Longitudinal In Vivo Changes in Retinal Ganglion Cell Dendritic Morphology After Acute and Chronic Optic Nerve Injury.

Purpose: To characterize in vivo dendritic changes in retinal ganglion cells (RGCs) after acute (optic nerve transection, ONT) and chronic (experimental glaucoma, EG) optic nerve injury.

Methods: ONT and EG (microbead model) were carried out in Thy1-YFP mice in which the entire RGC dendritic arbor was imaged with confocal fluorescence scanning laser ophthalmoscopy over two weeks in the ONT group and over two and six months, respectively, in two (groups 1 and 2) EG groups. Sholl analysis was used to quantify dendritic structure with the parameters: area under the curve (AUC), radius of the dendritic field, peak number of intersections (PI), and distance to the PI (PD).

GCaMP3 expressing cells in the ganglion cell layer of Thy1-GCaMP3 transgenic mice before and after optic nerve injury.

The genetically encoded green fluorescent protein-based calcium sensor, GCaMP, has been used to detect calcium transients and report neuronal activity. We evaluated the specificity of GCaMP3 expression to retinal ganglion cells (RGCs) of the transgenic Thy1-GCaMP3 mouse line in healthy control animals and in those after optic nerve transection (ONT). Retinas from control mice (n = 4) were isolated and stained for RNA-binding protein with multiple splicing (RBPMS) and choline acetyltransferase (ChAT), specific markers for RGCs and cholinergic amacrine cells, respectively.

Effects of 3D Stratification of Retinal Ganglion Cells in Sholl Analysis.

Background: Sholl analysis is used to quantify the dendritic complexity of neurons. Differences between two-dimensional (2D) and three-dimensional (3D) Sholl analysis can exist in neurons with extensive axial stratification of dendrites, however, in retinal ganglion cells (RGCs), only 2D analysis is typically reported despite varying degrees of stratification within the retinal inner plexiform layer. We determined the impact of this stratification by comparing 2D and 3D analysis of the same RGCs.

Morphological multivariate cluster analysis of murine retinal ganglion cells selectively expressing yellow fluorescent protein.

Optic neuropathies, such as glaucoma, lead to retinal ganglion cell (RGC) death. Transgenic mouse strains that express fluorescent proteins under the control of the Thy1 promoter have permitted single RGC imaging. Specifically, in one strain of mice expressing yellow fluorescent protein (Thy1-YFP), fluorescence is expressed in only 0.

Retinal Characterization of the Thy1-GCaMP3 Transgenic Mouse Line After Optic Nerve Transection.

Purpose: GCaMP3 is a genetically encoded calcium indicator for monitoring intracellular calcium dynamics. We characterized the expression pattern and functional properties of GCaMP3 in the Thy1-GCaMP3 transgenic mouse retina.

Methods: To determine the specificity of GCaMP3 expression, Thy1-GCaMP3 (B6; CBA-Tg(Thy1-GCaMP3)6Gfng/J) retinas were processed for immunohistochemistry with anti-green fluorescent protein (anti-GFP, to enhance GCaMP3 fluorescence), anti-RBPMS (retinal ganglion cell [RGC]-specific marker), and antibodies against amacrine cell markers (ChAT, GABA, GAD67, syntaxin).

Simultaneous in vivo confocal reflectance and two-photon retinal ganglion cell imaging based on a hollow core fiber platform.

We have developed a compact hollow core fiber (HCF)-based imaging platform capable of simultaneous in vivo confocal reflectance and two-photon imaging through the mouse pupil. We demonstrate the performance of this platform by imaging retinal ganglion cells (RGCs) in which the fluorophores YFP and GCaMP3 are expressed in Thy1-YFP-16 and Thy1-GCaMP3 transgenic mice, respectively. Confocal reflectance images of the mouse retina served as a reference for the simultaneous acquisition of the two-photon signals that clearly showed RGCs with single-cell resolution.

Assessment of retinal ganglion cell damage in glaucomatous optic neuropathy: Axon transport, injury and soma loss.

