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.

Rapid warping of two-photon illumination wavefronts

16 02 2011

A short paper in Optics Express looks interesting.  In A high speed wavefront determination method based on spatial frequency modulations for focusing light through random scattering media, Meng Cui presents a method for rapidly determining the optimal wavefront to ‘cancel out’ the scattering when 785nm light passes through turbid media.  In his example, a glass diffuser was used, but the clear goal for this work is to replace the glass with a brain.

To understand why this is so important for in vivo two-photon imaging, let’s review how 2-p imaging works. Light from a laser is focused to a point and swept across the field in a raster. The resulting fluorescence is of a different wavelength and can thus be filtered out from the excitation light. For each voxel, all the fluorescence that re-enters the objective is collected, regardless of its source.  The total amount of fluorescence collected for that timepoint in the sweep is assigned as the brightness of that voxel. Since the user knows where the laser was being aimed, scattering of fluorescence emission may reduce the brightness but will not blur the image.  However, scattering of the excitation light can dramatically reduce the excitation at the target voxel while increasing the off-target excitation of its neighbors. This causes a rapid increase in background fluorescence and blur at increasing brain depth.

The vasculature was labeled by injecting flourescein dextran into the circulatory stream. The light source was a regenerative amplifier. ‘‘0 mm’’ corresponds to the top of the brain. Left, XZ projection. Right, examples of XY projections. Note the increase in background fluo- rescence deeper than 600 mm in the brain due to out-of-focus 2PE. (Theer et al., 2003)

Previous reports work has shown that one can use adaptive optics to adjust the phase of the wavefront of the excitation light to correct for the scattering of the excitation.  However, determination of the optimal wavefront for a field of view took minutes, which could be problematic for imaging in an awake animal.  Any changes in the precise position of the brain might change the optimal wavefront.  Ideally, one would want a system that could optimize the wavefront every second, or even before every frame of acquisition (typically 4-8 Hz in a raster scan in vivo experiment)

Scattering in the brain warps two-photon excitation light, but adaptive optics can correct this.

I’ll let Meng Cui explain the technique in his own words

Elastic scattering is the dominant factor limiting the optical imaging depth in tissues. Take gray matter as an example, at 800 nm the scattering coefficient is 77 /cm and the absorption coefficient is 0.2 / cm. If there is a way to suppress scattering, the optical imaging depth could be greatly improved. Despite the apparent randomness, scattering is a deterministic process. A properly engineered wave can propagate inside scattering media and form a focus, a well understood phenomenon in the time reversal and optical phase conjugation (OPC) studies…

For applications on biological tissues, acquisition time on the order of one millisecond (ms) per degree of freedom is desired. Deformable mirrors can provide a high modulation speed. However the degrees of freedom are rather limited. A phase-only SLM can provide about one million degrees of freedom at a much lower modulation speed. In this work, I present a novel method, capable of providing as many degrees of freedom as a SLM with a data acquisition time of one ms per degree of freedom. The method was employed to focus light through a random scattering medium with a 400 ms total data acquisition time, ~three orders of magnitude faster than the previous report [25].

The essence of a COAT system is to phase modulate different input spatial modes while detecting the output signal from the target. To greatly improve the operation speed, the experiment requires a device that can provide fast phase modulation and can access a large number of spatial modes very quickly. To meet these two requirements, a pair of scanning Galvanometer mirrors was used to quickly visit different modes in the spatial frequency domain or k space, and a frequency shifted reference beam was provided for a heterodyne detection. The wavefront profile was first determined in k space and then transformed to the spatial domain. The spatial phase profile was displayed on a SLM to focus light onto the target. In such a design, the number of degrees of freedom is limited by the number of pixels on the SLM and the experiment speed is determined by the scanning mirror speed…

Compared to existing techniques, the reported method can provide both a high operation speed and a large number of degrees of freedom. In the current design, the operation speed is limited by the scanning mirror speed and the maximum number of degrees of freedom is limited by the SLM pixel number. In this demonstration, 400 spatial modes in k space were visited and the determined phase profile was displayed on the SLM. Depending on the scattering property of the media, more (up to 1920 x 1080) or less number of degrees of freedom can be used to optimize the focus quality and the operation speed.

Using a stepwise position scanning, the method achieves an operation speed of one ms (400 μs transition time + 600 μs recording time) per spatial mode, ~three orders of magnitude faster than the previous report. Using a continuous position scanning and a faster position scanner such as resonant scanning mirrors, polygon mirror scanners, or acousto-optic deflectors, the operation speed can be potentially increased by at least one order of magnitude. It is anticipated that the reported technique will find a broad range of applications in biomedical deep tissue imaging.

UPDATE : DIADEM Final Results

15 09 2010

The DIADEM automated neuronal reconstruction contest has finished.  Accurate, fast, and high-resolution automated neuron reconstruction is of vital importance to cracking the mystery of how neural circuits perform. Even with perfect knowledge of the firing patterns of every cell in a circuit, our understanding of how these patterns are produced and how the information is processed would be quite limited.  True understanding requires knowledge of the precise wiring diagram.  This prize is a good first step towards bringing awareness of this tricky problem to the world’s best computer scientists.

$75,000 in prize money was to go to the group that was able to produce high-quality reconstructions of neuronal structures at least 20x faster than by-hand reconstructions.  In the finals, the fastest speed achieved was 10X the by-hand method. Some groups were hindered by slight variances in the source data formatting, which normally isn’t a big deal unless you only have 20 minutes to produce as much reconstruction as possible…

Since no group was able to beat the hard floor, but substantial progress was made, the money was distributed amongst these finalists.

Badrinath Roysam Team, $25,000
“for the better overall generality of their program in producing robust reconstructions by integration of human and machines interactions.”

Armen Stepanyants Team, $25,000
“for the better overall biological results in the spirit of pure automation.”

Eugene Myers Team, $15,000
“for the excellent quality and strength of their algorithm.”

German Gonzalez Team, $10,000
“for their deeper potential, more original approach, and ultimate scalability of their proposed solution.”

Deniz Erdogmus Team
“for elevating themselves above the current state of automated reconstructions…with a deep understanding of the technical and scientific problems.”

Congrats to the placing teams.

CNiFERS of Acetylcholine and Attention

10 03 2010

“If you find yourself needing to reread this paragraph, perhaps it’s not that well written. Or it may be that you are low on acetylcholine.” Acetylcholine (ACh) is a major modulator of brain activity in vivo and its release strongly influences attention. If we could visualize when and where ACh is released, we could more fully understand the large trial to trial variance found in many in vivo recordings of spike activity, and perhaps correlate that to attentional and behavioral states mediated by ACh transmission.

Back in grad school, when I was desperately trying to figure out what biological question to answer with my GluSnFR glutamate sensor, I ended up in a meeting with Kleinfeld, his grad student Lee Schroder and Palmer Taylor. We plotted a strategy to make a FRET sensor for acetylcholine.  Palmer had recently solved crystal structures of an acetylcholine binding protein bound to agonists and antagonists.  Snails secrete this binding protein into their ACh synapses to modulate their potency.  The structures showed a conformational change upon agonist binding.  The hope was that by fusing CFP and YFP to the most translocated bits of the protein, they would be able to see an ACh dependent FRET change.  I was skeptical that it would work, as the translocation was much less than with calmodulin-M13 or periplasmic binding proteins used in Cameleon and GluSnFR, but thought was at least worth a shot.  FRET efficiency is highly dependent on dipole orientation, not just dipole distance, and you never know how a small conformational change might rearrange the FP dipoles…

Of course, the simple idea didn’t work.  Instead of giving up on the first dozen attempts, they kept plugging away at alternative strategies for measuring ACh release, and eventually succeeded.  In this Nature Neuroscience report, An in vivo biosensor for neurotransmitter release and in situ receptor activity, Nguyen et al demonstrate a mammalian cell based system for optically measuring ACh levels in an intact brain.  They coexpressed M1 muscarinic receptors with the genetically-encoded calcium indicator TN-XXL in HEK293 cells.  ACh binding to the M1 receptor induced IP3-mediated calcium influx.  This calcium rise was then picked up by the TN-XXL and reported as a change in CFP/YFP fluorescence.  The crazy part is that they took this cell culture assay and implanted the cells into the brains of living rats!

The CNiFER in vivo experimental paradigm

In culture, the response was highly sensitive and monotonic (for phasic response section, EC50 of 11 nM, a Hill coefficient of 1.9 and a maximum of ΔR/R = 1.1). In vivo, using two-photon imaging through a cortical window, they were able to see clear ACh responses in frontal cortex from electrical stimulation of the nucleus basalis magnocellularis, typically 200-μs current pulses of 200 μA @ 100Hz for 20-500ms.

This was essentially a in vivo proof of principal experiment, showing that one could image ACh release in spatially and temporally precise regions of the brain.  However, the imaging was done under urethane anesthesia, which is a much different brain state than an awake, behaving animal.  Are CNiFERs sensitive, powerful and stable enough to determine behavioral states via imaging in an awake animal?  Would expressing GCaMP3 (an indicator with greater fluorescence dynamic range) improve the performance of the CNiFER system? We used a very similar assay with ACh applied to HEK cells during the initial screens for better GCaMPs. Or, is the performance more limited by the properties of the M1 receptor and the adapting nature of IP3-mediated calcium dynamics?  CNiFERS provide an interesting platform for looking at ACh and potentially other G-protein mediated signaling, but it remains to be seen if labs that aren’t as technically proficient with two-photon rig will find it more useful than cyclic voltammetry for measuring acetylcholine levels.

Nature Neuroscience, 13 (1), 127-132 DOI: 10.1038/
Nguyen, Q., Schroeder, L., Mank, M., Muller, A., Taylor, P., Griesbeck, O., & Kleinfeld, D. (2009). An in vivo biosensor for neurotransmitter release and in situ receptor activity

Adaptive Optics for In Vivo Microscopy

13 01 2010

Imaging fluorescence in an intact, living brain is difficult due to absorption and scattering of excitation and emission light.  Two photon microscopy uses excitation light in the narrow optical window (700-950nm) where water and hemoglobin do not significantly absorb, which allows structure determination and functional imaging down to depths of ~600nm from the surface of the brain.  However, scattering of the excitation light still occurs at these wavelengths, which distorts the excitation volume and causes a rapidly increasing fluorescent background at greater depths.

The vasculature was labeled by injecting flourescein dextran into the circulatory stream. The light source was a regenerative amplifier. ‘‘0 mm’’ corresponds to the top of the brain. Left, XZ projection. Right, examples of XY projections. Note the increase in background fluo- rescence deeper than 600 mm in the brain due to out-of-focus 2PE. (Theer et al., 2003)

In order to reduce this background and sharpen the two-photon excitation volume, Ji et al in Adaptive optics via pupil segmentation for high-resolution imaging in biological tissues adapt tricks from astronomy (see also Rueckel et al 2006). Depending on where each ray of excitation light enters the brain, the angles of scattering are different.  By sequentially illuminating with spatially restricted subsections of full illumination beam, they see how the brain warps the light from each part of the beam.  Then they use a flexible mirror and to change the angle and phase of the excitation beam so that each part of the beam comes together in the same place and phase. Since two-photon fluorescence scales as the square of the intensity of the excitation light, this dramatically improves both the resolution and the signal to noise of the fluorescence image.

Scattering in the brain warps two-photon excitation light, but adaptive optics can correct this.

Pre-warping the excitation light to cancel the scattering improves fluorescence localization and intensity.

How well will this work in the brain of a living animal?  It’s not clear. Large differences in scattering paths across a field of view may dramatically slow the determination of optimal excitation beam warping and make it difficult to scan across a field of view quickly. Motion of the brain may change the pattern of scattering faster than the system can adapt.  Still, the use of adaptive optics is one of the few promising techniques for increasing penetration depth and signal in optical brain imaging (not counting sucking off the bits of the brain that are “in the way” of the excitation beam.) There are other optical tricks in astronomy that have not yet been mined for neuroscience applications.  Hopefully these will one day allow the functional imaging of all layers of the mammalian cortex, not just layer 2/3.

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.

Annual Reviews worth reading

22 07 2009

Annual Reviews of Neuroscience published their 2009 issue recently.  These articles are usually a great way to catch up with a field, particularly when they are recently published.  Here are a few that might be of interest to the Brain Windows reader.

Daniel E. Feldman

Sensory experience and learning alter sensory representations in cerebral cortex. The synaptic mechanisms underlying sensory cortical plasticity have long been sought. Recent work indicates that long-term cortical plasticity is a complex, multicomponent process involving multiple synaptic and cellular mechanisms. Sensory use, disuse, and training drive long-term potentiation and depression (LTP and LTD), homeostatic synaptic plasticity and plasticity of intrinsic excitability, and structural changes including formation, removal, and morphological remodeling of cortical synapses and dendritic spines. Both excitatory and inhibitory circuits are strongly regulated by experience. This review summarizes these findings and proposes that these mechanisms map onto specific functional components of plasticity, which occur in common across the primary somatosensory, visual, and auditory cortices.

Heidi Johansen-Berg and Matthew F.S. Rushworth

Diffusion imaging can be used to estimate the routes taken by fiber pathways connecting different regions of the living brain. This approach has already supplied novel insights into in vivo human brain anatomy. For example, by detecting where connection patterns change, one can define anatomical borders between cortical regions or subcortical nuclei in the living human brain for the first time. Because diffusion tractography is a relatively new technique, however, it is important to assess its validity critically. We discuss the degree to which diffusion tractography meets the requirements of a technique to assess structural connectivity and how its results compare to those from the gold-standard tract tracing methods in nonhuman animals. We conclude that although tractography offers novel opportunities it also raises significant challenges to be addressed by further validation studies to define precisely the limitations and scope of this exciting new technique.

Nicholas G. Hatsopoulos and John P. Donoghue

The ultimate goal of neural interface research is to create links between the nervous system and the outside world either by stimulating or by recording from neural tissue to treat or assist people with sensory, motor, or other disabilities of neural function. Although electrical stimulation systems have already reached widespread clinical application, neural interfaces that record neural signals to decipher movement intentions are only now beginning to develop into clinically viable systems to help paralyzed people. We begin by reviewing state-of-the-art research and early-stage clinical recording systems and focus on systems that record single-unit action potentials. We then address the potential for neural interface research to enhance basic scientific understanding of brain function by offering unique insights in neural coding and representation, plasticity, brain-behavior relations, and the neurobiology of disease. Finally, we discuss technical and scientific challenges faced by these systems before they are widely adopted by severely motor-disabled patients.

Brian A. Wilt, Laurie D. Burns, Eric Tatt Wei Ho, Kunal K. Ghosh, Eran A. Mukamel, and Mark J. Schnitzer

Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists.