Search

1AbstractBehaviour involving two or more individuals within the same species is known as social behaviour. Sexual dimorphisms and environmental cues as well as age, experience and social hierarchy shape social behaviour. The study of this complex behaviour, however, has one essential boundary condition: the distance between individuals. Because social signals (e.g. acoustic, visual or olfactory) have limited reach the inter-individual distance is crucial for the correct transmission of such signals. In this study we present a newly developed assay to study group behaviour and inter-individual distance in Drosophila melanogaster. This assay consists of a circular observation space flanked by two automatic release devices that allow flies to enter individually. By releasing the flies one at a time, the observer can study different features of (intra-)group behaviour with different group compositions. Importantly, the observer can control (manually or through automatisation) the increase of group size over time.Over the last decades insects became more relevant as convenient model organisms to study the neurophysiological and genetic basis of (human) neuro-developmental disorders. Drosophila models are used to study the genetic and neuronal underpinnings of a wide range of neurological disorders. In some cases the studies revealed alterations in social behaviour consistent with descriptions of behavioural symptoms in human disorders.Social behaviours in Drosophila are well-studied and include courtship, mating, aggression and group interactions. This setup will facilitate the analysis of these aspects of group interactions in Drosophila, allowing for a deeper understanding of the neuronal circuits and genetic factors involved in those behaviours.2Contribution to the fieldSocial behaviour pertains to the most sophisticated behavioural feats, as it involves multiple, interacting individuals. These complex interactions often conceal the underlying neuronal and ethological mechanisms. One of the most basal ethological mechanisms is the inter-individual distance, which resembles a perimeter in which each individual needs to formulate a response to the approach of others.We introduce a device that allows to test the inter-individual distance under consistent circumstances, by automating the entry time and direction of conspecifics into the arena. We can further observe the composition, dynamics and forming of larger animal groups as well as their separating. We can also observe how the individual distances alter during the process. Also other behaviours can be easily observed, e.g. aggression, courtship, homosexual courtship, etc. We successfully employed this approach in [1] and could discriminate the role of different neuroligins in social behaviour. We provide a detailed description including building plans and material lists for this social observation device. The system can be run in an automatic mode to ensure the consistency of experiments or in a manual mode to test animals under more flexible social situations. We provide multiple back lighting systems to test animals in the dark (infra-red LEDs) or in illuminated environments (vis. range LEDs). The system is fully automated and can be linked with a number of animal trackers (e.g. T-Rex, deeplabcut, LACE, etc.) via simple videography. We hope that our experimental setup augments the variety of behaviours testable in ethological setups (T-maze, water-mazes, operant conditing setups, etc) with social interaction and group formation.

Sensing environmental temperatures is essential for the survival of ectothermic organisms. InDrosophila,two methodologies are used to study temperature preferences (TP) and the genes involved in thermosensation: two-choice assays and temperature gradients. Whereas two-choice assays reveal a relative TP, temperature gradients can identify the absolute Tp. One drawback of gradients is that small ectothermic animals are susceptible to cold-trapping: a physiological inability to move at the cold area of the gradient. Often cold-trapping cannot be avoided, biasing the resulting TPto lower temperatures. Two mathematical models were previously developed to correct for cold-trapping. These models, however, focus on group behaviour which can lead to overestimation of cold-trapping due to group aggregation. Here we present a mathematical model that estimates the behaviour of individualDrosophilain temperature gradients. The model takes the spatial dimension and temperature difference of the gradient into account, as well as the rearing temperature of the flies. Furthermore, it allows quantifying cold-trapping, reveals true TP, and differentiates between temperature preference and tolerance. Online simulation is hosted athttp://igloo.uni-goettingen.de. The code can be accessed athttps://github.com/zerotonin/igloo.

An inexpensive specimen holder for objects under a dissecting microscope is described which allows arbitrary orientation and rotation of the object within the visual field and focal plane of the microscope. Based on a spherical cap upon a magnet, this apparatus is easy to construct and permits single-handed manipulation of the specimen from some distance. Precise positioning is demonstrated for halteres of Drosophila under up to 120× magnification. Because of the restricted field of view and depth of focus of a dissecting microscope, it is often difficult to position tiny objects under study in varying orientation, especially if the specimen must simultaneously be dissected with forceps, microscissors or needles. The problem is solved most conveniently with a specimen holder based on a spherical cap which is held by a strong magnet on a steel ring. The specimen tilts vertically in any direction by up to 70° and-even in oblique orientation-rotates through 360° around the sphere's centre in the focal plane. By help of an interleaved plastic foil in a ring carrier, these movements can be controlled unimanually from outside the visual field.

