Development 116, 507-519 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
507
Membrane-bound molecules in rat cerebral cortex regulate thalamic
innervation
MAGDALENA GÖTZ, NINO NOVAK, MARTIN BASTMEYER and JÜRGEN BOLZ*
Friedrich-Miescher-Labor der Max-Planck-Gesellschaft, Spemannstrasse 37-39, 7400 Tübingen, Germany
*Author for correspondence
Summary
During development of the thalamocortical projection,
afferent fibers from the thalamus reach the cortex at a
time when their target cells have just been generated
but have not yet migrated to their final position. Thalamic axons begin to invade the cortex only shortly
before their target layer 4 is formed. The mechanisms
responsible for the innervation and termination of thalamic fibers in the cortex are not known. Here we show
that the growth of thalamic axons in vitro is influenced
by the age of cortical explants. Cortical explants of early
embryonic stages were not invaded by thalamic
explants, whereas thalamic fibers entered explants from
postnatal cortices and terminated properly in their
target layer 4 in vitro. Outgrowth assays on cortical cell
membranes prepared at different developmental stages
revealed that the growth of thalamic axons is selectively
influenced by growth-promoting molecules that are upregulated during development. Moreover, experiments
with postnatal cortical membranes isolated from distinct
layers revealed that the growth of thalamic axons is
selectively reduced on membranes prepared from layer
4. These results provide evidence that membrane-bound
molecules in the cortex are involved in both the regulation of thalamic innervation into the cortical layers and
their termination in the correct target layer.
Introduction
(Berry and Eayrs, 1963; Berry and Rogers, 1965; Hicks and
D’Amato, 1968; Rakic, 1972; Bayer et al., 1991). During
phylogenesis, the duration of cortical neuroblast migration
increases and there is a corresponding increase in the time
delay between the arrival of thalamic fibers and the formation of their target layer: thalamic fibers “pause” the
longest, for about two months, in the developing human
cortex (Bayer and Altman, 1991; Luskin and Shatz, 1985;
Rakic, 1990, Kostovic and Rakic, 1990). The mechanisms
that keep thalamic fibers out of the cortical plate during
early development and that trigger the invasion of cortical
layers at the appropriate time are not known.
Along with mechanisms that regulate the timing of afferent cortical innervation, information must also be available
for thalamic fibers to allow them to recognize their correct
target layer. It has been suggested that interactions with several cell types in the cortex are involved in the process of
target cell selection. For example, interactions between
growing thalamic axons and migrating layer 4 cells could
occur in the subplate zone (Bayer and Altman, 1991). In
addition, there is evidence that thalamic fibers make transient synaptic contacts with subplate cells (Shatz et al.,
1988; Friauf et al., 1990; Herrmann et al., 1991) and these
contacts might be essential for the successful recognition
of the later target cells in layer 4 (Shatz et al., 1988; Ghosh
et al., 1990).
In order to get some insights into guidance cues that
The establishment of specific connections between neuronal
populations and their distant target areas involves several
steps. Growing axons have to find the correct pathways in
order to reach the appropriate region of the brain and, once
they arrive in their target area, they must then navigate and
select the correct subsets of target cells. A striking feature
in the development of the neocortex is that afferent fibers
reach the cortex before most cortical neurons are born or
cortical layers have been established (Levitt and Moore,
1979; Schlumpf et al., 1980; Lund and Mustari, 1977;
Rakic, 1977; Shatz and Luskin, 1986; Uylings et al., 1990).
In several mammalian species, it has been shown that fibers
from the thalamus, the major afferent input to the cortex,
arrive prior to the generation of their cortical target neurons in layer 4 (Lund and Mustari, 1977; Luskin and Shatz,
1985; Shatz and Luskin, 1986; Rakic, 1977). Thalamic
axons are first confined to the subplate zone beneath the
developing cortical layers and enter the cortical grey matter
only after layer 4 cells have migrated to their final position
(Shatz and Luskin, 1986).
In the developing cortex, cells destined for one layer are
generated at about the same time in the ventricular zone
and then migrate towards the pia. Cortical layers are formed
in an inside-out fashion, with cells in the deep cortical
layers produced before the cells in the superficial layers
Key words: cerebral cortex, cortex development, axonal
pathfinding, slice cultures, rat, thalamic innervation.
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M. Götz and others
might influence the growth of thalamic axons, we studied
the interactions of thalamic fibers with cortical tissue using
different in vitro approaches. The establishment of thalamocortical connections was examined in co-cultures of slices
from rat thalamus and cortex prepared at different developmental stages. We found that the formation of thalamocortical connections in vitro depended crucially on the age
of the cortical, but not on the age of the thalamic explants.
Thalamic fibers of all ages tested did not invade cortical
explants prepared early during development, around embryonic day 16 (E16). However, when cortical explants were
prepared a few days later, thalamic fibers readily invaded
the cortical explants and in cortical slices from postnatal
cortex they terminated in their appropriate target layer in
vitro. Using a quantitative growth assay, we found that
membrane-bound molecules specifically promoting the
growth of thalamic fibers were upregulated in the developing cortex, in parallel to its innervation by thalamic fibers
in vivo and in cortical slice cultures. The invasion of thalamic fibers into cortical layers in vivo and in slice cultures
closely correlated with the distribution of lectin-binding
molecules suggesting that membrane-bound glycosylated
proteins in the cortex are responsible for its innervation by
thalamic fibers. In addition, the growth rate of thalamic
fibers was specifically reduced on membranes from their
isolated target layer. These results suggest that membranebound molecules in the cortex are involved in the innervation and termination of afferents from the thalamus.
buffer (PB) and sections were cut on a vibratome at 100-200 µm.
In thalamocortical co-cultures, small crystals of DiI were inserted
into the thalamic explant after 4-23 days in vitro and then kept in
PFA for several weeks. The trajectories of DiI-labeled fibers from
the thalamus in vivo and in co-cultures were examined with a fluorescence microscope, photographed and drawn with the aid of a
drawing apparatus.
