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The Journal of Neuroscience, June 15, 1998, 18(12):4616-4626
Involvement of Distinct Pioneer Neurons in the Formation of
Layer-Specific Connections in the Hippocampus
Hans
Supèr,
Albert
Martínez,
José A.
Del
Río, and
Eduardo
Soriano
Department of Animal and Plant Cell Biology, Faculty of Biology,
University of Barcelona, Barcelona 08028, Spain
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ABSTRACT |
During neural development, specific recognition molecules provide
the cues necessary for the formation of initial projection maps, which
are reshaped later in development. In some systems, guiding cues for
axonal pathfinding and target selection are provided by specific cells
that are present only at critical times. For instance, the floor plate
guides commissural axons in the spinal cord, and the subplate is
involved in the formation of thalamocortical connections. Here we study
the development of entorhinal and commissural connections to the murine
hippocampus, which in the adult terminate in nonoverlapping layers. We
show that two groups of pioneer neurons, Cajal-Retzius cells and
GABAergic neurons, form layer-specific scaffolds that overlap with
distinct hippocampal afferents at embryonic and early postnatal stages.
Furthermore, at postnatal day 0 (P0)-P5, before the
dendrites of pyramidal neurons develop, these pioneer neurons are
preferential synaptic targets for hippocampal afferents.
Birthdating analysis using 5'-bromodeoxyuridine (BrdU) pulses showed
that most such early-generated neurons disappear at late postnatal
stages, most likely by cell death. Together with previous studies,
these findings indicate that distinct pioneer neurons are involved in
the guidance and targeting of different hippocampal afferents.
Key words:
Cajal-Retzius cells; pioneer neurons; synapse formation; axonal guidance; anterograde tracers; mouse
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INTRODUCTION |
The development of specific neural
connections is a multifactorial process in which different recognition
signals acting at sequential developmental stages provide the cues
necessary for the development of axonal projections. Among these cues
there are diffusible factors, such as netrins and semaphorins, as well as membrane-anchored factors and extracellular matrix proteins. These
initial connections are later reshaped and pruned by mechanisms such as
neurotrophin signaling and neural activity (for review, see Katz and
Shatz, 1996 ; Tessier-Lavigne and Goodman, 1996).
In both vertebrates and invertebrates, guiding cues for axonal
pathfinding and target selection are often provided by specific transient cells, which are only present at critical times (Bentley and
Keshishian, 1982). For instance, the cells in the floor plate releasing
netrin-1 exert a chemoattractive influence on commissural axons in the
spinal cord (Kennedy et al., 1994; Serafini et al., 1994), whereas the
same factor exerts chemorepellent action on trochlear motor fibers
(Colamarino and Tessier-Lavigne, 1995; Culotti and Kolodkin, 1996). In
the forebrain, the subplate cells of the developing visual cortex are
involved in the guidance of ingrowing thalamocortical fibers and in
their later segregation into ocular dominance columns (McConnell et
al., 1989; Ghosh et al., 1990; Ghosh and Shatz, 1992; Allendoerfer and
Shatz, 1994). Similarly, a population of cells in the optic chiasm may
be essential for the ingrowth and decussation of retinal axons
(Sretavan, 1993; Wang et al., 1995).
One important feature of many regions of the adult brain is that neural
connections are highly laminated with different groups of axons
innervating specific target layers. In the hippocampus, for instance,
the main afferent systems terminate in a characteristic nonoverlapping
manner, with entorhinal fibers innervating outer pyramidal cell
dendrites in the stratum lacunosum-moleculare and with
commissural/associational fibers terminating in the stratum radiatum
and stratum oriens. A similarly segregated distribution of hippocampal
afferents occurs in the dentate gyrus, with entorhinal fibers
innervating the outer molecular layer and with
commissural/associational fibers terminating in the inner molecular
layer (Blackstad, 1956; Raisman et al., 1965; Hjorth-Simonsen and
Jeune, 1972; Amaral and Witter, 1995). Little is known about the
factors that govern the generation of such a precise pattern of
layer-specific connections during development. It has been suggested
that the pattern of hippocampal innervation is independent of temporal
factors such as the order of fiber arrival (Frotscher and Heimrich,
1993). Also, we have shown previously that both entorhinal and
commissural axons terminate in their specific target layers at
embryonic stages, which suggests the presence of layer-specific cues at
these stages (Supèr and Soriano, 1994). Using the organotypic
coculture approach, we have shown recently that a population of
hippocampal early neurons, the cells of Cajal-Retzius, is essential for
the ingrowth of entorhinal axons under these conditions in
vitro (Del Río et al., 1997). Further, we have identified
the reelin gene, which encodes an extracellular protein
highly expressed in Cajal-Retzius cells (D'Arcangelo et al., 1995,
1997; Hirotsune et al., 1995; Ogawa et al., 1995), as one of the
factors regulating the growth of entorhinal afferents (Del Río
et al., 1997).
In the present study, we have analyzed the development of entorhinal
and commissural/associational afferents to the mouse hippocampus
in vivo. We found that, at the time of ingrowth, these developing afferents are spatially related with specific populations of
hippocampal early neurons, which are arranged in specific laminae. Furthermore, at perinatal stages, hippocampal afferents establish preferential synaptic contacts with these layer-specific early neurons.
