Inhibition of protein synthesis blocks long-term enhancement of generator potentials produced by one-trial in vivo conditioning

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 87, pp , June 199 Neurobiology Inhibition of protein synthesis blocks long-term enhancement of generator potentials produced by one-trial in vivo conditioning in Hermissenda TRRY CROW* AND JAMS FORRSTR Department of Physiology, School of Medicine, University of Pittsburgh, Pittsburgh, PA Communicated by Neal F. Miller, January 18, 199 (received for review June 19, 1989) ABSTRACT A one-trial in vivo conditionig procedure produces short- and long-term cellular changes that can be detected in identified sensory neurons of the pathway mediating the conditioned stimulus. The memory of the associative experience in the conditioned stimulus pathway is expressed by short- and long-term enhancement of light-evoked generator potentials recorded from identified lateral and medial type B photoreceptors in the eyes of Hermissenda. To identify mechanisms of the induction and expression of short- and long-term enhancement in identified photoreceptors, we have investigated the effects of inhibiting protein synthesis during the application of the one-trial in vivo conditioning procedure. Anisomycin (1 plm) present during and after the conditioning trial blocked long-term enhancement out affecting the induction or expression of short-term enhancement. Application of a control compound, deacetyl (1 PzM), or delaying the application of until 1 hr after the conditioning trial did not block either long- or short-term enhancement. These results indicate that synthesis of proteins during or shortly after training may be a critical step in the formation of long-term memory of the associative experience. Behavioral and cellular neurophysiological studies of memory have provided support for the hypothesis that protein synthesis during or shortly after an experience is a necessary step for the formation of enduring forms of memory called long-term memory (review in ref. 1). Support for the hypothesis that, for long-term memory to be formed, proteins must be synthesized is provided by learning and memory studies of vertebrates where inhibition of protein synthesis and/or RNA synthesis interferes the formation of long-term memory while short-term memory remains relatively intact (2-5). Recently, studies of short- and long-term synaptic facilitation of monosynaptic connections between cultured sensory and motor neurons in Aplysia revealed that inhibitors of protein or RNA synthesis blocked long-term facilitation, while leaving short-term facilitation intact (6). Studies of short-term and long-term synaptic and cellular plasticity in invertebrate nervous systems are particularly attractive since such preparations provide for the opportunity of relating biochemical and molecular changes in identified neuronal cell types to learning and memory (6-11). The results of recent studies of Hermissenda have shown that a one-trial in vivo conditioning procedure produces a long-term change in phototactic behavior that depends upon pairing a conditioned stimulus (CS) the direct application of serotonin (5- hydroxytryptamine, 5-HT) to the exposed nervous system of otherwise intact animals (12). One site for cellular changes that may contribute to the long-term behavioral modification is the sensory pathway mediating the CS. We recently reported that one-trial in vivo conditioning produces both a The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance 18 U.S.C solely to indicate this fact. 449 short-term nonassociative enhancement and a long-term associative enhancement of generator potentials recorded from identified lateral and medial B photoreceptors (13). The induction of short-term enhancement is due in part to activation of the Ca2+/phospholipid-dependent kinase, protein kinase C (14-16). An essential role for protein kinase C in long-term enhancement has not been demonstrated; however, long-term enhancement can be produced by pairing the CS activation of protein kinase C by diacylglycerol analogues or phorbol esters (14). Although some progress has been made in understanding the events that may be critical for induction (i.e., the associative interaction between depolarization of photoreceptors and activation of protein kinases), the mechanisms responsible for the maintenance and expression of long-term enhancement are not understood. Here we report that the protein synthesis inhibitor, when present during and after one-trial in vivo conditioning, blocks long-term enhancement out affecting the induction or expression of short-term enhancement. We found that the application of 1 hr after in vivo conditioning was ineffective in blocking long-term enhancement, which is consistent earlier reports from studies of vertebrates suggesting that a critical "time window" operates for macromolecular synthesis. The results of this study indicate that retention of the memory for the associative experience depends upon protein synthesis during or shortly after the presentation of the conditioning trial. MATRIALS AND MTHODS A total of 78 adult Hermissenda crassicornis was used in the experiments. Hermissenda were obtained from Sea Life Supply (Sand City, CA). The animals were fed small pieces of scallops each day during the experiment and housed in artificial sea water (ASW) aquaria at 14 ±+ PC on a 12-hr light/dark cycle. The animals were prepared for the in vivo conditioning procedure as described (12). Before conditioning, animals were lightly anesthetized.25-ml injections of isotonic MgCl2, and a small dorsolateral incision was made to expose the circumesophageal nervous system. Surgically prepared animals were then transferred to a chamber containing 5 ml of normal ASW. The exposed nervous system was visualized in infrared illumination provided by a 45-W tungsten/halogen light source projected through an infrared filter (Schott model RG-85). A dissecting microscope formed an image of the nervous system upon a Dage MTi video camera connected to a video monitor and time-lapse video recorder. In Vivo Conditioning Procedure. The one-trial in vivo conditioning procedure consisted of presenting visible light (1 x Abbreviations: 5-HT, 5-hydroxytryptamine (serotonin); CS, conditioned stimulus; ASW, artificial sea water. *Present address: Department of Neurobiology and Anatomy, University of Texas Medical School at Houston, Houston, TX

2 Neurobiology: Crow and Forrester lo-4 W/cm2) paired the injection of.5 ml of 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 (17). The injection of 5-HT was applied to the desired region of the exposed nervous system by observing the image of the ganglion and injecting needle on a video monitor. The final concentration of 5-HT in the ASW was.1 mm. Anisomycin and deacetyl applications were adjusted to yield a final concentration in the ASW of 1,LM. Measurement of Protein Synthesis. We used the reversible protein synthesis inhibitor (18-2) at a concentration of 1 gm. The concentration of and deacetyl used in the studies was based upon the results of experiments that examined the incorporation of [3H]leucine (15,uCi/ml; 1 Ci = 37 GBq) into protein insoluble in trichloroacetic acid. The incorporation of [3H]leucine into newly synthesized proteins was terminated after 4 min by washing and homogenizing each nervous system in cold (4C) phosphate-buffered saline, which was then adjusted to 1% (wt/vol) trichloroacetic acid and held on ice for 3 min. Solutions were centrifuged at 1, x g for 1 min, and pellets were washed twice 1% trichloroacetic acid by resuspension and centrifugation. Radioactivities were quantified by scintillation counting. We found that 1,uM blocks protein synthesis by 86% in the Hermissenda circumesophageal nervous system. To address the problem of specificity of the inhibitor, we used a derivative of, deacetyl (2), which produced 3% inhibition of protein synthesis at a concentration of 1,uM. xperimental Protocol. As shown in the diagram of the experimental protocol in Fig. 1, four different treatments were examined. The paired group received light (CS) paired the injection of 5-HT in the presence of the protein synthesis inhibitor or a derivative, deacetyl, that does not significantly inhibit protein synthesis at the concentration used in this study (1,uM). The or deacetyl was applied 3 min prior to the presentation of the conditioning trial and was present during the application of light and 5-HT. The and deacetyl were washed out 3 min after the end of the one-trial conditioning procedure. An unpaired control group received light (CS) and 5-HT separated by 5 min in the presence of. The 5-HT was applied in the dark (infrared illumination) and washed out at the end of the 5-min period (see Fig. 1). An control group received only the 5-min light step (CS) in the presence of. The final treatment group received light paired 5-HT; Proc. Natl. Acad. Sci. USA 87 (199) 4491 however, the was applied 1 hr after the end of the conditioning trial (1 hr posttraining). The remained on the exposed nervous system for the same period (65 min) as was used for other treatment groups. After the various experimental treatments, the nervous systems were washed ASW and the animals were returned to marked cages in the sea water aquaria. Intracellular Recording. One hour or 24 hr after the different experimental treatments, animals were prepared for intracellular recording from identified medial and lateral type B photoreceptors. Recordings of light-evoked generator potentials were collected either 1 hr after the end of the conditioning trial (short term) or 24 hr after the conditioning trial (long term). The animals were coded so that the experimenter collecting the electrophysiological data did not know the treatment condition that a given preparation had received. The nervous systems were removed, and B photoreceptors were isolated by axotomy of the optic nerve as described (11, 22). After 15 min of dark adaptation, generator potentials elicited by a 2-min light step (1 x 1-4 W/cm2 at 51 nm) were recorded from visually identified B photoreceptors in the medial and lateral position in the eye by using standard procedures for intracellular recording and light stimulation. Microelectrodes used for intracellular recordings had a resistance between 4 and 6 Mfl when filled 4 M potassium acetate. Prior to intracellular recordings, the isolated nervous systems received a mild incubation in a protease solution (type VIII,.67 mg/ml, 5 to 6 min; Sigma) to facilitate microelectrode penetration of identified B photoreceptors. lectrophysiological experiments were conducted in buffered ASW (1 mm Hepes/46 mm NaCI/1 mm KCI/1 mm CaCl2/55 mm MgC12). The solution was brought to a final ph of 7.6 at C dilute NaOH. The temperature of the ASW in the recording chamber was maintained at 15 ±.5 C. The sea water temperature was monitored by a thermistor inserted into the recording chamber. Statistical Analysis. A two-way analysis of variance repeated measures (treatment x trials unweighted-means solution) was used to assess overall effects of the various treatments on the amplitude of generator potentials (21). Tests of significance of the peak and plateau phase of the generator potentials following a significant overall F test employed Newman-Keuls procedures (21). In cases where the interaction between treatment and trials was significant, the simple main effects were analyzed to determine significance. Within group comparisons of the change in amplitude of the peak and plateau phase of the generator potential PAIRD LIGHT & 5-HT Room( 8MIN) Dark( 2MIN), - (CS)&5-HT(5MIN) Dark( 2MIN) - Room( 8MIN) N or DACTYL UNPAIRD Room( 8MIN) Dark( 2MIN),l (CS)(5MIN) Dark( 2MIN) Room( 8MIN) LIGHT & 5-HT l'-i A O5-HT-N AN ISOMYCIN- CONTROL Room( 8MIN) 1 Dark( 2MIN) (CS)(5MIN) Dark( 2MIN) Room( 8MIN) DLAYD Room( I 8MIN) Dark( 2MIN) (CS)&5-HT(5MIN) Dark( 2MIN) Room( 1 8MIN) lh-u K hrpost-training-h FIG. 1. xperimental protocol for investigating the effects of protein synthesis inhibition produced by on short- and long-term enhancement. Animals prepared for in vivo conditioning were assigned to one of four different treatment conditions. The paired light (CS) and 5-HT group received one 5-min trial of light paired simultaneously the injection of 5-HT onto the exposed nervous system in the presence of either or deacetyl. The unpaired control group received the CS (5 min) followed by a 5-min period in the dark before applying 5-HT (5 min) to the nervous system in the dark (infrared illumination). The control group received only the 5-min CS in the presence of. The delayed group received the CS and 5-HT paired, and the was applied 1 hr after the termination of one-trial in vivo conditioning.

3 4492 Neurobiology: Crow and Forrester following the application of 5-HT to animals that had previously received pretreatment were made difference scores using a t test for correlated means. Proc. Natl. Acad. Sci. USA 87 (199) RSULTS Anisomycin Does Not Block Short-Term nhancement. We previously reported that one-trial in vivo conditioning produces both a short-term and a long-term enhancement of light-evoked generator potentials recorded from identified medial and lateral B photoreceptors (13). Short-term enhancement of generator potentials recorded from medial and lateral B photoreceptors is not dependent upon pairing the CS 5-HT, whereas long-term enhancement found only in lateral B photoreceptors is dependent upon pairing the CS 5-HT,(11, 13). Pretreatment 1 AuM did not block the induction or the expression of enhanced generator potentials produced by light and 5-HT recorded from medial or lateral B photoreceptors 1 hr after the conditioning trial. Representative examples of generator potentials evoked by a 2-min light step are shown in Fig. 2A for preparations that received light paired 5-HT (Fig. 2AI) or only the CS (light control) (Fig. 2A2). Both preparations had received identical pretreatment. The enhanced generator potential shown in Fig. 2AI as compared to the control in Fig. 2A2 indicates that pretreatment did not block short-term enhancement produced by the light and 5-HT. This conclusion is supported by the analysis of the group data shown in the graph in Fig. 2B comparing the paired and unpaired groups to the control group. The results of the treatment by trials analysis of variance revealed an overall significant difference between the groups (F2,27 = 3.42; P <.5). Short-term enhancement does not depend upon pairing light and 5-HT, which is consistent previously reported results (13), since the generator potentials recorded from both the paired (n = 1) and unpaired groups (n = 11) exhibited significant enhancement. The statistical results revealed that, while the paired and unpaired groups were not significantly different from each other, both were significantly larger as compared to the control group (n = 9), which received only