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J Neurophysiol (March 1, 2003). 10.1152/jn.00644.2002
Submitted on Submitted 7 August 2002; accepted in final form 14 November
2002
Departments of Neurology and Neurological Sciences, Stanford University Medical Center, Stanford, California 94305
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Bandrowski, A. E.,
J. R. Huguenard, and
D. A. Prince.
Baseline Glutamate Levels Affect Group I and II mGluRs in Layer V
Pyramidal Neurons of Rat Sensorimotor Cortex.
J. Neurophysiol. 89: 1308-1316, 2003.
Possible
functional roles for glutamate that is detectable at low concentrations
in the extracellular space of intact brain and brain slices have not
been explored. To determine whether this endogenous glutamate acts on
metabotropic glutamate receptors (mGluRs), we obtained whole cell
recordings from layer V pyramidal neurons of rat sensorimotor cortical
slices. Blockade of mGluRs with
(+)-
-amino-4-carboxy--methyl-benzeacetic acid (MCPG, a general
mGluR antagonist) increased the mean amplitude of spontaneous excitatory postsynaptic currents (sEPSCs), an effect attributable to a
selective increase in the occurrence of large amplitude sEPSCs. 2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495, a group II antagonist) increased, but
R(
)-1-amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid
(AIDA) and (RS)-hexyl-HIBO (group I antagonists) decreased sEPSC
amplitude, and (R,S)--cyclopropyl-4-phosphonophenylglycine (CPPG, a
group III antagonist) did not change it. The change in sEPSCs elicited
by MCPG, AIDA, and LY341495 was absent in tetrodotoxin, suggesting that
it was action potential-dependent. The increase in sEPSCs persisted in
GABA receptor antagonists, indicating that it was not due to
effects on inhibitory interneurons. AIDA and (S)-3,5-dihydroxyphenylglycine (DHPG, a group I
agonist) elicited positive and negative shifts in holding current,
respectively. LY341495 and
(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV, a
group II agonist) elicited negative and positive shifts in
holding current, respectively. The AIDA and LY341495 elicited currents
persisted in TTX. Finally, in current clamp, LY341495 depolarized cells
by ~2 mV and increased the number of action potentials to a given
depolarizing current pulse. Thus ambient levels of glutamate tonically
activate mGluRs and regulate cortical excitability.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Glutamate is rapidly
released from presynaptic terminals and exerts its action on both
ionotropic and metabotropic glutamate receptors, whose function has
been carefully defined over many years (Anwyl 1999
;
Collingridge and Lester 1989; Conn and Pin 1997). In addition to this clearly demonstrated fast, precise, and well-controlled release, there is abundant evidence for a persistent low-level baseline concentration of glutamate (0.5-2 µM/L) in the brain in vivo (Meldrum 2000) and in
perfusate from unstimulated brain slices (Bianchi et al.
1999; Kapetanovic et al. 1994). Although much is
known about glutamate released during synaptic transmission, the
function of steady-state low, but detectable, levels of glutamate in
brain remains relatively unexplored. Of particular interest in this
regard are metabotropic glutamate receptors (mGluRs), which are coupled
to G-proteins and have the ability to respond to low concentrations of
glutamate and modulate neuronal excitability. These mGluRs, which are
present in neocortex, are divided into three major groups (I-III) on
the basis of sequence homology and agonist affinity, and they have a
wide variety of effects on neuronal communication and cellular
excitability (Anwyl 1999; Conn and Pin
1997; Meldrum 2000).
The present set of experiments was undertaken to determine whether the
baseline extracellular levels of glutamate in unstimulated neocortical
slices, maintained under standard conditions, might tonically activate
mGluRs, and modulate spontaneous synaptic transmission. Portions of
this work have appeared in abstract form (Bandrowski et al.
