Group I mGluR Activation Turns on a Voltage-Gated Inward Current in Hippocampal Pyramidal Cells

Shih-Chieh Chuang, Riccardo Bianchi, and Robert K. S. Wong

Department of Physiology and Pharmacology, State University of New York Health Science Center, Brooklyn, New York 11203


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chuang, Shih-Chieh, Riccardo Bianchi, and Robert K. S. Wong. Group I mGluR Activation Turns on a Voltage-Gated Inward Current in Hippocampal Pyramidal Cells. J. Neurophysiol. 83: 2844-2853, 2000. A unique property of the group I metabotropic glutamate receptor (mGluR)-induced depolarization in hippocampal cells is that the amplitude of the depolarization is larger when the response is elicited at more depolarized membrane potentials. Our understanding of the conductance mechanism underlying this voltage-dependent response is incomplete. Through the use of current-clamp and single-electrode voltage-clamp recordings in guinea pig hippocampal slices, we examined the group I mGluR-induced depolarization in CA3 pyramidal cells. The group I mGluR agonists (S)-3-hydroxyphenylglycine and (S)-3,5-dihydroxyphenylglycine turned on a voltage-gated inward current (ImGluR(V)), which was pharmacologically distinct from the voltage-gated sodium and calcium currents intrinsic to the cells. ImGluR(V) was a slowly activating, noninactivating current with a threshold at about -75 mV. In addition to the activation of ImGluR(V), group I mGluR stimulation also produced a voltage-independent decrease in the K+ conductance. Our results suggest that the depolarization induced by group I mGluR activation is generated by two ionic mechanisms---a heretofore unrecognized voltage-gated inward current (ImGluR(V)) that is turned on by depolarization and a voltage-insensitive inward current that results from a turn-off of the K+ conductance. The low-threshold and noninactivating properties of ImGluR(V) allow the current to play a significant role in setting the resting potential and firing pattern of CA3 pyramidal cells.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Metabotropic glutamate receptors (mGluRs) have been classified into three groups based on structural homology and their link to intracellular second-messenger systems (Nakanishi 1994). Physiological studies suggest that the different subgroups of mGluRs have distinct roles in neuronal signal processing. Activation of group II and III mGluRs causes presynaptic inhibition (Baskys and Malenka 1991; Desai et al. 1994; Vignes et al. 1995). In contrast, activation of group I mGluRs affects postsynaptic function, causing neuronal depolarization and excitation (Bianchi and Wong 1995; Miles and Poncer 1993; Pozzo Miller et al. 1995; Whittington et al. 1995).

In the hippocampus, stimulation of group I mGluRs elicits neuronal population oscillations at the gamma  (30-50 Hz) and beta  (12-30 Hz) frequencies. gamma  oscillations are elicited primarily by an mGluR-induced excitation of inhibitory interneurons (Whittington et al. 1995), whereas beta  activities and the associated epileptiform afterdischarges are mediated by an excitation of pyramidal cells (Merlin and Wong 1997; Taylor et al. 1995). beta  oscillations, once induced, are long-lasting and cannot be reversed by washing out the agonist (Merlin and Wong 1997; Merlin et al. 1998). At present, the mechanisms whereby group I mGluRs excite hippocampal cells and elicit long-lasting beta  oscillations remain unclear. We begin to explore this issue by examining the action of group I mGluRs on individual pyramidal cells in hippocampal slices.

Group I mGluR activation produces depolarization of hippocampal neurons via a suppression of potassium conductances (Charpak et al. 1990; Guérineau et al. 1994; Harata et al. 1996; Lüthi et al. 1997; Stratton et al. 1989) and an activation of nonspecific cationic conductances (Congar et al. 1997; Guérineau et al. 1995; Pozzo Miller et al. 1995). It has been noted that the amplitude of the mGluR-mediated depolarization is enhanced significantly with cell depolarization (Charpak et al. 1990; Guérineau et al. 1994; Lüthi et al. 1997). One study suggests that the voltage dependency of the mGluR response is caused by an increase in potassium channel blockade as the cell is depolarized (Lüthi et al. 1997). We have carried out experiments to describe more fully the conductance mechanism underlying the mGluR-mediated depolarization in CA3 neurons and to evaluate whether the voltage dependency can be adequately explained by the mechanisms described in the preceding text.

Portions of this work have been presented in abstract form (Chuang et al. 1997, 1998).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Slice preparation

Transverse hippocampal slices 400-µm thick were prepared from adult guinea pigs as described previously (Bianchi and Wong 1995) and placed on nylon mesh in an interface recording chamber (Fine Science Tools, BC, Canada). The control solution consisted of (in mM) 157 Na+, 136 Cl-, 5 K+, 1.6 Mg2+, 2 Ca2+, 26 HCO3-, and 11 D-glucose. In some experiments, Mn2+-containing solutions were used. These solutions had added Mn2+ (1.5 mM) and reduced Ca2+ (0.5 mM). Perfusion media were bubbled with 95% O2-5% CO2 to maintain the pH near 7.4, and the temperature was at 34-36°C.

