TOTAL NUMBER AND DISTRIBUTION OF INHIBITORY AND EXCITATORY

SYNAPSES ON HIPPOCAMPAL CA1 PYRAMIDAL CELLS

M. MEGIÂAS, ZS. EMRI, T. F. FREUND and A. I. GULYAÂ S*

Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, P.O. Box, H-1450, Hungary

AbstractÐThe integrative properties of neurons depend strongly on the number, proportions and distribution of excitatoryand inhibitory synaptic inputs they receive. In this study the three-dimensional geometry of dendritic trees and the density ofsymmetrical and asymmetrical synapses on different cellular compartments of rat hippocampal CA1 area pyramidal cellswas measured to calculate the total number and distribution of excitatory and inhibitory inputs on a single cell.

A single pyramidal cell has ,12,000 mm dendrites and receives around 30,000 excitatory and 1700 inhibitory inputs, ofwhich 40% are concentrated in the perisomatic region and 20% on dendrites in the stratum lacunosum-moleculare. The pre-and post-synaptic features suggest that CA1 pyramidal cell dendrites are heterogeneous. Strata radiatum and oriens dendritesare similar and differ from stratum lacunosum-moleculare dendrites. Proximal apical and basal strata radiatum and oriensdendrites are spine-free or sparsely spiny. Distal strata radiatum and oriens dendrites (forming 68.5% of the pyramidal cells'dendritic tree) are densely spiny; their excitatory inputs terminate exclusively on dendritic spines, while inhibitory inputstarget only dendritic shafts. The proportion of inhibitory inputs on distal spiny strata radiatum and oriens dendrites is low(,3%). In contrast, proximal dendritic segments receive mostly (70±100%) inhibitory inputs. Only inhibitory inputsinnervate the somata (77±103 per cell) and axon initial segments. Dendrites in the stratum lacunosum-moleculare possessmoderate to small amounts of spines. Excitatory synapses on stratum lacunosum-moleculare dendrites are larger than thesynapses in other layers, are frequently perforated (,40%) and can be located on dendritic shafts. Inhibitory inputs, whosepercentage is relatively high (,14±17%), also terminate on dendritic spines.

Our results indicate that: (i) the highly convergent excitation arriving onto the distal dendrites of pyramidal cells isprimarily controlled by proximally located inhibition; (ii) the organization of excitatory and inhibitory inputs in layersreceiving Schaffer collateral input (radiatum/oriens) versus perforant path input (lacunosum-moleculare) is signi®cantlydifferent. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved.

Key words: synaptic convergence, dendrite geometry, serial reconstruction, 3D, electron microscopy, database for modeling.

The principal cells of the hippocampal CA1 area, thepyramidal cells, integrate information arriving fromseveral sources. Schaffer collaterals from the CA3 areaform synapses on dendrites located in strata radiatum andoriens; input from the entorhinal cortex and differentsubcortical structures (e.g. nucleus reuniens thalami,amygdala) innervate the distal apical dendrites in stratumlacunosum-moleculare, whereas recurrent collateralsfrom CA1 pyramidal cells innervate basal dendrites.3,36

Several functionally different interneuron populationscontrol pyramidal cell activity via axon terminals thattarget precise domains of the postsynaptic cells.7,14,17

Inhibitory cells terminating in the perisomatic regioncontrol the generation of Na1 spikes and thus the outputof the neurons. Inhibition in the dendritic region, in

contrast, controls the generation of dendritic Ca21 spikesand is associated with synaptic plasticity in dendrites.27

Although several studies have estimated the totalnumber of inputs converging onto a pyramidal cell bycounting spines,5,23,30,33 important aspects of pyramidalcell innervation may have gone unnoticed at the lightmicroscopic level. Measurements of spine density didnot reveal the detailed organization of excitatory inputson different cell domains. The number, distribution andratio of inhibitory synapses converging onto pyramidalcells has not been studied either. The arrangement ofexcitatory and inhibitory inputs relative to each other,their ratio and the distribution of the inputs on thecells' surface are important parameters in¯uencing inte-grative properties and cellular activity patterns. Thisinformation may shed light on how pyramidal cells trans-form signals arriving from different sources.

We recently described the number, ratio and distribu-tion of excitatory and inhibitory inputs on three subsetsof hippocampal interneurons in the CA1 area,15 andfound signi®cant differences as well as common prin-ciples in the organization of synaptic inputs amongthese cell populations. Interneurons and principal cellsshow rather different average activity levels10 and®ring patterns.11 Mapping the distribution of terminals

Synaptic convergence onto CA1 pyramidal cells 527

527

Neuroscience Vol. 102, No. 3, pp. 527±540, 2001q 2001 IBRO. Published by Elsevier Science Ltd

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*Corresponding author. Tel: 136-1-2100-819/246; fax: 136-1-313-9498.

E-mail address: gulyas@koki.hu (A. I. GulyaÂs).Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole-

propionate; BDA, biotinylated dextran amine; CA, cornu ammonis;CB, calbindin D28k; CCK, cholecystokinin; CR, calretinin; DAB,3,3 0-diaminobenzidine; PB, phosphate buffer; PV, parvalbumin;SLM, stratum lacunosum-moleculare dendrites; SRO, strata radia-tum and oriens dendrites; TBS, Tris-buffered saline; VIP, vaso-active intestinal polypeptide.

on pyramidal cells provides structural parameters that arenecessary to understand how interactions between exci-tatory and inhibitory inputs combine to shape cellularactivity.

To answer these questions, the complete dendritictrees of CA1 pyramidal cells in the adult rat hippocampuswere reconstructed in 3D at the light microscopic level,followed by serial electron microscopic reconstruction ofexcitatory and inhibitory synaptic inputs converging ontodifferent dendrites, somata and axon initial segments.The density, distribution and ratio of inhibitory and exci-tatory inputs were measured, and the total number ofconverging inputs was calculated.

EXPERIMENTAL PROCEDURES

Seven adult (300 g) male Wistar rats (Charles River, Budapest)were used in these experiments. The animals were anesthetizedwith Equithesin (chlornembutal, 0.3 ml per 100 g of bodyweight). Biotinylated dextran amine (BDA, Molecular Probes;10% in phosphate buffer, PB) was injected into the CA1 regionat the following coordinates: Bregma: 23.2 mm; lateral: 2 mm(left and right); 22.4 mm from pia mater; and Bregma:24.3 mm; lateral: 3 mm (left and right); 22.4 from pia mater.The BDA was iontophoretically applied by pulsed positivecurrent for 5 min, 5 mA, 7 s on/7 s off via a glass capillary.

