Biomechanics of transduction by mechanosensory cilia for prey detection in aquatic organisms.

Surface-feeding aquatic animals navigate towards the source of water disturbances and must differentiate prey from other environmental stimuli. Medicinal leeches locate prey, in part, using a distribution of mechanosensory hairs along their body that deflect under fluid flow. Leech's behavioral responses to surface wave temporal frequency are well documented. However, a surface wave's temporal frequency depends on many underlying environmental and fluid properties that vary substantially in natural habitats (e.g., water depth, temperature). The impact of these variables on neural response and behavior is unknown. Here, we developed a physics-based leech mechanosensor model to examine the impact of environmental and fluid properties on neural response. Our model used the physical properties of a leech cilium and was verified against existing behavioral and electrophysiological data. The model's peak response occurred with waves where the effects of gravity and surface tension were nearly equal (i.e., the phase velocity minimum). This suggests that preferred stimuli are related to the interaction between fundamental properties of the surrounding medium and the mechanical properties of the sensor. This interaction likely tunes the sensor to detect the nondispersive components of the signal, filtering out irrelevant ambient stimuli, and may be a general property of cilia across the animal kingdom.

Tyrosine hydroxylase immunolabeling reveals the distribution of catecholaminergic neurons in the central nervous systems of the spiders Hogna lenta (Araneae: Lycosidae) and Phidippus regius (Araneae: Salticidae).

With over 48,000 species currently described, spiders (Arthropoda: Chelicerata: Araneae) comprise one of the most diverse groups of animals on our planet, and exhibit an equally wide array of fascinating behaviors. Studies of central nervous systems (CNSs) in spiders, however, are relatively sparse, and no reports have yet characterized catecholaminergic (dopamine [DA]- or norepinephrine-synthesizing) neurons in any spider species. Because these neuromodulators are especially important for sensory and motor processing across animal taxa, we embarked on a study to identify catecholaminergic neurons in the CNS of the wolf spider Hogna lenta (Lycosidae) and the jumping spider Phidippus regius (Salticidae). These neurons were most effectively labeled with an antiserum raised against tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. We found extensive catecholamine-rich neuronal fibers in the first- and second-order optic neuropils of the supraesophageal mass (brain), as well as in the arcuate body, a region of the brain thought to receive visual input and which may be involved in higher order sensorimotor integration. This structure likely shares evolutionary origins with the DA-enriched central complex of the Mandibulata. In the subesophageal mass, we detected an extensive filigree of TH-immunoreactive (TH-ir) arborizations in the appendage neuromeres, as well as three prominent plurisegmental fiber tracts. A vast abundance of TH-ir somata were located in the opisthosomal neuromeres, the largest of which appeared to project to the brain and decorate the appendage neuromeres. Our study underscores the important roles that the catecholamines likely play in modulating spider vision, higher order sensorimotor processing, and motor patterning.

Plasmon resonance and the imaging of metal-impregnated neurons with the laser scanning confocal microscope.

The staining of neurons with silver began in the 1800s, but until now the great resolving power of the laser scanning confocal microscope has not been utilized to capture the in-focus and three-dimensional cytoarchitecture of metal-impregnated cells. Here, we demonstrate how spectral confocal microscopy, typically reserved for fluorescent imaging, can be used to visualize metal-labeled tissues. This imaging does not involve the reflectance of metal particles, but rather the excitation of silver (or gold) nanoparticles and their putative surface plasmon resonance. To induce such resonance, silver or gold particles were excited with visible-wavelength laser lines (561 or 640 nm), and the maximal emission signal was collected at a shorter wavelength (i.e., higher energy state). Because the surface plasmon resonances of noble metal nanoparticles offer a superior optical signal and do not photobleach, our novel protocol holds enormous promise of a rebirth and further development of silver- and gold-based cell labeling protocols.

Central-complex control of movement in the freely walking cockroach.

To navigate in the world, an animal's brain must produce commands to move, change direction, and negotiate obstacles. In the insect brain, the central complex integrates multiple forms of sensory information and guides locomotion during behaviors such as foraging, climbing over barriers, and navigating to memorized locations. These roles suggest that the central complex influences motor commands, directing the appropriate movement within the current context. Such commands are ultimately carried out by the limbs and must therefore interact with pattern generators and reflex circuits that coordinate them. Recent studies have described how neurons of the central complex encode sensory information: neurons subdivide the space around the animal, encoding the direction or orientation of stimuli used in navigation. Does a similar central-complex code directing movement exist, and if so, how does it effect changes in the control of limbs? Recording from central-complex neurons in freely walking cockroaches (Blaberus discoidalis), we identified classes of movement-predictive cells selective for slow or fast forward walking, left or right turns, or combinations of forward and turning speeds. Stimulation through recording wires produced consistent trajectories of forward walking or turning in these animals, and those that elicited turns also altered an inter-joint reflex to a pattern resembling spontaneous turning. When an animal transitioned to climbing over an obstacle, the encoding of movement in this new context changed for a subset of cells. These results indicate that encoding of movement in the central complex participates in motor control by a distributed, flexible code targeting limb reflex circuits.

Compensatory plasticity restores locomotion after chronic removal of descending projections.

