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September 27, 2017

Introduction to CNS neurobiology: focus on retina

The retina is part ofthe CNS

Calloway et al., 2009)

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Retinal circuits: neurons and synapses

Sherry, 2002

Rods and Cones

Bipolar cellsHorizontal cells(Mueller Glia)Amacrine cells

Retinal ganglioncells

1. Dendrites

2. Cell body -Soma

3. Axon hillock Axon initial segment

4. Synaptic terminal

Dendrites of post-synaptic cell

Neuron

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Single cell recordingRemote referenceelectrode, outside ofthe cell

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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

Depolarization: Greater than resting potential

Less than resting potential

Return toresting potential

Membrane potentials

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Composition: Extracellular & intracellular fluid

Resting Ca2+ concentration in the cytoplasm is 10-100 nM

10-7

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Transport across the cell membrane

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Membrane permeability

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AquaporinsWater channels

202003 Nobel Prize in Chemistry

Peter Agre & Roderick MacKinnon

tetramer

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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

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Ion concentrations and equilibrium potentials

The Nernst Equation

For calculating equilibrium potentials

Eion (mV)= - 61(mV) x log ( [ion conc]in/[ion conc]out)

-61 = RT/F

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The Nernst Equation

Electrochemical equilibriumfor an ion

Eion (mV)= - 61(mV) x log ( [ion conc]in/[ion conc]out)

potassium (K+): -61 X log (150 mM/5 mM) = -91 mVsodium (Na+): -61 X log (14.5 mM/145 mM) = +61 mVchloride (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

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Ohm’s Law E=I*RE = voltageI = currentR = resistance

R= 1/g Resistance (R) = 1/conductance (g)

E=I*1/g; I=E*g

The chord equation for membrane potential

g

+gNa+ X ENa+

g

The membrane potential will depend on the relative conductances for the major ions and the equilibrium potential for those ions.

gK+ X EK+

Em =

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The Donnan EquilibriumPrior 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+]AX [Cl-]A = [Cl-]B X [K+]B

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The Donnan equilibrium A B

[Y-] = 0.1 M[K+ ]= 0.133[Cl-] = 0.033

[K]= [Cl] =0.0666 M

Concentration ratios of diffusible ions are =# of positive and negative ions balance in each compartmentY- -- A nondiffusable large protein, e.g. albumin

.033 x .133 = .0044 .066 x .066 = .0044

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

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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

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• 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 opticnerve

Impulses and circuitshttp://hubel.med.harvard.edu/index.html

Local potential and action potential

Linear summation vs threshold

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Action potential: Tetrodotoxin (TTX) blockade of NaVs

There is no inward sodium (Na+) current

KugelfischPuffer fish

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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

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Propagation of action potentials

Hubel online book

Retina: cells and layersLocal potentials

Action potentials

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Myelin sheath

II. Synapses for neural transmission

Electrical – gap junctions

Chemical – classical pre and postsynaptic membrane- vesicular release

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Junctionsbetween cells

Gap Junctions

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Copyright ©2009 The American Physiological Society

Abd-El-Barr, M. M. et al. J Neurophysiol 102: 1945-1955 2009;doi:10.1152/jn.00142.2009

Schematic diagram of 6 rod and cone synaptic pathways(note the gap junctions (ww)

Chemical Synapse -axodendritic

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Chemical Synapse -axodendritic

Exocytosis - vesicular release and the importance of calcium

Sudhoff – In Ganong Review of Medical Physiology

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Receptors

G-protein-mediated signal transduction pathways

Second messengers

Ionotropic and metabotropic receptors

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Ionotropic & metabotropic glutamate receptors

Synaptic transmission – glutamate is the major neurotransmitter in CNS and retina

Glutamate

Ionotropic(GluR)KainateAmpaNMDA

MetabotropicmGluR

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Ionotropic and metabotropic glutamate receptors

Ionotropic Metabotropic

Retinal glutamate receptor types

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Ionotropic & Metabotropic Receptors(GABA receptors in this example)

Ganong, Review of Medical Physiology

Neurotransmitters

m: metabotropic i: ionotropic

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Heterotrimeric G-proteins

Examples of G-protein–coupled receptors and common effectors

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Signal transduction cascades:at each stage, amplification may occur

Gs and Gi: stimulation or inhibition of AC, and formation of cAMP

Beta receptorsEpinephrineNorepinephrine

Dopamine receptors(D1, D3)

Alpha-2Norepi

Dopamine r (D2,D4)

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Phototransduction – a well studied G-protein cascade

Rhodopsin2 adrenergic receptor

Rhodopsin is a G proteincoupled receptor (GPCR)

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Visual pigments: seven membrane-spanning loops

Photoreceptors in primates: humans and monkeys

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Spectral Sensitivity

Visual pigments: homologies in amino acid sequences

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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

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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

Retina

RPE FIGURE 3. Schematics of two visual cycles in vertebrate eye. The canonicalRPE visual cycle (left) recycles all-trans-retinol (at ROL) released from rods andcones following a bleach to 11-cis-retinal (11c ROL), which can be used byboth rods and cones for pigment regeneration. The retina visual cycle (right) relies on the Müller cells to recycle all-trans-retinol released from cones to11-cis-retinol, which only cones can move to their outer segments and oxidizeto 11-cis-retinal for regeneration of the pigment. IPM, interphotoreceptormatrix. h, photon of light

Kefalov, JBC 2012

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Biochemical steps in the phototransductioncascade

Phototransduction cascade

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Current flow around photoreceptors

Rod photocurrents:prolonged responses

Cone photocurrents:brief responses

Rods are 70-100 times more sensitive than cones

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

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