The morphological basis of intracellular measurements in the cockroach tactile spine neuron

Summary The femoral tactile spine of the cockroach (Periplaneta americana) contains a single sensory neuron, which adapts rapidly and completely to step deformations of the spine. Techniques for stable intracellular recording from the tactile spine neuron have recently been established, allowing electrophysiological investigation of mechanotransduction and adaptation in this sensory neuron. However, intracellular recordings from the neuron produce a wide range of action potential heights and thresholds, raising the possibility that some penetrations are in adjacent, but closely coupled supporting glial cells. This problem is exacerbated because the cell cannot be visualized during penetration.Systematic measurements of action potential heights and thresholds were made in tactile spine cells, together with identification of some penetrated cells by intracellular injection of Lucifer Yellow. All stained cells were clearly sensory neurons, although their action potentials amplitudes varied from 9 mV to 80 mV. Smaller action potentials were broader than larger action potentials, and the changes in height and shape could be explained by a simple cable conduction model using measured morphological and electrical parameters. The model could also account for the observed relationship between action potential height and threshold.These results indicate that reliable recording from the tactile spine neuron is possible, but that variability in the positions of the penetration or the spike initiating zone cause an apparently wide range of electrophysiological measurements.     

Encoding of forces by cockroach tibial campaniform sensilla: implications in dynamic control of posture and locomotion

Forces exerted by a leg in support and propulsion can vary considerably when animals stand upon or traverse irregular terrains. We characterized the responses of the cockroach tibial campaniform sensilla, mechanoreceptors which encode force via strains produced in the exoskeleton, by applying forces to the leg at controlled magnitudes and rates. We also examined how sensory responses are altered in the presence of different levels of static load. All receptors exhibit phasico-tonic discharges that reflect the level and rate of force application. Our studies show that: (1) tonic discharges of sensilla can signal the level of force, but accurate encoding of static loads may be affected by substantial receptor adaptation and hysteresis; (2) the absolute tonic sensitivities of receptors decrease when incremental forces are applied at different initial load levels; (3) phasic discharges of sensilla accurately encode the rate of force application; and (4) sensitivities to changing rates of force are strictly preserved in the presence of static loads. These findings imply that discharges of the sensilla are particularly tuned to the rate of change of force at all levels of leg loading. This information could be utilized to adapt posture and walking to varying terrains and unexpected perturbations. Accepted: 8 January 2000     

Antennal motor activity induced by pilocarpine in the American cockroach

The antennal motor system is activated by the muscarinic agonist pilocarpine in the American cockroach Periplaneta americana, and its output patterns were examined both in restrained intact animals and in isolated CNS preparations. The three-dimensional antennal movements induced by the hemocoelic drug injection were analyzed in in vivo preparations. Pilocarpine effectively induced prolonged rhythmic movements of both antennae. The antennae tended to describe a spatially patterned trajectory, forming loops or the symbol of infinity (∞). Such spatial regularity is comparable to that during spontaneous tethered-walking. Rhythmic bursting activities of the antennal motor nerves in in vitro preparations were also elicited by bath application of pilocarpine. Cross-correlation analyses of the bursting spike activities revealed significant couplings among certain motor units, implying the spatial regularity of the antennal trajectory. The pilocarpine-induced rhythmic activity of antennal motor nerves was effectively suppressed by the muscarinic antagonist atropine. These results indicate that the activation of the antennal motor system is mediated by muscarinic receptors.     

Structural and functional classification of antennal sensilla of the cockroach,Leucophaea maderae

Summary Antennal sensilla of Leucophaea maderae were investigated electrophysiologically, labeled and then examined with the scanning or transmission electron microscope. The sensilla can be classified into morphological types according to their external shape and the structure of their hair wall. Sensilla showing similar reaction spectra of their cells can be cathegorized into physiological groups. The morphological classification corresponds to the physiological grouping: one morphological type of sensillum comprises one or several groups of physiologically similar sensilla. In many of these groups constant combinations of physiologically different cells occur. The possible functional significance of the relationships found between the structural features of the sensilla and the physiological properties of their sensory cells is discussed.Supported by the Deutsche Forschungsgemeinschaft (Scha 292/1)     

Antennal and locomotor responses to attractive and aversive odors in the searching cockroach

The behavioral responses to attractive and aversive odors were examined in blinded adult male cockroaches under tethered-walking conditions. A sex pheromone-like stimulant derived from adult virgin females and artificially synthesized limonene were used as attractive and aversive odor sources, respectively. When a searching animal was stimulated with the attractive female-derived odor, the horizontal deflections of both the antennae were increased, and in most cases the vertical antennal positions were shifted downward. The stimulation also significantly decreased the walking speed of the animal. These behavioral changes imply a careful search in the immediate surroundings. The aftereffect of the sex pheromone was more pronounced on locomotion than on antennal movement. On the other hand, stimulation with the aversive odor (limonene) tended to suppress active antennal movement, and also increased the walking speed. Immediately after the withdrawal of the aversive odor, the active movement of the antennae was resumed, and the walking speed rapidly decreased to a level approximately the same as that of the control period. These results indicate that the responses to the qualitatively opposite types of odor are reciprocal to each other with regard to both antennal movement and locomotion.     

External structure of the sensillum capitulum,a hygro- and thermoreceptive sensillum of the cockroach,Periplaneta americana

Summary The antennal hygro- and thermoreceptive sensillum (sensillum capitulum) of the cockraoch, Periplaneta americana, has been identified electrophysiologically and examined by scanning electron microscopy. It appears basiconic and has a characteristic cuticular apparatus; a mushroom-shaped inner stem and a non-perforated outer wall. The head of the inner stem, which is a cap of about 0.5 m diameter, protrudes from the distal margin of the outer wall. The inner stem is not perforated, though in some cases a tiny hollow is observed in the apex of the cap. This kind of cuticular apparatus is not found in other sensilla and may be modality-specific to hygro- and/or thermoreception. The sensillum capitulum occurs singly on the distal part of each alternating segment of the flagellum and on each segment of some distal meristal segments.Supported by Grant-in-Aid for Special Project Research Mechanism of animal behavior from the Ministry of Education (Japan)     

Quantification of allatostatin receptor mRNA levels in the cockroach, Diploptera punctata, using real-time PCR

The cockroach allatostatin receptor (Dippu-AstR) is a 425 amino acid G-protein coupled receptor that is related to the mammalian galanin receptor. Using relative standard curve real-time PCR analysis, changes in Dippu-AstR mRNA expression levels were examined in tissues of adult mated and virgin female Diploptera punctata. Tissues were chosen that were either known targets of allatostatin (Dippu-AST) action or sites of Dippu-AST localization. Tissues examined included brain, corpora allata (CA), gut, ovaries, testes and abdominal ganglia. Dippu-AstR was expressed in all tissues examined for 7 days after adult emergence. Juvenile hormone (JH) biosynthesis is known to peak on day 5 post-emergence in mated females. In mated females, Dippu-AstR mRNA was at the highest levels on day 6 post-emergence in brain and CA and day 2 post-emergence in midgut. Dippu-AstR expression was found to correlate with the decline in JH biosynthesis noted on day 5 post-emergence and early inhibition of feeding. Dippu-AstR mRNA expression in virgin female midgut and CA was dramatically elevated on days 6 and 7, respectively. Expression of Dippu-AstR mRNA was found to be similar in the abdominal ganglia of mated or virgin females. Ovarian Dippu-AstR expression declined to low levels by day 4. Testes exhibited maximal Dippu-AstR mRNA expression on days 4 and 7 of adult life. A role for Dippu-AST in testes of Diploptera is unknown.     

