serial electron microscopic reconstruction of axon terminals on physiologically identified...

Serial Electron MicroscopicReconstruction of Axon Terminals on

Physiologically IdentifiedThalamocortical Neurons in the Cat

Ventral Lateral Nucleus

FUMI SATO,1* YASUHISA NAKAMURA,1 AND YOSHIKAZU SHINODA2

1Department of Anatomy, Faculty of Medicine,Tokyo Medical and Dental University, Tokyo 113, Japan

2Department of Physiology, Faculty of Medicine,Tokyo Medical and Dental University, Tokyo 113, Japan

ABSTRACTThe distribution of different types of terminals on different portions of single thalamocor-

tical neurons (TCNs) was quantitatively investigated in the cat ventral lateral nucleus (VL)by the application of computer-assisted three-dimensional reconstruction from serial ultra-thin sections. Single neurons in the VL were intracellularly penetrated with a glassmicropipette filled with horseradish peroxidase (HRP), and were electrophysiologicallyidentified as TCNs by their antidromic responses to stimulation of the motor cortex. TheseTCNs received monosynaptic excitation from the contralateral cerebellum. After electrophysi-ological identification, they were injected with HRP iontophoretically. The spatial distributionof terminals of different types on two identified TCNs was analyzed on serial ultrathinsections, some of which were stained by a postembedding immunogold technique by using ag-aminobutyric acid (GABA) antibody. Terminals that synapsed on the injected cells werecategorized as LR terminals (GABA-negative large axon terminals containing round vesicles),SR terminals (GABA-negative small axon terminals containing round vesicles), P terminals(GABA-positive axon terminals of various sizes containing pleomorphic vesicles), or PSDs(presynaptic dendrites). The order of dendritic branches of labeled TCNs was determined bycomputer-assisted reconstruction from serial sections. LR terminals made contacts mainlywith proximal dendrites of TCNs. SR terminals made contacts predominantly with distaldendrites, and were never found on somata or primary dendrites. P terminals were observedon somata and on every portion of the dendritic trees. Synapses formed by PSDs wereconcentrated on the proximal dendrites and sometimes formed synaptic triads with LRterminals. Only a few terminals were found on somata, all of which were P type. Therefore,terminals belonging to different classes were not uniformly distributed on the somata anddendrites of single TCNs. These results suggest that terminals originating from differentsources may preferentially contact specific regions of TCNs in the VL, and their topographicallocations reflect the electrophysiological response properties of the TCNs. J. Comp. Neurol.388:613–631, 1997. r 1997 Wiley-Liss, Inc.

Indexing terms: three-dimensional reconstruction; synapse; HRP; intracellular labeling; GABA

The ventral lateral nucleus (VL) of the thalamus re-ceives inputs from the cerebellum and cerebral cortex andprovides output to the motor, premotor, and parietalcortices in the cat (Strick and Sterling, 1974; Hendry et al.,1979; Nakano et al., 1980; Sugimoto et al., 1981; Wannieret al., 1992; Shinoda et al., 1985b, 1993) and in the monkey

Grant sponsor: Japanese Ministry of Education, Science, Sports, andCulture; Grant numbers: 05789561, 06780630.

*Correspondence to: Dr. F. Sato, Department of Anatomy, Faculty ofMedicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113, Japan. E-mail: [email protected]

Received 26 September 1996; Revised 9 June 1997; Accepted 1 July 1997

THE JOURNAL OF COMPARATIVE NEUROLOGY 388:613–631 (1997)

r 1997 WILEY-LISS, INC.

(Schell and Strick, 1983). Two classes of neurons have beenidentified in the VL on the basis of anatomical studies:thalamocortical neurons (TCNs) and local circuit neurons(LCNs) in the cat (Rinvik and Grofova, 1974a; Kultas-Ilinsky et al., 1985; Rinvik et al., 1987) and monkey(Kultas-Ilinsky and Ilinsky, 1991).

Axon terminals in the motor thalamus (VL and theventral anterior nucleus, VA) have been classified intoseveral types according to the size of the axon terminalsand the shape of synaptic vesicles in the cat (Rinvik andGrofova, 1974a; Kultas-Ilinsky et al., 1985) and the mon-key (Harding, 1971, 1973a; Harding and Powell, 1977;Kultas-Ilinsky and Ilinsky, 1991). There are two types ofsynaptic boutons with round vesicles in the VL of the catand monkey. One type, which is larger, originates from thecerebellar nuclei (Harding, 1973b; Rinvik and Grofova,1974b; Kultas-Ilinsky and Ilinsky, 1991; Mason et al.,1996; Sato et al., 1996), and the other smaller typeoriginates from the motor area of the cerebral cortex(Harding, 1973b; Grofova and Rinvik, 1974; Kultas-Ilinsky and Ilinsky, 1991). Synaptic boutons with flattenedvesicles have been classified into three types in the catmotor thalamus (Rinvik and Grofova, 1974a). In themonkey VL, Kultas-Ilinsky and Ilinsky (1991) definedthem as F1 type, which are believed to be of variousorigins. In addition, the dendrites of LCNs (i.e., presynap-tic dendrites) form dendrodendritic synapses on the den-drites of TCNs (Grofova and Rinvik, 1974; Kultas-Ilinskyand Ilinsky, 1991; Sato et al., 1996).

