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.
1. Drugs for Microiontophoresis
NOTE: The drugs used for microiontophoresis must have an electrical charge when dissolved in water. Check the literature to see if the drug of interest is appropriate for this procedure. Here, the procedure for gabazine, an antagonist of the GABAA receptor, is described.
<ol>
	<li>Filter distilled water using a 0.2 &#956;m syringe filter to sterilize it and ensure it contains no solutes. Using this distilled water, prepare ~1,000 &#956;l of 20 mM gabazine.</li>
	<li>Adjust the pH of the dilution to 4 with 0.2 &#956;m filtered NaOH using a fine-tip pH electrode.</li>
	<li>Store 100 - 200 &#956;l aliquots at -20 &#186;C. Defrost on the day of the recording. That day, before the animal is restrained, fill the barrels (3 - 5 &#956;l) of the multibarrel electrode with the drugs, using a 100 &#956;l microsyringe fitted with a flexible plastic needle.</li>
</ol>
2. Surgery and Headpost Implantation
<ol>
	<li>Perform experiments in two male CBA/J mice (Mus musculus) with weights of 27 and 30 g.</li>
	<li>In the days before the surgery, handle the animals and allow them to explore freely in the recording chamber to habituate them to it. Perform three sessions per day, each lasting at least 30 minutes. This will calm them and make them comfortable during the recordings.</li>
	<li>Anesthetize the mouse using a mix of ketamine (50 mg/kg) and xylazine (10 mg/kg) injected intramuscularly. This dose deeply anesthetizes the animal for around 1 hr. Give supplementary doses of one-third of the initial dose as needed.</li>
	<li>Place the animal on a heating blanket set to maintain a temperature of 38 &#177; 1 &#186;C and stabilize the animal&#39;s head in a stereotaxic frame using two ear bars and a bite bar. Be careful not to pierce the tympanic membrane with the ear bars.</li>
	<li>Protect the eyes by applying a drop of ophthalmic gel.</li>
	<li>Shave the scalp using scissors and apply povidone-iodine to disinfect the skin.</li>
	<li>Using a scalpel, make an incision along the midline to expose the skull and retract the periosteum covering the parietal and the more rostral part of the occipital bones.</li>
	<li>Glue a lightweight duralumin headpost (weight ~0.65 g, length 30 mm, Figure 1A) with a round and flat base (5 mm diameter) on the skull. Glue a silver wire to the middle of the headpost leaving its ends free. The silver wire will be used as the reference electrode for the electrophysiological recording.</li>
	<li>Apply a thin layer of light-curable adhesive on the skull and the base of the headpost. Wait 10 sec and place the headpost over the rostral area of the parietal bones, along the midline.</li>
	<li>Light cure the adhesive using a blue light source for 20 sec for an effective bonding strength.</li>
	<li>Drill three holes around and behind the base of the headpost using an electric drill and a small drill bit.</li>
	<li>Place two watch screws (~2 mm length) in the drilled holes to serve as additional contact points between the skull and the headstage. The screws require 1&#189; turns to achieve a snug fit. Test the screws with forceps to make sure they are not loose.</li>
	<li>Insert the tip of the silver ground wire into the third hole, ensuring it contacts the dura. Apply a small drop of water-resistant cyanoacrylate glue to bind the wire to the skull.</li>
	<li>Apply light-curing composite to reinforce the bond of the headpost with the skull. Cover the base of the headpost, the low part of the silver wire, and the screws, being careful not to extend behind lambda to allow access during recording. Cure using a blue light source.</li>
	<li>Retract the muscle at the back of the neck near its insertion on the skull. Using a small trephine (2.35 mm diameter), drill a round window just below lambda suture and lateral to the midline, to expose the inferior colliculus (IC). Pull up the covering bone with fine tweezers when the borders are loose. If bleeding occurs, rinse with cold sterile saline to stop it.</li>
	<li>Use composite to make a small (~1 mm wide and ~1 mm high) wall around the window and light-cure it.</li>
	<li>Cover the exposed skull and muscle with antibiotic ointment. Then, wet the exposed surface of the IC with sterile saline and cover it with petroleum jelly, filling the well generated after making the wall around the recording window.</li>
	<li>Inject buprenorphine subcutaneously (0.03 mg/kg, diluted 1:10 in sterile saline) as an analgesic.</li>
	<li>Keep the animal on the heating blanket until it awakes and return it to its housing cage to recover from surgery for three days before recording sessions. House animals in individual rat&#180;s housing cage to prevent cage mates from cleaning out the jelly and the head post touching the metal-grid cover. Place some enrichment in the cage when the animal is single housed. Also, change the sawdust daily to prevent infection and carefully check that the animal recovers properly and shows no signs of discomfort. Also, check if petroleum jelly is over the recording window protecting the exposed brain.</li>
</ol>
3. Electrophysiological Recording and Microiontophoresis
<ol>
	<li>After recovery, give the animal time to acclimate to the recording environment and have its head restrained. Acclimate the animal by fixing the headpost to the holder while the mouse sits on a custom-made cushioned foam restrainer carved to its body size (Figure 1B). This will limit the movements of the mouse and will contribute to its calmness.</li>
	<li>Start with 10 min sessions and progressively increase the duration up to 120 min. During the session, reward the animals with sweet liquids (condensed milk, diluted 30:70 in water) at given intervals. Later, during the recording session, give the same reward at the start and at the end of the session or while no neuron is isolated.</li>
