Probing Deep Brain Circuitry: New Advances in in Vivo Calcium

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Review

Probing deep brain circuitry: New advances in in vivo calcium measurement strategies Kasey S Girven, and Dennis Ryan Sparta ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00307 • Publication Date (Web): 16 Dec 2016 Downloaded from http://pubs.acs.org on December 18, 2016

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ACS Chemical Neuroscience

Probing deep brain circuitry: New advances in in vivo calcium measurement strategies Kasey S. Girven and Dennis R. Sparta

1

Department of Anatomy and Neurobiology 2 Program in Neuroscience University of Maryland School of Medicine Baltimore MD, 21201

#Address correspondence to: Dennis R. Sparta, Ph.D. Department of Anatomy and Neurobiology University of Maryland School of Medicine Tel: + 1 (410) 706 4778 Email: [email protected]

ACS Paragon Plus Environment


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Abstract

The study of neuronal ensembles in awake and behaving animals is a critical question in contemporary neuroscience research. Through the examination of calcium fluctuations, which are correlated with neuronal activity, we are able to better understand complex neural circuits. Recently, the development of technologies including two-photon microscopy, miniature microscopes, and fiber photometry has allowed us to examine calcium activity in behaving subjects over time. Visualizing changes in intracellular calcium in vivo has been accomplished utilizing GCaMP, a genetically encoded calcium indicator. GCaMP allows researchers to tag cell-type specific neurons with engineered fluorescent proteins that alter their levels of fluorescence in response to changes in intracellular calcium concentration.

Even with the evolution of GCaMP, in vivo calcium

imaging had yet to overcome the limitation of light scattering, which occurs when imaging from neural tissue in deep brain regions. Currently, researchers have created in vivo methods to bypass this problem; this review will delve into three of these state of the art techniques: (1) two-photon calcium imaging, (2) single photon calcium imaging, and (3) fiber photometry.

Here we discuss the

advantages and disadvantages of the three techniques. Continued advances in these imaging techniques will provide researchers with unparalleled access to the inner workings of the brain.

Keywords: calcium imaging, genetically encoded calcium indicators, two photon calcium imaging, fiber photometry, miniature microscope, behavior


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Introduction Ramon y Cajal once said, “If a photographic plate under the center of a lens focused on the heavens is exposed for hours, it comes to reveal stars so far away that even the most powerful telescopes fail to reveal them to the naked eye. In a similar way, time and concentration allow the intellect to perceive a ray of light in the darkness of the most complex problem1.” Both time and concentration have allowed researchers to shed light on the complex questions that underlie neuronal function. Particularly, discoveries made surrounding intracellular calcium signaling, a mechanism critical in neuronal processing, have illuminated some of the darkest and most complex regions of the brain. The importance of calcium and its role in neuronal signaling was first demonstrated by Katz and Miledi when they established that increases in extracellular calcium concentrations was correlated with neurotransmitter release2. Since neurotransmitter release is an intracellular process, these results implied that calcium influx is critical for release to occur2. Furthermore, studies proved that calcium influx at the presynaptic terminal of a neuron is required for exocytosis of all neurotransmitters through synaptic vesicles3, indicating calcium as a critical ion in neurotransmitter release. Within dendrites, calcium plays a necessary role in the induction of plasticity at the synapse4. Calcium also regulates cell proliferation and cell death5. For these activities to occur, calcium is either brought into the cell through voltage gated calcium channels that are activated depending on the membrane potential of the cell, or released from stores within the cell body such as the endoplasmic reticulum, or the sarcoplasmic reticulum in muscle cells6. Intracellular calcium stores are also involved in the release of neuropeptides at locations other than the axon terminals, such as the dendrites (Ludwig, 2002). Calcium measurement techniques allow researchers to examine the temporal differences in the intracellular calcium concentration that occur in a group of cells that can be monitored over a prolonged period of time such as days, or even weeks7. Therefore a population of cell’s calcium activity can be easily correlated with a behavioral task8 such as spatial learning9, memory



