Neurotransmitter Document PDF

Wayne Sossin MNI [email protected] Learning Objectives Neurotransmitters stored in synaptic vesicles – What are the neurotransmitters? – What determine whether a neuron uses a specific transmitter ? – Differences between fast and slow neurotransmission and which transmitters use which kind of n...

Wayne Sossin MNI [email protected] Learning Objectives Neurotransmitters stored in synaptic vesicles – What are the neurotransmitters? – What determine whether a neuron uses a specific transmitter ? – Differences between fast and slow neurotransmission and which transmitters use which kind of neurotransmission – How are each one degraded or removed? Vesicular Transporters – Which transporters transport which transmitters – How do they work? – How does changing levels of transporters affect synaptic transmission? – Co-release vs synergy for co-expression of transporters synaptic vesicles = key to neurotransmission Dense Core vesicles = release neuropeptides Presynaptic Terminal Post-synaptic Spine Synaptic Vesicles (Neurotransmitters) Active Zone Post-synaptic Density (PSD) NeurotransmitterG-protein Gated Channel -gated Receptor Dense Core Vesicle (Neuropeptides) Classical and Peptide Transmitters classical = first discovered The major difference between classical transmitters and peptide transmitters is the way that they are synthesized and where they are stored. Classical transmitters are synthesized in the cytoplasm and then packaged into synaptic vesicles by transporters (todays lecture). Peptide transmitters are synthesized by the translation of mRNAs and processed through the Golgi apparatus where they are packaged into regulated secretory vesicles (next lecture). Fast vs Slow Neurotransmission Classical transmitters are released at the active zone and can mediate both fast and slow neurotransmission, while neuropeptides are released from outside the active zone and only mediate slow neurotransmission. Fast Neurotransmission --> Ligand-gated Ion Channels; start electrical signal. Slow Neurotransmission --> G-protein linked receptors; start chemical signal by altering second messengers – Ligand gated channels in the presynaptic terminal mediate slow neurotransmission by modulating neurotransmitter release. Examples include presynaptic ligand-gated acetylcholine receptors and NMDA receptors (Glutamate). the point of slow neurotransmission is to modulate fast neurotransmission - almost all fast neurotrnasmission - the slow one is mediated by eyes failure of slow neurotranmission - is not noticing he eraser hitting you —> sleep, rest etc. 9 Transmitters known to be released from synaptic vesicles Transmitter Fast or Slow Acetylcholine (Ach) Both Protein specific to Comments neurons that use transmitter Choline Major fast neurotransmitter Acetlytransferase between neurons and muscle; (Chat); Vesicular In brain released from midAcetylcholine brain nuclei transporter (VACHT) Both Glutamate vesicular transporter (VGLUT) first one discovered nicotinic gated = fast Glutamate major excitatory transmitter G-AminoButyric Acid (GABA) Glycine Major excitatory transmitter in all cells make brain glutamate Both Glutamic Acid Decarboxylase (GAD) Major inhibitory transmitter in brain GABA and glycine use the same transporter to put it into a vesicle Fast Glycine plasma membrane transporter Inhibitory transmitter (mainly in spinal cord) if you block it you DIE Biogenic Amines -its the next enzyme to make N0repineph. Dopamine Slow Tyrosine Hydroxylase (TH); Dopadecarboxylase; not DBH Transmitter lost in Parkinson’s disease; midbrain nuclei like Substantia Nigra Norepinephrine Slow Dopamine betahydroxylase (DBH), not PNMT Localized to Locus Coeruleus Epinephrine Slow Phenylethanolamine N-methyltransferase (PNMT) Localized to Adrenal Medula Serotonin Slow (1 fast Receptor) Tryptophan hydroxylase; 5-HTP decarboxylase Localized to Raphe Nucleus Histamine Slow Histidine decarboxylase Hypothalamus Fast neurotransmission vs Slow/ Modulatory Neurotransmission There are anatomical differences between transmitters mainly used for fast neurotransmission (Glutamate, GABA, and Glycine) and transmitters used mainly for slow modulatory neurotransmission (Dopamine, Serotonin, Noradrenaline, Adrenaline, Histamine). Acetylcholine (Ach) shares properties with both classes Neurons that use modulatory neurotransmission are localized to midbrain nuclei that project widely to the whole neocortex (Modulatory systems) Acetylcholine (Ach) fits both categories; for motor neurons connecting to muscles it is the major fast transmitter, in the brain ACh is in nuclei that project widely to the whole neocortex Modulatory transmitters affect mood, attention, and most psychological disorders and psychoactive drugs mainly affect modulatory transmission, not fast neurotransmission. Modulatory neurotransmitters are localized in mid-brain Nuclei (Dopamine) Modulatory neurotransmitters are localized in