Expression of transgenes in midbrain dopamine neurons using the tyrosine hydroxylase promoter

Abstract

Billions of neurons are interconnected in the central nervous system (CNS). Identification of specific neuronal circuit is indispensable for understanding the relationship between structure and function in the CNS. The midbrain dopamine (DA) neuron system consists of the retrorubral area (A8), the substantia nigra (SN; A9) and the ventral tegmental area (VTA; A10). We hypothesized that genetic methods using cell-type-specific promoters may offer the possibility to express tracer molecules in DA neurons to facilitate neuronal tracing. To address this, we used the 2.5 kb rat tyrosine hydroxylase (TH) promoter in adenovirus or adeno-associated virus (AAV) to express tracers specifically in DA neurons. We found that stereotaxic injection of TH promoter containing adenoviral construct resulted in cell-type-specific transgene expression in the noradrenaline (NA) neurons of the locus coeruleus (LC). However, it caused a significant toxicity to DA neurons in the SN. In contrast, stereotaxic injection of TH promoter containing AAV to the SN resulted in cell-type-specific transgene expression in DA neurons with no detectable toxicity. Taken together, our results demonstrate that it is possible to selectively trace DA neuronal circuits in rodent brains using the TH promoter in the context of AAV.

Figure 1

The 2.5 kb rat TH promoter expresses WGA in CA neuronal cells. (a) Schematic drawing of adenoviral vector used in the study. Abbreviations; THp (2.5 kb rat TH promoter), WGA (wheat germ agglutinin), CMVp (cytomegalovirus promoter), GFP (green fluorescence proteins), pA (poly adenylation signal). (b) HeLa and SK-N-BE(2)M17 cells were analyzed 2 days after adenovirus infection. GFP was expressed by CMV promoter. Expression of WGA was detected with goat anti-WGA specific antibody (1: 500 dilution, Vector Lab). Alexa 594 anti-goat IgG (1:200 dilution, Molecular Probes) was used as a secondary antibody. Merged images of GFP and WGA are shown. Bar; 100 μm. Recombinant adenovirus was prepared as described . The WGA gene, which is under 2.5 kb rat TH promoter, was cloned into the pAdTrack vector. The resulting plasmid was linearized by digesting with PmeI and transformed into E. coli BJ5183 in which an adenoviral backbone plasmid (pAdEasy-1) was introduced. The recombinant adenoviral DNA was selected and transfected into the adenovirus packaging cell line, HEK293, using lipofectamine (Invitrogen). Viruses were harvested 5 days after transfection and concentrated by CsCl density-gradient ultracentrifugation. Viral titer was determined by quantifying the number of cells showing GFP signal (expression forming units, EFU). Cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Hyclone), 100 μg/ml of streptomycin, and 100 units/ml of penicillin in a CO₂ incubator.

Figure 2

Adenovirus showed toxicity to midbrain DA neurons of SN. Adenovirus in Figure 1a was delivered to LC and SN of adult rat by stereotaxic injection. Three days after injection, animals were analyzed. Delivery sites of adenovirus were detected by expression of GFP (a, d). Expression of WGA was detected with WGA specific antibody (b, c, e, f; 1:1,000 dilution, Vector Lab). Magnified view of boxed area of (b) and (e) are shown in (c) and (f), respectively. Bar; 200 μm (a, b, d, e), 100 μm (c, f). Ten week-old male Sprague-Dawley rats (280-320 g, Charles River) were used. Adenovirus (1.5 μl of 8 × 10¹⁰ EFU/ml) was unilaterally injected into the SN (AP -5.8 mm relative to bregma, L -2.0 mm from midline, and DV -6.0 mm below dura) and the LC (AP -3.9 mm relative to bregma, L -1.4 mm from midline, and DV -7.2 mm below dura) using a stereotaxic instrument (Stoelting Co.) equipped with an autoinjector (Stoelting Co.). After stereotaxic injection of adenovirus, rats were perfused with 4% PFA and the brains were sectioned coronally to 40 μm thickness using a cryostat. Brain sections were mounted on poly-L-lysine-coated glass slides. Antibodies were detected using the Vectastain kit (Vector Labs) and the signal was visualized using 3,3'-diaminobenzidine (DAB). Goat anti-WGA antibody was used to detect expression of WGA. All animal procedures were performed according to experimental protocols approved by the Institutional Animal Care and Use Committee of McLean Hospital and followed the National Institutes of Health guidelines.

Figure 3

Transgenes were expressed in SN of adult rat. AAV (1.5 μl of 2-5 × 10¹² genome copy/ml) which expresses GFP-WGA fusion protein (a) or DsRed2 (f) was delivered to the SN by stereotaxic injection as described in Figure 2. Seven or 17 days after injection, animals were analyzed. Expression of GFP-WGA (b) and DsRed2 (g) was detected by its fluorescence. TH expression was detected by immunostaining (c, h). Merged images of GFPWGA and TH (d) or DsRed2 and TH (i) are shown. Magnified view of boxed area of (d) and (i) are shown in (e) and (j), respectively. Expression transgenes in the non-DA cells are shown by white arrow heads. VTA and SN are shown. Bar; 100 μm (b, c, d, e), 200 μm (g, h, i, j). Goat anti-WGA antibody (1:200 dilution, Vector Lab) and rabbit anti-TH antibody (1:1,000 dilution, Pel-Freez) were used. For immunofluorescence labeling, Alexa 594 or 488 anti-rabbit or anti-goat IgG (1:200 dilution, Molecular Probes) were used as secondary antibodies. Recombinant AAV was obtained by using the AAV helper-free system according to manufacture's protocol (Stratagene). We replaced the CMV promoter of pAAV-MCS with the 2.5 kb rat TH promoter to obtain the pAAV2.5THp. The GFP-WGA fusion protein or DsRed2 were cloned in the pAAV2.5THp. This plasmid was used to transfect AAV-293 cells with pAAV-RC and pHelper using the calcium phosphate method to make recombinant viral particles. Viruses were harvested 3 days after transfection and purified using heparin-agarose column and concentrated by centrifugation using a filter membrane (Amicon). One ml of viral solution was treated with proteinase K and viral DNA was recovered by phenol extraction followed by ethanol precipitation. The genome copy of viral DNA was determined by PCR.