Demonstration of cholinergic ganglion cells in rat retina: expression of an alternative splice variant of choline acetyltransferase.

ABSTRACT

Acetylcholine acts as a neurotransmitter in the retina. Although previous physiological studies have indicated that some retinal ganglion cells may be cholinergic, several immunohistochemical studies using antibodies to choline acetyltransferase (ChAT) have stained only amacrine cells but not ganglion cells. Recently, we identified a splice variant of ChAT mRNA, lacking exons 6-9, in rat peripheral nervous system. The encoded protein was designated as ChAT of a peripheral type (pChAT), against which an antiserum was raised. In the present study, we examined expression of pChAT in rat retina, both at the protein level by immunohistochemistry using the antiserum and at the mRNA level by RT-PCR. Immunohistochemistry revealed that although no positive neurons were found in untreated intact retinas, many neurons became immunoreactive for pChAT after intravitreal injection of colchicine. Damage of the optic nerve was also effective in disclosing positive cells. Such positive neurons were shown to be ganglion cells by double labeling with a retrograde tracer that had been injected into the contralateral superior colliculus. Western blot analysis and RT-PCR revealed a corresponding band to the pChAT protein and to the amplified pChAT gene fragment, respectively, in retinal samples. In addition, ChAT activity was definitely detected in retinofugal fibers of the optic nerve. These results indicate the presence of cholinergic ganglion cells in rat retina.