GCaMP6 plasmids at addgene

8 11 2012

GCaMP6 variants are on addgene. Three flavors, fast kinetics or big signals. Bigger responses than OGB-1, some are MUCH bigger.  The responses to drifting gratings in visual cortex are spectacular. Sorry no pics for now. Hopefully the reviewers will be nice so we can all read about it soon. Still work to be done getting true 1AP resolution when simultaneously imaging large populations of neurons, but for single neuron imaging in vivo, this has 1AP resolution.  If you have been waiting for the GCaMPs that will blow your expectations away, these are them.

From the SfN abstract :

Using structure-guided mutagenesis and high-throughput screening, we increased the fluorescence change in response to single action potentials (APs) by >10-fold compared to GCaMP3. We also accelerated the kinetics by ~2-fold. These new GECIs reliably report single APs in single trials in vivo with near 100% accuracy. In the mouse visual cortex, we detected ~5-fold more visually responsive neurons. The sensitivity, dynamic range and speed of the new GECIs exceed those of the synthetic indicator OGB-1. The improved sensitivity further facilitated reliable measurement of synaptic calcium signals in the dendrites of pyramidal cells and parvabumin (PV)-positive interneurons in vivo. Hot spots of orientation-selective domains can be resolved both in single pyramidal cell spines and small segments of PV cell dendrites. These improved GECIs will permit a more complete description of neuronal circuit function and enable long-term functional imaging of single synapses.

Three ways of looking at touch coding

20 09 2012

At SfN, a block of three posters by myself, Simon Peron and Daniel O’Connor will showcase three ways to approach the problem of touch coding.

My work on whisker force measurements, and single cell and silicon probe based cortical recordings during active objection localization :

Program#/Poster#: 677.18/KK18
Presentation Title: Encoding whisking-related variables in the mouse barrel cortex during object localization
Location: Hall F-J
Presentation time: Tuesday, Oct 16, 2012, 2:00 PM – 3:00 PM
Janelia Farm Res. Campus, ASHBURN, VA

Simon Peron’s work on recording a complete representation of touch using in-vivo imaging with new G-CaMP variants during a similar behavior :

Program#/Poster#: 677.12/KK12
Presentation Title: Towards imaging complete representations of whisker touch in the mouse barrel cortex
Location: Hall F-J
Presentation time: Tuesday, Oct 16, 2012, 4:00 PM – 5:00 PM
Authors: *S. P. PERON1, V. IYER2, Z. GUO2, T.-W. CHEN2, D. KIM2, D. HUBER3, K. SVOBODA2;

Daniel O’Connor’s work on constructing synthetic perception of touch and object localization via cortical cell-type specific optogenetic stimulation during behavior :

Program#/Poster#: 677.06/KK6
Presentation Title: Neural coding for object location revealed using synthetic touch
Location: Hall F-J
Presentation time: Tuesday, Oct 16, 2012, 2:00 PM – 3:00 PM
Authors: *D. H. O’CONNOR1, S. A. HIRES1, Z. GUO1, Q.-Q. SUN2, D. HUBER1, K. SVOBODA1;

This is a must-see session for people interested in touch coding, the whisker system, in-vivo cortical imaging, or synthetic perception via optogenetics.

I hope to see you there.

GCaMP5 is out

15 11 2011

GCaMP3 is now officially old skool.  GCaMP5 is available at addgene.

Plasmid 31788: pCMV-GCaMP5G, a.k.a. GCaMP3-T302L R303P D380Y

Dimmer baseline F, higher dF/F, picks up significantly more activity in vivo cortex.  Still not holy grail level, but getting closer.

Cameleon-Nanos : High Affinity GECIs

9 08 2010

Takeharu Nagai’s lab has published in Nature Methods, Spontaneous network activity visualized by ultrasensitive Ca2+ indicators, yellow Cameleon-Nano, demonstrating a new set of calcium indicators based on yellow cameleon. Back when he was still Take-san, Take’s ability to churn out and manually screen hundreds of cameleon variants was impressive and inspiring. With high-throughput GECI pipelines now ramping up at Janelia, the idea of laboriously screening 200 variations on a theme (be it cameleons or GluSnFRs), seems a bit archaic. However, this paper is a good example of the progress that can still be made by understanding the needed sensor parameters and fiddling with the primary amino acid structure in a relatively low-throughput way. Take-sensei’s results are another example of the pramatic rule in protein design, “when in doubt, tinker with the linker.”

