Voltage-dependence of cobalt-induced blockade of synaptic transmission between photoreceptors and horizontal retinal cells

Synaptic transmission between photoreceptors and horizontal cells in the turtle retina blocked by Co²⁺ ions can be restored by passing constant radial current through the retina which depolarizes presynaptic receptor terminals. This finding is unassociated with current action on horizontal cells themselves, since polarization of these cells via an intracellular microelectrode did not restore response to light. The unblocking effect of depolarization at the receptor synaptic endings consists of two components: the opening of additional calcium channels not blocked by Co²⁺ at the presynaptic membrane and cobalt-induced voltage-dependent blockade of clacium channels. The latter may explain the paradoxical phenomenon of increased response to the action of moderate light in horizontal cells during cobalt-induced partial blockade of synaptic transmission.Institute for Research into Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 20, No. 3, pp. 374–383, May–June, 1988.     

Investigation of the nature of electrical responses of horizontal cells of the fish retina

On the basis of the syncytial structure of the layer of horizontal cells of the fish retina, a method is developed which effectively shifts the membrane potential of cells by means of an electrical current. It is shown that the response of L-type horizontal cells to light and electrical stimulation of the retina is reversed when the membrane of the horizontal cells is depolarized by a direct current. The equilibrium potential of the cells was near the zero level. Consequently, the depolarization response of the horizontal cells to disconnection of the light and to electrical stimulation of the retina is an excitatory postsynaptic potential, whereas hyperpolarization of the horizontal cells to light is a decrease of this potential. It is shown that the membrane of fish horizontal cells have pronounced nonlinear properties: in the case of strong depolarization and especially in the case of hyperpolarization its impedance drops markedly. The latter probably occurs due to an increase of the permeability of the nonsynaptic membrane of the horizintal cells for K⁺. This can also explain the decrease of membrane impedance during the hyperpolarization response of the horizontal cells to bright light. The available data indicate the presence of regenerative properties of the membrane of horizontal cells.Institute of Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 3, No. 1, pp. 89–98, January–February, 1971.     

Effect of polarization of horizontal cells of the pike retina on spread of their electrical potentials

The spread of electrical responses over the layer of horizontal cells of the pike retina was investigated at different levels of their membrane potential varied by application of a steady current. Depolarization of the membrane, accompanied by an increase in its resistance, led to an increase in its time constant and length constant, so that electrical waves spread further over the layer of horizontal cells. The effect of polarization was thus due to the nonlinear membrane properties of the horizontal cells, i.e., to the increase in their resistance on depolarization and its decrease on hyperpolarization. In some cases this nonlinearlity was manifested as a special type of regeneration: the same strength of steady current crossing the membrane of the horizontal cells corresponded to two stable levels of the membrane potential. The role of various factors (changes in resistance of the extrasynaptic and subsynaptic membranes of the horizontal cells, the presynaptic effect of the current) determining the spread of the potentials over the horizontal cells under natural conditions during photic stimulation is discussed.Institute of Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 4, No. 1, pp- 90–96, January–February, 1972.     

Electrical properties of subsynaptic and nonsynaptic membranes of horizontal cells of the turtle retina

Horizontal cells of the L-type in the turtle retina were polarized by passing a steady current through extracellular electrodes. In this way controlled changes in membrane potential can be effectively produced in the region of the cell body. The hyperpolarization response of the horizontal cell to light is reversed on depolarization of the cell membrane to about the zero level. Consequently, the response of the horizontal cell to light is the result of a decrease in the EPSP, the magnitude of which remains constant in darkness. The resistance of the cell membrane depends on the membrane potential. Hyperpolarization of horizontal cells produced by bright light or by passage of a steady current was accompanied by a decrease in their membrane resistance. This nonlinearity evidently depends on the properties of the nonsynaptic membrane of the horizontal cells, whose resistance falls considerably on hyperpolarization. The results are qualitatively similar to those demonstrated previously [10] in an investigation of the horizontal cells of the fish retina.Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 5, No. 4, pp. 423–431, July–August, 1973.     

