This research was supported by grants from the LuMind Foundation, Research Down Syndrome, and the Alzheimer&#8217;s Association (AS). CP was partially supported by a faculty development grant from the College of Nursing and Health Professions at Arkansas State University.

NOTE: Experiments were conducted in accordance with the ethical standards approved by the Committee on Animal Research at the Veterans Affairs Palo Alto Health Care System.
1. Golgi-Cox Staining
<ol>
	<li>Brain extraction and staining
	<ol>
		<li>On the 1st day, deeply anesthetize mice with 100 mg/kg ketamine and 10 mg/kg xylazine before euthanizing via exsanguination.</li>
		<li>Carefully remove the calvarium and dissect out the brain.
		<ol>
			<li>First remove the skin on the top of the skull, placing a curved scissor on the top of the cerebellum and gently cut through the calvarium parallel to the central sulcus with the tip of the scissor pointed outward, moving in the direction toward the olfactory bulb. Repeat this process on both sides.</li>
			<li>Use a surgical pincet to lift the calvarium while turning it toward the olfactory bulbs and break it off. Then, flip the skull upside down and use a pick to cut through the optic nerves and loosen the brain. Finally, use the pick to separate the brain from the spinal cord at the level of the foramen magnum.</li>
		</ol>
		</li>
		<li>Immediately immerse the brain in 15 ml of commercially-available, undiluted Golgi-Cox solution. Store the brain in Golgi solution in a capped, 20 ml glass bottle at room temperature in the dark.</li>
		<li>On the 2nd day, slowly pour out the solution and replace it with 15 ml fresh Golgi-Cox solution. Recap the bottle containing the brains and leave undisturbed for another 9 days in the dark.</li>
	</ol>
	</li>
	<li>Sectioning and embedment
	<ol>
		<li>On the 11th day, place the brains in 30% sucrose solution, prepared in ddH2O for dehydration. Change the solution with freshly prepared 30% sucrose after 12 hours. Incubate the brains in a 30% sucrose solution for 3 days at 4 &#176;C.</li>
		<li>On the 14th day, fill the reservoir of the vibratome with a 30% sucrose solution until the blade is covered. Set the speed to 1 mm/s with amplitude of 0.95 mm.</li>
		<li>Blot the brains with a Kimwipe to remove excess moisture and remove the cerebellum using a razor blade to cut coronally.</li>
		<li>Firmly mount the whole brain onto the vibratome platform using superglue and allow it to set for 2-4 min. Insert the platform with the adhered brain into the reservoir and adjust the blade to the top of the brain.</li>
		<li>Using the vibratome, slice the brain into 150 &#181;m thick sections at room temperature, remembering to change the blade after every 3rd brain.</li>
		<li>Pick up the sections from the reservoir using a small tipped brush and immerse them into &#160;a petri dish containing 0.3% gelatin made in ddH2O. While the sections remain in the petri dish, add 0.5 ml of 0.3% gelatin on each slide and use a brush to spread and coat the superfrost + slides.</li>
		<li>Gently pick up the immersed sections from the petri dish and place them onto the gelatinized slides. Orient the brain sections by touching them only on their sides and not on the tops.</li>
		<li>After about ten min, coat the sections with 30% sucrose before the tissue completely dries. Then air dry the prepared slides in a dark place in a slide rack for 3 days.</li>
		<li>Dilute commercially-available 10x Developing solution to 1x using ddH2O. Immerse the slides in developing solution for 7-10 min. Rinse the slides 3 times in ddH2O.</li>
	</ol>
	</li>
	<li>Dehydration
	<ol>
		<li>Prepare 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% ethanol solutions using distilled ddH2O.</li>
		<li>Soak the slides in increasingly higher ethanol solutions for 10 min each.</li>
		<li>Soak the slides in 100% ethanol solution 3 times for 10 min each time.</li>
		<li>Soak the slides in 100% xylene 3 consecutive times for 5 min each, keeping the sections in the final solution until they are cover-slipped.</li>
		<li>Place a generous amount of DPX (Di-n-butyl phthalate in xylene) mounting medium (about 1-1.5 ml) on each slide using a plastic transfer pipet. Carefully place coverslips to avoid bubbles.</li>
		<li>Air dry cover slipped-brain sections horizontally for 3 days.</li>
	</ol>
	</li>
</ol>
2. Doublecortin Staining
<ol>
	<li>Deeply anesthetize (see step 1.1.1) the animals.</li>
	<li>Perform cardiac perfusion with freshly prepared 4% paraformaldehyde made in phosphate buffer12.</li>
	<li>Extract the brains (see 1.1.2) and store in 45 ml of 4% paraformaldehyde at 4 &#176;C overnight on a rocking shaker.</li>
	<li>Transfer the brains to a 30% sucrose solution and wait for complete dehydration for 48 hr at 4 &#176;C. Remove the brains from sucrose and place them directly on copper blocks placed on dry ice.</li>
	<li>Fill the block with optimum cutting temperature (OCT) compound and mark the orientation of the brain using the olfactory bulb as a landmark.</li>
	<li>Using a cryostat, cut 70 micron-thick sections at -20 &#186;C and place them in cryoprotectant solution (25% ethylene glycol, 25% glycerol, in 0.05 M sodium phosphate buffer, pH 7.4) and keep at -20 &#176;C until use.</li>
	<li>Make a solution of 50% cryoprotectant and 50% methanol. To this solution add 0.5% hydrogen peroxide (vol/vol). Incubate floating sections for 30 min at room temperature.</li>
	<li>Warm sections in 37 &#186;C in TBS (Tris-buffered saline) for 30-40 min by placing them in a histological oven.</li>
	<li>Incubate the sections with 0.3% triton and 3% normal horse serum for 45 min at room temperature prior to immunostaining. Incubate the sections at 4 &#186;C overnight in 3 ml solution of the DCX antibody diluted 1:500 in TBS with 0.3% triton and 1% normal horse serum.</li>
	<li>The following day wash the sections 3 consecutive times in TBS (10 min each).</li>
	<li>Incubate sections with a biotinylated horse anti-goat (1:200 in TBS) for two hr at room temperature. Wash the sections 3 consecutive times in TBS (10 min each).</li>
