Virtual Cocaine Lab
BOOK 2: Dopamine and Cocaine

Paul Garris: Primary Author

Ch. 1: The dopamine neuron

A dopamine neuron is a neuron that uses the neurotransmitter dopamine for chemical neurotransmission. As will be discussed, dopamine neurons are important for motor control, motivated behavior, and in mediating the effects of drugs of abuse such as cocaine.

By binding to receptors, neurotransmitters act as a chemical messenger or link connecting the action potential from one neuron with a membrane potential in another. But unlike receptors found in "classic" chemical neurotransmission, dopamine receptors function a bit differently. In "classic" chemical neurotransmission, neurotransmitter binds to receptors that open ion channels. This allows ions to pass through the neuronal membrane. In this way, the chemical signal, the neurotransmitter, is transduced into an electrical signal, because ion flow will generate a change in voltage at the postsynaptic membrane. If this voltage change reaches threshold, the target neuron will fire an action potential. In contrast, the dopamine receptor is called a G-protein coupled receptor. Instead of directly opening an ion channel, dopamine binding to its receptor activates a G-protein that in turn activates a second messenger inside the target neuron. The second message can cause several changes in the postsynaptic neuron. These changes include opening and closing ion channels, but they also include gene transcription (the synthesis of RNA from DNA) and protein synthesis (the translation of RNA into amino-acids sequences to form proteins).

Chemical neurotransmission is terminated by removal of neurotransmitter from the cleft. For dopamine, like most neurotransmitters, this is done through transporter proteins on the presynaptic neuron. Once inside the neuron, dopamine is either re-packaged into vesicles for use again or degraded via degradative enzymes. These enzymes maintain intracellular levels of dopamine at safe levels. Interestingly, high concentrations of dopamine appear to be toxic, so only by degrading dopamine or repackaging it into vesicles is the dopamine neuron protected. Also, similar to the receptors found in the postsynaptic neuron, the presynaptic dopamine neuron contains dopamine receptors itself. These autoreceptors function as a "thermostat," either shutting down the dopamine neuron when it is too active or speeding it up when too lethargic.

 It is worth noting that the synapse between a dopamine neuron and its target appears to function rather differently than in the "classic" view. In the "classic" view of chemical neurotransmission, because transporters or degradative enzymes prevent neurotransmitter from escaping the synaptic cleft, the action of neurotransmitter is confined to the synaptic cleft. But at a dopamine chemical synapse however, dopamine released into the cleft readily diffuses out. For this reason, dopamine is often called an extrasynaptic messenger. Unlike "classic" chemical neurotransmission, the target of dopamine release is not restricted to the postsynaptic neuron. Instead, the target is any neuron with a dopamine receptor close enough to the dopamine synapse to be exposed to an effective dopamine concentration. Hence, in addition to "classic" chemical synapses at their terminals, dopamine neurons also form en passant ("in passing") synapses. These synapses allow a dopamine neuron to affect a target neuron without terminating the axon. The ability of dopamine to escape the synapse readily is why this neurotransmitter can be measured chemically in the brain without a sensor or probe that is small enough to be placed inside the synaptic cleft. To date, no such probe or sensor exists.

 

Ch. 2: Dopamine neuron systems

In addition to extensive overlap in brain anatomy, organization, and function, rats and humans also share similar dopamine neuron systems. A neuronal system describes the origins, projections, and terminations of a collection of like neurons. Thus, a dopamine neuron system is defined by the incoming or afferent neurons, the locations of dendrites, cell bodies, axons and terminals, and finally the outgoing or efferent neurons.

The brain contains several dopamine neuron systems. One important group originates in the hypothalamus. Consistent with the function of the hypothalamus, these dopamine neurons are involved in sexual behavior and the regulation of the pituitary gland. Although many of these dopamine neurons both begin and terminate within the hypothalamus, others project to and terminate in the spinal cord. This later subset of hypothalamic dopamine neurons would be said to be descending in anatomical terms, going from the forebrain to the spinal cord.

Another important group of dopamine neurons originates in the midbrain. These dopamine neurons are ascending because they project to and terminate in the forebrain. The ascending dopamine neurons originate in two regions of the midbrain, the substantia nigra and ventral tegmental area (see Figure 1).

Fig. 1

The brain is symmetrical in rats and humans, so each hemisphere contains a substantia nigra and striatum. The dopamine neurons originating in the substantia nigra terminate in the striatum. The striatum is a part of the brain located "under" the cerebral cortex. It receives projections from most, if not all, cortical areas. Because the striatum is an important region of the basal ganglia of the cerebrum, these dopamine neurons play an important role in movement. Motor diseases such as Parkinson's and Huntington's seriously affect the striatum.

 

Ch. 3: Approaches for assessing dopamine function

Several approaches have been used to assess the role of dopamine in behavior (e.g., lesioning of dopamine neurons, use of dopamine receptor drugs, etc.). This chapter will consider only certain monitoring techniques.

One type of monitoring approach is to use a microelectrode (a microscopic probe typically made of glass or metal) to record the firing rate. The firing rate is the frequency of action potentials a dopamine neuron generates over a period of time. This technique is called electrophysiology. Because the cell body is physically the largest portion of the neuron, it generates the largest electrical signals. Hence, electrophysiological recordings are usually performed in a region of the brain containing neuron cell bodies. For midbrain dopamine neurons, this region would be either the substantia nigra or ventral tegmental area. On the downside, to use electrophysiology one must assume that an action potential occurring at the cell body always causes dopamine release at the axon terminal. This may not always be true. For instance, dopamine autoreceptors can regulate dopamine release at the terminal independent of control by the cell body.

Other monitoring techniques have been developed to measure dopamine directly in terminal fields, which are those regions of the brain where dopamine neurons make synapses (or "terminate"). For instance, the striatum is the terminal field of midbrain dopamine neurons originating in the substantia nigra. One widely used monitoring technique is microdialysis. In general, dialysis is a procedure in which some but not all molecules move across a membrane. Molecules are typically excluded based on size. Such a membrane is said to be semi-permeable. Based on this principle, a dialysis machine is used to filter the blood of patients with kidney problems.

In microdialysis of the brain, a probe is implanted in a terminal field so dopamine can pass from extracellular fluid across a dialysis membrane and into the center of the probe. By pumping artificial extracellular fluid through the inside of the probe, dopamine is collected and measured outside of the animal using very sensitive and selective instrumentation.

Although microdialysis is used in animal experiments of many kinds, even to deliver drugs to specific brain regions via a procedure called reverse dialysis, it has two main disadvantages. First, samples are usually collected every few minutes. This is slow relative to many behaviors. Second, microdialysis probes are relatively large, about 300 microns (1000 microns = 1 millimeter) in diameter. Thus, a probe can cause damage to the region where dopamine is monitored.

Another technique for directly monitoring dopamine in terminal fields uses a chemical microsensor. A common type is made from a carbon fiber. Carbon is a biologically inert chemical, so it causes a minimal reaction when implanted in the brain. And carbon fibers can be made very small,  5 microns, which is 60 times smaller than the diameter of a microdialysis probe. Hence, a carbon-fiber microelectrode causes less damage than a microdialysis probe. The carbon fiber also provides an excellent surface for electrochemistry (the transfer of electrons between molecules), which is how chemical microsensors measure dopamine. Two events must occur before dopamine is monitored by a chemical microsensor. First, dopamine must come in contact with the carbon fiber, or at least within a few nanometers. Second, to pull off electrons from dopamine, the carbon fiber must be made positive electrically, just like the positive end of a battery. Electrons are small charged particles found in all molecules. The removal of electrons from a chemical is called oxidation. The rate of electrons flowing to the carbon fiber during oxidation is related to the concentration of dopamine near the microsensor. Consequently, monitoring dopamine using a chemical microsensor is called electrochemical measurement.

In addition to small size, electrochemical microsensors also have the important advantage of making dopamine measurements very quickly, even several times a second. Thus, chemical microsensors are a powerful tool for measuring dopamine changes during behavior. The downside is that many chemicals besides dopamine can be oxidized. This means that knowing what is measured by the chemical microsensor is an important consideration.

 

Ch. 4: Dopamine neurons and motivated behavior

Motivated behavior

Motivated behavior is behavior directed toward receiving a reward or goal. The reward may be natural (e.g., food, sex, etc.) or artificial (e.g., drugs of abuse). There are two components or phases of motivated behavior. The appetitive phase consists of those behaviors related to "approaching" the goal. In sexual behavior, for instance, the appetitive phase consists of behaviors that establish, maintain, or promote sexual interaction. Generally speaking, appetitive behaviors allow an animal to come into contact with its goal. The consummatory phase represents the actual "consuming" of the goal. In the case of sexual behavior, the consummatory phase is sexual intercourse. Collectively, appetitive and consummatory aspects characterize a sexual encounter, which is a motivated behavior. Addicted behavior is motivated behavior too.

The neurobiology of motivation is a field that seeks to identify the neural substrates (the brain regions, neuronal systems, neurotransmitters, receptors, etc.) that mediate motivated behavior. As described below, the classic experiment of intracranial self-stimulation demonstrated the existence of a brain reward system. The nucleus accumbens plays a central role in this system. Through dopamine neurons, it links motivational information processed in the cortex with emotional information processed in the limbic system, and then sends this combined information to regions of the brain controlling motor output, hormone release, and the fight-or-flight response. Thus, dopamine neurons terminating in the nucleus accumbens play an important role in motivated behavior. Not unexpectedly, the activity of these dopamine neurons changes during motivated behavior. And such changes can be monitored while an animal engages in motivated behavior.

Intracranial self-stimulation

One of the earliest experiments identifying a relationship between nucleus accumbens dopamine neurons and motivated behavior was intracranial self-stimulation. During this experiment, a stimulating electrode is implanted in the ventral tegmental area to activate dopamine neurons artificially using electrical pulses. The stimulating electrode and the instrument generating the electrical pulses are connected to the lever of a bar press machine. When the animal presses the lever, electrical pulses are delivered to the stimulating electrode. Thus, the animal controls stimulation of its dopamine neurons. (This type of control is called contingent. When a scientist controls the stimulation, the control is called non-contingent.) To obtain the "rewarding" electrical stimulation, rats lever press at astonishing rates, sometimes as fast a five times per second. They will also lever press continuously for hours.

Early studies with intracranial self-stimulation were very informative. Indeed, the highest rates of lever pressing during intracranial self-stimulation occurred with the stimulating electrode activated dopamine neurons directly. Collectively, such experiments led neuroscientists studying the neurobiology of motivated behavior to conclude that dopamine was the neural substrate of reward. In this view, dopamine is released when the animal consumes the reward, and the amplitude of dopamine release reflects the magnitude of the reward (or how good does this reward make it feel). Thus, dopamine is said to act as the neural substrate of reward during the consummatory phase of motivated behavior. Moreover, all rewards, whether natural (e.g., food, sex, etc) or artificial (e.g., electrical stimulation or drugs of abuse), were thought to be mediated by dopamine release.

Recent results challenge the traditional view that dopamine is only the neural substrate of reward. One of the key considerations with this new evidence is this: To understand fully the role of dopamine in motivated behavior, one must be able to monitor dopamine very quickly because behavior can be very fast as well. For example, microdialysis clearly shows that dopamine release increases when animals lever press for a rewarding electrical stimulation. But rats will bar press at rates upwards of 5 per second during intracranial self-stimulation. Microdialysis can in no way tell us what happens to dopamine release with each bar press. Nor can it tell us what happens to dopamine levels just before the animal bar presses. Both the bar press and the time leading up to it constitute the appetitive phase of this motivated behavior. Because of faster sampling rates, chemical microsensors can do both these things. One type of chemical microsensor technique, fast-scan cyclic voltammetry (or voltammetry), can measure dopamine 10 times per second.

