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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Setup and Preparation
<ol>
	<li>Solutions
	<ol>
		<li>Prepare artificial cerebrospinal fluid (aCSF) stock solutions according to the following recipes. Other recipes with concentration variations are available in the literature. Store stock solutions at 4 &#176;C for up to one month.
		<ol>
			<li>Salt solution: add 75.39 g of NaCl (129 mM final); 2.5 g of KCl (3.35 mM final); 0.81 g of NaH2PO4 (0.58 mM final); 2.33 g of MgCl2 (1.15 mM final); 1.85 g of CaCl2 (1.26 mM). Dissolve in 800 ml of distilled water then fill to 1 L with distillated water.</li>
			<li>Bicarbonate solution: add 17.65 g of NaHCO3&#160;(21 mM final). Dissolve in 900 ml of distillated water then fill to 1 L with distilled water. Variations of the bicarbonate concentration will result in pH variations.</li>
			<li>Glucose solution: add 54.06 g of glucose (30 mM final). Dissolve in 400 ml of distillated water then fill to 500 ml with distilled water.</li>
		</ol>
		</li>
		<li>Prepare aCSF by diluting 100 ml of salt solution, 100 ml of bicarbonate solution, and 50 ml of glucose solution in 1 L of distilled water. Store at 4 &#176;C until use.</li>
		<li>Prepare a glass electrode by warming and stretching a glass tube until it breaks. Sand down the tip before use. The electrode can be reused many time as long as it stays clean.</li>
	</ol>
	</li>
	<li>Experimental Setup 
	NOTE: The set-up details are presented in&#160;Figure 1.
	<ol>
		<li>Turn on the amplifier, moving averager, data acquisition system, temperature controller and pump. Check the signal amplification (gain = 10,000), the filtration (low threshold, 10 Hz; high threshold, 5 kHz), and the sampling rate of the analog to digital conversion of the raw signal (2.5 kHz).</li>
		<li>Fill a bottle with aCSF and bubble it with carbogen (95% O2, 5% CO2). Induce a bubbled- and warmed-aCSF flow in the recording chamber (volume 5 ml) at 4 ml/min. Hold the temperature in the recording chamber at 26 (&#177; 1) &#176;C. pH should be 7.4 in these standard conditions.</li>
		<li>Keep 50 ml of room temperature (RT) and carbogen-bubbled aCSF in a 50 ml syringe near the dissection chamber.</li>
		<li>Turn on the computer and start the recording software.</li>
	</ol>
	</li>
</ol>
2. Dissection
<ol>
	<li>Weigh and visually determine the sex of the animal (males have a longer&#160;ano-genital distance, while females have a short&#160;ano-genital distance; male genitals are often dark while female genitals are pink).</li>
	<li>Anesthetize the newborn rodents by one of the following options: cryoanesthesia (completely immerse the animal in ice for 4 &#8211; 5 min), injection (equitensine at 4 ml/kg)&#160;or inhalation of volatile anesthetics (isoflurane&#160;or ether). Confirm an adequate plane of anesthesia by the absence of a paw withdrawal reflex.</li>
	<li>Place the animal on the bench, ventral face down. Section the rostral part of the head coronally with a scalpel at the bregma level (visible through the skin and skull). Perform this step immediately after the animal is anesthetized.</li>
	<li>Section the body coronally with a scalpel under the anterior members.</li>
	<li>Remove skin, muscles, fatty tissues and viscus with surgical and thin scissors and pliers. Since bones are soft at this age, be cautious not to damage the nervous system. Perform this step on the bench.</li>
	<li>Place the preparation in the dissection chamber. Use the aCSF stored in the syringe to oxygenate the preparation. From this point to the end of the recording, use a microscope.</li>
	<li>On the dorsal face of the preparation, cut the skull and vertebras from the rostral to the caudal part along the medium axis with surgical and thin scissors and pliers. Open the cut skull and vertebras in order to expose the nervous tissue. Use the aCSF stored in the syringe to oxygenate the preparation.</li>
	<li>With the pliers, remove the arachnoid membrane, a thin tissue covering the surface of the nervous tissue. Retain the pia mater and blood vessels against the nervous tissue. Use the aCSF stored in the syringe to oxygenate the preparation.</li>
	<li>Place the preparation with the dorsal face down, and carefully bring down the brainstem and spinal cord by cutting the nerves and connective tissue with the scissors while holding the preparation in place. A dorsal approach is also possible. Keep the roots and nerves as long as possible. Remove the bones to isolate the brainstem and spinal cord. Use the aCSF stored in the syringe to oxygenate the preparation.</li>
	<li>Remove the cerebellum and remaining rostral structures by sectioning them with the scalpel. Use the aCSF stored in the needle to oxygenate the preparation.</li>
	<li>Optionally depending on experimental design, remove the pons with the scalpel by a coronal section anterior to the inferior cerebellar artery. Note that keeping the whole pons will slow down the rhythm in rats and fully inhibit it in mice. Preparations that include only the caudal part of the pons display a respiratory-like activity with a stable frequency at the C4 root.</li>
</ol>
3. Recording
<ol>
	<li>Place the preparation in the recording chamber, ventral face up. Fix the preparation with pins in the lowest part of the spinal cord and rostral-most part of the brainstem.</li>
	<li>Using a syringe linked to the electrode by its needle, induce a depression in the electrode (glass tip diameter: 150 - 225 &#181;m) by pulling out the syringe piston, in order to partially fill the electrode with aCSF.</li>
	<li>Using a micromanipulator, carefully place the electrode close to one of the fourth ventral roots. Other ventral roots could also present a respiratory-like activity (e.g.,&#160;XII cranial nerve, C1 ventral root).</li>
	<li>Induce a depression in the electrode tank via the syringe by pulling out the piston in order to gently aspirate the nerve rootlet. Then carefully move the electrode to apply it against the spinal cord. 
	NOTE: Ideally the size of the rootlet matches the size of the electrode opening and thus creates a seal between the internal and external compartments of the electrode. Since differential amplifiers are commonly used for such recordings, any opening between the inside of the electrode and the recording chamber reduces the quality of the signal and makes it difficult to eliminate background noise.</li>
	<li>Start the recording.</li>
	<li>Record the rhythm produced by the preparation under normoxic conditions (i.e.,&#160;aCSF bubbled with carbogen: 95% O2&#160;and 5% CO2) for at least 20 min to determine the baseline parameters of the preparation.</li>
	<li>Switch the perfusion from carbogen-bubbled aCSF to stimulus aCSF (i.e.,&#160;bubbled with 95% N2&#160;and 5% CO2&#160;for hypoxia stimulus) for 15 min. Alter exposure duration depending of the experimental protocol. Record the exposure duration on the recording and animal datasheet. Use stainless steel tubes between the aCSF bottle and the chamber whenever possible to avoid gas diffusion to the outside of the tube.</li>
	<li>Switch the perfusion back to standard carbogen-bubbled aCSF for at least 15 min for a recovery recording. Record this on the recording and animal datasheet.</li>
	<li>End the recording.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sylgard</td>
			<td>Sigma Aldrich</td>
			<td>761036-5EA</td>
			<td>Use under hood</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>SOD002</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bioshop</td>
			<td>POC888</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Bioshop</td>
			<td>CCL444</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Bioshop</td>
			<td>MAG510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Bioshop</td>
			<td>SPM306</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Bioshop</td>
			<td>GLU501</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Linde</td>
			<td>343-02-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction electrode</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 573000</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential AC amplifier</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Moving averager</td>
			<td>CWE, Ardmore, PA, USA</td>
			<td>model MA-821</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition system</td>
			<td>Dataq Instruments, Akron, OH, USA</td>
			<td>model DI-720</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart software</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism sofware</td>
			<td>Graphpad, La Jolla, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recording chamber</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instrument Inc, Sarasota, FL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson, Middleton, WI, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Home made</td>
			<td>MINIPULS 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiological recordings of brainstem-spinal cord preparations from a newborn rodent

Source: Rousseau, J., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/53071">Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording.</a> J. Vis. Exp. (2015).
This video demonstrates electrophysiological recordings of isolated brainstem-spinal cord preparations from newborn rodents. The preparation is placed in a recording chamber, and the inspiratory bursts from the brainstem are recorded under oxygenated and oxygen-deprived conditions using the fourth ventral nerve root in the spinal cord.

<img alt="Figure 1" src="/files/ftp_upload/22863/22863_Figure1.jpg" /> 
Figure 1.&#160;Recapitulative Schema of the Setup.&#160;The bubbled aCSF is warmed and sent by a pump in the recording chamber. aCSF excess in the recording chamber is aspirated by a pump and disposed of. The recording chamber is linked to the ground and the temperature controller. The electrode output is directly relayed to the amplifier, then to the moving averager and the data acquisition syst...

Electrophysiological Recordings of Brainstem-Spinal Cord Preparations from a Newborn Rodent

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Setup and Preparation

	
...

Secure a brainstem-spinal cord preparation from a newborn rodent in a recording chamber with a flow of oxygenated aCSF. Remove the thin tissue covering the preparation.
The brainstem&#39;s neuronal network generates a rhythmic electrical activity, termed inspiratory burst, transmitted via motor neurons of the spinal cord&#39;s fourth ventral, or C4, nerve root to control breathing.
Position a recording pipette near the nerve root and use suction to draw the root inside, forming a seal.
Intrinsic properties of the brainstem neurons cause sodium influx, depolarizing the membrane and generating an action potential, followed by potassium outflow for repolarization.
Ensuing hyperpolarization prevents new action potentials until the sodium-potassium ATPase utilizes ATP to restore the membrane potential.
The action potentials propagate to the nerve root as rhythmic bursts.
Flow oxygen-deprived aCSF to disrupt ATP production and impair ATPase function, delaying action potentials and decreasing rhythmic bursts.
Switching back to oxygenated aCSF recovers the number of rhythmic bursts.

electrophysiological-recordings-brainstem-spinal-cord-preparations

Universidad San Sebastian

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Electrophysiology Techniques

49 Views. Source: Rousseau, J., et al. Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording. J. Vis. Exp. (2015). This video demonstrates electrophysiological recordings of isolated brainstem-spinal cord preparations from newborn rodents. The preparation is placed in a recording chamber, and the inspiratory bursts from the brainstem are recorded under oxygenated and oxygen-deprived conditions using the fourth ventral nerv...

Video: Electrophysiological Recordings of Brainstem-Spinal Cord Preparations from a Newborn Rodent - Concept

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday products. They are classified as endocrine disrupting compounds (EDC) with estradiol-like properties. Long-term exposure to EDCs, even at low doses, has been linked with various health defects including cancer, behavioral disorders, and infertility, with greater vulnerability during early developmental periods. To study the effects of BPA on the development of neuronal function, we used an in vitro neuronal network derived from the early chick embryonic brain as a model. We found that exposure to BPA affected the development of network activity, specifically spiking activity and synchronization. A change in network activity is the crucial link between the molecular target of a drug or compound and its effect on behavioral outcome. Multi-electrode arrays are increasingly becoming useful tools to study the effects of drugs on network activity in vitro. There are several systems available in the market and, although there are variations in the number of electrodes, the type and quality of the electrode array and the analysis software, the basic underlying principles, and the data obtained is the same across the different systems. Although currently limited to analysis of two-dimensional in vitro cultures, these MEA systems are being improved to enable in vivo network activity in brain slices. Here, we provide a detailed protocol for embryonic exposure and recording neuronal network activity and synchrony, along with representative results.

Exposure to environmental toxins can acutely impact developing embryos. Endocrine disrupting chemicals such as bisphenols are known to adversely affect the nervous system. Here we describe a protocol using an in vitro vertebrate (chick embryo) neuronal network model to study the functional impact of toxin exposure on early embryos.

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

JoVE Journal

Developmental Biology

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...

Electrophysiological Recordings of Brainstem-Spinal Cord Preparations from a Newborn Rodent

Transcript

Electrophysiological Recordings of Brainstem-Spinal Cord Preparations from a Newborn Rodent

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology