We are grateful to Austin Ferro and Juao-Guilherme Rosa in the Cvetanovic lab for their help in troubleshooting dissections and in RNA extraction and RTqPCR. This research is funded by M. Cvetanovic, R01 NS197387; HHS | National Institutes of Health (NIH).

1. Setup
<ol>
	<li>Gather necessary equipment including decapitation scissors, blunt forceps, dissection scissors, vascular scissors, microspatula, sagittal mouse brain matrix, razor blades, 200 &#181;L&#160;pipet tips, glass petri dish, glass slide, and ice bucket. Lay out all equipment on an absorbent pad.</li>
	<li>Place petri dish, glass plate, and brain matrix on ice.</li>
	<li>Using a razor blade at a perpendicular angle, trim about 5 mm off the tip of one 200 &#181;L&#160;pipet tip. This makes t...

<ol>
	<li>Herculano-Houzel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glia/neuron+ratio:+How+it+varies+uniformly+across+brain+structures+and+species+and+what+that+means+for+brain+physiology+and+evolution.">The glia/neuron ratio: How it varies uniformly across brain structures and species and what that means for brain physiology and evolution.</a> Glia. 62 (9), 1377-1391 (2014).</li><li>Schmahmann, J. D., Caplan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognition,+enotion,+and+the+cerebellum.">Cognition, enotion, and the cerebellum.</a> Brain. 129 (2), 290-292 (2006).</li><li>Badura, A., 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=Normal+cognitive+and+social+development+require+posterior+cerebellar+activity.">Normal cognitive and social development require posterior cerebellar activity.</a> eLife. 7, 36401 (2018).</li><li>Diedrichsen, J., King, M., Hernandez-Castillo, C., Sereno, M., Ivry, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+Transform+or+Multiple+Functionality?+Understanding+the+Contribution+of+the+Human+Cerebellum+across+Task+Domains.">Universal Transform or Multiple Functionality? Understanding the Contribution of the Human Cerebellum across Task Domains.</a> Neuron. 102 (5), 918-928 (2019).</li><li>Strick, P. L., Dum, R. P., Fiez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellum+and+Nonmotor+Function.">Cerebellum and Nonmotor Function.</a> Annual Review of Neuroscience. 32 (1), 413-434 (2009).</li><li>King, M., Hernandez-castillo, C. R., Poldrack, R. A., Ivry, R. B., Diedrichsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+boundaries+in+the+human+cerebellum+revealed+by+a+multi-domain+task+battery.">Functional boundaries in the human cerebellum revealed by a multi-domain task battery.</a> Nature Neuroscience. 22, 1371-1378 (2019).</li><li>Buckner, R. L., Krienen, F. M., Castellanos, A., Diaz, J. C., Yeo, B. T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+the+human+cerebellum+estimated+by+intrinsic+functional+connectivity.">The organization of the human cerebellum estimated by intrinsic functional connectivity.</a> Journal of neurophysiology. 106 (5), 2322-2345 (2011).</li><li>Schmahmann, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+movement+to+thought:+Anatomic+substrates+of+the+cerebellar+contribution+to+cognitive+processing.">From movement to thought: Anatomic substrates of the cerebellar contribution to cognitive processing.</a> Human Brain Mapping. 4 (3), 174-198 (1996).</li><li>Kelly, R. M., Strick, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+Loops+with+Motor+Cortex+and+Prefrontal+Cortex+of+a+Nonhuman+Primate.">Cerebellar Loops with Motor Cortex and Prefrontal Cortex of a Nonhuman Primate.</a> The Journal of Neuroscience. 23 (23), 8432-8444 (2003).</li><li>Stoodley, C. J., Macmore, J. P., Makris, N., Sherman, J. C., Schmahmann, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Location+of+lesion+determines+motor+vs.+cognitive+consequences+in+patients+with+cerebellar+stroke.">Clinical Location of lesion determines motor vs. cognitive consequences in patients with cerebellar stroke.</a> NeuroImage: Clinical. 12, 765-775 (2016).