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.

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. The use of an HCF platform makes the system compact with future application in the longitudinal investigation into the structure and function of healthy and diseased RGCs.

The In Vivo Effects of the CB1-Positive Allosteric Modulator GAT229 on Intraocular Pressure in Ocular Normotensive and Hypertensive Mice.

PURPOSE: Orthosteric cannabinoid receptor 1 (CB1) activation leads to decreases in intraocular pressure (IOP). However, use of orthosteric CB1 agonists chronically has several disadvantages, limiting their usefulness as clinically relevant drugs. Allosteric modulators interact with topographically distinct sites to orthosteric ligands and may be useful to circumvent some of these disadvantages. The purpose of this study was to investigate the effects of the novel CB1-positive allosteric modulator (PAM) GAT229 on IOP. METHODS: IOP was measured using rebound tonometry in anesthetized normotensive C57Bl/6 mice and in a genetic model of ocular hypertension [nose, eyes, ears (nee) mice] before drug administration, and at 1, 6, and 12 h thereafter. RESULTS: In normotensive mice, topical administration of 5 µL GAT229 alone at either 0.2% or 2% did not reduce IOP. However, a subthreshold dose (0.25%) of the nonselective orthosteric CB1 agonist WIN 55,212-2, when combined with 0.2% GAT229, significantly reduced IOP compared with vehicle at 6 and 12 h. Similarly, combination of subthreshold Δ9-tetrahydrocannabinol (a nonselective orthosteric CB1 agonist; 1 mg/kg) with topical 0.2% GAT229 produced IOP lowering at 6 h. In nee mice, administration of topical 0.2% GAT229 or 10 mg/kg GAT229 alone was sufficient to lower IOP at 6 and 12 h, and 12 h, respectively. CONCLUSIONS: The CB1 PAM GAT229 reduces IOP in ocular hypertensive mice and enhanced CB1-mediated IOP reduction when combined with subthreshold CB1 orthosteric ligands in normotensive mice. Administration of CB1 PAMs may provide a novel approach to reduce IOP with fewer of the disadvantages associated with orthosteric CB1 activation.

The Effect of Glutamate Receptor Agonists on Mouse Retinal Astrocyte [Ca(2+)]i.

Calcium-imaging techniques were used to determine if mouse retinal astrocytes in situ respond to agonists of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA; N-methyl-D-aspartate, NMDA) and metabotropic (S-3,5-dihydroxyphenylglycine, DHPG; trans-1-amino-1,3-cyclopentanedicarboxylic acid, ACPD) glutamate receptors. In most cases we found no evidence that retinal astrocyte intracellular calcium ion concentration ([Ca(2+)]i) increased in response to these glutamate agonists. The one exception was AMPA that increased [Ca(2+)]i in some, but not all, mouse retinal astrocytes in situ. However, AMPA did not increase [Ca(2+)]i in mouse retinal astrocytes in vitro, suggesting that the effect of AMPA in situ may be indirect.

The Endocannabinoid System as a Therapeutic Target in Glaucoma.

Glaucoma is an irreversible blinding eye disease which produces progressive retinal ganglion cell (RGC) loss. Intraocular pressure (IOP) is currently the only modifiable risk factor, and lowering IOP results in reduced risk of progression of the disorder. The endocannabinoid system (ECS) has attracted considerable attention as a potential target for the treatment of glaucoma, largely due to the observed IOP lowering effects seen after administration of exogenous cannabinoids. However, recent evidence has suggested that modulation of the ECS may also be neuroprotective. This paper will review the use of cannabinoids in glaucoma, presenting pertinent information regarding the pathophysiology of glaucoma and how alterations in cannabinoid signalling may contribute to glaucoma pathology. Additionally, the mechanisms and potential for the use of cannabinoids and other novel agents that target the endocannabinoid system in the treatment of glaucoma will be discussed.

Histological confirmation of needle tip position during ultrasound-guided interscalene block: a randomized comparison between the intraplexus and the periplexus approach.

