Merged 2.pdf

Synapse and neurotransmitter release mechanism  Synapse The synapse is a specialized structure that allow the transmission of electrical signals (communication) between excitable cells. On the basis of the mechanism by which the transmission of signals occurs, synapses c...

Synapse and neurotransmitter release mechanism  Synapse The synapse is a specialized structure that allow the transmission of electrical signals (communication) between excitable cells. On the basis of the mechanism by which the transmission of signals occurs, synapses can be classified in: Electrical synapses Chemical synapses Electrical synapses In electrical synapses, the electrical signal passes directly from the presynaptic cell to the post synaptic cell At the level of electrical synapses the cell membranes of adjacent cells are connected by communicating junctions (gap junctions).  Electrical synapses The communicating junctions consist of connecting protein channels between two adjacent cells, forming a sort of cytoplasmic bridge between adjacent cells. These connection channels between adjacent cells are made up of joint pairs of hemichannels called connexons. Each connexon is composed of 6 connexins, arranged in a circle around a central axis along which the pore of the canal runs  Electrical synapses The connexons can rotate clockwise, modifying the lumen of the channel and therefore its degree of opening. Signals such as a lowering of pH or an increase in the intracellular concentration of Ca2+ in one of the two cells induces the closure of the channel. Electrical synapses Currents applied to one cell propagate into the other through the electrical synapse, flowing through the connexons  Electrical synapses Similarly to all gap junctions, also in electrical synapses the channels are wide enough (about 15Å) to allow the passage of all inorganic ions. Therefore, the electrical synapse allows the passage of electrotonic currents.  Characteristics of electrical synapses An action potential in a presynaptic neuron inevitably triggers an action potential in a postsynaptic neuron They do not allow the integration of multiple synaptic signals They allow rapid communications between adjacent excitable cells thus guaranteeing the synchronization of electrical activity.  Electrical synapses distribution Electrical synapses in the central nervous system are rare even though they have recently been found in many brain regions (the cerebellum, spinal cord, thalamus, hippocampus, olfactory bulb and retina). They are present when: speed in signal transmission is required synchronization of the electrical activity of multiple excitable cells is required (as in the case of the heart muscle, where the fiber cells are connected, through the intercalary discs, by electrical synapses) Chemical synapses The chemical synapse consists of three elements: the presynaptic terminal, or synaptic button the synaptic space, also called the inter-synaptic cleft, of about 20- 40nm the post-synaptic membrane. The neurotransmitter released by the presynaptic neuron diffuses into the synaptic cleft and binds to specific receptors located on the post synaptic membrane, modifying the ion permeability of the postsynaptic membrane which in turn trigger a change in the potential of the membrane, called the post-synaptic potential. Chemical synapses Synaptic contacts can occur in different parts of the postsynaptic cell: Axon-dendritic synapses between axon and dendrites Axon-somatic synapses between axon and soma Axon-axon synapses between axon and axon Chemical synapses  Chemical synapses At the level of the synaptic terminal neurotransmitters are contained in vesicles called synaptic vesicles. The action potential, generated at the level of the emergency cone, propagating along the axon, reaches the presynaptic membrane. Depolarization of the presynaptic terminal triggers the opening of voltage-gated Ca2+ channels. Therefore, a transmembrane flow of Ca2+ ions is generated according to its electrochemical gradient which produces a local increase in the intracellular concentration of Ca2+ at the level of the presynaptic terminals. The increase in the intracellular concentration of Ca2+ triggers the exocytosis of the synaptic vesicles.  The inflow of Ca2+ ions (through voltage-gated channels) within nerve terminals is foundamental for neurotransmitter release. The amplitude of the post-synaptic potential depends on the quantity of Ca2+ entering the nerve terminal. ↑Ca2+ ➔ ↑ quantity of released neurotransmitter. Mechanism of neurotransmitter release The mechanisms of regulated exocytosis, by which the vesicles merge with the presynaptic membrane and release the neurotransmitter, involve the interaction of proteins associated with the membrane of the vesicles, proteins bound to the plasma membrane and cytoplasmic proteins. The neuromuscolar junction (endplate) The neuromuscular junction or endplate was the first vertebrate synapse to be studied in detail The neuromuscolar junction (endplate) Chemical transmission occurs through a synaptic cleft, of about 20-30 nm, which separates the pre- and post-synaptic cells. The presynaptic terminal contains synaptic vesicles of 40nm in diameter containing 10,000-50,000 molecules of transmitter each  Quantal release of neurotransmitter Fatt & Katz 1950’s (discovery of miniature end-plate potentials MEPPs) Spontaneous small depolarization of muscle fiber resting potential Occurr randomly  1 per second Observed only at endplate Blocked by curare (competitive AChR antagonist) Abolished by denervation Enhanced by blocking ACh esterase (eserine) Therefore arises from spontaneous release of “packets” (quanta) of ACh from the nerve terminal  Neurotransmitters are released in unit packets (quanta) In the absence of nerve stimulation, random low-amplitude ( 0.5 mV) spontaneous post-synaptic depolarizations are recorded: miniature plate potentials (MPP). Eserine (a AChE blocker) increases the amplitude and duration, but not the frequency of MEPPs. MEPPs are due to the release of packets of neurotransmitter molecules called "quanta". A MEPP is the result of the Ach-dependent activation of approximately 2000 channels. The plaque potential is the result of many quanta, therefore it is a multiple of the elementary response. The quanta are contained in specialized structures: the synaptic vesicles (1 vesicle = 1 quantum of ACh = about 5000 molecules). The neurotransmitters are released by exocytosis from the synaptic vesicles, near the active areas.  In the absence of action potential there is a spontaneous, low frequency, quantal release: 1 quantum/sec responsible for the MPPs AP triggers Ca2+ inflow at pre-synaptic terminals which transiently increases the quantal release frequency by more than 100,000 folds, causing the release of ~ 150 quanta/msec  Synaptic vesicles are the storage organelles of neurotransmitter quanta. The vesicles fuse with the inner surface of the pre- synaptic terminal membrane at the level of specialized release sites (active zones). The release of the vesicles is an all or nothing phenomenon. The release probability depends on the amount of Ca2+ that enters the pre-synaptiv terminal during the AP  Exocytosis occurs through the transient formation of a fusion pore, which crosses the vesicular and pre- synaptic membranes. The influx of Ca2+ determines the opening and subsequent dilation of the pre-existing fusion pores, allowing the release of the neurotransmitter. Mechanism of neurotransmitter release The release of the neurotransmitter involves the passage of the synaptic vesicles through a series of preparatory steps: 1. release from cytoskeleton interaction 2. targeting and anchoring to active zones of the plasma membrane 3. preparation to the fusion with the membrane (priming) 4. fusion with the presynaptic membrane 5. vesicle recycling and recovery Mechanism of neurotransmitter release -release from cytoskeleton interaction- The reserve pool vesicles are anchored to the actin cytoskeleton through synapsins, which are extrinsic proteins associated with the cytoplasmic side of the vesicular membrane and are able to bind actin molecules. The entry of Ca2+ at presynaptic terminal level determines the phosphorylation of synapsin by the Ca2+/calmodulin-dependent protein kinase (CaMK). Phosphorylation reduces the affinity of synapsin for actin, thus promoting the detachment of the vesicles associated with actin filaments Mechanism of neurotransmitter release -targeting to active zones- The detachment of a vesicle from the reserve pool is followed by its mobilization and its targeting towards an active zone. This process requires the intervention of extrinsic proteins of the vesicular membrane called Rab3. Rab3 is a monomeric protein with GTPase activity. In the GTP-linked form, Rab3 marks the vesicles that need to be transported to the active zones. Rab3 allows anchoring to the active zones through interaction with the Rim protein (intrinsic protein of the active zones) Mechanism of neurotransmitter release -anchoring to the active zones- The SNARE complex proteins of the vesicles and the membrane interact ensuring the correct positioning of the vesicles near the voltage-gated Ca2+ channels. SNARE proteins are divided into v- SNARE proteins (vesicular membrane proteins) and t-SNARE proteins (presynaptic membrane proteins at the level of active zones). Main v-SNARE: synaptobrevin Main t-SNAREs: syntaxin and SNAP-25 Mechanism of neurotransmitter release -priming- SNARE proteins contact each other starting from the respective N- terminals and progressively in the direction of the C-terminals, with an interaction that involves a reciprocal helical winding (zip-lock model). The proceeding of this interaction develops a powerful pulling force that brings the vesicular membrane into contact with the presynaptic membrane Mechanism of neurotransmitter release -fusion with the presynaptic membrane- The fusion of the vesicle with the presynaptic membrane is promoted by the binding of Ca2+ to the synaptotagmin, a large transmembrane protein of synaptic vesicles. Following the influx of Ca2+ into the axonal terminal, the binding of Ca2+ to synaptotagmin induces a conformational change that allows it to interact with the proteins of the SNARE family that in the meantime have brought the two membranes closer together. Synaptotagmin binds to membrane phospholipids resulting in the formation of a fusion pore  Vescicular stages and neurotransmitter release 1. Mobilization: release from binding to the cytoskeleton (synapsins) 2.Traffic: targeting to active zones (G proteins: vesicular Rab3, membrane Rim) 3.Docking and priming: Anchoring to active areas and predisposition to fusion (SNARE complex: synaptobrevin vescicolar protein + syntaxin and Snap-25 membrane proteins) 4.Fusion: (Synaptotagmin + Ca2+) 5. Recycling of vescicle membrane and new vescicle formation  MOBILIZATION: release from binding to the cytoskeleton The vesicles far from the active areas (neurotransmitter reserve), are anchored to the cytoskeleton (actin filaments) through synapsin. The PK-Ca2+/Calmodulin dependent phosphorylation of synapsin, triggered by depolarization + Ca2+ inflow, release the vesicles, which move towards the active zones. Mechanism of neurotransmitter release -vescicle recycling and recovery- The exocytosis of the vesicles is followed by the removal of the vesicular membrane, which occurs through a process mediated by clathrin. Clathrin determines the curvature of the membrane and the formation of a coated vesicular structure. The detachment of the neovesicle from the membrane occurs by the dynamine, with consumption of GTP. The neorformed vesicle can then be directed to the reserve pool by specific vesicular transporters. During this process it is filled with neurotransmitter.  Physiological folding of α-synuclein. The protein is initially unstructured and monomeric. During the docking and priming process, its binding to vescicles induces a conformational change folding it into an α-helix and forming multimeric complexes. Thus α-synuclein promotes the assembly of the SNARE complex in the docking and priming phases of the synaptic vesicles. ©2014 by National Academy of Sciences Jacqueline Burré et al. PNAS 2014;111:40:E4274-E4283 After release, the neurotransmitter (or part of its molecule) undergoes: Reuptake in the pre-synaptic terminals: ▪ The neurotransmitter is brought back into synaptic vescicles by a vescicular transporter (H+-ATPase proton pump and H+-NT exchanger) ▪ metabolization Reuptake by glial cells Metabolization at extra-neuronal level Diffusion in the extra-synaptic zones Neurotransmitters and synaptic receptors  Neurotransmitters The neurotransmitters belong to two main categories Classic neurotransmitter: low molecular weight molecules Acetylcholine Monoamine (dopamine, noradrenaline, histamine, serotonin) Aminoacids (GABA, glycine, glutamate) ATP Neuropeptides (at least 50 identified, including): oppioids, P substance, neurohypophyseal hormones, secretin, insulin, somatostatins  Low molecular weight neurotransmitter synthesis Low molecular weight vescicola sinaptica Zone neurotransmitters are T H+ attive synthesized at synaptic terminals and stored in small vescicles (40-60 nm) ADP + ATP H Slow axonal transport: 0.5 – 5 mm/day  Neuropeptides synthesis The precursors are transported in vesicles along the microtubules and transformed into the definitive neurotransmitter by specific enzymes. Fast axonal transport: up to 400 mm/day The neuropeptides are stored in larger vesicles (90 - 250 nm), whose membranes, after endocytosis, are redirected to the soma and recycled (retrograde transport).  Nerve terminals can contain both types of vescicles When different neurotransmitters are present, the molecules in question are defined as co-transmitters Peptides are released and removed more slowly thus producing prolonged effects associated with modulatory functions.  Neurotransmitter removal The released neurotransmitter is removed from the synaptic cleft through three mechanisms: 1) Diffusion out of the synaptic cleft All neurotransmitters 2) Enzymatic degradation. Peptides 3) Reuptake in the presynaptic terminal. Small molecule neurotransmitters  Types of receptors Ionotropic- non-selective ion channels. Mediate rapid and short- lasting responses. Metabotropic- associated to the activation of a second messenger which modulates the activity of ion channels. Mediate slow responses. G-protein coupled receptors (different neurotransmitters and peptides) tyrosin-kinase receptors (hormones, neuropeptides, growth factors) Receptors for neurotransmitters can also be localized at the pre-synaptic level where they function as autoreceptors and modulate the release of the neurotransmitter. Double-control mechanisms  Structure and mechanism of action of metabotropic receptors Seven transmembrain domains (M1- M7). The second and third cytoplasmic loops between M3-M4 and M5-M6 contain the G-protein binding sites NT + R ➔ activation of G proteins (trimers: +GDP subunit,  and ) ➔ GDP- GTP exchange ➔ dissociation of the GTP- and - complexes that in turn act on target proteins (enzymes producing a second messenger). GTP is hydrolyzed to GDP + phosphate (Pi), and the three subunits interact again with the receptor G proteins and second messengers can induce the opening or the closure of ion channels, or modulate the opening of voltage-gated channels for K+, Na+ and Ca2+.  Second messenger mechanism The g proteins can act on ion channels by mean of different enzymes (adenylate cyclase, phospholipase C, phospholipase A2) leading to the formation of second messengers ➔ activation of protein-kinases ➔ phosphorylation of target proteins. Noradrenalina Long lasting effect The PKs activated by second messengers can: induce modification of already existing proteins induce new protein syntesis, modifying gene expression This type of activity can trigger long-lasting changes, important in the processes of neuronal development and long-term memory. Main synaptic neurotransmitter and their receptors  Acetylcholine (ACh) Neurotransmitter: Motor neurons Preganglionic neurons of the ANS Postganglionic neurons of the parasympathetic system Neurons of various areas of the CNS, where it plays an essential role in cognitive processes (cholinergic neuron degeneration ➔ Alzheimer’s disease). Receptors: Ionotropic (Nicotinic): periferic (Na+ e K+), central (high Ca2+ permeability) ➔ depolarization Metabotropic (Muscarinic M1-M5): M1, M3 e M5 ➔ activation of phospholipase C, M2 e M4 ➔ inhibition of adenylate cyclase  GABA and GLYCINE GABA: main inhibitory neurotransmitter of the CNS. Glycin: inhibitory neurotransmitter of spinal cord. It is involved in spinal reflex and in motor coordination. (B6)  -amino-butirric acid (GABA) GABA ➔ hyperpolarization of post-synaptic membrane. Receptors: GABAA ionotropic, Cl- channel GABAB metabotropic ➔ inhibits adenylate cyclase ➔ activation of K+ channels GABAC ionotropic, Cl- channel, expressed in the retina (bipolar cells) GABAA They are targeted by neuroactive substances, both exogenous (benzodiazepines, barbiturates and alcohol) and endogenous (neurosteroids), which by binding to specific sites increase the sensitivity of the receptor to GABA.  Glycine Glycine ➔ hyperpolarization of the post-synaptic membrane. Receptors: ionotropic 1, Cl- channel The activation of GABA and glycine receptors may have an excitatory effect during post-natal development, due to the higher intracellular concentration of Cl- during development than in adults.  