DNA-based MRI probes for specific detection of chronic exposure to amphetamine in living brains

Abstract

We designed phosphorothioate-modified DNA probes linked to superparamagnetic iron oxide nanoparticles (SPION) for in vivo magnetic resonance imaging (MRI) of fosB and Delta fosB mRNA after amphetamine (AMPH) exposure in mice. Specificity of both the fosB and Delta fosB probes was verified by in vitro reverse transcriptase-PCR amplification to a single fragment of total cDNA obtained from acutely AMPH-exposed mouse brains. We confirmed time-dependent uptake and retention profiles of both probes in neurons of GAD67-green fluorescent protein knock-in mice. MRI signal of SPION-labeled fosB probe delivered via intracerebroventricular route was elevated in both acutely and chronically AMPH-exposed mice; the signal was suppressed by dopaminergic receptor antagonist pretreatment. SPION-labeled Delta fosB probe signal elevation occurred only in chronically AMPH-exposed mice. The in vivo target specificity of these probes permits reliable MRI visualization of AMPH-induced differential elevations of fosB and Delta fosB mRNA in living brains.

Figure 1.

Probe design and specificity. A shows a relative location of sODN-fosB within the splice site of the fosB mRNA. B shows the location of sODN-ΔfosB flanking the spliced site of ΔfosB mRNA. C shows a combination of sODN-fosB to a common USP ODN in sense sequence allows amplification of 146 bp from fosB cDNA, but not ΔfosB cDNA by PCR. D shows one cDNA fragment was amplified from total cDNA of mouse striatum (C57black6 mice) when the sODN-fosB or sODN-ΔfosB was used (two of four mice tested were shown) (Cui et al., 1999). A combination of sODN-ΔfosB and USP-ODN allows amplification of 123-bp ΔfosB cDNA. Because of partial homology in this 123 bp fragment to FosB cDNA, we did not perform diagnostic sequencing, but the specificity of sODN-ΔfosB was tested by in vivo hybridization and MRI (see Fig. 3D).

Figure 2.

Delivery and uptake of rhodamine-sODN-FosB by confocal microscopy to normal resting mice. A shows the location of ICV and the relative location of tissue sample (box) in B. The direction of migration is indicated by open arrows in a migration front. C shows two types of sODN distribution: Rhd-sODN in green GABAnergic neurons can be seen as overlapping of red (Rhd-sODN-FosB) and GFP in neurons (behind the migration line) and Rhd-sODN in punctate formations (asterisk) ahead of the migration front.

Figure 3.

Distribution and retention of SPION-fosB and SPION-ΔfosB probes in vivo. A compares the R2* maps (caudal view) obtained from live animals at four time points referenced to ICV infusion of SPION-fosB probe. A thick arrow points to the site of ICV infusion. B depicts outlines of ROI for statistical analyses (Paxinos and Franklin, 2001). These regions include the mPFC, NAc, and CPU, HP, somatosensory (SSC), and MC. C shows quantitative analyses of fosB expression in ROI from R2* maps of acute and chronic AMPH in this study. Representative SPION-fosB signal maps are presented above the bar graph based on the following equation and signal cutoffs are 10–100%: ΔR2* = (R2* − R2*_baseline)/R2*_baseline× 100%. We observed significantly reduced fosB mRNA induction in the CPU after AMPH challenge dose after chronic AMPH exposure and a period of no drug (p = 0.03). D shows quantitative analyses of ΔfosB expression in ROI of SAL, acute AMPH exposure (A1), or chronic AMPH with challenge (A7/W/A) and without challenge (A7/W/S). Significant level (compared with the group received saline); *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4.

Distribution of FITC-sODN-fosB or FITC-sODN-ΔfosB in C57black mice. A and B show FITC-sODN-fosB in SAL or AI group. Overlapping of green (FITC-sODN) and red (antibodies to neuronal nuclear protein) indicate the distribution of sODN in the neurons. C, D show FITC-sODN-ΔfosB in SAL or A7/W/A group. Samples were obtained 4 h after AMPH (7 h after ICV infusion according to MRI protocol).

Figure 5.

A and B show elevated retention of FITC signal as punctate formations surrounding the nucleus (counterstained by PI) in the neuronal formation of the hippocampus of A7/W/A group (n = 4) compared with A1 group (n = 4). Samples were obtained 1 h after AMPH. Elevation of ΔFosB-positive cells is increased in A7/W/A mice (no ICV infusion of any probe), predominantly in the shell of the NAc (C) and the mPFC (E). Very few ΔFosB positive cells are present in A1 samples (D and F). N = 6 each group.