1Faculty of Pharmacy, Kuwait University, Safat 13110, Kuwait; 2Neuroscience Research Group and Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1; and 3CHUL Research Centre, Laval University, Sainte-Foy, Quebec G1V 4G2, Canada
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ABSTRACT |
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Kombian, Samuel B.,
Michiru Hirasawa,
Didier Mouginot,
Xihua Chen, and
Quentin J. Pittman.
Short-Term Potentiation of Miniature Excitatory Synaptic Currents
Causes Excitation of Supraoptic Neurons.
J. Neurophysiol. 83: 2542-2553, 2000.
Magnocellular neurons
(MCNs) of the hypothalamic supraoptic nucleus (SON) secrete vasopressin
and oxytocin. With the use of whole-cell and nystatin-perforated patch
recordings of MCNs in current- and voltage-clamp modes, we show that
high-frequency stimulation (HFS, 10-200 Hz) of excitatory afferents
induces increases in the frequency and amplitude of
2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide (NBQX)-sensitive miniature excitatory postsynaptic currents (mEPSCs) lasting up to 20 min. This synaptic enhancement, referred to as short-term potentiation (STP), could be induced repeatedly; required tetrodotoxin (TTX)-dependent action potentials to initiate, but not to
maintain; and was independent of postsynaptic membrane potential,
N-methyl-D-aspartate (NMDA) receptors, or
retrograde neurohypophyseal neuropeptide release. STP was not
accompanied by changes in the conductance of the MCNs or in the
responsiveness of the postsynaptic non-NMDA receptors, as revealed by
brief application of -amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) and kainate. mEPSCs showed similar rise times before and
after HFS and analysis of amplitude distributions of mEPSCs revealed one or more peaks pre-HFS and the appearance of additional peaks post-HFS, which were equidistant from the first peak. STP of mEPSCs was
not associated with enhanced evoked responses, but was associated with
an NBQX-sensitive increase in spontaneous activity of MCNs. Thus we
have identified a particularly long-lasting potentiation of excitatory
synapses in the SON, which has a presynaptic locus, is dissociated from
changes in evoked release, and which regulates postsynaptic cell excitability.
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INTRODUCTION |
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Magnocellular neurons (MCNs) of the supraoptic
nucleus (SON) synthesize the peptides vasopressin (AVP) and oxytocin
(OXT) that are released into the peripheral circulation, where they regulate important functions such as salt-water balance, blood pressure, parturition, and milk ejection. In response to increased demand for their products, MCNs not only display increased frequency of
firing, but also switch to a bursting or phasic activity pattern that
facilitates release (Armstrong 1995; Bourque and
Renaud 1990
). Both intrinsic conductances and synaptic
mechanisms have been proposed to underlie this change in activity
pattern. Although it has been shown that both inhibitory and excitatory
synaptic inputs onto MCNs can modulate their excitability (Hu
and Bourque 1991
; Kabashima et al. 1997
;
Kombian et al. 1996
; Mouginot et al.
1998
; Wuarin and Dudek 1993
), it is not clear
what role these afferent inputs play in the switching and shaping of
activity patterns that optimize peptide release. The majority of the
afferents to the MCNs have been shown to be GABAergic and glutamatergic in nature. Manipulation of the strength of these inputs can markedly influence the output of the SON; in particular, the unique phasic activity patterns displayed by the AVP-containing neurons of
the SON are dependent on the glutamatergic inputs to the nucleus
(Nissen et al. 1995
). Similarly, the ability of the
OXT-containing cells to respond appropriately to the peripheral
inputs associated with the milk ejection reflex requires afferent inputs.
In a hypothalamic slice containing the SON, it is possible to record
both action potential-dependent and -independent events (miniature
excitatory or inhibitory postsynaptic currents; mEPSCs or mIPSCs).
Electrophysiological studies of these miniature events have provided
information concerning the mode of action of various presynaptic
transmitters and pharmacological compounds on MCN activity. However,
the possible role of miniature events in the synaptic function of the
SON or indeed in any central neuron is poorly understood (Staley
1999). In the present study, we have monitored miniature
excitatory postsynaptic potentials/currents (mEPSPs/mEPSCs) and action
potential firing in MCNs of the SON, and have observed an
activity-dependent change in the properties of excitatory afferent
terminals that may trigger and help maintain firing patterns that
optimize peptide release from MCNs.
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METHODS |
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Coronal (400 µm) hypothalamic slices from adult male
Sprague-Dawley rats were perfused (submerged, 27-29°C) with
artificial cerebrospinal fluid (ACSF) as previously described
(Kombian et al. 1997). Recordings were done by the use
of both whole-cell recording (WCR) and the nystatin-perforated patch
technique by the use of electrodes with tip resistance of 4-10 M
and a series/access resistance of 10-40 M
. The internal recording
solution for WCR contained (in mM) K-gluconate (130); KCl (10); NaCl
(10); MgCl2 (1);
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES, 10), ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA, 10),
Mg-ATP (2), and (GTP, 0.3), whereas the nystatin patch solution
contained (in mM): K-Acetate (120), MgCl2 (5), EGTA (10), and HEPES (40). Nystatin was dissolved in dimethyl sulfoxide
(DMSO) with Pluronic F127 and added to the internal solution to yield a
final concentration of 450 µg/ml. The pH of both solutions was
adjusted to between 7.2 and 7.4. Tungsten-stimulating electrodes were
placed in the hypothalamic region dorsal-medial to the SON to evoke
synaptic responses and to apply the high-frequency stimulation (HFS; 10 to 200 Hz for 1 s, applied twice in a 10-s interval). Unless
indicated otherwise, all illustrated data used the 100-Hz frequency.