After two days survival, the animals were anesthetized withEquithesin (chlornembutal, 0.3 ml per 100 g of body weight) andperfused transcardially with 50 ml of physiological salinefollowed by 300 ml of ®xative containing 3% paraformaldehyde,1% glutaraldehyde and 15% saturated picric acid in 0.1 M PB(pH 7.4). All experiments were approved by the animal carecommittee of the Institute. Every effort was made to minimizeanimal numbers and suffering during the experiments.

The brains were removed from the skull and post®xed (1 h).Then 60-mm-thick sections were cut on a Vibratome. Thesections were kept in sequential order to allow serial reconstruc-tion of the dendritic arbors. To calculate possible shrinkageduring the experimental procedure, several sections were placedon a slide and their pro®les and cut capillaries were drawn using acamera lucida. To increase penetration of reagents, sections werecryoprotected by immersion in 25% sucrose and 10% glycerol in0.1 M PB for 30 min, and freeze-thawed three times over liquidnitrogen. Sections were treated with 1% NaBH4 for 30 min. BDAwas then visualized by incubation in Elite ABC (1:200, Vector,2 h), and developed using 0.03% DAB (3,3 0-diaminobenzidine-4HCl, Sigma) and hydrogen peroxide (0.01%, 5 min). Sectionswere treated with 1% OsO4 in 0.1 M PB for 40 min, dehydratedand embedded in Durcupan (ACM Fluka). The shrinkage wasestimated by checking the change in the size of the sectionsdrawn before the immunostaining.

Light microscopic sampling

The BDA injection sites contained large amounts of labeledneurons hindering the identi®cation of individual dendrites (Fig.1B). Therefore we selected neurons for reconstruction at adistance from the injection sites, where individually visualizedcells could also be found. We selected only those cells where allthe dendrites could be clearly followed until their natural end.The dendritic segments were classi®ed into different subclasses ineach layer, based on their order, spine density and distance fromthe soma. The division between subclasses was evident due tothe organization principles of pyramidal cell dendrites. Cells(n� 20) were reconstructed from six to 13 sections using cameralucida. The subclasses of dendrites and their location wererecorded. The 3D structure of the neurons and data on the lengthof dendritic subclasses were obtained after the digitalization ofthe reconstructed cells using the program ARBOR developed byS. PomahaÂzy and modi®ed by A. I. GulyaÂs.15,35

Sampling for electron microscopy. Four to six segments ofeach dendritic subclass were selected, re-embedded and seriallysectioned for electron microscopy (samples derive from ®veanimals, each dendritic subclass was sampled in at least twoanimals, at least two samples per animal). Before and after theserial ultrathin sectioning, dendrites were drawn using a cameralucida to determine the length of the sectioned segment. To mini-mize the error in the measurements we cut long series of ultrathinsections containing on average 25-mm-long segments of den-drites. The synapses were classi®ed as symmetrical or asym-metrical from the postsynaptic density and the type of vesicles.13

Spine densities shown in Table 2 derive from the electron micro-scopic measurements. We noticed differences in the size ofsynapses in different layers. We made a semi-quantitativemeasurement: in the case of 30 synapses from each dendriticsubclass we counted the number of sections the synapses occu-pied to gain a distribution of synapse size. To count the numberand types of synapses on somata and axon initial segments, longserial sections (300±400 sections, 55 nm thickness) were cutfrom the CA1 pyramidal cell layer. Unlabeled pyramidal cellbodies (n� 5, from two animals) were completely reconstructed.Axon initial segments of labeled cells with a long straight passagewere also reconstructed (n� 4, from three animals).

In case of the input density measurements of this paper, stan-dard errors are not given, since, due to the rather laborious natureof the sampling, the sample size for individual dendritesubclasses from each layer was small (three to ®ve segments).

Immunogold detection of GABA. The immunostaining proce-dure followed those described by Somogyi and Hodgson,32 withsmall modi®cations,15 using a well-characterized antiserumagainst GABA.21 The steps were carried out on droplets ofMillipore-®ltered solutions in humid Petri dishes, as follows:2% periodic acid (H5IO6) for 10 min; wash by dipping in severalchanges of double-distilled water; 2% sodium metaperiodate(NaIO4) for 10 min; wash as before; three times 2 min in TBS

M. MegõÂas et al.528

Fig. 1. CA1 pyramidal cells visualized by extracellular BDA injection. After BDA injection in CA1, a tightly packed group ofstained pyramidal cells was observed at the injection site (long arrow in B). At distances of 1 or 2 mm, some cells were labeledretrogradely (short arrow in B) and individual cells could be distinguished and reconstructed completely. (A) Apical dendrite. Thespine density increases from the proximal to the distal part of the dendrite. In the proximal part (P: radiatum/Thick/proximal) nospines are visible; they start to appear at around 100 mm from the soma, and increase progressively in number through the middlesegment (m, radiatum/Thick/medial) until they reach the distal third (d, radiatum/Thick/distal) of stratum radiatum where theirdensity is maximal. Open arrows label subclass boundaries. (C) Thin spiny dendrites (short arrows, radiatum/thin) branch from theapical dendrite in stratum radiatum. These dendrites are homogeneous in appearance and could be clearly distinguished from labeledinterneuron dendrites (long arrow). (E) In the stratum oriens, proximal dendrites bore no spines (curved arrow, oriens/proximal).Spine number increased gradually with the distance from the soma, reaching and maintaining a maximal density (arrows) on themost distal portions (oriens/distal). Axon initial segments were also labeled (arrowheads). (F) The density of spines decreasedconsiderably at the stratum radiatum (r, long arrow)/stratum lacunosum-moleculare (lm, short arrow) border. (G) Spines in thestratum lacunosum-moleculare are larger and less frequent, their number decreases from thick (large arrow, l-m/Thick) to mediumdendrites (short arrow, l-m/medium). (H) The most distal dendrites in the stratum lacunosum-moleculare are very thin and long (l-m/thin). They bear very few spines (large arrows) and show varicosities (short arrows). Scale bars� 4 mm (A), 250 mm (B), 20 mm (D),

10 mm (C, E±H).

