We thank Susanne S&#248;rensen for the skillful technical assistance. This work was supported by grants from The Velux Foundation; The Jascha Foundation; Aase and Ejnar Danielsens Foundation; Hartmann Brothers Foundation; Director Jacob Madsen and wife Olga Madsens Foundation; Doctor Sofus Carl Emil Friis and wife Doris Friis&#39; Trust; The Foundation of 1870; Torben and Alice Frimodts Foundation and The Foundation for Neurological Research. Manuscript editing was performed by Inglewood Biomedical Editing.

All the animal procedures were performed in accordance with guidelines from the Danish Animal Experimentation Inspectorate and approved by the local ethical committee for Experimental Animals.
1. Tissue processing
NOTE: Following transcardial perfusion and post fixation in 4% paraformaldehyde (PFA) diluted in 0.2 M phosphate buffer overnight at 4 &#176;C, brains are transferred to 30% sucrose in phosphate buffered saline (PBS) for three days at 4 &#176;C. Thereafter, the brains are frozen on powdered dry ice and stored at -80 &#176;C until sectioning.
<ol>
	<li>Separate the right and left hemispheres along the midline of the brain using a scalpel and then choose one or the other hemisphere randomly in the first brain, thereafter shift systematically between right and left hemisphere (Figure 1A). Mount the sampled hemisphere onto a specimen disc with the long axis perpendicular to the disc using a mounting medium.</li>
	<li>Place numbered multidish containers for pre-cooling in the cryostat.</li>
	<li>Mount the specimen disc into the cryostat and cut the entirety of the hippocampus (Bregma 1.80 to -&#160;7.0416) into 80 &#181;m thick sections in the coronal plane (Figure 1B).
	<ol>
		<li>Place all sampled sections consecutively in the cooled multidish containers, to ensure that the sections are kept in cutting-order.</li>
		<li>Cover the sections completely with cryoprotectant and hold the containers at -20 &#176;C until further use.</li>
	</ol>
	</li>
</ol>
2. Immunostaining
NOTE: To keep track of the individual sections throughout the staining procedure, place the sampled sections in 25-well staining nets. Use every 5th section, giving a mean final number of 12 (8-16) sections per hippocampus for immunostaining and subsequent cell counting.
<ol>
	<li>Prior to subsampling every nth section with a random start between section one and section n (Figure 1C) transfer the containers from -20 &#176;C to room temperature (RT).</li>
	<li>Use e.g. a paintbrush, to transfer the sections in numerical order to the 25-well staining nets placed in petri dishes filled with PBS. 
	NOTE: From this point, keep the sections in the staining nets which are placed in matching glass dishes placed on an orbital shaker (section 2.3 to 2.9).</li>
	<li>Transfer the staining nets to the matching glass dishes and wash the sections twice for 10 min in PBS at RT before incubation in 3% hydrogen peroxide (H2O2) diluted in distilled water (dH2O) for 20 min.</li>
	<li>Transfer the sections to three different solutions of hydrochloric acid (HCl) (1 M at 0 &#176;C for 10 min, 2 M at RT for 10 min, and 2 M at 37 &#176;C for 20 min) before neutralization in 0.1 M sodium tetraborate at RT for 20 min.</li>
	<li>After 3 &#215; 10 min incubations in 1% Triton X-100 diluted in PBS (washing buffer), transfer the sections to washing buffer containing 10% fetal bovine serum (blocking solution) for 1 h at RT.</li>
	<li>Incubate the sections for 48 h at 4 &#176;C in mouse-anti-BrdU diluted 1:100 in blocking solution.</li>
	<li>Wash the sections 3 &#215; 10 min in washing buffer, and incubate for 48 h in horse radish peroxidase diluted 1:10 in washing buffer.</li>
	<li>Transfer the sections to PBS for 5 &#215; 10 min, and then for 7 min in 0.01% diaminobenzidine (DAB) diluted in PBS before they are transferred to a similar DAB solution containing 0.02% H2O2 for 10 min at RT.</li>
	<li>Complete a series of washes (2 &#215; 10 min in PBS and 2 &#215; 10 min in phosphate buffer without added sodium chloride) and mount the sections in numerical order on microscope slides.</li>
	<li>Dry the sections for approximately 30 min before counterstaining with cresyl violet.</li>
	<li>Place the microscope slides in a slide rack.</li>
	<li>Rehydrate the sections for 10 min in slide staining dishes containing dH2O, and then transfer the slides to 0.02% cresyl violet in dH2O for 15 min.</li>
	<li>Repeat step 2.12 before the sections are dehydrated three times in ethanol (96% for 5 min and 99% for 2 &#215; 2 min).</li>
	<li>Place the sections in xylene for 2 &#215; 15 min and cover-slip the slides using a rapid drying medium for mounting.</li>
	<li>Finally, leave the cover-slipped slides for approximately 24 h before they can be used for microscopy.</li>
</ol>
3. Estimation of the total number of BrdU-labelled neurons using the optical fractionator
NOTE: Conduct a pilot study including a few animals to determine the optimal sampling parameters, such as the number of sections to be analyzed and the number of optical disectors within the sampled sections. This pilot also provides a preliminary CV (standard deviation/mean) and the possibility to adjust the CE (see section 3.9.3) to obtain a satisfactory precision of the estimates determined by the investigator (for more details see below). Likewise perform a z-distribution analysis to address the following points: shrinkage of the tissue, quality of the staining throughout the section and distribution of the cells in the z-axis14.
<ol>
	<li>Check the thickness of the tissue in the sections to confirm that they are suitable for the use of the optical fractionator, e.g. is thick enough for the chosen disector height including guard zones (see below).&#160;NOTE: The tissue thickness is measures in several places within the region of interest.
	<ol>
		<li>Place the slides on the motorized stage of the microscope and turn on the preferred stereological software.</li>
		<li>Delineate the area of interest using a low-magnification objective (2X or 4X) before changing to a 100X oil-immersion objective (see Figure 2A).</li>
		<li>Using a specific point of a counting frame (e.g. at or adjacent to a corner), locate the top of the section by moving along the focal plane until some feature of the section appears in focus. Register this z-position as 0 (see Figure 2B).</li>
		<li>Move the focal plane down through the tissue until the same specific point of the counting frame is at the last z-level of tissue in focus and mark this position. The local tissue thickness is defined from 0 to this endpoint and can be read on the z-axis. Register the tissue thickness (see Figure 2B).</li>
	</ol>
	</li>
	<li>Document full penetration of the stain and distribution of the cells in the full thickness of the section. 
	NOTE: The distribution of BrdU-labelled neurons is used as a guide for determining the required guard zones. Although a uniform distribution of cells is critical, it is acceptable to observe fewer cells near the top and bottom of the section, indicating the effect of lost caps (for further details see Dorph-Petersen et al., 2009).
	<ol>
		<li>Sample BrdU-labelled neurons and register their z-position together with the local section thickness measured in the selected corner of each counting frame.</li>
		<li>Plot the number of BrdU-positive neurons as a function of their z-position.</li>
	</ol>
	</li>
	<li>Fix the height of the disector and guard zones based on the mean section thickness and the distribution of cells (see 3.1 and 3.2). 
	NOTE: The height of the disector should be less than the section thickness to avoid artifacts close to the surfaces. This risk is circumvented by including guard zones to the top and bottom of the section.</li>
	<li>Determine the step length between the disectors.
	<ol>
		<li>Delineate the region of interest on all the sections to be analyzed and register the areas.</li>
		<li>Sum these areas and then divide by the number of disectors to be placed within this area. 
		NOTE: Based on previous experience a good starting point entails 75 probes. This will provide an approximation of the area and the corresponding sampling positions (Astep) (for further details see West, 2012).</li>
		<li>Take the square root of Astep, which will provide the x-y-step size.</li>
	</ol>
	</li>
	<li>Determine the size of the counting frame.
	<ol>
		<li>Set the size of the counting frame to an arbitrary unit and perform a pilot sampling of BrdU-positive neurons using the parameters obtained in section 3.3 and 3.4. 
		NOTE: The size has to be determined empirically by trial and error, but it is recommended that it is adjusted to enable sampling of 2-3 cells per disector8.</li>
	</ol>
	</li>
	<li>Following this final step of the pilot study begin cell sampling. 
	NOTE: The obtained sampling parameters should result in counts of approximately 150-200 cells in each animal which is sufficient to obtain an efficient stereological design8.</li>