the light (CS) (P <.1). The generator potentials recorded from the three groups exhibited adaptation, which was expressed by a significant reduction in the amplitude of the generator potential measured from peak onset to the end of the 2-min light step (F24,648 = 63; P <.1). A comparison of averaged generator potentials between the control group and a normal control group that did not receive (n = 1) revealed that at the concentration used in this study (1 jzm) does not significantly alter the amplitude ofthe generator potential (see data represented by asterisks in Fig. 2B). These results show that protein synthesis inhibition at the time of application of the CS and 5-HT does not block short-term enhancement produced by one-trial in vivo conditioning. Anisomycin Blocks Long-Term nhancement. After finding that short-term enhancement is not affected by inhibiting protein synthesis, we next examined the effect of application upon long-term enhancement, which is detected in only lateral B photoreceptors. Previous results have shown that long-term enhancement is expressed in lateral B photoreceptors by a decrease in the rate of adaptation of the generator potential in response to a light step, which produces a larger plateau phase while the initial transient peak is not changed (11, 13). Presenting the in vivo conditioning trial during the period of protein synthesis inhibition blocks longterm enhancement. Representative examples of generator potentials are shown in Fig. 3A and the group data are shown in Fig. 3B. The results of the treatment x trials analysis of variance showed no significant overall effect of the different treatments. This is due to the observation that long-term enhancement is expressed during the plateau phase of the light step and not in the early part of the light response. Indeed, the mean amplitude of the generator potential was virtually identical during the first 4 sec of the light response for all of the treatment groups (see Fig. 3B). The significant differences between groups expressed during the plateau phase of the light response is supported statistically by the significant interaction between the treatments and trials (the time from light onset) (F48,84 = 3.9; P <.1). This means that there are overall significant differences in the later component (plateau phase) of the generator potential amplitude between the group that received in vivo conditioning in the presence of (n = 15) and the groups that received either the control compound deacetyl (n = 11) or application delayed by 1 hr (n = 12). Al Paired A2 control 1 hr posttraining ----~~ -\ 1 sec 11 mv B 45 o6 3- -a c 15 co) d), 2 * Paired + (1 hr) * Unpaired + (1 hr) A Anisomycin (1 hr) FIG. 2. Anisomycin does not block short-term enhancement produced by light and 5-HT. (Al) Intracellular recording of a generator potential for an isolated medial B photoreceptor evoked by a 2-min light step (1 x 1o-4 W/cm2) 1 hr after the termination of one-trial in vivo conditioning. The conditioning trial was presented in the presence of 1,uM. (A2) Generator potential evoked by the light step recorded from an isolated medial B photoreceptor 1 hr after the presentation of the light (CS) in the presence of 1,uM. The dashed lines in Al and A2 represent the dark-adapted resting membrane potential. Pretreatment failed to block the enhancement of the generator potential produced by light and 5-HT. (B) Group data showing the mean amplitude ± SM (in mv) of the generator potential evoked by a 2-min light step for the groups that received the CS paired 5-HT in the presence of (n = 1), the unpaired CS and 5-HT in the presence of (n = 11), and only the CS in the presence of (n = 9). The paired and unpaired groups were not significantly different from each other; however, the generator potentials from both groups were significantly larger as compared to the control group (P <.5). The application of 1,uM by itself does not produce a diminished generation potential since the transient peak and plateau phase at light offset is virtually identical to normal controls (n = 1), as indicated by the asterisks at time and 12 sec after light onset. 12

4 Neurobiology: Crow and Forrester Proc. Natl. Acad. Sci. USA 87 (199) 4493 Al Paired delayed 24 hr posttraining B 45 a 3 11mV. A2 Paired -11 mv 1 sec co r_ 15 a) Paired + deacetyl (24 hr) Paired + delayed (24 hr) FIG. 3. Anisomycin blocks long-term enhancement in lateral B photoreceptors produced by one-trial in vivo conditioning. (Al) Intracellular recording of a generator potential from an isolated lateral B photoreceptor evoked by a 2-min light step 24 hr after the termination of one-trial in vivo conditioning. The application of 1,M was delayed 1 hr after the conditioning trial. (A2) Generator potential evoked by the light step recorded from an isolated lateral B photoreceptor 24 hr after the termination of one-trial in vivo conditioning. The conditioning trial was presented in the presence of 1 A±M. The dashed lines in Al