Inputs to parasol ganglion cells in the primate retina

Retinal ganglion cells carry signals from the eye to the brain. One of the most common types of ganglion cells is parasol cells. They have larger dendritic trees, somas and axons than other ganglion cells. While much was known about parasol cell light responses, little was known about how these responses are formed. One possibility is that they receive input from a unique set of local circuit neurons that have similar responses. The goal was to identify these presynaptic neurons and study their synaptic connectivity.^ Ganglion cells receive input from bipolar and amacrine cells, but there are numerous subtypes of each. To determine which of these were most likely to provide input to parasol cells, the parasol cells were intracellularly-injected and then various bipolar and amacrine cells were immunolabeled and the tissue analyzed using a confocal microscope. DB3 bipolar cells labeled with antibodies to calbindin made extensive contacts with OFF parasol cells. Antibodies to recover in labeled flat midget bipolar cells (FMB). They made only random contacts with OFF parasol cells, and they are not expected to provide significant input. Type DB2 bipolar cells and FMB cells labeled with antibodies to excitatory amino acid transporter-2 made extensive contacts with OFF parasol cells. This suggests that DB2 bipolar cells are likely to provide input to parasol cells.^ Two types of amacrine cells were labeled in material containing injected parasol cells. Cholinergic amacrine cells were labeled with antibodies to choline acetyltransferase, and they made extensive contacts with ON parasol cells. The large amacrine cells labeled with antibodies to a precursor of cholecystokinin were among the amacrine cells that are tracer-coupled to parasol cells.^ From electron microscopic (EM) analysis, most of the synapses made by DB3 axons were found on varicosities. Some postsynaptic and presynaptic amacrine cells resembled AII amacrine cells. Others were relatively electron-lucent and may be cholinergic amacrine cells or cholecystokinin-containing amacrine cells. Gap junctions were found between neighboring DB3 axons. They occurred whenever two axons contacted each other, and the junctions were as large as the area of contact. In double-label EM experiments, DB3 axons made synapses onto OFF parasol cells. ^

Confocal analysis of synaptic connectivity of the rod pathway in the rabbit retina

The retina is a specialized neuronal structure that transforms the optical image into electrical signals which are transmitted to the brain via the optic nerve. As part of the strategy to cover a stimulus range as broad as 10 log units, from dim starlight to bright sunlight, retinal circuits are broadly divided into rod and cone pathways, responsible for dark and light-adapted vision, respectively. ^ In this dissertation, confocal microscopy and immunocytochemical methods were combined to study the synaptic connectivity of the rod pathway from the level of individual synapses to whole populations of neurons. The study was focused on synaptic interactions at the rod bipolar terminal. The purpose is to understand the synaptic structure of the dyad synapse made by rod bipolar terminals, including the synaptic components and connections, and their physiological functions in the rod pathway. In addition, some additional components and connections of the rod pathway were also studied in these experiments. The major results can be summarized as following: At the dyad synapse of rod bipolar terminals, three postsynaptic components—processes of All amacrine cells and the varicosities of S1 or S2 amacrine cells express different glutamate receptor subunits, which may underlie the functional diversity of these postsynaptic neurons. A reciprocal feedback system is formed by rod bipolar terminals and S1/S2 amacrine cells. Analysis showed these two wide-field GABA amacrine cells have stereotyped synaptic connections with the appropriate morphology and distribution to perform specific functions. In addition, S1 and S2 cells have different coupling patterns and, in general, there is no coupling between the two types. Besides the classic rod pathway though rod bipolar cells and All amacrine cells, the finding of direct connections between certain types of OFF cone bipolar cells and rods indicates the presence of an alternative rod pathway in the rabbit retina. ^ In summary, this dissertation presents a detailed view of the connection and receptors at rod bipolar terminals. Based on the morphology, distribution and coupling, different functional roles were identified for S1 and S2 amacrine cells. Finally, an alternative to the classic rod pathway was found in the rabbit retina. ^

Depolarization-dependent synaptic potentiation and hyperexcitability induced by a Ca2+-independent trigger

Despite vast research efforts since Cajal's seminal thoughts on the adaptation of the nervous system, researchers have only recently begun to understand the diversity of forms of neuronal plasticity and its mechanisms. All known forms of activity-dependent neuronal plasticity utilize alterations in [Ca 2+]i as a signal of changes in the membrane voltage. Ca 2+ sensors trigger modifications in excitability or synaptic strength that last from seconds to weeks and presumably years. Intriguingly, Kunjilwar et al., (unpublished observations) discovered in peripheral sensory axons of Aplysia that the induction of depolarization-dependent long-term axonal hyperexcitability does not require Ca2+ transients. Here we show that induction of depolarization-dependent intermediate-term and long-term synaptic potentiation in Aplysia occurs in conditions that prevent Ca2+ entry through voltage-gated channels and elevation of [Ca2+]i. We found that the intermediate-term synaptic potentiation induced under conditions expected to prevent Ca 2+ transients is associated with increased excitability of sensory neuron axons near presynaptic terminals, suggesting that the synaptic potentiation involves a presynaptic locus. The Ca2+-independent intermediate- and long-term synaptic potentiation appeared similar to previously reported Ca2+-dependent modifications in Aplysia. ^

Spatial and temporal aspects of synaptic plasticity

The notion that changes in synaptic efficacy underlie learning and memory processes is now widely accepted even if definitive proof of the synaptic plasticity and memory hypothesis is still lacking. When learning occurs, patterns of neural activity representing the occurrence of events cause changes in the strength of synaptic connections within the brain. Reactivation of these altered connections constitutes the experience of memory for these events and for other events with which they may be associated. These statements summarize a long-standing theory of memory formation that we refer to as the synaptic plasticity and memory hypothesis. Since activity-dependent synaptic plasticity is induced at appropriate synapses during memory formation, and is both necessary and sufficient for the information storage, we can speculate that a methodological study of the synapse will help us understand the mechanism of learning. Random events underlie a wide range of biological processes as diverse as genetic drift and molecular diffusion, regulation of gene expression and neural network function. Additionally spatial variability may be important especially in systems with nonlinear behavior. Since synapse is a complex biological system we expect that stochasticity as well as spatial gradients of different enzymes may be significant for induction of plasticity. ^ In that study we address the question "how important spatial and temporal aspects of synaptic plasticity may be". We developed methods to justify our basic assumptions and examined the main sources of variability of calcium dynamics. Among them, a physiological method to estimate the number of postsynaptic receptors as well as a hybrid algorithm for simulating postsynaptic calcium dynamics. Additionally we studied how synaptic geometry may enhance any possible spatial gradient of calcium dynamics and how that spatial variability affect plasticity curves. Finally, we explored the potential of structural synaptic plasticity to provide a metaplasticity mechanism specific for the synapse. ^

THE FUNCTIONAL ORGANIZATION OF SYNAPTIC VESICLE POOLS IN A RETINAL BIPOLAR NEURON

Ribbon synapses are found in sensory systems and are characterized by ‘ribbon-like’ organelles that tether synaptic vesicles. The synaptic ribbons co-localize with sites of calcium entry and vesicle fusion, forming ribbon-style active zones. The ability of ribbon synapses to maintain rapid and sustained neurotransmission is critical for vision, hearing and balance. At retinal ribbon synapses, three vesicle pools have been proposed. A rapid pool of vesicles that are docked at the plasma membrane, and whose fusion is limited only by calcium entry, a releasable pool of ATP-primed vesicles whose size also correlates with the number of ribbon-tethered vesicles, and a reserve pool of non-ribbon-tethered cytoplasmic vesicles. However evidence of vesicle fusion at sites away from ribbon-style active zones questions this organization. Another fundamental question underlying the mechanism of vesicle fusion at these synapses is the role of SNARE (Soluble N-ethylmaleimide sensitive factor Attachment Protein Receptor) proteins. Vesicles at conventional neurons undergo SNARE complex-mediated fusion. However a recent study has suggested that ribbon synapses involved in hearing can operate independently of neuronal SNAREs. We used the well-characterized goldfish bipolar neuron to investigate the organization of vesicle pools and the role of SNARE proteins at a retinal ribbon synapse. We blocked functional refilling of the releasable pool and then stimulated bipolar terminals with brief depolarizations that triggered the fusion of the rapid pool of vesicles. We found that the rapid pool draws vesicles from the releasable pool and that both pools undergo release at ribbon-style active zones. To assess the functional role of SNARE proteins at retinal ribbon synapses, we used peptides derived from SNARE proteins that compete with endogenous proteins for SNARE complex formation. The SNARE peptides blocked fusion of reserve vesicles but not vesicles in the rapid and releasable pools, possibly because both rapid and releasable vesicles were associated with preformed SNARE complexes. However, an activity-dependent block in refilling of the releasable pool was seen, suggesting that new SNARE complexes must be formed before vesicles can join a fusion-competent pool. Taken together, our results suggest that SNARE complex-mediated exocytosis of serially-organized vesicle pools at ribbon-style active zones is important in the neurotransmission of vision.