This work was supported by National Institute of Neurological Disorders and Stroke grant NS081333 (to CMT), Alzheimer&#8217;s Association grant NIRG-10-171721 and National Institute of Mental Health grant MH096702 (to UH), and National Institute on Aging-funded Alzheimer&#8217;s Disease Research Center at Columbia University pilot grant AG008702 (to YYJ and JB).

Note: For all animal experimentation, Institutional and National guidelines for the care and use of laboratory animals were followed.
1. Prepare Surgical Instruments and Solutions for Surgery
<ol>
	<li>Autoclave all stainless steel surgical instruments.</li>
	<li>Prepare 70% ethanol by diluting 200 proof absolute ethanol with sterile molecular grade deionized distilled water.</li>
	<li>Attach the 29 G needle to the Hamilton syringe. Clean the interior of Hamilton syringe and needle by drawing up and ejecting nanopure water repeatedly for 1 min. Repeat this procedure with 70% ethanol.
	<ol>
		<li>Remove the plunger and needle from the Hamilton syringe. Air dry the parts in a laminar flow hood O/N.</li>
		<li>Irradiate the needle and syringe with ultraviolet light for 30 min before use.</li>
	</ol>
	</li>
	<li>Prepare saline solution by dissolving NaCl in molecular grade water to a final weight to volume concentration of 0.9%. Sterilize the solution by filtering it through 0.2 &#956;m pore filter.</li>
</ol>
2. Prepare Oligomeric A&#946;1-42
<ol>
	<li>Monomerize and resuspend recombinant human A&#946;1-42 in DMSO to 5 mM exactly as described in the publication of Fa&#39; and others13 .</li>
	<li>Dilute 5 mM A&#946;1-42 with sterile (1x) PBS to 100 &#181;M on the day before surgery.</li>
	<li>Mix the solution well by trituration.</li>
	<li>Incubate A&#946;1-42 solution for 12 hr at 4&#730;C. 
	Note: Control solutions such as diluting DMSO in PBS or scrambled/reverse A&#946;1-42 peptide should be prepared the same way as A&#946;1-42.</li>
</ol>
3. Determine Injection Coordinates
<ol>
	<li>Use the adult mouse brain atlas to determine the exact anterior-posterior (AP), medial-lateral (ML), and dorsal-ventral (DV) coordinates for the brain region of interest14. 
	&#8203;Note: To illustrate the technique we picked the dentate gyrus of the hippocampus as the target with the following coordinates from the bregma: AP, -2.00 mm; ML, &#177;1.3 mm; DV, -2.2 mm. The negative sign preceding the AP value indicates that it is 2.00 mm posterior of the bregma. The &#177; sign preceding the ML value indicates left and right direction from the center. Lastly, the negative sign preceding the DV indicates it is moving ventrally from the surface of the brain.</li>
</ol>
4. Stereotaxic Frame Setup
<ol>
	<li>Wear a surgical face mask, clean lab coat, and sterile surgical gloves.</li>
	<li>Wipe down stereotaxic instrument and mouse adaptor with 70% ethanol.</li>
	<li>Lay down sterile surgical drape on the counter.</li>
	<li>Place the stereotaxic instrument with mouse adaptor on top of the sterile surgical drape.</li>
	<li>Wipe the mouse heating pad with 70% ethanol. Position the heating pad over the stereotaxic frame bed. Use general purpose laboratory labeling tape to tape down the heating pad over the mouse adaptor bed.
	<ol>
		<li>Attach the heating pad to the warm water recirculator pump according to manufacturer&#8217;s directions.</li>
		<li>Turn on the warm water recirculator pump and set the temperature to 37 &#176;C, and then wait until it reaches temperature.</li>
	</ol>
	</li>
	<li>Affix the 50 &#181;l Hamilton syringe with a 29 G needle onto the stereotaxic frame by screwing it onto the motorized vertical injecting shaft according to manufacturer&#8217;s directions.</li>
