Tonically Active Kainate Receptors (tKARs) : A Novel Mechanism Regulating Neuronal Function in the Brain

Fast excitatory transmission between neurons in the central nervous system is mainly mediated by L-glutamate acting on ligand gated (ionotropic) receptors. These are further categorized according to their pharmacological properties to AMPA (2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid), NMDA (N-Methyl-D-aspartic acid) and kainate (KAR) subclasses. In the rat and the mouse hippocampus, development of glutamatergic transmission is most dynamic during the first postnatal weeks. This coincides with the declining developmental expression of the GluK1 subunit-containing KARs. However, the function of KARs during early development of the brain is poorly understood. The present study reveals novel types of tonically active KARs (hereafter referred to as tKARs) which play a central role in functional development of the hippocampal CA3-CA1 network. The study shows for the first time how concomitant pre- and postsynaptic KAR function contributes to development of CA3-CA1 circuitry by regulating transmitter release and interneuron excitability. Moreover, the tKAR-dependent regulation of transmitter release provides a novel mechanism for silencing and unsilencing early synapses and thus shaping the early synaptic connectivity. The role of GluK1-containing KARs was studied in area CA3 of the neonatal hippocampus. The data demonstrate that presynaptic KARs in excitatory synapses to both pyramidal cells and interneurons are tonically activated by ambient glutamate and that they regulate glutamate release differentially, depending on target cell type. At synapses to pyramidal cells these tKARs inhibit glutamate release in a G-protein dependent manner but in contrast, at synapses to interneurons, tKARs facilitate glutamate release. On the network level these mechanisms act together upregulating activity of GABAergic microcircuits and promoting endogenous hippocampal network oscillations. By virtue of this, tKARs are likely to have an instrumental role in the functional development of the hippocampal circuitry. The next step was to investigate the role of GluK1 -containing receptors in the regulation of interneuron excitability. The spontaneous firing of interneurons in the CA3 stratum lucidum is markedly decreased during development. The shift involves tKARs that inhibit medium-duration afterhyperpolarization (mAHP) in these neurons during the first postnatal week. This promotes burst spiking of interneurons and thereby increases GABAergic activity in the network synergistically with the tKAR-mediated facilitation of their excitatory drive. During development the amplitude of evoked medium afterhyperpolarizing current (ImAHP) is dramatically increased due to decoupling tKAR activation and ImAHP modulation. These changes take place at the same time when the endogeneous network oscillations disappear. These tKAR-driven mechanisms in the CA3 area regulate both GABAergic and glutamatergic transmission and thus gate the feedforward excitatory drive to the area CA1. Here presynaptic tKARs to CA1 pyramidal cells suppress glutamate release and enable strong facilitation in response to high-frequency input. Therefore, CA1 synapses are finely tuned to high-frequency transmission; an activity pattern that is common in neonatal CA3-CA1 circuitry both in vivo and in vitro. The tKAR-regulated release probability acts as a novel presynaptic silencing mechanism that can be unsilenced in response to Hebbian activity. The present results shed new light on the mechanisms modulating the early network activity that paves the way for oscillations lying behind cognitive tasks such as learning and memory. Kainate receptor antagonists are already being developed for therapeutic use for instance against pain and migraine. Because of these modulatory actions, tKARs also represent an attractive candidate for therapeutic treatment of developmentally related complications such as learning disabilities.

Bovine mastitis caused by coagulase-negative staphylococci : host response and bacterial factors

