Anaesthesia Research Department, McGill University, Montreal, Quebec H3G 1Y6, Canada
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Krnjevi, Kre
imir and
Yong-Tao Zhao.
2-Deoxyglucose-Induced Long-Term Potentiation of
Monosynaptic IPSPs in CA1 Hippocampal Neurons.
J. Neurophysiol. 83: 879-887, 2000.
In previous experiments on excitatory
synaptic transmission in CA1, temporary (10-20 min) replacement of
glucose with 10 mM 2-deoxyglucose (2-DG) consistently caused a marked
and very sustained potentiation (2-DG LTP). To find out whether 2-DG
has a similar effect on inhibitory synapses, we recorded
pharmacologically isolated mononosynaptic inhibitory postsynaptic
potentials (IPSPs; under current clamp) and inhibitory postsynaptic
currents (IPSCs; under voltage clamp); 2-DG was applied both in the
presence and the absence of antagonists of
N-methyl-D-aspartate (NMDA). In spite of
sharply varied results (some neurons showing large potentiation, lasting for >1 h, and many little or none), overall there was a
significant and similar potentiation of IPSP conductance, both for the
early (at
30 ms) and later (at
140 ms) components of IPSPs or
IPSCs: by 35.1 ± 10.25% (mean ± SE; for
n = 24, P = 0.0023) and
36.5 ± 16.3% (for n = 19, P = 0.038), respectively. The similar potentiation
of the early and late IPSP points to a presynaptic mechanism of LTP.
Overall, the LTP was statistically significant only when 2-DG was
applied in the absence of glutamate antagonists. Tetanic stimulations
(in presence or absence of glutamate antagonists) only depressed IPSPs
(by half). In conclusion, although smaller and more variable,
2-DG-induced LTP of inhibitory synapses appears to be broadly similar
to the 2-DG-induced LTP of excitatory postsynaptic potentials
previously observed in CA1.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There have been numerous studies of long-term
potentiation (LTP) at excitatory synapses (Bliss and
Collingridge 1993; Larkman and Jack 1995
;
Wang et al. 1997
), but only few on isolated inhibitory synapses (Komatsu 1994
, 1996
;
Komatsu and Iwakiri 1993
; Oda et al.
1995
; Xie et al. 1995
), including the
hippocampus from mature guinea pigs (Xie et al. 1995
)
and very young rats (McLean et al. 1996
). A major reason
for this dearth of information is that inhibitory postsynaptic
potentials (IPSPs), unlike excitatory ones (EPSPs), cannot be easily
recorded as extracellular fields. Compelling evidence requires
intracellular recording. Moreover, because inhibitory neurons can
seldom be stimulated selectively (without concomitant activation of
other cells and axons), IPSPs can be obtained independently of
excitatory synaptic activity only if EPSPs are suppressed by blockage
of glutamatergic synapses (Davies et al. 1990
;
Neuman et al. 1988
). Previous studies have reported that
such monosynaptic IPSPs in visual and hippocampal cortex of young
rodents can be potentiated by tetanic stimulation; but the resulting
LTP differs from LTP of EPSPs in being independent of
N-methyl-D-aspartate (NMDA) receptor activation
(Komatsu 1994
; McLean et al. 1996
; Xie et al. 1995
) and variably affected by chelation of
intracellular Ca2+ (cf. McLean et al. 1996
; Xie
et al. 1995
).
Temporary replacement of glucose by 2-deoxyglucose (2-DG) very
predictably elicits LTP of EPSPs in CA1 neurons (Tekkök
and Krnjevi 1995
, 1996
). Although 2-DG
suppresses glycolysis by selectively blocking hexokinase (Tower
1958
), even prolonged removal of glucose has no comparable
effect on EPSPs (Krnjevi
and Tekkök 1996
).
Like many other forms of LTP, 2-DG-induced LTP (2-DG LTP) is NMDA
receptor dependent, but it is not suppressed by intracellular application of a chelator (Zhao and Krnjevi
2000
).
Its very unusual characteristics (notably its unique independence of postsynaptic depolarization and [Ca2+]i increase) make 2-DG LTP an exceptionally interesting form of synaptic plasticity. Albeit without an obvious physiological correlate, the fact that a temporary metabolic disturbance causes such highly reproducible and sustained enhancement of synaptic transmission, apparently by a purely presynaptic mechanism, has a wider significance both for the understanding of LTP-type plastic changes in general and possible pathological manifestations resulting from metabolic disorders.
In the present experiments, we examined the effects of 2-DG on
monosynaptic inhibitory potentials (IPSPs) or currents (IPSCs; isolated pharmacologically by glutamate antagonists) recorded intracellularly from CA1 pyramidal layer neurons with sharp
microelectrodes. A preliminary report of these results has appeared as
an abstract (Krnjevi and Zhao 1998
).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Young Sprague-Dawley male rats (110-180 g) were obtained from
Charles River (St. Constant, Quebec, Canada). After
decapitation under deep halothane or ether anesthesia, the brain was
quickly removed and the hippocampus dissected out in ice-cold
oxygenated saline. Transverse slices (400 µm thick) were cut with a
Vibroslice (Campden Instruments, Loughborough, UK). They were kept for
at least 1 h at room temperature in artificial cerebrospinal fluid (ACSF) containing (in mM) 124 NaCl, 3.0 KCl, 2.0 CaCl2, 2.0 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose; being continually aerated with
carbogen (95% O2-5% CO2) the ACSF had a pH
7.3. Slices were then transferred to a recording chamber where they
were submerged under 0.1-0.2 mm of flowing carbogenated ACSF and kept
at 34 ± 0.5°C.
The sharp microelectrodes were pulled from thin-walled borosilicate
glass tubes (1.2 mm OD, WP Instruments, New Haven, CT). After filling
with 3 M KCl or 4 M K acetate, they had resistances of 60-80 M. In
some electrodes, 10-50 mM QX-222 or QX-314 (Astra Pharma, Ontario,
Canada) was also added.
Half-maximal synaptic responses were evoked by stimuli applied at
intervals of 20 s through insulated nickel-chromium wires placed
in the stratum radiatum. To obtain monosynaptic IPSPs/IPSCs, excitatory
synaptic transmission was suppressed by adding glutamate antagonists to
the superfusate (Davies et al. 1990; Neuman et al. 1988
). The agents used were either the wide-spectrum
blocker kynurenate (Sigma; 2-3 mM) or a combination of
6,7-dinitroquinoxaline-2,3-dione (DNQX; 25-50 µM) and
D,L-aminophosphonovalerate (APV; 40-50 µM). In several
experiments, we also attempted to elicit tetanic LTP of the
monosynaptic IPSPs/IPSCs by high-frequency stimulation of stratum
radiatum (2 100-Hz volleys, each lasting 1 s, separated by a 20-s interval).
For current-clamp recordings of IPSPs, the signals were amplified
by an Axoclamp 2 (Axon Instruments, Burlingame, CA) in bridge mode. For
voltage-clamp recordings of IPSCs, the signals were amplified in the
discontinuous clamp mode, operating at a frequency of 3 kHz, a gain of
25 nA/mV, and an upper bandwidth limit of 300 Hz; the usual precautions
were taken to optimize the efficacy of the clamp. In all experiments,
IPSPs/IPSCs were recorded over a range of potentials so that IPSP/IPSC
conductances (GIPSP and GIPSC) and reversal potentials
(VIPSP) could be compared before and after a
2-DG application. Thus GIPSC was obtained
directly from the slope of IPSC/Vm plots of
voltage-clamp data. But for current-clamp data,
GIPSP was calculated from the following
relation: IPSP/(Vm VIPSP) = GIPSP/(GIPSP + Grest) (Takeuchi 1977
), where Grest (resting conductance) was obtained
from plots of Vm as function of injected
currents. All slopes were calculated by fitting linear regressions to
the data. Changes in slopes after 2-DG applications were expressed as
percentage of the initial control slope; the standard error of the
ratio of post-2-DG to control GIPSP/C
(G2/G1) was taken
as [V2 + V1(G2/G1)2]1/2 /G1
(Mellor 1931
), where V is the variance of
the corresponding slope.
