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The Journal of Neuroscience, September 15, 1999, 19(18):7823-7833
Tetanic Stimulation Leads to Increased Accumulation of
Ca2+/Calmodulin-Dependent Protein Kinase II via Dendritic
Protein Synthesis in Hippocampal Neurons
Yannan
Ouyang1,
Alan
Rosenstein1,
Gabriel
Kreiman1,
Erin M.
Schuman1, 2, and
Mary B.
Kennedy1
1 Division of Biology and 2 Howard Hughes
Medical Institute, California Institute of Technology, Pasadena,
California 91125
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ABSTRACT |
mRNA for the  -subunit of CaMKII is abundant in dendrites of
neurons in the forebrain (Steward, 1997 ). Here we show that tetanic stimulation of the Schaffer collateral pathway causes an increase in
the concentration of -CaMKII in the dendrites of postsynaptic neurons. The increase is blocked by anisomycin and is detected by both
quantitative immunoblot and semiquantitative immunocytochemistry. The
increase in dendritic -CaMKII can be measured 100-200 µm away
from the neuronal cell bodies as early as 5 min after a tetanus. Transport mechanisms for macromolecules from neuronal cell bodies are
not fast enough to account for this rapid increase in distal portions
of the dendrites. Therefore, we conclude that dendritic protein
synthesis must produce a portion of the newly accumulated CaMKII. The
increase in concentration of dendritic CaMKII after tetanus, together
with the previously demonstrated increase in autophosphorylated CaMKII
(Ouyang et al., 1997), will produce a prolonged increase in
steady-state kinase activity in the dendrites, potentially influencing
mechanisms of synaptic plasticity that are controlled through
phosphorylation by CaMKII.
Key words:
long-term potentiation; protein phosphorylation; synapse; synaptic regulation; synaptic plasticity; immunocytochemistry; hippocampal slices
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INTRODUCTION |
The presence of polyribosomes in
neuronal dendrites in the CNS has been recognized for some time
(Steward and Banker, 1992; Steward, 1997). Recent studies have shown
that these polyribosomes can carry out protein synthesis (Crino and
Eberwine, 1996; Torre and Steward, 1996), and that membrane vesicles
containing protein components of the Golgi apparatus are also found in
dendrites (Gardiol et al., 1999). Furthermore, synaptic stimulation in
the presence of carbachol can increase incorporation of tritiated leucine into dendrites in area CA1 (Feig and Lipton, 1993). It has been
postulated that proteins synthesized in dendrites might contribute to
the input-specific nature of long-term potentiation (LTP) (Schuman,
1997).
A subset of mRNAs is present at high concentration far out into the
dendrites and is not selectively concentrated in neuronal cell bodies
(Steward, 1997). One of the most abundant of these in forebrain neurons
is the message encoding the -subunit of CaMKII (Burgin et al., 1990;
Mackler et al., 1992; Martone et al., 1996; Steward, 1997). The CaM
kinase II holoenzyme is an oligomer comprising two homologous catalytic
subunits, a 50 kDa -subunit and a 60 kDa  -subunit (Bennett et
al., 1983). The CaMKII protein is expressed at high levels in
excitatory principal neurons in the forebrain (Benson et al., 1992; Sik
et al., 1998; Zhang et al., 1999), particularly in the hippocampus
where it is ~2% of total protein (Erondu and Kennedy, 1985). Its
high concentration in forebrain is the result of a high level of
expression of the -subunit (Miller and Kennedy, 1985). The kinase is
present in cell bodies, axon terminals, and dendrites, where it
concentrates in postsynaptic densities opposite glutamatergic terminals
(Kennedy, 1998).
CaMKII becomes autophosphorylated upon activation by
Ca2+/calmodulin. The autophosphorylated
form remains active even in the absence of high
Ca2+ until it is dephosphorylated by
cellular phosphatases (Miller and Kennedy, 1986; Miller et al., 1988;
Hanson et al., 1989). In a recent study, we made use of a
semiquantitative immunohistochemical method for visualizing
autophosphorylated CaMKII (P-CaMKII) in fixed hippocampal slices
(Kindler and Kennedy, 1996; Ouyang et al., 1997) to demonstrate that 30 min after tetanization of the Schaffer collateral pathway, substantial
increases in autophosphorylation of CaMKII can be seen in dendrites and
cell bodies of principal neurons in the portion of area CA1 located
near the stimulating electrode (Ouyang et al., 1997). In the course of
that study, we also made the unexpected observation that immunostaining
for nonphospho-CaMKII (NP-CaMKII) was increased at 30 min after tetanus but only in apical dendrites in stratum radiatum, not in neuronal cell
bodies. The increase in both P-CaMKII and NP-CaMKII was blocked by APV,
a blocker of NMDA-type glutamate receptors, and did not occur in slices
in which LTP did not develop after tetanus (Ouyang et al., 1997). We
postulated that the increase in staining for NP-CaMKII triggered by the
tetanic stimulation might reflect either accumulation of -subunit of
CaMKII synthesized in dendrites or a change in the disposition of the
kinase holoenzyme making it more accessible to antibody labeling.
Here we have tested in two ways the hypothesis that the increase
reflects accumulation of newly synthesized -CaMKII. We examined the
effect of the protein synthesis inhibitor anisomycin on the increase in
staining for NP-CaMKII after tetanus. We also directly measured the
amounts of -subunit of CaMKII in tetanized and control halves of
stratum radiatum after microdissection of the slices. Both tests
support the conclusion that the increased staining for NP-CaMKII 30 min
after tetanus results from synthesis of new CaMKII in the stimulated
neurons. Furthermore, we report that an increase in CaMKII protein can
be visualized 100-200 µm out into the dendrites 5 min after the
tetanic stimulation. This result rules out the neuronal cell body as
the sole source of new CaMKII in the dendrites, because transport from
the cell body is not rapid enough to account for the increase in CaMKII
protein  100 µm from the cell body 5 min after tetanus (Brady and
Lasek, 1982; see Discussion). These experiments demonstrate for the
first time that tetanic stimulation of synapses can rapidly increase
the concentration of a signaling molecule in postsynaptic dendrites via
dendritic protein synthesis.
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MATERIALS AND METHODS |
Antibodies. Mouse monoclonal antibody 22B1
(anti-P-CaMKII; Affinity Bioreagents, Golden, CO; www.bioreagents.com)
recognizes CaM kinase II only when it is autophosphorylated at
threonine 286 (Patton et al., 1993). A rabbit polyclonal antibody that
recognizes CaM kinase II only when it is not phosphorylated at
threonine 286 (anti-NP-CaMKII) was prepared by injection of a synthetic peptide into rabbits and was affinity-purified as described (Patton et
al., 1993). Fluorescein-conjugated goat anti-mouse antibody (Cappel,
Organon Teknika, Durham, NC) was used to visualize bound 22B1.
Cy3-conjugated goat anti-rabbit antibody (Chemicon International, Temecula, CA) was used to visualize bound anti-NP-CaMKII. Dilutions of
reagents were as described previously (Ouyang et al., 1997).
Electrophysiology. Electrophysiological experiments were
conducted as described previously (Ouyang et al., 1997). Briefly, young
adult male Sprague Dawley rats (6-8 weeks old) were anesthetized with
halothane and then decapitated, and the brains were placed in ice-cold,
oxygenated artificial CSF (ACSF; 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO4, 2.5 mM CaCl2, 1.0 mM
NaH2PO4, 26.2 mM NaHCO3, and 11.0 mM
D-glucose). Hippocampal slices (500 µm) were prepared with a manual tissue chopper and maintained in an interface chamber gassed with 95% O2 plus 5%
CO2 at room temperature for at least 2 hr before
recording. Experiments were performed with slices from the middle third
of the hippocampus. For electrophysiological manipulations, slices were
transferred to a submersion chamber and superfused continuously with
oxygenated ACSF at room temperature. Electrophysiology was performed
according to a standard "two-pathway" paradigm. Two bipolar
stimulating electrodes were placed about 800 µm apart in stratum
radiatum of area CA1. A glass recording electrode filled with ACSF was
placed in the tissue between them to monitor synaptic potentials. Both
stimulating electrodes delivered a monitoring stimulus (single shock)
every 30 sec, and the slope of the field EPSPs was recorded. The
baseline slope was monitored for at least 30 min or until it became
stable, and then tetanic stimulation (four trains of 100 Hz for 1 sec
with an intertrain interval of 30 sec) was delivered through one of the
stimulating electrodes. Responses were monitored for an additional 5 or
30 min, after which the slices were fixed in ice-cold fixative as described below. An adjacent slice from the same animal was placed in
the recording chamber but received no electrical stimulation (chamber
control). For some experiments, 40 µM anisomycin (Sigma, St. Louis, MO) was added to the ACSF 30 min before the tetanus was applied.
Data from six slices stimulated in the absence of anisomycin were
reported previously (Ouyang et al., 1997); four test slices from the
same animals as those used for recording in the presence of anisomycin
were tetanized in the absence of anisomycin, developed LTP measured at
30 min, and were processed through the immunocytochemical analysis for
P- and NP-CaMKII. The results from these four slices were not
statistically different from the previously reported experiments, and
thus the two sets of numbers were pooled.
We found that the depth of placement of the stimulating electrodes
influenced the extent to which increases in staining were confined to
one region of area CA1 (data not shown). In a series of experiments,
stimulating electrodes were lowered toward the surface of the slice in
the half of stratum radiatum nearest area CA3, until the point at which
a small EPSP could first be recorded from a test pulse through the
electrode, and then advanced 100, 150, or 250 µm further into the
slice. Tetanic stimulation from electrodes advanced 100 µm into the
slice usually produced increased staining for phosphokinase largely
confined to the tetanized half of area CA1; those advanced 150 µm
produced increased staining that usually extended into the
"nontetanized" half of area CA1; and finally, electrodes advanced
250 µm often produced increased staining throughout area CA1; in
superficial sections of these slices, the increased staining was often
more pronounced in the half of CA1 opposite the tetanizing electrode.
