1Department of Physiology and 2Department of Pharmacology and Therapeutics, Trinity College, Dublin 2, Ireland
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ABSTRACT |
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Kilbride, John, Anthony M. Rush, Michael J. Rowan, and Roger Anwyl. Presynaptic Group II mGluR Inhibition of Short-Term Depression in the Medial Perforant Path of the Dentate Gyrus In Vitro. J. Neurophysiol. 85: 2509-2515, 2001. Inhibition of short-term plasticity by activation of presynaptic group II metabotropic glutamate receptors (group II mGluR) was investigated in the medial perforant path of the dentate gyrus in the hippocampus in vitro. Brief trains of stimulation (10 stimuli at 1-200 Hz) evoked short-term depression of field excitatory postsynaptic potentials (EPSPs). The steady-state level of depression, measured after 10 stimuli, was frequency dependent, increasing between 1 and 200 Hz. Activation of group II mGluR by the selective agonist LY354740 did not alter short-term depression evoked by frequencies up to 10 Hz, but did inhibit short-term depression evoked at higher frequencies in a frequency- and concentration-dependent manner. The time-averaged postsynaptic response (EPSP per unit time) was found to increase linearly with frequency up to ~20 Hz. At higher frequencies, the response plateaued, thereby becoming independent of frequency. Frequencies above this were differentiated only during the transient postsynaptic response that accompanies changes in firing rates. Activation of presynaptically located group II mGluR increased the frequency at which the EPSP per unit time plateaued up to 30-50 Hz.
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INTRODUCTION |
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Short-term depression is an
activity-dependent reduction in synaptic efficacy that has been
observed at a wide variety of synapses in the CNS (reviewed by
Zucker 1989). This depression is widely believed to
result from a reduction in the amount of neurotransmitter released from
presynaptic terminals. Short-term depression in cortical synapses has
been shown to operate as an automatic gain control mechanism that
unlike inhibitory or adaptive mechanisms has the advantage of being
input specific (Abbott et al. 1997
; Tsodyks and
Markram 1997
). As such, afferents that are very active are
handled at a lower gain, while the overall strength of the synapse,
determined postsynaptically, remains at the same level.
Presynaptically located G-protein-coupled autoreceptors are widespread
in the CNS, and many studies have shown that activation of such
presynaptic receptors results in inhibition of transmitter release at
very low test frequencies at which no short-term plasticity occurs
(Anwyl 1999; Thompson et al. 1993
;
Wu and Saggau 1997
). However, there have been few
studies investigating the effect of presynaptic autoreceptors at higher
stimulation frequencies at which short-term plasticity is evoked,
although it has recently been shown that activation of presynaptic
GABAB, ACh, and adenosine receptors results in a
reduction of the extent of short-term depression evoked by
high-frequency stimulation (Brenowitz et al. 1998
;
Isaacson and Hille 1997
; Tsodyks and Markram
1997
; Varela et al. 1997
).
In the present study, the effects of activation of group II mGluR on
short-term plasticity was investigated at medial perforant path-granule cell synapses in the dentate gyrus that contain a high
density of presynaptically located group II mGluRs (Shigemoto et
al. 1997). Previous studies have shown that at low stimulation test frequencies, activation of group II mGluRs with selective agonists
such as LY354740 (Kilbride et al. 1998
) or
(2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV)
(Brown and Reyman 1994
; Huang et al.
1999
; Kilbride et al. 1998
; Macek et al.
1996
; Yokai et al. 1996
) results in a
concentration-dependent reversible depression of the excitatory
synaptic transmission in the medial perforant path.
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METHODS |
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Slice preparation
All experiments were carried out on hippocampal slices obtained
from male Wistar rats (50-70 g; BioResources Unit, Trinity College,
Dublin, Ireland). Slices were obtained as described previously (Kilbride et al. 1998). Briefly, the brain was rapidly
removed after decapitation and placed in cold (5°C) oxygenated (95%
O2-5% CO2) artificial
cerebrospinal fluid (ACSF) containing (in mM) 120 NaCl, 26 NaHCO3, 1.25 NaH2
PO4, 2.5 KCl, 2 Mg2SO4, 2 CaCl2, and 10 glucose. Hippocampal slices (350 µM) were cut using a Campden vibroslice (Campden Group Instruments,
London) and transferred immediately to an incubation chamber where they
were maintained at room temperature, for a period of at least 60 min.
Single slices were then transferred to a submersion type recording
chamber perfused with ACSF at 30-31°C.
