Name: stereological estimation of cholinergic fiber length in the nucleus basalis of meynert of the mouse brain
Uploaded: 2020-02-05T12:00:00
Description: 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

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
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	<li>Anesthetize mice using an intraperitoneal injection with...

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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.

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			<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&#39;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>
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			<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>
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			<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 ...

Neurodenegration in many neurological disorders is a chronic process of which neuronal fiber loss often occurs prior to the cell body loss. The stereological quantification of neuronal fibers in three dimensions provides a sensitive and accurate method for detecting early neurodegenerative change. This technology utilizes the reliable and efficient computer-based unbiased fractionation of space balls to measure the lengths of line-like structures such as neuronal fibers in 3D.Demonstrating the procedure will be done by Prabhakar Singh, a post-doc and our expert in stereology, and David Peng, the research assistant in my lab. After confirming a lack of response to pedal reflex in the anesthetized mouse, transcardially perfuse with approximately 50 milliliters of ice cold 0.1 molar DPBS, followed by approximately 25 milliliters of 4%PFA in 0.1 molar PBS. At the end of the perfusion, post-fix the brain in a container of fresh 4%PFA for 24 hours at four degrees Celsius.The next day, wash the brain with three one to two-minute washes in fresh DPBS per wash and transfer the brain into a 15%sucrose in 0.1 DPBS solution overnight. The next morning, place the brain into a 30%sucrose solution and 0.1 molar DPBS for 48 hours at four degrees Celsius. At the end of the incubation, transfer the brain to a cryotome chamber pre-set to minus 20 degrees Celsius.Brains can be kept in sealed tubes and stored at minus 80 degrees Celsius. For sectioning, embed the tissue sample in optimum cutting temperature embedding medium and mount the sample on a specimen disc. Then acquire serial 30 micrometer thick sections in a coronal plane in the cryotome, transferring each section into individual wells of a 24-well culture plate containing cryoprotectant solution in preparation for minus 20 degrees Celsius storage.To identify the first and last sections of each brain, compare the morphological features with a standard mouse brain atlas. NBM begins at bregma minus 0.0 millimeters and ends at minus 1.6 millimeters. The selection of every eighth section yields a total of six to seven sections and allows an appropriate coefficient of error for both a volume and fiber length estimation.For immunohistochemical labeling of the tissue sections, after blocking any nonspecific binding with 10%normal bovine serum in 0.1%Triton X-100 in Tris-buffered saline, label the sections with the primary antibody of interest for 48 hours at four degrees Celsius. At the end of the incubation, wash the sections three times for three minutes in fresh Tris-buffered saline per wash, followed by incubation with an appropriate biotinylated secondary antibody for one hour at room temperature. At the end of the incubation, wash the sections three times in fresh Tris-buffered saline as demonstrated, followed by staining with Avidin biotin peroxidase complex and DAB peroxidase substrate solution according to the manufacturer's protocols.After washing, mount all of the sections from one brain tissue sample onto one gelatinized slide and allow the samples to air dry. To dehydrate the sections, immerse the samples two times for five minutes per dilution in sequential ascending ethanol solutions as indicated, followed by clearing with two 10-minute xylene washes. Then use an appropriate mounting medium to place coverslips onto the slides.For stereology, open a new study in the software. A study initialization dialogue box will open. Fill out the study information and confirm that multi-level fraction-based is selected.Double-click on volume to open the volume dialogue box. Name the feature of interest and select the region point counting probe and click next. Double-click on length and provide a name of the feature.Select sphere probe and click next. In the case initialization box, to code the groups before starting the study, set the total number of sections starting from the first section containing the area of interest to the last section containing the area of interest and the section sampling interval to eight. Click next to open the probe initialization dialogue box which will automatically set the lowest magnification for the region selection and volume estimation and confirm the settings.Click preview to check if the region of interest can be identified at the selected lower magnification. Enter 50, 000 micrometers cube per point for the region volume fraction and click preview and done. Under object high magnification, set the length to 63X and double-click length to open the length sphere dialogue box.Set the diameter of this sphere to 10 micrometers and click done. Set appropriate values for the frame area, frame height, guard zone, and frame spacing. Then click next and follow the instructions provided by the software.To insert the first section, place the slide onto the stage of the microscope under a 5X objective and click the green arrow to draw an arbitrary boundary around the NBM to define the region of interest. Click the green arrow and done to change the 63X objective to obtain the fiber measurements of the current fraction. The section thickness dialogue box will appear.Set the top and bottom surface of the section to measure the actual thickness of the section using the manual z-axis focus knob and click done. Move the z-axis slowly from top to bottom in the frame height of the section to mark all of the intersecting fibers on the surface of the virtual sphere probe. When all of the fibers have been marked, click next to advance to the next location.When all of the fractions have been completed, click the green arrow. The software will ask to have the next section inserted. Bring the next sample on the slide into focus using the 5X objective and repeat the fiber measurement as just demonstrated.When all of the sections have been measured, the software will generate a result for the case showing the coefficient of error values. If the coefficient of error is acceptable, proceed to the next case. If the coefficient of error is not acceptable, the software will provide recommendations to change some of the parameters.When all of the cases have been completed, generate results for each case and group. In this representative experiment, the Swedish mutant beta amyloid precursor protein group demonstrated a significantly lower fiber length and fiber length density compared to their wild-type litter mates. The results also showed that there was no significant difference in the volume of the NBM between the two groups analyzed.This technique requires proper the acquisition in the correct orientation and a good staining method with the incubation period. The stereology protocol can be used for the estimation of any linear profiles such as other neuronal fibers, astrocyte processes, or even vascular profiles.

