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Ciliary neurotrophic factor (Cntf) acts as a differentiation and survival factor for different types of neurons and glial cells. It is expressed by peripheral Schwann cells and astrocytes in the central nervous system and mediates its effects via a receptor complex involving CntfRα, LifRß and gp130, leading to downstream activation of Stat3. Recent studies by our group have shown that Cntf modulates neuronal microtubule dynamics via Stat3/stathmin interaction. In a mouse model for motor neuron disease, i.e. pmn, Cntf is able to rescue axonal degeneration through Stat3/stathmin signaling. While these findings suggest a role of Cntf in controlling axonal functions in the neuromuscular system, additional data indicate that Cntf might also play a role in synaptic plasticity in the hippocampus. Electrophysiological recordings in hippocampal organotypic cultures and acute slices revealed a deficit in long-term potentiation (LTP) in Cntf -/- mice. This deficit was rescued by 24 h stimulation with Cntf, combined with an acute application of Cntf during LTP-measurements indicating that Cntf is both necessary and sufficient for hippocampal LTP, and possibly synaptic plasticity. Therefore, Cntf knockout mice were investigated to elucidate this possible role of Cntf in hippocampal LTP and synaptic plasticity.
First, we validated the presence of Cntf in the target tissue: in the hippocampus, Cntf was localized in Gfap-positive astrocytes surrounding small blood vessels in the fissure and in meningeal areas close to the dentate gyrus. Laser micro-dissection and qPCR analysis showed a similar distribution of Cntf-coding mRNA validating the obtained immunofluorescent results. Despite the strong LTP deficit in organotypic cultures, in vivo behavior of Cntf -/- mice regarding hippocampus-dependent learning and anxiety-related paradigms was largely inconspicuous. However, western blot analysis of hippocampal organotypic cultures revealed a significant reduction of pStat3 levels in Cntf -/- cultures under baseline conditions, which in turn were elevated upon Cntf stimulation. In order to resolve and examine synaptic structures we turned to in vitro analysis of cultured hippocampal neurons which indicated that pStat3 is predominantly located in the presynapse. In line with these findings, presynapses of Cntf -/- cultures were reduced in size and when in contact to astrocytes, contained less pStat3 immunoreactivity compared to presynapses in wildtype cultures.
In conclusion, our findings hypothesize that despite of a largely inconspicuous behavioral phenotype of Cntf -/- mice, Cntf appears to have an influence on pStat3 levels at hippocampal synapses. In a next step these two key questions need to be addressed experimentally: 1) is there a compensatory mechanism by members of the Cntf family, possibly downstream of pStat3, which explains the in vivo behavioral results of Cntf -/- mice and can likewise account for the largely inconspicuous phenotype in CNTF-deficient humans? 2) How exactly does Cntf influence LTP through Stat3 signaling? To unravel the underlying mechanism further experiments should therefore investigate whether microtubule dynamics downstream of Stat3 and stathmin signaling are involved in the Cntf-induced modulation of hippocampal synaptic plasticity, similar to as it was shown in motoneurons.
Desert ants of the genus Cataglyphis have become model systems for the study of insect navigation. An age-related polyethism subdivides their colonies into interior workers and short-lived light-exposed foragers. While foraging in featureless and cluttered terrain over distances up to several hundred meters, the ants are able to precisely return back to their often inconspicuous nest entrance. They accomplish this enormous navigational performance by using a path integration system - including a polarization compass and an odometer - as their main navigational means in addition to landmark-dependent orientation and olfactory cues. C. fortis, being the focus of the present thesis, is endemic to the salt flats of western North Africa, which are completely avoided by other Cataglyphis species. The fact that Cataglyphis ants undergo a behavioral transition associated with drastically changing sensory demands makes these ants particularly interesting for studying synaptic plasticity in visual and olfactory brain centers. This thesis focuses on plastic changes in the mushroom bodies (MBs) - sensory integration centers supposed to be involved in learning and memory presumably including landmark learning - and in synaptic complexes belonging to the lateral accessory lobe (LAL) known to be a relay station in the polarization processing pathway. To investigate structural synaptic plasticity in the MBs of C. fortis, synaptic complexes (microglomeruli, MG) in the visual (collar) and olfactory (lip) input regions of the MB calyx were immunolabeled and their pre- and postsynaptic profiles were quantified. The results show that a volume increase of the MB calyx during behavioral transition is associated with a decrease of MG number - an effect called pruning - in the collar and, less pronounced, in the lip that goes along with dendritic expansion in MB intrinsic Kenyon cells. Light-exposure of dark-reared ants of different age classes revealed similar effects and dark-reared ants age-matched to foragers had MG numbers comparable to those of interior workers. The results indicate that the enormous structural synaptic plasticity of the MB calyx collar is primarily driven by visual experience rather than by an internal program. Ants aged artificially for up to one year expressed a similar plasticity indicating that the system remains flexible over the entire life-span. To investigate whether light-induced synaptic reorganization is reversible, experienced foragers were transferred back to darkness with the result that their MBs exhibit only some reverse-type characteristics, in particular differences in presynaptic synapsin expression. To investigate the structure of large synaptic complexes in the LAL of C. fortis and to detect potential structural changes, pre- and postsynaptic profiles in interior workers and foragers were immunolabeled and quantified by using confocal imaging and 3D-reconstruction. The results show that these complexes consist of postsynaptic processes located in a central region that is surrounded by a cup-like presynaptic profile. Tracer injections identified input and output tracts of the LAL: projection neurons from the anterior optic tubercle build connections with neurons projecting to the central complex. The behavioral transition is associated with an increase by ~13% of synaptic complexes suggesting that the polarization pathway may undergo some sort of calibration process. The structural features of these synaptic contacts indicate that they may serve a fast and reliable signal transmission in the polarization vision pathway. Behavioral analyses of C. fortis in the field revealed that the ants perform exploration runs including pirouette-like turns very close to the nest entrance for a period of up to two days, before they actually start their foraging activity. During these orientation runs the ants gather visual experience and might associate the nest entrance with specific landmarks or get entrained to other visual information like the polarization pattern, and, concomitantly adapt their neuronal circuitries to the upcoming challenges. Moreover, the pirouettes may serve to stimulate and calibrate the neuronal networks involved in the polarization compass pathway. Video recordings and analyses demonstrate that light experience enhanced the ants’ locomotor activity after three days of exposure. The fact that both the light-induced behavioral and neuronal changes in visual brain centers occur in the same time frame suggests that there may be a link between structural synaptic plasticity and the behavioral transition from interior tasks to outdoor foraging. Desert ants of the genus Cataglyphis possess remarkable visual navigation capabilities, but also employ olfactory cues for detecting nest and food sites. Using confocal imaging and 3D-reconstruction, potential adaptations in primary olfactory brain centers were analyzed by comparing the number, size and spatial arrangement of olfactory glomeruli in the antennal lobe of C. fortis, C. albicans, C. bicolor, C. rubra, and C. noda. Workers of all Cataglyphis species have smaller numbers of glomeruli compared to those of more olfactory-guided Formica species - a genus closely related to Cataglyphis - and to those previously found in other olfactory-guided ant species. C. fortis has the lowest number of glomeruli compared to all other species, but possesses a conspicuously enlarged glomerulus that is located close to the antennal nerve entrance. Males of C. fortis have a significantly smaller number of glomeruli compared to female workers and queens and a prominent male-specific macroglomerulus likely to be involved in sex pheromone communication. The behavioral significance of the enlarged glomerulus in female workers remains elusive. The fact that C. fortis inhabits microhabitats that are avoided by all other Cataglyphis species suggests that extreme ecological conditions may not only have resulted in adaptations of visual capabilities, but also in specializations of the olfactory system. The present thesis demonstrates that Cataglyphis is an excellent candidate for studying the neuronal mechanisms underlying navigational features and for studying neuronal plasticity associated with the ant’s lifelong flexibility of individual behavioral repertoires.