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). Results: Dendritic changes were observed after three days post-ONT with significant decreases in all parameters at two weeks. In group 1 EG mice, mean (SD) intraocular pressure (IOP) was 15.2 (1.1) and 9.8 (0.3) mmHg in the EG and untreated contralateral eyes, respectively, with a significant corresponding decrease in AUC, PI, and PD, but not radius. In group 2 mice, the respective IOP was 13.1 (1.0) and 8.8 (0.1) mmHg, peaking at two months before trending towards baseline. Over the first two months, AUC, PI, and PD decreased significantly, with no further subsequent changes. The rates of change of the parameters after ONT was 5 to 10 times faster than in EG. Conclusions: Rapid dendritic changes occurred after ONT, while changes in EG were slower and associated with level of IOP increase. The earliest alterations were loss of inner neurites without change in dendritic field.

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. GCaMP3 expression was enhanced with green fluorescent protein (GFP) immunoreactivity. In one subset of animals, ONT was performed 3, 7, or 14 days before sacrifice (n = 4, 4, 4, respectively). Cells positive for GCaMP3, RBPMS, ChAT, as well as the population of co-labeled cells, were quantified. In another subset of animals (n = 4), in vivo confocal scanning laser ophthalmoscope imaging was performed in the same mice at baseline and at 3, 7 and 14 days after ONT. The mean (SD) densities of GCaMP3, RBPMS, and ChAT expressing cells in control retinas were 2663 (110), 3401 (175), and 1041 (47) cells/mm2, respectively. Of the GCaMP3+ cells, 92 (1)% were co-labeled with RBPMS, while 72 (1)% of RBPMS-labeled cells expressed GCaMP3. ChAT expressing cells were not co-labeled with GCaMP3. The number of GCaMP3+ and RBPMS+ cells decreased dramatically after ONT; 78%, 39%, and 18% of GCaMP3+ and 80%, 40%, and 15% of RBPMS+ cells, relative to control retinas, survived at 3, 7, and 14 days after ONT. However, the number of ChAT+ cells did not change. There was a progressive decrease in GCaMP3 fluorescence after ONT in in vivo images. The majority of RGCs in the ganglion cell layer of Thy1-GCaMP3 mice express GCaMP3. There was an expected progressive and specific loss of GCaMP3 expression after ONT. Thy1-GCaMP3 transgenic mice have potential for longitudinal assessment of RGCs in injury models.

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. NEW METHOD: Twelve retinas of mice expressing yellow fluorescent protein in RGCs under the control of the Thy1 promotor were whole-mounted. The entire dendritic arbor of 120 RGCs was traced, after which 2D and 3D Sholl analysis was performed. Two parameters describing dendritic complexity; area under the curve (AUC) and peak number of intersections (PNI) were then derived and analyzed. RESULTS AND COMPARISON WITH EXISTING METHODS: The AUC and PNI were significantly higher with 3D analysis compared to 2D analysis with medians of 2805 and 2508 units, and 31 and 27, respectively (P < 0.01). Both 2D and 3D AUC increased with arbor thickness. The discrepancy in AUC between the two methods depended on mean AUC (with every 1 unit increase in mean AUC resulting in a discrepancy of 0.1 unit), but not arbor thickness. CONCLUSION: In RGCs imaged in vitro, there is a difference in AUC and PNI derived with 2D and 3D Sholl analysis. Where possible, 3D Sholl analysis of RGCs should be performed for more accurate quantitative analysis of dendritic structure.

