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: Synaptic Organization

LYNETTE T. NGUYEN,1 ROBERT BAKER,2 AND ROBERT F. SPENCER1,3*1Departments of Anatomy, Medical College of Virginia, Virginia Commonwealth University,

Richmond, Virginia 232982Department of Physiology and Neuroscience, New York University Medical Center,

New York, New York 100163Department of Otolaryngology-Head and Neck Surgery, Medical College of Virginia,

Virginia Commonwealth University, Richmond, Virginia 23298

ABSTRACTAbducens internuclear and ascending tract of Deiters (ATD) inputs to medial rectus

motoneurons in the oculomotor nucleus are important for conjugate horizontal movements. Inthe present study, the organization of these separate populations of neurons and theirsynaptic connections with medial rectus motoneurons in the cat oculomotor nucleus have beenexamined by light and electron microscopy by using retrograde and anterograde axonaltracers. Consistent with the patterns of retrograde horseradish peroxidase labeling, theabducens internuclear projection is predominantly, if not exclusively, contralateral, whereasthe ATD projection is exclusively ipsilateral, as demonstrated by anterograde autoradio-graphic and biocytin labeling. Both populations of synaptic endings contain spheroidalsynaptic vesicles and establish synaptic contacts with modest postsynaptic densifications. Inaddition, ATD synaptic endings frequently are associated with subjunctional dense bodies andsubsurface cisternae. The two populations of excitatory inputs differ, however, in theirsoma-dendritic distribution. The majority of abducens internuclear synaptic endings contactdistal dendrites, whereas the majority of ATD synaptic endings contact proximal dendrites orsomata. Abducens internuclear synaptic endings furthermore have a higher density ofmitochondria than ATD synaptic endings. The more proximal location of ATD synaptic endings isconsistent with the faster rise time and earlier reversal to polarizing currents of ATD excitatorypostsynaptic potentials in comparison to those evoked by the abducens internuclear pathway asdetermined electrophysiologically. Given the differences in the physiologic signals conveyed by theabducens internuclear (eye velocity and eye position) and ATD (head velocity) pathways, thefindings in this study suggest that the soma-dendritic stratification of the two inputs to medialrectus motoneurons may provide a means for the separate control of visuomotor andvestibular functions, respectively. J. Comp. Neurol. 405:141–159, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: horseradish peroxidase; autoradiography; biocytin; abducens nucleus; vestibular

nucleus; conjugate horizontal eye movements

The abducens nucleus is regarded as the center forconjugate horizontal eye movements (Bender, 1980). Le-sions involving the abducens nucleus produce a paralysisof ipsilateral ocular abduction and a paresis of contralat-eral ocular adduction on attempted conjugate horizontalgaze toward the side of the lesion. The basis for thesedeficits is the coexistence of two populations of neurons inthe abducens nucleus: motoneurons that innervate theipsilateral lateral rectus muscle and internuclear neurons

Grant sponsor: United States Public Health Service; Grant number:MERIT Award EY02191; Grant sponsor: National Eye Institute; Grantnumber: Research Grant EY02007.

Dr. Nguyen8s current address is: 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 14 September 1998; Accepted 24September 1998

THE JOURNAL OF COMPARATIVE NEUROLOGY 405:141–159 (1999)

r 1999 WILEY-LISS, INC.

whose axons ascend to the contralateral oculomotor nucleusby means of the medial longitudinal fasciculus (MLF;reviewed in Evinger, 1988). Within the oculomotor nucleus,the axons of abducens internuclear neurons establishmonosynaptic excitatory synaptic connections with medialrectus motoneurons (Baker and Highstein, 1975; High-stein and Baker, 1978; Yamamoto et al., 1978; Nakao andSasaki, 1980; Furuya and Markham, 1981; de la Cruz etal., 1989). Abducens internuclear neurons receive synapticinputs that are similar to those of lateral rectus motoneu-rons (Spencer and Sterling, 1977; Highstein and Baker,1978). Reciprocal excitatory and inhibitory synaptic inputsfrom second-order neurons in the medial and ventrallateral vestibular nuclei are related to the horizontalvestibulo-ocular reflex (Baker et al., 1969; Highstein,1973; Baker and Highstein, 1975; Maciewicz et al., 1977;Ishizuka et al., 1980; McCrea et al., 1980; Uchino et al.,1981, 1982; Nakao et al., 1982; Carleton and Carpenter,1983; Uchino and Suzuki, 1983; Langer et al., 1986;McCrea et al., 1987; Belknap and McCrea, 1988; Ohgaki etal., 1988; Escudero and Delgado-Garcia, 1988; Isu et al.,1991; Scudder and Fuchs, 1992). Reciprocal excitatory andinhibitory synaptic inputs from neurons in the pontomed-ullary reticular formation are related to the control ofconjugate horizontal gaze (Cohen and Henn, 1972; Buttner-Ennever and Henn, 1976; Highstein et al., 1976; Graybiel,1977; Hikosaka and Kawakami, 1977; Maciewicz et al.,1977; Hikosaka et al., 1978; Grantyn et al., 1980a,b;Hikosaka and Igusa, 1980; Igusa et al., 1980; Curthoys etal., 1981; Kaneko et al., 1981; Yoshida et al., 1982; Langeret al., 1986; Strassman et al., 1986a,b; Escudero andDelgado-Garcia, 1988; Scudder et al., 1988; Hepp et al.,1989). In addition, consistent with its postulated role inthe integration of head velocity and eye velocity signals toeye position signals (Cheron et al., 1986a,b; Cannon andRobinson, 1987), the prepositus hypoglossi nucleus hasextensive interconnections with the vestibular nuclei andpontomedullary reticular formation (McCrea and Baker,1985; Belknap and McCrea, 1988) and efferent connectionswith the extraocular motor nuclei, including the abducensnucleus (Graybiel and Hartwieg, 1974; Baker et al., 1977;Graybiel, 1977; Maciewicz et al., 1977; Steiger and Buttner-Ennever, 1979; Hikosaka and Igusa, 1980; Lopez-Barneoet al., 1981, 1982; McCrea and Baker, 1985; Langer et al.,1986; Belknap and McCrea, 1988; Delgado-Garcia et al.,1989). Like the vestibular and reticular inputs to abducensneurons, those from the prepositus hypoglossi nucleus alsohave reciprocal ipsilateral excitatory and contralateralinhibitory components, as demonstrated physiologically(Escudero and Delgado-Garcia, 1988) and pharmacologi-cally (Spencer et al., 1989). Both populations of abducensneurons exhibit eye velocity and eye position signalsassociated with horizontal eye movements, discharging aburst of spikes before the on-direction of an eye movementand maintaining a tonic activity that is related to eyeposition in the off-direction (King et al., 1976; Delgado-Garcia et al., 1977, 1986a,b; Furuya and Markham, 1981;Fuchs et al., 1988). However, abducens internuclear neu-rons differ from the motoneurons both morphologically(Baker and Highstein, 1975; Spencer and Sterling, 1977;Highstein et al., 1982; Carpenter and Batton, 1980) andphysiologically (Graybiel and Hartwieg, 1974; Baker andHighstein, 1975; Delgado-Garcia et al., 1977, 1986a,b;Fuchs et al., 1988; Stahl and Simpson, 1995a).

