Anat Cell Biol.  2010 Jun;43(2):157-164. 10.5115/acb.2010.43.2.157.

Evaluation of recombinant adenovirus-mediated gene delivery for expression of tracer genes in catecholaminergic neurons

• Kim ML
• Han S
• Lee SB
• Kim JH
• Ahn HK
• Huh Y

• KMID: 2168889
• DOI: http://doi.org/10.5115/acb.2010.43.2.157

Abstract

Selective labeling of small populations of neurons of a given phenotype for conventional neuronal tracing is difficult because tracers can be taken up by all neurons at the injection site, resulting in nonspecific labeling of unrelated pathways. To overcome these problems, genetic approaches have been developed that introduce tracer proteins as transgenes under the control of cell-type-specific promoter elements for visualization of specific neuronal pathways. The aim of this study was to explore the use of tracer gene expression for neuroanatomical tracing to chart the complex interconnections of the central nervous system. Genetic tracing methods allow for expression of tracer molecules using cell-type-specific promoters to facilitate neuronal tracing. In this study, the rat tyrosine hydroxylase (TH) promoter and an adenoviral delivery system were used to express tracers specifically in dopaminergic and noradrenergic neurons. Region-specific expression of the transgenes was then analyzed. Initially, we characterized cell-type-specific expression of GFP or RFP in cultured cell lines. We then injected an adenovirus carrying the tracer transgene into several brain regions using a stereotaxic apparatus. Three days after injection, strong GFP expression was observed in the injected site of the brain. RFP and WGA were expressed in a cell-type-specific manner in the cerebellum, locus coeruleus, and ventral tegmental regions. Our results demonstrate that selective tracing of catecholaminergic neuronal circuits is possible in the rat brain using the TH promoter and adenoviral expression.

Keyword

Tyrosine hydroxylase promoter; adenovirus; tracer; neuronal circuit; catecholamine

MeSH Terms

Adenoviridae
Adrenergic Neurons
Animals
Brain
Cells, Cultured
Central Nervous System
Cerebellum
Gene Expression
Lifting
Locus Coeruleus
Neurons
Phenotype
Proteins
Rats
Transgenes
Tyrosine 3-Monooxygenase
Proteins
Tyrosine 3-Monooxygenase

Figure

• 

  Fig. 1 Structure and recombinant adenovirus transfection in cultured cells and cerebellar injections in Sprague-Dawley rats. (A) Cell type specific promoter and tracer transgene of recombinant adenovirus. (B) HEK293 cells were cultured in 10-cm culture dishes. (C) Five days after transfection with the pAdeasy-TH-RFP-CMV-GFP construct, strong GFP expression was observed in HEK293 cells. (D) Three days after injection of the TH-RFP-CMV-GFP recombinant adenovirus into the cerebellum of male Sprague-Dawley rats, GFP was highly expressed in the cerebellar cortex. g, granular layer; P, Purkinje cell layer; m, molecular layer; (E) RFP was not expressed in the cerebellar cortex. Scale bars=100 µm.

• 

  Fig. 2 Transgene expression 3 days after injection of the TH-RFP-CMV-GFP recombinant adenovirus into the locus coeruleus. (A) Distribution of Hoechst 33342-stained nuclei in the midbrain. (B) GFP was highly expressed in the injected side of midbrain. (C) RFP was expressed in several neurons of the LC. (D) RFP expression colocalized with GFP-positive neurons. Scale bar=100 µm.

• 

  Fig. 3 Transgene expression 3 days after TH-WGA-CMV-GFP recombinant adenovirus injection into the VTA. (A) GFP was highly expressed in the injected side. (B) Magnified image of the indicated region of (A) (square). (C) WGA immunohistochemistry showed the presence of WGA-immunoreactive neurons in regions adjacent to the injected site. Scale bar=100 µm.

• 

  Fig. 4 Transgene expression 3 days after TH-WGA-CMV-GFP recombinant adenovirus injection into the LC. (A) Distribution of Hoechst 33342-positive nuclei in the midbrain. (B) GFP was highly expressed in the injected side of the LC and in adjacent regions. Some GFP-positive fibers were distributed in the lower lateral side of midbrain. (C) WGA immunohistochemistry showed that several neurons were positive for WGA staining. (D) Merged image of (A, B) and (C). (E~H) Magnified images of the indicated region of (D) (square). (E) Distribution of Hoechst 33342 stained nuclei. (F) GFP-expressing cells. (G) WGA-immunoreactive neurons. (H) Merged image. (I) WGA immunohistochemistry using DAB development showed WGA-immunoreactive neurons in the injected side of the locus coeruleus. (J) Contralateral side of the locus coeruleus. (K) Magnified image of the indicated region of (I) (square). (L) Magnified image of the indicated region of (J) (square). Scale bar=100 µm.

• 

  Fig. 5 Transgene expression 3 days after TH-WGA-CMV-GFP recombinant adenovirus injection in the VTA. (A) Distribution of Hoechst 33342-positive nuclei in the injected side of the VTA. (B) GFP was highly expressed in the injected side of the VTA. Many GFP-positive fibers were distributed among the GFP-positive cell bodies. (C) WGA immunohistochemistry showed that several neurons were WGA positive. WGA-positive fibers and cell bodies were distributed in adjacent regions. (D) Merged image of (A, B) and (C). Z-stack image of (E) GFP and WGA colocalized in the same cells. Scale bar=100 µm.

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