A High School Level Health and Disease-Focused Biology Curriculum Promotes Higher Level Skills in Nutrition Literacy.

OBJECTIVE: To determine the efficacy of a high school biology curriculum focused on promoting nutrition literacy skills. DESIGN: High school students participated in a six-week biology curriculum focused on the three subdomains of nutrition literacy: functional use of factual knowledge (FNL); interactive skills in seeking out information (INL); critical interpretation and analysis (CNL). We used a mixed-methods, change-over-time model that leverages longitudinal aspects of instructor practice and students' development. Pre- and posttest measures of FNL, INL and CNL were administered. Students were also given a retrospective pre-post online survey to measure interactive nutrition literacy and self-efficacy towards learning about nutrition topics. PARTICIPANTS: A total of 111 high school 11th and 12th grade students from four sections of a Biology II course participated. RESULTS: Students' overall NL scores improved (P<0.0001) and they also showed gains in each subdomain (FNL, INL and CNL, P<0.0001). Self-efficacy toward learning about nutrition also increased (P<0.0001). Students reported increased communication about the topics with family and peers who were neither classmates or friends (P<0.0001). CONCLUSIONS: Participation improved nutrition literacy in each of the subdomains, as well as self-efficacy. Self-efficacy was strongly related to increased communication.

Collaborative Curriculum Design as a Framework for Designing Teacher Professional Development that Produces the Content Knowledge for Teaching the Life Sciences.

Effective science teaching critically requires content focused professional development (PD), particularly in life sciences where content evolves rapidly. How subject matter knowledge related to teaching (SCK) is most effectively incorporated into PD has not been investigated. We studied how a professional learning community of high school teachers and scientists co-designing a bioscience curriculum produced the accompanying SCK-focused PD. SCK was level-specific but teachers could not generate it alone. Co-designing SCK with scientists was valuable to teachers, as evidenced by significant increases in their cognitive and attitudinal attributes toward the PD, in turn promoting change in practice and student learning gains, both within and outside the initial partnership. Surprisingly, social network analysis of how the collaborators interacted revealed that though the network was cognitively and effectively robust, it was behaviorally much sparser than anticipated for such a high functioning partnership, counter to commonly accepted PD best practices. We suggest that the scientist/educator facilitators who intentionally promoted collaboration in the context of distributed leadership were able to eliminate extraneous interactions, optimizing the process. The results are further evidence that developing content-focused PD relevant to 21st century life sciences requires dismantling the institutionalized segregation between practitioners of science and teaching.

Addressing Health Literacy Challenges With a Cutting-Edge Infectious Disease Curriculum for the High School Biology Classroom.

This study reports the secondary analysis of evaluation data from an innovative high school biology curriculum focused on infectious disease (ID) to examine the health literacy implications of teaching claims evaluation, data interpretation, and risk assessment skills in the context of 21st-Century health science. The curriculum was implemented between 2010 and 2013 in Biology II classes held in four public high schools (three in Massachusetts and one in Ohio), plus a private school in Virginia. A quasi-experimental design was used in which student participants (n = 273) were compared to an age-matched, nonparticipant, peer group (N = 125). Participants in each school setting demonstrated increases in conceptual content knowledge (Cohen's d > 1.89) as well as in understanding how to apply scientific principles to health claims evaluation and risk assessment (Cohen's d > 1.76) and in self-efficacy toward learning about ID (Cohen's d > 2.27). Participants also displayed enhanced communication about ID within their social networks relative to the comparison group (p < .05). The data show that integrating the claims evaluation, data interpretation, and risk assessment skills critical for 21st-century health literacy health into high school biology classrooms is effective at fostering both the skills and self-efficacy pertinent to health literacy learning in diverse populations.

Modeling for Fidelity: virtual mentorship by scientists fosters teacher self-efficacy and promotes implementation of novel high school biomedical curricula.