	<li>If the animal is excited before the recording, inject the mild sedative acepromazine (2 mg/kg, intraperitoneal). According to the neural system of interest, the sedative can affect your results.</li>
	<li>Let the animal freely explore the recording chamber for 5 - 10 min.</li>
	<li>Place the animal&#39;s body into the foam custom-made cushioned restrainer (Figure 1B).</li>
	<li>Secure the headpost to a holder attached to the stereotactic frame (Figure 1C). This is a good time to give the animal a liquid reward.</li>
	<li>Cover the animal&#39;s body with a cotton blanket and loosely fasten it using a plastic hemicylinder and adhesive tape.</li>
	<li>Remove the petroleum jelly from the recording window and rinse with warm sterile saline.</li>
	<li>Record neural activity and iontophoretic application of gabazine.
	<ol>
		<li>Place the multibarrel electrode on its holder, controlled by a microdrive and attached to a micromanipulator.</li>
		<li>Connect the wires for recording using small crocodile clips. Connect the positive terminal to the end of the tungsten electrode, and the ground terminal to the silver wire that contacts the dura.</li>
		<li>Place an uncoated silver wire inside each barrel so one end contacts the solution and the other is accessible. Connect those ends to the microiontophoresis device, following the manufacturer&#39;s instructions.</li>
		<li>Move the multibarrel electrode until the tip contacts the brain&#39;s surface. Apply warm sterile saline to prevent the desiccation of the brain tissue. At that point, apply a retention current (-10 nA for gabazine; check the literature for the particular drug) to avoid leakage of the drug from the barrel tip while looking for single-unit activity.</li>
		<li>Use standard techniques for isolating single neuron extracellular activity. Obtain the frequency response area (FRA) of the neuron, i.e., the combination of frequencies and intensities capable of evoking a response because the neuron is sensitive to these sounds.</li>
		<li>Choose two frequencies (f1 and f2) that evoke a similar firing rate and pattern. Present each of those frequencies as rare and repetitive stimulus under the oddball paradigm. 
		NOTE: Briefly, in the oddball paradigm, one frequency (f1) is presented as the repetitive stimulus with a high probability of occurrence, while the second (f2) is the rarely occurring deviant stimulus, interspersed randomly among the repetitive ones. In a second oddball sequence, the relative probabilities of the two stimuli are reversed.</li>
		<li>Release the gabazine by shifting the current to positive values (10 - 20 nA). Obtain the FRA again and repeat the oddball paradigm under the gabazine application. Maintain the ejection until a visible effect in the firing rate is observed; it usually takes 5 - 20 min, depending on the particular drug, its concentration, and the magnitude of the ejection current. Repeat the recording protocol to obtain the values under the drug&#39;s effect.</li>
		<li>Stop the injection by returning the current to retention values. Wait for the effect of the drug to wash away by monitoring whether the FRA or response to the oddball paradigm returns to control values. 
		NOTE: The recording sessions can last 2 - 3 hr per day and should be ended earlier if the animal shows any sign of discomfort or struggling. Give liquid reward and cover the recording chamber with petroleum jelly.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tungsten wire</td>
			<td>Harvard Apparatus LTD</td>
			<td>33-0099</td>
			<td>0.005 inches x 3 inches</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary</td>
			<td>Harvard Apparatus LTD</td>
			<td>30-0053</td>
			<td>Borosilicate standard wall without filament, 1.5 mm OD, 0.86 mm ID, 100 mm long</td>
		</tr>
		<tr>
			<td>Multibarrel glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>5B120F-4</td>
			<td>5-barrel capillary, 4 inches long, 1.2 mm OD, with filament</td>
		</tr>
		<tr>
			<td>Diaplus</td>
			<td>DiaDent</td>
			<td>2001-2101</td>
			<td>Light-curing adhesive, used to attach the tungsten electrode to the glas multibarrel pipette</td>
		</tr>
		<tr>
			<td>G-Bond</td>
			<td>GC Corporation</td>
			<td>2277</td>
			<td>Light-curing adhesive, used to attach the headpost to the animal&#39;s skull</td>
		</tr>
		<tr>
			<td>Charisma</td>
			<td>Heraeus Kulzer</td>
			<td>66000087</td>
			<td>Light-curing composite, used to reinforce the bond of the headpost with the skull</td>
		</tr>
		<tr>
			<td>Araldit Cristal</td>
			<td>Ceys</td>
			<td></td>
			<td>2-component expoxy, used to further secure the attachment of the tungsten electrode to the glass multibarrel pipette</td>
		</tr>
		<tr>
			<td>Heating blanket</td>
			<td>Cibertec</td>
			<td>RTC1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotactic frame</td>
			<td>Narishige</td>
			<td>SR-6N</td>
			<td>Modified for mice</td>
		</tr>
		<tr>
			<td>Microiontophoretic device</td>
			<td>Harvard Apparatus LTD</td>
			<td>Neurophore BH-2</td>
			<td>Including IP-2 iontophoresis pumps (one for each drug delivery channel) and a balance module</td>
		</tr>
		<tr>
			<td>Multibarrel glass pipette puller</td>
			<td>Narishige</td>
			<td>Model PE-21</td>
			<td></td>
		</tr>
		<tr>
			<td>LED lamp</td>
			<td>Technoflux</td>
			<td>CV-215</td>
			<td>5 W, 430-485 nm</td>
		</tr>
		<tr>
			<td>MicroFil</td>
			<td>World Precision Instruments</td>
			<td>MF34G-5</td>
			<td>Flexible plastic needle, 34 AWG</td>
		</tr>
		<tr>
			<td>Imalgene</td>
			<td>Merial</td>
			<td></td>
			<td>Ketamine, 100 mg/mL</td>
		</tr>
		<tr>
			<td>Rompun</td>
			<td>Bayer</td>
			<td></td>
			<td>Xylazine, 20 mg/mL</td>
		</tr>
		<tr>
			<td>Gabazine / SR-95531</td>
			<td>Sigma</td>
			<td>S106</td>
			<td>Prepare ~ 1000&#181;l of 20 mM gabazine in distilled water and adjust the pH to 4</td>
		</tr>
	</tbody>
</table>

