Organic osmolytes are small solutes used by cells of numerous water-stressed organisms and tissues to maintain cell volume. All known osmolytes are amino acids and derivatives, polyols and sugars, methylamines, and urea; unlike salt ions, most are “compatible,” i.e., do not perturb macromolecules. In addition, some stabilize macromolecules and are “counteracting” towards perturbants, e.g., methylamines can stabilize proteins and ligand binding against perturbations by urea in elasmobranchs and mammalian kidney, and (our latest findings) high hydrostatic pressure in deep-sea animals. Methylamines appear to coordinate water molecules tightly, resulting in osmolyte exclusion from hydration layers of peptide backbones. This makes unfolded protein conformations entropically unfavorable (work of Timasheff, Galinski, Bolen and coworkers). These properties have led to proposed uses in biotechnology, agriculture and medicine, including improved biochemical methods, in vitro rescue of misfolded proteins in cystic fibrosis and prion diseases (work of Welch and others), and plants engineered for drought and salt tolerance. These properties also explain some but not all of the considerable variation in osmolyte composition among species with different metabolisms and habitats, and among and within mammalian tissues in development.

The control of cell volume in all cell types is accomplished by the regulation of two general categories of osmolytes: inorganic ions, most commonly K and Cl⁻, and small molecular weight organic compounds, usually certain amino acids and certain quaternary ammonium compounds. The difference in who regulates what does not depend phylogeny, but instead upon the type of osmotic environment that a cell expects (in an evolutionary sense) to encounter. Cells that exist in extracellular osmotic concentrations up to 300–400 mosmol/kg (mosm) rely primarily on inorganic osmolytes for volume control, while cells that exist at greater osmotic concentrations rely more on organic osmolytes for volume control. Usually, strange or unique volume regulatory mechanisms are found in cells that exist in particularly demanding osmotic conditions. In order to provide further support the foregoing generalizations, the following paper will focus on comparisons between the hypoosmotically induced mechanism of taurine efflux regulation by red blood cells of the bivalve, Noetia ponderosa, probably the best understood “invertebrate” cell type in this regard, and taurine efflux from a variety of “vertebrate” cells.

Extrapolating from a body of work on isolated neurons, a model is suggested for how central nervous system (CNS) neurons in situ may handle local swelling and shrinking perturbations so as to interfere minimally with the primary role of neurons, synaptic information processing. The strategy used for osmoregulatory membrane adjustments may first come into play during ontogeny, when it could provide membrane tension sensitive membrane trafficking in arborized neurons subjected to morphogenetic forces.

In the mammalian kidney renal medullary cells use organic osmolytes such as sorbitol, myo-inositol, glycerophosphorylcholine, betaine, and taurine to adjust their intracellular osmolarity (and thereby their volume) to rapid and drastic changes in extracellular osmolarity. Using an immortalized cell line derived from rabbit thick ascending limb of Henle's loop (TALH cells) and primary cultures of rat inner medullary collecting duct (IMCD cells) the membrane transport systems activated during exposure to hypotonicity were investigated. In TALH cells an increase in sorbitol permeability of the (luminal) plasma membrane occurs by activation of a channel-like transporter involving a calcium/calmodulin-dependent protein kinase. A similar system seems to operate in IMCD cells. In addition, the latter cells possess a swelling-activated anion channel that is also permeable for taurine and myo-inositol and inhibited by “anion channel” blockers, such as NPPB and DIDS. The sorbitol permeability of the plasma membrane appears to be furthermore regulated by a transient insertion of active transporters into the basolateral cell surface by a membrane recycling mechanism.

