Introduction to CNS neurobiology: focus on retina

Transcription

1 Introduction to CNS neurobiology: focus on retina September 27, 2017 The retina is part of the CNS Calloway et al., 2009) 1

2 Retinal circuits: neurons and synapses Rods and Cones Bipolar cells Horizontal cells (Mueller Glia) Amacrine cells Retinal ganglion cells Sherry, 2002 Neuron 1. Dendrites 2. Cell body -Soma 3. Axon hillock Axon initial segment 4. Synaptic terminal Dendrites of postsynaptic cell 2

3 Single cell recording Remote reference electrode, outside of the cell 3

4 Membrane potentials Resting potential (example: -70 mv) Hyperpolarize (more negative than resting potential, e.g. -90 mv) Depolarize (less negative than resting, e.g -40 mv) may lead to action potentials. Repolarize return to resting potential Membrane potentials Depolarization: Greater than resting potential Return to resting potential Less than resting potential 4

5 Composition: Extracellular & intracellular fluid 10-7 Resting Ca 2 + concentration in the cytoplasm is nm 5

6 Transport across the cell membrane 11 Membrane permeability 12 6

7 Aquaporins Water channels tetramer Nobel Prize in Chemistry Peter Agre & Roderick MacKinnon 13 Primary active transport: Na + K + ATPase pump uses energy to pump ions against the concentration gradients. For every cycle of the pump, 3 Na + ions leave the cell and 2 K + ions enter the cell 7

8 Ion concentrations and equilibrium potentials The Nernst Equation For calculating equilibrium potentials E ion (mv)= - 61(mV) x log ( [ion conc] in /[ion conc] out ) -61 = RT/F 8

9 The Nernst Equation Electrochemical equilibrium for an ion E ion (mv)= - 61(mV) x log ( [ion conc] in /[ion conc] out ) potassium (K + ): -61 X log (150 mm/5 mm) = -91 mv sodium (Na + ): -61 X log (14.5 mm/145 mm) = +61 mv chloride (Cl - ): -61 X log (115 mm/3.6 mm) = -90 mv (for neg ion, Cl - ) ( [ion conc] in /[ion conc] out What is conductance, g? conductance g is the inverse of resistance (R) (g=1/r) If membrane ion channels are open, resistance (R) is low, and conductance (g) is high 9

10 Ohm s Law E=I*R E = voltage I = current R = resistance R= 1/g Resistance (R) = 1/conductance (g) E=I*1/g; I=E*g The chord equation for membrane potential E m = g K+ X E K+ g + g Na+ X E Na+ g The membrane potential will depend on the relative conductances for the major ions and the equilibrium potential for those ions. 10

11 The Donnan Equilibrium Prior to the equilibrium A B [Y - ] = 0.1 M [K + ]= 0.1 M [K + ]= [Cl - ] = 0.1 M Y- -- A nondiffusable large protein, e.g. albumin The Donnan Equilibrium Potential difference across the membrane must be equal for K + and Cl - K + Em = -61 * log ([K + ] A /[K + ] B ) Cl- Em = -61 * log ([Cl - ] B /[Cl - ] A ) [K + ] A /[K + ] B = [Cl - ] B /[Cl - ] A [K + ] A X [Cl - ] A = [Cl - ] B X [K + ] B 11

12 The Donnan equilibrium A B [Y - ] = 0.1 M [K + ]= [Cl - ] = [K]= [Cl] = M.033 x.133 = x.066 =.0044 Concentration ratios of diffusible ions are = # of positive and negative ions balance in each compartment Y- -- A nondiffusable large protein, e.g. albumin Donnan equilibrium would cause cells to swell More particles, i.e. higher osmolarity inside the cell (A) than outside the cell (A) Water would move (osmosis) into the cell to offset the difference in number of particles of solute per amount of solvent 12

13 Primary active transport: Na + K + ATPase keeps the Donnan equilbrium from occurring in cells. If it did occur, the cells would burst. Graded (local) potentials and action potentials Graded potentials are generated via ligand gated channels, They are small and can be hyperpolarizing or depolarizing and they scale in amplitude with the strength of the input. Action potentials are all or none events, that have a threshold, and rely on the presence of voltage-gated channels 13