2002).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Brain slices were prepared from 13- to 20-day-old male and
female Sprague-Dawley rats. Animals were anesthetized with
pentobarbital (50 mg/kg), and their brains rapidly removed and placed
in cold sucrose-artificial cerebrospinal fluid (ACSF) containing (in
mM) 230 sucrose, 2.5 KCl, 1.25 NaH2PO4, 26 NaHCO3, 0.5 CaCl2, 10 MgSO4, and 10 glucose. This "cutting
solution" was gassed with 95% O2-5% CO2 and had a pH of ~7.4. Coronal slices, 300 µm in thickness, were obtained from sensorimotor cortex
(Zilles et al. 1980) between the rostrocaudal landmarks
of the anterior commissure and anterior hippocampus, using a TPI
vibratome (St. Louis, MO). Slices were sequentially transferred into
half sucrose-ACSF and then a standard ACSF solution containing (in mM)
126 NaCl, 3 KCl, 1.25 NaH2PO4, 26 NaHCO3, 2 CaCl2, 2 MgSO4, and 10 glucose, that had an osmolarity of
298-302 mosmol and a pH of ~7.4 when gassed with 95%
O2-5% CO2. After ~1 h of
equilibration at 32°C in an interface holding chamber, temperature
was allowed to return to ~24°C, and slices were transferred into a
recording chamber where they were minimally submerged, perfused with
ACSF at a rate of 1.5-2 ml/min, and maintained at 32°C. Typically, a
light band ~550-650 µm from the pial surface, corresponding to
superficial layer V, was identified using the ×2.5 objective of a
Zeiss Axioskop (Carl Zeiss, Thornwood, NY; Fig.
1A), and unless otherwise
stated, recordings were made at that location. Pyramidal cells were
visualized with a ×60 water-immersion objective and identified as
neurons with a single emerging apical dendrite extending toward the
pial surface (Fig. 1A). A few such neurons were filled with
biocytin (5% in the pipette solution), and the processed sections
contained labeled typical layer V pyramidal neurons (see processing
methods in Horikawa and Armstrong 1988; Tseng et
al. 1991).
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Micropipettes for whole cell recording were pulled from borosilicate
glass capillary tubes (ID: 0.84 mm, OD: 1.5 mm; WPI, Sarasota, FL).
Pipettes had tip diameters of ~3 µm and were filled with a solution
containing (in mM) 120 K+ gluconate, 10 KCl, 1 MgCl2, 1 CaCl2, 10 ethylene
glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid
(EGTA), 10 N-2 hydroxyethylpiperazine-N'-2
ethanesulfonic acid (HEPES), 3 ATP, and 0.2 GTP, pH adjusted to pH 7.3 with KOH (1.0 M). Final osmolarity was 278-292 mosM, and DC
resistances were 1.5-3 M
. Series resistance
(Ra) was monitored every 1-5 min by
applying brief voltage steps, and data from a given cell were discarded
if changes of >15% were detected during the recording, or if values
exceeded 17 M. Membrane currents and potentials were monitored and
stored on a PC using Clampex8 software that interfaced with an Axopatch
200 amplifier via the Digidata 1322A (Axon Instruments, Union City,
CA). Clampex8 software was programmed for stimulus delivery and data
collection. The Axopatch 200 low-pass filter setting was 2 kHz, and the
digitization rate was 5 kHz.
