Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06 Ludwig-Maximilians-Universität München

Organisms respond to changes in their environment affecting their physiological or
ecological optimum by reactions called stress responses. These stress responses may
enable the organism to survive by counteracting the consequences of the environ-
mental change, the stressor, and usually consist of plastic alterations of traits related
to physiology, behaviour, or morphology. In the ecological model species Daphnia,
the waterflea, stressors like predators or parasites are known to have an important
role in adaptive evolution and have been therefore studied in great detail. However,
although various aspects of stress responses in Daphnia have been analysed, molecu-
lar mechanisms underlying these traits are not well understood so far. For studying
unknown molecular mechanisms, untargeted ‘omics’ approaches are especially suit-
able, as they may identify undescribed key players and processes.
Recently, ‘omics’ approaches became available for Daphnia. Daphnia is a cosmo-
politan distributed fresh water crustacean and has been in research focus for a long
time because of its central role in the limnic food web. Furthermore, the responses of
this organism to a variety of stressors have been intensively studied e.g. to hypoxic
conditions, temperature changes, ecotoxicological relevant substances, parasites or
predation. Of these environmental factors, especially predation and interactions with
parasites have gained much attention, as both are known to have great influence on
the structure of Daphnia populations.
In the work presented in this thesis, I characterised the stress responses of Daphnia
using proteomic approaches. Proteomics is particularly well suited to analyse bio-
logical systems, as proteins are the main effector of nearly all biological processes.
However, performing Daphnia proteomics is a challenging task due to high proteolytic activity in the samples, which most probably originate from proteases located
in the gut of Daphnia, and are not inhibited by proteomics standard sample pre-
paration protocols. Therefore, before performing successful proteomic approaches,
I had to optimise the sample preparation step to inhibit proteolytic activity in Daph-
nia samples. After succeeding with this task, I was able to analyse stress responses of
Daphnia to well-studied stressors like predation and parasites. Furthermore, I stud-
ied their response to microgravity exposure, a stressor not well analysed in Daphnia
so far.
My work on proteins involved in predator-induced phenotypic plasticity is de-
scribed in chapter 2 and 3. Daphnia is a textbook example for this phenomenon and is
known to show a multitude of inducible defences. For my analysis, I used the system
of Daphnia magna and its predator Triops cancriformis. D. magna is known to change its
morphology and to increase the stability of its carapace when exposed to the pred-
ator, which has been shown to serve as an efficient protection against T. cancriformis
predation. In chapter 2, I used a proteomic approach to study predator-induced traits
in late-stage D. magna embryos. D. magna neonates are known to be defended against
Triops immediately after the release from the brood pouch, if mothers were exposed
to the predator. Therefore, the formation of the defensive traits most probably oc-
curs during embryonic development. Furthermore, embryos should have reduced
protease abundances, as they do not feed inside the brood pouch until release. To
study proteins differing in abundance between D. magna exposed to the predator
and a control group, I applied a proteomic 2D-DIGE approach, which is a gel based
method and therefore enables visual monitoring of protein sample quality. I found
differences in traits directly associated with known defences like cuticle proteins and
chitin-modifying enzymes most probably involved in carapace stability. In addition,
enzymes of the energy metabolism and the yolk protein vitellogenin indicat

Eye movements are important to aid vision, and they serve two main functions: to stabilize a moving visual target on the retina and to stabilize gaze during own body movements. Six types of eye movements have been evolved fulfilling this function: saccades, smooth pursuit, vestibulo-ocular reflex, optokinetic response, convergence and gaze holding. In all vertebrates the eyes are moved by six pairs of extraocular muscles that enable horizontal, vertical and rotatory eye movements. The motoneurons of these muscles are located in the oculomotor (nIII), trochlear (nIV) and abducens (nVI) nucleus in the brainstem. Motoneurons of the lateral rectus muscle (LR) in nVI and of the medial rectus muscle (MR) in nIII provide horizontal eye movements, those of inferior oblique (IO) and superior rectus muscle (SR) in nIII upward eye movements. Motoneurons of the superior oblique (SO) and the inferior rectus muscle (IR) in nIII convey downward eye movements. Recently, it was shown that each extraocular muscle is controlled by two motoneuronal groups:
1. Motoneurons of singly innervated muscle fibers (SIF) that lie within the boundaries of motonuclei providing a fast muscle contraction (twitch) and 2. motoneurons of multiply innervated muscle fibers (MIF) in the periphery of motonuclei providing a tonic muscle contraction (non-twitch). Tract-tracing studies indicate that both motoneuronal groups receive premotor inputs from different brainstem areas. A current hypothesis suggests that pathways controlling twitch motoneurons serve to generate eye movements, whereas the non-twitch system is involved in gaze holding. Lesions of inputs to the twitch motoneuron system may lead to supranuclear gaze palsies, whereas impairment of the non-twitch motoneuron system may result in gaze holding deficits, like nystagmus, or strabismus. Up to date only limited data are available about the histochemical characteristics including transmitters to the SIF- (twitch) and MIF (non-twitch) motoneurons.
The present study was undertaken to investigate the histochemical profile of inputs to motoneuronal groups of individual eye muscles mediating horizontal and vertical eye movements including the inputs to MIF- and SIF motoneurons. The MIF motoneurons of the IR and MR are located in the periphery dorsolateral to nIII, close to the Edinger-Westphal nucleus (EW), which is known to contain preganglionic cholinergic neurons. Other scientists have found that the EW is composed of urocortin-positive neurons involved in food intake or stress. In order to delineate these different cell populations within the supraoculomotor area dorsal to nIII, a comparative study in different mammals was conducted to locate the cholinergic preganglionic neurons and urocortin-positive neurons. Only then, it became obvious that the cytoarchitecturally defined EW labels different cell populations in different species. In rat, ferret and human the cytoarchitecturally defined EW is composed of urocortin-positive neurons. Only in monkey the EW contains cholinergic preganglionic neurons, which lie close to the MIF-motoneurons of MR and IR in the C-group.
In monkey, I performed a systematic study on the histochemical profile and transmitter inputs to the different motoneuron subgroups, including MIF- and SIF motoneurons. Brainstem sections containing prelabelled motoneurons were immunostained for the calcium-binding protein calretinin (CR), gamma-aminobutyric acid (GABA) or glutamate decarboxylase (GAD), glycine transporter 2, glycine receptor 1, and the vesicular glutamate transporters (vGlut) 1 and 2.
The study on the histochemical profile of the motoneuron inputs revealed three main results: 1.The inhibitory control of SIF motoneurons for horizontal and vertical eye movements differs. Unlike previous studies in the primate a considerable GABAergic input was found to all SIF motoneuronal groups, but a glycinergic input was confined to motoneurons of the MR mediating horizontal eye movements