	<li>Count the BrdU-positive neurons using a 100&#215; oil-immersion objective and a final magnification of 2000-3000&#215;. In each optical disector, identify any BrdU-labelled neurons that are clearly recognized by the feature of interest (in the present study, the criterion is when the leading edge of the BrdU-labelled neuron comes into focus for the first time), are located inside the counting frame or touch the inclusion lines (Figure 2B). Do not count the BrdU-positive neurons that, when clearly recognized by the feature of interest inside the disector height, touch the exclusion lines. It is critically important to follow scrupulously these counting rules throughout the entire stereological quantification. 
	NOTE: As BrdU incorporates into all dividing cells, morphology is used to distinguish between neurons and other types of cells. Neuron characteristics are a clearly defined nucleus with a neutral cytoplasm and a dark nucleolus. BrdU-positive cells are recognized as newly formed neurons based on their relatively larger size compared to glial cells. It should be mentioned that in studies of short-term proliferation (e.g. &#60; 24 h) double-staining using immature neuronal markers may be a prerequisite17.</li>
	<li>Estimate the total number of BrdU-positive neurons in each brain by multiplying the total number of cells counted (&#931;Q-) with the reciprocal sampling fractions: 
	<img alt="Equation" src="/files/ftp_upload/55737/55737eq1.jpg" /> 
	for <img alt="Equation" src="/files/ftp_upload/55737/55737eq2.jpg" /> 
	where <img alt="Equation" src="/files/ftp_upload/55737/55737eq3.jpg" />Q- is the number-weighted mean section thickness, h is the height of the disector, asf is the area sampling fraction, ssf is the section sampling fraction and ti is the section thickness in the ith counting frame with a cell count of <img alt="Equation" src="/files/ftp_upload/55737/55737eq4.jpg" /> in the disector14,18.</li>
	<li>Calculate the coefficient of error (CE)8 which is the sampling variance of BrdU-positive neurons in systematic uniformly random sampling (SURS).
	<ol>
		<li>First, calculate the noise, which is equal to the total number of BrdU-positive neurons counted: 
		<img alt="Equation" src="/files/ftp_upload/55737/55737eq5.jpg" /></li>
		<li>Next, calculate the variance in SURS: 
		NOTE: We have judged the area and cell counts in the hippocampus to change smoothly from section to section and consequently used &#34;smoothness class&#34; m = 1 (1/240)8,19<img alt="Equation" src="/files/ftp_upload/55737/55737eq6.jpg" /> 
		where 
		<img alt="Equation" src="/files/ftp_upload/55737/55737eq7.jpg" /> 
		<img alt="Equation" src="/files/ftp_upload/55737/55737eq8.jpg" /> 
		<img alt="Equation" src="/files/ftp_upload/55737/55737eq9.jpg" /> 
		Pi is the number of points counted for the object in the ith section and n is the number of sections4,20.</li>
		<li>Finally, calculate the CE of the estimate: 
		NOTE: In general, optimal precision is typically achieved when the CE value is less than half of the observed CV, as OCV2 = ICV2 + CE2, where OCV is the observed CV and ICV is the inherent CV4. 
		<img alt="Equation" src="/files/ftp_upload/55737/55737eq10.jpg" /></li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55737/55737fig1.jpg" /> 
Figure 1: Schematic illustration showing tissue processing according to the fractionator principles used in the present study. 
Following experimentation, the hemispheres are separated and right or left hemisphere chosen systematically and randomly (A). The complete brain extending over the entire hippocampus is cut into 80-&#181;m-thick coronal sections (B), whereupon every 5th section is sub-sampled, starting randomly between section 1 to 5, for final immunostaining and stereological quantification (C). <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55737/55737fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55737/55737fig2.jpg" /> 
Figure 2: Illustration showing the stereological sampling procedure using optical disectors. 
(A) Following histological processing the hippocampal GCL/SGZ was delineate and optical disectors applied uniformly and randomly over the region. (B) In the optical disectors (left), the BrdU-positive neurons were counted only if their feature of interest were clearly recognized within the disector height, and located inside the counting frame or touching the inclusion line (right). Neurons touching the exclusion line were not counted. Scale bars = 10 &#181;m. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55737/55737fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have no competing financial interests.

