We wish to thank the administrative staff of CIBIO (University of Trento) and CNR Neuroscience Institute for assistance. Giovanni Provenzano is supported by a post-doctoral fellowship from Fondazione Veronesi (Milan, Italy). This work was funded by the Italian Ministry of University and Research (PRIN 2008 grant # 200894SYW2_002 and PRIN 2010-2011 grant # 2010N8PBAA_002 to Y.B.), University of Trento (CIBIO start-up grant to Y.B.) and Telethon Foundation (grant # GGP13034 to Y.B.).

All experiments were conducted in conformity with the European Community Directive 2010/63/EU and were approved by the Italian Ministry of Health.
1. Animal Care, Housing and Treatment
<ol>
	<li>Perform all experimental protocols using mice in accordance with the guidelines of the respective institutional animal care.</li>
	<li>Maintain animals in a 12 hr light/dark cycle with food and water available ad libitum.</li>
	<li>House the mice in groups of 2-4, in standard-sized polycarbonate mouse cages with sawdust and bedding material changed weekly.</li>
	<li>Use age and sex-matched mice, because susceptibility to disease can vary with age and gender in different mouse models of neurodevelopmental disorders.</li>
	<li>Choose an appropriate number of animals to reach statistical significance (a minimum of 10 mice per genotype for the behavioral study and 4 mice per genotype for the immunohistochemistry experiment).</li>
</ol>
2. Morris Water Maze (MWM)
<ol>
	<li>Use a Circular Tank (diameter 100 cm, height 50 cm).
	<ol>
		<li>Place some visual cues on the room dividers around the tank.</li>
		<li>Place a 10 cm diameter platform into the tank at a fixed location in the center of one of four imaginary quadrants. Keep the hidden platform in the same quadrant for all trials across all training sessions.</li>
		<li>Fill the pool with tap water until the platform is 1 cm above the water surface. Equilibrate water temperature should be close to 22 &#176;C; this step may take several hours. Add hot water to shorten the time required to equilibrate water temperature.</li>
		<li>Add approximately 1.5-2 L of white, non-toxic paint to make the water opaque.</li>
	</ol>
	</li>
	<li>Track Acquisition Procedure
	<ol>
		<li>Set up MWM&#8217;s tracking system with standard CCD cameras and video boards. Choose a video-tracking system to monitor and analyze several variables such as path length, escape latency, and time spent in each quadrant.</li>
		<li>Divide the arena in four quadrants and adjust detection settings to ensure optimal contrast between animals and background for live tracking.</li>
		<li>Specify the platform region.</li>
		<li>Set the maximum trial time as 60 sec.</li>
	</ol>
	</li>
	<li>MWM Testing
	<ol>
		<li>Prior to behavior testing, allow mice an adaptation period of 20-25 min in an area where they cannot see the pool or spatial cues.</li>
		<li>Train each mouse for 9 days (two consecutive trials a day, with 20-30 min interval between them).</li>
		<li>Switch on the computer and the camera and start the video-tracking system software.</li>
		<li>Take the mouse by its tail and place it into water, with its head pointed towards the side of pool, starting from one of the four start positions (north, south, east, west). For each mouse, the start position should be different and pseudorandomized across trials.</li>
		<li>Let the mouse swim and search for the platform for a maximum of 60 sec. Once the animal reaches the platform, allow it to stay on the platform for 20 sec.</li>
		<li>If mouse failed to locate the platform within that time, gently guide the mouse to the platform, holding it by its tail and let it to stay there for 20 sec. Once the animal has completed all two trials, dry it off with a towel and put it back into cage.</li>
		<li>Each day of the training period, repeat the same procedure (steps 2.3.4-2.3.6).</li>
		<li>Perform the probe test on day 9 after the last training trail.</li>
		<li>Remove the platform from the pool.</li>
		<li>Let the mouse swim and search for the platform for a maximum of 60 sec.</li>
		<li>Periodically, check the water temperature so that it should be the same (22 &#176;C) and clean out the pool.</li>
	</ol>
	</li>
</ol>
3. Preparation of Sections from Perfused Animals
<ol>
	<li>Sacrifice the mice at the end of MWM. Four mice per genotype must be anesthetized prior to euthanization according to local and international guidelines.</li>
	<li>Perfuse the animal transcardially (cut the right auriclewith a scissor to allow bleeding and introduce a butterfly cannula into the left ventricle to perfuse) with phosphate buffered saline (PBS) for 4-5 min, followed by 4% paraformaldehyde in PBS for 10-15 min22.</li>
