Probe design, binding, and specificity
The relative location of sODN-fosB or sODN-ΔfosB in their mRNA is shown in . Initial assessment in the binding specificity of fosB probe to the fosB cDNA showed that the sODN-fosB probe together with a USP-amplified 146 bp fragment using PCR from the fosB cDNA; this probe, however, did not amplify any fragment from the ΔfosB cDNA, a cDNA clone that shares homology with most of fosB cDNA (). We further evaluated the probe binding stringency against total cDNA obtained from the striatum of C57black6 mice, as the probe should exhibit selectivity against thousands of gene transcripts. We would expect to observe amplification of a smear or several fragments if it was not specific and bound to several cDNA of different sizes. demonstrates that these primers did in fact amplify only one fragment from the total striatal cDNA (n = 4). The same was observed with sODN-ΔfosB and the USP in reverse-transcription PCR, but in this case one 123 bp fragment was amplified, as predicted from the mRNA sequence in . The result supported specific antisense interaction between sODN-fosB (the ligand) and its mRNA target (the receptor). The specificity of these two probes is shown by an ability to distinguish fosB and ΔfosB mRNA in vivo, as described below.
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 (more ...)
Neuronal uptake of sODN after ICV delivery
We next examined the uptake and retention dynamics of sODN probe in vivo after ICV delivery of rhodamine (Rhd)-labeled sODN-fosB (8 nmol/kg) (). GAD67-GFP knock-in mice expressing GFP in GABAergic neurons were used for this study. shows that Rhd-sODN-fosB (red) uptake occurs as an apparent wave propagating away from the lateral ventricles into the parenchyma. Under higher magnification, we found that the Rhd-sODN-fosB signal was distributed homogenously within GFP-positive, presumptive GABAnergic neurons (green) as well as GFP-negative cells (, closed arrows) (n = 4), or as punctuate at perinuclear location (, asterisk). The homogeneous labeling was seen in cells immediately adjacent to the lateral ventricles while the punctate formations were seen in cells away from the lateral ventricles.
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. (more ...)
Probe dynamics in neural cells after ICV delivery
We delivered the sODN probe labeled with a strong MRI contrast agent (SPION) and acquired whole-brain MRI of anesthetized mice at four time points referenced to probe delivery (SPION-fosB, ICV, 120 pmol/kg). We calculated R2* maps to assess distribution of the SPION-containing probe (). While there is endogenous but relatively low R2* signals in the pre-ICV map, we observed elevated R2* signal in and around the infusion site (thick open arrow) and bilaterally within the ventricles (dotted arrows) at 30 min. At this early time point, the spread of R2* signal away from the ventricles may be attributed to localized distortion of the magnetic field because of high concentration of iron-containing probes, which did not cause R2* enhancement in the contralateral cortices (thin arrow). Animals were allowed to wake up after MRI in their home cages to recover with minimal disturbance. R2* maps acquired at the 3 h time point in the animals under anesthesia showed widespread enhancement in the striatum, septum, and cortex (thin arrows) of the cortex contralateral to infusion hemispheres, as previously elevated R2* signal in the infusion track and ventricles diminished. To mimic the experimental paradigm to study AMPH-induced fosB gene expression profiles, we injected saline (10 ml/kg, i.p.) to the mice after the MRI scans at this time point. Again, these animals were returned to their home cages and R2* maps were acquired at 7 h, under anesthesia; we observed a decline in these maps similar to the pre-ICV map (, fourth row). In conclusion, MRI R2* maps acquired at different time points after ICV delivery of SPION-sODN indicated that global availability of the probe distant to the infusion site had been achieved at 3 h (Liu et al., 2007a
). For subsequent statistical analysis, we determined the mean R2* values in several brain regions () of each animal and calculated group averages. ROIs in the contralateral hemisphere of the brain, covering −2.7 to 2.10 mm to the bregma based on the mouse brain atlas (Paxinos and Franklin, 2001
), included the medial pre-frontal cortex, nucleus accumbens, caudate putamen, hippocampus, somatorsensory, and motor cortices.
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 (more ...)
