slice preparation of adult rat spinal cord with an attached dorsal root that permits intracellular recording from identified substantia gelatinosa (s.g.) neurons and activation of A-delta and C fiber synaptic inputs to these neurons. II/ a preparation of isolated neonatal rat dorsal horn neurons that permits the post-synaptic actions of i-glutamate and substance P to be examined by whole cell recording and by measurement of intracellular Ca++ levels. These preparations will be used to determined the chemical identify and physiological actions of transmitters that mediate fine fiber input to s.g. neurons. the post-synaptic actions of I-glutamate on s.g. neurons will be examined in detail to determine the role of different excitatory amino acid receptors in mediating I-glutamate actions and afferent evoked excitation. The possibility that some primary afferents use fast transmitters other than I-glutamate will also be examined. In particular, the role of ATP as an excitatory transmitter in the dorsal horn will be determined. Intracellular dye injection will be used to determine the morphology and anatomical classification of s.g. neurons that have been characterized physiologically. The role of substance P in sensory transmission will be examined in two ways; I/ by determining the actions of substance P on s.g., neurons in the slice preparation, with attention focussed on the actions of the peptide on after potentials in these neurons, II/ by monitoring the intracellular events triggered by substance P receptor activation using Ca++ sensitive dyes and whole cell recording. Two distinct classes of dorsal horn neuronal responses to substance P have been defined through the use of Ca++ sensitive dyes. the mechanisms of signalling evoked by substance P in these two classes of neurons will be examined in detail.
