Notes

Neurotransmitters

Chemical Transmission Definition of Neurotransmitter

1. Synthesized and released from neurons Localization of synthesizing enzymes.
2. Released from nerve terminals in a chemically or pharmacologically identifiable form Must be possible to isolate it.
3. Reproduces events in the postsynaptic cells that are characteristically seen after stimulation of presynaptic neuron.
4. Its effect can blocked by competitive antagonist for the receptor, in a dose-dependent manner.
5. Presence of active mechanisms to terminate action of neurotransmitters. Uptake mechanisms and enzymatic inactivation.

Classical Neurotransmitters There are 5 classical neurotransmitters and are classified as classical because they satisfy the previously mentioned characteristics. Catecholamines (Excitatory)

• Dopamine (DA), Norepinephrin (Noradrenaline) and Epinephrin (Adrenaline).
• Derived from the same precursor, but require separate enzymes.
• Storage and Release of Catecholamines and their enzymes:
  • Vesicular Storage
  • Release of Catecholamines
    • "Usual" Ca2+ dependent exocytosis
    • Reversal of DA and NE transporters to extrude Catecholamines
    • Apparent Ca2+ independent dendritic release
  • Regulation by Autoreceptors

These are broad ways in which NT can operate on the postsynaptic neurons

• Synthesis-modulating autoreceptor
• Release-modulating autoreceptor
• Firing-rate-modulating autoreceptor
• Inactivation/Catabolism
  • Two enzymes: monoamine oxidase (MAO) and cathchol-0-methyltransfrease (COMT). MAOs are drug targets in neuropsychiatric disorders.
• Transporters (They might cross-bind or cross-activate with each other)
  • Facilitate transmitter re-uptake in an energy-dependent manner
  • High affinity transporters are neuron-specifi
  • DAT: Dopamine transporter
  • NET: Norepinephrin transporter
  • Transporters are drug targets: Cocaine and Amphetamine both increase extracellular level of Catcholamines by blocking transporters. Cocaine has very high affinity for DAT, and Amphetamine reverses the normal direction, resulting in release
  • The Euphoria level from drug consume derives from the high concentrations of Catecholamines.

Serotonin (Excitatory)

• In blood, induced powerful contraction of smooth muscle organs.
• The brain accounts for only approx. 1% of total body serotonin.
• Serotonin inactivation:
  • Inactived primarily by re-uptake through SERT, which belongs to the family of Catecholamine transporters.
  • Selective serotonin re-uptake inhibitors (SSRIs) block SERTs and widely used as antidepressants (such as fluoxetine aka Prozac).
  • MAOs (monoamine oxydases) enzymatically degrade serotonin MAO blockers elevate serotonin levels and are used as antidepressants.

Acetylcholine (Excitatory) It is important in neuromuscular junctions. Classic studies examining endplate potentials in neuromuscular junction.

• MAOs (monoamine oxydases) enzymatically degrade serotonin MAO blockers elevate serotonin levels (antidepressants).
• Many of the rules that govern Ach transmission have found to be generally applicable to many neurotransmitters.

GABA (Inhibitory)

• Amino acid transmitter gamma-aminobutyric acid
• Derived from glucose metabolism
• GABA inactivation - the GABA shunt:
  • GABA is inactivated by GABA-T (which is both synthetic and degradative enzyme), but only if alpha-Ketoglutarate is present to receive the amino group that is removed from GABA. This unusual GABA shunt serves to maintain supplies of GABA. GABA uptake is mediated by GABA transporter (GATs), which are non-specific to GABA and take up other amino acids as well.

Glutamate and Aspartate (Excitatory Amino Acid Transporters)

• Account for most fast excitatory synaptic transmission in the brain
• Neither crosses the blood-brain barrier; need to be derived by local synthesis from glucose.
• Glutamine is exported from glia and need to be taken up by nerve terminals before being converted to glutamate.
• Vescicular uptake transporters VGlut1, 2, and 3 identify glutamatergic neurons, in most cases.
• Glutamate uptake after transmission is mostly by astrocytes, which have high levels of glutamate transporters. Glia can also release glutamate.

Non-classical neurotransmitters

• Fail most classical criteria, but still function as classical transmitters over restricted spatial and temporal domains.
• Can be also classical neurotransmitters used in "non-classical" ways. For example, in non-junctional appositions.

Peptide Transmitters

• Biosynthesis is mostly non-enzymatic and non-axonal, as classical neurotransmitters
• Gene prohormone uptake to secretory vesicles peptidase on prohormone peptide transmitter, which is then transported down the axon.
• Because transcription is required, peptide transmitters respond to demand slower than classical transmitters.
• Packaged to dense core vesicles (approx. 100nm diameter, vs approx. 50nm of classical), and released in response to high frequency stimulation.
• Inactivation: there are no uptake mechanisms. They are inactivated enzymatically, or by diffusion. However, certain fragments may remain biologically "active".
• Synthesis, release, and termination of action of the neuropeptide transmitter neurotensin:
  • Single gene yields two mRNAs: neurotensin (NT) and neuromedin (NMN)
  • NT and NMN co-express, but molar ratios are different. Likely, because different processing of the precursor.
  • There are no membrane transporters, but peptides can still accumulate in neurons, via internalization of receptor bound peptide, and subsequent dissociation.
  • NT co-localizes with DA< but their release is reciprocally regulated by actions of release-modulating DA autoreceptors.

