Abram Akopian, Ph.D.

Masked excitatory crosstalk between the ON and OFF visual pathways in the mammalian retina
Farajian, Reza; Pan, Feng; Akopian, Abram; Volgyi, Bela; Bloomfield, Stewart A
2011 Sep 15;589(Pt 18):4473-4489, Journal of physiology
Abstract A fundamental organizing feature of the visual system is the segregation of ON and OFF responses into parallel streams to signal light increment and decrement. However, we found that blockade of GABAergic inhibition unmasks robust ON responses in OFF alpha-ganglion cells (alpha-GCs). These ON responses had the same centre-mediated structure as the classic OFF responses of OFF alpha-GCs, but were abolished following disruption of the ON pathway with l-AP4. Experiments showed that both GABA(A) and GABA(C) receptors are involved in the masking inhibition of this ON response, located at presynaptic inhibitory synapses on bipolar cell axon terminals and possibly amacrine cell dendrites. Since the dendrites of OFF alpha-GCs are not positioned to receive excitatory inputs from ON bipolar cell axon terminals in sublamina-b of the inner plexiform layer (IPL), we investigated the possibility that gap junction-mediated electrical synapses made with neighbouring amacrine cells form the avenue for reception of ON signals. We found that the application of gap junction blockers eliminated the unmasked ON responses in OFF alpha-GCs, while the classic OFF responses remained. Furthermore, we found that amacrine cells coupled to OFF alpha-GCs display processes in both sublaminae of the IPL, thus forming a plausible substrate for the reception and delivery of ON signals to OFF alpha-GCs. Finally, using a multielectrode array, we found that masked ON and OFF signals are displayed by over one-third of ganglion cells in the rabbit and mouse retinas, suggesting that masked crossover excitation is a widespread phenomenon in the inner mammalian retina
— id: 137837, year: 2011, vol: 589, page: 4473, stat: Journal Article,

Ca(v)1.3 L-type Ca2+ channels modulate depression-like behaviour in mice independent of deaf phenotype
Busquet, P; Nguyen, NK; Schmid, E; Tanimoto, N; Seeliger, MW; Ben-Yosef, T; Mizuno, F; Akopian, A; Striessnig, J; Singewald, N
2010 MAY ;13(4):499-513, International journal of neuropsychopharmacology
Mounting evidence suggests that voltage-gated L-type Ca2+ channels can modulate affective behaviour. We therefore explored the role of Ca(v)1.3 L-type Ca2+ channels in depression-and anxiety-like behaviours using Ca(v)1.3-deficient mice (Ca-v-1.3(-/-)). We showed that Ca(v)1.3(-/-) mice displayed less immobility in the forced swim test as well as in the tail suspension test, indicating an antidepressant-like phenotype. Locomotor activity in the home cage or a novel open-field test was not influenced. In the elevated plus maze (EPM), Ca(v)1.3(-/-) mice entered the open arms more frequently and spent more time there indicating an anxiolytic-like phenotype which was, however, not supported in the stress-induced hyperthermia test. By performing parallel experiments in Claudin 14 knockout mice (Cldn14(-/-)), which like Ca(v)1.3(-/-) mice are congenitally deaf, an influence of deafness on the antidepressant-like phenotype could be ruled out. On the other hand, a similar EPM behaviour indicative of an anxiolytic phenotype was also found in the Cldn14(-/-) animals. Using electroretinography and visual behavioural tasks we demonstrated that at least in mice, Ca-v-1.3 channels do not significantly contribute to visual function. However, marked morphological changes were revealed in synaptic ribbons in the outer plexiform layer of Ca(v)1.3(-/-) retinas by immunohistochemistry suggesting a possible role of this channel type in structural plasticity at the ribbon synapse. Taken together, our findings indicate that Cav1.3 L-type Ca2+ channels modulate depression-like behaviour but are not essential for visual function. The findings raise the possibility that selective modulation of Ca(v)1.3 channels could be a promising new therapeutic concept for the treatment of mood disorders
— id: 110151, year: 2010, vol: 13, page: 499, stat: Journal Article,

