8 Transmitters and receptors

Electrical Synapses

Electrical synapses are scarce in the mammalian nervous system. As seen in Figure 8.1, they consist of gap junctions (nexuses) between dendrites or somas of contiguous neurons, where there is cytoplasmic continuity through 1.5-nm channels. No transmitter is involved, and there is no synaptic delay.

Figure 8.1 Structure of an electrical synapse. (A) Synaptic contact between two dendrites. (B) Enlargement from (A). (C) The gap junction between the cell membranes is bridged by close-packed ion channels. (D) Each ion channel comprises a mirror image pair of connexons. (E) Each connexon comprises six connexins, each having a wedge-shaped subunit bordering the ion channel. (F) The subunits open the ion channel by synchronous rotation.

The gap junctions are bridged by tightly packed ion channels, each comprising mirror image pairs of connexons, which are transmembrane protein groups (connexins) disposed in hexagonal format around an ion pore. Wedge-shaped connexin subunits bordering each ion pore are closely apposed when the neurons are inactive. Action potentials passing along the cell membrane cause the subunits to rotate individually, creating a pore large enough to permit free diffusion of ions and small molecules down their concentration gradients.

The overall function of these gap junctions is to ensure synchronous activity of neurons having a common action. An example is the inspiratory center in the medulla oblongata, where all the cells exhibit synchronous discharge during inspiration. A second example is among neuronal circuits controlling saccades, where the gaze darts from one object of interest to another.

Chemical Synapses

Transmitter liberation (Table 8.1 and Figure 8.2)

At resting nerve terminals, synaptic vesicles accumulate in the active zones, where they are tethered to the presynaptic densities by strands of docking proteins including actin. With the arrival of action potentials, myriads of voltage-gated calcium channels located in the presynaptic membrane are opened, leading to instant flooding of the active zone with Ca²⁺ ions. These ions cause contraction of actin filaments to bring the nearest vesicles into direct contact with the presynaptic membrane.

Table 8.1 Some named proteins involved in transmitter transport and vesicle recycling

Named protein	Function
Actin	Brings vesicle into contact with presynaptic membrane
Synaptophysin	Creates the membrane fusion pore
Calmodulin	Expels vesicle content into synaptic cleft
Clathrin	Withdraws vesicle membrane from synaptic cleft
Dynamin	Pinches the neck of the developing vesicle to complete its separation
Ligand	Receptor protein that binds with the transmitter molecule

Figure 8.2 Sequence of events following depolarization of the presynaptic membrane. (1) Opening of calcium channels (arrows) causes synaptic vesicles to be pulled into contact with the presynaptic membrane by actin filaments. Matching pairs of fusion protein macromolecules (FPMs) in the vesicle and presynaptic membrane are aligned. (2) The FPMs separate (outward arrows), permitting transmitter molecules to enter the synaptic cleft. (3) Vesicle membrane is incorporated into the presynaptic membrane while transmitter is activating the specific receptors. (4) Clathrin molecules assist inward movement of vesicle membrane. Dynamin molecules (green) assist approximation of FPM pairs (inward arrows) and pinch the neck of the emerging vesicle. (5) The vesicle is now free for recycling.

When the vesicle protein synaptophysin contacts the vesicle membrane, it becomes embedded in it and hollows out to create a fusion pore through which the vesicle discharges its contents into the synaptic cleft. The act of expulsion appears to be a function of another vesicle protein, calmodulin, activated by Ca²⁺ ions. These and other local protein responses to calcium entry are extremely rapid, the time elapsing between calcium entry and transmitter expulsion being less than 1 ms. In the case of small synaptic vesicles such as those containing glutamate or γ-aminobutyric acid (GABA), single spikes are sufficient to yield some transmitter release. In the case of peptidergic neurons, impulse frequencies of 10 Hz or more are required to induce typically slow (delay of 50 ms or more) transmitter release from the large, dense-cored vesicles.

Target cell receptor binding

Transmitter molecules bind with receptor protein molecules in the postsynaptic membrane. The two great categories of receptor are known as ionotropic and metabotropic. Each category contains some receptors whose activation leads to opening of ion pores and others whose activation leads to closure.

Ionotropic receptors

Ionotropic receptors are characterized by the presence of an ion channel within each receptor macromolecule (Figure 8.2). The transmitter binds with its specific receptor facing the synaptic cleft, causing it to change its conformation so as to open or close its ion pore. Ionotropic receptor channels are said to be transmitter-gated, or ligand-gated (from the Latin ligandum, ‘binding’), signifying their capacity to bind a transmitter molecule or a drug substitute.

In Figure 8.3A, the excitatory channel has been opened by the transmitter, causing a major influx of sodium and a minor efflux of potassium; the excitatory postsynaptic potential (EPSP) has depolarized the cell membrane almost to firing point. In Figure 8.3B, the membrane can be hyperpolarized only to −70 mV, the chloride equilibrium potential. A greater level of depolarization requires a second messenger system (see below).

Figure 8.3 (A) A transmitter-gated excitatory ionotropic receptor. Binding of the transmitter (red, in this example representing glutamate) has opened the pore of a ‘mixed’ Na⁺−K⁺ cation channel. A large influx of sodium ions has depolarized the membrane, as shown by the excitatory postsynaptic potential (EPSP). (B) A transmitter-gated inhibitory ionotropic receptor. Binding of the transmitter (blue, representing GABA_A) has opened the pore of a chloride channel. Inward chloride conductance has been increased, and the inhibitory postsynaptic potential (IPSP) restores the membrane potential to its resting state.

Ionotropic receptors are called fast receptors on account of their immediate and brief effects on ion channels.

Metabotropic receptors

Metabotropic receptors are so called because many are capable of generating multiple metabolic effects within the cytoplasm of the neuron. The receptor macromolecule is an intrinsic membrane protein devoid of an ion channel. Its function is initiated by one or other of two subunits (α subunit, β subunit) that detaches in response to transmitter activation and moves along the inner surface of the membrane. The subunits are called G proteins, because most bind with guanine nucleotides such as guanine triphosphate (GTP) or guanine diphosphate. Their action on ion channels is usually indirect, via a second messenger system. However, some G proteins do not bind with nucleotides; instead, they activate ion channels directly (see later).

A G protein having a stimulatory effect is known as a G_s protein; a G_i protein is one with an inhibitory effect.

Three second messenger systems are well recognized.

1 The cyclic AMP system, responsible for phosphorylation of proteins.

2 The phosphoinositol system, responsible for liberating calcium from cytoplasmic stores.

3 The arachidonic acid system, responsible for production of arachidonic acid metabolites.

Cyclic AMP system

(The abbreviation cyclic AMP represents cyclic adenosine monophosphate.) In the examples shown in Figure 8.4, transmitter–receptor binding releases the alpha subunit of a G_s protein, leaving it free to link with GTP, which in turn facilitates adenosine cyclase to convert ATP to cyclic AMP. Protein kinase A in the membrane is stimulated by cyclic AMP to transfer phosphate ions from ATP to an ion channel, causing its pore to admit sodium ions, initiating depolarization of the target neuron. When the G_s protein is switched off, the membrane-attached enzyme protein phosphatase catalyzes extraction of the phosphate ions, resulting in pore closure.

Figure 8.4 The cyclic AMP (cAMP) system. The diagram shows the basic steps along the path from a G_s protein-linked receptor via cyclic AMP to an ion channel. (1) Transmitter is activating the receptor macromolecule. (2) G_s protein α subunit is freed to bind with guanosine triphosphate (GTP). (3) GTP links the unit to adenylate cyclase. (4) Adenylate cyclase catalyzes synthesis of cyclic AMP from ATP. (5) Cyclic AMP activates protein kinase A (PKA). (6) PKA transfers phosphate groups from ATP to a sodium ion channel, causing its pore to open and Na⁺ ions to rush into the cytosol, with a depolarizing effect. (7) Following inactivation of the G_s protein, dephosphorylation of the channel by a phosphatase enzyme allows the channel pore to close.

Phosphoinositol system

In the example shown in Figure 8.5, activation of another kind of G_s protein alpha subunit causes the effector enzyme phospholipase C to split a membrane phospholipid (PIP₂) into a pair of second messengers: diacylglycerol (DAG) and inositol phosphate (IP₃). DAG activates protein kinase C, which initiates protein phosphorylation. IP₃ diffuses into the cytosol, where it opens calcium-gated channels, mainly in nearby membranes of smooth endoplasmic reticulum. The Ca²⁺ ions activate certain calcium-dependent enzymes downstream, and may open calcium-gated K⁺ (outward) and Cl⁻ (inward) channels with excitatory effect.

Figure 8.5 The phosphoinositol system. The steps indicate the dual function of this system. (1) The transmitter activates the receptor macromolecule. (2) The G_s protein α subunit is freed to bind with guanosine triphosphate (GTP), which links it to phospholipase C (PLC). (3) PLC moves along the membrane and splits the enzyme phospholipase C (PLC) into diacylglycerol (DAG) and inositol triphosphate (IP₃). (4) DAG attracts enzyme protein kinase C (PKC) into the membrane, where (5) DAG is triggered to phosphorylate several proteins, potentially including ion channels. (6) IP₃ activates calcium channels in sacs of smooth endoplasmic reticulum. (7) Stored calcium spills out of the channel pore into the cytosol. (8) Ca²⁺-dependent enzymes are activated.

Arachidonic acid system

This is described later, in connection with histamine.

Metabotropic receptors are in general slow receptors. Membrane channel effects may continue for hundreds of milliseconds after a single stimulus.

Gene transcription effects

It is also well established that the reflex responses to repetitive stimuli may be either progressively increased (a state of sensitization, usually induced by noxious stimuli) or diminished (a state of habituation induced by harmless stimuli). Animal experiments involving reflex arcs involving sets of sensory, internuncial, and motor neurons have shown that a characteristic of sensitization is the development of additional synaptic contacts between the internuncials and the motor neurons, together with additional transmitter synthesis and release. Characteristic of habituation is a reduction of transmitter synthesis and release. All these changes result from alterations of gene transcription. Repetitive noxious stimuli cause cyclic AMP to increase its normal rate of activation of protein kinases involved in phosphorylation of proteins that regulate gene transcription. The outcome is increased production of proteins (including enzymes) required for transmitter synthesis, and of other proteins for construction of additional channels and synaptic cytoskeletons. Repetitive harmless stimuli merely reduce the rate of transmitter synthesis and release.

Gene transcription effects are especially important in the context of forming long-term memories (Ch. 34).

Transmitters and Modulators

Several criteria should be fulfilled for a substance to be accepted as a neurotransmitter.

• The substance must be present within neurons, together with the molecules, including enzymes, required to synthesize it.

• The substance must be released following depolarization of the nerve endings that contain it, and this release must be induced by entry of calcium.

• The postsynaptic membrane must contain specific receptors that will modify the membrane potential of the target neuron.

• The isolated substance must exert the same effect when applied to a target neuron via a micropipette (microiontophoresis).

• Specific antagonist molecules, whether delivered through the circulation or by iontophoresis, must block the effect of the putative (‘thought to be’) transmitter.

• The physiologic mode of termination of the transmitter effect must be identified, whether it be by enzymatic degradation or by active transport into the parent neuron or adjacent neuroglial cells.

Many transmitters limit their own rate of release by negative feedback activation of autoreceptors in the presynaptic membrane, having the effect of inhibiting further release. Ideally, the existence of specific inhibitory autoreceptors should be established.

The term neuromodulator (L. modulare, to regulate) has been subject to several interpretations. The most satisfactory appears to derive from the terms amplitude modulation and frequency modulation in electrical engineering, signifying superimposition of one wave or signal onto another. Figure 8.6 represents a sympathetic and a parasympathetic nerve ending, close to a pacemaker cell (modified cardiac myocyte). This neighborly arrangement of nerve endings is common in the heart, and allows the respective transmitters to modulate each other’s activity. The sympathetic nerve ending liberates norepinephrine (noradrenaline), which has a stimulatory effect. The three modulators shown exert their effects via second messenger systems.

Figure 8.6 Neuromodulation occurs at nerve endings in the sinuatrial node of the heart, where sympathetic and parasympathetic nerve endings often occur in pairs. In this representation, the sympathetic system is the more active, releasing the transmitter norepinephrine, which depolarizes cardiac pacemaker cells via β₁ pacemaker membrane receptors. Circulating epinephrine exerts positive modulation on the nerve ending by increasing transmitter release via β₂ presynaptic membrane heteroreceptors. Inhibitory modulation of excess norepinephrine release is expressed via α₂ presynaptic membrane autoreceptors. At the same time, release of the inhibitory transmitter acetylcholine (ACh) from the parasympathetic bouton is inhibited via α₂ heteroreceptors.

The figure caption also refers to autoreceptors and heteroreceptors. Receptors for a particular transmitter often occur in the presynaptic as well as in the postsynaptic membrane. They are called autoreceptors. These are activated by high transmitter concentration in the synaptic cleft, and they have a negative feedback effect, inhibiting further transmitter release from the synaptic bouton. Heteroreceptors occupy the plasma membrane of neurons that do not liberate the specific transmitter. In the example shown, activity at sympathetic nerve endings is accompanied by inhibition of parasympathetic activity through the medium of heteroreceptors located on parasympathetic nerve endings.

Fate of neurotransmitters

The ultimate fate of transmitters released into synaptic clefts is highly variable. Some transmitters are inactivated within the cleft, some diffuse away into the cerebrospinal fluid via the extracellular fluid, and some are recycled either by direct uptake or indirectly via glial cells.

The principal transmitters and modulators are shown in Table 8.2. Respective receptor types are in Table 8.3.

Table 8.2 Main types of transmitters and modulators, with examples of each ^a The neuropeptides are modulators

Type	Example(s)
Amino acids	Glutamate
	γ-Aminobutyric acid (GABA)
	Glycine
Biogenic amines	Acetylcholine
	Monoamines
	Catecholamines (dopamine, norepinephrine [noradrenaline], epinephrine [adrenaline])
	Serotonin
	Histamine
Neuropeptides	Vasoactive intestinal polypeptide
	Substance P
	Enkephalin
	Endorphins
	Many others
Adenosine	—
Gaseous	Nitric oxide