We interpret our findings to mean that in slices cut in our laboratory,
from approximately the middle third of the hippocampus, the average
trajectory of Schaffer collateral axons through area CA1 is not quite
parallel to the plane of the slice; rather, axons tend to travel from
one face of the slice in the region of stratum radiatum near CA3 toward the other face of the slice as they move toward the region of CA1 near
the subiculum. Thus, on average, axons in stratum radiatum near area
CA3 stimulated more superficially (by electrodes advanced 100 µm)
would be cut at the surface of the slice before traversing to the
opposite region of the slice near the subiculum. In contrast, axons
stimulated at a deeper level (by electrodes advanced 250 µm) more
often traverse the full-length of area CA1 before reaching the top of
the slice. For this reason, in most of the experiments reported here,
stimulating electrodes were advanced only 100 µm into the slice to
take advantage of the anatomical arrangement of axons.
Immunohistochemistry. Immunohistochemical staining was
performed as described previously (Ouyang et al., 1997). Briefly,
slices were fixed by rapid immersion in ice-cold 4% paraformaldehyde plus 0.2% glutaraldehyde in 0.1 M sodium phosphate buffer,
pH 7.4, and kept on ice for 1 hr. Fixed slices were stored in ice-cold 0.02 M phosphate buffer, pH 7.4, and 0.9% NaCl (PBS)
overnight. Five to six 50 µm sections were cut from each slice with a
vibratome (Pelco; Ted Pella, Redding, CA). Sections were permeabilized
with 0.7% Triton X-100 in PBS for 1 hr and then rinsed with 0.1 M glycine in PBS for another hour followed by 1% Na
borohydride in distilled water for 20 min. Sections were preblocked by
incubation with 5% normal goat serum in phosphate buffer plus 0.45 M NaCl (HSP) for 90 min. Sections were incubated with a
mixture of the two primary antibodies overnight at 4°C. After
washing, the sections were incubated with a mixture of two secondary
antibodies (fluorescein-labeled for P-CaMKII and Cy3-labeled for
NP-CaMKII) for 1 hr and washed free of unbound antibodies. Sections
were mounted with an anti-fade medium (4% n-propyl gallate
in 100 mM NaHCO3, pH 8.7, plus 80% glycerol).
Fluorescein and Cy3 fluorescence images of the CA1 region were obtained
from the central plane of each section with a Zeiss (Thornwood, NY) 310 laser-scanning confocal microscope with a 10× lens [numerical
aperture (NA), 0.3; pinhole, 20; theoretical optical section (OS),
~20 µm], 20× lens (NA, 0.6; pinhole, 20; OS, ~5 µm),
or 40× lens (NA, 1.3; pinhole, 20; OS, ~1.2 µm), as described
previously (Ouyang et al., 1997). Contrast and brightness settings were
optimized in each experiment for the image with the brightest staining
so the data filled the dynamic range of 256 brightness units without
saturation. The settings were then kept constant for all images in a
single experiment. Contrast and brightness settings must be determined
separately for the Cy3 and fluorescein channels; thus absolute image
brightnesses are not directly comparable between the NP and P images.
All data were analyzed as a ratio between the tetanized and
nontetanized region of each section (Ouyang et al., 1997). Images were
saved as tagged image file format (TIFF) files and transferred to a Macintosh computer. For illustration, images were colorized and assembled into montages with Photoshop software (Adobe Systems, Mountain View, CA).
Occasionally staining for P- and/or NP-CaMKII in a chamber control
slice showed >10% difference between the two regions of area CA1 in
three or more sections. When this occurred, slices from the same animal
were not analyzed further, and the data were not included in the pooled data.
Semiquantitative image analysis. Semiquantitative analysis
was carried out as described previously (Ouyang et al., 1997). Each
TIFF image obtained with the confocal microscope is composed of
512 × 512 pixels. Each pixel has a brightness value ranging from
0 to 255. To obtain quantitative data we used MacPhase software (Otter
Solution, Whitesboro, NY) to draw regions of interest (ROIs) in the
tetanized and control regions of the original images and calculate the
average brightness value of the pixels within each ROI. For data from
stratum radiatum, each ROI was a rectangle of 50 × 100 pixels
positioned as described previously (Ouyang et al., 1997). For data from
pyramidal cell bodies, each ROI was drawn free hand to encircle a
region of cell bodies. The data were transferred to Excel (Microsoft,
Redmond, WA) for statistical analysis. The ratio of average brightness
in the tetanized region of area CA1 to that in the control region was
calculated after summing averaged brightness data
(Bave) from three adjacent sections that showed the highest ratios. A corresponding ratio was calculated from each chamber control slice. The normalized ratio was obtained by
dividing the ratio for the tetanized slice by the ratio for the chamber
control slice.
