1 Department of Medical Biochemistry and Biophysics, Laboratory of Molecular
Neurobiology, Karolinska Institutet, Stockholm S-171 77, Sweden
2 Millennium Pharmaceuticals Incorporated, 75 Sidney Street, Cambridge, MA
02139, USA
Author for correspondence (e-mail:
ernest.arenas{at}mbb.ki.se)
Accepted 25 April 2003
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SUMMARY |
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Key words: TrkB, TrkC, BDNF, NT4, NT3, GDNF, Locus coeruleus, Neurotrophins, Rat primary cultures
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INTRODUCTION |
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There are, however, indications that LC NA neurons could respond to
neurotrophic factors of the GDNF family during development because GDNF
receptors are expressed by LC neurons and primary cultures of E13.5 LC NA
neurons respond to GDNF and to a second neurotrophic factor of the GDNF
family, neurturin (NTN) (Holm et al.,
2002). Interestingly, GDNF and NTN promoted the neuritogenesis of
NA neurons in serum-free cultures (Holm et
al., 2002
), suggesting that GDNF and NTN might regulate this
aspect of LC neuron development. However, when LC cultures were grown in the
presence of BMP2 or serum, GDNF or NTN enhanced the survival-promoting effects
of BMP2 on LC NA neurons (Reiriz et al.,
2002
). Thus, provided that the LC is exposed to BMPs in vivo
(Guo et al., 1999
;
Vogel-Hopker and Rohrer,
2002
), it is conceivable that GDNF and NTN may also regulate the
survival of LC NA neurons during development.
With regard to the neurotrophins, their high affinity tyrosine kinase
receptors TrkB (NTRK2 Mouse Genome Informatics) and TrkC (NTRK3
Mouse Genome Informatics) are expressed in the adult rat LC
(King et al., 1999;
Merlio et al., 1992
;
Smith et al., 1995
;
Tetzlaff et al., 1994
), but it
is not known whether they are also expressed in the developing LC NA neurons.
Primary LC cultures grown in the presence of serum and treated with
brain-derived neurotrophic factor (BDNF), NT3 or NT4 (NTF5 Mouse
Genome Informatics), but not NGF, have shown increased survival of NA neurons
and norepinephrine uptake (Friedman et
al., 1993
; Sklair-Tavron and
Nestler, 1995
; Sklair-Tavron
and Segal, 1993
), suggesting a function of neurotrophins in the
developing LC. However, we have recently reported that NT3 per se, in
serum-free culture conditions, does not increase the survival of LC neurons,
and that NT3 and BMP2 or NT3 and serum promote survival of LC NA neurons
(Reiriz et al., 2002
). It thus
remains to be determined whether other neurotrophins play a role per se and
whether they regulate the development of LC NA neurons in vivo.
In the present study we re-evaluated the function of BDNF, NT3, NT4 and GDNF in the development of LC NA neurons both in vitro and in vivo. We found that mice with null mutations for both Gdnf and Nt3 displayed intact LC cell number, neuropeptide expression and target innervation. This independence of GDNF and NT3 for proper LC development was further emphasized by the lack of effect of these molecules in rat primary NA cultures. We also found that LC neurons express higher levels of TrkB mRNA than TrkC mRNA through development, and TrkB, but not TrkC, null mutant mice showed a 25-30% loss of LC neurons at P0. Moreover, addition of either of the two TrkB ligands, BDNF or NT4, to primary cultures resulted in a tenfold increase in number of TH-positive cells by mechanisms involving both survival of NA neurons and induction or promotion of a NA phenotype.
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MATERIALS AND METHODS |
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In situ hybridization
In situ hybridization with 35S-labeled TrkB and TrkC riboprobes
(Funakoshi et al., 1993) and
Ret, GFR
1 and GFR
2 riboprobes
(Trupp et al., 1997
) was
performed as previously described, on fresh frozen sections
(Trupp et al., 1997
). In
brief, sections were fixed for 15 minutes in refrigerated 4% PFA and rinsed
three times in PBS. The tissue was deproteinated in 0.2 M HCl for 10 minutes,
acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine for 20
minutes, and dehydrated in increasing concentrations of ethanol. Slides were
incubated for 16 hours in a humidified chamber at 53°C with
1x106 cpm of probe in 200 µl of hybridization cocktail.
Washes were performed at 62°C. First, 2x15 minutes in 1xSSC,
30 minutes in 50% formamide/0.5xSSC and 15 minutes in 1xSSC. Then
the slides were subjected to a 30-minute RNase treatment (40 µg/ml) at
37°C before washing 2x15 minutes in 1xSSC with DTT and 1x5 minutes
in room temperature 1xSSC. Finally, the slides were dehydrated in
ethanol, air-dried, dipped in NTB-2 photoemulsion diluted 1:1 in water
(Eastman Kodak), exposed at 4°C for 6 weeks, developed with D19 (Eastman
Kodak), fixed with AL-4 (Agfa Gevaert), and counterstained with Cresyl
Violet.
Digoxigenin (DIG) in situ hybridization was performed using the same
protocol as for the 35S-labeled probe until the last washing step,
but with UTP labeled with DIG instead of 35S. Thereafter the tissue
was washed 2x30minutes in PBS with 0.1% Tween 20 and 2x15 minutes
in PBT (PBS with 0.1% Triton X-100 and 2 mg/ml BSA). Blocking was performed
for 3-5 hours in PBT with 20% Horse Serum (HS) (GIBCO). The sections were then
incubated with an -DIG alcaline phosphatase antibody 1:2000 (Boeringer
Manheim) in PBT with 20% HS at 4°C overnight. After 3x30-minute
washes in PBT, the alcaline phosphatase antibody was visualized by incubating
the slides in an alcaline phosphatase buffer containing levamisole, nitro blue
tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate, toluidine
salt (BCIP) for 1-2 days at room temperature. The buffer was changed every 12
hours. The reaction was stopped with EDTA and the slides were postfixed in 4%
PFA for 15 minutes and washed 3x15 minutes in PBS before let dry. After
5 minutes in xylene the slides were mounted with Pertex (Histolab Products AB)
and coverslipped.
In situ hybridization with 35S-labeled oligoprobes was performed
as previously described (Kresse et al.,
1995). Probes used were DBH
(Nakano et al., 1992
)
(nucleotides 481-528), NPY (Larhammar et
al., 1987
) (nucleotides 1671-1714), galanin
(Vrontakis et al., 1987
)
(nucleotides 152-199), BDNF (Maisonpierre
et al., 1991
) (nucleotides 645-694), and CGRP
(Amara et al., 1982
)
(aminoacids 5-23).
Immunohistochemistry
Pups were perfused with cold 4% PFA in PBS and the heads were left in PFA
for two hours before they were sequentially immersed in 10% sucrose (1 day)
and 20% sucrose the following day. Serial cryostat sections (14-µm thick)
were obtained covering the entire hindbrain, including the LC.
Immunohistochemistry was performed by first blocking with 20% HS in PBT for 30
minutes before incubating the slides in PBT at 4°C overnight with the
following primary antibodies: 1:300 mouse anti-TH (DiaSorin), 1:500 rabbit
anti-NOS (kind gift from Gerhard Skofitsch) and 1:300 mouse anti-ki67 (Abcam).
After washing with PBS, the slides were incubated with 1:200 donkey anti-mouse
rhodamine (Jackson) or 1:200 goat anti-rabbit rhodamine (Jackson) for 1-2
hours, washed with PBS again and finally mounted with Vectashield anti-fading
solution and coverslipped.
Cell counts in brain sections
Sections every 30 or 100 µm, throughout the entire LC, were processed
for DBH or BDNF in situ hybridization and counterstained with cresyl violet.
Cell counts were performed at x100 magnification in bright field.
Clearly identified cresyl-violet-positive cells covered by silver grains were
counted as positive, as described (Kresse
et al., 1995). Similarly, sections processed for TH or NOS
immunohistochemistry were analyzed. Locus coeruleus cells showing a clear
NOS-positive cytoplasm around a non-stained nuclei, were counted as positive
in blind determinations in a Zeiss microscope, at x100, as described
(Arenas et al., 1995
).
DiI tracing
The analysis of the projections from the LC into different parts of the
central nervous system in P0-GDNF/NT3 double null mutant mice was performed by
placing crystals of carbocyanine DiI (Molecular Probes) into perfusion-fixed
brains of null mutant and wild-type littermates. DiI crystals were placed with
glass micropipettes into the dorsal horn and intermediolateral column of the
cervical spinal cord as well as the area of the nucleus accumbens in the basal
ganglia. The brains were stored for 6 weeks in phosphate-buffered 4%
paraformaldehyde (PFA) pH 7.0 at 37°C to allow for retrograde transport of
the dye. After cryoprotection for 48 hours in 20% sucrose, 50 µm sections
were cut in a cryostat, mounted on glass slides, coverslipped with a
fluorescence-protecting mounting medium (Vectashield, Vector Laboratories),
and photographed under a microscope with epifluorescence illumination using
rhodamine optics.
Cell culture and treatments
Pregnant Sprague-Dawley rats (B&K Universal AB) were killed at E13.5 by
exposure to CO2, and then soaked in 70% ethanol for a minute.
Embryos were removed under sterile conditions and kept in ice-cooled PBS
during dissection. The whole proximal rhombencephalic ring (rhombomeres 1 and
2), between the distal part of the mesencephalic flexure and the proximal part
of the pontine flexure (Specht et al.,
1981), was dissected. The tissue was gently dissociated with a
fire-polished Pasteur pipette and plated on poly-D-lysine coated 12-well
plates (BD Falcon) in N2 media at a density of 1.25x105 cells
per cm2 (5x105 cells/well). Time elapsed between
the death of the mothers and plating of the cells was ≤1.5 hours. Trophic
factors were added to the wells just before plating the cells (if not stated
differently), at a concentration of 30 ng/ml. BDNF, NT4, GDNF, NT3 and bFGF
were all obtained from R&D Systems. Solutions used in tissue culture were
sterilized by filtration through 0.22 µm filters (Millipore). Cultures were
grown at 37°C in a water-saturated 5% CO2 and 95% air incubator
without changing media or supplementing the factors.
Immunocytochemistry and cell counts of primary cultures
Cultures were fixed in 4% PFA in phosphate buffer with pH 7.4 (PBS) for 50
minutes and then washed in PBS and incubated overnight with the following
primary antibodies: 1:1000 mouse anti-TH (DiaSorin), 1:50 mouse
anti-5-bromodeoxyuridine (BrdU) (DAKO), and 1:1000 rabbit anti-Phox2a
(generous gift from Dr Christo Goridis), diluted in PBS containing 1% BSA and
0.3% Triton X-100 (PBT), at 4°C. After washing, cultures were incubated
for 1-2 hours with biotinylated horse anti-mouse, or goat anti-rabbit, IgG
(Vector Laboratory), 1:500, in PBT. Immunostaining was visualized with the
Vector Laboratories ABC immunoperoxidase kit, using either gray (SG) or red
(AEC) substrates.
BrdU incorporation assay
To determine whether the cells in the primary cultures synthesized DNA,
cultures were incubated in 10 µM BrdU for a period of 6 hours (if not
stated differently) prior to fixation and then treated with 2M HCl for 20
minutes and visualized with a mouse anti-BrdU antibody (1/50, DAKO).
BDNF and NT4 ELISA
The production of BDNF or NT4 protein was analyzed in conditioned media
from LCr cultures grown in N2 medium. Conditioned media was collected after 12
and 24 hours in vitro and analyzed with a BDNF and NT4 ELISA kits
(Emax® ImmunoAssay System, Promega) according to the
manufacturer's recommendations. A standard curve of pure BDNF and NT4 proteins
provided in the kits were used to quantify the levels of neurotrophic factors
in the media. The detection limit of the ELISA assays was below 0.1 ng/ml of
BDNF or NT4. BDNF- and NT4-treated wells were used as positive controls.
Detection of nuclear DNA fragmentation by TUNEL staining
DNA fragmentation in primary cell cultures was examined by using the
DeadEndTM Colorimetric TUNEL System (Promega). Paraformaldehyde fixed
cultures were permeabilized with 0.2% Triton® X-100 solution in PBS,
washed with PBS, equilibrated and exposed to a reaction mix containing 1%
terminal deoxynucleotidyl transferase and 1% biotinylated nucleotide in
equilibration buffer for 60 minutes at 37°C. The reaction was terminated
with 2xSSC and the cells were washed with PBS. Endogenous peroxidases
were blocked with 0.3% hydrogen peroxide for 5 minutes at room temperature.
Following PBS wash, the cells were covered with Streptavidin horseradish
peroxidase solution 1:500 in PBS for 30 minutes at room temperature. The cells
were washed with PBS again and stained with gray (SG) chromogen (Vector
Laboratories).
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RESULTS |
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|
|
To further confirm the function of GDNF and NT3 on developing LC NA
neurons, primary cultures of rat E13.5 LCr were treated with GDNF, NT3 or both
factors (Fig. 3) in serum-free
conditions for 6 days in vitro (DIV). Counts of TH immunoreactive cells in
these conditions revealed no difference between treated and untreated wells in
neuronal survival (Fig. 3),
indicating that NT3 and/or GDNF are not sufficient to promote the survival of
LCr NA neurons or to induce a NA fate. However, consistent with a previous
report (Holm et al., 2002), we
found that GDNF promoted robust neurite extension of an axon-like process, an
activity that was neither observed in control cultures nor induced by NT3 (not
shown). Provided that GDNF and NT3 are neither sufficient nor required to
promote the survival of LC NA neurons, we next set out to examine whether
other neurotrophins could be involved in regulating the survival of LC NA
neurons and analyzed individual and double TrkB and TrkC
mutant mice.
|
|
BDNF or NT4 increases the number of TH-positive neurons in E13.5 rat
primary cultures of the LC region
Serum-free E13.5 LCr primary cultures were treated with either BDNF
(Fig. 5A,C) or NT4
(Fig. 5A,D) alone or in
combination with other factors, including GDNF and NT3. The cultures were
fixed after 6 days and immunostained for detection of TH
(Fig. 5B-D). Control wells
(Fig. 5A,B) contained an
average of 53±18.14 (n=6) TH-positive cells, whereas BDNF- and
NT4-treated wells contained an average of 517.75±135.00 (n=8)
and 541.43±119.597 (n=8) TH-positive cells, respectively. This
corresponds to an increase of 971% in the number of TH-positive neurons in
BDNF-treated wells and of 1015% in NT4-treated wells
(Fig. 5A). Combinations of BDNF
and NT4 with each other or with GDNF or NT3
(Fig. 5A) did not induce any
further significant increase in the number of TH-positive cells compared to
BDNF or NT4 alone. Thus our findings clearly show that either BDNF or NT4 is
sufficient to increase the number of LCr NA neurons in vitro by tenfold. We
next set out to determine the possible mechanism by which TrkB receptor and
ligands regulate the number of TH-positive cells in the LC.
|
We next examined whether the increase in TH-positive neurons in rat LCr primary culture at E13.5 resulted from increased proliferation induced by BDNF or NT4. To address this possibility BrdU was added to the culture at the following time points: (1) At the time of plating the cells, in wells that were fixed 6, 24 or 48 hours later. (2) At the time of plating, with a change of media after 6 hours, in wells that were fixed after 24 or 48 hours. (3) Six hours before fixation in cultures that were kept for 24 or 48 hours. In all the conditions and time points mentioned above, less than 5 TH-positive cells per well (0-2%) were found to be BrdU-positive (Fig. 6A) (data not shown). Moreover, comparison of the effects of BDNF and NT4 at 24 and 48 hours with those of NT3 or basic fibroblast growth factor (bFGF), two factors with mitogenic activity, revealed that the kinetics of the increase in the number of TH-positive LCr neurons in the cultures were completely different. Thus, despite the fact that proliferating cells were present in the cultures, our results suggested that the increase in numbers of TH-positive cells was not because of increased proliferation.
|
We next examined whether the late effect of TrkB ligands (between 2 and 12 hours, Fig. 6C) could also be related to survival, but several lines of evidence indicated that this was not the case: (1) The number of TH-positive cells did not decrease between 2 and 48 hours in untreated control cultures (Fig. 6C). (2) The number of TH-positive/TUNEL-positive neurons was not different in control and BDNF treated cultures after 3, 6, 12 or 24 hours (whereas no double positive cells were detected at 3 and 6 hours, the percentage of double positive cells in control and BDNF treated cultures at 12 hours were 0.52±0.06 and 0.49±0.07 respectively, and at 24 hours: 0.38±0.1 and 0.40±0.05 respectively). (3) Administration of NT4 with a 24-hour delay still increased the number of TH-positive neurons in as much as cultures treated with the TrkB ligand from the time of plating (Fig. 7). Interestingly, this late effect was not detected after a 48-hour delayed administration, suggesting that there is a window of time of approximately 24 hours during which delayed administration of TrkB ligands increase the numbers of TH-positive neurons in LCr cultures. Thus, combined, our results suggest that the increase in the number of LCr NA TH-positive cells between 2 and 12 hours results from an additional mechanism that does not involve proliferation or survival. We therefore examined whether Bdnf could work as an inductive signal for LCr NA neurons.
|
|
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DISCUSSION |
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We also examined whether other neurotrophic factors of the neurotrophin
family, in particular BDNF and NT4, were required and sufficient for the
development of LC NA neurons. The 30% decrease in the number of TH-positive LC
neurons in the TrkB-/- mice or the
TrkB-/- and TrkC+/-, but not in
TrkC-/- or TrkB+/- mice at P0,
indicated that the TrkB receptor was required for LC development. Moreover,
because the decrease of TH-positive LC cells in TrkB null mutants was
accompanied by a decrease in CV-positive LC cells, our results indicated that
LC NA neurons are lost by birth and suggest a physiological role of BDNF and
NT4 in LC development. In agreement with this possibility, addition of either
of these factors to E13.5 rat primary LCr cultures prevented the loss of NA
neurons that takes place during the first 2 hours in vitro and maintained
their survival for upto 6 days in vitro (the latest time-point tested).
Despite the fact that the function of TrkB ligands on NA neurons was analyzed
in both mutant mice and primary cultures, our results do not allow for the
determination of whether the effects of the TrkB ligands are direct or
indirect on LC NA neurons because cell compositions both in vitro and in vivo
are heterogeneous. However, the presence of TrkB receptors in LC neurons
during development, the rapid kinetics in the biological effects of BDNF and
NT4 in vitro, and the availability of TrkB ligands in the LC suggest that a
direct action is very probable. Interestingly, NA neurons are tightly packed
in the LC and produce high levels of BDNF
(Numan et al., 1998)
(Fig. 2G). Moreover,
overexpression of BDNF under the DBH promoter does not affect LC NA neurons
(Fawcett et al., 1998
),
suggesting that they are normally exposed to sufficiently high levels of BDNF
in vivo. In contrast, NA neurons in LCr primary cultures responded to the
administration of either NT4 or BDNF with increased survival after damage by
tissue dissociation and reduction in the availability of endogenous BDNF
levels by dilution in the culture media. This effect persisted for the
duration of the culture (6 days), indicating that the survival-promoting
effect is not transient and that either BDNF or NT4 maintained the survival of
LCr NA neurons. Thus, in vivo and in vitro data suggests that TrkB ligands are
both required and sufficient, respectively, to promote the survival of a
subset LC NA neurons.
In addition to the survival-promoting effects of BDNF and NT4, treatment of LCr NA neurons with either of these factors resulted in a massive 6-10-fold increase in the number of TH-positive LCr NA neurons between 2 and 12-24 hours in vitro. This effect did not involve proliferation, which was not increased in BDNF- or NT4-treated cultures, or survival, because no reduction in the number of TUNEL-positive cells was detected in BDNF-treated cultures and the increase in TH-positive cells in the culture could be induced by delayed administration of TrkB ligand. Interestingly, the effect of TrkB ligands involved an increase in the number of neurons expressing both Phox2a and TH. Surprisingly, the upregulation in both TH and Phox2a protein by TrkB ligand was simultaneous and had a very fast kinetic, because the effects were not detected at 3 hours and they were already maximal by 6 hours. Thus, our results indicate that TrkB ligands increase the number of LCr NA neurons by two different mechanisms that follow very different kinetics: (1) A very fast survival-promoting mechanism (first detected at 2 hours in vitro) which is sustained for 6 days in vitro and that is only elicited by administration of TrkB ligand with a short time window (<30 minutes) at the time of dissection. (2) A slower effect on the induction or maturation of a NA phenotype (first detected at 6 hours in vitro), which is sustained for upto 6 days in vitro and could be elicited by administration of TrkB ligand in a broad time window (from the time of dissection to upto 24 hours after plating).
The simultaneous increase in Phox2a/TH double positive cells in the
cultures also has interesting implications with regard to the mechanism by
which the NA phenotype is regulated. It is known that activation of the TrkB
receptor leads to an increase in cAMP response element-binding protein (CREB)
(Feng et al., 1999;
Pizzorusso et al., 2000
),
which in turn regulates the expression of TH
(Kim et al., 1993
;
Lane-Ladd et al., 1997
;
Piech-Dumas and Tank, 1999
).
Our results suggest that in addition to this pathway, activation of TrkB
receptor by BDNF may regulate the expression of Phox2a. This is interesting
because Phox2a is known to be part of a NA developmental pathway independent
from cAMP-CREB but involving BMPs, Mash1 and Phox2a/b
(Lo et al., 1999
). Thus, our
results suggest that there might be a link between the two pathways allowing a
regulation of Phox2a by signaling components downstream of TrkB.
Interestingly, the converse type of regulation, that BMP2 induces the
expression of BDNF has also been described in striatal cultures
(Gratacos et al., 2001
). Thus,
it is probable that both BMPs and BDNF cooperate by regulating the expression
of critical components of each other's signaling pathways.
In summary, the findings presented here show that TrkB is required for the development of mice LC NA neurons in vivo and that BDNF or NT4 are sufficient to promote both the survival and the NA phenotype of rat primary LCr neurons in vitro.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
---|
Present address: Laboratory of Cellular and Molecular Neurobiology, Fac. of
Medicine, University of Barcelona, 08036 Barcelona, Spain
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