Changes in Protein Kinase C (PKC) Activity, IsozymeTranslocation, and GAP-43 Phosphorylation in the RatHippocampal Formation After a Single-Trial ContextualFear Conditioning Paradigm

Elizabeth Young,1 Teresa Cesena,1 Karina F. Meiri,2

and Nora I. Perrone-Bizzozero1*

1Department of Neurosciences, University of New MexicoSchool of Medicine, Albuquerque, New Mexico2Department of Pharmacology, Tufts University, Boston,Massachusetts

ABSTRACT: The hippocampus plays an important role in spatial learn-ing and memory. However, the biochemical alterations that subserve thisfunction remain to be fully elucidated. In this study, rats were subjectedto a single-trial contextual fear conditioning (CFC) paradigm; the activa-tion of different protein kinase C (PKC) subtypes and the levels andphosphorylation of the plasticity-associated protein GAP-43 were assayedin the hippocampus at varying times after training. We observed a rapidactivation of hippocampal PKC (15 min through 24 h), with differentialtranslocation of the PKC isotypes studied. At early times after CFC (15–90min), PKC� and PKC� translocated to the membrane, while PKC�II andPKC� moved more transiently (15 to 30 min) to the cytosol. These PKCisotypes returned to the membrane at later time points after CFC. Corre-lating with these changes in PKC translocation and activity, there was anearly decrease in GAP-43 phosphorylation followed by a more sustainedincrease from 1.5-72 h. GAP-43 protein levels were also increased after3 h, and these levels remained elevated for at least 72 h. These changes inPKC and GAP-43 were specific to the CFC trained animals and no changeswere seen in animals exposed to the same stimuli in a non-associativefashion. Comparison of translocation of different PKC isotypes with thechanges in GAP-43 phosphorylation suggested that PKC�II and PKC�maymediate both the early changes in the phosphorylation of this protein andthe increases in GAP-43 expression at later times after CFC. Hippocampus2002;12:457–464. © 2002 Wiley-Liss, Inc.

KEY WORDS: PKC�II; PKC�; synaptic plasticity; learning and memory

INTRODUCTION

It has been demonstrated repeatedly that contextual learning is dependenton a functional hippocampus (Phillips and LeDoux, 1992; Kim et al., 1993;

Logue et al., 1997; Sacchetti et al., 1999; Anagnostarasand Fanselow, 2001). The contextual fear conditioning(CFC) task requires associative learning of a complex setof cues and the relationships between them to make arepresentation of the training environment. In this task,the unconditioned stimulus (US), a footshock, becomesassociated not only with a discrete conditioned stimulus(auditory cue, CS2), but also with the environment (con-text, CS1) in which the US was administered. After asingle training session, animals exhibit increased freezingbehavior when later returned to that environment. Thishas become a convenient tool for probing hippocampalfunction and has been used to study the effects of aging,drugs and genetic variations on hippocampal processing(Paylor et al., 1994; Lu and Wehner, 1997; Owen et al.,1997; Frankland et al., 1998; Oler and Markus, 1998).Despite the utility of this model, little is known about thebiochemical changes underlying the altered hippocampalprocessing after contextual learning.

Long-term potentiation (LTP) is a neurophysiologicalphenomenon thought to underlie specific forms of learn-ing and memory (Bliss and Collingridge, 1993). In thehippocampus, several links between LTP and spatiallearning have been made (Doyere and Laroche, 1992;Barnes et al., 1994; Izquierdo, 1994; Ishihara et al., 1997;Moser and Moser, 1999). A number of biochemicalchanges in the hippocampus have been described afterinduction of LTP. Activation of hippocampal proteinkinase C (PKC) (Akers et al., 1986; Malenka et al., 1986;Klann et al., 1993; Son et al., 1996) and GAP-43 phos-phorylation (Routtenberg et al., 1985; Gianotti et al.,1992; Pasinelli et al., 1995; Ramakers et al., 1999a) areboth promoted by the high-frequency stimulation re-quired to produce LTP. Increasingly, PKC activation hasbeen shown to be involved in multiple forms of learning(Bank et al., 1988; Wehner et al., 1990; Olds and Alkon,1991; Paylor et al., 1991). Recent evidence indicates thatGAP-43 may also be involved in certain forms of learning