	<li>Turn on the bead sterilizer and wait until it reaches the manufacturer&#8217;s preset temperature. 
	Note: This is used for sterilizing stainless steel instruments between animals. It takes instruments 20 sec of contact with the beads for sterilization.</li>
	<li>Turn on hot plate and set it to 42 &#176;C and place a clean empty cage atop.</li>
	<li>While the Hamilton syringe is fixed on the stereotaxic frame use the motorized stereotaxic injector to draw up the A&#946;1-42 solution. 
	&#8203;Note: Draw up more than 4 &#956;l of the solution into the syringe and then use the stereotaxic injector to set the injecting volume. Operate the injector according to manufacturer&#8217;s instructions.</li>
</ol>
5. Animal Preparation
<ol>
	<li>Determine the weight of the mouse using the weigh scale.</li>
	<li>Anesthetize mouse with ketamine/xylazine cocktail at 100 mg/kg ketamine and 10 mg/kg xylazine by injecting intraperitoneally (IP). Monitor the depth of anesthesia by the loss of toe pinch reflex.</li>
	<li>After the mouse is sedated take the hair clipper and shave its head to expose the skin over the skull.</li>
	<li>Place the mouse on top of the heating pad on top of the stereotaxic bed.</li>
	<li>Use the spatula to open the mouth and place the incisor teeth inside the teeth guard. Secure the nosepiece over the face of the mouse. Clamp it down gently and not too tight.</li>
	<li>Position the bilateral ear crossbars into auditory meatus to secure the head. Move each crossbar in until it hits the skull, and then turn the screw to lock it.
	<ol>
		<li>To make sure the head is secured use your index finger to gently push down on the head. If the head is properly secured it will not give out or move when pressed.</li>
	</ol>
	</li>
	<li>Apply a drop of eye cream or eye drop to the eyes to keep them moist. Make sure the eyes are moist throughout the surgical procedure.</li>
	<li>To disinfect the surgical site use sterile cotton swabs to apply betadine solution to the skin over the skull, followed by 70% ethanol. Perform the alternating betadine and 70% ethanol cleaning 2 more times.</li>
</ol>
6. Surgery and Infusion
<ol>
	<li>Use a scalpel to make a 2-3 mm incision in the midline of the scalp, and then use a straight fine scissors to extend the incision line to 1.0-1.5 cm to expose the sagittal suture, bregma, and lambda of the skull, landmarks which the stereotaxic coordinates are based on. Use micro clamps to keep the skin apart. 
	Note: The bregma and lambda positions are explained in the mouse brain atlas &#8220;The Mouse Brain in Stereotaxic Coordinates&#8221;14.</li>
	<li>Take a cotton swab, soak it in sterile saline, and use it to clean the skull. Then, use a clean dry cotton swab to dry the skull.</li>
	<li>Use a sterile fine point pen to mark a dot on the bregma. Use the stereotaxic micromanipulator to position the Hamilton syringe so that the tip of the needle just touches the dot on the bregma.
	<ol>
		<li>Record the DV coordinate.</li>
		<li>To check if the AP axis of the skull is level move the Hamilton syringe posteriorly so that the tip of the needle touches the lambda point.</li>
		<li>Record the DV position. Make sure the DV coordinates for bregma and lambda are within 0.5 mm. 
		Note: The goal is to minimize the DV difference between bregma and lambda.</li>
	</ol>
	</li>
	<li>Use the micromanipulator to return the Hamilton syringe needle tip back to the bregma dot.</li>
	<li>Record the starting AP position.</li>
	<li>Calculate the ending AP position by subtracting 2.00 mm from the starting AP coordinate.</li>
	<li>Use the micromanipulator to reposition the Hamilton syringe needle to the final AP coordinate.</li>
	<li>Record the current ML coordinate.</li>