Using ECS as a methodology to induce neurogenesis, we find an immediate increase of 260% in the formation of new BrdU-positive neurons in the hippocampus. In this pool of acutely generated neurons we found 40% attrition from day 1 to three months, with nearly 50% of the newly formed neurons surviving at least 12 months following treatment10,11. The counting of BrdU-labelled neurons followed a strict sampling scheme, whereby the entire hippocampus was cut into 80-&#181;m-thick sections followed by sub-sampling of every 5th section with a random sampling start between section one and section five. Providing these sections are chosen in a systematic random manner, this is demonstrably an excellent method to reduce the variance of the end result, without exhaustive counting1. This sampling scheme allowed us to count BrdU-positive neurons in an average of 12 (8-16) hippocampal sections in each rat brain, with a final precision of 9-11%.
When using the fractionator method, it is essential to know the fraction of the section height in which counting is performed, since tissue shrinkage and deformation frequently occurs during histological processing14. Indeed, we saw substantial shrinkage of thickness in this study. Furthermore, it should be noted that differential tissue shrinkage may occur, as presented in Dorph-Petersen et al. 2001. However, as long as the complete structure of interest is available for analysis, these limitations do not result in a bias of the total numbers of particles, i.e. BrdU-positive neurons. In studies where tissue shrinkage is a particular issue, it should always be stated that the results are obtained in deformed tissue. In this study, we counted in a disector height of 10 &#181;m. The sections of the present study had a final mean thickness of 26 &#181;m, a 5-&#181;m guard zone at the top and a 11-&#181;m (mean) guard zone at the bottom of the section. These parameters are acceptable since guard zones should be approximately the diameter of the sampled particles.
Following delineation of the GCL/SGZ, disectors were uniformly randomly placed within the delineated area. The disectors should be positioned with a fixed step length that optimizes sampling and counting to efficiently obtain estimates with a precision determined by the investigator. Optimal precision is typically achieved by counting up to 150-200 cells in each structure of interest5. In the present study, we counted a mean of 133 (range 33-372) BrdU-positive neurons in each rat hippocampus. Due to a low cell numbers in some of the control animals, our counts of BrdU-positive neurons fell below the generally acceptable number of cells required to obtain a suitable precision, which resulted in relatively high CE values for those cases. However, as we obtained CE-values of less than half of the CV-values (see representative result section) additional sampling and counting was not required. The high CV-values show that the greatest contributor to the observed variation originates from the biological variation. Indeed, we might assert that the average number of BrdU-positive neurons counted in the present study was higher than necessary. For example, in one group of rats we achieved a CE-value of 9%, and observed a CV-value of 43%. In this particular case, we could have aimed for a precision of approximately 20%. In summary, the precision of the total number of BrdU-positive neurons in the present study is sufficient in that it captures real treatment effects on the true numbers of particles. Due to the rather large biological variation in certain groups of animals the same estimates could have been obtained with an acceptable precision, despite less expenditure of effort. High biological variances within groups can only be compensated by increasing the number of animals.
One might argue that the gold standard for obtaining exact cell numbers entails exhaustive counting of all the objects of interest. However, in most studies of the brain this is not a possibility due to the vast numbers of cells. Although much more efficient than exhaustive cell counting, the optional fractionator is relatively time consuming in comparison to simple screening of differences in cell numbers which is preferred if the accurate number of cells is not essential. It is our experience that differences of more than 20-30% can be detected purely by screening procedures.
If performed correctly, sampling using design-based stereology provides unbiased and precise estimates in an efficient manner1. The unbiased property of stereology produces estimates that, when replicated, approximate the true population mean, and enhance the reproducibility of the estimate21. Initially, a representative sample of the entire structure of interest (in this study the hippocampal GCL/SGZ) must be obtained, allowing for estimation in a statistically valid subset of sections4. To obtain an appropriate number of sections and counting probes we apply SURS, which reduces the variance compared to random sampling8. Furthermore, SURS ensures that the particles of interest within the structure are sampled with the same probabilities, independent of their size, shape, orientation and distribution in the structure. As such stereology is to be highly recommended when obtaining precise and unbiased estimates is a central aspect of the project.