Glutamate-induced internalization of Cav1.3 L-type Ca2+ channels protects retinal neurons against excitotoxicity
Mizuno, Fengxia; Barabas, Peter; Krizaj, David; Akopian, Abram
2010 Mar 15;588(Pt 6):953-966, Journal of physiology
Glutamate-induced rise in the intracellular Ca(2+) level is thought to be a major cause of excitotoxic cell death, but the mechanisms that control the Ca(2+) overload are poorly understood. Using immunocytochemistry, electrophysiology and Ca(2+) imaging, we show that activation of ionotropic glutamate receptors induces a selective internalization of Ca(v)1.3 L-type Ca(2+) channels in salamander retinal neurons. The effect of glutamate on Ca(v)1.3 internalization was blocked in Ca(2+)-free external solution, or by strong buffering of internal Ca(2+) with BAPTA. Downregulation of L-type Ca(2+) 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 Ca(2+)-dependent reorganization of actin cytoskeleton. We showed that the Ca(v)1.3 is the primary L-type Ca(2+) channel contributing to kainate-induced excitotoxic death of amacrine and ganglion cells. Block of Ca(v)1.3 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 Ca(v)1.3 L-type Ca(2+) channels are subject to rapid glutamate-induced internalization, which may serve as a negative feedback mechanism protecting retinal neurons against glutamate-induced excitotoxicity
— id: 108428, year: 2010, vol: 588, page: 953, stat: Journal Article,

CaV1.3 L-type calcium channels modulate depression-like behavior in mice independent of deaf phenotype
Busquet P.; Nguyen N.K.; Schmid E.; Tanimoto N.; Seeliger M.W.; Ben-Yosef T.; Mizuno F.; Akopian A.; Singewald N.; Striessnig J.
2009 ;9:?-?, BMC pharmacology
Background: Mounting evidence suggests that neuronal voltage-gated Ca_VI.2 and Ca_VI.3 L-type calcium channels (LTCCs) can modulate mood and anxiety behaviors. In Ca_VI.2 dihydro- pyridine (DHP)-insensitive mice (Ca_VI.2DHP^-/- mice), systemic application of the DHP channel activator BAYK 8644 induced pro-depression-like behavior providing evidence for a possible role of Ca_VI.3 channels in mood behavior. We therefore explored the role of Ca_VI.3 LTCCs in depression- and anxietylike behaviors using Ca_VI.3-deficient mice (Ca_VI.3^-/-). However, Ca_VI.3^-/- mice are congenitally deaf and it is so far unclear how deafness affects emotional behavior in mice. We therefore used another mouse model suffering from congenital deafness, claudin I4-deficient mice (CldnI4^-/-) as a control to address this question. As Ca_VI.3 channels are expressed in the retina we also investigated Ca_VI.3^-/- mice for possible disturbances in retinal morphology and visual function that could interfere with behavioral analysis. Methods: Depression-like behavior was assessed using forced swim and tail suspension tests (FST and TST) whereas elevated plus maze (EPM) and stress-induced hyperthermia (SIH) were performed to test anxiety-like behavior. Morris water maze, electroretinography and immunofluorescence stainings were performed to evaluate the consequence on visual acuity and retinal morphology of Ca_VI.3deletion inCa_VI.3^-/- mice. Results: We showed that Ca_VI.3^-/- mice displayed less immobility in the FST as well as in the TST, indicating an antidepressant-like phenotype. In the EPM, Ca_VI.3^-/- mice entered the open arms more frequently and spent more time there indicating an anxiolytic-like phenotype which was, however not supported in the SIH test. By performing parallel experiments in Cldn14^-/- mice, an influence of deafness on the antidepressant-like phenotype could be ruled out. On the other hand, a similar EPM behavior indicative of an anxiolytic phenotype was also found in the Cldn14^-/- animals. Using electroretinography and visual behavioral tasks we demonstrated that in mice, Ca_Vl.3 channels do not significantly contribute to visual function. However, distinct morphological changes were revealed in synaptic ribbons in the outer plexiform layer of Ca_Vl.3^-/- retinas by immunohistochemistry. Although these changes have no major effects on visual function, they indicate a possible role of this channel type in structural plasticity at the ribbon synapse. Conclusion: Ca_Vl.3 LTCCs modulate depression-like behavior but are not essential for visual function. The findings raise the possibility that selective modulation of Ca_Vl.3 channels could be a promising new therapeutic concept for the treatment of mood disorders
— id: 122705, year: 2009, vol: 9, page: ?, stat: Journal Article,