We report on factors affecting the spontaneous firing pattern of the identified serotonin-containing Retzius neurons of the medicinal leech. Increased firing activity induced by intracellular current injection is followed by a 'post-stimulus-depression' (PSD) without spiking for up to 23 s. PSD duration depends both on the duration and the amplitude of the injected current and correlates inversely with the spontaneous spiking activity. In contrast to serotonin-containing neurons in mammals, serotonin release from the Retzius cells presumably does not mediate the observed spike suppression in a self-inhibitory manner since robust PSD persists after synaptic isolation. Moreover, single additional spikes elicited at specific delays after spontaneously occurring action potentials are sufficient to significantly alter the firing pattern. Since sub-threshold current injections do not affect the ongoing spiking pattern and PSD persists in synaptically isolated preparations our data suggest that PSD reflects an endogenous and 'spike-dependent' mechanism controlling the spiking activity of Retzius cells in a use-dependent way.

Tethered walking crickets often respond to single wind puffs (50 ms duration) directed from 45° left or right to the abdominal cerci with a short running bout of about 300 ms, followed by normal locomotion. To test for an effect of the current behavioral state on the running response, we applied wind stimuli when the insect attained a predefined translatorial and/or rotatorial velocity during spontaneous walking. The latency, duration, and velocity profile of the running bout always proved to be constant, representing a reflexlike all-or-nothing reaction, while the probability of this response was low after even brief standing and increased with the forward speed of spontaneous walking at the moment of stimulation. In contrast, the current rotatorial speed did not affect the stimulus response.

Using intracellular recording techniques in stationary walking crickets (Gryllus bimaculatus), we have investigated the relationship between locomotion and the activity of interneurones ascending from the terminal ganglion. Nine different types of giant interneurones (GI) were characterized during walking and standing. One third of them reduced their activity, while the others enhanced their spike rate, during walking. These physiological properties were strictly correlated with morphological characteristics such as axon position in the longitudinal tracts of the terminal ganglion. In general, ventral GIs reduced and dorsal GIs increased their spike frequency during walking. In some of them, there was a weak but significant correlation between the spike rate and translational speed, but no correlation with rotational speed. In all GIs except 10-3a, the changes in activity occurred at the start of walking. In GI 10-3a, an increase in membrane potential and spike rate was observed before the start of locomotion. Therefore, an intrinsic mechanism within the central nervous system operating on GI 10-3a is suggested. Additionally, the activities of filiform hair receptors and of previously undescribed small ascending interneurones (SAI) have been studied during walking. About 80% of the receptors slightly increased their spike rate during walking, while one SAI became more active during walking and another one was hardly affected. The physiological properties of ascending interneurones are discussed with respect to their modulation and particular function during walking.

We studied the effects of repetitive wind pulses on the escape behaviour of tethered male cockroaches and crickets walking on a styrofoam ball (open-loop stimulation). The movements of this sphere were recorded to measure duration, forward and turning speed of walking and standing phases during the insect's intended locomotion. All tested parameters were quantitatively similar for spontaneous walking in both species. During stimulation crickets generated a sequence of running bouts very regular both in duration and forward speed. Interposed were standing phases the mean duration of which was inversely correlated with the wind puff frequency. The mean distance run per period varied only between 100 and 150 mm for the tested frequencies. Cockroaches often showed continuous high speed running for seconds, variable in duration and followed by variable standing phases. Both parameters together with the mean forward speed of walking phases depended on the stimulus frequency in a non-linear manner. Therefore, the longest distance per period was run during stimulation with medium frequencies. Crickets were much better than cockroaches in turning away from the wind source in the range of 5–10 Hz stimulation. In cockroaches, the escape reaction was partly retained after cercus ablation, but ceased completely when the styli were also removed.

