The authors wish to thanks Ph.D. student Marta Bisio for culturing and maintaining neuronal networks over Micro-Electrode Arrays and Dr. Marina Nanni and Dr. Claudia Chiabrera from NBT-IIT for the technical assistance for the dissection and dissociation procedures. The research leading to these results has received funding from the European Union&#39;s Seventh Framework Programme (ICT-FET FP7/2007-2013, FET Young Explorers scheme) under grant agreement n&#176; 284772 BRAIN BOW (<a href="http://www.brainbowproject.eu">www.brainbowproject.eu</a>). The authors would also like to thank Silvia Chiappalone for her help in producing the graphics used in the introduction.

1. Preparation of Neuronal Culture over a MEA
<ol>
	<li>Plate neuronal cultures on MEA chips, as described 29. The description of a similar procedure is also provided 9 and in the Discussion section.</li>
	<li>Turn the MEA heating system on 5-10 min before starting the recording to minimize the thermal stress experienced by the cells: set temperature target of the temperature controller to 37 &#176;C and switch on the heating plate below the MEA itself (provided in most commercial MEA systems) and, if available, a heated cover in order to significantly reduce evaporation.</li>
	<li>Sterilize gas-permeable caps before each use in an autoclave with humid-heat (30 min, 130 &#176;C). To avoid evaporation and prevent changes in osmolarity, keep the cultures covered with caps during recording.</li>
	<li>Place the neuronal culture in the amplifier 30 min before actually starting the recording. 
	NOTE: Simply moving the cultures from the incubator to the amplifier will significantly disrupt spontaneous activity for about half an hour. During this time, culture medium temperature will stabilize.</li>
	<li>If a carbair circuit is available, start circulation of carbair (a mixture of 5% CO2 and 95% O2 + N2) for experiments longer than a couple of hours: cultures will require it in order to maintain both oxygen and pH levels stable.</li>
</ol>
2. Selection of Recording Parameters for MEA Acquisition
<ol>
	<li>Select software filter bandwidth to detect spikes (i.e., Multi-Unit Activity &#8211; MUA) 30: in the RawDataDisplay form mark the &#8216;300 Hz-3 kHz&#8217; checkbox .</li>
	<li>Start the acquisition of data: press the &#8216;Start&#8217; button in the RawDataDisplay form.</li>
	<li>Set Threshold Gain for spike detection in the RawDataDisplay to 7. 
	NOTE: Depending on the desired specificity/selectivity tradeoff and on the adopted detection algorithm, this threshold can be set between 6 and 10 times the computed standard deviation .</li>
	<li>Set Threshold Memory to 2 sec and press the &#8216;Lock&#8217; button when no spiking activity is visible on the display (both commands are in the RawDataDisplay form). Mark the &#8216;Estimate SD from median&#8217; to compute noise standard deviation from the median of the absolute value of the observed signal 31,32, if it is difficult to provide even short time windows without spiking activity. Remove the checkmark from this option after pressing the &#8216;Lock&#8217; button, as the underlying algorithm is computationally intensive and might cause the PC to lag.</li>
	<li>Turn on spike detection routine (&#8216;Spike Detection&#8217; checkbox in the RawDataDisplay form). If spike detection is already running (i.e., the checkbox is already marked), discard spikes detected up to this moment by clicking the &#8216;Reset&#8217; button in the Data Recording form.</li>
</ol>
3. Selection of MEA Electrodes to Stimulate the Neuronal Culture and Response Map Computation
<ol>
	<li>Record spontaneous activity of the neuronal cells cultured over the MEA for 30 min: save data to file by clicking the &#8216;Record&#8217; button, in the &#8216;Spikes&#8217; box of the DataRecording form, after the desired amount of time has elapsed (30 min, in this case).</li>
	<li>Identify the 10 most active channels (i.e., 10 channels with highest spike count), then select those channels in any of the MEA layouts (either in the Coding, Decoding or Connection Map forms) by dragging the mouse cursor over the desired areas. Once the channels are selected, right click anywhere on the MEA layout and select &#8216;Add to left sensory area&#8217; in the pop-up menu: these electrodes will be used to deliver electrical stimulation in step 3.5.</li>
	<li>Verify that stimulator and MEA amplifier are correctly connected: all configurations require two wires per desired stimulation channel, while an extra coaxial cable will be required to carry the synchrony signal (please refer to the manuals of the specifics instruments for connection diagrams). Proceed then to turn the stimulator on.</li>
	<li>Define stimulus parameters in the Connection Map form. All the stimulations delivered to the culture are biphasic square voltage waves. Set half-duration to 300 &#181;sec and amplitude to 1.5 Vpp33 . 
	NOTE: Stimuli large enough to reliably evoke neural responses will very likely compromise the recording of activity from the same sites. In the rest of the paper, electrodes used for stimulation delivery should not be selected for recording any relevant information.</li>
	<li>Record response to stimulation: press the Start button in the ConnectionMap form. A series of 30 stimuli, with an interval of 5 sec is, in turn, automatically delivered from each one of the selected electrodes, while responses are recorded from the remaining 59 electrodes.</li>
	<li>Compute a connection map (i.e., the probability of observing a response from an electrode within a time slot after delivery of stimulus from a different electrode) for each stimulating channel through any math software or SpyCode, an application developed in the past (and freely available upon request) to perform computations on neural data 28.</li>
	<li>From the connection maps, select best electrodes: discard all stimulating electrodes that did not evoke responses (i.e. in the time window following stimulation, firing rates are not significantly higher than during spontaneous firing).
	<ol>
		<li>Select, among the remaining electrodes, the pair with the least overleap in responses. Specifically for each stimulating electrode, compute the average spike count in each recording electrode, then compute the difference between corresponding electrodes for all stimulating electrodes pairs. Select the couple for which the sum of absolute values of response differences over all recording channels is highest.</li>
