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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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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 (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 M (±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 M
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. ![]() |
RESULTS |
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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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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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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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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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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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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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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).
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We thank Drs. John Cork, Andrei Derbenev, and David Linn for helpful discussions throughout the course of this study.
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FOOTNOTES |
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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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