The cameleon-nano family achieves greater apparent calcium affinity than YC2.60-4.60, reaching levels of up to 15nM.  They did this by increasing the flexibility of the linker by extending the standard Tsien/Miyawaki/Baird Gly-Gly-Ser linker with additional glycines.  In this case, the longer the linker between the CaM and M13 segments, the greater the apparent affinity. Interestingly, improvement by increasing linker flexibility is precisely the opposite the advice Atsushi and Take gave me for achieving high ratio changes with FRET reporters.  Back at RIKEN in 2002, they suggested I use short, stiff linkers to restrict the rotational freedom of the fluorescent pairs.  Then one could find orientations where relative rotation of dipole moment gave much greater FRET changes than would be expected from changes in FP distance alone. Take and Atsushi’s big YC2.60/3.60 paper strongly supported this idea!  However, as our understanding of the ideal parameters of calcium sensor’s for in vivo imaging has grown, development directions have adjusted.

Cameleon-Nanos achieve higher signal/noise for sparse action potentials at the expense of linearity.  Like Fluo-4, the signal saturates at relatively low AP frequencies.  I think the absolute affinities measured for this family (15, 30, 50 and 140nM) should be considered very rough estimates. They extrapolated these values from stopped-flow binding experiments, because

Although we would like to measure the koff of YC2.60 and its high affinity variants such as YC-Nano15, we could not do it because it was very difficult to precisely control free Ca2+ concentration at around few tens of nM as far as we used EGTA (Kd for Ca2+ = 151 nM in 0.1 M ionic strength, pH 7.2 at 25 oC). For this purpose, much stronger Ca2+ chelator with a smaller Kd value was required. However there is no such Ca2+ chelator available now.

I’m not sure why they didn’t just use the higher affinity, Mg++ insensitive, chelator BAPTA to make the Kd measurements the right way, with a linear regression of log-log fluorescence/concentration values.  Due to instrument dead time, and the high affinity, I didn’t like stopped-flow based Kd measurements in the early GCaMP papers, and I don’t like them now.  Also, the apparent calcium Kd will be highly dependent on solution ionic strength and [Mg++] which is unreported. Despite these quibbles, which are important only inasmuch as they give insight into the mechanism of improvement and the direction of future development, the cameleon-nano family looks promising for mammalian brain imaging.  I still wonder if, assuming the reported Kd values are relevant in vivo, YC2.60 would be the best of the bunch, since cortical neurons have a resting Kd of ~50nM, which implies that a single AP transient of say 200nM free [Ca++] increase would push the calcium levels right up into the sweet-spot of YC2.60’s sensitivity.

This is all the more interesting given the recent results in YC3.60 imaging from Maz Hasan’s group.  Previously, he had shown that transgenic YC animals were pretty bad for imaging.  However, AAV-mediated gene delivery of YC3.60 has significantly improved the responses of the YC family. I’m not sure if they are really up to GCaMP3 levels under identical in vivo conditions, but they might have better long-term protein stability (or that might depend on which viral serotype is used.) What about cameleon-nanos, what about YC2.60?

Journal Scan – Calcium Imaging in Auditory and Visual Cortex

4 03 2010

A few papers on in vivo calcium imaging have just come out and are worth a careful read.

The first two examine the fine organization of layer 2/3 of the mouse auditory cortex.  The canonical view of auditory cortex organization is that neurons are arranged in a tonotopic pattern, with a smooth gradient in auditory frequency tuning across the surface of the cortex.  Using two-photon imaging in anesthetized mice, the groups saw that, while there was an overall gradient, the tuning of neighboring neurons was highly variable.  These are similar results to what Sato et al and Kerr et al found in the whisker barrel cortex back in 2007.  Moral of the story : mapping brain organization by microstimulation or sparse sampling (as in the classic papers) can be very misleading.

UPDATE : David Kleinfeld kindly directed me to the 40 year old work by Moshe Abeles and others that showed a similar spread in frequency tuning using microelectrodes…

Now, back to the more recent papers…

Functional organization and population dynamics in the mouse primary auditory cortexRothschild GNelken IMizrahi A. Nat Neurosci. 2010 Mar;13(3):353-60. Epub 2010 Jan 31.

