Currents In Retinal Ganglion Cells Research Articles

Dual-Cell Patch-Clamp Recording Revealed a Mechanism for a Ribbon Synapse to Process Both Digital and Analog Inputs and Outputs.

A chemical synapse is either an action potential (AP) synapse or a graded potential (GP) synapse but not both. This study investigated how signals passed the glutamatergic synapse between the rod photoreceptor and its postsynaptic hyperpolarizing bipolar cells (HBCs) and light responses of retinal neurons with dual-cell and single-cell patch-clamp recording techniques. The results showed that scotopic lights evoked GPs in rods, whose depolarizing Phase 3 associated with the light offset also evoked APs of a duration of 241.8 ms and a slope of 4.5 mV/ms. The depolarization speed of Phase 3 (Speed) was 0.0001–0.0111 mV/ms and 0.103–0.469 mV/ms for rods and cones, respectively. On pairs of recorded rods and HBCs, only the depolarizing limbs of square waves applied to rods evoked clear currents in HBCs which reversed at −6.1 mV, indicating cation currents. We further used stimuli that simulated the rod light response to stimulate rods and recorded the rod-evoked excitatory current (rdEPSC) in HBCs. The normalized amplitude (R/Rmax), delay, and rising slope of rdEPSCs were differentially exponentially correlated with the Speed (all p < 0.001). For the Speed < 0.1 mV/ms, R/Rmax grew while the delay and duration reduced slowly; for the Speed between 0.1 and 0.4 mV/ms, R/Rmax grew fast while the delay and duration dramatically decreased; for the Speed > 0.4 mV/ms, R/Rmax reached the plateau, while the delay and duration approached the minimum, resembling digital signals. The rdEPSC peak was left-shifted and much faster than currents in rods. The scotopic-light-offset-associated major and minor cation currents in retinal ganglion cells (RGCs), the gigantic excitatory transient currents (GTECs) in HBCs, and APs and Phase 3 in rods showed comparable light-intensity-related locations. The data demonstrate that the rod-HBC synapse is a perfect synapse that can differentially decode and code analog and digital signals to process enormously varied rod and coupled-cone inputs.

Presynaptic SNAP-25 regulates retinal waves and retinogeniculate projection via phosphorylation

Patterned spontaneous activity periodically displays in developing retinas termed retinal waves, essential for visual circuit refinement. In neonatal rodents, retinal waves initiate in starburst amacrine cells (SACs), propagating across retinal ganglion cells (RGCs), further through visual centers. Although these waves are shown temporally synchronized with transiently high PKA activity, the downstream PKA target important for regulating the transmission from SACs remains unidentified. A t-SNARE, synaptosome-associated protein of 25 kDa (SNAP-25/SN25), serves as a PKA substrate, implying a potential role of SN25 in regulating retinal development. Here, we examined whether SN25 in SACs could regulate wave properties and retinogeniculate projection during development. In developing SACs, overexpression of wild-type SN25b, but not the PKA-phosphodeficient mutant (SN25b-T138A), decreased the frequency and spatial correlation of wave-associated calcium transients. Overexpressing SN25b, but not SN25b-T138A, in SACs dampened spontaneous, wave-associated, postsynaptic currents in RGCs and decreased the SAC release upon augmenting the cAMP-PKA signaling. These results suggest that SN25b overexpression may inhibit the strength of transmission from SACs via PKA-mediated phosphorylation at T138. Moreover, knockdown of endogenous SN25b increased the frequency of wave-associated calcium transients, supporting the role of SN25 in restraining wave periodicity. Finally, the eye-specific segregation of retinogeniculate projection was impaired by in vivo overexpression of SN25b, but not SN25b-T138A, in SACs. These results suggest that SN25 in developing SACs dampens the spatiotemporal properties of retinal waves and limits visual circuit refinement by phosphorylation at T138. Therefore, SN25 in SACs plays a profound role in regulating visual circuit refinement.

