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Somatic ribosomal changes induced by long-term potentiation of the perforant path-hippocampal CA1 synapses
Brain Research, 619 (1993) 331-333 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00
331
BRES 25762
Short Communications
Somatic ribosomal changes induced by long-term potentiation of the perforant path-hippocampal CA1 synapses J. W e n z e l , N . L D e s m o n d , a n d W . B L e v y Department of Neurological Surgery, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (USA) (Accepted 4 May 1993)
Key words: Hippocampus; Ribosome; Ultrastructure; Synaptic plasticity; Long-term potentiation; Protein synthesis
The present study quantified ribosomes, as an ultrastructural marker of neuronal protein synthesis, following long-term potentiation (LTP) in the hippocampal CA1 region in vitro. Sixty min after LTP-inducing, high-frequency stimulation of the perforant path, the total number of ribosomes, the number of polysomes, and the number of membrane-bound ribosomes increased significantly. These increases are a postsynaptic morphological correlate consistent with enhanced protein synthesis following the induction of LTP in the perforant path-CA1 system.
Protein synthesis is one of several mechanisms hypothesized to be involved in the increased synaptic efficacy known as long-term potentiation (LTP). While the early stages of LTP may be due to changes such as an increase in transmitter release 2 and the activation of protein kinases 13, the maintenance of LTP may involve protein synthesis1'3'6'8-12. Ribosomes, which can serve as an ultrastructural marker for protein synthetic activity 14, may also increase in number with LTP ~5 (but see ref. 7). In the present study we examined somatic ribosomal changes in hippocampal CA1 neurons after the induction of LTP in the perforant path-CA1 system in vitro. The experiments were performed on hippocampal slices prepared from anesthetized male rats (SpragueDawley, 150-200 g) as described previously 5. Following removal of the brain, the right hippocampus was dissected and sectioned transversely at 400 tzm. The slices were transferred to an interface recording chamber maintained at 31-34°C. The perfusion medium consisted of (in mM): NaC1 127, KCI 2, CaCI 2 2.0, MgSO4 1.5, NaHCO 3 26, KH2PO 4 3.1, and glucose 10. This medium was bubbled with 95% 0 2, 5% CO 2 and was delivered at 2 ml/min. To prevent possible seizure propagation from CA3, CA3 was dissected away from the remainder of the slice. The dentate gyrus was
dissected to prevent its potentials from contaminating the perforant path response evoked in CA1 stratum (s.) lacunosum-moleculare. The slices were then incubated for 1-2 h before beginning an experiment. There were two recording and two stimulating electrodes. One recording pipet was placed in CA1 s. radiatum to record Schaffer collateral responses, and another was placed in CA1 s. lacunosum-moleculare to record perforant path responses. One bipolar stimulating electrode was positioned in s. radiatum to activate the Schaffer collaterals. The s. radiatum responses served as a control for the quality and stability of the slices. A second bipolar stimulating electrode was placed in the myelinated fibers at the CAl-subicular border to activate the perforant path. Electrical stimulation consisted of brief (100 /zs), constant current (50-250/xA) square pulses delivered through the bipolar electrodes. Individual responses were tested 1/30 s. The basic experimental paradigm consisted of a preconditioning test period (60 min duration), a brief conditioning period, and a postconditioning test period (60 min duration). Bicuculline methiodide (Sigma, 10 tzM) was bath applied to all slices for the duration of the experiment. LTP was induced by high-frequency tetanic stimulation consisting of three sets of theta-burst trains (1
Correspondence: J. Wenzel, Department of Neurological Surgery, University of Virginia Health Sciences Center, Box 420, Charlottesville, VA 22908, USA. Fax: (1) (804) 982-3829.
332 s e t / 5 s). Each set consisted of 8 trains of 8 pulses at 100 Hz delivered every 200 ms. The intertrain interval was 200 ms. Test and conditioning stimulus intensities were identical. Control slices were unstimulated. (For further details, see ref. 5.) High-frequency stimulation potentiated the perforant path-CA1 population EPSP in all conditioned slices. The mean increase of the population EPSP slope was 66.5% ( _ 36.2%; median, 36%). Four to five hours after preparing the slices (1 h after conditioning), the slices were fixed with a phosphate-buffered mixed aldehyde solution. The slices were postfixed in osmium tetroxide the next day and processed for electron microscopy using conventional procedures. Four potentiated and three control slices were used for quantitative electron microscopy. (A fourth control slice was not analyzed because there was severe somatic shrinkage.) For each slice, a total of 50 / z m 2 of perikaryal cytoplasm from 12-15 neurons was randomly photographed (final magnification, × 90,000). The neurons sampled were located in the middle of the slice and showed no signs of shrinkage or swelling. Five parameters were evaluated p e r /a,m 2 of cytoplasm: (1) total number of ribosomes; (2) number of free ribosomes; (3) number of membrane-bound ribosomes; (4) number of ribosomes in polysomes; and (5) number of membrane-bound ribosomes p e r / z m of endoplasmic reticulum membrane. An average of 6680 +_ 281 ribosomes in 50 # m 2 cytoplasm were counted and classified from each of the 7 slices. The results were statistically evaluated with a multivariate analysis of variance using S P S S / P C ÷ and a two-tailed t-test. Values in the text are mean _+ S.E.M. Ribosomes were quantified in morphologically wellpreserved neurons without swelling or shrinkage of internal structures, i.e. organelles and membranes (see Fig. 1). The ultrastructure of these neurons corresponded well with that seen in vivo ~5. After 4-5 h in vitro, there was no disaggregation of polyribosomes, displacement of membrane-bound ribosomes, or general vacuolization of the endoplasmic reticulum. The total number of ribosomes averaged 118.4 _+ 1.5 ribosomes//zm 2 of cytoplasm in the control slices and 145.0 _ 3.2 ribosomes//zm 2 in the potentiated slices, a significant increase of 22.5% 1 h after the induction of L T P (F3, 3 = 18.4, P = 0.02). The number of free ribosomes did not change significantly with LTP (F~, 5 = 1.08, P = 0.35; see Fig. 2). However, the number of ribosomes in polysomes and of membrane-bound ribosomes both increased significantly with LTP ( F I . 5 = 30.52, P = 0.003; F~.5 = 79.84, P < 0.001, respectively). There was no correlation between the magnitude of
Fig. 1. CA1 neuronal cytoplasm from a potentiated slice incubated for 4 h before fixation, m, m e m b r a n e - b o u n d ribosome; p, polysome; f, free ribosome. Bar = 0.1 ~ m .
~ "
140
Control LTP
:g
12
120
~ so // // // // // //
20 k
Free Polysomes Bound Total Ribosomes Ribosomes Ribosomes
4
Z
Ribosomes
Fig. 2. Changes in the n u m b e r of ribosomes in CA1 neuronal somata 1 h after the induction of LTP in the perforant p a t h - C A 1 synapses. With LTP of the perforant p a t h - C A 1 synapses, the total n u m b e r of ribosomes increases, reflecting significant increases in the n u m b e r of ribosomes in polysomes and m e m b r a n e - b o u n d ribosomes. Values plotted are the mean ( + S . E . M . ) n u m b e r of ribosomes per 1 txm 2 of neuronal cytoplasm or the n u m b e r of ribosomes per 1 /xm of endoplasmic reticulum m e m b r a n e for the experimental (n = 4) and control (n = 3) slices. *, statistically significant differences (see text for values).
333 the electrophysiologically measured LTP and the total number of ribosomes (r = 0.17), the number of ribosomes in polysomes (r = 0.26), or the number of membrane-bound ribosomes (r = 0.35). The number of ribosomes in polysomes increased 22.9% in potentiated slices compared to the controls (see Fig. 2). In general, polysomes contained 4 or 5 ribosomes, and there was no visible change in the structure of the characteristic polysomal rosettes with LTP. We did, however, have the impression that these rosettes occurred more frequently in potentiated slices than in control slices. The number of membrane-bound ribosomes increased 27.8% with LTP (see Fig. 2). The density of these bound ribosomes per length of cross-sectional membrane also increased 19.6%, but this was not significant (see Fig. 2; t = 1.81, df = 5, P > 0.05). There was no significant difference in the length of endoplasmic reticulum membranes//xm 2 cytoplasm between the two groups. These results show significant increases in several ribosomal compartments as well as a trend for an enhanced density of ribosomes on endoplasmic reticulum membranes 1 h following LTP-inducing conditioning stimulation. Although LTP occurs rapidly and probably does not require protein synthesis immediately after tetanization, the synthesis of at least some proteins is necessary for LTP maintenance TM. Because an increased number of ribosomes in the neuronal cytoplasm may accompany enhanced protein synthesis, the present data support the involvement of protein synthesis in LTP maintenance. In addition, these results are consistent with previous reports of ribosomal changes with LTP in other hippocampal systems tS. The increased incidence of ribosomes with LTP in multiple hippocampal systems suggests that similar mechanisms are employed by these different synapses to maintain LTP. A particularly interesting aspect of the present results is that the trigger event for the increase in somatic ribosomes occurs in the most distal apical dendrites. Thus, the trigger for increased protein synthesis must travel down the dendritic tree from the distal perforant path synapses to the soma, a considerable distance (300-500 izm). It would be natural to assume that transient, synaptic depolarization is the trigger event except that such depolarization by the perforant path synapses has quite a weak effect on the CA1 somata 4. A more plausible candidate for this trigger is relatively prolonged (tens of ms) dendritic depolarization because repetitive spiking is rarely seen with the perforant path conditioning stimulation that produces LTP s. Alternatively, some type of chemical trigger, e.g.
localized calcium release from the smooth endoplasmic reticulum, might travel down the dendrite and enable the soma to distinguish a signal generated in s. moleculare from one in s. radiatum. Supported by NIH NS15488 and NIMH MH00622 to W.B.L., NIH NS26645 to N.L.D., and the Department of Neurological Surgery, Dr. John A. Jane, Chairman. The authors thank C.M. Colbert, C.P. Fall, J.M. Vallee and D.X. Zhang for their assistance in various phases of this study.
1 Abraham, C.W., Dragunow, M. and Tate, W.P., The role of immediate early genes in the stabilization of long-term potentiation, Mol. Neurobiol., 5 (1991) 297-314. 2 Bliss, T.V.P., Douglas, R.M., Errington, M.L. and Lynch, M.A., Correlation between long-term potentiation and release of endogenous amino acids from dentate gyrus of anaesthetized rats, J. Physiol., 377 (1986) 392-408. 3 Charriaut-Marlangue, C., Aniksztejn, L., Roisin, M.P. and BenAri, Y., Release of proteins during long-term potentiation in the hippocampus of the anaesthetized rat, Neurosci. Lett., 91 (1988) 308-314. 4 Colbert, C.M. and Levy, W.B, GABA-A inhibition opposes monosynaptic perforant-path excitation of CA1 pyramids, Soc. Neurosci. Abstr., 18 (1992) 1496. 5 Colbert, C.M. and Levy, W.B, Long-term potentiation of perforant path synapses in hippocampal CA1 in vitro, Brain Res., 606 (1993) 87-91. 6 Deadwyler, S.A., Dunwiddie, T. and Lynch, G., A critical level of protein synthesis is required for long-term potentiation, Synapse, 1 (1987) 90-95. 7 Desmond, N.L and Levy, W.B, Morphological correlates of longterm potentiation imply the modification of existing synapses, not synaptogenesis, in the hippocampal dentate gyrus, Synapse, 5 (1990) 139-143. 8 Duffy, C,, Teyler, T.J. and Shashoua, V.E., Long-term potentiation in the hippocampal slice: evidence for stimulated secretion of newly synthesized proteins, Science, 212 (1981) 1148-1151. 9 Fazeli, M.S., Errington, M.L., Dolphin, A.C. and Bliss, T.V.P., Long-term potentiation in the dentate gyrus of the anaesthetized rats is accompanied by an increase in protein efflux into push-pull cannula perfusates, Brain Res., 473 (1988) 51-59. 10 Fifkova, E., Anderson, C.L., Young, S.J. and van Harreveld, A., Effect of anisomycin on stimulation-induced changes in dendritic spines of the dentate granule cells, J. Neurocytol., 11 (1982) 183-210. 11 Otani, S., Marshall, C.J., Tate, W.P., Goddard, G.V. and Abraham, W.C., Maintenance of long-term potentiation in rat dentate gyrus requires protein synthesis but not messenger RNA synthesis immediately post-tetanization, Neuroscience, 28 (1989) 519526. 12 Otani, S., Roisin-Lallemand, M.-P. and Ben-Ari, Y., Enhancement of extracellular protein concentrations during long-term potentiation in rat hippocampal slice, Neuroscience, 47 (1992) 265-272. 13 Reymann, K.G., Postsynaptic mechanisms of hippocampal longterm potentiation. The involvement of glutamate receptors and protein kinases in its late maintenance. In B.S. Meldrum, F. Moroni, R.P. Simon and J.H. Woods (Eds.), Excitatory Amino Acids, Raven, New York, 1991, pp. 475-485. 14 Steward, O. and Reeves, T.M., Protein-synthetic machinery beneath postsynaptic sites on CNS neurons: association between polyribosomes and other organelles at the synaptic site, J. Neurosci., 8 (1988) 176-184. 15 Wenzel, J. and Matthies, H., Morphological changes in the hippocampal formation accompanying memory formation and longterm potentiation. In N.M. Weinberger, J.L. McGaugh and G. Lynch (Eds.), Memory Systems of the Brain, Guilford Press, New York, 1985, pp. 150-170.
331
BRES 25762
Short Communications
Somatic ribosomal changes induced by long-term potentiation of the perforant path-hippocampal CA1 synapses J. W e n z e l , N . L D e s m o n d , a n d W . B L e v y Department of Neurological Surgery, University of Virginia Health Sciences Center, Charlottesville, VA 22908 (USA) (Accepted 4 May 1993)
Key words: Hippocampus; Ribosome; Ultrastructure; Synaptic plasticity; Long-term potentiation; Protein synthesis
The present study quantified ribosomes, as an ultrastructural marker of neuronal protein synthesis, following long-term potentiation (LTP) in the hippocampal CA1 region in vitro. Sixty min after LTP-inducing, high-frequency stimulation of the perforant path, the total number of ribosomes, the number of polysomes, and the number of membrane-bound ribosomes increased significantly. These increases are a postsynaptic morphological correlate consistent with enhanced protein synthesis following the induction of LTP in the perforant path-CA1 system.
Protein synthesis is one of several mechanisms hypothesized to be involved in the increased synaptic efficacy known as long-term potentiation (LTP). While the early stages of LTP may be due to changes such as an increase in transmitter release 2 and the activation of protein kinases 13, the maintenance of LTP may involve protein synthesis1'3'6'8-12. Ribosomes, which can serve as an ultrastructural marker for protein synthetic activity 14, may also increase in number with LTP ~5 (but see ref. 7). In the present study we examined somatic ribosomal changes in hippocampal CA1 neurons after the induction of LTP in the perforant path-CA1 system in vitro. The experiments were performed on hippocampal slices prepared from anesthetized male rats (SpragueDawley, 150-200 g) as described previously 5. Following removal of the brain, the right hippocampus was dissected and sectioned transversely at 400 tzm. The slices were transferred to an interface recording chamber maintained at 31-34°C. The perfusion medium consisted of (in mM): NaC1 127, KCI 2, CaCI 2 2.0, MgSO4 1.5, NaHCO 3 26, KH2PO 4 3.1, and glucose 10. This medium was bubbled with 95% 0 2, 5% CO 2 and was delivered at 2 ml/min. To prevent possible seizure propagation from CA3, CA3 was dissected away from the remainder of the slice. The dentate gyrus was
dissected to prevent its potentials from contaminating the perforant path response evoked in CA1 stratum (s.) lacunosum-moleculare. The slices were then incubated for 1-2 h before beginning an experiment. There were two recording and two stimulating electrodes. One recording pipet was placed in CA1 s. radiatum to record Schaffer collateral responses, and another was placed in CA1 s. lacunosum-moleculare to record perforant path responses. One bipolar stimulating electrode was positioned in s. radiatum to activate the Schaffer collaterals. The s. radiatum responses served as a control for the quality and stability of the slices. A second bipolar stimulating electrode was placed in the myelinated fibers at the CAl-subicular border to activate the perforant path. Electrical stimulation consisted of brief (100 /zs), constant current (50-250/xA) square pulses delivered through the bipolar electrodes. Individual responses were tested 1/30 s. The basic experimental paradigm consisted of a preconditioning test period (60 min duration), a brief conditioning period, and a postconditioning test period (60 min duration). Bicuculline methiodide (Sigma, 10 tzM) was bath applied to all slices for the duration of the experiment. LTP was induced by high-frequency tetanic stimulation consisting of three sets of theta-burst trains (1
Correspondence: J. Wenzel, Department of Neurological Surgery, University of Virginia Health Sciences Center, Box 420, Charlottesville, VA 22908, USA. Fax: (1) (804) 982-3829.
332 s e t / 5 s). Each set consisted of 8 trains of 8 pulses at 100 Hz delivered every 200 ms. The intertrain interval was 200 ms. Test and conditioning stimulus intensities were identical. Control slices were unstimulated. (For further details, see ref. 5.) High-frequency stimulation potentiated the perforant path-CA1 population EPSP in all conditioned slices. The mean increase of the population EPSP slope was 66.5% ( _ 36.2%; median, 36%). Four to five hours after preparing the slices (1 h after conditioning), the slices were fixed with a phosphate-buffered mixed aldehyde solution. The slices were postfixed in osmium tetroxide the next day and processed for electron microscopy using conventional procedures. Four potentiated and three control slices were used for quantitative electron microscopy. (A fourth control slice was not analyzed because there was severe somatic shrinkage.) For each slice, a total of 50 / z m 2 of perikaryal cytoplasm from 12-15 neurons was randomly photographed (final magnification, × 90,000). The neurons sampled were located in the middle of the slice and showed no signs of shrinkage or swelling. Five parameters were evaluated p e r /a,m 2 of cytoplasm: (1) total number of ribosomes; (2) number of free ribosomes; (3) number of membrane-bound ribosomes; (4) number of ribosomes in polysomes; and (5) number of membrane-bound ribosomes p e r / z m of endoplasmic reticulum membrane. An average of 6680 +_ 281 ribosomes in 50 # m 2 cytoplasm were counted and classified from each of the 7 slices. The results were statistically evaluated with a multivariate analysis of variance using S P S S / P C ÷ and a two-tailed t-test. Values in the text are mean _+ S.E.M. Ribosomes were quantified in morphologically wellpreserved neurons without swelling or shrinkage of internal structures, i.e. organelles and membranes (see Fig. 1). The ultrastructure of these neurons corresponded well with that seen in vivo ~5. After 4-5 h in vitro, there was no disaggregation of polyribosomes, displacement of membrane-bound ribosomes, or general vacuolization of the endoplasmic reticulum. The total number of ribosomes averaged 118.4 _+ 1.5 ribosomes//zm 2 of cytoplasm in the control slices and 145.0 _ 3.2 ribosomes//zm 2 in the potentiated slices, a significant increase of 22.5% 1 h after the induction of L T P (F3, 3 = 18.4, P = 0.02). The number of free ribosomes did not change significantly with LTP (F~, 5 = 1.08, P = 0.35; see Fig. 2). However, the number of ribosomes in polysomes and of membrane-bound ribosomes both increased significantly with LTP ( F I . 5 = 30.52, P = 0.003; F~.5 = 79.84, P < 0.001, respectively). There was no correlation between the magnitude of
Fig. 1. CA1 neuronal cytoplasm from a potentiated slice incubated for 4 h before fixation, m, m e m b r a n e - b o u n d ribosome; p, polysome; f, free ribosome. Bar = 0.1 ~ m .
~ "
140
Control LTP
:g
12
120
~ so // // // // // //
20 k
Free Polysomes Bound Total Ribosomes Ribosomes Ribosomes
4
Z
Ribosomes
Fig. 2. Changes in the n u m b e r of ribosomes in CA1 neuronal somata 1 h after the induction of LTP in the perforant p a t h - C A 1 synapses. With LTP of the perforant p a t h - C A 1 synapses, the total n u m b e r of ribosomes increases, reflecting significant increases in the n u m b e r of ribosomes in polysomes and m e m b r a n e - b o u n d ribosomes. Values plotted are the mean ( + S . E . M . ) n u m b e r of ribosomes per 1 txm 2 of neuronal cytoplasm or the n u m b e r of ribosomes per 1 /xm of endoplasmic reticulum m e m b r a n e for the experimental (n = 4) and control (n = 3) slices. *, statistically significant differences (see text for values).
333 the electrophysiologically measured LTP and the total number of ribosomes (r = 0.17), the number of ribosomes in polysomes (r = 0.26), or the number of membrane-bound ribosomes (r = 0.35). The number of ribosomes in polysomes increased 22.9% in potentiated slices compared to the controls (see Fig. 2). In general, polysomes contained 4 or 5 ribosomes, and there was no visible change in the structure of the characteristic polysomal rosettes with LTP. We did, however, have the impression that these rosettes occurred more frequently in potentiated slices than in control slices. The number of membrane-bound ribosomes increased 27.8% with LTP (see Fig. 2). The density of these bound ribosomes per length of cross-sectional membrane also increased 19.6%, but this was not significant (see Fig. 2; t = 1.81, df = 5, P > 0.05). There was no significant difference in the length of endoplasmic reticulum membranes//xm 2 cytoplasm between the two groups. These results show significant increases in several ribosomal compartments as well as a trend for an enhanced density of ribosomes on endoplasmic reticulum membranes 1 h following LTP-inducing conditioning stimulation. Although LTP occurs rapidly and probably does not require protein synthesis immediately after tetanization, the synthesis of at least some proteins is necessary for LTP maintenance TM. Because an increased number of ribosomes in the neuronal cytoplasm may accompany enhanced protein synthesis, the present data support the involvement of protein synthesis in LTP maintenance. In addition, these results are consistent with previous reports of ribosomal changes with LTP in other hippocampal systems tS. The increased incidence of ribosomes with LTP in multiple hippocampal systems suggests that similar mechanisms are employed by these different synapses to maintain LTP. A particularly interesting aspect of the present results is that the trigger event for the increase in somatic ribosomes occurs in the most distal apical dendrites. Thus, the trigger for increased protein synthesis must travel down the dendritic tree from the distal perforant path synapses to the soma, a considerable distance (300-500 izm). It would be natural to assume that transient, synaptic depolarization is the trigger event except that such depolarization by the perforant path synapses has quite a weak effect on the CA1 somata 4. A more plausible candidate for this trigger is relatively prolonged (tens of ms) dendritic depolarization because repetitive spiking is rarely seen with the perforant path conditioning stimulation that produces LTP s. Alternatively, some type of chemical trigger, e.g.
localized calcium release from the smooth endoplasmic reticulum, might travel down the dendrite and enable the soma to distinguish a signal generated in s. moleculare from one in s. radiatum. Supported by NIH NS15488 and NIMH MH00622 to W.B.L., NIH NS26645 to N.L.D., and the Department of Neurological Surgery, Dr. John A. Jane, Chairman. The authors thank C.M. Colbert, C.P. Fall, J.M. Vallee and D.X. Zhang for their assistance in various phases of this study.
1 Abraham, C.W., Dragunow, M. and Tate, W.P., The role of immediate early genes in the stabilization of long-term potentiation, Mol. Neurobiol., 5 (1991) 297-314. 2 Bliss, T.V.P., Douglas, R.M., Errington, M.L. and Lynch, M.A., Correlation between long-term potentiation and release of endogenous amino acids from dentate gyrus of anaesthetized rats, J. Physiol., 377 (1986) 392-408. 3 Charriaut-Marlangue, C., Aniksztejn, L., Roisin, M.P. and BenAri, Y., Release of proteins during long-term potentiation in the hippocampus of the anaesthetized rat, Neurosci. Lett., 91 (1988) 308-314. 4 Colbert, C.M. and Levy, W.B, GABA-A inhibition opposes monosynaptic perforant-path excitation of CA1 pyramids, Soc. Neurosci. Abstr., 18 (1992) 1496. 5 Colbert, C.M. and Levy, W.B, Long-term potentiation of perforant path synapses in hippocampal CA1 in vitro, Brain Res., 606 (1993) 87-91. 6 Deadwyler, S.A., Dunwiddie, T. and Lynch, G., A critical level of protein synthesis is required for long-term potentiation, Synapse, 1 (1987) 90-95. 7 Desmond, N.L and Levy, W.B, Morphological correlates of longterm potentiation imply the modification of existing synapses, not synaptogenesis, in the hippocampal dentate gyrus, Synapse, 5 (1990) 139-143. 8 Duffy, C,, Teyler, T.J. and Shashoua, V.E., Long-term potentiation in the hippocampal slice: evidence for stimulated secretion of newly synthesized proteins, Science, 212 (1981) 1148-1151. 9 Fazeli, M.S., Errington, M.L., Dolphin, A.C. and Bliss, T.V.P., Long-term potentiation in the dentate gyrus of the anaesthetized rats is accompanied by an increase in protein efflux into push-pull cannula perfusates, Brain Res., 473 (1988) 51-59. 10 Fifkova, E., Anderson, C.L., Young, S.J. and van Harreveld, A., Effect of anisomycin on stimulation-induced changes in dendritic spines of the dentate granule cells, J. Neurocytol., 11 (1982) 183-210. 11 Otani, S., Marshall, C.J., Tate, W.P., Goddard, G.V. and Abraham, W.C., Maintenance of long-term potentiation in rat dentate gyrus requires protein synthesis but not messenger RNA synthesis immediately post-tetanization, Neuroscience, 28 (1989) 519526. 12 Otani, S., Roisin-Lallemand, M.-P. and Ben-Ari, Y., Enhancement of extracellular protein concentrations during long-term potentiation in rat hippocampal slice, Neuroscience, 47 (1992) 265-272. 13 Reymann, K.G., Postsynaptic mechanisms of hippocampal longterm potentiation. The involvement of glutamate receptors and protein kinases in its late maintenance. In B.S. Meldrum, F. Moroni, R.P. Simon and J.H. Woods (Eds.), Excitatory Amino Acids, Raven, New York, 1991, pp. 475-485. 14 Steward, O. and Reeves, T.M., Protein-synthetic machinery beneath postsynaptic sites on CNS neurons: association between polyribosomes and other organelles at the synaptic site, J. Neurosci., 8 (1988) 176-184. 15 Wenzel, J. and Matthies, H., Morphological changes in the hippocampal formation accompanying memory formation and longterm potentiation. In N.M. Weinberger, J.L. McGaugh and G. Lynch (Eds.), Memory Systems of the Brain, Guilford Press, New York, 1985, pp. 150-170.