Disruption of actin cytoskeleton causes internalization of Ca(v)1.3 (alpha 1D) L-type calcium channels in salamander retinal neurons
Cristofanilli, Massimiliano; Mizuno, Fengxia; Akopian, Abram
2007 ;13:1496-1507, Molecular vision
PURPOSE: To study the influence of actin cytoskeleton reorganization on the subcellular distribution of Ca(v)1.3 L-type Ca2+ channels in salamander retinal third-order neurons. METHODS: Immunocytochemistry with confocal microscopy was used to demonstrate internalization of the Ca(v)1.3 isoform of L-type voltage-gated Ca2+ channels in third-order retinal neurons. A specificity of antibody was confirmed with Western blotting and in control experiments preabsorbing antibody wit its respective peptide. Whole-cell patch clamp technique was applied to record L-type currents from ganglion cells in slice preparations in the presence of N- and P/Q type Ca2+ channel blockers. RESULTS: A high level of Ca(v)1.3 labeling was present in cone photoreceptor terminals in the outer plexiform layer (OPL), as aggregates of puncta. Punctate Ca(v)1.3 labeling was evident throughout the IPL and around the cell bodies in the outer nuclear (ONL), inner nuclear (INL) and on somas and axons of ganglion cells labeled with rhodamine-conjugated dextran. Doubly labeled sections for synaptophysin and Ca(v)1.3 revealed colocalization in the OPL and IPL. Depolymerization of the actin cytoskeleton caused a dynamin-dependent internalization of Ca(v)1.3 but not Ca(v)1.2 subtype of voltage-gated Ca2+ channels in dissociated neurons. In ganglion cells, the inhibition of L-type Ca2+ currents by F-actin disrupters was mediated by Ca2+ channel internalization. Treatment with cytochalasin D protected retinal neurons against kainate-induced excitotoxicity. CONCLUSIONS: Actin cytoskeleton dynamics plays an important role in the regulation of subcellular distribution and function of Ca(v)1.3 L-type Ca2+ channels in salamander retinal neurons. Ca2+-dependent actin depolymerization may serve as a negative feedback mechanism to reduce excessive Ca2+ influx and thereby protect neurons against glutamate-induced excitotoxicity
— id: 74463, year: 2007, vol: 13, page: 1496, stat: Journal Article,

Glutamate-induced Ca2+ influx in third-order neurons of salamander retina is regulated by the actin cytoskeleton
Akopian, A; Szikra, T; Cristofanilli, M; Krizaj, D
2006 ;138(1):17-24, Neuroscience
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
— id: 64196, year: 2006, vol: 138, page: 17, stat: Journal Article,

Calcium channel and glutamate receptor activities regulate actin organization in salamander retinal neurons
Cristofanilli, Massimiliano; Akopian, Abram
2006 Sep 1;575(Pt 2):543-554, Journal of physiology
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 4alpha-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
— id: 68980, year: 2006, vol: 575, page: 543, stat: Journal Article,

Actin filaments regulate voltage-gated ion channels in salamander retinal ganglion cells
Schubert, T; Akopian, A
2004 MAY ;125(3):583-590, Neuroscience
The regulation of voltage-activated K+, and Ca2+ currents by actin filaments was studied in salamander retinal ganglion cells, using the whole-cell patch clamp technique and Ca2+ imaging. Disruption of F-actin by cytochalasin B or latrunculin B resulted in a reduction of L-type Ca2+ current by 55 +/- 4%, and a sustained outward K+ current (I-k) by 41 +/- 3%. The effect was diminished when the F-actin stabilizing agent phalloidin was present in the patch pipette. In a group of cells where I-K exhibited a small degree of inactivation, the effect of F-actin disruption on current was dual; it increased it by 89 +/- 16%, at -10 mV, and reduced it by 37 +/- 5% at +50 mV voltage step from the same holding potential of -70 mV. This was accompanied by a shift in a voltage of half-maximal activation toward negative potentials by approximately 20 mV. In Ca2+ imaging experiments, 30 min incubation of isolated neurons with latrunculin A reduced a depolarization-induced Ca2+ accumulation by 45 5%. These results suggest a role for the actin cytoskeleton in regulating voltage-gated ion channels in retinal ganglion cells. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved
— id: 46634, year: 2004, vol: 125, page: 583, stat: Journal Article,

Activity-dependent phosphorylation of tyrosine hydroxylase in dopaminergic neurons of the rat retina
Witkovsky, Paul; Veisenberger, Eleonora; Haycock, John W; Akopian, Abram; Garcia-Espana, Antonio; Meller, Emanuel
2004 May 28;24(17):4242-4249, Journal of neuroscience
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 GABA(A) 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
— id: 45999, year: 2004, vol: 24, page: 4242, stat: Journal Article,

Role of actin Microfilaments in the regulation of voltage-gated ion channels in salamander retinal neurons
Akopian, A; Schubert, T
2003 MAY ;44(19):U734-U734, Investigative ophthalmology & visual science. IOVS
— id: 55470, year: 2003, vol: 44, page: U734, stat: Journal Article,

