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><tbody><tr><td>7 Tesla scanner</td><td>Agilent Technologies</td><td>Discovery MR901 System</td><td></td></tr><tr><td>Sprague Dawley rats</td><td>Charles River</td><td>Crl:SD</td><td>11 weeks old</td></tr><tr><td>fiber cleaver</td><td>Fujikura</td><td>CT-05</td><td></td></tr><tr><td>multimode optical fiber</td><td>Thor Labs</td><td>AFS105/125Y</td><td></td></tr><tr><td>fiber stripper&amp;nbsp;&amp;nbsp;</td><td>Thor Labs</td><td>T08S13</td><td></td></tr><tr><td>ceramic split sleeve</td><td>Precision Fiber Products</td><td>SM-CS1140S</td><td></td></tr><tr><td>epoxy glue</td><td>Thor Labs</td><td>G14250</td><td></td></tr><tr><td>cotton-tipped applicators</td><td>Stoelting Co.</td><td>50975</td><td></td></tr><tr><td>multimode ceramic zirconia ferrules</td><td>Precision Fiber Products</td><td>MM-FER2002</td><td></td></tr><tr><td>FC/PC multimode connector</td><td>Thor Labs</td><td>30128C3</td><td></td></tr><tr><td>fiber optic polishing disk</td><td>Precision Fiber Products</td><td>M1-80754</td><td></td></tr><tr><td>aluminum oxide lapping sheet, 0.3 &amp;micro;m</td><td>Thor Labs</td><td>LFG03P</td><td></td></tr><tr><td>aluminum oxide lapping sheet, 1 &amp;micro;m</td><td>Thor Labs</td><td>LFG1P</td><td></td></tr><tr><td>aluminum oxide lapping sheet, 3 &amp;micro;m</td><td>Thor Labs</td><td>LFG3P</td><td></td></tr><tr><td>binocular biological microscope 40X-1,000X</td><td>Amscope</td><td>B100</td><td></td></tr><tr><td>laser safety glasses</td><td>Kentek</td><td>KXL-62W01</td><td></td></tr><tr><td>473 nm DPSS laser</td><td>Laserglow</td><td>LRS-0473</td><td></td></tr><tr><td>594 nm DPSS laser</td><td>Laserglow</td><td>LRS-0594</td><td></td></tr><tr><td>Allen hex wrench set</td><td></td><td></td><td>2.0 mm (5/64&quot;) for alignment of fiber tip to focal point of coupler in the laser</td></tr><tr><td>power meter, Si Sensor, 400-1,100 nm</td><td>Thor Labs</td><td>PM121D</td><td></td></tr><tr><td>Isoflurane (Isothesia)</td><td>Henry Schein&amp;nbsp;</td><td>50033</td><td></td></tr><tr><td>isoflurane vaporizer with induction chamber</td><td>VetEquip</td><td>901806</td><td></td></tr><tr><td>NanoFil 100 &amp;micro;l syringe</td><td>World Precision Instruments</td><td>NANOFIL-100</td><td></td></tr><tr><td>UltraMicroPump with SYS-Micro4 Controller</td><td>World Precision Instruments</td><td>UMP3-1</td><td></td></tr><tr><td>function generator</td><td>A.M.P.I.&amp;nbsp;</td><td>Master-8</td><td></td></tr><tr><td>small animal stereotax</td><td>David Kopf Instruments</td><td>Model 940&amp;nbsp;</td><td></td></tr><tr><td>Model 683 small animal ventilator&amp;nbsp;</td><td>Harvard Apparatus</td><td>550000</td><td></td></tr><tr><td>Type 340 capnograph&amp;nbsp;</td><td>Harvard Apparatus</td><td>733809</td><td></td></tr><tr><td>dental drill (rotary micromotor kit)</td><td>Foredom Electric Co.</td><td>K.1070</td><td></td></tr><tr><td>ophthalmic ointment (Artificial Tears)</td><td>Rugby</td><td>00536-6550-91</td><td></td></tr><tr><td>instrument sterilizer</td><td>CellPoint Scientific</td><td>Germinator 500</td><td>glass bead sterilizer</td></tr><tr><td>antibiotic powder</td><td>Pfizer</td><td>NEO-PREDEF</td><td>neomycin sulfate, isoflupredone acetate and tetracaine hydrochloride</td></tr><tr><td>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

Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and molecular approaches allow the investigation of either different stimuli on few neurons, or one stimulus in large parts of the brain, blood oxygenation level dependent (BOLD) functional Magnetic Resonance Imaging (fMRI) allows combining both advantages, i.e. compare the neural activation induced by different stimuli in the entire brain at once. fMRI in songbirds is challenging because of the small size of their brains and because their bones and especially their skull comprise numerous air cavities, inducing important susceptibility artifacts. Gradient-echo (GE) BOLD fMRI has been successfully applied to songbirds 1-5 (for a review, see 6). These studies focused on the primary and secondary auditory brain areas, which are regions free of susceptibility artifacts. However, because processes of interest may occur beyond these regions, whole brain BOLD fMRI is required using an MRI sequence less susceptible to these artifacts. This can be achieved by using spin-echo (SE) BOLD fMRI 7,8 . In this article, we describe how to use this technique in zebra finches (Taeniopygia guttata), which are small songbirds with a bodyweight of 15-25 g extensively studied in behavioral neurosciences of birdsong. The main topic of fMRI studies on songbirds is song perception and song learning. The auditory nature of the stimuli combined with the weak BOLD sensitivity of SE (compared to GE) based fMRI sequences makes the implementation of this technique very challenging.

