THE JOURNAL OF COMPARATIVE NEUROLOGY 28839-50 (1989)

Relationship Between Isthmotectal Fibers and Other Tectopetal Systems

in the Leopard Frog

EDWARD R. GRUBERG, MARK T. WALMCE, AND ROBERT F. WALDECK Biology Department, Temple University, Philadelphia, Pennsylvania 19122

ABSTRACT We studied the relationship of isthmotectal input to other tectal afferent

fiber systems in three ways. 1) Using horseradish peroxidase (HRP) histo- chemistry, we determined the nonretinal inputs to the superficial tectum. In different sets of animals we a) applied HRP to the tectal surface; b) inserted HRP crystals into the tectum; c) injected small volumes of HRP solutions into the superficial tectum. N. isthmi accounts for more than 65 % of the nonretinal extrinsic input in the superficial tectal layers. One set of fibers from the con- tralateral n. isthmi projects to the most superficial layer. Fibers from posterior thalamus and tegmentum project to both superficial and deeper layers in the tectum, but not to the most superficial layer. The ipsilaterally projecting isth- motectal fibers terminate in the deeper superficial layers. 2) We investigated the relationship between retinofugal and contralaterally projecting isthmotec- tal pathways. We orthogradely labelled n. isthmi fibers by unilateral HRP injections into n. isthmi, and we also labelled retinal fibers by injecting tri- tiated 1-proline into both eyes. In such animals contralaterally projecting isth- motectal fibers cross in the dorsal posterior region of the optic chiasm. From the chiasm to the tectum isthmotectal fibers and retinofugal fibers are admixed. 3) We determined whether other fiber systems cross with contralat- erally projecting isthmotectal fibers. We cut the posterior part of the optic chiasm and applied HRP crystals to the cut. Only n. isthmi and retina are ret- rogradely labelled.

Key words: nucleus isthmi, optic tectum, posterior thalamus, mesencephalic tegmentum, HBP, retinal fibers, optic chiasm

Nucleus isthmi and the retina both project to the superfi- cial layers of the frog tectum (Gruberg and Udin, '78). The nucleus is located in the posterior mesencephalic tegmen- tum. It receives most of its input from the ipsilateral tectum and it projects bilaterally to the tectum. Unilateral ablation of n. isthmi leads to a loss of responsiveness to visually pre- sented prey and threat stimuli in the contralateral monocu- lar field (Caine and Gruberg, '85). The behavioral deficit is quite similar to the deficit that occurs after unilateral removal of the tectum (Ingle, '73). Nucleus isthmi thus appears to play an important role in influencing visually guided behavior mediated by the tectum. In order to better understand how n. isthmi influences tectal function it is important to know what other areas of the brain project to the superficial layers of the tectum.

Using horseradish peroxidase (HRP) histochemistry Wilczynski and Northcutt ('77) showed that the frog tectum receives projections from several posterior thalamic nuclei

and mesencephalic tegmental fields. It was not determined whether these inputs terminated in deep or superficial tec- tum. We assessed if other extrinsic inputs project to the superficial tectal layers in addition to the inputs from n. isthmi and the retina. We applied HRP to the surface of the tectum such that cell processes in the most superficial layers would selectively take up the enzyme. For comparison we injected solutions of HRP or inserted HRP crystals into the superficial tectum. We also injected HRP into the posterior thalamus and studied the distribution of orthogradely stained fibers in the tectum.

Earlier work has shown that contralaterally projecting n. isthmi fibers follow a lengthy pathway that brings them close to retinofugal tracts (Gruberg and Udin, '78). Fibers originating in the nucleus isthmi can be followed along the

Accepted May 1,1989.

0 1989 ALAN R. LISS, INC.

40

lateral margin of the mesencephalon and diencephalon. These fibers decussate ventrally in or near the optic chiasm and can be followed back dorsolaterally to the tectum. Since retinotectal fibers are almost entirely crossed, the contralat- erally projecting isthmotectal fibers enable each tectal lobe to receive binocular input (Glasser and Ingle, '78, Grobstein et al., '78). We investigated if there is any overlap of retino- fugal and isthmotectal pathways. We have orthogradely labelled fibers from n. isthmi with HRP and in the same preparations orthogradely labelled retinofugal fibers with binocular injections of tritiated 1-proline. We then com- pared the distribution of the two labels.

I t is not known if other systems cross with contralaterally projecting isthmotectal fibers. We have cut the crossed pro- jection at the site of decussation and then applied HRP crystals to the cut. We then determined the areas of the brain that were retrogradely labelled. A preliminary sum- mary of this work has been published (Gruberg et al., '87).

E.R. GRUBERG ET AL.

formaldehyde, 2.5 % glutaraldehyde). The brain (and in some cases the eye contralateral to the injected tectal lobe) was removed, postfixed for an additional 15-45 minutes, and then placed in a chilled 30% sucrose solution overnight. The brain was frozen in a cryostat, cut transversely a t 40 hm, mounted on subbed slides, dried, and stained by using either the chromagen benzidine dihydrochloride (Mesulam, '76) or tetramethyl benzidine (Mesulam, '78). In some cases the sections were subsequently counterstained with neutral red.

