This work was supported through funding from the NIH/NIBIB R00 Award (4R00EB008738), Okawa Foundation Research Grant Award, NIH Director's New Innovator Award (1DP2OD007265), the NSF CAREER Award (1056008), and the Alfred P. Sloan Foundation Research Fellowship. J.H.L. would like to thank Karl Deisseroth for providing the DNA plasmids used for the optogenetic experiments. The authors would also like to thank Andrew Weitz and Mankin Choy for editing the manuscript and all the Lee Lab members for their assistance with the ofMRI experiments.

Ethics Statement: Experimental procedures here have been approved by the Stanford University Institutional Animal Care and Use Committee (IACUC).
1. Preparing Patch Cables and Ferrule Implants
Note: Although patch cables and ferrule implants are available commercially, producing these in-house enables specialty designs and will cost less.
<ol>
	<li>To prepare a fiber patch cable for delivering light from the laser to the implant in the brain, first cleave the optical fiber to the desired length.
	<ol>
		<li>Using a fiber cleaver, terminate the optical fiber to produce a flat end at the termination point. If the fiber has been coated with a jacket, strip the jacket off with a fiber-stripping tool beforehand.</li>
		<li>Cleave the other end of the optical fiber to produce the desired length for the cable, ensuring that the cable is long enough to extend from the light source to the animal inside the bore of the scanner.</li>
	</ol>
	</li>
	<li>Mix a small amount of epoxy glue in a 1:1 ratio onto a piece of aluminum foil shortly before the next step, as the epoxy glue becomes too viscous for use 5 min&#160;after mixing.</li>
	<li>Using a small wooden stick, gently apply epoxy glue to the part of the fiber that will be placed inside the concave side of the ceramic ferrule and then apply a small drop onto the surface of the flat side of the ferrule. After inserting it into the ferrule, ensure that a small length of fiber (&#60;0.5 mm) protrudes from the flat side of the ceramic ferrule. Allow the epoxy glue to harden O/N&#160;for optimal results.</li>
	<li>On the side of the fiber optic patch cable that will connect to the laser light source, gently apply epoxy glue to the part of the fiber that will be placed inside the concave side of the ferrule of the FC/PC connector and then apply a small drop onto the surface of the flat side of the ferrule. After inserting it into the ferrule, ensure that a small length of fiber (&#60;0.5 mm) protrudes from the flat side of the ferrule. Allow the epoxy glue to harden O/N for optimal results.</li>
	<li>Polish the flat end of the ferrules for both sides of the cable using a polishing disk by using tweezers to apply gentle downward pressure on the ferrule while making figure-8 rotations on aluminum oxide lapping sheets (from 3 &#181;m to 1 &#181;m to 0.3 &#181;m grit).</li>
	<li>Examine the flat end of the ferrule with a microscope at 100X magnification. Ensure that the surface of the flat end, including the fiber optic surface itself, is free of any epoxy glue; continue polishing if epoxy glue remains on the surface. Ensure that the fiber optic surface has not broken or chipped. 
	CAUTION: Ensure personnel take appropriate laser safety training classes and wear laser safety goggles before handling laser equipment.</li>
	<li>Connect the fiber optic patch cable to the laser light source through the FC/PC connector and align the fiber tip to the focal point of the coupler. Measure the light transmission power of the fiber with a power meter to ensure adequate light output. 
	Note: The following steps are for preparing ceramic ferrules with fiber optics for chronic implantation into the brain; ceramic ferrules are hollow in the center and carry fiber optics to deliver light from a patch cable to a region of interest (ROI) within the brain.</li>
	<li>Using a fiber cleaver, terminate the optical fiber to produce a flat end at the termination point. If the fiber has been coated with a jacket, strip the jacket off with a fiber-stripping tool beforehand.</li>
	<li>Cleave the other end of the optical fiber to produce the desired length for implantation into the brain. Determine the length of the fiber using a stereotaxic atlas to target the ROI within the brain.
	<ol>
		<li>For example: to target dorsal hippocampus in rat which is 3.5 mm below bregma, ensure that the length of the fiber protruding from the ferrule is 3.5 mm + 0.25 mm, accounting for skull thickness and allowing for margin of error. Therefore, check that the final length of the fiber is 3.5 mm + 0.25 mm + 10.5 mm (length of the ferrule) = 14.25 mm.</li>
	</ol>
	</li>
	<li>Mix a small amount of epoxy glue in a 1:1 ratio onto a piece of aluminum foil shortly before the next step (the epoxy glue becomes too viscous for use 5 min&#160;after mixing).</li>
	<li>Using a small wooden stick, gently apply epoxy glue to the part of the fiber that will be placed inside the concave side of the ceramic ferrule and then apply a small drop onto the surface of the flat side of the ferrule. After inserting it into the ferrule, ensure that a small length of fiber (&#60;0.5 mm) protrudes from the flat side of the ceramic ferrule. Allow the epoxy glue to harden O/N for optimal results.</li>
	<li>Polish the flat end of the ferrule using a polishing disk by using tweezers to apply gentle downward pressure on the ferrule while making figure-8 rotations on aluminum oxide lapping sheets (from 3 &#181;m to 1 &#181;m to 0.3 &#181;m grit).</li>
	<li>Examine the flat end of the ferrule with a microscope at 100X magnification. Ensure that the surface of the flat end, including the fiber optic surface itself, is free of any epoxy glue; continue polishing if epoxy glue remains on the surface. Ensure that the fiber optic surface has not broken or chipped.</li>
	<li>Couple the polished ferrule to a fiber optic patch cable with a ferrule sleeve and connect the patch cable to a laser light source. Measure the light transmission power at the tip of the fiber with a power meter to ensure adequate efficiency.</li>
	<li>Keep a log of the power output required from the patch cable&#39;s ferrule for each ferrule implant to output the desired power level at the tip of the fiber optic (2.5 mW in this protocol). Discard ferrules with an attenuation rate of over 50% and with non-circular output pattern.</li>
