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BOOK 1: Nervous system anatomy and function
Ch. 1: Nervous system anatomy
Basic divisions of the nervous system
The nervous system coordinates the activity of the muscles, monitors the organs, constructs and
also stops input from the senses, and initiates action. The brain and spinal cord comprise the
central nervous system. All other components belong to the peripheral nervous system (table
1). These other components include motor neurons, which provides nerves in muscle tissue, and
sensory neurons, including those connected to pain- or temperature-sensitive receptors in the
skin, for example. The brain is further divided into six subdivisions: medulla oblongata, pons,
cerebellum, mesencephalon, diencephalon (or "in between brain"), and cerebrum (figure 1).
These brain subdivisions generally control different functions. Not unexpectedly, these
subdivisions come in varying sizes in different animals, depending upon specific needs or
features of the animal.
Table 1.
Central Nervous System
Brain
Spinal Cord
Peripheral Nervous System
Motor neurons
Sensory neurons
Figure 1.
(it would be nice if the picture above also included mesencephalon and cerebrum labels and
the forebrain and midbrain labels, or use the same vocabulary that is used in the writing)
Hindbrain, midbrain and forebrain
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Collectively, the medulla, pons and cerebellum are called the hindbrain and perform “lowerlevel functions.” Closest to the spinal cord, the medulla controls breathing and heart beat. On
the opposite side of the medulla towards the rest of the brain is the pons (or “bridge”). It relays
sensory information between the cerebellum and cerebrum. This subdivision controls fine motor
movement, coordination, and posture. The mesencephalon, which is involved in movement, and
visual and auditory processing, is also called the midbrain. The forebrain consists of the
diencephalon and cerebral hemispheres. The diencephalon is comprised of two prominent
structures: the hypothalamus and thalamus. The hypothalamus is located at the very bottom of
the brain, directly on top of the roof of the mouth. It controls a variety of involuntary functions,
including: blood pressure, temperature regulation, feeding, sexual behavior, and the pituitary
gland – the body's master gland. The thalamus resides on top of the hypothalamus and is the
major relay system in the brain. All sensory information except olfactory (i.e., smell) first comes
to the thalamus before being sent to other regions for processing. (Figure 1)
The cerebrum is the other major region of the forebrain. In humans, the cerebrum is by far the
largest structure of the brain. The cerebrum contains the cerebral cortices, which are involved in
cognition and house the primary motor and sensory areas, and basal ganglia, which is central to
the control of movement. Parkinson’s disease and
Huntington’s disease are diseases that result from
damage to the basal ganglia.
Figure 2
The four lobes of the cerebral cortex are the frontal, parietal, temporal and occipital (Figure 2).
As they are involved with planning, the frontal lobes are considered the site of executive
decisions and personality. They are also the primary seat of motor information processing. The
parietal lobes are involved with attention and somatosensory (i.e., “body senses”) information
processing. The temporal lobes are involved in recognition and auditory information
processing. The occipital lobes, which have no cognitive function, contain the primary visual
information centers.
Ch. 2: Neuronal signaling
Cells use a variety of mechanisms to communicate. Cells must communicate internally between
different parts within themselves and with other cells externally. In this general sense of cell
communication, neurons are no different than other cells. However, neurons have evolved
unique capabilities for intra-cellular signaling (communication within the cell) and intercellular signaling (communication between cells) to support the general function of the
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nervous system. This is called integration. Integration requires both rapid communication over
long distances and slower communication that is more selective. By comparison to neuronal
signaling, endocrine signaling is slow with widespread actions. To achieve long distance, rapid
communication, neurons have evolved special abilities for sending electrical signals called
action potentials along its axons (figure 3). This mechanism, called conduction is how a cell
body of a neuron communicates with its own terminal via an axon. Remember, axons can be
very long, so a very fast mechanism is necessary for intra-cellular signaling in neurons.
Selective inter-cellular communication is achieved by the process of transmission. This process
takes place at the synapse and is how two neurons communicate very quickly with each other.
This communication is called neurotransmission (Figure 3).
Figure 3 I’m not sure as this is showing what we are writing about. The vocabulary does not
match. The vocab in the picture needs to be in the writing, and either the picture is in the wrong
place, or different items need to be labeled. Otherwise it will confuse the reader. Maybe remove
presynaptic and postsynaptic, or describe it in the narrative.
