This work was supported by grants to W.Z.S. from the Medical Research and Development Service, Department of Veterans Affairs (Merit Review 1I01 BX001067-01A2), the Alzheimer&#39;s Association (NPSPAD-11-202149), and resources from the Midwest Biomedical Research Foundation.

All procedures for using these animals have been approved by the Kansas City Veterans Affairs Medical Center Institutional Animal Care and Use Committee. Eighteen-month-old mice overexpressing Swedish mutant beta-amyloid precursor protein (APPswe) and their C57/BL6 WT littermates were used for the experiments. Details of breeding and genotyping is given in He et al.8.
1. Perfusion and tissue processing
<ol>
	<li>Anesthetize mice using an intraperitoneal injection with ketamine (100 mg/kg) and xylazine (10 mg/kg). Pinch toes to confirm a lack of response before continuing9.</li>
	<li>Transcardially perfuse with ~50 mL of ice cold 0.1M Dulbecco&#39;s phosphate buffered saline (DPBS) followed by 4% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB)10. 
	CAUTION: PFA is toxic. Use personal protective equipment (PPE) when working with PFA.</li>
	<li>Remove brains10 and immerse in 4% PFA in 0.1 M PB at 4 &#176;C for postfixation for 24 h. Wash with DPBS 3x.</li>
	<li>Change to 15% sucrose solution in 0.1 DPBS overnight and then 30% sucrose solution in 0.1 M DPBS for another 48 h at 4 &#176;C.</li>
	<li>Remove the tissue from the sucrose solution and freeze in a cryotome chamber preset to a temperature of -20 &#176;C. Frozen samples can be stored in sealed tubes at -80 &#176;C until sectioning.</li>
	<li>Embed tissues in optimum cutting temperature embedding medium (see Table of Materials) and mount on a specimen disc. Cut serial 30 &#181;m thick sections in a coronal plane using a cryotome and collect all the sections consequently in 24 well culture plates filled with cryoprotectant solution (30% glycerol, 30% ethylene glycol, 40% DPBS; Figure 1A&#8211;C). Store in a -20 &#176;C freezer. 
	NOTE: Thicker sections (e.g., 50 &#181;m) are preferable when feasible. Make sure that the sections are kept in cutting order and all sections are completely in cryoprotectant. A 96 well plate can be used as an alternative to 24 well plates to collect sections.</li>
	<li>Cover the wells with plate sealer to prevent evaporation and drying. Store plates at -20 &#176;C until further use. Sections stored at -20 &#176;C are stable for several months for ChAT immunohistochemistry.</li>
</ol>
2. Systematic section selection for IHC
NOTE: A pilot study should be done to know the total number of sections required to achieve an acceptable coefficient of error (CE). The CE value is an expression of the total amount of error in the sampling procedure. The lowest value represents the minimal error and a CE value lower than 0.1 is considered acceptable by the software used here (see Table of Materials)11.
<ol>
	<li>Identify the first and last sections for each brain by comparing morphological features with a standard mouse brain atlas such as Franklin and Paxinos. NBM begins at bregma -0.0mm and ends at -1.6 mm. Therefore, about 50 sections contain NBM. The total number of sections is one of the parameters required during stereology. The selection of every 8th section (SSF = 1/8) gave a total of 6&#8211;7 sections for the analysis and yielded an acceptable CE for both volume estimation and fiber length estimation in our pilot study.</li>
	<li>Randomly start with one of the first eight sections and then systemically sample every 8th section until the last posterior section containing NBM (Figure 1C).</li>
</ol>
3. Immunohistochemistry
<ol>
	<li>Transfer the cryopreserved sections to room temperature in cryoprotectant and then 0.1M phosphate buffer (PB) in 6 well plates.</li>