Iontophoresis of HRP into posterior thalamus Using the same iontophoretic procedure described above,

we injected HRP into the posterior thalamus. The micropi- pette was inserted medial to the medial retinotectal tract and placed approximately 1 mm below the dorsal surface of the thalamus. Passing of current, survival time, and histo- chemistry are as described above.

Orthograde labelling of contralaterally projecting n. isthmi fibers and retinal fibers The tectal surface was exposed as described above. The

animal was allowed to recover and was subsequently cura- rized. The nucleus isthmi was located by electrophysiologi- cal recording (see Gruberg and Lettvin, '80). Immediately adjacent to the recording electrode was a micropipette filled with a 20% solution of HRP. One to 2 nl of HRP was pres- sure injected into n. isthmi. In other animals, under anes- thesia, the posterior pole of the tectum was aspirated uni- laterally, exposing the underlying isthmic tegmentum. A crystal of HRP was placed in n. isthmi. In both methods the anesthetized animals subsequently had both eyes injected with 8 ~1 of a saline solution of 10 &i tritiated 1-proline (ICN Radiochemicals, 56 Ci/mmol). Each animal was main- tained 3-4 days, reanesthetized, and fixed by perfusion (see above). The brain was cut in a cryostat as described above. The sections were stained for HRP activity by using a cobalt-intensified diaminobenzidine reaction (Adams, '77), dehydrated, and defatted in xylene for 1-2 hours. The sec- tions were rehydrated, dried, covered with Kodak NTB-2 radio track emulsion, and stored in the dark a t 4°C for 2-3 weeks. The sections were developed by the autoradiographic method of Cowan et al. ('72).

Application of HRP to the chiasm area Each animal was anesthetized as above. A midline inci-

sion was made in the skin of the roof of the mouth. A patch of the soft bone ventral to the optic chiasm area was cut out with a sharp scalpel. The optic chiasm was then visible as a light, flattened "x" against the darker ventral brain surface. We cut either anterior or posterior parts of the chiasm or the region immediately caudal to the chiasm. Two sharp pins,

MATERIALS AND METHODS Administration of HRP to the tectum

Leopard frogs, Rana pipiens (obtained from Hazen, Al- burg, VT), were anesthetized by immersing them in an aqueous solution of 3 g/liter tricaine (3-aminobenzoic acid ethyl ester methanesulfonate salt). In each animal the tec- tum was exposed by cutting a flap of skin and removing a patch of bone. Over one tectal lobe the dura and arachnoid were slit open and retracted. HRP was administered by one of three methods.

By electrostatic attraction we adhered small HRP crystals (Sigma, type VI) to the tip of a sharp pin. The pin tip was briefly dipped in distilled water so the crystals formed a larger adhesive crys- tal. The surface of the tectum was punctured and the crystal was inserted and held in place until it dissolved.

We saturated a piece of bibulous paper (approximately 0.5 mm by 0.5 mm) with a solution containing 20% HRP (Sigma, type VI) and 1 % lysolecithin (lysophosphatidylcholine). The paper was air dried and then applied to the tectal surface for 20 min- utes. To avoid possible diffusion to nontectal structures, we kept the paper away from the edge of the tectum. The paper was then removed and the surface of the brain was tamped gently with cotton wool.

The shank of a micropipette (tip diameter 15-20 hm) was filled with a 20% solution of HRP or wheat germ agglutinin-conjugated HRP (Sigma). The pipette was lowered into the tectum with the aid of a micromanipulator. Approximately 0.25-0.5 nl of the HRP solution was pressure injected. Usually one injec- tion was made per animal. In other animals we carried out iontophoretic injection. We used pipettes containing a 20% HRP solution in 0.05 M Tris-HC1 buffer (pH 8.6). We passed 0.5 FA current in 0.5 second square wave pulses (elec- trode positive with respect to ground) with a 50% duty cycle for 2 minutes.

After each of these methods the patch of bone and flap of skin were replaced. Each animal recovered a t 4OC overnight and was maintained at room temperature, approximately 20-23OC, for 3-7 days. Additional animals were maintained at room temperature for only 90 minutes to determine the initial spread of the HRP. Each animal was then reanesthe- tized and perfused intracardially with a saline solution and then with a pH 7.3 phosphate-buffered fixative (0.5% para-

Method 1. Insertion of HRP crystal.

Method 2. Application on paper.

Method 3. Administration by injection.

