Proteolipid protein gene mutation induces altered ventilatory response to hypoxia in the myelin-deficient rat.
ABSTRACT Pelizaeus Merzbacher disease is an X-linked dysmyelinating disorder of the CNS, resulting from mutations in the proteolipid protein (PLP) gene. An animal model for this disorder, the myelin-deficient (MD) rat, carries a point mutation in the PLP gene and exhibits a phenotype similar to the fatal, connatal disease, including extensive dysmyelination, tremors, ataxia, and death at approximately postnatal day 21 (P21). We postulated that early death might result from disruption of myelinated neural pathways in the caudal brainstem and altered ventilatory response to oxygen deprivation or hypercapnic stimulus. Using barometric plethysmography to measure respiratory function, we found that the MD rat develops lethal hypoxic depression of breathing at P21, but hypercapnic ventilatory response is normal. Histologic examination of the caudal brainstem in the MD rat at this age showed extensive dysmyelination and downregulation of NMDA and to a lesser extent GABA(A) receptors on neurons in the nucleus tractus solitarius, hypoglossal nucleus, and dorsal motor nucleus of the vagus. Unexpectedly, immunoreactive PLP/DM20 was detected in neurons in the caudal brainstem. Not all biosynthetic functions and structural elements were altered in these neurons, because phosphorylated and nonphosphorylated neurofilament and choline acetyltransferase expression were comparable between MD and wild-type rats. These findings suggest that PLP is expressed in neurons in the developing brainstem and that PLP gene mutation can selectively disrupt central processing of afferent neural input from peripheral chemoreceptors, leaving the central chemosensory system for hypercapnia intact.
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ABSTRACT: N-methyl-D-aspartate (NMDA) glutamate receptors mediate critical components of cardiorespiratory control in anesthetized animals. The role of NMDA receptors in the ventilatory responses to peripheral and central chemoreceptor stimulation was investigated in conscious, freely behaving rats. Minute ventilation (VE) responses to 10% O2, 5% CO2, and increasing intravenous doses of sodium cyanide were measured in intact rats before and after intravenous administration of the NMDA receptor antagonist MK-801 (3 mg/kg). After MK-801, eupcapnic tidal volume (VT) decreased while frequency increased, resulting in a modest reduction in VE. Inspiratory time (TI) decreased, whereas expiratory time remained unchanged. The VE responses to hypercapnia were qualitatively similar in control and MK-801 conditions, with slight reductions in respiratory drive (VT/TI) after MK-801. In contrast, responses to hypoxia were markedly attenuated after MK-801 and were primarily due to reduced frequency changes, whereas VT was unaffected. Sodium cyanide doses associated with significant VE increases were 5 and 50 microg/kg before and after MK-801, respectively. Thus 1-log shift to the right of individual dose-response curves occurred with MK-801. Selective carotid body denervation reduced VE during hypoxia by 70%, and residual hypoxic ventilatory responses were abolished after MK-801. These findings suggest that, in conscious rats, carotid and other peripheral chemoreceptor-mediated hypoxic ventilatory responses are critically dependent on NMDA receptor activation and that NMDA receptor mechanisms are only modestly involved during hypercapnia.Journal of Applied Physiology 04/1998; 84(3):853-61. · 3.48 Impact Factor
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ABSTRACT: Previous studies have indicated that newly formed oligodendrocytes are dynamic cells whose production, survival, and differentiation depend upon axonal influences. This study has characterized the appearance and fate of newly formed oligodendrocytes in developing rat brain. Oligodendrocytes appear in predictable locations and radially extend DM-20-positive processes that cover 80-microm domains in the cortex and 40-microm domains in the corpus callosum. These premyelinating oligodendrocytes have one of two fates: they myelinate axons or degenerate. Between 7 and 21 d after birth, approximately 20% of premyelinating oligodendrocytes identified in the cerebral cortex were degenerating. Oligodendrocytes that ensheathed axons expressed and selectively targeted proteolipid protein to compact myelin and did not degenerate. These observations support the hypothesis that axonal influences affect oligodendrocyte survival, differentiation, and expression of proteolipid protein gene products.The Journal of Cell Biology 05/1997; 137(2):459-68. · 10.82 Impact Factor
Pelizaeus Merzbacher disease is an X-linked dysmyelinating disorder of the CNS, resulting from mutations in the proteolipid protein
(PLP) gene. An animal model for this disorder, the myelin-deficient (MD) rat, carries a point mutation in the PLP gene and exhibits a
of NMDA and to a lesser extent GABAAreceptors on neurons in the nucleus tractus solitarius, hypoglossal nucleus, and dorsal motor
nucleus of the vagus. Unexpectedly, immunoreactive PLP/DM20 was detected in neurons in the caudal brainstem. Not all biosynthetic
functions and structural elements were altered in these neurons, because phosphorylated and nonphosphorylated neurofilament and
choline acetyltransferase expression were comparable between MD and wild-type rats. These findings suggest that PLP is expressed in
Pelizaeus-Merzbacher disease is an X-linked dysmyelinating dis-
order characterized by nystagmus, hypotonia, ataxia, spasticity,
and mental retardation (for review, see Seitelberger et al., 2002).
