| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 17, 14367-14369, April 26, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
§,¶, and
From the Medical Scientist Training Program, the
§ Neuroscience Program, and the ¶ Department of
Pharmacology, University of Colorado Health Sciences Center, Denver,
Colorado 80262
Received for publication, February 6, 2002, and in revised form, March 11, 2002
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Aging is known to alter many physiological
processes within the brain including synaptic responses, long-term
potentiation, learning, and memory. Aging has also been shown to
alter the expression and distribution of
N-methyl-D-aspartate (NMDA) receptors in many different brain regions, including the hippocampus. Additionally, we
have recently reported that young adult rats show an
activity-dependent increase in the surface expression of
NMDA receptors. We have extended these observations in the present
study in aged animals and have found that aged Fischer 344 rats fail to
show activity-dependent changes in the surface distribution
of NMDA receptors. In conjunction with this observation we have also
noted that aged rats show an expression deficit in the C2 splice
variant of the NR1 subunit. This subunit is preferentially shifted to
the surface following stimulation in young adult animals. As the NMDA
receptor is thought to play an important role in neuronal signaling,
these observations suggest possible new areas of dysfunction in this
receptor that might underlie age-related deficits in neuronal physiology.
Aging has long been known to have detrimental effects upon the
function of the nervous system, but the precise molecular mechanisms involved have not been elucidated. Multiple signaling pathways involved
in inflammation, antioxidant defenses, developmental gene regulation,
and cellular signaling have been implicated as playing a role in the
age-related deterioration (1-9). The
N-methyl-D-aspartate (NMDA)1 receptor is thought
to play an important role in neuronal signaling because of its ability
to flux calcium. This occurs in response to glutamate and concomitant
depolarization, thus allowing the NMDA receptor to function as a
"coincidence detector." This capability has led some to suggest
that the NMDA receptor could be involved in "Hebbian" synaptic
plasticity. We and others (5, 9-14) have noted that as part of the
aging process, a specific decrease in the expression and function of
the NMDA receptor occurs in multiple areas of the brain. Moreover, we
have recently demonstrated a correlation between the amount of NR2B
subunit of the NMDA receptor in the hippocampus and spatial learning
task performance in aged rats (15).
Long-term potentiation (LTP) is a long lasting increase in synaptic
efficacy, which can be produced by repeated stimulation of neurons in
many regions of the brain including the hippocampus. We have recently
reported that LTP stimulation produces a redistribution of NMDA
receptors from an intracellular pool to the surface of hippocampal
neurons from young adult rats (16). To extend these studies, we have
examined the effect of LTP stimulation upon the surface distribution of
NMDA receptors in Fischer 344 rats at 4, 16, and 24 months to examine
the effect of aging upon this activity-dependent
redistribution of receptor. Additionally, in young adult rats, there is
a preferential redistribution of the C2 cassette-containing NR1
splice variant subunits to the surface. We therefore also examined the
expression of NR1 splice variants in aged animals.
Animals--
Fisher 344 rats were obtained from the National
Institute of Aging breeding colony at Harlan Sprague-Dawley at
6, 16, and 24 months of age. After sacrifice the brains were removed
and the hippocampi dissected out. 400-µm slices were prepared on a McIlwain tissue slicer and allowed to recover at 32 °C in a
perfusion chamber containing ACSF (124 mM NaCl, 4 mM KCl, 1 mM MgSO4, 2.5 mM CaCl2, 10 mM dextrose, 1 mM KH2PO4, 25.7 mM
NaHCO3) at a rate of 4 ml/min for 90 min. For surface
expression experiments, CA1 mini-slices were prepared directly from
isolated CA1 as described previously (16).
