Molecular and Cellular Signaling - Martin Beckerman

492 20. Neural Rhythms

tle back its production of cAMP. As a result the activation function shifts to more negative membrane potentials and the heart rate slows down.

20.3 Synchronous Activity in the Central

Nervous System

Electroencephalographic (EEG) recordings of wave patterns in the brain are a well-established tool for identifying abnormal conditions and various stages of sleep and arousal. Several different kinds of wave patterns can be produced in the brain, each characterized by a specific brain wave frequency range (Table 20.2). These patterns fall into two groups—low frequency and high frequency. The low frequency group includes delta waves, theta waves, spindling oscillations, and alpha waves. These patterns are of large amplitude when observed in the EEG, and as such are produced by large populations of neurons undergoing synchronized firing. The high frequency group includes the beta and gamma bands. The amplitudes of these rhythms are far lower that those of the low frequency waves, indicative of participation by smaller populations of neurons. These patterns are not as sharply delineated in the EEG recordings.

As indicated in Table 20.2, delta waves are associated with deep sleep and with abnormal brain states such as coma and anesthesia. Theta oscillations are most often associated with drowsiness and the onset of sleep. The amplitude of theta waves is elevated in a number of attention-related disorders. Alpha waves are observed during drowsiness and relaxed activity, and spindle activity associated with early stages of quiescent sleep. In contrast to the low frequency regime, the high frequency activity is associated with arousal and alert, attentive states, with complex motor and sensory processing tasks, and with rapid eye movement (REM) sleep.

20.4 Role of Low Voltage-Activated

Calcium Channels

Neurons can fire action potentials in a number of ways. Neurons can be silent and not fire at all. Alternatively, they can act as spiking neurons and generate a small number, just one or two, of action potentials at isolated time intervals. Tonic firing is yet another mode. In tonic firing, a train of action potentials is generated. The action potentials may be generated at regular time intervals or they may be irregularly spaced in time. This type of firing pattern is illustrated in Figure 20.4(a) for the case of regular spacing. Yet another kind of firing pattern is rhythmic bursting. In this kind of firing, illustrated in Figure 20.4(b), multiple sequences of action potentials are produced. Each sequence, or burst, consists of a train of closely spaced action potentials. The intervals between each spike in the burst are small compared to the time intervals between successive bursts, which are

20.4 Role of Low Voltage-Activated Calcium Channels

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FIGURE 20.4. Rhythmic firing patterns, plots of membrane potential versus time: Stereotypic plots of current versus time are shown for

(a) tonic firing and (b) rhythmic bursting.

somewhat variable. The inverse of the average burst-to-burst time interval is the burst frequency. These frequencies can vary from less than one per second to 150 or 200 per second.

The creation of trains and bursts of action potentials rather than single spikes is an important component of the rhythmicity. One of the most important ion channels contributing to the rhythmicity is the low voltageactivated (T-type) calcium channel. Whereas high voltage-activated calcium channels require depolarization of about 40 mV away from resting membrane potentials in order to open, low voltage-activated calcium channels only need about 10 mV of depolarization to open. When they do open and let in calcium, the membrane is further depolarized, resulting in activation of sodium channels and the generation of bursts of action potentials.

The T-type calcium channels operate in a way closely resembling the sodium channels responsible for the upstroke of the action potential. That is, they possess activating and deactivating functions as do the sodium channels and like the sodium channels the T-type calcium channels are activated by depolarization. There are two main differences between the two kinds of channels. The first difference is that the calcium channel activation and deactivation functions are centered at larger hyperpolarizations than those for sodium. At the resting potential, the calcium channels are activating whereas the sodium channels are deactivating. This means that the calcium channels can open at and near the resting potential. The entry of calcium through these channels depolarizes the membrane and triggers the opening of the sodium channels leading to the firing of action potentials.

The second difference between the T-type calcium channel and the sodium channel is in how fast they respond to changes in membrane potential. The sodium channels respond quickly to changes in potential. The calcium channels activate more slowly, and deactivate considerably more slowly. As the membrane voltage moves up and down rapidly due to the sodium/potassium depolarization and repolarizations the calcium current

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increases and then decreases back to zero, terminating the burst of action potentials. The calcium channels that open to start the burst then require a large hyperpolarization in order to de-inactivate and start the next burst.

