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) histochemistry, 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 contralateral 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 isthmotectal fibers terminate in the deeper superficial layers. 2) We investigated
the relationship between retinofugal and contralaterally projecting isthmotectal pathways. We orthogradely labelled n. isthmi fibers by unilateral HRP
injections into n. isthmi, and we also labelled retinal fibers by injecting tritiated 1-proline into both eyes. In such animals contralaterally projecting isthmotectal 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 contralaterally 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 retrogradely 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 superficial layers of the frog tectum (Gruberg and Udin, '78). The
nucleus is located in the posterior mesencephalic tegmentum. 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 presented prey and threat stimuli in the contralateral monocular 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
0 1989 ALAN R. LISS, INC.
and mesencephalic tegmental fields. It was not determined
whether these inputs terminated in deep or superficial tectum. 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.
40
E.R. GRUBERG ET AL.
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 contralaterally 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 retinofugal 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 compared the distribution of the two labels.
It is not known if other systems cross with contralaterally
projecting isthmotectal fibers. We have cut the crossed projection 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 summary of this work has been published (Gruberg et al., '87).
MATERIALS AND METHODS
Administration of HRP to the tectum
Leopard frogs, Rana pipiens (obtained from Hazen, Alburg, 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 tectum 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.
Method 1. Insertion of HRP crystal. 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 crystal. The surface of the tectum was punctured and the crystal
was inserted and held in place until it dissolved.
Method 2. Application on paper. 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 minutes. 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.
Method 3. Administration b y injection. 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 injection 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 (electrode 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 reanesthetized and perfused intracardially with a saline solution and
then with a pH 7.3 phosphate-buffered fixative (0.5% para-
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 micropipette 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 histochemistry 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 curarized. The nucleus isthmi was located by electrophysiological 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 pressure injected into n. isthmi. In other animals, under anesthesia, the posterior pole of the tectum was aspirated unilaterally, 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 maintained 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 sections 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 incision 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,
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 application 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
Figure 1
41
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 crystals (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.
TABLE 1. Distribution of Stained Cells After HRP
Crystal Insertion Into Tectum
% of total stained cells
Brain
1C
2C
3C
4C
5C
Tectal
location
Rostra1
Midtectum
Midtectum
Midteeturn
Caudal
Total no. N. isthmi N. isthmi Posterior Tegmental
of cells
contra
ipsi
thalamus
nuclei
323
390
388
529
224
52
37
26
35
16
33
41
43
28
13
20
10
3
17
13
12
fi
63
1R
1.7
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 tectum that are retrogradely stained: 1) retina, contralaterally;
2) nucleus isthmi, bilaterally; 3) posterior thalamus, primarily ipsilateral; and 4) mesencephalic tegmentum, primarily ipsilateral (Fig. 1).Within the posterior thalamus the
structures that contain stained cells (followingthe anatomical 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 nomenclature 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 sections are relatively thick compared to the average diameter
of the cells we use the raw counts in our table. Approximately 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 contralatera1 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 application 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 approximately 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).
Application of HRP by bibulous paper. 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 uniform distribution of HRP through the tectum, the proportion of stained cells in different brain regions was quite different from the proportion of stained cells in cases with
HRP crystal insertion. We have counted the total number of
TABLE 2. Distribution of Stained Cells After Paper Application of HRP to
Tectal Surface
% of total stained cells
Brain
1A
2A
3A
4A
5A
6A
7A
8A
9A
10A
11A
12A
13A
14A
Total no
of cells
N. isthmi
contra
N. isthrni
ipsi
Posterior
thalamus
Tegmental
nuclei
283
94
618
308
207
406
211
212
1,021
623
652
1,088
899
620
100
0
0
3
3
1
2
5
1
1
0
2
13
6
40
0
0
0
0
0
4
0
4
12
44
38
43
59
7
0
0
0
4
7
3
4
17
10
100
96
92
92
91
91
77
77
41
37
30
19
51
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 extending 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 posterior 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, implying 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.
Administration by idection. 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 contralaterally (Table 3). The locations of the injection sites are shown
in Figure 4. While there were significant differences be-
ISTHMOTECTAL AND OTHER TECTOPETAL FIBERS
43
TABLE 3. Distribution of Stained Cells After Injection of HRP into
Tectum
% of total stained cells
Brain
1J
25
35
41
5J
61
75
8J
9J
1OJ
llJ
121
135
141
155
16J
175
185
19J
Total no.
of cells
N. isthmi
contra
N. isthmi
ipsi
Posterior
thalamus
Tegmental
nuclei
45
64
573
231
39
165
1,077
22
43
256
70
168
56
27
545
442
255
198
283
9
50
16
31
56
30
54
9
33
37
47
48
34
7
32
26
11
24
15
91
45
79
62
28
53
29
68
32
27
13
9
23
44
19
24
33
14
7
0
0
1
6
8
15
15
23
34
25
39
38
34
33
34
23
15
7
63
0
5
4
1
8
2
2
0
0
11
1
5
9
15
15
26
40
55
14
tween individual cases, on the average there was no obvious
difference in proportions of stained cells in the four principal areas stained by using pressure injection or iontophoresis. 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-termsurvival
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. It 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 perfused 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 minutes, dark staining was seen in much of the outer mesencephalon including the tegmentum (Fig. 5b). With a similar
size HRP crystal and 4-day survival only a rather circumscribed 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 injection 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 darkstained 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. Orthogradelylabelled tectoisthma1 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-
notectal tract. Scale 250 pm. b: Stained cells in posterior thalamic neuropil (ptn).Tectum is in the upper left. Stained fibers above labeled cells
are part of medial retinotectal tract. Scale 250 pm. c: Cells in anterodorsal tegmental nucleus (ad). Scale 250 pm. d Cells in nucleus profundus
mesencephali (pr). Scale 150 Nm.
Double labelling: isthmotectal fibers
and retinofugal fibers
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
We followed contralaterally projecting fibers orthogradely stained with HRP from n. isthmi to their decussation and then back to the contralateral tectum (Fig. 7 ) . In
the same animals retinofugal fibers were labelled by autoradiographic methods. In the region between n. isthmi and the
optic chiasm the contralaterally projecting isthmotectal fi-
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 immediately 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 isthmotectal fibers. Inserting an HRP crystal into the severed
anterior part of the chiasm results in the staining of only retinal fibers and a handful of n. isthmi cells. Applying HRP to
a sagittal cut of the postoptic commissure immediately caudal of the optic chiasm results in the staining of diencephalic
structures only.
Fig. 5. HRP staining after two methods of administration. Ninetyminute interval between administration and perfusion. a: HRP application 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 neurons 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.
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. 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 contralaterally 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 distribution 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
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 isthmotectal 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 diencephalon 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.
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 isthmotectal fibers are admixed with retinal fibers from the
chiasm to the tectum. Further, no other fiber system is present 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 contralateral n. isthmi. However, there are few stained cells in ipsilateral n. isthmi. These results suggest that there is a zone
between the outermost tectal layer and the bulk of ipsilaterally projecting n. isthmi fibers. Within this zone both tegmental 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 tectum contained two bands of immunoreactive fibers separated 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 disappeared ipsilaterally and the more superficial band disappeared contralaterally. Thus, the immunoreactive tectal fibers 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 homologue the parabigeminal nucleus have significant ipsilateral
reciprocal connections with the tectum in a variety of vertebrates 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; Baleydier and Magnin, '79; Sherk, '79; Roldan et al., '83; opossum: 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). However, since the parvicellular division of n. isthmi receives virtually no direct input from the tectum, it is not likely to be
homologous to the frog n. isthmi.
48
It appears to be a general rule that isthmotectal/parabigeminocollicular fibers terminate in or adjacent to retinorecipient 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 isthmotectal 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 (Gruberg and Udin, ’78). In the garter snake ipsilaterally projecting isthmotectal fibers terminate in retinorecipient tectal 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 superficial layers of the superior colliculus while nonvisual projections are deeper. For the most part the frog tectum is reminiscent of the superficial part of the mammalian superior
colliculus. The most superficial layer of the superior colliculus is lamina I; the deepest is lamina VII. Retinal fibers generally end in laminae 1-111 (see Huerta and Harting, ’84, for
review). In the cat, ipsilaterally projecting parabigeminocollicular 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 contralateral 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 collicular laminae I and 11. The termination of the collicular projection from the parabigeminal nucleus is superficial compared to geniculocollicular projections which end in some of
the deeper retinorecipient layers. Visual projections from
the cerebral cortex of some mammalian species also terminate 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 tectal 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 thalamus. 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 communication) 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 superficial 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 commissure to the ipsilateral tectum. They made lesions “aimed at
the caudo-dorsal part of the optic chiasma” in order to “interrupt the components of the post-optic commissural system.” When they subsequently recorded in the tectum they
could no longer find units that responded to visual stimulation 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 projecting 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 contralateral isthmotectal projection crosses in the postoptic commissure 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). However, 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 permeated 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 corresponds to the area of “dense and uniform deposits of reaction product throughout the neuropil at the time of microscopic examination.” The effective injection site is “the
volume of tissue which has sustained the uptake and subsequent 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 corresponds 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 HRPhysolecithinsoaked paper was applied to the tectal surface and the animal 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
49
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 virtual injection site is enormous compared to the probable
effective injection site. Thus, when HRP crystals are applied to the tectal lobes the virtual injection site probably
better corresponds to the effective injection site after several 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 consequences of ablating n. isthmi are distinct from the consequences 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 thalamic 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 Gruberg, ’85) and immunocytochemical (Desan et al., ’87) studies 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 acetylcholine 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 retinotectal 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). Retinotectal terminals do make serial synapses with elements
(type 3 of Szekely et al., ’73) interposed between retinal terminals and tectal dendrites. This intermediate is possibly an
isthmotectal terminal. However, using morphological criteria, 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 contralaterally projecting fibers are mixed with retinal fibers
along their subsequent pathway to the tectum.
ACKNOWLEDGMENTS
We thank William Harris for advice about HRP administration and Mark Hulsebosch and Dagmar Skee for technical help. This work was supported by NIH grant EY 04366.
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