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Journal of Physiology (2002), 541.1, pp. 113-121
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013309
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ABSTRACT |
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The group I metabotropic glutamate receptor agonist DHPG has been shown to produce two major effects on CA3 pyramidal cells at rest: a reduction in the background conductance and an activation of a voltage-gated inward current (ImGluR(V)). Both effects contribute to depolarising CA3 pyramidal cells and the latter has been implicated in eliciting prolonged epileptiform population bursts. We observed that DHPG-induced depolarisation was smaller in CA1 pyramidal cells than in CA3 cells. Voltage clamp studies revealed that while DHPG elicited ImGluR(V) in CA3 pyramidal cells, such a response was absent in CA1 pyramidal cells. Both mGluR1 and mGluR5 have been localised in CA3 pyramidal cells, whereas only mGluR5 has been detected in CA1 pyramidal cells. Using mGluR1 knockout mice, we evaluated whether the absence of an ImGluR(V) response can be correlated with the absence of mGluR1. In these experiments, DHPG failed to elicit ImGluR(V) in CA3 pyramidal cells. This suggests that the smaller depolarising effects of DHPG on wild-type CA1 pyramidal cells is caused, at least in part, by the absence of ImGluR(V) in these cells and that the difference in the responses of CA1 and CA3 cells may be attributable to the lack of mGluR1 in CA1 pyramidal cells.
(Received 20 September 2001; accepted after revision 11 February 2002)
Corresponding author R. K. S. Wong: SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Box 29, Brooklyn, NY 11203, USA. Email: bwong{at}netmail.hscbklyn.edu
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INTRODUCTION |
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Stimulation of group I metabotropic glutamate receptors (mGluRs) depolarises hippocampal cells. Studies on interneurones reveal that the amplitude of the mGluR-induced depolarisation varies among different interneurone types. Interneurones with large group I mGluR-mediated responses have cell bodies in stratum oriens/alveus and axonal ramifications in stratum lacunosum (McBain et al. 1994; van Hooft et al. 2000). In comparison, more modest depolarisations are elicited in another type of stratum oriens interneurone with axons innervating the somata and proximal dendrites of pyramidal cells (McBain et al. 1994; van Hooft et al. 2000). The interneurone type with the larger response to group I mGluR agonist expresses both receptor subtypes mGluR1 and mGluR5, whereas the other interneurone type only expresses mGluR5 (van Hooft et al. 2000).
Group I mGluR subtypes are also distributed differently in CA1 and CA3 pyramidal cells. CA3 cells possess both mGluR1 and mGluR5 while CA1 cells only express mGluR5 (Lujan et al. 1996; Shigemoto et al. 1997). Previous studies have shown that the broad spectrum mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD) depolarised both CA1 (Davies et al. 1995; Bianchi et al. 1999) and CA3 pyramidal cells (Bianchi et al. 1999), although the effects on CA1 pyramidal cells appeared to be smaller than on CA3 cells (Bortolotto & Collingridge, 1995). These observations, together with the results obtained from interneurones, suggest that mGluR5 may be less effective in mediating the depolarising responses than mGluR1.
At least six conductance mechanisms have been described that may contribute to the group I mGluR-mediated depolarisation in hippocampal pyramidal cells: (1) reductions in voltage-dependent and -independent K+ conductances (Guérineau et al. 1994; Lüthi et al. 1996; Wu & Barish, 1999; Chuang et al. 2001); (2) reduction in Ca2+-activated K+ conductance (Charpak et al. 1990; Desai & Conn, 1991); (3) activation of an intracellular Ca2+-gated non-specific cation channel (Congar et al. 1997); (4) activation of a G protein-dependent cationic conduct-ance (Pozzo-Miller et al. 1995); (5) activation of a G protein-independent and voltage-sensitive cationic conductance (Heuss et al. 1999); and (6) activation of a phospholipase C 
1 (PLC1)-dependent voltage-gated inward current (ImGluR(V); Chuang et al. 2000, 2001). In amygdala neurones, group I mGluRs also stimulate the Na+/Ca2+ exchanger (Keele et al. 1997). This additional effect could also contribute to the depolarisation of hippocampal pyramidal cells. Previous studies show that, near the resting potential, the two dominant effects of group I mGluR activation are a reduction in the background conductance and activation of ImGluR(V) (Chuang et al. 2001). In this paper, we compared the effects of group I mGluR agonist on CA1 and CA3 pyramidal cells. Our results are consistent with those reported by Bortolotto and Collingridge (1995) showing that mGluR agonist elicited a reduced response in CA1 pyramidal cells. We explored possible differences in conductance changes induced by (S)-3, 5-dihydroxyphenylglycine (DHPG, a selective group I mGluR agonist) in the two cell types that may account for the difference in response. Our results show that while stimulation of CA3 pyramidal cells by DHPG decreased the background conductance and activated ImGluR(V), ImGluR(V) is not elicited in CA1 cells. Additional results suggest that the absence of ImGluR(V) in CA1 cells may be due to the lack of mGluR1 in these cells.
