Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel.

The neurotransmitter serotonin (5HT) activates a variety of second messenger signaling systems and through them indirectly regulates the function of ion channels. Serotonin also activates ion channels directly, suggesting that it may also mediate rapid, excitatory responses. A complementary DNA clone containing the coding sequence of one of these rapidly responding channels, a 5HT3 subtype of the serotonin receptor, has been isolated by screening a neuroblastoma expression library for functional expression of serotonin-gated currents in Xenopus oocytes. The predicted protein product has many of the features shared by other members of the ligand-gated ion channel family. The pharmacological and electrophysiological characteristics of the cloned receptor are largely consistent with the properties of native 5HT3 receptors. Messenger RNA encoding this receptor is found in the brain, spinal cord, and heart. This receptor defines a new class of excitatory ligand-gated channels.

Calcium and calcium-dependent chloride currents generate action potentials in solitary cone photoreceptors.

Vertebrate rod and cone photoreceptors hyperpolarize when illuminated. However, synaptic input from horizontal cells can depolarize cones and even elicit action potentials. Using the whole-cell tight-seal recording technique, we determined that, in solitary cones isolated from a lizard retina, action potentials can be generated by depolarizing current steps under conditions where only two ionic currents are activated. A dihydropyridine-sensitive, inward Ca2+ current that activates at potentials positive to -40 mV can regeneratively depolarize the cell. Subsequently, a SITS-sensitive, Ca2(+)-dependent outward Cl- current repolarizes the cell. We suggest that these ionic currents may help explain lateral inhibition in the retina.

Neuronal control of locomotion in C. elegans is modified by a dominant mutation in the GLR-1 ionotropic glutamate receptor.

How simple neuronal circuits control behavior is not well understood at the molecular or genetic level. In Caenorhabditis elegans, foraging behavior consists of long, forward movements interrupted by brief reversals. To determine how this pattern is generated and regulated, we have developed novel perturbation techniques that allow us to depolarize selected neurons in vivo using the dominant glutamate receptor mutation identified in the Lurcher mouse. Transgenic worms that expressed a mutated C. elegans glutamate receptor in interneurons that control locomotion displayed a remarkable and unexpected change in their behavior-they rapidly alternated between forward and backward coordinated movement. Our findings suggest that the gating of movement reversals is controlled in a partially distributed fashion by a small subset of interneurons and that this gating is modified by sensory input.

The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion.

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is important for synaptic plasticity and nervous system development and function. We have used genetic and electrophysiological methods to demonstrate that NMR-1, a Caenorhabditis elegans NMDA-type ionotropic glutamate receptor subunit, plays a role in the control of movement and foraging behavior. nmr-1 mutants show a lower probability of switching from forward to backward movement and a reduced ability to navigate a complex environment. Electrical recordings from the interneuron AVA show that NMDA-dependent currents are selectively disrupted in nmr-1 mutants. We also show that a slowly desensitizing variant of a non-NMDA receptor can rescue the nmr-1 mutant phenotype. We propose that NMDA receptors in C. elegans provide long-lived currents that modulate the frequency of movement reversals during foraging behavior.

Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42.

In almost all nervous systems, rapid excitatory synaptic communication is mediated by a diversity of ionotropic glutamate receptors. In Caenorhabditis elegans, 10 putative ionotropic glutamate receptor subunits have been identified, a surprising number for an organism with only 302 neurons. Sequence analysis of the predicted proteins identified two NMDA and eight non-NMDA receptor subunits. Here we describe the complete distribution of these subunits in the nervous system of C. elegans. Receptor subunits were found almost exclusively in interneurons and motor neurons, but no expression was detected in muscle cells. Interestingly, some neurons expressed only a single subunit, suggesting that these may form functional homomeric channels. Conversely, interneurons of the locomotory control circuit (AVA, AVB, AVD, AVE, and PVC) coexpressed up to six subunits, suggesting that these subunits interact to generate a diversity of heteromeric glutamate receptor channels that regulate various aspects of worm movement. We also show that expression of these subunits in this circuit is differentially regulated by the homeodomain protein UNC-42 and that UNC-42 is also required for axonal pathfinding of neurons in the circuit. In wild-type worms, the axons of AVA, AVD, and AVE lie in the ventral cord, whereas in unc-42 mutants, the axons are anteriorly, laterally, or dorsally displaced, and the mutant worms have sensory and locomotory defects.

The differential effects of haloperidol and methamphetamine on time estimation in the rat.

Forty rats were trained to make a left lever response if a signal (white noise) was 2.5s and to make a right lever response if the signal was 6.3s. When seven intermediate signal durations, to which responses were not reinforced, were randomly interspersed the probability of a right-lever ('long') response increased as a function of signal duration. Methamphetamine shifted this psychometric function leftward and decreased its slope: haloperidol also decreased the slope but shifted the function rightward. A combination of haloperidol and methamphetamine led to a function similar to the saline control function. The leftward shift probably reflects an increase in the speed of an internal clock, and the rightward shift probably reflects a decrease in its speed. Since methamphetamine releases several catecholamines, including dopamine, and haloperidol blocks dopamine receptors, it is plausible that the horizontal location of the psychometric function (the speed of the clock) is related to the effective level of dopamine.

Methamphetamine and time estimation.

Three experiments were conducted to determine the effect of methamphetamine on the performance of rats in two timing tasks. When food sometimes followed the first response after T sec of a signal, the response rate increased to a peak near T sec and then declined. Methamphetamine decreased the time of the peak (Experiments 1 and 2). When one response (called a "short response") was reinforced following a short signal and a different response (a "long response") was reinforced following a long signal (where the short and long signals were 1 and 4, 2 and 8, or 4 and 16 sec), the probability of a long response increased as a function of signal duration. The point of indifference (50% long response) occurred near the geometric mean of the extreme durations, and methamphetamine decreased the point of indifference by about 10%. These results suggest that methamphetamine increases the speed of an internal clock used by rats in time discrimination tasks.

Building a synapse: genetic analysis of glutamatergic neurotransmission.

Ionotropic glutamate receptors (iGluRs) are a critical component of the vertebrate central nervous system and mediate the majority of rapid excitatory neurotransmission. However, iGluRs are not self-regulating molecules and require additional proteins in order to function properly. Understanding the molecular architecture of functional glutamatergic synapses is therefore an important challenge in neurobiology. To address this question, we combine the techniques of genetics, molecular biology and electrophysiology in the nematode Caenorhabditis elegans. To date, genetic analysis has identified a number of genes required to build a glutamatergic synapse, including the CUB-domain transmembrane protein, SOL-1, which is thought to act as an auxiliary subunit that directly modifies iGluR function. Identifying and characterizing new proteins, such as SOL-1, in the relatively simple nervous system of the worm can contribute to our understanding of how more complex vertebrate nervous systems function.

Potassium currents in the inner segment of single retinal cone photoreceptors.

1. The K+ currents of cone inner segments isolated from the retina of a lizard were studied with the use of tight-seal electrodes in the whole cell configuration. To conduct these studies other identified currents in the cell were blocked. Co2+ blocked a voltage-dependent Ca2+ current and a Ca2(+)-dependent Cl- current, and Cs+ blocked an inward-rectifying current partially carried by K+. 2. The cells sustained a voltage-dependent K+ current that was blocked by tetraethylammonium (TEA)+ and had characteristics typical of the delayed rectifier. However, we found no evidence for the existence of "A"-type K+ currents or Ca2(+)-dependent K+ currents. 3. The delayed-rectifier current was nearly ideally selective for K+. Increasing external K+ concentration 10-fold shifted the reversal potential by 55 mV. 4. Analysis of the voltage dependence of the activation of the delayed-rectifier current revealed the existence of two distinct subclasses of this current. We referred to them as IdrL and IdrH for low and high threshold of voltage activation. 5. IdrL activated at voltages above -70 mV. Its dependence on voltage was described by Boltzmann's function with average half-maximum activation at -51 mV and steepness factor k = 7.5 mV. IdrH activated at voltages above -50 mV. Its dependence on voltage was described by Boltzmann's function with average half-maximum activation at -4.6 mV and steepness factor k = 17.1 mV. 6. Of nine cells analyzed in detail, one demonstrated IdrH alone, whereas the remaining had a variable mixture of the two current subtypes. At maximum activation the current through IdrL ranged between 0.3 and 0.5 of the total delayed-rectifier current. 7. The kinetics of activation of the total delayed-rectifier current were described by the sum of two exponentials the amplitudes and time constants of which were voltage dependent. However, the kinetics of the current subtypes were not resolved individually. The current inactivated slowly with a single-exponential time course that was voltage dependent. 8. The voltage dependence of the delayed-rectifier current indicates the current is active in a cone photoreceptor in the dark. The current is 20-30 pA in amplitude at the dark-membrane potential and outwardly directed. 9. IdrL may generate a rapid relaxation of photovoltages activated by dim lights--those that hyperpolarize the membrane by only a few millivolts. The delayed-rectifier currents help shape the action potentials that can be generated in isolated cone photoreceptors.

