synapse restructuring associated with the maintenance phase of hippocampal long-term potentiation

THE JOURNAL OF COMPARATIVE NEUROLOGY 368:413-423 (1996)

Synapse Restructuring Associated With the Maintenance Phase of Hippocampal Long-Term Potentiation

YURI GEINISMAN, LEYLA DETOLEDO-MORRELL, FRANK MORRELL, INNA S. PERSINA, AND MICHAEL A. BEATTY

Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611 (Y.G., I.S.P., M.A.B.), and Department of Neurological Sciences,

Rush Medical College, Chicago, Illinois 60612 (L.dT.-M., F.M.)

ABSTRACT Synapses in the middle molecular layer of the rat dentate gyms were analyzed by electron

microscopy during the maintenance phase of long-term potentiation (LTP). LTP was induced by high-frequency stimulation of the medial perforant path carried out on each of 4 consecutive days. The dentate gyrus was examined electron microscopically 13 days following the fourth stimulation. At this time point, synaptic responses were still significantly enhanced relative to baseline, although the extent of their potentiation was lower than 1 hour after the last high-frequency stimulation. Stimulated, but not potentiated, rats served as controls. Using the stereological double disector method, estimates of the number of different morphological types of synapses per postsynaptic neuron were obtained. The number of asymmetrical axodendritic synapses increased (by 28%) during LTP maintenance, whereas the number of other synaptic types was not significantly altered. Our previous work demonstrated that the induction of LTP is followed by a selective increase in the number of axospinous perforated synapses with multiple, completely partitioned, transmission zones. Thus, the induction and maintenance phases of LTP are characterized by different structural synaptic alterations. These alterations may be related to each other as indicated by another finding of the present study regarding the existence of perforated synapses that appear to be transitional between axospinous and axodendritic junctions. This suggests a model of structural synaptic plasticity associated with LTP in which some axospinous perforated synapses increase in numbers shortly after the induction of LTP and are then converted into axodendritic ones during LTP maintenance. D 1996 Wiley-Liss, Inc.

Indexing terms: synaptic plasticity, synapse ultrastructure, double disector, rat dentate gyrus

Hippocampal long-term potentiation (LTP) is widely regarded as a synaptic model of memory (Bliss and Collin- gridge, 1993). LTP is manifested by a persistent enhancement of synaptic responses following brief high-frequency stimulation (Bliss and Gardner-Medwin, 1973; Bliss and L@mo, 1973). One of the basic properties of LTP is its long-lasting duration. Although enhanced synaptic responses decay with time, they can be maintained at an elevated level for days or weeks without further reinforce- ment (Bliss and Gardner-Medwin, 1973; Barnes, 1979, 1983; Barnes and McNaughton, 1980; Racine et al., 1983; Staubli and Lynch, 1987). It has been proposed that the unusually long duration of synaptic enhancement, which is at the core of LTP, may be due to structural synaptic modifications such as an increase in the number of synapses (Lynch et al., 1988; Lynch and Baudry, 1991; Bliss and Collingridge, 1993).

Earlier electron microscopic studies examined the effect of LTP induction on synaptic morphology during a period of minutes to hours following cessation of high-frequency stimulation. Quantitative analyses of synapses in the CA1 region of the hippocampus and the dentate gyrus have shown that the induction phase of LTP is associated with an increase in the number of certain morphological types of synaptic contacts (Lee et al., 1980, 1981; Desmond and Levy, 1983,1986; Chang and Greenough, 1984; Wenzel and Matthies, 1985; Schuster et al., 1990; Trommald et al., 1990; Chang et al., 1991; Geinisman et al., 1991, 1992, 1993,1994).

Accepted December 15,1995. Address correspondence to Yuri Geinisman, Department of Cell and

Molecular Biology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611.

O 1996 WILEY-LISS, INC.

414 Y. GEINISMAN ET AL.

It remained unknown, however, whether or not the maintenance of LTP is also associated with structural synaptic alterations. The present work was designed to address this issue by examining potentiated animals several days after cessation of high-frequency stimulation. We report that the maintenance phase of LTP is, indeed, accompanied by synapse restructuring in the potentiated synaptic field and that the pattern of this restructuring is different from that observed shortly after the induction of LTP.

