Most physiological and ecological approaches to animal locomotion are based on steady state assumptions, yet movements of many animals are interspersed with pauses lasting from milliseconds to minutes. Thus, pauses, along with changes in the duration and speed of moves, form part of a dynamic system of intermittent locomotion by which animals adjust their locomotor behavior to changing circumstances. Intermittent locomotion occurs in a wide array of organisms from protozoans to mammals. It is found in aerial, aquatic and terrestrial locomotion and in many behavioral contexts including search and pursuit of prey, mate search, escape from predators, habitat assessment and general travel. In our survey, animals exhibiting intermittent locomotion paused on average nearly 50% of their locomotion time (range 6–94%). Although intermittent locomotion is usually expected to increase energetic costs as a result of additional expenditure for acceleration and deceleration, a variety of energetic benefits can arise when forward movement continues during pauses. Endurance also can be improved by partial recovery from fatigue during pauses. Perceptual benefits can arise because pauses increase the capacity of the sensory systems to detect relevant stimuli. Several processes, including velocity blur, relative motion detection, foveation, attention and interference between sensory systems are probably involved. In animals that do not pause, alternative mechanisms for stabilizing the perceptual field are often present. Because movement is an important cue for stimulus detection, pauses can also reduce unwanted detection by an organism's predators or prey. Several models have attempted to integrate energetic and perceptual processes, but many challenges remain. Future advances will require improved quantification of the effects of speed on perception.

A number of bird species swim underwater by wing propulsion. Both among and within species, thrust generated during the recovery phase (upstroke) varies from almost none to more than during the power phase (downstroke). More uneven thrust and unsteady speed may increase swimming costs because of greater inertial work to accelerate the body fuselage (head and trunk), especially when buoyant resistance is high during descent. I investigated these effects by varying relative fuselage speed during upstroke vs. downstroke in a model for wing-propelled murres which descend at relatively constant mean speed. As buoyant resistance declined with depth, the model varied stroke frequency and glide duration to maintain constant mean descent speed, stroke duration, and work per stroke. When mean fuselage speed during the upstroke was only 18% of that during the downstroke, stroke frequency was constant with no gliding, so that power output was unchanged throughout descent. When mean upstroke speed of the fuselage was raised to 40% and 73% of mean downstroke speed, stroke frequency declined and gliding increased, so that power output decreased rapidly with increasing depth. Greater inertial work with more unequal fuselage speeds was a minor contributor to differences in swimming costs. Instead, lower speeds during upstrokes required higher speeds during downstrokes to maintain the same mean speed, resulting in nonlinear increases in drag at greater fuselage speeds during the power phase. When fuselage speed was relatively higher during upstrokes, lower net drag at the same mean speed increased the ability to glide between strokes, thereby decreasing the cost of swimming.

The evolutionary history of marine mammals involved marked physiological and morphological modifications to change from terrestrial to aquatic locomotion. A consequence of this ancestry is that swimming is energetically expensive for mammals in comparison to fish. This study examined the use of behavioral strategies by marine mammals to circumvent these elevated locomotor costs during horizontal swimming and vertical diving. Intermittent forms of locomotion, including wave-riding and porpoising when near the water surface, and prolonged gliding and a stroke and glide mode of propulsion when diving, enabled marine mammals to increase the efficiency of aquatic locomotion. Video instrumentation packs (8-mm camera, video recorder and time-depth microprocessor) deployed on deep diving bottlenose dolphins (Tursiops truncatus), northern elephant seals (Mirounga angustirostris), and Weddell seals (Leptonychotes weddellii) revealed exceptionally long periods of gliding during descent to depth. Glide duration depended on depth and represented nearly 80% of the descent for dives exceeding 200 m. Transitions in locomotor mode during diving were attributed to buoyancy changes with compression of the lungs at depth, and were associated with a 9–60% reduction in the energetic cost of dives for the species examined. By changing to intermittent locomotor patterns, marine mammals are able to increase travelling speed for little additional energetic cost when surface swimming, and to extend the duration of submergence despite limitations in oxygen stores when diving.

