| dc.description.abstract | Recently, genetically encoded optical indicators have emerged as noninvasive tools of
high spatial and temporal resolution utilized to monitor the activity of individual neurons and
specific neuronal populations. The increasing number of new optogenetic indicators, together
with the absence of comparisons under identical conditions, has generated difficulty in
choosing the most appropriate protein, depending on the experimental design. Therefore, the
purpose of our study was to compare three recently developed reporter proteins: the calcium
indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These
probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp
recordings and optical imaging. The three groups (each one expressing a protein)
exhibited similar values of membrane potential (in mV, GCaMP3: -56 ±8.0, R-GECO1: -57
±2.5; VSFP: -60 ±3.9, p = 0.86); however, the group of neurons expressing VSFP showed a
lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ±18.3;
GECO1-R: 128 ±15.3; VSFP: 94 ±14.0, p = 0.02). Each neuron was submitted to current
injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their
fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons
expressing VSFP showed detectable fluorescence signal in response to action potentials
(APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz)
was small (1.3 ± 0.21), however the rapid kinetics of the VSFP allowed discrimination of
APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz
and subthreshold signals were undetectable due to high noise. On the other hand, calcium
indicators showed the greatest change in fluorescence following the same protocol (five APs
at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an
average SNR value of 21 ±6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the
neurons had signal, with a mean SNR value of 52 ±19.7 (soma). For protocols at 10 Hz, 54%
of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were
detectable in all the analyzed frequencies and fluorescence signals were detected from
subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield
fluorescence signal and with high SNR, some experiments were performed only with this
probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+
influx), with high spatial and temporal resolution. Differences were also observed between
the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of
groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and
reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results
indicate that the presently available calcium indicators allow detailed studies on neuronal
communication, ranging from individual dendritic spines to the investigation of events of
synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs
represent a promising technology for monitoring neural activity and, although still to be
improved, they may become more appropriate than calcium indicators, since neurons work
on a time scale faster than events of calcium may foresee | |