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recall10, fear conditioning11, and/or goal-directed behavior12,13. Visualizing calcium fluctuations in cells has been accomplished throughout the years utilizing in vitro and ex vivo techniques because of the creation of calcium indicator dyes, particularly the invention of GCaMPs, genetically encoded calcium indicators14,15. The evolution of GCaMPs has become the major driving force for imaging cellular activity in vivo16. However, imaging from neural tissue results in light scattering, which is not conducive for observing the activity of a neuronal population particularly in deeper brain regions17. Currently, researchers have devised in vivo methods to bypass this problem, this review will delve into three of these state of the art techniques (Fig. 1): (1) two-photon calcium imaging, (2) single photon calcium imaging utilizing a miniature microscope, and (3) fiber photometry, and how they differ from one another, the limitations that come along with each of the three techniques, and current research utilizing the three techniques.

Figure 1. Three in vivo techniques for measuring calcium in a behaving animal A) Two-photon calcium imaging of a headfixed animal where the objective is placed directly above the imaging plane. While with two-photon, both cell bodies and dendrites can be visualized, the animal is head-fixed and therefore cannot freely move B) Calcium imaging with a miniature microscope that attaches to a surgically implanted baseplate, which secures the microscope above the implanted microendoscope allowing free-movement of the


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animal. With this technique, subjects are not restrained, but due to poorer resolution, only cell bodies can be visualized. C) Measuring calcium using fiber photometry, here subjects have a pair of optical fibers (one for delivering excitation, and one for recording emissions) attached to the implanted fiber probe, this option is the most lightweight, but records calcium from populations of cells and not individual neurons.

Calcium Indicators The evolution of calcium imaging has been limited by two factors: (1) the quality of the calcium indicator, and (2) the advancement of deep brain imaging technology18. The most considerable advancement for performing calcium recordings in living mammalian tissue came from the development of genetically encoded calcium indicators (GECIs)14. GECIs allow researchers to target distinct cell-types utilizing transgenic indicator animals16. This technique achieves a level of specificity unknown to previously adopted cell-loading techniques used to deliver the Fura-219,20, a calcium chelator combined with a fluorescent chromophore. There are two main types of GECIs: (1) indicators that use Förster resonance energy transfer (FRET) and (2) Single-fluorophore GECIs such as GCamPs18. FRET-based calcium indicators have the advantage of simple donor/acceptor ratioing21. This is crucial for motion artifact control21, which is imperative for accurately measuring changes in distance. The principal behind FRET is similar to that of how a TV broadcasts: (1) The FRET donor must be close enough to the acceptor (less than 50 Å), (2) the donor emission must match the acceptor absorbance spectrum, and (3) the donor emission spectrum must overlap with the acceptor absorbance spectrum22. An example of how FRET is used for indicating the presence of calcium is demonstrated with YC 3.60, a member of the cameleon family of GECIs, which contains two fluorescent proteins: enhanced cyan fluorescent protein (ECFP) (donor), and the venus protein (acceptor) (Fig. 2A)23,24,25. Calmodulin, calcium binding protein, and M13 (a calmodulin binding peptide) are responsible for tethering the two fluorescent



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proteins together24. When calcium is present, there is a reduction in the spatial distance between ECFP and the Venus protein causing the emission of photons by the Venus protein due to FRET activation at an approximate wavelength of 530nm18. When calcium is no longer present, the distance between the proteins increases once again and fluorescent excitation is dominated by the blue ECFP at a wavelength of 480nm18. The ratio between the Venus and ECFP fluorescence is what indicates the changes in the calcium signal. GCaMPs are the most widely used single-fluorophores26. Using GCaMPs, researchers are able to tag cell type specific neurons with engineered fluorescent proteins that alter their fluorescent levels in response to changes in intracellular calcium concentration giving an insight to neuronal processing16. The GCaMP sensor consists of a circularly permuted green fluorescent protein (cpGFP), calmodulin, and CaM-binding peptide M1315. When calcium is present, it will bind to CaM causing the M13 and CaM domains to interact. These are in close proximity to the chromophore from the cpGFP causing the it to reorganize leading to an increase in the fluorescence at an emitted wavelength of 515 nm (Fig. 2B)16,26. This change in fluorescence is the result of water-mediated interactions between calmodulin and the chromophore27. However, different cell types have different firing rates meaning inferring in vivo spike activity accurately from the fluorescent changes of GECIs requires knowledge about the frequency of firing from the neuron being imaged, and likely electrophysiological characterization. The most recent version of GCaMPs; GCaMP6, was developed using structure-based mutagenesis and neuron-based screening26, critical because of the rapid calcium dynamics with low peaks of intracellular calcium accumulation within the neurons28. GCaMP6 indicators are more sensitive to calcium concentrations and can detect individual action potentials due to their fast dynamics with high accuracy26. Research has demonstrated that the three variants of GCaMP6: 6s, 6m, 6f, meaning slow, medium and fast kinetics respectively, can reliably detect a single action potential from the soma of neurons29, thus, making GCaMP6 the most widely used GECI for in vivo imaging currently. However, it is important to note, that as the speed of the