Electrophysiological recordings

Intracellular recordings of CA3 neurons were carried out using an Axoclamp 2A amplifier (Axon Instruments; Foster City, CA). Electrodes were pulled with thin-wall glass tubings and had resistance of 30-40 MOmega when filled with K-acetate (2 M) solution. Voltage and current signals were digitized and stored in an Intel 386-based computer using a 12-bit A/D converter controlled by pClamp software (Axon Instruments). Voltage-clamp experiments were performed using the single-electrode discontinuous clamp mode. The headstage output was monitored continuously on an oscilloscope, and the switching frequency (4-6 kHz) and gain (0.5-1.0 nA/mV) were adjusted so that the decay of voltage transients was complete between switch cycles. Synaptic stimulation was applied using tungsten bipolar electrodes (100 µs; 0.1-1 mA).

Pharmacological agents

To optimize the conditions for studying the mGluR-mediated inward current, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM), 3-((R,S)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 20 µM), and tetrodotoxin (TTX, 0.6 µM) were added to the perfusing solution. The mGluR agonists and antagonists (S)-3-hydroxyphenylglycine (3HPG), (S)-3,5-dihydroxyphenylglycine (DHPG), (+)-alpha -methyl-4-carboxyphenylglycine (MCPG), and (S)-4-carboxyphenylglycine (4CPG) also were added to the perfusate. About 15-20 min was required for added drugs to equilibrate in the recording chamber. The peak inward current amplitude induced by Group I mGluR agonists was usually reached 45-60 min after the addition of the agonist. This delay is probably caused by the slow development of the inward current after mGluR stimulation. Glutamate receptor ligands were purchased from Tocris Cookson (Ballwin, MO); all the other chemicals were from Sigma (St. Louis, MO).

Estimation of the reversal potential of ImGluR(V)

Maximum outward current (I1, indicated in Fig. 2C) activated during a hyperpolarizing pulse (V2, indicated in Fig. 2A) is given by
<IT>I</IT><SUB><IT>1</IT></SUB><IT>=</IT>(<IT>g</IT><SUB><IT>2</IT></SUB><IT>−</IT><IT>g</IT><SUB><IT>1</IT></SUB>)(<IT>V</IT><SUB><IT>2</IT></SUB><IT>−</IT><IT>E</IT><SUB><IT>mGluR</IT>(<IT>V</IT>)</SUB>) (1)
where EmGluR(V) is the reversal potential for ImGluR(V) and g1 and g2 are the membrane conductances at the holding potential (steady-state value) and at the end of the hyperpolarizing pulse, respectively. g = gm + gmGluR(V), where gm is the voltage-independent input conductance of the cell and gmGluR(V) is the membrane conductance contributed by the mGluR agonist.

Peak inward ImGluR(V) (I2) observed after the return of the membrane potential back to the holding level (V1) after the hyperpolarization is given by
<IT>I</IT><SUB><IT>2</IT></SUB><IT>=</IT>(<IT>g</IT><SUB><IT>1</IT></SUB><IT>−</IT><IT>g</IT><SUB><IT>2</IT></SUB>)(<IT>V</IT><SUB><IT>1</IT></SUB><IT>−</IT><IT>E</IT><SUB><IT>mGluR</IT>(<IT>V</IT>)</SUB>) (2)
Dividing Eq. 1 by Eq. 2 yields
<IT>I</IT><SUB><IT>1</IT></SUB><IT>/</IT><IT>I</IT><SUB><IT>2</IT></SUB><IT>=</IT>−(<IT>V</IT><SUB><IT>2</IT></SUB><IT>−</IT><IT>E</IT><SUB><IT>mGluR</IT>(<IT>V</IT>)</SUB>)<IT>/</IT>(<IT>V</IT><SUB><IT>1</IT></SUB><IT>−</IT><IT>E</IT><SUB><IT>mGluR</IT>(<IT>V</IT>)</SUB>)
By rearranging the equation, the reversal potential can be calculated
<IT>E</IT><SUB><IT>mGluR</IT>(<IT>V</IT>)</SUB><IT>=</IT>(<IT>I</IT><SUB><IT>1</IT></SUB><IT>V</IT><SUB><IT>1</IT></SUB><IT>+</IT><IT>I</IT><SUB><IT>2</IT></SUB><IT>V</IT><SUB><IT>2</IT></SUB>)<IT>/</IT>(<IT>I</IT><SUB><IT>1</IT></SUB><IT>+</IT><IT>I</IT><SUB><IT>2</IT></SUB>) (3)
For the preceding equations to be true, the assumption is that when the membrane potential jumps from rest to a hyperpolarized level or vice versa, the membrane conductance remains unchanged for an instant. This assumption is probably justified because the changes in voltage-dependent conductance occurred slowly compared with the instantaneous current response. For the example illustrated in Fig. 2, the time constant of development of the outward current (I1) at -80 mV was 543.5 ms and that of the inward current (I2) at -45 mV was 894.3 ms. I1, I2, V1, and V2 were measured from the data to estimate EmGluR(V).

Student's t-test was applied and differences between data sets were considered to be significant when P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of group I mGluR agonists on the subthreshold properties of CA3 pyramidal cells

In CA3 pyramidal cells with resting potentials of -60 to -65 mV, bath perfusion of the selective group I mGluR agonists 3HPG (50 µM, n = 6) or DHPG (50 µM, n = 32) produced depolarization of 7-18 mV and altered neuronal responses to hyperpolarizing current injection. Before agonist application, current-induced hyperpolarizations developed with an exponential time course (Fig. 1A, Control). In the presence of an mGluR agonist, responses to hyperpolarizing current developed in two steps---an initial fast hyperpolarization followed by a slower second phase (Fig. 1, DHPG). Recovery from hyperpolarization at the end of the current pulse mirrored that recorded at the onset of the hyperpolarization, consisting of an initial fast depolarization followed by a slow phase (Fig. 1, DHPG). The amplitude and duration of the slow phase during repolarization increased in proportion to the amplitude of the hyperpolarization.



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Fig. 1. Effects of group I metabotropic glutamate receptor (mGluR) agonist on the intracellular response of a CA3 pyramidal cell to hyperpolarizing current pulses. A, left: control voltage responses (top) to injected current pulses. Right: responses of the same cell to the same current injections as in control after the addition of (S)-3,5-dihydroxyphenylglycine (DHPG; 50 µM) to the perfusing solution. left-arrow , point where the fast responses were followed by the slow responses. B, left: control responses of the same cell displayed on a slower time base. Right: responses of the cell after addition of DHPG. Note the prolonged duration of the slow responses elicited after the hyperpolarization.

Group I mGluR agonist-induced responses examined under voltage clamp

Because the subthreshold responses elicited by group I mGluR agonists exhibit voltage- and time-dependent changes, we examined the agonist effects under voltage clamp. All voltage-clamp experiments were carried out in the presence of TTX (0.6 µM) to suppress Na+ current.

Under the control condition, a hyperpolarizing command pulse from -45 to -80 mV elicited a brief surge of inwardly directed capacitive current followed by a sustained inward ionic current (Fig. 2A). At the end of the hyperpolarizing pulse, an outwardly directed surge of capacitive current was observed before the membrane current returned to the holding level (Fig. 2A). The time courses of the capacitive current at the onset and release of the hyperpolarizing pulse were fitted with single exponential functions with time constants of 24.7 and 29.5 ms, respectively.



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Fig. 2. Effects of group I mGluR agonist on a CA3 hippocampal pyramidal cell examined under voltage clamp. A, top: membrane current elicited by a hyperpolarizing command pulse. Data points are presented in dots. Solid traces through the dots represent exponential fits to the data points. Bottom: time course of the command pulse. B, top: current responses in the same cell to the same command pulse in the presence of 50 µM DHPG. Data points are fitted by a biexponential function. C: same record as in B now fitted with single exponentials (solid traces) for the ionic currents developed during and after the hyperpolarizing pulse. In A and B, the fitting lines closely follow the data points and cannot be discerned easily.

Addition of the group I mGluR agonist DHPG produced an inward shift of the holding current (Fig. 2B). A hyperpolarizing command pulse elicited the capacitive current and an inward ionic current as in the control condition. However, the inward current was not maintained for the duration of the hyperpolarizing pulse. A slow outward current appeared that was not observed in the control condition (Fig. 2B). At the end of the hyperpolarizing pulse, an initial outward surge of capacitive current was followed by a slow inward ionic current I2, returning the current to the holding level. The current responses activated during and after the hyperpolarization could be fitted with biexponential functions. The initial shorter time constants, largely reflecting the capacitive currents, were 24.5 and 32.6 ms for the responses at the onset and release of the hyperpolarization, respectively. These values for the capacitive current are similar to those of the control response. The longer time constants, reflecting the slow ionic current elicited in the presence of the agonist, had values of 543.5 and 894.0 ms for the responses during and after the hyperpolarization, respectively. In the presence of the mGluR antagonists MCPG (0.5-1 mM; n = 3) and 4CPG (0.5 mM; n = 2) in the bath, the effects of DHPG were prevented.

Conductance mechanism underlying the hyperpolarization-activated outward current induced by group I mGluR agonist

The outward ionic current I1 observed during the hyperpolarizing pulse may represent the turn-off of an inward current (involving a conductance decrease) or the turn-on of an outward current (involving a conductance increase). We measured the conductance changes accompanying the outward current to distinguish between the two possibilities.

The amplitude of the instantaneous current elicited by command voltage step is directly proportional to the cell conductance. The instantaneous current elicited at the onset of the hyperpolarization (providing an indication of the cell conductance at rest, prior to the hyperpolarizing step) was obtained by extrapolating the single-exponential fit for the outward current to the start of the hyperpolarizing pulse (Fig. 2C; I1 + I'). The instantaneous current associated with the termination of the hyperpolarizing step was similarly obtained (Fig. 2C; I2 + I'). Assuming that there is no significant shift in the relevant ionic concentrations during the hyperpolarizing pulse, the difference in the instantaneous currents elicited at the onset and at the termination of the hyperpolarizing pulse represents the difference in cell conductances at the onset of the hyperpolarization and at its termination. This difference in instantaneous currents is determined by subtracting (I2 + I') from (I1 + I') or, simply, I2 from I1. If I1 is larger than I2, the cell conductance at the start of the hyperpolarizing pulse is larger than that at the termination of the pulse.

We measured I1 and I2 of cells in response to a series of hyperpolarizations (Fig. 3). The values of the currents were measured from traces obtained after subtraction of the responses recorded in the presence of the agonist from those recorded in the control condition (Fig. 3, C and D). For any given hyperpolarization, I1 was always larger than I2 (Fig. 3, E and F), indicating that the cell conductance was always larger at the beginning of the hyperpolarizing pulse than at the end. The data indicate that the outward current activated during the hyperpolarization was associated with a conductance decrease, suggesting that group I mGluR stimulation activates an inward current (ImGluR(v)) that is turned off by hyperpolarization and turned on by depolarization.



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Fig. 3. Current-voltage relationship of ImGluR(V). A: current responses of a CA3 pyramidal cell to a series of voltage-clamp hyperpolarizing pulses from -40 to -110 mV at 5-mV intervals. Holding voltage: -45 mV. For display clarity, only responses to -40-, -60-, -80-, and -100-mV command pulses are shown. *, 0 holding current. B: current responses of the same cell after the addition of DHPG (50 µM). C: ImGluR(V) time course obtained by subtracting current traces in A from those in B. Single-exponential fits to the curves are shown as solid lines. Time constants of development of the inward current at -45 mV after hyperpolarizing pulses to -60, -80, and -100 mV were 852.5, 894.2, and 818.8 ms, respectively. D: illustration of the measurement of I1 and I2 using as an example the exponential fit to the subtracted record of the responses to -100 mV (the trace with the largest outward current shown in C). I1 is the maximum outward current activated by the hyperpolarization and is measured as the coefficient of the exponential fit of the data extrapolated to the time of onset of the hyperpolarization. Note that the outward current (I1) represents a turn-off of an inward current (see RESULTS). I2 is the maximum inward current, obtained in a similar manner from the subtracted responses after the hyperpolarizing pulse. Delta Ion and Delta Ioff are the differences between the control and DHPG-modified instantaneous currents at the onset and offset of the hyperpolarization, respectively. E: plot of the peak outward ImGluR(V) (I1; ) elicited by different levels of hyperpolarizations and of the peak inward ImGluR(V) (I2; open circle ) activated by the turning off of the hyperpolarization for the cell shown in A-D. F: mean current-voltage relationship (n = 5 cells) for the peak outward ImGluR(V) (I1; ) recorded during the hyperpolarizing pulse and the peak inward ImGluR(V) (I2; open circle ) recorded after the hyperpolarizing prepulse. Error bars = SE. In E and F, the y axis displays the values of I1 and I2 at various voltage levels normalized to the maximum I1 and I2, respectively.

The values of I1 and I2 also allowed an estimation of the reversal potential for ImGluR(V) (see METHODS). For the cell shown in Fig. 3, corresponding I1 and I2 measurements were obtained for seven levels of hyperpolarizations (from -80 to -110 mV in 5-mV steps) yielding seven values for EmGluR(V). The mean ImGluR(V) reversal potential for this cell was -12.3 ± 3.0 mV. The mean reversal potential value obtained in this way for a total of five cells was -9.8 ± 1.1 mV.

In addition to the activation of ImGluR(V), stimulation of group I mGluRs also produced a voltage-independent decrease in the baseline conductance of the cell. The group I mGluR agonist reduced the instantaneous currents elicited both at the onset (Delta Ion) and release (Delta Ioff) of the hyperpolarizing pulses compared with the control response. This was reflected in subtracted traces where the instantaneous currents (Delta Ion and Delta Ioff) were always outward (e.g., Fig. 3D). Figure 3D also shows that Delta Ioff is larger than Delta Ion. This observation is consistent with the hypothesis that an outward current is turned off by the hyperpolarizing pulse.

Relationship between ImGluR(V) and ICa

CA3 pyramidal cells have robust voltage-gated Na+ and Ca2+ conductances. Because the preceding experiments were carried out in TTX, the contribution of intrinsic Na+ conductance to ImGluR(V) appears unlikely. We explored a possible relationship between ImGluR(V) and intrinsic voltage-gated Ca2+ currents.

Nimodipine (10 µM), a blocker of L-type calcium channels, was applied to the perfusing solution after group I mGluR stimulation (n = 4; Fig. 4). The agent blocked the generation of Ca2+ spikes (Fig. 4C). The amplitude of ImGluR suppressed by hyperpolarization was not affected by nimodipine (Fig. 4, A and B). In addition, current-clamp experiments carried out in the same cell showed that the slow hyperpolarization responses induced by group I mGluR agonists persisted in the presence of nimodipine (Fig. 4C, right).



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Fig. 4. Suppression of Ca2+ current did not affect the generation of ImGluR(V). A: current responses to a series of hyperpolarizations. (i): cell was held at -40 mV and steps of hyperpolarization at 5-mV increments were applied to the cell. (ii) and (iii): responses of the same cell to the hyperpolarization after indicated drug treatments. B: net ImGluR(V) generated after DHPG () and after DHPG and nimodipine (black-triangle). C: current-clamp records of responses to current injection; same cell as shown in A and B. (i): suprathreshold depolarization elicited Ca2+ spikes after DHPG treatment (TTX was present in the solution throughout the experiment). (ii): addition of nimodipine (10 µM) blocked Ca2+ spike generation (left), but the mGluR-activated slow hyperpolarizing responses elicited by current injection persisted (right).

In other experiments, Mn2+ (1.5 mM), a nonspecific Ca2+ conductance blocker, was added to a solution with reduced Ca2+ (0.5 mM) after ImGluR(V) was elicited. Mn2+ blocked Ca2+ spikes in the recorded cell without affecting the amplitude of ImGluR(V). The hyperpolarization-activated outward current induced by DHPG persisted under Mn2+ treatment (n = 3; data not shown).

Effects of group I mGluR agonists on the baseline conductance of CA3 pyramidal cells

On occasion, the two effects of DHPG, namely the reduction in cell conductance and the activation of ImGluR(V), appeared separately during the wash in of the agonist. DHPG first produced a decrease in instantaneous current responses to hyperpolarizing steps (early response; Fig. 5, A(ii) and B). Over time and with continued agonist perfusion, the slowly developing hyperpolarization-activated outward current appeared (delayed response; Fig. 5A(iii)).



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Fig. 5. Temporal separation of the development of the dual effects of DHPG. A(i): control current responses to hyperpolarizations of -60, -80, and -100 mV from a holding potential of -45 mV. (ii): responses to the same voltage protocol after 20 min of perfusion with 50 µM DHPG. An inward shift in the holding current occurred, accompanied by a decrease in cell conductance. The difference between A, (i) and (ii), is displayed in C. (iii): responses after 56 min. B: I-V plots from records shown in A, (i) and (ii). C-E show subtracted traces as indicated.

Figure 5B shows a plot of the current-voltage (I-V) relationship of the cell in response to hyperpolarizing pulses. The I-V relationship for both the control () and early (black-triangle) test responses were linear. The two lines cross at about -90 mV, close to the K+ equilibrium potential of the cell. The separation of early and delayed responses was observed in two other cells. Extrapolated reversal potentials obtained from these two cells were -123 and -127 mV.

Activation properties of the group I mGluR-induced current

The data suggest that ImGluR(V) is an inward current activated by depolarization. We examined the activation threshold of the current (Fig. 6). In the presence of DHPG, cells were held at -45 mV. A conditioning pulse of -90 mV was applied for 750 ms to deactivate ImGluR(V) (Fig. 6B). This was followed by a series of 2-s test pulses from -90 to -20 mV in 5-mV increments. Inward current (ImGluR(V)) first appeared when the test pulses were at about -75 mV (Fig. 6, D and E). The amplitude of ImGluR(V) progressively increased with increasing levels of test depolarization and peaked between -30 to -20 mV. Once activated, ImGluR(V) persisted for the duration of the depolarization without showing inactivation.



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Fig. 6. Activation properties of ImGluR(V). A: current responses recorded from a CA3 cell in response to the voltage protocol presented in the inset. From a holding voltage of -45 mV, a conditioning prepulse to -90 mV was delivered and then followed by test pulses to various amplitudes. B: current responses obtained in the presence of DHPG (50 µM). C: time course of ImGluR(V) obtained by subtracting current traces in A from those in B. *, beginning and the end of the segment of current responses displayed. The time constant for activation decreased with depolarization. For this cell, tau  was 1,272.6 ms at -55 mV; 1,023.3 ms at -50 mV; 978.1 ms at -45 mV; 947.3 ms at -40 mV; 952.8 ms at -35 mV; 825.0 ms at -30 mV; 684.1 ms at -25 mV; and 628.2 ms at -20 mV. D: activation curve, consisting of a plot of the maximum amplitude of ImGluR(V) (I2 in the inset) obtained during the test pulse vs. the different voltage levels of test pulse. Maximum ImGluR(V) (I2) for a given test pulse is given by the coefficient of the exponential fit for the current. E: mean activation curve; data were normalized to the respective maximum I2 in each cell (n = 5 cells). Error bars = SE. Note that at test pulses below -70 mV I2 is in the outward direction (D and E). This reflects the continuing deactivation of ImGluR(V), which was activated at the holding potential of -45 mV. The conditioning pulse at -90 mV deactivated the current producing the outward current. The deactivation process was not complete at the end of the conditioning pulse and continued into the test pulses. The presence of this residual deactivation may cause an underestimate of the amplitude of I2 at all levels of depolarization.

Effects of group I mGluR stimulation on the resting potential and firing pattern of CA3 pyramidal cells

Because ImGluR(V) is a persistent current and it had a threshold close to the resting potential, we observed interesting modulations of the resting potential by group I mGluR agonists. In current-clamp recordings, cells exhibiting stable depolarized resting potentials in the presence of DHPG occasionally shifted their resting potential to a more hyperpolarized level. The hyperpolarized resting potential was brought about by an incomplete recovery of the cell response from a hyperpolarizing pulse (Fig. 7A). Figure 7B shows that an inhibitory postsynaptic potential (IPSP) could reset the resting potential of the cell to a more hyperpolarized level in a manner similar to that elicited by intracellular current injection.



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Fig. 7. Bistable resting potentials in a CA3 cell in the presence of DHPG. Two consecutive records are superimposed in each panel. A: a hyperpolarizing current pulse applied to the cell resting at -51 mV when indicated (bar) caused a stable shift of the resting potential to a more hyperpolarized level. B: in the same cell resting at -54 mV, a synaptic stimulus (up-arrow ) applied to the stratum radiatum in the CA1 region induced an inhibitory postsynaptic potential (IPSP). The IPSP elicited a hyperpolarizing shift of the resting potential. C: a depolarizing pulse (bar) applied to the same cell resting at -84 mV caused an intense phase of firing and elicited a sustained depolarization that outlasted the duration of the applied current.

For cells resting at the more hyperpolarized state in the presence of the group I mGluR agonist, depolarizing currents could reset the resting potential to a more depolarized level (Fig. 7C). In such instances, the fast phase depolarization elicited by the applied current was succeeded by a slow phase depolarization usually causing phasic cell firing. After the phasic firing, the membrane potential often stayed at the depolarized level. In six cells examined, bilevel swings of the resting potential ranged from 8 to 19 mV. This phenomenon of bilevel resting potential was not observed in cells under the control condition.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data indicate that stimulation of group I mGluRs activates a novel inward current (ImGluR(V)) with a reversal potential at about -10 mV. A distinct property of ImGluR(V) is its voltage dependency: the amplitude of ImGluR(V) increases monotonically when the cell is depolarized (Fig. 6). The threshold of activation of ImGluR(V) is between -75 and -70 mV and maximum activation of ImGluR(V) occurs at about -30 mV. In addition to the activation of ImGluR(V), group I mGluR agonists also elicit a voltage-independent reduction in K+ conductance.

The low activation threshold of ImGluR(V) (-75 to -70 mV) facilitated our studies of the current. ImGluR(V) can be examined using hyperpolarizing pulses from -45 mV. Hyperpolarizing pulses at this range also should activate Iq (Perkins and Wong 1995), the only other time- and voltage-dependent persistent current operating at this voltage range. Activation of Iq would produce a slow inward current opposing the development of ImGluR(V). Current- (Fig. 1) and voltage-clamp (e.g., Figs. 2 and 3) recordings show that no noticeable time-dependent current developed in response to hyperpolarizing pulses under the control condition. This observation is consistent with previous findings suggesting that Iq is not prominently expressed in CA3 cells (Bianchi et al. 1999) in contrast to its significant presence in CA1 cells (Perkins and Wong 1995). To further ensure that the properties of ImGluR(V) were examined in isolation from other contaminating responses, all the data used for analysis were obtained by subtracting the control responses from those recorded in the presence of an agonist.

Group I mGluRs activate a voltage-dependent depolarizing current

There are two major mechanisms by which stimulation of mGluRs has been shown to depolarize hippocampal cells. These are an increase in a nonspecific conductance to cations (Congar et al. 1997; Guérineau et al. 1995) and a decrease in conductance to potassium ions (Charpak et al. 1990; Guérineau et al. 1994; Harata et al. 1996; Lüthi et al. 1997; Stratton et al. 1989). A recent study also shows that group I mGluR stimulation elicited an inward current in basolateral amygdala neurons via an activation of the Na+-Ca2+exchange (Keele et al. 1997). This current differs from ImGluR(V) in that it is not accompanied by conductance change and is insensitive to voltage (Keele et al. 1997). The mGluR-activated opening of nonspecific cationic channels involves the direct action of G proteins (Pozzo Miller et al. 1995) or intracellular Ca2+ increase (Congar et al. 1997) and does not show voltage-gated properties. In contrast, voltage-dependent mGluR-induced depolarizations have been noted by others (Charpak et al. 1990; Guérineau et al. 1994; Lüthi et al. 1997) and have been attributed to an mGluR-mediated suppression of potassium conductances. The voltage dependency of the response was explained by assuming that the agonist-mediated conductance block itself is voltage dependent and is markedly increased at depolarized potentials (Lüthi et al. 1997). However, the voltage-dependent depolarization induced by group I mGluR activation in our studies is accompanied by a conductance increase and therefore cannot be attributed to a blockade of potassium channels. Instead, the present data indicate that the depolarization is mediated by a novel, voltage-gated inward current that requires cell depolarization for its activation.

A similar dichotomy exists for the explanation of the muscarinic receptor-mediated depolarization. Although a decrease in potassium conductance had been suggested to be a primary action mediated by muscarinic receptors (Benardo and Prince 1982; McCormick and Prince 1986), more detailed examination revealed that the fundamental mechanism for the muscarinic depolarization of cortical neurons involves the activation of a voltage-dependent inward current with a reversal potential near -15 mV (Haj-Dahmane and Andrade 1996). The authors suggest that the voltage-dependent properties of the current led to the apparent conductance decrease observed under voltage- or current-clamp measurements.

A recent study by Wu and Barish (1999) showed that group I or group II mGluR agonists suppressed ID (Wu and Barish 1992) by acceleration of its inactivation. The activation threshold of ID is depolarized to -45 mV, thus the effects of mGluR agonists on ID should not affect most of our study demonstrating the deactivation of I mGluR(V) by hyperpolarizing pulses from a holding potential of -45 mV (Figs. 2-5). The action of mGluR agonists on ID may introduce overestimation in our measurements of ImGluR(V) at depolarizing pulses above -45 mV (Fig. 6). However the peak activation of ID is depolarized to +30 mV, whereas ImGluR(V) is maximally activated at around -30 to -20 mV. This suggests that the inward current caused by an accelerated inactivation of ID may not be a major factor affecting the measurement of ImGluR(V). However, the detailed effects of the mGluR agonist modulation of ID on the kinetic properties of ImGluR(V) can only be derived from studies using dendrotoxin and 4-aminopyridine.

Because of the complex structure of hippocampal pyramidal cells, the data obtained in this study will be affected by space-clamp errors. However, these errors may not be a major factor in determining the measurements of the time course of ImGluR(V) because different amplitudes of ImGluR(V) appearing at the same membrane potential developed with comparable time constants (Fig. 3), whereas systematic variations in the activation time constant were observed when ImGluR(V) developed at different levels of depolarization (Fig. 6).

Although we emphasize the existence of a voltage-dependent inward current turned on by group I mGluR activation, we also observed the coexistence of a voltage-independent conductance decrease contributing to the production of mGluR-mediated depolarization. The reduction in cell conductance can occur separately over time from the development of ImGluR(V) during agonist wash in (Fig. 5) and is expected to amplify the ImGluR(V)-mediated effects on the membrane potential and firing pattern of pyramidal cells.