Semiquantitative Western Blotting--
Tissue samples for
protein expression were sonicated into Tris-buffered SDS (10 mM Tris, pH 8.0, 2 mM EDTA, 1% SDS). Protein concentrations were determined using a modified BCA assay (Pierce). Samples were loaded onto 7.5% SDS-PAGE along with a five-point standard curve for comparative analysis. Whole rat brain homogenate was
used as the standard with aliquots from the same batch freshly prepared
before each set of gels was run. Gels were transferred to
polyvinylidene difluoride membrane using a submarine transfer apparatus
(Owl Scientific). Following transfer, blots were dried and blocked with
5% milk or 3% BSA in TTBS (10 mM Tris/HCl, pH 7.4, 100 mM NaCl, 0.1% Tween 20). Primary antibody was applied for
16 h at 4 C in 1% milk or 1% BSA in TTBS. Secondary antibody was
applied for 2 h at 25° in 1% milk or 1% BSA in TTBS. Gels were
developed using SuperSignal (Pierce) and were imaged and analyzed using
an Alpha Imager (Alpha Innotech). Quantitation was performed using
Excel (Microsoft), and statistics were performed using StatView (SAS
Institute). Antibodies to NR2A, NR2B, the NR1 splice variants
(N-terminal cassette, C1 cassette, C2 cassette, C2' alternate reading
frame), and synapsin were produced in our laboratory and have been
described previously (16). Antibodies were obtained commercially, NR1
from PharMingen and GluR2 from Chemicon.
Electrophysiology--
Field EPSPs were obtained by the usual
protocols. In brief, the Schaeffer collateral pathway was stimulated
using a bipolar nichrome electrode, and recordings were made from the
dendritic layer of CA1 using a silver electrode contained in a
finely drawn glass capillary tube containing ACSF. These field
EPSPs were considered as representing mainly AMPA receptor responses.
To obtain NMDA receptor responses, AMPA EPSPs were blocked by the
administration of 2-µm NBQX. For electrophysiological studies,
1 train of 1-s 100-Hz stimulation was delivered at 50% of the maximal
NMDA receptor response. For biochemical studies, LTP was induced in the
slice using a multi-rake electrode, which we have described
previously. In short, 16 rake electrodes were fixed to a rigid support
in a 4 × 4 matrix arrangement allowing for the stimulation of 4 slices at 4 rake positions each (16 stimulation points/mini-slice).
Columns of 4 rake electrodes on the same slice were oriented parallel to each other and separated by 500 µm. Adjacent columns were
separated by 2.5 mm. LTP was induced by the sequential delivery of four 1-s trains of 100-Hz stimulation per rake electrode separated by
30 s. Rake positions were stimulated in turn across the slice so
that only one rake/slice was stimulating at any given time.
Cross-linking for Surface Expression Analysis--
To assay
basal and stimulation-dependent surface expression, we
employed the membrane impermeable cross-linker,
bis(sulfosuccinimidyl)suberate (BS3, Pierce) as described
previously (16). For determination of basal surface expression values,
slices were allowed to recover for a minimum of 90 min and transferred
to either ACSF or ACSF containing 1 mg/ml BS3 for 45 min at
4 °C. To assay stimulation-dependent changes in surface
expression, slices were harvested at 30 min post-LTP, and both
stimulated and matched control slices were treated with cross-linker. A
minimum of three slices per control or treatment groups was included
for each animal. To terminate the reaction and quench remaining
BS3, slices were washed three times in cold ACSF containing
20 mM Tris, pH 7.6. Surface expression was determined
following semiquantitative Western blot analysis and a comparison of
cross-linked samples to nontreated controls.
We have previously reported that aging is associated with a
selective decrease in the expression of the NR1 and NR2B subunits of
the NMDA receptor with no change in the expression of the NR2A subunit
of the NMDA receptor or in the expression of the GluR2 subunit of the
AMPA receptor (10). Other groups have found similar results, although
age-related decreases in the expression of the NR2A subunit have also
been reported (9, 11, 12, 14). In this report we have extended our
previous studies by analyzing the surface expression of NMDA receptors
in the CA1 region of the hippocampus across the life span of the
Fischer 344 rat. We saw no significant differences in basal surface
expression in any of the examined targets in rats at 6, 16, and 24 months of age (data not shown). Thus, in marked contrast to the
expression of NMDA receptors in aged animals, which is down-regulated,
the percentage of the receptor pool that is basally expressed at the surface remains relatively constant with age.
We next sought to examine the effect of aging upon LTP-related changes
in surface expression of glutamate receptors in Fischer 344 rats at 6, 16, and 24 months using a multiple "rake" stimulation device (Fig.
