©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Temporal Differences in the Phosphorylation State of Pre- and Postsynaptic Protein Kinase C Substrates B-50/GAP-43 and Neurogranin during Long Term Potentiation (*)

Geert M. J. Ramakers , Pierre N. E. De Graan (§) , Ivan J. A. Urban , Dick Kraay , Tong Tang (¶) , Piera Pasinelli (**) , A. Beate Oestreicher , Willem H. Gispen

From the (1) Department of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, University of Utrecht, Utrecht, The Netherlands

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The phosphorylation state of two identified neural-specific protein kinase C substrates (the presynaptic protein B-50 and the postsynaptic protein neurogranin) was monitored after the induction of long term potentiation in the CA1 field of rat hippocampus slices by quantitative immunoprecipitation following P labeling in the recording chamber. B-50 phosphorylation was increased from 10 to 60 min, but no longer at 90 min after long term potentiation had been induced, neurogranin phosphorylation only at 60 min. Increased phosphorylation was not found when long term potentiation was blocked with the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate, when only low frequency stimulation was applied or tetanic stimulation failed to induce long term potentiation. Our data show that both B-50 and neurogranin phosphorylation are increased following the induction of long term potentiation, thus providing strong evidence for pre- and postsynaptic protein kinase C activation during narrow, partially overlapping, time windows after the induction of long term potentiation.


INTRODUCTION

Hippocampal long term potentiation (LTP)() is the most extensively studied model of activity-dependent synaptic plasticity and is proposed to be a model for certain forms of memory (1) . The major form of LTP in the CA1 subfield of the hippocampus can be induced by application of a brief train of high frequency pulses to afferent fibers (2) and requires activation of N-methyl-D-aspartate (NMDA) receptors (3) and an elevation of the internal Ca concentration (4, 5) .

Activation of PKC is thought to be one of the steps in the molecular mechanisms underlying increased synaptic efficacy (for review, see Ref. 6). This notion is based on different lines of evidence: (i) pharmacological manipulation of PKC activity alters LTP (7, 8, 9, 10, 11) ; (ii) mice lacking the -PKC gene have altered LTP (12) ; (iii) measurement of total PKC activity after tetanic stimulation either using exogenous artificial or neuron-specific substrates shows increased activity (13-15); (iv) tetanic stimulation is paralleled by a PKC translocation as measured by Western blotting (16) or an increase in the amount of a persistent active form of PKC (17) . Limited information is available about the temporal and spatial distribution of PKC activation during LTP. Interestingly, phorbol ester- and tetanus-induced LTP were shown to be accompanied by an increased phosphorylation of protein B-50 (16, 18, 19), confirming previous post-hoc phosphorylation data implying an involvement of presynaptic PKC in LTP (20) .

B-50 (also known as GAP-43, F1, or neuromodulin) (for review, see Ref. 21) is nervous tissue-specific in adult animals, has a strict presynaptic localization (22, 23) , and has been implicated in neurotransmitter release (24, 25) . Recently, a postsynaptic, neuron-specific substrate for PKC has been identified, named neurogranin (also known as RC3, BICKS) (26, 27, 28, 29, 30) . Rat neurogranin shares an 18-amino acid sequence with B-50 which comprises the single PKC phosphorylation site (the only phosphorylation site reported) and the atypical calmodulin binding domain (26, 27, 31) . Taking advantage of this sequence identity in B-50 and neurogranin, we developed a quantitative procedure to precipitate B-50 and neurogranin from a single hippocampal slice, thus enabling us to monitor the phosphorylation state of a pre- and postsynaptic PKC substrate simultaneously. We show that phosphorylation of both PKC substrates is increased in a narrow time window around 60 min after LTP induction and that the increase in B-50 phosphorylation precedes that of neurogranin phosphorylation.


EXPERIMENTAL PROCEDURES

Transversal hippocampal slices (450 µm) were made from hippocampi from male Wistar rats (100-120 g) on a McIlwain tissue-chopper, collected in Petri dishes containing constantly gassed with 95% O, 5% CO phosphate-free artificial cerebrospinal fluid (phosphate-free ACSF: NaCl, 124 mM; KCl, 4.5 mM; MgSO, 1.3 mM; glucose, 10 mM; NaHCO, 20 mM; and CaCl, 2.5 mM, pH 7.4) and stored at room temperature until the start of the experiments.

