Purpose:
Amacrine cells in the inner nuclear layer are difficult to access with patch electrodes, and patch recording cannot measure local asynchronous activity in the processes. We developed electroporation protocols for high resolution morphological identification and recording from local regions of cell processes. We sought to determine whether ON and OFF signals in narrow field amacrine cells are segregated to specific sublamina or intermixed throughout processes spanning the inner plexiform layer.
Methods:
Oregon Green Bapta (OGB) was electroporated into individual amacrine cells in the whole mount retina under visual control using an Olympus confocal microscope. A series of 20-40 V pulses at a concentration of 1 mM and a pressure of 10 mBar filled soma and dendrites with a success rate of 80 percent. Cells remained viable even after 4 hours of measurement. A miniature LED illuminator presented patterned white light stimuli. We measured changes of fluorescence of 20 percent at the cell body and 5-10 percent at local regions along the processes. We identified cell types by comparing their OGB-filled morphology with earlier rabbit studies. The figure shows both process levels of a bistratified narrow field amacrine cell. This technique offers a significant improvement over current patch technology for studying previously inaccessible regions of cells and signals in the inner retina.
Results:
Narrow field amacrine cells were identified by their narrow spread of processes and diffuse stratification throughout the IPL. We identified them as ON, OFF or ON-OFF based upon their responses at the soma to ON and OFF center and surround stimuli. We measured OFF activity in processes in the ON sublamina, but we also measured ON activity in the OFF sublamina in the same cell representing an unexpected mixing of ON and OFF activity.
Conclusions:
Using electroporation of OGB, we can morphologically-identify most retinal cell types that have soma in the INL. Electroporation also allows high spatial resolution recording from the processes restricted to individual levels within the ON or OFF sublaminae of the IPL. The finding of ON and OFF activity in amacrine cell processes in both the ON and OFF sublamina introduces an unexpected processing complexity.
Keywords: retinal connections, networks, circuitry • amacrine cells • calcium