The following pharmacological agents were used: from Sigma/RBI (St
Louis, MO):
R()-1-amino-2,3-dihydro-1H-indene-1,5-dicarboxylic acid
(AIDA), 2-amino-5-phosphonovaleric acid (APV),
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX),
(+)--amino-4-carboxy--methyl-benzeacetic acid (MCPG), 6-imino-3-(4-methoxyphenyl)-1(6H)-pyridazinebutanoic acid (SR 95531 hydrobromide or gabazine); from Tocris (Ellisville, MO): 2S-2-amino-2-(1S,2S-2carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid (LY341495), (R,S)--cyclopropyl-4-phosphonophenylglycine (CPPG),
(S)-3,5-dihydroxyphenylglycine (DHPG),
(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV); from
Almone Labs: tetrodotoxin (TTX); from CGP: CGP55845. (RS)-hexyl-HIBO
(HIBO) was generously donated by Dr. Ulf Madsen (compound 9 in
Madsen et al. 2001). These drugs were prepared as a
concentrated stock solution in distilled water, 0.1 M NaOH, solution
(for MCPG, AIDA, HIBO, and LY341495) or a 50% water-50% DMSO solution
(for CNQX) and diluted to final concentration in ACSF. Drugs were
stored in aliquots, each of which was used only once to avoid loss of
potency by the freeze-thaw process. Drugs were either added to the
bathing medium (TTX, CNQX, APV, and gabazine) or applied locally to the
slice using a perfusion pipette placed ~500 µm from the recording
pipette (see also Sun et al. 2001). For local perfusion
experiments, typically two barrels of the pipette were filled, one with
ACSF and the other with the desired drug dissolved in ACSF. The area of
the slice containing the recorded cell was perfused with a stream of
ACSF from one barrel in the control and wash conditions, and with a
stream of drug-containing ACSF from the second barrel for drug
conditions. The time to "wash on" the drug was typically 1-4 s,
with some variation due to placement of perfusion pipette.
Spontaneous excitatory postsynaptic currents (sEPSCs) were analyzed
using detector software (Ulrich and Huguenard 1996).
They had average amplitudes of 9-27 pA, or about two to four times the
baseline noise level of 2-8 pA, and were divided by the software into
three types of events. Rise times, decay constants, amplitudes, and
time of occurrence were recorded for each event. Type 1 sEPSCs were
those that were not preceded or followed by another sEPSC within 20 ms,
whereas types 2 and 3 sEPSCs were components of compound sEPSCs in
which one event was closely preceded or followed by another. Holding
potential (Vh), and membrane potential
(Vm) measurements were made using
Clampfit Software (Axon Instruments) by sampling a point every 10 s for the duration of data collection. Data were pooled, and averages
were determined using Microsoft Excel. Graphs were generated using
Microcal Origin software. Data are presented as means ± SE.
Differences between means were evaluated using the t-test
for unpaired samples, or where appropriate, t-test for
paired samples. Statistical significance was set at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We recorded from 104 visually identified superficial layer V
pyramidal cells. Neurons included in the present data set had an
Ra < 17 Ms (mean: 11.7 ± 0.4 M), input resistance of 58.6 ± 2.6 M, and resting membrane
potential of 60.4 ± 0.6 mV.
mGluR antagonism increases the amplitude of sEPSCs
To determine whether mGluRs were activated in unstimulated slices
of neocortex, we applied MCPG, a general antagonist of mGluRs, and
monitored ongoing spontaneous synaptic activity. sEPSCs had 10-90%
rise times of 0.85 ± 0.09 ms, weighted decay time constants (
D,W) of 4.6 ± 0.4 ms, and were blocked
by perfusion of APV/CNQX. Application of MCPG to 15 cells gave rise to
a significant increase in sEPSC amplitude of 18.0 ± 5.6%
(P < 0.01) that lasted for the duration of MCPG
application (Fig. 1, B-E). This effect was reversible, but
variable, ranging from a 0 to 56% increase. These data suggest that
mGluRs are tonically activated in unstimulated neocortical slices,
where they reduce sEPSC amplitudes in a significant proportion of
neurons. Analysis of rise times and weighted decay time constants (D,W) in responsive cells during MCPG
application revealed no detectable change from control. Figure
1D shows an average of all type 1 events (see
METHODS) obtained from one neuron during 100-s periods
before (48 events) and during MCPG perfusion (54 events). Although
sEPSCs increased in amplitude, no difference in rise or decay times was
present when mean sEPSCs were scaled to the same amplitude (Fig.
1D). To determine whether all events were larger or whether
larger amplitude events were more numerous, cumulative probability
plots were generated. These cumulative probability distributions
revealed that MCPG mainly increased the largest ~40% of events (Fig.
