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

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