abducens internuclear and ascending tract of deiters inputs to medial rectus motoneurons in the cat...

Abducens Internuclear and AscendingTract of Deiters Inputs to Medial Rectus

Motoneurons in the Cat OculomotorNucleus: Neurotransmitters

LYNETTE T. NGUYEN1 AND ROBERT F. SPENCER2*1Department of Anatomy, Medical College of Virginia, Virginia Commonwealth University,

Richmond, VA 232982Department of Otolaryngology—Head and Neck Surgery, Medical College of Virginia,

Virginia Commonwealth University, Richmond, VA 23298

ABSTRACTThe abducens internuclear and ascending tract of Deiters (ATD) pathways are the

principal excitatory inputs to medial rectus motoneurons in the oculomotor nucleus and arerelated to the control of conjugate horizontal eye movements. Differences in the morphologyand soma-dendritic distribution of abducens internuclear and ATD synaptic endings arecorrelated with known differences in the physiological properties of these independent inputs.The present study extends these observations to the ultrastructural localization of theexcitatory amino acid neurotransmitters, glutamate and aspartate, using a postembeddingimmunogold procedure combined with the pre-embedding immunoperoxidase localization ofanterogradely transported biocytin from the abducens nucleus and the ventral lateralvestibular nucleus. Consistent with their spheroidal synaptic vesicle content and theasymmetric pre/postsynaptic membrane profile, both the abducens internuclear and ATDsynaptic endings are labeled with glutamate and aspartate. However, quantitative analysis ofthe density of colloidal gold particles associated with mitochondria versus synaptic vesicles/axoplasmic matrix reveals significant differences in the metabolic versus neurotransmitterpools of the amino acids in the two populations of synaptic endings. The findings indicate thatboth aspartate and glutamate, possibly co-localized, are the excitatory neurotransmittersutilized by abducens internuclear synaptic endings whose burst-tonic physiological activityconveys information related to eye position to medial rectus motoneurons. By contrast,glutamate is the excitatory neurotransmitter associated with ATD synaptic endings whosehigh frequency burst activity is related to head velocity. J. Comp. Neurol. 411:73–86,1999. r 1999 Wiley-Liss, Inc.

Indexing terms: biocytin; glutamate; aspartate; immunohistochemistry; abducens nucleus;

vestibular nucleus

Conjugate horizontal eye movements require the coordi-nated activation of the lateral rectus muscle in one eye andthe medial rectus muscle in the other eye. Most of thesignals that are necessary for the generation of conjugatehorizontal vestibulo-ocular, optokinetic, smooth pursuit,and saccadic eye movements are provided by brainstemafferent neurons that establish synaptic connections withlateral rectus motoneurons and internuclear neurons inthe abducens nucleus (reviewed in Nguyen et al., 1999).Abducens internuclear neurons, whose axons coursethrough the medial longitudinal fasciculus (MLF), are thesource of the eye position signal to medial rectus motoneu-rons in the contralateral oculomotor nucleus. In addition,

second-order neurons in the ventral lateral vestibularnucleus that receive direct horizontal semicircular canalinput provide a head velocity and weak eye position signal

Grant sponsor: U.S. Public Health Service MERIT Award; Grant number:EY02191; Grant sponsor: National Eye Institute; Grant number: EY02007.

Dr. Nguyen’s current address: The Smith-Kettlewell Eye ResearchInstitute, 2232 Webster Street, San Francisco, CA 94115-1821.

*Correspondence to: Dr. Robert F. Spencer, Department of Anatomy,Medical College of Virginia, Virginia Commonwealth University, 1101 EastMarshall Street, Richmond, VA 23298-0709. E-mail: [email protected]

Received 24 February 1998; Revised 21 January 1999; Accepted 11 March1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 411:73–86 (1999)

r 1999 WILEY-LISS, INC.

to medial rectus motoneurons in the ipsilateral oculomotornucleus. The axons of these second-order vestibular neu-rons course rostrally through the brainstem in the ascend-ing tract of Deiters (ATD), which is located lateral to theMLF.

The abducens internuclear and ATD synaptic inputs tomedial rectus motoneurons are both excitatory, but exhibitphysiological (Baker and Highstein, 1978; Highstein andBaker, 1978; Yamamoto et al., 1978; Reisine and High-stein, 1979; Nakao and Sasaki, 1980; Furuya andMarkham, 1981; Highstein and Reisine, 1981; Reisine etal., 1981; de la Cruz et al., 1989) and morphological(Carleton and Carpenter, 1983; Carpenter and Carleton,1983; Markham et al., 1986; McCrea et al, 1987; Nguyen etal., 1999) differences. Vestibulo-ocular neurons that projectto the oculomotor and trochlear nuclei are immunoreactivetoward glutamate and/or aspartate (Kevetter and Hoff-man, 1991; Carpenter et al., 1992). Recent findings indi-cate that the vertical saccade-related premotor excitatoryneurons in the rostral interstitial nucleus of the MLF thatproject to the oculomotor and trochlear nuclei for verticaleye movement also utilize glutamate and/or aspartate(Spencer and Wang, 1996). Furthermore, consistent withother studies (e.g., Decavel and van den Pol, 1992; Helfertet al., 1992), type of neurotransmitter has been correlatedwith distinct ultrastructural features (e.g., synaptic vesicleshape, type of postsynaptic specialization) of the synapticending (Spencer and Wang, 1996). Consequently, the differ-ences in the morphology and soma-dendritic distributionof abducens internuclear and ATD synaptic endings(Nguyen et al., 1999) might be related to differences inneurotransmitter utilization of the two inputs.

The identification of the putative neurotransmitters ofpremotor neurons that are related to the control of conju-gate horizontal eye movements has fundamental impor-tance toward understanding the pharmacology of theoculomotor system in relation to its neuronal and synapticorganization (Leigh et al., 1994). In the present study, apostembedding immunogold procedure has been used tolocalize the excitatory amino acid neurotransmitters, glu-tamate and aspartate, in relation to abducens internuclearand ATD synaptic endings labeled by a pre-embeddingimmunoperoxidase procedure.

