Books in Desk area
BOOK 1: Nervous system anatomy and function
Ch. 1: Nervous system anatomy
Basic divisions of the nervous system
Hindbrain and midbrain
Forebrain
Rats versus humans
Ch. 2: Neurons
Overview of cells in the nervous system
Structure of neurons
Ch. 3: Neuronal signaling
Neuronal conduction
Neuronal transmission (or neurotransmission)
BOOK 2: Dopamine and cocaine
Ch. 1: The neurotransmitter dopamine
Ch. 2: Anatomy and function of dopamine neurons
Ch. 3: Approaches for assessing dopamine function
Ablation
Receptor drugs
Brain Dopamine Content
Monitoring Techniques
Imaging Techniques
Ch. 4: Dopamine neurons and motivated behavior
Reflex behavior
Complex behavior
Motivated behavior
Intracranial self-stimulation
Sexual behavior
Ch. 5: Dopamine neurons and drugs of abuse
BOOK 3: The experiment and lab procedures
Ch. 1: The hypothesis and overview
Introduction
Significance
Objective, hypothesis, and prediction
Technical issues
Experimental design
Ch. 2: Prep area lab procedures
Ch. 3: Surgery area lab procedures
Ch. 4: Cocaine-self-administration training
Ch. 5: Experiment area lab procedures
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BOOK 1: Nervous system anatomy and function
Ch. 1: Nervous system anatomy
Basic divisions of the nervous system
In rats, as in humans and other mammals, the nervous system is divided into the central
nervous system and peripheral nervous system. The brain and spinal cord comprise
the central nervous system. All other components belong to the peripheral nervous
system. These other components include motor neurons, which innervate muscles, and
sensory neurons, including those connected to pain- or temperature-sensitive receptors
in the skin. The brain is further divided into six subdivisions: medulla oblongata, pons,
cerebellum, mesencephalon, diencephalon (or "in between brain"), and cerebrum.
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.
[INSERT IMAGE – whole human body with brain, spinal cord and peripheral nerves]
Hindbrain and midbrain
Collectively, the medulla oblongata, pons and cerebellum are called the hindbrain and
perform “lower-level functions.” Closest to the spinal cord and resembling it in gross
anatomy, the medulla oblongata controls breathing and heart beat. On the opposite
side of the medulla is the pons (or “bridge”). It relays sensory information between the
cerebellum and cerebrum. The cerebellum is the ball-like structure resting on the back
of the brain. 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 [because it lies between the hindbrain (medulla
oblongata, pons and cerebellum) and forebrain (diencephalon and cerebrum)].
[INSERT IMAGE – focus human brain and spinal cord]
Forebrain
The forebrain consists of the diencephalon and cerebral hemispheres. The
diencephalon (“in between” brain) 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.
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The cerebrum or cerebral hemispheres 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, central to the control of movement.
Neuropathologies of movement, such as Parkinson’s disease and Huntington’s disease,
result from damage to the basal ganglia. The four lobes of the cerebral cortex are the
frontal, parietal, temporal and occipital. 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 somatasensory (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.
[INSERT CEREBRAL CORTEX IMAGE]
Rats versus humans
Rats and humans share all of the major subdivisions of the brain and their general
functions. As mammals and compared to other animals, the midbrain is greatly
reduced, as the cerebrum has evolved to become the primary center for coordinating
responses to auditory and visual stimuli. 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 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 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.
[INSERT COMPARISON IMAGE]
Ch. 2: Neurons
[The tone of this chaper is more conversational than that of Ch. 1. We shall need to
tweek this chapter to make it consistent in tone with the others.]
Overview of cells in the nervous system
The nervous system is composed of two kinds of specialized cells: neurons and glia.
Neurons are the basic information processing structures, whereas glia (or glial cells)
are the cells that provide support to the neurons. Because our main interest lies in
exploring how information processing occurs in the nervous system, [having already
intrduced the distinction between the CNS and PNS, should we say 'the nervous
system' here?] we will only introduce a few basic ideas about glia. In much the same
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way that the foundation, framework, walls, and roof of a house prove the structure
through which run various electric, cable, and telephone lines, along with various pipes
for water and waste, not only do glia provide the structural framework that allows
networks of neurons to remain connected, they also attend to the brain's various house
keeping functions (such as removing debris after neuronal death). Glia also perform
functions of the immune system within the central nervous system. Although we will
focus our attention next on how neurons function, it is worth noting that there are as
many as 50 times more glia than neurons in our central nervous system!
[INSERT GLIA IMAGE]
The function of a neuron is to receive INPUT "information" from other neurons, to
process that information, then to send "information" as OUTPUT to other neurons. For
the record, synapses are connections between neurons through which "information"
flows from one neuron to another, but you will hear more about this in a moment.
Hence, neurons process all of the "information" that flows within, to, or out of the
nervous system. All of it! All of the motor information through which we are able to
move; all of the sensory information through which we are able to see, to hear, to smell,
to taste, and to touch; and of course all of the cognitive information through which we
are able to reason, to think, to dream, to plan, to remember, and to do everything else
that we do with our minds. Processing so many kinds of information requires many
types of neurons; there may be as many as 10,000 types of them. Processing so much
information requires a lot of neurons. How many? Well, "best estimates" indicate that
there are around 200 billion neurons in the human brain alone! And as each of these
neurons is connected to between 5,000 and 200,000 other neurons, the number of
ways that information flows among neurons in the brain is so large, it is greater than the
number atoms that exist in the entire universe!
Before we see how neurons process information (and what that means), you need to
know a few things about the structure of neurons.
Structure of neurons
While there are as many as 10,000 specific types of neurons in the human central
nervous system, generally speaking, neurons come in three flavors: motor neurons (for
conveying motor information), sensory neurons (for conveying sensory information), and
interneurons (which convey information between different types of neurons). The
following image identifies how neurons come in various shapes and sizes. (It is based
on drawings made by Cajal.)
Don't worry. You are not responsible
for being able to recognize any of
these neurons or to know what, say,
the reticular formation is or does.
After all, this is your introduction to
neurons. And by way of introduction,
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you should know that a "typical" neuron has four physically distinct parts (or more
accurately, morphologically defined regions): cell body, dendrites, axons and
terminals. As can be send in the figure, neurons are unusually shaped and complex
cells, in that they have a very long process called an axon linking the terminal at one
end to the cell body at the other end. Additionally, both the dendrites at one end and
the terminals at the other extensively branch like a tree. This branching is called
arborization, and enables one neuron to receive many input connections from other
neurons at the dendritic arborization and to make many output connections to other
neurons at the terminal arborization.
[INSERT IMAGE of GENERIC NEURON]
The largest part of the neuron is the cell body (or soma). The typical cell body is round
in shape and between 5 and 20 microns in diameter (a micron is one-millionth of a
meter and one meter is about one year or three feet). The cell body is not only the
"control center" of the neuron, integrating thousands of inputs signals, it is also its
"manufacturing and recycling plant." For instance, it is within the cell body that neuronal
proteins are synthesized.
The second and third parts of the neuron are processes -- structures that extend away
from the cell body. Generally speaking, the function of processes is to be conduits
through which information flows to the cell body and away from the cell body. Incoming
information from other neurons is (typically) received through its dendrites. The
outgoing information to other neurons flows along its axon. A neuron may have many
thousands of dendrites, but it will have only one axon. Dendrites and axons become
very thin processes far away from the cell body. Axons are typically longer than
dendrites, especially those of the single neurons connecting the spinal cord to muscles
or sensory receptors in the leg. Perhaps even up to a meter in length, axons typically
have a diameter less than 1 nanometer (one nanometer is one billionth of a meter). The
electrical signal of the neuron called an action potential is conducted very quickly
along the axon.
