Mielinhüvely degradációt követő lipid törmelék eloszlás vizsgálata a központi idegrendszerben CARS mikroszkópiával és Agykérgi neurogliaform sejthálózat által kiváltott ionotróp és metabotróp gátló posztszinaptikus válaszok eltérő összegződése

In my doctoral dissertation, I present two independent research topics: 1. Degradation of the myelin sheath is thought to be the cause of neurodegenerative diseases, such as multiple sclerosis (MS), but definitive agreement on the mechanism of how myelin is lost is currently lacking. Autoimmune initiation of MS has been recently questioned by proposing that the immune response is a consequence of oligodendrocyte degeneration. To study the process of myelin breakdown, we induced demyelination with cuprizone and applied coherent anti-Stokes Raman scattering (CARS) microscopy, a non-destructive label-free method to image lipid structures in living tissue. We confirmed earlier results showing a brain region-dependent myelin destructive effect of cuprizone. In addition, high resolution in situ CARS imaging revealed myelin debris forming lipid droplets along with myelinated axon fibers. Quantification of lipid debris with custom-made software for segmentation and three-dimensional reconstruction revealed brain region-dependent accumulation of lipid drops inversely correlated with the thickness of myelin sheaths. Finally, we confirmed that in situ CARS imaging is applicable to living human brain tissue in brain slices derived from a patient. Thus, CARS microscopy is a potent tool for quantitative monitoring of myelin degradation in unprecedented spatiotemporal resolution during oligodendrocyte damage. We think that the accumulation of lipid drops around degrading myelin might be instrumental in triggering subsequent inflammatory processes. 2. The integration properties of synaptic inputs scattered over the neuronal surface are crucial to shaping the input-output characteristics of neurons. However, contrary to the summation of ionotropic receptor-mediated responses, experiments addressing the interaction of postsynaptic metabotropic signals arriving from multiple neuronal sources are scarce. Characterization of inhibitory connections established by individual layer 1 neurogliaform cells (NGFCs) revealed that NGFCs effectively activate postsynaptic GABA receptors in ~1.5um distance from the release site. This wide-spreading mode of inhibition and structural features combined with the NGFC network cooperation observed in vivo provide a contingency for converging outputs. Electrophysiological measurements of IPSPs arriving at the same postsynaptic neuron from two presynaptic NGFC showed that GABAA receptor-mediated responses summated sublinearly, while the interaction of GABAB receptor-mediated responses was linear. Next, We further investigated the possible influence of the receptor-channel complexes spatial distribution on the integration properties of inhibitory metabotropic receptor-mediated responses. Our results indicate that the spatial distribution itself has an effect on the integration properties: tight localization of the receptor-channel favors to the supralinear molecular interaction, while, segregated placement likely shifts the interaction towards sublinear

A combinatory genetic strategy for targeting neurogliaform neurons in the mouse basolateral amygdala

The mouse basolateral amygdala (BLA) contains various GABAergic interneuron subpopulations, which have distinctive roles in the neuronal microcircuit controlling numerous behavioral functions. In mice, roughly 15% of the BLA GABAergic interneurons express neuropeptide Y (NPY), a reasonably characteristic marker for neurogliaform cells (NGFCs) in cortical-like brain structures. However, genetically labeled putative NPY-expressing interneurons in the BLA yield a mixture of interneuron subtypes besides NGFCs. Thus, selective molecular markers are lacking for genetically accessing NGFCs in the BLA. Here, we validated the NGFC-specific labeling with a molecular marker, neuron-derived neurotrophic factor (NDNF), in the mouse BLA, as such specificity has been demonstrated in the neocortex and hippocampus. We characterized genetically defined NDNF-expressing (NDNF+) GABAergic interneurons in the mouse BLA by combining the Ndnf-IRES2-dgCre-D transgenic mouse line with viral labeling, immunohistochemical staining, and in vitro electrophysiology. We found that BLA NDNF+ GABAergic cells mainly expressed NGFC neurochemical markers NPY and reelin (Reln) and exhibited small round soma and dense axonal arborization. Whole-cell patch clamp recordings indicated that most NDNF+ interneurons showed late spiking and moderate firing adaptation. Moreover, ∼81% of BLA NDNF+ cells generated retroaxonal action potential after current injections or optogenetic stimulations, frequently developing into persistent barrage firing. Optogenetic activation of the BLA NDNF+ cell population yielded both GABAA- and GABAB receptor-mediated currents onto BLA pyramidal neurons (PNs). We demonstrate a combinatory strategy combining the NDNF-cre mouse line with viral transfection to specifically target adult mouse BLA NGFCs and further explore their functional and behavioral roles

Predominantly linear summation of metabotropic postsynaptic potentials follows coactivation of neurogliaform interneurons

Summation of ionotropic receptor-mediated responses is critical in neuronal computation by shaping input-output characteristics of neurons. However, arithmetics of summation for metabotropic signals are not known. We characterized the combined ionotropic and metabotropic output of neocortical neurogliaform cells (NGFCs) using electrophysiological and anatomical methods in the rat cerebral cortex. These experiments revealed that GABA receptors are activated outside release sites and confirmed coactivation of putative NGFCs in superficial cortical layers in vivo. Triple recordings from presynaptic NGFCs converging to a postsynaptic neuron revealed sublinear summation of ionotropic GABA(A) responses and linear summation of metabotropic GABA(B) responses. Based on a model combining properties of volume transmission and distributions of all NGFC axon terminals, we predict that in 83% of cases one or two NGFCs can provide input to a point in the neuropil. We suggest that interactions of metabotropic GABAergic responses remain linear even if most superficial layer interneurons specialized to recruit GABA(B) receptors are simultaneously active