Fig. 1. Transverse sections showing the distribution of HRP-stained cells after application of a crystal of HRP to the tectum (te). Brain 4C of Table 1. Left: Photomicrographs of cresyl-violet-stained hemisections. Right: Camera lucida drawings. Dark area in section d shows applica- tion site. Section a: Most posterior. Section h Most anterior. Cells of nucleus istbmi (ni) are stained on both sides. Other stained tegmental nuclei include anterodorsal nucleus (ad), dorsolateral neuropil (dl), nucleus profundus mesencephali (pr), and magnocellular nucleus of the torus semicircularis (tmc). Stained posterior thalamic nuclei include posterior neuropil (ptn), posterior nucleus (p), and posterodorsal (lpd) division of the lateral nucleus.

ISTHMOTECTAL AND OTHER TECTOPETAL FIBERS 41

Figure 1

42 E.R.GRUBERGETAL.

one anterior of the other, were placed approximately 600 pm into the chiasm in the sagittal plane. The two pins were then moved together severing the intervening fibers. HRP crys- tals (prepared as above) were applied to the cut. The bone patch was replaced and the incision sutured. The animals were maintained for 2-5 days. Their brains were fixed, cut, and stained as described above.

RESULTS Tectal administration of HRP

Insertion of HRP crystals into the tectum. There is dark staining of all tectal layers in the region of the insertion site. There are four major areas extrinsic to the labelled tec- tum that are retrogradely stained: 1) retina, contralaterally; 2) nucleus isthmi, bilaterally; 3) posterior thalamus, pri- marily ipsilateral; and 4) mesencephalic tegmentum, pri- marily ipsilateral (Fig. 1). Within the posterior thalamus the structures that contain stained cells (following the anatomi- cal nomenclature of Neary and Northcutt, '83) are posterior nucleus (p), posterodorsal division of lateral nucleus (lpd), and posterior neuropil (ptn). Within the tegmentum the structures that contain stained cells (using the nomencla- ture of Potter, '65; and Nieuwenhuys and Opdam, '76) are anterodorsal nucleus (ad), nucleus profundus mesencephali (pr), magnocellular nucleus of the torus semicircularis (tmc), and a dorsolateral neuropil (dl) that is lateral of the tmc. We counted the number of nonretinal/nontectal stained cells from each major area (Table 1). Since the sec- tions are relatively thick compared to the average diameter of the cells we use the raw counts in our table. Approxi- mately 70% of all the stained nonretinal cells are in nucleus isthmi. In the case with the most rostra1 application site there are approximately three times as many stained cells in contralateral n. isthmi as in ipsilateral n. isthmi. In the cases with more central application sites there are approximately equal numbers of stained cells in ipsilateral and contralat- era1 n. isthmi. In the most caudal application site there are approximately ten times as many stained cells in ipsilateral n. isthmi as in contralateral n. isthmi. For a midtectal appli- cation we found that there are approximately nine times as many stained cells in the retina as there are in n. isthmi bilaterally. Of the tegmental nuclei, ad contained approxi- mately half of the stained cells. The other tegmental areas (dl, pr, and tmc) contained approximately equal numbers of stained cells. In some cases we see a few stained cells in the posterodorsal tegmental nucleus. Of the posterior thalamic nuclei, ptn, lpd, and p had approximately equal numbers of stained cells. In some cases we see a few stained cells in the posteroventral division of the lateral thalamic nucleus and the central thalamic nucleus. In our preparations we did not see stained cells in deep layers of the contralateral tectum, the suprapeduncular nucleus, the dorsal gray columns of the cervical spinal cord, or the ventral preoptic hypothalamus, as was previously reported (Wilczynski and Northcutt, '77).

Without the addition of lysolecithin there was virtually no diffusion of HRP from the paper into the tectum. With the addition of lysolecithin, dark staining was found in all tectal layers underlying the paper (Fig. 2). Despite this seemingly uni- form distribution of HRP through the tectum, the propor- tion of stained cells in different brain regions was quite dif- ferent from the proportion of stained cells in cases with HRP crystal insertion. We have counted the total number of

Application of HRP by bibulous paper.

TABLE 1. Distribution of Stained Cells After HRP Crystal Insertion Into Tectum

% of total stained cells

Tectal Total no. N. isthmi N. isthmi Posterior Tegmental Brain location of cells contra ipsi thalamus nuclei

1C Rostra1 323 52 16 28 3 2C Midtectum 390 37 33 13 17

26 41 20 13 3C Midtectum 388 35 43 10 12 4C Midteeturn 529

5C Caudal 224 fi 63 1 R 1.7

TABLE 2. Distribution of Stained Cells After Paper Application of HRP to Tectal Surface