Together with the above studies, such a precise sequence of
developmental events indicates that these transient early neurons provide the laminar cues necessary for the targeting of hippocampal afferents, which constitutes a mechanism by which layer-specific neural
connections can be specified. On the basis of their early neurogenesis
and maturation and their participation in the formation of hippocampal
connections, we regard these early neurons as pioneer cells.
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MATERIALS AND METHODS |
Tracing studies. Time-pregnant mice (NMRI
strain) were obtained from IFFA Credo (Abresle, France). Embryos were
removed by caesarean section after ether anesthesia of the mother and
were stored in cooled PBS. Postnatal mice were anesthetized with ether or ice. Embryonic [embryonic day 15 (E15)-E19; n = 47] and postnatal [postnatal day 0 (P0)-P10; n = 21] mice were transcardially perfused with 4% paraformaldehyde. After
dissection, brains were injected with
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) or
4-(p-dihexadecylaminostyryl)-N-methylquinoloinium
iodide (DiQ) into the entorhinal cortex or the hippocampus as described (Supèr and Soriano, 1994). After 3-7 weeks in paraformaldehyde, 80-µm-thick vibratome sections were obtained, counterstained with bisbenzimide, and coverslipped. For tracing associational
connections, we injected 500-µm-thick vibratome slices with DiI in
the CA3 region; after 2 weeks, slices were sectioned as above. For the simultaneous labeling of pyramidal cells and entorhinal
fibers, DiQ and
4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide (DiA)
were injected in the entorhinal cortex and contralateral hippocampus.
Sections were examined in an epifluorescence microscope.
Embryonic (E19; n = 7) and postnatal (P0;
n = 14; P5; n = 17) mice were also
injected with crystals of biocytin in the entorhinal cortex or
hippocampus. After ether anesthesia, mice were placed onto a
stereotaxic frame mounted on chilled ice. After removal of the skin and
parietal bone, a crystal of biocytin was positioned in the entorhinal
cortex or hippocampus with the help of a glass micropipette. Eighteen
to twenty-four hours later, the animals were perfused with
paraformaldehyde, and their brains were dissected out. Older animals
(P10, P21, and young-adult mice) were traced by iontophoresis of
biocytin. After chloral hydrate anesthesia, mice were fixed to a
stereotaxic frame, a glass micropipette (tip, 25-20 µm) was
positioned in the entorhinal cortex or dorsal hippocampus, and biocytin
was injected (positive current; 7 sec on-off cycle; 20-25 min). After
1-2 d of survival, mice were perfused with 4% paraformaldehyde, and
their brains were dissected out and post-fixed in the same solution.
Coronal sections (50 µm thick) were obtained with a vibratome and
incubated overnight with the avidin-biotin-peroxidase complex (ABC;
Vector Laboratories, Burlingame, CA) diluted 1:200. After development
with 0.03% diaminobenzidine (DAB) and 0.01% hydrogen peroxide in the
presence of nickel (Del Río et al., 1997), sections were
counterstained and coverslipped.
Immunocytochemistry. Embryos (E14-E18) and postnatal
animals (P0-P45 and adults) were transcardially perfused with 4%
paraformaldehyde (plus 0.25% glutaraldehyde for GABA immunostaining).
Coronal vibratome sections (50 µm thick) were immunostained using
well characterized calretinin (Swant, Bellinzona, Switzerland),
calbindin (Swant), and GABA (Incstar) antibodies raised in rabbits and
the ABC-peroxidase complex (Soriano et al., 1994; Del Río et
al., 1995). Sections in which the primary antibody was omitted did not
reveal immunolabeling. For double-labeling studies, sections were
incubated with a mixture of goat anti-calretinin (Swant) and rabbit
anti-GABA or -calbindin antibodies and then with Texas Red- and
fluorescein-coupled secondary antibodies.
Double-labeling biocytin studies. E19 embryos, postnatal
mice (P0-P21), and adult animals were injected with biocytin in the entorhinal cortex or hippocampus as described above. After perfusion, the brains were sectioned with a vibratome (50 µm). Biocytin was then
visualized with the ABC reagent developed with DAB-nickel (black
reaction product); sections were then immunostained for neural antigens
(calretinin, calbindin, or GABA), which were visualized with DAB (brown
reaction product). In some cases, sections were incubated with Texas
Red-streptavidin for biocytin and with fluorescein-conjugated antibodies for neural antigens.
5'-Bromodeoxyuridine studies. For the
5'-bromodeoxyuridine (BrdU) experiments, time-pregnant dams were
injected with a single BrdU pulse at E10, E11, E12, E13, E15, or E16
(n = 2-3 dams per age). Offspring were killed at E18,
P0, P15, P15, and P45 (Soriano and Del Río, 1991). After
perfusion with paraformaldehyde, brains were fixed in Carnoy's
solution, embedded in paraffin, and coronally sectioned at 15 µm. After DNA denaturation, sections were immunolabeled for
BrdU as described, using a DAB-nickel-enhanced reaction (Soriano and Del Río, 1991), counterstained with hematoxylin, and
coverslipped.