1). Surface expression changes following
HFS were measured by cross-linking of both stimulated and nonstimulated matched control slices 30 min post-LTP. Samples were then used for
semiquantitative Western blotting; as the cross-linker (BS3) is
membrane-impermeable, only surface expressed molecules are cross-linked, rendering them unable to enter polyacrylamide gels. The
internal pool of receptor, which is not cross-linked, is measured directly. Equal amounts of total protein are loaded, so that changes in
surface expression with treatment are reflected in alterations in the
levels of intracellular proteins. As previously described, LTP
stimulation led to an increase in surface expression of NR2A, 2B, and
NR1 in young adult animals (16 months). However, no LTP-related changes
were seen in the surface expression of NR1, 2A, or 2B subunits in the
24-month group (Fig. 2). An intermediate
effect was seen in the 16-month group. In this group, NR2A shows a
trend toward an increase in surface expression with LTP
(p = 0.11) (Fig. 2). NR2B was not significantly
effected (p = 0.39), nor was NR1 (p = 0.52). There were no LTP-related effects on GluR2 in any age group
(Fig. 2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 1.
Multi-rake electrode stimulation.
Hippocampal CA1 slices were stimulated with a multi-rake
electrode to saturate the amount of LTP within slices. This saturation
is intended to produce sufficient tissue for biochemical analysis and
reduce the amount of unstimulated tissue included in the biochemical
analyses. A description of the multi-rake electrode is found under
"Experimental Procedures."
View larger version (51K):
[in a new window]
Fig. 2.
Surface expression changes with LTP.
Surface expression was assayed with the use of a membrane-impermeable
cross-linking agent (BS3), which only cross-links
proteins on the surface of cells. The cross-linked product is unable to
enter polyacrylamide gels, so that only the intracellular pool is
resolved by Western blotting. Changes in surface expression are
detected by observing changes in this directly detected intracellular
pool. A, no significant changes were seen in the surface
expression of NR1 (mean % change = 3.34 ± 7.26%), 2A (mean
% change = 2.38 ± 6.94%), 2B (mean % change = 
3.96 ± 7.61%), or GluR2 (mean % change = 7.26 ± 8.20%) in the 24-month group (n = 7, all p
values > 0.9). In the 16-month group, NR2A shows a trend toward a
significant change indicating an increase in surface expression with
LTP (mean % change = 26.58 ± 6.86%, n = 7, p = 0.11). NR2B was not significantly effected
(mean % change = 14.58 ± 16.64%, n = 7, p = 0.39) nor was NR1 (mean % change = 9.61 ± 6.02%, n = 7, p = 0.52) or GluR2
(p = 1). Six-month-old animals showed a significant
increase in the surface expression of NR1 (mean % change = 24.37 ± 6.72%, n = 6, p < 0.05)
and NR2A (mean % change = 21.23 ± 1.68%, n = 6, p < 0.05) and no change in the surface expression
of NR2B (mean % change = 14.76 ± 9.31, n = 6, p = 0.34) or GluR2 (mean % change = 3.69 ± 7.28%, n = 6, p = 0.88).
Statistical analyses were performed by ANOVA with Fisher's PLSD
post-hoc test. B, representative
immunoblots.
As shown previously, the LTP-related increase in surface expression of
the NMDA receptor is accompanied by an increase in the function of the
NMDA receptor in the CA1 region of the hippocampus. We have replicated
this result in young adult animals, as delivery of a single train of
100-Hz stimulation leads to a potentiation of NMDA receptor current of
28% (p < 0.05) (Fig.
3). In 16-month-old animals, stimulation
results in a trend toward an increase in NMDA receptor-mediated current
(20%, p = 0.14) (Fig. 3). However, in aged animals (24 months), no increase in NMDA receptor current is seen (Fig. 3). All
NMDA receptor responses were blocked by the addition of APV
(2-amino-5-phosphopentanoic acid).
|
We have also shown that the increase in NR1 surface expression seen
with activity in young adult rats is specific for the C2 cassette
containing splice variants (16). Thus, as the NR1 subunit is not
redistributed in aged animals, we sought to measure the total
expression level of the NR1 subunit splice variants in aging. We find
that there is a significant decrease in the total expression of the C2
splice variant in aged animals (Fig. 4).
Interestingly, the total expression of subunits containing the N1, C1,
and C2' cassettes is not altered with age.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
NMDA receptor dysfunction in the hippocampus of aged animals has been demonstrated by multiple groups using a variety of techniques (5, 13). We and others have also demonstrated age-related reductions in the expression of the NMDA receptor. In this report we extend those findings by examining the surface expression of the NMDA receptor subunits in the CA1 region of the hippocampus in 6-, 16-, and 24-month-old Fischer 344 rats. We have found that aging does not affect the basal surface expression of NMDA receptors. Although aged animals express less of the receptors, the percentage of total cellular receptor on the surface does not change.