Differential modulation of light-evoked on- and off-EPSCs by paired-pulse stimulation in salamander retinal ganglion cells
Akopian, Abram
2003 Mar 28;967(1-2):235-246, Brain research
Short-term plasticity of On- and Off-EPSPs, and its potential role in regulation of signal processing was studied in salamander retinal On-Off ganglion cells by whole-cell recording. Paired-pulse light stimulation resulted in a depression of On-, and an enhancement of Off-EPSCs. Recovery from depression and enhancement was exponential and complete by 20 s. Paired-pulse enhancement, but not depression, was abolished with increasing stimulus duration. Blockade of On-EPSC by L-2-amino-4-phosphonobutyrate (AP-4), an agonist at group III mGluRs, significantly increased Off-EPSCs evoked by short (<2 s) duration conditioning light stimuli, resulting in a reversal of the paired-pulse enhancement to depression. The acetylcholinesterase inhibitor eserine reduced Off-EPSC1 and increased the ratio of enhancement. An opposite effect was observed in the presence of the nACh receptor antagonist d-tubocurarine. AP-7, an antagonist of NMDA receptors attenuated the enhancement of Off-EPSCs. In current clamp mode paired-pulse stimulation resulted in a modulation of light evoked, as well as the depolarization-induced spike firing pattern of ganglion cells. The present study suggests that paired light stimulation differently modulates On and Off EPSPs, and the light-evoked spike firing pattern of On-Off ganglion cells
— id: 39264, year: 2003, vol: 967, page: 235, stat: Journal Article,

Effect of hypothalamic proline-rich-polypeptide on voltage-gated Ca2+ currents in retinal ganglion cells
Akopian, Abram; Galoyan, Armen
2003 Dec;28(12):1867-1871, Neurochemical research
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
— id: 46291, year: 2003, vol: 28, page: 1867, stat: Journal Article,

Paired-pulse modulation of On- and Off-EPSCs in retinal ganglion cells
Akopian, A
2002 MAY ;43(19):U1343-U1343, Investigative ophthalmology & visual science. IOVS
— id: 55464, year: 2002, vol: 43, page: U1343, stat: Journal Article,

Calcium and retinal function
Akopian, Abram; Witkovsky, Paul
2002 Apr;25(2):113-132, Molecular neurobiology
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 Cl-, 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
— id: 39683, year: 2002, vol: 25, page: 113, stat: Journal Article,

Intracellular calcium reduces light-induced excitatory post-synaptic responses in salamander retinal ganglion cells
Akopian A; Witkovsky P
2001 Apr 1;532(Pt 1):43-53, Journal of physiology
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. 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. The inhibitory effect of depolarization on light-evoked EPSCs was strongly reduced in the presence of 10 mM BAPTA. The degree of EPSC inhibition by the prepulse holding potential followed the current-voltage relationship of the Ca2+ current found in the ganglion cell. 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. 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. 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. 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. 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
— id: 26758, year: 2001, vol: 532, page: 43, stat: Journal Article,

Glutamate-induced calcium accumulation in salamander retinal neurons
Akopian, A; Schubert, T; Weiler, R
2001 MAR 15 ;42(4):S677-S677, Investigative ophthalmology & visual science. IOVS
— id: 54983, year: 2001, vol: 42, page: S677, stat: Journal Article,

Neuromodulation of ligand- and voltage-gated channels in the amphibian retina
Akopian A
2000 Sep 1;50(5):403-410, Microscopy research & technique
To understand information processing in the retina, it is important to identify and characterize the types of synaptic receptors and intrinsic ion channels in retinal neurons. In order to achieve a high degree of adaptability, retinal synapses have evolved multiple neuromodulatory mechanisms. Light or modulatory agents can alter the efficacies of both electrical and chemical synaptic transmission in the retina. Recent studies indicate that interaction of voltage-gated channels with those activated by neurotransmitters plays a significant role in shaping the light-evoked postsynaptic responses of retinal neurons. The fact that both types of channels are subject to modulation by multiple second messenger-mediated intracellular processes is a clear indicator of the importance of neuromodulation in retinal function. The whole-cell patch clamp technique provides a means to study mechanisms of regulation of ion channels by controlling intracellular as well as the extracellular environment. This review describes the experimental evidence, mostly obtained in our laboratory, which indicates the important role of Ca-dependent neuromodulatory processes in the regulation of signal transmission in the vertical pathway of the amphibian retina.
— id: 11551, year: 2000, vol: 50, page: 403, stat: Journal Article,