	<li>Dissect and post-fix the brains in 4% paraformaldehyde at 4 &#176;C for at least 12 hr.</li>
	<li>Place the brains in 50 ml polystyrene conical tubes containing 15 ml of 0.02% Sodium Azide in 0.1 M PBS and store at 4 &#176;C until required for slicing.</li>
	<li>Cut and remove the cerebellum before vibratome cutting.</li>
	<li>Glue the brain upright with the cutting site, using superglue, onto the holder plate of the vibratome.</li>
	<li>Cut the brain into 20-40 &#181;m thick coronal sections, in serial order throughout the dorsal hippocampus, on appropriate vibratome.</li>
	<li>Collect vibratome slices with a paint brush and transfer to a 24 multi-well plate containing 1 ml of 0.1 M PBS for each well.</li>
	<li>Store sections for short term in PBS or long term in 0.02% Sodium Azide in PBS at 4 &#186;C (tissue might be used for up to one year).</li>
</ol>
4. Immunohistochemistry
<ol>
	<li>Transfer 3-5 dorsal hippocampal sections for each mouse, into 24 multi-well plate containing 500 &#181;l of 0.1 M PBS for each well.</li>
	<li>Wash the sections contained in each well with 500 &#181;l of 0.1 M PBS (5 min,&#160; 3 times with gentle shaking).</li>
	<li>Quench endogenous peroxidase activity, by incubating sections in 1% H2O2 in PBS for 20 min with gentle agitation.</li>
	<li>Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).</li>
	<li>Permeabilization of tissue: incubate sections for 5 min in 0.2% Triton X-100 in PBS.</li>
	<li>During the above steps (4.4; 4.5), make enough blocking buffer (10% Goat Serum and 0.1% Triton-X100 in PBS) for the entire experiment (number of sections x 100 &#181;l x 3 steps).</li>
	<li>Note: Use serum from the same species in which the secondary Ab (conjugated to biotin) was made.</li>
	<li>Prevent non-specific binding of the antibody, by incubating sections in blocking buffer (100 &#181;l/section) for 1 hr at room temperature (RT) with gentle agitation.</li>
	<li>Incubate sections in primary Ab (pERK) diluted in blocking buffer, overnight at 4 &#176;C, with gentle agitation.</li>
	<li>Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).</li>
	<li>Incubate with the secondary Ab conjugated with biotin (1:250) diluted in blocking buffer (same as used in step 4.6) for 1 hr at RT with gentle agitation.</li>
	<li>Prepare ABC reagent at the same time, because Avidin and biotin require at least 30 min at room temperature to complex.</li>
	<li>Prepare the required volume of ABC reagent according to manufacturers&#8217; protocol.</li>
	<li>Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).</li>
	<li>Incubate sections for 45 min at RT with ABC Reagent.</li>
	<li>Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).</li>
	<li>Prepare DAB working solution, every step involving DAB must be done in the fume hood as DAB is carcinogenic and teratogenic.</li>
	<li>Transfer DAB solution to a new plate by plastic transfer pipette.</li>
	<li>Place sections in the plate containing DAB working solution and slowly swirl plate by hand, to allow maximum exposure to sections.</li>
	<li>Monitor DAB reaction on microscope until adequate signal develops (2-5 min).</li>
	<li>Stop the reaction at the desired color intensity by washing the tissue in 0.1 M PBS (3 x 5 min).</li>
	<li>Use bleach to deactivate any remaining DAB substrate solution in the fume hood overnight and dispose of it according to laboratory guidelines.</li>
	<li>Mount sections on gelatin coated slides by floating in PBS. Then dry them at room temperature at least 3-4 hr.</li>
	<li>Dehydrate the sections sequentially in 50%, 70%, 95% and 100% ethanol for 2 min each, and clear in 100% xylene for 5 min.</li>
	<li>Coverslip using mounting medium and leave in fume hood to dry overnight.</li>
</ol>
5. Cell Count
<ol>
	<li>Count pERK-positive cells, three to five sections at the level of the dorsal hippocampus should be analyzed per animal.</li>
	<li>Acquire multiple bright-field images from each section at 200X magnification using an appropriate microscope, and then assemble them using an image processing software.</li>
	<li>Perform cell counts on tiff-converted mosaic images using the ImageJ free software.</li>
	<li>Count pERK-stained cells in the hilus and granule cell layer over two to three counting boxes of 100 &#215; 100 &#181;m each.</li>
	<li>Cell densities will be expressed as the number of labeled cells per counting window (100 &#215; 100 &#181;m).</li>
	<li>Perform statistical analysis to assess significant difference between genotypes. This can be accomplished with Student&#39;s t test or ANOVA followed by appropriate post hoc test using commercial softwares. Set statistical significance level at p &#60;0.05.</li>
</ol>

