Targeting GCaMP3 expression to genetically defined cell types in rodent retina
In search of a concise set of vectors that gives genetic access to each neuron class in the mouse retina, we injected a series of AAVs with different serotype and promoter combinations into the mouse eye. The gene expression pattern was assessed after 10–14 d [summarized (see Notes)]. As shown in , neurons in each of five major cell classes of the retina can be successfully labeled with GCaMP3 driven by an appropriate combination of serotype and promoter. AAV2/9 with the mouse synapsin-1 (syn1) promoter expressed GCaMP3 in both rod and cone photoreceptors (). Horizontal cells were also labeled with AAV2/9 and the syn1 promoter (). These two cell classes could be independently targeted for optical recording because of their distinct morphologies and adequate spatial separation. Attempts to transfect ON-type bipolar cells with a viral vector and the mGluR6 promoter (AAV2/1-mGluR6-SV40) were not successful (data not shown). This is possibly attributable to incompatibility of the bipolar cell surface receptors with the AAV2/1 capsid serotype. However, when the same mGluR6 construct was electroporated into P0 mouse pups, it gave broad labeling exclusively in ON-type bipolar cells () (see Notes). AAV2/1 with a synthetic mGluR1 promoter (see Materials and Methods) (see Notes) expressed strongly and almost exclusively in type AII amacrine cells (). Ganglion cells were labeled with both AAV2/1 and 2/9 combined with the syn1 promoter.
Summary of vectors and their target neurons in mouse retina
To test whether GCaMP3 is functional in each of the cell classes, we presented a brief blue light flash (125 ms duration; 458 nm LED; 100 µW/cm2), delivered 2 s after two-photon scan onset. Strong (>20% ΔF/F) light stimulus-evoked fluorescence changes were detected in cone photoreceptors, ON-type bipolar cells, AII amacrine cells and ganglion cells, but not in rod spherules or horizontal cells () (see Notes).
Characterization of GCaMP3 expression in ganglion cells
Intravitreal injection of AAV2/1-syn1-GCaMP3 into an adult mouse retina typically gave patchy labeling, with 10–25 clusters of 20–100 strongly labeled cells distributed across the retina (). Within a cluster, >70% of all somas were labeled (). In addition to distributed clusters, often a “hot spot” of strongly labeled neurons was visible around the injection site (see Notes). Larger injection volumes (up to 2.0 µl) labeled a similar fraction of cells, but over a larger area (). We characterized the performance of GCaMP3 in ganglion cells in terms of expression pattern, cytotoxicity, fluorescence response to light stimuli, and correlation to neural signaling. Furthermore, we compared properties of GCaMP3 with those of the calcium indicator OGB.
AAV2/1-synapsin-1-mediated expression patterns in the mouse retina
The ganglion cell layer contains ganglion cells as well as numerous somas of starburst amacrine cells. We found that GCaMP3 was selectively targeted to ganglion cells because all labeled cells were axon bearing. Amacrine cells, whose somas are smaller than those of most ganglion cells, were absent or grossly underrepresented in the labeled population, in agreement with known selectivity of the syn1
promoter (Mandell et al., 1992
). In the inner plexiform layer (IPL), GCaMP3 resolved individual dendritic processes that could typically be traced back to the soma of a neuron ().
AAV2/1-syn1-GCaMP3 labels a subset of neurons in the ganglion cell layer and permits imaging with tolerably low laser power
Bulk loading of OGB under the inner limiting membrane, however, labeled all ganglion cells and amacrine cells in the imaged volume indiscriminately (). It also labeled the entire IPL diffusely, with almost no discernable structure (). The fluorescence intensity of both GCaMP3- and OGB-labeled neurons was similar at the same laser power and wavelength (25 mW at the sample; λ = 910 nm) ().
