Triggering a Closed-Loop Stimulation by Neuron Spiking Activity

研究方案

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

1. Surgery

NOTE: The exact details of the surgery for closed-loop stimulation depend on the type of recording probes used and the brain areas targeted. In most cases, however, a typical surgery will consist of the following steps.

1. Bring a cage with a rat to the surgery room to be implanted with a silicone probe or electrode array to record neuronal activity.
2. Anesthetize the rodent with 2-2.5% isoflurane and fix the head in a stereotaxic frame. Ensure that the animal is unconscious during surgery by observing any motoric reaction to mild tactile stimuli.
3. Apply an eye ointment to minimize dryness during the surgery.
4. Shave the surgical area and disinfect the skin with 2% chlorhexidine solution and 70% isopropyl alcohol.
5. Inject lidocaine (5 mg/kg) under the scalp over the brain area where electrodes will be implanted.
6. Make an incision of the scalp over the area of the future implant, and use a scalpel and a cotton swab to clear the periosteum from the exposed skull.
7. Drill 4-8 holes in the skull for implantation of anchor screws (~0.5 mm) as structural support for the implant. Attach the screws to the skull by inserting them in the holes and ensure that they are held firmly in place.
8. Drill the craniotomy at the specified coordinates and inset the microdrive/probe implant.
   NOTE: The described protocol for closed-loop stimulation will work for any brain region in which the electrodes are inserted.
9. Fix the microdrive/probe and any required electrical interface connector to the skull using dental acrylic. The amount of dental acrylic should be enough to attach the implant firmly, but it should not come in contact with the surrounding soft tissue.
10. After surgery, closely monitor the animal until it has regained sufficient consciousness to maintain sternal recumbency. For the subsequent three days, administer subcutaneously an analgesic (e.g., Metacam, 1 mg/kg) and an antibiotic to prevent infection (e.g., enrofloxacin, 10 mg/kg).
    NOTE: Animals are typically left to recover from surgery for one week prior to any testing or recording.

2. Software installation

NOTE: This was tested on Windows 10, 64-bit version.

1. Install data acquisition and processing software.
2. Install the data acquisition system Cheetah 6.4 (https://neuralynx.com/software/category/sw-acquisition-control), which includes libraries to interact with the Cheetah Acquisition System.
3. Install SpikeSort3D (https://neuralynx.com/software/spikesort-3d) or any other software that uses KlustaKwik27 for spike sorting. The online detection software uses the cluster definitions from the KlustaKwik engine. This software may run on the same computer or on separate computers on the same network.
4. Install the NetComDevelopmentPackage (https://github.com/leomol/cheetah-interface/blob/master/NetComDevelopmentPackage_v3.1.0), which can also be downloaded from https://neuralynx.com/software/netcom-development-package.
5. Install Matlab (https://www.mathworks.com/downloads/; code was tested on Matlab version R2018a). Make sure that Matlab is enabled in the Windows firewall. Normally, a pop-up will appear during the first connection.
6. Log in to a Matlab account. Choose the license. Choose the version. Choose the operating system.
7. Download the following library for online event triggering from https://github.com/leomol/cheetah-interface and extract files to the computer's 'Documents/Matlab' folder. A copy of the code is provided in the accompanying Supplemental Materials.

3. Initial data acquisition

1. Start data acquisition using Cheetah software.
2. Record a few minutes of spiking data to populate template waveforms.
3. Stop the data acquisition and perform spike sorting on the recorded data.
4. Open SpikeSort3D, click File | Menu | Load Spike File and select a spike file from the folder with recorded data.
5. Click Cluster Menu, then Autocluster using KlustaKwik, leaving the default settings, and click Run.

4. Closed-loop experiment

1. Resume data acquisition in Cheetah.
2. Open Matlab.
3. Open ClosedLoop.m and click on Run. Alternatively, in the Matlab command window, execute ClosedLoop(). Ensure that ClosedLoop.m is on the Matlab path. If the user wants to employ a custom function to call on every trigger, execute ClosedLoop('-callback', customFunction) instead, where customFunction is a handle to that function.
4. Load the spike information defined on the initial recording by clicking on Load, browsing the recording folder, and selecting one of the spiking data files (.ntt, .nse).
5. Select one or multiple neurons that will trigger stimulation by clicking the checkbox under the plotted waveforms.
6. Define the minimum number of neurons that will trigger stimulation by typing an integer in the "min matches" text box. Then, define the time window in which spikes matching different waveforms are considered co-active by typing a number in the "window" text box.
7. Click Send to start. This will begin online triggering of events (tones as default) based on the spiking activity of selected neurons.

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材料

Name	Company	Catalog Number	Comments
Baytril	Bayer, Mississauga, CA	DIN 02169428	antibiotic; 50 mg/mL
Cheetah 6.4	NeuraLynx, Tucson, AZ	6.4.0.beta	Software interfaces for data acquisition
Digital Lynx 4SX	NeuraLynx, Tucson, AZ	4SX	recording equipment
Headstage transmitter	TBSI	B10-3163-GK	transmits the neural signal to the receiver
Isoflurane	Fresenius Kabi, Toronto, CA	DIN 02237518	inhalation anesthetic
Jet Denture Powder & Liqud	Lang Dental, Wheeling, US	1230	dental acrylic
Lacri-Lube	Allergan, Markham, CA	DIN 00210889	eye ointment
Lido-2	Rafter 8, Calgary	DIN 00654639	local anesthetic; 20 mg/mL
Matlab	Mathworks	R2018b	software for signal processing and triggering external events
Metacam	Boehringer, Ingelheim, DE	DIN 02240463	analgesic; 5 mg/mL
Netcom	NeuraLynx	v1	Application Programming Interface (API) that communicates with Cheetah
Silicone probe	Cambridge Neurotech	ASSY-156-DBC2	implanted device
SpikeSort 3D	NeuraLynx, Tucson, AZ	SS3D	spike waveform-to-cell classification tools
Wireless Radio Receiver	TBSI	911-1062-00	transmits the neural signal to the Digital Lynx