Peculiarities of Tetraethylammonium-Induced Blocking of Potassium Currents in Rat Retinal Ganglion Cells

We studied the sensitivity of integral potassium currents in retinal ganglion cells (RGCs) of 21-day-old rats to the blocking action of tetraethylammonium (TEA) applied in concentrations of 0.25 and 1.0 mM. The currents were recorded using a patch-clamp technique in the whole-cell configuration using a voltage-clamp mode; retinal preparations were not subjected to enzymatic treatment. Potassium currents were induced by long-lasting (400 msec) depolarizing current pulses of increased intensities. Under the action of 0.25 mM TEA, the integral potassium current was blocked by 18 ± 5%, on average; the amplitude of the TEA-sensitive (blocked) component at the membrane potential of +40 mV was 0.75 ± ± 0.02 nA (n = 6). At 1.0 mM TEA, 32 ± 5% of the current was blocked, and the mean amplitude of the TEA-sensitive component was 1.12 ± 0.02 nA (n = 9). The effect of TEA was reversible; the relative intensity of the TEA-sensitive component in different cells varied within a wide range (from 3 to 38% at 0.25 mM TEA and from 12 to 54% at 1.0 mM). A comparison of the findings with the data reported earlier showed that the dependence of the intensity of TEA blocking within a 0.25 to 1.0 mM range is nonlinear; it appears that the 0.5 mM concentration is relatively close to the saturating one. Analysis of the obtained data shows that TEA applied in the tested concentrations blocks mostly the potassium current through Kv3.1/Kv3.2 channels; the effects of the blocker on other types of channels (Kv3.3/Kv3.4, Kv1.1, and BK) are, in any case, much less significant.

Synthetic conantokin peptides potently inhibit N-methyl-D-aspartate receptor-mediated currents of retinal ganglion cells.

Retinal ganglion cells (RGCs), which are the sole output neurons of the retina, express N-methyl-D-aspartate receptors (NMDARs), rendering these cells susceptible to glutamate excitotoxicity, with implications for loss of normal RGC excitatory responses in disorders such as glaucoma and diabetic retinopathy. Therefore, antagonists that inhibit NMDAR-mediated currents specifically by targeting the GluN2B component of the ion channel have the potential to serve as a basis for developing potential therapeutics. The roles of peptidic conantokins, which are potent brain neuronal NMDAR inhibitors, were studied. By using patch-clamp whole-cell analyses in dissociated RGCs and retinal whole-mount RGCs, we evaluated the effects of synthetic conantokin-G (conG) and conantokin-T (conT), which are small γ-carboxyglutamate-containing peptides, on NMDA-mediated excitatory responses in mouse RGCs. Both conG and conT inhibited the NMDA-mediated currents of dark-adapted dissociated and whole-mount RGCs in a dose-dependent, reversible, noncompetitive manner. Inhibition of NMDA-mediated steady-state currents by NMDAR nonsubunit-selective conT was approximately threefold greater than GluN2B-selective conG or ifenprodil, demonstrating its potential ability to inhibit both GluN2A- and GluN2B-containing ion channels in RGCs. Because the extent of inhibition of NMDA-evoked currents by conG and the pharmacologic GluN2B-selective inhibitor ifenprodil were similar (40-45%) to that of the GluN2A-selective antagonist NVP-AAM0077, we conclude that the levels of GluN2A and GluN2B subunits are similar in RGCs. These results provide a novel basis for developing effective neuroprotective agents to aid in the prevention of undesired glutamatergic excitotoxicity in neurodegenerative diseases of the retina and demonstrate functional assembly of NMDARs in RGCs.

