Article Figures & Data
Figures
- Figure 1.
5-Bromo-4-chloro-3-indolyl-β-D-galactoside analysis of tPA expression in PlatβGAL reporter mice. Representative images (10×) from a heterozygous PlatβGAL reporter mouse (mice, n = 5) stained for β-Gal. Sections (50 μm) were cut coronally starting from the frontal cortex around bregma +2.5 and progressing caudally to the cerebellum around bregma -8.0. tPA/β-Gal activity is strongly present in the cortex (A–F), in the granule and pyramidal cell layers of the hippocampus (D–F), and in the molecular and granular layers of the cerebellum (G, H). More diffuse tPA/β-Gal staining is observable in subcortical regions, such as the medial (B) and lateral septal nuclei (C), the bed nucleus of the stria terminalis (C), caudate/putamen (D), the basolateral and centromedial nuclei of the amygdala (D), and thalamus and hypothalamus (D, E). The locus coeruleus, where neuroserpin, the neuronal inhibitor of tPA is highly concentrated, is largely devoid of tPA/β-Gal staining (G). There are also distinct β-Gal clusters in midbrain, pontine, and medulla structures (F, G), such as the interpeduncular nucleus (F). Coronal reference atlas images (were taken from the Allen Developing Mouse Brain Atlas; http://mouse.brain-map.org/static/atlas). From A–H, the following thumbnails were used: 32, 47, 53, 71, 82, 89, 109, and 123, respectively. MS, medial septal nuclei; LS, lateral septal nuclei; BNST, bed nucleus of the stria terminalis; HPF, hippocampal formation; TH, thalamus; CP, caudate/putamen; BLA, basolateral nucleus of the amygdala; CeA, central nucleus of the amygdala; MeA, medial nucleus of the amygdala; HY, hypothalamus; MB, midbrain; IPN, interpeduncular nucleus; P, pons; LC, locus coeruleus; CB, cerebellum; MY, medulla. Scale bars: 1 mm. Reference atlas images credit: Allen Institute.
- Figure 2.
Immunohistochemical analysis of tPA protein expression in the hippocampus of PlatβGAL reporter mice reveals a differential expression pattern between the sites of tPA synthesis and tPA trafficking. A, Representative image (10×) of a 50-μm hippocampal section from a heterozygous PlatβGAL reporter mouse (mice, n = 5). tPA is shown to be strongly expressed in the granule cell layer of the dentate gyrus and the pyramidal cell layer of hippocampal regions CA1–CA3. There are also scattered β-Gal puncta (arrows) in the hilus, SR, and SO; β-Gal staining is also present in blood vessels in the hippocampus (filled arrows). B, Immunohistochemical analysis of a representative image (10×) from a 50-μm hippocampal section from a heterozygous PlatβGAL reporter mouse (mice, n = 5) using antibodies directed against murine tPA (orange). In contrast to the β-Gal stain (cyan), tPA is not expressed in the cell body layers, but in the mossy fiber axons of dentate granule cells in the hilus and stratum lucidum lamina (B’, B’’, 40×). To visualize the colored β-Gal stain in the immunofluorescent image captured showing tPA-immunoreactivity, a negative of the fluorescent image was generated and pseudo-colored cyan. SLu, stratum lucidum; Hi, hilus; DG, dentate gyrus. Scale bars: 500 μm (A, B) and 50 μm (B’, B’’).
- Figure 3.
Generation and global tPA protein characterization of tPABAC-Cerulean transgenic mice. A, Founder lines for tPABAC-Cer transgenic mice (863 and 876) were generated using BAC technology. To generate tPABAC-Cer transgenic mice, a kanamycin-resistance recombineering cassette containing a cerulean fluorescent gene fused to the C-terminus of exon 14 of the tPA gene was recombineered into a BAC. The tPA-Cer fusion gene is under control of endogenous regulatory elements contained in the Plat locus. B–D, Protein expression profile of tPABAC-Cer transgenic mice. Brains from transgene-positive and transgene-negative adult tPABAC-Cer mice were harvested and homogenized for total and active protein quantification using a bead-based Luminex assay (B) and ELISA (C). Data are presented as the mean ± SEM fold change from tPA protein levels in transgene negative littermate controls (mice n = 6-7). No statistical difference was noted in total and active tPA protein levels between line 863 and 876. D, Zymographic analysis of whole-brain homogenates from tPABAC-Cer transgene positive and negative mice (lines 863 and 876) visually delineates endogenous tPA (lower molecular weight bands, E-tPA) and the tPA-Cer from the BAC (higher molecular weight bands, tPA-Cer). Not only is there increased levels of tPA protein in both tPABAC-Cer transgenic lines, but the tPA-Cer protein is proteolytically active. FRT, flippase recognition target; HM, homology arms; r-mtPA, recombinant-murine tPA; E-tPA, endogenous-tPA.