Cortical processing of auditory stimuli involves large populations of neurons with distinct individual response profiles. However, the functional organization and dynamics of local populations in the auditory cortex have remained largely unknown. Using in vivo two-photon calcium imaging, we examined the response profiles and network dynamics of layer 2/3 neurons in the primary auditory cortex (A1) of mice in response to pure tones. We found that local populations in A1 were highly heterogeneous in the large-scale tonotopic organization. Despite the spatial heterogeneity, the tendency of neurons to respond together (measured as noise correlation) was high on average. This functional organization and high levels of noise correlations are consistent with the existence of partially overlapping cortical subnetworks. Our findings may account for apparent discrepancies between ordered large-scale organization and local heterogeneity.

In vivo two-photon calcium imaging from dozens of neurons simultaneously in A1.

Dichotomy of functional organization in the mouse auditory cortexBandyopadhyay SShamma SAKanold PO. Nat Neurosci. 2010 Mar;13(3):361-8. Epub 2010 Jan 31.

The sensory areas of the cerebral cortex possess multiple topographic representations of sensory dimensions. The gradient of frequency selectivity (tonotopy) is the dominant organizational feature in the primary auditory cortex, whereas other feature-based organizations are less well established. We probed the topographic organization of the mouse auditory cortex at the single-cell level using in vivo two-photon Ca(2+) imaging. Tonotopy was present on a large scale but was fractured on a fine scale. Intensity tuning, which is important in level-invariant representation, was observed in individual cells, but was not topographically organized. The presence or near absence of putative subthreshold responses revealed a dichotomy in topographic organization. Inclusion of subthreshold responses revealed a topographic clustering of neurons with similar response properties, whereas such clustering was absent in supra-threshold responses. This dichotomy indicates that groups of nearby neurons with locally shared inputs can perform independent parallel computations in the auditory cortex.

Tonotopy exists in A1 and AAF on a large scale, but not on small spatial scales.

The third paper uses a GECI (YC3.6) to do chronic imaging in visual cortex. Their results are noteworthy in that they look at visual responses to both a passive viewing and an ACTIVE discrimination task in an awake, head-fixed mouse.  The patterns of neural activity between anesthetized, awake but passively receiving sensory input, and awake while paying attention and using the sensory input are likely to be hugely different. Recording from neurons that are actively involved in a discrimination task is essential to understanding how the cortex is actually processing information.  Although this paper is more focused on simply presenting the technique rather than in depth analysis of the activity, we will be seeing more of this style of neuroscience in high-profile journals very soon…

Chronic cellular imaging of mouse visual cortex during operant behavior and passive viewing  –  Andermann ML, Kerlin AM and Reid RC, Front. Cell. Neurosci. 4:3.

Nearby neurons in mammalian neocortex demonstrate a great diversity of cell types and connectivity patterns. The importance of this diversity for computation is not understood. While extracellular recording studies in visual cortex have provided a particularly rich description of behavioral modulation of neural activity, new methods are needed to dissect the contribution of specific circuit elements in guiding visual perception. Here, we describe a method for three-dimensional cellular imaging of neural activity in the awake mouse visual cortex during active discrimination and passive viewing of visual stimuli. Head-fixed mice demonstrated robust discrimination for many hundred trials per day after initial task acquisition. To record from multiple neurons during operant behavior with single-trial resolution and minimal artifacts, we built a sensitive microscope for two-photon calcium imaging, capable of rapid tracking of neurons in three dimensions. We demonstrate stable recordings of cellular calcium activity during discrimination behavior across hours, days, and weeks, using both synthetic and genetically-encoded calcium indicators. When combined with molecular and genetic technologies in mice (e.g., cell-type specific transgenic labeling), this approach allows the identification of neuronal classes in vivo. Physiological measurements from distinct classes of neighboring neurons will enrich our understanding of the coordinated roles of diverse elements of cortical microcircuits in guiding sensory perception and perceptual learning. Further, our method provides a high-throughput, chronic in vivo assay of behavioral influences on cellular activity that is applicable to a wide range of mouse models of neurologic disease.

