Cytochrome oxidase and NADPH-diaphorase on the afferent relay branch of the optokinetic reflex in...

Cytochrome Oxidase andNADPH-Diaphorase on the Afferent Relay

Branch of the Optokinetic Reflexin the Opossum

C.D. VARGAS,* A.O. SOUSA, F.L.R. BITTENCOURT, C.M. SANTOS, A. PEREIRA, JR.,

R.F. BERNARDES, C.E. ROCHA-MIRANDA, AND E. VOLCHAN

Laboratory of Neurobiology II, Institute of Biophysics Carlos Chagas Filho, Federal Universityof Rio de Janeiro. CCS BL. G Ilha do Fundao. 21 949- 900 Rio de Janeiro, R.J., Brazil

ABSTRACTIn the present study, histochemical techniques combined with more conventional

anatomical methods were used to refine the identification of the nucleus of the optic tract andthe nuclei of the accessory optic system in the opossum. The distribution of the enzymecytochrome oxidase (CO) was examined in the cells and the neuropil of the opossum’smesodiencephalic region. Strong CO labeling was present in the nucleus of the optic tract(NOT)-dorsal terminal nucleus (DTN). Alternate sections, taken from animals that hadreceived bilateral injections of horseradish peroxidase centered in the region of the inferiorolive, were subjected to assays for CO and horseradish peroxidase. The region occupied byCO-labeled cells in the NOT-DTN superimposed with the one defined by retrogradely labeledcells. Cell counts along the NOT-DTN anteroposterior axis revealed that although the olivaryand CO-positive cells were confined within similar boundaries, the latter are up to twofoldmore numerous than the former. As revealed by cytochrome oxidase histochemistry, theoutlines of the NOT-DTN, the other pretectal nuclei and the nuclei belonging to the accessoryoptic system coincided with those revealed by the histochemistry for nicotinamide dinucleo-tide phosphate diaphorase (NADPH-d). After an intraocular injection of cholera toxin betasubunit and alternate sections processing for NADPH-d and CO, the distribution of labeledretinal terminal fields in the mesodiencephalic region was shown to be coincident with regionsof high levels of histochemical labeling. These results are discussed in the light of previousanatomofunctional assessments of the pretectum and accessory optic system. J. Comp.Neurol. 398:206–224, 1998. r 1998 Wiley-Liss, Inc.

Indexing terms: nucleus of the optic tract; accessory optic system; pretectum; inferior olive;

metabolic activity

The pretectal nucleus of the optic tract (NOT) can bedefined as an ensemble of cells sparsely distributed withinthe optic tract at the level of the dorsal mesodiencephalictransition, extending back to rostrolateral levels of thesuperior colliculus (SC). Caudal to the NOT, the dorsalterminal nucleus (DTN) of the accessory optic system(AOS) has been considered as forming a complex with theNOT (Simpson, 1984; Hoffmann et al., 1988; Nunes Car-dozo and Van der Want, 1990; Zhang and Hoffmann, 1993).The medial terminal nucleus (MTN), the lateral terminalnucleus (LTN), and the interstitial nucleus of the superiorfascicle (INSFp) are other components of the AOS, placedat the periphery of the midbrain tegmentum (Hayhow,1959, 1966; Simpson, 1984). Evidence for the role of thesenuclei in the generation of the optokinetic reflex (OKR)

is manifold (reviews in Simpson, 1984; Simpson et al.,1988).

In some species, the NOT-DTN has been considered ashardly distinguishable from the other pretectal nucleiwhen conventional histologic techniques are used (Simp-son et al., 1988). The revelation of retinal terminal fieldscontributed to the definition of nuclei borders in this region(Giolli and Guthrie, 1969; Scalia, 1972; Kanaseki and

Grant sponsors: CNPq, FINEP, CEPG.*Correspondence to: Claudia Domingues Vargas, Laboratorio de Neuro-

biologia II, Instituto de Biofısica Carlos Chagas Filho, CCS BL. G., 2°andar, Ilha do Fundao, Cidade Universitaria, U.F.R.J., 21949–900, Rio deJaneiro, R.J., Brasil. E-mail: [email protected]

Received 19 November 1997; Revised 2 April 1998; Accepted 15 April1998.

THE JOURNAL OF COMPARATIVE NEUROLOGY 398:206–224 (1998)

r 1998 WILEY-LISS, INC.

Sprague, 1974; Lent et al., 1976; Scalia and Arango, 1979;Avendano and Juretschke, 1980; Klooster et al., 1983;Hutchins and Weber, 1985b; Koontz et al., 1985; Narborsand Mize, 1991; Zhang and Hoffmann, 1993). Tracerinjections in the inferior olive (IO) region and analysis ofthe labeled cells in the pretectal region (cat: Saint-Cyr andCourville, 1981; Walberg et al., 1981; Robertson, 1983;Horn and Hoffmann, 1987; Narbors and Mize, 1991;Schmidt et al., 1995, 1996; monkey: Horn and Hoffmann,1987; Cohen et al., 1990; opossum: Vargas et al., 1996), aswell as the immunocytochemical detection of calbindin-labeled neuropil and cell bodies (cat: Narbors and Mize,1991), made additional contributions to delimit the pretec-tal nuclei in these species. Nicotinamide dinucleotidephosphate diaphorase (NADPH-d) associated with theacetylcholinesterase histochemistry has also been pro-posed as a tool to identify the limits of pretectal nuclei inrabbit (Caballero-Bleda et al., 1992). Although consider-able progress has been made in the identification of thepretectal nuclei, disagreement persists in the literature,particularly when interspecies comparisons are made.

Electrophysiologic studies performed in the NOT ofseveral mammals (rabbit: Collewijn, 1975; rat: Cazin etal., 1980; cat: Hoffmann and Schoppmann, 1975, 1981;monkey: Hoffmann and Distler, 1989; opossum: Volchan etal., 1989; Pereira Jr., et al., 1994; ferret: Klauer et al.,1990; wallaby: Ibbotson et al., 1994), and in the DTN (cat:Grasse and Cynader, 1984) have demonstrated the pres-ence of cells with large receptive fields and high spontane-ous activity. Many of them exhibit a strong directionalselectivity for large checker board stimuli moving horizon-tally. Similarly, directionally selective units have also beenfound in the LTN and MTN, albeit with a directionalpreference tuned to the vertical axis (Simpson et al., 1979;Grasse and Cynader, 1982, 1984; Simpson et al., 1988).

Cytochrome oxidase (CO) is a mitochondrial enzymethat constitutes the final path of the oxidative aerobicmetabolism, supplying cells with adenosine triphosphate(ATP). It is thus considered to be essential for tissues thatdepend critically on oxidative metabolism (Wong-Riley,

1989). In 1968, Seligman et al. demonstrated in fresh andfixed non-nervous tissue that CO histochemical (COH)activity could be revealed in the presence of diaminobenzi-dine (DAB). Later on, this method was used to studynormal patterns of CO labeling in the brainstem (Wong-Riley, 1976), as well as to evaluate the effects of sensorydeafferentation on CO levels in the auditory (Wong-Rileyet al., 1978) and visual systems (Wong-Riley, 1979). Sincethen, many studies have been able to map distinct levels ofCO activity in various regions of the nervous system(Kageyama and Wong-Riley, 1982, 1994, 1985; Carroll andWong-Riley, 1984; Tootell et al., 1985; Kageyama andMeyer, 1988; Wong-Riley and Norton, 1988; Wong-Riley,1989; Chandrasekaran et al., 1993; Hevner et al., 1995).Particular attention has been devoted to the blob-likepattern of CO labeling in the supragranular layers of themonkey primary visual area (V1; Hendrikson et al., 1981;Horton and Hubel, 1981; Carroll and Wong-Riley, 1984;Horton, 1984; Livingstone and Hubel, 1984; Tootell et al.,1985; Silverman et al., 1989; Condo and Casagrande, 1990;Rosa et al., 1991; DeYoe et al., 1995; Farias et al., 1997).Hendrikson et al. (1981) have shown in monkeys that V1blobs superimpose with glutamic acid decarboxylase (GAD)blob-like structures. The same was shown for other en-zymes of the respiratory chain (Horton, 1984), NADPH-d(Sandell, 1986; Franca et al., 1997), and parvalbumin(Blumke et al., 1990; Johnson and Casagrande, 1995).

