Synaptic Plasticity II: Introduction to Long-Term Potentiation

Introduction

• Until this unit, we have focused almost exclusively on the electrical properties of the nervous system.

• Aspects of neurons that are just as important are their chemical properties.

• In this unit, we will focus on the chemicals that neurons use to transmit messages, focusing primarily on small molecule transmitters. Because these were discovered first, they are often referred to as conventional transmitters. Other transmitters - peptides composed of chains of amino acids, and diffusible gases - were discovered later, and are often referred to as unconventional transmitters. We will discuss them in the next unit.

• After discussing some of the conventional transmitters and their receptors, you will explore a form of plasticity - long term potentiation - that involves the interplay of different glutamate receptors, and provides one possible mechanism for the formation of associative memories.

Establishing that a Compound is a Neurotransmitter

• There are many chemicals associated with cells in general, and with neurons. How can one determine that a compound is a neurotransmitter?

• Over the years, a series of criteria have been established to answer this question. As we will see, not all transmitters satisfy all the criteria, but enough do that they are still useful for guiding experimental work to establish whether a compound is or is not a transmitter.
  • Postsynaptic action: When applied to the postsynaptic neuron in concentrations similar to those found under physiological conditions, does it mimic the effect of the natural (endogenous) transmitter?
  • Release: Is the transmitter released by the presynaptic neuron in sufficient amounts to induce the physiological effects?
  • Presence: Is the transmitter present in the presynaptic neuron?
  • Synthesis: Is the transmitter synthesized by the presynaptic neuron?
  • Pharmacology: When drugs that specifically block the actions of the natural transmitter (antagonists) are applied, do they also block the actions of the compound when it is applied to the postsynaptic neuron?
  • Molecular biology: Are specific receptors for the compound present in the postsynaptic neuron?
  • Metabolism: Are there specific mechanisms for removing the transmitter from its site of action? For example, is there an enzyme that specifically degrades the compound, or is there a transport system that specifically re-uptakes it into the presynaptic neuron?

Classes of Neurotransmitters

• Acetylcholine
  • Acetylcholine was one of the earliest transmitters identified.
  • It is made from choline and acetyl-CoA by the enzyme choline acetyl-transferase.
  • It is broken down by acetylcholine esterase (AChE).
  • The actions of acetylcholine can be prolonged by using drugs that block AChE, such as the nerve gas sarin or the insecticide parathion.
  • Acetylcholine is the transmitter at the neuromuscular junction
  • It serves as a transmitter in both the peripheral and central nervous system
  • In the central nervous system, it is the transmitter in basal forebrain neurons that project to the hippocampus and the neocortex, and has been implicated in memory.

• Biogenic amines (or monoamines)
  • Each of these transmitters are made from amino acids.
    • The transmitters made from the amino acid tyrosine are known as catecholamines. The transmitter released by a given neuron is determined by the presence or absence of the last of the sequence of synthetic enzymes.
      • Tyrosine $\Rightarrow$ di-hydroxy phenylalanine (DOPA) $\Rightarrow$ Dopamine $\Rightarrow$ Norepinephrine $\Rightarrow$ Epinephrine
      • Dopamine plays an important role in the striatum; loss of dopaminergic neurons in the nigro-striatal tract leads to Parkinson's disease. Dopamine also plays an important role in the ventral tegmental area as a monitor of reward.
      • Norepinephrine (also known as noradrenaline) plays an important role in responding to stress both in the periphery (for example, through its actions on the heart), and in the central nervous system.
      • Epinephrine (also known as adrenaline) is synthesized by the adrenal glands, and plays an important role in response to emergency threats to an animal.
    • Tryptophan $\Rightarrow$ 5 hydroxy Tryptophan $\Rightarrow$ Serotonin (5 hydroxy tryptamine, 5HT)
      • Selective serotonin re-uptake inhibitors (SSRI) have been found to be effective in treating severe depression; they act by blocking the re-uptake of serotonin from the synaptic cleft, thus prolonging the actions of serotonin.
    • Histidine $\Rightarrow$ Histamine
      • In the central nervous system, histamine plays an important role in regulating sleep; as a consequence, when many antihistamine drugs are taken to reduce the effects of allergic reactions, they can make the patient very sleepy.

• Amino Acids
  • Glutamate
    • This is the main excitatory transmitter of the central nervous system.
    • In glutamatergic neurons, glutamate is stored in vesicles and released when the neurons are excited.
    • Excessive glutamate can lead to excitotoxicity, which can destroy neurons.
  • Gamma amino butyric acid (GABA)
    • GABA is a major inhibitory neurotransmitter in the central nervous system.
    • GABA is synthesized from glutamate via glutamic acid decarboxylase (GAD) which is localized to GABA neurons.
    • A major target of many tranquilizers are GABA receptors.
  • Glycine
    • Glycine has a mainly inhibitory effect in the spinal cord, the brainstem and in the retina.

• Neuropeptides
  • Neuropeptides are synthesized in the cell body, and transported down the axon to the presynaptic terminal.
  • Neuropeptides are stored in larger vesicles than are usually used for "classical" transmitters.
  • Peptide release often occurs when the presynaptic neuron is strongly activated.
  • The effects of peptides are often terminated by diffusion away from the synaptic cleft, so they may act over longer periods and longer spatial regions.
  • We will have more to say about them in the next unit.

• Purines
  • Adenosine triphosphate
  • Adenosine

• Gases
  • Nitric oxide
  • Carbon monoxide
  • Hydrogen sulfide

Neurotransmitters in the Peripheral Nervous System

• Skeletal motor transmission: motor neurons have their cell bodies in the spinal cord, extend axons to muscles in the periphery, and use acetylcholine as their transmitter at the neuromuscular junction.

• The autonomic nervous system, which controls non-skeletal muscles, such as glands, the digestive system, and the heart, has two major subdivisions: the sympathetic nervous system and the parasympathetic nervous system.

• The sympathetic nervous system generally causes overall activation, or what is known as the "fight or flight" response. Its neurons have their cell bodies in the spinal cord, and then send an axon to a peripheral ganglion (usually close to the spinal cord), and the synapse within that ganglion uses acetylcholine. The postsynaptic neuron then sends an axon to the peripheral target, and at that synapse, it releases norepinephrine.

• The parasympathetic nervous system generally causes a reduction in excitation, aiding in digestion and rest. Like the sympathetic nervous system, its neurons have their cell bodies in the spinal cord, and then send an axon to a peripheral ganglion (which is usually close to the target organ), and the synapse within that ganglion uses acetylcholine. The postsynaptic neuron then sends an axon to the peripheral target, and at that synapse, it release acetylcholine.

• The balance of sympathetic and parasympathetic input is important for normal responses. For example, the sympathetic inputs to the heart can accelerate heart rate; in contrast, parasympathetic inputs to the heart can slow heart rate. Both processes are important for normal response to ongoing changes in the environment.

Receptors and the Effects of Transmitters: Ionotropic versus Metabotropic

• Although investigators often refer to a transmitter as "excitatory" or "inhibitory", this is rarely correct, because what determines the actions of a transmitter are its postsynaptic receptors and their effects on ion channels.

• It may sometimes be the case that a particular neurotransmitter has a uniform effect, but this reflects the uniform distribution of a particular kind of receptor to the transmitter, not an inherent property of the neurotransmitter itself.

• As we mentioned in the previous units on chemical synaptic transmission, the effects of receptors are broadly divided into two classes: ionotropic and metabotropic.

• Ionotropic receptors are directly linked to ion channels, as their name suggests. Once transmitter binds, the ion channel may begin to conduct, or may cease to conduct. The termination of the effect may be due to unbinding of the transmitter, or to inactivation of the channel.

• Metabotropic receptors work through second messenger systems, and thus their actions are often both slower and more widespread than those of ionotropic receptors. A very important class of metabotropic receptors are those that are coupled to molecules known as G proteins. G-protein coupled receptors (often abbreviated as GPCRs) play important roles in many systems. Working through the cAMP and phosphatidylinositol pathways, they can have a very wide variety of intracellular effects over both short and long time scales.

