Abstract
Voltage imaging with cellular resolution in mammalian brain slices is still a challenging task. Here, we describe and validate a method for delivery of the voltage-sensitive dye ANNINE-6plus (A6+) into tissue for voltage imaging that results in higher signal-to-noise ratio (SNR) than conventional bath application methods. The not fully dissolved dye was injected into the inferior olive (IO) 0, 1, or 7 days prior to acute slice preparation using stereotactic surgery. We find that the voltage imaging improves after an extended incubation period in vivo in terms of labeled volume, homogeneous neuropil labeling with saliently labeled somata, and SNR. Preparing acute slices 7 days after the dye injection, the SNR is high enough to allow single-trial recording of IO subthreshold oscillations using wide-field (network-level) as well as high-magnification (single-cell level) voltage imaging with a CMOS camera. This method is easily adaptable to other brain regions where genetically-encoded voltage sensors are prohibitively difficult to use and where an ultrafast, pure electrochromic sensor, like A6+, is required. Due to the long-lasting staining demonstrated here, the method can be combined, for example, with deep-brain imaging using implantable GRIN lenses.
Highlights
Investigating neuronal activity on network and single-cell level with the use of fluorescent voltage indicators has in recent years been regaining popularity
The inferior olive (IO) subthreshold oscillations (STOs) are regular, sinusoidal fluctuations of the membrane voltage, ranging between 3 and 12 Hz in frequency and occasionally reaching up to 15 mV peak-to-peak amplitudes but most commonly ranging between 2 and 10 mV (Llinás and Yarom, 1986; Chorev et al, 2007; Khosrovani et al, 2007). While they are dramatically smaller in amplitude on single-cell level than other commonly-studied oscillatory phenomena in mammalian brains that reflect synchronization of suprathreshold action potentials, the IO STOs are considered a key component for input integration and subsequent complex spike activity in the cerebellum (Jacobson et al, 2009; Llinás, 2009; Negrello et al, 2019)
The stock solution is stored at −20◦and diluted in phosphate-buffered saline (PBS) around 20 min before the injection to obtain the final solution of 100 μg/ml A6+ (140 μM) in 1% DMSO. 3% DMSO solutions were obtained by diluting A6+ stock solution in PBS, and adding 2% DMSO 20 min before experiment
Summary
Investigating neuronal activity on network and single-cell level with the use of fluorescent voltage indicators has in recent years been regaining popularity. The IO STOs are regular, sinusoidal fluctuations of the membrane voltage, ranging between 3 and 12 Hz in frequency and occasionally reaching up to 15 mV peak-to-peak amplitudes but most commonly ranging between 2 and 10 mV (Llinás and Yarom, 1986; Chorev et al, 2007; Khosrovani et al, 2007) While they are dramatically smaller in amplitude on single-cell level than other commonly-studied oscillatory phenomena in mammalian brains that reflect synchronization of suprathreshold action potentials (such as gamma or theta oscillations; Cunningham et al, 2004; Hummos and Nair, 2017), the IO STOs are considered a key component for input integration and subsequent complex spike activity in the cerebellum (Jacobson et al, 2009; Llinás, 2009; Negrello et al, 2019). IO spikes are the source of synchronous cerebellar complex spike activity that in turn underpin the construction of overall cerebellar output (Wise et al, 2010; Streng et al, 2018; Tang et al, 2019; Arlt and Häusser, 2020)
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