(pH 7.4); 30 min in 1% ovalbumin dissolved in TBS; three times10 min in TBS containing 1% normal goat serum; 1±2 h in arabbit anti-GABA antiserum (Code No. 9, diluted 1:1000 innormal goat serum/TBS); two times 10 min TBS; 10 min in0.05 M Tris buffer (pH 7.4) containing 1% bovine serum albuminand 0.5% Tween 20; goat anti-rabbit IgG-coated colloidal gold(12 nm, Jackson) for 2 h (diluted 1:20 in the same buffer); twotimes 5 min wash in double-distilled water; saturated uranyl

acetate for 30 min; wash in four changes of double-distilledwater; staining with lead citrate; wash in distilled water. Pro-®les showing a density of colloidal gold particles at least ®vetimes above background level, in two to three adjacent sec-tions, were considered GABA-immunoreactive. Axon terminalsforming asymmetrical synapses (presumed glutamatergic) wereused to establish background density. Replacement of the GABAantiserum with normal rabbit serum in the postembedding

Synaptic convergence onto CA1 pyramidal cells 529

Fig. 1.

immunogold reaction resulted in a loss of speci®c staining; i.e. nosigns of colloidal gold accumulation could be detected over anypro®les.

RESULTS

Light microscopy

Biotinylated dextran amine (BDA) injected into theCA1 area labeled a dense group of cells in the centerof the injection site, as well as cells several hundredmicrometers away from the injection site, probably byretrograde transport. These solitary cells (Fig. 1D)showed very dense precipitation of DAB so that den-drites could be followed and morphological featuresdescribed in detail (Fig. 1C). The labeling visualizedthe ®ne dendritic spines and the dendrites could befollowed until their natural ends terminated abruptly,without fading within the sections. We used these soli-tary cells for the reconstructions.

During the experimental procedures the shrinkage ofthe slices was checked. The dimensions of the sectionsbefore and after the BDA visualization and embeddingremained the same, so no corrections were applied forshrinkage. Note also that for the calculation of the totalnumber of synapses, no correction was necessary, sincelight and electron microscopic data were obtained fromthe same material.

Dendritic features and types. Although several studiesand databases have quanti®ed the dendritic length ofCA1 pyramidal cells,5,23,30,33 we needed to classify thedendrites and to measure the length of each subclassseparately in order to calculate the total synaptic conver-gence. We reconstructed 20 pyramidal cells from theCA1 region of the dorsal hippocampus (the detailedgeometry of the cells together with additional data, notpresented in the paper, can be downloaded from: http://www.koki.hu/,gulyas/ca1cells). As shown in Figs 1 and2, pyramidal cell dendritic trees are rather heterogeneous.The dendrites were classi®ed into nine subclassesaccording to their location, diameter, and the density ofspines on them. Dendritic subclasses and their character-istic diameters are listed in Fig. 2 and Table 1.

Pyramidal cells have a basal dendritic tree consistingof three to ®ve (3.55^ 0.82) primary dendrites and anapical dendritic tree which arises in the form of a thickapical dendrite ascending until stratum lacunosum-moleculare and emitting secondary branches in stratumradiatum. Two dendritic subclasses were distinguished in

the basilar dendritic tree: (i) thick dendrites, close to thesoma with few or no spines in upper stratum oriens (oriens/proximal); and beyond that (ii) thinner distal dendritesdensely covered with spines (oriens/distal). The spinedensity progressively increased from spine free segments(up to 30±50 mm from the soma) until the ®rst branch-point, where they reached their maximum density (Figs1E, 2).

The thick apical trunk of the pyramidal cells emitsmany secondary thinner dendrites in stratum radiatum,and upon reaching stratum lacunosum-moleculare bifur-cates several times to form thinner branches of differentappearance. The bifurcating apical dendrites turn, andrun parallel with the hippocampal ®ssure spanning aconsiderable distance. The diameter (lateral spread) ofthe apical dendritic tree in stratum lacunosum-moleculareis thus often larger (350±750 mm, 462.4^ 132.5 mm)than the diameter of the dendritic arbor in stratum oriens(170±320 mm, 250.5^ 54.5 mm) and stratum radiatum(200±350 mm, 255.6^ 54.2 mm).

Thick apical dendrites were divided into threesubclasses, based on spine density: (i) a proximal seg-ment of approximately 100 mm with no spines (radiatum/Thick/proximal); (ii) a medial segment (,150 mm) witha low spine density (radiatum/Thick/medial); and (iii) athick distal segment (,200 mm) with very high densityof spines (radiatum/Thick/distal, Figs 1A and 2). Thinnerbranches, originating from the apical dendrites in stratumradiatum formed the fourth group (radiatum/thin). Thesedendrites represented the majority of dendrites in thislayer and were similar in appearance to oriens/distaldendrites.

The spine density decreased on thick (bifurcated)apical dendrites entering stratum lacunosum-moleculare(Fig. 1F). Here we distinguished three dendrite sub-classes: (i) the prolongation of the thick apical dendrite(l-m/Thick, ,1.1 mm) that were covered by fewer butlarger spines than in stratum radiatum (Fig. 1G, longarrow); (ii) after further bifurcations the segments carriedfewer spines of smaller size (l-m/medium, Fig. 1G, shortarrow); and ®nally (iii) thin distal dendrites (Fig. 1H)representing the majority of the dendrites in stratumlacunosum-moleculare (l-m/thin). These dendrites pos-sessed very few spines and also showed varicositiesand segments without spines.

