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Hippocampus. Author manuscript; available in PMC 2010 August 3.
Published in final edited form as:
PMCID: PMC2914311
NIHMSID: NIHMS221175

Myristoylated Alanine Rich C Kinase Substrate (MARCKS) Heterozygous Mutant Mice Exhibit Deficits in Hippocampal Mossy Fiber-CA3 Long-Term Potentiation

Abstract

The myristoylated alanine-rich C kinase substrate (MARCKS) is a primary protein kinase C (PKC) substrate in brain thought to transduce PKC signaling into alterations in the filamentous (F) actin cytoskeleton. Within the adult hippocampus, MARCKS is highly expressed in the dentate gyrus (DG)-CA3 mossy fiber pathway, but is expressed at low levels in the CA3-CA1 Schaffer collateral-CA1 pathway. We have previously demonstrated that 50% reductions in MARCKS expression in heterozygous Marcks mutant mice produce robust deficits in spatial reversal learning, but not contextual fear conditioning, suggesting that only specific aspects of hippocampal function are impaired by reduction in MARCKS expression. To further elucidate the role of MARCKS in hippocampal synaptic plasticity, in the present study we examined basal synaptic transmission, paired-pulse facilitation, post-tetanic potentiation, and long-term potentiation (LTP) in the hippocampal mossy fiber-CA3 and Schaffer collateral-CA1 pathways of heterozygous Marcks mutant and wild-type mice. We found that LTP is significantly impaired in the mossy fiber-CA3 pathway, but not in the Schaffer collateral-CA1 pathway, in heterozygous Marcks mutant mice, whereas basal synaptic transmission, paired-pulse facilitation, and post-tetanic potentiation are unaffected in both pathways. These findings indicate that a 50% reduction in MARCKS expression impairs processes required for long-term, but not short-term, synaptic plasticity in the mossy fiber-CA3 pathway. The implications of these findings for the role of the mossy fiber-CA3 pathway in hippocampus-dependent learning processes are discussed.

Keywords: MARCKS, protein kinase C, mossy fibers, Schaffer collateral, filamentous actin, hippo-campus, LTP, paired-pulse facilitation, post-tetanic potentiation, synaptic plasticity

INTRODUCTION

Protein kinase C (PKC) is a family of calcium- and phospholipid-dependent isozymes with differing tissue expression, subcellular localization, and responsiveness to second messenger activators (reviewed in Tanaka and Nishizuka, 1994; Mellor and Parker, 1998). In the adult rodent hippocampus, several different PKC isozymes are expressed, and they exhibit differential as well as overlapping distributions within individual hippocampal pathways and neuronal compartments (reviewed in Tanaka and Nishizuka, 1994). For example, PKCε is enriched in mossy fiber axons, whereas PKCα is enriched in Schaffer collateral axons (McGinty et al., 1991). Although PKC inhibitors have differential effects on synaptic transmission in these hippocampal pathways (Hussain and Carpenter, 2003), PKC inhibitors impair LTP induced by high-frequency stimulation (HFS) in both the mossy fiber-CA3 pathway (Son and Carpenter, 1996; Son et al., 1996; Hussain and Carpenter, 2005) and the Schaffer collateral-CA1 pathway (Wang and Feng, 1992; Hvalby et al., 1994; Hussain and Carpenter, 2005). Furthermore, the induction of LTP in the mossy fiber-CA3 pathway (Son et al., 1996) and Schaffer collateral-CA1 pathway (Ramakers et al., 1999) is associated with PKC isozyme membrane translocation or increased phosphorylation of specific PKC substrates, two indices of PKC activation. Finally, exogenous activation of PKC with phorbol esters induces long-term elevations in synaptic responses in the Schaffer collateral-CA1 pathway (Reymann et al., 1988) and the mossy fiber-CA3 pathway (Son et al., 1996) and in mossy fiber synaptosomes (Terrian et al., 1993). Although these and other data suggest that PKC plays a role in the long-term modulation of synaptic efficacy (reviewed in Vaughan et al., 1998), the precise role of PKC in the cascade of second messenger events mediating long-term alterations in synaptic plasticity remains controversial, owing in part to the lack of specificity of PKC activators and inhibitors employed in many of these studies (Lee et al., 1998; Brose and Rosenmund, 2002). Knockout mice not expressing PKCγ, a postsynaptic-specific isozyme, exhibit deficits in Schaffer collateral LTP induced by HFS (Abeliovich et al., 1993), but not by phorbol esters (Goda et al., 1996), and exhibit normal mossy fiber LTP (see Fig. 3 in Hsia et al., 1995).