Body size, pectoralis composition, aspect ratio of the wing, and forward speed affect the use of intermittent flight in birds. During intermittent non-flapping phases, birds extend their wings and glide or flex their wings and bound. The pectoralis muscle is active during glides but not during bounds; activity in other primary flight muscles is variable. Mechanical power, altitude, and velocity vary among wingbeats in flapping phases; associated with this variation are changes in neuromuscular recruitment, wingbeat frequency, amplitude, and gait. Species of intermediate body mass (35–158 g) tend to flap-glide at slower speeds and flap-bound at faster speeds, regardless of the aspect ratio of their wings. Such behavior may reduce mechanical power output relative to continuous flapping. Smaller species (<20 g) with wings of low aspect ratio may flap-bound at all speeds, yet existing models do not predict an aerodynamic advantage for the flight style at slow speeds. The behavior of these species appears to be due to wing shape rather than pectoralis physiology. As body size increases among species, percent time spent flapping increases, and birds much larger than 300 g do not flap-bound. This pattern may be explained by adverse scaling of mass-specific power or lift per unit power output available from flight muscles. The size limit for the ability to bound intermittently may be offset somewhat by the scaling of pectoralis composition. The percentage of time spent flapping during intermittent flight also varies according to flight speed.

Hypotheses explaining the use of intermittent bounding and undulating flight modes in birds are considered. Existing theoretical models of intermittent flight have assumed that the animal flies at a constant speed throughout. They predict that mean mechanical power in undulating (flap-gliding) flight is reduced compared to steady flight over a broad range of speeds, but is reduced in bounding flight only at very high flight speeds. Lift generated by the bird's body or tail has a small effect on power, but is insufficient to explain observations of bounding at intermediate flight speeds. Measurements on starlings Sturnus vulgaris in undulating flight in a wind tunnel show that flight speed varies by around ±1 m/sec during a flap-glide cycle. Dynamic energy is used to quantify flight performance, and reveals that the geometry of the flight path depends upon wingbeat kinematics, and that neither flapping nor gliding phases are at constant speed and angle to the horizontal. The bird gains both kinetic and potential energy during the flapping phases. A new theoretical model indicates that such speed variation can give significant savings in mechanical power in both bounding and undulating flight. Alternative hypotheses for intermittent flight include a gearing mechanism, based on duty factor, mediating muscle power or force output against aerodynamic requirements. This could explain the use of bounding flight in hovering and climbing in small passerines. Both bounding and undulating confer other adaptive benefits; undulating may be primitive in birds, but bounding may have evolved in response to flight performance optimization, or to factors such as unpredictability in response to predation.

Seven subjects walked on a programmable treadmill both at constant (3.5 ± 0.0 and 5.0 ± 0.0 km/hr) and oscillating speeds (±0.5, ±1.0, ±1.5, ±2.0 km hr⁻¹), set to sinusoidally change between the two limits in 3 sec. In each condition oxygen consumption measurements were taken. The same experimental protocols were replicated on a walkway by asking subjects to adapt their stride frequency to an audio signal corresponding to the sinusoidal stride frequency changes measured on the treadmill. Differently from what expected, only the ±2.0 km hr⁻¹ oscillation resulted to be metabolically different from the constant speed walking, both for the treadmill and the walkway conditions. The time course of the mechanical energy of the body centre of mass could reveal that a strategy devoted to benefit from the usual energy fluctuations occurring at “constant speed,” is likely to be used to cope with speed varying sequences. From the energy curve observed at constant speed, it is possible to derive an energetically equivalent curve by cumulating acceleration portions, and deceleration ones, of a group of strides as to produce a single acceleration and a single deceleration phase, as it is observed in oscillating speed walking. Being aware of the bias introduced by using a non-inertial frame (the treadmill protocol), we are replicating the experiments with a laser beam projected on a wide radius circular path at oscillating speeds, that the subjects have to follow. The preliminary data seem to confirm the invariance of the metabolic requirements in oscillatory walking up to ±1.5 km hr⁻¹.

When locomotor activity is brief, physiological steady state conditions are not attained. It is therefore difficult to model the energetic costs of intermittent activity using standard methods. This difficulty is addressed by considering as reflective of the metabolic costs of activity not only the oxygen consumed during the activity itself, but also the excess post-exercise oxygen consumption (EPOC) and any excess metabolites persisting at the end of EPOC. This paper briefly reviews the metabolic events associated with EPOC, and then examines how this approach can be applied to address questions of how behavioral variables associated with locomotion (activity duration, intensity, frequency) can influence the energetic costs to the animal per unit distance. Using data for lizards, mice, and others, EPOC can be shown to be the major component of energetic costs when durations are short, regardless of exercise intensity. Brief activity is much more expensive by this measure than is steady state locomotion, regardless of phylogeny or body mass. Three studies of intermittent locomotion provide evidence that brief behaviors can be undertaken at lower metabolic costs than predicted from single bouts of activity when repeated in a frequent, repeated pattern. Metabolic savings appear greatest when the pause period between behaviors is short relative to EPOC duration, the time for organismal metabolic rate to return to pre-exercise levels, although longer pause periods may increase endurance.

