Biomedical subjects

R M Harris-Warrick

Publications and source records attributed to R M Harris-Warrick.

At least 19 recordsLinked to original sources

PubMed2000

Contributions of intrinsic motor neuron properties to the production of rhythmic motor output in the mammalian spinal cord.

Motor neurons are endowed with intrinsic and conditional membrane properties that may shape the final motor output. In the first half of this paper we present data on the contribution of I(h), a hyperpolarization-activated inward cation current, to phase-transition in motor neurons during rhythmic firing. Motor neurons were recorded intracellularly during locomotion induced with a mixture of N-methyl-D-aspartate (NMDA) and serotonin, after pharmacological blockade of I(h). I(h) was then replaced by using dynamic clamp, a computer program that allows artificial conductances to be inserted into real neurons. I(h) was simulated with biophysical parameters determined in voltage clamp experiments. The data showed that electronic replacement of the native I(h) caused a depolarization of the average membrane potential, a phase-advance of the locomotor drive potential, and increased motor neuron spiking. Introducing an artificial leak conductance could mimic all of these effects. The observed effects on phase-advance and firing, therefore, seem to be secondary to the tonic depolarization; i.e., I(h) acts as a tonic leak conductance during locomotion. In the second half of this paper we discuss recent data showing that the neonatal rat spinal cord can produce a stable motor rhythm in the absence of spike activity in premotor interneuronal networks. These coordinated motor neuron oscillations are dependent on NMDA-evoked pacemaker properties, which are synchronized across gap junctions. We discuss the functional relevance for such coordinated oscillations in immature and mature spinal motor systems.

Animals↗

PubMed2000

Molecular underpinnings of motor pattern generation: differential targeting of shal and shaker in the pyloric motor system.

The patterned activity generated by the pyloric circuit in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus, results not only from the synaptic connectivity between the 14 component neurons but also from differences in the intrinsic properties of the neurons. Presumably, differences in the complement and distribution of expressed ion channels endow these neurons with many of their distinct attributes. Each pyloric cell type possesses a unique, modulatable transient potassium current, or A-current (I(A)), that is instrumental in determining the output of the network. Two genes encode A-channels in this system, shaker and shal. We examined the hypothesis that cell-specific differences in shaker and shal channel distribution contribute to diversity among pyloric neurons. We found a stereotypic distribution of channels in the cells, such that each channel type could contribute to different aspects of the firing properties of a cell. Shal is predominantly found in the somatodendritic compartment in which it influences oscillatory behavior and spike frequency. Shaker channels are exclusively localized to the membranes of the distal axonal compartments and most likely affect distal spike propagation. Neither channel is detectably inserted into the preaxonal or proximal portions of the axonal membrane. Both channel types are targeted to synaptic contacts at the neuromuscular junction. We conclude that the differential targeting of shaker and shal to different compartments is conserved among all the pyloric neurons and that the channels most likely subserve different functions in the neuron.

Amino Acid Sequence↗

PubMed2000

Highly localized Ca(2+) accumulation revealed by multiphoton microscopy in an identified motoneuron and its modulation by dopamine.

Calcium is essential for synaptic transmission and the control of the intrinsic firing properties of neurons; this makes Ca(2+) channels a prime target for neuromodulators. A combination of multiphoton microscopy and voltage-clamp recording was used to determine the localization of voltage-dependent Ca(2+) accumulation in the two pyloric dilator (PD) neurons of the pyloric network in the spiny lobster, Panulirus interruptus, and its modulation by dopamine. We monitored [Ca(2+)](i) in fine distal branches in the neuropil >350 microm below the surface of the ganglion during controlled voltage steps in voltage clamp. Ca(2+) accumulation originated mostly from small, fairly rare, spatially restricted varicosities on distal neuritic arborizations. Ca(2+) diffused from these point sources into adjacent regions. Varicosities with similar morphology in the PD neuron have been shown previously to be sites of synaptic contacts. We have demonstrated in earlier studies that dopamine inhibits activity and greatly reduces synaptic transmission from the PD neuron. In approximately 60% of the varicosities, the voltage-activated Ca(2+) accumulation was reduced by exogenous dopamine (DA) (10(-4) M). DA decreased the peak amplitude of Ca(2+) accumulation but had no effect on the rise and decay time. We conclude that DA reduces chemical synaptic transmission from the PD neurons at least in part by decreasing Ca(2+) entry at neurotransmitter release sites.

Animals↗

PubMed2000

Patterns of shaker family gene expression in single identified neurons of the American lobster, Homarus americanus.

The patterns of expression of voltage gated potassium channel genes of the Shaker family have been mapped in identified neurons of the lobster (Homarus americanus) ventral nerve cord using a single cell reverse transcriptase polymerase chain reaction procedure. Using specific oligonucleotides derived from the sequences of the shaker, shab, and shaw genes of the spiny lobster, Panulirus interruptus, we detected the corresponding potassium channel DNA fragments from Homarus americanus. The Homarus DNA fragments are 87-98% identical at the nucleotide level to the Panulirus DNA fragments. We used the Panulirus primers to measure the complement of RNAs for shaker, shab, and shaw in single identified cells that use GABA, glutamate, octopamine or serotonin as chemical messengers. Shaker and shaw RNAs were found in all four identified neuron types but shab RNA was not detected in serotonin cells under the present experimental conditions. All cells expressed alpha-tubulin RNA, which serves as an internal control suggesting that cells are intact after dissection. In glial cells that surround the neuronal cell bodies, the potassium channel genes are expressed at low to non-detectable levels.

Animals↗

PubMed1999

Monoamine control of the pacemaker kernel and cycle frequency in the lobster pyloric network.

The monoamines dopamine (DA), serotonin (5HT), and octopamine (Oct) can each sculpt a unique motor pattern from the pyloric network in the stomatogastric ganglion (STG) of the spiny lobster Panulirus interruptus. In this paper we investigate the contribution of individual network components in determining the specific amine-induced cycle frequency. We used photoinactivation of identified neurons and pharmacological blockade of synapses to isolate the anterior burster (AB) and pyloric dilator (PD) neurons. Bath application of DA, 5HT, or Oct enhanced cycle frequency in an isolated AB neuron, with DA generating the most rapid oscillations and Oct the slowest. When an AB-PD or AB-2xPD subnetworks were tested, DA often reduced the ongoing cycle frequency, whereas 5HT and Oct both evoked similar accelerations in cycle frequency. However, in the intact pyloric network, both DA and Oct either reduced or did not alter the cycle frequency, whereas 5HT continued to enhance the cycle frequency as before. Our results show that the major target of 5HT in altering the pyloric cycle frequency is the AB neuron, whereas DA's effects on the AB-2xPD subnetwork are critical in understanding its modulation of the cycle frequency. Octopamine's effects on cycle frequency require an understanding of its modulation of the feedback inhibition to the AB-PD group from the lateral pyloric neuron, which constrains the pacemaker group to oscillate more slowly than it would alone. We have thus demonstrated that the relative importance of the different network components in determining the final cycle frequency is not fixed but can vary under different modulatory conditions.

Animals↗

PubMed1999

Identification and localization of Ca(2+)-activated K+ channels in rat sciatic nerve.

To understand the physiology of Schwann cells and myelinated nerve, we have been engaged in identifying K+ channels in sciatic nerve and determining their subcellular localization. In the present study, we examined the slo family of Ca(2+)-activated K+ channels, a class of channel that had not previously been identified in myelinated nerve. We have determined that these channels are indeed expressed in peripheral nerve, and have cloned rat homologues of slo that are more than 95% identical to the murine slo. We found that sciatic nerve RNA contained numerous alternatively spliced variants of the slo homologue, as has been seen in other tissues. We raised a polyclonal antibody against a peptide from the carboxyl terminal of the channels. Immunocytochemistry revealed that the channel proteins are in Schwann cells and are associated with canaliculi that run along the outer surface of the cells. They are also relatively concentrated near the node of Ranvier in the Schwann cell outer membrane. This staining pattern is quite similar to what we previously reported for the voltage-dependent K+ channel Kv 1.5. We did not observe staining of axons or connective tissue in the nerve and so it seems likely that most or all of the splicing variants are located in the Schwann cells. The localization of these channels also suggests that they may participate in maintaining the resting potential of the Schwann cells during K+ buffering.

Amino Acid Sequence↗

PubMed1999

The evolution of neuronal circuits underlying species-specific behavior.

The nervous system is evolutionarily conservative compared to the peripheral appendages that it controls. However, species-specific behaviors may have arisen from very small changes in neuronal circuits. In particular, changes in neuromodulatory systems may allow multifunctional circuits to produce different sets of behaviors in closely related species. Recently, it was demonstrated that even species differences in complex social behavior may be attributed to a change in the promoter region of a single gene regulating a neuromodulatory action.

Animals↗

PubMed1999

Dopamine modulates two potassium currents and inhibits the intrinsic firing properties of an identified motor neuron in a central pattern generator network.

The two pyloric dilator (PD) neurons are components [along with the anterior burster (AB) neuron] of the pacemaker group of the pyloric network in the stomatogastric ganglion of the spiny lobster Panulirus interruptus. Dopamine (DA) modifies the motor pattern generated by the pyloric network, in part by exciting or inhibiting different neurons. DA inhibits the PD neuron by hyperpolarizing it and reducing its rate of firing action potentials, which leads to a phase delay of PD relative to the electrically coupled AB and a reduction in the pyloric cycle frequency. In synaptically isolated PD neurons, DA slows the rate of recovery to spike after hyperpolarization. The latency from a hyperpolarizing prestep to the first action potential is increased, and the action potential frequency as well as the total number of action potentials are decreased. When a brief (1 s) puff of DA is applied to a synaptically isolated, voltage-clamped PD neuron, a small voltage-dependent outward current is evoked, accompanied by an increase in membrane conductance. These responses are occluded by the combined presence of the potassium channel blockers 4-aminopyridine and tetraethylammonium. In voltage-clamped PD neurons, DA enhances the maximal conductance of a voltage-sensitive transient potassium current (IA) and shifts its Vact to more negative potentials without affecting its Vinact. This enlarges the "window current" between the voltage activation and inactivation curves, increasing the tonically active IA near the resting potential and causing the cell to hyperpolarize. Thus DA's effect is to enhance both the transient and resting K+ currents by modulating the same channels. In addition, DA enhances the amplitude of a calcium-dependent potassium current (IO(Ca)), but has no effect on a sustained potassium current (IK(V)). These results suggest that DA hyperpolarizes and phase delays the activity of the PD neurons at least in part by modulating their intrinsic postinhibitory recovery properties. This modulation appears to be mediated in part by an increase of IA and IO(Ca). IA appears to be a common target of DA action in the pyloric network, but it can be enhanced or decreased in different ways by DA in different neurons.

Action Potentials↗

PubMed1998

Distributed effects of dopamine modulation in the crustacean pyloric network.

It is now clear that neuromodulators can reconfigure a single motor network to allow the generation of a family of related movements. Using dopamine modulation of the 14-neuron pyloric network from the crustacean stomatogastric ganglion as an example, we describe two major mechanisms by which network output is modulated. First, the baseline electrophysiological properties of the network neurons can be altered. Dopamine can affect the activity of each neuron independently. For example, DA modulates IA in nearly every neuron in the pyloric network, but in opposite directions in different cells. Furthermore, DA usually modulates combinations of ionic currents. In some cases, currents with opposing actions on cell excitability are simultaneously affected, and the net response reflects the sum of these opposing effects. Second, neuromodulators can alter the strength of synaptic interactions within the network, quantitatively "rewiring" the network. Every synapse in the network is affected by DA, with some increased and others decreased in strength. DA acts both pre- and postsynaptically to affect transmission: these actions are frequently opposing in sign, and the net response arises as the sum of these opposing actions. Finally, spike-evoked and graded transmission at the same synapse can be oppositely affected by DA. These results emphasize the distributed nature of modulation in motor networks.

Animals↗

PubMed1998

Differential expression and targeting of K+ channel genes in the lobster pyloric central pattern generator.

A molecular analysis of motor pattern generation is an essential complement to electrophysiological and computational investigations. In arthropods, A-channels are posttranslationally modified multimeric proteins containing Shaker family alpha-subunits that may interact with beta-subunits, gamma-subunits, and other auxiliary proteins. One consequence of A-channel structure is that several mechanisms could underlie the cell-specific differences in pyloric IAs including differential gene expression, alternate splicing, and posttranslational modifications. Oocyte expression studies, single-cell RT-PCR, and immunocytochemistry suggest that differential alpha-subunit gene expression is not a mechanism for creating pyloric IA heterogeneity, and that the same gene, shal, encodes the alpha-subunits for the entire family of somatic IAs in the pyloric network. Changes in the level of shal gene expression alter A-channel density between cells, but cannot account for the differences in the biophysical properties of the six pyloric IAs. Preliminary data suggest that the shal gene also encodes the A-channel alpha-subunits for the coarse and fine neuropil but not for most axons. A second gene, shaker, encodes the A-channel alpha-subunits in the majority of axons and at the neuromuscular junction. The distinct properties of the two types of A-channels are consistent with the different roles of IA at the different locations. Both the shaker and shal genes are alternately spliced, and investigations are under way to determine whether alternate splicing is a mechanism for generating pyloric IA heterogeneity.

Animals↗

PubMed1998

Interaction of dopamine and cardiac sac modulatory inputs on the pyloric network in the lobster stomatogastric ganglion.