Consistent with the proposed role of PKC in the regulation of synaptic efficacy, exogenous activation of PKC with phorbol esters induces the movement of neurotransmitter vesicles from reserve pools to the readily releasable pool at active release sites (Stevens and Sullivan, 1998; Brager et al., 2002; Shoji-Kasai et al., 2002; Kumakura et al., 2004), which is positively correlated with synaptic release probability (Schikorski and Stevens, 1997). The maintenance of synaptic vesicles in reserve pools, and their movement to the readily releasable pool, is also regulated by the filamentous (F) actin cytoskeleton (Gotow et al., 1991; Doussau and Augustine, 2000; Morales et al., 2000; Trifaro et al., 2002), and accumulating evidence suggests that the maintenance phase of LTP requires synaptic F-actin cytoskeletal assembly–disassembly dynamics (Kim and Lisman, 1999; Krucker et al., 2000; Fukazawa et al., 2003; Lang et al., 2004; Matsuzaki et al., 2004). The myristoylated alanine-rich C kinase substrate (MARCKS) is a primary substrate of PKC that binds and crosslinks filamentous (F)-actin in a PKC phosphorylation-reversible manner (Hartwig et al., 1992; Bubb et al., 1999; Yarmola et al., 2001; Larsson, 2006). MARCKS also binds the cytoplasmic surface of the plasma membrane and synaptic vesicle membranes via electrostatic attraction and N-terminal myristoylation, and is displaced from these membranes following phosphorylation by PKC (Thelen et al., 1991; Blackshear, 1993; Taniguchi and Manenti, 1993). MARCKS therefore serves as a transducer of receptor-generated PKC signaling into the relaxation of the F-actin cytoskeleton to regulate PKC-mediated vesicle trafficking as well as pre- and postsynaptic synaptic morphology (Calabrese and Halpain, 2005; McNamara and Lenox, 2004). In the rodent hippocampus, MARCKS is developmentally regulated (McNamara and Lenox, 1998) and MARCKS mRNA levels remain highly expressed in granule cells, but not in CA3 neurons, in the adult rat and mouse brain (McNamara and Lenox, 1997; McNamara et al., 1998). The MARCKS protein is highly expressed in granule cell axons and axon collaterals (mossy fibers), but not in granule cell dendrites or in CA3 neurons, and is colocalized with synaptic vesicles (Ouimet et al., 1990; Lu et al., 1998).