Three hippocampal slices from three different rats were transferred to tubes containing 900 µl of phosphate-free ACSF at 30 °C. After 30 min, 100 µCi of P (40 mCi/ml, Amersham, Buckinghamshire, United Kingdom) was added, and slices were labeled for 90 min. Slices were treated for 30 min with 4--phorbol 12,13-dibutyrate (PDB, 10M; Sigma), 4--phorbol 12,13-didecanoate (PDD, 10M; Sigma), or a combination of PDB (10M) with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7, 10M; Sigma), polymyxin B (PMXB, 10,000 IU/ml; Sigma), chelerythrine (10M; Sigma), calphostin C (5 10M; gift from H. Boddeke, Sandoz AG, Basel, Switzerland) or staurosporine (10M; Sigma). The PKC antagonists were added 15 min prior to PDB. Phorbol esters, chelerythrine, calphostin C, and staurosporine were dissolved in MeSO (0.01%), H-7, and PMXB in phosphate-free ACSF. MeSO did not affect B-50 or neurogranin phosphorylation. After treatment the slices were washed twice in 2 ml of ice-cold ACSF buffer containing 100 mM NaF, 1.2 mM NaHPO, 10 mM EDTA, 5 mM EGTA, and homogenized in 100 µl of HO containing 100 mM NaF, 10 mM EDTA, and 5 mM EGTA. Of the total homogenate 75 µl was added to 50 µl stop-mix solution containing (final concentration) 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol (v/v), 5% -mercaptoethanol (v/v), 0.001% bromphenol blue, and samples were stored at -20 °C until trichloroacetic acid precipitation and immunoprecipitation. The remainder of the homogenate was used to determine protein content (32) with bovin serum albumin as a standard. To determine quantitative conditions for neurogranin immunoprecipitation slices were P labeled, washed, and homogenized. A linear relationship between the amount of hippocampal tissue added to the precipitation mixture and the amount of immunoprecipitated neurogranin was found in the range of 0.15-2.5 µg of hippocampal tissue (data not shown). Therefore all immunoprecipitations to determine neurogranin phosphorylation were performed with 0.625 µg slice homogenate protein. Purified neurogranin was phosphorylated with PKC as described earlier (33) .

For electrophysiological recordings three slices were transferred to a recording chamber and superfused (1 ml/min) for at least 60 min with gassed phosphate-free ACSF at 30 °C. Bipolar stimulation electrodes, stainless steel wires of 50 µm diameter (insulated except for the tip), were placed on the radial layer of the CA1 field. Glass microelectrodes with a tip diameter of 3-5 µm, resistance 2-5 M, filled with phosphate-free ACSF were used to record field excitatory postsynaptic potentials (fEPSPs) in the radial layer of CA1 field in response to orthodromic stimulation. In all three slices the maximal fEPSP was determined first. Only slices in which the maximal fEPSP was bigger than 1 mV were selected. Two slices were randomly chosen to be used for electrophysiological recordings. Stimulation intensity was adjusted to evoke half-maximal responses and was kept constant throughout the experiment. Thirty min after placement of the electrodes the superfusion medium was changed to ACSF containing 100 µCi/ml P, and after 90 min, base-line recordings of fEPSPs were started (0.05 Hz, 25 min). Subsequently, one slice received a short train of high frequency stimulation (100 Hz, 1 s, test intensity), and recordings were continued for another 10, 30, 60, 90, or 120 min. The other stimulated slice did not receive high frequency stimulation and the third slice served as an unstimulated control. In some experiments slices were treated with 50 µMD-2-amino-5-phosphonovalerate (D-APV; Cambridge Research Biochemicals, Norwich, UK) 10 min before high frequency stimulation was applied. At the end of the experiments, all three slices were removed from the recording chamber, washed, and homogenized. Evoked fEPSPs were recorded using a conventional AC-coupled amplifier, stored on magnetic tape, and digitized using pCLAMP (Axon Instruments) software. Data were analyzed using the same software, and the initial slope of the fEPSPs was calculated. For graphical presentation, the data are expressed as percentages of the average of the base-line slope of the fEPSPs.