The mechanosensory apparatus of the femoral tactile spine of the cockroach,Periplaneta americana

Summary Tactile spines are large cuticular sense organs that appear to provide insects with a sense of touch which is spatially coarse but of great sensitivity. Cockroach legs have a number of these spines on each leg and a particularly prominent spine on the end of each femur, the femoral tactile spine. The ease of recording afferent activity from this spine during mechanical stimulation has made it one of the most thoroughly studied insect mechanoreceptors and yet it has never been examined by electron microscopy.We report here the results of an examination of the femoral tactile spine by both scanning and transmission electron microscopy, as well as by light microscopy. The spine is shown to be innervated by a single sensory bipolar neuron with its soma located in the base of the spine. A canal through the wall of the spine leads to the outside and emerges just above the junction between the base of the spine and its articulating socket membrane. The sensory dendrite of the neuron passes from the soma through this canal and forms a modified ciliary sensory ending with the typical dendritic sheath and dense tubular body that is characteristic of insect mechanosensory cuticular sensilla. The tubular body is embedded in a cuticular terminal plug which closes the exterior end of the canal but appears to be fastened to the spine by a very flexible ring of cuticle. This plug is connected to the socket membrane by a specialized socket attachment which presumably serves to move the plug relative to the wall of the spine during movement of the spine within the socket. The morphology of this sensillum is discussed in terms of the possible ways in which it is stimulated by movements of the spine and also in light of the dynamic behaviour of the receptor which is now very well described.Supported by the Canadian Medical Research Council. The authors gratefully acknowledge the expert technical assistance of Sita Prasad and Rodney Gramlich     

Embryonic development of the sensory innervation of the antenna of the grasshopper Schistocerca gregaria

The establishment of the sensory nervous system of the antenna of the grasshopper Schistocerca gregaria was examined using immunocytochemical methods and in the light of the appendicular and articulated nature of this structure. The former is demonstrated first by the expression pattern of the segment polarity gene engrailed in the head neuromere innervating the antenna, the deutocerebrum. Engrailed expression is present in identified deutocerebral neuroblasts and, as elsewhere in the body, is continuous with cells of the posterior epithelium of the associated appendage, in this case the antenna. Second, early expression of the glial homeobox gene reversed polarity (repo) in the antenna is by a stereotypic pair of cells at the antenna base, a pattern we show is repeated metamerically for each thoracic appendage of the embryo. Subsequently, three regions of Repo expression (A1, A2, A3) are seen within the antenna, and may represent a preliminary form of articulation. Bromodeoxyuridine incorporation reveals that these regions are sites of intense cell differentiation. Neuron-specific horseradish peroxidase and Lazarillo expression confirm that the pioneers of the ventral and dorsal tracts of the antennal sensory nervous system are amongst these differentiating cells. Sets of pioneers appear simultaneously in several bands and project confluent axons towards the antennal base. We conclude that the sensory nervous system of the antenna is not pioneered from the tip of the antenna alone, but in a stepwise manner by cells from several zones. The early sensory nervous systems of antenna, maxilla and leg therefore follow a similar developmental program consistent with their serially homologous nature.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Effect of opioid compounds on feeding and activity of the cockroach, Periplaneta americana

Opioid peptides have been implicated in regulation of feeding in invertebrates. Studies have suggested that receptors for opioids are present in cockroaches and that these receptors play roles in affecting both behaviour and feeding. We examined the effect of µ, δ, and κ opioid receptor agonists and antagonists on feeding, mass changes and activity in the cockroach, Periplaneta americana. The κ antagonist, nor-binaltorphimine, significantly increased food intake, while naltrexone (general antagonist) and naloxonazine (µ antagonist) both reduced feeding. A large mass loss was observed in cockroaches treated with nor-binaltorphimine, despite the increased food intake. Males did not lose as much mass during the 3 h as females, although drug treatment did have some effect on the loss. Time of activity (%) was not influenced by any drug. Water loss experiments suggested that nor-binaltorphimine increased water loss, accounting for the mass loss despite the increased feeding. We suggest that two populations of opioid receptors are present as previously reported, with one affecting feeding and the other involved with evaporative water loss.     