Previous studies have demonstrated that homozygous Marcks mutant (knockout) mice exhibit neuronal lamination abnormalities, particularly in the hippocampal CA3 pyramidal cell layer, colossal and commissural agenesis, and perinatal lethality (Stumpo et al., 1995). Quantitative western blot and RNase protection analyses indicate that MARCKS protein and mRNA expression, respectively, are reduced by 50% in the hippocampus of Marcks heterozygous mutant mice relative to wild-type littermates (McNamara et al., 1998). Furthermore, in situ hybridization suggests that MARCKS mRNA expression is reduced by ~50% in each of the hippocampal cell fields of Marcks heterozygous mutant mice relative to wildtype littermates (McNamara et al., 1998). However, hippocampal PKC isozyme (α, β, γ, δ, ε), pre-(GAP-43), and postsynaptic (RC3) PKC substrate expression do not differ between Marcks heterozygous mutant mice and wild-type littermates (McNamara et al., 1998). Heterozygous Marcks mutant mice do not exhibit cortical or hippocampal lamination abnormalities, and quantitative histological analysis indicate that the distribution and length of the suprapyramidal mossy fiber pathway, the infrapyramidal mossy fiber pathway, and the suprapyramidal: infrapyramidal ratio of Marcks mutant mice do not differ significantly from either wild-type littermates or inbred C57BL/6J mice (Stumpo et al., 1995; McNamara et al., 1998). Nevertheless, heterozygous Marcks mutant mice do exhibit deficits in spatial learning in the Morris water maze that are more robust during spatial reversal learning, e.g., following prior training to a different spatial location (McNamara et al., 1998). Moreover, we have recently reported that transgenic mice overexpressing MARCKS by ~80% above wild-type controls similarly exhibit more severe deficits in spatial reversal learning (McNamara et al., 2005). Interestingly, neither heterozygous Marcks mutant mice (McNamara et al., 2004) nor MARCKS transgenic mice (McNamara et al., 2005) exhibit deficits in contextual fear conditioning, a task that is impaired in C57BL/6 mice following hippocampal lesions (Logue et al., 1997). These findings suggest that specific aspects of hippocampal function, e.g., those required for spatial reversal learning, are particularly sensitive to alterations in MARCKS expression.

Based on the relative distribution of MARCKS expression in the different hippocampal pathways (mossy fiber ≥ Schaffer collateral), the role of MARCKS in specific aspects of hippocampus function, and the proposed role of MARCKS in the regulation of PKC-mediated alterations in the F-actin cytoskeleton, we hypothesized that 50% reductions in MARCKS expression would induce more severe deficits in synaptic plasticity in the granule cell-CA3 (mossy fiber) pathway than the CA3-CA1 (Schaffer collateral) pathway. To test this hypothesis, we examined basal synaptic transmission (input/output curves), paired-pulse facilitation, post-tetanic potentiation, and the induction and maintenance of LTP in the mossy fiber-CA3 and Schaffer collateral-CA1 pathways in hippocampal slices from heterozygous Marcks mutant mice and wild-type littermates. We show that LTP is impaired in the mossy fiber-CA3 pathway, but not in the Schaffer collateral-CA1 pathway, of heterozygous Marcks mutant mice, whereas basal synaptic transmission and short-term synaptic plasticity are not affected. The implications of these finding for understanding the role of the mossy fiber-CA3 pathway in hippocampus-dependent learning processes are discussed. A portion of these data have previously been presented in abstract form (McNamara et al., 2004).

MATERIALS AND METHODS

Animals

The generation and characterization of Marcks mutant mice is described in detail elsewhere (Stumpo et al., 1995; McNamara et al., 1998). Briefly, heterozygous Marcks mutant mice were created by homologous recombination using 129Sv (E14TG2a) ES stem cells, and chimeric mice were backcrossed to C57BL/6J mice for nine generations to reduce 129 genes to 0.2% (Silver, 1995). Experimental animals were housed with same sex littermates with 4–5 mice per cage in SPF Murine Facilities maintained on a 12:12 light/dark cycle. All experiments were conducted in adult (10–12 weeks) male mice during the light portion of the cycle. Food and water were available ad libitum. Genotyping by Southern blot was conducted using DNA extracted from tail clips, as previously described (Stumpo et al., 1995), following data collection to maintain blinding. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania and carried out in accordance with National Institute of Health guidelines on animal experimentation.

