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BME 502 / handout #5 BME 502: Handout on Synaptic Transmission #2 Synaptic Transmission II: Postsynaptic Mechanisms relation between receptor binding, channel state, synaptic current, and synaptic potential: hydrolysis(C )(O ) uptake A T B k1 + R _ T R _ T R* k2 diffusion concentration of neurotransmitter, T, in the synaptic cleft that is available to bind to the postsynaptic receptor, R, is dependent on the concentration of transmitter in the vesicles, the number of vesicles released, and the cleft geometry decline of neurotransmitter in the synaptic cleft is dependent on diffusion and either hydrolysis or reuptake by pre- and postsynaptic elements and glial cells one or more of the transmitter molecules will bind to the receptor, forming T • R, a bound but closed state of the channel, (C) binding to the receptor will occur with a rate constant of k1 unbinding from the receptor will occur with a rate constant of k2 the channel will make a transition from the closed state to the open state, (O), which is a different state of the bound receptor, T • R* the transition from closed to open will occur with a rate constant of the transition from open back to closed will occur with a rate constant of during the open state, the amount of current flowing through the channel is dependent on the single-channel conductance and the driving force; the current per channel is given by: Install Equation Editor and doubleclick here to view equation. where g' is the single channel conductance, and I' is the current flowing through a single channel 1 BME 502 / handout #5 2 BME 502 / handout #5 if all channels opened simultaneously and stayed open, then the total synaptic conductance would be the sum of all of the individual channel conductances: Install Equation Editor and doubleclick here to view equation. thus, given multiple populations of channels, each with different properties, a multiple, parallel conductance model is most appropriate: considering again the a change in conductance for a single channel, when a channel is open (switch closed), I' charges the membrane capacitance, Cm (current flows across the membrane conductance, Gr ) the direction of I' , and thus the polarity of the potential developed across Cm , is determined by Es relative to Er when the transmitter concentration is low and the channels close (switches open), the potential developed across Cm decays with a rate determined by m for synaptic events faster than m, decay of the PSP potential is governed by m for synaptic events slower than m, the decay of the PSP is governed by the kinetics of the channel the vast majority of excitatory postsynaptic potentials (EPSPs) are "fast" synaptic events, i.e., conductances mediated by channels that have a rapid transition rate from the closed to the open state and from the open to the closed state also true of the majority of inhibitory postsynaptic potentials (IPSPs) 3 BME 502 / handout #5 the consequence is that the membrane m is the primary determinant of the rise time and fall time of the synaptic potential, resulting in the following relation between the synaptic current and the synaptic potential: where gs is the total synaptic conductance, shown here with a polarity reversed relative to that traditionally used synaptic inputs are classified as excitatory or inhibitory depending on whether or not they increase or decrease the probability of action potential generation -- and not on whether or not they are depolarizing or hyperpolarizing, because whether or not a given synaptic current results in depolarization or hyperpolarization depends on Vm at the time of the synaptic input, and on E for the ion species carrying the synaptic current: 4 BME 502 / handout #5 5 BME 502 / handout #5 Excitatory Amino Acids: Glutamate glutamate is the most prevalent exctitatory neurotransmitter in the nervous system binds to three different subtypes of receptors, all of which have very different consequences for cell excitability receptor subtypes are identified by the agonists that bind most selectively to each: AMPA (-amino-3-hydroxy-5-methyl-4-isoxazoleproprianate) subtype fast kinetics; time-to-peak: 20 msec; duration: 50 msec receptor linked directly to the channel to cause a change in conductance conductance is carried by Na+ and K+ ions antagonist: 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) I-V curve constructed in the presence of agonist 6 BME 502 / handout #5 7 BME 502 / handout #5 NMDA (N-methyl-D-aspartate) subtype slow kinetics; time-to-peak: 70 msec; duration: 120 msec receptor also linked directly to channel to cause a change in conductance conductance is Ca2+, Na+ and K+ ions antagonist: D-5-aminophosphonovalerate (APV) but channel conductance requires more than just the presence of the ligand, glutamate also requires depolarization not because channel is voltage-sensitive but because Mg2+ ions bind to another binding site inside the channel so as to block conductance by any other ions the blockade is voltage-dependent, so that depolarization is required to remove Mg2+ from the channel site when both the requirements of glutamate and depolarization to remove the channel block are met, channel opens and Ca2+ ions are allowed to flow 8 BME 502 / handout #5 thus, I-V curve substantially different than I-V curve for AMPA receptor-channel, and shape of I-V curve markedly dependent on concentration of extracellular Mg2+ 9 BME 502 / handout #5 time courses of AMPA and NMDA components are substantially different: 10 BME 502 / handout #5 relative contributions of each receptor-channel subtype to the amplitude-time course of the EPSP depends on Vm : major functional consequence is that magnitude of NMDA conductance is frequency-dependent due to temporal summation 11 BME 502 / handout #5 12 BME 502 / handout #5 13 BME 502 / handout #5 at least one other requirement for NMDA channel conductance a binding site on the receptor-channel complex exists for glycine (inhibitory neurotransmitter in spinal cord; not in brain) glycine must be present for NMDA channel to function at all, regardless of the amount of glutamate or the level of depolarization thus, multiple mechanisms for regulating the NMDA receptor channel; contrasts with relative simplicity of AMPA receptor channel other binding sites inside the NMDA channel PCP, drug that has psychotomimetic effects MK-801 Zn2+ 14 BME 502 / handout #5 Metabotropic receptor (mGlu) identified by most effective agonist, quisqualate receptor is not linked to a channel, but instead has the effect of releasing calcium from intracellular storage sites, i.e., mitochrondria, and endoplasmic reticulum 15 BME 502 / handout #5 second messenger mediated synaptic transmission: example of the glutamate metabotropic receptor fast, i.e., receptor-channel complex slow, i.e., second messenger activated conductances second messenger-activated conductances mediated by G-proteins, which bind guanine nucleotides. G-proteins are trimers of three subunits, , , and the subunits bind and hydrolyze guanosine triphosphate (GTP) and interact with receptor and effector proteins the and subunits anchor the subunit to the membrane (and thus are required) in resting state, GDP (guanine diphosphate) is bound to the subunit and the three subunits are associated as a trimer when the receptor is activated by a neurotransmitter, GTP replaces GDP, resulting in the dissociation of the subunit, which then is free to diffuse through the membrane to interact with various target proteins, which can be components of channels, enzymes or pumps, eventually changing the conductance state of a channel 16 BME 502 / handout #5 17 BME 502 / handout #5 G-proteins have been groups into classes depending on their target: Gs Gi Gt Gk Gp activates adenylyl cyclase inhibits adenylyl cyclase activates cyclic GMP phosphodiesterase activates potassium channels activates phospholipase C Direct effects of G-protein -subunits on channel conductance a number of instances have been identified in which the -subunit acts directly on a channel protein to alter channel conductance example 1: ACh effect on cardiac muscle: closes K+ channels (figure above applies, with ACh as neurotransmitter, and in this case with channel closing) example 2: similar effect in CNS with ACh effects on muscarinic subtype of ACh receptor two ACh receptor subtypes: nicotinic and muscarinic nicotinic is the type at the neuromuscular junction permeable to both Na+ and K+ muscarinic receptors localized at other synapses in peripheral nervous system and at CNS synapses action is to close K+ channels -- voltage-dependent K+ channels that are normally open at rest or that open rapidly at voltages near rest referred to as M-current; rapidly activating, but very slow to no inactivation 18 BME 502 / handout #5 Effect of metabotropic receptor on intracellular stores of Ca2+ is an example of an indirect effect of G-protein -subunits on channel conductance G-protein activation of phospholipase C activated -subunit hydrolyzes membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), producing inositol 1,4,5-triphosphate (IP3) and diacyglycerol (DAG) IP3 releases Ca2+ from intracellular stores (endoplasmic reticulum): 19 BME 502 / handout #5 Inhibitory Amino Acids: GABA and Glycine GABA (gamma-aminobutyric acid) -- primary inhibitory neurotransmitter in brain; glycine is the primary inhibitory neurotransmitter in spinal cord almost always found in interneurons; cells having local connections with principal neuron that provides the output from a structure; thus, acts to modify excitability of principal neuron; involved in feedforward and feedback inhibitory circuitry acts through two classes of receptors - GABAA and GABAB GABAA receptor conductance: Clconcentration of Cl- approx 10x greater outside than inside, so when channels opened, inward flow of Cl- ions, resulting in hyperpolarization of neuron, IPSP postsynaptic hyperpolarization associated with a marked decrease in membrane resistance pharmacology: receptor blocker: bicuculline; channel blocker: picrotoxin 20 BME 502 / handout #5 kinetics: fast kinetics; rapid onset and offset time-to-peak: 50 ms; duration: 100-200 msec when part of a negative feedback pathway, IPSP follows directly after the repolarization of the membrane by Ic and gk(v), so partially merges with the hyperpolarization caused by those currents also partially overlaps with hyperpolarization of AHP; "hump" in hyperpolarization inactivation: primary inactivation is uptake mechanism for location: postsynaptic modulatory sites: allosteric action of pentobarbital pentobarbital increases the probability of channel opening to GABA, with little effect in isolation of the agonist, similar to glycine action at the NMDA receptor channel 21 BME 502 / handout #5 allosteric action of hormones: progesterone derivatives, e.g., pregnanolone functional role: feedback vs. feedforward circuits feedback: how might feedback inhibition be revealed in the activity of a principal neuron? assume circuit below: principal neuron #1 and #2 are excitatory; epsp time to peak in #2 and interneuron is 5 msec; ipsp in principal neuron caused by interneuron activation of GABAA receptor has a time to peak of 10 msec and a decay time of 30 msec summary: feedback inhibition will have a "low-pass filtering" effect on the activity of neuron #2; or, the interneuron acts like a "high-pass filter" with respect to the output of neuron #2 22 BME 502 / handout #5 paired pulse stimulation: in the real nervous system, inhibitory feedback is rarely if ever distributed to only one neuron; because principal neurons almost always out-number interneurons, the output of principal neurons converges onto interneurons; output of the interneuron diverges with respect to principal neurons 23 BME 502 / handout #5 thus, it is possible to have feedback inhibition to a cell which exhibits only a depolarization EPSP to an excitatory input, i.e., which does not drive the feedback; the excitatory input is suprathreshold to some other principal neuron of the population consequence is that IPSP can limit the duration of a simultaneous EPSP, i.e., 24 BME 502 / handout #5 precise effect of interneuron output on principal neuron depends to a large extent on the intrinsic membrane properties of the interneuron as well as on the kinetics of the channels, etc. 25 BME 502 / handout #5 feedforward inhibition: have found that interneurons involved with feedforward inhibition often have lower thresholds and thus, discharge with an earlier latency to same excitatory afferent that drives principal neuron thus, can "select" that subpopulations which will be susceptible to subsequent excitatory signal depolarizing ipsp's apply GABA to cell body; observation is hyperpolarization; bicuculline blockade indicates GABAA receptor mediation; picrotoxin blockade indicates Cl- conductance apply GABA to dendrites; observation is depolarization; bicuculline blockade and picrotoxin effects consistent with mediation by GABAA receptor; raises the question of whether or not the GABA effect is inhibitory -- maybe it is excitatory which would be more consistent with the depolarization; test hypothesis by stimulating excitatory input and applying GABA -effect is blockade of stimulation-induced action potential discharge how to explain depolarization: leads back to concepts of driving force -- two relevant variables are membrane potential and equilibrium potential how to explain inhibitory effect: leads back to same concept -- effect of a given conductance can be seen as "clamping" membrane potential at the equilibrium potential of the ion carrying the conductance activation of GABAA receptors usually associated with increased Cl- conductance and resulting hyperpolarization in some cases, however, have found that activation of GABAA receptors leads to a depolarization, even though conductance still is carried by Clremains inhibitory because ECl below threshold; thus, activation of GABAA receptors 26 BME 502 / handout #5 effectively "clamps" membrane potential below threshold for action potential 27 BME 502 / handout #5 GABAB receptor linked to a K+ conductance inhibition due to GABAA receptor activation illustrates a mechanism of inhibition that relies on an influx of negatively-charged ions, into the cell an alternative mechanism could involve the efflux of postively-charged ions, out of the cell the inhibtion caused by the GABAB receptor subtype induces inhibition by increasing K+ conductance conductance: K+ conductance; outward current, resulting in hyperpolarization of neuron, IPSP pharmacology: receptor blocker: saclofen; receptor agonist: baclofen (see below) kinetics: slow kinetics; time-to-peak: 200 msec; duration: 600 ms very slow onset and offset due to the fact that the GABAB receptor induces a change in K+ conductance indirectly through action of a G-protein receptor is linked to a G-protein inactivation: no distinction with respect to receptor subtype; inactivation 28 BME 502 / handout #5 location: pre- and postsynaptic GABAB receptors found postsynaptically, but also found presynaptically leads to inhibition of neurotransmitter release can inhibit either excitatory or inhibitory neurotransmitters, and because can inhibit GABA release, represents a form of auto-inhibition action of saclofen (antagonist) and baclofen (agonist) 29 BME 502 / handout #5 30 BME 502 / handout #5 Catecholamines norepinephrine (NE) and dopamine (DA) as examples 1. biosynthetic pathway in the case of NE, biosynthetic pathway consists of tyrosine dopa dopamine NE, with the specific enzymes responsible for the conversion of each protein, tyrosine hydroxylase for conversion from tyrosine dopa; dopa decarboxylase for conversion from dopa DA; dopamine--hydroxylase for the conversion from dopa DA; the synthetic enzymes, not the substrate proteins, are the rate-limiting step in the synthetic process 2. synaptic morphology different synaptic morphology than the characteristics of the other neurotransmitters some synapses appear to be relatively normal in the sense of the presence of a small synaptic cleft, vesicles, pre- and postsynaptic densities, etc other synapses, however, can be identified only on the basis of the presynaptic release site; no apparent postsynaptic receptor in the immediate vicinity mechanism of inactivation includes both degradative enzymes and uptake; for NE, degradative enzymes include monoamine oxidase (MAO) and catechol-o-methyl-transferase (COMT), though uptake appears to be more important than the degradative enzymes 3. receptor subtypes -receptors 1 receptors located postsynaptically lead to opening of K+ channels, and thus, hyperpolarization of the neuron (act through IP turnover) 2 receptors are located presynaptically, and are inhibitory to NE release, so that blockade of 2 receptors leads to prolonged release of NE in response to electrical stimulation, and increased basal levels of NE release (acts to inhibit cAMP) -receptors stimulation of the adrenergic receptor subtype catalyzes the formation of cAMP, which then activates a substrate protein, protein kinase A (PKA) 31 BME 502 / handout #5 PKA acts on the gk(AHP) channel to block conductance of K+ ions indirect second messenger systems cAMP system receptor ligand binding leads to activation of the -subunit, which then catalyzes the formation of cAMP cAMP activates a substrate enzyme, protein kinase A (PKA) PKA acts on the channel to change conductance 32 BME 502 / handout #5 example: -adrenergic decrease in gK(AHP) channel conductance left top panel: response of cell to intracellular current injection; note that cell eventually fails to exhibit action potential (called accomodation) due to AHP (left bottom panel) in the presence of NE, cell continues to respond (top right panel) because AHP is blocked (bottom right panel) 33 BME 502 / handout #5 G-protein activation of phospholipase C activated -subunit hydrolyzes membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), producing inositol 1,4,5-triphosphate (IP3) and diacyglycerol (DAG) IP3 releases Ca2+ from intracellular stores (endoplasmic reticulum): example 1: metabotropic receptor example 2: NE activation of IP3 and DAG second messengers; increased Ca2+ due to IP3 and DAG together activate protein kinase C, which leads to decrease in voltage-dependent Ca2+ current 34 BME 502 / handout #5 35