Quantitative and Qualitative Representation of Introductory and Advanced EEG Concepts: An Exploration of Different EEG Setups.

Electroencephalograms (EEGs) are the gold standard test used in the medical field to diagnose epilepsy and aid in the diagnosis of many other neurological and mental disorders. Growing in popularity in terms of nonmedical applications, the EEG is also used in research, neurofeedback, and brain-computer interface, making it increasingly relevant to student learning. Recent innovations have made EEG setups more accessible and affordable, thus allowing their integration into neuroscience educational settings. Introducing students to EEGs, however, can be daunting due to intricate setup protocols, individual variation, and potentially expensive equipment. This paper aims to provide guidance for introducing students and educators to fundamental beginning and advanced level EEG concepts. Specifically, this paper tested the potential of three different setups, with varying channel number and wired or wireless connectivity, for introducing students to qualitative and quantitative exploration of alpha enhancement when eyes are closed, and observation of the alpha/beta anterior to posterior gradient. The setups were compared to determine their relative advantages and their robustness in detecting these well-established parameters. The basic 1- or 2-channel setups are sufficient for observing alpha and beta waves, while more advanced systems containing 8 or 16 channels are required for consistent observation of an anterior-posterior gradient. In terms of localization, the 16-channel setup, in principle, was more adept. The 8-channel setup, however, was more effective than the 16-channel setup with regards to displaying the anterior to posterior gradient. Thus, an 8-channel setup is sufficient in an education setting to display these known trends. Modification of the 16-channel setup may provide a better observation of the anterior to posterior gradient.

Electrophysiology and transcriptomics reveal two photoreceptor classes and complex visual integration in Hirudo verbana.

Among animals with visual processing mechanisms, the leech Hirudo verbana is a rare example in which all neurons can be identified. However, little is known about its visual system, which is composed of several pigmented head eyes and photosensitive non-pigmented sensilla that are distributed across its entire body. Although several interneurons are known to respond to visual stimuli, their response properties are poorly understood. Among these, the S-cell system is especially intriguing: it is multimodal, spans the entire body of the leech and is thought to be involved in sensory integration. To improve our understanding of the role of this system, we tested its spectral sensitivity, spatial integration and adaptation properties. The response of the S-cell system to visual stimuli was found to be strongly dependent on the size of the area stimulated, and adaptation was local. Furthermore, an adaptation experiment demonstrated that at least two color channels contributed to the response, and that their contribution was dependent on the adaptation to the background. The existence of at least two color channels was further supported by transcriptomic evidence, which indicated the existence of at least two distinct groups of putative opsins for leeches. Taken together, our results show that the S-cell system has response properties that could be involved in the processing of spatial and color information of visual stimuli. We propose the leech as a novel system to understand visual processing mechanisms with many practical advantages.

Giving invertebrates an eye exam: an ophthalmoscope that utilizes the autofluorescence of photoreceptors.

One of the most important functional features of eyes is focusing light, as both nearsightedness and farsightedness have major functional implications. Accordingly, refractive errors are frequently assessed in vertebrates, but not in the very small invertebrate eyes. We describe a micro-ophthalmoscope that takes advantage of autofluorescent properties of invertebrate photoreceptors and test the device on the relatively well-understood eyes of jumping spiders and flies. In each case, our measurements confirmed previous findings with a greater degree of accuracy. For example, we could precisely resolve the layering of the anterior median eyes and could map out the extensive retina of the anterior lateral eyes of the spider. Measurements also confirmed that fly ommatidia are focused into infinity, but showed that their focal plane is situated slightly below the receptor surface. In contrast to other approaches, this device does not rely on reflective tapeta and allows for precise optical assessment of diverse invertebrate eyes.

Embryonic development of the larval eyes of the Sunburst Diving Beetle, Thermonectus marmoratus (Insecta: Dytiscidae): a morphological study.

Stemmata, the larval eyes of holometabolous insects are extremely diverse, ranging from full compound eyes, to a few ommatidial units as are typical in compound eyes, to sophisticated and functionally specialized image-forming camera-type eyes. Stemmata evolved from a compound eye ommatidial ancestor, an eye type that is morphologically well conserved in regards to cellular composition, and well studied in regards to development. However, despite this evolutionary origin it remains largely unknown how stemmata develop. In addition, it is completely unclear how development is altered to give rise to some of the functionally most complex stemmata, such as those of the sunburst diving beetle, Thermonectus marmoratus. In this study, we used histological methods to investigate the embryonic development of the functionally complex principal stemmata Eye 1 and Eye 2 of the larval visual system of T. marmoratus. To gain insights into how cellular components of their sophisticated camera-type eyes might have evolved from the cellular components of ommatidial ancestors, we contrast our findings against known features of ommatidia development, which are particularly well understood in Drosophila. We find many similarities, such as the early presence of a pseudostratified epithelium, and the order in which specific cell types are recruited. However, in Thermonectus each cell type is represented by a large number of cells from early on and major tissue re-orientation occurs as eye development progresses. This study provides insights into the timing of morphological features and represents the basis for future molecular studies.

Electrophysiology Meets Ecology: Investigating How Vision is Tuned to the Life Style of an Animal using Electroretinography.

Students learn best when projects are multidisciplinary, hands-on, and provide ample opportunity for self-driven investigation. We present a teaching unit that leads students to explore relationships between sensory function and ecology. Field studies, which are rare in neurobiology education, are combined with laboratory experiments that assess visual properties of insect eyes, using electroretinography (ERG). Comprised of nearly one million species, insects are a diverse group of animals, living in nearly all habitats and ecological niches. Each of these lifestyles puts different demands on their visual systems, and accordingly, insects display a wide array of eye organizations and specializations. Physiologically relevant differences can be measured using relatively simple extracellular electrophysiological methods that can be carried out with standard equipment, much of which is already in place in most physiology laboratories. The teaching unit takes advantage of the large pool of locally available species, some of which likely show specialized visual properties that can be measured by students. In the course of the experiments, students collect local insects or other arthropods of their choice, are guided to formulate hypotheses about how the visual system of "their" insects might be tuned to the lifestyle of the species, and use ERGs to investigate the insects' visual response dynamics, and both chromatic and temporal properties of the visual system. Students are then guided to interpret their results in both a comparative physiological and ecological context. This set of experiments closely mirrors authentic research and has proven to be a popular, informative and highly engaging teaching tool.

How aquatic water-beetle larvae with small chambered eyes overcome challenges of hunting under water.

A particularly unusual visual system exists in the visually guided aquatic predator, the Sunburst Diving Beetle, Thermonectus marmoratus (Coleoptera: Dytiscidae). The question arises: how does this peculiar visual system function? A series of experiments suggests that their principal eyes (E1 and E2) are highly specialized for hunting. These eyes are tubular and have relatively long focal lengths leading to high image magnification. Their retinae are linear, and are divided into distinct green-sensitive distal and UV and polarization-sensitive proximal portions. Each distal retina, moreover, has many tiers of photoreceptors with rhabdomeres the long axis of which are peculiarly oriented perpendicular to the light path. Based on detailed optical investigations, the lenses of these eyes are bifocal and project focused images onto specific retinal tiers. Behavioral experiments suggest that these larvae approach prey within their eyes' near-fields, and that they can correctly gauge prey distances even when conventional distance-vision mechanisms are unavailable. In the near-field of these eyes object distance determines which of the many retinal layers receive the best-focused images. This retinal organization could facilitate an unusual distance-vision mechanism. We here summarize past findings and discuss how these eyes allow Thermonectus larvae to be such successful predators.

Multitasking in an eye: the unusual organization of the Thermonectus marmoratus principal larval eyes allows for far and near vision and might aid in depth perception.

Very few visual systems diverge fundamentally from the basic plans of well-studied animal eyes. However, investigating those that do can provide novel insights into visual system function. A particularly unusual system exists in the principal larval eyes of a visually guided aquatic predator, the sunburst diving beetle, Thermonectus marmoratus (Coleoptera: Dystiscidae). These eyes are characterized by complex layered distal and proximal retinas. We previously reported that their principal eye E2 has a bifocal lens, and previous behavioral experiments suggested that these larvae have a unilateral range-finding mechanism that may involve their bizarre eye organization. In the present study, we expanded our optical measurements and found that: (1) E1 also has a bifocal lens, (2) E1 is best suited for far vision while E2 is best suited for near vision and (3) throughout their typical hunting range, the positions of focused images shift across specific retinal layers. This anatomical and optical organization in principle could support unilateral range finding. Taken together, our findings outline an unusual visual mechanism that is likely to be essential for the extraordinary hunting ability of these larvae.

Unilateral range finding in diving beetle larvae.

One of the biggest challenges that predators, such as the larvae of the diving beetle Thermonectus marmoratus (Coleoptera: Dytiscidae), are faced with is to accurately assess the distance of their prey. Most animals derive distance information from disparities of images that are viewed from different angles, from information that is obtained from well-controlled translational movements (motion parallax) or from the image size of known objects. Using a behavioral assay we demonstrated that T. marmoratus larvae continue to accurately strike at artificial prey, even if none of these typical distance estimation cues are available to them. Specifically, we excluded bilateral binocular stereopsis by occlusion, confounded possible motion parallax cues with an artificially moving prey, and excluded the possibility that beetle larvae simply approached their targets based on known prey size by presenting different prey sizes. Despite these constraints, larvae consistently struck our artificial targets from a distance of ~4.5 mm. Based on these findings we conclude that T. marmoratus likely employ an unusual mechanism to accurately determine prey distances, possibly mediated by the object-distance-dependent activation of specific subsets of their many-tiered and peculiarly positioned photoreceptors.

Nutrition-induced macular-degeneration-like photoreceptor damage in jumping spider eyes.

Age-related macular degeneration (AMD) is a leading cause of vision loss in humans. Despite its prevalence and medical significance, many aspects of AMD remain elusive and treatment options are limited. Here, we present data that suggest jumping spiders offer a unique opportunity for understanding the fundamentals underlying retinal degeneration, thereby shedding light on a process that impacts millions of people globally. Using a micro-ophthalmoscope and histological evidence, we demonstrate that significant photoreceptor damage can occur during development in the image-forming anterior lateral eyes of the jumping spider Phidippus audax. Furthermore, we find that this photoreceptor degeneration is exacerbated by inadequate nutrition and is most prevalent in the high-density region of the retina, like AMD in humans. This suggests that similar to those in vertebrates, the retinas in P. audax are challenged to meet high-energy cellular demands.

Electrophysiological evidence for polarization sensitivity in the camera-type eyes of the aquatic predacious insect larva Thermonectus marmoratus.

Polarization sensitivity has most often been studied in mature insects, yet it is likely that larvae also make use of this visual modality. The aquatic larvae of the predacious diving beetle Thermonectus marmoratus are highly successful visually guided predators, with a UV-sensitive proximal retina that, according to its ultrastructure, has three distinct cell types with anatomical attributes that are consistent with polarization sensitivity. In the present study we used electrophysiological methods and single-cell staining to confirm polarization sensitivity in the proximal retinas of both principal eyes of these larvae. As expected from their microvillar orientation, cells of type T1 are most sensitive to vertically polarized light, while cells of type T2 are most sensitive to horizontally polarized light. In addition, T3 cells probably constitute a second population of cells that are most sensitive to light with vertical e-vector orientation, characterized by shallower polarization modulations, and smaller polarization sensitivity (PS) values than are typical for T1 cells. The level of PS values found in this study suggests that polarization sensitivity probably plays an important role in the visual system of these larvae. Based on their natural history and behavior, possible functions are: (1) finding water after hatching, (2) finding the shore before pupation, and (3) making prey more visible, by filtering out horizontally polarized haze, and/or using polarization features for prey detection.

Optically transparent multi-suction electrode arrays.

Multielectrode arrays (MEAs) allow for acquisition of multisite electrophysiological activity with submillisecond temporal resolution from neural preparations. The signal to noise ratio from such arrays has recently been improved by substrate perforations that allow negative pressure to be applied to the tissue; however, such arrays are not optically transparent, limiting their potential to be combined with optical-based technologies. We present here multi-suction electrode arrays (MSEAs) in quartz that yield a substantial increase in the detected number of units and in signal to noise ratio from mouse cortico-hippocampal slices and mouse retina explants. This enables the visualization of stronger cross correlations between the firing rates of the various sources. Additionally, the MSEA's transparency allows us to record voltage sensitive dye activity from a leech ganglion with single neuron resolution using widefield microscopy simultaneously with the electrode array recordings. The combination of enhanced electrical signals and compatibility with optical-based technologies should make the MSEA a valuable tool for investigating neuronal circuits.

Biological bifocal lenses with image separation.

Almost all animal eyes follow a few, relatively well-understood functional plans. Only rarely do researchers discover an eye that diverges fundamentally from known types. The principal eye E2 of sunburst diving beetle (Thermonectus marmoratus) larvae clearly falls into the rarer category. On the basis of two different tests, we here report that it has truly bifocal lenses, something that has been previously suggested only for certain trilobites. Our evidence comes from (1) the relative contrast in images of a square wave grating and (2) the refraction of a narrow laser beam projected through the lens. T. marmoratus larvae have two retinas at different depths behind the lens, and these are situated so that each can receive its own focused image. This is consistent with a novel eye organization that possibly comprises "two eyes in one." Moreover, we find that in contrast to most commercial bifocal lenses, the lens of E2 exhibits asymmetry, which results in separation of the images both dorsoventrally and rostrocaudally within the layered retina. Visual contrast might thus be improved over conventional bifocal lenses because the unfocused version of one image is shifted away from the focused version of the other, an organization which could potentially be exploited in optical engineering.