NEUROTRANSMITTERSYSTEMS

Mary ET Boyle, Ph. D.Department of Cognitive ScienceUCSD

Chemical Synapses can______

A have variable signaling

B mediate complex information (excitatory/inhibitory)

C amplify neuronal signals

D All of the above are correct

Electrical Synapses ______

A have variable signaling

B have gap junction channels

C amplify neuronal signals

D have significant synaptic delay

Three classes of neurotransmitters Amino acids, amines, and peptides

Many different neurotransmittersDefining particular transmitter systems

molecule, synthetic machinery, packaging, reuptake and degradation, etc.

Acetylcholine (Ach) First identified neurotransmitter (Otto Loewi)

Nomenclature (-ergic) Cholinergic and noradrenergic

Introduction

Ligands

Ligand-binding methods Identify natural receptors using radioactive ligands Can be: agonist, antagonist, or chemical

neurotransmitter

Molecular analysis two receptor protein classes Transmitter-gated ion channels

GABA receptors 5 subunits, each made with 6 different subunit

polypeptidespotential for enormous diversity

G-protein-coupled receptors - metabotropic

Ligand: any chemical compound that binds to a specific site on a receptor

Neurotransmitter Chemistry

Evolution of neurotransmitters Neurotransmitter molecules

Amino acids, amines, and peptides ACh is derived from acetyl CoA

Dale’s Principle One neuron, one neurotransmitter

Co-transmitters Two or more transmitters released from one nerve

terminal An amino acid or amine plus a peptide

Studying Neurotransmitter Systems1. Synthesis and storage in presynaptic neuron

Presynaptic neuron should contain a transmitter and the appropriate enzymes needed to synthesize the neurotransmitter.

2. Released by presynaptic axon terminalOne should be able to isolate the transmitter and

characterize its structure using biochemical or molecular biological techniques.

3. Produces response in postsynaptic cellMimics response produced by release of

neurotransmitter from the presynaptic neuron

4. Removal of the transmitter from the synaptic cleft

Neurotransmitter– four steps

Synthesis in presynaptic neuron

Precursors should be present in the appropriate places

Enzymes involved in the process should be in the active form

Enzymes should be localized to the appropriate compartment

Synthesis of the xmtr in presynaptic neuron

2

3

1

Neurotransmitters must have ____

A Synthesis and storage of putative xmtr

B Released by the terminal under normal conditions

C Have a response in the postsynaptic terminal

D All of the above

Transmitter Localization

Transmitters and Transmitter-Synthesizing EnzymesImmunocytochemistry –localize molecules to cells

Primary antibody: Rabbit anti-laminin a1 (basement membrane marker), 1:400

Primary antibody: Rabbit anti-laminin a1 (basement membrane marker), 1:400

In situ hybridizationLocalize synthesis of protein or peptide to a cell (detect mRNA)

Storage of neurotransmitter in terminal

Classical and Peptide transmitters are stored in vesicles

(to protect them from enzymatic degradation)

In vesicles make them available for quick release

A mechanism must be present to transport the xmtr or peptide into the

vesicles (e.g. vesicular transporter)

Storage of the xmtr and/or precursors in the presynaptic nerve terminal

2

3

1

Release of the neurotransmitter into the synaptic cleft

Vesicle fuses with cell membrane to release contents into cleft

Release into the synaptic cleft

Transmitter Release Transmitter candidate: Synthesized and localized in terminal and

released upon stimulation CNS contains a diverse mixture of synapses that use different

neurotransmitters Brain slice as a model

Kept alive in vitro Stimulate synapses, collect and measure released chemicals

Buchner funnel

Binding and recognition of xmtr by target receptors

Neurotransmitters that are released interact with receptors

Two types of receptors on target cells: xmtr-gated and metabotropic

Autoreceptor on pre-synaptic neuron

Binding and recognition of xmtr by target receptors 2

3

1

Removing transmitter is critical to synaptic transmission

Removing transmitter enables a new signal to get through

The synapse would become refractory because of receptor

desensitization due to exposure

Three removal mechanisms: diffusion, degradation and reuptake

Removal of transmitter from the synaptic cleft terminates synaptic transmission

2

3

1

Synaptic Mimicry

Qualifying condition: Molecules evoking same response as neurotransmitters

Microionophoresis:Assess the postsynaptic actions

Microelectrode: Measures effects on membrane potential

AgonistA drug that facilitates the effects of a

particular neurotransmitter on the postsynaptic cell.

AntagonistA drug that opposes or inhibits the effects of a particular neurotransmitter on the postsynaptic cell.

An antagonist drug

A Facilitates the effects of a neurotransmitter

B Opposes or inhibits the effects of a neurotransmitter

C All of the above

D None of the above

Direct agonists and antagonists act directly on the neurotransmitter binding site

Indirect agonists and antagonists act on an alternative binding site and modify the effects of the neurotransmitter

on opening of the ion channel.

Sites on transmitter receptors can bind:• Transmitter• Agonist• Antagonist

Agonist-1 Agonist-2

transmitteragonist-2

antagonist-2

One neurotransmitter can bind to many different receptors.No two neurotransmitters bind to the same receptor

Receptor subtypes can be distinguished by the action of different drugs.

One neurotransmitter can bind to many receptors?

A True

B False

C Don’t know

Cholinergic receptor subtypes -

Responds to Nicotine Tobacco plant derivative Agonist – (means that it mimics ACh)

Blocked by Curare (poison darts) Antagonist – (means that it blocks ACh and Nicotine)

Skeletal muscle & CNS

ACh

nicotinic

• Responds to Muscarine• Mushroom (e.g. Amanita muscaria)• Agonist – (means that it mimics ACh)

• Blocked by Atropine (belladona plant)• Antagonist – (means that it blocks ACh & Muscarine)

• Cardiac muscle & CNS

ACh

muscarinic

The tale of two receptors:

nicotine vs. muscarine

Which type of cholinergic receptor is at the endplate?

A Muscarinic

B Nicotinic

C Both Muscarinic and Nicotinic

D There is no difference they both use ACh

“Black widow spider venom stimulates abnormal release of acetylcholine

The bite of the black widow spider initially produces an increase in neuromuscular activity that leads to painful skeletal muscle spasms and rigidity.

This phase of hyperexcitability is rapidly followed by progressive failure of neuromuscular transmission and paralysis.

The venom in ACh followed by a progressive decline and failure of both spontaneous and induced transmitter release and depletion of presynaptic vesicles.”

Barchi RL. (1999) Defects in Neuromuscular Transmission Can Interrupt Normal Muscle Function

ACh Biosynthesis

Acetyl coenzyme A

choline Choline acetyltransferase

(ChAT)

coenzyme A

Acetylcholine

Which enzyme is used to synthesize ACh?

A AChE

B ChAT

C AChM

D None of the above

Cholinergic (ACh) Neurons

Botulium and the venom of the black widow spider, affect the release of acetylcholine.

Botulinum toxin is produced by clostridium botulinum, a bacterium.

Black widow spider venom has the opposite effect: it stimulates the release of ACh.

ACh Deactivation

AcetylcholinesteraseAChE

Choline gets recycled back to the presynaptic

terminal.

Agonists:

Receptors:Rece

ptor

sub

type

s

Each named for a different chemical agonist

Catecholaminergic Neurons Involved in movement,

mood, attention, and visceral function

Tyrosine: Precursor for three amine neurotransmitters that contain catechol group

Dopamine (DA) Norepinephrine (NE) Epinephrine (E, adrenaline)

Dopamine

Produces both excitatory and inhibitory PSPs

Movement, attention, learning, rewards

AMPT inactivates

tyrosine hydroxylase

Parkinson

Disease

SubstantiaNigra

Ventral Tegmental Area

Norepinephrine

Also in ANS –adrenalin

epinephrine noradrenalin

Final synthesis step is in the

vesicle

Fusaric acid inhibits dopamine-

B-hydroxylase

Excess NE in terminal is

destroyed by MAO-A

Serotonergic (5-HT) Neurons

Amine neurotransmitter

Derived from tryptophan

Regulates mood, emotional behavior, sleep Selective

serotonin reuptake inhibitors (SSRIs) - Antidepressants

Synthesis of serotonin

Serotonin

Mood regulation

Released from varicosities rather

than terminal boutons (like NE)

At least 9 different types of

receptors and autorecptors

SSRI – block reuptake receptors

4. Drug stimulates release of NT (e.g. black widow venom – ACh)

2. Drug inactivates synthetic enzyme; inhibits synthesis of NT(e.g. PCPA – serotonin)3. Drug prevents storage of NT in

vesicles(e.g. reserpine – monoamines)

1. Drug serves as a precursor (e.g. L-Dopa – dopamine dopamine)

5. Drug inhibits release of NT (e.g. botunlinum toxin – ACh )

6. Drug stimulates postsynaptic receptors

(e.g. nicotine, muscarine – ACh )

7. Drug blocks post synaptic receptors (e.g. curare, atropine – ACh )

8. Drug stimulates autoreceptors; inhibits synthesis/release of NT(e.g. apomorphine – dopamine)

9. Drug blocks autoreceptors; increases synthesis/release of NT

(e.g. idazoxan – norepinephrine)

10. Drug blocks reuptake(e.g. cocaine – dopamine)

11. Drug inactivates acetylcholinesterase

(e.g. physostigmine – ACh)

They each have their own receptors and do not interact with each other.

amino acids have an amino group and a carboxyl group in

their chemical structures

Amino Acidergic NeuronsDifferences among amino acidergic neurons quantitative NOT

qualitative Glutamic acid decarboxylase (GAD)Key enzyme in GABA synthesisGood marker for GABAergic neuronsGABAergic neurons are major of synaptic inhibition in the CNS

Synthesized from glucose and other precursors.Exist in all cells.Major excitatory transmitter!

Synthesized only by neurons that use it.Major source of synaptic inhibition in the brain.

ATP: Excites neurons; Binds to purinergic receptors

Both transmitter and G-protein coupled receptors

Concentrated in vesicles at many synapses (CNS + PNS) Ca++ dependent release Co-localized with catecholamines

EndocannabinoidsEndogenous

cannabinoidsAnandamide

(“internal bliss”)Arachidonylglycerol

(2-AG)

High Ca++ concentration

Synthesis of endocannabinoidmolecules from membrane

lipids

Small and membrane permeable

Once synthesized they can diffuse to neighboring cells

Bind selectively to the CB1 type of cannabinoid

receptor

CB1 is G-protein coupled receptors

Main effect raise VGCC threshold

Amino Acid-Gated ChannelsGlutamate-Gated ChannelsAMPA, NMDA, kainite

one neuron stimulates another by releasing glutamate into the

synapse

1

the glutamate binds to AMPA receptors on the postsynaptic

neuron

2

receptors open ion channels that allow sodium into the

postsynaptic neuron

3

The sodium influx depolarizes the cell to some degree.

4

Depolarization affects postsynaptic glutamate NMDA

receptors which control ion channels for calcium

5

The depolarization pops the magnesium gatekeepers out of the ion channels associated with the NMDA receptors, allowing calcium ions to flow into the postsynaptic neuron.

6

Amino Acid-Gated ChannelsGlutamate-Gated ChannelsAMPA, NMDA, kainite

Voltage dependent NMDA channels

How does ionic current flow

through the NMDA-gated channel?

GABA-Gated and Glycine-Gated Channels

GABA mediates inhibitory

transmission

Glycine mediates non-GABA inhibitory

transmission

There are two classes of GABA

receptors: GABAAand GABAB

GABAA are ligand gated ion channels

GABAB are G-protein coupled

receptors

This is interesting because a single molecule binds to

receptors which function in completely different ways

G-Protein-Coupled Receptors and Effectors

Three steps Binding of the

neurotransmitter to the receptor protein

Activation of G-proteins Activation of effector systems

The Basic Structure of G-Protein-Coupled Receptors (GPCRs) Single polypeptide with seven

membrane-spanning alpha-helices

• The Ubiquitous G-Proteins • GTP-binding (G-)

protein• Signal from

receptor to effector proteins

Neurotransmitters Transmit information between neurons Essential link between neurons and effector cells

Signaling pathways Signaling network within a neuron somewhat

resembles brain’s neural network Inputs vary temporally and spatially to increase

and/or decrease drive Delicately balanced Signals regulate signals- drugs can shift the balance

of signaling power

Concluding Remarks

“Dr. Snyder pioneered the labeling of receptors by reversible ligand binding in the identification of opiate receptors and extended this technique to all the major neurotransmitter receptors in the brain.”

He also worked out intracellular messenger systems and established gases (nitric oxide and carbon monoxide) as a new class of neurotransmitters.

http://www.ohsu.edu