This study has been supported by the Edmond and Lily Safra Center for Brain Sciences (SB fellowship), The Legacy Heritage Biomedical Science Partnership Program of the Israel Science Foundation (Grant No. 378/11, to HS) and the German Israeli Foundation for Scientific Research and Development (G.I.F) (Grant No. 1093-32.2/2010, to HS).

1. Lentivirus Preparation
<ol>
	<li>Grow HEK-293FT cells to 90% confluence.</li>
	<li>On the day of transfection change cell medium to serum-free DMEM supplemented with 1 mM glutamine and 50 mg/ml penicillin-streptomycin.</li>
	<li>Co-transfect the cells with a pLKO.1-Puro vector and with plasmids coding for the delta R8.2 and VSV-G moieties and the miRNA of interest, using 10 &mu;l of 1 mg/ml polyethylenimine as a carrier 9.</li>
	<li>Collect packaged lentiviruses at 24 hr and 48 hr post-transfection, filter through 0.45 &mu;m filter.</li>
	<li>Concentrate the lentiviruses using ultracentrifugation in 70,000 x g, for 2 hr and 15 &deg;C, aliquot and store at -70 &deg;C.</li>
	<li>Measure virus titer by infecting HEK-293T cells with serially diluted virus preparations (1 to 10-6 ml of vector per well). The resulting titer (a minimum of ~1&times;109 infectious particles per ml is recommended) is evaluated for expression of the gene of interest, using puromycin selection and by quantifying GFP expressing viruses through counting fluorescent cells.</li>
</ol>
For more detailed protocol of lentivirus preparation see 10.
2. Preparing Animals for Surgery
<ol>
	<li>Surgeons garb should include surgical gown, sterile gloves, cap and mask.</li>
	<li>Weigh the animal, then anesthetize it with an IP-injection of a Ketamine mixture, with the volume proportional to the animal weight as specified for each drug. For a Ketamine/Xylazine mixture, a dose range of 80-200 mg/kg Ketamine and 7-20 mg/kg Xylazine is usually used for mice.</li>
	<li>Wait until the animal is fully anesthetized, then check for lack of withdrawal reflex in the hind limb of the animal by extending the limb and then pressing it with your finger.</li>
	<li>Inject the animal subcutaneously with Rimadyl, as specified on the box, for pain relief. Typical dose range for Rymadil is 5-10 mg.</li>
	<li>Place the animal on a heating pad to keep a steady body temperature and moisten the eyes with ointment.</li>
	<li>Shave the area between the two ears with a trimming machine.</li>
	<li>Sanitize the skin with betadine.</li>
</ol>
3. Exposure of the Skull and Drilling
<ol>
	<li>Place the animal inside the stereotact by adjusting the rods into the crevices just anterior to the animal's ears.</li>
	<li>Use a scalpel to make an anterior-posterior incision of about 1.5 cm between the ears and keep it open with surgery clamps (serfines).</li>
	<li>Clean the surface of the skull with a cotton swab until the intersection between the coronal suture and sagittal sutures; i.e. the bregma and the intersection between the coronal and lambdoid sutures; i.e. lambda, are visible.</li>
	<li>Point the tip of the syringe, held by the stereotact, to the bregma point, at all three axes. Write down the coordinates. This point would be considered as the zero point in all three axes.</li>
	<li>Lift the syringe in the vertical axis so that a planar movement would not scratch the skull and move the syringe head to the correct location. Hippocampal CA1 injections require the following coordinates relative to the bregma in mm: -2 at the anterior/posterior axis, &plusmn;1.8 at the lateral/medial axis and -1.5 at the dorsal/ventral axis.</li>
	<li>Lower the tip of the syringe until it touches the skull and mark the spot with a marker. Remove the syringe back to avoid stabbing.</li>
	<li>Drill a shallow hole, only in the skull bone using a fine driller. Hold the driller steady so it would not continue to drill into the soft tissue underneath. You can prevent this by steadying one hand with the other and by drilling in short pulses.</li>
</ol>
4. Injection of the Lentivirus
<ol>
	<li>Withdraw 0.5 ml of the concentrated lentivirus solution.</li>
	<li>Place the syringe above the hole and slowly lower it vertically until it reaches the surface of the skull. Continue to lower the syringe into the brain very slowly. At this step some bleeding might occur which does not necessarily indicate a failed penetration. If bleeding occurs mop it up with a cotton swab.</li>
	<li>Set the digital pump to 0.02 ml/min (0.5 &mu;l would be injected in 25 min) and start infusion. Slow infusion allows for effective spreading of the virus into the tissue and prevents back flow. In some syringe types consider inserting the syringe for an extra depth of 0.5 mm before retreating to original coordinates and infusing.</li>
	<li>After infusion is completed wait for additional 5 min to allow the material to spread into the brain instead of retreating back into the canal formed by the syringe.</li>
	<li>Remove the syringe very slowly and watch for back flows. If a back flow is observed at this step, a fraction of the injected material was probably lost.</li>
</ol>
5. Wound Sealing and Recovery
<ol>
	<li>Seal the wound with histoacryl. Be sure to avoid leaking of the histoacryl into the eyes.</li>
	<li>Inject the animal intraperitoneally (IP) with 1 ml pre-heated saline, to avoid dehydration.</li>
	<li>Place the animal in a recovery cage that is positioned on a heated pad. Watch the animal while it recovers for an additional hour.</li>
</ol>
In case of weight loss, inappetance, weakness/inability to obtain feed or water, moribund state or infection, euthanasia of the animal should be performed. Acceptable methods for euthanasia of rodents are barbiturates, inhalant anesthetics, CO2, CO, or potassium chloride in conjunction with general anesthesia.
After 4 to 6 weeks, assess the animal's navigation memory is assessed in the Morris Water Maze. The lentivirus will usually infect most cells within the injection sphere.
6. Behavioral Assessment in the Morris Water Maze
<ol>
	<li>Train the animals in the Morris Water Maze. For exact training and testing protocol see 11. After each trial, dry the animal with a dry towel, replace it in its cage and refresh the water in the tank.</li>
	<li>Test the animals in the Morris Water Maze for 3 consecutive days, 4 trials each day. Each day insert the animal into the maze in each of the 4 directions of the maze (north, south, east and west) in a changing order, for example day one: east-west-north-south, day two: west-south-north-east. Wipe the animals after each trial.</li>
	<li>During the test trials track the animal with a tracking system such as the Noldus tracking system.</li>
	<li>After the 12th test trial, insert the animal into the water tank without the platform for one minute. This would be used to analyze the searching strategy the animal is taking.</li>
	<li>Analyze the data as suggested in 12.</li>
</ol>