Dendritic lengths. Following the light microscopicreconstruction, the total dendritic length as well as the

M. MegõÂas et al.530

Fig. 2. Dendritic structure of a CA1 pyramidal cell ®lled with BDA. The drawing illustrates the subclasses of dendrites distinguishedand sampled in the study (illustrated by light micrographs in Fig. 1). Two types of dendrites were classi®ed in stratum oriens.Proximal basal dendrites carried no spines or were sparsely spinous (oriens/proximal). Spine density increased until the ®rst branchpoint (,50 mm), where the second subclass of dendrites (oriens/distal) began; these carried large numbers of spines throughout theirextent. In stratum radiatum, four subclasses of dendrites were distinguished. The thick apical dendritic trunk was divided into threesegments: a proximal part with no spines (radiatum/thick/proximal), a medial sparsely spiny part (radiatum/Thick/medial) and adensely spiny distal part (radiatum/Thick/distal). The fourth type corresponded to the thin side branches (radiatum/thin) arising fromthe thick apical dendrite. In stratum lacunosum-moleculare, three subclasses of dendrites were identi®ed on the basis of diameter andspine density: dendrites which were a continuation of the radiatum thick dendrites possessed fewer spines than the radiatumdendrites, and were relatively thick (l-m/Thick). These dendrites became thinner and sparsely spinous (l-m/medium). In themore distal apical parts, the dendrites were nearly spine-free, and often became beaded and very thin (l-m/thin). For every dendriticsubclass the density of asymmetrical, symmetrical (left and middle numbers, respectively, number/1 mm) and the proportion of

symmetrical synapses (right number) are shown. Scale bar� 100 mm.

Fig. 2.

length of dendrites in each layer were calculated (Fig.3B, Table 1). The total dendritic length was11.5^ 2.0 mm and it was distributed unimodally. Thecontribution of the strata radiatum and oriens dendritesto the total dendritic length was similar (4.63^ 1.0 mmand 4.2 ^ 1.0 mm, respectively), whereas the stratumlacunosum-moleculare dendrites represented approxi-mately 2.7 ^ 0.8 mm. Thin dendrites, densely coveredby spines in strata oriens and radiatum, represented morethan 60% of the total dendritic length. The contributionof l-m/thin dendrites is also signi®cant. The apicalbranches form about two thirds of the total dendritic tree.

Electron microscopy

The number and ratio of excitatory and inhibitoryinputs terminating on each subclass of dendrite wascalculated from electron microscopic observations ofserial sections. `Excitatory' and `inhibitory' synapseswere identi®ed in two ways. In a smaller set of samples,postembedding GABA immunostaining was made onserial sections of all subclasses of dendrites. Inhibitoryterminals were identi®ed on the basis of accumulatedimmunogold particles. On a larger set of samples thesymmetrical or asymmetrical nature of the synapseswas identi®ed. The two methods gave similar results.All symmetrical synaptic contacts were formed byGABA-positive (Figs 4D, E, 5F, 7E, F) terminals,whereas the GABA-negative terminals (Figs 4D, E,

5D) were associated with asymmetrical synaptic special-izations. We found a large difference in synaptic organi-zation between stratum lacunosum-moleculare dendritesand the relatively similar strata oriens and radiatumdendrites (Figs 4, 5). Therefore, the two sets of dendriteswill be described separately.

Strata radiatum and oriens dendrites. In agreementwith previous studies19 the asymmetric synapses termin-ated on spines, regardless of the subclass of dendrite(Figs 4, 9B). Every spine received only one synapticcontact that was always asymmetrical. The morpho-logical heterogeneity of spines was similar to thatreported in previous studies.5,20 No correlation wasapparent between the dendrite subclasses and the spinesize or shape. The size of the synapses also varied (Fig.4C). By counting the number of sections spanned by asingle synapse we compared the size of asymmetricalsynapses on different dendritic subclasses. Analysis of30 synapses for each dendritic subclass resulted in simi-lar, unimodal distributions, with extents ranging fromtwo sections (approximately 20%) to six sections(approximately 15%). Synapses most often occupiedthree to four sections (approximately 30%).

The density of spines and thus the density of asym-metrical synapses varied among the different dendritesubclasses. On oriens/proximal and radiatum/Thick/proximal dendrites, there were no asymmetrical contacts.In contrast, on radiatum/Thick/distal dendrites, the siteof the highest spine (synapse) density, there were 6.98asymmetrical synapses (spine) per micrometer. The oriens/distal and radiatum/thin dendrites were extensivelycovered by a rather similar density of spines (3.08 and3.52 synapses/mm, respectively; Figs 2, 6A and Table 3).

Perforated (or partitioned) asymmetrical synapses

M. MegõÂas et al.532

Table 1. Length and proportion of different dendritic subclasses

Length (mm) Contributionto total

Dendritediameter (mm)

Ori/dist 3815^ 1023 33.0% 0.3; 0.25±0.45Ori/prox 382.8^ 129.1 3.3% 0.7; 0.50±0.9Rad/T/prox 114.2^ 19.4 1.0% 2.1; 1.8±2.5Rad/T/med 117.4^ 47.6 1.0% 2.0; 1.6±2.2Rad/T/dist 311.3^ 137.5 2.7% 1.2; 1.0±1.5Rad/t 4095^ 895 35.5% 0.5; 0.45±0.55L-M/T 283.0^ 174.8 2.5% 1.1; 0.8±1.2L-M/M 610.2^ 216.6 5.3% 0.6; 0.3±0.8L-M/t 1818^ 631 15.7% 0.2; 0.15±0.4Total 11549^ 2010Ori 4198^ 1056 36.3%Rad 4638^ 1022 40.2%L-M 2712^ 873 23.5%Proximal 497.0^ 140.9 4.3%Distal 11052^ 1992 95.7%Apical 7350^ 1494 63.6%Basal 4198^ 1056 36.4%

T, Thick; M, medium; t, thin dendrites, respectively. The most char-acteristic diameter value is followed by the range of diameterswithin a dendrite subclass. Rad, stratum radiatum; Ori, stratumoriens; L-M, stratum lacunosum-moleculare; prox, med, dist repre-sent proximal, medial and distal parts, respectively.

Fig. 3. Dendritic length of CA1 pyramidal cells. (A) Total dendriticlength, length by layers, by distance from the soma and length ofapical versus basal dendritic tree. (B) Length of each dendriticsubclass examined. Note that the majority of dendrites belong to

the densely spiny thin oriens and radiatum dendrites.

were observed on spines of different dendritic subclasses.Because such structures have been implicated in synapticplasticity,12 we recorded their occurrence. We found nodifference in the ratio of perforated synapses on spines ofdifferent dendritic segments in these layers. Theyappeared to distribute randomly along the differentspiny dendrites and represented 10.7% of the asym-metrical synapses.

