• Ca²⁺ sensitive dyes flourece at a different wavelength when they bind Ca²⁺ ions, first used to detect intracellular Ca²⁺ was aequorin, chemical that gives jellyfish luminescent glow, inject neuron with dye to monitor Ca²⁺ distribution in cell
• During synaptic transmission, local very high concentrations of Ca²⁺ develop, enables Ca²⁺ to exert rapid effect, delay between Ca²⁺ entry and neurotransmitter release only 0.1 to 0.2 msec, not enough time for long distance diffusion or complex sequence of biochemical reactions

Diseases due to breakdown of docking and fusion

Tetanus and botulinum neurotoxins affect docking and fusion prevent release of neurotransmitter

• Tetanus toxin prevents release of neurotransmitter by inhibitory neurons in spinal cord, causes uncontrolable muscle contractions
• Botulinum prevents neurotransmitter release from spinal neurons causes flaccid paralysis

Fate of synaptic vesicles

• Synaptic vesicle fuses with cell membrane and becomes part of it, measurement of membrane capacitance during and following synaptic transmission indicates increase in surface area of cell membrane
• Theoretical calculations indicate that if all vesicle membrane remained in cell membrane surface area increase should be much greater, therefore mechanism for removing membrane must exist
• Removed membrane recycled back to synaptic vesicles, patch of membrane that came from particular vesicle removed from synapse to specialized region where new vesicles are pinched off into cell, demonstrated by tagging v-SNARE proteins with flourescent antibodies
• 45-90 seconds to recycle synaptic vesicles

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Filling vesicles with neurotransmitter

Two kinds of vesicles

1. clear small 40-50 nm, oval or spherical in shape

• some filled in cell body and transported to synapse, others filled from transmitter available in terminal
• reformed clear vesicles empty of transmitter
• new transmitter molecules synthesized in terminal and transported into vesicle
• ATP is expended to transport free protons (H+) into vesicle
• Proton gradient used to power movement of neurotransmitter into vesicle by transporter proteins in embedded in membrane

1. dense core 70-200 nm, solid looking dense core, spherical shape

• usually filled with peptide neurotransmitter
• peptide must be synthesized by Golgi apparatus in cell body
• synthesis and filling occur together
• filled vesicle moved by axonal transport down axon to release site

Transmitter release from non-spiking neurons

Non-spiking neurons cannot generate AP, present in retina, inner ear, lateral line of fish, and olfactory bulb, in invertebrates some sensory neurons and many interneurons involved in generating motor patterns don't spike

• AP not necessary for release of neurotransmitter, only facilitate necessary depolarization
• For non-spiking neurons release of neurotransmitter proportional to degree of depolarization, with even slight fluctuations having significant effect graded transmission,
• release in spiking cells is also graded to as indicated by presynaptic inhibition and by effects of experimental reduction in size of AP
• at some synapses both graded transmission and triggered release occur, for taste cells low level stimulation induces graded release, high level stimulation triggers AP

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Chemistry of neurotransmitters

Hard to determine whether particular chemical serves as neurotransmitter at given synapse because synapses are not readily accessible for experimentation

Four criteria for identification of substance as neurotransmitter:

1. 1) must be synthesized in neuron from which released

• biochemical techniques used to demonstrate enzymatic pathways necessary synthesis of putative neurotransmitter

1. 2) must be stored in neuron from which it is released

• Anatomical labeling techniques such as immunohistochemistry may be used to determine the distribution of a putative transmitter, and in particular whether it is present in presynaptic terminals

1. 3) must be released from terminals of these neurons

• may be possible to collect and identify suspected neurotransmitter from vicinity of active synapses

1. 4) when applied exogenously, must mimic action of natural neurotransmitter

• ionophoresis may be used apply putative neurotransmitter to postsynaptic cell
• drugs known to block binding of natural neurotransmitter, should also block effects of iontophoretic application of chemical

Applying full battery of tests at particular synapse seldom possible, but application of technique to many different synapses allows many substances to be identified as neurotransmitters based on good evidence

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Neurotransmitter chemicals

Most known or suspected neurotransmitters fall into three groups

1. 1) amino acids

• building blocks of proteins, possess an acid and an amine (NH₂ containing) chemical group
• many amino acids are neurotransmitters
• glutamate an excitatory neurotransmitter in vertebrate brain and in most excitatory neuromuscular junctions in insects
• aspartate also an excitatory neurotransmitter
• Gamma-aminobutyric acid (GABA) and glycine inhibitory

