Expression of functional GABAc receptors in the brain

γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.

Spatial ecology of a specialist insect herbivore : the leaf-mining moth Tischeria ekebladella on the pedunculate oak Quercus robur

Herbivorous insects comprise a major part of terrestrial biodiversity, and their interactions with their host plants and natural enemies are of vast ecological importance. A large body of research demonstrates that the ecology and evolution of these insects may be affected by trophic interactions, by abiotic influences, and by intraspecific processes, but so far research on these individual aspects has rarely been combined. This thesis uses the leaf-mining moth Tischeria ekebladella and the pedunculate oak (Quercus robur) as a case study to assess how spatial variation in trophic interactions and the physical distribution of host trees jointly affect the distribution, dynamics and evolution of a host-specific herbivore. With respect to habitat quality, Tischeria ekebladella experiences abundant variation at several spatial scales. Most of this variation occurs at small scales notably among leaves and shoots within individual trees. While hypothetically this could cause moths to evolve an ability to select leaves and shoots of high quality, I did not find any coupling between female preference and offspring performance. Based on my studies on temporal variation in resource quality I therefore propose that unpredictable temporal changes in the relative rankings of individual resource units may render it difficult for females to predict the fate of their developing offspring. With respect to intraspecific processes, my results suggest that limited moth dispersal in relation to the spatial distribution of oak trees plays a key role in determining the regional distribution of Tischeria ekebladella. The distribution of the moth is aggregated at the landscape level, where local leaf miner populations are less likely to be present where oaks are scarce. A modelling exercise based on empirical dispersal estimates revealed that the moth population on Wattkast an island in south-western Finland is spatially structured overall, but that the relative importance of local and regional processes on tree-specific moth dynamics varies drastically across the landscape. To conclude, my work in the oak-Tischeria ekebladella system demonstrates that the local abundance and regional distribution of a herbivore may be more strongly influenced by the spatial location of host trees than by their relative quality. Hence, it reveals the importance of considering spatial context in the study of herbivorous insects, and forms a bridge between the classical fields of plant-insect interactions and spatial ecology.