Scanning Laser Optical Tomography Resolves Structural Plasticity during Regeneration in an Insect Brain

Background: Optical Projection Tomography (OPT) is a microscopic technique that generates three dimensional images from whole mount samples the size of which exceeds the maximum focal depth of confocal laser scanning microscopes. As an advancement of conventional emission-OPT, Scanning Laser Optical Tomography (SLOTy) allows simultaneous detection of fluorescence and absorbance with high sensitivity. In the present study, we employ SLOTy in a paradigm of brain plasticity in an insect model system. Methodology: We visualize and quantify volumetric changes in sensory information procession centers in the adult locust, Locusta migratoria. Olfactory receptor neurons, which project from the antenna into the brain, are axotomized by crushing the antennal nerve or ablating the entire antenna. We follow the resulting degeneration and regeneration in the olfactory centers (antennal lobes and mushroom bodies) by measuring their size in reconstructed SLOTy images with respect to the untreated control side. Within three weeks post treatment antennal lobes with ablated antennae lose as much as 60% of their initial volume. In contrast, antennal lobes with crushed antennal nerves initially shrink as well, but regain size back to normal within three weeks. The combined application of transmission-and fluorescence projections of Neurobiotin labeled axotomized fibers confirms that recovery of normal size is restored by regenerated afferents. Remarkably, SLOTy images reveal that degeneration of olfactory receptor axons has a trans-synaptic effect on second order brain centers and leads to size reduction of the mushroom body calyx. Conclusions: This study demonstrates that SLOTy is a suitable method for rapid screening of volumetric plasticity in insect brains and suggests its application also to vertebrate preparations.

Gewebeansatz-Strukturen auf pyritisierten Steinkernen von Ammonoideen

3400 pyritized internal moulds of Upper Devonian, Triassic, Jurassic and Lower Cretaceous ammonoids show various soft tissue attachment structures. They are preserved as regularly distributed black patterns on the moulds. All structures can be interpreted as attachment areas of muscles, ligaments and intracameral membranes. Paired structures are developed along the umbilicus and on the flanks of the moulds, unpaired ones appear on the middle of their dorsal and ventral sides. Strong lateral muscles cause paired twin lines on the flanks of the phragmocone and of the body chamber. A ventral muscle is deduced from small rounded or crescent shaped spots in front of each septum on the ventral side. These spots are often connected, forming a band-like structure. Broad dark external bands on the ventral side of the phragmocone, ventral preseptal areas in the posterior part of the living chamber, small twin lines or oval shaped areas on the ventral side of the living chamber represent paired or unpaired attachment areas of the hyponome muscle. A middorsal muscle is documented by small roughened areas in front of each dorsal lobe. Dark spots along the umbilicus, often connected and thus forming a band-like structure (tracking band), are remains of a pair of small dorsolateral muscles at the posterior end of the soft body. Dark bands, lines and rows of small crescent shaped structures behind the tips of sutural lobes are due to spotlike fixation places of the posterior part of the mantle and their translocation before subsequent septal secretion. Devonian goniatites had a paired system of lateral and ventrolateral muscles preserved on the moulds as black or incised lines on the flanks of the living chamber and as dark preseptal areas, ventrally indented. These structures represent the attachment areas of paired lateral cephalic and paired ventral hyponome retractors. Fine black lines on the phragmocone situated parallel to the sutures (pseudosutures) represent a rhythmical secretion of camera! membranes during softbody translocation. Goniatites had a paired system of lateral and ventrolateral muscles, whilst Neoammonoids have a paired lateral and dorsolateral system, and, additionally, an unpaired system on the ventral and on the dorsal side. Mesoammonoids show only a paired lateral and an unpaired dorsal one. Fine black lines situated parallel to the saddles and behind the lobes of the suture line can be interpreted as structures left during softbody translocation and a temporary attachment of rhythmical secreted cameral membranes. Cameral membranes had supported the efficiency of the phragmocone. Only some of the observed structures are also present in recent Nautilus. Differences in the form and position of attachment sites between ammonoids and recent Nautilus indicate different soft body organizations between ammonoids and nautiloids. The attachment structures of goniatites especially of tornoceratids can be compared with those of Nautilus which indicates Richter - Gewebeansatz-Strukturen bei Ammonoideen 3 a comparable mode of life. Differences in the form and position of attachment structures between goniatites and ammonites may indicate an increasing differentiation of the muscular system in the phylogeny of this group. Different soft body organization may depend on shell morphology and on a different mode of life. On the modification or reduction of distinct muscle systems ammonoids can be assigned to different ecotypes. Based on shell morphology and the attachment areas of cephalic and hyponome retractor muscles two groups can be subdivided: - Depressed, evolute morphotypes with longidome body-chambers show only small ventral hyponome retractor muscles. Lateral cephalic retractors are not developed. These morphotypes are adapted to a demersal mode of life. Without strong cephalic retractor muscles no efficient jet propulsion can be produced. These groups represent vertical migrants with efficient phragmocone properties (multilobate sutures, cameral membranes, narrow septal spacing). - Compressed, involute moiphotypes with brevidome body-chambers show strong cephalic and hyponome retractor muscles and represent a group of active swimmers. These morphotypes were able to live at different depths, in the free water column or/and near the seafloor. They are not confined only to one habitat. Most of the examined genera and species belong to this group. Changes of the attachment structures in the course of ontogeny confirm that juveniles of Amaltheus and Quenstedtoceras lived as passive planche drifters in upper and intermediate parts of the free water column after hatching. At the end of the juvenile stage with a shell diameter of 0,3 - 0,5 cm cephalic retractor muscles developed. With the beginning of an active swimming mode of life (neanic stage) the subadult animals left the free water column and moved into shallow water habitats. Fuciniceras showed no marked changes in the attachment structures during ontogeny. This indicates that there occur no differences in the mode of life between juvenile and adult growth stages. Based on attachment structures and shell morphology of Devonian goniatites their relation to the systematic position permits statements about probable phylogenetic relationships between the Cheiloceratidae and Tornoceratidae. In some cases attachment structures of ammonites permit statements about phylogenetic relationships on family and genus level.