eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;
1. Assembly of the Recording-injection Pipette
<ol>
	<li>Pull an approximately 7 cm long glass capillary (1 mm outer diameter) using a pipette puller.</li>
	<li>Break the capillary tip and check the aperture under a light microscope. Confirm that the inner diameter is between 30 &#181;m to 40 &#181;m.</li>
	<li>Insert a 7 cm long silver wire into the glass capillary with approximately 1 cm protruding from the non-tapered end of the glass pipette.</li>
	<li>Bend the excess filament orthogonally to the glass capillary.</li>
	<li>Apply a droplet of flexible plastic adhesive on the shaft of a 30 G hypodermic needle.</li>
	<li>Insert a 30 G hypodermic needle in the glass pipette according to the schematics presented in Figure 1.</li>
	<li>Add a second glue coating to ensure a proper seal from the junction between the glass pipette and the hypodermic needle.</li>
	<li>Leave the pipette to dry with the tip facing upward for about 12 hr to ensure proper glue curing. The finished result is shown in Figure 2.</li>
</ol>
NOTE: This procedure is done in acute experiments, and sterilization of the pipette tip is not required.
2. Animal Preparation
<ol>
	<li>Place the rat in an anesthesia box.</li>
	<li>Induce anesthesia using 4% isoflurane for 5 to 10 min.</li>
	<li>Place the animal on a stereotaxic table with a heating pad and rectal probe to maintain a body temperature of 37 &#176;C. Use a nose cone with 2% isoflurane to maintain anesthesia. Secure the rat&#39;s head using ear bars and a teeth holder.</li>
	<li>Apply ophthalmic ointment or eye drops, including 1% Atropine drops, to aid in pupil dilation. To prevent dryness, apply lubricating drops approximately every 30 minutes.</li>
	<li>Shave the head and clean it with 10% povidone-iodine.</li>
	<li>For local anesthesia, inject 0.5 ml of 2% lidocaine under the scalp in 2-3 locations by lifting the skin and inserting the tip of the needle.</li>
	<li>Confirm an adequate level of anesthesia by performing a toe pinch and observing the lack of movement. In addition, the heart rate must be monitored to ensure it is within normal values (300 to 400 beats/min).</li>
	<li>Incise the scalp in a straight line along the median with a #10 scalpel blade to expose both the coronal and sagittal sutures.</li>
	<li>Reveal the Lambda and Bregma points by pushing aside the tissue covering the cranium with a surgical spatula.</li>
	<li>Level the cranium so Bregma and Lambda positions are on the same plane.</li>
	<li>To set the reference point, use a stereotaxic device with a mounted glass tube to set it right above Bregma. This will be the &#34;zero&#34; for the anteroposterior and medial-lateral measurement coordinates.</li>
	<li>Set the point of interest by moving the stereotaxic mount to the required coordinates, note the stereotaxic coordinates, and draw a square around the target area that marks where the craniotomy will be performed.</li>
	<li>Use a surgical drill with a sterilized drill along the marked square slowly without pressure to remove the bone material. Be careful not to drill too long in the same area, as it will produce heat and cause lesions on the cortex.</li>
	<li>When the bone delimiting the craniotomy has become sufficiently thin, carefully remove the cranial section with tweezers to expose the cortex.</li>
	<li>Frequently irrigate the exposed cortex with artificial cerebral spinal fluid to prevent tissue desiccation. 
	NOTE: Dura mater removal is unnecessary on the rat as the tip of the injectrode is sturdy enough to penetrate.</li>
</ol>
3. Filling and Mounting of the Injection System
<ol>
	<li>Fill the 5-10 &#181;l microsyringe by aspiration with mineral oil.</li>
	<li>Fill the hypodermic needle with the fixed glass pipette with a solution of 0.5% Chicago Sky Blue (CSB) and 300 &#181;M &#947;-aminobutyric acid (GABA) or a solution of 2% lidocaine with 0.5% CSB 9. Dilute all solutions with saline.
	<ol>
		<li>In the case of an abundant substance, fill using a regular syringe and use the usual preventive techniques to avoid the formation of air bubbles.</li>
		<li>In the case of more expensive substances, mineral oil can be used to fill the injectrode, and the chemical agent can then be introduced by aspiration. The density difference between mineral oil and water is relatively high, so this substance is a good candidate for injecting aqueous solutions. 
		NOTE: A dye can be added to confirm the separation between both liquids.</li>
	</ol>
	</li>
	<li>To fill the injection pipette, aspirate a 1 ml syringe with the solution and slowly inject it into the pipette.</li>
	<li>Pay careful attention to leaks in regions indicated in Figure 1 by swabbing these areas clean and observing leakages by injecting the solution slowly with the syringe.</li>
	<li>Remove the 1 ml syringe. When doing so, keep light pressure on the plunger so that the vacuum does not remove the solution from the injection pipette.</li>
	<li>Aspirate the microsyringe with mineral oil and attach it firmly to the filled injection pipette. Then, carefully wipe away any excess solution from the assembled injectrode with gauze.</li>
	<li>Inject a very small volume, enough to see a small drop form at the tip of the glass pipette, to verify that the tip is not blocked.</li>
	<li>Mount the injectrode on the micropump system and ensure it is well fixed.</li>
	<li>Carefully position the injectrode tip at the target coordinates and lower the tip to the surface of the cortex.</li>
	<li>Slowly lower the injectrode using the stereotaxic apparatus to the target structure (superior colliculus) using the appropriate anatomical coordinates.</li>
	<li>Cover the exposed cortex with warm agar to prevent tissue desiccation.</li>
</ol>
4. Injection and Reversible Inactivation
<ol>
	<li>Set the microinjection pump to inject 400 to 800 nl at 40 nl/min and press Run to start the injection. Note that the spike rate will show a reduction during the injection. 
	NOTE: In the experimental setup, neural activity recovered within an hour after the end of GABA delivery. Any calibrated mechanical device can be used to apply pressure to the microinjection syringe and conduct the injection.</li>
	<li>After acquiring the electrophysiological data, euthanize using a method approved by the local Animal Ethics Community.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Injection pump (UltraMicroPump III)</td>
			<td>WPI</td>
			<td>#UMP3</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection console (Micro4 Controller)</td>
			<td>WPI</td>
			<td>#SYS-MICRO4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamliton</td>
			<td>(80301) 701LT 10 &#181;L SYR</td>
			<td>Syringes between 5 and 10 &#956;L used</td>
		</tr>
		<tr>
			<td>Gel cyanoacrylate adhesive</td>
			<td>Krazy Glue</td>
			<td>KG86648R</td>
			<td>The gel form is easier to apply on the shaft of the 30G hypodermic needle</td>
		</tr>
		<tr>
			<td>Glass pipettes</td>
			<td>WPI</td>
			<td>#TW100F-4</td>
			<td>Thin wall, 1mm OD, 0.75mm ID with filament pipettes used</td>
		</tr>
		<tr>
			<td>720 Needle Pipette Puller</td>
			<td>Kopf</td>
			<td>720</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>A-M Systems, Inc.</td>
			<td>782500</td>
			<td>Bare 0.010&#34;</td>
		</tr>
		<tr>
			<td>Agar&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chicago Sky Blue (CSB)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#947;-aminobutyric acid (GABA)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lidocaine&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine drops</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Isoflurane</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic table</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>#10 scalpel blade</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mineral oil&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

