Objectives
- 1.
Describe the structure and function of electrical synapses.
- 2.
Describe the structure of a representative chemical synapse.
- 3.
Explain the quantal nature of neurotransmitter release.
- 4.
Describe the mechanism of transmitter release and the role of calcium.
- 5.
Describe the synaptic vesicle cycle.
- 6.
List the mechanisms that underlie short-term synaptic plasticity.
The synapse is a junction between cells that is specialized for cell-cell signaling
In Section II, we learned how the action potential (AP) is generated and conducted in neurons and muscle cells. The critical issue in the nervous system is to get the right signal to the right place in the body at the right time. A key question then is, “How is the signal communicated from cell to cell, that is, from neuron to neuron, or from neuron to neuroeffector (muscle or gland) cell?” The intercellular junction through which the signals are transmitted is called the synapse , a
Charles Sherrington, the physiologist who coined the term synapse in the late 19th century, was a recipient of the 1932 Nobel Prize in Physiology or Medicine for his seminal work on spinal reflexes.
and the communication across this junction is therefore called synaptic transmission. In this section (Chapters 12 and 13 ), we elucidate the cellular and molecular mechanisms that underlie synaptic transmission.Approximately 100 billion neurons are present in the human brain. Moreover, neurons branch like trees, and the average neuron has approximately 1000 branches, each ending in a small swelling, the presynaptic portion of the synapse, which is known as the presynaptic terminal or synaptic bouton . Thus the human nervous system has on the order of 100 trillion (10 14 ) synapses! Adding to the complexity is the fact that most neurons receive inputs from multiple neurons. The average neuron receives many more than 1000 synaptic inputs; indeed, a cerebellar Purkinje neuron may receive as many as 200,000! These neurons and synapses play essential roles in an enormous number of bodily activities from the control of respiration, blood circulation, and renal and gastrointestinal function to sensory perception, body movements, and learning and memory. Our task here is to understand the mechanisms by which neurons communicate with one another.
Synaptic transmission can be either electrical or chemical
In the 19th century, the classic morphological studies of Santiago Ramón y Cajal demonstrated that the nervous system, like other organs, is composed of cells (the neuron doctrine ). b
Cajal and Camillo Golgi shared the 1906 Nobel Prize in Physiology or Medicine for their seminal work on neuronal structure. Ironically, Golgi, whose staining methods proved crucial for elucidating neuronal structure, favored the idea that the nervous system was a continuous reticulum rather than a network of discrete cells.
During the late 19th and early 20th centuries there was fierce debate over two divergent views of synaptic transmission, dubbed the “war of soups and sparks.” aValenstein ES: The war of the soups and the sparks: the discovery of neurotransmitters and the dispute over how nerves communicate, New York, 2005, Columbia University Press.
As a result of the demonstration that nerves and muscle cells conduct electrical signals, one popular idea was that an electric “spark” at the end of a presynaptic neuron directly triggered the electrical signal in the postsynaptic neuron or muscle cell (i.e., synaptic transmission was thought to be purely electrical). Conversely, studies on the paralytic action of curare , bCurare, or D-tubocurarine, is an alkaloid toxin from the bark of a South American liana vine.
and on the autonomic nervous system , hinted at the idea of chemical transmission.The discovery of chemical synaptic transmission, and recognition that most synapses are chemical, nearly led to the demise of the concept of electrical transmission. Nevertheless, some synapses in the mammalian central nervous system (CNS) are electrical. We will consider the mechanism of transmission at electrical synapses before turning to the more prevalent and diverse chemical synapses.
Electrical synapses are designed for rapid, synchronous transmission
Chemical and electrical synapses have distinct morphological features that are related to their differing functional properties. Electrical synapses are designed to allow current to flow directly from one neuron to another. At electrical synapses, the presynaptic and postsynaptic membranes are separated by only 3 to 4 nm ( Fig. 12.1 A). At these narrow gaps the two neurons are connected by gap junction channels, each of which consists of two hemichannels: one in the presynaptic and one in the postsynaptic membrane. Each hemichannel, called a connexon , is an annular assembly of six polypeptide subunits, called connexins . The connexon in the presynaptic membrane docks face-to-face with a connexon in the postsynaptic membrane to form a conducting channel that connects the cytoplasm of the two neurons ( Fig. 12.1 B). Gap junction channels allow the passage of nutrients, metabolites, ions, and other small molecules (≤1000 daltons). More than 20 connexin isoforms have been identified, and mutations in about half of the genes that encode these proteins are linked to human disease ( Box 12.1 ).
Mutations in about half of the genes that encode the connexin family of proteins have been linked to several diseases. In some of these diseases, the connexin mutations result in dysfunctional gap junctions between glial cells. Mutations in the gene encoding connexin-32 (Cx32), for example, are associated with the X-linked form of Charcot-Marie-Tooth disease, one of the most common hereditary neurological disorders. Charcot-Marie-Tooth disease is a motor and sensory neuropathy characterized by muscle weakness and various sensory defects. Many of the Cx32 mutants fail to form functional gap junctions between Schwann cells, and this leads to demyelination and axonal degeneration. Recessive mutations in the gene encoding connexin-47 (Cx47) are linked to Pelizaeus-Merzbacher–like disease, a rare disorder characterized by lack of CNS myelin development. The Cx47 mutants also fail to form functional gap junction channels. Mutations in Cx26 are implicated in deafness. This connexin is normally expressed in the nonsensory epithelial cells in the cochlea, and not in the hair cells. The exact function of Cx26 in the cochlea is unknown, but it has been proposed to play a role in the recycling of K + .
In the majority of connexin mutants that have been studied, the altered connexin subunits reach the cell surface and form gap junction-like structures. However, these structures either are nonfunctional or they form channels that function poorly compared with normal gap junction channels. In another class of mutants, the altered connexin subunits are retained in the endoplasmic reticulum and never reach the cell surface.
The first description of electrical synaptic transmission was based on studies of the crayfish giant motor synapse. In this preparation, the presynaptic and postsynaptic axons are large enough to allow placement of intracellular stimulating and recording electrodes close to the synapse. These experiments demonstrated that an AP in the presynaptic neuron produces a depolarization in the postsynaptic neuron after a negligible synaptic delay ( Fig. 12.2 ), which is much shorter than the delay at chemical synapses. Such nearly instantaneous transmission can be caused only by direct current flow between the cells. This current flows from the presynaptic cell through the gap junction channels and into the postsynaptic cell . Such direct flow of current does not occur at chemical synapses. Most electrical synapses are bidirectional: signals can be transmitted from either one of the connected cells to the other. In contrast, chemical synapses are unidirectional. The conductance of gap junction channels is regulated by two distinct gating mechanisms ( Box 12.2 ).
The conductance of gap junction channels is physiologically regulated. This is accomplished through channel gating, and at least two distinct gating mechanisms operate within each gap junction hemichannel. The first is Vj-gating, which depends on the junctional voltage ( V j ) across the gap junction. Vj-gating is responsible for rapid transitions between high and low conducting states of the channel. The low-conductance state that is entered as a result of Vj-gating does not completely close the channel. Hemichannels formed by some connexin isoforms close with depolarization; others close with hyperpolarization. The second type of gating mechanism involves slow transitions (10–30 msec) between the fully open and fully closed states. These slow transitions can be mediated by three distinct processes. First, slow transitions can occur in response to changes in voltage: this is called loop gating because it involves the extracellular loops that connect adjacent transmembrane domains in connexin. The loop gating voltage sensor and the Vj-gating voltage sensor are independent structures. Second, slow transitions can be caused by changes in pH or Ca 2+ ; this is called chemical gating. In cells that are normally coupled electrically and metabolically through gap junctions, an increase in [Ca 2+ ] i or a decrease in pH can close gap junction channels and uncouple the cells. This can serve as a protective mechanism, uncoupling damaged cells, which have elevated [Ca 2+ ] i or [H + ] i , from healthy cells. Finally, slow transitions can be mediated by the docking or undocking of two hemichannels.
Electrical synapses between neurons in the mammalian CNS play a role in neuronal synchronization because they allow the direct, bidirectional flow of current from one cell to the other. For example, electrical synapses coordinate spiking among clusters of cells in the thalamic reticular nucleus. Similarly, electrical synapses in the suprachiasmatic nucleus help synchronize spiking that may be necessary for normal circadian rhythm. Direct electrical communication between cells is also physiologically important outside the nervous system: for example, gap junction channels between heart cells enable the cells to depolarize and contract synchronously ( Chapter 14 ).
Most synapses are chemical synapses
At chemical synapses , the AP in the presynaptic nerve terminal releases neurotransmitter molecules that generate electrical or biochemical signals in the postsynaptic cells. Early in the 20th century, Henry Dale and Otto Loewi obtained critical evidence that dispelled doubts about chemical transmission. Dale showed that acetylcholine (ACh) was the most potent agent capable of mimicking parasympathetic nerve activation. His observation that the effects of ACh, injected into the bloodstream, were very rapid but short-lived led him to suggest that ACh was rapidly hydrolyzed. This presaged the discovery that the enzyme acetylcholinesterase terminates ACh action at synapses. Loewi subsequently demonstrated that stimulation of the vagus nerve to a frog heart released a substance into the bathing solution. When a different frog heart was immersed in this bathing solution, its rate slowed. a
Dale and Loewi shared the 1936 Nobel Prize in Physiology or Medicine for the discovery of chemical neurotransmission.
This chemical neurotransmitter, released by the vagus nerve, was later shown to be ACh. These discoveries laid the foundation for most of neuropharmacology and neurotherapeutics: agents that stimulate neurotransmitter release, mimic neurotransmitters, or interfere with their actions are among the most useful tools in the physician’s arsenal.The application of electron microscopy and ultracentrifugation methods in the 1950s and 1960s led to important advances in understanding the structure and chemistry of synapses. Fig. 12.3 shows key structural features of a representative chemical synapse. The presynaptic terminal contains many small (∼40 nm diameter) round structures, the synaptic vesicles (SVs) , which contain high concentrations of neurotransmitters. SVs tend to concentrate at or near the active zone , a specialized region of the presynaptic plasma membrane (PM) that is involved in transmitter release. This region is closely apposed to the postsynaptic cell, with its own postsynaptic density region that is enriched with neurotransmitter receptors ( Chapter 13 ). At the synapse, the two cells are separated by a synaptic cleft 20 to 40 nm wide.
Neurons communicate with other neurons and with muscle by releasing neurotransmitters
When a nerve AP is conducted down the axon to the presynaptic terminal, the resulting depolarization triggers the Ca 2+ -dependent release of SV contents into the synaptic cleft. This process of exocytosis involves fusion of the SV membrane with the PM and the consequent emptying of the vesicular contents into the synaptic cleft. The SV membrane is then recycled.
Released neurotransmitter molecules diffuse across the synaptic cleft and interact with specific receptor molecules that are integral proteins in the PM of the postsynaptic neuron or neuroeffector cell. The interaction between the transmitter and its receptor can be characterized as a lock-and-key mechanism in which the transmitter (key) unlocks the receptor. This activates the receptor so that, depending on the receptor type ( Chapter 13 ), it either directly affects membrane conductance in the postsynaptic cell or, alternatively, initiates an intracellular signaling cascade that regulates a wide range of cellular processes including membrane conductance changes. These mechanisms modulate the postsynaptic neuron’s excitability (i.e., the ability to fire an AP).
Most synapses in the mammalian nervous system are chemical synapses. The number of presynaptic neurons that synapse onto one postsynaptic cell varies widely. For example, one skeletal muscle fiber is usually innervated by only a single motor neuron, whereas each motor neuron usually innervates more than one muscle cell. In contrast, as already noted, many presynaptic neurons may synapse onto a single postsynaptic neuron. Synapses are not static structures: new synapses can form, synaptic connections can be strengthened or weakened, and some synapses can be eliminated. This flexibility contributes to the enormous complexity, rich diversity, and remarkable plasticity of synaptic transmission that underlies higher brain function.
The neuromuscular junction is a large chemical synapse
The large synapse formed between a spinal motor neuron and a skeletal muscle fiber is called the neuromuscular junction (NMJ) . Studies of neuromuscular transmission by Bernard Katz a
a Katz shared the 1970 Nobel Prize in Physiology or Medicine for this work.
and his collaborators greatly enriched our understanding of how chemical synapses work. The axon of the motor neuron contacts the muscle fiber at a region called the end plate ( Fig. 12.4 ). As the axon approaches the muscle it divides into several small branches, and each branch terminates in a knoblike swelling, the synaptic bouton.
Each synaptic bouton contains numerous SVs filled with the neurotransmitter ACh. The vesicles cluster around active zones. At the NMJ, the synaptic boutons are separated from the postsynaptic membrane by a 100-nm synaptic cleft, which is wider than the synaptic clefts between neurons (typically, ∼20–40 nm). Within the NMJ cleft is a basement membrane that anchors the enzyme acetylcholinesterase. This enzyme hydrolyzes ACh and thereby helps limit the duration of ACh action. Each active zone in each synaptic bouton lies directly opposite a junctional fold, which is a deep invagination of the muscle cell membrane ( Fig. 12.4 ). A high density of ACh receptors (AChRs; ∼20,000/μm 2 ) is localized near the top of each junctional fold. These NMJ AChRs, which are multimeric nonselective cation channels, are also activated by nicotine, the addictive drug from the tobacco plant—hence the name nicotinic AChRs (nAChRs) .
