The authors would like to acknowledge the contributions of Kyle Gilbert, who designed the custom RF coil, and Kevin Barker, who designed the MRI-compatible sled. This work was supported by the Canadian Institutes of Health Research (CIHR), Natural Sciences and Engineering Research Council of Canada (NSERC), and Canada Foundation for Innovation (CFI).

The following procedure can be applied to any imaging experiment in which anesthetized cats are used. Steps which are specifically required for auditory experiments (steps 1.1-1.7, 2.8, 4.1) can be modified to accommodate other sensory stimulus protocols.
All experimental procedures received approval from the Animal Use Subcommittee of the University Council on Animal Care at the University of Western Ontario and followed the guidelines specified by the Canadian Council on Animal Care (CCAC)44. The outlined experiment requires approximately 150 min from animal preparation to recovery. The time course of the experiment is illustrated in Figure 1.
1. Stimulus Equipment Preparation
Figure 2 shows the electronic components and corresponding connections required for generating an auditory stimulus in the MRI scanner. The requirements are as follows: a computer, an external sound card, a stereo power amplifier and an fMRI-compatible earphone system.
<ol>
	<li>Connect the computer which will be used to present the auditory stimulus to the external sound card via the Universal Serial Bus (USB) cable.</li>
	<li>Attach the cables connecting the output ports of the external sound card to the input ports of the stereo power amplifier.</li>
	<li>Attach the cables connecting the output ports of the stereo power amplifier to the input ports of the transformer box of the fMRI-compatible earphone system.</li>
	<li>Connect the binaural earphones to the output ports of the transformer box.</li>
	<li>Use shielded coaxial cables with BNC connections to connect the transformer box to the penetration panel outside the scanner room.</li>
	<li>Connect the earphone cable assembly to the corresponding BNC ports on the penetration panel inside the scanner room.</li>
	<li>Connect the foam ear tips to the earphones then connect the earphones to the cable assembly. Run a test auditory stimulus to confirm that sound is being transmitted from the computer to the earphones. Disconnect the earphones and insert the foam ear tips securely into the cat&#39;s ears during the animal preparation phase (step 2.7).</li>
</ol>
2. Animal Preparation
<ol>
	<li>To premedicate the cat, administer a sedative mixture of atropine sulphate (0.02 mg/kg) and acepromazine (0.02 mg/kg) via a subcutaneous (SC) injection.</li>
	<li>After 20 min, administer ketamine (4 mg/kg) and dexmedetomidine hydrochloride (0.02-0.03 mg/kg) via an intramuscular (IM) injection to induce anesthesia. Ketamine is usually combined with a sedative and muscle relaxant, in this case, dexmedetomidine hydrochloride, to reduce the tremors and muscle rigidity commonly observed when ketamine is used alone45. This anesthetic combination typically induces approximately 150 min of sedation and is frequently used in veterinary practice to induce anesthesia in small animals.</li>
	<li>Once the cat has lost its righting reflex, apply ophthalmic ointment to the eyes to prevent dryness during the procedure. Place an indwelling catheter in the medial saphenous vein for intravenous delivery of ketamine.</li>
	<li>Test for successful anesthetic induction by pinching a toe on the forepaw then observing whether the cat withdraws its paw. Once the pedal reflex disappears, suppress the gag reflex by spraying lidocaine onto the pharyngeal walls then intubate the cat with a 4.0-4.5 endotracheal tube.</li>
	<li>Maintain anesthesia throughout the imaging session with a constant rate infusion of ketamine (0.6-0.75 mg/kg/hr) and inhaled isoflurane (0.4-0.5%) delivered in 100% oxygen at 1-1.5 L/min. Combine 60 ml of saline and 0.07 ml of ketamine in a 60 ml syringe then place the syringe in the syringe pump. This step can be performed before premedicating the cat.</li>
	<li>Place warm wax-filled heating pads on the floor of the MRI-compatible sled (Figures 3a and 3c) then layer the insulating plastic bubble wrap around the interior walls of the sled.</li>
	<li>Place the cat in a sternal position within the insulating bubble wrap in the MRI-compatible sled (Figure 3c).</li>
	<li>Once the cat is positioned, adjust the head to gain access to the ears. Roll the foam ear tips into the smallest diameter possible then insert each ear tip deep into the ear canal. Once inserted, the foam ear tips should expand to fill the space within the ear canals.</li>