Somatostatin modulates voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors of the salamander retina
Akopian A; Johnson J; Gabriel R; Brecha N; Witkovsky P
2000 Feb 1;20(3):929-936, Journal of neuroscience
We investigated the cellular localization in the salamander retina of one of the somatostatin [or somatotropin release-inhibiting factor (SRIF)] receptors, sst(2A), and studied the modulatory action of SRIF on voltage-gated K(+) and Ca(2+) currents in rod and cone photoreceptors. SRIF immunostaining was observed in widely spaced amacrine cells, whose perikarya are at the border of the inner nuclear layer and inner plexiform layer. sst(2A) immunostaining was seen in the inner segments and terminals of rod and cone photoreceptors. Additional sst(2A) immunoreactivity was expressed by presumed bipolar and amacrine cells. SRIF, at concentrations of 100-500 nM, enhanced a delayed outwardly rectifying K(+) current (I(K)) in both rod and cone photoreceptors. SRIF action was blocked in cells pretreated with pertussis toxin (PTX) and was substantially reduced by intracellular GDP(beta)S. Voltage-gated L-type Ca(2+) currents in rods and cones were differently modulated by SRIF. SRIF reduced Ca(2+) current in rods by 33% but increased it in cones by 40%, on average. Both effects were mediated via G-protein activation and blocked by PTX. Ca(2+)-imaging experiments supported these results by showing that 500 nM SRIF reduced a K(+)-induced increase in intracellular Ca(2+) in rod photoreceptor terminals but increased it in those of cones. Our results suggest that SRIF may play a role in the regulation of glutamate transmitter release from photoreceptors via modulation of voltage-gated K(+) and Ca(2+) currents
— id: 11856, year: 2000, vol: 20, page: 929, stat: Journal Article,

Relationship between intracellular Ca2+ and light-evoked EPSCs in retinal ganglion cells
Akopian, A; Witkovsky, P
2000 MAR 15 ;41(4):S935-S935, Investigative ophthalmology & visual science. IOVS
— id: 54625, year: 2000, vol: 41, page: S935, stat: Journal Article,

Effects of glutamate-related ligands on the electroretinogram (ERG) of the Xenopus retina
Szikra, T; Akopian, A; Witkovsky, P
2000 NOV ;12(11):486-486, European journal of neuroscience
— id: 54454, year: 2000, vol: 12, page: 486, stat: Journal Article,

Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina
Akopian A; Gabriel R; Witkovsky P
1998 Sep;80(3):1105-1115, Journal of neurophysiology
We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters > 15 microns and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 microM gamma-aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 microM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 microM), an inhibitor of intracellular calcium pumps, and ryanodine (20 microM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 microM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3'5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 microM) and cyclosporin A (20 microM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 micrograms/ml) and calcineurin (300 micrograms/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases
— id: 12072, year: 1998, vol: 80, page: 1105, stat: Journal Article,

Both high- and low voltage-activated calcium currents contribute to the light-evoked responses of luminosity horizontal cells in the Xenopus retina
Akopian A; Krizaj D; Witkovsky P
1997 Jul 11;762(1-2):121-130, Brain research
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). HC's 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 HC's 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
— id: 56919, year: 1997, vol: 762, page: 121, stat: Journal Article,

Intracellular calcium induces inactivation of A-current in fish retinal horizontal cells
Akopian, A; Witkovsky, P
1997 MAR 15 ;38(4):2875-2875, Investigative ophthalmology & visual science. IOVS
— id: 53238, year: 1997, vol: 38, page: 2875, stat: Journal Article,

Gain of rod to horizontal cell synaptic transfer: relation to glutamate release and a dihydropyridine-sensitive calcium current
Witkovsky P; Schmitz Y; Akopian A; Krizaj D; Tranchina D
1997 Oct 1;17(19):7297-7306, Journal of neuroscience
We related rod to horizontal cell synaptic transfer to glutamate release by rods. Simultaneous intracellular records were obtained from dark-adapted rod-horizontal cell pairs. Steady-state synaptic gain (defined as the ratio of horizontal cell voltage to rod voltage evoked by the same light stimulus) was 3.35 +/- 0.60 for dim flashes and 1.50 +/- 0.03 for bright flashes. Under conditions of maintained illumination, there was a measurable increment of horizontal cell hyperpolarization for each light-induced increment of rod hyperpolarization over the full range of rod voltages. In separate experiments we studied glutamate release from an intact, light-responsive photoreceptor layer, from which inner retinal layers were removed. Steady light reduced glutamate release as a monotonic function of intensity; spectral sensitivity measures indicated that we monitored glutamate release from rods. The dependence of glutamate release on rod voltage was well fit by the activation function for a high-voltage-activated, dihydropyridine-sensitive L-type calcium current, suggesting a linear dependence of glutamate release on [Ca]i in the synaptic terminal. A simple model incorporating this assumption accounts for the steady-state gain of the rod to horizontal cell synapse
— id: 7283, year: 1997, vol: 17, page: 7297, stat: Journal Article,