Slice Preparation and Field Recordings

Each animal was euthanized by cervical dislocation, and the brain was quickly removed and immersed in semi liquid (0–1°C) oxygenated (95%O2/5%CO2) normal Krebs Ringer solution or modified Krebs Ringer solution with sucrose substituted for NaCl (Lipton et al., 1995). The Krebs Ringer solution (pH 7.3) was composed of (in mM) NaCl 125, KCl 3.5, NaH2PO4 1.2, CaCl2 2.4, MgSO4 3.5, NaHCO3 26, and glucose 10. For sucrose Krebs Ringer, NaCl was replaced with sucrose (218 mM). The right or left hemisphere was blocked and glued to a vibratome (FHC, OTS 4,000) stage for sectioning in a plane transverse to the long axis of hippocampus to get maximal mossy fiber input. Combined slices of hippocampus and entorhinal cortex (EC) (400 μm thick) were cut and incubated in normal Krebs Ringer at 30–31°C at least for 1 h in a submerged chamber and then incubated in an interface chamber for another hour. Slices were continuously superfused with normal Krebs Ringer at a rate of 2–3 ml/ min and maintained at 30–31°C while recording. A concentric bipolar stimulating electrode, with a 12.5 μm tip (Frederick Haer Co. Brunswick, ME), was placed on afferents, and an extracellular recording electrode, a micropipette (2–5 MΩ) filled with Krebs Ringer solution, was placed near the synaptic site. A stimulation frequency of 0.1 Hz was used throughout the experiments except during tetanus. Extracellular field excitatory postsynaptic potentials (fEPSPs) were recorded and subsequently analyzed using pClamp 7 (Axon Instruments). Baseline responses were recorded at 30–40% of the maximum response over a 20–60 min period prior to tetanus.

Short-Term Plasticity

To test for paired-pulse facilitation (PPF), two stimuli were delivered at different interpulse intervals (from 20 to 140 ms apart) at the recording stimulation intensity in all experiments in CA1 and CA3. PPF ratios were calculated by measuring the peak amplitude of the fEPSP evoked by each shock and taking the ratio of the second to the first. Post-tetanic potentiation (PTP) was measured as the first fEPSP 10 s following tetanus stimulation. The first fEPSP recording after tetanus was calculated as a percent of the baseline response prior to tetanus.

Long-Term Potentiation

After a stable baseline had been obtained, pre-tetanus input–output curves were generated by changing the presynaptic shock intensity (input) and measuring the fEPSP responses (output). Stimulation intensity varied from 2 to 50 V and HFS used a stimulation intensity that was approximately half that required to induce a maximal response. In some experiments, second input–output curves were generated 60 min post-tetanus. LTP in the mossy fiber-CA3 pathway was induced by four trains of 100 Hz applied for 1 s separated by 20 s at twice the pulse duration. The fEPSP was recorded for >65 min post-tetanus for all experiments. For each experiment, the negative peak amplitude (at 90% of maximum) and slope (between 10 and 90% of max) of the fEPSP were analyzed as measures of synaptic strength. These values were normalized to the mean value obtained over the last 10 min of the baseline period and expressed as a percent of this baseline value. All mossy fiber LTP recordings were made in the presence of an NMDA receptor antagonist (50 μM D-AP5, Tocris). LTP experiments in the Schaffer collateral-CA1 pathway were performed as described (Abel et al., 1997). LTP was induced by two tetanic stimuli of 100 Hz (1 s duration and 20 s apart). Statistical differences between wild-type and heterozygous mutant mice were assessed using a two-tailed Student’s t-test. All values are indicated as the mean ± standard error of the mean (SEM). For all analyses, P < 0.05 was accepted as evidence of statistical significance.

RESULTS

Basal Synaptic Transmission

Basal synaptic transmission was not altered in Schaffer collateral-CA1 pathway in heterozygous Marcks mutant mice (n = 5 mice, 9 slices) relative to wild-type controls (n = 4 mice, 8 slices), as assessed by repeated measures analysis of variance (ANOVA) of peak amplitudes across different stimulation intensities, F(1,238) = 1.58, P = 0.23 (Fig. 1A), and initial slope of the fEPSP across different stimulation intensities, F(1,238) = 1.79, P = 0.20 (Fig. 1B). Moreover, there was no significant alteration in basal synaptic transmission in the mossy fiber-CA3 pathway of heterozygous Marcks mutant mice (n = 5 mice, 8 slices) relative to wild-type controls (n = 4 mice, 5 slices), as assessed by repeated measures ANOVA of peak amplitudes across different stimulation intensities, F(1,156) = 0.25, P = 0.63 (Fig. 1C), and initial slope of the fEPSP across different stimulation intensities, F(1,156) = 0.13, P = 0.91 (Fig. 1D).

FIGURE 1
Reduction in MARCKS expression does not affect basal synaptic transmission in either Schaffer collateral-CA1 or mossy fiber-CA3 pathways. Basal synaptic transmission is not altered in Schaffer collateral-CA1 as measured by the peak amplitude (A) or the ...

Paired-Pulse Facilitation

The PPF ratios measured from the Schaffer collateral-CA1 pathway (wild-type, n = 4 mice, 7 slices; heterozygous mutant, n = 5 mice, 8 slices) were not altered in heterozygous Marcks mutant mice relative to wild-type controls as assessed by repeated measures ANOVA, F(1,90) = 1.19, P = 0.29 (Fig. 2A). The PPF ratios measured from the mossy fiber-CA3 pathway (wildtype, n = 6 mice, 10 slices; heterozygous mutant, n = 5 mice, 8 slices) were not altered in heterozygous Marcks mutant mice relative to wild-type controls as assessed by repeated measures ANOVA, F(1,234) = 0.249, P = 0.62 (Fig. 2B).

FIGURE 2
Reduction in MARCKS expression does not affect paired-pulse facilitation (PPF) in either Schaffer collateral-CA1 or mossy fiber-CA3 pathways. PPF ratios (second peak/first peak) ± SEM are plotted as a function of interpulse interval (ms) for slices ...

Schaffer Collateral LTP

Schaffer collateral-CA1 LTP was unaltered in heterozygous Marcks mutant mice (n = 5 mice, 10 slices) relative to wild-type controls (n = 4 mice, 8 slices)(Fig. 3). There was no significant difference between mean fEPSP slopes for wild-type (173.3% ± 16.9%) and heterozygous Marcks mutant mice (181.1% ± 23.9%) up to 60 min after tetanization (P > 0.05).

FIGURE 3
Fifty percent reduction in MARCKS expression has no effect on Schaffer collateral-CA1 LTP. Schaffer collateral-CA1 LTP was examined in hippocampal slices prepared from heterozygous Marcks mutant mice and wild-type controls. LTP was induced by two 100 ...

Mossy Fiber LTP

A significant reduction in mossy fiber LTP was observed in hippocampal slices from heterozygous Marcks mutant mice (n = 5 mice, 9 slices) relative to slices from wild-type controls (n = 6 mice, 10 slices)(Fig. 4). At 60 min following tetanization, the level of potentiation observed in wild-type slices (mean fEPSP slope at 60 min, 146.2% ± 15.5%) was significantly greater than that observed in slices from heterozygous Marcks mutant mice (mean fEPSP slope at 60 min, 111.9% ± 7.3%; P = 0.02).

FIGURE 4
LTP in the mossy fiber-CA3 pathway is impaired in heterozygous Marcks mutant mice. Mossy fiber LTP was examined in hippocampal slices from heterozygous Marcks mutant mice and wild-type controls. LTP was induced by four 100 Hz trains of 1 s duration (20 ...

Post-Tetanic Potentiation

To examine post-tetanic potentiation (PTP), we compared the first fEPSP recorded 10 s following tetanus. PTP in the Schaffer collateral-CA1 pathway of heterozygous Marcks mutant mice did not differ significantly from wild-type mice (wild-type PTP, 295% ± 51%, n = 6 mice, 10 slices; heterozygous Marcks mutant PTP, 300.3% ± 43.0%, n = 5 mice, 10 slices; P > 0.05). PTP in the mossy fiber-CA3 pathway of heterozygous Marcks mutant mice was reduced relative to those obtained from wild-type mice (~30%), but this decrease was not statistically significant (wild-type PTP, 325.0% ± 52.7%, n = 6 mice, 10 slices; heterozygous Marcks mutant PTP, 230.3% ± 59.0%, n = 5 mice, 9 slices; P > 0.05).