Homeostatic plasticity is an important attribute of neurons and their networks, enabling functional recovery after perturbation. Furthermore, the directed nature of this plasticity may hold a key to the restoration of locomotion after spinal cord injury. Here we studied the recovery of crawling in the leech Hirudo verbana after descending cephalic fibers were surgically separated from crawl central pattern generators shown previously to be regulated by dopamine. We observed that immediately after nerve cord transection leeches were unable to crawl, but remarkably, after a day to weeks, animals began to show elements of crawling and intersegmental coordination. Over a similar time course, excessive swimming due to the loss of descending inhibition returned to control levels. Additionally, removal of the brain did not prevent crawl recovery, indicating that connectivity of severed descending neurons was not essential. After crawl recovery, a subset of animals received a second transection immediately below the anterior-most ganglion remaining. Similar to their initial transection, a loss of crawling with subsequent recovery was observed. These data, in recovered individuals, support the idea that compensatory plasticity directly below the site of injury is essential for the initiation and coordination of crawling. We maintain that the leech provides a valuable model to understand the neural mechanisms underlying locomotor recovery after injury because of its experimental accessibility, segmental organization, and dependence on higher-order control involved in the initiation, modulation, and coordination of locomotor behavior.

Scanning behavior in the medicinal leech Hirudo verbana.

While moving through their environment, medicinal leeches stop periodically and wave their head or body back and forth. This activity has been previously described as two separate behaviors: one called 'head movement' and another called 'body waving'. Here, we report that these behaviors exist on a continuum, and provide a detailed description of what we now call 'scanning'. Scanning-related behavior has been thought to be involved in orientation; its function has never before been assessed. While previous studies suggested an involvement of scanning in social behavior, or sucker placement, our behavioral studies indicate that scanning is involved in orienting the leech towards prey stimuli. When such stimuli are present, scanning behavior is used to re-orient the leech in the direction of a prey-like stimulus. Scanning, however, occurs whether or not prey is present, but in the presence of prey-like stimuli scanning becomes localized to the stimulus origin. Most likely, this behavior helps the leech to gain a more detailed picture of its prey target. The display of scanning, regardless of the presence or absence of prey stimuli, is suggestive of a behavior that is part of an internally driven motor program, which is not released by the presence of sensory stimuli. The data herein include first steps to understanding the neural mechanisms underlying this important behavior.

Discontinuous locomotion and prey sensing in the leech.

The medicinal leech, Hirudo verbana, is an aquatic predator that utilizes water waves to locate its prey. However, to reach their prey, the leeches must move within the same water that they are using to sense prey. This requires that they either move ballistically towards a pre-determined prey location or that they account for their self-movement and continually track prey. We found that leeches do not localize prey ballistically. Instead, they require continual sensory information to track their prey. Indeed, in the event that the prey moves, leeches will approach the prey's new location. While leeches need to continually sense water disturbances to update their percept of prey location, their own behavior is discontinuous--prey involves switching between swimming, crawling and non-locomoting. Each of these behaviors may allow for different sensory capabilities and may require different sensory filters. Here, we examined the sensory capabilities of leeches during each of these behaviors. We found that while one could expect the non-locomoting phases to direct subsequent behaviors, crawling phases were more effective than non-locomotor phases for providing direction. During crawling bouts, leeches adjusted their heading so as to become more directed towards the stimulus. This was not observed during swimming. Furthermore, in the presence of prey-like stimuli, leeches crawled more often and for longer periods of time.

Deciding which way to go: how do insects alter movements to negotiate barriers?

Animals must routinely deal with barriers as they move through their natural environment. These challenges require directed changes in leg movements and posture performed in the context of ever changing internal and external conditions. In particular, cockroaches use a combination of tactile and visual information to evaluate objects in their path in order to effectively guide their movements in complex terrain. When encountering a large block, the insect uses its antennae to evaluate the object's height then rears upward accordingly before climbing. A shelf presents a choice between climbing and tunneling that depends on how the antennae strike the shelf; tapping from above yields climbing, while tapping from below causes tunneling. However, ambient light conditions detected by the ocelli can bias that decision. Similarly, in a T-maze turning is determined by antennal contact but influenced by visual cues. These multi-sensory behaviors led us to look at the central complex as a center for sensori-motor integration within the insect brain. Visual and antennal tactile cues are processed within the central complex and, in tethered preparations, several central complex units changed firing rates in tandem with or prior to altered step frequency or turning, while stimulation through the implanted electrodes evoked these same behavioral changes. To further test for a central complex role in these decisions, we examined behavioral effects of brain lesions. Electrolytic lesions in restricted regions of the central complex generated site specific behavioral deficits. Similar changes were also found in reversible effects of procaine injections in the brain. Finally, we are examining these kinds of decisions made in a large arena that more closely matches the conditions under which cockroaches forage. Overall, our studies suggest that CC circuits may indeed influence the descending commands associated with navigational decisions, thereby making them more context dependent.

Developmentally regulated multisensory integration for prey localization in the medicinal leech.

Medicinal leeches, like many aquatic animals, use water disturbances to localize their prey, so they need to be able to determine if a wave disturbance is created by prey or by another source. Many aquatic predators perform this separation by responding only to those wave frequencies representing their prey. As leeches' prey preference changes over the course of their development, we examined their responses at three different life stages. We found that juveniles more readily localize wave sources of lower frequencies (2 Hz) than their adult counterparts (8-12 Hz), and that adolescents exhibited elements of both juvenile and adult behavior, readily localizing sources of both frequencies. Leeches are known to be able to localize the source of waves through the use of either mechanical or visual information. We separately characterized their ability to localize various frequencies of stimuli using unimodal cues. Within a single modality, the frequency-response curves of adults and juveniles were virtually indistinguishable. However, the differences between the responses for each modality (visual and mechanosensory) were striking. The optimal visual stimulus had a much lower frequency (2 Hz) than the optimal mechanical stimulus (12 Hz). These frequencies matched, respectively, the juvenile and the adult preferred frequency for multimodally sensed waves. This suggests that, in the multimodal condition, adult behavior is driven more by mechanosensory information and juvenile behavior more by visual. Indeed, when stimuli of the two modalities were placed in conflict with one another, adult leeches, unlike juveniles, were attracted to the mechanical stimulus much more strongly than to the visual stimulus.