Quantitative immunoblots. Thirty minutes after a tetanus
slices were flash frozen. Stratum radiatum of area CA1 (lacking cell bodies) was microdissected under a dissecting microscope by one investigator (Y.O.) and cut in half so that one half contained the
region surrounding the tetanizing electrode and the other half
contained the region surrounding the control electrode. Halves of
stratum radiatum were homogenized in SDS-PAGE sample buffer containing
3% SDS, 2% -mercaptoethanol, and 5% glycerol in 60 mM
Tris buffer, pH 6.7. The homogenates of individual halves of stratum
radiatum were labeled with a code number, boiled for 5 min, and stored
at  20°C. A second investigator (A.R.) then determined the protein
concentration by a modified Lowry method (Peterson, 1983). Equal
amounts of protein were loaded in triplicate on gels for SDS-PAGE.
Electrophoresed proteins were transferred to polyvinylidene difluoride membranes. After incubation with a primary antibody, 6G9 (1:1000; Affinity Bioreagents), that recognizes both phosphorylated and nonphosphorylated -CaMKII, the membranes were labeled with secondary antibody conjugated to fluorescein (1:100; Amersham, Arlington Heights, IL). After washing and drying, the membranes were
scanned with a STORM system (Molecular Dynamics; Sunnyvale, CA). The
resultant data were digitized and then analyzed with ImageQuant
software provided by Molecular Dynamics. Data from lanes containing an
unknown sample were compared with standard curves made with forebrain
CaMKII purified as previously described (Miller and Kennedy, 1985), and
the amount of -subunit (nanograms per microgram of total protein)
was determined. Finally, the data were decoded, and the ratios of
-subunit in tetanized and nontetanized regions of slices were
calculated (see Table 2).
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RESULTS |
The protein synthesis inhibitor anisomycin blocks the increase in
NP-CaMKII in dendrites 30 min after a tetanus
Seven slices were tetanized in the presence of anisomycin, and 10 were tetanized in the absence of anisomycin in a two-pathway paradigm
as described in Materials and Methods. All 17 of the control and
anisomycin-perfused slices developed LTP of magnitude 15% in the
tetanized pathway measured 30 min after tetanus, whereas the EPSP in
the nontetanized pathway remained stable. "Chamber control" slices
(16 slices) were superfused alongside the stimulated slices from the
same animal but received no electrical stimulation. Slices were fixed,
stained, and examined by laser-scanning confocal microscopy to
visualize the distribution of P-CaMKII and NP-CaMKII as described
previously (Ouyang et al., 1997).
Figure 1 is an example of images obtained
with a 10× lens. The slice shown in Figure 1, A and
B, was not treated with anisomycin. The slice shown
in Figure 1, C and D, was incubated in anisomycin before and during the tetanus. The presence of anisomycin did not
affect the increase in staining for P-CaMKII in the tetanized region of
the slice (compare tetanized with control regions), but it suppressed
the increased staining for NP-CaMKII. These results were analyzed
semiquantitatively as described previously (Ouyang et al., 1997) and in
Materials and Methods (Table 1, Fig.
2). In the absence of anisomycin, the
increase in staining for both P-CaMKII and NP-CaMKII in the tetanized
region of stratum radiatum 30 min after the tetanus is statistically
significant, as observed previously (Ouyang et al., 1997). In neuronal
cell bodies, again as observed previously, only staining for P-CaMKII is significantly increased in the tetanized region. In the presence of
anisomycin, the increase in staining for P-CaMKII is similar to that in
the absence of anisomycin. However, the increase in staining for
NP-CaMKII in stratum radiatum is abolished, consistent with the notion
that the increase of NP-CaMKII in dendrites requires protein
synthesis.
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Figure 1.
Staining for P-CaMKII and NP-CaMKII in
area CA1 from representative sections of hippocampal slices fixed 30 min after tetanic stimulation in the absence or presence of anisomycin.
A, B, Slices were fixed 30 min after a
tetanus was delivered (see Materials and Methods) through a stimulating
electrode located in the regions of area CA1 marked T. A
stimulating electrode that delivered only test stimulation was located
in regions marked c. Sections (50 µm) cut from the
slices were double-immunolabeled for P-CaMKII (A)
and NP-CaMKII (B) as described in Materials and
Methods. Montages of images were converted into color according to the
color look-up table depicted at the bottom. The figure
shows labeling of one representative section with rectangular ROIs used
to compute the ratio of staining between tetanized and control regions
(see below and Materials and Methods). Increased labeling for both
P-CaMKII (A) and NP-CaMKII
(B) is visible in dendrites in stratum radiatum
in the region that received tetanic stimulation, decreasing with
distance from the tetanizing electrode as described previously (Ouyang
et al., 1997). (Compare the region of stratum radiatum labeled
T with that labeled c.) The cell bodies
of pyramidal neurons in the tetanized region also show stronger
labeling for P-CaMKII but not for NP-CaMKII. C,
D, Images of a section from a different slice tetanized
in the presence of anisomycin. Labeling for P-CaMKII
(C) is increased in cell bodies and dendrites in
the tetanized region of the section (Compare the region of stratum
radiatum labeled T with that labeled c.)
In contrast, no increase in labeling for NP-CaMKII
(D) is visible in dendrites or cell bodies in the
tetanized region compared with those in the control region. Note in
D that staining for NP-CaMKII is higher in cell bodies
in the control region than in the tetanized region. This pattern was
observed occasionally and is the complement of the pattern of staining
for P-CaMKII in the same section (C); it likely
reflects a reduction in staining for NP-CaMKII in the tetanized cell
bodies caused by increased autophosphorylation of CaMKII without a net
increase in amount of CaMKII. It is important to note that absolute
brightness is not directly comparable between sections, because the
microscope contrast settings were chosen in each experiment to fill the
8 bit scale in the brightest of all the sections and then held constant
for that experiment. Furthermore, contrast settings are set separately
for each fluorophore. Comparison of brightness values is only
meaningful between tetanized and control regions of the individual
sections averaged over many sections. To make this comparison,
ROIs shown as black rectangles were chosen as described
in Materials and Methods. The average brightness value in each ROI was
recorded as shown to the right. The ratios
T/C were calculated for the three brightest sections
from each slice and averaged (Table 1, Fig. 2). Scale bar, 250 µm.
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Figure 2.
Quantitative analysis of the ratio of staining in
the tetanized region of area CA1 to that in the control region 30 min
after tetanic stimulation in the presence and absence of anisomycin.
The data from Table 1 are plotted as percent deviation from 1.0 of the
ratio of brightness in the tetanized region to brightness in the
control region in stratum radiatum and in the cell body layer of area
CA1. Ratios from chamber control slices and from slices in which LTP
was induced by tetanus in the presence and absence of anisomycin are
shown side by side. The data are the average ± SEM of 27 sections
from nine chamber control slices and 30 sections from 10 tetanized
slices treated in the absence of anisomycin and 14 sections each from
seven chamber control slices and seven tetanized slices treated in the
presence of anisomycin. A, Percent change in NP-CaMKII
between tetanized and control regions of sections. ANOVA followed by
t test showed that the change in NP-CaMKII in stratum
radiatum after induction of LTP by tetanus is abolished in the presence
of anisomycin. B, Percent change in P-CaMKII between
tetanized and control regions of sections. No significant differences
were observed between brightness values for P-CaMKII in the presence
and absence of anisomycin. Solid bars, Control without
anisomycin; open bars, with anisomycin;
*p < 0.002.
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The amount of -subunit of CaMKII increases in dendrites 30 min
after a tetanus as measured by quantitative Western blot
To substantiate that the increase in staining for NP-CaMKII after
tetanus reflects a true increase in amount of CaMKII rather than, for
example, unmasking of the antibody epitope, we measured the amount of
CaMKII in microdissected halves of hippocampal slices after
physiological treatments. Experiments were performed to generate pairs
of tetanized slices and chamber controls as described above. Thirty
minutes after the tetanus, stratum radiatum was dissected from these
slices and divided in half. The amount of -CaMKII in each half was
determined by comparison with standard lanes containing CaMKII purified
from forebrain as described in Materials and Methods. Figure
3 shows an example of one such
immunoblot, demonstrating that measurements of standard -CaMKII and
-CaMKII in the unknown samples were made in the linear range of the
assay. Ratios of -CaMKII concentration in the tetanized and control regions of each slice were determined for seven pairs of experimental and chamber control slices (Table 2, Fig.
4). In six of the seven slices, an
increase in the amount of -CaMKII in the tetanized region was
measured when compared with chamber controls. The data reveal a
statistically significant increase in the amount of -CaMKII induced
by tetanus. Indeed the average percent increase (29%) measured by this
technique is higher than the average percent increase calculated from
the immunocytochemical data gathered from sister slices (11%). In
performing the quantitative immunoblots, we were able to ensure that
measurements were obtained in the linear range of the assay. It is more
difficult to make a determination of the full linear range for the
immunocytochemical assay, and it may be that immunocytochemical
measurements move out of the linear range above a 10-15% increase.
The antibody that we used for immunocytochemistry detects only
NP-CaMKII, whereas the antibody used for quantitative blots detects the
total -subunit of CaMKII. Although previous measurements suggest
that NP-CaMKII varies from ~70 to 97% of total CaMKII (Molloy and
Kennedy, 1991), this percentage may fall lower in dendrites or cell
bodies after tetanic stimulation. Therefore, immunocytochemical
measurements of NP-CaMKII may underestimate the amount of total CaMKII
in the tetanized region of the slice. For this reason, we continue to
describe the immunocytochemical method as semiquantitative.
Nevertheless, the quantitative immunoblots confirm that we are
detecting a true increase in total -CaMKII in dendrites induced by
tetanic stimulation.
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Figure 3.
Quantitative immunoblot of the -subunit of
CaMKII. A, Example of a quantitative immunoblot. Slices
were tetanized as described in Materials and Methods and then frozen.