and A2 represent the dark-adapted resting membrane potential. Delaying the application of resulted in long-term enhancement of the generator potential while pretreatment blocked long-term enhancement. (B) Group data showing the mean amplitude of the generator potential + SM (in mv) evoked by a 2-min light step recorded 24 hr after the conditioning trial for the groups that received light paired 5-HT in the presence of deacetyl (n = 11), light paired 5-HT delayed 1 hr after one-trial conditioning (n = 12), or light paired 5-HT in the presence of (n = 15). The mean amplitude of the transient peak and plateau phase of the generator potential for an unpaired control group (n = 5) in the presence of is denoted by the asterisks at time and 12 sec from light onset. The plateau phase of the generator potential was significantly enhanced for both the paired deacetyl and paired delayed groups as compared to the group that received the light paired 5-HT or the unpaired light and 5-HT in the presence of (P <.1). The amplitude of the transient peak and plateau phase of the generator potential was not significantly different from the paired and unpaired groups in the presence of. Pretreatment blocked long-term enhancement (plateau phase of the generator potential) recorded from lateral B photoreceptors. The effect of light adaptation on the amplitude of the generator potential was significant for all groups (F24,M = 64.7; P <.1). A separate statistical analysis of generator potential amplitudes for a group that received unpaired light and 5-HT in the presence of (n = 5) and the paired light and 5-HT group (n = 15) in the presence of revealed that there were no significant differences between the two groups. The average peak amplitude and average amplitude of generator potentials at the end of the 2-min light step for the unpaired control group in the presence of is indicated by the asterisks in Fig. 3B. Since previously published results have suggested that a time window must exist for protein synthesis inhibition to be effective in blocking long-term memory (see ref. 1), we examined the effect of delaying application. We found that delaying the application of the by 1 hr resulted in long-term enhancement that was identical to enhancement observed if the in vivo conditioning trial was presented in the presence of the control compound (deacetyl) (see Fig. 3B). Two examples ofgenerator potentials recorded from lateral B photoreceptors in a preparation that received the 1 hr after in vivo conditioning and a preparation that received conditioning in the presence of are shown in Fig. 3A, and the group data are presented in Fig. 3B. These results show that blocks the enhanced plateau phase of the generator potential if it is present during the presentation ofthe conditioning trial; however, inhibition of protein synthesis is ineffective if delayed 1 hr after conditioning. Although the mechanisms for the expression of long-term enhancement are not known, it could be argued that interferes the expression of enhancement and not induction by interfering second-messenger activation. To test this, preparations that received paired light and 5-HT pretreatment (n = 5) were tested at 24 hr to again verify that enhancement was blocked. After the light step, the preparations received an application of5-ht to test if short-term enhancement could be induced. The analysis of the results showed that short-term enhancement could be induced by 5-HT application in preparations where had been shown to block long-term enhancement. This is shown by the group data of average generator potential amplitudes before and after 5-HT application (Fig. 4). Both the peak amplitude (t = 4.14; P <.1) and plateau phase (t = 5.89; P <.25) were enhanced following the application of 5-HT to the same preparations where had blocked long-term enhancement. Our results indicate that inhibiting protein synthesis during the presentation of a one-trial in vivo conditioning procedure is sufficient to block long-term enhancement while leaving short-term enhancement intact. The effects of inhibiting protein synthesis show specificity since a derivative of, deacetyl, which does not inhibit protein synthesis at the concentration used in this study (1 jxm), does not block long-term enhancement. The results indicate that a time 45 a, 3- *. 7 - Cun a 15 Cu a) i S\S~~~~ o Paired + (24 hr) + 5-HT o Paired + (24 hr) FIG HT produces short-term enhancement in lateral B photoreceptors of preparations where long-term enhancement is blocked by. o, Average generator potential amplitude ± SM (in mv) evoked by a 2-min light step 24 hr after one-trial in vivo conditioning (n = 5). The conditioning trial was presented in the presence of 1 1LM. Consistent the data shown in Fig. 3, blocks long-term enhancement. However, the same lateral B photoreceptors can express significant short-term enhancement of the peak (P <.1) and plateau phase of the generator potentials (P <.25) after the application of 5-HT ().