Glutamate It is produced from glucose by ketoglutarate transamination or by hydrolysis of glutamine. It is the main excitatory neurotransmitter of the CNS After it has been released, glutamate undergoes reuptake by specific glutamate transporter localized in neurons and glia. Excess of glutamate (for example in ischemia) causes excitotoxic effects that can lead to cell death.  Glutamate receptors Ionotropic: can be classified in NMDA (N-metil-D-aspartate): high affinity for glutamate, permeable to K+, Na+ and Ca2+ with high Ca2+ permeability. At resting membrane potential they are closed for the presence of a Mg2+ ion, they activate after the voltage-dependent removal of Mg2+ block. Mediate slow synaptic responses. non-NMDA: permeable to Na+ e K+, low Ca2+ permeability ✓AMPA ( α-amino-3-idrossi-5-metil-isossazol-4-propionic acid): mediate fast synaptic transmission ✓ Kainate mediate slow synaptic transmission Metabotropic: eight types divided into three groups I (mGluR1, R5) ➔ activation of phospholipase C II (mGluR2, R3) and III (mGluR4, R6, R7, R8) ➔ inhibition of adenylate cyclase. The biogenic amines  Catecholamines Dopamine- Substantia nigra (midbrain) and arcuate nucleus (hypothalamus). The nigrostrial pathway is altered in Parkinson's disease and other motor disorders. Metabotropic receptors divided into two classes: D1 (D1, D5), D2 (D2, D3, D4) activation and inhibition of AC respectively  Catecholamines Noradrenaline- CNS: locus coeruleus nuclei, with diffuse projection (cortex, cerebellum, spinal cord). ANS: sympathetic postganglionic nuclei. Metabotropic receptors divided into two classes:  (1 ➔ PLC activation, 2 ➔ AC inhibition) and  (1, 2, 3) ➔ AC activation  Catecholamines Serotonin (5-HT)- Nuclei of the raphe (brain stem) projection on different cerebral and medullary nuclei, involved in complex cognitive functions and in the sleep-wake rhythm. Involved in the pathogenesis of depressive forms. Receptors: 7 subtypes ionotropic (5-HT3) and metabotropic (5-HT1 -T7)  Catecholamines Histamine: Tuberomammyl nucleus (posterior hypothalamus) projection on almost all structures of the CNS. Involved in the regulation of the state of alertness and in neuroendocrine control. Metabotropic receptors divided into three classes: H1 and H2 (excitatory postsynaptic), H3 (presynaptic) Purinergic receptors ATP and adenosine They are used in the CNS and in some parts of the ANS (sympathetic). Important for pain transmission. ATP receptors: - ionotropic (P2X1-7, permeable to Ca2+). - metabotropic (P2Y1-6) Adenosine receptors: -metabotropic (A1, A2, A3) They can be located at the pre-synaptic level (they control the release of other neurotransmitters).  Retrograde messengers They can easily diffuse across the membranes (transcellular messengers). They are synthesized at post-synaptic level and diffuse to the pre- synaptic terminal where they modulate the release of neurotransmitter. Gas: NO and CO (involved in synaptic potentiation)  Retrograde messengers Endocannabinoids: arachidonic acid and / or its metabolites (2- AG, anandamide) Dopamine (DA) is a neurotransmitter released by neurons involved in various functions of the nervous system Quaak et al., 2009 DA affects: movement Learning and memory reward/pleasure attention and cognitive performance The dopaminergic system has behavioral facilitation functions. The main dopaminergic circuits identified in our brain originate from the substantia nigra and in the ventral tegmental area (VTA) of the midbrain. The dopaminergic neurons of the stubstantia nigra project to the striatum, in the base of the telencephalon, in a control system of the coordination of voluntary movements https://currentmedicalresearch.wordpress.com (nigrostriatal system). Motor execution depends on the DA released by the substantia nigra, a nucleus belonging to the basal ganglia. Impaired DA release causes motor problems. for example, an DA deficiency causes disease states including Parkinson's disease DA mediates the pleasure and reward mechanism. The neurons of the dopaminergic area most involved in reward and pleasure mechanisms are those of the nucleus accumbens and of the prefrontal cortex. an excess of DA causes pathological states of addiction www.pinterest.com Nicotine intake stimulates an excessive release of DA from the VTA to the nucleus accumbens causing states of addiction D'Souza & Markou, Addict Sci Clin Pract. 2011 Central synapses and synaptic integration  Differences between neuromuscular synapses and central synapses Neuromuscular synapses use only Ach as a neurotransmitter. Central synapses use several neurotransmitters. Plaque potentials are excitatory only. Central postsynaptic signals can be both excitatory (EPSP) and inhibitory (IPSP). At the neuromuscular level there is a 1: 1 ratio between pre- and post-synaptic AP. The plaque potentials are always above threshold, with each AP of the motor neuron following a AP in the muscle fiber cell. At central level, single EPSPs (amplitude K+ K+>Na+ Na+ Na+ + + + + + + + + + + + + - - - - - - - - - - - - - 60 mV - 50 mV K+ K+ Non-selective channel opening: The electro-chemical strength of Na+ prevails at the resting potential. Input of Na+ exceeds output of K+. As the membrane potential shifts towards less negative values (depolarization) the electrochemical strength of K+ increases. The output of K+ exceeds the input of Na+ and the potential returns to the rest values. The AP is characterized by the polarity inversion of the Na+ inflow K+ outflow membrane and depends on the opening of selective voltage-gated channels, for Na + and K +, in sequence. In a neuron, the Threshold depolarization threshold for the onset of an AP is determined by the synaptic EPSPs. EPSP graded in amplitude (higher amount of neurotransmitter released → higher EPSP amplitude) AP always has the same amplitude (all or nothing) EPSPs propagate with decrement (their amplitude decreases as they move away from the point where they were generated) AP propagates without decrement because it is continuously regenerated Neurons of the CNS receives many excitatory and inhibitory synaptic contacts, on dendrites and cell body that need to be integrated to produce adequate responses. Types of synapses  Morphological types of synapses The localization of synapses in the CNS reflects their function Type I generally excitatory Type II generally inhibitory The localization of inhibitory synapses is a very important Axon-axon synapse mediates factor in determining their effectiveness. The inhibitory pre-synaptic inhibition or activity localized on the cell body will open Cl channels which will facilitation. Involved in the short-circuit much of the depolarization. Conversely, the control of neurotransmitter inhibitory activities that arise in the distal portions of the release by an axon terminal. dendrites are less effective. For this principle the inhibitory afferents are localized on the cell body of neurons  The signal propagation distance depends on the space constant . ↑➔↑distance The synapses are concentrated on the dendrites and soma of a neuron (high AP threshold), while the AP arises in the emergency cone of the axon (trigger zone, low threshold). The EPSPs (0.2-0.4 mV) propagate with a decrement and reach the emergency cone below the threshold. Since the depolarization necessary to reach the threshold is ~ 10mV, synaptic summation phenomena are necessary.  Segmento iniziale punto di nascita del PA To trigger an AP, synaptic signals must be added together (spatial and temporal synaptic summation) After the summation process the depolarization from excitatory synapses on dendrites and soma must reach the initial portion of the axon with intensity big enough to reach the threshold for AP. Since the EPSPs propagate reducing their intensity, the final depolarization can or cannot reach the threshold for AP. The ability of the graded potential to propagate along the membrane depends on the intrinsic passive properties of the membrane (membrane R and internal / axial R). The distance reached depends on the space constant 𝜆, the distance in which the amplitude of the EPSP is reduced by 63%. 𝜆 increases if Rm increases and if Ri/axial decreases. This occurs when: 1) the axon is large in diameter (Ri decreases); 2) the axon is covered by the myelin sheath (Rm increases in proportion to the number of number of layers of the myelin sheath). 𝜆 of an unmyelinated fiber is 1 mm, that of a myelinated fiber can reach 7 mm. The propagation of the graded potential also depends on the time constant 𝜏, the time required for Vm to increase or decrease until it reaches or loses 63% of its final value. Cells in which  is high have greater ease of spatial summation.  Spatial summation Different excitatory signals produced at the same time in different sites of the neurons are added together to produce a bigger depolarization that may be able to reach the trigger zone with intensity large enough to trigger an AP.  Spatial summation Simultaneous activation of excitatory synapses located in different points ➔ summation of currents associated with EPSP. The resulting current is sufficient to reach the threshold. The AP can be generated. The  and  influence the spatial course of the synaptic currents. In neurons with greater  and , spatial summation is facilitated.  Sommazione temporale sopra soglia Temporal summation Sequences of many EPSPs can be summed together to generate a larger EPSP that may be able to reach the trigger zone with intensity large enough to trigger an AP.  Temporal summation The single synapse activated repeatedly ➔ it is possible to generate EPSPs in succession and add them because they have a duration greater than the AP. The resulting current is sufficient to reach the threshold. The AP can be generated. The  influences the time course of the synaptic responses. In neurons with greater , temporal summation is facilitated, it can be obtained with lower activation frequencies.  Competitive effects Simultaneous activation of excitatory and inhibitory synapses leads to summation of EPSP and IPSP. The hyperpolarization produced by inhibitory signals reduces the depolarization of the excitatory synapses. If a depolarization that reaches the trigger zone is too small, it will not overcome the threshold for AP and the information of the excitatory synapses is blocked.  Competitive effects The simultaneous activation of excitatory and inhibitory synapses leads to the summation of EPSP and IPSP, the result of which depends on the magnitude of the excitatory and inhibitory signals. The inhibitory synapses (mainly on the soma) can effectively counteract up to cancel the excitatory effects. The summation is the basis of synaptic integration, which allows the neuron, reached by multiple signals, to generate different AP frequencies, and therefore different responses. Competitive effects Inhibitory effects  Less information More information Synaptic activation The amplitude of the depolarization, resulting from the summation of the synaptic currents, which reaches the emergency cone, determines the frequency of the AP generated by the neuron and transmitted along the axon: depolarization ➔  AP Frequency ➔  neurotransmitter release at the level of the axonal terminal ➔  information transmitted.  Axon-axon synapses The modulatory activity is performed by modulating Ca2+ flow into the terminal of the neuron a, pre-synaptic to b. Inhibitory neuron Excitatory neuron  Convergence and divergence of the nerve pathways A neural circuit is a population of neurons interconnected by synapses to perform a specific function when activated. Neural circuits are interconnect with each other to form large-scale brain networks. There are various configurations of neural circuits that we can distinguish in the following fundamental circuits: Converging circuits in which information from different neurons is conveyed to a single neuron. Converging circuits can sum up information from different sources, such as the eyes, tactile corpuscles and hearing organs; this contributes, for example, to the recognition - perceptual categorization - of objects in the environment.  Convergence and divergence of the nerve pathways A neural circuit is a population of neurons interconnected by synapses to perform a specific function when activated. Neural circuits are interconnect with each other to form large-scale brain networks. There are various configurations of neural circuits that we can distinguish in the following fundamental circuits: Divergent circuits in which information from a single nerve cell is conveyed to several neurons. An example of this is the eye whose information is sent to different brain districts or maps. Pre-synaptic modulation Pre-synaptic modulation Inhibition -- Pre-synaptic modulation Facilitation + https://youtu.be/FFY3OfwJFfs  Synaptic plasticity Synaptic plasticity represents one of the most fundamental and important functions of the brain, which is the ability of the neural activity, generated by an experience, to modify neural circuit function and thereby modify subsequent thoughts, feelings, and behavior https://www.youtube.com/watch?v=xvqCvFxjz88  Synaptic plasticity Synaptic plasticity refers to the activity-dependent modification of the strength or efficacy of synaptic transmission at preexisting synapses, and has been proposed to play an essential role in the capacity of the brain to integrate transient experiences into persistent memory traces. Furthermore, synaptic plasticity is thought to play an important role in the early development of neural circuitry, and accumulating evidence suggests that impairments in synaptic plasticity may contribute to pathophysiology of multiple neuropsychiatric disorders  Synaptic plasticity: Synaptogenesis Synaptic remodeling: structural and functional  Functional synaptic plasticity phenomena The efficacy of a synapse (extent of the synaptic response) can vary in relation to the frequency of the nerve impulses that affect it. ▪ Short-term phenomena (duration 1 ms - 5 min) due to modification of neurotransmitter release. ▪ Long-term phenomena (30 min - weeks) associated with functional and structural changes of the post- and pre-synaptic element.  Synaptic facilitation (short term) Two synaptic currents evoked at a distance of 50ms Rapporto S2/S1 Ratio between the amplitude of the second and first current  Post-tetanic potentiation (short term) Pre-synaptic neuron Action High frequency stimulation potentials The activation of a nerve terminal with repeated, high- frequency stimuli causes an increase in the amplitude of the post- synaptic potential Post-synaptic neuron (EPSP), which lasts for a Post-tetanic potentiation EPSPs certain time after stimulation.  Long-term plasticity Experience of any sort modifies subsequent behavior at least in part through activity-dependent, long-lasting modifications of synaptic strength. The brain encodes external and internal events as complex, spatio- temporal patterns of activity in large groups of neurons that constitute ‘neural circuits’. The behavior of any given neural circuit is defined by the pattern of synaptic strenghts that connect the individual neurons that comprise the circuit. information is stored (ie, memories are generated) when activity in a circuit causes a long-lasting change in the pattern of synaptic strenghts associative memories are formed in the brain by a process of synaptic modification that strengthens connections when presynaptic activity correlates with postsynaptic firing. This proposed function for synaptic plasticity, forming a memory trace following the detection of two coincident events, suggests an appealing cellular basis for behavioral phenomena  Long-term potentiation (LTP) memorization process The high-frequency stimulation of a nerve terminal causes an increase in the amplitude of the post-synaptic EPSP, which lasts for a long time after stimulation. Stimolazione elevata frequenza High frequency stimulation applied at this time  Long-term depression (LTD) memory cancellation process Low frequency stimulation (LFS) can cause a long-lasting reduction of the EPSP. LFS applied after LTP can abolish the synaptic potentiation. LFS HFS LFS  Long-term potentiation as a neuronal substrate for memory formation Facilitation of the the High frequency excitatory synapse stimulation transmission A phenomenon underlying synaptic plasticity, learning and memory. A typical time-course recording of the EPSP amplitude from a single neuron EPSPs of different amplitudes are evoked by the same electric stimulus Time (minutes of recording) potentiated normal EPSP after HFS EPSP HFS  Hippocampus We can measure EPSPs in CA1 CA1 Stimulation of SC Fornix From entorhinal cortex Hippocampal long-term potentiation EPSP recorded EPSP amplitude potentiation respect to baseline baseline CA1 neuron Time Tetanic stimulation (HFS) EPSP Long-term changes in synaptic strength and circuit refinement have been extensively studied at excitatory glutamatergic synapses. Activity-driven structural changes include: elimination and formation of synapses, actin-dependent stabilization and enlargement of the postsynaptic density, alterations of the composition of ionotropic glutamate receptors Plasticity at inhibitory synapses has received much less attention, despite its contribution to the maintenance of the stability, dynamic range, and flexibility of neuronal circuits  Long-term memory mechanism Glutamate is involved in synaptic transmission and in different forms of synaptic plasticity such as LTP and LTD of synaptic transmission. Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved.  A glutamatergic, axo-dendritic synapse consisting of a presynaptic terminal and a postsynaptic dendritic spine The nerve terminal contains synaptic vesicles with glutamate transporters (red dot), mitochondria (blue) with glutaminase in nerve terminal (yellow dot), metabotropic glutamate receptors, and transporters for glutamate (EAAT2) and glutamine (SA transporters). The postsynaptic dendritic spine contains glutamate receptors, both ionotropic (AMPA and NMDA type) and metabotropic, and glutamate transporters (EAAT3 and EAAT4, the latter in cerebellar Purkinje cells). Surrounding the synapse are astrocytic processes with glutamate (EAAT1 and EAAT2) and glutamine transporters (SN transporters), glutamate receptors and even glutamate-filled vesicles. Green rectangles in plasma membrane of the axon terminal represent EAAT2 and of the astrocyte represent EAAT1/EAAT2 glu transporters. Glutamate that escapes out of the synapse without being cleared by transporters may spill over into neighboring synapses. AMPA-R, AMPA receptors; EAAT1–4, excitatory amino acid transporters 1–4; Gln, glutamine; Glu, glutamate; mGlu-R, metabotropic glutamate receptors; SA and SN, system A and system N transporters respectively (for glutamine). Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved.  Organization of the postsynaptic membrane Schematic representation of a dendritic spine of a glutamatergic synapse with some proteins of the postsynaptic density (PSD). Lipid rafts, scaffolding proteins (such as PSD95, GRIP, shank and homer) and cytoskeletal proteins (F-actin) help to concentrate and stabilize effector proteins at the spine. Effector proteins are glutamate receptors (AMPA-R, NMDA-R, mGlu- R), protein kinases and protein phosphatases, enzymes for the production of second messengers (nitric oxide, cAMP). AdCyc, adenylate cyclase; AKAP, (protein kinase) A-kinase anchoring protein; AMPA-R, AMPA receptor; CaMK II, Ca2/calmodulin- dependent kinase II; CaN, calcineurin (protein phosphatase 2B); DAG, diacylglycerol; F-actin, filamentous actin; GRIP, glutamate receptor-interacting protein; IP3, inositol-1,4,5-triphosphate; mGlu receptor, metabotropic glutamate receptor; NMDA-R, NMDA-receptor; NOS, nitric oxide synthase; PKA and PKC, protein kinase A and C respectively (activated by cAMP and Ca2/DAG respectively); PLC, phospholipase C; PSD95, postsynaptic density protein with a molecular weight of 95 kDa; SER, smooth endoplasmic reticulum. Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved. Molecular families of glutamate receptors. Each of the two main glutamate receptor divisions comprises three functionally defined groups (classes) of receptor. These are made up of numerous individual subunits, each encoded by a different gene. Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved.  Schematic view of four types of glutamate receptor Two ionotropic receptors are shown, the NMDA and AMPA receptors, as well as group I and group II metabotropic receptors. Both classes of metabotropic receptor are coupled via G proteins (G) to intracellular enzymes, phosphoinositide-specific phospholipase C (PI-PLC) for group I receptors and adenylate cyclase (AC) for group II receptors. PI-PLC catalyzes the production of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) from phosphatidylinositol-4,5-bisphosphate (PIP2). The resulting increase in cytoplasmic IP3 triggers release of Ca2 from intracellular stores. Activation of group II metabotropic glutamate receptors typically results in inhibition of AC. The cytoplasmic proteins PSD-95, GRIP and Homer anchor the receptors to the PSD complex. Copyright © 2012, American Society for Neurochemistry. Published by Elsevier Inc. All rights reserved.  Long-term memory mechanism Glutamate receptors are involved in synaptic transmission and in different forms of synaptic plasticity such as LTP and LTD of synaptic plasticity. Glu NMDA receptor Mg2+ AMPA Glu receptor Glu Na+ Ca2+ before HFS Vm = -80 mV  Long-term memory mechanism Glutamate receptors are involved in synaptic transmission and in different forms of synaptic plasticity such as LTP and LTD of synaptic plasticity. Glu NMDA receptor Mg2+ AMPA Glu receptor Glu Na+ Ca2+ LTD after HFS Vm = -80 mV  Long-term memory mechanism Glutamate receptors are involved in synaptic transmission and in different forms of synaptic plasticity such as LTP and LTD of synaptic plasticity. Glu NMDA receptor Mg2+ AMPA Glu receptor Glu Na+ Ca2+ EPSP NMDA component before HFS Vm = -80 mV  Long-term memory mechanism Glutamate receptors are involved in synaptic transmission and in different forms of synaptic plasticity such as LTP and LTD of synaptic plasticity. Glu NMDA receptor Mg2+ AMPA Glu receptor Glu Na+ Ca2+ LTD LTP after HFS Vm = -80 mV NMDA receptor is an associative molecule Only if the NMDA-R is activated LTP can occour HFS NMDAR: a molecular switch of synaptic plasticity; Is a detector of coincidence Is activated by depolarization + binding with glutamate Messenger of short- and long-term synaptic changes  LTP INDUCTION 1. HFS Glutamate vesicles NMDA-R K+ AMPA-R mGluR-I 2. Membrane PLC depolarization Na+ Na+ Ca2+ 3. Activation of NMDA receptor by removal of Mg2+ block  LTP Presynaptic changes Glutamate Increases glutamate release Retrograde messengers Na+ NO, CO PAF, AA mGluR-I NMDA AMPA PLC K+ ↑ Ca2+ concentration Postsynaptic changes: phosphorilation of receptors and enzymes -modulation of present AMPARs CaMKII and other -insertion of new AMPA-Rs by kinases activation regulating their transport into the proper synaptic sites Produces retrograde signals  LTP induction If the AMPAR is activated and the NMDAR is blocked, the synapse normally works producing a normal synaptic response that can not be potentiated. Phase 1: Glutamate binds AMPARs and triggers an AP  LTP induction When the Mg2+ block is removed by the NMDAR and Ca2+ is allowed to flow through the NMDA channel, LTP is produced. Phase 2: The AP depolarizes the cell, removes Mg2+ from NMDARs allowing Ca2+ inflow  NMDAR as “coincidence detector” NMDAR contribution to postsynaptic reponses requires both presynaptic release of glutamate and postsynaptic depolarization due to the simultaneous activation of a population of synapses. Basic properties of LTP Cooperativity: LTP can be induced by the coincident activation of a critical number of synapses Associativity: is the capacity to potentiate a weak input (a small number of synapses) when it is activated in association with a strong input (a larger number of synapses). Cooperativity and associativity occur because of the requirement for multiple synapses to be activated simultaneously to generate adequate postsynaptic depolarization to remove the magnesium block of the NMDAR. Input specificity is due to the compartmentalized increase in calcium, which is limited to the postsynaptic dendritic spine and does not influence adjacent spines  LTP, early phase (short-term memory) Protein-kinases activation causes receptors phosphorylation. In ex., phosphorylation of AMPARs  stronger post-synaptic response phosphorylation AMPAR *CaMK release *PKC processes *PKC NMDAR Ca++ Retrograde messenger * = persistent activation  LTP, delayed phase (long-term memory) A strong high frequency stimulation spine dendrite induces long-lasting LTP. cAMP CaMK New proteins are synthetized PKA meaning structural synaptic MAPK plasticity. cell body proteic synthesis CREB nucleus gene expression CREB = Cyclic AMP-Response Element Binding Protein MAPK = mitogen-activated protein kinase Long-term structural synaptic reorganization Developmental changes in synaptic organization This remodeling involves removal or addition of dendritic spines This remodeling involves removal or addition of dendritic spines Incoming signals Spine maturity progresses This remodeling involves removal or addition of dendritic spines Image of spines in a striatal neuron Growth of a Dendritic Spine Spine with large heads tend to have a greater volume, larger PSD, more glutamatergic receptors, and greater signaling efficacy Mechanisms at the basis of the LTP and LTD expression Post-synaptic changes: Phosphorylation (LTP) and de- phosphorylation (LTD) of existing AMPA receptors. Exocytosis (LTP) and endocytosis (LTD) of AMPARs. Presynaptic changes: Increase / decrease of glutamate release by retrograde messengers (PAF, NO, CO, AA, eCBs).  LTP induction HFS HFS = high frequency stimulation Di Filippo et al., Trends Pharmacol Sci, 200 1. Many brain structures are involved in memory formation: hippocampus cortex basal ganglia cerebellum Amygdala 2. Repeated stimulation of neurons induces LTP / LTD 3. Role of AMPA and NMDA glutamate receptors in the formation of LTP 4. NMDA receptor as a coincidence detector 5. Intracellular Ca2+ mediates the early and late stages of LTP  ACFS O2 + glucose Electrophysiology in rat brain slice Bipolar stimulating electrode Recording electrode Cortex Glu Glu NMDA Striatum AMPA receptor Glu receptor Mg2+ Na+ Spiny neuron AMPA mediated EPSP The NMDAR component of evoked striatal EPSP is seen in the absence of Mg2+ but not in the control medium HFS Membrane potential LTD induction RMP -80mV HFS LTP induction Membrane potential RMP -80mV Note: RMP of striatal spiny projection neurons = -80 mV  HFS Note: RMP of striatal spiny projection neurons = -80 mV  NMDAR antagonist prevents LTP and unmasks the LTD HFS Note: RMP of striatal spiny projection neurons = -80 mV AMPAR antagonist does not prevent LTP induction HFS  Under physiological conditions, tetanic activation of cortico-striatal inputs (HFS) leads to LTD of synaptic transmission in the striatum. Conversely, after removal of the voltage-dependent Mg2+ block of NMDA channels, the same repetitive activation of cortico-striatal inputs (HFS) does not generate LTD, but LTP.  During striatal synaptic plasticity different neurotransmitter signaling systems are involved D1 MIDRBRAIN DA NEURON LTP D2 Dopamine synaptic learning LTD M1 STRIATAL synaptic forgetting ACh Depotentiation INTERNEURON M2 Acetylcholine nACh Facilitatory action Inhibitory action Calabresi et al., Lancet Neurology, 2006  6-OHDA La denervazione completa unilaterale di DA previene l’induzione dell’LTP e LTD della trasmissione cortico-striatale 140 Sham normal LTP HFS 6-OHDA 120 EPSP amplitude (% of control) no LTD 100 no LTP 80 normal LTD 60 40 -10 -5 0 5 10 15 20 25 30 35 40 45 Time (min) Calabresi et al.,1992, J Neurosci, Centonze et al., 1999 J Neurophysiol. Picconi et al., Nat Neurosci 2003 BDNF and other neurotrophins can regulate neuronal connectivity and modulate synaptic efficacy. Neurotrophins regulate long-term survival and neuron differentiation. They are involved in synaptic development and plasticity in various neuronal populations. For example, it has been shown that BDNF plays a role in LTP by acting both at the presynaptic and postsynaptic level. In CA1, BDNF enhances high-frequency transmission by modulating the number of anchored vesicles at the level of the vesicular proteins synaptobrevin and synaptophysin. Neuronal activity regulates neurotrophins at three different levels: i) synthesis, ii) secretion and iii) signaling. Synaptic transmission and connectivity are modified as a result of specific changes in pre- and postsynaptic neurons. TrkB, a tyrosine kinase receptor for neurotrophins well known for its functions during the development of the nervous system, is a powerful regulator of hippocampal LTP and learning.