Cell counts. For all cell counts, the total number of
immunoreactive (or BrdU-labeled) neurons present in the entire stratum lacunosum/moleculare, stratum radiatum, pyramidal layer, and stratum oriens of single sections (from the CA3 region to the subiculum) was
counted in coronal sections of the dorsal hippocampus of mice aged P0,
P5, P15, P21, and P45. Data were expressed as the total number of cells
per section. All sections counted corresponded to equivalent
rostrocaudal levels [F, 1.5-2.0, according to Slotnick and
Leonard (1975)]. This procedure, i.e., counting the total number of
cells in a section, avoids cell dilution attributable to the
mediolateral and dorsoventral growth of the telencephalon (for details,
see Del Río et al., 1995; Valverde et al., 1995). Crude cell
counts were then corrected for rostrocaudal dilution by multiplying by
a factor of rostrocaudal increase of the hippocampus (see Del
Río et al., 1995) (P0, being 1; P5, ×1.3; P15, ×1.8; P21,
×2.2; and P45, ×2.3). Sections from two to four animals were counted
at each stage (12-16 sections per age).
Electron microscopy studies. Postnatal mice (P0-P5)
and adult animals were injected with biocytin as described above.
Animals were then perfused with 4% paraformaldehyde and 0.2-1%
glutaraldehyde in 0.12 M phosphate buffer; their brains
were sectioned in a vibratome and processed for the detection of
biocytin with the ABC reagent as described above. For entorhinal
injections, biocytin was developed with DAB-nickel, and the sections
were subsequently immunostained for calretinin, which was visualized
with DAB alone (Gulyás et al., 1993). Sections were then osmified
and embedded in Araldite. Ultrathin sections were stained with lead
citrate and examined in the electron microscope. For commissural
injections, vibratome slices were developed with DAB to visualize
biocytin, osmified, and embedded in Araldite. Ultrathin sections were
then immunostained using the GABA-9 antibody and gold (10 nm)-coupled
secondary antibodies as described (Somogyi et al., 1985). For the
quantitative data, ultrathin sections mounted onto single slot grids
were positioned in the stratum lacunosum-moleculare or stratum
radiatum, and the microscope was moved at random over a given layer.
Every synaptic contact formed by a biocytin-positive bouton was
photographed and recorded. Two animals were used at each stage for
either commissural and entorhinal injections (115-131 synapses
harvested at each age).
Delimitation of layers. The laminar boundaries in the
developing hippocampus were delimited according to the
cytoarchitectonic criteria described in our previous studies (Soriano
et al., 1994; Supèr and Soriano, 1994; Del Río et al.,
1997) using Nomarski interference optics (in immunohistochemical
preparations) or bisbenzimide staining (for DiI tracing). At embryonic
and perinatal stages, the following layers were distinguished:
ventricular zone, white matter (intermediate zone), stratum oriens
(subplate zone), pyramidal cell layer (hippocampal plate), and stratum
radiatum and stratum lacunosum-moleculare (both in the marginal zone).
The embryonic white matter appeared as a fiber-rich layer located just
above the ventricular layer. The stratum oriens was comprised between the white matter and the pyramidal cell layer, which at these stages
(E15-E18) was five to eight cells thick. The stratum radiatum (inner
marginal zone) was located just above the pyramidal layer and, at these
stages, corresponded to the inner half of the marginal zone; the
stratum radiatum was populated by many more cells than was the outer
marginal zone. The stratum lacunosum-moleculare (outer marginal zone)
was located between the stratum radiatum and the hippocampal fissure,
approximately corresponding to the outer half of the marginal zone.
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RESULTS |
Developing hippocampal afferents are targeted in a
layer-specific manner
The development of hippocampal connections in the mouse was
studied with injections of lipophilic dyes (DiI) and biocytin. In
agreement with a previous study (Supèr and Soriano, 1994), entorhinal axons labeled after injections made in the entorhinal area
reached the target hippocampus at embryonic day 15 (E15). One day
later, entorhinal fibers grew selectively into the outer marginal zone
(prospective stratum lacunosum-moleculare), where they arborized
densely by E17-E19 (Fig.
1A). There was
virtually no invasion of ingrowing entorhinal fibers into other
hippocampal layers such as the stratum radiatum or the hilar region.
Conversely, CA3-to-CA1 associational axons labeled after local DiI
injections in the CA3 region were seen to terminate specifically in the
inner marginal zone (future stratum radiatum) with no invasion of the stratum lacunosum-moleculare (Fig. 1B), as previously
shown for commissural fibers (Supèr and Soriano, 1994). Thus,
developing hippocampal afferents innervate their specific target layer
from the very beginning without exuberant growth in inappropriate
layers, indicating that cues allowing target-layer selection are
present at these embryonic ages.
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Figure 1.
Layer-specific termination of developing
connections in the hippocampus. A, Distribution of
entorhinal fibers at E18 in the slm and in the
wm, after an entorhinal injection of DiI.
B, Distribution of associational fibers at E18 in the
sr of the CA1 region after an injection of DiI in the
CA3 region. C, E, Visualization of
anterogradely traced entorhinal fibers and retrogradely labeled
pyramidal cells in the CA1 region at E18 and P2, showing entorhinal
afferents running above pyramidal cell dendrites. In C,
entorhinal axons were labeled with DiA (green),
and pyramidal neurons were labeled with DiQ (red); the
reverse combination was used in E. D,
Distribution of DiI-stained pyramidal cell apical dendrites at P0. Note
that these dendrites terminate before the slm. Sections
in A and B are counterstained with
bisbenzimide. Scale bars: A, 250 µm; B,
100 µm; C-E, 50 µm. CA1,
CA3, Hippocampal subfields; DG, dentate
gyrus; F, fimbria; slm, stratum
lacunosum-moleculare; sp, stratum pyramidale;
sr, stratum radiatum; wm, white
matter.
