Schichtenspezifische Charakterisierung der VIPcre/tdTomato-Mauslinie mittels neurochemischer Marker

Abstract

The neurons of the neocortex can be subdivided into excitatory and inhibitory neurons. The inhibitory neurons, which make up 20-30% of the neurons are GABAergic interneurons, which can be distinguished by morphological, electrophysiological and molecular characteristics from each other. There are three distinct groups of GABAergic interneurons, the parvalbumin (PV)-expressing, the somatostatin (SOM)-expressing and the ionotropic serotonin receptor 5HT3a-expressing interneurons. The 5HT3a-receptor-expressing interneurons represent a very heterogeneous group. 40% of this group express vasoactive intestinal polypeptide (VIP). In the present study, the transgenic VIPcre/tdTomato mouse was used. The transgenic mouse was generated by using the Cre/loxP technology. In this mouse, VIP-expressing cells are marked with the fluorescent protein tdTomato. The aim was to characterize the VIP-expressing neurons in the somatosensory cortex (barrel cortex) by immunohistochemistry and fluorescence-in-situ-hybridization. The proteins vasoactive intestinal polypeptide, somatostatin, parvalbumin, glutamate decarboxylase (GAD67) and the vesicular glutamate transporter 1 (VGLUT1) were used as molecular markers. Statements about the cell density and cell distribution in the layers I-VI of the barrel cortex could be made for a layer-specific characterization of the VIPcre/tdTomato mouse. It was also searched for possible colocalisation between VIP and SOM and VIP and PV. By using the probes Gad1 and Vglut1, conclusions about the excitatory or inhibitory properties of VIP-expressing interneurons could be drawn. Through the use of two different VIP antibodies and a Vip probe it could be demonstrated that the tdTomato fluorescent cells are indeed VIP-expressing interneurons. Between the VIP/tdTomato positive cells and the PV antibody or Pvalb probe no colocalization was detected. For the SOM antibody or Sst probe only a very small number of colocalisation could be shown with the VIP/tdTomato cells. This confirms that the VIPcre/tdTomato mouse is a reliable mouse model. The Vglut1 probe had never marked a VIP/tdTomato cell, so that excitatory properties of the VIP cells could not be detected. In contrast the Gad1 probe marked the majority of VIP/tdTomato cells. This confirms that the VIP cells are inhibitory GABAergic interneurons. The largest population of GABAergic interneurons in the VIPcre/tdTomato mouse are the PV-expressing interneurons. In the layers IV and Vb, most PV positive cells were detected. The SOM-expressing interneurons are the second largest cell population. The majority of SOM positive cells are located in the neocortical layers Vb and VI. For the VIP-expressing interneurons the largest number of cells could be found in layer II/III.