</li><li>Guo, C. C., Tan, R., Hodges, J. R., Hu, X., Sami, S., Hornberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network-selective+vulnerability+of+the+human+cerebellum+to+Alzheimer's+disease+and+frontotemporal+dementia.">Network-selective vulnerability of the human cerebellum to Alzheimer's disease and frontotemporal dementia.</a> Brain. 139 (5), (2016).</li><li>Bocchetta, M., Cardoso, M. J., Cash, D. M., Ourselin, S., Warren, J. D., Rohrer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+regional+cerebellar+atrophy+in+genetic+frontotemporal+dementia.">Patterns of regional cerebellar atrophy in genetic frontotemporal dementia.</a> NeuroImage: Clinical. 11, 287-290 (2016).</li><li>Sillitoe, R. V., Fu, Y., Watson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellum.">Cerebellum.</a> The Mouse Nervous System. , 360-397 (2012).</li><li>Nguyen-Minh, V. T., Tran-Anh, K., Luo, Y., Sugihara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+Excitability+and+Parallel+Fiber+Synaptic+Properties+of+Zebrin-Positive+and+-Negative+Purkinje+Cells+in+Lobule+VIII+of+the+Mouse+Cerebellar+Slice.">Electrophysiological Excitability and Parallel Fiber Synaptic Properties of Zebrin-Positive and -Negative Purkinje Cells in Lobule VIII of the Mouse Cerebellar Slice.</a> Frontiers in Cellular Neuroscience. 12, 513 (2019).</li><li>Manto, M., Oulad Ben Taib, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebellar+nuclei:+Key+Roles+for+Strategically+Located+Structures.">Cerebellar nuclei: Key Roles for Strategically Located Structures.</a> The Cerebellum. 9 (1), 17-21 (2010).</li><li>Driessen, T. M., Lee, P. J., Lim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathway+analysis+towards+understanding+tissue+vulnerability+in+spinocerebellar+ataxia+type+1.">Molecular pathway analysis towards understanding tissue vulnerability in spinocerebellar ataxia type 1.</a> eLife. , (2018).</li><li>Grabert, K., 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=Microglial+brain+region+-+dependent+diversity+and+selective+regional+sensitivities+to+aging.">Microglial brain region - dependent diversity and selective regional sensitivities to aging.</a> Nat Neurosci. 19 (3), 504 (2016).</li><li>Ingram, M., 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=Cerebellar+Transcriptome+Profiles+of+ATXN1+Transgenic+Mice+Reveal+SCA1+Disease+Progression+and+Protection+Pathways.">Cerebellar Transcriptome Profiles of ATXN1 Transgenic Mice Reveal SCA1 Disease Progression and Protection Pathways.</a> Neuron. 89 (6), 1194-1207 (2016).</li><li>Cendelin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> From mice to men : lessons from mutant ataxic mice. , 1-21 (2014).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+RNA+Using+TRIzol+(TRI+Reagent).">Purification of RNA Using TRIzol (TRI Reagent).</a> Cold Spring Harbor Protocols. 2010 (6), (2010).</li><li>Kim, J. H., Lukowicz, A., Qu, W., Johnson, A., Cvetanovic, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astroglia+contribute+to+the+pathogenesis+of+spinocerebellar+ataxia+Type+1+(SCA1)+in+a+biphasic,+stage-of-disease+specific+manner.">Astroglia contribute to the pathogenesis of spinocerebellar ataxia Type 1 (SCA1) in a biphasic, stage-of-disease specific manner.</a> Glia. 66 (9), 1972-1987 (2018).</li><li>Chopra, R., 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=Altered+Capicua+expression+drives+regional+Purkinje+neuron+vulnerability+through+ion+channel+gene+dysregulation+in+spinocerebellar+ataxia+type+1.">Altered Capicua expression drives regional Purkinje neuron vulnerability through ion channel gene dysregulation in spinocerebellar ataxia type 1.</a> Human Molecular Genetics. , (2020).</li></ol>

The method described here makes it possible to assess the underlying gene expression and molecular mechanisms within four distinct cerebellar regions &#8211; the deep cerebellar nuclei (DCN), the anterior cerebellar cortex of the vermis (CCaV), the posterior cerebellar cortex of the vermis (CCpV), and the cerebellar cortex of the hemispheres (CCH). The ability to assess these regions separately will expand our knowledge of the heterogeneity of specific cerebellar regions and possibly shed light on their contribution to v...

