Synaptic Origins of the Complex Receptive Field Structure in Primate Smooth Monostratified Retinal Ganglion Cells.

Considerable progress has been made in studying the receptive fields of the most common primate retinal ganglion cell (RGC) types, such as parasol RGCs. Much less is known about the rarer primate RGC types and the circuitry that gives rise to noncanonical receptive field structures. The goal of this study was to analyze synaptic inputs to smooth monostratified RGCs to determine the origins of their complex spatial receptive fields, which contain isolated regions of high sensitivity called "hotspots." Interestingly, smooth monostratified RGCs co-stratify with the well-studied parasol RGCs and are thus constrained to receiving input from bipolar and amacrine cells with processes sharing the same layer, raising the question of how their functional differences originate. Through 3D reconstructions of circuitry and synapses onto ON smooth monostratified and ON parasol RGCs from central macaque retina, we identified four distinct sampling strategies employed by smooth and parasol RGCs to extract diverse response properties from co-stratifying bipolar and amacrine cells. The two RGC types differed in the proportion of amacrine cell input, relative contributions of co-stratifying bipolar cell types, amount of synaptic input per bipolar cell, and spatial distribution of bipolar cell synapses. Our results indicate that the smooth RGC's complex receptive field structure arises through spatial asymmetries in excitatory bipolar cell input which formed several discrete clusters comparable with physiologically measured hotspots. Taken together, our results demonstrate how the striking differences between ON parasol and ON smooth monostratified RGCs arise from distinct strategies for sampling a common set of synaptic inputs.

Trogocytosis of neurons and glial cells by microglia in a healthy adult macaque retina.

Microglial cells are the primary resident immune cells in the retina. In healthy adults, they are ramified; that is, they have extensive processes that move continually. In adult retinas, microglia maintain the normal structure and function of neurons and other glial cells, but the mechanism underlying this process is not well-understood. In the mouse hippocampus, microglia engulf small pieces of axons and presynaptic terminals via a process called trogocytosis. Here we report that microglia in the adult macaque retina also engulf pieces of neurons and glial cells, but not at sites of synapses. We analyzed microglia in a volume of serial, ultrathin sections of central macaque retina in which many neurons that ramify in the inner plexiform layer (IPL) had been reconstructed previously. We surveyed the IPL and identified the somas of microglia by their small size and scant cytoplasm. We then reconstructed the microglia and studied their interactions with other cells. We found that ramified microglia frequently ingested small pieces of each major type of inner retinal neuron and Müller glial cells via trogocytosis. There were a few instances where the interactions took place near synapses, but the synapses, themselves, were never engulfed. If trogocytosis by retinal microglia plays a role in synaptic remodeling, it was not apparent from the ultrastructure. Instead, we propose that trogocytosis enables these microglia to present antigens derived from normal inner retinal cells and, when activated, they would promote antigen-specific tolerance.

Stephen Massey: a career in visual neuroscience.

This review is a memoir by Dr. Stephen C. Massey's longtime collaborator, Dr. Stephen L. Mills, and written, for the most part, in the first person. It also serves as an introduction to the virtual festschrift to celebrate Dr. Massey's retirement. and. The references cited here are only a few of the highlights of Dr. Massey's distinguished career. A complete list is found here: https://pubmed.ncbi.nlm.nih.gov/?term=massey+sc+%28retina+or+photoreceptors%29&sort=date.

Synaptic inputs to displaced intrinsically-photosensitive ganglion cells in macaque retina.