BrdU-birthdating
In order to label cells at their birthday, timed pregnant rats were
injected intraperitoneally with 250 mg bromodeoxyuridine (BrdU,
Serva) per kg body weight. The brains were removed, immediately frozen on dry ice and cut on a cryostat at a thickness of 10
µm. Sections were fixed for 10 minutes in ice-cold methanol and
washed several times in PB. Cortical slice cultures were prepared
from embryonic or postnatal animals of BrdU-injected timed-pregnant rats, and fixed after 2 or more days in vitro (DIV) with 4%
paraformaldehyde in PB for 30 minutes. After several washings
in the same buffer, fixed cultures and sections were incubated in
2N HCl for 30 minutes, followed by incubation in borate buffer,
pH 8.5, twice for 15 minutes. BrdU-antiserum (Bioscience) was
used at a 1:10 dilution in PB at 4°C overnight. After several
washes, sections and cultures were incubated with a biotinylated
secondary antibody solution (1:250) for 30 minutes at 4°C and
further processed using avidin-biotin-peroxidase (Vectastain Kit),
which was visualized by an enhanced DAB-reaction. Cultures and
sections then were dehydrated in alcohol, cleared in xylene and
embedded in Entellan. The location of BrdU-cells was drawn
using a drawing apparatus on a microscope equipped with
Nomarksi optics.
Lectin-histochemistry
Materials and methods
Preparation of slice cultures
Slice cultures were prepared using a roller culture technique (Gähwiler, 1981) as described in Caeser et al. (1989). Briefly, small
blocks of cortical or diencephalic tissue were isolated in Gey’s
balanced salt solution supplemented with glucose (6.5 mg/ml) and
cut with a McIlwain tissue chopper to a thickness of 200-300 µm.
The slices were stored for 30-45 minutes at 4°C in Gey’s balanced salt solution supplemented with glucose. The slices were
then placed on a glass coverslip and embedded in 20 µl chicken
plasma coagulated with 20 µl thrombin (0.2 mg/ml, 20 NIHU/ml
Hoffmann-La Roche). Slice cultures were maintained in 0.75 ml
medium consisting of 50% Eagle’s basal medium, 25% Hank’s
balanced salt solution and 25% horse serum, containing 0.1 mM
glutamine and 6.5 mg ml -1 glucose (all from Gibco) at 36°C under
continuous rotation.
DiI-tracing
The lipophilic tracers DiI and DiA were used to study thalamocortical projections in situ and in co-cultures (Godement et al.,
1987). Embryos were obtained by Cesarian section from deeply
anesthetized (400 mg/kg chloral hydrate), timed-pregnant rats
(Lewis, day of sperm detection = E1). After decapitation of the
embryos, brains were removed and stored in 4% paraformaldehyde in 0.1 M phosphate buffer (PFA), pH 7.4. Rat pups were
deeply anaesthetized with ether and transcardially perfused with
physiological saline followed by PFA. The brains were removed
and stored in the same fixative. Small crystals of DiI were inserted
into the thalamic region with a glass electrode. Brains were kept
in fixative at room temperature for up to several months to allow
diffusion of the lipophilic dye within the membranes. The brains
were then embedded in a 2% agar solution in 0.1 M phosphate
Cryostat sections were obtained as described above and were fixed
for 10 minutes (slice cultures for 30 minutes) in 2% glutaraldehyde and 2% paraformaldehyde in 0.15 M sodium-cacodylate
buffer, pH 7.3, followed by extensive washing in 1% (w/v) bovine
serum albumin (BSA) in PBS with 1 mM CaCl2 and 1 mM MnCl2,
pH 7.4, for several hours. Sections and cultures were then incubated overnight at 4°C in the same buffer containing either 50
µg/ml peanut lectin from Arachis hypogaea (Sigma), Jacalin from
Artocarpus integrifolia (Medac), Concanavalin A from Canavalia
ensiformis (Vektor), or the lectin from Erythrina cristagallis
(Medac). All lectins were biotinylated and further processed using
the avidin-biotin technique (Vectastain) and an enhanced DABreaction. Controls were incubated in the same lectin-solution containing the appropriate sugar concentration, e.g. 20 mM galactose
and 50 µg/ml PNA.
Membrane preparation
Blocks of cortex or in some control experiments from the hindbrain, were dissected in Gey’s-balanced-salt-solution supplemented with glucose (6.5 mg/ml), and the pia was removed. Slices
were cut with a McIlwain tissue chopper and pooled in the homogenization buffer consisting of 10 mM Tris-HCl, 1.5 mM CaCl2,
1 mM spermidine, 25 µg/ml aprotinine, 25 µg/ml leupeptine, 5
µg/ml pepstatine (all from Serva) and 15 µg/ml 2,3-dehydro-2desoxy-N-acetylneuraminic acid (Sigma), pH 7.4. For some experiments, layer 4 was isolated from each individual cortex slice by
two cuts made parallel to the pial surface. Both isolated layer 4
slices, and the ventral part consisting of white matter, layer 6 and
5, were then collected separately in homogenization buffer. The
homogenate was centrifuged for 10 minutes at 25,000 revs/minute
in a sucrose step gradient (upper phase 150 µl of 5% sucrose;
lower phase 350 µl of 50% sucrose) in a Beckman TLS 55 rotor.
The interband containing the membrane fraction was washed twice
in PBS without Ca 2+ and Mg 2+ at 14000 revs/minute in an Eppen -
Development of thalamocortical connections
dorf Biofuge and, after resuspension, the concentration of the purified membranes was determined either by its optical density at
220 nm with a spectrophotometer or according to the method of
Bradford (1976). For the enzyme treatment, the same amount of
cortical membrane was incubated in PBS containing 0.3% trypsin
(Serva) or, as a control, without trypsin for 45 minutes at 37°C.
The reaction was stopped by the addition of medium containing
25% serum and the membranes were washed several times with
PBS at 14,000 revs/minute in an Eppendorf centrifuge at 4°C.
After the concentration was determined again, equal amounts of
enzyme-treated and control membranes were used in the growth
assays. In these cell membrane preparations, molecules from the
extracellular matrix may co-purify. Therefore the term ‘membrane-bound’ molecules refers to the method of preparation and
does not necessarily imply that the molecules tested are located
exclusively on cellular membranes.