ImGluR(V): a novel, voltage-dependent inward current activated by group I mGluRs

Five unique features of ImGluR(V) distinguish this current from other known voltage-dependent inward currents, including Na+ and Ca2+ currents, intrinsic to hippocampal neurons: 1) ImGluR(V) is pharmacologically distinct from Na+ and Ca2+ currents because it persists in the presence of TTX and Ca2+ channel blockers (Fig. 4). 2) ImGluR(V) is noninactivating. In voltage-clamp experiments, after a hyperpolarizing test pulse (-80 mV) and on the return of the membrane potential to the holding level (-45 mV; e.g., Fig. 2), the current develops slowly to a maximum in ~1 s. This maximum current shows no sign of decay and is maintained for >= 10 s, the longest interval at which cells were held at the holding potential in our experiments before another test pulse. The extent to which ImGluR(V) persists greatly exceeds that demonstrated for intrinsic Na+ and Ca2+ conductances. 3) The voltage-dependent activation and deactivation of ImGluR(V) develops with time constants on the order of several hundred milliseconds---significantly longer than those of the Na+ and Ca2+ currents. 4) The threshold of ImGluR(V) is more hyperpolarized (between -75 to -70 mV) than that of other known noninactivating or slowly-inactivating voltage-dependent inward currents. The latter include L-, N-, P-, and Q-type Ca2+ currents, all of which require depolarization beyond -45 mV for activation (Bean 1989; Hess 1990; Hillman et al. 1991; Zhang et al. 1993). And 5) ImGluR(V) is not detectable under normal conditions and appears only when group I mGluRs are stimulated.

Bistable resting potentials associated with the generation of ImGluR(V)

The low-threshold and noninactivation properties of ImGluR(V) allowed it to contribute in an unexpected way to the maintenance of the resting membrane potential. The data suggest that persistent activation of ImGluR(V) sustained the pyramidal cells in a depolarized state. As a result, in current-clamp recordings, hyperpolarizing pulses turned off the persistent ImGluR(V) and produced the slow phase hyperpolarization (e.g., Fig. 1, A and B). Conversely, on release of the hyperpolarization, ImGluR(V) was turned on. Activation of ImGluR(V) caused regenerative depolarization of the cell, eliciting the slow phase depolarization (Fig. 1, A and B). A stable depolarized membrane potential then is attained when the inward ImGluR(V) is balanced by counteracting outward currents. Intrinsic Na+ and Ca2+ currents also can have a tonic influence on the resting potential within the range where the activation and inactivation curves overlap (e.g., Hughes et al. 1999). The contribution of these intrinsic currents to the group I mGluR-mediated changes in the resting potential is not significant because robust voltage-dependent shifts in membrane potential still are elicited by hyperpolarizing pulses in pyramidal cells exposed to DHPG when Na+ and Ca2+ channels are blocked (Fig. 4C). In this way, group I mGluR-mediated effects on pyramidal cells differ from those shown in other preparations (dorsal horn cells, Morisset and Nagy 1996; turtle motoneurons, Svirskis and Hounsgaard 1998), where Ca2+ channel antagonists blocked the responses mediated by mGluRs. Svirskis and Hounsgaard (1998) showed that group I mGluR action on the turtle motoneurons consisted solely of a depolarization associated with a reduction in input conductance and that this response was sufficient to promote regenerative events driven by intrinsic Ca2+ currents.

ImGluR(V) also enables the hippocampal cells to rest at two membrane potentials. Sufficient hyperpolarization can totally deactivate ImGluR(V) and reset the membrane potential to a hyperpolarized level. Physiologically, hippocampal cells can be driven to operate at the hyperpolarized state by inhibitory postsynaptic potentials (Fig. 7).

The properties of ImGluR(V) can be compared with those of the inward current activated by hyperpolarization (Iq) (Perkins and Wong 1995). Previous studies emphasized the importance of Iq in regulating the pacing activities of hippocampal neurons (Maccaferri and McBain 1996; Perkins and Wong 1995). The two features of Iq that allowed it to contribute in this manner are its activation at the subthreshold range and its noninactivation properties---properties similarly possessed by ImGluR(V). The major difference between these two currents is that Iq is activated by hyperpolarization, whereas ImGluR(V) is activated by depolarization. Accordingly, Iq may have a more dominant role in shaping the subthreshold properties of the cell, whereas ImGluR(V) may play a bigger role in regulating the pacing and firing activities of the cell. We have shown previously that via intrinsic and synaptic interactions, group I mGluR agonists induce hippocampal neurons into prolonged epileptiform afterdischarges (Taylor et al. 1995). Additional studies showed that these group I mGluR-mediated effects are long-lasting (Merlin and Wong 1997; Merlin et al. 1998) and may constitute an epileptogenic process. By eliciting periodic prolonged depolarizations of hippocampal pyramidal cells, ImGluR(V) may sustain the prolonged epileptiform afterdischarges. We used group I mGluR agonists to induce the slow inward current and the epileptiform activity. At present there is no information on whether synaptically released glutamate can induce these responses. Experiments involving tetanization of glutamatergic pathways will be needed to establish the duration and extent of synaptic stimulation that is required to induce ImGluR(V) and epileptiform afterdischarges.


    ACKNOWLEDGMENTS

We thank Dr. Lisa Merlin for providing helpful input to the manuscript.

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-35481.


    FOOTNOTES

Address for reprint requests: R.K.S. Wong, SUNY Health Science Center, 450 Clarkson Ave., Box 29, Brooklyn, NY 11203.

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.

Received 22 October 1999; accepted in final form 7 February 2000.


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