Functional Magnetic Resonance Imaging fMRI with Auditory Stimulation in Songbirds

This article shows an optimized procedure for imaging of the neural substrates of auditory stimulation in the songbird brain using functional Magnetic Resonance Imaging (fMRI). It describes the preparation of the sound stimuli, the positioning of the subject and the acquisition and subsequent analysis of the fMRI data.

The neurobiology of birdsong, as a model for human speech, is a pronounced area of research in behavioral neuroscience. Whereas electrophysiology and ...

Behavior

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

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 ...

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the interactions of these forebrain circuits has been aided recently by the development of optogenetic methods for light-mediated modulation of neuronal activity. Here, we describe a protocol for examining the functional organization of forebrain circuits&#160;in vitro using laser-scanning photostimulation of channelrhodopsin, expressed optogenetically via viral-mediated transfection. This approach also exploits the utility of cre-lox recombination in transgenic mice to target expression in specific neuronal cell types. Following transfection, neurons are physiologically recorded in slice preparations using whole-cell patch clamp to measure their evoked responses to laser-scanning photostimulation of channelrhodopsin expressing fibers. This approach enables an assessment of functional topography and synaptic properties. Morphological correlates can be obtained by imaging the neuroanatomical expression of channelrhodopsin expressing fibers using confocal microscopy of the live slice or post-fixed tissue. These methods enable functional investigations of forebrain circuits that expand upon more conventional approaches.

We describe a method for characterizing the functional topography and synaptic properties of forebrain circuits using an optogenetic approach to photostimulate neuronal populations&#160;in vitro.

The sensory forebrain is composed of intricately connected cell types, of which functional properties have yet to be fully elucidated. Understanding the ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and postsynaptic projections to the visual cortex. Based on its distinct anatomical structure and convenient accessibility, it has become the favored structure for studies on neuronal survival, axonal regeneration, and synaptic plasticity. Recent advancements in MR imaging have enabled the in vivo visualization of the retino-tectal part of this projection using manganese mediated contrast enhancement (MEMRI). Here, we present a MEMRI protocol for illustration of the visual projection in mice, by which resolutions of (200 &#181;m)3 can be achieved using common 3 Tesla scanners. We demonstrate how intravitreal injection of a single dosage of 15 nmol MnCl2 leads to a saturated enhancement of the intact projection within 24 hr. With exception of the retina, changes in signal intensity are independent of coincided visual stimulation or physiological aging. We further apply this technique to longitudinally monitor axonal degeneration in response to acute optic nerve injury, a paradigm by which Mn2+ transport completely arrests at the lesion site. Conversely, active Mn2+ transport is quantitatively proportionate to the viability, number, and electrical activity of axon fibers. For such an analysis, we exemplify Mn2+ transport kinetics along the visual path in a transgenic mouse model (NF-&#954;B p50KO) displaying spontaneous atrophy of sensory, including visual, projections. In these mice, MEMRI indicates reduced but not delayed Mn2+ transport as compared to wild type mice, thus revealing signs of structural and/or functional impairments by NF-&#954;B mutations.
In summary, MEMRI conveniently bridges in vivo assays and post mortem histology for the characterization of nerve fiber integrity and activity. It is highly useful for longitudinal studies on axonal degeneration and regeneration, and investigations of mutant mice for genuine or inducible phenotypes.

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

This video illustrates a method, using a clinical 3 T scanner, for contrast-enhanced MR imaging of the na&#239;ve mouse visual projection and for repetitive and longitudinal in vivo studies of optic nerve degeneration associated with acute optic nerve crush injury and chronic optic nerve degeneration in knock-out mice (p50KO).

The rodent visual system encompasses retinal ganglion cells and their axons that form the optic nerve to enter thalamic and midbrain centers, and ...