% of total stained cells

Brain Total no

of cells

1A 2A 3A 4A 5A 6A 7A 8A 9A

10A 11A 12A 13A 14A

283 94

618 308 207 406 211 212

1,021 623 652

1,088 899 620

N. isthmi contra

100 100 96 92 92 91 91 77 77 41 37 30 19 51

N. isthrni ipsi

0 0 3 3 1 2 5 1 1 0 2

13 6

40

Posterior thalamus

0 0 0 0 0 4 0 4

12 44 38 43 59

7

Tegmental nuclei

0 0 0 4 7 3 4

17 10 9

23 13 15 2

nonretinal/nontectal stained cells and have determined the proportion of such cells in contralateral n. isthmi, ipsilateral n. isthmi, posterior thalamus, and tegmentum (Table 2). In seven cases (#1A-#7A of table 2) over 90% of all the stained cells are located in contralateral n. isthmi (Fig. 2). A few scattered cells are seen in ipsilateral n. isthmi, posterior thalamus, and tegmentum. While the ipsilateral n. isthmi contains very few labelled cells, there are many densely stained tectoisthmal fibers (Fig. 2). This implies that tectal cells projecting to ipsilateral n. isthmi have dorsally extend- ing processes that reach layer A, the most superficial layer of the tectum. In six cases (#8A-l3A of Table 2), where there was presumably deeper diffusion of the HRP, there was a significant increase in the proportion of cells stained in pos- terior thalamus and tegmentum (Fig. 3) but little staining of ipsilateral n. isthmi cells. In general the same thalamic and tegmental cell groups stain with HRP paper applications as with HRP crystal insertions. The only exception is that with paper applications we do not see stained cells in tmc, imply- ing that tmc does not project as superficially as the other areas. These six cases suggest that the HRP had diffused deeper than the first seven cases hut very little into the superficial tectal layers that receive ipsilaterally projecting isthmotectal fibers. We have one case where we backfilled n. isthmi bilaterally (#14A of Table 2) but had relatively little staining of cells in the posterior thalamus and tegmentum.

We injected HRP by pressure or by iontophoresis. A t the injection sites staining was seen in all tectal layers. On the average of 19 cases about 67% of the stained cells were located in nucleus isthmi with approximately equal numbers ipsilaterally and contralater- ally (Table 3). The locations of the injection sites are shown in Figure 4. While there were significant differences be-

Administration by idection.

ISTHMOTECTAL AND OTHER TECTOPETAL FIBERS 43

TABLE 3. Distribution of Stained Cells After Injection of HRP into Tectum

% of total stained cells

Total no. N. isthmi N. isthmi Posterior Tegmental Brain of cells contra ipsi thalamus nuclei

1J 45 9 91 0 0 25 64 50 45 0 5 35 573 16 79 1 4 41 231 31 62 6 1 5J 39 56 28 8 8 61 165 30 53 15 2 75 1,077 54 29 15 2 8J 22 9 68 23 0 9J 43 33 32 34 0

1OJ 256 37 27 25 11 l l J 70 47 13 39 1 121 168 48 9 38 5 135 56 34 23 34 9 141 27 7 44 33 15 155 545 32 19 34 15 16J 442 26 24 23 26 175 255 11 33 15 40 185 198 24 14 7 55 19J 283 15 7 63 14

tween individual cases, on the average there was no obvious difference in proportions of stained cells in the four princi- pal areas stained by using pressure injection or iontophore- sis. Nor were there any consistent differences seen when the injections were in different regions of the tectum. The tmc contained labelled cells in the deeper injections but not in the more superficial injections. On the average, about 24% of the stained cells were in the posterior thalamus and 8 % of the stained cells were in the tegmentum.

Short-term survival In the three methods of HRP application all tectal layers

are darkly stained after 3-7 days survival. Thus one cannot predict the extent to which each tectopetal cell group will backfill with HRP by viewing the distribution of stain in the tectum after several days survival. I t has been suggested that shorter survival time would more accurately reveal the effective distribution of tectal HRP (Vanegas et al., '78; Mesulam, '82). When HRPAysolecithin was applied to the tectal surface with bibulous paper and the animal was per- fused after 90 minutes, the most superficial tectal layer was more densely stained than the underlying layers (Fig. 5a). Such staining is more indicative of the effective uptake zone of the HRP. However, when a crystal of HRP was inserted into the tectum and the animal was perfused after 90 min- utes, dark staining was seen in much of the outer mesen- cephalon including the tegmentum (Fig. 5b). With a similar size HRP crystal and 4-day survival only a rather circum- scribed area of the tectum stains (see Fig. Id).

Staining of thalamotectal fibers The injection sites extended from near the dorsal surface

of the thalamus to a depth of approximately 1 mm. They included much of the dorsal posterior thalamus. The injec- tion sites did not extend into the tectum to any significant degree since virtually no stained cells were found in nucleus isthmi. Stained fibers were found in both the superficial and deeper tectal layers (Fig. 6). Most of the superficial tectum above layer 8 had some stained fibers with the exception of layer A which had very little staining. We also backfilled tectal cells, primarily in layer 6.

Fig. 2. Tectal HRP application using bibulous paper soaked in HRPilysolecithin. Brain 9A of Table 2. a: Application site. All tectal layers under paper show heavy staining. Layer A is the most superficial tectal layer. The location of tectal layers 8 and 6 is also shown. The dark- stained material above layer A is pigment located on the pial surface. Medial to right. Survival time 3% days. Scale 250 pm. b Nucleus isthmi ipsilateral to tectal application site of a. Orthogradely labelled tectoisth- ma1 fibers are densely stained. In this brain only 1 % of the nonretinal/ nontectal stained cells are in ipsilateral n. isthmi. Scale 200 pm. c: Nucleus isthmi contralateral to tectal application site of a. In this brain 77% of the nonretinal/nontectal stained cells are in contralateral n. isthmi. Scale 200 pm.

