Synaptic Transmission and Cellular Signaling: an Overview

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Synaptic Transmission and Cellular Signaling
an Overview

• Department of Pharmacology
• Jin-Chung Chen, Ph.D.
• Room 664 x 5282

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Synaptic Transmission historic view

• 1. Histologist Golgi and Ramon Cajal neuronal
  connection via Synapse
• 2. Oliver, Shafer, Langley and Elliot (1890)
  Chemical transmission
• 3. Experimental biology by Otto Loewi electrical
  stimulation on vagus n. decrease heart
  contraction

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Criteria for chemical transmission

• 1. Synthesis of the neurotransmitter in the
  presynaptic nerve terminals
• 2. Storage of the neurotransmitter in secretory
  vesicles.
• 3. Regulated release of neurotransmitter in the
  synaptic space between the pre- and post-synaptic
  neurons.
• 4. Presence of receptors on the postsynaptic
  membrane mimics the effect of nerve stimulation
• 5. A means for termination of the action of the
  released neurotransmitter.

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Eukaryotic neurotransmitter release

• 1. Classic neurotransmitter ACh, DA, NE, EPI and
  5-HT are low-molecular-weight substance that have
  no other physiological function
• 2. They store in synaptic vesicles acidic
  interior (pH 5.5), maintained by a
  vacuolar-type, proton-translocating ATPase driven
  by H pump and specific vesicular transporter

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3. Peptides and proteins can also be released
from the n. terminals. They utilize the ER, Golgi
and trans-Golgi network in the cell body (not the
terminal) and transport into the nerve terminal
by axonal transprot.
4. Constitutive secretion and endocytosis in
yeast and mammalian cells are similar in
presynaptic events of synaptic transmission
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Methods to study exocytosis

• 1. Membrane capacity (Cm)
• Cm QAm/V (Q is the charge across the
  membrane)
• specific capacitance is mainly determined by
  the thickness and dielectric constant of the
  phospholipid bilayer. Therefore, the increase in
  plasma membrane area due to exocytosis is
  proportional to the increase in Cm.
• Patch clamp micropipet has tight seal to allow
  low noise, high sensitivity, electric measurement

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Electrochemical amperometric
Electrophysiological capacity
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• 2. Electrochemical detection (carbon fiber
  amperometric electrode) directly measure the
  release of oxidizable neurotransmitter, such as
  catecholamines and serotonin.
• 3. Synaptosome homogenize brain tissues to shear
  off the nerve terminals. Each of the synaptosome
  is about 0.5 1.0 ?m in diameter, contains
  hundreds of synaptic vesicles, trace mitochondria
  and postsynaptic membrane. It can function for
  several hours.

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Synaptosomal preparation
Superfusion apparatus
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4. Tissue culture adrenal medullary chromaffin
cells have the same precursor cells as
postganglionic sympathetic neurons 5. Cell
culture collagenase digestion of bovine adrenal
glands or using PC12 cells to study the
neurochemical nature of neurotransmitter release.
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Neuromuscular junction ACh release and
postsynaptic effect

• 1. There are approximately 300 active zones per
  NMJ 500,000 vesicles in all of the active zones
  at one NMJ. It is estimated a vesicle contains
  20,000 ACh molecules.
• 2. The specialized postsynaptic membrane
  structure consists a high density of nicotinic
  ACh receptors (nAChRs)
• 3. Basal lamina matrix proteins are important for
  the formation and maintenance of the NMJ and are
  concentrated in the cleft enriched with AChE.

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Quantum release mechanism of release as
Exocytosis

1. Fatt and Katz (1952) observed motor neuron
   stimulation results in spontaneous potentials of
   approximately 1 mV at the motor end plate
   (miniature end-plate potentials MEPPs).
2. Measurement of quantum release amplitude fixed
   but number variated.

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Calcium is necessary for transmission at the NMJ
and other synapses

• 1. In most cases in the CNS and PNS, chemical
  transmission does not occur unless Ca2 is
  present.
• 2. EPPs could not be elicited if the calcium
  pulse immediately followed the depolarization
  but should be beforehand (pulse as short as 1
  msec preceded the depolarization).
• 3. Extracellular calcium 2 mM vs. intracellular
  0.1 ?M the requirement to activate calcium
  channels 50-100 ?M.
• 4. Origin of calcium could be extracellular (thru
  voltage-dependent Ca2 channel) or intracellular
  GPCR-coupled PI-PLC activation release IP3, thus
  Ca2 release from ER

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Top trace illustrates the postsynaptic membrane
potential
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Pre-synaptic event and time scale

• 1. The time between calcium influx and exocytosis
  in the n. terminal is very short (0.5 -1.0 msec
  at NMJ 200 ?sec at squid axon 60 ?sec in CNS
  neuron)
• 2. In such short time (due to docking and fusion
  of synaptic vesicle), synaptic vesicle can not
  move significantly (calcium diffuse only 850 Å)
• 3. The supply of synaptic vesicles in the
  terminal in limited. Rapid recycled from the
  synaptic membrane with plasma membrane via
  clathrin-mediated endocytosis is essential
  (endocytosis is occurred away from active zones).

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Calcium-dependent proteins are required for
vesicle docking
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The use of fluorescent dye FM1-43 to label the
membrane and visualize endocytosis and exocytosis
at the NMJ 1. Complete cycle of exocytosis and
endocytosis app. 1 min 2. A single n. impulse
release 0.1 of the recycling pool. 3.
Endocytosis follows app. 20 sec. after exocytosis.
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Endocytosis and clathrin cage
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Fast synaptic transmission vs. peptide/protein
release at the nerve terminals

• 1. Fast transmission catecholamines, amino acid
  transmitters can be synthesized within the n.
  terminals (synaptic vesicle) or transported
  rapidly across the membrane (uptake carrier).
• 2. In contrast, peptide/protein are inserted in
  the secretory granules, then transport down to n.
  terminals (no carrier)
• 3. Peptidergic granules are far less numbers than
  vesicles involved in fast exocytosis and are not
  localized at active zone (maybe function to
  maintain the long-lasting effect on postsynaptic
  neuron)

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Function of ATP in presynaptic secretion ?

• 1. ATP usually is released along with transmitter
  in the vesicle and important in maintaining the
  exocytosis mechanism.
• 2. ATP is necessary for the function of NSF (an
  ATPase) to dissociate complexes of the SNARE
  protein VAMP.
• 3. Maintain PIP2 and PIP by phosphorylation of
  phosphatidylinositol 4-kinaes and PIP kinase
  (PIP2 binds to synaptotagmin and rabphilin 3a and
  regulate cytoskeleton profilin, gelsolin,
  scinderin and myosin).

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Cellular signaling mechanisms

• Langley (1900s) noted the high specificity and
  potency of biological response and postulated the
  existence of receptor or acceptor.
• Three phases of receptor-mediated signaling
• 1. Polar molecule forms a complex with
  cell-surface receptor
• 2. Activated receptor-ligand complex elicits an
  increase of 2nd messenger, or channel open/close
• 3. Signal cascades

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Cell-surface receptors utilize four distinct
molecular mechanisms for transmembrane signaling
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Signal transduction cascade mediated by GPCR
(G-protein coupled receptor)
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Signal transduction cascade mediated by RTK
(receptor tyrosine kinase)
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Cross-talk between GPCR, ligand-gated ion
channels and receptor tyrosine kinase