Foundations I Fall, 2016 Overview of Different Techniques for - - PowerPoint PPT Presentation

Foundations I Fall, 2016 Overview of Different Techniques for Monitoring Neuronal Activity Basics of Electrical Circuitry

Intracellular Recording

Sir John Eccles (1903 -1997)

Small-tipped glass micropipettes (<1 µm) were introduced by Ling and Gerard in 1949 for in vivo intracellular recording in the spinal cord. Intracellular microelectrodes are filled with potassium acetate, methysulfate, gluconate or (more rarely) chloride (0.5-3 M) Intracellular micropipettes have high resistance (50-100 MΩ)

Today this technique is termed “Sharp Electrode Recording” and remains the most common method for intracellular recording in vivo, although it is used less and less for in vitro recordings.

Intracellular recording measures the difference between the potential (voltage) inside of a cell and an extracellular reference, take to be 0 mV. It can also be used to measure the current flowing through the cell’s membrane.

Current Clamp vs. Voltage Clamp Recording

measures transmembrane voltage measures transmembrane current(s)

Current Clamp vs. Voltage Clamp Recording

dendrites OK dendrites bad biophysics/kinetics bad biophysics/kinetics good can measure firing activity no firing activity less sensitive more sensitive C-Clamp V-Clamp

voltage clamp recordings

PD 14 PD 19 PD 29 PD 32 Adult

20 mV 80 ms

• 48 mV
• 61 mV
• 66 mV
• 64 mV
• 74 mV

Current Clamp recordings

excitatory postsynaptic potentials (EPSPs)

20 ms 10 mV

PD10 PD14 PD22 PD32 Adult monosynaptic polysynaptic

Current clamp recordings

power test for monosynapticity

monosynaptic vs. polysynaptic?

20 mV

• 80 mV
• 59 mV

"Up" State "Down" State

0.5 sec subthreshold membrane oscillations

spikes

Current clamp recordings

250 pA 100 mV 20 ms

presynaptic neuron (stimulus) postsynaptic neuron (response)

EPSCs

(inward current)

Voltage clamp recordings

current clamp voltage clamp

25 mV 1 nA 100 ms

0.400 0.200

• 0.00
• 0.20
• 0.40
• 15.00

0.000 15.000 30.000 45.000 Current (nA) Voltage (mV)

intracellular current pulses (I) IV Plot membrane potential (V) (stimulus) (response)

slope=V/I= R = membrane (input) resistance

m

Ohm’s Law: V=IR

R i 1

= 200 MΩ

R i 2 =100 MΩ R i 3

=60 MΩ most neurons are non-linear

Intracellular micropipettes can be filled with various substances that will stain the entire neuron

in vivo biocytin filled neuron intracellular recording

HRP-labeled dendrite unlabeled presynaptic terminal

...not the method of choice for spontaneous activity measurements since impalement may damage or otherwise affect the neuron - “somatic shunt” problem. ...recordings may be difficult to obtain and maintain particularly in small CNS neurons in vivo.

Sharp Electrode Intracellular Recording

Disadvantages

Most modern ex vivo (slice) recordings now use a newer technique

Patch clamp recording

uses large tipped (1-2 µm), low resistance (3-6 MΩ) micropipettes

Neher Sakmann

very stable can access very small neurons used mostly in slices or dissociated cells but can be used in vivo as well

Different types of patch clamp recording

In vitro recordings

acute brain slices acutely dissociated cells cell cultures

voltage clamp dissociated cells slice culture

• rganotypic slice cultures

In vivo versus In vitro recordings

network connectivity? pharmacological manipulation? ease of recording? anatomical studies?

✔ ✔ ✔ ✔ ? ✔

B

20 mV 0.5 nA 40 ms

14.600

• 15.80
• 31.00
• 46.20

105 210 315 420

Current (nA) ΔVoltage from rest (mV)

* * * * * * * * * *

A C D

ΔVoltage from rest (mV) Input Resistance (MΩ)

0.100

• 0.02
• 0.15
• 0.27
• 0.40
• 50.00
• 33.75
• 17.50

15.000

IR-DIC Whole Cell Recording

A

micropipette cell

Transgenic mice with genetically engineered reporter genes

enhanced green fluorescent protein EGFP DIC can be made cell-type specific

cell soma

pipette tip

Single cell RT-PCR

allows genetic and neurochemical phenotyping of recorded cell

Biocytin Fills

Whole Cell Recording

Disadvantages Intracellular dialysis - cell rundown

10 20 100 200 300 1 2 3 1 2 3 time (min) Amplitude (pA)

B A

time (min) Amplitude (pA) 100 200 10 20 4 nA 25 ms 10 pA 1 2 3 1 2 3 5/20 15/20

Presynaptic whole cell Presynaptic Perforated-Patch

10 pA 25 ms 0.4 nA

Solution - use perforated patch technique

gramicidin (anion-impermeant) amphotericin (anion-impermeant) nystatin (anion permeant, some cation per

Zecevic, 1996

JPW1114 Voltage-sensitive dye

Calcium Imaging

GCaMP photometry ex vivo

GCamp6 expressed in dopamine axons

Extracellular Recording

What kind of information can one get from extracellular recordings?

wire electrodes microelectrodes filled with electrolyte usually NaCl platinum-iridium or tungsten in glass Low resistance - 0.2 mΩ - 20 mΩ Extracellular recording measures the highly localized field surrounding a neuron