DISCUSSION

In the present study, we found that mossy fiber LTP, but not Schaffer collateral LTP, is significantly impaired in heterozygous Marcks mutant mice in the absence of significant alterations in basal synaptic transmission, PPF or PTP. The absence of deficits in basal synaptic transmission in either the mossy fiber-CA3 or Schaffer collateral-CA1 pathways of heterozygous Marcks mutant mice relative to wild-type mice suggests that the synaptic connectivity of these two hippocampal pathways is not compromised by 50% reductions in MARCKS expression during postnatal synaptogenesis. This is further supported by the finding that PPF, a presynaptic form of short-term plasticity resulting from residual calcium remaining in the presynaptic terminal following the first of two closely spaced pulses (Zucker and Regehr, 2002), is similarly unaffected in either pathway. Furthermore, the failure to observe PPF deficits in heterozygous Marcks mutant mice suggest that intracellular calcium homeostasis is not impaired by 50% reductions in MARCKS expression, even though MARCKS binds calcium-calmodulin at elevated calcium levels in a PKC phosphorylation-reversible manner (Porumb et al., 1997). PTP refers to the transient increase in glutamate release immediately following HFS, and is associated with the depletion of the readily releasable vesicle pool in a PKC-regulated manner (Brager et al., 2002, 2003). In the present study, heterozygous Marcks mutant mice exhibited a nonsignificant reduction (~30%) in PTP in the mossy fiber-CA3 pathway, but not in the Schaffer collateral-CA1 pathway, relative to wild-type mice. Collectively, these findings suggest that basal synaptic transmission and two forms of short-term synaptic plasticity are not significantly affected by 50% reductions in MARCKS expression in either pathway.

The finding that LTP is not altered in the Schaffer collateral-CA1 pathway of heterozygous Marcks mutant mice was some-what unexpected in light of evidence that PKC inhibitors impair LTP in the Schaffer collateral-CA1 pathway (Wang and Feng, 1992; Hvalby et al., 1994; Hussain and Carpenter, 2005) and our previous findings of a transient (<10 min), but NMDA receptor-independent, elevation in MARCKS phosphorylation following the induction of LTP in the rat Schaffer collateral-CA1 pathway (Ramakers et al., 1999). However, we have also demonstrated that basal synaptic transmission, PPF, PTP, and LTP, are unimpaired in the Schaffer collateral-CA1 pathway of MARCKS transgenic mice overexpressing MARCKS at ~80% above wild-type controls (McNamara et al., 2005). Although the present results do not rule out a role for MARCKS in Schaffer collateral LTP, they do indicate that LTP in the mossy fiber-CA3 pathway is more sensitive to 50% reductions in MARCKS expression, which is consistent with the relative distribution of MARCKS in these pathways (mossy fiber ≥ Schaffer collateral) (Ouimet et al., 1990; McNamara and Lenox, 1997). It may be relevant, therefore, that GAP-43, a presynaptic-specific substrate of PKC that is highly expressed in Schaffer collateral axons but is not expressed in mature mossy fiber axons (De la Monte et al., 1989; McNamara and Lenox, 1997), is phosphorylated for a protracted period (60 min) following LTP-inducing HFS of the Schaffer collateral-CA1 pathway, both of which are prevented by prior treatment with an NMDA receptor antagonist (Ramakers et al., 1999). Schaffer collateral LTP may therefore be more dependent on PKC-GAP-43 mediated processes than PKC-MARCKS processes, and potential deficits in Schaffer collateral LTP in heterozygous Marcks mutant mice may be compensated for by GAP-43.