Evolutionarily, adaptation to hyperosmotic stress through accumulation of osmotically active organic solutes (organic osmolytes) is a highly conserved mechanism. Hyperosmotic accumulation of organic osmolytes is transcriptionally regulated: e.g., betaine in bacteria (e.g., Escherichia coli), glycerol in yeast (e.g., Saccharomyces cerevisiae), betaine in plants (e.g., Spinacea oleracea L.) and sorbitol, betaine, and inositol in cells of the mammalian renal medulla. Renal medullary cells, among mammalian cells, are uniquely exposed to hyperosmotic stress; in these cells, hyperosmotic stress results in accumulation of sorbitol as one of the predominant osmolytes. Sorbitol accumulates due to a rise in the synthesis rate of aldose reductase (AR), which catalyzes the conversion of glucose to sorbitol. Hyperosmotic stress increases transcription of the AR gene which leads to a rise in AR mRNA levels. In cloning and characterizing the rabbit AR gene, the first evidence of a eukaryotic osmotic response element (ORE) was found. Since then, several mammalian OREs (also called TonE) have been discovered. Sequence containing an ORE was identified for the canine Na - and Cl⁻-coupled betaine transporter gene as well as the Na /myo-inositol cotransporter gene. Because it is possible to find homology between the OREs of the AR genes and those of the betaine and inositol genes, a consensus for the mammalian ORE was derived by functional assessment. Most recent studies have resulted in the discovery of other cis-elements that potentiate the ORE response and a trans-activating factor that binds to the ORE.

Mitogen-activated protein (MAP) kinases constitute a large family of proteins with many functions. They are represented by a multitude of paralogous isoforms in yeast, vertebrates, and other eukaryotes. A phylogenetically conserved function of MAP kinases is to carry osmotic signals from sensory to target elements of cells. Even though this function of MAP kinases is ubiquitous and characteristic of unicellular and multicellular eukaryotes alike the contingencies between individual MAP kinases, sensor elements, and target elements have been subject to vast modification during evolution. Extensive networking of MAP kinase cascades with other signaling pathways is reflected by the large number of diverse signals that can be carried by a single MAP kinase pathway and flexible activation kinetics. It is emerging that the most important function of MAP kinase networks may not be signal amplification but integration of information about the setpoint of environmental parameters (including osmolality) with other physiological processes to control cell function. Insight into how this cellular integration of information is achieved by MAP kinase networks will shed light on the principles of cell dynamics and adaptation.

Drought and salinity stress are the major causes of historic and modern agricultural productivity losses throughout the world. Both drought and salinity result in osmotic stress that may lead to inhibition of growth. Salinity causes additional ion toxicity effects mainly through perturbations in protein and membrane structure. In contrast to animals, which rely on Na /K -ATPases for the expulsion of osmotica, plants rely on plasma membrane and endosomal ATPase activities to generate proton gradients to drive ion extrusion and intracellular sequestration. Consequently, most angiosperms, including all major crop species, have a diminished capacity for Na transport and tolerance to high salinity. New insights into the molecular mechanisms of Na /K discrimination, Na extrusion and compartmentation, water transport, and osmolyte biosynthesis and function have led to genetically engineered plants with improved salt, drought, and cold tolerance. A deeper understanding of the complex signal transduction and regulatory responses to osmotic stress promises novel strategies for improving salinity and drought tolerance that will be of practical benefit to agriculture.

The physiological mechanisms by which aquatic animals regulate the osmoconcentration of their body fluids remain unclear despite many excellent studies of tissue and cell function. This review summarizes the current status of an ongoing molecular biological approach to investigating transporters and transport-related enzymes in ion-transporting gills of osmoregulating crustaceans. We have identified cDNAs coding for six candidate proteins in gills of the blue crab Callinectes sapidus and the green shore crab Carcinus maenas, including a Na K -ATPase α-subunit, a V-type H -ATPase B-subunit, a Na /H exchanger, a Na /K /2Cl⁻ cotransporter, two isoforms of carbonic anhydrase, and arginine kinase. Although our account is far from complete, examination of mRNA abundance by quantitative reverse transcription/polymerase chain reaction (RT/PCR) has identified candidates that are preferentially expressed in gill epithelium, including the Na K -ATPase α-subunit and Na /H exchanger. The osmoregulatory response to salinity reduction includes enhanced mRNA expression of at least one form of carbonic anhydrase.

As the primary link between environmental change and physiological response, the neuroendocrine system is a critical part of osmoregulatory adaptations. Cortisol has been viewed as ‘the’ seawater-adapting hormone in fish and prolactin as ‘the’ fresh water adapting hormone. Recent evidence indicates that the growth hormone/insulin-like growth factor I axis is also important in seawater adaptation in several teleosts of widely differing evolutionary lineages. In salmonids, growth hormone acts in synergy with cortisol to increase seawater tolerance, at least partly through the upregulation of gill cortisol receptors. Cortisol under some conditions may promote ion uptake and interacts with prolactin during acclimation to fresh water. The osmoregulatory actions of growth hormone and prolactin are antagonistic. In some species, thyroid hormones support the action of growth hormone and cortisol in promoting seawater acclimation. Although a broad generalization that holds for all teleosts is unlikely, our current understanding indicates that growth hormone promotes acclimation to seawater, prolactin promotes acclimation to fresh water, and cortisol interacts with both of these hormones thus having a dual osmoregulatory function.

Biologists must remember that physiology is the product of natural selection on organisms interacting with heterogeneous environments. “Behaving” organisms may alter the osmotic conditions they experience and achieve results unexpected from laboratory studies. Their ability to exploit environmental heterogeneity depends on its temporal/spatial scale relative to that of the organism, and the correspondence between the osmotic differences and the organism's sensory and osmoregulatory physiology. “Behaviors” include evasion of stressful habitats, selection among differing microenvironments, changing body characteristics that affect salt/water uptake/loss, manipulating fluids differing in osmolytes, and modification of osmotic microenvironments (especially for vulnerable offspring). To draw “comparative and integrative” inferences, investigators must strive to understand an organism's actual challenges by “seeing” the world from its perspective, and then making observations and performing experiments in the context of the “real world” experienced by that organism.

Ecologists have long been interested in factors that control the structure of communities and the relative importance of top-down effects of predators versus bottom-up effects of resources. There is a growing body of evidence that microbial symbioses are important determinants of plant community structure and indirectly affect herbivore and predator trophic levels. Studies with mycorrhizal fungi, N-fixing bacteria and endophytes of grasses have demonstrated that they can affect competition, coexistence, soil nutrient dynamics and plant-herbivore interactions. Long-term field experiments with one grass/endophyte interaction suggest that grassland community structure is determined by the fungus. While total plant productivity of experimental plots was similar, the composition of the vegetation was altered by endophyte symbiosis. The host grass tall fescue (Festuca arundinacea) dominated plots when infected while other grasses greatly increased in uninfected plots. Indirect evidence suggests that changes in prairie vole (Microtus ochrogaster) grazing patterns and reproductive physiology may be in part responsible for vegetational changes. These results provide evidence that, in addition to bottom-up and top-down forces, microbial symbionts of plants are important determinants of community structure.

Mutualisms are of central importance in biological systems. Despite growing attention in recent years, however, few conceptual themes have yet to be identified that span mutualisms differing in natural history. Here I examine the idea that the ecology and evolution of mutualisms are shaped by diverse costs, not only by the benefits they confer. This concept helps link mutualism to antagonisms such as herbivory, predation, and parasitism, interactions defined largely by the existence of costs. I first briefly review the range of costs associated with mutualisms, then describe how one cost, the consumption of seeds by pollinator offspring, was quantified for one fig/pollinator mutualism. I compare this cost to published values for other fig/pollinator mutualisms and for other kinds of pollinating seed parasite mutualisms, notably the yucca/yucca moth interaction. I then discuss four issues that fundamentally complicate comparative studies of the cost of mutualism: problems of knowing how to measure the magnitude of any one cost accurately; problems associated with using average estimates in the absence of data on sources of variation; complications arising from the complex correlates of costs, such as functional linkages between costs and benefits; and problems that arise from considering the cost of mutualism as a unilateral issue in what is fundamentally a reciprocal interaction. The rich diversity of as-yet unaddressed questions surrounding the costs of mutualism may best be investigated via detailed studies of individual interactions.

We have shown that eastern gray squirrels and other animals consistently prefer to store intact acorns from the red oak group rather than those from the white oak group. We hypothesized that the ultimate advantage to this behavior comes from the dormancy of red oak acorns. Acorns of the white oak group germinate early in the autumn; thus, we hypothesize that avoiding germination is the primary selective advantage to the preference for caching red oak acorns. Here, we test two alternative hypotheses to explain the benefits of this caching preference: 1) storing red oak acorns allows the high tannin concentrations in red oak acorns to decline (making them more palatable), and 2) storing red oak acorns minimizes losses to insects, presuming they are less infested with insects. We also test the effect of germination schedule on squirrel caching preferences directly, by presenting them with dormant red oak acorns, and red oak acorns about to germinate. We find no evidence that tannin concentrations in red oak acorns decline, although tannin levels did decline in our white oak acorns. We found high losses to insect infestations in one white oak species, but a second white oak species lost very little mass to insects. Finally, we found that germination schedule directly affects squirrel caching preferences: red oak acorns that are near germination are treated like white oak acorns. We conclude that the primary advantage to the preference for caching red oak acorns is that they are less perishable, due to their dormancy. We discuss the effects of this preference on the dispersal of red and white oak acorns, and the subsequent effects of differential dispersal on the ecology and evolution of oaks.

Previously we have shown how a range of physical and chemical characteristics of acorns influences the behavioral decisions of food-hoarding rodents which in turn affects the dispersal, establishment and spatial arrangement of oaks. One such behavior involves the selective caching of acorns of red oaks (subgenus: Erythrobalanus) over those of white oaks (Quercus) because of reduced perishability that results from delayed germination of acorns in the red oak group. In this study, we sought to identify the specific proximate cues (visual and olfactory) that eastern gray squirrels (Sciurus carolinensis) use when making these decisions. In two series of field experiments, we presented individual, free-ranging animals with pairs of experimentally altered acorns (that differed with respect to a single chemical or visual characteristic) and recorded their feeding and caching responses. Squirrels cached artificial acorns with pericarps (shells) of red oak acorns and ate those with shells of white oak regardless of the internal chemical composition of either type of acorn. Only when the shells of artificial acorns were first soaked in acetone (to remove potential chemical odors) did animals eat artificial acorns made with the shells of red oak acorns. Squirrels also ate one-year old red oak acorns that had broken dormancy, even when they exhibited no signs of germination. We argue that a chemical cue in the shell of acorns is important in the detection of seed dormancy and the decision to cache acorns, and that such a cue might ultimately contribute to the differential dispersal of red and white oaks by rodents.

The Cactus-Microorganism-Drosophila Model System of the Sonoran Desert represents an excellent paradigm of the role of chemistry in plant-animal interactions. In this system, four species of endemic Drosophila feed and reproduce in necrotic tissue of five species of columnar cacti. Studies over the past 35 yr have characterized a myriad of interactions between the three major components of the model system. The cacti contain a variety of allelochemicals which are primarily responsible for the highly specific pattern of host plant utilization exhibited by the desert Drosophila. Plant chemistry, through its effect on the microbially produced volatile patterns, is further involved in host specificity because the flies use the volatile pattern to cue in on necroses in the appropriate species of cactus. The metabolic activities of microorganisms (bacteria and yeasts) living in the necrosis can affect the substrate chemistry in both positive and negative ways (i.e., acting to increase or to decrease the toxicity of the substrate). Finally, cactus chemistry may affect drosophilid mating behavior since larval rearing substrate has been shown to influence adult hydrocarbon epicuticular composition. In D. mojavensis, adult hydrocarbon profile has been implicated as a determinant of mate choice leading to premating isolation between geographically isolated populations that use chemically different cactus substrates. Current research is focused on the evolution and regulation of genes whose products (cytochrome P450 enzymes) are involved in the specific insect-host plant relationships which exist between the Drosophila species and the cactus species.

There are many reasons why investigators choose to focus their research efforts on what are referred to as “model systems.” Typically included among these would be the idea that model systems are easier to study because they are less complex than other scientific situations. At the same time, model systems should be representative of more complex, natural systems so that information that is obtained from their study is broadly applicable. For almost a century, the fruit fly, Drosophila melanogaster, has served as a model organism for the study of genetics. As a genetic paradigm, Drosophila is more tractable to scientific investigation than most organisms and has provided important insights into a wide variety of human maladies from alcohol abuse to neurological brain disorders (Bellen, 1998). Similarly, the interrelationships of the columnar cacti and the cactophilic Drosophila species of the Sonoran Desert have, for the past 35 yr, provided an excellent model system with which to study relevant questions in evolution, ecological genetics, and chemical ecology. The intent of this article is to briefly review and characterize the chemical interactions between the plants (cacti) and animals (Drosophila) of this model system, and, in addition, provide some thoughts on possible future directions for integrative approaches in this research area.

Plant secondary compounds are deterrents and toxins to a variety of herbivores. The effect of secondary compounds on water balance of herbivores is virtually unexplored, yet many secondary compounds are renowned for their diuretic effects in humans and laboratory rats. We review data from the ethnopharmocological literature on plants with diuretic effects. We also present our data from experiments on water intake of specialist (Neotoma stephensi) and generalist woodrats (N. albigula) consuming plant secondary compounds from their natural diet. We measured effects of dietary secondary compounds on voluntary water consumption, urine volume and urine osmolarity. Ingestion of secondary compounds increased water intake and urine output and decreased urine osmolarity in both species. However, the generalist was more impacted by dietary secondary compounds than the specialist. Our results combined with that from the literature suggest that diuresis may be a prevalent consequence of ingestion of secondary compounds. Many herbivores live in arid habitats with limited access to free-standing water, thus an increase in the desire for water may have profound consequences on foraging behavior and fitness.

Nectar-feeding vertebrates respond to variation in nectar sugar content by modulating volumetric intake. In some nectar feeding animals, the intake response to sugar concentration can be accurately predicted from simple mathematical models that rely on knowledge of gut morphology, in vitro rates of sugar digestion, and daily energy expenditures. Because most of the floral nectars consumed by vertebrates are dilute, these animals ingest large amounts of water while feeding. The water turnover rates of hummingbirds feeding on dilute nectar are more similar to those of amphibious and aquatic organisms than to those of terrestrial vertebrates. Dilute nectars can pose osmoregulatory challenges for nectarivores. Nectarivorous birds exhibit renal traits that are well suited to dispose of large water loads and that appear inadequate to produce concentrated urine. Nectar-feeding birds prefer concentrated over dilute sugar solutions. However, the concentration difference that they can discriminate is smaller at low than at high concentration. We hypothesize that this pattern is a consequence of the functional form of intake responses that often results in decelerating sugar intakes with increasing sugar concentration. The diminishing returns in floral attractivity that may result from increased nectar concentration may be one of the reasons why the nectars of hummingbird pollinated flowers are dilute in spite of the preference of birds for higher concentrations. The intake responses of nectar-feeding birds capture the integration of a behavioral response with the physiological processes that shape it. Because the behavior of nectar-feeding birds can have consequences for the plants that they visit, the intake response may also have coevolutionary effects.

Almost forty years ago, Ehrlich and Raven (1964) hypothesized that the great diversity of plants and the herbivores that feed on them arose from a process of coevolution. Plants do possess an amazing diversity of traits that are easily imagined as having arisen from an antagonistic interaction between plants and herbivores. Two basic assumptions lie at the root of most theories of coevolution between plants and their herbivores. First, herbivores are agents of natural selection on plant resistance traits. Second, plants incur a significant fitness cost for possessing these resistance traits. An ecological genetic approach can provide rigorous evidence for these coevolutionary assumptions. In this paper, I present new experimental work on the subject of costs of resistance and review and discuss my own previous work bearing directly on these questions. Using both field experiments on natural populations of the mouse-ear cress (Arabidopsis thaliana) and laboratory experiments using genetically modified plants, I demonstrate that herbivores are exerting selection on both a chemical and physical resistance trait and that there are significant fitness costs to possessing these two traits. These results provide direct confirmation that our current models of the evolution of plant defenses are appropriate.

Host shifts and subsequent host-race formation likely play a more common role in the speciation of herbivorous insects than has generally been recognized. Our studies of the interactions of goldenrod host plants (Solidago: Compositae), the gall fly Eurosta solidaginis (Diptera: Tephritidae), and the stem- and gall-boring Mordellistena convicta (Coleoptera: Mordellidae) provide behavioral, ecological, and genetic evidence of insect host races that may represent incipient species formed via sympatric speciation. Eurosta solidaginis has developed genetically differentiated and reproductively isolated host races that are associated with the ancestral host Solidago altissima and the derived host S. gigantea. Conventional wisdom suggests that shifts even to closely related host plants are limited by host preferences or the inability to utilize a chemically and developmentally distinct host. However, our preliminary work with Eurosta from S. gigantea implies that host choice and gall induction do not deter a shift to S. canadensis. The galling of Solidago by Eurosta created a new resource that has led to a subsequent host range expansion by the stem-boring beetle. Mordellistena convicta from stems and galls are genetically distinct and likely shifted from stems to galls. Beetles from S. altissima versus S. gigantea galls exhibit assortative mating and higher preference for and/or performance on their natal host. The present-day distributions of the Eurosta host races and their behavioral isolating mechanisms do not suggest that geographic isolation was required for their formation; rather these characteristics suggest a sympatric mode of differentiation. Our findings lend credence to recent assertions that sympatric speciation may be an important source of biodiversity.

After opisthobranch molluscs dislodge from the substrate during onset of swimming, the ensuing flexion or undulatory motions are usually not well oriented with respect to predators, prey or suitable substrate. Swimming motions are effective in launching animals off the substrate and elevating them into the water column where they are primarily transported passively by ambient waves and tidal currents. Both active swimming and passive transport on ambient currents may provide escape from predators, search for food and mates, and dispersal to new and potentially adaptive locations. However, loss of contact with the substrate and launching into the water column may also bring a high cost in terms of exposure to diverse risks. I illustrate several forms of opisthobranch swimming and describe their mechanisms and roles. In addition, adaptations of some opisthobranchs to reduce the risks of exposure to predators during swimming are suggested. These adaptations include small size, transparency or inconspicuous color to reduce predation while swimming, and neurobehavioral mechanisms of rheotaxis and geomagnetic sensitivity.

Two groups of interneurons, Tr1 and DRI, have been identified in the escape swim circuit of the marine mollusc Tritonia diomedea that have important roles in behavioral initiation. DRI functions as a command neuron, receiving direct excitatory input from the afferent neurons, and in turn directly exciting the DSI neurons of the central pattern generator. DRI fires throughout the swim motor program, and activity in DRI is both necessary and sufficient for sensory input to elicit the swim motor program. Tr1 is an excitatory interneuron that fires briefly in response to sensory input and then remains silent during the motor program. Tr1 excites DRI with an excitatory connection that has fast and slow components and thus appears to have a role in converting brief afferent neuron activity to long-lasting firing in downstream circuit elements. These neurons complete the description of a continuous synaptic pathway from afferent to flexion neurons in the Tritonia swim circuit. Their identification should facilitate studies of motor program initiation, as well as of how various forms of experience, including simple forms of learning, act to influence neuronal decision-making processes.

Comparisons of the nervous systems of closely related invertebrate species show that identified neurons tend to be highly conserved even though the behaviors in which they participate vary. All opisthobranch molluscs examined have a similar set of serotonin-immunoreactive neurons located medially in the cerebral ganglion. In a small number of species, these neurons have been physiologically and morphologically identified. In the nudibranch, Tritonia diomedea, three of the neurons (the dorsal swim interneurons, DSIs) have been shown to be members of the central pattern generator (CPG) underlying dorsal/ventral swimming. The DSIs act as intrinsic neuromodulators, altering cellular and synaptic properties within the swim CPG circuit. Putative homologues of the DSIs have been identified in a number of other opisthobranchs. In the notaspid, Pleurobranchaea californica, the apparent DSI homologues (As1–3) play a similar role in the escape swim and they also have widespread actions on other systems such as feeding and ciliary locomotion. In the gymnosomatid, Clione limacina, the presumed homologous neurons (Cr-SP) are not part of the swimming pattern generator, which is located in the pedal ganglia, but act as extrinsic modulators, responding to noxious stimuli and increasing the frequency of the swim motor program. Putative homologous neurons are also present in non-swimming species such as the anaspid, Aplysia californica, where at least one of the cerebral serotonergic neurons, CC3 (CB-1), evokes neuromodulatory actions in response to noxious stimuli. Thus, the CPG circuit in Tritonia appears to have evolved from the interconnections of neurons that are common to other opisthobranchs where they participate in arousal to noxious stimuli but are not rhythmically active.

The within-swim pattern of cycle periods in Tritonia swimming changed when the behavior was repeatedly elicited suggesting that an excitatory process reaches a ceiling or wanes over repeated trials. Exposure to subthreshold stimuli enhanced swimming in response to a subsequent super-threshold stimulus, perhaps using a similar excitatory process. In reduced preparations, subthreshold stimuli increased action potential activity in identified serotonergic neurons. Finally, stimulating serotonergic neurons enhanced a fictive swimming pattern, much like subthreshold stimuli enhanced the swimming behavior. Both within-swim and across-swim changes in the swimming behavior may be caused by increased activity in identified serotonergic neurons. Comparative study suggests that ancestral serotonergic systems facilitated network oscillations for the production of rhythmic behaviors such as feeding and locomotion. This concept of serotonin as oscillatizer is used to explain the role of serotonergic neurons in Tritonia. Implications for human mental health are discussed.

The escape swimming pattern generator of the notaspid opisthobranch Pleurobranchaea drives a high threshold, override behavior. The pattern generator is integrated with neural networks of other behaviors so as to coordinate unitary behavioral expression and to promote general behavioral arousal. These functions are separately produced by different swim network elements. One set of swim premotor neurons, the A1/A10 ensemble, A3 and I_VS, generate the swim pattern and, through corollary activity, suppress potentially conflicting feeding behavior by exerting broad inhibition at major feeding network interneurons. A second set of swim neurons, the serotonergic As1–4 neurons, provides intrinsic neuromodulatory excitation to the swim pattern generator that sustains the escape swim episode through multiple cycles. The As1–4 also provide neuromodulatory excitation to important modulatory, serotonergic cells in the feeding motor network and locomotor network, and may have a general regulatory role in the distributed serotonergic arousal network of the mollusk. The As1–4 appear to be also necessary to both avoidance and orienting turning, and are therefore likely to be critical, multi-functional components upon which much of the organization of the animal's behavior rests.

Initiation of a particular behavior requires not only activation of the neural center directly involved in its control but also inhibition of the neural networks controlling competing behaviors. In the pteropod mollusc, Clione limacina, many identified serotonergic neurons activate or modulate different elements of the swimming system resulting in the initiation or acceleration of the swimming behavior. Cerebral serotonergic neurons are described here, which produce excitatory inputs to the swimming system as well as inhibitory inputs to the neural centers that control competing behaviors. Whole-body withdrawal behavior is incompatible with swimming activity in Clione. The main characteristic of whole-body withdrawal is complete inhibition of swimming. Cerebral serotonergic neurons were found to produce a prominent inhibition of the pleural neurons that control whole-body withdrawal behavior. By inhibiting pleural withdrawal cells, serotonergic neurons eliminate its inhibitory influence on the swimming system and thus favor increased swimming speed. Serotonergic neurons also produce a prominent inhibition of the Pleural White Cell, which is presumably involved in reproductive or egg-laying behavior. Thus the serotonergic system directly activates swimming system and, at the same time, alters a variety of other neural systems preventing simultaneous initiation of incompatible behaviors.

Three primary factors contribute to locomotory speed changes in the pteropod mollusk Clione limacina. (1) An increase in cycle frequency of locomotory appendages is associated with a baseline depolarization and enhancement of postinhibitory rebound in central pattern generator (CPG) interneurons, and a reorganization of the CPG through recruitment of additional interneurons. (2) An increase in the force of appendage movements is generated through enhancement of activity of active motoneurons, recruitment of additional motoneurons and peripheral modulation of swim musculature. (3) Changes in biomechanical aspects of appendage movements are presumably achieved, at least in part, through changes in the activity of motoneurons and swim muscle. All changes associated with non-startle swim acceleration are produced by a serotonergic arousal system that acts at all three levels of the swimming system: CPG interneurons, motoneurons and swim musculature.

Muscle fibers in the swim appendages of the mollusk Aplysia brasiliana are innervated by cholinergic motoneurons. Serotonin (5-HT) causes an increase in amplitude of junctional potentials and muscle contractions at this neuromuscular synapse. We studied motoneurons with intracellular current-clamp recording and single-electrode voltage-clamp analysis to determine the effects of 5-HT on somatic currents in these presynaptic neurons. Serotonin was found to have no effect on action potential duration in motoneurons bathed in normal seawater, and no effect of 5-HT could be detected on K currents, indicating that 5-HT does not indirectly enhance calcium currents by prolonging the action potential. Calcium currents were isolated by replacing extracellular sodium with TEA and adding tetrodotoxin and other K -channel blockers. Under these conditions motoneuron action potentials were greatly prolonged and could be blocked with Co² or Cd² . Addition of 5-HT increased the duration of these Ca² spikes by about 35%. In motoneurons studied with voltage clamp, the amine produced a 58% increase in total inward calcium current. Use of the calcium channel blockers nifedipine, nimodipine, ω-conotoxin GVIA, and ω-agatoxin TK revealed that motoneurons express varying amounts of L-, N- and P-like calcium channels, but only an agatoxin-sensitive, P-type channel is sensitive to 5-HT. It is concluded that 5-HT acts directly to increase a P-type Ca² current during a normal spike. The resulting increase in intracellular calcium could contribute to an increase in transmitter release and account for the increase in junctional potentials in swim muscles.

The nudibranch Melibe leonina swims by rhythmically flexing its body from side to side at a frequency of 1 cycle every 2–5 sec. Melibe swim spontaneously, when they are dislodged from the substrate, or when they come in contact with predatory seastars, such as Pycnopodia helianthoides. Intracellular recordings obtained from semi-intact swimming Melibe reveal a population of ∼15 swim motoneurons (SMNs) in each pedal ganglion. In general, SMNs in one pedal ganglion fire out-of-phase with SMNs in the opposite pedal ganglion, resulting in rhythmic side-to-side bending movements. In isolated brains, recordings from SMNs yield similar results, indicating the existence of a swim central pattern generator (CPG). There is no evidence for synaptic interactions between SMNs and either inhibiting or exciting SMNs has no impact on the swim pattern. The SMNs are driven by a CPG consisting of 4 interneurons; 2 in the cerebropleural ganglia and 1 in each pedal ganglion. Appropriate bursting activity in the swim interneurons is necessary for swimming to occur. Either hyperpolarization or depolarization of any of the 4 CPG interneurons disrupts the normal swim pattern. Swimming behavior, and the fictive swim motor program expressed by the isolated brain, are inhibited by light and nitric oxide donors. NADPH-diaphorase staining and nitric oxide synthase (NOS) immunocytochemistry of Melibe brains suggests the source of nitric oxide might be a pair of bilaterally symmetrical cells located in the cerebropleural ganglia.

Postinhibitory rebound (PIR) is an intrinsic property of many neurons but the underlying mechanism is not well understood. We studied PIR and its relationship to spike adaptation in B-cells isolated from the buccal ganglia of Aplysia. These neurons exhibit PIR following inhibitory synaptic input and following direct membrane hyperpolarization. Hyperpolarizing and depolarizing voltage clamp pulses from the resting potential evoke slow changes in membrane current that persist in the form of tail currents following the pulses. A subtraction method was used to isolate slow tail currents for study. Current-voltage measurements indicate that slow outward tail currents following depolarizing pulses result from increases in membrane conductance, while inward tail currents following hyperpolarizations to −50 and −60 mV result from conductance decreases. The reversal potential of both outward and inward tail current is between −60 and −70 mV. Tail currents activated by pulses more positive than −60 mV are sensitive to the external K concentration and blocked by injection of Cs and TEA. When Ca² influx is prevented by bathing cells in Ca² free saline or by adding Co² or Ni² , the tail currents are reduced but a significant fraction of the current is insensitive to these treatments. More negative conditioning pulses activate a second component of inward tail current that is weakly sensitive to K but more strongly effected by substitution of N-methyl glucamine or Li for external Na . We conclude that both PIR and adaptation result from slow changes in a voltage dependent, non-inactivating K conductance that is active at voltages near the resting potential and is not tightly coupled to Ca² influx. In addition, a second inward current is activated by large hyperpolarizing pulses that results from an increase in Na and K conductance. This second process is likely to contribute to PIR under particular circumstances.