This small-scale comparison case study evaluates the impact of an innovative approach to teacher professional development designed to promote implementation of a novel cutting edge high school neurological disorders curriculum. 'Modeling for Fidelity' (MFF) centers on an extended mentor relationship between teachers and biomedical scientists carried out in a virtual format in conjunction with extensive online educative materials. Four teachers from different diverse high schools in Massachusetts and Ohio who experienced MFF contextualized to a 6-week Neurological Disorders curriculum with the same science mentor were compared to a teacher who had experienced an intensive in-person professional development contextualized to the same curriculum with the same mentor. Fidelity of implementation was measured directly using an established metric and indirectly via student performance. The results show that teachers valued MFF, particularly the mentor relationship and were able to use it effectively to ensure critical components of the learning objectives were preserved. Moreover their students performed equivalently to those whose teacher had experienced intensive in-person professional development. Participants in all school settings demonstrated large (Cohen's d>2.0) and significant (p<0.0001 per-post) changes in conceptual knowledge as well as self-efficacy towards learning about neurological disorders (Cohen's d>1.5, p<0.0001 pre-post). The data demonstrates that the virtual mentorship format in conjunction with extensive online educative materials is an effective method of developing extended interactions between biomedical scientists and teachers that are scalable and not geographically constrained, facilitating teacher implementation of novel cutting-edge curricula.

The Great Diseases Project: a partnership between Tufts Medical School and the Boston public schools.

Medical schools, although the gatekeepers of much biomedical education and research, rarely engage formally with K-12 educators to influence curriculum content or professional development. This segregation of content experts from teachers creates a knowledge gap that limits inclusion of current biomedical science into high school curricula, affecting both public health literacy and the biomedical pipeline. The authors describe how, in 2009, scientists from Tufts Medical School and Boston public school teachers established a partnership of formal scholarly dialogue to create 11th- to 12th-grade high school curricula about critical health-related concepts, with the goal of increasing scientific literacy and influencing health-related decisions. The curricula are based on the great diseases (infectious diseases, neurological disorders, metabolic disease, and cancer). Unlike most health science curricular interventions that provide circumscribed activities, the curricula are comprehensive, each filling one full term of in-class learning and providing extensive real-time support for the teacher. In this article, the authors describe how they developed and implemented the infectious disease curriculum, and its impacts. The high school teachers and students showed robust gains in content knowledge and critical thinking skills, whereas the Tufts scientists increased their pedagogical knowledge and appreciation for health-related science communication. The results show how formal interactions between medical schools and K-12 educators can be mutually beneficial.

GAP-43 dependency defines distinct effects of netrin-1 on cortical and spinal neurite outgrowth and directional guidance.

Growth-associated protein-43 (GAP-43) is a major nervous system protein whose phosphorylation by protein kinase C regulates growth cone responses to extracellular guidance cues via F-actin. GAP-43 is essential for axon pathfinding in both cortical afferents and efferents: when it is genetically deleted, somatosensory, auditory and visual somatotopic maps fail to form, and telencephalic commissural axons fail to cross the midline. Here we investigated whether the midline guidance cue netrin-1 depends on GAP-43 for its functions in neurite growth and guidance. We used 3-dimensional collagen gel co-cultures to show that both endogenous netrin-1, expressed by the spinal cord floor plate, and recombinant netrin-1, expressed by transfected COS7 cells, stimulate neurite outgrowth and chemotropic guidance of neocortical callosal axons. In contrast both were significantly inhibited in GAP-43 (-/-) neocortical callosal axons, mimicking the in vivo phenotype. Conversely, neither netrin-1-stimulated neurite outgrowth nor guidance of dorsal spinal cord commissure axons were affected when GAP-43 was absent, again consistent with in vivo phenotype but suggesting fundamental differences in how neocortical and spinal cord axons respond to netrin-1. In addition, differences in GAP-43 dependency also distinguished how ventrolateral cortical efferents respond to netrin-1: in contrast to callosal neurites, in which netrin-1 required GAP-43 in order to stimulate both outgrowth and guidance, in ventrolateral efferents, netrin-1 required GAP-43 only to stimulate outgrowth, but not guidance. Moreover, netrin-1 increased the numbers of both types of cortical, but not spinal neurites. The results demonstrate previously unappreciated diversity in how different classes of neurons respond to the same guidance cue.

Coordination between extrinsic extracellular matrix cues and intrinsic responses to orient the centrosome in polarizing cerebellar granule neurons.

