Relative Number of Cells Projecting FromContralateral and Ipsilateral NucleusIsthmi to Loci in the Optic Tectum IsDependent on Visuotopic Location:

Horseradish Peroxidase Study in theLeopard Frog

ELIZABETH A. DUDKIN AND EDWARD R. GRUBERG*Biology Department, Temple University, Philadelphia, Pennsylvania 19122

ABSTRACTThe leopard frog optic tectum is the principal target of the contralateral retina. The

retinal terminals form a topographic map of the visual field. The tectum also receives bilateraltopographic input from a midbrain structure called nucleus isthmi. In this study wedetermined the relative strength of n. isthmi projections to different loci in the tectum.Horseradish peroxidase (HRP) was applied at single superficial tectal locations in a series ofleopard frogs. The application sites were distributed across the tectum. Retrogradely filledcells were counted in ipsilateral and contralateral nucleus isthmi. Although all regions of thetectum receive input from both n. isthmi, the relative number of labeled cells in the two n.isthmi is dependent on visuotopic location. Input to the rostromedial tectum representing thevisual field ipsilateral to the labeled tectum comes primarily from the contralateral n. isthmi.Input to the caudolateral tectum representing the visual field contralateral to the labeledtectum originates mostly from the ipsilateral n. isthmi. Tectal application sites representingthe visual midline had approximately equal numbers of labeled cells in the two n. isthmi. Theresults are similar at postapplication survival times ranging from 2 to 14 days. Usingapplication of HRP to rostral tectum and application of nuclear yellow to caudal tectum, weshow that the anisotropy in isthmi labeling is not due to take up of these labels by isthmotectalfibers passing through the application sites that terminate elsewhere. J. Comp. Neurol.414:212–216, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: retrograde labeling; nuclear yellow; visual field; Rana pipiens

Each eye of the leopard frog has a visual field at groundlevel of about 240°. The view includes the full 180° of theipsilateral field and approximately 60° in the frontalcontralateral field. These fields have been determined bothby visual perimetry (the observer determines the range ofhorizontal eccentricities over which the animal’s pupil canbe seen) and behaviorally (one eye of the frog is coveredwith an opaque patch, and the region where the animalresponds to prey stimuli is mapped out).

The optic tectum mediates responses to prey stimuli(Bechterew, 1884; Ingle, 1973). In leopard frogs, each of thetwo tectal lobes receives direct retinal input almost en-tirely from the contralateral eye. The retinotectal projec-tion terminates in distinct laminae and appears relativelyuniform in the different regions of the tectum (Potter,1969; Hughes, 1990). There are no obvious discontinuities

or anisotropies in the retinotectal projection: the projectionto rostral areas is similar to caudal areas; the projection tolateral areas is similar to medial areas. Furthermore,ganglion cells that are near each other in the retina projectto nearby locations in the tectum. That is, there is apoint-to-point map of the retina onto the tectum (Gaze,1958).

When one optic tract is cut near the optic chiasm, thereis complete interruption of the direct retinotectal input to

Grant sponsor: NIH; Grant number: EY4366; Grant sponsor: TempleUniversity.

*Correspondence to: Edward R. Gruberg, Biology Department, TempleUniversity, Philadelphia, PA 19122. E-mail: V5381e@VM.Temple.edu

Received 5 March 1999; Revised 20 July 1999; Accepted 26 July 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 414:212–216 (1999)

r 1999 WILEY-LISS, INC.

the tectal lobe on the side of the lesion. Such a cut leavesintact the retinotectal projection to the other tectal lobe.For ease of the subsequent description, we will assumethat the left optic tract is cut and the right optic tract isspared. When a frog with a cut left optic tract is tested forresponse to prey stimuli, the animal (predictably) does notrespond in the monocular region of the right eye (Weber etal., 1996). The frog does respond to prey stimuli in theipsilateral field of the left eye. Curiously, however, the frogdoes not respond to prey stimuli in the anterior contralat-eral field of the left eye (Weber et al., 1996). Cutting the leftoptic tract leaves intact the retinotectal projection to theright tectal lobe: there is still a full visuotopic map fromthe left eye representing visual regions on both sides of themidline. However, when the left optic tract is cut, inputfrom the left nucleus isthmi to the right tectal lobe is lost.

Nucleus isthmi is a tegmental structure in the posteriormidbrain that gives rise to cholinergic isthmotectal axons.There is a visuotopic projection from the tectum to theipsilateral nucleus isthmi and a reciprocal visuotopic mapof n. isthmi onto a superficial lamina of the entire ipsilat-eral tectum. Furthermore, there is a topographic projec-tion from nucleus isthmi to the entire contralateral tectumas well. Evidence exists that nucleus isthmi fibers have anexcitatory effect on retinotectal terminals (Schmidt, 1995).Eliminating nucleus isthmi input to the optic tectumresults in a loss of visually elicited responses to preystimuli in a portion of the visual field.

