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First published online 3 May 2006
doi: 10.1242/dev.02382
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1 Graduate School of Frontier Biosciences, Osaka University, and SORST, Japan
Science and Technology Corporation (JST), Yamadaoka 1-3, Suita, Osaka
560-8531, Japan.
2 Department of Genetic and Behavioral Neuroscience, Gunma University Graduate
School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan.
3 Neural Circuit Mechanism Group, BSI, RIKEN, Hirosawa 2-1, Wako, 351-0198,
Japan.
* Author for correspondence (e-mail: murakami{at}fbs.osaka-u.ac.jp)
Accepted 29 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Neuronal migration, Cortical interneuron, Time-lapse imaging, DiD labelling, Multidirectional tangential migration
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cerebral cortex is responsible for higher brain functions. Its
structure results from the orderly migration of two major types of neuron, the
glutamatergic projection neurons and the GABAergic interneurons. Glutamatergic
projection neurons arise in the germinal zones lining the lateral ventricle
and migrate radially within the cortex toward the pial surface (for reviews,
see Nadarajah and Parnavelas,
2002
; Kriegstein and Noctor,
2004). The migration of GABAergic interneurons involves a more
complex process, because most cortical interneurons originate from the
ganglionic eminences (GEs) located in the subpallial telencephalon. These
neurons first migrate tangentially toward the pallium and then populate the
cortex (for reviews, see Marín and
Rubenstein, 2001; Marín
and Rubenstein, 2003;
Parnavelas, 2000).
Although the migration from the GEs to the cortex is well documented,
relatively little is known about the migration of interneurons within the
cortex. During early development (from about E13.5 in mice), many interneurons
in the lower intermediate zone/subventricular zone (IZ/SVZ) migrate
dorsomedially towards the hippocampus
(Anderson et al., 2001;
Jimenez et al., 2002;
Tanaka et al., 2003). Some of
these cells appear to change their migratory direction, moving obliquely or
radially toward the MZ (Tanaka et al.,
2003). With the development of the cortical plate (CP; after about
E15.5), various modes of radial migration can be seen: from the IZ and CP
toward the pial surface (Tanaka et al.,
2003); from the IZ-ventricular zone (VZ) to the ventricular
surface (Nadarajah et al.,
2002); and from the MZ toward the ventricle
(Ang, Jr et al., 2003;
Tanaka et al., 2003). These
migration modes may be crucial in determining the final locations of the
cortical interneurons. Most of these previous observations, however, were made
in the coronal plane and therefore provide only fragmentary information about
intracortical neuronal migration, particularly with respect to the tangential
plane.
To clarify how cortical interneurons achieve appropriate locations within
the mature cortex, a three-dimensional examination of interneuronal migrations
is required. We, and others, recently carried out time-lapse imaging of MZ
interneurons using a flat-mount cortical preparation and unexpectedly found
that MZ interneurons migrate in all directions within the tangential plane
(Ang, Jr et al., 2003;
Tanaka et al., 2003). This
finding tempted us to examine this migratory behaviour in a more systematic
manner and quantitatively. Here, we show that multidirectional tangential
(MDT) migration of interneurons takes place in multiple zones of the cortex,
including the VZ. We further show that MDT migration occurs over long
distances.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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neo) mice
(Tamamaki et al., 2003;
Tsunekawa et al., 2005), which
will be referred to as GAD67-GFP mice. Experimental animals were obtained by
mating female ICR mice with heterozygous male mice. GAD67-GFP embryos could be
recognized by their GFP fluorescence. Noon of the day of vaginal plug
detection was termed embryonic day (E) 0.5, and E19.5 was referred to as
postnatal day (P) 0.
Although GAD65 (GAD2 – Mouse Genome Informatics), another isoform of
GAD, is expressed at early corticogenesis
(Lopez-Bendito et al., 2004)
and could also contribute to GABA synthesis, we confirmed that more than 96%
of GABA-immunoreactive neurons expressed GFP signal (n=458/471 and
1219/1270 at E13.5 and E15.5, respectively). Thus, GFP signal is expressed by
almost all interneurons at these developmental stages. For some experiments,
wild-type littermates or ICR mice (Clea Japan or SLC) were also used. All
experiments were performed in accordance with the Osaka University Guidelines
for the Welfare and Use of Laboratory Animals.