The cerebellum contains over half of the neurons in the brain and has historically been referred to as a motor control and balance center in the brain1. More recently, studies have demonstrated that the cerebellum plays a key role in various other functions including cognition, reward processing, and affect2,3,4,5.
The cerebellum has well-described anatomy: the cortex region is composed of granule, Purkinje, and molecular layers. Granule cells form the granule cell layer and send input via parallel fibers to the Purkinje cell dendrites of the molecular layer which also receive input from climbing fibers that originated in the inferior olive. Purkinje cells send inhibitory projections to cells in the deep cerebellar nuclei (DCN), which serves as the main output from the cerebellum. The output of this cerebellar circuit is further modulated by the activity of the inhibitory interneurons in the cerebellar cortex, including Golgi, stellate, and basket cells4. This cerebellar functional unit is distributed throughout all the lobules of the cerebellar cortex. Despite this relatively uniform circuitry across &#160;the cerebellum, evidence from human neuroimaging literature and patient studies indicates functional heterogeneity of the cerebellum6,7.
The cerebellar cortex can be divided into two main regions: the midline-defined vermis, and the lateral hemispheres. The vermis can be further divided into anterior and posterior lobules. These distinct regions of the cerebellum have been implicated in contributing to different behaviors. Task-evoked or task-free activity patterns implicated that anterior regions of the vermis contribute more to motor function while posterior vermis contributes more to cognition6,7. The vermis is also linked with affect and emotions, while cerebellar hemispheres contribute to executive, visual-spatial, language, and other mnemonic functions8. In addition, anatomical studies provided evidence that functionally distinct cerebellar regions are connected with different cortical regions9. Lesion-symptom mapping revealed that patients with strokes affecting the anterior lobules (extending into lobule VI) had poorer performance on fine motor tasks, while patients with damage to posterior lobe regions and hemispheres exhibited cognitive deficits in the absence of cerebellar motor syndrome10. Finally, regional cerebellar pathology in disease indicates that functionally distinct cerebellar regions are also differently susceptible to disease11,12.
While much less explored, preliminary evidence demonstrates distinct gene expression signatures across cerebellar cortical regions. Purkinje cell expression of Zebrin II shows region specific patterning in the vermis such that there are more Zebrin II positive cells in the posterior lobules and fewer in the anterior lobules13. This also correlates with regionally distinct physiological function as Zebrin II negative Purkinje cells display higher frequency of tonic firing than Purkinje cells that are Zebrin II positive14.
In addition to the cerebellar cortex, the cerebellum includes the deep cerebellar nuclei (DCN) which serve as the primary output for the cerebellum. The nuclei are made up of the medial (MN), interposed (IN), and lateral nuclei (LN). Functional imaging and patient studies have demonstrated that the DCN also participate in various behaviors15, but very few studies examine gene expression change in DCN.
Advances in molecular techniques have made it possible to assess regional gene expression in the brain and have uncovered heterogeneity across and within different brain regions in both physiological and disease states16. Such studies implicate that the cerebellum is different from other brain regions. For example, the ratio of neurons to glial cells is inverted in the cerebellum&#160; compared to other brain regions1. Even in normal physiological conditions, the expression of proinflammatory genes is upregulated in the cerebellum compared to the other brain regions17. Molecular techniques have also been very useful in identifying the pathways that contribute to the pathogenesis of cerebellar diseases. For instance, RNA sequencing of the whole cerebellar extracts identified genes altered in a Purkinje cell specific transgenic mouse model of spinocerebellar ataxia type 1 (SCA1) as compared to their wild type controls. Such evidence has revealed key molecular pathways underlying pathogenesis in cerebellar Purkinje cells and has helped identify potential therapeutic targets18. However, recent studies suggest that there are differences in the vulnerability to diseases across the cerebellar regions11,12,19. This could indicate that there are key changes occurring in distinct cerebellar regions, which may be masked or undetected with whole cerebellar extracts. Thus, there is a need to develop techniques which allow researchers to examine molecular profiles in different cerebellar regions.
The technique proposed here describes a reproducible method to dissect four distinct regions of the mouse cerebellum in order to isolate RNA from those regions and explore regional differences in gene expression. The schematic of the mouse cerebellum in Figure 1A highlights the vermis in blue, and hemispheres in yellow. Specifically, this technique makes it possible to isolate four regions:&#160; deep cerebellar nuclei (DCN) (red-dotted boxes in Figure 1A), the cerebellar cortex of anterior vermis (CCaV) (dark blue in Figure 1A), the cerebellar cortex of the posterior vermis (CCpV) (light blue in Figure 1A), and the cerebellar cortex of the hemispheres (CCH) (yellow in Figure 1A). By assessing gene expression of these regions separately, it will be possible to investigate molecular mechanisms underlying discrete functions of these different regions as well as potential differences in their vulnerability in disease.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >1.5 Microcentrifuge tubes</td>
			<td >ThermoScietific</td>
			<td >3456</td>
			<td ></td>
		</tr>
		<tr >
			<td >100% Isopropyl Alcohol</td>
			<td >VWR Life sciences</td>
			<td >1106C361</td>
			<td></td>
		</tr>
		<tr >
			<td >200 ul Pipet tips</td>
			<td >GeneMate</td>
			<td >P-1237-200</td>
			<td></td>
		</tr>
		<tr >
			<td >Adult Mouse Brain Matrix Sagittal</td>
			<td >Kent Scientific Corporation</td>
			<td >RBMA-200S</td>
			<td></td>
		</tr>
		<tr >
			<td >Blunt forceps</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Chloroform</td>
			<td >Macron</td>
			<td >220905</td>
			<td></td>
		</tr>
		<tr >
			<td >Decapitation Scissors</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Dissecting Scissors</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Ethyl Alcohol</td>
			<td >Pharmco</td>
			<td >111000200</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Slide (for electrophoresis)</td>
			<td >BIORAD</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Homogenizer</td>
			<td >Kimble</td>
			<td >6HAZ6</td>
			<td></td>
		</tr>
		<tr >
			<td >Ice Bucket</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Insulin Syringe (.5ml)</td>
			<td >BD</td>
			<td >329461</td>
			<td></td>
		</tr>
		<tr >
			<td >iScript Adv cDNA kit for RT-qPCR</td>
			<td >BIORAD</td>
			<td >1725037</td>
			<td></td>
		</tr>
		<tr >
			<td >Micro Spatula</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Needle Nose forceps</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri Dish</td>
			<td >Pyrex</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Primetime Primer for Aldolase C</td>
			<td >IDT</td>
			<td >Mm.PT.58&#62;43415246</td>
			<td></td>
		</tr>
		<tr >
			<td >Primetime Primer for Kcng4</td>
			<td >IDT</td>
			<td >Mm.PT.56a.9448518</td>
			<td></td>
		</tr>
		<tr >
			<td >Primetime Primer for Parvalbumin</td>
			<td >IDT</td>
			<td >Mm.PT.58.7596729</td>
			<td></td>
		</tr>
		<tr >
			<td >Primetime Primer Rps18&#160;</td>
			<td >IDT</td>
			<td >Mm.PT.58.12109666</td>
			<td></td>
		</tr>
		<tr >
			<td >Single Edge Rzor Blades</td>
			<td >Personna GEM</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Sterile, sigle-use pestles</td>
			<td >FisherScientific</td>
			<td >12141364</td>
			<td></td>
		</tr>
		<tr >
			<td >TRIzol Reagent</td>
			<td >Ambion by Life technologies</td>
			<td >15596018</td>
			<td></td>
		</tr>
		<tr >
			<td >Vascular Scissors</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

