This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC: RGPIN-2017-06131) to C.P. C. P. is a FRSQ Chercheur-Boursier. We also thank the Plateforme d&rsquo;Outils Mol&eacute;culaires (https://www.neurophotonics.ca/fr/pom) for the production of the viral vectors used in this study.

All experiments were done in accordance with the Institutional Animal Care and Use Committees of the University of California, San Diego, and the Canadian Guide for the Care and Use of Laboratory Animals and were approved by the Universit&#233; Laval Animal Protection Committee.&#160;
1. Alignment of the optical path between the CMOS (complementary metal oxide semiconductor) camera and the individual or branching patch cord
<ol>
	<li>Loosen all screws on the 5-axis translator (11, <strong cl...

<ol>
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J., Deisseroth, K., Lin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothalamic+control+of+male+aggression-seeking+behavior.">Hypothalamic control of male aggression-seeking behavior.</a> Nature Neuroscience. 19 (4), 596-604 (2016).</li><li>Ren, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomically+Defined+and+Functionally+Distinct+Dorsal+Raphe+Serotonin+Sub-systems.">Anatomically Defined and Functionally Distinct Dorsal Raphe Serotonin Sub-systems.</a> Cell. 175 (2), 472-487 (2018).</li><li>Barnett, L. M., Hughes, T. 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Fiber photometry is an accessible approach that allows researchers to record bulk-calcium dynamics from defined neuronal populations in freely-moving animals. This method can be combined with a wide range of behavioral tests, including &#8220;movement heavy&#8221; tasks such as forced swim tests2, fear-conditioning18, social interactions1,4, and others7,8<su...

The ability to correlate neural responses with specific aspects of an animal&rsquo;s behavior is critical to understand the role a particular group of neurons plays in directing or responding to an action or stimulus. Given the complexity of animal behavior, with the myriad of internal states and external stimuli that can affect even the simplest of actions, recording a signal with single-trial resolution equips researchers with the necessary tools to overcome these limitations.
Fiber photometry has become the technique of choice for many researchers in the field of systems neuroscience because of its relative simplicity compared to other in vivo recording techniques, its high signal-to-noise ratio, and the ability to record in a variety of behavioral paradigms1,2,3,4,5,6,7,8. Unlike traditional electrophysiological methods, photometry is the optical approach most commonly used in conjunction with genetically-encoded calcium indicators (GECIs, the GCaMP series)9. GECIs change their ability to fluoresce based on whether or not they are bound to calcium. Because the internal concentration of calcium in neurons is very tightly regulated and voltage-gated calcium channels open when a neuron fires an action potential, transient increases in internal calcium concentration, which result in transient increases in the ability of a GECI to fluorescence, can be a good proxy for neuronal firing9.
With fiber photometry, excitation light is directed down a thin, multimode optic fiber into the brain, and an emission signal is collected back up through the same fiber. Because these optic fibers are lightweight and bendable, an animal can move largely unhindered, making this technique compatible with a wide array of behavioral tests and conditions. Some conditions, such as rapid movements or bending of the fiber-optic patch cord beyond the radius at which it can maintain total internal reflection, can introduce signal artifacts. To disambiguate signal from noise, we can exploit a property of GCaMP known as the &ldquo;isosbestic point.&rdquo; Briefly, with GCaMP, as the wavelength of the excitation light is shifted to the left, its emission in the calcium-bound state decreases and the emission in the calcium-unbound state marginally increases. The point at which the relative intensity of these two emissions are equal is termed the isosbestic point. When GCaMP is excited at this point, its emission is unaffected by changes in internal calcium concentrations, and variance in the signal is most often due to attenuation of the signal from overbending of the fiber-optic patch cord or movement of the neural tissue relative to the implanted fiber.
Single unit electrophysiology is still the gold standard for freely-moving in vivo recordings due to its single-cell and single-spike level resolution. However, it can be difficult to pinpoint the molecular identity of the cells being recorded, and the post-hoc analysis can be quite laborious. While fiber photometry does not have single-cell resolution, it does allow researchers to ask questions impossible to address with traditional techniques. Combining viral strategies with transgenic animals, the expression of GECIs can be directed to genetically-defined neuronal types to record population- or projection-defined neural activity, which can be performed by monitoring calcium signal directly at axon terminals10,11. Moreover, by implanting multiple fiber-optic cannulas, it is possible to simultaneously monitor neural activity from several brain regions and pathways in the same animal12,13.
In this manuscript, we describe a technique for single and multi-fiber photometry, how to correct for calcium-independent artifacts, and detail how to perform mono- and dual-color recordings. We also provide examples of the types of questions it enables one to ask and their increasing levels of complexity (see Figure 1). The fiber photometry setup for multi-fiber recordings detailed in this protocol can be built using a list of materials found at https://sites.google.com/view/multifp/hardware (Figure 2).
It is essential that the system be equipped for both 410 nm and 470 nm excitation wavelengths for calcium-independent and calcium-dependent fluorescence emission from GCaMP6 or its variants. For custom-built setups or if there is no available software to run the system, the free, open source program Bonsai (http://www.open-ephys.org/bonsai/) can be used. Alternatively, fiber photometry can be run through MATLAB (e.g., https://github.com/deisseroth-lab/multifiber)12 or other programming language14. The software and hardware of the system should allow manipulation of both the 410 nm and 470 nm LEDs and the camera, extraction of images (Figure 2), and calculation of the mean fluorescent intensity in the regions of interest (ROIs) drawn around the fibers on the images. The output should be a table of mean intensity values recorded with the 470 nm and 410 nm LEDs from each fiber in the patch cord. When performing multi-fiber experiments, 400 &micro;m bundled fibers may limit the movement of mice. In such cases, we recommend using 200 &micro;m patch cords, which provide more flexibility. It may also be possible to use smaller dummy cables during training of mice.
It is crucial to be able to extract time points for events of interest during fiber photometry acquisition. If the system does not readily provide a built-in system to integrate TTLs for specific events, an alternative strategy is to assign a time stamp to individual time points recorded to align with specific times and events during the experiment. Time stamping can be done using the computer clock.

