Recording Gamma Band Oscillations From Pedunculopontine Nucleus Neurons in a Brain Slice

Abstract

Source: Urbano, F. J., et al. Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons. J. Vis. Exp. (2016).

The video demonstrates the method to record gamma-band oscillations in PPN neurons. Using a sagittal brain section with synaptic blockers in aCSF, it employs positive current pulses via a patch pipette to open calcium channels, inducing crucial subthreshold activity for wakefulness.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation of Standard-artificial Cerebrospinal Fluid (aCSF)

1. Preparation of Stock Solution A
   1. Add 700 ml of distilled water to a clean 1 L beaker before adding chemicals.
   2. While continuously stirring a volume of 500 ml, add 136.75 g of NaCl, 6.99 g of KCl, 2.89 g of MgSO₄, and 2.83 g of NaH₂PO₄.
   3. Add more distilled water to reach a final volume of 1 L. After dilution, the final concentration of each com....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sucrose	Sigma-Aldrich	S8501	C12H22O11, molecular weight = 342.30
Sodium Bicarbonate	Sigma-Aldrich	S6014	NaHCO3, molecular weight = 84.01
Potassium Chloride	Sigma-Aldrich	P3911	KCl, molecular weight = 74.55
Magnesium Chloride Hexahydrate	Sigma-Aldrich	M9272	MgCl2 · 6H2O, molecular weight = 203.30
Calcium Chloride Dihydrate	Sigma-Aldrich	C3881	CaCl2 · 2H2O, molecular weight =147.02
D-(+)-Glucose	Sigma-Aldrich	G5767	C6H12O6, molecular weight = 180.16
L-Ascorbic Acid	Sigma-Aldrich	A5960	C6H8O6, molecular weight =176.12
Sodium Chloride	Acros Organics	327300025	NaCl, molecular weight = 58.44
Potassium Gluconate	Sigma-Aldrich	G4500	C6H11KO7, molecular weight = 234.25
Phosphocreatine di(tris) salt	Sigma-Aldrich	P1937	C4H10N3O5P · 2C4H11NO3, molecular weight = 453.38
HEPES	Sigma-Aldrich	H3375	C8H18N2O4S, molecular weight = 238.30
EGTA	Sigma-Aldrich	E0396	[-CH2OCH2CH2N(CH2CO2H)2]2, molecular weight = 380.40
Adenosine 5'-triphosphate magnesium salt	Sigma-Aldrich	A9187	C10H16N5O13P3 · xMg2+, molecular weight = 507.18
Guanosine 5'-triphosphate sodium salt hydrate	Sigma-Aldrich	G8877	C10H16N5O14P3 · xNa+, molecular weight = 523.18
Tetrodotoxin citrate	Alomone Labs	T-550	C11H17N3O8, molecular weight = 319.27
DL-2-Amino-5-Phosphonovaleric Acid	Sigma-Aldrich	A5282	C5H12NO5P, molecular weight = 197.13
CNQX disodium salt hydrate	Sigma-Aldrich	C239	C9H2N4Na2O4 · xH2O, molecular weight = 276.12
Strychnine	Sigma-Aldrich	S0532	C21H22N2O2, molecular weight = 334.41
Mecamylamine hydrochloride	Sigma-Aldrich	M9020	C11H21N · HCl, molecular weight = 203.75
Gabazine (SR-95531)	Sigma-Aldrich	S106	C15H18BrN3O3, molecular weight = 368.23
Ketamine hydrochloride	Mylan	67457-001-00
Microscope	Nikon	Eclipse E600FN
Micromanipulator	Sutter Instruments	ROE-200
Micromanipulator	Sutter Instruments	MPC-200
Amplifier	Molecular Devices	Multiclamp 700B
A/D converter	Molecular Devices	Digidata 1440A
Heater	Warner Instruments	TC-324B
Pump	Cole-Parmer	Masterflex L/S 7519-20
Pump cartridge	Cole-Parmer	Masterflex 7519-85
Pipette puller	Sutter Instruments	P-97
Camera	Q-Imaging	RET-200R-F-M-12-C
Vibratome	Leica Biosystems	Leica VT1200 S
Refrigeration system	Vibratome Instruments	900R
Equipment
microscope	Nikon	Eclipse E600FN
micromanipulator	Sutter Instruments	ROE-200
micromanipulator	Sutter Instruments	MPC-200
amplifier	Molecular Devices	Multiclamp 700B
A/D converter	Molecular Devices	Digidata 1440A
heater	Warner Instruments	TC-324B
pump	Cole-Parmer	Masterflex L/S 7519-20
pump cartridge	Cole-Parmer	Masterflex 7519-85
pipette puller	Sutter Instruments	P-97
camera	Q-Imaging	RET-200R-F-M-12-C