Cellular Physiology in the Middle
By Jason Socrates
Bardi
"Midway
upon the journey of our life
I found myself within a forest dark,
For the straightforward pathway had been lost..."
———Dante
Alighieri, The Divine Comedy, Inferno: Canto I, From
the early 14th Century.
Cellular physiology, says Paul Schweitzer, who is assistant
professor of Neuropharmacology at The Scripps Research Institute
(TSRI), is situated somewhere between the individual molecules
of molecular biology and the whole organs and organisms of
physiology. And a cellular physiologist like Schweitzer occupies
a middle ground between the molecular biologists, on the one
hand, and the physiologists, on the other.
The work that he does each day, Schweitzer adds, is a good
example of this.
On one recent morning, he was looking at the hippocampus—a
small area near the front of the brain that is critical for
forming memories. To do this, Schweitzer took a tissue slice
of the hippocampus about 300 microns thick and perfused it
with a solution meant to mimic cerebrospinal fluid. The artificial
cerebrospinal solution is basically water, salts, and other
additives.
"Anything needed to keep the neurons alive for the rest
of the day," Schweitzer says.
Alive is the key here. Schweitzer looks at living neurons
and how they respond to certain stimuli by measuring this
response directly using a tiny electrode to connect to and
measure the conductance of a single neuron's soma (the cell
body) or dendrite (the branching "process" of a neuron). Alternatively,
a slightly thicker electrode can be used to measure the response
of a network of neurons. In either case, the measurements
are only valid if the neurons are healthy and remain connected
within the thin slice.
In the course of one of his studies, Schweitzer might look
at two to three neurons a day over several months. He might
examine the effect of some drug of interest on these neurons,
using electrodes and a series of chemicals and pharmacological
tools to tease out the detailed cellular interactions between
the drug and the neuron.
This sort of study, says Schweitzer, is usually referred
to as ex vivo. And, like cellular physiology, it lies
somewhere between the in vivo whole organism studies of the
physiologist and the in vitro cell culture experiments
of the cell or molecular biologist.
THC and the Brain
Schweitzer is funded by a National Institutes of Health
grant entitled, Cannabinoids and Central Neuronal Activity,
the purpose of which is to ask what role the brain's endogenous
cannabinoid system plays in memory formation and how this
system may be disrupted by the consumption of marijuana.
Marijuana contains as a principle active ingredient the
cannabinoid tetrahydrocannabinol (THC), which binds to the
same receptors as the body's natural endogenous cannabinoids.
This fact has made marijuana the subject of heated debate
in the last decade because THC is able to mimic the action
of natural cannabinoids that the body produces in signaling
cascades in response to a peripheral pain stimulus. THC binds
to cannabinoid receptors called "CB1" on cells of the spinal
cord and pain-modulating centers of the brain to decrease
sensitivity to pain.
Patients with multiple sclerosis, cancer, AIDS, and a number
of other conditions have sought marijuana for years to treat
their various symptoms. And public interest groups have taken
up this cause and fought successfully in certain states, including
California, to establish medical marijuana clubs and other
vehicles for providing the drug for ill patients. The issue
is far from settled, however, because the position of the
federal government remains unchanged regarding marijuana use.
Unfortunately, the brain's cannabinoid system is vast. The
CB1 receptors—the proteins that detect the release of
cannabinoids or the presence of THC—can be found all
over the body, and they are widely expressed throughout the
brain. In fact, CB1 receptors are concentrated in the memory
and information processing centers of the hippocampus.
Binding to nerve cells of the hippocampus and other cells
elsewhere in the body, THC creates a range of side effects
as it activates CB1-mediated signaling, including, according
to the National Institute of Drug Abuse, distorted perception,
difficulty in problem-solving, loss of coordination, increased
heart rate and blood pressure, anxiety, and panic attacks.
The work of the cellular physiologist, says Schweitzer,
is to determine the cascade by which THC and natural cannabinoids
have their effects.
"Our goal," he says, "is to determine the cellular outcome
of exposure to cannabinoids."
Schweitzer also studies the effect of neuropeptides on the
brain, and in the past few years, he has elucidated the neuronal
mechanisms of action of somatostatin, a tetradecapeptide implicated
in several physiologic and pathophysiologic processes. Recently,
Schweitzer began collaborating with Assistant Professor Luis
de Lecea in TSRI's Department of Molecular Biology to do a
similar investigation on a new peptide they call cortistatin.
Measuring Conductance
In his studies, Schweitzer makes tiny electrodes by heating
up and pulling apart capillary glass tubes so that they form
a microscopic tapered end that he can carefully place under
the microscope. Making these electrodes is more of an art
than a science, and he often has to go through several capillaries
before he gets one good electrode.
But when he does, he hooks one end to an amplifier that
will boost the tiny response signal coming from the neuron,
and he connects the other end directly to the soma of the
neuron or to whichever part of a neuron he wants to measure.
His goal is to measure the conductance or current due to
sodium or potassium influx. Neurons are excitable cells and
alter their activity by changing their potential, which is
determined by the fluctuating concentrations of ions inside
and outside. In general, a hyperpolarized neuron shows less
activity than a neuron that is depolarized.
Regulating the potential of neurons (and thus their excitability)
are different types of ion channels on neurons' surfaces.
There are an array of different potassium channels, for instance,
and a few sodium channels as well. These transport ions across
the membrane to control the excitability of the neurons and,
together with calcium channels, such important functions as
the release of neurotransmitters at the synapses.
The conductances that Schweitzer measures can tease apart
which particular ion channels are being affected by, say,
one particular interaction between a cannabinoid and a receptor
like CB1 on the surface of the neuron. When a cannabinoid
like THC binds to the CB1 receptor, this binding event starts
a cascade of reactions involving intracellular messengers
and other molecular signals that modulate the flow of ions
on one or more channel types on the neuron, and modify neuron
excitability.
The net effect of this cascade of events following ingestion
of THC is well known at the level of the whole organism. The
organism experiences a high. But the cellular details of this
cascade are not so well understood. Where in the brain are
the cannabinoids binding? Where on the neuron are they binding?
Which neurotransmitters are affected and how? How do the cannabinoids
work and how do they affect cellular activity? How do they
affect ensembles of neurons? How do they affect the hippocampus
function or the function of other areas of the brain?
"Even simple questions like these are difficult to answer
at this point," says Schweitzer. And, he adds, there are many
more complicated questions he is interested in as well. How
does THC interact with alcohol and other drugs of abuse? How
does the effect of THC or other cannabinoids affect the levels
of neuropeptides in the brain? How do these levels affect
pain sensation or appetite? How can these effects be controlled
or mimicked?
The goal of Schweitzer's sensitive measurements is to explain
in basic terms what happens at the cellular level when THC
hits the brain.
"You try to point out which specific conductances and synapses
are affected," he says. By looking at the characteristic response
of the conductance, he can relate this to the particular kinetic
or action potential profiles of the various ion channels on
the neurons and see which are turned on or off by the binding
of a cannabinoid to its receptor, and overall what this binding
event does to the neuron.
This allows him to study topics like the long-term potentiation,
or the synergistic effect of combining a cannabinoid like
THC with another drug, such as cocaine, methamphetamines,
heroin, or alcohol.
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