In the VL, TCNs projecting to the motor and premotorcortices receive excitation from the cerebellum and inhibi-tion from at least two sources (Uno et al., 1970; Deschenesand Hammond, 1980; Shinoda et al., 1985a; Rispal-Padelet al., 1987; Ando et al., 1995). One source of theseinhibitory inputs is LCNs in the VL, and the other is thethalamic reticular nucleus. Detailed information regard-ing synaptic relationships among cerebellar afferents,TCN dendrites, and dendrites and axons of LCNs isrequired to understand the mode of synaptic transmissionin the VL. Therefore, we previously analyzed the quantita-tive and three-dimensional properties of synaptic relation-ships among cerebellar terminals, TCN dendrites, andpresynaptic dendrites (PSDs; Sato et al., 1996). That studyfocused on cerebellar terminals, and we quantitativelyanalyzed short segments of TCN dendrites that werecontacted by cerebellar terminals by using serial ultrathinsections. However, in addition to cerebellar terminals,various other terminals are concentrated on TCNs (Rinvikand Grofova, 1974a; Harding and Powell, 1977; Kultas-Ilinsky et al., 1985; Kultas-Ilinsky and Ilinsky, 1991;Mason et al., 1996; Sato et al., 1996). Therefore, informa-tion about the distribution of the synapses formed byvarious afferents should aid in revealing how motor infor-mation is processed in the VL. However, there has been nosystematic attempt to determine quantitatively the distri-bution of particular types of synapses on the surface of asingle identified cell in the VL. Previous studies haveinvolved isolated electron photomicrographs or a shortseries of electron photomicrographs of isolated dendriticsegments (Kultas-Ilinsky and Ilinsky, 1991; Mason et al.,1996; Sato et al., 1996). Therefore, these previous studiesdo not provide quantitative information regarding howterminals of different origins are distributed, or howexcitatory and inhibitory terminals are distributed on thesomata and different portions of dendrites of single TCNs

of the VL. Recently, Liu et al. (1995) demonstrated thedistribution of several types of terminals on the soma-dendritic surfaces of physiologically identified TCNs in thecat ventral posterolateral nucleus (VPL) by extensivelyanalyzing serial ultrathin sections. Similar attempts havealso been made in the cat lateral geniculate nucleus (LGN;Wilson et al., 1984; Hamos et al., 1985; Raczkowski et al.,1988).

The goal of the present study was to analyze therelationship between the morphology and relative fre-quency of different types of terminals on different portionsof single identified TCNs. The present investigation wascarried out on two intracellularly stained TCNs in the catVL. By electron microscopy, we classified the types of axonterminals that synapsed on HRP-labeled TCNs and deter-mined their morphological types. By using a computer-assisted reconstruction system, we reconstructed the cellbodies and dendrites of single HRP-labeled TCNs fromserial ultrathin sections to determine the spatial distribu-tion of the classified terminals and evaluated the numberand location of the terminals that synapsed on the cellbodies and different portions of dendrites of single TCNs.Preliminary results have been presented in abstract form(Sato et al., 1992).

MATERIALS AND METHODS

Experimental procedure

The data were obtained from two adult cats weighing 2.8and 3.5 kg. The animals were anesthetized with pentobar-bital sodium (40 mg/kg, i.p.) and mounted in a stereotaxicframe. Blood pressure was monitored at the femoral arteryand maintained between 100 and 130 mmHg. Rectaltemperature was maintained between 37 and 38°C by aheating pad and an infrared lamp. The surgical proceduresand pre- and postoperative care of the animals conformedto principles approved by the American PhysiologicalSociety and followed protocols approved by the Committeefor Animal Experimentation at Tokyo Medical and DentalUniversity. The details of the experimental procedureshave been previously described (Shinoda et al., 1985a,1985b; Sato et al., 1996). Briefly, a craniotomy was madeover the left pericruciate cortex and over the right cerebel-lar hemisphere, and the dura was opened. The left parietalcortex over the thalamus was aspirated, and the dorsalsurface of the thalamus was exposed. To stimulate TCNaxons, stimulating electrodes were inserted into the graymatter of the left pericruciate cortex. To stimulate thecerebellar nuclei, two electrodes were placed in the dentatenucleus (DN) and the interpositus nucleus (IN). Negativepulses of 0.2 msec in duration were passed through aconstant-current generator. Currents were usually lessthan 200 µA, with a maximum of 500 µA. A recording glassmicropipette filled with a 10% horseradish peroxidase(HRP) solution in 0.2 M KCl-Tris buffer (pH 8.6) wasinserted into the thalamus. Single neurons in the VL wereintracellularly penetrated and electrophysiologically iden-tified as TCNs by their antidromic responses to stimula-tion of the pericruciate cortex. Subsequently, their re-sponses to stimulation of the DN and IN were examined.Stimulation of the IN and the DN evoked monosynapticexcitatory postsynaptic potentials (EPSPs), and theseEPSPs were evoked in an all-or-none manner at thethreshold. Three TCNs, which received inputs from the

614 F. SATO ET AL.

cerebellar nuclei, were injected with HRP iontophoreti-cally in each experiment.

Tissue processing

Four to 6 hours after the intracellular injection of HRP,the animals were perfused with a 0.1 M phosphate buffersolution (pH 7.4) containing 10% sucrose, and then with asolution containing 1.0% glutaraldehyde and 1.25% para-formaldehyde in 0.1 M phosphate buffer (pH 7.4). Thethalamus was removed and stored in the same fixativeovernight at 4°C. Serial transverse sections (100-µm-thick) of the removed thalamus were made by using amicroslicer, and HRP was visualized by a diaminobenzi-dine (DAB) method (Graham and Karnovsky, 1966). SixHRP-filled TCNs were stained well. After the specimenswere trimmed for electron microscopy, the rest of thetrimmed sections were stained lightly with cresyl violetand observed by light microscopy for cytoarchitectonicidentification. We always confirmed that the trimmedspecimens for electron microscopy were taken from the VL,by referring to the nuclear limits established in cytoarchi-tectural studies of the feline thalamus (Niimi and Kuwa-hara, 1973; Berman and Jones, 1982; Jones, 1985). Sec-tions containing a labeled cell body and dendritic profileswere post-fixed in 1% osmium tetraoxide for 1 hour andthen dehydrated in graded alcohol. They were flat-embedded in Epon between silicon-coated glass slides andcoverslips.

The embedded specimens were examined under a lightmicroscope, and dendritic trees of two labeled neurons(TCN 1 and TCN 2) were drawn with the aid of a Nikonmicroscope equipped with a drawing tube and thentrimmed for electron microscopy (Fig. 1). Under a dissect-ing microscope, small pieces that contained an HRP-labeled cell body and dendrites were cut from Epon-embedded sections. The dissected pieces were glued to ablock of blank resin and trimmed for ultrathin sectioning

(Fig. 2). Serial ultrathin sections displaying pale-goldinterference color (approximately 100-nm-thick) were cutby using a Reihert-Jung ULTRACUT microtome and pickedup on single-slot, formvar-coated nickel grids. Every othergrid was stained with 2% aqueous uranyl acetate and leadcitrate.

