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The
primary focus of the Mitchell laboratory concerns
mechanisms of neuroplasticity, specifically in
the respiratory motor control system. We investigate
fundamental mechanisms of compensatory plasticity
elicited by alterations in respiratory gases (adult
and developing animals), exercise, spinal injury
or motor neuron disease (ie. ALS). An emergent
theme from our work is that serotonin is a key
molecule, initiating and orchestrating important
forms of plasticity at the level of respiratory
motor nuclei. One important role of serotonin is
to regulate the synthesis of key proteins in the
underlying plasticity, such as the neurotrophin
brain derived neurotrophic factor (BDNF). Our basic
studies of respiratory plasticity may yield novel
insights concerning pathogenic mechanisms of respiratory
control disorders, such as obstructive sleep apnea
and sudden infant death syndrome. On the other
hand, our basic research has suggested novel strategies
in the treatment of sleep disordered breathing
or respiratory insufficiency caused by cervical
spinal injury or motor neuron disease.
Members
of our group perform experiments using a wide range
of techniques, allowing inferences at multiple levels of
biological organization (cell and molecular approaches
such as RNAi, neurophysiological recordings, neuroanatomical
techniques and whole animal physiological measurements).
Areas currently receiving major funding include:
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MOTOR
NEURON PLASTICTIY INDUCED BY INTERMITTENT HYPOXIA:
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Cellular/synaptic
mechanisms of respiratory long-term facilitation
following acute intermittent hypoxia. We
continue to develop a comprehensive model of
cellular/synaptic mechanisms that underlie
phrenic long-term facilitation (pLTF) following
brief exposures to intermittent hypoxia. pLTF
is a pattern-sensitive, progressive increase
in phrenic activity for hours following acute
intermittent, but not acute sustained hypoxia.
pLTF requires serotonin-receptor activation,
new BDNF synthesis within the phrenic motor
nucleus and the formation of reactive oxygen
species. Okadaic acid-sensitive protein phosphatases
constrain pLTF, preventing its expression following
sustained hypoxia; when these phosphatases
are inhibited in the cervical spinal cord,
pLTF is revealed following sustained hypoxia.
Our working hypothesis is that differential
ROS formation between intermittent versus sustained
hypoxia inhibits the relevant phosphatases,
thereby accounting for pLTF pattern-sensitivity.
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Phrenic
motor facilitation can also be induced by
trans-activation of the high affinity BDNF
receptor tyrosine kinase, TrkB .
Activation of metabotropic, Gs protein coupled
receptors (such as the adenosine 2A receptor)
induces new TrkB synthesis, TrkB signaling
and phrenic motor facilitation. This finding
represents an important ability to harness
intermittent hypoxia induced phrenic plasticity
using small molecules that activate the fundamental
mechanism without the need to apply intermittent
hypoxia.
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Phrenic
and hypoglossal long-term facilitation (pLTF
and XII LTF) exhibit metaplasticity. Both
phrenic and XII LTF exhibitmetaplasticity
following repetitive exposures to acute intermittent
hypoxia (10 episodes daily for one week,
or three times a week for 10 weeks). Repetitive
acute intermittent hypoxia enhances LTF and
up-regulates key proteins in its underlying
mechanism (serotonin receptors, BDNF, TrkB
and downstream kinases).
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Age,
gender and genetics influence pLTF expression .
Phrenic and XII LTF exhibit profound age
and sex specific patterns in rats, consistent
with age and sex-specific alterations in
serotonergic function. Futher, both pLTF
and XII LTF vary widely among rat strains,
or even sub-strains. Details of age/sex and
genetic influences on serotonin-dependent
LTF are under investigation.
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RNAi
investigations of neuroplasticity .
We were among the first to demonstrate that siRNA
can be used effectively to control gene expression
and protein synthesis (via translational regulation)
in the mammalian nervous system in vivo.
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Neuron-glial
interactions in respiratory plasticity .
A new initiative concerns the supportive
role of microglia and astroglia in phrenic
motor plasticity. Both in vivo and in vitro
experiments are underway, with current focus
on the potential roles of microglia in motor
neuron plasticity. In collaboration with
J. Watters, cell co-culture experiments have
been initiated using a model for motoneurons
(NSC 34 cells) and microglia to investigate
interactions among these cell types when confronted
with episodic versus continuous serotonin and/or
ATP receptor activation.
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RESIRATORY
PLASTICITY FOLLOWING SPINAL INJURY:
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Strategies
to strengthen silent spinal synaptic pathways
following cervical spinal injury .
Our goal is to harness respiratory plasticity
as a means of strengthening spared but ineffective
(silent) synaptic pathways to respiratory
motor neurons, thereby improving respiratory
function following cervical spinal injury.
For example, we have demonstrated that ineffective
crossed-spinal synaptic pathways to phrenic
motor neurons can be converted to effective
synaptic pathways by application of repetitive
acute intermittent hypoxia. Futher, strengthening
these synaptic pathways is associated with
nearly complete restoration of respiratory
function in rats with cervical hemisection.
Detailed mechanisms of these effects will
extensively investigated in the next few
years.
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Activation
of Gs protein coupled receptors induces TrkB
receptor trans-activation and strengthens
spinal synaptic pathways to phrenic motor
neurons, thereby restoring respiratory function
in rats with cervical spinal hemisection .
We are investigating the ability of Adenosine
2A receptor agonists (as well as agonists
of other Gs protein coupled metabotropic
receptors) to induce functional recovery
via TrkB trans-activation.
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Microglial
involvement in spontaneous and induced respiratory
functional recovery following cervical spinal
injury .
We have begun investigations concerning the potential
of microglial activation below cervical spinal
injury to enable spontaneous and evoked functional
recovery of respiratory motor output. For example,
microglia are activated within the phrenic motor
nucleus below C2-hemisection. However, we do
not yet know whether they play a beneficial versus
deleterious roles in functional recovery at this
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COMPENSATORY
PLASTICITY DURING MOTOR NEURON DISEASE (ALS):
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Respiratory
plasticity in the SOD1 G93A rat .
ALS is a devastating disease leading to progressive
motor neuron degeneration and death. Most
ALS patients develop severe respiratory insufficiency,
and the most frequent cause of death is ventilatory
failure. Our laboratory is investigating
respiratory motor function in a rodent model
of familial ALS, the transgenic rat over-expressing
mutated superoxide dismutase-1 (SOD1 G93A
rat). Our fundamental hypothesis is that
compensatory spinal neuroplasticity offsets
severe respiratory motor neuron degeneration,
preserving the ability to breathe adequately
until late in disease progression. We
are investigating cellular mechanisms of
spontaneous compensatory plasticity, and
are investigating the potential to enhance
plasticity with repetitive acute intermittent
hypoxia. We have also begun investigations
concerning the contributions of two trophic
factors postulated to play key roles in respiratory
plasticity or the pathogenesis of ALS: brain
derived neurotrophic factor (BDNF) and vascular
endothelial growth factor (VEGF). |
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Do
trophic factor secreting neural progenitor
transplants enhance phrenic motor output
and prolong motor neuron survival? In
collaboration with the laboratory of C.
Svendsen, we are using transplants of stem
cells capable of producing and secreting
relevant trophic factors (eg. BDNF, VEGF,
GDNF) to protect and prolong survival of
phrenic motor neurons during disease progression.
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