Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

Summary

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Abstract

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

Introduction

Breathing is a complex and vital activity controlled by the brain, allowing dioxygen (O₂) uptake and carbon dioxide (CO₂) elimination. The central respiratory drive is generated by a complex network located in the brainstem in both mammals ¹, amphibians ², reptiles ³, birds ⁴ and fishes ⁵. Even if the study of breathing can be processed in vivo, precise mechanistic investigations require direct access to the respiratory control network. To this end, Adrian and Buytendijk developed a reduced goldfish preparation, in which electrodes placed on the brainstem surface record the generated....

Protocol

This method required the use of animal subjects, allowed by Laval University Animal Ethics Committee (protocol # 2012-170).

1. Setup and Preparation

1. Solutions
   1. Prepare aCSF stock solutions according to the following recipes ^7,17. Other recipes with concentration variations are available in the literature. Store stock solutions at 4 °C for up to one month.
      1. 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 ....

Results

As mentioned in the introduction, one of the most important advantages of this technique is the direct access to the brainstem to apply various stimuli. As an example, hypoxia was applied here. Figure 1.A.B. displays a full protocol recording, with both normoxic and hypoxic conditions. Figure 1.C.E. displays the rhythm recorded in normoxic conditions (i.e., aCSF bubbled with 95% O₂ and 5% CO₂ at 26 °C). As previously demonstrated in this ex.......

Discussion

Accurate quantification of respiratory activity can be challenging. Indeed, breathing is a function that can be both automatic and voluntary, and that is modulated according to the environment, the body’s needs, the emotional state and the behavior. The advantage of this technique is the isolation of the neural elements responsible for producing the respiratory command. Thus, electrophysiological recordings of brainstem-spinal cord preparations and plethysmography are complementary techniques to study the whole neu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sylgard	Sigma Aldrich	761036-5EA	Use under hood
NaCl	Bioshop	SOD002
KCl	Bioshop	POC888
CaCl2	Bioshop	CCL444
MgCl2	Bioshop	MAG510
NaHCO3	Bioshop	SOB999
NaH2PO4	Bioshop	SPM306
D-glucose	Bioshop	GLU501
Carbogen	Linde	343-02-0006
Temperature Controller	Warner Instruments, Hamden, CT, USA	TC-324B
Suction electrode	A-M Systems, Everett, WA, USA	model 573000
Differential AC amplifier	A-M Systems, Everett, WA, USA	model 1700
Moving averager	CWE, Ardmore, PA, USA	model MA-821
Data acquisition system	Dataq Instruments, Akron, OH, USA	model DI-720
LabChart software	ADInstruments, Colorado Springs, CO, USA
Prism sofware	Graphpad, La Jolla, CA, USA
Dissection chamber	Plastic box (e.g. petri box) will do
Recording chamber	Home made
Base	Kanetec, Bensenville, IL, USA	MB
Micromanipulator	World Precision Instrument Inc, Sarasota, FL, USA	KITE-R
Base	Kanetec, Bensenville, IL, USA	MB
Peristaltic pump	Gilson, Middleton, WI, USA	MINIPULS 3
Faraday Cage	Home made
Computer