1Department of Physiology and 2Division of Neuroscience, University of Alberta School of Medicine, Edmonton, Alberta T6G 2H7, Canada; and 3Department of Biology and Neuroscience Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Woo, Newton H.,
Steven N. Duffy,
Ted Abel, and
Peter V. Nguyen.
Genetic and Pharmacological Demonstration of Differential
Recruitment of cAMP-Dependent Protein Kinases by Synaptic
Activity.
J. Neurophysiol. 84: 2739-2745, 2000.
cAMP-dependent protein kinase (PKA) is believed to play a
critical role in the expression of long-lasting forms of hippocampal long-term potentiation (LTP). Can distinct patterns of synaptic activity induce forms of LTP that require different isoforms of PKA? To
address this question, we used transgenic mice that have genetically
reduced hippocampal PKA activity, and a specific pharmacological inhibitor of PKA, Rp-cAMPS. Transgenic mice [R(AB) mice] that express
an inhibitory form of a particular type of regulatory subunit of PKA
(type-I) showed significantly reduced LTP in area CA1 of hippocampal
slices as compared with slices from wild-type mice. This impairment of
LTP expression was evident when LTP was induced by applying repeated,
temporally spaced stimulation (4 1-s bursts of 100-Hz applied once
every 5 min). In contrast, LTP induced by applying just 60 pulses in a
theta-burst pattern was normal in slices from R(AB) mice as compared
with slices from wild-type mice. We found that Rp-cAMPS blocked the
expression of LTP induced by both spaced tetra-burst and
compressed theta-burst stimulation in hippocampal slices of wild-type
and R(AB) mice, respectively. Since Rp-cAMPS is a PKA inhibitor that is
not selective for any particular isoform of PKA and these R(AB) mice
show reduced hippocampal PKA activity resulting from genetic
manipulation of a single isoform of PKA regulatory subunit, our data
support the idea that distinct patterns of synaptic activity can
produce different forms of LTP that significantly engage different
isoforms of PKA. In particular, theta-burst LTP significantly recruits
isoforms of PKA containing regulatory subunits other than the mutant
RI
subunit, whereas tetra-burst LTP requires PKA isoforms containing the mutant RI
subunit. Thus, altering both the total amount of imposed synaptic activity and the temporal spacing between bursts of
imposed activity may subtly modulate the PKA dependence of hippocampal
LTP by engaging distinct isoforms of PKA. In a broader context, our
findings suggest that synaptic plasticity in the mammalian brain might
be importantly regulated by activity-dependent recruitment of different
isoforms of key signal transduction molecules.
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INTRODUCTION |
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Hippocampal
long-term potentiation (LTP) is an activity-dependent enhancement of
synaptic transmission believed to be a cellular mechanism for some
forms of learning and memory in the mammalian brain (Bliss and
Collingridge 1993; Martin et al. 2000
). One
particular protein kinase that is critical for hippocampal LTP is
cAMP-dependent protein kinase (PKA) (Abel et al. 1997
;
Blitzer et al. 1995
; Frey et al. 1993
;
Huang and Kandel 1994
; Weisskopf et al.
1994
). In area CA1 of hippocampal slices, strong, repeated
stimulation using temporally spaced bursts of activity (e.g., 4 1-s
bursts of 100-Hz, interburst interval 5 min) induces long-lasting LTP
that requires PKA (Huang and Kandel 1994
). Transgenic
mice with genetically reduced hippocampal PKA activity also show
impairment of synaptic potentiation induced by temporally spaced
tetra-burst stimulation (Abel et al. 1997
). Although the
PKA dependence of hippocampal LTP has been characterized through the
application of pharmacological and genetic approaches, an unresolved
question is whether various forms of LTP induced by different patterns
of synaptic stimulation may require distinct types of PKA holoenzyme.
PKA is a tetrameric enzyme composed of two catalytic and two regulatory
subunits (Reimann et al. 1971). At least two
distinct types of catalytic subunits and four different types of
regulatory subunits are expressed in the mouse hippocampus (Cadd
and McKnight 1989
). Thus in principle, there may exist as many
as 30 different tetrameric subunit combinations within the PKA
holoenzyme in situ (i.e., 30 different "isoforms"). Mice with
genetic deletions of the C
1 catalytic subunit
of hippocampal PKA showed perturbed LTP and defective long-term
depression (LTD) in area CA1 of hippocampal slices (Qi et al.
1996
), whereas genetic deletion of the RI
regulatory subunit impaired LTD but did not affect LTP (Brandon et al.
1995
). These results suggest that different forms of
hippocampal synaptic plasticity may be regulated by distinct isoforms
of PKA subunits, but it should be noted that in both of these mouse
models, measurable levels of hippocampal PKA activity were unaffected
by the genetic manipulations employed to delete these particular
subunits (Brandon et al. 1995
; Qi et al.
1996
). Hence, these two "knock-out" mouse models
(Brandon et al. 1995
; Qi et al. 1996
)
cannot provide evidence as to whether the dependence of LTP expression
on PKA enzymatic activity involves differential recruitment
of distinct isoforms of PKA. An essential requirement for testing the
hypothesis that the amount and temporal pattern of synaptic activity
may regulate the PKA dependence of LTP by recruiting different PKA
isoforms is to look for LTP expression in the presence of substantial
reductions in the enzymatic activity of hippocampal PKA.
Are different isoforms of PKA involved in types of LTP induced by
distinct temporal patterns of synaptic stimulation? One way of
addressing this question is to use pharmacological and genetic
approaches to examine the dependence of different types of LTP on PKA.
Transgenic mice expressing an inhibitory form of the RI regulatory
subunit of PKA [termed R(AB) mice] show significantly reduced levels
of hippocampal PKA activity (approximately 40-50% of basal activity)
(Abel et al. 1997
). This reduction of PKA activity results from the suppression of the enzymatic activity of tetrameric holoenzymes containing the mutant inhibitory form of the RI
subunit of PKA (Abel et al. 1997
; Woodford et al.
1989
). R(AB) transgenic mice therefore provide an appropriate
and effective means of exploring the hypothesis that different patterns
of synaptic activity may induce forms of LTP that require distinct
isoforms of PKA holoenzyme (or more specifically, different isoforms of
regulatory subunits). Additionally, pharmacological inhibition of PKA
can be used to establish the PKA dependence of forms of LTP induced by
different patterns of synaptic activity. Together these two approaches, one genetic and the other pharmacological, can be combined to explore
the roles of different patterns of synaptic activity in regulating the
PKA dependence of LTP through putative recruitment of particular
isoforms of PKA.
In the present study, we have used R(AB) mice and a specific inhibitor
of PKA, Rp-cAMPS, to test the hypothesis that different forms of LTP
may require distinct isoforms of PKA. It should be noted that at the
concentration used here (100 µM), Rp-cAMPS does not preferentially
inhibit specific isoforms of PKA (Cadd et al. 1990;
Dostmann 1995
; Woodford et al.
1989
), but it does suppress total PKA activation by binding to
regulatory subunits (Dostmann 1995
). We report that LTP
induced by temporally spaced, repeated stimulation (a tetra-burst
protocol) was defective in area CA1 of hippocampal slices from R(AB)
mice, and this form of LTP was blocked in wild-type slices by Rp-cAMPS.
This same drug also blocked LTP induced by a compressed, theta-burst
pattern of synaptic stimulation in R(AB) mutant slices. Thus both forms
of LTP, induced by distinct patterns of synaptic stimulation, are PKA
dependent as defined by their sensitivity to a specific pharmacological
inhibitor of PKA. However, we found that theta-burst LTP was intact in
hippocampal slices from R(AB) mutant mice as compared with control
slices from wild-type mice. These findings suggest that theta-burst and tetra-burst stimulation may engage distinct isoforms of PKA. In particular, although theta-burst LTP was shown to be PKA dependent, normal expression of theta-burst LTP in R(AB) mutant slices indicates that this pattern of stimulation may significantly recruit isoforms of
PKA holoenzyme that do not contain the mutant RI
subunit. Our data
provide evidence to support the idea that distinct patterns of synaptic
activity can regulate the PKA dependence of LTP by engaging distinct
isoforms of PKA.
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METHODS |
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Experiments were performed on R(AB) transgenic mice (aged 6-8
mo) that were previously characterized for hippocampal PKA activity, synaptic plasticity, learning, and memory (Abel et al.
1997). In R(AB) transgenic mice, reduced hippocampal PKA
activity results from expression of a dominant negative form of the
RI
regulatory subunit of PKA (Abel et al. 1997
;
Clegg et al. 1987
). We examined equal numbers of male
and female mice from two transgenic lines, R(AB)-1 and R(AB)-2
(Abel et al. 1997
). Transgenic mice were maintained in
the hemizygous state on a C57BL/6J background. Currently the R(AB)
transgenic colony is at the 8th-10th backcross generation onto a
C57BL/6J background. These mutant mice show reductions of hippocampal
basal PKA activity of approximately 40-50%. Mice were housed at the
University of Pennsylvania and at the University of Alberta under IACUC
and CCAC guidelines. Age-matched wild-type littermates of the mutants
were used for controls. Separate slices from each animal were used to
test the effects of different patterns of stimulation. Experimenters
were blind to the genotypes of the animals. For genotyping, tail DNA
was prepared and analyzed by Southern blotting using a
transgene-specific probe as described (Abel et al.
1997
).
Transverse hippocampal slices (400-µm thickness) were prepared and
maintained in an interface chamber at 28°C (for details, see
Nguyen and Kandel 1997). Slices were perfused with
standard artificial cerebrospinal fluid (ACSF, 1 ml/min flow rate) with ionic composition as described in Nguyen and Kandel
(1997)
. A microelectrode (2-4 M
resistance, filled with
ACSF) was used to record field excitatory postsynaptic potentials
(fEPSPs) from stratum radiatum of area CA1 during extracellular
stimulation with a bipolar nickel-chromium electrode (130 µm diam,
A-M Systems) that was also positioned in s. radiatum. Stimulation
intensity (0.08-ms pulse width) was adjusted to give fEPSP amplitudes
approximately 40% of maximum sizes, and baseline responses were
elicited once per minute at this intensity. LTP was induced by applying
four 100-Hz bursts (1-s duration) spaced 5-min apart (tetra-burst
protocol) or by using theta-burst stimulation. The theta-burst
stimulation protocol consisted of 15 bursts of four pulses at 100-Hz,
delivered at a 200-ms interburst interval. In some experiments, 100 µM Rp-cAMPS, an inhibitor of PKA (Dostmann 1995
), was
bath-applied to wild-type and mutant slices to test for the PKA
dependence of tetra-burst and theta-burst LTP. Statistical comparisons
between mean fEPSP slopes measured from wild-type and R(AB) slices were
performed using Student's t-test. We compared the fEPSP
slopes measured 2 h following LTP induction and averaged across
slices within a group.
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RESULTS |
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LTP induced by temporally spaced stimulation is defective in R(AB) mutant mice
We compared LTP induced by four 100-Hz bursts of stimulation
(5-min interburst interval) in R(AB) mutant and wild-type slices (Fig.
1). In wild-type slices, this stimulation
regimen induced a robust and long-lasting form of LTP in area CA1: the
mean fEPSP slope measured 2 h after LTP induction was 169 ± 22% (mean ± SE) of pre-LTP baseline. In contrast, the
same amount and pattern of stimulation elicited a gradually decaying
LTP in R(AB) mutant slices: the mean fEPSP slope measured 2 h
after LTP induction was only 106 ± 9% of pre-LTP baseline
(P < 0.02 for comparison with wild types, Fig. 1).
These data replicate the previous observations reported by Abel
et al. (1997). A noteworthy difference between the present data
and those in Abel et al. (1997)
is the consistently reduced levels of potentiation observed in R(AB) mutants here at all
time points following LTP induction. This may be the result of the
higher levels of potentiation observed in slices from wild-type mice in
the present study. In the original study of Abel et al. (1997)
, the early stage of potentiation measured minutes after induction was unaffected in mutants. The larger potentiation seen here
may result from the fact that the genetic background of the mice used
in the current study is more completely C57BL/6J, a mouse strain known
to exhibit robust LTP (Nguyen et al. 2000
). Regardless
of the actual reason for the present study's stronger inhibition of
LTP expression in mutants, our results are consistent with those
reported previously by Abel et al. (1997)
. Our data also
support the idea that PKA may indeed play important roles in both early
and late phases of LTP in area CA1 (Blitzer et al. 1995
;
Huang and Kandel 1994
; see also Otmakhova
et al. 2000
, for cAMP data).
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Altering the amount and temporal spacing of synaptic stimulation induces robust LTP in R(AB) mutants
Previous studies on R(AB) mice have used temporally spaced,
repeated bursts of stimulation to induce persistent forms of
hippocampal LTP (Abel et al. 1997). This strong regimen
of stimulation is reliable in eliciting long-lasting forms of LTP that
are PKA dependent (Abel et al. 1997
; Huang and
Kandel 1994
), but it is not clear whether altering the amount
and temporal spacing of stimulation can modulate the PKA dependence of
hippocampal LTP by recruiting different isoforms of PKA. To address
this hypothesis, we first measured LTP induced by reducing both the
amount and the temporal spacing of imposed synaptic activity in R(AB)
and wild-type slices (Fig. 2). A
theta-burst pattern of stimulation (15 bursts of 4 pulses at 100 Hz,
delivered at a 200-ms interburst interval) elicited a substantial
amount of synaptic potentiation in wild-type slices: the mean fEPSP
slope measured 2 h after theta-burst stimulation was 142 ± 8% of pre-LTP baseline (Fig. 2A,
). In R(AB) mutants, theta-burst LTP was almost identical in time course and amplitude to
that seen in wild types (Fig. 2A). The mean fEPSP slope
measured in mutant slices 2 h postinduction was 147 ± 12%
of pre-LTP baseline. This was not significantly different from the
value observed in wild types (P > 0.2). Hence unlike
the temporally spaced protocol that yielded an LTP deficit in mutants,
the sharply reduced amount of imposed synaptic activity (total of 60 pulses of stimulation vs. 400 pulses in the spaced tetra-burst
protocol) and the decreased temporal spacing between bursts (200-ms
interburst interval vs. 5 min) in the theta-pattern protocol fully
rescued LTP in R(AB) mutants.
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These data, obtained from experiments performed on PKA mutant mice,
suggest that the amount and temporal pattern of synaptic activity can
modulate the PKA dependence of LTP in animals with a genetic reduction
of hippocampal PKA activity. However, these results alone do not
provide evidence for activity-dependent recruitment of different
isoforms of PKA following synaptic stimulation leading to LTP. The
theta-burst LTP described here may still be PKA dependent because the
R(AB) mutation predominantly affects activation of PKA tetramers that
contain the mutant RI isoform of the regulatory subunit. In other
words, theta-burst stimulation may recruit primarily PKA tetramers that
contain regulatory subunit isoforms other than the mutant RI
subunit. If this is true, then LTP induced by theta-burst stimulation
should be blocked by generic pharmacological inhibition of PKA (there
are presently no pharmacological inhibitors available that can
preferentially block specific isoforms of PKA). Hence the aim of our
next experiment was to determine whether theta-burst LTP in R(AB)
mutants is PKA dependent as defined by sensitivity to block by a
specific pharmacological inhibitor of PKA.
Specific pharmacological inhibitor of PKA blocks expression of LTP induced by both tetra- and theta-burst stimulation
Our observation that theta-burst LTP was intact in R(AB) mutant
slices prompted further investigation to try to establish whether this
form of LTP was PKA dependent in R(AB) mutant mice. Previous studies,
performed on area CA1 of hippocampal slices from C57BL/6J mice, have
shown that a theta-burst stimulation protocol (identical to that used
in our present study) induced LTP that was blocked by inhibitors of PKA
(Nguyen and Kandel 1997). Thus the normal theta-burst
LTP seen in R(AB) mutant slices could still be PKA dependent as defined
by its putative sensitivity to block by a specific pharmacological
inhibitor of PKA. Also the PKA dependence of tetra-burst LTP, which was
defective in R(AB) mutant slices, needs to be confirmed in wild-type slices.
To test these hypotheses, we first determined whether tetra-burst
stimulation of wild-type slices induces PKA-dependent LTP. We found
that a specific inhibitor of PKA, Rp-cAMPS (Dostmann 1995), blocked expression of tetra-burst LTP in wild-type
slices without affecting synaptic transmission in an adjacent,
untetanized pathway (Fig. 3A).
The mean fEPSP slope measured 2 h after the end of tetra-burst
stimulation was 227 ± 18% of pre-LTP baseline values in
wild-type control slices, whereas the corresponding mean fEPSP slope
measured in slices treated with 100 µM Rp-cAMPS was only 100 ± 2% (P < 0.01, Student's t-test). In an
adjacent untetanized pathway, the mean fEPSP slope 2 h after
tetra-burst stimulation of the neighboring pathway was 92 ± 9%
(Fig. 3A,
). Hence these results establish that
tetra-burst stimulation produces a form of LTP that is blocked by a
specific pharmacological inhibitor of PKA.
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We next tested the hypothesis that the theta-burst LTP that was intact
in R(AB) mutant slices may still be PKA dependent. In R(AB) mutant
slices, theta-burst stimulation (15 bursts of 4 pulses at 100 Hz,
delivered at a 200-ms interburst interval) induced robust LTP: the mean
fEPSP slope measured 2 h after the end of theta-burst stimulation
was 155 ± 12% in drug-free slices (Fig. 3B). In
contrast, Rp-cAMPS blocked expression of theta-burst LTP in R(AB)
mutant slices: the mean fEPSP slope measured 2 h after theta-burst
stimulation in drug-treated slices was only 97 ± 8% of pre-LTP
baseline values (P < 0.02, Fig. 3B).
Rp-cAMPS had no significant effect on fEPSP slopes recorded during
stimulation of an adjacent untetanized pathway (Fig. 3B,
).
These data suggest that theta-burst LTP in R(AB) mutant slices is
indeed PKA dependent (see also Nguyen and Kandel 1997
for wild-type data) as defined by its block by Rp-cAMPS. When
considered alongside our finding that theta-burst LTP was normal in
R(AB) mutant slices that express an inhibitory form of the RI
regulatory subunit, these pharmacological data suggest that although
both tetra-burst and theta-burst patterns of stimulation elicit
PKA-dependent forms of LTP in R(AB) slices, each may recruit different
isoforms of PKA. In particular it appears that theta-burst LTP requires significant amounts of PKA isoforms that contain regulatory subunits other than the mutant RI
subunit, whereas tetra-burst LTP requires isoforms of PKA that contain the mutant RI
subunit.
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DISCUSSION |
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Synaptic activity is an important regulator of plastic changes in
hippocampal synaptic transmission (Madison et al. 1991; Martin et al. 2000
). Protein kinases are also key
effectors of synaptic plasticity, and it is generally believed that the
activation of particular kinases may be modulated by specific patterns
of electrical activity (Chapman et al. 1995
;
Lisman 1989
; Micheau and Riedel 1999
).
There is biochemical evidence that some kinases may be tuned to
discrete patterns of impulse activity for optimal activation (see
De Koninck and Schulman 1998
, for CaMKII data). However,
in vitro biochemical assays of kinase activation kinetics cannot
address how synaptic activity influences kinase-mediated synaptoplastic
processes, such as LTP, in living neurons. In contrast, genetic
modification of the hippocampal activities of specific kinases in mice
can be used to study the in situ kinase dependence of different forms
of LTP that are induced by distinct temporal patterns of synaptic
activity (Abel et al. 1997
; Mayford et al. 1995
).
We have shown that decreasing both the amount and the temporal spacing
of imposed synaptic activity (theta-burst stimulation) in CA1 neurons
in hippocampal slices can fully restore LTP in R(AB) mutant mice that
have genetically reduced levels of hippocampal PKA activity. The
simplest explanation for this result is that theta-burst stimulation
rescued LTP by recruiting other subcellular processes or signaling
pathways that are not dependent on, or not linked to, a significant
activation of the cAMP-PKA signaling pathway. However, it is clear that
theta-burst LTP is critically dependent on the activation of PKA as
evidenced by the block of theta-burst LTP by Rp-cAMPS in R(AB) slices
(the present study) and in slices from wild-type mice (Nguyen
and Kandel 1997).
R(AB) mice have hippocampal basal PKA activity approximately 40-50%
less than that of wild-type mice (Abel et al. 1997).
This reduced PKA activity resulted from expression of a dominant
negative form of the RI
regulatory subunit of PKA (Clegg et
al. 1987
). One possible mechanism for the observed rescue of
LTP in R(AB) mutants is that theta-burst stimulation may have produced
modest increases in cAMP, not enough to substantially recruit isoforms of PKA containing the mutant RI
regulatory subunit (see following text for discussion of cAMP activation constants of regulatory subunits). However, the block of theta-burst LTP by Rp-cAMPS in these
mutants suggests that theta-burst stimulation still activated significant amounts of PKA isoforms that were critical for expression of this form of LTP. These isoforms likely did not contain mutant RI
subunits because theta-burst LTP was normal in R(AB) mutants. Theta-burst stimulation might recruit tetramers of PKA-containing RII
subunits, the subcellular localization of which is mediated by
interactions with A-kinase anchoring proteins (Carr et al. 1991
; Scott et al. 1990
). Although hypothetical,
these considerations underscore the need for further research, using
molecular biological and biochemical techniques, to specifically
identify and measure the amounts of particular isoforms of PKA that are
recruited by these different patterns of synaptic activity in the slice preparation.
It is noteworthy that another isoform of PKA, containing a different
type of regulatory subunit, RI, is 2.5-fold more sensitive to
activation by cAMP than RI
(Cadd et al. 1990
). It is
therefore plausible that the theta-burst rescue of LTP seen in R(AB)
mutants may have resulted from the activation of PKA isoforms
containing RI
following a modest increase in intracellular cAMP
elicited by theta-burst stimulation. Strong stimulation protocols, such as the tetra-burst regimen used here, produced an LTP deficit in R(AB)
mutant slices probably because such stimulation engaged significant
amounts of PKA isoforms containing the mutant RI
subunit (in
addition to tetramers containing RI
) or because the amount of PKA
activated by tetra-burst stimulation is not sufficient to support this
type of LTP in mutant slices. Levels of expression of PKA tetramers
containing the mutated form of RI
are likely higher than the
expression levels of tetramers containing RI
. In support of this
notion is our observation that tetra-burst stimulation, which should
activate PKA more strongly than our weaker theta-burst protocol, still
produces defective LTP in R(AB) mutant slices. Tetra-burst stimulation
should still activate isomers containing the RI
subunit, but the
presence of isomers containing mutant RI
in high or saturating
concentrations may buffer PKA activation sufficiently to block
tetra-burst LTP in R(AB) mice. Also the markedly lower sensitivity to
activation by cAMP of tetramers containing the mutant RI
subunit
(Woodford et al. 1989
) would prevent significant
activation of these isoforms by the more modest increases in cAMP that
may result from weaker theta-burst stimulation. Hence tetra-burst, but
not theta-burst, LTP likely requires PKA tetramers containing the
mutant form of RI
regulatory subunit.
It is interesting that RI knockout mice (Brandon et al.
1995
) showed normal LTP induced by a spaced stimulation
protocol similar to the regimen that we have used here to demonstrate
deficient tetra-burst LTP in R(AB) mutants. This may have resulted from selective, or substantial, recruitment of PKA isoforms containing native RI
subunits in these RI
knockout mice. Indeed the
expression of RI
subunits is believed to increase in compensation
for the genetic knockout of the RI
subunit (Brandon et al.
1995
). Thus LTP induced by spaced 100-Hz stimulation in these
RI
knockout mice does not appear to require tetramers containing the
RI
subunit. In R(AB) mice, no compensatory changes in the
hippocampal expression of other PKA subunits have been detected (T. Abel, unpublished observations). It would be interesting to see whether
theta-burst LTP is affected by the knockout of the RI
subunit.
However, it should be noted that the knockout mice of Brandon et
al. (1995)
did not show measurable changes in hippocampal PKA
activity unlike the R(AB) mice used in the present study, which showed
reductions in hippocampal PKA activity (Abel et al.
1997
).
The observed block of both forms of LTP by Rp-cAMPS lends
credence to the idea that although these distinct patterns of
stimulation elicited forms of LTP that are PKA dependent (as defined by
their sensitivities to Rp-cAMPS), this PKA dependence may be subtly regulated by the activity-dependent recruitment of different tetramers of PKA. Rp-cAMPS inhibits PKA activity by binding to the regulatory subunit of PKA, thereby preventing the cAMP-induced release of catalytic subunits (Dostmann 1995). At the concentration
of Rp-cAMPS used here (100 µM), inhibition of PKA by this drug should
show no isoform specificity, as the half-maximal activation constants for cAMP activation of wild-type and mutant regulatory subunits are in
the range of 40 nM to 5 µM (Cadd et al. 1990
;
Woodford et al. 1989
). However, genetic reduction of
hippocampal PKA activity in R(AB) mice, through directed expression of
an inhibitory form of just one type of regulatory subunit, was
sufficient to impair LTP induced by spaced tetra-burst, but not by
compressed theta-burst, stimulation. Hence our data, obtained by
combining pharmacological, electrophysiological, and genetic
approaches, support the notion that different patterns of synaptic
activity may induce PKA-dependent forms of LTP that require distinct
PKA isoforms.
Another interpretation of our results is that tetra- and
theta-burst stimulation require different amounts of the same
isoform(s) of PKA in R(AB) mutant mice. Because R(AB) mutant mice show
less hippocampal PKA activity, tetra-burst LTP (which is defective in
mutants) might require activation of larger quantities of PKA isoforms
identical to those needed for theta-burst LTP. In contrast, theta-burst
stimulation still elicits normal LTP in mutant slices because the
amount of PKA present in mutant mice may be sufficient to support
theta-LTP. This scenario may be improbable for the following reasons.
Tetra-burst stimulation (consisting of 4 1-s bursts of 100 Hz) should
lead to a larger increase in intracellular cAMP and stronger activation
of existing PKA isoforms in mutant slices than theta-burst
stimulation (consisting of only 20 pulses in the 1st second of our 3-s
theta protocol). Thus one would have to explain how stronger activation
of the same isoform(s) of available PKA (following tetra-burst
stimulation) can lead to defective LTP in mutant slices, whereas less
robust activation of the same isoform(s) of PKA (resulting from weaker
theta-burst stimulation) produces normal LTP in mutant slices. It
should be noted that the cAMP activation constants (which reflect the
concentration of cAMP needed to bind to and activate PKA) of tetramers
containing mutant RI subunits are considerably higher than those
measured from tetramers containing wild-type subunits (Cadd et
al. 1990
; Woodford et al. 1989
). Hence
tetra-burst stimulation should more readily engage larger quantities of
isoforms containing mutant RI
subunits than theta-burst stimulation.