Tonic firing and rhythmic bursting are widely encountered in the brain, and neurons can switch from one form to the other. Large populations of neurons in the brain undergo slow rhythmic bursting during natural, slow wave sleep, but switch their firing patterns from bursting to tonic firing in waking states and rapid eye movement (REM) sleep. In order to transition from a slow wave sleep state to either awake or REM sleep states, the delta, alpha, and spindling patterns have to be abolished and replaced by the high frequency tonic firing in the beta band (Table 20.1).

The waking up and REM sleep tasks are carried out by collections of neurons that the wake-up system comprises, the reticular formation, located in the brain stem. Neurons in the reticular formation release a number of neuromodulators, the most prominent of which are acetylcholine (ACh), norepinephrine (NE), and serotonin (5-HT). These neuromodulators influence the activities of the neurons generating brain rhythms and also influence muscle states. In the brain, high frequency activity replaces the low frequency waves. The membrane potential is depolarized in response to the neuromodulators, and the T-type calcium channels responsible for bursting are shut down.

20.5 Neuromodulators Modify the Activities of

Voltage-Gated Ion Channels

Up to this point, the discussion has been limited to voltage-gated ion channels and to how individual neurons fire. Except for two brief discussions of the effect of neuromodulators such as adrenaline and acetylcholine, no mention has been made of the role networks play in generating rhythms.

Network properties such as the manner in which neurons are connected to one another and what signals are sent and exchanged, together with the voltage-gated ion channels intrinsic to the individual neurons, jointly determine the nature of the neural rhythms. Three forms of cell-to-cell communication contribute to rhythmicity. These are communication by neuromodulators, by neurotransmitters at chemical synapses, and by small signaling molecules at electrical synapses (gap junctions).

Neuromodulators act on ion channels to produce slow, long-lasting changes in the excitability of the neurons. Neuromodulators are secreted by many different neurons and bind to G protein-coupled receptors on target cells, in contrast to neurotransmitters, which bind to ion channels.

Diffusible signaling molecules such as acetylcholine can act either as a neurotransmitter or as a neuromodulator. Acetylcholine acts as a neurotransmitter when binding to nicotinic (ion channel) receptors and as a neuromodulator when binding to muscarinic (G protein-coupled) recep-

20.6 Connexins Mediate Rapid Signaling Between Cells

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tors. Similarly, glutamate and GABA each can function either as a neurotransmitter or as a neuromodulator, depending on whether it acts through ionotropic or metabotropic (G protein-coupled) receptors. The functional characterization of a molecule as a hormone, as a neurotransmitter, or as a neuromodulator is determined by the nature of its receptor and what kind of cellular response is elicited upon binding.

Recall from Chapter 12 that G protein-coupled receptors activate heterotrimeric G proteins. Neuromodulators can exert their influences on ion channels in two ways. In direct regulation, G protein subunits, particularly the Gbg subunits, diffuse along the inner face of the plasma membrane and bind to the cytoplasmic portions of ion channel subunits, thereby modifying channel conductances. Alternatively, the neuromodulators can exert their influences indirectly by activating second messenger systems. The second messengers either bind the ion channels, thereby modifying their conductances, or they activate protein kinases and protein phosphatases, which, in turn, phosphorylate or dephosphorylate ion channel subunits, thereby modifying their conductances. The modified states of the ion channel subunits have lifetimes that are long compared to the time that it takes to generate and propagate action potentials, and thus the modulatory influences are long-lasting ones.

20.6 Gap Junctions Formed by Connexins Mediate Rapid Signaling Between Cells

Gap junctions are specialized communication channels that permit the rapid exchange of metabolites, ions, and second messengers such as Ca2+ and cAMP between pairs of neurons. The channels are nonselective and when open allow ions of mass less than about 1 kDa to pass directly from the cytoplasm of one cell to another. These communication channels are often found in opposing dendritic processes and are widely distributed among neurons. Because of their direct connectivity, communication through gap junctions, or electrical synapses, as these pores are often called, is more rapid than is possible through chemical synapses. This rapid means of exchanging cytoplasmic signaling elements enables the communication partners to coordinate their firing activities. This synchronizing capability is utilized heavily during the embryonic development of sensory circuits such as those found in the retina. Although there is no sensory input at that time, the neurons are spontaneously active. They continually communicate with one another in order to refine the circuits formed during the initial connecting stages. Communication through gap junctions plays a similar role during development of motor systems, and contributes to adult motor behavior as well.