MATERIALS AND METHODS

The data in this study were derived from the sameexperimental animals from which data for the pre-embedding immunoperoxidase studies (Nguyen et al., inpress) were obtained. All surgical and euthanasia proce-dures were performed in compliance with the NIH Guidefor the Care and Use of LaboratoryAnimals.All experimen-tal procedures were reviewed and approved by the Institu-tional Animal Care and Use Committee of Virginia Com-monwealth University. Briefly, biocytin was injected intothe abducens nucleus or the ventral portion of the lateralvestibular nucleus in adult cats anesthetized with sodiumpentobarbital (40 mg/kg) to label anterogradely the abdu-cens internuclear (n 5 six cats) or ATD (n 5 three cats)synaptic endings, respectively, in the oculomotor nucleus(see Fig. 2 in Nguyen et al., 1999). Groups of sections fromthose experiments were processed for light and electronmicroscopy using a pre-embedding immunoperoxidasemethod. Sections processed for electron microscopy wereused in the present study in combination with a postembed-

ding immunogold procedure to localize the excitatoryamino acid neurotransmitters, glutamate and aspartate,as described below.

Pre-embedding immunoperoxidaselocalization of biocytin-labeled synapticendings and postembedding immunogold

localization of excitatory neurotransmitters

Sections through the midbrain were cut at 50-mmthickness with a Vibratome (Technical Products Interna-tional, St. Louis, MO) and collected serially in 0.1 Msodium phosphate buffer, pH 7.2. The sections were di-vided into three groups, each group representing 150-mmintervals through the oculomotor nucleus, and processedfor light microscopy and pre- and postembedding electronmicroscopy. The light microscopy sections provided a refer-ence for areas that were selected for ultrastructural exami-nation using the postembedding immunogold procedure.

Sections through the oculomotor nucleus were incu-bated for 2 hours in avidin D-HRP (1:500; Vector Laborato-ries, Burlingame, CA) in 0.1 M sodium phosphate buffercontaining 0.1% Triton X-100. For the histochemical dem-onstration of HRP, sections were reacted in 0.05% 3,38-diaminobenzidine tetrahydrochloride (DAB;Aldrich Chemi-cal Co., Milwaukee, WI) and 0.01% hydrogen peroxide with0.005% cobalt acetate and 0.005% nickel chloride in 0.1 Msodium phosphate buffer, pH 7.2. After washing throughseveral changes of buffer, sections were processed forelectron microscopy. Sections were postfixed in 1.0% os-mium tetroxide in 0.1 M phosphate buffer with 7% dex-trose at 4°C for 1 hour and stained en bloc with 2% uranylacetate in 0.05 M maleate buffer at 4°C for 1 hour. Sectionsthen were dehydrated in graded methanols and propyleneoxide, infiltrated with plastic resin (Fullam, Latham, NY),and flat-embedded between glass microscope slides coatedwith a water-soluble release agent (Electron MicroscopySciences, Fort Washington, PA). After curing, the embed-ded sections were attached to labeled BEEM capsules andtrimmed to include only regions that were determined bylight microscopy to contain areas of anterogradely labeledterminals in the dorsolateral medial rectus subdivision ofthe oculomotor nucleus. Semithin (0.1-mm) sections werecut with glass knives on an ultramicrotome, stained with0.1% toluidine blue in 1.0% sodium borate, and examinedwith a light microscope for use as a reference for theelectron microscopic analysis. Ultrathin (60–80 nm) sec-tions were cut with a diamond knife on the ultramicro-tome, collected on Formvar-coated, single-slot gold grids,and then processed for the localization of neurotransmit-ters by a postembedding immunogold procedure.

The grids were first wetted in distilled water for 5minutes and then pretreated in 0.05–0.10% Triton X-100in 0.02 M Tris-buffered saline (TBS), pH 7.6, for 10minutes. The grids then were floated on drops of rabbitanti-glutamate or anti-aspartate (Arnel, New York, NY) atdilutions of 1:3,000 and 1:2,000, respectively, in ceramicdishes and incubated overnight at 4°C with constantagitation. The following day, the grids were allowed tocome to room temperature and then washed in TBS pH7.6, then 8.2. The grids next were incubated on drops ofgoat anti-rabbit IgG attached to 15-nm colloidal goldparticles (1:25; Amersham, Arlington Heights, IL) for 2hours with constant agitation. After the incubation period,the grids were washed extensively in TBS pH 8.2, then 7.6,followed by distilled water. The grids then were counter-

74 L.T. NGUYEN AND R.F. SPENCER

stained with 2.0% aqueous uranyl acetate and 0.1% leadcitrate, and sections were examined and photographedwith a Zeiss EM-10CA electron microscope (Oberkochen,Germany).

Control experiments

Several sections from the same blocks containing abdu-cens internuclear and ATD synaptic endings labeled withthe pre-embedding immunoperoxidase method were usedfor control experiments to test the specificity of the pri-mary and secondary antibodies. Negative controls wereperformed by excluding the primary antibody from theincubation schedule with all other processing steps remain-ing the same. As a positive preabsorption control, rabbitanti-glutamate and rabbit anti-aspartate antibodies wereincubated with L-glutamic acid (303 ml/mg; Sigma, St.Louis, MO) and L-aspartic acid (252 ml/mg; Sigma), respec-tively (Hepler et al., 1988), overnight with agitation at 4°Cand then centrifuged at 100,000 G for 30 minutes. Thesupernatant was collected and in each case was used inplace of the primary antibody. The remainder of theimmunohistochemical procedure was performed in thesame manner as described above.

