From 2D slices to a 3D model: Training students in digital microanatomy analysis techniques through a 3D printed neuron project.

The reconstruction of two-dimensional (2D) slices to three-dimensional (3D) digital anatomical models requires technical skills and software that are becoming increasingly important to the modern anatomist, but these skills are rarely taught in undergraduate science classrooms. Furthermore, learning opportunities that allow students to simultaneously explore anatomy in both 2D and 3D space are increasingly valuable. This report describes a novel learning activity that trains students to digitally trace a serially imaged neuron from a confocal stack and to model that neuron in 3D space for 3D printing. By engaging students in the production of a 3D digital model, this learning activity is designed to provide students a novel way to enhance their understanding of the content, including didactic knowledge of neuron morphology, technical research skills in image analysis, and career exploration of neuroanatomy research. Moreover, students engage with microanatomy in a way that starts in 2D but results in a 3D object they can see, touch, and keep. This discursive article presents the learning activity, including videos, instructional guides, and learning objectives designed to engage students on all six levels of Bloom's Taxonomy. Furthermore, this work is a proof of principle modeling workflow that is approachable, inexpensive, achievable, and adaptable to cell types in other organ systems. This work is designed to motivate the expansion of 3D printing technology into microanatomy and neuroanatomy education.

Classics in Chemical Neuroscience: Selegiline, Isocarboxazid, Phenelzine, and Tranylcypromine.

The discovery of monoamine oxidase inhibitors (MAOIs) in the 1950s marked a significant breakthrough in medicine, creating a powerful new category of drug: the antidepressant. In the years and decades that followed, MAOIs have been used in the treatment of several pathologies including Parkinson's disease, Alzheimer's disease, and various cancers and as anti-inflammatory agents. Despite once enjoying widespread use, MAOIs have dwindled in popularity due to side effects, food-drug interactions, and the introduction of other antidepressant drug classes such as tricyclic antidepressants (TCAs) and selective serotonin reuptake inhibitors (SSRIs). The recently published prescriber's guide for the use of MAOIs in treating depression has kindled a resurgence of their use in the clinical space. It is therefore timely to review key aspects of the four "classic" MAOIs: high-dose selegiline, isocarboxazid, phenelzine, and tranylcypromine. This review discusses their chemical synthesis, metabolism, pharmacology, adverse effects, and the history and importance of these drugs within the broader field of chemical neuroscience.

Crosstalk: The diversity of melanopsin ganglion cell types has begun to challenge the canonical divide between image-forming and non-image-forming vision.

Melanopsin ganglion cells have defied convention since their discovery almost 20 years ago. In the years following, many types of these intrinsically photosensitive retinal ganglion cells (ipRGCs) have emerged. In the mouse retina, there are currently six known types (M1-M6) of melanopsin ganglion cells, each with unique morphology, mosaics, connections, physiology, projections, and functions. While melanopsin-expressing cells are usually associated with behaviors like circadian photoentrainment and the pupillary light reflex, the characterization of multiple types has demonstrated a reach that may extend far beyond non-image-forming vision. In fact, studies have shown that individual types of melanopsin ganglion cells have the potential to impact image-forming functions like contrast sensitivity and color opponency. Thus, the goal of this review is to summarize the morphological and functional aspects of the six known types of melanopsin ganglion cells in the mouse retina and to highlight their respective roles in non-image-forming and image-forming vision. Although many melanopsin ganglion cell types do project to image-forming brain targets, it is important to note that this is only the first step in determining their influence on image-forming vision. Even so, the visual system has canonically been divided into these two functional realms and melanopsin ganglion cells have begun to challenge the boundary between them, providing an overlap of visual information that is complementary rather than redundant. Further studies on these ganglion cell photoreceptors will no doubt continue to illustrate an ever-expanding role for melanopsin ganglion cells in image-forming vision.

The M6 cell: A small-field bistratified photosensitive retinal ganglion cell.

We have identified a novel, sixth type of intrinsically photosensitive retinal ganglion cell (ipRGC) in the mouse-the M6 cell. Its spiny, highly branched dendritic arbor is bistratified, with dendrites restricted to the inner and outer margins of the inner plexiform layer, co-stratifying with the processes of other ipRGC types. We show that M6 cells are by far the most abundant ganglion cell type labeled in adult pigmented Cdh3-GFP BAC transgenic mice. A few M5 ipRGCs are also labeled, but no other RGC types were encountered. Several distinct subnuclei in the geniculate complex and the pretectum contain labeled retinofugal axons in the Cdh3-GFP mouse. These are presumably the principle central targets of M6 cells (as well as M5 cells). Projections from M6 cells to the dorsal lateral geniculate nucleus were confirmed by retrograde tracing, suggesting they contribute to pattern vision. M6 cells have low levels of melanopsin expression and relatively weak melanopsin-dependent light responses. They also exhibit strong synaptically driven light responses. Their dendritic fields are the smallest and most abundantly branched of all ipRGCs. They have small receptive fields and strong antagonistic surrounds. Despite deploying dendrites partly in the OFF sublamina, M6 cells appear to be driven exclusively by the ON pathway, suggesting that their OFF arbor, like those of certain other ipRGCs, may receive ectopic input from passing ON bipolar cells axons in the OFF sublayer.

Where You Cut Matters: A Dissection and Analysis Guide for the Spatial Orientation of the Mouse Retina from Ocular Landmarks.

Accurately and reliably identifying spatial orientation of the isolated mouse retina is important for many studies in visual neuroscience, including the analysis of density and size gradients of retinal cell types, the direction tuning of direction-selective ganglion cells, and the examination of topographic degeneration patterns in some retinal diseases. However, there are many different ocular dissection methods reported in the literature that are used to identify and label retinal orientation in the mouse retina. While the method of orientation used in such studies is often overlooked, not reporting how retinal orientation is determined can cause discrepancies in the literature and confusion when attempting to compare data between studies. Superficial ocular landmarks such as corneal burns are commonly used but have recently been shown to be less reliable than deeper landmarks such as the rectus muscles, the choroid fissure, or the s-opsin gradient. Here, we provide a comprehensive guide for the use of deep ocular landmarks to accurately dissect and document the spatial orientation of an isolated mouse retina. We have also compared the effectiveness of two s-opsin antibodies and included a protocol for s-opsin immunohistochemistry. Because orientation of the retina according to the s-opsin gradient requires retinal reconstruction with Retistruct software and rotation with custom code, we have presented the important steps required to use both of these programs. Overall, the goal of this protocol is to deliver a reliable and repeatable set of methods for accurate retinal orientation that is adaptable to most experimental protocols. An overarching goal of this work is to standardize retinal orientation methods for future studies.

A novel map of the mouse eye for orienting retinal topography in anatomical space.

Functionally distinct retinal ganglion cells have density and size gradients across the mouse retina, and some degenerative eye diseases follow topographic-specific gradients of cell death. Hence, the anatomical orientation of the retina with respect to the orbit and head is important for understanding the functional anatomy of the retina in both health and disease. However, different research groups use different anatomical landmarks to determine retinal orientation (dorsal, ventral, temporal, nasal poles). Variations in the accuracy and reliability in marking these landmarks during dissection may lead to discrepancies in the identification and reporting of retinal topography. The goal of this study was to compare the accuracy and reliability of the canthus, rectus muscle, and choroid fissure landmarks in reporting retinal orientation. The retinal relieving cut angle made from each landmark during dissection was calculated based on its relationship to the opsin transition zone (OTZ), determined via a custom MATLAB script that aligns retinas from immunostained s-opsin. The choroid fissure and rectus muscle landmarks were the most accurate and reliable, while burn marks using the canthus as a reference were the least. These values were used to build an anatomical map that plots various ocular landmarks in relationship to one another, to the horizontal semicircular canals, to lambda-bregma, and to the earth's horizon. Surprisingly, during normal locomotion, the mouse's opsin gradient and the horizontal semicircular canals make equivalent 6° angles aligning the OTZ near the earth's horizon, a feature which may enhance the mouse's ability to visually navigate through its environment.

The Development of Mid-Wavelength Photoresponsivity in the Mouse Retina.

PURPOSE: Photoreceptors in the mouse retina express much of the molecular machinery necessary for phototransduction and glutamatergic transmission prior to eye opening at postnatal day 13 (P13). Light responses have been observed collectively from rod and cone photoreceptors via electroretinogram recordings as early as P13 in mouse, and the responses are known to become more robust with maturation, reaching a mature state by P30. Photocurrents from single rod outer segments have been recorded at P12, but no earlier, and similar studies on cone photoreceptors have been done, but only in the adult mouse retina. In this study, we wanted to document the earliest time point in which outer retinal photoreceptors in the mouse retina begin to respond to mid-wavelength light. METHODS: Ex-vivo electroretinogram recordings were made from isolated mouse retinae at P7, P8, P9, P10, and P30 at seven different flash energies (561 nm). The a-wave was pharmacologically isolated and measured at each developmental time point across all flash energies. RESULTS: Outer-retinal photoreceptors generated a detectable response to mid-wavelength light as early as P8, but only at photopic flash energies. a-wave intensity response curves and kinetic response properties are similar to the mature retina as early as P10. CONCLUSION: These data represent the earliest recorded outer retinal light responses in the rodent. Photoreceptors are electrically functional and photoresponsive prior to eye opening, and much earlier than previously thought. Prior to eye opening, critical developmental processes occur that have been thought to be independent of outer retinal photic modulation. However, these data suggest light acting through outer-retinal photoreceptors has the potential to shape these critical developmental processes.

Use and perceptions of plastination among medical anatomy educators in the United States.

Traditionally, medical schools have maintained collections of tissues/organs to engage students in anatomy. Such collections are often stored in volatile and toxic preservatives. Plastination is an alternative tissue preservation technique in which polymers replace water and lipids resulting in benign, dry, and anatomically authentic specimens. Plastination is used in medical education internationally; however, its use within U.S. medical schools is not widely discussed in the anatomical literature. This study aimed to determine the knowledge, use, and perceptions of plastination as a teaching tool among U.S. anatomy medical educators. A total of 98 medical anatomy educators who fit inclusion criteria and teach allopathic (MD) students and/or osteopathic (DO) students in the United States completed a national survey, representing 77 medical schools across 37 states. Of these, 100% had heard of plastination, 57% correctly defined plastination, but only 39% currently utilize plastinates for anatomy education. The most frequent explanation for nonuse of plastinates was a preference for the dissection experience, followed by lack of resources and negative past experiences related to durability and quality. A majority (75%) of U.S. medical anatomy educators perceived plastination as a good supplement to, but not a replacement for, cadaveric dissection, 19% indicated no curiosity to use plastination or considered it not useful, and 14% expressed ethical concerns. These findings suggest plastinates are more widely used in the United States than reflected by the literature; however, perceptions regarding their utility indicate a dominant theme for their use to supplement, not replace, cadaveric dissection. Clin. Anat, 2017. © 2017 Wiley Periodicals, Inc.

The M5 Cell: A Color-Opponent Intrinsically Photosensitive Retinal Ganglion Cell.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) combine direct photosensitivity through melanopsin with synaptically mediated drive from classical photoreceptors through bipolar-cell input. Here, we sought to provide a fuller description of the least understood ipRGC type, the M5 cell, and discovered a distinctive functional characteristic-chromatic opponency (ultraviolet excitatory, green inhibitory). Serial electron microscopic reconstructions revealed that M5 cells receive selective UV-opsin drive from Type 9 cone bipolar cells but also mixed cone signals from bipolar Types 6, 7, and 8. Recordings suggest that both excitation and inhibition are driven by the ON channel and that chromatic opponency results from M-cone-driven surround inhibition mediated by wide-field spiking GABAergic amacrine cells. We show that M5 cells send axons to the dLGN and are thus positioned to provide chromatic signals to visual cortex. These findings underscore that melanopsin's influence extends beyond unconscious reflex functions to encompass cortical vision, perhaps including the perception of color.

Novel dissection of the central nervous system to bridge gross anatomy and neuroscience for an integrated medical curriculum.

Medical schools in the United States continue to undergo curricular change, reorganization, and reformation as more schools transition to an integrated curriculum. Anatomy educators must find novel approaches to teach in a way that will bridge multiple disciplines. The cadaveric extraction of the central nervous system (CNS) provides an opportunity to bridge gross anatomy, neuroanatomy, and clinical neurology. In this dissection, the brain, brainstem, spinal cord, cauda equina, optic nerve/tract, and eyes are removed in one piece so that the entire CNS and its gateway to the periphery through the spinal roots can be appreciated. However, this dissection is rarely, if ever, performed likely due to time constraints, perceived difficulty, and lack of instructions. The goals of this project were (i) to provide a comprehensive, step-by-step guide for an en bloc CNS extraction and (ii) to determine effective strategies to implement this dissection/prosection within modern curricula. Optimal dissection methods were determined after comparison of various approaches/tools, which reduced dissection time from approximately 10 to 4 hours. The CNS prosections were piloted in small group sessions with two types of learners in two different settings: graduate students studied wet CNS prosections within the dissection laboratory and medical students used plastinated CNS prosections to review clinical neuroanatomy and solve lesion localization cases during their neurology clerkship. In both cases, the CNS was highly rated as a teaching tool and 98% recommended it for future students. Notably, 90% of medical students surveyed suggested that the CNS prosection be introduced prior to clinical rotations. Anat Sci Educ 11: 185-195. © 2017 American Association of Anatomists.

Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia.

Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that is caused by a mutation in the gene for inhibitor of kappa B kinase complex-associated protein (IKBKAP). Although FD patients suffer from multiple neuropathies, a major debilitation that affects their quality of life is progressive blindness. To determine the requirement for Ikbkap in the developing and adult retina, we generated Ikbkap conditional knockout (CKO) mice using a TUBA1a promoter-Cre (Tα1-Cre). In the retina, Tα1-Cre expression is detected predominantly in retinal ganglion cells (RGCs). At 6 months, significant loss of RGCs had occurred in the CKO retinas, with the greatest loss in the temporal retina, which is the same spatial phenotype observed in FD, Leber hereditary optic neuropathy, and dominant optic atrophy. Interestingly, the melanopsin-positive RGCs were resistant to degeneration. By 9 months, signs of photoreceptor degeneration were observed, which later progressed to panretinal degeneration, including RGC and photoreceptor loss, optic nerve thinning, Müller glial activation, and disruption of layers. Taking these results together, we conclude that although Ikbkap is not required for normal development of RGCs, its loss causes a slow, progressive RGC degeneration most severely in the temporal retina, which is later followed by indirect photoreceptor loss and complete retinal disorganization. This mouse model of FD is not only useful for identifying the mechanisms mediating retinal degeneration, but also provides a model system in which to attempt to test therapeutics that may mitigate the loss of vision in FD patients.

Development and assessment of a new 3D neuroanatomy teaching tool for MRI training.

A computerized three-dimensional (3D) neuroanatomy teaching tool was developed for training medical students to identify subcortical structures on a magnetic resonance imaging (MRI) series of the human brain. This program allows the user to transition rapidly between two-dimensional (2D) MRI slices, 3D object composites, and a combined model in which 3D objects are overlaid onto the 2D MRI slices, all while rotating the brain in any direction and advancing through coronal, sagittal, or axial planes. The efficacy of this tool was assessed by comparing scores from an MRI identification quiz and survey in two groups of first-year medical students. The first group was taught using this new 3D teaching tool, and the second group was taught the same content for the same amount of time but with traditional methods, including 2D images of brain MRI slices and 3D models from widely used textbooks and online sources. Students from the experimental group performed marginally better than the control group on overall test score (P = 0.07) and significantly better on test scores extracted from questions involving C-shaped internal brain structures (P < 0.01). Experimental participants also expressed higher confidence in their abilities to visualize the 3D structure of the brain (P = 0.02) after using this tool. Furthermore, when surveyed, 100% of the students in the experimental group recommended this tool for future students. These results suggest that this neuroanatomy teaching tool is an effective way to train medical students to read an MRI of the brain and is particularly effective for teaching C-shaped internal brain structures.

Melanopsin ganglion cells extend dendrites into the outer retina during early postnatal development.

Melanopsin ganglion cells express the photopigment melanopsin and are the first functional photoreceptors to develop in the mammalian retina. They have been shown to play a variety of important roles in visual development and behavior in the early postnatal period (Johnson et al., 2010; Kirkby and Feller, 2013; Rao et al., 2013; Renna et al., 2011). Here, we probed the maturation of the dendritic arbors of melanopsin ganglion cells during this developmental period in mice. We found that some melanopsin ganglion cells (mainly the M1-subtype) transiently extend their dendrites not only into the inner plexiform layer (where they receive synaptic inputs from bipolar and amacrine cells) but also into the outer plexiform layer, where in mature retina, rod and cone photoreceptors are thought to contact only bipolar and horizontal cells. Thus, some immature melanopsin ganglion cells are biplexiform. This feature is much less common although still present in the mature retina. It reaches peak incidence 8-12 days after birth, before the eyes open and bipolar cells are sufficiently mature to link rods and cones to ganglion cells. At this age, some outer dendrites of melanopsin ganglion cells lie in close apposition to the axon terminals of cone photoreceptors and express a postsynaptic marker of glutamatergic transmission, postsynaptic density-95 protein (PSD-95). These findings raise the possibility of direct, monosynaptic connections between cones and melanopsin ganglion cells in the early postnatal retina. We provide a detailed description of the developmental profile of these processes and consider their possible functional and evolutionary significance.