SPION-fosB signal elevation after AMPH has regional specificity and is suppressed by SCH23390 pretreatment
We examined the feasibility of detecting AMPH-induced fosB gene transcription using SPION-fosB probe and MRI. Four groups of animals were used: (1) no AMPH (saline control or SAL, n = 8), (2) acute AMPH (A1, n = 6), (3) repeated AMPH followed by a period of drug abstinence and a subsequent AMPH challenge dose (4 mg/kg, i.p., A7/W/A, n = 4), and (4) acute AMPH with SCH23390 (0.1 mg/kg, s.c.) pretreatment 40 min before AMPH (SCH23390/A1, n = 4). SCH23390 is an effective D1/D5 receptor antagonist that depresses AMPH-induced neuronal activity within the dopaminergic pathway. The MRI experimental paradigm was similar in all animal groups (i.e., AMPH or SAL at 3 h and MRI at 7 h after probe delivery). Representative averaged maps of percentage R2* signal increase (10–100%) from baseline of SAL, A1, and SCH23390/A1 groups are displayed above the bar graph in . We observed sporadic SPION-fosB signal in the SAL group and robust signal elevation in localized brain regions in the A1 group, most notably in the stratum. The elevation was substantially depressed in SCH23390/A1. We observed significant R2* elevations in A1 and A7/W/A in all ROIs except the hippocampus (HP) and motor cortex (MC). We also observed significant reductions in AMPH-induced SPION-fosB signals in the striatum (p = 0.03) of the A7/W/A group compared with the A1 group (). This result suggests that SPION-fosB reports changes in gene transcription as a result of neuronal activation by AMPH exposure in vivo.
MRI detects SPION-ΔfosB signal elevation only in chronically AMPH-exposed animal brains
We examined AMPH-induced ΔfosB gene transcription using SPION-ΔfosB probe and MRI after the protocol established above. Here, we included animals groups similar to the previous SPION-fosB study: (1) SAL (n = 6), (2) A1 (n = 7), and (3) A7/W/A (n = 5). We also included an additional group of animals that received chronic exposure to AMPH followed by a period of abstinence; this sensitized group was given SAL (as a placebo) instead of AMPH challenge on the day of MRI acquisition (denoted as A7/W/S, n = 5). Representative averaged maps of percentage R2* signal increase (10–100%) from baseline of A1, A7/W/A and A7/W/S groups are displayed above the bar graph in . We observed robust elevation of the SPION-ΔfosB signal only in the A7/W/A group and in some restricted brain regions such as the striatum and cortex of the A7/W/S group (). ROI analysis showed significant R2* elevation in A7/W/A group compared with the SAL group in all brain regions under investigation including the HP. Furthermore, we observed a slight but significant R2* elevation in the striatum [caudate–putamen (CPU)] of A7/W/S group (p = 0.04).
Histological confirmation of MRI observations
To validate our MRI observations, we examined the uptake of FITC-labeled sODN-fosB or FITC-labeled sODN-ΔfosB (120 pmol/kg, ICV) in SAL, A1, or A7/W/A groups as in the MRI studies. We observed FITC-sODN-fosB signal in the cytoplasm in the nucleus accumbens (NAc) of A1 brains, with overlapping of FITC and anti-NeuN-Cy3 in the perinuclear regions () (n = 4); however, we observed little FITC signal, most likely of background nature, and little overlap between FITC and anti-NeuN-Cy3 in the SAL group received sOND-fosB () (n = 4) or sODN-ΔfosB () (n = 4). Similarly, we observed much enhanced FITC-sODN-ΔfosB signal in the cytoplasm of the A7/W/A group compared with the SAL control group. In another groups of 8 mice, we delivered FITC-sODN-ΔfosB (8 nmol/kg, ICV), then AMPH at 3 h, but collected brain samples 1 h later. Consistent with the result in , we observed elevated retention of FITC signal, as punctate formations surrounding the nucleus [counterstained by propidium iodide (PI)] in the neuronal formation of the hippocampus of A7/W/A group compared with A1 group ().
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. (more ...)
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. (more ...)
We further performed immunohistochemistry using antibodies against ΔfosB to validate our combination assay on mRNA elevation. We observed very little ΔFosB expression in the A1 group [A1, n = 6, left panels, for the NAc and medial prefrontal cortex (mPFC), respectively], but we did detect elevated ΔFosB expression in some of the cells in the A7/W/A group (n = 6, right panels, within the same ROI). This observation was consistent with MRI detection of different ΔfosB mRNA induction profiles for the A1 and A7/W/A groups. Based on the different R2* maps of SPION-ΔfosB in vivo, we conclude that SPION-ΔfosB and MRI detected a different gene expression profile for ΔfosB mRNA in chronically AMPH-exposed animal brains compared with acutely AMPH-exposed brains. Because sODN-fosB or sODN-ΔfosB binds to one mRNA (), we further conclude that the in vivo target specificity of our SPION-sODN probes allows MRI visualization of differential inductions of fosB and ΔfosB mRNA in living brains under AMPH influence.