Successful axon targeting during development is critically dependent on directionality of axon extension and requires coordination between the extrinsic cues that provide spatial information to the axon and the intrinsic responses that regulate structural specification of the axon during neuronal polarization. How these responses are coordinated is unclear but are known to involve aligning the centrosome with the base of the emerging axon. We have used a novel in vitro micropatterning assay that spatially segregates the extrinsic cues used by polarizing cerebellar granule cells to orient axon extension and used it to investigate the signaling mechanisms responsible for coordinating centrosome positioning with intrinsic responses. The results show that, when laminin and/or vitronectin are used as spatially restricted cues in association with substrate-associated sonic hedgehog, they are sufficient to induce cell cycle arrest, that laminin and vitronectin then induce integrin-mediated signaling that upregulates phosphoinositide-3 kinase and PKC function to produce phosphatidylinositol 3,4,5-trisphosphate (PIP3) that is associated with the centrosome, that this PIP3 can interact with PKC-phosphorylated growth-associated protein GAP-43, and that PKC-phosphorylated GAP-43 in turn is required for positioning Par6, Cdc42, and IQGAP1, all intrinsic response components, in proximity to the centrosome, such that, in the absence of GAP-43, they are mislocalized and microtubules are not oriented appropriately. We conclude from these results that GAP-43 plays an important role in coordinating extrinsic signaling and intrinsic responses in polarizing cerebellar granule neurons.

Regulation of GAP-43 at serine 41 acts as a switch to modulate both intrinsic and extrinsic behaviors of growing neurons, via altered membrane distribution.

GAP-43 is the major neuronal substrate of protein kinase C (PKC). Its phosphorylation status dictates the severity of pathfinding errors by GAP-43 (+/-) growth cones in vivo, as well as its modulation of actin dynamics in vitro. These experiments show that stably overexpressing cDNAs mutant at its single PKC phosphorylation site at serine41 in retinoic acid treated SH-Sy5Y neuroblastoma cells regulates intrinsic and extrinsic behaviors of growing neurons. Intrinsically, only Wt and pseudophosphorylated GAP-43Ser41Asp precipitated with F-actin and potentiated F-actin - regulated filopodia formation. GAP-43Ser41Asp inhibited neurite outgrowth whereas only unphosphorylatable GAP-43Ser41Ala precipitated neurotubulin, potentiated neurotubulin accumulation in neurites and increased outgrowth. When PI3-kinase was inhibited GAP-43Ser41Asp-mediated filopodia formation was inhibited whereas GAP-43Ser41Ala-mediated neurite extension was potentiated. Extrinsically, only Wt and GAP-43Ser41Asp potentiated both homotypic adhesion and neurite outgrowth on NCAM-expressing monolayers and promoted NCAM stability. With respect to the underlying mechanism, more F-actin and NCAM colocalized with Wt and GAP-43Ser41Asp in detergent resistant membranes (DRMs) isolated from live cells and GAP-43Ser41Asp-mediated functions were insensitive to cholesterol depletion. In contrast, GAP-43Ser41Ala-mediated functions were sensitive to cholesterol depletion. Neither GAP-43Ser41Asp nor GAP-43Ser41Ala was able to protect against growth cone collapse mediated by PIP2 inhibitors. The results show that modification of GAP-43 at its PKC phosphorylation site directs its distribution to different membrane microdomains that have distinct roles in the regulation of intrinsic and extrinsic behaviors in growing neurons.

Both cell-autonomous and cell non-autonomous functions of GAP-43 are required for normal patterning of the cerebellum in vivo.

Growth-associated protein 43 (GAP-43) is required for development of a functional cerebral cortex in vertebrates; however, its role in cerebellar development is not well understood. Recently, we showed that absence of GAP-43 caused defects in proliferation, differentiation, and polarization of cerebellar granule cells. In this paper, we show that absence of GAP-43 causes defects in cerebellar patterning that reflect both cell-autonomous and non-autonomous functions. Cell-autonomous effects of GAP-43 impact precursor proliferation and axon targeting: In its absence, (1) proliferation of granule cell precursors in response to sonic hedgehog and fibroblast growth factor is inhibited, (2) proliferation of neuroepithelial precursors is inhibited, and (3) targeting of climbing fibers to the central lobe is disrupted. Cell non-autonomous effects of GAP-43 impact differentiated Purkinje cells in which GAP-43 has been downregulated: In its absence, both maturation and mediolateral patterning of Purkinje cells are inhibited. Both cell-autonomous and non-autonomous functions of GAP-43 involve its phosphorylation by protein kinase C. GAP-43 is phosphorylated in granule cell precursors in response to sonic hedgehog in vitro, and phosphorylated GAP-43 is also found in proliferating neuroepithelium and climbing fibers. Phosphorylated GAP-43 is specifically enriched in the presynaptic terminals of parallel and climbing fibers that innervate Purkinje cell bodies and dendrites. The cell-autonomous and non-autonomous effects of GAP-43 converge on the central lobe. The multiple effects of GAP-43 on cerebellar development suggest that it is a critical downstream transducer of signaling mechanisms that integrate generation of cerebellar structure with functional parcellation at the central lobe.