GCaMP3 has no apparent effect on ganglion cell function
To test whether GCaMP3 expression interferes with cell function (e.g., through interference with endogenous calcium dynamics), we compared electrophysiological recordings from GCaMP3-labeled cells with unlabeled neurons of the same type (ON α ganglion cells). First, spike responses to a brief light flash were similar (). Second, spatiotemporal filters recorded with a white noise stimulus matched (). Finally, using a white noise stimulus and spike-triggered averaging methods (Chichilnisky, 2001
), we found that unlabeled and GCaMP3-expressing neurons showed no significant differences in the receptive field and time-to-peak of their temporal filter characteristics () (120 ± 8.8 ms, n
= 15; vs 120 ± 13.5 ms, n
= 13, respectively; t
= 0.34). These data suggest that buffering of intracellular Ca2+
by the sensor did not affect the spatiotemporal response properties of a ganglion cell, consistent with a previous report (Margolis et al., 2010
). Temporal filters recorded from OGB-labeled neurons, however, peaked slightly later than control neurons and this difference was significant (136 ± 10.4 ms, n
= 12; ~10% increase; t
= 0.002) ().
Adapting light reduces scan laser-evoked light responses
In the dark-adapted retina, photoreceptor activation by the infrared scan laser evoked significant spike responses from ON- and OFF-type ganglion cells (). Scanning retinas in the absence of fluorophore gave the same pattern and strength of activation (data not shown), demonstrating that the scan laser, and not fluorescence emission, is responsible for the scan artifact. Because the dark-adapted retina is extremely sensitive (Barlow et al., 1971
), adding light-adapting background illumination reduced photoreceptor activation by the scan laser. With a constant background stimulus set at the measured laser-equivalent intensity (2.5 µW/cm2
) (), the laser-evoked response never exceeded 10 spikes s−1
and in >80% of cells was 1 spike s−1
or less ().
Light-adapting background minimized scan laser-evoked light responses
GCaMP3 shows functional differences between ganglion cells in a labeled population
To measure light-evoked calcium dynamics in a neural population, we stimulated the whole-mount retina with full-field stimuli and measured the fluorescence response of a population of GCaMP3- or OGB-labeled ganglion cells.
Fluorescence responses during light stimulation showed a variety of response dynamics: slow and fast responses, and responses of opposite sign (), thus reflecting the known diversity of ganglion cell types that includes sluggish and brisk, ON and OFF (Masland, 2001
). Averaged across populations, the amplitude of a light flash-evoked response signaled by GCaMP3 and OGB was similar, 22 ± 1.8% (n
= 816) and 17 ± 0.6% ΔF
= 449), respectively (). Of all GCaMP3-labeled neurons, 27% showed a light response >15% ΔF
, compared with 28% of neurons for OGB. Amplitude distributions were nearly identical, except for a group of cells with very large response amplitude (>80% ΔF
) in the GCaMP3 population that was not present in the OGB population. Accordingly, of cells that showed >15% ΔF
, the average response amplitudes were 59 ± 5.7% (GCaMP3; n
= 221) and 35 ± 1.2% (OGB; n
Light-evoked response dynamics of GCaMP3- and OGB-labeled neuronal populations are similar
Temporal kinetics of the GCaMP3 fluorescence response in ganglion cells was slightly slower than those of OGB (half-rise: 200 ± 8 ms for GCaMP3, n = 139; 170 ± 30 ms for OGB, n = 11; not significant, p = 0.39, t test; half-decay: 710 ± 8 ms for GCaMP3, n = 854; 630 ± 10 ms for OGB, n = 464; significant, p < 0.0001, t test) ().
GCaMP3 was more photostable than OGB. Repetitive scanning of GCaMP3-labeled ganglion cells at adequate laser power (25 mW at specimen) gave a minor reduction in baseline fluorescence. In most cells, average fluorescence decreased <2% per trial (), and 40 continuous, 5 s frame scans, separated by 1.5 s nonscanning intervals, reduced average fluorescence intensity <7% (n = 42 cells) (). The same experiment with bulk-loaded OGB showed much more pronounced bleaching, >40% ().
