Cognitive Sciences Research Techniques Chapter 7
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Questions and Answers

A scientist can introduce a marker while the process occurs in live ______.

tissue

BrdU is a synthetic analog of the DNA base ______.

thymidine

Two biological functions commonly assayed include cell proliferation and ______ trafficking.

protein

IHC can be used to detect ______ in cells.

<p>BrdU</p> Signup and view all the answers

3H-thymidine can be detected using ______.

<p>autoradiography</p> Signup and view all the answers

Neural activity leads to the rapid transcription of immediate early ______.

<p>genes</p> Signup and view all the answers

These genes encode a diverse range of proteins, including transcription factors like ______.

<p>Fos</p> Signup and view all the answers

Cytoskeletal-interacting proteins such as ______ are also encoded by immediate early genes.

<p>Arc</p> Signup and view all the answers

Phosphorylated ribosomal subunits represented as ______ are part of the protein diversity from IEGs.

<p>pS6</p> Signup and view all the answers

IEG patterns can be used to screen neurons for the presence of activities that correlate with specific ______.

<p>behaviors</p> Signup and view all the answers

In the striatum and hippocampus, ______ expression is indicative of neuronal activity.

<p>Fos</p> Signup and view all the answers

The transcription of immediate early genes occurs ______ after neural activity.

<p>within minutes</p> Signup and view all the answers

IEGs provide insights into the molecular basis of ______ and learning processes.

<p>memory</p> Signup and view all the answers

Techniques for visualizing activity and function in fixed tissue include measuring neural activity using immediate early ______.

<p>genes</p> Signup and view all the answers

Static measures of activity and function can involve measuring cell proliferation with ______ analogs.

<p>thymidine</p> Signup and view all the answers

Dynamic neural activity can be visualized using voltage-sensitive ______ or genetically encoded voltage indicators.

<p>dyes</p> Signup and view all the answers

Calcium-sensitive dyes and genetically encoded ______ indicators are used to visualize dynamic neural activity.

<p>calcium</p> Signup and view all the answers

To visualize protein function, researchers may use techniques such as fluorescence resonance energy transfer (FRET) and ______ fluorescence complementation.

<p>bimolecular</p> Signup and view all the answers

Incorporating a marker into cells can indicate the presence of activity during subsequent ______ examination.

<p>histological</p> Signup and view all the answers

Static markers of neural activity involve measuring byproducts that accumulate during specific processes in active ______.

<p>neurons</p> Signup and view all the answers

Visualizing synaptic transmission can involve the use of FM dyes or ______ which monitor synaptic activity.

<p>synaptopHluorin</p> Signup and view all the answers

Nicotine-induced activation of the ______ nucleus is studied in relation to addiction.

<p>interpeduncular</p> Signup and view all the answers

The phosphorylation process is mediated by ______/MAPK-dependent phosphorylation.

<p>ERK</p> Signup and view all the answers

Voltage-sensitive ______ channels are integral to neural signaling.

<p>calcium</p> Signup and view all the answers

The habenular ______ receptor is implicated in nicotine addiction mechanisms.

<p>α5</p> Signup and view all the answers

Phosphorylation of SRF and ______ occurs via ribosomal S6 kinase (RSK).

<p>CREB</p> Signup and view all the answers

Knockout mice lacking the habenular α5 nicotinic receptor demonstrate altered responses to ______.

<p>nicotine</p> Signup and view all the answers

The effect of high nicotine doses was almost completely abolished in ______ mice.

<p>knockout</p> Signup and view all the answers

The habenula-IPN pathway is important in the regulation of ______ addiction.

<p>nicotine</p> Signup and view all the answers

Assaying cell proliferation often involves the use of ______ analogs.

<p>thymidine</p> Signup and view all the answers

One common method to analyze cell growth is through a ______.

<p>cell proliferation assay</p> Signup and view all the answers

BrdU is a type of ______ used in cell proliferation assays.

<p>thymidine</p> Signup and view all the answers

Cell proliferation can be measured in response to various ______.

<p>injections</p> Signup and view all the answers

Researchers often look for a gap in ______ to determine cell division rates.

<p>proliferation</p> Signup and view all the answers

Flashcards

Static Measures of Neural Activity

Techniques that indirectly measure neural activity by examining byproducts accumulated after activity has occurred.

Immediate Early Genes (IEGs)

Genes that are rapidly expressed in neurons following activation, serving as markers for recent neuronal activity.

Measuring Cell Proliferation

Techniques using thymidine analogs to track the division and growth of new neurons.

Protein Trafficking with Pulse-Chase Labeling

A technique that uses labeled molecules to track the movement of proteins within cells over time.

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Visualizing Dynamic Neural Activity

Techniques that directly observe changes in neural activity as they happen.

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Voltage-Sensitive Dyes

Fluorescent dyes that change their fluorescence intensity based on the voltage across the neuronal membrane.

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Genetically Encoded Calcium Indicators (GECIs)

Fluorescent proteins that change their fluorescence intensity based on calcium levels within the neuron.

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Synaptic Transmission Sensors

Molecules that detect changes in synaptic activity, like FM dyes and synaptopHluorin.

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Fos

A transcription factor encoded by an IEG, involved in regulating gene expression in response to neural activity.

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Arc

A cytoskeletal-interacting protein encoded by an IEG, involved in processes like synaptic plasticity.

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pS6

A phosphorylated ribosomal subunit encoded by an IEG, involved in protein synthesis.

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IEG Patterns

The unique combination of IEGs expressed in different types of neurons, indicating their activity.

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How IEGs Are Used

IEG expression patterns can be analyzed to study neural activity in response to specific stimuli or behaviors.

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Striatum

A brain region involved in reward processing, movement planning, and learning.

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Hippocampus

A brain region involved in memory formation and spatial navigation.

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Assaying Cellular Function in Fixed Tissue

A method to study cellular processes within preserved tissue by introducing a marker during an active process in live tissue, and subsequently detecting it in fixed tissue.

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Examples of Assayed Cellular Processes

Two common cellular processes assessed using marker-based methods in fixed tissue are cell proliferation (growth) and protein trafficking (movement within cells).

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What are BrdU and 3H-thymidine used for?

These are thymidine analogs, which are synthetic or radioactive molecules similar to thymidine, a DNA base. They are used to monitor cell proliferation.

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How is BrdU detected?

BrdU, a thymidine analog, is detected using immunohistochemistry (IHC).

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How is 3H-thymidine detected?

3H-thymidine, a radioactive thymidine analog, is detected using autoradiography.

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What is the role of SRF in gene expression?

SRF (Serum Response Factor) is a transcription factor that plays a crucial role in regulating gene expression, particularly in response to growth factors.

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How does RSK affect the Fos promoter?

Ribosomal S6 Kinase (RSK) is an enzyme that phosphorylates CREB, a transcription factor involved in the expression of the Fos gene. This phosphorylation activates the Fos promoter, ultimately leading to increased Fos expression.

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What are VSCC's?

VSCCs (Voltage-Sensitive Calcium Channels) are protein channels embedded in the neuronal membrane that open in response to changes in membrane voltage, allowing calcium ions to flow into the cell.

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Nicotine and the Habenular α5 nicotinic receptor

The Habenular α5 nicotinic receptor is a type of acetylcholine receptor found in the habenula, a brain region involved in reward and motivation. Nicotine binds to and activates this receptor, triggering downstream signaling events.

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What is the effect of habenular α5 nicotinic receptor knockout on nicotine addiction?

Mice lacking the habenular α5 nicotinic receptor (knockout mice) show reduced sensitivity to nicotine's rewarding effects, suggesting this receptor plays a crucial role in nicotine addiction.

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How does the habenula-IPN pathway regulate nicotine addiction?

The habenula-IPN pathway, involving the habenula and the interpeduncular nucleus (IPN), is crucial for regulating nicotine addiction. Activation of this pathway by nicotine contributes to its addictive properties.

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What happens with high doses of nicotine in knockout mice?

High doses of nicotine in knockout mice (lacking the habenular α5 nicotinic receptor) have a significantly reduced effect compared to normal mice, highlighting the importance of this receptor in nicotine's actions

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What are the implications of these findings for studying nicotine addiction?

Understanding the role of the habenular α5 nicotinic receptor in nicotine addiction provides insights into the mechanisms underlying this complex behavior and potentially informs strategies for developing new treatment approaches.

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Study Notes

Basics of Brain and Cognitive Sciences Research

  • Study guide for research techniques in neuroscience by Carter and Shieh.
  • Chapter 7, Sooyoung Chung.

Goal

  • Describe techniques for visualizing activity and function in fixed tissue.
  • Describe non-electrophysiological methods of measuring neural activity.
  • Describe techniques for visualizing protein function.

Techniques Covered

  • Static measures of activity and function: Measuring neural activity using immediate early genes, cell proliferation with thymidine analogs, and measuring protein trafficking with pulse-chase labeling.
  • Visualizing dynamic neural activity: Voltage sensors (voltage-sensitive dyes, genetically encoded voltage indicators), calcium sensors (calcium-sensitive dyes, genetically encoded calcium indicators), and synaptic transmission sensors (FM dyes, synaptopHluorin).
  • Visualizing protein function: Reporter genes, fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC), fluorescence recovery after photobleaching (FRAP), and photoactivation/photoconversion.

Neural Activity in Fixed Tissue

  • Static markers of activity:
    • Measuring activity indirectly by measuring byproducts that accumulate during specific processes in active neurons.
    • Incorporating a marker into cells to indicate activity during histological examination.
  • Assaying neural activity in fixed tissue:
    • Immediate early genes (IEGs) are transiently and rapidly transcribed following neural activity.
    • IEGs encode various proteins, including transcription factors (Fos), cytoskeletal-interacting proteins (Arc), and phosphorylated ribosomal subunits (pS6).
    • IEG patterns can identify neurons correlated with specific behaviors.
  • IEG and Neural Activity: Fos expression mediated by ERK/MAPK-dependent phosphorylation of SRF and phosphorylation of CREB.
  • Nicotine-induced activation of IPN (interpeduncular nucleus):
    • Habenula-IPN pathway is important in nicotine addiction regulation.
    • High nicotine dose effect almost completely abolished in knockout mice.

Assaying Cellular Function in Fixed Tissue

  • Measuring some functional processes in fixed tissue using markers introduced during live tissue, then detected in histological experiments.
  • Common assays include cell proliferation and protein trafficking.

Assaying Cell Proliferation with Thymidine Analogs

  • BrdU (bromo-deoxyuridine), a synthetic DNA base analog, or radioactive tritiated thymidine (3H-thymidine) can be used.
  • BrdU can be detected using immunohistochemistry (IHC) and 3H-thymidine through autoradiography.
  • Proliferation markers don’t reveal whether or not a cell becomes functional.
  • Additional IHC experiments for cell cycle or cell-type-specific proteins are important.

Dynamic Neural Activity

  • Visualizing neural activity relies on specialized fluorescent probes to detect changes in membrane potential, calcium concentration, or synaptic vesicle fusion.
  • Dyes tend to have better temporal properties and signal-to-noise characteristics than proteins.
  • Genetically encoded proteins can be targeted to specific cells, allowing observation of activity in defined circuits.

Imaging Voltage

  • Voltage-sensitive dye imaging (VSDI) is the primary method for visualizing voltage changes.
  • Genetically encoded voltage indicators (GEVIs) enable membrane potential imaging in specific cell types.

Voltage-Sensitive Dyes

  • Fluorescence changes according to membrane potential.

Imaging Calcium Dynamics

  • Intracellular calcium plays a critical role in physiological processes (e.g., neurotransmitter release, ion channel gating, and second messenger pathways).
  • Fluorescent calcium indicators (dyes & genetically encoded) exist to track calcium changes.

Calcium Indicator Dyes

  • Ratiometric dyes report changes in Ca2+ based on wavelength differences.
  • Non-ratiometric dyes report changes directly via intensity changes.

Genetically Encoded Calcium Indicators

  • Aequorin (jellyfish protein) emits light in response to calcium binding without external excitation.
  • GCaMP (GFP fusion protein) is a non-ratiometric GECI.

Imaging Synaptic Transmission

  • Fluorescent dyes and proteins identify synaptic vesicle activity for studying synaptic transmission.

Synaptic Vesicle

  • Description of the process of synaptic vesicle release from the presynaptic cell to the postsynaptic cell.

FM Dyes

  • Lipophilic styryl dyes that fluoresce when bound to membranes.
  • Used to track neurotransmitter release, vesicle recycling, and vesicle movement.

pH-Sensitive Fluorescent Proteins

  • Neurotransmitter release can be studied using synapto-pHluorins (pH-sensitive GFP mutants).
  • Synapto-pHluorins provide multiple rounds of vesicle release/recycling information (also synaptic transmission).

Visualizing Protein Function

  • Scientists use fluorescent probes to visualize protein activity and interactions.
  • Time-lapse imaging allows tracking subcellular protein localization and interactions.

Fluorescence Resonance Energy Transfer (FRET)

  • FRET monitors protein interactions by proximity.
  • Fluorophores are linked so if proteins approach closely, light energy transfers, resulting in a change in emitted light.

Bimolecular Fluorescence Complementation (BiFC)

  • BiFC splits a fluorescent protein into two fragments.
  • Recombination of fragments on interacting proteins results in fluorescence.

Fluorescence Recovery After Photobleaching (FRAP)

  • FRAP monitors protein mobility by photobleaching a region.
  • Measuring recovery in fluorescence intensity around the bleached region reveals proteins' diffusion, binding, or transport kinetics.

Photoactivation

  • Some fluorescent proteins require light activation to become fluorescent.

Photoconversion

  • Photoconversion changes the fluorophore's emission spectrum using light.

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Basics Of Brain And Cognitive Sciences Research PDF

Description

Explore the essential techniques for visualizing neural activity and protein function as outlined in Chapter 7 of Carter and Shieh's neuroscience study guide. This quiz covers both static and dynamic methods of measuring neural activity and methodologies for assessing protein function, providing a comprehensive overview for cognitive science research.

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