Bi 150 Lecture 9
Friday, October 18, 2013
Advanced electrophysiology
1. Inward rectifiers
2. Glia
3. A potpourri of contemporary recording and stimulating techniques
Henry Lester
Kandel has very little material on today’s topics; but see p. 24-27, p. 107, p. 115
1
Inward Rectification:
the only voltage-dependent “gating” mechanism in some K+ channels
Total intracellular conc.
spermine
> 1 mM in most cells.
Binding rate constant
spermidine ~ 108 /M/s x 10-3 M ~ 105/s,
Therefore block occurs
In ~ 10 μs.
extracellular
cytosol
Unblocked channel,
Inward current
Polyamine-blocked channel,
Swept in by outward current
2
From Lecture 1
H2O
carbonyl
K+ ion
3
If many channels are open, much current flows . . .
and the ions must be pumped back, using energy
GNa
ENa
GK
=
gNa
gK
EK
(- 60 mV)
(+60 mV)
gNa
E K GK + E NaGNa + ECl GCl
DV =
GK + GNa + GCl
mostly K+
gK
4
If inward rectifier K+ channels close,
the cell requires fewer Na+ channels,
saving energy
GNa
ENa
GK
=
gNa
gK
EK
(- 60 mV)
(+60 mV)
gNa
E K GK + E NaGNa + ECl GCl
DV =
GK + GNa + GCl
mostly K+
gK
5
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.
~1s
An inward rectifier functions like a “latch on a cabinet door” (Hille).
We’ll discuss G protein-gated inward rectifier K+ channels next week.
6
Three types of glial cells
glue
several branches
A.
Oligodendrocyte (CNS)
produces myelin
In white matter
B.
Schwann cell
(Peripheral NS)
produces myelin
C.
Astrocyte (CNS)
Plays several support
roles
Figure 2-5
7
One rarely sees a bare neuron.
There is usually a surrounding glial cell
(in this case, a Schwann cell)
Figure 11-1
8
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
9
Glial end feet surround brain capillaries, but they don’t form the blood-brain barrier
Blood vessel
Blood
Glial end foot
“Tight junctions”
form the blood-brain barrier
Endothelial cells lining the capillary
Red blood cells
10
Transport properties of astrocyte membranes
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.
11
Advanced (electro)physiology
Extracellular recording with pipette electrodes
Tetrodes
Wireless recording
Microdevice arrays
Direct imaging
Single-unit recording in humans
12
Frequency, Hz
Single-unit recordings can sometimes distinguish neuronal types in vivo
Dopamine neuron, ~ 1700 spikes
6
4*, 6*, and/or 7
mouse
Nicotine
injection
4
VTA
0.050.05mV
mV
2 ms
0.05 mV 2 ms
2
A
B
C
2 ms
0.05 mV
DAergic
D
2 ms
0
25
4*
only
Frequency, Hz
GABAergic
20
0.5
0.1 ms
mV
15
V
0.1 mV
10
0.5 ms
GABAergic neuron
(5 s smoothing), ~ 8300 spikes
5
0
0
100
200
300
400
s
500
600
700
13
Tetrode carrier (Thanos Siapas)
14
Tetrodes (Thanos Siapas)
15
Highly Stable Prefrontal Cortex Tetrode Recordings (Thanos Siapas)
16
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
17
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
(Harvard -> Caltech),
2011
Signal shows no degradation
when transmitted 60 m
40 g total, using 2005 technology . . .
Could presumably be 4 g now
(light enough for a mouse)
18
Inventor of fMRI
Dombeck et al
19
Single-cell activity in forelimb motor cortex of awake running and grooming mice
Dombeck et al
Two-photon microscopy
image from a bolus
loaded region; neuron
somata appear as green
discs.
significant
Ca
transients
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 offline motion correction.
microscope
objective
lens
headrestraint bar
Running
Grooming
http://www.jneurosci.org/content/vol29/issue44/images/data/13751/DC1/Movie_S1.mov
http://www.jneurosci.org/content/vol29/issue44/images/data/13751/DC1/Movie_S2.mov
20
Advanced stimulation
1. Electrical stimulation:
Pacemakers
Transcutaneous stimulation for back pain
Deep brain stimulation for Parkinson’s disease
Cochlear implants
Retinal prostheses
2. Transcranial magnetic stimulation
3. Pharmacological neuronal silencing
4. Optogenetics
21
Deep brain stimulation for Parkinson’s Disease
Tremor may arise in a
malfunctioning
feedback loop:
substantia nigra,
striatum, and other
structures.
dopaminergic
neurons die in PD
Implanted stimulating
electrodes retune this
loop.
Before the videos were shot, stimulating electrodes were implanted surgically.
Midway through each video, the stimulators were programmed magnetically;
then stimulation started.
More about the mechanism, later in today’s lecture.
22
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.
23
The “channelohm” is 2% of the human genome,
and many other organisms expand the repertoire
Voltage (actually, ΔE ~107 V/m)
External transmitter
Internal transmitter
Light
Temperature
Force/ stretch/ movement
Blockers
Switches
Binding
region
Membrane
region
Colored by
subunit
(chain)
=
Resistor
1/r = 0.1 – 100 pS
Battery
Cytosolic
region
(incomplete)
A nicotinic acetylcholine receptor / channel:
~ 2200 amino acids in 5 chains (“subunits”)
Nernst potential for
Na+,
K+,
Cl-,
Ca2+,
H+
24
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
0 nM IVM
1 nM IVM
20 nM IVM
Slimko, McKinney, Anderson, Davidson, Lester (2002) J Neurosci; Frazier, Cohen, Lester (2013) J Biol Chem.
More engineering of the “channelohm” with Light
“Optogenetics”
1Department
of Bioengineering,
2Program in Neuroscience, 3Department of Neurosurgery,
4Department of Psychiatry and Behavioral Sciences,
Stanford University, Stanford, CA94305, USA.
channelrhodopsin
halorhodopsin
Shapiro MG, Frazier SJ, and Lester HA (2012)
ACS chemical neuroscience
26
Illumination evokes photocurrents in ChR2-positive cortical neurons
Wang H et al. PNAS 2007;104:8143-8148
27
Illumination controls number and frequency of action potentials
Wang H et al. PNAS 2007;104:8143-8148
28
Deep brain stimulation for Parkinson’s Disease
Earlier today
Cortex
Tremor arises in a
malfunctioning
feedback loop:
substantia nigra,
striatum, and other
structures.
INs
ACh
Implanted stimulating
electrodes retune this
loop.
INs
MSN D2R MSN D1R
Indirect
pathway
ACh
PPTg
?
INs
dorsal
striatum
Direct
Pathway
SNc
=
Thalamus
GPe
Axons
passing through
GPi
STN
SNr
Excitation
(Regardless of color)
Transmitters
ACh
GABA
Glu
+
DA
Inhibition
29
Science, 2009
Optical Deconstruction of Parkinsonian Neural Circuitry
Viviana Gradinaru, (Caltech Bi 2005),
Murtaza Mogri, Kimberly R. Thompson,
Jaimie M. Henderson, Karl Deisseroth
(Bioengineering, Stanford)
Viviana Gradinaru, Assistant Professor of Biology at Caltech
30
“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
31
Transgenic mice (Thy1) expressing channelrhodopsin in
layer V cortical neurons & their axons . . .
Similar effects with cortical stimulation.
Tentative Conclusion:
DBS works via stimulating passing axons
Optical 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
32
Some requirements for further progress
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
33
End of Lecture 9
34