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity-regulated+gene+transcription+in+synapse+development+and+cognitive+function.">Neuronal activity-regulated gene transcription in synapse development and cognitive function.</a> Cold Spring Harbor perspectives in biology. 3, (2011).</li><li>Goorden, S. M., van Woerden, G. M., van der Weerd, L., Cheadle, J. 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A., 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=En2+knockout+mice+display+neurobehavioral+and+neurochemical+alterations+relevant+to+autism+spectrum+disorder.">En2 knockout mice display neurobehavioral and neurochemical alterations relevant to autism spectrum disorder.</a> Brain research. 1116, 166-176 (2006).</li><li>Dawson, G., 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=Defining+the+broader+phenotype+of+autism:+genetic,+brain,+and+behavioral+perspectives.">Defining the broader phenotype of autism: genetic, brain, and behavioral perspectives.</a> Development and psychopathology. 14, 581-611 (2002).</li><li>Gage, G. 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The authors have nothing to disclose

Here, we provide a pERK immunohistochemistry protocol for revealing neuronal activation following MWM in En2-/- mice, a mouse model of neurodevelopmental disorders. Decreased levels of pERK were detected in the CA3 subfield of En2-/- mutants compared to WT. Differently from what observed in CA3 subfield, a general increase of pERK-positive neurons was instead detected in both hilus and GCL of En2-/- mice compared to WT. A possible explanation of this opposite trend in ERK phosphorylation ...