Inward rectification in the inner segment of single retinal cone photoreceptors.

1. Single cone photoreceptors were dissociated from the retina of a lizard with the aid of papain. The majority of the cells lost their outer segments but had well-preserved, large synaptic pedicles. Electrical properties of the cells were studied with tight-seal electrodes in the whole cell configuration. On the average, cone inner segments had a resting potential of -55 mV, and at this potential their input resistance was 2.6 G omega and their capacitance was 8 pF. 2. Under current clamp the cones exhibited a pronounced anomalous voltage rectification in response to hyperpolarizing currents. The voltage rectification was eliminated by external Cs+. 3. The Cs(+)-sensitive current underlying voltage rectification was isolated by blocking other currents present in the cone. Co2+ blocked a voltage-dependent Ca2+ current and a Ca2(+)-dependent Cl- current, and tetraethylammonium (TEA)+ blocked a delayed-rectifier K+ current. 4. The Cs(+)-sensitive current was activated by hyperpolarization to potentials more negative than -50 mV, and its current-voltage (I-V) relationship exhibited inward rectification. 5. The inward-rectifying current was selective for K+, but not exclusively. Increasing external K+ concentration 10-fold shifted the reversal potential by 13 mV. If Na ions also permeate through the inward-rectifying channels, the ratio of permeabilities (PK+/PNa+) in normal solution is approximately 3.9. 6. The kinetics of the inward-rectifying current were described by the sum of two exponentials, the amplitudes and time constants of which were voltage dependent. 7. The voltage dependence of the inward-rectifying current was described by Boltzmann's function, with half-maximum activation at -79 mV and a steepness parameter of 7.5 mV. 8. The voltage dependence and kinetics of the inward-rectifying current suggest that it is inactive in a cone photoreceptor in the dark. However, it becomes activated in the course of large hyperpolarizations generated by bright-light illumination. This activity will modify the waveform of the photovoltage--the current will generate a depolarizing component that opposes the light-generated hyperpolarization.

Nervous system distribution of the serotonin 5-HT3 receptor mRNA.

The serotonin 5-HT3 receptor subtype has been implicated in many brain functions. Antagonists of this receptor have anxiolytic and antiemetic effects in humans and in animal models. To determine with cellular resolution the distribution of 5-HT3 receptor mRNA, in situ hybridization was performed in sections of mouse brain and dorsal root ganglia. Scattered labeled cells were observed throughout cortical regions, with highest densities in the piriform, cingulate, and entorhinal areas. Strong hybridization signals were seen in the hippocampal formation, where expression appeared primarily in interneurons. Labeled cells were most abundant in the posteroventral hippocampal region, particularly in the lacunosum moleculare layer of CA1. This distribution suggests that 5-HT3 receptors may mediate the known serotonergic inhibition of pyramidal cell populations via excitation of inhibitory interneurons. Labeled cells were also observed in the major subdivisions of the amygdaloid complex, the olfactory bulb, the trochlear nerve nucleus, the dorsal tegmental region, the facial nerve nucleus, the nucleus of the spinal tract of the trigeminal nerve, and the spinal cord dorsal horn. In the periphery, intense hybridization signals were seen in a subpopulation of cells in dorsal root ganglia. The data correlate generally with physiological, behavioral, and receptor autoradiographic studies, provide cellular resolution, and reveal regions of receptor expression not previously observed. The distribution of 5-HT3 receptor mRNA is consistent with roles for the receptor in cognition and affect and in the modulation of sensory input.

