1

Advanced electrophysiology

1. Inward rectifiers (mentioned, not explained in Kandel)

2. Glia (pp 24-27 & 88-95)

3. A potpourri of contemporary recording and stimulating techniques

(Not treated in Kandel)

Henry Lester

Bi / CNS / BE 150

Lecture 11

Wednesday, October 20, 2015

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Unblocked channel,Inward current

Polyamine-blocked channel,Swept in by outward current

cytosol

extracellular

Inward Rectification:

the only voltage-dependent “gating” mechanism in some K+ channels

spermine

spermidine

Total intracellular conc.> 1 mM in most cells.Binding rate constant

~ 108 /M/s x 10-3 M ~ 105/s,Therefore block occurs

In ~ 10 μs.

+H3N

H2+

N NH3+

+H3NNH2

+

H2+

N NH3+

3

H2O K+ ion

carbonyl

From Lecture 1

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If many channels are open, much current flows . . . and the ions must be pumped back, using energy

Na

Na

mostly K+

K

K

ENa

(+60 mV)

=GNa

EK (- 60 mV)

GK

VE G E G E G

G G GK K Na Na Cl Cl

K Na Cl

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If inward rectifier K+ channels close,

the cell requires fewer Na+ channels,saving energy

Na

Na

mostly K+

ENa

(+60 mV)

=GNa

EK (- 60 mV)

GK

K

K

VE G E G E G

G G GK K Na Na Cl Cl

K Na Cl

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~ 1 s

Cardiac tissue is depolarized for ~ 50% of one’s life

Most cardiac K channels are inward rectifiers.

The “plateau” requires very few open Na+ channels, saving pump energy.

An inward rectifier functions like a “latch on a cabinet door” (Hille).

We’ll discuss G protein-gated inward rectifier K+ channels next week.

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A. Oligodendrocyte (CNS)

produces myelinIn white matter

B. Schwann cell

(Peripheral NS) produces myelin

Three types of glial cells

C. Astrocyte (CNS)

Plays several support roles

Figure 2-5

several branchesglue

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One rarely sees a bare neuron.There is usually a surrounding glial cell

(in this case, a Schwann cell)

Figure 11-1

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There is very little extracellular space in the CNS

Astrocytes occupy ~ 5% of the volume and provide supporting pathways to maintain the extracellular space

1 μm

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Endothelial cells lining the capillary

Red blood cells

Astrocyte end feet surround brain capillaries, but don’t form the blood-brain barrier

“Tight junctions” form the blood-brain barrier

Blood vessel

Blood

astrocyte end foot

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Transporters for glutamate, GABA, and several other neurotransmitters.

This eliminates transmitter molecules from the restricted extracellular space.

Transporters for glucose, lactate, and other nutrients.

This brings nutrients from the capillaries to neurons.

Permanently open K+ channels.

This removes K+ from the extracellular space, where it might depolarize

neurons, and takes K+ to capillaries.

Transport properties of astrocyte membranes

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Extracellular recording with pipette electrodes

Tetrodes

Wireless recording

Microdevice arrays

Direct imaging

Single-unit recording in humans

Advanced (electro)physiology

Extracellular single-unit recordings sometimes distinguish neuronal types in vivo

Dopamine neuron

Nicotineinjection

GABAergic neuron (5 s smoothing)

0.05 m V2 m s

0.05 m V2 m sF

requ

ency

, H

z

0 100 200 300 400 500 600 700

0

2

4

6

0

5

10

15

20

25

tims

Fre

quen

cy,

Hz

0.1 m V

0.5 m s

0.1 mV0.5 ms

0.05 m V2 m s

0.05 mV2 ms

V

GABAergic

DAergic

mousemidbrain

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14

Tetrode carrier (Thanos Siapas)

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Tetrodes (Thanos Siapas)

Highly Stable Prefrontal Cortex Tetrode Recordings (Thanos Siapas)

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Daniel Kegel ‘86statistical analysis;

bit-mapped graphics displays

http://www.kegel.com/

Christmas ‘83“May I borrow your

notebook computer over vacation, please? I’m installing a radio link.”

Jan 1, 1984

Blacker House

http://www.nytimes.com/2014/01/01/sports/ncaafootball/100-glorious-years-of-the-rose-bowl.html?_r=0

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Nanofabricated Multiplexed Electrode Arrays

Du J, Blanche TJ, Harrison RR, Lester HA, and Masmanidis SC (2011) PLoS ONE

Scott KM, Du J, Lester HA, and Masmanidis SC (2012) J Neurosci Methods

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A wireless multi-channel neural amplifier for freely moving

animals

Tobi A Szuts . . . Evgueniy V Lubenov (Caltech postdoc),

Athanassios G Siapas (Caltech Prof) Markus Meister (now at Caltech),

2011

40 g total, using 2005 technology . . .Could presumably be ~ 2 g now

(light enough for a mouse)

Signal shows no degradation when transmitted 60 m

20Dombeck et al

Inventor of fMRI

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head-restraint bar

microscope objective

lens

Single-cell activity in forelimb motor cortex of awake running and grooming mice

Two-photon microscopy image from a bolus loaded region; neuron somata appear as green discs.

green, Ca green-1 fluorescence; (labels both neurons and astrocytes)

red, SR101 (labels only astrocytes). This allowed authors to differentiate neurons from astrocytes and provided a constant intensity image for off-line motion correction.

significant Ca transients

Running Grooming

Dombeck et al

http://www.jneurosci.org/content/vol29/issue44/images/data/13751/DC1/Movie_S2.mov

http://www.jneurosci.org/content/vol29/issue44/images/data/13751/DC1/Movie_S1.mov

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1. Electrical stimulation:PacemakersTranscutaneous stimulation for back painDeep brain stimulation for Parkinson’s diseaseCochlear implantsRetinal prostheses

2. Transcranial magnetic stimulation 3. Pharmacological neuronal silencing4. Optogenetics

Advanced stimulation

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dopaminergic neurons die in PD

Before the videos were shot, stimulating electrodes were implanted surgically. Midway through each video, the stimulators were programmed magnetically;

then stimulation started.

Deep brain stimulation for Parkinson’s Disease

Tremor may arise in a malfunctioning feedback loop:

substantia nigra, striatum, and other

structures.

Implanted stimulating electrodes retune this

loop.

More about the mechanism, later in today’s lecture.

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Transcranial magnetic stimulation, Used in Shimojo lab at Caltech

A changing magnetic field produces an electric field.This produces current flow in the brain.

This stimulates or silences spiking in neurons.Resolution ~ 5 mm. Maximum safe frequency, 1 Hz

Not yet approved for therapeutic use in US.

Binding region

Membrane region

Cytosolicregion

(incomplete)

Colored by subunit(chain)

The “channelohm” is 2% of the human genome,

A nicotinic acetylcholine receptor / channel:~ 2200 amino acids in 5 chains (“subunits”)

Voltage (actually, ΔE ~107 V/m)External transmitterInternal transmitter

LightTemperature

Force/ stretch/ movementBlockers

Nernst potential forNa+,

K+,Cl-,

Ca2+,H+

Switches

Resistor

Battery

=1/r = 0.1 – 100 pS

and many other organisms expand the repertoire

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Pharmacological neuronal silencing: Re-engineering a Cys-loop receptor channel

Ivermectin (IVM) made by

bacteria,

used as antiparasitic in

animals and humans (“River

blindness” / Heartgard™)

Allosterically activates GluCl

channels

O

O

O

OO

O

O

O

OO

O

OO

O

H

H

H H

H

H

HH

H

HH HH

H

H

HH

H

HH H

H

HH

H

Slimko, McKinney, Anderson, Davidson, Lester (2002) J Neurosci; Frazier, Cohen, Lester (2013) J Biol Chem.

0 nM IVM 1 nM IVM 20 nM IVM

1Department of Bioengineering, 2Program in Neuroscience, 3Department of Neurosurgery,

4Department of Psychiatry and Behavioral Sciences,Stanford University, Stanford, CA94305, USA.

channelrhodopsin halorhodopsin

“Optogenetics”

More engineering of the “channelohm” with Light

27Shapiro MG (now Caltech Prof), Frazier SJ, and Lester

HA (2012) ACS chemical neuroscience

Illumination evokes photocurrents in ChR2-positive cortical neurons

Wang H et al. PNAS 2007;104:8143-8148

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Illumination controls number and frequency of action potentials

Wang H et al. PNAS 2007;104:8143-8148

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Axons passing through

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ACh

ACh

GPi

DirectPathway

GPe

Thalamus

Cortex

Excitation

Inhibition

(Regardless of color)

INs

ACh GABA Glu

INs

Transmitters

dorsalstriatum

STN +SNr

=SNc

?

PPTg

MSN D1RMSN D2R

Indirect pathway

INs

DA

Tremor arises in a malfunctioning feedback loop:

substantia nigra, striatum, and other

structures.

Implanted stimulating electrodes retune this

loop.

Deep brain stimulation for Parkinson’s DiseaseEarlier today

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Optical Deconstruction of Parkinsonian Neural Circuitry

Viviana Gradinaru, (Caltech Bi 2005), Murtaza Mogri, Kimberly R. Thompson,Jaimie M. Henderson, Karl Deisseroth

(Bioengineering, Stanford)

Science, 2009

Viviana Gradinaru, Assistant Professor of Biology at Caltech

“We used optogenetics and solid-state optics to systematically drive or inhibit an array of distinct circuit elements in freely moving parkinsonian rodents and found that therapeutic effects within the subthalamic nucleus can be accounted for by direct selective stimulation of afferent axons projecting to this region.”

Toxin-treated mice (one side only), confirmed by tyrosine hydroxylase staining.Behavioral assay: rotation & head position.

Promoter-driven constructs: halorhodopsin driven by CAM kinase II promoter.“Electrical DBS was highly effective in reducing pathological rotational behavior, but despite precise targeting and robust physiological efficacy of halorhodopsin inhibition, the hemiparkinsonian animals did not show even minimal changes in rotational behavior

with direct true optical inhibition of the local excitatory STN neurons .”

Channelrhodopsin driven by CAM kinase II promoter, also ineffective. c-fos (biochemical marker of neuronal activation) showed that at > 0.7 mm3 , nearly the entire STN is recruited by light stimulation.

Glial promoter-drive channelrhodopsin, Also ineffective.

Gradinaru et al, 2009

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Transgenic mice (Thy1) expressing channelrhodopsin in layer V cortical neurons & their axons . . .

Similar effects with cortical stimulation.Tentative Conclusion:

DBS works via stimulating passing axonsOptical HFS (130 Hz, 5-ms pulse) of the STN region in an anesthetized Thy1::channelrhodopsin-YFP toxin-treated mouse inhibited STN large-amplitude spikes. Optical LFS (20 Hz produced reliable spiking at 20 Hz.

Whereas HFS prevented bursting, LFS had no significant effect on burst frequency nor on spikes per burst.

Optical HFS to STN in these five animals (100 to 130 Hz) produced robust therapeutic effects, reducing ipsilateral rotations and allowing animals to freely switch directions. In contrast, optical LFS (20 Hz) exacerbated pathologic effects, causing increased ipsilateral rotations. Both effects were reversible (post).

Gradinaru et al, 2009 33

magnetoencephalography +

event-related potentials

functional magneticresonance imaging

positron emission tomography

Modern Neuroscience Techniques:Time scales, Distance Scales, and Invasiveness

Intracellular Patch/Sharp

Extracellular Single Unit or Tetrode

Optical Dyes

Silicon ArrayMicrolesions

2-deoxyglucose

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Help with biochemistry (several advances required):

Help with chemistry (both good ideas & technology required):Requires industrial-scale drug screening, “chemical neurobiology”

Help with mice:Techniques for more efficient genome engineering.

Most important:Talented, excited young people

Some requirements for further progress

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End of Lecture 11