This study was financially supported by the Fonds de la recherche en sant&#233; du Qu&#233;bec, Canadian Institutes of Health Research, Canadian Foundation for Innovation and the Canada Research Chairs Secretariat (SCC). The Sherbrooke Molecular Imaging Center is part of the FRQS-funded &#201;tienne-Le Bel Clinical Research Center. The authors thank M&#233;lanie Fortier, Jennifer Tremblay-Mercier, Alexandre Courchesne-Loyer, Dr. Fabien Pifferi, Dr. M&#39;hamed Bentourkia, Dr. Otman Sarrhini, Dr. Jacques Rousseau, Caroline Mathieu, and M&#233;lanie Archambault for generous support and technical assistance. The authors would like to thank the image analysis and visualization platform (http://pavi.dinf.usherbrooke.ca) for their help.

All experiments were completed in accordance with the Animal Care and Use Committee at the Universit&#233; de Sherbrooke and with the Canadian Council on Animal Care. The experimental protocol&#160;was approved by the Institutional Animal Research Ethics Review Board (protocol #011-09).
1. Brain Anatomy with MRI
<ol>
	<li>Let rats acclimatize in the animal facility for a minimum of 7 days prior to the protocol. Perform brain MRI scans 1-2 weeks prior to the dual tracer PET protocol to allow full recovery from the anesthesia.</li>
	<li>Anesthetize the rat in an induction chamber. Use 2% isoflurane and 1.5 L/min oxygen throughout the protocol for anesthesia. Pinch the hind leg to ensure the animal is fully anesthetized. CAUTION: inhalation of isoflurane can cause headache, dizziness, or unconsciousness in some cases; it should always be used in the presence of an air exchange system in a suitably ventilated room.</li>
	<li>Position the rat on the MRI examination table in the head-first prone position with a nose cone for isoflurane. Position the respiration and rectal probes. Monitor the respiration rate during the experiment and maintain body temperature at 37 &#176;C with an automated air warming system.</li>
	<li>Acquire T2-weighted MR images using a fast spin-echo pulse sequence with acquisition parameters as detailed previously11. Place the rat on a heated mat during recovery from anesthesia.</li>
</ol>
2. Dual Tracer PET Acquisitions
<ol>
	<li>Use a small-animal PET scanner equipped with avalanche photodiode detectors, an axial field of view of ~7.5 cm and an isotropic spatial resolution of 1.2 mm17. Due to its short physical half-life, prepare&#160;11C by proton bombardment of natural nitrogen (through the 14N(&#961;,&#945;)11C nuclear reaction) in a cyclotron on-site before each experiment.</li>
	<li>Fast the rat for 18 hr prior to PET scanning. Fasting allows higher brain uptake of 11C-AcAc and 18F-FDG, thereby improving signal-to-noise ratio.</li>
	<li>Anesthetize the rat in an induction chamber. Transfer the rat onto a heating mat with a nose cone for isoflurane. Start 11C-AcAc synthesis at this time. The synthesis takes 18 min from the end of bombardment, as previously described1.</li>
	<li>Position the rat on its side. Prepare a PE50 polyethylene catheter filled with heparinized 0.9% sodium chloride solution (saline). Install the catheter in the tail vein for tracer injection. Install a second catheter in the mid-ventral tail artery for blood sampling throughout the acquisitions.</li>
	<li>Rapidly transfer the rat to the scanner table in the head-first prone position with a nose cone for isoflurane. Position the respiration and rectal probes. Monitor the respiration rate during the experiment and maintain body temperature at 37 &#176;C with an automated warming air system. Move the scanner table forward into the scanner to ensure the appropriate imaging of the brain and the heart simultaneously. As an anatomical landmark, position the edge of the field of view on the rat&#39;s eyes (with the help of laser lines).</li>
	<li>Using a concentrated 11C-AcAc solution (~1 GBq/ml), prepare a syringe of ~50 MBq of radioactivity (a range of 45-55 MBq is acceptable). Adjust the volume to 300 &#956;l with saline. CAUTION: Radioactivity can be harmful for the health. It should always be handled behind a lead shield. The experimenter should be wearing a body and ring dosimeter. A Geiger counter should also be on during the experiment.</li>
	<li>Install the syringe on an injection pump. Start the bolus injection of 11C-AcAc at a rate of 1 ml/min (injection duration: ~19 sec). On a second pump, immediately after terminating the 11C-AcAc injection, start the injection of 300 &#956;l of saline at a rate of 1 ml/min, which is important for an optimal injection of tracer into the blood circulation.</li>
	<li>Start a dynamic PET data acquisition 30 sec before starting the bolus injection for a total duration of 20.5 min. The 30 sec data acquisition before the tracer injection provides a measure of the ambient background to be subsequently subtracted from the PET data. Set the regular sampling mode and the energy window at 250-650 keV.</li>
	<li>Collect two 200 &#956;l blood samples at ~15 and 18 min after starting the 11C-AcAc injection. Inject heparinized saline in the catheter after each sampling to avoid blood clotting. Take note of the time at the beginning and the end of blood sampling and use the mean time. Centrifuge at 6,000 RPM for 5 min and collect plasma. Measure radioactivity counts in plasma using a gamma counter calibrated with the PET scanner.</li>
	<li>After the 11C-AcAc scan, allow a waiting period of 20 min to ensure that most of radioactivity has decayed. The period can vary from 15-30 min. Do not move the scanner bed and leave the rat under isoflurane throughout this period.</li>
	<li>During this time, using a concentrated 18F-FDG solution (~5.5 GBq/&#956;l), prepare a syringe of ~50 MBq. Proceed exactly as mentioned in steps 2.6 and 2.7. Start a dynamic acquisition of a total duration of 40.5 min, including the 30 sec before the injection. Collect two 200 &#956;l blood samples at ~30 and 35 min after starting the 18F-FDG injection.</li>
