Abstract
Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for in vivo brain studies with two-photon fluorescence (TPF) microscopy. Mice of the thy1GFP-M line have been engineered for selective expression of green fluorescent protein (GFP) in neuronal populations. Here, we report that TPF microscopy reveals, at the brain surface of these mice, also motile non-neuronal GFP+ cells. We have analyzed the behavior of these cells in vivo and characterized in brain sections their immunophenotype.With TPF imaging, motile GFP+ cells were found in the meninges, subarachnoid space and upper cortical layers. The striking feature of these cells was their ability to move across the brain parenchyma, exhibiting evident shape changes during their scanning-like motion. In brain sections, GFP+ cells were immunonegative to antigens recognizing motile cells such as migratory neuroblasts, neuronal and glial precursors, mast cells, and fibroblasts. GFP+ non-neuronal cells exhibited instead the characteristic features and immunophenotype (CD11c and major histocompatibility complex molecule class II immunopositivity) of dendritic cells (DCs), and were immunonegative to the microglial marker Iba-1. GFP+ cells were also identified in lymph nodes and blood of thy1GFP-M mice, supporting their identity as DCs. Thus, TPF microscopy has here allowed the visualization for the first time of the motile behavior of brain DCs in situ. The results indicate that the thy1GFP-M mouse line provides a novel animal model for the study of subsets of these professional antigen-presenting cells in the brain. Information on brain DCs is still very limited and imaging in thy1GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions.
Highlights
The generation of transgenic mice expressing fluorescent proteins in subsets of cells in the brain, such as glia, neurons, and monocyte-derived cells, together with the increasing use of two-photon fluorescence (TPF) microscopy have allowed in vivo investigations of different cell types at the single cell level [1,2,3]
The apical dendrites of green fluorescent protein (GFP)-tagged neurons, their dendritic spines and apical tufts were visualized in cortical layers I and II, together with cell bodies of pyramidal neurons residing in layers III and neuronal elements in layer IV
Motile green fluorescent cells with morphological features and distribution different from those of neurons were observed in the TPF microscopy investigation (Figure 1)
Summary
The generation of transgenic mice expressing fluorescent proteins in subsets of cells in the brain, such as glia, neurons, and monocyte-derived cells, together with the increasing use of two-photon fluorescence (TPF) microscopy have allowed in vivo investigations of different cell types at the single cell level [1,2,3]. The thy1GFP-M knock-in mice express green fluorescent protein (GFP) in about 10% of neurons. In these mice, the regulatory element Thy has been modified by deletion of the intron required for the expression of the insert gene in nonneuronal cells [12,13]. Thy1-expressing non-neuronal cell types (thymocytes, peripheral T cells, myoblasts, epidermal cells, keratinocytes) [14] and dendritic cells (DCs) [15] should not express GFP. In the initial description of this mouse line, the occurrence of a few fluorescent-tagged mononucleated cells in the brain was reported as occasional finding, though with no details on their identification and characterization [13]
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