The degree of B-50 phosphorylation was determined using the method described by De Graan et al.(34) , and the degree of neurogranin phosphorylation was determined with a slightly modified procedure (see below). For B-50 immunoprecipitation, 10 µg of protein homogenate was incubated overnight at 4 °C with 1:200 (final dilution) antibody 8919 or 8420 in 400 µl of radioimmunoassay buffer of the following composition: 200 mM NaCl, 10 mM EDTA, 10 mM NaHPO, 0.5% Nonidet P-40 (v/v). For neurogranin immunoprecipitation 0.625 µg of protein homogenate was incubated overnight at 4 °C with 1:66.7 (final dilution) antibody 8420 in 400 µl of radioimmunoassay buffer. Antigen complexes were precipitated with washed pansorbin (Calbiochem), solubilized in stop-mix solution, and boiled for 10 min. Immunoprecipitates were analyzed by 11% (B-50) or 18% (neurogranin) SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in duplicate or triplicate. Incorporation of P into proteins was detected using a Fuji BAS1000 imaging system (Raytest, Straubenhardt, Germany) and quantified using PDI Quantity One software.

Total P incorporation into proteins was determined by trichloroacetic acid precipitation. In short, 2.5 µg of protein homogenate was spotted on 3MM Whatmann paper, the filters were washed three times with ice-cold 10% trichloroacetic acid solution and thereafter with ethanol, ethanol/acetone (1:1), and acetone alone. Filters were dried and counted in a Packard 2000 CA liquid scintillation counter. The analyzed amounts of P incorporation into B-50 and neurogranin were normalized for total P incorporation into proteins as determined by trichloroacetic acid precipitation and are expressed as percentages of the unstimulated controls. Statistical analysis were performed using Student's t test for unpaired or paired samples as appropriate.


RESULTS

To assess whether neurogranin is a substrate for PKC in the hippocampal slice, we quantified B-50 and neurogranin phosphorylation in slices treated with either phorbol ester, PKC inhibitors, or both. After treatment of slices for 30 min with 10M PDB neurogranin as well as B-50 phosphorylation was significantly increased compared with buffer controls (Fig. 1): 134 ± 44% (mean ± S.E.) and 182 ± 36%, respectively. The PDB-induced increase in neurogranin and B-50 phosphorylation could be antagonized by the kinase inhibitors H-7 (10M), PMXB (10,000 IU/ml), calphostin C (5 10M), chelerythrine (10M), or staurosporine (10M). Neither antagonists had an effect on basal neurogranin or B-50 phosphorylation (data not shown). The phorbol ester PDD (10M), inactive with respect to PKC activation, affected neither B-50 nor neurogranin phosphorylation. These results show that it is possible to quantify changes in the in situ phosphorylation state of neurogranin and B-50 in a single hippocampal slice and that neurogranin is an in situ PKC substrate.


Figure 1: Phorbol ester-induced increase in neurogranin and B-50 phosphorylation can be inhibited by kinase inhibitors. Hippocampal slices were P-labeled and treated with PDB (10M), PDB (10M) + H-7 (10M), PDB (10M) + PMXB (10,000 IU/ml), PDB (10M) + chelerythrine (Chel; 10M), PDB (10M) + calphostin C (Cal C; 5 10M), PDB (10M) + staurosporine (Stau; 10M) or PDD (10M) for 30 min, and B-50 (A) and neurogranin (B) phosphorylation was determined (n = 2 or 4, for details see ``Experimental Procedures''). Antagonists were applied 15 min prior to phorbol ester treatment. PDB induces an increase in both neurogranin (134 ± 44%) and B-50 (182 ± 36%) phosphorylation, which could be antagonized by all kinase inhibitors. The inactive phorbol ester PDD and the inhibitors alone did not influence B-50 or neurogranin phosphorylation. Asterisk, p < 0.05 (n = 4, comparing PDB with PDB + inhibitor.