Quantitative growth assay
Following the method of Vielmetter and Stuermer (1989), an equal
amount of membranes (100 µl of membrane suspension containing 2.5 µg/ml protein per microwell) was centrifuged for 20 minutes at 4,000 revs/minute onto the bottom of wells in 96-microwell dishes (NUNC) in a Heraeus Biofuge. For some experiments,
microwells had been previously incubated for 30 minutes at 37°C
with 0.4 µg laminin (Gibco/Sigma) in 50 µl Gey’s solution. After
washing with medium, a single thalamic explant was pipetted into
each well containing about 50 µl medium with 0.4% methylcellulose. In some experiments, the medium contained 50 µg/ml
Jacalin or peanut agglutinin. Thalamic explants had been prepared
as described for the slice cultures and chopped into pieces of about
200×200×200 µm on a McIlwain tissue chopper. After 2-4 DIV
the explants were fixed in PFA. The number of outgrowing neuronal processes was counted using an inverted microscope with a
phase-contrast ×10 objective. To confirm the neuronal origin of
processes counted in phase contrast, we used antibodies against
neuronal markers, SMI31 (Sternberger and Meyer Inc.) and MAP5
(Sigma), and glial markers, vimentin (Sigma) and GFAP (Bioscience). Explants from the hindbrain region were stained with
antibodies against the transmitter serotonin (Incstar) to identify a
subset of cortical afferents (Uylings et al., 1990).
Preparation of membrane borders
Purified membranes were applied to laminin-coated glass coverslips following a method described by Kuromi (1991). Coverslips
were first incubated for 30 minutes at 37°C with 1 µg laminin per
coverslip in 50 µl Gey’s solution and then dried for 30 minutes
after several washes in distilled water. Then about 30 µl of membrane suspension containing 100 µg protein per ml was pipetted
on each side of the coverslip and a small glass capillary (diameter 40−80 µm) was put into these drops across the coverslip. After
incubation at 37°C in an incubator with a humid atmosphere for
2-4 hours, membranes adhered to the coverslip, the capillary was
removed and the coverslip washed with PBS. In experiments with
two membrane stripes, the same procedure was repeated with the
other membrane suspension and the second glass capillary was
placed as near as possible to the first stripe. To identify and to
localize the cortical membranes, fluorescein- or rhodamine-labeled
microspheres (Duke Scientific) were added in a 1:25,000 dilution
to the membrane suspension. After the membrane stripes were
located using an inverted Zeiss fluorescence microscope, the cover
slips were placed in Petriperm dishes (Multimed) and medium
containing 0.4% methylcellulose was added. Thalamic explants of
about 200 ×200×200 µm were then pipetted in the regions of the
membrane stripes. The axonal outgrowth was repeatedly checked
and photographed with phase-contrast optics in an inverted Zeiss
microscope.
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Results
Development of the thalamic projection in the cortex in
vivo
As a first step, we examined the establishment of the thalamocortical projection in vivo in relation to the location of
their target cells from layer 4 and the developmental pattern of lectin-staining in the cortex (Fig. 1). The invasion
of thalamic afferents in the cortex is depicted in Fig. 1B.
As has been shown previously (Lund and Mustari, 1977;
Catalano et al., 1991), the first thalamic afferents arrived at
E17 in rat cortex and initially did not invade the cortical
plate, but were restricted ventral to it in the subplate and
intermediate zones (Fig. 1B). Around E20, individual thalamic fibers redirected their growth perpendicularly and
started to enter the deep cortical layers (Fig. 1B). After several days, around P2-4, thalamic fibers then arrived at their
target layer where they terminated their growth and formed
terminal arbors. Thus, different phases of thalamic fiber
growth can be distinguished during innervation of rat cortex
which are also found during the development of the thalamocortical projection in other mammals (Shatz et al., 1988;
Rakic, 1977; Kostovic and Rakic, 1990): initially, thalamic
axons are restricted to the subplate zone; thereafter, they
change their direction of growth, invade the cortical layers
and finally arborize in layer 4. Since interactions of thalamic afferents with their target cells could influence the differential axonal growth, we studied the relation of thalamic
fiber growth to the position of their target cells. Cortical
target neurons destined for layer 4 were labeled with BrdU
at their generation time, i.e. E17 in rat cortex (Miller, 1988).
When the first fibers from the thalamus have reached the
cortex, future layer 4 cells have just been generated in the
ventricular zone (Fig. 1A,B). Then, BrdU-labeled cells destined for layer 4 migrated towards the pial surface for about
a week and completed their migration several days after
birth (Fig. 1A). At the time when thalamic axons redirected
their growth and left the subplate zone, their target cells
were already distributed throughout the whole cortex (Fig.
1A,B), indicating that thalamic fibers do not immediately
follow their migrating future layer 4 cells.
Another possible influence on the growth of thalamic
fibers in the cortex might be the existence of guidance molecules in the developing cortex. Since the guidance molecules for axonal growth so far identified are glycosylated
(for review: Jessell, 1988; Stahl et al., 1990; Schwab, 1990;
Furley et al., 1990) and various lectin-binding molecules
have been described to influence axonal growth (Davies et
al., 1990; Stahl et al., 1990), we examined the distribution
of lectin-binding molecules during cortical development in
vivo. We found a close correlation between the peanut
agglutinin (PNA) staining and the growth of thalamic fibers
throughout development in the cortex (Fig. 1B,C). In sections through the cortex at E17, staining with PNA was
confined to a small band in the subplate/intermediate zone
and in the marginal zone (Figs 1C and 4A; Raedler et al.,
1981; Brückner et al., 1985). When thalamic fibers started
to invade the cortical layers (E20), the PNA-positive band
in the cortex had extended from the subplate zone into the
deeper cortical layers (Figs 1C and 4B). During further
development, PNA-staining spread into the superficial
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Fig. 1. (A) Camera-lucida drawings of cells labeled with BrdU at embryonic day 17 (E17), (B) DiI-labeled fibers from the thalamus and
(C) staining with the peanut agglutinin (PNA) in frontal sections of the cortex of different developmental stages. The thick lines delineate
the pial surface of the cortical sections, and the thin lines the border between grey/white matter and cortical/subplate respectively. BrdU
was injected at E17 to label predominantly cells of layer 4 and the brains were processed at the developmental stages indicated in the
figure. Note that the ingrowth of thalamic fibers into the cortical layers (B) does not follow the migration of BrdU-labeled cells of layer 4
(A), but rather corresponds closely to the spread of PNA-staining during cortical development (C). Scale bar, 500 µm.
layers until the whole cortex was uniformly stained with
PNA in the first postnatal week (Figs 1 and 4C). In sections from adult cortices, PNA-staining was again very
weak. A similar labeling pattern was observed with the
lectins Concanavalin A and Jacalin, whereas no staining in
the cortex could be detected with the lectin from Erythrina
cristagalli (data not shown). The close correlation between
the growth of thalamic fibers and the distribution of PNAbinding molecules was confirmed in double-labeling experiments which revealed directly that DiI-labeled fibers from
the thalamus did not enter cortical layers with weak lectinstaining. Instead thalamic axons were always confined to
those regions of the cortex that exhibited intense PNAstaining and fibers were observed in the cortical plate only
after the PNA-staining had spread from the subplate zone
into the deep cortical layers.
