The Ultrastructural And Biochemical Evidences For That Capsaicin-sensitive Primary Afferent C Fiber Excites HAP1-containing Neurons In The Spinal Dorsal Horn

Huntingtin-associated protein1 (HAP1) is first identified as one of the huntingtin-associated proteins for its interaction with huntingtin (a gene product for huntington's disease.) while its functions have been unknown yet. There are two isoforms of HAP1 at least----HAP1A and HAP1B which are widely distributed in nervous system. HAP1A is generally located in pericaryon and axon terminal while HAP1B is mainly located in pericaryon and dendrite. Our previous study demonstrated that HAP1 is intensely expressed in the superficial spinal dorsal horn in rat with weak expression level in the ventral horn. Peripheral pain stimulus increase the expression level of the HAP1 and its mRNA in the superficial spinal dorsal horn, suggesting that the HAP1 in spinal dorsal horn may be involved in sensory information processing, particularly the primary input and/or modulation of nociception (algaesthesis). To prove this hypothesis, we used immuno-electron microscope to observe the possible synaptic connections between HAP1-containing neuron and primary afferent C fiber (shorter formed as C fiber), Western blot and RT-PCR were also applied to detect whether or not C fiber participates in the process of expression of HAP1 and its mRNA in the superficial spinal dorsal horn following a peripheral pain stimulus.The lumbar spinal cord in normal rat was immunohistochemically stained with ABC method using HAP1B antibody. Under light microscope, it was observed that HAP1 immunoreactants is mainly distributed in the pericaryon and neuropil in superficial lamella of the gray nucleus with little or no expression in other layers of the spinal horn. The substantia alba show no HAP1B immunoreactants. In another experiment, a catheter had been surgically implanted into spinal subarachnoid space closer to the lumbar intumescentia upon the perforation.of the atlanto-occipital membrane before capsaicin was introduced to selectively degenerate C-fibers in an adult rat. After 3 to 5 days, immuno-electron microscopy was applied to label the lumbar sections using HAP1B antibody. In the superficial lamella, numerous degenerated C-fibers characterize the axon terminals as blurred structure in Vesicle and increased electron density of axoplasm; HAP1B immunoreactant is mainly distributed in pericaryon and neurodendrite. The degenerative axon terminal of C-fiber is wrapped by one or several HAP1B-positive dendrites, showing asymmetric axodendritic synapse or synaptic glomeruli.24 hours after one side pain stimulus (administration of 4% formalin on unilateral hind paw of the rat), both Western blot and RT-PCR detected no statistically significant difference on the expression level of HAP1B and its mRNA between two sides of the spinal cord in two kinds of model rats. (One was subcutaneously treated with capsaicin on the second day of life; the other was injected with capsaicin in spinal subarachnoid space.) In the spinal dorsal horn of the adult rats, which was subcutaneously received dissolvent during neonatal period, the expression level of HAP1B and its mRNA on stimulated side increased significantly than that of the other side.Capsaicin possesses selective neurotoxicity toward C-fibers. Subcutaneously injecting capsaicin during neonatal period or introducing capsaicin through spinal subarachnoid space during adult stage all result in degeneration of the most C-fibers. Thus, it is generally accepted as a research tool for destruction of C-fibers. HAP1B-containing neurons can be activated by the nociceptive signal of C-fibers, which has been proved in this study by the fact that dissymmetric axodendritic synapse or synaptic glomeruli, being formed by HAP1B-positive dendrite and degenerative axon terminal of C-fibers from the rats administered with capsaicin in spinal subarachnoid space, does exist. Furthermore, pain stimulus can facilitate the expression of HAP1B and its mRNA in spinal dorsal horn. After C-fibers were destructed by capsaicin, however, this facilitation disappeared or remarkably reduced. This phenomenon reveals that C-fibers increase the expression of HAP1B and its mRNA in spinal dorsal horn in the processing of peripheral pain stimulus. The HAP1 in spinal dorsal horn may be involved in the message modulation of nociceptive input.