Neuromodulation of flight initiation by octopamine in the cockroach Periplaneta americana

We have tested the effect of a known insect neuromodulator, octopamine, on flight initiation in the cockroach. Using minimally dissected animals, we found that octopamine lowered the threshold for windevoked initiation of flight when applied to either of two major synaptic sites in the flight circuitry: 1) the last abdominal ganglion, where wind-sensitive neurons from the cerci excite dorsal giant interneurons, or 2) the metathoracic ganglion, where the dorsal giant interneurons activate interneurons and motoneurons which are involved in producing the rhythmic flight motor pattern in the flight muscles (Fig. 2).Correlated with this change in flight initiation threshold, we found that octopamine applied to the last abdominal ganglion increased the number of action potentials produced by individual dorsal giant interneurons when recruiting the cereal wind-sensitive neurons with wind puffs (Figs. 3, 4, 5) or with extracellular stimulation of their axons (Fig. 6). Octopamine increases the excitability of the giant interneurons (Figs. 7, 8). Also, when we stimulated individual dorsal giant interneurons intracellularly, the number of action potentials needed to initiate flight was reduced when octopamine was applied to the metathoracic ganglion (Fig. 9).Abbreviations EMG electromyogram - dGIs dorsal giant interneurons - GI giant interneuron - A6 sixth abdominal ganglion - T3 third thoracic ganglion - EPSP excitatory postsynaptic potential     

Autoradiographic localization of [3H]-GABA accumulation in the sixth abdominal ganglion of the cockroach, Periplaneta americana L.

Localization of [³H]-GABA accumulation in the sixth abdominal ganglion of the cockroach was achieved using autoradiography.Light and electron microscope autoradiographic results provide direct evidence for glial accumulation of [³H]-GABA via a Na⁺-dependent carrier system. The functional role of the glial uptake of GABA is discussed.     

A molecular marker for epithelial morphogenesis in the cockroach

Summary A molecular marker has been identified in embryos of the cockroach, Periplaneta americana, that is localized among epithelial cells to those directly involved in morphogenesis. A monoclonal antibody has been developed that selectively binds to epithelial cells undergoing any of three very different morphogenetic movements-invagination, evagination or epiboly. Neighboring cells not involved in these developmental processes are not labeled by the antibody. The antigen is transiently present on the cells for a period just prior to and during the morphogenetic activity. It is localized on the apical surface of the cells. The spatial, temporal and subcellular distributions of antibody binding during development indicate a role for the antigen in epithelial morphogenesis different from that of any previously described molecule.     

Tuning posture to body load: decreases in load produce discrete sensory signals in the legs of freely standing cockroaches

Decreases in load are important cues in the control of posture and walking. We recorded activities of the tibial campaniform sensilla, receptors that monitor forces as strains in the exoskeleton, in the middle legs of freely moving cockroaches. Small magnets were attached to the thorax and body load was changed by applying currents to a coil below the substrate. Body position was monitored by video recording. The tibial sensilla are organized into proximal and distal subgroups that have different response properties and reflex effects: proximal sensilla excite extensor motoneurons while distal receptors inhibit extensor firing. Sudden load decreases elicited bursts from distal sensilla, while increased load excited proximal receptors. The onset of sensory discharges closely approximated the time of peak velocity of body movement in both load decreases and increases. Firing of distal sensilla rapidly adapted to sustained unloading, while proximal sensilla discharged tonically to load increases. Load decreases of small amplitude or at low rates produced only inhibition of proximal activity while decrements of larger size or rate elicited distal firing. These response properties may provide discrete signals that either modulate excitatory extensor drive during small load variations or inhibit support prior to compensatory stepping or initiation of swing.     

Circadian rhythm in olfactory response in the antennae controlled by the optic lobe in the cockroach

The olfactory response in antennae of the cockroach, Leucophaea maderae, was investigated by measuring electroantennograms (EAGs) in restrained animals. The amplitude of the EAG response to pulses of ethyl acetate, octanol, or fenchone, exhibited a robust, light entrained, circadian rhythm that persisted at least 14 days in constant darkness. Dilution-response curves measured at the peak and trough of the rhythm indicated there was a 10-fold change in sensitivity. The EAG rhythm was abolished by severing the optic tracts, while entrainment was abolished by ablation of the compound eyes. The results indicate that the circadian system modulates olfactory sensitivity in the antennae and that the rhythm is driven by a circadian pacemaker in the optic lobes that is entrained by photoreceptors in the compound eyes.     

Filter characteristics of cercal afferents in the cockroach

Summary The response dynamics of cercal afferents in the cockroach, Periplaneta americana, were determined by means of a cross-correlation technique using a Gaussian white noise modulation of wind as a stimulus. The white noise stimulus could evoke sustained firing activity in most of the afferents examined (Fig. 1). The spike discharges were unitized and then cross-correlated with the stimulus to compute 1st- and 2nd-order Weiner kernels. The Ist-order kernels from a total of 28 afferents were biphasic and closely matched the time differential of a pulse (Figs. 1, 3 and 4). The amplitude and waveform of the kernels depended on the stimulus angle in such a way that the kernels were the mirror image of those on the polar opposite side (Figs. 2 and 3). The 2nd-order kernels were also differential. They had 2 diagonal peaks and 2 off-diagonal valleys in a 2-dimensional plot with 2 time axes (Figs. 1, 5 and 6). This 4-eye configuration was basically invariant irrespective of the stimulus angle, although the kernels varied in amplitude when the stimulus angle was changed. The time between the peak and a following trough of the 1st-order kernel was constant and had a mean of 4.6±0.1 ms, whereas the time between 2 diagonal peaks of the 2nd-order kernels was 4.7±0.1 ms (Figs. 4 and 6), suggesting that wind receptors (filiform sensilla) on cerci act as a band-pass filter with a peak frequency of about 106 Hz. The peak time, however, varies from 2.3 to 6.9 ms in both kernels, which may reflect the spatial distribution of the corresponding hairs on the cercus. The summation of the 1st- (linear) and 2nd-order (nonlinear) models precisely predicted the timing of the spike firing (Fig. 8). Thus, these 2 lower-order kernels can totally characterize the response dynamics of the wind receptors. The nonlinear response explains the directional sensitivity of the sensory neurons, while the differentiating 1st-order kernel explains the velocity sensitivity of the neurons. The nonlinearity is a signal compression in which one of the diagonal peaks of the 2nd-order kernel always offsets the downward phase of the 1st-order kernel (Fig. 7) and obviously represents a half-wave rectification property of the wind receptors that are excited by hair movement in only one direction and inhibited by hair movement in the polar opposite direction.     

Exploitation of food resources by the cockroach Blattella germanica in an urban habitat

The exploitation of food resources by the German cockroach, Blattella germanica (L.) (Dictyoptera: Blattellidae) was investigated experimentally in relation to distance from shelters and depletion of neighbouring food patches. In addition, the dynamics of exploitation of a patch were analysed. Observations were made after dark in a public swimming baths building and each one lasted 3 h. Food patches were placed in rows, at different distances from the shelters. The number of cockroaches in food dishes, in a 20 cm diameter circle round each food dish and in a 60 cm diameter circle round this first circle were recorded.Food items nearest the shelters were exploited first. Exploitation of row 2 and of row 3 food items started later, after row 1 food patches had been depleted. Under these conditions, the moment a food patch was exploited was related to its distance from shelter. Exploitation of food patches occurred in a step-by-step manner, one patch attracting animals when a nearby patch had been depleted, and not following a model of ideal free distribution.Although our experimental food patches were exploited in relation to their distance from shelter, we were able to demonstrate that distance did not influence the dynamics of exploitation of a food item. The mean number of cockroaches on a food patch, whatever its spatial position, increased regularly, reached a maximum at t=–10 min, and then decreased rapidly after all the food had been completely consumed, at t=0 min. The mean number of animals in the 20 cm diameter circle round a food source peaked at t=0 min, then decreased rapidly. This area appeared to be a transit area. The mean number of animals in a 60 cm diameter circle round the food source peaked later, and then decreased slowly. Animals remained in this area longer than in the area closer to the food dish, but their presence there was concomitant with the depletion of the food box.