Conduction
To begin conduction, an action potential is generated near the cell body portion of the axon. An
action potential is an electrical signal very much like the electrical signals in electronic devices.
But whereas an electrical signal in an electronic device occurs because electrons move along a
wire, an electrical signal in a neuron occurs because ions move across the neuronal membrane
and along the outside and inside of the membrane. Neurons generate action potentials by
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providing cells with the ability to generate a small electrical potential across their membrane.
This membrane potential is just like a battery, with the outside the positive pole and the inside
the negative pole, only much smaller. For example, a D battery commonly used in flash lights
generates a potential of 1.5 V between the positive and negative poles. In contrast, the potential
across a cell membrane is typically on the order of about 60 millivolts (or a thousandth of a volt).
Using the battery analogy again, the battery itself can not change its voltage output. Similarly,
most cells are “non-excitable”, which means that they can not change the voltage across their
membrane. However, neurons are unique and are called “excitable”, because they can alter the
membrane potential very precisely. It is this dynamic membrane potential that forms the basis
for the action potential. In other words, the action potential is essentially the result of the neuron
changing its membrane potential in a controlled manner. Moreover, conduction of the action
potential is achieved by virtue of the neuron precisely altering membrane potential along the
length of its axon. Just like electricity, the membrane potential can be changed very rapidly.
This is why the action potential can be conducted very quickly along the axon, moving at rates of
several meters per second (up to 150 m/s; one meter is roughly equivalent to one yard or three
fee).
To simplify the explanation of this process, the neuronal membrane first allows positive charges
to enter, making the membrane potential become more positive, then allows positive charges to
leave, restoring the membrane potential to its resting negative value.
More specifically, the cell membrane of a neuron is semi-permeable to ions, which means that
some but not all ions can move across. The neuron can additionally change which ions move
across. When ions move across the cell membrane, the membrane potential changes due to the
addition (or subtraction) of charges. Thus, during an action potential, the neuronal membrane
allows positively charged ions for sodium (Na+) to come inside initially, which makes the
membrane potential positive (the upswing or rising phase of the action potential). A little later in
time, actually one or two milliseconds later, the neuronal membrane prevents Na+ from coming
in, but allows another positively charged ion, K+ (the ion for potassium), to leave the neuron. As
K+ leaves the neuron, the membrane potential becomes more negative (the downswing or falling
phase of the action potential) and returns to its resting value. The neuronal membrane actually
does not determine the direction ions flow, as it only becomes “open” or “closed” for an ion to
move across. Rather, concentration gradients inside and outside the cell determine which
direction ions flow. It just so happens that the concentration of Na+ is higher on the outside, so
when the neuronal membrane “opens up”, this ion flows in. Conversely, the concentration of K+
is higher on the inside, so it flows out.
Ch. 3: Rats vs. Humans
Rats are often used in research because of the similarities they have with humans. In rats, as in
humans and other mammals, the nervous system is divided into the central nervous system and
peripheral nervous system. Rats and humans share all of the major subdivisions of the brain and
their general functions. As mammals and compared to other non-mammalian animals, the
midbrain is greatly reduced, because the cerebrum has evolved to become the primary center for
coordinating responses to auditory and visual stimuli. In addition to extensive overlap in the
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anatomy, organization, and function of the central nervous system, rats and humans are also
share similar dopamine neuron systems.
However, important differences exist between rat and human. For example, humans have a
considerably larger cerebral cortex, while rats have a more prominent olfactory bulb. This latter
region of the forebrain is important for smell, a sense that is extremely keen in rats, but less so in
humans. Another important difference between the rat and human is the physical relationship
between brain and spinal cord. As an animal standing upright, the brain and spinal cord in a
human roughly form a right angle, with the spinal cord extending down from the base of the
brain. Because rats “stand” on all four paws, the relationship is more linear, as the spinal cord
leaves the back of the brain.
(not sure if this is the best graphic for the vocabulary…nervous system, central nervous system,
peripheral nervous system, midbrain, cerebral cortex, olfactory bulb, forebrain, and spinal cord
need to be in this
picture.)
Figure 4
BOOK 2: Dopamine and cocaine
Ch. 1: Anatomy and function of dopamine neurons
A neuronal system describes the origins, projections, and terminations of a collection of like
neurons. Thus, a dopamine 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.
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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 also terminate or end within the hypothalamus, thus they are entirely
contained within this brain subdivision, 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.
Figure 5
This should have the items labeled (forebrain, spinal cord for the descending and substantia nigra
and ventral tegmental area for the ascending. Nucleus accumbens and caudate putamen are not
talked about so should not be labeled)
Another important group of dopamine neurons originates in the midbrain, the region of the brain
between the forebrain and hindbrain. 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 (Figure 5).
Ch. 2: Approaches for assessing dopamine function
Several approaches have been used to assess the role of dopamine in behavior. These
approaches include: (1) ablation or lesioning of dopamine neurons, (2) dopamine receptor
drugs that block or augment the action of dopamine, (3) determination of brain dopamine
content or the brain content of dopamine metabolites, (4) monitoring techniques, and (5)
imaging techniques.
Monitoring Techniques
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One type of monitoring approach is to use a microelectrode -- a small, microscopic probe
typically made of glass or metal -- to record the number of action potentials a dopamine neuron
generates. This technique is called electrophysiology or monitoring the “electrical functioning”
of the neuron. The frequency of action potentials or firing rate (the number of action potentials
“fired” or delivered in a given time) is typically measured. Because the cell body is physically
the largest portion of the neuron, and therefore generates the largest electrical signals on the
neuron, electrophysiological recordings are usually performed in the region of the brain
containing neuron cell bodies. For midbrain dopamine neurons, this region would be either the
substantia nigra or ventral tegmental. One downside of electrophysiology is that one must
assume that an action potential occurring at the cell body always causes dopamine release at the
presynapse. This may not always be true, as, for example, 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 the “terminal
fields,” those regions of the brain in which dopamine neurons make synapses (or “terminate”).
For example, the striatum is the terminal field of midbrain dopamine neurons originating in the
substantia nigra. One widely used monitoring technique is microdialysis. Dialysis is a general
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 or selectively
permeable. Based on the same principle, a dialysis machine is used to filter blood of patients
which kidney problems.
In microdialysis of the brain, a probe is implanted in the terminal field so released dopamine can
pass from extracellular fluid across a dialysis membrane and into the center of the probe. By
pumping artificial brain extracellular fluid through the inside of the probe, dopamine is collected
and measured outside of the animal using very sensitive and selective instrumentation. Artificial
brain extracellular fluid is pumped through the probe so that the composition of real brain
extracellular fluid does not change when dopamine is sampled. Most general components of
extracellular fluid such as salts readily pass through the dialysis membrane, but large proteins or
cells can not.
The instrument typically used to measure dopamine collected by microdialysis is high
performance liquid chromatography with electrochemical detection. This instrument is very
sensitive and selective. While microdialysis is very powerful and widely used in animal
experiments of many kinds, even to deliver drugs to specific brain regions in a procedure called
reverse dialysis, it has two main disadvantages. First, samples are usually collected every few
minutes, which is slow relative to many behaviors. Second, microdialysis probes are relatively
large, about 300 microns in diameter, and thus may cause some damage to the brain region in
which dopamine is monitored.
(Figure 6 here instead?)
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 that it causes a minimal reaction when implanted in the brain. Carbon fibers can
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also be made very small, for example, with a diameter of about 5 microns, so that a carbon-fiber
microelectrode causes less damage than a microdialysis probe, which is about 50-times larger.
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 angstroms (10 nanometers).
Second, the carbon fiber must be made positive electrically, just like the positive end of a
battery, to pull off electrons from dopamine. 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. The monitoring of dopamine using a chemical microsensor is therefore called an
electrochemical measurement.
The same electrochemical approach is used to measure dopamine using high performance liquid
chromatography with electrochemical detection. Chromatography separates the contents of the
microdialysis sample into individual components. A carbon-based detector then measures the
amount of each component once separated.
In addition to small size, electrochemical microsensors also have the important advantage of
making dopamine measurements very quickly, for example, several times a second. Thus,
chemical microsensors are a powerful tool for measurement dopamine changes during behavior.
The downside is that many chemicals in the brain besides dopamine can be oxidized. This
means that knowing what is measured by the chemical microsensor is an important
consideration.
Ch. 3: Dopamine neurons and motivated behavior
Motivated behavior
Motivated behavior is divided into two components, appetitive and consummatory. The
appetitive phase of motivated behavior consists of those behaviors related to “approaching” the
goal. In sexual behavior, for example, the appetitive phase consists of behaviors that establish,
maintain, or promote sexual interaction. Generally speaking, appetitive behaviors allow the
animal to come into contact with the goal. In contrast, the consummatory phase of motivated
behavior represents the actual “consuming” of the goal. In the case of sexual behavior, the
consummatory phase is sexual intercourse. Collectively, appetitive and consummatory aspects
of sexual behavior characterize a sexual encounter, which is a motivated behavior.
The neurobiology of motivation is a field of study that seeks to identify the neural substrates,
e.g., brain regions, neuronal systems, neurotransmitters, receptors, etc., that mediate motivated
behavior. As described below, the classic experiment of intracranial self-stimulation, in which
an animal lever presses to obtain rewarding electrical stimulation directly applied to the brain,
demonstrated that existence of a brain reward system. As we know now, there is not one single
brain region that serves the role of the brain reward system. Rather, several systems acting in
concert as a brain network or circuit, a collection of neuronal systems, does this job.
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Nevertheless, this same brain reward system presumably would mediate the response to natural
rewards such as food, water and sex, and would additionally be the site of action of drugs of
abuse.
Although not technically part of the limbic system, the nucleus accumbens plays a central role
in motivated behavior nonetheless, by virtue of where this region resides in the overall
organization of the brain. Indeed, the nucleus accumbens links motivational and emotional
information processed in the limbic system and cortex to the regions of the brain controlling
motor output. In addition to behavior per say, the nucleus accumbens also sends information to
the endocrine system, to control hormone release from the pituitary gland and other endocrine
organs, and the autonomic nervous systems, which controls the fight-or-flight response. Both
systems contribute to the overall physiological response of an animal during motivated behavior.
It is for these reasons that the nucleus accumbens is considered the gateway of the limbic system.
Not unsurprising, mesolimbic dopamine neurons originating in the ventral tegmental area and
terminating in the nucleus accumbens also play an important role in motivated behavior. It is the
action of dopamine on the neuron inputs from the limbic system and neuron outputs to motorrelated areas that enables the nucleus accumbens to interface limbic and motor systems. Not
unexpectedly as well, the activity of mesolimbic dopamine neurons also changes during
motivated behavior.
Intracranial self-stimulation
One of the earliest experiments identifying a relationship between nucleus accumbens dopamine
and motivated behavior was intracranial self-stimulation. During this behavior, a stimulating
electrode is implanted in the ventral tegmental area to activate mesolimbic dopamine neurons
artificially using electrical pulses. The stimulating electrode and the instrument generating the
electrical pulses is in turn connected to a lever so that when the animal presses the lever, the
electrical pulses are delivered to the stimulating electrode. Thus, the animal controls stimulation
of the mesolimbic dopamine neurons. This type of control is called contingent as opposed to
non-contingent, when the scientist controls the stimulation. Amazingly, rats lever press at
astonishing rates, sometimes as fast a five times per second and continuously for hours, to obtain
the “rewarding” electrical stimulation.
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
implanted in brain regions to activate dopamine neurons directly. Ablation of dopamine neurons
by 6-hyrdroxydoapmine and blocking the actions of released dopamine using dopamine receptor
antagonists also reduced lever pressing rates. Moreover, studies showed that released dopamine
measured in the brain by microdialysis increased during intracranial self-stimulation.
It was these types of experiments with intracranial self-stimulation that collectively led
neuroscientists studying the neurobiology of motivated behavior to conclude that dopamine was
the neural substrate of reward. In this view, brain 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
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substrate of reward during the consummatory phase of motivated behavior. Moreover, all
rewards, whether natural, such as food, water or sex, or artificial, such as electrical stimulation or
drugs of abuse, were thought to be mediated by dopamine similarly.
More recently, new results have been obtained to challenge the traditional view that dopamine is
the neural substrate of reward. One of the key considerations with this new evidence is that 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 the rewarding
electrical stimulation. However, animals will bar press at rates upwards of 5 per second during
intracranial self-stimulation. Thus, microdialysis can in now way tell us what happens to
dopamine release with each bar press or even during the time before the animal bar press, which
would constitute the appetitive phase of this motivated behavior. Because of faster sampling
rates, chemical microsensors can. One type of chemical microsensor technique called fast-scan
cyclic voltammetry can measure dopamine 10 times per second, which is faster than the animal
bar presses in intracranial self-stimulation.
When voltammetry was used to monitor dopamine released during intracranial self-stimulation,
some very unusual findings have were obtained. For example, a single electrical stimulus, the
same stimulus that animals will lever press for, when applied by the experimenter, will cause
dopamine release 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 selfstimulation did not release dopamine, i.e., 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 animal received the same number and timing of the
electrical stimulation as during intracranial self-stimulation but in this case, dopamine release
was observed. This procedure is called non-contingent electrical stimulation, because the animal
is not controlling delivery. In contrast, intracranial self-stimulation is called contingent electrical
stimulation because the animal controls delivery. Remarkably, non-contingent but not
contingent electrical stimulation, albeit identical, caused dopamine release. What this interesting
experiment demonstrates is that dopamine is not absolutely necessary for the consumption of an
award, but 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. 4: Dopamine neurons and drugs of abuse
One of the more prominent drugs of abuse in the modern history of the United States 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
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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 vasoconstriction, or causing blood vessels constrict or narrow. 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 and 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, in injected with the
test substance. After sufficient time for the animal to learn the association between the quadrant
and the drug injection, the animal is allowed to enter the chamber but without any drug injection.
If the drug is to be considered rewarding, the animal will tend to localize in the quadrant in
which the drug injection was received. If the drug is to be considered aversive, the animal will
tend to localize in the other quadrants in which no drug injection was received. If the drug is to
be considered neutral, no pattern of localization will occur.
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 a few seconds. There are
several methods for achieving cocaine self-administration, but training methods are typically
divided into manual shaping and autoshaping.
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. Lever pressing rates for drug self-administration
are considerably lower that for intracranial self-stimulation or even lever pressing for food
reward. The reason is that drugs have a long period when they are active in the brain, so they did
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 pressing for the
drug using the technique of successive approximation (or simply, reinforces behavior
increasingly similar to the wanted behavior). In contrast, autoshaping lets chance do the work,
with a little help of course. Both manual shaping and autoshaping are time-consuming
processes, often taking several days to train an animal to self-administer a drug.
The pharmacological effects of cocaine on dopamine, norepinephrine and serotonin neurons are
mediated by blocking the neurotransmitter transporter found on the presynapse. Dopamine,
norepinephrine and serotonin neurons have their own specific transporters to take up dopamine,
norepinephrine or serotonin released into the synaptic cleft. Cocaine binds to and blocks the
action of the dopamine, norepinephrine and serotonin transporters. Thus, by preventing the
removal of released neurotransmitter, cocaine increases the extracellular levels of dopamine,
norepinephrine and serotonin in the brain. Indeed, administration of cocaine to a rat will
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increase extracellular dopamine levels measured by microdialysis using a probe implanted in the
nucleus accumbens.
BOOK 3: The experiment and lab procedures
Ch. 1: The hypothesis and overview
Introduction
This experiment will investigate how cocaine acts on dopamine neurons in the brain. Cocaine is
a drug of abuse that increases extracellular dopamine levels in the nucleus accumbens by
blocking the dopamine transporter. Cocaine is considered an artificial reward, because this drug
supports conditioned placed preference and will be self-administered in laboratory animals such
as the rat. An important question in the neurobiology of cocaine concerns the role of dopamine:
If cocaine increases brain dopamine when consumed, does dopamine only play a role during the
consummatory phase of cocaine use? If so, is dopamine solely related to the pleasurable aspects
of cocaine consumption? Or, similar to other motivated behaviors like sex and intracranial selfstimulation, is dopamine involved in other aspects of motivated behavior such as during the
anticipatory phase? Check for repeat information from previous chapter and cut cut cut
Microdialysis has clearly shown that administration of cocaine to a laboratory animal causes an
increase in dopamine release in the nucleus accumbens. This action of cocaine is thought to be
mediated by the drug blocking the ability of the dopamine neuron to take up released dopamine
by the dopamine transporter. By blocking dopamine uptake, extracellular levels of dopamine in
the nucleus accumbens increase. Thus, “consumption” of cocaine increases dopamine release in
an area of the brain, the nucleus accumbens, which links the limbic system (the brain network
controlling with motivation) to behavior. Animals self-administering cocaine will experience the
same effect, cocaine-induced increases in dopamine release, which are thought to be associated
with the pleasurable aspects of cocaine use in humans as well.
Also recall that in microdialysis experiments with sexual behavior, an important type of
motivated behavior, an increase in dopamine release was observed in both male and female rats
when they were exposed to each other but prevented from copulating by a barrier. This result
suggests that dopamine is released during the anticipatory phase. However, dopamine still may
be released during the consummatory phase as well, but microdialysis can not answer this
question. In this experiment, you will measure how brain dopamine release changes during
cocaine self-administration using fast-scan cyclic voltammetry. This chemical microsensor
technique can monitor dopamine very quickly (10 times per second) and should provide great
insight into the role of dopamine in cocaine use.
Objective, hypothesis, and prediction
The overall objective of this experiment is to investigate the role of dopamine neurons in cocaine
use. Three important questions will be asked:
(1) What happens to brain dopamine levels when cocaine is used?
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(2) When do changes in brain dopamine levels occur when cocaine is used?
(3) How do changes in brain dopamine levels affect behavior?
With most research it is often best to focus on one or two questions at a time by simplifying the
experiment. Therefore, in this experiment, we shall investigate what cocaine does to dopamine
neurons during the appetitive phase of motivated behavior and what this action of dopamine
neurons means for behavior. Thus, we will be examining how cocaine affects the behaviors of
the animal directed towards obtaining cocaine. These behaviors have collectively been called
'cocaine seeking'. Because cocaine seeking is appetitive behavior, we will not be testing
whether cocaine acts on dopamine neurons during the consummatory phase of motivated
behavior. This is an important question that must be left to future experiments. Thus, what we
conclude about the role of dopamine neurons in cocaine seeking will not address the role of
dopamine neurons in cocaine consuming.
The hypothesis to be tested by our experiment is the following:
Hypothesis: Dopamine is involved in cocaine seeking.
To test our hypothesis, we will make a prediction regarding the outcome of our experiments. If
the outcome of the experiment agrees with out prediction, then our hypothesis is supported. The
prediction we shall make is this:
Prediction: Dopamine release will increase during cocaine seeking.
If our prediction is verified, then there are two other questions our experiment can answer:
(1) When during cocaine seeking is dopamine release increased?
(2) Does the increase in dopamine release cause a subsequent change in behavior?
Technical issues
Determining whether dopamine is involved in cocaine seeking requires us to consider several
technical issues. The first is that we need a laboratory animal to engage in cocaine seeking. This
issue is not too difficult, because laboratory rats will self-administer cocaine, similar to humans.
Perhaps the most challenging technical issue is the actual monitoring of dopamine during
cocaine seeking. Several measurement requirements must be considered, e.g., temporal
resolution (how fast is the measurement?), selectivity (what is being measured?) and spatial
resolution (over what area of the brain is the measurement collected?). As described below, the
chemical microsensor technique that will be used in our experiment, fast-scan cyclic
voltammetry at a carbon-fiber microelectrode, has suitable measurement characteristics for
monitoring dopamine during cocaine seeking.
First, the monitoring of dopamine must be sufficiently fast to capture changes during the cocaine
seeking portion of cocaine self-administration. Rats self-administer cocaine at a rate of about
once every 10 min on average. At this rate, even slow monitoring techniques could assess
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dopamine changes before and after a lever press for cocaine. However, the goal of this
experiment is to monitor dopamine during the behavior(s) just prior to a lever press for cocaine
self-administration. During this time, dopamine neurons may be responding to external and
internal cues to direct the rat to move towards the lever and press it. Moreover, the type of
dopamine neurotransmission that is thought to be involved with responding to cues, which is
called phasic signaling, results in short-lived increases in extracellular dopamine (called
dopamine transients) that lasts for about one second. Thus, a fast measurement technique will
be necessary to monitor dopamine levels during the time just before the lever press. Fortunately,
fast-scan cyclic voltammetry, which monitors dopamine several times a second, is well suited for
these measurements of dopamine during cocaine seeking in the laboratory rat. Voltammetry will
therefore have the appropriate temporal resolution to capture changes in brain dopamine levels
during this time.
Figure 6
Not sure if this is the correct location for this image, maybe page
Second, there are many chemicals in the brain that, like dopamine, are electroactive (which
means they can be measured electrochemically). These electroactive substances therefore
potentially interfere with the measurement of dopamine using voltammetry and the chemical
microsensor (Figure 6). Fortunately, fast-scan cyclic voltammetry is a unique type of
voltammetry that collects a “chemical signature“ called a voltammogram to identify the
chemical being measured the microsensor. The method for collecting the voltammogram is
beyond the goals of this experiment, but it is important to note that the measurement of
dopamine in the rat brain by fast-scan cyclic voltammetry is selective. However, in the present
experiment, the substance measured during cocaine seeking will be chemically compared to
dopamine that is released by directly activation dopamine neurons with electrical pulses applied
to stimulating electrode implanted in the ventral tegmental area. Decades of research have
established that this type of electrical stimulation elicits “authentic dopamine” in the rat brain.
Moreover, the chemical signature for this authentic dopamine will be collected by the same
microsensor in the same area of the brain as the chemical signature recorded during cocaine
seeking behavior for direct comparison.
Finally, it is necessary to consider where in the brain dopamine will be monitored and the extent
of damage the implanted microsensor will cause. While several regions could potentially be
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NIH Book Chapters, page 15
sampled, perhaps the best place to record dopamine is the nucleus accumbens. Indeed, this
region is part of one of the major midbrain dopamine systems, the mesolimbic dopamine system,
which originates in the ventral tegmental area and terminates in the nucleus accumbens. The
chemical microsensor technique, fast-scan cyclic voltammetry, additionally provides an
advantage for monitoring dopamine in this region. Moreover, this region of the brain links
reward-related information processed in the limbic system and cortex with motor output. Thus,
the nucleus accumbens occupies a key role in the brain circuitry supporting motivated behavior.
It is also well established that cocaine self-administration will increase dopamine release in the
nucleus accumbens as measured by microdialysis. However, the probe associated with this
technique is rather large compared to the size of the nucleus accumbens and in fact, may measure
dopamine for nearby areas of the forebrain. Additionally, the microdialysis probe is
demonstrated to cause damage to the adjacent tissue. Fortunately, the carbon-fiber
microelectrode used with fast-scan cyclic voltammetry is considerably smaller, so that its tip can
be readily implanted within the nucleus accumbens to measure dopamine only in this region.
Also, because of smaller size, the carbon-fiber microelectrode causes considerably less tissue
damage when implanted.
Experimental design
The experimental design will be divided into three parts:
PART 1: Training a rat to self-administer cocaine.
PART 2: Preparation and surgery for taking voltammetry measurements.
PART 3: Monitoring dopamine during cocaine seeking.
Let's explore each of these parts in detail.
PART 1: Training a rat to self-administer cocaine.
In the present experiment, all animals will already be capable of self-administering cocaine selfadministration.
PART 2: Preparation and surgery for voltammetry measurements of dopamine.
In preparation for brain surgery, a rat must be weighed, anesthetized, and have its scalp retracted
to expose the skull. In this experiment, you will walk through the steps as the research assistant
prepares the rat and performs the surgery. When surgery is completed, the rat will have a hub
with electrodes attached so that dopamine levels can be recorded during cocaine seeking
activities.
PART 3: Monitoring dopamine during cocaine seeking.
Two weeks after surgery, the rat is ready to take part in the experiment. Just before the rat is
allowed to engage in cocaine seeking, a microdrive will be loaded with a microsensor, it will be
attached to the skull, and the microsensor will be lowered into the nucleus accumbens. The
electrically evoked signal, which is dopamine because dopamine neurons are activated by the
stimulating electrode, will be used to identify the signals measured during cocaine selfadministration. For dopamine measurements during cocaine self-administration, a wireless piece
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NIH Book Chapters, page 16
of equipment will be attached to the animal as a backpack. The wireless backpack will perform
several functions, including: (1) collecting dopamine measurements at the microsensor, (2)
applying the electrical stimulation, and (3) injecting the cocaine.
The wireless backpack communicates with the “home base” computer you will use to run the
experiment and to observe the dopamine changes and behavior using radio waves or telemetry
just like your remote controls the television or your radio receives music to play. In this way,
dopamine can be monitored in the brain of the animal without being connected or “hardwired” to
the equipment. Using the home base computer, you will be able to command the wireless
backpack to stimulate the animal. The home base computer will also register the lever press by
the animal and send a signal to the wireless backpack to administer cocaine as well as control the
other stimuli associated with cocaine injection, such as delivery of an auditory tone and
illuminating a cage light. The wireless backpack will send the dopamine measurements back to
the home base computer for your viewing.
Completing the experiment will require that you perform specific tasks in each of the lab areas.
Ch. 2: Prep area lab procedures
[Don't worry about this "Chapter" for now.] If this section is described during the experiment, it
should not have to be described here.
Ch. 3: Experiment area lab procedures
If this experiment were to be completed in real time instead of through the virtual world, one
would have to perform certain tasks before beginning the actual experiment. As the
experimenter, you would be responsible for verifying that the hardware attached to the rat’s
bring is secure and the animal is healthy. If this is not done, hardware failure will prevent
dopamine measurement or cocaine injection. If a rat is unhealthy, it could die from cocaine
injection, and the data would be skewed.
Once rat has been chosen, it will be placed in the operant chamber and the telemetry unit is
attached to the animal. The jugular injection line is flushed with saline to verify that the line is
not clogged. The microdrive is then loaded with the dopamine microsensor to verify the
microsensor functions correctly. The protective cap covering the brain is removed from the
cemented hub and the microdrive is then attached to the hub which is attached to the rat’s brain.
The connections are then attached between the telemetry unit and the reference electrode, the
stimulating electrode, the microsensor and the jugular cannula. The microsensor is lowered to
the desired depth at the appropriate speed and the correct depth so as not be break the
microsensor or provide appropriate measurement of the dopamine in the nucleus accumbens. .
The rat must now be allowed animal to habituate to the operant chamber in order to feel
comfortable with its surroundings and not be stressed. A stressed animal may behave erratically
and have an adverse reaction to cocaine. The microsensors must then be allowed to equilibriate
in the brain so the microsensors will not perform erratically. A test stimulation will be
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NIH Book Chapters, page 17
performed to check the functionality of the microsensor and have it replaced if necessary, and
verify the microsensor has been placed in a dopamine-rich area so that appropriate readings will
take place.
Next, the stimulating electrodes must be checked to make sure there is not a bad connection, a
bad stimulation unit, or the stimulating electrode has been moved. Wireless communication
must also be checked to verify there is no problem with the battery, or the telemetry home-based
computer is not functioning.
The next step is to turn on the video camera in order to record the rat’s behavior. It is now time
to begin the experiment and monitor dopamine during cocaine self-stimulation. The following
steps will take place during the lab experiment.
a) session starts with extension of lever and illumination of light above lever
b) every bar press results in
i) six second injection of cocaine
ii) dimming of light above lever and illumination of operant chamber general light for 20
s
iii) auditory tone for 20 s
iv) during 20 s, additional lever presses will not result in additional injections of cocaine
c) session ends
i) lever is retracted and light above lever is extinguished
ii) typical session duration is 120 min
(a) does not need to be this length
(b) could just do a few with variable results
d) repeat test stimulation
1. check for functioning microsensor
Once you have completed the experiment, it is time for you to write up your analysis in your lab
report. The following questions should be answered: Are these questions identifiable in the lab
experiment that are being observed? I’m not sure…
1. Were/are animals lever pressing adequately?
2. What is the behavior of animals between and during lever pressing?
3. Is dopamine released during session?
4. What behavior is associated with dopamine release?
5. What cues are associated with dopamine release?
Think of additional experiments that may need to take place following the one that has just been
completed. For example,
6. Working with trained vs. untrained rats
7. changing the cues in dopamine for trained rats
8. changes in dopamine release and lever pressing behavior
What other experiments might be found useful?
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