Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Summary

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Abstract

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preBötzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

Introduction

The respiratory neural network of the brainstem provides a fertile domain for understanding the general characteristics of rhythmic neural networks. In particular, the interest is in the development of neonatal rodent breathing and understanding how the breathing rhythm develops. This may be done using a multi-level approach, including in vivo whole animal plethysmography, in vitro en bloc nerve recordings, and in vitro slice recordings that contain the breathing rhythm generator. Reductionist in vitro en bloc and slice recordings are an advantageous method to use when interrogating the mechanisms behind respiratory rhythmogenesis and neural circuitry in the brainstem....

Protocol

The following protocol has been accepted and approved by the Institutional Animal Care and Use Committee (IACUC) of Loma Linda University. NIH guidelines for the ethical treatment of animals are followed in all animal experiments performed in the laboratory. All ethical standards were upheld by individuals performing this protocol.

1. Solutions

1. Prepare artificial cerebral spinal fluid (aCSF).
   1. Prepare fresh aCSF the evening before an experiment in 1 L batches using the following recipe: 7.250 g NaCl (124 mM), 0.224 g KCl (3 mM), 2.100 g NaHCO₃ (25 mM), 0.069 g NaH₂PO₄&....

Results

The method presented here allows a researcher interested in obtaining rhythmically active slices of brainstem to reproducibly and reliably cut a viable, robust slice that will allow recording of fictive motor output for many hours. All of the minimally necessary neural circuit elements for generating and transmitting inspiratory rhythm can be captured in a thin slice using this method. These elements include: the preBötzinger Complex, premotor neurons projecting to the hypoglossal motor n.......

Discussion

Adapting the protocol presented here into an en bloc or slice workflow is advantageous for laboratories and studies that would like to utilize either en bloc brainstem-spinal cord and/or thin slice preparations for electrophysiology recordings. The dissection and slice method presented, combined with methods previously reported by others^17,18,19, will allow reproducible preparation of robust and viable tissue that is widely adap.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Fisher Scientific	S271-500
KCl	Sigma Aldrich	P5405-1KG
NaHCO3	Fisher Scientific	BP328-1
NaH2PO4 •H2O	Sigma Aldrch	S9638-25G
CaCl2•2H2O	Sigma Aldrich	C7902-500G
MgSO4•7H2O	Sigma Aldrich	M7774-500G
D-Glucose	Sigma Aldrich	G8270-1KG
Cold-Light source Halogen lamp 150 W	AmScope	H2L50-AY
Dissection Microscope	Leica	M-60
Vibratome 1000 Plus	Vibratome	W3 69-0353
Magnetic Base	Kanetic	MB-B-DG6C
Isoflurane, USP	Patterson Veterinary	NDC 14043-704-06
Sword Classic Double Edge Blades	Wilkinson	97573
Histoclear	Sigma-Aldrich	H2779
Dumont #5 Fine Forceps	Fine Science Tools	11254-20
Dumont #5/45 Forcep	Fine Science Tools	11251-35
Scalpel Blades #10	Fine Science Tools	10010-00
Scalpel Handel #3	Fine Science Tools	10003-12
Spring Scissors Straight	Fine Science Tools	15024-10
Narrow Pattern Forcep Serrated/straight	Fine Science Tools	11002-12
Castroviejo Micro Dissecting Spring Scissors; Straight	Roboz	RS-5650
Vannas Scissors 3" Curved	Roboz	RS-5621
Insect pins, 0	Fine Science Tools/8840604	26000-35
Insect pins, 0, SS	Fine Science Tools	26001-35
Insect pins, 00	Fine Science Tools	26000-30
Insect pins, 00, SS	Fine Science Tools	26001-30
Insect pins, 000	Fine Science Tools	26000-25
Insect pins, 000, SS	Fine Science Tools	26001-25
Minutien pins, 0.10 mm	Fine Science Tools	26002-10
Minutien pins, 0.15 mm	Fine Science Tools	26002-15
Minutien pins, 0.2 mm	Fine Science Tools	26002-20
Fisher Tissue prep Parafin	fisher	T56-5
Graphite	fisher	G67-500
Delrin Plastic	Grainger	3HMT2
18 Gauge Hypodermic Needle	BD	305195