We thank Souvik Maiti for help in designing the antagomirs. We also acknowledge Rangeetha J. Naik, Rakesh Dey, and Bijay Pattnaik for their help with experimental methods. This work was funded by the Council of Scientific and Industrial Research (BSC0123). HS, MV and RR acknowledge fellowship from the Council of Scientific and Industrial Research, India. MAS acknowledge fellowship from the University Grants Commission, India.

Note: All the procedure including animal subjects have been approved by Institutional Animals Ethics Committee (IAEC) at the Institute of Genomics and Integrative Biology, New Delhi (IGIB/AEC/10/2013). This protocol is specifically adjusted for targeted delivery of Antagomir-29 in the brain and knockdown of miR-29.
1. Antagomir Design Strategy
<ol>
	<li>Retrieve the mature miRNA sequence from miRBase11 (<a href="http://www.mirbase.org/">http://www.mirbase.org/</a>).</li>
	<li>Retrieve the sequences of related miRNA family members using the &#8220;Gene Family&#8221; link in the miRBase record 
	(<a href="http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009">http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000009</a> ).</li>
	<li>Reverse complement the sequence: <a href="http://www.bioinformatics.org/sms/rev_comp.html">http://www.bioinformatics.org/sms/rev_comp.html</a> provides a convenient tool to reverse complement sequences.</li>
	<li>Mark the positions to be modified by LNA using the following guidelines:
	<ol>
		<li>Choose Thymine residues placed 3-4 bases apart for the LNA modification, since LNA pyrimidines are more stable than purines12.</li>
		<li>Choose Cytosine residues separated by 3-4 residues if previous step produces less than five modifications.</li>
		<li>Prioritize the five modifications to the 5&#8217; end of the antagomir, since this avoids the seed sequence.</li>
	</ol>
	</li>
</ol>
2. Antagomir-neuropeptide Complex Preparation
Note: This is the most critical step in the protocol and it needs standardization. To standardize the protocol any fluorescently labeled oligonucleotide (FLO) can be used in place of the antagomir9. The peptides should be of high purity (HPLC grade, 98% purity).Sequences and charge of peptide and antagomir are given in Table 1.
<ol>
	<li>Decide the number of moles of antagomir to be injected based on molar charge on antagomir, its toxicity and weight of the mouse. Calculate the number of moles of peptide to be injected based on molar charge ratio of antagomir : peptide. 
	NOTE: Standardize molar charge ratio of antagomir : peptide for toxicity and effective delivery in the mouse brain. Use different molar charge ratios of fluorescently labeled oligonucleotide: peptide for standardization. We found that Antagomir-29 and control were both toxic at 4microgram/gram body weight of mouse.&#160; Antagomir-29 and control were used at 2microgram/gram body weight, but this effective concentration is likely to vary for each microRNA.</li>
	<li>Dilute the peptide and antagomir in separate microfuge tubes from stock solutions with sterile 10% D-glucose to the desired final concentration as calculated in step 1.</li>
	<li>Keep the microfuge tube with the peptide solution on a vortex at moderate vortexing speed.</li>
	<li>Add 1/3rd of the antagomir solution to the peptide solution tube, slowly, drop wise, while it is mixed thoroughly on a vortex mixer.</li>
	<li>Continue mixing of solutions on the vortex for next 1 min and then allow the mixture to stand for 1 min.</li>
