Journal of the American Chemical Society
Article
of 6.7 ms ± 1.5 ms (SD n = 60 pairs of spikes) and
than 10 Hz (Figure 5c). Neurons in the brain and retina can
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.6 ms ± 1.3 ms (S.D., n = 20 out of 60 spikes), respectively.
fire action potentials at rates up to several hundred Hz, for
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However, the data reveals differences in the relative strengths
of these connections with cell 1 triggering firing in cell 4 for
example, in interneurons of the hippocampus, Purkinje cells
86 87
of the cerebellum, and ganglion cells of the retina,
emphasizing the need for indicators with fast response kinetics.
1
00% of action potentials, and only 33% for cell 5.
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+
Interestingly, bursts of spontaneous activity indicate strong
recurrent connectivity between all neurons (Figure 4e,f, gray
shaded areas), but firing initiated in the ChR2-expressing
neuron (cell 1, Figure 4a−d) activates only a subset of these
neurons (Figure 4e,f): notably, cell 4, nearly 50 μm away from
cell 1, and cell 5, over 120 μm distant from ChR2-expressing
cell 1.
The combination of poRhoVR and ChR2 enables inter-
rogation of subthreshold potentials. In a few cases, during cyan
light stimulation, we observe slower, subthreshold potentials in
Ca indicators are often characterized against varying
numbers of action potentials arriving at a constant frequency.
However, neural information is often encoded in the form of
spike rates. Therefore, resolution of individual spikes and firing
frequency is critical to an understanding of the underlying
physiology of the system under observation. Even with very
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+
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+
able to resolve differences at 5, 10, 20, or 30 Hz firing rates. In
contrast, the optically recorded voltage transients revealed by
poRhoVR 14 to monitor subthreshold voltage changes (Figure
2
+
3
a−c). The use of cyan light to stimulate ChR2 does not cross-
Simultaneous voltage and Ca imaging in the same cells can
also be achieved alongside genetically encoded indicators, like
GCaMP6. We again stained neurons with poRhoVR 14
(1 μM). This time, a subset of hippocampal neurons expressed
GCaMP6s. Again, poRhoVR localizes to membranes (Figure
5d), while GCaMP6s fluorescence appears cytosolic (Figure
excite poRhoVR 14, as indicated by the lack of stimulus artifact
4
poRhoVR 14 as a powerful complement for all-optical
electrophysiology utilizing NIR absorbing indicators.
Two Color Imaging with poRhoVR 14 and Ca2+
Indicators. In addition to deployment alongside light-
activated actuators, poRhoVR can be used with optical
indicators. Fluorescent sensors for Ca2+ are among the most
widely used optical sensors. Despite some three decades since
the initial reports of fluorescent indicators for this critical
5e). Simultaneous voltage and Ca imaging of spontaneous
2+
activity in hippocampal neurons reveals fast-spiking bursts
resolved in voltage (Figure 5f−h, magenta trace), followed by
slower, sustained increases in GCaMP6-associated fluorescence
(Figure 5f−h, green trace). Notably, voltage imaging with
poRhoVR 14 exhibits sufficiently high temporal resolution to
distinguish individual action potentials in spike volleys (Figure
6
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2+
intracellular messanger,
most Ca indicators utilize
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+
excitation and emission profiles firmly centered in the blue/
5g, 8 spikes; Figure 5h, 9 spikes), while Ca imaging does not.
Together, these experiments establish the utility of poRhoVR
dyes for monitoring fast spiking in neurons alongside
green region of the visible spectrum (for example, Oregon
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Green BAPTA, OGB,7 and the GCaMP family of genetically
1
2+
2+
encoded indicators). Although promising new Ca in-
commonly used synthetic and genetically encoded Ca
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76−79
dicators, both synthetic
and genetically encoded,
indicators and emphasizes the care needed when interpreting
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+
possess red-shifted excitation and emission spectra, circularly
permuted (cp) GFP-based indicators, like the GCaMP
Ca imaging data.
Voltage and Ca2+ Imaging and Electrode Recording
in a Mouse Model of Retina Degeneration. The NIR
(>700 nm) excitation and emission spectra of poRhoVR dyes,
along with their good voltage sensitivity, compatibility with
commonly used optogenetic sensors and actuators, and ready
uptake into cell membranes (Figure S12), make poRhoVR 14 a
promising candidate for mapping voltage dynamics in intact
neural tissue like retinas. The retina is a highly organized and
accessible outpost of the central nervous system. Light
responses initiated in rods and cones are synaptically
transmitted to bipolar cells, which activate retinal ganglion
cells (RGCs). RGCs generate the action potentials that carry
visual information to the brain. In normally functioning retinas,
the intrinsic light sensitivity of photoreceptors in rods and
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1
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family, dominate the landscape of functional imaging.
Therefore, fluorescent voltage indicators with orthogonal
wavelengths are required.
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+
We performed two-color, simultaneous voltage and Ca
imaging in the same cells using poRhoVR 14 and the synthetic
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+
Ca indicator, OGB (Figure 5a−c). We treated hippocampal
neurons with both poRhoVR 14 (500 nM) and OGB (1 μM)
simultaneously and imaged using an image-splitting device to
project two emission wavelengths onto the same camera chip.
Under these conditions, we observe clear membrane-associated
fluorescence for poRhoVR 14 (Figure 5a) and cytosolic
cross-excitation exists under these conditions (Figure S10).
Using field-stimulation electrodes, we evoked a series of 10
action potentials, across a range of frequencies, and
simultaneously recorded voltage (Figure 5c, magenta traces)
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+
cones complicates optical imaging of both voltage and Ca
transients in RGCs, because visible light (or high intensity two
photon excitation) used to excite the indicators triggers
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+
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and Ca (Figure 5c, green traces) dynamics.
physiological responses. We applied poRhoVR 14 to
Both poRhoVR 14 and OGB clearly resolve single action
potentials when activity is evoked at rates of either 5 or 10 Hz.
poRhoVR 14 clearly resolves action potentials at firing rates of
investigate membrane potential dynamics in retinas from a
mouse model of retina degeneration.
In particular, retinas from rd1 mice are an attractive model
system in which functional imaging can be applied to explore
mechanisms occurring in inherited visual disorders, including
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+
2
0 and 30 Hz (Figure 5c). OGB, despite its fast Ca response
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kinetics (<5 ms to action potential peak) compared to
GCaMP6f (∼45 ms to peak) and other genetically encoded
indicators,
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the degenerative disorder retinitis pigmentosa (RP). Lacking
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2−84
fails to accurately report individual action
a functional β subunit of rod cGMP phosphodiesterase
potential-evoked Ca2 transients at firing frequencies higher
+
(βPDE), rd1 mice suffer rapid loss of rod cells, followed
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J. Am. Chem. Soc. 2021, 143, 2304−2314