Transcript 7Nt Release
Neurotransmitter Release Two principal kinds of synapses: electrical and chemical Gap junctions are formed where hexameric pores called connexons connect with one between cells Electrical synapses are built for speed Contrast with chemical synapse Delay of about 1 ms Electrical coupling is a way to synchronize neurons with one another Electrical synapses are not presently considered to be the primary means of communication between neurons in the mammalian nervous system, but they may prove to be more important than presently recognized Rectification and uni-directionality of electrical synapses …not just simple bidirectional bridges between cells Conductance through gap junctions may be sensitive to the junctional potential (i.e the voltage drop between the two coupled cells), or sensitive to the membrane potential of either of the coupled cells Glial cells can also be connected by gap junctions, which allows synchronous oscillations of intracellular calcium http://users.umassmed.edu/michael.sanderson/mjslab/MOVIE.HTM Chemical synapses: the predominant means of communication between neurons An early experiment to support the neurotransmitter hypothesis Synaptic Release I: Criteria that define a neurotransmitter: 1. Must be present at presynaptic terminal 2. Must be released by depolarization, Ca2+-dependent 3. Specific receptors must be present Neurotransmitters may be either small molecules or peptides Mechanisms and sites of synthesis are different Small molecule transmitters are synthesized at terminals, packaged into small clear-core vesicles (often referred to as ‘synaptic vesicles’ Peptides, or neuropeptides are synthesized in the endoplasmic reticulum and transported to the synapse, sometimes they are processed along the way. Neuropeptides are packaged in large dense-core vesicles Neurotransmitter is released in discrete packages, or quanta Failure analysis reveals that neurons release many quanta of neurotransmitter when stimulated, that all contribute to the response Quantal content: The number of quanta released by stimulation of the neuron Quantal size: How size of the individual quanta Quanta correspond to release of individual synaptic vesicles EM images and biochemistry suggest that a MEPP could be caused by a single vesicle EM studies revealed correlation between fusion of vesicles with plasma membrane and size of postsynaptic response Probabilistic Basis of Quantal Transmission gsyn (t) g p tet a n x nx n! p (1 p) P(a b) x!(n x)! b x 0 ( 1 e 2x k x 2 ) dk According to the Quantum Hypothesis of Synaptic Transmission, neurotransmitter is discharged in the form of integral numbers of multimolecular packets, called quanta. The magnitude of the postsynaptic response is a probability distribution given by P(κ). The time course of the synaptic response has a duration given by the alpha function shown. 4-AP was used to vary the efficiency of release Calcium influx is necessary for neurotransmitter release Voltage-gated calcium channels Calcium influx is sufficient for neurotransmitter release Synaptic Release II The synaptic vesicle release cycle 1. Tools and Pools 2. Molecular biology and biochemistry of vesicle release: 1. Docking 2. Priming 3. Fusion 3. Recovery and recycling of synaptic vesicles The synaptic vesicle cycle How do we study vesicle dynamics? Morphological techniques Electron microscopy to obtain static pictures of vesicle distribution; TIRFM (total internal reflection fluorescence microscopy) to visualize movement of vesicles close to the membrane Physiological studies Chromaffin cells Neuroendocrine cells derived from adrenal medulla with large dense-core vesicles. Can measure membrane fusion (capacitance measurements), or direct release of catecholamine transmitters using carbon fiber electrodes (amperometry) Neurons Measure release of neurotransmitter from a presynaptic cell by quantifying the response of a postsynaptic cell Genetics Delete or overexpress proteins in mice, worms, or flies, and analyze phenotype using the above techniques Synaptic vesicle release consists of three principal steps: 1. Docking Docked vesicles lie close to plasma membrane (within 30 nm) 1. Priming Primed vesicles can be induced to fuse with the plasma membrane by sustained depolarization, high K+, elevated Ca++, hypertonic sucrose treatment 2. Fusion Vesicles fuse with the plasma membrane to release transmitter. Physiologically this occurs near calcium channels, but can be induced experimentally over larger area (see ‘priming’). The ‘active zone’ is the site of physiological release, and can sometimes be recognized as an electron-dense structure. . Synaptic vesicles exist in multiple pools within the nerve terminal (Release stimulated by flash-photolysis of caged calcium) (reserve pool) Becherer, U, Rettig, J. Cell Tissue Res (2006) 326:393 Morphologically, vesicles are classified as docked or undocked. Docked vesicles are further subdivided into primed and unprimed pools depending on whether they are competent to fuse when cells are treated with high K+, elevated Ca++, sustained depolarization, or hypertonic sucrose treatment. In CNS neurons, vesicles are divided into Reserve pool (80-95%) Recycling pool (5-20%) Readily-releasable pool (0.1-2%; 5-10 synapses per active zone) Rizzoli, Betz (2005). Nature Reviews Neuroscience 6:57-69) A small fraction of vesicles (the recycling pool) replenishes the RRP upon mild stimulation. Strong stimulation causes the reserve pool to mobilize and be released Vesicle release requires many proteins on vesicle and plasma membrane Docking: UNC-18 (or munc-18) is necessary for vesicle docking (Weimer et al. 2003, Nature Neuroscience 6:1023) 1. unc-18 mutant C. elegans have neurotransmitter release defect 2. unc-18 mutant C. elegans have reduction of docked vesicles Unc-18 mutants are defective for evoked and spontaneous release Unc-18 mutants are defective for calcium-independent release primed vesicles occasionally fuse in the absence of calcium; a calcium-independent fusion defect suggests a lack of primed vesicles UNC-18 (munc18) is required for docking: unc-18 mutants have fewer docked vesicles Summary: Unc-18 mutants are unable to dock vesicles efficiently. Impaired docking leads to fewer primed vesicles; fewer primed vesicles leads to reduced overall neurotransmitter release. Priming Vesicles in the reserve pool undergo priming to enter the readilyreleasable pool At a molecular level, priming corresponds to the assembly of the SNARE complex The SNARE complex UNC-13 is a critical priming factor Richmond and Jorgensen (1999) Nature Neuroscience 2:959 unc-13 mutants have higher levels of synaptic vesicles than normal normal unc-13 mutants No docking defect was observed unc-13 mutants have evoked release defect Calcium-indepenent release is also defective, indicating that the defect is in priming Munc-13 function in priming Inhibitory domain, folds back on itself “open” syntaxin doesn’t fold properly unc-13 defect can be bypassed by providing an “open” form of syntaxin Model for unc-13, unc-18, syntaxin interaction in priming Synaptotagmin functions as a calcium sensor, promoting vesicle fusion Synaptic vesicles recycle post-fusion Modern methods to track recycling membrane Endocytosis retrieves synaptic vesicle membrane and protein from the plasma membrane following fusion The ATP-ase NSF disassembles the SNARE complex There are Numerous Neurotransmitters in the CNS AMINO ACIDS • Excitatory: – Glutamate – Aspartate – L-Homocystate • Inhibitory: – GABA – Glycine MONOAMINES, PEPTIDES, etc. • Modulatory: – Serotonin (5-HT) • – – – – – – – (5-hydroxytryptamine) Histamine Epinephrine, Norepinephrine Dopamine Nitric Oxide Substance P Endomorphins, enkephalins Acetylcholine Multiple Neurotransmitters can be Released from the same Synaptic Terminal •Neurotransmitters in the CNS can act on numerous sub-types of receptors Nature Neuroscience 8, 257 - 258 (2005) Summary of Presynaptic Differences • Many presynaptic axons converge on a single postsynaptic cell • Connections can be axon-dendritic, axo-somatic, or axo-axonic • There are many different neurotransmitter substances in the CNS, and sometimes a presynaptic element releases more than one • Transmitter is typically removed by neurotransmitter transporters, and is not always taken up into the presynaptic terminal On the Postsynaptic Side… • There are some similarities: – Transmitter binds to postsynaptic receptors – Postsynaptic receptors can couple directly to ion channels On the Postsynaptic Side… • But there are more differences – Many different types of neurotransmitter receptors are often on the postsynaptic membrane – The same neurotransmitter can act on numerous subtypes of neurotransmitter receptors, and can have dramatically different actions – Receptors can depolarize (excite), OR hyperpolarize (inhibit) a postsynaptic cell – Receptors can couple to ion channels indirectly, via a G-protein cascade – Activation of receptors can sometimes have effects unrelated to membrane potential What do Some of the CNS Neurotransmitters do? • Glutamate is excitatory, and (typically) mediates a “depolarizing” response called an EPSP (excitatory postsynaptic potential) •Glutamate can act on numerous types of glutamate receptors •D-AP5 blocks NMDA-type glutamate receptors Kinney et al, J Neurophysiology, 1993 Currents underlying an EPSP http://www.chrisparsons.de/Chris/images/AMPA.jpg What do some of the CNS neurotransmitters do? • GABA is inhibitory, and (typically) mediates a “hyperpolarizing” response called an IPSP (Inhibitory postsynaptic potential) http://psyche.knu.ac.kr/notebook/ima What are the conductance changes that occur during an IPSP? http://www.cnsforum.com/content/pictures/imagebank/hirespng/hrl_rcpt_sys_gab.png What are the conductance changes that occur during an IPSP? http://www.blackwellpublishing.com/matthews/neurotrans.html What do some of these CNS neurotransmitters do? • Modulatory neurotransmitters have numerous effects on synaptic transmission and neuronal firing Foehring et al, J Neuroscience, 2002 Receptors can be coupled to ion channels directly or indirectly Ligand Binding to G-Protein Coupled Receptors can cause transmitter release http://www.blackwellpublishing.com/matthews/neurotrans.html G-protein mediated synaptic actions differ from direct transmitter actions on ligandgated channels • Slower • May act through intracellular second messengers • May have actions other than changing membrane potential – Control calcium entry or release from intracellular stores – Affect gene expression The GABAA receptor is the site of action of many important drugs and compounds •Barbiturates •Benzodiazepines •Alcohol http://web.lemoyne.edu/~hevern/psy340/grap Many Drugs and Toxins Affect Synaptic Transmission • Excitatory transmission depressants – Toxins from spiders, wasps, and cone snails – Ketamine (“special K”) – Phencyclidine (PCP) • Excitatory transmission stimulants – Plant alkaloids from betel nuts, amino acids from mushrooms, algae, seeds, seaweed • Inhibitory transmission depressants (produce seizures) – Strychnine, plant alkaloids from Dutchman’s breeches, insecticides (dieldrin) • Inhibitory transmission enhancers (i.e., depressants) – Alcohol, benzodiazepines (Valium), barbiturates (Phenobarbital) – General Anesthetics: propofol, pentobarbital – Mushroom toxin: muscimol Many Drugs and Toxins Affect Synaptic Transmission • Modulatory Neurotransmitters – Prozac, Celexa, etc. – MDMA (Ecstasy) – Methamphetamine (crystal meth)