44 E.R.GRUBERGETAL.

Fig. 3. Retrogradely filled HRP-stained cells after paper application of HRP to tectum in which a significant number of cells were stained in posterior thalamic nuclei and mesencephalic tegmentum. Brain 12A of Table 2. Survival time 4 days. Medial to right in all photomicrographs. a: Cells in posterior thalamic nucleus (p) and posterodorsal division of lateral nucleus (lpd). Stained fibers in upper left are part of medial reti-

Double labelling: isthmotectal fibers and retinofugal fibers

We followed contralaterally projecting fibers ortho- gradely stained with HRP from n. isthmi to their decussa- tion and then back to the contralateral tectum (Fig. 7 ) . In the same animals retinofugal fibers were labelled by autora- diographic methods. In the region between n. isthmi and the optic chiasm the contralaterally projecting isthmotectal fi-

notectal tract. Scale 250 pm. b: Stained cells in posterior thalamic neu- ropil (ptn). Tectum is in the upper left. Stained fibers above labeled cells are part of medial retinotectal tract. Scale 250 pm. c: Cells in anterodor- sal tegmental nucleus (ad). Scale 250 pm. d Cells in nucleus profundus mesencephali (pr). Scale 150 Nm.

bers course along the lateral margin of the mesencephalon. The fibers are ventral to the lateral retinotectal tract. Through most of the diencephalon the fibers remain at the lateral margin segregated from the principal part of the optic tract, which is more dorsal, and the accessory optic tract, which is more ventral. Thus, in this region there is a spatial separation of retinofugal and isthmotectal fibers. The isthmotectal fibers cross in the dorsal posterior optic chiasm where they are mixed with crossing retinofugal

ISTHMOTECTAL AND OTHER TECTOPETAL FIBERS 46

Fig. 4. Dorsal view of tectal lobe showing locations of centers of HRP injection sites corresponding to brains of Table 3. Rostra1 (R) is up and medial (M) is to the left.

fibers. In this region autoradiographic counts overlay the HRP-stained fibers. After the isthmotectal fibers decussate they continue to be admixed with retinofugal fibers (Fig. 8). How finely mixed these two fiber systems are (fiber by fiber or fascicle by fascicle) must await ultrastructural studies.

Other fiber systems in the posterior optic chiasm?

In earlier experiments we had made midline cuts immedi- ately caudal to the optic chiasm. We then applied HRP to the tectum unilaterally and found that we could still backfill the contralateral n. isthmi. Thus, such cuts did not interrupt contralaterally projecting isthmotectal fibers. Cuts to the posterior optic chiasm did interrupt this projection since we could no longer backfill contralateral n. isthmi after tectal HRP application. In the current experiments we cut the posterior part of the optic chiasm and applied HRP crystals to the cut (Fig. 9a). Only retinal fibers and n. isthmi fibers and cells are stained (Fig. 9b). Thus, the only nonretinal fibers within the posterior part of the optic chiasm are isth- motectal fibers. Inserting an HRP crystal into the severed anterior part of the chiasm results in the staining of only ret- inal fibers and a handful of n. isthmi cells. Applying HRP to a sagittal cut of the postoptic commissure immediately cau- dal of the optic chiasm results in the staining of diencephalic structures only.

DISCUSSION Previous work has shown that both retina (Scalia, '73)

and contralateral n. isthmi (Gruberg and Udin, '78) project to the most superficial layer of the tectum. Currently, when

Fig. 5. HRP staining after two methods of administration. Ninety- minute interval between administration and perfusion. a: HRP applica- tion site using bibulous paper soaked in HRPhysolecithin. Note the most superficial tectal layers are more heavily stained. There is also staining of ependymal cells whose cell bodies are in the periventricular layer and whose processes extend to the dorsal surface. Scattered neu- rons are also stained. Scale 200 pm. b: Midbrain section of animal into whose left tectal lobe an HRP crystal had been inserted. Note extensive spread of HRP to contralateral tectum and to underlying tegmentum. The effective zone of uptake of HRP is much smaller than the virtual zone shown in this figure. Scale 500 pm.

Fig. 6. Orthograde filling of HRP-stained fibers in the tectum after HRP injection into the posterior thalamus. Both superficial and deep layers show staining hut layer A (above dashed line) is relatively free of stained fibers. Medial is to the left. Scale 100 pm.