Coagulase-negative staphylococci (CNS) are the most common bacteria isolated in bovine subclinical mastitis in many countries, and also a frequent cause of clinical mastitis. The most common species isolated are Staphylococcus (S) chromogenes, S. simulans, S. epidermidis, and S. xylosus. One half of the intramammary infections (IMI) caused by CNS persist in the udder. The pathogenesis of IMI caused by CNS is poorly understood. This dissertation focuses on host response in experimental intramammary infection induced by S. chromogenes, S. epidermidis and S. simulans. Model for a mild experimental CNS infection was developed with S. chromogenes (study I). All cows were infected and most developed subclinical mastitis. In study II the innate immune response to S. epidermidis and S. simulans IMI was compared in eight cows using a crossover design. A larger dose of bacteria was used to induce clinical mastitis. All cows became infected and showed mild to moderate clinical signs of mastitis. S. simulans caused a slightly stronger innate immune response than S. epidermidis, with significantly higher concentrations of the interleukins IL-1beta and IL-8 in the milk. The spontaneous elimination rate of the 16 IMIs was 31%, with no difference between species. No significant differences were recorded between infections eliminated spontaneously or remaining persistent, although the response was stronger in IMIs eliminated spontaneously, except the concentration of TNF-α, which remained elevated in persistent infections. Lactoferrin (Lf) is a component of the humoral defence of the host and is present at low concentrations in the milk. The concentration of Lf in milk is high during the dry period, in colostrum, and in mastitic milk. The effect of an inherent, high concentration of Lf in the milk on experimental IMI induced with S. chromogenes was studied in transgenic cows that expressed recombinant human Lf in their milk. Human Lf did not prevent S. chromogenes IMI, but the host response was milder in transgenic cows than in normal cows, and the former eliminated infection faster. Biofilm production has been suggested to promote persistence of IMI. Phenotypic biofilm formation and slime producing ability of CNS isolates from bovine mastitis was investigated in vitro. One-third of mastitis isolates produced biofilm. Slime production was less frequent for isolates of the most common mastitis causing species S. chromogenes and S. simulans compared with S. epidermidis. No association was found between the phenotypic ability to form biofilm and the persistence of IMI or severity of mastitis. Slime production was associated with persistent infections, but only 8% of isolates produced slime.

A proteomic view of probiotic Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG is a probiotic bacterium that is known worldwide. Since its discovery in 1985, the health effects and biology of this health-promoting strain have been researched at an increasing rate. However, knowledge of the molecular biology responsible for these health effects is limited, even though research in this area has continued to grow since the publication of the whole genome sequence of L. rhamnosus GG in 2009. In this thesis, the molecular biology of L. rhamnosus GG was explored by mapping the changes in protein levels in response to diverse stress factors and environmental conditions. The proteomics data were supplemented with transcriptome level mapping of gene expression. The harsh conditions of the gastro-intestinal tract, which involve acidic conditions and detergent-like bile acids, are a notable challenge to the survival of probiotic bacteria. To simulate these conditions, L. rhamnosus GG was exposed to a sudden bile stress, and several stress response mechanisms were revealed, among others various changes in the cell envelope properties. L. rhamnosus GG also responded in various ways to mild acid stress, which probiotic bacteria may face in dairy fermentations and product formulations. The acid stress response of L. rhamnosus GG included changes in central metabolism and specific responses related to the control of intracellular pH. Altogether, L. rhamnosus GG was shown to possess a large repertoire of mechanisms for responding to stress conditions, which is a beneficial character of a probiotic organism. Adaptation to different growth conditions was studied by comparing the proteome level responses of L. rhamnosus GG to divergent growth media and to different phases of growth. Comparing different growth phases revealed that the metabolism of L. rhamnosus GG is modified markedly during shift from the exponential to the stationary phase of growth. These changes were seen both at proteome and transcriptome levels and in various different cellular functions. When the growth of L. rhamnosus GG in a rich laboratory medium and in an industrial whey-based medium was compared, various differences in metabolism and in factors affecting the cell surface properties could be seen. These results led us to recommend that the industrial-type media should be used in laboratory studies of L. rhamnosus GG and other probiotic bacteria to achieve a similar physiological state for the bacteria as that found in industrial products, which would thus yield more relevant information about the bacteria. In addition, an interesting phenomenon of protein phosphorylation was observed in L. rhamnosus GG. Phosphorylation of several proteins of L. rhamnosus GG was detected, and there were hints that the degree of phosphorylation may be dependent on the growth pH.