Sensorische Grundlagen der thermischen Orientierung bei Blattschneiderameisen

Leaf-cutting ants have a highly developed thermal sense which the insects use to regulate the own body temperature and also to optimize brood and fungus development. Apart from the already described temperature guided behaviors inside the nest it is unknown to what extent the ants may use their thermal sense outside the nest. As part of the present thesis, the question was addressed whether leaf-cutting ants (Atta vollenweideri) are able to learn the position of a warm object as landmark for orientation during foraging. Using absolute conditioning, it was shown that ten training trials are sufficient to elicit the association be-tween food reward and the temperature stimulus. In the test situation (without reward) a significantly higher amount of ants preferred the heated site compared to the unheated con-trol. Importantly, thermal radiation alone was sufficient to establish the learned association and served as orientation cue during the test situation (chapter IV). Based on the experi-mental design used in the previous chapter, the localization of thermosensitive neurons, which detect the underlying thermal stimuli, is restricted to the head or the antennae of the ants. The antennal sensillum coeloconicum is a potential candidate to detect the thermal stimuli during the orientation behavior. In chapter V the sensillum coeloconicum of Atta vollenweideri was investigated concerning its gross morphology, fine-structure and the phy-siology of the associated thermosensitive neuron. The sensillum is predominantly located on the apical antennal segment (antennal tip) where around 12 sensilla are clustered, and it has a peg-in-pit morphology with a double walled, multiporous peg. The sensory peg is deeply embedded in a cuticular pit, connected to the environment only by a tiny aperture. The sen-sillum houses three receptor neurons of which one is thermosensitive whereas the sensory modality of the other two neurons remains to be shown. Upon stimulation with a drop in temperature, the thermosensitve neuron responds with a phasic-tonic increase in neuronal activity (cold-sensitive neuron) and shows rapid adaptation to prolonged stimulation. In ad-dition, it is shown that thermal radiation is an effective stimulus for the thermosensitive neuron. This is the first evidence that sensilla coeloconica play an important role during the thermal orientation behavior described in chapter IV. During the test situation of the classic-al conditioning paradigm, the ants showed rapid antennal movements, indicating that they scan their environment in order to detect the heated object. Rapid antennal movements will result in rapid discontinuities of thermal radiation that re-quire thermosensitive neurons with outstanding sensitivity and high temporal resolution. In Chapter VI the question was addressed whether the thermosensitive neuron of the sensilla coeloconica fulfils these preconditions. Extracellular recordings revealed that the neuron is extremely sensitive to temperature transients and that, due to the response dynamics, an estimated stimulus frequency of up to 5 Hz can be resolved by the neuron. Already a tem-perature increase of only 0.005 °C leads to a pronounced response of the thermosensitive neuron. Through sensory adaptation, the sensitivity to temperature transients is maintained over a wide range of ambient temperatures. The discovered extreme sensitivity, the high temporal resolution and the pronounced adaptation abilities are further evidence support-ing the idea that sensilla coeloconica receive information of the thermal environment, which the ants may use for orientation. In order to understand how the ants use their thermal environment for orientation, it is ne-cessary to know where and how thermal information is processed in their central nervous system. In Chapter VII the question is addressed where in the brain the thermal information, specifically received by the thermosensitive neuron of sensilla coeloconica, is represented. By selectively staining single sensilla coeloconica, the axons of the receptor neurons could be tracked into the antennal lobe of Atta vollenweideri workers. Each of the three axons termi-nated in a single functional unit (glomerulus) of the antennal lobe. Two of the innervated glomeruli were adjacent to each other and are located lateral, while the third one was clear-ly separate and located medial in the antennal lobe. Using two-photon Ca2+ imaging of an-tennal lobe projection neurons, the general representation of thermal information in the antennal lobe was studied. In 11 investigated antennal lobes up to six different glomeruli responded to temperature stimulation in a single specimen. Both, warm- and cold-sensitive glomeruli could be identified. All thermosensitive glomeruli were located in the medial half of the antennal lobe. Based on the correlative evidence of the general representation of thermal information and the results from the single sensilla stainings, it is assumed that thermal information received by sensilla coeloconica is processed in the medial of the three target glomeruli. This part of the thesis shows the important role of the antennal lobe in temperature processing and links one specific thermosensitive neuron to its target region (a single glomerulus). In chapter V it was shown that the sensilla coeloconica are clustered at the antennal tip and have an extraordinary peg-in-pit morphology. In the last chapter of this thesis (Chapter VIII) the question is addressed whether the morphology of the sensilla coeloconica predicts the receptive field of the thermosensitive neuron during the detection of thermal radiation. The sensory pegs of all sensilla coeloconica in the apical cluster have a similar orientation, which was not constraint by the shape of the antennal tip where the cluster is located. This finding indicates that the sensilla coeloconica function as a single unit. Finally the hypothesis was tested whether a single sensillum could be direction sensitive to thermal radiation based on its eye-catching morphology. By stimulating the thermosensitive neuron from various angles around the sensillum this indeed could be shown. This is the last and most significant evi-dence that the sensilla coeloconica may be adapted to detect spatially distributed heated objects in the environment during the thermal landmark orientation of ants.Blattschneiderameisen besitzen einen hochgradig entwickelten Temperatursinn, den sie hauptsächlich zur Regulation ihrer Körpertemperatur, aber auch zur Optimierung der Brut- und Pilzentwicklung einsetzen. Abgesehen von temperaturgesteuerten Verhaltensweisen innerhalb