Вы находитесь на странице: 1из 13

Ophthalmic Genetics, 31(2), 5365, 2010 Copyright 2010 Informa Healthcare USA, Inc.

. ISSN: 1381-6810 print/ 1744-5094 online DOI: 10.3109/13816811003698117

ORIGINAL ARTICLE

Characterization of Ca2+ Signalling in Postnatal Mouse Retinal Ganglion Cells: Involvement of OPA1 in Ca2+ Clearance
Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

Govindan Dayanithi1,2,3,4,*, Murielle Chen-Kuo-Chang1,2,3,*, Cedric Viero1,5, Christian Hamel1,2,3, Agns Muller1,2,3, and Guy Lenaers1,2,3
Institut des Neurosciences de Montpellier, INSERM U583, Montpellier, France 2 Universit de Montpellier, Mdicine 1, Montpellier France 3 Universit de Montpellier 2, Montpellier, France 4 Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic 5 Wales Heart Research Institute, Cardiff, United Kingdom
1

Abstract Purpose: The regulation of Ca entry and removal is a fine-tuned process which remains not well understood in mouse retinal ganglion cells (RGCs). The latter are known to be sensitive to dysfunctions of mitochondria, organelles playing a pivotal role in Ca2+ reuptake. Methods: We first described the Ca2+ signals of RGCs in response to varied drugs with Fura-2 imaging, and secondly tested the role of optic atrophy 1 or OPA1, the gene responsible for Auto somal Dominant Optic Atrophy, on mitochondrial ability to capture intracellular Ca2+ in cells transfected with the OPA1 small interfering ribonucleic acids (siRNAs). Results: In control RGCs, K+-evoked [Ca2+]i increase was blocked by the Ca2+ channel antagonists (Ni2++ Cd2+) and GABAA receptor agonist muscimol-induced [Ca2+]i responses were attenuated by the GABAA receptor antagonists, picrotoxin and gabazine. We also prove the presence of NMDA and AMPA/Kainate (glutamate receptor agonists) responsive receptors in this model. Application of cyclopiazonic acid, an inhibitor of Ca2+-ATPase pumps of the intracellular Ca2+ stores, induced an increase in [Ca2+]i while ryanodine or caffeine had no effect on resting [Ca2+]i. Spontaneous Ca2+ oscillations in contacting neurons highlighted the importance of cross-talks between RGCs during maturation. The mitochondrial respiration uncoupler, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), induced robust raises of intracellular Ca2+ after K+ application, with a more pronounced effect in cells silenced for OPA1, which could lead to cell death. Conclusions: Our results indicate an important role of OPA1 in mitochondrial dependent Ca2+ homeostasis and cell survival in RGCs, suggesting a possible patho-physiological mechanism involved in inherited optic neuropathies.
2+

KEYWORDS: Retinal ganglion cells; Ca2+ homeostasis; Ca2+ clearance; Mitochondria; Optic atrophies

INTRODUCTION
Optic neuropathies are a common cause of vision
Received 24 November 2009; Revised 08 January 2010; Accepted 11 February 2010 *Equal contributors to this work. Correspondence: Guy Lenaers, INSERM U583, Institut des Neurosciences de Montpellier, BP 74103, 80 Rue Augustin Fliche, F-34091 Montpellier cedex 5, France. E-mail: guy.lenaers@inserm.fr

loss and blindness. Indeed, functional impairment of the optic nerve affects the transmission of the visual information, collected and encoded by the retina, to the brain cortical region devoted to visual information analysis. Retinal ganglion cell (RGC) death is the unifying feature of all diseases of the optic nerve, including glaucomatous optic neuropathy, one of the most common causes of blindness. 53

54 G. Dayanithi etal. In glaucoma, there may be more than one pathway leading to RGC death, but much attention has been focused on glutamate-mediated excitotoxicity.1 In non-glaucomatous optic neuropathies, mitochondrial dysfunction is considered as the central hallmark of the neuronal degeneration. This is particularly evident for inherited optic neuropathies (ION), that to the present day are all related to genes encoding mitochondrial proteins.2 For maternally transmitted Leber ION, three principal mutations in the mitochondrial DNA, mainly in ND1, ND4 and ND6 genes, affecting the complex I (NADH oxydo-reductase) from the respiratory chain, are responsible for severe acute optic nerve dysfunction followed by RGC degeneration.3,4 In the case of Dominant Optic Atrophies, which are chronic IONs, mutations in the optic atrophy 1 (OPA1) and OPA3 genes have been identified, both encoding mitochondrial proteins acting on the inner membrane.5,6 [Ca2+]i plays a major role in cellular signalling7 and in the control of several neuronal functions,8,9 during development of peripheral nervous system,10 aging11 and cell death.12 The increase in [Ca2+]i leads to activation of specific cellular functions depending on several cellular factors, which co-ordinate the control of Ca2+ homeostasis. Over four decades, mitochondria has been shown to be an important organelle for Ca2+ homeostasis, acting as a major Ca2+ reservoir by taking up large Ca2+ loads.13 Affecting mitochondrial function can lead to many neurodegenerative diseases as demonstrated in MERFF syndrome (or Myoclonic Epilepsy with Ragged Red Fibers) and Huntington diseases.14 Although mitochondrial dysfunction in RGC is the purpose of many studies, the role of [Ca2+]i in ION patho-physiological conditions has modestly been addressed so far,15 and unfortunately the understanding of the Ca2+ signalling underlying these defects still remains poor. [Ca2+]i regulation in neurons involves Ca2+ entry by different receptors, voltage-gated Ca2+ channels localized to the plasma membrane, and Ca2+ release from intracellular stores, followed by different buffering mechanisms to restore basal [Ca2+]i level.16 Such mechanisms include Ca2+ pumps in both plasma membrane and endoplasmic reticulum (ER),17 plasma membrane Na+/Ca2+ exchangers,18 as well as mitochondrial Ca2+ uptake.19 To achieve the mitochondrial clearance of cytoplasmic Ca2+, the mitochondrial network has to be properly and specifically distributed as spatial buffers close to Ca2+ enriched micro domains, situated in the vicinity of plasma membrane Ca2+ channels and the ER.20,21 In addition, for proper Ca2+ clearance, the maintenance of the inner mitochondrial membrane integrity and in particular of the inner membrane potential is required by the low affinity mitochondrial uniporter, to import Ca2+ in the matrix.13 Consequently, we can expect that dysfunction of OPA1, an inner mitochondrial dynamin, which is involved in fusion of the mitochondrial network, in maintenance of the mitochondrial inner membrane potential (m) and in preventing pro-apoptotic cytochrome c release,22 could seriously affect the mitochondrial Ca2+ clearance23 in RGCs. Though [Ca2+]i responses to conventional agonists, developmental changes of voltage-gated Ca2+ currents and global Ca2+ dynamics were previously described in retinal ganglion cells,2426 little is known about the mechanisms underlying the Ca2+ handling in these sensory neurons, especially during degenerative remodelling. In the present study, we first characterized the [Ca2+]i increase induced by various ionotropic and metabotropic agonists and then analyzed the mechanisms involved in [Ca2+]i regulation in mouse RGCs primary cultures, isolated from post natal (P2) animals, at a stage when the retina is still developing and RGCs are immature, in which we could knock down OPA1 by gene silencing. In these cells, we demonstrated different plasma membrane channels and intracellular [Ca2+]i stores, and highlighted the critical involvement of mitochondria and OPA1 in Ca2+ clearance. We suggest that patho-physiological mitochondrial defects in Ca2+ clearance could be responsible of RGC degeneration in OPA1 patients.