The earliest studies of intermittent exercise physiology noted that moving intermittently (i.e., alternating brief movements with brief pauses) could transform a heavy workload into a submaximal one that can be tolerated and sustained. The brief pauses that characterize intermittent locomotion permit at least partial recovery from prior activity. This research provided the foundation for the development of interval training and more recently for the re-evaluation of steady-state paradigms for comparative animal locomotion. In this paper I review key concepts underlying the performance of repeated activity. I provide examples from human athletics and training and comparative animal locomotion. To explore the limits of intermittent exercise performance, I examine the performance limits for continuous exercise and the rate and extent of the recovery of performance capacity following activity. While it is evident that altering locomotor behavior (i.e., moving intermittently) can alter the capacity of an animal to perform work, mathematical models of intermittent exercise could predict strategies (i.e., exercise intensity, exercise duration, and pause duration) that will increase performance limits for intermittent activity.

The study of the kinetics of O₂ consumption (V̇o₂) at the onset and offset of constant-load submaximal exercise (V̇o₂ on- and off-kinetics) is useful from a practical point of view (a faster adjustment of oxidative metabolism following an increased metabolic demand reduces the need for substrate level phosphorylation, with implications on exercise tolerance and muscle fatigue) and can give valuable insights into the regulation of oxidative metabolism in skeletal muscle. Measurements have been carried out both in man and in animals, at the tissue and at the whole body level. At the tissue level, the V̇o₂ on- and off-kinetics were determined: a) Directly, by dynamic solution of the Fick equation throughout the transients; attempts were also made to obtain similar informations by near-infrared spectroscopy. b) Indirectly, from the kinetics of phosphocreatine hydrolysis and resynthesis, by chemical methods or by ³¹P magnetic resonance spectroscopy. At the whole body level, V̇o₂ on- and off-kinetics are determined from breath-by-breath measurements of pulmonary gas exchange. The V̇o₂ = f(t) function is a complex one, particularly during the on-transient. The so-called “phase 2” of the V̇o₂ on-response, as well as the V̇o₂ off-response, yield relevant metabolic informations. In muscle the V̇o₂ on- and off-kinetics are characterized by half-times (t½) of 15–20 sec. At the whole-body level, t½ of the V̇o₂ on-kinetics show a wider variability, related to the experimental protocol and to other factors. The V̇o₂ off-phase is more constant, and its kinetic parameters appear closer to those obtained at the tissue level. The study of the V̇o₂ kinetics is valuable for a functional evaluation of skeletal muscle oxidative metabolism. In ordinary conditions muscle V̇o₂ kinetics appears mainly imposed by intrinsic (metabolic) rather than extrinsic (O₂ delivery) factors.

The presence of nitric oxide synthase (NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida and in its symbiotic dinoflagellate algae, Symbiodinium bermudense. Enzyme activity was assayed by measuring the conversion of arginine to citrulline. Biochemical characterization of NOS from Aiptasia was characterized with respect to cellular localization, substrate and cofactor requirements, inhibitors, and kinetics. In response to acute temperature shock, anemones retracted their tentacles. Animals subjected to such stress had lower NOS activities than did controls. Treatment with NOS inhibitors caused tentacular retraction, while treatment with the NOS substrate L-arginine inhibited this response to stress, as did treatment with NO donors. These results provide a preliminary biochemical characterization of, and suggest a functional significance for, NOS activity in anthozoan-algal symbiotic assemblages.

Several neuroactive compounds have been implicated as playing roles in the circuitry that controls larval metamorphosis in marine molluscs. For the caenogastropod Ilyanassa obsoleta, results of neuroanatomical studies suggest that the production of nitric oxide (NO) increases throughout the planktonic stage and that NO production is necessary for the maintenance of the larval state, especially as it becomes metamorphically competent. Bath application or injection of exogenous serotonin (5HT) can initiate metamorphosis in competent larvae, and exogenous NO can inhibit such serotonergically-induced metamorphosis. Inhibition of endogenous nitric oxide synthase (NOS) can also trigger larval metamorphosis. The production of endogenous NO appears to decrease concurrently with the initiation of metamorphosis, but the specific interactions between serotonergic and nitrergic neurons are unknown. Evidence in support of NO acting to up-regulate the enzyme guanylyl cyclase (GC) is still equivocal. Thus, we do not yet know if NO exerts its effects through the actions of cyclic 3′,5′-guanosine monophosphate (cGMP) or by a cGMP-independent mechanism. The ubiquity of nitrergic signalling and its significance for developing molluscan embryos and larvae are still the subject of speculation and require further investigation.

