Name: induction of an isoelectric brain state to investigate the impact of endogenous synaptic activity on neuronal excitability in vivo
Uploaded: 2016-03-31T15:00:00
Description: Watch this Scientific Journal Video about Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo at JoVE.com

This work was supported by grants from the Fondation de France, the Institut National de la Sant&#233; Et de la Recherche M&#233;dicale, the Pierre &#38; Marie Curie University and the program &#39;Investissements d&#39;avenir&#39; ANR-10-IAIHU-06.

All procedures were carried out in accordance with the guidelines of the European Union (directive 2010/63/EU) and approved by the Charles Darwin Ethical Committee on Animal Experimentation. We describe here the procedure we routinely use in our laboratory, however most steps can be adapted to match everyone&#39;s specific needs.
1. Surgical Preparation
Note: All incision and pressure points should be repeatedly infiltrated with local anesthetic (lidocaine or bupivaca...

We describe here a new method to suppress in vivo spontaneous cerebral electrical activity at both network and cellular levels. This procedure leads to an extreme brain state, known as isoelectric comatose 41. From a clinical point of view, such an electrocerebral inactivity is the most severe abnormality that can be seen on the EEG. It is mostly associated with an irreversible coma, with all patients either dying or continuing in a persistent vegetative state 42, but can be at least partia...

In the absence of any environmental stimuli or behavioral tasks, the &#34;resting&#34; brain generates a continuous stream of electrical activity that can be recorded from the scalp, as electroencephalographic (EEG) waves. The intracellular correlate of this endogenous cerebral activity is characterized by background membrane voltage fluctuations (also known as &#34;synaptic noise&#34;), which are composed of a combination of excitatory and inhibitory synaptic potentials that reflect the ongoing activity of afferent networks 1,2. This spontaneous activity varies in frequency and amplitude with the different states of vigilance. Elucidating the impact of network activity on the excitability and responsiveness of single neurons is one of the major challenges of neurosciences 3,4.
Many experimental and computational studies have explored the functional impact of ongoing synaptic activity on the integrative properties of neurons. However, the role of the different neuronal parameters impacted by the background synaptic noise remains elusive. For instance, the mean level of membrane depolarization has been found positively 5,6 or negatively 7-9 correlated with the ability of sensory inputs to trigger action potentials. Moreover, whereas some investigations suggest that fluctuations of the membrane potential, resulting from a continuously varying stream of afferent synaptic inputs, strongly affect the responsiveness of single neurons by modulating the gain of their input-output relationship 3,10-13, others indicate that changes in membrane input conductance mediated by shunting inhibition are sufficient to modulate the neuronal gain regardless of the magnitude of membrane fluctuations 14,15. Finally, recent studies performed on awake animals stressed how the processing of sensory information in single neuron critically depends upon the state of vigilance and the current behavioral demand 16,17.
A straightforward strategy to elucidate the functional role of a given process in a highly interconnected system is to determine how its absence specifically alters the functioning of the system. This method has been extensively used in neuroscience research, for example using experimental lesions or inactivation of different brain areas 18-21, or pharmacological blockade of specific ion channels 22,23. Notably, it has been applied in vivo to unveil how functional connectivity and network dynamics affect single cell computation 24-27. However, to date local manipulations intended to block the firing of neurons and/or perturb their basic biophysical properties can be partially effective and are limited to relatively small brain volumes 28.
To overcome these limitations, we developed a new in vivo experimental approach in the rat to compare the electrophysiological properties of single neurons recorded in a given brain state, i.e., embedded in a particular network dynamic, to those obtained after complete suppression of the whole brain synaptic activity 29. In the control conditions, two distinct cortical dynamics could be generated. Sleep-like electrocorticographic (ECoG) patterns were induced by injection of moderate doses of sodium pentobarbital. Alternatively, fast ECoG waves of small amplitude comparable to the cortical activity underlying the waking state (waking-like pattern) could be produced by injection of fentanyl. Subsequently, while maintaining the same ECoG and intracellular recording, a complete silencing of endogenous brain electrical activity was obtained by systemic injection of a high dose of sodium pentobarbital, characterized by isoelectric ECoG and intracellular activities. Because the induction of such an extreme comatose could potentially have fatal consequences on biological functions, a careful and continuous monitoring of the physiological variables was essential. Therefore, we meticulously followed the heart beat frequency, the end-tidal CO2 concentration (EtCO2), the O2 saturation (SpO2) and core temperature of the rat throughout the experiments.
We evaluate single neurons properties during these different states using sharp microelectrodes, which are particularly suited for long and stable recordings in vivo. The procedure described here, can be combined with other electrophysiological and imaging approaches and could be extended to other animal models.

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			<td>Sodium Pentobarbital</td>
			<td>Centravet</td>
			<td>Pentobarbital</td>
			<td></td>
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		<tr>
			<td>Ketamine 500</td>
			<td>Merial</td>
			<td>Imalg&#232;ne 500</td>
			<td></td>
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			<td>Fentanyl&#160;</td>
			<td>Janssen-Cilag</td>
			<td>Fentanyl</td>
			<td></td>
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			<td>Xylocaine</td>
			<td>Centravet</td>
			<td>Xylovet</td>
			<td></td>
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			<td>Gallamine triethiodide</td>
			<td>Sigma</td>
			<td>G8134</td>
			<td></td>
		</tr>
		<tr>
			<td>ECoG amplifier</td>
			<td>A-M Systems</td>
			<td>AC amplifier, Model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular amplifier</td>
			<td>Molecular Devices</td>
			<td>Axoclamp 900A</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition interface</td>
			<td>Cambridge Electronic Design</td>
			<td>CED power 1401-3&#160;</td>
			<td></td>
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		<tr>
			<td>Data analysis software</td>
			<td>Cambridge Electronic Design</td>
			<td>Spike2 version 7</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>Scientifica</td>
			<td>IVM-3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Puller</td>
			<td>Narishige</td>
			<td>PE-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC150F-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire 0.125mm (intracellular recording)</td>
			<td>WPI</td>
			<td>AGT0525</td>
			<td></td>
		</tr>
		<tr>
			<td>Ag-AgCl reference</td>
			<td>Phymep</td>
			<td>E242</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire 0.25mm (ECoG recording)</td>
			<td>WPI</td>
			<td>AGT1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial respiration system</td>
			<td>Minerve</td>
			<td>Alpha Lab</td>
			<td></td>
		</tr>
		<tr>
			<td>Physiological parameters monitoring</td>
			<td>Digicare</td>
			<td>LifeWindow Lite</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Blanket</td>
			<td>Harvard Apparatus</td>
			<td>507215</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>FST</td>
			<td>15005-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5</td>
			<td>FST</td>
			<td>11295-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps Dumont #5SF</td>
			<td>FST</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr>
			<td>IP Polyurethane catheter - 0.43x0.69mm&#160;&#160;</td>
			<td>Instech</td>
			<td>BTPU-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon elastomere</td>
			<td>WPI</td>
			<td>KWIK-CAST</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental drill</td>
			<td>NSK</td>
			<td>Y1001151 and P496</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical glue</td>
			<td>3M</td>
			<td>vetbond</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of an isoelectric brain state to investigate the impact of endogenous synaptic activity on neuronal excitability in vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Inducing and maintaining an isoelectric brain state is a delicate in vivo experimental procedure. It has been proved to be a powerful tool to directly study the impact of cortical network activity on neuronal excitability and transfer function 29. Figure 1 shows the multi-parameter monitoring, including ECoG and vital constants, of the animal&#39;s physiological state before (Figure 1A) and after (Figure 1B) induction ...

Watch this Scientific Journal Video about Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo at JoVE.com

