Developing and Implementing Low-Cost Remote Laboratories for Undergraduate Biology and Neuroscience Courses.

The global pandemic caused by the novel coronavirus (SARS-COV-2) has forced many universities to abruptly change the delivery of courses from in-person to online. This change to remote learning requires creating new ways to deliver lectures, exams, and discussion groups through online meeting platforms. An often-overlooked challenge is performing lab courses that require access to specialized equipment and resources typically found in the undergraduate laboratory classrooms. Here we discuss some strategies for developing and implementing a full semester neuroscience laboratory course that allows students to fully participate in laboratory exercises at home or in their dorm rooms. Performing lab exercises remotely and independently was shown to significantly improve participant's self-efficacy and confidence that they can learn complex neuroscience material, when compared to participants who passively watch experiments online. We review best practices to ensure that lessons can be successfully demonstrated by the instructor and carried out by all students. Finally, we discuss the need to provide a level playing field such that all students may succeed, regardless of their current technology resources at home.

An Electrophysiological Investigation of Power-Amplification in the Ballistic Mantis Shrimp Punch.

Mantis shrimp are aggressive, burrowing crustaceans that hunt using one the fastest movements in the natural world. These stomatopods can crack the calcified shells of prey or spear down unsuspecting fish with lighting speed. Their strike makes use of power-amplification mechanisms to move their limbs much faster than is possible by muscles alone. Other arthropods such as crickets and grasshoppers also use power-amplified kicks that allow these animals to rapidly jump away from predator threats. Here we present a template laboratory exercise for studying the electrophysiology of power-amplified limb movement in arthropods, with a specific focus on mantis shrimp strikes. The exercise is designed in such a way that it can be applied to other species that perform power-amplified limb movements (e.g., house crickets, Acheta domesticus) and species that do not (e.g., cockroaches, Blaberus discoidalis). Students learn to handle the animals, make and implant electromyogram (EMG) probes, and finally perform experiments. This integrative approach introduces the concept of power-amplified neuromuscular control; allows students to develop scientific methods, and conveys high-level insights into behavior, and convergent evolution, the process by which different species evolve similar traits. Our power-amplification laboratory exercise involves a non-terminal preparation which allows electrophysiological recordings across multiple days from arthropods using a low-cost EMG amplifier. Students learn the principles of electrophysiology by fabricating their own electrode system and performing implant surgeries. Students then present behaviorally-relevant stimuli that generate attack strikes in the animals during the electrophysiology experiments to get insight into the underlying mechanisms of power amplification. Analyses of the EMG data (spike train burst duration, firing rate, and spike amplitude) allow students to compare mantis shrimp with other power-amplifying species, as well as a non-power-amplifying one. The major learning goal of this exercise is to empower students by providing an experience to develop their own setup to examine a complex biological principle. By contrasting power-amplifiers with non-power-amplifiers, these analyses highlight the peculiarity of power amplification at multiple levels of analysis, from behavior to physiology. Our comparative design requires students to consider the behavioral function of the movement in different species alongside the neuromuscular underpinnings of each movement. This laboratory exercise allows students to develop methodology, problem-solving and inquisitive skills crucial for pursuing science.

Correction: open labware: 3-d printing your own lab equipment.

[This corrects the article DOI: 10.1371/journal.pbio.1002086.].

Open Labware: 3-D printing your own lab equipment.

The introduction of affordable, consumer-oriented 3-D printers is a milestone in the current "maker movement," which has been heralded as the next industrial revolution. Combined with free and open sharing of detailed design blueprints and accessible development tools, rapid prototypes of complex products can now be assembled in one's own garage--a game-changer reminiscent of the early days of personal computing. At the same time, 3-D printing has also allowed the scientific and engineering community to build the "little things" that help a lab get up and running much faster and easier than ever before.

Grasshopper DCMD: An Undergraduate Electrophysiology Lab for Investigating Single-Unit Responses to Behaviorally-Relevant Stimuli.