To study neuromuscular transmission, a muscle and its attached nerve (e.g., the diaphragm and phrenic nerve) can be removed and placed in an experimental chamber for recording. Stimulating electrodes are placed on the nerve trunk to initiate APs, and microelectrodes are placed in the muscle cell at the end-plate region to measure changes in V m . After an AP in the presynaptic neuron, a transient depolarization, called the end-plate potential (EPP) , occurs in the muscle cell ( Fig. 12.5 ). The EPP is normally large enough to reach threshold for generating an AP in the skeletal muscle cell. To study the time course of the EPP, its size must be reduced to less than the AP threshold. This can be accomplished by lowering [Ca 2+ ] o and thus reducing the amount of transmitter released ( Chapter 13 ), or by blocking some of the nAChRs (e.g., with curare). Under these conditions the EPP has a rapid rising phase and a slower exponential decay ( Fig. 12.5 ). The rapid depolarization results from the sudden release of ACh from the presynaptic nerve terminal in response to the AP. The ACh diffuses rapidly across the synaptic cleft and binds to the postsynaptic nAChRs. The binding of two molecules of ACh opens the nAChR channel gate to conduct inward current, thereby depolarizing the muscle fiber (the postsynaptic cell of the NMJ). As the ACh diffuses away and is hydrolyzed by acetylcholinesterase, the concentration of ACh in the synaptic cleft quickly declines to zero, even before the nAChRs close. The slow exponential decline in the EPP largely reflects the rate of closure of the nAChR channels. Some diseases of neuromuscular transmission are caused by defective ACh release, others involve impaired hydrolysis of ACh, and several result from defects in the nAChR channel ( Chapter 13 ).
The significant synaptic delay between the arrival of the presynaptic AP at the nerve terminal and the beginning of the postsynaptic response ( Fig. 12.6 ) is a characteristic of all chemical synapses. The following events all contribute to the synaptic delay: (1) the presynaptic AP causes voltage-gated Ca 2+ channels (VGCCs) to open; (2) Ca 2+ enters the cell through the open Ca 2+ channels and triggers neurotransmitter release; and (3) the neurotransmitter rapidly diffuses across the synaptic cleft, binds to postsynaptic receptors, and opens ion channels in the postsynaptic membrane. The opening of VGCCs is the slowest process and thus the major contributor to the synaptic delay. The synaptic delay at chemical synapses contrasts with the near-absence of synaptic delay at electrical synapses ( Fig. 12.2 ).
Transmitter release at chemical synapses occurs in multiples of a unit size
Neurotransmitters are released in discrete packets, called quanta . Initial evidence for this was obtained by Katz and his colleagues from electrical recordings at the NMJ. Small spontaneous depolarizations of the muscle cell can be observed, even in the absence of presynaptic APs ( Fig. 12.7 ). These spontaneous depolarizations have many features in common with the EPPs but are normally much smaller in amplitude and are therefore called miniature end-plate potentials (MEPPs) . MEPPs are identical in time course to the EPP, with a rapid rising phase and a slower exponential falling phase. Like EPPs, MEPPs are largest when recorded at the end-plate region of the muscle cell. Both signals are reduced in amplitude by drugs (e.g., curare) that block nAChRs, and both are augmented by drugs that interfere with ACh hydrolysis. MEPP amplitude is approximately 2000 times larger than the depolarization resulting from the opening of a single nAChR. Because two molecules of ACh are required to open each channel, and not all the released ACh binds to postsynaptic receptors, each quantum must contain more than 4000 molecules of ACh. In fact, investigators have shown that approximately 5000 to 10,000 molecules of ACh are required to produce an MEPP. This implies that, in an SV with an outer diameter of 40 nm, the ACh concentration could be as high as 500 mM.
The presynaptic AP triggers release of neurotransmitters in quantal packets that are identical in size to the spontaneously released quanta. This can be demonstrated by studying neuromuscular transmission after decreasing [Ca 2+ ] o to reduce the EPP amplitude from its normal size of approximately 70 mV to approximately 1 to 2 mV in amplitude. The EPP size then fluctuates randomly from one stimulus to the next ( Fig. 12.7 ). Occasionally, nerve stimulation elicits no EPP; this is called a failure. After recording the responses to many stimuli, the number of EPPs of a given amplitude can be plotted in a histogram ( Fig. 12.8 ). The amplitude distribution shows that EPP amplitudes occur in integer multiples of the smallest EPP amplitude, and the smallest EPP is identical in size to the spontaneous MEPP amplitude ( Box 12.3 ). Thus both spontaneous and nerve-evoked release of neurotransmitter at the neuromuscular junction are quantal.
The EPP amplitude histogram ( Fig. 12.8 ) shows several peaks in the distribution. The first peak, at 0 mV, represents the number of failures. The next peak is centered at 0.4 mV, which is the same as the mean miniature end-plate potential (MEPP) amplitude and thus reflects the nerve-evoked release of a single quantum of transmitter. The other peaks in the distribution occur at integer multiples of 0.4 mV. This suggests that the second peak results from the release of two quanta, the third peak results from the release of three quanta, and so on. The smooth curve drawn over the amplitude histogram is a theoretical distribution based on quantal release, and it clearly gives a good fit to the data.
The number of events in one of the peaks of the amplitude distribution divided by the total number of events is an estimate of the probability that the corresponding number of quanta are released. For example, the peak centered at 0.8 mV represents the release of two quanta. Thus the number of events in this peak divided by the total number of events is a measure of the probability that two quanta are released in response to an action potential. If the release of a quantum of transmitter is an independent, random event, then this probability should fit a binomial distribution. A binomial distribution describes a process in which an experimental trial results in two possible outcomes, success or failure. A binomial distribution has two parameters: p , the probability of success (i.e., the release of a quantum), and n , the number of “trials” (i.e., the number of sites that can release a quantum). Using a binomial distribution, the probability that x quanta will be released when n sites are available can be calculated as follows:
P(x:n)=n!x!(n-x)!px(1-p)n-x
By fitting a binomial distribution to the data in the EPP amplitude distribution we can estimate p and n . The product of p and n is the mean number of quanta released, and this is referred to as the quantal content . The quantal content can be as high as 300 at the NMJ or as low as 1–10 at some CNS synapses. The probability of release, p , is as high as 0.7–0.9 at the NMJ and as low as 0.1 at some central synapses, and n ranges from 1000 at the NMJ to 1 in the CNS.
SVs are the morphological correlates of the physiological quanta. Each vesicle stores one quantum of ACh, and the entire content of the vesicle is released by exocytosis when the vesicle fuses with the presynaptic membrane at the active zone. Quantal release also has been demonstrated at a variety of CNS chemical synapses. The most extensively studied is the calyx of Held, an unusually large glutamatergic a
a The suffix – ergic is used to describe a neuron or synapse that uses a particular neurotransmitter. For example, a glutamatergic neuron releases glutamate, and neurotransmission at a glutamatergic synapse is activated by glutamate. Neurons or synapses that use dopamine, noradrenaline, and acetylcholine are described as, respectively, dopaminergic, noradrenergic, and acetylcholinergic (usually shortened to “cholinergic”).
synapse in the brainstem. Glutamate is stored in SVs in the presynaptic terminal, and release of the content of a single SV evokes a miniature excitatory postsynaptic current (EPSC) in the postsynaptic neuron. The quantal analysis used to describe transmitter release at the NMJ ( Box 12.3 ) is also used to describe glutamate release at the calyx of Held. Spontaneous and AP-evoked release are both quantal and Ca 2+ -dependent. At the frog NMJ, approximately 100 to 300 quanta, is normally released in response to a presynaptic AP. Depending on stimulation frequency, from 10 to a few hundred quanta may be released at the calyx of Held. In stark contrast, at most CNS synapses, which are much smaller, presynaptic APs often fail to trigger neurotransmitter release, and when release is triggered successfully, only one or two quanta are released.Ca 2+ is essential in transmitter release
As just described, lowering [Ca 2+ ] o decreases the size of the EPP. The smaller size results from the fact that fewer quanta are released in the presence of low [Ca 2+ ] o . Because transmitter release is an intracellular process, this result implies a pathway for Ca 2+ entry into the presynaptic neuron. Additional insights into how Ca 2+ regulates transmitter release were obtained from studies of the squid giant synapse, whose large size has the distinct advantage of allowing both presynaptic and postsynaptic neurons to be voltage-clamped. Application of this method provided direct evidence for the existence of VGCCs in presynaptic membranes. These studies showed that the amount of transmitter that is released depends on the amount of Ca 2+ that enters the cell; blocking VGCCs abolished transmitter release. These and similar studies at the NMJ and other synapses have elucidated the essential role of VGCCs in quantal release of neurotransmitters. Subsequent studies, including some with neurotoxins from spiders and predatory snails ( Box 12.4 ), revealed that two subtypes of VGCCs are involved in transmitter release in neurons: P/Q- and N-type Ca 2+ channels ( Chapter 8 ).
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