Evaluation of Hemisphere Lateralization with Bilateral Local Field Potential Recording in Secondary Motor Cortex of Mice

Summary

We present in vivo electrophysiological recording of the local field potential (LFP) in bilateral secondary motor cortex (M2) of mice, which can be applied to evaluate hemisphere lateralization. The study revealed altered levels of synchronization between the left and right M2 in APP/PS1 mice compared to WT controls.

Abstract

This article demonstrates complete, detailed procedures for both in vivo bilateral recording and analysis of local field potential (LFP) in the cortical areas of mice, which are useful for evaluating possible laterality deficits, as well as for assessing brain connectivity and coupling of neural network activities in rodents. The pathological mechanisms underlying Alzheimer's disease (AD), a common neurodegenerative disease, remain largely unknown. Altered brain laterality has been demonstrated in aging people, but whether or not abnormal lateralization is one of the early signs of AD has not been determined. To investigate this, we recorded bilateral LFPs in 3-5-month-old AD model mice, APP/PS1, together with littermate wild type (WT) controls. The LFPs of the left and right secondary motor cortex (M2), specifically in the gamma band, were more synchronized in APP/PS1 mice than in WT controls, suggesting a declined hemispheric asymmetry of bilateral M2 in this AD mouse model. Notably, the recording and data analysis processes are flexible and easy to carry out, and can also be applied to other brain pathways when conducting experiments that focus on neuronal circuits.

Introduction

Alzheimer's disease (AD) is the most common form of dementia^1,2. Extracellular beta amyloid protein (β-amyloid protein, Aβ) deposition and intracellular neurofibrillary tangles (NFTs) are the main pathological features of AD^3,4,5, but the mechanisms underlying AD pathogenesis remain largely unclear. Cerebral cortex, a key structure in cognition and memory, is impaired in AD⁶, and motor deficits such as slow walking, difficulty navigating the environment and gait disturbances occur with advancing age⁷. Aβ deposition and neurofibrillary tangles have also been observed in the premotor cortex (PMC) and supplementary motor area (SMA) in AD patients⁸ and cognitively impacted older adults⁹, indicating the involvement of an impaired motor system in AD pathogenesis.

The brain is formed by two distinct cerebral hemispheres that are divided by a longitudinal fissure. A healthy brain exhibits both structural and functional asymmetries¹⁰, which is called "lateralization", allowing the brain to efficiently deal with multiple tasks and activities. Aging results in a deterioration in cognition and locomotion, together with a reduction in brain laterality^11,12. The motor abilities of the left hemisphere are readily apparent in the healthy brain¹³, but in the AD brain aberrant laterality occurs as a consequence of the failure of left hemisphere dominance associated with left cortical atrophy^14,15,16. Therefore, an understanding of a possible alteration of brain lateralization in AD pathogenesis and the underlying mechanisms may provide new insights into AD pathogenesis and lead to identification of potential biomarkers for treatment.

Electrophysiological measurement is a sensitive and effective method of evaluating changes in the neuronal activities of animals. The reduction of hemispheric asymmetry in elders (HAROLD)¹⁷ has been documented by electrophysiological research with synchronized interhemispheric transfer time, which shows weakening or absence of hemispheric asymmetry to monaurally presented speech stimuli in the elderly¹⁸. Utilizing APP/PS1, one of the most commonly used AD mouse models^19,20,21,22, in combination with in vivo bilateral extracellular recording of LFPs in both left and right M2, we evaluated possible laterality deficits in AD. In addition, with simple parameter settings, the built-in function of data analysis software (see the Table of Materials) provides a faster and more straightforward way to analyze the synchronization of electrical signals than mathematically complex programming language, which is friendly to beginners with in vivo electrophysiology.

Protocol

All animals were paired-housed under standard conditions (12 h light/dark, constant temperature environment, free access to food and water) according to the Chinese Ministry of Science and Technology Laboratory Animals Guidelines and experiments were approved by the local ethical committee of Guangzhou University. This is a non-survival procedure. 

NOTE: For data shown in the representative results, APP/PS1 (B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/J) double-transgenic mice and littermate wild-type (WT) controls at 3-5 months of age, were used for recordings (n = 10, per group).

1. Animal anesthesia and surgery

1. Weigh and anesthetize the mouse by your approved anesthesia regimen from your local animal care committee. 
2. Perform a tail or toe pinch with forceps to confirm deep anesthesia prior to surgery.
3. Position the mouse in a stereotaxic apparatus and fix its head.
4. Apply eye ointment on both eyes to keep moist. Follow your local animal care guidelines regarding pre- and postoperative analgesia. 
5. Shave the hair using surgical clippers. Make a small incision (12-15 mm) in the middle of the exposed surgical area with scissors. Using forceps, gently pull the scalp away from the midline.
6. Separate the skin gently and remove residual tissue. Clean the skull using hydrogen peroxide-coated cotton buds.
7. Drill two small holes of radii 1.0-1.5 mm on both left and right sides of the skull to allow insertion of the recording microelectrodes into the M2 regions under a stereomicroscope (Figure 1A).
   NOTE: Stereotaxic locations of bilateral M2: 1.94 mm anterior to the bregma, 1.0 mm lateral to the midline, and 0.8-1.1 mm ventral to the dura.
8. Remove the dura mater carefully with a tungsten needle.
9. Pull glass borosilicate micropipettes (outer diameter: 1.0 mm) as recording microelectrodes with resistance of 1-2 MΩ.
10. Insert two separate recording microelectrodes filled with 0.5 M NaCl into the holes using mechanical micromanipulators (at 60°, Figure 1B).

2. LFP recordings in bilateral M2 of mice

1. Lower the left and right glass electrodes slowly into appropriate coordinates of bilateral M2 (Figure 1C).
2. For quality control, test the resistance of each electrode using the differential amplifier before capturing LFPs.
3. Set the recording process at 0.1 Hz high-pass and 1,000 Hz low-pass with 1,000x amplification.
4. Collect digitized raw LFP data of at least 60 s spontaneous activities in stable state, with mice breathing evenly at a respiratory rate of 2 breaths per second under anesthesia.
5. After recording, slowly raise the electrodes out of the brain, then euthanize the mice by fast cervical dislocation.
6. Save the data and analyze offline.

3. Cross-correlation analysis

1. Click Analysis - Waveform correlation in the analysis software and import the data.
2. Parameter settings
   1. Define one waveform channel signal as the first channel and the other as the reference. Set width as 2 and offset as 1 (Figure 2A).
   2. Set the duration of both LFPs for 100 s by selecting the start time and end time. Press the Process button to perform cross-correlation analysis (Figure 2B).
      NOTE: Simultaneous bilateral signals with such durations would be long enough to show neuronal spontaneous activities, thereby revealing the basic properties of synchronization.
3. Click File - Export As, then save the cross-correlation results corresponding to the resulting pop-up chart in .txt format.
4. Open the .txt file (Figure 2C), remove the correlation values at time lags ranged 0 ± 0.01 s (since two continuous gamma waves have at least 0.01 s interval), then average the rest of the cross-correlation data in the negative time lag part or average the rest of the cross-correlation data in the positive time lag part.

4. Coherence analysis

1. Import and run the data in the analysis software.
2. Assign the two LFP signals to be the first and second waveform channels separately. Then set the block size value (Figure 3A).
   NOTE: Block size means the number of data points used in the FFT. The larger the block size, the better the frequency resolution. Here we recommend setting it as 4096.
3. Move the dotted lines manually to ensure the time accuracy for signals in both channels are being set as the same period (Figure 3B). Press the Add Area button to load the area and perform coherence analysis.
4. Click File - Save As to save the coherence results corresponding to the resulting pop-up chart in .txt format (Figure 3B).

Results

To see whether early AD pathology impairs the capacity of hemisphere lateralization, we conducted bilateral extracellular LFP recordings in the left and right M2 of APP/PS1 mice and WT controls (aged 3-5 months), and analyzed the cross-correlation of these left and right LFPs. In WT mice, the results demonstrated that the mean correlation between left and right LFPs at positive time lags differed significantly from that at negative time lags, implicating the existence of hemispheric asymmetries in M2 areas of WT controls...

Discussion

We report here the procedure for in vivo bilateral extracellular recording, along with analyzing the synchronization of dual-region LFP signals, which is both flexible and easy to conduct for estimating brain hemisphere lateralization, as well as the connectivity, directionality or coupling between neural activities of two brain areas. This can be widely used to reveal not only group-neuronal activities, but also some basic properties of interregional electrophysiology, especially for labs which are interested i...

Materials

Name	Company	Catalog Number	Comments
AC/DC Differential Amplifier	A-M Systems	Model 3000
Analog Digital converter	Cambridge Electronic Design Ltd.	Micro1401
Glass borosilicate micropipettes	Nanjing spring teaching experimental equipment company	161230	Outer diameter: 1.0mm
Microelectrode puller	Narishige	PC-10
NaCl	Guangzhou Chemical Reagent Factory	7647-14-5
Pin microelectrode holder	World Precision Instruments, INC.	MEH3SW10
Spike2	Cambridge Electronic Design Ltd.
Stereomicroscope	Zeiss	435064-9020-000
Stereotaxic apparatus	RWD Life Science	68045
Urethane	Sigma-Aldrich	94300