investigating auditory neuronal responses in an awake mouse in the presence of an inhibitory neurotransmitter blocker

Source: Ayala, Y. A., et al. <a href="https://app.jove.com/v/53914">Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice.</a> J. Vis. Exp. (2016).
This video demonstrates the study of neuronal activity in a specific region of the mouse brain that processes auditory stimuli. The technique involves applying an inhibitory neurotransmission blocker and comparing neuronal responses to auditory stimuli before and after the drug is applied to the localized brain region.

<img alt="Figure 1" src="/files/ftp_upload/22845/22845_Figure1.jpg" /> 
Figure 1. Accessories for Awake Mice Recordings. (A) Headpost. (B) Custom-made cushioned foam restrainer. Place the mouse in between both pieces, leaving the head outside. (C) Modifications to the stereotaxic frame. The headpost holder (top bar) assists during headpost implantation and restrains the head during the recordings. The bite piece (lower bar) is only used during the headpost implantati...

Investigating Auditory Neuronal Responses in an Awake Mouse in the Presence of an Inhibitory Neurotransmitter Blocker

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

Secure an awake mouse in a stereotactic frame, with a silver wire inserted into its dura and an opening exposing a brain region that processes auditory stimuli.
Fill a drug solution into a multibarrel glass pipette fitted with a tungsten electrode and attach it to the stereotactic frame holder.
Insert uncoated silver wires into the barrels, ensuring one end contacts the solution while the other connects to a device controlling drug release.
Connect the positive and ground terminals of the recording wires to the tungsten electrode and the silver wire contacting the dura, respectively.
Place the multibarrel electrode on the brain surface and apply auditory stimuli.
Record the neuronal activity.
Next, apply a positive current to release the drug.
The drug enters the brain and blocks specific receptors, preventing inhibitory neurotransmitter binding and increasing neuronal excitability.
Finally, apply the auditory stimuli again and identify the increased neuronal response post-drug treatment.

investigating-auditory-neuronal-responses-an-awake-mouse-presence-an

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

35 조회수. 출처: Ayala, Y. A., et al. 깨어 있는 쥐에서 신경 활성 물질의 Microiontophoretic 적용과 결합된 신경 활동의 세포 외 기록. J. Vis. 특급 (2016). 이 비디오는 청각 자극을 처리하는 마우스 뇌의 특정 영역에서 신경 세포 활동에 대한 연구를 보여줍니다. 이 기술은 억제성 신경 전달 차단제를 적용하고 약물이 국소 뇌 영역에 적용되기 전과 후의 청각 자극에 대한 신경 반응을 비교하는 것을...