1E). Analysis of the frequency of sEPSCs revealed no
significant change between pre-drug and MCPG conditions (Fig.
1F, all sEPSCs). However, when only events >25 pA were
examined, a significant increase in sEPSC frequency was present in MCPG
(P < 0.01; Fig. 1F, large sEPSCs). Thus
MCPG selectively increases the frequency of larger-amplitude events. To
test for nonspecific pH or osmolarity effects, we locally perfused ACSF
containing 1.5 mM NaOH and found that this concentration NaOH,
equivalent to that in local MCPG-containing perfusate, had no effect on
the amplitude of sEPSCs (96.3 ± 4.6%, P > 0.05, n = 3, not shown).
Group II, but not I or III, mGluRs increase sEPSC amplitude
To determine which of the subtypes of mGluRs are responsible for the modulation of sEPSCs, we tested several mGluR group-specific antagonists. The effect of MCPG was mimicked by LY341495 (a group II antagonist), but HIBO and AIDA (group I antagonists) decreased sEPSC amplitude and CPPG (a group III antagonist) did not affect sEPSCs (Fig. 2). The effect of the specific group II mGluR antagonist, LY341495 (2 µM) was larger and less variable than that of MCPG, in that it increased sEPSC amplitude by an average of 42.0 ± 10.3% (P < 0.05, n = 11), as opposed to the mean 18% increase seen in MCPG. Individual cumulative probability plots show that, like MCPG, the change in amplitude in LY341495 was predominantly due to an increase in the number of large events with very little effect on the small sEPSCs (Fig. 3Aii). The effects of group I mGluR antagonists were examined in 14 neurons. AIDA (1mM) decreased sEPSC amplitude by 27.5 ± 6.0% (P < 0.05, n = 9; representative cell in Fig. 2A, top) and HIBO (200 µM) reduced sEPSC amplitude by 28.7 ± 11.8% (P < 0.05, n = 5; P > 0.05 AIDA vs. HIBO). The pooled data of Fig. 2B show the effects of AIDA and HIBO on sEPSC amplitude. Individual cumulative probability plots showed that the change in sEPSC amplitude in AIDA and HIBO was predominantly due to a decrease in the number of large events (Fig. 3Ai). Effects of MCPG, AIDA, and LY341495 were reversible during the washout period (amplitude: MCPG 95.4 ± 3.0%; AIDA 100.9 ± 20.0%; and LY341495 103.2 ± 4.6% of control levels following wash, P > 0.05 for all). These data suggest that the more modest effect of MCPG described in the preceding text may have resulted from a simultaneous increase of sEPSC amplitude due to antagonism of group II mGluRs, together with a decrease via blockade of group I mGluRs. The group III mGluR antagonist, CPPG (100 µM), was ineffective in changing amplitude or frequency of sEPSCs (n = 7, Figs. 2 and 3). These data indicate that mGluRs of groups I and II are activated in unstimulated slices and have a role in modulating sEPSCs.
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Increase in sEPSCs is not due to disinhibition of pyramidal neurons
It has been shown that fast-spiking (GABAergic) cells can be
depolarized and generate rhythmic activity when exposed to mGluR agonists (Boddeke et al. 1997; McBain et al.
1994; Whittington et al. 1995). Thus
inactivation of mGluRs might result in hyperpolarization of these
neurons, a reduction in their activity, and a secondary increase in the
activity of downstream pyramidal cells. If the effects of mGluR
antagonists on sEPSCs in pyramidal cells are indirect, i.e., due to
disinhibition, the actions of these agents should be occluded by
GABAA and GABAB
antagonists. In the presence of the GABAA
receptor antagonist gabazine (8 µM), which should block both phasic
and tonic GABAA receptor-mediated inhibition (Stell and Mody 2002), LY341495 (2 µM) produced a
significant (P < 0.05) increase in sEPSC amplitude in
eight of eight cells (Fig. 4). This
response was generally similar to that seen with perfusion of LY341495
alone (cf. Figs. 2A and 4B) with the exception that spontaneous bursts of EPSCs occasionally occurred, probably representing spontaneous epileptiform events (e.g., Fig. 4B,
~900 s). A similar significant effect of LY341495 was seen in seven of eight cells exposed to the GABAB receptor
antagonist CGP 55845 (0.2-1 µM) (Blake et al. 1993).