The remaining grids, which had not been lead-stained,were processed with a postembedding immunogold tech-nique (Liu et al., 1989) by using a g-aminobutyric acid(GABA) antibody (GABA antiserum was the generous giftof Dr. P. Streit; Matute and Streit, 1986). The reaction wascarried out on droplets of Millipore-filtered solutions in ahumid box. The grids were pretreated with 1% HIO4, 1%NaIO4, and 1% NaBH4 to etch the resin and removeosmium. They were washed in Tris-phosphate bufferedsaline (TPBS; pH 7.4) for 10 minutes and then incubatedin 7.5% normal bovine serum diluted in TPBS (BS-TPBS)for 20 minutes. After washing in 1% BS-TPBS for 5minutes, the grids were incubated in mouse anti-GABAantiserum (diluted 1:20,000–100,000 in 1% BS-TPBS) for2 to 3 hours at room temperature and washed three timesin 1% BS-TPBS for 15 minutes. To reveal GABA-likeimmunoreactivity, the grids were incubated in goat anti-mouse IgG conjugated with 15-nm colloidal gold particles(Amersham, Buckinghamshire, England) diluted 1:10 in50 mM Tris buffer containing 0.5% polyethylene glycol for2 hours at room temperature. After washing in distilledwater, the grids were stained with 2% aqueous uranylacetate and lead citrate. The grids were examined andphotographed with a JEOL 100C electron microscope.

Three-dimensional reconstruction

Three-dimensional reconstructions of the labeled cellbodies and dendrites of two TCNs, and axon terminals ofvarious types that were in contact with them, were madeby using a computer-assisted reconstruction method from236 sections for TCN 1 and 391 sections for TCN 2. Axon

Fig. 1. Partial reconstructions of two horseradish peroxidase (HRP)-labeled thalamocortical neurons(TCNs; A for TCN 1 and B for TCN 2). Both drawings were made from three 100-µm serial sections. Scalebar 5 100 µm.

SPATIAL DISTRIBUTION OF TERMINALS ON VL NEURON 615

terminals on the labeled dendrites and somata of TCNswere photographed in each serial ultrathin section. Elec-tron photomicrographs (final magnification, 37,500) wereused to determine the types of axon terminals and to tracelabeled dendrites and classified axon terminals that con-tacted them. Tracings of the electron photomicrographswere aligned by using HRP-labeled dendrites of TCNs,blood vessels, and large myelinated axons as fiducialpoints, and superimposed to produce reconstructions byusing a graphic program, TRI (Ratoc System Engineering,Co. Ltd., Tokyo) running on a Dell computer. Three-dimensionally reconstructed structures were examinedwith an Indigo2-XZ workstation (Silicon Graphics, Moun-tain View, CA) at different view angles. Photographs weretaken directly from the display.

RESULTS

The present results were derived from two of six TCNsthat were well labeled with HRP and isolated. Dendritesand somata of these labeled TCNs were traced in Epon-embedded specimens under light microscopy. Each recon-struction of the two labeled TCNs (TCN 1 and TCN 2 ) inFigure 1 was obtained from three 100-µm sections. Restingmembrane potentials were 265 mV (TCN 1) and 260mV(TCN 2). Both TCNs were antidromically activated bystimulation of the motor cortex at a latency of 0.9 msec(TCN 1) and 0.7 msec (TCN 2). Unitary EPSPs wereelicited by stimulation of both the DN and IN in both TCNs(Shinoda et al., 1985a). The amplitudes of the unitaryEPSPs ranged from 0.3 to 4.5 mV, and their latenciesranged from 0.9 to 1.6 msec. Stronger stimulation of theDN and the IN evoked larger EPSPs followed by inhibitorypostsynaptic potentials (IPSPs). These IPSPs were re-garded as disynaptic from the cerebellum (Ando et al.,1995). Each TCN had an extensive dendritic field of450–500 µm from the soma, with five (TCN 1) and seven(TCN 2) thick primary dendrites that divided further intosecondary branches at short distances from the soma. Thesecondary branches gave rise to profuse branches of ter-

tiary and higher-order dendrites. Some dendrites pos-sessed several dendritic appendages.

Under electron microscopy, the labeled cell bodies anddendrites were filled with electron-dense reaction productof HRP. As shown in a low-magnification electron photomi-crograph (Fig. 3), HRP-filled dendrites were scattered atvarious sites in a single section. Although we could esti-mate the probable correspondence of dendrites drawn fromthe light microscopic observation (inset) with those in thiselectron photomicrograph, observation of serial ultrathinsections and three-dimensional reconstructions of the den-drites made from them were required to obtain an accuratecorrespondence. An axon terminal is considered to form asynapse if there are plasma membrane thickenings onboth the pre- and postsynaptic sides, and a clustering ofsynaptic vesicles at that on the presynaptic side. However,with regard to terminals synapsing on the HRP-labeledTCNs, postsynaptic thickening was usually obscured, sincea reaction product adhered to the inner surface of theplasma membrane of the TCNs. It was more difficult tolocate the synaptic zone on labeled TCNs than on unla-beled ones. For quantitative analyses of axon terminalsthat contacted labeled TCNs, we selected terminals inwhich synaptic vesicles were clustered along a presynapticthickening and the synaptic cleft was widened. Further-more, we confirmed the continuation of synaptic contact inadjacent sections through serial-section analysis.

Classification of terminals synapsingon TCNs

Four morphological types of terminals were identified byelectron microscopy on the somatodendritic surfaces ofintracellularly labeled TCNs. The first type of terminalswas designated as LR type. LR terminals were character-ized by their large size (maximum diameter of 2–8 µmmeasured in a single section) and round clear synapticvesicles (30–50 nm in diameter) in moderate density (Figs.4A, 5C,D, 6A). They resembled cerebellar afferent termi-nals, which were identified by electron microscopy after

Fig. 2. A: Light photomicrograph of TCN 2 in a 100-µm section. The section was incubated withdiaminobenzidine (DAB) to reveal the dense reaction product, and then osmicated and embedded flat inEpon. B: Light photomicrograph of TCN 1 in a 100-µm section trimmed to a trapezoid for ultrathinsections after being glued to an Epon block. Scale bars 5 100 µm in A,B.