Glaucoma is a disease characterized by progressive axonal pathology and death of retinal ganglion cells (RGCs), which causes structural changes in the optic nerve head and irreversible vision loss. Several experimental models of glaucomatous optic neuropathy (GON) have been developed, primarily in non-human primates and, more recently and commonly, in rodents. These models provide important research tools to study the mechanisms underlying glaucomatous damage.

Somatostatin receptor subtype 4 modulates L-type calcium channels via Gβγ and PKC signaling in rat retinal ganglion cells.

Somatostatin subtype-4 receptors (sst4) inhibit L-type calcium channel currents (ICa) in retinal ganglion cells (RGCs). Here we identify the signaling pathways involved in sst4 stimulation leading to suppression of ICa in RGCs. Whole cell patch clamp recordings were made on isolated immunopanned RGCs using barium as a charge carrier to isolate ICa.

Modulation of voltage-gated Ca2+ channels in rat retinal ganglion cells by gabapentin.

The α2δ auxiliary subunits of voltage-gated Ca2+ channels (VGCCs) are important modulators of VGCC function. Gabapentin interacts with α2δ1 and α2δ2 subunits and is reported to reduce Ca2+ channel current amplitude (ICa). This study aimed to determine the effects of gabapentin on VGCCs in retinal ganglion cells (RGCs).

Modulation of voltage-gated ion channels in rat retinal ganglion cells mediated by somatostatin receptor subtype 4.

Somatostatin (somatotropin release-inhibiting factor [SRIF]) is known to modulate the excitability of retinal ganglion cells, but the membrane mechanisms responsible and the extent to which intracellular calcium signaling is affected have not been determined. We show that somatostatin receptor subtype 4 (sst(4)) is expressed specifically in rat ganglion cells and that the generation of repetitive action potentials by isolated ganglion cells is reduced in the presence of L-803,087, a selective sst(4) agonist (10 nM). Under voltage clamp, L-803,087 increased outward K(+) currents by 51.

Sex differences in mechanisms of cardiac excitation-contraction coupling in rat ventricular myocytes.

Components of excitation-contraction (E-C) coupling were compared in ventricular myocytes isolated from 3-mo-old male and female rats. Ca(2+) concentrations (fura-2) and cell shortening (edge detector) were measured simultaneously (37 degrees C). Membrane potential and ionic currents were measured with microelectrodes.

The age-related decrease in catecholamine sensitivity is mediated by beta(1)-adrenergic receptors linked to a decrease in adenylate cyclase activity in ventricular myocytes from male Fischer 344 rats.

This study determined whether reduced sensitivity to catecholamines in aged myocytes resulted from deficits in the beta-adrenergic receptor (beta-AR) signaling pathway. Contractions and intracellular Ca(2+) were measured simultaneously in field-stimulated (2Hz, 37 degrees C, fura-2) ventricular myocytes isolated from young adult ( approximately 3 months) and aged ( approximately 24 months) male Fischer 344 rats. Higher concentrations of a beta(1)-AR agonist were required to increase contraction amplitudes in aged compared to younger cells; however, Ca(2+) transients were similar in both groups.

The effects of isoproterenol on abnormal electrical and contractile activity and diastolic calcium are attenuated in myocytes from aged Fischer 344 rats.

We investigated whether the age-related decrease in sensitivity of the heart to catecholamines was accompanied by changes in Ca(2+) homeostasis and abnormal electrical and contractile activity caused by beta-adrenergic receptor (beta-AR) stimulation. Ventricular myocytes were isolated from young adult (3 months) and aged (24 months) male Fischer 344 rats. Unloaded cell shortening was measured in field-stimulated myocytes (2Hz, 37 degrees C); membrane currents and action potentials were measured with microelectrodes.

Novel jadomycins: incorporation of non-natural and natural amino acids.

Electrospray ionization mass spectrometry of extracts from Streptomyces venezuelae ISP5230 cultures grown on chemically synthesized non-natural L-amino acids, D-amino acids or any of the 20 natural amino acids demonstrated incorporation of the amino acid into a jadomycin B analogue.