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

MATERIALS AND METHODS


All animal experimentations were performed in accordance to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research FrenchEuropean regulations. Unless otherwise stated, all the standard chemicals were purchased from Sigma-Aldrich-France.

Retinal Ganglion Cell Purification and Culture


Post natal (P2) C57BL/6J mouse (Janvier Institute, Le Genest-St-Isle, France) were decapitated, the retinas were removed and transferred in warm (37C) phosphate buffered saline (PBS, Gibco). Retinas were then dissociated enzymatically in PBS, for 30min at 37C, containing papain (33U/ml, Worthington Biochemical Corporation, NJ) and then mechanically and gently triturated in PBS containing 1% ovomucoid (Worthington Biochemical Corporation, NJ) and DNase (100U/ml). The dissociated RGCs were then incubated for 15min at 4C with mouse anti-mouse CD90 (Thy1.2) antibodies conjugated to microbeads (Macs Cells Separation Columns, Miltenyi Biotech, France) in PBS with 0.2%
Ophthalmic Genetics

Ca2+ handling in mouse retinal ganglion neurones 55 BSA. After washing, cells were eluted using a plunger (Miltenyi Biotech). Approximately 20 000 cells per cm2 of purified RGCs were cultured onto glass bottom dishes (HBSt or GWSt-3522 series; 22mm diameter, 0.17mm thickness; WillCo Wells BV, Netherlands) coated with poly-D-lysine (20 g/ml for 2h at 37C). Cells were incubated in supplemented Neurobasal as previously described27 at 37C in a 5% CO2 humidified atmosphere. [Ca2+]i measurements were performed after 7 days in culture. Spontaneous Ca2+ oscillations were observed on 15 day-old cultures inoculated at a density of 2 million cells per cm2. siRNA transfections were performed after 4 days of culture, and cells were processed 7 days later for the [Ca2+]i measurements. (Molecular Probes-Invitrogen, France) for 30min, then fixed and stained for DAPI. Mitochondrial membrane potential was assessed using JC1 labelling (5g/ml) on life cells for 30min in normal growth conditions. TUNEL identification of apoptotic cells was performed according to the manufactures protocol (Apoptag Apoptosis Detection Systems-Chemicon). Fluorescence staining was recorded with a confocal system (Biorad MRC 1024) equipped with a Zeiss Axiovert 100 microscope. Image acquisition was undertaken with the software LaserSharp (Biorad).

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

Intracellular [Ca2+]i Measurements


Stock solutions of all drugs used were prepared in appropriate solvents as recommended by the suppliers, and then diluted to working solutions before use. For dye loading and measurement of [Ca2+]i on single cells, the cultured RGCs were loaded by incubation with 2 M Ca2+-sensitive dye Fura-2/AM (dissolved in DMSO) plus 0.2% pluronic F-127 (dissolved in water; Molecular Probes Inc. Eugene, OR, USA) in Lockes buffer which contains (mM): NaCl, 140; KCl, 5; MgCl2, 1.2; CaCl2, 1.8; glucose, 10; HEPES, 10; pH 7.25 adjusted with Tris. Buffer osmolarity was 300 mosmol/l. Dye loading was carried out for 40min at room temperature (22C). The [Ca2+]i in single neurons was measured as previously described.30 Briefly, fluorescence measurements of [Ca2+]i were recorded with a Zeiss Microscope Photometer System (FFP, Zeiss, Oberkochen, Germany), coupled to an inverted microscope (Axiovert 100, Zeiss) equipped for epifluorescence and 100 X oil immersion objective (Plan-Neofluar /1.30, Zeiss). With fluorescence values corrected for background and dark current, [Ca2+]i was calculated from the ratio between 340 and 380nm recordings. The calculation of the fluorescence signal, in terms of free-Ca2+ concentration, was based on a well-known procedure.31 In RGCs, after calibration at room temperature, the parameters Kd=224nm, Rmin=0.196, Rmax=5.362 and =6.871 were used to calculate [Ca2+]i values from measured data.

Gene Silencing Strategy


For siRNA experiments, we followed a well-established method as previously described by our group.22,28 Briefly, target-specific siRNA (AA(N19)UU) were designed (5'-aagucaucagucugagccagguu-3') corresponding to position c.1811-1833 of the mouse OPA1 open reading frame (GenBank accession number AY510274) and synthesized in duplex form (Dharmacond Research, CO, USA). The scramble siRNAs (5'- agguaguguaaucgccuuguu -3') were used as a negative control.

Biochemistry Analysis: Western Blot


Western blotting was performed on aliquots containing 50 g of proteins mixed with gel loading buffer, separated on 7.5% SDS-polyacrylamide gels electrophoresis and transferred onto PVDF (Polyvinylidene Difluoride Hybond P membrane, Amersham Biosciences) membranes. Membranes were blocked with 5% fat-free dry milk in between (0.5%) Tris buffer at pH 7.5, incubated overnight with antibodies against monoclonal mouse -actin (1/50 000), and OPA1 (1/300),29 then incubated with appropriate anti-mouse and anti-rabbit IgG antibodies conjugated to alkaline phosphatase (1/5 000) at room temperature for 1 hour. Finally, antibodies were revealed with BCIP-NBT (Sigma-Aldrich) substrate and band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Results are based on four independent siRNA transfection experiments.

