What are 3 types of experiments that need to be carried out in order to gain a causal and mechanistic understanding of brain function?
CORRELATION: electrophysiology (patch-clamp recordings in-vivo), optical imaging (fluorescent microscopy, fMRI)
CAUSAL: optogenetics where we perturb the activity of neurons!
MECHANICAL: computational modelling
Why does Karl Popper propose that hypotheses can only be falsified?
We can only improve on evidence; make it more significant. As a result, science only ever finetunes its understanding.
What similarities exist in the organisation of human and mouse brains?
- 99% genetically identical.
- 1g vs 1.5kg
- Regional organisation is similar
- L1-L6 in neocortex quite similar
What is the Cre-LoxP system and what is it good for?
Allows for the knock-out of particular genes. We surround the gene of interest with LoxP, the two LoxP combine in the presence of Cre-recombinase and ejects that gene.
Morris water maze can therefore be measured with and without gene, pre and post mutation
What is Translational Neuroscience?
Engineer a disease in a mouse, try to cure, then translate this to humans
How does two-photon microscopy allow high-resolution brain imaging?
- Red wavelength reduces the scattering of light in brain.
- Prob of receiving protons back once light is shone into brain is proportional to density^2
- The light we receive back is therefore spatially dependent.
What is calcium imaging? Why is it important?
- Fluorescence can be calcium sensitive in some Genetically-encoded calcium indicators (GECI), such as GCaMP proteins.
- Is relevant because each action potential in a neuron evokes an immediate calcium signal in the soma, because of the activation
of voltage-gated calcium channels. - Also relevant at synapses
What types of signals can be recorded with extracellular electrodes?
Neural firings. Voltage in EXTRAcellular space is not just constantly zero (there is 80% neurons in brain so lots of resistance in e.c. fluid).
The summation of the synaptic currents around the electrode results in a signal in the low-frequency range
(~ 0.5 – 100 Hz) called the local field potential (LFP) that is highly correlated to the subthreshold membrane
potential fluctuations of nearby neurons, but is of much smaller amplitude and reversed polarity.
How is it possible to record the membrane potential of a sparse cell-type in vivo?
- Drill the skull
- lower in a glass electrode…
- filled with Potassium solution
guide the recording pipette under 2-photon microscope to target a cell population labelled with a fluorophore. => 2-photon targeted whole-cell recording
Describe the difference between firing rates in cells.
Median = 0.1Hz, Mean = 1Hz.
Quieter behaviour usually corresponds to more in sync and slower activity in brain.
AP firing in neocortex is relatively asynchronous because (it is thought) that APs are mostly caused by the convergence of the few large unitary EPSPs in neighbouring cells.
What is channelrhodopsin-2?
Relevant for optogenetics:
- Blue Light-activated cation channel
- Results in fast inward current
- millisecond precision
- can work in vivo, probably requires two-photon excitation for same reasons as optical imaging
What optogenetic tools exist for inhibiting a targeted population of cells?
NpHR (Hallorhodopsin): 1 photon = pump in 1 x Cl-
Arch (Archaerhodopsin): 1 photon = pump in 1 x H+
iClC2: Cl- ion channel, opens with photon
Can be introduced using either viral approach or Cre-LoxP system
What is spike sorting?
Complex multi-electrode recording and signal processing approaches have been developed allowing for the extraction of single-neuron action potential signals from extracellular recordings