- Figure 4.
tPA-Cer fusion protein is prominently expressed in limbic structures and blood vessels in the adult murine brain. Images shown are representative of stitched serial coronal sections from tPABAC-Cer transgenic mice (n = 3) captured on a widefield microscope (A–H, 10×; I–L, 20×; M, 40×) or confocal microscope (N, 63×). Cryosections stained for the neuronal marker NeuN (red) clearly distinguishes the faint tPA-Cer (cyan) cells bodies observed in the piriform (A–D) and entorhinal cortex (E), and the tPA-Cer fluorescent nerve fibers found in the medial and lateral septal nuclei (B), the bed nucleus of the stria terminals (C, I), the paraventricular nucleus of the thalamus (C) and hypothalamus (D), the central (D, J) and medial (D) nuclei of the amygdala, the external globus pallidus (D) and internal globus pallidus (M) of the basal ganglia, the substantia nigra pars reticulata, (E), the periaqueductal gray (F, K), and the parabrachial nucleus (G, L). tPA is also robustly expressed in the hilus and mossy fiber pathway of the hippocampus (D–F). G, H, In contrast to the PlatβGAL reporter mice, tPA expression is not observable in the cerebellum. N, Brightly positive tPA-Cer puncta are noticeable throughout all brain regions in blood vessels using the endothelial cell marker, CD31 (magenta). PIR, piriform cortex; ENT, entorhinal cortex; LS, lateral septal nuclei; MS, medial septal nuclei; BNST, bed nucleus of the stria terminalis; PVT, paraventricular nucleus of the thalamus; HY, hypothalamus; HPF, hippocampal formation; CeA, central nucleus of the amygdala; MeA, medial nucleus of the amygdala; BLA, basolateral nucleus of the amygdala; PAG, periaqueductal gray; PBN, parabrachial nucleus; GPe, globus pallidus external segment; GPi, globus pallidus internal segment; SNr, substantia nigra pars reticulata; MB, midbrain; P, pons; CB, cerebellum; MY, medulla. Scale bars: 1 mm (A–H), 100 μm (I–M), and 50 μm (K, N).
- Figure 5.
tPA-Cer fluorescence is not observed in tPABAC-Cer transgene negative littermate controls. A–D, Images shown are representative of stitched serial coronal sections from tPABAC-Cer transgene negative littermate mice (n = 2) captured on a widefield microscope (10×). Cyrosections (14 μm) stained for the neuronal marker NeuN (red) show no cerulean fluorescence in cell bodies in the piriform (A–C) or entorhinal cortex (D). Cerulean fluorescence is also not observable in the medial and lateral septal nuclei (B), the bed nucleus of the stria terminals (B), the amygdala (C), globus pallidus (C), or the thalamus and hypothalamus (B, C). Midbrain and pontine brain structures are devoid of any cerulean fluorescence (D, E), as are the medulla and cerebellum (F). G, H, Representative images from widefield microscopy (4×) of the hippocampus from a tPABAC-Cer transgene-positive (Tg+) mouse and its transgene-negative (Tg-) littermate. Cerulean fluorescence is clearly observable in the mossy fiber pathway of a tPABAC-Cer transgene-positive mouse, but completely absent in the transgene-negative control. Cerulean fluorescent artifacts from edge effects or folds are apparent in panels C, E, F. PIR, piriform cortex; ENT, entorhinal cortex; LS, lateral septal nuclei; MS, medial septal nuclei; BNST, bed nucleus of the stria terminalis; TH, thalamus; HPF, hippocampal formation; AMYG, amygdala; HTH, hypothalamus; CP, caudate/putament; GP, globus pallidus; MB, midbrain; P, pons; CB, cerebellum; MY, medulla. Scale bars: 1 mm (A–F) and 500 μm (G, H).
- Figure 6.
tPA-Cer is localized to large MFBs and astrocytes in the CA3 stratum lucidum lamina of the hippocampus. Subcellular colocalization of tPA in stratum lucidum of the hippocampus was investigated using high-resolution confocal microscopy. Images (63×) are representative regions of interest from the stratum lucidum lamina of tPABAC-Cer transgenic mice and are presented as 5-μm z-stacks (Δz = 0.5 μm) visualized in orthogonal YZ and XZ slices (A–C) and magnified 3D maximum intensity projections (A’–C’), for each of the respective colocalization markers. For quantification of colocalization using the Manders coefficient (D, E) entire images were concatenated from 5-μm z-stacks (512 × 512 pixels; Δz = 0.5 μm) that were independently acquired two to four times (ZnT3: mice n = 10, lines 863 and 876; EAAT2: mice n = 6–8, lines 863 and 876, respectively; MAP2: mice n = 4, lines 863 and 876). tPA-Cer puncta was found to colocalize with the zinc transporter-3 (ZnT3; red), which has previously been observed exclusively in MFBs, as indicated visually by the white overlay in the orthogonal sections and 3D max projection (Manders M1: 0.704 and 0.649 for lines 863 and 876, respectively; Costes probability p value ≥ 95%; D, E). Partial colocalization was weakly observed in astrocytes visualized with the astrocytic glutamate transporter EAAT2 (orange; B, B’), which is in agreement with the lower quantified colocalization coefficient (Manders M1: 0.183 and 0.198 for lines 863 and 876, respectively; Costes probability p value ≥ 95%; D, E). To confirm the presynaptic localization of tPA to MFBs, sections were also stained for the dendritic marker MAP2, which detects the dendritic thorny excrescences of CA3 pyramidal neurons that MFBs encase. Orthogonal YZ and XZ slices and 3D max projections of tPA-Cer and MAP2 (yellow; C, C’) showed no colocalization (Manders M1: 0.032 and 0.060 for lines 863 and 876, respectively; Costes probability p value = 0.0%; D, E). ZnT3, zinc-transporter 3; EAAT2, excitatory amino acid transporter 2; MAP2, microtubule associated protein 2. Scale bars: 5 μm (A–C) and the units on the scale boxes are given in μm (A’–C’).