	<li>Calculate the left and right ending ML coordinates by adding or subtracting 1.3 mm from the starting ML coordinate.</li>
	<li>Use the micromanipulator to move the Hamilton syringe needle to either the ending ML coordinate.
	<ol>
		<li>Touch the needle tip to the surface of the skull on both ending ML coordinates and record the DV coordinates and make sure the values are within 0.5 mm. 
		Note: The goal is to minimize the DV difference between the two ending ML coordinates.</li>
		<li>While at an ending coordinate pull the needle up 0.5-1 cm over the skull. Take a sterile fine point pen to mark a dot on the surface of the skull. Repeat this step for the contralateral side of the skull.</li>
	</ol>
	</li>
	<li>Swing the Hamilton syringe clear out of the way.</li>
	<li>Attach 0.8 mm drill head to the drill. Take the drill with both hands, set elbows on the surface of the table for stability, and position the drill head slightly above the sharpie dot on either the left or right ML coordinate. Activate the drill, lower the drill tip onto the skull surface to introduce a hole in the skull. Repeat this step for the contralateral side. 
	Note: Some bleeding may occur from the newly introduced holes. If there is bleeding then use a clean dry cotton swab to dab the blood.</li>
	<li>Move the Hamilton syringe back into position over either of the newly introduced ML holes. Lower the Hamilton needle just past the skull. At this point be careful not to puncture the brain. To make sure the needle has passed the skull take your index finger and gently push the needle against the skull to make sure the needle doesn&#8217;t exit the hole.</li>
	<li>Record the starting DV coordinate.</li>
	<li>Calculate the ending DV coordinate by subtracting 2.2 mm from the starting DV coordinate.</li>
	<li>Lower the needle down to the ending DV coordinate.</li>
	<li>Activate the stereotaxic injector pump to pump 4 &#181;l of A&#946;1-42 into the dentate gyrus at a rate of 0.5 &#181;l/min.</li>
	<li>When the infusion is complete let the needle remain in place for an additional 1 min to minimize backflow of solution out of the injection site.</li>
	<li>Move the needle to the other side of the brain and repeat steps 6.13-6.18.</li>
</ol>
7. Animal Removal and Postoperative Care
<ol>
	<li>Unscrew the ear bars and face/nose guard. Remove the mouse from the apparatus.</li>
	<li>Use a student standard pattern forceps to pull close the scalp and then seal the wound with suture.</li>
	<li>Inject 1 ml of sterile saline into the mouse via IP for hydration.</li>
	<li>Inject buprenorphine (0.1 mg/kg) subcutaneously to relieve pain.</li>
	<li>Maintain the mouse in the clean empty cage set on top of 42 &#176;C hot plate until it wakes, then return it to its housing cage with ample food and water.</li>
	<li>Administer buprenorphine via subcutaneous injection every 12 hr for 3 days for pain relief.</li>
</ol>
8. Suture Removal
<ol>
	<li>Anesthetize mouse with ketamine/xylazine cocktail at 100 mg/kg ketamine and 10 mg/kg xlyazine by IP. Note: This is done 7-10 days after surgery.</li>
	<li>Use a student standard pattern forceps and straight fine scissors to remove the suture.</li>
	<li>Inject 1 ml of sterile saline into the mouse via IP for hydration.</li>
	<li>Maintain the mouse in a clean empty cage set on top of 42 &#176;C hot plate until it wakes, and then return it to its housing cage with ample food and water.</li>
</ol>
9. Animal Sacrifice and Tissue Processing
<ol>
	<li>Anesthetize the mouse and perfuse it with 4% paraformaldehyde15, remove the brain, and process it for cryosectioning16. 
	Note: The timing of animal sacrifice depends on the experimenter. However, for our studies we chose 7 and 14 days post surgery.</li>
</ol>