	<li>Repeat the step 4 and 5 two more times to mix the remaining antagomir solution with the peptide solution in the same microfuge tube. The slow addition is critical for the formation of mono-disperse complexes that are effective in transfection. Rapid addition can lead to aggregation of the complexes and their precipitation.</li>
	<li>Incubate the complex for 30 min at RT without vortexing. During this time of&#160;&#160;&#160; incubation, acclimatize the mice to the lab where injections will be performed.</li>
</ol>
3. Tail Vein Injection of Antagomir-neuropeptide Complex
<ol>
	<li>To restrain the animal, place the mouse in a restrainer or decapicone of proper size.</li>
	<li>Clean the surface of the tail using cotton swab pre-soaked in warm water (about 40&#176;C). This will promote vasodilation and increase the visibility of the vein.</li>
	<li>Approach the tail with a small volume (0.5 &#8211; 1.0 cc) insulin syringe at a 15-20&#176; angle. Be careful not to introduce any air into the syringe. Start at the distal portion of the tail. Inject the complex. Remove the needle and apply an antiseptic swab directly to the injection site (approximately 5-10 sec) to stop any bleeding.</li>
	<li>Replace the mouse in an individually ventilated cage in the group of 3-5, with the corncob and tissue paper as enrichment. Do not keep uninjected and injected mice in same cage.</li>
</ol>
4. Behavioural Assays
Note: Keep the time interval of 3-4 hr in between injection and behavioural assays to allow recovery after injection. Bring the mouse to a quiet room.&#160; Do not disturb the animals during this period of acclimatization.
<ol>
	<li>Acclimatize the mouse to the new room for 30 min and then start the following behavioural assays. Keep a gap of 10 min in between each behavioural assay.</li>
	<li>Hindlimb clasping: 
	NOTE: This test indicates motor dysfunction and neurological impairment.
	<ol>
		<li>Lift the mouse gently by holding near base of the tail in the clear background.</li>
		<li>Check the hindlimb position for 10-15 sec. Wild type mouse splays hindlimbs away from abdomen, while an ataxic mouse tends to retract one or both hindlimbs towards the abdomen more than 50% of time suspended13.</li>
		<li>Return the mouse to the cage</li>
	</ol>
	</li>
	<li>Ledge test:
	<ol>
		<li>Lift the mouse gently by holding the tail and keep it on the ledge of a cage.</li>
		<li>Observe the mouse specifically at the corner of the cage. Wild type mouse can pass the corners easily without fear and losing the balance. Ataxic mouse lose its balance, freeze or shakes while walking along the cage ledge and at the corners.</li>
	</ol>
	</li>
	<li>Mouse foot print assay: 
	NOTE: For this assay keep the absorbent sheet ready, on the floor of a narrow runway (~70-cm-long, ~5-cm-wide with ~5-cm-high walls.)
	<ol>
		<li>Hold the mouse gently by one hand and apply ink on the hind limbs by a brush.</li>
		<li>Place the absorbent sheet on the floor of a narrow runway.</li>
		<li>Allow mouse to walk or run over an absorbent sheet in a straight line in a runway from one end to other.</li>
		<li>Repeat the process two more times with each mouse using fresh absorbent sheet.</li>
		<li>Measure the distance between two consecutive steps in the forward movement. Do not include first and last few footprints where the animal is just initiating and finishing its run, respectively.</li>
	</ol>
	</li>
</ol>