Loss of Photoreceptors Results in Upregulation of Synaptic Proteins in Bipolar Cells and Amacrine Cells

Deafferentation is known to cause significant changes in the postsynaptic neurons in the central nervous system. Loss of photoreceptors, for instance, results in remarkable morphological and physiological changes in bipolar cells and horizontal cells. Retinal ganglion cells (RGCs), which send visual information to the brain, are relatively preserved, but show aberrant firing patterns, including spontaneous bursts of spikes in the absence of photoreceptors. To understand how loss of photoreceptors affects the circuitry presynaptic to the ganglion cells, we measured specific synaptic proteins in two mouse models of retinal degeneration. We found that despite the nearly total loss of photoreceptors, the synaptophysin protein and mRNA levels in retina were largely unaltered. Interestingly, the levels of synaptophysin in the inner plexiform layer (IPL) were higher, implying that photoreceptor loss results in increased synaptophysin in bipolar and/or amacrine cells. The levels of SV2B, a synaptic protein expressed by photoreceptors and bipolar cells, were reduced in whole retina, but increased in the IPL of rd1 mouse. Similarly, the levels of syntaxin-I and synapsin-I, synaptic proteins expressed selectively by amacrine cells, were higher after loss of photoreceptors. The upregulation of syntaxin-I was evident as early as one day after the onset of photoreceptor loss, suggesting that it did not require any massive or structural remodeling, and therefore is possibly reversible. Together, these data show that loss of photoreceptors results in increased synaptic protein levels in bipolar and amacrine cells. Combined with previous reports of increased excitatory and inhibitory synaptic currents in RGCs, these results provide clues to understand the mechanism underlying the aberrant spiking in RGCs.

Photoswitching HCN Channels in Degenerated Retina Neurons

Some degenerative retinal diseases involve death of rods and cones, while the remaining retinal cells remain intact, yet nonresponsive to light. An azobenzene-photoswitch ion-channel-trans-blocker DENAQ restores retina light sensitivity in mouse model of photoreceptor degeneration (rd1), without affecting wild type (WT) animals. We compared ion currents in retinal ganglion cells (RGCs) of the WT and rd1 mice, and their regulation by light following application of DENAQ. Voltage-gated K-currents were regulated by DENAQ similarly in RGCs of the WT and rd1-retinas (19% vs 21%-increase by 480nm-light). However, DENAQ photosensitized hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels in rd1 RGCs, but not WT retinas. The WT-RGCs revealed mainly fast-activating (tens ms) Ih, while most rd1-RGCs showed a substantial portion of slow Ih (τa>100ms). Illumination produced 21%-increase of Ih (1.3pA/pF at −100mV) within hundreds ms in DENAQ-pretreated (150μM) neurons. Same light in DENAQ-treated WT-RGCs produced no fast changes, while some neurons showed small slow decrease of Ih (<5% developing within 5sec). We hypothesize that slow HCN channels up-regulated in degenerated retina, might be more sensitive to DENAQ than the fast ones in the WT, but do not exclude other targets mediating light-effects on Ih.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Voltage-Operated Calcium Currents in Retinal Ganglion Cells of the Rat Eye

Using a voltage-clamp technique in the whole-cell configuration, we studied voltage-operated calcium currents in retinal ganglion cells (RGCs) obtained from the rat eye. In all examined cells, we recorded high-threshold calcium currents with the mean maximum amplitude 285 ± 33 pA (104-593 pA; n = 22) at the membrane potential –10 to 0 mV. In a considerable part of the neurons (10 of 22; 45%), we also observed low-threshold calcium currents (published data on the presence of such currents in mammalian RGCs are contradictory). The mean maximum amplitude of the latter (low-threshold) currents was 99 ± 11 pA (54-157 pA; n = 10) at the membrane potential –35 mV. The observed differences in the patterns of voltage-dependent calcium currents in various RGCs can be related to functional differentiation of these cells.

