 |
INTRODUCTION |
Since the pioneering work of Bliss and Lømo
(1973)
, long-term potentiation (LTP) has been extensively
studied in various brain regions as a model for learning and memory
(for review, see Bliss and Collingridge 1993
). The
molecular processes leading to a prolonged increase in synaptic
efficacy have been attributed to presynaptic and/or postsynaptic
mechanisms (for review, Madison and Schuman 1991
). A
favorite hypothesis postulates the existence of a retrograde messenger
that travels from the postsynaptic site back to the presynapse to cause
changes in the release of excitatory transmitters (Arancio et
al. 1996
). Arachidonic acid, nitric oxide (NO), and carbon
monoxide are candidate molecules to fulfill the criteria of a
retrograde messenger (O'Dell et al. 1991
; Zhuo
et al. 1993
). Inhibition of NO synthase (NOS) has been shown to
suppress LTP, both under in vivo (Iga et al. 1993
;
Mizutani et al. 1993
), and in vitro (Bon et al.
1992
; Haley et al. 1992
) conditions (but see
also Bannerman et al. 1994
). In addition, the
involvement of the different NOS isoforms, neuronal (type I, nNOS),
inducible (type II, iNOS), and endothelial (type III, eNOS), in
hippocampal LTP has been recently studied in more detail in mutant mice
deficient of nNOS (nNOS
) (O'Dell et al.
1994
). These data indicate that eNOS, rather than nNOS, is the
primary source of NO in the postsynaptic neuron during LTP. This
hypothesis has been confirmed by two different groups using recombinant
adenovirus vectors containing a truncated eNOS (Kantor et al.
1996
) and mutant mice deficient of eNOS (eNOS
)
(Wilson et al. 1997
). In contrast, Son et al. (1996)
demonstrated that hippocampal LTP was normal in eNOS
mice, but reduced in CA1 stratum radiatum of doubly mutant mice (nNOS
/eNOS
). We were interested in the
extent that eNOS contributes to neocortical synaptic plasticity and
studied the expression of LTP in somatosensory cortical slices of
eNOS
mice.
 |
METHODS |
Twenty-six control mice and seven eNOS-deficient mice with an
age of 8-12 wk were used for this study. eNOS knockout mice were
constructed by deletion of exons 24825 of the eNOS gene, which resulted
in the disruption of the essential NADPH binding site. Homozygous
animals have no eNOS activity and develop hypertension (for details of
construction and characterization of this mouse, see
Gödecke et al. 1998
). For all experiments, control
and knockout mice were siblings derived from F1 heterozygous
intercrosses.
The methods for preparing and maintaining neocortical slices in vitro
were similar to those described previously (Luhmann et al.
1995
). In brief, 400-µm-thick coronal slices of the primary somatosensory cortex were cut on a vibratome in ice-cold artificial cerebrospinal fluid (ACSF) consisting of (in mM) 124 NaCl, 5 KCl, 1.25 NaH2PO4, 1 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 glucose with a pH of
7.4 when saturated with 95% O2-5% CO2. Slices
were transferred to an incubation-storage chamber or to an
interface-type recording chamber and kept at 31-32°C. Slices were
allowed to recover for at least 1.5 h before recording began.
Extracellular recordings were performed with 2-5 M
electrodes
filled with ACSF. Extracellular field potential responses in layers
II/III to orthodromic synaptic stimulation of the underlying layer IV
were elicited at intervals of 60 s. For obtaining input-output
curves, the stimulus duration was increased in steps of 20 µs from 40 to 300 µs. For the LTP experiments, the pulse duration was fixed at
200 µs, and the stimulus strength was adjusted to an intensity that
evoked a submaximal field response of at least 1 mV in amplitude. Only
slices with stable responses showing variations in amplitude <5%
during baseline recording were used for analysis. After 15 min baseline
recording, 200-ms-long 100-Hz stimulus trains were delivered every
5 s to the afferent pathway for 10 min. This high-frequency
stimulation (HFS) reliably produced LTP by 20-30% (Aroniadou
and Teyler 1992
). After this 10-min high-frequency stimulation,
synaptic responses were recorded every 60 s for a period of 60 min. Data were digitized on-line and analyzed off-line using the TIDA
software program (HEKA, Lambrecht, Germany). The field amplitude was
measured between the negative- and positive-going peak of the
orthodromic response. For pharmacological analyses, the selective
N-methyl-D-aspartate (NMDA) receptor antagonist
DL-amino-phosphonovaleric acid (APV, Sigma, Basel) was
applied locally in a concentration of 120-200 µM (dissolved in ACSF)
with a broken micropipette placed on the slice surface near the
recording site. In experiments designed to investigate the effect of
NOS inhibition, slices were incubated for >1.5 h in ACSF containing
200 µM NG-nitro-L-arginine methyl
ester (L-NAME; Sigma) and transferred to the recording
chamber containing the same bathing solution. For statistical analyses,
five subsequent responses before and 50 min after HFS were compared by
the Student's t-test. If not otherwise noted, values
throughout this report are expressed as means ± SE.
 |
RESULTS |
Neocortical slices prepared from wild type and eNOS
mice were analyzed in basal excitatory synaptic transmission by
recording field potential responses to electrical stimulation of the
afferents with constant intensity and various durations (Fig.
