World - November/December 2009
A new tool for brain discovery
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
dyea 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
"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 he
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 signa
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 (see 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 (sec
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
metro-logical 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
Similarly, during cardiac fibrillation, spiral waves form
in the heart emitting rotating and scroll waves in two and
three dimensions. As a leading life-threatening situation,
these rotating waves can kill the patient instantly as the
pumping function of the heart is disrupted by the 5 to l0Hz
rotations that drive chaotic and abnormally rapid cardiac
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.
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.
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
"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
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 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
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."
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.jfeorgetown.edu/faculty/ wuj/
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