"Blind" patch recordings were made in the SON by the use of an
Axopatch 1D amplifier. MCNs were identified on the basis of the delayed
onset to action potential generation in response to depolarizing
current injection, as originally reported for paraventricular neurons
(Tasker and Dudek 1991), and now thought to be
characteristic also of SON neurons (Armstrong 1995
);
however, no attempts were made to assign a phenotype to the neurons on
the basis of their current-voltage responses (see Stern and
Armstrong 1995
). Most experiments were done on cells
voltage-clamped at
80 mV. Input resistance
(Rinput) and access resistance
(Ra) of all cells were monitored regularly
throughout each experiment by applying a 20-mV hyperpolarizing pulse
for 75-100 ms. Rinput was calculated
from the steady-state current obtained during the pulse. The decay rate
(
) of the capacitance transient was taken as a measure of Ra. Data from cells that showed >15%
(estimated inherent variability in evoked responses) change in these
parameters were excluded from further analysis. Pharmacologically
isolated spontaneous and evoked EPSCs (sEPSCs and eEPSCs) were recorded
in the presence of 50 µM picrotoxin. All cells had a graded evoked
synaptic response to increasing stimulation intensity; and an intensity
giving 50-60% of the maximum evoked EPSC was used to elicit HFS or
evoked responses. All data were acquired with the use of pClamp
Software (Clampex 5.5 and 7; Axon Instruments). sEPSCs were acquired at
a 2- to 5-kHz sampling rate, filtered at 500-1,000 Hz, digitized at 10 kHz, and stored for off-line analysis. Hard-copy chart records were
also captured on a Gould Recorder.
Frequency-time plots were generated by taking the mean number of events in 16-s epochs. All values are stated as means ± SE. One-way ANOVA and post hoc tests, as indicated, were used to compare different curves and points. P < 0.05 was taken as significant.
For experiments that involved changing holding potential, control
frequencies were taken at 80 mV. Cells were then held at the
appropriate test potential for about 1 min. HFS was applied at the test
potential and the cell membrane returned to
80 mV and data collection
resumed. Steady-state current-voltage relationships (I-V
curves) were generated by applying slow-voltage ramps from
120 to
40 mV at a rate of 4.5 mV/s. The resulting steady-state current was
then captured and stored for off-line analysis.
sEPSCs were detected and counted visually (in Clampfit) and by a
commercially available Mini Analysis software (Synaptosoft, Inc.) and
counted if amplitude 3 pA with fast rise times (Tr; 1-4 ms measured from baseline to peak) and exponential decay. For the
measurement of amplitude, Tr, and decay constant (
), only
events with a clearly defined baseline (>5 ms) and that did not have
shoulders on the rising and falling phases, on visual inspection on an
expanded scale, were used. Cumulative probability plots, frequency
distribution histogram, graphs, and statistical analysis were done with
the use of Mini Analysis, Sigmaplot, and GraphPad software.
Amplitude-distribution histograms of sEPSCs were fitted with either one
or the sum of several Gaussian curves, by Simplex nonlinear
least-squares algorithm (Graphpad Prism 3.0). The number of Guassian
curves fitted to each distribution was determined by eye. For
multimodal distribution, mean modal separation q was
calculated from the peak value of each Gaussian curve according to
q =
(xk/k)/n,
where xk refers to the mean value of each
Gaussian function numbered k = 1, ... ,
n, and n is the total number of curves fitted
(Edwards et al. 1990
). The quantal coefficient of
variation (c.v.) of sEPSC amplitude was calculated as (variance of
amplitudes in mode 1
noise
variance)1/2/q × 100, assuming
that noise variance stays constant throughout peaks, independent of the
quantal variance and added linearly (Edwards et al.
1990
). The noise variance was calculated as the standard
deviation of current recorded in between sEPSCs, for a total of more
than 200 ms per sample.
The firing rate of the cells was monitored under current clamp at the
resting membrane potentials. Those cells that had no spontaneous
activity were injected with constant positive currents (50 pA) to
obtain baseline activity.
All drugs were bath-perfused at final concentrations indicated by
dissolving aliquots of stock in the ACSF. All drugs and chemicals were
from Sigma, except for
2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo(f)quinoxaline-7-sulfonamide (NBQX) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; RBI), Pluronic F127 (BASF), and the oxytocin receptor antagonist ([des-glycinamide9,d(CH2)5,
O-Me-Tyr2, Thr4,
Orn8]-vasotocin) and Manning Compound (Bachem).
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RESULTS |
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In this study, 136 MCNs were recorded by the use of either
nystatin patch or whole-cell recording (WCR) modes. Recordings made by
the use of either technique gave similar access and input resistance
(Kombian et al. 1996); however, in our hands, MCNs recorded in WCR mode were less stable, did not last long enough (<30
min from break-in) to perform prolonged experiments, and the evoked
EPSCs routinely ran down. To avoid the possibility of washout of
essential cellular substances [shown to be important in many
plasticity studies (Malenka and Nicoll 1993
)], the
majority (132 cells) of experiments in this study were done with the
use of the nystatin patch-recording technique (Kombian et al.
1996
,1997
; Mouginot et al. 1998
).
Picrotoxin-resistant and NBQX-sensitive sEPSCs or sEPSPs were routinely
recorded in MCNs as has been reported (Boudaba et al.
1997; Gribkoff and Dudek 1990
; Kabashima
et al. 1997
; Schrader and Tasker 1997
). The
recorded sEPSCs had amplitudes, frequencies, and kinetics (decay time)
similar to those reported (Kabashima et al. 1997
).
Because the recorded sEPSCs could arise either from action potentials
in local interneurons or from action potential-independent events in
the presynaptic terminal, we determined the nature of the sEPSCs by the
use of tetrodotoxin (TTX, 1 µM). In six cells tested, TTX completely
abolished the evoked EPSCs, but the frequency of the sEPSCs was not
altered by TTX (1.95 ± 0.66 Hz in control vs. 2.38 ± 0.99 Hz in TTX, n = 6; P > 0.05; paired
Student's t-test). Thus sEPSCs recorded in MCNs represent
mEPSCs, as has been reported before (Kabashima et al.
1997
).
After high-frequency stimulation (HFS; 100 Hz) of afferents to the SON, 94% of MCNs tested responded with a marked increase in the frequency of mEPSCs (Fig. 1, A1, A2, and B). These mEPSCs remained sensitive to NBQX after HFS. Similar responses were obtained when MCNs were recorded by the use of WCR (n = 4). Analysis of the increase in mEPSC frequency after HFS showed that all cells responded with an initially large increase in events (3,141 ± 615% of control after two 100-Hz stimuli, n = 15) that then declined exponentially back to near baseline frequency in 5-20 min (Fig. 1C). The magnitude and duration of the potentiation were not correlated to the basal mEPSC frequency (r = 0.17 and 0.4, respectively; P > 0.05; n = 18; Spearman rank-order correlation). To test the ability of these cells to consistently respond to repeated HFS, we determined whether this short-term potentiation (STP) in mEPSCs could be induced repeatedly. Repeated HFS (two times at 100 Hz, 1 s) consistently induced robust STP with no significant difference between the peak response and the time course of the two STPs (Fig. 1D, n = 7, P > 0.05, Kruskal-Wallis one-way ANOVA). In some cells, similar response patterns could be evoked with up to five repetitions. As the previous experiments were carried out with picrotoxin in the bath, it was possible that STP occurred only in situations of blocked GABAergic transmission. Therefore in five neurons, spontaneous events were recorded without picrotoxin in the bath and we observed that HFS induced increases in the frequency of these currents similar to those previously observed after HFS with GABAergic transmission blocked (552.2 ± 263% without picrotoxin and 475 ± 114% in picrotoxin; P > 0.05; data from 2 min post-HFS). Thus excitatory inputs onto MCNs are capable of undergoing STP, even in the presence of opposing inhibitory transmission.
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Because potentiation of mEPSCs after HFS of this magnitude and duration has not previously been reported in central neurons, we asked whether it could be seen at more physiological frequencies. As seen in Fig. 2, HFS at frequencies as low as 10 Hz was capable of eliciting a short-lasting STP, which increased in magnitude and duration as HFS frequency increased. Above 100 Hz, STP magnitude and duration began to decrease. We then carried out further studies to identify the nature of the potentiation. As would be expected, HFS in the presence of TTX did not produce any STP (Fig. 3A, n = 6, P < 0.05 vs. control; Kruskal-Wallis one-way ANOVA), indicating that depolarization of afferent axons is necessary to generate STP of mEPSCs. Because action potentials are required to produce STP, it is possible that the observed enhanced synaptic activity arose from both mEPSCs and sEPSCs because of invasion of the terminals by action potentials arising from interneurons that may participate in afferent excitation onto SON MCNs. To investigate this possibility, TTX was applied immediately after HFS and both the evoked and mEPSCs were monitored. Within 2 min of 1 µM TTX application, the evoked EPSC was completely abolished, whereas the mEPSC frequency remained enhanced and followed a similar time course as in control experiments (Fig. 3B, n = 5, P > 0.05, Kruskal-Wallis one-way ANOVA). These TTX experiments indicate that sodium-dependent action potentials are not required for maintenance of STP and point to the synapse as the site for the effect. Furthermore, they indicate that the induction and maintenance of STP are two separate processes that, although causally related, may be independently regulated.
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Our experiments would appear to have established the synapse as the
locus for these changes. The next question we addressed was to
determine whether it is the pre- or postsynaptic side of the activated
synapses that is critical for the initiation of STP? We used
several approaches to examine the contribution of MCNs to the
generation of STP. First, we tested for the participation of
postsynaptic N-methyl-D-aspartate (NMDA)
receptors in STP, because these receptors are known to be involved in
several forms of synaptic plasticity in other brain regions
(Bliss and Collingridge 1993; Malenka and Nicoll
1993
). Furthermore, they are voltage dependent and any
significant postsynaptic depolarization would be expected to activate
them, thus recruiting them to modify synaptic strength. Application of
the NMDA receptor antagonist D-2-amino-5-phosphonovaleric acid (D-APV; 50 µM) affected neither the evoked EPSC
amplitude (data not shown) nor control mEPSC frequency (Fig.
4A; 1.5 ± 0.75 Hz in control
vs. 1.4 ± 0.75 Hz in D-APV; n = 4;
P > 0.05). Furthermore, it affected neither the
induction nor the time course of STP of the mEPSC frequency (Fig.
4A, n = 4, P > 0.05, Kruskal-Wallis one-way ANOVA). This finding shows that the induction
and maintenance of STP do not involve NMDA receptors that are present
in MCNs (Hu and Bourque 1991
). During HFS, the
postsynaptic MCNs and presumably the presynaptic terminals are all
depolarized, but it is not known whether depolarization at one or both
loci is required to induce STP. To determine whether the membrane
potential of the postsynaptic MCN influences the ability to induce STP,
we did experiments in which MCNs were held at different holding
potentials during HFS. However, holding the postsynaptic cell at
potentials either depolarized or hyperpolarized to the regular holding
potential of
80 mV did not affect the induction and time course of
the STP (Fig. 4B; n = 4-6,
P > 0.05, Kruskal-Wallis one-way ANOVA).
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Next, we asked whether a brief depolarization of the postsynaptic cell alone is sufficient to induce STP. Therefore, in 16 MCNs, we briefly exited from voltage clamp to inject two or three depolarizing currents, by the use of the same duration and interval as the HFS, to initiate action potentials. This treatment did not affect the frequency of mEPSCs recorded when we immediately returned to voltage clamp (Fig. 4C); nonetheless, the synapses onto these neurons were amenable to STP as subsequent HFS applied to five of these cells induced robust STP. The results from these postsynaptic manipulations strongly suggest that it is the presynaptic terminal and not the MCN that is the site of induction of STP.
In addition to the marked increase in the frequency of mEPSCs, HFS
appears to produce an increase in the mean mEPSC amplitude and this was
substantiated by analysis (11.3 ± 1.23 pA pre-HFS and 14.6 ± 0.95 pA post-HFS, P < 0.05, n = 9;
see Fig. 1A). Because this effect is often associated with a
postsynaptic change, we performed a series of experiments and post hoc
analysis of mEPSCs to determine whether postsynaptic cell properties
were altered during STP. First of all, we asked whether there was a
post-HFS change in the conductance of the recorded MCN in a voltage
range that may contribute to the changes in mEPSCs, but that may not have been detected by the previous postsynaptic manipulations. In five
MCNs, we applied slow-voltage ramps before and 2 min post-HFS and the
steady-state currents were recorded. The two I-V curves were
superimposable over the voltage range 100 to
40 mV, indicating that
there was no conductance change in the physiologically relevant voltage
range after HFS (Fig. 5A).
Furthermore, neither the input resistance
Rinput, measured around resting
membrane potential (766 ± 65 M
before, 736 ± 56 M
after, n = 7, P > 0.05 by paired t-test), nor the access resistance
Ra, both monitored regularly throughout
each experiment, changed after the HFS (n = 5; Fig. 5B).
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Next, we asked whether the properties of the postsynaptic non-NMDA receptors underlying the mEPSCs changed after STP. Repeated brief (5-15 s) bath applications of AMPA (5 µM) or kainate (5 µM) at intervals of 2 to 5 min produced consistent steady-state currents (3.8 ± 2.7% between application variation, n = 7; P > 0.3 for AMPA and 11.1 ± 8.0, n = 6; P > 0.4 for kainate; Fig. 5C). The AMPA-induced inward currents before and 30 s to 2 min post-HFS were identical (15 ± 10.2% change; n = 5, P > 0.05, paired t-test; Fig. 5C). Even though we detected an increase in the amplitude of the mEPSCs, the lack of a detectable increase in postsynaptic steady-state AMPA response suggests that HFS does not affect steady-state properties of postsynaptic AMPA receptors.
Despite this lack of available evidence in support of postsynaptic
changes in AMPA receptors on MCNs during STP, others have reported
possible activation of previously silent kainate receptors after HFS of
hippocampal mossy fibers (Castillo et al. 1997). We
could not discount this possibility, because kainate will activate postsynaptic receptors on magnocellular neurons (Hu and Bourque 1991
) and the blockade of mEPSCs by NBQX after HFS would not
differentiate between AMPA and kainate receptors (Wilding and
Huettner 1996
). However, application of kainate before and
after HFS resulted in similar currents (
4.0 ± 11.4% change;
n = 4, P > 0.05), indicating that
postsynaptic kainate receptors are unlikely to be responsible for the
appearance of the new and big events.