We have recently documented activity-dependent changes in the surface expression of NMDA receptors (16). Extending those findings, we find that aged animals are deficient in the ability to increase the quantity of NMDA receptor at the surface of the neuron in response to activity. Young animals show a significant increase in the amount of NR1 and NR2A subunits at the surface of the neuron (NR2B increases are seen but are not significant), which is not seen in aged animals. Similarly, aged animals fail to show an increase in NMDA receptor-mediated current post-LTP stimulation as is seen in young animals. These findings, in combination, are suggestive of a plasticity deficit in the function of aged neurons, whereby aged animals are unable to increase the amount of NMDA receptor at the surface in response to stimulation. If NMDA receptors are important for forming a "synaptic tag" for synapse formation or enlargement, as has been suggested previously, aged animals should be compromised in their ability to increase synaptic contacts in this fashion. Thus, this described defect in activity-dependent regulation may indicate a mechanism for decreased plasticity in aging mediated by a decrease in the formation or enlargement of synapses with activity. Cycling of receptors and other proteins into membranes occurs both constitutively and with activation of various signaling cascades. Our data suggest that constitutive cycling of NMDA and AMPA receptors is not altered with age. In contrast, activity-dependent cycling of NMDA receptors appears to be altered profoundly as a function of age. The mechanism of this selective deficit in receptor trafficking is unknown; however, one clue may be found in our previous observation that the increase in surface expression of the NR1 subunit is preferential for NR1 subunit splice variants containing the C2 cassette. In examining the expression of NR1 splice variant proteins in aged animals, we find that there is a specific deficit in the expression of the C2 cassette containing subunits. It has been suggested that the cellular quantity of the NR1 subunit protein is determined in part by assembly with other subunits and that the differential assembly of subunits may determine the intracellular distribution of receptor (17-19). Thus, the age-related reduction in the amount of C2 splice variant NR1 subunit may impair the activity-dependent cycling of NMDA receptors. Age-related decreases in the expression of NR2 subunits could also contribute to this scenario, reducing the amount of total functional receptor that could be assembled.
These findings extend our understanding of the cellular distribution of
NMDA receptors in aged animals and describe a new deficit in the
activity-dependent regulation of the cellular distribution of these receptors in aged animals. Further studies are needed to
elucidate the importance of activity-dependent alterations in the surface expression of NMDA in synaptic plasticity in aged animals.
| |
FOOTNOTES |
|---|
* 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.
To whom correspondence should be addressed: University of
Colorado Health Sciences Center, Box C236, 4200 E. Ninth Ave.,
Denver, CO 80262. Tel: 303-315-6936; Fax: 303-315-0137; E-mail:
Michael.Browning@uchsc.edu.
Published, JBC Papers in Press, March 12, 2002, DOI 10.1074/jbc.C200074200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: NMDA, N-methyl-D-aspartic acid; LTP, long-term potentiation; BSA, bovine serum albumin; AMPA, aminomethyl phosphonic acid; ANOVA, analysis of variance; EPSP, excitatory post-synaptic potential; PLSD, predicted least-square distance determination; HFS, high-frequency stimulation; GluR2, glutamate receptor subunit-2; NBQX, 2-3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Lynch, M. A.
(1998)
Prog. Neurobiol.
56,
571-589[CrossRef][Medline]
[Order article via Infotrieve] |
| 2. |
deToledo-Morrell, L.,
Geinisman, Y.,
and Morrell, F.
(1988)
Neurobiol. Aging
9,
581-590[Medline]
[Order article via Infotrieve] |
| 3. |
Herman, J. P.,
Chen, K. C.,
Booze, R.,
and Landfield, P. W.
(1998)
Neurobiol. Aging
19,
581-587[CrossRef][Medline]
[Order article via Infotrieve] |
| 4. |
Bach, M. E.,
Barad, M.,
Son, H.,
Zhuo, M., Lu, Y. F.,
Shih, R.,
Mansuy, I.,
Hawkins, R. D.,
and Kandel, E. R.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5280-5285 |
| 5. |
Eckles-Smith, K.,
Clayton, D.,
Bickford, P.,
and Browning, M. D.
(2000)
Brain Res. Mol. Brain Res.
78,
154-162[Medline]
[Order article via Infotrieve] |
| 6. |
McGahon, B. M.,
Martin, D. S.,
Horrobin, D. F.,
and Lynch, M. A.