Activation of metabotropic glutamate receptors decreases a high-threshold calcium current in spiking neurons of the Xenopus retina
Akopian A; Witkovsky P
1996 May-Jun;13(3):549-557, Visual neuroscience
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
— id: 12605, year: 1996, vol: 13, page: 549, stat: Journal Article,

D2 dopamine receptor-mediated inhibition of a hyperpolarization-activated current in rod photoreceptors
Akopian A; Witkovsky P
1996 Sep;76(3):1828-1835, Journal of neurophysiology
1. Using the whole cell patch clamp method, we investigated the effect of dopamine on a hyperpolarization-activated current (Ih) in the inner segments of rod photoreceptors of the Xenopus retina. 2. Ih was elicited by hyperpolarizing voltage steps to -120 mV from a holding potential of -40 mV. Dopamine reversibly reduced Ih in a dose-dependent manner. Dopamine-mediated inhibition of Ih was blocked by the D2 dopamine antagonist sulpiride. 3. The D2 dopamine agonist quinpirole (0.1-20 microM) inhibited Ih whereas the D1 agonist SKF-38393 (100 microM) had no effect on Ih. Quinpirole-induced inhibition of Ih was blocked by sulpiride, but not by the D4 antagonist, clozapine. The D3 agonists (+/-)-7-hydroxy-2-dipropylaminotetralin hydrochloride and trans-7-hydroxy-2[N-propyl-N-(3'-iodo-2'-propenyl)amino]-tetralin maleate were, respectively, 5 and 100 times less effective than quinpirole in inhibiting Ih. 4. Quinpirole failed to reduce Ih when the internal solution contained GDP beta S (500 microM). Internal application GTP gamma S (300 microM) progressively and irreversibly reduced Ih and blocked a further reduction by quinpirole, indicating that the inhibition of Ih by quinpirole involves a G protein. 5. The inhibition of Ih by quinpirole was not affected by intracellularly applied adenosine 3',5'-cyclic monophosphate (cAMP) or by the protein kinase inhibitor H-7, indicating that a cAMP-mediated second messenger cascade does not participate in the dopamine-mediated inhibition. 6. Ih was not altered when the patch pipette contained a nominally Ca(2+)-free internal solution, but the inhibition of Ih by quinpirole was abolished, suggesting an involvement of Ca(2+) in the quinpirole-induced effect. 7. We conclude that a D2 dopamine receptor modulates Ih through the activation of a G protein and that intracellular Ca2+, but not cAMP, plays a key role in this process. 8. The reduction of Ih by dopamine may reduce the ability of rods to signal time-modulated light stimuli
— id: 12548, year: 1996, vol: 76, page: 1828, stat: Journal Article,

D2 dopamine receptor modulates hyperpolarization activated current in xenopus rods: A role for [Ca]
Akopian, A; Witkovsky, P
1996 FEB 15 ;37(3):24-24, Investigative ophthalmology & visual science. IOVS
— id: 53018, year: 1996, vol: 37, page: 24, stat: Journal Article,

DOPAMINE MODULATES DISTINCT HYPERPOLARIZATION-ACTIVATED CURRENTS IN RODS AND GLIAL-CELLS
AKOPIAN, A; WITKOVSKY, P
1995 MAR 15 ;36(4):S289-S289, Investigative ophthalmology & visual science. IOVS
— id: 87332, year: 1995, vol: 36, page: S289, stat: Journal Article,