	<li>Adjust the cat until its head is properly positioned within the 3-channel radio-frequency (RF) coil (Figure 3b). Stabilize the head with acoustic dampening memory foam (Figure 3d). Place foam around the ears to provide additional attenuation of the scanner noise.</li>
	<li>Wrap the cat in the blanket of insulating plastic bubble wrap then secure and transport the sled to the scanner bed.</li>
	<li>Connect the infusion lines, anesthetic delivery tubes and monitoring equipment to the cat. Connect the earphones to the earphone cable assembly attached to the penetration panel.</li>
	<li>Start the ketamine infusion at the base flow rate of 0.6 mg/kg/hr then increase the flow rate as required based on the depth of anesthesia. Set the initial isoflurane dose to 0.5% then decrease to 0.4% once the anatomical scans have been collected.</li>
	<li>Monitor and record the cat&#39;s blood oxygen saturation, end-tidal CO2 levels, heart rate, respiration and rectal temperature (if possible) throughout the experiment using MRI-compatible monitoring equipment positioned at an appropriate distance from the scanner bore. Table 1 lists the mean values and ranges of the physiological measurements for successful execution of this procedure. Steady increases in heart rate and respiration are usually associated with imminent recovery from anesthesia.</li>
	<li>After the session is completed, remove the cat from the sled. Continue to provide supplemental heating with heating pads and towels until the animal fully recovers. Once the gag reflex returns, remove the endotracheal tube. Monitor the cat until the righting reflex is restored then return the animal to the facility. Assess the animal the day after the procedure to ensure no adverse effects from the experiment.</li>
</ol>
3. Brain Imaging
<ol>
	<li>Collect anatomical scans of the cat&#39;s brain in an axial slice orientation. Use the following imaging parameters for the anatomical reference volume: FLASH imaging sequence with TR = 750 msec, TE = 8 msec, matrix = 256 x 256, acquisition voxel size = 281 &#956;m x 281 &#956;m x 1.0 mm. The duration of the anatomical scan is approximately 6 min. Figure 4 (left panel) provides a sample anatomical image slice obtained using the specified parameters.</li>
	<li>Use the following imaging parameters for the functional volumes: segmented interleaved echo-planar acquisition (EPI) with TR = 1,000 msec, TE = 15 msec, 3 segments/plane, 21 x 1 mm slices; matrix = 96 x 96; field-of-view = 72 mm x 72 mm; acquisition voxel size = 0.75 mm x 0.75 mm x 1.0 mm; acquisition time = 3 sec/volume. Figure 4 (right panel) provides a sample functional image slice obtained using the specified parameters.</li>
</ol>
4. Stimulus Presentation
<ol>
	<li>Present a broadband white noise stimulus (0-25 kHz, 100 msec bursts with 5 msec rise/fall time, 1 presentation every 200 msec, 90-100 dB SPL) in a block design in which the auditory stimulus is played for 30 sec and alternated with a 30 sec baseline (no-stimulus) condition (Figure 5). Repeat this step until acoustically-evoked BOLD activity is observed in the auditory cortex. The duration of each functional run using a block design is approximately 4.5 min for 90 volumes.</li>
	<li>Present the stimulus in the appropriate block design configuration for the desired number of functional runs.</li>
</ol>
5. Data Analysis
<ol>
	<li>Select appropriate fMRI analysis software (e.g. SPM, FSL) to process the acquired functional volumes.</li>
	<li>Realign each functional volume to the volume acquired closest in time to the reference anatomical scan. Save the resulting motion correction values for use in step 5.6. Exclude any functional runs in which rotational head movements exceed 1&#176; or translational head movements exceed 1 mm.</li>
	<li>Coregister each volume to the reference anatomical scan.</li>
	<li>Smooth each volume with a 2 mm full-width half-maximum (FWHM) Gaussian filter.</li>
	<li>Incorporate a square-wave (boxcar) function which corresponds to the ON-OFF stimulus block design as a regressor to the general linear model (GLM).</li>
	<li>Incorporate motion correction values as regressors of no interest to account for movement-related artifacts. Apply an uncorrected statistical threshold of p = 0.001 to the GLM results to view clusters of BOLD activation. Determine the size of the smallest cluster which meets a corrected (family-wise error: FWE) threshold of p &#60; 0.05 at the cluster level. Set the cluster extent threshold to this value to view statistically significant clusters in regions of interest.</li>
	<li>Define the BOLD percent signal change (PSC) at each voxel as the difference between the mean BOLD signal during the stimulation blocks and the mean BOLD signal during the baseline blocks.</li>
</ol>

The authors declare no conflicts of interest, financial or otherwise.