Quantitative morphometry

The cross-sectional areas of biocytin-labeled synapticendings localized by the postembedding procedure and thediameters of the postsynaptic profiles were measured fromelectron micrographs at a magnification of 324,000 usinga BIOQUANT (R&M Biometrics, Nashville, TN) digitalimage analysis system operating on a microcomputer withstandard morphometric, spreadsheet, and statistical soft-ware. Only pre- and postsynaptic profiles that exhibited asynaptic contact zone identified by pre- and/or postsynap-tic membrane densifications and the invariable presence ofan intermediate dense line in the intersynaptic zone(Spencer et al., 1982) were analyzed. All synaptic endingsthat were analyzed quantitatively furthermore exhibitedan approximately equivalent density of immunoperoxi-dase reaction product for the localization of biocytin tominimize the possibility that the reaction product mightinterfere with the subsequent immunogold localization ofthe neurotransmitters. Colloidal gold particles overlyingsynaptic endings labeled by the postembedding methodwere counted, and their density was calculated as thenumber of particles per µm2 overall, with an additionaldistinction being made between particles overlying synap-tic vesicles versus mitochondria. The determination ofbackground labeling due to nonspecific binding or meta-bolic pools of the amino acids was made by samplingregions of blood vessel lumina and motoneuron somataand dendrites, respectively. Descriptive statistics werecalculated using commercially available software and ex-pressed as the mean 6 standard error (Table 1). Differ-ences within and between different populations of synapticendings were tested statistically using a single factoranalysis of variance (ANOVA).

RESULTS

Neurotransmitter localization inbiocytin-labeled abducens internuclear

synaptic endings in the oculomotor nucleus

Biocytin-labeled abducens internuclear synaptic end-ings were recognized by the dense peroxidase reaction

product that permitted their unequivocal identificationamong unlabeled synaptic endings. The morphology andsoma-dendritic distribution of biocytin-labeled abducensinternuclear synaptic endings labeled by the pre-embed-ding immunoperoxidase method were similar to thosedescribed in a previous study (Nguyen et al., 1999). Mostsynaptic endings usually occurred in isolation in theneuropil surrounding the motoneurons in the dorsolateralmedial rectus subdivision of the oculomotor nucleus, weredome-shaped in appearance, and contained a uniformpopulation of spheroidal synaptic vesicles that were distrib-uted throughout the terminal and numerous mitochondriathat were clustered toward the center. When combinedwith the postembedding immunogold procedure to localizethe neurotransmitters, glutamate and aspartate, colloidalgold particles associated with the labeling of both neuro-transmitters were observed overlying the mitochondriaand synaptic vesicles in the biocytin immunoperoxidase-labeled abducens internuclear synaptic terminals (Figs. 1and 2). However, as also noted in another study (Spencerand Wang, 1996), both glutamate- and aspartate-immuno-reactive labeling were associated with most neuronalprofiles, both pre- and postsynaptic, in the oculomotornucleus, even though background labeling (e.g., associatedwith capillary lumina, myelin, glial elements, bare Form-var film) was low.

Aspartate. Abducens internuclear synaptic endingslabeled with aspartate established synaptic contact withsmall- (Fig. 1B), medium- (Fig. 1A,C), and large- (Fig. 1D)diameter dendrites. Synaptic contact zones were character-ized by a modest or prominent postsynaptic densification.The presynaptic areas of aspartate-immunoreactive synap-tic endings (n 5 86) ranged from 0.39 to 11.15 µm2, with amean of 1.57 6 0.15 µm2 (Fig. 3). The number of mitochon-dria ranged from 0 to 22, with a mean of 6.07 6 0.47. Thedensity of mitochondria ranged from 0 to 19.10/µm2, with amean of 4.28 6 0.29 mitochondria/µm2.

The diameters of profiles that were postsynaptic to theaspartate-immunoreactive abducens internuclear synap-tic endings ranged from 0.30 to 10.30 µm, with a mean of2.70 6 0.26 µm (Fig. 4). Within this range, synapticendings established contacts with somata (9%), proximaldendrites (38%), distal dendrites (51%), and spine-likeappendages (2%), each characterized on the basis of theirultrastructural features (e.g., size and organelle content;Fig. 5). When the soma-dendritic distributions based onultrastructural features were coded to numerical values

TABLE 1. Quantitative Measures of Presynaptic and Postsynaptic ProfilesAssociated With Aspartate- and Glutamate-Immunoreactive AbducensInternuclear and Ascending Tract of Deiters (ATD) Synaptic Endings in

the Medial Rectus Subdivision of the Cat Oculomotor Nucleus1

Abducens internuclear ATD

Aspartate(n 5 86)

Glutamate(n 5 98)

Aspartate(n 5 75)

Glutamate(n 5 103)

Presynaptic area (µm2) 1.57 6 0.15 1.45 6 0.08 1.36 6 0.09 1.53 6 0.11Postsynaptic diameter

(µm) 2.70 6 0.26 2.39 6 0.23 6.16 6 0.45 6.48 6 0.37Weighted distribution 2.55 6 0.08 2.49 6 0.08 3.28 6 0.08 3.36 6 0.07No. mitochondria/µm2 4.28 6 0.29 4.10 6 0.19 3.45 6 0.22 3.50 6 0.19Labeling density—syn-

aptic vesicles (particles/µm2) 33.60 6 1.46 53.15 6 2.17 14.65 6 0.52 19.74 6 1.21

Labeling density—mito-chondria (particles/µm2) 33.58 6 2.05 55.58 6 2.77 10.27 6 0.73 21.13 6 1.34

v/m ratio 1.80 6 0.38 3.52 6 1.21 3.28 6 0.77 2.24 6 0.65

1Spine 5 1, Distal 5 2, Proximal 5 3, Soma 5 4.

ABDUCENS INTERNUCLEAR AND ATD NEUROTRANSMITTERS 75

(i.e., spine-like appendage 5 1, distal dendrite 5 2, proxi-mal dendrite 5 3, soma 5 4), aspartate-immunoreactiveabducens internuclear synaptic endings had a meanweighted soma-dendritic distribution of 2.55 6 0.08.