</ol>
2. Stereotaxic Implantation Surgery and Virus Injection
<ol>
	<li>Ensure that any experimental procedures involving the use of animals are approved by the local IACUC. Maintain aseptic conditions during survival surgeries by following aseptic procedures, including using sterile gloves, sterile masks, sterile surgical drapes and sterilized surgical instruments. 
	CAUTION: Ensure that surgeons are wearing proper personal protective equipment (PPE) including safety goggles before beginning the procedure. Follow standard biosafety procedures when working with adeno-associated vector (AAV), taking care to avoid splashing. Dispose of AAV waste in a biohazard container.</li>
	<li>Load a microliter syringe with enough AAV for injection into the animal plus extra to account for potential volume losses (total of 4 &#181;l&#160;per animal), keeping the syringe on ice before use. In this protocol, AAV5-CaMKIIa-hChR2(H134R)-EYFP at a titer of 4x1012 vg/ml is used. Place the animal under isoflurane anesthesia with an induction chamber, connected to a precision isoflurane vaporizer set at 3 - 4% with an oxygen gas source.</li>
	<li>Shave the head with an electric razor and perform a triple surgical scrub on the skin using betadine and a 70% ethanol rinse.</li>
	<li>Once the animal is in deep anesthesia (check toe reflex and breathing rate), immobilize the animal&#39;s skull in a stereotaxic apparatus with intra-aural positioning studs and tooth bar. 
	Note: The entire procedure will take 1 - 2 hr, from anesthesia induction to recovery.</li>
	<li>Set the anesthesia to an appropriate level (1&#160;- 3% isoflurane on the vaporizer) and continually monitor the animal&#39;s vital signs, adjusting anesthesia as necessary to maintain a breathing rate of ~40 breaths/min. Administer ophthalmic ointment on the eyes of the animal to prevent dryness while under anesthesia.</li>
	<li>Make a 15 - 20 mm midline scalp incision with a scalpel and retract the scalp using surgical hemostats attached to the periosteum. Reference lambda and bregma to position the drill bit over the coordinates for the ROI.</li>
	<li>Drill a small craniotomy (2 - 3 mm) over the ROI with a dental drill, taking care not to puncture the brain. Slowly insert needle attached to the microliter syringe through the craniotomy to the ROI in the brain.</li>
	<li>With a microsyringe pump controller, inject 2 &#181;l&#160;of the vector solution into the ROI. Use a flow rate of 150 nl/min to avoid tissue damage. After the injection is complete, wait 10 min&#160;before removing the syringe slowly, at a rate of 0.5 mm/min.</li>
	<li>Following injection, dry the surface of the skull. Reference lambda and bregma to confirm coordinates and then insert the ferrule implant to the target depth (for example: 3.5 mm below bregma for dorsal hippocampus) at a rate of about 0.5 mm/min. Mount the ferrule implant to the skull using dental cement. After the dental cement has solidified, seal the incision with sutures (size 5-0 for rats) around the dental cement cap.</li>
	<li>After surgery, place the animal in its cage singly housed with half of the cage on top of a heater for anesthesia recovery. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not place the animal in the company of other animals until it has fully recovered.</li>
	<li>For post-surgical management of pain, administer buprenorphine subcutaneously every 12 hr&#160;at a dosage of 0.05 mg/kg for 24 hr. Administer antibiotic powder daily over the incision site for 3 days.</li>
	<li>Remove the sutures approximately two weeks after the surgery to prevent scabbing.</li>
	<li>Wait 4 - 6 weeks after the virus injection for sufficient expression of optogenetic genes before performing experiments.</li>
</ol>
3. Optogenetic Functional MRI
CAUTION: Exercise caution around the permanent magnetic field of an MRI scanner. Secure equipment, including the function generator, light source, ventilator, capnograph and gas tanks, sufficiently far away (at least beyond the 5 Gauss limit).
<ol>
	<li>Place the animal under gas anesthesia with an induction chamber, connected to a precision isoflurane vaporizer set at 5% with an oxygen gas source.</li>
	<li>Once the animal is in deep anesthesia (check toe reflex and breathing rate), intubate the animal according to the protocol detailed in Rivard et al. (2006) to allow monitoring of carbon dioxide by capnography26. Note: intubation is critical in maintaining proper levels of expiratory CO2 during imaging.</li>
	<li>Secure the animal in the scanner cradle. 
	Note: In this protocol, the cradle was custom produced but such cradles are also commercially available. The rat cradle functions to secure the animal within the scanner for delivery of anesthesia, heated air and for restriction of motion.</li>
	<li>Deliver a mix of isoflurane (ranging from 1.2 - 1.5%) in approximately 60% nitrous oxide and 40% oxygen via tubing through the cradle. Ensure that the animal&#39;s head is securely fixed in order to avoid motion artifacts.</li>
	<li>Provide heated air through tubing in the cradle and insert a rectal thermometer with lubricant to monitor body temperature. Administer ophthalmic ointment on the eyes of the animal to prevent dryness while under anesthesia.</li>
	<li>Connect the fiber optic patch cable to a laser light source and measure the output at the tip of the patch cable&#39;s ferrule with a power meter.</li>
	<li>Adjust to the appropriate power level (determined previously in step 1.15) to produce the desired output (2.5 mW in this protocol) at the tip of the fiber optic implanted inside the brain. Since excessive power output from the fiber optic can potentially cause tissue damage inside the brain or cause tissue heating that will produce signal artifacts, do not significantly increase the power output of the laser beyond the intended output.</li>
	<li>Prevent light leakage from the implant with a cone of black electrical tape and cover the eyes of the animal.&#160;Couple the fiber optic cable to the ferrule implant on the animal with a ferrule sleeve.&#160;</li>
	<li>Place the coil over the head of the animal. Insert the cradle with animal into the bore of the scanner. 
