The authors would like to thank Ikiel Ptito for his continued technical support. NSERC grant to MP.

Part 1: Pre-processing of tissue should be done according to Burke et al. (2009)5.
<ol>
<li>Briefly, tissue should be well 	perfused with paraformaldehyde, glutaraldehyde, or formalin. This 	can be achieved through standard transcardial perfusion typically 	used to harvest other organs. In the present study the subject was 	deeply sedated with ketamine hydrochloride (10 mg/kg, i.m.), 	euthanized with an overdose of sodium pentobarbital (25 mg/kg, i.v.) 	and perfused transcardially with 0.1 M PBS until completely 	exsanguinated. This is followed by a 4% paraformaldehyde solution in 	PBS for 5 min (~1 liter). Tissue should be well perfused with 	paraformaldeyhde, gluteraldehyde, or formalin. The brain should be 	stereotaxically blocked, removed from the skull, weighed, volume 	determined, cryoprotected, and frozen5.</li>
</ol>
Part 2: Systematic Sampling of tissue, according to Burke et al. (2009)6.
<ol>
<li>In this example we focused on the 	frontal lobe of the vervet brain. The reference space was defined to 	include cortical tissue from the central sulcus to the frontal pole 	of the left hemisphere. For our purposes the series were set at 1/10 	sections throughout the cortex, sectioned at 50&micro;m. One complete 	series was stained with cresyl violet (series #1). Series 2 was 	split so that half of the sections were stained with gold chloride 	for myelin revelation and the other half stained for acetylcholine 	esterase (for delineation of certain subcortical areas). All other 	series were banked in antigen preserve as part of our long-term 	research plans. </li>
</ol>
Part 3: Stereology (for more details, see Mouton 2002)4
<ol>
<li>The total estimation of cell 	numbers (N) is calculated based on the following equation: 
N = ssf-1 x asf-1 x tsf-1 x &Sigma;Q-
 Where ssf is the section sampling fraction, asf is the area sampling fraction, tsf if the thickness sampling fraction (where the measured thickness of the tissue is divided by the dissector height), and &sum;Q- is the total number of objects of interest counted within the dissector. The next sections of this protocol show how to determine the ssf, asf, tsf and &sum;Q. 
</li>
<li>Section Sampling Fraction (non-computer based)<ol style="list-style-type:lower-alpha;">
<li>The entire reference space must be well defined from anterior to posterior and dorsal to ventral. In this example we were interested in the frontal lobe and have defined it as the region from the tip of the frontal pole (anterior) to the central sulcus (posterior) and the lateral sulcus (ventral) and excluded the insula.</li>
<li>For the frontal lobe of this particular subject a total of 760 sections were systematically collected with 76 stained for cresyl violet. Since the target was 10 sections throughout the reference space 1/6 cresyl violet stained sections were sampled, leading to a Section Sample Fraction of about 1/60. We randomly started with one of the first 6 cresyl violet stained sections and systematically sampled 1/6 thereafter. A total of 13 sections were sampled for the selected subject (Figure 2).</li>
</ol></li>
<li>
 Area Sample Fraction 	(computer-based) 
<ol style="list-style-type:lower-alpha;">
<li>A pilot study will indicate the optimal grid size, the Area Sampling Fraction, to sample the reference space with about 100-300 disectors. Within the frontal lobe, a grid size of 2500 &micro;m2 yielded an average of 254 disectors for this subject (Figure 2). The size of the disector should yield between about 0-5 counted objects, in this case, neurons.</li>
</ol> </li>
<li>Thickness Sample Fraction 	(computer-based)<ol style="list-style-type:lower-alpha;">
<li>At each disector location, the height of the tissue is measured, and a fraction of this height is sampled; this is known as the Thickness Sampling Fraction. To determine the measured thickness, focus through the z-plane until the first cell comes into focus, then back up slightly to the top of the section, i.e., until the last object appears just out of focus. To determine the bottom of the tissue, focus through the z-plane until the cells are barely out of focus.</li>
<li>Focusing through the z-plane, cells should be stained at every depth; if not, this may indicate an incomplete penetration of the stain or non-uniform dehydration of the tissue, in which case the sections should be re-stained. This precaution is especially important for immunostained tissue that requires penetration of immunoprobes through relatively thick tissue sections. For this reason, immunostained tissue should be lightly counterstained with a basophilic stain (e.g., cresyl violet, hematoxylin) in order to accurately determine top and bottom of the section and to confirm penetration of the antibody. In this study the sections were sliced at a microtome setting of 50&micro;m. After all tissue processing was complete, the measured tissue thickness averaged 17.9&micro;m (Figure 2), with a mean shrinkage of about 65%. Note that these results represent typical shrinkage for tissue processed for routine histological preparation7.</li>
