AG Entwicklungsneurobiologie, Fakultät für Biologie, Ruhr-Universität, ND 6/56a, D-44780 Bochum, Germany
* Author for correspondence (e-mail: marcus.wirth{at}ruhr-uni-bochum.de)
Accepted 18 August 2003
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SUMMARY |
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In pyramidal neurons, both TrkB ligands increased dendritic length and number of segments without affecting maximum branch order and number of primary dendrites. In the early time window, only infragranular neurons were responsive. Neurons in layers II/III became responsive to NT4/5, but not BDNF, during the later time window. BDNF and NT4/5 transfectants at 10 days in vitro had still significantly shorter dendrites than adult pyramidal neurons, suggesting a massive growth spurt after 10 days in vitro. However, segment numbers were already in the range of adult neurons. Although this suggested a role for BDNF, long-term activity-deprived, and thus BDNF-deprived, pyramidal cells developed a dendritic complexity not different from neurons in active cultures except for higher spine densities on neurons of layers II/III and VI. Neutralization of endogenous NT4/5 causes shorter and less branched dendrites at 10 days in vitro suggesting an essential role for NT4/5. Neutralization of BDNF had no effect. Transfected multipolar interneurons became identifiable during the second time window. Both TrkB ligands significantly increased number of segments and branch order towards the adult state with little effects on dendritic length. The results suggested that early in development BDNF and NT4/5 probably accelerate dendritogenesis in an autocrine fashion. In particular, branch formation was advanced towards the adult pattern in pyramidal cells and interneurons.
Key words: Gene gun, Dendrites, Neurotrophic factors, Autocrine actions, Rodent cortex, Spontaneous activity
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Introduction |
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Activity and glutamatergic transmission play a pivotal role depending on
receptor status (Rajan and Cline,
1998; McAllister,
2000
; Matus, 2000
;
Inglis et al., 2002
). The
complexity of basal dendrites of pyramidal neurons increases in the presence
of afferents delivering more excitation in cocultured cortical explants
(Baker et al., 1997
;
Baker and van Pelt, 1997
), and
spiny stellates in deprived-eye columns extend dendrites into open-eye columns
(Kossel at al., 1995
).
Hippocampal cell dendritogenesis in contrast depends on afferent connectivity
rather than activity (Kossel et al.,
1997
; Frotscher et al.,
2000
), whereas activity-deprived CA1 neurons build fewer, but not
shorter dendrites (Groc et al.,
2002
). Synaptic activity evokes further structural changes [e.g.
controlling spine formation and synaptic plasticity
(Maletic-Savatic et al., 1999
;
Lendvai et al., 2000
;
Knott et al., 2002
;
Frotscher et al., 2000
;
Thoenen, 2000
)]. Transmitters
also influence interneuronal morphogenesis
(Ross and Porter, 2002
;
Porter et al., 1999
;
Jones et al., 2000
;
Durig and Hornung, 2000
).
In pyramidal neurons, exogenous BDNF, NT4/5 (NTF5 Mouse Genome
Informatics) and NT3 (NTF3 Mouse Genome Informatics), as well as
neutralization of endogenous neurotrophins exert layer-specific effects on
dendritic growth and branching, with BDNF affecting only active neurons
(McAllister, 2000;
Baker et al., 1998
;
Niblock et al., 2000
). BDNF
also increases dendritic length of hippocampal interneurons
(Marty et al., 1996
). However,
exogenous applications are rather non-physiological in terms of dosages and
routes of acquisition. Moreover, the neurochemical markers employed to
visualize the interneuronal morphology are often downstream of neurotrophin
signaling (Marty et al., 1997
;
Gorba and Wahle, 1999
;
Wahle et al., 2000
).
Furthermore, the neutralization of endogenous TrkB (Ntrk2 Mouse Genome
Informatics) ligands was usually achieved by TrkB-IgGs, which do not
distinguish between BDNF and NT4/5
(McAllister, 2000
).
An important issue, therefore, is whether neurotrophins can promote
dendritic growth in an autocrine fashion. Horch et al.
(Horch et al., 1999) reported
that ferret pyramidal neurons overexpressing BDNF develop extra dendrites, as
do hippocampal granule cells in BDNF-overexpressing mice
(Tolwani et al., 2002
)
suggesting that BDNF even alters primary neurite patterning. We therefore
analyzed in organotypic cultures (OTC) the dendritogenesis of early postnatal
rat cortical pyramidal cells and interneurons overexpressing BDNF and NT4/5.
