Anaesthesia Research Department, McGill University, Montreal, Quebec H3G 1Y6, Canada
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ABSTRACT |
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Zhao, Yong-Tao and
Kreimir Krnjevi
.
2-Deoxyglucose-Induced Long-Term Potentiation in CA1 Is Not
Prevented by Intraneuronal Chelator.
J. Neurophysiol. 83: 177-180, 2000.
In hippocampal slices, temporary
(10-20 min) replacement of glucose with 10 mM 2-deoxyglucose is
followed by marked and very sustained potentiation of EPSPs (2-DG LTP).
To investigate its mechanism, we examined 2-DG's effect in CA1 neurons
recorded with sharp 3 M KCl electrodes containing a strong chelator, 50 or 100 mM ethylene glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA). In most cases, field EPSPs were simultaneously recorded and conventional LTP was also elicited in some cells by tetanic stimulation of stratum radiatum. 2-DG potentiated intracellular EPSP
slopes by 48 ± 5.1% (SE) in nine cells recorded with plain KCl
electrodes and by 52 ± 6.2% in seven cells recorded with
EGTA-containing electrodes. In four of the latter cells, tetanic
stimulation (twice 100 Hz for 1 s) failed to evoke LTP (2 ± 1.1%), although field EPSPs were clearly potentiated (by 28 ± 6.9%). Thus unlike tetanic LTP, 2-DG LTP is not readily prevented by
postsynaptic intraneuronal injection of EGTA. These findings agree with
other evidence that the rise in postsynaptic (somatic)
[Ca2+]i caused by 2-DG is not the principal
trigger for the subsequent 2-DG LTP and that it may be a purely
presynaptic phenomenon.
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INTRODUCTION |
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Although there is much disagreement about
underlying mechanisms, it is widely agreed that the induction of
conventional long-term potentiation (LTP) is initiated by a rise in
postsynaptic intracellular Ca2+ concentration
([Ca2+]i) (Baudry
1998; Bliss and Collingridge 1993
;
Larkman and Jack 1995
; Nicoll and Malenka
1995
). The principal evidence for this is the finding that
intraneuronal injections of a strong chelator, such as ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), consistently prevent the subsequent induction of LTP
induced by tetanic stimulation in CA1 pyramidal cells (Lynch et
al. 1983
; Parfitt and Madison 1993
) and other
types of neurons (Komatsu and Iwakiri 1992
;
Ouardouz and Lacaille 1995
; Shindou et
al. 1993
). Even some unconventional forms of LTP can be
prevented by similar postsynaptic injections of chelators. For example, LTP induced by NMDA receptor-independent Ca2+ influx
(Kullmann et al. 1992
) and selective LTP of the
N-methyl-D-aspartate (NMDA) receptor
component of EPSPs induced by anoxia combined with aglycemia
(Crépel and Ben-Ari 1996
). By contrast, the LTP of
mossy fiber EPSPs, which is believed to have a presynaptic mechanism,
is not sensitive to postsynaptic injections of chelators (Katsuki et al. 1991
; Zalutsky and Nicoll
1990
).
Another unconventional type of LTP is one produced very reliably in CA1
neurons by 2-deoxyglucose (Tekkök and Krnjevi
1995
), which blocks glycolysis by competing with glucose for
phosphorylation by hexokinase (Tower 1958
). Albeit NMDA
receptor-dependent, this form of LTP (2-DG LTP) is very atypical in
that 2-DG causes postsynaptic hyperpolarization, not depolarization
(Zhao et al. 1997
). Nevertheless, it is also
Ca2+-dependent (Tekkök and Krnjevi
1996
) and 2-DG raises [Ca2+]i in
hippocampal neurons (Tekkök et al. 1999
). It was
therefore of interest to see whether similar injections of EGTA into
CA1 pyramidal cells would also prevent the subsequent induction of 2-DG
LTP. A preliminary report of these results has appeared as an abstract
(Zhao and Krnjevi
1997
).
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METHODS |
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Young Sprague-Dawley male rats (110-180 g) obtained from
Charles River, Québec, were decapitated under full halothane
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, U. K.). They were kept for 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 (pH
7.3) and were
continually aerated with carbogen (95% O2-5% CO2). 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 (SE).