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J., Van Dongen, S., Bateman, A., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+microRNA+sequences,+targets+and+gene+nomenclature.">miRBase: microRNA sequences, targets and gene nomenclature.</a> Nucleic Acids Research. 34, D140-D144 (2006).</li><li>Kaur, H., Babu, B. R., Maiti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+Chemistry+and+Therapeutic+Applications+of+Locked+Nucleic+Acid+(LNA).">Perspectives on Chemistry and Therapeutic Applications of Locked Nucleic Acid (LNA).</a> Chemical Reviews. 107, 4672-4697 (2007).</li><li>Guyenet, S. 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The authors have nothing to disclose.

Here we demonstrate a widely accessible methodology to study the effects of miRNA modulation. Currently, most attempts at in vivo characterization of miRNA functions involve the creation of knockout mice or a transgenic that expresses a miRNA sponge. Most miRNAs, even the cell type specific ones are expressed in more than one organ. For instance, miRNAs initially thought to be specific to the hematopoietic system are also expressed in the brain, due to the presence of microglia. Thus even a cell type specific knock-down strategy, expressing anti-sense agents against miRNAs under the control of cell type specific promoters is likely to produce pleotropic effects. miR-29 family of miRNAs for instance are expressed in the brain, blood cells and several other sites in the body. Thus genetic tools for modulation of miRNA are not widely applicable. It is also not amenable to drug development or testing of potential therapeutic strategies. 
The ability to deliver the cargo through simple tail vein injections makes the technique widely accessible. It also circumvents the use of lentiviruses for neurotropic delivery. This method, originally developed for knockdown of a neurotropic virus, is amenable to modular changes in the three domains that produced cell type specific knockdown of miRNA, namely, the cell penetrating peptide, the neurotropic peptide and the anti-sense molecule. For instance, we found that using a different miR-29 specific antagomir leads to knockdown but fails to produce the behavioural effects9. Besides cell penetrating peptides, non-viral methods of gene delivery to neuronal cells include nano-particles, lipids and cationic polymers. However, many viral vectors and cationic lipids still need invasive methods of administration due to the blood brain barrier. One approach to this problem has been to use mimics of endogenous proteins that can cross the blood brain barrier. 
Monoclonal antibodies against the insulin receptor and transferrin receptor have been used effectively as in vivo brain delivery vectors since the blood brain barrier expresses these receptors14. The Rabies virus glycoprotein derived, RVG peptide is known to bind to the nicotinic acetylcholine receptor and has been used in combination with liposomes and nano-particles. The unique ability of this peptide to cross the blood brain barrier with minimal toxicity and carry siRNA, small molecules, anti-sense molecules or plasmid for delivery to the brain can be very useful in drug delivery to the CNS8,15,16. More specifically, a simple conjugate of the peptide with a modified anti-sense molecule, for CNS delivery of microRNA inhibitors by-passing the blood brain barrier can accelerate in vivo functional studies on neuronal miRNAs. 
The most critical step in this protocol is antagomir-neuropeptide complex preparation. Rapid addition of antagomir to the peptide solution may cause precipitation. This reduces the targeting efficiency. Also dose of peptide and antagomir has to be standardized because low dose of peptide or antagomir may not be efficient for the effective knockdown of target molecule while higher dose may be toxic to the mouse due to off-targets. 
A minor limitation of this technique is that repeated injection of the complex has to be given for at-least 3 days to achieve phenotypic effect. The treatment regimen is likely to vary from microRNA to microRNA. Also the effect of knockdown of the molecule is transient, lasting for a few days.We found that a treatment regimen of single daily dose of antagomir:peptide for 3 continuous days resulted in knockdown for five days after which expression of miR-29 was restored (unpublished observation). 
The applicability of this method to knockdown of larger molecules like long non-coding RNAs need to be tested since it is not clear if effective complexes can be formed with larger RNA. A potential limitation could be the high cumulative negative charge on long RNAs, and consequently, the large amount of peptide required to achieve the required charge ratio.
Using a fluorescent, non-targeting oligonucleotide we found that the peptide effectively delivers the cargo to the cortex, cerebellum and the hippocampus. Thus pan-brain delivery was achieved. The knockdown of the miRNA was also comparable in all three parts of the brain. The modular nature of the method allows it to be adopted in future for delivery of miRNA mimics to restore pathologically down-regulated miRNAs.

MicroRNAs have emerged as novel therapeutic targets due to their universal role in the regulation of gene expression and direct evidence for involvement in disease. MiRNAs are being actively explored for their potential as drug targets1,2. Further, alterations in miRNA expression are associated with several diseases3 and simulation of these changes by artificial perturbation of miRNA expression can be used to study the cellular pathways involved in disease manifestation. Tissue specific delivery of miRNA targeting drugs is currently a major challenge for miRNA based drug development. Antagomirs and miRNA mimics are promising agents for perturbing miRNA levels4&#8211;6. However, special features that enhance their specificity and efficacy have to be incorporated into the design of antagomirs before they can be used for in vivo perturbation of miRNA expression.
MicroRNAs are especially relevant as targets in currently incurable neurodegenerative and neuro-developmental diseases. The blood-brain barrier poses a restriction to the delivery of antagomirs in the brain. Stereotactic injections are widely used in rodent models to deliver molecules to specific locations in the brain7. It requires skill, extensive investment in instrumentation and time. Stereotactic injections are invasive, involve surgery, cause at least minor injury and are restricted to local delivery. The use of cell penetrating peptides with a preference for targeting neurons can counter these limitations since they can be delivered through the trans-vascular route but breach the blood brain barrier. Such a peptide derived from the Rabies Virus Glycoprotein (RVG), was previously used to deliver siRNA against Japanese Encephalitis Virus in mice8. We found that using the peptide for antagomir delivery, miRNAs can be effectively knocked down in the mouse brain9.
The second major challenge of miRNA knock-down arises from the small size of miRNAs and the presence of closely related sequence isoforms. We take the example of mmu-miR-29 family which consists of three closely related isoforms, miR-29a, b and c. Antagomirs are also generally modified along the backbone to increase their stability and render them resistant to attack by nucleases. Locked Nucleic Acids (LNAs) offer a further advantage that they enhance thermal stability and even lead to target degradation over and beyond steric hindrance10. Introducing modifications all along the backbone can be effective but expensive. We have earlier seen that modifications beyond an optimal number may not further enhance the efficacy. The design of the antagomir therefore involves the optimal modification of the antagomir.
To complex the antagomir non-covalently with the neurotropic peptide, a charged hepta- to nona-arginine extension is used. D-Arginine residues are used since they confer higher stability as they are not susceptible to cleavage by proteases. Hepta- to nona-arginine stretches act as efficient cell penetrating agents, although they do not confer cell type specificity. By covalently linking the RVG peptide to the nona-arginine linker, a neurotropic, cell penetrating peptide was generated. The positively charged residues of the peptide interact with the negatively charged nucleic acid backbone, to form complexes. These complexes can be used to effectively transfect DNA or RNA into cultured cells and in vivo into tissues.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Restrainer or Decapicone</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Narrow runway</td>
			<td></td>
			<td></td>
			<td>~70-cm-long, ~5-cm-wide with ~5-cm-high walls.</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescently labelled oligonucleotides (siGLO)</td>
			<td>GE Healthcare Dharmacon INC</td>
			<td>D0016300120</td>
			<td></td>
		</tr>
		<tr>
			<td>10% sterile D-glucose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Antagomir-control</td>
			<td>Exiqon</td>
			<td>custom synthesis</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuropeptide RVG</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td>Neuropeptide RVM</td>
			<td>G.L.Biochem (Shanghai) Ltd.</td>
			<td>custom synthesis</td>
			<td>&#62;98% purity</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Other</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Warm water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent sheets</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ink</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple alternative to stereotactic injection for brain specific knockdown of mirna

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

Using the procedure presented here, complexes of 50microgram fluorescently labeled oligonucleotide (FLO) and ~850microgram RVG peptide of 1:15 molar charge ratio (FLO : peptide) were prepared and injected only once through tail vein. Complex of non-neurotropic Rabies Virus Matrix (RVM) peptide and FLO was used as a delivery control. Next day, mice brain and liver were isolated and single cell suspensions were prepared. Cells were observed under microscope for green fluorescence. FLO-RVG complex was successfully delivered with a shortest time of one day and detected as a green fluorescence in the brain cells but not in the liver (Figure 2B,2D) while RVM peptide delivered FLO to the liver but not in the brain cells (Figure 2A,2C).
To knockdown miR-29 in the mouse brain antagomir against miR-29 were designed with five LNA modifications. The scrambled, non-miRNA targeting sequence used as an Antagomir-control. Complex of 50microgram Antagomir-control or Antagomir-29 with ~850microgram RVG peptide was prepared and injected through tail vein over a 3 day time course, followed by daily behaviour assays as shown in figure 1. In order to achieve maximum knockdown effect, complexes were injected once per day for 3 continuous days. On fourth day behaviour assays were performed without injection. Mouse footprint analysis showed significant reduction in the step length of Antagomir-29 RVG injected mice (Figure 3A-3B). Hindlimb clasping assay of Antagomir-29 RVG injected mice clearly showed ataxic behaviour with tendency to retract one or both hindlimbs towards the abdomen. In the ledge test Antagomir-29 RVG injected mice showed freezing or shaking behaviour with loss of balance at the corner of the cage. Mice were euthanized by injection of xylazine hydrochloride (16milligram/kg) and thiopentone sodium (80milligram/kg) on fourth day (24 hr after the last injection of complex). Brain samples were used to perform molecular and cellular analysis. Total RNA collected from cortex, cerebellum and hippocampus. Quantitative real time PCR assay showed clear reduction in the expression levels of both the isoforms of miR-29, miR-29a (Figure 4A) and miR-29b (Figure 4B), in the Antagomir-29 RVG injected mice brain parts.
<img alt="Figure 1" src="/files/ftp_upload/53307/53307fig1.jpg" /> 
Figure 1. Schema for mouse tail vein injection and behaviour. 
In order to target miR-29 in the brain, Antagomir-control or Antagomir-29 with RVG peptide was injected once a day for 3 consecutive days through tail vein. Behavioural assays were performed daily, after a 3 hr period, to allow the mouse to recover from injection. On fourth day only behavioural assays were performed and mice were euthanized and brain tissue was collected for real time PCR and cellular assays. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53307/53307fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53307/53307fig2.jpg" /> 
Figure 2. RVG peptide delivers fluorescently labeled oligonucleotide specifically to the mouse brain. 
Using the method described in this article, complex of fluorescently labeled oligonucleotides (FLO) and peptide RVG/RVM were prepared and injected only once through tail vein. FLO-RVG complex was detected as a green fluorescence in the brain cells but not in the liver (B,D) while RVM peptide delivered FLO to the liver but not the brain (A,C)9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53307/53307fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53307/53307fig3.jpg" /> 
Figure 3. miR-29 knock down leads to the ataxic behaviour. 
Mouse footprint assays (A) on fourth day showed clear reduction in distance between two consecutive steps for the mouse treated with Antagomir-29 (B) (**) p-value &#60; 0.001; n=3 (Figure modified from9.) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53307/53307fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53307/53307fig4.jpg" /> 
Figure 4. Antagomir-29 and RVG complex down-regulates miR-29 in mouse brain. 
Total RNA collected from three brain parts. Quantitative real time PCR assay was performed to estimate expression levels of both the isoforms of miR-29, miR-29a (A) and miR-29b (B), and normalized to miR-92a. (Figure modified from9.) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53307/53307fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Sequence</td>
			<td>Charge</td>
			<td>Length</td>
		</tr>
		<tr>
			<td>RVG-peptide</td>
			<td>YTIWMPENPRPGTPCDIFTNSRGK 
			RASN-GGGG-RRRRRRRRR*</td>
			<td>11 +</td>
			<td>41</td>
		</tr>
		<tr>
			<td>RVM-peptide</td>
			<td>MNLLRKIVKNRRDEDTQKSSPASA 
			PLD-GGGG-RRRRRRRRR*</td>
			<td>11 +</td>
			<td>40</td>
		</tr>
		<tr>
			<td>Antagomir-control (Scrambled)</td>
			<td>5&#8217; ACCAATCGACCAAC 3&#8217;</td>
			<td>14 -</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Antagomir-29</td>
			<td>5&#8217; CACTGATTTCAAATGG 3&#8217;</td>
			<td>16 -</td>
			<td>16</td>
		</tr>
		<tr>
			<td></td>
			<td>*Arginine residues at the C-terminal end of RVG and RVM peptides are in D-form.&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>LNA modified bases in the Antagomir sequences are underlined.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1. Sequences of peptide and Antagomirs