Above detection threshold, the GCaMP3 signal is linear with spike response amplitude
To further characterize the fluorescence changes in response to neural signaling [action potential (AP) firing], we targeted labeled ganglion cells for simultaneous electrophysiological and optical recording (). The number of APs (spikes) evoked by a brief light flash varied from trial to trial, in accordance with known neural response properties of retinal ganglion cells (Dhingra et al., 2003
) (). Average fluorescence intensity of the soma covaried with the number of spikes fired during each 5 s trial. Average SNR (ratio of fluorescence response amplitude to SD of the baseline fluorescence) (see Materials and Methods) of the light-evoked response for cells that showed a >5% change in spike rate was 19.7 ± 21.2 (range, 0.98–114; n
GCaMP3 signal gain and linearity vary across the recorded ganglion cell population
In most cells, the relationship was distinctly linear (), evidenced by a value of R > 0.7 in 25 of 42 cells. In some cells, responses comprising less than ~10 spikes did not show a significant response in the fluorescence signal (). This apparent rectification of the sensor at low spike rates was not ubiquitous, as we encountered at least one case in which the average fluorescence response for trials with zero and two spikes differed significantly (p = 0.006, t test) ().
Next, we compared the gain of the sensor (change in fluorescence for a given change in spiking) for all recorded cells. We tested three related measures of the gain: (1) peak fluorescence response against the flash-dependent change in the total number of spikes fired (), (2) peak fluorescence response against the flash-dependent change in spike rate (), and (3) sensor gain against baseline fluorescence (). All three measures varied strongly between cells. The third measure produced a weakly linear relationship (), suggesting that, at high expression levels, free Ca2+ is in excess relative to the sensor binding sites.
Because intracellular calcium dynamics might vary between ganglion cell types, we compared the fluorescence gain of cells of the same apparent functional type. Recorded cells (n
= 42) were grouped based on the similarity, S
, of their flash-evoked spike responses, where
represent the respective spike rates of cell x
and cell y
, broken into n
time bins (50 ms time bins, 5 s trials). A conservative threshold applied to S
(>5) grouped only highly similar cells, gave four groups of two or more cells and left >50% of recorded cells unsorted (). Fluorescence response amplitude within a group could differ by more than threefold—despite near-identical spike responses (). The cause of this variation is not known and may reflect expression level changes resulting from stochastic differences in viral infection, sensor transcription and translation rates, or physiological heterogeneity between cells with similar spike response patterns (i.e., same apparent functional type).
Sensor gain varied across ganglion cells of the same functional type
Fluorescence responses in dendrites are larger and faster than in soma
Dendritic arbors of GCaMP3-expressing neurons could be resolved at high spatial resolution (<1 µm) and traced from the ganglion cell into the specific sublamina in the IPL where they stratified () (see Notes). To compare dendritic and somatic GCaMP3 signals, we simultaneously recorded light-evoked fluorescence responses from the soma and primary dendrites of a cell. The response amplitude in the primary dendrites was always larger than in the soma (1.89 ± 0.61-fold difference; n = 7). The slope of a linear fit to the data constrained to pass through the origin was 1.6 (). The fluorescence signal in the dendrites also peaked earlier and had faster temporal dynamics (see Notes), reflected by a nearly twofold shorter decay time constant in dendrites compared with their respective somas (1.79 ± 0.32-fold difference; n = 7). Here, the slope of a linear fit to the data constrained to pass through the origin was 0.52 (). Stimulated with a full-field flash, the speed and amplitude of the fluorescence response scaled such that the total response (integral of the fluorescence change over time) in soma and dendrites was the same (Fdendrite:Fsoma = 0.97 ± 0.42; n = 7) ().
Calcium responses in dendrites are twofold larger and faster than in somas
GCaMP3 response can identify known functional types
To efficiently study a specific neural circuit (or part thereof), one would ideally label just a single type of neuron within a class with a genetically encoded calcium indicator for optical measurement. For want of genetic markers, most neuron types cannot currently be targeted exclusively and specifically. An alternate approach to achieve specificity uses the fluorescence response of a broadly expressed neural activity indicator to detect a specific functional type in a larger labeled population. Extracellular spike recordings confirmed that ganglion cells with respectively ON- and OFF-type fluorescence responses (as in ) were indeed ON- and OFF-type cells based on their spike response (). Since GCaMP3 labeled all ganglion cells, we tested whether we could differentiate DS ganglion cells based on their fluorescence response evoked by motion stimuli.
Stimulus-evoked GCaMP3 responses identify distinct functional types
DS ganglion cells represent a subset of ganglion cells that respond selectively to contrast edges moving in a preferred direction (Barlow and Levick, 1965
; Oyster, 1968
) (for review, see Demb, 2007
). To detect DS ganglion cells among their non-DS neighbors, we presented low spatial frequency square wave gratings drifting in four orthogonal directions, and compared the fluorescence response of ~30 GCaMP3-expressing cells in the stimulated region (). Although cells differed in the modulation amplitude (F1
) of their fluorescence response, most cells showed no significant difference in the modulation amplitude evoked by the different directions of motion. However, among the recorded population, two cells did show a significant (>1.5 SD) asymmetry in their response to the different motion directions (, white arrowheads), suggesting that these are DS ganglion cells.
Next, we tested the feasibility of optically measuring spatiotemporal tuning curves, which vary between ganglion cell types and are an established means to discriminate between retinal ganglion cells of different types in cat (X vs Y) (Derrington and Lennie, 1984
) and primate (P vs M) (Frishman et al., 1987
). We presented drifting sine wave gratings of different spatial and temporal frequencies, recorded the fluorescence response of a cell, and compared it with the simultaneously recorded spike response ().
The peaks of spatiotemporal tuning curves recorded optically and electrophysiologically broadly matched (). Tuning curves were similar except at low spike rates, where response modulation that was apparent in the spike response could not be resolved from the fluorescence signal. Furthermore, because GCaMP3 has a fluorescence decay time constant of ~700 ms (see above) (Tian et al., 2009
), frequencies above ~2 Hz were increasingly attenuated. The absence of modulation in the fluorescence response at high temporal frequencies may be attributable to signal integration by the sensor (see Notes). Signal integration is apparent in the recordings () (see Notes) and can be used to discriminate between a cell that is not activated by the stimulus and a cell whose response modulation exceeds the temporal bandwidth of the sensor (see Notes).
Compartmentalized calcium dynamics in a nonspiking neuron
As shown in , AAV2/1 with a synthetic mGluR1 promoter drove expression strongly in type AII amacrine cells. Because these were the only labeled neurons with processes in the IPL, the dendritic arbor of each cell could be traced unambiguously from the soma to the distal dendrites (), and calcium responses could be recorded at different distances from the soma.
Type AII amacrine cells feature calcium dynamics localized to subcellular compartments that can operate in two apparently distinct response modes
Light-evoked fluorescence responses recorded from labeled AII amacrine cells differed distinctly between different subcellular compartments (n
= 12). In response to a brief light flash, a small decrease was observed in the soma, a large increase in the lobular appendages, and a small decrease in the distal dendrites (). Our data agree with an earlier measurement of calcium dynamics in lobular appendages and distal dendrites in a slice preparation (Habermann et al., 2003
), with the reported distribution of L-type voltage-gated calcium channels of the AII (Habermann et al., 2003
), and with the presence of glycinergic synapses in the lobes of type AII amacrine cells (Xin and Bloomfield, 1999
; Wässle et al., 2009
). Calcium signals measured in the lobular appendages likely reflect the presynaptic calcium influx that drives synaptic glycine release onto OFF bipolar and OFF-type ganglion cells (Liang and Freed, 2010
For the first time, we were able to observe two distinct calcium responses in the lobular appendages, which differed in time course and amplitude. In one apparent response mode, the fluorescence response amplitude was high, the response time-to-peak was short (<1.0 s), and the signal returned to baseline with a time constant <2 s (, dashed lines). In the other, the calcium response recorded in the same dendrite was slower (time to peak, >1.5 s), and for a light flash paired with scan onset showed a dip followed by a slow rise (half-rise time, >1 s) (, solid lines). In all recordings, GCaMP3-expressing AII cells in a local population shared the same response dynamic. This dynamic switched over time, possibly after changes in the light-adapted state of the retina, which could change from dark-adapted (rod-mediated) signaling to light-adapted (cone-mediated) signaling during experiments. The exact relationship between light adaptation and response mode has not yet been resolved. The function of the two apparent “response modes” and the neuronal mechanism that underlies them are unknown, but can now be studied.