Abram Akopian

Activity-Dependent Phosphorylation of Tyrosine Hydroxylase in Dopaminergic Neurons of the Rat Retina

We studied in vivo activity-dependent phosphorylation of tyrosine hydroxylase (TH) in dopaminergic (DA) neurons of the rat retina. TH phosphorylation (TH-P) was evaluated by immunocytochemistry, using antibodies specific for each of three regulated phosphorylation sites. TH synthesis rate was measured by dihydroxyphenylalanine (DOPA) accumulation in the presence of NSD-1015, an inhibitor of aromatic amino acid decarboxylase. TH-P was increased markedly by light or after intraocular injection of GABAA and glycine inhibitors. All three phosphospecific antibodies responded similarly to test drugs or light. A 30 min exposure to light increased DOPA accumulation by threefold over that seen after 30 min in darkness. Immunostaining to an anti-panNa channel antibody was found in all parts of the DA neuron. TTX blocked TH-P induced by light or GABA/glycine inhibitors but only in varicosities of the DA axon plexus, not in perikarya or dendrites. Veratridine increased TH-P in all parts of the DA neuron. The distribution of the monoamine vesicular transporter 2 was shown by immunocytochemistry to reside in varicosities of the DA plexus but not in dendrites, indicating that the varicosities are sites of dopamine release. Collectively, these data indicate that, in the retina, dopamine synthesis in varicosities is affected by the spiking activity of retinal neurons, possibly including that of the DA neurons themselves.

Calcium and retinal function

We survey the primary roles of calcium in retinal function, including photoreceptor transduction, transmitter release by different classes of retinal neuron, calcium-mediated regulation of gap-junctional conductance, activation of certain voltage-gated channels for K+ and C1−, and modulation of postsynaptic potentials in retinal ganglion cells. We discuss three mechanisms for changing [Ca2+]i, which include flux through voltage-gated calcium channels, through ligand-gated channels, and by release from stores. The neuromodulatory pathways affecting each of these routes of entry are considered. The many neuromodulatory mechanisms in which calcium is a player are described and their effects upon retinal function discussed.

Activation of metabotropic glutamate receptors decreases a high-threshold calcium current in spiking neurons of the Xenopus retina

Two types of spiking neuron were identified among acutely dissociated neurons from the Xenopus retina by their responses to a depolarizing current step: single spikers and multiple spikers. In culture, multiple spikers had perikaryal diameters > 15 microns, whereas single spikers had smaller somata, 5-10 microns in diameter. Using a conventional whole-cell patch-clamp technique, both T- and L-type calcium currents were identified in multiply spiking cells whereas only an L-type current was present in singly spiking cells. The metabotropic glutamate receptor (mGluR) agonist trans-(1S-3R)-1-amino-1,3-cyclopentane-dicarboxylic acid (trans-ACPD) significantly decreased the L-type calcium current by 46 +/- 3% (mean +/- S.E.M.) in both types of cell but had only a minor effect on the T-type current in multiply spiking neurons. In the presence of 50 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 microM quisqualate (a potent mGluR1/5 agonist) decreased the L-type calcium current by 47 +/- 9% but had no effect on the T-type current. The selective mGluR4/6/7 agonist (+/-) 2-amino-4-phosphonobutyric acid (L-AP4, 100 microM), and the mGluR2/3 agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG1, 100 microM) decreased the L-type calcium current by 12 +/- 3% and 14 +/- 2%, respectively. The inhibition of calcium current by trans-ACPD was reduced when the patch pipette contained the G-protein inhibitor, GDP beta S. The presence of the G-protein activator GTP gamma S in the patch pipette irreversibly reduced the L-type calcium current, but was without effect on the T-type current. Heparin applied intracellularly significantly reduced the inhibitory effect of quisqualate, indicating an involvement of the inositol triphosphate (IP3) pathway in the mGluR-induced reduction of calcium current. Replacement of internal EGTA with BAPTA significantly reduced the inhibitory effect of quisqualate. In contrast, internal application of cAMP did not prevent an inhibition of calcium current by quisqualate. Thus, the mechanism by which calcium current is inhibited by mGluR seems not to involve an intracellular cAMP cascade. Our findings indicate that activation of mGluR1/5 results in the inhibition of a high-threshold calcium current. This process is mediated by the activation of a G-protein and is consistent with inhibition occurring by an IP3-stimulated release of internal calcium.

Calcium Homeostasis and Cone Signaling Are Regulated by Interactions between Calcium Stores and Plasma Membrane Ion Channels

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca2+ entry (SOCE) to Ca2+ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn2+ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca2+ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca2+ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca2+ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca2+ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca2+ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca2+ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca2+ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse.

Masked excitatory crosstalk between the ON and OFF visual pathways in the mammalian retina

Non‐technical summary  An organizing principle of the visual system is the segregation of ON and OFF responses into parallel streams to signal light increment and decrement. This segregation begins in the retina where the output ganglion cells can be divided into ON and OFF subtypes based on their responses to light. Here we show that blockade of GABAergic inhibition reveals opposite polarity responses in ganglion cells whereby OFF cells display ON responses and ON cells display OFF responses. This crossover excitation was found in both the rabbit and mouse, indicating that it is a common synaptic mechanism in the mammalian retina. Overall, these results challenge the idea that light increment and decrement is signalled by different visual pathways. Moreover, our findings suggest that release of inhibition under certain light conditions can enable single ganglion cells to carry both ON and OFF signals, thereby allowing additional information to be propagated across the limited bandwidth of the optic nerve.

Calcium channel and glutamate receptor activities regulate actin organization in salamander retinal neurons

Intracellular Ca2+ regulates a variety of neuronal functions, including neurotransmitter release, protein phosphorylation, gene expression and synaptic plasticity. In a variety of cell types, including neurons, Ca2+ is involved in actin reorganization, resulting in either actin polymerization or depolymerization. Very little, however, is known about the relationship between Ca2+ and the actin cytoskeleton organization in retinal neurons. We studied the effect of high‐K+‐induced depolarization on F‐actin organization in salamander retina and found that Ca2+ influx through voltage‐gated L‐type channels causes F‐actin disruption, as assessed by 53 ± 5% (n= 23, P < 0.001) reduction in the intensity of staining with Alexa‐Fluor488‐phalloidin, a compound that permits visualization and quantification of polymerized actin. Calcium‐induced F‐actin depolymerization was attenuated in the presence of protein kinase C antagonists, chelerythrine or bis‐indolylmaleimide hydrochloride (GF 109203X). In addition, phorbol 12‐myristate 13‐acetate (PMA), but not 4α‐PMA, mimicked the effect of Ca2+ influx on F‐actin. Activation of ionotropic AMPA and NMDA glutamate receptors also caused a reduction in F‐actin. No effect on F‐actin was exerted by caffeine or thapsigargin, agents that stimulate Ca2+ release from internal stores. In whole‐cell recording from a slice preparation, light‐evoked ‘off’ but not ‘on’ EPSCs in ‘on–off’ ganglion cells were reduced by 60 ± 8% (n= 8, P < 0.01) by cytochalasin D. These data suggest that elevation of intracellular Ca2+ during excitatory synaptic activity initiates a cascade for activity‐dependent actin remodelling, which in turn may serve as a feedback mechanism to attenuate excitotoxic Ca2+ accumulation induced by synaptic depolarization.

Intracellular calcium reduces light-induced excitatory post-synaptic responses in salamander retinal ganglion cells

1 The whole‐cell patch clamp technique was used to study the effect of intracellular Ca2+ on light‐evoked EPSCs in on‐off ganglion cells in salamander retinal slices. Both AMPA and NMDA receptors contributed to the light‐evoked responses. 2 In the presence of strychnine and picrotoxin, ganglion cells responded to light onset and offset with transient inward currents at ‐70 mV. These currents were reduced by 35 ± 3 % when the light stimulus was preceded by a depolarizing step from ‐70 to 0 mV. 3 The inhibitory effect of depolarization on light‐evoked EPSCs was strongly reduced in the presence of 10 mm BAPTA. 4 The degree of EPSC inhibition by the prepulse holding potential followed the current‐voltage relationship of the Ca2+ current found in the ganglion cell. 5 In the presence of the NMDA receptor antagonist AP‐7, glutamate‐dependent current was nearly abolished when high Ca2+ was substituted for high Na+ solution. 6 The release of Ca2+ from internal stores by caffeine or inositol trisphosphate reduced the EPSCs by 36 ± 5 and 38 ± 11 %, respectively, and abolished the inhibitory effect of depolarization. 7 The inhibitory effect of depolarization on EPSCs was reduced 5‐fold in the presence of AP‐7, but was not reduced by the AMPA receptor antagonist CNQX. 8 Neither inhibition of Ca2+‐calmodulin‐dependent enzymes, nor inhibition of protein kinase A or C had any significant effect on the depolarization‐induced inhibition of EPSCs. 9 Our data suggest that elevation of [Ca2+]i, through voltage‐gated channels or by release from intracellular stores, reduced primarily the NMDA component of the light‐evoked EPSCs.

Glutamate-induced internalization of Cav1.3 L-type Ca2+ channels protects retinal neurons against excitotoxicity

Glutamate‐induced rise in the intracellular Ca2+ level is thought to be a major cause of excitotoxic cell death, but the mechanisms that control the Ca2+ overload are poorly understood. Using immunocytochemistry, electrophysiology and Ca2+ imaging, we show that activation of ionotropic glutamate receptors induces a selective internalization of Cav1.3 L‐type Ca2+ channels in salamander retinal neurons. The effect of glutamate on Cav1.3 internalization was blocked in Ca2+‐free external solution, or by strong buffering of internal Ca2+ with BAPTA. Downregulation of L‐type Ca2+ channel activity in retinal ganglion cells by glutamate was suppressed by inhibitors of dynamin‐dependent endocytosis. Stabilization of F‐actin by jasplakinolide significantly reduced the ability of glutamate to induce internalization suggesting it is mediated by Ca2+‐dependent reorganization of actin cytoskeleton. We showed that the Cav1.3 is the primary L‐type Ca2+ channel contributing to kainate‐induced excitotoxic death of amacrine and ganglion cells. Block of Cav1.3 internalization by either dynamin inhibition or F‐actin stabilization increased vulnerability of retinal amacrine and ganglion cells to kainate‐induced excitotoxicity. Our data show for the first time that Cav1.3 L‐type Ca2+ channels are subject to rapid glutamate‐induced internalization, which may serve as a negative feedback mechanism protecting retinal neurons against glutamate‐induced excitotoxicity.

Both high- and low voltage-activated calcium currents contribute to the light-evoked responses of luminosity horizontal cells in the Xenopus retina

We examined the contribution of two intrinsic voltage-dependent calcium channels to the light-evoked responses of a non-spiking retinal neuron, the horizontal cell (HC). HCs isolated from the Xenopus retina were studied by the whole cell version of the patch clamp. In a mixture of agents which suppressed Na- and K-dependent currents, we identified a transient, low voltage-activated Ca current suppressed by Ba2+ and blocked by Ni2+ (T-type) and a sustained, high voltage-activated, dihydropyridine-sensitive Ca current that was enhanced by Ba2+ (L-type). We made simultaneous intracellular recordings from rods and HCs in the intact, dark-adapted Xenopus retina. Under certain stimulus conditions, transient oscillations appeared in HC responses but were absent in rod light-evoked waveforms. One type of transient was seen at relatively hyperpolarized potentials (< -45 mV), was enhanced by Sr2+ and inhibited by Ni2+. It thus appears to depend on a T-type Ca-current. A second type of oscillation was seen to be superimposed on a prolonged depolarizing wave following light off in the HC and as spike-like depolarizations in rods. These oscillations were enhanced by Ba2+ and Sr2+, but blocked by the dihydropyridine, nifedipine, indicating their dependence on an L-type calcium conductance. All calcium-dependent oscillations were suppressed by 0.05-0.5 mM Co2+. Suppression of glutamate neurotransmission with CNQX or kynurenate, or glycine neurotransmission with strychnine, enhanced the HC oscillations.

Glutamate-induced Ca2+ influx in third-order neurons of salamander retina is regulated by the actin cytoskeleton

Ligand-gated ion channels (ionotropic receptors) link to the cortical cytoskeleton via specialized scaffold proteins and thereby to appropriate signal transduction pathways in the cell. We studied the role of filamentous actin in the regulation of Ca influx through glutamate receptor-activated channels in third-order neurons of salamander retina. Staining by Alexa-Fluor 488-phalloidin, to visualize polymerized actin, we show localization of filamentous actin in neurites, and the membrane surrounding the cell soma. With Ca(2+) imaging we found that in dissociated neurons, depolymerization of filamentous actin by latrunculin A, or cytochalasin D significantly reduced glutamate-induced intracellular Ca(2+) accumulation to 53+/-7% of control value. Jasplakinolide, a stabilizer of filamentous actin, by itself slightly increased the glutamate-induced Ca(2+) signal and completely attenuated the inhibitory effect when applied in combination with actin depolymerizing agents. These results indicate that in salamander retinal neurons the actin cytoskeleton regulates Ca(2+) influx through ionotropic glutamate receptor-activated channels, suggesting regulatory roles for filamentous actin in a number of Ca(2+)-dependent physiological and pathological processes.