Kinetics of activation of acetylcholine receptors in a mouse muscle cell line under a range of acetylcholine concentrations.

We studied, using the patch-clamp technique, the kinetics of single acetylcholine (ACh)-activated channels in a mouse muscle cell line. In the presence of high ACh concentrations we estimated the rate of channel isomerization into the open state (beta) from the dwell time between openings. Also, we obtained estimates for beta under low agonist concentrations by assuming a linear sequential model of channel activation and applying burst analysis. If the linear model is correct, then the two estimates of beta should agree since beta should be independent of ACh concentration. However, the estimates of beta obtained under low ACh concentrations were slower than those obtained independently under high ACh concentrations. The discrepancy in the estimates of beta suggests that the linear model is inadequate, but the discrepancy can be explained if open channels can close through two separate pathways. Two alternative kinetic models that can account for our data are discussed.

The effects of a myasthenic serum on the acetylcholine receptors of C2 myotubes. II. Functional inactivation of the receptor.

We have investigated the effect of antibodies from a myasthenic serum on the physiological properties of acetylcholine receptors (AChRs) in myotubes of a mouse muscle cell line, C2. The antibodies in this serum blocked the binding of 125I-alpha-bungarotoxin to the myotubes to an extent of about 50%. The antibodies also inhibited the increase in 22Na influx caused by carbamylcholine (CARB). At a concentration of antibody that blocked about 50% of toxin binding, greater than 80% of the AChR-mediated 22Na influx was blocked. The apparent KD for CARB, estimated from the dose-response curve for 22Na influx, was unaffected. The effect of the antibodies was further examined by patch-clamp recording. In greater than 30% of the patches from antibody-treated cells, no channel activity in response to acetylcholine was seen; in contrast, every patch from control cells showed activity. The channels that were seen after antibody treatment were indistinguishable from those seen in normal cells, both in their single-channel conductance and in the kinetic constants used to describe channel opening and closing. We conclude that the antibodies in this serum inhibit the functional response of AChRs in C2 myotubes to acetylcholine and do so by inactivating individual receptors.

Mechanosensory signalling in C. elegans mediated by the GLR-1 glutamate receptor.

NEURONAL signalling across synapses involves activation of many neurotransmitter receptors on postsynaptic cells. glr-1 encodes a potential glutamate receptor in the nematode Caenorhabditis elegans which is most similar to vertebrae AMPA-type ionotropic glutamate receptors. glr-1 is expressed in motor neurons and interneurons, including interneurons implicated in the control of locomotion. Here we investigate the contribution of glr-1 to the normal signalling of these neurons, by generating a deletion mutation in glr-1. We find that mutant worms are deficient in their ability to withdraw backwards when mechanically stimulated, but they withdraw normally in response to chemical repellents. The ASH sensory neurons mediate withdrawal responses both to mechanical stimuli and to repellents, and ASH makes chemical synapses with glr-1-expressing interneurons. Our results suggest that postsynaptic interneurons use different neurotransmitter receptors to process two sensory stimuli detected by one sensory neuron.

The C. elegans gene vab-8 guides posteriorly directed axon outgrowth and cell migration.

The assembly of the nervous system in the nematode C. elegans requires the directed migrations of cells and growth comes along the anteroposterior and dorsoventral body axis. We show here that the gene vab-8 is essential for most posteriorly directed migrations of cells and growth cones. Mutations in vab-8 disrupt fourteen of seventeen posteriorly directed migrations, but only two of seventeen anteriorly directed and dorsoventral migrations. For two types of neurons that extend axons both anteriorly and posteriorly, vab-8 mutations disrupt only the growth of the posteriorly directed axon. vab-8 encodes two genetic activities that function in the guidance of different migrations. Our results suggest that most posteriorly directed cell and growth cone migrations are guided by a common mechanism involving the vab-8 gene.