Electrophysiologic recordings performed on CO-rich re-gions of V1 in monkeys have revealed the presence of cellswith a high spontaneous activity and a poor selectivity tooriented visual patterns (Livingstone and Hubel, 1984).These regions were also shown to prefer visual stimulationof low spatial frequencies when compared with those foundin interblobs (Silverman et al., 1989; Edwards et al., 1995).In 1990, Allman and Zucker have postulated that theCO-rich regions in V1 might be responsible for the process-ing of luminous flow, nonoriented patterns and texture,which they called ‘‘scalar intensity variables.’’ These au-thors have also remarked that intensity coding might beconceptually viewed as a more primitive form of visualprocessing that is commonly described in the brainstemoculomotor system. A blob-like pattern has also beenidentified in primate visual cortical area MT (Tootell et al.,1985). In this area, Born and Tootell (1992) have shownthat the majority of units that are driven preferentially bywide moving checkerboard patterns are located withinregions of high metabolic activity.

The above considerations encouraged us to refine theidentification of the NOT-DTN, the other pretectal nucleiand the nuclei of the AOS by the use of metabolic methods,confronting the results with those using more conven-tional techniques. The objectives of this study can be thussummarized as follows: (1) to check whether the NOT-DTNand other nuclei of the afferent branch of the OKR arereactive for CO histochemistry; (2) to establish a compari-son of the distribution of CO-positive cells in the opossumNOT-DTN with those retrogradely labeled by inferior oliveinjections; (3) to verify whether the regions histochemi-cally reactive for NADPH-d and CO are coincident; and (4)to compare the distribution of cholera toxin b-subunit(CTB)-labeled retinal terminals in the pretectum and AOSwith the CO and NADPH-d histochemical labeling in theseregions.

Abbreviations

AOS accessory optic systemAP anteroposteriorAPN anterior pretectal nucleusCTB cholera toxin beta sub-unitCO cytochrome oxidaseCOH cytochrome oxidase histochemistryDTN dorsal terminal nucleusdLGN dorsal lateral geniculate nucleusHRP horseradish peroxidaseINSFp interstitial nucleus of the superior fasciculus, posterior fi-

bersIO inferior oliveLTN lateral terminal nucleusLP lateral posterior nucleusML mediolateralMGN medial geniculate nucleusMTN medial terminal nucleusNADPH-d nicotinamide adenine dinucleotide phosphate diaphoraseNOT nucleus of the optic tractNPC nuclei of the posterior commissureNRTP nucleus reticularis tegmenti pontisOKR optokinetic reflexOPN olivary pretectal nucleusPC posterior commissurePH prepositus hypoglossiPPN posterior pretectal nucleusSC superior colliculus

CO ON THE AFFERENT RELAY BRANCH OF THE OKN 207

MATERIALS AND METHODS

This research followed the guidelines for the use ofanimal in experimental research established by the Brazil-ian Society of Neuroscience and Behavior and the Instituteof Biophysics Carlos Chagas Filho of the Federal Univer-sity of Rio de Janeiro, Brazil. Ten adult opossums (Didel-phis aurita), each weighing from 1.2 to 1.5 kg, were used.

CO and NADPH-D histochemistry

Care was taken to perfuse the animals at the same timeof the day to avoid metabolic variations. The animals weredeeply anesthetized with sodium pentobarbital (90 mg/mlper kg, Knoll) and perfused through the aorta with 500 mlof a 5% sucrose solution in phosphate buffer (PB), pH 7.4,with heparin (0.5 cc/l) and lidocaine (1.0 ml/l) added.Alternatively, 500 ml of phosphate buffered saline (PBS),pH 7.4, was used. This procedure was followed by 400 ml of4% paraformaldehyde in 0.1 M PB, pH 7.4, for 30 minutes.Finally, 10 and 30% sucrose-PB at 0.1 M, pH 7.4, was usedfor cryoprotection. The brains were subsequently removedand stored in 30% sucrose-PB at 4°C. On the next day,coronal sections cut at 25, 30, or 40 µm were obtained in acryostat and collected in PB. After two washes in PB, thesections were incubated at 37°C during 12 hours in thedark in a solution containing 0.5 mg/ml of DAB (Sigma, St.Louis, MO), 0.3 mg/ml of cytochrome C (Sigma), and 0.2mg/ml of catalase (Sigma), dissolved in PB. After severalwashes in PB, the sections were mounted in gelatin-coatedglass slides, dehydrated, and cover-slipped with Entellan.

In two of these animals, alternate sections were incu-bated free floating for about 1 hour at 37°C in 0.1%b-NADPH (Sigma), 0.03% nitroblue tetrazolium (NBT,Sigma) and 0.3% Triton X-100 diluted in 0.1 M Tris-HCL,pH 7.4 (direct method, Sandell, 1986). In one animal,sections were incubated in 1% NADP, 0.03% NBT, 0.6%malic acid, 0.02% MnCl, 1% DMSO, and 1% Triton X-100diluted in 0.1 M Tris HCL, pH 8.2 (indirect method,Scherer-Singler et al., 1983). In two other animals, includ-ing the one used to illustrate the NADPH-d labelingpattern in the pretectum and AOS, we used the indirectmethod, but NADP was replaced by NADPH. Except for aslight difference in intensity of neuropil and cell labeling inthe regions of interest, the latter protocol gave about thesame results as the direct method. The reacted sectionswere washed in 0.1 M Tris-HCL buffer, pH 7.4, andmounted on gelatin-coated slides.

Intraocular injection

In one animal, CTB was injected in one eye. The animalwas kept anesthetized with sodium pentobarbital (30mg/kg/h, Knoll) injected intraperitoneally, after an initialdose twice as large. The eye was rendered mydriatic bytopical application of atropine sulfate (1%, Oculum) andphenylephrine chlorate (10%, Oculum). A total volume ofabout 7 µl of a solution of 0.5% CTB (Sigma) was pressureinjected through three retrolimbic sites after puncturingthe eyeball. Penicillin G (Benzetacil, Wyeth, Sao Paulo,Brazil) 150,000 units/kg was given intramuscularly on thefirst two days. After a survival of 9 days, the animal wasdeeply anaesthetized and perfused as described above. Theinjected retina was flattened and processed for evaluationof the injection.

Coronal sections of the mesodiencephalic region cut at40 µm were obtained on a cryostat and collected in PB. Two

of four consecutive sections were reacted immunocyto-chemically as follows: sections were washed in PBS andincubated in a blocking medium (5% normal goat serumdiluted in PBS) for 30 minutes. The sections were thentransferred to rabbit anti-CTB serum (Sigma) diluted1/10.000 in PBS-Triton X-100 0.05%, for 12 to 18 hours.After several washes in PBS, the sections were incubatedin biotinylated goat anti-rabbit immunoglobulin, followedby three washes in PBS and incubation with avidin-biotinhorseradish peroxidase (HRP) complex (kit ABC Elite,Vector Labs, Burlingame, CA). Finally, the sections werewashed in PBS and incubated in a solution preparedfollowing the Kit Vector (PK 4700) for 8 minutes to revealHRP. After several washes in PBS, they were dehydratedand mounted in gelatin-coated slides. The other two seriesof sections were reacted alternatively for CO and NADPH-d.In half of these sections, double labeling was achieved byprocessing for CTB.

IO injection

One animal was used to compare the distribution ofCO-labeled cells in the NOT-DTN with that of cells thatproject to the IO. A detailed description of this procedurewas given elsewhere (Nasi et al., 1997; Vargas et al., 1996).Briefly, the animal was kept anesthetized with sodiumpentobarbital (30 mg/kg per hour, Knoll) injected intraper-itoneally after an initial dose twice as large. Throughoutthe experiment, the heart rate and temperature weremonitored. Six injections of 30% HRP (Sigma, type 6)diluted in 2% DMSO were placed stereotaxically (Oswaldo-Cruz and Rocha-Miranda, 1968) in the region of the IO.Penicillin G (Benzetacil, Wyeth) 150,000 units/kg wasgiven intramuscularly. After a survival of 48 hours, theanimal was deeply anesthetized with sodium pentobarbi-tal (90 mg/kg, Knoll) and perfused through the aorta asdescribed above. Coronal sections of 40 µm from themesodiencephalic and bulbopontine regions were obtainedin a cryostat and reacted following the protocol of Mesulam(1978). Alternate sections were histochemically reacted toreveal CO activity.