des Nests ist nicht bekannt, ob die Tiere ihren Temperatursinn auch außerhalb des Nests einsetzen können. Im ersten Teil der vorliegenden Arbeit wird der Frage nachgegan-gen, ob Blattschneiderameisen (Atta vollenweideri) die Position eines warmen Objektes de-tektieren können und ob sie das Objekt anschließend als erlernte Landmarke zur Orientie-rung während des Furagierens nutzen können. Mit Hilfe eines absoluten Konditionierungs-paradigmas konnte gezeigt werden, dass nach zehn Trainingsdurchläufen die Assoziation zwischen Futter und einem thermischem Stimulus von den Tieren gebildet wird. In der unbe-lohnten Testsituation entscheiden sich die signifikant höhere Anzahl der Tiere für die er-wärmte Seite. Alleine die thermische Strahlung des erwärmten Körpers ist bereits ausrei-chend, um die Assoziation zu bilden und während des Tests als Orientierungssignal zu dienen (Kapitel IV). Durch die Art und Weise der Durchführung des Experiments im vorangegangenen Kapitel, konnte der Ort, an dem sich die nötigen thermosensitiven Neurone befinden, auf den Kopf bzw. die Antennen der Tiere beschränkt werden. Aufgrund ihrer Position auf den Antennen gelten die Sensilla coeloconica als potentielle Kandidaten für die Detektion der notwendigen Stimuli während des thermischen Orientierungsverhaltens. In Kapitel V dieser Arbeit wird das Sensillum coeloconicum in Bezug auf seine Morphologie, seine Ultrastruktur und die Physiologie des assoziierten thermosensitiven Neurons untersucht. Sensilla coeloconica be-finden sich hauptsächlich auf dem letzen Antennensegment, der Antennenspitze, in einer Gruppe von bis zu 12 einzelnen Sensillen. Morphologisch kann das Sensillum als Grubensensillum klassifiziert werden und es enthält einem doppelwandigen Zapfen, der von zahlreichen Poren durchzogen ist. Der Zapfen ist tief in eine kutikuläre Grube eingelassen und nur über eine winzige Apertur mit der Umwelt verbunden. Das Sensillum beherbergt drei Rezeptorneurone, von denen eines thermosensitiv ist, während die sensorische Modali-tät der anderen beiden Neurone bis auf weiteres unklar ist. Als Antwort auf eine Abnahme in der Stimulustemperatur generiert das thermosensitive Neuron eine phasisch-tonische Erhö-hung der neuronalen Aktivität (kältesensitives Neuron) und adaptiert sehr schnell an anhaltende Stimulationen. Zusätzlich kann gezeigt werden, dass thermische Strahlung ein wirksa-mer Stimulus für das thermosensitive Neuron ist. Die Ergebnisse dieser Untersuchungen sind ein erster Hinweis darauf, dass die Sensilla coeloconica eine wichtige Rolle während des thermischen Orientierungsverhaltens spielen. Bei der klassischen Konditionierung wurden schnelle Antennenbewegungen bei den Ameisen festgestellt, die sich in der Testsituation zwischen dem warmen Objekt und dem Kontrollob-jekt entscheiden mussten. Diese schnellen Bewegungen könnten bedeuten, dass die Tiere Ihre Umgebung nach dem konditionierten warmen Objekt absuchen. Solche schnellen An-tennenbewegungen führen zu schnellen Temperaturänderungen und die Detektion dieser Stimuli setzt thermosensitive Neurone mit besonderer Sensitivität und erhöhtem zeitlichen Auflösungsvermögen voraus. In Kapitel VI wird untersucht, ob das thermosensitive Neuron der Sensilla coeloconica diese Voraussetzungen erfüllt. Extrazelluläre Ableitungen zeigen, dass das Neuron extrem sensitiv auf Temperaturänderungen reagiert und dass aufgrund der Antwortdynamik Stimulationsfrequenzen von bis zu fünf Hertz aufgelöst werden können. Schon eine Temperaturänderung von 0.005 °C führt zu einer ausgeprägten Antwort des thermosensitiven Neurons. Durch sensorische Adaption bleibt diese erhöhte Sensitivität über einen großen Umgebungstemperaturbereich erhalten. Die außergewöhnliche Sensitivi-tät, die hohe zeitliche Auflösung sowie die Adaptionsfähigkeit des thermosensitiven Neurons sind weitere Hinweise darauf, dass die Sensilla coeloconica in der Lage sind Stimuli zu rezi-pieren, welche zur thermischen Orientierung genutzt werden könnten. Um zu verstehen, wie sich die Tiere anhand ihrer thermischen Umwelt orientieren, ist es nötig zu wissen, wo im Zentralnervensystem die thermische Information prozessiert wird. In Kapitel VII wird analysiert, in welchem Bereich des Gehirns die thermische Information der Sensilla coeloconica repräsentiert ist. Mittels selektiver Färbung einzelner Sensilla coeloconica können die Axone der Rezeptorneurone im Gehirn verfolgt werden. Jedes der drei Axone endet in jeweils einer funktionellen Einheit (Glomerulus) im Antennallobus. Zwei der innervierten Glomeruli sind direkt benachbart und liegen im lateralen Teil des Antennallobus während der dritte Glomerulus im medialen Bereich zu finden ist. Mit Hilfe von zwei-Photonen Ca2+ Imaging der Projektionsneurone wurde die Repräsentation von thermischer Information im Antennallobus untersucht. In 11 untersuchten Antennalloben antworten bis zu sechs einzelne Glomeruli auf die Temperaturstimulation. Sowohl warm- als auch kalt-sensitive Glomeruli konnten identifiziert werden. Alle thermosensitiven Glomeruli tende Stimulationen. Zusätzlich kann gezeigt werden, dass thermische Strahlung ein wirksa-mer Stimulus für das thermosensitive Neuron ist. Die Ergebnisse dieser Untersuchungen sind ein erster Hinweis darauf, dass die Sensilla coeloconica eine wichtige Rolle während des thermischen Orientierungsverhaltens spielen. Bei der klassischen Konditionierung wurden schnelle Antennenbewegungen bei den Ameisen festgestellt, die sich in der Testsituation zwischen dem warmen Objekt und dem Kontrollob-jekt entscheiden mussten. Diese schnellen Bewegungen könnten bedeuten, dass die Tiere Ihre Umgebung nach dem konditionierten warmen Objekt absuchen. Solche schnellen An-tennenbewegungen führen zu schnellen Temperaturänderungen und die Detektion dieser Stimuli setzt thermosensitive Neurone mit besonderer Sensitivität und erhöhtem zeitlichen Auflösungsvermögen voraus. In Kapitel VI wird untersucht, ob das thermosensitive Neuron der Sensilla coeloconica diese Voraussetzungen erfüllt. Extrazelluläre Ableitungen zeigen, dass das Neuron extrem sensitiv auf Temperaturänderungen reagiert und dass aufgrund der Antwortdynamik Stimulationsfrequenzen von bis zu fünf Hertz aufgelöst werden können. Schon eine Temperaturänderung von 0.005 °C führt zu einer ausgeprägten Antwort des thermosensitiven Neurons. Durch sensorische Adaption bleibt diese erhöhte Sensitivität über einen großen Umgebungstemperaturbereich erhalten. Die außergewöhnliche Sensitivi-tät, die hohe zeitliche Auflösung sowie die Adaptionsfähigkeit des thermosensitiven Neurons sind weitere Hinweise darauf, dass die Sensilla coeloconica in der Lage sind Stimuli zu rezipieren, welche zur thermischen Orientierung genutzt werden könnten

