A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags

Abstract

Summary Fibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.

Gephyrin-Lacking PV Synapses on Neocortical Pyramidal Neurons

“…Gephyrin is an attractive molecular candidate for pan-inhibitory synaptic labeling, due to the presumed ubiquity of its expression at inhibitory synapses [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Transgenic overexpression of fluorescent proteins fused to gephyrin and Gephryin.FingRs have previously been used to label cortical inhibitory synapses, as well as track their plasticity on dendrites [ 1 , 6 , 30 , 34 ].…”

“…Gephyrin is widely thought to be structurally important at inhibitory synapses [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ] and has been used as an indicator for GABAergic synapses in quantitative analyses [ 1 , 6 , 30 , 31 , 32 ]. Genetically-encoded fibronectin intrabodies generated with mRNA display against gephyrin (Gephyrin.FingR) have been used for the fluorescent labeling of gephyrin in living cells [ 1 , 33 , 34 , 35 ], where levels of FingR fluorescence are thought to be proportional to the number of gephyrin multimers at synapses. Consistent with prior studies [ 33 , 34 , 35 ], we found that EGFP-tagged Gephyrin.FingR showed strong colocalization with immunolabeled gephyrin in virally-transduced neurons.…”

“…Genetically-encoded fibronectin intrabodies generated with mRNA display against gephyrin (Gephyrin.FingR) have been used for the fluorescent labeling of gephyrin in living cells [1,[33][34][35], where levels of FingR fluorescence are thought to be proportional to the number of gephyrin multimers at synapses. Consistent with prior studies [33][34][35], we found that EGFP-tagged Gephyrin.FingR showed strong colocalization with immunolabeled gephyrin in virally-transduced neurons.…”

NMDA receptor-targeted enrichment of CaMKIIα improves fear memory

“…We next examined colocalization and interaction between GluN1 and the three intrabody candidates in a heterologous system. To circumvent the overproduction of intrabodies that results in diffusion throughout cells, we adopted a previously established system of CCR5TC transcriptional repression 17,18 . When an intrabody candidate expresses and binds to less than 100% of GluN1 (if unsaturated), protein expression continues for further GluN1 binding (Fig.…”

Recombinant Antibodies in Basic Neuroscience Research

“…Highly specific FingRs were developed against the excitatory synapse scaffolding protein PSD‐95 and the inhibitory synapse scaffolding protein gephyrin, allowing for selective targeting of fluorescent proteins to these sites in cells expressing these genetically encoded antibody mimetics. FingRs have been introduced through transfection, in utero electroporation, or viral vectors to label synapses in living cells (Bensussen et al., 2020; Kannan, Gross, Arnold, & Higley, 2016; Kwon et al., 2019; Sinnen et al., 2017; Walker et al., 2017). The ability to target fluorescent proteins or other cargo to specific sites in neurons through the expression of these FingRs opens many possibilities for reporting and/or manipulating synaptic structure and function.…”