Membrane potential clamping in horizontal cells of the fish retina

The membrane potential of horizontal cells of the retina was clamped by uniform polarization of the layer of these cells by a current passed through extracellular electrodes. The volt-ampere characteristic curve of the synaptic membrane of the horizontal cells in some cases had segments with negative slope. With a sharp change in the level of voltage clamping the time taken for the resistance of the membrane to change was under 20 msec. Comparison of responses to photic stimulation recorded with and without voltage clamping showed that participation of the nonsynaptic membrane in the generation of responses to photic stimulation can affect their shape substantially.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 9, No. 4, pp. 402–407, July–August, 1977.     

Peculiarities of synaptic transmission between photoreceptors and horizontal cells

A previously advanced hypothesis, according to which the transmitter which depolarizes the membrane of horizontal cells is continually liberated in the dark, and ceases to be liberated in the light, is tested experimentally. The data presented show that a current acting on presynatic receptor endings evokes a depolarizing response in horizontal cells to short current impulses passing through the retina (anode on receptor surface, cathode in vitreous body). These receptor endings are depolarized, which evidently leads to liberation of the transmitter from the receptors. Experiments with electrical stimulation of the retina have shown that treatment of the retina with potassium cyanide disrupts synaptic transmission between the receptor and horizontal cell. A potential equal to their membrane potential is established in horizontal cells in bright light; this potential is evidently the true rest potential of these cells. The relative stability of the membrane potential of horizontal cells in light with change in temperature is evidence in support of this assumption. In the dark, the membrane potential increases considerably with increase in temperature; this effect is possibly due to a rise in the rate of decomposition of the depolarizing transmitter. Evidence in support of this hypothesis is the rise in steepness of the falling phase of the response of the horizontal cells to electrical stimulation observed on elevation of the temperature.Institute for Problems of Information Transmission of the Academy of Sciences of the USSR, and Institute of Higher Nervous Activity and Neurophysiology of the Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 2, No. 1, pp. 79–86, January–February, 1970.     

Midkine-a Protein Localization in the Developing and Adult Retina of the Zebrafish and Its Function During Photoreceptor Regeneration

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.     

Local changes of resistance in the retinal horizontal cell evoked by changes in membrane potential

A steady current (10·10^–10–6·10^–9 A) was passed by means of a bridge circuit through a recording microelectrode inserted into a horizontal cell of the turtle retina. Illumination of the retina caused an increase in the resistance of the microelectrode circuit (by 10–80 M), causing a change in the shape of the recorded response of the horizontal cell to light. The change in resistance was shown to take place, not on the cell membrane itself, but inside the cell close to the microelectrode tip. The effect described can be reproduced by passing a current through one barrel of a double-barreled microelectrode alongside the recording barrel, but the strength required for this current was greater than that passed through the recording barrel. If the membrane potential of the horizontal cell was made equal to the equilibrium potential (by means of a steady current passed through extracellular electrodes) the hyperpolarization response to light and the effect of the increase in resistance of the microelectrode circuit disappeared simultaneously. On the other hand, artificial hyperpolarization of the cell membrane caused an increase, but depolarization caused a decrease in the resistance of the microelectrode circuit. It is postulated that the observed effect is due to blocking of the microelectrode tip by an intracellular structure whose resistance varies with a change in membrane potential.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol.5, No.4, pp.432–441, July–August, 1973.     

Dynamic model of interaction between cones and horizontal photic cells in the carp retina

A mathematical model is examined of interaction between cones and horizontal L-type cells (HC) in the carp retina using data from intracellular recording of HC spectral response. This model, describing the operation of ionic channels at the membrane of cones and HC, is based on a numerical analog solution to Hodgkin-Huxley differential equations [11] and enables predictions of spectral response levels in HC to be made as a function of time.M. L. Lomonosov State University, Moscow. Translated from Neirofiziologiya, Vol. 21, No. 4, pp. 461–466, July–August, 1989.     