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 0.2% of RGCs. This reduced expression allows visualization of the full dendritic arbour of YFP-expressing RGCs, facilitating the investigation of structural changes. As susceptibility amongst RGCs varies with morphology and subtype, labelling methods should ideally non-discriminately label RGCs to accurately determine the effects of experimental glaucoma. This study therefore sought to determine morphological subtypes of RGCs in the Thy1-YFP mouse strain. Retinas from Thy1-YFP mice were imaged ex vivo with fluorescence microscopy. With Sholl analysis, a technique for quantifying the morphology of individual neurons, the dendritic field (DF), area under the curve (AUC), normalized AUC (Nav), peak number of intersections (PNI), and skew for single RGCs were computed. The distance of the RGC from the optic nerve head (dONH) was also measured. These morphological parameters were inputted into a multivariate cluster analysis to determine the optimal number of clusters to group all RGCs analyzed, which were then grouped into "Small", "Medium", and "Large" sized cluster groups according to increasing DF size. A total of 178 RGCs from 10 retinas of 8 mice were analyzed from which the cluster analysis identified 13 clusters. Eighty-eight (49%), 77 (43.2%), and 13 (7.3%) RGCs were grouped into small, medium and large clusters, respectively. Clusters 1-6 had small DFs. Clusters 1 and 3 had the lowest AUC and Nav. Clusters 2, 3, and 5 had asymmetric DFs while Clusters 3, 5, and 6 were distal to the ONH. Clusters 7-11 had medium DFs; of these, Clusters 7 and 10 had the lowest AUC, Clusters 8 and 10 had the highest skew, and Clusters 7 and 11 were closest to the ONH. Clusters 12 and 13 had large DFs. Both had low skew and high AUC. High PNI and dONH distinguished Cluster 12 from Cluster 13. We present the largest study to date examining YFP expression in RGCs of transgenic Thy1-YFP mice. Among the 13 clusters, there was a wide range of morphological features with further variation within size categories. Our findings support the notion that YFP is expressed non-discriminatingly in RGCs of Thy1-YFP transgenic mice and this strain is a valuable tool for studies of experimental optic neuropathies.

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). Calcium imaging was used to characterize functional responses of GCaMP3-expressing (GCaMP+) cells by recording calcium transients evoked by superfusion of kainic acid (KA; 10, 50, or 100 µM). In a subset of animals, optic nerve transection (ONT) was performed 3, 5, or 7 days prior to calcium imaging. Results: GFP immunoreactivity colocalized with RBPMS but not amacrine cell markers in both ONT and non-ONT (control) groups. Calcium transients evoked by KA were reduced after ONT (50 µM KA; ΔF/F0 [SD]; control: 1.00 [0.67], day 3: 0.50 [0.35], day 5: 0.31 [0.28], day 7: 0.35 [0.36]; P < 0.05 versus control). There was also a decrease in the number of GCaMP3+ cells after ONT (cells/mm2 [SD]; control: 2198 [453], day 3: 2224 [643], day 5: 1383 [375], day 7: 913 [178]; P < 0.05). Furthermore, the proportion of GCaMP3+ cells that responded to KA decreased after ONT (50 µM KA, 97%, 54%, 47%, and 58%; control, 3, 5, and 7 days, respectively). Conclusions: Following ONT, functional RGC responses are lost prior to the loss of RGC somata, suggesting that anatomical markers of RGCs may underestimate the extent of RGC dysfunction.

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. Moreover, experimental GON provides the ability to quantify and monitor risk factors leading to RGC loss such as the level of intraocular pressure, axonal health and the RGC population. Using these experimental models we are able to gain a better understanding of GON, which allows for the development of potential neuroprotective strategies. Here we review the advantages and disadvantages of the relevant and most often utilized methods for evaluating axonal degeneration and RGC loss in GON. Axonal pathology in GON includes functional disruption of axonal transport (AT) and structural degeneration. Horseradish peroxidase (HRP), rhodamine-B-isothiocyanate (RITC) and cholera toxin-B (CTB) fluorescent conjugates have proven to be effective reporters of AT. Also, immunohistochemistry (IHC) for endogenous AT-associated proteins is often used as an indicator of AT function. Similarly, structural degeneration of axons in GON can be investigated via changes in the activity and expression of key axonal enzymes and structural proteins. Assessment of axonal degeneration can be measured by direct quantification of axons, qualitative grading, or a combination of both methods. RGC loss is the most frequently quantified variable in studies of experimental GON. Retrograde tracers can be used to quantify RGC populations in rodents via application to the superior colliculus (SC). In addition, in situ IHC for RGC-specific proteins is a common method of RGC quantification used in many studies. Recently, transgenic mouse models that express fluorescent proteins under the Thy-1 promoter have been examined for their potential to provide specific and selective labeling of RGCs for the study of GON. While these methods represent important advances in assessing the structural and functional integrity of RGCs, each has its advantages and disadvantages; together they provide an extensive toolbox for the study of GON.