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GCaMP6 variant increases, the accuracy for action potential detection decreases29. Also, even the fastest variant, GCaMP6f lacks the ability to accurately detect spike frequencies associated with burst firing. The evolution of calcium indicators has become the major driving force for imaging cellular activity in vivo leaving light scattering as the main limitation17. However, optical tools now exist that circumvent this limitation.

Figure 2. FRET and Single-Fluorophore GECI Schematics A) FRET based calcium indicator schematic. When calcium is absent the ECFP and Venus proteins are apart and not interacting and fluorescent excitation is dominated by the blue ECFP at a wavelength of 480 nm. In the presence of calcium, the distance between ECFP and the Venus protein decreases causing photon emission by the Venus protein and FRET activation at an approximate wavelength of 530 nm. B) Singlefluorophore calcium indicator schematic. Calcium binds to CaM and causes the M13 and CaM domains to interact. They are in close proximity to the chromophore from the cpGFP causing reorganization and an increase in fluorescence emitting a wavelength of 515 nm.



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Two-Photon In Vivo Calcium Imaging In the past, the majority of in vivo calcium imaging has been accomplished using two-photon microscopy30. Two-photon microscopy is a fluorescence imaging technique where a coherent laser beam is focused through the objective of the microscope down to a size defined by a point spread function31 to selectively excite fluorescent molecules similar to that of confocal microscopy32. The principal difference between two-photon microscopy and confocal microscopy is the physical process of light absorption and fluorescence emission32. In two-photon microscopy, two photons are absorbed practically instantaneously. The single photon absorption has a shorter wavelength than the excitation light, which has a wavelength between 700-1000nm nearing the infared spectrum30,32. This brings forth the first advantage of two-photon in vivo imaging: longer-wavelength light will have less light scattering in tissue30,32. Another critical feature of two-photon microscopy is that the fluorescent signal depends on the square of the excitation light intensity indicating a nonlinear relationship30,32. Thus, the fluorescence produced where the laser beam is focused is far greater than any fluorescence detected where the laser beam is more diffuse; making a confocal pinhole stationed in front of the detector unnecessary. In fact, the detector should pick up as much fluorescence as possible because the photons can be correctly assigned to their original point of origin32. This feature provides two-photon microscopy with superior resolution, which allows the visualization of not only individual neurons, but also calcium fluctuations at the axon terminals. Laser scanning technology in 2-photon microscopy is also used to detect the fast calcium dynamics when imaging neurons in vivo32. The speed at which two-photon microscopy can detect calcium dynamics is also dependent on the binding kinetics of the fluorescent calcium indicators causing challenges for the measurements to have high temporal accuracy32. It is also important for laser scanning to occur not only in the two dimensional horizontal plane as done using line-scan technologies, but also to expand to the three dimensional plane so as to better monitor the activity occurring in a population of cells32. Researchers