Experimental protocols
IN CURRENT-CLAMP MODE.
After a period of stable recording of control isolated IPSPs,
2-deoxy-D-glucose (2-DG; Sigma) was applied by equimolar
replacement of 10 mM glucose in ACSF. Superfusion with standard
glucose-containing ACSF was later resumed, and recording continued for
30-60 min. In some experiments, glutamate antagonists were present
throughout. In others, the slice was "washed" with ACSF for 20-30
min to remove the antagonists before applying 2-DG, to minimize
interference with any NMDA-sensitive effects of 2-DG. Although a longer
period of wash might have been desirable, previous evidence shows that the LTP-blocking action of APV can be reversed by washing for periods
of only 20-30 min (Wigström et al. 1986). The
glutamate antagonists were reapplied subsequently. Before ending,
picrotoxin (Sigma; 100-200 µM) was applied to confirm that the early
IPSP was indeed GABAA receptor mediated;
typically, the late IPSP was very small or absent after such prolonged recordings.
UNDER VOLTAGE CLAMP. The pharmacologically isolated IPSCs were recorded over a range of holding potentials (Vh) before the start of the 2-DG application, and for 30-60 min after the return of glucose-containing ACSF. In several experiments, the slices were "washed" to remove kynurenate or APV and the voltage-clamp interrupted during the 2-DG application to minimize block of any NMDA receptor and/or voltage-sensitive effects of 2-DG. The glutamate antagonists and the voltage clamp were then reapplied.
Means ± SE are given throughout. The significance of differences was assessed by Student's t-test. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intracellular penetrations into the stratum pyramidale of CA1 yielded a total of 24 stable recordings, adequate for the purpose of these experiments: 11 were under current clamp and 13 under voltage clamp. As described under METHODS, monosynaptic IPSPs or IPSCs were isolated by bath application of glutamate antagonists, and 2-DG was applied for periods of 11-22 min, either after washing out antagonists or in their presence.
Observations under current clamp
The recordings were made with K acetate- or KCl-containing sharp microelectrodes.
2-DG APPLIED IN THE ABSENCE OF GLUTAMATE ANTAGONISTS.
Figure 1 illustrates IPSPs recorded from
a CA1 neuron. The initial trace (A), obtained at the initial
resting membrane potential (Vm) of
69 mV while superfusing with standard ACSF, shows a characteristic EPSP/IPSP sequence evoked by stratum radiatum stimulation. After addition of DNQX (45 µM) and APV (50 µM), only a biphasic IPSP remained (B), which was recorded at several levels of
Vm (see IPSP/Vm plots in Fig.
2, A and B).
Superfusion with blocker-free ACSF was resumed for 20 min to wash out
APV before 10 mM 2-DG (replacing glucose) was applied for 15 min (Fig.
1, C and D); note the hyperpolarization typically
elicited by 2-DG (Zhao et al. 1997
). In the present
case, it did not fully reverse after the return of standard
(glucose-containing) ACSF (E). The glutamate antagonists
were then reapplied to eliminate any EPSP, and further traces were
recorded for over 1 h from the end of the 2-DG application. The
traces F-H clearly show a marked potentiation of the
monosynaptic IPSP, in spite of some persistent hyperpolarization. A
true and sustained potentiation is further suggested by the steeper
IPSP/Vm slopes for data obtained at 25 and 65 min post-2-DG, both for the early and the late IPSP (measured
at 20 and 190 ms from the stimulus artifact, Fig. 2, B and
A, respectively): from these slopes, we calculated (see
METHODS) the values of
GIPSP illustrated in Fig.
2C.
|
|
|
|
2-DG APPLIED IN THE PRESENCE OF GLUTAMATE ANTAGONISTS.
In six cells, post-2-DG changes in
GIPSP ranged from 0 to 107% for the
early IPSP and from 49 to 83% for the late IPSP. From Table 1, where
these data are also summarized, the mean increases were by 43% for the
early IPSP (just significant) and only 3.8% for the late IPSP.
Observations under voltage clamp
All these recordings were done with KCl-containing sharp
microelectrodes [in some cases with QX-314 added to depress the late, GABAB receptor-mediated event (Nathan et
al. 1990)]. To have more comprehensive information about
2-DG-induced changes, IPSCs were routinely recorded over a wide range
of holding potentials, before and at several intervals after the usual
applications of 2-DG (substituted for glucose for 10-20 min).
2-DG APPLIED IN THE ABSENCE OF GLUTAMATE ANTAGONISTS. Twelve neurons were studied. Representative data are illustrated by the traces (A-C) and IPSC/Vm plots (D-F) of Fig. 4. These monosynaptic IPSCs were all recorded from one neuron while applying DNQX and APV: in A is a control series; B and C, obtained 25 and 65 min after superfusing 2-DG (in antagonist-free ACSF) for 21 min, show marked and lasting enhancement of the IPSCs. From these and other data, the amplitude of the IPSCs (measured at 3 points: 6, 30, and 100 ms after the stimulation artifact) was plotted as function of Vm: the corresponding plots for data obtained at 25, 45, and 65 min post-DG [as well as the initial, pre-2-DG control data (open circles and thicker lines)] are shown for the very early IPSC (D, measured at 6 ms), the early IPSC (E, at 30 ms), and the later IPSC (F, at 100 ms). The changes in GIPSC calculated from the slopes of these plots are given by the histograms of Fig. 5: LTP-like major increases of the earlier components were sustained for >1 h; but the small late component, although it also increased initially, later ran down owing to the presence of QX-314 in the electrode.
|
|
|
2-DG APPLIED IN THE PRESENCE OF GLUTAMATE ANTAGONISTS. Two of the three cells in this group showed 30-80% increases in GIPSC. The IPSCs of the third cell were sharply and increasingly depressed post-2-DG; IPSC/Vm plots of these data are illustrated in Fig. 7, A-C, as a possible example of 2-DG-induced long-term depression (LTD). The mean change for these three cells was not significant (Table 1).
|
Overall effects of 2-DG on both IPSPs and IPSCs
Notwithstanding the great variability of individual results, ranging from apparent LTD to large LTP, overall there was a clear indication of a significant and similar post-2-DG potentiation of both early and late components of GIPSP (Table 2).
|
EARLY IPSP/C. Changes in early GIPSP observed >30 min post-2-DG in 24 neurons (under either current or voltage clamp) gave a very significant mean increase by 35.1 ± 10.25% (mean ± SE; for t = 3.42, P = 0.0023). Although 2-DG was applied for times varying between 10 and 20 min (15.7 ± 0.61 min), there was no correlation between the changes in GIPSP and the duration of 2-DG application (r = 0.034; P = 0.875).
For 15 of these cells, 2-DG was applied in the absence of glutamate antagonists: the corresponding mean increase was by 34.9 ± 13.52% (P = 0.022). For the other nine cells, exposed to 2-DG in the presence of the antagonists, the mean change was very similar (35.4 ± 16.5), but its significance was dubious (P = 0.064). These data are summarized in Table 3.
|
LATE IPSP/C.
Changes in late GIPSP were observed
35 ± 2.3 min post-2-DG in 19 neurons (not 24 because in 5 neurons late IPSPs were too small to be analyzed). Although even more
variable than the data for the early IPSP/C, these estimates gave a
virtually identical overall mean increase by 36.5 ± 16.3%
(P = 0.038). These changes also showed no correlation
to the duration of 2-DG applications (r = 0.237;
P = 0.33). On the other hand, there was a highly significant correlation between changes in late and early IPSP/Cs: for
n = 19, r was 0.974 (P = 10
6).