The role of abducens internuclear neurons in relayingsignals related to conjugate horizontal eye movements iswell established (Evinger et al., 1977; Gamlin et al., 1989).Lesions of the MLF rostral to the abducens nucleusproduce the syndrome of internuclear ophthalmoplegia,which is characterized by paresis or paralysis of ocularadduction on attempted lateral gaze and nystagmus in theabducted eye, but preservation of convergence (Zee, 1994).Although MLF lesions interrupting the excitatory abdu-cens internuclear pathway affect all types of conjugatehorizontal versional (i.e., vestibulo-ocular, saccadic, optoki-netic, smooth pursuit) eye movements, residual horizontalvestibulo-ocular movements in the adducting eye havebeen attributed to a direct excitatory second-order vestibu-lar input to medial rectus motoneurons that originatesfrom neurons in the ventral portion of the lateral vestibu-lar nucleus. The axons of these vestibular neurons courseipsilaterally by means of the ascending tract of Deiters(ATD; Baker and Highstein, 1978; Furuya and Markham,1981; Highstein and Reisine, 1981; Reisine et al., 1981;Carleton and Carpenter, 1983; Carpenter and Carleton,1983; Markham et al., 1986), which is located between theMLF and the brachium conjunctivum. ATD neurons re-ceive excitatory inputs from the ipsilateral labyrinth andconvey head velocity signals in the horizontal plane to themedial rectus motoneurons (Baker and Highstein, 1978;Reisine and Highstein, 1979; Reisine et al., 1981; McCreaet al., 1987). The neuronal activity of medial rectusmotoneurons during conjugate horizontal eye movements,however, is similar to that of abducens internuclear neu-rons (de la Cruz et al., 1989).

Although physiologic studies have demonstrated theaxonal trajectories and actions of the abducens inter-nuclear and ATD inputs to medial rectus motoneurons,little anatomical information is available regarding theirsoma-dendritic distribution, as well as their morphologyand mode of termination within the oculomotor nucleus. Inthe present study, the locations of abducens internuclearand ATD neurons labeled by retrograde transport ofhorseradish peroxidase have been used as reference for theplacement of injections of [3H]leucine and biocytin for therespective autoradiographic and immunohistochemicalidentification of the axons and terminal arborizations ofthe two populations of synaptic inputs to medial rectusmotoneurons in the oculomotor nucleus. The findings fromlight and electron microscopic analyses demonstrate bothsimilarities and differences in regard to the morphologyand mode (single vs. multiple synaptic contact zones),pattern (single vs. multiple postsynaptic profiles), andsoma-dendritic distribution of the synaptic endings fromthese two different pathways that relate to their functionalsignificance in conjugate horizontal eye movements.

MATERIALS AND METHODS

Experiments were performed on adult cats (2.1 to 2.7 kg)under sodium pentobarbital (Nembutal, 40 mg/kg) anesthe-sia administered intravenously. All surgical and euthana-sia procedures were performed in compliance with theNational Institutes of Health Guide for the Care and Useof Laboratory Animals. All experimental procedures werereviewed and approved by the Institutional Animal Careand Use Committee of Virginia Commonwealth Univer-sity. For the surgical procedures, animals were positionedin a Kopf stereotaxic frame and a small (3–5 mm) crani-

142 L.T. NGUYEN ET AL.

otomy was performed by using a surgical drill under theguidance of a surgical microscope. Body temperature wasmaintained at 37°C with a circulating water heating padthat was placed under the animal. At the completion ofeach injection, skin and muscle incisions were closed withinterrupted silk sutures, and appropriate analgesics (bu-prenorphine) and antibiotics (Flo-Cillin) were adminis-tered. After an 18- to 24-hour postinjection survival period,animals were euthanized by a lethal intravenous dose ofbarbiturate and perfused transcardially with a fixativesolution. The fixative solution was preceded by a vascularwash with physiologic saline. Heparin sodium (2,000 IU)and sodium nitrite (10 mg) were administered 20 minutesand 30 seconds, respectively, before the perfusion bymeans of a catheter in a femoral vein. In some animals,artificial respiration was provided through an endotra-cheal tube with 95% oxygen/5% carbon dioxide. The overly-ing skull was removed, and the brainstem was blocked inthe stereotaxic coronal plane. Brainstem blocks wereplaced overnight in cold (4°C) 0.1 M phosphate buffer with0.02% calcium chloride, pH 7.4. Brainstems were sectionedwith a Vibratome at 50 µm thickness. Sections werecollected serially in the same buffer and processed forautoradiography, horseradish peroxidase (HRP) histochem-istry, and/or biocytin immunohistochemistry, as describedbelow.

Retrograde HRP labeling

Six cats were used for the identification of brainstemafferent neurons to the oculomotor nucleus by retrogradetransport of HRP. A glass microelectrode with a 20- to25-µm tip diameter and containing 25% HRP (Boehringer-Mannheim, Indianapolis, IN) in 0.05 M Tris-HCL buffer,pH 7.6, was lowered through a craniotomy into the brain-stem by using a micromanipulator. The location of theoculomotor nucleus was determined by recording anti-dromic field potentials elicited by electrical stimulation ofthe IIIrd nerve in the orbit by using bipolar silver elec-trodes positioned near the nerve entry zone into the medialrectus muscle. HRP was injected by iontophoresis with 2µA positive pulses of 250-msec duration delivered at 2 Hzfor 20 to 30 minutes.

After 18 to 24 hours, animals were euthanized andperfused with a fixative solution containing 1.0% parafor-maldehyde and 1.25% glutaraldehyde in 0.1 M phosphatebuffer with 0.02% calcium chloride, pH 7.4, as describedabove. Vibratome sections of the brainstem were processedfor the histochemical localization of HRP by using 3,38,5,58-tetramethylbenzidine dihydrochloride (Sigma, St. Louis,MO) as the chromogen (Mesulam, 1978). After the reactionwith hydrogen peroxide, sections were washed in acetatebuffer and collected on microscope slides pretreated withchrom-alum gelatin. Sections were counterstained withneutral red and dehydrated in ethanols and xylenes.Coverslips were affixed with Permount. Sections wereexamined and photographed by light microscopy by usingbrightfield or darkfield illumination. The locations of retro-gradely labeled neurons were charted by using a microcom-puter-interfaced digitizer (Minnesota Datametrics, Minne-apolis, MN) connected to X- and Y-axis encoders attachedto the stage of the microscope.

Anterograde autoradiographic labeling

Injections of [3H]leucine were made into the abducensnucleus (four cats) or ventral lateral vestibular nucleus (4

cats) by using the plots of neurons labeled by retrogradetransport of HRP identified in the previous experiments asa guide for their placement. The posterior brainstem wasapproached visually after aspiration removal of the poste-rior vermis of the cerebellum to expose the floor of the IVthventricle. Injections were made with calibrated glass micro-pipettes with 10- to 15-µm tip diameters and containingL-[4,5-3H]leucine (135–160 Ci/mmol; Amersham, ArlingtonHeights, IL). Leucine was prepared by evaporation of stocksolution under nitrogen atmosphere and reconstitution insterile 0.9% saline to a concentration of 100 µCi/µl. Injec-tions of 0.2 to 0.3 µl were made by positive pressure (10 to20 psi) applied through the tip of the micropipette. In someanimals, the medial rectus muscles were injected bilater-ally with 10 µl of 25% HRP in 0.9% saline for thedelineation of the motoneuronal boundary of the medialrectus subdivision of the oculomotor nucleus.

After 18 to 24 hours, animals were euthanized andperfused with fixative solution as described above. Vibra-tome sections of the brainstem were processed for thehistochemical localization of HRP with 3,38-diaminobenzi-dine tetrahydrochloride (DAB) as the chromogen (Spenceret al., 1980, 1982; Spencer and Baker, 1986). For lightmicroscopic autoradiography, alternate sections throughthe abducens nucleus and the remainder of the brainstemwere washed thoroughly in 0.1 M phosphate buffer andcollected on pretreated microscope slides. Sections weredefatted, hydrated, and dried overnight at 40°C. Slideswere coated with Kodak NTB-2 nuclear track emulsiondiluted 1:1 with distilled water. After exposures of 6 to 8weeks at 4°C, slides were developed in Kodak D-19 devel-oper for 2 minutes at 17°C and fixed for 6 minutes inKodak Ektaflo fixer. Sections were counterstained in 0.25%thionin and dehydrated in ethanols and xylenes. Cover-slips were affixed with Permount. Sections were examinedand photographed by light microscopy with brightfield anddarkfield illumination.