	<li>Wash 3x in PB.</li>
	<li>Incubate in 0.3% H2O2 in methanol for 15 min.</li>
	<li>Wash 3x in Tris-buffered saline (TBS).</li>
	<li>Incubate in 0.25% Triton X-100 in TBS for 30 min.</li>
	<li>Block with 10% normal bovine serum (NBS) in 0.1% Triton X-100 in TBS for 30 min.</li>
	<li>Incubate with a 1:1,000 dilution of goat anti-human ChAT primary antibodies (see Table of Materials) in TBS with 0.1% Triton X-100 and 1% NBS for 48 h at 4 &#176;C.</li>
	<li>Wash 3x in TBS at room temperature. Perform all further incubation and washing at room temperature.</li>
	<li>Incubate with biotinylated bovine anti-goat secondary antibody (see Table of Materials) for 1 h.</li>
	<li>Wash 3x in TBS.</li>
	<li>Incubate with the avidin-biotin&#8211;peroxidase complex (see Table of Materials).</li>
	<li>Wash 3x in TBS.</li>
	<li>Develop using an enhanced DAB peroxidase substrate solution (see Table of Materials) according to the manufacturer&#39;s recommendations. 
	CAUTION: DAB is a suspected carcinogen. It is toxic by contact and inhalation. Use PPE when working with DAB.</li>
	<li>Wash the section a couple of times with distilled water and keep in Tris pH = 7.6.</li>
	<li>Mount the sections on the gelatinized slides. All sections from one tissue can be put on the same slide. Air dry the sections, dehydrate 2x for 5 min each with 70%, 90%, 95%, and 100% ethanol, and then clear the sections with two 10 min xylene washes. Coverslip the sections using mounting medium (see Table of Materials). 
	NOTE: The processing time of dehydration and clearing affects the thickness of the sections. Therefore, the same conditions should be used for all sections. In the current study, the mean value for the final thickness was 21.11 &#177; 0.45 &#181;m.</li>
	<li>Keep the sections in the fume hood to dry. The dry sections are ready for stereology.</li>
</ol>
4. Stereology
NOTE: See the Table of Materials for the microscope and software used. An immersion objective with a numerical aperture (NA) &#62; 1.2 will be useful and should be used if required. Slides should be grouped according to genotype or treatment group and coded. The complete stereology for one study should be performed by the same person and the person performing the stereology should be blind to the identity of the individual slides or group examined1,12,13.
<ol>
	<li>Open a new study in the software. (File &#62; New Study). A &#39;Study Initialization&#39; dialog box will open. Fill out the study information, using Multi-level (Fraction Based) (Figure 2A).</li>
	<li>Double click on &#39;Volume&#39; under Parameters, which will open the Volume dialog box. Name the feature of interest and select &#39;Region Point Counting&#39; probe. Click Next.</li>
	<li>Double click on the next parameter, &#39;Length&#39;.
	<ol>
		<li>Provide a name of the feature (e.g., &#39;L&#39;) Select &#39;Sphere&#39; probe and click Next.</li>
	</ol>
	</li>
	<li>Next, click on the &#39;Study Initialization&#39; dialog box, which will open the &#39;Case Initialization&#39; box. Fill in the case information. Groups must be coded before starting the study. The total number of sections is the number of sections starting from the first section containing the area of interest to the last section containing area of interest (see step 2.1). The section sampling interval is eight, because every 8th section was selected for the IHC staining.</li>
	<li>Clicking Next opens the &#39;Probe Initialization&#39; dialog box, which will automatically set the lowest magnification for the region selection and volume estimation. Confirm the settings and check if the region of interest can be identified at the selected lower magnification. Double click on &#39;Volume&#39; and fill 50,000 &#181;m3 per point for the region volume fraction. Click Done.</li>
