BIPN140 Lecture 8: Synaptic Transmission II 1. Postsynaptic Receptors: Metabotropic & Ionotropic 2. Postsynaptic Responses (Postsynaptic Potentials, PSPs) 3. Neurotransmitters Su (FA16) Chemical Synapse: an Overview (Fig. 5.3) Possible fates for neurotransmitters at the synaptic cleft: 1. Function as ligands for a receptor which directly gates an ion channel (ionotropic) 2. Function as ligands for a receptor which regulates the function of enzymes or ion channels (metabotropic). 3. Degraded by enzymes 4. Sequestered by transporters. 5. Diffuse away
Two Types of Neurotransmitter Receptors (Fig. 5.16) (1) Membrane proteins (2) Embedded in the postsynaptic membrane (3) Having an extracellular NT binding site Metabotropic Receptors (Fig. 5.16) In some synapses, a neurotransmitter binds to a receptor that is metabotropic (G-protein coupled receptors). In this case, movement of ions through a channel depends on one or more metabolic steps. Binding of a neurotransmitter to a metabotropic receptor activates a signal transduction pathway in the postsynaptic cell involving a second messenger (e.g. camp, Ca 2+, etc.) Compared to ligand-gated channels, the effects of second-messenger systems have a slower onset but last longer (hundreds of milliseconds to minutes), allowing more opportunity for temporal integration. Amplifying effect: can affect many channels 2 nd messengers can diffuse outside the postsynaptic sites.
Ionotropic Receptors (Fig. 5.16) Ligand-gated ion channels: (1) having a pore that allows a particular type of ion to flow through (cation or anion) upon activation (binding to NTs). (2) mediate rapid postsynaptic events. Postsynaptic potentials (PSPs) usually arise within a millisecond or two of an presynaptic AP, and last for only a few tens of milliseconds or less. (3) can allow either positively charged ion (cation) or negatively charged ion (anion) to go through. (4) mainly affects PSPs (localized effect) Postsynaptic Responses: ACh Receptors at the NMJ (Fig. 5.17) Activation of postsynaptic ionotropic receptors: (1) NT binding to the receptor opens the channel (ligand-gated ion channel) (2) Single channel event: patch-clamp recording, voltage-clamp mode to measure currents microscopic current macroscopic inward current Depolarization By ~ 30 mv (not voltageclamped)
The Influence of PSP on End Plate Currents (Fig. 5.18) I = g x V (Ohm s law) EPC = g ACh x (V m -E rev ) V m < E rev => inward current V m = E rev => no current V m > E rev => outward current Net driving force on the ion flowing: the difference between membrane potential and the reversal potential (potential where there is no net current flow) More negative inside, drawing cation inside the cell => Depolarization effect More positive inside, pushing cation outside the cell => Hyperpolarization effect E rev = 0 mv, between E Na and E K, suggesting that the channel is permeable to both Na + and K +. Reversal Potential of a Non-selective Cation Channel For a neuron that is permeable to multiple types of ions, the equilibrium membrane potential depends on the relative permeability of the ions (GHK equation). At equilibrium, I K = I Na ; I K = g K x (V m -E K ); I Na = g Na x (V m -E Na ) g K x (V m -E K ) = g Na x (V m -E Na ) Tug-of-war E Na = +58 mv V m = g Na E Na + g K E K (g Na + g K ) For a channel that is permeable to both Na + and K + (non-selective cation channel), the reversal potential (E rev ) is a linear sum of the weighted conductance times the respective reversal potential of the permeable ions: E rev = g Na E Na + g K E K (g Na + g K ) g Na /g K = (E K E rev )/(E rev -E Na ) Reversal potential is determined experimentally (in between the equilibrium potentials of the permeable ion species) E K = -90 mv
Postsynaptic Potentials (PSPs) For a neuron that has two types of conductance (g m, from leak channels and g ACh, e.g. conductance of ACh gated ion channel), the membrane potential is determined by the ratio of these two types of the conductance and the reversal potential of the ligand-gated ion channel. Tug-of-war V m = g m E rest + g ACh E rev (g m + g ACh ) E rev = 0 mv The degree to which the voltage approaches the reversal potential of the ligand-gated ion channel is determined by the relative conductance (g m & g ACh ). PSPs generated from an single synapse usually do not reach E rev. In other words, neurons with small g m require fewer ionotropic receptor to reach the same EPSP. E rest = -60 mv Reversal Potential (Figs. 5.18 & 5.19) Reversal Potential: voltage at which EPC reverse direction (the equilibrium potential for the ligand-gated ion channel) Reducing E Na Increasing E K E AChR = ~0 mv (the channel is permeable to multiple types of cations, nonselective cation channel) E AChR = ~0 mv The channel is permeable to both Na + and K +, therefore, E AChR is between E Na and E K At rest, EPC is primarily carried by Na + influx. Why? V rest = -60 mv E Na = +70 mv; E K = -90 mv Net driving force for Na + : 130 mv Net driving force for K + : 30mV
Na + and K + Movements during EPCs and EPPs (Fig. 5.20) V m < E rev => inward current more negative inside, drawing Na + inside => depolarization Net driving force for Na + : 160 mv Net driving force for K + : 10 mv V m = E rev => no current (no effect on membrane potential) V m > E rev => outward current more positive inside, pushing K + outside => hyperpolarization Net driving force for Na + : 0 mv Net driving force for K + : 170 mv Reversal Potentials and Threshold Potentials Determine Postsynaptic Excitation and Inhibition (Fig. 5.21) EPSP; depolarizing IPSP; hyperpolarizing IPSP; depolarizing E Cl = -70 mv E Cl = -50 mv Activation of an ionotropic receptor tend to bring the postsynaptic potential towards the reversal potential of the ionotropic receptor (E rev ). Excitatory Postsynaptic Potential (EPSP): E rev > AP threshold (usually ~ -40 mv) Inhibitory Postsynaptic Potential (IPSP): E rev < AP threshold The relationship between resting membrane potential and E rev does not impact whether a PSP is excitatory or inhibitory! It does not matter whether the PSP is depolarizing or hyperpolarizing.
Summation of Postsynaptic Potentials (Fig. 5.22) V = I x R; I = g x (V m -E rev ); g: number of opened channel x conductance per channel That is, the magnitude of PSP is determined by the size of PSC, i.e. how many postsynaptic channels are activated by NTs. NMJ synapse is very strong, i.e. lots of postsynaptic AChRs. Most CNS synapses are not strong, but neurons are usually innervated by thousands of synapses. PSPs can sum in space and time (PSP duration >> AP duration). The summation of EPSPs and IPSPs by a postsynaptic neuron permits a neuron to integrate the electrical information provided by all the inhibitory and excitatory synapses acting on it at any moment. Synapses closer to axon hillock will have a greater impact on AP generation. Location of Synapses Matters If the neuron responds to two identical GABAergic inputs at location (1) and (2) with two identical IPSCs, which of the following is true? A. IPSC at location 1 is more effective in inhibiting action potentials than location 2. B. IPSC at location 1 is less effective in inhibiting action potentials than location 2. C. IPSC at location 1 is as effective in inhibiting action potentials as location 2. D. None of the above. It depends on the threshold of the neuron.
Size of Neurons Matters a b If neuron (a) and neuron (b) respond to glutamatergic inputs with EPSCs of identical amplitude, which of the following is true? (Remember V m = i x R input ) A. The EPSC at neuron (a) is more effective in generating action potentials than at neuron (b). B. The EPSC at neuron (a) is less effective in generating action potentials than at neuron (b). C. The EPSC at neuron (a) is equally effective in generating action potentials as at neuron (b). From NT Release to Postsynaptic Excitation or Inhibition (Fig. 5.23) NT release at all presynaptic terminals on a cell results in receptor binding, which causes the opening or closing of specific ion channels. The resulting conductance change (g; I = g x V) causes current (I) to flow, which may change the membrane potential ( V = I x R). The postsynaptic cell integrates all of the EPSPs and IPSPs, resulting in moment-to-moment control of action potential generation (neuronal output, signal that can be propagated to another neuron).
Major Neurotransmitters (Fig 6.1) Many types of neurotransmitters (>100) A given neurotransmitter can bind to different types of receptor (a large number of combinations). Therefore, a given neurotransmitter can excite postsynaptic cells expressing one receptor and inhibit others expressing a different receptor. The effect of a given neurotransmitter is determined by the receptor it activates. Neurotransmitter receptors can be regulated by other compounds (drugs!). For example, nicotine can activate certain acetylcholine receptors. Curare (arrow poison) inhibit certain acetylcholine receptor to paralyze muscles. Biogenic Amines Biogenic amines include Dopamine, Norepinephrine, Epinephrine (adrenaline) Serotonin Histamine Affect coordination of body movement, sleep, mood, reward, attention and learning (some also function as hormones). They are active in the CNS and PNS Biogenic amines have a central role in a number of nervous system disorders and treatments (Parkinson s disease, psychiatric disorders, addiction, depression etc.).
Biogenic Amines (Figs. 6.10 & 6.14) Rate-limiting Rate-limiting Found in neurons in the hypothalamus, mediating arousal and attention. Side effect of Benadryl (antihistamine): drowsiness. Removal: reuptake by transporters. Catabolism of catecholamine NTs requires monoamine oxidase (MAO) and catechol-o-methyltransferase (COMT). Termination of 5-HT by serotonin transporter (SERT) Local synthesis: using amino acids as precursors (catecholamines: tyrosine; Histamine: histidine; Serotonin: tryptophan). Modern neuropharmacology: development of many drugs affecting the synthesis, receptor binding or catabolism of these neurotransmitters. (Cocaine inhibits dopamine transporter, DAT, thereby increasing DA concentrations in the synaptic cleft. So is amphetamine, which also affects norepinephrine.) Antidepressants: MAO inhibitors (MAOI) & selective serotonin reuptake inhibitors (SSRI, e.g. Prozac). Unconventional NTs: Gases (Fig. 6.20) NO synthase is regulated by Ca 2+ /Calmodulin Unconventional: Unlike most neurotransmitters, these are not stored in vesicles but are instead synthesized as needed (local regulator). Release is regulated by Ca 2+. Mediate inter-neuronal communication: Can be release pre-synaptically or post-synaptically (retrograde signaling) Once synthesized, they can permeate the plasma membrane to enter and act on nearby cells. Can activate guanylyl cyclase to generate cgmp or modify protein directly via nitrosylation (addition of a nitrosyl group to select amino acid)
Unconventional NTs: Endocannabinoids (Fig. 6.18, Box 6G) agonist Endocannabinoids: Anandamide and 2- arachidonylglycerol (2-AG) Unsaturated fatty acids produced by our nervous system to act on the cannabinoid receptors (target of 9 -tetrahydrocannabinol (THC, active component of marijuana). endocannabinoid antagonist Agonists (e.g. WIN 55,212-2) and antagonists (e.g. rimonabant) are available to study endocannabinoid receptors (CB1 type in the CNS, metabotropic receptor). endocannabinoid Unconventional NTs: Endocannabinoids (Fig. 6.19) Reduced IPSC In the hippocampus, CB1 is found mainly in the presynaptic terminals of the inhibitory interneurons Depolarization-induced Suppression of Inhibition (DSI) CB1 antagonist: blocks the reduction of IPSCs > 90% reduction Production is stimulated by a second messenger in the postsynaptic neurons (usually Ca 2+ ). Functions as a retrograde signaling molecule (from postsynaptic neuron to presynaptic neuron). Likely released via diffusion (membrane permeable). Not stored in SVs. Terminated by enzyme hydrolysis (fatty acids hydrolase, FAAH). Best known to inhibit the communication between postsynaptic target cells and their presynaptic inputs.
Unconventional NTs: Endocannabinoids (Fig. 6.19) In the hippocampus, CB1 is found mainly in the presynaptic terminals of the inhibitory interneurons Depolarization-induced Suppression of Inhibition (DSI) Reduced IPSC Background: Endogenous cannabinoids, e.g. anandamide (as well as THC from marijuana), diffuse across cell membranes and activate receptors in hippocampal interneurons that depress GABA release. Depolarization of hippocampal pyramidal neurons suppresses GABAergic IPSPs the neurons receive, a phenomenon called Depolarization-induced Suppression of Inhibition or DSI. DSI was thought to represent a retrograde effect of the postsynaptic neuron onto presynaptic inhibitory terminals, but the mechanism was unknown. Experiments: Induce DSI by depolarizing a hippocampal pyramidal neuron with a patch pipette, and record the changes that occur in IPSPs elicited by stimulating interneuron input to the neuron. (1) Test the effects of compounds that block cannabinoid receptors to see if they block DSI. (2) Similarly, test compounds that activate cannabinoid receptors to see if they mimic DSI. (3) Determine if the cannabinoid effect is likely to be presynaptic, e.g. affects mepsc frequency (how frequent are SVs released by the presynaptic neuron) but not mepsc amplitude (unitary activation of postsynaptic receptors).
Fig. 1. DSI requires endogenous cannabinoids CB1 antagonist Control experiment: DSI after 30 min of recording eipsc: evoked inhibitory postsynaptic current CB1 antagonist: prevents DSI CB1 agonist: mimics DSI (occlusion) Fig. 3. DSI and a CB1 agonist suppress IPSCs by the same mechanism Paired-Pulse Ratio: the ratio of two PSPs or PSCs evoked in close succession. Changes in PPR are usually interpreted as presynaptic changes, reflecting the differences in the availability of readily releasable SVs (the first pulse, or stimulus, may deplete a lot of SVs) mipsc frequency: miniature IPSC frequency, (reflecting how often a SV is released) Cd 2+ : blocks VGCC, thereby blocking SV release (positive control) CB1 agonist Markedly reduced the first PSC (i.e. not so many SVs were released due to CB1 antagonist or DSI) Recovery: due to removal of endogenous ligand (e.g. 2-AG) by a transporter TTX: block voltage gated Na + channel to block action potentials. KCl: to depolarize presynaptic terminal to favor SV release
Results: Blockers of cannabinoid receptors blocked DSI expression; agonist of the receptors mimicked DSI. Both the agonists and DSI acted on presynaptic machinery in GABAergic (inhibitory) terminals innervating the pyramidal neuron. Finally, DSI was shown to spread locally, affecting IPSP amplitude in nearby neurons, exactly as predicted for a diffusible membrane-permeant regulator like cannabinoids. Take-home message: These DSI relies on the retrograde (back across the synapse) transfer of cannabinoids which inhibit GABA release from presynaptic terminals, thereby reducing the evoked IPSC seen in the postsynaptic pyramidal neuron. Globally applied cannabinoids, e.g. THC, can be expected to globally diminish inhibitory activity and alter information processing and system output.