Anterograde biocytin labeling

Injections of 5% biocytin (Sigma) in 0.05 M Tris-HClbuffer, pH 7.6, were made into the abducens nucleus (sixcats) or ventral lateral vestibular nucleus (three cats) witha Hamilton microsyringe equipped with a 30-gauge needleand attached to a microinjector (Edwards and Shalna,1974). Injections of 0.15 to 0.30 µl of biocytin were madestereotaxically into the abducens nucleus (P 6.0, H -4.3, L1.3) or the ventral lateral vestibular nucleus (P 6.0 to 7.5,H -4.0 to -4.1, L 2.5 to 3.5) at a rate of 0.005 µl/15–30seconds. After an 18- to 24-hour postinjection survivalperiod, the animals were euthanized and perfused transcar-dially with a fixative solution containing 4.0% paraformal-dehyde and 0.5% glutaraldehyde in 0.1 M phosphate bufferwith 0.002% calcium chloride, pH 7.2. Upon completion ofthe perfusion, the overlying skull was removed and thebrain was blocked in the stereotaxic coronal plane toinclude in separate slabs the injection sites (vestibularnucleus or abducens nucleus) in the posterior brainstemand the oculomotor nucleus in the midbrain. The slabswere removed and immersed overnight in 0.1 M sodiumphosphate buffer, pH 7.2, at 4°C.

Pre-embedding immunoperoxidaselocalization of biocytin

Sections through the midbrain were cut at 50 µmthickness with a Vibratome and collected serially in 0.1 M

ABDUCENS INTERNUCLEAR AND ATD SYNAPTIC CONNECTIONS 143

sodium phosphate buffer, pH 7.2. For light microscopy,alternate sections were incubated for 2 hours in avidin-D-HRP (1:500; Vector, Burlingame, CA ) in 0.1 M sodiumphosphate buffer, pH 7.2, containing 0.3% Triton X-100(Wang and Spencer, 1996a). For the histochemical demon-stration of HRP, sections were incubated in 0.05% DABand 0.01% hydrogen peroxide with 0.005% cobalt acetateand 0.005% nickel chloride in 0.1 M sodium phosphatebuffer, pH 7.2, for 5 to 10 minutes. Sections were mountedon microscope slides pretreated with chrom-alum gelatin,counterstained with cresyl violet, and dehydrated in gradedethanols and xylenes. Sections were examined and photo-graphed with a light microscope by using brightfield andNomarski differential interference contrast optics.

A second group of adjacent sections through the oculomo-tor nucleus was processed for the pre-embedding electronmicroscopic localization of biocytin (Wang and Spencer,1996a). Sections were incubated for 2 hours in avidin-D-HRP (1:500; Vector Laboratories, Burlingame, CA) in 0.1M sodium phosphate buffer containing 0.1% Triton X-100.For the histochemical demonstration of HRP, sectionswere incubated in 0.05% DAB and 0.01% hydrogen perox-ide with 0.005% cobalt acetate and 0.005% nickel chloridein 0.1 M sodium phosphate buffer, pH 7.2. After washingthrough several changes of buffer, sections were processedfor electron microscopy. Sections were post-fixed in 1.0%osmium tetroxide in 0.1 M phosphate buffer with 7%dextrose at 4°C for 1 hour and stained en bloc with 2%uranyl acetate in 0.05 M maleate buffer at 4°C for 1 hour.Sections then were dehydrated in graded methanols andpropylene oxide, infiltrated with plastic resin (Fullam,Latham, NY), and flat-embedded between glass micro-scope slides coated with a water-soluble release agent(Electron Microscopy Sciences, Fort Washington, PA). Af-ter curing, the embedded sections were attached to prela-beled BEEM capsules and trimmed to include only regionsin the oculomotor nucleus that were determined by lightmicroscopy to contain areas of anterogradely labeled termi-nals. Semithin (0.2 µm) sections were cut with glass kniveson an ultramicrotome, stained with 0.1% toluidine blue in1.0% sodium borate, and examined with a light microscopefor use as a reference for the electron microscopic analysis.Ultrathin (60–80 nm) sections were cut with a diamondknife on the ultramicrotome, collected on Formvar-coated,single-slot copper grids, and stained with 2.0% uranylacetate in methanol and 0.1% lead citrate in 0.1 N sodiumhydroxide. Sections were examined and photographedwith a Zeiss EM-10CA electron microscope.

Quantitative morphometry

The cross-sectional areas of biocytin-labeled synapticendings and the diameters of the postsynaptic profileswere measured from electron photomicrographs by using aBIOQUANT (R&M Biometrics, Nashville, TN) digital im-age analysis system operating on a microcomputer withstandard morphometric and statistical software. The cross-sectional area was measured for each labeled synapticending that exhibited a synaptic contact zone identified bypre- and/or postsynaptic membrane densifications and theinvariable presence of an intermediate dense line in theintersynaptic zone (Spencer et al., 1982). The diameter ofthe postsynaptic profile also was measured to provide anindication of the overall soma-dendritic distribution ofdifferent types of synaptic inputs. For obliquely and longi-tudinally sectioned dendrites, diameters were measured

perpendicular to the long axis. Diameter measurements ofthe proximal dendrites of medial rectus motoneurons,stained by choline acetyltransferase (ChAT) immunohisto-chemistry in a previous study (McHaffie et al., 1991), weremade directly from a light microscope with images cap-tured from a videocamera with a frame grabber boardinterfaced to the microcomputer and image analysis soft-ware. Descriptive statistics were calculated by using com-mercially available software and expressed as the mean 6standard error (Table 1). Differences within and betweendifferent populations of synaptic endings were testedstatistically with a single factor analysis of variance.

RESULTS

Origin, course, and pattern of terminationof abducens internuclear projections

Injections of HRP into the oculomotor nucleus largelyconfined to one side but not necessarily restricted to asingle motoneuron subdivision (see Fig. 6 in Wang andSpencer, 1996b) produced extensive retrograde labeling ofinternuclear neurons in the contralateral abducens nucleus(Fig. 1A). Abducens internuclear neurons were distributedthroughout the rostral-caudal extent of the nucleus andoverlapped the distribution of unlabeled, presumed moto-neurons within the neuronal boundary of the nucleus.Retrogradely labeled neurons also occasionally were ob-served in the ipsilateral abducens nucleus and the regionof the prepositus hypoglossi nucleus overlying the genu ofthe facial nerve.

Injections of [3H]leucine into the abducens nucleus (Fig.1C) produced dense autoradiographic labeling of axons inthe MLF and their termination within the dorsolateralregion of the oculomotor nucleus as well as in the regionventrolateral to the MLF (Fig. 1F), both sites correspond-ing to the locations of medial rectus motoneurons asdemonstrated by ChAT immunohistochemistry (Fig. 1E).Although injection sites appeared to be confined to theneuronal boundary of the abducens nucleus on one side,terminal labeling was observed bilaterally in the oculomo-tor nucleus, but with overwhelming contralateral predomi-nance.

