What happens to neurotransmitters after a message is passed on to another neuron?

Introduction

Neurotransmitter release from presynaptic nerve terminals is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Synaptic vesicle fusion is tightly coupled to voltage-induced Ca2 + influx and has a latency on the microsecond timescale. This rapidity suggests that within a nerve terminal a population of synaptic vesicles is competent, or primed, to undergo membrane fusion immediately upon Ca2 + entry.

Synaptic vesicle priming and fusion requires members of the conserved soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein families, specifically, the plasma membrane-associated SNAREs (t-SNAREs, also called Q-SNAREs) syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25) and the vesicle membrane-associated SNARE (v-SNARE, also called R-SNARE) synaptobrevin. These SNARE proteins interact through their SNARE domains to form a parallel four α-helical bundle termed the SNARE complex. SNARE complex assembly in trans is predicted to bring the vesicle membrane into close proximity to the plasma membrane, a prerequisite for membrane fusion.

Because SNARE interactions can be detected prior to neurotransmitter release, interactions between SNARE proteins are thought to represent the molecular underpinnings of vesicle priming. By extension, proteins that interact with the SNAREs are candidates for promoters and regulators of vesicle priming. UNC-13/Munc13 is a presynaptic protein that interacts with syntaxin. Here we review the evidence implicating UNC-13/Munc13 in vesicle priming and discuss the current models for how UNC-13/Munc13 functions in vesicle priming and how this core function is regulated.

INTRODUCTION

The process of neurotransmitter release in synapses has been known to depend on extracellular Ca2+ ions for many years (del Castillo and Katz, 1954). More recently it became evident that the action of Ca2+ in promoting neurotransmitter release comes from inside the cell (Katz and Miledi, 1977; Llinás et al., 1981). Based on these and similar experiments, the concept that Ca2+ is not only required, but is also sufficient to evoke release on its own, became widely accepted (Stockbridge and Moore, 1984; Simon and Llinás, 1985; Fogelson and Zucker, 1985). However, there is no conclusive evidence that Ca2+ is indeed the only limiting factor in the process of neurotransmitter release.

The assumption that Ca2+ is required and sufficient to start the release process is in fact the essence of the ‘Ca2+ hypothesis’ for release. An unavoidable conclusion from this hypothesis is that after an impulse, release should last as long as the Ca2+ concentration near crucial domains of release is above a certain level. Moreover, release after an impulse should last longer if more Ca2+ enters or if its removal is slowed (Parnas and Segel, 1984). In contrast, the time course of release after an impulse was found to be insensitive to variations in extracellular Ca2+ concentration (Datyner and Gage, 1980), to repetitive stimulation (Datyner and Gage, 1980; Barrett and Stevens, 1972; H. Parnas et al., 1986a), or to other treatments that are known to modulate intracellular Ca2+ concentration (Matzner et al., 1988) and its regulation. Therefore a conflict exists between predictions from the classical Ca2+ hypothesis and experimental results pertaining to the time course of release.

Two main approaches were advanced for solving this conflict. The first remains in the boundaries of the classical Ca2+ hypothesis, but adds refinements associated with the spatio-temporal changes in intracellular Ca2+ concentration (Simon and Llinás, 1985; Fogelson and Zucker, 1985). In particular, these authors still assume that Ca2+ is the only limiting factor in the release process. Other factors, such as release sites or vesicles, are not only assumed to be ready and constant during release, but to be in excess. These authors developed detailed mathematical models to describe the entry of Ca2+ through patches of Ca2+ channels and away from the channels by diffusion.

Diffusion is the key process in keeping the time course of release short and insensitive to conditions that alter intracellular Ca2+ concentration.

A second approach questions the legitimacy of the foundation of the Ca2+ hypothesis, namely that Ca2+ is the only limiting factor in the release process. It suggests that while Ca2+ is certainly required for release, it is nevertheless unable to support evoked release by itself. This approach assumes that another factor, together with Ca2+, accumulates during the natural stimulus, and that both this factor and Ca2+ are required to start the chain of events leading to release. In the case of the neuromuscular junction, the natural stimulus is membrane depolarization, and therefore this second factor, called S, must be produced by membrane depolarization and must rapidly disappear with membrane repolarization. Thus the classical Ca2+ hypothesis was extended, becoming the ‘Ca2+−voltage’ hypothesis (H. Parnas et al., 1986a; I. Parnas et al., 1986). According to the Ca–voltage hypothesis, the amount of release or quantal content depends both on intracellular Ca2+ concentration and the amount of S, while the time course of release, after an impulse, depends mainly on the time course of disappearance of the membrane potential-dependent factor, S. Therefore, there is no need to correlate the time course of release to the time course of the increase and decrease in intracellular Ca2+ concentration after an impulse, and there is no more conflict between the hypothesis and the experimental results which show independence of the time course of release on conditions which manipulate intracellular Ca2+ concentration.

The most direct way to test the Ca2+ hypothesis is to monitor the changes in Ca2+ concentrations using Ca2+ indicators. However, even with the most sophisticated new developments in the field of Ca2+ indicators, the present spatio-temporal resolution is not refined enough to monitor changes in Ca2+ concentration near the Ca2+ channels and release sites. In the same way, there are no clues as to what that missing factor S could be. We therefore used a theoretical approach to distinguish between these two hypotheses.

In the present chapter, we show some of the main predictions from the two hypotheses. These predictions are compared with key experimental results, followed by a discussion as to the ability of these two models to cope with the experiments.

Taste Receptors Project to the Cortex Through the Solitary Nucleus and the Thalamus

The TRCs are not neurons, even though they release neurotransmitters, because they have no dendritic of axonal processes. They make synapses onto dendritic processes of primary afferent sensory neurons whose cell bodies reside in three cranial nerve ganglia. The anterior two-thirds of the tongue and the palate are innervated by the facial nerve or cranial nerve VII. The cell bodies for the taste fibers are in the geniculate ganglia. The posterior third of the tongue is supplied by the glossopharyngeal nerve or cranial nerve IX. The cell bodies for this sensory nerve are located in the inferior glossopharyngeal ganglia. The vagus nerve, cranial nerve X, supplies the scattered taste receptors in the throat regions, including the glottis, epiglottis, and pharynx. The cell bodies of the vagus reside in the inferior vagal ganglia.

Sensory fibers from all three of these cranial nerves enter the lateral medulla and make synapses on cells in the gustatory division of the solitary nucleus in the medulla. The second-order neurons in the solitary nucleus send fibers up to the ventral posterior medial nucleus of the thalamus, where they make synapses on third-order neurons. These thalamic neurons then project to the primary gustatory cortex located in the insular and orbitofrontal regions.

Summary

The adrenal medulla is essentially a ganglion of the sympathetic nervous system that releases neurotransmitter into the blood instead of near local targets. Preganglionic sympathetic nervous fibers originating mainly in the thoracic spinal cord reach the adrenal medulla through the splanchnic nerves, and release acetylcholine onto chromaffin cells in the adrenal gland, causing release of epinephrine into the blood; the epinephrine is then transported to distant targets. Epinephrine is synthesized from tyrosine in the sequence tyrosine–dihydroxyphenylalanine–dopamine–norepinephrine–epinephrine. Circulating epinephrine and norepinephrine are degraded by catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO). A variety of stimuli increase epinephrine secretion including hypoglycemia, hypovolemia, hypotension, fear and anxiety, pain, and trauma.

The effects of epinephrine and norepinephrine are mediated through adrenergic receptors, of which there are at least five types. The α1 receptors work through a Gq mechanism that activates smooth muscle contraction mainly in the arterioles of skin, GI system and kidney, and the urethral sphincter. Adrenergic α2 receptors activate a Gi mechanism. All of the β adrenergic receptors exert their effects through a Gs mechanism.

The overall effect of adrenergic stimulation is to prepare the body for emergency action. The pupils dilate (α1 receptors), blood pressure increases, bronchioles dilate (β2 receptors), blood flow to inessential organs is reduced, heart rate and contractility are increased, and stored metabolic fuels, glycogen and triglycerides, are mobilized to increase plasma glucose and free fatty acids for metabolism.

Overview

Presynaptic active zones consist of a highly specialized network of proteins that acts in neurotransmitter release. The key function of the active zone is to organize the fusion process that is mediated by SNARE proteins and triggered by calcium to render it highly temporally and spatially precise. Major insight into the molecular functions of individual active zone proteins have been gained over the past 15 years, but many important issues remain unsolved. Crucial questions like the molecular nature of the tight membrane association, the link to the cytoskeleton, and the docking mechanisms of synaptic vesicles are only partially understood. The profound diversity in the morphology of active zones suggests a high level of functional variety. However, it is not well understood what accounts for their diverse architecture, and how such morphological diversity relates to functional specification. Another fascinating question that we are only beginning to address is the use-dependent structural reorganization of active zones, and how such molecular adaptations are involved in synaptic plasticity and neural circuit dynamics.

