Protein Synthesis-Dependent and mRNA Synthesis-Independent Intermediate Phase of Memory in Hermissenda

Terry Crow, Juan-Juan Xue-Bian, and Vilma Siddiqi

Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77225


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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Crow, Terry, Juan-Juan Xue-Bian, and Vilma Siddiqi. Protein Synthesis-Dependent and mRNA Synthesis-Independent Intermediate Phase of Memory in Hermissenda. J. Neurophysiol. 82: 495-500, 1999. The conditioned stimulus pathway in Hermissenda has been used to examine the time-dependent mechanisms of memory consolidation following one-trial conditioning. Here we report an intermediate phase of memory consolidation following one-trial conditioning that requires protein synthesis, but not mRNA synthesis. In conditioned animals, enhanced excitability normally expressed during an intermediate phase of memory was reversed by the protein synthesis inhibitor anisomycin, but not by the mRNA synthesis inhibitor 5,6-dichloro-1-beta -D-ribobenzimidazole (DRB). Associated with the intermediate phase of memory is an increase in the phosphorylation of a 24-kDa protein. Anisomycin present during the intermediate phase blocked the increased phosphorylation of the 24-kDa phosphoprotein, but did not block the increased phosphorylation of other proteins associated with conditioning or significantly change their baseline phosphorylation. DRB did not reverse enhanced excitability or decrease protein phosphorylation expressed during the intermediate phase of memory formation, but it did reverse enhanced excitability 3.5 h after conditioning. Phosphorylation of the 24-kDa protein may support enhanced excitability during the intermediate phase, in the transition period between short- and long-term memory.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Studies of the time-dependent development of cellular and synaptic plasticity have identified multiple phases in the formation of memory (DeZazzo and Tully 1995; Freeman et al. 1995; McGaugh 1966, 1968; Ng and Gibbs 1991; Rosenzweig 1993). The components of memory consolidation can be differentiated based on the contribution of signal transduction pathways, protein synthesis, and gene induction (Kane et al. 1997; Nguyen et al. 1994; Otani et al. 1989). Examples of plasticity have been reported that exhibited an immediate (Kang and Schuman 1996) or intermediate requirement for protein synthesis (Ghirardi et al. 1995); however, specific proteins have not been identified. Short- and long-term memory in the conditioned stimulus (CS) pathway of conditioned Hermissenda can be dissociated based on the contribution of mRNA synthesis, protein synthesis, and protein kinases (Crow and Forrester 1990, 1993; Crow et al. 1997, 1998; Farley and Schuman 1991; Matzel et al. 1990; Ramirez et al. 1998). One-trial conditioning of Hermissenda (Crow and Forrester 1986) results in the biphasic development of enhanced cellular excitability detected in the lateral type-B photoreceptors. Enhanced excitability reaches asymptotic levels 3 h postconditioning, followed by a decrease in excitability at 5-6 h postconditioning, which leads to a second phase of enhanced excitability at 16-24 h (Crow and Siddiqi 1997). The short-term phase of enhancement (<= 1 h) has been shown to be independent of protein and mRNA synthesis, whereas the long-term phase is blocked by both mRNA and protein synthesis inhibitors (Crow and Forrester 1990; Crow et al. 1997). In the present study, a requirement for protein synthesis, but not mRNA synthesis during the intermediate phase of memory was observed between 1.5 and 2.5 h postconditioning.


    METHODS
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INTRODUCTION
METHODS
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Experimental procedures

Three types of preparations were used; an exposed, but otherwise intact nervous system, an isolated intact nervous system, and isolated components of the CS pathway consisting of the eye and proximal optic nerve. Conditioning was conducted with exposed nervous systems followed by electrophysiological studies of isolated nervous systems at different times postconditioning. Adult Hermissenda crassicornis were maintained in artificial sea water (ASW) aquaria at 14 ± 1°C on a 12-h light/dark cycle. Before conditioning, animals were anesthetized with a 0.25-ml injection of isotonic MgCl2, and a small dorsolateral incision was made to expose the circumesophageal nervous system. Surgically prepared animals were transferred to a chamber containing 50 ml of normal ASW or 50 ml of ASW containing 10-4 M 5,6-dichloro-1-beta -D-ribobenzimidazole (DRB), 10-7 M DRB, or 10-5 M anisomycin. The one-trial conditioning procedure consisted of a 5-min presentation of light, the CS (10-4 W/cm2) paired with the application of serotonin (5-HT) to the region of the cerebropleural ganglion where previous immunocytochemistry revealed 5-HT reactive processes near the optic nerve and photoreceptor terminals in the neuropil (Land and Crow 1985). The final concentration of 5-HT in the ASW was 0.1 mM. Animals were placed in a chamber with normal ASW or ASW containing the different inhibitors 15 min before presenting the conditioning trial. The conditioning trial was presented in the presence of DRB, anisomycin, or normal ASW. Unpaired control groups received the CS and 5-HT separated by 5 min. For the unpaired control group, the 5-HT was applied in the dark (infrared illumination) and washed out after the 5-min exposure. Following the conditioning trial, animals were maintained in ASW containing the different inhibitors before assessing excitability.

