Novel Transmitters I: Introduction to Nitric Oxide

Introduction

• In the previous unit, we began to focus on the chemical as well as the electrical properties of the nervous system by learning about key neurotransmitters and their receptors.

• We focused on compounds that had relatively low molecular weights, contained amine groups, and were stored in vesicles in the presynaptic terminals. These characteristics are true for acetylcholine, amino acid transmitters and transmitters derived from amino acids (like the catecholamines), and transmitters such as histamine and serotonin.

• Although these "classical" transmitters were first to be discovered, a very large group of additional transmitters was found to consist of chains of amino acids, known as neuropeptides.

• Still more recently, small molecular weight gases were found to be synthesized by the nervous system: nitric oxide, carbon monoxide and hydrogen sulfide. What made these results both surprising and controversial was that the latter two molecules were well known to have highly toxic effects. A recent review summarizes the evidence for their role as transmitters, and their involvement in brain and behavior.

• In this unit, we will briefly describe a few neuropeptides whose functional roles in the nervous system have begun to be clarified, and then turn to a description of the role of nitric oxide in the function of a model glutamatergic synapse, the calyx of Held. With the background we have developed in these two units, we will also briefly discuss the roles of transmitters and their receptors in the effects of drugs, and their basis for certain addictive processes. You will then have the opportunity to use a simulation to explore the role of nitric oxide at a synapse.

Peptidergic Transmission

• Short chains of amino acids (usually between 2 and 20) are referred to as peptides. Many of these peptides have been found to serve as neurotransmitters.

• Unlike conventional transmitters, which are usually packaged in clear vesicles, peptidergic transmitters are usually packaged in dense core vesicles.

• Several features often distinguish peptidergic transmission from neurotransmission that uses conventional transmitters:
  • Peptidergic transmitters are often released in response to more intense neural activity.
  • Many peptidergic transmitters work through receptors that are coupled to second messenger systems (i.e., metabotropic receptors), and thus they may act over much longer time periods.
  • Peptides are often not broken down by specific enzymes within the synaptic cleft, as is typical for conventional transmitters. Instead, they can diffuse out of the synaptic cleft, and begin to affect much larger numbers of neurons that have appropriate receptors that are within the vicinity of the original presynaptic terminal. Peptidergic effects are often terminated by nonspecific enzymes that break down peptides (peptidases).
  • Thus, the nervous system can use forms of communication that range in both their spatial and temporal extent, as well as flexibly grouping together components - even those that are not anatomically contiguous - into dynamic neural assemblies, based on their receptors. A useful analogy is a private phone conversation (transmission from one neuron via a single synapse to another neuron), a speakerphone conversation that allows several people in the same room to participate (transmission within a larger volume), to radio communication (hormonal actions throughout the body determined by whether or not a target tissue has a receptor for that hormone). Even though the nervous system is protected by the blood/brain barrier, many peptides that are synthesized in the periphery, and have extensive actions there, are also synthesized and act within the brain.

• We will briefly discuss several peptides whose actions have been well-characterized; the number of peptides that have been identified and their complex effects continue to increase each year, so this is an active and growing area of research.

• Sensory neurons contain and release a variety of peptides. An eleven amino acid long peptide, Substance P, has been implicated as a transmitter involved in the sensing and reporting of pain (nociception). It is one of a class of peptides that are released in response to pain, and much effort has been expended in trying to develop novel pain-blocking (analgesic) drugs that act to block receptors to these peptides. A recent review describes several peptides, including Substance P, that are released by sensory nerves, and recent efforts to use this knowledge to develop new therapies.

• Another class of peptides that have been intensively studied are the endogenous opiates. The first to be characterized, the enkephalins, are five amino acids long, and bind to the same receptors that are activated by opiate drugs such as morphine. It was initially hoped that these compounds could be effective suppressors of addictive behavior, but it was found that they could themselves induce addiction. Studying the effects of the endogenous opiates, their receptors and potential antagonists is an area of intensive investigation. A recent review for the year 2009 briefly summarizes results from 815 papers published on the topic in that year alone.

• The peptide oxytocin consists of a sequence of nine amino acids. It plays important roles in lactation and in uterine contractions during birth. The peptide has attracted wide attention because of evidence in animals and in humans that it affects social behaviors such as pair bonding. A recent review summarizes some of the exciting work in this area, emphasizing the therapeutic potential of oxytocin in a variety of psychiatric disorders such as social anxiety and autism.

• The interplay among conventional and peptidergic transmitters has been intensively studied in the lateral hypothalamic region, which plays important roles in the regulation of food and water intake. A recent review discusses some of the most important peptides that have been implicated in feeding, and are also serving as possible therapeutic targets for treatment of disorders such as anorexia or obesity.

A Gas Transmitter: Nitric Oxide

• The idea that a gas could serve as a transmitter between neurons was initially very controversial.

