Neuro Research- Final Exam

Horizontal plane

Dorsal and ventral

(Also referred to as superior and inferior)

Coronal planes

Anterior and posterior (front and back)

Saggital plane

Medial and lateral (relative to the midline of the brain)

Organization of encephalon in terms of complexity (high to low)

Prosencephalon (front)

Mesencephalon (middle)

Rhombencephalon (back)

Organization of encephalon in terms of plasticity (high to low)

Rhombencephalon

Mesencephalon

Prosencephalon

Components of prosencephalon, ceiling and floor

Telencephalon (cortex, basal ganglia, and limbic system (inc. hippocampus, amygdala, and septum))

Diencephalon (thalamus and hypothalamus)

Components of mesencephalon, ceiling and floor

Tectum (superior and inferior caliculi)

Tegmentum

Components of rhombencephalon, ceiling and floor

Metencephalon (cerebellum and pons)

Myelencephalon (medula)

Glands and location (2)

Pituitary (connected to the hypothalamus, although not directly part of neural tube)

Pineal (connected to thalamus by the hybenula)

Breakdown of the cortex, components and functions (5)

Cingulate cortex (connected to hippocampus and amygdala)

Lingual gyrus (important for vision)

Perehippocampal gyrus

Rectal gyrus

Insular cortex (viceral reactions, sense of comfort/internalness, bodily discomfort)

Broad functions of brain regions (bottom to top)

Myelencephalon: breathing/HR/respiration

Metencephalon: coordination/sleep cycles

Tegmentum: salience (dopamine)

Tectum: auditory reflexive/visual reflexive

Diencephalon: drives

Telencephalon: habits/emotions

Tools for imaging brain structure (4)

Cerebral Angiography

CT

MRI

DTI

Tools for imaging brain function (5)

fMRI

SPECT

EEG

MEG

fNIRS

Cerebral angiography

Tool for investigating brain structure that involves dying the blood and using blood vessels to learn about circulation in the brain

CT scan

Tool for imaging brain structure that uses X-ray to look at brain

Good for big things (ex: stroke, brain shrinkage, tumor), but too low of resolution to see smaller things

MRI

Tool for looking at brain structure that uses magnetic fields and the presence/absence of magnetic elements (iron)

Good for getting more detailed look at soft tissue of brain

DTI

Tool for imaging brain structure that involves the presence/absence of amounts of water — looking for presence of tracts to show where the connections and brain circuits are

Where water is, myelin isn’t (and vice versa)

fMRI

Tool for imaging brain function that employs repeated pictures over and over, traces the movement of iron

PET scan

Tool for imaging brain fuction that uses radioactive dyes to trace the movement of glucose

Glucose is a more precise indicator of cell activity than oxygen, but want to avoid repeated use due to radioactive substances

SPECT scan

Tool for imaging brain function that is a more precise version of a PET scan

EEG

Tool for imaging brain function that uses electrodes to trace waves of cellular activity in the presence of a particular stimuli (infer what brain is doing from it’s electrical activity)

MEG

Tool for imaging brain function that used magnetic detectors (simular to EEG)

fNIRS

Tool for imaging brain function that uses flashing light and sees how it is absorbed.

Light is absorbed through deoxygenated and oxygenated hemoglobin at different rates, so function can be inferred by where oxygen is being used

Tools for brain stimulation (4)

TMS

tDCS

ECT

DBS

TMS

Method of brain stimulation that involves pumping magnetic current into regions (generally used therapeutically)

tDCS

Method of brain stimulation that pumps electrical current into regions (generally used therapeutically)

ECT

Method of brain stimulation that involves using large amounts of electricity

Much less refined, and therefore less common today

DBS

Method of brain stimulation that involves placing electrodes deep inside the brain in order to prompt them to fire

Clinical applications for Parkinson’s, Huntington’s

Experimental design (5)

1. Purpose

2. IRB and IACUC

3. Task Paradigms

4. Gain/loss of function

5. Regions of interest (ROI)

Types of experimental purposes (3)

1. Diagnostic

2. Correlation

3. Experimental

Types of task paradigms (3)

1. Event-related

2. Blocked (chunks of time working on an activity

3. Mixed

Coordinate systems, types and importance

Standardize the pixels for different sized brains, etc.

1. Montreal Neurological Institute Template

2. Talairach

3. Brodmann

Steps for using animal model, guidelines

1. Selecting a model: replace/reduce/refine

2. Neuroethology: observing natural behavior

3. Housing: deprived vs. enriched

4. Validity: face, construct, predictive

5. Appropriate paradigm selection

Types of validity, definitions

Face (value): does it initially seem to measure what you want to measure?

Construct: is the build/environment suited to demonstrate x?

Predictive: does the experiment generate data to show that it is a good measure for x?

Behavioral animal model tools (4)

1. Locomotor

2. Running wheel

3. Homecage activity

4. Open field

Coordination animal model tools

1. Rotorod

2. Footprint pattern

3. Hanging wire/grip

4. Vertical pole

Sensory animal model tools (4)

1. Visual cliff

2. Startle response

3. Conditioned taste

4. Olfaction assay

Pain animal model tools

1. Tail flick

2. Hot plate

3. Formalin injection (for chronic pain)

4. Eyedrop/airpuff

Spatial learning animal model tools

1. Morris water maze (moving the underwater platform)

2. Barnes maze (circular table w/ holes, one of which tunnels)

3. VR maze

4. elevated plus maze

Stimulus detection animal model tools (7)

1. Operant/classical conditioning protocols

2. Novel objection detection/response

3. Go/no go task (discrimination)

4. Approach/avoidance

5. Escape

6. Self-administration

7. Real time/conditioned place preference

Social animal model tools (3)

1. Resident-intruder

2. Approach/avoidance

3. Mating protocols

Affective animal model tools

1. State (how they are) v. trait

2. Open field exploration

3. Learned helplessness (shock)

4. Restraint (repeated)

5. Forced swim (posolt test)

6. Tail suspension

Other animal models

1. Drosophila (fly)

2. C. elegans (worm)

3. Cichlid (fish)

4. Non-human primates

Stereotaxic surgery, definition and importance

Three-dimensional technique used to inject substances or implant devices into precise brain regions by immobilizing the animal’s head and using anatomical landmarks like bregma and lambda

Essential for experiments involving electrophysiology, neuroanatomical tracing, optical imaging, neuromodulation, and gene delivery

Steps for stereotaxic surgery with details (6)

1. Creating sterile environment (using autoclaves, bead sterilizers, sterile fields, and disinfectants for all tools and implants)

2. Anesthesia (typically ketamine/xylazine mix for short operations, or isoflurane gas for longer procedures; confirm depth of anesthesia before incision)

3. Head positioning (uses skull sutures and anatomical reference points to maintain consistent coordinates)

4. Craniotomy (dental drill used to expose the desired brain surface, something requiring durotomy for probe access)

5. Delivery/implantation (injection pumps allow precise reagent delivery; permanent implants (sealable chambers, cannulae, electrodes, optical fibers) are attached with dental acrylic)

6. Recovery (incision closure, cleaning, analgesic/antibiotic administration, observation for distress or complications during healing phase)

Types of implants, definitions (4)

1. Sealable chambers: used for chronic access to cortical areas

2. Cannulae: used for repeated substance delivery, including intracerebroventricular infusion

3. Electrophysiology implants: used for recording neural activity, including arrays and probes

4. Optical implants: used for imaging and optogenetics, using fibers, microendoscopes, or cranial windows

BOLD signal changes problem

Up to 40% of our assumptions about oxygen usage may be wrong.

fMRI technology doesn’t account for blood vessel expansion/shrinkage, so additional oxygen in an area may be due to expanded blood vessels rather than increased neural activity

Top-down v. bottom-up neuroscience

Researching what really matters is most important; investigating what we want even if we can’t have certainty about the details

Precision is most important; saying what we can even if we have to narrow our questions

Primary cells

Cells or tissues removed directly from a living animal rather than from dividing cell lines

Provide a more realistic physiological environment to study cell behavior, signaling, and disease models

Three types of primary cells

Slice cultures

Explant cultures

Dissociated cultures

Explant Cultures

Intact tissue fragments are submerged in media to study cell mixtures, neurite outgrowth, and migration

Dissociated cell cultures

Tissue is broken down into single cells on 2D/3D substrates

Isolated neurons retain their identity, grow processes, form synapses, and show electrical activity

Limitations of primary cells

Incapable of dividing indefinitely and produce a lower volume of cells

Not great for high-yield biochemical experiments

Stem Cells

Cells that have stopped dividing and are capable of generating any cell type, while continuously producing more stem cells

Types of stem cells (3)

Embryonic stem cells (ethical challenges)

Neural stem cells (can become neurons/astrocytes/oligodendrocytes)

Induced pluripotent stem cells (reprogrammed backwards)

Steps of tissue preparation

1. Fixation

2. Embedding

3. Sectioning

4. Clearing

5. Staining/labeling

Tissue fixation

Using chemicals to preserve, stabilize, and strengthen biological specimen

Can be immersed or perfused (delivered through cardiovascular system)

Tissue embedding

Tissue is surrounded by a protective shell in order to stabilize the structure

Tissue sectioning

Slicing brain into thin slices

Can use microtome (frozen brains and medium slice), cryostat (frozen brains and thin slice), or vibratome (non-frozen, thick slice)

Tissue clearing

Making tissue transparent (can use clarity technique to remove light-scattering lipids)

Stains for visualizing morphology (3)

Basophilic stain

Fiber stain

Golgi stain

In Situ Hybridization (ISH)

Visualizes nucleic acid expression (mRNA) to determine when are where a specific gene is expressed in the nervous system

Works by identifying sequence of mRNA and creating a probe with a complementary base-pair sequence. Tag the probe with radioactive nucleotides, and then expose the tissue of tagged probe

Fluorescent in situ hybridization (FISH)

Antisense probes are labeled with fluorescent dyes, and multiple probes can be used in the same tissue section to determine whether certain cells coexpress genes of interest

Immunohistochemistry (IHC)

Visualize expression of proteins by using antibodies to recognize and bind to specific proteins (antigens).

Can be direct (primary antibody binding) or indirect (secondary antibody amplifies signal of primary antibody)

Enzymatic histochemistry

Sample is incubated in chromogenic chemical solution, and enzyme presence is indicated by colored by-product

Reporter proteins

Fluorescent protein/enzyme that visibly marks cells of interest. Uses genetically encoded promoter sequences to target specific cells

Anterograde v. retrograde tracers

Transport from cell body to presynaptic terminal vs. transport from synaptic terminal back to cell body

Immediate Early Genes (IEGs)

a class of genes that activate rapidly and transiently within minutes to an hour of cellular or neuronal stimulation, without requiring de novo (new) protein synthesis

Both ISH and IHC can be used for IEGs

Pulse-chase labeing

Pulse (labeled component such as radioactive amino acid) and chase (unlabeled component so cell does not continuously label everything)

Thymidine Analogs

BrdU replaces thymine molecules in DNA synthesis, indicating whether a cell was dividing

Voltage-sensitive dye imaging (VSDI)

Different membrane potentials correspond to different fluorescence or absorption levels of dye

Calcium indicator dyes

Ratiometric dyes that emit different wavelengths when bound to calcium, and non-ratiometric dyes that emit different intensities of fluorescence

Fluorescence Resonance Energy Transfer (FRET)

Tool for imaging protein function that assigns emission spectrum to donor and absorption spectrum to acceptor

Bimolecular Fluorescence Complementation

Tool for imaging protein function that assigns each protein ½ of a fluorphore, so that two proteins together makes a whole fluorophore

Reasons that delivering genes to the brain is difficult

1. Neurons are post-mitotic, so can’t exploit nuclear envelope breakdown to transfect genes

2. Tight junctions, extracellular packing, and blood-brain barrier

3. Heterogenous brain tissue requires precise cell-type targeting

Microinjection

Tool for inserting genes that pierces cell membrane with a small glass needle

Useful for fertilized eggs and GMOs, but labor intensive and low throughput

Electroporation

Electrical pulse is used to transfect cells with DNA

Induces temporary pores in cell membrane, allowing DNA uptake

Low throughput

Biolistics

Particle-mediated gene transfer, shoots DNA fragments into cells

Useful for thick tissue, but can cause physical damage

Calcium phosphate transfection

Calcium choride is used to make DNA precipitate out of solution

Useful for cell cultures, but not for neurons

Lipid transfection

Lipid complex is used to deliver DNA to cells

Works in cell cultures, but nothing else

Adeno-Associated Virus (AAV)

Used to infect dividing and postmitotic cells to deliver DNA

Used for carrying CRISPR gene-editing tools

Labor intensive, but stable and useful long-term

Lentivirus

Used to infect dividing and post-mitotic cells

Uses RNA to produce cDNA that migrates to the nucleus and integrates

Useful for long-term gene expression

Canine adenovirus (CAV)

Used for introducing DNA into a cell, employs retrograde properties (presynaptic membrane to the cell soma)

Rabies Virus (RV)

Singled stranded RNA virus used to insert genes into a cell

Toxic, due to it’s high level of replication

Restriction enzymes

Useful for isolating DNA fragments; work by recognizing specific DNA and cutting it at specific recognition/restriction sites

Blunt ends v. sticky/cohesive ends

Straight cuts without overhang vs. staggered cuts with overhangs

Sticky ends are useful for allowing complementary ends to pair together

Polymerase Chain Reaction (PCR)

Involves separating DNA strands using heat, then hybridizing primers using cooling, than synthesizing DNA

Gel electrophoresis

Used to isolate, identify, and characterize properties of DNA fragments

Loads DNA into wells in a gel, then exposed them to electrical field such that they are pulled towards + end based on size

Vector

Nucleic acid storage system capable of carrying an isolated DNA sequence

Examples include plasmids and bacteriophages

DNA sequencing

Determining whether the nucleotide sequence is correct

Hybridization

Binding two complementary nucleic acid strands to each other

(Southern blot and northern blot)

Southern blot vs. northern blot

Identifying known DNA sequence vs. identifying known mRNA sequences

Monoclonal v. polyclonal antibodies

Same epitope (region of antigen recognized by an antibody) vs. different epitope of same antigen

Chromotography

Proteins separated into columns based on characteristics (size, charge, affinity)

Western blot

Tool for investigating protein expression that makes proteins soluble and negatively charged such that they move towards an electrode

Enzyme-linked immunosorbent assay (ELISA)

Measures low quantities of protein expression

Radioimmunoassay

Tool for investigating protein expression that has tagged and regular proteins in different concentrations, then uses binding curve to determine unknown concentrations

Tools for investigating protein-protein interactions

Co-immunoprecipitation

Protein affinity chromotography

Yeast two-hybrid analysis

Proximity labeling

Fluorescent visualization

Luciferase Assay

Tests whether a protein can activate or repress the expression of a target gene

Unable to determine whether DNA and the protein directly interact, but can establish a functional connection

Electrophoretic mobility shift assay (EMSA)

Detects whether a protein is able to interact with a short sequence of DNA