Posttetanic LTP
While recording monosynaptic IPSPs or IPSCs from six cells, we attempted to elicit LTP by tetanic stimulation. In four cases, the tetani were applied in the presence of glutamate antagonists, in the other two, in their absence. Whether tested after 2-DG (as in the examples of Figs. 1 and 6) or before any other tests (as in the case of the IPSPs illustrated by the traces and the IPSP/Vm plot in Fig. 7D), all the tetani had only sustained depressant effects (51 ± 2.9%).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Unlike 2-DG-LTP of EPSPs (Tekkök and Krnjevi
1995
; Zhao et al. 1997
; Zhao and
Krnjevi
2000
), 2-DG-LTP of IPSPs proved to be a quite
variable phenomenon. In every group studied, at least one or two cells
showed a sharp LTP (marked enhancement of
GIPSP, lasting without major decrement
for >30 min), but many did not yield such convincing results. Hence
the early IPSC was significantly enhanced only in the group recorded by
voltage clamp and exposed to 2-DG in the absence of glutamate
antagonists. At least part of the variability can be ascribed to the
much greater technical hurdles that have to be overcome to obtain
meaningful data from "pure" IPSPs, uncontaminated by EPSPs, which
limits the numbers of useful recordings, especially when comparing with experiments on field EPSPs.
Although an unevenly reproducible phenomenon, 2-DG-LTP of these
inhibitory synapses is more consistent than appears at first sight.
When all the changes in GIPSP/C
obtained under current and voltage clamp were viewed together,
significant increases were evident for both the early and the late
IPSP/C. The relatively modest potentiation [on the average (35%)
only ~1/2 that typically seen with field EPSPs
(Tekkök and Krnjevi
1995
)] may indicate a
less pronounced action of 2-DG on inhibitory terminals, but it may be a
reflection of the technical problems and long duration of the experiments.
NMDA receptor dependence of 2-DG-LTP
In some cells, IPSPs or IPSCs were potentiated even when 2-DG was
applied in the presence of glutamate antagonists. But when all the data
were grouped accordingly (Table 3), although the mean potentiation was
similar (35%) in both groups, it was significant (for both early
and late IPSP/C) only for the cells exposed to 2-DG in the absence of
antagonists. The less consistent changes seen when antagonists were
present suggest that NMDA receptors play some role in 2-DG-LTP
induction of IPSPs; this conclusion, however, can only be tentative in
view of the somewhat smaller number of observations in the second group
(9 vs. 15 for the early IPSP/C). These results are thus only in partial
agreement with the experiments on EPSPs, where 2-DG-LTP was
consistently prevented by APV (Tekkök and Krnjevi
1995
). Some residual block of NMDA receptors, unavoidable
because of the relatively short periods of wash out, may in part be
responsible for the smaller mean LTPs observed in the present experiments.
Mechanism of 2-DG-LTP of IPSPs
Is 2-DG-LTP initiated in the postsynaptic neuron? We know that
2-DG raises the cytoplasmic [Ca2+] of CA1
pyramidal neurons (Tekkök et al. 1999). Therefore
Wang et al.'s (1995)
finding that injections of the
-subunit of the calcium/calmodulin-dependent protein kinase II
(CAMKII) into CA1 pyramidal cells elicits a lasting potentiation of
monosynaptic fast IPSPs could explain our observations. Because of
several discrepancies, we concluded that the rise in
[Ca2+]i was probably not
the main trigger for 2-DG-LTP of EPSPs; but this does not exclude an
essential role in the induction of 2-DG-LTP of IPSPs.
Mediation by CAMKII is fully in keeping with much evidence that
phosphorylation modulates the function of GABAA
receptors (Kano and Konnerth 1992; Stelzer et al.
1988
). Admittedly, it is well-known that a rise in
[Ca2+]i can depress IPSPs
(Inoue et al. 1986
; Mouginot et al. 1991
; Stelzer and Shi 1995
). But whether LTP or LTD is induced
may depend on the magnitude and time course of the change in
[Ca2+]i [as appears to
be the case with tetanic LTP (Yang et al. 1999
)] and
perhaps a corresponding shift in relative kinase and phosphatase activity (Stelzer et al. 1988
; Wang et al.
1995
).
