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Volume 16, Number 17,
Issue of September 1, 1996
pp. 5361-5371
Copyright ©1996 Society for Neuroscience
Mice Lacking Brain-Derived Neurotrophic Factor Exhibit Visceral
Sensory Neuron Losses Distinct from Mice Lacking NT4 and Display a
Severe Developmental Deficit in Control of Breathing
Jeffery T. Erickson1,
Joanne C. Conover2,
Veronique Borday3,
Jean Champagnat3,
Mariano Barbacid4,
George Yancopoulos2, and
David M. Katz1
1 Department of Neurosciences, Case Western Reserve
University School of Medicine, Cleveland, Ohio 44106, 2 Regeneron Pharmaceuticals, Tarrytown, New York 10591, 3 Institut Alfred Fessard, 91198 Gif-Sur-Yvette Cedex,
France, and 4 Bristol-Myers Squibb, Princeton, New Jersey
08543
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
The neurotrophins brain-derived neurotrophic factor (BDNF) and
neurotrophin-4/5 (NT4) act via the TrkB receptor and support survival
of primary somatic and visceral sensory neurons. The major visceral
sensory population, the nodose-petrosal ganglion complex (NPG),
requires BDNF and NT4 for survival of a full complement
of neurons, providing a unique opportunity to compare gene dosage
effects between the two TrkB ligands and to explore the possibility
that one ligand can compensate for loss of the other. Analysis of
newborn transgenic mice lacking BDNF or NT4, or BDNF and
NT4, revealed that survival of many NPG afferents is proportional to
the number of functional BDNF alleles, whereas only one
functional NT4 allele is required to support survival of all
NT4-dependent neurons. In addition, subpopulation analysis revealed
that BDNF and NT4 can compensate for the loss of the other to support a
subset of dopaminergic ganglion cells. Together, these data demonstrate
that the pattern of neuronal dependencies on BDNF and NT4 in
vivo is far more heterogeneous than predicted from previous
studies in culture. Moreover, BDNF knockout animals lack a subset of
afferents involved in ventilatory control and exhibit severe
respiratory abnormalities characterized by depressed and irregular
breathing and reduced chemosensory drive. BDNF is therefore required
for expression of normal respiratory behavior in newborn animals.
Key words:
chemoreceptor;
neurotrophin;
nodose;
petrosal;
respiration;
Sudden Infant Death Syndrome
INTRODUCTION
The neurotrophins comprise a multigene family that
includes nerve growth factor (NGF), brain-derived neurotrophic factor
(BDNF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4), and
neurotrophin-6 (NT6) (Davies, 1994 ; Snider, 1994; Lewin and Barde,
1996). These factors signal through the Trk family of protein tyrosine
kinases; NGF acts primarily via TrkA, BDNF and NT4 via TrkB, and NT3
via TrkC, although all bind with equal affinity to the low-affinity
neurotrophin receptor p75LNR (for review, see Barbacid,
1994).
The fact that BDNF and NT4 both bind to TrkB with high affinity raises
the question of whether these two ligands serve distinct or redundant
functions in vivo. The two TrkB ligands seem
indistinguishable in their ability to support survival of primary
sensory neurons in culture (Davies et al., 1993; Ibáñez et
al., 1993; Hertzberg et al., 1994), yet they seem capable of eliciting
differential effects on survival and morphological development of some
CNS neurons (Cohen-Cory and Fraser, 1995; McAllister et al., 1995;
Riddle et al., 1995).
We recently demonstrated that cells in the nodose-petrosal ganglion
complex (NPG), the principal visceral sensory population, require
both BDNF and NT4 for survival of a full complement of
neurons (Conover et al., 1995; Liu et al., 1995). Analysis of cell loss
in the NPG complex as a whole demonstrated additivity of the BDNF and
NT4 null mutations, suggesting that the two TrkB ligands support
primarily separate populations of ganglion cells (Conover et al., 1995;
Liu et al., 1995). However, these studies did not examine survival of
identified NPG neurons and therefore could not exclude the possibility
that some of the same cells were affected by loss of both ligands. If
this were true, it could indicate that BDNF and NT4 can compensate for
each other to support survival of some TrkB neurons in vivo,
as in vitro. In the present study, therefore, we examined
similarities and differences in the biological activities of
endogenous BDNF and NT4 by comparing survival requirements
of an identified subpopulation of NPG neurons, distinguished by
expression of dopaminergic (DA) phenotypic traits, as well as total
neuron survival, in mice lacking functional BDNF, NT4, BDNF
and NT4, or TrkB alleles. Survival of dopaminergic NPG
neurons in culture is supported by both BDNF and NT4 but not other
neurotrophins (Hertzberg et al., 1994). By analyzing both hetero- and
homozygous animals, we also were able to examine potential gene dosage
effects on NPG neuron survival. Although gene dosing has been reported
for neuronal populations supported by BDNF, NGF, and NT3 (Crowley et
al., 1994; Ernfors et al., 1994b, 1995; Bianchi et al., 1996), it is
not known whether survival of NT4-dependent neurons is regulated
similarly.
The fact that the BDNF and TrkB null mutations are lethal, whereas
animals lacking NT4 are viable (Klein et al., 1993; Ernfors et al.,
1994a; Jones et al., 1994; Conover et al., 1995; Liu et al., 1995),
argues for distinct physiological roles for the two TrkB ligands
in vivo. Previously, we suggested that the lethality of the
BDNF and TrkB mutations may be related specifically to loss of NPG
neurons (Hertzberg et al., 1994; Conover et al., 1995). These cells
provide sensory innervation to visceral tissues and thereby mediate
cardiovascular, respiratory, and gastrointestinal reflexes critical for
homeostasis. In particular, we proposed that loss of dopaminergic NPG
neurons, associated with afferent pathways controlling respiration,
could result in potentially lethal respiratory disturbances (Hertzberg
et al., 1994). To examine this possibility, the present study
characterized development of respiratory reflexes in BDNF-deficient
mice by using whole-body plethysmography. These experiments
demonstrated that loss of BDNF results in a severe developmental
deficit in reflex control of respiration.
MATERIALS AND METHODS
Cell culture
Pregnant dams (Sprague Dawley strain, Zivic-Miller, Zelienople,
PA) were killed by exposure to carbon dioxide. The uterine horns were
removed and placed into PBS containing 10% glucose, and the embryos
were excised. To assign gestational ages, the day after mating was
designated Embryonic Day (E) 0.5. Fetal (E16.5) nodose ganglia were
isolated and digested enzymatically in Dispase (Collaborative Research,
Bedford, MA; diluted 1:1 in PBS) for 1 hr at 37°C, followed by
trituration through fire-polished Pasteur pipettes. Cells were plated
(one ganglion per well) onto glass coverslips coated with polylysine
(0.1 mg/ml) and laminin (0.3 g/ml) and grown for 3-5 d in Leibovitz's
L-15/CO2 medium containing 10% NuSerum (Collaborative
Research), 5% heat-inactivated rat serum, fresh vitamin mixture (Mains
and Patterson, 1973), penicillin (50 IU/ml), and streptomycin (50 µg/ml). Cultures were grown in the absence or presence of either BDNF
alone (10 ng/ml), NT4 alone (10 ng/ml), or both BDNF and NT4 (10 ng/ml
each). Cultures subsequently were fixed in 4% paraformaldehyde (in 0.1 M sodium phosphate buffer, pH 7.4) overnight at 4°C and
processed for immunocytochemical staining as described below.
Immunocytochemistry
Cell cultures. Double-immunostaining was performed as
follows with polyclonal anti-tyrosine hydroxylase (TH; 1:200;
Pel-Freez, Rogers, AR), monoclonal anti-neurofilament protein
(NF200,160,68 1:100; Sigma, St. Louis, MO), goat
anti-rabbit IgG-FITC (1:200; Boehringer Mannheim, Indianapolis, IN),
and goat anti-mouse IgG-Rhodamine (1:200; Cappel Research Products,
Organon Teknika, Durham, NC): Cells were (1) incubated overnight at
room temperature in anti-TH and anti-NF, diluted in PBS containing
0.5% Triton X-100; (2) washed three times in PBS; (3) incubated for 1 hr at room temperature in goat anti-rabbit IgG-FITC plus goat
anti-mouse IgG-Rhodamine diluted in PBS-Triton containing 10% goat
serum and 10% rat serum; (4) washed in PBS; (5) incubated in
 -phenylenediamine (1 mg/ml) for 1 min; (6) washed in PBS; and (7)
coverslipped with glycerol gel.
Intact ganglia. Newborn animals were anesthetized
deeply and perfused through the heart with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The head of the animal
was hemisected along the midline, infiltrated with 30% sucrose for
24-48 hr, placed in a 1:1 mixture of 30% sucrose and Tissue Tek
embedding medium (Baxter Scientific, McGraw Park, IL) for 24 hr,
embedded and frozen in Tissue Tek, and stored at  80°C until use.
Frozen sections were cut (10 µm), thaw-mounted onto gelatin-coated
microscope slides, and then processed immunohistochemically as
described above.
Cell counts
Dissociate cell cultures. The number of
TH+ and NF+ neurons was counted in ~10% of
the area of each dissociate culture. Data were derived from three
cultures per condition (control, +BDNF, +NT4, +BDNF and NT4) from three
separate experiments and analyzed by ANOVA ( = 0.05), followed by
Tukey's multiple comparison procedure (Kleinbaum and Kupper,
1978).
Transgenic mice. BDNF-, NT4-, and BDNF/NT4-deficient
mice (Conover et al., 1995) were obtained from Regeneron
Pharmaceuticals, and TrkB-deficient animals (Klein et al., 1993) were
provided by Bristol-Myers Squibb.  : Mice
were perfused transcardially with 4% paraformaldehyde, and hemisected
heads were paraffin-embedded, sectioned (7 µm) in the sagittal plane,
and stained with hematoxylin and eosin. All sections containing the NPG
were identified, and every second or third section was analyzed to
determine the volume of the complex occupied by neurons, using image
analysis software (National Institutes of Health Image, v.1.55).
Volumes (µm3 × 106) were calculated from the
cross-sectional area of measured sections occupied by neurons, section
thickness, and the total number of NPG sections. Total neuron number
for each NPG was estimated by sampling ~20% of the sections; all
sections from the beginning to the end of the NPG were parcellated into
groups containing 4-5 serial sections. Then one section from each
group was selected randomly for analysis. The number of neuronal nuclei
within a measured area of the section was counted, the neuronal density
[number of neurons/(measured area × section thickness)] was
calculated, and the mean of these measurements was multiplied by
the volume of the NPG occupied by neurons. Corrected total counts were
obtained from each animal by using the mean nuclear diameter derived
from 75 individual measurements (Abercrombie, 1946).
 : All
TH-immunostained neuronal profiles with a nucleus in the plane
of section were counted in every other section from each ganglion. No
correction factor was applied. Total neuron and TH+ profile
counts from transgenic animals were analyzed by ANOVA ( = 0.05),
followed by Scheffé's multiple comparison procedure (Kleinbaum
and Kupper, 1978).
Plethysmography
Resting ventilation in normoxia (21% O2) and
ventilatory responses to hyperoxia (100% O2) and
hypercapnia (5% CO2) were measured in
bdnf+/+ and bdnf /
mice, using a whole-body plethysmograph (Bartlett and Tenney, 1970)
adapted for use with neonatal animals (Schweitzer et al., 1990).
Individual, unanesthetized animals were placed in a chamber (20 ml of
vol) that was connected to one side of a differential pressure
transducer (model DP103-14, Validyne Engineering, Northridge, CA).
Pressure changes were measured with reference to a second chamber of
identical volume connected to the other side of the transducer.
Temperature inside the chamber was measured with a thermocouple
thermometer and maintained constant (mean ± SE = 31.1 ± 0.25°C). Each chamber communicated with atmospheric pressure
through a slow leak (27 gauge hypodermic needle) to minimize pressure
differences between the chambers because of fluctuations in atmospheric
pressure during measurements. The analog signal from the transducer was
demodulated (model CD-15 carrier demodulator, Validyne Engineering),
amplified, filtered, passed through an analog-to-digital converter
(Labmaster TL-1), and then captured and stored to disk by computer
(pClamp, v.5.5.1, Axon Instruments, Foster City, CA). Signals were
calibrated by recording pressure changes associated with known volumes
of air injected into the chambers through a microliter syringe.
Barometric pressure was obtained before each recording session.
The baseline ventilatory pattern in normoxia was recorded from each
animal for 2-3 min before the presentation of each test gas. Then the
animal was exposed to the test gas for 2 min, and a continuous
recording of ventilation was obtained for an additional 2-3 min
period. Baseline ventilatory measurements were made on postnatal days
2.5 and 4.5. Responses to hyperoxia were obtained on postnatal day 2.5, whereas responses to elevated CO2 levels were obtained on
postnatal day 3.5. The hypercapnic gas mixture was obtained by mixing
pure CO2 with room air. The composition of the warmed and
humidified gas mixtures was measured with gas analyzers (models OM-11
O2 and LB-2 CO2 analyzers, Beckman Instruments,
Fullerton, CA) calibrated with gases of precisely known O2
or CO2 content. Data were analyzed by paired t
tests or ANOVA ( = 0.05), followed by Scheffé's multiple
comparison procedure (Kleinbaum and Kupper, 1978).
RESULTS
Analysis of total neuron survival revealed that both BDNF and NT4
were required for survival of a full complement of NPG neurons in
newborn transgenic mice, consistent with previous observations in fetal
and 2-week-old animals (Conover et al., 1995; Liu et al., 1995). Loss
of either BDNF or NT4 alone reduced the total number of NPG neurons by
~50% (Table 1C,E; Fig.
1B,C); the sum of the reductions observed in
mice lacking either BDNF or NT4 individually (97%) was similar to the
reduction observed after disruption of both neurotrophin genes
simultaneously (90%; Table 1H) or after loss of TrkB (94%; Table 1I).
Thus, for the majority of newborn NPG neurons, endogenous BDNF and NT4
act in a primarily noncompensatory and nonredundant manner and target
predominantly separate subpopulations of ganglion cells in
vivo.
Fig. 1.
Photomicrographs of hematoxylin- and
eosin-stained sections through the nodose ganglion from
(A) wild-type, (B)
bdnf/nt4+/+,
(C)
bdnf+/+nt4/,
(D)
bdnf/nt4/,
and (E) trkb/ mice.
Compared with the wild-type, ganglion size and cell number were reduced
significantly in
bdnf/nt4+/+
and
bdnf+/+nt4/
mice and further reduced in
bdnf/nt4/
and trkb/ animals. Scale bar, 50 µm.
[View Larger Version of this Image (156K GIF file)]
Subpopulation analysis reveals compensation between BDNF
and NT4
Although the pattern of total neuron survival in BDNF and NT4
knockout mice suggested these factors support predominantly separate
populations of ganglion cells, we could not rule out the possibility
that small subsets of ganglion cells were, in fact, targeted by both
ligands. To explore this possibility, we examined survival of a
subpopulation of ganglion cells distinguished by their expression DA
transmitter traits. These cells, which comprise ~15-20% of all NPG
neurons, can be distinguished from other ganglion neurons by expression
of the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH).
Loss of both BDNF alleles led to a 58% reduction in the number of
TH-immunoreactive (TH+) neurons in the NPG (Table
2C; compare Fig.
2A,C), similar to the reduction in
total neuron survival, indicating that a subset of DA neurons depend
exclusively on BDNF. In contrast, there was no significant change in
the number of TH+ neurons in the absence of both NT4
alleles (Table 2E; Fig. 2B), indicating that these cells do
not require NT4 for survival. The intensity of TH immunoreactivity was
comparable in ganglia from wild-type, BDNF, and NT4 knockout animals
(compare Fig. 2A-C). The fact that both the number
of TH+ neurons and total NPG cell counts decreased by
approximately the same amount in
bdnf/nt4+/+
animals indicates that the loss of TH+ neurons was caused
by decreased survival rather than by diminished TH expression.
Fig. 2.
Photomicrographs of TH-immunostained sections
through the petrosal ganglion, taken at the level of the
glossopharyngeal nerve (arrow), from (A)
wild-type, (B)
bdnf+/+nt4/,
(C)
bdnf/nt4+/+,
(D)
bdnf/nt4/,
and (E) trkb/ mice.
Compared with the wild-type, the number of TH+ profiles was
reduced significantly in
bdnf/nt4+/+,
bdnf/nt4/,
and trkb/ mice but was unchanged in
bdnf+/+nt4/animals.
Scale bar, 100 µm.
[View Larger Version of this Image (116K GIF file)]
Although TH cell counts were unchanged in
bdnf+/+nt4/
mice compared with wild-type controls, loss of BDNF plus NT4
led to a 25% greater reduction in TH cell number than loss of BDNF
alone. These data indicate that, in the absence of BDNF, a substantial
subset of DA neurons can be supported by NT4 (compare Table 2C,F,H).
Therefore, BDNF and NT4 can compensate for the loss of the other to
promote survival of at least this subset of ganglion cells. These data
contrast sharply with the results of in vitro experiments
indicating that BDNF and NT4 support the same populations of nodose and
other sensory neurons in culture (Fig. 3; Davies et al.,
1993; Ibáñez et al., 1993).
Fig. 3.
BDNF and NT4 support survival of DA nodose neurons
to the same degree in vitro. Values are means ± SEM of cell counts from three separate experiments with three cultures
per condition per experiment. The presence of BDNF and NT4 together
does not increase TH+ neuron number, as compared with
cultures supplemented with either neurotrophin alone. Moreover, total
neuronal survival was similar in the presence of BDNF, NT4, or BDNF
plus NT4 (BDNF alone, 3570 ± 268; NT4 alone, 3525 ± 300;
BDNF plus NT4, 3628 ± 181 neurons, respectively).
[View Larger Version of this Image (16K GIF file)]
Sensory neuron survival is proportional to the number of functional
BDNF, but not NT4, alleles
To compare gene dosage effects between BDNF and NT4, we analyzed
neuron survival in heterozygous animals. Our data demonstrate that a
large proportion of NPG neurons depends on the number of BDNF alleles
in a dose-dependent manner. The percentage reduction in either total
neuron number or the number of DA neurons in
bdnf+/ mice was approximately half
that observed in bdnf/ animals
(Tables 1A-C and E,G,H, 2A-C and E,G,H), implying that these neurons
normally are supported by limiting amounts of BDNF. In contrast, loss
of one NT4 allele had no effect on total NPG neuron survival, either in
the presence or absence of functional BDNF alleles; rather, survival of
NT4-dependent neurons was decreased only after disruption of
both NT4 alleles (Table 1A,D,E and C,F,H). Similarly, a
decrease in TH+ neurons was observed only after loss of
both NT4 alleles, in the absence of BDNF (compare Table 2C,F,H). These
data indicate that a full complement of NT4-dependent neurons can be
supported by a single functional NT4 allele, highlighting a fundamental
difference between BDNF- and NT4-mediated survival in
vivo.
BDNF-deficient mice exhibit severe developmental deficits in
respiratory control
Our subpopulation analysis demonstrated that DA neurons in the NPG
are severely depleted in trkb/ and
bdnf/, but not in
nt4/, mice. A large proportion of these
cells innervate the carotid body (Katz et al., 1983; Katz and Black,
1986; Finley et al., 1992), a chemoreceptor organ that provides tonic
excitatory drive to ventilation (Dejours, 1962) and the primary site at
which hypoxemia triggers homeostatic cardiorespiratory reflexes
(Heymans and Heymans, 1927; Heymans and Bouckaert, 1930). We therefore
hypothesized that loss of these cells would result in abnormal
breathing, possibly contributing to the lethality associated with loss
of BDNF or TrkB.
To determine whether respiratory control was altered by loss of BDNF,
we first compared resting ventilation in wild-type and BDNF-deficient
mice with whole-body plethysmography. All animals exhibited
irregularities in ventilation during the first 1-2 d of life, as is
typical of newborn animals (Mortola, 1984); however, differences in
breathing pattern between bdnf+/+ and
bdnf/ mice were apparent as early as
6-12 hr after birth (Fig. 4). By 2 d of age, when
the respiratory rhythm in bdnf+/+
animals was well established and regular, the ventilatory pattern in
bdnf/ animals remained irregular,
with end-expiratory pauses and occasional apneas (>2-3 sec intervals
between breaths). Quantitative analysis at this age revealed that tidal
volume (VT; a measure of the amount of air
moving in and out of the lungs) and breathing frequency
(f) in
bdnf/ mice were reduced by 43 and
49%, respectively, compared with
bdnf+/+ animals (Table
3A), resulting in a 70% decrease in minute ventilation
( E = VT × f). Two days later, on postnatal day 4.5, E was still 52% below the wild-type
control value (Table 3A), demonstrating that the ventilatory depression
in bdnf/ animals was not a transient
phenomenon associated with initial adjustments to postnatal life. A
cycle-by-cycle analysis of the breathing pattern at this age
demonstrated a twofold increase in variability of
VT and f in
bdnf/ animals (Table
4). Thus, disruption of both bdnf
alleles resulted in a drastic reduction in resting ventilation and
highly irregular breathing.
Fig. 4.
Plethysmograph records of resting ventilation in
normoxia from individual bdnf+/+ and
bdnf/ littermates during the
first 4 d of postnatal life (P0.5-P4.5). Differences in the
pattern of resting ventilation between wild-type and knockout animals
were apparent as early as 6-12 hr after birth. Scale bars: vertical,
20 µl; horizontal, 2 sec.
[View Larger Version of this Image (25K GIF file)]
Input from the peripheral chemoreceptors provides a tonic excitatory
drive to breathing, allowing continuous reflex adjustments to changes
in arterial oxygen tension (Dejours, 1962). We hypothesized, therefore,
that loss of this input might account for the reduced and irregular
ventilation of bdnf/ mice. To assess
whether oxygen-sensing mechanisms were altered in BDNF-deficient mice,
we measured resting ventilation in
bdnf+/+ and
bdnf/ animals before and during a
short exposure to 100% O2 (hyperoxia). In normal newborn
rodents, hyperoxia depresses ventilation, presumably by decreasing
activity in the peripheral chemoreceptors (Hertzberg et al., 1990).
Moreover, the magnitude of the hyperoxia-induced ventilatory depression
provides a measure of chemoafferent drive (Dejours, 1962; Hertzberg et
al., 1990). As expected, hyperoxia decreased
E on postnatal day 2.5 in
bdnf+/+ mice by 30% during periods of
quiet uninterrupted breathing, indicating the presence of a significant
chemoafferent drive to ventilation in normal animals. In contrast,
100% O2 produced no significant decrease in
E in
bdnf/ mice during quiet breathing
(Fig. 5A; Table 3B), consistent with a loss
of hypoxic drive in these animals. However,
bdnf/ mice did exhibit increased
incidence of apneas during hyperoxia, as compared with wild-type
animals.
Fig. 5.
Ventilatory responses of individual neonatal mice
to hyperoxia (A) and hypercapnia (B).
Records show periods of quiet breathing without apneas.
A, Hyperoxia depresses ventilation in P2.5 wild-type
(+/+) mice, primarily via a decrease in VT,
but had no significant effect on either VT
or f in knockout (/) animals. B, P3
knockout mice were capable of increasing respiratory output above
resting normoxic levels in response to 5% CO2. Scale bars:
vertical, 20 µl; horizontal, 2 sec.
[View Larger Version of this Image (19K GIF file)]
To examine whether bdnf/ mice were
simply refractory to all chemical inputs to ventilation, we also
evaluated responsiveness to increased inspired CO2, a
physiological stimulus that increases respiration by activating both
peripheral and central chemoreceptors (Cherniack and Longobardo, 1981).
Mild hypercapnia (5% CO2) increased breathing frequency in
bdnf/ mice by 59% (Fig.
5B; normoxia, 120 ± 4 vs hypercapnia, 191 ± 10 breaths/min; n = 5; p < 0.01),
demonstrating that the respiratory controller was responsive to
increased CO2 and that BDNF-deficient mice were
capable of increasing ventilation above normoxic resting
levels.
DISCUSSION
The present study demonstrates a spectrum of neuronal dependencies
on TrkB ligands in vivo that contrasts sharply with results
obtained from in vitro assays of neuronal survival. Thus, we
have shown that some NPG subpopulations depend exclusively on BDNF for
survival and others depend on NT4, whereas yet others can be supported
by either BDNF or NT4. The finding that BDNF and NT4 can substitute for
the other in supporting survival of some neurons provides the first
evidence of compensatory or redundant actions of endogenous TrkB
ligands on neuronal survival. Moreover, our analysis of heterozygous
animals has revealed a fundamental difference between BDNF- and
NT4-mediated survival of visceral sensory neurons in vivo.
Finally, we have demonstrated that loss of BDNF results in depressed
and irregular breathing and reduced chemosensory drive that correlates
with a severe loss of chemoafferent neurons in the NPG.
The difference between in vitro survival responses to
exogenous factors (Fig. 3) and in vivo responses to
neurotrophin gene deletions (Tables 1, 2) was striking and may be
related to the diversity of target tissues innervated by NPG neurons
in situ. For example, subsets of ganglion cells may project
to targets that provide either one or the other, or both, TrkB ligands.
Indeed, BDNF and NT4 mRNAs have been detected in developing visceral
tissues with overlapping, although not identical, spatial and temporal
distributions (Timmusk et al., 1993). It is also possible, however,
that neurotrophins could act selectively on subsets of NPG neurons
in vivo by activating different TrkB isoforms, a selectivity
that may be masked by saturating levels of growth factor in culture. At
least eight different mRNA transcripts encoding at least three
different TrkB isoforms have been identified in mammals (Klein et al.,
1989, 1990; Middlemas et al., 1991), and additional isoforms have been
demonstrated in the developing avian visual system (Garner et al.,
1996). Moreover, recent studies have shown that BDNF and NT4 can
mediate distinct biological effects on the same population of neurons.
For example, BDNF and NT4 each promote distinct, lamina-specific
patterns of dendritic growth in pyramidal neurons in developing ferret
visual cortex (McAllister et al., 1995), and NT4, but not BDNF,
prevents death of lateral geniculate neurons after monocular
deprivation (Riddle et al., 1995). In addition, exogenous BDNF and NT4
produce distinctive axonal morphologies in developing retinal ganglion
cell projections in Xenopus optic tectum (Cohen-Cory and
Fraser, 1995). We cannot rule out the possibility, therefore, that
activation of different TrkB isoforms, expressed selectively by subsets
of NPG neurons, could account for the diversity of requirements for
BDNF and NT4 exhibited by NPG neurons in vivo.
A prediction of the neurotrophic hypothesis is that growth factor
concentration is limiting for neuron survival in vivo
(Levi-Montalcini, 1987). Mice heterozygous for the NGF gene display an
intermediate responsiveness to pain (Crowley et al., 1994), and NT3
heterozygotes contain an intermediate number of muscle spindles
(Ernfors et al., 1994b), as compared with wild-type and homozygous
mice. Moreover, approximately half as many neurons are lost in the
vestibular ganglion of mice as compared with animals lacking both
bdnf alleles (Ernfors et al., 1995; Bianchi et al., 1996).
These results suggest that the availability of NGF, NT3, and BDNF
in vivo is limiting for survival of neurons dependent on
these factors. We have demonstrated that approximately half of all NPG
neurons depend on BDNF for survival in a dose-dependent manner,
implying that these neurons normally are supported by limiting amounts
of BDNF. In contrast, the NT4-dependent population of NPG neurons can
be supported fully by a single, functional nt4 allele. It is
possible that NT4 availability in wild-type animals is not limiting for
neuron survival. Alternatively, NT4 may be limiting, yet, in the
absence of one allele, NT4 synthesis is up-regulated from the remaining
allele to levels sufficient to support all NT4-dependent neurons. It is
also possible that loss of one allele increases NT4 responsiveness in
dependent neurons, perhaps via up-regulation of the low-affinity
neurotrophin receptor p75LNR or TrkB itself. The precise
function of p75LNR in neurotrophin signaling is not known
(Chao, 1994; Chao and Hempstead, 1995). Although this receptor seems
capable of Trk-independent signal transduction through the
sphingomyelin pathway (Dobrowsky et al., 1994), p75LNR also
may interact with Trk receptors to promote neurotrophin receptor
discrimination (Benedetti et al., 1993; Clary and Reichardt, 1994),
increase Trk responsiveness (Hantzopoulos et al., 1994), and
participate in retrograde transport of neurotrophins (Johnson et al.,
1987; von Bartheld et al., 1994; Curtis et al., 1995). Indeed, recent
studies indicate that p75LNR plays an important role in
modulating biological responsiveness to NT4 (Curtis et al., 1995;
Rydén et al., 1995). Whether NT4 can regulate expression of
p75LNR, however, remains to be defined.
Recent studies have indicated that the specificities of neurotrophin
interactions with their preferred Trk receptors may not be absolute. In
particular, NT3 has been shown to activate both TrkA and TrkB receptors
in vitro, but the extent to which this occurs in
vivo is not known (Cordon-Cardo et al., 1991; Klein et al., 1991;
Soppet et al., 1991; Squinto et al., 1991; Ip et al., 1993). In the
NPG, although neuron numbers are depleted by ~90% in
bdnf/nt4/ mice
(Table 1H; Fig. 1D), BDNF and NT4 are not the only trophic
factors that support survival of NPG neurons in vivo. NPG
neurons (30-50%) also are lost in mice lacking NT3 (Ernfors et al.,
1994b; Fariñas et al., 1994), indicating that NT3 and TrkB
ligands must act either sequentially (Buchman and Davies, 1993) or
simultaneously to support survival of overlapping populations of NPG
neurons. Davies et al. (1995) have provided evidence that NT3 may act
via TrkB to support survival of nodose neurons in culture. However, the
fact that neuronal losses are comparable in
bdnf/nt4/
and trkb/ mice (Tables 1H,I, 2H,I) indicates
that few, if any, cells in this ganglion are supported
exclusively by NT3 acting via the TrkB receptor in
vivo.
NT4-deficient mice appear normal, are long-lived, and produce viable
offspring, whereas BDNF and TrkB knockout mice die by 3 weeks of age
(Klein et al., 1993; Ernfors et al., 1994a; Jones et al., 1994; Conover
et al., 1995; Liu et al., 1995). Therefore, BDNF- but not NT4-dependent
neurons seem to be critical for survival after birth. In the present
study we show that bdnf/ mice
display depressed and irregular resting ventilation from birth.
Moreover, hypoxic chemosensory drive in these animals is reduced
severely. These respiratory abnormalities correlate with a severe loss
of DA neurons in the NPG, a large proportion of which are believed to
transmit chemosensory information from the carotid body (Katz et al.,
1983; Katz and Black, 1986; Finley et al., 1992). Because ventilation
and metabolic rate also are influenced by body temperature,
particularly in newborn animals, further studies will be required to
evaluate potential contributions of these factors to the knockout
respiratory phenotype. Taken together, however, our findings indicate
that loss of chemosensory input could account for the reduced and
irregular breathing of BDNF-deficient mice. Indeed, this idea is
strengthened by the observation that surgical denervation of the
peripheral chemoreceptors in newborn rats produces a highly irregular
pattern of breathing, characterized by arrhythmic low amplitude
breaths, frequent end-expiratory pauses, and apneas (Hofer, 1984, 1986)
similar to that observed in BDNF knockout animals. Our finding that
bdnf/ mice can respond to a
hypercapnic stimulus indicates that the deficit in ventilatory control
produced by loss of BDNF may be specific to the afferent
pathway-mediating hypoxic ventilatory drive, although further studies
are necessary to rule out other possible loci. It is unlikely that loss
of motor output to the respiratory musculature contributed to the
depressed ventilation, because motor neurons in the brainstem and
spinal cord are unaffected by the BDNF null mutation (Ernfors et al.,
1994a; Jones et al., 1994; Conover et al., 1995; Liu et al., 1995).
However, our studies do not rule out the possibility of more subtle
changes in motor output to the respiratory muscles.
Interestingly, BDNF heterozygous animals did not differ significantly
from wild-type controls with respect to the ventilatory parameters we
measured, although the mean values we obtained for this genotype were
nearly always intermediate between
bdnf+/+ and
bdnf/ animals (Tables 3A,B, 4). It
is possible that a more sensitive measure of ventilatory drive, such as
direct neural recordings of respiratory motor output, might define more
clearly the heterozygous phenotype. On the other hand, it is likely
that the afferent populations that subserve reflex modulation of
ventilation contain a substantial physiological ``reserve,'' such
that deficits are apparent only after neuronal losses below a threshold
level, as in bdnf/ mice. Moreover,
the long-term physiological consequences of BDNF knockout clearly are
linked to the severity of the deficit, because heterozygous animals are
long-lived, whereas homozygotes die within 3 weeks of birth (Ernfors et
al., 1994a; Jones et al., 1994; Conover et al., 1995; Liu et al.,
1995). It is likely that a prolonged depression of breathing severely
compromises oxygen-dependent metabolic processes during this period of
rapid growth and contributes to the lethality of the
bdnf/ mutation.
In addition to defining a physiological role for BDNF in development of
normal respiratory behavior, our data also may be important for
understanding clinical syndromes of abnormal respiratory control in the
neonatal period. For example, increased incidence of periodic breathing
and apnea (Steinschneider, 1972; Guilleminault et al., 1979; Kelly et
al., 1986), impaired regulation of alveolar ventilation (Shannon and
Kelly, 1977), abnormal fluctuations in heart rate and respiratory
patterns (Gordon et al., 1984; Schechtman et al., 1988, 1990, 1992),
and abnormal development of vagal nerve fibers (Becker et al., 1993)
have all been implicated as factors contributing to the Sudden Infant
Death Syndrome. Impaired hypoxic ventilatory responsiveness may also
contribute to some forms of congenital chronic hypoventilation syndrome
(Marcus et al., 1991; Weese-Mayer et al., 1992; Ogawa et al., 1993). As
shown in the present study, bdnf/
mice display many of these same deficits. Therefore, defining the
relationship between growth factor dysfunction and autonomic
pathophysiology in bdnf/ mice as
well as in other neurotrophin mutants may help to shed light on the
molecular pathogenesis of these and related developmental
disorders.
FOOTNOTES
Received April 25, 1996; revised June 6, 1996; accepted June 12, 1996.
This work was supported by HL-24131 to D.M.K. and the Francis Families
Foundation Parker B. Francis Fellowship in Pulmonary Research to
J.T.E. We gratefully acknowledge the assistance of Dr. Richard Smeyne
(formerly of Bristol-Myers Squibb; currently at Hoffman-LaRoche) in
obtaining TrkB knockout mice. We would also like to acknowledge the
thoughtful comments of Drs. Lynn Landmesser and Tom Large. We thank Hua
Jun He and Li Pan for expert technical assistance, Teresa Brosenitsch
for quantitation of the in vitro studies, and Emily Buck
and Charles Kunos for assistance with morphometric analyses.
Correspondence should be addressed to Dr. David M. Katz, Department of
Neurosciences, Case Western Reserve University School of Medicine,
10900 Euclid Avenue, Cleveland, OH 44106.
Dr. Conover's present address: Rockefeller University, 1230 York
Avenue, New York, NY 10021.
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H. Wang, S.-a. Chan, M. Ogier, D. Hellard, Q. Wang, C. Smith, and D. M. Katz
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M. Thoby-Brisson, B. Cauli, J. Champagnat, G. Fortin, and D. M. Katz
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E. A. Fox, R. J. Phillips, E. A. Baronowsky, M. S. Byerly, S. Jones, and T. L. Powley
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Y. Qian, B. Fritzsch, S. Shirasawa, C.-L. Chen, Y. Choi, and Q. Ma
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C. E. HUNT
Sudden Infant Death Syndrome and Other Causes of Infant Mortality . Diagnosis, Mechanisms, and Risk for Recurrence in Siblings
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H. Burnet, M. Bevengut, F. Chakri, C. Bou-Flores, P. Coulon, S. Gaytan, R. Pasaro, and G. Hilaire
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A. Roosen, A. Schober, J. Strelau, M. Bottner, J. Faulhaber, G. Bendner, S. L. McIlwrath, H. Seller, H. Ehmke, G. R. Lewin, et al.
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J. T. Erickson, T. A. Brosenitsch, and D. M. Katz
Brain-Derived Neurotrophic Factor and Glial Cell Line-Derived Neurotrophic Factor Are Required Simultaneously for Survival of Dopaminergic Primary Sensory Neurons In Vivo
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A. Balkowiec and D. M. Katz
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D. F. Donnelly
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D. M. Robinson, H. Kwok, B. M. Adams, K. C. Peebles, and G. D. Funk
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J Peyronnet, J C Roux, A Geloen, L Q Tang, J M Pequignot, H Lagercrantz, and Y Dalmaz
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C Baudet, A Mikaels, H Westphal, J Johansen, T. Johansen, and P Ernfors
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M. B. Lips and B. U. Keller
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A. Braun, M. Lommatzsch, A. Mannsfeldt, U. Neuhaus-Steinmetz, A. Fischer, N. Schnoy, G. R. Lewin, and H. Renz
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M. Lommatzsch, A. Braun, A. Mannsfeldt, V. A. Botchkarev, N. V. Botchkareva, R. Paus, A. Fischer, G. R. Lewin, and H. Renz
Abundant Production of Brain-Derived Neurotrophic Factor by Adult Visceral Epithelia : Implications for Paracrine and Target-Derived NeurotrophicFunctions
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A. M. LeMaster, R. F. Krimm, B. M. Davis, T. Noel, M. E. Forbes, J. E. Johnson, and K. M. Albers
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K. L. Boeshore, C. N. Luckey, R. E. Zigmond, and T. H. Large
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R. Brady, S. I. A. Zaidi, C. Mayer, and D. M. Katz
BDNF Is a Target-Derived Survival Factor for Arterial Baroreceptor and Chemoafferent Primary Sensory Neurons
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M. E. Palko, V. Coppola, and L. Tessarollo
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C. L. Stucky, T. DeChiara, R. M. Lindsay, G. D. Yancopoulos, and M. Koltzenburg
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A. Balkowiec and D. M Katz
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J. T Erickson, C. Mayer, A. Jawa, L. Ling, E B. Olson Jr, E. H Vidruk, G. S Mitchell, and D. M Katz
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K. Kolandaivelu and C.-S. Poon
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W. M. ElShamy and P. Ernfors
Brain-Derived Neurotrophic Factor, Neurotrophin-3, and Neurotrophin-4 Complement and Cooperate with Each Other Sequentially during Visceral Neuron Development
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C. Nosrat, J Blomlof, W. ElShamy, P Ernfors, and L Olson
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R. A. Johnson, A. J. Okragly, M. Haak-Frendscho, and G. S. Mitchell
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Gene