14 Impulses and circuits Neurons sum and integrate information from their inputs and pass information to the next cell. Action potentials (brief impulses) are necessary for signals to travel long distances. Information is coded in local potentials when axons are short, such as for all cells within the retina except for retinal ganglion cells whose axons form the optic nerve Local potential and action potential Linear summation vs threshold 14

15 Action potential: Tetrodotoxin (TTX) blockade of Na V s There is no inward sodium (Na + ) current Kugelfisch Puffer fish 15

16 Action Potential - initiated by depolarization: Conductance (g) changes in voltage-gated channels Action potential: Sea water experiments There is no inward sodium (Na + ) current in sodium-free sea water Only the outward potassium (K + ) current remains 16

17 Propagation of action potentials Hubel online book Retina: cells and layers Local potentials Action potentials 17

18 Myelin sheath II. Synapses for neural transmission Electrical gap junctions Chemical classical pre and post synaptic membrane - vesicular release 18

19 Junctions between cells Gap Junctions 19

20 Schematic diagram of 6 rod and cone synaptic pathways (note the gap junctions (ww) Abd-El-Barr, M. M. et al. J Neurophysiol 102: ; doi: /jn Copyright 2009 The American Physiological Society Chemical Synapse -axodendritic 20

21 Chemical Synapse -axodendritic Exocytosis - vesicular release and the importance of calcium Sudhoff In Ganong Review of Medical Physiology 21

22 Receptors G-protein-mediated signal transduction pathways Second messengers Ionotropic and metabotropic receptors 22

23 Ionotropic & metabotropic glutamate receptors Synaptic transmission glutamate is the major neurotransmitter in CNS and retina Glutamate Ionotropic (GluR) Kainate Ampa NMDA Metabotropic mglur 23

24 Ionotropic and metabotropic glutamate receptors Ionotropic Metabotropic Retinal glutamate receptor types 24

25 Ionotropic & Metabotropic Receptors (GABA receptors in this example) Ganong, Review of Medical Physiology Neurotransmitters m: metabotropic i: ionotropic 25

26 Heterotrimeric G-proteins Examples of G-protein coupled receptors and common effectors 26

27 Signal transduction cascades: at each stage, amplification may occur G s and G i : stimulation or inhibition of AC, and formation of camp Beta receptors Epinephrine Norepinephrine Dopamine receptors (D1, D3) Alpha-2 Norepi Dopamine r (D2,D4) 27

28 Phototransduction a well studied G-protein cascade Rhodopsin is a G protein coupled receptor (GPCR) 2 adrenergic receptor Rhodopsin 28

29 Visual pigments: seven membrane-spanning loops Photoreceptors in primates: humans and monkeys 29

30 Spectral Sensitivity Visual pigments: homologies in amino acid sequences 30

31 AVA-322 : gene for L-opsin - protan defects. AVA-323 : gene for M-opsin - tdeutan defects. Breakthrough non-surgical intravitreal injection method to deliver genes directly to cone cells at the back of the eye. Phototransduction Leads to closure of a cation channel in the plasma membrane. This interrupts the dark current, and hyperpolarizes of the rod or cone photoreceptor The opsin in the outer segments, rhodopsin in rods, catches light and is activated when 11-cis retinal is attached to it 31

32 The visual cycle conversion of all-trans retinol (from the blood) to 11-cis retinal in the retinal pigment epithelium (RPE) Details of the visual cycle RPE Retina FIGURE 3. Schematics of two visual cycles in vertebrate eye. The canonical RPE visual cycle (left) recycles all-transretinol (at ROL) released from rods and cones following a bleach to 11-cis-retinal (11c ROL), which can be used by both rods and cones for pigment regeneration. The retina visual cycle (right) relies on the Müller cells to recycle all-trans-retinol released from cones to 11-cis-retinol, which only cones can move to their outer segments and oxidize to 11-cis-retinal for regeneration of the pigment. IPM, interphotoreceptor matrix. h, photon of light Kefalov, JBC

33 Biochemical steps in the phototransduction cascade Phototransduction cascade 33

34 Current flow around photoreceptors Rod photocurrents: prolonged responses Cone photocurrents: brief responses Rods are times more sensitive than cones 34

35 RPE cells phagocytize outer segments entire OS turns over in less than two weeks 35

36 The retina has two blood supplies: outer (PCA) and inner (CRA) retinal (pg. 9) 36