1. 2) Amines

• Contain amine group but lack acid group characterizing amino acids
• Histamine, octopamine, serotonin, octopamine important in invertebrate nervous systems
• Catacholamines amines containing catacol group (six carbon ring with 2 OH groups) main ones dopamine, epinephrine (adrenaline), norepinephrine (noradrenaline)
• serotonin and dopamine have influence on a variety of behaviors from mood to movement, neurons using them widespread
• norephinephrine released by sympathetic postganglionic neurons in autonomic NS, also important neurotransmitter in CNS

1. 3) neuropeptides

• small chains of amino acids, neurotransmitter role, discovered more recently
• substance P involved in conveying pain information in vertebrates
• endorphins important in amelieorating pain
• most are co-transmitters, substances released along with another neurotransmitter from a synapse, proctolin released along with glutamate at invertebrate neuromuscular junction

1. 4) other- a micellany of substances with no special chemical relationship

• acetylcholine- neurotransmitter at neuromuscular junctions of vertebrate somatic muscles, parasympathetic postganglionic neurons, and all preganglionic neurons in autonomic NS, also involved in insect nervous system, also may act presynaptically in vertebrate brain
• adenosine triphosphate (ATP)- coreleased with catacholamines, may have separate transmitter function, especially in autonomic NS
• the gases nitric oxide (NO) and carbon monoxide (CO), soluble gases neither stored nor released from vesicles, but synthesized as needed, and diffuses away, easily crossing cell membrane, effect is neuromodulatory, interact directly with biochemical machinery of target neuron rather than with a specific receptor

Many other substances may act as neurotransmitters, with new candidates identified every year

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Neurotransmitter synthesis

Chemically related neurotransmitters also have synthetic pathways in common, and some co-exist in the same neuron, in some cases co-transmitters in separate vesicles, in other cases in same vesicles

Amino acid neurotransmitters

• obtained from diet or synthesized as in other cells
• glutamate and aspartate obtained from Kreb's cycle
• GABA formed by decarboxylation of glutamate by glutamic acid decarboxylase (GAD)
• Glycine derived from glucose via amino acid serine

Amines

• Catacholamines contain catachol ring, all three catacholamine neurotransmitters, dopamine, norephinephrine, and epinephrine all derived from single synthetic pathway
• Serotonin, an amine but not a catacholamine derived from amino acid triptophane

Peptides

• Consist of chains of amino acids, and therefore probably all synthesized in soma where chemical machinery for assembling, some synthesized directly, others cleaved from longer protein chains

Acetylcholine

Synthesized from choline and acetyl-CoA

Synthesis takes place mainly in terminals of axon

Nitric oxide by product of conversion of amino acid arginine to citrulline, as gas can pass through any membrane and thus cannot be stored

Carbon monoxide split from heme by heme oxygenase, present in hippocampus and olfactory bulb

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Neurotransmitters nervous system

Why so many?

The response of a neuron to a neurotransmitter determined by the receptor to which transmitter binds, therefore no obvious functional explanation for why so many needed

• explanation may be evolutionary, brain evolved by random variation and natural selection, mutations in membrane proteins may have yeilded molecules that respond to various chemical substances that nerves happen to release, particular neurotransmitter systems evolved because different chemicals happened to best serve neurons that had specific functions

Neurotransmitters have specific distributions in the brain

Examples:

• ACh released by parasympathetic postganglionic neurons, and by all preganglionic neurons of autonomic nervous system
• Norephinephrine released by sympathetic postganglionic neurons
• GABA released by local neurons in cortex and cerebellum
• Peptides often co-localized in GABAergic neurons
• Dopamine used largely by brainstem neurons

Neurotransmitters in ANS

• ACh neurotransmitter of all preganglionic autonomic neurons, and postganglionic parasympathetic neurons, ephinephrine neurotransmitter of postganglionic sympathetic neurons
• At least 3 other classical neurotransmitters, dopamine, serotonin, and GABA are involved
• A bewildering variety of co-transmitters, including neuropeptides, ATP, and nitrous oxide

Determining neurotransmitter distribution

Histoflourescence

Certain compounds flouresce when amines, chatecholamines or serotonin, used to map distribution in the brain

Immunohistochemistry

Inject protein of interest into mammal (often rabbit) thereby induce to make antibodies

Antibodies extracted from rabbit, bound to marker, spread over slice of tissue containing protein of interest, a two step proceedure of making an antibody to the antibody can be used to amplify signal

In situ hybridization

Label messenger RNA containing instructions for the protein of interest, tends to be more reliable than immunohistochemistry

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