Spontaneous walking and escape running in response to wind puffs directed to the abdominal cerci were quantitatively studied in tethered walking crickets. An apparatus for optically recording rotations of an air-supported sphere was developed to measure the intended locomotion of insects with high linear and temporal resolution but without mechanically imposed bias. During spontaneous locomotion without sensory cues for orientation, alternate pauses of 0.35–2.2 s and walking phases of 0.5–6 s resulted in a highly variable pattern of locomotion on a meandering path. A single air puff to one or both of the wind-sensitive cerci evoked a short run, whereas a continuous sequence of puffs caused sustained escape running with a tendency to turn away from the stimulus source. Escape running was characterized by a series of stereotyped running bouts and pauses, both significantly shorter than those recorded during spontaneous locomotion. Forward speed and angular speed of escape running correlated linearly with the wind puff frequency between 5 and 10Hz. This was caused by a shortening of the standing phases, while the durations of the running bouts were constant. The reflex-like running bouts and the pattern of escape running were largely independent of the duty cycle of the wind puff series and the wind speed. Neither individual steps nor running bouts were synchronized with the stimulus pattern. The behavioural modes of spontaneous walking and escape running were maintained with a minor reduction in general activity in partly dissected specimens during intracellular recording in the prothoracic ganglion. Each impaled local interneurone with locomotion-related activity generated action potentials in the actual step rhythm of walking and running bouts, but did not show specific activity during escape running. Some of these local neurones, however, showed modulations of spike frequency before or during intended turns and may participate in the coordination of the prothoracic legs.

The cerci, paired appendages at the rear end of the cricket abdomen, are covered with a variety of sense organs: the mechanosensitive filiform, clavate and long bristle hairs as well as campaniform sensilla and the gustatory short bristle hairs (Gnatzy and Schmidt, 1972, 1972; Schmidt and Gnatzy 1972). In this multimodal receptor system (Murphey and Chiba 1990), the most numerous are the exclusively mechanosensitive receptor types: filiform hairs and campaniform sensilla.

In crickets, neurogenesis persists throughout adulthood for certain local brain interneurons, the Kenyon cells in the mushroom bodies, which represent a prominent compartment for sensory integration, learning, and memory. Several classes of these neurons originate from a perikaryal layer, which includes a cluster of neuroblasts, surrounded by somata that provide the mushroom body's columnar neuropil. We describe the form, distribution, and cytology of Kenyon cell groups in the process of generation and growth in comparison to developed parts of the mushroom bodies in adult crickets of the species Gryllus bimaculatus. A subset of growing Kenyon cells with sprouting processes has been distinguished from adjacent Kenyon cells by its prominent f-actin labelling. Growth cone-like elements are detected in the perikaryal layer and in their associated sprouting fiber bundles. Sprouting fibers distant from the perikarya contain ribosomes and rough endoplasmic reticulum not found in the dendritic processes of the calyx. A core of sprouting Kenyon cell processes is devoid of synapses and is not invaded by extrinsic neuronal elements. Measurements of fiber cross-sections and counts of synapses and organelles suggest a continuous gradient of growth and maturation leading from the core of added new processes out to the periphery of mature Kenyon cell fiber groups. Our results are discussed in the context of Kenyon cell classification, growth dynamics, axonal fiber maturation, and function.

Responses to sensory stimuli and spike activity uring walking were investigated in bilaterally symmetrical dorsal unpaired median (DUM) neurons of the cricket. Intracellular recordings within the prothoracic ganglion were made either in restrained animals or in stationary walking specimens whilst parameters of their intended locomotion were measured. Three types of DUM cells were distinguished morphologically and physiologically. DUMa neurons send axons through segmental nerves. They often generated spontaneously large action potentials with low frequencies. Most DUMa neurons showed multimodal sensitivity, preferentially to cereal wind puffs and 15 kHz sound. Mean latencies ranged from 25 to 349 ms. Their large intraindividual variability could be correlated with behavioral modes during walking. Generally, the spike frequency increased with increased forward speed, while it was not related to turning. DUMb neurons projected either through the anterior or posterior connectives, but seemed physiologically similar to DUMa neurons. DUMc neurons were H-shaped with axons in both pairs of connectives. No external stimulus led to discrete spikes, but the regular spontaneous activity was modulated following cereal wind puffs to a restrained animal. During wind evoked escape the spike activity of another DUMc cell was modulated in phase with the rhythmic running behavior. The possibly different functions of DUMa and DUMc neurons during walking are discussed.