	<li>Incubate them with ABC Lite (1:1,000) for 1.5 hr at room temperature.</li>
	<li>Add 5 &#181;l of 30% H2O2 to one tablet of 10 mg of DAB (3,3&#8221;-Diaminobenzidine) solution dissolved in 15 ml of 1 M Tris-HCl (pH 7.6). Incubate the sections immediately in this solution for 5 min.</li>
	<li>Terminate the reaction by washing them with ice-cold TBS three times followed by one wash in TBS at room temperature.</li>
	<li>Dehydrate sections using a series of ethanol solutions (50%, 60%, 70%, 80%, 90%, 95%, 100%, 5 min each), clear in xylene, and then cover slip using DPX as in step 1.3.</li>
</ol>
3. Visualization
<ol>
	<li>Following complete drying of the sections, remove&#160;excess DPX on top of the slides with a sharp blade, and place them on the scanning stage of the microscope. The system is composed of a microscope equipped with scanning stage, joystick, and a color 12-bit camera.</li>
	<li>Start the program. Open a new data file.</li>
	<li>Place a reference point on the screen by clicking with the mouse pointer at any place. This will activate all the icons from the tool panel of program window.</li>
	<li>Click on the &#8220;Joystick Free&#8221; icon on the toolbar and then use the joystick to locate the dentate gyrus of the hippocampus in the first section.</li>
	<li>In the &#8220;Tools&#8221; tab of the program window, select &#8220;Serial Section Manager&#8221;. Click on the &#8220;New Section&#8221; icon on the lower left corner of the window. This will open the &#8220;Serial Section Setup&#8221; window.</li>
	<li>Select the Serial Section Setup window and then enter the total number of sections that contain hippocampal regions. Select the evaluation interval. Enter the section cut thickness (70 &#181;m for DCX and 150 &#181;m for Golgi-stained sections).</li>
	<li>Start tracing the dentate gyrus region by clicking on the &#8220;Freehand Contour Drawing&#8221; icon in the tool bar. Outline the dentate granular cell layer.</li>
	<li>Select &#8220;100X&#8221; from the &#34;Magnification&#34; menu. Add a drop of immersion oil on the section and switch the objective to 100X. Locate the dentate granule cell bodies and dendrites under this objective.</li>
	<li>Focus on a selected neuron and click on the &#8220;Neuron Tracing&#8221; icon on the toolbar. Trace the circumference of the dentate granule cell body. When finished tracing, right click to select &#8220;Finish Cell Body&#8221;.</li>
	<li>Start tracing the dendrites manually in x, y, and z directions and follow each branch using the joystick and z-motor knob. At a bifurcation or trifurcation node, select the respective option from the dropdown menu upon right-click. Trace each of the branches that arise from these nodes. At the end of each branch right click and select &#8220;Ending&#8221; from the dropdown menu.</li>
	<li>Using the arrow key icons in the software window randomly select another dentate granule cell and follow the same procedure as described in step 3.9-3.10. Save the entire tracing.</li>
	<li>Start the neuronal tracing software.</li>
	<li>Open the first NRX data file from the first mouse of the experimental group. Append the NRX file of the second mouse of the experimental group. Continue until all the NRX files from this group are appended.</li>
	<li>Under the Analysis tab, select &#8220;Fan In-diagram&#8221;. This opens the Fan In-analysis window. Check mark &#8220;dendrites&#8221; and click Display, which depicts the lengths and branching patterns of dendrites for this group of mice (Figure 1).</li>
</ol>
4. EDFI
NOTE: This method enables the user to rapidly record a large number of images through the z-axis of each field and generate a 2D image that contains all the focused pixels throughout the z-axis. The result will be a composite image that contains all focused pixels from collected images.
<ol>
	<li>Connect the microscope to a digital camera. Place the sections first on the scanning stage connected to the microscope and then switch to a 10X objective. Move the stage to the area of interest.</li>
	<li>Start a video capturing program.</li>
	<li>Press the record button. As soon as the record button is pressed, use the macro-focus knob to move from the top to the bottom of the section for a total of 4 sec. Save the resulting video file.</li>
	<li>Use ImageJ freeware to convert the AVI video files into an uncompressed format by opening the file and saving them in uncompressed file format.</li>
	<li>Start an image analysis program and open the AVI file. Go to &#8220;Process&#8221; menu and click on &#8220;Extended Depth of Field&#8221;. Select the corresponding video file. In &#8220;Output&#8221; options, select &#8220;Generate Composite Best-Focus Image&#8221;.</li>
	<li>In &#8220;Focus Analysis&#8221; options, select &#8220;Normalized Illumination&#8221; and &#8220;Max Local Contrast&#8221; options. Then, select &#8220;Create&#8221;. The resulting image represents all the focused pixels throughout the z-axis (Figure 2).</li>
	<li>Save the resulting image.</li>
</ol>
5. Generating Extremely-high Resolution Images
NOTE: This method allows the user to automatically generate low magnification and extremely high-resolution images from high-magnification-high resolution images in an automatized fashion.
<ol>
	<li>Place the sections first on the scanning stage connected to the microscope. The microscope is connected to a digital camera. Move the stage to the area of interest.</li>
	<li>Start an image acquisition program.</li>
	<li>Use a 10X objective and acquire and save high-resolution (4076 x 3116) images from the region of interest making sure to have at least 10% overlap.</li>
	<li>Run Image Composite Editor to stitch images.</li>
	<li>Go to File menu, and click on &#8220;New Panorama&#8221;. Select the folder in which the images are stored. Select the images that belong to the same region and press &#8220;ok&#8221;.</li>
	<li>Press &#8220;Export to Disk&#8221; button and save the image. This image represents an extremely high-resolution picture of the area of interest that could be used for analysis and/or demonstration (Figure 3).</li>
</ol>