<table>
	<tbody>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 1&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S100</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 1/2&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S050</td>
			<td></td>
		</tr>
		<tr>
			<td>1/4&#34;-20 Stainless Steel Cap Screw, 3/8&#34; Long</td>
			<td>Thorlabs</td>
			<td>SH25S038</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#181;m, 0.50 NA, SMA-SMA Fiber Patch Cable</td>
			<td>Thorlabs</td>
			<td>M59L01</td>
			<td></td>
		</tr>
		<tr>
			<td>12.7 mm Optical Post</td>
			<td>Thorlabs</td>
			<td>TR30/M</td>
			<td></td>
		</tr>
		<tr>
			<td>12.7 mm Pedestal Post Holder</td>
			<td>Thorlabs</td>
			<td>PH20EM</td>
			<td></td>
		</tr>
		<tr>
			<td>15 V, 2.4 A Power Supply Unit with 3.5 mm Jack Connector for T-Cube</td>
			<td>Thorlabs</td>
			<td>KPS101</td>
			<td></td>
		</tr>
		<tr>
			<td>20x objective</td>
			<td>Thorlabs</td>
			<td>RMS20X</td>
			<td>#10 in Figure 2, #11 in Figure 5</td>
		</tr>
		<tr>
			<td>30 mm Cage Cube with Dichroic Filter Mount</td>
			<td>Thorlabs</td>
			<td>CM1-DCH/M</td>
			<td>#8-9 in Figure 2, #8-10 in Figure 5</td>
		</tr>
		<tr>
			<td>405 nm LED</td>
			<td>Doric Lenses</td>
			<td>CLED_405</td>
			<td>#2 in Figure 2</td>
		</tr>
		<tr>
			<td>410 nm bandpass filter</td>
			<td>Thorlabs</td>
			<td>FB410-10</td>
			<td>#5 in Figure 2; #7 in Figure 5</td>
		</tr>
		<tr>
			<td>465 nm. LED</td>
			<td>Doric Lenses</td>
			<td>CLED_465</td>
			<td>#1 in Figure 2</td>
		</tr>
		<tr>
			<td>470 nm bandpass filter</td>
			<td>Thorlabs</td>
			<td>FB470-10</td>
			<td>#4 in Figure 2; #6 in Figure 5</td>
		</tr>
		<tr>
			<td>560 nm bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-560/14-25</td>
			<td>#5 in Figure 5</td>
		</tr>
		<tr>
			<td>560 nm LED</td>
			<td>Doric Lenses</td>
			<td>CLED_560</td>
			<td>#1 in Figure 3</td>
		</tr>
		<tr>
			<td>5-axis kinematic Mount</td>
			<td>Thorlabs</td>
			<td>K5X1</td>
			<td>#11 in Figure 2, #12 in Figure 5</td>
		</tr>
		<tr>
			<td>Achromatic Doublet</td>
			<td>Thorlabs</td>
			<td>AC254-035-A-ML</td>
			<td>#7 in Figure 2</td>
		</tr>
		<tr>
			<td>Adaptor for 405 collimator</td>
			<td>Thorlabs</td>
			<td>AD11F</td>
			<td>#3 in Figure 2; #4 in Figure 5</td>
		</tr>
		<tr>
			<td>Adaptor for ajustable collimator</td>
			<td>Thorlabs</td>
			<td>AD127-F</td>
			<td>#3 in Figure 2; #4 in Figure 5</td>
		</tr>
		<tr>
			<td>Aluminum Breadboard</td>
			<td>Thorlabs</td>
			<td>MB1824</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamping Fork</td>
			<td>Thorlabs</td>
			<td>CF125</td>
			<td></td>
		</tr>
		<tr>
			<td>Cube connector</td>
			<td>Thorlabs</td>