PURPOSE: Ultrasound-guided interscalene block can be performed using either periplexus or intraplexus needle placement. In this novel study, we histologically examined the needle tip position in relation to the neural tissues with the two techniques. Our objective was to investigate the variable risk of subepineurial needle tip placement resulting from the two ultrasound-guided techniques. METHODS: In an embalmed cadaveric model, periplexus or intraplexus interscalene injections were performed with the side, order, and technique assigned randomly. Under real-time ultrasound guidance, the block needle was placed next to the hyperechoic layer of the plexus (periplexus) or between the hypoechoic nerve roots (intraplexus). Once positioned, 0.1 mL of black acrylic ink was injected. The brachial plexus tissues were then removed and histology sections were prepared and then coded in order to blind two reviewers to group allocation. The area of ink staining was used to determine needle tip location, and the groups were compared for the presence of subepineurial ink. RESULTS: Twenty-six cadavers had each of the blocks performed on either brachial plexus (i.e., 52 injections). No subepineurial ink deposits were observed in the periplexus group (0%), but subepineurial ink deposition was observed in 3/26 (11.5%) intraplexus injections (odds ratio, 0; 95% confidence interval, 0 to 2.362; P = 0.235). Furthermore, in the intraplexus group, two (of the three) subepineurial ink deposits were observed under the perineurium. CONCLUSION: Although our study was somewhat underpowered due to a lower than previously reported rate of subepineurial needle tip positioning, our results suggest that there may be an increased likelihood of subepineurial needle tip position with the intraplexus approach. The periplexus technique resulted in no subepineurial spread of ink, suggesting that this approach may be less likely to result in mechanical trauma to nerves from direct needle injury.

Controlling the number of melanopsin-containing retinal ganglion cells by early light exposure.

A small percentage of retinal ganglion cells (RGCs) express melanopsin and are intrinsically photosensitive (ipRGCs). Whether light can affect the development of ipRGCs is not clear. In the rat retina, we found constant light exposure during the first postnatal week significantly increased the number of melanopsin immunopositive ipRGCs. This increase was durable and specific for melanopsin immunopositive ipRGCs. BrdU labeling showed no proliferation of the melanopsin immunopositive ipRGCs during constant light exposure. Retrograde labeling from the superior colliculus showed that no other types of RGCs were induced to express melanopsin. Light exposure was effective in increasing melanopsin immunopositive ipRGCs only when it coincided with the apoptotic phase of RGC development. However, daily intravitreous injection of tetrodotoxin, blocking action potentials, abolished the light induced increase of melanopsin immunopositive ipRGCs. These findings indicate that early light exposure can increase the number of melanopsin immunopositive ipRGCs through a process dependent on intrinsic photosensitive spiking activity. Furthermore, the increase of melanopsin immunopositive ipRGCs is potentially induced by apoptosis suppression in ipRGCs or enhanced expression of melanopsin.

Functional evidence for D-serine inhibition of non-N-methyl-D-aspartate ionotropic glutamate receptors in retinal neurons.

D-Serine is an important signaling molecule throughout the central nervous system, acting as an N-methyl-D-aspartate (NMDA) receptor coagonist. This study investigated the D-serine modulation of non-NMDA ionotropic glutamate receptors expressed by inner retinal neurons. We first identified that the degradation of endogenous retinal D-serine, by application of D-amino acid oxidase, caused an enhancement of kainate- and α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor-mediated calcium responses from the ganglion cell layer of the isolated rat retina and light-evoked responses obtained by multi-electrode array recordings from the guinea pig retina. Approximately 30-45% of cells were endogenously inhibited by D-serine, as suggested by the effect of D-amino acid oxidase. Conversely, bath application of D-serine caused a reduction in multi-electrode array recorded responses and decreased kainate, but not potassium-induced calcium responses, in a concentration-dependent manner (IC(50), 280 µm). Using cultured retinal ganglion cells to reduce network influences, D-serine reduced kainate-induced calcium responses and AMPA induced whole-cell currents. Finally, the inhibitory effect of D-serine on the kainate-induced calcium response was abolished by IEM 1460, thereby identifying calcium-permeable AMPA receptors as a potential target for D-serine. To our knowledge, this is the first study to address specifically the effect of D-serine on AMPA/kainate receptors in intact central nervous system tissue, to identify its effect on calcium permeable AMPA receptors and to report the endogenous inhibition of AMPA/kainate receptors.

Endothelin-1-induced proliferation is reduced and Ca²âº signaling is enhanced in endothelin B-deficient optic nerve head astrocytes.

PURPOSE: To characterize the influence of endothelin-1 (ET-1) on optic nerve head astrocyte (ONHA) proliferation and Ca²âº signaling in ONHAs lacking functional endothelin B (ETB) receptors. METHODS: ONHAs were isolated from adult wild type (WT) and transgenic spotting lethal (TSL) rats, lacking functional ETB receptors. ONHA specificity was confirmed by positive glial fibrillary acidic protein (GFAP), negative A2B5 (a marker for type II astrocytes located outside the optic nerve head) and myelin basic protein (MBP) labeling. The mitogenic effects of 10⁻7 or 10⁻9 M ET-1, or vehicle were investigated for 48 or 72 hours in WT and TSL ONHAs. Intracellular calcium levels ([Ca²âº](i)) were assessed in ONHAs loaded with fura-2 calcium indicator dye. RESULTS: ET-1-induced proliferation of TSL ONHAs was blunted at 48 hours (by 37% at 10⁻7 M and by 33% at 10⁻9 M) and 72 hours (by 117% at 10⁻7 M and by 100% at 10⁻9 M) compared with WT cells. ET-1-induced ONHA fura-2 ratio increases were significantly greater in TSL ONHAs (by 20% at 10⁻7 M and by 48% at 10⁻9 M) compared with WT ONHAs. ET-1-induced fura-2 ratio increases were blocked after pretreatment with BQ-610 (ETA antagonist) in WT and TSL ONHAs, but not by BQ-788 (ETB antagonist) in WT ONHAs. CONCLUSIONS: ET-1-induced ONHA proliferation is reduced in cells lacking functional ETB receptors, ET-1-induced [Ca²âº](i) increases are enhanced in the absence of functional ETB receptors, and ETA, but not ETB, is required for ET-1-induced [Ca²âº](i) elevation.

The light-induced reduction of horizontal cell receptive field size in the goldfish retina involves nitric oxide.

Horizontal cells of the vertebrate retina have large receptive fields as a result of extensive gap junction coupling. Increased ambient illumination reduces horizontal cell receptive field size. Using the isolated goldfish retina, we have assessed the contribution of nitric oxide to the light-dependent reduction of horizontal cell receptive field size. Horizontal cell receptive field size was assessed by comparing the responses to centered spot and annulus stimuli and from the responses to translated slit stimuli. A period of steady illumination decreased the receptive field size of horizontal cells, as did treatment with the nitric oxide donor (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (100 µM). Blocking the endogenous production of nitric oxide with the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester (1 mM), decreased the light-induced reduction of horizontal cell receptive field size. These findings suggest that nitric oxide is involved in light-induced reduction of horizontal cell receptive field size.

Tracer coupling of intrinsically photosensitive retinal ganglion cells to amacrine cells in the mouse retina.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are a subtype of ganglion cell in the mammalian retina that expresses the photopigment melanopsin and drives non-image-forming visual functions. Three morphological subtypes of ipRGCs (M1, M2, and M3) have been described based on their dendritic stratifications in the inner plexiform layer (IPL), but the question of their potential interactions via electrical coupling remains unsettled. In this study, we have addressed this question in the mouse retina by, injecting the tracer Neurobiotin into ipRGCs that had been genetically labelled with the fluorescent protein, tdTomato. We confirmed the presence of the M1-M3 subtypes of ipRGCs based on their distinct dendritic stratifications. All three subtypes were tracer coupled to putative amacrine cells situated within the ganglion cell layer (GCL) but not the inner nuclear layer (INL). The cells tracer coupled to the M1 and M2 cells were shown to be widefield GABA-immunoreactive amacrine cells. We found no evidence of homologous tracer coupling of ipRGCs or heterologous coupling to other types of ganglion cells.

The effect of aminosulfonate buffers on the light responses and intracellular pH of goldfish retinal horizontal cells.