Therefore it is unlikely that mGluR actions on GABAergic cells are
necessary for the observed increase in sEPSC amplitude.
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Changes in sEPSC amplitude do not occur in TTX-containing solutions
Because mean action potential-dependent EPSCs may be larger in
amplitude than miniature EPSCs (Berretta and Jones
1996), a change in presynaptic firing could account for the
above effects of LY341495 and AIDA. When TTX (1-3 µM) was included
in the bathing medium to block action potentials, neither MCPG
(n = 7), AIDA (n = 6), nor LY341495
(n = 6) induced a change in sEPSC amplitudes (P > 0.05, Fig. 4A). Furthermore, in four
cells where MCPG elicited an average increase in sEPSC amplitude of
31.1 ± 16.4%, TTX treatment during the MCPG exposure reduced the
sEPSCs by 25.8 ± 8.8% (P < 0.05). Therefore the
changes in the number of large events seen after block of group I or II
mGluRs likely result from action-potential-evoked EPSCs. These data
suggest that spike firing in at least some spontaneously active cells
is apparently modified under resting conditions by activation of group
I and II mGluRs.
Holding current and membrane potential are changed by mGluRs
Application of group I and II mGluR agonists and antagonists
elicited reversible shifts in holding currents in layer V pyramidal cells. AIDA, a group I antagonist, shifted holding current by an
average 108 pA in the positive direction (Fig.
5Aii), indicating a negative
or hyperpolarizing shift in Vm. The
reversal potential for the shift, obtained from injection of voltage
ramps, was 71 ± 6 mV (n = 4). A group I
agonist, DHPG (100 µM), elicited an average 126-pA
negative shift in holding current (Fig. 5Ai). In contrast to AIDA, the group II antagonist, LY341495, caused an average
28-pA negative shift of holding current (Fig.
5Bii). The group II agonist, DCG IV (1 µM) shifted the
holding current by an average 209 pA in the positive
direction (Fig. 5Bi). To determine whether the shifts in
holding current elicited by LY341495 or by AIDA were dependent on
impulse-related spontaneous synaptic activity, each antagonist was
applied to six cells from slices bathed in TTX-containing perfusate.
Under these conditions, AIDA elicited a positive shift (56 ± 24 pA, n = 6) and LY341495 elicited a negative shift in
holding current (110 ± 33 pA, n = 6). These results suggest that blockade of group I and II mGluRs directly changes
membrane potential (Vm), independent
of an effect on impulse-related transmitter release. The reversal
potential of the LY341495-induced current was 76 ± 4 mV
(n = 4).
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To further test the hypothesis that endogenous activation of group II
mGluRs directly affects cellular excitability, current-clamp recordings
were obtained from six cells with resting Vms between 57
and 61 mV, during LY341495 application. LY341495 (2 µM) reversibly depolarized Vm by 1-4 mV (6 of 6 cells; average 2.3 ± 1.2 mV, P < 0.01; Fig.
