Electrophysiology of Ctenophore Smooth Muscle.

Unlike in the Cnidaria, where muscle cells are coupled together into an epithelium, ctenophore muscles are single, elongated, intramesogleal structures resembling vertebrate smooth muscle. Under voltage-clamp, these fibers can be separated into different classes with different sets of membrane ion channels. The ion channel makeup is related to the muscle's anatomical position and specific function. For example, Beroe ovata radial fibers, which are responsible for maintaining the rigidity of the body wall, generate sequences of brief action potentials whereas longitudinal fibers, which are concerned with mouth opening and body flexions, often produce single longer duration action potentials.Beroe muscle contractions depend on the influx of Ca2+. During an action potential the inward current is carried by Ca2+, and the increase in intracellular Ca2+ concentration generated can be monitored in FLUO-3-loaded cells. Confocal microscopy in line scan mode shows that the Ca2+ spreads from the outer membrane into the core of the fiber and is cleared from there relatively slowly. The rise in intracellular Ca2+ is linked to an increase in a Ca2+-activated K+ conductance (KCa), which can also be elicited by iontophoretic Ca2+ injection. Near the cell membrane, Ca2+ clearance monitored using FLUO3, matches the decline in the KCa conductance. For light loads, Ca2+ is cleared rapidly, but this fast system is insufficient when Ca2+ influx is maintained. Action potential frequency may be regulated by the slowly developing KCa conductance.

Phylogenetics of swimming behaviour in Medusozoa: the role of giant axons and their possible evolutionary origin.

Although neural tissues in cnidarian hydroids have a nerve net structure, some cnidarian medusae contain well-defined nerve tracts. As an example, the hydrozoan medusa Aglantha digitale has neural feeding circuits that show an alignment and condensation, which is absent in its relatives Aequorea victoria and Clytia hemisphaerica. In some cases, neural condensations take the form of fast propagating giant axons concerned with escape or evasion. Such giant axons appear to have developed from the fusion of many, much finer units. Ribosomal DNA analysis has identified the lineage leading to giant axon-based escape swimming in Aglantha and other members of the Aglaura clade of trachymedusan jellyfish. The Aglaura, along with sister subclades that include species such as Colobonema sericeum, have the distinctive ability to perform dual swimming, i.e. to swim at either high or low speeds. However, the form of dual swimming exhibited by Colobonema differs both biomechanically and physiologically from that in Aglantha and is not giant axon based. Comparisons between the genomes of such closely related species might provide a means to determine the molecular basis of giant axon formation and other neural condensations. The molecular mechanism responsible may involve 'fusogens', small molecules possibly derived from viruses, which draw membranes together prior to fusion. Identifying these fusogen-based mechanisms using genome analysis may be hindered by the many changes in anatomy and physiology that followed giant axon evolution, but the genomic signal-to-noise ratio may be improved by examining the convergent evolution of giant axons in other hydrozoa, such as the subclass Siphonophora.

Two swimming modes in Trachymedusae; bell kinematics and the role of giant axons.

Although members of the Rhopalonematidae family (Cnidaria, Hydrozoa, Trachymedusae) are known to exhibit unusually powerful jet swimming in addition to their more normal slow swimming behaviour, for the most part, reports are rare and anecdotal. Many species are found globally at depths of 600-2000 m, and so observation and collection depend on using remotely operated submersible vehicles. With a combination of in situ video footage and laboratory measurements, we have quantified kinematic aspects of this dual swimming motion and its electrophysiology. The species included are from two Rhopalonematidae clades; they are Colobonema sericeum, Pantachogon haeckeli, Crossota millsae and two species of Benthocodon. Comparison is made with Aglantha digitale, a species from a third Rhopalonematidae clade brought to the surface by natural water movement. We find that although all Rhopalonematidae appear to have two swimming modes, there are marked differences in their neural anatomy, kinematics and physiology. Giant motor axons, known to conduct impulses during fast swimming in A. digitale, are absent from C. sericeum and P. haeckeli. Slow swimming is also different; in C. sericeum and its relatives it is driven by contractions restricted to the base of the bell, whereas in A. digitale it is driven by contractions in the mid-bell region. These behavioural differences are related to the position of the different clades on a ribosomal DNA-based phylogenetic tree. This finding allows us to pinpoint the phylogenetic branch point leading to the appearance of giant motor axons and escape swimming. They place the remarkable dual swimming behaviour of members of the Rhopalonematidae family into an evolutionary context.

Structure and function of the nervous system in nectophores of the siphonophore Nanomia bijuga.