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have tackled this problem utilizing mirrors to create 3D scan technologies as well as acousto-optical deflectors that use sound-wave-generated diffraction of a laser beam at a precise angle, which then can be altered in a matter of microseconds causing jumps from focused point to point33,34,35. Any decisions made in regards to the laser scanning technology must also consider the strength of photobleaching that can occur with different sampling rates32. Photobleaching is important to consider because LED intensities needed to differentiate signal to noise are often high enough to induce a degree of bleaching that overtime can negatively impact the image quality36. Differences between experiments that utilize two-photon microscopy for in vivo calcium imaging often deal with the objective’s field of view (FOV)34. Most brain regions that are examined during a specific behavior are much larger than the FOV of a high-resolution microscope, and large FOV microscopes tend to have difficulty in resolving individual neurons34. However, the Svoboda lab developed a technique they refer to as “mesoscale microscopy” that allows the two-photon microscope to have a FOV of 5mm allowing the visualization of multiple brain areas, while maintaining subcellular resolution34. This technique is incredibly useful, because not only is the FOV larger, but also the resolution of the imaged tissue remains superior enough to visualize axon terminals. Subcellular resolution is obtained by combining lateral and axial scanning maximizing the total number of neurons that can be detected34. This mesoscale microscope also does not use galvo mirrors, which is used by other large FOV microscopes (Tsai, 2015; Stieman, 2014), and is occasionally too slow in some types of functional imaging34. Imaging depth utilizing two-photon microscopy is somewhat of a balancing act. Microendoscopes can be implanted to visualize deeper brain structures, however, because of the size of the objective, substantial portions of the cortex, or other structures above the region of interest, are often obliterated entirely to make room for the implant37, which could significantly impact behavior. Therefore, when imaging with two-photon microscopy, doing so from superficial



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layers of the brain is quite advantageous due to minimal damage and superior resolution. The expense of two-photon calcium imaging, precludes it from being used as a common imaging technique. Ignoring the cost of the microscope, an experimenter needs a light source that is ultrafast and high powered as well as a basic two-photon microscope model. It is also imperative to consider a “tunable laser” meaning the wavelength of operation can be altered over a range of wavelengths38. Once one is complete with purchasing all the necessary equipment, the lab could be looking at a half a million-dollar investment. However, in a developing field such as this, costs are likely to evolve and become more affordable along with the advancing technology. Another disadvantage associated with in vivo two-photon microscopy is that the animal must be head-fixed due to the size of the objective. Experimenters compensate for this by allowing the animal to be somewhat freely moving using cylindrical, or even spherical treadmills37,39. Research done using Drosophila melanogaster at the Janelia research campus takes this compensation one step further by using a virtual reality arena that corresponds to the movement of the head-fixed Drosophila on a freely moving ball in order to demonstrate the relationship between landmark-based orientation and the population response of neurons40. Findings utilizing in vivo two-photon microscopy have helped answer critical questions in neuroscience research such as the role of phasic dopamine signaling in the dorsal striatum. Here research demonstrated that phasic dopamine release in the dorsal striatum targeted specific axons capable of inducing locomotor movement in mice37. Researchers also demonstrated that these dopaminergic axons were separate from those that respond to an unexpected reward37. This demonstrated that slow-variations in dopaminergic firing are not the only neuronal control in place for shaping an animal’s movement37,41–44. Two-photon microscopy was integral in imaging the activity of the dopaminergic projections in the dorsal striatum.


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Two-photon microscopy was also used to image and reliably track calcium dynamics from adult-born granule cells (abGC) in behaving mice over multiple training days45. Researchers recorded activity of abGCs in vivo by selecting theses cells using NestinCreERT2 mice46 crossed with a conditional tdTomato reporter line. Window implantation occurred over the dentate gyrus, which was imaged in head fixed mice that were able to explore a virtual environment in a linear direction45,47,48. Their methodology allowed them to record from both abGCs and mature granule cells (mGCs) simultaneously and indicated that abGCs are more active than mGCs, and less spatially tuned45.