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intra-axonal staining with HRP or anterograde labelingwith WGA-HRP (Sato et al., 1996). They did not showGABA-like immunoreactivity (Figs. 5C, 6A). The secondtype of terminal was designated as SR type. SR terminalswere characterized by their small size (0.5–1.0 µm indiameter measured in a single section) and densely packedround clear vesicles (Figs. 4B, 6F, 7B). They sometimescontained one or two dense-cored vesicles and at most onemitochondria. Although their postsynaptic targets wereheavily filled with HRP reaction product, they showedpronounced thickening on the cytoplasmic face of thepostsynaptic membrane (Fig. 7B). Therefore, it was pre-sumed that these synaptic sites were asymmetric. GABA-like immunoreactivity was not observed in them (Figs. 4B,7B). The third type of terminal, readily distinguished fromthe previous two, was designated as P type. P terminalswere small to medium-sized (0.5–3.0 µm in maximumdiameter measured in a single section) and containedpleomorphic vesicles (Figs. 4C, 5B,C, 7A,B). In markedcontrast to the LR and SR types, P terminals showedGABA-like immunoreactivity (Figs. 4C, 5C, 7A,B). GABA-immunopositive terminals were identified by a density ofgold particles that was greater than that over otheradjacent structures, such as capillaries and glial pro-cesses, and the presence of a significant number of goldparticles in subsequent thin sections of the same struc-

ture. The fourth type of terminal seemed morphologicallyvery similar to the P type, and GABA-like immunoreactiv-ity was also positive in these terminals. These profilesshowed an average maximum diameter of 0.5–3.0 µmmeasured in a single section, and they contained pleomor-phic vesicles. In contrast to the P terminals, these termi-nals were postsynaptic to other terminals (Figs. 4D, 6C,D,7C–E, 8). Profiles of this type that were postsynaptic toother neuronal elements were regarded as PSDs. Strictlyspeaking, a PSD also includes ribosomes or a granularendoplasmic reticulum (Ralston and Herman, 1969; Rals-ton, 1971). Most profiles of this type, which were postsyn-aptic to other terminals, lacked ribosomes, even thoughthey were traced in serial sections (Sato et al., 1996). Inseveral previous studies in which serial sections were usedto identify synaptic boutons, postsynaptic vesicle-filledelements were invariably determined to be of dendriticorigin, and this feature has come to be a reliable means ofidentifying PSDs (Ralston, 1969; Ohara and Lieberman,1993). Therefore, we regarded profiles of this type thatcontained pleomorphic vesicles that were postsynaptic toother neuronal elements as PSDs, whereas profiles withpleomorphic vesicles that showed only presynaptic fea-tures were called P terminals. PSDs were more electron-lucent than P-type terminals, and the distribution of

Fig. 3. Low-magnification electron photomicrograph of TCN 1. HRP-labeled dendrites (arrows withletters) correspond to the dendrites indicated by the same letters in the inset drawing. The contour oflabeled dendrites is demarcated with ink. Scale bar 5 10 µm.


synaptic vesicles was somewhat sparser in PSDs than in Pterminals.

Distribution of terminals of different typeson single labeled TCNs

Because labeled dendrites of a single TCN were distrib-uted at various sites in individual ultrathin sections, asshown in Figure 3, and it was hard to identify the order ofTCN dendrites in a single section, we superimposed thetracings of electron photomicrographs and reconstructedthe morphology of a single labeled TCN to determine theorder of its dendrites. We determined the order of thedendrites as follows: we defined a dendrite that continuedfrom the neuronal soma as a primary dendrite as far as itstarted branching, and the branched dendrites from the

primary dendrite were regarded as secondary dendrites asfar as they started branching again. Tertiary and moredistal dendrites were also determined in the same manner.Three-dimensional computer-assisted reconstruction wasperformed by using 236 consecutive sections for TCN 1(Fig. 9) and 391 sections for TCN 2 (Fig. 10). For TCN 1,the reconstruction was made from the soma to tertiarydendrites (Fig. 9A–C). One of its primary dendrites gaverise to four secondary dendrites at a distance of 40 µmfrom the soma. One of these secondary dendrites bifur-cated into two tertiary dendrites, one of which was tracedalmost to its end. Two other separate branches near thistertiary dendrite were also reconstructed (Fig. 9B,C).Although they could not be continuously traced from thecell body, they were identified as belonging to the same

Fig. 4. Classification of terminals on labeled TCNs. Arrowheadsindicate synaptic contacts. A: LR-type axon terminal (GABA-negative,large axon terminal containing round vesicles). B: SR-type axonterminals (GABA-negative, small axon terminals containing roundvesicles). C: P-type axon terminal (GABA-positive axon terminal ofvarious sizes containing pleomorphic vesicles). This terminal containsa high density of colloidal gold particles. D: PSD (GABA-positive

dendrite postsynaptic to other neuronal elements). Arrows indicatepostsynaptic densities of the adjacent SR terminals. (Ultrathin sec-tions in B, C, and D were immunoreacted with GABA antibody.)GABA, g-aminobutyric acid; PSD, presynaptic dendrite. Scale bars 50.5 µm in A–D.

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Fig. 5. Electron photomicrographs of terminals on a soma and aprimary dendrite of a labeled TCN. A: Low-magnification electronphotomicrograph showing a labeled cell body contacted by a Pterminal (arrow) and another P terminal in the framed area. This cellbody is continuous from the primary dendrite shown in Figure 3 (thedendrite indicated by a small letter a). B: Higher magnification of theframed area in A. An arrowhead indicates a synaptic contact. P, P

terminal. C: One LR terminal and one P terminal contacting aproximal dendrite. This P terminal shows a high density of goldparticles. D: Two LR terminals, two PSDs, and one P terminal incontact with a primary dendrite. The ultrathin section in C wasimmunoreacted with GABA antibody. Abbreviations as in Figure 4.Scale bars 5 5 µm in A, 1 µm in B–D.