	Note: In this protocol, the single-loop transmit-receive coil was custom produced and pre-tuned to receive the optimal radio frequency from brain tissue.</li>
	<li>Monitor breathing rate and body temperature throughout this process, adjusting the artificial ventilator and heater as necessary to keep physiological values within limits (ensure that expiratory CO2 is 3 - 4% by rotating the knobs on the ventilator to adjust stroke frequency and volume and that temperature is 37 &#176;C by clicking the arrows for temperature setting).</li>
	<li>Select a positioning sequence to image the animal head location. If the brain is not at the iso-center, adjust the animal head location and repeat the positioning scan until the brain is at the iso-center. Select a linear shimming sequence and click load in the sequence selection window. Next, click start to reduce inhomogeneities of the magnetic field. 
	Note: Shimming is a critical step that will directly influence the integrity of the fMRI data.</li>
	<li>Select a T2-weighted sequence and click load in the sequence selection window. Next, click start to acquire T2-weighted high-resolution coronal anatomical images prior to fMRI to check on the overall integrity of the brain and to confirm the location of the optical fiber implant. 
	Note: These images can be used as anatomy overlays for the ofMRI scan series.</li>
	<li>Connect BNC cables from the triggering port of the MRI scanner to the function generator so that the laser light source is driven according to an experimental stimulation paradigm. 
	Note: In this protocol, the stimulation paradigm is 30 sec&#160;of baseline followed by 20 sec&#160;on/40 sec&#160;off for six min.</li>
	<li>Select a multi-slice gradient recalled echo (GRE) sequence and click load in the sequence selection window. Next, click start to acquire 35 mm x 35 mm (2D FOV) in-plane coronal slices with 0.5 mm x 0.5 mm x 0.5 mm spatial resolution. 
	Note: The GRE sequence used here has a repetition time (TR) and echo time (TE) of TR/TE = 750/12 msec and a 30&#176; flip angle.</li>
	<li>At the conclusion of the scan, remove the animal from the scanner and monitor until it has awakened from anesthesia and can maintain sternal recumbency.</li>
</ol>
4. ofMRI Data Analysis
Note: The following steps are performed in MATLAB as described in a publication on high-throughput ofMRI27.
<ol>
	<li>After the raw scan data has been transferred to the computer used for analysis, use sliding window reconstruction to update the image every TR, with a four-interleave spiral readout, 750 msec TR and 12 msec echo time to acquire 23 slices per scan series. Instead of conventional reconstruction where new fMRI images are reconstructed only after all interleaves are acquired for each slice, the sliding window reconstruction method reconstructs images after the acquisition of each interleaf27.</li>
	<li>Process the raw data for two-dimensional gridding reconstruction, motion correction, and calculation of time series as described previously27.</li>
	<li>Use a threshold coherence method to determine activated voxels by calculating the percent modulation of the BOLD signal of each voxel relative to the baseline period collected prior to stimulation as described previously27. Calculate coherence values as the magnitude of the Fourier transform at the frequency of repeated stimulation cycles divided by the sum-of-squares of all frequency components8.</li>
	<li>Using the time-series data for each voxel, average motion-corrected images that belong to consecutive scans of the same stimulation paradigm first. Then align the average 4D images to a common coordinate frame with a six degree-of-freedom rigid body transformation plus isotropic scaling for comparison within and between animals as described previously27.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agent-selective+Effects+of+Volatile+Anesthetics+on+GABA+A+Receptor+-+mediated+Synaptic+Inhibition+in+Hippocampal+Interneurons.">Agent-selective Effects of Volatile Anesthetics on GABA A Receptor - mediated Synaptic Inhibition in Hippocampal Interneurons.</a> Anesthesiology. 94 (2), 340-347 (2001).</li><li>Jackson, J. I., Meyer, C. H., Nishimura, D. G., Macovski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+a+convolution+function+for+Fourier+inversion+using+gridding.">Selection of a convolution function for Fourier inversion using gridding.</a> IEEE Trans. Med. Imag. 10 (3), 473-478 (1991).</li><li>Glover, G. H., Lee, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motion+artifacts+in+fMRI:+comparison+of+2DFT+with+PR+and+spiral+scan+methods.">Motion artifacts in fMRI: comparison of 2DFT with PR and spiral scan methods.</a> Magn. Reson. Med. 33 (20), 624-635 (1995).</li><li>Kim, D. H., Adalsteinsson, E., Spielman, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+analytic+variable+density+spiral+design.">Simple analytic variable density spiral design.</a> Magn. Reson. Med. 50, 214-219 (2003).</li><li>Friston, K. J., Harrison, L., Penny, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+causal+modeling.">Dynamic causal modeling.</a> Neuroimage. 19, 1273-1302 (2003).</li><li>Alkire, M. T., McReynolds, J. R., Hahn, E. L., Trivedi, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalamic+microinjection+of+nicotine+reverses+sevoflurane-induced+loss+of+righting+reflex+in+the+rat.">Thalamic microinjection of nicotine reverses sevoflurane-induced loss of righting reflex in the rat.</a> Anesthesiology. 107 (2), 264-272 (2007).</li><li>Zincarelli, C., Soltys, S., Rengo, G., Rabinowitz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+AAV+serotypes+1-9+mediated+gene+expression+and+tropism+in+mice+after+systemic+injection.">Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.</a> Mol. Ther. 16 (6), 1073-1080 (2008).</li><li>Aschauer, D. F., Kreuz, S., Rumpel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Transduction+Efficiency,+Tropism+and+Axonal+Transport+of+AAV+Serotypes+1,+2,+5,+6,+8+and+9+in+the+Mouse+Brain.">Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and 9 in the Mouse Brain.</a> PLoS ONE. 8 (9), 1-16 (2013).</li><li>Liu, 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=Frequency-selective+control+of+cortical+and+subcortical+networks+by+central+thalamus.">Frequency-selective control of cortical and subcortical networks by central thalamus.</a> eLife. 4, e09215 (2015).</li></ol>