<ol>
	<li>Siegel, 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=A+functional+screen+implicates+microRNA-138-dependent+regulation+of+the+depalmitoylation+enzyme+APT1+in+dendritic+spine+morphogenesis.">A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis.</a> Nature Cell Biology. 11, 705-716 (2009).</li><li>Jin, P., 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=Biochemical+and+genetic+interaction+between+the+fragile+X+mental+retardation+protein+and+the+microRNA+pathway.">Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway.</a> Nature Neuroscience. 7, 113-117 (2004).</li><li>Simon, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-1+regulates+a+MEF-2-dependent+retrograde+signal+at+neuromuscular+junctions.">The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions.</a> Cell. 133, 903-915 (2008).</li><li>Consiglio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+therapy+of+metachromatic+leukodystrophy+by+lentiviral+vectors:+correction+of+neuropathology+and+protection+against+learning+impairments+in+affected+mice.">In vivo gene therapy of metachromatic leukodystrophy by lentiviral vectors: correction of neuropathology and protection against learning impairments in affected mice.</a> Nature Medicine. 7, 310-316 (2001).</li><li>Jakobsson, J., Lundberg, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+for+use+in+the+central+nervous+system.">Lentiviral vectors for use in the central nervous system.</a> Molecular Therapy: The Journal of the American Society of Gene Therapy. 13, 484-493 (2006).</li><li>Berson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic-associated+loss+of+hnRNP-A/B+in+Alzheimer's+disease+impairs+cortical+splicing+and+cognitive+function+in+mice.">Cholinergic-associated loss of hnRNP-A/B in Alzheimer's disease impairs cortical splicing and cognitive function in mice.</a> EMBO Molecular Medicine. , (2012).</li><li>Haramati, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+as+repressors+of+stress-induced+anxiety:+the+case+of+amygdalar+miR-34.">MicroRNA as repressors of stress-induced anxiety: the case of amygdalar miR-34.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 31, 14191-14203 (2011).</li><li>Shaltiel, 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=Hippocampal+microRNA-132+mediates+stress-inducible+cognitive+deficits+through+its+acetylcholinesterase+target.">Hippocampal microRNA-132 mediates stress-inducible cognitive deficits through its acetylcholinesterase target.</a> Brain Structure &amp; Function. , 10-1007 (2012).</li><li>Boussif, O., 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=A+versatile+vector+for+gene+and+oligonucleotide+transfer+into+cells+in+culture+and+in+vivo:+polyethylenimine.">A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.</a> Proceedings of the National Academy of Sciences of the United States of America. 92, 7297-7301 (1995).</li><li>Mendenhall, A., Lesnik, J., Mukherjee, C., Antes, T., Sengupta, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Packaging+HIV-+or+FIV-based+lentivector+expression+constructs+&amp;+transduction+of+VSV-G+pseudotyped+viral+particles.">Packaging HIV- or FIV-based lentivector expression constructs &amp; transduction of VSV-G pseudotyped viral particles.</a> J. Vis. Exp. (62), e3171 (2012).</li><li>Nunez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+Water+Maze+Experiment.">Morris Water Maze Experiment.</a> J. Vis. Exp. (19), e897 (2008).</li><li>Bromley-Brits, K., Deng, Y., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze+test+for+learning+and+memory+deficits+in+Alzheimer's+disease+model+mice.">Morris water maze test for learning and memory deficits in Alzheimer's disease model mice.</a> J. Vis. Exp. (53), e2920 (2011).</li><li>Regev, L., Ezrielev, E., Gershon, E., Gil, S., Chen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+approach+for+intracerebroventricular+delivery.">Genetic approach for intracerebroventricular delivery.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 4424-4429 (2010).</li></ol>