The sharp microelectrodes were pulled from thin-walled borosilicate
glass tubes (1.2 mm OD, WP Instruments, New Haven, CT). After being
filled with 3 M KCl, the electrodes had resistances of 60-80 M. To
inject the chelator by diffusion into neurons, 50 or 100 mM EGTA
(Sigma, St. Louis, MO) was added to the 3 M KCl electrode-filling
solution. To prevent distortion of EPSPs by spiking, 10 mM QX-222 or
QX-314 (Astra Pharma, Ontario) was also added to many of the electrodes.
Field recordings from CA1 stratum radiatum were made with low resistance 2 M NaCl-containing electrodes. Half-maximal synaptic responses were evoked by stimuli applied at intervals of 20 s through insulated nickel-chromium wires placed in the s. radiatum. The signals were amplified by an Axoclamp 2 (Axon Instruments, Burlingame, CA) in current-clamp mode.
Tetanic LTP was elicited by s. radiatum stimulation with two 100 Hz volleys, each lasting 1 s and repeated after 20 s. 2-Deoxy-D-glucose (2-DG, Sigma) was applied for 10-20 min by equimolar replacement of 10 mM glucose in ASCF.
Means ± SE are given throughout. The significance of differences between means was assessed by Student's t-test (whenever possible for paired results).
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RESULTS |
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Intracellular penetrations into the stratum pyramidale of CA1 yielded stable recordings from nine control neurons with plain 3 M KCl electrodes and from seven test neurons with similar KCl electrodes containing 50 or 100 mM EGTA.
Effects of 2-DG
ACSF glucose was replaced by 2-DG for periods of 10-20 min, which regularly elicit 2-DG LTP in field recordings. The mean times were not significantly different (P = 0.11) for control cells (15 ± 1.2 min) and EGTA-injected cells (13 ± 0.80 min).
ON MEMBRANE POTENTIAL.
Initially, the resting potentials were similar in the two groups:
68.6 ± 0.41 mV for the nine control cells and
68.0 ± 0.58 mV for the seven EGTA-injected cells. However, in contrast to the
control cells, which were significantly hyperpolarized during the 2-DG
applications (by 4.9 ± 1.69 mV; P = 0.019), the
EGTA-injected cells showed no significant change in potential
(
1.0 ± 0.58 mV).
ON EPSPS.
As observed earlier (Tekkök and Krnjevi 1995
,
1996
; Zhao et al. 1997
), 2-DG had a biphasic
effect; after a 10 min delay EPSPs were sharply reduced, reaching a
minimum shortly after the return of glucose. This was followed by
recovery and then a well-sustained potentiation (lasting >30 min). In
nine control cells recorded with plain KCl electrodes, EPSPs were
enhanced by 48 ± 5.14%. As illustrated in Fig.
1, simultaneously recorded field EPSPs showed quite comparable changes. In three such double recordings, the
LTPs of field and intracellular EPSPs differed by only 2.7 ± 2.9% (paired data).
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DISCUSSION |
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The main findings can be summarized as follows. The extracellular
recordings showed quite conventional LTP after both tetanic stimulation
and applications of 2-DG, which could be depotentiated by low-frequency
stimulation. In intracellular recordings with plain KCl-containing
sharp electrodes, similar changes were seen as well as the
characteristic membrane hyperpolarization elicited by 2-DG (Zhao
et al. 1997). However, in cells recorded with electrodes also
containing EGTA, though sufficient EGTA leaked to prevent 1)
significant tetanic LTP, 2) 2-DG-induced hyperpolarization, and 3) depotentiation of 2-DG LTP by low-frequency
stimulation, nevertheless marked LTP was consistently induced by 2-DG.
These results are fully in agreement with previous studies that
diffusion of EGTA from recording microelectrodes readily prevents the
tetanic induction of either LTP (Lynch et al.
1983; Parfitt and Madison 1993
) or LTD
(Domenici et al. 1998
; Oliet et al.
