Optimization of a GCaMP calcium indicator for neural activity imaging

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Journal of Neuroscience 32 (2012): 13819-13840, doi:10.1523/JNEUROSCI.2601-12.2012.Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.A.F. has been supported by a European Molecular Biology Organization long-term fellowship. Work in H.B.’s laboratory was funded by the National Institutes of Health (NIH) Nanomedicine Development Center “Optical Control of Biological Function,” and work in S.S.-H.W.’s laboratory was funded by NIH R01 NS045193

Temporally Diverse Excitation Generates Direction-Selective Responses in ON- and OFF-Type Retinal Starburst Amacrine Cells

The complexity of sensory receptive fields increases from one synaptic stage to the next. In many cases, increased complexity is achieved through spatiotemporal interactions between convergent excitatory and inhibitory inputs. Here, we present evidence that direction selectivity (DS), a complex emergent receptive field property of retinal starburst amacrine cells (SACs), is generated by spatiotemporal interactions between functionally diverse excitatory inputs. Electrophysiological whole-cell recordings from ON and OFF SACs show distinct temporal differences in excitation following proximal compared with distal stimulation of their receptive fields. Distal excitation is both faster and more transient, ruling out passive filtering by the dendrites and indicating a task-specific specialization. Model simulations demonstrate that this specific organization of excitation generates robust DS responses in SACs, consistent with elementary motion detector models. These results indicate that selective integration of spatiotemporally patterned excitation is a computational mechanism for motion detection in the mammalian retina

LRIT3 expression in cone photoreceptors restores post-synaptic bipolar cell signalplex assembly and partial function in Lrit3−/− mice

Summary: Complete congenital stationary night blindness (cCSNB) is a heterogeneous disorder characterized by poor dim-light vision, myopia, and nystagmus that is caused by mutations in genes critical for signal transmission between photoreceptors and depolarizing bipolar cells (DBCs). One such gene, LRIT3, is required for assembly of the post-synaptic signaling complex (signalplex) at the dendritic tips of DBCs, although the number of signalplex components impacted is greater in cone DBCs (all components) than in rod bipolar cells (only TRPM1 and Nyctalopin). Here we show that rAAV-mediated expression of LRIT3 in cones results in robust rescue of cone DBC signalplex components and partially restores downstream visual function, as measured by the light-adapted electroretinogram (ERG) b-wave and electrophysiological recordings of bipolar cells (BCs) and RGCs. These data show that LRIT3 successfully restores partial function to cone DBCs most likely in a trans-synaptic manner, potentially paving the way for therapeutic intervention in LRIT3-associated cCSNB

Dynamics of directional selectivity in area 18 and PMLS of the cat

Visual latencies and temporal dynamics of area 18 and PMLS direction-selective complex cells were defined with a reverse correlation method. The method allowed us to analyze the time course of responses to motion steps, without confounding temporal integration effects. Several measures of response latency and direction tuning dynamics were quantified: optimal latency (OL), latency of first and last significant responses (FSR, LSR), the increase and decrease of direction sensitivity in time, and the change of direction tuning in time. FSR, OL and LSR values for PMLS and area 18 largely overlapped. The small differences in mean latencies (3-4 ms for FSR and OL and 11.9 ms for the LSR) were not statistically significant. All cells in area 18 and the vast majority of cells in PMLS showed no systematic changes in preferred direction (monophasic neurons). In PMLS 5 out of 41 cells showed a reversal of preferred direction after ∼56 ms relative to their OL (biphasic neurons). Monophasic cells showed no systematic changes in direction tuning width during the interval from FSR to LSR. In both areas, development of direction sensitivity was significantly faster than return to the non direction sensitive state, but no significant difference was found between the two areas. We conclude that, for the monophasic type of direction-selective complex cells, the dynamics of primary motion processing are highly comparable for area 18 and PMLS. This suggests that motion information is predominantly processed in parallel, presumably based on input from the fast conducting thalamocortical Y-pathway.</p