Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments

Podsumowanie

This protocol demonstrates how to use an electrophysiological system for closed-loop stimulation triggered by neuronal activity patterns. Sample Matlab code that can be easily modified for different stimulation devices is also provided.

Streszczenie

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems are already found in clinical applications, and are important tools for basic brain research. A particularly interesting recent development is the integration of closed-loop approaches with optogenetics, such that specific patterns of neuronal activity can trigger optical stimulation of selected neuronal groups. However, setting up an electrophysiological system for closed-loop experiments can be difficult. Here, a ready-to-apply Matlab code is provided for triggering stimuli based on the activity of single or multiple neurons. This sample code can be easily modified based on individual needs. For instance, it shows how to trigger sound stimuli and how to change it to trigger an external device connected to a PC serial port. The presented protocol is designed to work with a popular neuronal recording system for animal studies (Neuralynx). The implementation of closed-loop stimulation is demonstrated in an awake rat.

Wprowadzenie

The goal of this protocol is to demonstrate how to implement closed-loop stimulation in neurophysiological experiments. The typical setup for closed-loop experiments in neuroscience involves triggering stimuli based on the online readout of neuronal activity. This, in turn, causes modifications in the brain activity, thus closing the feedback loop^1,2. Such closed-loop experiments provide multiple benefits over standard open-loop setups, especially when combined with optogenetics, which allows researchers to target a specific subset of neurons. For example, Siegle and Wilson used closed-loop manipulations to study the role of theta oscillations in information processing³. They demonstrated that stimulating hippocampal neurons on the falling phase of theta oscillations had different effects on behavior than applying the same stimulation on the rising phase. Closed-loop experiments are also becoming increasingly important in preclinical studies. For instance, multiple epilepsy studies have shown that neuronal stimulation triggered on seizure onset is an effective approach to reduce the severity of seizures^4,5,6. Moreover, systems for automated seizure detection and the contingent delivery of therapy^7,8 showed significant benefits in epilepsy patients^9,10,11,12. Another application area with rapid advancement of closed-loop methodologies is the control of neuroprosthetics with cortical brain–machine interfaces. This is because providing instantaneous feedback to users of prosthetic devices significantly improves accuracy and capability¹³.

In recent years, several labs have developed custom systems for the simultaneous electrical recording of neuronal activity and delivery of stimuli in a closed-loop system^{14,15,16,17,18}. Although many of those setups have impressive characteristics, it is not always easy to implement them in other labs. This is because the systems often demand experienced technicians to assemble the required electronics and other necessary hardware and software components.

Therefore, in order to facilitate the adoption of closed-loop experiments in neuroscience research, this paper provides a protocol and Matlab code to convert an open-loop electrophysiological recording setup^19,20,21,22 into a closed-loop system^2,6,23. This protocol is designed to work with the Digital Lynx recording hardware, a popular laboratory system for neuronal population recordings. A typical experiment consists of the following: 1) Recording 5-20 minutes of spiking data; 2) Spike sorting to create neuronal templates; 3) Using these templates to perform online detection of neural activity patterns; and 4) Triggering stimulation or experimental events when user-specified patterns are detected.

Protokół

All procedures described here were performed under an Animal Research Protocol approved by the University of Lethbridge Animal Welfare Committee.

1. Surgery

NOTE: The surgery procedures used to implant probes for neurophysiological recordings have been presented in other publications^24,25,26. 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 to the surgery room a cage with a rat 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 stimuli²⁵.
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 future implant, and use a scalpel and cotton swab to clear the periosteum from the exposed skull²⁵.
7. Drill 4-8 holes in the skull for implantation of anchor screws (~0.5 mm) as structural support for the implant²⁵. 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 firmly attach the implant, but it should not come in contact with the surrounding soft tissue²⁵.
10. After surgery, closely monitor the animal until it has regained sufficient consciousness to maintain sternal recumbency²⁵. For the subsequent 3 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.
   1. 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.
   2. 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 it may run on separate computers that are on the same network.
   3. Install the NetComDevelopmentPackage (https://github.com/leomol/cheetah-interface/blob/master/NetComDevelopmentPackage_v3.1.0), which can be also downloaded from https://neuralynx.com/software/netcom-development-package.
2. 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 come up during the first connection.
   1. Log in to a Matlab account. Choose the licence. Choose the version. Choose the operating system.
3. 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.
   1. Open SpikeSort3D, click File | Menu | Load Spike File, and select a spike file from the folder with recorded data.
   2. Click Cluster Menu and then Autocluster using KlustaKwik, leaving the default settings and click Run.

4. Closed-loop experiment

1. Resume data acquisition in Cheetah.
2. Open Matlab.
   1. 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.
   2. Load the spike information defined on the initial recording by clicking on Load, browsing to the recording folder, and selecting one of the spiking data files (.ntt, .nse).
   3. Select one or multiple neurons that will trigger stimulation by clicking the checkbox under the plotted waveforms.
   4. Define the minimum number of neurons that will trigger stimulation by typing an integer in the “min matches” text box; and define the time window in which spikes matching different waveforms are considered co-active by typing a number in the “window” text box.
   5. Click Send to start. This will begin online triggering of events (tones as default) based on spiking activity of selected neurons.

Wyniki

Fisher-Brown Norway rats born and raised on-site were habituated to handling for two weeks prior to the experiment. A recording drive was surgically implanted, similar to methods described previously^{28,29,30,31,32,33,34}. The neuronal signals were recorded at 32 kHz with a digital acquisition...

Dyskusje

The protocol described here, shows how to use a standard neurophysiological recording system to perform closed-loop stimulation. This protocol allows neuroscientists with limited expertise in computer science to rapidly implement a variety of closed-loop experiments with little cost. Such experiments are often necessary to study causal interactions in the brain.

After preparing an animal and installing the software (Steps 1 & 2), the closed-loop experiment consists of two separate stages. ...

Materiały

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