The authors have nothing to disclose.

Motion of the subject during imaging is a significant source of artifact that can lead to data corruption. Appropriately securing the animal on the imaging cradle can minimize such artifacts as will maintaining appropriate anesthesia levels. Here, we used isoflurane but alternative anesthetics, such as medetomidine or ketamine and xylazine, should also be considered.&#160;However, the levels and choice of anesthetic can influence many parameters in the brain, including the BOLD response28. Isoflurane can cause changes in neuronal excitability29. Other anesthetics can also affect GABA synaptic inhibition30. Thus, the choice of anesthesia is important when performing ofMRI given its ability to affect neuronal activity. ofMRI in the absence of anesthesia is possible but can be challenging with increased motion from the animal, which can be reduced if the animal is habituated; such awake ofMRI studies have previously been performed and would avoid the confounding effect of anesthesia on the brain9,10. Post-processing motion correction algorithms can be used to greatly mitigate the effects of motion. Several of these methods exist, including the inverse Gauss-Newton algorithm employed in this protocol, which minimizes the sum of squares cost function of the reference image and image under correction. The algorithm is useful because it enables fast and robust motion correction, using a GPU parallel platform design to reduce processing times27.
For data reconstruction in this protocol, custom written software in a MATLAB environment was used for two-dimensional gridding reconstruction, where spiral samples are reconstructed in k-space into gridded images31-33. Time series data were generated by calculating the percent modulation of the BOLD signal of each voxel relative to the baseline period collected prior to stimulation. Voxels whose time series were synchronized to blocks of optogenetic stimulation with a coherence value of 0.35 or greater were defined as activated voxels; this coherence value corresponds to a less than 10-9 P value8. Coherence values were calculated as the magnitude of the Fourier transform at the frequency of repeated stimulation cycles divided by the sum-of-squares of all frequency components8,27. Familywise error can be controlled using the Bonferroni correction for multiple comparisons. Alternative methods of analysis can be used, including parametric statistical tests such as the general linear models (GLMs). The coherence method requires less prior knowledge of the HRF compared to the conventional general linear model. Therefore, it is advantageous when exploring data using ofMRI. However, the coherence method can only be used for data with block designs or selected event-related designs with a fixed interstimulus interval and may not be used in ofMRI data with other event-related designs or mixed designs. Subsequently, dynamic causal modeling (DCM) can be used to analyze interactions between brain regions identified through ofMRI. DCM is a Bayesian statistical technique developed for analysis of functional connectivity from system responses to experimental inputs during fMRI34.
Additional technical concerns for ofMRI are discussed here. Implants can be damaged or fall off, leading to the removal of the affected animal from the study. Re-implantation surgeries are not recommended due to the additional uncertainty of targeting the same ROI as in the original implantation surgery and due to animal welfare issues. Because of the significant amount of time and resources invested into each animal subject, consideration of the strength of the material is a significant concern when choosing a suitable dental cement for use in ofMRI studies. The implantation surgery is a critical factor in maximizing the longevity of the implant and animal subject. For example, ensuring that the skull is dry before applying the dental cement and placing an adequate amount of cement around the ceramic ferrule implant can ensure stability over the potential months-long timeline of the animal during the study. Additionally, alternative cage designs can be explored and discussed with the local animal care facility to avoid cages with wire tops holding the food and water that often protrude into the cage and provide opportunities for the animal to damage the implant. Importantly, the dental cement must be chosen carefully to reduce artifacts that affect imaging and alternative cements can be tested by application onto a phantom and imaging in a scanner before use in animal experiments. Trial and error with various dental cements has shown that the cement used in this protocol gives relatively few artifacts. Another technical challenge in performing ofMRI is the accuracy of fiber optic placement at the intended ROI, given the extremely small distances that can exist between nuclei in the brain35. After completing the implantation surgeries, T2-weighted anatomical scans can be used to determine correct placement by overlaying onto a brain atlas. The skill of the surgeon and practice performing these surgeries can improve correct placement rates. The specificity and expression of the opsin at the intended ROI can be verified at the conclusion of the study by perfusing the animal and fixing the brain, using immunohistochemistry or the endogenous fluorescence of a reporter-protein tagged to the opsin for visualization. These reporter proteins can also be colocalized with other proteins to ensure that the opsin is expressed in the desired neural cell types1,8,15,25. As mentioned previously, artifacts can arise when performing ofMRI due to tissue heating from light delivery22. The tissue heating causes modification of relaxation times, resulting in false BOLD signal. To ensure that activation resulting from light stimulation during ofMRI is not due to this artifact, opsin-negative controls should be performed in which either saline injected animals or animals injected with control fluorophore vectors (such as AAV-CaMKIIa-EYFP) undergo ofMRI. Additionally, only well-constructed fiber optic implants with good light transmission efficiency should be used to remove the need to use high laser powers. ofMRI studies have been performed in which false activation due to tissue heating has not been an issue1,6-8,10,11.
Regarding the choice of vector to introduce the required optogenetic genes into neurons for expression, AAVs are not known to cause disease in humans and are therefore a convenient option, given the lower biosafety level required to use these agents (BSL-1). In addition, a multitude of vector cores carry AAVs packaged with various optogenetic genes in stock and with multiple serotypes. The serotype of AAV must be chosen based on the intended cell population target to ensure optimal expression levels36,37. Lentiviruses can also be used but require a higher biosafety level. The time period required for sufficient expression of the optogenetic genes is variable depending on the specific animal model used, on the particular AAV used and on the specific experimental paradigm. In this protocol, Sprague Dawley rats at 11 weeks old are used and optogenetic studies begin four to six weeks after virus injection. Transgenic mice can also be used in optogenetic studies. It is necessary to perform pilot experiments to determine the specific amount of time required for sufficient expression of the opsins. Stimulation paradigms can vary depending on the specific opsin used. In this protocol, AAV5-CaMKIIa-hChR2(H134R)-EYFP is used and the stimulation paradigm is 20 sec&#160;on/40 sec&#160;off. If using an SSFO, the stimulation paradigm will vary because the SSFO requires only a brief pulse of light to be activated and then a brief pulse of light at another wavelength to be terminated.
An additional critical concern when performing ofMRI is preventing light leakage from the ferrule implant interface with the fiber optic patch cable during optogenetic stimulation to prevent a confounding brain signal originating from visual stimulation, even when the animal is anesthetized. Cones of black electrical tape can be used to block the light from the ferrules and to cover the eyes of the animal. Importantly, physiological values including expiratory CO2 and body temperature of the subject must be properly maintained throughout the duration of the imaging. Expiratory CO2 should be kept between 3 - 4% and body temperature at 37 &#176;C. In addition, the shimming sequences to reduce as much inhomogeneity as possible in the magnetic field prior to starting ofMRI scans greatly determines the quality of the resulting BOLD data. Control of these factors is critical in producing reliable ofMRI data. In this protocol, DPSS lasers are used as the light source for optogenetic stimulation. Because laser light is coherent, more than enough power can be easily supplied through the fiber optic. LED light sources coupled to fiber optics are available from commercial vendors, but have the disadvantage of decreased power of light transmission. The laser light source does require alignment to each particular fiber optic patch cable, but with practice, the alignment can be accomplished within seconds to minutes.
Future applications of ofMRI include the use of next-generation opsins such as red-shifted opsins to enable non-invasive stimulation during imaging. Additionally, the implantation of MRI-compatible EEG or similar recording electrodes along with the fiber optic implant could allow for the acquisition of high temporal resolution data in addition to the high spatial resolution data of MRI. ofMRI with electrophysiological recording could provide extensive information on the functional connectivity of the brain. In summary, the power of ofMRI to monitor the entire brain in response to the stimulation of specific cell populations defined by genetic or anatomical identity makes ofMRI a critical tool to use in the study of neurological diseases and of the connectomics of the healthy brain.