Gap junctions are formed by connexons, hemipores the span the plasma membrane of a cell.A gap junction between two adjacent cells is created when a connexon hemipore embedded in the plasma membrane of a cell aligns and

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docks with a connexon situated in the plasma membrane of an opposing cell. Once joined the two connexons form a communication channel permitting oneand two-way flow of small charged and uncharged molecules. The size restriction prevents the passage of larger molecules such as nucleic acids and proteins. Gap junctions tend to cluster together into local regions of the plasma membranes of adjacent cells. The two cells remain separated by a gap of from 2 to 4 nm, hence the name “gap junction.”

Each connexon is erected from six connexins arranged in a symmetric fashion to form a hollow pore through which small molecules can diffuse. Some connexons contain a single type of connexin and the connexon mates with an identical connexon. In these cases two homomeric connexons form a homotypic gap junction. Alternatively, a connexon can be assembled from more than one kind of connexin to form a heteromeric connexon. Such a connexon can dock with either the same kind of connexon or with a different kind of connexon on the opposing cell membrane. Thus, there are a variety of homomeric and heteromeric connexons, and these can combine in different homotypic and heterotypic ways to form gap junctions.

FIGURE 20.5. Connexin topology and assembly into gap junctions: (a) Connexins pass back and forth through the plasma membrane four times. Starting from the N- terminal end the transmembrane segments are designated as M1 through M4. The N- and C-terminal portions of the polypeptide chains are situated in the cytoplasm of the host cell. The C-terminal end is longer than the N-terminal end and may form a loop. The two extracellular loops, E1 and E2, mediate connexin to connexin contacts while the three cytoplasmic portions—the N- and C-terminal ends and the cytoplasmic loop (CL) connecting M2 to M3—are sites for regulation of the pore.

(b) Six connexins arrange themselves in a symmetric fashion to form a hemipore called a “connexon.” Two connexons, one from each cell, form the conduction pore, or gap junction. These remain separated from one another by a 2- to 4-mm gap, denoted in the figure by the pair of parallel lines.

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Like ion channels, gap junctions are voltage-gated. Gap junctions will open and close, and change their permeabilities, in response to alterations in potential difference. The state of the pore is responsive to differences in membrane potential of the two cells (transjunctional voltage differences) and to differences in potential across plasma membranes, i.e., between the cytoplasm and extracellular spaces (inside-outside voltage differences). Gap junctions are also responsive to biochemical changes in the intracellular envi- ronment—for instance, the pores will close when exposed to low pH levels.

In addition, gap junctions are subject to regulation by phosphorylation.

20.7 Synchronization of Neural Firing

Gamma-aminobutyric acid (GABA) is the preeminent inhibitory neurotransmitter in the brain. As indicated in Table 19.2, GABA binds to receptors belonging to the cys-loop family of ion channels. When these ion channels open, Cl- anions enter the cell and drive the membrane potential towards more negative, hyperpolarizing values. There are three kinds of GABA receptors: GABAA and GABAC receptors are chloride ion channels, while GABAB receptors are 7TM, G protein-coupled receptors. GABA-releasing neurons are found throughout the CNS interspersed among a larger population of excitatory, glutamate-releasing neurons. Glutamate acts through members of the glutamate family of receptor ion channels to allow Na+, K+, and Ca2+ cations to enter the cell. Most cells in the central nervous system (CNS), inhibitory and excitatory, express GABA receptors. These along with glutamate-binding NMDA and AMPA receptor ion channels are the predominant receptor ion channels.

GABA-releasing inhibitory neurons, referred to as interneurons, make contacts with other interneurons to form interneuron circuits. They also make contacts with excitatory cells, especially those expressing N-methyl-

D-aspartate (NMDA) receptors and capable of repetitive bursting behavior. They excitatory cells make contacts with each other, as well, and together the excitatory and inhibitory cells form large networks of interconnected cells that exhibit the population oscillations seen in the EEGs.

Inhibition mediated by GABAergic neurons and by gap junctions synchronizes the firing of neurons. The GABAergic interneurons synchronize their firing through release of GABA and the subsequent activation of the chloride channels. Their synchrony is greatly improved, and in some situations predominantly mediated, by gap junctions. There are a number of different kinds of interneurons. Each population of interneurons expresses a particular kind of gap junction that enables it to communicate with other interneurons of the same kind. For example, one kind of interneuron, the fast spiking (FS) interneuron, will communicate through gap junctions with other FS interneurons. The networks of interneurons collectively act as a pacemaker circuit for the larger circuits involving the excitatory cells. In these situations, the gap junctions operate in combination with NMDA

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receptors that endow the neuron with intrinsic rhythmic bursting properties. In the absence of the gap junctions and the inhibitory connections, each excitatory neuron would carry out its program of rhythmic bursting in a distinct and uncoordinated manner. When the inhibitory circuitry is present, the cells synchronize their firing patterns. They can undergo rhythmic discharges at low frequencies and also at the higher frequencies characteristic of the beta and gamma bands of the EEG.

20.8 How Spindling Patterns Are Generated

Spindle waves (Figure 20.6) are generated during the early stages of quiescent sleep. Large populations of neurons in the thalamus and cortex undergo rhythmic discharges in the 7 to 14 Hz range. These oscillations wax and wane with a 1- to 3-second period. That is, the oscillations steadily increase in magnitude up to a maximum and then decrease until they vanish some 1 to 3 seconds after they started. After a silent period of 3 to 20 seconds they start up again, and the waxing and waning pattern repeats itself.

Spindle rhythms are generated in thalamic neurons. Two populations of cells are involved—inhibitory neurons (GABAergic) in the thalamic reticular nucleus that make reciprocal connections with excitatory neurons in the thalamic relay nucleus. When pulses of GABA are released at the presynaptic terminal of thalamic reticular neurons, inhibitory postsynaptic potentials (IPSPs) are produced in the relay nuclei. These hyperpolarizing events activate the Ih channels, which permit the generation of action potentials in the relay neurons, which through their reciprocal synapses with the reticular neurons trigger membrane depolarization and re-excite the reticular cells.

20.9 Epileptic Seizures and Abnormal Brain Rhythms

Excitation mediated through glutamergic cationic channels must be in balance with inhibition mediated by GABAergic anionic channels. One of the consequences of an imbalance between excitation and inhibition is the

FIGURE 20.6. Plot of membrane potential versus time for 7- to 14-Hz spindle oscillations: The train of action potentials first waxes and then wanes in amplitude.

20.10 Swimming and Digestive Rhythms in Lower Vertebrates

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occurrence of epileptic seizures. These abnormal events are generated when there is too much excitation (hyperexcitabilty) leading to aberrant rhythms and epileptic seizures.

Because of their ability to generate rhythmic discharges, neurons in the neocortex, thalamus, and hippocampus are ideal sites for aberrant rhythms leading to epileptic events and seizures. These regions of the brain contain large numbers of excitatory, intrinsically bursting pyramidal cells that are massively interconnected with one another, and smaller populations of several different kinds of inhibitory interneurons that, as just discussed, not only make connections with one another but also make contact with large numbers of excitatory pyramidal cells.

One of the best-studied forms of epilepsy is the absence seizure,or petit mal epilepsy.This type of seizure appears in EEGs as an aberrant 3-Hz spike-and- wave pattern.Absence seizures occur most often in children and adolescents, and are characterized behaviorally as brief (~10 s), nonconvulsive interruptions in consciousness. The spike-and-wave patterns are generated by the general thalamic/thalamocortical circuitry that produces the spindling patterns. One of the likely causes of this type of aberrant brain wave pattern is malfunctioning GABAA receptors. These receptors are erected from mixtures of different GABA subunits. The different kinds of GABAA receptor subunits are designated as a, b, g, d, e, p, or r. Several different isoforms exist for many of the subunit types. Ion channels built from r subunits are called GABAC ion channels.The GABA channels are constructed from five subunits and, like the other ion channels, its subunits are arranged to form a hollow conducting pore. A stoichiometric combination that is widely distributed throughout the brain is one asubunit,two bsubunits and two gsubunits. Mutations in the g2 subunit have been implicated as a possible cause of absence seizures in several experimental studies, while theoretical modeling efforts have demonstrated how malfunctions in GABAA and GABAB receptors can generate aberrant 3-Hz spike-and-wave rhythms.

That mutations in an ion channel subunit can cause aberrant rhythms and have behavioral consequences is not unique to the GABA and absence seizures. Rather, mutations in other ion channel subunit exits have similar behavioral consequences. These include mutations in the channel-forming a subunit of high voltage-activated, P/Q-type calcium channels, in other GABA subunits, and in potassium and sodium channel subunits. Thus, there is an entire ensemble of ion channel disorders, or “channelpathies.”

20.10 Swimming and Digestive Rhythms in

Lower Vertebrates

Central pattern generators located in the spinal cord of lower vertebrates generate swimming and digestive rhythms. The rhythmic movements of muscles are controlled by neurons located in the brain stem and spinal cord.

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These neurons are organized into small neural circuits called central pattern generators (CPGs). The CPGs may be thought of as elementary circuits, autonomous modules built from small numbers of neurons that are used to drive locomotion activities such as walking and swimming, breathing, and chewing and digestion. Many of the principles governing their operation are shared by other rhythm-producing systems such as those responsible for sleep and alert behavior. Processes that generate rhythms, processes such as reciprocal connections involving inhibition, and burst-promoting voltage-gated ion channels, are common to all rhythm-producing circuits.

Motor neurons supply input to muscles that drives their contractions. They are the last stage of neural connectivity before the muscles. Inputs from different drivers such as the CPGs converge upon these cells. Some of the regulatory inputs come directly to the motor neurons while others converge upon the CPGs to regulate their firing patterns.

Invertebrates are widely used as model systems for studying central pattern generators. The invertebrate CPGs exhibit all of the main properties of vertebrate CPGs, yet are small enough to be well characterized at the network and ion channel levels. The invertebrates selected as model systems include the nudibranch Tritonia diomedea, the sea snail Aplysia californica, the decapod crustaceans Panulirus interruptus (spiny lobster) and Cancer borealis (rock crab), and the medicinal leech Hirudo medicinalis.

The stomatogastric ganglion (STG) of the lobster and crab is the site of a pair of central pattern generators that supply digestive rhythms. These crustaceans swallow their food whole. The material ingested is broken down in the stomach through rhythmic contractions of gastric teeth and the stomach. Several different kinds of rhythms are produced during digestion. One of these is the gastric rhythm that controls the gastric teeth, and another is the pyloric rhythm that regulates the food sorting and sifting machinery. The rhythms are supplied by two CPGs. The CPG responsible for the pyloric rhythm contains 14 neurons. Of these 13 are motor neurons whose output drives the muscle contractions, and one is a pacemaker neuron. The CPG responsible for gastric rhythms contains just 11 neurons. However, its wiring and operation is more complex than that of the pyloric circuit, and the discussion will be limited to the latter.

The pyloric circuit is presented in Figure 20.7. There are six different kinds of neurons in the circuit. One each of these neuron types and its connectivity is shown in the figure. Connection leading to cells outside the CPG are omitted to maintain clarity. As can be seen in the diagram the neurons in the pyloric central pattern generator are connected to one another by electrical synapses and by chemical synapses. The AB neuron functions as the pacemaker neuron for the circuit. It produces bursts of action potentials at regular intervals. Electrical synapses link the AB and PD neurons to one another. As a result the PD neuron fires in synchrony with the AB neuron, and these two neurons may be regarded jointly as the network pacemaker. As is shown in Figure 20.7, these two neurons establish contact with all the downstream motor neurons.

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FIGURE 20.7. Crustacean pyloric circuit: The central pattern generator responsible for pyloric rhythms consists of anterior burster (AB), pyloric dilator (PD), ventricular dilator (VD), inferior cardiac (IC), lateral pyloric (LP), and pyloric (PY) neurons. Resistor symbols denote electrical synapses, and lines terminating in filled circles represent inhibitory chemical synaptic connections.

FIGURE 20.8. Mutual inhibition: Two cells, A and B, are coupled to one another by means of inhibitory synaptic connections. Because of this coupling the two cells alternatively fire synchronized sequences of action potentials. The dashed lines denote the gradual depolarization of the membrane potential towards the plateau potential and threshold for firing action potentials.

The second main feature of the network is wide usage of mutual inhibition in which pairs of cells are reciprocally connected to each other and inhibit one another. Mutual inhibition produces alternative firing pattern that can drive the phased contractions of opposing muscles. A stereotypic pattern of alternating firing by a pair of cells mutually inhibiting one another is depicted in Figure 20.8. In the pyloric circuit, the AB and PD neurons fire first, inhibiting the firing of the downstream neurons until the bursting is completed. The downstream neurons then fire in a sequence