The authors declare that they have no competing financial interests.

Stereotactic injection of a lentivirus is a relatively rapid method for in vivo assessment for both up- or down- regulation of different genes and miRNAs. The main alternative is a genetically engineered mouse which is a much more laborious and time consuming technique then direct lentivirus injection. In addition, the up regulation, in lentivirus injection, occurs at a specific time in the adult mouse and does not include any possibility of leakiness during development, as is most often the case in the genetica...

The role of particular miRNAs in nervous system functioning has recently been challenged by lentiviral injection in several studies. MiRNAs have been found to be crucial for maintaining and re-shaping synapse structure 1, synaptogenesis 2 and synapse remodeling and maintenance 3. These studies strongly suggest that miRNAs are engaged, via multileveled regulatory effects in both the shaping up and in maintaining the main output of the nervous system, cognitive function. Stereotactic injection of lentivirus particles into specific regions in the rodent brain enables searching for alterations in synapse morphology and neuronal activity, and was used for establishing the functional significance of over-expressed transcripts 4,5. Direct infection of neurons at well-defined brain areas with miRNA-expressing lentiviruses may be implicated in studies of aging, brain disorders and neurodegeneration; Studies of miRNAs in the realm of behavioral regulation 6-8 are at a far less advanced state, and stereotactic injection of miRNA-expressing lentivirus particles followed by behavioral tests may be useful for such purposes. A considerably more laborious method for inducing over- or under-expression involves genetically engineered knock-in or knockout mice. Genetic systems can further allow for conditional and temporal control on expression (e.g. Cre-Lox, Tet systems), but these hardly offer the spatial specificity of the injection procedure and there is almost always a certain amount of leakiness. Also, the engineering procedures do not require surgery and can be used across laboratories with relatively good reproducibility; however, they are slower and require much more manpower and financial resources. In addition, the temporal control of the over- or under-expression in injected mice is far more accurate compared to genetically engineered mice.

<table border="0" cellpadding="0" cellspacing="0" width="748">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21">Rodent weigh scale</td>
			<td>Burtons (UK)</td>
			<td>115-455</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="24">
			<td height="24">heating pad</td>
			<td>FIRstTechnology</td>
			<td>DCT-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">trimming machine</td>
			<td>Stoelting</td>
			<td>51465</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">stereotact</td>
			<td>Stoelting</td>
			<td>51730</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Scalpel and blades</td>
			<td>Kent scientific</td>
			<td>INS500348</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Harland syringe</td>
			<td>Hamilton</td>
			<td>7632-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">driller</td>
			<td>Stoelting</td>
			<td>51449</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">digital pump</td>
			<td>Harvard apparatus</td>
			<td>704507</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Water tank and platform</td>
			<td>Stoelting</td>
			<td>60135</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
<table border="0" cellpadding="0" cellspacing="0" width="682">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td></td>
			<td></td>
			<td></td>
			<td>Reagents</td>
		</tr>
		<tr height="21">
			<td height="21">ketamine </td>
			<td>Vetoquinol(Lure France)</td>
			<td>3055503</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">domitor</td>
			<td>Orion pharma</td>
			<td>107140-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">Rimadyl</td>
			<td>Pfizer animal health</td>
			<td>24751</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="118">
			<td height="138" rowspan="2">moisture ointment - Synthomycine 5%</td>
			<td rowspan="2">Rekah Pharmaceutical</td>
			<td rowspan="2">195</td>
			<td rowspan="2"> </td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="21">
			<td height="21">histoacryl</td>
			<td>Braun</td>
			<td>112101</td>
			<td>&nbsp;</td>
		</tr>
		<tr height="21">
			<td height="21">saline</td>
			<td>Sigma Aldrich</td>
			<td>D8662</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an entire gene expression pathway by inducing destruction and/or inhibiting translation of these targets. Several miRNAs play key roles in maintaining neuronal structure and function and in higher-level brain functions, and methods are sought for manipulating their levels for exploring these functions. Here, we present a direct in vivo method for examining the cognitive consequences of enforced miRNAs excess in mice by stereotactic injection of miRNA-encoding virus particles. Specifically, the current protocol involves injection into the hippocampal CA1 region, which contributes to mammalian memory consolidation, learning, and stress responses, and offers a convenient injection site. The coordinates are measured according to the mouse bregma and virus perfusion is digitally controlled and kept very slow. After injection, the surgery wound is sealed and the animals recover. Lentiviruses encoding silencers of the corresponding mRNA targets serve to implicate the specific miRNA/target interaction responsible for the observed effect, with na&iuml;ve mice, mice injected with saline and mice injected with "empty" lentivirus vectors as controls. One month post-injection, the animals are examined in the Morris Water Maze (MWM) for assessing their navigation learning and memory abilities. The MWM is a round tank filled with colored water with a small platform submerged 1 cm below the water surface. Steady visual cues around the tank allow for spatial navigation (sound and the earth's magnetic field may also assist the animals in navigating). Video camera monitoring enables measuring the route of swim and the time to find and amount the platform. The mouse is first taught that mounting the hidden platform offers an escape from the enforced swimming; it is then tested for using this escape and finally, the platform is removed and probe tests examine if the mouse remembers its previous location. Repeated tests over several consecutive days highlight improved performance of tested mice at shorter latencies to find and mount the platform, and as more direct routes to reach the platform or its location. Failure to show such improvement represents impaired learning and memory and/or anxiety, which may then be tested specifically (e.g. in the elevated plus maze). This approach enables validation of specific miRNAs and target transcripts in the studied cognitive and/or stress-related processes.

Injection of 0.5 &mu;l lentivirus into the CA1 region in the mouse hippocampus, with the flow rate indicated in the protocol section yields an infected sphere of about 1 mm in the rostral-caudal axis, and about 0.5 mm in the medial-lateral and the anterior-posterior axes (Figure 3).
<img alt="Figure 1" src="/files/ftp_upload/50170/50170fig1.jpg" /> 
 Figure 1. Bregma point and injection apparatus. (a)...

Watch this Scientific Journal Video about Stereotactic Injection of MicroRNA-expressing Lentiviruses to the Mouse Hippocampus CA1 Region and Assessment of the Behavioral Outcome at JoVE.com

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 regions.

MicroRNAs (miRNAs) are small regulatory single-stranded RNA molecules around 22 nucleotides long that may each target numerous mRNA transcripts and dim an ...

stereotactic-injection-microrna-expressing-lentiviruses-to-mouse

Research

JoVE Journal

Behavior

22.7K Views.  The Hebrew University of Jerusalem. 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 regions.