MATERIALS AND METHODS Animals and electrophysiological procedures Young adult (5 months old at the end of experiment) male

rats of the Fischer-344 strain were examined. All animals were anesthetized with Nembutal (50 mgikg i.p.1 and implanted chronically with bipolar, stainless steel stimulating electrodes into the right medial perforant path and recording electrodes into the hilus of the ipsilateral dentate gyrus. Placement of the stimulating and recording electrodes was accomplished by using stereotaxic coordinates initially and then correcting their position until single- pulse stimulation of the electrode inserted into the angular bundle yielded a population spike within the dentate gyrus that arose from the rising phase of the extracellularly recorded field potential (EPSP). The population spike elicited by stimulation of the ventromedial track of the angular bundle (i.e., of the medial perforant path) has a shorter latency than that evoked by stimulation of the ventrolateral track (McNaughton and Barnes, 1977). The coordinates for the perforant path electrode were 8 mm posterior to bregrna, 4.4 mm lateral to the midline, and 4.2 mm ventral, with the horizontal zero plane being tangent to the upper incisor bar and 5 mm above the interaural line. The hilar electrode was introduced at a 35" angle to the vertical plane, 1.4 mm posterior to bregrna, 2 mm lateral to the midline, and 4.2 mm ventral. All leads were connected to an Amphenol plug, which was fixed to the skull with screws and dental cement.

Following a %week recovery period, animals were randomly assigned to two groups of nine rats each. One group consisted of potentiated animals that received high- frequency stimulation of the medial perforant path as specified below. The second group included coulombic controls stimulated at a low frequency (0.2 Hz) that does not elicit LTP. In the present work, unstimulated but implanted control rats were not examined because our previous studies showed that unstimulated and coulombic controls did not differ significantly from each other with respect to the number of synapses per neuron when either the entire synaptic population or various synaptic subtypes were compared (Geinisman et al., 1991, 1993).

The procedure of potentiation was very similar to that described by Barnes (1979). Initially, the level of baseline responses was determined on each of 2 consecutive days by stimulating with single pulses every 5 seconds (0.2 Hz) and averaging four sets of 10 responses. For each animal, the intensity and duration of a stimulating pulse was set at a level that initially produced a minimal population spike. High-frequency stimulation, which consisted of fifteen 20-ms bursts of 400 Hz delivered at 0.2 Hz, was then carried out on each of 4 consecutive days. A repetition of daily episodes of high-frequency stimulation was used because this stimulation protocol increases the magnitude

of synaptic responses and, most importantly, leads to more enduring LTP (Barnes, 1979). The extent of potentiation was assessed with single pulses (applied at 0.2 Hz) 2 and 10 minutes, 1, 6, and 24 hours following each high-frequency stimulation and 7 and 13 days after the fourth high- frequency stimulation. The last test was conducted immediately before perfusion fixation of animals for electron microscopy. A similar stimulation procedure was used for coulombic control rats except that all stimuli were delivered at 0.2 Hz. Each animal from the coulombic control group was matched with a respective potentiated rat according to the current level and the total amount of current delivered. Electrophysiological data collection and off-line analyses were performed with the aid of a Nicolet Med 80 computer.

Tissue preparation For electron microscopy, animals were coded, deeply

anesthetized with Nembutal (75 mgikg, i.p.1, and perfused transcardially with paraformaldehyde-glutaraldehyde fixa- tives as described previously (Geinisman et al., 1991). The right hippocampal formation was dissected free and sectioned, perpendicular to its septotemporal axis, to obtain 1-mm-thick tissue slabs. The latter were sampled only from the dorsal hippocampal formation, which terminates cau- dally at the occipital bend, because electrophysiological recordings were made from the dorsal, but not from the ventral, dentate gyrus. The most rostral portion of the latter (within 2.5 mm from the septal pole of the hippocampal formation) was not used for ultrastructural analyses in order to sample only tissue undamaged by the recording electrode track. Three consecutive slabs were prepared with the aid of a tissue chopper equipped with a microdrive. The position of the most rostral cut within the first 1-mm interval along the septotemporal axis of the hippocampal formation (i.e., between the points located 2.5 and 3.5 mm caudal to the septal pole) was selected by a random number from 1 to 100 using 0.01-mm settings of the microdrive. Subsequent cuts were made at uniform intervals of 1 mm. Tissue slabs were assigned code numbers, treated with Os04, dehydrated in ethanols, and flat embedded in Araldite.

From the rostral face of each tissue slab, a semithin (1-ym-thick) section was obtained and stained with methy- lene blue-azure I1 (Richardson et al., 1960). The dorsal (hidden) blade of the dentate gyrus was identified in semithin sections and trimmed down to prepare a complete series of 29-46 (mean = 36) ultrathin sections. The position of the first (medial) trimming cut was chosen at random along the mediolateral extent of the dorsal blade by using numbered divisions of an eyepiece reticule in a dissecting microscope. Each ultrathin section spanned the entire ventrodorsal extent (i.e., the width) of the molecular and granule cell layers in the dorsal blade of the dentate gyrus. A section series was stained with uranyl acetate and lead citrate, mounted on a single-slot grid coated with collodion-carbon, and examined in a JEOL lOOX electron microscope.