Advancing Visual Cortex Mapping for Enhanced Evaluation of Vision Disorders

Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation

High-resolution retinotopic blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) with a wide-view presentation can be used to functionally map the peripheral and central visual cortex. This method for measuring functional changes of the visual brain allows for functional mapping of the occipital lobe, stimulating &gt;100&#176; (&plusmn;50&#176;) or more of the visual field, compared to standard fMRI visual presentation setups which usually cover &lt;30&#176; of the visual field. A simple wide-view stimulation system for BOLD fMRI can be set up using common MR-compatible projectors by placing a large mirror or screen close to the subject's face and using only the posterior half of a standard head coil to provide a wide-viewing angle without obstructing their vision. The wide-view retinotopic fMRI map can then be imaged using various retinotopic stimulation paradigms, and the data can be analyzed to determine the functional activity of visual cortical regions corresponding to central and peripheral vision. This method provides a practical, easy-to-implement visual presentation system that can be used to evaluate changes in the peripheral and central visual cortex due to eye diseases such as glaucoma and the vision loss that may accompany them.

Author Spotlight: Insights into Visual Cortex Research Through Wide-View fMRI Mapping 

We have developed techniques for mapping the visual cortex function utilizing more of the visual field than is commonly used. This approach has the potential to enhance the evaluation of vision disorders and eye diseases.

High-resolution retinotopic blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) with a wide-view presentation can be ...

Neuronavigated tDCS for Enhanced Electrode Placement Accuracy in fMRI Studies

Neuronavigated Focalized Transcranial Direct Current Stimulation Administered During Functional Magnetic Resonance Imaging

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that allows the modulation of the excitability and plasticity of the human brain. Focalized tDCS setups use specific electrode arrangements to constrain the current flow to circumscribed brain regions. However, the effectiveness of focalized tDCS can be compromised by electrode positioning errors on the scalp, resulting in significant reductions of the current dose reaching the target brain regions for tDCS. Electrode placement guided by neuronavigation based on the individual&#39;s head and brain anatomy derived from structural magnetic resonance imaging (MRI) data may be suited to improve positioning accuracy.
This protocol describes the method of neuronavigated electrode placement for a focalized tDCS setup, which is suitable for concurrent administration during functional MRI (fMRI). We also quantify the accuracy of electrode placement and investigate electrode drift in a concurrent tDCS-fMRI experiment. Critical steps involve the optimization of electrode positions based on current modeling that considers the individual&#39;s head and brain anatomy, the implementation of neuronavigated electrode placement on the scalp, and the administration of optimized and focal tDCS during fMRI.
The regional precision of electrode placement is quantified using the Euclidean norm (L2&#160;Norm) to determine deviations of the actual from the intended electrode positions during a concurrent tDCS-fMRI study. Any potential displacement of electrodes (drift) during the experiment is investigated by comparing actual electrode positions before and after the fMRI acquisition. In addition, we directly compare the placement accuracy of neuronavigated tDCS to that achieved by a scalp-based targeting approach (a 10-20 Electroencephalography (EEG) system). These analyses demonstrate superior placement accuracy for neuronavigation compared to scalp-based electrode placement and negligible electrode drift across a 20 min scanning period.

This protocol describes the method of neuronavigated electrode placement for focal, transcranial direct current stimulation (tDCS) administered during functional magnetic resonance imaging (fMRI).

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that allows the modulation of the excitability and plasticity ...

fMRI: Functional Magnetic Resonance Imaging

Functional magnetic resonance imaging (fMRI) is a non-invasive neuroimaging technique used to investigate human brain function and cognition in both healthy individuals and populations with abnormal brain states. Functional MRI utilizes a magnetic resonance signal to detect changes in blood flow that are coupled to neuronal activation when a specific task is performed. This is possible because hemoglobin within the blood has different magnetic properties depending on whether or not it is bound to oxygen. When a certain task is performed, there is an influx of oxygenated blood to brain regions responsible for that function, and this influx can then be detected with specific MRI scan&#160;parameters. This phenomenon is termed the blood oxygen level ependent (BOLD) effect, and can be used to create maps of brain activity.
This video begins with a brief overview of how MRI and fMRI signal is obtained. Then, basic experimental design is reviewed, which involves first setting up a stimulus presentation that is specifically designed to test the function that will be mapped. Next, key steps involved in performing the fMRI scan are introduced, including subject safety and setting up at the scanner. Commonly used steps for data processing are then presented, including pre-processing and statistical analysis with the general linear model. Finally, some specific applications of fMRI are reviewed, such as investigations into abnormal function in psychological disorders, and combining fMRI with complimentary imaging modalities, such as diffusion tensor imaging (DTI).

Functional magnetic resonance imaging (fMRI) is a non-invasive neuroimaging technique used to investigate human brain function and cognition in both ...

Optogenetic Functional MRI

Summary

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Functional Magnetic Resonance Imaging (fMRI) with Auditory Stimulation in Songbirds

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

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

Laser-scanning Photostimulation of Optogenetically Targeted Forebrain Circuits

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In vivo Imaging of Optic Nerve Fiber Integrity by Contrast-Enhanced MRI in Mice

Functional Magnetic Resonance Imaging (fMRI) of the Visual Cortex with Wide-View Retinotopic Stimulation

Neuronavigated Focalized Transcranial Direct Current Stimulation Administered During Functional Magnetic Resonance Imaging

fMRI: Functional Magnetic Resonance Imaging