Single-Photon In Vivo Calcium Imaging Using Miniature Microscopes Up to this point, researchers were hindered by the constraints of headfixed behavior that is necessary for two-photon imaging. However both with the invention of microendoscopic lenses, and miniature microscopes, researchers are able to apply behaviors that require a freely moving animal to calcium imaging techniques. The microendoscopic lenses used are a gradientrefractive-index (GRIN) lens17, which can be readily interfaced with miniature microscopes that one can attach and detach to monitor population neuronal activity over extended periods of time49. They are also small enough to sit on top of an animal’s head without drastically hindering their movement50. This alone makes the advent of miniature microscopes a useful tool for research exploring development, neural plasticity, and degenerative disease. The weight of the miniature microscope, which is approximately 1.9g, is possible because of the technological developments in optoelectronics50. They use miniature light-emitting diodes (LEDs) combined with highly sensitive, complementary metal-oxide semiconductor (CMOS) image sensors (Fig. 3)50,51. Image sensors are responsible for transducing the light waves into bursts of current conveying the image51. The process for how the miniature scope works is as follows: (1) the LEDs are activated (2) a drum lens collects the LED’s emissions (3) those collected emissions then pass through the excitation filter and ricochet off a dichroic mirror (a mirror with different reflection properties at



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two different wavelengths) (4) the emissions then pass into the imaging pathway where a cylindrical, GRIN objective lens focuses the illumination on the tissue (5) from here fluorescence from the tissue will return back through the objective, an emission filter, and then an achromatic doublet lens (6) the achromatic doublet lens then focuses the image onto the CMOS sensor50. The limiting factor for resolution is not set by the optical limit of the lens, but instead the camera’s pixel pitch, therefore advancements in CMOS sensors will bring about better resolution imaging50.

Figure 3. Cartoon diagram of a miniature microscope attached to a microendoscope surgically implanted in the brain. Here the miniature microscope has an excitation source, dichroic mirror, and an objective that attaches to a GRIN lens to focus the illumination on the region of interest. There is a fluorescent sensor component on the miniature


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microscope that is necessary for detection of the emitted photons from deep brain GCaMP-expressing cells.

Although use of a miniature microscope to image calcium in freely moving animals has tremendous utility, there are several limitations to the methodology to consider. When compared with two-photon in vivo calcium imaging, single photon imaging has a higher background fluorescence, and is slightly more prone to light scattering59. This is because traditional fluorescence microscopy uses a single photon to excite fluorescent markers using mostly visible excitation wavelengths (390-700 nm)52. After excitation from the LED, the electron will return to its stable state and release a photon of light with slightly less energy than the excitation photon52. This returns to the point that longer-wavelength light will have less light scattering in tissue thereby making two-photon microscopy superior when taking resolution into account. There are also limits from the GRIN lens on imaging depth due to optical distortions at the edge of the lens53. Another disadvantage is that, although the miniature microscope is usable in many behavioral tasks10,17,54, it cannot be submerged in water. Therefore, use of the scope during the morris water maze55, or a forced swim task56 is not possible. In addition, due to the diameter of the GRIN lens implant, ranging from .5mm - ≥1mm, tissue damage occurring is unavoidable, which could affect circuitry critical to the study depending on the brain region of interest50. These miniature microscopes are also limited in their ability to detect farred shifted indicators50,17. The advantages of these red-fluorescent indicators include lowered phototoxicity, decreased light scattering allowing for deeper brain illumination57, and are also thought to be better suited for combing in vivo imaging with optogenetics17,58,59. Another limitation when using miniature microscopes is limited frame rate during imaging. The camera is unable to keep up on the real time membrane dynamics of a neuron60 hindering the real time readout of neuronal activity. However despite its limitations, single photon imaging using miniature microscopes has been integral for discovering new insights in the cellular activity



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of deep brain regions17. In a recent study, researchers tracked thousands of pyramidal cells in the CA1 region of the hippocampus over weeks10. These targeted pyramidal neurons are known as place cells because of their involvement in spatial memory. Researchers had the question of how place cell activity and their representation of space changes over an extended period of time10. To examine this question, researchers targeted these place cells in the CA1 and imaged them during a spatial task. Because of their use of miniature microscopes, researchers were able to examine GCaMP3-infected place cells in freely moving mice visiting a familiar track over a period of 45 days10. Miniature microscopes were also used to image GABAergic neurons in the lateral hypothalamus (LH) of freely moving mice to examine their involvement in either appetitive, or consummatory behaviors12. To examine this question, researchers utilized GCaMP6 to infect LH GABA neurons in combination with microendoscopic imaging strategies and recorded calcium activity from hundreds of cells in food deprived mice during a free feeding behavior task12. Neurons were categorized as either responsive to consumption, or nose poke, and results indicated that individual neurons that responded to reward consumption were independent from those that strongly responded to appetitive nose pokes12. Therefore, this data agrees with their hypothesis that LH GABAergic neurons are functionally segregated into two subpopulations that evoke either consummatory, or appetitive behaviors12.

Data Analysis in Calcium Imaging Imaging calcium either through two photon microscopy, or single-photon microscopy with a miniature microscope, generates a substantial amount of data, and although the capabilities of calcium imaging have advanced greatly, there is a disconnect between the evolution of the technique and the analysis of the data65. Common computational techniques for analyzing the cellular calcium levels are mainly manual- to semi-automated techniques33,66–68, which is not conducive for handling the large scale data sets associated with the technique65. Region of interest (ROI) analysis is commonly used for extracting signals from


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calcium imaging data, but lacks a set of general principles and instead relies on practicality and one’s own experience and knowledge for decomposing a data set33,68,69. This is rather limiting when imaging what can be hundreds of individual cells over days, or weeks at a time in multiple animals. Not only does ROI analysis provide issues due to the large quantity of cells, it also is inadequate in distinguishing signals from overlapping populations of cells within dense structures of the brain. It is difficult to create an automated procedure for the analysis needed because cell types are not easily characterized in vivo65. The calcium activity waveforms are mainly dependent on the buffering of intracellular calcium as well as the fluorescent marker’s binding kinetics32. These alone do not provide good criteria to determine the cell type being recorded from because different cells will have varying levels of calcium binding proteins, such as calbindins, calretinin, or calmodulin, which all affect the rate at which calcium is buffered70. Also, the presence of fluorescence through pixels on a screen can represent very complex events occurring, and it is not trivial to decompose the fluorescent signals65. Researchers, however, are striving to create more automated approaches at analyzing calcium-imaging results. The Schnitzer laboratory has worked to develop an algorithm that will estimate the imaged cell’s location as well as the cell’s activities using independent component analysis (ICA)65. ICA is a computational method used to separate a signal with multiple variables into additive subcomponents that are independent of each other71,72. ICA follows principal component analysis (PCA), a method that locates and deletes dimensions in the recordings that are mainly encoding noise65. Although PCA is incredibly useful for dimensional reduction, alone it is unable to isolate calcium signals from individual cells65. Therefore, combining PCA, followed by ICA provides an automated procedure that allows the breakdown of data into separate independent signals65. Another potential issue with data analysis is separating overlapping fluorescence due to insufficient spatial resolution of the 3D image causing two different cells to overlap each other73. The Paninski laboratory addressed this issue as well as the issues of denoising and the deconvolution of calcium



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imaging data with an approach that can be applied to large quantities of data, with little need of human intervention. It is computationally efficient, and can be used by non-mathematicians73. This approach utilizes nonnegative matrix factorization, an effective algorithm for separating image data into constitutive parts74,75,76. Researchers have applied this matrix decomposition method to calcium imaging by requiring a background constraint: “the bleaching line of the background fluorescence intensity74.” This limit is meant to separate the background component from the image data. As new advances in machine learning and AI occur, researchers will be able to increase their yield in data analysis through the creation of more automated methods, with increased accuracy.

Fiber Photometry While two-photon calcium imaging has superior resolution, and singlephoton calcium imaging from a miniature microscope has the benefit of mobility, the two methods both possess limitations. Two-photon requires the animal to be head fixed and can really only image calcium activity at the axon terminals in superficial layers of the brain. Miniature microscopes, while able to image cell bodies, requires the implantation of a GRIN lens that is .5 - 1mm in diameter inevitably resulting in tissue damage50. To avoid these issues, fiber photometry was developed that utilizes time-correlated single-photon counting (TCSPC)based fiber optics77. This technique is useful for measuring presynaptic calcium activity in populations of axon terminals and causes less damage to neighboring structures than that of other techniques78. Here subjects are injected with a GECI and a hybrid fiber probe is implanted directly above the brain region of interest. During behavior, subjects are tethered to a pair of fiber optics (both optical fibers are only 125µm in diameter); one for delivering excitation pulses to excite the fluorophore, and another that is responsible for collecting the emitted photons77,78. These fibers connect to the end of the implanted fiber probe and are lightweight and flexible77. This system also uses a polychromator to spread the fluorescent emission and a 16-channel photomultiplier array to detect photons77,78. This allows researchers to separate channels dominated by


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GCaMP from those dominated by autofluorescence, which is useful for controlling changes in fluorescence that are due to movement, or other artifacts that may occur within a given subject78. Researchers also developed the option for a single multimodal fiber system to make the probe even more lightweight, however, using the multimodal fiber for both delivering excitation, and emission collection causes it to be more prone to bending77. Photons collected by this system can be plotted in a 3D space over time and are represented as spectra77. These spectra convey important information such as fluorescent intensity of the individual photons, or the fluorescent timeline77. However, fiber photometry is imaging from bulk fluorescent signals in a population of neurons and does not have single cell resolution, unlike two-photon microscopy, and imaging with a miniature microscope, which examine single-cell calcium dynamics17,32,77. Fibered fluorescent microscopy (FFM) is another technique that has been used by many groups to obtain actual images of fluorescence, instead of photon collection as seen in TCSPC-based fiber optics79,80. This technique uses a flexible, fiber-optic probe that captures real-time images of the fluorescent expressing cell populations. It can also be used to image the same nerve fiber over a span of days, or weeks and is a non-invasive approach due to the small diameter size of the probe, currently as small as 0.30mm80,79. This technique has a better resolution than other fiber photometry techniques because the fiber probe contains many optical micro-fibers that form a bundle, improving the resolution to just under that of a GRIN lens79. Therefore, these probes are able to detect fluorescence from individual cell bodies in deep regions of the brain79. There are many advantages for utilizing fiber photometry. First, fiber photometry can be used to measure FRET by utilizing the technique: fluorescence lifetime imaging microscopy (FLIM)81. FLIM is an imaging technique that uses differences in the exponential decay rate of fluorescence from a sample to create an image82. Second, TCSPC-based fiber optics measures single photons and therefore are ultrasensitive for detecting light. This allows for a decrease in the intensity of the emitted excitation pulses and therefore decreases the occurrence of photobleaching; thus extending the



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recording time of the subject77. Third the dual fiber optic design provides stable illumination since the laser output is through a single fiber. This makes it more stable compared to multimodal fibers, since they are more susceptible to bending77. This technique is also significantly more affordable than the previously mentioned techniques, and is straightforward to manufacture in the lab77. Lastly, due to the size and weight of the probes, researchers could potentially record from multiple brain regions simultaneously83. A limitation of fiber photometry is that the technique is unable to monitor the activity of individual cells; instead, fluorescent detection is coming from a population of neurons in a close proximity to the tip of the probe77. The area of detection for the probe is determined by the overlap between the two acceptance cones formed at the single mode and multimode fibers78. Therefore if activity is being recorded from a functionally diverse set of neurons, activity might be lost if at too great of a distance, but still within the brain region of interest77. Fiber photometry is also limited by the researcher’s ability to tease-apart background autofluorescence from calcium signals because cellular structures cannot be resolved. Subjects also must be tethered during the single photon counting meaning extreme care must be taken for studies taking place over a prolonged period of time77. However, even with these limitations, fiber photometry has proven to be a valuable technique in examining the activity of populations of cells. The Deisseroth laboratory developed an alternative to the multiple photosensor approach previously described, This technique, known as frameprojected independent-fiber photometry (FIP), is unique because it projects the cell population activity onto a CMOS sensor eliminating the use of multiple photosensors and therefore making recording from multiple brain regions possible83. However, although this technique is useful, it does not eliminate the need for a multiple photosensor system, as this technique can detect photons from a broader range of the spectrum for a more accurate detection of the fluorescent emission source77. Nonetheless, FIP is capable of measuring fluorescence from GCaMP6f activity in vivo83, and researchers developed a