cerebellar regional dissection for molecular analysis

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies indicate that distinct cerebellar regions contribute to these different functions. Molecular studies examining regional cerebellar differences are lagging as they are mostly done on whole cerebellar extracts thereby masking any distinctions across specific cerebellar regions. Here we describe a technique to reproducibly and quickly dissect four different cerebellar regions: the deep cerebellar nuclei (DCN), anterior and posterior vermal cerebellar cortex, and the cerebellar cortex of the hemispheres. Dissecting out these distinct regions allows for the exploration of molecular mechanisms that may underlie their unique contributions to balance, movement, affect and cognition. This technique may also be used to explore differences in pathological susceptibility of these specific regions across various mouse disease models.

For these experiments, four eleven-week-old female wild type C57/Black6 mice were used. One mouse was used to conduct a full cerebellar dissection which is referred to as &#39;bulk cerebellum&#39; and allowed for the comparison of RNA levels in dissected regions to a full dissection. The other three mice were used to conduct the cerebellar dissection described in this protocol. Using three mice makes it possible to ensure that the trends detected in the levels of RNA are reproducible acro...

Watch this Scientific Journal Video about Cerebellar Regional Dissection for Molecular Analysis at JoVE.com

Cerebellar Regional Dissection for Molecular Analysis

Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene expression.&#160;

Cerebellum plays an important role in several key functions including control of movement, balance, cognition, reward, and affect. Imaging studies ...

Traditional molecular studies of the cerebellum have been done on whole cerebellar extracts, which may mask any distinctions across specific cerebellar regions. This protocol makes it possible to assess distinct regions of the cerebellum separately and allows for the exploration of molecular mechanisms that may underlie their unique contributions to a variety behaviors and disease progression. The main advantage of this technique is that it allows for the reproducible and quick dissection of four cerebellar regions, the deep cerebellar nuclei, the anterior and posterior cerebellar cortex of the Vermis and the cerebellar cortex of the hemispheres.After decapitating the euthanized mouse, make an incision with a razor blade along the medial sagittal line of the head, starting at the nose and continuing all the way back. Separate the skin and use the razor blade to cut away the muscle on each side, cutting down past the ear canals. Using dissecting scissors, trim many spinal cord regions up to where the brain stem meets the cerebellum, taking care to not damage the cerebellum.Insert one of the vascular scissor blades into the space between the brainstem and vertebral column and cut toward the ear canal, lifting upward while cutting to limit damage to the tissue. Continue to cut along the edge of the skull up toward the olfactory bulbs. Using the blunt forceps, gently peel off the back of this skull to uncover the posterior region of the brain and cerebellum.Position the blunt forceps along the cut edge of the skull and peel the skull up and over the brain. Trim the rest of the skull with the vascular scissors and blunt forceps, clearing most of it from the top of the brain. Slightly lift the brain with the micro spatula to remove the olfactory bulbs from the remaining skull and disconnect optic tract fibers.The brain should come free easily at this point. Place the brain into the Petri dish, sitting on ice and remove any remaining skull or other debris. Gently place the brain into the brain matrix with the dorsal side up.Make sure that it is set level in the matrix and that the midline is in the center. Place one razor blade along the sagittal midline, making sure that the blade pushes all the way to the bottom of the matrix. Place another razor blade one millimeter to the side of the first blade.Place two more blades resulting in three blades placed on one side of the brain, all one millimeter apart. Repeat this on the other side. carefully grab the front and back end to the razorblades, and lift them straight up out of the matrix.Discard the tissue on the outside of the razor blades. Slowly, separate one razor blade at a time from the others being careful not to damage the tissue sections. Slide the tissue section off the razor blade and onto the glass slide with the micro spatula.Six sagittal brain sections should be collected. The foremost lateral sections will have DCN