Retinal horizontal cell feedback acts as a gain control at the first synapse in the visual system and generates center-surround receptive fields in the outer retina. One model of feedback proposes that elevation of protons in the photoreceptor synaptic cleft produces feedback. Most evidence supporting the proton model has depended on the effect of proton buffers, in particular aminosulfonates, but these agents could potentially have effects other than external pH regulation. We therefore determined if the effects of aminosulfonates on horizontal cell rollback, an indicator of feedback, were consistent with external proton buffering. Intracellular recording from horizontal cells in isolated goldfish retina revealed that rollback was blocked only by aminosulfonates with an acid dissociation constant suited for buffering at the pH (7.5) of the Ringer's solution. In isolated goldfish horizontal cells, aminosulfonates, even those that did not block rollback, altered intracellular pH. This suggests that the effect of aminosulfonates on rollback is not because of changing intracellular pH. Measures of both intracellular and extracellular pH revealed that treatment with either glutamate or kainate resulted in acidification. As glutamate produced both internal and external acidification, intracellular and extracellular horizontal cell pH would be expected to increase in response to light, a change consistent with the proton model of feedback.

Cyan fluorescent protein (CFP) expressing cells in the retina of Thy1-CFP transgenic mice before and after optic nerve injury.

We investigated the specificity of cyan fluorescent protein (CFP) expression in retinal ganglion cells (RGCs) of the transgenic Thy1-CFP (B6.Cg-Tg(Thy1-CFP)23Jrs/J) mouse line, and the characteristics of these cells after optic nerve injury. RGCs of adult Thy1-CFP mice were retrogradely labeled with fluorochrome (2% fluorogold [FG]) from the superior colliculi (SC). Animals were sacrificed 7 days after RGC labeling. Retinas were fixed and whole-mounted. CFP and FG-positive cells were visualized and imaged separately. Cells positive for CFP, FG, or co-labeled were counted. In another group of animals, the left optic nerves were transected 7 days after FG labeling. They were sacrificed 7 or 21 days after transection. The retinas were whole-mounted and the characteristics of CFP-expressing cells examined. CFP-expressing cells were distributed evenly throughout the retinas of Thy1-CFP mice. The average densities of CFP and FG-positive cells in the retina were 2778+/-216 and 3230+/-157 cells/mm(2), respectively. 93.2+/-1.6% of CFP-expressing cells were also labeled with FG. However, only 79.9+/-2.5% of FG-labeled RGCs expressed CFP. The number of CFP-expressing cells decreased dramatically after transection. Cells with spindle shape, immunohistochemically identified as microglia, were seen in the retina with CFP expression at both 7 and 21 days after optic nerve transection. In retinas of Thy1-CFP mice, CFP is expressed by the large majority of RGCs, but not exclusively by RGCs. CFP is internalized by phagocytosing cells after injury to RGCs.

d-Serine enhancement of NMDA receptor-mediated calcium increases in rat retinal ganglion cells.

NMDA receptor (NMDAR) activation is enhanced by d-serine or glycine acting at a specific binding site. Previous work has shown d-serine enhancement of NMDAR currents in retinal ganglion cells. One of the major functions of most NMDA channels is to permit calcium influx into cells. We show that d-serine enhances glutamate-induced calcium responses in immunopanned retinal ganglion cells. This effect was specific to NMDA receptors as similar results were found with NMDA, but not kainate, and was reduced or blocked by modulators of the NMDAR coagonist binding site. d-Serine and glycine enhanced glutamate-induced calcium responses in a dose-dependent manner and at equimolar concentrations there was no difference in the efficacy of the coagonists. In isolated retinas NMDA-induced calcium responses were enhanced by d-serine coapplication in 46% of ganglion cells. Endogenous d-serine degradation by treatment with d-amino acid oxidase caused a approximately 45% decrease in the NMDA-induced response that could be reversed by coapplication with d-serine. d-Serine and glycine were equally effective in enhancing glutamatergic calcium responses. Endogenous d-serine contributes to NMDAR activation in retinal wholemounts and some but not all retinal ganglion cells may experience saturating levels of d-serine or glycine.

Slow excitation of cultured rat retinal ganglion cells by activating group I metabotropic glutamate receptors.