Fig. 6. Electron photomicrographs of terminals on secondary den-drites of a labeled TCN. Arrowheads indicate synaptic contacts. A: TwoLR and two P terminals contacting a secondary dendrite. B: An LRterminal contacting a secondary dendrite. This LR terminal also formsa synapse with a PSD (arrow). C,D: A PSD which is postsynaptic to anadjacent LR terminal (C) is presynaptic to a secondary dendrite (D). Cand D are separated by three sections. Arrows point to synaptic sites.

E: Low-magnification electron photomicrograph showing fragments ofa thin secondary dendrite (right three arrows) arising from a primarydendrite (left-most arrow). F: Higher magnification of the area out-lined in E. An SR terminal makes a synaptic contact with a secondarydendrite. Ultrathin sections in C and D were immunoreacted withGABA antibody. Abbreviations as in Figure 4. Scale bars 5 1 µm inA–D,F, 5 µm in E.

Fig. 7. Electron photomicrographs of terminals on tertiary den-drites of a labeled TCN. Arrowheads indicate synaptic contacts. A: OneLR and one P terminal contacting a tertiary dendrite. B: One P and sixSR terminals contacting a tertiary dendrite. C–F: Four sectionsthrough a PSD forming a synaptic contact on a tertiary dendrite. This

PSD is also postsynaptic to three SR terminals (arrows). The left SRterminal shown in F is the same one as in E. Section numbers areshown on the lower left corners. Ultrathin sections in A–E wereimmunoreacted with GABA antibody. Abbreviations as in Figure 4.Scale bars 5 1 µm in A–C (applies to A–F).


Fig. 8. A–D: A triad formed by an LR terminal, a PSD, and a TCNdendrite. B is a higher magnification electron photomicrograph of A. InA, two SR terminals are also contacting a PSD (arrows). Synapticcontacts between an LR terminal and a TCN dendrite (arrowhead; B),a PSD and the TCN dendrite (arrowhead; C), and the LR terminal andthe PSD (arrow; D) are shown on serial sections. Section numbers areshown on the lower left corners. E–G: A triad formed by a P terminal, a

PSD, and a TCN dendrite. In G, the same PSD is also postsynaptic toan SR terminal. Section numbers are shown on the lower left corners.A PSD contacts a labeled TCN dendrite (arrowhead) in E, and thisPSD is contacted by a P terminal (arrow) in F. Section F wasimmunoreacted with GABA antibody. Abbreviations as in Figure 4.Scale bars 5 2 µm in A, 1 µm in B–G.

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Fig. 9. Three-dimensional reconstruction of part of the soma anddendrites of TCN 1. This reconstructed image was obtained from 236ultrathin sections. The graphic image in A is rotated and shown fromdifferent viewing angles to obtain a complete picture of a neuron (Band C). B: A top view of A; C: a mirror image of the back view of B. D:Three-dimensional representation of the proximal portion of thedendrites and distribution of terminals of different types contacting

proximal dendrites. E: A top view of the same portion of the dendritesas in D. F: Three-dimensional reconstruction of the distal portion of adendrite of TCN 1 and terminals of different types contacting it. G:The outlined areas are enlarged in D, E, and F. Yellow-brown, TCNdendrite; green, LR terminal; red, SR terminal; blue, P terminal;dark-blue, PSD. Abbreviations as in Figure 4.

Fig. 10. A: Three-dimensional reconstruction of part of the somaand dendrites of TCN 2. Two primary dendrites (shown in yellow-brown and gray) arise from the soma. This reconstruction wasobtained from 391 sections. Aa: Dendrites shown in yellow-brown arerotated 90 degrees away from the viewer. The primary dendrite givesrise to four secondary dendrites that further divide into more distalbranches. Ab: The dendrite shown in gray is rotated 90 degrees towardthe viewer. The primary dendrite bifurcates into two secondarydendrites. One of them further gives rise to more distal branches. B,C:

Distribution of terminals of various types on the dendrites shown inyellow-brown in A (Aa). B and C show the distribution of terminals onthe dendrites of the left side and the right side, respectively. D,E:Distribution of terminals of various types contacting a dendrite shownin gray in A (Ab). D shows the distribution of terminals on the leftportion of the dendrites in A, and E shows the distribution of terminalson the right portion of the dendrites in A. Yellow-brown, TCN dendrite;green, LR terminal; red, SR terminal; blue, P terminal; dark-blue,PSD. Abbreviations as in Figure 4.

TCN by light microscopy (Fig. 3, inset) and these dendriteswere considered tertiary dendrites. For TCN 2, two pri-mary dendrites arose from the soma (Fig. 10A). One ofthem (shown in yellow-brown) gave rise to four secondarybranches, which in turn gave rise to two, two, two, andthree tertiary dendrites, respectively. One of the secondarydendrites was far longer than the other three. Amongthese nine tertiary dendrites, one further ramified intothree quaternary dendrites. The other primary dendrite(shown in gray) gave rise to two secondary dendrites, oneof which bifurcated into two tertiary dendrites. One of thetertiary dendrites gave rise to three quaternary dendrites,one of which bifurcated into two dendrites of the fifthorder.

The cell bodies of the labeled TCNs received few syn-apses: only one or two terminals were observed on a cellbody in a single section (Fig. 5A). All of the axosomaticsynapses were formed by P terminals (Fig. 5B). Althoughthe labeling was not so prominent in the cell body in Figure5A,B, this cell body was continuously traced from theprimary dendrite shown in Figure 3 (the dendrite indi-cated by a small letter a) and the sparse reaction productwas identified in the cell body by observing serial ultrathinsections. However, it is not clear why the labeling densityhas become diminishing around the cell body. Synapticcoverage on proximal parts of primary dendrites wassimilar to that on the cell bodies. Most of the surface of theproximal parts of the primary dendrites was devoid ofterminals. The number of terminals gradually increasedon the more distal parts of the primary dendrites. P andLR terminals were common, and PSDs were also found onthe primary dendrites (Fig. 5C,D). There were twice asmany P terminals as LR terminals. PSDs received syn-apses by LR terminals that also contacted labeled TCNdendrites. No SR terminals were found on the primarydendrites (Figs. 9D,E, 10B–D).