Tetanized and control halves of individual slices were dissected and
homogenized separately in SDS sample buffer. After determination of the
protein concentration of each homogenate, samples of each (0.25 and 0.5 µg) were loaded in triplicate onto SDS-PAGE gels as described in
Materials and Methods. CaMKII purified from forebrain (40 and 80 ng of
-subunit) was loaded in triplicate onto adjacent lanes as a
standard. Immunoblots were prepared with a fluorescein-conjugated
secondary antibody and imaged with a FluorImager. Immunoblot of a
homogenate from a tetanized half of stratum radiatum is labeled
T. The immunoblot of the corresponding control half of
stratum radiatum is labeled C. B,
Standard curve of fluorescence intensity plotted against nanograms of
purified CaMKII. Quantitative measurements of fluorescence were made as
described in Materials and Methods. C, Fluorescence
intensity of -subunit bands from tetanized (T)
and control (C) samples shown in
A, plotted against a microgram protein sample. The
concentration of -subunit in each homogenate (Fig. 4) was calculated
as nanograms per microgram of protein by comparison with the standard
curve. Both the standard curve (B) and values for
the unknown samples (C) were measured in the
linear range of the assay.
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Figure 4.
Comparison of increase in CaMKII in tetanized
regions of slices measured by immunofluorescence and by quantitative
immunoblot. Data from the seven experiments summarized in Table 2 are
plotted after normalization to chamber controls. The average percent
change measured by immunofluorescent labeling is 11.5 ± 4.0 (p < 0.02 compared with chamber controls).
The average percent change measured by quantitative immunoblot is
29.6 ± 8.3 (p < 0.01 compared with
chamber controls).
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An increase of NP-CaMKII is visible in dendrites 5 min
after tetanus
How soon after a tetanus can we detect the increase in amount of
-CaMKII in dendrites? We performed a set of experiments identical to
those reported in Figures 1 and 2, except that slices were placed in
ice-cold fixative 5 min after delivery of the tetanus. To control for
general health of the slices, in each experiment a slice from the same
animal was first tested for development of LTP by recording for 30 min
after a tetanus. If LTP was not observed in the test slice, the
experiment was not continued with slices from that animal. After we
obtained LTP in the test slice, successive slices that showed an
increase over baseline in slope of the EPSP at 5 min after the tetanus,
as shown in Figure 5, were fixed and
processed for immunocytochemistry. Examples of images of slices taken
with a 10× lens in these experiments are shown in Figure
6. The semiquantitative imaging data from
11 experiments performed without anisomycin, and seven experiments
performed in the presence of anisomycin are summarized in Table
3 and Figure 7. The results show that increases in NP-
and P-CaMKII are both visible 5 min after a tetanus. Increased staining
in the tetanized region of the slice can be seen throughout the length
of dendrites from near the cell bodies to >200 µm way from the cell
bodies (Fig. 8, middle). The
magnitude of the effect on NP-CaMKII appears lower at 5 min than at 30 min (compare Figs. 2, 7). In contrast to 30 min after tetanus, staining
for NP-CaMKII 5 min after tetanus is higher in both cell bodies and
dendrites in the tetanized region. The increase at 5 min in NP-CaMKII
in both cell bodies and dendrites is blocked in the presence of
anisomycin. Examination at high magnification of sections of slices
fixed 5 min after tetanus reveals that the increases in NP-CaMKII are
confined to patches along dendrites and are occasionally visible in
spines (Fig. 8A). This distribution of high NP-CaMKII
in tetanized neurons 100-200 µm from the cell bodies is consistent
with new synthesis of -subunit by polyribosomes in dendrites near
sites of synaptic activation followed by movement of some of the newly
synthesized subunit into adjacent spines.
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Figure 5.
Electrophysiological recording from a slice that
was fixed for immunolabeling 5 min after tetanic stimulation. The
stimulation paradigm was as described in Materials and Methods.
Baseline EPSPs were monitored for 30 min, and then four trains of
tetanic stimulation (100 Hz, 1.0 sec; 30 sec intertetanus interval)
were applied to one pathway (top). The slice was fixed 5 min after the first tetanus as described in Materials and Methods. The
response of the control pathway (bottom) remained
stable.
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Figure 6.
Staining for P-CaMKII and NP-CaMKII in area CA1
from representative sections of hippocampal slices fixed 5 min after
tetanic stimulation in the absence or presence of anisomycin.
Experimental treatment and analyses were exactly as described in Figure
1, except that slices were fixed 5 min after a tetanus was delivered
through one electrode (see Materials and Methods). A,
B, Images of a section tetanized in the absence of
anisomycin and fixed 5 min after the tetanus. Increased labeling for
P-CaMKII (A) and NP-CaMKII
(B) is measured in both cell bodies and in
dendrites in stratum radiatum in the region that received tetanic
stimulation. C, D, Images of a section
from a different slice tetanized in the presence of anisomycin and
fixed 5 min after the tetanus. Labeling for P-CaMKII
(C) is increased in cell bodies and dendrites in
the tetanized region of the section. In contrast, no increase in
labeling for NP-CaMKII (D) is measurable in
dendrites or cell bodies in the tetanized region compared with
those in the control region. Scale bar, 250 µm.
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Figure 7.
Quantitative analysis of the ratio of staining in
the tetanized region of area CA1 to that in the control region 5 min
after tetanic stimulation in the presence and absence of anisomycin.
The data from Table 3 are plotted as percent deviation from 1.0 of the
ratio of brightness in the tetanized region to brightness in the
control region in stratum radiatum and in the cell body layer. Ratios
from chamber control slices and from slices fixed 5 min after tetanus
in the presence and absence of anisomycin are shown side by side. The
data are the average ± SEM of 33 sections each from 11 chamber
control slices and 11 tetanized slices treated in the absence of
anisomycin and 21 sections each from seven chamber control slices and
seven tetanized slices treated in the presence of anisomycin as
described in Materials and Methods. A, Percent change in
NP-CaMKII between tetanized and control regions of sections. ANOVA
followed by t test indicated that staining for NP-CaMKII
was significantly brighter in tetanized regions of stratum radiatum and
in tetanized cell bodies 5 min after tetanus. The increases in
NP-CaMKII 5 min after tetanus in the absence of anisomycin were
abolished in the presence of anisomycin. B, Percent
change in P-CaMKII between tetanized and control regions of sections.
No significant differences were observed between brightness values for
P-CaMKII in the presence and absence of anisomycin. Solid
bars, Control without anisomycin; open bars,
with anisomycin; *p < 0.005;
**p < 0.04.
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Figure 8.
High-resolution images of staining for P-CaMKII
and NP-CaMKII from a section of a slice fixed 5 min after tetanus.
Images were recorded with a 40× lens (NA, 1.3) from several areas of a
section from a tetanized slice fixed 5 min after tetanus and
double-labeled for P- and NP-CaMKII as described in Materials and
Methods. Images of the section recorded with a 20× lens are shown for
reference (middle); letters
(A-H) on the reference images mark the locations
where the corresponding 40× images were recorded. Staining for
NP-CaMKII is shown on the left; staining for P-CaMKII of
the same area is shown on the right. Note that high
levels of immunolabeling for P- and NP-CaMKII coincide in some segments
of dendrites (A, E, black
arrowheads) in the tetanized region but not in others
(A, E, white arrowheads).
Scale bar: A-H, 15 µm; 20× reference images
(middle), 200 µm.
|
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| |
DISCUSSION |
Here we show that strong tetanic stimulation of the Schaffer
collateral pathway stimulates accumulation of newly synthesized -subunit of CaMKII in the dendrites of the postsynaptic neurons. The
increase can be visualized in patches along dendrites by
immunocytochemistry just 5 min after the tetanus and persists for at
least 30 min. Suppression of protein synthesis by anisomycin blocks the
increased accumulation, showing that it is dependent on synthesis of
new protein. The increase is corroborated by quantitative immunoblots by which we detect an average 29% increase in -CaMKII protein in
the tetanized region of hippocampal slices 30 min after tetanus. We
have previously shown that the increased staining for CaMKII is
abolished after addition of NMDA receptor blockers (Ouyang et al.,
1997); thus the increase requires activation of NMDA receptors.
The increase in CaMKII is clearly visible in dendrites 100-200 µm
from the cell bodies by 5 min after the tetanus (Fig. 8). A well
established body of evidence on protein transport mechanisms has
established that proteins associated with the cytoskeleton are
transported into axons at a rate of 2-4 mm/d, or 1.4-2.8 µm/min. Only membrane-bound organelles are transported at a faster rate (Brady
and Lasek, 1982). The molecular machinery subserving dendritic transport is identical or homologous to that involved in axonal transport (Saito et al., 1997). One difference is that dendrites contain a mixture of microtubules polarized in opposite directions, whereas axons contain microtubules polarized primarily in one direction
(Baas et al., 1989). This difference would not produce faster rates of
transport into dendrites, and indeed the few measurements made of
dendritic transport rates for macromolecules in hippocampal neurons
have yielded rates similar to those of axonal transport (Davis et al.,
1987, 1990; Overly et al., 1996). Axonal transport rates of proteins
are not increased by neuronal activity (Edwards and Grafstein, 1984;
Hammerschlag and Bobinski, 1992). The CaMKII holoenzyme is associated
with the cytoskeleton in dendrites and is not associated with
membranous organelles (Kennedy, 1998; Shen et al., 1998). Thus, it
would be transported at rates not exceeding 2.8 µm/min; therefore,
protein synthesis in the cell body cannot be the source of the
increased dendritic CaMKII observed 100-200 µm away from the cell
body 5 min after a tetanus. The -subunit of CaMKII is not expressed
in glial cells or in interneurons (Sik et al., 1998; Zhang et al.,
1999); thus, protein synthesis in these cells cannot be the source of
new -CaMKII.
In this issue, Steward and Halpain (1999) report that stimulation of a
pathway ending in synapses that are confined to one lamina of the
dentate gyrus in live rats increases immunostaining intensity for MAP2
and CaMKII only in the portion of dendrites in the stimulated lamina.
The increase in staining for MAP2 was reduced in the presence of
cycloheximide, suggesting the involvement of dendritic protein
synthesis. Increased staining for CaMKII was not measurably reduced by
cycloheximide in this study; thus it is not clear whether synaptic
activity can alter accumulation of newly synthesized CaMKII in the
dentate gyrus. In contrast, our results indicate that tetanic activity
of the Schaffer-collateral pathway can increase accumulation of newly
synthesized CaMKII in dendrites in area CA1 via dendritic protein
synthesis. The synaptic input into stratum radiatum via the Schaffer
collateral pathway is not highly laminated (Ishizuka et al., 1990).
Individual axons take tortuous paths through stratum radiatum, making
en passant synapses along their lengths. Therefore, strong stimulation of the Schaffer collateral pathway in slices would not be expected to
produce laminar activation of synapses within stratum radiatum. The
increase in CaMKII that we observe in stimulated pyramidal neurons at
30 min after tetanus is, however, confined to the portion of apical
dendrites in stratum radiatum. At 5 min after tetanus, small increases
in total CaMKII that are blocked by anisomycin are also observed in the
cell bodies. No significant changes in CaMKII are observed at any time
in the basal dendrites in stratum oriens.
Increased accumulation of CaMKII could result, in theory, from a direct
increase in biosynthetic rate in the dendrites, from a decrease in
degradation rate, or from a combination of the two. Each of these
mechanisms would require dendritic protein synthesis to produce the
observed increased accumulation in dendrites 100-200 µm away from
the cell body 5 min after tetanic stimulation. Wu et al. (1998)
recently presented evidence that binding of CPEB protein to CPE sites
located in the 3'-end of the RNA message for -CaMKII can stimulate
its translation rate. Thus, one possible mechanism by which tetanic
stimulation might increase -CaMKII synthesis is via phosphorylation
of the CPEB protein, which is indeed present in hippocampal dendrites
(Wu et al., 1998). An important next step will be to determine whether
this mechanism is involved in the effect of tetanus on CaMKII
concentration in CA1 pyramidal neurons and whether the degradation rate
of the -subunit is slowed after tetanus.
Regulation of the concentration of dendritic CaMKII will influence
control of synaptic plasticity in the hippocampus. Mutant mice lacking
the -subunit or bearing an -subunit gene that cannot be
autophosphorylated at threonine 286 show severely impaired plasticity
at Schaffer collateral synapses (Silva et al., 1992; Stevens et al.,
1994; Giese et al., 1998). Several potential mechanisms by which
phosphorylation by activated CaMKII could mediate a change in synaptic
efficacy have been postulated, including modification of the current
through AMPA receptors (Barria et al., 1997) and potentiation of MAP
kinase activation at the synapse (Chen et al., 1998). An
increase in concentration of CaMKII in dendrites would contribute to a
relatively long-lasting increase in the steady-state activity of CaMKII
and would thus influence the magnitude and time course of all
regulatory processes controlled by CaMKII.
Protein synthesis during tetanic stimulation is necessary for
development of long-lasting or "late" LTP (Krug et al., 1984; Frey
and Morris, 1997). Frey and Morris (1997) found evidence that late LTP
requires both a synapse-specific "tag" induced by relatively weak
tetanus and one or more proteins whose synthesis is stimulated more
globally in the neuron during a strong tetanus. They reported that
strong tetanus to one pathway onto a neuron permits the development of
late LTP when a weak tetanus is applied 35 min later at a second
pathway after addition of protein synthesis inhibitors. The synthesis
and increased accumulation of -CaMKII in dendrites that we report
here may contribute to such a non-synapse-specific protein
synthesis-dependent mechanism. Frey and Morris (1997) suggest that the
required protein synthesis could be occurring in the neuronal cell
body; however, their data do not exclude a contribution from dendritic
protein synthesis. Proteins synthesized within dendrites, like those
synthesized in the cell body, could move away from their site of
synthesis in the dendritic shaft and become concentrated at tagged
synapses. The data presented here indicate that CaMKII is one protein
whose accumulation by new protein synthesis is induced by tetanic
stimulation and may be necessary for late LTP.
| |
FOOTNOTES |
Received March 10, 1999; revised July 1, 1999; accepted July 2, 1999.
This work was supported by National Institutes of Health Research
Service Award NS10660 (Y.O.), Grants MH49176 and NS17660 (M.B.K.) and
NS32792 (E.M.S.), and grants from the Alfred P. Sloan Foundation,
Beckman Foundation, John Merck Fund, and PEW Charitable Trusts
(E.M.S.). We thank Dr. Scott Fraser, director of the Caltech Biological
Imaging Resource Center, for valuable technical advice and for use of
the confocal microscope.
Correspondence should be addressed to Mary B. Kennedy, Division of
Biology 216-76, California Institute of Technology, Pasadena, CA 91125.
| |
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TOP
ABSTRACT
INTRODUCTION
Compound via MeSH
