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Redistribution of synaptic vesicles during long-term potentiation in the hippocampus
Brain Research, 41)1 (1987) 4/11 406 Elsevier
401
BRE 21987
Redistribution of synaptic vesicles during long-term potentiation in the hippocampus Michael D. Applegate, Douglas S. Kerr and Philip W. Landfield Department of Physiology and Pharmacology, Bo w'rnan Gray School of Medicine, Winston-Salem. NC 2 7103 ( U. S. A. )
(Accepted 9 September 1986) Key words. Synaptic vesicle; Long-term potentiation: Hippocampus; Dendritic spine; Presynaptic: Stcrcology
Synaptic vesicles were quantified 20 min after the induction of long-term potcntiation (LTP) in the Schatfer-commissural system of the hippocampus. With LTP, significant increases were found in vesicles attached to the active zone membrane, and in the percentage of vesicles adjacent to the active zone. In addition, as others have reported, overall vesicle density was decreascd and spine area was increased. These results suggest that an increased probability of vesicle release may contribute to brain LTP.
Following brief bursts of high frequency stimulation of m a m m a l i a n h i p p o c a m p a l pathways a remarkably long-duration (e.g. hours-to-days) increase in synaptic efficacy occurs, which is usually termed 'long-lasting' or qong-term' potentiation (LTP) s. Since persistent synaptic changes clearly might function as biological substrates of m e m o r y , the mechanisms and sites of such central LTP have generated considerable research interest. H i p p o c a m p a l LTP is associated with increased synaptic conductance 5, but the precise pre- or postsynaptic locus of LTP is still unknown. A number of studies have suggested that the primary site of LTP is postsynaptic (see reviews in refs, 7 and 23), and several postsynaptic ultrastructural correlates of LTP have been described, among which are altered dendritic spine volume or shape 1215"22, increased concave spines ~2, and increased dendritic shaft synapses ~,z2. Nevertheless, there is also some evidence of a presynaptic contribution to h i p p o c a m p a l L T P 27" ~ and nearly all well-studied forms of potentiation in peripheral systems, including LTP in the crayfish 6, have been found to be presynaptic (e.g. ref. 24). Although the specific relations of synaptic vesicles
to transmitter quanta are still unresolved (see reviews in refs. l0 and 30), recent studies employing rapid freeze techniques provide extremely strong support for the view that vesicles participate in the release of packets of transmitter at the neuromuscular junction (NMJ) and may be identical to quanta t3'lo. However, only a handful of studies have examined synaptic vesicle alterations in response to repetitive monosynaptic stimulation in a vertebrate CNS p a t h w a y 11"15'21,25. Of two such studies on brain LTP, one found a decrease in overall vesicle density ~5, whereas the other found no change in density in a limited region adjacent to the center of the active zone I 1. In addition to overall vesicle density changes, repetitive stimulation in peripheral systems alters the intraterminal distribution of vesicles w'17"27 but such
redistribution has not been studied in the m a m m a l i a n CNS. Further, the time course of peripheral posttetanic potentiation has been correlated with an increase in the fraction of vesicles near to or in contact with the active zone ( p a r a m e m b r a n o u s density) 27. Thus, if presynaptic changes in transmitter release are important in hippocampal LTP, such changes might be
Correspondence: P.W. Landfield, Bowman Gray School of Medicine, Department of Physiology and Pharmacology, 300 South Hawthorne Road, Winston-Salem, NC 27103, U.S.A.
0(1(16-8993/87/$03.50 © 1987 Elsevier Science Publishers B,V. (Biomedical Division)
402 partly reflected in intraterminal vesicle distribution. The results of the present study have been described in preliminary form 3. Ten male, young-mature (3-5 months old), Fischer 344 rats were divided into implanted control (sham) and repetitive stimulated (LTP) groups of 5 animals each. Surgical and electrode placement procedures have been described in detail in previous reports >'e~. Briefly, after a recording microelectrode had been placed in the CA 1 cell somal layer (stratuln pyramidale), a stimulating electrode was lowered into the Schaffer collateral-commissural fibers of field CA3 which project to the apical dendrites ol CA129. The recording microelectrode was not placed directly in the synaptic terminal fields (stratum radiaturn), in order to avoid tissue damage to regions that were used for ultrastructural analysis, and since both the population spike and the {inverted) population EPSP can be recorded from the stratum pyramidale ~. After the monosynaptic Schaffer-commissural response was obtained, according to established physiological and stereotaxic criteria t,2°, the stimulation intensity was set at 150% of threshold for the population spike (an extracellular summation of action potentials; cf. refs. 1 and 2, and Fig. 1) and LTP animals were subjected to 5, one-second bursts of 100 Hz stimulation (5-s intervals between bursts). The development of LTP thereafter was monitored by stimulating at the same intensity at 0.2 Hz for 20 min. The 20-rain period is well beyond that at which previously defined forms of short-term potentiation can be observed (e.g. ref. 24) and the LTP at 20 rain was maximal, showing no evidence of the onset of a decay. For the spike, the mean increase over control levels was 75% (_+ 28cA S.E.M.) whereas field EPSP initial slope (measured between EPSP onset and 1 ms after onset) was increased by an average of 27~ (_+ 6~i S.E.M.). Twenty minutes following the repetitive stimulation trains, animals were sacrificed by intracardial perfusion for 15 min with a mixed aldehyde solution (2% glutaraldehyde/2% paraformaldehyde; 0.15 M sodium cacodylate buffer). Sham controls were subjected to the same procedures, but were not given the five 1-s bursts of 100 Hz stimulation. No attempts were made to equate total stimulation pulses given to controls (at 0.2 Hz), since this would have required that the controls be maintained under anesthesia for a considerably longer
period than the LTP animals, and since we have previously found that stimulation frequency is a substantially more significant factor in vesicle distribution than is total pulses a. After perfusion, brains were removed and stored in cold fixative overnight, and were serially sectioned into 251)-itm sections (vibratome) until the electrode tracks were located. The tissue was then trimmed to yield a block containing the apical dendrites of the CA I pyramidal cells adjacent to the recording electrode, and was processed according to standard EM procedures. Twenty-five micrographs were shot on 3 ultrathin sections from each animal, in a programmed sequence through a strictly defined region of the CA 1 stratum radiatum (e.g. the midpoint between the CA~ pyramidal cell layer and the perforant path) at a point of densest Schaffer collateral-commissural termination >. Micrographs were photographically enlarged from an initial magnification of ×25,000 to ×70,000 for quantitative analysis. Approximately 10(1 synapses, and 351Xt vesicles were analyzed for each of the 10 animals. Synaptic terminals and associaled dendritic spines were analyzed using a digitizing pad and a computerized morphometric analysis system (Bioquant) to obtain area and perimeter measurements. Each synaptic complex fully included on the micrographs was analyzed for the length of the active zone, the total presynaptic terminal area, and the terminal area within 150 nm of the active zone. Each spine head associated with a quantified terminal was analyzed for area and perimeter (the spine neck was infrequently present on the micrographs and was therefore excluded from the analysis). Synaptic vesicles were counted m the whole terminal (total vesicles: TV), and in the area within 150 nm of any point of the active zone (local vesicles: LV) Ive~. The distant vesicles tDV) were derived from the other counts (TV-LV). Vesicles in direct contact with the presynaptic membrane of the active zone {attached vesicles: AV) were also counted. The LV, DV and TV were converted to densities (LVD, DVD and TVD) by dividing vesicle counts by relevant areas, and the AV were corrected by length of the active zone. The size of the vesicles can substantially affect density analyses, yet, with few exceptions, prior quantitative studies of vesicles have not considered
403 possible vesicle diameter changes during stimulation.
of the total) and included these values in a stereologi-
In the present study, therefore, we measured the
cal formula for estimating synaptic vesicle density in a volume is'31
diameters of a randomly selected sample (grid overlay method) of synaptic vesicles (approximately 5%
,/
L__
Fig. 1. A, B: examples of electrophysiological responses from the same animal before (A) and 20 rain after (13) LTP stimulation paradigm. Both the population spike and the EPSP slope were increased after stimulation. Calibration: 2 mV, 5 ms. C. D: micrographs of synapses from a control animal (C) and from an LTP animal (D) showing an overall depletion and shift of remaining vesicles toward the active zone during LTP. ×70,000.
404
NA N V =
((4/~).d) + t - 2 h
2500 A.
B.
C.
where Nv is n u m b e r per volume, N A is n u m b e r pet area, d is the average measured diameter, t is section thickness (60 nm, silver-gray interference patterns) and h is the height of invisible fragments (determined from the formula r 2 = ( r - h ) 2 + k 2, where k is the estimated radius of invisible fragments, which was measured as 7 n m in this study). This formula corrects for section thickness, vesicle diameter, and missing or invisible caps Is. We did not use corrections for shape or
ol_
TVD
-D
1 LVD
1 DVD
-E
100~
7 I)~
F
/
200J
I
150! i
for polydispersion31 because of the close approximation of vesicles to spheres, and because the coefficient of variation for vesicle diameters was well below the 20% cut-off at which it is thought necessary
E 50-
~
l il
to correct for polydispersion (e.g. ref. 31). All analyses were performed 'blind' on coded micrographs, and all parameters were averaged across terminals to provide a single value per animal. The statistical population for each variable therefore was n u m b e r of animals. Comparisons between LTP and control animals were made using two-tailed t-tests. We found that animals in which LTP was induced showed declines in both the LVD (P < 0.005) and the D V D (P < 0.005) (and consequently, the T V D (P < 0.005) (Fig. 2). However, the density of vesicles within 150 nm of the active zone (LVD) was proportionately greater than in the distant area ( D V D ) , resulting in a significant increase in the L V D / D V D ratio for LTP animals (P < 0.025) (Fig. 2F). This suggests that there is a migration of the remaining vesicles toward the synaptic active zone during LTP. In addition, the n u m b e r of vesicles attached to the presynaptic m e m b r a n e of the active zone (AV) increased significantly (P < 0.025) in LTP animals (Fig. 2D). Further, if the AV are viewed as a component of the LV, then the fraction of local vesicles attached to the active zone m e m b r a n e (AV/LV) exhibited a highly significant increase (P < 0.005) in LTP animals (Fig. 2El. Examples of these changes are seen in Fig. l, although, of course, not all synapses in this region are equally affected by the stimulation paradigm. Both average area and perimeter per spine were significantly greater (P < 0.025 and P < 0.05 respectively) in the LTP group, while terminal area, perimeter or shape factor were not significantly changed
AV AV LV% LVD DVDo Fig. 2. Synaptic vesicle densities and rattos. A-C show that the decrease m total vesicle density (TVD) during LTP was influenced by decreases in both the local (LVDj and the distant (DVD) vesicle densities. Attached vesicles (AV) are expressed as vesicles per length of active zone and exhibited a significant increase during LTP (D). This increase, in conlunctton with the LVD decrease, resulted in a highly significant increase of the AV/LV ratio (E). The LVD/DVD ratio was also significantly increased (F) in the LTP group, indicating that a greater proportion of the remaining vesicles were situated close to the active zone. All data are expressed as means ~_:S.E.M. TABLE 1 Synaptic ultrastructural variables after L ]!P induction
Summary of quantitative ultrastructural variables, other than vesicle densities. Shape factor was computed according to the formula (4.marea)/(perimeter 2) which gives a value of 1 when the profile is a circle and a value of 0 when the profile is a straight line. See table for all other units (area and perimeter units are per terminal or per spine). Values are expressed as means _+S.E.M. Control
Spine area (urnz) 0.114 + 0.0114 Spine perimeter (um) 1.357_+0.032 Terminal area (jtm2) 0. 181 _+0.008 Terminal perimeter (,urn) 1.8116_+0.022 Vesicle diameter (nm) 41.8611 ± (}.220 Synaptic density length ~um) 0.275 ± 0.0% Terminal shape factor 0.707_+0.023
L TP
P
0. t38 _+0.007 0.02 1.511 _+0.050 0.[)3 0.176+_0.011 n.s. 1.789+0.061
n.s.
42.7ll] +_0.520 n.s. U.270 +_0.078 n.s. [).683 _+0.006 n.s.
405 (Table 1). Vesicle diameter and the length of the active zone were also not significantly different between the control and LTP groups (Table I). The present study replicates earlier reports of an increase in spine volume ~:'~5, and a decrease in total vesicle density ~5 following repetitive hippocampal stimulation (either shown or presumed to induce LTP). In addition, however, the present study shows that a redistribution of synaptic vesicles within the terminal occurs during LTP. Although electrophysiological recordings showed that the stimulation used in this paradigm induced LTP, results from such studies are correlational, and of course cannot demonstrate that the observed ultrastructural changes are the basis of LTP. Nevertheless, the observed increase in attached vesicles, and in the proportion of vesicles adjacent to the active zone, are indicants of vesicle shifts that, under current concepts of synaptic function 131617'2527, would be anticipated to contribute to increased synaptic efficacy. Moreover, increased vesicle attachment could be a basis for the previously observed increases in glutamate release ~4. Thus, our findings provide ultrastructural evidence that presynaptic factors mav contribute to LTP (quite possibly in combination with postsynaptic factors). Recent studies also examined vesicle changes in relation to hippocampal frequency potentiation (FP; increased hippocampal synaptic efficacy during repetitive stimulation1). The present results for LTP were similar in two respects to those for FP, in that an increase of the LVD/DVD ratio and of total AV were seen in both types of potentiation 4. However, there were also two notable differences, since during LTP, total vesicle density was decreased, whereas during FP it was not. and in fact the DVD and LVD were both increased in FP. In addition, the AV/LV ratio was increased during LTP (Fig. 2E), but not during Fp ~. In peripheral systems, vesicles adjacent to the active zone appear to be more available for release, and a shift in vesicles toward the active zone is correlated with greater release m.13.17.27. By analogy, then,
the data suggest that hippocampal frequency potentiation is associated with an increase in more readily releasable (proximal) vesicles (increased LVD), and a small increase in vesicle release probability (increased LVD/DVD) 4, whereas LTP appears to be associated with a decrease in readily releasable vesicles (decreased LVD), and a greater increase in vesicle release probability (an increase of both AV/LV and LVD/DVD). Whether the above vesicle ratios and LVD are related to the quantal binomial parameters p and n respectively, is of course highly problematic, since the association of vesicles with quanta is itself still somewhat controversial L<3{~. Moreover, several workers have found that the binomial parameter n correlates more closely with number of active zones than with number of vesicles, at least in some systems tg'>. On the other hand, it has been shown that binomial analyses can be flawed by major effects of non-stationarity and non-uniformity of p~,_,s, and therefore, reported estimates of n may not be accurate in all studies. Consequently, while it is obviously not possible at this point to propose that vesicle distribution ratios are relevant to binomial parameters, we suggest that viewing these ratios as indicants of the probability of vesicle release may provide insights into the implications of dynamic vesicle shifts for brain synaptic physiology. In summary, the present data show that presynaptic morphological alterations which could increase synaptic efficacy are found during LTP. Since others have shown that potentially relevant postsynaptic changes also occur lL1215'22, the evidence suggests that structural changes on both sides of the synapse may play a role in the induction and/or maintenance of LTP.
We appreciate the valuable and extensive technical assistance of S. Vinsant and L. Cadwallader and the excellent assistance of D. Lefler in preparing the manuscript. This research was supported in part by Grant AG 04542 from the NIH.
406 1 Andersen, P., Organization of hippocampal neurons and their interconnections. In R.L. Isaacson and K.H. Pribram (Eds.), The Hippocampus, Vol. 1, Plenum, New York, 1975, pp. 155-175. 2 Andersen, P., Avoli, M. and Hvalby, O., Evidence for both pre- and postsynaptic mechanisms during long-term potentiation in hippocampal slices. Exp. Brain Res. (Suppl. i, 9 (1984) 315-324. 3 Applegate, M.D., Kerr, D.S. and Landfield, P.W., Hippocampal synaptic vesicle density and spine alterations during long-term potentiation. Soc. Neurosci, Abstr., 12 11986) 506. 4 Applegate, M.D. and Landfield, P.W., Vesicle mobilization and depletion during frequency potentiation and depression in the hippocampus of aged and young rats, Soc. Neurosci. Abstr., 11 (1985) 895. 5 Barrionuevo, G., Kelso, S.R., Johnston, D. and Brown, T.H., Conductance mechanism responsible for long-term potentiation in monosynaptic and isolated excitatory synaptic inputs to hippocampus, J. Neurophysiol., 55 (19861 540-550. 6 Baxter, D.A., Bittner, G.D. and Brown, T.H., Quantal mechanism of long-term synaptic potentiation, Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 5978-5982. 7 Bliss, T.V.P. and Dolphin, A.C., Where is the locus of long-term potentiation?. In G. Lynch, J.L. McGaugh and N.M. Weinberger (Eds.), Neurobiology of Learning and Memory, Plenum, New York, 1984, pp. 451-458. 8 Bliss, T.V.P. and Lomo, T., Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path, J. Physiol. (London), 232 (1973) 331-356, 9 Brown, T.H., Perkel, D.H. and Feldman, M.W., Evoked neurotransmitter release: statistical effects of nonuniformity and nonstationarity, Proc. Natl. Acad. Sci. U.S.A., 73 11976) 2913-2917. 10 Ceccarelli, B. and Hurlbut, W.P., Vesicle hypothesis of the release of quanta of acetylcholine, Physiol. Rev., 60 (19801 396-441. 11 Chang, F.-L.F. and Greenough, W.T., Transient and enduring morphological correlates of synaptic activity and efficacy change in the rat hippocampal slice, Brain Research, 309 (1984) 35-46. 12 Desmond, N.L. and Levy, W.B., Synaptic associative potentiation/depression: an ultrastructural study in the hippocampus, Brain Research, 265 (1983) 21-30. 13 Dickinson-Nelson, A. and Reese, T.S., Structural changes during transmitter release at synapses in the frog sympathetic ganglion, J. Neurosci., 3 (1983) 42-52. 14 Dolphin, A.C., Errington, M.D. and Bliss, T.V.P.. Longterm potentiation of the perforant path in vivo is associated with increased glutamate release, Nature (London), 297 (1982) 496-498. 15 Fifkova, E. and van Harreveld, A., Long-lasting morphological changes in dendritic spines of dentate granular cells following stimulation of the entorhinal area, J. Neuroo, tol.. 6 (1977) 211-2311.
16 Haimann, C., Torri-Tarelli, F., Fesce, R, and Ceccarelli, B., Measurement of quantat secretion induced by ouabain and its correlation with depletion of synaptic vesicles, J. Cell Biol., I01 (19851 1953-1965. 17 Heuser, J.E., Reese, T.S, and Landis D.M.D., Functional changes in frog neuromuscular junctions studied with freeze-fracture, J. Neurocytol., 3 119741 1119--131 18 Konigsmark, B.W., Methods for the counting of neurons, In W, Nauta and S.O.E. Ebbesson (Eds.). Contempora O' Research Methods in Neuroanatomv, Springer, New York, 19711, pp. 315-340. 19 Korn, H.. Triller, A., Mallet, A. and Faber. D.S., Flucluating responses at a central synapse: n of binomial fit predicts number of stained presynaptic boutons, Science, 213 ( 19811 898-900. 2(1 Landfield. P,W., McGaugh, J.L. and l,ynch. G.. Impaired synaptic potentiation in the hippocampus of aged. memorydeficient rats, Brain Research, 150 ( 10781 85-101. 21 Landfield, P.W., Wurtz, C. and Lindsey, J.D., Quantification of synaptic vesicles in hippocampus of aging rats and initial studies of possible relations to neurophysiology. Brain Res. Bull., 4 (19791 757-763. 22 Lee. K.S., Schottler, F., Oliver, M and Lynch. G.. Brief bursts of high frequency stimulation produce two types of structural change in rat hippocampus, ,L Neurophysiol,, 44 ( 19801 247-248. 23 Lynch, G. and Baudry, M., The biochemistry of memory: a new and specific hypothesis, Sciem'e, 224 (19841 1057-1063. 24 Martin, A.R., Presynaptic mechanisms. In ,I.M, Brookhart and V.B. Mountcastle (Eds.), Handbook of Physiology. 1. ]'he Nervous System, American Physiological Society, Bethesda, 1977. pp. 329-355. 25 Model, P.G., Highstein, S.M. and Bennett, M.V.L.. Depletion of vesicles and fatigue of transmission at a vertebrate central synapse, Brain Research, 98 (1975) 2119-228. 26 Neale, E.A.. Nelson, P.G., MacDonald, R.L., Christian. C.N. and Bowers, L.M., Synaptic interactions between mammalian central neurons in cell cutmre. III. Morphophysiological correlates of quantal synaptic transmission, ,1. Neurophysiol., 49 (1983) 1459-1468, 27 Quilliam. J.P, and Tamarind, D.I,. l,ocal vesicle populalions in rat superior cervical ganglia and the vesicle hypothesis, J. Neurocytol., 2 (1973) 59-75 28 Smith, D.O., Variable activation oi synaptic release sites at the neuromuscular junction, Exp Neurol., 8(1 (1983) 520-528. 29 Swanson, L.W., Wyss, J.M. and Cowan, W.M.. An autoradiographic study of the organization of intrahippocampal association pathways in the rat, J. Comp. Neurol., 181 (1978) 681-716. 3/) Taut. 1,.. Nonvesicular release ot neurotransmitter, Physiol. Rev., 62 (1982) 857-893. 31 Weibel, E,R., Stereological methoda, Vol. l, Practical Methods for Biological Morphometrv, Academic Press, New York, 1979, pp. 232.
401
BRE 21987
Redistribution of synaptic vesicles during long-term potentiation in the hippocampus Michael D. Applegate, Douglas S. Kerr and Philip W. Landfield Department of Physiology and Pharmacology, Bo w'rnan Gray School of Medicine, Winston-Salem. NC 2 7103 ( U. S. A. )
(Accepted 9 September 1986) Key words. Synaptic vesicle; Long-term potentiation: Hippocampus; Dendritic spine; Presynaptic: Stcrcology
Synaptic vesicles were quantified 20 min after the induction of long-term potcntiation (LTP) in the Schatfer-commissural system of the hippocampus. With LTP, significant increases were found in vesicles attached to the active zone membrane, and in the percentage of vesicles adjacent to the active zone. In addition, as others have reported, overall vesicle density was decreascd and spine area was increased. These results suggest that an increased probability of vesicle release may contribute to brain LTP.
Following brief bursts of high frequency stimulation of m a m m a l i a n h i p p o c a m p a l pathways a remarkably long-duration (e.g. hours-to-days) increase in synaptic efficacy occurs, which is usually termed 'long-lasting' or qong-term' potentiation (LTP) s. Since persistent synaptic changes clearly might function as biological substrates of m e m o r y , the mechanisms and sites of such central LTP have generated considerable research interest. H i p p o c a m p a l LTP is associated with increased synaptic conductance 5, but the precise pre- or postsynaptic locus of LTP is still unknown. A number of studies have suggested that the primary site of LTP is postsynaptic (see reviews in refs, 7 and 23), and several postsynaptic ultrastructural correlates of LTP have been described, among which are altered dendritic spine volume or shape 1215"22, increased concave spines ~2, and increased dendritic shaft synapses ~,z2. Nevertheless, there is also some evidence of a presynaptic contribution to h i p p o c a m p a l L T P 27" ~ and nearly all well-studied forms of potentiation in peripheral systems, including LTP in the crayfish 6, have been found to be presynaptic (e.g. ref. 24). Although the specific relations of synaptic vesicles
to transmitter quanta are still unresolved (see reviews in refs. l0 and 30), recent studies employing rapid freeze techniques provide extremely strong support for the view that vesicles participate in the release of packets of transmitter at the neuromuscular junction (NMJ) and may be identical to quanta t3'lo. However, only a handful of studies have examined synaptic vesicle alterations in response to repetitive monosynaptic stimulation in a vertebrate CNS p a t h w a y 11"15'21,25. Of two such studies on brain LTP, one found a decrease in overall vesicle density ~5, whereas the other found no change in density in a limited region adjacent to the center of the active zone I 1. In addition to overall vesicle density changes, repetitive stimulation in peripheral systems alters the intraterminal distribution of vesicles w'17"27 but such
redistribution has not been studied in the m a m m a l i a n CNS. Further, the time course of peripheral posttetanic potentiation has been correlated with an increase in the fraction of vesicles near to or in contact with the active zone ( p a r a m e m b r a n o u s density) 27. Thus, if presynaptic changes in transmitter release are important in hippocampal LTP, such changes might be
Correspondence: P.W. Landfield, Bowman Gray School of Medicine, Department of Physiology and Pharmacology, 300 South Hawthorne Road, Winston-Salem, NC 27103, U.S.A.
0(1(16-8993/87/$03.50 © 1987 Elsevier Science Publishers B,V. (Biomedical Division)
402 partly reflected in intraterminal vesicle distribution. The results of the present study have been described in preliminary form 3. Ten male, young-mature (3-5 months old), Fischer 344 rats were divided into implanted control (sham) and repetitive stimulated (LTP) groups of 5 animals each. Surgical and electrode placement procedures have been described in detail in previous reports >'e~. Briefly, after a recording microelectrode had been placed in the CA 1 cell somal layer (stratuln pyramidale), a stimulating electrode was lowered into the Schaffer collateral-commissural fibers of field CA3 which project to the apical dendrites ol CA129. The recording microelectrode was not placed directly in the synaptic terminal fields (stratum radiaturn), in order to avoid tissue damage to regions that were used for ultrastructural analysis, and since both the population spike and the {inverted) population EPSP can be recorded from the stratum pyramidale ~. After the monosynaptic Schaffer-commissural response was obtained, according to established physiological and stereotaxic criteria t,2°, the stimulation intensity was set at 150% of threshold for the population spike (an extracellular summation of action potentials; cf. refs. 1 and 2, and Fig. 1) and LTP animals were subjected to 5, one-second bursts of 100 Hz stimulation (5-s intervals between bursts). The development of LTP thereafter was monitored by stimulating at the same intensity at 0.2 Hz for 20 min. The 20-rain period is well beyond that at which previously defined forms of short-term potentiation can be observed (e.g. ref. 24) and the LTP at 20 rain was maximal, showing no evidence of the onset of a decay. For the spike, the mean increase over control levels was 75% (_+ 28cA S.E.M.) whereas field EPSP initial slope (measured between EPSP onset and 1 ms after onset) was increased by an average of 27~ (_+ 6~i S.E.M.). Twenty minutes following the repetitive stimulation trains, animals were sacrificed by intracardial perfusion for 15 min with a mixed aldehyde solution (2% glutaraldehyde/2% paraformaldehyde; 0.15 M sodium cacodylate buffer). Sham controls were subjected to the same procedures, but were not given the five 1-s bursts of 100 Hz stimulation. No attempts were made to equate total stimulation pulses given to controls (at 0.2 Hz), since this would have required that the controls be maintained under anesthesia for a considerably longer
period than the LTP animals, and since we have previously found that stimulation frequency is a substantially more significant factor in vesicle distribution than is total pulses a. After perfusion, brains were removed and stored in cold fixative overnight, and were serially sectioned into 251)-itm sections (vibratome) until the electrode tracks were located. The tissue was then trimmed to yield a block containing the apical dendrites of the CA I pyramidal cells adjacent to the recording electrode, and was processed according to standard EM procedures. Twenty-five micrographs were shot on 3 ultrathin sections from each animal, in a programmed sequence through a strictly defined region of the CA 1 stratum radiatum (e.g. the midpoint between the CA~ pyramidal cell layer and the perforant path) at a point of densest Schaffer collateral-commissural termination >. Micrographs were photographically enlarged from an initial magnification of ×25,000 to ×70,000 for quantitative analysis. Approximately 10(1 synapses, and 351Xt vesicles were analyzed for each of the 10 animals. Synaptic terminals and associaled dendritic spines were analyzed using a digitizing pad and a computerized morphometric analysis system (Bioquant) to obtain area and perimeter measurements. Each synaptic complex fully included on the micrographs was analyzed for the length of the active zone, the total presynaptic terminal area, and the terminal area within 150 nm of the active zone. Each spine head associated with a quantified terminal was analyzed for area and perimeter (the spine neck was infrequently present on the micrographs and was therefore excluded from the analysis). Synaptic vesicles were counted m the whole terminal (total vesicles: TV), and in the area within 150 nm of any point of the active zone (local vesicles: LV) Ive~. The distant vesicles tDV) were derived from the other counts (TV-LV). Vesicles in direct contact with the presynaptic membrane of the active zone {attached vesicles: AV) were also counted. The LV, DV and TV were converted to densities (LVD, DVD and TVD) by dividing vesicle counts by relevant areas, and the AV were corrected by length of the active zone. The size of the vesicles can substantially affect density analyses, yet, with few exceptions, prior quantitative studies of vesicles have not considered
403 possible vesicle diameter changes during stimulation.
of the total) and included these values in a stereologi-
In the present study, therefore, we measured the
cal formula for estimating synaptic vesicle density in a volume is'31
diameters of a randomly selected sample (grid overlay method) of synaptic vesicles (approximately 5%
,/
L__
Fig. 1. A, B: examples of electrophysiological responses from the same animal before (A) and 20 rain after (13) LTP stimulation paradigm. Both the population spike and the EPSP slope were increased after stimulation. Calibration: 2 mV, 5 ms. C. D: micrographs of synapses from a control animal (C) and from an LTP animal (D) showing an overall depletion and shift of remaining vesicles toward the active zone during LTP. ×70,000.
404
NA N V =
((4/~).d) + t - 2 h
2500 A.
B.
C.
where Nv is n u m b e r per volume, N A is n u m b e r pet area, d is the average measured diameter, t is section thickness (60 nm, silver-gray interference patterns) and h is the height of invisible fragments (determined from the formula r 2 = ( r - h ) 2 + k 2, where k is the estimated radius of invisible fragments, which was measured as 7 n m in this study). This formula corrects for section thickness, vesicle diameter, and missing or invisible caps Is. We did not use corrections for shape or
ol_
TVD
-D
1 LVD
1 DVD
-E
100~
7 I)~
F
/
200J
I
150! i
for polydispersion31 because of the close approximation of vesicles to spheres, and because the coefficient of variation for vesicle diameters was well below the 20% cut-off at which it is thought necessary
E 50-
~
l il
to correct for polydispersion (e.g. ref. 31). All analyses were performed 'blind' on coded micrographs, and all parameters were averaged across terminals to provide a single value per animal. The statistical population for each variable therefore was n u m b e r of animals. Comparisons between LTP and control animals were made using two-tailed t-tests. We found that animals in which LTP was induced showed declines in both the LVD (P < 0.005) and the D V D (P < 0.005) (and consequently, the T V D (P < 0.005) (Fig. 2). However, the density of vesicles within 150 nm of the active zone (LVD) was proportionately greater than in the distant area ( D V D ) , resulting in a significant increase in the L V D / D V D ratio for LTP animals (P < 0.025) (Fig. 2F). This suggests that there is a migration of the remaining vesicles toward the synaptic active zone during LTP. In addition, the n u m b e r of vesicles attached to the presynaptic m e m b r a n e of the active zone (AV) increased significantly (P < 0.025) in LTP animals (Fig. 2D). Further, if the AV are viewed as a component of the LV, then the fraction of local vesicles attached to the active zone m e m b r a n e (AV/LV) exhibited a highly significant increase (P < 0.005) in LTP animals (Fig. 2El. Examples of these changes are seen in Fig. l, although, of course, not all synapses in this region are equally affected by the stimulation paradigm. Both average area and perimeter per spine were significantly greater (P < 0.025 and P < 0.05 respectively) in the LTP group, while terminal area, perimeter or shape factor were not significantly changed
AV AV LV% LVD DVDo Fig. 2. Synaptic vesicle densities and rattos. A-C show that the decrease m total vesicle density (TVD) during LTP was influenced by decreases in both the local (LVDj and the distant (DVD) vesicle densities. Attached vesicles (AV) are expressed as vesicles per length of active zone and exhibited a significant increase during LTP (D). This increase, in conlunctton with the LVD decrease, resulted in a highly significant increase of the AV/LV ratio (E). The LVD/DVD ratio was also significantly increased (F) in the LTP group, indicating that a greater proportion of the remaining vesicles were situated close to the active zone. All data are expressed as means ~_:S.E.M. TABLE 1 Synaptic ultrastructural variables after L ]!P induction
Summary of quantitative ultrastructural variables, other than vesicle densities. Shape factor was computed according to the formula (4.marea)/(perimeter 2) which gives a value of 1 when the profile is a circle and a value of 0 when the profile is a straight line. See table for all other units (area and perimeter units are per terminal or per spine). Values are expressed as means _+S.E.M. Control
Spine area (urnz) 0.114 + 0.0114 Spine perimeter (um) 1.357_+0.032 Terminal area (jtm2) 0. 181 _+0.008 Terminal perimeter (,urn) 1.8116_+0.022 Vesicle diameter (nm) 41.8611 ± (}.220 Synaptic density length ~um) 0.275 ± 0.0% Terminal shape factor 0.707_+0.023
L TP
P
0. t38 _+0.007 0.02 1.511 _+0.050 0.[)3 0.176+_0.011 n.s. 1.789+0.061
n.s.
42.7ll] +_0.520 n.s. U.270 +_0.078 n.s. [).683 _+0.006 n.s.
405 (Table 1). Vesicle diameter and the length of the active zone were also not significantly different between the control and LTP groups (Table I). The present study replicates earlier reports of an increase in spine volume ~:'~5, and a decrease in total vesicle density ~5 following repetitive hippocampal stimulation (either shown or presumed to induce LTP). In addition, however, the present study shows that a redistribution of synaptic vesicles within the terminal occurs during LTP. Although electrophysiological recordings showed that the stimulation used in this paradigm induced LTP, results from such studies are correlational, and of course cannot demonstrate that the observed ultrastructural changes are the basis of LTP. Nevertheless, the observed increase in attached vesicles, and in the proportion of vesicles adjacent to the active zone, are indicants of vesicle shifts that, under current concepts of synaptic function 131617'2527, would be anticipated to contribute to increased synaptic efficacy. Moreover, increased vesicle attachment could be a basis for the previously observed increases in glutamate release ~4. Thus, our findings provide ultrastructural evidence that presynaptic factors mav contribute to LTP (quite possibly in combination with postsynaptic factors). Recent studies also examined vesicle changes in relation to hippocampal frequency potentiation (FP; increased hippocampal synaptic efficacy during repetitive stimulation1). The present results for LTP were similar in two respects to those for FP, in that an increase of the LVD/DVD ratio and of total AV were seen in both types of potentiation 4. However, there were also two notable differences, since during LTP, total vesicle density was decreased, whereas during FP it was not. and in fact the DVD and LVD were both increased in FP. In addition, the AV/LV ratio was increased during LTP (Fig. 2E), but not during Fp ~. In peripheral systems, vesicles adjacent to the active zone appear to be more available for release, and a shift in vesicles toward the active zone is correlated with greater release m.13.17.27. By analogy, then,
the data suggest that hippocampal frequency potentiation is associated with an increase in more readily releasable (proximal) vesicles (increased LVD), and a small increase in vesicle release probability (increased LVD/DVD) 4, whereas LTP appears to be associated with a decrease in readily releasable vesicles (decreased LVD), and a greater increase in vesicle release probability (an increase of both AV/LV and LVD/DVD). Whether the above vesicle ratios and LVD are related to the quantal binomial parameters p and n respectively, is of course highly problematic, since the association of vesicles with quanta is itself still somewhat controversial L<3{~. Moreover, several workers have found that the binomial parameter n correlates more closely with number of active zones than with number of vesicles, at least in some systems tg'>. On the other hand, it has been shown that binomial analyses can be flawed by major effects of non-stationarity and non-uniformity of p~,_,s, and therefore, reported estimates of n may not be accurate in all studies. Consequently, while it is obviously not possible at this point to propose that vesicle distribution ratios are relevant to binomial parameters, we suggest that viewing these ratios as indicants of the probability of vesicle release may provide insights into the implications of dynamic vesicle shifts for brain synaptic physiology. In summary, the present data show that presynaptic morphological alterations which could increase synaptic efficacy are found during LTP. Since others have shown that potentially relevant postsynaptic changes also occur lL1215'22, the evidence suggests that structural changes on both sides of the synapse may play a role in the induction and/or maintenance of LTP.
We appreciate the valuable and extensive technical assistance of S. Vinsant and L. Cadwallader and the excellent assistance of D. Lefler in preparing the manuscript. This research was supported in part by Grant AG 04542 from the NIH.
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