*Bi 150 Lecture 9 Friday, October 18, 2013Advanced electrophysiologyInward rectifiersGliaA potpourri of contemporary recording and stimulating techniquesHenry LesterKandel has very little material on todays topics; but see p. 24-27, p. 107, p. 115

*Unblocked channel,Inward currentPolyamine-blocked channel,Swept in by outward currentcytosolextracellularInward Rectification:the only voltage-dependent gating mechanism in some K+ channelsspermine

spermidineTotal intracellular conc.> 1 mM in most cells.Binding rate constant ~ 108 /M/s x 10-3 M ~ 105/s,Therefore block occursIn ~ 10 s.

*

From Lecture 1

*If many channels are open, much current flows . . . and the ions must be pumped back, using energy gNagNamostly K+gKgK

*If inward rectifier K+ channels close, the cell requires fewer Na+ channels,saving energy gNagNamostly K+gKgK

*Cardiac tissue is depolarized for ~ 50% of ones lifeMost 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).

Well discuss G protein-gated inward rectifier K+ channels next week.

*A. Oligodendrocyte (CNS)produces myelinIn white matter

B. Schwann cell(Peripheral NS) produces myelin Three types of glial cellsC. Astrocyte (CNS)Plays several support rolesFigure 2-5

*One rarely sees a bare neuron.There is usually a surrounding glial cell(in this case, a Schwann cell)Figure 11-1

*There is very little extracellular space in the CNSAstrocytes occupy ~ 5% of the volume and provide supporting pathways to maintain the extracellular space 1 m

*Endothelial cells lining the capillaryRed blood cellsGlial end feet surround brain capillaries, but they dont form the blood-brain barrierBlood vesselBlood Glial end foot

*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

*Extracellular recording with pipette electrodes

Tetrodes

Wireless recording Microdevice arrays

Direct imaging

Single-unit recording in humans

Advanced (electro)physiology

Single-unit recordings can sometimes distinguish neuronal types in vivomouse*

*Tetrode carrier (Thanos Siapas)

*Tetrodes (Thanos Siapas)

Highly Stable Prefrontal Cortex Tetrode Recordings (Thanos Siapas)*

*Nanofabricated Multiplexed Electrode ArraysDu J, Blanche TJ, Harrison RR, Lester HA, and Masmanidis SC (2011) PLoS ONEScott KM, Du J, Lester HA, and Masmanidis SC (2012) J Neurosci Methods

*A wireless multi-channel neural amplifier for freely moving animalsTobi A Szuts . . . Evgueniy V Lubenov (Caltech postdoc), Athanassios G Siapas (Caltech Prof) Markus Meister (Harvard -> Caltech), 201140 g total, using 2005 technology . . .Could presumably be 4 g now (light enough for a mouse)

*Dombeck et al

*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 transientsRunning Grooming Dombeck et alhttp://www.jneurosci.org/content/vol29/issue44/images/data/13751/DC1/Movie_S2.movhttp://www.jneurosci.org/content/vol29/issue44/images/data/13751/DC1/Movie_S1.mov

*1. Electrical stimulation:PacemakersTranscutaneous stimulation for back painDeep brain stimulation for Parkinsons diseaseCochlear implantsRetinal prostheses

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

Advanced stimulation

*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 Parkinsons DiseaseImplanted stimulating electrodes retune this loop.More about the mechanism, later in todays lecture.

*Transcranial magnetic stimulation, Used in Shimojo lab at CaltechA 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 HzNot yet approved for therapeutic use in US.

The channelohm is 2% of the human genome, A nicotinic acetylcholine receptor / channel:~ 2200 amino acids in 5 chains (subunits)Voltage (actually, E ~107 V/m)External transmitterInternal transmitterLightTemperatureForce/ stretch/ movementBlockersNernst potential forNa+,K+,Cl-,Ca2+,H+1/r = 0.1 100 pSand many other organisms expand the repertoire*

Pharmacological neuronal silencing: Re-engineering a Cys-loop receptor channelIvermectin (IVM) made by bacteria,used as antiparasitic in animals and humans (River blindness / Heartgard) Allosterically activates GluCl channels

Slimko, McKinney, Anderson, Davidson, Lester (2002) J Neurosci; Frazier, Cohen, Lester (2013) J Biol Chem. 0 nM IVM1 nM IVM20 nM IVM

1Department of Bioengineering, 2Program in Neuroscience, 3Department of Neurosurgery, 4Department of Psychiatry and Behavioral Sciences,Stanford University, Stanford, CA94305, USA.More engineering of the channelohm with Light*Shapiro MG, Frazier SJ, and Lester HA (2012) ACS chemical neuroscience

Illumination evokes photocurrents in ChR2-positive cortical neuronsWang H et al. PNAS 2007;104:8143-8148*

Illumination controls number and frequency of action potentialsWang H et al. PNAS 2007;104:8143-8148*

*AChAChGPiDirectPathwayGPeThalamusCortexExcitationInhibition(Regardless of color)INsAChGABAGluINsTransmittersdorsalstriatumSTN+SNr=SNc?PPTgMSN D1RMSN D2RIndirect pathwayINsDATremor arises in a malfunctioning feedback loop: substantia nigra, striatum, and other structures.

Implanted stimulating electrodes retune this loop.Deep brain stimulation for Parkinsons DiseaseEarlier today

*Optical Deconstruction of Parkinsonian Neural Circuitry

Viviana Gradinaru, (Caltech Bi 2005), Murtaza Mogri, Kimberly R. Thompson,Jaimie M. Henderson, Karl Deisseroth(Bioengineering, Stanford)Science, 2009Viviana 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*

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*

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 peopleSome requirements for further progress *

*End of Lecture 9

***Illumination evokes photocurrents in ChR2-positive cortical neurons. (A) Prolonged light flash (top trace) generated a photocurrent (middle trace) in a neuron that expressed ChR2. No photocurrent was observed in a ChR2-negative neuron from a wild-type mouse (bottom trace). (B) The peak amplitude of photocurrents (lower trace) increased as a function of light intensity (upper trace). (C) Relationship between photocurrent amplitude and light intensity (100-ms duration); points indicate means SEM for seven neurons. Curve is a fit of the Hill equation. (D and E) Photocurrent (bottom traces) varied with light duration (top traces). (D) Responses to brief light pulses (1- to 8-ms durations). (E) Responses to longer duration flashes (10- to 100-ms durations). (F) Relationship between photocurrent amplitude and photostimulus duration (9.2 mW/mm2 luminance). Curve is fit of the Hill equation.Illumination controls number and frequency of action potentials. (A) Light of varying luminance elicited graded changes in membrane potential and varying numbers of action potentials. (B) Relationship between light intensity and number of light-evoked action potentials (n = 4). Curve is fit of the Hill equation. (C) Varying the frequency of brief light flashes (4 ms duration; 9.2 mW/mm2) caused proportional changes in action potential frequency. (D) Mean probability of evoked action potentials decreased with light pulse frequency (n = 4). Curve is a Lorentzian function, with a roll-off frequency of 35 Hz.*