Electrophysiology
Field excitatory postsynaptic potentials (EPSPs) were recorded
using standard glass electrodes filled with ACSF. Field EPSPs were
generated by a Master 8 eight-channel, programmable pulse generator
(A.M.P.I., Jerusalem, Israel) driven by pCLAMP 6 software (Axon
Instruments, Foster City, CA) on an IBM-compatible PC. Stimulation pulses (0.1 ms duration) were delivered via a bipolar insulated tungsten wire electrode, adjusted to give about 30% of the maximal response (~1 mV). The EPSPs were amplified by a Grass P16
microelectrode DC amplifier (Grass Instruments, Quincy, MA), converted
from A/D form (Axon Instruments, Digidata 1200) before being stored on a Dell dimension 466 PC for subsequent off-line data processing. Trains
of 10-20 stimuli were delivered to the medial perforant path. Where
stated, the frequency at which the stimulation was being delivered was
instantaneously increased after 10 stimuli, either by a fixed
percentage (50 or 100%) or by a fixed increment (5 Hz) of the
initial stimulation frequency (see Abbott et al. 1997).
Data analysis and statistics
Summarized results are expressed as EPSP mean amplitude ± SE. The amplitude of each EPSP in a train was expressed as a percentage of the first control EPSP, and the "normalized" EPSP (%) was calculated by normalizing every value in a particular experimental protocol to the first EPSP amplitude in that protocol. Data were analyzed using Student's paired t-test, and repeated measures ANOVA, at the 5% level of significance.
The rate of onset of depression in the medial perforant path was
calculated using Graphpad Prism software and verified on Jandel
Sigmaplot software. The summary data from seven experiments at a range
of frequencies between 0.5 and 200 Hz were fitted with a one-phase
exponential decay curve using the following equation: Y = Span * exp(K * x) + plateau. Starts at span
and decays to plateau with a rate constant K. The time
constant of decay (
) was calculated as 1/K.
The reduction in short-term depression by activation of group II mGluR was estimated as follows. Data points were constructed by expressing the steady-state EPSP amplitude in LY354740 as a percentage of the steady-state EPSP amplitude in control conditions, over a range of frequencies. The arrows in Fig. 3 show the threshold frequency of significant LY354740-induced attenuation of the short-term depression. This was established by comparing at each frequency, the steady-state EPSP amplitude in LY354740, as a percentage of control, with the test frequency (0.0166 Hz), using Student's t-test, at the 5% level of significance (mean ± SE, n = 4-11).
Compounds
LY354740 ((+)-2-aminobicyclo[3.1.0]hexane-2-6-dicarboxylic acid), which was a generous gift from Eli Lily (Indianapolis, IN), was dissolved in freshly made NaOH (0.1 N) and was added directly to the perfusate after establishing a steady baseline. This was accompanied by no observable change in the pH of the ACSF.
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RESULTS |
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Brief trains of stimuli at 1-200 Hz evoke a reversible depression of synaptic transmission
Field EPSPs remained at a stable amplitude when evoked at the test
frequency of 0.0166 Hz. However, trains of 10-20 stimuli delivered at
frequencies of 1-200 Hz resulted in a reversible depression of the
amplitude of EPSPs (Fig. 1A).
The depression occurred rapidly during the initial five stimuli and
then stabilized at a steady-state level during the subsequent five
stimuli. Trains were limited to 10-20 stimuli to minimize a long-term
form of synaptic depression (Dittman and Regehr 1998;
Galarreta and Hestrin 1998
). The extent of steady-state
depression increased as the frequency was increased between 1 and 200 Hz (Fig. 1B). For example the steady-state EPSP amplitude at
1, 50, and 200 Hz was 74.8 ± 2.9% (mean ± SE,
n = 5), 32.4 ± 2.3% (n = 9), and
8.4 ± 2.2% (n = 5) of the initial EPSP amplitude
respectively (ANOVA, P < 0.05). The rate of onset of
short-term depression also increased as the frequency was raised,
having time constants of decay of 813, 210, and 17 ms for 1, 10, and 50 Hz, respectively (n = 5-9). The rate of onset and
extent of short-term depression were found to be independent of
stimulation intensity as test EPSPs that varied between 0.4 and 2.2 mV
elicited almost identical results (data not shown).
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Short-term depression at frequencies above 10 Hz is inhibited by activation of group II mGluRs
LY354740 is a recently synthesized potent and specific agonist at
group II mGluRs (Monn et al. 1997; Schoepp et al.
