Basic

neurochemistry
Colm Cunningham

Basic Neurochemistry

Dr. Colm Cunningham

colm.cunningham@tcd.ie

The brain contains neurons

GLIA la#n glue (Virchow, 1850s)

neurons @ 85 billion, about 10% Macroglia
Astrocytes (most abundant 55%) Oligodendrocytes 30%

Microglia - about 5%

Meet the family

1 oligodendrocyte 2 axon hillock 3 neuronal soma 4 myelin sheath 5 microglia 6 astrocyte 7 synapse 8 blood vessel

Glial funcUons
Oligodendrocytes (& Schwann cells) - myelinaUon Microglia - immune funcUon scavengers Astrocytes - NT reuptake - isolaUon/insulaUon - BBB - migraUon (radial) - NMJ synapUc reg.

The cells The Neurons The Neuron The contents

bioenergeUcs

Cajal, neurons, glia, glial metabolism types, projecUons, tracers structure, regions neurotransmi[ers, metabolism, vesicles,

The acUon (presynapUc) Na+-K+ ATPase, acUon potenUal, depolarisaUon,

Ca2+ channels, synapUc release

The acUon (post-synapUc) receptors, signalling (ionotropic, metabotropic) diversity of receptors and responses DysfuncUon incidental; paralysis, depression, excitotoxicity,

recreaUonal drugs, disease

 Neuronal types

Neurons of the hippocampus

Cajal 1893

Neuronal communicaUon
Structual - cell, axon, synapse Chemical - neurotransmi[er release

Glutamate and GABA

Major Excitatory, inhibitory NTs respecUvely Short axons and Interneurons - local signalling

COO-

+ H

I CH2 I CH2 I COO-

3N-CH2

I +H N-C-H 3 I CH2 I CH2 I COO-

GABA glutamate

Tract tracing (1970s)

Anterograde (soma - terminal) Phaseolus vulgaris leucoaggluUnin (10 days) BiocyUn (48 hrs) Retrograde (terminal - soma) Horseradish peroxidase (axons & synapses) Wheatgerm aggluUnin-HRP Cholera toxin-HRP (more specic)

Immunohistochemistry
BiosyntheUc enzymes characterisUc of specic populaUons

Dopaminergic system

 Noradrenergic projecUons

5HT (serotonin) projecUons

5 criteria for designaUon as a neurotransmi[er

Synthesis in neuron Veriable release from neuron Eect on post-synapUc neuron
Exogenous applicaUon should have same eect on post-synapUc cells as electrical sUmulaUon of the pre-synapUc terminal

Specic post-synapUc receptor

Blocked by specic antagonists and mimicked by agonists

Appropriate terminaUon mechanisms Reuptake, degradaUon

Neurotransmi[ers
Biogenic amines Amino acid neurotransmi[ers Other types: Acetylcholine, NO, D-serine, neuropepUdes Acetyl CoA + choline = CH3-C-O-CH2-CH2-N-(CH3)3
O

Noradrenaline & Dopamine biosynthesis

PD

catecholamines

Serotonin (5-HT) biosynthesis

indoleamines

GABA & glutamate

GABA made from Glutamate by decarboxylase GABA transaminase GABA + 2-oxoglutarate to succinic semialdehyde + glutamate
GAD
-OOCCCH

if

2CH2CCOO

=O

=O

GABA
GABA made from Glutamate by decarboxylase GABA transaminase GABA + 2-oxoglutarate to succinic semialdehyde + glutamate
2
-OOCCCH

if

2CH2CCOO

=O

=O

Neurotransmi[er vesicles

SSV - small synapUc vesicles - 35 - 60 nm - classical neurotransmi[ers - packed anywhere in cell - synapse LDCV - large dense core vesicles

- larger size - NTs and neuropepUdes (co-release) - cell body - ER - golgi - secretory granules - harder to release, not reusable (not classical NTs)

Vesicular uptake

BioenergeUcs - proton electrochemical gradient - vesicular ATPase - drives protons in (pH 5.5) - this drives NT against its chemical gradient - 2 protons for one NT - up to 100 mM NT - low anity (0.3 mM)

Uptake and storage inhibitors

Bind to transporter Inhibit uptake anU-psychoUc drugs
tetrabenazine

Amphetamine and Ecstasy also deplete Tyramine false transmi[er - its Similar enough to get Packaged, but Decrease ecacy Treat Hypertension PEA

5 criteria for designaUon as a neurotransmi[er

Synthesis in neuron Veriable release from neuron Eect on post-synapUc neuron
Exogenous applicaUon should have same eect on post-synapUc cells as electrical sUmulaUon of the pre-synapUc terminal

Specic post-synapUc receptor

Blocked by specic antagonists and mimicked by agonists

Appropriate terminaUon mechanisms Reuptake, degradaUon

SynapUc vesicle cycle

1 2 3 4 5 6 acUve lling of vesicles Clustering in region of acUve zone Docking of a subset of these at AZ Priming to make competent for Ca2+-dependent exocytosis Ca2+ triggering of exocytosis (fusion pore opening) Recycling

Axon conductance
ResUng membrane potenUal of -65mV w.r.t. to extracellular Membrane channels permeable to K+ so it leaks out and leaves localised negaUve charge, Likewise Na+ leaks in Na+-K+ Pump maintains high intracellular K+ and low Na+ despite this leakage (against electrochemical gradients (Hydrolysis of ATP)) Upon sUmulaUon, voltage-gated Na+ channels open, Na+ ows in, depolarizing the cell (-65 to -55). This opens nearby Na+ channels and the acUon potenUal is propagated along axon.

Conc gradient

SynapUc potenUal
High intracellular K+ (400mM vs 20) High extracellular Na+ (440mM vs 50) Leakage of both by passive diusion down concentraUon gradient
2
ResUng potenUal = -60 to -70mV

ATP
AcUve (ATP) pumping of both against gradient - electrogenic (3+ vs 2+)
3

AcUon potenUal arrives and voltage-gated Na+ channels open (Na+ in) - depolarisaUon Causes voltage gated K+ channels to open (K+ out) - repolarisaUon

IniUally thought that Na+ inux was the chemical trigger for release

Experimental evidence
Katz & Miledi, 1967

(chapter 14 Kandel)

Na+ entry is important (expts with tetrodotoxin :Na+ channel blocker). But only in so far as it depolarises the membrane suciently to generate the acUon potenUal that facilitates transmi[er release. Direct applicaUon of a similar acUon potenUal produces normal release K+ channel blockers like tetraethylamonium do not prevent release (infact extend release period by blocking repolarizaUon). Ca2+ : increasing extracellular Ca increase NT release (opp for decrease) 10,000 fold excess of Ca extracellularly with respect to cellular Therefore Ca would need to get into the cell Voltage-dependent Ca channels discoved (Llinas et al., 1977)

Neurotransmi[er release
Na+ channels open Voltage change above 40mV (depolarisaUon) Voltage-dependent Ca2+ channels open - Ca2+ ows down concentraUon gradient Low anity Ca2+ sensors in acUve zone (100m) bind Ca2+ Ca2+-dependent exocytosis And rapid turn o as Ca2+ diuses away from acUve zone
3 2
ResUng potenUal = -60 to -70mV

Ca2+ 104 higher

Neurotransmi[er release

SNARE proteins

SNARE complex and exocytosis

SNARE - SNAP receptor proteins SNAP-soluble NSF attachment Prot NSF-N-ethylmaleimide-sensitive fusion protien

The SNARE complex is a four-helix bundle, formed between the vesicle and cell membranes. Synaptobrevin (blue) is partly inside the vesicle (R SNARE) Syntaxin (red) is partly within the cell membrane (Qa SNARE). SNAP-25, a third SNARE complex protein, is shown in green and yellow (two SNARE motifs, Qb & c).

