NMD Lecture 5 PDF

NEUROCHEMICAL SYSTEM NMD Lecture 5  Neurotransmitters: Neurotransmitters are more diverse and their release more complex than originally thought There is 50 known and maybe up to 100 neurotransmitters Often release from the presynaptic sites, they can however be released from other sites...

NEUROCHEMICAL SYSTEM NMD Lecture 5  Neurotransmitters: Neurotransmitters are more diverse and their release more complex than originally thought There is 50 known and maybe up to 100 neurotransmitters Often release from the presynaptic sites, they can however be released from other sites. Chemical signals are the primary means of communication between two neurons Their synthesis, release and degradation are tightly control by varied molecular mechanisms Criteria to define neurotransmitters. Classical NTs are: 1. Synthetized form the neuron from which it is release 2. Identifiable substances (chemically or pharmacologically) 3. Exogenous application of the NT should elicit changes in the postsynaptic neuron (mimic effect of stimulating presynaptic neuron) 4. NTs should act on specific receptors (and action should be block by antagonists of the receptors or genetic ablation) 5. Active mechanism to terminate NTs action should exist  Majors class of Neurotransmitters Monoamines (Catecholamines, serotonin, melatonin…) Amino acids (Glutamate, GABA, Glycine…) Peptide transmitters (endorphin, somatostatin, oxytocin…) Acetylcholine (organic compound released at the neuromuscular junction) Gasotransmitters (nitric oxide (NO), carbon monoxide (CO)..) Unconventional transmitters Endocannabinoids (lipid transmitter involved in retrograde neurotransmission) Regulation of the Monoamines Monoamine neurotransmitters or neuromodulators contain one amino group connected to an aromatic ring by a two-carbon chain (such as -CH2-CH2-). Epinephrine (=adrenaline) Monoaminergic systems, (i.e., the networks of neurons that utilize monoamine neurotransmitters), are involved in the regulation of cognitive processes such as emotion, arousal, and certain types of memory. Classical monoamines: Imidazoleamines Indolamines Histamine We will focus Serotonin (5-HT) on these Catecholamines: Dopamine, DA Epinephrine, Epi (=Adrenaline, Ad) Norepinephrine NE (=Noradrenaline NAd) Dopaminergic projection system in the brain Major nuclei: Substansia nigra ( striatum) Ventral tegmental area ( frontal and cingulate cortex, accumbens..) Arcuate nucleus ( pituitary)  Human brain frontal (coronal) section Cortex Norepinephrine and epinephrine projection systems in the brain: Norepinephrine cells are located in Epinephrine cells are located in two nucleus in the the medulla and pons medulla Neuroscience, Fourth edition Serotonin, projections in the brain: Neuroanatomical projections in human brain. Depicted here are the projections (shown as red arrows) from the caudal raphé nuclei (CRN) and one of the rostal raphé nuclei (RRN), i.e. the dorsal raphé nucleus. There are also projections from the median raphé nucleus (another one of the RRN), for example to the hippocampus (not shown). C, cerebellum; Th, thalamus; A, amygdala; TL, temporal lobe; ST, striatum; PFC, prefrontal cortex; OC, occipital cortex. Cools et al. Trends in Cognitive Sciences 2008  Catecholamines: (dopamine, norepinephrine, epinephrine) Organic compounds derivate from Tyrosine. Neurons will only express the Dopaminergic neuron enzymes necessary for the synthesis of the (DA) Noradrenergic neuron neurotransmitter they release = can (NE) Adrenergic neuron be used as a cell identity marker. (EPI) Expression of a specific set of biosynthetic enzymes defines neuronal identity In Situ Hybridization ISH is used to detect and localized specific DNA or RNA fragment within chromosome preparation, fixed cell or tissue section mRNA or or mRNA  In Situ Hybridization ISH is used to detect and localized specific DNA or RNA fragment within chromosome preparation, fixed cell or tissue section (c) Immunohistochemistry, Immunofluorescence, Immunostaining  Immunohistochemistry Incubation with secondary antibody (fluorescent) Tissue preparation (fixation) Incubation with primary antibody Immunohistochemistry of Parvalbumin neuron in the dentate Gyrus  Immunohistochemistry combined with expression of a fluorescent reporter Reticular thalamic nucleus Dentate Gyrus Red: Sparse tagging of granular cells with tdT Red: Parvalbumin neurons (tdt from (using viral vector) transgenic mice) Green: PV+ neuron morphology (PV antibody ) Magenta: Slit2 antibody, Green: Dapi Distribution of TH and GAD1 mRNA in the mouse brain: In the images, in situ hybridization using probe (antisense for the mRNA) for TH and GAD1 reveal an astonishing difference in the See http://mouse.brain-map.org/gene/show/21582 distribution of both enzymes (see images on the left). cortex hippocampus Question 1: Specific cell-types (hair cells, or muscle cells, or Schwann cells, or dopaminergic cells...) express a very specific set of genes that provide them with a cell function, morphologies, metabolic processes or any other features unique to that cell types (e.g. dopaminergic neurons express and release dopamine). The specificity of this expression pattern can in turn be useful for the scientist as the mRNA and corresponding proteins are unique to that cell-types and can be used to identify these cells (and localize them). This molecules are referred as cell identity marker. For instance, in the nervous system, because enzymes See http://mouse.brain-map.org/experiment/show/480 necessary to synthesize specific neurotransmitters (NTs) are only hippocampus cortex expressed in the neurons releasing such NTs, the localization of the mRNA coding for an enzymes can reliably help scientist to localize the class of neuron expressing said NTs within the brain. Knowing that TH and GAD1 are the rate-limiting enzyme for the synthesis of the catecholamines (DA, Eph, NE) and GABA respectively, what do the results presented in these images tell you about the localization of the GABAergic and the catecholaminergic neurons respectively? You don't need to be precise about the brain region here. Just give the main difference in term of the distribution of the distinct populations (catecholaminergic vs GABAergic neurons). Distribution of TH and GAD1 mRNA in the mouse brain: See http://mouse.brain-map.org/gene/show/21582 In the images, in situ hybridization using probe cortex hippocampus (antisense for the mRNA) for TH and GAD1 reveal an astonishing difference in the distribution of both enzymes (see images on the left). Question 2: Biochemical assays were used to measure the level of the catecholamines (DA, NE and Eph) and GABA in mouse cell lysates (brain homogenate) obtained from different brain regions. All neurotransmitters (NTs) were found in large See http://mouse.brain-map.org/experiment/show/480 amount in the cortex as well as in the hippocampus hippocampus. This strongly demonstrate that the cortex NTs are all released in these brain regions. In light of this biochemical analysis and based on your previous observations (question 1), what prediction can you make about the distinctive axonal morphologies of GABAergic versus catecholaminergic neurons? Don't extrapolate too much. Only make prediction about the features (e.g. axons or dendrites) that you can argue for based on the information provided. “Birth and death” of classical NTs From their synthesis to their final degradation, many steps regulating NTs are tightly controlled with specific molecular mechanisms and signaling pathways. Regulation of the catecholamines 1. Biosynthesis: Amount and activity of TH determine the rate of DOPA synthesis For example: Rate-limiting step - In norepinephric neurons of the Locus coeruleus, increases of TH gene expression is seen in response to increase demand of NE. Rate-limiting step In Dopaminergic neurons of the midbrain, TH is phosphorylated in response to increase demand of DA. Increase synthesis of catecholamines can in turn inhibit TH Regulation of the catecholamines 2. Storage of catecholamines. Catecholamines are store in vesicles. Prepared for release and also protected against toxin and degradations Vesicular monoamine transporter does the job (2 genes): VMAT2 found in catecholamine and serotonin neurons. Target of psychotropic drugs (e.g. Reserpine = reduce psychotic symptoms) Target of stimulant drugs (e.g. amphetamines, The transport of DA into synaptic vesicles from cocaine, MDMA) may cause mood disorders the cytoplasmic space through VMAT2, facilitated by H+-ATPase function. associated with chronic use Christopher L. German et al. Pharmacol Rev 2015;67:1005-1024 3. Release and activation of postsynaptic receptors. A. What are the neuronal properties that could affect the release of catecholamines? A1- intrinsic excitability (e.g. threshold AP, firing properties…) A2- release probability (e.g. change in calcium conductance…) B1- change in receptors (e.g. The synapse (article) | Human biology | Khan Academy B. What are the molecular changes that could affect numbers, sensitivity…) the effect of the catecholamines on the postsynaptic sites (e.g. change in postsynaptic potential) ? B2- intrinsic excitability (e.g. increase Na+ conductance…) 4. Inactivation of released catecholamines. Once released in the synaptic cleft, neurotransmitters are rapidly removed: a) Re-uptake via transporters = dopamine (DAT) and norepinephrine (NET) transporters. Present in DA and NE neurons, respectively. Cocaine blocks DA, NE and serotonin transporters and induces an increase of extracellular monoamines levels. Tricyclic antidepressants inhibit NE and serotonin reuptake with weak effect on DATs. = Fluoxetine is the most widely prescribe antidepressant The movement of DA from the extracellular to cytoplasmic space through transition of DAT from an outward-facing to inward-facing state Christopher L. German et al. Pharmacol Rev 2015;67:1005-1024 b) Enzymatic inactivation. Two enzymes sequentially metabolize catecholamines: Monoamine oxidase (MAO) and Catechol-O-methyltransferase (COMT) COMT is the primary mode of terminating the action of catecholamines in the blood (in the brain reuptake appears to be more important) Also important for regulating DA in PFC. COMT gene polymorphism associated with SCZ, BD, AD and increase aggressive behavior Two MAO genes have been cloned: MAOa and MAOb MAOa displays high affinity for NE and serotonin MAOa inhibitors are effective antidepressants (clorgylin, tranylcypromine) Those drugs have serious side effects (increase blood pressure associated with high tyramines diet) 5. Autoreceptors and feedback loop. Synthesis and release of catecholamines as well as neuronal firing can be regulated by feedback mechanisms activated by autoreceptors (located on the presynaptic neuron). D2 receptors (one of the five DA receptors) appears to be involved in all three pathways. What does this suggest knowing that D2 is a RCPG? This suggest different G proteins are coupled with it depending on the pathway it regulates = different intracellular transduction cascades. Other examples of catecholamines autoreceptors are 2- adrenergic (CNS) and -adrenergic (PNS) NE receptors Note that postsynaptic regulations (downstream of metabotropic receptors) can also adjust neurotransmission! Summary example Transport = VMAT2 DA conversion to NE = DBA with NE Reserpine = antipsychotic drug (rarely used nowadays) Activation of autoreceptors induces negative feedback loops Antipsychotics drugs: Reserpine inhibit transport of DA inside the vesicle Beneficial effect of typical and atypical antipsychotic drugs depends on blockade of D2 receptors in the mesolimbic system Based on these observations, what can you predict about the “neurotransmitter imbalance” in individual with psychotic disorders? How do amphetamines work? Amphetamine is a central nervous (CNS) system stimulant that functions by increasing the amounts of dopamine, norepinephrine, and serotonin (to a lesser extent) in the synaptic cleft through a variety of mechanisms. In this diagram, amphetamine enters the presynaptic neuron across the neuronal membrane or through the dopamine transporter (DAT). Once inside, it binds to TAAR1 or enters synaptic vesicles through vesicular monoamine transporter 2 (VMAT2). When amphetamine or a trace amine binds to TAAR1, it reduces postsynaptic dopamine neuron firing rate (via mechanisms that are not shown) and triggers protein kinase A (PKA) and protein kinase C (PKC) signaling, resulting in DAT phosphorylation. Phosphorylated DAT then either operates in reverse or withdraws into the presynaptic neuron and ceases transport. When amphetamine or a trace amine enters the synaptic vesicles through VMAT2, dopamine is released into the cytosol (light tan area). (https://en.wikipedia.org/wiki/Amphetamine) How does cocaine work? Cocaine in the brain: In the normal neural communication process, dopamine is released by a neuron into the synapse, where it can bind to dopamine receptors on neighboring neurons. Normally, dopamine is then recycled back into the transmitting neuron by a specialized protein called the dopamine transporter. If cocaine is present, it attaches to the dopamine transporter and blocks the normal recycling process, resulting in a buildup of dopamine in the synapse, which contributes to the pleasurable effects of cocaine. (https://www.drugabuse.gov/publications/resea rch-reports/cocaine/how-does-cocaine-produce- its-effects)  Study of dopamine (DA) regulation Researcher have generated a hyperdopaminergic mouse model. They have deleted the gene coding for DAT (DA reuptake transporter). Extensive investigation of the DAT- KO mouse brain revealed significant increases dopaminergic neurotransmission believed to be a result of increase level of DA at the synapses. The model of this finding is illustrated in this figure: Questions : 1- Explain why there is an elevation of DA in the synapse of the Tyrosine Tyrosine DAT-KO mice? +AACD +AACD 2- Notice the reduction of DA receptors at the postsynaptic membranes in the DAT-KO. How do you interpret such result? What mechanism could explain the removal of the receptors? 3- Briefly describe the three mechanisms affecting the presynaptic neuron (seen in class) that could have participated in attenuating the observed increase of DA in the synaptic cleft but doesn’t seem to be operating in this case? 4- What pharmacological treatment could have mimicked genetic removal of DAT? http://www.med.tohoku.ac.jp/nsgcoe/en/eng/member/sora/index.html 1- Explain why there is an elevation of DA in the synapse of the DAT-KO mice? A. The presynaptic neuron produces more DA because the postsynaptic neuron is no longer activated B. The presynaptic neuron continues to release DA as in the control mice, but the lack of DAT prevent the reuptake and clearance of DA in synaptic cleft C. There is not enough receptors at the postsynaptic terminals to clear DA from the synaptic cleft. Notice the reduction of DA receptors at the postsynaptic membranes in the DAT-KO. How do you interpret such result? What mechanism could explain the removal of the receptors? A. The excess DA triggers a feedback signaling mechanism at the postsynapse leading to trafficking and removal of the DA receptors B. Similar to what is seen in cocaine addicts, DA cytotoxicity attacks the postsynaptic neurons and lead to defect in DA receptors trafficking C. The genetic manipulation affects both the DAT and the DA receptors because the genes are both related to dopamine neurotransmission.  Study of dopamine (DA) regulation Researcher have generated a hyperdopaminergic mouse model. They have deleted the gene coding for DAT (DA reuptake transporter). Extensive investigation of the DAT- KO mouse brain revealed significant increases dopaminergic neurotransmission believed to be a result of increase level of DA at the synapses. The model of this finding is illustrated in this figure: Questions : 1- Explain why there is an elevation of DA in the synapse of the Tyrosine Tyrosine DAT-KO mice? +AACD +AACD 2- Notice the reduction of DA receptors at the postsynaptic membranes in the DAT-KO. How do you interpret such result? What mechanism could explain the removal of the receptors? 3- Briefly describe the three mechanisms affecting the presynaptic neuron (seen in class) that could have participated in attenuating the observed increase of DA in the synaptic cleft but doesn’t seem to be operating in this case? 4- What pharmacological treatment could have mimicked genetic removal of DAT? http://www.med.tohoku.ac.jp/nsgcoe/en/eng/member/sora/index.html Regulation of Serotonin: Major processes that regulate serotonin are similar than those regulating catecholamines. 1. Synthesis: 2. Storage and regulation: Accumulation in vesicles is done by VMAT2 (same as catecholamines). Autoreceptors play major roles in the regulation of 5-HT synthesis and release. 5-HT1a = autoreceptors present on somatodendritic region of serotonin neurons 5-HT1b = autoreceptors present on axon terminals (and some non-serotoninergic neurons) (signaling pathways downstream of these autoreceptors are involved in the pathophysiology of depression) Serotonin and antidepressant drugs: Reuptake of released 5-HT by serotonin transporter (SERT) is the major means of terminating 5-HT’s action SERT is the target of the most commonly used antidepressants, the serotonin-selective reuptake inhibitor (SSRI) Like catecholamines, serotonin can also be inactivated by MAO, therefore MAO inhibitors antidepressant’s effects is also mediated by 5-HT regulation How extasy (MDMA) works Amino acid transmitters: GABA and Glutamate They are the most abundant NTs in the brain Their synthesis are related: In mammals, GAD exists in two isoforms with molecular (GAD) weights of 67 and 65 kDa (GAD67 and GAD65), which are encoded by two different genes on different chromosomes (GAD 1 and GAD2 genes, chromoso mes 2 and 10 in humans, respectively). GABA and glutamate reuptake: The major way amino acid NTs are inactivated is by reuptake of the released transmitter through high affinity transporters found on glial cells GAD as well as transporters found on VGAT neurons GABA transporters found in the brain: Glial cell GABA transporter type 1 (GAT1) GABA transporter type 3 (GAT3) Vesicular GABA transporter (VGAT) Glutamate transporters found in the brain: protein gene tissue distribution EAAT1 SLC1A3 astroglia EAAT2 SLC1A2 Mainly astroglia; mediates >90% of CNS glutamate reuptake EAAT3 SLC1A1 all neurons – located on dendrites and axon terminals EAAT4 SLC1A6 neurons EAAT5 SLC1A7 retina VGLUT1 SLC17A7 neurons VGLUT2 SLC17A6 neurons VGLUT3 SLC17A8 neurons EAAT = Excitatory amino acid transporter VGLUT = Vesicular glutamate transporters Glutamate transporters found in the brain:  The excitatory / inhibitory balance is essential for normal brain function 80% 20% Excitatory neurons Inhibitory neurons Glutamate GABA (Gamma amino Butyric Acid) Carry neuronal information Overexcitation Locally control excitatory Long range projection = Epilepsy neuron activity (= Interneuron) Which antibody would you use to label and quantify inhibitory synapse in brain tissue samples? An antibody against: A. VGLUT1 B. EAAT2 C. GABA D. VGAT Which antibody would you use to label and quantify inhibitory synapse in brain tissue samples? An antibody against: A. VGLUT1 B. EAAT2 C. GABA D. VGAT  20 schizophrenia and 20 matched normal control subjects (A) Photomicrographs showing vGluT1-immunoreactive boutons in layer 3 of the PFC. Scale bar = 100 μm. (B) Density of vGluT1-immunoreactive boutons is significantly decreased in layer 3 of the PFC in schizophrenia. Bitanihirwe et al., BMC Psychiatry2009 What other labeling technique could be used to quantify excitatory synapse in postmortem tissue? A. Spine count following Golgi stain B. GABAa receptor staining C. AMPA receptor current recording D. NMDA receptor current recording E. EPSC recording What other labeling technique could be used to quantify excitatory synapse in postmortem tissue? A. Spine count following Golgi stain B. GABAa receptor staining C. AMPA receptor current recording D. NMDA receptor current recording E. EPSC recording Fusion proteins with fluorescent marker enable precise quantification of synapses in animal models (not humans!). Pre or postsynaptic protein can be fused to a fluorescent protein using genetic method or targeted with antibody for immunohistochemistry. Fusion proteins with fluorescent marker enable precise quantification of synapses in animal models. Homer GABA-Arec Synaptophysin-GFP Polleux lab. (https://polleuxlab.com/) Pieraut et al, 2014