Neuroscience

Cyber Technology

Laboratory Manual

Terence Bazzett

SUNY Geneseo

2013

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Table of Contents

Chapter 1: Organelle Staining

Introduction:

A wide range of cell staining techniques are used in Neuroscience. Simple staining techniques can be used to visualize neuronal cell bodies, neuronal projections, or glial cells. More sophisticated techniques can also be used to visualize functional elements of cells. In particular, most organelles within the cell can be individually stained allowing researchers to visualize, and in some cases quantify, the integrity of intracellular structures.Characterizing changes within neurons during the progression of neurodegenerative diseases can be invaluable for verification of a diagnosis. Furthermore, if those changes are found to be contributing factors to the degenerative process, they may become a starting point for testing treatments to slow or halt progression of an illness. As such, research designed to elucidate organelle changes in degenerative disorders is a potentially valuable form of applied research in Neuroscience.Read the following article, paying particular attention to the Introduction and Discussion sections, which provide the rationale for the methods used. Within the Experimental Procedures section focus on the Immunocytochemistry section which gives a brief summary of the methodology required for staining of the Golgi apparatus and the mitochondria within the neuron.

Background reading:

Nakagomi,Barsoum, Bossy-Wetzel, Sütterlin,VivekMalhotra, and Liptona (2008).A Golgi fragmentation pathway in neurodegeneration.Neurobiology of Disease, 29, 221-231.

ERIN resource:

Authors: Invitrogen

Topic / Subtopic: B. Neural Excitability, Synapses, and Glia: Cellular Mechanisms; I. Neuroanatomy / I.05. Anatomical Techniques Neuroanatomy

Resource Type: Image - Photograph, Simulation

Educational Level: 1 Introductory undergraduate, 2 Intermediate undergraduate, 3 Advanced undergraduate

Listing Date: October 23, 2012

Although the images are not from a neuron, this clever and realistic simulation allows students to stain specific cellular structures using fluorescent antibodies. Up to four structures can be stained simultaneously. Stains can be deleted so that other structures can be stained instead, a flexibility that encourages exploration. Cellular structures include the membrane, cytoskeletal components, mitochondria, the nucleus, Golgi, ER, and others. Using real images rather than diagrams makes the simulation especially persuasive.

Lab exercise:

Consider the immunocytochemistry methods described by Nakagomi et al. Use the resource link above to select appropriate stains for this laboratory preparation. Note: you do not need to use the same stains cited in the Nakogomi et al. article, but you must select stains that would be appropriate for your sample.

Student quiz questions:

  1. When is Golgi fragmentation anticipated and possibly even functional within the cell?

Mitosis

  1. Nakagomi and colleagues cite Gonatas et al., 2006 when noting that Golgi fragmentation is believed to be associated with neuronal degeneration in what two disorders?
  1. Alzheimer
  2. ALS
  1. Using the immunocytochemistry methods of Nakagomi et al., approximately how much time would you anticipateneeding to complete the staining procedure used in their paper?
  1. 5 minutes
  2. 15 minutes
  3. 1 hour
  4. 14-16 hours
  5. 16-19 hours
  1. In Nakagomi et al. fragmentation was quantified after Golgi staining. What was the effect of NMDA on the fragmentation process?
  1. Increased fragmentation
  2. Decreased fragmentation
  3. Had no effect on fragmentation
  1. In your own words, describe how the image of the Golgi apparatus from the control group differs from that of the image of the Golgi apparatus from the NMDA group.

The staining is more diffuse (fragmented) in the NMDA image.

  1. What is general effect of NMDA on cell activity?
  1. Increases activity
  2. Decreases activity
  3. Has no effect on activity
  1. In the experiment by Nakagomi et al., NMDA exposure was used to mimic which of the following? (circle all that apply)
  1. Effects of a neurodegenerative disorder
  2. Excitotoxicity
  3. Neuroprotection
  4. Neuronal sprouting
  5. Mitosis
  1. Which green Golgi stain(s) available from Invitrogen would be appropriate to use for this experiment?

Golgin-97 is the only stain for fixed tissue as describe in the article.

  1. Which orange-red mitochondrial stain(s) available from Invitrogen would be appropriate to use for this experiment?

The two Oxphos stains are the only stains for fixed tissue as described in the article.

  1. What doe Nakagomi and colleagues suggest can be accomplished by inhibiting Golgi fragmentation such as that seen in response to NMDA stimulation?

They believe it candlayapoptiotic-like cell death

  1. Generate a useful, concise question from the background reading for this Chapter.
  1. Generate a useful, concise question from the cyber laboratory exercise for this Chapter.

Chapter 2: Epigenetics

Introduction:

There has been long standing debate about the relative importance of genetics versus the importance of environment on phenotype expression. Since the origins of this “nature vs. nurture” debate, many researchers have argued vehemently that the one or the other of these factors reigns as most influential. In particular, the famous behavioral psychologist John B. Watson is oft cited for his 1930 statement promoting the importance of environmental influences:

"Give me a dozen healthy infants, well-formed, and my own specified world to bring them up in and I'll guarantee to take any one at random and train him to become any type of specialist I might select–doctor, lawyer, artist–regardless of his talents, penchants, tendencies, abilities, vocations and race of his ancestors"

More recently, there seems to be a growing consensus among researchers that, for most behaviors, it may be difficult to determine a comparable difference in contributions of genetics and environmental influences. Perhaps more importantly, researchers are now finding that an equally influential factor might, in some cases, be the effect of a an interaction between these two elements. Epigentics is the study of modifications to the genome that do not involve a change in the nucleotide sequence. Such modifications are brought about by variables in the environment in which the genome resides. Such variables may, in turn, be directly influenced by the environment in which an organism resides, behaviors of the organism, experiences of the organism, and so on.

Background reading:

Roth and Sweatt(2011).Annual Research Review: Epigeneticmechanisms and environmental shaping of thebrain during sensitive periods of development. J Child Psychology and Psychiatry 52, 398–408.

ERIN resource:

Authors: Genetic Science Learning Center, University of Utah

Topic / Subtopic: A. Development / A.02. Neurogenesis and Gliogenesis, A.03. Postnatal Neurogenesis, A.06. Synaptogenesis and Activity-Dependent Development; C.Disorders of the Nervous System / C.16.Cognitive, Emotional, and Behavioral State Disorders; E. Homeostatic and Neuroendocrine Systems / E.01.Neuroendocrine Processes, E.03. Behavioral Neuroendocrinology, E.04. Autonomic Regulation, E.05. Stress and the Brain; F. Cognition and Behavior / F.02.Language; G. Novel Methods and Technology Development / G.07. Computation, Modeling, and Simulation

Resource Type: Animation, Simulation

Educational Level: 1 Introductory undergraduate, 2 Intermediate undergraduate

Listing Date: March 22, 2012

A series of web links to readings and interactive videos describing epigenetics. Links provide information directly related to the assigned reading. Emphasis is on developmental periods in animals, but information about lifespan changes and human research is also included.

Lab exercise:

After completing the background reading, begin by watching the video “The Epigenome at a Glance” followed by the interactive lab “Gene Control”. These two exercises will give you some basic information on epigenomics. Next, view the video “Insights from Identical Twins” and finish the lab by completing the interactive lab entitled “Lick Your Rats”. In this exercise, take the time to lick several rat pups with a range of vigor. Begin with what you feel would be a “normal” level of licking. Then try another pup with very vigorous licking. Finally, try a pup with only a few licks. See what differences occur in the epigenome, and listen to the explanation given for your licking activity in each case.

Student quiz questions:

  1. From the Roth and Sweatt (2011) article, a rat model of an “abusive” caretaker, is described. In this model, what behaviors exhibited by mother rats toward their pups that are considered, and quantified as, abusive.

Frequent stepping on, dropping, dragging, actively rejecting, rough handling.

  1. From the Roth and Sweatt (2011) article, rats raised by mothers who displayed high levels of pup licking/grooming had significantly less methylation of glucocorticoid receptor DNA in their ____ than did rats raised by mothers who displayed low levels of pup licking/grooming.
  1. Amygdala
  2. Hypothalamus
  3. Hippocampus
  4. Thyroid gland
  5. Entire brain
  1. have been found to show decreases in which of the following?
  1. Glucocorticoid receptor mRNA
  2. Dopamine receptor mRNA
  3. Cell number
  4. Tissue volume
  5. All of the above
  1. From the movie, The Epigenome at a Glance, you learned that epigenome does which of the following?
  1. Changes the DNA code
  2. Alters the sequence of genes within the DNA
  3. Stimulates DNA self-replication
  4. Shapes the physical structure of the DNA
  5. All of the above
  1. While the epigenome in identical twins may initially be nearly identical, factors throughout life can create significant differences between the epigenome of twins as they age. As a result, while identical twins may continue to share physical features established during early development, behavioral characteristics tend to be less similar over time. From the laboratory video, list the four categories of environmental factors that were cited as potentially contributing to such epigenomic changes.
  1. Diet
  2. Physical Activity
  3. Toxins
  4. Stress
  1. From the interactive lab Gene Control, you learned that growth of cancerous tumors is mediated in part by out of control cell growth that has two primary effects on gene coding. Describe those two effects in very basic terms.

Out of control growth leads to cancer by both turning off genes coding for proteins that slow cell growth, and turning on genes coding for proteins that speed up cell growth.

  1. From the interactive lab Gene Control, list the effects on the following factors when you increase and decrease the external signal using the control knob.

Stimulation

Increase Decrease

GFP Gene

Structurecoils/tightensuncoils/loosens

GFP mRNAIncreasesDecreases

GFPIncreasesDecreases

Cell imageBrightensDims

  1. List from highest percentage (1) to lowest percentage (5), the traits listed below that may be shared by identical twins.

2 Autism

4 Bipolar disorder

1 Height

3Hypertension

5Stroke

  1. In adulthood, rats that were vigorously licked as pups tend to have more GR protein in the hippocampus. This is particularly important because GR protein binds circulating ______released during stressful periods, and helps to reduce the brain’s stress response resulting in a calmer, generally more adaptive, animal.
  1. Dopamine
  2. Glutamate
  3. Cortisol
  4. Adrenaline
  5. Oxytocin
  1. After completing the Lick Your Rat interactive lab, briefly describe the difference in appearance of the epigenome in a pup that was licked vigorously versus a put that received very little licking. Also describe the primary behavioral difference that will be seen between these two rats as a result of this licking, when they become adults.

Vigorous Licking Little Licking

Epigenome appearance Loosely coiledTightly coiled

Adult Behavior Easily relaxes Does not relax easily

  1. Generate a useful, concise question from the background reading for this Chapter.
  1. Generate a useful, concise question from the cyber laboratory exercise for this Chapter.

Chapter3: Pedigree Mapping

Introduction:

Understanding inheritance patterns of disorders is one of the first, and most basic, steps in establishing genetic influences that may cause, or contribute to a disorder. Generating a family pedigree chart allows researchers to summarize information about inheritance patterns over several generations. The pedigree chart is also an easily interpreted visual aid that offers a concise summary of basic and essential information. In simple terms, a pedigree chart is a great example of the old adage “a picture is worth a thousand words”. In reviewing a simple pedigree chart, researchers can identify, at a glance, family members as living or deceased, male or female, affected or unaffected, and so on. Pedigree charts are generally considered a staple for studying qualitative traits, such as single gene disorders. However, they also provide useful information for studying quantitative traits, including diseases that are influence by multiple genes. With the advent of the Human Genome Project, and the subsequent increase in the prevalence and utility of pedigree charts, the National Council of Genetic Counselors assembled a PedigreeStandardization Task Force (PSTF)in the early 1990s and charged its members with creating a universal coding system of symbols to be used in pedigree charts. The standardization of pedigree symbol usage helped legitimize the use of these charts for medical and research purposes. Today, regardless of the area of neuroscience, the pedigree chart is a useful if not essential tool for understanding inheritance patterns, co-morbidity, genetic contributions and more.

Background readings:

Ahearn, Steffens, Cassidy, Van Meter, Provenzale, Seldin, Weisler, Krishnan (1998).Familial leukoencephalopathy in bipolar disorder.American Journal of Psychiatry, 155, 1605-7.

Bennett, Steinhaus, Uhrich, O'Sullivan, Resta, Lochner-Doyle, Markel, Vincent, & Hamanish (1995).Recommendations for standardized human pedigree nomenclature.American journal of human genetics, 56, 745-52.

ERIN resource:

Authors: Genetic Science Learning Center, University of Utah

Topic / Subtopic: B. Neural Excitability, Synapses, and Glia: Cellular Mechanisms / B.01. Neurotransmitters and Signaling Molecules, B.02. Ligand Gated Ion Channels, B.03. G-Protein Linked Receptors, B.06. Neurotransmitter Release, B.07. Synaptic Transmission; C.Disorders of the Nervous System / C.17.Drugs of Abuse and Addiction, C.18.Behavioral Pharmacology; H. Teaching, History and Societal Impacts of Neuroscience / H.03. Public Awareness of Neuroscience

Resource Type: Animation

Educational Level: 1 Introductory undergraduate, 2 Intermediate undergraduate, 3 Advanced undergraduate

Listing Date: March 22, 2012

This web site is expansive, with many related web links. You are encouraged to browse about some of the other links when your assignment is complete. There are audio links, some video links of related lectures, several animated exercises (including the Pedigree Investigator lab assigned for this chapter), and links to other related sites.

Lab exercise:

Read the Ahearn et al., article as background information and to help you understand how familial studies are conducted and how pedigree charts can be used to represent results. You do not need to read the Bennett et al. article, but instead you should use it to help develop a greater understanding of the depth and utility of pedigree charts. Understanding the possible complexity of such charts is important for this lab assignment, since Ahearn et al., and the cyber lab both use highly simplistic representations that may give an overly simplistic impression of this area of research. After reading the Adhearn et al. article and reviewing Bennett et al., visit the web link listed above and from there the Pedigree Investigator link. Read all of the information associated with the Pedigree Investigator lab exercise and view all interview videos. After completing the Pedigree Investigator lab exercise, return to the original web link page, and listen to both audio recordings by Dr. Glen Hanson.

Student quiz questions:

  1. Adhearn et al. (1998) note the use of SCID in their research. What does SCID stand for?

Structured Clinical Interview for DSM III-R

  1. From the Ahearn et al. (1998) article, it was noted that Dupont, Jernigan and Butters were the first to report white matter hyperintensities in a large number of young patients diagnosed with bipolar disorder. In what year was that early report published?
  1. 1980
  2. 1985
  3. 1990
  4. 1995
  5. The year of that publication is not known
  1. In reviewing the pedigree chart from Adhearn et al. (1998), indicate the following:

Number of males showing Unipolar Disorder symptoms __3___

Number of deceased females showing Bipolar symptoms __1__

Number of living females showing Unipolar Disorder symptoms__4__

Number of sets of twins evaluated__0__

  1. What was the earliest age of Bipolar Disorder symptom onset for a participant in this study? At what age was that participant interviewed for this pedigree. Is that participant male or female?

Age of onset __8 years old__

Age at time of interview __20 years old__

Sex of participant __Female__

  1. When gathering information from a client about severe combined immune deficiency syndrome (SCIDS) you are told that she had elected to have an earlier pregnancy terminated when it was discovered through a genome analysis that the fetus she was carrying tested positive for SCIDS. With consideration to Bennett et al. (1995), what would be the appropriate symbol to use in your pedigree chart to note this pregnancy termination if the fetus was female? What would be the symbol used if the fetus was male? (note, symbols can be created using the Insert Shapes function of your Word program).

Female Male

  1. In some cases, dizygotic twins may closely resemble each other in physical appearance, making it difficult to determine if they are dizygotic or monozygotic. Actresses Mary Kate and Ashley Olsen, for example, are often believed to be monozygotic twins, but are reportedly dizygotic. Without genetic testing to positively establish twin status, pedigree charts may necessarily include some level of uncertainty. With consideration to Bennett et al. (1995) what would be the appropriate way to represent twin sisters if zygote status is unknown? Also indicated that one of the sisters is affected and the other unaffected for the condition of interest.