In the present study, we found that mossy fiber LTP was significantly impaired in heterozygous Marcks mutant mice. Mossy fiber-CA3 responses are potentially confounded by associational and commissural (A/C) afferents inputs to CA3 dendrites, and a set of criteria have been proposed to evaluate the specificity of mossy fiber recordings (Claiborne et al., 1993). The following characteristics of our mossy fiber fEPSPs suggest minimal A/C contamination: (1) fEPSPs exhibit a negative waveform restricted to the stratum lucidum, (2) fEPSPs exhibit a rapid onset latency (<3.5 ms) and rapid rise time (<5 ms), (3) fEPSPs exhibit a smooth exponential decay, (4) HFS changed the amplitude but not the overall shape of the fEPSP. Additionally, all mossy fiber LTP recordings were performed in the presence of the NMDA receptor antagonist D-AP5 to block NMDA receptor-dependent recurrent inputs from A/C fibers (Zalutsky and Nicoll, 1990). fEPSPs were recorded from the stratum lucidum region of CA3 and the stimulating electrode was placed close to the cell body layer to maximize recording from mossy fiber inputs (Claiborne et al., 1993), and mossy fiber fEPSPs exhibit a large PPF (ratio of ~2.0 at 40 ms interstimulus interval; Hussain and Carpenter, 2003). Collectively, these data suggest that the mossy fiber fEPSPs recorded in the present study had minimal A/C fiber contamination.

Deficits in LTP in the mossy fiber-CA3 pathway of heterozygous Marcks mutant mice is consistent with a role of PKC in processes contributing to mossy fiber LTP (Kamiya et al., 1988; Son and Carpenter, 1996; Son et al., 1996; Hussain and Carpenter, 2005). Several studies have demonstrated that PKA-dependent processes also play a role in mossy fiber LTP (Huang et al., 1994, 1995; Weisskopf et al., 1994; Castillo et al., 1997; Wang et al., 2003), suggesting that both signaling pathways may act in concert to support mossy fiber LTP. Because MARCKS is not a substrate of PKA (Graff et al., 1991), the present results suggest that if the PKA signaling pathway is intact in these mice, it cannot by itself sustain LTP. Similarly, studies demonstrating impaired LTP in PKA-substrate mutant mice suggest that the PKC signaling pathway cannot by itself sustain LTP (e.g., Castillo et al., 1997). It is possible, therefore, that both PKC and PKA signaling pathways interact in a cooperative manner to modulate synaptic efficacy (e.g., Hollingsworth et al., 1986), and mediate mossy fiber synaptic plasticity in separate mossy fiber synapses. For example, PKA activity appears to be critical for mossy fiber-CA3 pyramidal cell synaptic plasticity but not for mossy fiber-interneuron synaptic plasticity (Maccaferri et al., 1998). This raises the possibility that PKC and MARCKS may play a more central role in long-term synaptic plasticity at mossy fiber-interneuron terminals, which is supported by the finding that PKC inhibitors, but not PKA inhibitors, impair LTP at mossy fiber-interneuron synapses (Alle et al., 2001).

One potential mechanism by which 50% reductions in MARCKS expression could impair mossy fiber LTP, while sparing short-term synaptic plasticity (basal synaptic responses, PPF, PTP), is through impairing PKC-mediated alterations in F-actin cytoskeletal structure in the regulation of vesicle trafficking or synaptic remodeling. F-actin cytoskeletal plasticity is required for vesicular trafficking (Doussau and Augustine, 2000; Morales et al., 2000; Trifaro et al., 2002), dendritic motility (Matus, 2000), and the maintenance phase of LTP (Kim and Lisman, 1999; Krucker et al., 2000; Fukazawa et al., 2003). MARCKS binds and crosslinks F-actin in a PKC phosphorylation-dependent manner (Hartwig et al., 1992; Bubb et al., 1999; Yarmola et al., 2001), and PKC-mediated phosphorylation of MARCKS results in the movement of synaptic vesicles from reserve pools to active release sites in cultured cells and synaptosomes (Vitale et al., 1995; Stevens and Sullivan, 1998; Walaas and Sefland, 2000; Rose et al., 2001; Yang et al., 2002). Furthermore, anti-MARCKS antibodies, or F-actin destabilizing agents, increase spontaneous neurotransmitter release in PC12 cells (Shoji-Kasai et al., 2002), whereas MARCKS overexpression (two-fold) impaired PKC-induced enhancement of neurotransmitter release in human neuroblastoma SH-SY5Y cells (Hartness et al., 2001). Therefore, a 50% reduction in MARCKS in mossy fiber axon terminals may compromise the regulation of F-actin cytoskeletal rigidity, resulting in less restriction in the movement of synaptic vesicles from reserve pools to active release sites. Since PTP is associated with the initial depletion of the readily-release vesicle pool (Brager et al., 2002) and is followed by a redistribution of synaptic vesicles from reserve pools to the readily-release pool (Applegate et al., 1987; Brager et al., 2002), a failure to maintain vesicles in reserve/surplus pools would be predicted to impair long-term, but not short-term, elevations in neurotransmitter release probability (Markram and Tsodyks, 1996; Stevens and Sullivan, 1998; Yawo, 1999; Brager et al., 2002). If this model is correct, future ultrastuctural analysis of mossy fiber terminals should reveal a paucity of vesicles in reserve pools in heterozygous Marcks mutant mice. Additionally or alternatively, 50% reductions in MARCKS expression in mossy fiber terminals may impair alterations in other aspects of synaptic morphology mediated by the F-actin cytoskeleton and associated with LTP, including synaptogenesis or the curvature of exiting synapses (Adams et al., 1997; Marrone and Petit, 2002; Lang et al., 2004; Matsuzaki et al., 2004; Calabrese and Halpain, 2005).