Secondary dendrites were predominantly contacted by Pand LR terminals (Figs. 6A,B, 9D,E, 10B–E). The numberof P terminals, however, had declined and instead, aconsiderable number of PSDs were observed to synapse onthese parts of the dendrites (Fig. 6C,D). LR terminalscontacted both TCN dendrites and PSDs, which in turnsynapsed on the same TCN dendrites (Fig. 8A–D). Becausetriads of this type were frequently observed in the VL, wequantitatively analyzed the relationships among thesethree elements in detail in a separate paper (Sato et al.,1996). On rare occasions, triads were found among a Pterminal (instead of an LR terminal), a PSD, and a TCNdendrite (Fig. 8E,F). On the distal parts of the secondarydendrites, the number of LR terminals gradually declined.Instead, a small number of SR terminals appeared, whichwere frequently found on the distal parts of the secondarydendrites (Figs. 6F, 9D,E, 10B–D).

On tertiary and more distal dendrites, SR terminalswere the predominant type (Figs. 7B, 9F, 10B–E). Thenumber of LR terminals rapidly declined, and they wereonly found near the branching points of the tertiarydendrites. Compared with SR terminals, there were consid-erably fewer PSDs and P terminals. The number of PSDswas particularly reduced. In contrast to the proximaldendrites, PSDs were generally postsynaptic to SR termi-nals, which did not form triads like LR terminals (Fig.7C–F); when SR terminals were presynaptic to PSDs, theydid not synapse with TCN dendrites. Compared with theproximal dendrites, the synaptic density on the dendritic

surface increased and a considerable number of SR termi-nals covered almost the entire dendritic surface (Figs. 9,10). We traced three labeled primary dendrites (one forTCN1 and two for TCN2) as far as their distal portions,and counted the number of terminals of different types per10-µm segment (Table 1). By dividing the total length of adendrite of an order by 10 µm, the remainder was added tothe most distal 10-µm segment of that dendrite, and werestarted to measure the length of the next-order dendriteby 10 µm and count the number of terminals on it from itsbranching point.

The distribution of terminals of different types on differ-ent portions of dendrites of single TCNs is summarized inFigure 11 and Table 2. All of the axosomatic synapses weremade by P terminals. On primary dendrites, P terminalsoccupied almost half of presynaptic profiles, and LR termi-nals formed approximately 35% of the synapses. The restof the synapses on the primary dendrites were made byPSDs. On secondary dendrites, SR terminals comprised20% of the observed presynaptic elements, LR and Pterminals comprised approximately 30% each, and PSDscomprised the remaining 20%. On tertiary and more distaldendrites, SR terminals increased in number and com-prised 70% of the observed synapses, P terminals com-prised 25%, and LR and PSDs constituted the rest. InTable 2, we calculated the mean synaptic density (thenumber of terminals/µm) of primary, secondary and moredistal dendrites of two TCNs analyzed in this study. Thesynaptic density of distal dendrites was greater than thatof proximal dendrites, and the average synaptic density fordendrites of all orders was 1.1/µm.

LR terminals were predominantly found on the primaryand secondary dendrites of TCNs, whereas SR terminalswere predominantly found on more distal dendrites. Pterminals were widely distributed on the somata and on allportions of the dendrites of the TCNs. The ratio of Pterminals to the total number of terminals on a singleneuron decreased at increasing distances from the somaalong the dendrites. PSDs appeared to be concentrated onproximal dendrites, whereas P terminals were distributedrather randomly throughout all portions of the dendrites.

DISCUSSION

The present analysis provides quantitative data on thedistribution of different synapses on identified portions ofdendrites of single HRP-labeled TCNs in the cat VL. Theseresults indicate that afferent axon terminals from differentsources are selectively distributed on either the soma andthe proximal portions of dendrites, or the distal portions ofdendrites of single TCNs. Although the sample size of thesynapses we analyzed was quite large, we studied only twoTCNs in detail. Nevertheless, we believe that the presentresults are reasonably representative of the innervationpatterns of different synapses on single TCNs in the VL,because the results obtained from the two TCNs showsimilar distribution patterns for the synapses (Figs. 9–11).Quantitative data on the density and overall distributionof synapses on physiologically identified neurons wereobtained from four TCNs in the cat VPL by using serialelectron microscopic analysis (Liu et al., 1995). Similarattempts to assess terminal distribution have been per-formed on four TCNs (Wilson et al., 1984), two TCNs(Raczkowski et al., 1988), and one LCN (Hamos et al.,1985) in the cat LGN. Although previous studies implied


TABLE 1. Number of Terminals of Different Types on Each 10-µm Segment of Labeled Dendrites1

Branch of TCN1 shown in Figure 9orderlength (µm)

1 2-110 20 30 40 42 10 13

P 3 2 10 2 7 6 5PSD 0 0 0 10 3 9 4LR 0 1 3 8 5 13 4SR 0 0 0 0 0 0 0orderlength (µm)

2-210 20 23

P 8 4 2PSD 2 4 1LR 3 2 0SR 2 4 2orderlength (µm)

2-3 3-110 10 20 30 40 50 60 70 80 90 100 110 119

P 5 4 7 3 6 5 5 3 3 5 2 5 5PSD 2 1 1 1 0 1 0 0 0 0 0 0 0LR 9 2 0 0 0 0 0 0 0 0 0 0 0SR 0 0 12 19 16 19 20 15 14 15 13 10 12orderlength (µm)

3-210

P 6PSD 1LR 1SR 2orderlength (µm)

2-410 20 30 40 56

P 2 3 4 3 2PSD 0 0 1 0 0LR 1 2 1 1 0SR 0 0 3 4 8Gray branch of TCN2 shown in Figure 10orderlength (µm)

1 2-110 20 30 40 42 10 20 30 39

P 0 1 1 1 1 3 3 2 1PSD 0 0 0 0 1 1 1 0 1LR 0 1 1 1 0 4 2 1 2SR 0 0 0 0 0 0 0 2 3orderlength (µm)

2-2 3-110 20 24 10 20 30 37

P 2 3 1 1 4 3 1PSD 0 1 1 0 0 0 0LR 1 4 1 0 0 0 0SR 0 0 0 3 17 19 12orderlength (µm)

