This work was supported by NSERC Discovery grants to AL and AG.

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 publications24,25,26. 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.
<ol>
	<li>Bring to the surgery room a cage with a rat to be implanted with a silicone probe or electrode array to record neuronal activity.</li>
	<li>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.</li>
	<li>Apply an eye ointment to minimize dryness during the surgery.</li>
	<li>Shave the surgical area and disinfect the skin with 2% chlorhexidine solution and 70% isopropyl alcohol.</li>
	<li>Inject lidocaine (5 mg/kg) under the scalp over the brain area where electrodes will be implanted.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Software installation
NOTE: This was tested on Windows 10, 64 bit version.
<ol>
	<li>Install data acquisition and processing software.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Log in to a Matlab account. Choose the licence. Choose the version. Choose the operating system.</li>
	</ol>
	</li>
	<li>Download the following library for online event triggering from: https://github.com/leomol/cheetah-interface and extract files to the computer&#8217;s &#8216;Documents/Matlab&#8217; folder. A copy of the code is provided in the accompanying Supplemental Materials.</li>
</ol>
3. Initial data acquisition
<ol>
	<li>Start data acquisition using Cheetah software.</li>
	<li>Record a few minutes of spiking data to populate template waveforms.</li>
	<li>Stop the data acquisition and perform spike sorting on the recorded data.
	<ol>
		<li>Open SpikeSort3D, click File | Menu | Load Spike File, and select a spike file from the folder with recorded data.</li>
		<li>Click Cluster Menu and then Autocluster using KlustaKwik, leaving the default settings and click Run.</li>
	</ol>
	</li>
</ol>
4. Closed-loop experiment
<ol>
	<li>Resume data acquisition in Cheetah.</li>
	<li>Open Matlab.
	<ol>
		<li>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(&#8216;-callback&#8217;, customFunction) instead, where customFunction is a handle to that function.</li>
		<li>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).</li>
		<li>Select one or multiple neurons that will trigger stimulation by clicking the checkbox under the plotted waveforms.</li>
		<li>Define the minimum number of neurons that will trigger stimulation by typing an integer in the &#8220;min matches&#8221; text box; and define the time window in which spikes matching different waveforms are considered co-active by typing a number in the &#8220;window&#8221; text box.</li>
		<li>Click Send to start. This will begin online triggering of events (tones as default) based on spiking activity of selected neurons.</li>
	</ol>
	</li>
</ol>

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Authors do not have any conflict of interests related to this work.

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 &amp; 2), the closed-loop experiment consists of two separate stages. ...

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 loop1,2. 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 seizures4,5,6. Moreover, systems for automated seizure detection and the contingent delivery of therapy7,8 showed significant benefits in epilepsy patients9,10,11,12. Another application area with rapid advancement of closed-loop methodologies is the control of neuroprosthetics with cortical brain&#8211;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 system14,15,16,17,18. 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 setup19,20,21,22 into a closed-loop system2,6,23. 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.

