1. 1 Bi / CNS 150 Lecture 10 Synaptic inhibition; cable properties of neurons; electrical integration in cerebellum Monday, October 19, 2015 Henry Lester Chapter 2 (p. 22-30); Chapter 10 (223-234)

2. The pentameric GABA A and glycine receptors look like ACh receptors; but they are permeable to anions (mostly Cl -, of course) 1.  -amino-butyric acid (GABA) is the principal inhibitory transmitter in the brain. 2. Glycine is the dominant inhibitory transmitter in the spinal cord & hindbrain. GABA A receptors are more variable than glycine receptors in subunit composition and therefore in kinetic behavior... Cation channels become anion channels with only one amino acid change per subunit, in this approximate location Like a previous lecture

3. 3 Homomer of 5 GABA A β3 subunits Overall structure similar to nicotinic receptor Crystal structure of GABA A Receptor (Miller et al, 2014)

4. A Synapse “pushes” the Membrane Potential Toward the Reversal Potential (E rev ) for the synaptic Channels 4 At E rev, the current through open receptors is zero. Positive to E rev, current flows outward Negative to E rev, current flows inward ACh and glutamate receptors flux Na + and K +, (and in some cases Ca 2+ ), and E rev ~ 0 mV. -20 -50 -80 -100 -5 +20 +40 +60 +80 Membrane potential Resting potential E K E Na At GABA A and glycine receptors, E rev is near E Cl ~ -70 mV Like Figure 10-11

5. 5 Benzodiazepines (= BZ below): Valium (diazepam), (Ambien, Lunesta are analogs) Pharmacology of GABA A Receptors: activators phenobarbital site is unceratin The natural ligand binds at subunit interfeces (like ACh at ACh receptors) More on neuropharmacology: Bi 155, January 2017

6. 6 GABA A and Glycine blockers bind either at the agonist site or in the channel Agonist site Picrotoxin (GABA A & glycine receptors) Strychnine (glycine receptor) Bicuculline (GABA A receptor

7. How does the receptor transduce binding into channel gating? (prev. lecture) 7 OPEN CLOSED Twist? Corringer, J Physiol 2010 Swivel? Miyazawa, Nature 2003... Both ideas are also in play for GABA or glycine receptors

8. We have Completed our Survey of Synaptic Receptors 8 A. ACh, Serotonin 5-HT3, GABA, (invert. GluCl, dopamine, tyrosine) receptor-channels Most ^ Figure 10-7

9. 9 Like Figure 2-1 (rotated) Parts of two generalized CNS neurons synaptic cleft direction of information flow apical dendrites Excitatory terminal cell body (soma) nucleus axon presynaptic terminal postsynaptic dendrite Inhibitory terminal presynaptic terminal axon hillock neuron Presynaptic neuron Postsynaptic basal dendrites initial segment node of Ranvier myelin (apex) (base) little hill

10. 10 Molecular layer Purkinje cell layer Ganule cell layer White matter Figure 42-4 10% of the neurons in the CNS are cerebellar granule cells The cerebellum: a famous circuit in neuroscience. In today’s lecture, it exemplifies pre- and postsynaptic structures.

11. 11 A plurality of synapses in the CNS (> 10 13 ) occur between parallel fiber axons and Purkinje cell dendritic spines 500 nm Molecular layer Like Figure 42-5

12. 12 Types of synapses (Don’t mind the Type I, Type II stuff) Figure 10-3

13. 13 1. Temporal A. Molecular lifetimes B. Capacitive filtering 2. Spatial 3. Excitatory-inhibitory Types of synaptic integration

14. 14 Concentration of acetylcholine at NMJ (because of acetylcholinesterase, turnover time ~ 100 μs) Number of open channels ms 0 high closed open State 1State 2 k 21 all molecules begin here at t= 0 units: s -1 Synaptic integration 1A. Molecular lifetimes Previous lecture

15. 15 What causes the ~ δ-function of glutamate & GABA at CNS synapses? Na + -coupled transporters for glutamate & GABA are present at densities of > 1000 / μm 2 near each synapse, probably high enough to sequester each transmitter molecule as it leaves a receptor (more on this topic, later today) At the nerve-muscle synapse, acetylcholinesterase is present at densities of > 1000 / μm 2 near each synapse, high enough to destroy each transmitter molecule as it leaves a receptor Figure 4-17

16. 16 Synaptic Integration 1B. Capacitive filtering Figure 9-6

17. 17 1B. Temporal Summation 2. Spatial summation Recording Synaptic Current Synaptic Potential Long time constant (100 ms) Short time constant (20 ms) Axon Synaptic Current Synaptic Potential Long length constant (1 mm) Short length constant (0.33 mm) VmVm VmVm 2 mV 25 ms Improved from Figure 10-14 ~ 100 pA

18. 18 1. If dendrites were passive, they would act like leaky cables... Gulledge & Stuart (2005) J. Neurobiol 64:75, V EPSP measured in soma V EPSP measured in dendrite Excitatory synapses

19. 19... and passively integrate inputs... Gulledge & Stuart (2005) J. Neurobiol 64:75, Δt = 0 Simultaneous, colocalized EPSPs (two individual trials) V Nearly simultaneous, colocalized EPSPs (two individual trials) V Δt = 5 ms Simultaneous, Spatially distinct EPSPs V Δt = 0 Prolonged rising phase http://www.neuron.yale.edu/neuron/static/about/stylmn.html Inspect the simulation, and run the movie, at

20. 20... but two-photon microscopes allow researchers to visualize patch-clamped dendrites in living animals... Gulledge & Stuart (2005) J. Neurobiol 64:75,

21. 21 immunocytochemistry 25 μm Whitaker, Brain Res, 2001 Magee & Johnston, J Physiol (1995) Now break the patch, to fill the cell with dye: Averaged traces * = axon hillock... dendrites are not passive. They have Na channels

22. 22... voltage-gated Na + and Ca 2+ channels in dendrites lead to partial “backpropagation” of action potentials, implying that parts of cells can process signals semi-independently. Stay tuned! Gulledge & Stuart (2005) J. Neurobiol 64:75, brain slice

23. 23 3.Excitatory-inhibitory integration: The “veto principle” of inhibitory transmission Inhibitory synapses work best when they are “near“ the excitatory event they will inhibit. “Near” means < one cable length. A. Inhibitory synapses on dendrites do a good job of inhibiting EPSPs on nearby spines B. Inhibitory synapses on cell bodies and initial segments do a good job of inhibiting spikes

24. 24 “Veto” inhibition at the axon initial segment: Schematic of a GABAergic “chandelier cell” in human cerebral cortex Ch terminals from Felipe et al, Brain (1999) 122, 1807 Ch. axon Inhibitory Chandelier Cell Ch terminals Pyramidal Cells

25. 25 Molecular layer Purkinje cell layer Ganule cell layer White matter Now we localize the inhibitory “vetos” of cerebellar Purkinje cells by “pinceaux” (paintbrushes) of basket cells Figure 42-4

26. 26 NH 2 A fusion protein: GABA transporter (GAT1)-GFP How to localize and quantify inhibitory synapses

27. 27 cerebellum

28. 28 Molecular layer (basket cells stain) Purkinje cell layer “pinceux” (paintbrushes) stain heavily Granule cell layer <Immunocytochemistry For GABA transporter

29. 29 Molecular layer (basket cells stain) Purkinje cell layer “pinceaux” stain heavily, showing soma-hillock “veto” Granule cell layer mGAT1 GFP knock-in fluorescence > <Immunocytochemistry For GABA transporter

30. 30 GAT1-GFP expression in cerebellum: basket cell terminals in molecular layer, Showing dendritic “veto” GABA transporter density is ~1000/( μ m 2 ) 50  m

31. 31 End of Lecture 10 HAL’s Office Hours, as usual Mon 1:15-2 Red Door