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

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

Behavioral Assessment of the Aging Mouse Vestibular System

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of studies show changes in balance phenotype with age in rodents, very few isolate the vestibular contribution to balance under either normal conditions or during senescence. We use two standard behavioral tests to characterize the balance performance of mice at defined age points over the lifespan: the rotarod test and the inclined balance beam test. Importantly though, a custom built rotator is also used to stimulate the vestibular system of mice (without inducing overt signs of motion sickness). These two tests have been used to show that changes in vestibular mediated-balance performance are present over the murine lifespan. Preliminary results show that both the rotarod test and the modified balance beam test can be used to identify changes in balance performance during aging as an alternative to more difficult and invasive techniques such as vestibulo-ocular (VOR) measurements.

Motor control and balance performance are known to deteriorate with age. This paper presents a number of standard noninvasive behavioral tests with the addition of a simple rotary stimulus to challenge the vestibular system and show changes in balance performance in a murine model of aging.

Age related decline in balance performance is associated with deteriorating muscle strength, motor coordination and vestibular function. While a number of ...

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2.
In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells.
The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies.

This manuscript describes a protocol that applies comprehensive profiling for analysis of transcriptional programs induced in specific brain nuclei of rodents following behavioral paradigms. Herein, this approach is illustrated in the context of profiling genes induced in the nucleus accumbens (NAc) of mice following acute cocaine exposure, utilizing microfluidic qPCR arrays.

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the test mouse is placed in a standardized apparatus and relevant behaviors are assessed in three different sessions (called session I, II, and III).
The apparatus has three compartments (see Figure 1), the left and right compartments contain an inverted cup which can house a mouse (called &#34;stimulus mouse&#34;).
In session I, the test mouse is placed in the cage and its mobility is characterized by the number of transitions made between compartments. In session II, a stimulus mouse is placed under one of the inverted cups and the sociability of the test mouse is quantified by the amounts of time it spends near the cup containing the enclosed stimulus mouse vs. the empty inverted cup. In session III, the inverted cups are removed and both mice interact freely. The sociability of the test mouse in session III is quantified by the number of social approaches it makes toward the stimulus mouse and by the number of times it avoids a social approach by the stimulus mouse.
The automated evaluation of the movie detects the nose of the test mouse, which allows the determination of all described sociability measures in session I and II (in session III, approaches are identified automatically but classified manually). To find the nose, the image of an empty cage is digitally subtracted from each frame of the movie and the resulting image is binarized to identify the mouse pixels. The mouse tail is automatically removed and the two most distant points of the remaining mouse are determined; these are close to nose and base of tail. By analyzing the motion of the mouse and using continuity arguments, the nose is identified.
<img alt="Figure 1" src="/files/ftp_upload/52508/52508fig1.jpg" /> 
Figure 1. Assessment of Sociability During 3 sessions. Session I (top): Acclimation of test mouse to the cage. Session II (middle): Test mouse moving freely in the cage while the stimulus mouse is enclosed in an inverted cup. Session III (bottom): Both test mouse and stimulus mouse are allowed to move freely and interact with each other.

This protocol describes a method to quantify mouse sociability. Mice are videotaped as they move and interact in a special cage. Movie processing allows for the automated quantification of sociability with excellent accuracy and reliability.

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.

Stereotactic injection of lentiviruses expressing cDNAs or shRNAs can modulate gene expression in specific brain areas of mice. Here, we present a protocol to combine stereotactic injections with behavioral tasks, such as the Open Field Test (OFT) and the Forced Swim Test (FST).

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Behavioral Assessment of Hearing in 2 to 4 Year-old Children: A Two-interval, Observer-based Procedure Using Conditioned Play-based Responses

Collecting reliable behavioral data from toddlers and preschoolers is challenging. As a result, there are significant gaps in our understanding of human auditory development for these age groups. This paper describes an observer-based procedure for measuring hearing sensitivity with a two-interval, two-alternative forced-choice paradigm. Young children are trained to perform a play-based, motor response (e.g., putting a block in a bucket) whenever they hear a target signal. An experimenter observes the child&#39;s behavior and makes a judgment about whether the signal was presented during the first or second observation interval; the experimenter is blinded to the true signal interval, so this judgment is based solely on the child&#39;s behavior. These procedures were used to test 2 to 4 year-olds (n = 33) with no known hearing problems. The signal was a 1,000 Hz warble tone presented in quiet, and the signal level was adjusted to estimate a threshold corresponding to 71%-correct detection. A valid threshold was obtained for 82% of children. These results indicate that the two-interval procedure is both feasible and reliable for use with toddlers and preschoolers. The two-interval, observer-based procedure described in this paper is a powerful tool for evaluating hearing in young children because it guards against response bias on the part of the experimenter.