Ganglion cells are the projection neurons of the retina. Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and also receive input from rods and cones via bipolar cells and amacrine cells. In primates, multiple types of ipRGCs have been identified. The ipRGCs with somas in the ganglion cell layer have been studied extensively, but less is known about those with somas in the inner nuclear layer, the "displaced" cells. To investigate their synaptic inputs, three sets of horizontal, ultrathin sections through central macaque retina were collected using serial block-face scanning electron microscopy. One displaced ipRGC received nearly all of its excitatory inputs from ON bipolar cells and would therefore be expected to have ON responses to light. In each of the three volumes, there was also at least one cell that had a large soma in the inner nuclear layer, varicose axons and dendrites with a large diameter that formed large, extremely sparse arbor in the outermost stratum of the inner plexiform layer. They were identified as the displaced M1 type of ipRGCs based on this morphology and on the high density of granules in their somas. They received extensive input from amacrine cells, including the dopaminergic type. The vast majority of their excitatory inputs were from OFF bipolar cells, including two subtypes with extensive input from the primary rod pathway. They would be expected to have OFF responses to light stimuli below the threshold for melanopsin or soon after the offset of a light stimulus.

Synaptic inputs to broad thorny ganglion cells in macaque retina.

In primates, broad thorny retinal ganglion cells are highly sensitive to small, moving stimuli. They have tortuous, fine dendrites with many short, spine-like branches that occupy three contiguous strata in the middle of the inner plexiform layer. The neural circuits that generate their responses to moving stimuli are not well-understood, and that was the goal of this study. A connectome from central macaque retina was generated by serial block-face scanning electron microscopy, a broad thorny cell was reconstructed, and its synaptic inputs were analyzed. It received fewer than 2% of its inputs from both ON and OFF types of bipolar cells; the vast majority of its inputs were from amacrine cells. The presynaptic amacrine cells were reconstructed, and seven types were identified based on their characteristic morphology. Two types of narrow-field cells, knotty bistratified Type 1 and wavy multistratified Type 2, were identified. Two types of medium-field amacrine cells, ON starburst and spiny, were also presynaptic to the broad thorny cell. Three types of wide-field amacrine cells, wiry Type 2, stellate wavy, and semilunar Type 2, also made synapses onto the broad thorny cell. Physiological experiments using a macaque retinal preparation in vitro confirmed that broad thorny cells received robust excitatory input from both the ON and the OFF pathways. Given the paucity of bipolar cell inputs, it is likely that amacrine cells provided much of the excitatory input, in addition to inhibitory input.

Molecular and functional architecture of the mouse photoreceptor network.

Mouse photoreceptors are electrically coupled via gap junctions, but the relative importance of rod/rod, cone/cone, or rod/cone coupling is unknown. Furthermore, while connexin36 (Cx36) is expressed by cones, the identity of the rod connexin has been controversial. We report that FACS-sorted rods and cones both express Cx36 but no other connexins. We created rod- and cone-specific Cx36 knockout mice to dissect the photoreceptor network. In the wild type, Cx36 plaques at rod/cone contacts accounted for more than 95% of photoreceptor labeling and paired recordings showed the transjunctional conductance between rods and cones was ~300 pS. When Cx36 was eliminated on one side of the gap junction, in either conditional knockout, Cx36 labeling and rod/cone coupling were almost abolished. We could not detect direct rod/rod coupling, and cone/cone coupling was minor. Rod/cone coupling is so prevalent that indirect rod/cone/rod coupling via the network may account for previous reports of rod coupling.

Wide-field amacrine cell inputs to ON parasol ganglion cells in macaque retina.

Parasol cells are one of the major types of primate retinal ganglion cells. The goal of this study was to describe the synaptic inputs that shape the light responses of the ON type of parasol cells, which are excited by increments in light intensity. A connectome from central macaque retina was generated by serial blockface scanning electron microscopy. Six neighboring ON parasol cells were reconstructed, and their synaptic inputs were analyzed. On average, they received 21% of their input from bipolar cells, excitatory local circuit neurons receiving input from cones. The majority of their input was from amacrine cells, local circuit neurons of the inner retina that are typically inhibitory. Their contributions to the neural circuit providing input to parasol cells are not well-understood, and the focus of this study was on the presynaptic wide-field amacrine cells, which provided 17% of the input to ON parasol cells. These are GABAergic amacrine cells with long, relatively straight dendrites, and sometimes also axons, that run in a single, narrow stratum of the inner plexiform layer. The presynaptic wide-field amacrine cells were reconstructed, and two types were identified based on their characteristic morphology. One presynaptic amacrine cell was identified as semilunar type 2, a polyaxonal cell that is electrically coupled to ON parasol cells. A second amacrine was identified as wiry type 2, a type known to be sensitive to motion. These inputs likely make ON parasol cells more sensitive to stimuli that are rapidly changing outside their classical receptive fields.

An S-cone circuit for edge detection in the primate retina.

Midget retinal ganglion cells (RGCs) are the most common RGC type in the primate retina. Their responses have been proposed to mediate both color and spatial vision, yet the specific links between midget RGC responses and visual perception are unclear. Previous research on the dual roles of midget RGCs has focused on those comparing long (L) vs. middle (M) wavelength sensitive cones. However, there is evidence for several other rare midget RGC subtypes receiving S-cone input, but their role in color and spatial vision is uncertain. Here, we confirm the existence of the single S-cone center OFF midget RGC circuit in the central retina of macaque monkey both structurally and functionally. We investigated the receptive field properties of the S-OFF midget circuit with single cell electrophysiology and 3D electron microscopy reconstructions of the upstream circuitry. Like the well-studied L vs. M midget RGCs, the S-OFF midget RGCs have a center-surround receptive field consistent with a role in spatial vision. While spectral opponency in a primate RGC is classically assumed to contribute to hue perception, a role supporting edge detection is more consistent with the S-OFF midget RGC receptive field structure and studies of hue perception.

Synaptic inputs from identified bipolar and amacrine cells to a sparsely branched ganglion cell in rabbit retina.

There are more than 30 distinct types of mammalian retinal ganglion cells, each sensitive to different features of the visual environment. In rabbit retina, they can be grouped into four classes according to their morphology and stratification of their dendrites in the inner plexiform layer (IPL). The goal of this study was to describe the synaptic inputs to one type of Class IV ganglion cell, the third member of the sparsely branched Class IV cells (SB3). One cell of this type was partially reconstructed in a retinal connectome developed using automated transmission electron microscopy (ATEM). It had slender, relatively straight dendrites that ramify in the sublamina a of the IPL. The dendrites of the SB3 cell were always postsynaptic in the IPL, supporting its identity as a ganglion cell. It received 29% of its input from bipolar cells, a value in the middle of the range for rabbit retinal ganglion cells studied previously. The SB3 cell typically received only one synapse per bipolar cell from multiple types of presumed OFF bipolar cells; reciprocal synapses from amacrine cells at the dyad synapses were infrequent. In a few instances, the bipolar cells presynaptic to the SB3 ganglion cell also provided input to an amacrine cell presynaptic to the ganglion cell. There was apparently no crossover inhibition from narrow-field ON amacrine cells. Most of the amacrine cell inputs were from axons and dendrites of GABAergic amacrine cells, likely providing inhibitory input from outside the classical receptive field.

Wavy multistratified amacrine cells in the monkey retina contain immunoreactive secretoneurin.