Synapse quantitation Synapses were analyzed in the middle molecular layer of

the dentate gyrus because it is predominantly innervated by axons of the medial perforant path (Hjorth-Simonsen, 1972; Hjorth-Simonsen and Jeune, 1972; Steward, 1976; Wyss, 19811, which were stimulated during the induction of LTP. In the molecular layer, perforant path axons make asymmetrical synaptic contacts on dendritic shafts and

415 SYNAPSE RESTRUCTURING DURING LTP MAINTENANCE

spines (Nafstad, 1967; Fifkova, 1975; Matthews et al., 1976; Nieto-Sampedro et al., 1982; Steward and Vinsant, 1983). The terminal synaptic field of perforant path fibers in the dentate molecular layer is of an exceptionally high density: more than 90% of all synaptic contacts degenerate in the middle and outer molecular layer of the rat dentate gyrus following ablation of the ipsilateral entorhinal cortex (Matthews et al., 1976; Nieto-Sampedro et al., 1982; Stew- ard and Vinsant, 1983; Anthes et al., 1993).

Estimates of the number of synapses per postsynaptic granule cell were obtained with the unbiased method of double disector (Braendgaard and Gundersen, 1986; Gun- dersen, 1986; Gundersen et al., 1988). This method was used because it does not require knowledge of the precise thickness of ultrathin sections. For counting synapses, each series of ultrathin sections was initially examined at an electron microscope magnification of 4,OOOX. In the first section of a series, the middle molecular layer was defined as the middle third of the entire molecular layer width and divided into rows and columns of counting fields by using a field delineator of the microscope screen. Each of these was numbered (starting from the top left-hand corner) for a random selection of a counting field to be photographed in all consecutive sections. For this purpose, only straight ribbons of serial sections were used. The sections were aligned by choosing a tissue landmark (such as a trans- versely sectioned myelinated fiber) and placing it into the same position on the microscope screen. Electron micrographs of the middle molecular layer were taken from all sections of a given series at an initial magnification of 8 ,000~ and enlarged photographically to a final magnification of 21,000~. For counting postsynaptic neurons, the granule cell layer was photographed in the first, several intermediate and the last sections of each series at an initial magnification of 1,000 x , and the negatives were enlarged to a final magnification of 2 , 5 0 0 ~ . A magnification stan- dard (grating replica) was photographed and printed with each series of electron micrographs.

Each disector for synapse counting consisted of two adjacent ultrathin sections, a reference section, and a look-up one immediately above it. With the aid of electron micrographs of consecutive serial sections, synapses were identified by the presence of synaptic vesicles in a presynaptic axon terminal and of a postsynaptic density (PSD) in a postsynaptic element. Subtypes of axospinous synapses were classified as described elsewhere (Geinisman et al., 1991, 1993; Geinisman, 1993). Synaptic counts were performed by using the unbiased counting rule and the unbiased two-dimensional counting frame of Gundersen (1977). A synapse was counted if its PSD was observed within the frame (or intersected by its inclusion edges) in the reference section but was not seen in the look-up section of a disector. From each section series, 20 (in the case of axodendritic and axospinous perforated synapses) or 10 (in the case of axospinous nonperforated synapses) disectors were randomly selected to count synaptic contacts. For counting granule cells, the first and the last sections of each series were alternately used as a reference and a look-up section of disectors, the cell nucleus being employed as a counting unit.

The number of synapses per postsynaptic neuron (n1N) was calculated for an individual animal by the formula (Brzndgaard and Gundersen, 1986): n/N = [(Zq- . X4 . Ck)/(BQ ~ . h)] . (Zw/ZW). In this formula, q- and Q- indicate numbers of synapses and neurons counted in an

area a or A, respectively; k designates the number of sections in a series minus one, which is a measure of the double disector height; w and W represent the width, i.e., the ventrodorsal extent, of the middle molecular layer (expressed as one-third of the entire molecular layer width) or of the granule cell layer, respectively (the widths were measured in semithin sections); and E is the summation over all disectors and section series analyzed in an animal. The wlW ratio, which is an estimator of the ratio of two reference volumes, provided a correction for possible differ- ential volumetric alterations of the synaptic and cell body layers (Braendgaard and Gundersen, 1986). Estimates obtained for each individual animal were based on counting a total of 249 - 317 (mean = 285) nonperforated axospinous synapses (in a total average area of 2,050 km2), 55 - 81 (mean = 70) perforated axospinous and 36-68 (mean = 48) axodendritic synaptic contacts (in a total average area of 4,100 km2), as well as a total of 2 3 4 6 (mean = 35) granule cells (in a total average area of 13,260 pm2).