Quantification of cells in a three-dimensional (3-D) structure may require obtaining estimates based on unbiased principles. Even today, studies frequently use two-dimensional (2-D) methods to report data of 3-D structures. However, these methods do not consider the heterogeneous nature of the structure in terms of shape and distribution of cells resulting in methodical limitations; they make assumptions about the 3-D structure of interest and the results do not refer to the complete structure. These limitations reduce the sensitivity and accuracy of the methods as well as increase the risk of errors1.
Bias in cell counting can be avoided by the application of design-based stereology, which is of general importance for obtaining quantitative information about the number of cells in a given tissue or structure. While stereology has formerly been a very time-consuming method, advances in procedures, software and imaging, has made it&#160;become much more efficient and widely applicable. Stereology entails statistical sampling principles and stochastic geometric theory to provide efficient tools for estimation of volume, surface area, length, and number of objects in a 3-D structure by sampling in 2-D sections2. Thus, stereology allows one to obtain unbiased quantitative data on structural changes in tissue sections, which give average estimations of the true numbers, without exhaustive analysis1. Stereology is defined as the statistical inference of geometric parameters from sampled information. This can be achieved by unbiased sampling, that is to say systematic random sampling, of sections as well as counting rules that ensure that all objects have equal probabilities of being counted3. While stereological results can have varying degrees of precision as indicated by the coefficient of error (CE), this can be adjusted to suit the inherent biological variance (coefficient of variance (CV)) by adjusting the amount of sampling as required4,5.&#160;A few previous stereological studies have examined the short-term effects of electroconvulsive stimulation (ECS) on hippocampal plasticity in rodents6,7. Data from these studies show an initial increase in the total number of neurons as well as elevated synaptic differentiation following repeated ECS. However, these studies do not estimate neurogenesis directly, as they do not use specific markers for cell proliferation.
Here we present an application of a stereological method, the optical fractionator technique8,9, for quantifying the total number of newly-formed neurons in the rat hippocampal granule cell layer (GCL), including the sub-granular zone (SGZ) as well as their long-term survival10,11. We subjected rats to a clinical relevant schedule of ECS (three times a week for 3 weeks), which is one of the most potent stimulators of neurogenesis12. Control animals were treated using the same procedure, but without passing of current. On each of the 21 days of experimentation, all rats were injected intraperitoneally with 5-bromo-2`-deoxyuridine (BrdU) that incorporates into DNA in place of thymidine during cell division13. Following experimentation, the rats were maintained for four different time periods (24 hours, 3 months, 6 months and 12 months). Using the fractionator principle, we obtained a known fraction of the hippocampus through uniform random sampling of sections, and applied optical disectors (3-D probes) to the sampled and immunostained thick tissue sections. It is especially noted that frozen sections usually collapse in the z-axis during processing, and that optical disectors based upon the number-weighted mean section thickness should be used in this particular case14. As the focal plane of the optical disectors was moved a known distance down through the section, BrdU-positive neurons were counted when their feature of interest was clearly recognized. In the field of stereology there is some debate about the total number of particles that should be counted15; typically 150-200 neurons are counted in the structure of interest to obtain adequate precision i.e. a coefficient of 7-8%8. The BrdU-positive neuronal counts were completed using stereological software with imaging by a standard microscope equipped with camera and the following objectives: 2X, 4X and 10X as well as a 100X oil-immersion objective (numerical aperture = 1.40), and a motorized stage. Movements in the z-direction were measured with a digital microcator. The final magnification was 3000X.