Feedback from luminosity horizontal cells mediates depolarizing responses of chromaticity horizontal cells in the Xenopus retina
Witkovsky P; Gabriel R; Krizaj D; Akopian A
1995 Apr 11;92(8):3556-3560, Proceedings of the National Academy of Sciences of the United States of America
It has been proposed that the depolarizing responses of chromaticity horizontal cells (C-HCs) to red light depend on a feedback signal from luminosity horizontal cells (L-HCs) to short-wavelength-sensitive cones in the retinas of lower vertebrates. In this regard we studied the C-HCs of the Xenopus retina. C-HCs and L-HCs were identified by physiological criteria and then injected with neurobiotin. The retina then was incubated with peanut agglutinin, which stains red-but not blue-sensitive cones. Electron microscopic examination revealed that L-HCs contact all cone classes, whereas C-HCs contact only blue-sensitive cones. Simultaneous recordings from C-HC/L-HC pairs established that when the L-HC was saturated by a steady bright red light, C-HCs alone responded to a superimposed blue stimulus. In response to red test flashes, the C-HC response was delayed by approximately 30 msec with respect to the L-HC response. Isolated HCs of both subtypes were examined by whole-cell patch clamp. Both responded to kainate with sustained inward currents and to quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) with desensitizing currents from a negative holding potential; i.e., both have AMPA-type glutamate receptors. gamma-Aminobutyric acid or glycine opened a chloride channel in the L-HC, whereas the C-HC was unresponsive to either inhibitory amino acid. Since glycine has been shown to abolish selectively the depolarizing response of the C-HC, this finding and other pharmacological data strongly implicate the L-HC in the underlying circuit. Moreover, because the C-HC does not respond to gamma-aminobutyric acid, the neurotransmitter of the L-HC, by elimination, a feedback synapse from L-HC to blue cone is the most plausible mechanism for the creation of depolarizing responses in C-HCs
— id: 56728, year: 1995, vol: 92, page: 3556, stat: Journal Article,

Modulation of transient outward potassium current by GTP, calcium, and glutamate in horizontal cells of the Xenopus retina
Akopian A; Witkovsky P
1994 May;71(5):1661-1671, Journal of neurophysiology
1. Membrane currents of luminosity horizontal cells (L-HCs) and chromatic horizontal cells (C-HCs) isolated from the Xenopus retina were characterized using the whole-cell patch-clamp technique. 2. The current-voltage curve for the L-HC had a characteristic negative slope conductance in the voltage range of -30 to -10 mV that was not evident in the C-HC. 3. A transient outward 4-aminopyridine-sensitive potassium current (A-current) was the most prominent current in C-HCs but was also present in L-HCs. A-current characteristics in the two horizontal cell (HC) classes were closely similar. Its threshold of activation was above -45 mV. The half-voltage of inactivation was close to -70 mV. The decay of the A-current was fit by a single exponential with time constants of 30 and 40 ms at depolarizing voltage steps to -10 and +30 mV, respectively. 4. The voltage for 50% A-current inactivation shifted toward negative potentials shortly after we established the whole-cell configuration. This shift was changed to more positive potentials by internal application of guanosine 5'-triphosphate, resulting in a significant overlap of A-current activation and inactivation functions near -40 mV, which is well within the normal operating range of the HC. 5. Internal application of the G-protein activator GTP gamma S shifted the voltage-dependent inactivation of the A-current toward positive potentials by +15 mV. In contrast, GDP beta S shifted the inactivation curve by about -10 mV, similar to what was observed in untreated cells. 6. GTP and GTP gamma S increased the rate of recovery from inactivation and slowed down the rate of inactivation of the A-current enabled by a depolarizing prepulse. 7. Glutamate superfused in the bath solution significantly accelerated the rate of inactivation of A-current induced by depolarizing prepulses. The rate of A-current recovery from inactivation, however, was not affected by glutamate. 8. Removal of calcium from the bath solution reversibly decreased the amplitude of the A-current without a significant shift in its threshold of activation
— id: 12975, year: 1994, vol: 71, page: 1661, stat: Journal Article,

The effects of L-glutamate, AMPA, quisqualate, and kainate on retinal horizontal cells depend on adaptational state: implications for rod-cone interactions
Krizaj D; Akopian A; Witkovsky P
1994 Sep;14(9):5661-5671, Journal of neuroscience
We studied the responses of isolated and intact luminosity-type horizontal cells (L-HC) in the Xenopus retina to L-glutamate (L-glu) and its analogs. Isolated L-HCs studied with whole-cell patch clamp responded to L-glu, kainate (KA), AMPA, or quisqualate (quis) with inward currents from a holding potential of -60 mV, associated with a conductance increase. The current elicited by KA was relatively large and sustained, whereas AMPA or quis evoked a desensitizing current. Coapplication of quis and KA resulted in a smaller current and conductance change than that evoked by a pulse of either alone at the same concentration. This finding suggests that the L-HC has a single subtype of glutamate receptor that responds to both quis and KA. Prior exposure to dopamine enhanced the KA-evoked current about twofold. In the superfused eyecup we found that L-HC responses to quinoxalinediones (CNQX or DNQX) and to L-glu, KA, AMPA, and quis varied as a function of adaptational state. When driven exclusively by either cones or by rods, CNQX/DNQX hyperpolarized the L-HC and reduced its light response, without altering response kinetics, indicating that both rods and cones communicate with L-HCs at ionotropic glutamatergic synapses. Under mesopic conditions, however, as CNQX or DNQX reduced cone input, the rod input to the L-HC increased up to fivefold in magnitude and had slowed kinetics. The depolarizing response of the L-HC to L-glu, AMPA, or quis was relatively small and transient under photopic conditions, but was much larger and sustained when the eyecup was dark adapted. The D1 dopamine antagonist SCH 23390 potentiated the response to quis. In contrast, responses to KA were largest in light-adapted eyecups, were potentiated by a D1 dopamine agonist, SKF 38393, and were reduced by SCH 23390. We hypothesize that the segregated populations of glutamate receptors in the L-HC opposite cone and rod synaptic endings can be separately modulated to respond differentially to the native transmitter, glutamate. In photopic and mesopic states the dominant cone input tonically inhibits rod to L-HC communication. This inhibition appears to occur at the postsynaptic membrane and may be mediated by second messengers
— id: 8332, year: 1994, vol: 14, page: 5661, stat: Journal Article,