Application of a short train of high frequency stimulation (100 Hz, 1 s, test intensity) induced an increase in the slope of the fEPSPs in the CA1 field of the hippocampus (Fig. 2). Ten min after the tetanus was applied the increase was 71.3 ± 17.3%; at 30 min, 56.2 ± 12.8%; at 60 min, 60.8 ± 14.1%; at 90 min, 59.7 ± 7%; and at 120 min, 53 ± 6.2%. Typical examples of evoked responses are shown in Fig. 2F. Slices not receiving high frequency stimulation showed stable responses throughout the experiments (fig. 2 , A-E, open circles).


Figure 2: Induction of LTP by high frequency stimulation. A-E, to examine the development of LTP after application of the conditioning train electrophysiological recordings were stopped 10 (A), 30 (B), 60 (C), 90 (D), or 120 (E) min after induction of LTP. Presented are means ± S.E. from 5 (E), 6 (A, B, and D), or 13 (C) experiments. Filled circles represent slices receiving high frequency stimulation at t = 0 min, and open circles represent slices only receiving low frequency stimulation. F, typical examples of evoked responses in the dendritic region of area CA1 of the hippocampus. The upper plot is a response 5 min before induction of LTP, and the middle plot is a response 55 min after induction of LTP. Both responses are plotted together in the lower panel. Calibration: 2 mV and 10 ms.



A typical example of phosphorimage analysis of immunoprecipitates from hippocampal slices 60 min after tetanic stimulation is shown in Fig. 3 . B-50 immunoprecipitation reveals a single phospho band at 48 kDa on 11% SDS-PAGE (Fig. 3A). Phosphorylated neurogranin is not seen under these conditions, because it runs of the gel in the dye front. Neurogranin immunoprecipitation (Fig. 3B) reveals a single band at 17 kDa on 18% SDS-PAGE. Under these conditions B-50 phosphorylation is not detectable, because it is much less prominent than neurogranin phosphorylation. The 17-kDa phospho-band was identified as neurogranin by three independent techniques. 1) The 17-kDa band comigrated with PKC phosphorylated purified neurogranin on 18% SDS-PAGE. 2) Unlabeled hippocampal tissue displaced PKC-phosphorylated purified neurogranin in the immunoprecipitation in a concentration-dependent manner. 3) No 17-kDa phosphoprotein was detectable when the immunoprecipitation was performed after preabsorption of the antibodies with purified neurogranin (data not shown). Quantification of these phosphorimages revealed that neurogranin phosphorylation (Fig. 3B) in the LTP slice was 168%, in the slice receiving only low frequency stimulation 98.7% compared with the unstimulated control slice (100%). For B-50 phosphorylation (Fig. 3A), these data were 166.3 and 94.3%, respectively. Total P incorporation as determined with trichloroacetic acid precipitation was not different between the three slices in this experiment.


Figure 3: Increase in B-50 and neurogranin phosphorylation 60 min after induction of LTP. A, typical example of phosphorimage analysis of B-50 immunoprecipitation after 11% SDS-PAGE, showing the amount of P incorporation into B-50 in an unstimulated control slice (CON), a slice in which LTP is induced with high frequency stimulation (LTP), and a slice only receiving low frequency stimulation (LFS). B, typical example of phosphorimage analysis of neurogranin immunoprecipitation after 18% SDS-PAGE (same experiment as for A). Arrows indicate the position of phosphorylated B-50 and neurogranin, respectively.



One hour after LTP induction neurogranin phosphorylation was increased by 77.9 ± 17.5% in slices showing LTP (n = 6, p < 0.01) and B-50 phosphorylation was increased by 31 ± 6.5% (n = 13, p < 0.001) (Fig. 4, A and C). To test if this increase was specific for LTP expression, induction of LTP was prevented by application of 50 µMD-APV (electrophysiological data not shown). Slices in which LTP was blocked with D-APV did not show an increase in neurogranin or B-50 phosphorylation 60 min after high frequency stimulation (Fig. 4, A and C). D-APV itself had no effect on B-50 or neurogranin phosphorylation in low frequency stimulated slices. In four slices, the tetanic stimulation induced only a short (less than 5 min) increase in the evoked responses (data not shown). In these four slices, recordings were continued for 60 min and than B-50 and neurogranin phosphorylation were determined. In these slices where high frequency stimulation failed to produce LTP, there was also no change in B-50 or neurogranin phosphorylation (Fig. 4, A and C).


