Name: a dual tracer pet-mri protocol for the quantitative measure of regional brain energy substrates uptake in the rat
Uploaded: 2013-12-28T14:00:00
Description: 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

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
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	<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 ...

<ol>
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L., 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=Abnormal+in+vivo+myocardial+energy+substrate+uptake+in+diet-induced+type+2+diabetic+cardiomyopathy+in+rats.">Abnormal in vivo myocardial energy substrate uptake in diet-induced type 2 diabetic cardiomyopathy in rats.</a> Am. J. Physiol. Endocrinol. Metab. 298, 1049-1057 (2010).</li><li>Liistro, T., 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=Brain+glucose+overexposure+and+lack+of+acute+metabolic+flexibility+in+obesity+and+type+2+diabetes:+a+PET-[18F]FDG+study+in+Zucker+and+ZDF+rats.">Brain glucose overexposure and lack of acute metabolic flexibility in obesity and type 2 diabetes: a PET-[18F]FDG study in Zucker and ZDF rats.</a> J. Cereb. Blood Flow Metab. 30, 895-899 (2010).</li><li>Croteau, E., 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=Image-derived+input+function+in+dynamic+human+PET/CT:+methodology+and+validation+with+11C-acetate+and+18F-fluorothioheptadecanoic+acid+in+muscle+and+18F-fluorodeoxyglucose+in+brain.">Image-derived input function in dynamic human PET/CT: methodology and validation with 11C-acetate and 18F-fluorothioheptadecanoic acid in muscle and 18F-fluorodeoxyglucose in brain.</a> Eur. J. Nucl. Med. Mol. Imaging. 37, 1539-1550 (2010).</li><li>Bergeron, M., Cadorette, J., Beaudoin, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+evaluation+of+the+LabPETTM+APD-based+digital+PET+scanner.">Performance evaluation of the LabPETTM APD-based digital PET scanner.</a> IEEE Trans. Nucl. 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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 height="21" style="height:21px;width:227px;">MRI scanner</td>
			<td style="width:207px;">Varian</td>
			<td style="width:181px;"></td>
			<td style="width:224px;">7 Tesla</td>
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			<td height="21" style="height:21px;">Small-animal PET scanner</td>
			<td>Gamma Medica</td>
			<td></td>
			<td>Lab-PET/-Triumph</td>
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			<td height="21" style="height:21px;">Heat mat</td>
			<td>Sunbeam</td>
			<td>PN 143937</td>
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			<td height="21" style="height:21px;">Heater system</td>
			<td>SA Instruments</td>
			<td>761 100 Rev B</td>
			<td></td>
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			<td height="24" style="height:24px;">Respiratory gating</td>
			<td>SA Instruments</td>
			<td>SAII&#39;s P-resp</td>
			<td></td>
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			<td height="21" style="height:21px;">Clinical chemistry analyzer</td>
			<td>Siemens Healthcare Diagnosis</td>
			<td>&#160;765000.931</td>
			<td>Dimension Xpand Plus</td>
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			<td height="21" style="height:21px;">Polyethylene tubing 50&#160;</td>
			<td>Becton Dickinson</td>
			<td>427411</td>
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			<td>KD Scientific</td>
			<td>Model 210</td>
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			<td height="21" style="height:21px;">Gamma-counter</td>
			<td>GMI</td>
			<td>Packard Cobra II</td>
			<td></td>
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			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>75002416</td>
			<td>Heraeus Pico 21</td>
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			<td height="21" style="height:21px;">PMOD software</td>
			<td>PMOD Technologies</td>
			<td></td>
			<td>PMOD 3.2 version</td>
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			<td height="21" style="height:21px;">Geiger counter</td>
			<td>Fluke Biomedical&#160;</td>
			<td>ASM-990</td>
			<td>Advanced Survey Meter</td>
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			<td height="22" style="height:22px;">Reagent</td>
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			<td height="22" style="height:22px;width:227px;">Name of the Reagent</td>
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			<td height="21" style="height:21px;">Isoflurane</td>
			<td>Abbott Laboratories, Ltd</td>
			<td>B506</td>
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			<td height="21" style="height:21px;">0.9% NaCl solution</td>
			<td>Hospira</td>
			<td>4888010</td>
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			<td>Sandoz</td>
			<td>1004336</td>
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			<td height="21" style="height:21px;">Isopropenyl acetate</td>
			<td>Aldrich</td>
			<td>11778</td>
			<td>99%</td>
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			<td height="21" style="height:21px;">Methyllithium</td>
			<td>Aldrich</td>
			<td>197343</td>
			<td>1.6 M</td>
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			<td height="21" style="height:21px;">THF</td>
			<td>Aldrich</td>
			<td>87371</td>
			<td></td>
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			<td height="21" style="height:21px;">Flex reagent cartridge glucose</td>
			<td>Siemens Healthcare Diagnosis</td>
			<td>DF40</td>
			<td></td>
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			<td height="21" style="height:21px;">Trizma base</td>
			<td>Sigma</td>
			<td>T6066-500G</td>
			<td>prepare tris buffer 100 mM pH 7.0</td>
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			<td height="21" style="height:21px;">Sodium oxamate</td>
			<td>Sigma</td>
			<td>O2751</td>
			<td>20 mM</td>
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		<tr height="21">
			<td height="21" style="height:21px;">NADH</td>
			<td>Roche</td>
			<td>10128015001</td>
			<td>0.15 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-Hydroxybutyrate dehydrogenase</td>
			<td>Toyobo</td>
			<td>HBD-301</td>
			<td>1 U/ml</td>
		</tr>
	</tbody>
</table>

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

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

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  ...

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.