	<li>At the end of the 18F-FDG acquisition, take one final 200 &#956;l blood sample. Centrifuge and collect plasma. Keep plasma samples at -80 &#176;C for glucose and AcAc analysis.</li>
	<li>Finally, place the rat on a heating mat during the waking period. Keep the animal for a longitudinal follow-up, or alternatively, euthanize the rat for brain sampling and further biochemical studies.</li>
	<li>Reconstruct the PET images according to the following time frame sequences: 1 &#215; 30 sec; 12 &#215; 5 sec; 8 &#215; 30 sec; and n &#215; 300 sec, where n = 3 for 11C-AcAc acquisition and n = 7 for 18F-FDG acquisition.</li>
</ol>
3. Plasma Glucose and AcAc Analysis
<ol>
	<li>Measure plasma glucose and AcAc with a clinical chemistry analyzer. Perform assays within 48 hr of the blood sampling to minimize AcAc decarboxylation into acetone. Measure glucose concentration with DF40 kit as previously described18 and AcAc with an open channel2. This type of analyzer is more accurate than strips (0.1 &#956;M) and has a broad detection window.</li>
</ol>
4. Quantitative PET Analysis
<ol>
	<li>Use PMOD&#160;software or an equivalent system for small-animal PET image analysis.</li>
	<li>Plasma time-activity curve (TAC)
	<ol>
		<li>Load&#160;18F-FDG data. Sum image frames of the first 60 sec following injection (when tracer is mainly in blood).</li>
		<li>Using a manual drawing tool, draw a volume of interest (VOI) on the left ventricular cavity blood pool (a large pool centered in the bottom part of images). Draw the VOI &#160;~1 mm inside of the edge of the blood pool (red voxels) to ensure no inclusion of tissue and avoid tissue radioactivity spill into the blood pool (Figure 1A). Copy the VOI to the entire dynamic image series and generate a curve of radioactivity as a function of time (TAC).</li>
		<li>In a spreadsheet,&#160;use radioactivity counts of the two plasma samples taken during the acquisition to correct plasma TAC (Figure 1B). Use the following equation: 
		<img alt="" fo:content-width="5in" src="/files/ftp_upload/50761/50761eq1.jpg" width="500" /> 
		Some smoothing of the plasma TAC can be performed.</li>
		<li>Verify if residual radioactivity from 11C-AcAc is present in the first 30 sec of 18F-FDG scan, prior to injection. If so, subtract the following factor from all the subsequent time frames of the TAC: 
		<img alt="" fo:content-width="1.5in" src="/files/ftp_upload/50761/50761eq2.jpg" width="150" />, 
		where R is the residual radioactivity, t1/2&#160;is the half-life of 11C (20.4 min) and T is the time frame2,13. Residual radioactivity from 11C-AcAc after a waiting period of 20 min can be up to 1.5% of maximal 18F-FDG counts.</li>
	</ol>
	</li>
	<li>18F-FDG/MRI coregistration
	<ol>
		<li>Apply coregistration obtained with 18F-FDG images to the11C-AcAc images. This is necessary because&#160;11C-AcAc images have a low signal-to-noise ratio and automatic coregistration is difficult.</li>
		<li>Load the corresponding MRI data. Load all 28&#160;18F-FDG time frames in the same orientation as the MR images (on three axes).</li>
		<li>Compute the 18F-FDG summed image of the 28 time frames. This generates an image with higher counts, so it is easier to work with for coregistration with MRI than individual image frames.</li>
		<li>Perform the automatic coregistration of the&#160;18F-FDG summed image on MR images (Figure 2). Apply the transformation to all individual&#160;18F-FDG image frames. Save the transformation which will later be applied to 11C-AcAc images.</li>
	</ol>
	</li>
	<li>Volumes of interest (VOIs)
	<ol>
		<li>Using segmentation software such as PMOD, select MRI data and choose preferred plane for manual drawing. Choose the manual or the semi-automatic drawing tool, depending on brain region size and image contrast. Locate brain structures according to a standard rat brain atlas19.</li>
		<li>Draw the VOIs. As shown in Figure 3, segment the whole brain, cortex, hippocampus, striatum and cerebellum, but other regions could be chosen. Save the VOIs, which will be applied to the coregistered&#160;18F-FDG and&#160;11C-AcAc images.</li>
		<li>Apply VOIs to 18F-FDG coregistered images. Select the VOI statistics to visualize brain TAC. If needed, correct TAC by subtracting the decay-corrected mean radioactivity in the first 30 sec from all the subsequent time frames.</li>
	</ol>
	</li>
	<li>Cerebral metabolic rate calculation
	<ol>
		<li>Load brain and the corresponding plasma TACs.</li>
		<li>Select Patlak plot kinetic model20,21. Set the lumped constant to 0.4822 and the corresponding plasma glucose concentration (Figure 4B). Set the maximal relative deviation from the Patlak plot to 5%. Fit the data to the model.</li>
	</ol>
	</li>
	<li>11C-AcAc images
	<ol>
		<li>Compute the 18F-FDG summed image of the 24 first time frames to have the same number of frames as the 11C-AcAc scan. Otherwise, 11C-AcAc/MRI coregistration fails. Perform the automatic coregistration of 18F-FDG on MRI. Save the transformation process which will be used for 11C-AcAc image coregistration.</li>
		<li>Repeat steps 4.2 and 4.3 for 11C-AcAc images. Use 18F-FDG transformation in step 4.6.1 and apply it to all individual 11C-AcAc image frames. Repeat steps 4.4 and 4.5 and use 18F-FDG saved VOIs. Set the lumped constant to 1.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Critical steps
A critical step in this dual tracer PET protocol is to be able to simultaneously scan the heart&#39;s left ventricle and the brain at the same time. This requires a PET scanner with a sufficient axial length, i.e. a minimum of 7.5 cm. A few test scans are needed to determine the exact position of the scanner table (x, y, and z&#160;values), where the brain and the heart are scanned correctly.
Tracer injection is also a crucial point for a succes...