Light-Evoked Currents in Retinal Ganglion Cells from Dystrophic RCS Rats

Purpose: To study the electrophysiological properties of the light-evoked currents in ganglion cells in situations of retinal degeneration. Methods: We investigated light-evoked currents in ganglion cells by performing whole-cell patch-clamp recordings from ganglion cells using a retina-stretched preparation from Royal College of Surgeons (RCS) rats, a model of retinal degeneration and congenic controls at different ages. Pharmacological inhibitors of the AMPA receptor (NBQX), GABA receptor (BMI), and sodium channels (TTX) were used to identify the components of the light-evoked currents in ON, OFF and ON-OFF retinal ganglion cells. Results: We found that the light-evoked currents in ganglion cells from control rats were inhibited by NBQX, BMI and TTX, suggesting that AMPA receptors, GABA receptors and sodium channels contribute to these currents in ganglion cells. However, only AMPA receptor-mediated currents were recorded in RCS rats. Light-evoked inward currents were absent in the majority of ganglion cells from RCS rats, particularly at the later stages of retinal degeneration. At earlier stages of retinal degeneration, we found that both the timing and amplitude of light-evoked currents are significantly different in ganglion cells from RCS and control rats. Conclusions: Our study furthers the understanding of the electrophysiological characteristics of retinal ganglion cells during retinal degeneration, and provides insight into the optimal timing for the treatment of retinal degeneration.

Spikes in Mammalian Bipolar Cells Support Temporal Layering of the Inner Retina

In the mammalian retina, 10-12 different cone bipolar cell (BC) types decompose the photoreceptor signal into parallel channels, providing the basis for the functional diversity of retinal ganglion cells (RGCs). BCs differing in their temporal properties appear to project to different strata of the retina's inner synaptic layer, based on somatic recordings of BCs and excitatory synaptic currents measured in RGCs. However, postsynaptic currents in RGCs are influenced by dendritic morphology and receptor types, and the BC signal can be transformed at the axon terminals both through interactions with amacrine cells and through the generation of all-or-nothing spikes. Therefore, the temporal properties of the BC output have not been analyzed systematically across different types of mammalian BCs. We recorded calcium signals directly within axon terminals using two-photon imaging and show that BCs can be divided into ≥eight functional clusters. The temporal properties of the BC output were directly reflected in their anatomical organization within the retina's inner synaptic layer: faster cells stratified closer to the border between ON and OFF sublamina. Moreover, ≥three fastest groups generated clear all-or-nothing spikes. Therefore, the systematic projection pattern of BCs provides distinct temporal "building blocks" for the feature extracting circuits of the inner retina.

Serine racemase deletion abolishes light‐evoked NMDA receptor currents in retinal ganglion cells

Glycine and/or D-serine are obligatory coagonists of the N-methyl-D-aspartate receptor (NMDAR). Serine racemase, the D-serine-synthesizing enzyme, is expressed by astrocytes and Müller cells of the retina, but little is known about its role in retinal signalling. In this study, we utilize a serine racemase knockout (SRKO) mouse to explore the contribution of D-serine to inner-retinal function. Retinal tissue levels of D-serine in SRKO mice are reduced by 85%. Whole-cell recordings from SRKO retinal ganglion cells showed markedly reduced coagonist occupancy of NMDARs and consequently a dramatic reduction in the NMDAR component of light-evoked responses. NMDAR currents in SRKOs could be rescued by applying exogenous coagonist, but SRKO ganglion cells still displayed lower NMDA/AMPA receptor ratios than wild-type (WT) controls when the coagonist site was saturated. Despite having abnormalities in synaptic glutamatergic transmission, SRKO mice displayed no obvious signs of visual impairment in behavioural testing. These findings raise interesting questions about the role of D-serine in inner-retinal function and development.

Nimodipine Enhancement of α2 Adrenergic Modulation of NMDA Receptor via a Mechanism Independent of Ca2+Channel Blocking

To further understand alpha2 receptor signaling in the retina and the mechanisms that mediate ocular beneficial effects of brimonidine (an alpha2 agonist) and nimodipine (an L-type Ca(2+) channel blocker). The authors used in situ retinal ganglion cells (RGCs) in the isolated rat retina to characterize alpha2 modulation of NMDA receptor function and a rabbit retinal NMDA excitotoxicity model to verify in vitro findings under in vivo conditions. Electrophysiological (whole-cell patch clamp) recordings and Ca(2+) imaging were used to characterize NMDA receptor function and to verify the effect of various Ca(2+) channel blockers. In vivo drug application in rabbits was achieved by intravitreal injections. Application of NMDA elicited a robust whole-cell inward current in individual in situ RGCs voltage clamped at -70 mV. Pretreatment with brimonidine significantly reduced NMDA-elicited currents in RGCs. This suppressive effect of brimonidine was substantially enhanced by background addition of nimodipine or isradipine, but not by diltiazem, verapamil, or cadmium. This effect of nimodipine was blocked by either a selective alpha2 antagonist, a cyclic adenosine monophosphate (cAMP) analogue, or an adenylate cyclase activator, indicating that nimodipine acts through the alpha2 receptor-G(alphai)-coupled pathway. Brimonidine protects RGCs in the rabbit excitotoxicity model. This brimonidine protection is also enhanced significantly by application of nimodipine but not of diltiazem. These in vitro and in vivo findings demonstrate a novel neural mechanism involving nimodipine enhancement of alpha2 signaling in RGCs. This nimodipine effect appears to be independent of its classic L-type Ca(2+) channel-blocking action.

