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1.
Figure 10

Figure 10. Viral co-expression of tTA and tdTomato induces expression of tet-responsive genes and fluorescently labels transgenic cells. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

TetO-LacZ/GFP reporter mice were injected at P0 with AAV8-tdTomato-2A-tTA (5.0×109 particles per hemisphere) to determine if virally-expressed transactivator was capable of activating transgene expression from the tetO promoter and whether the transgenic cells could be reliably identified by the viral fluorescent label. Representative images from cerebral cortex (A), hippocampal CA1 (B), CA3 (C), and cerebellum (D) showing the viral reporter tdTomato in red (top row) and the transgenic reporter GFP in green (middle row), along with their co-localization (bottom row).

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
2.
Figure 9

Figure 9. Viral transduction can be used to genetically modify and fluorescently label neurons. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

Representative images show the secondary motor cortex layer 5 (left column), hippocampus (middle column), and striatum (right column) of R26R Cre-reporter mice 3 wk after co-injection at P0 with AAV8-iCre-2A-tdtomato (2.0×109 particles per hemisphere) and AAV8-triple-YFP (4.0×108 particles per hemisphere). Sections were immunostained for β-galactosidase (β-gal) to determine if tdTomato fluorescence accurately labels genetically manipulated cells. The top 3 rows show native fluorescence for tdTomato (red, A–C) and YFP (green, D–F), along with β-galactosidase immunostaining (blue, G–I). The lower 3 rows show overlap between tdTomato + YFP (J–L), YFP + β-gal (M–O), and tdTomato + β-gal (P–R). Reliable co-localization between tdTomato and β-gal is seen in the hippocampus (P) and cerebellum (Q), while reliable overlap in the cortex requires a lower titer of virus (R). White arrows indicate representative cells expressing YFP alone; yellow arrows indicate cells that were transduced by both viruses and which express YFP, tdTomato, Cre, and β-gal.

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
3.
Figure 7

Figure 7. Multiple transgenes can be delivered simultaneously by AAV co-injection. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

Representative images show the transduction pattern found in whole brain (A, D), cerebral cortex (B, E), and cerebellum (C, F) 3 weeks after co-injection with two different AAVs. Subtle differences in the pattern of transgene expression were found following co-injection of two viruses of the same serotype (AAV8-YFP and AAV8-tdTomato; A, B, and C) or of different serotypes (AAV1-YFP and AAV8-tdTomato; D, E and F). High-magnification views of pyramidal neurons in cortical layers 4–6 (B, E) and Purkinje neurons in the cerebellum (C, F) reveal that cells can be transduced by one or both viruses, although a greater proportion of neurons express both fluorescent labels after co-injection of AAV8 than after a combination of AAV8 and AAV1. AAV8-YFP and AAV1-YFP were delivered at 2.0×1010 particles/hemisphere; AAV8-tdTomato was delivered at 2.0×109 particles/hemisphere. YFP fluorescence is shown in green, tdTomato in red. Cortical layers II/III-VI are marked; ML: molecular layer, PL: Purkinje cell layer, GL: granule cell layer

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
4.
Figure 6

Figure 6. AAV8 offers a broad dynamic range for mosaicism. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

Serial dilution of AAV8 or AAV1 yielded decreasing transduction densities. Representative images show transduction patterns in coronal sections taken 3 weeks after P0 injection of serially diluted AAV8 or AAV1 (A). When injected at the same concentration, AAV8 produced much more efficient transduction compared to AAV1. Co-injection of 2.0×109 particles AAV8-tdTomato and 2.0×109 particles AAV1-YFP per hemisphere provided a direct comparison of their respective infection efficiencies. Sagittal section from a co-injected mouse shows that AAV8 spread throughout the parenchyma, while AAV1 at this titer was largely restricted to the choroid plexus (B), consistent with its distribution when injected alone at this concentration (A, bottom right panel). The inset in B shows a higher magnification view of labeled cells in the third ventricle. Higher magnification images of cortex (upper row) and cerebellum (lower row) show the cellular pattern of transgene mosaicism achieved by serial dilution of AAV8 (C). Sagittal sections were collected 3 weeks after P0 injection.

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
5.
Figure 5

Figure 5. Timing of AAV8 injection alters the pattern and cell-specificity of transduction. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

Representative images of whole-brain sagittal sections from mice harvested 3 weeks after P0, P2, or P3 injection show diminished spread of virus with age, particularly in distant structures such as the olfactory bulb and cerebellum (A, C, E). Higher magnification images of coronal sections shows the transduction of non-pyramidal cells in the deep layers of the cortex following delayed AAV8 injection (compare B with D and F). Immunostaining with cell-type-specific markers for neurons (NeuN), astrocytes (S100β), microglia (Iba1), and GABAergic interneurons (GAD67) was used to identify the transduction preference of AAV8 from P0-injected mice (G–J) and P3-injected mice (K, L). Green = YFP native fluorescence (first column); Red = immunomarker (second column); overlay of the two channels is shown in third column of G and H, I, J. Representative images are from hippocampal CA1 (G) or deep layers of cerebral cortex (H–L). Immunofluorescence staining for NeuN (G) shows that AAV8 primarily transduced NeuN-positive neurons in P0 injected mice. In contrast, P3 injection resulted in viral transduction of S100β-positive astrocytes (K). All images were taken from mice bilaterally injected