			<td>CM1-CC</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual 493/574 dichroic</td>
			<td>Semrock</td>
			<td>FF493/574-Di01-25x36</td>
			<td>#10 in Figure 5</td>
		</tr>
		<tr>
			<td>Emission filter for GCaMP6</td>
			<td>Semrock</td>
			<td>FF01-535/22-25</td>
			<td>#6 in Figure 2</td>
		</tr>
		<tr>
			<td>Enclosure with Black Hardboard Panels</td>
			<td>Thorlabs</td>
			<td>XE25C9</td>
			<td></td>
		</tr>
		<tr>
			<td>Externally SM1-Threaded End Cap for Machining</td>
			<td>Thorlabs</td>
			<td>SM1CP2M</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast-change SM1 Lens Tube Filter Holder</td>
			<td>Thorlabs</td>
			<td>SM1QP</td>
			<td>#4-6 in Figure 2, #5-7 in Figure 5</td>
		</tr>
		<tr>
			<td>Fixed Collimator for 405 nm light</td>
			<td>Thorlabs</td>
			<td>F671SMA-405</td>
			<td>#3 in Figure 2; #4 in Figure 5</td>
		</tr>
		<tr>
			<td>Fixed collimator for 470 and 560 nm light</td>
			<td>Thorlabs</td>
			<td>F240SMA-532</td>
			<td>#3 in Figure 2; #4 in Figure 5</td>
		</tr>
		<tr>
			<td>Green emission filter</td>
			<td>Semrock</td>
			<td>FF01-520/35-25</td>
			<td>In light beam splitter</td>
		</tr>
		<tr>
			<td>High-Resolution USB 3.0 CMOS Camera</td>
			<td>Thorlabs</td>
			<td>DCC3260M</td>
			<td>#13 in Figure 2, #15 in Figure 5</td>
		</tr>
		<tr>
			<td>Light beam splitter</td>
			<td>Neurophotometrics</td>
			<td>SPLIT</td>
			<td>#14 in Figure 5</td>
		</tr>
		<tr>
			<td>Longpass Dichroic Mirror, 425 nm Cutoff</td>
			<td>Thorlabs</td>
			<td>DMLP425R</td>
			<td>#8 in Figure 2, #9 in Figure 5</td>
		</tr>
		<tr>
			<td>Longpass Dichroic Mirror, 495 nm Cutoff</td>
			<td>Semrock</td>
			<td>FF495-Di03</td>
			<td>#9 in Figure 2, #8 in Figure 5</td>
		</tr>
		<tr>
			<td>Metabond dental cement</td>
			<td>C&#38;B</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M8 - M8 cable</td>
			<td>Doric Lenses</td>
			<td>Cable_M8-M8</td>
			<td></td>
		</tr>
		<tr>
			<td>Optic fiber cannulas</td>
			<td>Doric Lenses</td>
			<td></td>
			<td>Need to specify that these will be used to photometry experiments requiring low autofluorescence</td>
		</tr>
		<tr>
			<td>Optic fiber Patchcords</td>
			<td>Doric Lenses</td>
			<td></td>
			<td>Need to specify that these will be used to photometry experiments requiring low autofluorescence</td>
		</tr>
		<tr>
			<td>Red emission filter</td>
			<td>Semrock</td>
			<td>FF01-600/37-25</td>
			<td>In light beam splitter</td>
		</tr>
		<tr>
			<td>T7 LabJack</td>
			<td>LabJack</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T-cube LED Driver</td>
			<td>Thorlabs</td>
			<td>LEDD1B</td>
			<td></td>
		</tr>
		<tr>
			<td>USB 3.0 I/O Cable, Hirose 25, for DCC3240</td>
			<td>Thorlabs</td>
			<td>CAB-DCU-T3</td>
			<td></td>
		</tr>
	</tbody>
</table>

multi-fiber photometry to record neural activity in freely-moving animals

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and smaller functional subgroups, it becomes paramount to record from projections and/or genetically-defined subpopulations of neurons. Fiber photometry is an accessible and powerful approach that can overcome these challenges. By combining optical and genetic methodologies, neural activity can be measured in deep brain structures by expressing genetically-encoded calcium indicators, which translate neural activity into an optical signal that can be easily measured. The current protocol details the components of a multi-fiber photometry system, how to access deep brain structures to deliver and collect light, a method to account for motion artifacts, and how to process and analyze fluorescent signals. The protocol details experimental considerations when performing single and dual color imaging, from either single or multiple implanted optic fibers.

Neural correlates of behavioral responses can vary depending on a variety of factors. In this example, we used in vivo fiber photometry to measure the activity of axon terminals from the lateral hypothalamic area (LHA) that terminate in the lateral habenula (LHb). Wild type mice were injected with an adeno-associated virus (AAV) encoding GCaMP6s (AAV-hSyn-GCaMP6s) in the LHA and an optic fiber was implanted with the tip immediately above the LHb (Figure 4A). GCaMP6s expressi...

Watch this Scientific Journal Video about Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals at JoVE.com

Multi-Fiber Photometry to Record Neural Activity in Freely-Moving Animals

This protocol details how to implement and perform multi-fiber photometry recordings, how to correct for calcium-independent artifacts, and important considerations for dual-color photometry imaging.

Recording the activity of a group of neurons in a freely-moving animal is a challenging undertaking. Moreover, as the brain is dissected into smaller and ...

Fiber photometry CSOM imaging combines genetically encoded custom indicator and optical fibers to monitor neural activity in freely-moving animal. Which is critical to understand how a specific group of neurons play in directing or responding to an action or a stimulus. This technique is an accessible way to record from specific brain regions that are defined by their connection or genetic profiles, with the built in control to separate signal to noise.Demonstrating the processor will be, Ekaterina Martianova, a graduate student in my lab. Begin by loosening all screws on the five axis translator. Screw in the patch cord to the adaptor that is affixed to the five axis translator.Then turn on the 470 nanometer excitation light at low power and position the tip of the fiber pointing to an auto fluorescent plastic slide. Next record from the complimentary metal-oxide semiconductor or CMOS camera in live mode. Increase the gain or adjust the lookup table until the image is visible at the focal point of the objective.Advance the five axis translator towards the objective ensuring that the 470 nanometer light is centered on the fiber at the SMA or FC end of the patch cord, until an image can be resolved on the camera. Finally, adjust the X and Y axes until the image is centered and well resolved. Visualize the light emitted from the variole end of the patch cord, as it should appear as an isotropic circle.Start by turning on all the excitation lights to better visualize the fibers. And adjust the camera gains such that no pixels are saturated, and a clear image of the fibers are present. Then take a preliminary image.Draw regions of interest or ROIS around the fibers. And keep them for the measurement of the mean intensity values during recordings. For multiple fiber recordings, test for independents by live recording from all fibers.Point one fiber towards a light source, and tap with a finger. Observe fluctuations in the channel. Then apply colored tape to the end of the fibers to label which ROI corresponds to which fiber.Finally, set up the recording area by hanging the patch cord above the arena using stands, clamps, and holders. Lastly, make sure that the animal will not be limited in movement by the length of the fiber and can freely move throughout the entire arena. Begin by inspecting the distal end of the fibers of the patch cord by eye, and with a mini fiber microscope.If the surface of the fibers is scratched, re-polish the fibers using fiber polishing film with fine grit. Then clean the distal ends of the patch cord with 70%ethanol and a cotton tip applicator. Clean the fiber optic cannulas using 70%ethanol and a cotton tip applicator.Connect the variole end of the patch cord to the implanted fiber using a ceramic split sleeve covered with a black shrink tube. During the connection, make sure that the sleeve is tight. Allow the animal to recover for a few minutes.Then start recording the optical signal, and run the experiment. While recording keep a careful eye on the live trace to ensure quality recordings. And watch for any jumps in signal, indicating the sleeve is not tight enough.For the dual color recordings, add a 516 nanometer LED to the photometry system to excite the red fluorescent calcium sensor, and appropriate dichroic mirrors and filters. Then add an image splitter between the objective and the CMOS camera to separate the green and red emission wave lengths. Finally, draw ROIS around all fibers in both colors, red and green.Make sure to clearly identify each ROI with the corresponding fiber and channel. Trigger simultaneous excitation with 470 nanometer, and 560 nanometer LEDs, and alternate them with a 410 nanometer LED. Results indicate, the measured fluorescence significantly increased concurrently with the administration of air puffs for mice with activation of the LHA-LHB pathway.However, in mice expressing green fluorescent protein or GFP, no change in the signal was detected during the administration of air puffs. This technique allows researchers to reasonably record from specific type of cells in different brain regions. While an animal is freely behaving.This furthers the ability to dissect the functions of these brain regions as they relate to behavior.

multi-fiber-photometry-to-record-neural-activity-freely-moving

Research

JoVE Journal

Neuroscience

34.9K visualizações.  Université Laval. A imagem CSOM de fotometria de fibra combina indicador personalizado geneticamente codificado e fibras ópticas para monitorar a atividade neural em animais em movimento livre. O que é fundamental para entender como um grupo específico de neurônios joga na direção ou resposta a uma ação ou um estímulo. Esta técnica é uma maneira acessível de registrar a partir de regiões cerebrais específicas que são definidas por sua conexão ou perfis genéticos, com o controle incorporado par...