Immortalization of neural precursors when telomerase is overexpressed in embryonal carcinomas and stem cells.

The DNA repair enzyme telomerase maintains chromosome stability by ensuring that telomeres regenerate each time the cell divides, protecting chromosome ends. During onset of neuroectodermal differentiation in P19 embryonal carcinoma (EC) cells three independent techniques (Southern blotting, Q-FISH, and Q-PCR) revealed a catastrophic reduction in telomere length in nestin-expressing neuronal precursors even though telomerase activity remained high. Overexpressing telomerase protein (mTERT) prevented telomere collapse and the neuroepithelial precursors produced continued to divide, but deaggregated and died. Addition of FGF-2 prevented deaggregation, protected the precursors from the apoptotic event that normally accompanies onset of terminal neuronal differentiation, allowed them to evade senescence, and enabled completion of morphological differentiation. Similarly, primary embryonic stem (ES) cells overexpressing mTERT also initiated neuroectodermal differentiation efficiently, acquiring markers of neuronal precursors and mature neurons. ES precursors are normally cultured with FGF-2, and overexpression of mTERT alone was sufficient to allow them to evade senescence. However, when FGF-2 was removed in order for differentiation to be completed most neural precursors underwent apoptosis indicating that in ES cells mTERT is not sufficient allow terminal differentiation of ES neural precursors in vitro. The results demonstrate that telomerase can potentiate the transition between pluripotent stem cell and committed neuron in both EC and ES cells.

GAP-43 is key to mitotic spindle control and centrosome-based polarization in neurons.

In neurons, the position of the centrosome during final mitosis marks the point of emergence of the future axon. However, the molecular underpinnings linking centrosome position to axon emergence are unknown. GAP-43 is a calmodulin-binding IQ motif protein that regulates neuronal cytoskeletal architecture by interacting with F-actin in a phosphorylation dependent manner. Here we show that GAP-43 is associated with the centrosome and plays a critical role in mitosis and acquisition of neuronal polarity in cerebellar granule neurons. In the absence of GAP-43, the centrosome position is delinked from process outgrowth and is only capable of mediating morphological polarization, however molecular specification of the axonal compartment does not take place. These results show that GAP-43 is required to link centrosome position to process outgrowth in order to generate neuronal polarity in cerebellar granule cells.

P-GAP-43 is enriched in horizontal cell divisions throughout rat cortical development.

Asymmetric cell divisions are correlated to neurogenesis in the mammalian cortex and occur often with a horizontal orientation of cell division. However, the molecular mechanisms of spindle orientation or asymmetric cell divisions are not well understood in the developing mammalian central nervous system. Here we show a new molecular marker for horizontally dividing precursors in the mammalian telencephalon. The antibody 2G12 directed against phosphorylated serine of growth associated protein 43 (GAP-43) labels postmitotic neurons and a subset of cells in mitoses in the developing rat telencephalon. 2G12 immunoreactivity was found at a high frequency in mitotic cells dividing parallel to the ventricular surface throughout neurogenesis (embryonic day 13-17) in the cerebral cortex and ganglionic eminence. Interestingly, we detected the same predominance of 2G12 immunoreactivity in horizontally dividing cells in the subventricular zone, the second proliferative layer that has recently been involved in the generation of neurons. Moreover, 2G12 immunostaining is no longer detectable in mitotic cells of the ventricular zones at E21, the onset of gliogenesis in rat telencephalon. These data imply GAP-43 phosphorylation in the phase of neuronal commitment during M-phase and present to our knowledge the first molecular correlate to horizontally dividing precursors in mammalian neurogenesis.

Lipid rafts and regulation of the cytoskeleton during T cell activation.

