Foundations I Fall, 2017 I. Overview of Different Techniques for - PowerPoint PPT Presentation

Foundations I Fall, 2017 I. Overview of Different Techniques for Monitoring Neuronal Activity II. Basics of Electrical Circuitry

Intracellular Recording Intracellular recording measures the difference between the potential (voltage) inside of a cell and an extracellular reference, take to be 0 mV. This is equivalent to saying that it can also be used to measure the current flowing through the cell’s membrane. Small-tipped glass micropipettes (<1 µ m) were introduced by Ling and Gerard in 1949 for in vivo intracellular recording in the spinal cord. Sir John Eccles (1903 -1997) Intracellular microelectrodes are filled with potassium acetate, methysulfate, gluconate or (more rarely) chloride (0.5-3 M). Why not Na+? Intracellular micropipettes have high resistance (50-100 M Ω ) Today this technique is termed “Sharp Electrode Recording” and remains a common method for intracellular recording in vivo, although it is being supplanted by whole cell patch clamp recordings and is now rarely used in vitro recordings.

Current Clamp vs. Voltage Clamp Recording measures transmembrane voltage measures transmembrane current(s)

Current Clamp vs. Voltage Clamp Recording PD 14 -48 mV PD 19 -61 mV PD 29 -66 mV PD 32 voltage clamp recordings -64 mV C-Clamp V-Clamp Adult dendrites OK dendrites bad biophysics/kinetics bad biophysics/kinetics good -74 mV can measure firing no firing activity 20 mV activity 80 ms less sensitive more sensitive Current Clamp recordings

Current clamp recordings PD10 excitatory postsynaptic potentials (EPSPs) monosynaptic vs. polysynaptic? PD14 PD22 polysynaptic power test for monosynapticity PD32 monosynaptic Adult 10 mV 20 ms

Current clamp recordings spikes "Up" State -59 mV subthreshold membrane oscillations 20 mV -80 mV "Down" State 0.5 sec

Voltage clamp recordings postsynaptic neuron (response) EPSCs voltage clamp (inward current) 250 pA 100 mV 20 ms current clamp presynaptic neuron (stimulus)

Ohm’s Law: V=IR Voltage (mV) 45.000 30.000 = 200 M Ω R i 1 membrane potential (V) 15.000 R i 2 =100 M Ω (response) 0.000 -0.40 -0.20 -0.00 0.200 0.400 R i 3 =60 M Ω Current (nA) -15.00 25 mV 1 nA intracellular current 100 ms pulses (I) IV Plot (stimulus) most neurons are non-linear

Intracellular micropipettes can be filled with various substances that will stain the entire neuron intracellular recording in vivo biocytin filled neuron

unlabeled presynaptic HRP-labeled dendrite terminal

Sharp Electrode Intracellular Recording Disadvantages ...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.

Most modern ex vivo (slice) recordings now use a newer technique Patch clamp recording Neher Sakmann uses large tipped (1-2 µ m), low resistance (3-6 M Ω ) micropipettes 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 voltage clamp cell cultures dissociated cells slice culture organotypic slice cultures

In vivo versus In vitro recordings network connectivity? ✔ ? ✔ pharmacological manipulation? ✔ ease of recording? ✔ anatomical studies? ✔

IR-DIC Whole Cell Recording A * cell A B * * 20 mV * * 0.5 nA 40 ms micropipette * * * * * C D 420 Input Resistance (M Ω ) 15.000 Δ Voltage from rest (mV) 315 -0.40 -0.27 -0.15 -0.02 0.100 210 Current (nA) -17.50 105 -33.75 -46.20 -31.00 -15.80 14.600 -50.00 Δ Voltage from rest (mV)

Transgenic mice with genetically engineered reporter genes cell soma pipette tip Infrared Differential interference enhanced green fluorescent protein contrast (IR-DIC) TH-EGFP can be made cell-type specific with viral mediated gene transfer and Cre recombinase

