Selective localization of collybistin at a subset of inhibitory synapses in brain circuits

Abstract

Collybistin is a brain-specific guanine nucleotide exchange factor (GEF) that is crucial for the postsynaptic accumulation of gephyrin and γ-aminobutyric acid A receptors (GABA(A) Rs) at a specific subset of inhibitory synapses. Our understanding of the in vivo function of collybistin has been hampered by lack of information about the synaptic localization of this protein in brain circuits. Here we describe the subcellular localization of endogenous collybistin by using antibodies raised against distinct molecular domains that should recognize the majority of endogenous collybistin isoforms. We show that collybistin co-clusters with gephyrin and GABA(A) Rs in synaptic puncta and is recruited to postsynaptic specializations early during synapse development. Notably, collybistin is present in only a subset of gephyrin-positive synapses, with variable co-localization values in different brain regions. Moreover, collybistin co-localizes with GABA(A) Rs containing the α1, α2, or α3 subunits, arguing against a selective association with specific GABA(A) R subtypes. Surprisingly, we found that collybistin is expressed only transiently in Purkinje cells, suggesting that in these cerebellar neurons collybistin plays a selective role during the initial assembly of postsynaptic specializations. These data reveal a remarkable heterogeneity in the organization of GABAergic synapses and provide an anatomical basis for interpreting the variable effects caused by disruption of the collybistin gene in human X-linked intellectual disability and mouse knockout models.

Molecular and functional heterogeneity of GABAergic synapses

“…The relative abundance of CB splice variants in the CNS and their expression pattern across different brain regions or developmental stages have not been investigated systematically. A recent report using antibodies recognizing the majority of CB isoforms (notably CB3) confirmed the localization of CB at GABAergic PSDs in most brain regions [41].…”

“…4) are elaborated on the basis of observations made in α1-KO, α2-KO, NL2-KO, CB-KO, and mdx mice and take the following considerations into account: 1) In wild-type mice, while gephyrin, NL2, and CB likely are present in the majority of GABAergic synapses [41,65,32,14], a major distinction is brought about by the presence or absence of the DGC, as exemplified by thalamic relay neurons and cerebellar neurons ( Fig. 4A-B).…”

Gephyrin, the enigmatic organizer at GABAergic synapses

“…Therefore, synapses containing GABA A Rs with an α3 subunit do not need collybistin as a cofactor, while collybistin is mandatory for synapses with α2-containing GABA A Rs (see Figures 2A,B). The theory of a selective association of CB with certain GABA A R subtypes has been rejected in the paper by Patrizi et al, yet the interaction of CB with the synaptic complexes seems to be only transient (Patrizi et al, 2011). Therefore, not all synaptic puncta are associated with CB immunoreactivity.…”

A method for the three-dimensional reconstruction of Neurobiotin™-filled neurons and the location of their synaptic inputs

“…For inhibitory post-synaptic specializations, we have used antibody against GABA-A receptor subunit α1, a post-synaptic receptor component of inhibitory GABA-A synapses (Vicini et al, 2001). These primary antibodies have been applied to the mouse central nervous system and validated extensively, VGLUT2 (Graziano et al, 2008; Jakovcevski et al, 2009), PSD-95 (Gazula et al, 2010; Soiza-Reilly and Commons, 2011; Spangler et al, 2011), VGAT (Dudanova et al, 2007; Panzanelli et al, 2007; Fortune and Lurie, 2009; Jakovcevski et al, 2009) and GABA-A receptor subunit α1 (Panzanelli et al, 2007; Belichenko et al, 2009; Patrizi et al, 2012). This subsequently allowed us to use commercially available software Imaris (BitPlane, South Windsor, CT, USA) to reconstruct the filled neuron and to determine the distribution and number of glutamatergic or GABAergic synapses contacting it.…”