Cholinesterases precede "ON-OFF" channel dichotomy in the embryonic chick retina before onset of synaptogenesis

Retinal physiology is balanced by the interplay between an ON and an OFF channel. The formation of this wiring dichotomy is not understood. Two neuropil sublayers of the inner plexiform layer (IPL) represent levels of cholinergic synaptic circuitry. These two sublayers are better segregated in avians than in most vertebrates; in the chick they are called a and d and participate in the OFF and ON channel, respectively. Both levels can be detected easily in the mature retina by choline acetyltransferase (ChAT) or by acetylcholinesterase (AChE); however, the usefulness of these enzymes as developmental markers is restricted, since ChAT is detected too late, while AChE labels not only cholinergic neuropil. To establish that individual AChE+ cells indeed project into cholinergic subbands a or d (and thus are involved in either channel), in the first part of this study we used the AChE-specific monoclonal antibody 1A2 plus confocal microscopy. We show that at embryonic stage E17 two AChE+ cell types of the inner nuclear layer project into the cholinergic subband a, and another one projects into d. Moreover, two others project into noncholinergic subbands, b and c, which develop only from E14 onwards. In contrast to immunocytochemistry, sensitive AChE histochemistry allows us to trace back the establishment of subbands a and d before E10, with the first AChE subband appearing along with IPL differentiation at E7. The establishment of AChE subbands is preceded by butyrylcholinesterase subbands; with differentiation following a central-peripheral gradient. Since synapses do not develop before E12 in the chick, we conclude that retinal ON-OFF dichotomy is laid down long before the formation of chemical synapses.