Two-Photon Microscopy Imaging of <em>thy1</em>GFP-M Transgenic Mice: A Novel Animal Model to Investigate Brain Dendritic Cell Subsets <em>In Vivo</em>

<div><p>Transgenic mice expressing fluorescent proteins in specific cell populations are widely used for <em>in vivo</em> brain studies with two-photon fluorescence (TPF) microscopy. Mice of the <em>thy1</em>GFP-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 <em>in vivo</em> and characterized in brain sections their immunophenotype.</p> <p>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 <em>thy1</em>GFP-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 <em>in situ</em>. The results indicate that the <em>thy1</em>GFP-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 <em>thy1</em>GFP-M mice has a great potential for analyses of DC-neuron interaction in normal and pathological conditions.</p> </div

GFP-DCs in cervical lymph nodes of a <i>thy1</i>GFP-M mouse.

<p>(A) Confocal analysis of cryosectioned cervical lymph node shows the presence of numerous GFP-tagged cells (green). They surround the B cell follicles (dashed lines) and extend in the T cell zone (on the upper right of the figure), while they rarely occur in the subcapsular zone. CD3+ cells, visualized by immunohistochemistry, are here shown in red. (B) High magnification of CD3+ cells (red) and GFP+ cells (green) in the T cell zone. (C) Immunopositivity of GFP-tagged cells (green) to CD11c (red; white arrowheads). Note that the GFP is expressed in the cytoplasm of GFP+DCs.</p

<i>In vivo</i> observation of motile GFP-labeled cells in the cortex of <i>thy1</i>GFP-M mice.

<p>(A) GFP+ cell (white arrowhead) above the pial surface rapidly changing its morphology. Time-lapse sequence of maximum intensity projections of a set of optical sections acquired at 2 µm z-step. The right column shows the depth of the cell through a digital rotation of the corresponding images on the left. The white dotted lines indicate the dura and pia mater.The frame acquired at 45′ shows fluorescent cells (blue arrowheads) passing above the pia through the CSF. (B) GFP+ cell rolling inside a blood vessel on the pial surface. The blood vessel walls (shown in red) were labeled by the intravital dye SR101. Fluorescent cells showing motility at the pial surface (C) and deep in the brain parenchyma (D–E). E) GFP+ cells showing translation across the field of view. Scale bars 10 µm.</p

Immunophenotypic analysis of the non-neuronal GFP-tagged cells in the brain of <i>thy</i>GFP-M mice.

<p>Confocal images of coronal brain sections showing non-neuronal GFP+ cells in several locations. (A–C) GFP+ cell into the lumen of a blood vessel (arrowhead) in layer II of the parietal cortex. Neuronal nuclei were stained with anti-NeuN, (B, C, red); the blood vessel is visualized in bright-field (insert A, C). The inset in A represents the area shown an high magnification in A–C. (D–F) GFP+ cell in the ependyma between hippocampus and thalamus. This cell is immunopositive to the dendritic cell marker anti-CD11c (E, red). Cell nuclei are stained with DAPI in F and represented in blue. (G–I) In the anterior hypothalamus a high number of small-sized GFP+ cells shows immunopositivity to the lymphocytes marker CD3+. Different morphologies of GFP+/CD3+ cells in the anterior hypothalamus. A small round (H, high magnification) GFP+/CD3+ cell and a GFP+/CD3+ cell showing an irregular shape (I, high magnification). Scale bars 10 µm.</p

Immunophenotypic profile of NeuN-/GFP+ cells in <i>thy1</i>GFP-M mice.

A<p>Subset of positive cells in higher number in meninges than in choroid plexus ;^BSubset of positive cells mainly round shaped and without ramifications.</p

Distribution of the NeuN-/GFP+ cells in the brain of <i>thy1</i>GFP-M mice and quantitative analyses

<p>. Confocal microscopy images of brain sections showing non-neuronal GFP+ cells with different morphologies in the meninges. (A–D) Ramified non-neuronal GFP+ cell at the pia/parenchyma interface visualized in bright-field in B. (E–H) Elongated non-neuronal GFP+ cell in the subarachnoid space; blood vessels and pia mater were visualized by anti-laminin immunostaining (F, red). (I–J) Non-neuronal/GFP+ cells with ramifications or round-shaped (J), do not express the microglial marker Iba-1 (red). The insert in I represents an high magnification of the area in the dashed box. (K–L) The glial/monocyte marker CD11b (red) was not expressed by ramified cells in the leptomeninges (K) but is instead expressed by small-size round cells (example shown in L), which are therefore identified as monocytes. (M–P) A non-neuronal GFP+ cell in the meninges covering the optic tract (cell nuclei are stained with DAPI, blue). This cell exhibits the morphological features of a migratory dendritic cell, whose cytoplasmic organization in a “veil-like” structure provides a more efficient motility (veiled cell); the immunopositivity to dendritic markers CD11c (N, red) and major histocompatibility complex class II (O, magenta), together with its morphology, confirms the identity of dendritic cell. Scale bars 20 µm. The graph shows the percentage of GFP+ cells immunopositive to anti-MHCII, anti CD11c or both these markers. Data were obtained from 83 and 118 of GFP+ cells counted in 6 animals in meninges and choroid plexus, respectively.</p

Cytofluorimetric analysis of GFP expression in blood cells from WT and <i>thy1</i>GFP-M mice.

<p>(A) GFP expression was evaluated in monocytes as CD11b positive, GR-1 (Ly6C, Ly6G) intermediate, F4/80 positive cells (CD11b⁺ GR1^int F4/80⁺); in granulocytes as CD11b positive, GR-1 highly positive cells (CD11b⁺ GR1^high); in dendritic cells as CD11b positive, GR-1 negative, F4/80 negative, CD11c positive cells (CD11b⁺ GR1^- F4/80^- CD11c⁺). (B) Lymphocyte population were phenotypically characteryzed as B220 positive, CD3 negative cells (B lymphocytes, B220⁺ CD3^-); B220 negative, CD3 positive, CD8 positive, CD4 negative cells (T cytotoxic cells, B220^- CD3⁺ CD8⁺ CD4^-) and B220 negative, CD3 positive, CD8 negative, CD4 positive (T helper, B220^- CD3⁺ CD8^- CD4⁺). Percentages inside the gates represent positive cells of parent populations. The figures shown are a representative experiment of 3 other separate experiments from 3 different mice performed with similar results.</p