When voltammetry was used to monitor dopamine release, some very unusual findings were obtained. For example, when the electrical stimulus was applied by the experimenter, the same stimulus that animals will lever press for, dopamine levels increase in the nucleus accumbens. This result suggested that each lever press during intracranial self-stimulation appeared to be rewarding in the same way as other rewarding stimuli. During training of lever press behavior, when the animal learns to associate the lever with the rewarding electrical stimulation, voltammetry showed that dopamine is also released during intracranial self-stimulation. However, in well trained animals, intracranial self-stimulation did not release dopamine; that is, animals lever pressed and received electrical stimulation but dopamine release did not increase.

Moreover, the same record of lever press activity, when replayed to the same and other animals, caused dopamine release. Remarkably, these animals received the same number and timing of the electrical stimulation as during intracranial self-stimulation, but in this case, dopamine release was observed. Remarkably, non-contingent but not contingent electrical stimulation caused dopamine release. What this interesting experiment demonstrates is that dopamine is not absolutely necessary for the consumption of an award. Instead, it appears to play a role perhaps related to learning of the cues associated with reward. In intracranial self-stimulation, the cue would be the lever press. This type of learning is called associative learning.

 

Ch. 5: Dopamine neurons and drugs of abuse

In the United States, one of the more prominent drugs of abuse is cocaine. Cocaine has multiple effects on the body, both peripherally outside the brain and centrally within the brain. Cocaine was originally used in medicine as a local anesthetic during eye surgery. The effect as a local anesthetic is independent of any action cocaine has on the brain. Cocaine also has potent effects directly on the cardiovascular system, which consists of the heart and all of the blood vessels. In general, cocaine increases blood pressure by stimulating the beating of the heart and by causing blood vessels to constrict. Large doses of cocaine can result in cardiac failure. The actions of cocaine on the brain are generally thought to be mediated by three neurotransmitters: dopamine, norepinephrine, and serotonin.

Two classic experiments are used to demonstrate that cocaine and other drugs of abuse are rewarding: conditioned place preference and drug self-administration. In conditioned place preference, an animal is released into a chamber that is demarcated into different quadrants. Over a period of time, the animal, when venturing into a specific quadrant, is injected with the test substance. After sufficient time, the animal learns the association between a quadrant and the drug injection. The animal is then allowed to enter the chamber but without any drug injection. If a drug is considered rewarding, the animal will tend to localize in the quadrant causing the drug injection. If a drug is considered aversive, the animal will tend to localize in the other quadrants. If a drug is considered neutral, no pattern of localization will occur.

Drug self-administration is analogous to intracranial self-stimulation, except that instead of a lever press delivering a rewarding electrical stimulation, an injection of a drug is administered. The drug is typically delivered intravenously. The goal of cocaine self-administration training is to teach a laboratory rat to press a lever in order to obtain an injection of cocaine. Cocaine will be injected into the jugular vein by a cannula, a small hollow tube inserted into the blood vessel. The jugular vein will carry the cocaine to the heart, which will then pump it to the brain in a matter of 30 seconds. Lever pressing rates for drug self-administration are considerably lower than for intracranial self-stimulation or even lever pressing for food reward. The reason for this is that drugs have a long period when they are active in the brain, so they do not need to be administered as often.

Two regimens are used to train animals to self-administer drugs: manual shaping and autoshaping. In manual shaping, the experimenter guides the animal to lever press for the drug using the technique of successive approximation. In other words, the researcher reinforces behavior increasingly similar to the wanted behavior. In contrast, autoshaping lets chance do most of the work. Both manual shaping and autoshaping are time-consuming processes, often taking several days to train an animal to self-administer a drug.

The pharmacological effect of cocaine on dopamine neurons is that cocaine blocks the dopamine transporter. This prevents dopamine from being transported back into the presynaptic neuron from the synaptic cleft. Norepinephrine neurons and serotonin neurons have their own specific transporters too. Cocaine binds to and blocks the action of the norepinephrine and serotonin transporters as well. Thus, by preventing the removal of released neurotransmitter, cocaine increases the extracellular levels of dopamine, norepinephrine, and serotonin in the brain.