1997
). Previous electrophysiological studies carried out in
this laboratory have shown that LY354740 has a potent presynaptic
reversible inhibitory action on EPSPs evoked at the test frequency in
the medial perforant path, with an IC50 of ~100
nM, and maximal inhibition of 80% at ~5 µM (Kilbride et al.
1998
). To investigate whether the activation of group II mGluRs
by LY354740 modulates short-term plasticity, brief trains of 10 stimuli
were applied at frequencies of 1-200 Hz after the inhibition of the
test EPSP by LY354740 had attained equilibrium (20 min perfusion). No
desensitization of the inhibition generated by LY354740 was observed
within the time period for which LY354740 was applied, usually up to 40 min.
At frequencies up to 10 Hz, short-term depression was unaffected by either an intermediate (100 nM) or a high (5 µM) dose of LY354740. For example, at 1 Hz, when the data were normalized to the first EPSP in each condition, no significant difference in the normalized steady-state EPSP was observed in the presence of a low or high concentrations of LY354740. Thus the normalized steady-state EPSP amplitude was 74.8 ± 2.9% (n = 4), 72.3 ± 2.0% (n = 4), and 71.3 ± 5.2% (n = 4) in control, 100 nM, and 5 µM LY354740, respectively; values were not significantly different (Fig. 2A).
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At frequencies >10 Hz, short-term depression was inhibited by the activation of group II mGluRs with LY354740, and strong facilitation was often observed. For example, at 50 Hz, the short-term depression that occurred in control was strongly reduced in both the intermediate (100 nM) and high (5 µM) dose of LY354740, and facilitation occurred in the high dose of LY354740 (Fig. 2B). The normalized steady-state depressed amplitude of the EPSP was 32.4 ± 2.3% (n = 9), 51.1 ± 1.8% (n = 7), and 91.6 ± 4.6% (n = 10) in control, 100 nM, and 5 µM LY354740, respectively (ANOVA, P < 0.05).
The concentration and frequency dependency of the group II mGluR-induced inhibition of short-term depression is shown in Fig. 3A, which displays the amplitude of the steady-state EPSP in LY354740 (100 nM and 5 µM) as a percentage of the steady-state control EPSP between 0.0166 and 200 Hz. Above a certain threshold frequency, the amplitude of the steady-state EPSP amplitude in LY354740, relative to control, was increased as the stimulation frequency became higher, demonstrating inhibition of short-term depression. The threshold frequency at which LY354740 reduced short-term depression was 50 Hz for 100 nM LY354740 and 20 Hz for 5 µM LY354740. Interestingly at 100 Hz, the attenuation of short-term depression was so pronounced that the steady-state EPSP amplitude in LY354740 was actually larger than in control conditions.
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Time-averaged postsynaptic response becomes independent of frequency for input rates greater than ~20 Hz
An estimate of the amount of transmitter released per unit time
can be established by plotting the steady-state EPSP amplitude times
frequency as a function of stimulation frequency (Abbott et al.
1997; Curtis and Eccles 1960
; Richards
1972
; Tsodyks and Markram 1997
). In these
studies, the mobilization of transmitter became rate limiting above a
certain limiting frequency. A rate limitation of the mobilization of
transmitter was also found to occur in the present study. Thus the EPSP
amplitude per unit time was approximately linearly related to frequency
up to ~20 Hz but approached a constant value at higher frequencies
(Fig. 3B). The modulation of short-term depression by
activation of group II mGluRs is further revealed in the curves of the
normalized EPSP per unit time to frequency, plotted in the presence and
absence of LY354740 (Fig. 3B). LY354740 shifted the curve
upward from control in a concentration-dependent manner due to a shift
to higher frequency of the point at which the normalized EPSP per unit
time became independent of frequency (Fig. 3B). Thus in
LY354740 (5 µM), the limiting frequency was shifted upward to ~50 Hz.
Short-term depression at frequencies above 10 Hz is reduced by lowering extracellular Ca2+
Previous studies have suggested that the inhibitory action of presynaptic mGluRs on EPSPs elicited at low frequencies is a result of a reduced influx Ca2+ into the presynaptic terminals. To investigate whether the presynaptic action of LY354740 may be due to a reduction of Ca2+ influx into the presynaptic terminal, the effect of low extracellular concentrations of Ca2+ were investigated on short-term depression.
At the test frequency of 0.0166 Hz, the steady-state EPSP amplitude was reduced as the level of extracellular Ca2+ was lowered. In 1.6, 1.2, and 0.8 mM Ca2+ the test (0.0166 Hz) EPSP amplitude was measured as being 63.9 ± 16.8% (n = 3), 49.8 ± 10.3% (n = 4), and 29.6 ± 8.6% (n = 5) of control (2 mM; ANOVA, P < 0.05). Trains of stimuli at low frequency, up to ~10 Hz, were not inhibited by the low Ca2+ media. For example, at 1 Hz the curves relating the short-term depression in 2 mM (control), 1.6, 1.2, and 0.8 mM Ca2+ overlap, and moreover, the normalized steady-state EPSP amplitudes were not significantly different: 70.6 ± 2.4%, (n = 8), 76.6 ± 2.4%, (n = 3), 77.9 ± 3.0% (n = 6), and 69.9 ± 18.1% (n = 4), respectively (Fig. 4A). However, high-frequency stimulation in low extracellular Ca2+ resulted in a reduction of the short-term depression. For example at 50 Hz, the reduction in the level of extracellular Ca2+ was accompanied by a slowing down of the rate of onset of depression and a reduction in the extent of short-term depression. The normalized steady-state EPSP amplitudes were 33.0 ± 2.0% (n = 11), 43.8 ± 2.3% (n = 3), 56.1 ± 4.8% (n = 7), and 126.2 ± 32.1% (n = 4) in 2.0, 1.6, 1.2, and 0.8 mM Ca2+ (P < 0.05, Fig. 4B). The curves relating the normalized EPSP per unit time to frequency were shifted upward as the limiting frequency was extended from 10 to 20 Hz in control to over 50 Hz in 0.8 mM Ca2+ (Fig. 4C).
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Effect of an instantaneous increase in the stimulation frequency in control and following activation of group II mGluR
In the absence of short-term depression, the response to fixed
percentage frequency increments would increase linearly as a function
of the initial input frequency. Short-term depression at excitatory
synapses in the cortex has previously been shown to exert a type of
synaptic gain control, equalizing the response to fixed percentage
frequency changes at higher frequencies while amplifying the response
to lower frequencies (Abbott et al. 1997). The effects
of instantaneous frequency changes were investigated in the present
study on the medial perforant path to dentate granule cell synapse, and
modulation of the effects following the activation of group II mGluR
also studied. The frequency was increased instantaneously by a fixed
percentage (50 or 100%) after an initial 10 stimuli at frequencies
between 0.5 and 50 Hz, and a further 10 stimuli delivered at the new
rate. Measurements were made of the effects of the instantaneous
increase in frequency on both the EPSP and EPSP per unit time.
An instantaneous fixed percentage increase of 100% in the frequency of stimulation following the initial 10 stimuli at frequencies of 0.5-50 Hz resulted in further depression of the EPSP amplitude to a new steady-state amplitude, reached after a further 5-10 stimuli, with the extent of the additional depression being dependent on the frequency (Fig. 5A). In contrast to this further depression of the EPSP amplitude on an instantaneous rise in frequency, the EPSP per unit time was actually initially increased by the instantaneous frequency rise. The increase in the EPSP per unit time in response to fixed percentage increases was approximately proportional to the magnitude of the frequency change for frequencies over ~20 Hz, i.e., the time-averaged postsynaptic response to a 100% change in firing rate was almost twice that of a 50% change (Fig. 6A). Furthermore the absolute increase in the time-averaged postsynaptic response to a 100% increase in the afferent firing rate did not differ significantly at frequencies above 10 Hz (Fig. 6A).
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Activation of group II mGluRs was found to reduce the responsiveness to instantaneous frequency changes of 100% at frequencies lower than 25 Hz, but not at higher frequencies. Thus a significant reduction in the absolute increase in the EPSP per unit time from control occurred in the presence of LY354740 with instantaneous increases from 0.05 to 1 Hz, 5 to 10 Hz, 10 to 20 Hz, and 25 to 50 Hz, but not 40 to 80 Hz or 50 to 100 Hz (Figs. 5B and 6B).
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DISCUSSION |
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Short-term depression and frequency of stimulation
The results of this study have shown that short-term plasticity in the medial perforant path of the dentate gyrus is a context-dependent and dynamic phenomenon. Under normal physiological conditions, the predominant response to an increased frequency of stimulation is short-term depression, whereas when the probability of release is lowered, for example, during the activation of presynaptic group II mGluRs, facilitation becomes more prominent, especially at higher frequencies. Both the rate of onset and extent of short-term depression were found to increase with stimulation frequency.