Activation of Protein Kinase C Modulates Light Responses in Horizontal Cells of the Turtle Retina
Akopian A; McReynolds J; Weiler R
1992 ;4(8):745-749, European journal of neuroscience
The effect of phorbol esters on the light-evoked responses of horizontal cells were studied in the turtle eyecup preparation. Phorbol esters caused a reduction in receptive field size and a significant decrease in the amplitude of responses to annular and full-field illumination; however, they caused only minor changes in responses to small spots in the receptive field centre. The dark membrane potential was not affected. The results suggest that phorbol esters may affect both coupling resistance and membrane resistance in horizontal cells. The effects of phorbol esters were blocked by the protein kinase C inhibitor staurosporine, and inactive phorbol ester had no effect, making it very likely that the phorbol ester effects were mediated through activation of protein kinase C. The above effects of the phorbol esters were considerably reduced by the dopamine antagonists haloperidol and fluphenazine, suggesting that they were in part mediated by release of dopamine
— id: 66504, year: 1992, vol: 4, page: 745, stat: Journal Article,

BACKGROUND ILLUMINATION MODULATES DOPAMINERGIC ACTIVITY AND THE RECEPTIVE-FIELD SIZE OF HORIZONTAL CELLS IN THE TURTLE RETINA
AKOPIAN, A; WEILER, R; KOLBINGER, W
1992 MAR 15 ;33(4):1406-1406, Investigative ophthalmology & visual science. IOVS
— id: 52058, year: 1992, vol: 33, page: 1406, stat: Journal Article,

Effects of background illuminations on the receptive field size of horizontal cells in the turtle retina are mediated by dopamine
Weiler R; Akopian A
1992 Jun 8;140(1):121-124, Neuroscience letters
Intracellular recordings from luminosity-type horizontal cells of the turtle retina were used to analyze the effects of steady and flickering background illumination on the size of their receptive fields. Both types of background illumination reduce the size of the receptive field to about the same extent. The reduction seems largely due an increase in the coupling resistance between horizontal cells. The effects of both types of background illumination are sensitive to the dopamine antagonist fluphenazine. This suggests that steady and flickering illuminations stimulate the release of endogenous dopamine
— id: 66503, year: 1992, vol: 140, page: 121, stat: Journal Article,

Short-term potentiation of off-responses in turtle horizontal cells
Akopian A; McReynolds J; Weiler R
1991 Apr 12;546(1):132-138, Brain research
Depolarizing responses to light off were studied in turtle horizontal cells using intracellular recording in the everted eyecup preparation. In many cells the off-response showed two components (fast and slow) which could overshoot beyond the steady-state dark level. The peak amplitudes of the fast and slow components increased with increasing duration of the light stimulus. A similar enhancement of the off-responses could also be produced by repetitive stimulation with brief flashes. However, the degree of enhancement produced by repetitive stimulation was greater than could be produced by increasing stimulus duration, and the latency of the onset of depolarization was longer, suggesting that the enhancement produced by repetitive stimulation involves an additional mechanism. Dramatic enhancement of the off-response by stimuli which did not affect the on-response during light indicates that the off-response may contain information not present in the on-response. The fast component of the off-response was suppressed to a greater degree than other components by reducing extracellular calcium or in the presence of 500 microM cobalt, suggesting that this component may involve a calcium current
— id: 66505, year: 1991, vol: 546, page: 132, stat: Journal Article,