Lecture 8 - Research Methods PDF

Introduction to Behavioral Neuroscience PSYC 211 Lecture 8 of 24 – Research Methods (Chapter 1) End of midterm 1 material. Professor Jonathan Britt Questions? Concerns? Please write to [email protected] 1 MIDTERM 1 – Monday, Sept. 30th...

Introduction to Behavioral Neuroscience PSYC 211 Lecture 8 of 24 – Research Methods (Chapter 1) End of midterm 1 material. Professor Jonathan Britt Questions? Concerns? Please write to [email protected] 1 MIDTERM 1 – Monday, Sept. 30th First letter of Midterm 1 covers lectures 1-8. Room your last name It has 56 multiple-choice questions. A The exam will start at 4:05 pm (no admittance after 4:30 pm) B It should take about an hour, but you have until 5:45 pm. C D E F G Please take the test LEA 132 H I in your assigned room  J K L M N O Sit every other seat P Q MCMED 1034 R S LEA 232 T Bring a pencil and an eraser U V W ARTS W- X 120 Y Z 2  Quiz 1 – Online Now Be sure to take Quiz 1 on MyCourses, which covers lectures 2-5. This quiz is worth 5% of you class grade and is a good practice for Midterm 1. You can take this quiz as many times as you want. Only your highest grade is recorded. Full credit is awarded for correctly answering at least 21 out of 25 questions in one attempt (which corresponds to a grade of 17.64 or higher in the grade book on MyCourses). The quiz will be open until the end of the semester, but you should aim to get full credit on it before Monday, because at least ten of the midterm exam questions come directly from the Quiz 1 question bank. 3  The Meninges of the CNS The brain and spinal cord are wrapped by 3 protective layers of tissue called meninges (whereas the peripheral nervous system is only surrounded by regular connective tissue). Moreover, the CNS is encased in bone (skull and vertebrae), whereas the PNS is not. The 3 types of meninges that surround the CNS: a) The outer layer is dura mater. It is thick, tough, unstretchable tissue. b) The middle layer is the arachnoid membrane. Its web-like extensions (arachnoid trabeculae) create a soft, spongy layer that is filled with cerebrospinal fluid (the extracellular fluid of the CNS). c) The third layer is pia mater. This layer sits closest to the brain and is a bit like Saran-Wrap. Large blood vessels course through the subarachnoid space (arachnoid trabeculae). Smaller blood vessels (capillaries) branch off and dive into the brain to provide nutrients and oxygen. 4 Meninges in the Human Brain 5  THE VENTRICLES OF THE BRAIN The brain floats in cerebrospinal fluid (CSF). CSF is made from blood by tissue called choroid plexus, which are in each of the brain’s four ventricles (i.e., the interconnected hollow spaces in the center of the brain). The two large lateral ventricles sit underneath the cerebrum (cerebral cortex). The third ventricle lies between the two thalamic nuclei at the center of the brain. The cerebral aqueduct is a long, tube-like structure that connects the third and fourth ventricle. The fourth ventricle is in the hindbrain, between the pons and cerebellum. The central canal of the spinal cord connects to the fourth ventricle. CSF is made continuously and is fully exchanged about 4 times per day. It circulates around and into the brain providing nutrients and removing waste. CSF exits the CNS by passing through holes in the dura mater, where it is absorbed into the blood supply. 6 The Ventricular System of the Brain CSF flows over immune system cells in the dura mater before returning to the blood supply central canal of the spinal cord 7  Cerebral Cortex The cerebral cortex contains 4 lobes. Each lobe has a Primary Area (where information leaves/enters the cerebral cortex). Other areas are Association Cortex, where we interpret sensory information and plan movements. 8  OTHER FOREBRAIN STRUCTURES The basal ganglia and limbic system are subcortical structures; they sit beneath the cerebral cortex. 9  THE FOREBRAIN: BASAL GANGLIA The basal ganglia are a collection of nuclei in the forebrain (located beneath the lateral ventricles). As a circuit, they regulate intentional movements, motivation, reinforcement learning, and habits. Inputs to the basal ganglia come from all over the forebrain, especially from the frontal lobe. There is also a strong dopamine input from the midbrain. Some outputs of the basal ganglia descend to midbrain and hindbrain to regulate movement. Other outputs ascend to the cerebral cortex (via the thalamus) to regulate sensory processing and decision making. Many neurological (movement) disorders are associated with basal ganglia dysfunction. For example, Parkinson’s disease relates to the loss of dopamine signaling in the basal ganglia. 