Heterogeneous Presynaptic Distribution of Munc13 Isoforms at Retinal Synapses and Identification of an Unconventional Bipolar Cell Type with Dual Expression of Munc13 Isoforms: A Study Using Munc13-EXFP Knock-in Mice.

Kaspar Gierke,Hanna Regus-Leidig,Jenny Atorf,Anneka Joachimsthaler,Benjamin H Cooper,Frederique Varoqueaux,Jan Kremers,Johann Helmut Brandstätter,Julia Von Wittgenstein,Maike Hemmerlein

doi:10.3390/ijms21217848

Abstract

Munc13 isoforms are constituents of the presynaptic compartment of chemical synapses, where they govern important steps in preparing synaptic vesicles for exocytosis. The role of Munc13-1, -2 and -3 is well documented in brain neurons, but less is known about their function and distribution among the neurons of the retina and their conventional and ribbon-type chemical synapses. Here, we examined the retinae of Munc13-1-, -2-, and -3-EXFP knock-in (KI) mice with a combination of immunocytochemistry, physiology, and electron microscopy. We show that knock-in of Munc13-EXFP fusion proteins did not affect overall retinal anatomy or synapse structure, but slightly affected synaptic transmission. By labeling Munc13-EXFP KI retinae with specific antibodies against Munc13-1, -2 and -3, we found that unlike in the brain, most retinal synapses seem to operate with a single Munc13 isoform. A surprising exception to this rule was type 6 ON bipolar cells, which expressed two Munc13 isoforms in their synaptic terminals, ubMunc13-2 and Munc13-3. The results of this study provide an important basis for future studies on the contribution of Munc13 isoforms in visual signal processing in the mammalian retina.

Highlights

Transgenic mice expressing genetically encoded fluorescent fusion proteins have become a valuable tool in neurobiology research for the analysis of the distribution, dynamics, and interactions of neuron-specific proteins in health and disease
We present a detailed description of the distribution of the Munc13 isoforms in retinal neurons and their chemical synapses, which provides a basis for future studies on the synaptic function of Munc13 isoforms in the mammalian retina
Until now it has not been examined in detail whether the expression of the Munc13-EXFP fusion proteins in Munc13-1, Munc13-2 and Munc13-3 KI mice affects the overall structure and function of the

Summary

Introduction

Transgenic mice expressing genetically encoded fluorescent fusion proteins have become a valuable tool in neurobiology research for the analysis of the distribution, dynamics, and interactions of neuron-specific proteins in health and disease. High expression levels of the tagged proteins are beneficial for their visualization, but can disturb the biochemical homeostasis of the target cells by interfering with the endogenous gene regulatory mechanisms. This may result in artificial conditions, which are difficult to interpret [2]. To study fluorescently tagged proteins in their native environment under the control of their endogenous regulatory elements, the generation of knock-in (KI) mice in which target proteins are manipulated in their endogenous loci is better suited

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Conclusion