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Research: The Neural Origins of Breathing
Systems Neuroscience
The College of Wi iiiam and Mary
The most important issues in contemporary
biology are interdisciplinary, spanning multiple
levels of analysis from
genes and molecules to
the complex properties of
networks. Nowhere is the
interdisciplinary nature of
modern-day life sciences
more relevant than in
neuroscience. Brain
function underlies the vast
repertoire of human
behaviors and dysfunction
of the brain and central nervous system causes
many of society's most serious health problems.
I am Professor Christopher A. Del Negro and my
Systems Neuroscience laboratory in the
Department of Applied Science provides a unique
environment for neuroscience research and
training at the graduate and postdoctoral level. My
team and I have assembled state-of-the art
experimental workstations for intracellular and
patch-clamp electrophysiology combined with in
vitro live-cell videomicroscopy and fluorescence
imaging. We also have a vigorous computational
neuroscience branch; we support parallel
computing facilities for modeling and simulation.
Our strategy to elucidate new knowledge in
neuroscience blends theory and experiment: we
use modeling to generate testable predictions,
which are systematically evaluated via hypothesisdriven experimental studies.
The masters and doctoral degree programs in
Applied Science offer flexible curricula that are
individually tailored by students, in consultation
with their faculty advisors, to accomplish each
student's unique and often interdisciplinary
educational goals.
Breathing behavior in mammals begins in utero
and continues without lapse for the entire lifespan
of the animal, which in humans can last up to, or
exceed, 100 years. Diseases that affect the neural
control of breathing can strike at any age, but
newborns and premature babies are particularly
susceptible to various forms of apnea and SIDS.
We aim to provide new knowledge about how the
neurons, synapses and networks of the brain stem
assemble the rhythm-generating systems that
drive breathing movements and control respiratory
physiology.
Breathing is an especially advantageous model
system for this type of analysis because it is a
behavior that can be studied under controlled
conditions in vitro, using reduced brain stem 'slice'
preparations. Our 'breathing slices' retain
functional respiratory networks and generate
spontaneous motor output during the inspiratory
phase of the respiratory cycle.
Figure 2. Students and faculty work together, using
cutting-edge technology, to discover new principles
in neuroscience at The College of William and Mary.
One ongoing project in the laboratory examines
calcium-activated nonspecific cationic membrane
currents (i.e., 'CAN)' The ion channels underlying 'CAN
are putatively from the transient receptor potential,
or TRP, superfamily and have the unique ability to
integrate voltage-dependent calcium influx with
synaptically activated intracellular calcium release
mechanisms. TRP channels can transform these
In mammals, the rhythm for breathing originates in
the brain stem nucleus called the preBotzinger
Complex (preBotC). We isolate the preBotC and
.A
B
110 mV
Figure 1. The 'breathing slice' preparation
in vitro.
Figure 3. Two preBotC neurons recorded in vitro
using patch-clamp and fluorescent calcium imaging.
its constituent rhythmogenic neurons in slice
preparations (Fig. 1A). Rhythmic inspiratory motor
activity can be measured via the hypoglossal
cranial nerve output (Xlln) in vitro, while we
examine cellular and synaptic properties in the
context of network function.
convergent calcium signals into high-amplitude,
long-lasting neuronal activity states, which are
suitable for driving inspiratory bursts and rhythm
generation.
During fictive inspiration, neurons in the preBotC
generate robust bursts coincident with XII motor
output (Fig. 1B). We aim to discover the cellular
and synaptic mechanisms that produce rhythmic
inspiratory bursts in preBotC neurons.
Another thrust of our research examines the
patterns of network connectivity among preBotC
neurons. Here we use dual intracellular recordings
to quantify synaptic coupling between preBotC
neurons, as well as epifluorescence, confocal, and
multi-photon imaging to measure calcium
dynamics. Calcium imaging represents a
convenient way to monitor the activity of many
neurons simultaneously, which helps reveal
network properties such as connectivity (Fig. 3).
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Figure 4. Voltage-clamp analysis of preBtitC neuron
sensitivity to neuropeptides such as substance P.
Our efforts to unravel the neural basis for
respiratory rhythm also emphasize mathematical
modeling (Fig. 5). We are developing biophysically
realistic models of preBotC neurons that account
for morphology, the known complement of ion
channels in preBotC neurons, as well as the novel
biochemical signaling pathways that integrate
synaptic and transmembrane calcium fluxes to
evoke the burst-generating current ,CAN• Modeling
at the cellular level forms the basis for network
simulations that explore the role of connectivity:
both the strength of coupling and the pattern of
synaptic interconnections in the preBotC.
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Del Negro, CA et al. (2002) Biophysical Journal
82, 206-214.
Del Negro, C.A., Morgado-Valle, C., & Feldman,
J.L. (2002) Neuron 34, 821-830.
Del Negro, C.A. et al. (2002) Journal of
Neurophysiology 88, 2242-2250.
Del Negro, C.A. et al. (2001) Journal of
Neurophysiology 86, 59-74.
Del Negro, C.A. et al. (1999) Journal of
Neurophysiology 81, 1478-1485.
Del Negro, C.A. et al. (1998) Biophysical Journal
75,174-182.
Del Negro, C.A. & Chandler, S.H. (1998) Journal of
Neuroscience 18, 9216-9226.
Del Negro, C. A. & Chandler, S.H. (1997) Journal of
Neurophysiology 77,537-553.
Selected Recent Publications
Applied Science at William and Mary
Feldman, J.L. & Del Negro, C.A. (2006) Nature
Reviews Neuroscience 7, 232-241.
Del Negro, C.A. et al. (2005) Journal of
Neuroscience 25, 446-453.
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This intrinsic current (lCAN) amplifies synaptic
excitation and allows for the creation of robust
inspiratory bursts via a positive feedback process
often called recurrent excitation, which is a form of
self-organized behavior in biology. Inspiratory
bursts end once all of the preBotC neurons have
become fully excited: this extinguishes recurrent
excitation, deactivates ,CAN' and leads to burst
termination. Some preBotC neurons spike tonically
at low rates and subsequently restart the cycle,
leading to network rhythmicity (Fig. 6). Our overall
goal is to evaluate the group-pacemaker
hypothesis by determining its key mechanisms at
the cellular and synaptic level, and then use
mathematical models to test and refine our
understanding.
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Figure 5. Ordinary differential equations that describe
membrane potential trajectory in preBtitC neurons
during respiratory-like network activity. These equations
are a subset of the full model.
Our framework for analyzing respiratory
rhythmogenesis is the group-pacemaker
hypothesis which posits that 'CAN is a dendritically
sited burst-generating current with an activation
mechanism coupled to glutamatergic synapses.
The College of William and Mary is the second
oldest institution of higher education in the nation
and has been repeatedly honored for its
excellence by annual ran kings in US News &
World Report. The Department of Applied Science
is at the forefront of interdisciplinary research and
its graduate students enjoy a flexible curriculum
and yearly stipends of $20,040 plus tuition and
health insurance. Finally, William & Mary is located
in the town of Williamsburg, with its historic
Colonial village and nearby tourist attractions like
the Chesapeake bay and Atlantic beaches.
Figure 6. An illustrated cycle of respiratory activity
according to the group pacemaker hypothesis of
rhythm generation. Panels 1 - 4 show the evolution of
one inspiratory burst: from the onset of spontaneous
activity (1), to the spread of excitation through the
network (2-3), to the culmination of recurrent
excitation, which evokes intrinsic currents and causes
a robust network-wide inspiratory burst (4).
Christopher A. Del Negro, Ph.D.
Department of Applied Science
The College of William and Mary
Williamsburg, VA 23185
757 -221-7808
[email protected]
http://people.wm.edu/-cadeln/
WILLIAM
&MARY