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
6.
Figure 1

Figure 1. Intracranial injection of adeno-associated virus into the cerebral lateral ventricles of neonatal mice. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

The three landmarks used to target the lateral ventricle for injection are easily visible through the translucent skin of P0 C57 neonatal pups (A). Diagrammatic view of mouse head shows the two different target sites (arrows) that can be used for viral injection relative to the eyes and the suture intersections of lambda and bregma. One injection site is located approximately two-fifth of the distance between lambda and each eye; the other site is located approximately 1 mm lateral to the sagittal suture, halfway between lambda and bregma. Both coordinates reliably target the lateral ventricles (B). Neonatal whole brain (C) and coronal cross-section of frozen brain (D) harvested immediately after bilateral dye injection to illustrate the extent and localization of the fill. The two injection sites can be seen at the brain surface from dye that escaped along the needle path, while the filled ventricles are visible through the tissue and can be seen along the rostral-caudal extent of contiguous chambers. LV-lateral ventricle: 3V-third ventricle.

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
7.
Figure 3

Figure 3. Rapid onset of a fluorescent Cre reporter following P0 injection of AAV8. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

Representative images show complete sagittal sections (left column) and high-magnification views of hippocampal dentate gyrus (middle column) and cerebellum (right column) following P0 injection of AAV8-EF1α-iCre into the Cre reporter line Ai3 (Madisen et al., 2010). Animals were harvested at P2, P4, P7, and P14 to examine the onset and spread of transduction using native YFP fluorescence as an indicator of virally-expressed Cre activity. All images show YFP fluorescence derived from the Cre-activated transgene; no immunostaining was needed to visualize the label. Widespread expression is apparent within days after injection, allowing observation of neuronal migration (dentate gyrus) and neurite maturation (cerebellum) during early postnatal development. Fluorescence increases across ages as more neurons inherit the active reporter, to normalize the image, longer exposures were used for P2 and P4 than P7 and P14. Exposure times for whole brain images were determined based on the cortex and hippocampus, which fluoresce most brightly, while exposure times for the magnified panels were adjusted to show patterns within each region rather than the intensity of expression between them.

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
8.
Figure 8

Figure 8. Serotype and titer can be independently adjusted to control the pattern of mosaicism obtained by co-injection of multiple viruses. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

Representative images show the transduction pattern found in whole brain (A, E), CA1 hippocampus (B), cerebral cortex layer 4, 5 (C, F), cerebral cortex layer 2, 3 (G) and cerebellum (C, F) 3 weeks after co-injection of two different AAVs at reduced titer. High-magnification views of neurons transduced following co-injection of the same serotype (4.0×108 particles of AAV8-YFP and 4.0×108 particles AAV8-tdTomato per hemisphere) in CA1 hippocampus (B), cerebral cortex (C) and cerebellar lobe (D) reveals a greater number of singly-transduced cells than observed when both viruses are injected at high titers. High-magnification views of neurons transduced by co-injection of two different AAV serotypes (2.0×109 particles of AAV1-YFP and 8.0×108 particles AAV8-tdTomato per hemisphere) in cerebral cortex layer 4, 5 (F) and layer 2, 3 (G) and cerebellar lobe (H) demonstrates that both serotype and titer can be independently adjusted to control the density of cells expressing each virally-encoded protein. Cortical layers II/III-VI are marked; SO: stratum oriens, SP: stratum pyramidale, SR: stratum radiatum, GL: granule cell layer, PL: Purkinje cell layer, ML: molecular layer

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
9.
Figure 2

Figure 2. Neonatal intraventricular AAV injection produces widespread and long-lasting transgene expression in the brain. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

Images were taken from mice harvested 3 weeks (A–H, K, L) or 10 months (I, J) after intraventricular injection at birth (A–J) or stereotaxic injection as a young adult (K, L). All images were taken from mice injected with 2 µl of AAV8 to deliver 2.0×1010 particles/hemisphere. Transduction is visualized by native YFP fluorescence. Whole-brain sagittal sections and high-magnification views of transduced regions show widespread transduction with dense neuronal labeling (A–H). Transgene expression is maintained for at least 12 months after P0 injection, as shown in this whole brain sagittal section and high magnification view of the hippocampal CA1 region. In contrast to the widespread expression attained at P0, viral injection into the adult brain results in a significantly more restricted spread, with transduction localized within several mm of the injection site (K, L). Scale bar in L applies to panels E, G, H, and J; scale bar in K applies to D and I. Exposure times for whole brain images have been determined based on cortex and hippocampus which fluoresce most brightly, while exposure times for higher magnification panels have been adjusted to match the intensity of labeling in each brain region.

