Synaptic transmission and neuropharmacology

Slow axoplasmic transport rate = .2-4 mm/day (actin, tubulin)
Intermediate axoplasmic transport rate = 15-50 mm/day (mitochondrial protein)
Fast axoplasmic transport rate = 200-400 mm/day (peptides, glyolipids)
Number of molecules of neurotransmitter in one synaptic vesicle = 10,000-100,000
Diameter of synaptic vesicle = 50 nanometer (small); 70-200 nanometer (large)

Otto Loewi's Experiment Confirming the Existence of Chemical Neurotransmission

In 1920 the Austrian neuroscientist Otto Loewi performed a now-classic experiment. Loewi knew that stimulation of the vagus (X) nerve caused a slowing of the heart. Question was whether this was the result of a direct electrical or an indirect chemical effect? Loewi's Ingenious experiment (which came to him in a dream!) involved placing the heart of a frog into a beaker (with oxygenated isotonic saline solution). He stimulated the vagus nerve of the first heart to produce a reduced heartrate. The chamber which held the first heart was connected to a second chamber which contained a second heart. Loewi allowed fluid from the first chamber to flow into the second. Loewi observed that after a delay the second heart slowed down as well. Loewi hypothesized that the electrical stimulation of the vagus nerve which slowed the first heart released a chemical into the fluid of the first chamber which, when allowed to flow into the second chamber, also slowed the second heart. He called this chemical "Vagusstoff". We now know this chemical as the neurotransmitter called acetylcholine.

Loewi's pioneering work gave birth to the field of Neuropharmacology: the science of how drugs affect behavior through their action on the nervous system.

Transmitters are generally classified as either neuromediators or neuromodulators.

Mediating synapses open ion channels and mediate instantaneous transmembrane potential and have brief effects (measured in milliseconds).

Modulating synapses work via second messenger system (internal transmitter), and can have both membrane potential effects (although usually longer-lasting – measured in seconds), and can produce permanent structural changes in the neuron via interaction with genetic expression.

	Synthesis and storage of substance in presynaptic terminals Substance is released upon depolarization of presynaptic terminal Application mimics normal postsynaptic action (i.e., substance binds with receptors and produces an effect) Known reuptake or enzymatic inactivation and/or degradation systems Application of antagonist blocks normal postsynaptic action
Steps in neurotransmission	Criteria for identification as neurotransmitter

The action of neurotransmitters can be stopped by three different mechanisms
Diffusion The neurotransmitter drifts away, out of the synaptic cleft where it can no longer act on a receptor.
Enzymatic degradation (deactivation) A specific enzyme changes the structure of the neurotransmitter so it is not recognized by the receptor. For example, acetylcholinesterase is theenzyme that breaks acetylcholine into choline and acetate.
Reuptake The whole neurotransmitter molecule is taken back into the axon terminal that released it. This is a common way the action of norepinephrine, dopamine and serotonin is stopped...these neurotransmitters are removed from the synaptic cleft so they cannot bind to receptors.

Biosynthesis: The dietary amino acid choline is combined with Acetyl coenzyme A, and is converted to Acetylcholine and coenzyme A by the action of the enzyme choline acetyltransferase (ChAT). Can use regional distribution of ChAT to visualize ACh.

Receptors: Nicotinic (named after nicotine--a poison found in tobacco leaves) and muscarinic (named after muscarine--a poison found in certain mushrooms) receptor types named for primary agonists, nicotine and muscarine, respectively. Noteworthy antagonists include curare and atropine, respectively. Many subtypes of nicotinic and muscarinic receptors. Generalizations include: nicotinic receptors found at neuromuscular junction (excitatory effect on skeletal muscles. Control sodium ion channels and produces depolarizations), and both nicotinic and muscarinic receptors are found at peripheral autonomic ganglia. Inhibitory effect on the muscle fibers of the heart (controls potassium ion channels and produces hyperpolarizations). Cholinergic neurotransmission is primarily nicotinic in spinal cord, whereas both nicotinic and muscarinic receptors are involved throughout the brain; central action of muscarinic ACh receptors is probably through modulating synapses; nicotinic receptors are mediating. ACh also plays role in learning and remembering and controls the onset of REM sleep.

ACh action is terminated by the enzyme acetylcholinesterase (AChE). Acetylcholine is the only neurotransmitter destroyed in the synapse. Choline itself is reuptaken. Plasma choline does not cross the blood brain barrier. AChE is also present in the cytoplasm to destroy any transmitter substance produced by the cell that exceeds the storage capacity of the synaptic vesicles.

Regional Distribution: Synthesized fairly widely. Pyramidal cortical cells; caudate nucleus, hippocampus, LGN, posteriomedial thalamus, midbrain reticular formation & tectal nuclei; hypothalamus, cerebellum.

Physiological Functions: movement -- Myasthenia gravis (destruction of ACh receptors; autoimmune), botulism; autonomic regulation (nerve gas and SLUD syndrome; Black widow spider venom); learning & memory -- Alzheimers; infusion increases neural plasticity; arousal; possible role in pain perception (application of ACh to wound increases pain). Chronic administration of anticholinesterase inhibitors (to suffers of myasthenia gravis, for example) causes nightmares, confusion and hallucinations. Short-term exposure to organophosphates (like insecticides) which are ACh agonists, causes agitation, tenseness and confusion, and a slowing of intellectual and motor processes.

The Monoamines: Catecholamines (DA, NE and E) and Indolamines (5-HT)

There are four transmitters considered monoamines because their molecular structure is similar. Because these are so similar, some drugs will affect each of these receptors to a degree. Within the mammalian central nervous system, there is evidence that the monoamine transmitters are found in pathways essential for sensory and motor performance as well as for higher brain functions. However, out of the total cells in the human brain, relatively few appear to contain these transmitters-thousands rather than millions or billions. What is more, most of the cells containing these transmitters are frequently clustered together in discrete regions of the brain.

Three of the monoamine neurotransmitters are classified in a subclass, catecholamines. These are epinephrine, norepinephrine, and dopamine. Most neurons that release catecholamines do not do so through terminal buttons on the ends of axonal branches. Instead, they usually release them through axonal varicosities, beadlike swellings of the axonal branches. These varicosities give the axonal branches the appearance of beaded chains.

Dopamine Varicosities

Norepinephrine Varicositeis

Biosynthesis: tyrosine is base AA ® L-DOPA ® Dopamine (packaged in vesicles).

Receptors: D1 and D2, both of which activate 2^nd messenger systems. D1 (found mostly in retina) causes increase in cAMP, D2 (nigrostriatal and mesolimbic) causes decrease in cAMP. Also DA autoreceptors (inhibitory feedback). Action of DA terminated by reuptake.

DA synapses.JPG (28185 bytes)

(1) synthesis by enzymatic pathway from Tyr to DOPA to DA; (2) transport and storage (storage inhibited by reserpine, Res); (3) release of DA by exocytosis; corelease of a neuropeptide such as cholecystokinin (CCK); (4) binding to D₁ receptor, acting through stimulatory G protein (G) to increase levels of cAMP, or to D₂ receptor, acting through inhibitory G protein to lower levels of cAMP (antipsychotic drugs such as butyrophenones block D₂ receptors); G protein can also have a direct action on K⁺ channels at some synapses; (5) binding of DA to presynaptic receptors [typically D_2; DA receptors are stimulated by psychoactive drugs such as apomorphine (APO), blocked by haloperidol (Halo)]; (6) reuptake terminates DA action; (7) degradation by MAO and inactivation by COMT.

Regional Distribution: Two distinct sites of primary synthesis: in substantia nigra and ventral tegmental area, the former having projections to the basal ganglia (forming nigrostriatal pathway) , and the latter projecting to the amygdala and prefrontal cortex (forming mesolimbic pathway) . Some intrinsic synthesis in hypothalamus, with projection to pituitary. Each DA neuron gives rise to 500,000 terminals.