Injections of biocytin into the abducens nucleus pro-duced a dense focus of staining at the site of the injection(Fig. 2A). Injection sites were characterized by diffuseperoxidase reaction product that filled the somata, den-drites, and axons of abducens neurons. Injections wereconfined to the neuronal boundary of the abducens nucleuswith little, if any, spread to adjacent structures, particu-larly the medial vestibular nucleus laterally and theunderlying reticular formation ventrally. Diffusion of biocy-tin into the needle track through the overlying internal

TABLE 1. Quantitative Measures of Presynaptic and Postsynaptic ProfilesAssociated With Abducens Internuclear and Ascending Tract of Deiters

(ATD) Synaptic Endings in the Medial Rectus Subdivisionof the Cat Oculomotor Nucleus

ParameterAbducens internuclear

(n 5 323)ATD

(n 5 330)

Presynaptic area (µm2) 1.71 6 0.05 1.63 6 0.06No. mitochondria 4.56 6 0.20 4.14 6 0.22No. mitochondria/µm2 3.80 6 0.26 2.34 6 0.09Postsynaptic diameter (µm) 2.97 6 0.14 4.87 6 0.17Weighted soma-dendritic distribution1 2.54 6 0.04 3.08 6 0.04

1Spine, 1; distal, 2; proximal, 3; soma, 4.


Fig. 1. Darkfield photomicrographs of retrogradely labeled neu-rons in the left (contralateral) abducens nucleus (A) and the right(ipsilateral) ventral lateral vestibular nucleus (B) after injections ofhorseradish peroxidase into the right oculomotor nucleus. Retro-gradely labeled neurons in the ipsilateral medial vestibular nucleuspresumably correspond to excitatory posterior canal-related second-order vestibular neurons that project to inferior oblique motoneuronsbilaterally. C,D: Brightfield photomicrographs of injection sites of[3H]leucine into the left abducens nucleus and right ventral lateraland ventrolateral medial vestibular nuclei, respectively. Darkfieldphotomicrographs demonstrate anterograde autoradiographic label-ing of the abducens internuclear (F) and ascending tract of Deiters

(ATD) (G) projections to the medial rectus subdivision of the oculomo-tor nucleus, defined by choline acetyltransferase–immunoreactivelabeling in E. Note that the abducens internuclear projection occupiesthe entire soma-dendritic extent of medial rectus motoneurons withcontralateral predominance (F), whereas the ATD projection is con-fined to somatic regions of the motoneurons and is exclusivelyipsilateral (G). 3, oculomotor nucleus; 6, abducens nucleus; 7G, genu offacial nerve; das, dorsal acoustic stria; mlf, medial longitudinalfasciculus; LVN, lateral vestibular nucleus; MVN, medial vestibularnucleus; MR, medial rectus subdivision; PH, prepositus hyperglossinucleus. Scale bars 5 0.5 mm in B (applies to A,B), 2.0 mm in D(applies to C,D), 1 mm in G (applies to E–G).


Fig. 2. Brightfield photomicrographs of biocytin injection sites inthe right abducens nucleus (A) and right ventral lateral vestibularnuclei (D). B: Anterograde labeling of the abducens internuclearprojection is predominantly contralateral with a widespread distribu-tion throughout the medial rectus (MR) subdivision. C: Terminalarborizations are distributed diffusely in the neuropil surrounding

presumed motoneurons. E: By contrast, anterograde labeling of theascending tract of Deiters projection is exclusively ipsilateral.F: Preterminal and terminal boutons are clustered in the vicinity ofthe somata of presumed motoneurons. Abbreviations as in Figure 1.Scale bars 5 1 mm in D (applies to A,D), 500 µm in E (applies to B,E),100 µm in F (applies to C,F).


genu of the facial nerve, however, produced retrograde andanterograde labeling of axons in the ascending and descend-ing limbs of the facial nerve, respectively. In addition,diffusion also involved the overlying rostral pole of theprepositus hypoglossi nucleus. The labeled axons of abdu-cens motoneurons coursed from the ventral aspect of thenucleus and descended through the tegmentum towardtheir exit from the pontomedullary junction lateral to thepyramid. By contrast, the labeled axons of abducensinternuclear neurons emerged from the medial part of thenucleus and coursed dorsally toward the MLF, where mostof them crossed the midline and entered the dorsal regionof the contralateral MLF. The abducens internuclear axonsoccupied a dorsal location in the MLF throughout theirrostral course in the pons and caudal midbrain and thenshifted to a dorsolateral location as the MLF approachedthe trochlear nucleus. At the level of the oculomotornucleus (Fig. 2B), anterogradely labeled abducens inter-nuclear axons emerged from the MLF and arborizeddiffusely in the dorsolateral and ventrolateral medialrectus subdivisions. The projections to these subdivisionswere predominantly contralateral, but a modest ipsilat-eral projection was apparent in all cases. Both axonal andpreterminal and terminal bouton labeling was observed inthe vicinity of presumed medial rectus motoneurons withinthese subdivisions (Fig. 2C). Boutons were characterizedas varicosities or swellings at the distal ends of smalldiameter axonal arborizations.

Morphology, soma-dendritic distribution,and mode and pattern of synaptic

connections of abducens internuclearsynaptic endings

At the electron microscopic level, biocytin-labeled abdu-cens internuclear synaptic endings were recognized by thedense peroxidase reaction product that permitted theirunequivocal identification among unlabeled synaptic end-ings (Figs. 3, 4). Abducens internuclear synaptic endingswere encountered in the greatest concentration in theneuropil surrounding medial rectus motoneurons in thedorsolateral region of the oculomotor nucleus. Most synap-tic endings were dome shaped in appearance and usuallyoccurred in isolation (Fig. 3), rather than clustering on asingle postsynaptic process. Labeled myelinated axonsoccasionally were seen in the neuropil in proximity to thelabeled synaptic endings. In some instances, labeled synap-tic endings were observed in continuity with the labeledpreterminal myelinated axon (Fig. 4A). The synaptic end-ings (n 5 323) ranged from 0.23 to 6.46 µm2 in cross-sectional area, with a mean of 1.71 6 0.05 µm2 (Fig. 5).

Abducens internuclear synaptic endings contained auniform population of spheroidal synaptic vesicles thatwere distributed throughout the terminal and numerousmitochondria that were clustered toward the center. Thenumber of mitochondria in abducens internuclear synapticendings ranged from 0 to 16, with a mean of 4.56 6 0.20.The density of mitochondria in abducens internuclearsynaptic endings ranged from 0 to 30.43/µm2, with a meanof 3.80 6 0.26/µm2.

Synaptic contact zones associated with abducens inter-nuclear synaptic endings were characterized by an accumu-lation of synaptic vesicles along the presynaptic mem-brane and a modest postsynaptic densification (Figs. 3A–E,4B–C). Most synaptic endings were associated with onlyone postsynaptic process, and only one synaptic contact

zone was observed for each postsynaptic process. Abducensinternuclear synaptic endings established synaptic con-tacts with postsynaptic profiles of varying size. Theseincluded small- (Figs. 3A,D, 4A,C), medium- (Figs. 3E, 4B),and less frequently large- (Figs. 3A,C,E, 4C) caliber den-drites and somata (Figs 3B,D). Occasionally, synapticendings made contact with spine-like appendages thatemerged from dendrites (Fig. 4D). The postsynaptic diam-eter profiles of abducens internuclear synaptic endingsranged from 0.15 to 12.38 µm, with a mean of 2.97 6 0.14µm (Fig. 6). For comparison, the diameters of 572 proximaldendrites in the medial rectus subdivision stained withChAT were measured by light microscopy to use as areference for the classification of the soma-dendritic distri-bution. The diameters of ChAT-immunoreactive proximaldendrites ranged from 2.72 to 14.50 µm (Fig. 6), with amean of 6.59 6 0.08 µm. The 95% confidence interval ofthis range allowed for the characterization of the postsyn-aptic profiles to be somata (.10.95 µm), proximal den-drites (3.85 to 10.95 µm), or distal dendrites includingdendritic spines (,3.85 µm). Based on this criterion, 76%of abducens internuclear synaptic endings contacted thedistal dendrites, 22% contacted the proximal dendrites,and 2% contacted the somata of medial rectus motoneu-rons. Spine-like appendages were not included in thiscategorization.

