Video Microscopy for Teaching: Optimizing the Field of View.

Whether teaching online or in person, video microscopy can demonstrate and document procedures such as dissection and electrophysiology. Such videos can streamline in-person lab work or make online material more interesting and lifelike. Microscope video can also be streamed live over Zoom or other services for live online demonstration. It can be difficult, however, to match a microscope and camera such that the field of view (FOV) captured by the camera encompasses the entire FOV seen by the microscope user. Standard recommendations usually give a camera FOV much smaller than the user's FOV. This paper explains how to work with three variables (camera sensor size, microscope coupler magnification, and ocular diameter) to achieve a good FOV match.

Reducing the Cost of Electrophysiology in the Teaching Laboratory.

Electrophysiology is a fundamental part of neuroscience and there are many published laboratory exercises suitable for undergraduates. However, the cost of equipping a lab is often a barrier to implementing these exercises. In this paper, we outline lab needs, suggest strategies for building a lab incrementally by adding equipment as budgets permit, and suggest specific areas for cost-cutting. We also point out instances in which it makes most sense to purchase or borrow research-grade equipment. A linked Google document lists specific items, prices, and purchase links.

A Protocol Demonstrating 60 Different Drosophila Behaviors in One Assay.

The fruit fly Drosophila melanogaster performs many behaviors, from simple motor actions to complex social interactions, that are of interest to neurobiologists studying how the brain controls behavior. Here, an undergraduate laboratory exercise uses cutting-edge methods to activate sets of neurons thermogenetically, triggering 60 different behaviors. Students learn how to recognize this large repertoire of behaviors from 16 fly strains that are publicly available, and from a large set of training videos provided here. A full protocol, timeline and handouts are included. Instructors need not have any experience working with flies. Student feedback is reported; in our experience, students are fascinated and highly engaged by watching animals perform such a broad array of behaviors. This exercise teaches fly husbandry and crossing, careful scientific observation, and principles of behavioral screening.

Documenting Laboratory Procedures with Video.

Demonstrating laboratory procedures in person during class time can be time-consuming. When procedures are done under a microscope, live demonstration is also impractical because of the limited number of students who can view the demonstration at once. Creating videos beforehand, which students can watch before class and review during lab sessions, solves both of these problems. This article suggests ways to make and distribute high quality video of microscopic procedures.

Mosquito (Aedes aegypti) flight tones: frequency, harmonicity, spherical spreading, and phase relationships.

Mosquito flight produces a tone as a side effect of wing movement; this tone is also a communication signal that is frequency-modulated during courtship. Recordings of tones produced by tethered flying male and female Aedes aegypti were undertaken using pairs of pressure-gradient microphones above and below, ahead and behind, and to the left and right over a range of distances. Fundamental frequencies were close to those previously reported, although amplitudes were lower. The male fundamental frequency was higher than that of the female and males modulated it over a wider range. Analysis of harmonics shows that the first six partials were nearly always within 1 Hz of integer multiples of the fundamental, even when the fundamental was being modulated. Along the front-back axis, amplitude attenuated as a function of distance raised to the power 2.3. Front and back recordings were out of phase, as were above and below, while left and right were in phase. Recordings from ahead and behind showed quadratic phase coupling, while others did not. Finally, two methods are presented for separating simultaneous flight tones in a single recording and enhancing their frequency resolution. Implications for mosquito behavior are discussed.

Associative learning in the dengue vector mosquito, Aedes aegypti: avoidance of a previously attractive odor or surface color that is paired with an aversive stimulus.

Associative learning has been shown in a variety of insects, including the mosquitoes Culex quinquefasciatus and Anopheles gambiae. This study demonstrates associative learning for the first time in Aedes aegypti, an important vector of dengue, yellow fever and chikungunya viruses. This species prefers to rest on dark surfaces and is attracted to the odor of 1-octen-3-ol. After training in which a dark surface alone or a dark surface with odor was paired with electric shock, mosquitoes avoided the previously attractive area. The association was stronger when odor was included in training, was retained for at least 60 min but not for 24 h, and was equal for males and females. These results demonstrate the utility of a bulk-training paradigm for mosquitoes similar to that used with Drosophila melanogaster.

Exploring sensory neuroscience through experience and experiment.

Many phenomena that we take for granted are illusions - color and motion on a TV or computer monitor, for example, or the impression of space in a stereo music recording. Even the stable image that we perceive when looking directly at the real world is illusory. One of the important lessons from sensory neuroscience is that our perception of the world is constructed rather than received. Sensory illusions effectively capture student interest, but how do you then move on to substantive discussion of neuroscience? This article illustrates several illusions, attempts to connect them to neuroscience, and shows how students can explore and experiment with them. Even when (as is often the case) there is no agreed-upon mechanistic explanation for an illusion, students can form hypotheses and test them by manipulating stimuli and measuring their effects. In effect, students can experiment with illusions using themselves as subjects.

Classical conditioning through auditory stimuli in Drosophila: methods and models.

The role of sound in Drosophila melanogaster courtship, along with its perception via the antennae, is well established, as is the ability of this fly to learn in classical conditioning protocols. Here, we demonstrate that a neutral acoustic stimulus paired with a sucrose reward can be used to condition the proboscis-extension reflex, part of normal feeding behavior. This appetitive conditioning produces results comparable to those obtained with chemical stimuli in aversive conditioning protocols. We applied a logistic model with general estimating equations to predict the dynamics of learning, which successfully predicts the outcome of training and provides a quantitative estimate of the rate of learning. Use of acoustic stimuli with appetitive conditioning provides both an alternative to models most commonly used in studies of learning and memory in Drosophila and a means of testing hearing in both sexes, independently of courtship responsiveness.

Neural responses to one- and two-tone stimuli in the hearing organ of the dengue vector mosquito.

Recent studies demonstrate that mosquitoes listen to each other's wing beats just prior to mating in flight. Field potentials from sound-transducing neurons in the antennae contain both sustained and oscillatory components to pure and paired tone stimuli. Described here is a direct comparison of these two types of response in the dengue vector mosquito, Aedes aegypti. Across a wide range of frequencies and intensities, sustained responses to one- and two-tone stimuli are about equal in magnitude to oscillatory responses to the beats produced by two-tone stimuli. All of these responses are much larger than the oscillatory responses to one-tone stimuli. Similarly, the frequency range extends up to at least the fifth harmonic of the male flight tone for sustained responses to one- and two-tone stimuli and oscillatory responses at the beat frequency of two-tone stimuli, whereas the range of oscillatory response to a one-tone stimulus is limited to, at most, the third harmonic. Thresholds near the fundamental of the flight tone are lower for oscillatory responses than for sustained deflections, lower for males than for females, and within the behaviorally relevant range. A simple model of the transduction process can qualitatively account for both oscillatory and sustained responses to pure and paired tones. These data leave open the question as to which of several alternative strategies underlie flight tone matching behavior in mosquitoes.

Psychophysics in insect hearing.

Psychophysics has much to offer the study of insect hearing. Not only is there a rich set of experimental methods to apply, there is a large body of experimental work on vertebrate hearing that can suggest topics for investigation and provide material for cross-species comparisons. We present an overview of the methods of psychophysics, followed by specific examples of their use in insects. Topics covered include intensity discrimination, frequency analysis and discrimination, temporal integration and acuity, and localization. We conclude by pointing out additional areas of research suggested by the reviewed work and areas in which a psychophysical approach would be useful.

Action potentials in a giant algal cell: a comparative approach to mechanisms and evolution of excitability.

The giant alga Chara corallina generates action potentials (APs) in response to mechanical stimulation, injury, or direct electrical stimulation. Students examine the waveform characteristics of these APs using standard intracellular recording techniques. Intracellular recording is easier than with neurons because of the large size of the Chara cell. Students observe very negative resting potentials (up to -250 mV), large AP amplitudes with depolarizing peaks approaching 0 mV, AP durations of seconds, and refractory periods up to several minutes. Students calculate Nernst potentials for the ions distributed across the Chara cell membrane to hypothesize the ions responsible for the resting potential and for the depolarizing phase of the AP. These calculations suggest that K(+) is responsible for the resting potential and that Ca(2+) influx and Ca(2+)-activated Cl(-) efflux are responsible for depolarizing phases of the AP, which they are. Comparison of the Chara AP characteristics with animal neuron and muscle APs reinforces understanding of mechanisms of excitability in animals, demonstrates that multiple solutions exist for action potential generation, and leads to discussion of the evolution of ion channels and excitability.

Tools for physiology labs: inexpensive equipment for physiological stimulation.

We describe the design of inexpensive equipment and software for physiological stimulation in the neurobiology teaching laboratory. The core component is a stimulus isolation unit (SIU) that uses DC-DC converters, rather than expensive high-voltage batteries, to generate isolated power at high voltage. The SIU has no offset when inactive and produces pulses up to 100 V with moderately fast (50 µs) rise times. We also describe two methods of stimulus timing control. The first is a simplified conventional, stand-alone analog pulse generator. The second uses a digital microcontroller interfaced with a personal computer. The SIU has performed well and withstood intensive use in our undergraduate physiology laboratory. This project is part of our ongoing effort to make reliable low-cost physiology equipment available for both student teaching and faculty research laboratories.