1). In both experimental groups, short
stimulus pulses (40-80 µs) elicited a small orthodromic response
(Fig. 1A). An increase in stimulus duration evoked a larger
field response that reached a maximum at a stimulus duration of
240-300 µs (Fig. 1B). The input-output curves for both
groups were very similar, and no statistically significant difference
could be detected between wildtype and eNOS
mice. In
wildtype mice, HFS for 10 min produced a gradual increase in the
orthodromic field potential response, which after 50 min reached a
value of 122.7 ± 3.6% (mean ± SE; n = 11, P < 0.001) of the baseline control (Fig.
2, A1 and A2 and
Fig. 2B,
). Local application of the selective NMDA
antagonist APV to the potentiated slice caused in wildtype mice a
significant (P < 0.05) reduction in the field
potential amplitude to 94.8 ± 10.6% (Fig. 2A3 and APV
in Fig. 2B, 10 min after APV application), indicating that the potentiated response was mediated by an NMDA receptor-dependent synaptic component. In contrast, slices from eNOS
mice
did not reveal any significant increase in the field potential response
(Fig. 2B,
). Fifty minutes after HFS stimulation, the average field amplitude in eNOS
mice amounted to only
102.1 ± 2.8% (n = 10). In agreement with our
observations on wildtype mice, application of the NMDA antagonist APV
to slices prepared from eNOS
animals produced an
insignificant reduction in the field response to 97 ± 8.2% (APV
in Fig. 2B).

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Fig. 1.
Input-output relationship of stimulus-evoked field potential responses
in wildtype and eNOS mice. A: responses to
electrical stimuli of various duration (40, 100, 200, and 300 µs) in
wildtype (top row) and eNOS mice
(bottom row). B: absolute field potential
amplitudes to stimuli ranging from 40 to 300 µs in wildtype
( , n = 7 slices) and
eNOS mice ( , n = 8).
Data are expressed as means ± SE. No significant difference
between both groups could be obtained over the whole range of stimulus
durations. eNOS, endothelial NOS.
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Fig. 2.
Neocortical long-term potentiation (LTP) in adult wildtype
( ) and eNOS ( ) mice.
A: characteristic field potential responses in
layers II/III to orthodromic synaptic stimulation before
(1) and 50 min after high-frequency stimulation (HFS;
2) recorded in somatosensory cortex of a wildtype mouse.
Application of the selective N-methyl-d-aspartate (NMDA)
antagonist DL-amino-phosphonovaleric acid (APV) causes a
blockade of the potentiated response (3). Time points
1-3 are also indicated in B. B:
expression of LTP in wildtype mice ( ,
n = 11) and lack of LTP in eNOS mice
( , n = 10). Local application of
APV causes a blockade of LTP in wildtype mice and small decrease of
synaptic responses in eNOS mice. C:
induction of LTP in wildtype mice ( ,
n = 10) is not prevented by local application of
APV. A 2nd HFS does not induce a further potentiation.
|
|
The role of NMDA receptors in the induction of LTP was studied in
parietal cortical slices from wildtype mice. Application of APV during
the HFS did not prevent the induction of LTP (Fig. 2C).
After termination of the HFS and wash out of APV, the postsynaptic response gradually increased significantly (P < 0.02)
to 125.6 ± 8.8% (n = 10). A second HFS 60 min
after termination of the first HFS did not induce any further
potentiation in the field potential response (124 ± 11.9%, Fig.
2C). These data suggest that the stimulus protocol induced
an NMDA receptor-independent form of LTP. In another control
experiment, we tested the effects of an NOS inhibitor on the induction
of LTP in neocortical slices obtained from wildtype mice. Bath
application of the NOS inhibitor L-NAME prevented the
induction of LTP in wildtype mice (n = 10, Fig.
3), indicating that the stimulus protocol
produced a potentiation that was completely mediated by the
effects of NO.

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Fig. 3.
Blockade of neocortical LTP by NOS inhibition with 200 µM
NG-nitro-L-arginine methyl ester
(L-NAME). A: single field potential
responses before (1) and 50 min after HFS
(2). Note identical amplitude of both responses when
traces are superimposed (1&2). B: average
field potential amplitudes recorded in neocortical slices exposed to
L-NAME (n = 10).
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|
 |
DISCUSSION |
The major conclusions from our study are that 1) mouse
neocortical slices reveal a slow-onset LTP, which in its induction is
NMDA receptor independent and in its maintenance NMDA receptor dependent; 2) that this form of LTP can be prevented by
blockade of NO synthase in the postsynaptic neuron; and 3)
that this NO synthase is predominantly mediated by the eNOS isoform.
Because the induction of NO-mediated LTP is strongly stimulus dependent (Chetkovich et al. 1993
; Wilson et al.
1997
), the lack of LTP in eNOS
mice may have been
attributed to an impairment in basal excitatory synaptic transmission.
However, the input-output response curves did not differ between both
experimental groups, suggesting that our protocol produced the same
orthodromic synaptic stimulation. The gradual increase in the response
amplitude in wildtype mice is in good agreement with earlier
observations obtained in adult rodent hippocampal (Bashir et al.
1993
) and neocortical slices (Aroniadou and Teyler
1992
; Kirkwood et al. 1993
). Furthermore, a
slow-onset form of LTP not requiring the activation of NMDA receptors
for its induction has been previously reported in area CA1 on HFS
(Grover and Teyler 1990
) and after application of
metabotropic glutamate receptor agonist (Bashir et al.
1993
). Whereas the former type of LTP was suppressed by a
voltage-dependent calcium antagonist (Grover and Teyler
1990
), the latter form of LTP was prevented by an antagonist
acting at the metabotropic glutamate receptor (Bashir et al.
1993
). These data suggest that the HFS protocol used in the
present report induced an NMDA receptor-independent form of LTP, which
may depend on calcium entry via voltage-gated calcium channels or
inositol trisphosphate-mediated calcium release from intracellular
stores after activation of metabotropic glutamate receptors
(Wilsch et al. 1998
). The discrepancy between ours and the recent observations of Hensch et al. (1998)
in mouse
visual cortical slices demonstrating the prerequisite of NMDA receptor for the induction of a fast-onset LTP may be explained by the different
stimulus protocols (see Grover and Teyler 1990
) or
different ages of the animals (see Crair and Malenka
1995
).
Our observation on the pronounced APV sensitivity of the potentiated
response is surprising. A potentiation of an NMDA receptor-mediated response with a latency of 4-7 ms has been occasionally observed in
the rat motor cortex (Aroniadou and Keller 1995
), but
the magnitude of this APV-sensitive potentiated field potential
component was considerably smaller. The relatively low magnesium
concentration of 1 mM used in our experiments certainly contributes to
a stronger potentiation of an NMDA receptor-mediated component. In
agreement with previous reports on hippocampal (O'Dell et al.
1991
) and neocortical (Nowicky and Bindman 1993
)
slices, demonstrating a blockade of LTP in the presence of NOS
inhibitors, we also observed in wildtype mice a prevention of LTP by
the NOS inhibitor L-NAME, indicating that the stimulus
protocol produced a potentiation that was completely mediated by the
effects of NO. These data are also compatible with immunocytochemical
studies illustrating moderately high levels of NOS in the supragranular
levels of the rodent cerebral cortex (Bredt et al.
1990
). Taken together, these data suggest that NO plays an
important role in the induction of LTP in the cerebral cortex of adult
mice.
The experimental analysis of neocortical slices obtained from mice
deficient of eNOS allowed the investigation of the question whether
this isoform is the critical synthase in mediating this form of
neocortical LTP. Slices from eNOS
mice showed the usual
posttetanic depression but did not reveal any significant increase in
the field response, suggesting that eNOS, rather than nNOS, is the
dominant synthase of NO production in the postsynaptic neuron during
LTP. This result corresponds well to previous in vitro data in
nNOS
mice, which expressed normal hippocampal LTP
(O'Dell et al. 1994
), and recent observations on
eNOS
mice, which showed no hippocampal LTP to weak
stimuli (Wilson et al. 1997
). Furthermore, Kantor
et al. (1996)
reported that rat hippocampal slices treated with
an adenovirus vector containing a truncated eNOS revealed no LTP in
stratum radiatum. These data strongly suggest that the eNOS isoform
plays an important role in hippocampal synaptic plasticity. However,
Son et al. (1996)
have shown that LTP in stratum
radiatum was most profoundly reduced in doubly mutant mice
(nNOS
/eNOS
), but normal in
nNOS
animals and eNOS
mice.
Our observations suggest that eNOS is also involved in
neocortical LTP, but that the magnitude of potentiation is smaller in
the cerebral cortex than in the hippocampus. Immunocytochemical studies
have shown that staining for eNOS is much weaker in the rodent
neocortex as compared with the hippocampus (Dinerman et al.
1994
). This difference in the localization of eNOS may be one
of the reasons why neocortical LTP shares many features with hippocampal LTP but usually reaches a much smaller amplitude
(Kirkwood et al. 1993
).
This work was supported by Deutsche Forschungsgemeinschaft Grants
Lu 375/3-1 and 3-2 to H. J. Luhmann, DFG Grant "Synaptic Interactions" to H. L. Haas, and a grant from the
Fritz-Thyssen-Stiftung Köln to J. Schrader.
Address for reprint requests: H. J. Luhmann, Institute of
Neurophysiology, University of Düsseldorf, PO Box 101007, D-40001
Dusseldorf, Germany.