5 4494 Neurobiology: Crow and Forrester window must operate in which inhibiting protein synthesis will be effective in blocking long-term enhancement, since delaying the application of by 1 hr results in normal long-term enhancement. DISCUSSION We have found that inhibiting protein synthesis during onetrial in vivo conditioning blocks long-term enhancement but not short-term enhancement of light-evoked generator potentials recorded from identified B photoreceptors in the eyes of Hermissenda. In previous studies of in vivo conditioning, we showed that long-term enhancement in lateral B photoreceptors is expressed by a decrease in the rate of adaptation and/or accommodation of light-evoked generator potentials, which results in larger amplitudes of the steady-state or plateau phase of the generator potential (13). Our preliminary studies of intact B photoreceptors capable ofgenerating action potentials indicate that the magnitude of enhancement of the amplitude of the plateau phases of generator potentials observed after in vivo conditioning would be sufficient to produce an increase in the frequency of action potentials recorded from intact photoreceptors during light stimulation. An increase in the light-elicited spike discharge rate of B photoreceptors could contribute to the reported suppression of normal phototactic behavior produced by in vivo conditioning (12) since Hermissenda are capable of behaviorally discriminating between light intensities that vary by as little as 1 logarithmic unit of attenuation (22). Our results would indicate that inhibiting protein synthesis during the conditioning experience blocks the induction of events necessary for long-term enhancement, rather than interfering receptor and/or second-messenger mediated steps that may be necessary for maintenance or expression of enhancement. This hypothesis is supported by our results demonstrating that 5-HT application is sufficient to produce short-term enhancement in the same identified photoreceptors where pretreatment blocked long-term enhancement. Moreover, the short-term enhancement that we observed 24 hr after treatment was virtually identical to the enhancement detected at 1 hr (short-term enhancement) following 5-HT application. The results showing that inhibition of protein synthesis after in vivo conditioning is ineffective in blocking long-term enhancement suggests that protein synthesis during or shortly after the conditioning trial is necessary for the formation of an enduring memory of the associative experience. This observation suggests that a critical time window must exist for protein synthesis and is consistent studies in vertebrates where deficits of long-term memory were assessed by changes in behavior (1-5). Our results are consistent the findings of recent studies of a 5-HT-induced short- and long-term facilitation in cultured Aplysia neurons (6). In Aplysia, protein synthesis inhibitors blocked long-term facilitation when given during 5-HT application but not when presented before or after the 5-HT application (6). More recently it was reported that inhibiting protein synthesis blocked both camp-evoked long-term facilitation (23) and long-term behavioral sensitization in the isolated gilldrawal reflex of Aplysia (8). Consistent the results of studies of synaptic and cellular plasticity in invertebrates is the observation that maintenance of long-term potentiation in the rat dentate gyrus requires protein synthesis, which may be completed as soon as 15 min after tetanization (24). As is the case vertebrates, short-term memory for the conditioning experience in Hermissenda appears to be intact following the treatment. Our results showing that inhibiting protein synthesis during and after one-trial in vivo conditioning does not affect the induction or expression of short-term enhancement indicates that the expression of gene Proc. Natl. Acad. Sci. USA 87 (199) products essential for long-term enhancement are not required for short-term enhancement. Long-term enhancement may operate through an independent and parallel signaling system, or alternately, both short-term enhancement and long-term enhancement could operate in series such that some of the same steps important for induction may be common to both. Recent work in Aplysia has shown that short- and long-term facilitation produced by 5-HT or camp (6, 23, 25-27) involves the phosphorylation of the same 17 substrate proteins (9). Although we do not know the cellular signaling systems essential for long-term enhancement, we have evidence that activation of protein kinase C may be an important step for induction of short-term enhancement (14-16). It is not known if the pathways essential for the induction of short- and long-term enhancement are independent; however, recent results have indicated that conditions that are sufficient to block short-term enhancement do not block long-term enhancement, which suggests that different and independent pathways may be involved (15). 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