visible. To isolate the DCN, hold a trim to 200 microliter pipette tip perpendicularly over the DCN and push down through the tissue firmly rocking in all directions.Lift the pipette up and visually confirm the presence of the tissue in the tip. Place one finger at the top of the tip and push down, causing the tissue to bulge out. Insert the tip into a correctly labeled microfiche tube and ensure that the tissue punch is placed in the bottom of the tube.Repeat this for the remaining three sections, placing the DCN punches in the same tube. Flash freeze the tube in liquid nitrogen. Push away the rest of the brain tissue around the cerebellum in the sections where the DCN was extracted.Use blunt forceps to gently pick up these hemisphere cerebellar cortex sections and place them into respective microfiche tubes, then flash freeze them. For the last two vermal sections, push away surrounding brain tissue to leave only the cerebellum. Using a razor blade, make a cut separating the anterior labials from the posterior labials.Ensure that the cut is just after the formation of labial six and does not include labial 10. Using blunt forceps, carefully placed the anterior cerebellar cortex sections and posterior cerebellar cortex sections into their respective microfiche tubes and flash freeze them by leaving the tubes in liquid nitrogen for five minutes. Place the microfiche tubes on ice to keep the tissue from thawing too quickly and apply 150 microliters of cold RNA isolation solution in the microfiche tube, then homogenize with a sterilized pestle.Once the tissue is homogenized pipette the solution up and down to ensure there is no remaining intact tissue. Further, break up any small tissue pieces by pulling it up into an insulin syringe a few times. Add another 350 microliters of the reagent, pipette up and down to mix thoroughly and let it sit at room temperature for five minutes.Add 150 microliters of chloroform to the tube and shake it vigorously, then let it rest for two to three minutes. Centrifuge at 12, 000 times G at 15 degrees Celsius for 10 minutes. Carefully remove the tubes from the centrifuge and set the temperature of the centrifuge to four degrees Celsius.Transfer only the clear aqueous phase into a new tube, taking care not to disrupt the opaque interphase. Save or discard the remaining solution in the tubes. Add 100%isopropyl alcohol at a one to two ratio and mix thoroughly by pipetting up and down.Let the sample rest at room temperature for 10 minutes to precipitate the RNA out of the solution. Centrifuge at 12, 000 times G and four degrees Celsius for 10 minutes, placing all tubes in the same orientation to make it easier to visualize the pellet. After centrifugation, carefully remove the tubes and remove the supernatant with the pipette, making sure to not disrupt the pellet.The pellet is gel like and difficult to see, so estimate where it is based on the orientation of the tubes in the centrifuge. After removing all supernatant, add 500 microliters of 75%ethanol, vortex briefly and centrifuge at 7, 500 times G and four degrees Celsius for five minutes. Remove the supernatant carefully, without disrupting the pellet.Leave the caps open to dry the sample for five to 10 minutes. Once dry resuspend the pellet in DNA's free water at 20 microliters to the samples for DCN and 30 microliters for all others. Store the samples at minus 80 degrees Celsius until further analysis.This protocol was used to dissect four distinct regions of the mouse cerebellum and to explore regional differences in gene expression. The expression levels of Aldolase C, parvalbumin and KCNG4 were assessed using real-time quantitative PCR. Aldolase C is more highly expressed in the posterior cerebellar vermis.But lower in the DCN and the anterior region of the vermis when compared to the bulk cerebellar dissection. Parvalbumin is similarly present in the DCN anterior vermis, posterior vermis and hemisphere cerebellar cortices, when compared to bulk cerebellar extracts. KCNG4 is significantly enriched in the DCN and the interior vermis, but not in the posterior vermis or hemispheres when compared to the bulk extraction.To directly compare the expression of aldolase C across the cerebellar cortex, the expression levels were compared to the anterior vermis. The expression level of aldolase C was significantly higher in the posterior Vermis and trending higher in the cerebellar hemispheres. The RNA extracted from these regions can also be used in RNA sequencing, experiments.RNA seq comparing these for cerebellar regions would provide more detailed information on molecular mechanisms, underlying discrete functions of these regions. As well as potential differences in their vulnerability in disease.