Retrograde tracers such as horseradish peroxidase (HRP)injected into the superficial layers of any locus in thetectum will back-fill cells in the contralateral and ipsilat-eral nucleus isthmi. We report here that, although allregions of the optic tectum receive input from both nucleiisthmi, the relative number of isthmic cells projecting to aparticular tectal locus from each n. isthmi varies greatlywith tectal location. Loci in the part of the left tectumcorresponding to the region representing the left visualfield receive input from a much greater number of cellsfrom the right (contralateral) n. isthmi than from the left(ipsilateral) n. isthmi. Conversely, loci in the part of theleft tectum representing the right visual field receive inputfrom a much greater number of cells from the left n. isthmithan the right n. isthmi. We will argue that these resultsare consistent with the behavioral results described aboveand suggest the importance of nucleus isthmi input to theoptic tectum.

MATERIALS AND METHODS

Leopard frogs, Rana pipiens pipiens, 5–7 cm (s to v) wereobtained from northern Vermont. To map the relativestrength of tectal input from the two n. isthmi to thetectum, we made single HRP injections into individualfrogs. Collectively, the injection sites were scattered overthe tectum.

Each frog was anesthetized by immersion in a 0.3%aqueous solution of 3-aminobenzoic acid ethyl ester (Sigma,St. Louis, MO). A flap of skin was cut and retracted, and apatch of bone was cut out, exposing the dorsal surface ofthe optic tectum. A micropipette filled with 10% HRPsolution was lowered into the tectum to a depth of approxi-mately 150 µm. Current was 6 µA for 8 minutes (electrodepositive). Since a significant part of the tectal surface islocated ventrolaterally (and thus is not visible with adorsal exposure), we located other sites for injection by

first recording extracellularly with a laterally placed metalmicroelectrode to gauge depth. The injection sites werechosen to be at the level of type 2 and type 3 retinotectalphysiological units (Lettvin et al., 1959). Post-HRP injec-tion survival time ranged from 2 to 16 days, with mostmaintained for 8 days. All animals were kept at a tempera-ture of about 22°C.

In one animal pellets of HRP were placed in threelocations in the rostral tectum. The pellets were spacedabout 400 µm apart. A nuclear yellow (Sigma) pellet wasplaced in the caudal tectum approximately 600 µm moreposterior than the HRP pellets.

After surviving at 22°C, the animals were anesthetizedand perfused via the conus arteriosus, first in saline, andthen fixed with 3% paraformaldehyde and 1% glutaralde-hyde in a 0.1 M phosphate buffer, pH 7.3. The brains wereremoved and placed in a solution of 30% sucrose in 0.1 Mphosphate buffer, pH 7.3, overnight at 4°C. Serial 40 µmsections were cut on a cryostat and mounted on subbedslides, where they were dried overnight, and then stainedusing the procedure of metal intensified DAB (Straus,1982).

All labeled cells were counted in each nucleus isthmi bytwo people independently. Because the cells were smallcompared to section thickness and we were interested inthe relative number of labeled cells from the two nuclei, wemade no postcounting correction. The locations of the HRPinjection sites in the tectum were also identified indepen-dently by two people. The data were normalized as if allHRP injections were into a left tectal lobe and thenmapped.

We mapped the tectal locations of points on the frog’ssagittal visual field by extracellular recording of multiunitreceptive fields in the superficial tectum using visualstimulation with small dark disks. At each tectal locationcorresponding to the visual midline, current was passedthrough the recording electrode (10 µA for 10 seconds,electrode negative) making small electrolytic lesions. Theanimal was then anesthetized and perfused, first withsaline, then with 3% paraformaldehyde in 0.1 M phos-phate buffer, pH 7.3. The brain was removed, dehydrated,suffused with paraffin, and cut into 15 µm serial sections,which were then stained with cresyl violet. The locations oflesions were verified by two people, then normalized andmapped. All procedures were performed in accordancewith the Institutional Animal Care and Use Committee ofTemple University.

RESULTS

Single tectal sites were injected in a total of 25 animals.Figure 1A,B shows a typical tectal injection site. All thesuperficial layers are darkly stained, and the lateral extent ofthe site is approximately 400 µm. Figure 1C,D showsback-filled cells stained by HRP histochemistry in ipsilat-eral and contralateral n. isthmi. The relative number ofcells stained in the two n. isthmi is dependent on thevisuotopic location of the tectal injection site (Fig. 2A,B).In loci (n 5 9) in the rostral tectum corresponding to thevisual field ipsilateral to the injected tectum, an average of4.5 times more cells are labeled in contralateral n. isthmithan ipsilateral n. isthmi. In loci (n 5 11) in caudal tectumcorresponding to the visual field contralateral to theinjected tectum, an average of 3.3 times more cells arelabeled in ipsilateral n. isthmi than contralateral n. isthmi.

