Research in neuroscience relies to a large extent on the use of genetically modified animals. Extensive validation of new and existing models is a requirement for the acquisition of trustworthy data and to enable generalization to human physiology and disease. This thesis includes, as one part (project I and II), validation of a transgenic mouse model with the expression of the enzyme Cre-recombinase restricted to neurons in a band in the deepest layer of the cerebral cortex. Secondly, in project III we use this mouse model to study the process of short-term plasticity in neuronal cultures. Lastly, we investigate synaptic plasticity by studying the effect that the developmental signaling factor Hedgehog (Hh) has on mature hippocampal cultures (project IV). 

In project I and II, we identified the transgenic mouse Neurotensin receptor 1-Cre GN220 (Ntsr1-Cre) to have Cre expression targeted to the corticothalamic (CT) pyramidal neuron population in neocortical layer 6. Further, we identified a small group of Ntsr1-Cre positive neurons present in the white matter that is distinct from the CT population. We also identified that the transcription factor Forkhead box protein 2 (FoxP2) was specifically expressed by CT neurons in neocortex. In project I, we further explored the sensitivity of CT neurons to cholinergic modulation and found that they are sensitive to even low concentrations of acetylcholine. Both nicotinic and muscarinic acetylcholine receptors depolarize the neurons. Presenting CT neurons as a potential target for cholinergic modulation in wakefulness and arousal. 

In project III we studied Ntsr1-Cre neurons in cortical cultures and found that cultured neurons have similar properties to CT neurons in the intact nervous system. Ntsr1-Cre neurons in culture often formed synapses with itself, i. e. autapses, with short-term synaptic plasticity that was different to ordinary synapses. By expressing the light-controlled ion channel channelrhodopsin-2 (ChR2) in Ntsr1-Cre neurons we could compare paired pulse ratios with either electrical or light stimulation. Electrical stimulation typically produced paired-pulse facilitation while light stimulation produced paired pulse depression, presumably due to unphysiological Ca2+ influx in presynaptic terminals. Thus, cultured Ntsr1- Cre neurons can be used to study facilitation, and ChR2 could be used as a practical tool to further study the dependence of Ca2+ for short-term plasticity. 

In project IV we investigated the role of Hedgehog (Hh) for hippocampal neuron plasticity. Non-canonical Hh-signaling negatively regulated NMDA- receptor function through an unknown mechanism resulting in changes in NMDA-receptor mediated currents and subsequent changes in AMPA- receptors in an LTP/LTD manner in mature neurons. Proposing Hh as a slow-acting factor with ability to scale down excitation for instances of excessive activity, e.g. during an epileptic seizure, as a mechanism to make the activity in the neuronal networks stable. 

PURPOSE: Mutations in apolipoprotein A-I (apoA-I) may affect plasma high-density lipoprotein (HDL) cholesterol levels and the risk for cardiovascular disease but little is known about the presence and effects of circulating apoA-I variants. This study investigates whether the apoA-I mutations, apoA-I(L202P) and apoA-I(K131del) , are present on plasma HDL particles derived from heterozygote carriers and whether this is associated to changes in HDL protein composition.

EXPERIMENTAL DESIGN: Plasma HDL of heterozygotes for either apoA-I(L202P) or apoA-I(K131del) and family controls was isolated using ultracentrifugation. HDL proteins were separated by 2DE and analyzed by MS.

RESULTS: ApoA-I peptides containing apoA-I(L202P) or apoA-I(K131del) were identified in HDL from heterozygotes. The apoA-I(L202P) mutant peptide was less abundant than wild-type peptide while the apoA-I(K131del) mutant peptide was more abundant than wild-type peptide in the heterozygotes. Two-dimensional gel electrophoresis analyses indicated that, compared to controls, HDL in apoA-I(L202P) carriers contained less apoE and more zinc-α-2-glycoprotein while HDL from the apoA-I(K131del) heterozygotes contained more alpha-1-antitrypsin and transthyretin.

CONCLUSIONS AND CLINICAL RELEVANCE: Both apoA-I(L202P) and apoA-I(K131del) were identified in HDL. In heterozygotes, these mutations have markedly differential effects on the concentration of wild-type apoA-I in the circulation, as well as the HDL proteome, both of which might affect the clinical phenotype encountered in the heterozygous carriers.

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