	<li>Under Object (High) Magnification, set Length at 63x or 100x. Double click on &#39;Length&#39; to open the Length-Sphere dialog box and set the diameter of the sphere to 10 &#181;m. Click Done. 
	NOTE: The guard zone should be determined based on the actual thickness of the sections. The operator must check the thickness of the sections at multiple sites to avoid any damage. If necessary, adjust the guard zone thickness based on section thickness.</li>
	<li>Set appropriate values for frame area, frame height, guard zone, and frame spacing. 
	NOTE: These values depend on the heterogeneity of the object profiles (fibers) in the region of study. A frame area of 400 &#181;m2, frame height of 10 &#181;m, guard zone of 2 &#181;m, and frame spacing of 300 &#181;m gives acceptable CE values for fiber length estimation in NBM. A pilot study must be performed with these values before heading to the next case.</li>
	<li>Follow the instructions provided by the software after each step. The instructions appear either as a dialog box and/or at the bottom of the screen.</li>
	<li>Insert Section 1.</li>
	<li>At low magnification (5x), define the region of interest by making an arbitrary boundary around the NBM. Click the Next button at the left corner of the video window. Follow the instructions and confirm all green points are in the NBM. The points can be included or excluded by clicking on the points. 
	NOTE: There is no definite, set boundary around the NBM. The selection is mostly investigator-defined. The ChAT+ cholinergic neurons of the NBM can be observed in the internal capsule (ic) and globus pallidus (GP). In this protocol, the ic with ChAT+ cholinergic neurons and their projections (fibers) and whole GP is included in the NBM (Figure 1D).</li>
	<li>Follow the instructions and change to 63x for fiber measurements at the current fraction. The &#39;Section Thickness&#39; dialog box appears on the screen. Set the top and bottom surface of the section to measure the actual thickness of the section. The manual Z axis movement should be used. Click on Done. 
	NOTE: If there is no fiber in the area, the step can be skipped to go to the next fraction location.</li>
	<li>Move the Z axis slowly from top to bottom in the frame height of the section and mark all intersecting fibers on the surface of the virtual sphere probe (Figure 3). When done, click Next to go to the next location.</li>
	<li>After completing all the fractions, the software will ask to insert the next section. Repeat steps 4.9&#8211;4.12 for all six or seven sections of the tissue.
	<ol>
		<li>At the end, the software will generate a result for the case showing the CE values (Figure 4). If the CE is acceptable, proceed to the next case. File &#62; New Case. If the CE is not acceptable (Figure 4A), the software provides recommendations to change some of the parameters. In many cases, decreasing the frame spacing decreases the CE values to an acceptable range (Figure 4B).</li>
	</ol>
	</li>
	<li>After completing all the cases, generate results for each case and group (File &#62; Results).</li>
</ol>
5. Analysis and statistics
<ol>
	<li>The software provides data for each sample and each group. The software itself calculates fractions such as SSF, ASF, and TSF, and provides the total reference volume (VREF) and total length (L) of the fibers in the reference region (in this case, NBM) (Figure 4B). Export the data and save or copy to the statistical software of choice for between group analysis. Table 1 shows typical result data for statistical analysis. Analyze fiber density (Lv) by dividing the total fiber length with the reference volume.</li>
</ol>