mid-brain Nuclei (Serotonin) Dale’s Hypothesis - we define the neuron by the transmitter that they make - even tho its not correct Neurons use only one transmitter. – One can call a neuron dopaminergic or glutaminergic. While a useful concept, lots of exceptions. – True for classical transmitters only; neuropeptides are co-transmitters in many cells. – GABA and glycine can be released from the same synaptic vesicle – A glutamate transporter is found in many cells with other classical transmitters; e.g. GABA, dopamine and 5-HT; in some cases this leads to co-release. – Dopamine and norepinephrine are released from the same neuron in different brain areas. What is the evidence that we need to call something a classical neurotransmitter? when you casue that neuron to fire, and if its a transmitter and it mediates that function -presynaptic neuron fires, post synaptic side does smth, mediated by a neurotramsitter -has to be made in the cytoplasm, then have a vesicular transporter that packages into synaptic vesicles -has to be released from the synaptic vesicles at active zones Should it be 10? (ATP) ATP has both fast and slow ligand-gated channels A Specific transporter ATP has a transporter to put it into vesicles Applying ATP has effects on the postsynaptic cell it could be Dense It is controversial whether the ATP core vesicles transporter is sorted into synaptic vesicles -it isn’t released synaptic ATP is likely released from secretory from vesicles granules. Should there be more? we have lost lots of neurotransmitters over evolution There are arguments that Creatine, Aspartate and Taurine fit the criteria for classical transmitters Higher deuterostomes have lost the common neurotransmitters, tyramine and octopamine that were present in the common ancestor to all bilaterians and are used by insects and molluscs. Many species have expansions of vesicular transporters suggesting they have evolved new SV transmitters. What does a neuron need to release a transmitter from a synaptic vesicle ALWAYS -these are the critical thing – A vesicular transporter to concentrate the transmitter into a vesicle USUALLY glutamate and glycine are examples where you don’t – Biosynthetic enzymes to synthesize the neurotransmitter (glutamate and glycine are not specifically synthesized in cells that use them) RARELY neurons that have glycine have it on the plasma membrane transporter it is critical for them – Uptake transporters in plasma membrane (only glycine absolutely requires this, but for many biogenic amines, this is an important source of transmitter) Vesicular and Plasma Membrane Transporters Vesicular Protein ATPase ATP ADP -its job is to transport H ions into the vesicle -it makes the inside more positive (+) and it increases the concentration of hydrogen inside (voltage gradient vs conc. gradient) H+ 5-HT = serotonin Na + 5-HT VMAT takes 1 H+ ion and will leave the cell, and that energy will be used to transport serotonin across its chemical gradient H+ 5-HT many drugs work on plasma membrane transporters not on vesicular transporters Vesicular Transporter SSRis act on this: Plasma Membrane this is a Transporter =specific serotinin transporter uses sodium to push serotonin into the cytoplasm Neurotransmitter Transporters Two types; Plasma Membrane transporters for transport from extracellular medium to cytoplasm (Important for removal of transmitter, uptake); Vesicular transporters for transport from cytoplasm into vesicle Requires energy of some form to transport protein against concentration gradient Transporters are different from channels; they are slower and do not contain a pore, transport mechanisms are still not completely understood, but requires binding and unbinding of transmitter. transporters are not channels = theyre much much slower 4 types of vesicular transporters Vesicular Monoamine Transporter (VMAT1 and VMAT2) transport all the modulatory transmitters (Serotonin, dopamine, norepinephrine, epinephrine, histamine) Vesicular Acetylcholine Transporter (VACHT) Homologous to VMAT; Vesicular GABA and Glycine Transporter (VGAT). Lower affinity for glycine, requires glycine plasma membrane transporter to get glycine levels high enough in the cytoplasm. Vesicular Glutamate Transporter (VGLUT): Three isoforms; VGLUT1 and 2 are expressed mainly in glutaminergic neurons. VGLUT3 is mainly expressed in cells releasing other transmitters, although one exception is in some auditory neurons. Lack of VGLUT3 leads to deafness. (VATP is related to VGLUT) 3 families or transporters were co-opted independently for synaptic vesicles One might imagine that when synapses evolved (probably in Cnidarians (coral, anemones, hydra and jelly-fish), a transporter was co-opted for transmitter uptake that then evolution evolved into separate transporters (VMAT/VACHT; the of different transporters VGAT; VGLUT). This is NOT true. happened Actually, these three transporters come from distinct differently they were transporter families that had separated long before different even metazoans evolved. VGAT and VGLUT come from families back then transporter families that were distinct in bacteria. Three independent evolutionary events. VGAT and VGLUT are present in Cnidaria; VMAT/VACHT was coopted later. VATP is actually the oldest; predates synaptic vesicles Synaptic Strength Synaptic Strength (M) can be defined as the number of release sites (N) multiplied by the probability of release (P) multiplied by the synaptic effect from the release of a single vesicle (Q). M=NPQ To determine mechanisms for an increase in synaptic strength one often looks at miniature excitatory post synaptic currents (minis) that are observed in the absence of an action potential and are due to the spontaneous release of one synaptic vesicle. An increase in the frequency of minis is due to a change in P or N, while an increase in the amplitude of a mini is a change in Q. EPSPs = potential = measuring voltage change EPSC = current = by not letting voltage change (putting voltage clamp) measuring amount of current needed to keep vp;tage EPSPs vs EPSC vs EPPs Potential vs Current is due to a difference in how the measurement is being made. – Potentials mean the cell is in current clamp Usually sharp electrode in cell, measure voltage changes. (mV) – Current means the cell is in voltage clamp Usually whole cell patch clamp, inject current to keep cell at constant voltage; measure how much current is needed. (pA) – Neuromuscular junctions are called end plate potentials (just because) if its A = you know its current if its V = you know its voltage What mini-EPSCs look like. the time scale is huge here What would happen if we expressed the VGLUT in a cell that normally releases GABA? Would you release glutamate? YES! Would they be released from the same could be, depends on sorting of vesicle? vesicular transporters and the amount that are available Do you think you would generate glutamate you are able to see EPSPs - this is because sorting isnt as good as you would EPSPs ??? yes, think between the receptors What about the reverse: express VGAT transporter in glutaminergic neuron? -NO bc you have to make GABA (vs glutamate that exists in every cell) so you need an enzyme to create GABA Effects of VGLUT1 overexpression on quantal excitatory transmission. -there is an increase in amplitude of mini EPSCs -the frequency is unchanged this means you have more glutamate is going into each vesicle ©2005 by Society for Neuroscience Society for Neuroscience J. Neurosci. 2005;25:7100 Transporter levels regulate synaptic strength Increased number of VGLUT transporters leads to increased molecules of glutamate stored in a synaptic vesicle. – When the vesicle is released, more glutamate is released, larger amplitude of mini-EPSCs (Q) – Leaky bathtub model explains why more transporters lead to more stored transmitter. Leaky Bathtub model the spead at which the glutamate comes in determines how strong that faucet is the more transporters = the more glutamate you actually store in the vesicle Number of transporters represent the strength of the faucet. Without a leak, the level of water reached is independent of the strength of the faucet, but with a leak the strength matters. Sometimes levels of Vglut affects mini frequency… decreasing amount of vesiculer glutamate transporter - but found that freq of minis went down there are going to be synaptic vesicles with noglutamate in them bc there are so little VGLUT transporters, so there will be some that release no glutamate. SO frequency will be decreased Daniels et al, 2006 Neuron 49:11-16 there are VGLUTs in lots of cells that don’t express glutamate, and don’t have EPSPs Levels of VGLUT can affect number of vesicles with transmitter In some cases when VGLUT levels are low (i.e. around 1 molecule/vesicle), increasing VGLUT levels will increase the number of vesicles that contain neurotransmitter. This will affect frequency of transmitter release (one cannot functionally tell a difference between the release of an empty vesicles or the failure to release a vesicle; thus less empty vesicles will be measured as an increase in frequency of minis, not an increase in amplitude). – Neurons do not seem to have a quality control pathway to prevent release of empty vesicles. Loss of transporters do not lead to changes in synaptic vesicle release. VGLUTs allow co-release VGLUTs are present in many cells that were not thought to be glutaminergic. For dopaminergic and serotonergic neurons, co-release of glutamate from a subset of terminals has important roles. There are now a number of examples of co-release of glutamate and GABA from same neuron. An interesting variation of how to regulate excitation/inhibition. When there is co-expression, there are three possibilities – Storage of two transmitters in same vesicle – Two separate populations of synaptic vesicles in same release site – Separate release sites with distinct populations of synaptic vesicles Neurotransmitter Co-release Two separate populations of synaptic vesicles in same release site Storage of two transmitters in same vesicle Separate release sites with distinct populations of synaptic vesicles Synergy between VACHT and VGLUT3 VACHT - cares about concentration of H+ ions Vesicular ATPase doesnt have much of a charge graident if it doesnt have a Gras et al, Nature Neuroscience 11, 292 - 300 (2008) Positively charged Ach builds up in vesicle making it harder to pump in H+ ions and build up pH gradient. Pumping in negatively charged Glutamate lowers this charge gradient, enabling the ATP pump to work better. VGLUT3 synergizes storage of other transmitters Removing VGLUT3 reduces Acetylcholine release when two are co-expressed in same vesicle. One can measure in-vitro that adding glutamate increases Acetylcholine uptake into some synaptic vesicles and this requires VGLUT3. There are three things one needs to know to understand this: – VGLUT is more sensitive to charge than PH; while other transporters are more sensitive to PH than charge. – Glutamate is partially negatively charged, while most other transmitters are partially positively charged – The Vesicular ATPase cares about both the charge and pH of the vesicle. How common is synergy? Biochemical experiments find that vesicles with two transporters are rare. – Most common is Vglut and Zn transporter – In this case,the role of Zn in facilitation Vglut transport would be to dissipate the charge difference (i.e. vesicle would get too negatively charged with just Vglut. – Other pairs do happen (i.e. Dopamine and Vglut2, but this study finds that co-expression is rare. ) LDopa gets turned into dopamine - Tyrosine L-DOPA Tyrosine Hydroxylase (Rate-Limiting) DopaDecarboxylase Beta blocker blocks beta noradrenaline Nor-epinephrine Dopamine b-Hydroxylase (vesicular enzyme) Dopamine Is all the dopamine converted to NE in the vesicle? Noradrenaline neurons were releasing dopamine It has been known for a while that dopamine release in the hippocampus is important for memory However, there are almost no axons from dopaminergic neurons in the hippocampus Recently, it has been established that the dopamine important for learning is coming from the locus coeruleus neurons that were through to release norepinephrine. – – – – Is dopamine beta hydroxylase (DBH) not that efficient? Are there inhibitors of DBH in the hippocampus? Is there mis-sorting of DBH when vesicles are reused? Is NE converted to dopamine after release? Removal of neurotransmitters is a critical step in neurotransmission For fast neurotransmission need to send many signals at fast pace (e.g. 100 Hz). If neurotransmitter sticks around, new release cannot be sensed For slow neurotransmission, removal of transmitter is essential in regulating the effect of the modulatory neurotransmitter. How are neurotransmitters removed Fast neurotransmission – Degradation (Ach) – Uptake Slow neurotransmission – Uptake – Degradation regulates level of transmitters. How are neurotransmitters removed Fast neurotransmission: – Acetylcholinesterase is located in the synaptic cleft; degrades Acetylcholine. The deadly poisonous gases Sarin and Novichok acts by inhibiting this enzyme, so yes, degradation is important. – Glutamate is taken up mainly by glial cells that wrap synapses. – GABA are Glycine are taken up by both neurons and glial cells Slow neurotransmission – Biogenic amines are also taken up by cells that secrete them; uptake can regulate both activation of receptors and regulate level of transmitter (i.e. transmitter is reused). – Blockade of biogenic amine plasma membrane transporters is a site of action of many drugs; both pharmaceutical (Prozac; other serotonin selective re-uptake inhibitors (SSRIs) and illegal (Cocaine (mainly blocks dopamine uptake transporters). Recycling pathway for GABA and Glutamate extracellular glutamate is toxic - so u don’t want it just floating around Patel, Anant B. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 5588-5593 glial cells take up glutamate and release glutamine, and release them to be taken up and created into glutamate Copyright ©2005 by the National Academy of Sciences Why not just take up glutamate? You can, there are glutamate uptake transporters on neurons as well. However, like other aspects of fast transmission we will discuss later, you’ve got to be fast. It’s faster for astrocytes, wrapped around the synapse to pick up the glutamate. Energetic costs of glutamate/glutamine At a minimum; 30% of energy (glucose) used in the brain is to support this cycle (includes contribution from GABA-Glutamine shunt as well). Some think this an underestimate. The energy used is proportional to the neurotransmitter released. This may be the basis for functional imaging; the increased use of glucose to support transmitter recycling is what is visualized in fMRI.