Considerable variation was observed with the gold par-ticle labeling of the mitochondrial versus the synapticvesicle/axoplasmic matrix compartments of the aspartate-immunoreactive abducens internuclear synaptic endings.The number of gold particles overlying mitochondria rangedfrom 0 to 182, with a mean of 50.14 6 4.34 particles. Bycontrast, the number of colloidal gold particles overlyingsynaptic vesicles ranged from 5 to 358, with a mean of49.42 6 4.68 particles. The density of gold particle labelingassociated with the mitochondria ranged from 0.85 to83.19 particles/µm2, with a mean of 33.61 6 2.04 particles/µm2; while the labeling density associated with the synap-

tic vesicles/axoplasmic matrix ranged from 1.49 to 109.80particles/µm2, with a mean of 33.60 6 1.46 particles/µm2.The ratio between the labeling density associated with themitochondrial versus the synaptic vesicle/axoplasmic ma-trix compartments (v/m ratio) ranged from 0.12 to 29, witha mean of 1.80 6 0.38 (Fig. 6).

Glutamate. Abducens internuclear synaptic endingslabeled with glutamate established synaptic contact pre-dominantly with small- (Fig. 2C) and medium- (Fig.2A,B,E–F) diameter dendrites, as well as occasionally withdendritic spine-like appendages (Fig. 2D). Synaptic con-tact zones were characterized by a modest or prominentpostsynaptic densification. The presynaptic areas of gluta-mate-immunoreactive synaptic endings (n 5 98) rangedfrom 0.35 to 3.86 µm2, with a mean of 1.45 6 0.08 µm2 (Fig.3). The number of mitochondria ranged from 0 to 17, with a

Fig. 1. Electron micrographs of aspartate-immunoreactive (postem-bedding immunogold) abducens internuclear synaptic endings (pre-embedding immunoperoxidase). Aspartate-immunoreactive abducensinternuclear synaptic endings (s) contain spheroidal vesicles and

establish synaptic contact with small- (B), medium- (A,C), and large-(D) diameter dendrites (d). Synaptic contact zones are characterizedby modest (B,D) or prominent (A) postsynaptic densifications (arrows).Scale bars 5 0.5 µm in A–D.


mean of 5.96 6 0.41. The density of mitochondria ranged from0 to 8.81/µm2, with a mean of 4.10 6 0.19 mitochondria/µm2.

The diameters of profiles that were postsynaptic to theglutamate-immunoreactive abducens internuclear synap-tic endings ranged from 0.18 to 10.48 µm, with a mean of2.2.39 6 0.23 µm (Fig. 4). When the postsynaptic profilewas characterized on the basis of ultrastructural criteria,synaptic endings established synaptic contacts with so-mata (9%), proximal dendrites (25%), distal dendrites(55%), and spine-like appendages (11%, Fig. 5). The meanweighted soma-dendritic distribution of glutamate-immu-noreactive abducens internuclear synaptic endings was2.49 6 0.08.

Like the aspartate-immunoreactive synaptic endings,the gold particle labeling of the mitochondrial versus the

synaptic vesicle/axoplasmic matrix compartments of theglutamate-immunoreactive abducens internuclear synap-tic endings exhibited substantial variation. The number ofgold particles overlying mitochondria ranged from 0 to253, with a mean of 79.54 6 5.12 particles. By contrast, thenumber of colloidal gold particles overlying synapticvesicles ranged from 11 to 307, with a mean of 72.94 6 4.62particles. The density of gold particle labeling associatedwith the mitochondria ranged from 0.75 to 123.53 particles/µm2, with a mean of 55.69 6 2.75 particles/µm2; while thelabeling density associated with the synaptic vesicles/axoplasmic matrix ranged from 16.01 to 114.04 particles/µm2, with a mean of 53.15 6 2.17 particles/µm2. The ratiobetween the labeling density associated with the mitochon-drial versus the synaptic vesicle/axoplasmic matrix com-

Fig. 2. Electron micrographs of glutamate-immunoreactive(postembedding immunogold) abducens internuclear synaptic endings(pre-embedding immunoperoxidase). Glutamate-immunoreactive ab-ducens internuclear synaptic endings (s) contain spheroidal synapticvesicles and establish synaptic contacts with small- and medium-

(B,D) diameter dendrites (d) and dendritic spine-like appendages (spin A). Synaptic contact zones (arrows) are characterized by modest(A,C) or prominent (B–D) postsynaptic densifications. Scale bars 5 0.5µm in A–D.


partments (v/m ratio) ranged from 0.23 to 89, with a meanof 3.52 6 1.21 (Fig. 6).

Neurotransmitter localization inbiocytin-labeled ATD synaptic endings in the

oculomotor nucleus

The morphology and soma-dendritic distribution of bio-cytin-labeled ATD synaptic endings labeled by the pre-embedding immunoperoxidase method were similar tothose described in a previous study (Nguyen et al.,1999). Most synaptic endings were associated with thesomata and proximal dendrites of motoneurons in thedorsolateral medial rectus subdivision of the oculomotornucleus and contained a uniform population of spheroidalsynaptic vesicles that were distributed throughout theterminal and numerous mitochondria that were clusteredtoward the center. When combined with the postembed-ding immunogold procedure to localize the neurotransmit-

ters, glutamate and aspartate, colloidal gold particlesassociated with the labeling of both neurotransmitterswere observed overlying the mitochondria and synapticvesicles in the biocytin immunoperoxidase-labeled ATDsynaptic terminals (Figs. 7 and 8). However, like withabducens internuclear synaptic endings, both glutamate-and aspartate-immunoreactive labeling are associated withmost neuronal profiles, both pre- and postsynaptic, in theoculomotor nucleus—even though background labelingwas low.

Aspartate. ATD synaptic endings labeled with aspar-tate established synaptic contact predominantly with large-diameter dendrites (Fig. 7A–E) and somata. Synapticcontact zones were characterized by a modest or promi-

Fig. 3. Histograms of the presynaptic area distributions of aspar-tate- and glutamate-immunoreactive abducens internuclear (top) andDeiters (ATD) (bottom) synaptic endings. The differences within andbetween the different neurotransmitter-specific populations of synap-tic endings are not statistically significant.