46 E.R. GRUBERG ET AL.

d on

Fig. 7. Camera lucida drawings of transverse sections of a brain demonstrating the relationship between retinal fibers and contralater- ally projecting n. isthmi fibers. An HRP crystal had been inserted into n. isthmi unilaterally. Both eyes had been injected with tritiated 1-proline. Section a, at level of n. isthmi (ni), is most caudal. Section g, at level of anterior optic chiasm (ch), is most rostra1 and shows proximal parts of

optic nerves (on); te is tectum; tel is telencephalon. Small dots represent distribution of autoradiographic counts. Large dots represent distribu- tion of HRP. Bold lines and solid black areas represent loci where there is both HRP staining and autoradiographic counts. For clarity tectal HRP staining is not shown.

ISTHMOTECTAL AND OTHER TECTOPETAL FIBERS 47

we applied HRP to the tectal surface we found in half our cases that over 90% of the nonretinal stained cells of the brain were in contralateral n. isthmi. This suggests that retina and contralaterally projecting n. isthmi are the only two fiber systems significantly represented in the most superficial tectal layer. Our results are corroborated, in part, by our staining of thalamotectal fibers which are absent from the outermost tectal layer. The intimate relationship between retinal fibers and contralaterally projecting n. isthmi fibers begins at the optic chiasm. Our double label experiments reveal that the contralaterally projecting isth- motectal fibers are admixed with retinal fibers from the chiasm to the tectum. Further, no other fiber system is pres- ent in the chiasm.

In most of the other cases of HRP paper application to the tectal surface significant numbers of stained cells are seen in tegmental and posterior thalamic loci in addition to contra- lateral n. isthmi. However, there are few stained cells in ipsi- lateral n. isthmi. These results suggest that there is a zone between the outermost tectal layer and the bulk of ipsilater- ally projecting n. isthmi fibers. Within this zone both teg- mental and posterior thalamic inputs terminate. This is in accord with an earlier immunocytochemical study which used a polyclonal antibody to choline acetyltransferase to show that isthmotectal fibers were the only cholinergic inputs to the tectum (Desan et al., '87). The superficial tec- tum contained two bands of immunoreactive fibers sepa- rated by a 50 pm zone of little staining: the more superficial band immediately below the pial surface was densely stained; the deeper band of diffusely stained fibers extended through much of the remaining superficial tectum. When n. isthmi was ablated unilaterally the deeper band disap- peared ipsilaterally and the more superficial band disap- peared contralaterally. Thus, the immunoreactive tectal fi- bers are likely to be of n. isthmi origin: the superficial band a projection of the contralateral n. isthmi and the deeper band a projection of the ipsilateral n. isthmi.

Our tectal HRP studies indicate that a majority of the nonretinal'neurons that project to the tectum are located in n. isthmi. The nucleus isthmi and its mammalian homo- logue the parabigeminal nucleus have significant ipsilateral reciprocal connections with the tectum in a variety of verte- brates including fish (filefish: Sakamoto et al., '81; carp: Luiten, '81; longnose gar: Northcutt, '82), amphibia (leopard frog: Gruberg and Udin, '78; clawed toad: Udin and Keating, '81; European fire salamander, Rettig, '88), reptiles (iguana: Foster and Hall, '75; lacerta: Wang et al., '83; garter snake: Dacey and Ulinski, '86; turtle: Kunzle and Schnyder, '84; Sereno and Ulinski, '87), birds (pigeon: Hunt and Kunzle, '76; Hunt et al., '77; Brecha, '78), and mammals (rat: Kunzle and Schnyder, '84; Linden and Perry, '83; Watanabe and Kawana, '79; hamster: Jen et al., '84; cat: Graybiel, '78; Bal- eydier and Magnin, '79; Sherk, '79; Roldan et al., '83; opos- sum: Mendez-Otero et al., '80; tree shrew: Hashikawa et al., '86). Contralateral isthmotectal projections are commonly found in amphibia and mammals but so far have been rarely described in other vertebrate classes. One species of fish, the weakly electric Apteronotus leptorhynchus, has been shown to have such a projection (Sas and Maler, '86). Among reptiles the isthmotectal projection is bilateral in the python (Welker et al., '83). In turtles the parvicellular region of n. isthmi projects bilaterally to tectal layers that do not receive retinal input (Kunzle and Schnyder, '84). How- ever, since the parvicellular division of n. isthmi receives vir- tually no direct input from the tectum, it is not likely to be homologous to the frog n. isthmi.

Fig. 8. Transverse section of lateral thalamus showing admixture of retinofugal fibers and contralaterally projecting n. isthmi fibers (boxed area of section d of camera lucida drawing of Fig. 7). Retinal fibers are labelled with tritiated 1-proline (dark grains). Isthmotectal fibers are labelled with HRP. Double exposure taken at two depths (since grains are above the section). Note admixture of retinofugal fibers and isthmo- tectal fascicles in region within arrowheads. Scale 100 pm.

Fig. 9. Insertion of HRP crystal into posterior part of optic chiasm leading to retrogradely stained cells of n. isthmi. a: Ventral diencepha- lon at the level of the posterior part of the optic chiasm. This area of the chiasm has been transected and an HRP crystal inserted. Three-day survival. Scale 500 pm. b: Nucleus isthmi from same brain as a. N. isthmi contains the only stained cells outside the retina. Scale 200 pm.

48

It appears to be a general rule that isthmotectal/parabi- geminocollicular fibers terminate in or adjacent to retinore- cipient layers. In leopard frogs the ipsilaterally projecting isthmotectal fibers terminate in several of the superficial tectal layers which receive retinal input (Gruberg and Udin, ’78), although probably not to any great extent in the most superficial layers. The frog’s contralaterally projecting isth- motectal fibers project to the most superficial tectal layer (which is retinorecipient) and a deeper superficial layer (layer 8) which is adjacent to a retinorecipient layer (Gru- berg and Udin, ’78). In the garter snake ipsilaterally pro- jecting isthmotectal fibers terminate in retinorecipient tec- tal layers which appear to contain no other extrinsic input (Dacey and Ulinski, ’86). In pond turtles the magnocellular component of n. isthmi projects primarily to the ipsilateral retinorecipient tectal layers (Sereno and Ulinski, ’87).

In general, mammals have visual projections to the super- ficial layers of the superior colliculus while nonvisual projec- tions are deeper. For the most part the frog tectum is remi- niscent of the superficial part of the mammalian superior colliculus. The most superficial layer of the superior collicu- lus is lamina I; the deepest is lamina VII. Retinal fibers gen- erally end in laminae 1-111 (see Huerta and Harting, ’84, for review). In the cat, ipsilaterally projecting parabigeminocol- licular fibers project to a wide band of retinorecipient layers while the contralateral parabigeminal fibers project only to the superficial part of lamina I1 “closely mimicking the con- tralateral retinotectal projection” (Graybiel, ’78). Roldan et al. (’83) showed that the cat parabigeminal nucleus is the only structure in the mesencephalon and rhombencephalon which is labelled when HRP injections are placed in collicu- lar laminae I and 11. The termination of the collicular pro- jection from the parabigeminal nucleus is superficial com- pared to geniculocollicular projections which end in some of the deeper retinorecipient layers. Visual projections from the cerebral cortex of some mammalian species also termi- nate superficially. However, in rodents and the opossum they tend to be below the parabigeminal projection (Huerta and Harting, ’84). In frogs there appears to be no direct tec- tal projection from the forebrain.

In our study we found thalamic inputs to the tectum only from posterior areas of the thalamus. In a degeneration study, Trachtenberg and Ingle (’74) found that electrolytic lesions in either the anterior or posterior dorsal thalamus of Rana pipiens resulted in degenerating axons throughout the deep and superficial tectum. Subsequent studies have not confirmed a tectal projection from anterior dorsal thala- mus. Perhaps some of the degenerating fibers they found in the superficial tectum after anterior thalamic lesions could have their origin in the contralateral nucleus isthmi. Such fibers pass through the thalamus and could be damaged by diencephalic lesions. Recently Lazar (personal communica- tion) has injected cobalthysine into the posterior thalamus and found staining of tectopetal fibers in superficial and deeper tectal layers (layer 6 and below). Kuljis and Karten (’83) have found peptide-like immunoreactivity in the su- perficial tectum which could be of pretectal origin (Karten, personal communication).

In the leopard frog the projection from eye to tectum is almost entirely crossed. However, by electrical recording it is easy to find units in the tectum that respond to visual stimulation of the ipsilateral eye. Keating and Gaze (’70) were the first to describe (in part) the pathway by which information goes from the retina to the ipsilateral tectum. They discovered that the pathway is from retina to the con-

E.R. GRUBERG ET AL.

tralateral tectum, then to an intermediate structure (which we now know is n. isthmi), then via the postoptic commis- sure to the ipsilateral tectum. They made lesions “aimed at the caudo-dorsal part of the optic chiasma” in order to “in- terrupt the components of the post-optic commissural sys- tem.” When they subsequently recorded in the tectum they could no longer find units that responded to visual stimula- tion of the ipsilateral eye. They showed two transverse brain sections from such an animal, one section “revealing the lesion of the post-optic commissures, the other showing that the optic chiasma itself was spared.” Gruberg and Udin (’78) also described the contralaterally projecting isthmotectal fibers decussating in the postoptic commissure/supraoptic decussation. Based on our present results (double label cases and cases with HRP inserted into the cut optic chiasm), we suggest that the isthmotectal fibers are in fact decussating in the dorsocaudal part of the optic chiasm and not in the postoptic commissure. After crystals of HRP were applied to the cut postoptic commissure we backfilled cells of several diencephalic areas but not nucleus isthmi.

All other studies of the crossing of contralaterally pro- jecting isthmotectal and parabigeminocollicular fibers have used single label techniques. Rettig (’88) injected HRP into the postoptic commissure in two salamander species and found labelled neurons bilaterally in nucleus isthmi. In addition, McCart and Straznicky (’88) state that the contra- lateral isthmotectal projection crosses in the postoptic com- missure in Xenopus. Graybiel(’78) mentions that in the cat parabigeminal fibers decussate in Gudden’s commissure, a part of the supraoptic decussations (Crosby et al., ’62). How- ever, Hashikawa et al. (’86) describe parabigeminal fibers in the tree shrew crossing in the caudal portion of the optic chiasm.

