Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Summary

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Abstract

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest¹. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness^2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.

One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram^5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state⁷. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell⁸; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.

Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.

We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

Protocol

1. Surgical Preparation of Animals

1. Experimental Subjects

Use mice of the B6.Cg-Tg(Thy1-COP4/eYFP)18Gfng/J transgenic line⁹; JAX strain #7612) or other mice expressing the blue light-sensitive cation channel, Channelrhodopsin-2, in cerebral cortical neurons. Application of blue light to the cerebral cortex of the B6.Cg-Tg(Thy1-COP4/eYFP)18Gfng/J transgenic line causes the pyramidal neurons expressing Channelrhodopsin-2 to depolarize and undergo action potentials^9,10. As a consequence of pyramidal cell activation, local interneurons are activated, and the change in potential is propagated to neu....

Results

As shown in Figure 2, a mouse equipped for optogenetic stimulation and lactate/EEG/EMG data collection underwent spontaneous sleep/wake state transitions while EEG, EMG and cerebral lactate concentration were monitored continuously. Current at the lactate sensor increased during periods of low amplitude EEG and decreased during periods of high amplitude EEG. As shown in Figure 3, both channels of the EEG are responsive to optogenetic stimuli delivered in the frontal cort.......

Discussion

The methods presented here allow one to measure the relationship between sleep and changes in the brain concentration of the glycolytic intermediate lactate on a time scale not previously possible. Animals undergo spontaneous transitions between wake, NREMS and REMS. Furthermore, we are able to apply optogenetic stimuli while animals undergo these transitions. Data collected to date demonstrate that both spontaneous and induced waves impact on the readout of a lactate oxidase-based biosensor.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BASi Mouse Guide Cannula	Pinnacle Technology/BASi Inc	7032
Lactate Biosensor	Pinnacle Technology	7004
Head Mount	Pinnacle Technology	8402
Sleep/Biosensor Recording system	Pinnacle Technology	8400-K1-SL	2 EEG channels, 1 EMG channel, & 1 biosensor
Tethered Mouse in-vitro Calibration kit	Pinnacle Technology	7000-K1-T
Fiber Optic Guide Cannula	Plastics One	C312G	21 Gauge Guide Cannula
Dummy Cannula	Plastics One	C312DC	21 Gauge Dummy
Diamond Fiber Scribe	Thorlabs	S90W
Fiber Connector Crimp Tool	Thorlabs	CT042
Furcation Tubing	Thorlabs	FT030	03.0 mm
	Thorlabs	T10S13	Max Dia. 0.012
Furcation Tube Stripper	Thorlabs	FTS3
Bare Hard Cladding Multimode Fiber	Thorlabs	BFL37-200	200 μm Core, 0.37 NA
Wire Snips/Kevlar Shears	Thorlabs	T865
Fiber Optic Epoxy	Thorlabs	F112
Fiber Stripper Tool	Thorlabs
Glass Polishing Plate	Thorlabs	CTG913
Rubber Polishing Pad	Thorlabs	NRS913
Eye Loupe	Thorlabs	JEL10
Kim Wipes	Thorlabs	KW32
Compressed Air	Thorlabs	CA3
Polishing Puck	Thorlabs	D50-xx
Fiber Inspection scope	Thorlabs	CL-200
Polishing Films	Thorlabs	LFG5P, LFG3P, LFG1P, LFG03P
FC/PC connector end	Thorlabs	30126G2-240	240 μm Bore, SS Ferrule
MC Stimulus Unit	Multi-Channel Systems	STG-4002
MC Stimulus Software	Multi-Channel Systems	MC-Stimulus V 2.1.5
Blue Laser	CrystaLaser	CL473-050-0
Laser Power supply	CrystaLaser	CL2005
Fiber Optic Rotary Joint	Doric Lenses	FRJ-v4
Table 2. Supplies and equipment.