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

EEGsRecording spontaneous brain (voltage volume conductance) activity from the scalp, described in rhythmic activity: Delta (<4 Hz), theta (4-7 Hz), gamma (30-100 Hz)

Clinical Neuroscience: epilepsy, coma, tumors, stroke,focal brain damage, depth of anesthesia

Coordinate cortical activity = high contributionDeep 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

Inside-Out Outside-Out

Whole-Cell

Suction

PullQuickly

PullSlowly

>1 GΩ seal – going ‘whole-cell’ does not compromise the seal: prevents leak current & extracellular buffer from entering the neuron

BindingSite?

Perforated Patch Recording

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

start

~10-15 min

~20-30 min

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 recordedvoltage-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

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 recordedAP waveform, plasticity of EPSPs, intrinsic excitability

Voltage vs. Current Clamp

sEPSC Somatic current injection producing AP firing

A.

B.Stimulation

Local Field Potentials - fEPSPs

SA = stimulus artifact

* = presynaptic fiber volley – presynaptic activity generated by stimulation

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

SA*

fEPSP

PS

A.

B.

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), low-background 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

Helios Gene Gun – can be used to load gold particles coated with cDNA into cells on the day of culturing to change protein expression

Multiple brain regions (hpc, co-cultures) grown on porous membrane inserts

Prepared from 2-8 day old rodent pups

Maintained for months

Dissociated Cultures

Autaptic/MicroislandCultured Primary

Dissociated Neurons

Acutely Dissociated Neurons - the neurons preserve their dendritic structure proximal to the soma, maintain intact synaptic boutons, and are largely devoid of glial ensheathments.

Typically prepared in low- or high-density from embryonic or <24 h old pups

Hippocampal, Cortical, Striatal cultures are common

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

Cultures

Cell Lines

HEK 293 CellsXenopus oocytesPC-12 Adrenal Cells

Pros: excellent for answering certain ?’sExpress select proteinsPoint mutation studiesModel system for neuronal differentiation

Cons: Non-mammalian , non-CNS cellsLack complete neuronal constituents

(e.g., signaling complexes)

Performed under anesthesia or in freely-moving rodents

In Vivo Recordings

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, dataAP: 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 eventchanges in frequency: presynaptic release

Spike Sorting – used in multi-array recording to assign spikes to different neurons based on their spike properties

Pharmacological & Electrical Isolation of distinct currents

Data, data, data