Representation of Thermal Information in the Antennal Lobe of Leaf-Cutting Ants

Insects are equipped with various types of antennal sensilla, which house thermosensitive neurons adapted to receive different parameters of the thermal environment for a variety of temperature-guided behaviors. In the leaf-cutting ant Atta vollenweideri, the physiology and the morphology of the thermosensitive sensillum coeloconicum (Sc) has been thoroughly investigated. However, the central projections of its receptor neurons are unknown. Here we selectively stained the three neurons found in single Sc and tracked their axons into the brain of Atta vollenweideri workers. Each of the three axons terminates in a single glomerulus of the antennal lobe (Sc-glomeruli). Two of the innervated glomeruli are adjacent to each other and are located laterally, while the third one is clearly separated and located medially in the antennal lobe. Using two-photon Ca2+ imaging of antennal lobe projection neurons, we studied where in the antennal lobe thermal information is represented. In the 11 investigated antennal lobes, we found up to 10 different glomeruli in a single specimen responding to temperature stimulation. Both, warm- and cold-sensitive glomeruli could be identified. The thermosensitive glomeruli were mainly located in the medial part of the antennal lobe. Based on the general representation of thermal information in the antennal lobe and functional data on the Sc-glomeruli we conclude that temperature stimuli received by Sc are processed in the medial of the three target glomeruli. The present study reveals an important role of the antennal lobe in temperature processing and links a specific thermosensitive neuron to its central target glomerulus

Detection of minute temperature transients by thermosensitive neurons in ants

The antennae of leaf-cutting ants are equipped with sensilla coeloconica that house three receptor neurons, one of which is thermosensitive. Using convective heat (air at different temperatures), we investigated the physiological characteristics of the thermosensitive neuron associated with the sensilla coeloconica in the leaf-cutting ant Atta vollenweideri. The thermosensitive neuron very quickly responds to a drop in temperature with a brief phasic increase (50 ms) in spike rate and thus classifies as cold receptor (ambient temperature = 24°C). The short latency and the brief phasic response enable the thermosensitive neuron to follow temperature transients up to an estimated frequency of around 5 Hz. Although the neuron responds as a cold receptor, it is extremely sensitive to warm stimuli. A temperature increase of only 0.005°C already leads to a pronounced decrease in the resting activity of the thermosensitive neuron. Through sensory adaptation, the sensitivity to temperature transients is maintained over a wide range of ambient temperatures (18 30°C). We conclude that the thermosensitive neuron of the sensilla coeloconica is adapted to detect minute temperature transients, providing the ants with thermal information of their microenvironment, which they may use for orientation

Thermal radiation as a learned orientation cue in leaf-cutting ants (Atta vollenweideri)

We explored the ability of leaf-cutting ants (Atta vollenweideri) to learn the location of a food reward by using thermal information as an orientation cue. During training of single workers, the conditioned stimulus was a distant thermal source placed frontally, 15 mm away from a platform having a leaf fragment as reward. After training, single workers were confronted with the choice between two sides, one being coupled, in a pseudo-randomized design, with a thermal stimulus heated 5 °C above environmental temperature. After 10 learning trials, workers significantly chose the side with the thermal stimulus. This showed that workers can use thermal information for spatial orientation in the context of foraging, which may help them to locate, for instance, highly attractive sun-exposed leaves. Thermal radiation alone as orientation cue was sufficient to allow learning, since preclusion of thermal convection during training and test did not impair workers’ response. Shielding of both thorax and gaster from the thermal source did not weaken learning, suggesting the sole participation of head and antennae in temperature reception. A thermal stimulus heated 1 °C above environmental temperature could not be used as a learned orientation cue, even when foragers were allowed to directly contact the thermal source

Clearing pigmented insect cuticle to investigate small insects' organs in situ using confocal laser-scanning microscopy (CLSM)

Various microscopic techniques allow investigating structures from submicron to millimeter range, however, this is only possible if the structures of interest are not covered by pigmented cuticle. Here, we present a protocol that combines clearing of pigmented cuticle while preserving both, hard and soft tissues. The resulting transparent cuticle allows confocal laser-scanning microscopy (CLSM), which yields high-resolution images of e.g. the brain, glands, muscles and fine cuticular structures. Using a fluorescent dye, even single labeled neurons can be visualized and resolved up to an imaging depth of 150 μm through the cleared cuticle. Hydrogen-peroxide, which was used to clear the cuticle, does not preclude immunocytochemical techniques, shown by successful labeling of serotonin-immunoreactive neurons (5HT-ir) in the ants' brain. The ‘transparent insect protocol’ presented here is especially suited for small arthropods where dissection of organs is very demanding and difficult to achieve. Furthermore, the insect organs are preserved in situ thus allowing a more precise three-dimensional reconstruction of the structures of interest compared to, e.g., dissected or sectioned tissue

The thermo-sensitive sensilla coeloconica of leaf-cutting ants (Atta vollenweideri)

Social insects show a variety of temperature-guided behaviors. Depending on whether heat reaches the sensillum via air movements (convective heat) or as radiant heat, specific adaptations of thermo-sensitive sensilla are expected. In the present study the morphology and the physiology of thermo-sensitive peg-in-pit sensilla (S. coeloconica) of the leaf-cutting ant Atta vollenweideri were investigated. S. coeloconica are located predominantly in a single cluster on the apical antennomere, and connect to the outside through a small aperture. The sensory peg is double-walled, embedded in a chamber and innervated by three unbranched dendrites. Using tungsten electrodes, activity of the sensory neurons was measured. In most cases, the neuron with the largest spike amplitude responds to changes in air temperature (convective heat) as well as to radiant heat. In response to a drop in air temperature, the neuron shows a phasic-tonic response followed by a complete adaptation within 1 min (cold-sensitive neuron). Based on their morphology and physiology, it is suggested that the S. coeloconica are involved in the recently described thermal orientation behavior of A. vollenweideri leaf-cutting ants