Nov 1, 2009
The ability to measure micron-level neuronal activity patterns
in the mammalian neocortex is enabling insight into brain
sensory and motor processing functions related to cardiac
fibrillation and epilepsy. Voltage-sensitive dye, optical
recording techniques, and vibration isolation are key to
the work.
By Jim McMahon
Professor Jian-Young Wu has been conducting research
on waves of neuronal activity in the neocortex of the brain.
Wu and his colleagues at the Department of Physiology and
Biophysics at Georgetown University Medical Center visualize
wave-like patterns in the brain cortex using optical imaging
and voltage-sensitive dye-a method that depends on robust
vibration isolation. Their neuronal specimens are derived
from slices of rat neocortex, the outer layer of a mammal's
brain, which is involved in higher functions such as sensory
perception and generation of motor commands and, in humans,
language.
The neurons of the neocortex are arranged in vertical structures
called neocortical columns that measure about 0.5 mm in
diameter and 2 mm in depth. Each column typically responds
to a sensory stimulus representing a certain body part,
or region of sound or vision. In the human neocortex, it
is postulated that there are about a half-million of these
columns, each of which contains approximately 60,000 neurons.
The neocortex can be viewed as a huge web, consisting of
billions to trillions of neurons and hundred of trillions
of interconnections. While individual neurons are too simple
to have intelligence, the collective behavior of the billions
of interneuronal interactions occurring each second can
be highly intelligent.
Brain activity investigation
Traditionally, scientists have studied brain activity
by placing a few electrodes in the brain and measuring the
electrical signals of the neurons close to the electrodes.
This method is workable for understanding the function of
the cortex and interactions between individual neurons,
but it is not suitable for studying the emerging properties
of the nervous system.
"It is like viewing a few pixels on a television screen
and trying to figure out the story," explains Dr. Wu.
"Now, with optical methods and voltage-sensitive dyes,
we can visualize the activation in a large area of the neocortex
when the brain is processing sensory information, similar
to watching the whole television screen."
"Voltage-sensitive dye is a compound that stains neuronal
membrane and changes its color when the neuron is excited,"
continues Dr. Wu. "This allows us to visualize population
neuronal activity dynamically in the cortex. We study how
individual neurons in the neocortex interact to generate
population neuronal activities that underlie sensory and
motor processing functions. Population activities are composed
of the coordinated activity of up to billions of neurons.
Currently, we study how oscillations and propagating waves
can be generated by small ensembles of neocortical neurons."
FIGURE 1. Tangential slices of rat occipital cortex,
stained with NK3630 dye, change color (absorption)
as the transmembrane potential of the neurons varies.
Imaging was performed by a 464-element photodiode
array at a rate of 1600 frames /s. The oscillations
(4-12 Hz) were induced by perfusing slices with 100
mM carbachol and 10 mM bicuculline. The optical signal
on each optical detector is a summation of depolarization/hyperpolarization
of about 1000 neurons. The optical signal of the oscillations
was about 0.01% of the resting light intensity. The
signal on each detector is normalized to its own max-min
and assigned to a color according to a linear color
scale. The oscillations were organized spatially as
propagating waves. Wave patterns shown in the movies
(bioopticsworld.com/articles/370602) were formed spontaneously.
Viewing the spatiotemporal patterns of neuronal population
in the cortex is markedly different from recording
individual neurons. Here the cortical activity is
viewed as "population activity" that can
be more complex than the linear addition of an individual
neuron's activity. Voltage-sensitive dye and optical
recording techniques give the neuroscientist a new
tool for figuring out how the brain cortex works (see
Fig. 1).
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Spirals highlight cardiac fibrillation, epilepsy
Wu's imaging team has uncovered spiraling wave patterns
resembling little hurricanes in the brain. He believes that
this hurricane-like spiral pattern is an emergent behavior
of the network. "A metrological hurricane is an emergent
behavior of a large volume of air molecules," says
Wu. "If you were to dissect a hurricane into individual
air molecules you would not find any special process that
generates a hurricane. Similarly, in the nervous system,
spiral waves are an emergent process of the neuronal population
and there might be no special cellular process attributed
to spirals." But like a hurricane, spiral waves can
be a powerful force for organizing the activity of a neuronal
population. Spirals generated in a small area can send out
a powerful storm that invades large, normal brain areas
and starts a seizure attack. This hypothesis would mean
that epilepsy could be viewed not just as mis-wiring in
the brain, but as an abnormal wave pattern that invades
normal tissue.
Wu believes that propagating waves are a basic pattern of
cortical neuronal activity, and that these wave patterns
may play an important role in initiating and organizing
brain activity involving millions to billions of neurons.
Studying the spatiotemporal patterns of neuronal population
activity may provide insight into normal brain functions
and pathological disorders. This research has the potential
to help scientists understand abnormal waves that are generated
in the brains of patients with epilepsy.
FIGURE 2. Negative-stiffness mechanism (NSM) isolators
use a completely mechanical concept in low-frequency
vibration isolation. Such passive isolators can be highly
compact, and capable of very low vertical and horizontal
natural frequencies, and very high internal structural
frequencies. |
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Vibration isolation
Because voltage-sensitive dye signals are small,
usually a change of 0.1 to 1.0 percent of the illumination
intensity, Wu's team has used a high-dynamic-range
camera, photodiode array to detect the voltage-sensitive
dye signals of the cortical activity. The photodiode
array can resolve extremely small changes in light,
usually one part of ten thousands. (Human eyes and
ordinary digital cameras register light changes
of one part to a hundred). Detecting such small
signals requires a high level of vibration isolation.
The lab had to contend with low frequency vibrations
from air conditioning equipment, people walking,
and wind blowing against the building. Vibrations
as low as 1Hz were inhibiting the integrity of the
images and data.
"At first, we used high-quality air tables,
but they were not adequate for isolating low frequency
vibrations," Dr. Wu continues. "We tried
putting a second air table on top of the first one,
but that still did not give us the isolation we
needed. Then we tried an active, electronic system,
but we were still spending much time fighting with
floor vibrations. We were dealing with an unresolved
vibration problem for many years."
The Georgetown lab eventually tested, and settled
upon negative-stiffness mechanism systems from Minus
K Technology (Inglewood, CA; www.minusk.com), which
reduced vibration isolation to 1Hz and cancelled
out noise difficulties that were inhibiting image
and data readings (see Fig. 2). Negative-stiffness
mechanism (NSM) isolators have the flexibility of
custom tailoring resonant frequencies vertically
and horizontally. Vertical-motion isolation is provided
by a stiff spring that supports a weight load, combined
with a NSM. The net vertical stiffness is made very
low without affecting the static load-supporting
capability of the spring. Beam-columns connected
in series with the vertical-motion isolator provide
horizontal-motion isolation. The horizontal stiffness
of the beam-columns is reduced by the "beam-column"
effect. A beam-column behaves as a spring combined
with a NSM.
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NSM also offers substantial transmissibility improvement
over air systems (whose resonant frequency can match that
of floor vibrations and actually worsen problems) and over
active systems (which have a limited dynamic range and are
vulnerable to electronic dysfunction and power modulations).
Although active isolation systems have fundamentally no
resonance, their transmissibility does not roll off as fast
as negative-stiffness isolators. Transmissibility is a measure
of the vibrations that transmit through the isolator relative
to the input vibrations. The NSM isolators, when adjusted
to 0.5Hz, achieve 93 percent isolation efficiency at 2Hz;
99 percent at 5Hz; and 99.7 percent at 10Hz.
Continuing research
Within the past ten years, Wu's team has documented
a variety of waveforms (e.g., plane wave and spirals) in
brain slices during artificially induced oscillations. Using
neocortical slices and mathematic models, they are studying
the initiation of the waves and the factors that control
their propagating direction and velocity.
The lab is also involved in the development of new optical
imaging techniques (also dependent on vibration isolation)
such as imaging propagating waves in vivo, in intact brains.
"This is technically difficult," says Wu. "Other
imaging methods (MRI, PET, or MEG) provide inadequate spatiotemporal
resolution. Large scale neuronal activity is a hallmark
of a living brain. We are improving the optical imaging
techniques with a hope to visualize the waves in the cortex
in vivo during sensory processes and in awake animals while
behavioral and cognitive tasks are performed."
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Jim McMahon writes on instrumentation and controls technology.
(For more information, contact Jian-Young Wu, professor,
Department of Physiology and Biophysics, at Georgetown University
Medical Center, www.georgetown.edu/faculty/wuj/ wuj@georgetown.edu.)