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We next examined whether the dendrites of pyramidal neurons, the main
targets of entorhinal and commissural/associational fibers in the
adult, could play a role in such a guidance. Retrograde tracing
experiments with DiI, however, showed that at E16-P2 the growing
apical dendrites of pyramidal neurons terminated at the stratum
radiatum and stratum lacunosum-moleculare interphase, with no growth
into the outer marginal zone, long before reaching the hippocampal
fissure (Fig. 1D). Double-labeling retrograde and
anterograde experiments with two lipophilic dyes confirmed that most
entorhinal axons ran above pyramidal cell dendrites (Fig.
1C,E), indicating that positional cues for the
targeting of entorhinal fibers are unlikely to be present in these
dendrites. However, the dendrites of pyramidal neurons in the stratum
radiatum could potentially be in a suitable position to influence or
target commissural/associational fibers.
Layer-specific arrangement of pioneer neurons in
the hippocampus
In the neocortex the subplate is involved in the development of
thalamocortical connections (McConnell et al., 1989; Ghosh et al.,
1990; Allendoerfer and Shatz, 1994). We next studied the developmental
history of hippocampal pioneer neurons (Nowakowski and Rakic, 1979;
Soriano et al., 1994) and their putative involvement in the formation
of hippocampal circuits. From E15 to P5, two main subsets of pioneer
neurons were found in the marginal zone of the hippocampus.
Cajal-Retzius cells, identified using a calretinin antibody marker
(Soriano et al., 1994; Del Río et al., 1995), were densely
packed in the outer marginal zone or stratum lacunosum-moleculare (Fig.
2A,C).
In contrast, GABA-positive neurons were located in the inner marginal
zone or stratum radiatum (Fig. 2E). Most such GABA-positive neurons (73% at P0; n = 612) also
displayed calbindin 28K, a more suitable antibody cell marker than
GABA, immunoreactivity (Fig. 2B,D).
Double immunolabeling confirmed that at perinatal stages Cajal-Retzius
cells and GABA-positive or calbindin-positive neurons were distributed
in distinct, nonoverlapping layers (Fig. 2F).
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Figure 2.
Distribution and neurogenesis of pioneer neurons
in the hippocampus. A, B, Panoramic views
of the hippocampus at E18 immunolabeled with calretinin and calbindin
antibodies. In A, Cajal-Retzius cells
(arrows), identified with calretinin antibodies, are
located in the stratum lacunosum-moleculare or outer marginal zone; the
immature granule cells in the dentate gyrus also display weak
calretinin immunolabeling. In contrast, calbindin-positive neurons
(B) are located in the stratum radiatum or inner
marginal zone but not in the stratum lacunosum-moleculare.
C-E, Higher views of the hippocampus at E18 showing the
distinct laminar distributions of calretinin-positive Cajal-Retzius
cells (C) and calbindin- and GABA-immunoreactive
neurons (D, E, respectively).
F, Double-immunofluorescence confocal photomicrograph
showing nonoverlapping distribution of calretinin-positive
Cajal-Retzius cells (red) and calbindin-positive neurons
(green) at P0. G, Distribution of
BrdU-positive neurons in the E18 hippocampus after a BrdU pulse at E11.
BrdU-immunoreactive cells (black) are mainly present in
the stratum radiatum and stratum lacunosum-moleculare, as well as in
the stratum oriens. Note that pyramidal neurons in the pyramidal layer
are devoid of labeling after a BrdU pulse at E11. H,
Confocal fluorescence photomicrograph showing BrdU labeling
(red) of calretinin-positive Cajal-Retzius cells
(green) in the stratum lacunosum-moleculare after
a BrdU pulse at E11; arrows point to double-labeled
cells. I, BrdU immunolabeling of a pyknotic cell
(arrow) and a healthy neuron (open arrow)
in the stratum lacunosum-moleculare at P15, after an injection of BrdU
at E10. Sections in G and I are
counterstained with hematoxylin. J, BrdU immunostaining
of a section from a P15 hippocampus after a BrdU injection administered
at E11. Note the dramatic decrease in the number of BrdU-positive cells
(arrows) present in the stratum radiatum and stratum
lacunosum-moleculare compared with that at E18
(G). Scale bars: A,
B, 300 µm; C-E, G, 200 µm; F, 25 µm; H, J,
100 µm; I, 50 µm. DG, dentate gyrus;
F, fimbria; gl, granular layer;
ml, molecular layer; slm, stratum
lacunosum-moleculare; so, stratum oriens;
sp, stratum pyramidale; sr, stratum
radiatum.
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To investigate the birthdates of these pioneer neurons in the
hippocampus, mouse embryos were labeled with a single-pulse injection
of BrdU (Soriano and Del Río, 1991). At E18-P0, most BrdU-positive neurons born at E10-E12 were in the outer marginal zone
or stratum lacunosum-moleculare and in the inner marginal zone or
stratum radiatum (Figs. 2G,
3C); they were also observed, to a lesser extent, in the subplate or stratum oriens. In contrast, the
neurons in the pyramidal layer were primarily devoid of BrdU labeling
after early BrdU injections at E10-E12 (Fig. 2G). Double immunostaining with calretinin and calbindin 28K antibodies showed that
most hippocampal neurons born at E10-E11 were Cajal-Retzius cells
(Fig. 2H) and GABA-positive neurons (data not shown).
