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Immunoreactivity for m2 and m4 muscarinic acetylcholine receptors (mAChRs) was demonstrated in the adult newt retina. The m2 mAChR was localized to somata on either side of the inner plexiform layer (IPL), especially ganglion cells, and also distributed into two bands within the IPL. The distal band at a depth of 0-15% IPL co-localized with one of two choline acetyltransferase (ChAT) immunoreactive bands, while the proximal band at 85-100% depth did not overlap with either of the ChAT-ir bands. The m4 mAChR was localized to somata closely apposed to either side of the IPL, probably amacrine cell somata, and no immunoreactivity was detectable throughout the IPL. The time course of appearance of the m2 and m4 mAChRs was examined in both developing and regenerating retinas. Like acetylcholinesterase (AChE), the m2 was first detected in somata located at the most proximal level of the retina well before ChAT-ir cholinergic neurons appeared, while the m4 was detected at the time of appearance of ChAT, in both developing and regenerating retinas. When the outer plexiform layer (OPL) began to form, somata in the horizontal cell layer became transiently immunoreactive to the m2. The discrepancy in distribution of the m2 and ChAT in the IPL suggests that mAChR may play a role other than cholinergic neurotransmission. Furthermore, the similarity in time course of appearance of the m2 and m4, as well as other cholinergic system components [4], in both developing and regenerating retinas would suggest that the mechanisms that control neuronal differentiation during retinal development and regeneration are similar.

The presence of the choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) was demonstrated in the adult newt retina using immunocytochemical and histochemical techniques. Within the inner plexiform layer (IPL), two ChAT-positive bands were detected at relative depths of 0-15% and 45-60% of the total thickness (100%) of the IPL. AChE-positive band occupied approximately 0-60% of the IPL width with an intensive AChE-positive band at a depth of 20-40% within the IPL. Localizations of maximum ChAT and AChE activity were not exactly the same in the IPL of the mature retina. To elucidate whether retinal regeneration follows the same sequence of cellular differentiation steps that occur in retinal development, we examined the time course of appearance of the cholinergic neurons and AChE activity in both developing and regenerating retinas. The ChAT-positive cells were first detected in the retina just before or at the beginning of the morphological development of the IPL in both developing and regenerating retinas. AChE activity first became detectable in somata located at the most proximal layer of the retina before the ChAT-positive cells could be detected and well before the IPL developed in both developing and regenerating retinas. During subsequent development and regeneration, the outer plexiform layer, the IPL, and somata close to either side of the IPL became AChE-positive. The fact that the time course of the appearance of ChAT and AChE molecules during regeneration was similar to that observed during development suggests that common mechanisms may control both the development and the regeneration of the newt retina., (Copyright 1999 Elsevier Science B.V.)

The adult newt regenerates a functional retina following removal or destruction of the original retina. We studied the order of appearance of cell types in the regenerating retina by using immunohistochemical techniques. An antibody that recognizes the alpha subunit (260 kDa) of voltage-dependent Na+ channels was found to label a 255-kDa band in Western blots of crude membrane fractions from the normal retina. Cryosections of normal retina revealed intense Na+ channel immunoreactivity in somata and axons of ganglion cells, weaker immunoreactivity in somata of amacrine cells, and no immunoreactivity in the inner plexiform layer. In the same sections, immunoreactivity to a monoclonal antibody (RB-1) specific to newt cones was intense in the photoreceptor layer. In regenerating retinas, double staining with the Na+ channel antibody as a possible marker of ganglion cells and RB-1 antibody first revealed immunoreactive cells at the intermediate stage (three to five cells thick), which does not exhibit segregated synaptic layers. Na+ channel-immunoreactive ganglion cells appeared before the RB-1-immunoreactive photoreceptors. Because ganglion cells also appear before photoreceptor cells in normal development, common mechanisms may control both the generation and the regeneration of the newt retina.