Optogenetic functional magnetic resonance imaging (ofMRI) is a novel technique that combines the spatial resolution of high-field fMRI with the precision of optogenetic stimulation1-11,38, enabling cell type-specific mapping of functional neural circuits and their dynamics across the whole brain. Optogenetics allows for specific cell types to be targeted for stimulation by the introduction of light-sensitive trans-membrane conductance channels, called opsins. Specific elements of neural circuits are genetically modified to express these channels, enabling millisecond-timescale modulation of activity in the intact brain1-15. fMRI provides a non-invasive method of determining the brain&#39;s global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal16-18, which provides an indirect measurement of neuronal activity.
The combination of these two techniques, termed optogenetic functional magnetic resonance imaging (ofMRI), is advantageous over other methods of recording brain activity during stimulation such as electrophysiology because it can provide a view of the entire brain at relatively high spatial resolution. This enables the detection of neuronal activity in response to targeted stimulation at great distances from the site of stimulation without the need for implantation of invasive recording electrodes1-11. ofMRI is advantageous over the more traditional method of performing electrical stimulation during fMRI, which can recruit different cell types near the electrode and thus confound the causal influence of each population19. In addition, the electrodes used for electrical stimulation and the generated current can produce artifacts during MR imaging20. Indeed, ofMRI enables the observation of the influence on global brain activity from the specific modulation of a wide variety of cell types through the use of advanced genetic targeting techniques such as the Cre-Lox system in transgenic animals or the use of promoters. Combinatorial optical control with whole-brain monitoring is possible with ofMRI through the use of both NpHR to inhibit and ChR2 to excite specific cell types. The optogenetic toolkit available for use in ofMRI is also rapidly improving over time with the introduction of opsins with increased light-sensitivity or improved kinetics, of stabilized step function opsins (SSFOs) or of red-shifted opsins that may negate the requirement for implanted fiber optics, enabling non-invasive stimulation during imaging21. These possibilities are not available with electrical stimulation.
However, signal artifacts resulting from tissue heating due to light delivery in the brain have been reported22, where temperature-induced modification of relaxation times has been shown to produce pseudo activation. Researchers performing ofMRI should therefore be aware of this potential confound. With the proper setup and controls, the issue can be addressed. Additionally, relatively low temporal resolution of measuring the hemodynamic response in fMRI may be a limiting factor for certain applications of this technique.
This protocol first describes the construction of the fiber optic implants that enable delivery of specific wavelengths of light deep into the brain in vivo. The protocol then describes the delivery of the opsin-encoding viral vector to a precise brain region using stereotactic surgery. Next the protocol describes the process of whole-brain functional MRI during simultaneous light stimulation. Finally, the protocol outlines basic data analysis of the acquired data.
Of note, the optogenetics described here require a chronic implant for light delivery. However, the fiber optic implants are stable and bio-compatible, allowing for longitudinal scanning and investigation of neural circuitry over a period of months23,24.
In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain. In addition, it can provide novel insight into the mechanisms of neurological diseases25 when coupled with different animal models. Indeed, ofMRI has been used to elucidate the network activity of distinct hippocampal subregions associated with seizures8. Therefore, laboratories interested in answering systems-level neuroscience questions will find this technique of importance.

