Transcript Cons

Techniques in Electrophysiology
What you are expected to gain from this lecture:
1. Approaches
2. In-vivo vs. in-vitro preparations
3. Advantages & Pitfalls
4. Types of Measures
5 Common Ephys Approaches:
1. EEG
2. Extracellular/Local Field Potentials
3. Intracellular – Sharp Electrode
4. Patch-Clamp Configurations
5. Multi-Unit Array Recordings
EEGs
Recording spontaneous brain (voltage volume conductance) activity from the
scalp, described in rhythmic activity: Delta (<4 Hz), theta (4-7 Hz), gamma (30100 Hz)
Clinical Neuroscience: epilepsy, coma, tumors, stroke,
focal brain damage, depth of anesthesia
Coordinate cortical activity = high contribution
Deep structure activity = low contribution
Application to Cognitive Psychology:
Evoked Potentials: time lock of EEG to presentation
of stimuli
Event Related Potentials: average of EEG over many
trials of higher processing conditions
(e.g., memory, attention)
N1 or P3 = coma recovery
Typical Slice/Culture Ephys Rig
Patch-Clamp Electrophysiology
Apply positive pressure (2-6 MΩ)
Clear tissue as you move down
Near cell membrane > ‘bubble’
Apply negative pressure > suction
until 1 GΩ seal
4 Common Patch-Clamp Configurations
Cell-Attached
Whole-Cell
Suction
Pull
Quickly
Inside-Out
Binding
Site?
Pull
Slowly
Outside-Out
>1 GΩ seal – going ‘whole-cell’ does not compromise the seal: prevents
leak current & extracellular buffer from entering the neuron
Perforated Patch Recording
~20-30 min
~10-15 min
start
Back-filling – nystatin, gramicidin, or amphotericin B (antibiotic/antifungal)
– creates pores for select ions to pass
Pros: Prevent dialysis of the intracellular contents & current run-down,
used for hard to patch cells
Cons: slow, high access resistance, weak membrane which leads to
whole-cell configuration
Voltage vs. Current Clamp
Voltage Clamp: holding the cell at a predetermined value (e.g., -70 mV)
the amount of current (e.g., mA) required to maintain that value
is recorded
voltage-dependent K+ channels, spontaneous EPSCs
Cons: Space Clamp (i.e., inability to adequately maintain holding command in
distal dendrites) & washout of cytosolic factors in whole-cell
sEPSC
Somatic current injection
producing AP firing
Current Clamp: can be used to measure the ‘resting membrane potential’
current is injected into the cell to maintain a predetermined
membrane potential (e.g., -80 mV)
the injected current is constant and free fluctuations in the membrane
potential are recorded
AP waveform, plasticity of EPSPs, intrinsic excitability
Local Field Potentials - fEPSPs
SA = stimulus artifact
A.
Stimulation
* = presynaptic fiber volley – presynaptic activity
generated by stimulation
B.
fEPSP = field excitatory postsynaptic
potential
PS = somatic population spike – coordinated
spiking activity
The initial slope of the fEPSP (mV/ ms) in the s.r.
is a widely used measure in LTP studies
PS
A.
B.
SA
*
fEPSP
Intracellular/Sharp Recording
Intracellular recording – used ‘sharp’ glass electrodes with > 25 MΩ
resistance
(#1) records the change in membrane potential that the incoming
current causes
(#2) fEPSP without a clear presynaptic fiber volley
Single Channel Recordings
Cell-attached (CA), inside-out (IO), and outside-out (OO) patches
Patch typically contains one or a few channels
Measure channel open probability, open time at different voltages or in the
presence of a test compound
CA: stable (>20GΩ seal), lowbackground but less control over
holding potential
IO: access to intracellular sites &
signaling pathways, difficult to obtain,
must replace bath solution from
external to internal
OO: repetitive & different doses, but
less stable, disruption of cytoskeleton
Preparations
1. Acute slices
2. Organotypic cultures
3. Dissociated cultures
4. Cell Lines
5. In vivo
Acute Slices
Widely used technique
Usually from adolescent rodents, coronal sections
Used the day they are made
Best to do cardiac perfusion to maintain slice viability
Buffer must be oxygenated and at the correct pH/osmolarity
Pros: treatments can be done in vivo, numerous brain regions can be
prepared, slices are not too excitable, can combine ephys with confocal
imaging, versatile (voltage or current clamp, fields, intracellular, plasticity, etc)
Cons: difficult to get viable slices in adult rodents, confound of recordings in
adolescents …translatation to adults, afferents are severed, there are changes
in instrinsic excitability over the day of recording, bath application of drugs
Organotypic Slice Cultures
Multiple brain regions (hpc, co-cultures) grown on porous membrane inserts
Prepared from 2-8 day old rodent pups
Maintained for months
Helios Gene Gun – can be used to
load gold particles coated with cDNA
into cells on the day of culturing to
change protein expression
Dissociated Cultures
Typically prepared in low- or high-density from embryonic or <24 h old pups
Hippocampal, Cortical, Striatal cultures are common
Cultured Primary
Dissociated Neurons
Autaptic/Microisland
Acutely Dissociated Neurons - the neurons preserve their dendritic structure proximal to
the soma, maintain intact synaptic boutons, and are largely devoid of glial ensheathments.
Cultures
Pros: Self-cleaning after insult during preparation, highly controllable experimental
conditions, ease & success of growing & maintaining, can be used almost
anytime, gene gun & lentiviral expression is easy, combine with imaging, focal
drug application & whole-cell currents in dissociated neurons, glutamate
uncaging/calcium transients in dendritic spines (dissociated neurons), versatile
(current & voltage clamp, fEPSPs, etc)
Cons: Thin over time, loss of afferents (except hpc), developmental differences,
contamination, highly excitable (transections), dissociated neurons don’t have
intrinsic networks or glial cells, de novo expression of excitatory connections
Cell Lines
HEK 293 Cells
Xenopus oocytes
PC-12 Adrenal Cells
Pros: excellent for answering certain ?’s
Express select proteins
Point mutation studies
Model system for neuronal differentiation
Cons: Non-mammalian , non-CNS cells
Lack complete neuronal constituents
(e.g., signaling complexes)
In Vivo Recordings
Performed under anesthesia or in freely-moving rodents
Intra- & extra-cellular, whole-cell, single or multi-unit array recordings
Network Properties: Can stimulate in one region and record in another
(e.g., mPFC influence on NAc plasticity)
Phase locking to brain rhythms
(e.g., mPFC neurons & hippocampal theta)
In Vivo Recordings
Lee et al., 2006, Neuron, v51, p399
In Vivo Recordings
Multi-unit Array Recordings
Pros: recording from an in vivo situation, network activity, population &
single cell activity, phase locking of gamma & theta rhythms,
correlation of neuronal or network activity with ongoing behavior,
becoming more common
Cons: Technically difficult, confound of anesthesia, application of
mathematics to isolate data, probes are time-consuming to fabricate
Data, data, data
AP: waveform, peak, half-width, AHP, frequency, back-propagating AP
Subthreshold excitatory postsynaptic potentials: LTP, LTD
Current-Voltage relationships: Mg unblock of NMDA receptors, shifts in voltage
activation & inactivation curves
Paired-pulse facilitation: second event that follows is up to 5X as large due to
increased probability of presynaptic vesicle release
miniEPSPs – recorded in presence of TTX:
changes in amplitude: postsynaptic event
changes in frequency: presynaptic release
Data, data, data
Spike Sorting – used in multi-array recording to assign spikes to different neurons
based on their spike properties
Pharmacological & Electrical Isolation of distinct currents