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

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.

Cell Types and Synapses Expressing the SNARE Complex Regulating Proteins Complexin 1 and Complexin 2 in Mammalian Retina

“…As each amacrine cell type fulfills a specific role in the processing of visual signals, the presence of a given Cplx isoform at their synapses might contribute to shaping the response of ganglion cells to visual signals and thus the output of the retina to the brain [ 20 ]. The concept of specificity of the presynaptic molecular machinery applies not only to the Cplxs but also to the Munc13s, which operate as single isoforms at amacrine cell synapses [ 56 ].…”

“…Preparation of the retinae of male and female mice and antibody incubation for light microscopic immunocytochemistry were done as described previously with minor modifications [ 56 ]. Briefly, mice were deeply anesthetized by inhalation of isoflurane (Abbott Laboratories, Chicago, IL, USA) and killed by cervical dislocation.…”

Molecular Assembly and Structural Plasticity of Sensory Ribbon Synapses—A Presynaptic Perspective

“…Likewise, SNARE regulators such as complexins I-IV [ 120 ] and vesicular priming factors of the Munc13 and CAPS families appear to be absent from IHC AZs [ 108 ]. This latter finding is of particular interest since all other neuronal and neurosecretory systems—in invertebrates and vertebrates alike—and even other mammalian ribbon-type synapses in the retina and pineal gland [ 11 , 121 , 122 ], seem to employ these proteins to render SVs fusion competent (reviewed in Reference [ 123 ]). Therefore, future experiments will have to clarify which molecule(s) may substitute for Munc13 function in IHCs.…”