A library of electrophysiological responses in plants - a model of transversal education and open science.

Electrophysiology in plants is understudied, and, moreover, an ideal model for student inclusion at all levels of education. Here, we report on an investigation in open science, whereby scientists worked with high school students, faculty, and undergraduates from Chile, Germany, Serbia, South Korea, and the USA. The students recorded the electrophysiological signals of >15 plant species in response to a flame or tactile stimulus applied to the leaves. We observed that approximately 60% of the plants studied showed an electrophysiological response, with a delay of ~ 3-6 s after stimulus presentation. In preliminary conduction velocity experiments, we verified that observed signals are indeed biological in origin, with information transmission speeds of ~ 2-9 mm/s. Such easily replicable experiments can serve to include more investigators and students in contributing to our understanding of plant electrophysiology.

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.

The construction of high-magnification homemade lenses for a simple microscope: an easy "DIY" tool for biological and interdisciplinary education.

The rise of microscopy in the seventeenth century allowed scientists to discover a new world of microorganisms and achieve great physiological advances. One of the first microscopes of the epoch was Antonie van Leeuwenhoek's microscope, a deceptively simple device that contains a single ball lens housed in a metal plate allowing the observation of samples at up to ×250 magnification. Such magnification was much greater than that achieved by rudimentary compound microscopes of the era, allowing for the discovery of microscopic, single-celled life, an achievement that marked the study of biology up to the nineteenth century. Since Leeuwenhoek's design uses a single ball lens, it is possible to fabricate variations for educational activities in physics and biology university and high school classrooms. A fundamental problem, however, with home-built microscopes is that it is difficult to work with glass. We developed a simple protocol to make ball lenses of glass and gelatin with high magnification that can be done in a university/high school classroom, and we designed an optimized support for focusing and taking photographs with a smartphone. The protocol details a simple, easily accessible, low-cost, and effective tool for the observation of microscopic samples, possible to perform anywhere without the need for a laboratory or complex tools. Our protocol has been implemented in classrooms in Chile to a favorable reception.

Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering.

The advent of easy-to-use open source microcontrollers, off-the-shelf electronics and customizable manufacturing technologies has facilitated the development of inexpensive scientific devices and laboratory equipment. In this study, we describe an imaging system that integrates low-cost and open-source hardware, software and genetic resources. The multi-fluorescence imaging system consists of readily available 470 nm LEDs, a Raspberry Pi camera and a set of filters made with low cost acrylics. This device allows imaging in scales ranging from single colonies to entire plates. We developed a set of genetic components (e.g. promoters, coding sequences, terminators) and vectors following the standard framework of Golden Gate, which allowed the fabrication of genetic constructs in a combinatorial, low cost and robust manner. In order to provide simultaneous imaging of multiple wavelength signals, we screened a series of long stokes shift fluorescent proteins that could be combined with cyan/green fluorescent proteins. We found CyOFP1, mBeRFP and sfGFP to be the most compatible set for 3-channel fluorescent imaging. We developed open source Python code to operate the hardware to run time-lapse experiments with automated control of illumination and camera and a Python module to analyze data and extract meaningful biological information. To demonstrate the potential application of this integral system, we tested its performance on a diverse range of imaging assays often used in disciplines such as microbial ecology, microbiology and synthetic biology. We also assessed its potential use in a high school environment to teach biology, hardware design, optics, and programming. Together, these results demonstrate the successful integration of open source hardware, software, genetic resources and customizable manufacturing to obtain a powerful, low cost and robust system for education, scientific research and bioengineering. All the resources developed here are available under open source licenses.

The Missing Manuscript of Dr. Jose Delgado's Radio Controlled Bulls.

Neuroscience systems level courses teach: 1) the role of neuroanatomical structures of the brain for perception, movement, and cognition; 2) methods to manipulate and study the brain including lesions, electrophysiological recordings, microstimulation, optogenetics, and pharmacology; 3) proper interpretation of behavioral data to deduce brain circuit operation; and 4) the similarities, differences, and ethics of animal models and their relation to human physiology. These four topics come together quite dramatically in Dr. Jose Delgado's 1960s famous experiments on the neural correlates of aggression in which he stopped bulls in mid-charge by electrically stimulating basal ganglia and thalamic structures. Technical documentation on these experiments is famously difficult to find. Here I translate and discuss a Spanish language article written by Dr. Delgado in 1981 for an encyclopedia on bull fighting published in Madrid. Here Dr. Delgado appears to give the most complete explanation of his experiments on microstimulation of bovine brains. Dr. Delgado's motivations, methods, and his interpretation of the bull experiments are summarized, as well as some accompanying information from his 1970 English language book: "Physical Control of the Mind." This review of Dr. Delgado's written work on the bull experiments can provide a resource to educators and students who desire to learn more about and interpret the attention-calling experiments that Dr. Delgado did on a ranch in Andalucía over 50 years ago.

Leg Regrowth in Blaberus discoidalis (Discoid Cockroach) following Limb Autotomy versus Limb Severance and Relevance to Neurophysiology Experiments.

BACKGROUND: Many insects can regenerate limbs, but less is known about the regrowth process with regard to limb injury type. As part of our neurophysiology education experiments involving the removal of a cockroach leg, 1) the ability of Blaberus discoidalis cockroaches to regenerate a metathoracic leg was examined following autotomy at the femur/trochanter joint versus severance via a transverse coxa-cut, and 2) the neurophysiology of the detached legs with regard to leg removal type was studied by measuring spike firing rate and microstimulation movement thresholds. LEG REGROWTH RESULTS: First appearance of leg regrowth was after 5 weeks in the autotomy group and 12 weeks in the coxa-cut group. Moreover, regenerated legs in the autotomy group were 72% of full size on first appearance, significantly larger (p<0.05) than coxa-cut legs (29% of full size at first appearance). Regenerated legs in both groups grew in size with each subsequent molt; the autotomy-removed legs grew to full size within 18 weeks, whereas coxa-cut legs took longer than 28 weeks to regrow. Removal of the metathoracic leg in both conditions did not have an effect on mortality compared to matched controls with unmolested legs. NEUROPHYSIOLOGY RESULTS: Autotomy-removed legs had lower spontaneous firing rates, similar marked increased firing rates upon tactile manipulation of tibial barbs, and a 10% higher electrical microstimulation threshold for movement. SUMMARY: It is recommended that neurophysiology experiments on cockroach legs remove the limb at autotomy joints instead of coxa cuts, as the leg regenerates significantly faster when autotomized and does not detract from the neurophysiology educational content.

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.

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.

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.

Use of a Bayesian maximum-likelihood classifier to generate training data for brain-machine interfaces.

Brain-machine interface decoding algorithms need to be predicated on assumptions that are easily met outside of an experimental setting to enable a practical clinical device. Given present technological limitations, there is a need for decoding algorithms which (a) are not dependent upon a large number of neurons for control, (b) are adaptable to alternative sources of neuronal input such as local field potentials (LFPs), and (c) require only marginal training data for daily calibrations. Moreover, practical algorithms must recognize when the user is not intending to generate a control output and eliminate poor training data. In this paper, we introduce and evaluate a Bayesian maximum-likelihood estimation strategy to address the issues of isolating quality training data and self-paced control. Six animal subjects demonstrate that a multiple state classification task, loosely based on the standard center-out task, can be accomplished with fewer than five engaged neurons while requiring less than ten trials for algorithm training. In addition, untrained animals quickly obtained accurate device control, utilizing LFPs as well as neurons in cingulate cortex, two non-traditional neural inputs.

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.

Lower layers in the motor cortex are more effective targets for penetrating microelectrodes in cortical prostheses.

Improving cortical prostheses requires the development of recording neural interfaces that are efficient in terms of providing maximal control information with minimal interface complexity. While the typical approaches have targeted neurons in the motor cortex with multiple penetrating shanks, an alternative approach is to determine an efficient distribution of electrode sites within the layers of the cortex with fewer penetrating shanks. The objective of this study was to compare unit activity in the upper and lower layers of the cortex with respect to movement and direction in order to inform the design of penetrating microelectrodes. Four rats were implanted bilaterally with multi-site single-shank silicon microelectrode arrays in the neck/shoulder region of the motor cortex. We simultaneously recorded unit activity across all layers of the motor cortex while the animal was engaged in a movement direction task. Localization of the electrode array within the different layers of the cortex was determined by histology. We denoted units from layers 2 and 3 and units as upper layer units, and units from layers 5 and 6 as lower layer units. Analysis of unit spiking activity demonstrated that both the upper and lower layers encode movement and direction information. Unit responses in either cortical layer of the cortex were not preferentially associated with contralateral or ipsilateral movement. Aggregate analysis (633 neurons) and best session analysis (75 neurons) indicated that units in the lower layers (layers 5, 6) are more likely to encode direction information when compared to units in the upper layers (layers 2, 3) (p< 0.05). These results suggest that electrode sites clustered in the lower layers provide access to more salient control information for cortical neuroprostheses.

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.

Suitability of the cingulate cortex for neural control.

Recent neuroprosthetic work has focused on the motor cortex as a source of voluntary control signals. However, the motor cortex can be damaged in upper motor neuron degenerative diseases such as primary lateral sclerosis and amyotrophic lateral sclerosis. The possibility exists that prefrontal areas may also be used in neuroprosthetic devices. Here, we report the use of the cingulate cortex in a neuroprosthetic model. Seven rats were able to significantly modulate spiking activity in the cingulate cortex in order to receive reward. Furthermore, experiments with single neurons provide evidence that the cingulate cortex neuronal modulation is highly flexible and thus useful for a neuroprosthetic device.

Laminar analysis of movement direction information in local field potentials of the rat motor cortex.

Local field potentials (LFPs) have been proposed for use in controlling neural prosthetic devices because they can provide reliable motor and sensory-related information, and can easily be recorded over long periods of time. While studies have shown that directional information about motor movements can be inferred from LFPs, it is not known at what depth these signals should be recorded from in order to maximize the amount of movement information. Towards this end, we used a directional motor task in Long Evans rats, while sampling LFPs with an electrode consisting of 16 vertical recording sites that were evenly-spaced 100 microm apart. This allowed for simultaneous recording of all layers of the motor cortex. The frequency components of LFPs were then analyzed using k-means clustering to determine directional information as a function of depth. Here we report our initial findings that superficial layers (II/III) of motor cortex may provide more information about movement directions then deeper layers (V).

Humans to Mars: a feasibility and cost-benefit analysis.

Mars is a compelling astrobiological target, and a human mission would provide an opportunity to collect immense amounts of scientific data. Exploration alone, however, cannot justify the increased risk. Instead, three factors drive a human mission: economics, education, and exploration. A human mission has a unique potential to inspire the next generation of young people to enter critically needed science and engineering disciplines. A mission is economically feasible, and the research and development program put in place for a human mission would propel growth in related high-technology industries. The main hurdles are human physiological responses to 1-2 years of radiation and microgravity exposure. However, enabling technologies are sufficiently mature in these areas that they can be developed within a few decade timescale. Hence, the decision of whether or not to undertake a human mission to Mars is a political decision, and thus, educational and economic benefits are the crucial factors.

Real-time Detection of Unitary Events For Cortical Control.

Traditional brain-machine interfaces have typically focused on methods that use rate-based codes as a source for control signals. Opposed to rate, timing of firing across different neurons and within each neuron could also provide information that can be used for controlling brain-machine interfaces or neuroprosthetic devices. Findings have indicated that synchronization of individual spike discharges may help serve the organization of cortical motor processes. We are investigating neural firing synchrony in the context of using it for real-time control for neuroprostheses systems. Our results with rats suggest that subjects can be trained to synchronize neural firing and increase unitary events i.e. spike coincidence patterns that are significantly above chance. Temporal coding methods could be used as additional or alternative cortical control signals for neuroprostheses and brain machine interfaces.