Sensory interaction in the snailHelix lucorum

The activity of hair cells of statocysts inHelix lucorum was investigated by means of intra- and extracellular recording, applying appropriate stimulation of the organs of balance, optic photoreceptors, and the chemoreceptors of the optic tentacle bulb. Mechanical stimulation of the statocysts evoked a firing reaction in the hair cells as a result of generator potentials occurring at the receptors. The amplitude of generator potentials was proportional to the intensity of the reaction. Stimulating the optic photoreceptors by switching on a light produced a spike response in the hair cells with a short latency of 0.3–2 sec. The latent period of this response was inversely proportional to the intensity of the light. Appropriate stimulation of the chemoreceptors of the optic tentacle bulb caused a faint spike response with a long latent period of 20–40 sec in the hair cells. Illumination and stimulation of the chemoreceptors produced an inhibitory response in the form of bursts of IPSP in 2 out of more than 50 hair cells.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 17–26, January–February, 1986.     

Changes in direct current input resistance in horizontal cells of fish retina during excitation

The input resistance of pike retinal horizontal cells was measured by means of coaxial electrodes under various conditions of illumination. With moderate intensities of illumination, the resistance (determined from a potential drop caused by the current passed through the microelectrode) increases, whereas at high saturating intensities it decreases, as compared with its value in darkness. Such changes in resistance of the horizontal cells explain the effects of input signals interaction in these cells, such as enhancement and complete saturation, observed earlier. Some properties of the horizontal cell response permit us to assume that the "active" cell response to polarization makes a substantial contribution to the measured resistance of these cells. Possible mechanisms of such changes in input resistance of horizontal cells are discussed.Institute of Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 3, No. 2, pp. 210–216, March–April, 1971.     

Hyperpolarizing photoreceptors in the eyes of the giant clamTridacna: physiological evidence for both spiking and nonspiking cell types

Summary Intracellular studies on photoreceptors in the eyes of the giant clamTridacna give evidence for two types of light-sensitive cells, both of which are hyperpolarized by light. These cells are distinguished by the presence or absence of spikes and corresponding characteristics of the receptor potential. In non-spiking (NS) receptors, the average resting potential in the dark is low (-15 mV) and peak receptor potentials are large (to 100 mV) and adapt rapidly to light. Spiking (S) receptors have higher average resting potentials (-45 mV), but receptor potentials do not exceed 20 mV and also do not adapt to light. The spikes in S-receptors are small (3–8 mV), occur spontaneously at low levels of illumination and are inhibited by light. Bursts of spikes arise on the repolarizing off-component of the receptor potential. Light adaptation increases the excitability of S-receptors in terms of a higher frequency and shorter latency of the off response burst. The receptor potential in both cells is due to a light-activated increase in membrane conductance to potassium ions. Membrane conductance decreases in NS-receptors in relation to light adaptation. Unlike the scallop eye, no depolarizing photoreceptors are present.Abbreviations NS non-spiking photoreceptors - S spiking photoreceptors - SW seawater     

Identification and regulation of whole-cell Cl− and Ca2+-activated K+ currents in cultured medullary thick ascending limb cells