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METHODS |
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Animals
Experiments were carried out using 5-12-week-old wild-type and mutant mice. Mutant mice lacking the gene for mGluR1 (mGluR1 knockout) were generated from C57B1/6 mice by a method described in detail previously (Conquet et al. 1994).
Slice preparation
Transverse slices (400 µm thick) were prepared from mutant and wild-type C57B1/6 mice. Wild-type mice included mGluR1 +/+ littermates of the mutant. Mice were anaesthetised by inhalation of halothane before decapitation with an animal guillotine, in conformation with the guidelines of the Institutional Animal Care and Use Committee (protocol No. 9808069). After removal of the brain and isolation of the hippocampus, slices were prepared as described previously (Bianchi & Wong, 1995) and placed on the nylon mesh in an interface recording chamber (Fine Science Tools, BC, Canada). The control solution contained (mM): 150 Na+, 136 Cl-, 5 K+, 1.6 Mg2+, 2 Ca2+, 26 HCO3-, and 10 D-glucose. Cs+ 2 mM was added to the extracellular solution in voltage clamp experiments to suppress Iq (McCormick & Pape, 1990; Maccaferri et al. 1993). Perfusion media were bubbled with 95 % O2-5 % CO2 to maintain the pH near 7.4, and the temperature was maintained at 34-36 °C.
Electrophysiological recording
Intracellular recordings were carried out using an Axoclamp 2A amplifier (Axon Instruments, Union City, CA, USA). Electrodes were pulled with thin-walled glass tubing and had resistances of 30-40 M
when filled with potassium acetate (2 M). Voltage and current signals were digitised and stored in an Intel Pentium-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-1) were adjusted so that the decay of voltage transients was complete between switch cycles.
Pharmacological agents
To optimise the conditions for studying the mGluR-mediated inward current, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 µM) and 3-((R,S)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 20 µM) were added to the perfusing solution. Tetrodotoxin (TTX, 0.6 µM) was also added in some experiments as noted. The mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) was bath-applied (50 µM) for 1-2 h. The responses observed in this study were recorded at steady-state conditions and presumably reflect the fraction of the receptor population that was in the active (non-desensitised) state. DHPG, CNQX and CPP were purchased from Tocris Cookson (Ballwin, MO, USA). All the other chemicals were from Sigma (St Louis, MO, USA).
Data analysis
Average values are expressed as means ± standard error of means (S.E.M.). Data groups were compared by using Student's t test for paired or unpaired data and considered significantly different when P < 0.05.
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RESULTS |
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Effects of group I mGluR activation on cellular properties
Intracellular recordings of pyramidal cells in hippocampal slices were performed in the presence of ionotropic glutamate receptor (iGluR) blockers CNQX and CPP (20 µM each). Addition of DHPG (50 µM) to the perfusing solution depolarised CA3 cells by up to 20 mV (16.2 ± 3.3 mV; n = 15). In the presence of DHPG, the membrane potential of CA3 cells oscillated at 1-2 Hz (Fig. 1Ab). Action potential firing often occurred at the crest of each oscillatory cycle.
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Figure 1. Effects of DHPG on the spontaneous activities of CA3 and CA1 pyramidal cells Current-clamp intracellular recordings were carried out in the presence of CNQX and CPP (20 µM each) to block iGluR-mediated synaptic transmission. A, the pattern of spontaneous firing in a CA3 cell was recorded before (Aa) and after (Ab) the addition of DHPG (50 µM). Resting membrane potential: -57 mV (Aa) and -48 mV at the trough (Ab). Ac, voltage responses (upper traces) of another CA3 pyramidal cell to injected hyperpolarising current pulses (bottom traces; current intensities: from -0.8 to 0 nA, in 0.2 nA steps). Left and right arrows in the lowest trace indicate the onset of the slow hyperpolarising and depolarising responses, respectively. B, recordings in the same conditions from a pyramidal cell in a mini-slice containing only the CA1 region (in Bc note the absence of the slow responses). Resting membrane potential: -60 mV (Ba) and -55 mV (Bb and Bc). | ||
DHPG also altered CA3 pyramidal cell responses to hyperpolarising current pulses applied near the resting membrane potential (-62 to -55 mV). In the presence of DHPG, an active component appeared superimposed on the passive charging curve elicited by current injection (Fig. 1Ac). This active component caused an additional, delayed, slow hyperpolarisation following an initial phase of fast, passive hyperpolarisation. At the termination of the hyperpolarising current pulse, the return of the membrane potential to the resting level also proceeded in a fast and a slow phase. In control condition, only the fast, passive phases were observed (not shown). The DHPG-induced, slow active hyperpolarisation has been shown to be due to the deactivation of ImGluR(V), a group I mGluR-mediated, voltage-dependent, non-inactivating inward current (Chuang et al. 2000).
Responses of the CA1 pyramidal cells to DHPG were studied using segments of slices with the CA3 region cut off. In the presence of iGluR blockers, DHPG produced a gradual depolarisation of the cell. The depolarisation elicited in CA1 neurones was smaller in amplitude compared to that recorded in CA3 pyramidal cells (6.8 ± 1.5 mV; n = 17; and 16.2 ± 3.3 mV; n = 15; respectively). In addition, the depolarisation induced by DHPG in CA1 cells was usually maintained at a stable level and did not undergo oscillations (Fig. 1Bb).
The responses of CA1 cells to hyperpolarising current pulses (Fig. 1Bc) were also different from those of the CA3 cells. DHPG did not alter the passive charging time course of the hyperpolarising responses in CA1 pyramidal cells. These responses followed a single, fast exponential time course both before and after DHPG.
Voltage clamp studies on the DHPG-induced responses in CA3 and CA1 pyramidal cells
Neurones were held at -45 mV and the current responses to command pulses to -55 and -65 mV were recorded (Fig. 2A). Tetrodotoxin (0.6 µM) and Cs+ (2 mM) were added to the perfusing solution to prevent the activation of Na+ current and Iq, respectively.
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Figure 2. Effects of DHPG on CA1 and CA3 pyramidal cells recorded in voltage clamp Recordings were performed in the presence iGluR blockers as in Fig. 1. A, cells were held at -45 mV and command pulses to -55 and -65 mV were applied. Current responses elicited in CA3 (top traces) and CA1 (middle traces) pyramidal cells from wild-type mice before (Control) and after (DHPG) application of DHPG (50 µM) are displayed. Bottom row (CA3 mGluR1 -/-), recordings obtained from a CA3 pyramidal cell in a slice prepared from an mGluR1 knockout mouse. B, the inward current elicited in a CA3 pyramidal cell by a depolarisation from -90 mV to -45 mV (ImGluR(-45)) was measured. Top traces, responses recorded before (a) and after (b) DHPG. Bottom traces, segment of the subtracted trace (b)-(a) between the two points marked by asterisks shown in the voltage clamp protocol. The amplitude of the inward current was obtained by fitting the trace with a single exponential and by extrapolating the current to the onset of the depolarisation. Bottom graph, average current amplitude measured in this way for the three cell types. n for CA1, CA3 and CA3 mGluR1 -/- cells are 9, 9 and 4, respectively. The current amplitude elicited in CA3 pyramidal cells is significantly larger than those activated in the other cell types (for both comparisons, CA3 vs. CA1 and CA3 vs. CA3 mGluR1 -/-, * P < 0.001). The current amplitude elicited in CA3 mGluR1 -/- cells is not significantly different from that recorded in CA1 cells. C, inward currents elicited by depolarisations from -90 mV to -80, -50 and -20 mV in a CA3 pyramidal cell and in a CA1 neurone of wild-type mouse slices. Currents were obtained by subtracting the control responses from those recorded in the presence of DHPG and were measured as described in B. Bottom graph, activation curves for ImGluR(V) elicited in CA3 and CA1 cells from wild-type hippocampal slices (n = 3 each group). The amplitude of ImGluR(V) in CA3 cells was significantly larger than that in CA1 cells at membrane potentials | ||
Under the control condition, the hyperpolarising command pulses applied to a CA3 cell elicited a brief surge of inwardly directed capacitive current followed by a sustained inward ionic current (Fig. 2A, top left). At the end of the hyperpolarising command pulse, an outwardly directed capacitive current was observed before the current returned to the holding level. Addition of DHPG caused an inward shift of the holding current in the CA3 cell (Fig. 2A, top right). A hyperpolarising command pulse elicited a capacitive current followed by an inward ionic current. In the presence of DHPG, the inward ionic current was not maintained but showed a gradual shift in the outward direction (Fig. 2A, top right). At the end of the hyperpolarising pulse, a capacitive current was followed by a gradually developing inward current that brought the current amplitude back to the holding level. Previous studies (Chuang et al. 2000) indicated that the outward current elicited during the hyperpolarisation and the inward current activated following the hyperpolarisation reflect the turn-off and turn-on of the DHPG-activated inward current ImGluR(V), respectively.
In recordings from CA1 pyramidal cells, the time course of the ionic current responses to hyperpolarising command pulses was not altered by DHPG (Fig. 2A, middle). However, the amplitude of the current response to the hyperpolarising command pulse was reduced in the presence of DHPG indicating that the agonist caused an increase in the input resistance of the cell. In five cells the input resistance changed from 58.0 ± 4.6 M in control to 74.4 ± 3.0 M after DHPG (P < 0.01).
The group I mGluR consists of the mGluR1 and mGluR5 subtypes (Nakanishi, 1994). Both subtypes are present in CA3 pyramidal cells, whereas only mGluR5 is detected in CA1 pyramidal cells (Lujan et al. 1996; Shigemoto et al. 1997). To evaluate the role of mGluR1 in the induction of ImGluR(V), we examined CA3 pyramidal cells from mGluR1 -/- knockout mice. The addition of DHPG caused an inward shift in the holding current in CA3 pyramidal cells from mGluR1 -/- mutant mice (Fig. 2A, bottom). DHPG also caused an increase in input resistance in these cells. Input resistance changed from 51.2 ± 3.9 M in control to 62.2 ± 3.7 M after DHPG (n = 4; P < 0.01). However, the outward current shift elicited by the hyperpolarising pulse observed in CA3 wild-type pyramidal cells was no longer activated in the mutant cells. Likewise, the inward current elicited in wild-type cells following the hyperpolarising pulse was absent in the mutant cells. In CA1 pyramidal cells from mGluR1 -/- knockout mice, as observed in mGluR1 -/- CA3 cells, DHPG induced an inward shift of the holding current of 34.7 ± 4.6 pA and an increase in input resistance (from 62.3 ± 3.2 M in control to 70.1 ± 1.4 M after DHPG; n = 4; P < 0.05), but it did not induce ImGluR(V). Thus, ImGluR(V) responses were not elicited in the mGluR1 -/- CA3 and CA1 pyramidal cells.
Figure 2B summarises the results obtained in the three cell types. The amplitude of the DHPG-induced inward current elicited at -45 mV following a hyperpolarisation to -90 mV was measured in the wild-type CA3 and CA1 pyramidal cells as well as in CA3 pyramidal cells of mGluR1 -/- mutant mice. The mean amplitude elicited in the wild-type CA3 pyramidal cells was -246.3 ± 23.0 pA (Fig. 2B). Negligible current was elicited in wild-type CA1 cells and mutant CA3 cells (-1.4 ± 12.0 pA and -28.3 ± 3.2 pA, respectively; Fig. 2B, graph).
Activation curves for ImGluR(V) in CA3 and CA1 cells of wild-type mouse slices were obtained by measuring the subtracted currents (DHPG-control) elicited by voltage pulses from the holding potential of -90 mV to depolarised levels up to 0 mV in 10 mV increments (Fig. 2C). ImGluR(V) was activated only in CA3 cells at membrane potentials more depolarised than -70 mV and reached a maximum value at about -20 mV. In contrast, almost no current was elicited in CA1 cells at all the membrane potentials tested (Fig. 2C, graph).
DHPG-induced population activities
DHPG produced prolonged (2-10 s) rhythmic population discharges in mouse hippocampal slices (Fig. 3Ab; n = 8). ImGluR(V) has been proposed to play a critical role in maintaining the population discharges (Chuang et al. 2001). As ImGluR(V) responses were suppressed in CA3 pyramidal cells of the mGluR1 -/- mutant (Fig. 2A and B), DHPG-induced long rhythmic discharges may also be affected in the mutant preparation. Figure 3 compares the population activities elicited by DHPG in hippocampal slices prepared from the wild-type versus the mGluR1 -/- mutant mice. To examine population activities, the slices were perfused in control solution with no added iGluR blockers.
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Figure 3. Population events evoked by DHPG A, spontaneous activities recorded intracellularly from a CA3 pyramidal cell of a wild-type mouse hippocampal slice perfused in normal solution (Aa). Periodic, prolonged bursts were recorded in the same cell 1 h after DHPG addition to the perfusing solution (Ab). Asterisks mark short bursts that precede an episode of prolonged burst discharge. B, intracellular recordings from a CA3 pyramidal cell obtained from an mGluR1 -/- mutant mouse hippocampal slice in normal solution (Ba). After 1 h and 50 min of perfusion of the same slice with DHPG, periodic bursts of about 1 s duration were recorded (Bb). Periodic prolonged bursts were recorded from the same cell 45 min after the introduction of carbachol (50 µM; Bc). Prolonged discharges were preceded by brief bursts (asterisks). C, frequency distribution of burst duration induced by DHPG in a wild-type slice. Events were counted over a 5 min period. Two distinct populations of bursts appeared on the basis of duration. The record shown in the inset is an expanded trace of the segment indicated by the horizontal bar under the bottom trace in A. D, frequency histograms of burst duration in a slice obtained from an mGluR1-/- mouse. DHPG-induced bursts show a single group of duration distribution (Db). The data represent events occurring over a 5 min period. The trace shown in the inset is an expanded segment of the record marked by the horizontal bar below the trace in Bb. Dc, frequency distribution of the burst duration over a 5 min period observed after addition of carbachol. The trace shown in the inset represents the segment marked by the bar below the trace in Bc. | ||
Intracellular recordings in wild-type CA3 pyramidal cells showed stereotypical long bursts (5-7 s duration) recurring once every 12-15 s (Fig. 3Ab and 3C). Previous data showed that the long bursts are synchronised population events because the frequency and duration of these bursts are not affected by changes in the membrane potential of the recorded cell and the bursts are accompanied by simultaneous extracellular discharges of similar time course and frequency (Taylor et al. 1995).