In designing an fMRI experiment for an anesthetized animal model of auditory function, the following issues should be given careful consideration: (i) the impact of anesthesia on cortical responses, (ii) the effect of background scanner noise, and (iii) the optimization of the data collection phase of the experimental procedure.
While an anesthetized preparation offers the important advantage of producing a prolonged period of sedation and minimizing potential head motion during a functional i...

Current understanding of auditory processing in mammals is mainly derived from invasive electrophysiological studies in monkeys1-5, ferrets6-10, bats11-14, rodents15-19, and cats20-24. Electrophysiological techniques commonly utilize extracellular microelectrodes to record the activity of single and multiple neurons within a small area of neural tissue surrounding the electrode tip. Established functional imaging methods, such as optical imaging and functional magnetic resonance imaging (fMRI), serve as useful complements to extracellular recordings by providing a macroscopic perspective of simultaneous driven activity across multiple, spatially distinct regions of the brain. Intrinsic signal optical imaging facilitates visualization of evoked activity in the brain by measuring activity-related changes in the reflectance properties of surface tissue while fMRI utilizes the blood-oxygen level-dependent (BOLD) contrast to measure stimulus-evoked hemodynamic changes in brain regions which are active during a particular task. Optical imaging requires direct exposure of the cortical surface to measures changes in surface tissue reflectance that are related to stimulus-evoked activity25. In comparison, fMRI is noninvasive and exploits the paramagnetic properties of deoxygenated blood to measure both cortical surface26-28 and sulcus-based27,29 evoked activity within an intact skull. Strong correlations between the BOLD signal and neuronal activity in nonhuman primate visual cortex30 and in human auditory cortex31 validate fMRI as a useful tool to study sensory function. Since fMRI has been used extensively to study features of the auditory pathway such as tonotopic organization32-36, lateralization of auditory function37, patterns of cortical activation, identification of cortical regions38, effects of sound intensity on auditory response properties39,40, and characteristics of the BOLD response time course29,41 in human, monkey, and rat models, the development of a suitable functional imaging protocol to study auditory function in the cat would provide a useful complement to the functional imaging literature. While fMRI has also been used to explore various functional aspects of the visual cortex in the anesthetized cat26-28,42, few studies have used this technique to examine sensory processing in cat auditory cortex. The purpose of the present protocol is to establish an effective method of using fMRI to quantify function in the auditory cortex of the anesthetized cat. The experimental procedures outlined in this manuscript have been successfully used to describe the features of the BOLD response time course in the adult cat auditory cortex43.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>Atropine sulphate injection 0.5 mg/mL</td>
 <td>Rafter 8 Products</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Acepromazine 5 mg/mL</td>
 <td>Vetoquinol Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine hydrochloride 100 mg/mL</td>
 <td>Bimeda-MTC</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Dexmedetomidine hydrochloride (Dexdomitor 0.5 mg/mL)</td>
 <td>Orion Pharma</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Isoflurane 99.9%</td>
 <td>Abbott Laboratories</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lidocaine (Xylocaine endotracheal 10 mg/metered dose)</td>
 <td>Astra Zeneca</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lubricating opthalmic ointment (Refresh Lacri Lube)</td>
 <td>Allergan Inc.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Saline 0.95%</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>IV Catheter 22g (wings)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>IV Extension Set</td>
 <td>Codan US Corp.</td>
 <td>BC 269</td>
 <td></td>
 </tr>
 <tr>
 <td>IV Administration Set 10 drips/mL</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Endotracheal tube 4.0</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Heating pads (Snuggle Safe)</td>
 <td>Lenric C21 Ltd.</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe 60 mL</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>External sound card</td>
 <td>Roland Corporation</td>
 <td>Cakewalk UA-25EX</td>
 <td></td>
 </tr>
 <tr>
 <td>Stereo power amplifier</td>
 <td>Pyle Audio Inc.</td>
 <td>Pyle Pro PCAU11</td>
 <td></td>
 </tr>
 <tr>
 <td>MRI-compatible insert earphone system</td>
 <td>Sensimetric Corporation</td>
 <td>Model S14</td>
 <td></td>
 </tr>
 <tr>
 <td>Foam ear tips for insert earphones</td>
 <td>E-A-R Auditory Systems</td>
 <td>Earlink 3B</td>
 <td></td>
 </tr>
 <tr>
 <td>End-tidal CO2 monitor</td>
 <td>Nellcor&nbsp;</td>
 <td>N-85</td>
 <td></td>
 </tr>
 <tr>
 <td>MRI-compatible pulse oximeter</td>
 <td>Nonin Medical Inc.</td>
 <td>Model 7500</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe pump</td>
 <td>Harvard Apparatus</td>
 <td>70-2208</td>
 <td></td>
 </tr>
 </tbody>
 </table>

