Correlated Variations in the Parameters That Regulate Dendritic Calcium Signaling in Mouse Retinal Ganglion Cells

Gartland, Andrew J.; Detwiler, Peter B.

doi:10.1523/jneurosci.4212-11.2011

Cited by 5 publications

(5 citation statements)

References 45 publications

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“…In vivo, RGCs respond to light by opening plasma membrane voltage-gated Ca 2+ channels with a corresponding increase of intracellular calcium, generating an action potential that is transmitted to the brain (Gartland and Detwiler, 2011); while in vitro, RGCs can be directly simulated by small molecules. Here, we use two different molecules: potassium chloride, which in high concentrations is known to cause an influx of Ca 2+ into neurons, and glutamate, an excitatory neurotransmitter secreted by bipolar cells in vivo which increases cytosolic calcium by activating NMDA Summary of both immunopanning and low-pressure cell sorting protocols for isolation of RGCs from postnatal rodents.…”

Section: Calcium Imaging Resultsmentioning

confidence: 99%

Purification of retinal ganglion cells using low-pressure flow cytometry

Mcloughlin,

Aladdad,

Payne

et al. 2023

Front. Mol. Neurosci.

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Purified Retinal Ganglion Cells (RGCs) for in vitro study have been a valuable tool in the study of neural regeneration and in the development of therapies to treat glaucoma. Traditionally, RGCs have been isolated from early postnatal rats and mice, and more recently from human in vitro derived retinal organoids using a two-step immunopanning technique based upon the expression of Thy-1. This technique, however, limits the time periods from which RGCs can be isolated, missing the earliest born RGCs at which time the greatest stage of axon growth occurs, as well as being limited in its use with models of retinal degeneration as Thy-1 is downregulated following injury. While fluorescence associated cell sorting (FACS) in combination with new optogenetically labeled RGCs would be able to overcome this limitation, the use of traditional FACS sorters has been limited to genomic and proteomic studies, as RGCs have little to no survival post-sorting. Here we describe a new method for RGC isolation utilizing a combined immunopanning-fluorescence associated cell sorting (IP-FACS) protocol that initially depletes macrophages and photoreceptors, using immunopanning to enrich for RGCs before using low-pressure FACS to isolate these cells. We demonstrate that RGCs isolated via IP-FACS when compared to RGCs isolated via immunopanning at the same age have similar purity as measured by antibody staining and qRT-PCR; survival as measured by live dead staining; neurite outgrowth; and electrophysiological properties as measured by calcium release response to glutamate. Finally, we demonstrate the ability to isolate RGCs from early embryonic mice prior to the expression of Thy-1 using Brn3b-eGFP optogenetically labeled cells. This method provides a new approach for the isolation of RGCs for the study of early developed RGCs, the study of RGC subtypes and the isolation of RGCs for cell transplantation studies.

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Section: Calcium Imaging Resultsmentioning

confidence: 99%

Purification of retinal ganglion cells using low-pressure flow cytometry

Mcloughlin,

Aladdad,

Payne

et al. 2023

Front. Mol. Neurosci.

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“…These were G3, G5, G6, G7, G11, G12, G15, and G17 ganglion cells [32] , which represent a wide range of physiological and structural properties. For example, G3 cells are the OFF alpha ganglion cells [36] – [38] , G12 cells are large bistratified ganglion cells [39] , G15 cells are the OFF type of direction selective ganglion cell [40] , [41] , and G17 cells are the ON-OFF type of direction selective ganglion cell [39] , [42] – [44] .…”

Section: Resultsmentioning

confidence: 99%

Spatial Distribution of Excitatory Synapses on the Dendrites of Ganglion Cells in the Mouse Retina