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Materials&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>5&#039;-bromo-2&#039;deoxyuridine - BrdU</td><td>Sigma-Aldrich</td><td>19-160</td><td></td></tr><tr><td>Cresyl Violet</td><td>Sigma-Aldrich</td><td>C5042</td><td>Contains the following materials for 1 L: 0.2 g cresyl violet; 2.0 g sodiumacetate, 3 H2O; 3 ml glacial acetic acid; 1000 ml destilled water&amp;nbsp;</td></tr><tr><td>Cryoprotectant</td><td>Capital Region Pharmacy, DK</td><td>861334</td><td>Contains the following materials: Sucrose; polyvinylpyrrolidon PVP 40; sodiumphosphatebuffer (0.2 M);&amp;nbsp; ethylenglycol; demineralized water.</td></tr><tr><td>dH&lt;sub&gt;2&lt;/sub&gt;O</td><td>Capital Region Pharmacy, DK</td><td>817205</td><td></td></tr><tr><td>Diaminobenzidine &amp;ndash; DAB</td><td>Sigma-Aldrich</td><td>D5637</td><td></td></tr><tr><td>Envision-HRP</td><td>DAKO</td><td>K4001</td><td>Peroxidase labeled polymer conjugated to goat anti-mouse immunoglobulins</td></tr><tr><td>Ethanol - 70 %</td><td>Plum A/S</td><td>201098</td><td></td></tr><tr><td>Ethanol - 96 %</td><td>Plum A/S</td><td>201104</td><td></td></tr><tr><td>Ethanol - 99.9 %</td><td>Plum A/S</td><td>201111</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>In Vitro</td><td>BI-04-003-1A</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; 30%</td><td>Capital Region Pharmacy, DK</td><td>896456</td><td></td></tr><tr><td>HCl</td><td>J. T. Baker</td><td>6081</td><td></td></tr><tr><td>Mounting medium</td><td>Sakura</td><td>4583</td><td>Tissue Tek</td></tr><tr><td>Mouse-anti-BrdU</td><td>Becton-Dickinson</td><td>347580</td><td></td></tr><tr><td>PBS buffer</td><td>Capital Region Pharmacy, DK</td><td>853436</td><td>pH = 7.4</td></tr><tr><td>PFA</td><td>VWR</td><td>28,794,295</td><td>pH = 7.4</td></tr><tr><td>Phosphate buffer&amp;nbsp;</td><td>Capital Region Pharmacy, DK</td><td>866533</td><td>0.1 mol/l, pH 7.4</td></tr><tr><td>Rapid drying mounting medium</td><td>Histolab Products AB</td><td>801</td><td>Pertex</td></tr><tr><td>Sodium Tetraborate</td><td>Merck A/S</td><td>1,063,080,500</td><td></td></tr><tr><td>Sucrose</td><td>Merck A/S</td><td>1,076,515,000</td><td></td></tr><tr><td>Triton X-100</td><td>Merck A/S</td><td>1,086,031,000</td><td></td></tr><tr><td>Xylene</td><td>VWR</td><td>28,973,363</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Equipment&lt;/em&gt;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cover slips</td><td>Menzel-Gl&amp;auml;ser</td><td>24x50 mm #0</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>&amp;nbsp;CM1860</td><td></td></tr><tr><td>Digital Microcator</td><td>Heidenhain</td><td>VRZ 401</td><td></td></tr><tr><td>Glass dishes</td><td>Brain Resaerch Laboratories</td><td>1256</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse 80i</td><td></td></tr><tr><td>Microscope camera</td><td>Olympus</td><td>DP72</td><td></td></tr><tr><td>Microscope slides</td><td>VWR</td><td>48311-703</td><td>SuperFrost+</td></tr><tr><td>Motorized stage</td><td>Prior</td><td>H101A</td><td></td></tr><tr><td>Multidish Containers</td><td>Sigma-Aldrich</td><td>D6315-1CS</td><td>&amp;nbsp;Nuclon 12-wells</td></tr><tr><td>New-Cast software</td><td>Visiopharm</td><td>ver. 4.5.6.440</td><td></td></tr><tr><td>Objective 2x</td><td>Nikon</td><td>CFI Plan UW 2X</td><td></td></tr><tr><td>Objective 4x</td><td>Nikon</td><td>CFI Plan Fluor 4X</td><td></td></tr><tr><td>Objective 10x</td><td>Nikon</td><td>CFI Plan Fluor 10X</td><td></td></tr><tr><td>Objective 100x oil immersion</td><td>Nikon</td><td>CFI Plan Apo Lambda DM 100X Oil</td><td>Numerical aperture 1.40</td></tr><tr><td>Orbital shaker</td><td>Gemini BV</td><td>CM-9</td><td>Sarstedt</td></tr><tr><td>Slide rack</td><td>Sakura</td><td>4465</td><td></td></tr><tr><td>Slide staining dishes</td><td>Sakura</td><td>4457</td><td>Tissue-Tek</td></tr><tr><td>Specimen disc</td><td>Leica</td><td>14037008637</td><td></td></tr><tr><td>Staining nets</td><td>Brain Research Laboratories</td><td>2512</td><td>25-wells</td></tr></tbody></table>

