Abstract
Rapidly progressing development of optogenetic tools, particularly genetically encoded optical indicators, enables monitoring activities of neuronal circuits of identified cell populations in longitudinal in vivo studies. Recently developed advanced transgenic approaches achieve high levels of indicator expression. However, targeting non-sparse cell populations leads to dense expression patterns such that optical signals from neuronal processes cannot be allocated to individual neurons. This issue is particularly pertinent for the use of genetically encoded voltage indicators whose membrane-delimited signals arise largely from the neuropil where dendritic and axonal membranes of many cells intermingle. Here we address this need for sparse but strong expression of genetically encoded optical indicators using a titratable recombination-activated transgene transcription to achieve a Golgi staining-type indicator expression pattern in vivo. Using different transgenic strategies, we also illustrate that co-expression of genetically encoded voltage and calcium indicators can be achieved in vivo for studying neuronal circuit input–output relationships.
Highlights
To understand the dynamic interactions of neuronal circuits, covering large neuronal populations across multiple spatially distant brain regions, is a key focus in systems neuroscience
Using a mouse line that expresses dCre under Rasgrf2A promoter [6], here we show that titratable recombination probability can be used to achieve strong intensity, but sparsely distributed (Golgi staining-like) indicator expression pattern in cortical layer II/III pyramidal neurons with either GCaMP6f (GECI) [10] or the voltage sensitive fluorescent protein (VSFP) VSFP Butterfly 1.2 (GEVI) [11,12]
We show that, using these fluorescent proteins, the modular transgenic approach produced dual GEVI/GECI neuronal labelling, shown here in cortical layer II/III pyramidal neurons, which will allow monitoring of concurrent voltage and calcium activity in either the same neuron or in neighbouring neurons
Summary
To understand the dynamic interactions of neuronal circuits, covering large neuronal populations across multiple spatially distant brain regions, is a key focus in systems neuroscience. GEVI expression needs to be strictly targeted to the plasma membrane to sense transmembrane voltage transients, and because membrane of the soma is only a small fraction of total membrane, the membrane-limited signals of GEVIs arise predominantly from dendritic and axonal membranes For this reason, it is very difficult to attribute optical voltage signals from intermingled processes of many neurons to individual neurons. To achieve a strong cell class-specific but sparse expression of GECIs and GEVIs, we took advantage of the recently developed strategy to control Cre-dependent recombination via the use of a destabilized Cre variant, dCre [6] This modified recombinase can be stabilized (that is, “de-destabilized”) using the antibiotic trimethoprim (TMP) that has no natural targets in mammals and penetrates the blood brain barrier [9]. We show that, using these fluorescent proteins, the modular transgenic approach produced dual GEVI/GECI neuronal labelling, shown here in cortical layer II/III pyramidal neurons, which will allow monitoring of concurrent voltage and calcium activity in either the same neuron or in neighbouring neurons
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