Neurodevelopmental disorders include a wide and heterogeneous group of disorders such as Down syndrome, fragile X syndrome (FXS), Rett syndrome, neurofibromatosis, tuberous sclerosis and ASD, in which the development and maturation of the central nervous system (CNS) is disturbed early during the prenatal period1. These developmental brain dysfunctions can cause profound, lifelong effects on motor function, language, learning and memory process. A plethora of genetic and environmental factors have been implicated in the pathogenesis of neurodevelopmental disorders during the last few years2,3. Even if the molecular mechanisms underlying the clinical phenotype remain unknown, the above mentioned findings have allowed the development of several mouse models of these disorders. Learning and memory deficits have been identified in a number of these mouse models such as Tsc1+/-, Tsc2+/-, Nf1+/- and En2-/- mice2,4-7. An important challenge in the field of neurodevelopmental disorders is the identification of cellular and molecular processes underlying memory and learning dysfunction. Selected signaling pathways activated during learning or memory can induce the transcription of specific genes and ultimately lead to de novo protein synthesis. Immediate-early genes (IEGs) activation and protein-dependent synaptic modifications are rapidly induced in brain neurons in response to neuronal activity and behavioral training8,9.
Deficits in signaling pathways involving neurofibromin have been associated with impaired learning in neurodevelopmental disorders. Neurofibromin is the product of the NF1 gene, whose mutation causes neurofibromatosis type 1, a complex genetic syndrome characterized by nervous system tumors, behavioral and motor delays, and cognitive disabilities10. Mice heterozygous for Nf1 deletion restricted to inhibitory neurons show deficits in the early phase of long-term potentiation (LTP), as well as compromised spatial learning in MWM5,11,12. Interestingly, Nf1 deficiency in this mouse model leads to an over-activation of Ras signaling in inhibitory interneurons during learning, resulting in increased ERK phosphorylation and finally in an abnormal enhancement of GABA release from these neurons5.
Based on these findings, the visualization of neuronal activity after behavioral tasks represents a way to reconstruct specific circuits involved in neurodevelopmental diseases. The immunohistochemistry protocol described here aims to assess and quantify hippocampal ERK phosphorylation levels following MWM in an ASD mouse model with cognitive deficits. MWM is widely used to investigate hippocampal dependent spatial learning and memory in rodents13,14. We decide to use ERK phosphorylation as molecular readout of task-dependent hippocampal learning, since ERK was shown to have an essential role in learning and memory formation15. Moreover, the ERK pathway is necessary for experience-dependent plasticity in the developing visual cortex16. Finally, mice lacking one of the two ERK isoforms (ERK2) in the CNS show marked anomalies in cognitive, emotional and social behaviors17, indicating that ERK signaling might play a critical role in the pathogenesis of neurodevelopmental disorders such as ASD.
We used Engrailed 2 knockout (En2-/-) mice as a model of neurodevelopmental disorders. En2-/- mice show anatomical and behavioral &#8220;ASD-like&#8221; features, including loss of forebrain interneurons18, reduced expression of ASD-related genes19, decreased sociability, and impaired cognitive flexibility6,7,20. Spatial learning and memory defects, such as those detected in MWM, are especially robust in En2-/- mice6,7 and might be relevant to the cognitive impairments observed in ASD patients21. Furthermore, we showed that impaired spatial learning in MWM is associated with reduced neurofibromin expression and increased pERK levels in the hilus of En2-/-adult mice7. Here we present the detailed protocol for the immunohistochemical characterization of pERK following MWM in this ASD mouse model.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EthoVision XT 8</td>
			<td>Noldus Information Technology</td>
			<td></td>
			<td>This software platform is not a requirement &#8211; there are many other behavioral softwares on the market.</td>
		</tr>
		<tr>
			<td>Tempera Paint</td>
			<td>Giotto - Fila Group Company</td>
			<td></td>
			<td>White and liquid, non toxic. Used to prepare opaque water in the Morris water maze.</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>VT1200</td>
			<td>Equivalent models from other companies can be used.</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sigma</td>
			<td>CLS3524</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>Fisher Scientific</td>
			<td>A406-20</td>
			<td>Used to make ethanol gradient for dehydration prior to slide mounting.</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>66004-950</td>
			<td>Toxic - to be used under hood. Change xylene every month depending on use.&#160;</td>
		</tr>
		<tr>
			<td>Sodium Azide</td>
			<td>Sigma</td>
			<td>&#160;S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma</td>
			<td>P3813-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma&#160;</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Hydrogen Peroxide</td>
			<td>Sigma</td>
			<td>H1009-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Abcam</td>
			<td>G9023-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>ABC kit Vectastain&#160;</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>Add in a volume of 5 ml of PBS 2 drops of reagent A, mix and then add 2 drops of reagent B and mix.</td>
		</tr>
		<tr>
			<td>DAB peroxidase substrate</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>Add in a volume of 5 ml ddH2O: 2 drops of buffer stock solution and mix; 4 drops of DAB and mix; 2 drops of H2O2 and mix.</td>
		</tr>
		<tr>
			<td>pERK antibody</td>
			<td>Cell Signaling Technologies&#160;</td>
			<td>4370</td>
			<td>Dilution 1:500</td>
		</tr>
		<tr>
			<td>Biotinylated Goat Anti-Rabbit IgG Antibody</td>
			<td>Vector Laboratories</td>
			<td>BA-1000&#160;</td>
			<td>Dilution 1:250</td>
		</tr>
		<tr>
			<td>SuperFrost Slides&#160;</td>
			<td>Carl Roth</td>
			<td>1879</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher</td>
			<td>12-548-B</td>
			<td></td>
		</tr>
		<tr>
			<td>DPX</td>
			<td>Sigma</td>
			<td>317616</td>
			<td>Mounting medium for slides. Equivalent mounting medium can be used.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>&#160;Zeiss&#160;</td>
			<td>Axio Imager.M2</td>
			<td>Equivalent microscope can be used.</td>
		</tr>
		<tr>
			<td>Adobe Photoshop</td>
			<td>Adobe Systems, San Jose, CA</td>
			<td></td>
			<td>To assemble images.</td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institute of Health</td>
			<td></td>
			<td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>SigmaPlot 11.0</td>
			<td>Systat Software Inc. (USA)</td>
			<td></td>
			<td>Equivalent softwares for statistical analysis can be used.</td>
		</tr>
		<tr>
			<td>Prism 6</td>
			<td>GraphPad Software, Inc. (La Jolla, CA, USA)</td>
			<td></td>
			<td>Equivalent softwares for statistical analysis can be used.</td>
		</tr>
	</tbody>
</table>

immunohistochemical visualization of hippocampal neuron activity after spatial learning in a mouse model of neurodevelopmental disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

The protocol described here was designed to visualize, by immunohistochemistry, the expression of a specific marker of hippocampal neuron activity after MWM in a mouse model of neurodevelopmental disorders. All the experimental data shown herein were taken from our recent work7. Male and female WT and En2-/- age-matched adult littermates (3-5 months old; weight = 25-35 g) obtained from heterozygous matings were used. Twenty-two mice (11 per genotype) were used for the MWM. Eight mice (4 per genotype...

Watch this Scientific Journal Video about Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders at JoVE.com

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

immunohistochemical-visualization-hippocampal-neuron-activity-after

Research

JoVE Journal

Neuroscience

11.2K Views.  University of Trento.  Мы опишем протокол иммуногистохимии для изучения профиля активации гиппокампа нейронов после воздействия пространственной учебной задачи в модели мыши характеризуется когнитивных дефицитов развития нервной системы координат. Этот протокол может быть пр?...

