A Single Vector Platform for High-Level Gene Transduction of Central Neurons: Adeno-Associated Virus Vector Equipped with the Tet-Off System

<div><p>Visualization of neurons is indispensable for the investigation of neuronal circuits in the central nervous system. Virus vectors have been widely used for labeling particular subsets of neurons, and the adeno-associated virus (AAV) vector has gained popularity as a tool for gene transfer. Here, we developed a single AAV vector Tet-Off platform, AAV-SynTetOff, to improve the gene-transduction efficiency, specifically in neurons. The platform is composed of regulator and response elements in a single AAV genome. After infection of Neuro-2a cells with the AAV-SynTetOff vector, the transduction efficiency of green fluorescent protein (GFP) was increased by approximately 2- and 15-fold relative to the conventional AAV vector with the human cytomegalovirus (CMV) or human synapsin I (SYN) promoter, respectively. We then injected the AAV vectors into the mouse neostriatum. GFP expression in the neostriatal neurons infected with the AAV-SynTetOff vector was approximately 40-times higher than that with the CMV or SYN promoter. By adding a membrane-targeting signal to GFP, the axon fibers of neostriatal neurons were clearly visualized. In contrast, by attaching somatodendritic membrane-targeting signals to GFP, axon fiber labeling was mostly suppressed. Furthermore, we prepared the AAV-SynTetOff vector, which simultaneously expressed somatodendritic membrane-targeted GFP and membrane-targeted red fluorescent protein (RFP). After injection of the vector into the neostriatum, the cell bodies and dendrites of neostriatal neurons were labeled with both GFP and RFP, whereas the axons in the projection sites were labeled only with RFP. Finally, we applied this vector to vasoactive intestinal polypeptide-positive (VIP+) neocortical neurons, one of the subclasses of inhibitory neurons in the neocortex, in layer 2/3 of the mouse primary somatosensory cortex. The results revealed the differential distribution of the somatodendritic and axonal structures at the population level. The AAV-SynTetOff vector developed in the present study exhibits strong fluorescence labeling and has promising applications in neuronal imaging.</p></div

Differential distributions of the dendrites and axons of L2/3 VIP+ neurons in the S1BF.

(A) Construction of the vector plasmid, pAAV2-SynTetOff-FLEX-FGL-2A-palmRFP1. (B–E) Coronal sections immunostained for mRFP1 (B), GFP (C), and VGluT2 (D). Scale bar in B applies to B–E. (F, G) Binarization of the images for GFP and mRFP1. The binary image of GFP signals (F) represents the somatodendritic distribution of L2/3 VIP+ neurons. (H) Axonal distribution was estimated by subtracting the binarized image for GFP (F) from that for mRFP1 (G). (I) The distribution probabilities of the somatodendrites and axons of L2/3 VIP+ neurons across cortical layers. (J) VGluT2-immunofluorescence intensity in the tangential direction in L4 of the S1BF. Blue indicates barrel locations. (K, L) The distribution probabilities of the dendrites (K) or axons (L) of L2/3 VIP+ neurons in L4 of the S1BF. (M) Mean distribution probabilities of the dendrites and/or axons of L2/3 VIP+ neurons in L4 of the S1BF. The probabilities are plotted between the centers of adjacent barrels. The tangential width was divided into 10 bins. Blue bars represent VGluT2 immunofluorescence intensities. (N) Axonal distributions of L2/3 VIP+ neurons in the S1BF. The vertical widths of L2/3, L5, and L6 were divided by a factor of 2 at the center of each layer. The distribution probability of the axons in L4 was higher in septa than in barrels, whereas those in the other layers were not significantly different between the barrel- and septa-related columns. Error bars, ± SEM. *p p < 0.01.</p

Dual-color labeling of the somatodendritic and axonal structures of neostriatal neurons with the AAV-SynTetOff vectors.

(A) Construction of the vector plasmid, pAAV2-SynTetOff-FGL-2A-palmRFP1. (B) Sagittal view of brain sections with injection of AAV2/1-SynTetOff-FGL-2A-palmRFP1 into the CPu. This AAV vector expressed both FGL and palmRFP1 in the infected neurons. GFP-NF was restricted to the CPu, and mRFP1-NF was observed not only in the CPu but also the GPe and SNr, where neostriatal neurons project. (C) After immunofluorescence staining for MAP2 (blue), the sections were observed under a confocal laser scanning microscope in the CPu. (D) High-magnification images in (C). Arrowheads and arrows indicate the dendritic and axonal structures, respectively. Scale bar in B1 applies to B1–B3. Scale bar in C1 applies to C2–C4. Scale bar in D1 applies to D2–E4.</p

Axon labeling of neostriatal neurons with the AAV-SynTetOff vectors.

<p>(<b>A</b>) Construction of the vector plasmids, pAAV2-SynTetOff-GFP, pAAV2-SynTetOff-palGFP, pAAV2-SynTetOff-myrGFP, and pAAV2-SynTetOff-FGL. (<b>B–E</b>) Sagittal views of brain sections with injections of the AAV vectors. By addition of the palmitoylation site of the GAP-43 N-terminus (palGFP; C) or the myristoylation/palmitoylation site of the Fyn N-terminus (myrGFP; D), axon fibers in the GPe and SNr were more clearly visualized than with GFP without membrane-targeting signal (B). On the other hand, when a somatodendritic-targeting signal, LDLRct, was added to the C-terminus of myrGFP (FGL; E), axon fiber labeling in the GPe and SNr was mostly suppressed (E₄, E₅). Scale bar in B₁ applies to B₁–E₁, B₂–E₂, and B₃–E₃. Scale bar in B₄ applies to B₄–E₄ and B₅–E₅.</p

Neuron-specific and high-level transgene expression with the AAV-SynTetOff-GFP vector <i>in vivo</i>.

<p>(<b>A</b>_<b>1</b><b>–C</b>_<b>4</b>) One week after the injection of the AAV vectors, GFP-NF was observed in the caudate-putamen (CPu). Cells infected with the vector AAV2/1-SynTetOff-GFP (C) were more strongly labeled with GFP than those infected with the vectors AAV2/1-CMV-GFP-BGHpA (A) and AAV2/1-SYN-GFP-BGHpA (B). Almost all GFP-positive cells were also immunoreactive for NeuN with AAV2/1-SYN-GFP-BGHpA (B₂–B₄, arrowheads) and AAV2/1-SynTetOff-GFP (C₂–C₄, arrowheads), whereas some GFP-positive cells were negative for NeuN with the AAV2/1-CMV-GFP-BGHpA vector (A₂–A₄, arrow; a putative glial cell). Scale bar in A₁ applies to A₁–C₁; Scale bar in A₂ applies to A₂–A₄, B₂–B₄, and C₂–C₄. (<b>D</b>) Specificities of GFP expression in neostriatal neurons. AAV2/1-SYN-GFP-BGHpA and AAV2/1-SynTetOff-GFP displayed specific expression in neuronal cells, while the expression of GFP with AAV2/1-CMV-GFP-BGHpA was not neuron-specific. (<b>E</b>) GFP-NF intensities in neostriatal neurons. The mean GFP-NF intensity with AAV2/1-CMV-GFP-BGHpA was standardized as 1 AU. AAV2/1-SynTetOff-GFP transduced much stronger GFP expression in neurons than AAV2/1-CMV-GFP-BGHpA and AAV2/1-SYN-GFP-BGHpA (factors of 43.3 and 34.3, respectively). Error bars, ± SEM. *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Efficient gene-transduction with AAV-SynTetOff vector <i>in vitro</i>.

<p>(<b>A</b>) Construction of the vector plasmids, pAAV2-CMV-GFP-BGHpA, pAAV2-SYN-GFP-BGHpA, and pAAV2-SynTetOff-GFP. (<b>B</b>) One week after infection of Neuro-2a cells with AAV2/1-CMV-GFP-BGHpA, AAV2/1-SYN-GFP-BGHpA, and AAV2/1-SynTetOff-GFP, GFP-NF intensities were measured in the infected cells (outlined by dotted lines with ImageJ). (<b>C</b>) The gene-transduction efficiency with AAV2/1-CMV-GFP-BGHpA, AAV2/1-SYN-GFP-BGHpA, or AAV2/1-SynTetOff-GFP was examined quantitatively. The GFP-mRNA/GFP-DNA ratio and the GFP-NF intensity of cells infected with AAV2/1-CMV-GFP-BGHpA were standardized as 1 arbitrary unit (AU). A qRT-PCR assay revealed that GFP-mRNA expression was 1.7- and 15.6-fold higher with AAV2/1-SynTetOff-GFP than with AAV2/1-CMV-GFP-BGHpA and AAV2/1-SYN-GFP-BGHpA, respectively (normalized to GFP-DNA levels). GFP-NF intensities showed similar ratios to AAV2/1-CMV-GFP-BGHpA and AAV2/1-SYN-GFP-BGHpA (factors of 1.8 and 14.4, respectively), indicating that the GFP-NF intensities reflected GFP-mRNA expression levels. Error bars, ± standard error of the mean (SEM). *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001.</p