Avoiding capture from a fast-approaching predator is an important survival skill shared by many animals. Investigating the neural circuits that give rise to this escape behavior can provide a tractable demonstration of systems-level neuroscience research for undergraduate laboratories. In this paper, we describe three related hands-on exercises using the grasshopper and affordable technology to bring neurophysiology, neuroethology, and neural computation to life and enhance student understanding and interest. We simplified a looming stimuli procedure using the Backyard Brains SpikerBox bioamplifier, an open-source and low-cost electrophysiology rig, to extracellularly record activity of the descending contralateral movement detector (DCMD) neuron from the grasshopper's neck. The DCMD activity underlies the grasshopper's motor responses to looming monocular visual cues and can easily be recorded and analyzed on an open-source iOS oscilloscope app, Spike Recorder. Visual stimuli are presented to the grasshopper by this same mobile application allowing for synchronized recording of stimuli and neural activity. An in-app spike-sorting algorithm is described that allows a quick way for students to record, sort, and analyze their data at the bench. We also describe a way for students to export these data to other analysis tools. With the protocol described, students will be able to prepare the grasshopper, find and record from the DCMD neuron, and visualize the DCMD responses to quantitatively investigate the escape system by adjusting the speed and size of simulated approaching objects. We describe the results from 22 grasshoppers, where 50 of the 57 recording sessions (87.7%) had a reliable DCMD response. Finally, we field-tested our experiment in an undergraduate neuroscience laboratory and found that a majority of students (67%) could perform this exercise in one two-hour lab setting, and had an increase in interest for studying the neural systems that drive behavior.

Portable conduction velocity experiments using earthworms for the college and high school neuroscience teaching laboratory.

The earthworm is ideal for studying action potential conduction velocity in a classroom setting, as its simple linear anatomy allows easy axon length measurements and the worm's sparse coding allows single action potentials to be easily identified. The earthworm has two giant fiber systems (lateral and medial) with different conduction velocities that can be easily measured by manipulating electrode placement and the tactile stimulus. Here, we present a portable and robust experimental setup that allows students to perform conduction velocity measurements within a 30-min to 1-h laboratory session. Our improvement over this well-known preparation is the combination of behaviorally relevant tactile stimuli (avoiding electrical stimulation) with the invention of minimal, low-cost, and portable equipment. We tested these experiments during workshops in both a high school and college classroom environment and found positive learning outcomes when we compared pre- and posttests taken by the students.

Using crickets to introduce neurophysiology to early undergraduate students.

Anatomy and physiology instructors often face the daunting task of teaching the principles of neurophysiology as part of a laboratory course with very limited resources. Teaching neurophysiology can be a difficult undertaking as sophisticated electrophysiology and data acquisition equipment is often financially out-of-reach for two-year institutions, and for many preparations, instructors need to be highly skilled in electrophysiology techniques when teaching hands-on laboratories. In the absence of appropriate laboratory tools, many undergraduate students have difficulty understanding concepts related to neurophysiology. The cricket can serve as a reliable invertebrate model to teach the basic concepts of neurophysiology in the educational laboratory. In this manuscript, we describe a series of hands-on, demonstrative, technologically simple, and affordable laboratory activities that will help undergraduate students gain an understanding of the principles of neurophysiology. By using the cerci ganglion and leg preparation, students can quantify extracellular neural activity in response to sensory stimulation, understand the principles of rate coding and somatotopy, perform electrical microstimulation to understand the threshold of sensory stimulation, and do pharmacological manipulation of neuronal activity. We describe the utility of these laboratory activities, provide a convenient protocol for quantifying extracellular recordings, and discuss feedback provided by undergraduate students with regards to the quality of the educational experience after performing the lab activities.

Gills Just Want to Have Fun: Can Fish Play Games, Just like Us?