3-2 4-110 15 10 20 22

P 0 5 5 2 2PSD 0 0 0 0 0LR 4 2 5 1 0SR 0 2 0 9 3orderlength (µm)

4-2 5-110 0 20 29

P 1 1 2 1PSD 0 0 1 0LR 0 0 0 0SR 12 21 18 9orderlength (µm)

5-210 20 30 40 43


4-310 20 23

P 1 0 0PSD 0 1 1LR 0 0 0SR 7 7 3Yellow-brown branch of TCN2 shown in Figure 10orderlength (µm)

1 2-1 3-1110 10 20 30 40 48 10 20 26

P 6 3 2 2 5 5 2 4 2PSD 2 2 0 0 1 0 0 1 0LR 6 3 1 1 4 0 0 0 0SR 0 0 0 0 0 15 12 18 12orderlength (µm)

3-1210

P 6PSD 0LR 0SR 17

(Continued.)

626 F. SATO ET AL.

that afferents to TCNs are spatially segregated in the VL,this implication was derived from observations of shortand limited segments of dendrites (Rinvik and Grofova,1974a; Kultas-Ilinsky et al., 1985; Kultas-Ilinsky andIlinsky, 1991; Sawyer et al., 1994; Mason et al., 1996; Satoet al., 1996). Precise quantitative information about thedistribution of different types of synapses on single identi-fied VL neurons was previously lacking.

The general distribution pattern of various terminals inthe VL, except PSDs, is very similar to that in otherthalamic subnuclei such as the LGN and VPL. LR and Pterminals formed the majority of synapses on proximaldendrites of TCNs (Fig. 11). The number of synapses

formed by LR terminals declined as the distance from thesoma along a dendrite increased. Instead, SR terminalswere predominantly distributed on distal dendrites. Pterminals were distributed rather randomly along theentire lengths of dendrites. PSDs formed approximately20% of synapses on primary and secondary dendrites inthe present study. Kultas-Ilinsky and Ilinsky (1991) re-ported that LR terminals and PSDs are the predominanttypes on primary and secondary dendrites in the monkeyVL. Although this result was not obtained from serialsections and their precise percentages were not described,it seemed that PSDs occupied a rather large percentage onproximal dendrites. However, there were few PSDs (,6%)

TABLE 1. (continued).

Yellow-brown branch of TCN2 shown in Figure 10 (continued)orderlength (µm)

2-2 3-2110 10 20 24


3-2210 20 30


2-3 3-31 4-31110 10 10 20


4-31210 16

P 2 5PSD 5 3LR 3 0SR 0 8orderlength (µm)

4-31310 20 22


3-3210 17

P 2 4PSD 3 1LR 0 2SR 2 1orderlength (µm)

3-33 4-3314 10 20 25


4-33210 20 23


2-4 3-412 10 20 30 32


3-4210 20 30

P 3 2 6PSD 1 2 1LR 2 3 2SR 0 1 4

1P, g-aminobutyric (GABA)-positive axon terminals of various sizes containing pleomorphic vesicles; PSD, presynaptic dendrites; LR, GABA-negative large axon terminalscontaining round vesicles; SR, GABA-negative small axon terminals containing round vesicles; TCN, thalamocortical neurons.


in the cat VPL (Liu et al., 1995). This difference in thesubnuclei is one of the reasons for the paucity of PSDs inthe VPL compared with the VL. Although Wilson et al.

(1984) regarded both PSDs and P terminals as a singlecategory (which they called f terminals) in the cat LGN,they noted that some f terminals formed triads. Therefore,

Fig. 11. The number (A) and proportions (B) of terminals of thefour types on different portions of identified dendrites for TCN 1 andTCN 2. The numbers on the right in B indicate the total number ofterminals on each portion of the identified dendrite of a TCN. SR,

small axon terminal containing round vesicles; LR, large axon termi-nal containing round vesicles; PSD, presynaptic dendrite; P, terminalcontaining pleomorphic vesicles; TCN, thalamocortical neurons.

628 F. SATO ET AL.

f terminals that formed triads were considered PSDs in thepresent study. They further reported that f terminals thatsynapsed on X cells often formed triads, but those on Ycells did not. This finding suggests that the difference inthe frequency of PSDs might be due to the type of TCNsthat PSDs contact. It is possible that TCNs in the VPLdescribed by Liu et al. (1995) correspond to Y cells in theLGN. Furthermore, in the monkey VPL, medial lemniscalafferents frequently form triadic or glomerular arrange-ments, whereas spinothalamic tract afferents do not (Rals-ton and Ralston, 1994). Therefore, the paucity of PSDs inthe cat VPL might depend on the type of main excitatoryinputs TCNs receive. The synaptic density of distal den-drites was greater than that of proximal dendrites. Theaverage synaptic density on dendrites of all orders is1.1/µm in this study and similar to that previously re-ported in cat LGN and VPL. It was 1/µm for the X cell and0.9/µm for the Y cell in the cat LGN (Wilson et al., 1984),and 0.6–0.9/µm for the TCN in the cat VPL (Liu et al.,1995). Synaptic density was lower at 0.4/µm for the W cellin the cat LGN (Raczkowski et al., 1988).

LR terminals in the present study resemble cerebellarafferent terminals that were previously identified in thecat and monkey by degeneration (Harding, 1973b; Rinvikand Grofova, 1974b; Harding and Powell, 1977), WGA-HRP labeling (Kultas-Ilinsky and Ilinsky, 1991; Mason etal., 1996; Sato et al., 1996), or HRP intracellular labeling(Sato et al., 1996). Although it has been reported thatcerebellar terminals contacted both cell bodies and proxi-mal dendrites in the monkey (Kultas-Ilinsky and Ilinsky,1991), LR terminals predominantly synapsed on primaryand secondary dendrites, but never on cell bodies in thepresent study. This result was in good agreement withprevious data obtained in the cat VL (Kultas-Ilinsky et al.,1980, 1985; Sato et al., 1996) and in the rat VL (Aumann etal., 1994). Physiological studies have revealed that stimu-lation of the cerebellum evokes strong excitation in TCNs(Uno et al., 1970; Shinoda et al., 1985a; Bava et al., 1986;Rispal-Padel et al., 1987); stimulation of the DN or INevokes large unitary EPSPs in an all-or-none manner atthe threshold, and the slope of the rising phase of theEPSPs is sharp and the amplitude of unitary EPSPs is0.3–5.6 mV (Shinoda et al., 1985a). These physiologicalresults suggest that cerebellar terminals are located oncell bodies or proximal portions of TCN dendrites, and alarge number of cerebellar terminals or a large boutonoriginating from a single cerebellar nucleus neuron shouldsynapse on them. The present electron microscopic find-ings are consistent with these electrophysiological sugges-tions in that LR terminals form synaptic contacts predomi-nantly with primary and secondary dendrites of TCNs.