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Publication fees for this article are sponsored by MBF Bioscience.

Here, two methods were described to quantify the extent of dendritic arborization in mature and newly-born neurons using conventional staining methods in conjunction with RTI and EDFI. The acquisition of high resolution images of neurons provides an extremely useful method for testing the deleterious effects of neurodegenerative disorders and, in turn, provides a means to assess therapeutic strategies that target hippocampal neurons.
While the RTI method was utilized to capture in-depth data p...

Dynamic alterations in the number and structure of synapses are hallmarks of development, aging, and numerous neurodegenerative disorders1-3. The ability of neurons to receive and integrate synaptic information depends upon dendritic morphology and dynamic alterations in synaptic connections. Indeed, a positive correlation exists between dendritic spine and synapse number, which both impact cognitive function4. Thus, it is not surprising that decrements in dendritic spine number have been associated with cognitive dysfunction in a number of neurological disorders5-7, prompting great interest in dendritic spine quantification. Nevertheless, the quantification of spine density remains a time-consuming and tedious task that fails to generate useful information regarding the topography and distribution of synapses across the dendritic tree. Fortunately, staining methods (e.g., Golgi-Cox and doublecortin (DCX)) in conjunction with sophisticated imaging techniques can be utilized to overcome current barriers and produce high-resolution images of dendritic arborization in a reliable and expeditious manner. While Golgi-Cox staining method can be deployed to assess the state of dendritic arborization in all neurons8, DCX can be deployed to label newly-born neurons particularly in the dentate gyrus and subventricular zone9, an important consideration given that neurogenesis occurs in both these regions throughout the lifespan10,11.
Following staining, two imaging methods were deployed to assess dendritic characteristics: i) real-time imaging (RTI) and ii) extended depth of field imaging (EDFI). The RTI technique provides a mean to trace and quantify the length and order of arborization along the individual dendritic segments and branches. Thus it enables one to estimate the total area and volume occupied by each dendritic tree. More specifically, in the RTI method the user continuously identifies the segments and refocuses iteratively as the neuron tracing software collects the x, y, and z coordinates of the dendritic structure and reconstructs the trajectory of the dendritic structure in 3D. Comparatively, the EDFI method provides a rather simple and expedited means for assessing dendritic density in rather thick tissue specimens by generating a composite image, providing information on the entire z-axis. To do so, the user records high definition video files throughout the thickness of the section and then uses software to search the video frames to identify points wherein a pixel is completely in focus. Subsequently, the focused pixels are merged and integrated into a high-resolution, composite 2D image. This composite image contains all pixels that were in-focus regardless of their position in the z axis. Qualitative and quantitative analysis of these 2D images can be used subsequently to determine the density of dendritic branching in each field.
Lastly, we present a panoramic method for generating extremely high-resolution images for the analysis and assessment of dendrites in an entire region of interest. This technique can be deployed to overcome the lack of access to very high-resolution and expensive digital cameras. Using this method, one captures serial images at different locations along the x- and y-axes and then automatically stitches them together using a freeware (e.g., Image Composite Editor). Notably, this method can be used for qualitative and quantitative assessment of dendritic arborization in a rather wide area.