cerebellar-regional-dissection-for-molecular-analysis

Research

JoVE Journal

Neuroscience

3.8K ビュー.  University of Minnesota. Traditional molecular studies of the cerebellum have been done on whole cerebellar extracts, which may mask any distinctions across specific cerebellar regions. This protocol makes it possible to assess distinct regions of the cerebellum separately and allows for the exploration of molecular mechanisms that may underlie their unique contributions to a variety behaviors and disease progression. The main advantage of this technique is that it allows for the reproducible and quick dissection of ...

ビデオ: Cerebellar Regional Dissection for Molecular Analysis

Different cerebellar regions have been implicated to play a role in distinct behavioral outputs, yet the underlying molecular mechanisms remain unknown. This work describes a method to reproducibly and quickly dissect cerebellar cortex of the hemispheres, anterior and posterior regions of the vermis, and the deep cerebellar nuclei in order to probe for molecular differences by isolating RNA and testing for differences in gene...

whole vitreous humor dissection for vitreodynamic analysis

Whole Vitreous Humor Dissection for Vitreodynamic Analysis

The goal of this protocol is to show an effective technique to isolate whole, intact vitreous core and cortex from post mortem enucleated porcine...

transoral robotic total thyroidectomy and bilateral central regional lymph node dissection for papillary thyroid carcinoma