Thalamic projections to co-cultured cortical slices of
different developmental stages
In order to examine the relationship between cortex maturation and thalamic fiber growth in vitro, we co-cultured
thalamic explants with cortical slices prepared at different
times during development. When thalamic axons encountered cortical slices prepared at E16, in 88% of all cases
(Table 1; n=111) not a single thalamic fiber grew into the
E16 cortex explant (Figs 2D, 3), whereas axons from the
very same thalamic slice readily invaded co-cultured cortical slices prepared at later developmental stages (Fig. 2AC). Axons extending from older thalamic explants (E20,
P0) also did not invade E16 cortical slices, but did innervate explants prepared later during development (Table 1).
Table 1. Percentage of cortical cultures invaded by
thalamic axons
Thal
E16
E19-20
E21-P2
E16
E19-21
P0-6
12%
26%
17%
88%
91%
100%
90%
88%
87%
Ctx
The direction from which growing axons approached the
cortical explants was not critical, since thalamic fibers grew
around an E16 cortical slice irrespective of whether they
approached the explant from the ventricular or the pial side.
Thalamic fibers exhibited a different growth in cortical
slices prepared at E19, where they grew randomly throughout the explant and extended up to the pial side of the cortical slice (Fig. 2B). Again, the growth of thalamic axons
in E19 cortical slice cultures was independent of the position of the thalamic explant relative to the cortical slice
(Fig. 2B,C), and also independent of the age of the thalamic explants (Table 1). Previous birthdating studies have
shown that cells of layer 4 are scattered throughout the cortical thickness at E19 and layer 4 is not formed in slice cultures from E19 cortex during the time in vitro (Götz and
Bolz, 1992). In postnatal cortical explants, however, layer
4 is preserved as an organotypic layer (Götz and Bolz,
1992) and thalamic fibers invaded these explants, stopped
and arborized in their appropriate target layer in vitro (Fig.
2A; Yamamoto et al., 1989; Götz et al., 1990, Molnár and
Blakemore, 1991; Bolz et al., 1992). Thus, similar to the
growth of thalamic fibers during innervation of the cortex
Development of thalamocortical connections
511
Fig. 2. (A-D) Camera-lucida drawings of DiI-labeled fibers from embryonic thalamic explants confronted with cortical slices at different
developmental stages (A, P6; B,C, E19; D, E16). In A,B and D, cortical slices are oriented with the white matter and ventricular side,
respectively, towards the thalamic explant, whereas in C the E19 cortical culture faces the thalamic slice with the pial surface. The dashed
line in A indicates the border between the grey and white matter. (E) Camera lucida drawing of cells in an E16 cortical explant (pial side
up) retrogradely labeled with DiI from a thalamic explant of the same age. For the sake of clarity, fibers emanating from the thalamic
explant were not drawn. (F) Camera-lucida drawing of cells labeled with BrdU at E13 in a cortical slice culture (pial side up) prepared at
E16. Cultures were fixed after 9 (A), 10 (B,C), 5 (D), 7 (E) and 5 (F) days in vitro respectively. Scale bars, (A,B) 500 µm, (C-F) 200 µm.
Fig. 3. (A) Fluorescence and (B) corresponding phase-contrast micrographs of a thalamocortical co-culture prepared at E16 after 4 days
in vitro. The cortical slice is located in the centre of the micrograph, beneath the border of the thalamic explant. DiI-labeled thalamic
fibers do not invade the embryonic cortical slice. Scale bar, 200 µm.
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M. Götz and others
Fig. 4. Immunohistochemical localization of peanut agglutinin (PNA) in cortical sections in vivo (A-C) and in cortical slice cultures (DF). Frontal sections of cortex were cut at E17 (A), E19 (B) and P5 (C). In (A,B) the border between the subplate/cortical plate and, in (C),
between the white/grey matter is indicated. Slice cultures were prepared from cortices at E19 (D,E) and E16 (F) and fixed after 1 (D), 4
(E) and 6 (F) days in vitro. Note that during the in vivo development, PNA-labeling spreads from the subplate zone into the cortical
layers. The same development of PNA-labeling occurs in cortical cultures prepared at E19 (D,E), but not in cultures prepared at E16 (F).
Scale bar, 200 µm (A-E), 100 µm (F).
in vivo, differential growth of axons from thalamic explants
was observed in cortical explants of different developmental stages in vitro.
Lectin-staining in cortical slice cultures
The close correlation between the developmental spread of
PNA-staining and the invasion of thalamic fibers into the
cortex in vivo prompted us to examine the distribution of
PNA-binding molecules in cortical slice cultures. Fig. 4
illustrates the development of PNA-staining in cortical slice
cultures (D-F) in comparison to the development in the
cortex in vivo (A-C). In slice cultures prepared from E16
cortices, PNA-staining remained restricted to a small band
in the subplate zone, similar to its distribution at the time
of preparation (Fig. 4A,F). Most of the cortical thickness
of E16 cortical slice cultures did not bind PNA. In contrast,
intense staining with PNA was present throughout the cortical thickness of cultures prepared at E19 after few days
in vitro (Fig. 4E). Since PNA-staining was not yet distributed throughout all cortical layers at the time of preparation (compare Fig. 4B-E), PNA-staining must have spread
into the cortical plate in slice cultures prepared from E19
cortices. These results show that PNA-binding molecules
cannot be located exclusively on thalamic axons or be
secreted solely by afferent fibers, since thalamic axons are
absent in isolated cortical slice cultures. Thus, at least a
proportion of PNA-binding molecules must be located on
cortical cells itself and spread in cortical slices in vitro.
Moreover, the pattern of PNA-staining closely corresponds
to the ingrowth of thalamic fibers in cortical slice cultures.
Development of thalamocortical connections
In cortical slice cultures prepared at E16, PNA-staining is
restricted to a small band and these slices are not invaded
by thalamic fibers, whereas in slice cultures from E19 cortices PNA-staining is present throughout the cortical layers
and thalamic fibers enter these explants from all directions.