In as much as the synaptic structural remodeling observed following LTP induction is also observed following behavioral learning (reviewed in Geinisman, 2000), and the impairment of mossy fiber LTP, but not Schaffer collateral LTP, in heterozygous Marcks mutant mice suggest that the pattern of hippocampus-dependent learning deficits exhibited by these mice may be a consequence of selective deficits in mossy fiber long-term synaptic plasticity. The learning deficit exhibited by heterozygous Marcks mutant mice appears to be specific to spatial navigation and does not extend to contextual aspects of hippocampal function (McNamara et al., 2004) and is most apparent following prior training to a different spatial location (spatial reversal learning) (McNamara et al., 1998). Previous studies suggest that mossy fiber synaptic plasticity may be important for hippocampus-dependent spatial learning processes (Ishihara et al., 1997; Lassalle et al., 2000; Ramirez-Amaya et al., 2001), and naturally occurring variations in hippocampal infrapyramidal mossy fiber distribution in inbred mice are specifically correlated with spatial reversal learning performance (Schopke et al., 1991; Bernasconi-Guastalla et al., 1994). In agreement with the pattern of synaptic and behavioral deficits observed in heterozygous Marcks mutant mice, mice lacking the synaptic vesicle binding protein Rab3a also exhibit deficits in mossy fiber LTP, but not PPF or basal synaptic transmission (Castillo et al., 1997), and deficits in spatial reversal learning, but not initial spatial acquisition or contextual fear conditioning (Hensbroek et al., 2003; D’Adamo et al., 2004). Collectively, these data suggest that long-term plasticity in the mossy fiber-CA3 synapses may play a specific role in diminishing synaptic strengths formed by a previously acquired spatial response. Interestingly, low-intensity (no after-discharge) electrical stimulation of hippocampal granule cells, but not area CA1, produced retrograde amnesia for a previously acquired spatial response, leading to the suggestion that the mossy fiber-CA3 pathway performs an erasure function (Collier et al., 1987). The failure of previous studies to observe initial spatial acquisition deficits in mutant mice exhibiting deficits in mossy fiber LTP (e.g., Hensbroek et al., 2003) is consistent with this model of mossy fiber function, and future studies examining the role of mossy fiber synaptic plasticity in spatial learning process should routinely assess spatial reversal learning performance.

Acknowledgments

NIMH; Grant number: RO1 MH59959, RO1 MH60244;

Grant sponsors: National Institute of Environmental Health Science, NIH, National Alliance for Research on Schizophrenia and Depression; Grant sponsor: NIA; Grant number: RO1 AG18199; Grant sponsors: Whitehall Foundation, the University of Pennsylvania Research Foundation; Grant sponsor: Mental Retardation and Development Disabilities Research Center at Children’s Hospital of Philadelphia; Grant number: HD26979.

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