Dopamine and cardiac sac network activity each have strong, and different, modulatory actions on the pyloric rhythm in the stomatogastric ganglion of the spiny lobster. When combined, the two modulatory inputs have a complex effect. Dopamine and cardiac sac activity cancel one another's effects to restore normal pyloric activity to 4 of the 6 classes of pyloric neurons. In the remaining two pyloric neurons, dopamine's strong modulatory effects are completely overruled during cardiac sac network related activity. Possible cellular mechanisms underlying these interactions are discussed.

Animals↗

PubMed1998

Dopamine modulates graded and spike-evoked synaptic inhibition independently at single synapses in pyloric network of lobster.

Bath application of dopamine (DA) modifies the rhythmic motor pattern generated by the pyloric network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. Synaptic transmission between network members is an important target of DA action. All pyloric neurons employ both graded transmitter release and action-potential-mediated synaptic inhibition. DA was previously shown to alter the graded synaptic strength of every pyloric synapse. In this study, we compared DA's effects on action-potential-mediated and graded synaptic inhibition at output synapses of the lateral pyloric (LP) neuron. At each synapse the postsynaptic cell tested was isolated from other descending and pyloric synaptic inputs. DA caused a reduction in the size of the LP spike-evoked inhibitory postsynaptic potentials (IPSPs) in the pyloric dilator (PD) neuron. The change in IPSP size was significantly and linearly correlated with DA-induced reduction in the input resistance of the postsynaptic PD neuron. In contrast, graded inhibition, tested in the same preparations after superfusing the stomatogastric ganglion (STG) with tetrodotoxin (TTX), was consistently enhanced by DA. DA shifted the amplitude of spike-evoked IPSPs in the same direction as the alteration of the postsynaptic cell input resistance at two additional synapses tested: DA weakened the LP spike-mediated inhibition of the ventricular dilator (VD) and reduced the VD input resistance, while strengthening the LP --> pyloric constrictor (PY) synapse and increasing PY input resistance. As previously reported, graded inhibition was enhanced at these two LP output synapses. We conclude that DA can differentially modulate the spike-evoked and graded components of synapses between members of a central pattern generator network. At the synapses we studied, actions on the presynaptic cell predominate in the modulation of graded transmission, whereas effects on postsynaptic cells predominate in the regulation of spike-evoked IPSPs.

Action Potentials↗

PubMed1998

Expression of Panulirus shaker potassium channel splice variants.

In Drosophila shaker voltage-dependent potassium channels, alternative splicing at the amino and carboxy termini produces currents with different electrophysiological characteristics. We have cloned alternatively spliced forms of shaker from the spiny lobster Panulirus interruptus. Alternative exons were found at three sites of the gene; eight different 5' exons, two alternative exons encoding the pore-forming P region, and an alternative 3' exon. Two of the different amino terminal splice forms were expressed with two alternatively spliced pore forms to produce channels with markedly different characteristics. One of the amino termini produced a channel with transient characteristics while the other produced a delayed rectifier-type channel. The effects of alternative exons at the amino terminus and in the P region appear to be additive. Our results provide new information on the structural requirements for rapid N-type inactivation.

Alternative Splicing↗

PubMed1997

Alternative splicing in the pore-forming region of shaker potassium channels.

We have cloned cDNAs for the shaker potassium channel gene from the spiny lobster Panulirus interruptus. As previously found in Drosophila, there is alternative splicing at the 5' and 3' ends of the coding region. However, in Panulirus shaker, alternative splicing also occurs within the pore-forming region of the protein. Three different splice variants were found within the P region, two of which bestow unique electrophysiological characteristics to channel function. Pore I and pore II variants differ in voltage dependence for activation, kinetics of inactivation, current rectification, and drug resistance. The pore 0 variant lacks a P region exon and does not produce a functional channel. This is the first example of alternative splicing within the pore-forming region of a voltage-dependent ion channel. We used a recently identified potassium channel blocker, kappa-conotoxin PVIIA, to study the physiological role of the two pore forms. The toxin selectively blocked one pore form, whereas the other form, heteromers between the two pore forms, and Panulirus shal were not blocked. When it was tested in the Panulirus stomatogastric ganglion, the toxin produced no effects on transient K+ currents or synaptic transmission between neurons.

Amino Acid Sequence↗

PubMed1997

Quantitative single-cell-reverse transcription-PCR demonstrates that A-current magnitude varies as a linear function of shal gene expression in identified stomatogastric neurons.

Different Shaker family alpha-subunit genes generate distinct voltage-dependent K+ currents when expressed in heterologous expression systems. Thus it generally is believed that diverse neuronal K+ current phenotypes arise, in part, from differences in Shaker family gene expression among neurons. It is difficult to evaluate the extent to which differential Shaker family gene expression contributes to endogenous K+ current diversity, because the specific Shaker family gene or genes responsible for a given K+ current are still unknown for nearly all adult neurons. In this paper we explore the role of differential Shaker family gene expression in creating transient K+ current (IA) diversity in the 14-neuron pyloric network of the spiny lobster, Panulirus interruptus. We used two-electrode voltage clamp to characterize the somatic IA in each of the six different cell types of the pyloric network. The size, voltage-dependent properties, and kinetic properties of the somatic IA vary significantly among pyloric neurons such that the somatic IA is unique in each pyloric cell type. Comparing these currents with the IAs obtained from oocytes injected with Panulirus shaker and shal cRNA (lobster Ishaker and lobster Ishal, respectively) reveals that the pyloric cell IAs more closely resemble lobster Ishal than lobster Ishaker. Using a novel, quantitative single-cell-reverse transcription-PCR method to count the number of shal transcripts in individual identified pyloric neurons, we found that the size of the somatic IA varies linearly with the number of endogenous shal transcripts. These data suggest that the shal gene contributes substantially to the peak somatic IA in all neurons of the pyloric network.

Animals↗

PubMed1997

Amine modulation of glutamate responses from pyloric motor neurons in lobster stomatogastric ganglion.

The amines dopamine (DA), serotonin (5-HT), and octopamine (Oct) each elicit a distinctive motor pattern from a quiescent pyloric network in the lobster stomatogastric ganglion (STG). We previously have demonstrated that these amines alter the synaptic strength at multiple, distributed sites within the pyloric network that could contribute to the amine-induced motor patterns. Here, we examined the postsynaptic contribution to these changes in synaptic strength by determining how the amines modify responses of pyloric motor neurons to glutamate (Glu), one of the network transmitters, applied iontophoretically into the STG neuropil. Dopamine reduced the Glu responses of the pyloric dilator (PD), ventricular dilator (VD), and inferior cardiac (IC) neurons and enhanced the Glu responses of the lateral pyloric (LP) and pyloric constrictor (PY) neurons. The only effect of 5-HT was to reduce the Glu response of the VD neuron. Oct enhanced the Glu responses of the LP and PY neurons but did not affect the PD, VD, and IC responses. We also examined amine effects on the depolarizing responses to iontophoresed acetylcholine (ACh) in the PD and VD and found that they paralleled the amine effects on Glu responses in these neurons. This suggests that amine modulation of PD and VD responses to Glu and ACh may be explained by general changes in the ionic conductance of these neurons. We compare our results with our earlier work describing amine effects on synaptic strength and input resistance to show that amines act at both pre- and postsynaptic sites to modify graded synaptic transmission in the pyloric network.

Acetylcholine↗

PubMed1996

The lobster shaw gene: cloning, sequence analysis and comparison to fly shaw.

We cloned and sequenced the cDNA for the shaw gene, encoding a voltage-dependent potassium (K+) channel, from the spiny lobster, Panulirus interruptus. The deduced amino acid sequence has a high degree of homology to the Drosophila melanogaster Shaw protein. In addition, lobster Shaw has several putative sites for post-translational modifications.

Amino Acid Sequence↗

PubMed1996

Lobster shal: comparison with Drosophila shal and native potassium currents in identified neurons.

The transient potassium (K+) current, or A-current (IA), plays an essential role in shaping the firing properties of identified neurons in the 14-cell pyloric network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. The different cells in the pyloric network have distinct IAs. To begin to understand the molecular basis for IA heterogeneity, we examined the relationship between the Panulirus shal current, the IAs in the lateral pyloric (LP) and pyloric dilator (PY) cells, and the Drosophila shal current. After isolating a complete open reading frame for lobster shal 1, which shows significant sequence homology to the fly, mouse, and rat shal homologs, we used a single-cell reverse transcription polymerase chain reaction method to demonstrate that the shal 1 gene was expressed in the LP and PY cells. Next, we compared the lobster shal 1 current generated in a Xenopus oocyte expression system to the IAs in the LP and PY neurons as well as to the Drosophila shal current in Xenopus oocytes. While the transient K+ lobster shal 1 current was similar to the IAs in pyloric neurons, a detailed comparison shows that they are not identical and differ in kinetic and voltage-dependent parameters. The highly homologous lobster and fly shal genes also produce currents with some significant similarities and differences in an oocyte expression system.

Amino Acid Sequence↗