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Neuroscience

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

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With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

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With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.
In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.
Compared to the traditional TRAP assay that utilize 32P labeled radioactive dCTP&#39;s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol TRAP Assay

Telomerase is expressed in the neonatal brain and also in distinct regions of the adult brain. We present a non-toxic time saving TRAP assay for the analysis of telomerase activity in various regions of the mouse brain and detection of differences in telomerase activity between male and female mouse brains.

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Microelectrode Guided Implantation of Electrodes into the Subthalamic Nucleus of Rats for Long-term Deep Brain Stimulation

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, dystonia or tremor. DBS is based on the delivery of electrical stimuli to specific deep anatomic structures of the central nervous system. However, the mechanisms underlying the effect of DBS remain enigmatic. This has led to an interest in investigating the impact of DBS in animal models, especially in rats. As DBS is a long-term therapy, research should be focused on molecular-genetic changes of neural circuits that occur several weeks after DBS. Long-term DBS in rats is challenging because the rats move around in their cage, which causes problems in keeping in place the wire leading from the head of the animal to the stimulator. Furthermore, target structures for stimulation in the rat brain are small and therefore electrodes cannot easily be placed at the required position. Thus, a set-up for long-lasting stimulation of rats using platinum/iridium electrodes with an impedance of about 1 M&#937; was developed for this study. An electrode with these specifications allows for not only adequate stimulation but also recording of deep brain structures to identify the target area for DBS. In our set-up, an electrode with a plug for the wire was embedded in dental cement with four anchoring screws secured onto the skull. The wire from the plug to the stimulator was protected by a stainless-steel spring. A swivel was connected to the circuit to prevent the wire from becoming tangled. Overall, this stimulation set-up offers a high degree of free mobility for the rat and enables the head plug, as well as the wire connection between the plug and the stimulator, to retain long-lasting strength.

A method for implanting electrodes into the subthalamic nucleus (STN) of rats is described. Better localization of the STN was achieved by using a microrecording system. Furthermore, a stimulation set-up is presented that is characterized by long-lasting connections between the head of the animal and the stimulator.

Deep brain stimulation (DBS) is a widely used and effective therapy for several neurologic disorders, such as idiopathic Parkinson&#8217;s disease, ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

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 ...

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene reporters. This new and easy to use in vivo fluorescence monitoring system was developed to record the transcriptional rhythm of the clock gene, Cry1, in the suprachiasmatic nucleus (SCN) of freely moving mice. To do so, a Cry1 transcription fluorescence reporter was designed and packaged into Adeno-associated virus. Purified, concentrated virus was injected into the mouse SCN followed by the insertion of an optic fiber, which was then fixed onto the surface of the brain. The animals were returned to their home cages and allowed a 1-month post-operative recovery period to ensure sufficient reporter expression. Fluorescence was then recorded in freely moving mice via an in vivo monitoring system that was constructed at our institution. For the in vivo recording system, a 488 nm laser was coupled with a 1 &#215; 4 beam-splitter that divided the light into four laser excitation outputs of equal power. This setup enabled us to record from four animals simultaneously. Each of the emitted fluorescence signals was collected via a photomultiplier tube and a data acquisition card. In contrast to the previous bioluminescence in vivo circadian clock recording technique, this fluorescence in vivo recording system allowed the recording of circadian clock gene expression during the light cycle.

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

This newly developed fluorescence-based technology enables long-term monitoring of the transcription of circadian clock genes in the suprachiasmatic nucleus (SCN) of freely moving mice in real-time and at a high temporal resolution.