<table><tbody><tr><td>Modified Golgi-cox staining solution&amp;nbsp;</td><td>Weill Cornell Medical College</td><td>NA</td><td>store at 4&amp;deg;C till use</td></tr><tr><td>1x Developing Solution (Stock 10x)</td><td>Weill Cornell Medical College</td><td>NA</td><td>store at 4&amp;deg;C till use</td></tr><tr><td>30% Sucrose,</td><td>Sigma</td><td>CAS # 57-50-1</td><td>make fresh &amp;nbsp;in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>0.3% Gelatin</td><td>Sigma</td><td>CAS # 9000-70-8</td><td>NA</td></tr><tr><td>Graded Ethanol Solutions (20%, 30%, 40%, 50%, 80%. 90%, 95%. 100%)</td><td>Sigma</td><td>CAS 603-003-00-5</td><td>NA</td></tr><tr><td>Xylene</td><td>Sigma</td><td>CAS # 1330-20-7</td><td>NA</td></tr><tr><td>DPX Medium</td><td>EMS&amp;nbsp;</td><td>#13510</td><td>NA</td></tr><tr><td>Superfrost (+) white</td><td>Electron Microscopy Sciences</td><td>71869-10</td><td>NA</td></tr><tr><td>Coverslip 22x50mm (VWR #48393-059)</td><td>VWR&amp;nbsp;</td><td>#4811-703</td><td>NA</td></tr><tr><td>DCX Antibody</td><td>Santa Cruz Biotechnology</td><td>sc-8066</td><td>4 C</td></tr><tr><td>DAB</td><td>Sigma</td><td>CAS Number 91-95-2 &amp;nbsp;</td><td>-20</td></tr><tr><td>OCT</td><td>Tissue-tek</td><td>4583</td><td>NA</td></tr><tr><td>Tris</td><td>Sigma</td><td>CAS Number 77-86-1 &amp;nbsp;</td><td>NA</td></tr><tr><td>ABC Lite</td><td>Vector</td><td>PK4000</td><td>NA</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse 80i</td><td></td></tr><tr><td>Digital Camera</td><td>Nikon</td><td>DS-Ri1</td><td></td></tr><tr><td>12 bit Camera&amp;nbsp;</td><td>QImaging&amp;nbsp;</td><td>01 MBF2000RF-CLR-12</td><td></td></tr><tr><td>Neurolucida System</td><td>MBF Bioscience</td><td>V.10</td><td></td></tr><tr><td>Image Composite Editor</td><td>Microsoft</td><td>1.4.4.0</td><td></td></tr><tr><td>NIS Elements</td><td>Nikon</td><td>F 3.0</td><td></td></tr><tr><td>Image Pro Plus</td><td>Mediacy</td><td>Versin 7.00</td><td></td></tr></tbody></table>

assessment of dendritic arborization in the dentate gyrus of the hippocampal region in mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

The extent of arborization arising from extant and newly born dentate granule cells was analyzed in wild type mice using either Golgi-Cox or DCX staining (Figure 1). Dendritic segments of DCX-positive cells were found to be 13-36 microns long. The normal distribution of dendritic length was tested using Kolmogorov-Smirnov test (D = 0.1217, p &#60;0.01, Liliefors p &#60;0.001; Figures 4 and 5).
In the analysis of length of segments per order of...

Watch this Scientific Journal Video about Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice at JoVE.com

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

assessment-dendritic-arborization-dentate-gyrus-hippocampal-region

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JoVE Journal

Neuroscience

10.2K Views.  VA Palo Alto Health Care System. We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Video: Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

Behavior

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Biology

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

Biochemistry

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.