Drugs
Unless otherwise stated, all chemicals (glutamate, 100 M; NMDA, 100 M; GABA, 100 M; picrotoxin, 50 M; gabazine, 10 M; CCCP, 10 M; and all the other compounds mentioned below) were purchased from Sigma-France. Concentrated stock DMSO solutions of cyclopiazonic acid (CPA, 500nM and 1 M) and ryanodine (100nM; Alomone Labs, Israel) were stored at -20C. Caffeine (20mM; Almone Labs, Isreal) was dissolved directly in the working buffer

Confocal Analysis
For mitochondrial structure identification, cells were incubated with 100nM CMXros Mitotracker Red
2010 Informa Healthcare USA, Inc.

56 G. Dayanithi etal. at appropriate concentrations. Muscimol (used at 10 and 50 M) was diluted with 0.05 N HCl. Test solutions were prepared daily using aliquots from frozen stocks to obtain the working concentrations. Drug Application The control and test solutions were applied using a multiple capillary perfusion system (200 m inner diameter capillary Tygon tubing, flow rate 250 l/min) placed in the close proximity of each cell (<0.2mm). After mounting the culture dish onto the microscope stage, selected neurons were subjected to a close proximal constant buffer perfusion with an identical inflow and outflow rates in order to keep constant the buffer volume of about 400 l in the culture dish. Each capillary was fed by a reservoir situated 60cm above the sample and connected to a temperature control device (Harvard-France). [Ca2+]i measurements were performed at 35C. With this device, switching the flow from one capillary to the next resulted in complete solution change within a few seconds. After each drug application, cells were washed with control buffer as described above. the [Ca2+]i increase induced by exposure to high K+ (50mM KCl) at two different time intervals. Increasing the K+ concentration in the perfusion solution from 5 to 50mM for 30s caused a fast and large increase in [Ca2+]i in all cells tested (Figure 1A). The response was monophasic and presented a clear fast transient high K+-evoked [Ca2+]i increase reaching 70223nM (n=14) with a fast transient [Ca2+]i decay the K+ concentration was returned to 5mM. Reduction of external Ca2+ from 2.2mM to 100nM in the perfusion solution (buffered with 2mM EGTA) did not affect resting [Ca2+]i but drastically reduced (90%; data not shown) the high K+induced [Ca2+]i increase, suggesting that the major part of the [Ca2+]i increase depends on the Ca2+ influx and resulted from Ca2+ entry through VGCC.

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

Effect of Ca2+ Channel Blockers


To investigate the presence of high and low voltagegated (HVA / LVA) Ca2+ channels in RGCs, we tested the high K+-evoked [Ca2+]i increase in the presence of 100 M Cd2+, which antagonizes HVA Ca2+ channels and 50 M Ni2+, which blocks LVA Ca2+ channels. Figure 1A shows a typical recording of the [Ca2+]i increase induced by exposure to high K+. The cells were first depolarized with high K+ as control stimulus and then pre-incubated during 2min with the blockers before challenging with high K+. The high K+-induced responses were similar to those observed previously (peak 1 & 2, mean peak value: 754 66nM, n=6), while in the presence of Cd2+ and Ni2+, the high K+-induced response was decreased by more than 90% (peak 3, peak value: 52 7nM; p<0.001; n=6). After washing the Cd2+ and Ni2+, the response to high K+ was restored (peak 4, mean peak value: 722 87 nM; n=6). These results confirm the existence of voltage-gated Ca2+ channels in RGCs.24

Data Analysis
The results are expressed as means S.E.M. The sample size is given in brackets. Statistical significance was analyzed using one-way ANOVA followed by Scheffes post-hoc comparisons. The figures (traces) show online measurements of the [Ca2+]i levels before and after application of test substances. Differences at the p<0.05 level were considered statistically significant. To avoid desensitization phenomenon, a typical experiment on a single cell comprised at least two control stimulations before and after the test stimulation with drugs. The results were then expressed with respect to the first control stimulation.

RESULTS
Cell Culture and [Ca2+]i Increase Evoked by High K+
We tested the specificity of the microbead method and found that the purity of the cell culture was higher than 98%. Indeed almost all cells were Thy1.2 positive few days after starting the RGC culture (data not shown). In micee developing RGCs isolated from post natal (P2) animals from primary cultures, the mean resting state value of [Ca2+]i was 7812nM (n=37). To check for the presence of VGCC (Voltage-Gated Calcium Channels) on plasma membranes of RGCs, we first recorded

Presence of Ionotropic Glutamate Receptors in RGCs


We further investigated the presence of ionotropic glutamate receptors in these neurons using both a non-selective physiological agonist (glutamate) and more selective exogenous agonists (NMDA and Kainate). The mean resting [Ca2+]i in these neurons was 9822nM. An initial 50mM K+ stimulation induced a robust increase in [Ca2+]i (879101 nM ; n=5) and was followed by three consecutive exposures to 100 M glutamate that generated an increase in [Ca2+]i ( Figure 1B, peak value: 36120nM; n=5). The glutamate-induced response was bi-phasic, an initial low increase in [Ca2+]i was followed by an high amplitude second abrupt [Ca2+]i response. These results suggest the presence of functional ionotropic glutaOphthalmic Genetics

Ca2+ handling in mouse retinal ganglion neurones 57


A 1000 800 K+ 600 Cd2+ + Ni2+ 400 200 (s) 0 C 400 [Ca2+]i (nM) 300 200 100 (s) 0 100 (s) 200 300 200 400 600 D K+ K+ K+ B 800 [Ca2+]i (nM) 600 Glutamate 400 200 0 0 K+ 200 400 600 (s) K+

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

[Ca2+]i (nM)

NMDA

NMDA [Ca2+]i (nM)

400

Kainate

Kainate

200

0 0 200 400 600

(s)

FIGURE 1 [Ca2+]i response induced by high K+ and glutamate receptor agonists in retinal ganglion cells in primary cultures. (A) RGCs in Locke buffer were stimulated twice with K+ (50mM) for 30 s, then pre-incubated with Cd2+ (100 M) plus Ni2+ (50 M) for 2 min and challenged with K+. After washing off of Cd2+ and Ni2+, the K+-induced [Ca2+]i was fully restored (n=6). (B) Typical [Ca2+]i profiles observed after one K+ and three successive glutamate (100 M) stimulations at different time intervals. (C) Typical [Ca2+]i response to NMDA (100 M; n=4). (D) Trace showing high K+ response followed by two successive exposures to 100 M kainate (n=5).

mate receptors in RGCs. In another set of experiments, we tested for the presence of NMDA receptors using 100 M NMDA. We observed a fast increase of [Ca2+]i (mean NMDA values: 3189nM; n=4), followed by a second response in the absence of NMDA probably due to release of Ca2+ from intracellular Ca2+ stores (Figure 1C). Furthermore, we stimulated the neurons with kainate (100 M) in order to test for the presence of AMPA receptors. Application of kainate (Figure 1D) induced a single monophasic fast [Ca2+]i increase (mean peak values: 34622nM; n=5), followed by a fast decay. Our data show that functional NMDA and AMPA/ kainate responsive receptors coexist in these neurons.

GABA and GABAA Agonist Induced [Ca2+]i Increases in Immature RGCs


The effect of GABAA receptor activation on [Ca2+]i was tested on RGCs. Figure 2A shows the [Ca2+]i response
2010 Informa Healthcare USA, Inc.

induced by the application of 30s 100 M GABA in a selected RGC that was responsive to high K+. GABA induced a fast transient [Ca2+]i release, followed by a slow decay when compared to that of high K+ stimulus. The mean peak values for GABA, first high K+ and second high K+ after GABA were 22813nM, 62238nM and 57244nM (n=3), respectively. We then tested the effect of two concentrations of muscimol, a specific GABAA receptor agonist, (peak values 10 M: 19718nM; 50 M: 29738nM; n=4) which induced [Ca2+]i responses in the similar (type/ age) culture. When the RGCs were perfused 2min before and during muscimol stimulation with 50M picrotoxin (Figure 2C), a GABA receptor antagonist, the muscimol induced [Ca2+]i response (peak value, 497123nM; n=3) was reversibly inhibited by 65% 12 by picrotoxin (p<0.01). When the cells were perfused with 10 M gabazine, another specific GABAA receptor antagonist, 2min before and during muscimol stimulation, a complete inhibition

58 G. Dayanithi etal.
A 800 [Ca2+]i (nM) 600 400 200 (s) GABA K+ K+ [Ca2+]i (nM) B Muscimol 300 10M 50M

200

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

100 (s) 0 100 200 300 0 200 400 600 Muscimol

C 500 400 [Ca2+]i (nM) 300 200 100

Muscimol

D 300

Muscimol

Muscimol

Muscimol [Ca2+]i (nM) Picrotoxin(50M) 200

Muscimol Gabazine (10M)

100

300

600

(s)

200

400

(s) 600

FIGURE 2 Involvement of GABAA receptors in [Ca2+]i homeostasis in RGCs. (A) [Ca2+]i profiles after high K+, GABA (100 M) and a second K+ stimulation. (B) Traces show the effect of muscimol (10 M and 50 M), a GABAA agonist, on [Ca2+]i increase. (C) Inhibition of 50 M muscimol-induced [Ca2+]i increase by picrotoxin (50 M), an antagonist of GABAA receptor. (D) Traces showing [Ca2+]i responses induced by 50 M muscimol pre-incubated or not with 10 M gabazine, a GABAA blocker. Cells were then challenged with muscimol after washing off the antagonist (n=3).

of muscimol-induced response was observed. After washing off the gabazine, the muscimol response was nearly restored (Figure 2D, 1st peak value for muscimol: 25514nM; n=5). Neurones from older cultures (up to 2 weeks) did not respond to GABA or muscimol (data not shown). These results suggest that in RGCs isolated from post natal (P2) animals, GABA exerts an excitatory effect.

Intracellular [Ca2+]i Activators in RGCs


The biphasic [Ca2+]i responses to glutamate and NMDA stimuli observed in this study suggest the participation of both Ca2+ influx and Ca2+ release from RGC intracellular Ca2+ compartments. To examine and characterize these stores, drugs acting on intracellular Ca2+ pumps were used. The response to 500nM (30s application) and 1 M (60s application) cyclopiazonic acid (CPA), an inhibitor of the Ca2+-ATPase pump from the ER and therefore a depletor of the inositol 1,2,3 triphosphate (InsP3)-sensitive intracellular Ca2+ stores [10], showed

a strong monophasic fast [Ca2+]i increase (39267nM; n=6) (Figure 3A). The Ca2+ recapture was fast at CPA concentration of 500nM and apparently slower at 1 M. However, when CPA was applied at higher concentration (1025 M) over a longer period, the decay phase was very slow and lasted several minutes even after retrieval of the agonist (data not shown). These results suggest that InsP3-sensitive Ca2+ stores are important in RGCs for controlling the Ca2+ signals under physiological conditions. In order to further characterize the involvement of Ca2+ stores, we then tested the effect of 20mM caffeine (Figure 3B) and 100nM ryanodine (Figure 3C), or the successive combination of these drugs (Figure 3D) which are known to deplete caffeine/ ryanodine Ca2+ stores and thereby suppress subsequent Ca2+ release from such stores. Although high K+ control stimulus induced typical [Ca2+]i responses, as shown before, (Figures 3B, C and D), our data clearly show that neither caffeine (Figure 3B), nor ryanodine (Figure 3C), nor ryanodine / caffeine (Figure 3D) had a significant effect on resting [Ca2+]i, suggesting that the ryanodine receptor is absent or inactive in these RGCs.
Ophthalmic Genetics

Ca2+ handling in mouse retinal ganglion neurones 59


A CPA 500nM CPA 1M B 800 [Ca2+]i (nM) 600 400 200 Caffeine K+ K+

[Ca2+]i (nM) Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

400

200

0 C 800 [Ca2+]i (nM) 600 400 200 K+

200

400

600

(s) 0 D 800 [Ca2+]i (nM) 600 400 Ryanodine 200 Caffeine K+ K+ 200 400 600

(s)

K+

Ryanodine

200

400

600

(s)

200

400

600

(s)

FIGURE 3 Effect of intracellular endoplasmic reticulum Ca2+ activators in RGCs. (A) [Ca2+]i profiles observed after stimulations of InsP3-activated Ca2+ stores by cyclopiazonic acid (CPA, 500nM or 1 M). (B) [Ca2+]i profiles show high K+-induced responses and then exposed to caffeine (20mM), a ryanodine receptor agonist. Cells were then subjected to a second K+ stimulus. (C) Traces showing the absence of [Ca2+]i changes after stimulation with ryanodine (100 nM, an agonist of its receptors at low concentration). (D) [Ca2+]i responses in cells responding to high K+, absence of [Ca2+]i changes after exposure to 20mM caffeine followed by 100 nM ryanodine (n=6).

Spontaneous Ca2+ Oscillations in RGCsCa2+


In parallel experiments, with dense two week-old RGC cultures, we monitored remarkable spontaneous [Ca2+]i oscillations in many neurons (Figure 4). These oscillations were observed only when neuronal dendrites had established connections with the neighboring cells, but were not found on individual neurons devoid of interconnected dendritic arborizations. Interestingly, these spontaneous oscillations with an average of one spike every 30sec, were amplified and accelerated by applications of 100 M NMDA (Figure 4), suggesting a possible involvement of NMDA receptors, especially in the light of the importance of these receptors for the survival of rodent RGCs.24 No further characterization of such [Ca2+]i oscillations in OPA1 silenced cells (see next paragraph in the Results section) were performed due to the restriction in obtaining high density primary cultures with connected neurons. Indeed the number of oscillating
2010 Informa Healthcare USA, Inc.
400 [Ca2+]i (nM) 300 200 100 0 5 10 15 (min)

NMDA

20

25

30

FIGURE 4 Spontaneous [Ca2+]i oscillations in retinal ganglion neurones. Purified RGC were seeded at a higher concentration (2 million cells / cm2) and cultured for two weeks. The recordings show a typical profile observed in a single neurone over a long period of spontaneous [Ca2+]i oscillations. NMDA (100 M) was applied at various time intervals which induced an amplification of [Ca2+]i oscillation frequency and amplitude. These types of oscillations were observed in at least 6 d ifferent cells.

60 G. Dayanithi etal. neurons in OPA1 siRNA cultures was too low to be able to get a meaningful amount of i nformation. and showed considerable fission of the mitochondrial network (Figure 5B). The Mitotracker dye was specifically staining the mitochondrial network. In OPA1 siRNA treated cells, we observed that the mitochondrial network, instead of being somewhat uniformly spread along the neuritis similar to what was the case in control cells, was aggregated in dots, suggesting that the fusion mechanisms were impaired by OPA1 absence. In parallel, the mitochondrial membrane potential dissipation (illustrated in Figure 5C) was assessed by the reduction of the orange fluorescence of the JC1 dye in RGC soma and dendrites. Indeed the JC1 dye present a normal green fluorescence in mitochondria, which is turned in red/orange when the membrane potential is high. Hence, we demonstrated, using the JC1 dye that the OPA1 silenced RGCs had a drastic decrease of m (Figure 5C). Finally we found that the silencing of OPA1 induced a significant increase of apoptosis as judged by counting

RGC Phenotype Associated with Mouse OPA1 Silencing


In order to assess the role of mitochondria in Ca2+ uptake, we used cells transfected with a siRNA which silences OPA1 expression or by a control scramble siRNA. Silencing of OPA1 was assessed by western blot (Figure 5A) and demonstrated a strong inhibition of OPA1 expression. The reduction of OPA1 expression in the whole culture was greater than 90%, as calculated by western blot scanning (see Material and Methods section). The western blot (Figure5A) showed the complete dissipation of the bands revealed by OPA1 antibodies in neurons treated with the OPA1 siRNA. In the corresponding cells, we analyzed the mitochondrial network structure using the Mitotracker dye
A siRNA scr. siRNA OPA1 OPA1 B

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

actin

scramble siRNA C D
Percentage of tunel positive cells

OPA1-siRNA

* 40 30 20 10 0-

scramble siRNA

OPA1-siRNA

TUNEL positive RGCs

siRNA scr.

siRNA OPA1

FIGURE 5 Down regulation of OPA1 induces mitochondrial fragmentation, loss of m and increases apoptosis. Primary cultures of RGCs were transfected after 4 days of culture, with the scrambled siRNAs and the OPA1 siRNAs, and analyzed 7 days later (see Ref.s 22 and 28 for detailed protocols). (A) Western blot using OPA1 and actin antibodies on total protein extracts. (B) Pictures of the mitochondrial network labeled with the Mitotracker dye, and of the nucleus stained with DAPI. (C) RGCs stained with membrane potential sensitive JC1 dye. (D) TUNEL positive (green fluorescence) wild-type cells and their percentages in the RGC cultures transfected with the scrambled and the OPA1 siRNAs. Ophthalmic Genetics

Ca2+ handling in mouse retinal ganglion neurones 61 TUNEL positive cells in the cultures (Figure 5D). The histogram shows the percentage of TUNEL positive RGCs transfected with the OPA1 siRNAs (black) and the scramble siRNAs (gray), 7 days after transfection (ANOVA test * p<0.005; n=5). p<0.05; trace not shown) and induced a slight raise of the baseline after high K+ stimulation, indicating that mitochondria play a role in Ca2+ clearance. Surprisingly, in OPA1 siRNA transfected cells, no difference was observed in the [Ca2+]i responses observed after high K+ stimulation in absence of CCCP or during CCCP application alone (data not shown). Conversely, treatment with CCCP during the recovery phase after high K+ stimulation, affected Ca2+ release with varying intensities from a low effect (Figure 6B), to drastic effects (Figure 6C), that ultimately resulted in a run down and probably leading to cell death (Figure6D). These results point that according to cells and probably to the amount of OPA1 residual expression, mitochondria have a critical role in RGC Ca2+ clearance that requires normal activity of OPA1. Thus, these data suggest that mitochondrial Ca2+ content can be enhanced in cells silenced for OPA1 (with R being the ratio between the amplitudes of the K+ stimulation and the CCCP application, R=1.890.33 for scramble siRNA, and R=0.930.12 for OPA1 siRNA, p<0.05, n=6 cells for each condition).

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

Effects of the Mitochondrial Integrity on [Ca2+]i Response


To assess the role of mitochondrial Ca2+ uptake, the uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) of mitochondrial respiratory chain was used. In control scrambled siRNA transfected cell cultures, we measured first the [Ca2+]i responses to high K+ stimulation with or without the subsequent application of 10 M CCCP (Figure 6A). CCCP in this way causes a rapid increase in Ca2+ which can reasonably be interpreted as release of Ca2+ from mitochondria following their uptake during the initial Ca2+ transient. In these cells, CCCP caused a small increase in resting [Ca2+]i (from 729nM to 13622nM; n=12;
A 800 [Ca2+]i (nM) 600 400 200 (s) CCCP scramble siRNA B

OPA1 siRNA

K+ CCCP

K+

K+

800 600 400 200

K+

200

400

600

200

400 Time (s)

600

800

(s)

C 800 [Ca2+]i (nM) 600 400 200

OPA1 siRNA

CCCP K+

D 1500 1000 500 0

OPA1 siRNA

K+

CCCP

K+

200

400

(s) 600

200

400

600

(s)

FIGURE 6 Effect of CCCP on K+-induced [Ca2+]i responses on RGCs transfected with the scrambled siRNAs and the OPA1 siRNAs. RGCs were processed for [Ca2+]i response, 7 days after transfections with the scrambled siRNAs (A) and the OPA1 siRNAs (B, C and D). The effect of the mitochondrial uncoupler, CCCP, was assessed on the K+-induced [Ca2+]i response (n=12). Traces show 3 different types of [Ca2+]i responses in RGC transfected with the OPA1 siRNAs (n=6 for each different stimulation protocol). In (D), the cell died during the sustained Ca2+ over load (n=4). 2010 Informa Healthcare USA, Inc.

62 G. Dayanithi etal.

DISCUSSION
In the present work, we investigated cytosolic Ca2+ entry and clearance mechanisms in RGCs from 2 dayold mouse pups. At this stage, the retina is undergoing maturation, with an inner nuclear layer of differentiated RGCs and a large outer nuclear layer of retinoblasts and undifferentiated retinal neurons.32 RGCs, which extend their axons through the optical paths, have not achieved full maturation. During the mouse retinal development, GABA provided excitatory action through a chloride efflux, which will later at P6 shift to an inhibitory stimulus.33 Our results confirmed that GABA increases intracellular [Ca2+]i by an influx of Ca2+ through the plasma membrane. This effect was mediated by GABAA receptor since muscimol mimicked the GABA-evoked Ca2+ rise and GABAA receptor antagonists blocked muscimol activity. Through depolarization and Ca2+ elevation, GABA could play a crucial role in the differentiation, survival and synaptogenesis34 of young RGCs. Glutamate is the principal neurotransmitter in the retina and mediates synaptic communication between bipolar cells and RGCs.35 In purified RGC cultures, glutamate increased [Ca2+]i in a biphasic manner suggesting a direct activation of NMDA-gated channels and a subsequent rapid activation of VGCC due to NMDA and non NMDA glutamate receptors. NMDA alone induced a signal compatible with an increase in [Ca2+]i due to external Ca2+ influx, followed by a slower Ca2+ mobilization from internal stores. Moreover, in connecting cells, NMDA stimulated spontaneous Ca2+ oscillations, which are typical of Ca2+ release from InsP3-sensitive internal stores.36 Based on our current knowledge of the phenomenon, we are unable to demonstrate a direct effect of OPA1 on Ca2+ oscillations. At this stage of our investigation, we can only indirectly link mitochondrial OPA1 to spontaneous Ca2+ oscillations. In fact, the number of connected neurons is decreased in OPA1 silenced cell cultures. This is probably due to an increase of the apoptotic rate as we show in the present work. Moreover, when the density of cells is low as it is when OPA1 is silenced, the level of connections between neurons is also low and therefore the probability to observe spontaneous Ca2+ oscillations is decreased, since we could only see these oscillations in interconnected neurons and not in isolated neurons in wild type cultures. In the retina, spontaneous electrical activity has been described as rhythmic bursts of action potentials that spread between adjacent cells and produce transient elevations in [Ca2+]i.3739 These waves seem necessary during development for the formation of appropriate synaptic connections by ganglion cells within the lateral geniculate nucleus and optic tectum.40 In embryonic

chick retina, this spontaneous activity of RGCs was regulated by ionotropic glutamate receptors as well as GABAA receptors.39 Retinal waves characterized by calcium imaging are suggested to be important for early activity dependent formation of functional circuits. Oscillations are present during the period when the inner plexiform layer becomes stratified. Retinal waves propagate slowly across retina, therefore communication between close neurons is more important than between distantly located neurons.32 In most cell types, Ca2+ entry occurs both by receptoroperated and voltage-gated Ca2+ channels. In RGCs, we demonstrated the presence of voltage-gated channels, because the high K+-evoked [Ca2+]i peak was inhibited by a mixture of Cd2+ and Ni2+, inhibitors of HVA and LVA Ca2+ channels respectively. In 1999, Schmid and Guenther proposed in an ex vivo model of RGCs that voltage-gated Ca2+ currents were exclusively of a LVA type at the embryonic stage E17, composed of HVA and LVA at the postnatal stage, and solely of a HVA type in adult RGCs.25 This assumption based only on electrophysiological recordings is consistent with our data on postnatal RGCs displaying a block of 20% of the high K+ response in the presence of Ni2+. Increase in [Ca2+]i results from Ca2+ entry through plasma membrane and from Ca2+ release from intracellular stores. The response to high K+ showed a residual increase of [Ca2+]i in the presence of Cd2+/Ni2+, most likely suggesting the contri bution of internal stores during this response. The two major possibilities of internal Ca2+ mobilization involve the InsP3-sensitive and ryanodine-sensitive Ca2+ channels present on the ER membrane. To check their respective possible contribution to RGC Ca2+ increase, we tested the effect of direct application of CPA, a drug which depletes the InsP3-sensitive pool, and caffeine and/or ryanodine on resting [Ca2+]i. Surprisingly, CPA, but not caffeine nor ryanodine nor their association, increased the [Ca2+]i, in these cells at this stage of development. Consequently, although ryanodine receptors have been described in other sensory neurons early during development10 and the presence of ryanodine receptors in retina has been suggested by recent studies,41 we showed that the contribution of ryanodine receptors in this model of RGCs is not significant for the Ca2+ handling. The role of mitochondria as Ca2+-buffering systems has been demonstrated in several models by many studies (see for example [42,43]). Furthermore, this was shown to be true also for retinal cells, especially for amacrine cells.44 Though the likelihood that mitochondria behave like Ca2+-sequestering organelles in RGCs as well was reasonably high, no direct evidence so far could be found in the literature. Therefore a noteworthy finding here is the demonstration and confirmation that mitochondria play a major role as Ca2+-buffering system in RGCs. The
Ophthalmic Genetics

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

Ca2+ handling in mouse retinal ganglion neurones 63 itochondrial uncoupler, CCCP, showed significant m effects on the basal Ca2+ level and especially when applied during the recovery phase of K+-induced [Ca2+]i, suggesting that in these cells mitochondria represent an important organelle participating in the Ca2+ clearance mechanism. Taking into account that the causative inherited optic neuropathies (ION) genes are ubiquitously expressed, the patho-physiological hypotheses that have been proposed to explain the restricted degeneration of the RGCs in IONs, are based on processes focusing on RGC mitochondrial particularities.4 The first hypothesis is based on an RGC exceptional requirement for energy resources. These cells are highly solicited during the visual process, and the RGC axon in the ocular globe is devoid of myelin, requiring higher energy to transduce visual information.45 The second hypothesis, related to the first one, is based on an irregular distribution of mitochondria along the RGC axon.46 In the case of mitochondrial defects, low ATP production can decrease the rate of axoplasmic transport and consequently compromise the transport of mitochondria, as well as of neurotrophic factors, jeopardizing cell survival. Finally, the third hypothesis is related to the unusual localization of RGCs in the eye. These cells are permanently exposed to the pro-apoptotic effects of the light and consequently, any mitochondrial defects that would affect susceptibility to apoptosis or ATP production, would specifically favor RGC degeneration.47 Because OPA1 displays a prominent role not only in maintaining the membrane potential required for Ca2+ entry into mitochondria, but also in producing ATP and in the dynamics of the mitochondrial network,22 we monitored its role in RGC Ca2+ clearance. It is noteworthy that La3+, an inhibitor of the Na+/Ca2+ exchangers of the plasma membrane, did not affect the high K+-induced depolarization, indicating no contribution of the Na+/Ca2+ exchangers in the Ca2+ clearance of postnatal RGCs (data not shown). Down-regulation of OPA1 expression was induced by specific OPA1 siRNA transfection and led to a fragmented mitochondrial phenotype, which can be summarised by a significant dissipation (decrease) of the m (using the JC-1 probe and assessing the red to green ratio), punctuated mitochondrial network, and increased apoptosis. In RGCs, the relative silencing of OPA1 allowed normal Ca2+ clearance after high K+ stimulation, suggesting that the global mechanisms required for Ca2+ recapture still function. Conversely, addition of CCCP on cells silenced for OPA1 emphasized the enhanced role of mitochondria on Ca2+ capture, with different patterns probably resulting from the different amounts of OPA1 protein remaining per cell. Nevertheless, these results suggest that in the absence of a normal m, and in the absence of OPA1 activity, Ca2+ recapture pathways are
2010 Informa Healthcare USA, Inc.

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

highly defective, leading to drastic impairment in Ca2+ clearance and ultimately to cell death. In this respect, the increase of Ca2+ recapture showed by the presence of CCCP might result from the complete disturbance of the m, possibly combined with the reduction of the ATP synthesis (which could decrease the activity of the other reuptake systems within the cell) or with the deregulated mitochondria distribution in the cell, induced by OPA1 silencing. These hypotheses should be addressed now in animal models with mutated OPA1. Noteworthy is that OPA1 and OPA3 proteins are located in the mitochondrial inner membrane, and in optic atrophy, a similar mechanism leads to RGC dysfunction via disruption of the mitochondrial respiratory chain. Whether OPA1 silencing affects OPA3 function in mitochondrial Ca2+ regulation is questionable, and the role of OPA3in particular in Ca2+ clearanceis under investigation right now. However it is important to mention that OPA1 is the major gene involved in Dominant Optic Atrophy (>80% of all cases), whereas OPA3 is only involved in less than 2% of all cases.48 Taken together, our data indicate that RGCs are critically dependent on OPA1 for Ca2+ clearance during mouse retinal development. Since mutations of the OPA1 gene are responsible for progressive degeneration of RGCs in patients suffering from dominant optic atrophy, it seems still speculative but highly conceivable that Ca2+ clearance could be impaired as well, hence taking part in the degeneration of ganglion cells in patients with the mutated OPA1 gene. Therefore, we propose here a new foundation for a patho-physiological mechanism that might account for RGC degeneration observed in some young OPA1 patients. However this requires obviously further investigations, particularly the establishment of more physiological animal models.

ACKNOWLEDGMENTS
We thank Dr. H. Aptel (Institut de Genomique Fonctionelle, Montpellier, France) for assistance and helpful discussions and Dr. Keith Langley ( Montpellier, France) for critical reading and language editing of the manuscript. Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

REFERENCES
1. Bonne C, Muller A, Villain M. Free radicals in retinal ischemia. General Pharmacology 1998;30:275280.

64 G. Dayanithi etal.
2. Carelli V, Ross-Cisneros FN, Sadun AA. Optic nerve degeneration and mitochondrial dysfunction: genetic and acquired optic neuropathies. Neurochemistry International 2002;40:573584. Chinnery PF. Inheritance of mitochondrial disorders. Mitochondrion 2002;2:149155. Carelli V, Ross-Cisneros FN, Sadun AA. Mitochondrial dysfunction as a cause of optic neuropathies. Progress in Retinal and Eye Research 2004;23:5389. Delettre C, Lenaers G, Griffoin JM, et al. Nuclear gene OPA1, encoding a mitochondrial dynamin-related protein, is mutated in dominant optic atrophy. Nature Genetics 2000;26:207210. Reynier P, Amati-Bonneau P, Verny C, et al. OPA3 gene mutations responsible for autosomal dominant optic atrophy and cataract. Journal of Medical Genetics 2004;41:e110. Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nature Reviews Molecular Cell Biology 2000;1:1121. Petersen OH, Michalak M, Verkhratsky A. Calcium signalling: past, present and future. Cell Calcium 2005;38:161 169. Verkhratsky A. Physiology and pathophysiology of the calcium store in the endoplasmic reticulum of neurons. Physiological Review 2005;85:201279. Dayanithi G, Mechaly I, Viero C, et al. Intracellular Ca2+ regulation in rat motoneurons during development. Cell Calcium 2006;39:237246. Toescu EC, Verkhratsky A. Neuronal ageing from an intraneuronal perspective: roles of endoplasmic reticulum and mitochondria. Cell Calcium 2003;34:311323. Berridge MJ, Bootman MD, Lipp P. Calciuma life and death signal. Nature 1998;395:645648. Duchen MR. Mitochondria and calcium: from cell signalling to cell death. Journal of Physiology 2000;529:5768. Schapira AH. Mitochondrial disease. Lancet 2006;368: 7082. Brini M, Pinton P, King MP, Davidson M, Schon EA, Rizzuto R. A calcium signaling defect in the patho genesis of a mitochondrial DNA inherited oxidative phosphorylation deficiency. Nature Medicine 1999;5: 951954. Solovyova N, Veselovsky N, Toescu EC, Verkhratsky A. Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry. The EMBO Journal 2002;21:622630. Sasaki N, Dayanithi G, Shibuya I. Ca2+ clearance mechanisms in neurohypophysial terminals of the rat. Cell Calcium 2005;37:4556. Lee SH, Kim MH, Park KH, Earm YE, Ho WK. K+-dependent Na+/Ca2+ exchange is a major Ca2+ clearance mechanism in axon terminals of rat neurohypophysis. Journal of Neuroscience 2002;22:68916899. Simpson PB, Russell JT. Mitochondrial Ca2+ uptake and release influence metabotropic and ionotropic cytosolic Ca2+ responses in rat oligodendrocyte progenitors. Journal of Physiology 1998;508:413426. Rizzuto R, Brini M, Murgia M, Pozzan T. Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria. Science 1993;262:744747. Hajnoczky G, Csordas G, Madesh M, Pacher P. The machinery of local Ca2+ signalling between sarco- endoplasmic reticulum and mitochondria. Journal of Physiology 2000;529:6981. 22. Olichon A, Baricault L, Gas N, etal. Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis. Journal of Biological Chemistry 2003;278:77437746. 23. Spat A. Calcium microdomains and the fine control of cell function: an introduction. Cell Calcium 2006;40:403404. 24. Sucher NJ, Lei SZ, Lipton SA. Calcium channel antagonists attenuate NMDA receptor-mediated neurotoxicity of retinal ganglion cells in culture. Brain Research 1991;551:297302. 25. Schmid S, Guenther E. Voltage-activated calcium currents in rat retinal ganglion cells in situ: changes during prenatal and postnatal development. Journal of Neuroscience 1999;19:34863494. 26. Firth SI, Wang CT, Feller MB. Retinal waves: mechanisms and function in visual system development. Cell Calcium 2005;37:425432. 27. Meyer-Franke A, Kaplan MR, Pfrieger FW, Barres BA. Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture. Neuron 1995;15:805819. 28. Kamei S, Chen-Kuo-Chang M, Cazevieille C etal. Expression of the OPA1 mitochondrial protein in retinal ganglion cells: its downregulation causes aggregation of the mitochondrial network. Investigative Ophthalmology & Visual Science 2005;46:42884294. 29. Olichon A, Emorine LJ, Descoins E, et al. The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space. FEBS Letters 2002;523:171176. 30. Viero C, Mechaly I, Aptel H etal. Rapid inhibition of Ca(2+) influx by neurosteroids in murine embryonic sensory neurons. Cell Calcium 2006;40:383391. 31. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 1985;260:34403450. 32. Cepko CL, Austin CP, Yang X, Alexiades M, EzzeddineD. Cell fate determination in the vertebrate retina. Proceedings of the National Academy of Science USA 1996;93: 589595. 33. Zhang LL, Pathak HR, Coulter DA, Freed MA, Vardi N. Shift of intracellular chloride concentration in ganglion and amacrine cells of developing mouse retina. Journal of Neurophysiology 2006;95:24042416. 34. Ben-Ari Y. Excitatory actions of gaba during development: the nature of the nurture. Nature Revies Neuroscience 2002;3:728739. 35. Hartwick AT, Lalonde MR, Barnes S, Baldridge WH. Adenosine A1-receptor modulation of glutamate-induced calcium influx in rat retinal ganglion cells. Investigative Ophthalmology & Visual Science 2004;45:37403748. 36. Berridge MJ. Inositol trisphosphate and calcium oscillations. Biochemical Social Symposia 2007;74: 4147. 37. Meister M, Wong RO, Baylor DA, Shatz CJ. Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina. Science 1991;252: 939943. 38. Catsicas, M., Bonness, V., Becker, D., Mobbs, P., 1998. Spontaneous Ca2+ transients and their transmission in the developing chick retina. Current Biology 8(5), 283286. 39. Wong WT, Sanes JR, Wong RO. Developmentally regulated spontaneous activity in the embryonic chick retina. Journal of Neuroscience 1998;18:88398852. 40. Wong RO. Retinal waves and visual system development. Annual Review of Neuroscience 1999;22:2947. Ophthalmic Genetics

3. 4. 5.
Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

6.

7. 8. 9. 10. 11. 12. 13. 14. 15.

16.

17. 18.

19.

20. 21.

Ca2+ handling in mouse retinal ganglion neurones 65


41. Shoshan-Barmatz V, Orr I, Martin C, Vardi N. Novel ryanodine-binding properties in mammalian retina. International Journal of Biochemical Cell Biology 2005;37: 16811695. 42. Sergeant GP, Bradley E, Thornbury KD, McHale NG, Hollywood MA. Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra. Journal of Physiology 2008;586:46314642. 43. Coburn CG, Currs-Collazo MC, Kodavanti PR. In vitro effects of environmentally relevant polybrominated diphenyl ether (PBDE) congeners on calcium buffering mechanisms in rat brain. Neurochemical Research 2008;33:355364. 44. Medler K, Gleason EL. Mitochondrial Ca(2+) buffering regulates synaptic transmission between retinal amacrine cells. Journal of Neurophysiology 2002;87:14261439. 45. Yu Wai Man CY, Chinnery PF, Griffiths PG. Optic neuropathiesimportance of spatial distribution of mitochondria as well as function. Medical Hypotheses 2005;65: 10381042. 46. Andrews RM, Griffiths PG, Johnson MA, Turnbull DM. Histochemical localisation of mitochondrial enzyme activity in human optic nerve and retina. British Journal of Ophthalmology 1999;83:231235. 47. Osborne NN, Lascaratos G, Bron AJ, Chidlow G, Wood JP. A hypothesis to suggest that light is a risk factor in glaucoma and the mitochondrial optic neuropathies. British Journal of Ophthalmology 2006;90:237241. 48. Ferr M, Bonneau D, Milea D etal. Molecular screening of 980 cases of suspected hereditary optic neuropathy with a report on 77 novel OPA1 mutations. Human Mutation 2009;30:E692705.

Ophthalmic Genet Downloaded from informahealthcare.com by Wales College of Medicine Biology and Health Science on 12/12/12 For personal use only.

2010 Informa Healthcare USA, Inc.

Вам также может понравиться