	</ol>
	</li>
	<li>Select one of these electrodes to code sensory information from the left side of the robot and the other to code readings from the right side: in order to do this, drag the mouse cursor over one electrode, right-click on the MEA layout then select &#8216;Add to left sensory area&#8217; (or &#8216;Add to right sensory area&#8217;).</li>
</ol>
4. Interfacing the Neuronal Culture with the Robot: Selection of Coding and Decoding Schemes
<ol>
	<li>Set &#8216;Coding Type&#8217; in the Coding form to Linear.</li>
	<li>Define minimum and maximum stimulation rates in the Coding form. Use the default range of 0.5-2 Hz.</li>
	<li>Set the &#8216;Jitter&#8217; parameter in the Coding form to 0.</li>
	<li>Set the decoding algorithm parameters in the Decoding Form (weight and extinction coefficient) to 1, for a moderately active culture (~1 spike/sec per channel). Define a new parameter pair if firing rates strongly and constantly deviate from this value. See discussion for the exact function of decoding parameters.</li>
	<li>Set the decoding algorithm burst parameters in the Decoding Form. Set weight to 0 (decay time is then irrelevant), unless distinction between spikes and bursts is the subject of study. 
	NOTE: The values suggested in step 4.4 will produce smooth robot movements and speeds compatible with robot reaction times for moderately active rat primary cultures. The burst parameters have exactly the same function as those described in the step 4.4, but the triggering event is the detection of a burst instead of a spike: the actual speed of each wheel is simply the sum of the contributions of detected spikes and bursts.</li>
</ol>
5. Design of Navigation Arena for the Robot
<ol>
	<li>In the Virtual Arena Designer form, choose between bounded (the boundaries of the arena are visible to the robot and impassable) or boundless (if the robot exits from one side of the arena, it will instantly re-enter from the opposite one) arena and set the arena size in pixels.
	<ol>
		<li>Do not use bounded arenas smaller than 100 x 100 pixels, in order to allow significant movement. Do not use obstacles with a radius smaller than 5 pixels as they can simply fall between the lines of sight of the robot.</li>
		<li>Keep in mind that very large arena sizes might cause computer performance to degrade: if a large arena is required, test software performance with the desired arena before starting hard-to-repeat experiments.</li>
	</ol>
	</li>
	<li>Set robot starting position either manually (click the &#8216;Manual Selection&#8217; button in the Virtual Arena Designer form, then the desired location) or by specifying the coordinates of the robot at experiment start in the &#8216;Robot Starting Position&#8217; fields.</li>
	<li>Add any number of impassable obstacles within the arena. Either place them manually in the arena after clicking the &#8216;Add Obstacle Manually&#8217; button or set number and size range.</li>
	<li>Click the &#8216;Generate Arena&#8217; button to generate the arena with the selected features. No changes will occur until this button is pressed.</li>
	<li>Save the designed arena and load the relative file, before use in an experiment, with the command buttons in the lower part of the Virtual Arena Designer form.</li>
</ol>
6. Selection of MEA Electrodes to Record Neuronal Activity from the Culture
<ol>
	<li>Select as temporary recording electrodes those on which different responses, in step 3.8, have been observed: electrodes that showed larger responses following stimulation from the &#8216;left&#8217; (or &#8216;right&#8217;) electrode will constitute the &#8216;left&#8217; (or &#8216;right&#8217;) recording electrodes. Do not use electrodes that did not show significant responses to either stimulating electrode to control the robot. From the MEA layout pop-up menu, select &#8216;Add to left motor area&#8217; (or &#8216;Add to right motor area&#8217;) to define recording electrodes.</li>
	<li>Select features to record in the Experiment Manager form. At this stage, spikes and stimulation time stamps are the only relevant information.</li>
	<li>Perform a 10 min test drive: start the robot run by clicking the Start Experiment button in the Experiment Manager form, click it again after 10 min have elapsed. 
	NOTE: Once all the parameters have been set (coding and decoding, robot arena, stimulating and recording electrodes, features to record), a file name selection will ask for recorded files name and destination, then the robot will start moving in its arena, following the defined rules. All the selected features are automatically saved in real time.</li>
	<li>Repeat steps 3.6 and 6.1 on data acquired in step 6.3, in order to select recording electrodes from data collected during an actual robot run (see Discussion for the rationale of this two-step approach).</li>
</ol>
7. Performing a Neuro-robotic Experiment
<ol>
	<li>Select in the Experiment Manager form the data to be recorded: mark the Spike, Robot and Stimuli Data checkboxes.</li>
	<li>Launch a pre-learning robot run: click the &#8216;Start Experiment&#8217; button in the Experiment Manager form. Select new file names for data files when prompted. When 30 minutes have elapsed, click the &#8216;Start Experiment&#8217; button again to stop the robot run.</li>
	<li>Switch on the learning protocol (mark the &#8216;Deliver Tetanic Stimulation After Hit&#8217; check box in the Experiment Manager form) and perform the training robot run, of the same length as the pre-training phase (i.e., 30 min). Remember to input different file names when prompted to avoid overwriting data from step 7.2.</li>
	<li>Switch off the learning protocol again (unmark the &#8216;Deliver Tetanic Stimulation After Hit&#8217; check box) and perform the post-learning robot run. Once again, remember to change file names to prevent overwriting.</li>
</ol>
8. Second Response Map Computation
<ol>
	<li>Repeat steps 3.1-3.6. Use data from these recordings to test if any changes have been induced either in the spontaneous or evoked activity patterns by the learning protocol.</li>
</ol>