In the most severe form (connatal), symptoms may develop
shortly after birth, with death within the first decade (Boulloche
(including duplications and deletions) have been found in the
proteolipid protein (PLP) gene (Cremers et al., 1987; Raskind et
al., 1991; Ellis and Malcom, 1994; Harding et al., 1995,
Yamamoto et al., 1998; Garbern et al., 1999; Inoue et al., 1999).
PLP is the predominant myelin protein in the CNS, accounting
for ?50% of total myelin protein. Spontaneous mutations of the
PLP gene have been described in diverse animals, including the
rat [PLP myelin-deficient (MD)], dog, rabbit, and mouse
( jimpy) (Nave, 1994; Knapp, 1996; Garbern et al., 1999; Yool et
al., 2000). Almost all rodent PLP mutants, including the MD rat,
exhibit a phenotype similar to connatal Pelizaeus-Merzbacher
disease, developing tremor and ataxia at 10–12 d, seizures at
16–21 d, and death at 21–28 d. In the MD rat, a point mutation
of the PLP gene (A to C transversion) (Boison and Stoffel, 1989),
which induces dramatic hypomyelination and early cell death of
oligodendrocytes in the CNS. How mutation of the PLP gene
results in early mortality in humans and affected animals is
In the rat, control of breathing undergoes significant matura-
tion in the first 2–3 weeks of life, with dramatic changes in the
ventilatory response to changes in CO2and O2concentration in
the blood. Respiratory rate is primarily driven by blood CO2
concentration, and, during postnatal life, sensitivity to this stim-
sic ventilatory response, consisting of an increase in breathing,
followed by a decline. As the animal matures, O2deprivation
elicits a more sustained increase in breathing (for review, see
life, neuronal pathways in the brainstem that regulate breathing
also undergo rapid myelination (Jacobson, 1963; Csiza and De
Lahunta, 1979). The impact of dysmyelination on postnatal de-
velopment of the central neural pathways that control breathing
has not been investigated.
We speculated that early death characteristic of the MD rat
might result from dysmyelination of brainstem pathways neces-
nia) or hypoxia. To test this hypothesis, we compared these re-
sponses during development in affected MD pups and wild-type
(WT) littermates and found that hypoxia induces a dramatic
inhibition of respiration at postnatal day 21 (P21) in the MD rat,
although the hypercapnic response was normal. We then inves-
tigated histopathology of the caudal brainstem in the MD rat at
P21 and found that extensive dysmyelination was accompanied
by downregulation of the glutamatergic NMDA NR1receptor
Correspondence should be addressed to Dr. Martha J. Miller, Division of Neonatology, Rainbow Babies and
TheJournalofNeuroscience,March15,2003 • 23(6):2265–2273 • 2265
protein accumulated in neuronal cell bodies as well as in oligo-
PLP mutation selectively disrupts autonomic control of respira-
tion during hypoxia, possibly through expression of the mutant
PLP/DM20 protein in neurons of the caudal brainstem.
Male MD rat pups were bred at the Lerner Research Institute of the
Cleveland Clinic Foundation by mating MD/? females with normal
tion of the 12 hr light/dark cycle. All protocols for physiologic research
met with previous approval of the Institutional Animal Care and Use
Committees of Case Western Reserve University and the Cleveland
Clinic Foundation in compliance with the Public Health Services Policy
on humane care and use of animals.