Because the diameters measured from single ultrathinsections were dependent on the plane of section, thepostsynaptic profiles were classified subjectively on thebasis of their ultrastructural features. For example, adistinction was made between proximal and distal den-drites on the basis of the presence of granular endoplasmicreticulum, Golgi apparatus, and polyribosomes in proxi-mal dendrites. Proximal dendrites were distinguishedfrom somata on the basis of their parallel arrays ofmicrotubules. Spines were identified by their sac-likeprotrusion from dendrites or somata. Based on theseultrastructural criteria, the majority of abducens inter-nuclear synaptic endings (50%) established contact withdistal dendrites (Fig. 7). A smaller proportion of synapticendings established synaptic contact with proximal den-drites (33%), and fewer synaptic endings contacted cellsomata (12%) and spine-like appendages (4%). When thesoma-dendritic distributions based on ultrastructural fea-tures were coded to numerical values based on size (i.e.,spine-like appendage 5 1, distal dendrite 5 2, proximaldendrite 5 3, soma 5 4), abducens internuclear synapticendings had a mean weighted soma-dendritic distributionof 2.54 6 0.04.

Origin, course, and pattern of terminationof ATD projections

In the same experiments in which abducens inter-nuclear neurons were labeled, extensive retrograde label-ing of neurons was observed in the ipsilateral and contra-lateral vestibular nuclei with a distribution similar to thatdescribed in previous studies (Gacek, 1977; Belknap andMcCrea, 1988; Carleton and Carpenter, 1983; Carpenter,1988; Graybiel and Hartwieg, 1974; Langer et al., 1986;Maciewicz et al., 1977; McCrea et al.,1980, 1987; Robinsonet al., 1994). Specifically, retrogradely labeled neuronswere observed predominantly in the superior and medialvestibular nuclei bilaterally. At the level of the caudal halfof the internal genu of the facial nerve, medium-sizedneurons were located in the ventrolateral extension of the


Fig. 3. A–E: Electron photomicrographs of biocytin-labeled abdu-cens internuclear synaptic endings (s) associated with small- (A,D),medium- (E) and large- (A,C,E) caliber dendrites (d) and somata (somain B,D) in the contralateral medial rectus subdivision of the oculomo-tor nucleus. Individual synaptic contact zones (arrows in A–E),

characterized by an accumulation of spheroidal synaptic vesicles alongthe presynaptic membrane and a modest postsynaptic densification,are associated with only one postsynaptic profile. Scale bars 5 1.0 µmin A,E (scale bar in E applies to B–E).

Fig. 4. A–D: Electron photomicrographs of biocytin-labeled abdu-cens internuclear synaptic endings (s) contacting small-caliber den-drites (d) and a spine-like dendritic appendage (sp in D) in thecontralateral medial rectus subdivision of the oculomotor nucleus.

Individual synaptic endings are associated with only one postsynapticprofile. A: Note the continuity of a synaptic ending with a preterminalmyelinated axon (a). Synaptic contact zones are indicated by arrows.Scale bars 5 1.0 µm in C (applies to A–C), 0.5 µm in D.

medial vestibular nucleus and presumably correspondedto vertical semicircular canal- and/or utricle-related excita-tory second-order vestibular neurons that project to supe-rior rectus motoneurons on the midline of the oculomotornucleus. In addition, only at this level large multipolarlabeled neurons were located along the medial border ofthe ventral lateral vestibular nucleus (Fig. 1B).

By using the location of HRP retrogradely labeled ATDneurons as a reference, injections of [3H]leucine into thismedial region of the ventral lateral vestibular nucleus(Fig. 1D) produced anterograde autoradiographic labelingof axons lateral to the MLF throughout its course in therostral pons and caudal midbrain. Within the oculomotornucleus, terminal labeling occupied discrete regions of theipsilateral dorsolateral and ventrolateral medial rectusmotoneuron subdivisions (Fig. 1G), in contrast to the morewidespread distribution of the abducens internuclear pro-jection (Fig. 1F). Labeling on the contralateral side wasassociated with the inferior rectus, inferior oblique, andsuperior rectus subdivisions corresponding to the excita-tory second-order vestibular projection to vertical motoneu-rons originating from the medial vestibular nucleus, whichalso was involved in the injection site (Fig. 1D). Nolabeling was observed in either of the medial rectussubdivisions of the contralateral oculomotor nucleus.

Injections of biocytin into the right ventral lateralvestibular nucleus (Fig. 2D) produced a dense focus ofstaining at the site of injection. Injections invariably alsoinvolved the adjacent medial vestibular nucleus, but didnot invade the neuronal boundary of the abducens nucleus.Labeled axons emanating from the injection site followedseveral trajectories. At the level of the injection site, axonswere observed coursing through the ipsilateral abducensnucleus and crossing the midline to enter the contralateralMLF or continuing toward the contralateral abducens andvestibular nuclei. These axons presumably representedexcitatory second-order vestibulo-ocular projections to theoculomotor, trochlear, and abducens nuclei, as well as

vestibular commissural projections. Terminal labeling wasobserved in the ipsilateral and contralateral abducensnuclei and in the contralateral homotopic vestibular nu-clei. Another group of axons coursed ventromedially fromthe injection sites and appeared to represent the ipsilat-eral lateral vestibulospinal projection. A third group ofaxons coursed dorsolaterally and entered the inferiorcerebellar peduncle. A fourth group of axons coursedrostrally and medially from the injection site and ascendedipsilaterally through the pons and caudal midbrain lateralto the MLF, corresponding to the ATD. Within the oculomo-tor nucleus, biocytin-labeled axonal arborizations andpreterminal and terminal boutons were observed in thecontralateral inferior oblique and inferior rectus subdivi-sions and the superior rectus subdivision bilaterally (Fig.2E). This pattern of labeling corresponded to the verticalcanal-related excitatory second-order vestibular input tovertical motoneurons that arises from neurons located inthe medial vestibular nucleus. On the ipsilateral side,dense anterograde labeling was observed in both thedorsolateral and ventrolateral medial rectus subdivisions,representing the ATD termination. Preterminal and termi-nal boutons were located in close proximity to presumedmotoneuron somata within the medial rectus subdivisions(Fig. 2F).

Morphology, soma-dendritic distribution,and mode and pattern of synaptic

connections of ATD synaptic endings

Like the abducens internuclear synaptic endings, biocy-tin-labeled ATD synaptic endings localized by electronmicroscopy were recognized by the diffuse electron denseperoxidase reaction product that filled the terminals (Figs.

Fig. 6. Histogram of the postsynaptic diameter distributions ofbiocytin-labeled abducens internuclear (AbdIN) and ascending tract ofDeiters (ATD) synaptic endings in comparison to the diameter distribu-tion of proximal dendrites of ChAT-immunoreactive medial rectusmotoneurons. The postsynaptic diameter profiles of abducens inter-nuclear synaptic endings (n 5 323) range from 0.15 to 12.38 µm, with amean of 2.97 6 0.14 µm. The postsynaptic diameter profiles of ATDsynaptic endings (n 5 330) range from 0.10 to 10.86 µm, with a meanof 4.87 6 0.17 µm. By contrast, the diameters of ChAT-immunoreac-tive proximal dendrites (n 5 572) range from 2.72 to 14.5 µm, with amean of 6.59 6 0.08 µm.

Fig. 5. Histogram of the presynaptic area distributions of biocytin-labeled abducens internuclear (AbdIN) and ascending tract of Deiters(ATD) synaptic endings. Abducens internuclear synaptic endings (n 5323) range from 0.23 to 6.46 µm2 in cross-sectional area, with a meanof 1.71 6 0.05 µm2. By contrast, ATD synaptic endings (n 5 330) rangefrom 0.10 to 6.99 µm2 in cross-sectional area, with a mean of 1.63 60.06 µm2.


8–10). ATD synaptic endings were dome shaped in appear-ance and ranged from 0.26 to 6.99 µm2 in cross-sectionalarea, with a mean of 1.63 6 0.06 µm2 (Fig. 5).

ATD synaptic endings contained spheroidal synapticvesicles that were loosely distributed throughout the termi-nal except at sites of synaptic contact, where dense accumu-lations were observed along the presynaptic membrane.Synaptic endings also contained numerous small- to me-dium-sized mitochondria that were clustered toward thecenter. The number of mitochondria in synaptic endings(n 5 330) ranged from 0 to 20, with a mean of 4.14 6 0.22.The density of mitochondria in synaptic endings rangedfrom 0 to 8.04/µm2, with a mean of 2.34 6 0.09/µm2.

ATD synaptic endings most frequently were observed insynaptic relationship to the somata of presumed motoneu-rons (Fig. 8). Synaptic contacts also were observed withlarge- (Fig. 9A–C) and medium- (Fig. 10B–E) caliberdendrites and infrequently with small-caliber dendrites(Figs. 9D, 10A) and spine-like somatic (Fig. 8E,F) anddendritic (Fig. 9C) appendages. There was a tendency forlabeled axosomatic synaptic endings to occur in isolation,whereas those on proximal dendrites were arranged inclusters. Labeled myelinated axons occasionally were ob-served in close proximity to the labeled synaptic endings,and in some instances arose at sites of nodes of Ranvier(Fig. 9D). Synaptic contact zones were characterized by anaccumulation of synaptic vesicles along the presynapticmembrane and a postsynaptic densification that was moreprominent when associated with axodendritic synapticcontacts than axosomatic synaptic contacts. Axodendriticsynaptic endings in particular also commonly were associ-ated with subjunctional dense bodies that were locatedbeneath the postsynaptic densification (Fig. 10E,F). Onthe other hand, labeled axosomatic synaptic endings fre-quently were associated with subsurface cisternae thatextended internally from close proximity to the postsynap-tic membrane at the synaptic contact zone (Fig. 8B–D). Inall cases, each ATD synaptic ending established synaptic

contact with only one postsynaptic profile, and only onesynaptic contact zone was associated with each postsynap-tic profile.

The diameters of the profiles that were postsynaptic toATD synaptic endings ranged from 0.10 to 10.86 µm with amean of 4.87 6 0.17 µm (Fig. 6). When compared with thesize distribution of ChAT-immunoreactive proximal den-drites of medial rectus motoneurons, ATD synaptic end-ings exhibited a bimodal distribution with one weightingtoward smaller diameter profiles and a second peak to-ward larger diameter profiles (Fig. 6). By using the quanti-tative data derived from measurements of ChAT-immuno-reactive proximal dendrites of medial rectus motoneurons(see above), 49% of ATD synaptic endings establishedsynaptic connections with the distal dendrites and 51%contacted the proximal dendrites of medial rectus moto-neurons.

As was the case for abducens internuclear synapticendings, the quantitative measurements of postsynapticdiameter from single ultrathin sections were dependent onthe plane of the section. Consequently, the measureddiameters may not be representative of the actual soma-dendritic distribution of the ATD synaptic endings. Byusing the same subjective criteria that were applied for thecharacterization of postsynaptic profiles associated withabducens internuclear synaptic endings, ATD synapticendings established synaptic connections predominantlywith the proximal dendrites (49%) and somata (30%) ofmedial rectus motoneurons (Fig. 7). A smaller populationterminated in relation to distal dendrites (20%) and spine-like appendages (1%). When the soma-dendritic distribu-tions based on ultrastructural features were coded tonumerical values (see above), ATD synaptic endings had amean weighted soma-dendritic distribution of 3.08 6 0.04.

Comparisons between abducens internuclearand ATD synaptic endings

When the quantitative data for abducens internuclearand ATD synaptic endings were compared, the differencesbetween the two populations in regard to presynaptic areaand number of mitochondria were not statistically signifi-cant. However, the density of mitochondria in abducensinternuclear synaptic endings was significantly greaterthan that in ATD synaptic endings (P , 0.01). The mostsignificant differences between the two populations ofsynaptic endings were in the diameter of the postsynapticprofile and the weighted soma-dendritic distribution basedon ultrastructural criteria. Abducens internuclear synap-tic endings established synaptic contact with significantlysmaller diameter profiles that correlated with a moredistal dendritic location than the ATD synaptic endings(P , 0.001).

DISCUSSION

In the present study, the locations of neurons in theabducens and ventral lateral vestibular nuclei labeled byretrograde transport of HRP from the oculomotor nucleushave provided a reference for the placement of injectionsfor the anterograde autoradiographic and biocytin labelingof the abducens internuclear and ATD projections. Thefindings in this study have demonstrated not only theaxonal projections from the abducens nucleus and ventrallateral vestibular nucleus, respectively, but also the mor-phology and synaptic organization of their respective

Fig. 7. Histogram of the soma-dendritic distributions of abducensinternuclear (AbdIN) and ascending tract of Deiters (ATD) synapticendings. Postsynaptic profiles are classified as distal and proximaldendrites, soma, and spine on the basis of their ultrastructuralfeatures. The majority of abducens internuclear synaptic endingscontact distal dendrites, whereas the majority of ATD synaptic end-ings contact proximal dendrites and somata.


Fig. 8. A–D: Electron photomicrographs of biocytin-labeled ascend-ing tract of Deiters (ATD) synaptic endings (s) establishing synapticcontacts (arrows) with somata (soma) and somatic spine-like append-ages (sp in E,F) in the ipsilateral medial rectus subdivision of the

oculomotor nucleus. Note subsurface cisternae (sc in B–D) that areassociated with the postsynaptic membrane at sites of axosomaticsynaptic contacts. Scale bars 5 1.0 µm in A,D, 0.5 µm in B,C,E,F.

Fig. 9. A–D: Electron photomicrographs of biocytin-labeled ascend-ing tract of Deiters (ATD) synaptic endings establishing synapticcontacts (arrows) with large- (A–C) and small- (D) diameter dendrites(d) in the ipsilateral medial rectus subdivision of the oculomotor

nucleus. D: Synaptic ending arises from a node of Ranvier betweenthinly myelinated segments of the axon (a). sp, spine-like dendriticappendage. Scale bars 5 1.0 µm in A, 0.5 µm in B–D.

Fig. 10. Electron photomicrographs of biocytin-labeled ascendingtract of Deiters synaptic endings establishing synaptic contacts (ar-rows) with small- (A–C) and medium- (D–F) caliber dendrites (d) inthe ipsilateral medial rectus subdivision of the oculomotor nucleus.

E,F: Note the subjunctional dense bodies (sdb) associated with theprominent postsynaptic densifications (psd) at synaptic contact zones.Scale bars 5 0.5 µm in A–E, 0.25 µm in F.

terminations on medial rectus motoneurons in the oculomo-tor nucleus. Although no attempt was made to identify themotoneurons directly by retrograde labeling, the differentmotoneuron subdivisions of the cat oculomotor nucleushave been defined adequately in previous studies (Akagi,1978; Spencer et al., 1980, 1992; Miyazaki, 1985) to permittheir identification with reference to ChAT immunohisto-chemical staining (Wang and Spencer, 1996b). Further-more, motoneurons differ both morphologically and physi-ologically from internuclear neurons within the oculomotornucleus (Maciewicz and Spencer, 1977; Maciewicz et al.,1977; May et al., 1987). Oculomotor internuclear neuronsexhibit little overlap with medial rectus motoneurons, aresmaller than and ultrastructurally distinct from the moto-neurons, and do not receive either abducens internuclearor vestibular inputs.

Course and pattern of termination ofabducens internuclear and ATD axons

The findings in the present study regarding the originand course of abducens internuclear projections to theoculomotor nucleus are consistent with those from previ-ous anatomical (Graybiel and Hartwieg, 1974; Gacek,1977; Spencer and Sterling, 1977; Bienfang, 1978; Carpen-ter and Batton, 1980; Buttner-Ennever and Akert, 1981;Furuya and Markham, 1981; Highstein et al., 1982; Car-penter and Carleton, 1983; Langer et al., 1986; McCrea etal., 1986; Evinger et al., 1987; Cabrera et al., 1988) andphysiologic (Highstein and Baker, 1978; Nakao and Sasaki,1980; Furuya and Markham, 1981; Fuchs et al., 1988). Atthe level of the oculomotor nucleus, the abducens inter-nuclear axons emerge from the MLF and arborize diffuselythroughout the neuropil in the ventrolateral and dorsolat-eral regions of the oculomotor nucleus, which correspondto the locations of medial rectus motoneurons (Agaki,1978; Baker and Highstein, 1978; Spencer et al., 1980,1992; Spencer and Porter, 1981; Miyazaki, 1985). How-ever, in contrast to previous studies, the findings from boththe autoradiographic and the biocytin anterograde label-ing experiments have demonstrated a bilateral, albeitoverwhelming contralateral, abducens internuclear projec-tion to medial rectus motoneurons. It is unlikely that theipsilateral projection might be attributed to spread ofeither the [3H]leucine or the biocytin to the adjacentmedial vestibular nucleus, because the bilateral labelingin the oculomotor nucleus was confined only to the medialrectus subdivision. Involvement of the medial vestibularnucleus would have resulted in extensive labeling of theexcitatory second-order vestibular projection to verticalmotoneurons in the oculomotor and trochlear nuclei (re-viewed in Spencer and Baker, 1992; Spencer et al., 1992). Amore likely possibility is spread of the tracers to the rostralpole of the prepositus hypoglossi nucleus overlying thegenu of the facial nerve. The prepositus hypoglossi nucleushas extensive efferent connections with the extraocularmotor nuclei (Graybiel and Hartwieg, 1974; Baker et al.,1977; Graybiel, 1977; Maciewicz et al., 1977; Steiger andButtner-Ennever, 1979; Carpenter and Batton, 1980; Hiko-saka and Igusa, 1980; Lopez-Barneo et al., 1981, 1982;McCrea and Baker, 1985; Langer et al., 1986; Belknap andMcCrea, 1988; Robinson et al., 1994), particularly to themedial rectus subdivision of the oculomotor nucleus bilat-erally (McCrea and Baker, 1985; Belknap and McCrea,1988). Connections arising from the rostral half of theprepositus hypoglossi nucleus that might have been in-

volved by the injection site, however, target vertical moto-neurons in the oculomotor and trochlear nuclei (Lopez-Barneo et al., 1981).

By contrast, consistent with previous studies (Gacek,1971, 1977; Baker and Highstein, 1978; Furuya andMarkham, 1981; Highstein and Reisine, 1981; Reisine etal., 1981; Carleton and Carpenter, 1983; Carpenter andCarleton, 1983; Markham et al., 1986), the ATD arisesfrom a restricted population of neurons in the ventrallateral vestibular nucleus whose axons course lateral tothe MLF and terminate in the ipsilateral oculomotornucleus. Within the oculomotor nucleus, the ATD termi-nates in the dorsolateral and ventrolateral medial rectussubdivisions. Although intracellular HRP studies havedemonstrated bilateral terminations of individual ATDaxons (Furuya and Markham, 1981), only an ipsilateralprojection has been demonstrated in the present study byusing two different tracers. Furthermore, in contrast to thewidespread distribution of the abducens internuclear pro-jection, the present study has demonstrated that the ATDterminates in a more restricted pattern. These differentpatterns of termination reflect the differential soma-dendritic termination of the two inputs in relation tomedial rectus motoneurons.

Morphology of abducens internuclearand ATD synaptic endings

Although previous anatomical and electrophysiologicstudies have mapped the independent courses of theabducens internuclear and the ATD pathways and havedemonstrated that these two pathways are excitatory,little information is available regarding the synaptic orga-nization of these projections onto their target motoneu-rons. The findings in the present study provide significantinsight into both the similarities and differences in themorphology, mode, and pattern of synaptic connections,and soma-dendritic distribution of the two inputs to me-dial rectus motoneurons. Both the abducens internuclearand ATD synaptic endings contain spheroidal synapticvesicles and have synaptic contact zones with postsynapticdensifications that provide an asymmetrical pre-/postsyn-aptic membrane profile. The morphology of ATD synapticendings, as shown in the present study, is similar to thoseidentified electrophysiologically and characterized by elec-tron microscopy in a previous study (Markham et al.,1986). These ultrastructural features generally are re-garded as being associated with excitatory synapses (Uchi-zono, 1965; Larramendi et al., 1967) and are consistentwith electrophysiologic studies that have demonstratedthat both the abducens internuclear and ATD pathwaysevoke excitatory postsynaptic potentials (EPSPs) in me-dial rectus motoneurons (Baker and Highstein; 1978;Highstein and Baker, 1978; Highstein and Reisine, 1981;Reisine et al., 1981). A comparison of the presynaptic areasof abducens internuclear and ATD synaptic endings showsthat both populations also are similar in size. The mode(single versus multiple contact zones) and pattern (singleversus multiple postsynaptic processes) of termination ofboth inputs also is the same. Individual abducens inter-nuclear and ATD terminals exhibit only a single synapticcontact zone with each postsynaptic process, and, in mostcases, establish synaptic contacts with only one postsynap-tic profile. Combined with the findings from previousintracellular HRP reconstructions (Spencer and Baker,1983), this feature appears to be a general rule for the


excitatory vestibular as well as reticular (Wang and Spen-cer, 1996a) inputs to extraocular motoneurons.

One difference between abducens internuclear and ATDsynaptic endings is in relation to mitochondrial content.Although both populations of synaptic endings containsimilar numbers of mitochondria, abducens internuclearsynaptic endings have a significantly greater density ofmitochondria than is found in ATD synaptic endings.These differences in mitochondrial density presumably arerelated to the known differences in the physiologic activityof the abducens internuclear (burst-tonic) and ATD (burst)neurons.

Another difference that is apparent between abducensinternuclear and ATD synaptic endings is the presence ofpostsynaptic cytoplasmic inclusions that are associatedwith the synaptic contact zones. Abducens internuclearsynaptic endings typically are associated with only amodest postsynaptic densification at sites of synapticcontact. By contrast, many synaptic contact zones of ATDsynaptic endings exhibit either subjunctional dense bodiesor subsurface cisterns in association with a prominentpostsynaptic membrane densification. In the present study,subsurface cisterns are associated with synaptic endingson the soma, whereas subjunctional dense bodies gener-ally are observed in relation to axodendritic synapticendings. Both types of inclusions have been observed inassociation with a variety of synaptic endings in differentregions of the central nervous system, including the oculo-motor (Waxman and Pappas, 1979; Spencer et al., 1982;Spencer and Baker, 1983) and trochlear (Bak and Choi,1974) nuclei. In the spinal cord, subsurface (or subsynap-tic) cisterns are associated with one specific type of synap-tic ending, the C-terminal, on the somata and proximaldendrites of motoneurons (Conradi, 1969; Bodian, 1975),specifically alpha-motoneurons (Johnson, 1986; Johnsonand Sears, 1988; Destombes et al., 1992), derived fromspinal interneurons or propriospinal neurons (Pullen andSears, 1983). When present, the subsurface cistern islocated postsynaptically in the vicinity of the synapticcontact zone and appears to correspond to modified agranu-lar endoplasmic reticulum closely applied to the inneraspect of the plasma membrane of cell bodies and proximaldendrites and in close relation to granular endoplasmicreticulum (Rosenbluth, 1962). Although the function ofsubsurface cisterns is unknown, its intimate relationshipto the cell surface and granular endoplasmic reticulummight be a suitable structure for the rapid and widedissemination of substances from their site of synthesis oruptake (Rosenbluth, 1962). In addition, subsurface cis-terns have a high affinity for concanavalin A, suggestingthat they may contain a reservoir for glycoprotein for useat the plasma membrane (Wood et al., 1974). If so, thepresence of subsurface cisterns may be indicative of ahigher turnover rate of the postsynaptic surface mem-brane, possibly related to the high frequency burst physi-ologic activity of the ATD synaptic input to medial rectusmotoneurons associated with rapid head accelerations.Presumably related to this, a role of subsurface cisterns inbuffering Ca12 at the subsynaptic zone has been postu-lated (Li et al., 1995). Furthermore, subsurface cisterns ofmammalian extraocular and spinal motoneurons expressthe gap junction protein connexin32 (Yamamoto et al.,1991). It is tempting to speculate, therefore, that subsur-face cisterns associated with the excitatory ATD input tomedial rectus motoneurons, as well as the second-order

vestibular inputs to other extraocular motoneurons (e.g.,Spencer and Baker, 1983), in the cat may be much morethan a functional remnant of the gap junctions thatmediate electrotonic coupling of the vestibular inputs toextraocular motoneurons in teleosts (Graf et al., 1997).

Subjunctional dense bodies are an array of electrondense plaques arranged in a linear manner beneath andparallel to the postsynaptic densification. Like the subsur-face cisternae, the function of the subjunctional densebodies is unknown. In the oculomotor nucleus, subjunc-tional dense bodies also are associated with motoneuronaxon collateral synaptic endings (Spencer et al., 1982), aswell as with excitatory second-order vertical vestibularsynaptic endings (Spencer and Baker, 1983). Conse-quently, subjunctional dense bodies do not appear to be adistinguishing feature of an afferent synapse that isderived from a specific source, uses a particular neurotrans-mitter, or is associated with a specific type of postsynapticreceptor.

Soma-dendritic distribution of abducensinternuclear and ATD synaptic endings

The present study has demonstrated that both theabducens internuclear and ATD synaptic endings have awidespread soma-dendritic distribution in relation to me-dial rectus motoneurons. Quantitatively, however, al-though the two populations of synaptic endings overlap,they differ significantly in their preferential location ondistal versus proximal regions of the soma-dendritic tree.Abducens internuclear synaptic endings are located pre-dominantly on small- and medium-diameter distal den-drites. By contrast, the majority of the ATD synapticendings are located on proximal dendrites and somata.The proximal location of ATD synaptic endings on medialrectus motoneurons, however, differs from the predomi-nantly distal termination of second-order vestibular in-puts to other extraocular motoneurons (Bak et al., 1976;Dememes and Raymond, 1980; Spencer and Baker, 1983).

The soma-dendritic distributions of abducens inter-nuclear and ATD synaptic endings are likely to have asignificant role in influencing the postsynaptic physiologicresponses of medial rectus motoneurons. Given the differ-ences in the electrotonic location of the two excitatoryinputs onto medial rectus motoneurons demonstrated inthe present study, the shape of the EPSPs produced byactivation of the different populations of premotor neuronsmight be expected to be different (Luscher and Clamann,1992). The time course of the EPSPs also would bedifferent for the two inputs, based on their spatial andtemporal dispersion (Walmsley and Stuklis, 1989). Otherfactors, especially in the cat, such as the size-relatedrheobase and input resistance of the postsynaptic motoneu-rons (Koerber and Mendell, 1991), would be influenced bythe modulation of the EPSP amplitude driven by high-frequency stimulation. All of these synaptic propertiespresumably play a role in the threshold frequency recruit-ment of the motoneurons during eye movements (Delgado-Garcia et al., 1986a,b).

Previous electrophysiologic studies have suggested asimilar soma-dendritic distribution for the abducens inter-nuclear and ATD synaptic terminals (Baker and High-stein, 1978; Highstein and Baker, 1978; Reisine et al.,1981). However, consistent with the morphologic findingsin the present study, the EPSPs evoked from stimulation ofthe ATD reverse earlier with depolarizing currents, have


faster rise times (Highstein and Reisine, 1981), and re-verse with lower currents (Reisine et al., 1981) than thoseevoked from the abducens internuclear pathway, suggest-ing a more proximal location of this input on the soma-dendritic tree.

Functional significance of the abducensinternuclear and ATD inputs to medial

rectus motoneurons

Electrophysiologic studies have shown that the abdu-cens internuclear and ATD pathways differ in the signalsthat are conveyed to medial rectus motoneurons. Abducensinternuclear neurons discharge in relation to eye velocityand eye position during all types of eye movements (Kinget al., 1976; Delgado-Garcia et al., 1977, 1986b; Markhamet al., 1986; Fuchs et al., 1988; Stahl and Simpson, 1995a).By contrast, ATD neurons, like other second-order vestibu-lar neurons (Stahl and Simpson, 1995b), have head veloc-ity signals, but exhibit only a weak eye position signal(Reisine and Highstein, 1979; Highstein and Reisine,1981; Markham et al., 1986). The ATD, however, lacks theintensity in firing and has an insufficient eye velocitysignal that is needed to move the eye past the midline(Reisine and Highstein, 1979; Highstein and Reisine,1981). Consequently, the summation of signals conveyedindependently by the abducens internuclear and ATDpathways must account for the similarities in the positionsensitivities of medial rectus and lateral rectus motoneu-rons (de la Cruz et al., 1989; Delgado-Garcia et al., 1986a),both of which in the cat are significantly less than those ofabducens internuclear neurons (Delgado-Garcia et al.,1986b). The more proximal location of the ATD excitatorysynaptic input onto medial rectus motoneurons may re-duce the threshold for activation by the more distal andspatially distributed input from the abducens internuclearpathway during conjugate horizontal eye movements (Del-gado-Garcia et al., 1986b). Coupled with the faster mechani-cal properties of the medial rectus muscle (Meredith andGoldberg, 1986), another function of the ATD input may beto reduce the synaptic delay of the abducens internuclearinput to ensure the conjugacy of horizontal eye move-ments. Given the differences in the physiologic signalsconveyed by the abducens internuclear (eye velocity andeye position) and ATD (head velocity) pathways, the find-ings in this study suggest that the limited soma-dendriticinteraction of the two inputs to medial rectus motoneuronsmay provide a means for the separate control of visuomo-tor and vestibular functions, respectively. In this context,the ATD pathway could function as a fast, independentlyregulated feed-forward, high-pass filter. By contrast, theabducens internuclear may relay a more dynamicallybalanced combination of velocity and position signals thatmediate conjugate horizontal gaze in light of vergenceangle.

ACKNOWLEDGMENTS

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

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