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.

Melatonin modulates glycine currents of retinal ganglion cells in rat

Melatonin is a hormone participating in the modulation of various physiological functions via binding to specific melatonin receptors. In the retina, melatonin is synthesized and released by photoreceptors and may play a neuromodulatory role. By using patch clamp techniques, we demonstrate for the first time that glycine-induced currents from a population of isolated ganglion cells in the rat retina are potentiated by melatonin of nanomolar concentrations by increasing the efficacy and the channel conductance of the strychnine-sensitive glycine receptor. The melatonin effect is blocked by 4-P-PDOT, indicating the mediation of the MT2 receptor. These results suggest that melatonin, along with the MT2 receptor, may be involved in retinal information processing by modulating glycine receptor-mediated inhibition.

GABA(A) receptor-mediated signaling alters the structure of spontaneous activity in the developing retina.

Ambient GABA modulates firing patterns in adult neural circuits by tonically activating extrasynaptic GABA(A) receptors. Here, we demonstrate that during a developmental period when activation of GABA(A) receptors causes membrane depolarization, tonic activation of GABA(A) receptors blocks all spontaneous activity recorded in retinal ganglion cells (RGCs) and starburst amacrine cells (SACs). Bath application of the GABA(A) receptor agonist muscimol blocked spontaneous correlated increases in intracellular calcium concentration and compound postsynaptic currents in RGCs associated with retinal waves. In addition, GABA(A) receptor agonists activated a tonic current in RGCs that significantly reduced their excitability. Using a transgenic mouse in which green fluorescent protein is expressed under the metabotropic glutamate receptor subtype 2 promoter to target recordings from SACs, we found that GABA(A) receptor agonists blocked compound postsynaptic currents and also activated a tonic current. GABA(A) receptor antagonists reduced the holding current in SACs but not RGCs, indicating that ambient levels of GABA tonically activate GABA(A) receptors in SACs. GABA(A) receptor antagonists did not block retinal waves but did alter the frequency and correlation structure of spontaneous RGC firing. Interestingly, the drug aminophylline, a general adenosine receptor antagonist used to block retinal waves, induced a tonic GABA(A) receptor antagonist-sensitive current in outside-out patches excised from RGCs, indicating that aminophylline exerts its action on retinal waves by direct activation of GABA(A) receptors. These findings have implications for how various neuroactive drugs and neurohormones known to modulate extrasynaptic GABA(A) receptors may influence spontaneous firing patterns that are critical for the establishment of adult neural circuits.

Effect of hypothalamic proline-rich-polypeptide on voltage-gated Ca2+ currents in retinal ganglion cells.

Neurotrophins are molecules that regulate neuronal survival, nervous system plasticity, and many other physiological functions of neuronal and glial cells. Here we studied the physiological action of a novel neurosecretory polypeptide proline-rich polypeptide (PRP), isolated from bovine neurohypophysis neurosecretory granules, on voltage-gated Ca currents and spike firing activity of retinal ganglion cells. PRP reversibly increased high voltage-activated L-type Ca current, but was without effect on low voltage-activated T-type current. PRP also increased the spike after hyperpolarization and reduced the frequency of spike firing, most likely by affecting a Ca-dependent potassium current.