Somatostatin receptor subtype 4 modulates L-type calcium channels via Gßγ 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. Application of the selective sst4 agonist, L-803 (10 nM), reduced ICa by 41.2%. Pretreatment of cells with pertussis toxin (Gi/o inhibitor) did not prevent the action of L-803, which reduced ICa by 34.7%. To determine the involvement of Gßγ subunits after sst4 activation, depolarizing pre-pulse facilitation paradigms were used to remove voltage-dependent inhibition of calcium channels. Pre-pulse facilitation did not reverse the inhibitory effects of L-803 on ICa (8.4 vs. 8.8% reductions, ctrl vs. L-803); however, pharmacologic inhibition of Gßγ reduced ICa suppression by L-803 (23.0%, P < 0.05). Inhibition of PKC (GF109203X; GFX) showed a concentration-dependent effect in preventing the action of L-803 on ICa (1 µM GFX, 34.3%; 5 µM GFX, 14.6%, P < 0.05). When both PKC and Gßγ were inhibited, the effects of L-803 on ICa were blocked (1.8%, P < 0.05). These results suggest that sst4 stimulation modulates RGC calcium channels via Gßγ and PKC activation. Since reducing intracellular Ca(2+) is known to be neuroprotective in RGCs, modulating these sst4 signaling pathways may provide insights to the discovery of unique therapeutic targets to reduce intracellular Ca(2+) levels in RGCs.

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). Whole cell patch clamp was used to record ICa in isolated RGCs, and calcium imaging was used to measure Ca2+ transients from RGCs in situ. Immunohistochemistry was used to detect the presence of α2δ1-containing VGCCs in isolated RGCs in the absence and presence of gabapentin pretreatment. Acute administration of gabapentin reduced ICa and Ca2+ transients compared to control conditions. In isolated RGCs, pretreatment with gabapentin (4-18 h) reduced ICa, and cell surface α2δ1 staining was reduced compared to nonpretreated cells. Acute administration of gabapentin to isolated RGCs that had been pretreated further reduced ICa. These results show that gabapentin has both short-term and long-term mechanisms to reduce ICa in isolated RGCs. Some Ca2+ channel blockers have been shown to protect RGCs in retinal trauma suggesting that modulation of VGCCs by gabapentin may prevent the deleterious effects of elevated Ca2+ levels in RGCs in trauma and disease.

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 51.1 ± 13.1% at 0 mV and suppressed Ca(2+) channel currents by 32.5 ± 9.4% at -10 mV in whole cell patch-clamped ganglion cells. The N-type Ca(2+) channel blocker ω-conotoxin GVIA (CTX, 1 µM) reduced L-type Ca(2+) current (I(Ca)) in ganglion cells by 43.5 ± 7.2% at -10 mV, after which addition of L-803,087 further reduced I(Ca) by 28.0 ± 16.0% . In contrast, ganglion cells treated first with nifedipine (NIF, 10 µM), which blocked 46.1 ± 3.5% of the control current at -10 mV, did not undergo any further reduction in I(Ca) in the presence of L-803,087 (-3.5 ± 3.8% vs. NIF), showing that stimulation of sst(4) reduces Ca(2+) influx through L-type Ca(2+) channels. To assess the effects of sst(4) stimulation on intracellular Ca(2+) levels ([Ca(2+)](i)) in ganglion cells, fura-2 was used to measure changes in [Ca(2+)](i) in response to depolarization induced by elevated [K(+)](o). [Ca(2+)](i) was increased to a lesser extent (86%) in the presence of L-803,087 compared with recordings made in the absence of the sst(4) agonist and this effect was blocked by NIF (10 µM). Suppression of spiking and Ca(2+) signaling via sst(4) may contribute to the reported neuroprotective actions of somatostatin and promote ganglion cell survival following ischemia and axonal trauma.