Data analysis

The sections were observed on Zeiss Microscope (Ax-ioplan). The outlines of chosen sections were drawn aidedby a morphometric computer program. Some landmarksthat could help in the identification of the stereotaxic levelwere represented and the CO and cells retrogradely la-beled from the IO were plotted. Likewise, sections assayedfor CTB, CO, and NADPH-d were observed on a Nikonmicroscope (Optiphot-2) and drawn with the aid of aNeurolucida program (MicroBrightField Inc., Colchester,VT). In both cases, the approximate stereotaxic levels wereestimated from the cytoarchitectonic atlas of the opos-sum’s brain (Oswaldo-Cruz and Rocha-Miranda, 1968).When necessary, the posterior commissure (PC) closure, aswell as the rostral edge of the optic layers of the superiorcolliculus (SC) and the lateral pole of the medial geniculatenucleus (MGN), were used as reference for adjustments inthe anteroposterior (AP) and mediolateral (ML) axis tocompensate for individual variations. NOT-DTN area mea-surements were made in the drawings and correspond tothe region in the optic tract where the olivary and CO-labeled cells were found.

208 C.D. VARGAS ET AL.

RESULTS

The first part of this study will focus on the anatomicaldelimitation of the NOT-DTN through the comparison ofthe distribution of CO-labeled cells with that of theneurons that project to the IO. The second part will beconcerned with the correlation of the CO and NADPH-dhistochemical labeling with the CTB labeled retinal termi-nal fields in the pretectum and AOS.

COH in the NOT-DTN

A distinctive pattern of CO labeling is observed through-out the whole extent of the pretectal complex: Groups oflabeled cells immersed in a strongly labeled neuropil canbe distinguished surrounded by less labeled regions. Agroup of strongly labeled cell bodies and neuropil lyingwithin the brachium of the SC (Fig. 1), identified as theNOT, stand out in marked contrast with the background.At more posterior levels, the DTN stands out very clearlyin this preparation as a dense agglomerate of labeled cellsand neuropil.

IO and CO-labeled cells in the NOT-DTN

In this and in previous studies, multiple tracer injec-tions were used to allow a complete labeling of NOT-DTNcells that project to the IO region (Vargas et al., 1996; Nasiet al., 1997). Figure 2 shows drawings of alternate sectionsreacted to reveal the distribution of these cells in thebrachium (A) and those histochemically reacted for CO(B). Both populations occupy the same region. The area inthe NOT defined by the olivary and CO-labeled cells ateach coronal plane is illustrated in Figure 2C. The goodmatch between the areas defined by these two methods canbe observed.

The number of cells in the two populations along the APaxis is plotted in Figure 3. It can be noted that theCO-labeled cells are more numerous than the olivary cellsover the whole NOT-DTN extent.

Retinal terminal fields labeled by CTB in thepretectum and AOS compared with the areas

labeled by CO and NADPH-d

The retina of the animal with CTB eye injection showedthe presence of labeled cells and fibers at all retinaleccentricities, indicating that the tracer was taken up byganglion cells across the entire retina. A small lesion of theretina by the needle just above the optic nerve headresulted in the presence of a corresponding region devoidof labeled terminals in the dorsal lateral geniculate nucleusdLGN.

The distribution of retinal terminal fields (A), CO (B),and NADPH-d (C) labeling is shown in the dorsal pretec-tum (Fig. 4) and the MTN (Fig. 5). Comparison of thelabeling obtained by the histochemical techniques re-vealed that, although there is a strong CO labeling of theneuropil and cells in the pretectal and AOS nuclei, theNADPH-d labeling is found mainly in the neuropil.

In Figure 6, sequences of adjacent sections processed toreveal the distribution of retinal terminal fields (bottomright) and regions of high CO (middle) and NADPH-d (top)activity in pretectum and AOS have been drawn. It can beobserved that limits drawn for the CO-rich regions are, ingeneral, coincident with those for NADPH-d labeling.

Retinorecipient regions in the pretectum

As described in a previous work using tritiated prolineas an anterograde tracer (Lent et al., 1976), retinal termi-nal fields of one eye are observed contralaterally in theNOT and bilaterally in the olivary pretectal nucleus (OPN)(Figs. 4, 6, 7) and the posterior pretectal nucleus (PPN)(Fig. 6). Besides these projections, the CTB intraocularinjection allowed the identification of some pretectal reti-nal terminal fields that were not previously described inthis species (Lent et al., 1976; Rocha-Miranda and Lent,1978; Linden and Rocha-Miranda, 1981; Vargas et al.,1996; Nasi et al., 1997). For instance, with this method, asparse but consistent ipsilateral projection to the NOT(Fig. 7B,C) localized symmetrically with respect to thecontralateral labeling (Fig. 7A) can be observed. Likewise,as shown in Figure 6, labeled terminals are found bilater-ally at superficial portions of a region previously identifiedby Lent et al. (1976) as the anterior pretectal nucleus(APN), pars compacta, previously considered by theseauthors as devoid of retinal terminal fields. At more rostralplanes and lateral to the pretectum, bilateral retinalterminal fields are found in the thalamus within a regionidentified as the lateral posterior nucleus (LP). Ventral tothe pretectum, sparse, delicate labeled fibers and fewterminal-like structures can be found bilaterally in thenuclei of the posterior commissure (NPC).

Besides drawings of the distribution of retinal terminalfields in the pretectal region, Figure 6 also shows regions ofheavier histochemical labeling outlined on drawings ofalternate sections reacted for NADPH-d and CO. Whenoutlines are drawn by reference to regions highly CO andNADPH-d reactive, some of the borders defined by thepresence of dense retinal terminal fields appear coincidentwith those defined by the histochemical markers. Forinstance, the ‘‘isles’’ of retinal terminal fields in the NOTseem to correlate with regions strongly labeled for CO andNADPH-d (Figs. 4A–C, 6A–D): at these planes, a stripe ofhigher metabolic labeling can be seen in the more ventralaspects the optic tract, coincident with the region of densecontralateral retinal terminal fields.

At rostral levels (Figs. 4A–C, 6A), the borders of theOPN as defined by the overall pattern of distribution ofretinal terminal fields coincide with the NADPH-d label-ing. However, the OPN region defined by CO labeling issmaller. At more posterior levels, the retinal terminalfields seem organized in two foci of labeling on both sides(Fig. 6B,C) that can be correlated to regions of dense COand NADPH-d labeling. OPN at its caudal end is repre-sented by a small region (Fig. 6D), the limits of which arebest defined by the distribution of retinal terminal fields.

In the pretectal region previously defined as PPN (Lentet al., 1976), the contralateral retinal terminal field, thatbegins to be observed ventral to the visual layers of therostral SC and extends caudolaterally in the pretectum,has its counterpart on a topographically similar region ofstrong histochemical labeling (Fig. 6C–E). In the PPN, onthe ipsilateral side, one can identify clumps of retinalterminal fields.

Finally, in the APN, pars compacta (Lent et al., 1976),one can identify the presence of bilateral retinal terminalfields that are scattered on a region of high histochemicallabeling (Fig. 6A–C).


Fig. 1. Photomicrographs of coronal sections reacted for cyto-chrome oxidase histochemistry (COH) showing the labeling in thepretectal nuclei. Insets indicate the stereotaxic coordinates and illus-trate the distribution of cytochrome oxidase (CO)-labeled cells in the

nucleus of the optic tract-dorsal terminal nucleus (NOT-DTN). Otherstrongly labeled pretectal nuclei are identified in the insets byabbreviations. For abbreviations, see list. Scale bars 5 200 µm.

Fig. 1 (Continued)


Fig. 2. A: Drawings of coronal sections illustrating the distributionof cells that project to the inferior olive in the nucleus of the optictract-dorsal terminal nucleus (NOT-DTN). B: Drawings of consecutivesections showing the distribution of cytochrome oxidase (CO)-labeled

cells in the NOT-DTN. C: Average of the area in the optic tract on bothsides defined by the olivary and CO-labeled cells in each stereotaxicplane. For abbreviations, see list.


AOS

In the opossum, the MTN receives a dense contralateralretinal projection (Cavalcante et al., 1975; Lent et al.,1976). By the use of HRP, retinal terminal fields havebeen observed in the ipsilateral MTN (Nasi, 1993). Thisretinal field can also be observed by a CTB intraocularinjection (Figs. 5A, 6C,D,E). Many CO-labeled cells and aprofusely CO-labeled neuropil are apparent in the MTN(Fig. 5B). In alternate sections reacted for NADPH-d,faintly labeled cells and strongly labeled neuropil (Fig.5C) can also be identified. Note that at its more rostralportions (Fig. 6C,D), the MTN is located at the ventraledge of the mesodiencephalon. More caudally (Fig. 6E),though, it shifts dorsally, becoming placed dorsomediallyto the cerebral peduncle.

The INSFp was defined by Giolli et al. (1984) in therabbit and the rat as a diffuse group of cells lying betweenthe DTN, the LTN, and the MTN. In the opossum, thisnucleus can be identified after an intraocular CTB injec-tion by the presence of labeled fibers and terminals ofcontralateral origin at the ventral border of the cerebralpeduncle (Fig. 6C–F). At more posterior stereotaxic levels,clumps of labeled terminals are identified between theposterior LTN and the DTN. Both histochemical tech-niques yield labeled cells and neuropil dispersed in theseregions.