Effects of β-adrenergic blockers on glutamate-induced calcium signals in adult mouse retinal ganglion cells

Betaxolol, a selective β 1-adrenoceptor antagonist, is an antiglaucoma drug commonly used to lower the intraocular pressure (IOP) in treatment of glaucoma. Recent evidence has also shown that it attenuates ligand- and voltage-gated currents in retinal ganglion cells, which may lead to reduction of intracellular calcium and prevention of glutamate-induced ganglion cell damage in glaucoma. In the present study, we examined the effectiveness of betaxolol and other β-adrenergic blockers on glutamate-induced calcium signals. Dissociated adult mouse retinal ganglion cells were immuno-labeled with antibody CD90.2 and loaded with Fura-2AM. Calcium signals were recorded with optical recording techniques. Low doses of glutamate cause an increase in intracellular calcium that may result in pathological changes in ganglion cells. The action of glutamate could be reversibly suppressed by β-adrenergic blockers and the order of inhibitory potency is ( s)(−)-propranolol>betaxolol≫timolol, with average IC 50 of 78.05, 235.7 and 2167.05, μM, respectively. Betaxolol compressed the dose–response curve of glutamate. The EC 50 of glutamate was shifted from 6.19 to 23.53 μM, indicating that betaxolol acts as a non-competitive inhibitor of glutamate response in retinal ganglion cells. Our data are consistent with previous reports that betaxolol and other β-adrenergic blockers may exert its neuroprotective action by suppression of glutamate-induced intracellular calcium increase in retinal ganglion cells.

Reduction of Potassium Currents and Phosphatidylinositol 3-Kinase-Dependent Akt Phosphorylation by Tumor Necrosis Factor-α Rescues Axotomized Retinal Ganglion Cells from Retrograde Cell DeathIn Vivo

Tumor-necrosis-factor-alpha (TNF-alpha) prevented secondary death of retinal ganglion cells (RGCs) after axotomy of the optic nerve in vivo. This RGC rescue was confirmed in vitro in a mixed retinal culture model. In accordance with our previous findings, TNF-alpha decreased outward potassium currents in RGCs. Antagonism of the TNF-alpha-induced decrease in outward potassium currents with the potassium channel opener minoxidilsulfate (as verified by electrophysiology) abolished neuroprotection. Western blot analysis revealed an upregulation of phospho-Akt as a consequence of TNF-alpha-induced potassium current reduction. Inhibition of the phosphatidylinositol 3-kinase-Akt pathway with wortmannin decreased TNF-alpha-promoted RGC survival. These data point to a functionally relevant cytokine-dependent neuroprotective signaling cascade in adult CNS neurons.

Development and Organization of the Retina: From Molecules to Function

Development of Vision and the Pre-Visual System S.S. Easter, Jr., et al. Cellular and Molecular Aspects of Photoreceptor Differentiation R. Adler. Embryonic Patterning of Cone Subtypes in the Mammalian Retina K.C. Wikler, D.L. Stull. Development of Cone Distribution Patterns in Mammals A. Szel, et al. Genesis of Topographic and Cellular Diversity in the Primate Retina P. Rakic. Development of ON and OFF Retinal Ganglion Cell Mosaics L.M. Chalupa, et al. Getting to Grips with Neuronal Diversity: What is a Neuronal Type? J.E. Cook. Glio-Neuronal Interactions in Retinal Development A. Reichenbach. Comparative Anatomy and Function of Mammalian Horizontal Cells L. Peichl, et al. Parallel Pathways of Primate Vision: Sampling of Information in the Fourier Space by M and P Cells L. Peichl, et al. Parallel Pathways of Primate Vision: Sampling of Information in the Fourier Space by M and P Cells L.C.L. Silveira, H.D. De Mello, Jr. Transient and Persistent Na+, Ca2+, and Mixed-Cation Currents in Retinal Ganglion Cells A.T. Ishida. Synaptic Transmission Between Retinal Neurons M. Wilson. Scaling the Retina, Micro and Macro B.L. Finlay, R.L. Snow. Retinal Ganglion Cell Axonal Transport: Moving Down the Road to Functional Connections K.L. Moya. 5 Additional Articles. Index.