10  THE LIMBIC SYSTEM The limbic system is a collection of subcortical brain areas that regulate emotions and the formation of episodic memories. Its principal areas include the hippocampus, amygdala, and cingulate cortex. (Some areas of the cerebral cortex, thalamus, and hypothalamus that interconnect the hippocampus and amygdala are often considered to be part of the limbic system as well.) 11  THE FOREBRAIN: LIMBIC SYSTEM The cingulate cortex is a large area that overlies the corpus callosum. Cingulate means encircling. This region interconnects many limbic areas of the brain. The hippocampus and amygdala are hidden in the temporal lobe. The hippocampus is critical for explicit memory formation. The amygdala is critical for processing emotion, especially fear. (Don’t worry about other parts of the limbic system, such as the mammillary bodies, septum, and fornix.) 12 BRAIN DEVELOPMENT 13  BRAIN DEVELOPMENT A hollow, enclosed neural tube forms during the first month of human development in the womb. The first cells in this tube are neural progenitor cells. Up until the 8th week of development, these cells only undergo symmetrical cell division: each neural progenitor cell becomes two neural progenitor cells. Asymmetrical cell division starts around the 8th week of development. Over the next 3 months, when a neural progenitor cell divides, one of the daughter cells migrates away from the center of the neural tube. The next time that cell divides, it will produce either two neurons or two glia cells. By the end of the fifth month, there are 85 billion neurons in the human brain, the most we ever have. Many of these neurons die 14 before birth, seemingly because they can’t find a place in the network.  BRAIN DEVELOPMENT Neurogenesis Production of new neurons. - Neural progenitor cells produce neurons and glia after they undergo asymmetrical cell division. Human neurogenesis largely stops five months after conception when neural progenitor cells undergo apoptosis. - There may be a little neurogenesis in some adult mammals, but whether this occurs in humans is controversial. Apoptosis A process of programmed cell death that occurs in multicellular organisms. - Apoptosis is a highly regulated and controlled form of cell suicide that ensures a dying cell does not cause problems for its neighbors. - Human neural progenitor cells undergo apoptosis around the fifth month of development in the womb. This is the moment when (most or all) neurogenesis stops. 15  HOW TO STUDY THE BRAIN If you want to know what an area of the brain is good for, you should find someone who has brain damage in that specific area. Someone goes to the doctor with a problem. Maybe they had a stroke or a serious brain injury. The doctor will want to take a photo of their brain. 16  Photographing the Living Human Brain Computerised Tomography (CT scan) CT scans are relatively cheap and fast, but the resolution is not great for soft tissue like brain. A computer assisted X-ray procedure used to take a “photograph” of the brain. The patient places their head in the center of a large cylinder. An X-ray beam (i.e., high energy light) is projected through the head to an X-ray detector. The X-ray beam is delivered from all angles. A computer translates the information received from the X-ray detector into a series of pictures of the skull and brain. A series of CT scans from a patient with a lesion in the occipital parietal area 17  Photographing the Living Human Brain Magnetic Resonance Imaging (MRI) Uses a strong magnetic field and radio waves (instead of X-rays). A patient lies in a large cylinder, and a strong magnetic field is applied to the body, which causes the proton of every hydrogen atom in the body to orient in a particular direction (in line with the magnetic field). The orientation of these atoms (the direction they are facing) is inconsequential to the chemical reactions of life. While in the scanner, radio waves (i.e., low energy light) are administered to the body. This energy is absorbed by protons, changing the direction they are facing. But each proton immediately flips back to the position determined by the magnet. When this happens, the protons emit their own radio waves, which are detected by the scanner. By triangulating where radio waves came back from, the scanner provides an estimate of the relative density of hydrogen atoms throughout the body. Since hydrogen atoms are especially prevalent in fat (lipids), MRI provides a high spatial resolution, three- dimensional image of the brain, which is mostly fat (myelin). 18 Magnetic Resonance Imaging (MRI) Hydrogen atom protons (arrows indicate spin direction) 19 Photographing the Living Human Brain Magnetic Resonance Imaging (MRI) Normal MRI scans reveal the density of lipid molecules (primarily). This is because the settings of the magnet and the radiofrequencies used are optimized to detect the protons of hydrogen atoms in lipids. But, by adjusting the magnet and other variables, the MRI can be optimized to better detect the hydrogen atoms in water molecules. This approach is used for DTI imaging (on the next slide). 20  A VARIATION OF THE MRI TECHNIQUE Diffusion Tensor Imaging (DTI) an MRI technique that measures the direction and speed of the diffusion of water molecules used to identify axon tracts Colors indicate the direction of water molecule diffusion 21  Functional magnetic resonance imaging (fMRI) of spontaneous human brain activity. Oxygenated blood slightly distorts the magnetic field. We can infer the movement of oxygenated blood around the brain by rapidly collecting a series of images and measuring the movement of these magnetic field distortions over time. 22  Functional Magnetic Resonance Imaging (fMRI) fMRI uses a rapid series of MRI scans. The amount of oxygen in blood distorts the local magnetic field. With a series of MRI scans, it is possible to detect changes in blood oxygenation, which reflects blood flow and correlates with neural activity. When a brain area is active, blood flow to that region quickly increases (~5s lag). fMRI is popular because it doesn’t involve needles, surgery, or radioactivity. It provides both structural and functional information with decent spatial resolution (1 to 5 mm) and temporal resolution (several seconds). Researchers are now trying to modify the fMRI technique to measure fluctuations in neurotransmitter signaling. This approach uses “enzyme-activated magnetic resonance contrast agents,” which are molecules that distort the magnetic field differently when they bind to neurotransmitter. 23  Recording Neural Activity in the Human Brain Positron Emission Tomography (PET) PET scans involve injecting a person with a radioactive compound. Radioactive sugar molecules (like 2-DG) are commonly used to detect changes in energy use in the brain. 2-DG is similar to glucose, in that it is taken up by active cells in the body. However, 2-DG is not broken down as easily as sugar is, so it stays around for hours. The scanner identifies where radioactive 2-DG molecules are located over time. The main disadvantage of PET scanners is their operating costs. For safety reasons, the PET scan of human brain showing brain radioactive molecules are designed to decay at rest (top row) and when clenching rapidly (over hours), thus they have to be right fist (bottom row). 2- DG uptake is made on site the morning of the experiment. indicated by greater yellow/orange/red. 24  Recording Neural Activity in the Human Brain Positron Emission Tomography (PET) The PET approach of using 2-DG as a measure of neural activity has been superseded by fMRI, but researchers still use PET with other radioactive tracers. Below are PET images of radioactive L-Dopa given to healthy people and people with Parkinson’s disease. Radioactive L-Dopa is picked up by dopamine neurons, converted into dopamine, and released as normal. There are fewer dopamine neurons in the brains of people with Parkinson’s disease. PET is also used to measure changes in the expression levels of neurotransmitter receptors across weeks. These studies use radioactive drugs (agonists or antagonists). 25 Recording Neural Activity in the Human Brain Macroelectrodes An electroencephalogram (EEG) is a measure of electrical activity in the brain that uses macroelectrodes (metal discs) attached to the scalp. It records the summed population-level activity of millions of neurons. It can be used as a diagnostic tool, since specific patterns of EEG activity are associated with different states of consciousness, stages of sleep, and types of cerebral atrophy. Some typical EEG’s and their psychological correlates 26  HOW TO STUDY THE ANIMAL BRAIN If you want to know what an area of the brain is good for, lesion it in an animal and see how their behaviour changes. Experimental ablation (lesion study) involves the removal or destruction of a portion of the brain. The functions that can no longer be performed following the surgery are probably controlled by that brain region. 27  EXPERIMENTAL ABLATION: BRAIN LESIONS How do scientist create small lesions in the brain (without using a knife or icepick)? One approach is to burn the tissue… Radiofrequency Lesions Small lesions can be made by passing radiofrequency current through a metal wire that is insulated everywhere but the tip. This electric current produces heat that burns cells around the tip of the wire. The size and shape of the lesion is determined by the duration and intensity of the current. A downside to this approach is that axons just passing through will also be burned. 28  EXPERIMENTAL ABLATION: BRAIN LESIONS Excitotoxic Brain lesion produced by injection of a lesion glutamate receptor agonist, such as kainic acid. These drugs cause so much excitation (and calcium influx) that the affected neurons undergo apoptosis, whereas axons just passing through (fibers of passage) are usually spared. Sham “Placebo” procedure that duplicates lesion all steps of producing a brain lesion except for the step that causes extensive brain damage. Reversible A temporary brain “lesion” can be achieved by injecting drugs that block lesion or reduce neural activity in a given region. Common drugs include… - Voltage-gated sodium channel blockers (stops all action potentials) - GABA receptor agonists (which hyperpolarize cell bodies) 29  Recording Neural Activity The most direct measurements of neural activity are made with metal wires placed in the brain. Microelectrodes are thin metal wires with a fine tip that can record the electrical activity of individual neurons (known as single-unit recordings). They can be used in behaving animals to record every action potential from a given neuron. And with the newest microelectrodes, it is possible to record from hundreds of single neurons simultaneously. Microelectrodes are implanted in the brains of animals during stereotaxic surgery. The wires are connected A permanently attached set of to a socket on the animal’s head so that they can be electrodes, with a connecting ‘plugged in’ to a recording system at any time. socket cemented to the skull Chronic electrical recordings are made over an extended period of time. Acute recordings are made over a relatively short period of time (often during surgery when the animal is anesthetized). 30  Manipulating Neural Activity We often want to know how the activity of specific receptors or cell populations influence behaviour. Electrical stimulation Involves passing an electrical current through a wire inserted into the brain. This will affect everything in the area (cell bodies and fibers of passage). Very fast stimulation frequencies counterintuitively produce the same behavioral effects as lesioning the brain area. Chemical stimulation Is achieved with drugs. In rodents, drugs are often administered through a guide cannula (hollow tube) implanted in a particular brain region. Anesthetics can be injected to shut down all neural activity. Alternatively, receptor agonist/antagonist can be used, which should not affect fibers of passage (i.e., axons just passing through the area) since there are no neurotransmitter receptors in the middle of an axon. 31  Manipulating Neural Activity We have also developed ways to use light to depolarize and hyperpolarize neurons with millisecond precision. So, we can turn up or down the activity of specific cells or receptors in any given brain region or neural pathway. Optogenetics refers to the use of light to control neurons that have been made sensitive to light through the introduction of foreign DNA. This foreign DNA provides instructions to make light-sensitive proteins. Proteins that are activated by light are called opsins. The opsins we have in our eye are metabotropic receptors that operate with a 30-millisecond delay. The opsins we use to manipulate neural activity (optogenetic techniques) are often ion channels that open and close instantly in response to light. The first opsins that were used to manipulate neuronal activity were discovered in bacteria. Scientists are now intelligently designing and modifying opsins for research purposes. 32 Lots of different photosensitive ion channels evolved in bacteria and algae. A popular excitatory one, named channelrhodopsin-II (ChR2), is permeable to sodium ions. When activated with blue light, it depolarizes neurons, causing them to spike. Other opsins, such as IC++ (designed by humans), are inhibitory light-gated ion channels. They pass chloride and hyperpolarize neurons when activated by blue light. 33 Optogenetics With excitatory opsins such as ChR2, you can pulse light or leave it on to generate action potentials. With inhibitory opsins such as halorhodopsin, continuous light delivery can prevent action potentials. 34 Viral-Mediated Gene Delivery a hollowed-out virus A virus is type of DNA delivery system. Viruses normally replicate by injecting viral DNA into a host organism. Virus DNA contains instructions on how to make more virus. 35 Viral-Mediated Gene Delivery A virus is a small infectious agent that replicates by injecting its DNA into normal cells. Virus DNA is the instructions for how to make more virus. We know how to remove the DNA from a virus, which renders the virus “replication- deficient”. We can also add foreign DNA to a virus, DNA that encodes proteins we want a cell to express, like fluorescent proteins or optogenetic proteins. When a modified virus is injected into an animal’s brain, it will infect all the cells in the area. Some viruses infect cell bodies (e.g., AAV); others infect axon terminals (e.g., rabies). Once a virus gets its DNA into the nucleus of a cell, that cell will start to transcribe the viral DNA and make the associated proteins. Almost all lab-made viral constructs contain a section of DNA that encodes a fluorescent protein (e.g., GFP). Fluorescent proteins are used to later identify which cells got infected. To study the function of neurons, we use viruses to deliver DNA to them. We often make neurons express proteins that will change their resting membrane potential, or that will generate action potentials in response to light, or anything we can think of. 36 FLUORESCENT CALCIUM IMAGING THROUGH A BRAIN IMPLANTED FIBER OPTIC CABLE green fluorescent protein Researchers modified the fluorescent protein GFP (discovered in jellyfish), causing it bind calcium and fluoresce much brighter when it does. This protein is called GCaMP. Since a little calcium influx always occurs during action potentials (even in the cell body), monitoring GCaMP fluorescence is good way to measure neural activity (in cells made to express GCaMP protein). 37  What are the afferents? What are the efferents? 38  Tracing Neural Connections Retrograde labeling (tracing afferent axons) Anterograde labeling (tracing efferent axons) What brain areas send their axons here? Where do the axons from these cells go? Retrograde labeling is used to label the cells Anterograde labeling is used to label where that innervate (project to) a given region. axons from a particular location go to. Various chemicals such as fluorogold can Various chemicals such as PHA-L can be be used as retrograde tracers. used as anterograde tracers. Fluorogold is taken up by axon terminals PHA-L is taken up by cell bodies and and transported back to the cell body. transported down to axon terminals. 39 40  Stereotaxic Surgery Stereotaxic surgery is a surgical intervention that uses a stereotaxic apparatus. Using this device, we can put something into a very specific part of the brain. We use stereotaxic surgery to inject things into the brain, such as drugs, viruses, or tracers (dyes). It is also used to permanently implant things, like cannula, electrodes, or fiber optic cable. 41 Bregma: The junction where pieces of skull fuse together. Bregma is often used as a reference point for stereotaxic brain surgery. 42  Common Reasons for Stereotaxic Surgery: Stereotaxic surgery is commonly used for one-time injections of drug or virus to: Lesion a brain area (e.g., excitotoxic lesion) Lesion a specific type of cell in a particular brain area (e.g., inject a compound that specifically kills dopamine neurons) To change gene expression (e.g., to prevent a protein from being made or to deliver DNA that codes for a foreign protein). Changes in gene expression often involved viral-mediated gene delivery. Stereotaxic surgery is also used to: Implant a guide cannula (hollow tube) to allow for later infusions of drugs. E.g., temporary (reversible) lesions can be made by infusing a local anaesthetic (e.g., lidocaine) which blocks action potentials. Implant microelectrodes for stimulation or recording experiments. Implant fiber optic cables to allow for imaging or stimulation using optogenetic techniques. 43  MEASURING NEUROTRANSMITTER LEVELS How do we measure fluctuations in neurotransmitter levels in behaving animals? For example, how can we determine if there is a change in serotonin signaling in the amygdala as an animal moves around? The old-fashioned approach: microdialysis The modern approach: man-made fluorescent reporter proteins 44 MEASURING SIGNALING MOLECULES IN THE BRAIN Microdialysis used to be a popular technique for measuring changes in neurotransmitter levels in a brain region in behaving animals. Dialysis refers to the use of a semipermeable membrane to either deliver molecules to or measure the amount of molecules in some solution (or brain area). Microdialysis probe Small metal tube that holds dialysis tubing, which can be placed in an animal’s head. It takes time for the concentration of molecules to equilibrate across dialysis membrane, so the fastest sampling rate possible is once per minute. More typically the sampling rate is once every 10 minutes. 45  MAN-MADE FLUORESCENT RECEPTORS Scientist have started to get good at designing new proteins, such as receptors that become fluorescent when bound to neurotransmitter. Scientists now use viral-mediated gene delivery to get neurons to express man-made fluorescent sensors, which allow us to visualize neurotransmitter release in a living brain. In this video, a few neurons were made to express a man-made fluorescent glutamate receptor. These receptors fluorescence whenever glutamate binds to them. Scientists have started to make fluorescent reporters for all the different neurotransmitters. 46  IMMUNOHISTOCHEMISTRY I may not have time to cover the remaining slides, but I encourage you to read them if you are interested in how scientists identified which cells in the body and brain express the different proteins encoded in the genome. 47 Immunohistochemistry: A method to label proteins and peptides in slices of biological tissue (non-living). The most common technique for identifying which cells make a specific protein is called immunohistochemistry. This technique takes advantage of antibodies, which are proteins made by the immune systems of mammals. Antibodies, by nature, are designed to selectively bind to a single type of protein. Researchers have made fluorescent antibodies that selectively bind to all different types of proteins. When these fluorescent antibodies are washed over a brain slice, the protein of interest will be fluorescently labeled, and under a microscope it is easy to identify which cells contain these proteins. Immunohistochemistry is used to identify protein expression patterns around the body and brain, which in turn tells us where in the brain each of the neurotransmitters are released and which cells contain the relevant receptors. 48  IMMUNOHISTOCHEMISTRY The job of our immune system is to destroy infectious agents that may harm the body. One way the immune system recognizes infectious agents is by looking for foreign proteins (i.e., proteins that aren’t made by the host animal). Proteins that are recognized as foreign by the immune system are called antigens. The immune system destroys antigens (and all invading organisms that express antigens), so it must be careful not to mistake normal (self) proteins for antigens. One type of cell in the immune system is tasked with generating novel proteins. These novel proteins are immediately destroyed if they bind to any self proteins. But if they survive that screening process, the immune system keeps them around to see if they bind to any foreign proteins (i.e., antigens). If they selectively bind to an antigen, they will be mass produced by the immune system to be used as antibodies (proteins that bind selectively recognize antigens, not self proteins). Because the immune system has evolved to continuously generate novel proteins (antibodies) that selectively recognize foreign proteins (antigens), we can use the immune system of an animal to generate antibodies for us that selectively bind to a protein (from another species) that we are interested in studying. 49 You don’t need to know these details  IDENTIFYING THE LOCATION OF PROTEINS IN THE BRAIN USING IMMUNOHISTOCHEMISTRY The adaptive immune system is exceptionally complex, but it generally works in this way: 1) Some cells of the immune system have instinctive ways to identify invading organisms. When they do, they phagocytose (swallow) them and digest the proteins into small pieces. Some of these pieces might be good antigens (proteins foreign to the host animal). 2) The immune system then “presents” this collection of potential antigens to T cells. There are millions of unique T cells in the immune system; each one randomly rearranges a portion of its DNA so it can create a novel protein, one that just might happen to bind to an antigen with perfect specificity. T cells are doing this all the time, even when no antigens are present. T cells are destroyed if they make a novel protein that binds to an endogenous (self) protein. 3) The novel protein that a T cell makes and expresses on its membrane is called an antibody. Most antibodies (and consequently most T cells) won’t actually be useful for anything, but sometimes a T cell happens to make the perfect protein (antibody) that binds to an antigen (foreign protein) and does not bind to anything normally found in the body (no self proteins). 4) When the antibody of a T cell selectively binds to an antigen (a successful match!), it triggers the immune system to mass produce this antibody. 5) Invading organisms soon get covered with these antibodies, which is the sign that they are foreign, don’t belong in the body, and should be destroyed. 50 You don’t need to know these details How to make an antibody for a specific protein (like a serotonin receptor) to label that protein in a slice of brain: Synthesize (or purify) the protein you want to label (e.g., the mouse serotonin receptor 5HT2B). Attach (conjugate) this protein to a known antigen, which is simply a protein that is known to elicit a large immune response in a mammal (such as rabbit). This step is necessary to ensure the protein will be recognized as foreign. Inject a mammal (rabbit) with this combo protein (the antigen conjugated to the serotonin receptor). Wait for the animal’s immune system to create antibodies that target this foreign combo protein. These antibodies will bind to different parts of the serotonin receptor-antigen combo protein. Draw the animal’s blood and extract the antibodies that bind to your protein of interest (i.e., the serotonin receptor). Attach (conjugate) these purified antibodies to a dye or fluorescent marker. Now you have a fluorescent antibody that can be used to label your protein of interest in a brain slice. So, simply wash this antibody over a brain slice and all the serotonin 5HT2B receptor proteins will become visible. 51 You don’t need to know these details  IMMUNOHISTOCHEMISTRY While it is relatively easy to make an antibody that binds to a specific protein, it is often not possible to make an antibody that selectively binds to a small molecule, such as an individual amino acid or classical neurotransmitter. To identify cells that make and release classical neurotransmitters, researchers often use antibodies that bind to the enzymes that make these neurotransmitters. For example, cells must make serotonin if they plan to release it. To do so, they must express the enzyme tryptophan hydroxylase, which converts the amino acid tryptophan into serotonin. Therefore, the antibodies that label tryptophan hydroxylase are used to identify serotonin neurons. 52 Abrupt changes in neural activity often trigger changes in gene expression. Immediate Early Genes are genes that tend to be expressed following periods of elevated spiking activity. c-Fos is an immediate early gene. When a neuron experiences a sharp increase in spiking activity, levels of c-Fos protein become elevated in the nucleus within minutes. c-Fos protein levels can be measured in brain slices using immunohistochemistry to identify neurons that were highly active the hour or two before the animal died. 53