Electrophysiological fingerprints of OFF bipolar cells in rat retina

In the mammalian retina, five classes of neurons provide extensive processing and filtering of raw data input, extracting spatial, temporal and chromatic information from the visual scene. The discovery of parallel processing of visual signals by ON and OFF channels in the retina, responding antagonistically to light stimulus increments or decrements, has been a milestone in vision research1,2. This division of visual input is achieved at the first retinal synapse, formed by photoreceptors, horizontal and bipolar cells (BCs). While ON BCs respond to glutamate liberation from photoreceptors with sign-inverting membrane hyperpolarization mediated by mGluR6 receptors coupled to TRPM1 channels3,4, OFF BCs express ionotropic glutamate receptors at this synapse, generating sign-conserving depolarizing cationic currents in response to glutamate5,6,7. Within OFF BCs, the information is shaped by differential contributions of voltage-dependent Na+, Ca2+ 8,9 and K+ conductances10, including hyperpolarization-activated cyclic nucleotide-gated (HCN) channels11. Inhibitory signalling from different types of amacrine cells, mediated by GABA and glycine receptors12, as well as retinal neuromodulators like acetylcholine (ACh) and nitric oxide (NO) contribute to the conditioning of BC responses13,14. Finally, the processed information is passed on via glutamate release to ganglion cells in the outer half of the inner plexiform layer (IPL). A large body of evidence, accumulated during the last two decades, has revealed significant differences among mammals regarding the number and relative percentage of OFF BC types, their morphology and glutamate receptor subunit composition at the photoreceptor synapse15,16,17,18. In mouse, the five established types of OFF BCs are labelled 1, 2, 3a, 3b and 4, and their axonal arbours stratify in sublayers 1 and 2 of the IPL19,20, comprising about 40% width of this synaptic stratum21,22. Although important progress in the functional differentiation of OFF BCs in different species, including mouse, ground squirrel and rat, has been achieved during recent years15,16, the organizational scheme of BCs is still mostly based on morphological criteria, particularly axonal arbour shape and localization with respect to the IPL sublayers. Physiological studies of BC function are hindered by the fact that these cells cannot be reliably identified in retinal whole mounts and slice preparations without dye filling or immunohistochemical processing, and transgenic mouse lines expressing fluorescent markers in specific BC types, although becoming increasingly popular, may not always be available or experimentally suitable. Here, we present an electrophysiological approach to unequivocally distinguish OFF BCs in rat retina, based on their voltage-gated currents and responses to glutamate stimuli under voltage and current clamp. This study expands the number of known OFF BC types from 46,11,18 to 5 in rat, as in ground squirrel23 and mouse15,21, and presents a variation of BC-2 expressing prominent Na+ currents. Moreover, our results show that glutamate responses in BC-2, 3b and 4 depend on both AMPA and kainate receptor activation and are subject to inhibitory modulation by NO.