Retinal Ganglion Cell Dysfunction Induced by Hypoxia and Glutamate: Potential Neuroprotective Effects of β-Blockers

The objective of this study was to examine the effects of hypoxia, glutamate, and beta-blockers on the electrical activities of retinal ganglion cells. Single-unit extracellular and whole-cell voltage clamp recording techniques were used to record electrical activities from ganglion cells in the tiger salamander retina. This was performed under physiologic conditions, hypoxia, or elevated exogenous or endogenous glutamate levels. Light-evoked spike activities, glutamate-induced currents, and voltage-gated sodium and calcium currents were measured in the presence of the beta-1 selective antagonist betaxolol or the nonselective antagonist timolol. Hypoxia resulted in suppressing or blocking the OFF responses in the majority of ON-OFF ganglion cells tested, whereas the ON responses were only slightly affected. The presence of increased glutamate had similar findings and demonstrated an increase in the spontaneous firing rate of retinal ganglion cells. Betaxolol (2–50 μM) reduced the rate of spontaneous firing of retinal ganglion cells induced by glutamate. At 2 to 50 μM, betaxolol reversibly reduced the voltage-gated sodium currents and calcium currents in retinal ganglion cells. Timolol (up to 100 μM) did not demonstrate any detectable action on these currents. The physiologic responses of retinal ganglion cells to hypoxia or elevated glutamate levels in this animal model appear to be very similar. Although short-term exposure to hypoxia and glutamate used in this study exerts reversible actions on ganglion cells and does not induce permanent cell damage, such initial physiologic actions are likely to be precursors of permanent cell damage. Thus, hypoxia and elevated glutamate levels in the retina may represent a final pathway in diseases affecting retinal ganglion cells, such as glaucoma. Similar damage could result from different factors, such as decreased perfusion-induced ischemia or anomalous neuronal processing of glutamate. Betaxolol exerts its primary neuronal actions on retinal ganglion cells. It reversibly blocked voltage-gated calcium current and reduced the spontaneous firing rate by suppressing glutamate-gated currents and sodium currents in ganglion cells. These actions may protect ganglion cells from damage caused by ischemia or elevated glutamate levels.

Alterations in channel density and kinetic properties of the sodium current in retinal ganglion cells of the rat during in vivo differentiation

Changes in the kinetic properties of voltage-activated sodium currents (I Na) were studied in rat retinal ganglion cells during in vivo differentiation. Whole-cell recordings from cells maintained as retinal slices or whole-mounts were examined using the patch-clamp technique in the perforated patch mode. Voltage-clamp recordings revealed significant ontogenetic modifications in key properties of I Na and the present study described for the first time the detailed time course of such alterations. I Na was first expressed on embryonic day 17/18 (E17/18). Current density increased during development from an average of −81 pA/pF on E17/18 to a maximum of −747 pA/pF on postnatal day 10/12 (P10/12). Simultaneously, the activation of I Na shifted towards more negative potentials, reflected by a shift in the potential of half-activation from −14.1 mV on E17/18 to −37.5 mV on P10/12. No significant changes in these parameters were observed after P10/12. Steady-state inactivation shifted first towards more positive potentials, reflected by a shift in the potential of half-inactivation from −51 mV on E17/18 to −38 mV on P3/5, but shifted back towards more negative values thereafter (−44 mV in the adult). The most striking feature of I Na in rat RGCs was a transient slowing of I Na kinetics that was never described before. Time to peak and decay time constants increased between E20 and P5, resulting in slow and broad sodium currents within a developmental period that is characterized by intensive synaptogenesis in the target structures of retinal ganglion cells and maximum retinal ganglion cell death. Thereafter, time to peak and decay time constants decreased again to values found before E20, resulting in rapid sodium spikes. In conclusion, sodium currents in rat retinal ganglion cells displayed substantial electrophysiological changes during pre- and postnatal development. These changes in the sodium system had different temporal time patterns, indicating that they may play specific roles during the development of the visual system.