Digital reconstruction, quantitative morphometric analysis, and membrane properties of bipolar cells in the rat retina.

Abstract

A basic principle of neuroscience is that structure reflects function. This has led to numerous attempts to characterize the complete morphology of types of neurons throughout the central nervous system. The ability to acquire and analyze complete neuronal morphologies has advanced with continuous technological developments for over 150 years, with progressive refinements and increased understanding of the precise anatomical details of different types of neurons. Bipolar cells of the mammalian retina are short-range projections neurons that link the outer and inner retina. Their dendrites contact and receive input from the terminals of the light-sensing photoreceptors in the outer plexiform layer and their axons descend through the inner nuclear and inner plexiform layers to stratify at different levels of the inner plexiform layer. The stratification level of the axon terminals of different types of bipolar cells in the inner plexiform layer determines their synaptic connectivity and is an important basis for the morphological classification of these cells. Between 10 and 15 different types of cone bipolar cells have been identified in different species and they can be divided into ON-cone bipolar cells (that depolarize to the onset of light) and OFF-cone bipolar cells (that depolarize to the offset of light). Different types of cone bipolar cells are thought to be responsible for coding and transmitting different features of our visual environment and generating parallel channels that uniquely filter and transform the inputs from the photoreceptors. There is a lack of detailed morphological data for bipolar cells, especially for the rat, where biophysical mechanisms have been most extensively studied. The work presented in this thesis provides the groundwork for the future goal of developing morphologically realistic compartmental models for cone and rod bipolar cells. First, the contribution of gap junctions to the membrane properties, specifically input resistance, of bipolar cells was investigated. Gap junctions are ubiquitous within the retina, but it remains to be determined whether the strength of coupling between specific cell types is sufficiently strong for the cells to be functionally coupled via electrical synapses. There are gap junctions between the same class of bipolar cells, and this appears to be a common circuit motif in the vertebrate retina. Surprisingly, our results suggested that the gap junctions between OFF-cone bipolar cells do not support consequential electrical coupling. This provides an important first step both to elucidate the potential roles for these gap junctions, and also for the development of compartmental models for cone bipolar cells. Second, from image stacks acquired from multiphoton excitation microscopy, quantitative morphological reconstructions and detailed morphological analysis were performed with fluorescent dye-filled cone and rod bipolar cells. Compared to previous descriptions, the extent and complexity of branching of the axon terminals was surprisingly high. By precisely quantifying the level of stratification of the axon terminals in the inner plexiform layer, we have generated a reference system for the reliable classification of individual cells in future studies that are focused on correlating physiological and morphological properties. The workflow that we have implemented can be readily extended to the development of morphologically realistic compartmental models for these neurons.