| dc.description.abstracteng | In the severely disorganized reeler primary somatosensory cortex, thalamocortical axons manage to target layer 4-fated neurons. Although the reeler somatosensory cortex maintains proper activation in response to sensory input, the main thalamic input, from the ventral posteromedial (VPm) nucleus onto layer 4-fated spiny stellates (SpS), was found to be weakened compared to wild-type. These findings suggest that an intracortical mechanism compensates the weakened thalamic input to the reeler cortex. In this study, putative compensatory mechanisms were examined by following two dedicated hypotheses. The first hypothesis tested for enhanced recurrent excitation between SpS neurons. The second hypothesis proposed a reduced GABAergic transmission between SpS neurons and fast spiking (FS) interneurons. In order to study layer 4 in the disorganized reeler cortex, the Scnn1a-tdTomato-Reeler mouse line was used, where layer 4-excitatory neurons express cre-dependent tdTomato. In vitro paired whole-cell voltage clamp recordings of FS interneurons and SpS neurons allowed for assessing connection probability, reliability and strength in reeler versus WT mice. Prior to testing these hypotheses, previous findings of the lab that were fundamental to this study were replicated. Using optogenetic stimulation of thalamocortical axons in layer 4, thalamic input was measured in SpS neurons. Like the aforementioned results, it was concluded that reeler SpS neurons receive a weakened direct thalamic input, whereas the overall network excitation was increased. Before examining the connections between SpS neurons and FS interneurons, it was experimentally verified that the morphological and electrophysiological properties of SpS neurons and FS interneurons were not affected by improper lamination in the reeler cortex. Whole-cell patch clamp recordings between pairs of SpS neurons demonstrated overall comparable connection parameters in WT and reeler mutant mice, hence it is unlikely that enhanced recurrent excitation rescued the weakened thalamic input. Subsequently, it was tested whether a decreased GABAergic transmission compensated for the weakened thalamic input. In the reeler S1BF, a drastic decrease in connection probability and strength was found between FS interneurons and SpS neurons when using standard potassium-based intracellular solution. Remarkably, repeating these recordings with cesium-based intracellular solution in reeler revealed aWT-level connectivity rate and connection strength. Neuronal reconstructions of recorded neurons and immunolabeling for synaptotagmin-2, marking presynaptic boutons of FS, parvalbumin-expressing interneurons, showed that inhibitory synapses targeting reeler and WT SpS neurons have a similar somatodendritic distribution. These results suggest a proper connection probability, connection strength and synapse location between SpS neurons and FS interneurons in the reeler S1BF. This led me to examine the dendritic integration of inhibitory postsynaptic potentials (IPSPs) in reeler SpS neurons. Dendritic excitability is regulated by the hyperpolarization-activated current (Ih), an inward current mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Ih-mediated depolarization ensures a sufficient driving force for the propagation of dendritic inhibitory inputs. In addition, HCN channel activity has an excitatory effect where Ih depolarizes the membrane potential of the presynaptic terminal, bringing it closer to the threshold for Ca2+ entry. Thus, a reduced HCN channel expression in reeler SpS neurons was hypothesized to impair proper inhibitory and excitatory signal integration. First, the role of Ih on temporal summation of EPSPs in WT and reeler SpS neurons was studied, before and after pharmacological blockade of HCN channels. The slightly smaller effect of HCN channel blockade on the temporal summation of EPSPs in reeler SpS neurons, compared to WT, indicate a potentially reduced Ih intensity. In a different approach, paired recordings were performed between FS interneurons and SpS neurons in the presence of FK506 in order to increase HCN channel activity. Enhancing HCN channel activity restored connection probability and strength between reeler SpS neurons and FS interneurons. Further confirming a role for Ih, pharmacological blockade of HCN channels made inhibitory connection less reliable. These results suggest a reduced Ih in the reeler S1BF, resulting in reduced inhibitory and excitatory signaling. Taken together, the increased network activity, compromised GABAergic transmission, and decreased HCN channel expression, cause hyperexcitability in the reeler S1BF. These same features are known to cause epileptogenesis, hence the reeler mutant mouse presents to be potential model for epilepsy studies. | de |