6, A and C). In
each cell, depolarizing current pulses evoked more spikes during
LY341495 application than in control solution (Fig. 6, A and
B). The occurrence of action potentials in 8-s intervals
between current pulses was analyzed. LY341495 application induced
action potential firing in two of six cells. The TTX data of Fig. 4
suggest that these changes in excitability likely resulted in
alterations in the frequency of spontaneous action potentials.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Our results show that antagonists of group I and II, but not group III, mGluRs alter cellular excitability and impulse-related EPSCs in layer V pyramidal neurons of neocortical slices. These findings suggest that the low concentrations of ambient glutamate known to be present in brain slices (see references in INTRODUCTION) are sufficient to activate these receptors. Judging from effects of antagonists, activation of group II mGluRs reduces excitability, whereas activation of group I mGluRs increases excitability. The effects of mGluR antagonists persist in GABAA or GABAB receptor blockers and are therefore not due to indirect actions resulting from disinhibition. These findings support the conclusion that spontaneous firing of layer V neurons is enhanced and suppressed by activated group I and II mGluRs, respectively.
The finding that group I mGluRs increase and group II mGluRs decrease
excitability, when activated, is not entirely consistent with previous
results obtained with application of exogenous mGluR agonists (for
review see Anwyl 1999; Bockaert and Pin
1999; Schoepp 2001). For example, it has been
previously reported that the group II agonist DCG IV does not affect
membrane potential in layer V cells (Cho et al. 2000;
Otani et al. 2002). Yet in our experiments, DCG IV and
LY341495 had opposite effects on holding current, indicating that
activation of group II mGluRs decreases cellular excitability. However,
our data do agree with those of previous studies showing that DHPG
produces a negative-going shift in holding current (for review see
Wisniewski and Carr 2002), and we show that opposite effects are produced by antagonists of group I mGluRs, suggesting that
group I mGluRs are activated in brain slices in the absence of overt
stimulation. Our data support the conclusion that group I and II, but
not III, mGluRs can directly alter membrane excitability and also
affect release of glutamate in unstimulated slices through modulation
of pyramidal cell spike frequency.
The reduction of sEPSCs is most likely to be a group-I-mediated effect.
The reduction can be seen with both AIDA (1 mM) and HIBO (0.2 mM),
molecules with different chemical structures that both antagonize group
I mGluRs, ruling out some of the arguments for nonspecific effects of
either drug. Furthermore, the effects of a group I mGluR agonist
(DHPG), are opposite to the effects of AIDA, at least on the holding
current, adding credibility to the claim that AIDA is acting as a group
I antagonist. Additionally, the decrease does not appear to be an
artifact of the drug application process, as sEPSCs are increased by
group II antagonists and unchanged by NaOH application. Finally, the
concentration of AIDA used, 1 mM, should affect both subtypes of group
I mGluRs as this was reported to be the EC50 for mGluR5 (EC50 for
mGluR1 is 0.2 mM) (see Pellicciari et al. 1995).
LY341495 is not a selective group II mGluR antagonist when used at
higher concentrations (see Fitzjohn et al. 1998;
Kingston et al. 1998; Ornstein et al.
1998; Schaffhauser et al. 1998); however, the
concentration used in this study (2 µM) likely only affects group II
mGluRs. Several lines of evidence suggest a specific role for group II
mGluRs in the LY341495-mediated response. First, the increase in sEPSCs
was also found for MCPG, a compound most active at group II and I
mGluRs and almost inactive at group III sites (Jane et al.
1993; Pellicciari et al. 2001), indicating that
the increase is not an effect of group III mGluRs. Second, group I
mGluR antagonists AIDA (Moroni et al. 1997) and HIBO
(Madsen et al. 2001) reduce, rather than enhance, sEPSC
amplitudes, suggesting that the increase is not group I mediated.
Third, the group III antagonist, CPPG (Jane et al.
1996), had no effect on sEPSCs, again suggesting that the
increase is not group III mediated. Therefore the increase in sEPSCs is
mediated via group II mGluRs, and the decrease is group I mediated.
Because group I and II, but not group III, mGluRs have been found in
extrasynaptic locations, it has been concluded that group I and II
mGluRs are activated under conditions of lateral diffusion of glutamate
or "spillover" (for review, see Anwyl 1999). It is
possible that these receptors are activated in the presence of
high-frequency neuronal activity that increases the ambient level of
glutamate; however, the present results suggest that these receptors
are also activated under resting conditions in brain slices.
The nature of the increase in sEPSCs is likely to be related to changes
in spike frequency of cells within the neocortex. This conclusion is
based on data showing that mGluR-mediated changes in the frequency of
large-amplitude sEPSCs are absent in TTX. Previous reports show that
TTX-insensitive miniature EPSCs (mEPSCs) are decreased in frequency by
agonists of group II mGluRs in several preparations (Scanziani
et al. 1995; Schoppa and Westbrook 1997; Tyler and Lovinger 1995), suggesting that group II mGluR
antagonists should affect mEPSCs. However, mEPSCs are not significantly
affected by LY341495 in our experiments, making it unlikely that mGluRs on the axon terminal are activated by endogenous glutamate in slices.
Another possible interpretation of this result is that TTX reduces the
concentration of ambient glutamate present in slices, resulting in a
lower occupation of receptors and a decrease in the effects of both
AIDA and LY341495. However, our data also show that AIDA and LY341495
continue to have an effect on the holding current of cells in the
presence of TTX even though the effect of these drugs on sEPSCs is
eliminated. Therefore it is unlikely that reduced ambient glutamate
concentration in TTX accounts for the effect of these antagonists on
large sEPSCs. The source of the excitatory synaptic events that are
affected by mGluR antagonists is not clear from our results, although
current-clamp recordings reveal that layer V cells, known to innervate
neighboring pyramidal neurons (Gilbert 1985;
Kisvarday et al. 1986; Lubke et al. 1996; Markram et al. 1997; Schwark and Jones
1989; for review see Markram 1997) did fire more
action potentials in the presence of LY341495. It has not been
determined if cells in other layers of the cortex or other brain
regions are similarly affected by group I and II mGluR antagonists.
The nature of the conductance underlying the shift in membrane
potential remains unexplored. The reversal potential of this current,
about 70 mV, is not near EK or
ECl, calculated to be 95 and 57
mV, respectively, in our experiments. This indicates that more than one
conductance is activated by ambient glutamate as has been proposed for
mGluR1 actions in CA1 cells (Crepel et al. 1994). The
conductance activated by ambient glutamate would increase the
electrotonic length of the recorded pyramidal neurons, and blockade of
the resting conductance would be associated with a decreased
electrotonic length and an improved space clamp. The resultant improved
voltage-clamp fidelity would be expected to increase the amplitude of
all distal synaptic events. However, we observed a specific increase in
the frequency of large events, suggesting that altered voltage-clamp
conditions cannot explain our results.
In a recent review, it was suggested that group II presynaptic mGluRs
may be partially occupied (activated) at rest because these receptors
are sensitive to glutamate in the micromolar range, and extracellular
levels of glutamate readily reach these concentrations (Schoepp
2001). The present observations tend to support this conclusion
because neither MCPG nor LY341495 should have any effect without some
level of activation of mGluRs by an endogenous agonist in unstimulated
slices. Furthermore, the findings that HIBO and AIDA decrease sEPSC
amplitude and shift holding current suggest that group I mGluRs are
also at least partially occupied at rest, whereas group III are not.
Another possibility, at least for group I mGluRs, is that they may be
constitutively active in the absence of glutamate, as was suggested by
Ango and colleagues (2000). This constitutive activity
may account for a portion of the present data obtained with
antagonists; however, it is unlikely that constitutive activity
accounts for a large portion of the effect as there is very little
activity in wild-type cells containing Homer1a protein (Ango et
al. 2000). The above-described effects of mGluRs should be
greatly exaggerated in the intact brain as the levels of spontaneous activity, and therefore the extracellular concentration of glutamate, should be much higher (e.g., Pare et al. 1998;
Steriade 2001). Therefore we believe that our results
may underestimate the importance of mGluRs in the tonic control of
excitability within cortical circuits.