However, few cortical explants prepared at E16 were
invaded by thalamic fibers (Table 1). In these co-cultures
(n=13), only axons from thalamic explants that approached
the cortical slice parallel to the pial surface entered the cortical slice and these fibers extended exclusively in the
region of the subplate zone. Thus, the only thalamic fibers
that invaded E16 cortical explants grew along the PNApositive subplate zone in vitro as is the case in the cortex
in vivo.
The subplate zone in cortical slice cultures
During development of the cortex, the subplate zone is
related to the establishment of the thalamocortical projections in two aspects: first, the growth of thalamic fibers is
restricted to the subplate zone in early developing cortex
(Fig. 1B) and, second, the first projection neurons that send
their axons to the thalamus are cells of the subplate zone
(McConnell et al., 1989). We therefore examined the subplate zone in embryonic slice cultures with BrdU-injections
at E13/14, the time when subplate cells are generated in rat
cortex (Bayer and Altman, 1990; Götz and Bolz, 1992). As
depicted in Fig. 2F, cells labeled with BrdU at E13/14
formed a horizontal band in embryonic cortical slice cultures that was visible after several days in vitro. Since neurogenesis and migration ceases in this in vitro system, granular and supragranular layers are missing in E16-E20
cortical slice cultures (Götz and Bolz, 1992). Those layers,
however, that already had been formed at the time of preparation, like e.g. the subplate zone, were equally well preserved in slice cultures from embryonic or postnatal cortices in this in vitro system (Götz and Bolz, 1992). Despite
the presence of the subplate zone in E19 cortical slice cultures, thalamic fibers were not confined to this layer, but
rather extended beyond it regardless whether they invaded
the cortical slice from the ventricular or the pial side (Fig.
2B,C). In contrast, the few thalamic fibers that entered an
E16 cortical explant in vitro all grew along the region of
the subplate zone (n=13). As described above, the distribution of PNA-staining in E16 cortical slice cultures remains
restricted to the subplate zone (Fig. 4F), whereas it spreads
into the cortical plate in slice cultures from E19 cortex (Fig.
4D,E). Thus, the growth of thalamic fibers corresponds to
the location of PNA-binding molecules, but not to the subplate zone by itself.
Similar to the situation in the cortex in vivo (McConnell
et al., 1989), cells located in the subplate zone of slices
from embryonic cortices were found to project to thalamic
slices in vitro. In contrast to fibers from the thalamic explant
that avoided the E16 cortical slice or extended only along
the narrow subplate zone, axons from the cortical slice readily invaded E16 thalamic explants (Fig. 2E). About 65% of
the corticothalamic projection neurons in E16 slice cultures
(n=26) were located in the same region as cells of the subplate zone. Thus, despite the presence of corticothalamic
projections from subplate cells, thalamic fibers only rarely
invaded the E16 cortical slice. However, there were several
513
examples where thalamic fibers invaded older cortical
slices, but no corticofugal fibers were detected. Thus, it
appears that efferent cortical projections are neither necessary nor sufficient for the invasion of cortical cultures by
thalamic axons.
Thalamic fiber growth on cortical cell membranes from
different developmental stages
The results obtained with the thalamocortical co-cultures
suggested the presence of glycosylated molecules in isolated cortical slices that influence the growth of thalamic
axons. Molecules that affect the growth of axonal processes
can be either located in the extracellular matrix, or bound
to cell membranes, or exist in a soluble form (Levi-Montalcini 1987; Stahl et al., 1990; Schwab, 1990; Furley et al.,
1990; Davies et al., 1990; for review: Dodd and Jessell,
1988). Since diffusible molecules are stabilized in a graded
way in our co-culture system (Bolz et al., 1990), we used
cortical cell membranes to test whether soluble or membrane-bound molecules influence the growth of thalamic
axons. Membranes prepared from cortices at different
developmental stages were tested as substrata for thalamic
axons in a quantitative growth assay (Vielmetter and
Stürmer, 1989). Thalamic explants placed on E16 cortical
membranes showed very little, if any, fiber outgrowth (Figs
5A, 6A), in contrast to explants placed on cortical membranes prepared at P7 that were surrounded by a dense fiber
network after 2-4 days in vitro (Fig. 5B). An intermediate
rate of thalamic fiber growth was observed on cortical membranes prepared at developmental stages E19 and 20 (Fig.
6A). On membrane substrata prepared from adult cortices,
the rate of thalamic fiber growth was again reduced to a
lower level (Fig. 6A). Thus, purified cortical membranes
show growth-supporting properties for thalamic afferents
(see below) that increase during development and peak in
early postnatal development.
To test whether this differential effect on axonal growth
was specific for thalamic fibers, we placed explants from
the E16 cortex and hindbrain on cortical membranes prepared at E16 and P7. The number of cortical axons extending on cortical membranes far exceeded the axonal outgrowth from hindbrain explants (Fig. 6B). In contrast to
thalamic axons, however, both of these axonal populations
exhibited the same rate of outgrowth on embryonic and
postnatal cortical membranes (Figs 5C,D; 6B). Since serotonergic fibers from raphe nuclei in the hindbrain establish
projections to the cortex in vivo (Uylings et al., 1990),
explants from the hindbrain were also stained with an antibody directed against serotonin. Fibers immunoreactive for
serotonin also showed no preference for one of the cortical
substrata (on E16 cortex membranes: 6.8 axons (n=23); on
P7 cortex membranes: 7.3 axons (n=32)). The large number
of cortical axons extending on E16 cortical membranes
(Fig. 5C) indicates that embryonic cortical membranes do
not inhibit axonal outgrowth in general. Moreover, thalamic fibers do not avoid extending onto embryonic brain
membranes in all cases, since more thalamic axons were
found to extend on membranes prepared from E16 hindbrain than from E16 cortex (data not shown). Thus, there
is a difference between embryonic and postnatal cortical
514
M. Götz and others
Fig. 5. Phase-contrast micrographs of fiber growth from thalamic (A,B,E,F) and cortical (C,D) explants on cortical membranes of
different ages. All explants were prepared at E16 and placed on membranes from E16 cortices (A,C) or P6 cortices (B,D). The arrow in E
indicates the border between P6 cortex membranes mixed with laminin and laminin alone (top), in F the border between mixed E16 and
P6 membranes (bottom) and E16 membranes alone (top). Cultures were fixed after 2 (A-D), 3 (E) and 7 (F) days in vitro. Scale bars in AE, 200 µm; in E,F, 100 µm.
membranes which is specifically recognized by thalamic
axons.