Context and Rationale
Positron emission tomography (PET) enables the minimally-invasive study of functional processes in the brain. Glucose is the brain&#39;s main energy substrate, but in conditions of glucose deficiency, ketones (acetoacetate [AcAc] and &#946;-hydroxybutyrate) are the main alternative energy substrates. Brain energy metabolism has been widely studied by PET using the most common PET tracer,&#160;18F-fluorodeoxyglucose (18F-FDG), a glucose analog. Our group recently developed a novel radiotracer -11C-AcAc - to measure brain ketone metabolism1. Magnetic resonance imaging (MRI) is a much higher resolution technique (0.1 mm &#215; 0.1 mm in-plane resolution) than PET, and is needed to clearly localize anatomical brain regions required for the regional PET analysis of brain energy metabolism.
PET data are commonly expressed as standardized uptake values (SUV)2-6. SUV are the tissue activity concentration normalized by the fraction of the injected dose/unit weight, as initially proposed over 70 years ago7. These units are still widely used because they require simpler PET acquisition and image analysis methodologies. However, an important limitation is that SUV are relative not absolute units, making it difficult to compare results across different studies. This difficulty of comparison may contribute to contradictory findings in the literature on brain glucose uptake in the elderly8. &#160;Therefore, the quantitative cerebral metabolic rate (CMR; &#956;mol/100 g/min) has particular advantages9-11. Generating CMR values requires a dynamic PET acquisition and the plasma radioactivity counts as a function of time, i.e. the plasma time-activity curve (TAC) or input function. The input function can be obtained by multiple blood samplings throughout the PET acquisition9,12&#160;or by the image-derived input function (IDIF) method, in which a region of interest is drawn on a blood pool (heart&#39;s left ventricle or major artery)11,13-16.
Goal
The aim of our method was to quantitatively compare for the first time the uptake of the brain&#39;s two key energy substrates, glucose and ketones, using PET and MRI in rodents. 11C-AcAc and 18F-FDG were used sequentially in the same animal. The protocol was designed to measure regional uptakes in different relevant brain structures (cortex, hippocampus, striatum, and cerebellum) clearly visible in the MR images. The protocol was also specifically intended to permit quantitative analysis of tracer brain uptake, i.e. CMR of both 18F-FDG and 11C-AcAc. Although this protocol was developed to study brain energy substrates, the radiotracers we used could be replaced by others, and the same methodology can be used to study different brain functional processes.
Advantages over existing methods
PET and MRI do not require the animal to be sacrificed after the acquisition. Therefore, follow-up studies of treatments are possible. Thus, baseline data followed by an experimental condition can be measured within the same animal, thereby reducing both biological variability and the number of animals required. A key advantage of our dual tracer PET protocol is to compare both tracers uptake in the same animal under the same physiological conditions within the same imaging session, thereby reducing even more biological variability and systematic discrepancies. This dual tracer PET protocol is feasible primarily because of the short physical half-life of 11C (20.4 min) and the fast biological washout of 11C-AcAc, which leave minimal residual 11C radioactivity during the second acquisition with 18F-FDG. The MRI scan is an important feature of this protocol as it enables the tracer uptake to be studied in specific brain areas. In addition, this method enables an absolute quantitative measure of brain tracer uptake in contrast to relative units obtained by the SUV method. Finally, 11C-AcAc images have a low signal-to-noise ratio because of relatively low brain AcAc uptake under physiological conditions, which makes automatic&#160;11C-AcAc and MR images registration challenging. Hence, because 11C-AcAc and 18F-FDG acquisitions are sequential (no motion of the animal), the 18F-FDG to MRI alignment can be applied to 11C-AcAc images.
Key papers where the protocol has been used
We have used the dual tracer PET protocol in a study involving the comparison of the fasted state and the ketogenic diet (KD) in young rats2. We showed that both fasting and the KD increase significantly both 11C-AcAc and 18F-FDG brain uptake. However, these were not quantitative results as we did not use the dynamic PET imaging and tracer kinetic modeling methodology at that time. Thereafter, we undertook a regional and quantitative study of brain metabolism in aged rats, where the effect of aging and a KD were evaluated on brain 11C-AcAc and 18F-FDG uptake11. We also showed that the percentage of distribution across brain regions was different between 11C-AcAc and 18F-FDG. Furthermore, not only the CMR of 11C-AcAc but also that of 18F-FDG was increased in the whole brain as well as in the striatum of aged rats on the KD.