• The neurotransmitters described above couple to many different receptors. Names of receptors are therefore often based on the drugs that bind and may activate them more strongly than the native ligand (these drugs are called agonists), or on their molecular biological properties.

• Knowing the details of receptors is very important, especially if one wants to develop targeted treatments. A novel drug that binds selectively to one class of receptors will have far fewer deleterious side effects than one that binds indiscriminately to all receptors of a class, causing multiple unwanted side effects.

• The endogenous transmitter acetylcholine binds to different receptors, which are known by the names of their agonists: nicotinic receptors, and muscarinic receptors.

• Nicotinic receptors are ionotropic, are made of five subunits, and mainly allow sodium entry and potassium exit (generally leading to a net depolarization), though depending on their subunit composition, they can also permeate calcium ions (these forms of the receptor are mainly found in the central nervous system). They are antagonized by toxins that act to paralyze prey by impairing the function of the neuromuscular junction, such as tubocurarine (curare), α-bungarotoxin and α-conotoxin. These receptors are not only found in the neuromuscular junction but also in the brain and in autonomic ganglia.

• In contrast, muscarinic receptors are metabotropic, are coupled to G-proteins, are antagonized by atropine (among many other agents), and are found in the brain and in autonomic targets.

An Amino Acid Transmitter: Glutamate and its Receptors

• The amino acid transmitter glutamate exerts its actions through its receptors, which are classified by their agonists: AMPA and NMDA receptors (ionotropic receptors), and metabotropic glutamate receptors (mGluR).

• AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) glutamate receptors (often abbreviated further as AMPARs) are classified further based on the subunits that make them up. Those AMPA receptors containing the subunit designated GluR2 are permeable to sodium and potassium ions, but not calcium ions.

• NMDA (N-methyl D-aspartate) receptors are examples of both voltage and ligand-gated channels. In response to depolarization and the presence of glutamate, the channel, which is blocked by magnesium ions at the resting potential, is capable of permeating cations, including calcium ions. Thus, it serves as monitor, at the level of an ion channel, of both the presence of transmitter from a presynaptic neuron and of depolarization in a postsynaptic neuron, and can thus be used to monitor conjunctive activity in the neuronal circuit surrounding it.

• Metabotropic glutamate receptors, mGluR, which are G-protein coupled, are also widespread, and have been implicated in several forms of plasticity. A recent review describes their important role in long term depression (LTD).

Long Term Potentiation and Long Term Depression at Glutamatergic Synapses

• In the late 1960s and early 1970s, investigators found that applying a tetanic stimulus to inputs to the hippocampus could generate a long-lasting facilitation of the synaptic connections. This long-lasting facilitation is called long-term potentiation, or LTP.

• A great deal of effort has been put into the analysis of the mechanism by which LTP is formed.

• The current consensus is that, at glutamatergic synapses, the early phase of LTP is due to strong depolarization of the postsynaptic neuron, which frees the NMDA receptor of its magnesium ion block, so that it can allow calcium ions to flow into the postsynaptic terminal. Binding of calcium to various proteins, such as calmodulin, then triggers a cascade of responses, including the phosphorylation of AMPA receptors, and the insertion of additional AMPA receptors into the postsynaptic membrane. Over longer periods of time, protein synthesis is induced, leading to remodeling of the synapse, making that synapse more excitable over a very long time.

• Although this continues to be controversial, there is evidence that a retrograde signal is generated in the postsynaptic neuron and can affect the expression of the strength of the presynaptic neuron. In studies of LTD due to metabotropic glutamate receptors, it appears that an endogenous cannabinoid, anandamine, may travel from the postsynaptic to the presynaptic neuron, altering the amount of transmitter released by the presynaptic terminal.

Exploring Long Term Potentiation in a Glutamatergic Synapse

You now have enough information to begin to explore a form of associative plasticity, long term potentiation, in a model of a glutamatergic synapse.

• Question 1: Glutamate binds a class of receptors that are also activated by the drugs AMPA or kainate. Let's look at the properties of this class of receptors first. Begin with the simulation that allows you to apply current clamp to a glutamatergic synapse. To eliminate the NMDA receptors, under Synapse properties, set the NMDA maximum conductance to 0. Run the simulation. What is the effect of the presynaptic neuron on the postsynaptic neuron, both in terms of the postsynaptic neuron's potential and its calcium concentration? Turn the NMDA maximum conductance back to 6 $\mu S$, and again run the simulation. What is the effect of the presynaptic neuron on the postsynaptic neuron, both in terms of the postsynaptic neuron's potential and its calcium concentration? What might this suggest about one of the actions of the conductance that you just blocked? Explain.

• Question 2. To see the underlying postsynaptic potential (PSP) due to AMPA more clearly, block action potentials in the postsynaptic neuron. After setting the NMDA maximum conductance to 0 under the Synapse properties, set Fast transient sodium conductance and Delayed rectifier potassium conductance to 0 under the Postsynaptic Cell Properties. Run the simulation. What is the effect of the presynaptic neuron on the postsynaptic neuron?

• Question 3. It is possible to determine the reversal potential of a synapse using current clamp. By injecting sufficient current, it is possible to raise or lower the membrane potential of the postsynaptic neuron, and thus affect how much current is induced in the postsynaptic membrane by activation of the synaptic receptors. To simulate this, under Postsynaptic Cell Properties, change the Leak potential to +10 mV (since you are focusing on the AMPA PSP, keep the other conductances that you set to 0 in Question 2 at 0). Run the simulation. What happens to the PSP? Please measure the magnitude of the first PSP. Explain. Vary the Leak potential value of the postsynaptic neuron to find the reversal potential. What is the voltage of the postsynaptic neuron at which the synapse generates no net current? What does this imply about the ions that are likely to contribute to generating the PSP?

• Question 4. Restore the leak potential to its original values (under Postsynaptic Cell Properties, change the Leak potential back to -70 mV). Check that the NMDA maximum conductance is still 0, as are the maximum conductances of potassium and sodium in the postsynaptic model neuron. Now increase the firing frequency of the presynaptic neuron: under Presynaptic current clamp, change both the Stimulus current first pulse and the Stimulus current subsequent pulses to 50 nA, change the Inter-stimulus interval to 5 ms, and change the Number of pulses to 15. What happens to the firing pattern of the presynaptic neuron? Explain (this is a partial review of material from earlier in the semester). What happens to the PSPs in the postsynaptic neuron? Explain (this is also a partial review of material from earlier in the semester).

• Question 5: Now restore the maximum potassium and sodium conductances to their prior values in the postsynaptic model neuron (50.0 $\mu S$ and 260.0 $\mu S$, respectively). Describe and explain what you observe in the postsynaptic neuron.

• Question 6. Restore the default values for the simulation (press the Default simulation button). We will now examine the effects of the NMDA receptors. Set the AMPA max conductance and the AMPA minimum conductance to zero. Increase the duration of the simulation to 300 ms, and the number of pulses in the presynaptic neuron (under the Presynaptic Current Clamp menu) to 12. Describe what is happening to the size of the PSPs in the postsynaptic neuron and its effect on the ability of the postsynaptic neuron to generate an action potential. Contrast this with what you observed when only AMPA receptors were present. What role would you expect AMPA versus NMDA receptors to play in retaining a memory of the inputs that have just previously occurred?

• Question 7. Channels activated by NMDA receptors allow calcium to flow into the cell. To examine the effects of the calcium influx, first block the action potentials by setting the maximum potassium and sodium conductances under the Postsynaptic properties to 0. Leave the other parameters of the simulation that you set up in the previous question the same (AMPA conductances: 0 µS, simulation duration: 300 ms, number of presynaptic pulses: 12). Run the simulation. What happens to the membrane potential and to the levels of calcium in the postsynaptic neuron? Measure the peak calcium concentration. Now, under the Postsynaptic Cell Properties, set the Calcium buffering time constant to 1 ms. Run the simulation. Measure the peak calcium concentration in the postsynaptic neuron. How does this level compare to the postsynaptic calcium levels when the buffering time constant was 25? Explain.

• Question 8. Restore the simulation parameters (press the Default simulation button). Under Presynaptic Current Clamp, set the Stimulus current first pulse and Stimulus current subsequent pulses to 50 nA. Set the Inter-stimulus interval to 5 ms. Set the Total duration to 400 ms. Run the simulation. Look at the membrane potential of the postsynaptic neuron and the level of calcium within it after the stimulation ends. What would you predict about the probability of transmitter release from the postsynaptic neuron in response to inputs within 200 ms of the initial burst you just provided? Now set the NMDA maximum conductance to 0, and again run the simulation. Look at the membrane potential of the postsynaptic neuron and the level of calcium within it after the stimulation ends. What would you predict about the probability of transmitter release from the postsynaptic neuron in response to inputs within 200 ms of the initial burst you just provided?

• Question 9: Depolarizing a neuron with the NMDA receptor relieves the block of the receptor by magnesium ions. We can reproduce the effect of the relief of the magnesium block by changing the extracellular magnesium concentration in the simulation. Press the Default simulation button. Change the duration of the simulation to 400 ms, the number of pulses in the presynaptic neuron to 1 (under the Presynaptic Current Clamp menu), and change the AMPA maximum conductance and the AMPA minimum conductance to 0. Also, change the Fast transient sodium conductance and the Delayed rectifier potassium conductance in the postsynaptic neuron to zero. Run the simulation. Measure the peak calcium level. What happens? Explain. Now, change the Extracellular magnesium concentration (under the Synapse Properties) from 1 mM to 0 mM. Run the simulation. Measure the peak calcium level. What happens? Explain. Repeat both of these experiments after restoring the maximum potassium and sodium conductances in the postsynaptic neuron to their normal levels (50 and 260 $\mu S$, respectively). What happens? Explain.

• Questions 10: Perhaps the most interesting feature of the synapse we have been studying is its ability to maintain a longterm memory that associates two events, one of which activated the presynaptic neuron, and one of which activated the postsynaptic neuron. The resulting long term increase in the synaptic strength is referred to as long-term potentiation, or LTP. In this question, you will study some of the mechanisms underlying this phenomenon, which will build on the understanding you have developed in the previous questions. Press the button labeled "LTP simulation".
  • Please look at the resulting simulation carefully. Note that the presynaptic and postsynaptic neurons were activated simultaneously.
    • How many action potentials do you observe in the postsynaptic neuron in response to the second current pulse?
    • What is the size of the AMPA current during the first action potential in response to the pair of presynaptic and postsynaptic stimulating current pulses?
    • What is the size of the AMPA current during the first action potential in response to the second presynaptic stimulating current?
    • Note the change in the calcium levels after the first set of action potentials. Measure the calcium level just before the second current pulse begins. Increases in calcium that are large enough can activate a second messenger cascade that leads to adding phosphate groups (i.e., phosphorylation) to AMPA-activated ion channels, allowing these additional channels to become active; over longer time periods, new AMPA-activated ion channels may be inserted into the neuronal membrane.
  • Now set the number of pulses in the Postsynaptic Current Clamp to 0. Run the simulation.
    • How many action potentials do you observe in the postsynaptic neuron in response to the second current pulse?
    • What is the size of the AMPA current during the first action potential in response to the first presynaptic stimulating current pulse?
    • What is the size of the AMPA current during the first action potential in response to the second presynaptic stimulating current pulse?
    • Again measure the calcium level just before the second current pulse begins. What has changed? Explain.
  • For associative learning to work, one needs a mechanism that will enhance transmission when (1) the presynaptic neuron and (2) the postsynaptic neuron have both been simultaneously excited, but not otherwise. Explain how the NMDA, AMPA conductances, and the magnesium block (and its relief by depolarizing the postsynaptic neuron) could work together to generate associative learning.