Measurement of respiration. At postnatal days 14, 18, and 21–24, pups
were placed in a heated, flow-through barometric plethysmograph
(BUXCO Electronics, Troy, NY). This apparatus allows non-invasive
quantitative measurement of breathing. Affected male pups (n ? 18)
were compared with normal littermate males (n ? 19). Air flow was
maintained at 0.6 l/min. The pups were allowed to adapt to the plethys-
movement. Then, after baseline respiration was measured, the respira-
ing to an increase in inhaled CO2above this level (hypercapnia), the
inflow to the plethysmograph was switched to the test gas: 10% CO2,
balance N2and 30% O2for 10 min. To evaluate the response of the rat
At the conclusion of the test, the plethysmograph gas flow was returned
minute of gas expired), and minute ventilation (the volume of air ex-
changed through the lungs; the product of respiratory rate and tidal
volume) was recorded. Minute ventilation was averaged over 20 sec in-
tervals before and during the test gas exposure. For comparison between
multiple individuals, minute ventilation was expressed as percentage
change from the control period before the test gas, as in previous studies
SE; statistical analysis was by ANOVA. p ? 0.05 was considered
or PLP-enhanced green fluorescent protein (EGFP) mice (Mallon et al.,
with 2 ml/gm room temperature (RT) PBS, containing 2 U/ml heparin
sulfate and then with ice-cold 2, 4, or 4% paraformaldehyde plus 0.2%
glutaraldehyde buffered with PBS. For detection of the GABAAreceptor
?2 subunit and the glutamatergic NMDA NR1receptor subunit, perfu-
sion with 2% paraformaldehyde was used, with 24 hr postfixation at 4°C
in 2% paraformaldehyde. For detection of choline acetyltransferase
aldehyde in PBS. Brains were postfixed overnight at 4°C in 2% parafor-
dehyde perfusion was used, and brains were postfixed for 24 hr at 4°C in
rostral to the decussation of the pyramids, were cut under ice-cold PBS
on a Leica (Nussloch, Germany) VT 1000S vibratome and stored at 0°C
in cryostorage solution. For each immunohistochemical comparison,
brains from two to nine individual WT and MD rats were studied.
Free-floating sections were rinsed with PBS at room temperature and
then incubated for 60 min in 3% normal goat serum (NGS) or milk in
PBS. After rinsing in PBS, sections were incubated overnight at 4°C in
PBS with the following primary antibodies: mouse monoclonal anti-
NMDA NR1, 6021A (1:1000; BD PharMingen, San Diego, CA); mouse
monoclonal anti-GABAA?2 subunit, MAB341 (1:1000; Chemicon, Te-
mecula, CA); mouse anti-neuronal nuclei (NeuN), MAB377 (1:1000;
Chemicon); PLP/DM20 antibody, clone AA3 (1:1000; gift from Steven
Pfeiffer, University of Connecticut Health Science Center, Farmington
CT); mouse anti-SMI31 monoclonal antibody (1:10,000; Sternberger
Monoclonals, Lutherville, MD); mouse anti-SMI32 monoclonal anti-
body (1:3000; Sternberger Monoclonals); and goat anti-ChAT AB144p
(1:500; Chemicon). Sections were then rinsed three times in PBS and
incubated for 60 min in 3% NGS or milk with secondary antibody. For
immunofluorescence, the incubation with secondary antibody was 45
For bright-field microscopy, sections were washed in PBS and then
incubated for 30 min in 1% H2O2and 10% Triton X-100 at room tem-
perature; then sections were incubated with primary antibody. The sec-
tions were then washed and incubated with the appropriate biotinylated
secondary antibody (Vector Laboratories, Burlingame, CA) in 3% NGS
or milk in PBS for 60 min at RT. After rinsing with PBS three times,
sections were incubated for 60 min in ABC solution (1:1000; Vecta-
shield), rinsed in PBS, incubated 2 min in DAB, rinsed in PBS, post-
treated with 0.08% osmium tetroxide in water for 20 sec, rinsed in PBS,
incubated 5 min in 60% glycerol, mounted in PBS, air dried, and cover-
slipped with glycerol. For ChAT, sections from tissue prepared as de-
scribed above were washed with PBS and incubated 30 min in PBS con-
taining 1% H2O2and 10% Triton X-100. After washing in PBS, the
washed in PBS and then incubated at 4°C for 72 hr with the primary
antibody. After washing in PBS, the sections were incubated at RT with
son ImmunoResearch, West Grove, PA). After rinsing in PBS, sections
were incubated for 60 min at RT in ABC solution (1:1000), washed in
PBS, and incubated for 5 min in 1 M Tris buffer, pH 7.6. Sections were
then incubated in DAB in 1 M Tris, with 2% nickel ammonium sulfate,
and 0.4% H2O2. Sections were then washed in PBS, incubated for 20 sec
in 4% OsO4, washed in PBS, and incubated for 5 min in 60% glycerol
For staining with cresyl violet, tissues were first washed in PBS,
mounted onto slides, and then dehydrated in an 80°C oven before incu-
bation for 3 min in 0.1% cresyl violet. Slides were then rinsed in deion-
mounting in Permount.
Quantification of cells in nuclei of the caudal brainstem. To determine
total cell density and neuronal cell number, sections were stained with
DAPI or with NeuN antibody. Cells were quantified in the nucleus trac-
tus solitarius (NTS), the dorsal motor nucleus of the vagus (DMV), and
the hypoglossal nucleus in five WT and five MD rats at P21. Location of
these nuclei was defined as in The Rat Brain in Stereotaxic Coordinates
(Paxinos and Watson, 1986). Cell counts were verified by two investiga-
paired t test.
Immunoblot. Tissue lysates were made using lysis buffer: 0.15 M NaCl,
0.05 M Tris, 0.5 mM EDTA, 1% Triton X-100, and 0.05% SDS, pH 7.5,
supplemented with a protease inhibitor cocktail (20 ?g/ml leupeptin,
hr on ice, samples were centrifuged at 15,000 ? g for 10 min to remove
by the bicinchoninic acid method (Sigma, St. Louis, MO).
Lysates were separated on 8.5% SDS-PAGE, blotted to the polyvinyli-
dene difluoride membrane, blocked with 5% nonfat dry milk in TBS-T
Gudz et al. (2002). Immunoreactive bands were visualized using an en-
hanced chemiluminescence kit (ECL-Plus; Amersham Biosciences, Pis-
At P14, P18, and P21–P24, there was no difference in respiratory
2266 • J.Neurosci.,March15,2003 • 23(6):2265–2273Milleretal.•ProteolipidGeneMutationAltersRespiratoryControl
(the product of tidal volume and respiratory rate) between MD
and WT littermate males at rest. No prolonged apnea, defined as
prolongation of expiratory duration ?2 sec, was detected in ei-
ther group. When the respiratory response to an increase in in-
haled carbon dioxide (10% CO2, 30% O2, and balance N2for 10
min) was tested at P14, P18, or P21, there was no difference
between the responses of the MD males and normal male litter-
mate controls. At P14 and P18, there was also no significant dif-
ference in the ventilatory response to hypoxia (8% O2and bal-
ance N2for 5 min) (Fig. 1A,B). However, when the ventilatory
hypoxic depression in the MD pups resulted in death of five of
seven animals during hypoxic exposure at this age.
The dramatic hypoxic depression of breathing coupled with a
normal hypercapnic response in the MD rat at P21–P24 sug-
gested that expression of the abnormal PLP gene in this mutant
could have selectively affected the central neural pathways that
govern the response of breathing to hypoxia. Therefore, we fo-
cused our investigation on the neuronal nuclei within the caudal
from the peripheral sensors of hypoxia (the carotid bodies). Spe-
cifically, we studied the caudal brainstem at the area of the com-
missural nucleus of the NTS, which is the predominant site for
synapse of afferent fibers from the carotid bodies. From this nu-
cleus, adjacent areas, such as the DMV and the hypoglossal nu-
cleus, receive efferent input from the NTS.
Initial analysis of the areas of interest in the medulla by cresyl
2), although a reduction in intensity was noted. To determine
whether maturation of the brainstem in the MD rat was accom-
MD and WT rat (Table 1). To determine the number of neurons
Although there were apparent decreases in NeuN-positive neu-
rons in the NTS, DMV, and hypoglossal nuclei, these differences
were not statistically significant.
medulla in the MD rat, we compared neurofilament expression
in MD and WT rats. A similar pattern of staining for nonphos-
phorylated (Fig. 4) or phosphorylated (data not shown) neuro-
filament expression in the caudal brainstem was found in MD
and WT rats. Thus, the MD mutation does not appear to alter
distribution of either phosphorylated or nonphosphorylated
neurofilaments at this age.
Dysmyelination of the CNS and decreased synthesis of PLP/
DM20, myelin basic protein (MBP), and myelin-associated gly-
coprotein have been described previously in the CNS in the MD
rat (Dentinger et al., 1982, Kumar et al., 1990), but no data are
Cells stained with cresyl violet were evenly distributed at the level of the area
Milleretal.•ProteolipidGeneMutationAltersRespiratoryControlJ.Neurosci.,March15,2003 • 23(6):2265–2273 • 2267
available on the caudal brainstem. When PLP immunoreactivity
was compared in MD and WT littermates, as expected, there was
a striking decrease in immunoreactive fibers in the caudal brain-
in the MD pup, PLP/DM20 protein was detected in apparent
neurons in the hypoglossal nucleus and ventrolateral to that area
(Fig. 5B). Thus, it appeared that the PLP gene is transcribed and
translated in specific neurons in the respiratory neuraxis of the
caudal brainstem in the MD pup.
rats were double labeled for NeuN and PLP/DM20 (Fig. 5).
NeuN-positive neurons also expressing PLP/DM20 protein were
detected in the hypoglossal motor nucleus and in scattered neu-
rons ventrolateral to this area (Fig. 5D), but no double-labeled
neurons were detected in normal WT males (Fig. 5C). Thus, in
the MD rat, a neuronal population in the caudal brainstem ex-
presses PLP/DM20 protein, and the mutated protein accumu-
bodies (Gow et al., 1998). It was striking that, within the caudal
brainstem, a subset of neurons expressed the PLP gene, while
intermingled in the same region were neurons that appeared to
have no PLP in the cell body. The double-labeled neurons were
accounted for 55% (range of 37–69%, in four animals) of neu-
rons in the hypoglossal motor nucleus. This suggests the exis-
that do not express the PLP gene and are unaffected by the
To determine whether the PLP gene can be expressed in the
caudal brainstem during normal rodent development, we ana-
lyzed this area of the brain in the transgenic PLP-EGFP mouse
mouse expresses EGFP under the control of the PLP promoter.
Neurons weakly expressing the PLP promoter were identified in
nontransgenic mice had no green fluorescent signal in neurons
(Fig. 6). This observation lends support to our premise that the
PLP gene is transcribed and translated in neurons within the
The ventilatory response to hypoxia requires integrity of afferent
sensory input from the carotid bodies to the NTS (Cottle, 1964;
Panneton and Loewy, 1980; Ciriello et al., 1981; Finley and Katz,
1992; Jordan, 1994). At this nucleus, glutamatergic NMDA re-
c-fos expression (Haxhiu et al., 1995) and for the increase in
ventilation after exposure to hypoxia (Ang et al., 1992; Ogawa et
al., 1995; Lin et al., 1996; Ohtake et al., 1998, 2000). We selected
the NMDA receptor subunit R1for analysis because this subunit
may be required for function of the NMDA receptor (Nakanishi,
1992). When we compared the expression of the NMDA NR1
subunit in the caudal brainstem at the level of the area postrema,
relative to littermate controls. Loss of this receptor subunit was
particularly evident in the NTS, DMV, and hypoglossal nuclei
(Fig. 7). The downregulation of NMDA NR1receptor subunits
2268 • J.Neurosci.,March15,2003 • 23(6):2265–2273Milleretal.•ProteolipidGeneMutationAltersRespiratoryControl
NR1in the forebrain was normal in the P21 MD rat (data not
a role in the net balance of ventilatory response to hypoxia. This
class of receptors in the caudal ventrolateral medulla is tonically
active (Miyawaki et al., 2002; Zuperku and McCrimmon, 2002),
and activity increases in response to hypoxia-induced GABA re-
lease, thereby contributing to depression of ventilation by pro-
longed or recurrent hypoxia (Melton et al., 1990; Miller et al.,
2000; Tabata et al., 2001). On the basis of our physiologic find-
ings, we postulated that profound hypoxic inhibition in the MD
rat could reflect a relative imbalance of stimulatory (glutamater-
gic) and inhibitory (GABAergic) influences on respiratory drive.