Electrophysiology

Intracellular recordings were collected from lateral type B photoreceptors at times between 0.5 and 3.5 h after the conditioning trial. A total of 175 animals from the experimental and control groups were prepared for intracellular recording and stimulation with extrinsic current using previously published standard procedures (Crow and Forrester 1990, 1993; Crow et al. 1997). Experiments with the isolated circumesophageal nervous system were conducted in ASW maintained at 15 ± 0.5°C and had the following composition (in mM): 460 NaCl, 10 KCl, 10 CaCl2, and 55 MgCl2, buffered with 10 mM HEPES and brought to pH 7.6 with NaOH. Excitability was assessed with 30-s, 20-mV depolarizing current steps and in a second group of animals at 1.5 and 3 h postconditioning using 2-s depolarizing pulses at three current levels: 0.1, 0.15, and 0.2 nA. Averages of spike frequency were determined by subtracting spontaneous baseline activity from the total number of action potentials elicited by the 30-s extrinsic current and dividing by the duration of the current pulse. Membrane potential was not controlled in experiments examining excitability with 20-mV depolarizing steps. In experiments using 2-s pulses, spikes were elicited by the three current pulses from a membrane potential of -60 mV.

Protein phosphorylation and two-dimensional gel electrophoresis

Protein phosphorylation in normal controls and conditioned groups was examined in components of the CS pathway of preparations exposed to anisomycin or DRB during a 2-h incubation in 0.125 mCi of 32PO4. To minimize potential animal-to-animal variability in 32PO4 uptake, the eyes and proximal optic nerves from three animals were used for each treatment and control procedure in each experimental replication. After the 2-h incubation the samples were rinsed in an isotonic ice-cold wash solution (in mM: 460 NaCl, 10 KCl, 5 EDTA, and 100 Tris-HCl, pH 7.8) and lysed in a modified lysis solution containing 9.2 M urea, 2% Nonidet P-40, 5% beta -mercaptoethanol, and 2% carrier ampholytes (1.6% pH 5-8, 0.4% pH 3.5-10), 100 mM NaF, 1 mM sodium orthovandate, 0.1 mM okadiac acid and stored frozen at -80°C. Samples were analyzed by two-dimensional gel electrophoresis using a first-dimension isoelectric focusing (IEF) gel with an immobilized pH gradient (4-7) and a precast SDS polyacrylamide (8-18% linear gradient) second-dimension gel. Gels were exposed to storage phosphor screens for a period of 24 h. Phosphor screens were computer scanned and analyzed using ImageQuant software (Molecular Dynamics, Sunnyvale, CA) for quantitative analysis.

Statistical analysis

A two-way analysis of variance (ANOVA) was used to assess the main effects of the treatments on excitability and membrane potential, and a one-way ANOVA assessed the effects of inhibitors on protein phosphorylation. After the determination of overall significant effects, paired comparisons consisted of Tukey tests. Comparisons of the differences in the ratios between experimental treatments and control gels involved t-tests for correlated means.