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The authors declare that they have no competing financial interests.

To achieve a successful A&#946;1-42 injection the experimenter or surgeon must: 1) use aseptic technique; 2) correctly identify the brain region of interest with accurate coordinates; 3) be able to properly secure the mouse in the stereotaxic frame with the brain leveled in the AP and ML axis; 4) have the ability to operate the micromanipulator with precision; 5) ensure proper post-operative care. If these key steps are followed the mouse should survive the surgery with no observable infection.
<p class="j...

Amyloid plaque deposits, which are composed of amyloid-beta (A&#946;1-42), are a key feature of the pathology of Alzheimer&#8217;s disease (AD). Numerous studies have shown that high or toxic levels of recombinant oligomeric A&#946;1-42 elicit neuronal death, synaptic dystrophy, loss and dysfunction; as well as learning and memory deficits1-4. Brain regions affected include the hippocampus, the cortex, and subcortical structures such as the basal forebrain and the amygdala5,6. To date, there are multiple transgenic mouse models that attempt to simulate the A&#946;1-42 pathology of AD. Depending on the strain these animals prove to be useful in examining select pathological features of AD. Unfortunately, with the exception of 2 transgenic lines, APP23 and 5XFAD, these mice never fully replicate neuronal loss, a key aspect of AD. Even with the neuronal loss observed in APP23 and 5XFAD, the neuronal death observed was subtle, age dependent, and isolated to a few select regions7,8.
The direct infusion of oligomeric A&#946;1-42 into the wild-type mouse brain provides an excellent in vivo model which replicates the neuronal death aspect of amyloidopathy1,9,10. Unlike the commonly utilized transgenic mouse models the oligomeric A&#946;1-42 infusion model is ideal for acutely elevating A&#946;1-42 levels in a spatial and temporal manner. The advantage of using wild-type mice for this model obviates potential compensation or side effects from the mutations introduced in transgenic mouse lines. Past studies have shown that infusing toxic levels of A&#946;1-42 into the hippocampus elicits neuron death in the vicinity of the injection site within 1 week1. Moreover, consistent with the observation that A&#946;1-42 is toxic for cholinergic neurons11 the basal forebrain cholinergic neuron (BFCN) population which projects to the hippocampus is decreased 20-50% within 7-14 days following beta-amyloid infusion1,10 in mice, effectively allowing for the examinations of isolated neuronal circuitry in the brain. Since BFCN project ipsilaterally to the dentate gyrus of the hippocampus12, for the most part control/vehicle and oligomeric A&#946;1-42 solutions can be injected on either side of the brain allowing comparisons to be made between the left and right hemispheres1.
In this report we will provide a detailed surgical and injection methodology for adult wild-type C57BL/6J mice. This mouse strain is chosen because of its wide use in research. Technically, any brain region can be targeted for infusion, however here we will use the dentate gyrus of the hippocampus as the target to illustrate the technique.

