Name: immunohistochemical visualization of hippocampal neuron activity after spatial learning in a mouse model of neurodevelopmental disorders
Uploaded: 2015-05-12T15:00:00
Description: 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

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 polycarbo...

<ol>
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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 ...

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