The principal neurons of the cerebellar nuclei (CN), the sole output of the olivo-cerebellar system, receive a massive inhibitory input from Purkinje cells (PCs) of the cerebellar cortex. Morphological evidence suggests that CN principal cells are also contacted by inhibitory interneurons, but the properties of this connection are unknown. Using transgenic, tracing, and immunohistochemical approaches in mice, we show that CN interneurons form a large heterogeneous population with GABA/glycinergic phenotypes, distinct from GABAergic olive-projecting neurons. CN interneurons are found to contact principal output neurons, via glycine receptor (GlyR)-enriched synapses, virtually devoid of the main GABA receptor (GABAR) subunits ␣1 and ␥2. Those clusters account for 5% of the total number of inhibitory receptor clusters on principal neurons. Brief optogenetic stimulations of CN interneurons, through selective expression of channelrhodopsin 2 after viral-mediated transfection of the flexed gene in GlyT2-Cre transgenic mice, evoked fast IPSCs in principal cells. GlyR activation accounted for 15% of interneuron IPSC amplitude, while the remaining current was mediated by activation of GABAR. Surprisingly, small GlyR clusters were also found at PC synapses onto principal CN neurons in addition to ␣1 and ␥2 GABAR subunits. However, GlyR activation was found to account for Ͻ3% of the PC inhibitory synaptic currents evoked by electrical stimulation. This work establishes CN glycinergic neurons as a significant source of inhibition to CN principal cells, forming contacts molecularly distinct from, but functionally similar to, Purkinje cell synapses. Their impact on CN output, motor learning, and motor execution deserves further investigation.

Endocytosis is considered as an important mechanism for regulating cell surface numbers and thereby signaling strength of G protein-coupled receptors. Currently, little is known about the endocytotic pathways of GABA B receptors in neurons. Here we report that GABA B receptors are constitutively internalized presumably via clathrin-dependent endocytosis in cultured cortical neurons. Colocalization of GABA B receptors with endosomal marker proteins indicated sorting of GABA B receptors from early endosomes to recycling endosomes and to lysosomes. Cell surface biotinylation experiments revealed fast constitutive recycling

Transgenic mice are highly valuable tools for biological research as they allow cell type-specific expression of functionally instrumental genes. In this protocol, the generation of bacterial artificial chromosome (BAC) transgenic constructs is described. We give an overview of different transgenic inserts, such as fluorescent proteins (alone or in combination with Cre variants), diphtheria toxin receptor, lacZ, and light-activated ion channels. The most reliable and versatile approach to express these genes is by using BACs, which allow "highjacking" of the expression pattern of a gene without characterizing its transcriptional control elements. Here, we describe the necessary cloning techniques compared with conventional transgenesis. With the provided "toolbox" of already available transgene constructs, the generation of the BAC transgenes is made easy and rapid. We provide a comprehensive outline how to insert the different transgenes into a chosen BAC by either ET cloning or recombineering. We also describe in detail the methods to identify the correct insertion and the integrity of the final BAC construct, and finally, the preparation of the BAC DNA for oocyte injection is described.

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