<li>The default disector height for the typical study is 10&micro;m. The difference between the disector height and the measured section thickness is the guard height, the volume of tissue where no biological features are counted. The use of a guard height avoids tissue damage at the sectioning surfaces (e.g., lost caps).</li>
</ol></li>
<li>The total number of objects 	counted, &Sigma;Q-<ol style="list-style-type:lower-alpha;">
<li>To avoid bias from recognition errors, it is imperative that a standard definition is followed for the particular biological feature of interest. In this case, we were interested in counting neurons. Therefore, a neuron was defined as having a visible centrally located nucleolus and a clearly defined cytoplasm, whereas glial cells generally lacked visible nucleoli and cytoplasm. For this subject 457 neurons were counted. </li>
</ol></li>
</ol>
Part 4: Stereologer -- A Computerized Stereology System (Stereology Resource Center, Chester, MD)
<ol>
<li>The Stereologer System prompts the user to fill out the necessary information in a step-by-step fashion. In the &ldquo;Study Information&rdquo; section the 	parameters of the study are established. Since a reference space must be defined for the study, the volume parameter should be 	selected (for the reference space the Cavalieri estimator should be selected). Object volume may also be selected here to estimate the 	number-weighted volume for the population of objects of interest. 	For our example, only the volume of the reference space was selected. For each object, select the number and define the feature of interest, in this case Neurons.</li>
<li>Case Initialization: In this 	section the sampling information is established. Enter the slab sampling interval (if separate slabs of tissue were sliced 	exhaustively and each section is placed in sequential order then enter 1 here). Enter the total number of sections taken through the reference space (in this case 760). Then enter the section sampling interval (in this case 59). The system then calculates the number of sections to be sampled (in this case 13).</li>
<li>Probe Parameters: This section 	defines the grid and disector size. For the volume, use a low 	magnification 2.5x-10x to define the grid spacing under the edit 	menu. Object magnification should be performed at 100x (N.A. 1.3 or 	1.4). Frame area is the size of the disector, in this case we used 	50% Screen. The frame height is the thickness of the disector; set 	at 10&micro;m here. The frame spacing is the size of the grid. In our 	pilot study we found that 2500&micro;m yields between 150-200 frames 	spaced in a systematic-uniform manner through a relatively large 	reference space. For smaller areas, a smaller grid size should be 	used. A pilot study will identify the optical probe parameters for 	each particular study.</li>
<li>Once the study parameters have 	been established, sampling through the tissue may proceed. The 	program will prompt you to sample the first section. Step 1: The program will prompt the user, under low magnification, to trace 	the reference space on the section. The system will then place a 	grid over the section based on the probe parameters and the user 	will then verify that points fall within the reference space. If a 	point is not within the reference space simply click on the point 	and will not be calculated into the volume. Step 2: The 	system will place a new grid over the reference space, based on the 	frame spacing parameters. Verify that the intersections fall within 	the reference space. Step 3: The system will then prompt the 	user to switch to the higher magnification objective and move the 	stage to the first dissector. Step 4: At this point the 	system prompts the user to define the top and bottom of the section 	then the system sets the sampling space in the z-axis. Step 5: The user will then click on each object that falls within the 	disector through the z-plane. Objects that touch the red lines or 	the bottom of the disector may not be counted. Once each object is 	counted, click next. The stage will move to the next disector and 	steps 4 and 5 will be repeated. Once all of the disectors for the 	section have been counted, the system will prompt the user to insert 	the next sequential section to be probed. Steps 1-5 will be repeated 	until all of the sequential sections have been sampled. The system 	will then provide the calculations for asf, ssf, tsf, 	and &sum;Q. Based on these parameters, the system will generate 	an estimated N, reference volume and CE&rsquo;s for both the estimated 	number and volume. It will also give recommendations to become more 	efficient or to reduce the CE (Figure 2).</li>
</ol>
Part 5: Representative Results:
Unbiased stereology provides efficient and reliable estimates of cell populations within a reference space. There are a number of computer-based stereological systems available, all of which rely on systematic sampling and a defined reference space. We have used the BioQuant Life Sciences system to estimate the total neuronal population of the cerebral cortex of 2-year-old vervets to be over 828 millions (Figure 3) with an average CE of 0.042. We have subsequently used the Stereologer system to estimate that the frontal lobe accounts for about half the number of cortical neurons8.
<img src="/files/ftp_upload/1262/1262fig1.jpg" alt="figure 1" /> Figure 1 Computer-based Stereology Systems. The basic set-up of the stereology systems is the same, a microscope with a motorized stage (x-y-z), a stage controller, video camera, computer, and software. The system that is ultimately chosen should be based on efficiency, needs, and cost analysis.
<div style="border:1px #000000 solid">
		<table border="0">
			<tbody>
				<tr>
					<td colspan="2">STUDY INFORMATION</td>
				</tr>
				<tr>
 <td>Study Name</td>
 <td>Frontal Cortex</td>
				</tr>
				<tr>
 <td>Principal Investigator</td>
 <td>MB</td>
 </tr>
 <tr>
 <td>Species</td>
 <td>AG</td>
 </tr>
 <tr>
 <td>Reference Space	Frontal</td>
 <td>Cortex</td>
 </tr>
 <tr>
					<td colspan="2">Notes</td>
 </tr>
 <tr>
 <td colspan="2">CASE INFORMATION</td>
				</tr>
				<tr>
					<td>Data Collector</td>
					<td>MB</td>
 </tr>
 <tr>
					<td>Date</td>
					<td>Wednesday, April 30, 2008</td>
				</tr>
				<tr>
 <td>Group</td>
 <td>Control</td>
 </tr>
				<tr>
 <td>Subject</td>
 <td>O26</td>
				</tr>
				<tr>
 <td colspan="2">Notes</td>
				</tr>
				<tr>
					<td colspan="2">SAMPLING CHARACTERISTICS</td>
				</tr>
				<tr>
 <td>Slab Sampling Interval</td>
 <td>1</td>
 </tr>
 <tr>
 <td>Total Number of Sections</td>
 <td>760</td>
 </tr>
 <tr>
 <td>Section Sampling Interval</td>
 <td>59</td>
 </tr>
 <tr>
 <td>Starting Section</td>
 <td>2</td>
 </tr>
 <tr>
 <td colspan="2">FRACTIONS</td>
 </tr>
 <tr>
 <td>ASF</td>
 <td>0.0002</td>
 </tr>
 <tr>
 <td>SlabSF</td>
 <td>1.0000</td>
 </tr>
 <tr>
 <td>SSF</td>
 <td>0.0169</td>
 </tr>
 <tr>
 <td>TSF</td>
 <td>0.5587</td>
 </tr>
			</tbody>
	</table>
	<table border="0">
		<tbody>
 <tr>
		<td colspan="6">RESULTS SUMMARY</td>
	</tr>
	<tr>
		<td>Parameter</td>
 <td>Probe</td>
 <td>Name</td>
 <td>Result</td>
 <td>CE</td>
 <td>SD</td>
	</tr>
	<tr>
 <td>Thickness</td>
 <td>---</td>
 <td>---</td>
 <td>17.8996 &micro;</td>
 <td>---</td>
 <td>---</td>
	</tr>
	<tr>
 <td>Number</td>
 <td>Disector</td>
 <td>neuron</td>
 <td>243833769.1473</td>
 <td>0.0474</td>
 <td>N/A</td>
	</tr>
	<tr>
 <td>Volume</td>
 <td>Cavalieri Point Grid</td>
 <td>volume</td>
 <td>1719769397954.1284 &micro;^3</td>
 <td>0.0078</td>
 <td>N/A</td>
	</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
	<tr>
		<td colspan="2">RECOMMENDATIONS</td>
	</tr>
	<tr>
		<td>Probe</td>
		<td>Object Number (neuron)</td>
	</tr>
	<tr>
 <td>CE</td>
 <td>0.0474</td>
	</tr>
	<tr>
 <td>Recommendations</td>
 <td>CE is acceptable.</td>
	</tr>
</tbody>
</table>
<table border="0">
<tbody>
	<tr>
		<td colspan="2">Number of Disectors Viewed is Too High, Increase Disector Spacing.</td>
	</tr>
	<tr>
		<td>Probe</td>
		<td>Region Volume (volume)</td>
	</tr>
	<tr>
		<td>CE</td>
		<td>0.0078</td>
	</tr>
	<tr>
 <td>Recommendations</td>
 <td>CE is acceptable.</td>
	</tr>
	</tbody>
</table>
<table border="0">
	<tbody>
	<tr>
		<td colspan="3">SECTION RESULTS</td>
	</tr>
	<tr>
		<td>Section</td>
 <td>neuron NumObjects neuron NumCorners</td>
 <td>volume RegPoints</td>
	</tr>
	<tr>
 <td>1.	40</td>
 <td>48</td>
 <td>34</td>
	</tr>
	<tr>
 <td>2.	26</td>
 <td>52</td>
 <td>44</td>
	</tr>
	<tr>
 <td>3.	29</td>
 <td>56</td>
 <td>48</td>
 </tr>
 <tr>
 <td>4.	39</td>
 <td>100</td>
 <td>83</td>
	</tr>
	<tr>
 <td>5.	49</td>
 <td>112</td>
 <td>83</td>
 </tr>
 <tr>
 <td>6.	53</td>
 <td>156</td>
 <td>120</td>
 </tr>
 <tr>
 <td>7.	65</td>
 <td>128</td>
 <td>106</td>
 </tr>
 <tr>
 <td>8.	45</td>
 <td>108</td>
 <td>100</td>
 </tr>
 <tr>
 <td>9.	52</td>
 <td>100</td>
 <td>77</td>
 </tr>
 <tr>
 <td>10.	26</td>
 <td>64</td>
 <td>60</td>
	</tr>
	<tr>
 <td>11.	21</td>
 <td>44</td>
 <td>44</td>
 </tr>
 <tr>
 <td>12.	11</td>
 <td>36</td>
 <td>32</td>
	</tr>
	<tr>
 <td>13.	1</td>
 <td>12</td>
 <td>6</td>
	</tr>
</tbody>
</table>
</div>
Figure 2 Results from the Fontal Lobe Obtained with the Stereologer System. For the frontal lobe, the longest part of the stereological process was in the systematic sampling and sectioning. Topography, volume and neuronal estimate were completed in about a day for each subject. The left hemisphere was sampled and the number is doubled in the text to approximate an estimate for both hemispheres.
<img src="/files/ftp_upload/1262/1262fig3.jpg" alt="figure 3" /> Figure 3 Cortex Neuronal Population Estimate Obtained with BioQuant. The BioQuant system requires an exact topography prior to cell counting. Here an average of 13 sections were sampled. Cortical topography and subsequent volume estimation took between 15-20 hours for each subject. Topography was performed under a 2.5x objective and counting was done using a 100x oil immersion objective (N.A. 1.3). Every 100th section was chosen throughout the left hemisphere with an average of 180 disectors sampled. Once the topography was completed neuronal counting took about a day. The left hemisphere was sampled and the number is doubled in the text to approximate an estimate for both hemispheres.

<ol>
	<li>West, M., Slomianka, L., Gundersen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+stereological+estimation+of+the+total+number+of+neurons+in+the+subdivisions+of+the+rat+hippocampus+using+the+optical+fractionator.">Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator.</a> Aat Rec. 231, 482-497 (1991).</li><li>Saper, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Any+way+you+cut+it:+a+new+journal+policy+for+the+use+of+unbiased+counting+methods.">Any way you cut it: a new journal policy for the use of unbiased counting methods.</a> J Comp Neurol. 364, 5-5 (1996).</li><li>Sterio, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unbiased+estimation+of+number+and+sizes+of+arbitrary+particles+using+the+disector.">The unbiased estimation of number and sizes of arbitrary particles using the disector.</a> J Microsc. 134, 127-136 (1984).</li><li>Mouton, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practices of Unbiased Stereology. , (2002).</li><li>Burke, M. W., Zangenehpour, S., Boire, D., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+non-human+primate+brain+in+stereotaxic+space.">Dissecting the non-human primate brain in stereotaxic space.</a> J Vis Exp. 29, (2009).</li><li>Burke, M. W., Zangenehpour, S., Boire, D., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+banking:+Making+the+most+of+your+research.">Brain banking: Making the most of your research.</a> J Vis Exp. 29, (2009).</li><li>Manaye, K. F., Wang, P., O'Neil, J., Huafu, S., Tizabi, Y., Thompson, N., Ottinger, M. A., Ingram, D. K., Mouton, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+Quantification+Of+Dtg+APP/PS1:+Neuroimaging,+Stereology,+And+Biochemistry.">Neuropathological Quantification Of Dtg APP/PS1: Neuroimaging, Stereology, And Biochemistry.</a> AGE. 29, 87-96 (2009).</li><li>Burke, M. W., Palmour, R. M., Ervin, F. R., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+reduction+in+frontal+cortex+of+primates+after+prenatal+alcohol+exposure.">Neuronal reduction in frontal cortex of primates after prenatal alcohol exposure.</a> Neuroreport. 20, 13-17 (2009).</li><li>Zangenehpour, S., Burke, M. W., Chaudhuri, A., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Batch+immunostaining+for+large-scale+protein+detection+in+the+whole+monkey+brain.">Batch immunostaining for large-scale protein detection in the whole monkey brain.</a> J Vis Exp. 29, (2009).</li></ol>