In intracellular recordings from CA3 pyramidal cells obtained from mGluR1 knockout mice, DHPG (1.7-2 h exposure, n = 4) elicited bursts that were significantly shorter in duration than those recorded from the wild-type cells (Fig. 3Bb and 3Db). The bursts were considered synchronised population activities because their frequency was not affected by changes in membrane potential (not shown). We then added carbachol (50 µM) to the perfusing solution. Longer bursts began to emerge within 30 min, and at 45 min of carbachol application prolonged rhythmic bursts of 2.5-6.5 s duration occurred regularly (Fig. 3Bc and 3Dc; n = 3). In mGluR1 -/- CA3 cells, the average duration of the group of longest bursts was 0.53 ± 0.51 s after DHPG (n = 4) and 4.76 ± 1.08 s after carbachol (n = 3). The burst duration after DHPG in mGluR1 -/- was significantly shorter than that after DHPG in wild-type (5.37 ± 0.67 s; n = 8; Student's t test for unpaired data: P < 0.001), whereas the burst duration after DHPG in wild-type and that after carbachol in mGluR1 -/- were not significantly different (P = 0.65).
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DISCUSSION |
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Our results confirm a previous finding suggesting that activation of mGluRs produces a larger depolarisation in CA3 than in CA1 pyramidal cells (Bortolotto & Collingridge, 1995). Here, we show that the presence of ImGluR(V) in CA3 cells accounts, at least in part, for the larger depolarisation induced in these neurones by group I mGluR stimulation at the resting membrane potential compared to that elicited in CA1 cells. The kinetic properties of ImGluR(V) can explain the intrinsic oscillatory burst observed in CA3 cells (Fig. 1A; see also Chuang et al. 2001). The absence of ImGluR(V) in CA1 cells at resting membrane potentials was not due to a shift in its voltage dependency, because negligible current was elicited by depolarisations up to 0 mV. In contrast to ImGluR(V), the inward shift in holding current and the membrane conductance decrease were elicited by DHPG in both CA1 and CA3 cells. These effects have been studied in detail previously and have been attributed, in part, to decrease in background K+ conductance (Guérineau et al. 1994; Davies et al. 1995; Gereau & Conn, 1995; Bianchi et al. 1999; Chuang et al. 2000, 2001). Our results also suggest that the difference in group I mGluR-induced responses between CA1 and CA3 pyramidal cells may be caused by the lack of mGluR1 in CA1 pyramidal cells as demonstrated anatomically (Lujan et al. 1996; Shigemoto et al. 1997; Ferraguti et al. 1998). The lack of mGluR1 in CA1 pyramidal neurones was also supported by physiological evidence: Lu and colleagues (1997) showed that CA1 pyramidal cells in mGluR5 -/- mice lacked ACPD-induced responses. However, other authors (Mannaioni et al. 2001) provided contrary evidence showing that both mGluR1 and mGluR5 responses were elicited in rat CA1 pyramidal cells. The reason for this discrepancy is not known. As the latter group speculated, the different results might depend on species and/or age differences between the rats and mice used in these studies. Also, alterations in mGluR distribution or function may have occurred in the mutant mice. On the other hand, a clear selective blockade of the two receptor subtypes by the antagonists, at the concentrations used in the study by Mannaioni and colleagues (2001), has not been shown. Thus, it is possible that the mGluR5-mediated responses observed in their study were suppressed by relatively high concentrations of the mGluR1 antagonist.
Previous studies suggest that ImGluR(V) plays a critical role in generating the prolonged rhythmic bursts in the hippocampal CA3 pyramidal cell population (Chuang et al. 2001). Prolonged population bursts were not observed in hippocampal slices prepared from mGluR1 knockout mice (Fig. 3Bb). This observation is consistent with the finding that ImGluR(V) generation is suppressed in CA3 pyramidal cells from the knockout mice (Fig. 2A and B). The shorter synchronised bursts which were elicited by DHPG in the knockout slices may have been generated by a diminished, residually activated ImGluR(V), or they may be sustained by an entirely different mechanism. Preliminary data favour the second explanation. Fig. 3A and C show that DHPG elicited prolonged bursts (5-8 s) as well as short bursts (up to 1 s) in the wild-type hippocampal preparation. Our initial data suggest that interfering with the intracellular Ca2+ stores using thapsigargin suppresses the prolonged bursts while the short bursts persist (Zhao et al. 2001). Thapsigargin also suppresses ImGluR(V) generation in wild-type CA3 pyramidal cells (S. C. Chuang & R. K. S. Wong, unpublished observation). These data suggest that the generation of short bursts does not require a contribution by ImGluR(V); instead, the events may be sustained by other group I mGluR-mediated responses such as the suppression of voltage-dependent background K+ conductances and afterhyperpolarisations.