The differential outgrowth of thalamic fibers on E16 and
P7 cortex can be explained in two ways: growth of thalamic axons may be either inhibited by molecules bound to
membranes from E16 cortex or, conversely, membranes
from E16 cortex may not contain growth-supporting molecules for thalamic axons. To address this issue, we mixed
membranes prepared from E16 and postnatal cortex. If
inhibitory molecules were associated with E16 cortical
membranes, their addition should reduce the outgrowth of
thalamic fibers on a growth-permissive substratum, as has
been described previously for other inhibitory molecules
(Davies et al., 1990). However, an equal number of axons
extended on mixed cortical membranes as on P7 cortical
membranes alone (Fig. 6C). Similarly, when the plastic
dishes were coated with laminin, the addition of E16 cortical membranes did not reduce the rate of outgrowing
fibers, but the rate of fiber outgrowth on postnatal cortical
membranes exceeded the number of thalamic axons on
laminin alone (Fig. 6A). These data suggest that postnatal
cortical membranes possess growth-promoting molecules
for thalamic axons that are not present in the early embryonic cortex. To test this hypothesis further, cortical membranes of both ages were trypsinized for 45 minutes. The
same number of thalamic axons extended on the trypsinized
as on the control membranes prepared from E16 cortices
(Fig. 6D). In contrast, the rate of thalamic fiber outgrowth
was strongly reduced after trypsinization of P7 cortex membranes, almost to the low level found on E16 cortex membranes (Fig. 6D).
To examine the influence of PNA-binding molecules in
cortical cell membranes on the outgrowth of thalamic fibers,
we applied 50 µg/ml PNA in the quantitative growth assay.
The rate of thalamic fiber growth on membrane substrata
from postnatal cortices was diminished by more than 50%
Development of thalamocortical connections
515
Fig. 6. Histograms of axonal outgrowth from explants prepared at E16 on cortical membranes. Error bars indicate the standard error of the
mean (s.e.m.). (A) The rate of thalamic growth increases on substrata from cortical membranes during development and decreases again
on membranes from adult cortices. (B) Explants from E16 thalamus, cortex and hindbrain were placed on membranes from E16 or P7
cortices, respectively. Whereas the growth rate of thalamic fibers exhibits a pronounced difference between these substrata, axons from
cortex and hindbrain do not recognize a difference between cortical membranes of different ages. (C) The rate of thalamic fiber growth on
membrane substrata from both E16 and P7 cortices corresponds to their rate of outgrowth on postnatal cortical membranes alone. (D)
Trypsinization of postnatal cortical membranes strongly reduces the outgrowth of thalamic fibers compared to their growth rate on control
membranes, whereas trypsinization of embryonic cortical membranes does not exhibit any effect on the growth of axons from thalamic
explants. (E) The growth rate of thalamic fibers on postnatal cortical membranes is strongly reduced after the addition of peanut
agglutinin (PNA), but not after addition of Jacalin. (F) Many more thalamic fibers extend on membrane substrata from the deeper layers
including the white matter of a postnatal cortex, compared to their growth rate on layer 4 membranes or a mixture of all cortical layers
including the white matter.
in the presence of PNA (Fig. 6E). In contrast, the addition
of the lectin Jacalin did not influence the number of thalamic axons extending on postnatal cortical membranes (Fig.
6E). Thus, binding of the peanut agglutinin to glycosylated
molecules present in membrane preparations from postnatal cortices seems to block the growth-promoting properties of this substratum for thalamic fiber growth.
The influence of different substrata on the direction of
thalamic fiber growth
So far we have shown that the rate of thalamic fiber growth
can be influenced by membrane-bound molecules in the
cortex. However, changes in the direction of axonal
elongation are also required, e.g. to confine the growth of
thalamic axons to the subplate zone in early development
(Fig. 1B,C). In order to examine which substrata might
cause a redirection of axonal growth, thalamic axons were
confronted with borders of different substrata. At the border
between a pure laminin substratum and laminin with membranes from postnatal cortices in 95% of all thalamic
explants (n=43) sharp turns of the growing axons were
observed (Fig. 5E). Most of the fibers (97%;n=270) that
encountered the border remained on the side where the postnatal cortical membranes were located and only 3% of thalamic axons entered the laminin substratum. In contrast,
thalamic fibers that extended on pure laminin crossed the
border to the region coated in addition with membranes
from postnatal cortices in all cases examined (n=12). However, fibers showed almost no preference for membranes
prepared from E16 cortex since, in only 2 out of 10 cultures examined, thalamic fibers respected the border
between E16 cortical membranes and pure laminin. When
axons from thalamic explants (n=15) approached the border
between stripes containing E16 and P7 cortical membranes,
92% of all thalamic fibers (n=370) performed sharp turns
and remained in regions where postnatal cortical mem-
516
M. Götz and others
branes were available (Fig. 5F). In control experiments,
where two stripes of postnatal cortical membranes were
applied adjacent to one another (n=15), no change in the
number and direction of growing thalamic axons was
observed at the border between the two postnatal membrane
substrata. Thus, growing thalamic fibers show a strong preference for cortical membranes of postnatal origin and perform sharp turns to stay on this growth-supporting substratum while they avoid E16 cortex membranes.
Thalamic fiber growth on membranes from isolated cortex
layers
These results revealed the presence of developmentally regulated membrane-bound molecules in the cortex that influence the growth of thalamic fibers. To elucidate a potential
contribution of membrane-bound molecules to the layerspecific termination of thalamic fibers, we separated layer
4 of early postnatal cortex from the deeper layers, through
which thalamic fibers grow in vivo. The same number of
thalamic fibers extended on membranes from isolated layer
4 as on membranes derived from all cortical layers and the
white matter (Fig. 6F). However, more than twice as many
fibers extended from thalamic explants placed on membranes prepared only from the deeper cortical layers and
the white matter (Fig. 6F). Thus, membrane-bound molecules that are differentially distributed in the cortical layers
influence the growth of thalamic axons and specifically
diminish the rate of thalamic fiber growth on membranes
from layer 4, the target layer for thalamic afferents.
Discussion
There has been much discussion of how afferent fibers from
thalamus make their appropriate connections with cortical
targets (Wise and Jones, 1978; Lund and Mustari, 1977;
Shatz et al., 1988; Ghosh et al., 1990). Along with the selection of their target cells, cortical afferents face the additional problem of a temporal mismatch between their early
arrival and the late formation of their cortical target layer.
Our in vitro results provide evidence that membrane-bound
guidance molecules in the cortex are involved in both the
regulation of thalamic innervation into the cortical layers
and their termination in layer 4.
Innervation of thalamic fibers into the cortex
Thalamic fibers of all ages tested (E16-P1) recognize guidance cues in the cortex: whereas axons from thalamic
explants terminated in layer 4 of postnatal cortical slices,
they exhibited a random growth in cortical explants where
layer 4 had not yet been established, and they avoided E16
cortical explants. Thus, the growth of thalamic fibers
depends crucially on the age of the cortical tissue but not
on the age of the thalamic explants. Preparations of cell
membranes revealed that membrane-bound molecules in the
cortex are responsible for the differential growth of thalamic axons and influence the growth of thalamic fibers in a
developmentally regulated fashion: only few axons extend
on cortical membranes isolated during early development
and an increasingly higher rate of outgrowth was observed
on cortical membranes prepared at later developmental
stages. This effect on axonal growth rate was specific for
axons from the thalamus since axons from other regions of
the brain grew equally well on membranes prepared from
embryonic and postnatal cortex. Thus, either distinct guidance cues for different axonal subsets are present in the
cortex or, alternatively, the same cues act differently on
axonal subpopulations, which should then possess different
receptors for these molecules (Reichardt and Tomaselli,
1991; Neugebauer and Reichardt, 1991; DeCurtis, 1991).
This selectivity is not surprising in view of the large variety of adhesion and extracellular matrix molecules in the
developing cortex (Stewart and Pearlman, 1987; Chun and
Shatz, 1988; Nakanishi, 1983; Bignami and Delpech, 1985;
Sheppard et al., 1991). A recent study combining fiber-tracing with immunocytochemistry showed that thalamic and
cortical axons differ in their growth along the chondroitin
sulfate proteoglycan (CSPG)-containing subplate layer, in
that axons labeled from the thalamus were found to be
restricted to this region, whereas cortical axons crossed the
CSPG-rich subplate and performed sharp turns beneath it
(Bicknese et al., 1991). In contrast to the increase and
spread of PNA-staining from the subplate zone into the
developing cortical layers, the CSPG-staining disappeared
during further cortical development, similar to the developmental expression of fibronectin in the cortex (Bicknese
et al., 1991; Stewart and Pearlman, 1987; Chun and Shatz,
1988). In addition to such guidance molecules for the
specific growth of axonal subpopulations, differential fiberfiber interaction might also play a role in the establishment
of separate afferent and efferent paths in the cortical white
matter (Woodward et al., 1990). In our thalamocortical cocultures, however, we could not detect interactions such as
axonal fasciculation or axonal avoidance. Moreover, the
presence of an efferent corticothalamic projection in vitro
was neither necessary nor sufficient for the ingrowth and
termination of thalamic fibers in cortical explants.
Axonal populations from the cortex and the hindbrain
grew equally well on embryonic and postnatal cortical
membranes in vitro, in contrast to the lower growth rate of
thalamic axons on embryonic compared to their growth rate
on postnatal cortical membranes. This in vitro observation
corresponds to the behaviour of these axonal subpopulations in the cortex in vivo: both cortical axons and fibers
from the hindbrain extend through the cortical thickness in
vivo during early development (Uylings et al., 1990; Bicknese et al., 1991), whereas the growth of thalamic axons is
still confined to the subplate zone (Fig. 1B; Catalano et al.,
1991). The low growth rate of thalamic axons on embryonic cortical membranes in vitro indicates that most of the
cortex does not permit the growth of thalamic axons at early
developmental stages. Thus, the confinement of thalamic
fibers to a narrow band in the cortex during early development seems to be achieved by the non-permissive substratum properties of the surrounding tissue. Consistent with
this idea is the observation that thalamic fibers reorient their
growth as they encounter less permissive substrata in vitro.
As development progresses, the growth rate of thalamic
fibers on cortical membranes increases and reaches a peak
at early postnatal ages. This increase in the substratum quality for thalamic fiber growth coincides with the invasion of
thalamic fibers into the cortical layers in vivo, suggesting
Development of thalamocortical connections
that substratum properties in the cortex regulate the
ingrowth of thalamic fibers during development. Enzyme
treatment of the cortical membranes and experiments where
cortical membranes from different developmental stages
were mixed show no indication of inhibitory molecules on
embryonic cortical membranes. These experiments reveal
the presence of growth-supporting molecules for thalamic
axons on postnatal cortical membranes that are rare or
absent on membranes prepared from E16 cortex. Thus, molecules that specifically support the growth of thalamic afferents appear to be up-regulated during cortical development,
and establish a growth-supportive substratum that allows
thalamic fiber ingrowth only when layer 4 is about to be
formed. Since layer 4 cells themselves constitute only a
small percentage of the membrane fractions, it seems
unlikely that the growth-supporting molecules are located
only on the thalamic target cells. On the contrary, thalamic
fiber growth was reduced on cortical membranes prepared
from layer 4 cells alone. Taken together, this suggests that
those molecules that establish a growth-promoting substratum for thalamic fibers are widely distributed in early postnatal cortex.
Which molecules that are up-regulated during cortical
development could be candidates for these membranebound proteins that support the growth of thalamic fibers?
Staining with peanut agglutinin (PNA) and some other
lectins revealed a close association between the lectin-staining pattern and the cortical invasion of thalamic fibers, both
in vivo and in slice cultures. Furthermore, the growth rate
of thalamic fibers on postnatal cortical membranes was
reduced by the addition of PNA to the membranes. At least
a portion of these PNA-binding molecules must be of cortical origin, since they are expressed and up-regulated in
isolated cortical slice cultures. A staining pattern similar to
that obtained with lectins has also been observed for various extracellular matrix (ECM) molecules (Godfraind et al.,
1988; Sheppard et al., 1991). For example, staining for
undefined glycosaminoglycans, tenascin and L2 showed a
similar distribution during early cortical development
(Nakanishi, 1983; Godfraind et al., 1988; Sheppard et al.,
1991). Initially, these ECM components are expressed in
the subplate zone, but later they gradually spread from the
white matter zone towards the pial side of the cortex
(Nakanishi, 1983; Godfraind et al., 1988; Sheppard et al.,
1991). Thus, a variety of glycosylated molecules develop
in concert with the sequential maturation of the cortical
layers and the invasion of thalamic fibers. Since glycoproteins and proteoglycans are varied both in their protein core
and their glycosylated side chains (Esko, 1991; Gallagher,
1989), future studies will have to characterize the exact
nature of the glycosylated proteins that regulate the invasion of the cortex by thalamic afferents. For further isolation and characterization of these growth-promoting molecules in the cortex, their PNA-binding properties should
serve as a convenient tool (Davies et al., 1990).
In the light of their axon guidance function, the mechanisms that regulate the expression of adhesion molecules
and ECM components during development become of particular interest. Since PNA-binding increases in isolated
cortical slice cultures, intrinsic mechanisms in the cortex
seem to be involved in the spread of lectin-binding mole-
517
cules. However, this development of lectin-binding was
only observed in slice cultures prepared towards the end of
cortical neurogenesis, but not in slice cultures prepared earlier. These results suggest that influences that appear later
during cortical development are crucial for the spread of
lectin-binding molecules in the cortex. From these experiments, it is not possible to distinguish whether these factors are of intrinsic cortical or extrinsic origin. It has been
shown, however, that cortical cells are at least required for
the developmental regulation of ECM molecules, since
lesioning of the subplate cells prevents the expression of
some ECM molecules (Chun and Shatz, 1988). Moreover,
Ghosh et al. (1990) reported that lesion of the subplate
during early development in cat cortex prevented thalamic
fibers from entering the cortex, suggesting that subplate
cells interfere with the regulation of molecules that support
the ingrowth of thalamic fibers. Thus, cells situated in the
cortex are at least required, if not alone responsible, for the
developmental regulation of these guidance cues.
Layer-specific termination of thalamic fibers in the cortex
Following the increasing expression of growth-promoting
molecules in the cortex, thalamic fibers invade the cortical
layers and terminate specifically in layer 4. In co-cultures
with slices from postnatal cortex, where cells of layer 4 are
located at the appropriate position, axons from thalamic
explants are able to terminate in their correct target layer
(Yamamoto et al., 1989, Götz et al., 1990; Molnár and
Blakemore, 1991; Bolz et al., 1992). This appropriate projection to layer 4 is established in vitro irrespective of
whether thalamic fibers invaded the cortex from the pia or
the white matter side (Götz et al., 1990; Molnár and Blakemore, 1991; Bolz et al., 1992). The earliest thalamic axons
emanating from explants isolated at E16, i.e. shortly after
neurogenesis is finished in the thalamus (Jones, 1985), recognized their cortical target cells in vitro (Yamamoto et al.,
1989; Götz et al., 1990; Molnár and Blakemore, 1991).
These fibers had neither made contacts in the subplate zone
nor experienced bypassing layer 4 cells. Thus, thalamic
fibers do not need external influences to find their way
through the cortex; rather they seem to have an intrinsic
mechanism that allows them to recognize their target cells
in cortex.
Here we have shown that membrane-bound molecules
differentially distributed in the cortical layers seem to be
involved in the layer-specific termination of thalamic fibers.
The growth rate of thalamic axons was reduced on membranes from layer 4 compared to the growth rate on membranes prepared from the deeper cortical layers through
which thalamic fibers have to grow in vivo. This decrease
in the growth of thalamic axons could be due to a reduced
amount of growth-supporting molecules in layer 4. Alternatively, since thalamic outgrowth on membranes prepared
from all cortical layers including layer 4 was also less than
the growth rate on membranes prepared from the deep
layers, the presence of inhibitory molecules might explain
the low growth rate on cortical membranes including layer
4 compared to tissue excluding it. Thus, a membrane-bound
stop-signal for growing thalamic axons in layer 4 might initiate their termination. The question remains whether these
molecules are located on cellular membranes of the target
518
M. Götz and others
neurons in layer 4, or if other components, for example
processes of neuronal or glial cells that pass through this
layer, are responsible for the reduced growth rate of thalamic fibers. Norris and Kalil (1991) have recently presented
evidence that axonal growth cones extend along radial glial
fibers in the cortex. Moreover, this mechanism could
explain the ordered vertical ingrowth of both thalamic and
callosal axons into the cortical layers. On the other hand,
results obtained in dissociated cell cultures of the cerebellum have shown that contact-mediated stop-signals for
afferent mossy fibers are located on the target cells themselves (Baird et al., 1992). Future studies have to address
whether these putative target-related stop-signals act in the
same way as guidance molecules that induce growth cone
collapses of growing axons (Schwab, 1991; Davies et al.,
1991; Cox et al., 1990) or whether there are different mechanisms that initiate the termination of elongating fibers.
In summary, the present study revealed that the invasion
of cortical layers by thalamic afferents is controled by membrane-bound molecules in the cortex. The differential
expression of these molecules appears to prevent thalamic
fibers from entering the cortical plate early in development
when their target cells are not yet generated and allows thalamic ingrowth only after layer 4 is about to be formed.
Thus, the cortex orchestrates the timing of thalamic fiber
ingrowth by the regulation of growth-permissive molecules
for thalamic axons, and thereby solves the problem of the
temporal mismatch between the arrival of afferents and the
generation of their target cells during development. These
molecules are specifically recognized by axons from the
thalamus. Other axonal populations whose target cells are
already present do not recognize developmental changes in
the cortical substratum. After layer 4 has been established,
membrane-bound molecules specifically located in this
target layer reduce the growth of thalamic fibers and might
be involved in their termination in the cortex. The developmental status of thalamic fibers does not seem to play a
significant role in the establishment of thalamocortical connections, because thalamic fibers of all ages recognize the
different membrane-bound properties of cortical tissue and
terminate in their cortical target layer. Thus, guidance cues
in the cortex are responsible for the proper innervation and
termination of thalamic fibers.
We thank Sandra Poesdorf and Renate Thanos for excellent
technical assistance, Iris Kehrer for help with the illustrations and
Mary Behan, Ysander v.Boxberg and Mark Hübener for valuable
comments on the manuscript.
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(Accepted 14 August 1992)