Transoral Robotic Total Thyroidectomy and Bilateral Central Regional Lymph Node Dissection for Papillary Thyroid Carcinoma

The transoral robotic total thyroidectomy and bilateral central regional lymph node dissection using three ports without axillary incision are feasible and safe for early-stage papillary thyroid carcinoma. Here we present the operative techniques of this...

dissection of human retina and rpe-choroid for proteomic analysis

Dissection of Human Retina and RPE-Choroid for Proteomic Analysis

The human retina is composed of functionally and molecularly distinct regions, including the fovea, macula, and peripheral retina. Here, we describe a method using punch biopsies and manual removal of tissue layers from a human eye to dissect and collect these distinct retinal regions for downstream proteomic...

dissection of human vitreous body elements for proteomic analysis

Dissection of Human Vitreous Body Elements for Proteomic Analysis

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem...

planarian ovary dissection for ultrastructural analysis and antibody staining

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously...

large-scale zebrafish embryonic heart dissection for transcriptional analysis

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific...

quantitative analysis of synaptic vesicle pool replenishment in cultured cerebellar granule neurons using fm dyes

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described.  Two rounds of SV recycling are monitored in the same nerve terminals providing an internal...

dissection of the mouse pancreas for histological analysis and metabolic profiling

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic...

understanding cerebellar pattern formation

Understanding Cerebellar Pattern Formation

purkinje cell survival in organotypic cerebellar slice cultures

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug...

organ-specific dissection of mosquitoes for microbiota and metagenomic analysis

Organ-Specific Dissection of Mosquitoes for Microbiota and Metagenomic Analysis

dissection of the drosophila pupal retina for immunohistochemistry, western analysis, and rna isolation

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

crispr-mediated loss of function analysis in cerebellar granule cells using in utero electroporation-based gene transfer

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

dissection of mosquito ovaries, midgut, and salivary glands for microbiome analyses at the organ level

Author Spotlight: Optimizing Mosquito Organ Dissection for Studying Symbionts and Vector Microbiomes

Microbial communities in mosquitoes hold great promise for vector biocontrol strategies. Most symbionts are uncultivable, requiring metagenomic analyses. We describe a method to dissect female mosquitoes and separate ovaries, midgut, and salivary glands preventing cross-contamination, facilitating microbiome studies at the organ level, and enhancing understanding of microorganisms' roles in mosquito...

High-Precision Mosquito Organ Isolation for Microbiome Analyses

https://cloudfront.jove.com/CDNSource/teasers/67128_b.jpg

preparing developing peripheral olfactory tissue for molecular and immunohistochemical analysis in drosophila

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

laser nanosurgery of cerebellar axons in vivo

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in...

Laser Nanosurgery of Cerebellar Axons In Vivo

dissection of the transversus abdominis muscle for whole-mount neuromuscular junction analysis

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

whole rabbit eye dissection and ocular tissue preparation for histological analysis

Whole Rabbit Eye Dissection and Ocular Tissue Preparation for Histological Analysis

A Simple Composite Phenotype Scoring System for Evaluating Mouse Models of Cerebellar Ataxia

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously published phenotype assessments in several disease models, including spinocerebellar ataxias, Huntington s disease and spinobulbar muscular atrophy. Measures include hind limb clasping, ledge test, gait and kyphosis. Each measure is recorded on a scale of 0-3, with a combined total of 0-12 for all four measures. The results effectively discriminate between affected and non-affected individuals, while also quantifying the temporal progression of neurodegenerative disease phenotypes. Measures may be analyzed individually or combined into a composite phenotype score for greater statistical power. The ideal combination of the four described measures will depend upon the disorder in question. We present an example of the protocol used to assess disease severity in a transgenic mouse model of spinocerebellar ataxia type 7 (SCA7).
Albert R. La Spada and Gwenn A. Garden contributed to this manuscript equally.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebellar ataxia. Measures include hind limb clasping, ledge test, gait and kyphosis. This protocol effectively discriminates between affected and non-affected individuals, and detects the progression of affected individuals over time.

We describe a protocol for the rapid and sensitive quantification of disease severity in mouse models of cerebella ataxia. It is derived from previously ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

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.

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 ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...