We could not confirm the presence of several tectal inputs described previously (Wilczynski and Northcutt, ’77). We cannot easily account for the discrepancy. One possibility is that these other inputs project to the deepest tectal layers and in our methods of administration the HRP did not effectively penetrate to the deepest layers of the tectum. It is difficult to determine the true uptake zone for the HRP. We were surprised that after tectal application of paper per- meated with HRP and lysolecithin all tectal layers stained densely yet, as we described above, in half our cases virtually the only extrinsic stained structures were contralateral retina and contralateral n. isthmi. We assume that most of the deeper staining was due to intracellular uptake of the HRP by ependymoglia. These cells take up HRP as part of the mechanism for clearing the enzyme. Mesulam (’82) has discussed the distinction between “virtual” and “effective” injection sites. The virtual injection site of HRP corre- sponds to the area of “dense and uniform deposits of reac- tion product throughout the neuropil at the time of micro- scopic examination.” The effective injection site is “the volume of tissue which has sustained the uptake and subse- quent transport of the tracer.” Vanegas et al. (’78) showed that between 10 minutes and 2 hours after injection into the visual cortex of cats the primary distribution of HRP corre- sponds to the effective injection site. Between 2 and 18 hours there is a “dramatic” increase in the virtual injection site. This is followed by a gradual contraction of the virtual site over several days. In our study when HRPhysolecithin- soaked paper was applied to the tectal surface and the ani- mal was maintained for only 90 minutes before perfusion, the most superficial layers are more densely stained than deeper layers (Fig. 5a). With this short survival time there is

ISTHMOTECTAL AND OTHER TECTOPETAL FIBERS

a much closer correspondence between the virtual injection site and the effective injection site than after several days survival. However, when an HRP crystal is applied to the tectum and the animal is maintained for 90 minutes before perfusion much of the outer midbrain is stained (Fig. 5b). In this case even after a relatively short survival time the vir- tual injection site is enormous compared to the probable effective injection site. Thus, when HRP crystals are ap- plied to the tectal lobes the virtual injection site probably better corresponds to the effective injection site after sev- eral days of survival. Such disparate results for paper and crystal insertion indicate the difficulty in determining the effective size of the administration site. Observation after short survival time (less than 2 hours) is insufficient to assure reliable determination of the effective administration site.

There is still much that is unknown about the functional roles for the various tectal inputs. The behavioral conse- quences of ablating n. isthmi are distinct from the conse- quences of ablating the posterior thalamus (which contains three tectopetal structures). After unilateral ablation of n. isthmi the frog has a scotoma to prey and threat stimuli in the contralateral monocular visual field. After ablation of posterior thalamus the frog responds to prey and threat stimuli everywhere. However, the frog is “disinhibited”; it attacks threat stimuli with the same vigor that it attacks prey stimuli (Ewert, ’70; Ingle, ’73). Because posterior thal- amic and tegmental inputs to the tectum originate from a number of areas, sorting out their individual contributions to influencing tectal function remains a difficult task.

There is evidence to suggest that n. isthmi may directly affect retinal input. Earlier biochemical (Ricciuti and Gru- berg, ’85) and immunocytochemical (Desan et al., ’87) stud- ies have shown that n. isthmi is the only significant source of cholinergic input to the tectum. Henley et al. (’86) have found that goldfish retinal ganglion cells synthesize acetyl- choline receptors and transport them to the optic tectum. Sargent (personal communication) has found similar results in R. pipiens. These results imply that at least some retino- tectal fibers could be cholinoceptive. Cholinergic n. isthmi fibers could then synapse onto these retinal fibers. However, ultrastructural observations of frog tectum do not reveal synapses on to retinal elements (Szekely et al., ’73). Retino- tectal terminals do make serial synapses with elements (type 3 of Szekely et al., ’73) interposed between retinal ter- minals and tectal dendrites. This intermediate is possibly an isthmotectal terminal. However, using morphological crite- ria, Szekely et al. suggest that type 3 terminals are more likely to be dendritic appendages. The ultrastructural locus of isthmotectal terminals is still obscure.

In summary, we conclude that the predominant extrinsic inputs to the superficial layers of the frog tectum are from retina and n. isthmi. Contralaterally projecting isthmotectal fibers cross in the posterior part of the optic chiasm and no other nonretinal fiber systems are present there. These con- tralaterally projecting fibers are mixed with retinal fibers along their subsequent pathway to the tectum.

49

ACKNOWLEDGMENTS We thank William Harris for advice about HRP adminis-

tration and Mark Hulsebosch and Dagmar Skee for techni- cal help. This work was supported by NIH grant EY 04366.

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