(1999)
Neurobiol. Aging
20,
655-664[CrossRef][Medline]
[Order article via Infotrieve] |
| 7. |
Joseph, J. A.,
Shukitt-Hale, B.,
Denisova, N. A.,
Bielinski, D.,
Martin, A.,
McEwen, J. J.,
and Bickford, P. C.
(1999)
J. Neurosci.
19,
8114-8121 |
| 8. |
Desjardins, S.,
Mayo, W.,
Vallee, M.,
Hancock, D., Le,
Moal, M.,
Simon, H.,
and Abrous, D. N.
(1997)
Neurobiol. Aging
18,
37-44[CrossRef][Medline]
[Order article via Infotrieve] |
| 9. |
Sonntag, W. E.,
Bennett, S. A.,
Khan, A. S.,
Thornton, P. L., Xu, X.,
Ingram, R. L.,
and Brunso-Bechtold, J. K.
(2000)
Brain Res. Bull.
51,
331-338[CrossRef][Medline]
[Order article via Infotrieve] |
| 10. |
Clayton, D. A.,
and Browning, M. D.
(2001)
Neurobiol. Aging
22,
165-168[CrossRef][Medline]
[Order article via Infotrieve] |
| 11. |
Magnusson, K. R.
(2000)
J. Neurosci.
20,
1666-1674 |
| 12. |
Kuehl-Kovarik, M. C.,
Magnusson, K. R.,
Premkumar, L. S.,
and Partin, K. M.
(2000)
Mech. Ageing Dev.
115,
39-59[CrossRef][Medline]
[Order article via Infotrieve] |
| 13. |
Barnes, C. A.,
Rao, G.,
and Shen, J.
(1997)
Neurobiol. Aging
18,
445-452[CrossRef][Medline]
[Order article via Infotrieve] |
| 14. |
Wenk, G. L.,
and Barnes, C. A.
(2000)
Brain Res.
885,
1-5[CrossRef][Medline]
[Order article via Infotrieve] |
| 15. | Clayton, D. A. M. M., Alvarez, E., Bickford, P. C., Browning, M. D. (2002) J. Neurosci., in press |
| 16. |
Grosshans, D. R.,
Clayton, D. A.,
Coultrap, S. J.,
and Browning, M. D.
(2001)
Nat. Neurosci.
5,
27-33 |
| 17. |
Huh, K. H.,
and Wenthold, R. J.
(1999)
J. Biol. Chem.
274,
151-157 |
| 18. |
Winkler, A.,
Mahal, B.,
Kiianmaa, K.,
Zieglgansberger, W.,
and Spanagel, R.
(1999)
Brain Res. Mol. Brain Res.
72,
166-175[Medline]
[Order article via Infotrieve] |
| 19. |
Okabe, S.,
Miwa, A.,
and Okado, H.
(1999)
J. Neurosci.
19,
7781-7792 |
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  C. M. Norris, E. M. Blalock, O. Thibault, L. D. Brewer, G. V. Clodfelter, N. M. Porter, and P. W. Landfield Electrophysiological Mechanisms of Delayed Excitotoxicity: Positive Feedback Loop Between NMDA Receptor Current and Depolarization-Mediated Glutamate Release J Neurophysiol, November 1, 2006; 96(5): 2488 - 2500. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-M. Samuelsson, E. Jennische, H.-A. Hansson, and A. Holmang Prenatal exposure to interleukin-6 results in inflammatory neurodegeneration in hippocampus with NMDA/GABAA dysregulation and impaired spatial learning Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2006; 290(5): R1345 - R1356. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Boudreau and M. E. Wolf Behavioral Sensitization to Cocaine Is Associated with Increased AMPA Receptor Surface Expression in the Nucleus Accumbens J. Neurosci., October 5, 2005; 25(40): 9144 - 9151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-X. Tao, G. Rumbaugh, G.-D. Wang, R. S. Petralia, C. Zhao, F. W. Kauer, F. Tao, M. Zhuo, R. J. Wenthold, S. N. Raja, et al. Impaired NMDA Receptor-Mediated Postsynaptic Function and Blunted NMDA Receptor-Dependent Persistent Pain in Mice Lacking Postsynaptic Density-93 Protein J. Neurosci., July 30, 2003; 23(17): 6703 - 6712. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
