Voltage Clamp Method 3 1 Current flow across an axon membrane during a voltage clamp experiment Hyperpolarization elicits only a capacitative current transient and a small leakage current current flowing into the cell is shown as a negative value flowing out positive value TEA blocks K channels TTX blocks Na channels voltage clamp finds conductances A version of Ohms Law can be written for each ion flowing across the membrane during the action potential iNa gNa Vm ENa ik gk Vm Ek ileak gL Vm EL gion represents the number of open channels that carry that ion It is a measure of the status of the channel gating process The term Vm Eion represents a DRIVING FORCE that drives an ion through through a channel once the channel is open Interpretation of V I curves in terms of the electrical model 1 Both iNa and iK are activated when the membrane is depolarized 2 The early current is iNa iNa gNa Vm ENa so the early current reverses from inward negative to outward positive when Vm exceeds ENa 58 mV 3 The late current is IK iK gK Vm Ek so the late current is outward positive at all membrane potentials more positive than Ek 70mV The sodium and potassium conductances are time and voltage dependent lecture 10 8 Criteria That Define a Neurotransmitter 1 The substance must be present in the presynaptic terminals a Corroborated by presence of the enzymes that synthesize it or break it down b Demonstrated e g by immunohistochemistry if the NT is a peptide or protein or for enzymes immunogold labeling of proteins under the electron microscope 2 The substance must be released upon the arrrival of a presynaptic action potential and its release must be calcium dependent a This is tough to demonstrate since must devise a way of measuring NT release from specific living cells 3 Receptors exist for the substance on the postsynaptic cell and stimulation of the receptors must result in the natural response of postsynaptic cell to the NT a immunohistochemical labeling of receptor proteins most often used b Application of exogenous NT to the synapse e g puffing NT onto the synapse from a micropippette to see whether the postsynaptic cell responds appropriately c Application of agonists mimics or antagonists pharmacological agents drugs that block both the natural response and the response to the exogenously applied NT also most often used as evidence Dale s Principle A given neuron only secretes one NT chemical from all of its synapses o A good simplifying statement allows to classify a neuron or even a brain pathway containing many similar neurons by it s NT Cholinergic neurons use acetylcholine Aminergic neurons use a biological amine like noradrenaline Peptidergic neurons uses small peptides o Exceptions some synapses found to secrete both a main small molecule and a peptide NT a co neurotransmitter o Otherwise this principle is proven to be resilliant Metabolism of small molecule and peptide transmitters Small molecule NT s are synthesized in the axon terminal from precursors The synthetic enzymes however have to be carried down from the cell body via slow axonal transport Small clear core vesicles in the axon terminal typically contain small molecule NT s vesicles Peptide NT s are synthesized in the cell body and transported down the axon in Dense core vesicles in the axon terminal typically contain peptide NT s Sequence of events involved in transmission at a typical chemical synapse AP arrives V dependent Ca2 channels open Ca2 promotes NT vesicle fusion with plasma membrane NT released into Synaptic cleft NT binds to NT receptor proteins on surface of postsynaptic cell opens ligand gated channels Ions enter Postsynaptic cell Changes Postsynaptic Vm Evidence that a rise in presynaptic Ca2 concentration triggers transmitter release Direct injection of Ca2 into the presynaptic terminal elicits a postsynaptic response due to NT release Increase in presynaptic Ca2 monitored from a squid giant synapse in which the presynaptic terminal was injected with Ca sensitive fluorescent dyes during stimulation by a train of a p s Evidence that a rise in presynaptic Ca2 concentration triggers transmitter release Injection of a Ca chelator into the presynaptic terminal blocks transmission Molecular mechanisms of neurotransmitter release Cyc Cycling of pre synaptic membrane Differential release of neuropeptide and small molecule co transmitters The Neuromuscular junction was the classic preparation for understanding the basis for synaptic transmission in experiments in the 1960 s by Katz Miledi and collaborators EPP is a graded potential whose amplitude is dependent on the number of synaptic boutons activated and the conc of Ca2 in the extracellular fluid Use of patch clamp to measure activity of acetylcholine gated channels at the neuromuscular junction flux of na into cell causes depolarization Reversal potential of end plate currents If current was carried solely by na ions what vm would the voltage reverse 50 b c that s the Nernst pot for na Why does current through the Ach gated channel reverse at 0mV at neither EK or ENa Because the Ach gated channel is permeable to BOTH sodium and potassium ions Influence of reversal potential on postsynaptic membrane potential When the Ach receptor protein channel is open the postsynaptic membrane permeability changes from mostly potassium and perhaps chloride permeable at rest to sodium and potassium permeable The postsynaptic Vm will move towards Erev for the Ach induced current Excitatory and inhibitory synapses The movement of postsynaptic Vm towards the reversal potential for current flow through active NT gated receptor channels is true of all postsynaptic responses mediated by NT gated channels So if the postsynaptic channels are sodium selective calcium selective or cation non selective then Erev will be 0mV or greater and the postsynaptic Vm will rise beyond threshold to trigger an action potential These are excitatory synapses However if the postsynaptic channels are potassium selective or chloride selective then Erev will be 60 mV or less and the postsynaptic Vm will be forced below threshold inhibiting action potentials These are inhibitory synapses