the optical fractionator technique to estimate cell numbers in a rat model of electroconvulsive therapy

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells or structures. These methods have been revolutionary for quantitative analysis of the mammalian brain, in which volumetric cell populations are too high to count manually, and stereology is now the technique of choice whenever estimates of three-dimensional quantities need to be extracted from measurements on two-dimensional sections. All stereological methods are in principle unbiased; however, they rely on proper knowledge about the structure of interest and the characteristics of the tissue. Stereology is based on Systematic Uniformly Random Sampling (SURS), with adjustment of sampling to the most efficient level in respect to precision, providing reliable, quantitative information about the whole structure of interest. Here we present the optical fractionator in conjunction with BrdU immunohistochemistry to estimate the production and survival of newly-formed neurons in the granule cell layer (including the sub-granular zone) of the rat hippocampus following electroconvulsive stimulation, which is among the most potent stimulators of neurogenesis. The optical fractionator technique is designed to provide estimates of the total number of cells from thick sections sampled from the full structure. Thick sections provide the opportunity to observe cells in their full 3-D extent and thus, allow for easy and robust cell classification based on morphological criteria. When correctly implemented, the sensitivity and efficiency of the optical fractionator provides accurate estimates with a fixed and predetermined precision.

Sampling parameters
By analyzing the z-distribution in a pilot study (Figure 3), we found a uniform distribution of BrdU-positive neurons in the disector height. Shrinkage of the sections was measured; final mean thickness was 26.4 &#956;m (19.0-32.0 &#956;m) in sections originally cut at 80 &#956;m. Based on the tissue thickness and distribution of BrdU labelled neurons the disector height was set to 10 &#956;m, and the guard zones at the top and bottom of the section was set to 5-&#956;m and 4-17 &#956;m respectively. Depending of the density of the cells, the area of the counting frames was 1414 or 5210 &#956;m2. The step length was 220 &#956;m in both the x and y directions and cell counting was performed in an average of 12 (range 8-16) sections per animal. These sampling parameters resulted in a mean of 133 (range 33-372) BrdU-positive cells counted in 173 (107-204) disectors in each brain hemisphere.
As an example, we obtained the following CV and CE values for BrdU-positive neuronal estimations in ECS treated rats: 24 hours: CV = 0.59, CE = 0.11; 3 months: CV = 0.34, CE = 0.12; 6 months: CV = 0.43, CE = 0.09; 12 months: CV = 0.22, CE = 0.09.
We quantified the total number of newly-formed neurons and their long-term survival in the rat hippocampus following ECS using the optical fractionator in conjunction with a BrdU staining technique10,11. The final results showed a basal neurogenesis in the four groups of control animals, which did not significantly differ as a function of survival time (Figure 3). Furthermore, ECS induced a significant increase in the total number of BrdU-positive neurons at 24 hours (259%, p&#60;0.0001), 3 (229%, p&#60;0.001), 6 (152%, p&#60;0.05) and 12 months (162%, p&#60;0.05) following experimentation, as compared to controls animals.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55737/55737fig3.jpg" /> 
Figure 3: Quantitative data of the total number of BrdU-positive neurons in the GCL/SGZ of the rat hippocampus10,11. 
Horizontal bars represent the mean values. Abbreviations: GCL, granule cell layer; SGZ, sub-granular zone; h, hours; m, months. ****p&#60;0.0001, ***p&#60;0.001 and *p&#60;0.05 versus control (n=11-12 per group). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55737/55737fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy at JoVE.com

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

the-optical-fractionator-technique-to-estimate-cell-numbers-rat-model

Nanyang Technological University

Research

JoVE Journal

Neuroscience

11.6K Views.  Bispebjerg-Frederiksberg Hospital.  Hier presenteren we een stereologische methode, de optische fractionator, gebruikt om de vorming van nieuwe neuronen te kwantificeren, en hun overleving, in de rathippocampus na elektroconvulsieve stimulatie. Bij correcte implementatie zorgt de gevoeligheid en efficiëntie van stereologische methoden voor een nauwkeurige schatting met een vaste en vooraf bepaalde precisie. 

Video: The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Stem Cell Transplantation Strategies for the Restoration of Cognitive Dysfunction Caused by Cranial Radiotherapy

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation can induce a progressive and debilitating decline in cognition that may, in part, be caused by the depletion of neural stem cells. Given the increased survival of patients diagnosed with brain cancer, quality of life in terms of cognitive health has become an increasing concern, especially in the absence of any satisfactory long-term treatments.
To address this serious health concern we have used stem cell replacement as a strategy to combat radiation-induced cognitive decline. Our model utilizes athymic nude rats subjected to cranial irradiation. The ionizing radiation is delivered as either whole brain or as a highly focused beam to the hippocampus via linear accelerator (LINAC) based stereotaxic radiosurgery. Two days following irradiation, human neural stem cells (hNSCs) were stereotaxically transplanted into the hippocampus. Rats were then assessed for changes in cognition, grafted cell survival and for the expression of differentiation-specific markers 1 and 4-months after irradiation. Our cognitive testing paradigms have demonstrated that animals engrafted with hNSCs exhibit significant improvements in cognitive function. Unbiased stereology reveals significant survival (10-40%) of the engrafted cells at 1 and 4-months after transplantation, dependent on the amount and type of cells grafted. Engrafted cells migrate extensively, differentiate along glial and neuronal lineages, and express a range of immature and mature phenotypic markers.
Our data demonstrate direct cognitive benefits derived from engrafted human stem cells, suggesting that this procedure may one day afford a promising strategy for the long-term functional restoration of cognition in individuals subjected to cranial radiotherapy. To promote the dissemination of the critical procedures necessary to replicate and extend our studies, we have provided written and visual documentation of several key steps in our experimental plan, with an emphasis on stereotaxic radiosurgey and transplantation.

Brain tumor patients routinely undergo cranial radiotherapy, and while beneficial, this treatment often results in debilitating cognitive dysfunction. This serious unresolved problem has at present, no clinical recourse, and has driven our efforts to devise stem cell based therapies for the recovery of radiation-induced cognitive decrements.

Radiotherapy often provides the only clinical recourse for those afflicted with primary or metastatic brain tumors. While beneficial, cranial irradiation ...

Medicine

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham &#34;no seizure&#34; rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein &#945;-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy for severe depression. ECS globally stimulates activity in the hippocampus, leading to synaptogenesis and synaptic plasticity. Here, we describe methods for ECS induction in rats and for subcellular fractionation of their hippocampi to examine seizure-induced changes in synaptic proteins.

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS ...

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the substantia nigra, is essential. To estimate the total dopaminergic neuron number, unbiased stereology using the optical fractionator method is currently considered the gold standard. Because the theory behind the optical fractionator method is complex and because stereology is difficult to achieve without specialized equipment, several commercially available complete stereology systems that include the necessary software do exist, purely for cell counting reasons. Since purchasing a specialized stereology setup is not always feasible, for many reasons, this report describes a method for the stereological estimation of dopaminergic neuronal cell counts using standard microscopy equipment, including a light microscope, a motorized object table (x, y, z plane) with imaging software, and a computer for analysis. A step-by-step explanation is given on how to perform stereological quantification using the optical fractionator method, and pre-programmed files for the calculation of estimated cell counts are provided. To assess the accuracy of this method, a comparison to data obtained from a commercially available stereology apparatus was performed. Comparable cell numbers were found using this protocol and the stereology device, thus demonstrating the precision of this protocol for unbiased stereology.

This work presents a step-by-step protocol for the unbiased stereological estimation of dopaminergic neuronal cell numbers in the mouse substantia nigra using standard microscopy equipment (i.e., a light microscope, a motorized object table (x, y, z plane), and public domain software for digital image analysis.

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...

A Pipeline using Bilateral In Utero Electroporation to Interrogate Genetic Influences on Rodent Behavior

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.

The role of recently discovered disease-associated genes in the pathogenesis of neuropsychiatric disorders remains obscure. A modified bilateral in utero electroporation technique allows for the gene transfer in large populations of neurons and examination of the causative effects of gene expression changes on social behavior.

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution ...

Biology

Quantifying Newly Generated Neurons in Rat Brain Tissue Using the Optical Fractionator Technique

Source:<a style='text-decoration:none;' href='https://app-jove-com.remotexs.ntu.edu.sg/v/55737/the-optical-fractionator-technique-to-estimate-cell-numbers-in-a-rat-model-of-electroconvulsive-therapy'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> Olesen, M. V.,&#160;et al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2017).</a> &#160;The video demonstrates the technique for quantifying newly generated neurons in brain tissue. Hippocampal tissue slices containing Bromodeoxyuridine (BrdU)-labeled neurons were stained using immunohistochemistry and subsequently quantified across the tissue thickness using the optical fractionator technique.

Source: Olesen, M. V.,&#160;et al. The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy.&#160;J. Vis. ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

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