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. Action potentials were similar in male and female myocytes, but contractions were smaller and slower in females. In voltage-clamped cells, peak contractions were smaller in females than in males (5.1 +/- 0.7% vs. 7.7 +/- 0.8% diastolic length, P < 0.05). Similarly, Ca(2+) transients were smaller in females than in males and the rate of rise of the Ca(2+) transient was slower in females. Despite smaller contractions and Ca(2+) transients in females, Ca(2+) current density was similar in both groups. Sarcoplasmic reticulum Ca(2+) content, assessed with caffeine, did not differ between the sexes. However, E-C coupling gain (rate of Ca(2+) release/Ca(2+) current) was smaller in females than in males (157.0 +/- 15.6 vs. 338.4 +/- 54.3 (nM/s)/(pA/pF), P < 0.05). To determine whether the reduced gain in female cells was due to changes in unitary Ca(2+) release, spontaneous Ca(2+) sparks were evaluated (fluo-4, 37 degrees C). Spark frequencies and widths were similar in both groups, but spark amplitudes were smaller in females than in males (0.56 +/- 0.01 vs. 0.64 +/- 0.01 DeltaF/F(0), P < 0.05). Spark durations also were shorter in females than in males (full duration at half-maximum = 14.86 +/- 0.17 vs. 16.25 +/- 0.27 ms, P < 0.05). These observations suggest that decreases in the size and duration of Ca(2+) sparks contributes to the decrease in E-C coupling gain in female myocytes. Thus, differences in cardiac contractile function arise, in part, from differences in unitary Ca(2+) release between the sexes.

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. There was no age-related difference in contraction or Ca(2+) transient amplitudes in response to a beta(2)-AR agonist. The direct adenylate cyclase agonist forskolin caused smaller increases in contraction and Ca(2+) transient amplitudes in aged compared to younger cells. Phosphodiesterase inhibitors did not reverse the age-related deficit in positive inotropy caused by beta-AR stimulation. Direct measurement of cAMP showed significantly less cAMP formation in response to either beta-AR or adenylate cyclase stimulation in aged compared to younger cells. However, responses to dibutyryl cAMP were similar in young adult and aged myocytes, suggesting that events downstream of cAMP formation are not affected by age. The age-related decrease in catecholamine sensitivity is mediated by beta(1)-ARs, resulting in a defect in cAMP production.

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. Contractile responses to the non-selective beta-AR agonist, isoproterenol were significantly decreased in aged myocytes compared to younger myocytes and aged myocytes were less sensitive to isoproterenol. In contrast, Ca(2+) transients measured simultaneously with contractions were similar between groups. Isoproterenol increased sarcoplasmic reticulum Ca(2+) stores in both groups, but the increase was larger in aged cells. However, signs of Ca(2+) overload induced by isoproterenol were reduced with age. Diastolic Ca(2+) accumulation, contracture and the incidences of transient inward current, oscillatory afterpotentials (OAPs), aftertransients and aftercontractions induced by isoproterenol also were reduced with age. These results demonstrate that aged myocytes exhibit fewer signs of Ca(2+) overload in response to isoproterenol than young adult myocytes. These age-related changes in intracellular Ca(2+) may protect the aging heart against induction of arrhythmias initiated by OAPs.(1).