Chen

Chiao

2014

PLoS ONE

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Excitatory glutamatergic inputs from bipolar cells affect the physiological properties of ganglion cells in the mammalian retina. The spatial distribution of these excitatory synapses on the dendrites of retinal ganglion cells thus may shape their distinct functions. To visualize the spatial pattern of excitatory glutamatergic input into the ganglion cells in the mouse retina, particle-mediated gene transfer of plasmids expressing postsynaptic density 95-green fluorescent fusion protein (PSD95-GFP) was used to label the excitatory synapses. Despite wide variation in the size and morphology of the retinal ganglion cells, the expression of PSD95 puncta was found to follow two general rules. Firstly, the PSD95 puncta are regularly spaced, at 1–2 µm intervals, along the dendrites, whereby the presence of an excitatory synapse creates an exclusion zone that rules out the presence of other glutamatergic synaptic inputs. Secondly, the spatial distribution of PSD95 puncta on the dendrites of diverse retinal ganglion cells are similar in that the number of excitatory synapses appears to be less on primary dendrites and to increase to a plateau on higher branch order dendrites. These observations suggest that synaptogenesis is spatially regulated along the dendritic segments and that the number of synaptic contacts is relatively constant beyond the primary dendrites. Interestingly, we also found that the linear puncta density is slightly higher in large cells than in small cells. This may suggest that retinal ganglion cells with a large dendritic field tend to show an increased connectivity of excitatory synapses that makes up for their reduced dendrite density. Mapping the spatial distribution pattern of the excitatory synapses on retinal ganglion cells thus provides explicit structural information that is essential for our understanding of how excitatory glutamatergic inputs shape neuronal responses.

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“…Within the retina, κ endo in ganglion cell somata is reported to decline from 575 in early postnatal retinas to ∼120 in adult RGCs (Mann et al, ). In the dendrites of ganglion cells, however, κ endo varies from cell‐to‐cell, ranging from 2 to 123 (Gartland and Detwiler, ). These values are correlated with other parameters of Ca 2+ handling such as extrusion rate constant and total Ca 2+ influx, suggesting that they work together in a homeostatic fashion to ensure uniform Ca 2+ handling in RGC dendrites.…”

Section: Discussionmentioning

confidence: 99%

Endogenous calcium buffering at photoreceptor synaptic terminals in salamander retina

Hook

Thoreson

2014

Synapse

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Calcium operates by several mechanisms to regulate glutamate release at rod and cone synaptic terminals. In addition to serving as the exocytotic trigger, Ca2+ accelerates replenishment of vesicles in cones and triggers Ca2+-induced Ca2+ release (CICR) in rods. Ca2+ thereby amplifies sustained exocytosis, enabling photoreceptor synapses to encode constant and changing light. A complete picture of the role of Ca2+ in regulating synaptic transmission requires an understanding of the endogenous Ca2+ handling mechanisms at the synapse. We therefore used the “added buffer” approach to measure the endogenous Ca2+ binding ratio (κendo) and extrusion rate constant (γ) in synaptic terminals of photoreceptors in retinal slices from tiger salamander. We found that κendo was similar in both cell types - approximately 25 and 50 in rods and cones, respectively. Using measurements of the decay time constants of Ca2+ transients, we found that γ was also similar, with values of approximately 100 s−1 and 160 s−1 in rods and cones, respectively. The measurements of κendo differ considerably from measurements in retinal bipolar cells, another ribbon-bearing class of retinal neurons, but are comparable to similar measurements at other conventional synapses. The values of γ are slower than at other synapses, suggesting that Ca2+ ions linger longer in photoreceptor terminals, supporting sustained exocytosis, CICR, and Ca2+-dependent ribbon replenishment. The mechanisms of endogenous Ca2+ handling in photoreceptors are thus well-suited for supporting tonic neurotransmission. Similarities between rod and cone Ca2+ handling suggest that neither buffering nor extrusion underlie differences in synaptic transmission kinetics.

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Correlated Variations in the Parameters That Regulate Dendritic Calcium Signaling in Mouse Retinal Ganglion Cells

Cited by 5 publications

References 45 publications

Purification of retinal ganglion cells using low-pressure flow cytometry

Purification of retinal ganglion cells using low-pressure flow cytometry

Spatial Distribution of Excitatory Synapses on the Dendrites of Ganglion Cells in the Mouse Retina

Endogenous calcium buffering at photoreceptor synaptic terminals in salamander retina

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