Mapping visual responses in identified excitatory and inhibitory neurons in awake mice

Monte Carlo Calcium Spike Detection

9 02 2010

I somehow missed that Josh Vogelstein’s method on action potential detection was published last summer. In Spike Inference from Calcium Imaging Using Sequential Monte Carlo Methods, the authors use a Monte Carlo approach to determine spike times from calcium imaging with superior performance to other deconvolution methods.  It does a great job on simulated and in vitro data, I’d love to see performance on real in vivo recordings.  If you are serious about calcium imaging, you should definitely get in touch with Josh and see what magic he can do with all that math.  You should also ask him about the benefits of linen pants vs. denim, he’s got strong opinions on that subject as well…

Using only strongly saturating and very noisy in vitro fluorescence measurements to infer precise spike times in a ‘‘naturalistic’’ spike train recorded in vitro

Three Cheers for GCaMP : Optogenetic Brain Reading

9 11 2009

Three papers are out online in Nature Methods that show big improvements in calcium imaging with genetically encoded sensors.  They are are based on the fluorescence intensity indicator, GCaMP.   GCaMP, first developed by Junichi Nakai, consists of a GFP that has been circularly permuted so that the N and C termini are fused and new termini are made in the middle of the protein.  Fused to one terminus is calmodulin and the other is a peptide, M13, that calmodulin (CaM) binds to in the presence of calcium. The name is supposed to look like GFP with a CaM inserted into it, G-CaM-P.  Normally the GFP is dim, as there is a hole from the outside of its barrel into the chromophore.  Upon binding calcium, this hole is plugged and fluorescence increases.

Crystal structure of GCaMP2

The first paper, A genetically encoded reporter of synaptic activity in vivo, from Leon Lagnado’s group, targets GCaMP2 to the outer surface of synaptic vesicles. This localization allows the fluorescence signal to be confined to the presynaptic terminal, where calcium fluxes in response to action potentials are high.  This targeting improves the response magnitude of GCaMP2 and permits the optical recording of synaptic inputs into whatever region of the brain one looks at.  They demonstrate the technique in live zebrafish.

In the second paper, Optical interrogation of neural circuits in Caenorhabditis elegans, from Sharad Ramanathan’s group, GCaMP2 has been combined with Channelrhodopsin-2 to perform functional circuit mapping in the worm.   Since the worm’s structural wiring diagram has been essentially solved, functional data could say much about how “thick” the wires between each cell are.  Unfortunately, with GCaMP2, the responses are too slow and weak to distinguish direct from indirect connections.

Finally, we have published a paper, Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators, describing the improved GCaMP3.  This indicator has between 2-10x better signal to noise than GCaMP2, D3cpv and TN-XXL, depending on the system you are using.  It’s kinetics are faster and it is more photostable than FRET indicators, and the responses are huge.  When expressed in motor cortex of the mouse, neuronal activity is easily seen directly in the raw data.  Furthermore, the sensor can be expressed stably for months, making it a potential tool for observing how learning reshapes the patterns of activity in the cortex.

Screen shot 2009-11-09 at 7.19.27 PM

Imaging of mouse motor cortex (M1) expressing the genetically-encoded calcium indicator GCaMP3 through a cortical window. After 72 days of GCaMP3 expression, large fluorescence transients can be seen in many neurons that are highly correlated with mouse running.

GCaMP3 is not perfect. It cannot reliably detect single action potential in vivo in mammals, though I doubt that any existing GECI can. Work continues on future generations of GCaMP that may achieve 100% fidelity in optical reading of the bits in the brain. However, there is considerable evidence from a number of groups that have been beta-testing the sensor, including the Tank lab of “quake mouse” fame, that it is a significant leap forward and unlocks much of the fantastic and fantasized potential of genetically-encoded calcium indicators.

Screen shot 2009-11-09 at 7.20.12 PM

Comparison of fluorescence changes in response to trains of action potentials in acute cortical slices.

I will try to post a more complete writeup of GCaMP3 for Brain Windows soon, with an unbiased eye to its strengths and weaknesses.  We worked very hard to carefully characterize this sensor’s effects on cellular and circuit properties.  If you have any questions about GCaMP3, please post them to the comments.

For further info about strategies for GECI use and optimization, check out our previous paper, Reporting neural activity with genetically encoded calcium indicators in Brain Cell Biology.

The official press release from HHMI regarding GCaMP3 is available here.