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Questions and Answers
A scientist can introduce a marker while the process occurs in live ______.
A scientist can introduce a marker while the process occurs in live ______.
tissue
BrdU is a synthetic analog of the DNA base ______.
BrdU is a synthetic analog of the DNA base ______.
thymidine
Two biological functions commonly assayed include cell proliferation and ______ trafficking.
Two biological functions commonly assayed include cell proliferation and ______ trafficking.
protein
IHC can be used to detect ______ in cells.
IHC can be used to detect ______ in cells.
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3H-thymidine can be detected using ______.
3H-thymidine can be detected using ______.
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Neural activity leads to the rapid transcription of immediate early ______.
Neural activity leads to the rapid transcription of immediate early ______.
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These genes encode a diverse range of proteins, including transcription factors like ______.
These genes encode a diverse range of proteins, including transcription factors like ______.
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Cytoskeletal-interacting proteins such as ______ are also encoded by immediate early genes.
Cytoskeletal-interacting proteins such as ______ are also encoded by immediate early genes.
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Phosphorylated ribosomal subunits represented as ______ are part of the protein diversity from IEGs.
Phosphorylated ribosomal subunits represented as ______ are part of the protein diversity from IEGs.
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IEG patterns can be used to screen neurons for the presence of activities that correlate with specific ______.
IEG patterns can be used to screen neurons for the presence of activities that correlate with specific ______.
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In the striatum and hippocampus, ______ expression is indicative of neuronal activity.
In the striatum and hippocampus, ______ expression is indicative of neuronal activity.
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The transcription of immediate early genes occurs ______ after neural activity.
The transcription of immediate early genes occurs ______ after neural activity.
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IEGs provide insights into the molecular basis of ______ and learning processes.
IEGs provide insights into the molecular basis of ______ and learning processes.
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Techniques for visualizing activity and function in fixed tissue include measuring neural activity using immediate early ______.
Techniques for visualizing activity and function in fixed tissue include measuring neural activity using immediate early ______.
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Static measures of activity and function can involve measuring cell proliferation with ______ analogs.
Static measures of activity and function can involve measuring cell proliferation with ______ analogs.
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Dynamic neural activity can be visualized using voltage-sensitive ______ or genetically encoded voltage indicators.
Dynamic neural activity can be visualized using voltage-sensitive ______ or genetically encoded voltage indicators.
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Calcium-sensitive dyes and genetically encoded ______ indicators are used to visualize dynamic neural activity.
Calcium-sensitive dyes and genetically encoded ______ indicators are used to visualize dynamic neural activity.
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To visualize protein function, researchers may use techniques such as fluorescence resonance energy transfer (FRET) and ______ fluorescence complementation.
To visualize protein function, researchers may use techniques such as fluorescence resonance energy transfer (FRET) and ______ fluorescence complementation.
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Incorporating a marker into cells can indicate the presence of activity during subsequent ______ examination.
Incorporating a marker into cells can indicate the presence of activity during subsequent ______ examination.
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Static markers of neural activity involve measuring byproducts that accumulate during specific processes in active ______.
Static markers of neural activity involve measuring byproducts that accumulate during specific processes in active ______.
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Visualizing synaptic transmission can involve the use of FM dyes or ______ which monitor synaptic activity.
Visualizing synaptic transmission can involve the use of FM dyes or ______ which monitor synaptic activity.
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Nicotine-induced activation of the ______ nucleus is studied in relation to addiction.
Nicotine-induced activation of the ______ nucleus is studied in relation to addiction.
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The phosphorylation process is mediated by ______/MAPK-dependent phosphorylation.
The phosphorylation process is mediated by ______/MAPK-dependent phosphorylation.
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Voltage-sensitive ______ channels are integral to neural signaling.
Voltage-sensitive ______ channels are integral to neural signaling.
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The habenular ______ receptor is implicated in nicotine addiction mechanisms.
The habenular ______ receptor is implicated in nicotine addiction mechanisms.
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Phosphorylation of SRF and ______ occurs via ribosomal S6 kinase (RSK).
Phosphorylation of SRF and ______ occurs via ribosomal S6 kinase (RSK).
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Knockout mice lacking the habenular α5 nicotinic receptor demonstrate altered responses to ______.
Knockout mice lacking the habenular α5 nicotinic receptor demonstrate altered responses to ______.
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The effect of high nicotine doses was almost completely abolished in ______ mice.
The effect of high nicotine doses was almost completely abolished in ______ mice.
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The habenula-IPN pathway is important in the regulation of ______ addiction.
The habenula-IPN pathway is important in the regulation of ______ addiction.
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Assaying cell proliferation often involves the use of ______ analogs.
Assaying cell proliferation often involves the use of ______ analogs.
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One common method to analyze cell growth is through a ______.
One common method to analyze cell growth is through a ______.
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BrdU is a type of ______ used in cell proliferation assays.
BrdU is a type of ______ used in cell proliferation assays.
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Cell proliferation can be measured in response to various ______.
Cell proliferation can be measured in response to various ______.
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Researchers often look for a gap in ______ to determine cell division rates.
Researchers often look for a gap in ______ to determine cell division rates.
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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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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.