Vesicle docking: Toxins

TETANUS

SNARE complex
Bluesynaptobrevin Red-syntaxin-1A Green-SNAP-25B
Toxin cleavage sites are located: at the peptide bonds between Gln-76 and Phe-77 (TeNT and BoNT/B), between Ala-81 and Ala-82 (BoNT/G) for synaptobrevin-II, between Lys-253 and Ala-254 (BoNT/C) for syntaxin-1A, and between Gln-197 and Arg-198 (BoNT/A) for SNAP-25B. Fasshauer et al, 1998

Neurotransmi[er release

PhosphorylaUon Kinases DephosphorylaUon Phosphatases

Sudhof

Fusion/exocytosis

Toxins aecUng release/transmission

Tetrodoxin - puersh - Na+ channel blocker - death Clostridial toxins Botulinum A,B,C cleave SNAP 25, VAMP, syntaxin respecUvely (BOTOX: ACh) Tetanus toxin cleaves VAMP (zinc endopepUdase) Disrupt THE key interacUon in neurotransmi[er release (g 14-13) Dendrotoxin - black mamba - K+ channels Latrotoxin - black widow spider - massive acetylcholine release -bungarotoxin - snake venom- ACh nicoUnic receptor on skeletal muscle (paralysis)

Neurotransmi[er release - its fast - motor funcUon

Whole cycle has to be repeated rapidly for repeat acUons !!! RECYCLING

SNARE disassembly

5 criteria for designaUon as a neurotransmi[er

Synthesis in neuron Veriable release from neuron Eect on post-synapUc neuron
Exogenous applicaUon should have same eect on post-synapUc cells as electrical sUmulaUon of the pre-synapUc terminal

Specic post-synapUc receptor

Blocked by specic antagonists and mimicked by agonists

Appropriate terminaUon mechanisms Reuptake, degradaUon

NT acUons/Receptor diversity
Ionotropic - direct gaUng by NT binding Metabotropic - metabolic/signalling changes lead indirectly to ion ux - G protein coupled - many NTs - Receptor tyrosine kinases - hormones, neuropepUdes ACh: nicoUnic (I), muscarinic (M) Glutamate recptors: ionotropic: NMDA, KA, AMPA, metabotropic mGLUR D1-D5 Alpha, beta-adrenergic 5HT: many types Dierent receptors for the same NT on dierent neurons: dierent eects

Acetylcholine
Excitatory or inhibitory Receptors: NicoUnic - ionotropic Muscarinic - metabotropic Visible acUon at NMJ

Neuromuscular juncUon - ACh

NicoUnic Acetylcholine receptor - Myasthenia Gravis

Alpha-bungarotoxin

Acetylcholinergic terminal

Metabolism - ChAT, AChE, no-reuptake AD, Learning and memory, mostly muscarinic (metabotropic receptors)

ionotropic
permeability

metabotropic

Glutamate receptors

Signalling G protein mechanisms cAMP

5HT binding Exposure of G protein binding domain GDP exchanged for GTP Gs alpha subunit released AcUvaUon of adenylyl cyclase ProducUon of cAMP, Hydrolysis of GTP (to terminate cAMP producUon) cAMP-dependent Protein Kinase Reforming of G , , complex DissociaUon of 5HT

Signalling mechanisms
G alpha acUvates PLC Cleaves phospholipids Makes DAG & IP3 Releases Ca2+ from ER

Ca2+ acUvates: PKC Ca2+/calmodulin-dependent PK PhosphorylaUon & cellular response

Receptor diversity

Receptor diversity

5 criteria for designaUon as a neurotransmi[er

Synthesis in neuron Veriable release from neuron Eect on post-synapUc neuron
Exogenous applicaUon should have same eect on post-synapUc cells as electrical sUmulaUon of the pre-synapUc terminal

Specic post-synapUc receptor

Blocked by specic antagonists and mimicked by agonists

Appropriate terminaUon mechanisms Reuptake, degradaUon

Re-uptake
Rapidly diluUng neurotransmi[er

Q
Co-transport with Na+ (high) Counter transport of K+(balance) Balance is achieved with Cl- for other transmi[ers Dierent transporter families No reuptake for ACh - AChE

Glutamate uptake
Release GLT1/EAAT1 uptake Glutamine synthetase GLU to GLN Export Glutaminase in neuron