It is common to observe play in dogs, cats, and birds, but have we been ignoring play in one of the most common house pets of all… fish? Aquarium fish are often used as meditative decoration in family households, but it could be that fish have similarly diverse behavioral repertoires as mammals and birds. To examine this theory, we conducted field tests at local pet stores where a range of aquarium fish species was tested for responsiveness to laser pointer stimuli. Out of 66 species of fish tested, over 80% showed a tendency to be interested in the moving laser spots, particularly red ones. Whether this behavior constitutes play is an active topic of investigation that we examine in this work.

Local dynamics of gap-junction-coupled interneuron networks.

Interneurons coupled by both electrical gap-junctions (GJs) and chemical GABAergic synapses are major components of forebrain networks. However, their contributions to the generation of specific activity patterns, and their overall contributions to network function, remain poorly understood. Here we demonstrate, using computational methods, that the topological properties of interneuron networks can elicit a wide range of activity dynamics, and either prevent or permit local pattern formation. We systematically varied the topology of GJ and inhibitory chemical synapses within simulated networks, by changing connection types from local to random, and changing the total number of connections. As previously observed we found that randomly coupled GJs lead to globally synchronous activity. In contrast, we found that local GJ connectivity may govern the formation of highly spatially heterogeneous activity states. These states are inherently temporally unstable when the input is uniformly random, but can rapidly stabilize when the network detects correlations or asymmetries in the inputs. We show a correspondence between this feature of network activity and experimental observations of transient stabilization of striatal fast-spiking interneurons (FSIs), in electrophysiological recordings from rats performing a simple decision-making task. We suggest that local GJ coupling enables an active search-and-select function of striatal FSIs, which contributes to the overall role of cortical-basal ganglia circuits in decision-making.

Neurorobotics Workshop for High School Students Promotes Competence and Confidence in Computational Neuroscience.

Understanding the brain is a fascinating challenge, captivating the scientific community and the public alike. The lack of effective treatment for most brain disorders makes the training of the next generation of neuroscientists, engineers and physicians a key concern. Over the past decade there has been a growing effort to introduce neuroscience in primary and secondary schools, however, hands-on laboratories have been limited to anatomical or electrophysiological activities. Modern neuroscience research labs are increasingly using computational tools to model circuits of the brain to understand information processing. Here we introduce the use of neurorobots - robots controlled by computer models of biological brains - as an introduction to computational neuroscience in the classroom. Neurorobotics has enormous potential as an education technology because it combines multiple activities with clear educational benefits including neuroscience, active learning, and robotics. We describe a 1-week introductory neurorobot workshop that teaches high school students how to use neurorobots to investigate key concepts in neuroscience, including spiking neural networks, synaptic plasticity, and adaptive action selection. Our do-it-yourself (DIY) neurorobot uses wheels, a camera, a speaker, and a distance sensor to interact with its environment, and can be built from generic parts costing about $170 in under 4 h. Our Neurorobot App visualizes the neurorobot's visual input and brain activity in real-time, and enables students to design new brains and deliver dopamine-like reward signals to reinforce chosen behaviors. We ran the neurorobot workshop at two high schools (n = 295 students total) and found significant improvement in students' understanding of key neuroscience concepts and in students' confidence in neuroscience, as assessed by a pre/post workshop survey. Here we provide DIY hardware assembly instructions, discuss our open-source Neurorobot App and demonstrate how to teach the Neurorobot Workshop. By doing this we hope to accelerate research in educational neurorobotics and promote the use of neurorobots to teach computational neuroscience in high school.

The Case for Neuroscience Research in the Classroom.

Neuroscience courses, largely relegated to advanced undergraduate or graduate universities, are now being offered in high schools and middle schools. Low-tech versions of advanced neuroscience research tools are being used in hands-on labs. In this NeuroView, I will argue the need for and provide an overview of neuroscience research beyond academia.

Wavelet filtering before spike detection preserves waveform shape and enhances single-unit discrimination.

The isolation of single units in extracellular recordings involves filtering. Removing lower frequencies allows a constant threshold to be applied in order to identify and extract action potential events. However, standard methods such as Butterworth bandpass filtering perform this frequency excision at a cost of grossly distorting waveform shapes. Here, we apply wavelet decomposition and reconstruction as a filter for electrophysiology data and demonstrate its ability to better preserve spike shape. For the majority of cells, this approach also improves spike signal-to-noise ratio (SNR) and increases cluster discrimination. Additionally, the described technique is fast enough to be applied real-time.

Naive coadaptive cortical control.

The ability to control a prosthetic device directly from the neocortex has been demonstrated in rats, monkeys and humans. Here we investigate whether neural control can be accomplished in situations where (1) subjects have not received prior motor training to control the device (naive user) and (2) the neural encoding of movement parameters in the cortex is unknown to the prosthetic device (naive controller). By adopting a decoding strategy that identifies and focuses on units whose firing rate properties are best suited for control, we show that naive subjects mutually adapt to learn control of a neural prosthetic system. Six untrained Long-Evans rats, implanted with silicon micro-electrodes in the motor cortex, learned cortical control of an auditory device without prior motor characterization of the recorded neural ensemble. Single- and multi-unit activities were decoded using a Kalman filter to represent an audio "cursor" (90 ms tone pips ranging from 250 Hz to 16 kHz) which subjects controlled to match a given target frequency. After each trial, a novel adaptive algorithm trained the decoding filter based on correlations of the firing patterns with expected cursor movement. Each behavioral session consisted of 100 trials and began with randomized decoding weights. Within 7 +/- 1.4 (mean +/- SD) sessions, all subjects were able to significantly score above chance (P < 0.05, randomization method) in a fixed target paradigm. Training lasted 24 sessions in which both the behavioral performance and signal to noise ratio of the peri-event histograms increased significantly (P < 0.01, ANOVA). Two rats continued training on a more complex task using a bilateral, two-target control paradigm. Both subjects were able to significantly discriminate the target tones (P < 0.05, Z-test), while one subject demonstrated control above chance (P < 0.05, Z-test) after 12 sessions and continued improvement with many sessions achieving over 90% correct targets. Dynamic analysis of binary trial responses indicated that early learning for this subject occurred during session 6. This study demonstrates that subjects can learn to generate neural control signals that are well suited for use with external devices without prior experience or training.

The SpikerBox: a low cost, open-source bioamplifier for increasing public participation in neuroscience inquiry.

Although people are generally interested in how the brain functions, neuroscience education for the public is hampered by a lack of low cost and engaging teaching materials. To address this, we developed an open-source tool, the SpikerBox, which is appropriate for use in middle/high school educational programs and by amateurs. This device can be used in easy experiments in which students insert sewing pins into the leg of a cockroach, or other invertebrate, to amplify and listen to the electrical activity of neurons. With the cockroach leg preparation, students can hear and see (using a smartphone oscilloscope app we have developed) the dramatic changes in activity caused by touching the mechanosensitive barbs. Students can also experiment with other manipulations such as temperature, drugs, and microstimulation that affect the neural activity. We include teaching guides and other resources in the supplemental materials. These hands-on lessons with the SpikerBox have proven to be effective in teaching basic neuroscience.

Whole animal perfusion fixation for rodents.

The goal of fixation is to rapidly and uniformly preserve tissue in a life-like state. While placing tissue directly in fixative works well for small pieces of tissue, larger specimens like the intact brain pose a problem for immersion fixation because the fixative does not reach all regions of the tissue at the same rate (5,7). Often, changes in response to hypoxia begin before the tissue can be preserved (12). The advantage of directly perfusing fixative through the circulatory system is that the chemical can quickly reach every corner of the organism using the natural vascular network. In order to utilize the circulatory system most effectively, care must be taken to match physiological pressures (3). It is important to note that physiological pressures are dependent on the species used. Techniques for perfusion fixation vary depending on the tissue to be fixed and how the tissue will be processed following fixation. In this video, we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain for immunohistochemistry. The main advantage of this technique (vs. gravity-fed systems) is that the circulatory system is utilized most effectively.

