We thank Dr. Chris Rogers (Tufts University) for programming and manuscript suggestions. We thank Alex Giuliano and Deborah Lee for video production support. 
Funding provided by an NSF Graduate Research Fellowship and an ONR MURI in Synthetic Biology.

1. Building the Robot Model 
<ol>
 <li>Choose a model organism to study that is well represented in the neuroethological literature. Invertebrates generally make good candidates because their relatively simple nervous systems have been well studied and are primarily comprised of innate reflexes. We will demonstrate this approach using the American Lobster, Homarus americanus. </li>
 <li>Select well-studied reflexive behaviors for modeling purposes. We have chosen the lobster's responses to antennal bend and to claw bump detection as the animal reflexively responds to bending of antennae to elicit rheotaxis (orientation to water flow) while claw contacts mediate obstacle avoidance. Simple reflexes relying on decussating neural connections from bilaterally symmetrical sensors are well suited for this type of study1.</li>
 <li>Build or choose a robotic platform with appropriate sensors for the selected behaviors in order to run the neural network simulation. While a dedicated robot can be constructed such as RoboLobster12 or the Sprawl (cockroach) robot family13, here we use a commercially available kit to expedite the experimental process. The LEGO Mindstorms NXT 2.0 kit, providing templates, modular components and established sensors, allows for the rapid physical construction of the robot. </li>
 <li>Select or build sensors to match the modalities of the behaviors selected in step 1.2. Premade sensors from the LEGO Mindstorms kit can be used or homebrew sensors can be engineered14,15. We have selected the included LEGO touch sensor and a custom antennal bend sensor consisting of a FlexPoint bend sensor spliced to an NXT connector. To build a custom resistive connector, cut an NXT connector wire and solder the black and white cables to the leads of the sensor. </li>
 <li>Use previously published neuroethological studies to find or develop a hypothetical neural network for modeling purposes. The literature has proposed various neural networks to explain behavior in various model organisms, from lobsters16 to lamprey17. Functional neural units should be identified and their synaptic connections theorized. Novel neuroethological experiments can also be developed and implemented if laboratory circumstances allow.
 </li>
</ol>
2. Programming the Nervous System
<ol>
 <li>Use LabVIEW software along with the LabVIEW Module for LEGO MINDSTORMS NXT to create Virtual Instruments (VIs) that run equations for a mathematical neuron and synapse model. While any published model can be used, we recommend the discrete time map-based (DTM) model18. This allows for computationally efficient real time operation while maintaining a variety of neuronal firing output regimes. Two control parameters, alpha and sigma, determine the dynamics of the simulated neuron to produce varied neural output including tonic spiking, chaotic firing, bursting and intrinsic silence. LabVIEW VIs for LEGO Mindstorms for this neuron model can be found online here: <a href="http://myfiles.neu.edu/blustein.d/neurobots" target="_blank">http://myfiles.neu.edu/blustein.d/neurobots</a></li>
 <li>Place VIs into a While Loop so that the network will update iteratively. </li>
 <li>Connect presynaptic neuron spike outputs through synapses to postsynaptic neurons. </li>
 <li>Pass iteratively updating parameters through shift registers to the next calculation cycle. The DTM model updates fast (x) and slow (y) dynamical variables that are passed to the next iteration of calculations. </li>
 <li>Set synapse parameters to define network characteristics. For each synapse, the synaptic strength (gsyn), the relaxation rate (gamma), and the reversal potential (xrp) are coefficients in the DTM model equations that determine synaptic behavior. The value of gsyn is a multiplier that determines the magnitude of the synaptic current effect from a spike event, ranging from 0.0 (no effect) to 1.0 (100% effect). The value of gamma is a multiplier that determines the rate of decay of the synaptic current regardless of spike events, ranging from 0.0 (complete decay) to 1.0 (no decay). The value of xrp determines the level of excitation or inhibition of the synapse, values typically range from -3.0 to 3.0 with positive values representing excitation and negative values representing inhibition. Values can be tuned for your specific network characteristics. Here we use the following settings: gamma= 0.95, gsyn= 0.60 and xrp = 2.2 (excitatory synapse) or -1.8 (inhibitory synapse). </li>
 <li>Adjust alpha and sigma control parameters to modify baseline neuron activity. The dynamics of the model for varied control parameters are described in detail elsewhere18 but typically, alpha &lt; 4 produces intrinsically silent neural activity for low sigma values or tonic spiking for high sigma values. The transition between 'low' and 'high' sigma values generally occurs between -1.0 and 1.0 depending on the alpha value. Bursting neuron activity is produced for alpha &gt; 4 when sigma values are near zero. Here we use alpha = 4.05 and sigma = -3.10. </li>
 <li>Use LabVIEW VIs to input sensor information into your network. For provided sensors, the VIs are already available within the Mindstorms LabVIEW module. For custom resistive sensors, use the Light Sensor VI as a programming framework. Information about creating homebrew sensors can be found elsewhere14.</li>
 <li>Insert graphical charts on the front panel of the LabVIEW program to visualize neuronal activity. </li>
 <li>Temporarily replace sensor inputs with front panel control boxes in order to manually manipulate the sensor information going to the neural network. </li>
 <li>Run the neuronal network and manually adjust the front panel controls to present varied sensory input. Confirm that the network qualitatively functions as predicted for known conditions. </li>
 <li>Adjust the network as needed by changing the neuron and synapse parameters. First attempts at tuning the network should be done by changing synaptic strength (gsyn) of the various synapses. Other parameters may need to be adjusted as well. </li>
 <li>Once the network functions with qualitative accuracy, replace the front panel control boxes with the sensor input VIs. </li>
</ol>
3. Nervous System Simulation Testing
<ol>
 <li>Set up a controlled environment in which to observe the animal and the robot under similar conditions. We use a rectangular tank containing rock obstacles (Figure 1). The tank can be emptied to accommodate the LEGO robot in the same space. </li>
 <li>Mount a video camera overhead to record the animal/robot behavior. Make sure the contrast between the subject and the background is high to allow for automatic tracking. We painted the bottom of the test tank white. </li>
 <li>Place the animal in the controlled environment and video record the behavior. Be sure to limit the sensory capabilities of the lobster to match those of the robot. Here we restrict the lobster's sensory capabilities by covering the eyes with an aluminum foil mask and deafferenting chemoreceptors with freshwater19. </li>
 <li>Adjust the environment to suit a LEGO robot (removing water if necessary) and run the robot in the same controlled environment.</li>
 <li>Use a MATLAB script to automatically track the markers on the specimens. We use MouseLabTracker20. </li>
 <li>Create and compare vector plots of the animal and robot movements. </li>
 <li>Modify the nervous system and repeat steps 3.3-3.6 to observe how different aspects of the electronic nervous system affects the robot's behavior. Here we adjust the synaptic strength between the claw bump sensor neurons and the motor system and monitor robot performance compared to that of a lobster. Alternatively, the sensory conditions in the test arena can be modified and the effects observed in robot and animal. For example, the arena described here could be subjected to different water flow rates.</li>
</ol>

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crayfish+Swimming:+Alternating+Motor+Output+and+Giant+Fiber+Activity.">Crayfish Swimming: Alternating Motor Output and Giant Fiber Activity.</a> Science. 169 (3946), 698-700 (1970).</li><li>Kanz, J. E., Eberly, L. B., Cobbs, J. S., Pinsker, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+correlates+of+siphon+withdrawal+in+freely+behaving+Aplysia.">Neuronal correlates of siphon withdrawal in freely behaving Aplysia.</a> J. Neurophysiol. 42 (6), 1538-1556 (1979).</li><li>Webb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validating+biorobotic+models.">Validating biorobotic models.</a> Journal of Neural Engineering. 3, 1-20 (2006).</li><li>Wessnitzer, J., Asthenidis, A., Petrou, G., Webb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cricket-controlled+robot+orienting+towards+a+sound+source.">A cricket-controlled robot orienting towards a sound source.</a> Proceedings of the 12th Annual conference on Towards autonomous robotic systems. , 1-12 (2011).</li><li>Grasso, F., Atema, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+flow+and+chemical+sensing+for+guidance+of+autonomous+marine+robots+in+turbulent+flows.">Integration of flow and chemical sensing for guidance of autonomous marine robots in turbulent flows.</a> Environ. 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The authors declare that they have no competing financial interests.

When initiating biorobotic nervous system simulation experiments, there are a few important guidelines to follow. Choosing the right model organism is critical: pick an organism that is easy to obtain and maintain. Invertebrates are ideal because they do not usually require institutional approval for experimentation and their husbandry needs are often less demanding than those of vertebrates. From a scientific perspective, it is beneficial to choose an animal that has an established neuroethological literature record fro...

Neurophysiological investigations over the past 100 years have broadened tremendously our knowledge of nervous system structure and function. However, the majority of nervous system research to date has relied on the use of isolated preparations or restrained subjects. While there have been many successful efforts to record neural activity from freely behaving animals2-5, the biorobotic approach provides a valuable tool to allow for nervous system manipulation in order to test systems level neuroscience hypotheses6. Simulated nervous systems operating on robots can be experimentally manipulated and allow for the extension of software modeling to the physical world. This approach has been well implemented in the academic world7,8 but the process of building a biomimetic robot for hypothesis testing can be expensive and time consuming. We present a method to execute the biorobotic approach using a commercially available robotics kit (LEGO Mindstorms NXT 2.0). The goal of this method is to provide a rapid and efficient way to test systems level neuroscience hypotheses on robotic9 or bio-hybrid10 embodied neural network simulations. Accelerating the process from hypothesis to experiment improves research productivity. The simple LEGO Mindstorms platform provides a test bed for biomimetic sensors and neural networks which we demonstrate using the American Lobster (Homarus americanus) as a model organism. The method also provides a powerful hands-on educational tool in the classroom as students can design and manipulate nervous systems for their own robots11.

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.

Inputs from a lobster's claws into its nervous system mediate obstacle negotiation in a novel environment. Figure 1 shows a screenshot of the video used to analyze the behavior of a LEGO robot (Figure 1A) and a lobster (Figure 1B) in the test arena. The test arena was unmodified between animal and robot tests except that the water was emptied from the tank for the robot trials.
Video tracking results are displayed in Figure 2....

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Designing and Implementing Nervous System Simulations on LEGO Robots

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 ...

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15.0K Views.  Northeastern University. 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.

Video: Designing and Implementing Nervous System Simulations on LEGO Robots

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca2+) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca2+ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca2+ indicator such as Fluo-4 permits measurements of Ca2+ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca2+ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca2+ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca2+ responses in enteric neurons and glial cells.

In Situ Ca2+ Imaging of the Enteric Nervous System

The enteric nervous system (ENS) is a network of neurons and glia located in the gut wall that controls intestinal reflexes. This protocol describes methods for recording the activity of enteric neurons and glia in live preparations of ENS using Ca2+ imaging.

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ...

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique non-linear deformations, dramatically hampering one's ability to evaluate cellular morphology, distribution and connectivity in the central nervous system (CNS). However, optical clearing techniques are changing the way tissues are visualized. These approaches permit one to probe deeply into intact organ preparations, providing tremendous insight into the structural organization of tissues in health and disease. Techniques such as Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) achieve this goal by providing a matrix that binds important biomolecules while permitting light-scattering lipids to freely diffuse out. Lipid removal, followed by refractive index matching, renders the tissue transparent and readily imaged in 3 dimensions (3D). Nevertheless, the electrophoretic tissue clearing (ETC) used in the original CLARITY protocol can be challenging to implement successfully and the use of a proprietary refraction index matching solution makes it expensive to use the technique routinely. This report demonstrates the implementation of a simple and inexpensive optical clearing protocol that combines passive CLARITY for improved tissue integrity and 2,2&#8242;-thiodiethanol (TDE), a previously described refractive index matching solution.

Optical clearing techniques are revolutionizing the way tissues are visualized. In this report we describe modifications of the original Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) protocol that yields more consistent and less expensive results.

Traditionally, tissue visualization has required that the tissue of interest be serially sectioned and imaged, subjecting each tissue section to unique ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

Designing and Implementing Nervous System Simulations on LEGO Robots

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Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Neural Circuit Recording from an Intact Cockroach Nervous System

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

In Situ Ca²⁺ Imaging of the Enteric Nervous System

Optical Clearing of the Mouse Central Nervous System Using Passive CLARITY

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains