Genetic and Pharmacological Demonstration of Differential Recruitment of cAMP-Dependent Protein Kinases by Synaptic Activity

Newton H. Woo,1,* Steven N. Duffy,1,2,* Ted Abel,3 and Peter V. Nguyen1,2

 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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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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-Ialpha ) 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 RIalpha subunit, whereas tetra-burst LTP requires PKA isoforms containing the mutant RIalpha 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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Cbeta 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 RIbeta 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 RIalpha 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 RIalpha 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 RIalpha 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.


    METHODS
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ABSTRACT
INTRODUCTION
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 RIalpha 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 MOmega 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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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Fig. 1. Strong, spaced stimulation reveals a deficit in long-term potentiation (LTP) in R(AB) mutant slices. A tetra-burst stimulation protocol, consisting of four 100-Hz bursts (each 1 s in duration) with an interburst interval of 5 min, elicited robust LTP in wild-types (), but not in R(AB) slices (). Sample field excitatory postsynaptic potential (fEPSP) sweeps were recorded at the time points indicated as a and b on the graph.

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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Fig. 2. Reducing the amount and temporal spacing of imposed activity rescues LTP in R(AB) mutant slices. A: plot of mean fEPSP slopes recorded from mutant and wild-type slices. A 3-s period of theta-burst stimulation (TBS) rescued LTP in R(AB) slices. Sample fEPSP traces were recorded at times a and b marked on the graph. B: summary histogram of cumulative fEPSP slopes recorded 2 h after LTP induction by theta-burst and by tetra-burst ("spaced") stimulation. Individual points superimposed on bars represent measurements from single slices. Many of these points overlap each other within a bar. Shapiro-Wilk's test for normality of the distribution of sampled data yielded P > 0.1 for these data points, indicating that a parametric Student's t-test for statistical significance is appropriate.

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 RIalpha 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 RIalpha 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, open circle ). 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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Fig. 3. A pharmacological inhibitor of protein kinase A (PKA) blocks LTP induced by both tetra-burst and theta-burst stimulation. A: Rp-cAMPS blocked LTP in wild-type slices following tetra-burst stimulation (). In an adjacent untetanized pathway in drug-treated slices, fEPSP slopes were unaffected during stimulation at once per minute (open circle ). B: theta-burst stimulation elicits a form of LTP in R(AB) mutant slices that is PKA dependent. Rp-cAMPS blocked expression of LTP in mutant slices (), whereas baseline fEPSPs, measured in a 2nd adjacent untetanized pathway in drug-treated slices (), were unaffected. Sample fEPSP traces were recorded at 5 min before the start of LTP induction and 2 h after the end of tetra- or theta-burst stimulation.

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 RIalpha 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 RIalpha subunit, whereas tetra-burst LTP requires isoforms of PKA that contain the mutant RIalpha subunit.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
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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 RIalpha 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 RIalpha 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 RIalpha 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, RIbeta , is 2.5-fold more sensitive to activation by cAMP than RIalpha (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 RIbeta 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 RIalpha subunit (in addition to tetramers containing RIbeta ) 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 RIalpha are likely higher than the expression levels of tetramers containing RIbeta . 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 RIbeta subunit, but the presence of isomers containing mutant RIalpha 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 RIalpha 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 RIalpha regulatory subunit.

It is interesting that RIbeta 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 RIalpha subunits in these RIbeta knockout mice. Indeed the expression of RIalpha subunits is believed to increase in compensation for the genetic knockout of the RIbeta subunit (Brandon et al. 1995). Thus LTP induced by spaced 100-Hz stimulation in these RIbeta knockout mice does not appear to require tetramers containing the RIbeta 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 RIbeta 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 RIalpha 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 RIalpha 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 RIalpha 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 RIalpha regulatory subunit. There are 12 possible tetrameric forms of PKA that contain at least one RIalpha subunit. Thus, of 30 possible tetrameric combinations of catalytic and regulatory subunits, the contribution (to the total PKA complement) of tetramers containing the RIalpha 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 RIalpha 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 RIalpha subunit and the observed levels of inhibition of hippocampal PKA activity produced by genetic mutation of RIalpha 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 RIalpha .

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 RIalpha 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.


    ACKNOWLEDGMENTS

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).


    FOOTNOTES

* 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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ABSTRACT
INTRODUCTION
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