<table>
	<tbody>
		<tr>
			<td>Baytril</td>
			<td>Bayer, Mississauga, CA</td>
			<td>DIN 02169428</td>
			<td>antibiotic; 50 mg/mL</td>
		</tr>
		<tr>
			<td>Cheetah 6.4</td>
			<td>NeuraLynx, Tucson, AZ</td>
			<td>6.4.0.beta</td>
			<td>Software interfaces for data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Digital Lynx 4SX</td>
			<td>NeuraLynx, Tucson, AZ</td>
			<td>4SX</td>
			<td>recording equipment</td>
		</tr>
		<tr>
			<td>Headstage transmitter</td>
			<td>TBSI</td>
			<td>B10-3163-GK</td>
			<td>transmits the neural signal to the receiver</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Fresenius Kabi, Toronto, CA</td>
			<td>DIN 02237518</td>
			<td>inhalation anesthetic</td>
		</tr>
		<tr>
			<td>Jet Denture Powder &#38; Liqud</td>
			<td>Lang Dental, Wheeling, US</td>
			<td>1230</td>
			<td>dental acrylic</td>
		</tr>
		<tr>
			<td>Lacri-Lube</td>
			<td>Allergan, Markham, CA</td>
			<td>DIN 00210889</td>
			<td>eye ointment</td>
		</tr>
		<tr>
			<td>Lido-2</td>
			<td>Rafter 8, Calgary</td>
			<td>DIN 00654639</td>
			<td>local anesthetic; 20 mg/mL</td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td>R2018b</td>
			<td>software for signal processing and triggering external events</td>
		</tr>
		<tr>
			<td>Metacam</td>
			<td>Boehringer, Ingelheim, DE</td>
			<td>DIN 02240463</td>
			<td>analgesic; 5 mg/mL</td>
		</tr>
		<tr>
			<td>Netcom</td>
			<td>NeuraLynx</td>
			<td>v1</td>
			<td>Application Programming Interface (API) that communicates with Cheetah</td>
		</tr>
		<tr>
			<td>Silicone probe</td>
			<td>Cambridge Neurotech</td>
			<td>ASSY-156-DBC2</td>
			<td>implanted device</td>
		</tr>
		<tr>
			<td>SpikeSort 3D&#160;</td>
			<td>NeuraLynx, Tucson, AZ</td>
			<td>SS3D</td>
			<td>spike waveform-to-cell classification tools</td>
		</tr>
		<tr>
			<td>Wireless Radio Receiver</td>
			<td>TBSI</td>
			<td>911-1062-00</td>
			<td>transmits the neural signal to the Digital Lynx</td>
		</tr>
	</tbody>
</table>

using neuron spiking activity to trigger closed-loop stimuli in neurophysiological experiments

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.

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 previously28,29,30,31,32,33,34. The neuronal signals were recorded at 32 kHz with a digital acquisition...

Watch this Scientific Journal Video about Using Neuron Spiking Activity to Trigger Closed-Loop Stimuli in Neurophysiological Experiments at JoVE.com

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

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.

Closed-loop neurophysiological systems use patterns of neuronal activity to trigger stimuli, which in turn affect brain activity. Such closed-loop systems ...

using-neuron-spiking-activity-to-trigger-closed-loop-stimuli

Research

JoVE Journal

Neuroscience

6.9K Views.  University of Lethbridge. 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.

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

An Experimental Platform to Study the Closed-loop Performance of Brain-machine Interfaces

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine interface to assess the robustness of different control-laws applied to a closed-loop image stabilization task. Taking advantage of the well-characterized fly visuomotor pathway we record the electrical activity from an identified, motion-sensitive neuron, H1, to control the yaw rotation of a two-wheeled robot. The robot is equipped with 2 high-speed video cameras providing visual motion input to a fly placed in front of 2 CRT computer monitors. The activity of the H1 neuron indicates the direction and relative speed of the robot's rotation. The neural activity is filtered and fed back into the steering system of the robot by means of proportional and proportional/adaptive control. Our goal is to test and optimize the performance of various control laws under closed-loop conditions for a broader application also in other brain machine interfaces.

We use a closed-loop fly-machine interface to investigate general principles in neuronal control.

The non-stationary nature and variability of neuronal signals is a fundamental problem in brain-machine interfacing. We developed a brain-machine ...

Preterm EEG: A Multimodal Neurophysiological Protocol

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and monitoring of brain function in preterm and term babies. Most common indications are the diagnosis of epileptic seizures, assessment of brain maturity, and recovery from hypoxic-ischemic events. EEG recording techniques and the understanding of neonatal EEG signals have dramatically improved, but these advances have been slow to penetrate through the clinical traditions. The aim of this presentation is to bring theory and practice of advanced EEG recording available for neonatal units. 
In the theoretical part, we will present animations to illustrate how a preterm brain gives rise to spontaneous and evoked EEG activities, both of which are unique to this developmental phase, as well as crucial for a proper brain maturation. Recent animal work has shown that the structural brain development is clearly reflected in early EEG activity. Most important structures in this regard are the growing long range connections and the transient cortical structure, subplate. Sensory stimuli in a preterm baby will generate responses that are seen at a single trial level, and they have underpinnings in the subplate-cortex interaction. This brings neonatal EEG readily into a multimodal study, where EEG is not only recording cortical function, but it also tests subplate function via different sensory modalities. Finally, introduction of clinically suitable dense array EEG caps, as well as amplifiers capable of recording low frequencies, have disclosed multitude of brain activities that have as yet been overlooked.
In the practical part of this video, we show how a multimodal, dense array EEG study is performed in neonatal intensive care unit from a preterm baby in the incubator. The video demonstrates preparation of the baby and incubator, application of the EEG cap, and performance of the sensory stimulations.