- Figure 7.
Amplification of tPA-Cer signal reveals a population of cells in SO/alveus of the hippocampal CA1 and CA3 regions that coexpress SST. Representative images of hippocampal CA1 (A) and CA3 (E) subfields from tPABAC-Cer transgenic mice (mice, n = 8) exemplifying the localization and distribution of tPA-Cer cell bodies (tPA; green) that coexpress the inhibitory interneuronal marker SST (magenta). For localization and distribution statistics, see Table 1. With GFP tyramide signal amplification, somatic tPA is also detectable in the pyramidal cell layer of the CA3 subfield (E), although it is still largely absent in the CA1 pyramidal cell layer (A). Confocal (63×) maximum intensity projections focused on a region of interest (dashed box) highlighting tPA-Cer-positive cells (tPA; green) that coexpress SST (magenta) from the CA1 (B–D) and CA3 (F–H) SO/alveus lamina. Unlike the punctate nature of tPA in axonal projections, tPA’s somatic expression appears more diffuse. I, J, Immunostaining for SST and GFP with tyramide signal amplification in transgene negative controls only revealed SST-positive interneurons in the SO/alveus lamina of hippocampal CA1 (I) and CA3 (J). SLu, stratum lucidum. Scale bars: 50 μm (A), 10 μm (C, D), 100 μm (E), 10 μm (F–H), 50 μm (I), and 100 μm (J).
- Figure 8.
tPA-Cer cells are positive for immunocytochemical markers of O-LM inhibitory interneurons. Dorsal hippocampal sections from tPABAC-Cer transgenic mice were probed for previously confirmed immunocytochemical markers of O-LM inhibitory interneurons. While O-LM interneurons are known for expressing SST (A), other morphologically distinct interneuron subgroups have been shown to stain positive for the neuropeptide. O-LM interneurons, however, have been shown to be strongly decorated with the metabotropic glutamate receptor 1a (mGluR1a), and they have been shown to express the calcium-binding protein calbindin. High-resolution, confocal images (63×) of 10-μm-thick z-stack maximum intensity projections demonstrate that tPA-Cer-positive cells bodies in SO/alveus coexpress both mGluR1a (B) and calbindin (C). Sections were also stained for GAD65, confirming the GABAergic nature of the tPA-Cer-expressing cells (D). In agreement with this, O-LM immunocytochemical profile was the observation of axonal-like projections in SR, as axons of O-LM interneurons extend to stratum lacunosum moleculare (E). Pictures are representative images from SO/alveus of stainings from at least four transgenic mice (n = 4–8) per interneuronal marker. mGluR1a, metabotropic glutamate receptor 1a; GAD65, glutamic acid decarboxylase 65. Scale bars: 5 μm (A–E).
Tables
- Table 1.
Localization and coexpression of tPA-Cer-positive soma in hippocampus of tPABAC-Cer mice
Localization of tPA-Cer-positive soma Coexpression of tPA-Cer-positive soma CA1 SO alveus CA3 SO alveus CA1 and CA3
SP/SRtPA-Cer-positive neurons expressing SST (%) SST-positive interneurons expressing tPA (%) Line 863 11.68 ± 1.53 5.58 ± 1.14 3.32 ± 1.15 54.35 ± 6.32 53.89 ± 8.82 (n = 301) (n = 141) (n = 78) (n = 520) (n = 520) Line 876 10.90 ± 2.06 5.34 ± 1.98 2.28 ± 1.06 58.90 ± 6.74 44.66 ± 9.45 (n = 238) (n = 115) (n = 52) (n = 405) (n = 540) Results are presented as the mean ± CI(0.95) for a given hippocampal section. Cell count data were gathered from SO/alveus, SP, and SR; n refers to the total number of cells counted from two to four hippocampal sections per tPABAC-Cer transgenic mouse (lines 863 and 876; mice = eight per line).
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