Methods in neuroscience -Stephany
Content
1. introduction to electrophysiology ...................................................................................................2
Intracellular recording.....................................................................................................................3
Patch clamp recording ....................................................................................................................4
EEG .................................................................................................................................................5
Spikes .............................................................................................................................................7
2. Anatomy and connectomics ............................................................................................................8
Tracers ............................................................................................................................................9
Supragranular Labeled Neurons .................................................................................................... 11
Brain function theories.................................................................................................................. 12
Electron Microscopy ..................................................................................................................... 13
3. Imaging ......................................................................................................................................... 14
Two-photon microscopy ............................................................................................................... 15
Electron microscopy (EM) ............................................................................................................. 15
Structural imaging ......................................................................................................................... 17
Functional imaging ........................................................................................................................ 18
Two photon imaging in visual neuroscience .................................................................................. 20
4. Optogenetics l ............................................................................................................................... 21
5. Optogenetics ll .............................................................................................................................. 27
6. Behavioural experiments ............................................................................................................... 33
Open field (locomotion) test .......................................................................................................... 35
Morris water maze ........................................................................................................................ 36
Footprint pattern assay ................................................................................................................. 36
Conventional approach to behavioural testing: home cage testing ................................................. 36
Behaviour recognition ................................................................................................................... 37
7. Genetics for neuroscience ............................................................................................................. 38
Gene delivery methods.................................................................................................................. 38
Viral methods ............................................................................................................................ 39
Transgenic strategies ..................................................................................................................... 40
Gene knock-outs ........................................................................................................................... 43
,1. introduction to electrophysiology
• Extracellular: electrode is placed in the extracellular fluid in the proximity of the neuron.
Advantage: Link neural activity and behaviour.
Disadvantage: You don’t know specifically from which neuron the spikes comes from.
• Intracellular: electrode is inserted into the neuron.
Advantage: Specifically from one neuron.
Disadvantage: Damage of the membrane.
• Patch clamp: electrode makes a seal with a membrane patch.
Advantage: Specific from neuron-neuron interactions
Disadvantage: technically difficult.
Properties neurons:
• Depolarization: shift of the membrane potential from rest (-70mV inside the cell) towards less
negative values
• Hyperpolarization: shift of the membrane potential towards more negative values.
• Synaptic potentials: excitatory (depolarizing; EPSP) and inhibitory (hyperpolarizing; IPSP) post
synaptic potentials.
• Spikes: a regenerative process causing a membrane potential short pulse and initiating synaptic
transmission processes.
Configurations for electrophysiology
• in vitro: a slice or isolated brain portion is kept alive in a dish, typically under a microscopy setup.
• Acute in vivo: animal is anesthetized or immobilized under a microscope/micromanipulator.
• Chronic/freely moving in vivo: animal is implanted with a miniaturized micro-drive which contains
electrodes.
Components of electrophysiology
• Microelectrode: the transducer between biophysical currents (ionic) and currents in an electronic
circuit.
• Headstage: mechanical placement of electrodes (micromanipulator, microdrive) and first
amplification stage.
• Cables: must be shielded to protect from noise!
• Amplifier: low-noise, 100x-2000x gain.
• Digitization: translate analog signals into digital data.
• Visualization/storage: Computer, oscilloscope.
Microelectrodes
• Glass pipettes: mostly for intracellular/patch clamp
• Metal wires: insulated, thin, good plates wires, sharp or blunt tip.
• Silicon probes: C-MOS micro fabricated.
,Noise insulation
• electrical signals are tiny (microvolts, nano, pico amps).
• Electrical noise (50 Hz line noise) may swamp actual signals.
• Other electrical noise: cable movement artifact, electromyographic.
• differential recording: signal from a nearby electrode in a “neutral” brain location is subtracted.
• Cable shielding, Faraday cages.
• Active headstage, first amplifier stage on animal’s head, to increase signal before passing through
cables.
Digitization
• Sampling rate (must be at least twice the maximum frequency that we want to record) typically 20
30 kHz.
• Analog depth: the number of bits used to encode the signal: e.g. 16 bits: can output 2^16=65536
different values.
• A/D range: the difference between the lowest and highest voltage that can be sampled (saturation
outside of this range).
Integrated chips have been developed that do amplifying, digitizing, multiplexing on the animals
head
• Cut cost and complexity of (multi-channel) ephys systems.
Intracellular recording
• Voltage-Clamp: a feedback circuit is used to inject current in the neuron to keep the voltage
constant at a desired level.
• Neural dynamics can be analysed by measuring the current flowing through the electrode.
• I/V curve.
, Patch clamp recording
Content
1. introduction to electrophysiology ...................................................................................................2
Intracellular recording.....................................................................................................................3
Patch clamp recording ....................................................................................................................4
EEG .................................................................................................................................................5
Spikes .............................................................................................................................................7
2. Anatomy and connectomics ............................................................................................................8
Tracers ............................................................................................................................................9
Supragranular Labeled Neurons .................................................................................................... 11
Brain function theories.................................................................................................................. 12
Electron Microscopy ..................................................................................................................... 13
3. Imaging ......................................................................................................................................... 14
Two-photon microscopy ............................................................................................................... 15
Electron microscopy (EM) ............................................................................................................. 15
Structural imaging ......................................................................................................................... 17
Functional imaging ........................................................................................................................ 18
Two photon imaging in visual neuroscience .................................................................................. 20
4. Optogenetics l ............................................................................................................................... 21
5. Optogenetics ll .............................................................................................................................. 27
6. Behavioural experiments ............................................................................................................... 33
Open field (locomotion) test .......................................................................................................... 35
Morris water maze ........................................................................................................................ 36
Footprint pattern assay ................................................................................................................. 36
Conventional approach to behavioural testing: home cage testing ................................................. 36
Behaviour recognition ................................................................................................................... 37
7. Genetics for neuroscience ............................................................................................................. 38
Gene delivery methods.................................................................................................................. 38
Viral methods ............................................................................................................................ 39
Transgenic strategies ..................................................................................................................... 40
Gene knock-outs ........................................................................................................................... 43
,1. introduction to electrophysiology
• Extracellular: electrode is placed in the extracellular fluid in the proximity of the neuron.
Advantage: Link neural activity and behaviour.
Disadvantage: You don’t know specifically from which neuron the spikes comes from.
• Intracellular: electrode is inserted into the neuron.
Advantage: Specifically from one neuron.
Disadvantage: Damage of the membrane.
• Patch clamp: electrode makes a seal with a membrane patch.
Advantage: Specific from neuron-neuron interactions
Disadvantage: technically difficult.
Properties neurons:
• Depolarization: shift of the membrane potential from rest (-70mV inside the cell) towards less
negative values
• Hyperpolarization: shift of the membrane potential towards more negative values.
• Synaptic potentials: excitatory (depolarizing; EPSP) and inhibitory (hyperpolarizing; IPSP) post
synaptic potentials.
• Spikes: a regenerative process causing a membrane potential short pulse and initiating synaptic
transmission processes.
Configurations for electrophysiology
• in vitro: a slice or isolated brain portion is kept alive in a dish, typically under a microscopy setup.
• Acute in vivo: animal is anesthetized or immobilized under a microscope/micromanipulator.
• Chronic/freely moving in vivo: animal is implanted with a miniaturized micro-drive which contains
electrodes.
Components of electrophysiology
• Microelectrode: the transducer between biophysical currents (ionic) and currents in an electronic
circuit.
• Headstage: mechanical placement of electrodes (micromanipulator, microdrive) and first
amplification stage.
• Cables: must be shielded to protect from noise!
• Amplifier: low-noise, 100x-2000x gain.
• Digitization: translate analog signals into digital data.
• Visualization/storage: Computer, oscilloscope.
Microelectrodes
• Glass pipettes: mostly for intracellular/patch clamp
• Metal wires: insulated, thin, good plates wires, sharp or blunt tip.
• Silicon probes: C-MOS micro fabricated.
,Noise insulation
• electrical signals are tiny (microvolts, nano, pico amps).
• Electrical noise (50 Hz line noise) may swamp actual signals.
• Other electrical noise: cable movement artifact, electromyographic.
• differential recording: signal from a nearby electrode in a “neutral” brain location is subtracted.
• Cable shielding, Faraday cages.
• Active headstage, first amplifier stage on animal’s head, to increase signal before passing through
cables.
Digitization
• Sampling rate (must be at least twice the maximum frequency that we want to record) typically 20
30 kHz.
• Analog depth: the number of bits used to encode the signal: e.g. 16 bits: can output 2^16=65536
different values.
• A/D range: the difference between the lowest and highest voltage that can be sampled (saturation
outside of this range).
Integrated chips have been developed that do amplifying, digitizing, multiplexing on the animals
head
• Cut cost and complexity of (multi-channel) ephys systems.
Intracellular recording
• Voltage-Clamp: a feedback circuit is used to inject current in the neuron to keep the voltage
constant at a desired level.
• Neural dynamics can be analysed by measuring the current flowing through the electrode.
• I/V curve.
, Patch clamp recording