Symmetrical synapses on strata radiatum and oriensdendrites were always located on dendritic shafts (Figs4B±D, 7B, D, 9C). Their density and distributioncomplemented the distribution of the spines and thus of

asymmetrical synapses. They were most abundant onspineless proximal dendrites (oriens/proximal and radia-tum/Thick/proximal), where less asymmetrical synapseswere found. The number of symmetrical synapses permicrometer increased towards the soma, i.e. the radia-tum/Thick/proximal and oriens/proximal dendritesshowed 1.72 and 0.61 symmetrical synapses per micro-meter, respectively, whereas the radiatum/thin andoriens/distal showed 0.11 symmetrical synapses permicrometer (Table 2). Close to the soma, synapseswere nearly exclusively inhibitory (98.1% on radiatum/Thick/proximal and 48.6% on oriens/proximal dendrites),

Synaptic convergence onto CA1 pyramidal cells 533

Fig. 4. Synaptic inputs on dendrites in strata oriens and radiatum. (A) Thick apical dendrite (radiatum/Thick/distal) located in distalradiatum. The BDA staining reliably visualized all spines. (B) and (C) Consecutive sections from the ®eld shown by a rectangle in A.Asymmetrical synapses were always found on spines (curved arrows), whereas symmetrical synapses were located on the shafts(white arrowhead). Sometimes symmetrical synapses formed another synaptic contact with additional dendrites (double arrowhead).The asymmetrical synapses showed large differences in size and contacted spines of different types and sizes. The larger spinesspanned ®ve to seven sections (curved arrow on the left), whereas small spines can be seen only in two sections (curved arrow on theright). (D) and (E) In both strata oriens (D) and radiatum (E), all the terminals tested for GABA immunoreactivity were negative on

spines (curved arrow) and positive on the shafts (arrowhead). Scale bars� 1 mm (A), 0.25 mm (B±E).

whereas their ratio was only 3.3±2.9% on the thin spinydendrites in strata oriens and radiatum. Symmetricalsynapses were in general larger than asymmetricalsynapses, and were present in ®ve to 14 sections.

Stratum lacunosum-moleculare. As apical dendritesentered stratum lacunosum-moleculare, spine densitydropped to 1.72 spines per micrometer. This density wasdecreased to 0.60 spines per micrometer in l-m/mediumdendrites, and to 0.37 spines per micrometer in the l-m/thin dendrites (Fig. 1F±H; Table 2). In contrast to the strataradiatum and oriens, asymmetrical synaptic contacts couldalso be observed on dendritic shafts (Fig. 5C±E), mainly

on the l-m/medium and l-m/thin dendrites, so in this regionthe density of asymmetrical synapses was higher than spinedensity (Fig. 6; Table 2). In general the size of asym-metrical synapses was larger than in the strata radiatumand oriens. Asymmetrical synaptic contacts frequentlyspanned ®ve to six sections (30%), while small synapticcontacts ranging from one to two sections represented only10±15% of all asymmetrical synapses. On the l-m/thindendritic segments, where spines were infrequentlyobserved, asymmetrical synapses were located on dendriticvaricosities. The ratio of asymmetrical synapses showingperforated synaptic junctions was also higher than in strataoriens and radiatum. The perforated synapses were most

M. MegõÂas et al.534

Fig. 5. Pyramidal cell dendrites in stratum lacunosum-moleculare. The synaptic organization of stratum lacunosum-molecularedendrites differed from dendrites in other layers. (A), (B) Spines originating from dendritic shafts (S) in stratum lacunosum-moleculare were larger than in strata radiatum and oriens. Presynaptic boutons were also larger (curved arrow) and often formedperforated synapses (straight arrow). Symmetrical synapses making synaptic contact on spines could also be seen (arrowhead).These inhibitory terminals frequently contacted other postsynaptic targets (double arrowhead). (C), (D) Medium dendrites (l-m/medium) possessed few spines (black curved arrow in D) and often received asymmetrical synapses from GABA-negative terminalsonto their shafts (white curved arrow). Asterisks (in D) show GABA-positive pro®les. (E) Perforated synapses were observed bothon spines (straight arrow) and dendritic shafts (S, curved arrow). The spine shown was innervated by a terminal that formedsymmetrical synapses on it (arrowhead) and also on a dendritic shaft (double arrowhead). (F) Symmetrical synapses (arrowhead)

in stratum lacunosum-moleculare were formed by GABA-positive terminals. Scale bars� 1 mm (C), 0.25 mm (A, B, D±F).

abundant on l-m/Thick (34, 37%) and l-m/medium (47,89%) dendrites. They represented 28.7% of the total asym-metrical synapses in the stratum lacunosum-moleculare(Figs 5A, B, E, 8 and Table 2).

The ratio of symmetrical synapses was higher (15%,Figs 6, 8C and Table 2) on all subclasses of stratumlacunosum-moleculare dendrites than in the strata oriensand radiatum dendrites, excluding the proximal region.The number of symmetrical synapses decreased withdendritic thickness from 0.28^ 0.19 synapses per micro-meter on l-m/Thick dendrites to 0.09^ 0.06 synapses permicrometer on l-m/thin dendrites. Symmetrical synapseswere located mainly on shafts (Fig. 5F), but around 10%of them contacted spine heads (Fig. 5A, B, D).

Somatic input. The number of synapses on the pyra-midal cell bodies was obtained by serial reconstruction ofcomplete somata (n� 5). All synaptic inputs convergingonto the somata were symmetrical (Fig. 7A, C) andGABA-positive (Fig. 7E). On average, 91.6^ 12 syn-apses per cell body were found. With soma surfaces of465.4^ 50.2 sq. micrometer, the density of synapses isthus 19.91 synapse/100 sq. micrometer. Synapses weredistributed all over the soma surface without evidentclustering, although areas in close contact with othersomata were not innervated. CA1 pyramidal cellsreceive somatic inhibitory input from two distinct setsof basket cells:1 those containing parvalbumin and

those immunopositive for CCK/VIP. The axon terminalsof the two types of cells can be differentiated in theelectron microscope, because only CCK/VIP terminalscontain dense-core vesicles. In our sample, 18% of thepresynaptic terminals contained at least one dense-corevesicle when examined in the serial sections. Althoughthis might be a minimum estimate for the ratio of CCK/VIP terminals, it suggests that the majority of the somaticinput derives from parvalbumin-containing basket cells.