Unbiased stereology in a primate brain is similar to that of other biological specimens. However, given the size of the primate brain, a careful processing plan is recommended since a single hemisphere of the vervet monkey yields more than 1200 sections when sliced at 50&micro;m. First we recommend stereotaxically blocking the brain into 1cm blocks, which provides a standard plane of section between blocks and subjects, minimizes partial sections between blocks, and provides manageable sized blocks5. A crucial step for stereology is systematic sampling such that 6-10 sections are obtained through the reference area. For the primate brain, this leaves a significant amount of material unprocessed, so in order to maximize the potential data obtained from each brain we suggest banking the tissue in antigen preserve for long-term research plans6. When tissue is banked, a systematic immunodetection approach for identifying target proteins in the monkey brain can be prepared for stereology9. Larger areas such as the cortex or the lobes may require 10-14 sections, so prior to sectioning a plan should be made to have at least 10 sections through the smallest reference space of potential interest. A pilot study should also be performed on a control subject to set the parameters for the stereological study. Furthermore when presenting or interpreting published stereological data the asf, tsf, ssf, and &sum;Q should be reported along with the appropriate CE values.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Stereologer</td>
<td>&nbsp;</td>
<td>Stereology Resource Center (Chester, MD)</td>
<td>www.disector.com</td>
<td>&nbsp;</td>
<tr>
<td>Stereology Tool-Kit Stereologer system</td>
<td>&nbsp;</td>
<td>BioQuant Life Sciences (Nashville, TN)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>StereoInvestigator</td>
<td>&nbsp;</td>
<td>MicroBright Field (Willston, VT)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

knowing what counts: unbiased stereology in the non-human primate brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

Watch this Scientific Journal Video about Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain at JoVE.com

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

knowing-what-counts-unbiased-stereology-in-the-non-human-primate-brain

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13.7K Views.  University of Montreal. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1. 

Video: Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

Application of Light-cured Dental Adhesive Resin for Mounting Electrodes or Microdialysis Probes in Chronic Experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.

In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium.

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these ...

Micro-dissection of Rat Brain for RNA or Protein Extraction from Specific Brain Region

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates micro-dissection of four major brain regions include olfactory bulb, frontal cortex, striatum and hippocampus in fresh rat brain tissue. Useful tips for quick removal of respective regions to avoid RNA and protein degradation of the tissue are given.

Micro-dissection of rat brain into various regions is extremely important for the study of different neurodegenerative diseases. This video demonstrates ...

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. The distinct advantage of this technique is that experimental manipulations may be performed on individual cells while leaving the surrounding tissue unaltered, thereby distinguishing cell-autonomous effects from those resulting from global treatments. When combined with advanced in vivo imaging techniques, SCE of fluorescent markers permits direct visualization of cellular morphology, cell growth, and intracellular events over timescales ranging from seconds to days. While this technique is used in a variety of in vivo and ex vivo preparations, we have optimized this technique for use in Xenopus laevis tadpoles. In this video article, we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the albino Xenopus tadpole. We also discuss methods to optimize yield, and show examples of live two-photon fluorescence imaging of neurons fluorescently labeled by SCE.

Single-cell electroporation (SCE) is a specialized technique allowing delivery of DNA or other macromolecules into individual cells within intact tissue, including in vivo preparations. Here we detail the procedure for SCE of a fluorescent dye or plasmid DNA into neurons within the intact brain of the Xenopus laevis tadpole.

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Determination of Mammalian Cell Counts, Cell Size and Cell Health Using the Moxi Z Mini Automated Cell Counter

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A critical breakthrough in cell/particle counting technologies was the development of the Coulter technique by Wallace Coulter over 50 years ago. The technique involves the application of an electric field across a micron-sized aperture and hydrodynamically focusing single particles through the aperture. The resulting occlusion of the aperture by the particles yields a measurable change in electric impedance that can be directly and precisely correlated to cell size/volume. The recognition of the approach as the benchmark in cell/particle counting stems from the extraordinary precision and accuracy of its particle sizing and counts, particularly as compared to manual and imaging based technologies (accuracies on the order of 98% for Coulter counters versus 75-80% for manual and vision-based systems). This can be attributed to the fact that, unlike imaging-based approaches to cell counting, the Coulter Technique makes a true three-dimensional (3-D) measurement of cells/particles which dramatically reduces count interference from debris and clustering by calculating precise volumetric information about the cells/particles. Overall this provides a means for enumerating and sizing cells in a more accurate, less tedious, less time-consuming, and less subjective means than other counting techniques6.
Despite the prominence of the Coulter technique in cell counting, its widespread use in routine biological studies has been prohibitive due to the cost and size of traditional instruments. Although a less expensive Coulter-based instrument has been produced, it has limitations as compared to its more expensive counterparts in the correction for "coincidence events" in which two or more cells pass through the aperture and are measured simultaneously. Another limitation with existing Coulter technologies is the lack of metrics on the overall health of cell samples. Consequently, additional techniques must often be used in conjunction with Coulter counting to assess cell viability. This extends experimental setup time and cost since the traditional methods of viability assessment require cell staining and/or use of expensive and cumbersome equipment such as a flow cytometer.
The Moxi Z mini automated cell counter, described here, is an ultra-small benchtop instrument that combines the accuracy of the Coulter Principle with a thin-film sensor technology to enable precise sizing and counting of particles ranging from 3-25 microns, depending on the cell counting cassette used. The M type cassette can be used to count particles from with average diameters of 4 - 25 microns (dynamic range 2 - 34 microns), and the Type S cassette can be used to count particles with and average diameter of 3 - 20 microns (dynamic range 2 - 26 microns). Since the system uses a volumetric measurement method, the 4-25 microns corresponds to a cell volume range of 34 - 8,180 fL and the 3 - 20 microns corresponds to a cell volume range of 14 - 4200 fL, which is relevant when non-spherical particles are being measured. To perform mammalian cell counts using the Moxi Z, the cells to be counted are first diluted with ORFLO or similar diluent. A cell counting cassette is inserted into the instrument, and the sample is loaded into the port of the cassette. Thousands of cells are pulled, single-file through a "Cell Sensing Zone" (CSZ) in the thin-film membrane over 8-15 seconds. Following the run, the instrument uses proprietary curve-fitting in conjunction with a proprietary software algorithm to provide coincidence event correction along with an assessment of overall culture health by determining the ratio of the number of cells in the population of interest to the total number of particles. The total particle counts include shrunken and broken down dead cells, as well as other debris and contaminants. The results are presented in histogram format with an automatic curve fit, with gates that can be adjusted manually as needed.
Ultimately, the Moxi Z enables counting with a precision and accuracy comparable to a Coulter Z2, the current gold standard, while providing additional culture health information. Furthermore it achieves these results in less time, with a smaller footprint, with significantly easier operation and maintenance, and at a fraction of the cost of comparable technologies.

The Moxi Z miniature automated cell counter is a novel instrument that combines the Coulter Principle with patented thin-film sensor technology and a proprietary software algorithm to perform sizing and counting of a broad size range of particles as well as to determine the overall health of monodisperse mammalian cell cultures. This protocol describes the use of this instrument for counting and assessing the health of cell cultures.

Particle and cell counting is used for a variety of applications including routine cell culture, hematological analysis, and industrial controls1-5. A ...

Mapping the After-effects of Theta Burst Stimulation on the Human Auditory Cortex with Functional Imaging

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent progress, the functional properties and lateralization of the human auditory cortex are far from being fully understood. Transcranial Magnetic Stimulation (TMS) is a non-invasive technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses, and represents a unique method of exploring plasticity and connectivity. It has only recently begun to be applied to understand auditory cortical function 2. 
An important issue in using TMS is that the physiological consequences of the stimulation are difficult to establish. Although many TMS studies make the implicit assumption that the area targeted by the coil is the area affected, this need not be the case, particularly for complex cognitive functions which depend on interactions across many brain regions 3. One solution to this problem is to combine TMS with functional Magnetic resonance imaging (fMRI). The idea here is that fMRI will provide an index of changes in brain activity associated with TMS. Thus, fMRI would give an independent means of assessing which areas are affected by TMS and how they are modulated 4. In addition, fMRI allows the assessment of functional connectivity, which represents a measure of the temporal coupling between distant regions. It can thus be useful not only to measure the net activity modulation induced by TMS in given locations, but also the degree to which the network properties are affected by TMS, via any observed changes in functional connectivity.
Different approaches exist to combine TMS and functional imaging according to the temporal order of the methods. Functional MRI can be applied before, during, after, or both before and after TMS. Recently, some studies interleaved TMS and fMRI in order to provide online mapping of the functional changes induced by TMS 5-7. However, this online combination has many technical problems, including the static artifacts resulting from the presence of the TMS coil in the scanner room, or the effects of TMS pulses on the process of MR image formation. But more importantly, the loud acoustic noise induced by TMS (increased compared with standard use because of the resonance of the scanner bore) and the increased TMS coil vibrations (caused by the strong mechanical forces due to the static magnetic field of the MR scanner) constitute a crucial problem when studying auditory processing. 
This is one reason why fMRI was carried out before and after TMS in the present study. Similar approaches have been used to target the motor cortex 8,9, premotor cortex 10, primary somatosensory cortex 11,12 and language-related areas 13, but so far no combined TMS-fMRI study has investigated the auditory cortex. The purpose of this article is to provide details concerning the protocol and considerations necessary to successfully combine these two neuroscientific tools to investigate auditory processing. 
Previously we showed that repetitive TMS (rTMS) at high and low frequencies (resp. 10 Hz and 1 Hz) applied over the auditory cortex modulated response time (RT) in a melody discrimination task 2. We also showed that RT modulation was correlated with functional connectivity in the auditory network assessed using fMRI: the higher the functional connectivity between left and right auditory cortices during task performance, the higher the facilitatory effect (i.e. decreased RT) observed with rTMS. However those findings were mainly correlational, as fMRI was performed before rTMS. Here, fMRI was carried out before and immediately after TMS to provide direct measures of the functional organization of the auditory cortex, and more specifically of the plastic reorganization of the auditory neural network occurring after the neural intervention provided by TMS. 
Combined fMRI and TMS applied over the auditory cortex should enable a better understanding of brain mechanisms of auditory processing, providing physiological information about functional effects of TMS. This knowledge could be useful for many cognitive neuroscience applications, as well as for optimizing therapeutic applications of TMS, particularly in auditory-related disorders.

Auditory processing is the basis of speech and music-related processing. Transcranial Magnetic Stimulation (TMS) has been used successfully to study cognitive, sensory and motor systems but has rarely been applied to audition. Here we investigated TMS combined with functional Magnetic Resonance Imaging to understand the functional organization of auditory cortex.

Auditory cortex pertains to the processing of sound, which is at the basis of speech or music-related processing1. However, despite considerable recent ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...