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F., Sejnowski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+spike+timing+in+neocortical+neurons.">Reliability of spike timing in neocortical neurons.</a> Science. 268 (5216), 1503-1506 (1995).</li><li>Maccione, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+algorithm+for+precise+identification+of+spikes+in+extracellularly+recorded+neuronal+signals.">A novel algorithm for precise identification of spikes in extracellularly recorded neuronal signals.</a> Journal of neuroscience methods. 177 (1), 241-249 (2009).</li><li>DeMarse, T. B., Wagenaar, D. A., Blau, A. W., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurally+controlled+animat:+biological+brains+acting+with+simulated+bodies.">The neurally controlled animat: biological brains acting with simulated bodies.</a> Autonomous Robots. 11 (3), 305-310 (1023).</li><li>Bakkum, D. J., Chao, Z. C., Potter, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+electrical+stimuli+shape+behavior+of+an+embodied+cortical+network+in+a+goal-directed+learning+task.">Spatio-temporal electrical stimuli shape behavior of an embodied cortical network in a goal-directed learning task.</a> Journal of Neural Engineering. 5 (3), 310-323 (2008).</li><li>Truccolo, W., Hochberg, L. R., Donoghue, J. 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The authors declare they have no competing financial interests.

In this paper a neuro-robotic architecture based on a neural controller (i.e., a network of neurons coming from the cortex or the hippocampus of embryonic rats), bi-directionally connected to a virtual robot, is presented. The robot, which has sensors and wheels, is forced to move in a static arena with obstacles and its task consists of avoiding collisions.
The first and possibly most critical aspect of the described procedure is the preparation of the cultures themselves, as the failure rate will tend to be significant even in the best of technical conditions. A detailed description of culturing techniques is, however, outside of the scope of the present work. As a general guideline, recordings should occur when the network firing rate reaches a stable level, usually after 3 weeks in vitro. A rough indication of healthy cultures is the presence of spontaneous electrophysiological activity in several recording channels (at least 20 channels over 60 available). Such cultures are characterized by a high degree of neuronal inter-connectivity. Under such conditions, neural activity usually becomes strongly synchronized and, at times, it displays epileptiform events, with intense spiking followed by minutes-long silent periods 37. Both of these features present a problem: excessive synchronization will make it impossible to distinguish responses to stimuli from different electrodes, while cultures exhibiting epileptiform activity will respond to the first stimulus presented with a long outburst of activity, followed by a silent period, regardless of any successive stimuli delivered. Both these issues can be strongly ameliorated by the employment of patterned cultures 35, in which the neural population is split into two or more weakly interconnected sub-groups.
Another issue is that neural responses strongly depend on the distribution of incoming stimuli 38,39. In a closed-loop experiment, the delivered stimulation is a function of the sensor readings, which, in turn, are a consequence of the robot movement and therefore of the neural responses themselves. This implies that there is no easy way of establishing beforehand what responses will be observed during the actual experiment. As a result, the selection of input-output electrodes must rely on successive approximations. In the described protocol, a two-step process (namely, steps 5.5 and 6.4) is implemented to try to determine a connection map. In the first step, a regular sequence of stimulations is delivered and responses to such stimuli are used to derive a first connection map and a temporary set of recording channels. This configuration is then used to perform the test drive described in step 6.4 and select the recording channels that will be monitored during the actual experiment.
In the Results section, a representative navigation result and the immediate improvements brought about by the introduction of a learning paradigm are presented. Throughout the protocol description, several other possible experimental variants are mentioned. For instance, the two major coding systems implemented in HyBrainWare2 (linear and stochastic) where developed to investigate the exact role of temporal input variability on the neural code 38. In the linear case, instantaneous stimulation rate is a function of user-defined parameters and robot sensors recordings. In the stochastic case, each time instance has a given probability to be chosen to deliver a stimulus. Such probability is automatically computed by HyBrainWare2 so that the expected stimulation rates match that of the former case. The possibility to add jitter to linear coding provides a smooth transition between the two cases described above. In the same way, exploring different combinations of parameters in the decoding section might help shed light on the precise role of bursting in in vitro neural networks. The speed of each wheel of the robot increases proportionally to the weight parameter each time an event is detected in the corresponding output area, while the extinction coefficient indicates how much time, in seconds, one of the contributions takes to lose 50% of its value. The decay is a simple exponential. Those variants have already been taken into account into the current design of HyBrainWare2, but many more research possibilities are open if additional modifications in the software or experimental setup can be introduced.
A rather significant limitation of the protocol here described is the requirement of the developed custom software, HyBrainWare2 (freely available upon request to all interested users). This software has been designed for a set of devices (stimulator, acquisition board, MEA amplifier) of a specific model and manufacturer. While it is indeed possible to adapt it to work on different setups, the conversion will take some programming skill. Similarly, the included options cover only a limited set of all the experimental questions that could be investigated through such a setup. For example, the spike detection algorithm implemented in the presented architecture (precise time spike detection40) is completely defined by several hard-coded parameters that need to be changed if the subject of registration is very different from dissociated neural networks (e.g., cardiac cells or slices). Finally, the learning protocol consists of the delivery of a short, high frequency (2 sec of 20 Hz stimulation, each stimulus pulse is the same as those used to code sensory information) stimulation following each obstacle hit. If the robot hits an obstacle with its right side, the tetanic stimulation is delivered to the electrode that usually codes for right side information and the same holds true for left side hits. This protocol is hard-coded and cannot be changed by the user without modifying exiting software.
While the setup presented here is not the first embodied system for hybrid, closed-loop experiments 20,23,27,41,42, the ones designed in the past were focused on a single thesis supported by data from a limited number of analogous preparations. On the other hand, the described setup has been used for a large number of experiments (more than 100 cultures have been recorded since 2012) with preparations differing for modularity and origin, while the experiments themselves addressed different issues (e.g. impact of tetanic stimulation and relevance of bursting, as presented in the results). In the near future, new experimental sessions are foreseen to verify the lasting effects of both tetanic and closed-loop stimulation and the relationship between stimulation regularity and observed responses. Another issue to be addressed is the link between spontaneous and observed activity, with the idea of adding a new decoding algorithm that takes into account the history of past activity and stimulation 43.

Many features of brain function are currently impossible to replicate in an artificial system. The brain&#8217;s ability to quickly process complex sensory information and to generate, in response, precise motor commands is by itself already beyond the current state-of-the-art. But its ability to adapt to different conditions by learning from past experience makes it so vastly superior to human-developed control systems. So far, attempts to replicate or exploit this plasticity have met little success, and the comprehension of the inner workings of the brain has eluded the grasp of researchers. One of the main issues while investigating the relationship between brain and behavior is the inability to properly access all of the variables in the system: ideally, an optimal experimental setup would allow simultaneous recording and stimulation to a large numbers of neurons, long-term stability, monitoring of synapses positions and weights, and controllable bi-directional interaction with the environment. The difficulty in tracking all those variables simultaneously led to the study of the brain-behavior relationship at two very different scales: either with behaving animals, with no fine control over experimental conditions 1-7 or with small, isolated parts, such as portions of neuronal tissue, with no overall view of the system 8. In the latter case, while no devised experimental setup allows the complete monitoring of all the parameters involved in the workings of even a simple neural network, a good trade-off is provided by dissociated neurons grown over Micro-Electrode Arrays (MEAs) 9. Those devices, born at the end of the 70&#39;s 10, have several advantages over traditional electrophysiology techniques: firstly, the possibility of recording and stimulating a neural network in many different locations at once (usually 60 electrodes). Furthermore, the coupling of MEAs with cells is almost non-invasive, allowing the observation of the same network for long periods of time, up to several months 11. The physiological effects of electrical stimulation on dissociated cultures have been extensively studied thanks to those devices, revealing that many properties observed at higher scales (such as, for example, plasticity and simple memory processes 12-14) are conserved despite the loss of architecture. During culture growth, those networks start showing spontaneous activity at about 7 days in vitro (DIV) 15,16. Network activity tends to change radically with further growth; first as single spikes gather into bursts (towards the end of the second week) 17, later as it changes into a highly complex pattern of synchronized, non-periodic network bursts 18, which represents the mature state of a network. It has been suggested 19 that this synchronous behavior, somewhat similar to that observed in in vivo recordings on sleeping animals, is caused by the lack of sensory input.
A different approach attempted to gain a better understanding of information coding has been taken by performing closed-loop experiments, in which different types of signals were used to control the stimulation of the neuronal network itself 11,20-23. In these experiments, an external agent capable of interaction with the environment has been used to generate sensory information fed to the neural network, which, in turn, produced motor commands for an effector mechanism. This allowed observations of how dynamic and adaptive properties of neural systems evolved in response to induced changes in the environment.
A setup to perform &#8216;embodied neurophysiology&#8217; experiments was developed, where a wheeled sensor platform (a physical robot or its virtual model) moves about in an arena and its speed profiles are determined by the activity of a neuronal system (i.e., a population of rat neurons cultured over a MEA). The robot is characterized by the speed profiles of its two independently-controlled wheels and by the current readings of the distance sensors. The exact nature of the distance sensors is not relevant; they may be active or passive optical sensors or ultrasound sensors. Clearly, this issue does not apply in the case of virtual robots, in which sensors may be designed with any desired feature.
In the experiments herein described, the robot used is always the virtual implementation, with 6 distance sensors pointing at 30&#176;, 60&#176; and 90&#176; from the robot heading in both directions. The activity of the three left and right sensors is averaged and the activity of the biological culture is driven by the information collected by such &#8216;super-sensors&#8217; (which will just be referred to as &#8216;left&#8217; and &#8216;right&#8217; sensors in the rest of this work). The protocol described may actually be applied to the physical robot with fairly minor adjustments. The information collected by the robot (either physical or virtual) is encoded in a series of stimuli that are used to manipulate the activity of the biological neural network, which is physically separated by the robot. The stimuli themselves are all identical and thus do not code any information. What is relevant is their frequency: stimulation rate increases when the robot approaches an obstacle, with different delivery sites coding sensory information from the left and right &#8216;eyes&#8217; of the robot. The neural network will present different responses to the incoming train of stimulations: the task of the decoding algorithm is to translate the resulting network activity into commands used to control the wheels of the robot. Given a &#8216;perfect&#8217; network behavior (i.e., with reliable and totally separated responses to stimuli from different electrodes), this would result in the robot driving in its arena without hitting any obstacle. Most networks present a behavior very different from ideality, therefore a simple learning protocol is introduced: when activated, tetanic stimulation (brief spells of high-frequency stimulation, 20 Hz stimulation for 2 sec, inspired by protocols described in 24,25) following a collision with an obstacle is delivered. If the tetanic stimulation results in a local strengthening of network connectivity, this will result in a progressive increase in the navigational capabilities of the robot.
HyBrainWare2, an improved version of the custom software published in 26, is the core architecture developed to handle the control of the different devices of the system (stimulator, data acquisition, processing and visualization, robot communication or simulation). This software has been developed at our lab and is freely available on request. This software provides the interface with the data acquisition board: once the user starts data acquisition from the GUI, the software controls the acquisition board to start the sampling and A/D conversion of data coming from the recording electrodes. This data can then be recorded, displayed to screen or analyzed in real-time to detect spikes, according to the options set by the user (see Procedure section for details). Furthermore, within the software, the definition of coding (translation of sensory information into an electrical stimulation) and decoding (translation of recorded activity into motor commands for the robot) algorithms must be specified. In particular, our setup is relatively user-friendly compared to similar systems designed in the past 27, since almost all variables can be accessed by the user right before starting the actual experiment, while all the recorded information is automatically saved in a format compatible with a neural data analysis toolbox 28.
The following Procedure section describes a learning experiment on dissociated rat hippocampal cultures: all the culturing and experimental parameters are provided for this particular preparation and may need to be modified if a different biological substrate is to be used. Similarly, the described experiment takes advantage of the closed-loop architecture to investigate the learning effect of tetanic stimulation, but the architecture itself is flexible enough to be used in the study of different features of dissociated neural networks. Major variants of the proposed experiment are further explained in the Discussion section.

<table><tbody><tr><td>Stimulus Generator 4002</td><td>Multi Channel Systems</td><td></td><td></td></tr><tr><td>MEA1060-Inv-BC</td><td>Multi Channel Systems</td><td></td><td></td></tr><tr><td>TC02</td><td>Multi Channel Systems</td><td></td><td></td></tr><tr><td>NI 6255 Acquisition Card</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Microsoft Visual Studio 2008</td><td>Microsoft</td><td></td><td></td></tr><tr><td>2078P Multichannel System-National Instruments adapter board</td><td>Developed at University of Genova (Italy)</td><td></td><td></td></tr><tr><td>Matlab 2010</td><td>Mathworks</td><td></td><td></td></tr><tr><td>HyBrainWare2</td><td></td><td></td><td>&lt;a href=&quot;https://docs.google.com/forms/d/1ffdr_a5OhzuuAduiPvoLJIvR4x_fDfsKvuM6RcRPLhU/viewform&quot;&gt;HyBrainWare2: Contact Information&lt;/a&gt;</td></tr></tbody></table>

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.