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Exposure before and after one-trial conditioning to the translational inhibitor anisomycin (10-5 M) or the transcriptional inhibitor DRB (10-4 M and 10-7 M) produced time-dependent differential effects on enhanced excitability as shown in Fig. 1A. Significant overall differences between the treatment groups and the controls were detected by the results of the ANOVA (F4,96 = 63.6; P < 0.001). The main effect of time after conditioning was significant (F4,96 = 6.5; P < 0.001), and the interaction between treatments and time after conditioning was also significant (F16,96 = 1.9; P < 0.05). At 0.5 and 1 h postconditioning, none of the treatment groups were significantly different from each other (F3,26 = 0.3; NS; Fig. 1A). However, the groups conditioned and maintained postconditioning in anisomycin showed a significant reduction in excitability relative to the groups conditioned and maintained in normal ASW at 1.5 h (q = 4.9), 2 h (q = 4.2), 2.5 h (q = 7.9), 3 h (q = 6.7), and 3.5 h (q = 7.4) postconditioning (P < 0.05 for each comparison). The anisomycin groups were significantly different from the DRB groups (10-4) at 1.5 h (q = 5.6), 2 h (q = 5.7), and 2.5 h (q = 4.1; P < 0.05). The anisomycin groups were not significantly different from the unpaired controls at 1.5 h (q = 3.3), 2 h (q = 2.8), 2.5 h (q = 1.3), 3 h (q = 2.2), and 3.5 h (q = 0.12). In contrast, excitability measures from the groups conditioned and maintained in DRB (10-4 M) were not significantly reduced relative to the groups conditioned in normal ASW or the groups conditioned in DRB (10-7 M) when examined at 1.5 and 2 h postconditioning (q = 0.02, 0.6; q = 1.1, 0.5). However, inhibition of mRNA synthesis with DRB (10-4 M) reduced enhanced excitability measured 2.5, 3, and 3.5 h postconditioning as compared with the conditioned group (q = 4.03; q = 7.9; q = 5.2; P < 0.05 for all comparisons). The groups conditioned in 10-7 M DRB, which does not significantly inhibit mRNA synthesis (Crow et al. 1997), were not significantly different from the groups conditioned in normal ASW at any time period (Fig. 1A). The unpaired control group was not significantly different from the DRB (10-4 M) group at 3.5 h (q = 3.05). The analysis of dark-adapted membrane potential for the experimental and control groups did not reveal significant overall differences (F4,96 = 1.55; NS). We further examined enhanced excitability 1.5 h postconditioning at levels of depolarization <20 mV for the different experimental groups and the unpaired control group by measuring spike activity elicited by three levels of depolarizing extrinsic current (0.1, 0.15, 0.2 nA). As shown in Fig. 1Bb, at 1.5 h postconditioning, fewer spikes were elicited by the 0.2-nA current pulse for the anisomycin preparation as compared with the example from a conditioned group (Fig. 1Ba) or one from the group conditioned in 10-4 M DRB (Fig. 1Bc). The example from the unpaired control group (Fig. 1Bd) is similar to the anisomycin group and different from the conditioned group and conditioned DRB group. However, at 3 h postconditioning, the example from the group conditioned in DRB (10-4 M) showed fewer elicited spikes (Fig. 1Be) as compared with excitability assessed at 1.5 h for the DRB (10-4 M) preparation. The group data for excitability changes elicited by the three different current levels and four different treatments is shown in Fig. 1C. The results of the ANOVA revealed a significant overall effect of treatments (F3,16= 31.9; P < 0.01) and current levels (F2,32 = 44.9; P > 0.01). The group conditioned and maintained in anisomycin (n = 5) and the unpaired control group (n = 5) showed a reduction in excitability at all three current levels as compared with the conditioned ASW (n = 5) and DRB groups (n = 5) (q = 7.8; P < 0.05). The unpaired control group was not significantly different from the anisomycin group. Overall, these results show that at an intermediate time in memory consolidation (1.5-2.5 h) protein synthesis, but not mRNA synthesis is necessary to support excitability changes produced by one-trial conditioning.



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Fig. 1. Protein synthesis and mRNA synthesis inhibitors reverse enhanced excitability measured at different times following 1-trial conditioning. A: group data depicting mean frequency (±SE) in spikes/s elicited by 30-s, 20-mV depolarizing extrinsic current pulses. Excitability was assessed at only 1 time for each preparation, i.e., independent groups. Animals exposed to anisomycin (10-5 M) 15 min before conditioning and for different durations after conditioning exhibited a reversal of enhanced excitability 1st detected 1.5 h postconditioning. The groups conditioned and maintained postconditioning in anisomycin showed a significant reduction in excitability at 1.5, 2, 2.5, 3, and 3.5 h postconditioning compared with groups conditioned in normal artificial seawater (ASW), groups conditioned and maintained in a concentration of 5,6-dichloro-1-beta -D-ribobenzimidazole (DRB) that does not inhibit mRNA synthesis (10-7 M), and at 1.5, 2, and 2.5 h for groups conditioned and maintained in (10-4 M) DRB. The group conditioned and maintained postconditioning in DRB (10-4 M) was not significantly different from the unpaired control group at 3.5 h postconditioning. The DRB (10-4 M) groups showed a significant reduction in excitability at 2.5, 3, and 3.5 h as compared with animals conditioned in normal ASW, and at 3 and 3.5 h for groups conditioned and maintained in 10-7 M DRB. B: examples of enhanced excitability examined in lateral B photoreceptors from different groups of animals than shown in A, 1.5 and 3 h postconditioning. Excitability changes were examined in a 2nd experiment 1.5 h postconditioning using additional animals in the conditioned, anisomycin, DRB, and unpaired treatments. Excitability was assessed at voltages <20 mV by passing 2-s extrinsic constant current pulses from a membrane potential of -60 mV at 3 current levels; 0.1, 0.15, and 0.2 nA. The example shown in Bb from the group conditioned and maintained in anisomycin for 1.5 h showed a reduction in excitability elicited by the 0.2-nA pulse as compared with the example from the group conditioned in normal ASW at 1.5 h (Ba) or the example for the group conditioned and maintained in (10-4 M) DRB (Bc) or the unpaired example (Bd). As shown in the example in Be at 3 h postconditioning, the group conditioned and maintained in 10-4 M DRB exhibited a decrease in excitability elicited by the 0.2-nA pulse as compared with the example shown in Bc. C: group data (mean spikes/s ± SE) for excitability measured at the 3 current levels 1.5 h postconditioning for the group conditioned in ASW (n = 5), the group conditioned in 10-4 M DRB (n = 5), the group conditioned in 10-5 M anisomycin (n = 5), and the unpaired control group (n = 5; *P < 0.05).