1997
); from which it has been widely accepted that the
sustained changes in CA1 EPSPs induced tetanically are triggered by a
rise in postsynaptic [Ca2+]i (Baudry
1998
; Bliss and Collingridge 1993
;
Larkman and Jack 1995
; Nicoll and Malenka
1995
). Although the precise location of the critical rise in
[Ca2+]i is not known, it must be readily
accessible to EGTA leaking from a somatically-located sharp
microelectrode. This is evidently not the case where 2-DG LTP is concerned.
These results are thus in keeping with various tests on the 2-DG-evoked
rise in somatic [Ca2+]i, which revealed some
major discrepancies between its properties and those of 2-DG LTP
(Tekkök et al. 1999), and therefore led to the
conclusion that the observed [Ca2+]i rise
cannot be essential for the LTP. And yet 2-DG LTP does appear to be
Ca2+-dependent, being prevented by very prolonged lack of
Ca2+ or by dantrolene (Tekkök and
Krnjevi
1996
). If the significant [Ca2+]
change is not readily accessed by somatic injections of EGTA, where can
it be situated?
It might be argued that it occurs in small dendritic branches/spines, too far to be easily reached by diffusion of EGTA from the soma; but this must be the site of most of the NMDA receptor-mediated Ca2+ influx responsible for tetanic LTP, which is prevented by EGTA. A more likely explanation is that the critical events occur elsewhere, such as in adjacent glia or presynaptic terminals.
There is evidence that glia may alter the function of neighboring
neurons by a Ca2+-dependent release of arachidonic acid and
subsequent potentiation of glutamatergic transmission (Glowinski
et al. 1994). A comparable interaction, initiated by
2-DG-induced glutamate leakage and ATP depletion (Tower
1958
) could conceivably induce 2-DG LTP.
In view of the absence of NMDA receptors on most glia
(Steinhauser et al. 1994), a presynaptic event is more
likely. Our previous finding that 2-DG LTP virtually abolishes
paired-pulse facilitation (Tekkök and Krnjevi
1997
) suggested that 2-DG LTP is expressed presynaptically. A
simple explanation is that LTP is both induced and expressed in
afferent terminals, the induction being mediated by a
Ca2+-dependent dantrolene-sensitive mechanism, necessarily
inaccessible to postsynaptically EGTA.
There is an obvious parallel with the tetanic LTP of mossy fiber EPSPs,
which is also independent of postsynaptic depolarization and is not
affected by injecting chelators into the postsynaptic neurons
(Katsuki et al. 1991; Nicoll and Malenka
1995
; Zalutsky and Nicoll 1990
). This form of
LTP thus appears to be an essentially presynaptic phenomenon, initiated
by a rise in intraterminal [Ca2+] and activation of a
Ca2+-sensitive protein kinase A (PKA) (Nicoll and
Malenka 1995
; Villacres et al. 1998
;
Xiang et al. 1994
). The mechanism of 2-DG LTP, however, cannot be identical because 2-DG LTP does not seem to involve PKA
(Tekkök and Krnjevi
1997
) and it is NMDA
receptor-dependent.
A likely target for a presynaptic action of glutamate is syntaxin, a
protein that combines a role in synaptic vesicle docking (Bennett et al. 1992) with NMDA receptor properties
(Smirnova et al. 1993
). One can speculate that 2-DG LTP
is induced by the conjunction of several critical events initiated by
2-DG: glutamate release and presynaptic NMDA receptor-triggered
Ca2+ influx; intraterminal Ca2+ release from a
dantrolene-sensitive store (Katchman and Hershkowitz 1993
); and rapid depletion of ATP, which together initiate
lasting changes in phosphorylation at a site involved in transmitter release.
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ACKNOWLEDGMENTS |
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Y. T. Zhao was on leave from the Neurology Department, Liu Hua Qiao Hospital, Guang Zhou, China. We are grateful to Astra Pharma Inc., Ontario, for a supply of QX314.
This research was financially supported by the Medical Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests: K. Krnjevi, Room 1207, McIntyre
Building, 3655 Drummond St., Montréal, 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 24 May 1999; accepted in final form 2 September 1999.
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REFERENCES |
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