Although the bell-shaped nectophores of the siphonophore Nanomia bijuga are clearly specialized for locomotion, their complex neuroanatomy described here testifies to multiple subsidiary functions. These include secretion, by the extensively innervated 'flask cells' located around the bell margin, and protection, by the numerous nematocytes that line the nectophore's exposed ridges. The main nerve complex consists of a nerve ring at the base of the bell, an adjacent column-shaped matrix plus two associated nerve projections. At the top of the nectophore the upper nerve tract appears to have a sensory role; on the lower surface a second nerve tract provides a motor input connecting the nectophore with the rest of the colony via a cluster of nerve cells at the stem. N. bijuga is capable of both forward and backward jet-propelled swimming. During backwards swimming the water jet is redirected by the contraction of the Claus' muscle system, part of the muscular velum that fringes the bell aperture. Contractions can be elicited by electrical stimulation of the nectophore surface, even when both upper and lower nerve tracts have been destroyed. Epithelial impulses elicited there, generate slow potentials and action potentials in the velum musculature. Slow potentials arise at different sites around the bell margin and give rise to action potentials in contracting Claus' muscle fibres. A synaptic rather than an electrotonic model more readily accounts for the time course of the slow potentials. During backward swimming, isometrically contracting muscle fibres in the endoderm provide the Claus' fibres with an immobile base.

Electrogenesis in the lower Metazoa and implications for neuronal integration.

Electrogenic communication appears to have evolved independently in a variety of animal and plant lineages. Considered here are metazoan cells as disparate as the loose three-dimensional parenchyma of glass sponges, the two-dimensional epithelial sheets of hydrozoan jellyfish and the egg cell membranes of the ctenophore Beroe ovata, all of which are capable of generating electrical impulses. Neuronal electrogenesis may have evolved independently in ctenophores and cnidarians but the dearth of electrophysiological data relating to ctenophore nerves means that our attention is focused on the Cnidaria, whose nervous systems have been the subject of extensive study. The aim here is to show how their active and passive neuronal properties interact to give integrated behaviour. Neuronal electrogenesis, goes beyond simply relaying 'states of excitement' and utilizes the equivalent of a set of basic electrical 'apps' to integrate incoming sensory information with internally generated pacemaker activity. A small number of membrane-based processes make up these analogue applications. Passive components include the decremental spread of current determined by cellular anatomy; active components include ion channels specified by their selectivity and voltage dependence. A recurring theme is the role of inactivating potassium channels in regulating performance. Although different aspects of cnidarian behaviour are controlled by separate neuronal systems, integrated responses and coordinated movements depend on interactions between them. Integrative interactions discussed here include those between feeding and swimming, between tentacle contraction and swimming and between slow and fast swimming in the hydrozoan jellyfish Aglantha digitale.

Nerves in the endodermal canals of hydromedusae and their role in swimming inhibition.

N eoturris breviconis (Anthomedusae) has a nerve plexus in the walls of its endodermal canals. The plexus is distinct from the ectodermal nerve plexuses supplying the radial and circular muscles in the ectoderm and no connections have been observed between them. Stimulation of the endodermal plexus evokes electrical events recorded extracellularly as "E" potentials. These propagate through all areas where the plexus has been shown by immunohistology to exist and nowhere else. When Neoturris is ingesting food, trains of "E" potentials propagate down the radial canals to the margin and cause inhibition of swimming. This response is distinct from the inhibition of swimming associated with contractions of the radial muscles but both may play a part in feeding and involve chemoreceptors. Preliminary observations suggest that the "E" system occurs in other medusae including Aglantha digitale (Trachymedusae) where the conduction pathway was previously thought to be an excitable epithelium.

Non-neural reflexes: sponges and the origins of behaviour.

Sponges 'sneeze' without the benefit of nerves or muscles. While genomic analysis has uncovered a surprisingly complex set of molecular components in these ancient metazoans, physiological studies have revealed equally sophisticated cellular coordination.

A conductive pathway generated from fragments of the human red cell anion exchanger AE1.

Human red cell anion exchanger AE1 (band 3) is an electroneutral Cl-HCO3- exchanger with 12-14 transmembrane spans (TMs). Previous work using Xenopus oocytes has shown that two co-expressed fragments of AE1 lacking TMs 6 and 7 are capable of forming a stilbene disulphonate-sensitive (36)Cl-influx pathway, reminiscent of intact AE1. In the present study, we create a single construct, AE1Delta(6: 7), representing the intact protein lacking TMs 6 and 7. We expressed this construct in Xenopus oocytes and evaluated it employing a combination of two-electrode voltage clamp and pH-sensitive microelectrodes. We found that, whereas AE1Delta(6: 7) has some electroneutral Cl-base exchange activity, the protein also forms a novel anion-conductive pathway that is blocked by DIDS. The mutation Lys(539)Ala at the covalent DIDS-reaction site of AE1 reduced the DIDS sensitivity, demonstrating that (1) the conductive pathway is intrinsic to AE1Delta(6: 7) and (2) the conductive pathway has some commonality with the electroneutral anion-exchange pathway. The conductance has an anion-permeability sequence: NO3- approximately I- > NO2- > Br- > Cl- > SO4(2-) approximately HCO3- approximately gluconate- approximately aspartate- approximately cyclamate-. It may also have a limited permeability to Na+ and the zwitterion taurine. Although this conductive pathway is not a usual feature of intact mammalian AE1, it shares many properties with the anion-conductive pathways intrinsic to two other Cl-HCO3- exchangers, trout AE1 and mammalian SLC26A7.

Nitric oxide regulates swimming in the jellyfish Aglantha digitale.

The cnidarian nervous system is considered by many to represent neuronal organization in its earliest and simplest form. Here we demonstrate, for the first time in the Cnidaria, the neuronal localization of nitric oxide synthase (NOS) in the hydromedusa Aglantha digitale (Trachylina). Expression of specific, fixative-resistant NADPH-diaphorase (NADPH-d) activity, characteristic of NOS, was observed in neurites running in the outer nerve ring at the base of the animal and in putative sensory cells in the ectoderm covering its tentacles. At both sites, diphenyleneiodonium (10(-4) M) abolished staining. Capillary electrophoresis confirmed that the NO breakdown products NO2- and NO3- were present at high levels in the tentacles, but were not detectable in NADPH-d-negative areas. The NADPH-d-reactive neurons in the tentacles send processes to regions adjacent to the inner nerve ring where swimming pacemaker cells are located. Free-moving animals and semi-intact preparations were used to test whether NO is involved in regulating the swimming program. NO (30-50 nM) and its precursor L-arginine (1 mM) stimulated swimming, and the effect was mimicked by 8-Br-cGMP (50-100 microM). The NO scavenger PTIO (10-100 microM) and a competitive inhibitor of NOS, L-nitroarginine methyl ester (L-NAME, 200 microM), significantly decreased the swimming frequency in free-moving animals, while its less-active stereoisomer D-nitroarginine methyl ester (D-NAME, 200 microM) had no such effect. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 5-20 microM), a selective inhibitor of soluble guanylyl cyclase, suppressed spontaneous swimming and prevented NO-induced activation of the swimming program. We suggest that an NO/cGMP signaling pathway modulates the rhythmic swimming associated with feeding in Aglantha, possibly by means of putative nitrergic sensory neurons in its tentacles.

Fast Ca2+ signals at mouse inner hair cell synapse: a role for Ca2+-induced Ca2+ release.

Inner hair cells of the mammalian cochlea translate acoustic stimuli into 'phase-locked' nerve impulses with frequencies of up to at least 1 kHz. Little is known about the intracellular Ca2+ signal that links transduction to the release of neurotransmitter at the afferent synapse. Here, we use confocal microscopy to provide evidence that Ca2+-induced Ca2+ release (CICR) may contribute to the mechanism. Line scan images (2 ms repetition rate) of neonatal mouse inner hair cells filled with the fluorescent indicator FLUO-3, revealed a transient increase in intracellular Ca2+ concentration ([Ca2+]i) during brief (5-50 ms) depolarizing commands under voltage clamp. The amplitude of the [Ca2+]i transient depended upon the Ca2+ concentration in the bathing medium in the range 0-1.3 mM. [Ca2+]i transients were confined to a region near the plasma membrane at the base of the cell in the vicinity of the afferent synapses. The change in [Ca2+]i appeared uniform throughout the entire basal sub-membrane space and we were unable to observe hotspots of activity. Both the amplitude and the rate of rise of the [Ca2+]i transient was reduced by external ryanodine (20 microM), an agent that blocks Ca2+ release from the endoplasmic reticulum. Intracellular Cs+, commonly used to record at presynaptic sites, produced a similar effect. We conclude that both ryanodine and intracellular Cs+ block CICR in inner hair cells. We discuss the contribution of CICR to the measured [Ca2+]i transient, the implications for synaptic transmission at the afferent synapse and the significance of its sensitivity to intracellular Cs+.