Image_1_Kv4.2-Positive Domains on Dendrites in the Mouse Medial Geniculate Body Receive Ascending Excitatory and Inhibitory Inputs Preferentially From the Inferior Colliculus.pdf

The medial geniculate body (MGB) is the thalamic center of the auditory lemniscal pathway. The ventral division of MGB (MGV) receives excitatory and inhibitory inputs from the inferior colliculus (IC). MGV is involved in auditory attention by processing descending excitatory and inhibitory inputs from the auditory cortex (AC) and reticular thalamic nucleus (RTN), respectively. However, detailed mechanisms of the integration of different inputs in a single MGV neuron remain unclear. Kv4.2 is one of the isoforms of the Shal-related subfamily of potassium voltage-gated channels that are expressed in MGB. Since potassium channel is important for shaping synaptic current and spike waveforms, subcellular distribution of Kv4.2 is likely important for integration of various inputs. Here, we aimed to examine the detailed distribution of Kv4.2, in MGV neurons to understand its specific role in auditory attention. We found that Kv4.2 mRNA was expressed in most MGV neurons. At the protein level, Kv4.2-immunopositive patches were sparsely distributed in both the dendrites and the soma of neurons. The postsynaptic distribution of Kv4.2 protein was confirmed using electron microscopy (EM). The frequency of contact with Kv4.2-immunopositive puncta was higher in vesicular glutamate transporter 2 (VGluT2)-positive excitatory axon terminals, which are supposed to be extending from the IC, than in VGluT1-immunopositive terminals, which are expected to be originating from the AC. VGluT2-immunopositive terminals were significantly larger than VGluT1-immunopositive terminals. Furthermore, EM showed that the terminals forming asymmetric synapses with Kv4.2-immunopositive MGV dendritic domains were significantly larger than those forming synapses with Kv4.2-negative MGV dendritic domains. In inhibitory axons either from the IC or from the RTN, the frequency of terminals that were in contact with Kv4.2-positive puncta was higher in IC than in RTN. In summary, our study demonstrated that the Kv4.2-immunopositive domains of the MGV dendrites received excitatory and inhibitory ascending auditory inputs preferentially from the IC, and not from the RTN or cortex. Our findings imply that time course of synaptic current and spike waveforms elicited by IC inputs is modified in the Kv4.2 domains.</p

Study design and verification of reproducibility of induced labor model mice.

(A) Experimental design of induced labor model and pups. At gestational day 18.5 an osmotic pump was implanted subcutaneously in anesthetized mice. The male pups were analyzed at 24 h (P1) after birth. (B) Time until labor for each group. Time until labor of the oxytocin (OXT) group was significantly shorter than that of the phosphate-buffered saline (PBS) and Wild groups (P < 0.001, Tukey–Kramer method). There were no significant differences between the PBS and Wild groups. (C) Survival rate of OXT, PBS and Wild groups at P1. There were no significant differences between each group. (D) Body weight of OXT, PBS and Wild groups at P1. The body weight of the OXT group was significantly lower than that of the PBS and Wild groups (P < 0.001, Steel–Dwass test). There were no significant differences between the PBS and Wild groups.</p

Comparison of architecture of PL and IL of vmPFC between OXT and PBS groups at P40.

Nissl-stained sections of OXT (A) and PBS (B) groups. Scale bar: 100 μm. (TIF)</p

Ultrastructure of dying cells in forceps minor of corpus callosum and ventromedial prefrontal cortex of the male pups at 24 h after delivery.

(A–D) Electron micrographs of forceps minor (FMI) of the male pups at 24 h after delivery of the OXT (A–C) and PBS (D) groups. (A) In the OXT group, cells containing pyknotic nuclei and debris of dying cells were abundant (asterisks). (B) An enlarged image of the phagocytic cell (P) shown in A containing many pyknotic nuclei and debris of dying cells. (C) A dying cell with chromatin condensation observed in the OXT group. (D) An enlarged image of the phagocytic cell (P) containing pyknotic nuclei and debris of dying cells in the PBS group. (E–G) Electron micrographs of ventromedial prefrontal cortex (vmPFC) of the male pups at 24 h after delivery of the OXT group. Dying cells with pyknotic nuclei indicated by an arrow and an arrowhead in E were enlarged and shown in F and G, respectively. (F) A dying cell with pyknotic nuclei that was not phagocytosed (arrow). (G) A dying cell with pyknotic nuclei that was encircled by the cytoplasm of the phagocytic cell (arrow). Scale bars: 4 μm (A–E) and 2 μm (F, G).</p