- Bacteria / yeast cells

- Cell culture

- Tissue slice

- Invertebrates

(In vitro = in glass, isolated parts of organisms)

- More complex animals

- Humans

(In vivo = in living, alive cells or organisms)

- Cheap housing / maintenance / breading

- Easy to handle (very friendly!)

- Quite intelligent and agile

- Resistant to infection

- Many inbred strains availible

- A lot of background data

- Mechanical lesions: actually cutting the brain

- Electrolysis: using electrical pulses to locally destroy small regions of tissue

- Chemical lesions: using chemicals (like ibotenic acid) to destroy cells, fibers of passage are spared

- Systemic: inject into bloodstream or muscles (chemical will go everywhere)

- Intracerebroventicular: surgery, release substance into cerebrospinal fluid (chemical will spread throughout CSF and across brain)

- Local intracerebral: injection straight into the target area (chemical will affect target area)

- Under the skin

- Slow and controlled release of chemicals

- Electrophysiology (electrical)

- Microdialysis (chemical)

- Behavioural evaluation (tasks)

- Histology: assessing quantity of cellular components (like NMDA receptors) in slices of tissue using staining techniques

- Immunohistochemistry: specific kind of histology to detect chemical components (like serotonin)

- In situ hybridisation for mRNA or DNA: specific kind of histology to visualise genetic material (like mRNA)

- Using a device that allows you to keep the head still and perform surgery at a precise location

- Lesions

- Microinjections

- Cannula placements (cannula = small tube)

- Microdialysis

- Electrodes

- Headset (made of electrodes)

You insert a small catheter (tube) that contains a physiological salt solution and has a semi-permeable (allows gas/liquids to pass through) outer membrane.

Extracellular substances can flow into the tube, substances in the tube can flow out. The dialysate (outflow)/perfusate (inflow) is collected for later analysis.

 [image]

Fill in the red boxes.

 1) Perfusate, inflow, physiological salt solution (contains as many salt molecules as intracellular fluid)

2) Dialysate, outflow, contains analyte (substance being analysed) 

- Continuous monitoring of chemical events in living tissue

- Continuous drug delivery

- Simultaneous drug delivery and monitoring of drug effects

- Single ion channel

- Single unit (one cell)

- Multi unit

- Field potentials (synchronous activity of many cells)

- Patch-clamp technique

Cell-attatched mode: A micropipette is attached to the cell membrane. Suction is applied through the pipette to draw a piece of the membrane into the microelectrode tip.

Whole-cell mode: With more suction, the patch of membrane can be displaced. Allows for stable intracellular recording.

Perforated-patch technique: Tries to prevent intracellular fluid of the cell mixing with the solution inside the electrode. This is done by withdrawing the membrane from the rest of the cell. Allows for pharmacological analysis of membrane patch. 

- Patch-clamp technique

Cell-attatched mode: A micropipette is attached to the cell membrane. Suction is applied through the pipette to draw a piece of the membrane into the microelectrode tip.

Whole-cell mode: With more suction, the patch of membrane can be displaced. Allows for stable intracellular recording.

Perforated-patch technique: Tries to prevent intracellular fluid of the cell mixing with the solution inside the electrode. This is done by withdrawing the membrane from the rest of the cell. Allows for pharmacological analysis of membrane patch. 

[image]

- Use a sharp electrode that penetrates the cell membrane

- Records voltage/current over cell membrane

- Like single ion channel recording, here too you use a pipette that applies suction to the cell membrane, but the pore is much smaller.

- Place an electrode close to the cell

- Will record activity from one neuron 

- Reference electrode is grounded

- This is how all recordings of single neurons in conscious animals is done (think Hubel and Wiesel's cat measurements)

- If the tip is slightly larger --> multi-unit signals

- Records changes in activity of brain areas

- Field potential: activity generated by simultaneous activation of many neurons 

- Recorded using multiple electrodes with larger tips

- Synapse releases neurotransmitter onto postsynaptic cell

- Current sink is generated (net flow of current inward and outward gets more negative)

- Extracellular electrode detects this as negativity with respect to ground (2)

- Intracellular electrode records change in membrane potential, inside of cell becomes more positive (1)

[image]

- Postsynaptic potentials of cortical neurons

(Not ionic currents that generate fast action potentials)

Source:

- Extracellular ionic currents caused by dendritic electrical activity

- Always reflects summation of syncronous activity of thousands/millions of neurons with similar spatial orientation

- Measures oscillating potentials

- Blood-oxygen-level-dependent imaging

- Measures flow of oxyhemoglobin-rich arterial blood towards 'active' brain areas

- Sensitive to differences between two brain states / regions

- So not 'absolute'

- Modulatory activity

- Brain states

- Transgene: foreign DNA inserted into host organism

- Transgenetic organism: organism transformed with transgene

- Foreign DNA exists in the cell in the form of a plasmid

(Common in prokaryote hosts, unicellular organisms with simple cell structure, like bacteria)

- Foreign DNA is integrated into the host genome

(Always the case in mammals)

1. Isolation of gene of interest (this creates a PCR fragment)

2. Modification of the gene

3. Insertion of the gene into a vector

4. Insertion of the vector into organism's cells 

5. Test to isolate genetically modified organisms

1) Loss of function experiments:

Alter genes to cripple a function

2) Gain of function experiments:

Increase function of gene (via extra copies of increased synthesis)

3) Tracking experiments/expression studies:

- When and where is a protein produced?

- Fuse a gene with a reporting element (green fluorescent protein)

- Reintroduce this gene into organism, the GFP catalyses the production of a dye, allows us to see when and where proteins are produced

- Old-school: inject DNA and hope it integrates into their genome

- Not very effective, transgene may disrupt function of some gene critical to survival/development

- New-school: transfer new DNA into embryonic stem cells

- Allows for precise genetic modification

- Works for only one particular mouse strain 

- When DNA strands split, one DNA strand can pair with the 'guest' strand