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

The overall goal of this protocol is to induce a general state of cerebral inactivity in vivo to study single neurons integrated properties in the absence of synaptic inputs. The method we present here can help to answer key questions in neurophysiology, such as assessing the impact of the network activity on single cells computation or study the responsiveness of neuron in comatose states. The main advantage of this technique is that neuronal properties can be studied in isolation from the network in an intact in vivo preparation.To begin this procedure, place a fully anesthetized rat on a heating blanket. Insert a rectal probe in the animal to maintain core temperature around 37 degrees Celsius. To place a catheter in the peritoneal cavity to facilitate subsequent injection of anesthetic agents, first remove the hair over a small area on the stomach's lower right or left quadrant using depilatory cream.After injecting the local anesthetic, sterilize the surgical site and make a two to three millimeter incision on the skin with sharp scissors. Using blunt scissors, remove the fat and muscle layers until the peritoneal cavity is visible. Then insert about one to two centimeters of a small catheter in the cavity and close the wound with surgical glue.Make a loop and suture the catheter to the skin to secure it in place. To install a tracheal tube to control ventilation during artificial respiration, remove the hair, sterilize the site and incise the skin over the trachea right above the manubrium. Blunt dissect the first layer of fat and muscles.Move the salivary gland aside and expose the trachea by gently dissecting the last layer of muscles. Carefully isolate the trachea and slide a thread below it using small forceps. Then make a loose surgeons knot with the thread.Incise the trachea transversely between two cartilaginous rings. Next, insert a tracheal tube with the appropriate diameter and tighten the thread's knot in order to secure the tube. For additional stability, the thread can be further attached to a higher point of the trachea tube.Suture or use surgical staples to close the wound. Subsequently, transfer the animal to a stereotaxic frame and apply eye ointment to avoid desiccation. Then place appropriate probes to monitor its physiological variables such as the electrocardiogram, the blood oxygen saturation and the end tidal carbon dioxide pressure.After that, remove the hair over the scalp and sterilize the surgical site. Make a two centimeters longitudinal incision and resect the connective tissues overlying the skull using a scalpel. Then apply surgical glue on the skull and tissues to stabilize the preparation.Then make a small craniotomy of 1.5 millimeter diameter over the region of interest with a dental drill. Afterward, use a pair of extra fine forceps and vena scissors to gently make a small hole in the dura. Reserve of 0.5 millimeter region within the cranial trepanation to place the electrocorticographic electrode.Be sure to keep the cortex moist with 0.9%sodium chloride solution, or aCSF. Next, place a reference electrode on the contralateral scalp muscle, and place a low impedance electrocorticographic electrode on the dura. Adjust the artificial ventilation system so that the respiratory frequency and volume are similar to those of the rat's spontaneous breathing.If all the physiological variables and electrocorticographic patterns are within the normal values, inject gallamine triethiodide in each leg to paralyze the animal. Then connect the mechanical ventilation to the tracheal tube and verify the proper thoracic cage inflation on both sides. In this step, pull a glass micropipette with a 0.2 micrometer tip and fill it with two molar potassium acetate solution.Place the pipette in a specific holder with a silver/silver chloride wire to connect the pipette solution to an intracellular amplifier. Then place a silver/silver chloride reference electrode on the rat's neck muscles. Place the pipette above the region of interest.Slowly insert the pipette into the brain down to the desired depth using the buzz button of the amplifier to clear the pipette if needed. Compensate for the pipette resistance by zeroing the voltage drops in response to current steps using the bridge circuit of the amplifier. Next, use cotton swabs or synthetic absorption triangles to dry the craniotomy, and cover it with silicone elastomer or 4%agarose to reduce brain movements.Afterward, lower the pipette in one or two micrometer steps until it's resistance increases when approaching a cell. Use the buzz function of the amplifier to penetrate into the neuron. Once the recording is stable, compensate for the electrode resistance by adjusting the bridge parameter while small steps of currents are injected into the neuron.Then perform the appropriate experimental protocol, such as injecting current intracellularly to extract intrinsic properties of the neuron or perform sensory stimulations. Next, inject a high but infralethal dose of sodium pentobarbital through the intraperitoneal line. Within 15 to 20 minutes, the intracellular and electrocorticographic activities should slow down with intermittent electrical silences to transiently reach the so-called burst suppression profile which progressively collapses to a complete isoelectric state.It is expected that the heart rate significantly slows down but the SPO2 and EtCO2 should stay relatively steady. Then repeat the stimulation protocol to compare the impact of the network dynamics on the recorded neurons integrative properties. After the cessation of anesthetic injections, the electrical brain activity should fully recover within there to four hours.This figure shows spontaneous intracellular and electrocorticogram activities in two anesthetic conditions, mimicking the cortical dynamics endogenously generated during the early stages of sleep, or during waking. The sleeplike state is induced by sodium pentobarbital injection, and the waking-like state by fentanyl. In both cases, the subsequent injection of a high dose of sodium pentobarbital resulted in a complete abolition of spontaneous activity in the ECOG and the simultaneously recorded neurons, hence the term isoelectric.Suppressing the ongoing synaptic activity resulted in a significant study hyperpolarization of the neuronal membrane potential. Current evoked responses can be generated before and after the induction of the isoelectric state. This allows for the comparison of different cerebral activities on the input output transfer function of neurons, expressed as the main evoked firing rate in response to current steps of increasing intensity.While attempting this procedure, it's important to remember to keep the animal in the best physiological conditions to obtain good quality recording associated with our reliable cerebral dynamics. This approach can be further employed as a model to study neuron's responsiveness in irreversible or reversible deep comatose states.

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