functional imaging of auditory cortex in adult cats using high-field fmri

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal models, including monkeys, ferrets, bats, rodents, and cats. In order to draw suitable parallels between human and animal models of auditory function, it is important to establish a bridge between human functional imaging studies and animal electrophysiological studies. Functional magnetic resonance imaging (fMRI) is an established, minimally invasive method of measuring broad patterns of hemodynamic activity across different regions of the cerebral cortex. This technique is widely used to probe sensory function in the human brain, is a useful tool in linking studies of auditory processing in both humans and animals and has been successfully used to investigate auditory function in monkeys and rodents. The following protocol describes an experimental procedure for investigating auditory function in anesthetized adult cats by measuring stimulus-evoked hemodynamic changes in auditory cortex using fMRI. This method facilitates comparison of the hemodynamic responses across different models of auditory function thus leading to a better understanding of species-independent features of the mammalian auditory cortex.

Representative functional data were acquired in a 7T horizontal bore scanner and analyzed using the Statistical Parametric Mapping toolbox in MATLAB. Robust cortical hemodynamic responses to auditory stimulation have consistently been observed in cats using the described experimental protocol43. Figure 6 illustrates the BOLD activation in 2 animals in response to a 30 sec broadband noise stimulus presented in a block design. T-statistic maps of the broadband noise vs. baseline (no stimulus) co...

Watch this Scientific Journal Video about Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI at JoVE.com

Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Current knowledge of sensory processing in the mammalian auditory system is mainly derived from electrophysiological studies in a variety of animal ...

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11.4K Views.  University of Western Ontario. Functional studies of the auditory system in mammals have traditionally been conducted using spatially-focused techniques such as electrophysiological recordings. The following protocol describes a method of visualizing large-scale patterns of evoked hemodynamic activity in the cat auditory cortex using functional magnetic resonance imaging.

Video: Functional Imaging of Auditory Cortex in Adult Cats using High-field fMRI

Chronic Imaging of Mouse Visual Cortex Using a Thinned-skull Preparation

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The technique presented here allows the imaging of the same area of the brain at several time points (chronic imaging) with microscopic resolution allowing the tracking of dendritic spines which are the small structures that represent the majority of postsynaptic excitatory sites in the CNS. The ability to clearly resolve fine cortical structures over several time points has many advantages, specifically in the study of brain plasticity in which morphological changes at synapses and circuit remodeling may help explain underlying mechanisms. In this video and supplementary material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation. The thinned-skull preparation is a minimally invasive approach, which avoids potential damage to the dura and/or cortex, thus reducing the onset of an inflammatory response. When this protocol is performed correctly, it is possible to clearly monitor changes in dendritic spine characteristics in the intact brain over a prolonged period of time.

In this video and supplemental material, we show a protocol for chronic in vivo imaging of the intact brain using a thinned-skull preparation.

In vivo imaging using two-photon laser scanning microscopy (2PLSM) allows the study of living cells and neuronal processes in the intact brain. The ...