The ability of polarized cells to initiate and sustain directional responses to extracellular signals is critically dependent on direct communication between spatially organized signalling modules in the membrane and the underlying cytoskeleton. Pioneering work in T cells has shown that the assembly of signalling modules critically depends on the functional compartmentalization of membrane lipids into ordered microdomains or lipid rafts. The significance of rafts in T cell activation lies not only in their ability to recruit the signalling partners that eventually assemble into a mature immunological synapse but also in their ability to regulate actin dynamics and recruit cytoskeletal associated proteins, thereby achieving the structural polarization underlying stability of the synapse-a critical prerequisite for activation to be sustained. Lipid rafts vary quite considerably in size and visualizing the smallest of them in vivo has been challenging. Nonetheless it is now been shown quite convincingly that a surprisingly large proportion-in the order of 50%-of external membrane lipids (chiefly cholesterol and glycosphingolipids) can be dynamically localized in these liquid ordered rafts. Complementary inner leaflet rafts are less well characterized, but contain phosphoinositides as an important functional component that is crucial for regulating the behaviour of the actin cytoskeleton. This paper provides an overview of the interdependency between signalling and cytoskeletal polarization, and in particular considers how regulation of the cytoskeleton plays a crucial role in the consolidation of rafts and their stabilization into the immunological synapse.

Hippocampal-dependent memory is impaired in heterozygous GAP-43 knockout mice.

Cajal proposed that the rearrangement and growth of neurites and synaptic terminals is a substrate for the formation and storage of long-term memories. Proteins that regulate this learning-dependent growth are therefore likely to be "core determinants" (Sanes and Lichtman, Nat Neurosci 1999; 2:597-604) of such information storage processes. Although the growth-associated, protein kinase C (PKC) substrate GAP-43 has been oft-implicated in synaptic plasticity and memory, it has never been demonstrated that a reduction in the level of this protein has a deleterious effect on memory, because most homozygotes die perinatally. In this report, we observe significant memory impairments in heterozygous GAP-43 knockout mice with GAP-43 levels reduced by one-half. Impaired memory for a context was demonstrated in contextual fear conditioning. Importantly, no significant impairments in cued conditioning or on tests of nociceptive or auditory perception were observed in the heterozygous knockout, indicating that the observed impairments were unlikely related to performance or acquisition factors and are the result of reduced GAP-43 levels in the hippocampus. The present results, taken together with the prior demonstration of enhanced memory in transgenic mice overexpressing GAP-43, provide strong evidence for a pivotal role of hippocampal GAP-43 in the bidirectional regulation of mnemonic processing.

Membrane/cytoskeleton communication.

Accumulations of particular lipids in ordered arrays in the membrane (termed microdomains or lipid rafts) can attract proteins with specific targeting domains. Both the lipid and protein components of rafts communicate with the cytoskeleton directly thereby regulating cellular responses. Recent evidence implicating phosphoinositide 1,5 bisphosphate (PIP2) in cytoskeletal regulation shows that agonist sensitive regulation of PIP2 homoeostasis occurs specifically rafts, which appear to provide a major structural substrate for its function. The crucial role of PIP2 in generating cytoskeletal responses is chiefly achieved by regulating proteins that control actin dynamics directly. Many of these regulatory proteins are also specifically enriched in rafts either directly (by insertion into the lipid bilayer via acetylation motifs), or indirectly via interactions with other raft components. The notion that rafts form membrane platforms or modules that mediate signaling responses has been most extensively demonstrated in the immune synapse (IS) of T cells, a complex assemblage of rafts that integrates signaling cascades originating from the simultaneous activation of a wide variety of receptors. The IS is essential for both the amplification and maintenance of T-cell activation, and its assembly at the antigen presenting site depends on the interactions between rafts and the actin cytoskeleton that regulates coalescence of smaller raft components into the larger IS complex. Likewise the neuron, which represents the most highly polarized cell in the body, utilizes the regulation of actin dynamics in response to a plethora of extracellular signals to control axon pathfinding thereby sculpting nervous system cytoarchitecture with utmost precision. It is now becoming clear, that as in the T-cell, lipid rafts in the growing axon can assemble into highly specific, yet malleable and dynamic, signaling modules that regulate actin dynamics in a fashion that is also PIP2-dependent and that utilizes both familiar and novel regulatory mechanisms. It seems clear that raft mediated cytoskeletal regulation represents a highly conserved mechanism to integrate cellular responses to diverse signals.

Failure to express GAP-43 leads to disruption of a multipotent precursor and inhibits astrocyte differentiation.

The nervous system-specific protein GAP-43 is significantly upregulated in neurons and glia that are differentiating. In P19 EC cells that do not express GAP-43, neurogenesis is inhibited; many immature neurons apoptose and the survivors do not mature morphologically. Here we show that the initial defect is in an early precursor with characteristics of a neural stem cell, which failed to respond normally to retinoic acid (RA). As a consequence, its progeny had altered cell fates: In addition to the neuronal defects previously reported, RC1-labeled radial glia failed to exit the cell cycle, accumulated, and failed to acquire GFAP immunoreactivity. However, leukemia inhibitory factor (LIF) could stimulate GFAP expression suggesting that astrocytes not derived from radial glia are less affected by absence of GAP-43. Differentiation of radial glia-derived astrocytes was also inhibited in glial cultures from GAP-43 (-/-) cerebellum, and in GAP-43 (-/-) telencephalon in vivo, differentiation of astrocytes derived from both radial and nonradial glia lineages were both affected: In the glial wedge, GFAP-labeled radial glia-derived astrocytes were reduced consistent with the interpretation that they may be unable to deflect GAP-43 (-/-) commissural axons toward the midline. At the midline, both radial and nonradial glia-derived astrocytes were also decreased although it fused normally. The results demonstrate that GAP-43 expressed in multipotent precursors is required for appropriate cell fate commitment, and that its absence affects astrocyte as well as neuronal differentiation.

Fetal alcohol exposure alters GAP-43 phosphorylation and protein kinase C responses to contextual fear conditioning in the hippocampus of adult rat offspring.

BACKGROUND: The growth- and plasticity-associated protein GAP-43 plays a significant role in the establishment and remodeling of neuronal connections. We have previously shown that GAP-43 levels, protein kinase C (PKC) activity, and GAP-43 phosphorylation increase during contextual fear conditioning and that fetal alcohol exposure (FAE) decreases PKC activity and GAP-43 phosphorylation in the hippocampus of adult offspring. Drawing on these observations, we hypothesized that FAE manifests its cognitive impairment by disrupting PKC activation and membrane translocation, thereby decreasing GAP-43 phosphorylation and function. METHODS: Three groups of pregnant rat dams (FAE and two control diet groups) were placed on different diet regimens. Offspring from each of these groups were placed into each of four test groups, a contextual fear conditioned (CFC) group, a naïve unhandled group, and two nonlearning stress control groups. Hippocampi were dissected, homogenized, and used to prepare a cytosolic and a membrane fraction. These fractions were probed for total GAP-43, PKC-phosphorylated GAP-43, and several PKC subtypes. PKC activity also was measured in total homogenates. RESULTS: Compared with both control diet groups, FAE animals showed a deficit in the activation of PKC in the hippocampus at 24 hr but not at 1.5 hr after CFC. Likewise, we found that the amount of GAP-43 and its phosphorylation were decreased 24 hr after CFC in FAE rats but not at early times after training. Analysis of the translocation of various PKC isoforms revealed that FAE animals had decreased levels of membrane-bound PKC beta2 and PKC epsilon 24 hr after CFC. CONCLUSIONS: Considering the role of PKC activation and GAP-43 phosphorylation in synaptic plasticity, our results suggest that deficient translocation of PKC beta2 and PKC epsilon in the hippocampus may mediate the electrophysiological and behavioral deficits observed in fetal alcohol exposed animals.

Changes in protein kinase C (PKC) activity, isozyme translocation, and GAP-43 phosphorylation in the rat hippocampal formation after a single-trial contextual fear conditioning paradigm.

The hippocampus plays an important role in spatial learning and memory. However, the biochemical alterations that subserve this function remain to be fully elucidated. In this study, rats were subjected to a single-trial contextual fear conditioning (CFC) paradigm; the activation of different protein kinase C (PKC) subtypes and the levels and phosphorylation of the plasticity-associated protein GAP-43 were assayed in the hippocampus at varying times after training. We observed a rapid activation of hippocampal PKC (15 min through 24 h), with differential translocation of the PKC isotypes studied. At early times after CFC (15-90 min), PKCalpha and PKCgamma translocated to the membrane, while PKCbetaII and PKCepsilon moved more transiently (15 to 30 min) to the cytosol. These PKC isotypes returned to the membrane at later time points after CFC. Correlating with these changes in PKC translocation and activity, there was an early decrease in GAP-43 phosphorylation followed by a more sustained increase from 1.5-72 h. GAP-43 protein levels were also increased after 3 h, and these levels remained elevated for at least 72 h. These changes in PKC and GAP-43 were specific to the CFC trained animals and no changes were seen in animals exposed to the same stimuli in a non-associative fashion. Comparison of translocation of different PKC isotypes with the changes in GAP-43 phosphorylation suggested that PKCbetaII and PKCepsilon may mediate both the early changes in the phosphorylation of this protein and the increases in GAP-43 expression at later times after CFC.