Grant sponsor: National Institutes of Health; Grant number: AA 11336;Grant number: GM52576.*Correspondence to: Nora I. Perrone-Bizzozero, Department of Neuro-sciences, University of New Mexico School of Medicine, 915 Camino deSalud NE, BMSB Rm 145, Albuquerque, NM 87131-5223.E-mail: nbizzozero@salud.unm.eduAccepted for publication 24 August 2001DOI 10.1002/hipo.10015Published online 00 Month 2002in Wiley InterScience (www.interscience.wiley.com).

HIPPOCAMPUS 12:457–464 (2002)

© 2002 WILEY-LISS, INC.

(Routtenberg and Benson, 1980; Sheu et al., 1993; Zhao et al.,1995; Cammarota et al., 1997; Routtenberg et al., 2000; Young etal., 2000). These observations, although not conclusive in demon-strating a role for LTP in learning, suggest that there may becommon substrates for LTP and learning and memory. The firststep to understanding the role of these proteins in this process is tocharacterize changes in their activity or levels after the learningexperience. The contextual fear task used in this study offers severalbenefits for biochemical studies because it involves a single, simpletraining session, produces a robust behavior, and provides a de-fined starting point for such studies.

In this study, we sought to examine the time course of PKCactivation, as well as GAP-43 phosphorylation and expression afterCFC. In addition, the intracellular distribution of several PKCisotypes between the membrane and cytosolic fractions was as-sessed. We observed that PKC is rapidly activated in the hip-pocampus after the training session and there is differential trans-location of specific PKC isotypes. GAP-43 undergoes an earlydecrease in its phosphorylation and a later increase in both itsphosphorylation and levels of expression.

MATERIALS AND METHODS

Animals

Sprague-Dawley rats from Harlan Industries (Indianapolis, IN)were used for the experiments. Animals were maintained ad libi-tum on standard block chow and water, and kept on an 8:16-hlight/dark cycle. All were housed singly for at least 1 week beforetraining. All the experimental protocols were performed in accor-dance with the Guidelines for the Care and Use of LaboratoryAnimals established by the National Institutes of Health (NIH)and were approved by the University of New Mexico InstitutionalAnimal Care and Use Committee (IACUC).

Contextual Fear Conditioning

The training chamber was a rectangular box, 36 cm� 54 cm�24 cm in size, with a 7.5-W light mounted in the ceiling, and a gridfloor composed of 28 stainless steel rods, 4 mm in diameter, 2 cmapart. The grid floor was hooked to a shock generator, whichdelivered the 2-s, 1-mA footshock. The chamber was cleaned with70% EtOH between animals. Home cages for unpaired-control(UPC) rats were modified standard rat cages, in which the solidfloor was replaced with a grid floor similar to that of the trainingchamber.

Rats were randomly assigned to one of three training groups orto the unhandled naive group (N). Rats in the CFC group (alsoreferred to in the present study as tone-shock, or TS), were placedin the training chamber for 3 min before a 30-s presentation oftone, which co-terminated with the delivery of the footshock. Ratswere returned to their home cages 30 s after the tone-shock pre-sentation. Rats in the immediate-shock (IS) group were given asimilar foot shock immediately upon placement in the chamber,

and then allowed to remain there for 4 min. UPC rats were housedin the special home cages with a grid floor to allow the delivery ofthe footshock. On the training day, they were placed in the trainingchamber for 4 min (without presentation of tone or shock); 30 minlater, a 30-s tone was presented in their home cage. The shock wasadministered (through the grid floor of the home cage) 2 h later.Rats in the CFC (TS) group learn to associate the context (trainingenvironment) with the aversive footshock and display increasedfreezing behavior when tested 24 h later, with an average percent-age freezing to a context of 70.8 � 6.0% (Weeber et al., 2001).Rats in the IS and UPC groups, although receiving the same cues,do not exhibit freezing behavior in response to their training (Wee-ber et al., 2001).

