Targeted Transgene Expression in Cholinergic Interneurons in the Monkey Striatum Using Canine Adenovirus Serotype 2 Vectors

Abstract

The striatum, the main input structure of the basal ganglia, is critical for action selection and adaptive motor control. To understand the neuronal mechanisms underlying these functions, an analysis of microcircuits that compose the striatum is necessary. Among these, cholinergic interneurons (ChIs) provide intrinsic striatal innervation whose dysfunction is implicated in neuropsychiatric diseases, such as Parkinson's disease and Tourette syndrome. The ability to experimentally manipulate the activity of ChIs is critical to gain insights into their contribution to the normal function of the striatum and the emergence of behavioral abnormalities in pathological states. In this study, we generated and tested CAV-pChAT-GFP, a replication-defective canine adenovirus type 2 (CAV-2) vector carrying the green fluorescent protein (GFP) sequence under the control of the human choline acetyltransferase (ChAT) promoter. We first tested the potential specificity of CAV-pChAT-GFP to label striatal ChIs in a rat before performing experiments on two macaque monkeys. In the vector-injected rat and monkey striatum, we found that GFP expression preferentially colocalized with ChAT-immunoreactivity throughout the striatum, including those from local circuit interneurons. CAV-2 vectors containing transgene driven by the ChAT promoter provide a powerful tool for investigating ChI contributions to circuit function and behavior in nonhuman primates.

Keywords: acetylcholine; basal ganglia; microcircuit analysis; nonhuman primate; viral vector transduction.

Figure 1

Schematic representation of the CAV-2 construct. CAV-2 vector genomes (~32 kbp) are flanked by an inverted terminal repeat (ITR) of ~200 bp. “pChAT-eGFP-WRPE-pA” is the expression cassette that replaces the E1 region in the CAV-2 genome. “pChAT” is the promoter driving expression of choline acetylase gene, GFP is the optimized open reading frame (ORF) for green fluorescent protein (GFP) from jellyfish, WRPE is a sequence from a woodchuck hepatitis virus that increases mRNA stability and protein yield when placed 3′ to an ORF, and pA is the polyadenylation signal from human growth hormone gene (GH1). The CAV-2 E3 region is also deleted (ΔE3) to increase the packaging capacity.

Figure 2

Selective expression of GFP in cholinergic interneurons (ChIs) of the rat striatum with CAV-2 containing the human ChAT promoter (CAV-pChAT-GFP). (A) Coronal section of the rat brain stained with Cresyl violet showing the injection sites of CAV-pChAT-GFP and CAV-mCherry in the right and left striatum respectively. (B–U) Immunofluorescence labeling for simultaneous detection of ChAT (blue), GFP (green) and mCherry (red) in a coronal section. Images of the fluorophores obtained by sequential acquisition of separate wavelength channels from a single confocal slice. (B,L) Distributions of ChAT-containing neurons as well as GFP- (B, green) and mCherry- (L, red) expressing neurons within the right and left striatum. (C–K) Demonstrated that many cell bodies that were GFP+ (green, arrows) contained ChAT (blue arrows). A few GFP+ (green arrowhead) did not show a detectable level of ChAT. (E) Merge of (C,D). (H) Merge of (F,G). (K) Merge of (I,J). (M–U) Demonstrated that most mCherry+ neurons (red, arrowheads) were not labeled for ChAT. These neurons were distributed among many ChAT-containing neurons (Blue, arrows). (O) Merge of (M,N). (R) Merge of (P,Q). (U) Merge of (S,T). Scale bars: (A), 500 μm; (B–D,E,L,M–O), 100 μm; (F–K,P–U), 50 μm.

Figure 3

Selective expression of GFP in ChIs of the monkey striatum with CAV-2 containing the human ChAT promoter (CAV-pChAT-GFP). (A) Coronal section of a macaque brain stained with Cresyl violet showing the injection sites of CAV-pChAT-GFP in the striatum. (C–S) Immunofluorescence labeling for simultaneous detection of, GFP (green), mCherry (red) and ChAT (blue) in an adjacent coronal section. Images of the fluorophores obtained by sequential acquisition of separate wavelength channels from a single confocal slice. (B) Distributions of ChAT-containing neurons within the striatum. (C–J) In monkey C in which both CAV-2 vectors were injected in the same hemisphere, GFP and mCherry expression were restricted to the cell bodies. Most of these neurons that were labeled for both GFP (green, arrows) and mCherry (red, arrows) contained ChAT (blue) whereas neurons expressing mCherry only (red, arrowheads) were not labeled for ChAT. (K–S) In Monkey P in which CAV-pChAT-GFP and CAV-mCherry were injected in the right and left striatum, respectively most neurons expressing GFP (K,N,Q, green) or mCherry (S) displayed well-labeled cell bodies as well as dendritic and axonal (arrowheads) processes. (K–P) Most neurons expressing GFP (green, arrows) contained ChAT (Blue, arrows). (Q,R) A few neurons expressing GFP did not show detectable levels of ChAT. (F) Merge of (C–E). (J) Merge of (G–I). (M) Merge of (K,L). (P) Merge of (N,O). Scale bars: (A), 5 mm; (B), 600 μm; (C–S) 50 μm.