NROB60 Chapter 6.doc

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University of Toronto St. George
Human Biology
Janelle Le Boutillier

NROB60 – Chapter 6 Introduction - Cholinergic – used to describe the cells that produce and release ACh - Noradrenergic – Neurons that use the amine neurotransmitter norepinephrine (NE) - Glutamatergic – synapses that use glutamate - GABAergic – synapses that use GABA - Peptidergic – synapses that use peptides Studying Neurotransmitter Systems - Criteria that must be met for a molecule to be considered a neurotransmitter: o The molecule must be synthesized and stored in the presynaptic neuron o The molecule must be released by the presynaptic axon terminal upon stimulation o The molecule, when experimentally applied, must produce a response in the postsynaptic cell that mimics the responses produced by the release of a neurotransmitter from the presynaptic neuron Localization of Transmitters and Transmitter-Synthesizing Enzymes - Hypothesis that a molecule may be a neurotransmitter based on observing that the molecule is concentrated in brain tissue or that the application of the molecule to certain neurons alters their action potential firing rate - First step in confirming the hypothesis is to show that the molecule is localized in and synthesized by, particular neurons o Two important technique used to satisfy this criterion are  Immunocytochemistry – method for viewing the location of specific molecules, including proteins, in sections of brain tissue  In situ hybridization – method for localizing specific mRNA transcripts for mRNA Immunocytochemistry: - Used to anatomically localize particular molecules to particular cells - Once the neurotransmitter candidate has been chemically purified, it is injected into the bloodstream of an animal, where it stimulates an immune response o One aspect of the immune response is the generation of large proteins called antibodies  Antibodies can bind tightly to specific sites on the foreign molecule, the transmitter candidate in this case - It can also be used to localize any molecule for which a specific antibody can be generated In Situ Hybridization: - Useful for confirming that a cell synthesizes a particular protein or peptide o There is a unique mRNA molecule for every polypeptide synthesized by a neuron o The complementary strand is called a probe and the process by which the probe bonds to the mRNA molecule is called hybridization - For in situ hybridization, the probes are usually labeled by marking them radioactive o Hybridized probes are detected by laying the brain tissue on a sheet of special film that is sensitive to radioactive emissions o The technique for viewing the distribution of radioactivity is called autoradiography Studying Transmitter Release - Must show that the transmitter is actually released upon stimulation o A specific set of cells or axons can be stimulated while taking samples of the fluids bathing their synaptic targets o The biological activity of the sample can be tested to see if it mimics the effect of the intact synapses  Then the sample can be chemically analyzed to reveal the structure of the active molecule - Unlike the PNS, most regions of the CNS contain a diverse mixture of intermingled synapses using different neurotransmitters o This often makes it impossible to stimulate a single population of synapses, containing only a single neurotransmitter - Researchers must be content with stimulating many synapses in a region of the brain and collecting and measuring all the chemicals that are released o One way this is done is by using brain slices that are kept alive in vitro  To stimulate release, the slices are bathed in a solution containing a high K+ concentration  This treatment causes a large membrane depolarization, thereby stimulating transmitter release from the axon terminals in the tissue - Because transmitter release requires the entry of Ca2+ into the axon terminal, it must be shown that the release of the neurotransmitter candidate occurs only when Ca2+ ions are present in the bathing solution o However, we can’t be sure that the molecules collected in the fluids were released from the axon terminals; they may have been released as a secondary consequence of synaptic activation Studying Synaptic Mimicry - Establishing that a molecule is localized in, synthesized by, and released from a neuron is still not sufficient to qualify it as a neurotransmitter o A 3 criterion must be met:  The molecule must evoke the same response as that produced by the release of naturally occurring neurotransmitter from the presynaptic neuron - To assess the postsynaptic actions of a transmitter candidate, a method called microionophoresis is often used o A glass pipette with a very fine tip, is filled with the ionized solution and carefully positioned next to the postsynaptic membrane of the neuron  The transmitter candidate is ejected in very small amounts by passing electrical current through the pipette o Microelectrode in the postsynaptic neuron measures the effects of the transmitter candidate on the membrane potential - The molecule and the transmitter usually are considered to be the same chemical if o Ionophoretic application of the molecule causes electrophysiological changes that mimic the effects of transmitter released at the synapse o Other criteria of localization, synthesis and release have been met Studying Receptors - As a rule, no 2 neurotransmitters bind to the same receptors, however, one neurotransmitter can bind to many different receptors o Each of the different receptors a neurotransmitter binds to is called a receptor subtype - 3 approaches have proved to be useful for studying the different receptor subtypes o Neuropharmacological analysis of synaptic transmission o Ligand-binding methods o Molecular analysis of receptor proteins Neuropharmacological Analysis: - Using this method, we found that 2 ACh receptor subtypes can be distinguished by the actions of different drugs o Names were given of their agonists:  Nico-tinic ACh receptors – in skeletal muscle  Muscarinic ACh receptors – in the heart - Another way to distinguish receptor subtypes is to use selective antagonists o The poison, curare, inhibits the action of ACh at nicotinic receptors (causing paralysis)  Atropine antagonizes ACh at muscarinic receptors - Different drugs were used to distinguish several subtypes of glutamate receptors, which mediate the synaptic excitation in the CNS, 3 subtypes: o AMPA receptors o NMDA receptors o Kainite receptors - The neurotransmitter glutamate, activates all 3 receptor subtypes but AMPA acts only at the AMPA receptor, NMDA acts only at the NMDA receptor and so on Ligand-Binding Methods: - Opiates are a broad class of drugs and are hypothesized that they might be agonists at specific receptors in neuronal membranes o They radioactively labeled opiate compounds and applied them in small quantities to neuronal membranes that had been isolated from different parts of the brain o Found that receptors existed in the membrane since the labeled opiates bind tightly to them - Search was on to identify endogenous opiates, endorphins, the naturally occurring neurotransmitters that act on these receptors o 2 peptides called enkephalins were soon isolated from the brain and were proved to be opiate neurotransmitters - Any chemical compound that binds to a specific site on a receptor is called a ligand for that receptor - Technique of studying receptors using radioactively labeled ligands is called the ligand- binding method Molecular Analysis: - Receptor subtype diversity was expected from the actions of different drugs, but the broad extent of the diversity was not appreciated until researchers determined how many different polypeptides could serve as subunits of functional receptors Neurotransmitter Chemistry - Major neurotransmitters are amino acids, amines and peptides o Amino acids – building blocks of protein, essential to life o Amines – derived from amino acids o Peptides – constructed from amino acids  ACh is an exception, derived from acetyl CoA, a ubiquitous product of cellular respiration in mitochondria, and choline, important for fat metabolism - Amino acid and amine transmitters are generally each stored in and released by separate sets of neurons o The idea that a neuron has only one neurotransmitter is often called Dale’s principle  Peptide-containing neurons violate Dale’s principle because they release more than one neurotransmitter: a peptide + an amino acid or amine  When 2 or more transmitters are released from one nerve terminal, they are called co-transmitters Cholinergic Neurons (ACh) - Acetylcholine (ACh) is the neurotransmitter at the neuromuscular junction and is synthesized by all motor neurons in the spinal cord and brain stem o ACh synthesis requires a specific enzyme, choline acetyltransferase (ChAT), manufacted in the soma and transported to the axon terminal o Only cholinergic neurons contain ChAT, so this enzyme is a good marker for cells that use ACh as a neurotransmitter - ChAT synthesizes ACh in the cytosol of the axon terminal and the neurotransmitter is concentrated in synaptic vesicles by the actions of an ACh transporter o ChAT transfers an acetyl group from acetyl CoA to choline  The source of choline is the extracellular fluid o Choline is taken up by the cholinergic axon terminals via a specific transport - The availability of choline limits how much ACh can be synthesized in the axon terminal o Transport of choline into the neuron is said to be the rate-limiting step in ACh synthesis - Cholinergic neurons also manufacture the ACh degradative enzyme acetylcholinesterase (AChE) o AChE is secreted into the synaptic cleft and is associated with cholinergic axon terminal membranes o It is manufactured by some noncholinergic neurons, so this enzyme is not as useful a marker for cholinergic synapses as ChAT - AChE degrades ACh into choline and acetic acid quickly, due to fast catalytic rates o Much of the resulting choline is taken up by the cholinergic axon terminal and reused for ACh synthesis - Inhibition of AChE prevents the breakdown of ACh, disrupting transmission at cholinergic synapses on skeletal muscle and heart muscle o Acute effects include marked decreases in heart rate and blood pressure; however, death from the irreversible inhibition of AChE is a result of respiratory paralysis Catecholaminergic Neurons (Amines) - The amino acid tyrosine is the precursor for 3 different amine neurotransmitters that contain a chemical structure called a catechol o These neurotransmitters are collectively called catecholamines o The catecholamine neurotransmitters are:  Dopamine (DA)  Norepinephrine (NE)  Epinephrine (adrenaline) - Catecholaminergic neurons are found in regions of the nervous system involved in the regulation of movement, mood, attention and visceral function o They all contain the enzyme tyrosine hydroxylase (TH), which catalyzes the first step in catecholamine synthesis, the conversion of tyrosine to a compound called dopa - The enzyme’s activity is regulated by various signals in the cytosol of the axon terminal o Decreased catecholamine release by the axon terminal causes the catecholamine concentration in the cytosol to rise, thereby inhibiting TH  This type of regulation is called end-product inhibition o High rate release of catecholamines, causes elevation in [Ca2+]i triggering an increase in the activity of TH - Dopa is converted into the neurotransmitter dopamine by the enzyme dopa decarboxylase o Amount of dopamine synthesized depends on amount of dopa available - Neurons that use NE contain, in addition to TH and dopa decarboxylase, the enzyme, dopamine beta-hydroxylase (DBH), which converts dopamine to norepinephrine o Located within the synaptic vesicles - Adrenergic neurons contain the enzyme, phentolamine N-methyltransferase (PNMT), which converts NE to epinephrine o Located the cytosol of adrenergic axon terminals - The actions of catecholamines in the synaptic cleft are terminated by selective uptake of the neurotransmitters back into the axon terminal via Na+ dependent transporters o May be reloaded into synaptic vesicles for reuse or may be enzymatically destroyed by monoamine oxidase (MAO), an enzyme found on the outer membrane of mitochondria Serotonergic Neurons (Amines) - The amine neurotransmitter serotonin/5-HT, is derived from the amino acid tryptophan - Serotonergic neurons, few in number but help regulate mood, emotional behaviour and sleep o Synthesis of serotonin occurs in 2 steps  Tryptophan is converted first into an intermediary called 5-HTP by the enzyme tryptophan hydroxylase  The 5-HTP is then converted to 5-HT by the enzyme 5-HTP decarboxylase - The source of brain tryptophan is the blood, and the source of blood tryptophan is the diet o A dietary deficiency of tryptophan can lead to a depletion of serotonin in the brain - Following release from the axon terminal, 5-HT is removed from the synaptic cleft by the action of a specific transporter o The process of serotonin reuptake, like catecholamine reuptake, is sensitive to a number of different drugs o Once it is back in the cytosol of the serotonergic axon terminal, the transmitter is either reloaded into synaptic vesicles or degraded by MAO Amino Acidergic Neurons (Amino Acids) - The amino acids glutamate, glycine and GABA serve as neurotransmitters at most CNS synapses o Only GABA is unique to those neurons that use it as a neurotransmitter - Glutamate and glycine are synthesized from glucose and other precursors by the action of enzymes that exist in all cells o Differences among neurons in the synthesis of these amino acids are therefore quantitative rather than qualitative  Ex. average glutamate concentration of glutamatergic axon terminals are estimated to be 20 mM, 2 or 3 times higher than in nonglutamatergic cells • Distinction between glutamatergic and nonglutamatergic neurons is the transporter that loads the synaptic vesicles  In glutamatergic axon terminals, the glutamate transporter concentrates glutamate until it reaches a value of about 50 mM in the synaptic vesicles - Since GABA is not one of the 20 amino acids used to construct proteins, it is synthesized in large quantities only by the neurons that use it as a neurotransmitter o The precursor for GABA is glutamate, and the key synthesizing enzyme is glutamic acid decarboxylase (GAD)  GAD is a good marker for GABAergic neurons - The synaptic actions of amino acid neurotransmitters are terminated by selective uptake into the presynaptic terminals and glia via specific Na+ dependent transporters o Inside the terminal or glial cell, GABA is metabolized by the enzyme GABA transaminase Other Neurotransmitter Candidates and Intercellular Messengers - In addition to the amines and amino acids, a few other small molecules serve as chemical messengers between neurons o For instance, ATP is very likely to be a neurotransmitter - ATP is concentrated in vesicles at many synapses in the CNS and PNS, and it is released into the cleft by presynaptic spikes in a Ca2+ dependent manner o They are often packaged in vesicles along with another classic transmitter  Ex. catecholamine containing vesicles may have 100 mM of ATP, in addition to 400 mM of the catecholamine itself • In this case, the catecholamine and ATP are probably co- transmitters - ATP directly excites some neurons by gating a cation channel o Some of the neurotransmitter functions of ATP may be similar to those of glutamate - ATP binds to purinergic receptors some of which are transmitter-gated ion channels o There is also a large class of G-protein-coupled purinergic receptors - Small lipid molecules called endocannabinoids (endogenous cannabinoids) can be released from postsynaptic neurons and act on presynaptic terminals o Communication in this direction from post to pre is called retrograde signaling, thus endocannabinoids are retrograde messengers o Retrograde messengers serve as a feedback system to regulate the conventional forms of synaptic transmission, which go from pre to post - There are several unusual qualities about endocannabinoids: o They are not packaged in vesicles like other neurotransmitters; they are manufactured rapidly and on demand instead o They are small and membrane permeable; once synthesized, they can diffuse rapidly across the membrane of their cell of origin to contact neighboring cells o They bind selectively to the CB1 type of cannabinoid receptor, which is mainly located on certain pr
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