This video explains the background theory of the neonatal EEG activity and the sensory responses, followed by a live demonstration of their recording in neonatal intensive care unit.

Since its introduction in early 1950s, electroencephalography (EEG) has been widely used in the neonatal intensive care units (NICU) for assessment and ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation of a given stimulus might be dependent on context and history. If this is actually the case, bi-directional interactions between the brain (or if need be a reduced model of it) and sensory-motor system can shed a light on how encoding and decoding of information is performed. Here an experimental system is introduced and described in which the activity of a neuronal element (i.e., a network of neurons extracted from embryonic mammalian hippocampi) is given context and used to control the movement of an artificial agent, while environmental information is fed back to the culture as a sequence of electrical stimuli. This architecture allows a quick selection of diverse encoding, decoding, and learning algorithms to test different hypotheses on the computational properties of neuronal networks.

In this paper, an experimental framework to perform closed-loop experiments is presented, in which information processing (i.e., coding and decoding) and learning of neuronal assemblies are studied during the continuous interaction with a robotic body.

Information coding in the Central Nervous System (CNS) remains unexplored. There is mounting evidence that, even at a very low level, the representation ...

Using Single Sensillum Recording to Detect Olfactory Neuron Responses of Bed Bugs to Semiochemicals

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house single or multiple neurons inside, are considered to make the major contribution in responding to the chemical stimuli. By directly recording action potential in the olfactory sensillum after exposure to stimuli, single sensillum recording (SSR) technique provides a powerful approach for investigating the neural responses of insects to chemical stimuli. For the bed bug, which is a notorious human parasite, multiple types of olfactory sensillum have been characterized. In this study, we demonstrated neural responses of bed bug olfactory sensilla to two chemical stimuli and the dose-dependent responses to one of them using the SSR method. This approach enables researchers to conduct early screening for individual chemical stimuli on the bed bug olfactory sensilla, which would provide valuable information for the development of new bed bug attractants or repellents and benefits the bed bug control efforts.

Bed bugs rely on olfactory receptor neurons housed in their antennal olfactory sensilla to detect semiochemicals in the environment. Utilizing single sensillum recording, we demonstrate a method to evaluate bed bug response to semiochemicals and explore the coding process involved.

The insect olfactory system plays an important role in detecting semiochemicals in the environment. In particular, the antennal sensilla which house ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

Conducting Hyperscanning Experiments with Functional Near-Infrared Spectroscopy

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding of the neurobiological underpinnings of social interactions, and possibly interpersonal relationships. Functional near-infrared spectroscopy (fNIRS) is well suited for conducting hyperscanning experiments because it measures local hemodynamic effects with a high sampling rate and, importantly, it can be applied in natural settings, not requiring strict motion restrictions. In this article, we present a protocol for conducting fNIRS hyperscanning experiments with parent-child dyads and for analyzing brain-to-brain synchrony. Furthermore, we discuss critical issues and future directions, regarding the experimental design, spatial registration of the fNIRS channels, physiological influences and data analysis methods. The described protocol is not specific to parent-child dyads, but can be applied to a variety of different dyadic constellations, such as adult strangers, romantic partners or siblings. To conclude, fNIRS hyperscanning has the potential to yield new insights into the dynamics of the ongoing social interaction, which possibly go beyond what can be studied by examining the activities of individual brains.

The present protocol describes how to conduct fNIRS hyperscanning experiments and analyze brain-to-brain synchrony. Further, we discuss challenges and possible solutions.

Concurrent brain recordings of two or more interacting persons, an approach termed hyperscanning, are gaining increasing importance for our understanding ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...