The whole-cell patch-clamp technique has been used to study membrane currents in cultured rabbit medullary thick ascending limb (MTAL) epithelial cells. A Ca²⁺-activated K⁺ current was characterized by its voltage-dependent and Ca²⁺-dependent properties. When the extracellular K⁺ ion concentration was increased from 2 to 140 mm, the rereversal potential (E_k) was shifted from –85 to 0 mV with a slope of 46 mV per e-fold change. The Ca²⁺-activated K⁺ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca²⁺-activated K⁺ channel studied with the single channel patch-clamp technique. The results suggest that the Ca²⁺-activated K⁺ current is the predominant, large conductance and Ca²⁺-dependent K⁺ pathway in the cultured MTAL cell apical membrane. The biophysical properties and physiological regulation of a Cl^– current were also investigated. This current was activated by stimulation of intracellular cAMP using forskolin and isobutyl-1-methylxanthine (IBMX). The current-voltage (I–V) relationship of the Cl^– current showed an outward-rectifying pattern in symmetrical Cl^– solution. The Cl^– selectivity of the whole-cell current was confirmed by tail current analysis in different Cl^– concentration bath solutions. Several Cl^– channel blockers were found to be effective in blocking the outward-rectifying Cl^– current in MTAL cells. The cAMP-dependent Cl^– transport in MTAL cells was further confirmed by measuring changes in the intensity of Cl^– sensitive dye using fluorescence microscopy. These results suggest that the Cl^– channel in the apical or basolateral membrane of MTAL cells may be regulated by cAMP-dependent protein-kinase-induced phosphorylation.This study was supported by the National Institutes of Health grants GM46834 to L.L. and DK32753 to W.B.G., and by a Grant-in-Aid from the American Heart Association of Ohio to L.L.     

Reciprocal changes in center and surrounding S potentials of fish retina in response to dopamine

Effects of dopamine (DA) were examined on the intracellularly recorded potential from horizontal cells in the fish (Eugerres plumieri) retina. DA (100 M in the perfusate) augmented the center S potential in a response to a spot illumination and attenuated the surrounding S potential to an annular light by approximately 40%. These reciprocal changes in the S potentials were associated with a slight depolarization (2.5 mV) of the horizontal cell, and were reversible in 10–15 min. The results indicate that DA at this concentration does not affect directly the synaptic transmission from photoreceptors to horizontal cells, while it appears to interfere selectively with the lateral propagation of an S potential. The effects of DA observed may represent an aspect of function of DA-containing interplexiform cells in the retina.     

pH changes in the invaginating synaptic cleft mediate feedback from horizontal cells to cone photoreceptors by modulating Ca2+ channels

Feedback from horizontal cells (HCs) to cone photoreceptors plays a key role in the center-surround-receptive field organization of retinal neurons. Recordings from cone photoreceptors in newt retinal slices were obtained by the whole-cell patch-clamp technique, using a superfusate containing a GABA antagonist (100 microM picrotoxin). Surround illumination of the receptive field increased the voltage-dependent calcium current (ICa) in the cones, and shifted the activation voltage of ICa to negative voltages. External alkalinization also increased cone ICa and shifted its activation voltage toward negative voltages. Enrichment of the pH buffering capacity of the extracellular solution increased cone ICa, and blocked any additional increase in cone ICa by surround illumination. Hyperpolarization of the HCs by a glutamate receptor antagonist-augmented cone ICa, whereas depolarization of the HCs by kainate suppressed cone ICa. From these results, we propose the hypothesis that pH changes in the synaptic clefts, which are intimately related to the membrane voltage of the HCs, mediate the feedback from the HCs to cone photoreceptors. The feedback mediated by pH changes in the synaptic cleft may serve as an additional mechanism for the center-surround organization of the receptive field in the outer retina.     

Ion exchange in the horizontal cells of the retina

Experimental data indicate that the membrane potential of L-type horizontal cells of the retina to bright light (i.e., when synaptic inputs are completely closed) is close to the potassium equilibrium potential. From this observation the intracellular concentration of K⁺ and Na⁺ was estimated. The latter was found to be relatively high (tens of millimoles/liter), i.e., comparable with the intracellular K⁺ concentration. This result, coupled with data on closeness of the equilibrium potential of the photic response to zero, is evidence that besides sodium conductance, the potassium conductance of the subsynaptic membrane also participates in generation of the photic response by these cells. The steady-state sodium and potassium synaptic currents was shown to be relatively small and to vary only a little over the whole working range of potentials (from –72 to –16 mV), due to the nonlinear properties of the nonsynaptic cell membrane.Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 14, No. 1, pp. 3–10, January–February, 1982.     

Ionic mechanisms of nonlinearity of the retinal horizontal cell membrane

Changes in ionic conductance lying at the basis of nonlinearity of the current-voltage characteristic curve of the cell (nonsynaptic) membrane of horizontal cells were studied in experiments on the goldfish and turtle retina. All measurements were made during blocking of synaptic transmission by bright light or Co⁺⁺. An increase in the K⁺ concentration led to depolarization and to a reduction of the steepness of the hyperpolarization branch of the current-voltage curve, whereas a decrease in K⁺ had the opposite effect. Changes in the Cl^– or Na⁺ concentrations had no significant effect on membrane potential or on the shape of the current-voltage curve. The principal potential-forming ion in the horizontal cells is thus K⁺; conductance for Cl^– is absent or very low, and conductance for Na⁺ also is evidently small. In the presence of Ba⁺⁺ (2–5 mM) the steepness of the hyperpolarization branch of the current-voltage curve was increased and the whole curve became more linear. It is concluded that nonlinearity of the current-voltage curve of the horizontal cell membrane is due mainly to potential-dependent potassium channels, whose conductance increases during hyperpolarization; this increase in conductance is blocked by Ba⁺⁺. An increase in the Ca⁺⁺ concentration to 20 mM led to an increase in steepness of the depolarization branch of the current-voltage curve, suggesting that depolarization increases membrane conductance for Ca⁺⁺.Institute for Problems in Information Transmission, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 5, pp. 531–539, September–October, 1981.     

Changes in distribution of synaptic vesicles in endings of cones during electrical stimulation of the retina

Conditions (hypoxia) were chosen under which, judging from the reduction in the responses of the horizontal cells to electrical stimulation of the retina, mediator is exhausted in the presynaptic endings of the photoreceptors. Under these circumstances a number of "small" synaptic vesicles were shown to be reduced in those parts of the cones which are in direct contact with presynaptic membranes. No significant changes were found in the total number of "small" vesicles in the cone endings.Institute for Problems of Information Transmission, Academy of Sciences of the USSR, Moscow. Institute of Biological Physics, Academy of Sciences of the USSR, Pushchino-on-Oka. Translated from Neirofiziologiya, Vol. 8, No. 6, pp. 620–623, November–December, 1976.     

Mitochondrial glutathione peroxidase 4 is indispensable for photoreceptor development and survival in mice

Glutathione peroxidase 4 (GPx4) is known for its unique function in the direct detoxification of lipid peroxides in the cell membrane and as a key regulator of ferroptosis, a form of lipid peroxidation–induced nonapoptotic cell death. However, the cytosolic isoform of GPx4 is considered to play a major role in inhibiting ferroptosis in somatic cells, whereas the roles of the mitochondrial isoform of GPx4 (mGPx4) in cell survival are not yet clear. In the present study, we found that mGPx4 KO mice exhibit a cone–rod dystrophy-like phenotype in which loss of cone photoreceptors precedes loss of rod photoreceptors. Specifically, in mGPx4 KO mice, cone photoreceptors disappeared prior to their maturation, whereas rod photoreceptors persisted through maturation but gradually degenerated afterward. Mechanistically, we demonstrated that vitamin E supplementation significantly ameliorated photoreceptor loss in these mice. Furthermore, LC–MS showed a significant increase in peroxidized phosphatidylethanolamine esterified with docosahexaenoic acid in the retina of mGPx4 KO mice. We also observed shrunken and uniformly condensed nuclei as well as caspase-3 activation in mGPx4 KO photoreceptors, suggesting that apoptosis was prevalent. Taken together, our findings indicate that mGPx4 is essential for the maturation of cone photoreceptors but not for the maturation of rod photoreceptors, although it is still critical for the survival of rod photoreceptors after maturation. In conclusion, we reveal novel functions of mGPx4 in supporting development and survival of photoreceptors in vivo.     

Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing.