1Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794; and 2Department of Neuroscience, School of Medicine, University of New Mexico, Albuquerque, New Mexico 87131-5223
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ABSTRACT |
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Yuzaki, Michisuke and
John A. Connor.
Characterization of L-Homocysteate-Induced Currents
in Purkinje Cells From Wild-Type and NMDA Receptor Knockout
Mice.
J. Neurophysiol. 82: 2820-2826, 1999.
L-Homocysteic acid (HCA), an endogenous
excitatory amino acid in the mammalian CNS, potently activates
N-methyl-D-aspartate (NMDA) receptors in
hippocampal neurons. However, the responses to HCA in Purkinje cells,
which lack functional NMDA receptors, have been largely unexplored: HCA
may activate conventional non-NMDA receptors by its mixed agonistic
action on both NMDA and non-NMDA receptors, or it may activate a novel
non-NMDA receptor that has high affinity for HCA. To test these
possibilities, we compared the responses to HCA in cultured Purkinje
cells with those in hippocampal neurons by using the whole cell
patch-clamp technique. To clearly isolate HCA responses mediated by
non-NMDA receptors, we complemented pharmacological methods by using
neurons from mutant mice (NR/
) that lack functional
NMDA receptors. A moderate dose of HCA (100 µM) induced substantial
responses in Purkinje cells. These responses were blocked by non-NMDA
receptor antagonists but were insensitive to NMDA receptor antagonists.
HCA also activated responses mediated by non-NMDA receptors in both
wild-type and NR1
/
hippocampal neurons. HCA responses
in Purkinje cells had a pharmacological profile (EC50 and
Hill coefficient) very similar to that of non-NMDA receptor components
of HCA responses in hippocampal neurons. Moreover, the amplitude of the
non-NMDA receptor component of HCA responses was directly correlated
with that of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
(AMPA)- and kainate-induced responses in both types of neurons. Finally, in both types of neurons, HCA currents mediated by non-NMDA receptors were potently blocked by the AMPA receptor antagonist GYKI52466. These findings indicate that HCA-activated AMPA receptors in
Purkinje cells are similar to those in hippocampal neurons and that
there is no distinct HCA-preferring receptor in Purkinje cells. We also
found that in hippocampal neurons, the EC50s of HCA for
non-NMDA receptors and for NMDA receptors were more similar than
originally reported; this finding indicates that HCA is similar to
other mixed agonists, such as glutamate. HCA responses may appear to be
selective at NMDA receptors in cells that express these receptors, such
as hippocampal neurons; in cells that express few
functional NMDA receptors, such as Purkinje cells, HCA may appear to be
selective at non-NMDA receptors.
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INTRODUCTION |
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L-homocysteic acid (HCA), a sulfur-containing
analogue of L-glutamate, fulfills several criteria for an
endogenous neurotransmitter. It is released from rat brain slices
either by depolarization in a Ca2+-dependent
manner (Do et al. 1986) or by electrical stimulation (Klancnik et al. 1992
). It is also taken up by a
specific low-affinity uptake system (Cox et al. 1977
;
Ito et al. 1991
). Because HCA responses are potently
blocked by N-methyl-D-aspartate (NMDA) receptor
antagonists such as D,L-2-amino-5-phosphonovalerate (APV) but are little affected by non-NMDA receptor antagonists such as
6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and
6-nitro-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), HCA has been
considered to act predominantly via NMDA receptors in neurons from
various regions, including neocortex (Knopfel et al.
1987
), striatum (Do et al. 1986
), hippocampus (Ito et al. 1991
; Patneau and Mayer 1990
;
Provini et al. 1991
), olfactory cortex (Collins
and Brown 1986
), deep cerebellar nuclei (Audinat et al.
1990
), and spinal cord (Mayer and Westbrook
1984
).
In the cerebellum, HCA is thought to be involved in climbing
fiber-Purkinje cell synapses, because the elimination of climbing fibers led to decreased release of HCA in cerebellar slices from adult
rats (Vollenweider et al. 1990). Yet it has not been
clear what receptors are activated by HCA in Purkinje cells. If HCA acts predominantly on NMDA receptors, as it is thought to act in other
neurons, it should not activate significant currents in Purkinje cells,
which have few functional NMDA receptors (Audinat et al.
1990
; Kimura et al. 1985
; Yuzaki et al.
1996b
). However, the fact that HCA induces substantial currents
that are insensitive to APV but sensitive to CNQX in Purkinje cells
(Audinat et al. 1990
; Lee et al. 1988
)
indicates that HCA activates non-NMDA receptors. Thus a unique subtype
of glutamate receptor may exist in Purkinje cells (Vollenweider
et al. 1990
). Alternatively, because at high concentrations HCA
substantially activates non-NMDA receptors in hippocampal neurons
(Patneau and Mayer 1990
), the apparent activation of
non-NMDA receptors in Purkinje cells may reflect the mixed action of
HCA on both receptor types.
A comparison of non-NMDA receptor activation by HCA in the two types of neurons would differentiate between these possibilities. If the HCA responses in hippocampal neurons had no non-NMDA receptor component, or if the pharmacological properties of this component, such as dose-response relationship and antagonist profiles, were different from those in Purkinje cells, then these findings would indicate that Purkinje cells contain a specific receptor for HCA. However, there have been several technical obstacles to the pharmacological characterization of endogenous excitatory amino acid (EAA) candidates such as HCA. First, the action of endogenous EAA often has a mixed action involving both NMDA and non-NMDA receptors. Second, glutamate receptor antagonists are not always selective. For example, when mixed agonists are applied in the presence of NMDA receptor antagonists to identify the components mediated by non-NMDA receptors, NMDA receptor current may break through at higher concentrations of agonists.
Moreover, receptor classification based on pharmacological agonists and
antagonists is not infallible. For example, we previously demonstrated
that NMDA receptor antagonists, such as APV, show antagonistic effects
on aspartate responses in Purkinje cells from mice with disrupted
NMDAR1 genes (NR1/
mice) (Yuzaki et
al. 1996a
). However, because Purkinje cells express little NR2
protein (Monyer et al. 1994
),
NR1
/
Purkinje cells contain no proteins
related to conventional NMDA receptors. Thus these "NMDA
receptor-specific" antagonists may bind to proteins other than NMDA
receptors in Purkinje cells. Such potential pitfalls limit the
conclusiveness of studies that rely only on current pharmacological
antagonists. To circumvent these problems, we used neurons from
NR1
/
mice to complement conventional
pharmacological antagonists in characterizing the HCA responses in
Purkinje cells.
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METHODS |
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Culture preparation
Primary cultures were prepared from neonatal mice within 3-4 h
of birth as previously described (Forrest et al. 1994;
Yuzaki and Mikoshiba 1992
). Cultures were analyzed after
8-15 d in vitro. For unambiguous identification of Purkinje cells in
immature cultures, we routinely performed calbindinD28k staining as
previously reported (Yuzaki et al. 1996b
). NR1 geotype
was determined by polymerase chain reaction studies of DNA samples
prepared from mouse tail clips (Yuzaki et al 1996b
).
Recording system
Membrane currents were measured by using standard whole cell
patch-clamp methods with a List EPC-7 amplifier (Medical Systems, Greenvale, NY) as previously reported (Yuzaki et al
1996a,b
). Saline solution in the electrodes was composed of (in
mM) 130 CsMeSO3, 10 CsCl, 2 MgCl2, 0.1 CaCl2, 5.5 EGTA,
10 HEPES, and 2 Na2-ATP (pH was adjusted to 7.3 with CsOH). Dextran-conjugated rhodamine (0.625 mg/ml; Molecular
Probes, Eugene, OR) was added. Electrodes filled with the recording
saline had resistance of ~6-7 M
. Series resistance was 11-13
M
and was partially (50-60%) compensated. The bath solution
contained (in mM) 150 NaCl, 4.5 KCl, 2 CaCl2, 10 HEPES, and 20 glucose (pH 7.3). Mg2+ was excluded
from the external solution throughout the experiments unless otherwise
stated. Tetrodotoxin (TTX, 1 µM) and picrotoxin (100 µM) were added
to the solution to block spontaneous electrical activity and glycine or
-aminobutyric acid (GABA) channels. All drugs were dissolved in the
recording solution. The chamber was continuously perfused (1-2 ml/min)
at room temperature. Drugs were applied by the "Y-tube" method,
which had a time constant of 8-9 ms for solution exchange at the tip
of the electrode and 15-20 ms in the area surrounding the neuron
(Yuzaki et al. 1996b
). Currents were filtered at 1 kHz
and digitized at 3 kHz. Membrane potential was corrected for the liquid
junction potential.
Drugs
NMDA, kainate, HCA, glycine, TTX, and picrotoxin were obtained from Sigma (St. Louis, MO); 7-Cl-kynurenate (7-Cl-Kyn), AMPA, and 1-(4-aminophenyl)-4-methyl-7,8-methyl-enedioxy-5H-2,3-benzodizepine HCl (GYKI52466) were obtained from Research Biochemicals (Natick, MA); and 3-(DL)-2-carboxypiperazin-4-yl-propyl-1-phosphonic acid (CPP) and NBQX were obtained from Tocris Cookson (Ballwin, MO).
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RESULTS |
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HCA responses in neurons from wild-type and NR1-knockout mice
First, responses to HCA (100 µM) were pharmacologically
characterized in wild-type neurons. In voltage-clamped (60 mV)
wild-type Purkinje cells, HCA induced large currents (1.5 ± 0.2 nA, mean ± SE, n = 18) that were
insensitive to an NMDA receptor-antagonist mixture containing CPP (50 µM), 7-Cl-Kyn (0.5 µM), and Mg2+ (1 mM; Fig.
1A, top trace). These
currents were not blocked by Mg2+ at all holding potentials
(Fig. 1B), but they were completely blocked by the
non-NMDA receptor antagonist NBQX (5 µM; Fig. 1A). Thus HCA activated non-NMDA receptors in wild-type Purkinje cells.
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Earlier pharmacological studies of hippocampal neurons showed
that HCA activated non-NMDA receptors with an EC50 of 477 and a Hill coefficient of 1.4 (Patneau and Mayer 1990),
indicating that 100 µM HCA would activate only 10% of maximal
currents. If these results were applicable to Purkinje cells, maximal
currents would be expected to reach 15 nA, whereas observed maximal
steady-state non-NMDA receptor currents were ~3 nA (e.g., Fig.
2B). These results suggest
that the pharmacology of HCA responses in Purkinje cells may differ
from the reported responses in hippocampal neurons.
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In contrast, HCA-induced currents in wild-type hippocampal neurons were
substantially reduced by the NMDA receptor-antagonist mixture (14 ± 3% of currents in controls; n = 26) but were
relatively insensitive to NBQX (89 ± 4% of currents in controls,
n = 26; Fig. 1A, bottom trace). The
HCA-induced current was reduced by Mg2+ in a
voltage-dependent manner (Fig. 1B), as with classical
NMDA receptors. These results are consistent with earlier reports that HCA, at low doses, acts as a selective NMDA receptor agonist in hippocampal neurons (Patneau and Mayer 1990). However,
it should be noted that the fact that residual HCA-induced currents in
the presence of NMDA receptor antagonists were blocked by the addition of NBQX (Fig. 1C) suggests that non-NMDA receptors were
also activated by 100 µM HCA.
To confirm these results, we used neurons from NR1/
mice. As with wild-type mice, HCA induced large currents (1.8 ± 0.2 nA, n = 15) that were insensitive to the NMDA
receptor-antagonist mixture in Purkinje cells (Fig. 1C, top
trace). These currents were not blocked by Mg2+ at
all holding potentials (Fig. 1D), but they were
completely blocked by the non-NMDA receptor antagonist NBQX (5 µM;
Fig. 1C). Similarly, HCA activated substantial currents
in NR1
/
hippocampal neurons that showed no response to
100 µM NMDA (Fig. 1C, bottom trace). The currents were
not blocked by Mg2+ at all holding potentials (Fig.
1B) but were completely blocked by NBQX (Fig.