<table><tbody><tr><td>Ketamine HCl (100 mg/ml)</td><td>Henry Schein Medical</td><td>1049007</td><td>100 mg ketamine per 1 kg animal</td></tr><tr><td>Xylazine (20 mg/ml)</td><td>Henry Schein Medical</td><td>not available</td><td>10 mg xylazine per 1 kg animal</td></tr><tr><td>Buprenex (0.3 mg/ml)</td><td>Henry Schein Medical</td><td>1217793</td><td>0.1 mg buprenex per 1 kg animal</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>David Teplow/UCLA</td><td>not available</td><td>100 &amp;mu;M; This amyloid was used in the paper</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>Bachem</td><td>H-1368</td><td>Can be used in place of amyloid from the Teplow lab</td></tr><tr><td>A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>American Peptide</td><td>62-0-80B</td><td>Can be used in place of amyloid from the Teplow lab</td></tr><tr><td>Scrambled A&amp;beta;&lt;sub&gt;1-42&lt;/sub&gt;</td><td>American Peptide</td><td>62-0-46B</td><td>Can be used as control peptide for comparing A&amp;beta;1-42</td></tr><tr><td>NU4 Antibody (Oligomeric Amyloid Antibody)</td><td>Gift from William Klein/Northwestern U.</td><td>not available</td><td>1:2,000 dilution</td></tr><tr><td>Anti-Amyloid Oligomeric Antibody&amp;nbsp; (Polyclonal Rabbit)</td><td>EMD Millipore</td><td>AB9234</td><td>May be used in place of Nu4; needs to&amp;nbsp; be tested by the end user</td></tr><tr><td>6E10 Antibody (Monoclonal Mouse) (Amyloid Antibody)</td><td>Biolegend</td><td>sig-39320</td><td>1:1,000 dilution</td></tr><tr><td>ChAT Antibody (Polyclonal Goat)</td><td>Millipore</td><td>AB144P</td><td>1:100 dilution</td></tr><tr><td>DeadEnd Fluorometric TUNEL system</td><td>Promega</td><td>G3250</td><td>Follow manufacturer&#039;s directions for use</td></tr><tr><td>Prolong Gold Antifade Reagent with DAPI</td><td>Invitrogen</td><td>P36935</td><td>Use when coverslipping slides</td></tr><tr><td>Fluorogold</td><td>Fluorochrome, LLC</td><td>not available</td><td>2% solution</td></tr><tr><td>Absolute Ethanol (200 proof)</td><td>Fisher Scientific</td><td>BP2818-4</td><td>For making 70% ethanol for sanitizing and disinfecting</td></tr><tr><td>Novex 10-20% Tricine gel</td><td>Life Technologies</td><td>EC6625BOX</td><td>For separating A&amp;beta;1-42</td></tr><tr><td>Novex Tricine SDS Running Buffer (10X)</td><td>Life Technologies</td><td>LC1675</td><td>For running 10-20% Tricine gels</td></tr><tr><td>Novex Tris-Glycine Transfer Buffer (25X)</td><td>Life Technologies</td><td>LC3675</td><td>For transferring 10-20% Tricine gels</td></tr><tr><td>SuperSignal Western Blot Enhancer</td><td>Thermo Scientific</td><td>46640</td><td>For enhancing A&amp;beta;1-42 signal; follow manufacturer&#039;s protocol</td></tr><tr><td>Protran BA79 Nitrocellulose Blotting Membrane, 0.1 &amp;mu;m</td><td>GE Healthcare Life Sciences</td><td>10402088</td><td>For transferring 10-20% Tricine gels</td></tr><tr><td>Xcell SureLock Mini-Cell</td><td>Life Technologies</td><td>EI0001</td><td>Electrophoresis aparatus for running 10-20% Tricine gels</td></tr><tr><td>GenTeal Lubricant Eye Gel</td><td>Novartis</td><td>not available</td><td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores&lt;br /&gt;&amp;#160;&lt;br/&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Refresh Optive Lubricant Eye Drops</td><td>Allergan</td><td>not available</td><td>For keeping the mouse eyes moist during surgery; can be found in local pharmacy stores; Can be used in place of GenTeal</td></tr><tr><td>Betadine</td><td>Stoelting</td><td>50998</td><td>For sanitizing and disinfecting</td></tr><tr><td>Round/Tapered Spatula&amp;nbsp;</td><td>VWR</td><td>82027-490</td><td>For opening animal mouth</td></tr><tr><td>Bulldog Serrefines Clamps (Jaw Dims. 9X1.6 