Statistical analyses For electrophysiological and morphological data analy-

ses, a value for each parameter assessed in a given animal was used as an experimental observation. Statistical analyses of electrophysiological data were performed with the two-tailed randomization test for matched pairs (Siegel, 1956). This test utilizes all information about numerical data in two related samples and has the same power as the paired t test. Morphological data were treated with the two-tailed randomization test for two independent samples (Siegel, 1956). It is a powerful distribution-free technique for evaluating the significance of the difference between the means of two unrelated samples of numerical data. The exact probability values under the randomization tests were computed by using the software developed by Dr. Arkady Kanevsky at our request.

RESULTS Electrophysiology

The extent of potentiation of synaptic responses in the dentate gyrus following high frequency stimulation of the medial perforant path was assessed separately for the following three measures: (1) the slope of the extracellularly recorded EPSP defined as the peak amplitude divided by its latency, (2) the amplitude of the population spike determined as the distance from a tangent line between the two peaks of the EPSP to the population spike, and (3) the input-output (1-0) function (population spike amplitude/ EPSP slope). The degree of synaptic enhancement was determined by the formula T I - To/To. In this formula, T I represents the value for a given measure at a given time interval following high-frequency stimulation (or the equivalent of 120 pulses a t 0.2 Hz for coulombic controls) and To is the baseline value (i.e., the mean of the values obtained during the two baseline sessions prior to high- or low- frequency stimulation). The significance of the extent of potentiation at a given time interval following high- frequency stimulation versus baseline for each measure was assessed with the randomization test for matched pairs. A significance level of 0.01 was accepted because of the number of tests performed.

At the 13-day test interval following the fourth high- frequency stimulation, the EPSP slope was potentiated by 51 & 9% (mean & S.E.M.), the population spike amplitude

416

by 137 2 73%, and the 1-0 function by 55 2 44%. A paired randomization test comparing baseline values with those at the 13-day interval showed significant increases for the EPSP slope and the population spike amplitude (the number of possible outcomes = 512, the number of outcomes in the rejection region = 25, P = 0.0039 for both measures). The increase in the 1-0 function, however, was not significant (P = 0.305). Similar analyses carried out on coulombic control animals at an equivalent time interval following the last low-frequency stimulation versus baseline did not reveal any significant changes in the three measures used.

I t is important to note that, although synaptic responses were still potentiated relative to baseline 13 days following the induction of LTP, their augmentation had decayed over this time period. At the 1-hour test interval after the fourth high-frequency stimulation, the extent of potentiation was 111 2 25% for the EPSP slope, 695 ? 169% for the population spike amplitude, and 263 5 57% for the 1 - 0 function. A paired randomization test comparing these values with those obtained 13 days after LTP induction (see above) showed that the differences between them were significant ( P = 0.0039 for each of the three measures). During the maintenance phase of LTP, the population spike decayed relatively faster than the EPSP slope. This resulted in the relatively rapid decay of potentiation in the 1-0 function because the latter represents the ratio of the population spike amplitude to the EPSP slope.

Y. GEINISMAN ET AL.

Electron microscopy Quantitation of the entire synaptic population showed

that the total number of synapses per postsynaptic neuron was not significantly changed in the dentate middle molecular layer of potentiated rats examined 13 days following the last high-frequency stimulation as compared with coulombic controls (Fig. la). Synaptic contacts in the middle molecular layer are, however, morphologically heteroge- neous (Geinisman et al., 1987a,b, 1991, 1993; Geinisman, 1993). The majority of synapses in this synaptic field belong to the category of axospinous junctions that involve dendritic spines. The number of such synaptic contacts per neuron was not significantly altered during the maintenance phase of LTP (Fig. lb). The axospinous synaptic category consists of the so-called nonperforated synapses exhibiting continuous PSD profiles in all consecutive sections and perforated ones showing a discontinuous PSD profile in some serial sections (Peters and Kaiserman- Abramof, 1969; Cohen and Siekevitz, 1978; Calverley and Jones, 1990). Neither nonperforated (Fig. lc ) nor perforated (Fig. Id) axospinous synapses were significantly changed in number in the group of potentiated rats versus coulombic controls.

Perforated axospinous junctions can be subdivided further into several distinct morphological subtypes based on the configuration of their PSDs and the presence or absence of spine partitions (Nieto-Sampedro et al., 1982; Carlin and Siekevitz, 1983; Spacek and Hartmann, 1983; Dyson and Jones, 1984; Calverley and Jones, 1987; Geinisman et al., 1987b, 1991, 1993; Geinisman, 1993). The most numerous subtype of perforated axospinous synapses (Fig. 4d) is distinguished by multiple (2-41, completely partitioned transmission zones (Geinisman, 1993; Geinisman et al., 1993). Each transmission zone is formed presynaptically by a separate axon terminal protrusion and delineated postsyn- aptically by a separate PSD segment. Complete spine

partitions, which emanate from the spine head, entirely separate multiple transmission zones from each other. Spine partitions that differ from the complete ones are found in two other synaptic subtypes. These include synaptic contacts characterized by a sectional spine partition and horseshoe-shaped PSD (Fig. 4c) or a focal spine partition and fenestrated PSD (Fig. 4b). The partitioned synaptic subtypes are complemented by their nonpartitioned coun- terparts that have a segmented, horseshoe-shaped, or fenestrated PSD but lack spine partitions (Fig. 4e-g). Quantita- tive analysis of these subtypes of perforated axospinous junctions revealed that their number per neuron did not differ significantly between coulombic control and potentiated animals. This was true for synapses with multiple, completely partitioned transmission zones (Fig. le) and for other perforated subtypes when they were either combined (Fig. 10 or differentially quantified (data not shown).

Differences between control and potentiated rats with respect to the number of synapses per neuron did not exceed 9% when all axospinous synaptic contacts or their morphological varieties were examined (Fig. la-0. The number of axodendritic synapses, however, was 16% higher in potentiated animals than in coulombic controls (Fig. lg). The axodendritic synaptic population can be divided further into two morphologically distinct subtypes. Axodendritic synapses of one subtype, which are referred to as symmetrical synaptic contacts (Colonnier, 1968), have pre- and postsynaptic densities of equal thickness (Fig. 2, open arrows). In axodendritic junctions of the other subtype, the postsynaptic density is noticeably thicker than the presynaptic one (Fig. 2, closed arrows). This gives such synaptic contacts a marked asymmetry, and they are correspond- ingly classified as asymmetrical synapses (Colonnier, 1968). Quantitation of the two synaptic subtypes showed that symmetrical axodendritic junctions were not changed in numbers during the maintenance phase of LTP (Fig. lh). In contrast, the number of asymmetrical axodendritic synapses per neuron was substantially and significantly increased (by 28%) in the group of potentiated rats versus the coulombic control group (Fig. li , Table 1).

Examination of electron micrographs of serial ultrathin sections through the middle molecular layer revealed occa- sional unusual synaptic profiles belonging to perforated synapses that appeared to be intermediate between axodendritic and axospinous junctions. An example of such a synapse is demonstrated in Figure 3 (arrows). In one serial section through this synaptic contact (Fig. 3a), its postsynaptic element can be unequivocally identified as a spine because it contains a characteristic flocculate material, lacks mitochondria and microtubules, and is associated with a well-developed spine apparatus (Fig. 3, arrowheads). The postsynaptic spine has no neck, and it is seen in the following sections (Fig. 3b,c) to be retracted into the parent dendrite so that, finally, its upper surface becomes continuous with that of the dendrite (Fig. 3d). At this level (Fig. 3d), the postsynaptic element is represented by a dendritic shaft rather than by a spine. It is necessary to note that a perforated PSD was observed in all synaptic contacts of this kind. Such synapses were seldom encountered in the middle molecular layer of the dentate gyrus of either control or potentiated rats ( 0 4 synaptic junctions per total sampling area per animal). It was not possible, therefore, to obtain reliable estimates of the number per neuron for this synaptic subtype.

SYNAPSE RESTRUCTURING DURING LTP MAINTENANCE

a a cn !? cn 2 1500

lo00

500

r T

417

COULOMBIC CONTROLS

POTENTIATED RATS

a b C d e f a h i

MORPHOLOGICAL VARIETIES OF SYNAPTIC CONTACTS

a: entire synaptic population

b: all axospinous

c: nonperforated axospinous

d: all perforated axospinous

e: perforated axospinous with multiple,

f: other perforated axospinous

g: all axodendritic

h: symmetrical axodendritic

i: asymmetrical axodendritic

completely partitioned transmission zones

Fig. 1. Comparison of animals belonging to coulombic control and potentiated groups (n = 9 for each group) with regard to the number of synapses per postsynaptic neuron in the middle molecular layer of the hippocampal dentate gyrus. Rats were examined 13 days following the last potentiating or control stimulation. Data are group means 5 S.E.M. Differences between the mean group values for coulombic

control and potentiated animals are indicated by percentages. The only statistically significant change was an increase in the number of asymmetrical axodendritic synapses per neuron in potentiated animals versus coulombic controls. P < 0.05, two-tailed randomization test for two independent samples.

DISCUSSION Major finding of the present study

and its significance The data presented here demonstrate, for the first time,

that the maintenance phase of LTP is associated with synapse restructuring in the potentiated synaptic field. The major finding of this work is that there is a selective increase in the number of asymmetrical axodendritic synapses during LTP maintenance. In potentiated animals examined 13 days following the induction of LTP by high-frequency stimulation of the medial perforant path, each granule cell of the hippocampal dentate gyrus was estimated to acquire some 30 additional asymmetrical synapses involving its dendrites in the middle molecular layer (Table 1). This could underlie the enhancement of synaptic responses because asymmetrical axodendritic synapses are supposed to be excitatory in function and be relatively strong due to their strategic location directly on dendritic shafts rather than on spines. Moreover, the observed structural synaptic alteration results in a 1%

increase in the total number of synaptic contacts involving dendrites and spines of each granule cell in the middle molecular layer. It has been estimated that activation of only 1-5% of the total number of synaptic contacts in the middle molecular layer of the rat dentate gyrus is sufficient to evoke granule cell discharge (McNaughton et al., 1981). Thus, the magnitude of the LTP-associated increase in synapse number appears to be sufficient to exert a measur- able facilitating effect on the amplitude of synaptic responses elicited from the population of dentate granule cells.

Potential pitfalls of the procedures used for synapse quantitation

The morphological part of the present study has certain limitations, and it is necessary to evaluate the extent to which they may influence the interpretation of the results presented here. One of the limitations is that the tissue was obtained from the dorsal, but not from the entire, hippocampal formation and that synapses were sampled only from a


Fig. 2. Electron micrographs of consecutive serial sections (a-c) through the middle molecular layer of the rat dentate gyrus demonstrate a symmetrical axodendritic synapse (open arrows) and an asymmetrical axodendritic synapse (closed arrows) that involve the same dendritic shaft. The former synaptic subtype exhibits pre- and postsynaptic densities of approximately the same thickness in all serial sections, whereas the latter one has a postsynaptic density that is markedly thicker than the presynaptic density. Scale bar = 0.25 IJ-m.

TABLE 1. Number of Asymmetrical Axodendritic Synapses per Neuron in the Middle Molecular Layer of the Dentate Gyrus of Control

and Potentiated Rats

Pair of rats Coulombic controls Potentiated animals

1 2 3 4 5 fi 7 8 9 Group mean i S E M 5%hl PL

88 123 fil 89 101 127 125 111 96

102 ? 7

169 161 138 83 110 129 144 148 94

131 2 10 +28.4 0.0359

'%A, difference between group means. T h e exact probability value under the two-tailed randomization test for two independent samples.

segment of the dorsal dentate gyrus in the vicinity of the recording electrode tip. Being biased toward an area of interest, such sampling does not allow any conclusion to be made with regard to the entire structure under study (Bolender et al., 1991; Mayhew, 1992; West, 1993). How- ever, the results described above indicate structural synaptic changes the occur in that portion of the dentate gyrus, which was examined electrophysiologically.

It is also necessary to emphasize that the final parameter estimated in the process of synapse quantitation was the number of synaptic contacts per postsynaptic neuron, i.e., the synapse-to-neuron ratio. This parameter can be used as a measure of changes in the absolute number of synapses only when certain requirements are met. One of these is that the number of postsynaptic nerve cells should remain stable under experimental conditions so that alterations in the synapse-to-neuron ratio can be attributed to changes in the number of synapses (Gundersen et al., 1988). Such an assumption could be made safely, however, in the case of our experiment. The numerical density of granule cells corrected for the double disector height (i.e., ZQ-/CA . Zk) was practically the same in control and potentiated rats, the difference between the corresponding group mean values (206 t 13 and 204 ? 12 pm2 x being small (1.0%) and statistically not significant. I t appears, therefore, that the results of the morphological part of this study, which are based on the comparison of the two groups of animals with regard to the number of synapses per neuron, were not influenced by differences in neuronal numerical density between the groups.

When the number of synapses is related to the number of principal postsynaptic neurons, the latter should be the only source of postsynaptic elements involved in all synaptic contacts counted. This is certainly not the case for the hippocampal dentate gyrus. The molecular layer of the rat dentate gyms contains not only spinous dendrites of its principal neurons, granule cells, but also aspinous and sparsely spinous dendritic processes originating from hilar neurons and local basket cells (Amaral, 1978; Ribak and Seress, 1983; Lubbers and Frotscher, 1987; Zipp et al., 1989; Leranth et al., 1990; Frotscher et al., 1991). The middle molecular layer, however, represents a synaptic field that is suitable for estimating the number of synapses per granule cell for the following reasons. The granule-cell-to- hilar-cell ratio is 30:l (Gaarskjaer, 1978), and only some types of hilar cells send their dendrites into the dentate molecular layer (Amaral, 1978; Ribak et al., 1985; Ribak and Seress, 1988). Moreover, granule cells outnumber

SYNAPSE RESTRUCTURING DURING LTP MAINTENANCE 419

Fig. 3. Electron micrographs of consecutive serial sections (a-d) through the middle molecular layer of the rat dentate gyms demonstrate a special type of perforated synaptic contact (arrows). The postsynaptic element of this synapse can be classified as a neckless

dendritic spine that is associated with a spine apparatus (arrowheads). In some serial sections (c, d), however, the spine is retracted into the parent dendrite and becomes level with its surface. Scale bar = 0.25 +m.

basket cells by a factor of 150-200 (Seress and Pokorny, 1981). Therefore, the vast majority of postsynaptic elements in the dentate molecular layer are represented by dendritic shafts and spines of granule cells. It is, neverthe- less, possible that there may also be LTP-induced changes in synapse numbers on nongranule cells that contribute to the observed morphological alterations (and to the changes in the population EPSP).

Differences in the pattern of synapse restructuring between the maintenance

and induction phases of LTP In our previous work (Geinisman et al., 1991, 1993), the

effect of LTP induction on synaptic ultrastructure was

studied by using an experimental paradigm identical to that described above except that potentiation-related morphological alterations in synapses were examined 1 hour following the fourth high-frequency stimulation. The results showed that the induction of LTP is followed by a selective increase in the number of axospinous perforated synapses with multiple, completely partitioned transmission zones in the middle molecular layer (Geinisman et al., 1993). The data presented here indicate that the number of these synaptic contacts returns to the control level in potentiated rats examined 13 days after the fourth high-frequency stimulation, i.e., during the maintenance phase of LTP (Fig. le). The latter LTP phase, however, is accompanied by a selective increase in the number of asymmetrical axodendritic synapses in the potentiated synaptic field (Fig. li).

Y. GEINISMAN ET AL. 420

Interestingly enough, this alteration was not found to be associated with the induction of LTP. This observation needs emphasis because our earlier publication (Geinisman et al., 1991) reports data only for the total population of axodendritic synapses. The mean group values (kS.E.M.1 of the number of asymmetrical axodendritic junctions per neuron were estimated to be 96 f 10 for coulombic controls and 102 2 7 for potentiated animals examined 1 hour after the last potentiating stimulation. The difference between the group means (6.3%) was not statistically significant. These results provide evidence that the induction and maintenance phases of LTP are characterized by different patterns of synapse restructuring in the potentiated synaptic field.

Although the increases in the number of synapses observed following the induction and during the maintenance of LTP involve different synaptic subtypes, these changes may be related to each other. Such a possibility is suggested by the existence of a special type of perforated synaptic junction that appears to be transitional between axospinous and axodendritic ones. In these synapses (Fig. 31, the postsynaptic spine lacks a neck, and the spine head is lowered on one side to the level of the parent dendritic surface. The presynaptic terminal forms a single asymmetrical synaptic contact of the perforated variety, which ex- tends from the spine to its parent dendrite. It is conceivable that, under certain conditions, the postsynaptic spine may be completely retracted into the parent dendrite, and the axospinous asymmetrical synapse formerly associated with the spine becomes an axodendritic one.

Model of structural synaptic modifications underlying synaptic plasticity

Our previous results have suggested a model of structural modifications of axospinous synapses that may ac- count for synaptic plasticity associated with LTP (Geinis- man, 1993; Geinisman et al., 1993). The results of the present study indicate that this model needs to be expanded to incorporate axodendritic synapses based on the possibility of their formation from axospinous junctions. According to the revised model (Fig. 4), the sequence of synapse restructuring that may result in a marked augmentation of synaptic responses during the induction phase of LTP is postulated to commence with the conversion of nonperforated axospinous synapses (Fig. 4a) into perforated ones. It involves the consecutive formation of synaptic contacts that have initially a focal spine partition with a fenestrated PSD (Fig. 4b), then a sectional partition with a horseshoe- shaped PSD (Fig. 4c), and, finally, a complete partition(s) with a segmented PSD (Fig. 4d). Synapses belonging to the latter subtype have multiple (2-4) transmission zones instead of only a single one, as is usual. These synaptic contacts are presumed to be especially efficacious axospinous junctions: their transmission zones may represent functionally independent units, provided that complete spine partitions act as barriers for transmitter diffusion in the synaptic cleft and that each PSD segment is associated with a newly inserted or activated receptor cluster (Geinis- man et al., 1993; Edwards, 1995). It seems reasonable, therefore, to believe that an increase in the number of axospinous synapses with multiple, completely partitioned transmission zones following the induction of LTP could significantly augment synaptic transmission.

During the maintenance phase of LTP, which is accompanied by a decay of synaptic enhancement, the number of these synaptic contacts returns to the control level. The

reversal of the morphological change associated with the induction of LTP may reflect a disassembly of complete spine partitions in such synapses and their conversion into other synaptic subtypes. Among these are nonpartitioned axospinous synapses with a segmented (Fig. 4e), horseshoe- shaped (Fig. 40, and fenestrated (Fig. 4g) PSD. In addition, we propose that some axospinous synapses with multiple, completely partitioned transmission zones are remodeled into asymmetrical axodendritic junctions, which are increased in number during LTP maintenance. In the process of this remodeling, the postsynaptic spine would lose its neck (Fig. 4h) as it is gradually retracted into the parent dendrite (Fig. 4i) until the spine head becomes leveled with the dendritic surface (Fig. 4j). As a consequence of the retraction, the perforated axospinous synaptic contact that involved the spine becomes an asymmetrical axodendritic synapse with a perforated PSD.

Possible structural correlates of the decay of synaptic enhancement and its retention

at a relatively low level during the maintenance phase of LTP

The pattern of synaptic plasticity that characterizes the maintenance phase of LTP includes two basic phenomena: a decay of the maximum degree of synaptic enhancement that is observed during LTP induction and, simultaneously, the retention of a lesser degree of synaptic enhancement for a relatively long period of time. These two phenomena may be accounted for by different structural synaptic modifications. The decay may be a consequence of the remodeling of perforated axospinous junctions with multiple, completely partitioned transmission zones. As we reported elsewhere (Geinisman et al., 19931, these presumably most efficacious axospinous synapses are markedly increased in number (by about 50%) when the maximal enhancement of synaptic responses occurs following LTP induction. The data presented here indicate that this change is no longer present during LTP maintenance. The number of axospinous synapses with multiple transmission zones returns to the control level, probably due to their remodeling into other synaptic subtypes as described above. This may yield a reduction in the overall degree of synaptic enhancement.

The long-lasting retention of synaptic enhancement during the maintenance phase of LTP could be mediated by the more moderate increase (28%) in the number of asymmetrical axodendritic synapses, which we propose were formed from axospinous junctions with multiple, completely partitioned transmission zones (Fig. 4d,h-j). We speculate further that such a transformation in the morphological substrate of synaptic plasticity represents an efficient form of response conservation within the adaptive system. Be- cause dendritic shaft synapses are presumably more potent than axospinous ones, a smaller increase in their number might serve to sustain an adequate degree of synaptic enhancement, reflecting a significant and durable electrophysiological change. If the modification involving axospinous synapses we described earlier (Geinisman et al., 1993) is the structural basis for an initial stage of synaptic plasticity, then the formation of additional asymmetrical axodendritic synapses may represent the next stage in the sequence of synapse restructuring underlying long-term plasticity.

SYNAPSE RESTRUCTURING DURING LTP MAINTENANCE

A

4 - Fig. 4. The diagram illustrates the proposed model of structural

synaptic plasticity associated with long-term potentiation (LTP) as described in the text. The schematic drawing shows the following structural intermediates in synaptic plasticity: (a) nonperforated axospinous synapse; the subtypes of perforated axospinous junctions that have (b) a focal spine partition and fenestrated postsynaptic density (PSD); (c) a sectional spine partition and horseshoe-shaped PSD; or (d) complete spine partitionk) and segmented PSD; nonpartitioned coun- terparts of these synaptic subtypes that lack spine partitions and exhibit (e) a segmented, (f) horseshoe-shaped, or (g) fenestrated PSD;

421

.

1

h

(h) perforated axospinous synapse involving a dendritic spine that does not have a neck; (i) perforated axospinous synapse involving a dendritic spine that is partially retracted into the parent dendrite; and (j) asymmetrical axodendritic synapse with a perforated PSD. A remodeling of preexisting synaptic contacts is presumed to underlie the observed selective increases in the number of either axospinous synapses with multiple, completely partitioned transmission zones (d) following the induction of LTP or asymmetrical axodendritic synapses (j) during the maintenance phase of LTP.


ACKNOWLEDGMENTS We thank Dr. Igor Lifschitz for his valuable advice

regarding the statistical treatment of the data; Dr. Arkady Kanevsky for developing the software designed to compute the exact probability values under the randomization tests used; Dr. Bruce L. McNaughton for helpful discussion of some of our speculative ideas; Dr. Eddy Van der Zee for his criticism of the manuscript; William Goossens, Nicholas Kriho, and Marvin Rossi for their skillful technical assis- tance; and Kim C. Gersony for preparing Figure 4. This work was supported in part by grants AG 08794 and AG 09466 from NIA and by grant BNS-8912372 from NSF.

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