Figure 4: Increase in B-50 and neurogranin phosphorylation only occurs if LTP is induced. A and C, hippocampal slices received only low frequency test stimulation (open bars) or test stimulation and high frequency stimulation (filled bars). Sixty minutes after the tetanus slices were analyzed for B-50 (A) and neurogranin (C) phosphorylation. Slices expressing LTP (LTP) showed increased B-50 (31 ± 6.5%, p < 0.001, n = 13) and neurogranin (77.9 ± 17.5%, p < 0.01, n = 6) phosphorylation. After blockade of LTP by 50 µMD-APV (APV), there was no difference in B-50 and neurogranin phosphorylation between high frequency stimulated slices and slices only receiving low frequency stimulation (p > 0.1, n = 6 for both B-50 and neurogranin). In those slices in which high frequency stimulation failed to induce LTP (No LTP) no difference in B-50 or neurogranin phosphorylation was found between the two groups of slices. B and D, the increase in B-50 (B) and neurogranin (D) phosphorylation found 60 min after high frequency stimulation was plotted against the increase in the slope of the fEPSP of the same slice. The plot for B-50 as well as for neurogranin revealed a significant correlation (correlation coefficient = 0.80, p < 0.001, n = 13 for B-50 and correlation coefficient = 0.97, p < 0.001, n = 6 for neurogranin).



Subsequently, we determined whether there is a correlation between the increase in B-50 and neurogranin phosphorylation and the increase in the slope of the EPSPs. Sixty min after LTP induction (Fig. 4, B and D), the increase in B-50 as well as in neurogranin phosphorylation was significantly correlated with the increase in the slope of the EPSP (correlation coefficient = 0.80, p < 0.001, n = 13 for B-50 and correlation coefficient = 0.97, p < 0.001, n = 6 for neurogranin).

To study the temporal relationship between the increase in phosphorylation of both pre- and postsynaptic PKC substrates, experiments were stopped at different times after high frequency stimulation (Fig. 5). Neurogranin phosphorylation shows a significant increase in phosphorylation only 60 min after LTP was induced (77.9 ± 17.5%, n = 6). At no other time points significant differences could be detected between neurogranin phosphorylation in slices expressing LTP and slices receiving low frequency stimulation, although there was a robust potentiation at these time points (see Fig. 2, A-E). B-50 phosphorylation in potentiated slices is already increased 10 min after induction of LTP (62.6 ± 19.9%, n = 5) and remained elevated at 30 (66 ± 13.0%, n = 6) and 60 min (31 ± 6.5%, n = 13) after LTP induction, compared with slices receiving only low frequency stimulation. At all time points with a significant increase in B-50 phosphorylation (i.e. 10, 30, and 60 min after application of high frequency stimulation) this increase is significantly correlated with the increase in the evoked EPSP (correlation coefficient = 0.94, p < 0.01, n = 6 10 min after LTP; correlation coefficient = 0.81, p < 0.01, n = 6 30 min after LTP and correlation coefficient = 0.80, p < 0.001, n = 13 60 min after LTP). At 90 and 120 min after high frequency stimulation, no difference could be detected between B-50 phosphorylation in potentiated and low frequency stimulated slices (correlation coefficient = 0.19 and 0.21, not significant for 90 and 120 min after LTP, respectively), despite the robust potentiation of the fEPSP induced in these slices (see Fig. 2, D and E).