mm; Length 28 mm)</td><td>Fine Science Tools</td><td>18050-28</td><td>Optional; For keeping scalp skin apart during injection</td></tr><tr><td>Straight Fine Scissors (Cutting edge 25 mm; Length 11.5 cm)</td><td>Fine Science Tools</td><td>14060-11</td><td>For cutting scalp</td></tr><tr><td>#3 Scalpel Handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>#11 Surgical Blade</td><td>Fine Science Tools</td><td>10011-00</td><td>For making scalp incision</td></tr><tr><td>Student Standard Pattern Forcep (Tip Dims. 2.5x1.5 mm; Length 11.5 cm)</td><td>Fine Science Tools</td><td>91100-12</td><td>For holding scalp closed during suturing</td></tr><tr><td>Trimmer Combo Kit</td><td>Kent Scientific</td><td>CL9990-1201</td><td>For shaving hair</td></tr><tr><td>T/Pump Warm Water Recirculator&amp;nbsp;</td><td>Kent Scientific&amp;nbsp;</td><td>TP-700</td><td>For warming animal during surgery</td></tr><tr><td>Resusable Warmining Pad (5&quot; x 10&quot;)</td><td>Kent Scientific&amp;nbsp;</td><td>TPZ-0510FEA</td><td>For attaching it to the T/Pump warm water recirculator to warm the animal during surgery</td></tr><tr><td>Cordless Micro Drill</td><td>Stoelting</td><td>58610</td><td>Use 0.8 mm steel burrs to drill holes in the skull</td></tr><tr><td>Lab Standard Stereotaxic Instrument with Mouse &amp;amp; Neonatal Rat Adaptor</td><td>Stoelting</td><td>51615</td><td></td></tr><tr><td>Just for Mouse Stereotaxic Instrument</td><td>Stoelting</td><td>51730</td><td>Can use this in place of Stoelting Cat. #51615</td></tr><tr><td>Quintessential Stereotaxic Injector</td><td>Stoelting</td><td>53311</td><td></td></tr><tr><td>Dry Glass Bead Sterilizer</td><td>Stoelting</td><td>50287</td><td>For sterilizing stainless steel instruments</td></tr><tr><td>Sterile Surgical Drape (18&quot; x 26&quot;)</td><td>Stoelting</td><td>50981</td><td></td></tr><tr><td>Hamilton Syringe 50 ml, Model 705 RN SYR, NDL</td><td>Hamilton Company</td><td>7637-01</td><td>For brain injection; use different syringes for different solutions</td></tr><tr><td>29 G Needle, Small Hub RN NDL</td><td>Hamilton Company</td><td>7803-06</td><td>For attaching to the Hamilton syringe for brain injection</td></tr><tr><td>1 ml BD Tuberculin Syringes</td><td>VWR</td><td>BD309659</td><td>For administering anesthesia and saline</td></tr><tr><td>30 G Needle (0.5&quot;)</td><td>VWR</td><td>BD305106</td><td>For administering anesthesia and saline</td></tr><tr><td>Portable Electronic CS Series Scale (Ohaus)</td><td>VWR</td><td>65500-202</td><td>For weighing animals to determine anesthesia dose</td></tr><tr><td>Hot plate (Top Plate Dims. 7.25x7.25 in)</td><td>VWR</td><td>47751-148</td><td>For warming animals post-surgery</td></tr><tr><td>Sofsilk Silk Suture C-1 Cutting 3/8, 12 mm</td><td>Covidien</td><td>S1173</td><td>For closing wound</td></tr><tr><td>Vetbond Tissue Adhesive (3M)</td><td>Santa Cruz Biotechnology</td><td>sc-361931</td><td>Optional: for aiding in wound closure; Use with suture.</td></tr><tr><td>Cotton-Tipped Wooden-Shaft Sterile Applicators</td><td>Fisher scientific</td><td>22-029-488</td><td>For cleaning and drying surgical wound</td></tr><tr><td>Fisherbrand Superfrost Plus Microscope Slides</td><td>Fisher Scientific&amp;nbsp;</td><td>12-550-15</td><td>For collecting brain sections</td></tr><tr><td>VWR Micro Cover Glass 24 X 50 mm</td><td>VWR</td><td>48393241</td><td>For mounting microscope slides</td></tr><tr><td>Thermo Scientific Nalgene Syringe Filter 0.2 &amp;mu;m</td><td>Fisher Scientific</td><td>194-2520</td><td>For sterilizing saline solution</td></tr><tr><td>Sterile dual tip skin markers by Viscot Medical</td><td>Medline</td><td>VIS1422SRL91</td><td>For marking coordinates on the skull</td></tr></tbody></table>