We have previously shown that one-trial conditioning results in the phosphorylation of proteins that can be detected several hours after one-trial conditioning (Crow et al. 1996). To determine whether posttranslational modifications of proteins may be associated with the intermediate phase of memory, we examined changes in protein phosphorylation postconditioning. Phosphate incorporation into proteins from conditioned and unpaired controls was analyzed after separation by 2-D PAGE. We found an increase in phosphorylation of a 24-kDa phosphoprotein detected 2 h after one-trial conditioning that was not previously reported in our earlier study (Crow et al. 1996). Phosphate incorporation in the 24-kDa protein from a conditioned preparation (Fig. 2A) was increased as compared with an unpaired control (Fig. 2B). The analysis of the group data showed that phosphorylation was significantly greater for the conditioned group (n = 12) as compared with the unpaired control group (n = 12; t11 = 2.0; P < 0.05; Fig. 2C. To determine whether the increased 32PO4 labeling may reflect an increase in protein synthesis, we conducted control experiments using 35S-methionine labeling for 2-h postconditioning. The 35S-methionine labeling of the 24-kDa protein for the conditioned and unpaired controls was similar (t5 = 0.57; NS, n = 6). These results indicate that the increased 32PO4 labeling of the 24-kDa protein for the conditioned group does not reflect an increase in protein synthesis. In addition, there was a significant difference between conditioned and unpaired controls in the phosphorylation of the 55 kDa (t11 = 3.92; P < 0.01), 46 kDa (t8 = 4.35; P < 0.01), and 29 kDa (t11 = 4.42; P < 0.01) phosphoproteins that had been shown previously to increase with conditioning and 5-HT application (Crow et al. 1996).



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Fig. 2. 32PO4 incorporation in a 24-kDa protein from a conditioned group and unpaired control examined 2 h postconditioning. A and B: prints from a storage phosphor screen showing 32PO4 labeling of phosphoproteins after 2-D gel electrophoretic separation. A is from a conditioned group, and B is from an unpaired control group. Arrows indicate the location of the 24-kDa phosphoprotein. C: group data (n = 12) showing mean ± SE conditioned/unpaired control ratios of densitometric measurements for the 24-kDa phosphoprotein 2 h after 1-trial conditioning. Conditioning resulted in a significant increase in 32PO4 incorporation as compared with the unpaired control group (* P < 0.05). The hatched bar graph represents an E/C ratio of 1 where the conditioned group is identical to the unpaired control group.

We next examined the effect of anisomycin on protein phosphorylation during the intermediate phase of enhanced excitability following conditioning. Incubation of components of the isolated CS pathway in anisomycin (2 h) resulted in significant overall differences in 32PO4 incorporation into different phosphoproteins (F3,20 = 3.2; P < 0.05). Anisomycin produced a significant reduction in 32PO4 incorporation into the 24-kDa protein (n = 6; t5 = 3.9; P < 0.01; Fig. 3, B and C), whereas phosphorylation of three other proteins (55, 46, and 29 kDa) shown previously to exhibit increased 32PO4 incorporation after one-trial conditioning or 5-HT application were not significantly affected (Fig. 3C). Moreover, the same anisomycin treatment (2 h) did not produce a significant decrease in the baseline phosphorylation of the 55-, 46-, 29-, or 24-kDa proteins in normal untrained preparations (F3,8 = 0.28; NS). These results show that the effect of anisomycin during the intermediate phase of memory is specific to the 24-kDa phosphoprotein, and on the enhanced phosphorylation of the 24-kDa observed after one-trial conditioning. As a further control, we examined the effect of a 2-h exposure to DRB on protein phosphorylation because this period of postconditioning incubation did not reduce enhanced excitability when tested 2 h after one-trial conditioning (Fig. 1). The incubation of preparations for 2 h in DRB (10-4 M) did not change baseline phosphorylation of the 55-, 46-, 29-, or 24-kDa phosphoproteins in a normal control group (F3,8 = 0.13; NS), or significantly effect 32PO4 incorporation into the 55-, 46-, 29-, or 24-kDa phosphoproteins of conditioned animals (F3,12 = 1.1; NS).