Figure 5: Increases in B-50 and neurogranin phosphorylation show temporal differences during LTP. A, slices expressing LTP (filled circles) show a significant increase in the amount of phosphorylated B-50 compared with the matched low frequency stimulated slices at 10 (p < 0.01), 30 (p < 0.01), and 60 min (p < 0.001) after application of high frequency stimulation. Ninety and 120 min after LTP induction there is no difference between the two groups of slices (p > 0.1). Slices only receiving low frequency stimulation for all the time points studied show no change in B-50 phosphorylation (open circles). B, neurogranin phosphorylation is only increased 60 min after application of high frequency stimulation in slices expressing LTP (filled circles) compared with slices showing no potentiation (p < 0.01). At the other time points there is no difference between the two groups of slices (p > 0.1). Slices only receiving low frequency stimulation for all the time points studied show no change in neurogranin phosphorylation (open circles).




DISCUSSION

Our data demonstrate that in hippocampus slices neurogranin is an endogenous PKC substrate. Its phosphorylation is increased by the phorbol ester PDB, and this increase can be antagonized by kinase inhibitors. Furthermore, we show that 60 min after LTP induction in the CA1 field of the hippocampus the in situ phosphorylation state of presynaptic protein B-50 as well as postsynaptic protein neurogranin is significantly increased in potentiated as compared with unpotentiated slices. This increase in B-50 and neurogranin phosphorylation is dependent on LTP expression, because inhibition of LTP induction by the NMDA receptor antagonist D-APV also prevents the increase in phosphorylation of both proteins. The fact that slices in which tetanic stimulation failed to induce LTP do not exhibit increased phosphorylation shows that the increase in phosphorylation is related to the expression of LTP, rather than to the delivery of the high frequency stimulation. Moreover, the magnitude of the increase in B-50 and neurogranin phosphorylation measured in a single slice is highly correlated with the increase in the slope of the EPSPs measured in that slice.

Our experimental approach, using in situ phosphorylation in hippocampal slices combined with quantitative immunoprecipitation of B-50 and neurogranin, allows to distinguish between pre- and postsynaptic PKC activity and resolve temporal differences in their activation. Presynaptic B-50 phosphorylation is increased from 10 to 60 min, but no longer at 90 and 120 min after tetanic stimulation. These results are in line with data from previous experiments (19) obtained after static labeling of slices in an interface chamber, a setup limiting measurements to 60 min post-tetanus. Recently, an increase in B-50 phosphorylation (+26 ± 9%) at the PKC phosphorylation site 10 min post-tetanus was found (16) . This increase was measured by Western blotting, using monoclonal B-50 antibodies directed against the single PKC phosphorylation site (Ser-41). Since the increase in B-50 phosphorylation is strictly dependent upon NMDA receptor activation, a retrograde signal appears to be involved, the nature of which is at present unknown. Arachidonic acid (35) as well as nitric oxide (36) have been suggested to act as retrograde messengers during LTP. Recently, it has been shown that activation of PKC by arachidonic acid selectively enhances the phosphorylation of B-50 in nerve terminal membranes (37) , but the specific role of arachidonic acid and nitric oxide in stimulating B-50 phosphorylation during LTP remains to be investigated. The significance of increased presynaptic PKC activation and B-50 phosphorylation following LTP is not yet clear. Both PKC (38-40) and B-50 (25, 41, 42, 43, 44) have been implicated in the regulation of neurotransmitter release. Studies measuring glutamate release after LTP induction indicate an increased transmitter release until at least 60 min after tetanic stimulation (45, 46, 47) , a time window similar to that found for increased B-50 phosphorylation following tetanic stimulation. Although the use of quantal analysis for central synapses is still a matter of debate, studies using quantal analysis also indicate an increase in neurotransmitter release after LTP induction (48-50). However, changes in postsynaptic parameters have also been reported using this technique (51, 52, 53) .

