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Cellular Physiology in the Middle
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 BrainSchweitzer 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 ConductanceIn 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. Sticky BusinessMuch of the cannabinoid system is still a mystery to researchers in the field, largely because the cannabinoid system is a difficult one to study. Part of the problem is that cannabinoids are lipid molecules—one of a plethora of long-chain fatty acid molecules that are major constitutive components of the brain. Rather than looking for a needle in a haystack, looking for the cannabinoids in the brain is like liking for a particular type of hay in a haystack. Further complicating matters is that cannabinoids have degradation systems that remain uncertain, although TSRI Associate Professor Benjamin Cravatt has made great strides in recent years on elucidating the details of the degradation mechanism modulated by the enzyme fatty acid amide hydrolase (FAAH)—including solving the structure of FAAH last year. Even if scientists can separate the cannabinoid lipids from the other long-chain fatty acids, the molecules are still hard to work with because of the nature of these substances. "They stick to your tubing, your experimental apparatus," says Schweitzer. "To sort out and work with these molecules is difficult." For this reason, the field is still relatively new, even though it has grown rapidly in the last few years. It was only a decade ago, says Schweitzer, that the first cannabinoid receptor was discovered. And it was only in the mid-1990s that chemicals that could block these receptors were developed. Schweitzer himself started in the field by looking at the specific question of how THC works in the brain, but has since expanded his horizons to encompass a few larger questions. One of these is what purpose the endogenous cannabinoid system serves in the brain—especially given the vast number of CB1 receptors there and their concentration in vital parts of the brain, like the hippocampus. "How do cannabinoids work and what are they there for?" asks Schweitzer. "We still don't know why they are there." There is no shortage of opinions in the scientific community on this, says Schweitzer. And the implications of this field for politics and drug enforcement makes some of the debates as sticky as the lipidic molecules themselves. But scientists like Schweitzer are slowly gathering the tools and making the analyses needed to begin to unravel the complexities of the endogenous cannabinoid system and the effect that THC has on it. Hunger and Pain?One of Schweitzer's main goals is the potential applications that would follow if this endogenous cannabinoid system could be manipulated to achieve a desired effect. "This is of primary importance—the objective of pharmacological research," he says. When you feel pain, you release natural endocannabinoids, which provide some natural pain relief. For example, the body releases an endogenous cannabinoid called anandamide, a name derived from the Sanskrit word meaning "internal bliss." Recently, Schweitzer, in collaboration with Daniele Piomelli, who is now at the University of California, Irvine, characterized another endogenous cannabinoid found in the brain. This new cannabinoid, called 2-arachidonylglycerol, turned out to be present in much larger amounts than anandamide in the brain. When the body senses pain, these substances bind to CB1 and nullify pain by blocking the signaling. However, this effect is weak and short-lived as other molecules metabolize the endogenous cannabinoids. These compounds have a half-life of only a few minutes in vivo. The possible applications for designer chemicals that could inhibit the degradation of endogenous cannabinoids, inhibit their transport, or enhance their formation are tantalizing. If the right chemicals could be made, they might be developed into drugs for a number of clinical conditions—from appetite modulation to safer and more effective painkillers. The market for such compounds would be huge. The challenge for scientists is to use the cannabinoid system to produce effective, long-lasting relief from pain or viable appetite modulation without the deleterious side effects of marijuana use.
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