The fourth distinct part of a neuron lies at the end of the axon, the axon terminals (ore
pre-synapses). These are the structures that contain neurotransmitters. Because we
shall explore what neurotransmitters are shortly, for now you need to know only that
neurotransmitters are the chemical medium through which "information" flows from one
neuron to the next. You also need to know that information flows from one neuron to
another only at a synapse. While the neuron communicates with another neuron at the
synapse formed by its terminals, it also receives information from another neuron at the
synapse formed by its dendrites.
Before we turn our attention to how information is processed within a neuron, the
following image summarizes the story so far.
[INSERT IMAGE from neuroscience module]
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Ch. 3: Neuronal signaling
Cells use a variety of mechanisms to communicate internally between different
subcellular compartments (for example, between the cell membrane on the exterior and
the nucleus in the interior) 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 and inter-cellular signaling to
support the general function of the nervous system, integration. For example,
integration requires both rapid communication over long distances and selective inputs
and outputs. Neuronal signaling is contrasted with endocrine signaling, during which a
hormone is released into the blood stream and carried throughout the body to act on
multiple sites. 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. 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 [should this be
'neurotransmission' to be consistent with the section following the one below?] . This
process takes place at the synapse and is how two neurons communicate very quickly,
but only with each.
[INSERT IMAGE CONDUCTION VERSUS TRANSMISSION]
Neuronal conduction
To begin conduction, an action potential is generated near the cell body portion of the
axon. What is an action potential? 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. How do neurons generate action potentials? All
cells have 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). However, neurons are unique and called excitable
cells, because they can alter the membrane potential. [?] The action potential is
essentially the result of the neuron altering its 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.
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How does the neuronal control ions flowing across its membrane to generate an action
potential? Well, the protein membrane of a neuron acts as a barrier to electrically
charged particles called ions. When the concentration of ions on the inside of the
neuron changes, the membrane voltage changes. When the voltage of the membrane
reaches a point of no return -- called a threshold (as a result of ions entering and
leaving through ion channels in the membrane) -- the leakiness of the neuronal
membrane changes and allows many ions to pass (i.e, many ion channels temporarily
open). When many ions flow, a large electrical signal is generated. This signal is called
the action potential. This signal is then propagated along the axon (and not, say, back
to its dendrites) until it reaches its axon terminals. Thus, the action potential is also
directional, originating in the cell body and ending in the terminal. It is at axon terminals
where the neuron sends its OUTPUT to other neurons during the mechanism called
transmission. At electrical synapses, the OUTPUT will be the electrical signal itself.
At chemical synapses, the OUTPUT will be neurotransmitter.
Neuronal transmission (or neurotransmission)
Neurotransmission is communication of information between neurons as accomplished
by the movement of chemicals or electrical signals across a synapse. [Here we are not
being consistent. In the first section, you identify two processes: conduction and
transmission. Again, can we fix this by changing 'transmission' to 'neurotransmisson'?
We want to make sure that 'conduction' is reserved for intra-neuron communication and
'transmission' is reserved for inter-neuron communication'. If you think it is best to use
'transmission' instead of 'neurotrasmission', I can change the content on the website. I
will be editing it anyway.] Once again, the function of a neuron is to receive INPUT
"information," to process that information, then to send "information" as OUTPUT. This
is true regardless of whether the neuron is a sensory neuron, a motor neuron, or an
interneuron. But since the INPUT information to a sensory neuron will be a physical
stimulus (such as light on the retina, pressure on the skin, etc.), it is not the case that
that the only way a neuron can receive INPUT information is through synapses with
other neurons. And since the OUTPUT of motor neuron will be a signal sent to a
muscle, neither is it the case that every neuron sends its OUTPUT information to other
neurons. Nevertheless, since the aim of this section is to introduce you to how a
"typical" neuron does what it does, for now, let's ignore questions about how the
transduction of a physical stimulus occurs. Let's also ignore questions about how the
OUTPUT of a neuron can affect a muscle. Hence, let's consider only how a typical
interneuron processes information. [Change tone.]
For any interneuron, its function is to receive INPUT
"information" from other neurons through synapses, to
process that information, then to send "information" as
OUTPUT to other neurons through synapses.
Consequently, an interneuron cannot fulfill its function if
it is not connected to other neurons in a network. A
network of neurons (or neural network) is merely a
group of neurons through which information flows from
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one neuron to another. The image below represents a neural network. "Information"
flows between the blue neurons through electrical synapses. "Information" flows from
yellow neuron A, through blue neuron B, to pink neuron C via chemical synapses.
Although it is reasonable to think of dendrites and axons as conduits through which
information flows within a neuron, a neuron's dendrites and axon are part of its
structure. Synapses are not. Synapses are where information "flows" from one neuron
to another. And since the function of a neuron is to receive INPUT information, to
process it, then to send OUTPUT information, it would be impossible for a neuron to do
what it does without synapses. And before we explore exactly what it is that neurons
do, you should know that a neuron never receives INPUT information from just one
synapse. Remember, a typical neuron receives INPUT from thousands of synapses.
And INPUT synapses can occur anywhere, not just at dendrites.
So, the questions now are as follows: What is the nature of the INPUT information that
the blue neuron receives? How does it process this information? And what is the
nature of the OUTPUT information it sends to other neurons? The problem with the
order of these questions is that what occurs at a synapse determines what occurs within
a neuron, and vice versa. Since there is a bit of the chicken and the egg problem here,
let's first focus on what neurons do on the basis of the INPUT information they receive.
That's easy. They generate electrical signals called action potentials.
[This needs to be edited since there is no single blue neuron in the image. The
focus here is on simply setting up the distinction between electrical
neurotransmission and chemical neurotransmission. So, the subsections should
be as follows.]
Electrical neurotransmission
Electrical neurotransmission (typically) occurs between the dendrite of one neuron and
the dendrite of another. Click on the button below for an animation that will take you
through each step in this process.
Chemical neurotrasmission.
Neurotransmitters are ….
Over 100 different neurotransmitters have been identified to date, and new
neurotransmitters continue to be added to this long list. Neurotransmitters differ greatly
in their chemical properties. The classic neurotransmitters, such as dopamine,
norepinephrine, serotonin, glutamate, GABA (gamma-aminobutyric acid) and glycine,
are small chemicals the size of amino acids, the basic building blocks of proteins. In
fact, two neurotransmitters, glutamate and glycine, are amino acids, while others,
including dopamine, norepinephrine, and serotonin, are synthesized from amino acid
precursors. Other neurotransmitters are: (1) peptides, which are small proteins or
chains of amino acids linked together; (2) lipids or fat-like substances such as
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anandamide, which is an endogenous cannabinoid, a chemical mimicking the active
ingredient in marijuana; and (3) even gases, such as nitric oxide and carbon monoxide.
Neurons are furthermore classified by the neurotransmitter used to bridge the synaptic
cleft during chemical neurontransmisson. Hence, dopamine neurons use dopamine for
this purpose. The most numerous neuron in the human brain uses glutamate as a
neurotransmitter. Glutamate neurons comprise about half of the total number of
neurons in the mammalian brain. Glutamate is an excitatory neurotransmitter in that it
tends to cause the target neuron to generate more action potentials. The second most
numerous neuron uses the neurotransmitter GABA. In contrast to glutamate, GABA is
an inhibitory neurotransmitter in that it tends to cause the target neuron to generate less
action potentials. GABA is the most important inhibitory neurotransmitter in the brain,
while glycine is the most important neurotransmitter in the spinal cord. By comparison,
dopamine neurons are relatively few in number. As described in more detail later,
despite relatively low numbers, dopamine neurons play very important roles in brain
function.
"Classic" chemical neurotransmission occurs between the axon terminal of one neuron
and (typically) the dendrite of another. The following animation will take you through
each step in this process.
BOOK 2: Dopamine and cocaine
Ch. 1: The neurotransmitter dopamine
Dopamine is made from tyrosine, a non-essential amino acid (meaning the body can
synthesize it de novo or from scratch) that is also found in many foods. Dopamine
neurons form chemical synapses and thus use dopamine to bridge the synaptic cleft
between the end or the terminal of the dopamine neuron, which is sometimes called the
pre-synapse, and the beginning of the target neuron, which is sometimes called the
post-synapse. [The previous sentence needs to be broken up. There is another
inconsistency with terminology: in the animations, I lable the neurons 'presynaptic
neuron' and 'postsynaptic neuron'. We just need to be consistent.] A common
arrangement is for one neuron to make a synapse on the dendrite of another neuron. In
this case, neurons have both pre- and post-synaptic elements, but at opposite ends,
and electrical information flows from dendrite (post-synapse) to terminal (pre-synapse).
Although dopamine neurons form synapses at their terminals, they also from en
passant synapses. These are synapses “in passing”. This means that the dopamine
neuron can make a synapse with a target neuron without terminating the axon.
[INSERT IMAGE DOPAMINE NEUROTRANSMISSION]
When the action potential arrives at the pre-synaptic element, a series of biochemical
reactions occur that ultimately result in the release of dopamine into the cleft. The
majority of dopamine in the brain is found inside neurons packaged into synaptic
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vesicles or small, membrane-enclosed packets. At the electron microscope level,
vesicles appear as little spheres or balls in the pre-synaptic element immediately
adjacent to the cleft. About 2,000 dopamine molecules are present in each vesicle.
Because of its small size, packing this many molecules into a single vesicle results in a
very high concentration. However, when inside the neuron, dopamine is inactive. Only
when released into synaptic cleft, where it can act on the post-synaptic target neurons,
is dopamine active. The release of dopamine from the synaptic vesicle into the cleft,
which is a general process called exocytosis, is complex. However, the net result is that
a high concentration of dopamine is released and now becomes active or functional.
[If the user has already seen the animation of "classic" chemical neurotransmission,
then you can focus on what is different about dopamine chemical nerotransmission.]
Like other chemical synapses, the synapse between a dopamine neuron and a target
neuron is separated by a small cleft or gap. Compare this arrangement to electronic
devices, in which components are “hard-wired” or “soldered” together. The synaptic
cleft is also filled with a fluid called brain extracellular fluid that and is continuous with
the fluid bathing all neurons and glia in the brain. Although very small, typically on the
order of a few nanometers (a billionth of a meter), the synaptic cleft creates a physical
barrier for the electrical signal carried by one neuron to be conducted or transferred to
another neuron. By comparison, the dopamine synapse altogether is approximately
300 nanometers in diameter and each vesicle is about 15 nanometers across. In
electrical terms, the synaptic cleft would be considered a “short” in an electrical circuit.
The function of dopamine, as well as all other neurotransmitters then, is to overcome
this electrical short.
Once in the cleft, dopamine can diffuse across to the post-synaptic element of the target
neuron where it can bind to sensing proteins called receptors. These post-synaptic
receptors, which are specific to dopamine and hence are called dopamine receptors,
respond very quickly to dopamine binding by eliciting another series of biochemical
reactions that direct the target neuron to generate new action potentials or stop
generating action potentials. Thus, dopamine, as well as all neurotransmitters by
definition, acts as a chemical messenger or link connecting two electrical signals on
different neurons.
It should be noted that binding to a post-synaptic receptor does not destroy dopamine.
In chemical terms, this interaction is called an equilibrium reaction, where dopamine
temporarily attaches to the receptor and then breaks free to diffuse away. This
interaction takes place within the cleft, because the receptor protrudes into the
extracellular fluid. Though temporary, binding of dopamine to its post-synaptic receptor
is capable of producing very complex reactions in the target neuron, ultimately
controlling the activity of that neuron on many levels.
[INSERT IMAGE G-PROTEIN COUPLED RECEPTOR FUNCTION]
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The dopamine receptor also differs from post-synaptic receptors found in the classic
synapse. After binding by neurotransmitter, classic post-synaptic receptors form
channels that all 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 post-synaptic membrane. If this voltage
change is great enough, i.e., reaches threshold, the target neuron will fire an action
potential, thus completing neuronal signaling. 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 target neuron from opening and closing ion changes to gene transcription
[which is?] and protein synthesis.
Neurotransmission is terminated by removal of dopamine from the cleft, so that postsynaptic receptors are no longer activated. Like dopamine, most neurotransmitters are
cleared by a special protein on the pre-synaptic element called a transporter. This
protein binds to the neurotransmitter similar to the receptor, but physically moves or
translocates it back into the neuron. Thus, the same neuron that initiates
neurotransmission by releasing dopamine also terminates neurotransmission by
removing dopamine. The dopamine transporter has a very high affinity for binding
dopamine and can take up dopamine very quickly. Once inside the neuron, dopamine
can be re-packaged into vesicles and used again in a process called neurotransmitter
re-cycling. Instead of a transporter, some neurotransmitters like acetylcholine are
eliminated from the cleft by a degradative enzyme.
Dopamine neurotransmission is also described by several other important mechanisms.
For example, the pre-synaptic element can synthesize dopamine from the tyrosine that
it takes up from brain extracellular fluid using an amino acid transporter. To complete
the synthesis pathway, tyrosine is converted into another chemical called DOPA, which
is then made into dopamine, all in the pre-synaptic element of the dopamine neuron. In
this sense, the pre-synaptic element functions relatively autonomously by synthesizing,
releasing and re-cycling dopamine.
While not found in the synaptic cleft to remove released dopamine directly, enzymes
degrading dopamine are found inside the pre-synaptic element. These degradative
enzymes are thought to maintain the concentration of dopamine inside the neuron at
low levels, which can become quite high during active periods of rapid synthesis and
uptake before dopamine is packaged into vesicles. Interestingly, high concentrations of
dopamine appear to be neurotoxic, and only by degrading dopamine or packaging
dopamine into vesicles is the dopamine neuron protected. Similar to the post-synaptic
element, the pre-synaptic portion of the dopamine neuron also contains dopamine
receptors. These so called autoreceptors function as a “thermostat”, shutting down
the dopamine neuron when too active or speeding it up when too lethargic.
The synapse between a dopamine neuron and a target neuron appears to function
rather differently than the classic synapse depicted in textbooks. According to the
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classic view, the action of the neurotransmitter is confined to the synaptic cleft by
transporters or degradative enzymes, which prevent it from escaping. The dopamine
synapse, in sharp contrast, appears open such that dopamine released into the cleft
readily diffuses out. For this reason, dopamine is often called an extrasynaptic
messenger. The target of release dopamine then is not restricted to the post-synaptic
element, but rather any neuron with a dopamine receptor close enough to the dopamine
synapse to be exposed to a high dopamine concentration. Because of diffusion, the
farther way from a dopamine synapse, the lower the dopamine concentration. The
ability of dopamine to escape the synapse readily is why this neurotransmitter can be
measured chemically in the brain without a sensor that is small enough to be placed
inside the synaptic cleft. No such probe or sensor exists to date.
[INSERT IMAGE – DOPAMINE SYNAPSE AND SITES OF DRUG ACTION]
Mechanisms of dopamine neurotransmission are the site of action of many drugs used
to treat many neuropathologies. Haloperidol, clozapine and sulpiride, which block
dopamine receptors, are used as antipsychotic agents to treat schizophrenia. Made
form tyrosine, the biochemical precursor to dopamine, levodopa, alleviates symptoms of
Parkinson’s disease. Methylphenidate or Ritalin®, a blocker of the dopamine
transporter, is prescribed for attention-deficit hyperactivity disorder. Some
antidepressants are based on classes of drugs inhibiting dopamine uptake, such as
Welbutrin®, or drugs inhibiting degradative enzymes for dopamine, such as the MAOinhibitors. Interestingly, the same drug as Welbutrin®, but called Zybane®, is
prescribed to patients wishing to stop cigarette smoking. As will be addressed later,
dopamine neurotransmission is also the site of action of drugs of abuse such as
cocaine.
Ch. 2: Anatomy and function of dopamine neurons
In addition to extensive overlap in the anatomy, organization, and function of the central
nervous system, rats and humans are also share similar dopamine neuron systems. 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.
[INSERT IMAGE DOPAINE NEURONAL SYSTEMS rat or human]
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, some
project to and terminate in the spinal cord. This later subset of hypothalamic dopamine
neurons would be said to descend in anatomical terms, going from the forebrain to the
spinal cord.
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Another important group of dopamine neurons originates in the midbrain. These
dopamine neurons are ascending, because they project to and terminate in the
forebrain. [Should we modify the previous two sentences so we use 'decend' and
'ascend' or 'ascending' and 'decending'?] The ascending dopamine neurons originate in
two regions of the midbrain, the substantia nigra and ventral tegmental area.
[INSERT IMAGE?]
The dopamine neurons originating in the substantia nigra terminate in the striatum.
Neuroanatomists define neuron systems by the origin (prefix) and termination (suffix).
Thus, these dopamine neurons are classified as the nigrostriatal dopamine system.
Because the striatum is an important region of the basal ganglia, these dopamine
neurons play an important role in movement. Indeed, symptoms of Parkinson’s
disease, a movement disorder, are associated with the loss of nigrostriatal dopamine
neurons. When viewed post mortem, the substantia nigra in the human brain is darkly
stained naturally, hence the name 'nigra', which is Latin for 'black'. This coloration is
due to the presence of neuromelanin, a substance related to the skin pigment melanin
and a by-product of dopamine metabolism. First described in the 1930s, the loss of the
highly pigmented neurons in the substantia nigra was the earliest neurochemical deficit
associated with Parkinson’s disease. It took the scientific community another 30 years
to establish the neuropathology link further to the degeneration of nigrostriatal dopamine
neurons.
Two subsets of midbrain dopamine neurons originate in the ventral tegmental area.
The mesolimbic dopamine neurons project to and terminate in limbic areas of the
forebrain, such as the nucleus accumbens and amygdala. The limbic system is a
functional and anatomical organization of brain regions involved in motivation, emotions
and memory. As will be described below in more detail, the nucleus accumbens is
important for motivated behavior, because it integrates sensory input and motivational
status to direct behavioral output. The amygdala governs emotions. Drug addiction is
thought to be related to dysfunction of mesolimbic dopamine neurons.
The mesocortical dopamine neurons also originate in the ventral tegmental area but
terminate in several regions of the cortex. One prominent area of termination is the
medial prefrontal cortex, a region thought to play an important role in working or
“scratch pad” memory. This type of short-term memory is the most transient and has a
limited capacity. However, working memory is important, for example, when
understanding the meaning of a complex sentence requires one to remember what was
written in the first part of the sentence. Mesocortical dopamine neurons exhibit unusual
characteristics such as a very high rate of basal activity and more limited control by
autoreceptors. Schizophrenia is also thought to be associated with hyperactivity of the
mesoprefrontal cortical dopamine system.
[Should the following paragraph be moved to follow the one 2 paragraphs above this
one? Should we have an image?]
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The brain is symmetrical in the rat and human, and each side contains a substantia
nigra and striatum. About 8,000 dopamine neurons originate from each substantia nigra
in the rat. Although there are ~65,000 dopamine neurons in each human substantia
nigra, this region is larger in the human than the rat and the density of dopamine
neurons is the same in both animals. One dopamine neuron in the rat makes
approximately 500,000 synapses in the striatum due to extensive branching at the
terminal end. In additional to terminal synapses, dopamine neurons also make en
passant synapses (i.e., a synapse in passing). Thus, because each rat striatum
contains about 3 million neurons, dopamine neurons interact with almost all neurons in
this region. In the rat and human, the densest innervation by midbrain dopamine
neurons is in the striatum. The nucleus accumbens contains about half the number of
dopamine synapses, and the amygdala about one seventh. Although highest of the
cortical areas, the dopamine innervation in the medial prefrontal cortex is about one
hundred-time less than the striatum.
Ch. 3: 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.
Ablation
Historically, a successful way in which neuroscientists have assessed whether a
neuronal system is involved in a behavior is to study what happens to that behavior
when the neuronal system is prevented from functioning. One widely used method to
prevent a neuronal system from functioning is ablation or lesioning of neurons. A
neuronal pathway can be lesioned or destroyed by several procedures: a small knife
can be used to sever axons, drugs can be used to over-stimulate cell bodies, eventually
causing their death (a process called excitotoxicity), and neurotoxins can be used to
kill neurons.
Dopamine neurons can be killed by a neurotoxin called 6-hydroxydopamine. This
chemical resembles dopamine in structure with the exception of one small change. This
small changed, called hydroxylation, adds one carbon and one hydrogen molecule,
making dopamine, now 6-hydroxydopamine, very reactive. Even though dopamine has
been chemically modified, 6-hydroxydopamine can still be taken up by the dopamine
transporter. Thus, 6-hydroxydopamine acts by being taken up into dopamine neurons
through the dopamine transporter. Once inside the dopamine neuron, 6hydroxydopamine combines with oxygen and ascorbic acid (vitamen C), both of
which are in high concentrations in the brain, to generate a chemical called hydrogen
peroxide. This chemical, called a reactive oxygen species, attacks many
components of the neuron, eventually killing it.
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[INSERT IMAGE CHEMICAL STRUCTURES OF DOPAMINE AND 6HYDROXYDOPAINE AND ACTION OF 6-HYDROXYDOPAMINE]
Receptor drugs
Another successful way neuroscientists have used to prevent a neuronal system from
functioning is receptor antagonists. These drugs block the ability of neurotransmitters
to bind and activate post-synaptic receptors. These antagonists also bind to the
receptors but do not cause any effect on the target neuron. Rather, they prevent the
neurotransmitter from having an effect. In contrast, receptor agonists bind to the
receptor and mimic the effects of the neurotransmitters. Receptor agonists and
antagonists elicit opposite effects. Thus, if a behavior is suspected to involve the
neurotransmitter dopamine, a dopamine receptor antagonist can be administered to
determine of the behavior is reduced, while a dopamine receptor agonist can be
administered to determine if the behavior is enhanced.
[INSERT IMAGE ACTION OF RECEPTOR AGONISTS AND ANTAGONISTS]
Brain Dopamine Content
One of the earliest measures of dopamine neuron activity was to remove the brain after
an experiment quickly and determine the tissue amount of dopamine. However, the
measurement of tissue dopamine content did not prove very valuable, because the vast
majority of dopamine in the brain is found inside the neuron in synaptic vesicles. Only a
small amount of the total dopamine is actually released into the extracellular fluid of the
synaptic cleft at any given time.
Another confounding issue is that the dopamine neuron can increase synthesis in
response to increased release, so that tissue dopamine content changes little even
when dopamine neurons are very active. Tissue dopamine content therefore better
reflects the density of the dopamine innervation rather than being an index of functional
activity. A more useful measurement for brain tissue is to compare the levels of
dopamine metabolites, which are formed by the action of degradative enzymes on
dopamine, to dopamine levels. While tissue dopamine changes little, tissue metabolites
are more sensitive to activity.
The general disadvantage of brain tissue measurements of any kind is that only one
determination is taken per animal, and it occurs several seconds to minutes after the
event under study, whether that is a behavior or a drug injection. A superior approach
is to make a measurement of the activity of dopamine neurons during the event itself.
“Monitoring” techniques have been developed to make such measurements.
Monitoring Techniques
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
15
dopamine neuron generates. This technique is called electrophysiology or monitoring
the “electrical functioning” of the neuron. The action potential is an electrical signal or
more specific, a bioelectric signal. Instead of current carried by electrons as in an
electronic device, current during an action potential is carried by ions, so small charged
chemicals, along and through the neuron. [The user is required to read the material
in Book 1, so this is redundant.] 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, about 10 to 20
microns in diameter (a micron is a millionth of a meter), 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 pre-synapse. 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.
[INSERT IMAGE ELECTROPHSIOLOGICAL MICROELECTRODE AND THE
MEASUREMNT OF AN ACTION POTENITAL]
Other monitoring techniques have been developed to measure dopamine directly in the
“terminal fields,” those regions of the brain in which dopamine neurons 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 semipermeable or selectively permeable. Based on the same principle, a dialysis machine
is used to filter blood of patients which kidney problems.
[INSERT IMAGE MICRODIALYSIS PROBE]
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,
16
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.
[INSERT IMAGE CARBON-FIBER MICROSENSOR AND THE MEASUREMENT OF
DOPAMINE]
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 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. Although not relevant to dopamine monitoring, carbon
fibers are stronger than steel on a per weight basis and are used in the manufacturing
of tennis rackets, fishing poles and the shell of performance racing cars. For the same
reason, the new Boeing jet airliner is made from a carbon composite material.
The carbon fiber also provides an excellent surface for electrochemistry, 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.
Imaging Techniques
[INSERT IMAGE CAT, MRI AND fMRI SCAN]
17
Because of the need for non-invasive approaches, imaging not monitoring techniques
typically is used to measure the activity of neurons in the human brain. Imaging
techniques include computerized tomography or CT, magnetic resonance imaging or
MRI, and positron emission tomography or PET. A CT scan is essentially an x-ray of
the brain and provides no information about functional activity, only anatomy. MRI uses
a large magnet to vibrate water molecules in the brain and thus distinguishes regions on
the basis of water content, which differs greatly in the brain. The typical MRI scan
provides anatomical features of the brain. However, a special type of MRI, called
functional MRI of fMRI, measures oxygen levels in the brain. Since active neurons use
more oxygen than inactive neurons, changes in oxygen levels reflect the overall level of
neuron activity in a region. Thus, fMRI is used to study changes in neuron activity in the
human brain. Unfortunately, fMRI may not indicate which type of neuron is active or
inactive, only the brain region in which there is a change in activity.
[INSERT IMAGE PET SCAN]
To measure the activity of specific neurons, PET is typically used. In PET, the patient is
given a small amount of a radioactively labeled drug that interacts with a specific
neuron. PET then follows how the distribution of this labeled drug changes in the brain
with time. Changes in drug distribution reflect changes in neuron activity. For example,
the activity of dopamine neurons has been studied with PET using labeled DOPA, the
immediate biochemical precursor to dopamine that it also used in the treatment of
Parkinson’s disease. Dopamine neurons take up DOPA and convert it to dopamine.
Because more active dopamine neurons take up more DOPA than less active dopamine
neurons, the amount of labeled DOPA in a region is positively related to the activity of
dopamine neurons in that region.
Another class of drugs used to study dopamine neuron activity using PET is called
receptor antagonists. These drugs bind to dopamine receptors without causing an
effect on the neuron with the receptor. Dopamine receptor antagonists, such as
antipsychotics or neuroleptics used in the treatment of schizophrenia, block the action of
dopamine. Because antagonists and dopamine compete for the same receptor, there is
an inverse or negative or opposite relationship between labeled drug and dopamine
neuron activity in PET scans. As such, lower levels of labeled drug in a brain region
correspond to higher levels of released dopamine.
It should be noted that recent developments are blurring the typical distinction that
imaging is used to assess the human brain and monitoring techniques for the brain of
laboratory animals. Companies are now manufacturing smaller MRI and PET
instruments for use in rats, for example. The advantage of imaging is that a more
comprehensive view of the entire brain is provided compared to monitoring techniques,
which are typically limited to a specific region. On the other hand, because monitoring
techniques general provide more chemical information about changes in neuron activity
and better temporal resolution (faster measurements) than imaging, monitoring
techniques are now being used by clinicians. One important application is to monitor
the neuron activity of a specific region of the brain during neurosurgery. Implantable
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devices are also being developed that continuously monitor the brain and automatically
administer drugs in response to specific changes in neuron activity.
Ch. 4: Dopamine neurons and motivated behavior
Reflex behavior
Behavior in its simplex form is a reflex: a motor output in response to a sensory input.
Motor means that motion is produced. The “knee jerk’ response, elicited by a physician
tapping on the patella tendon of the knee during a physical examination, is a good
example of a reflex. In this case, the tap stretches the quadriceps muscle (the “quads”
or muscle on top of the thigh) which activates stretch receptors within the muscle.
Activation of stretch receptors increases the firing rate of sensory neurons originating in
the muscle and projecting to and terminating in the spinal cord. These sensory neurons
make a synapse with motor neurons which leave the spinal cord and innervate or
terminate on the quadriceps muscle. Activation of the motor neuron by the sensory
neuron ultimately causes contraction of the quadriceps muscle leading to the “knee
jerk”.
[INSERT IMAGE KNEE JERK REFLEX]
In its simplest form, the “knee jerk” response is mediated by two neurons in what is
called a monosynaptic circuit, a one synapse-two neuron pathway. In actually, the
sensory neuron also activates a second neuron that inhibits another motor neuron
innervating the hamstring muscle, the muscle on the back side of the thigh. Relaxing
the hamstring muscle permits the leg to move. Information about the “status” of the
muscles is also relayed by the spinal cord to the brain. The brain can also override the
“knee jerk” response by voluntary contraction of the quadriceps muscle.
Complex behavior
[INSERT IMAGE SCHEMATIC OF COMPLEX BEHAVIOR: MODIFICATION OF
SENSORY INPUT and MOTOR OUTPUT BY MOTIVATIONAL STATUS]
Complex animals such as rats and humans have evolved complex brains to control
behavior. Using the reflex as a model of behavior, the brain acts as a center for
integrating sensory input and motor output. Another important component of behavior is
the motivational status of the animal. Consider how an animal responds differently to
food, depending upon whether the animal is hungry or satiated. The hungry animal will
respond with behavior that ultimately results in acquiring and consuming the food. On
the other hand, the satiated animal may not respond at all to the identical stimulus. The
limbic system, which includes the hypothalamus, hippocampus, amygdala, and
olfactory bulb, plays an important role in motivated behavior.
Motivated behavior
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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
copulation. 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. The classic experiments using intracranial selfstimulation, in which an animal lever presses to obtain rewarding electrical stimulation
directly 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. 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.
[INSERT IMAGE HUMAN BRAIN DIAGRAM OF NUCLEUS ACCUMBENS AND
LIMBIC SYSTEM]
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 motor-related 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
[INSERT IMAGE PICTURE OF ANIMAL LEVER PRESSING FOR ELECTRICAL
STIMULATION]
20
One of the earliest experiments identifying a relationship between nucleus accumbens
dopamine and motivated behavior was intracrianal 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 noncontingent, 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 occured 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 massively 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 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, are 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,
voltammetry [which is what?] can. On the other hand, voltammetry can measure
dopamine 10 times per second, which is faster than the animal bar presses in
intracranial self-stimulation.
21
When voltammetry has been 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 shows that dopamine is also released during intracranial selfstimulation. However, in well trained animals, intracranial self-stimulation 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 animals, caused
dopamine release. Remarkably, the 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 selfstimulation 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.
Sexual behavior
Other types of motivated behavior have been show to activate mesolimbic dopamine
neurons similarly. One of the best studied is rat sexual behavior. Like intracranial selfstimulation, rats implanted with a microdialysis probe in the nucleus accumbens clearly
show an increase in extracellular dopamine levels increase during a sexual encounter.
Interestingly, dopamine levels also increase when the male and female rats are
separated by a barrier. Although copulation is prevented, the rats can interact to some
extent and engage in appetitive behavior, which for a female rat includes darting around
and wiggling its ears, and lordosis, during which the animal stops moving and arches it
back to expose its flank to the male. This result suggests that dopamine may be
involved in different aspects of motivated behavior besides consumption of the reward
as well.
Ch. 5: Dopamine neurons and drugs of abuse
Humans abuse a variety of drugs, including alcohol, cocaine, amphetamine, and heroin.
Each drug of abuse has both unique and general actions on the brain. The unique
actions give rise to the unique “experience” of taking that type of drug. Although the
mechanisms differ, the general action of all drugs of abuse is activation of mesolimbic
22
dopamine neurons. It is through this general effect that drugs of abuse become
addicting.
One current idea is that addiction represents a pathological control of the normal
functioning of brain dopamine related to learning. This pathology is driven by the ability
of drugs of abuse to increase levels of brain extracellular dopamine to a greater extent
than natural stimuli. Thus, the brain is “hijacked” to engage in behavior whose goal is to
obtain these drugs of abuse as opposed to obtain other rewards. The desire to obtain
drugs of abuse even overcomes obvious harmful consequences, such as job and family
loss, incarnation, poor heath, etc.
One of the more prominent drugs of abuse in the modern history of the United States is
cocaine. Derived from the coca plan grown in the mountains of South American,
cocaine is used in many forms, from chewing unprocessed leaves picked from the coca
plant, snorting a line of the white powder, intravenous injection of a cocaine solution, to
smoking coca paste or the free-base form of cocaine known as crack. The different
kinds of ingesting cocaine result in different quantities of cocaine ingested and different
speeds by which cocaine acts. Intravenous use, for example, results in an intense
“rush” within about one minute that lasts for about 30 minutes.
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
vasoconstriction, which means to narrow or close blood vessels. Large doses of
cocaine can result in cardiac failure.
Cocaine is also considered a psychomotor stimulant because it acts on the brain and
produces motor behavior. Other general effects of cocaine resulting from an action on
the brain include a reduction in food and water intake, an increase in aggression, and
increases in respiration rate and body temperature. The psychological effects of
cocaine are complex and include euphoria, depression, and anger. It is the euphoria
that is the sought by drug users. Cocaine can also cause feelings of increased mental
alertness, sexual desire, energy and self-confidence. 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
23
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.
Drug self-administration is analogous to intracranial self-stimulation exception 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 selfstimulation 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.
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
cause a massive increase in 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 selfadministered 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
self-stimulation, is dopamine involved in other aspects of motivated behavior such as
during the anticipatory phase?
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
24
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.
The problem with microdialysis is that dopamine release in the brain is sampled at a
rate much slower than the behavior. For example, rats lever press to self-administer
cocaine about one every 10 minutes. This is the same rate at which microdialysis
samples brain dopamine. Thus, microdialysis can not tell us what happens to dopamine
release during the behaviors in between each lever press. Recall that motivated
behavior is divided into appetitive and consummatory phases. What this means is that
microdialysis can not tell whether behaviors during the anticipatory phase, such as drug
seeking, or whether the consumption of cocaine during the consummatory phase
actually mediates the increase in measured dopamine release.
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 voltammetry.
This microsensor technique can monitor dopamine very quickly and should provide
great insight into the role of dopamine in cocaine use.
Significance
Because cocaine is a reward, understanding how this drug of abuse acts will provide
neuroscientists working on the neurobiology of motivation with insight into the brain
reward system. Some of the more interesting questions are:
(1) Do all rewards act similarly on the brain reward system?
(2) Do natural and artificial rewards act similarly on the brain reward system?
(3) Despite very different actions on the brain, do all drugs of abuse act similarly
on the brain reward system?
Cocaine, for example, blocks the transporter for three different neurotransmitters:
dopamine, norepinephrine and serotonin. Indeed, is only dopamine involved in
cocaine’s rewarding properties or do norepinephrine and serotonin also play a role? Or,
could it be that norepinephrine and serotonin mediate other subjective effects of cocaine
like increased energy and positive mood, as these two neurotransmitters have also
been linked to depression?
25
Understanding how cocaine acts on the brain is also important for the field of drug
addiction. One of the most perplexing questions in drug addiction research relates to
the phenomenon of stubborn persistence. Why, even after years of abstinence and at
great risk to one’s health and personal well being, will some users resume taking drugs?
One of the observations that may be critical to understanding stubborn persistence is
that drug use is driven by cues. These cues are stimuli associated with drug. Hence,
drug-related cues signal the users that drugs are potentially available in much the same
way that the bell triggered dogs to salivate with the expectation of food in Pavlov’s
experiments.
The proposed role of dopamine in associative learning may hold the key to
understanding the neural basis for stubborn persistence of drug use. One of the more
recent functions attributed to dopamine in the brain is controlling associative learning
related to reward. This special type of learning is involved in shaping or molding
behavior that is related to the acquiring of rewards and the cues that predict these
rewards. In this new way of thinking then, dopamine is less a neural substrate of
reward itself; i.e., dopamine is not the neurotransmitter of pleasure. Rather, dopamine is
more involved in the learning of cues that enable the animal to obtain rewards.
Drug addiction may therefore represent a pathological type of associative learning
related to rewards. Because drugs of abuse increase dopamine levels to a greater
extent than natural rewards, one idea is that the cues associated with drugs are more
strongly learned than cues associated with natural rewards. In this way, drugs of abuse
hijack the brain circuitry involved in the processing of rewards and their cues, rendering
drugs of abuse and their cues the highest priority.
Objective, hypothesis, and prediction
The overall objective of this experiment is to investigate the role of dopamine neurons in
cocaine use. Two important questions will be asked:
(1) What happens to brain dopamine levels when cocaine is used?
(2) Following coaine use, how do changes in brain dopamine levels affect behavior?
Cocaine could be acting on dopamine neurons to alter several aspects of motivated
behavior.
In science, it is sometimes 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
26
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 selfadminister cocaine, similar to humans.
This experiment also requires us to monitor dopamine during cocaine seeking. This
monitoring of dopamine must also be sufficiently fast to capture changes during the
cocaine seeking portion of cocaine self-administration. For example, it will be
necessary to monitor dopamine levels during the time just before the lever press.
Fortunately, voltammetry, which monitors dopamine several times a second, is well
suited for these measurements of dopamine during cocaine seeking in the laboratory
rat. These animals lever press for cocaine once every 10 minutes on average and will
engaging in cocaine seeking in the few seconds just before lever pressing.
Voltammetry will therefore have the ability to capture changes in brain dopamine levels
during this time.
Finally, it is necessary to consider where in the brain dopamine should be monitored.
While several regions could be sampled, perhaps the best place to record dopamine is
the nucleus accumbens. Indeed, this region receives a major dopamine innervation
originating in the ventral tegmental area (which in laymans terms is ...).
[INSERT PICTURE FROM PAGE 20?]
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
27
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.
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 performing cocaine
self-administration. Hence, only a description of the training will be provided.
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.
In manual shaping, the experimenter guides the animal to behavior using the
technique of successive approximation (or simply, reinforces behavior increasingly
similar to the wanted behavior). Thus, when the animal gets close to the lever, the
experimenter will deliver an injection of cocaine. Manual shaping for cocaine selfadministration can but not always involve first training for food reward. This is far
simpler (i.e., faster), because animals will bar press often for a food pellet up to 10 times
a minute, but only once every 10 minutes for cocaine.
In contrast, autoshaping lets chance do the work, with a little help of course. In
autoshaping, the animal is placed in a chamber with a retractable lever for several hours
a day. At various times, the lever is extended for a short of time. If the animal presses
the lever, cocaine is injected. At the end of the time, even if the animal does not lever
press, cocaine is still injected.
Both manual shaping and autoshaping are time-consuming processes, often taking
several days to train an animal to self-administer cocaine. Again, in the present
experiment, any rat you handle will be already trained to self-administer cocaine.
28
PART 2: Preparation and surgery for voltammetry measurements of dopamine.
In preparation for brain surgery, a rat must be weighed, anesthesised, and have its
scalp removed from its skull.
Brain surgery involves implanting two electrodes and a hub that will enable the lowering
of a microsensor for monitoring dopamine during cocaine seeking behavior. One
electrode is the reference electrode. The reference electrode supports the dopamine
measurement by the microsensor. Provided that the reference electrode is in the brain
and secured to the skull, it does not matter where it is implanted. The other electrode is
the stimulating electrode. Its location is critical, for it must be positioned to activate
dopamine neurons electrically. To achieve this, the stimulating electrode will be initially
positioned just above the ventral tegmental area. At the same time, a microsensor will
be lowered into the nucleus accumbens. A test pulse will then be applied to the
stimulating electrode while dopamine release is measured. If no dopamine release is
observed, the stimulating electrode will be lowered slightly and the test pulse repeated.
Once dopamine release is observed, the stimulating electrode will be cemented to the
skull along with the reference electrode.
The hub, which is cemented to the skull along with the reference and stimulating
electrodes, is actually a connector for a miniature manipulator or microdrive. This
piece of equipment can be loaded with a microsensor, attached to the hub, and then
precisely and accurately lower the microsensor into the brain, even when the animal is
awake. The microdrive also permits the experimenter to raise and lower the
microsensor during an experiment to find the best recording location for measuring
dopamine release.
PART 3: Monitoring dopamine during cocaine seeking.
Allow two weeks for the rat to recover from surgery.
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. A test pulse will be applied to the stimulating electrode to
make sure that the microsensor is in the correct position. 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 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. 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.
29
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. The procedures you must follow are outlined in the chapters to follow.
Ch. 2: Prep area lab procedures
[Don't worry about this "Chapter" for now.]
1)
2)
3)
4)
5)
6)
7)
8)
Put on non-sterile surgical gloves and lab coat.
Select a rat.
Weigh rat and then place the rat in the prep tray.
Determine how much anesthesia to draw.
a) per weight dose, e.g., for a pre-mixed cocktail of ketamine and xylazine, a
common anesthetic of laboratory rodents, 1 ml/kg ( inject 0.3 ml into a 300 g rat)
b) too much anesthesia, rat dies
c) too little anesthesia, need to inject again, but secondary doses are tricky and rats
may also die even with normal amount
Grab a vile of anesthesia and a syringe.
a) anesthesia is premixed
(1) stored in locked box (DEA regulated substance) in refrigerator
b) connect needle and syringe connect
Inject the needle into the vile and draw the correct amount of anesthesia.
a) remove bubbles from syringe
b) return anesthesia to lock box
c) record amount used in DEA notebook
Properly inject the anesthesia.
a) inject into peritoneal cavity
(1) too high (in thoracic cavity), cause pneumothorax, deflating lungs and
killing animal
(2) too low (in bladder) will also complications and perhaps death
b) properly dispose of needle in sharps container
Place the rat in the prep tray.
a) rat needs to be on a heating pad as anesthesia prevents thermoregulation
(1) rat can die from being too hot or too cold
Ch. 3: Surgery area lab procedures
[Don't worry about this "Chapter" for now.]
1. shave hair on neck and above skull and sterilize with Betadine (iodine solution
a. rat dies from infection if not done
30
b. expose jugular vein, and insert and secure canula (for cocaine injection)
2. choose artery and animal bleeds to death
a. run canula out back behind neck
b. suture neck
3. place rat in stereotaxic apparatus
a. if rat in crooked, electrodes will not be lowered properly
b. if ears bars too tight, skull is fractured (death)
c. on a heading pad
4. sterile surgical gloves and mask are worn
5. skin and muscle are cut above skull and separated exposing skull
6. skull is scrapped and cleaned
7. three holes are drilled through skull
a. reference electrode
b. stimulating electrode
c. sensing electrode
8. three holes are drilled into but not through skull for surgical screws (to hold cement
to skull)
9. a reference electrode is lowered into superficial cortex using a manipulator attached
to the stereotaxic apparatus
10. a hub is centered above the hole for the sensing electrode using another
manipulator attached to the stereotaxic apparatus
11. reference electrode and hub are cemented to the skull
12. after cement hardens, the hub and reference electrode are disconnected from their
manipulators
13. the reference electrode is connected to recording equipment
14. a microdrive armed with a sensing electrode is attached to the hub and the sensing
electrode is connected to recording equipment.
15. the sensing electrode is lowered to the nucleus accumbens
16. a stimulating electrode is lowered to just above the ventral tegmental area by a
manipulator attached to the stereotaxic apparatus
a. the stimulating electrode is connected to stimulating equipment
17. a train pulse is applied to the stimulating electrode
a. if no dopamine signal recorded in the nucleus accumbens, the stimulating
electrode is incrementally lowered and stimulation repeated.
18. once dopamine is recorded, the stimulating electrode is cemented
19. after cement hardens, the stimulating and reference electrodes are disconnected,
the sensing electrode withdrawn into the microdrive and the microdrive detached
from the hub
20. the hub is capped
21. the animal is removed from the stereotaxic apparatus and placed in postoperative
care on a heating pad
22. The animal is returned to housing when standing on all four paws
Ch. 4: Cocaine-self-administration training
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[I think we need to say more about this training in Book 2, Ch. 4: Dopamine neurons
and motivated behavior. In the first version of this lab, the user will not train any rat.
While I think it is important for the user to know what is required for training, I don't think
the user needs to complete this "Chapter" to meet the requirements of this lab. What do
you think?]
Training animals to self-administer cocaine is largely divided into “Manual Shaping’ and
“Autoshaping”. In manual shaping, the experimenter guides the animal to behavior
using the technique of successive approximation (or simply, reinforces behavior
increasingly similar to the wanted behavior). Manual shaping for cocaine selfadministration can but not always involve first shaping for food reward, which is far
simply (i.e., faster) because animals will bar press often for a food pellet (up to ~10
times a minute with a continuous reinforcement schedule – one lever press gets one
pellet). Because of pharmacokinetics and drug action, a rat will only lever press every 5
to 8 minutes for cocaine maximally. In contrast, autoshaping lets chance do the work,
with a little help of course. I don’t know enough to discuss the pros and cons of each,
but the caveat is that some people think that there are important differences between
shaping techniques. I would suggest not addressing this issue at all, but choose one or
the other, depending on which one is most convenient to incorporate. Both are lengthy,
taking several days to shape an animal to self-administer cocaine.
1) Cocaine-self-administration training – Option 1 – Manual Shaping
a) Note – this is a move generic manual shaping procedure applied to training for
cocaine self-administration that should be suitable for non-science students and
is consistent with the spirit of the technique
b) retrieve animal
i) check that hardware is secure and animal is healthy
(a) hardware failure will prevent subsequent dopamine measurement or
cocaine injection – reject animal
(b) unhealthy rat could die from cocaine injection – reject animal
(c) unhealthy rat could skew data – reject data
c) place rat in operant chamber
d) manually “prime” rat with one dose of cocaine
e) use method of successive approximation to train for cocaine self-administration
i) when rat is near the response lever (within 5 cm), manually infuse another
dose of cocaine
ii) when rat is reliably remaining within 5 cm of the response lever, alter required
distance to lever to 3 cm
iii) when rat is reliably remaining within 3 cm of response lever, alter criteria such
that rat must raise paw and hold above or on the response lever to receive
cocaine infusion
iv) when rat is reliably touching the lever, change criteria such that the rat must
apply pressure to lever to receive cocaine infusion
v) when the rat is reliably pressing lever itself and self-administrating, the rat is
now “shaped”
32
a. a typical cocaine self-administration session will begin with a priming dose
of cocaine, followed by three to four lever presses within the first few minutes
(“upload”) and finally a relatively stable lever pressing every 5 to 8 minutes
vi) N.B. In the real world, stable cocaine self-administration behavior may take
14-21 days of pretraining with this manual shaping paradigm (Carelli et al.,
1993, Brain Res. 626:14-22)
f) potential problems
i) be sure not to reinforce behavior which will be incompatible with bar pressing
(a) e.g., turning away from the bar is incompatible with pressing the bar,
so don't reinforce it.
ii) when you change your criterion, don't make too big a change. Otherwise,
extinction can occur.
(a) Extinction is a reduction in response rate which occurs when a
previously reinforced response is no longer reinforced.
(b) by far the most difficult transition to make is from "paws on bar" to
"pressing bar". It is best to use extremely small steps here.
iii) applying too many doses during training
(a) never exceed what a trained rat would normally self-administer
2) Cocaine-self-administration training – Option 2 – Autoshaping
a) From Lynch and Carroll, 1999, Psychopharmacology 144:77-82. Shaping will
take several days (10 to 20)
i) general design
(a) 12 hours of daily shaping, seven days a week
(b) first 6 hours, six one hour autoshaping periods
(i) lever extended and stimulus lights above levers illuminated for
variable length (mean 90 s)
(ii) lever retracted if animal presses lever or after 15 s, which ever
occurs first
(iii) cocaine injection with every retraction of lever or lever press
(iv) stop session after 10 cocaine infusions (13 to 15 min typically)
(c) second 6 hours, self-administration opportunity
(d) criterion – 100 level presses over five days of self-administration
33
Ch. 5: Experiment area lab procedures
[To be edited]
1) Recording dopamine during cocaine self-administration
a) retrieve animal
i) check that hardware is secure and animal is healthy
(a) hardware failure will prevent dopamine measurement or cocaine
injection – reject animal
(b) unhealthy rat could die from cocaine injection – reject animal
(c) unhealthy rat could skew data – reject animal
b) place animal in operant chamber
c) attach telemetry unit to animal
d) flush jugular injection line with saline
1. clogged line will not work
e) load microdrive with a dopamine microsensor
i) broken microsensor will not measure dopamine
f) remove protective cap from cemented hub
g) attach microdrive to hub
h) attach connections between telemetry unit and
i) reference electrode
ii) stimulating electrode
iii) microsensor
iv) jugular cannula
i) lower microsensor to the desired depth
i) if lower too fast, microsensor may break or become fouled and will not
measure dopamine
ii) if not lowered to correct depth, microsensor may not measure dopamine or
measure dopamine in the correct region (i.e., nucleus accumbens)
j) allow animal to habituate to the operant chamber
i) a stressed animal may behave erratically
ii) a stressed animal may have an adverse reaction to cocaine
k) allow microsensor to equilibrate in the brain
i) without equilibration, microsensor will perform erratically
l) perform test stimulation
i) check for functional microsensor
(a) if bad, will need to be replaced per above
ii) determine whether microsensor is placed in a dopamine-rich area
a. if signal if poor, incrementally lower microelectrode
iii) check for functional stimulating electrode
(a) if no outward response to test stimulation (slight head movement
followed by transient exploratory behavior), then there is a problem
(i) bad connection
(ii) bad stimulation unit
(iii) stimulating electrode has been moved – reject rat
iv) check for wireless communication
34
m)
n)
o)
p)
q)
r)
s)
t)
(a) could be bad
(i) low battery
(ii) telemetry home-base computer not functioning or program not setup properly
turn on video camera for recording behavior
once good dopamine “chemical signature” has been obtained from test
stimulation, begin the experiment to monitor dopamine during cocaine selfstimulation
session starts with extension of lever and illumination of light above lever
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
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
repeat test stimulation
1. check for functioning microsensor
analysis
i) are animals lever pressing adequately
(a) should be lever press every 5 to 8 min
ii) what is the behavior of animals between and during lever pressing
(a) stereotypy following cocaine injection
(b) end of stereotypy just prior to movement towards lever
iii) is dopamine released during session
(a) what behavior is associated with dopamine release
(b) what cues are associated with dopamine release
additional experiments
i) presentation of cocaine-associated visual and auditory cues
(a) trained rats versus untrained rats
(b) cue-evoked increase in dopamine only in trained rats
ii) effect of exogenous electrical stimulation (to release dopamine) on lever
pressing behavior
(a) electrical stimulation increases approach to lever and lever pressing
35