This scenario may explain why tetra-burst, but not theta-burst,
stimulation leads to defective LTP in mutant slices. Further research,
involving exact measurements of synaptic PKA isoform
expression and activities following defined patterns of stimulation, is
required to resolve this issue.
A key question that needs to be examined is: what proportion of
the total complement of hippocampal PKA is contributed by isoforms
containing the RI regulatory subunit? PKA is a tetrameric holoenzyme
composed of two catalytic and two regulatory subunits. At least two
distinct types of catalytic subunits and four different types of
regulatory subunits are known to be expressed in the mouse hippocampus
in situ and, particularly, in area CA1 (Cadd and McKnight
1989
). In principle, therefore, there may be as many as 30 different tetrameric combinations of these wild-type catalytic and
regulatory subunits in situ. It is not known whether all 30 tetrameric
isoforms actually exist in area CA1, and it is unclear whether there
are significant constraints on the formation of particular tetrameric
isoforms (e.g., spatial anchoring of regulatory subunits) (see
Colledge and Scott 1999
). Nonetheless one can calculate a theoretical estimate of the proportion of the total complement of PKA
holoenzyme that is contributed by tetrameric isoforms containing at
least one RI
regulatory subunit. There are 12 possible tetrameric forms of PKA that contain at least one RI
subunit. Thus, of 30 possible tetrameric combinations of catalytic and regulatory subunits, the contribution (to the total PKA complement) of tetramers containing the RI
subunit is, in principle, 12/30 or 40%. This figure assumes that all 30 possible tetrameric isoforms are present. How does this
theoretical calculation compare to the measured level of basal PKA
activity in R(AB) mutant mice? In these mice, basal PKA activity in
hippocampal extracts is reduced by approximately 40-50% (Abel
et al. 1997
) as a direct result of genetic expression of an
inhibitory form of the RI
regulatory subunit. Furthermore, no
compensatory changes in the hippocampal expression of other PKA
subunits have been detected in R(AB) mice (T. Abel, unpublished observations). Hence, there is a close correspondence between the
theoretical calculated proportion of the total PKA complement contributed by isoforms containing the RI
subunit and the observed levels of inhibition of hippocampal PKA activity produced by genetic mutation of RI
in R(AB) mice. This supports the notion that a significant amount (about 50%) of the total complement of hippocampal PKA available for recruitment in R(AB) mice consists of isoforms containing regulatory subunits other than RI
.
In summary, we have shown that altering the amount and temporal spacing
of synaptic stimulation fully restores LTP in hippocampal slices of
R(AB) mutant mice that have genetically reduced levels of hippocampal
PKA activity. These mice express an inhibitory form of the RI
regulatory subunit of PKA. Spaced tetra-burst, but not theta-burst, LTP
was impaired in R(AB) mutant slices. Together with our finding that
Rp-cAMPS, a specific inhibitor of PKA, blocked theta-burst LTP in R(AB)
slices, these experiments provide evidence for the notion that
different amounts and patterns of synaptic activity may elicit
PKA-dependent forms of LTP that require distinct isoforms of PKA.
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ACKNOWLEDGMENTS |
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N. H. Woo holds a Studentship from the Alberta Heritage Foundation for Medical Research (AHFMR). S. N. Duffy is a Postdoctoral Fellow of the AHFMR. P. V. Nguyen holds Scholar awards from the Canadian Institutes of Health Research (CIHR, formerly the Medical Research Council of Canada) and the AHFMR. T. Abel holds a Scholar award from the John Merck Fund.
This work was supported by grants from the National Institutes of Health and the Whitehall Foundation (T. Abel) and by the CIHR and the AHFMR (P. V. Nguyen).
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FOOTNOTES |
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* N. H. Woo and S. N. Duffy contributed equally to this work.
Address for reprint requests: P. V. Nguyen, Dept. of Physiology, University of Alberta School of Medicine, Medical Sciences Bldg., Edmonton, Alberta T6G 2H7, Canada.
Received 21 June 2000; accepted in final form 17 August 2000.
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REFERENCES |
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