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.

A small population of retinal ganglion cells express the photopigment melanopsin and function as autonomous photoreceptors. They encode global luminance levels critical for light-mediated non-image forming visual processes including circadian rhythms and the pupillary light reflex. There are five melanopsin ganglion cell subtypes (M1–M5). M1 and displaced M1 (M1d) cells have dendrites that ramify within the outermost layer of the inner plexiform layer (IPL). It was recently discovered that some melanopsin ganglion cells extend dendrites into the outer retina. Outer Retinal Dendrites (ORDs) either ramify within the outer plexiform layer (OPL) or the inner nuclear layer (INL), and while present in the mature retina, are most abundant postnatally. Anatomical evidence for synaptic transmission between cone photoreceptor terminals and ORDs suggests a novel photoreceptor to ganglion cell connection in the mammalian retina. While it is known that the number of ORDs in the retina is developmentally regulated, little is known about the morphology, the cells from which they originate, or their spatial distribution throughout the retina. We analyzed the morphology of melanopsin-immunopositive ORDs in the OPL at different developmental time points in the mouse retina and identified five types of ORD originating from either M1 or M1d cells. However, a pattern emerges within these: ORDs from M1d cells are generally longer and more highly branched than ORDs from conventional M1 cells. Additionally, we found ORDs asymmetrically distributed to the dorsal retina. This morphological analysis provides the first step in identifying a potential role for biplexiform melanopsin ganglion cell ORDs.

SUMMARY Newborn neurons follow molecular cues to reach their final destination, but whether early life experience influences lamination remains largely unexplored. As light is among the first stimuli to reach the developing nervous system via intrinsically photosensitive retinal ganglion cells (ipRGCs), we asked whether ipRGCs could affect lamination in the developing mouse retina. We show here that ablation of ipRGCs causes cone photoreceptors to mislocalize at different apicobasal positions in the retina. This effect is partly mediated by light-evoked activity in ipRGCs, as dark rearing or silencing of ipRGCs leads a subset of cones to mislocalize. Furthermore, ablation of ipRGCs alters the cone transcriptome and decreases expression of the dopamine receptor D4, while injection of L-DOPA or D4 receptor agonist rescues the displaced cone phenotype observed in dark-reared animals. These results show that early light-mediated activity in ipRGCs influences neuronal lamination and identify ipRGC-elicited dopamine release as a mechanism influencing cone position.

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.

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.

PurposeThree dimensional (3D) printing technology is being increasingly utilized in anatomy education, and its efficacy as a teaching tool is a growing area of education research. Most curriculum guides for 3D printing activities have students download pre‐designed digital anatomical 3D object files from websites (i.e. www.thingiverse.comor www.neuromorpho.org) rather than generate their own 3D models from original data. Recently a technique was published for tracing and 3D‐printing neurons, but it required expensive software, as well as extensive computer programing experience (McDougal and Shepard, 2015). The goal of our study was to develop a simple, intuitive, and inexpensive method to allow students to generate an original reconstructed neuron suitable for 3D‐printing, and to evaluate this method among targeted learners.MethodsNeurons from mouse brain and retina were labeled with fluorescent dye, fixed and imaged sequentially in 1 μm sections with a Zeiss LSM laser scanning microscope. The image stack was loaded into ImageJ, traced, and morphologically analyzed using the free ImageJ plugin Simple Neurite Tracer (SNT). Traced neurons were then exported as an object file into Blender, a free 3D graphics software program, edited, and printed with PLA, ABA, and gypsum powder material, in various sizes and color patterns. Based on this work, a “3D Printing Neurons Made Easy” instruction guide and 5‐part tutorial video was crafted; the raw neuron image stack was published in The Cell Image Library, a freely accessible online public repository that students can access from any computer and trace to create their own digital 3D model. The curriculum was piloted in a neuroanatomy laboratory session for high school students in a summer neuroscience outreach program. Students were given a pre‐ and post‐test on topics related to microscopy, neuroanatomy and image analysis, as well as an exit survey.ResultsOur “3D Printing Neurons Made Easy” curriculum guide enabled students to participate in an optional, self‐guided activity to learn principles in confocal microscopy, neuroanatomy, and image analysis. By using open source software (Image J and Blender) and a public repository, this self‐guided activity engages students in neuroanatomy research at a relatively low cost. 70% students attempted (31/44) and of the 31 students that attempted the project, 87% of the students successfully printed a 3D neuron. 97% of these students had a positive experience with the activity and 80% agreed that they would recommend the activity to their high school biology class. The curriculum is now being piloted among undergraduate and graduate anatomy students, and analysis is ongoing.ConclusionsWe have developed an easy method for students to reconstruct z‐stack images of neurons for 3D printing. These 3D models provide dramatic examples of the complex structure of neurons, and may help communicate complex 3‐dimensional concepts to students. The activity also allows students with no prior research experience to learn principles in microscopy and 3D image analysis in a way that is fun, engaging, and low cost. Our “3D Printing Neurons Made Easy” instructions guide and tutorial videos may be useful for crowd‐sourcing data collection, anatomy education, training students in anatomical research methods, or attracting students to careers in research.

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