<table border="0" cellpadding="0" cellspacing="0" width="1117">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">7 Tesla scanner</td>
			<td style="width:195px;">Agilent Technologies</td>
			<td style="width:205px;">Discovery MR901 System</td>
			<td style="width:325px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sprague Dawley rats</td>
			<td>Charles River</td>
			<td>Crl:SD</td>
			<td>11 weeks old</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fiber cleaver</td>
			<td>Fujikura</td>
			<td>CT-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">multimode optical fiber</td>
			<td>Thor Labs</td>
			<td>AFS105/125Y</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fiber stripper&#160;&#160;</td>
			<td>Thor Labs</td>
			<td>T08S13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ceramic split sleeve</td>
			<td>Precision Fiber Products</td>
			<td>SM-CS1140S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">epoxy glue</td>
			<td>Thor Labs</td>
			<td>G14250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cotton-tipped applicators</td>
			<td>Stoelting Co.</td>
			<td align="right">50975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">multimode ceramic zirconia ferrules</td>
			<td>Precision Fiber Products</td>
			<td>MM-FER2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">FC/PC multimode connector</td>
			<td>Thor Labs</td>
			<td>30128C3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fiber optic polishing disk</td>
			<td>Precision Fiber Products</td>
			<td>M1-80754</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aluminum oxide lapping sheet, 0.3 &#181;m</td>
			<td>Thor Labs</td>
			<td>LFG03P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aluminum oxide lapping sheet, 1 &#181;m</td>
			<td>Thor Labs</td>
			<td>LFG1P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">aluminum oxide lapping sheet, 3 &#181;m</td>
			<td>Thor Labs</td>
			<td>LFG3P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">binocular biological microscope 40X-1000X</td>
			<td>Amscope</td>
			<td>B100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">laser safety glasses</td>
			<td>Kentek</td>
			<td>KXL-62W01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:392px;">473 nm DPSS laser</td>
			<td>Laserglow</td>
			<td>LRS-0473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">594 nm DPSS laser</td>
			<td>Laserglow</td>
			<td>LRS-0594</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Allen hex wrench set</td>
			<td></td>
			<td></td>
			<td>2.0 mm (5/64&#34;) for alignment of fiber tip to focal point of coupler in the laser</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">power meter, Si Sensor, 400-1100 nm</td>
			<td>Thor Labs</td>
			<td>PM121D&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane (Isothesia)</td>
			<td>Henry Schein&#160;</td>
			<td align="right">50033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">isoflurane vaporizer with induction chamber</td>
			<td>VetEquip</td>
			<td align="right">901806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoFil 100uL syringe</td>
			<td>World Precision Instruments</td>
			<td>NANOFIL-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraMicroPump with SYS-Micro4 Controller</td>
			<td>World Precision Instruments</td>
			<td>UMP3-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">function generator</td>
			<td>A.M.P.I.&#160;</td>
			<td>Master-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">small animal stereotax</td>
			<td>David Kopf Instruments</td>
			<td>Model 940&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Model 683 small animal ventilator&#160;</td>
			<td>Harvard Apparatus</td>
			<td align="right">550000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type 340 capnograph&#160;</td>
			<td>Harvard Apparatus</td>
			<td align="right">733809</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dental drill (rotary micromotor kit)</td>
			<td>Foredom Electric Co.</td>
			<td style="width:205px;">K.1070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ophthalmic ointment (Artificial Tears)</td>
			<td>Rugby</td>
			<td>00536-6550-91</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">instrument sterilizer</td>
			<td>CellPoint Scientific</td>
			<td>Germinator 500</td>
			<td>glass bead sterilizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">antibiotic powder</td>
			<td>Pfizer</td>
			<td>NEO-PREDEF</td>
			<td>neomycin sulfate, isoflupredone acetate and tetracaine hydrochloride</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">buprenorphine painkiller</td>
			<td>Hospira</td>
			<td>NDC:0409-2012</td>
			<td>schedule III controlled substance , 0.3 mg/mL stock</td>
		</tr>
	</tbody>
</table>

optogenetic functional mri

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional magnetic resonance imaging (ofMRI), which is a novel technique that combines the relatively high spatial resolution of high-field fMRI with the precision of optogenetic stimulation. Fiber optics that enable delivery of specific wavelengths of light deep into the brain in vivo are implanted into regions of interest in order to specifically stimulate targeted cell types that have been genetically induced to express light-sensitive trans-membrane conductance channels, called opsins. fMRI is used to provide a non-invasive method of determining the brain's global dynamic response to optogenetic stimulation of specific neural circuits through measurement of the blood-oxygen-level-dependent (BOLD) signal, which provides an indirect measurement of neuronal activity. This protocol describes the construction of fiber optic implants, the implantation surgeries, the imaging with photostimulation and the data analysis required to successfully perform ofMRI. In summary, the precise stimulation and whole-brain monitoring ability of ofMRI are crucial factors in making ofMRI a powerful tool for the study of the connectomics of the brain in both healthy and diseased states.

Figure 1 and Figure 2 show representative data resulting from 20 Hz (15 msec pulse width, 473 nm, 30% duty cycle) optogenetic stimulation of the motor cortex. A stimulation paradigm of 30 seconds of baseline followed by 20 sec&#160;on/40 sec&#160;off for six min&#160;was used. Previous studies have shown that this paradigm produces robust BOLD signal from optogenetic stimulation1,8. Figure 1 shows activated voxels detected both at the local site of stimulation (motor cortex) and in the thalamus, as a result of the long-range synaptic connections between these regions. Figure 2 shows that temporal information can be gleaned from HRFs, as the thalamic response is delayed (lower initial slope) compared to the motor cortex response after optogenetic stimulation.
<img alt="Figure 1" src="/files/ftp_upload/53346/53346fig1.jpg" /> 
Figure 1.&#160;Activation Map of BOLD Signal Induced by Optogenetic Stimulation of CaMKIIa-expressing Cells in Motor Cortex. Coherence values of active voxels, identified as those significantly synchronized to repeated stimulations, are shown overlaid on a T2-weighted coronal anatomical slice. Data collected over a six&#160;min, 30&#160;sec&#160;period (initial 30 sec&#160;baseline and six stimulation cycles of 20 sec&#160;on/40 sec&#160;off with 473 nm light, 20 Hz, 15 msec pulse width) are condensed into one activation map. Sequential slices are 0.5 mm apart and the location of the fiber optic implant is denoted by the triangle. <a href="https://www.jove.com/files/ftp_upload/53346/53346fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53346/53346fig2.jpg" /> 
Figure 2.&#160;Hemodynamic Response Function. (Left) The percent modulation of the BOLD signal relative to baseline is shown for active voxels in motor cortex and thalamus during optogenetic stimulation of motor cortex (six stimulation cycles of 20 sec&#160;on/40 sec&#160;off with 473 nm light, 20 Hz, 15 msec&#160;pulse width). Shaded grey error bars denote standard error across activated voxels within ROI. (Right) Time-averaged responses are given by the hemodynamic response functions (HRF). The thalamus HRF shows a delayed response relative to the stimulated motor cortex. Blue bars indicate periods of 473 nm photostimulation. Shaded grey error bars denote standard error across six cycles. <a href="https://www.jove.com/files/ftp_upload/53346/53346fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Optogenetic Functional MRI at JoVE.com

Optogenetic Functional MRI

This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

The investigation of the functional connectivity of precise neural circuits across the entire intact brain can be achieved through optogenetic functional ...

Research

JoVE Journal

Neuroscience

14.7K Views.  Stanford University. This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.

Video: Optogenetic Functional MRI

Measuring the Subjective Value of Risky and Ambiguous Options using Experimental Economics and Functional MRI Methods

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition termed "risky". In other cases when probabilities cannot be estimated, this is a condition described as "ambiguous". While most people are averse to both risk and ambiguity1,2, the degree of those aversions vary substantially across individuals, such that the subjective value of the same risky or ambiguous option can be very different for different individuals. We combine functional MRI (fMRI) with an experimental economics-based method3 to assess the neural representation of the subjective values of risky and ambiguous options4. This technique can be now used to study these neural representations in different populations, such as different age groups and different patient populations.
In our experiment, subjects make consequential choices between two alternatives while their neural activation is tracked using fMRI. On each trial subjects choose between lotteries that vary in their monetary amount and in either the probability of winning that amount or the ambiguity level associated with winning. Our parametric design allows us to use each individual's choice behavior to estimate their attitudes towards risk and ambiguity, and thus to estimate the subjective values that each option held for them. Another important feature of the design is that the outcome of the chosen lottery is not revealed during the experiment, so that no learning can take place, and thus the ambiguous options remain ambiguous and risk attitudes are stable. Instead, at the end of the scanning session one or few trials are randomly selected and played for real money. Since subjects do not know beforehand which trials will be selected, they must treat each and every trial as if it and it alone was the one trial on which they will be paid. This design ensures that we can estimate the true subjective value of each option to each subject. We then look for areas in the brain whose activation is correlated with the subjective value of risky options and for areas whose activation is correlated with the subjective value of ambiguous options.