Watch this Scientific Journal Video about A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA at JoVE.com

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

a-simple-alternative-to-stereotactic-injection-for-brain-specific

Nanyang Technological University

Research

JoVE Journal

Neuroscience

12.6K Views.  CSIR-Institute of Genomics and Integrative Biology, New Delhi, India. MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

Video: A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the function of various brain regions for behavior or other experimental outcomes is to implement a localized ablation of function. In humans, patient populations with localized brain lesions are often studied for deficits, in hopes of revealing the underlying function of the damaged area. In rodents, one can experimentally induce lesions of specific brain regions.
Lesion can be accomplished in several ways. Electrolytic lesions can cause localized damage but will damage a variety of cell types as well as traversing fibers from other brain regions that happen to be near the lesion site. Inducible genetic techniques using cell-type specific promoters may also enable site-specific targeting. These techniques are complex and not always practical depending on the target brain area. Excitotoxic lesion using stereotaxic surgery, by contrast, is one of the most reliable and practical methods of lesioning excitatory neurons without damaging local glial cells or traversing fibers.
Here, we present a protocol for stereotaxic infusion of the excitotoxin, N-methyl-D-aspartate (NMDA), into the basolateral amygdala complex. Using anatomical indications, we apply stereotaxic coordinates to determine the location of our target brain region and lower an injection needle in place just above the target. We then infuse our excitotoxin into the brain, resulting in excitotoxic death of nearby neurons. While our experimental subject of choice is a rat, the same methods can be applied to other mammals, with the appropriate adjustments in equipment and coordinates.
This method can be used on a variety of brain regions, including the basolateral amygdala1-6, other amygdala nuclei6, 7, hippocampus8, entorhinal cortex9 and prefrontal cortex10. It can also be used to infuse biological compounds such as viral vectors1, 11. The basic stereotaxic technique could also be adapted for implantation of more permanent osmotic pumps, allowing more prolonged exposure to a compound of interest.

Targeted ablation of specific brain region(s) by infusion of an excitotoxin using stereotaxic coordinates is described. This technique could also be adapted for infusion of other chemicals into the rat brain.

Many behavioral functions in mammals, including rodents and humans, are mediated principally by discrete brain regions. A common method for discerning the ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to the interphotoreceptor/subretinal compartment that has direct exposure to photoreceptors and the retinal pigment epithelium (RPE). Subretinal injections of plasminogen as well as recent preclinical and clinical trials have demonstrated safety and/or efficacy of delivering viral vectors and stem cells to individuals with advanced retinal disease. Mouse models of retinal disease, particularly hereditary retinal dystrophies, are essential for testing these therapies. The most common injection procedure in rodents is to use small transcorneal or transcleral incisions with an anterior approach to the retina. With this approach, the injection needle penetrates the neurosensory retina disrupting the underlying RPE and on insertion can easily nick the lens, causing lens opacification and impairment of noninvasive imaging. Accessing the subretinal space via a transcleral, posterior approach avoids these problems: the needle crosses the sclera approximately 0.5 mm from the optic nerve, without retinal penetration and avoids disrupting the vitreous. Collateral damage is limited to that associated with the focal sclerotomy and the effects of a transient, serous retinal detachment. The simplicity of the method minimizes ocular injury, ensures rapid retinal reattachment and recovery, and has a low failure rate. The minimal damage to the retina and RPE allows for clear assessment of the efficacy and direct effects of the therapeutic agents themselves. This manuscript describes a novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, stem cells or induced pluripotent stem (iPS) cells to the subretinal space in mice with high efficacy, minimal damage, and fast recovery.

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

We describe the use of laser capture microdissection to obtain samples of distinct cell populations from different brain regions for gene and microRNA analysis. This technique allows the study of differential effects of traumatic brain injury in specific regions of the rat brain.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

Summary

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Chapters in this video

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Stereotaxic Surgery for Excitotoxic Lesion of Specific Brain Areas in the Adult Rat

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts