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

Summary

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.

Abstract

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' 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.

Introduction

In the absence of any environmental stimuli or behavioral tasks, the "resting" 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 "synaptic noise"), 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 net....

Protocol

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's specific needs.

1. Surgical Preparation

Note: All incision and pressure points should be repeatedly infiltrated with local anesthetic (lidocaine or bupivacaine). The present procedure is terminal, if an aseptic preparation is required several modifications should be implemented.

1. Anesthetize ....

Results

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's physiological state before (Figure 1A) and after (Figure 1B) induction of t.......

Discussion

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.......

Materials

List of materials used in this article

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