Tissue Preparation

All reagents were purchased from Sigma (St. Louis, MO) unlessotherwise noted. Animals were sacrificed by decapitation at varioustimes post-training. The brain was removed and the hippocampiwere dissected out and kept on ice for immediate use or stored at�80°C for later use.

Hippocampi were homogenized with a glass/Teflon homoge-nizer in cold buffered sucrose solution (20 mM Tris-HCl, pH 7.4,0.32 M sucrose, 2 mM EDTA, 2 mM EGTA, 0.3 mM PMSF, 0.2mM sodium vanadate, 50 mM sodium fluoride, 2 �g/ml leupep-tin, and 2 �g/ml aprotinin). The homogenate was centrifuged at100g for 4 min. Aliquots of the resulting supernatant (S1) wereused for PKC activity assays and for Western blotting. The remain-ing S1 supernatant was centrifuged at 17,000g, for 20 min at 4°C,to yield the S2 supernatant and the P2 pellet (cytosolic and mem-brane fractions, respectively). The P2 fraction was resuspended incold buffered sucrose solution supplemented with 0.1% TritonX-100. S2 and P2 fractions were also used for Western blotting.Protein determinations were carried out on all three fractions ac-cording to the method of Bradford (1976), using bovine serumalbumin (BSA) as the standard (Bio-Rad, Hercules, CA).

PKC Activity Assay

Assays were performed using the Peptag Assay for Non-radioac-tive Detection of PKC (Promega, Madison, WI). Briefly, aliquotsof the S1 supernatant were mixed with the assay buffer and dye-labeled PKC substrate and incubated at 30°C for 30 min. Thereaction was stopped by heating at 95°C for 10 min. Samples wererun on agarose gels to separate phosphorylated and nonphospho-rylated peptide. The phosphorylated substrate band was excisedfrom the gel, dissolved in buffer, and the amount of dye-labeledpeptide in each sample quantitated by spectroscopy.

Determination of GAP-43 and PKC Levels

Levels of total and phosphorylated GAP-43 and of the PKCisoforms �, �I, �II, � , and � were determined by a quantitativeWestern blot method as described by Perrone-Bizzozero et al.(1996). Measures of total PKC, GAP-43, and phospho-GAP-43were done in the S1 fraction. PKC isotype distribution was exam-ined in both the S2 and P2 fractions to try to assess translocation of

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the different isoforms between the cytosolic and membrane cellu-lar fractions. Aliquots containing 15 �g of total protein were runon 10% polyacrylamide gels and proteins electrophoretically trans-ferred to PVDF membranes (Bio-Rad) as previously described(Perrone-Bizzozero et al., 1996). To control for variations in pro-tein loading, the PVDF blots were first stained with 0.1% Coo-massie Brilliant Blue and scanned for densitometric analysis. Theblots were blocked in milk buffer (10% powdered milk in Tris-buffered saline containing 0.5% Tween, TBS-T), before beingincubated with the appropriate primary antibody. For GAP-43detection, a sheep polyclonal antibody (Benowitz et al., 1988) wasused at a 1:2,000 dilution. For phospho-GAP-43 detection, a 1:15dilution of 2G12 (monoclonal phospho-specific GAP-43 anti-body, Meiri et al., 1991) was used. All PKC isoforms were detectedusing a 1:2,000 dilution of each antibody (Santa Cruz Biotechnol-ogy, Santa Cruz, CA). Blots were then washed in TBS-T beforeincubation with a horseradish peroxidase (HRP)-conjugated sec-ondary antibody. After repeated TBS-T washes, the blots wereincubated with Renaissance enhanced chemiluminescence (ECL)reagents (DuPont–NEN, Boston, MA) according to the manufac-turer’s protocols, and then exposed to X-ray film (Kodak X-0MATLS). Image analysis of the resulting bands was done using thePhotoAnalyst I system (Fotodyne, as previously described (Per-rone-Bizzozero et al., 1996). Data are expressed as percentage ofnaive controls run in parallel on the same blot.