The developed experimental framework allowed the testing of whether an exchange of information between a culture of neurons and the virtual realization of a physical robot is possible 34. Figure 1 shows several samples of obtained paths travelled by a virtual robot in 20 min experiments in different conditions: from left to right a proper closed-loop experiment, an &#8216;empty MEA&#8217; robot experiment (no cells are plated on the MEA for this control experiment) and a open-loop robot experiment (stimulation rate is constant instead of coding sensor information) are represented. The representative traces confirm that a bi-directional interaction between the neuronal and artificial elements is necessary in order to obtain good navigation performances of the robot. Nevertheless, the robot experiences several hits against the obstacles.
In Figure 2, the navigation performance of the robot, expressed as pixels travelled between subsequent hits, is shown in different conditions. The first two columns display the distribution of distances travelled in the control experiments mentioned above (&#8216;empty MEA&#8217; and open-loop configurations), while the third and fourth columns display the performance without and with, respectively, the delivery of tetanic stimulation following each hit against an obstacle. The introduction of the tetanic stimulation (Cf. Procedure, point 7.2) improves significantly the distance travelled between two consecutive hits, thus improving the navigation performances of the robot 35.
In Figure 3, the navigation performance of a robot with different decoding conditions is presented. To this end, a different arena configuration has been adopted. This helped to quantify the successful navigation of the robot within the arena 36: as described in step 4.1 of the procedure, the robot is presented a series of short tracks. The success rate is simply the ratio of the number of successfully crossed tracks over the number of presented tracks. In particular, during the experiments a real-time identification of burst and isolated spikes was performed. The decoding paradigms differ from one another because of the relative weights of bursts and isolated spikes (Cf. Procedure, points 3.5-3.6 and Discussion).
<img alt="Figure 1" src="/files/ftp_upload/52341/52341fig1highres.jpg" width="700" /> 
Figure 1: A neuronal network and a robot have a bi-directional exchange of information. This figure shows three representative paths travelled by the robot over 20 minutes of experiment. In particular, light green areas are free for the robot to move in, whereas dark green pixels represent impassable obstacles that the robot can perceive through its distance sensors. In each trial, the robot starts in the upper left section of the arena and travels to its final position, depicted as a large pink dot. The smaller black dots represent hits against an obstacle, while the color-coded path provides an indication of the time evolution of robot movement. The color bar on the right expresses time elapsed from the beginning of the experiment in minutes. (A) Path followed during a closed-loop experiment. (B) Path followed during an &#8216;empty&#8217; MEA experiment (no cells are cultured on the MEA surface). (C) Path followed during an open-loop experiment (stimulation rate is constant throughout the experiment). <a href="/files/ftp_upload/52341/52341fig1highres.jpg" target="_blank">Please click here to view a larger version of the figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52341/52341fig2.jpg" /> 
Figure 2: Performance results are affected by closed-loop and tetanic stimulation. This graph reports the distributions of distances covered by the robot between subsequent hits in different conditions. In particular, the first two distributions represent control experiments (&#8216;Empty&#8217; for recordings with no neurons plated on the MEA, &#8216;Open loop&#8217; (OL) for experiment with a &#8216;blind&#8217; robot). The last two columns represent the &#8216;Closed loop&#8217; condition without (CL) and with (CL + TS) the learning protocol implemented through Tetanic Stimulation (TS), being delivered to the culture following hits against an obstacle. In each box, the central horizontal segment represents the median of the distribution, the empty square the mean value, the central bar extends to the first and third quartile and the whiskers extend to the 5th and 95th percentiles. Outliers are represented as diamonds. Statistics have been performed using the Kruskall-Wallis one way analysis of variance on ranks: the Student-Newman-Keuls pairwise comparison reveals that all median values are significantly different with p &#60; 0.05.
<img alt="Figure 3" src="/files/ftp_upload/52341/52341fig3.jpg" /> 
Figure 3: Decoding influences robotic performance. The graph above represents the probability, for a given decoding algorithm, of the robot to navigate successfully through a short track in a limited amount of time. Identification of bursts and isolated spikes is performed in real time during the experiment itself. In the first case (&#8216;Spikes&#8217;) all detected spikes present the same relative weight, for the second and third distributions a weight of 0 was set for, respectively, isolated spikes and spikes belonging to a burst. The last two columns represent the results obtained in the case where all the spikes are accounted for, but with a different relative weight according to their position. In particular in the fourth column isolated spikes were given higher relative weight than burst events, while the weighting is inverted for the decoding relative to the fifth column data. In each graph, the central line represents the median of the distribution, the empty square the mean value, while the central bar extends to the first and third quartile and the whiskers extend to the 5th and 95th percentiles. Statistics have been performed using the Kruskall-Wallis one way analysis of variance on ranks. The pairwise comparison (Student-Newman-Keuls method,) reveals that significant differences (p &#60;0.05) can be observed between columns 1 and 4, 2 and 4, 2 and 5, 2 and 3.

Watch this Scientific Journal Video about Closed-loop Neuro-robotic Experiments to Test Computational Properties of Neuronal Networks at JoVE.com

Closed-loop Neuro-robotic Experiments to Test 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 ...

closed-loop-neuro-robotic-experiments-to-test-computational

Research

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Neuroscience

10.2K Views.  Istituto Italiano di Tecnologia. 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.

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

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 ...

SSVEP-based Experimental Procedure for Brain-Robot Interaction with Humanoid Robots

Brain-Robot Interaction (BRI), which provides an innovative communication pathway between human and a robotic device via brain signals, is prospective in helping the disabled in their daily lives. The overall goal of our method is to establish an SSVEP-based experimental procedure by integrating multiple software programs, such as OpenViBE, Choregraph, and Central software as well as user developed programs written in C++ and MATLAB, to enable the study of brain-robot interaction with humanoid robots.
This is achieved by first placing EEG electrodes on a human subject to measure the brain responses through an EEG data acquisition system. A user interface is used to elicit SSVEP responses and to display video feedback in the closed-loop control experiments. The second step is to record the EEG signals of first-time subjects, to analyze their SSVEP features offline, and to train the classifier for each subject. Next, the Online Signal Processor and the Robot Controller are configured for the online control of a humanoid robot. As the final step, the subject completes three specific closed-loop control experiments within different environments to evaluate the brain-robot interaction performance.
The advantage of this approach is its reliability and flexibility because it is developed by integrating multiple software programs. The results show that using this approach, the subject is capable of interacting with the humanoid robot via brain signals. This allows the mind-controlled humanoid robot to perform typical tasks that are popular in robotic research and are helpful in assisting the disabled.

The overall goal of this method is to establish an SSVEP-based experimental procedure by integrating multiple software programs to enable the study of brain-robot interaction with humanoid robots, which is prospective in assisting the sick and elderly as well as performing unsanitary or dangerous jobs.

Brain-Robot Interaction (BRI), which provides an innovative communication pathway between human and a robotic device via brain signals, is prospective in ...

Engineering

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

Designing and Implementing Nervous System Simulations on LEGO Robots

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step of the method is to develop a nervous system simulation of specific reflexive behaviors of an appropriate model organism; here we use the American Lobster. Exteroceptive reflexes mediated by decussating (crossing) neural connections can explain an animal's taxis towards or away from a stimulus as described by Braitenberg and are particularly well suited for investigation using the NXT platform.1 The nervous system simulation is programmed using LabVIEW software on the LEGO Mindstorms platform. Once the nervous system is tuned properly, behavioral experiments are run on the robot and on the animal under identical environmental conditions. By controlling the sensory milieu experienced by the specimens, differences in behavioral outputs can be observed. These differences may point to specific deficiencies in the nervous system model and serve to inform the iteration of the model for the particular behavior under study. This method allows for the experimental manipulation of electronic nervous systems and serves as a way to explore neuroscience hypotheses specifically regarding the neurophysiological basis of simple innate reflexive behaviors. The LEGO Mindstorms NXT kit provides an affordable and efficient platform on which to test preliminary biomimetic robot control schemes. The approach is also well suited for the high school classroom to serve as the foundation for a hands-on inquiry-based biorobotics curriculum.

An approach to neural network modeling on the LEGO Mindstorms robotics platform is presented. The method provides a simulation tool for invertebrate neuroscience research in both the research lab and the classroom. This technique enables the investigation of biomimetic robot control principles.

We present a method to use the commercially available LEGO Mindstorms NXT robotics platform to test systems level neuroscience hypotheses. The first step ...

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 ...

Design, Surface Treatment, Cellular Plating, and Culturing of Modular Neuronal Networks Composed of Functionally Inter-connected Circuits

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire of dynamical motifs, which underlie most diverse brain functions, can emerge out of a fixed topological and modular organization of brain circuits. Compared to in vivo studies of neuronal circuits which present intrinsic experimental difficulties, in vitro preparations offer a much larger possibility to manipulate and probe the structural, dynamical and chemical properties of experimental neuronal systems. This work describes an in vitro experimental methodology which allows growing of modular networks composed by spatially distinct, functionally interconnected neuronal assemblies. The protocol allows controlling the two-dimensional (2D) architecture of the neuronal network at different levels of topological complexity.
A desired network patterning can be achieved both on regular cover slips and substrate embedded micro electrode arrays. Micromachined structures are embossed on a silicon wafer and used to create biocompatible polymeric stencils, which incorporate the negative features of the desired network architecture. The stencils are placed on the culturing substrates during the surface coating procedure with a molecular layer for promoting cellular adhesion. After removal of the stencils, neurons are plated and they spontaneously redirected to the coated areas. By decreasing the inter-compartment distance, it is possible to obtain either isolated or interconnected neuronal circuits. To promote cell survival, cells are co-cultured with a supporting neuronal network which is located at the periphery of the culture dish. Electrophysiological and optical recordings of the activity of modular networks obtained respectively by using substrate embedded micro electrode arrays and calcium imaging are presented. While each module shows spontaneous global synchronizations, the occurrence of inter-module synchronization is regulated by the density of connection among the circuits.