Given the similarity of both electrical and pharmacological properties of the MCNs before and after HFS, we believe that the most likely reason for the increase in large amplitude events is the increased probability of multiquantal mEPSCs; thus we next performed an analysis of the amplitude distribution of mEPSCs before and after HFS in a typical cell (Fig. 6A). As described in METHODS, only those that could be distinguished as individual events were used for this amplitude analysis. When the amplitude distribution histograms of the events before and after HFS were plotted, the latter showed a much greater skewing to the right in all seven cells tested. In control condition, mEPSC amplitude distribution was best fitted by one to three Gaussian curves with mean mode separation q of 8.17 ± 0.66 pA, c.v. = 16.1 ± 3.0% (n = 7). One to 2 min post-HFS, three to four Gaussian curves could be best fitted to mEPSC amplitude distribution. Mean mode separation q was 7.82 ± 0.71 pA (P = 0.14 vs. control), c.v. = 13.6 ± 1.0% (P = 0.45 vs. control). Mean standard deviation of q, calculated from each modal peak (xk/k) in these cells, was 6.51 ± 2.29% of q in control and 6.79 ± 0.93% after HFS (P = 0.78).
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Relatively low variance of the mean mode separation supports the equidistance of the modes, and suggests that mode 1 represents the quantal unit and the other modes are made up of summed quanta, released synchronously or near synchronously. The mean mode separation q (i.e., quantal unit) was the same before and after HFS, indicating the STP of mEPSCs did not result from an increase in the amplitude of small, previously undetected events because of changes in postsynaptic cell properties, or preferential recruitment of large vesicles after HFS. The putative multiquantal events used in the preceding analysis had a smooth rising phase, and thus from their waveform no indication of summation of small events could be ascertained (Fig. 6B, top trace). However, in some other putative multiquantal events, shoulders could be observed on their rising phase (Fig. 6B, middle and bottom traces), supporting the idea that the big events are indeed composed of smaller units. Thus the evidence from the preceding analysis suggests that the appearance of big events is the result of multiquantal release of transmitter.
The possibility remains that the spatial distribution of synapses
activated after HFS is different. For example, under basal conditions,
distally located synapses may predominate, whereas after HFS, the
synapses closer to the recording site in the cell body could be
preferentially activated, thereby giving bigger mEPSCs. We tested for
this possibility by examining the relationship among the amplitude,
rise time, and decay constant of mEPSCs before and after HFS. We
considered that mEPSCs arising from locations closer to the soma would
have a larger amplitude and faster rise and decay times than more
distant mEPSCs because of less dendritic filtering (Bekkers and
Stevens 1996). We calculated the rise time and decay constants
in a typical cell that showed a clear shift in both frequency and
amplitude distributions. As shown in Table 1 and Fig. 6C, despite the de
novo appearance of a population of large events after HFS, the rise
times and decay constants of both large- and small-amplitude events
after HFS were identical to those of the pre-HFS population. It is
therefore unlikely that big events, by comparison with small ones,
arose from different locations. Also, the lack of a significant
difference in
between small and big events would indicate that the
desensitization rate of non-NMDA receptors had not changed
(Isaacson and Nicoll 1991
; Trussell et al.
1993
).
|
HFS, such as that used in this study, is routinely used in plasticity
studies to induce long-lasting changes of evoked synaptic responses. In
this study, because HFS induces increases in mEPSC frequency and
amplitude, it is important to determine whether there is also a
parallel change in the evoked EPSC. To carry out these experiments, we
perfused the slice with a vasopressin/oxytocin antagonist, Manning
compound (10 µM), to block the actions of dendritically released
peptides that could influence synaptic inputs (Kombian et al.
1997). When HFS was applied, robust STP of mEPSC could be
induced in all eight cells tested (Fig.
7A). However, analysis of the
simultaneously collected evoked EPSC amplitude revealed that there was
no significant change in the evoked EPSC amplitude (71.5 ± 21.3%
of control; P > 0.05; Fig. 7, B and
C; value from 2 min post-HFS). This result indicates that
HFS has two different effects on evoked EPSC and mEPSCs.
|
Finally, to investigate a functional significance of this phenomenon, we asked whether there was a change in the excitability of MCNs after the induction of STP. We therefore measured the action potential firing rate, under current clamp, as an index of MCN excitability. In four spontaneously firing cells, HFS caused an increase in the firing rate (1,200 ± 214%; Fig. 8, A and B). This increase in frequency followed a time course that was similar to that of STP of the mEPSCs (Fig. 8C). Similar increases in frequency (1,036 ± 411% of control; n = 12; P < 0.001) were observed for cells that were held near spike threshold by constant positive current injection. In all cells included in this analysis, we initially or subsequent to action-potential measurements (current clamp), voltage-clamped the cells and showed that they exhibited STP of mEPSCs after HFS (n = 16; Fig. 8, A and C).
|
Because HFS causes both STP of mEPSCs and increases in spike frequency,
we asked whether these two events were causally related. Because
oxytocin is known to have postsynaptic excitatory effects within this
nucleus resulting from dendritic release (Ludwig 1998), it is possible that the increased activity of MCNs was subsequent to
depolarization caused by released oxytocin. When we repeated these
experiments during perfusion of an oxytocin receptor antagonist ([des-glycinamide9,d(CH2)5,
O-Me-Tyr2, Thr4,
Orn8]-vasotocin; 10 µM) (Elands et al.