Fig. 4. Histograms of the postsynaptic diameter distributions ofaspartate- and glutamate-immunoreactive abducens internuclear (top)and Deiters (ATD) (bottom) synaptic endings. The diameters of theproximal dendrites of choline acelyltransferase-immunoreactive me-dial rectus motorneurons (chat MR MN) are provided as references.The differences within the different populations of synaptic endings inregard to aspartate versus glutamate are not statistically significant,but the differences between abducens internuclear and ATD, irrespec-tive of neurotransmitter, are significant.


nent postsynaptic densification and occasionally were asso-ciated with subsurface cisterns (Fig. 7A). The presynapticareas of aspartate-immunoreactive synaptic endings (n 575) ranged from 0.38 to 5.41 µm2, with a mean of 1.36 60.09 µm2 (Fig. 3). The number of mitochondria ranged from0 to 13, with a mean of 4.33 6 0.31. The density ofmitochondria ranged from 0 to 8.41/µm2, with a mean of3.45 6 0.22 mitochondria/µm2.

The diameters of profiles that were postsynaptic to theaspartate-immunoreactive ATD synaptic endings rangedfrom 0.34 to 10.98 µm, with a mean of 6.16 6 0.45 µm (Fig.4). Within this range, synaptic endings established con-tacts with somata (41%), proximal dendrites (47%), anddistal dendrites (11%), each characterized on the basis oftheir ultrastructural features (Fig. 5). Aspartate-immuno-reactive ATD synaptic endings had a mean weightedsoma-dendritic distribution of 3.28 6 0.08.

Considerable variation was observed with the gold par-ticle labeling of the mitochondrial versus the synapticvesicle/axoplasmic matrix compartments of the aspartate-immunoreactive ATD synaptic endings. The number ofgold particles overlying mitochondria ranged from 0 to 49,with a mean of 13.61 6 1.23 particles. By contrast, thenumber of colloidal gold particles overlying synapticvesicles ranged from 3 to 71, with a mean of 19.76 6 1.50particles. The density of gold particle labeling associatedwith the mitochondria ranged from 0.30 to 29.91 particles/mm2, with a mean of 10.36 6 0.71 particles/mm2, while thelabeling density associated with the synaptic vesicles/axoplasmic matrix ranged from 2.56 to 26.14 particles/mm2, with a mean of 14.65 6 0.52 particles/mm2. The ratio

between the labeling density associated with the mitochon-drial versus the synaptic vesicle/axoplasmic matrix com-partments (v/m ratio) ranged from 0.09 to 49, with a meanof 3.28 6 0.77 (Fig. 6).

Glutamate. Like the aspartate-immunoreactive synap-tic endings, ATD synaptic endings labeled with glutamateestablished synaptic contact predominantly with large-diameter dendrites (Fig. 8C–D) and somata (Fig. 8A–B).Synaptic contact zones were characterized by a modest orprominent postsynaptic densification. Axosomatic synap-tic endings usually were associated with subsurface cis-terns. The presynaptic areas of glutamate-immunoreac-tive synaptic endings (n 5 103) ranged from 0.34 to 8.43µm2, with a mean of 1.53 6 0.11 µm2 (Fig. 3). The numberof mitochondria ranged from 0 to 14, with a mean of 5.19 60.35. The density of mitochondria ranged from 0 to 10.14/mm2, with a mean of 3.50 6 0.19 mitochondria/mm2.

The diameters of profiles that were postsynaptic to theglutamate-immunoreactive ATD synaptic endings rangedfrom 0.21 to 11.33 µm, with a mean of 6.48 6 0.37 µm (Fig.4). On the basis of the ultrastructural features of thepostsynaptic profiles, synaptic endings established con-tacts with somata (30%), proximal dendrites (53%), distaldendrites (14%), and spine-like appendages (3%, Fig. 5).The mean weighted soma-dendritic distribution of gluta-mate-immunoreactive ATD synaptic endings was 3.36 60.07.

Like the aspartate-immunoreactive synaptic endings,the gold particle labeling of the mitochondrial versus thesynaptic vesicle/axoplasmic matrix compartments of theglutamate-immunoreactive ATD synaptic endings exhib-ited substantial variation. The number of gold particlesoverlying mitochondria ranged from 0 to 362, with a meanof 33.59 6 4.06 particles. By contrast, the number ofcolloidal gold particles overlying synaptic vesicles rangedfrom 2 to 265, with a mean of 28.95 6 3.08 particles. Thedensity of gold particle labeling associated with the mito-chondria ranged from 0.25 to 56 particles/mm2, with amean of 21.20 6 1.33 particles/mm2; while the labelingdensity associated with the synaptic vesicles/axoplasmicmatrix ranged from 2.50 to 53.33 particles/mm2, with amean of 19.74 6 1.21 particles/mm2. The ratio between thelabeling density associated with the mitochondrial versusthe synaptic vesicle/axoplasmic matrix compartments (v/mratio) ranged from 0.25 to 56.00, with a mean of 2.27 60.65 (Fig. 6).

Comparison between abducens internuclearand ATD synaptic endings labeled with

different neurotransmitters

Similarities were noted among biocytin-labeled abdu-cens internuclear and ATD synaptic endings that wereimmunoreactive for the different neurotransmitters. Bydescriptive statistical analysis, all synaptic endings, irre-spective of source and neurotransmitter immunoreactiv-ity, had similar presynaptic areas. The soma-dendriticdistribution of aspartate- versus glutamate-immunoreac-tive abducens internuclear synaptic endings, as reflectedby quantitative measures of postsynaptic diameter andweighted distribution, were similar, as were those ofaspartate- versus glutamate-immunoreactive ATD synap-tic endings. However, by both measures, the distal soma-dendritic distribution of abducens internuclear synapticendings differed significantly from the proximal location ofATD synaptic endings (P , 0.01).