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Co-analysis of Brain Structure and Function using fMRI and Diffusion-weighted Imaging

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when studying the brain, as the functional role that a brain area fulfills may be largely defined by its connections to other brain areas. In this report, we describe a novel, non-invasive approach for relating brain structure and function using magnetic resonance imaging (MRI). This approach, a combination of structural imaging of long-range fiber connections and functional imaging data, is illustrated in two distinct cognitive domains, visual attention and face perception. Structural imaging is performed with diffusion-weighted imaging (DWI) and fiber tractography, which track the diffusion of water molecules along white-matter fiber tracts in the brain (Figure 1). By visualizing these fiber tracts, we are able to investigate the long-range connective architecture of the brain. The results compare favorably with one of the most widely-used techniques in DWI, diffusion tensor imaging (DTI). DTI is unable to resolve complex configurations of fiber tracts, limiting its utility for constructing detailed, anatomically-informed models of brain function. In contrast, our analyses reproduce known neuroanatomy with precision and accuracy. This advantage is partly due to data acquisition procedures: while many DTI protocols measure diffusion in a small number of directions (e.g., 6 or 12), we employ a diffusion spectrum imaging (DSI)1, 2 protocol which assesses diffusion in 257 directions and at a range of magnetic gradient strengths. Moreover, DSI data allow us to use more sophisticated methods for reconstructing acquired data. In two experiments (visual attention and face perception), tractography reveals that co-active areas of the human brain are anatomically connected, supporting extant hypotheses that they form functional networks. DWI allows us to create a "circuit diagram" and reproduce it on an individual-subject basis, for the purpose of monitoring task-relevant brain activity in networks of interest.

We describe a novel approach for simultaneous analysis of brain function and structure using magnetic resonance imaging (MRI). We assess brain structure with high-resolution diffusion-weighted imaging and white-matter fiber tractography. Unlike standard structural MRI, these techniques allow us to directly relate anatomical connectivity to functional properties of brain networks.

The study of complex computational systems is facilitated by network maps, such as circuit diagrams. Such mapping is particularly informative when ...

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 ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Real-Time fMRI Brain Mapping in Animals

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a real-time fMRI platform to guide experimenters to monitor fMRI responses instantaneously during acquisition, which can be used to modify the physiology of animals to achieve desired hemodynamic responses in animal brains. The real-time fMRI set-up is based on a 14.1T preclinical MRI system, enabling the real-time mapping of dynamic fMRI responses in the primary forepaw somatosensory cortex (FP-S1) of anesthetized rats. Instead of a retrospective analysis to investigate confounding sources leading to the variability of fMRI signals, the real-time fMRI platform provides a more effective scheme to identify dynamic fMRI responses using customized macro-functions and a common neuroimage analysis software in the MRI system. Also, it provides immediate troubleshooting feasibility and a real-time biofeedback stimulation paradigm for brain functional studies in animals.

Animal brain functional mapping can benefit from the real-time functional magnetic resonance imaging (fMRI) experimental set-up. Using the latest software implemented in the animal MRI system, we established a real-time monitoring platform for small animal fMRI.

Dynamic fMRI responses vary largely according to the physiological conditions of animals either under anesthesia or in awake states. We developed a ...

Whole-Brain 3D Activation and Functional Connectivity Mapping in Mice using Transcranial Functional Ultrasound Imaging

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ultrafast doppler angiography. As brain perfusion is strongly linked to local neuronal activity, this technique allows the whole-brain 3D mapping of task-induced regional activation as well as resting-state functional connectivity, non-invasively, with unmatched spatio-temporal resolution and operational simplicity. In comparison with fMRI (functional magnetic resonance imaging), a main advantage of fUS imaging consists in enabling a complete compatibility with awake and behaving animal experiments. Moreover, fMRI brain mapping in mice, the most used preclinical model in Neuroscience, remains technically challenging due to the small size of the brain and the difficulty to maintain stable physiological conditions. Here we present a simple, reliable and robust protocol for whole-brain fUS imaging in anesthetized and awake mice using an off-the-shelf commercial fUS system with a motorized linear transducer, yielding significant cortical activation following sensory stimulation as well as reproducible 3D functional connectivity pattern for network identification.

This protocol describes the quantification of volumetric cerebral hemodynamic variations in the mouse brain using functional ultrasound (fUS). Procedures for 3D functional activation map following sensory stimulation as well as resting-state functional connectivity are provided as illustrative examples, in anesthetized and awake mice.

Functional ultrasound (fUS) imaging is a novel brain imaging modality that relies on the high-sensitivity measure of the cerebral blood volume achieved by ...