stereotaxic infusion of oligomeric amyloid-beta into the mouse hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

The present method of preparing human recombinant oligomeric A&#946;1-42 yields soluble oligomeric species consisting of monomers, dimers, trimers, and tetramers (Figure 1A). These low molecular weight A&#946;1-42 species, but not the fibrils and plaques, have been shown in numerous settings to be most toxic to neurons1,4,9,17-19. To determine whether or not oligomeric A&#946;1-42 induces neuron death in the mouse brain A&#946;1-42 (4 &#181;l of 100 ...

Watch this Scientific Journal Video about Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus at JoVE.com

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

stereotaxic-infusion-oligomeric-amyloid-beta-into-mouse

Research

JoVE Journal

Neuroscience

18.1K Views.  Columbia University Medical Center. Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Video: Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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.

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 ...

Behavior

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Establishment of a Valuable Mimic of Alzheimer's Disease in Rat Animal Model by Intracerebroventricular Injection of Composited Amyloid Beta Protein

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure change. With the increase of AD patients worldwide, the pathology and treatment of the disease has become a focus in the International Pharmaceutical Industry. Thus, the establishment of the animal model to mimic AD in the laboratory is of great importance.
Here, we describe a detailed protocol for establishing a mimic of AD in a rat animal model though intracerebroventricular injection of amyloid beta protein 25-35 (A&#946;25-35) combined with aluminum trichloride (AlCl3) and anterodorsal thalamic nucleus injection of recombinant human transforming growth factor-&#946;1 (RHTGF-&#946;1) to rats. The related markers of AD were measured, including: spatial memory, neuronal structure and substructure, neuronal A&#946;, and neurofibrillary tangles (NFT) production. This rat model demonstrates spatial memory impairment, neuronal structure and substructure pathological changes, neuron intracellular A&#946; burden, and NFT aggregation, and provides a close mimic of the neuronal structure and function disorder to that of clinical AD patients. Thus, the presented AD rat modelprovides a valuable in vivo tool for exploring neuronal function, neuronal pathology, and drug screening of AD.

This is a protocol to mimic Alzheimer&#39;s Disease in rats by evaluation of spatial memory impairment, neuronal pathological changes, neuronal amyloid beta protein (A&#946;) burden, and neurofibrillary tangles aggregation, induced by the injection of A&#946;25-35 combined with aluminum trichloride and recombinant human transforming growth factor-&#946;1.

Alzheimer&#39;s disease (AD) is an irreversible, progressive brain disease that slowly destroys memory and is accompanied by neuron loss and structure ...

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 ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (A&#946;o) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble A&#946;o correlate with memory deficits in AD models and humans. The A&#946;o-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated A&#946;o infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning A&#946;o-induced memory deficits at the onset of AD. Since soluble A&#946;o aggregate relatively fast into insoluble A&#946; fibrils that correlate poorly with the clinical state of patients, soluble A&#946;o are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of A&#946;o and continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of A&#946;o in pre-dementia patients.

To study the effects of A&#946;o in vivo, we developed a model based on repeated hippocampal infusions of soluble A&#946;o coupled with continuous infusion of A&#946;o antibody (6E10) in the hippocampus using osmotic pumps to counteract the neurotoxic effect of A&#946;o.

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer&#39;s disease (AD). It ...

Medicine

Controlled Infusion of A&beta; Peptide-Specific Antibodies and A&beta; Oligomers into the Rat Hippocampus

Source: Sajadi, A., et al. <a href="https://app.jove.com/v/54215">Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps.</a> J. Vis. Exp. (2016).
This video demonstrates a technique for the continuous infusion of amyloid-beta (A&#946;) peptide-specific antibodies into the hippocampus of a rat. The study employs implanting a bilateral cannula system for a controlled infusion of A&#946; peptide-specific antibodies coupled with the infusion of soluble A&#946; oligomers (A&#946;o) into the rat hippocampus to study the interactions between the oligomers and the antibodies.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Direct Stereotaxic Brain Infusion of Amyloid-Beta Proteins for an Alzheimer's Disease Model

Source: Jean, Y. Y. et al., <a href="https://app.jove.com/v/52805">Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus.</a> J. Vis. Exp. (2015)
This video demonstrates the precise stereotaxic infusion of amyloid beta into the dentate gyrus to establish a mouse model of Alzheimer&#39;s disease. Accurate coordinate determination and controlled delivery of amyloid beta proteins result in neuronal degradation, disrupting memory processing and contributing to Alzheimer&#39;s disease development.

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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