This manuscript describes a protocol to grow in vitro modular networks consisting of spatially confined, functionally inter-connected neuronal circuits. A polymeric mask is used to pattern a protein layer to promote cellular adhesion over the culturing substrate. Plated neurons grow on coated areas establishing spontaneous connections and exhibiting electrophysiological activity.

The brain operates through the coordinated activation and the dynamic communication of neuronal assemblies. A major open question is how a vast repertoire ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

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.

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 ...

Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons to entire brain circuits. However, neural responses may vary due to prior experience, internal states, or stochastic factors, thus generating the need for methods that can assess neural function across many individuals at once. Whereas most recording techniques examine a single animal at a time, we describe the use of wide-field microscopy to scale up neuronal recordings to dozens of Caenorhabditis elegans or other sub-millimeter-scale organisms at once. Open-source hardware and software allow great flexibility in programming fully automated experiments that control the intensity and timing of various stimulus types, including chemical, optical, mechanical, thermal, and electromagnetic stimuli. In particular, microfluidic flow devices provide precise, repeatable, and quantitative control of chemosensory stimuli with sub-second time resolution. The NeuroTracker semi-automated data analysis pipeline then extracts individual and population-wide neural responses to uncover functional changes in neural excitability and dynamics. This paper presents examples of measuring neuronal adaptation, temporal inhibition, and stimulus crosstalk. These techniques increase the precision and repeatability of stimulation, allow the exploration of population variability, and are generalizable to other dynamic fluorescent signals in small biosystems from cells and organoids to whole organisms and plants.

We present a method for the flexible chemical and multimodal stimulation and recording of simultaneous neural activity from many Caenorhabditis elegans worms. This method uses microfluidics, open-source hardware and software, and supervised automated data analysis to enable the measurement of neuronal phenomena such as adaptation, temporal inhibition, and stimulus crosstalk.

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons ...

Multimodal Large-Scale Neural Recordings and Analysis on HD-MEA 

Recording and Analyzing Multimodal Large-Scale Neuronal Ensemble Dynamics on CMOS-Integrated High-Density Microelectrode Array

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from patterns of spatiotemporal neuronal activity. These dynamic patterns emerging from functional groups of interconnected neuronal ensembles facilitate precise computations for processing and coding multiscale neural information, thereby driving higher brain functions. To probe the computational principles of neural dynamics underlying this complexity and investigate the multiscale impact of biological processes in health and disease, large-scale simultaneous recordings have become instrumental. Here, a high-density microelectrode array (HD-MEA) is employed to study two modalities of neural dynamics - hippocampal and olfactory bulb circuits from ex-vivo mouse brain slices and neuronal networks from in-vitro cell cultures of human induced pluripotent stem cells (iPSCs). The HD-MEA platform, with 4096 microelectrodes, enables non-invasive, multi-site, label-free recordings of extracellular firing patterns from thousands of neuronal ensembles simultaneously at high spatiotemporal resolution. This approach allows the characterization of several electrophysiological network-wide features, including single/-multi-unit spiking activity patterns and local field potential oscillations. To scrutinize these multidimensional neural data, we have developed several computational tools incorporating machine learning algorithms, automatic event detection and classification, graph theory, and other advanced analyses. By supplementing these computational pipelines with this platform, we provide a methodology for studying the large, multiscale, and multimodal dynamics from cell assemblies to networks. This can potentially advance our understanding of complex brain functions and cognitive processes in health and disease. Commitment to open science and insights into large-scale computational neural dynamics could enhance brain-inspired modeling, neuromorphic computing, and neural learning algorithms. Furthermore, understanding the underlying mechanisms of impaired large-scale neural computations and their interconnected microcircuit dynamics could lead to the identification of specific biomarkers, paving the way for more accurate diagnostic tools and targeted therapies for neurological disorders.

Author Spotlight: Advancing Large-Scale Neural Dynamics Through HD-MEA Technology

Here, we employ HD-MEA to delve into computational dynamics of large-scale neuronal ensembles, particularly in hippocampal, olfactory bulb circuits, and human neuronal networks. Capturing spatiotemporal activity, combined with computational tools, provides insights into neuronal ensemble complexity. The method enhances understanding of brain functions, potentially identifying biomarkers and treatments for neurological disorders.

Large-scale neuronal networks and their complex distributed microcircuits are essential to generate perception, cognition, and behavior that emerge from ...