Single Unit Extracellular Recordings

• D. Hubel Nobel Lecture

evoked responses

spontaneous activity

spike noise signal to noise ratio ~10:1

spontaneous activity cell attached mode

2.0ms

*

4.0ms

A B C

2 ms

* C D

collision! collision! antidromic responses waveform information identification of projection neurons

initial segment component somatodendritic component

spontaneous (orthodromic) spikes

fixed latency conduction velocity

Intracellular spike 4 ms 25 mV Differentiated spike 4 ms 4 ms Extracellular spike

2 4 6 8 10 12 14 16 100 200 300

• 100

time (msec)

A B

20 msec

number of events

raster plot peri-stimulus time histogram (PSTH)

stimulus at time 0

inhibition rebound excitation

Synaptic responses

First Order Interspike Interval Histogram (ISH)

Text

Quantitative Descriptions of Firing Patterns

unimodal little ISI variance bimodal large ISI variance unimodal little ISI variance higher firing rate unimodal greater ISI variance

Computation of First Order Interspike Interval Histogram

i3 i4 i2 i1

16 32 48 64 80 96 112 128 200 400 600 800 time (msec)

Mean Firing Rate = 4.96 Spikes/sec

200 400 600 800 1000

Mean Firing Rate = 4.92 Spikes/sec

600 800

Mean Firing Rate = 5.84 Spikes/sec

Bursty Firing Mode Random Firing Mode Pacemaker Firing Mode

time (msec) time (msec)

3 sec

16 32 48 64 80 96 112 128 16 32 48 64 80 96 112 128

3 sec 3 sec

number of events

200 1000 1000 400

Autocorrelation Histograms

Computation of an Autocorrelogram

first pass second pass third pass etc through pass n-1 i2 i1 i3

~ 1 µm recording tip ~ 10 µm drug ejection Local Pressure Injection of Drugs

Text

Juxtacellular Recording and Labeling

• D. Pinault, 1996

Optrode recording in vivo

English et al., 2012

halorhodopsin3

20ms

200_m 20_m

Chronic recording in freely moving animals

Multisite recording

polarity reversal

Field Potentials

Power spectra

Text Text

single trial average of 32 trials

ERP

N1 P1 N2 N3

Basic Concepts in Electrical Circuitry

E=∫f(r) dr

1 V = work to move 1 coulomb 1 meter against 1 newton

Electrical potential (E or V) is a measure of work

E is the integral of force over distance

Current (I) is the rate of flow of charge

1 ampere(A) = 1 coulomb/sec

I= dq/dt

the charge on a proton or electron is 1.6X10 C

(this is pretty small)

• 19

Resistance is the frictional force against flow of current

V=IR Ohm’s Law

The unit of resistance is the Ω

Resistances connected in series add linearly:

e.g., Rtot= R1+ R2

R R1

2

Rtot 2 Ω 4 Ω 6 Ω Resistors In Series

2 + 4 = 6

Resistances connected in parallel add reciprocally:

e.g., 1/Rtot=1/R1+1/R2

Rtot 1.33 Ω R 2 Ω R 4 Ω

1 2

Resistors In Parallel

1/2 + 1/4 = 3/4 1/(3/4) = 4/3 = 1.3333

Sometimes it is more convenient to think of the relation between current and voltage in terms of the reciprocal of resistance, which is called conductance. Conductance (g) is defined as 1/R and is given in units called siemens (S). Thus, conductances in parallel add linearly and conductances in series add as their reciprocals.

(Once upon a time the units of conductance were just 1/Ω and were called “mhos”)

A capacitor is a device that separates and stores charges

A capacitor consists of two conductive plates separated by an insulating material Note that current doesn’t really flow through the capacitor since there is an insulator in the middle. Rather, the circuit behaves as though current is flowing as opposite charges move to the two plates of the capacitor. When the plates are fully charged, there is no more current flow in the circuit. That means that when the voltage is constant (dV/dt = 0), there is no capacitative

• current. Thus capacitors act as high pass filters.

Capacitance is defined as: C=q/V

+ + + + + + +

• battery

capacitor

where ε =dielectric constant (measure of insulatability) ε0 =polarizability of free space (9x10-14 f/cm2) A =surface area of plates d =distance between plates

The capacitance of a capacitor is given by C= ε ε0 A/d

Capacitance in parallel adds linearly

e.g., Ctot =C1 + C2

2 + 4 = 6 C1 Ctot 2 mf 4 mf 6 mf

+ + +

C2

Capacitance in series adds reciprocally

e.g., 1/Ctot = 1/C1 + 1/C2

1/2 + 1/4 = 3/4 1/(3/4) = 4/3 = 1.3333

C2 C1 C tot 2 mf 4 mf 1.33 mf + + +

Ideal capacitors will charge and discharge at speed of electrons flowing in a wire (close to c), but no capacitative circuit has no resistance. Instead, we consider capacitative circuits (as in a neuronal membrane) as consisting of a capacitance (C) and a resistance (R) in parallel.

The resistance slows the flow of current and the resulting voltage change

V = IR

Ohms Law

I = V R

Rearrange

q = CV

Rearrange

dq dt = C dV dt

differentiate

τ = RC

define the time constant, τ as and substitute

dV dt = −V τ

substitute for dq/dt

dV dt = −V /RC

current is rate of flow of charge, i.e. rate of change of charge so

I = dq dt = −V R

(V is negative because charge is decreasing over time)

What is the rate of change of voltage in a circuit with a resistance and a capacitance?

V = V

−tτ

e

integrate and solve for V

C = q/V

definition of capacitance

Because of the capacitance, the voltage rises (and decays) slowly, with a time course dictated by the resistance and the capacitance

I V

Time

V=V0e-t/τ

Text when t = τ

V=V0/e = 0.368V0