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seven-fiber patchcord to deliver excitation and collect fluorescence from each of the single fibers in vivo with a CMOS camera to image the bundled end83. The implanted fiber optics were positioned in different and specific brain regions of adult subjects83. Researchers also found that an advantage of FIP is the ability to record activity from two different populations of neurons from the same fiber using different GECIs83. The application of FIP alongside circuit perturbing techniques such as optogenetics84–87, allows for tuning of laser stimulation to match the natural level of activity in a population of cells83. Additionally, fiber photometry was used to further examine how D1 and D2 medium spiny neurons (MSN) are involved in the encoding of drug associated cues88. Previous research has provided evidence that stimulation of D1 MSNs in the nucleus accumbens (NAc) enhances reward, while stimulating only D2 MSNs prevents reward seeking through aversion89–91. Researchers used fiber photometry in a conditioned place preference task to observe populations of D1 and D2 MSN activity during exposure to drug associated contexts88. Their results further demonstrated that D1 MSNs are the population of neurons responsible for encoding drug associations and increased activity of these MSNs drive reward seeking88. The Deisseroth laboratory also utilized fiber photometry to examine social interaction and the involvement of a projection from the ventral tegmental area (VTA) to the NAc92. Here, researchers used a single multimodal fiber to both deliver excitation, and detect fluorescence with a sensitivity that can identify signaling at the axonal level92. Subjects were implanted with a chronic optical fiber in the NAc and an optical fiber in the VTA for optogenentic stimulation. Researchers indirectly measured dopamine release from a population of neurons, creating a new method for measuring neurotransmitter release unlike the previously used techniques such as cyclic voltammetry93, an indirect measurement of dopamine release at the axon terminals based on the chemical signature of the transmitter. Results from the study indicated that the projection from the VTA to the NAc could modulate social interactions while being uninvolved in novel-object interactions92. The development of fiber photometry



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was a critical step in examining deep brain structures while also allowing the animal free range of motion due to the lightweight design. David Lovinger, Rui Costa, and Steven Vogel implanted TCSPC-based fiber optics in the dorsal striatum to specifically examine the roles of MSNs in either the direct pathway, or indirect pathway and their effects on movement78. Here researchers specifically targeted the two types of MSN populations using the transgenic mouse lines D1-Cre for the direct pathway MSNs89,94, and A2ACre for the indirect pathway MSNs95,96 using the Cre-dependent GECI: GCaMP378,97. Previous research has indicated the role for the direct pathway to initiate movement, while the indirect pathway inhibits movement98,99. However, results from this study indicated increases in neuronal activity in both populations (direct and indirect SPNs) when subjects were initiating movement and were inactive when the subject was still78. These findings strengthened our understanding of the basal ganglia and the distinction between the direct and indirect pathway of SPNs in the dorsal striatum.

Conclusion The technology and methodologies underlying the measurement of in vivo calcium have evolved rapidly in the past decade and are continuing to do so with new developments in optoelectronics occurring often. As technology of CMOS sensors advance so will the resolution of images produced from in vivo recordings. It should also be expected that as technology advances the size of miniature microscopes are likely to become smaller, and less hindering of a subject’s movement. The field of in vivo calcium imaging will also continue to improve with the continued development of new rapid GECIs. Although the three different in vivo calcium-detection techniques differ in methodology and execution, all three have unique benefits that can be applied to research questions pertaining to neuronal processing. Each is revolutionary in the calciumimaging field and hold great promise for future research.


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Kasey S. Girven Department of Anatomy and Neurobiology HSF1 Rm 256 Baltimore, MD 21201 Dennis R Sparta Department of Anatomy and Neurobiology HSF1 Rm 280K Baltimore, MD 21201 Author Contributions: K.S.G. wrote and edited the manuscript D.R.S wrote and edited the manuscript Funding sources: K.S.G. NIH/NINDS T32 NS063391 D.R.S. NIAA R00 AA021417

No conflict of interest



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Acknowledgments: We would like to thank Michael R. Bruchas PhD for his constructive criticism and Miguel Plylar-Moore for his assistance with illustration.


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Probing Deep Brain Circuitry: New Advances in in Vivo Calcium

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