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 s...

<ol>
	<li>Bosch, M., Hayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+plasticity+of+dendritic+spines.">Structural plasticity of dendritic spines.</a> Curr Opin Neurobiol. 22 (3), 383-388 (2012).</li><li>Isaac, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synapse:+center+stage+for+many+brain+diseases.">The synapse: center stage for many brain diseases.</a> The Journal of Physiology. 587 (4), 727-729 (2009).</li><li>Sheng, M., Sabatini, B. L., S&#252;dhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapses+and+Alzheimer&#8217;s+disease.">Synapses and Alzheimer&#8217;s disease.</a> Cold Spring Harbor Perspectives in Biology. , a005777 (2012).</li><li>Alvarez, V. A., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+physiological+plasticity+of+dendritic+spines.">Anatomical and physiological plasticity of dendritic spines.</a> Annu Rev Neurosci. 30, 79-97 (2007).</li><li>Huttenlocher, 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=Dendritic+development+in+neocortex+of+children+with+mental+defect+and+infantile+spasms.">Dendritic development in neocortex of children with mental defect and infantile spasms.</a> Neurology. 24 (3), 203-210 (1974).</li><li>Marin-Padilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+origin+of+the+pericellular+baskets+of+the+pyramidal+cells+of+the+human+motor+cortex:+a+Golgi+study.">Double origin of the pericellular baskets of the pyramidal cells of the human motor cortex: a Golgi study.</a> Brain Res. 38 (1), 1-12 (1972).</li><li>Dang, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formoterol,+a+long-acting+&#946;2+adrenergic+agonist,+improves+cognitive+function+and+promotes+dendritic+complexity+in+a+mouse+model+of+Down+syndrome.">Formoterol, a long-acting &#946;2 adrenergic agonist, improves cognitive function and promotes dendritic complexity in a mouse model of Down syndrome.</a> Biol Psychiatry. 75 (3), 179-188 (2014).</li><li>Dobrovi&#263;, B., Curi&#263;, G., Petanjek, Z., Heffer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+morphology+and+spine+density+is+not+altered+in+motor+cortex+and+dentate+granular+cells+in+mice+lacking+the+ganglioside+biosynthetic+gene+B4galnt1+A+quantitative+Golgi+cox+study.">Dendritic morphology and spine density is not altered in motor cortex and dentate granular cells in mice lacking the ganglioside biosynthetic gene B4galnt1 A quantitative Golgi cox study.</a> Coll Antropol. 35 (Suppl 1), 25-30 (2011).</li><li>Dijkmans, T. F., van Hooijdonk, L. W., Fitzsimons, C. P., Vreugdenhil, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+doublecortin+gene+family+and+disorders+of+neuronal+structure.">The doublecortin gene family and disorders of neuronal structure.</a> Cent Nerv Syst Agents Med Chem. 10 (1), 32-46 (2010).</li><li>Guerra, E., Pignatelli, J., Nieto-Est&#233;vez, V., Vicario-Abej&#243;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+olfactory+bulb+neurogenesis.">Transcriptional regulation of olfactory bulb neurogenesis.</a> Anat Rec. 296 (9), 1364-1382 (2013).</li><li>Imayoshi, I., Shimojo, H., Sakamoto, M., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+visualization+of+notch+signaling+in+mammalian+neurogenesis.">Genetic visualization of notch signaling in mammalian neurogenesis.</a> Cell Mol Life Sci. 70 (12), 2045-2057 (2013).</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> J Vis Exp. (65), 3564-3510 (2012).</li><li>Das, G., Reuhl, K., Zhou, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Golgi-Cox+method.">The Golgi-Cox method.</a> Methods Mol Biol. 1018, 313-321 (2013).</li><li>Juraska, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+developmental+plasticity+in+the+visual+cortex+and+hippocampal+dentate+gyrus.">Sex differences in developmental plasticity in the visual cortex and hippocampal dentate gyrus.</a> Prog Brain Res. 61, 205-214 (1984).</li><li>Gao, X., Deng, P., Zao, C. X., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderate+traumatic+brain+injury+causes+acute+dendritic+and+synaptic+degeneration+in+the+hippocampal+dentate+gyrus.">Moderate traumatic brain injury causes acute dendritic and synaptic degeneration in the hippocampal dentate gyrus.</a> PLoS One. 6 (9), e24566 (2011).</li><li>Zhang, L., Hern&#225;ndez, V. S., Estrada, F. S., Luj&#225;n, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+CA+field+neurogenesis+after+pilocarpine+insult:+The+hippocampal+fissure+as+a+neurogenic+niche.">Hippocampal CA field neurogenesis after pilocarpine insult: The hippocampal fissure as a neurogenic niche.</a> J Chem Neuroanat. 56, 45-57 (2014).</li><li>Merz, K., Lie, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+Doublecortin+is+dispensable+for+the+development+of+adult+born+neurons+in+mice.">Evidence that Doublecortin is dispensable for the development of adult born neurons in mice.</a> PLoS One. 8 (5), e62693 (2013).</li><li>Hussaini, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-induced+antigen+retrieval:+an+effective+method+to+detect+and+identify+progenitor+cell+types+during+adult+hippocampal+neurogenesis.">Heat-induced antigen retrieval: an effective method to detect and identify progenitor cell types during adult hippocampal neurogenesis.</a> J Vis Exp. (78), (2013).</li></ol>

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.

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			<td height="22" style="height:22px;width:272px;">Modified Golgi-cox staining solution&#160;</td>
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			<td style="width:263px;">Santa Cruz Biotechnology</td>
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			<td style="width:159px;">-20</td>
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			<td height="22" style="height:22px;width:272px;">OCT</td>
			<td style="width:263px;">Tissue-tek</td>
			<td style="width:267px;">4583</td>
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			<td height="22" style="height:22px;width:272px;">ABC Lite</td>
			<td style="width:263px;">Vector</td>
			<td style="width:267px;">PK4000</td>
			<td style="width:159px;">NA</td>
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			<td height="22" style="height:22px;width:272px;">Microscope</td>
			<td style="width:263px;">Nikon</td>
			<td style="width:267px;">Eclipse 80i</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Digital Camera</td>
			<td style="width:263px;">Nikon</td>
			<td style="width:267px;">DS-Ri1</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">12 bit Camera&#160;</td>
			<td style="width:263px;">QImaging&#160;</td>
			<td>01 MBF2000RF-CLR-12</td>
			<td style="width:159px;"></td>
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			<td height="22" style="height:22px;width:272px;">Neurolucida System</td>
			<td>MBF Bioscience</td>
			<td style="width:267px;">V.10</td>
			<td style="width:159px;"></td>
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			<td height="22" style="height:22px;width:272px;">Image Composite Editor</td>
			<td style="width:263px;">Microsoft</td>
			<td>1.4.4.0</td>
			<td style="width:159px;"></td>
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			<td height="22" style="height:22px;width:272px;">NIS Elements</td>
			<td style="width:263px;">Nikon</td>
			<td style="width:267px;">F 3.0</td>
			<td style="width:159px;"></td>
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			<td height="22" style="height:22px;width:272px;">Image Pro Plus</td>
			<td style="width:263px;">Mediacy</td>
			<td>Versin 7.00</td>
			<td style="width:159px;"></td>
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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

Research

JoVE Journal

Neuroscience

10.2K ビュー.  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.

ビデオ: 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...

dissection of hippocampal dentate gyrus from adult mouse

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

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

assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method

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

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...

in vivo calcium imaging of granule cells in the dentate gyrus of hippocampus in mice 

Author Spotlight: Investigating Neural Activity of Dentate Gyrus Granule Cells with Miniature Microscope

The dentate gyrus of the hippocampus carries out essential and distinct functions in learning and memory. This protocol describes a set of robust and efficient procedures for in vivo calcium imaging of granule cells in the dentate gyrus in freely moving...

In Vivo Calcium Imaging of Granule Cells in the Dentate Gyrus of Hippocampus in Mice 

long-term potentiation of perforant pathway-dentate gyrus synapse in freely behaving mice

Long-term Potentiation of Perforant Pathway-dentate Gyrus Synapse in Freely Behaving Mice

Transgenic and knockout mouse models of neurological diseases are useful for studying the role of genes in normal and abnormal neurophysiology. This article describes methodologies which can be used to study long-term potentiation, a cellular mechanism which may underlie learning and memory, in transgenic and knockout freely&#160;behaving mouse models of...

a technique to isolate nuclei from non-neuronal cells in the hippocampal dentate gyrus

The video demonstrates a method for isolating intact nuclei from glial and stem cells in the mouse hippocampal dentate gyrus (DG). The DG tissue is mechanically dissociated in a cold homogenization buffer to release the nuclei. Tissue debris and other released organelles are discarded, and the nuclei are stained with a DNA stain and a fluorophore-conjugated antibody specific for the neuronal nuclear antigen (NeuN). Fluorescence-activated nuclei sorting is then employed to isolate the...

A Technique to Isolate Nuclei from Non-neuronal Cells in the Hippocampal Dentate Gyrus

Add 3 milliliters of HB to the 15-milliliter tube, and incubate for 5 minutes on ice. Mix this nuclei suspension twice by inverting the tube gently. Using 0.5 milliliters of HB, pre-wet the 70-micrometer strainer cap placed over a 50-milliliter test tube. Then, strain the nuclei suspension before washing the cell strainer with 0.5 milliliters of...

preparation of acute slices from dorsal hippocampus for whole-cell recording and neuronal reconstruction in the dentate gyrus of adult mice

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

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...

Real-Time Imaging for Neuron Activity in the Dentate Gyrus Cells of Mice 

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optimized virus injection and grin lens implantation in the dentate gyrus of mice for in vivo calcium imaging

Optimized Virus Injection and GRIN Lens Implantation in the Dentate Gyrus of Mice for In Vivo Calcium Imaging

an invasive method for the activation of the mouse dentate gyrus by high-frequency stimulation

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

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...

quantitative analysis of neuronal dendritic arborization complexity in drosophila

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

longitudinal two-photon imaging of dorsal hippocampal ca1 in live mice

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

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...

echocardiographic assessment of the right heart in mice

Echocardiographic Assessment of the Right Heart in Mice

This article provides a protocol for the echocardiographic assessment of right ventricular size and pulmonary hypertension in mice.  Applications include phenotype determination and serial assessment in transgenic and toxin-induced mouse models of cardiomyopathy and pulmonary vascular...

imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved...

optogenetic entrainment of hippocampal theta oscillations in behaving mice

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal...

stereotactic injection of microrna-expressing lentiviruses to the mouse hippocampus ca1 region and assessment of the behavioral outcome

Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome

MicroRNAs have significant roles in brain structure and function. Here we describe a method to enforce hippocampal miRNA over-expression using stereotactic injection of an engineered miRNA-expressing lentivirus. This approach can serve as a relatively rapid way to assess the in vivo effects of over-expressed miRNAs in specific brain...

horizontal hippocampal slices of the mouse brain

Horizontal Hippocampal Slices of the Mouse Brain

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological...

the analysis of neurovascular remodeling in entorhino-hippocampal organotypic slice cultures

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after...

assessment of memory function in pilocarpine-induced epileptic mice

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

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 ...

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 ...

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

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 ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

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.