drug microinjection with electrophysiological recording in a rat brain

Source: Lai, J., et al. <a href="https://app.jove.com/v/52271">Simultaneous electrophysiological recording and micro-injections of inhibitory agents in the rodent brain.</a> J. Vis. Exp. (2015)
This protocol outlines the assembly and use of an injectrode for precise drug delivery into targeted brain regions of an anesthetized rat, followed by monitoring of neuronal activity through recorded electrical signals.

<img alt="Figure 1" src="/files/ftp_upload/22711/22711_Figure1.jpg" /> 
Figure 1: Schematic representation of the injectrode assembly. A microsyringe (A) is attached to the recording-injection pipette, which consists of a 30 G hypodermic needle (B) adhered to a silver wire (C) inside a glass pipette (D). Regions circled (E-F) highlight areas that may be susceptible to leaks.
<img alt="Figure 2" src="/files/...

Drug Microinjection with Electrophysiological Recording in a Rat Brain

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

Start with an injectrode, an electrode-containing injection system. This features a microneedle and a silver wire electrode inserted within a glass micropipette.
Fill the injectrode with a test drug solution containing a dye to facilitate tracking of the drug&#39;s spread within the brain.
Attach a syringe filled with mineral oil to the injectrode. Wipe away any excess liquid.&#160;
Secure this injectrode onto a stereotaxic unit and connect it to a micropump system.
Position the injectrode on the target brain region of an anesthetized rat with a surgical hole in the skull.
Gradually lower the injectrode to penetrate the target region.
Halt the insertion upon receiving specific electrical signals that confirm the optimal injectrode placement.
Cover the exposed skull with warm agar to prevent tissue drying.
Allow the brain tissue to stabilize.
Inject the desired dosage of the drug.
Record electrical signals to monitor neuronal activity in response to the administered drug.

drug-microinjection-with-electrophysiological-recording-in-a-rat-brain

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

116 ビュー. 出典:Lai, J., et al.げっ歯類の脳における阻害剤の電気生理学的記録と微量注射の同時実施。J. Vis. Exp.(2015年) このプロトコルは、麻酔をかけたラットの標的脳領域への正確な薬物送達のための注射器の組み立てと使用を概説し、続いて記録された電気信号によるニューロン活動のモニタリングを行います。 

ビデオ: ラット脳における電気生理学的記録による薬物マイクロインジェクション - 概念

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

動機付け行動の分子基質を研究するために齧歯類の脳マイクロインジェクション

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

JoVE Journal

行動学

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

パーソナライズされた齧歯動物の脳でのマイクロインジェクション針

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

神経科学

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and deafferentation of neuronal groups, which are both invasive methods that permanently impair the integrity of the target area. These methods are accompanied by collateral effects that can affect the analysis of acute and temporal regulatory mechanisms. The stereotaxic implantation of guide cannulas aimed at specific brain regions, followed by a recovery period, allows researchers to microinject different drugs after the disappearance of the undesired effects of the surgery. Tetrodotoxin has been used to determine the roles of several brain areas in diverse physiological processes because it transiently inhibits the sodium-dependent action potentials, thus blocking all neural activity in the target region. This protocol combines this method with strategies for the assessment of the estrous cycle and ovulation to reveal the role of discrete brain regions in the regulation of ovulation at particular times of any given stage of the estrous cycle. Awake and unrestrained rats (Rattus norvegicus) were used to avoid the blocking effects that anesthetics and stress hormones exert on ovulation. This protocol can be easily adapted to other species, brain targets and pharmacological agents to study different physiological processes. Future improvements to this method include the design of a microinjection system using glass capillaries of small diameter instead of guide cannulas. This will reduce the amount of tissue damaged during the implantation and decrease the spread of the infused drugs outside the target area.

This protocol describes the construction of a low-cost microinjection system, its stereotaxic implantation into deep-brain structures and the procedure for timed microinjections of tetrodotoxin in awake and unrestrained rats. The goal is to reveal the participation of hypothalamic structures in the regulation of ovulation by inhibiting their neural activity.