Axon initial segments. Four axon initial segments wereserially sectioned (36-, 32-, 24- and 50-mm-longsegments were reconstructed) and received 25, 27, 22and 23 synaptic contacts, respectively (Fig. 7G), giving0.68 synapses per micrometer. However, the distribu-tions were not homogeneous; portions with differentcoverage of synaptic terminals could be observed. Oneof the initial segments was followed until the ®rst col-lateral branchpoint (,50 mm from the soma), and syn-aptic contacts were still apparent at this distance. Sinceaxons were not followed until the beginning of myelina-tion, these values may under-estimate the total number ofaxo-axonic synapses per pyramidal cell, and thereforewere not included when the total number of synapsesper cell was calculated.

Total number of synapses

The total number of synaptic inputs on CA1 pyramidalcells was calculated from the length of different dendritesubclasses and the associated values for synapse density.We estimate that a typical CA1 pyramidal cell receives32,351^ 5486 synaptic contacts on its dendritic tree(Fig. 8A, Table 3), of which 94.7% are asymmetricaland only 5.3% are symmetrical (Fig. 8B, Table 3). Themajority of the excitatory input is concentrated in stratumradiatum (55.1%, Table 3) and to a smaller extent inoriens (39.1%), because a considerable fraction of thetotal dendritic tree is formed by the densely spiny, thindendrites in these layers (Fig. 3B). The total number ofspines (30,382^ 5214) is less than the total number ofGABA-negative synapses (30,637^ 5259) since, in stra-tum lacunosum-moleculare, GABA-negative synapsesterminate on both dendritic shafts and spines. As withthe organization of the dendritic tree, a larger part ofthe synapses is located on the apical dendritic tree(60.9%), than on the basal dendritic tree (39.1%). For atypical pyramidal cell, perforated synapses form only10.2% of the total, and the majority of these contactsare on different subclasses of the stratum lacunosum-moleculare dendrites (7.4±48.0%, depending on sub-class; Fig. 9A). Unconventionally located asymmetricalsynapses on stratum lacunosum-moleculare dendriticshafts (10.7±19.2%) account for only 0.8% of the totalsynapses (Fig. 9B).

As shown in Fig. 8C, inhibitory synapses are concen-trated on the soma and proximal dendrites (40.5% of allsymmetrical synapses). GABAergic synapses terminat-ing on pyramidal cell soma and axon initial segments,the only inputs to these domains, form 7.2% of all inhi-bitory synapses. Inhibitory inputs predominate (98.1% of

Synaptic convergence onto CA1 pyramidal cells 535

Fig. 6. Density and ratio of excitatory and inhibitory synapses ondifferent subclasses of dendrite. (A) Density of asymmetrical andsymmetrical synapses expressed as number per mm. (B) Ratio ofasymmetrical and symmetrical synapses on different dendriticsubclasses. Note that on dendrites giving the majority of total dendri-tic length (ori/dist and rad/t) the GABA-negative inputs are much

more frequent than GABA-positive inputs.

synapses are symmetrical) on the radiatum/Thick/proximaldendrites, and represent around half of the synapses(48.6%) on oriens/proximal dendrites. These proximaldendrites, which form only 6.7% of the total dendritictree, carry 33.4% of the total symmetrical synapsesconverging onto a pyramidal cell. As shown above, theratio of symmetrical synapses on different subclassesof stratum lacunosum-moleculare dendrites is higher(14.1±17.0%) than on other parts of the dendritic tree.Another unique feature of symmetrical synapses in stratumlacunosum-moleculare is that they sometimes targetdendritic spines. These contacts form only 0.08% ofthe total synapses (Fig. 9C).

DISCUSSION

The main ®ndings of the present study are: (1) Pyra-midal cell dendrites are heterogeneous in diameter, spinedensity and in the number of asymmetrical and sym-metrical synaptic inputs. Strata radiatum and oriensdendrites (SRO) are similar to each other both at thelight and electron microscopic levels and are differentfrom stratum lacunosum-moleculare dendrites (SLM)with respect to dendritic shaft diameter, branchingpattern and spine density. (2) The ratio of symmetricalversus asymmetrical synapses on different parts of theCA1 pyramidal dendritic tree is highly variable butconsistent with dendrite type. (3) The majority of the

excitatory input arrives onto spiny SRO dendrites.Here, asymmetrical synapses terminate exclusively ondendritic spines, while symmetrical synapses contactdendritic shafts at much lower density. (4) Spine-free orsparsely spiny proximal dendritic shafts receive onlysymmetrical synapses, similar to somata and axon initialsegments, thus inhibition is concentrated in the perisomaticregion. (5) Asymmetrical synapses in stratum lacunosum-moleculare are larger than in other layers, they arefrequently perforated and also contact dendritic shafts. Inthis layer, symmetrical synapses, whose percentage is rela-tively high, also terminate on dendritic spines.

Pyramidal cells had a total dendritic length of approxi-mately 12 mm, consistent with previous results.2,5,28,31

Earlier estimates of the total synaptic inputs were basedon the observation that nearly all the asymmetricalsynapses terminate on spines of hippocampal pyramidalcells.19 Therefore, excitatory inputs have been largelyquanti®ed from light microscopic measurements ofspine density. This method may underestimate thenumber of spines, since smaller spines, and those locatedunder and above the dendritic shaft, may not be detectedin the light microscope. This might explain the reportedlower spine density values (0.7±1.9 spines per micro-meter; see Refs 2, 4) compared to the present measure-ments at the electron microscopic level (3.0±3.5 spinesper micrometer on spiny SRO dendrites). Our calculationsgave a density similar to that reported by Bannister and

M. MegõÂas et al.536

Fig. 7. The somatic and proximal dendritic region of a CA1 pyramidal cell is shown in (A). Open arrows identify segments enlargedin B±D. Pyramidal cells received exclusively symmetrical synapses in their perisomatic region; they were found on the soma (C) andon the proximal apical (B) and basal dendrites (D). All symmetrical synapses studied with postembedding GABA immunostainingwere positive (arrowheads) both on the somata (E) and the proximal apical dendrites (F). (G) The axon initial segments received only

symmetrical synapses (arrowhead). Scale bars� 2.5 mm (A), 0.25 mm (B±G).