Ambient levels of glutamate (0.5-2 µM) can also affect NMDA
receptors that are sensitive to glutamate concentrations in the range
of 2.5-3 µM (Meldrum 2000). Indeed, an APV-sensitive
steady-state inward current can be observed in cortical neurons if
Mg2+ is left out of the bathing medium (see
Blanton et al. 1990; LoTurco et al.
1990), suggesting that extracellular levels of glutamate in
brain slices are high enough to activate NMDA receptors under some
circumstances. Low levels of glutamate may be critical in early stages
of cortical development for activation of NMDA receptors present in
embryonic cortical cells that have migrated away from the ventricular
zone into the cortical plate (Blanton et al. 1990; LoTurco et al. 1991). These receptors are present before
the emergence of synapses, suggesting that they may guide development
of the immature neuron and be dependent on ambient levels of glutamate for their activation. mGluRs may also have an important developmental role during synaptogenesis as evidenced by changes in mGluR expression that accompany differentiation of cortical laminae (Furuta and Martin 1999; Muñoz et al. 1999). Also,
mGluR expression patterns in visual cortex are altered during
development and can be delayed by dark rearing (Reid et al.
1997). Many behavioral manifestations of brain function,
such as alertness, sleep, and affective states, are best described as
tonic in their time course. A variety of data from studies of
glutamatergic (Blanton et al. 1990; LoTurco et
al. 1990; present results) GABAergic (Hamann et al.
2002; Salin and Prince 1996) and perhaps other
types of neurotransmission in the brain (Bunin and Wightman
1999) suggest that ongoing tonic extrasynaptic receptor
activation is ubiquitous, raising the interesting hypothesis that
ongoing tonic receptor activation has important functional behavioral consequences.
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ACKNOWLEDGMENTS |
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We thank Dr. Ulf Madsen for the generous donation of hexyl-HIBO, Drs. Alberto Bacci and Viktor Kharazia for helpful comments during the preparation of this manuscript, and Dr. QQ Sun for helpful discussions of the data.
This research was supported by National Institute of Neurological Sciences and Stroke Grant NS-12151 and by the Pimley Research and Training Funds.
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FOOTNOTES |
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Address for reprint requests: D. A. Prince, Stanford University School of Medicine, Dept. of Neurology and Neurological Sciences, Room M016, 300 Pasteur Dr., Stanford, CA 94305-5122 (E-mail: daprince{at}Stanford.EDU).
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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). MCPG perfusion elicits a reversible increase in
large amplitude sEPSCs. D, top: MCPG
perfusion (0.2 mM) increases mean sEPSC amplitude. Pre-Drug and MCPG
traces are averages of 48 and 54 type 1 (see METHODS)
sEPSCs, respectively, obtained during 100-s periods. Calibration: 5 pA,
3 ms. Bottom: averaged sEPSCs of A are
scaled to the same amplitude to show that events Pre-Drug and in MCPG
do not differ in rise times or decay time constants. E:
a cumulative probability plot of sEPSC amplitudes from same cell.
Median sEPSC amplitude was larger in MCPG than control (K-S:
P < 0.01). F: graph of the mean
frequency of sEPSCs in control (Pre) and MCPG-containing solutions
(MCPG) in 15 cells. All sEPSCs, all detectible events (see
METHODS). Large sEPSCs: events with peak amplitude >25 pA.
*: P < 0.05. ns: P > 0.05.
), followed by washout. 
, a single sEPSC.
B: normalized mean sEPSC amplitude for groups of neurons
exposed to mGluR antagonists. MCPG increased amplitude of sEPSCs
(n = 15, P < 0.01); AIDA
and (RS)-hexyl-HIBO (HIBO) decreased sEPSC amplitude
(n = 14, results of 9 cells in AIDA and 5 cells in
HIBO pooled, P < 0.001); LY341495 increased sEPSC
amplitude (n = 11, P < 0.01),
while CPPG had no effect (-1.4 ± 4.4%, n = 7, P > 0.05). For wash values, see text.