A relevant parallel is the tetanic LTP of IPSPs in visual cortex,
extensively studied by Komatsu (1994,
1996
). This appears to have a postsynaptic mechanism:
Ca2+-enhanced GABAA
receptor sensitivity, probably mediated by activation of
GABAB receptors and the formation of inositol
trisphosphate (Komatsu 1996
). This would be in keeping
with our evidence that 2-DG (whose action is hyperpolarizing, not
depolarizing) causes a release of internal Ca2+
(Tekkök et al. 1999
); a major difference, however,
is that the tetanic LTP of cortical IPSPs is clearly not NMDA receptor
dependent; in the absence of an NMDA antagonist, tetanic stimulation
elicits LTD and not LTP (Komatsu and Iwakiri 1993
).
A postsynaptic mechanism of expression cannot be easily reconciled with
the strikingly similar 2-DG-LTP of the early and late components of the
IPSP/C. Although generated by quite different receptors and ionic
channels (Krnjevi 1997
), they showed strongly correlated changes. Because 2-DG is unlikely to have a similar effect
on the very different GABAA and
GABAB receptors, a presynaptic mechanism must be
viewed as a serious possibility; in keeping with a similar conclusion
about 2-DG-LTP of EPSPs, partly based on the finding that 2-DG-LTP of
EPSPs is associated with marked reduction of paired-pulse facilitation
(Tekkök and Krnjevi
1996
) and is not
sensitive to chelation of postsynaptic Ca2+
(Zhao and Krnjevi
2000
). A possible objection
that GABA receptors are saturated during normal transmission (De
Koninck and Mody 1994
) does not exclude a presynaptic mechanism
that enables previously "silent" synapses (Kullmann and
Siegelbaum 1995
).
How 2-DG might have such a presynaptic effect, whether it is directly
or indirectly mediated, and the location of relevant NMDA receptors
remain open to speculation. If the mechanism is indeed mainly
presynaptic and NMDA receptors do play a significant role, as appears
to be the case for 2-DG-LTP of EPSPs (Tekkök et al.
1999; Zhao and Krnjevi
2000
), the most
likely target is syntaxin, which is involved in synaptic vesicle
docking (Bennett et al. 1992
), as well having NMDA
receptor properties (Smirnova et al. 1993
).
Tetanic LTP of monosynaptic IPSPs
The main point of interest is that our attempts to elicit
posttetanic LTP failed: the consistent effect being sustained
depression. Because the majority of the tests were done in the presence
of an NMDA antagonist, these results partly disagree with those of previous studies of monosynaptic IPSPs. In visual cortex
(Komatsu 1994; Komatsu and Iwakiri 1993
)
and in CA1 (Xie et al. 1995
) or CA3 (McLean et
al. 1996
), tetanic LTP was seen more often than LTD in the
presence of NMDA antagonists. The apparent difference may be owing to
the small number of tests in our experiments; or a combination of
dissimilar recording site, species, temperature and age in the earlier experiments.
In conclusion, in these experiments on monosynaptic IPSPs and IPSCs (isolated by pharmacological block of glutamate ionotropic receptors), evidence was obtained that 10- to 20-min applications of 2-DG can induce a LTP-like sustained enhancement of inhibitory transmission. The comparable enhancement of early and late IPSPs favors a pre- rather than a postsynaptic mechanism of LTP. In this respect, and also the nonsignificant potentiation when 2-DG was applied in the absence of glutamate antagonists, this effect resembles that produced on EPSPs. Thus albeit less pronounced and more variable, these findings suggest that 2-DG has similar effects on excitatory and inhibitory synapses in the hippocampal CA1 region.
![]() |
ACKNOWLEDGMENTS |
---|
We are grateful to Astra Pharma Inc., Ontario for a supply of QX-314. Y. T. Zhao was on leave from the Neurology Department, Liu Hua Qiao Hospital, Guang Zhou, China.
This research was financially supported by the Medical Research Council of Canada.
![]() |
FOOTNOTES |
---|
Address for reprint requests: K. Krnjevi, Rm. 1207, McIntyre
Bldg., 3655 Drummond St., Montreal, Quebec H3G 1Y6, 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 2 July 1999; accepted in final form 8 October 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |