Isolated Carp Retina Research Articles

Enlarged gold-tipped silicon microprobe arrays and signal compensation for multi-site electroretinogram recordings in the isolated carp retina

In order to record multi-site electroretinogram (ERG) responses in isolated carp retinae, we utilized three-dimensional (3D), extracellular, 3.5-μm-diameter silicon (Si) probe arrays fabricated by the selective vapor–liquid–solid (VLS) growth method. Neural recordings with the Si microprobe exhibit low signal-to-noise (S/N) ratios of recorded responses due to the high-electrical-impedance characteristics of the small recording area at the probe tip. To increase the S/N ratio, we designed and fabricated enlarged gold (Au) tipped Si microprobes (10-μm-diameter Au tip and 3.5-μm-diameter probe body). In addition, we demonstrated that the signal attenuation and phase delay of ERG responses recorded via the Si probe can be compensated by the inverse filtering method. We conclude that the reduction of probe impedance and the compensation of recorded signals are useful approaches to obtain distortion-free recording of neural signals with high-impedance microelectrodes.

Nitric oxide release is induced by dopamine during illumination of the carp retina: serial neurochemical control of light adaptation

Several lines of indirect evidence have suggested that nitric oxide may play an important role during light adaptation of the vertebrate retina. We aimed to verify directly the effect of light on nitric oxide release in the isolated carp retina and to investigate the relationship between nitric oxide and dopamine, an established neuromodulator of retinal light adaptation. Using a biochemical nitric oxide assay, we found that steady or flicker light stimulation enhanced retinal nitric oxide production from a basal level. The metabotropic glutamate receptor agonist L-amino-4-phosphonobutyric acid, inhibited the light adaptation-induced nitric oxide production suggesting that the underlying cellular pathway involved centre-depolarizing bipolar cell activity. Application of exogenous dopamine to retinas in the dark significantly enhanced the basal production of nitric oxide and importantly, inhibition of endogenous dopaminergic activity completely suppressed the light-evoked nitric oxide release. The effect of dopamine was mediated through the D1 receptor subtype. Imaging of the nitric oxide-sensitive fluorescent indicator 4,5-diaminofluorescein di-acetate in retinal slices revealed that activation of D1 receptors resulted in nitric oxide production from two main spatial sources corresponding to the photoreceptor inner segment region and the inner nuclear layer. The results taken together would suggest that during the progression of retinal light adaptation there is a switch from dopaminergic to nitrergic control, probably to induce further neuromodulatory effects at higher levels of illumination and to enable more efficient spreading of the light adaptive signal.

Metabotropic glutamate receptor-mediated hetero-synaptic interaction of red- and green-cone inputs to LHC of carp retina

The role of presynaptic metabotropic glutamate receptor (mGluR) on the interaction of red- and green-cone signals was investigated in luminosity-type horizontal cell (LHC) of isolated carp retina. It was found that a dim red background could enhance LHC’s light response to green stimulus, and a dim green background was also able to increase the cell’s response to red flash. Such mutual color enhancement was eliminated by application of groups II and III mGluR antagonist ( S)-methyl-4-carboxyphenyl-glycine (MCPG). Furthermore, inhibition of glutamate uptake by using d-aspartate ( d-Asp) or dl-threo-β-hydroxy-aspartic acid (THA) completely blocked the mutual enhancement of color signals in LHC. However, the GABAergic feedback pathway in the outer retina was unnecessarily involved.

Stimulus pattern related plasticity of synapses between cones and horizontal cells in carp retina.

Stimulus pattern related synaptic plasticity in the luminosity-type horizontal cell (LHC) of isolated carp retina was investigated. The major findings were: (1) repetitive red flashes progressively strengthened the synaptic connection between red-cone and LHC, whereas weakened that between green-cone and LHC; (2) repetitive green flashes remarkably depressed the LHC's red response, but caused little changes in the cell's green response. A competitive depression between different cone signals is suggested.

Glutamate receptor mechanisms of cones signal pathways in isolated carp retina

Glutamate receptor mechanisms of cones signal pathways in isolated carp retina

Double-staining of horizontal and amacrine cells by intracellular injection with Lucifer Yellow and biocytin in carp retina

Horizontal and amacrine cells in the isolated carp retina were impaled with micropipette electrode, identified by their characteristic light responses, and injected iontophoretically with markers for morphological study. Both Lucifer Yellow CH and biocytin were injected simultaneously. Lucifer Yellow was seen by its own fluorescence while biocytin was visualized by binding with Texas Red-linked or horseradish peroxidase-conjugated avidin. For cone-connected horizontal cells, biocytin-coupled cells were found to be approximately five-times more numerous than Lucifer Yellow-coupled cells. Coupling for both tracers was consistently hampered by intravitreally applied dopamine. In untreated retinas, the injected Lucifer Yellow was restricted within one rod-connected horizontal cell, while biocytin revealed several coupled neighbors. Amacrine cells, labeled by the tracers, were morphologically grouped into eight types, based on our earlier classification. 29 Among them, amacrine cells, belonging to three types (Fnd, Pmb or Pma), were confirmed to be Lucifer Yellow-coupled, and the number of biocytin-coupled cells was more numreous (about 2.5 times) than that of Lucifer Yellow-coupled cells. Most amacrine cells (i.e. Pwd, Fnb and Fna) showed biocytin-coupling with no Lucifer Yellow-coupling. A few classified (i.e. Pwb and Fwa) and unclassified cells did not show any coupling. Since the tracer coupling takes place via gap junctions, the majority of amacrine cells, belonging to certain homologous types, appear to be functionally coupled with each other in the inner plexiform layer. However, dopamine did not influence the range of tracer coupling between amacrine cells in the carp retina under the present experimental conditions.

D-Aspartate potentiates the effects of both l-aspartate and l-glutamate on carp horizontal cells

l-Aspartate, l-glutamate and d-aspartate, alone or in combination, were applied by superfusion or by atomization to the isolated carp retina while recording from cone horizontal cells. Each of these agents when applied alone depolarized horizontal cells and reduced the size of their light responses, an action which mimics the effect of the endogenous photoreceptor transmitter. Application of d-aspartate in conjunction with either of the l-amino acids potentiated the effects of the l-amino acids so that the threshold concentration was reduced by about five-fold, compared to when the l-amino acids were applied alone. The potentiating effect of d-aspartate occurred with all types of cone horizontal cells—both l- and C-types. Furthermore, the potentiating effect of d-aspartate occurred not only in the dark but also in the presence of bright light backgrounds or Co 2+ ions, conditions during which the release of photoreceptor transmitter is reduced or blocked. d-Aspartate also potentiated the depolarizing effects of the acidic amino acid cysteine sulfinate. The potentiating effect of d-aspartate can be attributed to its action as an amino acid uptake blocker in the outer retina. Thus, these findings, in themselves, cannot eliminate l-aspartate, l-glutamate or cysteine sulfinate as candidates for the carp cone transmitter. However, other evidence, previously reported, strongly suggests that l-glutamate and not l-aspartate is the cone transmitter.

Analysis of synaptic inputs to on-off amacrine cells of the carp retina.

To elucidate the synaptic transmission between bipolar cells and amacrine cells, the effect of polarization of a bipolar cell on an amacrine cell was examined by simultaneous intracellular recordings from both cells in the isolated carp retina. When either an ON or OFF bipolar cell was depolarized by an extrinsic current step, an ON-OFF amacrine cell was transiently depolarized at the onset of the current but no sustained polarization during the current was detected. The current hyperpolarizing the OFF bipolar cell also produced the transient depolarization of the amacrine cell at the termination of the current. These responses had a latency of approximately 10 ms. The amplitude of the current-evoked responses changed gradually with current intensity within the range used in these experiments. They were affected by polarization of the amacrine cell membrane; the amplitude of the current-evoked responses as well as the light-evoked responses was increased when the amacrine cell membrane was hyperpolarized, while the amplitude was decreased when the cell was depolarized. These results confirm directly that ON-OFF amacrine cells receive excitatory inputs from both ON and OFF bipolar cells: the ON transient is due to inputs from ON bipolar cells, and the OFF transient to inputs from OFF bipolar cells. The steady polarization of bipolar cells is converted into transient signals during the synaptic process.

N-methyl d-aspartate acts as an antagonist of the photoreceptor transmitter in the carp retina

Glutamate, aspartate, and their agonists, kainate, quisqualate, cysteine sulfinate and N-methyl- d-aspartate (NMDA), were applied to the isolated carp retina while recording from horizontal cells. All these agents, except NMDA, depolarized horizontal cells membrane and reduced responses to light, thus mimicking the effect of the endogenous photorecepto transmitter. Application of NMDA, on the other hand, caused a membrane hyperpolarization of horizontal cells in the dark, an effect different from its depolarizing effect as observed elsewhere in the central nervous system. NMDA also reduced or blocked the light responses of these cells as well as the depolarizing responses to applications of glutamate, aspartate or kainate. Effects of NMDA on the spectral properties of the horizontal cell responses were identical to the effects of the acidic amino acid receptor antagonists α-methyl glutamate, and α-amino adipate. Thus, NMDA appears to act as a weak antagonist to the photoreceptor transmitter, whose receptors on the horizontal cell membrane interact with a glutamate-like substance but appear atypical of glutamate receptors described elsewhere in the brain.

Lateral actions at the inner plexiform layer of the carp retina: Effects of turning windmill pattern stimulus

Lateral action from amacrine to ganglion cells was studied in the isolated carp retina by using a truncated windmill pattern (TWP). About 25% of ganglion cells of both ȁonȁ and ȁoffȁ center types were suppressed or enhanced in firing activity in response to TWP turning. The suppressed cells were more sensitive to slow turning velocities of TWP than the enhanced cells. In the ȁon-offȁ type amacrine cells, a steady depolarizing or hyperpolarizing component (less than several mV) was maintained by stationary TWP, while the cells were exclusively depolarized by turning TWP at a wide range of velocities. These results suggest that individual responses of ganglion cells induced by both stationary and turning TWP are depending on a balance between two factors: the polarizing direction of steady components of the ȁon-offȁ amacrine cells and the polarizing direction of ganglion cells synaptically produced by the amacrine cells.

Inner plexiform circuits in the carp retina: Effects of cholinergic agonists, GABA, and substance P on the ganglion cells

Th effects on ganglion cell light responses and spontaneous activity of neurotransmitter candidates, applied by nebulizer spray and iontophoresis, were studied in the isolated carp retina. ACh, GABA, and substance P had strong effects on the ganglion cells; dopamine and the amino acids aspartate, glutamate, and glycine and only weak effects. ACh and substance P exerted their actions even when synaptic transmission was blocked by cobalt chloride, suggesting postsynaptic receptors for those agents on the ganglion cell membrane. The 3 amino acids and dopamine do not appear to act directly on the ganglion cells. The pharmacological sensitivity of ganglion cells was correlated with their physiological response type. About three-quarters of ON/OFF and half of other transiently responding ganglion cells were excited by micromolar concentrations of cholinergic agonists; most ON-center sustained ganglion cells were insensitive. The light response of some of the ACh-sensitive cells could be suppressed by cholinergic antagonists. Substance P generally excited ganglion cells with an ON-component in their light response. GABA inhibited cells of all response types, but affected least the OFF-center tonic cells. In view of these observations, and of corroborating histological evidence, we propose that ACh, GABA, and substance P are neurotransmitters that are released by amacrine cells and affect receptors located on ganglion cells.

Suppression of retinal spike discharge by dipropylacetate (depakene): A possible involvement of GABA

Effects of dipropylacetate (DPA) were studied on spike discharges in the isolated carp retina. (1) DPA, applied electrophoretically or by pressure-microinjection in the vicinity of the recording electrode at the inner plexiform layer, consistently activated spike discharges. (2) DPA introduced into the perfusate produced complex actions; some units responded to DPA with an initial activation followed by suppression of spike discharges. (3) When gamma-aminobutyric acid (GABA) was applied electrophoretically together with perfusion of DPA, the depressing effect was greater than that obtained with each of these compounds alone. Bicuculline in the perfusate markedly attenuated the depressing actions of GABA or DPA singly, or in combination. (4) It is concluded that the delayed suppression of spike discharges by perfused DPA appears to be mediated by GABA-receptors on ganglion cells in the carp retina.

Dual actions of some amino acids on spike discharges in the carp retina

The effects of some amino acids (Glu, Gly and GABA), applied in 3 different manners (electrophoretically, in the superfusate and by pressure-microinjection), were investigated on spontaneous and light-induced spike discharges in the isolated carp retina. When applied electrophoretically or by pressure-microinjection in the inner plexiform layer (IPL), the agents acted directly on spike-generating units. Electrophoretic application of Glu at IPL consistently increased while Gly and GABA always decreased spike discharges regardless of the light-induced response patterns, when the tangential distance between the recording and injection electrodes was 25–100 μm. Increasing the distance up to 400 μm diminished the effects, but did not invert them. When added to the superfusate, the amino acids produced a dual action (two different sequential effects); Glu (5m M) initially decreased and then increased spike discharges, while Gly and GABA (5m M) produced opposite effects. Gly and GABA tended to suppress selectively off-discharges (of ON-OFF units and certain OFF- center units), leaving on-discharges (of ON-OFF units and certain ON-center units) unaffected. The amino acids produced different effects on some units, when applied by pressure-microinjection into OPL or IPL. When injected in OPL Glu suppressed, while in IPL it activated spike discharges, whereas Gly and GABA caused opposite changes to those observed with Glu. Therefore, the action of the agents when pressure-microinjected in OPL is equivalent to the initial action of the agents applied in the superfusate. The dual actions of the agents are assumed to be mediated by bipolar cells, resulting in disfacilitation (Glu) or in disinhibition (Gly or GABA) of spike-generating units.

An electrophysiological study on the cholinergic system in the carp retina

The effects on spike discharges of acetylcholine chloride (ACh), applied electrophoretically at the inner plexiform layer, were examined in air-exposed and superfused preparations of the isolated carp retina. Most of spike-generating units examined (147 of 204 units; 72%) were sensitive to electrophoretically applied ACh. Among the ACh-sensitive ones, 125 units (85%), including all ON-center, most ON-OFF and two-thirds of OFF-center units, were activated, whereas 22 units (15%), including mainly OFF-center units, were suppressed by the agent. In retinas superfused with a medium containing 20m MMg 2+and0.5mMCa 2+, units ceased in their spontaneous and light-induced discharges, but they were activated by electrophoretically applied ACh. The ACh-induced activation was reduced in magnitude or abolished during an exposure of the retina to a medium containing hexamethonium chloride (25 μM), whereas the ACh-induced suppression was reduced by atropine sulfate (25 μM) more effectively than by hexamethonium. Therefore, the results suggest that nicotinic receptors are involved in the ACh-activation, whereas both, but predominantly muscarinic, receptors may participate in the ACh-suppression of spike discharges in the carp retina.

Effects of variations in the perfusate on the ERG and discharge of ganglion cells in carp retina

In order to determine the optimal condition of perfusion for maintaining a steady physiological state in the isolated carp retina, the biophysical and biochemical parameters of the perfusate were altered, and their reversible effects were investigated on the ERG and spike discharges of identified ganglion cells. A balanced salt solution for perfusion was made as the standard. When the isolated retina was perfused with the standard solution, keeping the pH at 8·0 by saturating with a gas mixture of 97% O 2 and 3% CO 2, the flow rate at 2·0 ml/min and the temperature at 20°C, a constant ERG was maintained for more than 6 hr. As the flow rate was increased from 0·6 to 13·5 ml/min, the amplitude of the b-wave was enlarged more than two times and the discharge frequency of ganglion cell also was increased. The amplitude of the b-wave was linearly related to temperature changes in the range of 14–28°C, and the Q 10 was 2–3. The spike discharge also increased until the temperature reached 25°C, beyond which the frequency decreased in most units. The changes in osmotic pressure between −30 mosmol (hypotonic) and +20 mosmol (hypertonic) in comparison with the standard solution did not cause any alterations in the retinal activities. An increase of more than 30 mosmol in osmotic pressure suppressed the retinal activities; the amplitude of the b-wave was markedly reduced and the spike discharges were abolished within 40 sec of exposing the retina to 100 mosmol-hypertonic solution. pH changes between 7·0 and 9·0 were achieved either by saturating the perfusate with gas mixtures containing 0, 3, 5 and 10% CO 2 in O 2, respectively, or by titration of HCl or NaOH. A pH shift to the alkaline side (8·0–9·0) enhanced the ERG and facilitated the spike discharge, and the opposite shift (8·0–7·0) suppressed the ERG and abolished the spike discharge. The effects of pH changes on the retinal activities were found to be the same regardless of the presence of CO 2 in the perfusate. The deprivation of O 2, glucose or both of them from the perfusate gradually reduced the ERG (especially the b-wave) and the spike discharge of ganglion cells. Phosphate-rich perfusate initially activated and subsequently suppressed the spike discharge; during this period the amplitude of the a-wave was remarkably enhanced whereas the b-wave was reduced and abolished.

Effects of locally applied chemicals on transretinal potential and horizontal cells in the isolated carp retina I. Amino acids.

Experiments were performed on isolated retinas of teh carp (Cyprinus carpio) to investigate the effects of locally applied amino acids on the transretinal potential and the membrane potential of horizontal cells. A minute quantity of each solution of an amino acid was applied locally at different layers by means of pressure-microinjection. The responses were induced by alternating central and annular stimuli of white light. Amino acids applied locally at the distal layer (receptor surface or outer plexiform layer) in the centrally stimulated area, caused, at concentrations of 25--50mM, rapid changes in the light-induced potential, whereas those microinjected into the inner plexiform layer did not. When applied locally at the distal layer, amino acids abolished the proximal PIII response and reduced the amplitude of S-potentials, particularly in response to a central stimulus. However, the resting potential of horizontal cells was depolarized only slightly by acidic amino acids. The results suggest that the passive membrane properties of horizontal cells are not altered significantly by the amino acids applied. Differential susceptibilities of the PII component to acidic and neutral amino acids were found; the former did not change or, in some cases, enlarged, whereas the latter abolished it. Possibly, neutral amino acids interfere with a neural mechanism generating the PII component.

Effects of locally applied chemicals on transretinal potential and horizontal cells in the isolated carp retina. II. Calcium-chelator.

Effects of locally applied chemicals on transretinal potential and horizontal cells in the isolated carp retina. II. Calcium-chelator.

Spreading depression in isolated carp retina

(1) Spreading depression (SD) could be elicited in isolated carp retina by KCl application, the concomitants of which were similar to those described in other vertebrates. (2) The threshold for generating SD was greatly reduced by brief immersion of the retina in low Cl − Ringer's solutions. The properties of SD waves were almost the same with treated and untreated retinas, except for intervals. (3) Extracellular negative potential shifts during SD, averaging 4.6 mV in amplitude and 27 sec in duration, were recorded in whole retinal layers with the maximum amplitude about at the inner plexiform layer. (4) The P IIIpotential of the local electroretinogram was virtually unaffected by SD. (5) Both L- and C-type S-potentials could be evoked with increase of 20–40% in amplitude around the peak of slow membrane depolarizations (mean value of maximal amplitude 5.8 mV) during SD in horizontal cells. (6) Increase in spike number was observed in both on- and off-center ganglion cells before and after the spike cessation during SD in the untreated retina. However, the off-discharges, which were a unique response to light in the immersed retina, only decreased during SD.

Effects of external ions on the synaptic transmission from photorecptors to horizontal cells in the carp retina.

1. Intracellular recordings were made from cones and horizontal cells of the isolated carp retina and the mechanisms of synaptic transmission from cones of horizontal cells were studied by changing the ionic composition of the external medium. 2. Cones were depolarized and their light responses enhanced in low Ca high-Mg medium. In the same medium, in which chemical transmission is suposed to be blocked, horizontal cells were hyperpolarized and their light responses disappeared. 3. When the synaptic input was removed, the membrane potential of horizontal cells agreed well with Ek. 4. In Na-free medium both cones and horizontal cells were hyperpolarized and response disappeared. On reaplication of normal Ringer, horizontal cells showed a trenaient membrane potential reversal (inside positive), indicating that the horizontal cell has a high Na permeability under the influence of the endogenous transmitter. 5. Application of La produced little change in cones, while it strongly depolarized horizontal cells...

Effects of some amino acids on light-induced responses in the isolated carp retina

The isolated retina of the carp ( Cyprinus carpio) was used to examine the effects of some amino acids on the extracellularly recorded transreceptoral, intraretinal and transretinal responses and on the horizontal cell membrane potential. A circular flashed spot (0.5 mm dia) of white light (65 lx) and diffuse background illumination (2 lx) were employed. Concentrated and neutralized solutions (1.5 m) of representative amino acids (L-glutamate, L-aspartate and glycine) as an aerosol jet over all the receptoral surface caused changes in all response types. Upon an application of any one of the agents, the transreceptoral response remained almost unchanged while the focal (or positive) intraretinal response rapidly disappeared or in some cases reversed polarity. The transretinal response changed accordingly. The S-potential was consistently abolished by these agents, though the resting potential increased (glycine) or decreased (other agents). The abolition of the S-potential was coincident in time with the abolition of the intraretinal response. These effects of the amino acids were reversible and reproducible. The ionic mechanisms underlying these effects are discussed and the tentative conclusion reached that it is unlikely that the amino acids are synaptic transmitters at the outer plexiform layer in the carp retina.