Noninvasive Evaluation of Cellular Proliferative Activity in Brain Neurogenic Regions in Rats under Depression and Treatment by Enhanced [18F]FLT-PET Imaging

Abstract

Neural stem cells in two neurogenic regions, the subventricular zone and the subgranular zone (SGZ) of the hippocampal dentate gyrus, can divide and produce new neurons throughout life. Hippocampal neurogenesis is related to emotions, including depression/anxiety, and the therapeutic effects of antidepressants, as well as learning and memory. The establishment of in vivo imaging for proliferative activity of neural stem cells in the SGZ might be used to diagnose depression and to monitor the therapeutic efficacy of antidepressants. Positron emission tomography (PET) imaging with 3'-deoxy-3'-[(18)F]fluoro-l-thymidine ([(18)F]FLT) has been studied to allow visualization of proliferative activity in two neurogenic regions of adult mammals; however, the PET imaging has not been widely used because of lower accumulation of [(18)F]FLT, which does not allow quantitative assessment of the decline in cellular proliferative activity in the SGZ under the condition of depression. We report the establishment of an enhanced PET imaging method with [(18)F]FLT combined with probenecid, an inhibitor of drug transporters at the blood-brain barrier, which can allow the quantitative visualization of neurogenic activity in rats. Enhanced PET imaging allowed us to evaluate reduced cell proliferation in the SGZ of rats with corticosterone-induced depression, and further the recovery of proliferative activity in rats under treatment with antidepressants. This enhanced [(18)F]FLT-PET imaging technique with probenecid can be used to assess the dynamic alteration of neurogenic activity in the adult mammalian brain and may also provide a means for objective diagnosis of depression and monitoring of the therapeutic effect of antidepressant treatment.

Significance statement: Adult hippocampal neurogenesis may play a role in major depression and antidepressant therapy. Establishment of in vivo imaging for hippocampal neurogenic activity may be useful to diagnose depression and monitor the therapeutic efficacy of antidepressants. Positron emission tomography (PET) imaging has been studied to allow visualization of neurogenic activity; however, PET imaging has not been widely used due to the lower accumulation of the PET tracer in the neurogenic regions. Here, we succeeded in establishing highly quantitative PET imaging for neurogenic activity in adult brain with an inhibitor for drug transporter. This enhanced PET imaging allowed evaluation of the decline of neurogenic activity in the hippocampus of rats with depression and the recovery of neurogenic activity by antidepressant treatment.

Keywords: PET imaging; SSRI; depression; drug transporters; neurogenesis.

Figure 1.

Effects of drug transporter inhibitors on the brain uptake of [¹⁸F]FLT with an ex vivo study. A, B, Representative [¹⁸F]FLT autoradiogram of the coronal brain slices with probenecid 50 mg/kg (Prob_50; A) or cyclosporine A 50 mg/kg (CsA_50; B). Magnified views are shown in red boxes. C, Autoradiogram of the coronal brain slices after the administration of [¹⁸F]FLT with vehicle (left) or probenecid 10 mg/kg (middle), or probenecid 30 mg/kg (right). Arrows show the SVZ of the lateral ventricle or the DG of the hippocampus.

Figure 2.

A–C, Time–activity curves in SVZ (A), DG (B), and thalamus (C) with or without probenecid in [¹⁸F]FLT-PET. Vehicle-treated group (n = 8), probenecid 30 mg/kg-treated group (n = 7), and probenecid 100 mg/kg-treated group (n = 7) are shown as white diamonds, gray diamonds, and black diamonds, respectively. Data are expressed as the mean ± SD.

Figure 3.

Enhanced [¹⁸F]FLT accumulation in two neurogenic regions of probenecid-treated rats. A, B, PET images were averaged from 45 to 90 min after the injection of [¹⁸F]FLT. These images represent SUVs. Comparison of averaged PET images of [¹⁸F]FLT in both the SVZ (A) and the DG (B) of the vehicle-treated group (left) and the probenecid (100 mg/kg)-treated group (right). Arrows point to PET signals in the SVZ or the DG of the hippocampus. Magnified coronal images are shown in white boxes. All images are adjusted to the same intensity scale (0.15–0.50 SUV). C, Mean SUVs in the two neurogenic regions (SVZ and DG) and reference region (Ref; thalamus) of the vehicle-treated group (white bar; n = 8), probenecid 30 mg/kg-treated group (gray bar; n = 7), and probenecid 100 mg/kg-treated group (black bar, n = 7). Data are expressed as the mean ± SD. Significantly different from vehicle-treated rats, ***p < 0.001.

Figure 4.

Assessment of cell proliferation in CORT- and/or FLX-treated hippocampal DG using [¹⁸F]FLT-PET with probenecid. A, Representative coronal averaged PET images (45–90 min) of [¹⁸F]FLT in the SVZ and DG of each group (control-, CORT-, FLX-, and CORT plus FLX-treated rats). Magnified images in DG are shown below. All images are adjusted to the same intensity scale (0.16–0.80 SUV). Arrows point to PET signals in the SVZ or the DG of hippocampus. B, Mean SUVs in two neurogenic regions (SVZ and DG) and the reference region (Ref; thalamus) of the control group (white bar; n = 6), CORT-treated rats (pink bar; n = 6), FLX-treated rats (green bar; n = 6), and both CORT plus FLX-treated rats (blue bar; n = 6). Data are expressed as the mean ± SD. Significantly different from CORT-treated group, *p < 0.05, **p < 0.01.

Figure 5.

Comparison of the number of Ki67⁺ dividing cells in the hippocampal DG of the CORT- and/or the FLX-treated rats. A, Confocal images of Ki67-expressing cells in the DG of each group (control-, CORT-, FLX-, and CORT plus FLX-treated rats). Arrowheads show Ki67-immunopositive cells in the SGZ of the DG. Scale bar, 100 μm. B, The number of Ki67-expressing proliferating cells in the SGZ and hilus of the DG in the control group (white bar; n = 6), CORT-treated rats (pink bar; n = 6), FLX-treated rats (green bar; n = 6), and CORT plus FLX-treated rats (blue bar; n = 5). Data are expressed as the mean ± SD. Significantly different from the control group, ***p < 0.001. C, Correlation between [¹⁸F]FLT accumulation and Ki67 expression in the DG of each group [control group, white diamonds (n = 6); CORT group, pink diamonds (n = 6); FLX group, green diamonds (n = 6); CORT plus FLX group, blue diamonds (n = 5)].

Figure 6.

No proliferating reactive astrocytes and microglia were observed in the DGs of the CORT- and/or FLX-treated rats. A, Ki67⁺ proliferating cell (red) was not immunopositive for GFAP (green) in the hilus of DG in CORT plus FLX-treated rats. B, BrdU-expressing proliferating cell (red) was not immunopositive for Iba1 (green) in the DG of CORT plus FLX-treated rats. Arrowheads show proliferating cells with expression of Ki67 (A) or BrdU (B). Scale bars, 100 μm.