Physiological Functions: Dopamine produces excitatory or inhibitory postsynaptic potentials, depending on the postsynaptic receptor. Interest in DA because it is involved in both intellectual and motoric functions. Neuroleptic drugs appear to block DA transmission at D2 receptors. Potent DA agonists, like amphetamine, can produce schizophrenic like behavior. The mesolimbic projection (to prefrontal cortex and amygdala) is the medial forebrain bundle: stimulation causes pleasure -- is reward system. DA antagonists applied to nucleus accumbens abolish operant reward systems. Death of SN neurons causes Parkinson's disease, a movement disorder characterized by tremors, rigidity of the limbs, poor balance, and difficulty in initiating movement.

Biosynthesis: tyrosine is base AA ® L-DOPA ® Dopamine (packaged in vesicles) ® converted to NE (and epinephrine) inside the vesicles.

Receptors: Alpha (a1 and a2) and Beta (b1 and b2) receptors. All operate via 2nd messenger systems. a1 receptors affect Ca²⁺ channels (mostly postsynaptic). a2 receptors are mostly presynaptic (but are also postsynaptic) and affect K⁺ channels (inhibitory). Beta receptors affect K⁺ channels, and are also mostly inhibitory.

NE synapses.JPG (46405 bytes)

a-Adrenergic synapse: (1) synthetic pathway is through tyrosine (Tyr) to 3, 4-dihydroxyphenylalanine (DOPA; catalyzed by tyrosine hydroxylase) to dopamine (DA; catalyzed by DOPA decarboxylase) to norepinephrine (NE; catalyzed by dopamine-b-hydroxylase); (2) transport and storage (storage is blocked by reserpine); (3) release by exocytosis (increased by amphetamine); corelease with neuropeptides such as enkephalin, Enk; (4) binding of NE to postsynaptic receptors. Examples are shown of binding to a₁ receptor which leads to modulation of Ca²⁺ channels, and binding to a₂ receptors, which are linked to adenylate cyclase and modulate K⁺ channels; there may also be direct actions of G proteins on K⁺ channels; (5) binding of NE to presynaptic a₂ receptors; (6) reuptake, which terminates NE action (blocked by tricyclic antidepressant drugs); (7) degradation by monoamine oxidase (MAO) [there may also be inactivation by catechol-O-methyltransferase (COMT)].

b-Adrenergic synapse: (1-3) synthesis, transport, storage, and release as for a-Adrenergic synapse; (4) binding of NE to b₁ receptor which leads to phosphorylation of ionic channels through cAMP; b₂ receptors are also found on glia; (5-7) presynaptic receptors, reuptake, degradation, and inactivation, as for a-Adrenergic synapse.

Regional Distribution: Largest site of synthesis is locus ceruleus, in midbrain -- just a few hundred cell, they send axons to almost every other region of the nervous system. Another site is lateral tegmental area, with projections which overlap those of the LC but which target the hypothalamus.

Physiological Functions: Wide projections of NE system make it ideal for gain-control of other neural systems. NE’s modulating action in CNS is primarily inhibitory. Acts like a CNS version of the sympathetic division of the autonomic nervous system. In the sympathetic nervous system norepinephrine has an excitatory effect. Released by nerves in internal organs, including the gut, spleen, and heart. Involved in many general functions, like sleep, wakefulness, vigilance, emotion, neuroendocrine function, temperature regulation. Catecholamine theory of depression (antidepressants work on NE -- MAOI's, tri-cyclics, reserpine, etc --but behavioral effect lags biochemistry; also, cocaine is effective agonist but poor antidepressant). Lithium is NE antagonist (facilitating reuptake of NE -- but why does it help w/depressive phase of bipolar affective disorder?).

Epinephrine (also known as adrenaline) is synthesized from norepinephrine in the adrenal medulla, the central core of the adrenal glands. In the CNS the role of epinephrine is not entirely known, but it is believed to play a role in the way the brain regulates blood pressure. In the periphery, it is the main circulating exciting transmitter released during the "fight or flight" stress reactions.

Biosynthesis: From tryptophan. Differs from catecholamines in having, in addition to the catechol ring, an indole ring. This is why it is called an indolamine rather than a catecholamine.

5-HT synapses.JPG (27306 bytes)

(1) synthetic pathway is from tryptophan to 5-hydroxytryptophan (catalyzed by tryptophan hydroxylase) to 5-hydroxytryptamine (5HT, or serotonin) (catalyzed by 5-hydroxytryptophan decarboxylase); (2) transport and storage (blocked by reserpine, Res); (3) release of 5HT by exocytosis; corelease with a neuropeptide, e.g. neurotensin, NT; (4) binding to a 5HT₁ postsynaptic receptor (coupled to G protein and cAMP), or to a 5HT₂ receptor (LSD is an agonist/antagonist);? (5) possible binding to presynaptic receptors; (6) reuptake terminates 5HT action (blocked by tricyclic antidepressant drugs such as imipramine, Ima); (7) degraded by MAO.

Regional Distribution: Synthesized mostly in midbrain, at raphe and in medulla, but has wide projection, much like NE. Highest concentration in pineal.

Physiological Functions: Mostly inhibitory effects on the post-synaptic membrane. Like NE, has widespread influence over sleep (suppresses REM sleep) arousal, sensory perception, including pain, emotion (particularly mood) and higher cognitive functions. LSD is antagonist, on 5HT-2 receptors (one theory of LSD is that it acts at Serotonin receptors to produce dreaming during wakefulness). Suicide victims have low serotonin levels. Certain anti-depressant drugs (Prozac) prevent the normal reuptake of serotonin to keep it in the synapse longer. Serotonin undergoes daily variations in level: like melatonin, high in day, low at night.

Amino Acid Neurotransmitters

Amino acid transmitters are located in the brain in far greater quantities than other neurotransmitters. Because amino acids are used for protein synthesis by all cells of the brain, it is difficult to prove that a particular amino acid is a transmitter substance. Eight amino acids have been discovered to serve as transmitters, but we will only discuss three of them.

Receptors: Mediating channels of three types: Quisqualate, Kainate and NMDA. NMDA receptor allows Na⁺ and Ca²⁺ influx. Excitotoxicity of Kanic acid (and CO & NO) via Ca²⁺.

glutamate synapses.JPG (23334 bytes)

(1) synthesis of glutamate (GLU) from glutamine; (2) transport and storage; (3) release of GLU by exocytosis or from cytosol; (4) binding of GLU to several types of receptors identified by specific antagonists [N-methyl-D-aspartate (NMDA), kainate (K), quisqualate (Q)]. The Q and K receptors gate Na⁺ and K⁺ flux; the NMDA receptor regulates a Ca²⁺-permeable conductance state which is normally blocked by Mg²⁺ and high resting membrane potential (-); when this block is relieved by membrane depolarization (+), Ca²⁺ flows in to depolarize the membrane further and activate other second messenger systems. The three receptor mechanisms illustrated in the figure represent different conductance states of the same receptor-channel complex.? (5) binding to presynaptic receptors; (6) reuptake; (7) degradation. A similar sequence is involved at synapses utilizing aspartate.

Physiological Functions: workhorse excitatory transmitter. Mediates excitotoxicity -- mention ALS and hypoxic ischemia; PCP is therapeutic. Appears to play a role in learning and memory. MSG (monosodium glutamate), a preservative found in many oriental foods, activates some glutamic acid receptors and may cause dizziness and numbness.

Receptors: Two types, GABA_A (most prevalent, mediating Cl^- channels only) and GABA_B (both mediating and modulating K⁺ and Ca²⁺ channels); produce inhibition.

gaba synapses.JPG (33351 bytes)

(1) Synthesis of g-aminobutyric acid (GABA) from glutamine (catalyzed by glutamic acid decarboxylase); (2) transport and storage of GABA; (3) release of GABA by exocytosis [corelease with a neuropeptide such as enkephalin (Enk) or somatostatin (SOM)]; (4) binding to a GABA-A receptor blocked by bicucylline (B), picrotoxin, or strychnine (S), which are coupled to a chloride channel; the GABA receptor also has a site for binding of benzodiazepines, such as valium (V); GABA-B receptors, by contrast, are linked via a G protein and/or cAMP to K⁺ and Ca²⁺ channels: these are blocked by baclofen; (5) binding to presynaptic receptors; (6) reuptake in presynaptic terminal, and uptake by glia; (7) transamination of GABA to a-ketoglutarate (catalyzed by GABA transaminase, GABA-T, regenerating glutamate and glutamine; glial glutamine then reenters the neuron.

Regional Distribution: spotty, high in striatum, medium level throughout cortex.

Physiological Functions: workhorse inhibitory transmitter. Too little GABA implicated in epilepsy. Alcohol though to have effects through GABA receptor (GABA agonist). Two cell types -- either short non-spinous stellates or projection fibers (to striatum). These latter are implicated in Huntington's disease. Certain drugs, such as barbituates or alcohol, can enhance the inhibitory effects of GABA. Valium is also a GABA agonist.

Inhibitory transmitter in the spinal cord and lower portions of the brain. Little is known about this transmitter, but if glycine synapses are blocked (like with the bacteria that causes Tetanus), we would undergo continuous contraction (e.g., lock-jaw).

bradykinin	insulin	motilin	galanin
beta-endorphin	gastrin	vasopressin	neuropeptide Y
bombesin	substance P	oxytocin	thyrotropin-releasing hormone
calcitonin	neurotensin	prolactin	gonadotropnin-releasing hormone
cholecystokinin	glucagon	thyrotropin	growth hormone-releasing hormone
enkephalin	secretin	angiotensin II	luteinizing hormone
dynorphin	somatostatin	sleep peptides	vasoactive intestinal peptide

Biosynthesis: Several different peptides are released by neurons of the central nervous system. In comparison with the other transmitters described, synthesis of peptides requires gene activation, DNA transcription, and RNA translation, with the final peptide being transported from soma to release sites. One of the most important family of peptides is the endogenous opioids.

peptide synapses.JPG (27334 bytes)

Molecular mechanism of a peptidergic synapse, as exemplified by opioid peptides: (1), synthesis in cell body; (2) transport and storage in vesicles; (3), release by exocytosis; corelease with neurotransmitters at many synapses; (4) binding to one of several types of receptor. M (µ) receptor preferentially binds morphine; it activates cAMP to open K⁺ channels and close Ca²⁺ channels. Delta (d) receptor is similar but binds enkephalin preferentially. K (K) receptor preferentially binds dynorphin (DN), and acts through second messenger to close Ca²⁺ channels. Not shown are two other types g and e.

Regional Distribution: spotty, high in hypothalamus (where most are made), pituitary and periaquaductal gray matter.

Physiological Functions: Some peptides are coreleased with NE, implicated in memory formation (endorphines enhance memory); regulates pain thresholds (strong analgesia, blocked by naloxone), regulates mood and emotional tone.

Name	Source	Neuronal Action
agotoxin	Funnel Web Spider	Blocks calcium channels
alpha-bungarotoxin	Krait	Blocks acetylcholine receptor
Anatoxin	Algae	Acetylcholine receptor agonist
Apamin	Honey bee	Blocks potassium channels
Batrachotoxin	Poison Arrow Frog	Prevents sodium channel closure
Botulinum toxin	Bacteria	Blocks acetylcholine release
Brevetoxin	Red Tide Dinoflagellate	Activates sodium channels
Capsaicin	Cayenne Pepper	Excites peripheral nerve endings
Charybdotoxin	Scorpion	Blocks potassium channels
Chlorotoxin	Scorpion	Blocks chloride channels
Conotoxin	Cone Snail	Blocks calcium channels
Crotoxin	Rattlesnake	Reduces acetylcholine release
Dendrotoxin	Green Mamba	Blocks voltage-gated potassium channels
Domoic acid	Blue mussel	Glutamate/kainate receptor agonist
Gonyautoxin	Dinoflagellate	Block sodium channels
Iberiotoxin	Scorpion	Blocks potassium channels
Joro spider toxin	Joro spider	Blocks glutamate receptors
Kaliotoxin	Scorpion	Blocks potassium channels
Latrotoxin	Black widow Spider	Enhances ACh release
Mandarntoxin	Wasp	Blocks sodium action potentials
Notexin	Tiger snake	Reduces ACh release
Noxiustoxin	Scorpion	Blocks sodium channels
Palytoxin	Soft coral	Activates sodium channels
Saxitoxin	Dinoflagellate	Blocks sodium channels
Tetrodotoxin (TTX)	Pufferfish	Blocks sodium channels

A good reference for NEUROTOXINS is: Trends in Neuroscience, June, 1996 (supplement).

Electrical synapses also exist (gap-junctions). Ironically, although their existence was hypothesized prior to chemical synapses, their function is less well-understood than chemical neurotransmission. Also, recently several gasses (nitric oxide, NO, and carbon monoxide, CO) have been identified as playing a role in neurotransmission.

Nitric oxide functions as a signaling molecule that tells the body to make blood vessels relax and widen. This physiological reaction is important when the body needs more blood--the brain signals to the blood vessels near the arms, for example, when the arms need more blood supply for muscle movement or for warmth. Thus, NO works as a signaling molecule in the cardiovascular system, the nervous system, and in other tissues. Since NO does not fit into the traditional form of neurotransmitters, it was surprising to find that it as a neurotransmitter.

Neurotransmitters are produced, stored, and used when needed by the nervous system. After the neurotransmitter is used, it is broken down by an enzyme or taken back up by the neuron that released it so it can no longer act as a signal. This means the effect is short lasting. A neurotransmitter released in the traditional way affects only neurons near the nerve cell that released it.

NO, by contrast, is not produced in advance or stored. It is made at the time it is needed, and it diffuses in all directions instead of having just a local effect. In part because NO is a gas, it can affect many cells, not just the cell where it was produced.

A typical neurotransmitter is like a letter. It sends information. You keep paper in your house for when you need to write a letter, then you send it (and could watch it travel to its destination, if you wanted to) and it is received by one person.

NO also transmits information, but it is more like e-mail. When you need to send an e-mail, you generate a new e-mail. They are not made ahead of time and stored until use. The transmission is invisible--you can not see your e-mail travel to its destination. And you can send e-mail to as many people as you want, quickly. So while both NO and the classical neurotransmitters carry information, they work differently.

NO is used as a signal in the cardiovascular system (heart) and in the nervous system (brain, spinal cord). It can be produced in large amounts by white blood cells, helping to fight infections by killing bacteria and parasites. Perhaps the toxic effects of NO could be used to combat cancer, too.

NO controls our blood pressure, giving us more blood when we're exercising or scared, and reducing the flow of blood when our body is at rest. It gives certain organs more blood at times: more blood to your stomach after a big meal helps you digest the food, for example. The anti-impotence drug Viagra similarly alters blood flow; it was developed based on the understanding of how NO works.

NO even plays a role in our sense a smell, which influences memory. So NO may affect memory and even learning.

Everything we learn about how our body works helps us to create treatments for diseases. Knowing that NO plays an important signaling role in the dilating (enlarging) blood vessels will be important in treating high blood pressure and other ailments that have to do with the flow of blood. Heart attacks, for example, happen when the blood cannot flow through the blood vessels of the heart (this is one reason nitroglycerine is used to treat angina). This blockage can be caused by spasms of the blood vessels or by the narrowing of blood vessels by a plaque of fatty substances. In either case, NO will help by relaxing the blood vessels, allowing them to widen and increasing blood flow.

Understanding how to control the contraction and dilation (getting smaller or larger) of blood vessels could help many patients with various diseases.


Typical Neurochemical Synapse