Activation of NMDA receptors linked to modulation of voltage-gated ion channels and functional implications

S. F. Davis1 and C. L. Linn2

1 Louisiana State University Health Sciences Center, Neuroscience Center of Excellence, New Orleans, Louisiana 70112; and 2 Western Michigan University, Department of Biological Sciences, Kalamazoo, Michigan 49008


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Catfish (Ictalurus punctatus) cone horizontal cells contain N-methyl-D-aspartate (NMDA) receptors, the function of which has yet to be determined. In the present study, we have examined the effect of NMDA receptor activation on voltage-gated ion channel activity. NMDA receptor activation produced a long-term downregulation of voltage-gated sodium and calcium currents but had no effect on the delayed rectifying potassium current. NMDA's effect was eliminated in the presence of AP-7. To determine whether NMDA receptor activation had functional implications, isolated catfish cone horizontal cells were current clamped to mimic the cell's physiological response. When horizontal cells were depolarized, they elicited a single depolarizing overshoot and maintained a depolarized steady state membrane potential. NMDA reduced the amplitude of the depolarizing overshoot and increased the depolarized steady-state membrane potential. Both effects of NMDA were eliminated in the presence of AP-7. These results support the hypothesis that activation of NMDA receptors in catfish horizontal cells may affect the type of visual information conveyed through the distal retina.

neuromodulation; excitatory amino acid; sensory system; ionotropic; patch-clamp technique


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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VOLTAGE-GATED CURRENTS have been associated with numerous biological functions in the nervous system. An important way in which voltage-gated currents are regulated is through the action of neurotransmitters and neuromodulators. In the present study, we examine the N-methyl-D-aspartate (NMDA)-type glutamate receptor in vertebrate horizontal cells and explore the effect of NMDA receptor activation on voltage-gated ion channel activity and horizontal cell function.

Horizontal cells are second order neurons in the retina that receive synaptic input from photoreceptors and interplexiform cells (47, 49) and make synaptic connections onto bipolar cells and photoreceptors (4, 49). Horizontal cells are electrically coupled by gap junctions (31) and are responsible for the lateral spread of information in the distal retina (30, 49) and the surround inhibition recorded from bipolar and photoreceptor neurons (4, 49). In response to light, glutamate released from the photoreceptors is reduced and horizontal cells respond with a graded hyperpolarization (17, 32, 49). When a light stimulus is turned off, glutamate release increases from the photoreceptors and the horizontal cells respond with a graded depolarization. In light-adapted retina, this depolarization produces an off-overshoot response similar in shape to an action potential. The depolarizing off-overshoot is followed by the attainment of a new depolarized dark resting membrane potential (43). Both the horizontal cell's response to light-on and the depolarizing overshoot response to light-off can be simulated with current-clamp techniques in isolated cells (43).

Voltage-clamp studies have identified four categories of voltage-dependent channels (42, 43) and several types of neurotransmitter-gated receptors (16, 33, 34, 38) on catfish cone horizontal cells. The voltage-gated ion channels include the classic delayed rectifier, the anomalous rectifier, a tetrodotoxin (TTX)-sensitive sodium channel (19, 42, 43), and an L-type, dihydropyridine-sensitive calcium channel (23, 42, 43).

Except for the anomalous rectifier, the voltage-gated channels have activation ranges well within the physiological operating range of the horizontal cell recorded from the intact retina (between -70 and -20 mV). It has been proposed that one or more of these voltage-gated channels contribute to shaping the physiological response to light and maintenance of the resting membrane potential in the dark (1, 43, 45, 50). Modulation of these channels may have considerable physiological implications as to the type of information conveyed throughout the retina.

Glutamate is the major excitatory neurotransmitter released from teleost photoreceptors onto second order horizontal cells (9, 13, 15, 18, 20, 21, 47, 51). The presence of both non-NMDA- and NMDA-type glutamate receptor has been pharmacologically and electrophysiologically characterized in isolated catfish cone horizontal cells (33, 34). However, the role of the NMDA receptor in the outer retina is unknown, even though anatomical evidence of the receptor exists on horizontal cells in a number of animal preparations, including catfish, turtle, rat, and human (12, 28, 33, 35, 37). In this study, we analyze a possible function of NMDA receptors in isolated catfish cone horizontal cells.

In the intact retina, changes in membrane potential during light stimulation result from changes in the amount of neurotransmitter released from the photoreceptors, as well as from activity of voltage-gated ion channels (1, 2, 10, 40, 44). A common mechanism used by cells to modulate voltage-gated ion channel activity is through activation of ligand-gated channels (5, 26). Therefore, we tested the hypothesis that NMDA receptor activation results in modulation of voltage-gated ion channels, as well as the neurons response to injected current, a paradigm designed to mimic the horizontal cell's physiological response to light (43). We demonstrate that activation of the NMDA receptor decreases the amplitude of voltage-gated sodium and calcium channels, alters the depolarizing off-overshoot, and shifts the depolarized steady-state plateau potential resulting from depolarizing current injection. These experiments provide the first step toward understanding a possible role for NMDA receptors in catfish cone horizontal cells and suggest that activation of NMDA receptors in vertebrate horizontal cells may be a mechanism used to modulate membrane potential, thereby altering visual information conveyed through the retina. These reports also provide the first evidence of NMDA modulation of TTX-sensitive, voltage-gated sodium channels.


    METHODS
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INTRODUCTION
METHODS
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Animals. Channel catfish (Ictalurus punctatus) were obtained from a local catfish farm. Experimental protocols used in this study were approved by Institutional Animal Care and Use Committee in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Experimental solutions. All voltage- and current-clamp experiments were performed in zero magnesium normal catfish saline containing 130 mM NaCl, 4 mM KCl, 3 mM CaCl2, 15 mM dextrose, and 2 mM HEPES. To pharmacologically isolate the voltage-gated calcium current, recordings were made in zero magnesium normal saline containing 1 µM of the dihydropyridine agonist, Bay K-8644, 10 µM of the sodium channel antagonist, TTX, and 10 mM 4-aminopyridine (4-AP) to block the outwardly rectifying potassium current. To pharmacologically isolate the voltage-gated delayed rectifier, zero magnesium normal catfish saline was used that contained 1 µM TTX and 10 µM nifendipine.

The patch pipettes used for whole cell recordings were uncoated and unpolished and were filled with a solution consisting of 120 mM K-gluconate, 4 mM NaCl, 11 mM EGTA, 1 mM CaCl2, 1 mM MgCl2, and 8.4 mM HEPES, buffered to pH 7.4. In some experiments, the patch solution contained an ATP-regenerating solution consisting of 120 mM K-gluconate, 1 mM CaCl2, 1.23 MgCl2, 8.4 mM HEPES, 11 mM ethylene glycol-bis (beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), 20 mM tetraethylammonium (TEA), 4 mM ATP-Tris salt, 20 mM phosphocreatine, and 50 µM/ml creatine phosphokinase to determine whether calcium current rundown had a role in voltage-gated calcium channel modulation (25). Results obtained within a 12-min testing period using the ATP-regenerating solution were not significantly different from results obtained using the non-ATP-regenerating solution.

Application of control and experimental solutions were achieved via a gravity-fed perfusion system. The complete exchange of solutions occurred within 500 ms. NMDA (100 µM) was used in these studies because it was the minimal dose yielding a maximal effect. Antagonist concentration was chosen due to its relative effectiveness against NMDA's effect on channel activity.

Horizontal cell isolation. Isolated catfish cone horizontal cells were prepared as previously described (24). Briefly, dark-adapted channel catfish were anesthetized using tricaine methanosulfonate (10 mg/ml). When the animal no longer responded to tactile stimulation, it was pithed and both eyes were removed under dim red light. After removal of the cornea and lens, the remaining eyecups were placed in magnesium free, low-calcium catfish saline containing hyaluronidase (0.1 mg/ml) for 4 min to digest the vitreous humor (pH adjusted to 7.4 using NaOH). The eyecups were then transferred to fresh low-calcium catfish saline containing cysteine-activated papain (0.7 mg/ml) for another 4 min. Papain-treated tissue was rinsed and the retina was manually peeled off the eyecup. Retinas were placed in fresh papain containing low-calcium catfish saline for another 4 min, rinsed, and cut into 8-10 pieces. Retinal pieces were stored in normal catfish saline containing 1 mg/ml bovine serum albumin. Consistent recordings can be obtained from these retinal pieces for 48 h (34). Before recording, a piece of retina was further dissociated to yield isolated catfish cone horizontal cells. This was accomplished by manual tituration of the retina through a series of progressively smaller tipped Pasteur pipettes. Once retinal pieces were broken down into isolated cells, a sample of cells was transferred to a recording chamber mounted on the stage on an inverted Nikon Diaphot 300 microscope. Cells were allowed to settle for 5 min before beginning an experiment. Individual cells were viewed using Hoffman contrast and easily identified based on characteristic morphology (29).

Electrophysiology. Catfish cone horizontal cells were voltage or current clamped as described by Hamill et al. (14). Under voltage-clamp conditions, the average input resistance of 30 catfish cone horizontal cells at resting membrane potentials measured 98.5 MOmega (±5.2), and the mean specific membrane capacitance measured 1.1 µF cm-2 (±0.15). Patch pipettes were pulled from borosilicate glass by a Narishige (Tokyo, Japan) vertical microelectrode puller. Electrode resistance was measured in normal catfish saline. Electrodes with resistances measuring between 3 and 8 MOmega were used in this study.

Voltage clamp. Once a cell was voltage clamped, the membrane potential was changed in either rampwise or stepwise manners to evoke voltage-gated ion currents. The rampwise stimulus paradigm consisted of changing the membrane potential of a voltage-clamped catfish cone horizontal cell between -60 and +50 mV over a 500-ms period of time to create a current-voltage (I/V) relationship for the cell. The stepwise stimulus paradigm consisted of stepping the membrane potential of a voltage-clamped cell from the resting membrane potential to various hyperpolarized or depolarized membrane potentials. When appropriate, both types of stimulus paradigms were used in the presence of pharmacological agents to isolate voltage-gated ion currents. From the current traces resulting from these stimulus paradigms, the following three parameters were routinely measured: 1) current's peak amplitude, 2) membrane potential corresponding to the current's peak amplitude, and 3) channels activation range, including the membrane potential corresponding to where a detectable inward current was measured and to where the current reversed. A paired t-test was performed on all data to determine whether experimental values differed significantly from controls.

Current clamp. Isolated catfish cone horizontal cells were current clamped to simulate in vivo responses to light flashes and to simulate the off-overshoot recorded from intact retina. A series of hyperpolarizing current injections were used to simulate light-on responses, whereas depolarizing current injections were used to simulate the off-overshoot recorded during light-off.

In hyperpolarizing current-clamp experiments, the following parameters were measured: 1) amplitude of hyperpolarization due to injected current and 2) rate of rise and fall of the hyperpolarizing response. When cells were injected with depolarizing currents, the following parameters were measured: 1) amplitude of the induced off-overshoot measured from the control depolarized steady-state plateau, 2) rates of rise and fall of the off-overshoot, 3) duration of the action potential, 4) membrane potential corresponding to the depolarized steady-state plateau, and 5) membrane potential corresponding to the peak of the off-overshoot.

Recordings were obtained using an Axon Instruments Axopatch 200A amplifier (Foster City, CA). Series resistance and capacitive artifacts were compensated for by using amplifier controls. No data were collected from cells with leakage currents >0.02 nA. A small junction potential was measured (<3 mV) and was therefore not compensated for. Data collection was controlled by a computer using a Digidata 1200 data acquisition board. Digitization and analysis was performed using Axon Instruments' pCLAMP program, version 8.0. Data were filtered at 1 kHz and sampled at 5 kHz.


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INTRODUCTION
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In the intact retina, the membrane potential of teleost cone horizontal cells fluctuates between -70 and -20 mV depending on the intensity of the light stimulus. Conveying information to the horizontal cell about light intensity is a function of glutamate release from the photoreceptors, as well as the voltage-gated ion channel activity that is activated within this range (19, 23, 43, 50). Three voltage-gated ion currents are activated in catfish cone horizontal cells between -70 and -20 mV and are illustrated in Fig. 1. They consist of TTX-sensitive sodium current, an L-type calcium current, and a delayed rectifying potassium current. However, in retinal horizontal cells, the voltage-gated delayed rectifier is unusually small over the in vivo operating range of the cell (43, 45), measuring only a few picoamperes.


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Fig. 1.   Activation range for voltage-gated ion currents in catfish cone horizontal cells. Three current traces were obtained from voltage-clamped catfish cone horizontal cells. Each current trace was obtained in pharmacological conditions that favored activity of the sodium current (A), calcium current (B), and delayed rectifier (C). Current-voltage (I/V) curves were obtained using the rampwise stimulus protocol and superimposed for comparison. The gray box represents the physiological operating range previously recorded from catfish cone horizontal cells in vivo.

In Fig. 1, each of the voltage-gated currents activated between -70 and -20 mV were obtained separately using the rampwise stimulus protocol outlined in METHODS and superimposed for comparison. Although the peak current for each channel type is outside the physiological operating range of the catfish cone horizontal cell, voltage-gated sodium and calcium channels produce a significant current within the physiological operating range of the cell (gray rectangle). This suggests that both the voltage-gated sodium and calcium current can potentially contribute to the physiological light response. The results below demonstrate that NMDA modulates voltage-gated sodium and calcium activity over a relatively long time period but has no modulatory effect on the delayed rectifier.

NMDA modulates the voltage-gated sodium current. As demonstrated in Fig. 1, the voltage-gated sodium current in catfish cone horizontal cell activates below -50 mV and reaches peak current at +10 mV. To determine whether NMDA had an effect on the voltage-gated sodium current in catfish cone horizontal cells, voltage-gated sodium currents were elicited using the stepwise stimulus paradigm by changing the membrane potential from -60 to +10 mV to evoke peak voltage-gated sodium current activity. In Fig. 2A, three superimposed current traces are demonstrated that were obtained under control conditions, 1 min after a 30-s NMDA pulse, and 10 min after rinsing out NMDA with control Ringer's solution (recovery). In the example shown in Fig. 2A, NMDA elicited an NMDA-induced inward current associated with the opening of the NMDA channel (arrow) and reduced the control sodium current by 61% (star), followed by a near-complete recovery. NMDA-induced reduction of the voltage-gated sodium current was observed in a total of 18 voltage-clamped cone horizontal cells in which NMDA caused a mean peak current reduction of 58% (SE ±5.2) from control current amplitudes (P < 0.01).


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Fig. 2.   N-methyl-D-aspartate (NMDA) reduced the amplitude of the voltage-gated sodium current in isolated horizontal cells. A: 3 superimposed current traces obtained from an isolated horizontal cell voltage-clamped under control conditions, 1 min after application of 100 µM NMDA (star), and after NMDA washout. Note the NMDA-induced current designated by the upward arrow. The membrane potential was stepped from -60 to +10 mV to elicit peak voltage-gated sodium current. B: 2 superimposed traces were obtained before and 1 min after NMDA application in the presence of 100 µM AP-7. C: the rampwise stimulus paradigm was used instead of the stepwise protocol. Three I/V relationship curves were obtained before application of 100 µM NMDA, 1 min after application of NMDA, and 10 min after NMDA wash out. D: voltage-gated sodium currents obtained under control conditions by changing the membrane potential from rest to +50 mV using depolarizing increments. The tail currents produced by these current traces were normalized and plotted against voltage using the Boltzmann function in E.

The NMDA antagonist, AP-7 (100 µM), eliminated both the NMDA-induced current and the effect of NMDA on channel activity. Figure 2B illustrates two superimposed current traces obtained from a different voltage-gated catfish cone horizontal cell before and 1 min after an NMDA pulse in the presence of AP-7. NMDA failed to reduce the voltage-gated sodium current amplitude when cells were pretreated with 100 µM AP-7 (star). Similar results were obtained from six other voltage-clamped catfish cone horizontal cells. In the presence of AP-7, 100 µM NMDA decreased the amplitude of the voltage-gated sodium current by an average of 4.2% (±5.1) compared with the 58% decrease typical of horizontal cells in the absence of AP-7. Results from these experiments suggest that NMDA receptor activation leads to reduction of the voltage-gated sodium channel amplitude in cone horizontal cells.

In Fig. 2C, the membrane potential of another voltage-clamped catfish cone horizontal cell was changed using the rampwise stimulus protocol. Current traces were obtained and superimposed under control conditions, 1 min after NMDA application (gray trace), and 10 min after NMDA wash out (recovery). As demonstrated from this figure, NMDA induced an inward current (arrow) that progressively decreased as the membrane potential reached NMDA's reversal potential (~0 mV). In addition, NMDA caused a significant decrease of the voltage-gated sodium current elicited between -30 and +30 mV (star). Similar results were obtained using the rampwise stimulus protocol from eight other voltage-clamped catfish cone horizontal cells where sodium I/V relationship curves were elicited before and 1 min after NMDA application. NMDA caused a significant decrease of current amplitude at all activated membrane potentials but had no effect on the membrane potential corresponding to the current's peak amplitude.

Figure 2D demonstrates superimposed voltage-gated sodium current traces generated under control conditions by depolarizing the membrane potential of a voltage-gated catfish cone horizontal cell in a stepwise manner from rest. To examine the voltage dependency of the sodium conductance, tail current amplitudes were normalized and plotted against voltage in Fig. 2E. Data points were best fit with a single Boltzmann function, with a half-activation of -10.8 mV and slope factor of 8.8 mV. Similar results were obtained from eight other voltage-clamped catfish cone horizontal cells in which the mean half-activation measured -10.4 (±0.8) mV and the mean slope factor measured 8.9 (±1) mV. The fact that the voltage dependence of activation could be described by a single Boltzmann function suggests that there is a single sodium channel subtype, although we have not ruled out that multiple subtypes could exist having similar voltage dependence characteristics.

To determine the dose-response characteristics of NMDA, various concentrations of NMDA were perfused over voltage-clamped isolated horizontal cells while the membrane potential was stepped to +10 mV from rest to elicit peak sodium current activity. The percent inhibition of the sodium current produced by NMDA was then plotted against NMDA concentration and illustrated in Fig. 3. As demonstrated by the concentration-response curve, NMDA concentrations as low as 5 µM inhibited voltage-gated sodium currents. The peak current amplitude was decreased by a mean of 60% (±10.3; n = 25) in the presence of 100 µM NMDA. The IC50 was calculated to be 36 µM. In this study, 100 µM NMDA was used in all other experiments to obtain maximum responses.


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Fig. 3.   Dose-response curve generated from voltage-clamped horizontal cells. Membrane potential was changed from rest to +10 mV to elicit voltage-gated sodium currents before and after various concentrations of NMDA were applied to voltage-clamped catfish cone horizontal cells. Data points represent the mean %inhibition of voltage-gated sodium current amplitude caused by NMDA compared with control conditions. Data points were obtained from between 5 and 18 horizontal cells. Error bars represent SE.

NMDA modulates current through the L-type calcium channel. Calcium currents are also a general feature of horizontal cells. In catfish cone horizontal cells, the L-type sustained calcium current activates at around -30 mV and reaches peak activity near 0 mV (Fig. 1). To determine whether NMDA receptor activation resulted in modulation of current through the dihydropyridine-sensitive L-type calcium channel, NMDA was perfused over voltage-clamped isolated horizontal cells in the presence of pharmacological agents that blocked other voltage-gated channels and enhanced the current through the L-type calcium channel (see METHODS). In Fig. 4A, the membrane potential of an isolated voltage-clamped horizontal cell was changed using the rampwise stimulus protocol to elicit calcium current activity. The three superimposed current traces were obtained under control conditions, 1 min after a 30-s NMDA pulse (gray trace), and 10 min after NMDA was rinsed out (recovery). NMDA had no effect on the membrane potential corresponding to the current's peak amplitude but did significantly decrease the peak amplitude of the current from -570 to -440 pA. This 22% decrease in calcium current amplitude was similar to results obtained from nine other voltage-clamped cells significant to a level of P < 0.05, in which the mean decrease equaled 23.5% (±2.9). In the presence of 100 µM AP-7, inhibition due to NMDA was decreased to only 3.2% (±1.2; n = 5) (Fig. 4B, gray trace) and was not significantly different from control levels. In Fig. 4C, voltage-gated calcium current traces were obtained by changing the membrane potential of a voltage-gated cone horizontal cell from rest to +50 mV using 10-mV increments under control conditions. The amplitudes of the tail currents generated in Fig. 4C were normalized and plotted against voltages to examine the voltage dependency of calcium conductance (Fig. 4D). Data points were best fit with a single Boltzmann function and suggest that all of the calcium channels in catfish cone horizontal cells are of a single calcium channel subtype or that multiple subtypes exist with similar voltage dependence. From the calcium current's activation curve, the calculated half activation measured -11.2 mV and the slope factor was 7.1 mV. Similar results were obtained from 10 other voltage-clamped catfish cone horizontal cells in which the mean half activation measured -10.8 (±1.4) mV and the mean slope factor for calcium measured 6.9 ± 0.3 mV.


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Fig. 4.   NMDA reduced L-type calcium channel activity. A: I/V relationships were obtained from a voltage-clamped catfish cone horizontal cell under control conditions, 1 min after application of 100 µM NMDA (gray trace), and after full recovery. Traces were superimposed for comparison. In B, I/V relationships were obtained before and after NMDA application from a cone horizontal cell incubated in the NMDA antagonist, AP-7. The superimposed current traces demonstrated in C were obtained by stepping a voltage-clamped catfish cone horizontal cell from rest to various depolarized membrane potentials under control conditions. Normalized tail current amplitudes obtained in this manner were plotted against voltage and fitted with a Boltzmann function in D to generate calcium current's activation curve.

Figure 5 demonstrates the effect of NMDA on the activity of the delayed rectifier. The three current traces superimposed in this figure were obtained by changing the membrane potential to elicit the delayed rectifier under pharmacological conditions that eliminated contribution of the voltage-gated sodium and calcium currents. In control conditions, little potassium current is apparent until the membrane potential is depolarized beyond 0 mV. Peak current under these stimulus conditions occurred when the membrane potential was changed to +50 mV. Immediately after application of NMDA (gray trace), NMDA induced an inward current between -60 and 0 mV, which added to the outward current when the membrane potential was between 0 and +50 mV. We will demonstrate that the NMDA-induced alterations in the delayed rectifier I/V curve results only from the addition of an NMDA ionotropic current and does not suggest any change in the delayed rectifier current. Full recovery of the NMDA-induced current occurred after a 10-min rinse. Because the delayed rectifier is not significantly active during the physiological operating range recorded from intact retina, it is unlikely to play a significant role in information processing in the retina.


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Fig. 5.   Effect of NMDA on the delayed rectifying potassium current. I/V curves were generated from a voltage-clamped catfish cone horizontal cell under pharmacological conditions that favor expression of the voltage-gated delayed rectifier using the rampwise stimulus paradigm. Three current traces were obtained under control conditions, 1 min after NMDA application (gray trace), and after full recovery.

Time course of NMDA's effect. When NMDA is applied to voltage-clamped cells, it opens nonselective cation channels that decrease the cell's input resistance. A change in input resistance could, by itself, explain NMDA's modulatory effect on ion channel activity. However, Fig. 6 demonstrates that NMDA's modulatory effect on calcium channel activity remains after complete recovery of the NMDA-induced current. In Fig. 6, the time course of the NMDA-induced current was compared with the time course of NMDA's effect on the amplitude of the voltage-gated calcium current. To generate this figure, NMDA was applied to a voltage-clamp catfish cone horizontal cell and the rampwise stimulus protocol was used to elicit voltage-gated calcium current activity every 30 s for 5 min. The amplitudes of the NMDA-induced current and the calcium current were measured at each interval and plotted in Fig. 6. Oval data points represent the amplitude of the NMDA-induced current, whereas squares represent the calcium current elicited at each time interval. Both sets of data points were curve fit. As apparent from this figure, the time course of the NMDA-induced current does not match the time course of calcium current modulation. In fact, calcium current modification due to NMDA application persists significantly longer than the NMDA-induced current, suggesting that NMDA's effect on voltage-gated channel activity may be due to the activation of intracellular signaling pathways that modulate ion channel activity (see DISCUSSION). Similar results were obtained from 30 voltage-clamped catfish cone horizontal cells. Although the NMDA-induced current recovered by 95% within ~60 s after application , reduction of the voltage-gated calcium channel persisted for an average of 135 additional s (±22.1) beyond this time. Persistence of the NMDA effect after recovery of the NMDA-induced current was also associated with modulation of the voltage-gated sodium current. Reduction of the voltage-gated sodium current persisted for an average of 126 s (±10.2) beyond recovery of the NMDA-induced current. However, NMDA did not have a long-term effect on the delayed rectifier's I/V curve. In fact, the I/V relationship curve returned to control conditions within 8 s (±4.2) after the NMDA-induced current recovered. There was no long-term effect of NMDA on the delayed rectifier's I/V curve. Together, these results suggest that NMDA has no modulatory effect on the delayed rectifier but does have a relatively long-term modulatory effect on the voltage-gated sodium and calcium channels.


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Fig. 6.   NMDA modulation of L-type calcium current persists after NMDA was washed out. NMDA (100 µM) was applied to a voltage-clamped catfish cone horizontal cell in saline that enhanced voltage-gated calcium current activity. The membrane potential was changed using the rampwise stimulus paradigm every 30 s to elicit voltage-gated calcium current activity. At each 30-s interval, the amplitude of the NMDA-induced current was measured, as well as the amplitude of the elicited voltage-gated calcium current. The amplitude of the NMDA-induced current is represented as ovals, whereas squares represent the amplitude of the calcium-induced current. Data points were curve fit.

If NMDA's effect on ion channel activity is not solely due to a change of input resistance, NMDA's modulatory effect should be apparent after the NMDA-induced current fully recovered. In Fig. 7A, the effect of NMDA on elicited voltage-gated sodium currents is demonstrated. The three superimposed current traces were obtained before application of 100 µM NMDA (control), 1 min after the NMDA-induced current had completely recovered (star), and after a 10-min washout (recovery). As demonstrated by this figure, even though the NMDA-induced current completely recovered back to control conditions, the amplitude of the voltage-gated sodium current remained significantly diminished compared with control conditions. Similar results were obtained from six other voltage-clamped catfish cone horizontal cells. Even after full recovery of the NMDA-induced current, the amplitude of the voltage-gated sodium current was reduced by an average of 36.2% compared with control conditions (±5.2; P < 0.01).


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Fig. 7.   NMDA's modulatory effect remained after NMDA-induced current recovered. Current traces were obtained from 3 separate voltage-clamped cone horizontal cells using stimulus paradigms to elicit the voltage-gated sodium current (A), the voltage-gated calcium current (B), and the delayed rectifier (C). Traces were obtained under control conditions, after full recovery of the NMDA-induced current (*), and 10 min after NMDA was washed out.

In Fig. 7B, the membrane potential of a voltage-clamped horizontal cell was changed in a rampwise fashion under pharmacological conditions to enhance the voltage-gated calcium current and block the voltage-gated sodium and potassium currents. Current traces were obtained under control conditions, 1 min after the NMDA-induced current fully recovered (star), and 10 min after NMDA was washed out (recovery). As demonstrated by the figure, the amplitude of the voltage-gated calcium current in the presence of NMDA was reduced by 22% compared with control conditions until recovery occurred. This typical response was repeated in eight other voltage-clamped cells in which application of NMDA affected voltage-gated calcium current activity even after the NMDA-induced current fully recovered to control levels. Calcium current decreased by a mean of 23% (±3.1; P < 0.01) compared with control conditions.

Unlike the voltage-gated sodium and calcium currents, the delayed rectifier's I/V curves returned to control conditions as soon as NMDA was washed out. Two superimposed current traces are illustrated in Fig. 7C and represent the delayed rectifying current obtained under control conditions and 1 min after recovery of the NMDA-induced current. There is no apparent difference between the two traces. This supports the hypothesis that NMDA has no modulatory effect on the delayed rectifying potassium current. Together, these results support the hypothesis that a change of input resistance due to opening of NMDA channels cannot be the mechanism of voltage-gated sodium and calcium channel modulation by NMDA. However, the opening of NMDA channels is sufficient to explain the effect of NMDA on the delayed rectifier's I/V curve in the presence of NMDA.

Current-clamp experiments simulate horizontal cell's light response. In the intact retina, horizontal cells depolarize in the dark and hyperpolarize in response to light. Both of these responses can be mimicked in isolated cells using current-clamp techniques. Injection of hyperpolarizing current into current-clamped horizontal cells simulate the cell's response to a light stimulus, whereas depolarizing current injection simulate the off-overshoot associated with cell depolarization and attainment of a second stable resting potential similar to the dark resting membrane potential recorded in intact retina (43). Figure 8A demonstrates an isolated catfish cone horizontal cell injected with hyperpolarizing current. Similar to responses recorded from the intact light-adapted retina (51, 52), the horizontal cell hyperpolarizes for the length of the current injection and then depolarizes back to the original resting membrane potential. Under these conditions, there is no overshoot of the membrane potential as the cell depolarized back to rest. Figure 8B demonstrates an isolated current-clamped horizontal cell injected with a brief pulse of depolarizing current. Depolarizing current injection resulted in an off-overshoot that peaked near 0 mV and decayed to a relatively depolarized steady-state membrane potential of around -20 mV until current pulse termination when the membrane returned to its original resting membrane potential. These depolarizing off-overshoots resemble the action potential-like overshoots recorded from dark-adapted intact retina. Similar results were obtained from all current-clamped catfish cone horizontal cells (n = 17).


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Fig. 8.   Simulated response to injected current in isolated horizontal cells. The response demonstrated in A was obtained from a current-clamped catfish cone horizontal cell injected with hyperpolarizing current. In B, the same cell was injected with depolarizing current. Note the depolarizing off-overshoot followed by a depolarized plateau potential. Hyperpolarizing current (-0.1 nA) was injected to obtain A, whereas +0.1 nA of depolarizing current was current injected to obtain B. Stimulus markers represent pulse durations.

Voltage-gated sodium and calcium channels involvement in the simulated light response. Previous work by Shingai and Christensen (43) demonstrated that sodium and calcium play a role in shaping the off-overshoot recorded from isolated horizontal cells under current clamp. This was based on pharmacological studies using TTX to block the voltage-gated sodium channels and dihydropyridine antagonists to block the L-type calcium channel. Our results support this hypothesis. In order to compare the effect of blocking voltage-gated sodium and calcium conductances to the effect of NMDA treatment on the off-overshoot and subsequent steady-state membrane potential, we perfused solutions containing 10 µM TTX, low-calcium (0.1 mM) catfish saline, or 10 mM cobalt onto current-clamped isolated horizontal cells (Fig. 8, A-C). TTX sharply reduced the peak amplitude of the off-overshoot by 11 mV and depolarized the steady-state membrane potential by +5 mV (Fig. 9A). This was a typical result recorded from five other current-clamped horizontal cells in which the addition of 10 µM TTX reduced the amplitude of the overshoot by an average of 10 mV (±1.1) and simultaneously depolarized the steady-state membrane potential by 4.5 mV (±0.41). All values are significant to P < 0.05. 


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Fig. 9.   Simulated off-overshoot recorded from isolated horizontal cells has a voltage-gated sodium and calcium component that is reduced by NMDA. A represents two voltage responses obtained from an isolated current-clamped horizontal cell before and after application of 10 µM tetrodotoxin (TTX) using 0.15 nA of depolarizing current. In B, 2 responses were obtained from an isolated current-clamped horizontal cell under control conditions and after normal saline was replaced with low calcium (0.1 mM) saline using 0.20 nA of depolarizing current. In C, responses to 0.15 nA of injected current from a third current-clamped horizontal cell was obtained under control conditions and after normal saline was replaced with 10 mM cobalt chloride to block the voltage-gated calcium channel. In D, 0.1 nA of depolarizing current was current injected to elicit the control voltage response. After a combination of 10 µM TTX and 10 mM cobalt chloride was added to the bathing saline, another 0.1 nA of depolarizing current was injected into the same cell to produce the voltage response demonstrated by the gray trace. In E, the depolarizing overshoot was produced by injecting 0.2 nA into a current-clamped horizontal cell under control conditions and after application of 100 µM NMDA. F demonstrates that NMDA's reduction of the depolarizing overshoot is eliminated if isolated cells are incubated in AP-7.

Figure 9, B and C, demonstrates two other current-clamped cells. In Fig. 9B, normal catfish saline was replaced with low-calcium saline (0.1 mM) and in Fig. 9C, 10 mM cobalt was added to saline to block current flow through the L-type calcium channels. Both of these substitutions decreased the amplitude of the off-overshoot from control conditions and changed the steady-state depolarized membrane potential. When low calcium was substituted for normal calcium in the extracellular bath, the peak of the off-overshoot decreased by an average of 10 mV (±1.1), and the depolarized steady-state plateau changed by a mean of +5.2 mV (n = 8 ± 1.4). When 10 mM cobalt was applied to the bath, the mean peak amplitude of the overshoot decreased by 8.1 mV (±1.4), and the steady-state depolarized membrane potential changed by a mean of 5.2 mV (n = 7 ± 1.2) compared with control conditions (P < 0.05). Together, these results support the hypothesis that both voltage-gated sodium and calcium conductances can potentially contribute to shaping the light response and help establish the depolarized steady-state membrane potential in catfish cone horizontal cells.

To determine whether the entire overshoot was due to activity of the voltage-gated calcium and sodium current, current-clamp experiments were repeated in saline containing TTX and cobalt to block the voltage-gated sodium and calcium current, respectively. In the presence of these two pharmacological agents, the off-overshoot was virtually eliminated (n = 6), but the steady-state membrane potential changed by a mean of 6.2 mV (±1.5). An example of this is demonstrated in Fig. 9D. The total elimination of the off-overshoot suggests that the overshoot is entirely a product of activated voltage-gated sodium and calcium currents. In addition, these two currents are involved in setting the depolarized steady-state membrane potential.

NMDA modulates horizontal cell response properties. Voltage-clamp results presented in this study provide evidence that NMDA decreases the activity of the voltage-gated sodium and calcium currents. Because both of these ion channels have been found to be involved in simulated light responses, we repeated current injection experiments using 100 µM NMDA. We found that application of NMDA had no affect on the shape of the simulated responses when cells were injected with hyperpolarizing current (n = 6, data not shown). However, depolarized horizontal cells responded to NMDA application by significantly reducing the height of the off-overshoot and changed the membrane potential corresponding to a new depolarized steady-state plateau. Figure 9E shows an example of an isolated current-clamped horizontal cell whose spike-like overshoot peaked at 5 mV under control conditions. In this typical case, after application of NMDA, the peak of the overshoot decreased by 10 mV. In addition, the cell achieved a new steady-state membrane potential that was more depolarized than the control trace by ~5 mV. The recovery trace was obtained 10 min after NMDA was washed out. This membrane response to NMDA was similar to results obtained from seven other current-clamped catfish cone horizontal cells. In these current-clamped cells, NMDA reduced the amplitude of the membrane potential overshoot by a mean of 9.2 mV (±1.5) compared with control conditions. In addition, the depolarized steady-state membrane potential changed after application of NMDA by a mean of +4 mV (P < 0.05). Recovery to control levels occurred within 5 min after stopping NMDA perfusion. AP-7 (100 µM) eliminated the effect of 100 µM NMDA on the horizontal cell's response properties (n = 8, Fig. 9F).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we conducted experiments to explore the effect of NMDA receptor activation on voltage-gated ion channels found within the physiological operating range of cone horizontal cells. Previous studies have suggested that modulation of these channels may be important in regulating the membrane potential of the cell and in shaping the physiological response to light (8, 43). Voltage-clamp results demonstrated that application of NMDA significantly reduced the amplitude of currents through the L-type calcium channel, as well as the voltage-gated sodium channel. This modulation was not solely due to a change of input resistance caused by activation of NMDA channels based on the findings that modulation of the voltage-gated sodium and calcium channels persisted well after the NMDA-induced current fully recovered, whereas there was no effect on the delayed rectifier. If a change of input resistance were the cause of persistent modulation, all currents would be affected in a similar manner.

Besides a time-dependent argument, other evidence supports the hypothesis that NMDA's modulation of voltage-gated sodium and calcium channels is not solely due to the opening of NMDA channels. This evidence involves the reversal potential of NMDA in catfish cone horizontal cells. As shown in Fig. 5, NMDA induces a current with a reversal potential of 0 mV. Therefore, because no current flows at the reversal potential, the NMDA current cannot contribute to the reduction of voltage-gated calcium current measured when the cells were stepped to 0 mV to elicit peak calcium current activity. In addition, when NMDA channels open, there is an influx of nonspecific cations that influences the shape of the I/V curve. If NMDA current were solely responsible for the changed sodium and calcium I/V curves, the end result would be an enhancement of the currents when the cells were hyperpolarized from 0 mV. Instead, as shown in Figs. 2 and 4, NMDA reduces the amplitude of the voltage-gated sodium and calcium current at all negative membrane potentials within the activating range. On the basis of these observations, the reduction of voltage-gated sodium and calcium currents caused by NMDA receptor activation must be due to a mechanism other than the opening of NMDA channels and subsequent change of input resistance. The mechanism linking NMDA receptor activation and modulation of voltage-gated ion channels in catfish cone horizontal cells is briefly discussed below and more thoroughly in a companion paper (7).

Modulation of the L-type calcium current. There is considerable evidence that activation of ionotropic and metabotropic glutamate receptors modulates voltage-gated calcium currents in a variety of preparations (3, 8, 23, 27, 41). In catfish cone horizontal cells, activation of group I and group III metabotropic glutamate receptor subtypes acts to increase the peak amplitude of the L-type calcium current in the isolated cell preparation (23). Here, we report that activation of ionotropic glutamate receptors reduced the activity of the voltage-gated L-type calcium channel. Therefore, catfish cone horizontal cells contain multiple glutamate receptors subtypes that have opposing effects on L-type calcium channel activity. Because both metabotropic and ionotropic glutamate receptors can be activated when photoreceptors release L-glutamate, it is likely that the modulation of the voltage-gated calcium current is, at least in part, regulated by the summed activity of metabotropic and ionotropic glutamate receptor activation. The resulting direction and magnitude of modulation would be due to temporal factors, the affinity of the receptor to glutamate, or to the strength of the visual stimuli and the corresponding amount of neurotransmitter released from the photoreceptors. Another possible scenario would preferentially activate one glutamate receptor subtype over another depending on the visual stimulus conditions. This has yet to be determined.

Modulation of the voltage-gated sodium current. Although there is evidence of neurotransmitter modulation of the TTX-sensitive voltage-gated sodium channel current in the literature (39), this is the first study to demonstrate modulation of the sodium channel by NMDA. We analyzed the effect of NMDA on voltage-gated sodium channel activity in this study because the voltage-gated sodium current's activation range is within the physiological operating range of the catfish's light response. Similar to the results we obtained when NMDA was applied to voltage-gated calcium channels, we found that NMDA significantly reduced the transient inward current elicited by activation of the voltage-gated sodium channel. Because voltage-gated sodium channels are associated with action potential generation and conductance, the fact that NMDA can modulate the current's amplitude suggests a possible role for the receptor in regulating neuronal excitability in other parts of the central nervous system.

Functional significance. The current-clamp studies presented in this paper are the first step toward an understanding of a role for the NMDA receptor in regulating the horizontal cell light response in the retina. Other groups have demonstrated that the off-overshoot elicited by injected depolarizing current into isolated horizontal cells is due to a combination of sodium and calcium conductances through voltage-gated ion channels. (42, 43). In this study, we have shown that activation of the NMDA receptor produces an affect on the simulated light response similar to blocking sodium and calcium conductance in these cells. Specifically, the amplitude of the off-overshoot is reduced and the cell arrives at a more depolarized steady-state membrane potential compared with control conditions. We have demonstrated that the amplitude of the off-overshoot was due to reduction of current through voltage-gated sodium and calcium channels. A change of the depolarized steady-state membrane potential in the depolarized direction could be due to inactivation of a sodium- or calcium-dependent pump in the plasma membrane that normally keeps the cell's membrane potential more hyperpolarized. Alternatively, the change of steady-state membrane potential may be attributed to NMDA-induced activation of an unidentified anion current.

Although these studies have been performed on isolated cells, a number of physiological implications can be made based on the evidence that physiological responses recorded from intact retina can be mimicked by current injection into isolated cells. One functional implication concerns NMDA's effect on the membrane potential associated with a light stimulus. If NMDA alters the shape of the physiological light response, visual information conveyed by the light response would be modified. For instance, in the intact retina, the depolarizing off-response is largest when the animal is in the light-adapted condition and gap junctions between adjacent horizontal cells are closed (53). Therefore, the off-response conveys information about dark and light adaptation in the retina. In the presence of NMDA, the off-response typically recorded in light-adapted preps would be converted to a response usually associated with dark-adapted retina. Therefore, NMDA receptors may play a role in modulating information about light and dark adaptation in the retina. In addition, the results obtained in isolated cells demonstrated that NMDA depolarized the steady-state membrane potential. This implies that NMDA receptor activation could play a role in maintenance of the dark resting membrane potential recorded from intact retina. A more depolarized membrane potential in the dark would result in greater transmitter release and affect the degree of surround inhibition in bipolar cells. An enhancement of the center-surround antagonism recorded from bipolar cells would increase visual acuity in the dark and would be an evolutionary advantage specific to catfish and other bottom dwelling teleosts that typically live in low light conditions.

Mechanism of NMDA-induced modulation. The mechanism by which NMDA receptor activation modulates voltage-gated sodium and calcium channels in catfish cone horizontal cells has been presented in a companion paper (7). In this paper, evidence is provided that calcium permeation through NMDA channels initiates calcium-induced calcium release from a ryanodine-sensitive intracellular calcium store present in catfish cone horizontal cells to increase intracellular calcium. The increase of intracellular free calcium was found to be linked to NMDA-modulation of voltage-gated sodium and calcium channel activity through a calcium-dependent pathway. Although numerous calcium-signaling pathways have been associated with modulation of voltage-gated channels (6, 11, 22, 24, 36, 46, 48), evidence is provided that calmodulin and calmodulin-dependent protein kinases are involved in modulating both the voltage-gated sodium and calcium channels in cone horizontal cells.


    ACKNOWLEDGEMENTS

We thank Drs. John Cork, Andrei Derbenev, and David Linn for helpful discussions throughout the course of this study.


    FOOTNOTES

This study was supported by National Eye Institute Grant EY-11133 awarded to C. L. Linn and by an LSU Board of Regents grant awarded to S. F. Davis.

Address for reprint requests and other correspondence: C. Linn, Dept. of Biological Sciences, 1903 W. Michigan Ave., Kalamazoo, MI 49008 (E-mail: clinn{at}unix.cc.wmich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 27, 2002;10.1152/ajpcell.00252.2002

Received 31 May 2002; accepted in final form 4 November 2002.


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