<ol>
	<li>Mouton, P. R., Gokhale, A. M., Ward, N. L., West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+length+estimation+using+spherical+probes.">Stereological length estimation using spherical probes.</a> Journal of Microscopy. 206, 54-64 (2002).</li><li>Whitehouse, P. J., Price, D. L., Clark, A. W., Long Coyle, J. T., DeLong, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer+disease:+evidence+for+selective+loss+of+cholinergic+neurons+in+the+nucleus+basalis.">Alzheimer disease: evidence for selective loss of cholinergic neurons in the nucleus basalis.</a> Annals of Neurology. 10 (2), 122-126 (1981).</li><li>Davies, P., Maloney, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+loss+of+central+cholinergic+neurons+in+Alzheimer's+disease.">Selective loss of central cholinergic neurons in Alzheimer's disease.</a> The Lancet. 2 (8000), 1403 (1976).</li><li>Bartus, R. T., Dean, R. L., Beer, B., Lippa, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cholinergic+hypothesis+of+geriatric+memory+dysfunction.">The cholinergic hypothesis of geriatric memory dysfunction.</a> Science. 217 (4558), 408-414 (1982).</li><li>Savonenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Episodic-like+memory+deficits+in+the+APPswe/PS1dE9+mouse+model+of+Alzheimer's+disease:+relationships+to+beta-amyloid+deposition+and+neurotransmitter+abnormalities.">Episodic-like memory deficits in the APPswe/PS1dE9 mouse model of Alzheimer's disease: relationships to beta-amyloid deposition and neurotransmitter abnormalities.</a> Neurobiology of Disease. 18 (3), 602-617 (2005).</li><li>Perez, S. E., Dar, S., Ikonomovic, M. D., DeKosky, S. T., Mufson, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+forebrain+degeneration+in+the+APPswe/PS1DeltaE9+transgenic+mouse.">Cholinergic forebrain degeneration in the APPswe/PS1DeltaE9 transgenic mouse.</a> Neurobiology of Disease. 28 (1), 3-15 (2007).</li><li>Stokin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonopathy+and+transport+deficits+early+in+the+pathogenesis+of+Alzheimer's+disease.">Axonopathy and transport deficits early in the pathogenesis of Alzheimer's disease.</a> Science. 307 (5713), 1282-1288 (2005).</li><li>He, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GRK5+Deficiency+Leads+to+Selective+Basal+Forebrain+Cholinergic+Neuronal+Vulnerability.">GRK5 Deficiency Leads to Selective Basal Forebrain Cholinergic Neuronal Vulnerability.</a> Scientific Reports. 6, 26116 (2016).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab+Animal+Research.+Anesthesia+Induction+and+Maintenance.">Lab Animal Research. Anesthesia Induction and Maintenance.</a> Journal of Visualized Experiments. , (2019).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> Journal of Visualized Experiments. (65), e3564 (2012).</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> Unbiased Stereology-A Concise Guide. , (2011).</li><li>West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+started+in+stereology.">Getting started in stereology.</a> Cold Spring Harbor Protocols. 2013 (4), 287-297 (2013).</li><li>West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Space+Balls+Revisited:+Stereological+Estimates+of+Length+With+Virtual+Isotropic+Surface+Probes.">Space Balls Revisited: Stereological Estimates of Length With Virtual Isotropic Surface Probes.</a> Frontiers in Neuroanatomy. 12, 49 (2018).</li><li>Nikolajsen, G. N., Kotynski, K. A., Jensen, M. S., West, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+the+capillary+network+of+aged+APPswe/PS1dE9+transgenic+mice.">Quantitative analysis of the capillary network of aged APPswe/PS1dE9 transgenic mice.</a> Neurobiology of Aging. 36 (11), 2954-2962 (2015).</li><li>Gutierrez-Jimenez, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbances+in+the+control+of+capillary+flow+in+an+aged+APP(swe)/PS1DeltaE9+model+of+Alzheimer's+disease.">Disturbances in the control of capillary flow in an aged APP(swe)/PS1DeltaE9 model of Alzheimer's disease.</a> Neurobiology of Aging. 62, 82-94 (2018).</li><li>Gundersen, H. J., Jensen, E. B., Kieu, K., Nielsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+systematic+sampling+in+stereology--reconsidered.">The efficiency of systematic sampling in stereology--reconsidered.</a> Journal of Microscopy. 193, 199-211 (1999).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+study+of+the+capillaries+within+the+white+matter+of+the+Tg2576+mouse+model+of+Alzheimer's+disease.">Quantitative study of the capillaries within the white matter of the Tg2576 mouse model of Alzheimer's disease.</a> Brain and Behavior. 9 (4), 01268 (2019).</li><li>McNeal, D. W. <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+Analysis+of+Reactive+Astrogliosis+to+Estimate+Age-Associated+Cerebral+White+Matter+Injury.">Unbiased Stereological Analysis of Reactive Astrogliosis to Estimate Age-Associated Cerebral White Matter Injury.</a> Journal of Neuropathology Experimental Neurology. 75 (6), 539-554 (2016).</li><li>Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+(amyloid-beta)+immunotherapy+attenuates+monoaminergic+axonal+degeneration+in+the+AbetaPPswe/PS1dE9+mice.">Passive (amyloid-beta) immunotherapy attenuates monoaminergic axonal degeneration in the AbetaPPswe/PS1dE9 mice.</a> Journal of Alzheimer's Disease. 23 (2), 271-279 (2011).</li><li>Gagnon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Sprouting+of+Dopamine+and+Serotonin+Axons+in+the+Pallidum+of+Parkinsonian+Monkeys.">Evidence for Sprouting of Dopamine and Serotonin Axons in the Pallidum of Parkinsonian Monkeys.</a> Frontiers of Neuroanatomy. 12, 38 (2018).</li><li>Boncristiano, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+changes+in+the+APP23+transgenic+mouse+model+of+cerebral+amyloidosis.">Cholinergic changes in the APP23 transgenic mouse model of cerebral amyloidosis.</a> Journal of Neuroscience. 22 (8), 3234-3243 (2002).</li></ol>

The authors have nothing to disclose.

Here we demonstrate a method to estimate the density of cholinergic fibers in the NBM using a space ball (sphere) probe. This probe estimates the total fiber length in the region of interest. The total length can be divided by the volume of the region to get the fiber density. To estimate the volume of the region, the Cavalieri point count method was used. The Cavalieri point count method is an unbiased and efficient estimator of a 3D reference volume for any region. The method calculates an estimate of the area on a cut...

Quantitative estimates of length and/or density of nerve fibers in the brain are important parameters of neuropathological studies. The length of cholinergic, dopaminergic, and serotonergic axons in various brain regions are often correlated with the specific functions of the region. Because the distribution of these axons is generally heterogeneous, design-based stereology is used to avoid bias during sampling. The space ball probe of stereology has been designed to provide efficient and reliable measures of line-like structures such as neuronal fibers in a region of interest1. The probe makes a virtual sphere that is imposed systematically in the tissue to measure line intersections with the surface of the probe. Because it is impossible to put sphere probes in the tissue for analysis, the commercially available software provides a virtual three-dimensional (3D) sphere, which is basically a series of concentric circles of various diameters that represent the surface of the sphere probe.
Selective cholinergic neurodegeneration is one of the consistent features of Alzheimer&#39;s disease (AD)2,3,4. Dysfunctional cholinergic transmission is considered a causative factor for cognitive decline in AD. Cholinergic dysfunction is also evident in many other mental disorders such as Parkinson&#39;s, addiction, and schizophrenia. Different aspects of cholinergic neurodegeneration are studied in animal models (e.g., reduction in acetylcholine5, ChAT protein6, cholinergic fiber neurodegeneration in the vicinity of amyloid plaques6, and decrease in cholinergic fibers and synaptic varicosities7,8). Fiber degeneration is believed to take place earlier than neuronal loss, because cholinergic neuronal loss is not always observed in studies. Most of the cholinergic neurons are in the basal forebrain and the brain stem, and their axons project to various brain regions such as the cortices and hippocampus. NBM is situated in the basal forebrain and found to be one of the commonly affected brain areas in AD.
The fractionator method of stereology is based on systematic random sampling of a tissue at multiple levels. Section sampling fraction (SSF) is the non-computer based systematic sampling of sections for the fractionator method of stereology. Area sampling fraction (ASF) is fractionation of an area of the region of interest in the section. Thickness sampling fraction (TSF) is the fractionation of the thickness of a section. The space ball probe allows us to quantify profiles of interest in a 3D sphere at fractionated locations. Here we use a space ball probe for estimation of the total length of cholinergic fibers in the NBM of mouse brain to illustrate the procedures. The current protocol provides details on tissue processing, sampling methods for stereology, immunohistochemical staining using the ChAT antibody, and unbiased stereology to estimate cholinergic fiber length and fiber density in the NBM of mouse brain.

<table><tbody><tr><td>ABC kit</td><td>Vector Laboratories</td><td>PK6100</td><td></td></tr><tr><td>Anti-ChAT Antibody</td><td>Millipore, MA, USA</td><td>AB144P</td><td></td></tr><tr><td>Bovine anti-goat IgG-B</td><td>Santacruz Biotechnology</td><td>SC-2347</td><td></td></tr><tr><td>Bovine Serum, Adult</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>B9433</td><td></td></tr><tr><td>Cryostat</td><td>Lieca Microsystems, Buffalo Grove, IL, USA</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>D5652</td><td></td></tr><tr><td>Ethylene Glycol</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>324558</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>G2025</td><td></td></tr><tr><td>Hydrogen Peroxide</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>H1009</td><td></td></tr><tr><td>Immpact-DAB kit</td><td>Vector Laboratories</td><td>SK4105</td><td>Enhanced DAB peroxidase substrate solution</td></tr><tr><td>Ketamine</td><td>Westward Pharmaceuticals, NJ, USA</td><td>0143-9509-01</td><td></td></tr><tr><td>Microscope</td><td>Lieca Microsystems, Buffalo Grove, IL, USA</td><td>AF6000</td><td>Equipped with motorized stage and IMI-tech color digital camera</td></tr><tr><td>Optimum cutting temperature (O.C.T.) embedding medium</td><td>Electron Microscopy Sciences, PA, USA</td><td>62550-12</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>P6148</td><td></td></tr><tr><td>Permount mounting medium</td><td>Electron Microscopy Sciences, PA, USA</td><td>17986-01</td><td></td></tr><tr><td>Stereologer Software</td><td>Stereology Resource Center, Inc. St. Petersburg, FL, USA</td><td>Stereologer2000</td><td>Installed on a Dell Desktop computer.</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>T8787</td><td></td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>T1503</td><td>Tris base</td></tr><tr><td>Trizma hydrochloride</td><td>Sigma-Aldrich, St. Louis, MO, USA</td><td>T5941</td><td>Tris hydrochloride</td></tr><tr><td>Xylazine</td><td>Bayer, Leverkusen, Germany</td><td>Rompun</td><td></td></tr><tr><td>Xylenes, Histological grade</td><td>Sigma-Aldrich, St. Louis, MO</td><td>534056</td><td></td></tr></tbody></table>

stereological estimation of cholinergic fiber length in the nucleus basalis of meynert of the mouse brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Representative results are shown in Table 1 and Figure 5. Group C, which was decoded as APPswe group (APP), had significantly lower fiber length (Figure 5B) and fiber length density (Figure 5C) compared to their wild type (WILD) littermates. The results showed that there was no significant difference in the volume of the NBM between the two groups analyzed (Figu...

Watch this Scientific Journal Video about Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain at JoVE.com

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...

stereological-estimation-cholinergic-fiber-length-nucleus-basalis

Research

JoVE Journal

Neuroscience

5.4K Views.  Kansas City Veterans Affairs Medical Center. Neurodenegratie bij veel neurologische aandoeningen is een chronisch proces waarvan neuronale vezelverlies vaak optreedt voorafgaand aan het verlies van het cellichaam. De stereologische kwantificering van neuronale vezels in drie dimensies biedt een gevoelige en nauwkeurige methode voor het detecteren van vroege neurodegeneratieve verandering. Deze technologie maakt gebruik van de betrouwbare en efficiënte computer-gebaseerde onpartijdige fractionatie van de ruimte ballen om de lengtes van ...

Video: Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Laser-guided Neuronal Tracing In Brain Explants

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake through electroporation in brain explants with targeted laser illumination and matching filter goggles during the procedure. The described technique of in vitro electroporation by itself yields relatively good visual control for targetting certain areas of the brain. By combining it with laser excitation of fluorescent genetic markers and their read-out through band-passing filter goggles, which can pick up the emissions of the genetically labeled cells/nuclei and the fluorescent tracing dye, a researcher can substantially increase the accuracy of injections by finding the area of interest and controlling for the dye-spread/uptake in the injection area much more efficiently. In addition, the laser illumination technique allows to study the functionality of a given neurocircuit by providing information about the type of neurons projecting to a certain area in cases where the GFP expression is linked to the type of transmitter expressed by a subpopulation of neurons.

We describe a technique to label neurons and their processes via anterograde or retrograde tracer injections into brain nuclei using an in vitro preparation. We modified an existing method of in vitro tracer electroporation by taking advantage of fluorescently labeled mouse mutants and basic optical equipment in order to increase labeling accuracy.

We present a technique which combines an in vitro tracer injection protocol, which uses a series of electrical and pressure pulses to increase dye uptake ...

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 ...

Morphological and Functional Evaluation of Ribbon Synapses at Specific Frequency Regions of the Mouse Cochlea

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have indicated that hair cell synapses may be the initial targets in sensorineural hearing loss (SNHL). Such studies have proposed the concept of cochlear &#34;synaptopathy&#34;, which refers to alterations in ribbon synapse number, structure, or function that result in abnormal synaptic transmission between IHCs and SGNs. While cochlear synaptopathy is irreversible, it does not affect the hearing threshold. In noise-induced experimental models, restricted damage to IHC synapses in select frequency regions is employed to identify the environmental factors that specifically cause synaptopathy, as well as the physiological consequences of disturbing this inner ear circuit. Here, we present a protocol for analyzing cochlear synaptic morphology and function at a specific frequency region in adult mice. In this protocol, cochlear localization of specific frequency regions is performed using place-frequency maps in conjunction with cochleogram data, following which the morphological characteristics of ribbon synapses are evaluated via synaptic immunostaining. The functional status of ribbon synapses is then determined based on the amplitudes of auditory brainstem response (ABR) wave I. The present report demonstrates that this approach can be used to deepen our understanding of the pathogenesis and mechanisms of synaptic dysfunction in the cochlea, which may aid in the development of novel therapeutic interventions.

This manuscript describes an experimental protocol for evaluating the morphological characteristics and functional status of ribbon synapses in normal mice. The present model is also suitable for noise-induced and age-related cochlear synaptopathy-restricted models. The correlative results of previous mouse studies are also discussed.

Cochlear inner hair cells (IHCs) transmit acoustic signals to spiral ganglion neurons (SGNs) through ribbon synapses. Several experimental studies have ...

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. This standard approach can be challenging for targets in the caudal brainstem and upper cervical cord due to specific anatomical challenges as these sites are remote from skull landmarks, leading to imprecision. Here we outline an alternative stereotaxic approach via the cisterna magna that has been used to target discrete regions of interest in the caudal brainstem and upper cervical cord. The cisterna magna extends from the occipital bone to the atlas (i.e., the second vertebral bone), is filled with cerebrospinal fluid, and is covered by dura mater. This approach provides a reproducible route of access to select central nervous system (CNS) structures that are otherwise hard to reach due to anatomical barriers. Furthermore, it allows for direct visualization of brainstem landmarks in close proximity to the target sites, increasing accuracy when delivering small injection volumes to restricted regions of interest in the caudal brainstem and upper cervical cord. Finally, this approach provides an opportunity to avoid the cerebellum, which can be important for motor and sensorimotor studies.

Stereotaxic Surgical Approach to Microinject the Caudal Brainstem and Upper Cervical Spinal Cord via the Cisterna Magna in Mice

Stereotaxic surgery to target brain sites in mice commonly involves access through the skull bones and is guided by skull landmarks. Here we outline an alternative stereotaxic approach to target the caudal brainstem and upper cervical spinal cord via the cisterna magna that relies on direct visualization of brainstem landmarks.

Stereotaxic surgery to target brain sites in mice is commonly guided by skull landmarks. Access is then obtained via burr holes drilled through the skull. ...

Immunolabeling and Counting Ribbon Synapses in Young Adult and Aged Gerbil Cochleae

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The most common method for detecting the loss of ribbon synapses is immunolabeling because it allows for quantitative sampling from several tonotopic locations in an individual cochlea. However, the structures of interest are buried deep inside the bony cochlea. Gerbils are used as an animal model for age-related hearing loss. Here, routine protocols for fixation, immunolabeling gerbil cochlear whole mounts, confocal imaging, and quantifying ribbon synapse numbers and volumes are described. Furthermore, the particular challenges associated with obtaining good material from valuable aging individuals are highlighted. 
Gerbils are euthanized and either perfused cardiovascularly, or their tympanic bullae are carefully dissected out of the skull. The cochleae are opened at the apex and base and directly transferred to the fixative. Irrespective of the initial method, the cochleae are postfixed and subsequently decalcified. The tissue is then labeled with primary antibodies against pre- and postsynaptic structures and hair cells. Next, the cochleae are incubated with secondary fluorescence-tagged antibodies that are specific against their respective primary ones. The cochleae of aged gerbils are then treated with an autofluorescence quencher to reduce the typically substantial background fluorescence of older animals' tissues. 
Finally, cochleae are dissected into 6-11 segments. The entire cochlear length is reconstructed such that specific cochlear locations can be reliably determined between individuals. Confocal image stacks, acquired sequentially, help visualize hair cells and synapses at the chosen locations. The confocal stacks are deconvolved, and the synapses are either counted manually using ImageJ, or more extensive quantification of synaptic structures is carried out with image analysis procedures custom-written in Matlab.

A protocol for processing young adult and aged gerbil cochleae by immunolabeling the afferent synaptic structures and hair cells, quenching autofluorescence in aged tissue, dissecting and estimating the length of the cochleae, and quantifying the synapses in image stacks obtained with confocal imaging is presented.

The loss of ribbon synapses connecting inner hair cells and afferent auditory nerve fibers is assumed to be one cause of age-related hearing loss. The ...

Artificial Intelligence Approaches to Assessing Primary Cilia

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. Cilia length is highly conserved, tightly regulated, and varies between different cell types and tissues and has been implicated in directly impacting their signaling capacity. For example, cilia have been shown to alter their lengths in response to activation of ciliary G protein-coupled receptors. However, accurately and reproducibly measuring the lengths of numerous cilia is a time-consuming and labor-intensive procedure. Current approaches are also error and bias prone. Artificial intelligence (Ai) programs can be utilized to overcome many of these challenges due to capabilities that permit assimilation, manipulation, and optimization of extensive data sets. Here, we demonstrate that an Ai module can be trained to recognize cilia in images from both in vivo and in vitro samples. After using the trained Ai to identify cilia, we are able to design and rapidly utilize applications that analyze hundreds of cilia in a single sample for length, fluorescence intensity and co-localization. This unbiased approach increased our confidence and rigor when comparing samples from different primary neuronal preps in vitro as well as across different brain regions within an animal and between animals. Moreover, this technique can be used to reliably analyze cilia dynamics from any cell type and tissue in a high-throughput manner across multiple samples and treatment groups. Ultimately, Ai-based approaches will likely become standard as most fields move toward less biased and more reproducible approaches for image acquisition and analysis.

The use of artificial intelligence (Ai) to analyze images is emerging as a powerful, less biased, and rapid approach compared with commonly used methods. Here we trained Ai to recognize a cellular organelle, primary cilia, and analyze properties such as length and staining intensity in a rigorous and reproducible manner.

Cilia are microtubule based cellular appendages that function as signaling centers for a diversity of signaling pathways in many mammalian cell types. ...

Biology

Immunohistochemical Staining of Nerve Fibers in the Nucleus Basalis of Meyner of the Mouse Brain

Source: Singh, P., et al. <a href="https://app.jove.com/v/60405">Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates the immunohistochemical staining of cholinergic fibers in the nucleus basalis of the Meynert region of mouse brain sections. Sections are blocked and treated with primary against choline acetyltransferase, followed by biotinylated secondary antibodies. Avidin-biotin-peroxidase complexes are added, which bind to secondary antibodies, followed by the chromogenic substrate. Sections are then dehydrated and mounted before visualizing under the microscope.

Source: Singh, P., et al. Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain. J. Vis. Exp. (2020)
...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

Samenvatting

Meer video's verkennen