The experiments in this study were performed in the presence of functional inhibitory synaptic input, however synaptic inhibition does not play a critical role in the DHPG-induced responses of pyramidal cells. The population prolonged discharges (Fig. 3Ab) were previously shown to persist unchanged after blockade of GABAergic synaptic transmission (Taylor et al. 1995; Merlin & Wong, 1997). These observations suggest that the absence of the population oscillations in mGluR1 -/- mice is not due to alterations in GABAergic inhibitory input. However, the effects of DHPG on interneurones of mGluR1 -/- mice remain to be investigated.
Activation of muscarinic receptors elicits prolonged population discharges resembling those induced by DHPG in hippocampal slices (MacVicar & Tse, 1989; McMahon et al. 1998). Figure 3B shows that, despite the ineffectiveness of DHPG in eliciting prolonged population discharges in the mGluR1 mutant preparation, the events were activated by carbachol. These results suggest that the failure of DHPG to elicit long bursts in the mutant hippocampal slices is not caused by an abnormal development of the synaptic circuit and cellular properties.
We have not examined the conductance mechanism underlying the carbachol-induced prolonged bursting. In cortical cells, Haj-Dahmane and Andrade (1996) have demonstrated that stimulation of muscarinic receptors elicits a voltage-dependent inward current with properties similar to ImGluR(V). Similar effects of muscarine have also been observed in sympathetic neurones (Delmas et al. 1996). At present, it is unclear whether the same channel type mediates the muscarinic ionic current and ImGluR(V). Stimulation of muscarinic receptors and group I mGluRs activates the same G protein (Gq; Berstein et al. 1992; Nakamura et al. 1994). Recent studies suggest that a common effect of stimulation of Gq protein-coupled receptors is the activation of the transient receptor potential C channels (TRPC channels; Li et al. 1999; Strübing et al. 2001). The TRPC channels, formed by type 1 and type 4 or 5 TRPC subunits, conduct an inward current that is voltage-sensitive and does not show inactivation (Strübing et al. 2001). The properties of the ionic current mediated through TRPC channels resemble those of ImGluR(V) and of the carbachol-activated current. TRPC channels may be the molecular substrate for both the group I mGluR- and the muscarinic receptor-operated channels. It is possible that ImGluR(V) and the muscarinic receptor-activated inward current are mediated by cations flowing through the same channel type. This possibility awaits further studies.
The lack of ImGluR(V) response in CA1 pyramidal cells and in mGluR1 knockout CA3 cells (both of which express mGluR5 only) suggests that ImGluR(V) is not an effector of mGluR5 activation in hippocampal pyramidal cells. It is possible that the activation of ImGluR(V) requires the stimulation of mGluR1. Alternatively, simultaneous stimulation of both mGluR1 and mGluR5 may be necessary for ImGluR(V) activation. Studies in interneurones reveal that the stimulation of group I mGluRs produces larger effects in cells with either mGluR1 and mGluR5 or mGluR1 alone compared to cells that only express mGluR5 (van Hooft et al. 2000). These results suggest that mGluR1 has a higher efficacy in eliciting the depolarising response than mGluR5. Activation of mGluR1 and mGluR5 produces different responses in other cell types as well (Kawabata et al. 1996, 1998). At present, knowledge of the transduction processes linking mGluR activation to ImGluR(V) is limited (e.g. Zhao et al. 2001). This information is required to evaluate whether mGluR1 and mGluR5 are differentially linked to ImGluR(V) as an effector.
Our studies demonstrate that in addition to differences in their local synaptic connectivity (Christian & Dudek, 1988), CA1 and CA3 pyramidal cells can also be distinguished by their distinct responses to group I mGluR stimulation. Our results focused on the study of conductance mechanisms that are activated close to the resting potential. It is possible that effects of group I mGluR elicited at voltage ranges other than the resting potential may also exhibit regional specificity.
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-50 mV (P < 0.01).