The goals of this study were to describe the morphology, neurotransmitter content and synaptic connections of neurons in primate retinas that contain the neuropeptide secretoneurin. Amacrine cells were labeled with antibodies to secretoneurin in macaque and baboon retinas. Their processes formed three distinct plexuses in the inner plexiform layer: one in the outermost stratum, one in the center and one in the innermost stratum. In light microscopic double immunolabeling experiments, GABA was colocalized with secretoneurin in these cells, but glycine transporter 1 and Substance P were not. ON bipolar cell axon terminals labeled with antibody to the cholecystokinin precursor, G6-gly, have ON responses to stimulation of short wavelength sensitive (S) cones. Axons of these bipolar cells made contacts with amacrine cell dendrites containing secretoneurin. Secretoneurin-IR amacrine cells also made contacts with retinal ganglion cell dendrites labeled with antibody to the photopigment melanopsin, which have OFF responses to stimulation of S cones. Using electron microscopic immunolabeling, 436 synapses from macaque retina were analyzed. Axons from bipolar cells were identified by their characteristic synaptic ribbons; their synaptic densities were asymmetric like those of excitatory synapses in the brain. Amacrine cells made and received conventional synapses with symmetric synaptic densities, like those of inhibitory synapses in the brain. Ganglion cell dendrites were identified by their absence of presynaptic specializations; they received inputs from both amacrine cells and bipolar cells. The majority of inputs to the secretoneurin-IR amacrine cells were from other amacrine cells, but they also received 21% of their input from bipolar cells. They directed most of their output, 54%, to amacrine cells, but there were many synapses onto bipolar cell axons and ganglion cell dendrites, as well. The synaptic connections were very similar in the three plexuses with one notable exception; output synapses to bipolar cells were significantly less common in the innermost one, where the S-ON bipolar cells terminate. Taken together, these findings suggest that the secretoneurin-IR amacrine cells in primates receive excitatory input from S-ON bipolar cells and, in turn, inhibit intrinsically photosensitive retinal ganglion cells.

Wide-field diffuse amacrine cells in the monkey retina contain immunoreactive Cocaine- and Amphetamine-Regulated Transcript (CART).

The goals of this study were to localize the neuropeptide Cocaine- and Amphetamine-Regulated Transcript (CART) in primate retinas and to describe the morphology, neurotransmitter content and synaptic connections of the neurons that contain it. Using in situ hybridization, light and electron microscopic immunolabeling, CART was localized to GABAergic amacrine cells in baboon retinas. The CART-positive cells had thin, varicose dendrites that gradually descended through the inner plexiform layer and ramified extensively in the innermost stratum. They resembled two types of wide-field diffuse amacrine cells that had been described previously in macaque retinas using the Golgi method and also A17, serotonin-accumulating and waterfall cells of other mammals. The CART-positive cells received synapses from rod bipolar cell axons and made synapses onto the axons in a reciprocal configuration. The CART-positive cells also received synapses from other amacrine cells. Some of these were located on their primary dendrites, and the presynaptic cells there included dopaminergic amacrine cells. Although some CART-positive somas were localized in the ganglion cell layer, they did not contain the ganglion cell marker RNA binding protein with multiple splicing (RBPMS). Based on these results and electrophysiological studies in other mammals, the CART-positive amacrine cells would be expected to play a major role in the primary rod pathway of primates, providing feedback inhibition to rod bipolar cells.

Melanopsin-expressing ganglion cells on macaque and human retinas form two morphologically distinct populations.

The long-term goal of this research is to understand how retinal ganglion cells that express the photopigment melanopsin, also known as OPN4, contribute to vision in humans and other primates. Here we report the results of anatomical studies using our polyclonal antibody specifically against human melanopsin that confirm and extend previous descriptions of melanopsin cells in primates. In macaque and human retina, two distinct populations of melanopsin cells were identified based on dendritic stratification in either the inner or the outer portion of the inner plexiform layer (IPL). Variation in dendritic field size and cell density with eccentricity was confirmed, and dendritic spines, a new feature of melanopsin cells, were described. The spines were the sites of input from DB6 diffuse bipolar cell axon terminals to the inner stratifying type of melanopsin cells. The outer stratifying melanopsin type received inputs from DB6 bipolar cells via a sparse outer axonal arbor. Outer stratifying melanopsin cells also received inputs from axon terminals of dopaminergic amacrine cells. On the outer stratifying melanopsin cells, ribbon synapses from bipolar cells and conventional synapses from amacrine cells were identified in electron microscopic immunolabeling experiments. Both inner and outer stratifying melanopsin cell types were retrogradely labeled following tracer injection in the lateral geniculate nucleus (LGN). In addition, a method for targeting melanopsin cells for intracellular injection using their intrinsic fluorescence was developed. This technique was used to demonstrate that melanopsin cells were tracer coupled to amacrine cells and would be applicable to electrophysiological experiments in the future. J. Comp. Neurol. 524:2845-2872, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

A tale of two neurotransmitters.

Amacrine cells are a diverse set of local circuit neurons of the inner retina, and they all release either GABA or glycine, amino acid neurotransmitters that are generally inhibitory. But some types of amacrine cells have another function besides inhibiting other neurons. One glycinergic amacrine cell, the Aii type, excites a subset of bipolar cells via extensive gap junctions while inhibiting others at chemical synapses. Many types of GABAergic amacrine cells also release monoamines, acetylcholine, or neuropeptides. There is now good evidence that another type of amacrine cell releases glycine at some of its synapses and releases the excitatory amino acid glutamate at others. The glutamatergic synapses are made onto a subset of retinal ganglion cells and amacrine cells and have the asymmetric postsynaptic densities characteristic of central excitatory synapses. The glycinergic synapses are made onto other types of ganglion cells and have the symmetric postsynaptic densities characteristic of central inhibitory synapses. These amacrine cells, which contain vesicular glutamate transporter 3, will be the focus of this brief review.

Synaptic connections of amacrine cells containing vesicular glutamate transporter 3 in baboon retinas.

The goals of these experiments were to describe the morphology and synaptic connections of amacrine cells in the baboon retina that contain immunoreactive vesicular glutamate transporter 3 (vGluT3). These amacrine cells had the morphology characteristic of knotty bistratified type 1 cells, and their dendrites formed two plexuses on either side of the center of the inner plexiform layer. The primary dendrites received large synapses from amacrine cells, and the higher-order dendrites were both pre- and postsynaptic to other amacrine cells. Based on light microscopic immunolabeling results, these include AII cells and starburst cells, but not the polyaxonal amacrine cells tracer-coupled to ON parasol ganglion cells. The vGluT3 cells received input from ON bipolar cells at ribbon synapses and made synapses onto OFF bipolar cells, including the diffuse DB3a type. Many synapses from vGluT3 cells onto retinal ganglion cells were observed in both plexuses. At synapses where vGluT3 cells were presynaptic, two types of postsynaptic densities were observed; there were relatively thin ones characteristic of inhibitory synapses and relatively thick ones characteristic of excitatory synapses. In the light microscopic experiments with Neurobiotin-injected ganglion cells, vGluT3 cells made contacts with midget and parasol ganglion cells, including both ON and OFF types. Puncta containing immunoreactive gephyrin, an inhibitory synapse marker, were found at appositions between vGluT3 cells and each of the four types of labeled ganglion cells. The vGluT3 cells did not have detectable levels of immunoreactive γ-aminobutyric acid (GABA) or immunoreactive glycine transporter 1. Thus, the vGluT3 cells would be expected to have ON responses to light and make synapses onto neurons in both the ON and the OFF pathways. Taken with previous results, these findings suggest that vGluT3 cells release glycine at some of their output synapses and glutamate at others.

Outcomes of a rotational dissection system in gross anatomy.

At the University of Texas Houston Medical School, a rotational dissection system was introduced to improve coordination between the Gross Anatomy and the Introduction to Clinical Medicine (ICM) courses. Six students were assigned to each cadaver and divided into two teams. For each laboratory, one team was assigned to dissect and the other to attend ICM or study independently. For the next laboratory, the assignments were reversed. At the start of the session, the team that had dissected previously spent 30 minutes teaching the other team. In 2012, the students were given three traditional practical examinations with 50 questions drawn equally from each laboratory. Students also completed three mid-course evaluations. There were no significant differences in overall performance between the two teams. Nevertheless, we wanted to determine how well individual students identified structures they had dissected compared with those they had not. For dissected structures, the mean percent correct was 80.0 ± 13.0 (mean ± standard deviation), and for undissected structures, it was 78.3 ± 14.1. The difference was small, but statistically significant (P = 0.0007). Although this result validated the concerns expressed by some students, it did not appear that a change in the system was justified. Students were generally enthusiastic about the opportunity to learn clinical skills in the first semester of medical school, and 91-96% of the students agreed that learning anatomy at the same time helped them understand the physical examination exercises in ICM.

Short-wavelength cone-opponent retinal ganglion cells in mammals.

In all of the mammalian species studied to date, the short-wavelength-sensitive (S) cones and the S-cone bipolar cells that receive their input are very similar, but the retinal ganglion cells that receive synapses from the S-cone bipolar cells appear to be quite different. Here, we review the literature on mammalian retinal ganglion cells that respond selectively to stimulation of S-cones and respond with opposite polarity to longer wavelength stimuli. There are at least three basic mechanisms to generate these color-opponent responses, including: (1) opponency is generated in the outer plexiform layer by horizontal cells and is conveyed to the ganglion cells via S-cone bipolar cells, (2) inputs from bipolar cells with different cone inputs and opposite response polarity converge directly on the ganglion cells, and (3) inputs from S-cone bipolar cells are inverted by S-cone amacrine cells. These are not mutually exclusive; some mammalian ganglion cells that respond selectively to S-cone stimulation seem to utilize at least two of them. Based on these findings, we suggest that the small bistratified ganglion cells described in primates are not the ancestral type, as proposed previously. Instead, the known types of ganglion cells in this pathway evolved from monostratified ancestral types and became bistratified in some mammalian lineages.

Histamine receptors of cones and horizontal cells in Old World monkey retinas.

In primates the retina receives input from histaminergic neurons in the posterior hypothalamus that are active during the day. In order to understand how this input contributes to information processing in Old World monkey retinas, we have been localizing histamine receptors (HR) and studying the effects of histamine on the neurons that express them. Previously, we localized HR3 to the tips of ON bipolar cell dendrites and showed that histamine hyperpolarizes the cells via this receptor. We raised antisera against synthetic peptides corresponding to an extracellular domain of HR1 between the 4th and 5th transmembrane domains and to an intracellular domain near the carboxyl terminus of HR2. Using these, we localized HR1 to horizontal cells and a small number of amacrine cells and localized HR2 to puncta closely associated with synaptic ribbons inside cone pedicles. Consistent with this, HR1 mRNA was detected in horizontal cell perikarya and primary dendrites and HR2 mRNA was found in cone inner segments. We studied the effect of 5 µM exogenous histamine on primate cones in macaque retinal slices. Histamine reduced I(h) at moderately hyperpolarized potentials, but not the maximal current. This would be expected to increase the operating range of cones and conserve ATP in bright, ambient light. Thus, all three major targets of histamine are in the outer plexiform layer, but the retinopetal axons containing histamine terminate in the inner plexiform layer. Taken together, the findings in these three studies suggest that histamine acts primarily via volume transmission in primate retina.

Testing to enhance retention in human anatomy.

Recent work in cognitive psychology has shown that repeatedly testing one's knowledge is a powerful learning aid and provides substantial benefits for retention of the material. To apply this in a human anatomy course for medical students, 39 fill-in-the-blank quizzes of about 50 questions each, one for each region of the body, and four about the nervous system, were developed. The quizzes were optional, and no credit was awarded. They were posted online using Blackboard, which provided feedback, and they were very popular. To determine whether the quizzes had any effect on retention, they were given in a controlled setting to 21 future medical and dental students. The weekly quizzes included questions on regional anatomy and an expanded set of questions on the nervous system. Each question about the nervous system was given three times, in a slightly different form each time. The second quiz was given approximately half an hour after the first one, and the third was given one week after the second to assess retention. The quizzes were unpopular, but students showed robust improvement on the questions about the nervous system. The scores increased by almost 9% on the second quiz, with no intervention except viewing the correct answers. The scores were 29% higher on the third quiz than on the first, and there was also a positive correlation between the grades on the quizzes and the final examination. Thus, repeated testing is an effective strategy for learning and retaining information about human anatomy.