ISTHMOTECTAL PROJECTIONS VARY WITH TECTAL TARGET 213

In loci (n 5 5) along a band immediately caudal to thevisual midline, there were roughly equal numbers of cellslabeled in the two n. isthmi.

Effect of survival time

One possible explanation for the regional difference inthe relative number of HRP-labeled cells in the two n.isthmi is the time it takes for HRP to reach n. isthmi cellbodies by retrograde transport. A longer pathway wouldtake a longer time. Conversely, if the survival time is toolong, the HRP that reached the closer target could havealready been cleared. To test this possibility, we injectedHRP into a few select locations and varied survival time.We found that survival times as short as 2 days and as longas 14 days gave ratios similar to those for the intermediatesurvival time (Fig. 2C).

Filling of fibers of passage?

There is another possible explanation for why the ratioof the number of HRP-stained cells in the ipsilateral n.isthmi vs. contralateral n. isthmi varies widely when HRP

is injected at different sites in the tectum. HRP injectionsinto the tectum could lead to uptake of HRP not only intolocal isthmotectal terminals but also into isthmotectalaxons passing through the local area. Isthmotectal fibersprojecting to the contralateral tectum take a path forwardfrom n. isthmi, decussate in the optic chiasm, and course ina dorsal-posterior direction to the optic tectum admixedwith retinal axons. They then spread out in a rostral toposterior direction before terminating in tectal layers Aand 8 (Gruberg and Udin, 1978). Isthmotectal fibersprojecting to the ipsilateral tectum project forward fromthe nucleus isthmi on the lateral surface of the midbraintegmentum ventral to the optic tectum. They then coursedorsally in the transverse plane into layers 7 and C of theoptic tectum before terminating more superficially. Thus,even if there were homogeneous projections from n. isthmito tectum, if isthmi fibers of passage took up HRP, therecould be relatively more cells back-filled in ipsilateral n.isthmi after posterior tectal HRP injections than afteranterior tectal HRP injections. If a retrograde tracer wereinjected into a posterior locus of the tectum, then it might

Fig. 1. HRP filling of n. isthmi cells from tectal injection.A: Transverse section showing injection site in rostrodorsal left optictectum. B: Top: Location of HRP injection site shown in A (solid circle).The dashed curve indicates the edge of the tectum seen from a dorsalview. The area outside the dashed curve is the more ventral surface ofthe tectum flattened onto the same plane. R, rostral; M, medial; C,

caudal; L lateral. Bottom: Lateral view of the brain showing location ofnucleus isthmi (n.i.); tect., tectum; cb., cerebellum; di, diencephalon;o.c., optic chiasm; tel., telencephalon. C: Transverse section of ipsilat-eral (left) n. isthmi showing labeled cells. D: Transverse section ofcontralateral (right) n. isthmi showing labeled cells. in C. Scale bars 5500 µm in A; 100 µm in C (also applies to D).

214 E.A. DUDKIN AND E.R. GRUBERG

retrogradely mark n. isthmi cells that project to theposterior part of the tectum and also fill n. isthmi cellsprojecting to the anterior part of the tectum. To test thispossibility, we injected HRP into three sites in the anteriorpart of the tectum and injected another retrograde tracer,nuclear yellow, into the posterior part of the tectum (Fig.3A). We found that the retrogradely labeled nuclear yellowcells were restricted to the dorsal part of n. isthmi,whereas the HRP-labeled cells were restricted to theventral part of n. isthmi. The two populations of stainedcells were clearly segregated (Fig. 3B). There were nodouble-labeled cells.

Furthermore, when single HRP injections are made inthe tectum, retrogradely labeled cells in the nucleus isthmiform a single locus. When we made pairs of HRP injectionsspaced 150 µm apart, we could distinguish two spatiallyseparate sets of labeled cells in each nucleus isthmiregardless of orientation of the pair of injection sites. Thus,because we could distinguish two populations of cells ineach pair of injections, it is unlikely that our results wereconfounded by uptake of HRP by fibers of passage.

DISCUSSION

The retina projects to the contralateral optic tectum andforms four precise retinotopic maps of the visual field(Lettvin et al., 1959). The maps are in register with eachother. The current results show that the relative number ofcells labeled with HRP in ipsilateral and contralateraltectum depends on the visuotopic location of the HRPinjection. Thus, after localized injections of HRP into thepart of the right tectal lobe representing the left eye’smonocular (left) visual field, more cells are stained in rightnucleus isthmi than left nucleus isthmi. After injections ofHRP into the part of the right tectal lobe representing theleft eye’s contralateral (right) visual field, more cells arestained in the left nucleus isthmi than the right nucleusisthmi. These results are consistent with earlier workshowing that more contralateral isthmotectal cells arestained when HRP injections are made into rostral tectal

Fig. 2. A: Left: Visual field of right eye of a frog at the center of anoverhead hemisphere. The field is divided into a binocular regionbeyond the sagittal plane (a), a binocular region ipsilateral to the righteye (b), and a monocular region (c). Right: The visuotopic locations ofthe three regions of the visual field of the right eye as projected ontothe left optic tectum. B: Summary diagram of tectal HRP injectionsites (circles) normalized to left tectal lobe. Postinjection survival timeof 8 days. The number next to each circle indicates the ratio of thenumber of labeled cells in the contralateral nucleus isthmi to thenumber of labeled cells in the ipsilateral nucleus isthmi. Dark circleshave ratios greater than 1.4. Hatched circles have ratios between 0.6and 1.4. Light circles have ratios less than 0.6. Solid squares representpoints on the tectum corresponding to the visual sagittal plane(vertical midline plane). C: Tectal injection sites and ratios for survivaltimes of 2, 6, and 14 days.

Fig. 3. A: Dorsal surface of left tectum showing rostral injectionsites of HRP (solid circles) and more caudal injection site for nuclearyellow (open circle). B: Transverse section through the ipsilateral (left)nucleus isthmi in double exposure of both dark field epifluorescenceand brightfield transillumination showing spatial separation of HRP-and nuclear yellow-labeled cells. Bright cells dorsomedial in thenucleus are labeled with nuclear yellow. Dark cells ventrolateral in thenucleus are labeled with HRP. The dark area between the two groupsof labeled cells is HRP-labeled tectoisthmal fibers and terminals. Scalebar 5 200 µm.

ISTHMOTECTAL PROJECTIONS VARY WITH TECTAL TARGET 215

loci. There are few contralateral nucleus isthmi cells filledwhen HRP is injected into caudal optic tectum, and manymore contralateral nucleus isthmi cells are filled whenHRP is injected into rostral tectum (Grobstein et al., 1978;Grobstein and Comer 1983; Gruberg et al., 1989). We havenow made explicit how the ratio of the number of back-filled cells in the two nuclei isthmi changes at differentvisuotopic locations in the tectum. We have also shownthat the ratio differences are not artifacts of survival time.Furthermore, there is no evidence of uptake of HRP infibers of passage.

In light of the current work, previous ablation andbehavioral work fit a pattern suggesting the importance ofnucleus isthmi in gating responses to prey stimuli. Unilat-eral ablation of (left) nucleus isthmi leads to loss ofresponsiveness to visually presented prey stimuli in themonocular field of the contralateral (right) eye. If the righteye is covered, the animal responds to stimuli only in theleft hemifield. Significant n. isthmi input was lost to theregion of the left tectal lobe corresponding to the rightmonocular visual field and to the region of the right tectallobe corresponding to the right half of the binocular field ofthe left eye. Loss of n. isthmi input to these areas reducesand may eliminate the effectiveness of retinal input tothese areas in evoking a response.

In recent work, Wiggers (1998) has intracellularly la-beled individual n. isthmi cells of plethodontid salamanderspecies Plethodon jordani and Hydromantes italicus withbiocytin. He discovered that virtually all of the labeledcells give rise to axons that project to the ipsilateral optictectum, where they each form an axonal tree. Each axonthen continues and crosses to the opposite side in theventral diencephalon and ultimately forms a second axo-nal tree in the equivalent visuotopic location of the contra-lateral optic tectum. Thus, each n. isthmi cell projects toboth tectal lobes. In recent preliminary work, Wiggers(personal communication) has found similar results in rimcortex cells of nucleus isthmi in frogs of the speciesDiscoglossus pictus. At first glance, Wiggers’s results mightsuggest that injection of HRP into any circumscribedregion of the tectum would result in the same numbers ofretrogradely labeled cells in the ipsilateral and contralat-eral n. isthmi. If the axonal trees of all isthmotectal cellswere uniformly spaced and of uniform size, there shouldindeed be equal numbers of cells retrogradely labeled ineach n. isthmi. Our results can be reconciled with Wig-

gers’s results if the density of isthmotectal axonal treesoriginating from each nucleus isthmi is not constant acrossthe tectum. Our results suggest that in the rostromedialpart of the tectum there is a higher density of axonal treesoriginating from cells of the contralateral n. isthmi. In thecaudolateral part of the tectum there is a higher density ofaxonal trees originating from the ipsilateral n. isthmi.

ACKNOWLEDGMENTS

We thank Michael Mote and Susan Udin for discussionand suggestions concerning this work.

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