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			<td>PMOD Technologies</td>
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			<td>87371</td>
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			<td>Siemens Healthcare Diagnosis</td>
			<td>DF40</td>
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			<td height="21" style="height:21px;">Trizma base</td>
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			<td>prepare tris buffer 100 mM pH 7.0</td>
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			<td>Toyobo</td>
			<td>HBD-301</td>
			<td>1 U/ml</td>
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a dual tracer pet-mri protocol for the quantitative measure of regional brain energy substrates uptake in the rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

As seen in Figure 2,11C-AcAc uptake is low within the brain itself. As mentioned earlier, ketones consumption by the brain is very low on a short-term fasting.&#160;11C-AcAc uptake is higher in the tongue and cheek muscles. Indeed, ketones are rapidly taken up by rat skeletal muscles23. In contrast, 18F-FDG uptake is mostly in the brain and the cheek muscles. Figure 2 shows that during the coregistration process, MR images are fixed and PET ima...

Watch this Scientific Journal Video about A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat at JoVE.com

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

a-dual-tracer-pet-mri-protocol-for-quantitative-measure-regional

Research

JoVE Journal

Neuroscience

7.0K Views.  Université de Sherbrooke. Small-animal positron emission tomography enables the assessment of the brain's two main energy substrates: glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

Video: A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

Zebrafish are a proven model for vision research, however many of the earlier methods generally focused on larval fish or demonstrated a simple response. More recently adult visual behavior in zebrafish has become of interest, but methods to measure specific responses are new coming. To address this gap, we set out to develop a methodology to repeatedly and accurately utilize the optokinetic response (OKR) to measure visual acuity in adult zebrafish. Here we show that the adult zebrafish's visual acuity can be measured, including both binocular and monocular acuities. Because the fish is not harmed during the procedure, the visual acuity can be measured and compared over short or long periods of time. The visual acuity measurements described here can also be done quickly allowing for high throughput and for additional visual procedures if desired. This type of analysis is conducive to drug intervention studies or investigations of disease progression.

Zebrafish have been a valuable tool for many research areas. Here we demonstrate a method to elicit a visual response and calculate a functional visual acuity in the adult zebrafish.

Zebrafish are a proven model for vision research, however many of the  earlier methods generally focused on larval fish or demonstrated a simple  ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.