Using functional MRI and behavioral methods to determine the neural representation of the subjective value of risky and ambiguous options in the human brain.

Most of the choices we make have uncertain consequences. In some cases the probabilities for different possible outcomes are precisely known, a condition ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Optogenetic Stimulation of the Auditory Nerve

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted deaf subjects1-6. Nonetheless, sound coding with current CIs has poor frequency and intensity resolution due to broad current spread from each electrode contact activating a large number of SGNs along the tonotopic axis of the cochlea7-9. Optical stimulation is proposed as an alternative to electrical stimulation that promises spatially more confined activation of SGNs and, hence, higher frequency resolution of coding. In recent years, direct infrared illumination of&#160;the cochlea has been used to evoke responses in the auditory nerve10. Nevertheless it requires higher energies than electrical stimulation10,11 and uncertainty remains as to the underlying mechanism12. Here we describe a method based on optogenetics to stimulate SGNs with low intensity blue light, using transgenic mice with neuronal expression of channelrhodopsin 2 (ChR2)13 or virus-mediated expression of the ChR2-variant CatCh14. We used micro-light emitting diodes (&#181;LEDs) and fiber-coupled lasers to stimulate ChR2-expressing SGNs through a small artificial opening (cochleostomy) or the round window. We assayed the responses by scalp recordings of light-evoked potentials (optogenetic auditory brainstem response: oABR) or by microelectrode recordings from the auditory pathway and compared them with acoustic and electrical stimulation.

Cochlear implants (CIs) enable hearing by direct electrical stimulation of the auditory nerve. However, poor frequency and intensity resolution limits the quality of hearing with CIs. Here we describe optogenetic stimulation of the auditory nerve in mice as an alternative strategy for auditory research and developing future CIs.

Direct electrical stimulation of spiral ganglion neurons (SGNs) by cochlear implants (CIs) enables open speech comprehension in the majority of implanted ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...

Automated Segmentation of Cortical Grey Matter from T1-Weighted MRI Images

Within neuroimaging research, a number of recent studies have discussed the impact of between-study differences in volumetric findings that are thought to result from the use of different segmentation tools to generate brain volumes. Here, processing pipelines for seven automated tools that can be used to segment grey matter within the brain are presented. The protocol provides an initial step for researchers aiming to find the most accurate method for generating grey matter volumes from T1-weighted MRI scans. Steps to undertake detailed visual quality control are also included in the manuscript. This protocol covers a range of potential segmentation tools and encourages users to compare the performance of these tools within a subset of their data before selecting one to apply to a full cohort. Furthermore, the protocol may be further generalized to the segmentation of other brain regions.

This protocol describes the process of applying seven different automated segmentation tools to structural T1-weighted MRI scans to delineate grey matter regions that can be used for the quantification of grey matter volume.

Within neuroimaging research, a number of recent studies have discussed the impact of between-study differences in volumetric findings that are thought to ...

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical methods for manipulating and monitoring the activity of neurons in vivo. These types of experiments rely on two main components: 1) implantable devices that provide optical access to the brain, and 2) light-sensitive proteins that change neuronal excitability or provide a readout of neuronal activity. There are a number of ways to express light-sensitive proteins, but stereotaxic injection of viral vectors is currently the most flexible approach because expression can be controlled with genetic, anatomical, and temporal precision. Despite the great utility of viral vectors, delivering the virus to the site of optical implants poses numerous challenges. Stereotaxic virus injections are demanding surgeries that increase surgical time, increase the cost of studies, and pose a risk to the animal's health. The surrounding tissue can be physically damaged by the injection syringe, and by immunogenic inflammation caused by the abrupt delivery of a bolus of high-titer virus. Aligning injections with optical implants is especially difficult when targeting small regions deep in the brain. To overcome these challenges, we describe a method for coating multiple types of optical implants with films composed of silk fibroin and Adeno-associated viral (AAV) vectors. Fibroin, a polymer derived from the cocoon of Bombyx mori, can encapsulate and protect biomolecules and can be processed into forms ranging from soluble films to ceramics. When implanted into the brain, silk/AAV coatings release virus at the interface between optical elements and the surrounding brain, driving expression precisely where it is needed. This method is easily implemented and promises to greatly facilitate in vivo studies of neural circuit function.

In Vivo Targeted Expression of Optogenetic Proteins Using Silk/AAV Films

Here, we present a method for delivering viral expression vectors into the brain using silk fibroin films. This method allows targeted delivery of expression vectors using silk/AAV coated optical fibers, tapered optical fibers, and cranial windows.

The quest to understand how neural circuits process information in order to drive behavioral output has been greatly aided by recently-developed optical ...