N371: in vitro approaches

Describe the framework for studying ANY behaviour?

Describe the behvaiour —> identify motor output —> identify motor neurons controlling that output —> localize brain region generating that behaviour —> determine map connectivity —> molecular properties of the system —> manipulate the system and hypothesis testing

What is the first step in studying any behavior?

Describe the behavior through observation. In vivo only

What types of data are collected when first describing behavior?

Observation, minor manipulation, correlative data

What is meant by identifying the motor output of a behavior?

Measuring the body’s physical responses. In vivo only

What techniques are commonly used to measure motor output?

EMGs (muscle) and ENGs (nerve)

Are EMGs and ENGs mechanistic or correlational?

Mostly correlational

What role do motorneurons play in behavior?

They execute behavior but do NOT generate it. Can be studied in vivo/in vitro

What methods are used to study motorneurons?

EMGs, ENGs, single-cell recordings, manipulation

How do we localize the brain region generating a behavior?

Lesions and stimulation in vivo or in vitro

Why are lesions/stimulation considered hypothesis testing?

They directly test causal mechanisms

What does mapping connectivity tell us?

How neurons in a network communicate

What does in vivo connectivity mapping show best?

Global connectivity

What does in vitro connectivity mapping allow?

Precise mapping of local connections

What kinds of properties are studied at the molecular level?

Ion channels, synapses, neuromodulators in vitro only

What techniques are used to study molecular properties?

Patch clamp, imaging, brain slices

What important rule applies to all in vitro molecular findings?

They must be verified in vivo

At what level do most cellular neuroscience questions sit?

Molecular / cellular level

Why is experimental design critical in neuroscience?

It guides the model, approach, and techniques. You must define the behaviou of interest/specific question before you choose methods!

What factors determine choice of animal model?

Long-term goal, question, tools, budget, ethics. Models must match the behavior and biology

Why are humans ideal for translational goals?

Direct relevance to human disease. Use limited due to ethical and practical constraints lol!

What methods are commonly used in humans?

Imaging, EEG, DBS, post-mortem tissue

Why are most human studies correlational?

Manipulative experiments are mostly not allowed

Why are rodents commonly used in neuroscience research?

They share ~99% of genes with humans and have powerful genetic tools + Extensive transgenic, optogenetic, and chemogenetic tools are available

Why might rats be preferred over mice for some experiments?

Rats are larger, making surgeries and implantations easier

Which species are most modern neuroscience tools optimized for?

Mice

What is the biggest advantage of in vitro experiments?

High experimental control since in vivo contains many uncontrolled variables (BP, arousal, breathing, hormones, temperature)

What is the main concern when interpreting in vitro results?

Physiological relevance + what happens if cells removed from their natural environments + gene expressnio drifting?

Why is synaptic relevance hard to determine in vitro?

Neurons normally receive thousands of inputs

As you move from acute slices to organoids, what becomes harder?

Identifying the cell type being recorded from

As in vitro models become more flexible (immortalized → iPSC → organoids), what generally happens?

Physiological relevance decreases and experimental flexibility increases

What are 2D cultures?

Cells grown as a monolayer in a dish

What are primary 2D neuronal cultures?

Neurons directly dissociated from real brain tissue

What ages can primary cultures/iPSCs be taken from?

Embryonic → adult

How long are primary neurons grown/studied?

Days to weeks

What are the advantages of primary 2D cultures?

Most “normal” physiology

Good for molecular and cellular questions

Can co-culture different cell types

What are the disadvantages of primary 2D cultures?

Need new batches → variability

Harder to maintain identity (gene expression changes)

Synapses present but don’t always reflect true behaviour

What are iPSCs?

Human skin or blood cells reprogrammed into stem cells. They can divide indefinitely and become almost any cell type

What are the advantages of iPSCs

Human-derived

Useful for disease modeling

Can control cell type with guided differentiation

What are the disadvantages of iPSCs

Very slow to generate

Cells are not fully identical to native neurons

Synapses form in vitro but are uncertain if “normal”. Can be guided or unguided

What is guided differentiation?

Forcing iPSCs to become one specific neuron type

What is unguided differentiation?

Cells spontaneously become many cell types

What are immortalized cell lines?

Engineered or cancer-derived cells that divide indefinitely. NOT true neurons but are human origin

What ages can immortalized lines originate from?

Any age

What are the advantages of immortalized cell lines?

Fast, easy, very reproducible

Excellent for ion channel structure/function studies

What are the disadvantages of immortalized cell lines?

Least physiological

Usually only 1 cell type

Often not representative of actual neural tissue

Some lines CAN form synapses, but have to consider: are they real synapses? Is signalling normal? Other questions

What defines a 3D culture?

Cells form tissue-like structures instead of a flat monolayer. They allow cell–cell interactions in all directions = closer to in vivo!

What are organoids?

Self-organizing 3D neural tissue grown from iPSCs. Develop multiple cell types and rudimentary brain-like structure

How long do organoids take to develop?

Months to form, but can be studied for months

What is the basic developmental sequence for organoids?

iPSCs → NECs (neuroepithelial cells) → NPCs (neural progenitor cells) → guided/unguided differentiation

What is guided differentiation in organoids?

Growth factors/small molecules force formation of one tissue type

What is unguided differentiation in organoids?

Spontaneous differentiation into heterogeneous tissues

What are the advantages of organoids?

Human-derived 3D tissue

Useful for developmental & disease modeling

Many cell types present

What are the disadvantages of organoids?

Diffusion limits = necrotic core (No vasculature)

Organization is only partially realistic

Very long prep time (months)

What are acute brain slices?

Thin slices of brain tissue kept alive in oxygenated solution

What ages can acute slices come from?

Embryo → adult (but best with young tissue)

What are the advantages of acute slices?

Most physiologically intact

Preserves spatial relationships and synaptic wiring

Ideal for studying synaptic function

Can see spontaneously active networks (young only!!)

Fast preparation

What are the disadvantages of acute slices?

No blood supply → diffusion limits

Short lifespan (hours)

Temperature kept below 37°C (physiology altered), needs 95% oxygen (unnatural and stressful)

What are organotypic slices?

Thin brain slices maintained in culture for weeks

What ages are organotypic slices taken from?

Embryo → neonate

How long do organotypic slices take to prepare? how long can they be studied for?

Weeks to months, can be studied for weeks

What are the advantages of organotypic slices?

Long-term study

Synapses and circuits more intact than 2D

Good for imaging and plasticity studies

What are the disadvantages of organotypic slices?

Still lacks blood supply

Cell types and gene expression may drift

Not identical to in vivo circuits

What is the goal of voltage clamp?

Hold membrane potential (Vm) constant and measure ionic current

How do voltage clamps work?

one internal electrode measures Vm and is connected to voltage clamp amplifiers (compares Vm to desired potential). If not at desired potential, amplifier ingests current to make them the same!

What is patch clamp?

A technique where a small piece of membrane is sealed into a glass pipette. Mosy commply used in 2D cultures

What is a cell-attached patch?

Pipette seals to membrane, membrane remains intact. Record ion channels in that small membrane patch

What is cell-attached patch best for?

Single ion channel recordings

What is an expression system?

Cells engineered to express ONE specific ion channel. Allow for clean, unambiguous channel analysis

How do you create an inside-out patch?

Pull pipette away → membrane patch detaches → the cytoplasmic side (inside) faces the bath

What is inside-out patch best for?

Studying intracellular modulation (Second messengers, kinases, Ca²⁺, phosphorylation)

How do you create an outside-out patch?

Go whole-cell → pull pipette back → membrane reseals → extracellular (outside) faces the bath

What is outside-out patch best for?

Studying extracellular modulation (Neurotransmitters, drugs, hormones)

What happens in whole-cell patch clamp?

The membrane patch is broken and the pipette interior becaomes continuos with the cell interior

Why can signaling be “weird” in whole-cell patch?

Loss of native intracellular signaling molecules

What is whole-cell patch best for?

Measuring all currents in the cell (Synaptic currents, firing patterns, emergent properties)

How does perforated patch differ from whole-cell patch?

Membrane is not fully broken

What is added to the pipette in perforated patch?

Pore-forming antibiotics so ions can pass though (not large molecules). This method preserves natural intracellular signalling