The effects of short-term depression on the limiting frequency in the
medial perforant path of the dentate gyrus are in broad agreement with
similar studies in the cortex (Abbott et al. 1997; Markram et al. 1998
; Tsodyks and Markram
1997
; Varela et al. 1997
). Thus in the present
study, the limiting frequency for transmitter mobilization was ~20
Hz, similar to that found in the visual cortex (10 Hz) (Abbott
et al. 1997
) and somatosensory cortex (10-25 Hz) (Tsodyks and Markram 1997
), although it should be noted
that previous studies made use of a high Ca2+ to
Mg2+ ratio, which has the effect of increasing
the rate of short-term depression and consequently lowering the
limiting frequency. The limiting frequency of ~20 Hz found in the
present study represents the upper ceiling to rate coding. At lower
frequencies the time-averaged EPSP increases with frequency and
therefore a neuron can decipher the information encoded by the rate of
presynaptic input. Above the limiting frequency, the time-averaged EPSP
is independent of the presynaptic firing rate, and therefore rate
coding is limited.
Inhibition of short-term depression by activation of presynaptic group II mGluRs
The results of the present study show that activation of
presynaptic group II mGluRs results in a concentration- and
frequency-dependent inhibition of the observed short-term depression. A
similar phenomenon has been observed following activation of other
presynaptic G-protein-coupled receptors, e.g., muscarinic ACh,
GABAB, and adenosine A1
(Barnes-Davies and Forsythe 1995; Brenowitz et
al. 1998
; Isaacson and Hille 1997
; Lev-Tov and Pinco 1992
; Pennartz and Lopes da
Silva 1994
; Shen and Horn 1996
; Tsodyks
and Markram 1997
; Varela et al. 1997
). In this
study, the inhibition of short-term depression by activation of group
II mGluRs only became significant above 50 and 20 Hz for concentrations
of 100 nM and 5 µM LY354740, respectively. These results reaffirm
that the effects of activation of autoreceptors are context dependent
and dynamic. For example, although an autoreceptor may be inhibitory
during low-frequency stimulation, facilitatory processes are unmasked
and become increasingly significant during high-frequency stimulation.
Activation of group II mGluR was found to extend the linear range of the curves relating the EPSP per unit time to frequency, which is consistent with its action on short-term depression. Thus the limiting was extended from ~20 to ~50 Hz. The response to temporal changes in input rate was also consistent with a shift toward a synapse displaying less synaptic depression. Under control conditions, small fluctuations in slowly firing afferents are handled at a higher gain; however, group II mGluR activation equalizes the postsynaptic response to small fluctuations on slow and rapidly firing afferents. In contrast there is less equality in the response to fixed percentage changes in input rates above ~20 Hz, and consequently, the curves relating the absolute increase in the EPSP per unit time to frequency, for fixed percentage rate changes in control and LY354740, converge at ~50 Hz. In effect this means that the ability of the postsynaptic cell to respond to fractional changes in rate equally at mid- and high-frequency input rates is diminished by group II mGluR activation.
Lowering extracellular Ca2+ had a very similar
action to that of activation of group II mGluR, with short-term
depression being relieved at relatively high, but not low, frequencies,
and the limiting frequency being shifted to a higher value as
extracellular Ca2+ was lowered. It is therefore
likely that inhibition of Ca2+ influx underlies
the mechanisms of activation of group II mGluR. The exact mechanism may
involve an inhibition of Ca2+ channels
(Takahashi et al. 1996; Wu and Saggau
1995
), a facilitation of K+ channels
(Sladeczek et al. 1993
), or the action may be at a site downstream from the site of Ca2+ entry
(Hille 1994
).
In conclusion, the results of the present studies in the medial
perforant path of the dentate gyrus demonstrate that short-term depression acts as an automatic gain control system, amplifying small
fluctuations on slowly firing afferents (Abbott et al.
1997). At low firing rates, when depression is mild, temporal
integration predominates, while at higher firing rates at which
depression becomes more prominent, temporal coherence becomes more
important, possibly acting as a complement to the limited rate coding
mechanism (Tsodyks and Markram 1997
). Activation of
group II mGluRs results in a reduction of short-term depression and
reduces the temporal changes on slowly firing afferents in preference
to more rapidly firing ones.
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ACKNOWLEDGMENTS |
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This research was supported by the Wellcome Trust and by Enterprise Ireland.
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FOOTNOTES |
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Address for reprint requests: R. Anwyl (E-mail: ranwyl{at}mail.tcd.ie).
* J. Kilbride and A. M. Rush contributed equally to this work.
Received 4 August 2000; accepted in final form 13 March 2001.
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REFERENCES |
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