As shown before for this species by intraocular prolineinjections (Cavalcante et al., 1975; Lent et al., 1976), theretinal projection to the contralateral DTN is spot-likeand almost absent for the ipsilateral nucleus. For bothhistochemical markers, labeling is observed to superim-pose with the region defined as DTN (Fig. 6F). The sameauthors have defined the LTN, wedged between theventral aspect of the MGN and the dorsolateral border ofthe cerebral peduncle. In the opossum, this nucleus issparsely innervated by retinal fibers of contralateral andipsilateral origin (Fig. 6B–E). The LTN stands out lessclearly than the MTN and INSFp on the CO and NADPH-dhistochemical material.

Double labeling experiments

Sections assayed simultaneously for CO and CTB orNADPH-d and CTB, although with lower resolution, con-firm that regions of heavier histochemical labeling in thepretectum and AOS are coincident with the regions ofdense contralateral retinal projection.

DISCUSSION

NOT-DTN

COH and the NOT-DTN. A strong labeling of the cellsand the neuropil in the pretectal region corresponding tothe NOT-DTN was obtained by histochemistry for CO. If,as proposed by Wong-Riley (1989), energy metabolism andneuronal activity are tightly coupled, then increased neu-ronal activity would be reflected in high levels of COHlabeling. In some species, electrophysiologic studies haveshown that the NOT-DTN cells that project to the IOexhibit a high spontaneous activity as well as a strongdirectional selectivity for large moving patterns (Hoff-mann et al., 1976) and could shape the hOKR by means ofthe IO (review in Simpson et al., 1988). In the cat, theseneurons, termed retinal slip, are innervated by slowlyconducting W fibers (Hoffmann and Shoppmann, 1975;Ballas and Hoffmann, 1985). In a previous report,Kageyama and Wong-Riley (1985) have verified that,contrasting with the moderate to low CO reactivity foundin the W-like cells of the cat dLGN, intensely labeled COpositive cells and neuropil could be found in the NOT. Theauthors argued that these differences could be because‘‘NOT neurons exhibit moderate to high levels of main-tained and/or evoked discharge rates, and may receiveconvergent input from other sensory pathways.’’

CO and IO labeled cells in the NOT-DTN. In this andprevious studies in the opossum (Vargas et al., 1996; Nasiet al., 1997), it was shown that large injections centered inthe IO yielded labeled cells in the NOT-DTN, the APN, andthe PPN. Also in the same species, IO projecting cells wereshown to be more abundant in the anterior half of theNOT-DTN. The present study has demonstrated that thedistribution of CO-labeled cells and of cells projecting tothe IO have similar profiles in the NOT-DTN, although theformer are more numerous than the latter.

Besides the IO, other mesodiencephalic and brainstemstructures are innervated by fibers coming from the NOT-DTN (cats: Berman, 1977; Schmidt et al., 1995; rats:Terasawa et al., 1979; Korp et al., 1989; Schmidt et al.,1995; tree shrews: Weber and Harting, 1980; rabbits:Holstege and Collewijn, 1982; Maekawa et al., 1984;monkeys: Baleydier et al., 1990; Watanabe et al., 1991;Mustari et al., 1994; Buttner-Ennever et al., 1996; opos-sums: Vargas et al., 1996). About 60% of the NOT-DTNneurons projecting to the nucleus prepositus hypoglossi(PH) also provide a bifurcation to the IO (cat: Magnin etal., 1989; rat: Schmidt et al., 1995). No double labeling wasshown by injecting simultaneously the dLGN and the IO(rat: Robertson, 1983). The same was shown for tripleinjections in dLGN, the IO, and the contralateral NOT-DTN (rat and cat: Schmidt et al., 1995). Furthermore, inan electrophysiologic study, Maekawa et al. (1984) haveshown that only a small percentage of the NOT cells thatproject to the nucleus reticularis tegmenti pontis (NRTP)can also be activated antidromically by stimulating the IO.Based on these studies, one can presume that the popula-

Fig. 3. Distribution of cytochrome oxidase-(CO)-positive cells andthose that project to the inferior olive (IO) in the nucleus of the optictract-dorsal terminal nucleus (NOT-DTN) anteroposterior axis on bothhemispheres. For abbreviations, see list.


Fig. 4. Photomicrographs of adjacent coronal sections reacted to reveal retinal terminal fields labeledwith cholera toxin beta subunit (CTB; A), cytochrome oxidase (CO) activity (B), and nicotinamidedinucleotide phosphate diaphorase (NADPH-d; C) in the pretectal region. Scale bar 5 150 µm (applies toall).

Fig. 5. Photomicrographs of adjacent coronal sections reacted toreveal retinal terminal fields labeled with cholera toxin beta subunit(CTB; A), cytochrome oxidase (CO) activity (B) and nicotinamide

dinucleotide phosphate diaphorase (NADPH-d; C) in the medialterminal nucleus (MTN). Scale bar 5 150 µm (applies to all).

Fig. 6. A–F: Groups of serial drawings of adjacent sections illustrat-ing the distribution of retinal terminal fields (bottom right), cyto-chrome oxidase (CO)-rich (middle), and nicotinamide dinucleotidephosphate diaphorase (NADPH-d) rich (top) regions in the pretectumand AOS. Sections are represented from rostral (A 3.8) to caudal (A 2.0)levels with stereotaxic estimates based on the atlas of the opossum’sbrain (Oswaldo-Cruz and Rocha-Miranda, 1968). At the bottom-right

drawings, the density of dots and of line fragments provide anapproximation of that of labeled retinal terminals and axons. On themiddle and top drawings, outlines were drawn by reference to regionshighly reactive for CO and NADPH-d. As can be observed, some of theregions defined by the presence of dense retinal terminal fields appearto be encompassed by those defined by the histochemical markers. Forabbreviations, see list. Scale bars 5 1 mm (applies to A–F).

Fig. 6 (Continued)

Fig. 7. Retinal terminal fields in the opossum pretectum. A: Contralateral to the injected eye.B: Ipsilateral to the injected eye. C: On a higher magnification, a sparse but consistent fiber and boutonlabeling can be identified at the ipsilateral nucleus of the optic tract (NOT). Scale bars 5 100 µm in B, 50µm in C.


tion of CO-positive cells identified in the opossum NOT-DTN is hodologically nonhomogeneous.

Notwithstanding, measurements of the area in the optictract occupied by the CO-labeled cells matched numeri-cally and coincided with that occupied by the IO projectingcells. Thus, CO cell labeling is in register with the distribu-tion of olivary cells in the opossum NOT-DTN.

In many mammalian species, tracer injections centeredin the IO have been used to help delimit the NOT(Robertson, 1983; Hoffmann et al., 1988; Cohen et al.,1990; Distler and Hoffmann, 1993). This procedure, particu-larly when allied to tracer injections in the retina (Ballasand Hoffmann, 1985; Nasi et al., 1997) or in extraretinicnuclei (Van der Togt and Van der Want, 1992; Vargas et al.,1996), has improved the definition of the NOT-DTN limits.NOT borders cannot be defined solely by this criterion,because, for instance, some of the other pretectal nucleialso project to neighboring regions of the IO (Walberg etal., 1981; Saint Cyr and Courville, 1981; Vargas et al.,1996).

Based on anatomical studies, Schmidt et al. (1996) haveargued that a region in the superficial pretectum, wheredLGN and IO projecting cells were found, should character-ize the NOT. The DTN, part of the PPN and of the OPNbecome incorporated within the NOT by this definition.However, the region of termination of these pretectalnuclei on the IO and dLGN is not the same (IO: Walberg etal., 1981; dLGN: Kubota et al., 1987). Sudkamp andSchmidt (1995) and Schmidt (1996) have also criticized the‘‘classic’’ borders within the dorsal pretectum based on thatcertain functional cell types are found within and beyondthe boundaries of the optic tract. This finding is the case ofjerk cells (cat: Hoffmann and Schoppmann, 1981; Ballasand Hoffmann, 1985; Sudkamp and Schmidt, 1995;wallaby: Ibbotson et al., 1994; Ibbotson and Mark, 1994)and saccade cells (Schmidt, 1996). In our view, that both inthe cat (Ballas and Hoffmann, 1985) and in the opossum(Volchan et al., 1989) directional selective cells are con-fined to the region of the optic tract argues in favor of theanatomical identity of this region.

As will be discussed below, in the opossum, the bordersbetween the NOT-DTN and other pretectal nuclei are quiteevident by the COH application and coincide with previousanatomofunctional criteria used to identify this complex(Lent et al., 1976; Volchan et al., 1989, 1996; Vargas et al.,1996; Nasi et al., 1997).

Pretectum

Definition of pretectal boundaries. Parcelation ofthe pretectum (rabbit, sheep, pig, and cat: Rose, 1942;mouse, rabbit, rat, tree shrew: Scalia, 1972; opossum: Lentet al., 1976; Linden and Rocha-Miranda, 1981; cat: Ka-naseki and Sprague, 1974; Avendano and Juretschke,1980; Koontz et al., 1985; Hutchins and Weber, 1985a;Narbors and Mize, 1991; monkey: Hutchins and Weber,1985b) has provided different interpretations (for review,see Scalia, 1972; Simpson et al., 1988).

Searching for alternative methods that could contributeto outline the pretectal borders, Narbors and Mize (1991)have used immunocytochemical methods to reveal thepresence of calbindin-D28K, a calcium binding protein, inthe pretectum. Koontz et al. (1985) had shown that theretinofugal projection to the cat pretectum, when viewedon horizontal sections, is distributed in four parallel stripsof terminal labeling running rostrocaudally that seem to

cross cytoarchitectonically defined pretectal boundaries.These strips, identified on coronal sections as foci ofterminal labeling, were shown to be coincident with clus-ters of calbindin D-28K (Narbors and Mize, 1991). More-over, calbindin-labeled cells have been found to double-label with part of the cells that project to the dLGN, butthey do not double-label with those that project to the IO(Narbors and Mize, 1991). Based on these results, theseauthors proposed that the conventional pretectal limitsshould be reexamined. Further evidence, however, isneeded to backup these claims.

Retinal terminal fields, CO, and NADPH-D. In themonkey primary visual cortex (Sandell, 1986; Franca etal., 1997), it has been shown that regions of high CO andNADPH labeling are superimposed. In the present study, asimilar coincidence was observed for the opossum pretec-tum. A positive correlation between certain types of retinalinputs and high CO levels has been described in retinore-cipient structures of some vertebrate species (LGNd, cat:Wong-Riley, 1979; Kageyama and Wong-Riley, 1984, 1985;rat: Land, 1987; tree shrew: Wong-Riley and Norton, 1988;optic tectum, goldfish: Kageyama and Meyer, 1988). Thecomparison of the retinal terminal field distribution withthe pattern of CO and NADPH-d labeling has revealedthat regions of high metabolic activity are in register withregions densely innervated by retinal afferents in theopossum pretectum.

It was shown that the CO labeling is distributed withinthe retinal terminal field in the optic tract. Moreover, bythe use of CTB intraocular injections, sparse ipsilateralterminal fields that went undetected before in Didelphisaurita (Lent et al., 1976; Nasi et al., 1997; Vargas et al.,1996), although described for Didelphis virginiana andMarmosa mitis (Royce et al., 1976), could be identifiedscattered throughout the NOT.

As shown before by Lent et al. (1976), the bilateralretinal terminal fields placed ventrally to the NOT inrostral sections can be assigned to the OPN. By thispreparation, the rostral OPN has an olive-like shape (Figs.4, 6A) and can be clearly identified with the histochemicaltechniques. Caudal to this region (Fig. 6B), one canidentify two foci of retinal terminal fields belonging to theOPN. When viewed dorsally (rat: Scalia and Arango, 1979;cat: Hutchins and Weber, 1985a), those fields assume atwo-tailed figure. At more posterior levels, as pointed outby Lent et al. (1976) and Scalia and Arango (1979), onlyone of the OPN ‘‘tails’’ subsists, barely discernible from theretinal terminal fields found in the ventral PPN, even withthe histochemical techniques.

The contralateral retinal terminal fields that beginunder the rostral pole of the SC (Fig. 6C), extending morelaterally as one runs toward the caudal pretectum (Fig.6D) and the bilateral retinal fields found medioventrally tothe NOT, once assigned by Linden and Rocha-Miranda(1981) as composing the subbrachial NOT, could, by com-parison with the CO and NADPH labeling at this region,be attributed to the retinorecipient region of the PPN (forfurther discussion, see Vargas et al., 1996). In fact, CO andNADPH-d yielded a strong pattern of labeling in theretinorecipient PPN, allowing its clear identification, espe-cially at rostral levels.

Finally, retinal terminal fields were observed bilaterallyin a superficial pretectal region identified as APN, parscompacta, and sparsely in the nuclei of the posteriorcommissure. In the first region, strongly labeled cells and


neuropil could be identified by application of CO andNADPH-d histochemical markers. The presence of bilat-eral retinal terminal fields in the APN has also beenreported in the tree shrew (Scalia, 1972) and the ferret(Zhang and Hoffmann, 1993), being absent in the rat,rabbit, and mouse (Scalia, 1972), and in a previous reportin the opossum (Lent et al., 1976). The sparse distributionof labeled fibers and terminals in the nuclei of the posteriorcommissure showed no correlation with the CO andNADPH-d labeling. To our knowledge, this is the firstreport of the existence of retinofugal elements in thisregion.

Accessory optic system

COH and NADPH-d histochemistry in association withthe analysis of retinal terminal fields have also here beenused to distinguish the MTN, LTN, and the INSFp. TheMTN was shown to be innervated by dense contralateraland sparse ipsilateral terminals and the INSFp, to receivea dense contralateral labeling. Both retinal componentswere sparse in the LTN. Although high levels of CO andNADPH-d labeling were present in the MTN and INSFp,the LTN displayed a weaker labeling with these tech-niques. Thus, in these regions, there was a clear parallelbetween the density of retinal terminal fields and theintensity of histochemical labeling.

In the opossum, the MTN was identified with Nisslstaining as nucleus tractus peduncularis transversi bycytoarchitectonic criteria (Oswaldo-Cruz and Rocha-Miranda, 1968). These authors identified two cell types inthis nucleus, but no subdivisions were reported. In thepresent study, the use of CTB intraocular injections associ-ated with the CO and NADPH-d histochemical markersallowed the recognition of both the dorsal (superior) andventral (inferior) subdivisions of the MTN as described forother mammals (Hayhow, 1959, 1966; Giolli and Guthrie,1969; Giolli et al., 1984; Coleman and Beazley, 1988; Vander Togt et al., 1993). Labeling intensity was similar inboth subdivisions of the opossum’s MTN.

Early electrophysiologic studies have reported a func-tional segregation between the dorsal and ventral MTN(Hamasaki and Marg, 1962). Recently, Van der Togt et al.(1993) have shown, in the rat, that upward directionselective (DS) units are found preferentially in the dorsaland ventromedial MTN regions, whereas downward DSunits are found ventrally and laterally in the MTN. Theseauthors correlated these distinct properties with the re-gions of termination of the inferior fascicle and the poste-rior bundle of superior fascicle of the accessory optic tract,respectively. Moreover, neurons retrogradely labeled fromthe IO region were found preferentially in the dorsal MTNand the medial portion of the ventral MTN, in correspon-dence with the regions whence upward DS neurons wererecorded (Van der Togt et al., 1993). The segregation ofefferents to the IO went previously unnoticed in studiesperformed in the same species (Clarke et al., 1989; Van derTogt and Van der Want, 1992). Inhibitory connectionsbetween the dorsal and ventral MTN portions were alsoshown (Van der Togt et al., 1993). If the existence of aventral MTN portion may be controversial in primates (seeWeber, 1985; Cooper and Magnin, 1986), the dorsal MTN ispresent in almost all mammalian species studied so far(Cooper et al., 1990).

The INSFp has been shown to share many commonanatomofunctional properties with the MTN, such as

metabolic activation by large random dot patterns movingin the vertical direction (Benassi et al., 1989), as well asnumerous connections with the NOT (Giolli et al., 1988;Vargas et al., 1996; Vargas et al., 1997). In the rat, rabbit,and gerbil (Giolli et al., 1985), as in the opossum (Vargas etal., 1997), many of the INSFp cells express GABA. In thelatter species (Vargas et al., 1997), contrary to what wasfound in the rat (Giolli et al., 1992), part of the INSFpprojecting cells are also GABAergic.

Similarly to the MTN, the LTN has been shown to beconnected with the NOT (rabbit and rat: Giolli et al., 1988;monkey: Baleydier et al., 1990; Mustari et al., 1994;opossum: Vargas et al., 1996, 1997). In the opossum LTN, asmall number of GABA-labeled cells and terminals wasidentified (Vargas et al., 1997). In the present study, theLTN was shown to receive a sparse retinal innervation andto express lower levels of CO and NADPH-d activity.Electrophysiologic recordings in the monkey LTN havesuggested that besides signaling the optokinetic move-ments, this nucleus could be involved in smooth pursuittasks (Mustari and Fuchs, 1989).