Fig. 5. Histogram of the soma-dendritic distributions of aspartate-and glutamate-immunoreactive abducens internuclear (AbdIn) andDeiters (ATD) synaptic endings. Postsynaptic profiles are classified asdistal and proximal dendrites, soma, and spine on the basis of theirultrastructural features. The majority of aspartate- and glutamate-immunoreactive abducens internuclear synaptic endings contact dis-tal dendrites, whereas the majority of aspartate- and glutamate-immunoreactive ATD synaptic endings contact proximal dendritesand somata. The differences within the different populations ofsynaptic endings in regard to aspartate versus glutamate are notstatistically significant, but the differences between abducens inter-nuclear and ATD, irrespective of neurotransmitter, are significant.


No significant differences were found in the number ordensity of mitochondria between aspartate- versus gluta-mate-labeled abducens internuclear synaptic endings.Similarly, the differences between aspartate- versus gluta-mate-labeled ATD synaptic endings in regard to the num-

ber and density of mitochondria were not significant.However, the aspartate-immunoreactive abducens inter-nuclear synaptic endings had a significantly larger num-ber and a greater density of mitochondria than the aspar-tate-labeled ATD synaptic endings (P , 0.05). The density

Fig. 6. Scatter-plots of synaptic vesicle versus mitochondrial gold(Au) labeling density associated with aspartate- and glutamate-immunoreactive abducens internuclear (top left and right) and Deiters(ATD) (bottom left and right) synaptic endings. Closed circles indicatevalues that exceed the labeling density associated with the postsynap-tic motoneuron somata and presumably represent neurotransmitterpools of the two amino acids. Open triangles indicate values that equal

or are less than the motoneuron labeling density and presumablyrepresent metabolic pools of the two amino acids. The overwhelmingmajority of abducens internuclear synaptic endings contain a neuro-transmitter pool of both aspartate and glutamate, whereas ATDsynaptic endings appear to contain a significant neurotransmitter poolof only glutamate.


Fig. 7. Electron micrographs of aspartate-immunoreactive (postem-bedding immunogold) Deiters (ATD) synaptic endings (pre-embeddingimmunoperoxidase). Aspartate-immunoreactive ATD synaptic end-ings contain clear spheroidal synaptic vesicles and establish synapticcontacts (arrows) with somata (A–C) and large-diameter proximal

dendrites (D,E). Synaptic contact zones are characterized by modest toprominent postsynaptic densifications (psd) and, in some instances,are associated with subsurface cisterns (sc in A). s, synaptic endings;sp, spine-like appendages; sc, subsurface cisterns; d, dendrites; sdb,subjunctional dense body. Scale bars 5 0.5 µm in A–E.

of mitochondria in glutamate-immunoreactive abducensinternuclear synaptic endings also was significantly greaterthan that of glutamate-labeled ATD synaptic endings (P ,0.05).

Significant differences also were noted between thedifferent neurotransmitter populations of abducens inter-nuclear and ATD synaptic endings with regard to thedensity of gold particles overlying synaptic vesicles/axoplasmic matrix versus those associated with mitochon-dria (Fig. 6). The density of gold particle labeling associ-ated with the mitochondrial and synaptic vesicle/axoplasmic matrix compartments in both the abducensinternuclear and ATD aspartate-immunoreactive synapticendings was significantly less than their glutamate-

labeled counterparts (P , 0.01). The density of goldparticle labeling associated with both the mitochondrialand synaptic vesicle/axoplasmic matrix compartments inboth the aspartate- and glutamate-immunoreactive ATDsynaptic endings was significantly less than those of theabducens internuclear synaptic endings (P , 0.01).

As a population, the labeling density of both the aspar-tate- and glutamate-immunoreactive abducens inter-nuclear synaptic endings differed significantly from thelabeling density of aspartate (40.60 6 2.66 particles/mm2)and glutamate (39.25 6 2.87 particles/mm2) associatedwith the motoneuron somata (P , 0.01). By contrast, onlythe glutamate-immunoreactive ATD synaptic endings dif-fered significantly from the postsynaptic labeling associ-

Fig. 8. Electron micrographs of glutamate-immunoreactive(postembedding immunogold) Deiters (ATD) synaptic endings (pre-embedding immunoperoxidase). ATD synaptic endings contain clearspheroidal synaptic vesicles and establish synaptic contacts withsomata (A,B) and large-diameter proximal dendrites (C,D). Synaptic

contact zones exhibit modest to prominent postsynaptic densifications(arrows). Note the subsurface cisterns associated with the axosomaticsynaptic endings (C,D). s, synaptic endings; sp, spine-like appendages;sc, subsurface cisterns; d, dendrites. Scale bars 5 0.5 µm in A–D.


ated with the motoneurons (P , 0.01). Using the motoneu-ron labeling as a reference for potentially distinguishingbetween metabolic versus neurotransmitter pools of theamino acids, 88% of the aspartate-immunoreactive and98% of the glutamate-immunoreactive abducens inter-nuclear synaptic endings had a labeling density exceedingthat of the postsynaptic motoneurons and thus wereconsidered to possess a neurotransmitter pool of the aminoacids (Fig. 6). By contrast, only 3% of the aspartate-immunoreactive and 44% of the glutamate-immunoreac-tive ATD synaptic endings had a labeling density that wasgreater than that of the motoneurons (Fig. 6).

Control experiments

Negative control sections in which the primary antibodywas omitted were virtually devoid of gold particles. Con-trol sections that were incubated with anti-glutamate andanti-aspartate antibodies that were preabsorbed with glu-tamate and aspartate, respectively, had significantly re-duced numbers of gold particles over the entire sectionwhen compared to experimental sections. The labelingwith preabsorbed glutamate exhibited a nonspecific pat-tern that was attributed to the nature of polyclonalantibody used. Preabsorption control experiments for as-partate contained more colloidal gold particles over thesections than the control glutamate sections, as also notedby Beltz and Burd (1989).

DISCUSSION

In the present study, abducens internuclear and ATDsynaptic endings labeled by the anterograde transport ofbiocytin and localized with a pre-embedding immunoper-oxidase method have been characterized further on thebasis of the colocalization of the putative excitatory neuro-transmitters, aspartate and glutamate, using a postembed-ding immunogold method. The morphological features ofboth the abducens internuclear and ATD synaptic endings,including their content of spheroidal synaptic vesicles andthe associated asymmetrical pre-/postsynaptic membranedensification at the synaptic contact zones, are consistentwith the classical criteria of excitatory synapses (Gray,1959; Uchizono, 1965; Larramendi et al., 1967). That bothpopulations of synaptic endings are immunoreactive toglutamate and/or aspartate is further consistent with thephysiological (Baker and Highstein, 1978; Highstein andBaker, 1978; Yamamoto et al., 1978; Reisine and High-stein, 1979; Nakao and Sasaki, 1980; Furuya andMarkham, 1981; Highstein and Reisine, 1981; Reisine etal., 1981; de la Cruz et al., 1989) and morphological(Markham et al., 1986; Nguyen et al., 1999) features ofthese excitatory inputs to medial rectus motoneurons.

As also noted in a previous study of mesencephalicreticular inputs to vertical motoneurons in the oculomotorand trochlear nuclei (Spencer and Wang, 1996), a highlevel of ‘‘background’’ labeling of glutamate and aspartateis associated with most pre- and postsynaptic profiles inthe medial rectus subdivision of the oculomotor nucleus.The labeling of postsynaptic profiles (e.g., motoneuronsomata and dendrites) is likely attributable to the highconcentration of the dipeptide, N-acetyl-aspartyl-gluta-mate (NAAG), in the cholinergic motoneurons, for whichboth glutamate and aspartate are requisite precursors(Forloni et al., 1987). Not surprisingly, a substantialamount of glutamate and aspartate immunolabeling is

associated with the granular endoplasmic reticulum andmitochondria in the motoneuron somata. Glutamate andaspartate are linked metabolically through the enzyme,aspartate aminotransferase (Merighi et al., 1991). Presum-ably related to their common synthesis by a process oftransamination, considerable variation has been observedin the density of glutamate and aspartate immunoreactiv-ity associated with the biocytin-labeled abducens inter-nuclear and ATD synaptic endings. Consequently, by quali-tative observation alone, it has not been possible todistinguish neurotransmitter versus metabolic labelingfor glutamate and aspartate in relation to the two popula-tions of synaptic endings or to determine whether differ-ences exist within and between the two populations ofsynaptic endings in regard to neurotransmitter utiliza-tion.

In the present study, a quantitative assessment of theglutamate- and aspartate-immunoreactive labeling of thebiocytin-labeled abducens internuclear and ATD synapticendings has been made on the basis of the gold particledensity associated with synaptic vesicles/axoplasmic ma-trix (neurotransmitter pool) and mitochondria (metabolicpool). The overall labeling densities of the two amino acidsin the different populations of synaptic endings further-more have been compared to their respective labelingdensities in the postsynaptic motoneuron somata, whichpresumably reflect only metabolic pools of the amino acidsthat are related to the synthesis of NAAG. By thesecriteria, the findings in the present study suggest that themajority of abducens internuclear synaptic endings areenriched with neurotransmitter pools of both aspartateand glutamate. By contrast, ATD synaptic endings have aneurotransmitter pool only for glutamate. Although ATDsynaptic endings exhibit immunoreactivity toward aspar-tate, the density of mitochondrial versus synaptic vesicle/axoplasmic matrix labeling with aspartate differs substan-tially from that associated with the glutamate labeling ofATD synaptic endings, as well as aspartate- and glutamate-immunoreactive abducens internuclear synaptic endings(Fig. 6). Consequently, aspartate appears to be associatedwith a metabolic pool of the amino acid in ATD synapticendings.

Substantial evidence exists for both aspartate and gluta-mate as the major excitatory neurotransmitters in thevestibular system (de Waele et al., 1995). Aspartate-(Kumoi et al., 1987; Carpenter et al., 1992) and glutamate-(Carpenter et al., 1990) immunoreactive neurons predomi-nate in the vestibular nuclei. Vestibulospinal neurons inthe superior, medial, descending (inferior), and particu-larly the lateral vestibular nuclei are immunoreactivetoward aspartate (Kevetter and Coffey, 1991), whereasvestibulo-ocular neurons that project to the oculomotorand trochlear nuclei exhibit glutamate and/or aspartateimmunoreactivity (Kevetter and Hoffman, 1991; Carpen-ter et al., 1992). Although glutamate- and aspartate-immunoreactive neurons (Ottersen and Storm-Mathisen,1985; Conti et al., 1987; Clements et al., 1987; Hepler etal., 1988; Spencer and Wang, 1996) and synaptic endings(Maxwell et al., 1990; Merighi et al., 1991; Spencer andWang, 1996), in at least some instances, are considered tobe distinct populations in other regions of the nervoussystem, it is possible that individual vestibular neurons(Yingcharoen et al., 1989; Walberg et al., 1990), likeneurons (Hepler et al., 1988) and synaptic endings (Traceyet al., 1991; Phend et al., 1992) in other regions of the


nervous system, may co-localize glutamate and aspartate.In the case of ATD neurons and their synaptic terminals inthe oculomotor nucleus, however, the co-localization ofaspartate and glutamate appears to represent distinctmetabolic and neurotransmitter pools of the amino acids,respectively. On the other hand, a more compelling argu-ment can be made for the co-localization of aspartate andglutamate as the neurotransmitters associated with indi-vidual abducens internuclear synaptic endings, given thesimilarities in the morphology, presynaptic area, mitochon-drial density, and soma-dendritic distribution of the aspar-tate- and glutamate-immunoreactive synaptic endings.

Recent studies (Shupliakov et al., 1992, 1995) haverelated the differences in the subcellular localization ofglutamate to not only the metabolic requirements, but alsothe physiological properties of different populations ofsynapses. A higher density of glutamate labeling is associ-ated with the mitochondrial and axoplasmic matrix com-partments in tonically active synapses than in phasicallyactive synapses. Although no attempt has been made inthe present study to distinguish between labeling associ-ated with synaptic vesicles versus axoplasmic matrix dueto the high density of synaptic vesicles in both populationsof synaptic endings, striking differences are apparentbetween aspartate- and glutamate-labeled ATD and abdu-cens internuclear synaptic endings when the density ofgold particles overlying mitochondria is compared to thedensity of gold particles overlying the extramitochondrialcompartment, including both synaptic vesicles and theaxoplasmic matrix (Fig. 6). These differences in labelingpatterns furthermore appear to be related to the knownphysiological differences between the two populations ofsynaptic inputs (Baker and Highstein, 1978; Highsteinand Baker, 1978; Yamamoto et al., 1978; Reisine andHighstein, 1979; Nakao and Sasaki, 1980; Furuya andMarkham, 1981; Highstein and Reisine, 1981; Reisine etal., 1981; de la Cruz et al., 1989). For example, the labelingdensity of glutamate-immunoreactive ATD synaptic end-ings is significantly less than the labeling density ofglutamate-immunoreactive abducens internuclear synap-tic endings, suggesting that the high frequency burstactivity of the ATD synaptic input conveying a headvelocity signal requires less neurotransmitter than theburst activity associated with the abducens internuclearinput that is providing an eye velocity signal. By contrast,aspartate may be the neurotransmitter that is associatedwith the tonic activity of the abducens internuclear synap-tic input that conveys the eye position signal and that ismediated by the same population of synaptic endings.Consistent with this hypothesis, both the previous (Nguyenet al., 1999) and current studies have demonstrated agreater number and a higher density of mitochondria inabducens internuclear endings in comparison to the mito-chondrial content of ATD synaptic endings.

Despite the possibility of the co-localization of putativeexcitatory amino acid neurotransmitters, in most in-stances only one or the other should have a synaptic effectthat is dictated by the presence and type of the postsynap-tic receptor with which the input is associated. Glutamateand aspartate are associated with various ionotropic andmetabotropic receptors. The ionotropic receptors generallyare characterized as NMDA and non-NMDA (AMPA,quisqualate/kainate) receptors, each of which produces aspecific and different synaptic effect and, in some cases, adifferent behavioral effect. While glutamate can act at

either receptor type, aspartate has a specific affinity forNMDA receptors. Within the extraocular motor nuclei, theexcitatory second-order vestibular inputs onto abducensneurons utilize glutamate as the neurotransmitter actingthrough AMPA receptors (Straka and Dieringer, 1993).Both glutamate and NMDA produce a depolarization ofabducens motoneurons (Durand et al., 1987). NMDA re-sponses are voltage-dependent and are characterized bybursts of action potentials followed by a stable repetitive,rhythmic firing (Durand, 1991). NMDA receptors appearto have a predominantly dendritic location, but are notassociated with the excitatory second-order vestibularinput to oculomotor motoneurons (Durand and Gueritaud,1990). Spencer and Wang (1996) have speculated that thevertical saccade-related riMLF excitatory synaptic inputto oculomotor and trochlear motoneurons is mediated byseparate populations of neurons that utilize aspartate as aneurotransmitter acting through NMDA receptors andglutamate acting through NMDA and/or non-NMDA (e.g.,kainate, quisqualate, AMPA) receptors. These separateneurotransmitter-specific populations of neurons may berelated more to their different projection targets in rela-tion to the control of eye movement (oculomotor andtrochlear nuclei) versus gaze (oculomotor and trochlearnuclei and cervical spinal cord) than to differences in thesignals conveyed by them. By contrast, the differences inneurotransmitter utilization of the abducens internuclearand ATD synaptic inputs to medial rectus motoneuronsappear to be more related to their differential roles in thecontrol of conjugate horizontal eye movements.

The findings from the previous (Nguyen et al., 1999) andpresent studies are summarized in Figure 9. The differ-ences between abducens internuclear and ATD synapticendings in regard to neurotransmitter localization may berelated to not only differences in their soma-dendriticlocation, but also physiological differences in the signalsthat are conveyed to medial rectus motoneurons by theindividual inputs. Abducens internuclear neurons exhibita burst-tonic discharge in relation to eye velocity and eyeposition (King et al., 1976; Delgado-Garcia et al., 1986;Markham et al., 1986; Fuchs et al., 1988; Stahl andSimpson, 1995). By contrast, ATD neurons convey a headvelocity signal (Reisine and Highstein, 1979; Highsteinand Reisine, 1981; Markham et al., 1986). These differ-ences in neurotransmitter localization furthermore maybe related to differences in the postsynaptic receptors withwhich the inputs are associated. NMDA receptors havebeen attributed to producing a slow and long-lastingresponse (Soto and Vega, 1988; Perez et al., 1991), whileAMPA receptors generate a rapid response with a quickdecay (Lester and Jahr, 1992). Consequently, the burstactivity of the ATD synaptic input as it relates to changesin head velocity is likely to be associated with glutamateacting through non-NMDA (e.g., AMPA, quisqualate/kainate) receptors, whereas the burst-tonic activity of theabducens internuclear synaptic input as it relates to eyeposition eye and velocity signals is likely to be associatedwith glutamate and aspartate acting through NMDA andnon-NMDA receptors, respectively, possibly co-localized atthe same postsynaptic site (Brodin and Shupliakov, 1994).This arrangement is consistent with the known soma-dendritic distribution of NMDA and non-NMDA receptorson both second-order vestibular (Cochran et al., 1987) andother extraocular (Durand et al., 1987; Durand, 1991;


Durand and Gueritaud, 1990; Straka and Dieringer, 1993)motoneurons.

ACKNOWLEDGMENTS

The technical assistance of Barbara Mann and MichaelNguyen is greatly appreciated.

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