This paper describes a procedure for measuring hearing sensitivity in 2 to 4 year-old children. Children are trained to perform play-based responses when they hear a target signal. Thresholds are then estimated in a two-interval, two-alternative forced-choice paradigm, based on observations of the child&#39;s behaviors.

Collecting reliable behavioral data from toddlers and preschoolers is challenging. As a result, there are significant gaps in our understanding of human ...

A Behavioral Test Battery for the Repeated Assessment of Motor Skills, Mood, and Cognition in Mice

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and dysfunctions in mood and cognition are common and often shared symptoms in neurodegenerative diseases. Shown here is a behavioral test battery for motor, mood, and cognition, which can be repeatedly tested in a longitudinal study. This battery assesses the overall behavioral phenotype in mice by examining each domain of behavior with at least two independent well-accepted tests (i.e., open-field test and rotarod test for motor function, social interaction test, elevated plus maze test, and forced swim test for emotional function, and Morris water maze test and novel object recognition test for cognitive function). Therefore, this sensitive and comprehensive test battery is a powerful tool for the study of behavioral alternation in neurodegeneration.

A comprehensive behavioral test battery of motor skills, mood&#8212;including social interaction, depression, and anxiety&#8212;and cognition is designed for the repeated assessment of neurodegeneration-related behavioral changes in mice.

Pharmacological and toxicological studies in neurodegeneration require comprehensive behavioral analysis in mice because motor dysfunctions and ...

Utilizing a Reconfigurable Maze System to Enhance the Reproducibility of Spatial Navigation Tests in Rodents

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze shape, thus requiring several separate mazes in different configurations. The maze geometry cannot be reconfigured in a single environment to accommodate scalability and reproducibility. The reconfigurable maze is a unique approach to address the limitations, allowing quick and flexible configurations of maze pathways in a repeatable manner. It consists of interlocking pathways and includes feeders, treadmills, movable walls, and shut-off sensors. The current protocol describes how the reconfigurable maze can replicate existing mazes, including the T-shaped, plus-shaped, W-shaped, and figure-eight mazes. Initially, the T-shaped maze was constructed inside a single experimental room, followed by modifications. The rapid and scalable protocol outlined herein demonstrates the flexibility of the reconfigurable maze, achieved through the addition of components and behavioral training phases in a stepwise manner. The reconfigurable maze systematically and precisely assesses the performance of multiple aspects of spatial navigation behavior.

The present protocol describes a reconfigurable maze, a unique system for testing spatial navigation and behavioral phenotypes in rodents. The adaptability of this maze system enables the execution of various experiments in a single physical environment. The ease of structural rearrangement generates reliable and reproducible experimental results.

Several maze shapes are used to test spatial navigation performance and behavioral phenotypes. Traditionally, each experiment requires a unique maze ...

Validating a Murine Model of Angelman Syndrome Using Behavioral Tests

Behavioral Characterization of an Angelman Syndrome Mouse Model

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of AS. We use the rotarod learning paradigm, detailed gait analysis, and nest building test to detect and characterize animal motor impairments. We test animal emotionality in the open field and elevated plus maze tests, as well as the affect in the tail suspension test. When AS mice are tested in the open field test, the results should be interpreted with care, since motor dysfunctions influence mouse behavior in the maze and alter activity scores.
The reproducibility and effectiveness of the presented behavioral tests has already been validated in several independent Uba3a mouse lines with different knockout variants, establishing this set of tests as an excellent validation tool in AS research. Models with the relevant construct and face validity will warrant further investigations to elucidate the pathophysiology of the disease and grant the development of causal treatments.

Author Spotlight: A Battery of Highly Reproducible Behavioral Tests to Validate an Angelman Syndrome Murine Model 

This manuscript presents a set of highly reproducible behavioral tests to validate an Angelman syndrome mouse model.

This manuscript describes a battery of behavioral tests available to characterize Angelman syndrome (AS)-like phenotypes in an established murine model of ...