CONCLUSIONS

The analysis of CO activity in the opossum mesodience-phalic region has revealed a high number of labeled cellsand neuropil in the NOT-DTN. This labeling was shown todefine an area in the optic tract that coincides with that ofcells which project to the IO region. Thus, this method hasproven to be useful in the NOT-DTN identification. Cellcounts in this region revealed that the number of COpositive cells can be up to twice that of olivary cells.Histochemistry for CO and NADPH-d have revealed astrong labeling in the pretectal and AOS nuclei thatmatched the regions of dense contralateral retinal projec-tion to these regions.

LITERATURE CITED

Allman, J. and S. Zucker (1990) Cytochrome oxidase and functional codingin primate striate cortex: A hypothesis. In Cold Spring Harbor Sympo-sium in Quantitative Biology, Vol LV, Cold Spring Harbor, NY: ColdSpring Harbor Lab. Press, pp. 979–982.

Avendano, C. and M.A. Juretschke (1980) The pretectal region of the cat: Astructural and topographic study with stereotaxic coordinates. J. Comp.Neurol. 193:69–88.

Ballas, I. and K.P. Hoffmann (1985) A correlation between receptive fieldproperties and morphological structures in the pretectum of the cat. J.Comp. Neurol. 238:417–438.

Baleydier, C., M. Magnin, and H.M. Cooper (1990) Macaque accessory opticsystem: Connections with the pretectum. J. Comp. Neurol. 302:405–416.

Benassi, C., G.P. Biral, F. Lui, C.A. Porro, and R. Corazza (1989) Theinterstitial nucleus of the superior fasciculus, posterior bundle (INSFp)in the guinea pig: Another nucleus of the accessory optic systemprocessing the vertical retinal slip signal. Vis. Neurosci. 2:377–382.

Berman, N. (1977) Connections of the pretectum in the cat. J. Comp.Neurol. 174:227–254.

Blumke, I., P.R. Hof, J.H. Morrisson, and M.R. Celio (1990) Distribution ofparvalbumin immunoreactivity in the visual cortex of old world mon-keys and humans. J. Comp. Neurol. 301:417–432.

Buttner-Ennever, J.A., B. Cohen, A.K.E. Horn, and H. Reisine (1996)Efferent pathways of the nucleus of the optic tract in monkey and theirrole in eye movements. J. Comp. Neurol. 373:90–107.

Born, R.T. and R.B.H. Tootell (1992) Segregation of global and local motionprocessing in primate middle temporal visual area. Nature 357:497–499.

Caballero-Bleda, M., B. Fernandez, and L. Puelles (1992) The pretectalcomplex of the rabbit: Distribution of acetylcholinesterase and reduced


nicotinamide adenine dinucleotide diaphorase activities. Acta Anat.144:7–16.

Cavalcante, L.A., C.E. Rocha-Miranda, and R. Lent (1975) Hypothalamic,tectal and accessory optic projections in the opossum. Brain Res.84:302–307.

Cazin, L., W. Precht, and J. Lannou (1980) Optokinetic responses ofvestibular nucleus neurons in the cat. Pflugers Arch. 384:31–38.

Carroll, E.W. and M.T.T. Wong-Riley (1984) Quantitative light and electronmicroscopic analysis of cytochrome oxidase rich zones in the striatecortex of the squirrel monkey. J. Comp. Neurol. 222:1–17.

Chandrasekaran, K., J. Stoll, S.I. Rapoport, and D. Brady (1993) Localiza-tion of cytochrome oxidase activity and COX mRNA in the perirhinaland superior temporal sulci of the monkey brain. Brain Res. 606:213–219.

Clarke, R.J., R.A. Giolli, R.H.I. Blanks, Y. Torigoe, and J.H. Fallon (1989)Neurons of the medial terminal accessory optic nucleus of the rat arepoorly collateralized. Vis. Neurosci. 2:269–273.

Cohen, B., D. Schiff, and J. Buttner (1990) Contribution of the nucleus ofthe optic tract to optokinetic afternystagmus in the monkey: Clinicalimplications. In B. Cohen and I. Bodis-Wollner (eds): Vision and theBrain. New York: Raven Press, pp. 233–255.

Coleman, L.A. and L.D. Beazley (1988) The accessory optic system of thewallaby, Setonix brachyurus: Anatomy of normal animals and afterearly unilateral eye removal. J. Comp. Neurol. 273:359–376.

Collewijn, H. (1975) Direction selective units in the rabbit’s nucleus of theoptic tract. Brain Res. 100:489–508.

Condo, G.J. and V.A. Casagrande (1990) Organization of cytochromeoxidase staining in the visual cortex of nocturnal primates (Galagocrassicaudatus and Galago senegalensis): I. Adult patterns. J. Comp.Neurol. 293:632–645.

Cooper, H.M. and M. Magnin (1986) A common mammalian plan ofaccessory optic system organization revealed in all primates. Nature324:457–459.

Cooper, H.M., C. Baleydier, and M. Magnin (1990) Macaque accessory opticsystem: I. Definition of the medial terminal nucleus. J. Comp. Neurol.302:394–404.

DeYoe, E.A., T.C. Trusk, and M.T.T. Wong-Riley (1995) Activity correlates ofcytochrome oxidase defined compartments in granular and supragranu-lar layers of primary visual cortex of macaque monkey. Vis. Neurosci.12:629–639.

Distler, C. and K.P. Hoffmann (1993) Visual receptive fields properties inkitten pretectal nucleus of the optic tract and dorsal terminal nucleus ofthe accessory optic tract. J. Neurophysiol. 70:814–827.

Edwards, D.P., K.P. Purpura, and E. Kaplan (1995) Contrast sensitivity andspatial frequency response of primate cortical neurons in and aroundthe cytochrome oxidase blobs. Vision Res. 11:1501–1523.

Farias, M.F., R. Gattass, M.C. Pinon, and L.G. Ungerleider (1997) Tangen-tial distribution of cytochrome oxidase-rich blobs in the primary visualcortex of macaque monkeys. J. Comp. Neurol. 386:217–228.

Franca, J.G., J.L.M. Nascimento, C.W. Picanco-Diniz, J.A.S. Quaresma,and A.L.C. Silva (1997) NADPH-diaphorase activity in area 17 of thesquirrel monkey visual cortex: Neuropil pattern, cell morphology andlaminar distribution Braz. J. Med. Biol. Res. 30:1093–1105.

Giolli, R.A. and M.D. Guthrie (1969) The primary optic projections in therabbit. An experimental degeneration study. J. Comp. Neurol. 136:99–126.

Giolli, R.A., R.H.I. Blanks, and Y. Torigoe (1984) Pretectal and brainstemprojections of the medial terminal nucleus of the accessory optic systemof the rabbit and rat as studied by anterograde and retrograde neuronaltracing methods. J. Comp. Neurol. 227:228–251.

Giolli, R.A., G.M. Peterson, C.E. Ribak, H.M. McDonald, R.H.I. Blanks, andJ.H. Fallon (1985) GABAergic neurons comprise a major cell type inrodent visual relay nuclei: An immunocytochemical study of pretectaland accessory optic nuclei. Exp. Brain Res. 61:194–203.

Giolli, R.A., Y. Torigoe, R.H.I. Blanks, and M.H. Mc Donald (1988) Projec-tions of the dorsal and lateral terminal accessory optic nuclei and of theinterstitial nucleus of the superior fascicles (posterior fibers) in therabbit and rat. J. Comp. Neurol. 277:608–620.

Giolli, R.A., R.J. Clarke, Y. Torigoe, R.H.I. Blanks, and J.H. Fallon (1992)GABAergic and non-GABAergic projections of optic nuclei including thevisual tegmental relay zone to the nucleus of the optic tract and dorsalterminal accessory optic nucleus in rat. J. Comp. Neurol. 319:349–358.

Grasse, K.L. and M.S. Cynader (1982) Electrophysiology of the medialterminal nucleus of the accessory optic system in the cat. J. Neuro-physiol. 48:490–504.

Grasse, K.L. and M.S. Cynader (1984) Electrophysiology of lateral anddorsal terminal nuclei of the cat accessory optic system. J. Neuro-physiol. 51:276–293.

Hamasaki, D. and E. Marg (1962) Microelectrode study of accessory optictract in the rabbit. Am. J. Physiol. 202:480–486.

Hayhow, W.G. (1959) An experimental study of the accessory optic fibersystem in the cat. J. Comp. Neurol. 113:281–313.

Hayhow, W.G. (1966) The accessory optic system in the marsupial phalanger,Trichosurus vupecula. J. Comp. Neurol. 126:653–672.

Hendrickson, A.E., S.P. Hunt, and J.Y. Wu (1981) Immunocytochemicallocalization of glutamic acid decarboxylase in monkey striate cortex.Nature 292:605–607.

Hevner, R.F, S. Liu, and M.T.T. Wong-Riley (1995) A metabolic map ofcytochrome oxidase in the rat brain: Histochemical, densitometric andbiochemical studies. Neuroscience 65:313–342.

Hoffmann, K.P. and C. Distler (1989) Quantitative analysis of visualreceptive fields of neurons in nucleus of the optic tract and dorsalterminal nucleus of the accessory optic tract in macaque monkey. J.Neurophysiol 62:416–428.

Hoffmann, K.P. and A. Schoppmann (1975) Retinal input to direction-selective cells in the nucleus tractus opticus in the cat. Brain Res.99:359–366.

Hoffmann, K.P. and A. Schoppmann (1981) A quantitative analysis of thedirection-specific response of neurons in the cat’s nucleus of the optictract. Exp. Brain Res. 42:146–157.

Hoffmann, K.P., K. Berend, and A. Schoppmann (1976) a direct afferentvisual pathway from the nucleus of the optic tract to the inferior olive inthe cat. Brain Res. 115:150–153.

Hoffmann, K.P., C. Distler, R. Erickson, and W. Mader (1988) Physiologicaland anatomical identification of the nucleus of the optic tract and dorsalterminal nucleus of the accessory optic tract in monkeys. Exp. Brain.Res. 69:635–644.

Holstege, G. and H. Collewijn (1982) The efferent connections of the nucleusof the optic tract and superior colliculus in the rabbit. J. Comp. Neurol.209:139–175.

Horn, A.E. and K.P. Hoffmann (1987) Combined GABA-immunocytochemis-try and TMB-HRP histochemistry of pretectal nuclei projecting to theinferior olive in rats, cats and monkeys. Brain Res. 409:133–138.

Horton, J.C. (1984) Cytochrome oxidase patches: A new cytoarchitectonicfeature of monkey visual cortex. Philos. Trans. R. Soc. Lond. 304:199–253.

Horton, J.C. and D.H. Hubel (1981) Regular patchy distribution of cyto-chrome oxidase staining on primary visual cortex of macaque monkey.Nature 292:762–764

Hutchins, B. and J.T. Weber (1985a) The pretectal olivary nucleus of thecat: Evidence for a two-tailed structure. Brain Res. 331:150–154.

Hutchins, B. and J.T. Weber (1985b) The pretectal complex of the monkey: Areinvestigation of the morphology and retinal terminations. J. Comp.Neurol. 232:425–442.

Ibbotson, M.R. and R.F. Mark (1994) Wide-field nondirectional visual unitsin the pretectum: Do they suppress ocular following of saccade-inducedvisual stimulation. J. Neurophysiol. 72:1448–1450.

Ibbotson, M.R., R.F. Mark, and T.L. Maddess (1994) Spatiotemporalresponse properties of direction-selective neurons in the nucleus of theoptic tract and dorsal terminal nucleus of the Wallaby, Macropuseugenii. J. Neurophysiol. 72:2927–2943.

Johnson, J.K. and V.A. Casagrande (1995) Distribution of calcium-bindingproteins within the parallel visual pathways of a primate (Galagocrassicaudatus) J. Comp. Neurol. 356:238–260.

Kageyama, G.H. and R.L. Meyer (1988) Histochemical localization ofcytochrome oxidase in the retina and optic tectum of normal goldfish: Acombined cytochrome oxidase-horseradish peroxidase study. J. Comp.Neurol. 270:354–371.

Kageyama, G.H. and M. Wong-Riley (1982) Histochemical localization ofcytochrome oxidase in the hippocampus: Correlation with specificneuronal types and afferent pathways. Neuroscience 7:2337–2361.

Kageyama, G.H. and M. Wong-Riley (1984) The histochemical localizationof cytochrome oxidase in the retina and lateral geniculate nucleus of theferret, cat and monkey, with particular reference to retinal mosaics andON/OFF center visual channels. J. Neurosci. 4:2445–2459.

Kageyama, G.H. and M. Wong-Riley (1985) An analysis of the cellularlocalization of cytochrome oxidase in the lateral geniculate nucleus ofthe adult cat. J. Comp. Neurol. 242:338–357.

Kanaseki, T. and J.M. Sprague (1974) Anatomical organization of pretectalnuclei and tectal laminae in the cat. J. Comp. Neurol. 158:319–338.


Klauer, S., F. Sengpiel, and K.P. Hoffmann (1990) Visual response proper-ties and afferents of nucleus of the optic tract in the ferret. Exp. BrainRes. 83:178–189.

Klooster, J., J.J.L. van der Want, and G. Vrensen (1983) Retinopretectalprojections in albino and pigmented rabbits: An autoradiographic study.Brain Res. 288:1–12.

Koontz, M.A., R.W. Rodieck, and S.G. Farmer (1985) The retinal projectionto the cat pretectum. J. Comp. Neurol. 236:42–59.

Korp, B.G., R.H.I. Blanks, and Y. Torigoe (1989) Projections of the nucleusof the optic tract to the nucleus reticularis tegmenti pontis andprepositus hypoglossi nucleus in the pigmented rat as demonstrated byanterograde and retrograde transport methods. Vis. Neurosci. 2:275–286.

Kubota, T., M. Morimoto, T. Kanaseki, and H. Inomata (1987) Projectionfrom the pretectal nuclei to the dorsal lateral geniculate nucleus in thecat: A wheat germ agglutinin-horseradish peroxidase study. Brain Res.421:30–40.

Land, P. (1987) Dependence of cytochrome oxidase activity in the rat lateralgeniculate nucleus on retinal innervation. J. Comp. Neurol. 262:78–89.

Lent, R., L.A. Cavalcante, and C.E. Rocha-Miranda (1976) Retinofugalprojections in the opossum. An anterograde degeneration and radioau-tographic study. Brain Res. 107:9–26.

Linden, R. and C.E. Rocha-Miranda (1981) The pretectal complex in theopossum: Projections from the striate cortex and correlation withretinal terminal fields. Brain Res. 207:267–277.

Livingstone, M. and D. Hubel (1984) Anatomy and physiology of a colorsystem in primate visual cortex. J. Neurosci. 4:309–356.

Maekawa, K., T. Takeda, and M. Kimura (1984) Responses of the nucleus ofthe optic tract neurons projecting to the nucleus reticularis tegmentipontis upon optokinetic stimulation in the rabbit. Neurosci. Res.2:1–25.

Magnin, M., H. Kennedy, and K.P. Hoffmann (1989) A double labelinginvestigation of the pretectal visuo-vestibular pathways. Vis. Neurosci.3:53–58.

Mesulam, M.M. (1978) Tetramethyl benzidine for horseradish peroxidaseneurohistochemistry: A non-carcinogenic blue reaction-product withsuperior sensitivity for visualizing neural afferents and efferents. J.Histochem. Cytochem. 26:106–117.

Mustari, M.J. and A.F. Fuchs (1989) Response properties of single units inthe lateral terminal nucleus of the accessory optic system in thebehaving primate. J. Neurophysiol. 61:1207–1219.

Mustari, M.J., A.F. Fuchs, C.R.S. Kaneko, and F.R. Robinson (1994)Anatomical connections of the primate nucleus of the optic tract. J.Comp. Neurol. 349:111–128.

Narbors, L.B. and R.R. Mize (1991) A unique neuronal organization in thecat pretectum revealed by antibodies to the calcium binding proteinCalbindin D-28K. J. Neurosci. 11:2460–2476.

Nasi, J.P. (1993) O reflexo optocinetico horizontal. Estudo anatomico eeletrofisiologico no gamba (Didelphis marsupialis aurita) normal e comenucleacao monocular precoce. PHD thesis at the Biophysics InstituteCarlos Chagas Filho, UFRJ, Brazil.

Nasi, J.P., Volchan, E., M.T. Tecles, R.F. Bernardes, and C.E. Rocha-Miranda (1997) The horizontal optokinetic reflex of the opossum(Didelphis marsupialis aurita): Physiological and anatomical studies innormal and early mononucleated specimens. Vis. Res. 37:1207–1216.

Nunes Cardozo, B. and J. Van der Want (1990) Ultrastructural organizationof the retino-pretecto-olivary pathway in the rabbit. J. Comp. Neurol.291:313–327.

Oswaldo-Cruz, E. and C.E. Rocha-Miranda (1968) The brain of the opossum(Didelphis marsupialis). A cytoarchitectonic atlas in stereotaxic coordi-nates. Biophysics Institute Carlos Chagas Filho, UFRJ, Brazil.

Pereira, Jr., A., E. Volchan, R.F. Bernardes, and C.E. Rocha-Miranda (1994)Binocularity in the nucleus of the optic tract of the opossum. Exp. BrainRes. 102:327–338.

Robertson, R.T. (1983) Efferents of the pretectal complex: Separate popula-tions of neurons project to lateral thalamus and to inferior olive. BrainRes. 258:91–95.

Rocha-Miranda, C.E. and R. Lent (1978) Opossum Neurobiology (Neurobio-logia do Gamba). Academia Brasileira de Ciencias, Rio de Janeiro,Brazil.

Rosa, M.G.P., R. Gattass, and J.G.M. Soares (1991) A quantitative analysisof cytochrome oxidase-rich patches in the primary visual cortex ofCebus monkeys: Topographic distribution and effects of late monocularenucleation. Exp. Brain Res. 84:195–209.

Rose, J.E. (1942) The thalamus of the sheep: Cellular and fibrous structureand comparison with pig, rabbit and cat. J. Comp. Neurol. 77:469–523.

Royce, G.G., J.P. Ward, and J.K. Harting (1976) Retinofugal pathways intwo marsupials. J. Comp. Neurol. 170:391–414.

Saint-Cyr, J.A. and J. Courville (1981) Sources of descending afferents tothe inferior olive from the upper brainstem in the cat as revealed by theretrograde transport of horseradish peroxidase. J. Comp. Neurol.198:567–581.

Sandell, J.H. (1986) NADPH diaphorase histochemistry in the macaquestriate cortex. J. Comp. Neurol. 251:388–397.

Scalia, F. (1972) The termination of retinal axons in the pretectal region ofmammals. J. Comp. Neurol. 145:223–258.

Scalia, F. and V. Arango (1979) Topographic organization of the projectionsof the retina to the pretectal region in the rat. J. Comp. Neurol.186:271–292.

Scherer-Singler, U., S.R. Vincent, H. Kimura, and E.G. McGeer (1983)Demonstration of a unique population of neurons with NADPH diapho-rase histochemistry. J. Neurosci. Methods 9:229–234.

Schmidt, M. (1996) Neurons in the cat pretectum that project to the dorsallateral geniculate are activated during saccades. J. Neurophysiol.76:2907–2918.

Schmidt, M., D. Schiff, and M. Bentivoglio (1995) Independent efferentpopulations in the nucleus of the optic tract: An anatomical andphysiological study in rat and cat. J. Comp. Neurol. 360:271–285.

Schmidt, M., G. Lehnert, R.G. Baker, and K.P. Hoffmann (1996) Dendriticmorphology of projection neurons in the cat pretectum. J. Comp.Neurol. 369:520–532.

Seligman, A.M., M.J. Karnowsky, H.L. Wasserkrug, and J.S. Hanker (1968)Nondroplet ultrastructural demonstration of cytochrome oxidase activ-ity with a polymerizing osmiophilic agent, diaminobenzidine (DAB). J.Cell Biol. 38:1–14.

Silverman, M.S., D.H. Grosof, R.L. De Valois, and S.D. Elfar (1989)Spatial-frequency organization in primate striate cortex. Proc. Natl.Acad. Sci. USA 86:711–715.

Simpson, J.I. (1984) The accessory optic system. Annu. Rev. Neurosci.7:13–41.

Simpson, J.I., R.E. Soodak, and R. Hess (1979) the accessory optic systemand its relation to vestibulocerebellum. In R. Granit and O. Pompeiano(eds): Reflex Control of Posture and Movement. Prog. Brain Res. Vol. 50.Amsterdam: Elsevier/North Holland Biomedical Press, pp. 715–724.

Simpson, J.I., R.A. Giolli, and R.H.I. Blanks (1988) The pretectal nuclearcomplex and the accessory optic system. In J.A. Buttner-Ennever (ed):Neuroanatomy of the Oculomotor System. Amsterdam: Elsevier, pp.335–364.

Sudkamp, S. and M. Schmidt (1995) Physiological characterization ofpretectal neurons projecting to the lateral posterior-pulvinar complexin the cat. Eur. J. Neurosci. 7:881–888.

Terasawa, K., K. Otani, and J. Yamada (1979) Descending pathways of thenucleus of the optic tract in the rat. Brain Res. 173:405–417.

Tootell, R.B., S.L. Hamilton, and M.S. Silverman (1985) Topography ofcytochrome oxidase activity in the owl monkey cortex. J. Neurosci.5:2766–2800.

Van der Togt, C. and J. Van der Want (1992) Variation in form and axonaltermination in the nucleus of the optic tract of the rat: The medialterminal nucleus input on the neurons projecting to the inferior olive. J.Comp. Neurol. 325:446–461.

Van der Togt, C., J. Van der Want, and M. Schmidt (1993) Segregation ofdirection selective neurons and synaptic organization of inhibitoryintranuclear connections in the medial terminal nucleus of the rat: Anelectrophysiological and immunoelectron microscopical study. J. Comp.Neurol. 338:175–192.

Vargas, C.D, E. Volchan, J.P. Nasi, R.F. Bernardes, and C.E. Rocha-Miranda (1996) The nucleus of the optic tract (NOT) and the dorsalterminal nucleus of opossums (Didelphis marsupialis aurita). BrainBehav. Evol. 48:1–15.

Vargas, C.D., E. Volchan, J.N. Hokoc, A. Pereira, Jr., R.F. Bernardes, andC.E. Rocha-Miranda (1997) On the functional anatomy of the nucleus ofthe optic tract-dorsal terminal nucleus commissural connection in theopossum (Dideplhis marsupialis aurita). Neuroscience 76:313–321.

Volchan, E., C.E. Rocha-Miranda, C.W. Picanco-Diniz, B. Zinsmeisser, R.F.Bernardes, and J.G. Franca (1989) Visual response properties of thepretectal nucleus of the optic tract in the opossum. Exp. Brain Res.78:380–386.

Volchan, E., A. Pereira, Jr., C.D. Vargas, and C.E. Rocha-Miranda (1996)The nucleus of the optic tract of the opossum. Rev. Bras. Biol. 56:373–380.


Walberg, F., T. Nordby, K.P. Hoffmann, and H. Hollander (1981) Olivaryafferents from the pretectal nuclei in the cat. Anat. Embryol. 161:291–304.

Watanabe, S., I. Kato, S. Sato, T. Okada, and M. Norita (1991) Descendingpathways from the nucleus of the optic tract in the Fuscata monkey.Soc. Neurosci. Abst. 17:113.

Weber, J.T. (1985) Pretectal complex and accessory optic system of pri-mates. Brain Behav. Evol. 26:117–140.

Weber, J.T. and J.K. Harting (1980) The efferent projections of the pretectalcomplex: An autoradiographic and horseradish peroxidase analysis.Brain Res. 194:1–28.

Wong-Riley, M.T.T. (1976) Endogenous peroxidatic activity in brainstemneurons as demonstrated by staining with diaminobenzidine in normalsquirrel monkeys. Brain Res. 108:257–277.

Wong-Riley, M.T.T. (1979) changes in the visual system of monocularlysutured or enucleated cats demonstrable with cytochrome oxidasehistochemistry. Brain Res. 171:11–28.

Wong-Riley, M.T.T. (1989) Cytochrome oxidase: An endogenous metabolicmarker of neuronal activity. Trends Neurosci. 12:94–102.

Wong-Riley, M.T.T. and T.T. Norton (1988) Histochemical localization ofcytochrome oxidase activity in the visual system of the tree shrew:Normal patterns and the effect of retinal impulse blockade. J. Comp.Neurol. 272:562–578.

Wong-Riley, M.T.T., M.M. Merzenich, and P.A. Leake (1978) Changes inendogenous enzymatic reactivity to DAB induced by neuronal inactiv-ity. Brain Res. 141:185–192.

Zhang, H.Y. and K.P. Hoffmann (1993) retinal projections to the pretectum,accessory optic system and superior colliculus in pigmented and albinoferrets. Eur. J. Neurosci. 5:486–500.


Cytochrome oxidase and NADPH-diaphorase on the afferent relay branch of the optokinetic reflex in...

Documents

Transcript of Cytochrome oxidase and NADPH-diaphorase on the afferent relay branch of the optokinetic reflex in...