Photoreceptors undergoing target selection in the optic lobe of Drosophila express a nitric oxide sensitive soluble guanylate cyclase (sGC). At the same time, cells in the target region of the optic lobe express nitric oxide synthase (NOS). Pharmacological inhibition of NOS, NO or sGC leads to disruption of the retinal projection pattern in vitro, and the extension of individual retinal axons beyond their appropriate targets. The disruptive effects of NOS inhibition in vitro are prevented by adding a cGMP analog. Mutations in the sGC alpha subunit gene, Gcα1, reduce sGC expression and attenuate NO-sensitive retinal cGMP production in the visual system. Although the retinal projection pattern is undisturbed in Gcα1 mutants, they lack positive phototaxis as adults, suggesting inappropriate connections exist between the photoreceptors and optic lobe interneurons in these flies. Preliminary results show that heat-shock expression of wild-type Gcα1 during metamorphosis can restore positive phototaxis in severe Gcα1 mutants. These in vivo results support the in vitro findings that NOS and sGC activity are required to promote the appropriate retinal innervation of the optic lobe.

Nitric oxide serves as an orthograde synaptic cotransmitter between identified neurons in the cerebral ganglion of Aplysia. Nitric oxide synthase, the enzyme that produces nitric oxide, is localized in a few specific neurons in the ganglia, including neuron C2. Guanylyl cyclase the target enzyme of nitric oxide, is found in neurons C4 and MCC, which are synaptic followers of C2. Stimulation of C2 causes a vsEPSP in these neurons that is reduced to 50% of its amplitude by nitric oxide synthase inhibitors and guanylyl cyclase inhibitors. The remaining portion of the vsEPSP is mediated by histamine. Thus, nitric oxide and histamine act as orthograde cotransmitters in producing the vsEPSP. Both cotransmitters cause closure of a background potassium channel, which depolarizes the neuron and enhances its response to synaptic inputs. Exogenous nitric oxide (released by nitric oxide donor molecules) and histamine mimic the vsEPSP's depolarization and decreased membrane conductance. When neurons C4 or MCC are isolated in cell culture they respond just as they do in the ganglion, i.e., the nitric oxide response but not the histamine response is blocked by guanylyl cyclase inhibitors, and the membrane conductance is decreased by both histamine and nitric oxide. Aplysia hemolymph partially suppresses the response to nitric oxide, due to nitric oxide scavenging by hemocyanin, which contains copper and is the equivalent of hemoglobin. Neuron C2 followers that are hyperpolarized by histamine are insensitive to nitric oxide. Thus, only select follower neurons respond to both transmitters.

The basic elements of the NO/cGMP signaling pathway have been identified in the nervous systems of animals from nearly all of the major phyla. In crustaceans, the NO/cGMP pathway is associated with certain fundamental neuronal processes, including sensory integration and the organization and production of motor behavior. Here I review the evidence for NO synthesis and action in crustacean neural networks, with an emphasis on the rhythmic motor circuits of the crab stomatogastric ganglion (STG). In the STG, NO appears to be released as an orthograde transmitter from descending projection neurons. NO's receptor, a cytopasmic isoform of guanylate cyclase (sGC), is expressed in a subset of the cells that participate in the gastric mill and pyloric central pattern generating networks. In spontaneously-active, in vitro preparations of the STG, pharmacological inhibitors of the NO/cGMP pathway cause the two rhythmic motor patterns to collapse into a single conjoint rhythm. Parallel motor output is restored when the ganglion is returned to normal saline. Although precise mechanisms have yet to be determined, these data suggest that NO and cGMP play an important role in the functional organization of STG networks. The STG, as well as other crustacean models, provides a promising context for studying the physiological and behavioral aspects of NO-mediated signaling in the nervous system.

This paper reviews comparative and evolutionary aspects of nitric oxide (NO) signaling in major systematic groups such as prokaryotes, plants, fungi and invertebrate animals. It appears that NO-mediated signaling can be as old as cellular organization itself. Both non-enzymatic and enzymatic (in addition to NOS) synthetic pathways can contribute to NO formation in living systems. The evolutionary roots of this means of gaseous signaling can be traced back to the role of NO in non-immune defensive mechanisms and the role of NO in control of gene expression, chemical ecology and, perhaps, symbiotic interactions in the ancient prokaryotic world. These functions of NO can be preserved in practically all modern taxons and be widely expressed in the nervous system. However, it is hypothesized that neuronal NO signaling is a relatively new evolutionary invention and it is likely to have happened several times during animal evolution. Although a comparative analysis of neuronal NO signaling is still in its early stages, the hypothesis is proposed that in many invertebrate lineages one of the primary neuronal functions of NO was regulation of feeding patterns, chemosensory processing and neurodevelopment.

Recent studies have investigated the source and target neurons for the diffusible neuronal messenger molecule nitric oxide (NO) in the nervous system of the locust. Here we compare the neuroarchitecture of NO signaling between different sensory systems. The available neuroanatomical data implicate NO in sensory processing for modalities as diverse as mechanoreception, vision, olfaction, gustation and hearing. All respective first-order sensory neuropils are innervated by NOS-containing interneurons. The corresponding sensory receptor neurons lack NOS but seem to express soluble guanylyl cyclase (sGC), the main receptor molecule for NO in the nervous system. The axonal projections of sensory neurons must therefore be considered the primary target of NO in these sensory neuropils. An exception is the antennal olfactory system where sGC is apparently expressed in interneurons, in partial colocalization with NOS.

We discuss these anatomical findings in relation to the spatiotemporal characteristics of NO signaling. Many sensory neuropils are organized into maps that reflect neuronal response properties (i.e., tuning or receptive fields). A local release of NO within such maps will therefore most strongly affect neurons with similar coding properties. If sensory receptor activity triggers NO synthesis locally in the map, this mechanism could link groups of similarly tuned receptors dynamically according to stimulus intensity. Furthermore, we explore the functional implications of differences between sensory systems in the anatomy of NOS-expressing interneurons, using the compound eye and the thoracic tactile system as examples.

The gaseous neurotransmitters nitric oxide (NO) and carbon monoxide (CO) are prominent and universal components of the array of neurotransmitters found in olfactory information processing systems. These highly mobile communication compounds have effects on both second messenger signaling and directly on ion channel gating in olfactory receptors and central synaptic processing of receptor input. Olfactory systems are notable for the plasticity of their synaptic connections, revealed both in higher-order associative learning mechanisms using odor cues and developmental plasticity operating to maintain function during addition of new olfactory receptors and new central olfactory interneurons. We use the macrosmatic terrestrial mollusk Limax maximus to investigate the role of NO and CO in the dynamics of central odor processing and odor learning. The major central site of odor processing in the Limax CNS is the procerebral (PC) lobe of the cerebral ganglion, which displays oscillatory dynamics of its local field potential and periodic activity waves modulated by odor input. The bursting neurons in the PC lobe are dependent on local NO synthesis for maintenance of bursting activity and wave propagation. New data show that these bursting PC interneurons are also stimulated by carbon monoxide. The synthesizing enzyme for carbon monoxide, heme oxygenase 2, is present in the neuropil of the PC lobe. Since the PC lobe exhibits two forms of synaptic plasticity related to both associative odor learning and continual connection of new receptors and interneurons, the use of multiple gaseous neurotransmitters may be required to enable these multiple forms of synaptic plasticity.

Rich redox chemistry of the diatomic NO gives this molecule the functional flexibility to interact with both metal and non-metal components of biological molecules. This important biological signaling and allosteric control has become evident in such varied applications as brain/nervous system function; immune response; growth and development; behavior; and gas transport. Many of the basic discoveries linking NO to biological systems have arisen from structure-function relationships in hemoglobin. For example, by analogy with hemoglobin, Lou Ignarro, in a now-classic paper on NO, proposed that the activation of soluble guanylate cyclase occurs via a NO-driven planar shift in the enzyme's heme iron (Ignarro et al., 1984). Many other proteins involved in NO biology are heme proteins where NO coordination plays an essential function. In this regard, we may view hemoglobin as a microcosm of NO biology.

Invertebrates provide rich examples in which to explore alternate functions, or even perhaps the original functions, of the globins. Oxygen-carrying proteins could well have evolved from metalloproteins that primarily functioned in nitrogen metabolism rather than reversible oxygen binding. Newly discovered aspects of Hb function relate to the signaling and control processes that nitric oxide shows in biological systems. The comparative approach to these processes has played an important role in their elucidation as well as providing rich, intellectual stimulation to those scientists interested in them.