Single cell RT-PCR allows genetic and neurochemical phenotyping of recorded cell

Biocytin Fills

Whole Cell Recording Disadvantages Intracellular dialysis - cell rundown

Solution - use perforated patch technique Presynaptic whole cell Presynaptic Perforated-Patch A B 0.4 nA 5/20 4 nA 200 300 15/20 1 1 2 3 1 Amplitude (pA) 1 Amplitude (pA) 2 200 2 100 2 100 3 3 3 0 10 pA 0 10 pA 25 ms 25 ms 0 10 20 0 10 20 time (min) time (min) gramicidin (anion-impermeant) amphotericin (anion-impermeant) nystatin (anion permeant, some cation per

Voltage-sensitive dye JPW1114 Zecevic, 1996

Calcium Imaging

GCaMP6 photometry ex vivo GCamp6 expressed in dopamine axons

GCaMP6 photometry ex vivo in striatal TH interneurons reward

Extracellular Recording Extracellular recording measures the highly localized field surrounding a neuron wire electrodes platinum-iridium or tungsten in glass microelectrodes filled with electrolyte usually NaCl Low resistance - 0.2 m Ω - 20 m Ω What kind of information can one get from extracellular recordings?

Single Unit Extracellular Recordings evoked responses D. Hubel Nobel Lecture

spontaneous activity spike noise signal to noise ratio ~10:1

spontaneous activity cell attached mode

spontaneous antidromic responses (orthodromic) spikes fixed latency A B conduction velocity * collision! initial segment somatodendritic component component C C D 4.0ms collision! * 2.0ms 2 ms waveform information identification of projection neurons

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

Synaptic responses A raster plot B 20 msec 16 14 number of events 12 rebound excitation 10 8 inhibition 6 peri-stimulus time 4 histogram (PSTH) 2 -100 0 100 200 300 time (msec) stimulus at time 0

Quantitative Descriptions of Firing Patterns First Order Interspike Interval Histogram (ISH) bimodal unimodal large ISI variance little ISI variance Text unimodal unimodal greater ISI variance little ISI variance higher firing rate

Computation of First Order Interspike Interval Histogram i1 i2 i3 i4

Autocorrelation Histograms Pacemaker Firing Mode Random Firing Mode Bursty Firing Mode 3 sec 3 sec 3 sec 128 128 128 112 number of events 112 112 96 96 96 Mean Firing Rate = 4.96 Mean Firing Rate = 4.92 Mean Firing Rate = 5.84 80 80 80 Spikes/sec Spikes/sec Spikes/sec 64 64 64 48 48 48 32 32 32 16 16 16 200 400 600 800 1000 200 400 600 800 1000 200 400 600 800 1000 time (msec) time (msec) time (msec)

Computation of an Autocorrelogram i1 i2 i3 first pass second pass third pass etc through pass n-1

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

Juxtacellular Recording and Labeling Text D. Pinault, 1996

Optrode recording in vivo halorhodopsin3 English et al., 2012

Chronic recording in freely moving animals Multisite recording 20_m 20ms 200_m

Field Potentials polarity reversal Power spectra

ERP single trial Text Text N3 N1 N2 average of 32 trials P1

Basic Concepts in Electrical Circuitry

Electrical potential (E or V) is a measure of work E= ∫ f(r) dr E is the integral of force over distance 1 V = work to move 1 coulomb 1 meter against 1 newton

Current (I) is the rate of flow of charge I = dq/dt 1 ampere(A) = 1 coulomb/sec -19 the charge on a proton or electron is 1.6X10 C (this is pretty small)

Resistance is the frictional force against flow of current The unit of resistance is the Ω V=IR Ohm’s Law

Resistances connected in series add linearly: e.g., Rtot= R1+ R2 Resistors In Series R 1 R R tot 2 4 Ω 2 Ω 6 Ω 2 + 4 = 6

Resistances connected in parallel add reciprocally: e.g., 1/Rtot=1/R1+1/R2 Resistors In Parallel R 1 R tot 2 Ω 4 Ω 1.33 Ω R 1/2 + 1/4 = 3/4 2 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). (Once upon a time the units of conductance were just 1/ Ω and were called “mhos”) Thus, conductances in parallel add linearly and conductances in series add as their reciprocals.

A capacitor is a device that separates and stores charges Capacitance is defined as: C=q/V A capacitor consists of two conductive plates separated by an insulating material capacitor - + - + - + - + + - - + - + battery 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.