As in many CNS neurons, retinal ganglion cells (RGCs) receive fast synaptic activation through postsynaptic ionotropic receptors. However, the potential role of postsynaptic group I metabotropic glutamate receptors (mGluRs) in these neurons is unknown. In this study we first demonstrated that the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) increased intracellular calcium concentration in neurons within the ganglion cell layer of the rat retina. This prompted us to use an immunopanned-RGC and cortical astroglia coculture preparation to explore the effect of group I mGluR activation on the electrophysiological properties of cultured RGCs. Using perforated patch-clamp recordings in current-clamp configuration, we found that application of DHPG increased spontaneous spiking and depolarized the resting membrane potential of RGCs. This boosting effect was attributed to an increase in membrane resistance due to blockade of a background K(+) conductance. Further experiments showed that the group I mGluR-sensitive K(+) conductance was not blocked by 3 mM Cs(+), but was sensitive to acidification. Pharmacological studies indicated that the effect of DHPG on RGCs was mediated by the mGluR1 rather than the mGluR5 receptor subtype. Our results suggest a facilitatory role for group I mGluR activation in modulating RGC excitability in the mammalian inner retina.

Glutamatergic calcium dynamics and deregulation of rat retinal ganglion cells.

A rise in intracellular calcium levels ([Ca(2+)](i)) is a key trigger for the lethal effects of the excitatory neurotransmitter glutamate in various central neurons, but a consensus has not been reached on the pathways that mediate glutamate-dependent increases of [Ca(2+)](i) in retinal ganglion cells (RGCs). Using Ca(2+) imaging techniques we demonstrated that, in the absence of external Mg(2+), the Ca(2+) signal evoked by glutamate was predominantly mediated by NMDA-type glutamate receptors (NMDA-Rs) in immunopanned RGCs isolated from neonatal or adult rats. Voltage-gated Ca(2+) channels and AMPA/kainate-Rs contributed a smaller portion of the Ca(2+) response at saturating concentrations of glutamate. Consistent with NMDA-R involvement, extracellular Mg(2+) inhibited RGC glutamate responses, while glycine had a potentiating effect. With Mg(2+) present externally, the effect of AMPA/kainate-R antagonists was enhanced and both NMDA- and AMPA/kainate-R antagonists greatly reduced the glutamate-induced increases of RGC [Ca(2+)](i). This finding indicates that the primary contribution of AMPA/kainate-Rs to RGC glutamatergic Ca(2+) dynamics is through the depolarization-dependent relief of the Mg(2+) block of NMDA-R channels. The effect of glutamate receptor antagonists on glutamatergic Ca(2+) signals from RGCs in adult rat retinal wholemounts yielded results similar to those obtained using immunopanned RGCs. Additional experiments on isolated RGCs revealed that during a 1 h glutamate (10-1000 microm) exposure, 18-28% of RGCs exhibited delayed Ca(2+) deregulation (DCD) and the RGCs that underwent DCD were positive for the death marker annexin V. RGCs with larger glutamate-evoked Ca(2+) signals were more likely to undergo DCD, and NMDA-R blockade significantly reduced the occurrence of DCD. Identifying the mechanisms underlying RGC excitotoxicity aids in our understanding of the pathophysiology of retinal ischaemia, and this work establishes a major role for NMDA-R-mediated increases in [Ca(2+)](i) in glutamate-related RGC death.

Acute endothelin-1 application induces reversible fast axonal transport blockade in adult rat optic nerve.

PURPOSE: To investigate the effects of endothelin (ET)-1 on fast axonal transport in the optic nerve. METHODS: Sterile sponge soaked in 1 nM ET-1 was applied to the optic nerve surface of adult Brown Norway rats for 1 hour, after which 20% horseradish peroxidase (HRP) was placed over the superior colliculi (SC). Rats were killed 2, 4, 6, 24, and 48 hours later; the retinas and optic nerves were removed and fixed; and cut sections were processed histochemically, to visualize the time course of HRP transport by light microscopy. Naive and saline controls were processed identically. Retinal ganglion cell (RGC) survival after acute ET-1 application was investigated in another group of animals. After retrograde labeling of RGCs with the fluorescent neurotracer Fluorochrome (FG; Fluorogold; Fluorochrome Inc., Denver, CO), 1 nM ET-1 was applied to the optic nerve. Rats were then killed 5, 10, and 21 days later. The retinas were whole mounted and FG-positive RGCs were imaged and quantified with fluorescence microscopy. RESULTS: In naive controls, HRP labeling was observed over the entire nerve at 2 hours but had cleared by 48 hours. HRP labeling of RGCs started at 6 hours, and by 48 hours, uniform labeling was seen throughout the retina. In ET-1-treated optic nerves, transport of HRP was arrested at the distal portion of the nerve at 2 hours. Recovery of transport was evident from 4 hours. At 6 and 24 hours, all nerves showed full recovery with HRP-positive RGCs in the retina, but the ratio of the RGC counts in treated versus fellow untreated eyes (0.66 +/- 0.13 and 0.67 +/- 0.17, respectively) was less than that of the naive control (1.02 +/- 0.28 and 1.05 +/- 0.13, respectively) animals. At 48 hours, recovery was complete, and there was no significant difference in the ratio of RGC counts between ET-1 and naive control groups. No RGC loss was observed after ET-1 application. CONCLUSIONS: Local acute application of ET-1 produces a reversible blockade of rapid axonal transport in optic nerve.