The anatomical organization of the two dorsal giant fiber systems of the earthworm Lumbricus terrestris is demonstrated in whole mounts and serial-section reconstructions based on backfillings of the ventral nerve cord with cobalt chloride. Both the medial and lateral fiber systems can be labeled selectively over more than ten body segments. They show a characteristic segmental pattern of collaterals with some modification in tail segments and of dorsal plasma protrusions in the unpaired medial giant fiber presumably representing openings in the myelin sheath. We found no multisegmental cobalt transport in other large neurons of the nerve cord. Cobalt passes through the segmentai septa between consecutive axonal elements of the metameric giant fibers and presumably also through commissural contacts between specific collaterals of the lateral giant fibers. Since these sites of contact are known to represent electrical synapses, cobalt coupling may, in L. terrestris, correlate with functional electrotonic coupling.

An algorithm is described to generate pictures in arbitrary perspective from serially sectioned biological material. Only those parts of the object which lie on a direct line of sight to the observer are included into the computed paper-plot, while hidden lines are omitted. The program was written for an inexpensive Apple II+ microcomputer as an extension of an earlier program library (NEUREC) for three-dimensional reconstruction. Application examples of neuronal and cardiac tissues are presented.

The distribution of serotonin-immunoreactive cell bodies and fibers in the ventral nerve cord of the earthworm has been investigated from whole-mount preparations and serial sections. Serotonin-immunoreactive neurons are organized in seven soma groups per ganglion; these are defined by cell number, soma shape, diameter and fiber projections. Positional variations of this pattern have been studied quantitatively. The number of labeled perikarya is constant in midbody ganglia, but increases markedly rostral to the posterior margin of the clitellum. Variability of position and cell number differs between the cell groups. Stained nerve fibers could only be partially traced; their distribution is described in relation to defined neuronal fiber bundles and segmental nerves. The distribution and morphology of serotonin-immunoreactive cells are compared with previous observations, based on pattern analysis of primary catecholamines and indolamines with the use of a formaldehyde-induced fluorescence technique. The possible role of serotonin-immunoreactive nervous elements in different nerve cord compartments is discussed with respect to physiological effects of serotonin in earthworms and other invertebrates.

Enregistrements intracellulaires des neurones intermediaires au cours de courses d'echappement provoquees par le vent chez les grillons, qui caracterisent des recepteurs sensibles au vent au niveau thoracique

1. 1. The chromatophores of the Diadematid sea urchin Centrostephanus longispinus react to changes in illumination: in darkness they have a round or ovoid shape. When subjected to light they change shape to a stellate form by stretching of pigment bearing cellular processes. 2. 2. The pigment cells show a different sensitivity to light between 370 and 650 nm with a maximum of sensitivity between 430 and 450 nm. 3. 3. The extinction curve of pigment shows a maximum between 500 and 540 nm and does not correspond to the spectral sensitivity of pigment dispersion.

On stimulation of the head, the caudal 20-30 segments of intact earthworms exhibit a marked dorso-ventral flattening reflex which is an element of the escape response. When the tail is removed this reflex is reconstituted gradually in segments anterior to the amputation level without regeneration of new tail segments. It takes longer to reestablish the flattening if larger parts of the body are cut off, but after 60 days p.o. all animals regained the full response independent of the number of segments amputated. Extirpation of three consecutive ganglia from the ventral nerve cord also suffices to develop flattening anterior to the operated segments, but the reaction is less pronounced and appears later than after tail amputation. Moreover, it vanishes at the same time the normal tail flattening is observed again, presumably after regeneration across the gap in the cord. In different regions of the body, area and perimeter distributions of transversely sectioned axons in the three pairs of segmental nerves (SN I-III) of the cord were measured. Characteristic changes along the length of the animal were found for SN I and SN III. In tail segments the axonal size distributions are more variable than "typical" segments. The flattening reflex is mediated by axons in SN III (Pallas and Drewes, 1981). In body segments having reconstituted behavioral characteristics of the lost tail segments, the area of the largest axons and the general size distribution in SN III approximate those found in normal tail segments.

A software package is described to reconstruct three-dimensional pictures in true perspective from a series of parallel sections using a low-cost computer system (Apple II plus). Data sampling via a graphic tablet and graphical output on the monitor screen or a digital plotter are assigned to different programs under control of a menu program. The number of data representing the object under study is unlimited. Originally written in BASIC, the programs were translated to machine language. As an application of the package, reconstructions of an identified large interneuron of the locust brain are presented.