Sy

nap

ticco

nverg

ence

onto

CA

1pyram

idal

cells537

Table 3. Total number of synapses terminating on distinct dendrite subclasses

Dendritesubclass

TotalSynapse number

TotalGABA2

TotalGABA1

TotalPerforated

Total GABA2on shaft

Total GABA1on spine

Spines GABA2(%)

GABA1(%)

% of allGABA2

% of allGABA1

Ori/dist 12141^ 3255 11735^ 3147 405^ 108 1047^ 281 11735^ 3147 96.7 3.3 38.3 23.7Ori/prox 479^ 161 246^ 83 233^ 78 22.0^ 7.4 246^ 83 51.4 48.6 0.8 13.6Rad/T/prox 196^ 33 3.8^ 0.6 193^ 32 0.4^ 0.1 3.8^ 0.6 1.9 98.1 0.0 11.3Rad/T/med 340^ 138 277^ 112 63.0^ 25.6 28.1^ 11.4 277^ 112 81.5 18.5 0.9 3.7Rad/T/dist 2219^ 980 2171^ 959 47.6^ 21.0 219^ 96 2171^ 959 97.9 2.1 7.1 2.8Rad/t 14862^ 3249 14425^ 3154 437.1^ 95.6 1456^ 318 14425^ 3154 97.1 2.9 47.1 25.5L-M/T 565^ 349 485.7^ 300.0 79.6^ 49.1 194^ 120 9.8^ 6.1 485^ 300 85.9 14.1 1.6 4.6L-M/M 493^ 175 418.0^ 148.4 75.5^ 26.8 237^ 84 52.8^ 18.8 5.8^ 2.1 365^ 129 84.7 15.3 1.4 4.4L-M/t 1051^ 365 873.0^ 303.0 178.6^ 62.0 77.5^ 26.9 202^ 70 11.9^ 4.1 670^ 232 83.0 17.0 2.8 10.4Total 32351^ 5486 30637^ 5259 1713^ 261 3283^ 587 255^ 82 27.6^ 10.1 30382^ 5214 94.7 5.3 100.0 100.0

LayersOri 12621^ 3292 11982^ 3164 639.2^ 146.6 1069^ 282 11982^ 3164 94.9 5.1 39.1 37.3Rad 17619^ 4085 16878^ 3964 740.8^ 125.9 1704^ 400 16878^ 3964 95.8 4.2 55.1 43.2L-M 2110^ 726 1776^ 613 333.6^ 113.0 509^ 193 255^ 82 27^ 10 1521^ 541 84.2 15.8 5.8 19.5

Rad, stratum radiatum; Ori, stratum oriens; L-M, stratum lacunosum-moleculare; T, Thick; M, medium; t, thin dendrites, respectively; prox, med, dist represent proximal, medial and distal parts respectively.

Table 2. Density (number per micrometer) and ratio of different synapses terminating on distinct dendrite subclasses

Total density Spine density GABA2 GABA 1 Perforated GABA2 shaft GABA1 spine GABA2 (%) GABA1 (%) Perforated (%)

Ori/dist 3.18 3.08 3.08 0.11 0.27 96.7 3.3 8.6Ori/prox 1.25 0.64 0.64 0.61 0.06 51.4 48.6 4.6Rad/T/prox 1.72 0.03 0.03 1.69 1.9 98.1 0.2Rad/T/med 2.90 2.37 2.37 0.54 0.24 81.5 18.5 8.2Rad/T/dist 7.13 6.98 6.98 0.15 0.70 97.9 2.1 9.9Rad(t) 3.63 3.52 3.52 0.11 0.36 97.1 2.9 9.8L-M/T 2.00 1.72 1.72 0.28 0.69 0.03 85.9 14.1 34.4L-M/M 0.81 0.60 0.69 0.12 0.39 0.09 0.01 84.7 15.3 48.1L-M/t 0.58 0.37 0.48 0.10 0.04 0.11 0.01 83.0 17.0 7.4

Rad, stratum radiatum; Ori, stratum oriens; L-M, stratum lacunosum-moleculare; T, Thick; M, medium; t, thin dendrites, respectively; prox, med, dist represent proximal, medial and distal parts, respectively.

Larkman,5 with correction for hidden spines. Spinedensity measurements may also underestimate totalasymmetrical synaptic input because, as we also showed,some of them arrive onto shafts of SLM dendrites. Inhi-bitory synapses were quanti®ed only in the present study.

Besides the glutamatergic and GABAergic local andextrinsic axons, axons of different transmitter contentascend from subcortical areas into the hippocampus.However, the axon terminals of subcortical projectionsusing monoamines or acetylcholine34 only occasionallyform classical synaptic specializations, and thereforetheir contribution to the total synaptic input of pyramidalcells is minimal.

Differences in inputs between strata radiatum/oriensversus stratum lacunosum-moleculare dendrites

Our light and electron microscopic data show that

distinct synaptic organization is associated with differentsubtypes of pyramidal cell dendrites. This suggest that, infunctional terms, the dendritic tree of pyramidal cellsshould be divided into SRO dendrites and SLM dendritesrather than into basal and apical trees. The distinct char-acteristics suggest that integration of synaptic inputsarriving from the CA3 area via the Schaffer collateralsin SRO differs from those arriving from entorhinal cortexand different subcortical structures (e.g. nucleus reuniensthalami, amygdala) onto SLM dendrites.3

A strong current sink has been shown in SLM25

following perforant path stimulation and during thetaactivity,6 suggesting that entorhinal input is powerful inspite of its distal location. This is supported by thepresent morphological data showing larger asymmetricalsynapses in SLM, and a horizontal trajectory of dendritesin this layer allowing multiple contacts from similarly-oriented entorhinal ®bers. It should also be noted thatlarger synapses are associated with more AMPA recep-tors,29 and that perforated (partitioned) synapses (havinga high incidence in this layer) are associated with moreeffective synaptic transmission following long-termpotentiation.12 The arrangement of the inhibitory inputsnicely matches the distribution of excitatory inputs. Thestronger excitation is coupled with an elevated inhibitorysynapse ratio, providing a strict inhibitory control inSLM. That might re¯ect the requirement of a moreprecise transmission of information from diverse affer-ents representing the sensory environment (thalamus,entorhinal cortex, and amygdala) for the formation ofreceptive ®elds (place cells).24

In addition to differences in synaptic inputs to SLM

M. MegõÂas et al.538

Fig. 9. Ratio of special types of synapses on subclasses of dendrites.(A) Proportion of perforated synapses. (B) GABA-negative terminalson dendritic shaft. (C) GABA-positive terminals on spines. Note thelarge difference between stratum lacunosum-moleculare dendrites

and the rest of the dendritic tree.

Fig. 8. Total number of different types of synapses converging onto aCA1 pyramidal cell. (A) Number of synapses and spines in differentlayers. (B) Ratio of GABA-negative and GABA-positive terminals ineach layer. (C) Distribution of synapse types among different domainsof a pyramidal cell. Note that, while a signi®cant portion of theGABA-positive terminals is concentrated on the soma and on the

proximal dendrites, excitatory synapses are devoid in these areas.

and SRO, we also observed a variation in the size ofasymmetrical synapses contacting distinct dendriticsubclasses. Rather small synapses spanning only twoconsecutive sections formed one ®fth of the synapticinputs on SRO dendrites. Similar proportions of smallsynapses were supposed to be `silent' synapses22 in theSchaffer-collateral/CA1 pyramidal cell input system bydemonstrating that they lack AMPA receptors.29 Thismay suggests that around one ®fth of the excitatorysynapses converging onto a pyramidal cell from theSchaffer-collateral system may be `silent'.

Inhibitory input is concentrated in the perisomatic region

The thick proximal dendrites represent the gateway forsynaptic potentials to reach the soma. Very few, if any,excitatory synapses are found on these dendrites, and thedensity of inhibitory contacts is the highest here. Thearrangement of synaptic inputs on these dendrites thusresembles that on the somata and axon initial segments,which receive exclusively inhibitory inputs. Thus, thethick, proximal apical dendrite is essentially equivalentto the cell body in terms of synaptic organization. Conse-quently, a large part of the cell's inhibitory terminalsconverges onto the perisomatic region (40%, Fig. 8C).This suggests that as dendritic inhibition is sparse, theperisomatically clustered inhibitionÐwhich controls theoutput of the neurons27Ðdoes not perform a selectivecontrol over individual dendritic excitatory synapticevents, but rather exercises a non-selective veto function.There is, however, speci®city associated with differentdendritic inhibitory cells. Their axons terminate in speci-®c layers and thus they control different sets of inputpathways.7,16±18 However, as shown by Miles et al.,26

the sparse dendritic inhibition can only be effective iflarge populations of dendritic inhibitory cells aresynchronously activated.

Comparison of interneurons and pyramidal cells

In a recent paper,15 we described the organization ofsynaptic inputs on three different types of CA1 area inter-neurons (parvalbumin-, PV, calbindin D28k-, CB, andcalretinin-, CR, containing cells) in much the same wayas in this study. There are clear differences and simi-larities in the organization of the inputs compared topyramidal cells.

The differences are that pyramidal cells receive severaltimes more (two to 10 times) synaptic inputs (,32,000)than interneurons (,3000±16,000, consistent with thefact that their total dendritic length is longer than forinterneurons), and the ratio of inhibitory terminals onthem is lower (,3% for pyramidal cells, ,4±30% forinterneurons). An important feature shared with inter-neurons is that the inhibitory terminals are concentratedin the perisomatic region. However, whereas inhibitorycells receive both excitatory and inhibitory somatic

inputs, pyramidal cell somata receive only inhibitoryinput.8 The innervation of the axon initial segment isalso different. Interneurons receive only a few (7±10)axo-axonic synapses onto the ®rst few micrometers oftheir axon initial segment, while the examined pyramidalcells received over 25 inhibitory terminals distributedalong the sampled length (up to 50 mm) of the axoninitial segment. These features may at least partly explainthat, despite the large number of converging excitatoryinputs, the average activity level in pyramidal cells issigni®cantly lower than in interneurons.9

We detected differences in synaptic inputs to distincttypes of interneurons. CB and CR cells received rela-tively fewer inputs (3800 and 2200, respectively) witha high ratio of inhibitory inputs (32 and 23%, respec-tively). In contrast, PV cells were contacted by a highernumber of terminals (16,300) and the ratio of inhibitoryinputs was rather low (6.4%). Thus, from the three inter-neuron subclasses, PV cells share the most commonfeatures with pyramidal cells: the density of excitatorysynapses is comparably high and the ratio of inhibitorysynapses is comparably low on the dendrites of the twocell populations. Another important similarity is that theratio of inhibitory inputs is higher on the SLM dendritesof both cell types, suggesting that their input from theentorhinal cortex and other subcortical structures is undera strong inhibitory control. Since these cells receiveinputs from similar sources, the excitatory and inhibitoryinnervation of PV cells and pyramidal cells is rathersimilar.

In conclusion, the present quantitative analysisdemonstrated a very high convergence of excitatoryinputs onto CA1 area pyramidal cells. Excitatory inputs,terminating primarily on distal spiny dendrites, arebalanced by inhibitory inputs that are largely concen-trated in the perisomatic region. This separation of exci-tatory and inhibitory inputs suggests that dendriticexcitatory events are vetoed by the potent perisomaticinhibition, irrespective of their origin. The relativelyhigh ratio of inhibitory terminals in SLM suggests thatentorhinal cortical input is under a stronger inhibitorycontrol than Schaffer collateral input. The similarity inthe synaptic inputs of pyramidal cells and PV-containinginterneuronsÐincluding basket and axo-axonic cellsÐsuggests that the dynamics of perisomatic inhibitioncontrolling the pyramidal cell's output are closelycoupled to the activation of the pyramidal cell itself.

AcknowledgementsÐWe are grateful to Drs L. AcsaÂdy, K. Kailaand R. Miles for helpful discussions and comments on themanuscript, and to Mrs E. BoroÂk, Mr G. Goda and Mrs E.Oswald for excellent technical assistance. This work wassupported by the Howard Hughes Medical Institute; theMcDonnell Foundation; NIH (MH 54671); FPI grants,Ministerio de EducioÂn y Ciencia, Spain; and OTKA (HungarianScienti®c Research Found, T23261), Hungary.

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(Accepted 18 October 2000)

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