We observed that PSDs were contacted by LR terminals,and that PSDs, in turn, contacted the same TCN dendritesthat the LR terminals contacted. Triads of this type arefrequently observed in the VL (Fig. 8A–D). Because tha-lamic LCNs are GABAergic (Sterling and Davis, 1980;

Ohara et al., 1983; Kultas-Ilinsky et al., 1985; Ohara et al.,1989), excitation of cerebellar terminals can release GABAfrom PSDs to TCNs, and thereby inhibit the TCNs. Thisagrees with the electrophysiological result that cerebellarstimulation evokes disynaptic inhibition in TCNs (Uno etal., 1970; Shinoda et al., 1985a; Rispal-Padel et al., 1987;Ando et al., 1995). Stimulation of the cerebellum usuallyevokes monosynaptic EPSPs followed by disynaptic IPSPsin single TCNs, and it is often difficult to isolate EPSPswithout evoking IPSPs (Shinoda et al., 1985a; Ando et al.,1995). This finding is consistent with the high frequency oftriads on proximal dendrites of TCNs. Triads formedamong a P terminal, a PSD, and a TCN dendrite werereported in the cat VPL (Liu et al., 1995) and in themonkey VL (Kultas-Ilinsky and Ilinsky, 1991). Althoughwe also observed them in the present study (Fig. 8E–G),they were encountered only rarely. The origin of P termi-nals is unknown and precise physiological analyses will benecessary to clarify their functional significance.

SR terminals that formed an asymmetric synapse werethe most common, primarily on distal dendrites of TCNs.SR terminals were similar to corticothalamic terminals(Grofova and Rinvik, 1974; Kultas-Ilinsky and Ilinsky,1991). Electrophysiologically, cortically evoked EPSPs hada slower rise time than cerebellar-evoked EPSPs, and itwas difficult to reverse cortically evoked EPSPs withintrasomatic current injection (Ando et al., 1995), whichsuggests that SR terminals are located on the distalportion of dendrites. These physiological results are ingood agreement with the location of SR terminals in thepresent study. Stimulation of the motor cortex evokesdisynaptic IPSPs in TCNs in the VL of the cat, and theseIPSPs have been assumed to originate from inhibitoryLCNs and thalamic reticular nucleus neurons (TRNs). Arecent electrophysiological study confirmed that theseIPSPs are at least partly mediated by inhibitory LCNs(Ando et al., 1995). This electrophysiological finding issupported by the present finding that GABAergic PSDs onlabeled TCN dendrites received synaptic contacts by SRterminals (Fig. 7C–F). Several studies have shown thatcorticothalamic terminals are of the LR type in varioussubnuclei (Kuroda and Price, 1991; Vidnyanszky et al.,1996). In the cat lateral posterior nucleus, LR terminalsderived from the cerebral cortex formed glomerulus-likearrangements (Vidnyanszky et al., 1996). Further investi-gations are required to determine whether corticothalamicterminals of this type exist in the VL.

The present results show that cell bodies and primarydendrites, which are not densely covered by axon termi-nals, receive a predominance of synapses from P termi-nals. The concentration of P terminals close to the cellbodies suggests that they strongly influence the firingproperties of the TCNs. P terminals showed GABA-likeimmunoreactivity and are therefore considered inhibitory;hence, they could render TCNs less excitable. P terminalsare believed to originate from axon terminals of TRNsand/or LCNs. TRNs are GABAergic (Houser et al., 1980;Hendrickson et al., 1983; Oertel et al., 1983) and send theiraxons to terminate in the most dorsal thalamic nuclei(Scheibel and Scheibel, 1966; Jones, 1975).Arecent electro-physiological study showed that cerebellar and cerebralinhibition of TCNs in the VL is partly mediated by TRNs(Ando et al., 1995). WGA-HRP-labeled TRN axon termi-nals have been reported to contact somata and each part ofdendrites of TCNs in the monkey LGN (Harting et al.,1991), and small and medium-sized dendrites in the cat

TABLE 2. Summary of Synaptic Density (Number of Terminals per µm)

Terminaltype

Dendritic order

AveragePrimary Secondary Distal

P 0.37 0.38 0.30 0.35PSD 0.17 0.18 0.07 0.14LR 0.28 0.32 0.07 0.22SR 0 0.18 0.87 0.35Total 0.82 1.06 1.31 1.06


lateralis medialis-suprageniculate complex (Norita andKatoh, 1987). A recent study of the distribution of TRNaxon terminals on partially reconstructed TCN dendritesin the cat LGN showed that most of the axon terminalsthat originated from the perigeniculate nucleus, which hasbeen thought to be the equivalent of the thalamic reticularnucleus in the visual segment, established synaptic con-tacts on the distal dendrites of TCNs (Cucchiaro et al.,1991). However, the morphological features of axon termi-nals that belong to a TRN have not yet been determined byelectron microscopy in the VL. To better understand theinhibitory interactions of TRN and LCN terminals onsingle TCNs, further investigation of the ultrastructuralcharacteristics and quantitative and three-dimensionalanalyses of the synaptic relationships of TRN and LCNterminals on single TCNs are required.

ACKNOWLEDGMENTS

We thank Dr. P. Streit for the generous gift of GABAantiserum. We also thank Dr. Patricia Morino Wannier forher advice on the postembedding procedure and Mrs. MieTaguchi and Mrs. Noriko Hattori for their technical assis-tance.

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