1A). Thus 100 µM HCA appears to activate non-NMDA receptors in both hippocampal neurons and Purkinje cells from NR1
/
mice. These results were comparable with those
obtained in wild-type neurons in the presence of NMDA receptor
antagonists, indicating the validity of pharmacological isolation of
non-NMDA receptor components in wild-type neurons. Moreover, the
suggestion that a subtype of the classic NMDA receptor is involved in
HCA responses in Purkinje cells (Vollenweider et al.
1990
) is unlikely to be valid, because HCA induced similar
responses in neurons lacking functional NMDA receptors.
Non-NMDA receptor activation in Purkinje cells may result from the mixed action of HCA on non-NMDA receptors. In wild-type hippocampal neurons, the potent activation of NMDA receptors, which are abundantly expressed, can mask the minor activation of non-NMDA receptors by 100 µM HCA. In contrast, HCA may induce large currents in Purkinje cells that express abundant non-NMDA receptors but have no functional NMDA receptors. It is also possible that Purkinje cells express distinct non-NMDA receptors that have higher affinity for HCA than do those in hippocampal neurons. To test these two possibilities, we next compared the pharmacological properties of HCA responses in Purkinje cells with those in hippocampal neurons.
Dose-response relationship of non-NMDA receptor component of HCA response
First, we analyzed the concentration-response curves of
HCA-activated responses mediated by non-NMDA receptors (Fig. 2). In wild-type neurons, HCA was applied in the presence of the NMDA receptor
antagonist cocktail to block NMDA receptor-mediated responses. Measurement of the peak current response at non-NMDA receptors was
complicated by the extremely rapid onset of desensitization, which can
have a time constant of <10 ms (Brorson et al. 1995; Patneau and Mayer 1990
) (greater than the speed of our
drug application) in whole cell recordings. Moreover, in large cells
such as Purkinje cells, the large electrotonic distance of dendrites
can potentially distort the rapid phase of desensitization.
Furthermore, the EC50 values obtained by
analyzing steady-state currents, but not the rapid inactivating phase,
have been shown to reflect the ligand affinity obtained from binding
assays (Patneau and Mayer 1990
) and biological assays
(Brorson et al. 1995
). Therefore we analyzed the
steady-state current to compare the receptors activated by HCA in the
two types of neurons.
The amplitudes of steady-state currents were well fit by the logistic
equation for wild-type and NR1/
hippocampal
neurons: wild-type neurons had an EC50 of
106 ± 12 µM (mean ± SE, n = 6) and a Hill
coefficient of 1.8 ± 0.2, whereas NR1
/
hippocampal neurons had an EC50 of 98 ± 11 µM (n = 7) and a Hill coefficient of 1.6 ± 0.1 (Fig. 2A). The logistic equation also showed a similar
dose-response relationship for Purkinje cells from wild-type and
NR1
/
mice: wild-type cells had an
EC50 of 96 ± 9 µM (n = 7)
and a Hill coefficient of 1.6 ± 0.1, whereas cells from
NR1
/
mice had an EC50
of 88 ± 10 µM (n = 9) and a Hill coefficient of
1.4 ± 0.1 (Fig. 2B). Conversely, HCA activated NMDA
receptors with an EC50 of 14 ± 4 µM
(n = 5) and a Hill coefficient of 1.4 ± 0.3, consistent with the previously reported EC50 of
12.9 µM (Patneau and Mayer 1990
). Thus non-NMDA
receptors in hippocampal and Purkinje neurons (both
NR1
/
and wild-type) have very similar
EC50s for HCA. These results support the
hypothesis that HCA is a mixed agonist that activates conventional
non-NMDA receptors in both Purkinje cells and hippocampal neurons.
It is unlikely that the EC50 value we obtained
for HCA at AMPA receptors, which was lower than previously reported
values (Patneau and Mayer 1990), was caused by the
voltage drop across the series resistance. If we had underestimated
EC50 because of the voltage-clamp error, we would
have obtained much lower values of EC50 for HCA
at NMDA receptors, because the larger HCA-induced NMDA receptor
currents would have caused larger voltage errors. However, the
EC50 of HCA at NMDA receptors in our study was
very similar to the reported value.
Although the differences were not statistically significant, the
EC50s for HCA at AMPA receptors tended to be
larger in wild-type neurons than in NR1/
neurons. Similarly, they were larger in hippocampal neurons than in
Purkinje cells. This finding suggests that EC50s
may increase in the presence of functional NMDA receptors. The
possibility that, at higher concentrations of HCA, NMDA receptors may
be activated even in the presence of blockers and thereby increase the
apparent EC50s is ruled out by the use of
NR1
/
neurons.
Relationship between HCA current and AMPA or kainate current
If HCA is a mixed agonist that activates similar non-NMDA receptors in both Purkinje cells and hippocampal neurons, cells that show large HCA responses mediated by non-NMDA receptors should have abundant non-NMDA receptors. We compared non-NMDA receptor currents induced by AMPA (20 µM), kainate (10 µM), and HCA (100 µM). The amplitude of HCA currents in all types of neurons was strongly correlated with that of AMPA currents (Fig. 3A) and kainate currents (Fig. 3B). In contrast, NMDA receptor currents induced by 100 µM NMDA were not correlated with HCA currents mediated by non-NMDA receptors (Fig. 3C). These results are consistent with the hypothesis that 100 µM HCA potently activates conventional non-NMDA receptors in both types of neurons.
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Interestingly, non-NMDA currents in NR1/
hippocampal neurons were larger than those in wild-type counterparts
(P < 0.01, Fig. 3D). This difference may be
caused by the up-regulation of non-NMDA receptors in the absence of
functional NMDA receptors. To test this possibility, we treated
cultures of wild-type hippocampal neurons with the NMDA receptor
antagonist cocktail from day 0 to day 14 in vitro
and compared AMPA-induced currents with those in untreated sister
cultures. Unlike NR1
/
neurons, neurons
treated with NMDA receptor antagonists displayed smaller responses to
20 µM AMPA (892 ± 107 pA, n = 25) than did control neurons (1,356 ± 185 pA, n = 17).
Moreover, if the loss of functional NMDA receptors were involved in the
up-regulation of functional non-NMDA receptors, the amplitude of
non-NMDA receptor currents would be inversely correlated with that of
NMDA receptor currents. However, there was no correlation between the
amplitudes of NMDA and non-NMDA currents (Fig. 3C). Thus the
larger AMPA current in NR1
/
hippocampal
neurons may reflect other factors. The presence of NR1 and some NR2
mRNAs as early as embryonic day 13 suggests that regulation
of non-NMDA receptors may be determined by the functions of NMDA
receptors at earlier stages in neurodevelopment. It is also possible
that the increase in non-NMDA currents may require complete loss of
NMDA receptor function, a condition that may not be achieved by
pharmacological blockade of NMDA receptors.
Sensitivity of HCA currents to GYKI52466
To further confirm the hypothesis that HCA activates conventional
non-NMDA receptors, and to test which non-NMDA receptors are activated
by HCA, we analyzed the dose-inhibition relationship of GYKI52466, a
potent and selective antagonist for AMPA receptors, in HCA responses in
NR1/
hippocampal and Purkinje cells. In
hippocampal cells, the IC50 was 5 ± 1 µM
(n = 7), and the Hill coefficient was 1.0 ± 0.1; in Purkinje cells, these values were 6 ± 2 µM
(n = 6) and 1.1 ± 0.2 (Fig. 1E).
GYKI52466 is reported to inhibit AMPA receptors in wild-type
hippocampal and Purkinje cells with IC50s between 7.5 and 10 µM, whereas it inhibits kainate receptors in Purkinje cells with an IC50 of 105 µM (Herrling
et al. 1989
; Provini et al. 1991
; Renard
et al. 1995
). Thus our findings indicate that HCA activated the
AMPA subtype of non-NMDA receptors in both hippocampal and Purkinje cells.
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DISCUSSION |
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Although it has been suggested that HCA is involved in
climbing-fiber-Purkinje cell synapses, the receptors activated by HCA in Purkinje cells have not been well characterized. In this study, by
taking advantage of NR1/
neurons, we have
unambiguously demonstrated that HCA activates AMPA receptors in
Purkinje cells by its mixed action on both NMDA and non-NMDA receptors.
First, non-NMDA receptors activated by HCA in Purkinje cells had
pharmacological profiles (EC50 and Hill coefficient) very similar to those of non-NMDA receptors activated by
HCA in hippocampal neurons (Fig. 2). Second, the amplitude of the
non-NMDA receptor component of HCA responses was directly correlated
with that of AMPA- and kainate-induced responses in both types of
neurons (Fig. 3). Finally, in both types of neurons, HCA currents
mediated by non-NMDA receptors were potently blocked by the AMPA
receptor antagonist GYKI52466, with similar IC50s (Fig. 1E). Thus unlike aspartate, which activates receptors
that are distinct from both NMDA and non-NMDA receptors, (Yuzaki
et al. 1996a
), HCA has no distinct preferential receptor in
Purkinje cells. In addition, HCA does not activate the aspartate
receptors (Yuzaki et al. 1996a
).
We have demonstrated that 100 µM HCA potently activates non-NMDA
receptors in Purkinje cells, whereas it appears to selectively activate
NMDA receptors in wild-type hippocampal neurons. This apparent
discrepancy can be explained by the mixed action of HCA on both NMDA
and non-NMDA receptors. The ratio of the EC50 for equilibrium responses at non-NMDA receptors to the
EC50 for equilibrium responses at NMDA receptors
reflects the degree of selectivity of the agonist at NMDA receptors.
L-glutamate, for example, which has mixed action on NMDA
and non-NMDA receptors, has a ratio of 8.2 (Patneau and Mayer
1990). Because the EC50 of HCA was 12.9 µM for NMDA receptors and 477 µM for AMPA receptors, yielding a
ratio of 36.9, HCA was reported to be a selective agonist at NMDA
receptors (Patneau and Mayer 1990
). However, we obtained a ratio of 7.5 in wild-type hippocampal neurons, in which the EC50 of HCA was 14 µM for NMDA receptors and
106 µM for AMPA receptors. Therefore we propose that HCA is similar
to other mixed agonists, such as glutamate, and that it has only modest
selectivity at NMDA receptors. Because the maximum amplitude of
equilibrium responses to non-NMDA receptor agonists is usually <10%
of the maximal response that can be evoked by saturating doses of NMDA
agonists (Patneau and Mayer 1990
), HCA responses may
appear to be selective at NMDA receptors in cells that express these
receptors, such as hippocampal neurons. In cells that express few
functional NMDA receptors, such as Purkinje cells and
NR1
/
hippocampal neurons, HCA may appear to
be selective at non-NMDA receptors.
Given that HCA is a mixed agonist that has selectivity very similar to
that of glutamate at NMDA and non-NMDA receptors, what could be the
functional role of this endogenous transmitter candidate? Several
important differences exist between glutamate and HCA. HCA is much less
effective than glutamate in activating metabotropic glutamate receptors
(Porter and Roberts 1993). HCA is taken up, at least
partially, by a specific uptake system that does not involve known
glutamate transporters (Ito et al. 1991
). In addition, unlike glutamate, which is released from both nerve terminals and
surrounding glia, HCA is released only from glia (Grandes et al.
1991
). Recently, glutamate released from glia has been shown to
modulate synaptic neurotransmission (Araque et al.
1999
). Thus HCA released from surrounding glia may also modify
basic neurotransmission, making further characterization of its release and actions on neurons important in understanding integrative functions.
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ACKNOWLEDGMENTS |
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We thank S. C. Sun and L. M. Verselis for technical support and S. Hestrin and T. Curran for critical reading of the manuscript.
This work was supported in part by National Cancer Institute Support Grant P30CA-21765 and by American Lebanese Syrian Associated Charities.
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FOOTNOTES |
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Address for reprint requests: M. Yuzaki, St. Jude Children's Research Hospital, Dept. of Developmental Neurobiology, 332 North Lauderdale, Memphis, TN 38105-2794.
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 8 March 1999; accepted in final form 22 July 1999.
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REFERENCES |
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