Surgical implantation of chronic neural electrodes for recording single unit activity and electrocorticographic signals.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

Basal ganglia beta oscillations accompany cue utilization.

Beta oscillations in cortical-basal ganglia (BG) circuits have been implicated in normal movement suppression and motor impairment in Parkinson's disease. To dissect the functional correlates of these rhythms we compared neural activity during four distinct variants of a cued choice task in rats. Brief beta (∼20 Hz) oscillations occurred simultaneously throughout the cortical-BG network, both spontaneously and at precise moments of task performance. Beta phase was rapidly reset in response to salient cues, yet increases in beta power were not rigidly linked to cues, movements, or movement suppression. Rather, beta power was enhanced after cues were used to determine motor output. We suggest that beta oscillations reflect a postdecision stabilized state of cortical-BG networks, which normally reduces interference from alternative potential actions. The abnormally strong beta seen in Parkinson's Disease may reflect overstabilization of these networks, producing pathological persistence of the current motor state.

Selective activation of striatal fast-spiking interneurons during choice execution.

Basal ganglia circuits are essential for the organization and execution of voluntary actions. Within the striatum, fast-spiking interneurons (FSIs) are thought to tightly regulate the activity of medium-spiny projection neurons (MSNs) through feed-forward inhibition, yet few studies have investigated the functional contributions of FSIs in behaving animals. We recorded presumed MSNs and FSIs together with motor cortex and globus pallidus (GP) neurons, in rats performing a simple choice task. MSN activity was widely distributed across the task sequence, especially near reward receipt. By contrast, FSIs showed a coordinated pulse of increased activity as chosen actions were initiated, in conjunction with a sharp decrease in GP activity. Both MSNs and FSIs were direction selective, but neighboring MSNs and FSIs showed opposite selectivity. Our findings suggest that individual FSIs participate in local striatal information processing, but more global disinhibition of FSIs by GP is important for initiating chosen actions while suppressing unwanted alternatives.

Development of closed-loop neural interface technology in a rat model: combining motor cortex operant conditioning with visual cortex microstimulation.

Closed-loop neural interface technology that combines neural ensemble decoding with simultaneous electrical microstimulation feedback is hypothesized to improve deep brain stimulation techniques, neuromotor prosthetic applications, and epilepsy treatment. Here we describe our iterative results in a rat model of a sensory and motor neurophysiological feedback control system. Three rats were chronically implanted with microelectrode arrays in both the motor and visual cortices. The rats were subsequently trained over a period of weeks to modulate their motor cortex ensemble unit activity upon delivery of intra-cortical microstimulation (ICMS) of the visual cortex in order to receive a food reward. Rats were given continuous feedback via visual cortex ICMS during the response periods that was representative of the motor cortex ensemble dynamics. Analysis revealed that the feedback provided the animals with indicators of the behavioral trials. At the hardware level, this preparation provides a tractable test model for improving the technology of closed-loop neural devices.

Laminar characterization of spiking activity in the rat motor cortex.

The neocortex is a six-layered tissue consisting of different cell types. How does unit activity in the different layers of the motor cortex relate to movement? Does implantation in a particular layer improve direction decoding ability for a neuroprosthetic device? We simultaneously recorded unit activity in different layers of the rat motor cortex using chronic multi-site silicon electrodes. We used a combination of histology and electrophysiological signatures of Local Field Potentials (LFPs) to accurately localize the electrode sites in the different layers of the cortex. We analyzed 142 units from two animals and found that 40 units (28%) in Layers II to V showed significant modulation with respect to movement. Of these units that showed significant modulation, 9/20 (45%) of units in Layers II/III encoded directional information as compared to 15/19 (79%) of the units in Layers IV/V. These preliminary results suggest that units in Layers IV/V relatively contain more directional information than other layers of the cortex.