Light-evoked calcium responses of isolated melanopsin-expressing retinal ganglion cells.

A small number (<2%) of mammalian retinal ganglion cells express the photopigment melanopsin and are intrinsically photosensitive (ipRGCs). Light depolarizes ipRGCs and increases intracellular calcium levels ([Ca2+]i) but the signaling cascades underlying these responses have yet to be elucidated. To facilitate physiological studies on these rare photoreceptors, highly enriched ipRGC cultures from neonatal rats were generated using anti-melanopsin-mediated plate adhesion (immunopanning). This novel approach enabled experiments on isolated ipRGCs, eliminating the potential confounding influence of rod/cone-driven input. Light induced a rise in [Ca2+]i (monitored using fura-2 imaging) in the immunopanned ipRGCs and the source of this Ca2+ signal was investigated. The Ca2+ responses were inhibited by 2-aminoethoxydiphenyl borate, SKF-96365 (1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole), flufenamic acid, lanthanum, and gadolinium, consistent with the involvement of canonical transient receptor potential (TRP) channels in ipRGC phototransduction. However, the contribution of direct Ca2+ flux through a putative TRP channel to ipRGC [Ca2+]i was relatively small, as most (approximately 90%) of the light-induced Ca2+ responses could be blocked by preventing action potential firing with tetrodotoxin. The L-type voltage-gated Ca2+ channel (VGCC) blockers verapamil and (+)-cis-diltiazem significantly reduced the light-evoked Ca2+ responses, while the internal Ca2+ stores depleting agent thapsigargin had negligible effect. These results indicate that Ca2+ influx through VGCCs, activated after action potential firing, was the primary source for light-evoked elevations in ipRGC [Ca2+]i. Furthermore, concurrent Ca2+ imaging and cell-attached electrophysiological recordings demonstrated that the Ca2+ responses were highly correlated to spike frequency, thereby establishing a direct link between action potential firing and somatic [Ca2+]i in light-stimulated ipRGCs.

Contribution of KCNQ1 to the regulatory volume decrease in the human mammary epithelial cell line MCF-7.

Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K(+) channel inhibitors Ba(2+), quinine, and tetraethylammonium chloride, implicating K(+) channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K(+) channel, and also a dominant-negative NH(2)-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K(+) current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K(+) secretion.

Prolonged costimulation is required for naive T cell activation.

Costimulation by members of the B7 family of molecules is critical for the activation of naive CD4+ T cells. While prolonged TCR signaling is necessary for T cell activation, the duration of costimulatory signals required has not been established. In this study, murine bone marrow-derived dendritic cells (DC) and naïve CD4+ T cells were used to determine the temporal costimulatory requirements for naive T cell activation. By blocking CD80/CD86 costimulation at various time points during DC-T cell interaction and using the CFSE technique to assess the dynamics of T cell proliferation, we found that prolonged costimulation was required for naive T cells to enter and progress through the cell cycle over a wide range of peptide concentrations. Prolonged costimulation was also important for IL-2 production and CD25/CD69 expression by naive T cells. Video microscopy demonstrated that DC and naive T cells formed stable conjugates that persisted for more than 6 h. Thus, persistent CD80/CD86 signaling during prolonged interactions with DC allows naive T cells to enter the cell cycle and programs the daughter cells to undergo subsequent divisions.