Effect on Neurotransmitter Receptors and Neurotransmitter Release

Cd may affect neurotransmission by different mechanisms. First of all it can suppress neurotransmitter release due to the blockade of CaV channels and, consequently, of Ca++ influx into synaptic terminals. This has first been well described for acetylcholine release at neuromuscular junction which was found to be inhibited by Cd in a Ca++ dependent manner (Cooper and Manalis, 1984; Toda, 1976). An additional mechanism of Cd interference with neurotransmission is the direct interaction of this ion with neurotransmitter receptors. This has been shown in the case of acetylcholine receptors which have been found to be activated by Cd in a subunit dependent manner (Hsiao et al., 2001), of GABA-A receptors, which, on the contrary, are inhibited by Cd in a subunit dependent way (Casagrande et al., 2003; Fisher and Macdonald, 1998). In addition, Cd blocks glycine (Wang et al., 2006) and kainite (Braitman and Coyle, 1987), metabotropic (Vignes et al., 1996) and glutamate receptors.

2.2.4 Lithium: Mechanism of action—Role of the phosphatidylinositol (PI) system and regulation of intracellular calcium

Calcium plays numerous roles in neurons including acting as a second messenger in cell bodies, triggering neurotransmitter release, mitochondrial function as well as plasticity and cell death (Alda, 2015). Several of lithium's mechanisms of action impact calcium metabolism, which may go a long way towards explaining the therapeutic specificity of lithium in treating bipolar disorder as altered intracellular calcium levels are a consistent finding in studies of bipolar disorder (Kato, 2008). Lithium inhibits inositol monophosphatase which ultimately leads to lowered levels of inositol-1,4,5-triphosphate (IP3). IP3 facilitates calcium release from endoplasmic reticulum stores and thus lower levels of IP3 decreases calcium burden on mitochondria potentially offsetting glutamate-induced calcium influx. The phosphoinositide signaling system also interacts with protein kinase C which contributes significantly to epigenetic and mitochondrial control (Strakowski, 2014).

3.15.4.1.3 Synaptic transmission

There is no direct physical connection between neurons, but they are close enough to communicate discontinuously. This discontinuous site of communication named after synapses which are the main structure that enable cell to cell communication among neurons or neuron to target cell. In typical neuronal communication in the CNS, chemical synapses play a very important role in transmitting the signals (Fig. 11). Electrical synapses [78] are not important for synaptic transmission in the CNS. However, it is by electrical synapses that action potentials are transmitted from one cell to the next in smooth and cardiac muscles. In electrical synapses, signals can be transmitted in either direction. However, chemical synapses always transmit the signals in one direction which is a very important and desirable feature for neurotransmission in the CNS.

Although electrical synapsis is a very important mechanism in cell to cell communication in smooth and cardiac muscles, the chemical synapsis is the major event in synaptic transmission. There are five major steps in the chemical synapses for transmission of signals: (1) synthesis of neurotransmitter, (2) neurotransmitter storage in synaptic vesicle (quanta), (3) release of the neurotransmitter to the synaptic space, (4) binding of the neurotransmitter to the specific receptors on postsynaptic cell membrane, and (5) generation of a new action potential in the postsynaptic neuron. The chemicals that transmit information between neurons are neurotransmitters. They are generally produced in the axon terminals (monoaminergic neurotransmitters) and in the cell body (peptidergic neurotransmitters). In the axon terminals, neurotransmitters are stored in the synaptic vesicles. The mechanism of neurotransmission in the chemical synapses is illustrated in Fig. 12. The first step in neurotransmission begins with the arrival of the action potential to the axon terminal in the presynaptic neuron. This rapid change in membrane potential leads to opening of the voltage-gated Ca+2 channels in the presynaptic nerve terminal. Through the concentration gradient between the extracellular and the intracellular compartments, the Ca+2 ions rapidly diffuse into the presynaptic nerve terminal. The elevated intracellular concentration of the Ca+2 ions initiates a signaling cascade, which results in release of the synaptic vesicles. Ca+2 ions cause fusion of the synaptic vesicles with the presynaptic cell membrane, and then the neurotransmitters are released into the synaptic cleft by exocytosis. A neurotransmitter binds to its specific receptor in the postsynaptic membrane that opens a ligand-gated Na+ channel, and Na+ ions diffuse into the neuron. Rapid diffusion of Na+ ions into the cytoplasm generates an action potential in the postsynaptic neuron. Once a neurotransmitter binds to its target (receptor), the remaining ones are degraded by enzymes in synaptic area to prevent postsynaptic cell to receive another redundant signal.

Most of the knowledge regarding the molecular structure of the chemical synapses come from the experiments related with neurotransmitter release at neuromuscular junctions during information transmission from a motor neuron to a skeletal muscle fiber. As discussed earlier, a neurotransmitter is released from quanta in response to an action potential in the presynaptic nerve terminal. The neurotransmitter released into a synapse is not exactly the same in amount in response to each stimulus. Although a typical synapse releases two vesicles per action potential, the actual number may differ depending on the probabilistic nature of the presynaptic neuron. It may be more, less or none in response to the next one. The average number of quanta and the quantum response size can be estimated. The quantitative approach of response called the “quantum hypothesis of Ca+2 release” or “quantal neurotransmitter release.” The earliest studies to understand the mechanism of the chemical synapses were experimented by Fatt and Katz [79] in neuromuscular junctions in frogs. In a neuromuscular junction, microelectrode studies showed that an action potential of a presynaptic cell leads to depolarization of a postsynaptic muscle fiber, called end-plate potential (EPP). The EPPs are large enough to initiate an action potential in a postsynaptic target and eventually cause muscle contraction. At a no action potential receiving presynaptic nerve ending, some quanta may spontaneously release neurotransmitters with a little depolarizing activity on the postsynaptic muscle fiber. This spontaneous potential called the miniature end-plate potential (MEPP). The MEPPs occur independently from an action potential and have the same shape in the potential-time scale with EPPs. Quantal analyses are very straight and reliable tools to understand the synaptic mechanism and plasticity. Synaptic plasticity is a very important feature of the neurons and basis of establishing neural networks. The synaptic plasticity contributes to learning and memory (cognitive functions) in the human brain [80]. To understand cognitive functions and underlying mechanisms in the human brain, synaptic plasticity need to be investigated. Statistical analysis has great importance as a quantitative study of chemical synapses. They provide valuable information about the probability of quanta release and size of the neurons. The evoked none spontaneous quanta release may be studied either with binomial or Poisson distribution models. In Binomial description of the probability of the quanta release to a response to action potential of presynaptic nerve cell, (n) stands for the number of quanta available in a nerve ending, each have a low (p) probability to release and number quanta which is not released is (q=1−p). The mean number of the quanta released quantitatively calculated by multiplying number of quanta available in nerve ending (n) and probability of the release (p), i.e., (m=n×p). The probability of the release of 3 quanta is p3. The probability of no quanta release become q3. The probability of only first or only second or only third in a three becomes 3q2p and the probability two of them released is 3p2q. When there are x quanta, with a release probability of (p) and no released probability of (q) binomial description is represented as:

(18)P(x)=n!(n−x)!x!pxq(n−x)

Consider a neuron terminal end that have n=3 number of quanta and each have p=0.1 probability to release neurotransmitter and no release q=1−p probability neurotransmitter and suppose there is no failure that mean number of quanta (m):

(19)m=n×p= 3×0.1=0.3

The three quanta release neurotransmitter became as:

(20)P(3)=p×p×p=p3=0.001

Similarly, no quanta release became as:

(21)P(0)=q×q×q=q3=0.729

Depending on probability rules on one quanta release at a time:

(22)P(1)=p×q×q+p×q×q+p×q×q=3pq2=0.243

The probability of two of three quanta release:

(23)P(2)=p×p×q+p×p×q+p×p× q=3p2q=0.027

P(x) also coefficients of polynomial expansion it becomes

(24)0.001+0.729z+0.243z2+0.027z3

where z is the dummy variable that have no function only used for coefficient calculation.

In the Poisson description of the probability of the quanta release as a response to action potential of presynaptic nerve cells (n) is very large, (p) is very small, and (m) is the mean quanta release. The mean number of the quanta is reached only from division of the mean EPP amplitude to mean MEPP amplitude. Then the Poisson description is represented as:

(25)P(x)=m xx!exp(−m)

Consider n becomes very large while p is very small probability starting from 0. When n try to go ∞ binomial model is limited. Suppose n=3 and p=0.4 and m becomes m=1.2. Now quanta release probability to observe 0, 1, …, can be solved by Poisson model (Eq. 25 ) where

(26)P(0)≅0.30P(1)≅0.36P (2)≅0.21P(3)≅0.08…

The importance of the quantum analysis can be pointed in the view of the synaptic energy management. Brain signaling relies on ATP consumption. Among the other energy consuming items; resting membrane potential, action potential and neurotransmitter recycling, pre- and postsynaptic mechanisms are the major processes that consumes nearly 50% of the total brain energy. The low probability (p) of neurotransmitter release in the synaptic transmission from the presynaptic terminal onto the postsynaptic neuron provides a dynamic behavior in enhancement transmission and/or storage of information to maximum level. The reaction of the postsynaptic cell depends on this low probability of release, and there is also a link between the energy usage and the probability of the release. The probability of release varies depending on the history of neuron. Lennie showed that increasing the probability of the release increases the energy utilization of the synapses [10]. In other words, energy consumption can be reduced by lowering the probability of release, in rat neocortex, energy used per spike is doubled when the probability of release increases from 0.25 to 1.00 [58]. Synaptic energy is supplied mainly by the mitochondrial energy metabolism, rather than glycolysis. In an adult brain, ATP is generated via aerobic respiration by direct oxidation of glucose. Mitochondrial oxidative phosphorylation produces nearly 93% of the total ATP [1]. Morphological studies showed that most of the mitochondria are highly localized in the pre- and postsynaptic areas, and consequently most of the ATP is utilized in these sites. The quantal analysis is very important to understand the energy management during synaptic transmission.

3.7.5 Neuronal-type nicotinic acetylcholine receptor antagonist

Nicotinic acetylcholine receptors (nAChRs) are ligand gated cation channels located mainly at presynaptic sites where they modulate neurotransmitter release, cell excitability and neuronal integration. They are named according to the endogenous neurotransmitter (acetylcholine) or exogenous ligand (nicotine) binding to them.94 Studies have shown that activation of nAChRs results in the release of multiple neurotransmitters in the spinal cord that activates descending inhibitory pathways of the brainstem. Involvement of these receptors in pain was first discovered when epibatidine (18), an alkaloid from Ecuadoran frog skin, showed its analgesic action partly through its action at nAChRs.95 Based on the structures of nicotine or acetylcholine, several nAChRs antagonists were developed for the management of pain. ABT-594 (19), a 3-pyridyl ether, designed on the basis of the structure of nicotine is now in phase II clinical trial for the treatment of peripheral neuropathic pain.95,96 Although a good number of nAChRs agonist and antagonist natural product has been reported so far, they lack their specificity and affects both muscle-type and neuronal-type nAChRs.97 The α-conotoxin class of cone snail venom peptides show analgesic action through their highly specific antagonistic action against neuronal-type nAChRs and represents a potential candidate for new analgesic drug development.27

Summary

Calcium plays a fundamentally important role in regulating cellular functions, exemplified by that which occurs in neurons, ranging from neurotransmitter release to synaptic plasticity to gene transcription. The calcium binding protein calmodulin plays the role of transducing increased cytoplasmic calcium to the activation of downstream enzymes such as the family of CaM-kinases. This family of enzymes phosphorylates Ser/Thr residues in substrate proteins, altering their function to regulate most every biochemical process in cells. Many of the CaM-kinases are themselves substrates for phosphorylation and in some cases, this phosphorylation is essential for their activity or for their biological function. Thus this family of enzymes forms an enzymatic network that leads to temporal integration of the calcium signal via the intermediary protein, calmodulin. Certain members of the CaM-kinase family can phosphorylate a variety of substrates providing the biochemical network a means of integrating well-coordinated responses through activation of a few key molecules that lie as hubs in the signaling network. Importantly, several of these phosphorylation events lead to activity that outlives the calcium signal that initiated phosphorylation providing a theoretical mechanism for short- and long-term plasticity in neurons; a property that can extend to signaling integration in all cells.