Video: Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Using MazeSuite and Functional Near Infrared Spectroscopy to Study Learning in Spatial Navigation

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted virtual 3D environments, track a participants' behavioral performance within the virtual environment and synchronize with external devices for physiological and neuroimaging measures, including electroencephalogram and eye tracking.
Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables continuous, noninvasive, and portable monitoring of changes in cerebral blood oxygenation related to human brain functions2-7. Over the last decade fNIR is used to effectively monitor cognitive tasks such as attention, working memory and problem solving7-11. fNIR can be implemented in the form of a wearable and minimally intrusive device; it has the capacity to monitor brain activity in ecologically valid environments. 
Cognitive functions assessed through task performance involve patterns of brain activation of the prefrontal cortex (PFC) that vary from the initial novel task performance, after practice and during retention12. Using positron emission tomography (PET), Van Horn and colleagues found that regional cerebral blood flow was activated in the right frontal lobe during the encoding (i.e., initial na&iuml;ve performance) of spatial navigation of virtual mazes while there was little to no activation of the frontal regions after practice and during retention tests. Furthermore, the effects of contextual interference, a learning phenomenon related to organization of practice, are evident when individuals acquire multiple tasks under different practice schedules13,14. High contextual interference (random practice schedule) is created when the tasks to be learned are presented in a non-sequential, unpredictable order. Low contextual interference (blocked practice schedule) is created when the tasks to be learned are presented in a predictable order.
Our goal here is twofold: first to illustrate the experimental protocol design process and the use of MazeSuite, and second, to demonstrate the setup and deployment of the fNIR brain activity monitoring system using Cognitive Optical Brain Imaging (COBI) Studio software15. To illustrate our goals, a subsample from a study is reported to show the use of both MazeSuite and COBI Studio in a single experiment. The study involves the assessment of cognitive activity of the PFC during the acquisition and learning of computer maze tasks for blocked and random orders. Two right-handed adults (one male, one female) performed 315 acquisition, 30 retention and 20 transfer trials across four days. Design, implementation, data acquisition and analysis phases of the study were explained with the intention to provide a guideline for future studies.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments. Functional near-infrared spectroscopy (fNIR) is an optical brain imaging technique that enables noninvasive and portable monitoring of cerebral blood oxygenation changes. This paper summarizes collective use of MazeSuite and fNIR within a cognitive processing learning paradigm.

MazeSuite is a complete toolset to prepare, present and analyze navigational and spatial experiments1. MazeSuite can be used to design and edit adapted ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies indicate that the severity of brain injury appear to be sex dependent, and that the male neonates are more susceptible to the effects of HI-related brain injury, resulting in more severe neurological outcomes as compared to females with comparable brain injuries. The development of reliable methods to isolate and maintain highly enriched populations of sexed hippocampal astrocytes is essential to understand the cellular basis of sex differences in the pathological consequences of neonatal HI. In this study, we describe a method for creating sex specific hippocampal astrocyte cultures that are subjected to a model of in-vitro ischemia, oxygen-glucose deprivation, followed by reoxygenation. Subsequent reactive astrogliosis was examined by immunostaining for the Glial Fibrillary Acidic Protein (GFAP) and S100B. This method provides a useful tool to study the role of male and female hippocampal astrocytes following neonatal HI, separately.

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Astrocytes are one of the most important key players in the central nervous system (CNS). Here, we are reporting a practical method of sexed hippocampal astrocyte culture protocol in order to study the mechanisms underlying the astrocyte function in male and female neonate pups after in-vitro ischemia.

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Assessing Spatial Learning and Memory in Small Squamate Reptiles

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, interest in the cognitive processes of nonmodel species, typically within an ecological context, has also become an emerging field of study. In particular, interest in the cognitive processes in reptiles is growing although experimental studies on reptilian cognition are sparse. The few reptilian studies that have experimentally tested for spatial learning and memory have used rodent paradigms modified for use in reptiles. However, ecologically important aspects of the physiology and behavior of this taxonomic group must be taken into account when testing for spatially based cognition. Here, we describe modifications of the dry land Barnes maze and associated testing protocol that can improve performance when probing for spatial learning and memory ability in small squamate reptiles. The described paradigm and procedures were successfully used with male side-blotched lizards (Uta stansburiana), demonstrating that spatial learning and memory can be assessed in this taxonomic group with an ecologically relevant apparatus and protocol.

This paper describes a modification of the Barnes maze, a standard rodent paradigm used to assess spatial memory and learning, for use in small squamate reptiles.

Clinical research has leveraged a variety of paradigms to assess cognitive decline, commonly targeting spatial learning and memory abilities. However, ...

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 ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.