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Fig. 3. Effect of anisomycin on protein phosphorylation examined during the intermediate phase of memory (2 h postconditioning). A and B are prints from a section of a storage phosphor screen showing 32PO4 labeling of phosphoproteins after 2-D gel electrophoretic separation. A is from a group conditioned and maintained in normal ASW, and B is an example from a group conditioned and maintained in 10-5 M anisomycin. The 4 phosphoproteins (55, 46, 29, and 24 kDa) are designated by numbers in A and B. C: group data (n = 6) showing mean ± SE (E/C) experimental/control ratios of densitometric measurements for 4 phosphoproteins (55, 46, 29, and 24 kDa) examined 2 h postconditioning. Anisomycin produced a significant reduction in 32PO4 incorporation into the 24-kDa protein and not in the other 3 phosphoproteins that were shown previously to exhibit increased phosphorylation after 1-trial conditioning or serotonin application (* P < 0.01). The hatched bar graphs represent an E/C ratio of 1 where the conditioned groups are identical to the unpaired control groups.


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We have shown that anisomycin and DRB effect enhanced excitability at different times following one-trial conditioning. The results showing that anisomycin, but not DRB, significantly reduces phosphorylation of a 24-kDa protein 2 h postconditioning provides additional evidence that protein synthesis is required in the intermediate phase of memory consolidation. Our finding that the intermediate phase of memory consolidation does not require mRNA synthesis suggests that activation of translation may occur without the activation of transcription. The requirement for protein synthesis in the intermediate phase of memory consolidation may depend on the learning paradigm, species, and the neural systems responsible for the maintenance and expression of memory. Increased excitability of Aplysia sensory neurons produced by protein kinase C activation measured 3 h after treatment was reported to be independent of protein synthesis (Manseau et al. 1998). In addition, there are examples from other species where an intermediate phase of memory may not be dependent on protein synthesis (DeZazzo and Tully 1995). However, an intermediate phase of synaptic facilitation in Aplysia sensory neurons produced by exposure to 5-HT has been shown to be protein synthesis dependent and mRNA synthesis independent (Ghirardi et al. 1995). Moreover, there are examples of synaptic plasticity that have an immediate requirement for protein synthesis (Kang and Schuman 1996).

The translational dependent intermediate phase does not appear only to involve an increase in the synthesis of the 24-kDa protein based on the results of the 35S-methionine labeling study. Therefore the requirement for protein synthesis may be necessary but not sufficient, or may reflect an indirect involvement in phosphorylation such that the change in the phosphorylation of the 24-kDa protein produced by conditioning could be due to changes in the synthesis of protein kinases in the signal transduction pathways or phosphatase inhibitors. One protein that has been previously identified in Hermissenda is calexcitin/cp20, a low molecular weight GTP- and Ca2+-binding protein (Ascoli et al. 1997) that is phosphorylated in conditioned Hermissenda (Neary et al. 1981). However, the evidence indicates that the 24-kDa phosphoprotein identified in this study is not calexcitin. The partial sequence of the 24-kDa protein produced peptides that were similar to the beta -thymosin family of actin-binding protein and did not show a sequence homology to calexcitin/cp20 based on a search of the National Center for Biotechnology Information protein databases that included calexcitin (unpublished observations). All known vertebrate and invertebrate beta -thymosins bind actin monomers (Nachmias 1993). Our results would thus support a potential role for beta -thymosin in cellular plasticity by modifications in actin assembly/diassembly in neurons of the CS pathway of conditioned animals.


    ACKNOWLEDGMENTS

We thank P. Dash for discussions and D. Parker for typing the manuscript.

This work was supported by National Institute of Mental Health Grants MH-40860 and MH-01363 to T. Crow.


    FOOTNOTES

Address for reprint requests: T. Crow, Dept. of Neurobiology and Anatomy, University of Texas Medical School, P.O. Box 20708, Houston, TX 77225.

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

Received 20 November 1998; accepted in final form 5 March 1999.


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