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
10.
Figure 11

Figure 11. P0 injection of AAV8-triple-YFP produces fluorescent neuronal labeling suitable for in vivo two-photon imaging. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

All images were taken from a 4-week old mouse injected at P0 with AAV8-triple-YFP (1.0×108 particles/hemisphere). (A) Z-projection of imaging volume from 70 to 85 µm cortical depth (seven 2-µm-thick optical slices). (B) Higher-magnification image of boxed section shown in panel A demonstrating labeled cell processes (three 1-µm-thick optical slices). Neuronal cell bodies (green arrow), apical dendrites (yellow arrow), basal dendrites (blue arrow), and axonal boutons (inset - red arrow) are all visible. (C) Three-dimensional projection of a 250 × 250 × 300 µm volume demonstrating the diversity of pyramidal neuron morphologies resolvable in P0-injected mice, with background fluorescence digitally masked in Imaris Bitplane. (D) Chronic time-lapse imaging of AAV8-triple-YFP-labeled apical dendrites, imaged at 5, 10, and 20 days post-surgery. Left panel: Z-projection of a chronically-imaged dendrite (0.3 µm-per-pixel in X–Y plane, 40 1-µm-thick optical slices). Right panel: Magnified view of a single optical section from the region boxed in panel D, imaged over subsequent days. New spines (green arrows), stable spines (yellow arrows), and changes in spine morphology (blue) are visible. Nonspecific background fluorescence was removed in C and D.

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
11.
Figure 12

Figure 12. Neonatal AAV transgenesis offers a new window into Purkinje cell development and in vivo dynamics. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

(A) High magnification images of sagittal sections through the cerebellum following P0 intraventricular injection with 2 µl of AAV8-triple-YFP (3.8×109 particles/hemisphere, top row) or AAV1-YFP (1.3×1010 particles/hemisphere, second row). Sparse fluorescent labeling provides a clear view of postnatal dendritic maturation from P2 until P14. (Bi) Low magnification view of a fixed sagittal section through the cerebellum. The images were taken from from an 8-week old mouse injected at P0 with AAV8-triple-YFP (1.0×108 particles/hemisphere) demonstrating sparse labeling of Purkinje cells. The surface of the brain is outlined and the border of the granule cell layer is dashed to provide structural landmarks. (Bii) High magnification projection of the Purkinje cell outlined in panel (Bi). (Biii) Magnified view of a single optical section resolves individual spines on a terminal dendrite. (C) Z-projections of a cerebellar Purkinje cell imaged in vivo by 2-photon microscopy. Top panel: Z-projection of the volume in the imaged orientation from 0 to 200 µm depth (220×60 pixels, 133×35 µm FOV). Bottom panel: Orthogonal view of the image stack displays the full dendritic arbor.

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.
12.
Figure 4

Figure 4. Both serotype and promoter influence the onset and pattern of transgene expression. From: Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualizing and manipulating neuronal circuits in vivo.

Representative images show transduction patterns in whole brain sagittal sections and high-magnification views of cerebral cortex and hippocampus after intraventricular injection of AAV1, AAV6, or AAV8 at P0. Mice were injected with 2 µl of AAV1-CBA-triple YFP to deliver 1.3 ×1010 particles/hemisphere (first column), AAV6-CBA-tdTomato to deliver 1.2 ×1010 (second column), AAV8-CBA-tdTomato to deliver 3.0×109 (third column), or AAV8-EF1α-tdTomato to deliver 1.2×109 (fourth column), and harvested at P2, P4, P7 and P14 for comparison of transduction efficiency and expression onset. High-magnification images were taken from somatosensory cortex for AAV1, primary motor cortex for AAV6 and AAV8. The hippocampal CA1 region is shown for all serotypes; dotted lines indicate the borders of the pyramidal cell layer. Immunostaining for NeuN in P14 tissue confirmed that all three serotypes primarily transduce neurons. Viral expression is visualized by native fluorescence in all panels (YFP for AAV1, tdTomato for AAV6, AAV8-CBA, and AAV8-EF1α) and shown as red in the color panels (AAV1-YFP has been pseudo-colored for this illustration). Immunostaining for NeuN is shown in green (middle row), and the merged channels below. Yellow arrows provide a fiduciary point in each image highlighting one example cell expressing both virally-delivered fluorescent protein and NeuN. Exposure times were determined based on fluorescence intensity within the hippocampus and cortex, and were longer for AAV1 and AAV6 than for AAV8. CBA: chicken β-actin promoter, EF1α: elongation factor 1α promoter.

Ji-Yoen Kim, et al. Eur J Neurosci. 2013 April;37(8):1203-1220.

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