• Earlier studies had shown that nitric oxide (NO) could play a role in the periphery, and was very important for such phenomena as vasodilation through its actions on cyclic guanosine monophosphate (cGMP), which induces relaxation of smooth muscle. Indeed, one of the most famous applications of the understanding of nitric oxide came from the development of a drug that could enhance its actions: sildenafil (known more popularly as Viagra) acts by blocking the degradation of cGMP, and thus allows a great deal of blood flow to a particular peripheral organ when necessary.

• A crucial step in realizing the role of nitric oxide in transmission was the identification of its synthetic enzyme, nitric oxide synthase (NOS), and its localization to neurons. Different forms of NOS have been identified, and given names reflecting their major roles: neuronal NOS (nNOS), endothelial NOS (eNOS) and cytokine inducible NOS (iNOS).

• Another amino acid plays a crucial role in NO synthesis: L-Arginine, whose terminal amine group provides the nitrogen for nitric oxide.

• The neuronal NOS is dependent on activation by calcium/calmodulin. Thus, the enzyme activity can be induced either by action potentials that activate voltage-dependent calcium channels, or by activation of receptors (such as the glutamate NMDA receptor) that allow calcium influx into the postsynaptic terminal. Through NO's actions on soluble guanylyl cyclase, NO affects cGMP synthesis, which in turn can act on a very wide variety of other molecular targets. A recent review summarizes the broad effects of nitric oxide on the nervous system.

• Once activated in the postsynaptic terminal, NO can diffuse out of the postsynaptic terminal, because it is not stopped by the cell membrane, and can then affect the pre-synaptic terminal, thus serving as an important retrograde signal. It can also affect surrounding neurons, thus serving as a volume transmitter (and thus having effects reminiscent of the peptide transmitters described above).

• A recent study of nitric oxide in the calyx of Held demonstrated that its synthesis was enhanced in the postsynaptic terminal in response to presynaptic stimulation, and also showed that its levels, and its effects on cGMP synthesis, could be measured in connected presynaptic neurons as well as in non-connected neighboring neurons. Moreover, NO inactivates postsynaptic voltage-gated potassium channels (Kv3 channels) through phosphorylation, and alters the responsiveness of both NMDA and AMPA receptors, reducing the strength of the synaptic connection, and the ability of the postsynaptic neuron to spike in response to excitatory inputs. Suppressing these potassium channels also broadens the action potential and slows repolarization after the action potential, increasing inactivation of voltage-gated sodium channels and thus reducing the efficacy of transmission.

• This may be another example of gain regulation in the nervous system: as levels of excitation change in a neuronal network, NO could act to turn down the gain within components of the network.

Neuropharmacology, Drugs of Abuse, and Addiction

• Neuropharmacology is the study of the actions of exogenous agents that act through the endogenous receptors. In many cases, understanding the mechanisms of action of a natural transmitter, and then identifying its receptors, and working out their genetic control, has opened up new realms for targeted therapies to alleviate diseases of the nervous system. Drugs used for treating psychiatric disorders, insomnia, memory loss, depression, or to reduce the damage due to stroke are all based on an understanding of the physiology of transmitter systems of the brain.

• As a consequence of mapping out the locations of transmitters, and understanding their physiological roles, it has become possible to understand phenomena that could only be understood previously at the level of the whole organism, such as motivated states or drives. Thus, for example, studies have shown that the brain contains elaborate systems that allow organisms to both recognize painful or pleasurable sensations, and to form associations between them and objects, events or actions.

• In particular, the dopaminergic system located in the ventral tegmental area (VTA) plays an important role in anticipating potential rewards, and directly acts to enhance behavior that allows an animal to gain rewards.

• Drugs of abuse hijack these systems, with the collusion of the brain's owner.

• A current hypothesis is that addiction is a disease of goal-directed learning processes. Drugs of abuse activate plasticity, and associate specific behaviors with the intense pleasure that the drugs can induce as they directly activate endogenous receptors. The behaviors are so strongly reinforced that they become compulsive. Furthermore, the context in which they occur creates powerful associations, so that being placed in the same context in which the person previously administered the drug to himself or herself induces powerful cravings that increase the probability of relapse.

• Ordinarily, dopamine signals serve to learn an association between an action and a reward; as the association becomes well-established, the dopamine signal is no longer evoked. Addictive drugs create excessive levels of dopamine, and induce a pathological form of synaptic association. A recent review implicates metabotropic glutamate receptors in this abnormal plasticity.

• Over time, the excess levels of the drug induce changes in receptors. They may be down-regulated in response to the high levels of their ligand, creating tolerance. This has two negative consequences. The original dose of the drug is no longer sufficient to induce its original effects. As a consequence, the addict may begin to take higher doses to achieve the same effects. Since many drugs of abuse can be lethal in large doses, this process can often be fatal. The other negative consequence is that if an addict attempts to withdraw from an addictive drug, the endogenous levels of normal transmitter are much less effective in activating the postsynaptic neurons. This is likely to be responsible, at least in part, for the cravings and other physiological effects of drug withdrawal, which are often severe enough to be life-threatening, and can also induce relapses.

• Many efforts are now underway to use the improved understanding of the mechanisms of drugs of abuse, and their effects on the normal physiology of the brain, to create new and more effective treatments to help wean addicts from drugs.

• It should be clear from this very brief description that the best way to avoid problems with addiction is not to start in the first place, whatever peer pressure you may get to "try it; it's fun!". Focusing on sources of pleasure that are not addictive is intrinsically a better way to be happy and successful.

Exploring a Nitrergic Synapse

• You now have enough information to begin to explore a specific synapse that utilizes both the conventional transmitter glutamate and the gas nitric oxide as part of its normal process of transmitting messages.

• In this simulation we are studying two neurons located close together with a synapse connecting them. The simulation is based on a paper describing nitric oxide as a volume transmitter by Steinert et al; (Neuron 60:642, 2008), which can be found here; however, instead of simulating the volume diffusion of NO, as was done in the original paper, the effect of NO is captured using an appropriate function of time (an alpha function, which is often used to simulate synaptic potentials).

• Question 1: From what you see in the simulation, is this an excitatory or inhibitory synapse? Please write down your hypothesis. To test your hypothesis, change the total duration of the simulation to 80 ms; in the Presynaptic Current clamp, change the Stimulus delay to 30 ms; in the Postsynaptic Current Clamp, change the stimulus delay to 5 ms, the stimulus current to 2 nA, the pulse duration to 70 ms, and the Number of pulses to 1.
  • Run the simulation. What do you observe? Explain the initial response of the neuron. Explain the reason that it does not continue to fire action potentials. What happens in the postsynaptic neuron when the presynaptic neuron fires an action potential? Is this result consistent with your hypothesis?
  • Now, in the Postsynaptic Current Clamp, change the stimulus current to 20 nA, and again run the simulation. What do you observe in the postsynaptic neuron? Did your observations support or refute your hypothesis? Explain.

Figure 1: Measuring the half-height width of an action potential

• Question 2: For some synapses, previous activity can have effects minutes, hours or even years later. To explore this, the simulation allows you to activate the synapse minutes or hours before the actual experiment by repeatedly stimulating the presynaptic neuron; we will call this early activation of the synapse through presynaptic stimulation the "pretreatment".
  • Reset the simulation to the default parameters. Set the number of presynaptic pulses to zero and the number of postsynaptic pulses to 1. Run the simulation to see what the postsynaptic action potential looks like.
  • Refer to Figure 1 for the following measurements. Use the cursor to measure the maximum height of the peak; also measure the resting value, which is given at the bottom left of the graph of the voltage. Use these numbers to calculate the half-height of the action potential (i.e., the potential at which the the membrane has reached half the height it will reach at the peak of the action potential).
    • For example, if the resting potential is at -60 mV, and the peak of the action potential is at +30 mV, then the half-height is ((+30) - (-60))/2 = 90/2 = 45 mV above the resting potential. Since the resting potential is at -60 mV, the half-height occurs at -60 mV + 45 mV = -15 mV.
    • Formulaically, we can represent this calculation of the half-height of the action potential as: $V_{^1/_2} = V_\text{rest} + \frac{V_\text{peak}-V_\text{rest}}{2}.$
  • Measure the half-height width of the action potential (i.e., the width of the action potential, in milliseconds, at the half-height potential, indicated in Figure 1 using the double-sided green arrow), and write down the value.
  • Next, under the list of Pretreatment parameters, change the duration of the prestimulation of the presynaptic neuron to 60 seconds, so that the synapse will be activated fifty times a second for one minute, then left to rest for 10 minutes before the experiment. What happens to the width of the action potential in the postsynaptic neuron? Again determine the maximum height, and again measure the width at half the maximum height. What do you observe? What is the percentage change?

• Question 3: How quickly do the effects of the pretreatment take place?
  • Measure the width of the postsynaptic action potential at its half-height with the same 60 seconds of 50 Hz prestimulation, but this time change the delay between the prestimulation and the experiment to 0, 1, 5, 10, 15, 30, 60 and 120 minutes.
  • Plot the time delay versus the width of the action potential at half its maximum height.
  • How long does it take for this effect to become maximal, and how long does it last after it reaches a maximum? How does the time it takes to reach a maximum and to wear off compare to the lengths of times we have seen for the actions of gates in ion channels opening and closing, like the Hodgkin and Huxley channels or the duration of action of AMPA receptors on the postsynaptic neuron? Can you suggest a mechanism that could account for the times that you observed in the plot?

• Question 4: For the synapses we've seen so far, the activation of the synapse does not significantly affect the presynaptic cell (unless the postsynaptic neuron is electrically coupled to the presynaptic neuron). What happens in this synapse?
  • Reset the simulation so that only the presynaptic cell is being stimulated during the experiment. Measure the action potential width at half of the maximum height in the presynaptic cell (note that your previous measurements have all been on the postsynaptic neuron). Now prestimulate the presynaptic neuron for 60 seconds at 50 Hz (thus activating the synapse many times) with a 10 minute delay before the experiment, and again measure the action potential width of the presynaptic neuron at half of its maximal height.
  • Do you see an effect on the presynaptic cell? What is the percentage change?