Real-time imaging in flat-mount preparations
Short-term (
2 hours) imaging
Real-time imaging of migrating GAD67-GFP-positive neurons (termed GFP
neurons hereafter) in flat-mount preparations was performed as described
previously (Tanaka et al.,
2003). Briefly, neocortical tissues from E13.5 or E15.5 GAD67-GFP
embryos were dissected and flat-mounted on membrane inserts with the
ventricular side down or up to analyse the GFP neurons in the MZ or VZ,
respectively, using a confocal microscope (MRC-1024; BioRad). Images were
taken from the tissue surface (at a depth of 0-50 µm for the MZ and 0-100
µm for the VZ) at 5 minutes intervals for 2-3 hours. A stack of images was
created from a series of four to ten consecutive images taken along the
z-axis at 10 µm intervals at every time point.
Long-term (>7 hours) imaging
Flat-mount preparations from neocortical wall were prepared as described
above. They were transferred to the temperature- and gas-controlled incubation
chamber (5% CO2, 95% O2 at 37°C) fitted onto a
microscope stage (BX50, Olympus). The chamber was filled with Opti-MEM I
reduced-serum medium containing 2.5% heat-inactivated fetal bovine serum, 2.5%
heat-inactivated horse serum, 1% N2 supplement (all from Invitrogen), 20 mM
D-glucose and 20 µg/ml streptomycin (Sigma). Labelled cells were
viewed through a water-immersion objective (20, NA=0.5 each). Images were
collected with a confocal microscope using 568 nm excitation and long-pass 585
nm emission filters. Images were taken from the tissue surface (at the depth
of 0-100 µm) at 20-minute intervals. A stack of images was created from a
series of 10 or 11 consecutive images taken along the z-axis at 12 or
14 µm intervals at every time point. Brightness and contrast were adjusted
for every frame using Adobe Photoshop 7.0 software.
Quantitative analysis of migratory behaviour in real-time imaging
Data of real-time imaging that showed notable tissue movement was
discarded. Because of the high density of GFP neurons, the analysis was
carried out for all individually distinguishable GFP neurons. To analyse the
rate and the direction of migration, a line was drawn connecting the position
of a cell at the outset with its position at the end of observations, and the
length and the angle of the line (medial direction was defined as 0° and
rostral as 90°) were measured. Because almost all neurons migrated in a
straight line during the observation period, the rate of migration was
calculated by dividing the length of the line by the observation period (2-3
hours). For the analysis of directionality, the horizontal plane was
subdivided into 24 or four sectors, and the proportion of migrating neurons in
each sector was scored. The angle and mean rate of migration were measured for
neurons with a rate of >5 µm/hour. Statistical analysis was done using
the Mann-Whitney U-test or 
2 test.
Observation of GFP neurons in tangential slices
Fixed GAD67-GFP brains (Tanaka et al.,
2003) were embedded in 4% low-melting point agarose (Nacalai
Tesque), and 50 µm-thick tangential slices were cut on a vibrating-blade
microtome (VT-1000; Leica Microsystems), at around the level of the MGE along
the rostrocaudal axis (Jimenez et al.,
2002), and then mounted onto slides. Images were captured using a
CCD camera (AxioCam; Zeiss) attached to an epifluorescence microscope (BX-60;
Olympus) or by a confocal microscope (MRC-1024 or TCS SP2 AOBS; Leica
Microsystems). The cortical zones were determined based on the density of GFP
neurons.
In vivo analysis of DiD-labelled neurons
Pregnant mice were deeply anaesthetized with sodium pentobarbitone
(Nembutal; Abbott; 100 mg/kg body weight) and laparotomies were performed to
expose their uteri. After removing the uterine wall and opening the yolk sac
of an embryo, a glass micropipette filled with
1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine
(DiD; Molecular Probes) solution (100 mg/ml in dimethylformamide) was fitted
to an injector (IM-30; Narishige); the pipe held vertically to the head of an
embryo (E15.5) and inserted into the frontal, parietal or the occipital
cortex, using a micromanipulator (Mk1; Singer instruments) under a dissecting
microscope. A small opening was made on the yolk sac around the injection
point to drain the amniotic fluid, which allowed us to make accurate
injections. The injection (<0.1 µl) was performed into the parietal
cortex so as not to label the LGE. After the injection procedure, embryos were
repositioned into the abdominal cavity; the abdominal wall and the skin were
then sutured.
Thirty-six or 72 hours after the injection procedure, embryos were removed and decapitated. Brains were fixed in 4% PFA in PB (0.1 M, pH 7.4) overnight at 4°C, and then transferred to phosphate-buffered saline (PBS; 0.1 M, pH 7.4) containing 0.1% EDTA and stored at 4°C. For flat-mount preparations, neocortical tissue encompassing the medial edge to the corticostriatal boundary was dissected and flat-mounted onto slides with the ventricular side down and coverslipped. For slice preparations, brains were embedded in 4% agarose (Nacalai Tesque), then sliced coronally or sagittally at 50 µm on a vibrating-blade microtome and stored at 4°C in PBS with 0.1% EDTA. The slices were transferred to slides and coverslipped.
Images were then captured using a confocal microscope (MRC-1024 or TCS SP2 AOBS). The following filter sets were used with the indicated wavelengths (in nm) for the excitation laser line and emission filters: GFP (green), 488/(504.5-539.5) with MRC-1024 or 488/(500-530) with TCS SP2 AOBS; DiD (far red), 647/(664-696) with MRC-1024 or 633/(650-730) with TCS SP2 AOBS. For the analysis of double labelling with DiD and GFP in individual neurons, the confocal pinhole was set to reduce the depth of optical sectioning as much as possible, and the same pinhole setting was used across channels to ensure equal section thickness for the different channels. Projection images were constructed from z-stacks (sets of stacked optical sections) for the analysis of morphology. Images were analysed using appropriate software supplied by the microscope manufacturer. Brightness and contrast were adjusted for every frame using Adobe Photoshop 7.0 software.
To examine the possibility that some DiD-labelled neurons had been labelled retrogradely, we injected DiD into fixed brains. For this, E18.5 GAD67-GFP embryos were removed from deeply anaesthetized pregnant mice and decapitated. Brains were then removed and DiD was injected into the dorsal cortex as described above. The brains were fixed immediately in 4% PFA in PB with 0.1% EDTA and kept in the fixative in the dark at room temperature (RT) over one month. Slices were then prepared and imaged as described above.
Quantitative analysis of DiD-labelled GFP neurons
Because a high density of labelled neurons near the injection site
interfered with the analysis, the analysis was carried out for all
DiD-labelled GFP neurons located >400 µm or >600 µm from the
injection site in slices or flat-mount preparations, respectively.
Slice preparations
The length of a straight line connecting the position of each DiD-labelled
GFP neuron and the injection site (the point in the same zone) was defined as
the migration distance. The distance thus measured should be an underestimate
because the curved contour of the cortex. The zonal position of DiD-labelled
GFP neurons was determined referring to the GAD67-GFP cell distribution
pattern, which could be correlated to the pattern by Nissl staining
(Tanaka et al., 2003).
Flat-mount preparations
The length and the angle of a straight line connecting the injection site
and the position of each DiD-labelled GFP neuron were measured. The
DiD-labelled GFP neurons located medial to the injection site could not be
analysed because the injection had to be made into the dorsal cortex, and the
region of the cortex medial to the injection site (including the cingulate
cortex) could not be flattened because of folding of the cortex. For the same
reason, a part of the rostral or caudal region could not be analysed when the
injection was administered into the frontal or occipital cortex, respectively.
The curved structure of the cortex also interfered with the analysis of
neurons located far from the injection site.
Exo utero electroporation and immunostaining of Red fluorescent protein-labelled cells
We performed exo utero electroporation on E12.0 embryos. Red fluorescent
protein (DsRed2; Clontech) expression vector with a CAG promoter
(Hatanaka et al., 2004;
Hatanaka and Murakami, 2002;
Niwa et al., 1991) was
dissolved in PBS at a concentration of 0.5 µg/µl. Immediately before
use, Fast Blue solution (0.1% in PBS; Nacalai Tesque) was added to the plasmid
solution at a ratio of 1:10 to monitor the injection. Plasmid solution (1
µl) was injected into the lateral ventricle using a glass micropipette
fitted to an injector (IM-30; Narishige). The embryo's head was clasped with
forceps-shaped electrodes (CUY650P2; Unique Medical) connected to a
square-pulse generator (CUY21; TOKIWA or ECM830; BTX) with an angle of
30-60° from the brain's horizontal plane for GE-directed electroporation.
Electric pulses (30 V, 50 mseconds, 5 times, 950 mseconds intervals) were then
delivered.
Three and half days after the electroporation, embryos were removed and decapitated. Brains were fixed in 4% PFA in PB overnight at 4°C, and subjected to cryosectioning. Cryosections (20-30 µm thickness) were mounted onto slides (12-550-15; Fisher Scientific) and incubated in 5% NGS and 0.3% Triton-X in PBS (NGS/TX/PBS) for 1 hour, followed by 4 hours incubation in a rabbit polyclonal anti-LHX6 antibody (1/500; generous gift from Dr V. Pachnis, University College, London) in NGS/TX/PBS at room temperature. Sections were then incubated with a goat FITC-conjugated anti-rabbit IgG antibody (1/25; Jackson ImmunoResearch) for 1 hour at room temperature in PBS and examined using confocal microscopy (TCS SP2 AOBS).
Co-culture of cortical slices
A cortical slice from a P0 wild-type mouse was co-cultured with that from a
P0 GAD67-GFP animal. P0 brains from wild-type or GAD67-GFP heterozygous mice
were dissected, embedded in 4% low-melting point agarose in PBS, and the
dorsolateral part of the cortex sliced coronally on a vibrating-blade
microtome at 250 or 300 µm in Hanks' solution. The MZ of a wild-type slice
was placed adjacent to that of a GAD67-GFP slice and cultured on a membrane
insert (Millicell-CM PICMORG50; Millipore) coated with rat tail collagen, in
Opti-MEM I reduced-serum medium containing 2.5% heat-inactivated fetal bovine
serum, 2.5% heat-inactivated horse serum, 1% N2 supplement (all from
Invitrogen, Grand Island, NY), 20 mM D-glucose, and 20 µg/ml
streptomycin (Sigma). After a culture period of one day, the slice was fixed
with 4% PFA in PB overnight, immersed in 20% sucrose in PB for six hours to
overnight (each at 4°C), put in glass tubes containing 20% sucrose in PB,
frozen with liquid nitrogen and thawed. Following washes with PBS, double
immunofluorescence labelling was performed. Slices were first incubated with a
rat anti-GFP monoclonal antibody (1:1000; Nacalai Tesque) overnight, followed
by incubation with a mouse anti-MAP2 monoclonal antibody (HM-2; 1:2000; Sigma)
overnight. Then, the slices were reacted in goat biotinylated anti-mouse IgG
(1/500; Jackson ImmunoResearch, West Grove, PA) for six hours, followed by six
hours incubation in goat Alexa488-conjugated anti-rat IgG antibody (1/200;
Jackson ImmunoResearch), then with Alexa594-conjugated streptavidin (1/200;
Jackson ImmunoResearch). All antibodies were diluted in PBS containing
0.3-0.5% Triton X-100. Normal goat serum (5%) was added to the primary and
secondary antibody solutions. All reactions were carried out at room
temperature. Immunofluorescent localization was examined using a confocal
microscope (TCS SP2 AOBS; Leica Microsystems).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This raises the possibility that migrations in directions
other than mediodorsal might also occur in deeper cortical zones. To address
this issue, we cut serial tangential sections from fixed cortices of E15.5
GAD67-GFP mice (Fig. 1A,B) and
examined the orientation of the leading processes of GFP neurons. As expected,
the leading processes of GFP neurons in the CP, subplate (SP) and VZ were
oriented in many directions (Fig.
1C2-C5,C10,C11), with most IZ processes oriented mediodorsally
(Fig. 1C5-C8). Although
recognition of the processes of individual neurons in the MZ and SVZ was
blurred because of the high cell density, it appeared that MZ interneurons
were oriented in all directions (Fig.
1C1), and SVZ neurons were only weakly oriented in the mediodorsal
direction (Fig. 1C9). Similar
results were obtained for E13.5, E14.5 and E16.5 animals, although the
orientation of most CP neurons at E16.5 could not be clearly determined,
perhaps because many were radially oriented (data not shown). These
morphological features raise the possibility that MDT migration would also
occur in deeper zones.
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A majority of interneurons in the MZ and VZ migrate rostrally or caudally
MZ interneurons show robust MDT migration in flat-mount cortical
preparations (Tanaka et al.,
2003). This observation is inconsistent with the view that the MZ
is a major pathway for mediodorsal migration
(de Carlos et al., 1996;
Jimenez et al., 2002;
Lavdas et al., 1999;
Polleux et al., 2002). One
possible explanation is that a significant number of MZ interneurons migrate
in directions other than mediodorsal, although most do migrate mediodorsally.
To examine this possibility, we performed a quantitative analysis of the
direction of the GFP migration on previously recorded images taken from the
dorsal cortex of E13.5 mice (Tanaka et
al., 2003). We first scored the migrating neurons by subdividing
the horizontal plane into 24 sectors, and we counted the proportion of the
migrating neurons in each sector (n=1132 neurons, five hemispheres).
The tendency for the population was rostrocaudal migration
(Fig. 3A). Comparisons of the
migrations in the four quadrants (medial, rostral, lateral and caudal
directions) indicated that the proportion of rostrally migrating neurons was
significantly greater than that of medially migrating neurons (U-test;
P<0.01; Fig. 3B,
left panel); The proportion of either rostrally or caudally migrating neurons
was significantly greater than that of either medially or laterally migrating
neurons (U-test; P<0.01; Fig.
3B, right panel). To examine whether migration in the VZ is also
biased in specific direction(s), a similar quantitative analysis for GFP
neurons was carried out in the VZ (n=474 neurons, four hemispheres).
Although the interneurons also migrated in all directions
(Fig. 3C), the proportion of
laterally migrating neurons was less than that in other directions
(Fig. 3D, left panel). VZ
interneurons also appeared to prefer rostrocaudal directions rather than
mediolateral directions (U-test; P<0.03;
Fig. 3D, right panel), although
VZ interneurons tended to migrate medially rather than laterally. These
results suggest that a majority of MZ and VZ interneurons migrate
rostrocaudally.
Cortical interneurons migrated faster (U-test; P<0.02) in the
VZ (23.7±0.5 µm/hour; four hemispheres) than in the MZ
(18.8±0.9 µm/hour; five hemispheres), suggesting that interneurons
have different motilities in different zones. Average migration rates did not
differ for neurons migrating in different directions within each zone (MZ:
2=3.01, P>9.99; VZ: 2=12.59,
P>0.9).
Long-distance migration of interneurons within the cortex
The physiological significance of cortical MDT migration is unclear. One
possibility is that it contributes to interneuron dispersion within the
cortex. MDT migration of interneurons should then occur over long distances, a
possibility that cannot be examined with time-lapse analysis in short time
windows. We therefore performed in vivo DiD injections into the parietal
cortices of E15.5 GAD67-GFP mice (Fig.
4A,B), and analysed the distribution of DiD-labelled GFP neurons
after survival of a few days. One and a half days after injection, some
DiD-labelled GFP neurons were found in sagittal sections far from the
injection site (>400 µm; Fig.
4B'). DiD-labelled GFP neurons were also observed medially
and laterally, far from the injection sites in coronal sections
(Fig. 4C,D). Some of these
DiD-labelled GFP neurons had a leading process oriented away from the
injection site (insets of Fig.
4B',C,D). These results suggest that tangential migration of
interneurons occurs over long distances within the cortex.
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To obtain an overview of tangential migration in the MZ, cortical flat
mounts were prepared from DiD-injected GAD67-GFP brains
(Fig. 6A). One and a half days
after injection (E17.0), a substantial number of DiD-labelled GFP neurons
(n=38 neurons, two brains) were seen in many directions, some more
than 600 µm from the injection sites
(Fig. 6B1,D). Most DiD-labelled
GFP neurons had a trailing process and a leading process with a few branches
(Fig. 6B2,B3), a morphology
typical for migrating MZ interneurons
(Tanaka et al., 2003)
(Fig. 9B, see also Movie 4 in
the supplementary material). Three days after the injection procedure (E18.5),
many DiD-labelled GFP neurons (n=119 neurons, six brains) were found
even further from the injection site (>800 µm;
Fig. 6C,D); some of these
neurons were more than 1.5 mm from the injection site
(Fig. 6C1,D). DiD-labelled GFP
neurons had many orientations (Fig.
6B,C). Many neurons were distributed in rostral, caudal and
lateral directions, although we could not compare the distribution of
DiD-labelled GFP neurons in all directions because of folding of the cortex
(see Materials and methods).
Similar dispersions of DiD-labelled GFP neurons were observed after DiD injections into the frontal (Fig. 7A) or the occipital cortices (Fig. 7B) (n=2 each). Many DiD-labelled GFP neurons were located caudolateral to frontal injection sites (Fig. 7A,C) but rostrolateral to the occipital injection sites (Fig. 7B,D). In both cases, a substantial number of neurons were found more than 2 mm from the injection site (Fig. 7C,D). Together, these results suggest that the MDT migration of interneurons takes place over long distances and across cortical areas.
Cortical interneurons appear to migrate toward the ventricle and enter the CP after dispersing throughout the MZ. Twelve per cent of DiD-labelled GFP neurons in parasagittal sections from DiD-injected brains had leading processes directed towards the ventricle (n=18/151 DiD-labelled GFP neurons, three brains; see Fig. S1A,B in the supplementary material). Similar results were obtained in coronal sections (n=6/145 DiD-labelled GFP neurons, two brains; see Fig. S1C in the supplementary material). All of these neurons were found in the MZ or upper CP, at various distances from the injection site (see Fig. S1D in the supplementary material). Ventricle-directed migration of MZ interneurons was also observed in co-culture experiments with postnatal neurons (see Fig. S2 in the supplementary material).
MGE-derived interneurons migrate in many directions within the MZ
Cortical interneurons have multiple origins
(Anderson et al., 2001;
Anderson et al., 1997;
Butt et al., 2005;
Lavdas et al., 1999;
Nery et al., 2002;
Wichterle et al., 2001;
Xu et al., 2004), raising the
possibility that directionality of MZ interneurons is correlated with their
origin. To test this, we introduced DsRed-coding plasmid into the
E12.0 MGE using the electroporation-based in vivo gene transfer technique
(Fig. 8A, inset). At E15.5, 3.5
days after the electroporation, DsRed-labelled cells have arrived at the
neocortex (Fig. 8A). Most of
them were positive for both GAD67-GFP (98.1±1%;
Fig. 8B) and LHX6
(91.4±7%, Fig. 8C),
indicating that these interneurons were derived from the MGE
(Anderson et al., 2001;
Lavdas et al., 1999;
Sussel et al., 1999).
Real-time imaging of E15.5 MZ (Fig. 9A) shows DsRed cells migrating in all directions (Fig. 9B-B'', see also Movie 4 in the supplementary material), with no obvious directional bias. This observation demonstrates that GABAergic interneurons from at least one specific site, the MGE, can migrate in many directions within the MZ.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Technical considerations
As DiD can be transported retrogradely, it is possible that GFP neurons
observed here were labelled retrogradely from the injection site. However, we
do not think it likely because: (1) most DiD-labelled GFP neurons extended
short trailing processes that did not reach the injection site (Figs
4,
6,
7); (2) DiD injection into the
cortex of E18.5 GAD67-GFP mice followed by immediate fixation caused many
retrogradely labelled GFP neurons, but most of them were confined to the
vicinity of the injection site (<400 µm) (K.M., D.H.T. and F.M.,
unpublished); (3) many DiD-labelled GFP neurons had morphological features
characteristic of migrating cortical neurons, extending a branched or
unbranched leading process and a trailing process
(Nadarajah et al., 2002;
Nadarajah et al., 2003;
Nadarajah et al., 2001;
Noctor et al., 2004;
Polleux et al., 2002;
Tabata and Nakajima, 2003;
Tanaka et al., 2003); (4) the
distance of DiD-labelled GFP neurons from the injection site observed after
three days survival (viz, 1-3 mm; Fig.
6C,D, Fig. 7)
translates into a rate of 14-42 µm/hour, which is comparable to the average
rate of GFP cell migration, observed in the present time-lapse imaging in the
MZ, of 19 µm/hour.
MDT migration of cortical interneurons in multiple zones
Our observation in fixed preparations suggested the occurrence of
multizonal MDT migration. This was directly demonstrated by performing
real-time analysis of VZ interneurons, which migrated in many directions
(Fig. 2,
Fig. 3C,D). Our findings agree
with a previous study in cortical explants that showed DiI-labelled cells near
the ventricular surface migrating in all directions
(Fishell et al., 1993).
Although most of the labelled cells in that study appeared to be progenitor
cells, it is possible that some were interneurons.
Because of technical limitations, it was not possible to carry out real-time analysis for interneurons in zones other than the MZ and VZ. Howerver, the orientation of interneurons in fixed preparations is consistent with the notion that MDT migration also occurs in the CP and SP (Fig. 1).
MDT migration of cortical interneurons over long distances
Our DiD-injection experiments demonstrate that cortical interneurons can
migrate long distances (1-3 mm) in the MZ (Figs
6,
7). Thus, it is likely that the
MZ GFP neurons observed with real-time imaging
(Tanaka et al., 2003)
(Fig. 3A,B) are undergoing
long-distance migrations rather than local movements.
VZ interneurons also appear to migrate long distances. DiD injections into
the cortex labelled only a small number of VZ neurons more than 400 µm from
the injection sites (Fig. 5A).
However, because we attempted to avoid DiD injections into the ventricle, the
centres of the injections tended to be located in the superficial parts of the
cortical wall. The poor DiD labelling in the VZ is perhaps therefore due to
injection sites that were not close enough to the VZ. Indeed, when DiI was
focally injected into the VZ and SVZ of the developing ferret cortex, neurons
in these zones, possibly including interneurons, tangentially migrated more
than 2.5 mm (O'Rourke et al.,
1997).
|
) and GAD67-GFP cells
(Tanaka et al., 2003).
Quantitative analysis, however, has not been performed in terms of the
directionality of their migration in real time. We show here that MZ GFP
neurons primarily migrate rostrocaudally rather than medially, by analysing
migrating neurons in cortical flatmounts
(Fig. 3). Our results may seem
to be inconsistent with the classical view that MZ interneurons migrate
dorsomedially (de Carlos et al.,
1996; Jimenez et al.,
2002; Lavdas et al.,
1999; Polleux et al.,
2002). This is not the case, however, because, in these previous
studies, dorsomedial migration of interneurons was observed in coronal slices,
in which rostrocaudal migration of cortical interneurons should not be
detected.
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;
Anderson et al., 1997;
Butt et al., 2005;
Lavdas et al., 1999;
Nery et al., 2002;
Wichterle et al., 2001;
Xu et al., 2004), raising the
possibility that migration directions might differ for interneurons with
different origins. This does not, however, explain the behaviour of
DsRed-labelled cells migrating in many directions in the MZ
(Fig. 9), because these cells
originated from progenitors at a specific site in the basal forebrain, the
MGE, and were labelled by electroporation carried out at a specific
developmental stage, E12.0. Therefore, although we cannot exclude the
possibility that the DsRed vector was transferred to several distinct
progenitor domains within the MGE, these results suggest that the migration
direction of MZ interneurons is independent of their origin.
There are numerous cortical interneuron subtypes with different calcium
binding-protein expression, neuropeptide expression, morphology and
physiological properties (Kawaguchi and
Kubota, 1997; Markram et al.,
2004). It is possible that interneurons of each subtype have
different migratory pathways, and that the direction of each pathway, which is
therefore different for different interneuron subtypes, is strictly regulated
by mechanisms present within the cortex. An alternative possibility is that
individual neurons can migrate in any direction irrespective of their subtype.
Subtype identification of interneurons whose migratory behaviour has been
analysed would be necessary to distinguish between these possibilities.
|
; Tanaka et al.,
2003). Co-culture experiments (see Fig. S2 in the supplementary
material) and a BrdU-labelling study
(Hevner et al., 2004) showed
postnatal shifts of interneurons from the MZ to the CP.
Waiting period?
DiD-labelling experiments suggest that GABAergic interneurons linger in the
MZ for a few days before approaching their final destination, the CP (Figs
6,
7). Several types of developing
axons are known to stop growing and wait for the maturation of their targets
(Ghosh and Shatz, 1992;
O'Leary and Terashima, 1988;
Wang and Scott, 2000). By
analogy, GABAergic MZ interneurons may require further maturation before
continuing their migration, or they may be waiting for the maturation of
nearby structures. The validity of this hypothesis awaits further studies.
Conclusions
Most cortical interneurons originate in the ventral forebrain and migrate
dorsally toward the cortex. The present study shows that some of these migrate
tangentially in all directions and in multiple zones, after entering the
dorsal cortex. The neurons do not merely wander around locally but migrate
over long distances before settling in the CP. This migratory behaviour may
contribute to the dispersing and intermixing of cortical interneuron subtypes;
it may therefore be important for achieving the final, even distribution of
cortical interneuron subtypes throughout the cortex.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/11/2167/DC1
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