비디오: 억제성 신경전달물질 차단제가 있는 상태에서 깨어 있는 쥐의 청각 신경 반응 조사 - 개념

Juxtacellular Monitoring and Localization of Single Neurons within Sub-cortical Brain Structures of Alert, Head-restrained Rats

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures in behaving animals remains a technical challenge, especially if the structures must be targeted stereotaxically. This protocol describes convenient surgical and electrophysiological techniques that maintain the animal&#8217;s head in the stereotaxic plane and unambiguously isolate the spiking activity of single neurons. The protocol combines head restraint of alert rodents, juxtacellular monitoring with micropipette electrodes, and iontophoretic dye injection to identify the neuron location in post-hoc histology. While each of these techniques is in itself well-established, the protocol focuses on the specifics of their combined use in a single experiment. These neurophysiological and neuroanatomical techniques are combined with behavioral monitoring. In the present example, the combined techniques are used to determine how self-generated vibrissa movements are encoded in the activity of neurons within the somatosensory thalamus. More generally, it is straightforward to adapt this protocol to monitor neuronal activity in conjunction with a variety of behavioral tasks in rats, mice, and other animals. Critically, the combination of these methods allows the experimenter to directly relate anatomically-identified neurophysiological signals to behavior.

This protocol describes the design and surgical implantation of a head-restraining mechanism to monitor neuronal activity in sub-cortical brain structures in alert rats. It delineates procedures to isolate single neurons in the juxtacellular configuration and to efficiently identify their anatomical locations.

경고, 헤드 억제 쥐의 하위 대뇌 피질의 뇌 구조 내에서 Juxtacellular 모니터링 및 현지화 단일의 뉴런

There are a variety of techniques to monitor extracellular activity of single neuronal units. However, monitoring this activity from deep brain structures ...

연구

JoVE Journal

신경과학

Real-time Iontophoresis with Tetramethylammonium to Quantify Volume Fraction and Tortuosity of Brain Extracellular Space

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the extracellular space (ECS) of the living brain. The ECS surrounds all brain cells and contains both interstitial fluid and extracellular matrix. The transport of many substances required for brain activity, including neurotransmitters, hormones, and nutrients, occurs by diffusion through the ECS. Changes in the volume and geometry of this space occur during normal brain processes, like sleep, and pathological conditions, like ischemia. However, the structure and regulation of brain ECS, particularly in diseased states, remains largely unexplored. The RTI method measures two physical parameters of living brain: volume fraction and tortuosity. Volume fraction is the proportion of tissue volume occupied by ECS. Tortuosity is a measure of the relative hindrance a substance encounters when diffusing through a brain region as compared to a medium with no obstructions. In RTI, an inert molecule is pulsed from a source microelectrode into the brain ECS. As molecules diffuse away from this source, the changing concentration of the ion is measured over time using an ion-selective microelectrode positioned roughly 100 &#181;m away. From the resulting diffusion curve, both volume fraction and tortuosity can be calculated. This technique has been used in brain slices from multiple species (including humans) and in vivo to study acute and chronic changes to ECS. Unlike other methods, RTI can be used to examine both reversible and irreversible changes to the brain ECS in real time.

This protocol describes real-time iontophoresis, a method that measures physical parameters of the extracellular space (ECS) of living brains. The diffusion of an inert molecule released into the ECS is used to calculate the ECS volume fraction and tortuosity. It is ideal for studying acute reversible changes to brain ECS.

뇌의 세포 외 공간의 부피비와 비틀림을 정량화하기위한 테트라 메틸 암모늄을 이용한 실시간 이온 도입

This review describes the basic concepts and protocol to perform the real-time iontophoresis (RTI) method, the gold-standard to explore and quantify the ...

Visualizing Astrocyte Morphology Using Lucifer Yellow Iontophoresis

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable&#160;protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

Astrocytes are morphologically complex cells, exemplified by their multiple processes and bushy territories. To analyze their elaborate morphology, we present a reliable protocol to perform intracellular Lucifer yellow iontophoresis in lightly fixed tissue.

루시퍼 황색 이온토포레시스를 사용하여 성상세포 형태 시각화

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, ...

Investigating Auditory Neuronal Responses in an Awake Mouse in the Presence of an Inhibitory Neurotransmitter Blocker

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