We therefore analyzed expression of the GABAAreceptor (?2
subunit) in the caudal brainstem of MD and WT rats at P21. In
the MD pup, there was a decrease in immunoreactivity for
GABAAreceptors in the NTS, hypoglossal nucleus, and DMV, as
well as the raphe pallidus (Fig. 8). Strong immunostaining was
still observed in the area postrema. The focal decrease in GABAA
receptor immunoreactivity was restricted to the dorsal aspect of
the medulla oblongata and raphe nuclei, because immunostain-
and was not correlated with a decrease in total GABAAreceptor
?2 or ? subunit proteins in brainstem (see below).
rons of the hypoglossal nucleus and the DMV, and cholinergic
neurotransmission has been implicated in the ventilatory re-
sponse to hypercapnia (Loeschcke, 1982). Given the normal re-
sponse of the MD rat to hypercapnia, we postulated that expres-
sion of the biosynthetic enzyme for acetylcholine, ChAT, would
be normal in the caudal medulla. As predicted, immunostaining
for this enzyme in motor neurons of the caudal medulla in the
MD rat at P21 was comparable with that in the WT rat (Fig.
9A,B). Thus, these data are consistent with the physiologic find-
ing of intact hypercapnic response in the MD mutation.
To determine whether the decrease in immunoreactivity for
NMDA NR1and GABAAreceptors was correlated with a quanti-
tative loss of receptor protein, we performed immunoblot anal-
yses for NMDA NR1, the GABAA?2 and ? subunit proteins, as
and WT rats (Fig. 10). We found that NMDA NR1receptor sub-
MBP. However, GABAA?2 and ? receptor subunits were not
and NMDA receptors are differentially regulated in the medulla
of the MD rat and that, although there are some cells that down-
regulate GABAAreceptors in the medulla, GABAAneurotrans-
mission is likely to be relatively intact in this tissue.
(A). Furthermore, neurons immunoreactive for PLP were present in the hypoglossal nucleus
Expression of PLP in the wild-type and MD rat at P21. In the caudal brainstem,
Milleretal.•ProteolipidGeneMutationAltersRespiratoryControlJ.Neurosci.,March15,2003 • 23(6):2265–2273 • 2269
for the hypoxic ventilatory response is altered in the MD rat, at
tion selectively alters signal transduction pathways in the brain-
stem involved in response to oxygen deprivation. Furthermore,
the fact that respiratory rhythm generation is preserved in the
dulla is not affected by the MD PLP mutation, nor is descending
outflow to spinal cord motoneurons regulating the activity of
chest wall pumping muscles.
It is quite striking that the MD mutation does not affect the
ventilatory response to hypercapnia. Hypercapnia activates a
subset of neurons along the neuraxis that have diverse functions.
It has been shown that cholinergic mechanisms in the ventrolat-
eral medulla play an important role in mediation of ventilatory
response to changes in CO2/H?concentration in the extracellu-
lar fluid (Loeschcke, 1982). Because ChAT expression in the me-
dulla oblongata of MD rats is comparable with that observed in
WT animals (Fig. 9), unaltered cholinergic mechanisms may ex-
the MD rat.
The ventilatory response to hypoxia in a normal unanesthe-
tized P21 rat consists of an initial increase in minute ventilation
within the first 1–2 min, followed by a sustained increase above
baseline, until the hypoxic stimulus is withdrawn (Fig. 1C, WT).
The initial phase of the hypoxic response depends on neural in-
puts from the carotid bodies mainly through the petrosal and
jugular ganglia to the caudal NTS. This excitatory input has been
shown to require release of glutamate and activation of NMDA
receptors on second-order neurons in the NTS (for review, see
Gozal and Gaultier, 2001). These neurons project to diverse ar-
eas, including the inspiratory rhythm-generating network in the
ventrolateral medulla (Loewy and Burton, 1978; Smith et al.,
1989). NMDA receptors are critical for the normal ventilatory
response to hypoxia in the adult rat (Ohtake et al., 1998, 2000),
and deletion of this glutamatergic receptor is lethal in the imme-
diate postnatal period in mice (Funk et al., 1997).
present in the nucleus tractus solitarius, dorsal motor nucleus of the vagus, and hypoglossal
2270 • J.Neurosci.,March15,2003 • 23(6):2265–2273Milleretal.•ProteolipidGeneMutationAltersRespiratoryControl
sion in the late phase of the hypoxic response. Thus, the initial
phase of the hypoxic response is intact in the MD rat, despite the
reduction in NMDA receptors in the NTS subnuclei receiving
inputs from peripheral chemoreceptors. Conceivably, sufficient
NMDA receptors remain for activation of respiratory output to
occur, or alternate pathways for generation of the initial phase of
the response may be present. The severe late hypoxic ventilatory
depression in these rats could be a consequence of reduced
NMDA activation of nitric oxide–cGMP signaling pathways in-
volved in regulating prolonged excitatory inputs at the NTS
(Haxhiu et al., 1995; Ogawa et al., 1995). Thus, we speculate that
The central effects of hypoxia may activate inhibitory GABAA
signaling pathways (Tabata et al., 2001) in the MD rat and con-
tribute to the profound late hypoxic ventilatory depression. Al-
of the caudal brainstem (Fig. 8), sufficient receptors may remain
in other areas of the brainstem for preservation of GABAergic
inhibitory influences. This is consistent with our Western blot
analysis showing that the overall protein content of the major
GABAAsubunits is normal in the brainstem of the P21 MD rat.
they may develop a relative imbalance of stimulatory and inhib-
itory neurotransmission in response to hypoxia, which results in
fatal hypoxic ventilatory depression.
CNS myelination in the rat proceeds in a caudal to rostral
direction and is essentially complete by P21 in the brainstem.
During late embryonic and early postnatal life in the rat, expres-
sion of the PLP gene also changes. The alternatively spliced iso-
nonmyelinating cells (Ikenaka et al., 1992; Timsit et al., 1992).
stage of oligodendrocyte differentiation, during myelination and
myelin compaction (Nadon et al., 1990; Trapp et al., 1997). MD
the oligodendrocyte and is poorly transported to cell processes,
resulting in reduced myelination and oligodendrocyte cell death
MD pup, hypomyelination, alters development of myelinated,
rapidly conducting neural pathways in the brainstem necessary
for rapid response to sensory input such as hypoxia.
On the other hand, this study also demonstrates that the MD
PLP gene is expressed in neurons in the medulla oblongata (Figs.
5, 6). This finding is consistent with previous observations that
the PLP gene is expressed in diverse cells, including neurons and
that the PLP promoter is expressed in the early postnatal period
in neurons in the NTS, hypoglossal nucleus, ventrolateral me-
PLP gene in neurons are unknown. It is possible that mutated
PLP/DM20 protein accumulates in neuronal cell bodies (Fig. 5),
later and/or less in most of the neurons because of reduced
amounts of the protein or reduced sensitivity of neurons to this
misfolded protein. Because most of these brainstem nuclei have
normal numbers of neurons, it is possible that defective PLP/
DM20 protein in neurons may affect their function before it ac-
tually leads to cell death, and the animal dies because of loss of
function in these critical neurons. It is quite striking that the
neurons in the respiratory neuraxis of the caudal brainstem have
diminished in the NTS, DMV, and hypoglossal nucleus supports
the concept that neurons in these areas are not (yet) dying but
have significantly altered function.
PLP/DM20 has structural similarities to proteins with four
transmembrane domains, some of which may function as ion
channels. A possible role of PLP/DM20 as an ion pore is sup-
ported by the elevated pH in mouse jimpy oligodendrocytes and
by a series of studies in lipid membranes (Ting-Beall et al., 1972;
Diaz et al., 1990; Knapp et al., 1993). Transcription of a new
also been shown to be upregulated in the ventral medulla by
expression of mutant PLP in CNS neurons that control ventila-
pH, resulting in diverse pathologic changes in cellular function.
In this study, we focused on ventilatory chemosensory re-
sponses in the MD rat. It is likely that other functions of the
caudal brainstem may be altered during postnatal development,
including autonomic control of heart rate and blood pressure, as
of the MD rat to hypoxia.
sayed by immunoblot in tissue from the brainstem in the wild-type and MD rat at P21.
Actin is present as a loading control. In the MD rat, there was downregulation of NMDA
Milleretal.•ProteolipidGeneMutationAltersRespiratoryControlJ.Neurosci.,March15,2003 • 23(6):2265–2273 • 2271
In summary, the results of this study show that the MD PLP
mutation causes pathologic alteration of respiratory control, ac-
to a lesser extent GABAergic receptors. These findings may be
relevant to understanding of the “connatal” or infantile form of
Pelizaeus Merzbacher disease. These children die in the first de-
cade of life of causes that are incompletely understood. It is con-
ceivable that expression of the mutant PLP gene in the connatal
of respiratory homeostasis, as in the MD pup, and contribute to
early death of affected children
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