1988
; Kombian et al. 1997
), the HFS was equally
effective in increasing action-potential frequency (1,471 ± 616%
of control, n = 4; P < 0.05, compared
with control). We also considered the possibility that the postsynaptic
depolarization itself elicited a long-lasting change in the
postsynaptic cell properties, but injection of depolarizing current
alone into the MCN did not alter subsequent spontaneous activity
(140 ± 27% of control; n = 4; P
0.1).
The similar time course of STP of mEPSC and action potential firing frequency suggests that the former may be responsible for the latter. If this is the case, then blockade of non-NMDA receptors that mediate the mEPSCs should also block any change in firing frequency. In five cells that were initially shown to respond to HFS with STP, bath application of NBQX (1 µM) eliminated all the mEPSCs. When these cells were subsequently subjected to HFS (under continuing NBQX blockade) in current clamp, there was no change in the spike frequency (78.7 ± 24.2%, P < 0.05 vs. no NBQX). Thus changes in mEPSC frequency and possible temporal and spatial summation of spontaneous events can lead to sustained changes in the excitability of MCNs and thus their firing pattern.
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DISCUSSION |
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We have shown that excitatory afferents to MCNs of the SON can undergo a type of plasticity that may contribute to the generation of increased spontaneous activity. We have observed that physiologically relevant high-frequency afferent stimulations lead to short-term (5-20 min) enhancement in frequency of TTX-resistant spontaneous excitatory postsynaptic responses. To the best of our knowledge, enhancement of this magnitude and duration has not previously been described. Salient features of this phenomenon include: 1) its induction is independent of postsynaptic NMDA receptors, membrane potential, or input resistance; 2) it is independent of GABAA receptors; 3) its expression is action potential independent; 4) it is not affected by retrograde endogenous neurohypophyseal peptides; and 5) it causes an increase in the spontaneous activity of the postsynaptic MCN. Synapses that undergo such an excitability change will be predicted to contribute to or influence the pattern of firing that optimizes peptide release.
Posttetanic potentiation of mEPSC frequency was reported at the
neuromuscular junction over 40 yr ago (Brooks 1956).
Since then, potentiation of both mEPSC and mIPSC frequencies has been reported intermittently in central synapses (Atluri and Regehr 1998
; Cummings et al. 1996
; Goda and
Stevens 1994
; Jensen et al. 1999a
,b
;
Mennerick and Zorumski 1995
; Oliet et al.
1996
). There is general agreement that posttetanic potentiation
is related to increased levels of intracellular calcium that persist
beyond the time of the stimulus train. What differentiates previously reported posttetanic potentiation from the phenomenon we report here is
the magnitude and duration of the response. To the best of our
understanding, the time course reported previously lasts for
milliseconds to a few seconds, whereas the STP in the SON is of at
least several minutes' duration. Because of this radically different
time course, we have carried out a number of experiments to define its
site of induction and to rule out possible postsynaptic contributions.
Locus of STP induction is presynaptic
The locus of induction and maintenance of long-term synaptic
enhancement in other brain regions has been intensively investigated. Evidence exists supporting both pre- and postsynaptic loci
(Bliss and Collingridge 1993; Nicoll and Malenka
1995
). Our experiments suggest that the induction of STP of
mEPSCs in the SON is presynaptic to the MCNs. First, the ability to
generate STP was independent of the voltage at which the postsynaptic
cell was held during HFS. In fact, STP could be induced while the
postsynaptic cell was voltage-clamped at a very negative potential
(
80 mV). Second, positive current injected into the postsynaptic
cell, to cause it to fire a burst of action potentials, did not induce STP.
In some types of synaptic plasticity, the phenomenon is dependent on
NMDA-receptor activation in the postsynaptic cell (Malenka and
Nicoll 1993). Because functional NMDA receptors are present in
this nucleus (Hu and Bourque 1991
; Nissen et al.
1995
), perhaps colocalized with AMPA receptors (Stern et
al. 1999
), we considered their participation in the induction
of STP. However, blockade of these receptors with D-APV did
not affect the magnitude or duration of the STP. Furthermore, changes
in postsynaptic holding potential, which will affect the contribution
of NMDA receptors to STP, did not affect its induction.
NMDA-receptor-independent induction of synaptic plasticity has been
reported in other selected brain regions (Nicoll and Malenka
1995
) but the accompanying change in mEPSC frequency has not
been reported.
HFS does not alter postsynaptic cell properties
HFS would be expected to cause massive activation of the SON, resulting in alterations in extracellular ion concentrations and dendritic release of neurohypophyseal peptides. We could detect no alteration in postsynaptic cell properties that might result from these events that could account for the expression of STP.
