Bi 150 Lecture 9 Friday, October 18, 2013 Advanced electrophysiology Inward rectifiers Glia

1. Bi 150 Lecture 9 Friday, October 18, 2013 • Advanced electrophysiology • Inward rectifiers • Glia • 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

2. Inward Rectification: the only voltage-dependent “gating” mechanism in some K+ channels 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. spermine spermidine extracellular cytosol Polyamine-blocked channel, Swept in by outward current Unblocked channel, Inward current

3. H2O K+ ion carbonyl From Lecture 1

4. + + E G E G E G D = K K Na Na Cl Cl V + + GNa G G G GK K Na Cl = ENa (+60 mV) EK (- 60 mV) If many channels are open, much current flows . . . and the ions must be pumped back, using energy gNa gK gNa mostly K+ gK

5. + + E G E G E G D = K K Na Na Cl Cl V + + GNa G G G GK K Na Cl = ENa (+60 mV) EK (- 60 mV) If inward rectifier K+ channels close, the cell requires fewer Na+ channels, saving energy gNa gK gNa mostly K+ gK

6. 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. ~ 1 s An inward rectifier functions like a “latch on a cabinet door” (Hille). We’ll discuss G protein-gated inward rectifier K+ channels next week.

7. glue several branches Three types of glial cells 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

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

9. 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

10. “Tight junctions” form the blood-brain barrier Glial end feet surround brain capillaries, but they don’t form the blood-brain barrier Blood vessel Blood Glial end foot Endothelial cells lining the capillary Red blood cells

11. 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.

12. Advanced (electro)physiology Extracellular recording with pipette electrodes Tetrodes Wireless recording Microdevice arrays Direct imaging Single-unit recording in humans

13. Dopamine neuron, ~ 1700 spikes 4*, 6*, and/or 7 6 Nicotine injection Frequency, Hz 4 2 A B C D VTA 0.05 mV 2 ms DAergic 0 25 4* only GABAergic 20 0.1 mV Frequency, Hz 0.5 ms 15 10 V GABAergic neuron (5 s smoothing), ~ 8300 spikes 5 0 100 200 300 400 500 600 700 0 s Single-unit recordings can sometimes distinguish neuronal types in vivo mouse

14. Tetrode carrier (Thanos Siapas)

15. Tetrodes (Thanos Siapas)

16. Highly Stable Prefrontal Cortex Tetrode Recordings (Thanos Siapas)

17. 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

18. 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)

19. Inventor of fMRI Dombeck et al

20. 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. 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 head-restraint bar microscope objective lens 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

21. 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

22. dopaminergic neurons die in PD 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. 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.

23. 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.

24. 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 Binding region Switches Resistor Battery = Membrane region 1/r = 0.1 – 100 pS Nernst potential for Na+, K+, Cl-, Ca2+, H+ Colored by subunit (chain) Cytosolic region (incomplete) A nicotinic acetylcholine receptor / channel: ~ 2200 amino acids in 5 chains (“subunits”)

25. 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.

26. 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. Shapiro MG, Frazier SJ, and Lester HA (2012) ACS chemical neuroscience halorhodopsin channelrhodopsin

27. Illumination evokes photocurrents in ChR2-positive cortical neurons Wang H et al. PNAS 2007;104:8143-8148

28. Illumination controls number and frequency of action potentials Wang H et al. PNAS 2007;104:8143-8148

29. Deep brain stimulation for Parkinson’s Disease Earlier today Cortex Tremor arises in a malfunctioning feedback loop: substantia nigra, striatum, and other structures. Implanted stimulating electrodes retune this loop. INs INs INs ? ACh MSN D2R MSN D1R dorsal striatum Indirect pathway Direct Pathway ACh = SNc Thalamus GPe PPTg Axons passing through GPi STN + SNr Excitation Transmitters (Regardless of color) DA Glu GABA ACh Inhibition

30. 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

31. “We used optogenetics and solid-state optics to systematicallydrive or inhibit an array of distinct circuit elements in freelymoving parkinsonian rodents and found that therapeutic effectswithin the subthalamic nucleus can be accounted for by directselective 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 reducingpathological rotational behavior, but despite precise targetingand robust physiological efficacy of halorhodopsin inhibition, the hemiparkinsoniananimals did not show even minimal changes in rotational behaviorwith direct true optical inhibition of the local excitatorySTN neurons .” Channelrhodopsin driven by CAM kinase II promoter, also ineffective. c-fos (biochemical markerof neuronal activation) showed that at > 0.7 mm3 , nearly the entire STN is recruitedby light stimulation. Glial promoter-drive channelrhodopsin, Also ineffective. Gradinaru et al, 2009

32. 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

33. 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

34. End of Lecture 9