GABA transaminase

GABA
GABA transaminase
-OOCCCH

if

GABA + 2-oxoglutarate to succinic semialdehyde + Glutamate SSADH deciency -GHB

2CH2CCOO

=O

=O

Q
2

Catabolism of amine neurotransmi[ers

COMT

COMT

5-HT catabolism

MAO - monoamine oxidase

Outer mitochondrial membrane Neurons, glia, endothelial cells, gut, liver MAO A - clorgyline (In), 5HT selecUve MAO B - selegiline (deprenyl) (In), PEA, Benzylamine Both - NA, dopamine, tyramine, tryptamine, adrenaline

MAO Inhibitors
First anU-depressants

2nd 1st

3rd

MAO inhibitors

Tyramine
Hypertensive crisis (the cheese reacUon) Irreversible A/B non-specic or MAO A inhibitors Reversible I - OK MAO B I - OK Brain selecUve - OK

Three classes of AnU-depressants

Serotonin acUon/inhibiUon

 RecreaUonal drugs

Noradrenergic synapse

MAO inhibitor

Tricyclics COMT inhibitor

Dopaminergic synapse

Ischemic stroke
Blood supply

Ischemic stroke
Blood supply Oxygen, glucose

Ischemic stroke
Blood supply Oxygen, glucose OxidaUve phosphorylaUon

Ischemic stroke
Blood supply Oxygen, glucose OxidaUve phosphorylaUon ATP, increased ROS (complex IV)

Ischemic stroke
Blood supply Oxygen, glucose OxidaUve phosphorylaUon ATP, increased ROS (complex IV) Collapse of membrane potenUal, reversal of pumps

Ischemic stroke
Blood supply Oxygen, glucose OxidaUve phosphorylaUon ATP, increased ROS (complex IV) Collapse of membrane potenUal, reversal of pumps Massive release of glutamate

Ischemic stroke
Blood supply Oxygen, glucose OxidaUve phosphorylaUon ATP, increased ROS (complex IV) Collapse of membrane potenUal, reversal of pumps Massive release of glutamate Massive ion imbalances, Ca2+ stores

Ischemic stroke
Blood supply Oxygen, glucose OxidaUve phosphorylaUon ATP, increased ROS (complex IV) Collapse of membrane potenUal, reversal of pumps Massive release of glutamate Massive ion imbalances, Ca2+ stores ROS, proteases

Ischemic stroke
Blood supply Oxygen, glucose OxidaUve phosphorylaUon ATP, increased ROS (complex IV) Collapse of membrane potenUal, reversal of pumps Massive release of glutamate Massive ion imbalances, Ca2+ stores ROS, proteases Protein, DNA, membrane damage

Ischemic stroke
Blood supply Oxygen, glucose OxidaUve phosphorylaUon ATP, increased ROS (complex IV) Collapse of membrane potenUal, reversal of pumps Massive release of glutamate Massive ion imbalances, Ca2+ stores ROS, proteases Protein, DNA, membrane damage Cell swelling, rupture

Glutamate & GABA

Excitotoxicity Epilepsy

LacUc acidosis - acidicaUon of cytoplasm

Reducing equivalents: NADH Ox Phos -> ATP NAD+ oxidising power for glycolysis

Glutamate uptake
Na down to 80 mM K up to 20 mM Reversal of GLT1/EAAT2 uptake Elevated [GLU] Further acUvaUon Ca2+ inux

Rossi, 2007

Glutamate & GABA

AnUconvulsants Na+ channels GABA agonists

The cells The Neurons The Neuron The contents

bioenergeUcs

Cajal, neurons, glia, glial metabolism types, projecUons, tracers structure, regions neurotransmi[ers, metabolism, vesicles,

The acUon (presynapUc) Na+-K+ ATPase, acUon potenUal, depolarisaUon,

Ca2+ channels, synapUc release

The acUon (post-synapUc) receptors, signalling (ionotropic, metabotropic) diversity of receptors and responses DysfuncUon incidental; paralysis, depression, excitotoxicity,

recreaUonal drugs, disease