Interneurons do not normally express BDNF and the question was whether they
could use it. As BDNF is expressed activity dependently in OTC
(Gorba and Wahle, 1999
), we
analyzed the degree of pyramidal cell differentiation in long-term
activity-deprived, and thus BDNF-deprived, OTC.
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Materials and methods |
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Expression plasmids
All plasmids were prepared as an endotoxin-free solution (Qiagen, Hilden,
Germany). The enhanced `green fluorescent protein' (EGPF) was used as reporter
(pEGFP-N1, CMV-promoter, Clontech, Heidelberg, Germany). The BDNF plasmid
(pCMV5-BDNF) was kindly provided by Dr Barde [MPI Martinsried
(Leibrock et al., 1989) for
BDNF, and Stratagene manual for pCMV]. The NT4/5 plasmid (pCMX-hNT4/5myc) was
kindly provided by Dr Yancopoulos (Regeneron, Tarrytown, USA). Both plasmids
carry the cytomegalovirus promoter for strong expression in eukaryotic cells.
Two independent plasmids were employed rather than bicistronic or chimeric
constructs in order to achieve maximal expression, independent processing and
targeting and conservation of biological activity of both polypeptides. By
imaging immunofluorescence intensities, Horch et al.
(Horch et al., 1999
) showed
that biolistics with similar constructs increases the neurotrophin content in
overexpressers within a physiological range.
DNA coating of the gold particles and preparation of the
cartridges
The cartridges were prepared as described
(Wahle et al., 2000;
Wirth and Wahle, 2003
). In
brief, particle size was 1.5-3.0 µm (Strem Chemicals, Kehl, Germany). The
density of gold was 0.2 mg per cartridge. The gold-particles were coated with
1 µg pEGFP-N1 per mg gold. For co-expression of neurotrophic factors and
EGFP the same gold-particles were coated with 1 µg pEGFP-N1 per mg gold and
2 µg per mg gold of the expression plasmids for one of the neurotrophic
factors. Tissue bombardment was performed as described
(Wahle et al., 2000
;
Wirth and Wahle, 2003
) using
the hand-held Helios Gene Gun (BioRad, Munich, Germany). The distance from the
muzzle to the culture was 1 cm. The helium pressure was 160-200 psi
(232x104 Pa to 290x104 Pa). To
avoid excitotoxicity by Ca2+ influx NMDA receptors were temporarily
blocked by adding 3 mM kynorenic acid (Sigma-Aldrich, Deisenhofen, Germany)
and 50 µM APV (DL-2-amino-5-phosphonovaleric acid, Sigma-Aldrich,
Deisenhofen, Germany) before blasting, and inhibitors were removed 6 hours
later. Cortical monocultures were transfected either at the day of
explantation (0 DIV=postnatal day 0) or at 5 DIV allowing 5 days for
expression. Cultures were harvested at 5 DIV and 10 DIV, respectively.
Detection of expression
EGPF expression reaches its maximum after 24 hours of cultivation and is
easily detected with a fluorescence-microscope (FITC filter, excitation at 490
nm/emission at 520 nm). Cultures were fixed in ice-cold 4% phosphate buffered
paraformaldehyde (pH 7.4) for 2 hours. Antibody penetration was enhanced with
0.5% Triton-X-100 in TBS for 30 minutes. followed by blocking in 1% bovine
serum albumin (Merck, Darmstadt, Germany) and 1% normal goat serum (Dakopatts,
Hamburg, Germany) in TBS for 60 minutes. The primary mouse antibody
(moGFP, Clontech, Heidelberg, Germany) was diluted 1:1000 in blocking
solution and incubated overnight at 11°C, followed by a 3 hour incubation
with a biotinylated secondary antibody (dilution 1:300, Dakopatts, Hamburg,
Germany) at room temperature. After several washing steps, OTC were incubated
in avidin-biotin-horseradish peroxidase-complex (Dakopatts, Hamburg, Germany),
developed with diaminobenzidine and H2O2 and mounted
with Depex (Sigma-Aldrich, Deisenhofen, Germany). The expression of
transfected genes at the mRNA and the protein level was previously shown, and
the degree of coexpression of two independent plasmids was 95%
(Wirth and Wahle, 2003
). Any
sampled EGFP-positive transfectant not concurrently overexpressing a
neurotrophin would work against our statistics and will not cause false
positive results. Both neurotrophin plasmids encode secreted, biologically
active peptides because both upregulate neuropeptide Y mRNA in thalamocortical
cocultures after the phenotype restriction [for leukemia inhibitory factor
(Wahle et al., 2000
); for BDNF
and NT4/5 (M.W., unpublished)], but the factors do not accumulate in the
medium of transfected cultures to levels, which would upregulate NPY mRNA in
untransfected cultures exposed to this medium (not shown).
Pharmacology
Activity deprivation was achieved by addition of 10 mM MgSO4 to
the medium. BDNF or NT4/5 was neutralized by addition of 300 ng/ml of
neutralizing antibodies (PeproTech, Rocky Hill, USA) to the medium. Tyrosine
kinases were inhibited by 40 nM K252a (Calbiochem/Merck, Bad Soden, Germany).
In both experimental conditions, the medium was changed every day from 5 to 10
DIV on.
Generation of cDNA libraries and PCR
Reactions were performed as described
(Gorba et al., 1999). In
brief, messenger RNA was isolated from at least three OTC for every time point
and experimental condition using DynaBeads mRNA Direct Kit (Dynal, Hamburg,
Germany). cDNA libraries were synthesized with Sensiscript reverse
transcriptase (20 U/ml, Qiagen, Hilden, Germany) at 37°C for 60 minutes.
Semiquantitative PCR was performed in a total volume of 50 µl with 5
U/µl Taq polymerase (Qiagen, Hilden, Germany). Amplified regions for BDNF,
NT4/5 and NT3 and glucose-6-phosphate dehydrogenase (GAPDH) were as described,
GAPDH was chosen as an activity-independent standard
(Gorba et al., 1999
). The
number of cycles was kept in the linear range determined for each product. The
amplificates were densitometrically analysed with the Eagle Eye system
(Stratagene, Amsterdam, The Netherlands) and normalized to GAPDH. Normalized
values from a total of 10 PCR reactions from two independent cDNA libraries
were used to construct the graphs with s.e.m.
Biocytin injections
Pyramidal cells and interneurons of spontaneously active and pyramidal
cells of chronically activity-deprived (with 10 mM MgSO4 in the
medium) OTC aged 30 to 60 DIV (summarized here as `adult') were analyzed.
Activity deprivation was initiated at 3 DIV and lasted until the time of
analysis. Cells were filled with biocytin after intracellular
electrophysiological recording followed by histochemical staining
(Klostermann and Wahle, 1999;
Gorba et al., 1999
).
Electrophysiological recording confirmed the lack of bioelectric activity in
OTC with 10 mM MgSO4 in the medium.
Analysis
Pyramidal neurons from layers II/III, V and VI and were reconstructed with
a Eutectics Neuron Tracing system (Raleigh, USA) at 1000x magnification
from cultures cut strictly perpendicular to the cortical surface as indicated
by apical dendrites of layer V cells connecting to layer I, by transfected
glia cells with radial processes extending into supragranular layers, by the
absence of transfectants with obviously truncated apical dendrites and by the
presence of supragranular pyramidal cell axons descending perpendicularly to
the white matter.
All pyramidal cells included here resided fairly isolated in order to trace all the processes of only one neuron. They had axons with a clear origin at the soma or a proximal dendrite. Supragranular neurons were included when apical dendrites extended into layer I. Layer VI neurons were included when apical dendrites ended in middle layers. Our sample of layer V neurons included only neurons with apical dendrites in layer I (corticotectal neurons). Interneurons were sampled from layers II/III and V/VI in the later time window and were pooled. We restricted the analysis to multipolar cells with smooth or sparsely spinous dendrites and locally branching axons this way enriching for presumptive basket neurons. Interneurons with a polarized appearance were excluded, because immature Martinotti or bi-tufted neurons were not always distinguishable from immature pyramidal cells.
Total dendritic length, length of apical and basal dendrites, number of
segments, maximum branch order, number of primary dendrites were determined
for neurons of layers II/III, V and VI. For the biocytin-labeled neurons the
spine density was determined for the entire dendrite and is given in the
Fig. 7 as density per 100
µm. Data are presented as means with s.e.m. For statistical analysis, the
nonparametric Mann-Whitney U-test was performed since often the data did not
pass the tests for equal variance and normality (Kolmogorov-Smirnov-test for
normality, both tests applied via SigmaStat program, SPSS). For multiple
testing the -value was corrected according to Holm
(Holm, 1979
).
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Results |
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Layer VI pyramidal neurons had one apical dendrite and three or four basal dendrites (Figs 1, 2) and a maximum branch order of 4-6. BDNF and NT4/5 transfectants displayed significantly longer apical dendrites (P<0.05 for both) and basal dendrites (BDNF: P<0.05; NT4/5: P<0.01; Fig. 2). Furthermore, the mean number of apical segments was significantly increased by BDNF and NT4/5 when compared with EGFP control (BDNF: P<0.05; NT4/5: P<0.01; Fig. 2). Basal segment numbers were only increased by NT4/5 (P<0.05).
Thus, both neurotrophins increased dendritic length and branching during the early time window with NT4/5 appearing more effective than BDNF, but effects were only in infragranular layers.
The later time window: supragranular neurons become responsive
Overall, the 10 DIV neurons were clearly more mature than 5 DIV neurons
(Fig. 1) indicating the
expected progressive differentiation in OTC with time (compare 5 with 10 DIV
in Table 1;
Fig. 3). In layers II/III, the
length of apical dendrites of EGFP control was still around 400 µm.
Compared with 5 DIV EGFP transfectants, the basal dendritic length of 10 DIV
EGFP transfectants had almost doubled. Total basal dendritic length was in the
range previously reported for P10 (Table
1).
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|
In layer VI, neurons (Fig. 3) NT4/5 significantly increased the length of apical (P<0.05) and basal dendrites (P<0.01). BDNF had weak effects on apical dendrites but significantly increased the length of basal dendrites (P<0.01; Fig. 3). Segment numbers of basal and apical dendrites were significantly increased by BDNF (apical, P<0.05; basal, P<0.05) and NT4/5 (apical, P<0.05; basal, P<0.05; Fig. 3).
Thus, both TrkB ligands strongly promoted dendritic maturation of infragranular neurons and supragranular neurons became responsive to NT4/5.
Comparison to adult pyramidal cells in vitro
A central question was whether the structural changes evoked by the factors
were really advancing differentiation towards an adult state. We therefore
analyzed a set of biocytin-filled pyramidal cells obtained previously in
spontaneously active adult OTC (30-60 DIV of age)
(Klostermann and Wahle, 1999)
with the assumption that they represent an end point of maturation in OTC.
Indeed, their morphometric parameters were quite similar to in vivo pyramidal
cells in the adult cortex. The neurons displayed a mean of 5 primary
dendrites, which was in line with previously published data
(Schröder and Luhmann,
1997
; Miller,
1981
; Juraska,
1982
). The total length of basal dendrites of layer II/III and of
layer V pyramidal neurons was about 2300 µm and 2600 µm, respectively.
Length and the number of dendritic tips were in the range of adult pyramidal
cells in vivo (Table 1). This
suggested that pyramidal cells in spontaneously active OTC acquire a degree of
differentiation quite similar to in vivo pyramidal neurons in the adult
cortex.
When comparing dendritic length and complexity of transfectants at 10 DIV to adult neurons there was a clear difference between the two sets of cells. Apical and basal dendrites at 10 DIV were shorter (Table 1; also compare Fig. 3 with Fig. 7). Even the most advanced transfectants at 10 DIV were still immature when compared with adult neurons aged 30-60 DIV suggesting major growth spurts after day 10.
The interesting parameter was basal dendritic segment number (Table 1). Not surprising, layer II/III neurons had highly significantly fewer segments at 10 DIV compared with adult. Although the neurotrophins doubled the total basal dendritic segment number, they failed to evoke the adult dimensions. By contrast, total basal dendritic segment numbers of neurotrophin transfectants in layer V and layer VI were fairly close to the adult pattern. Statistically, layer V cells were no longer different from adult (BDNF, P=0.56; NT4/5, P=0.72), whereas in layer VI the BDNF transfectants were close to adult (P=0.06) while NT4/5 transfectants still were significantly smaller than adult (P<0.01). Together this suggested that the early neurotrophin transfections precociously advance branch formation and evoke segments numbers, which at 10 DIV were in the range of adult pyramidal neurons.
Interneuronal dendritic complexity increased with overexpression of
TrkB ligands
Multipolar interneurons (Fig.
4) became recognizable during the first postnatal week in vivo and
in OTC (Obst and Wahle, 1995).
Dendritic length and complexity steadily increases during the second and third
week (Mathers, 1979
;
Uylings et al., 1990
;
Miller, 1986
), which overlaps
with our 5-10 DIV transfection period. Fig.
3 shows the interneuronal data in direct comparison to adult
multipolar interneurons in vitro.
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Compared with our adult interneurons in vitro, the transfectants were not different with respect to the number of primary dendrites (Fig. 5). The 10 DIV neurotrophin transfectants had much shorter dendrites and total dendritic length (P<0.0001). The mean total dendritic length of the EGFP transfectants was about 1000 µm, BDNF transfectants had 1600 µm, and NT4/5 transfectants had 1400 µm at 10 DIV as compared with about 4000 µm of adult interneurons (Fig. 5). However, maximum branch order and number of segments were statistically no longer different from adult (Fig. 5). This suggested that the TrkB ligands precociously evoke the adult number of segments, whereas the adult length develops after 10 DIV.
Activity blockade does not prevent dendritic maturation of pyramidal
neurons
The study so far revealed that TrkB ligands secreted by the transfectants
accelerated dendritogenesis in some aspects close to the adult in vitro
pattern. The important question now concerned the role of endogenous factors.
As BDNF is activity-dependently synthesized and released
(Androutsellis-Theotokis et al.,
1996; West et al.,
2001
; Tao-Cheng et al.,
2002
; Balkowiec and Katz,
2002
) (for a review, see
Lessmann et al., 2003
) we took
advantage of activity-deprived OTC in which the endogenous BDNF mRNA
expression, but not the NT4/5 and NT3 mRNA expression is strongly reduced
(Gorba et al., 1999
; Ichisaka
et al., 2003). Furthermore, the activity-dependent component of neurotrophin
release should be eliminated. If BDNF were essential, pyramidal neurons from
activity-deprived and thus BDNF-deprived OTC should display less
differentiated dendrites.
We compared 30-60 DIV adult pyramidal neurons of layers II/III, V and VI in
chronically activity-deprived OTC to adult neurons from active OTC. All were
pyramidal by morphological and electrophysiological classification
(Gorba et al., 1999) with
polarized dendritic trees covered by spines
(Fig. 6A-C for active and
Fig. 6D-F for activity-deprived
pyramidal neurons). In fact, the deprived neurons were surprisingly well
differentiated (compare Fig.
6A-D). Neither apical nor basal dendritic length differed
significantly (Fig. 7). In
layers II/III the number of primary dendrites (a mean of 5), maximum branch
order (of 10) and number of segments per dendrite of pyramidal neurons from
activity-deprived OTC were not different from those of active OTC
(Fig. 7). Layer V pyramidal
neurons were unchanged. In layer VI pyramidal cells, the number of primary
dendrites (a mean of 5), the maximum branch order (of 10) and the number of
segments per dendrite were not different.
The major difference was the spine density (compare
Fig. 6B,C with 6E,F; Fig. 7). Active neurons had
about 15-30 spines per 100 µm dendrite which was much lower than reported
for adult pyramidal neurons in the cortex in vivo [data extracted from other
(Miller, 1981;
Juraska, 1982
;
Schröder and Luhmann,
1997
; Kolb et al.,
1997
) and calculated per 100 µm dendrite; layers II/III basal,
65-110; layers II/III apical, 60-102; layer V basal, 60-100; layer V apical,
60-120 spines). By contrast, apical and basal dendrites of activity-deprived
neurons from layers II/III and layer VI, but not layer V, had significantly
increased spine densities (all P<0.01;
Fig. 7), which, however, were
still lower than densities reported for pyramidal neurons in vivo.
NT4/5 but not BDNF plays a physiological role for pyramidal cell
dendritogenesis
We have shown that overexpressed BDNF and NT4/5 accelerate dendritogenesis.
However, neurons grown under activity deprivation and therefore in the absence
of BDNF showed surprisingly `normal' dendrites. Thus, the question is whether
BDNF or NT4/5 is more important for dendritogenesis. We focused on development
of dendritic length and segment number of layer VI pyramidal neurons because
they displayed a strong response to both TrkB ligands during the second time
window.
To exclude the possibility that activity-deprived pyramidal neurons from OTC aged 30 to 60 DIV just compensated for the lack of BDNF simply by having enough time for dendritogenesis, we analyzed neurons activity-deprived from 5-10 DIV. The dendritic length and the number of segments were not significantly different from spontaneously active controls (Fig. 8) suggesting that short-term deprivation of activity and thus of BDNF does not impair dendritogenesis.
|
Both factors failed to promote dendritic growth in transfected layer VI pyramidal neurons in the presence of K252a (Fig. 8) suggesting that the action of overexpressed BDNF and NT4/5 depends on Trk receptors. However, EGFP-transfected neurons had significantly smaller and less complex dendrites in the presence of K252a than did untreated EGFP control neurons (Fig. 8) suggesting that endogenous neurotrophins acting via Trk receptors contribute to dendritic growth.
As expected, the BDNF mRNA expression remained very low in activity-deprived OTC (Fig. 9A). By contrast, activity-deprived OTC displayed a much higher expression of NT4/5 mRNA and to a lesser extend also of NT3 mRNA at 10 and 20 DIV, but no longer at 45 DIV (Fig. 9B,C) suggesting that NT4/5 and NT3 are present in higher amounts during the main period of dendritic development. This supported the view that the dendritic differentiation in activity-deprived OTC is primarily governed by NT4/5.
|
To summarize, dendritic maturation of pyramidal neurons and multipolar nonpyramidal neurons overexpressing BDNF and NT4/5 was accelerated in a layer- and time-dependent manner, suggesting an autocrine role for the TrkB ligands. However, pyramidal cells deprived of activity and of endogenous BDNF differentiate with high fidelity probably owing to an activity-independent action of NT4/5.
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Discussion |
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Neurotrophin transfectants displayed at 5 DIV a degree of differentiation
similar to control transfectants at 10 DIV (see
Fig. 1). Neurotrophin
transfectants at 10 DIV displayed an almost adult degree of branching, but not
yet length. The neurotrophins apparently accelerated dendritogenesis in a
step-by-step manner without evoking a precocious hypertrophy. A gradual
acceleration was also observed for somatic development of pyramidal cells in
mice postnatally overexpressing activated p21Ras acting as an intracellular
neurotrophin, and somatic hypertrophy was persisting with adult neurons having
thicker and more branched dendrites
(Heumann et al., 2000) (P.W.,
unpublished). Similar to some other G proteins
(Threadgill et al., 1997
),
activated Ras thus mimics the effects reported here suggesting that they are
evoked by autocrine neurotrophin signaling known to occur in neurons
(Miranda et al., 1993
;
Acheson and Lindsay, 1996
;
Giehl, 2001
). Paracrine
actions cannot be excluded, but are considered unlikely for the following
reasons. It would imply that two neurotrophin overexpressers interact
reciprocally to drive each other's differentiation. When considering an axonal
route of delivery one must take into account that the intracortical axonal
connections are fairly underdeveloped at the ages investigated (DIV 5 and
DIV10). When considering a somatodendritic release, the partner cells must be
in very close by because Horch and Katz
(Horch and Katz, 2002
) have
shown that BDNF released from overexpressers can modify dendrites only within
a 4.5 µm distance. Yet, our overexpressers had been selected for
reconstruction by their fairly isolated position, which is a prerequisite for
tracing all the fine processes of only one neuron.
Despite methodical differences and the difficulty to extrapolate
developmental time-course between rat and ferret, our results are comparable
with studies by McAllister et al.
(McAllister et al., 1995;
McAllister et al., 1996
;
McAllister et al., 1997
). The
authors found strong effects of exogenous BDNF and NT4/5 for infragranular
pyramidal neurons, although in contrast to ferret, rat layer V neurons
strongly responded to BDNF and layer VI neurons responded to both factors,
without evidence for a growth-inhibitory BDNF action. We further showed that
the most immature layer II/III neurons were still unresponsive. As exogenous
BDNF affects only active neurons as has been shown for its dendritogenic
(McAllister et al., 1996
) and
phenotype-promoting actions (Marty et al.,
1997
; Wirth et al.,
1998
), the lack of BDNF effects in, for example, 5-10 DIV
supragranular transfectants appeared due to low activity or a failure to
activity-dependently release overexpressed BDNF. Niblock et al. (Niblock et
al., 2002) found little effects of exogenous BDNF in P11 rat cortical layer II
pyramidal cells, which had just finished migration. Only basal dendritic
branching increases, possibly owing to the high doses used. Apparently, very
immature neurons fail to react to BDNF either due to a lack of local activity
or an immaturity of intrinsic excitability. An intriguing possibility is that
the onset of BDNF action correlates with the acquisition of specific
Na+ channel function (Kafitz et
al., 1999
). By contrast, the effectiveness of NT4/5 is due to its
activity-independent action (Wirth et al.,
1998
), and our results strongly suggest an activity-independent
release of NT4/5.
BDNF and NT4/5 failed to induce primary dendrites
In contrast to studies by McAllister et al.
(McAllister et al., 1995) and
Horch et al. (Horch et al.,
1999
) in ferret, we did not find dramatic increases in primary
dendrites. This could be due to species differences. Rodent neurons are for
instance less affected by gangliosides, which evoke aberrant dendrites in
higher mammals (Walkley et al., 2000). In addition, Jin et al.
(Jin et al., 2003
) did not
observe extra dendrites in BDNF-treated interneurons of mouse cortex. However,
the developmental time-course seems more important. It is possible that ferret
neurons at the ages analyzed were prone to sprout extra dendrites, whereas rat
neurons were already beyond the period of malleability of primary neurite
patterning. Evidence comes from two recent studies. Danzer et al.
(Danzer et al., 2002
) reported
extra dendrites after virally driven BDNF overexpression in hippocampal
granule cells at the inner blade of the fascia dentata, not in outer parts or
in CA pyramidal cells, suggesting that this structural plasticity can be
evoked only in cells of a certain developmental stage. By contrast, Tolwani et
al. (Tolwani et al., 2002
)
found all granule cells affected in mice overexpressing BDNF from actin
promoter. The neurons have more dendrites with more branches rather than being
longer. The promoter is active from embryonic time onwards overlapping with
continuous granule cells neurogenesis, and newly generated neurons cannot
avoid exposure to higher BDNF levels with the consequence that even primary
neurite patterning becomes altered.
BDNF and NT4/5 accelerate dendritic differentiation of
interneurons
Some hippocampal interneurons express NT3 or NGF
(Pascual et al., 1998) and
thus can process and release neurotrophins. BDNF is not in interneurons
although they heavily express TrkB receptors
(Rocamora et al., 1996
;
Gorba and Wahle, 1999
) for
consumption of BDNF-derived from pyramidal producers. However, pyramidal cell
degeneration evokes BDNF expression in interneurons suggestive of an autocrine
rescue response compensating the loss of target-derived factor
(Wang et al., 1998
). We now
showed that BDNF and NT4/5 equipotently increased interneuronal complexity
with less influence on dendritic elongation.
Concerning NT4/5, our results differ from recent results of Jin et al.
(Jin et al., 2003), who found
no effect of NT4/5 on the total dendritic length and the total number of
dendritic branch points. The authors analyzed OTC prepared from mice, which
were about 5 days older than ours. Taking into account that the time course of
development is shorter in mice, the age difference might be important. In line
with our data we rather suggest that the neurons grow with NT4/5 at times when
electrical activity is still low, but switch to BDNF as soon as activity
increases.
Our results suggest that interneurons, when made into BDNF/NT4/5 producers,
could use the factors for dendritic differentiation presumably in an autocrine
fashion. An additional trans-synaptic
(VonBartheld et al., 1996;
Kohara et al., 2001
) or
paracrine action of TrkB ligands from pyramidal transfectants in the same
culture could not be excluded, but appeared less likely (see above). In
particular, intrinsic axonal connections of pyramidal cells are underdeveloped
in an early stage as is the expression of synapse proteins
(Kierstein et al., 1996
). In
vivo, adult synapse densities are reached around P20
(Blue and Parnavelas, 1983
) and
even adult pyramidal cell axons form only few synapses with a given multipolar
interneuron (Buhl et al.,
1997
). Moreover, the multipolar neurons analyzed here mainly
represent presumptive basket cells and their typical axosomatic contacts
become recognizable during the third postnatal week, which largely rules out
retrograde signaling in DIV 5-10 neurons. Furthermore, synaptic neurotrophin
release requires activity, which is low in young OTC
(Klostermann and Wahle, 1999
).
Together, this suggested TrkB ligands as potent autocrine mediators of
interneuronal and pyramidal cell dendritogenesis.
Endogenous BDNF is not essential for pyramidal cell
dendritogenesis
Owing to the complex interdependence of neuronal activity, afferents and in
particular BDNF, activity deprivation studies delivered opposing results
depending on developmental stage of the cell class, manner and timing of
manipulation, and experimental conditions (see Introduction) (for a review,
see McAllister, 2000). In our
OTC, the lack of activity and BDNF did not compromise dendritic growth and
complexity. However, the lack of endogenous NT4/5 retarded dendritogenesis
suggesting that NT4/5 is more important than BDNF at least during the first
two postnatal weeks. The surprisingly normal neocortical dendritogenesis
without BDNF appeared to contradict the results from the overexpression study.
Yet, the lack of BDNF was presumably compensated for by the action of NT4/5
and possibly also of NT3. In the absence of electrical activity the NT4/5 mRNA
expression is upregulated. Previous studies also showed that NT4/5 and NT3 are
activity-independently expressed (Gorba et
al., 1999
; Ichisaka et al., 2003).
Both factors are released via the constitutive secretory pathway in the
absence of BDNF (for a review, see
Lessmann et al., 2003). NT3
increases dendritic complexity of hippocampal and cortical pyramidal cells
independent of glutamate-mediated transmission
(Morfini et al., 1994
;
Baker et al., 1998
), and NT4/5
promotes the dendritic development (this study) and the neuropeptide Y
expression in the absence of activity
(Wirth et al., 1998
). Although
apical dendrites are fairly promiscuous, NT4/5 and NT3 are highly effective
(e.g. on layer VI basal dendrites), and NT3 actions even improve in the
absence of TrkB ligands (McAllister et
al., 1995
; McAllister et al.,
1997
). Furthermore, layer II/III dendrites develop normally in
dark-reared visual cortex (Tieman et al.,
1995
) despite reduced expression of BDNF, but not of other
neurotrophins (Schoups et al.,
1995
; Ichisaka et al., 2003), and reduced Trk receptor activation
(Viegi et al., 2002
).
The lower spine density in adult active OTC presumably reflected the fact
that monocultures develop only the intrinsic wiring. Densities remained
equivalent to the spine equipment displayed around P10 in vivo
(Miller, 1981;
Petit et al., 1988
).
Hippocampal OTC also have lower spine densities because OTC lack
spine-targeting afferents and afferent drive known to promote spine
development (Engert and Bonhoeffer,
1999
; Toni et al.,
1999
; Knott et al.,
2002
). The higher spine densities in deprived OTC were surprising.
However, Harris (Harris, 1999
)
proposes that lower excitation increases and higher excitation decreases spine
density. Hippocampal slices instantly produce excessive spine densities owing
to the sudden fall in activity (Kirov et
al., 1999
), as do activity-deprived Purkinje
(Bravin et al., 1999
) and
thalamic neurons (Dalva et al.,
1994
; Rocha and Sur,
1995
). Our neurons developed and maintained more spines for weeks
under blockade and displayed higher synaptophysin expression
(Kierstein et al., 1996
). The
lack of activity does not prevent circuit formation and indeed neurons resume
synaptically evoked action potential activity upon recovery
(Gorba et al., 1999
). However,
it might prevent activity-dependent pruning of spines and intrinsic
connectivity.
Growing dendrites and shaping dendrites are thus two separate consecutive
processes. Our OTC were prepared at birth and largely before cortical
glutamatergic afferents and intrinsic connections become established. During
these early stages, neurite growth appears activity independent. With time,
activity-dependent control mechanism dominate to use-dependently shape
cortical structure and function (Katz and
Shatz, 1996), and activity-dependent BDNF starts to control speed
and mode of dendritogenesis (Yacoubian and
Lo, 2000
). However, neurons that are activity deprived from birth
never experience the activity-dependent mechanisms and instead continue to
grow with high fidelity by activity-independent mechanisms. It is possible
that young neurons, once they have switched to activity-dependent control,
suffer under deprivation because they lose the competence to grow by
activity-independent mechanisms.
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ACKNOWLEDGMENTS |
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