Morphology of antennal hair field afferents, descending interneurons and mesothoracic motoneurons, and in situ calcium imaging of retrogradely labeled retractor coxae neurons in the stick insect.

The phasmid species Carausius morosus serves as an established animal for studying terrestrial locomotion and antennal movements for tactile exploration of the near-range environment, but neuroanatomical data is only limited available. In the first part of this thesis, fluorescent dye stainings of antennal hair field sensilla, which are important antennal joint angle sensors, and stainings of all antennal motor nerves were performed to receive detailed insights into the neuroanatomical organization of the stick insects’ antennal sensorimotor system. It was shown that afferents of all seven antennal hair fields (HF) send collaterals into the dorsal lobe, a deutocerebral neuropil region in the brain, and further descend through the circumoesophageal connective to the suboesophageal ganglion (SOG), where these afferents terminate. Moreover, double stainings of HFs located on different antennal segments and of HFs with different direction properties showed similar staining patterns, but differences were found in a spatially different formation of synaptic boutons within the entire depth of the dorsal lobe and within the SOG. In the dorsal lobe, HF afferents and projections of antennal motoneurons overlap, which could be an indication for direct synaptic contacts. Interestingly, stainings of the antennal nerve of the flagellum revealed another deutocerebral neuropil separated from the dorsal lobe, the ventral area of flagellum afferents described for crickets. Besides, thoracic connectives were stained to reveal somata of descending interneurons (DIN) located in the brain and SOG. Axonal backfills of cervical (neck) connectives revealed up to 410 cell bodies of DINs (brain: 205, SOG: 205), and backfills of connectives between the pro- and mesothoracic ganglia stained up to 173 DINs (brain: 83, SOG: 90). Furthermore, in double stainings of connectives and scapal HF sensilla it was observed that neurites of DINs seem to be in vicinity to afferents of HFs, which suggests that antennal HF signals are transmitted via a fast cephalothoracic pathway to thoracic motor centers. In the second part, the morphology of leg motoneurons, dorsal unpaired median neurons (DUM), and sensory neurons in the stick insect mesothoracic ganglion were examined. For this, cut ends of lateral nerves were retrogradely labeled with fluorescent dyes. Numbers of motoneurons and details of their structure by far exceed previously published data. Somata of excitatory motoneurons form clusters in the lateral ganglion cortex and their dendrites branch mainly in the dorsal neuropil. Nine DUM neurons were identified, six of which have axons in nervus lateralis 5, which innervates the retractor coxae muscles. Most sensory fibers terminate in the ventral association center (VAC). Twenty-three small cell bodies located close to the soma of the fast extensor tibiae motoneuron (FETi) likely belong to strand receptors. Stained structures are compared with previously published data from stick insects and other orthopteran insects. In the third part, a calcium (Ca2+) imaging method is introduced for retrograde loading cut ends of stick insect retractor coxae motoneuron axons with a Ca2+-sensitive fluorescent indicator. Intracellular Ca2+ changes were induced either by the application of the muscarinic acetylcholine agonist pilocarpine into the thoracic cavity and by tactile stimulation of the animal’s abdomen. The measured intracellular Ca2+ dynamics are very similar and temporarily uniform across primary and secondary motoneuron neurites. Calculated integrals of measured intracellular Ca2+ changes and integrals of retractor coxae spike activity revealed a linear correlation. This suggests that the observed changes in intracellular Ca2+ result from activation of voltage-gated Ca2+ channels by membrane depolarizations. Moreover, Ca2+ changes of lateral DUM neurites could be measured. A first analysis of pilocarpine-induced Ca2+ transients in presumed three different DUM neurites revealed regular and in-phase Ca2+ oscillations. These DUM neurite Ca2+ elevations have no fixed phase relation to either the protractor coxae nor the retractor coxae spike activity