Quantal analyses of amplitude-frequency distributions of miniature
events have been used as indicators of pre- and postsynaptic loci of
synaptic changes (Bekkers and Stevens 1990;
Malinow and Tsien 1990
). In this study, mEPSCs showed
clear frequency and amplitude increases suggesting both pre- and
postsynaptic changes may be involved. However, the steady-state and
rapid kinetic properties and apparent distribution of the non-NMDA
receptors that underlie the STP appeared unchanged.
First of all, the AMPA-induced steady-state whole-cell currents were
identical before and during STP. It could be argued that our inability
to detect significant changes in these currents may be the result of
the rapid desensitization of AMPA receptors over the several
seconds-long applications. However, no difference in postsynaptic
current was observed with kainate, an agonist that activates both AMPA
and kainate receptors, but does not cause as rapid desensitization of
the AMPA current (Patneau et al. 1993). Thus if
desensitization of the AMPA receptor was responsible for the fact that
AMPA currents were identical post-HFS, the kainate experiment would
likely have revealed any such changes in receptor properties. However,
it is still possible that nonsynaptic receptors or postsynaptic
receptors not receiving afferents activated by HFS are much more
plentiful and their response swamps that of the postsynaptic receptors
altered directly by HFS.
Second, as the unitary amplitude, rise times, and decay rates of the
mEPSCs remained unaltered, we can state that neither the kinetics of
the receptors nor the distribution of the activated synapses was
changed post-HFS. Thus if there was an appearance of new non-NMDA
receptors at previously silent synapses, as has been reported elsewhere
after long-term potentiation (LTP) (Isaac et al. 1995;
Kullmann 1994
; Liao et al. 1995
), they
would have to be spatially very close to the population activated
before STP so that the rise times are identical.
We also considered the possibility that STP could unmask silent kainate
receptors (Castillo et al. 1997), but if this were the
case, they would have to contribute to the mEPSCs only, as steady-state
whole-cell kainate-activated currents remained unchanged. A
contribution of kainate receptors is unlikely, because they have
different kinetics compared with AMPA receptors (Frerking et al.
1998
) and we did not detect any changes in the kinetics of
mEPSCs in either small or big events compared with control.
STP is the result of increased presynaptic glutamate release
The increase in mEPSC frequency after HFS is consistent with an
increased probability of transmitter release. Whereas individual events
can randomly summate with this high release probability to generate
large events, the fact that the amplitude distribution of mEPSC
post-HFS can be fitted with multiple Gaussian curves with equidistant
peaks indicates increased synchronous multiquantal release
(Auger et al. 1998; Paulsen and Heggelund
1996
). Such multiquantal events could be the result of
recruitment of previously inactive terminals and/or facilitation of
release leading to "spillover" of presynaptic glutamate to activate
postsynaptic silent receptors (Kullmann 1994
).
The mechanism responsible for the increased transmitter release after
HFS is yet undetermined. As described for the much more transient
posttetanic potentiation in other preparations (see Kamiya and
Zucker 1994), a likely mechanism is the prolonged elevation of
intracellular calcium (Inenaga et al. 1998
). However,
this would suggest that the presynaptic terminal can maintain elevated intracellular calcium for tens of minutes, a phenomenon not previously demonstrated, to the best of our knowledge. A number of second messengers and presynaptic kinases have been implicated in the facilitation of spontaneous neurotransmitter release (Arancio et
al. 1995
; Capogna et al. 1995
; Malenka et
al. 1987
; Publicover 1985
); future work must
address the possibility that HFS activates specific kinases or
nucleotides in the presynaptic terminal as a result of depolarization
or by the action of some other compound released as a result of the
HFS. Although this is unlikely to be vasopressin or oxytocin, because
the STP was not altered in the presence of an antagonist to their
receptors, there are many other neuroactive compounds in the SON that
could precipitate or maintain STP. Further studies are required to
explore these possibilities.
STP of mEPSCs is independent of GABAergic transmission
The ability to induce STP in the presence of opposing GABAergic
transmission makes it novel, because GABAergic transmission is known to
strongly influence the generation of other forms of synaptic
plasticities (Davies et al. 1991; Kano et al.
1992
; Malenka and Nicoll 1993
). However, there
was no qualitative or quantitative difference between the STP in the
presence and absence of picrotoxin, a GABAA
receptor channel blocker. Nonetheless, because functional presynaptic
GABAB receptors are present on excitatory
terminals in this nucleus (Kabashima et al. 1997
;
Kombian et al. 1996
), it is possible that a presynaptic
action of GABA on GABAB receptors will limit the
duration of an otherwise long-term change to a short-term change. This
possibility remains to be explored.
STP is not a property of a network
The SON receives afferent fibers not only from a number of distant
nuclei (Tribollet et al. 1985) but also from local
interneurons. The focal stimulation used in our experiments could
activate either or both inputs. Previous studies (Boudaba et al.
1997
) have shown that repetitive stimulation of the excitatory
interneurons can elicit afterdischarges in MCNs, possibly resulting
from metabotropic glutamate receptor activation. However, those
afterdischarges were abolished by TTX, indicating that they were the
result of increased spike generation in presynaptic cells. The STP
reported in the present experiments appears to be quite different.
Although initiation of STP requires action potentials, presumably to
invade the presynaptic terminal after stimulation of the afferent
axons, maintenance of the STP is not sensitive to TTX; thus it appears to be the axon terminal or some process limited to the terminal area
that is altered after HFS to cause STP.
HFS differentially modulates mEPSCs and evoked EPSCs
We have earlier reported that HFS, such as that used in this
study, depresses evoked EPSCs as a consequence of dendritically released oxytocin and vasopressin (Kombian et al. 1997).
Despite this effect on evoked EPSCs, HFS causes a massive increase in miniature events, that is, STP of mEPSCs. Thus dendritically released oxytocin and vasopressin appear to have different effects on the two
events. We next examined whether, in the absence of these peptides'
effect, HFS differentially affected evoked and mEPSCs. In the presence
of Manning compound, an oxytocin and vasopressin receptor antagonist
(Kombian et al. 1997
), the evoked EPSC remained unchanged, whereas we could still induce robust STP of mEPSCs with HFS.
These findings suggest that spontaneous and evoked release of glutamate
in the SON can be differentially regulated. Similar observations
concerning the differential modulation of evoked and spontaneous
synaptic responses have been reached in several other systems
(Auger et al. 1998
; Bao et al. 1998
;
Cummings et al. 1996
). Although the mechanism for this
differential regulation is still obscure, it could have its basis in
different calcium requirements of evoked and mEPSCs in the SON; that
is, evoked EPSCs require calcium entry through voltage-gated calcium
channels (unpublished observations), whereas mEPSCs are thought to be
more dependent on intracellular stores of calcium (Inenaga et
al. 1998
). Alternately, the mEPSCs seen after STP may involve
active zones/synapses that are not utilized in evoked transmitter
action or release at the 0.1 Hz stimulation frequency we used. It
remains to be seen whether evoked EPSCs elicited at higher frequencies
would be increased.
Physiological relevance
The physiological relevance of the massive, short-term increase in
mEPSCs is underscored by our observation that the postsynaptic MCNs
increase their firing rate during this period of enhanced presynaptic
activity. Because individual mEPSCs in the SON have been reported to be
capable of eliciting action potentials (Inenaga et al.
1998), the increase in the excitability of MCNs during STP can
result from big, multiquantal events. This is further underscored by
the observation that elimination of the mEPSC with a specific
antagonist of the non-NMDA receptors prevents the change in
excitability of the MCN. Such an increase in clustered events could
arise from afferents activated by changes in osmolality (Richard
and Bourque 1995
) and lead to changes in firing patterns to
optimize peptide release (Nissen et al. 1995
).
There is little doubt that the spectacular phasic activity of
vasopressinergic neurons is generated by intrinsic conductances (Armstrong 1995). Nonetheless, these cells also must
attain a threshold firing rate before these regenerative mechanisms
come into play (Poulain et al. 1988
). This may be
accomplished through a glutamate-induced depolarization, because
glutamate application to MCNs can activate phasic bursts
(Wakerley and Noble 1982
) and maintenance of spontaneous
phasic firing requires tonic synaptic activation involving glutamate
receptors (Jourdain et al. 1998
; Nissen et al.
1995
). We suggest that the STP seen in response to strong
synaptic stimulation could provide the background glutamate activity
reported to be necessary for phasic activity. Whereas the STP is most
evident at higher frequencies, most likely above the physiological
range, it can also be seen at more physiological frequencies (i.e.,
<50 Hz). The persistence of the enhanced synaptic activity seen after
HFS could also provide the ideal condition for temporal summation of
EPSCs, whereby afferent input from different activated pathways could
summate, or indeed predispose, the neuron to other types of plasticity
(cf. Abraham and Bear 1996
). Although the molecular
mechanisms underlying the STP are not yet known, the present data
indicate that a unique type of synaptic potentiation may be found in a
nucleus in which such changes can be correlated with known
physiological functions. It has recently been proposed that miniature
events may encode information in postsynaptic cells (Staley
1999
). We have shown in this study that, in the SON, changes in
the occurrence of miniature events remarkably influence the excitability of MCNs.
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ACKNOWLEDGMENTS |
---|
We thank Drs. K. Lukowiak, R. French, W. Wildering, and S. Oliet for helpful discussions and L. Bauce and Y. Takahashi for technical assistance. S. B. Kombian and M. Hirasawa contributed equally to the manuscript.
This work was supported by the Medical Research Council (Canada) and by personnel awards from the MRC (to S. B. Kombian, X. Chen, and Q. J. Pittman), the Alberta Heritage Foundation for Medical Research (to Q. J. Pittman and D. Mouginot), the Japan Society for the Promotion of Science (to M. Hirasawa), the Neuroscience Canada Foundation (to Q. J. Pittman), and Kuwait University Grant FDT 116 (to S. B. Kombian).
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FOOTNOTES |
---|
Address for reprint requests: Q. J. Pittman, Neuroscience Research Group and Dept. of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada.
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 6 October 1999; accepted in final form 8 December 1999.
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REFERENCES |
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