Conversely, hippocampal pioneer neurons were unlabeled when BrdU
injections were administered at E15-E16, which resulted in massive
labeling of pyramidal neurons in both the CA3 and CA1 regions (Fig.
3C), in agreement with earlier studies reporting that
pyramidal cell neurogenesis in the mouse hippocampus peaks by E14-E16
(Soriano et al., 1989). We therefore conclude that the hippocampal
marginal zone, the prospective stratum lacunosum-moleculare and stratum radiatum, contains two different subsets of early-generated pioneer neurons arranged in a nonoverlapping manner.
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Figure 3.
Quantitative analyses on the neurogenesis
and fate of pioneer neurons in the hippocampus. Data are expressed as
the number of labeled cells per section. A,
B, Number of calretinin-positive Cajal-Retzius cells
(A) in the stratum lacunosum-moleculare and of
GABA-positive neurons (B) in the stratum radiatum
at different postnatal ages (P0-P21) and in the adult. Solid
bars are crude cell counts, whereas hatched bars
are values corrected for a factor of cortical growth (see Materials and
Methods). C, Laminar distribution of BrdU-positive cells
at P0 after a BrdU pulse injection at E10, E11, E12, or E16. Pioneer
neurons in the stratum radiatum and stratum lacunosum-moleculare are
generated at E10-E12, whereas the far more numerous pyramidal neurons
are born later. D, E, Comparison of the
numbers of BrdU-positive cells in the stratum lacunosum-moleculare and
stratum radiatum in littermates aged P0 and P45 after BrdU injections
administered at E10, E11, and E12. Values at P45 are corrected for a
factor of cortical growth as in A and B
(** indicates p  0.01; ANOVA, Scheffé's test).
AD, Adult; CALR, calretinin;
SLM, stratum lacunosum-moleculare; SO,
stratum oriens; SP, stratum pyramidale;
SR, stratum radiatum.
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Pioneer neurons in the hippocampus are transient
As counted from calretinin-immunostained sections, the number of
Cajal-Retzius cells in the stratum lacunosum-moleculare decreased gradually between P5 and adult stages, mostly after P15 (Fig. 3A). Few calretinin-positive Cajal-Retzius cells could be
observed in the adult stratum lacunosum-moleculare (82% decrease
respect to P5). During the period of disappearance, most Cajal-Retzius cells showed shrunken perikarya and atrophic dendrites, which are
morphological correlates of neuronal death (Valverde and
Facal-Valverde, 1987; Del Río et al., 1995). To substantiate
the notion that Cajal-Retzius cells disappear by naturally occurring
cell death, we counted the number of BrdU-positive cells generated at
E10-E12 that are present in hippocampal sections from P0 onward. In
the stratum lacunosum-moleculare, we found a 60-79% decrease in the number of BrdU-labeled cells present at P45 compared with that at P0
(Figs. 2J, 3D), with most of this decrease
occurring between P15 and adults (data not shown). A marked reduction,
although less dramatic than that for Cajal-Retzius cells, was also
noted for the GABA-positive neurons in the stratum radiatum, by cell counts made in both GABA- and BrdU-immunoreacted sections (Fig. 3B,E). Such a decrease occurred
mainly between P5 and P15. Consistent with the notion that many pioneer
neurons die, we often observed at P5-P15 pyknotic cells exhibiting
BrdU immunostaining (Fig. 2I) in the stratum radiatum
and stratum lacunosum-moleculare, after BrdU injections administered at
E10-E12. This observation demonstrates unequivocally that at least
some early-generated neurons in the hippocampus disappear by cell
death. We thus conclude that most pioneer neurons in the marginal zone
of the hippocampus, in particular Cajal-Retzius cells, are transient
and disappear by cell death at the end of postnatal development.
Relationship of developing hippocampal afferents and
pioneer neurons
To investigate whether hippocampal pioneer neurons participate in
the formation of hippocampal connections in vivo, we
combined anterograde tracing of biocytin with immunostaining for
pioneer cells. After injections of biocytin in the entorhinal cortex at E19-P5, the distribution of entorhinal axons in the stratum
lacunosum-moleculare matched that of Cajal-Retzius cells (Fig.
4A). Often, single
fibers impinged onto the dendrites and perikarya of clusters of
Cajal-Retzius cells and silhouetted their shapes (Fig.
4B). Conversely, the earliest commissural axons
labeled by biocytin injections in the contralateral hippocampus at
P0-P5 were observed to run and arborize specifically in the stratum
radiatum and stratum oriens, where they overlapped with the
GABA/calbindin-positive neurons (Fig. 4D,E).
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Figure 4.
Overlapping distribution of developing hippocampal
afferents and pioneer neurons in the hippocampus. A,
Fascicles of entorhinal axons (black arrows) traced with
biocytin overlap with Cajal-Retzius cells in the stratum
lacunosum-moleculare identified by calretinin immunostaining
(brown open arrows) at E19. B, High-power
fluorescence photomicrograph shows entorhinal axons
(red) intermingled with calretinin-positive
Cajal-Retzius cells (green) at P0 in the stratum
lacunosum-moleculare of the CA3 region. C, In the
dentate gyrus, ingrowing entorhinal axons (black) at P2
are restricted to the outer molecular layer (close to the
HF), forming patches (arrows)
around the clusters of Cajal-Retzius cells (brown) in
this layer. D, E, Photomicrographs show
that commissural fibers traced with biocytin at P2 (D,
black; E, red) arborize in
the stratum oriens and in the stratum radiatum, where
calbindin-positive neurons (D, brown;
E, green) are located. F,
G, In the dentate gyrus, commissural axons
(black) are restricted to the inner molecular layer at
P5, where they overlap with GABA-positive neurons
(brown; arrows in G). The
section in F was slightly stained with thionin. Sections
were photographed under Nomarski (A, C,
D, F, G) or
epifluorescence (B, E) optics. Scale
bars: A, 300 µm; B and
E, C, F and
G, 50 µm; D, 100 µm.
DG, dentate gyrus; gl, granular layer;
H, hilus; HF, hippocampal fissure;
ml, molecular layer; slm, stratum
lacunosum-moleculare; so, stratum oriens;
sp, stratum pyramidale; sr, stratum
radiatum.
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Similar populations of pioneer neurons were found in the late-formed
dentate gyrus at perinatal stages. Thus, Cajal-Retzius cells were
located in the outer half of the dentate molecular layer, close to the
hippocampal fissure, and GABA-positive neurons formed a tight band of
neurons restricted to the inner molecular layer (Fig.
4C,G). Again, the first entorhinal afferents
entering the dentate gyrus on P0-P2 were restricted to the outer
molecular layer, where axons ran and intermingled in close apposition
to dentate Cajal-Retzius cells (Fig. 4C). Similarly,
ingrowing commissural fibers were restricted to the inner molecular
layer, where they overlapped with the GABA-positive cells present in
this target layer (Fig. 4F,G). Both
in the hippocampus proper and in the dentate gyrus, the close
relationship of developing afferent fibers and pioneer neurons seen at
perinatal stages was progressively lost after P5, as the pioneer
neurons disappear and the afferent connection systems mature (data not
shown). We thus conclude that at the time of axon arrival and
layer-specific invasion, developing hippocampal afferents are spatially
related to distinct pioneer neurons.
Hippocampal pioneer neurons are transient synaptic targets for
developing afferents
We next used electron microscopy to ascertain whether pioneer
neurons could be synaptic targets for developing axons at perinatal stages. We used a double DAB reaction to identify entorhinal axons and
calretinin-positive target Cajal-Retzius cells at the electron microscope. There are relatively few synaptic contacts in the stratum
lacunosum-moleculare at P0. In agreement with this, biocytin-labeled entorhinal axons were seen to run within this layer, sometimes in
axonal bundles, and they displayed few differentiated axon terminals
along their length. These presynaptic axon terminals showed
accumulations of synaptic vesicles and established asymmetric synapses
with both Cajal-Retzius cells (Fig.
5A,B)
and other unlabeled profiles. At P0, up to 51% of entorhinal synaptic
contacts observed in the stratum lacunosum-moleculare were established
on calretinin-positive processes and perikarya corresponding to
Cajal-Retzius cells (Fig. 6A). This value is
likely to represent an underestimation because not all
calretinin-immunoreactive processes could be labeled by pre-embedding
immunostaining. The percentage of entorhinal boutons in synaptic
contact with calretinin-positive elements decreased to 28% at P5 and
to 3% in the adult (Fig. 6A).
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Figure 5.
Cajal-Retzius cells and GABA-positive neurons are
transient synaptic targets of entorhinal and commissural axons.
A, B, Electron micrographs show
biocytin-labeled entorhinal axon terminals (AT)
at P0 in asymmetric synaptic contact (arrows) with the
dendrites of Cajal-Retzius cells immunostained for calretinin.
Biocytin-positive boutons are identified by the homogenous,
electron-dense reaction product (DAB-nickel), whereas calretinin
immunostaining shows a granular, less dense precipitate (DAB).
C, D, Biocytin-positive commissural axon
terminals (AT) at P2 (D)
and P5 (C) establish asymmetric synaptic contacts
(large arrows) with GABA-positive dendrites identified
by the presence of gold particles (small arrows). Scale
bar, 0.4 µm.
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|
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Figure 6.
Percentage of entorhinal (A)
and commissural (B) boutons that are in synaptic
contact on calretinin-positive Cajal-Retzius cells and on GABA-positive
elements at different postnatal stages. AD, Adult;
CR, Cajal-Retzius.
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|
To analyze the targets of developing commissural axons, we combined
anterograde tracing of biocytin visualized by a DAB reaction and
postembedding immunogold staining for GABA. Synapses made by
commissural fibers in the stratum radiatum at P2 were also relatively
rare at P2. As for entorhinal axons, biocytin-labeled commissural axons
established asymmetric synaptic contacts (Fig. 5C,D). At these early postnatal stages, most
GABA-immunoreactive profiles were dendrites and perikarya, but a few
GABA-positive axon terminals forming symmetric synapses were recognized
(data not shown). As reported in other studies (Somogyi et al., 1985), immunogold particles were frequently associated with diverse cell membranes and mitochondria (Fig.
5B,C). At P2, up to 61% of
biocytin-labeled commissural synapses in the stratum radiatum were on
GABA-positive elements (Fig. 6B). This value
decreased to 52% at P5. In the adult hippocampus, GABA-immunoreactive
targets of commissural axon terminals represented less that 4% (Fig.
6B). Taken together, the above findings show that
Cajal-Retzius cells are main transient synaptic targets for ingrowing
entorhinal afferents in the stratum lacunosum-moleculare and that early
commissural axons prefer GABA-positive neurons as targets instead of
the far more numerous pyramidal cell dendrites that are present in the
stratum radiatum. Later in development, most pioneer neurons disappear,
and synaptogenesis takes place onto the late-maturing principal cells,
pyramidal neurons, and dentate granule cells.
| |
DISCUSSION |
In the present study, we have shown that the prospective
hippocampus contains two distinct populations of pioneer neurons, namely Cajal-Retzius cells and GABA-positive neurons, which are arranged from very early stages in different, nonoverlapping sublayers in the marginal zone. These distinct subsets of pioneer neurons form
early layer-specific scaffolds that are used as guides or "blueprints" for the ingrowth and layer-specific targeting of entorhinal and commissural/associational afferents. Developing hippocampal afferents are not only tightly related spatially to distinct subsets of pioneer neurons but also form synaptic contacts preferentially with them. Such a precise sequence of developmental events strongly indicates that these pioneer neurons participate in the
normal development of hippocampal connections (Fig.
7). This conclusion is supported by an
earlier study reporting that the laminar specification of hippocampal
connections does not depend on the temporal order of fiber arrival
(Frotscher and Heimrich, 1993) and by our parallel study showing that
the ablation of Cajal-Retzius cells in organotypic slice cocultures
prevents the ingrowth and formation of entorhinohippocampal connections
in vitro (Del Río et al., 1997). Thus, we propose
here a developmental mechanism by which different afferents could be
targeted to distinct dendritic domains of the same neurons, thereby
forming a layer-specific pattern of connectivity (Fig. 7).
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Figure 7.
Schematic representation summarizing the main
findings of the present study. At late embryonic (E16-E19) and early
postnatal stages, developing hippocampal afferents invade the target
hippocampus in a layer-specific manner. These ingrowing fibers overlap
with Cajal-Retzius cells in the stratum lacunosum-moleculare and with
GABA-positive neurons in the stratum radiatum and establish transient
synaptic contacts with them. At later postnatal stages and in the
adult, most Cajal-Retzius cells and approximately half the population
of GABA-positive neurons disappear, and hippocampal afferents form
synaptic contacts with the principal pyramidal cells.
IMZ, Inner marginal zone; OMZ, outer
marginal zone; SLM, stratum lacunosum-moleculare;
SP, stratum pyramidale; SR, stratum
radiatum; C/A, commissural/associational fibers;
EC, entorhinal cortex axons; HP, hippocampal
plate.
|
|
What is the significance of this process? In the murine hippocampus,
neurogenesis of pyramidal neurons peaks on E14-E15 in the CA3 region
and on E14-E16 in the CA1 subfield (Caviness, 1973; Soriano et al.,
1986, 1989). Thus, at the time of afferent ingrowth (e.g., E15-E17 for
entorhinal axons), most target pyramidal neurons are still
proliferating or migrating in the intermediate zone, which virtually
eliminates their participation in the targeting of hippocampal
afferents to the upper marginal zone. Indeed, considering that a few
pyramidal neurons are already positioned at these (or immediately
subsequent) stages in the pyramidal cell layer, it is unlikely that
distinct recognition cues could be segregated in a segment-specific
manner over the membranes of pyramidal cell dendrites, which at this
time are undergoing rapid remodeling and growth. The early ingrowth and
synaptogenesis of hippocampal afferents, in turn, is likely to be
needed to regulate other aspects of hippocampal development and
maturation (Mattson et al., 1988; Frotscher et al., 1995). A similar
principle might operate in the development of laminated connections in
other brain regions such as the optic tectum (e.g., Cheng et al., 1995;
Drescher et al., 1995; Yamagata et al., 1995).
The molecules involved in the ingrowth, layer-specific targeting and
specification of hippocampal circuits are unknown. In a previous study,
we have shown that the extracellular matrix protein reelin regulates
collateral branching and elongation of entorhinal axons, although it is
not essential for the ingrowth of developing entorhinal afferents (Del
Río et al., 1997). Similarly, recent analysis of the
development of entorhinal and commissural projections to the
hippocampus of trkB- and trkC-deficient mice indicates that neurotrophin signaling is not essential either for the
ingrowth or for the layer-specific targeting of hippocampal afferents
(A. Martínez, S. Alcántara, J. A. Del Río,
V. Borrell, M. Barbacid, I. Silos-Santiago, and E. Soriano, unpublished
observations), which is in agreement with a recent study in the optic
tectum (Inoue and Sanes, 1997). However, consistent with current views on neurotrophin action in development (Cabelli et al., 1995, 1997; Cohen-Cory and Fraser, 1995; Bonhoeffer, 1996; Inoue and Sanes, 1997),
both TrkB and TrkC receptors influence the branching and maturation of
hippocampal afferents. The laminar precision shown by developing
hippocampal axons from the earliest invasion of the target region
together with their tight relationships with pioneer neurons suggest
that, as in other brain regions, both chemoattractant and repulsive
factors, either diffusible or membrane-bound, may play a role (Goodman
and Shatz, 1993; Kennedy et al., 1994; Serafini et al., 1994; Cheng et
al., 1995; Drescher et al., 1995; Kennedy and Tessier-Lavigne, 1995;
Matthes et al., 1995; Messersmith et al., 1995; Chan et al., 1996;
Flenniken et al., 1996; Keino-Masu et al., 1996). In addition, some
extracellular matrix proteins, such as reelin, and adhesion molecules
have also been implicated in the lamina-specific connectivity in the
brain (Del Río et al., 1997; Inoue and Sanes, 1997). Some of
these cues are likely to be expressed by the present pioneer neurons.
Recent studies have reported, in fact, that developing hippocampal
tissue prevents the axonal outgrowth from neocortical explants
(Halloran and Kalil, 1996) and that several eph-receptors and ligands
are highly expressed in the developing hippocampus (Gao et al., 1996;
Zhang et al., 1996). Also, the phenotype of both netrin-1
and deleted in colorectal cancer (DCC) null-mutant mice,
with complete absence of the hippocampal commissure, suggests the
participation of netrin-1 in the development of hippocampal connections
(Serafini et al., 1996; Fazeli et al., 1997).
Our cell counts on both BrdU- and neural antigen-immunoreacted sections
show that ~75% of Cajal-Retzius cells disappear by cell death in the
hippocampus, which is consistent with the sparsity of neurons in the
adult stratum lacunosum-moleculare (Freund and Buzsaki, 1996). However,
the disappearance of Cajal-Retzius cells occurs slightly later in the
hippocampus than in the neocortex (Derer and Derer, 1992; Del
Río et al., 1995), consistent with the relatively late
development of the hippocampus and dentate gyrus. In contrast,
~40-50% of the early GABA-positive pioneer neurons located in the
stratum radiatum persist at adult stages. This is also consistent with
the abundant population of GABA-positive neurons present in the adult
stratum radiatum (Freund and Buzsaki, 1996) and with birthdating
analysis showing that most such neurons are generated early (Soriano et
al., 1989). Although discovered more than one century ago, the
functions of Cajal-Retzius cells in cortical development have remained
elusive (Marín-Padilla, 1972, 1978, 1988; Del Río et
al., 1995). Recently, studies reporting that reelin, the gene product
affected in the reeler mutation, is expressed by
Cajal-Retzius cells have strongly indicated a role for these neurons in
migration (D'Arcangelo et al., 1995; Hirotsune et al., 1995; Ogawa et
al., 1995). The present findings and those of our parallel study
in vitro (Del Río et al., 1997) show that
Cajal-Retzius cells are also involved in axonal growth and target-layer
selection, at least in the hippocampal region. This finding supports
the notion that these pioneer neurons subserve different and essential
functions in corticogenesis (Marín-Padilla, 1988).
The role proposed here for the pioneer neurons in the marginal zone of
the hippocampus, i.e., target selection by developing afferents, is
strikingly similar to that subserved in the neocortex by subplate
neurons for thalamocortical axons (Rakic, 1976, 1977; McConnell et al.,
1989; Ghosh et al., 1990; Allendoerfer and Shatz, 1994). Many studies
have indicated previously that in the hippocampus the subplate is
poorly developed, whereas the marginal zone contains many more neurons
than does the subplate (e.g., Stensaas, 1967; Nowakowski and Rakic,
1979; Soriano et al., 1994). The opposite holds true for the developing
neocortex, in which the marginal zone forms a very thin layer, whereas
the subplate, especially in carnivores and primates, is one of the most
prominent layers (Marín-Padilla, 1978; Kostovic and Rakic,
1980, 1990; Chun et al., 1987; Allendoerfer and Shatz, 1994). Given the
parallel functional significance of the subplate and marginal zone, it
is tempting to speculate that relative differences in the abundance and
location of the two main groups of pioneer neurons (marginal zone
neurons vs subplate cells) could be essential in directing the route of entry and pattern of termination of cortical afferents and the resulting cortical organization. It is noteworthy, for instance, than
in the neocortex of the reeler mutant mouse, in which
subplate neurons are located directly underneath the cortical surface, thalamocortical axonal tracts perforate the cortical gray matter to
reach the subplate neurons and then descend toward their target neurons
(Molnár and Blakemore, 1995). Thus, in conjunction with current
views of cortical evolution (Rakic, 1988; Northcutt and Kaas, 1995),
major developmental differences in the preplate populations may be a
relevant parameter to understand cortical ontogeny and phylogeny,
especially neo- versus archicortical specification and evolution.
| |
FOOTNOTES |
Received Oct. 7, 1997; revised March 20, 1998; accepted March 26, 1998.
This study was supported by grants from Comisión Interministerial
de Ciencia y Tecnología, Spain (SAF94-743, SAF96-1356-E, and
SAF98-106), Dirección General de Investigacion Científica y
Tecnológica (P.M.95-102), and Comissiò Interdepartamentol de Recerca i Tecnología (GR94-534) to E.S. and J.A.D.R. and from The Ramón Areces Foundation and the International Institute for Research in Paraplegia to E.S. and by a European Community fellowship to H.S. We thank G. Raisman (London), R. Rycroft (Barcelona), and
H. B. M. Uylings (Amsterdam) for critical reading of an
earlier version of this manuscript and P. Somogyi (Oxford) for the gift of GABA-9 antibody.
Correspondence should be addressed to Dr. Eduardo Soriano, Department
of Animal and Plant Cell Biology, Faculty of Biology, University of
Barcelona, Diagonal 645, Barcelona 08028, Spain.
| |
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Top
Abstract
Introduction