Postsynaptic PKC activity, as monitored by in situ neurogranin phosphorylation, was increased only in a short time window around 60 min after tetanic stimulation. No effects on neurogranin phosphorylation were found 10, 30, and 120 min after LTP induction. Studies in which PKC inhibitors were introduced into CA1 neurons support an involvement of postsynaptic PKC in the induction and early phases (<5 min) of LTP (9, 54, 55, 56) . Due to experimental limitations, we were unable to measure neurogranin and B-50 phosphorylation at time points shorter than 10 min. Therefore we have no information on a possible activation of postsynaptic PKC within the first 10 min following tetanic stimulation. Our data clearly show an increase in in situ neurogranin phosphorylation 60 min post-tetanus, indicating postsynaptic PKC involvement in a narrow time window overlapping with the tail of the time window of presynaptic PKC activation, as observed by the increase in B-50 phosphorylation. This is in line with the findings of Wang and Feng (56) , who show that postsynaptic PKC is important for the maintenance of LTP. However, their data and ours are not supported by those of Huang et al.(54) , who reported that micropressure injection of the inhibitors H-7 or polymyxin B in CA1 pyramidal cells, 10 min prior to tetanic stimulation, caused the potentiated EPSP slope to decay back to base line within 30-50 min after LTP induction, whereas injection 5 min post-tetanus was ineffective (54) . The most likely explanation for this apparent discrepancy is that a microinjection 5 min post-tetanus, as used by Huang et al.(54) , is no longer effective 60 min after tetanic stimulation (see also, Ref. 56). Alternatively, the increased phosphorylation state of neurogranin could be caused by a decrease in phosphatase activity. Phosphatases have recently been implicated in long term depression, another form of synaptic plasticity (57). Phosphorylation of neurogranin by other kinases than PKC seems unlikely, because it does not contain consensus phosphorylation sites for such kinases.

The functional significance of neurogranin in LTP has recently been demonstrated: microinjection of functionally interfering antibodies into CA1 pyramidal cells inhibited the induction of LTP (58) . Its precise function in the dendritic spine is not yet known. In a recent study (59) evidence was presented that PKC-mediated phosphorylation of neurogranin expressed in Xenopus oocytes increases the mobilization of intracellular Ca, probably by an interaction with the inositol triphosphate/diacylglycerol signal transduction pathway. Interestingly, B-50 and neurogranin share an 18-amino acid sequence containing the unique PKC phosphorylation site (Ser-41) within the atypical calmodulin binding domain (residues 39-51; Refs. 25, 31, 60, and 61). Since both a rise in intracellular Ca and PKC phosphorylation reduce calmodulin binding, B-50 as well as neurogranin may serve as local calmodulin stores, thus triggering local Ca/calmodulin-dependent processes such as calcium/calmodulin-dependent protein kinase II, which has been implicated in both pre- and postsynaptic mechanisms underlying LTP (9, 62) .

In conclusion, we present data showing that during expression of LTP in the CA1 field of the hippocampus, the in situ phosphorylation state of both a pre- and postsynaptic PKC substrate is increased, in narrow time windows within the first hour after LTP induction. Presynaptic PKC activation, as evidenced by B-50 phosphorylation, precedes postsynaptic PKC activation measured by neurogranin phosphorylation. Our data suggest that the increase in B-50 as well as neurogranin phosphorylation are part of the physiological changes occurring during the maintenance phase of LTP, thus implicating both pre- and postsynaptic PKC activation during the maintenance phase of LTP. Our finding strongly supports the idea that both pre- and postsynaptic changes occur during the expression of LTP.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Medical Pharmacology, Rudolf Magnus Institute for Neurosciences, P. O. Box 80040, 3508 TA Utrecht, The Netherlands. Tel.: 31-30-532662; Fax: 31-30-539032.

Present address: NIHHD, Section of Growth Factors, 9000 Rockville Pike, Bethesda, MD 20892.

**
Supported by European Neuroscience Program Grant ENP 16/3 from European Science Foundation.

The abbreviations used are: LTP, long term potentiation; ACSF, artificial cerebrospinal fluid; D-APV, D-2-amino-5-phosphonovalerate; fEPSP, field excitatory postsynaptic potential; H-7, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine; NMDA, N-methyl-D-aspartate; PDB, 4--phorbol 12,13-dibutyrate; PDD, 4--phorbol 12,13-didecanoate; PKC, protein kinase C; PMXB, polymyxin B; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We are grateful to Dr. H. Zwiers (University of Calgary, Canada) for supplying the purified neurogranin (BICKS).


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