Statistical Analysis

Statistical analysis of the data was done using two-tailed one-way analysis of variance (ANOVA). Post hoc tests consisted of theNewman-Keuls test and Bonferroni’s multiple comparison test.

RESULTS

Animals were given a single contextual training session with onepresentation of tone and shock (TS) or were subjected to the un-paired (UPC) or immediate shock (IS) training protocols as de-scribed in Materials and Methods. Initial experiments examinedthe changes in total PKC activity, and PKC isotype translocationin the hippocampus at various times after fear conditioning. Figure1 shows representative Western blots of several PKC isotypes in themembrane fraction from the hippocampus of naive, IS, and CFC(TS) rats. Statistical analysis of the data from these experiments isshown in Figures 2 and 3. For comparison, total PKC activity inthe hippocampus was measured at the same time points after train-ing. Significant increases in kinase activity were already observable15 min after training and remained elevated through 24 h afterCFC (Fig. 2A). In contrast, no significant changes were seen ineither the UPC or IS groups at any of the time points assayed.Therefore, data from these animals have been pooled for presenta-tion.

This measure of total PKC activity does not indicate which PKCisotypes may be activated. Because translocation to the membraneis necessary to activate PKC, an increase in the membrane fraction

may be indicative of activation of that particular isotype. To ad-dress this question, we measured the intracellular distribution offive major hippocampal PKC isotypes (� �I, �II, �, �). Total PKClevels (membrane plus cytosolic) did not change for any traininggroup or any time point (data not shown). For the UPC and ISgroups, no significant changes in the levels of any of the PKCisotypes were seen at any time point assayed. Even in the IS/UPC1 h post-training group, the apparently lower level of PKC� in themembrane fraction was not significantly different from that ofnaive rats (76.0� 1.5, P 0.152). In the CFC rats, PKC� levelsincreased in the membrane at 15–90 min and then returned tobaseline levels (Fig. 2B). PKC�I levels in the membrane showed nosignificant changes, except for a mild decrease at 48 h (Fig. 2C),while PKC� increased significantly in the membrane both at earlytimes (30 and 90 min) and at late times (48–72 h) after CFC (Fig.2E). In contrast with the other isotypes examined, the levels ofPKC�II and PKC� in the membrane fraction decreased at 15–30min and returned to this fraction at later time points after training(Fig. 2D,F).

Analysis of the relative distribution of the various PKC isotopesbetween the cytosolic and membrane fractions showed that theobserved decreases of PKC�II and PKC� in the membrane frac-tion at early times after CFC correlated with increases in the cyto-solic fraction (Fig. 3). Levels of PKC� in the cytosolic fractiondecreased at the same times in which we found increases in themembrane fraction (Fig. 3). Using PKC translocation to the mem-brane as a measure of activation would suggest that PKC�,PKC�I, and PKC� are responsible for most PKC activity observedat early time points, while PKC�II, PKC�, and PKC� contributeto this activity at later time points after CFC.

With the immediate activation of PKC after training, it wasexpected that GAP-43 phosphorylation would also increase. How-

FIGURE 1. Representative Western blots of PKC isotypes in themembrane fraction from hippocampus. Rats were contextual fearconditioned (CFC) using a tone and shock (TS) or trained with im-mediate shock (IS); the hippocampi were dissected at different timepoints after training. Naive animals (N) were left untrained. PKCisotypes were measured in the membrane fraction as described inMaterials and Methods.

____________________________________ GAP-43 AND PKC IN CONTEXTUAL FEAR CONDITIONING 459

ever, we found a significant decrease in GAP-43 phosphorylationat 15 and 30 min after training (Figs. 4, 5A). By 60 min, phos-phorylation had returned to basal levels and then showed signifi-cant increases from 90 min through our terminal time point at72 h. GAP-43 levels were also significantly increased from 3 h to72 h (Figs. 4, 5B). As with the PKC studies, no significant changesin either GAP-43 levels or phosphorylation state were seen in ratsfrom the IS or UPC groups at any of the time points observed.Analysis of the proportion of phosphorylated GAP-43 in the totalprotein pool indicated that the fraction of phosphorylated GAP-43was significantly decreased at 30 min and elevated at 72 h (Fig.5C).

DISCUSSION

The learning process is complex, involving multiple brain struc-tures. From lesioning studies, it is clear that the hippocampus is

FIGURE 2. Contextual fear conditioning (CFC) alters protein kinase C (PKC) activity and translocation of PKC subtypes. PKC activitywas measured in the S1 fraction of the hippocampus at various times after CFC, immediate-shock (IS), or unpaired-control (UPC) training. Analiquot of the S1 fraction was further purified to yield the membrane (P2) fraction for Western analysis of PKC isotypes. A: PKC activity isincreased by CFC, but not by IS or UPC training. Levels of PKC� (B), PKC�1 (C), PKC�II (D), PKC� (E), and PKC� (F) were measured inthe P2 fraction after CFC. All values are mean� SEM (n� 5–8). PKC isotype levels are expressed as percentage of naive control. *P < 0.05compared with naive.

FIGURE 3. Distribution of protein kinases PKC�, PKC�II, andPKC� between the membrane (P2) and cytosol (S2) fractions at earlytimes after contextual fear conditioning (CFC) training. All values aremean� SEM (n� 5–8). PKC isotype levels are expressed as percent-age of naive control. *P < 0.05 compared with naive.

460 YOUNG ET AL.

involved in contextual learning (Kim et al., 1993; Maren et al.,1997; Anagnostaras et al., 1999; Sacchetti et al., 1999). Within thehippocampus, specific changes in PKC activation and GAP-43phosphorylation have been observed in several learning paradigms.Recently, Young et al. (2000) showed that CFC differences inC57BL/6J vsDBA/2 mice correlated with changes GAP-43 phos-phorylation and expression. In this study, we demonstrated thatchanges in GAP-43 phosphorylation after CFC training correlatewith PKC activity and translocation of specific isotypes in the rathippocampal formation. These alterations are specific to ratstrained to associate the context with the footshock. It has beenshown repeatedly that delivery of a footshock immediately uponplacement in the training chamber results in no association be-tween the footshock and the chamber (Fanselow, 1986, 1990). Inthe absence of this association, as demonstrated by the IS rats, nochanges in PKC or GAP-43 were observed. Similarly, no changeswere seen in the UPC rats in which the context, tone, and foot-shock are presented individually in a non-associative fashion. Bothgroups serve as controls for the stresses of being subjected to a novelenvironment, either by itself or in conjunction with the tone, andto the delivery of an aversive footshock. Comparison of the CFCversus UPC and IS groups demonstrated that the changes inGAP-43 and PKC activity reported here are both context- andlearning-specific.

PKC activation has been shown to play an important role inlearning. For instance, in several inbred mouse strains endogenousPKC activity has been positively correlated with performance onthe Morris water maze (Wehner et al., 1990). Also, phorbol esters,which activate PKC, were shown to improve performance in theMorris water maze (Paylor et al., 1991). However, the contribu-

tions of the various PKC isotypes and substrates to LTP and learn-ing have been more difficult to assess. Consistent with the role ofPKC in other forms of associative learning and in the inductionand maintenance of LTP, we observed prolonged elevations ofPKC activity after fear conditioning. Using the crude measure oftranslocation for monitoring activation, it appears that PKC�,PKC�, and PKC� are largely responsible for this activity. PKC�has been shown to redistribute to the membrane rapidly after theinduction of LTP in CA1 (Leahy et al., 1993). Similarly, PKC�levels and activity were found to correlate with both LTP (Angen-stein et al., 1994) and the animal’s learning ability in various spatialtasks (Colombo et al., 1997, Douma et al., 1998). Studies inPKC� knockout mice revealed mild deficits in spatial and contex-tual learning (Abeliovich et al., 1993a) and a profound deficit inLTP (Abeliovich et al., 1993b). These mice also show altered basaland/or stimulated patterns of phosphorylation of the neuronal-specific PKC substrates GAP-43, MARCKS and neurogranin (Ra-makers et al., 1999b). Likewise, PKC� knockout mice were foundto have severe deficits in CFC (Weeber et al., 2000).

FIGURE 4. Representative Western blots of phosphorylatedGAP-43 (GAP-P) and total GAP-43 protein (GAP-T). The hip-pocampi of naive (N) and contextual fear conditioned (CFC) animalswere dissected at different time points after training. PhosphorylatedGAP-43 and total GAP-43 levels were measured in the S1 fraction asdescribed in Materials and Methods.

FIGURE 5. Contextual fear conditioning (CFC) alters GAP-43phosphorylation (A) and levels (B). GAP-43 phosphorylation andGAP-43 levels are expressed as percentage of naive control. The ratioof phosphorylated GAP-43 to total GAP-43 (GAP-P/GAP-T) is alsoaltered by CFC training (C). All values are mean � SEM (n � 5–8).*P < 0.05 compared with naive.

____________________________________ GAP-43 AND PKC IN CONTEXTUAL FEAR CONDITIONING 461

Additional support for the role of PKC in contextual learningcomes from studies comparing the behaviors of C57BL/6J andDBA/2 mice in various spatial learning tasks. The poor perfor-mance of DBA mice in these tasks correlated with a decrease intotal hippocampal PKC activity (Paylor et al., 1994, 1996), pre-sumably due to a decreased level of PKC� in the DBA mice (Payloret al., 1996). It also appears that differences in GAP-43 phosphor-ylation and levels are part of this strain difference (Young et al.,2000). However, since GAP-43 is a presynaptic PKC substrate andPKC� is localized in the postsynaptic terminal (Kose et al., 1990;Ramakers et al., 1999b), additional PKC isotypes must account forthese differences.

GAP-43 is important for synaptic plasticity mechanisms bothduring the initial development and the remodeling of synapticconnections (Benowitz and Routtenberg, 1997). Indeed, GAP-43expression remains high in the adult brain in the hippocampus andforebrain structures thought to be involved in learning (Benowitzet al., 1989). In addition, GAP-43 phosphorylation is altered incertain regions of chick brain after passive avoidance training(Zhao et al., 1995) and imprinting (Sheu et al., 1993). GAP-43 isalso altered in the rat cortex and striatum 24 h after footshock orlearning (Erlich et al., 1977; Routtenberg and Benson, 1980).More recently, inhibitory avoidance tasks were shown to increasePKC activity and GAP-43 phosphorylation in rat hippocampus 30min after training (Cammarota et al., 1997). LTP causes short-term increases (Meberg et al., 1995), followed by later decreases(Meberg et al., 1993) in GAP-43 mRNA levels, presumably allow-ing stabilization of the plastic changes of LTP. Thus, it appears thatchanges in GAP-43 phosphorylation and gene expression may beimportant to the learning process. In fact, recent work by Rout-tenberg and colleagues demonstrates that transgenic mice overex-pressing wild-type GAP-43 protein, or protein with a mutationmimicking constitutive PKC phosphorylation, exhibit increasedhippocampal LTP in vivo and enhanced spatial learning abilities(Routtenberg et al., 2000). Moreover, PKC activation or expres-sion of constitutively phosphorylated GAP-43 were capable of res-cuing LTP from NMDA receptor blockade (Routtenberg et al.,2000; Kleschevnikov and Routtenberg, 2001).

In this study, we found that a decrease in GAP-43 phosphory-lation at early times after CFC was followed by long lasting in-creases in phosphorylation, coupled with increased GAP-43 levels.A similar biphasic change in GAP-43 phosphorylation is also seenin rat hippocampus during LTP (Son et al., 1997). Comparison ofthe GAP-43 phosphorylation and protein level changes observedafter fear conditioning in adult rats appear to be somewhat differ-ent from those observed using a similar paradigm in C57BL/6 mice(Young et al., 2000). That study showed an early increase inGAP-43 phosphorylation and a more transient increase in GAP-43levels (returned to baseline by 48 h) in the trained C57 mice. Thesedifferences in GAP-43 phosphorylation between rat and mousemay be species-specific. In fact, McNamara et al. (1996) reportedspecies-related differences in GAP-43 gene expression, demon-strating that the ratio of GAP-43 mRNA in CA1 and CA3 areas ofthe hippocampus in the rat and mouse are markedly dissimilar.GAP-43 expression in mouse and rat also responded differently inresponse to kainic acid-induced seizures. These differences in

GAP-43 phosphorylation and levels emphasize the importance ofexamining the role of this protein in learning using different spe-cies.

Upon comparison of the time course of GAP-43 phosphoryla-tion and PKC translocation, it appears unlikely that either PKC�,PKC�I, or PKC� are responsible for the observed changes inGAP-43 phosphorylation. First, the timing of activation of thesesubtypes does not coincide with that for GAP-43 phosphorylation.Also, as indicated above, PKC� is postsynaptically localized inhippocampal neurons which express GAP-43 at the presynapticsite (Saito et al., 1989).

In contrast, PKC isoforms �II and PKC� show a similar timecourse for the activation as GAP-43 phosphorylation, suggestingthat they may be responsible for the phosphorylation of GAP-43 invivo. Supporting this idea, PKC�II and PKC� have been shown tohave high activity towards GAP-43 in vitro (Sheu et al., 1990;Oehrlein et al., 1996), and both PKCs are highly expressed in thehippocampal formation (Saito et al., 1989; Gschwendt et al.,1991). While PKC�II immunoreactivity is observed in theperikarya, dendrites, and axons of hippocampal pyramidal neuronsin CA1, PKC�, like GAP-43, is localized primarily to presynapticterminals. At the subcellular level, PKC�II is associated with Tri-ton-insoluble cytoskeletal elements, where it was found to phos-phorylate a 45-kDa protein, most likely GAP-43 (Tanaka et al.1991). Similarly, PKC� is bound to actin filaments (Prekeris et al.,1996), which provides a site for interaction with GAP-43. Consis-tent with these findings, both the initial translocation of these PKCsubtypes to the soluble fraction and their later return to the mem-brane coincide with changes in GAP-43 phosphorylation afterCFC.

In addition to changes in GAP-43 phosphorylation, it is clearthat at longer times after CFC, animals express greater levels ofGAP-43. GAP-43 protein levels were found to increase 3 h afterthe training session. These relatively rapid increases in GAP-43levels are consistent with the idea that GAP-43 levels are regulatedprimarily through post-transcriptional mechanisms (Benowitz andPerrone-Bizzozero, 1991). We have also shown that PKC activa-tion increased GAP-43 mRNA levels without affecting the rate oftranscription, thus suggesting that PKC-dependent mRNA turn-over may be a primary point of control for GAP-43 expression(Perrone-Bizzozero et al., 1993). Since PKC is activated after fearconditioning, it seems likely that this mechanism would underliethe observed increases in GAP-43 levels. In support of this idea, werecently found an increase in the levels of the GAP-43 mRNA andof the GAP-43 mRNA-binding protein HuD in the rat hippocam-pus after CFC (Merhege et al., 2001).

Evidence is accumulating for the roles of hippocampal PKC andGAP-43 in several learning paradigms. Here we show that alter-ations in GAP-43 phosphorylation and protein levels are coupledwith activation of specific PKC subtypes after CFC training. Dif-ferent PKC isotypes become activated and move to and from themembrane with different time courses, suggesting that each isotypemay have different functions. PKC�II and PKC� have transloca-tion profiles and intracellular distributions that would put them inposition to phosphorylate GAP-43. The relationship betweenPKC activity and GAP-43 phosphorylation is important not only

462 YOUNG ET AL.

during normal synaptic plasticity but also it may participate inmechanisms underlying cognitive deficits. Recent work from ourlaboratory indicates that PKC activity and GAP-43 phosphoryla-tion are compromised in the hippocampus of rats prenatally ex-posed to moderate levels of alcohol (Perrone-Bizzozero et al.,1998). These animals also show deficits in LTP and glutamaterelease from hippocampal slices (Sutherland et al., 1997; Savage etal., 1998). Future studies should help define the specific neuro-chemical deficits and the PKC isoforms affected by prenatal etha-nol exposure.

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