Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Summary

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Abstract

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Introduction

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. It inhibits excitatory postsynaptic potentials (EPSPs) in monosynaptically excited motoneurons without changing the postsynaptic membrane potential and the excitability of the motoneurons^1-3. Primary afferent depolarization (PAD) induced by GABAergic axo-axonal synapses onto sensory presynaptic fibers is the underlying mechanism^4-7 (see also Figure1a). These synapses contain GABA_A- and GABA_B-receptors (GABA_AR and GABA_BR). GABA_AR activity leads to an increase in chloride conductance wh....

Protocol

All experimental procedures mentioned in the following protocol were approved by the Thuringian state authorities (Thüringer Landesamt für Verbraucherschutz, Reg.-Nr. 02-044/12).

1. Preparations for Experiment

1. Fabrication of suction electrodes
   1. Pull a micropipette using a standard borosilicate glass capillary with a micropipette puller, e.g. a standard patch electrode.
   2. Brake the electrode to tip diameter of 0.5-1 mm (slightly larger than the diameter of the dorsal roots) using a diamond file.
   3. Heat polish the tip so it will not harm the dorsal root when it is sucked in. A standa....

Results

Typical DRP traces are shown in Figure 3. The prominent stimulation artifact is usually followed by a short downward deflection. Thereafter a slow, long-lasting upward deflection, representing the DRP is clearly distinguishable. In a subset of recordings, dorsal root reflexes are visible as small spikes on top of the DRP. In normal wild-type mice, dorsal root reflexes appear most often when stimulation voltage is excessive. As the dorsal root reflexes cannot be elicited with a high reproducibility in thi.......

Discussion

Extra- and intracellular electrophysiological recordings of neuronal activity and synaptic potentials in vivo are state of the art techniques in investigating CNS neuronal functions and pathophysiology. Spinal integration is critical for motor function, e.g. limb movement and for multimodal sensory perception. Presynaptic inhibition is one critical mechanism in this computational process ensuring appropriate responses to sensory inputs. GABAergic synapses on Ia afferent fibers inhibit the excitation of .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glass tubing (inner diameter 1.16 mm)	Science Products (Hofheim, Germany)	GB200F-10	Other glass tubing might also be suitable
Superfusion solution (sterile, 0,9% NaCl)	Braun Melsungen AG	3570350
Rompun 2% (Xylazine)	Bayer Animal Health GmbH (Leverkusen, Germany)
Ketamine 10%	Medistar GmbH (Ascheberg, Germany)	KETAMIN 10%
30 G Microneedle/ Sterican	Braun Melsungen AG	4656300
Salts for aCSF	Sigma-Aldrich	Diverse
S88 Dual Output Square Pulse	Grass Technologies (Warwick, USA)	S88X
SIU5 RF Transformer Isolation Unit	Grass Technologies (Warwick, USA)	SIU-V
InstruTECH LIH 8+8	HEKA (Lambrecht, Deutschland)	LIH 8+8 + Patchmaster software
Universal amplifier	NPI (Tamm, Deutschland)	ELC-03X
Micropipette puller	Sutter Instruments (Novato, USA)	P-1000
Dissecting microscope	Olympus (Tokyo, Japan)
Micromanipulator	Sutter Instruments (Novato, USA)	MPC-200/MPC-325	Mechanical micromanipulators also possible
Homeothermic Blanket System	Stoelting (Wood Dale, USA)	50300V
Intra-/extracellular recording electrode holder	Harvard Apparatus (Holliston, USA)	641227