Unconventional Transmitters

• Radically different from classical transmitters
• The original definition did not account for:
  • Multiple time scales (some transmitters can be present in low amounts and act on slow time scales).
  • Communication between neurons and non-neurons, such as glia.
  • Unconventional roles for neurotransmitters, such as regulation of neuronal development and intracellular signaling.
• No signaling and gaseous neurotransmitters ("gasotransmitters").
• Endocannabinoid signalling
  • Major psychoactive component of marijuana is delta-9-tetrahydrocannabinol (THC); if psychoactive, there must be a receptor.
  • Search identifies CB1 receptor, if there is receptor, there must be endogenous ligand.
  • Search identifies 2-AG, and subsequently multiple other endocannabinoids, if there are ligands, there must be enzymes involved in synthesis and inactivation.
  • Search identifies respective enzymes.
  • ECs act as retrograde transmitters in synapses.

Release of Neurotransmitters

Transmitter Release is Quantal (Chemical signals can work as amplifiers)

• Chemical transmitter based synapses are highly diverse and modifiable (as opposed to electric coupling).
• Post-synaptic effect can be excitatory or inhibitory.
• Transmission has to be fast.
• A consequence of releasing transmitter by the exocytosis of vesicles is that synaptic transmission is quantal.

Transmission in the neuromuscular junction

• Profusion and docking of synaptic vesicles at approx. 1000 active zones.
• Action potential triggers release of transmitter of approx. 300 quanta into the synaptic cleft (approx. 100nm wide).
• Diffusion to postsynaptic receptors and reaching concentrations of 1mM within 2ms.
• Activation of up to 2000 receptors, resulting in ion channel opening.
• Action potential induced release of 300 quanta results in a peak of few 10mVs, enough to generate an action potential in the muscle fiber.

Transmission in Central Synapses (as opposed to the neuromuscular junctions)

• A typical central synapse contains 1-4 active zones.
• Action potential triggers release of transmitter 5 to 10 vesicles.
• Cleft concentration also goes up to approx. 1mM, but typically there are less receptors, each vesicle activating approx. 30 receptors.
• Single evoked responses are in the range of few millivolts at most, clearly sub-threshold for excitatory responses to evoke postsynaptic action potential.

Excitation - Secretion Coupling

• Centrality of Ca2+.
• Coupling is achieved by the use of Ca2+ as intracellular messenger.
• Normal intracellular Ca2+ is buffered very low. Extracellular Ca2+ is in the mM range large driving force after Ca2+ channels open up to 1000-fold increase in intracellular concentration (change intra/extra concentrations to test dependency).
• Probing with Ca2+ loading
• Probing with ca2+ buffering
• Where is the Ca2+ sensor?

Ca2+ Microdomains

• Single active zone may have 100 Ca2+ channels.
• A single vesicle may be <5-nm from as many as 10 Ca2+ channels.
• Most prevalent Ca2+ channels for release are N- and P/Q-type channels.
• The exocytosis trigger must be fast (=fast on/off rate for speed).
• Cooperative Ca2+ binding; multiple Ca2+ sites need to be occupied for efficiency.

Molecular Mechanisms at the Nerve Terminal

• Neurons need an extremely efficient mechanism to recycle and reload vesicles within the terminal. (Otherwise they run out of readily releasable pool of vesicles).
• For most neurotransmitters, vesicles are refilled in approx. 30sec.
• A CNS synapse have only 2-20 fusion ready vesicles. These need to be replaced within seconds.

Structure and Topology of Major Synaptic Vesicle Membrane Proteins

• Protein compositions of synaptic vesicles are remarkably similar, independent of the neurotransmitter. (Extreme richness of different proteins).

SNARE Proteins and Core Complex are Key to Membrane Fusions

Quantal Analysis: Probing Synaptic Physiology (example: Neuromuscular Junction) In the 60's they noticed that by recording synaptic signals in the neuromuscular junction, an histogram of the distribution of potential amplitudes showed multiple peaks. It depends on the number of vesicles released during a synaptic release.

Short-Term Synaptic Plasticity Repetitive activation of the synapse, depending on the interval between the two stimulations, we can see a larger or smaller action potential amplitude in the second event, which are called "facilitation" (depends on the already higher concentration of calcium) and "depression" (which could be caused by a desensibilization of the synaptic site or depletion of the neurotransmitters vesicles).