Evaluation of F-nifene binding to α4β2 nicotinic receptors in the rat brain using microPET imaging

Abstract

MicroPET imaging studies using (18)F-nifene, a new positron emission tomography (PET) radiotracer for nicotinic acetylcholinergic receptors (nAChR) α4β2 receptors in rats, have been carried out. Rats were imaged for 90 min after intravenous injection of (18)F-nifene (0.8 to 1 mCi), and binding potential (BP(ND)) was measured. (18)F-Nifene binding to thalamic and extrathalamic brain regions was consistent with the α4β2 nAChR distribution in the rat brain. Using the cerebellum as a reference, the values for the thalamus varied less than 5% (BP(ND) = 1.30, n = 3), confirming reproducibility of (18)F-nifene binding. (18)F-Nifene microPET imaging was also used to evaluate effects of nicotine in a group of Sprague-Dawley rats under isoflurane anesthesia. Nicotine challenge postadministration of (18)F-nifene demonstrated reversibility of (18)F-nifene binding in vivo. For α4β2 nAChR receptor occupancy (nAChR(OCC)), various doses of nicotine (0, 0.02, 0.1, 0.25, and 0.50 mg/kg nicotine free base) 15 min prior to (18)F-nifene were administered. Low-dose nicotine (0.02 mg) reached > 80% nAChR(OCC) while at higher doses (0.25 mg) > 90% nAChR(OCC) was measured. The small amount of (18)F-nifene binding with reference to the cerebellum affects an accurate evaluation of nAChR(OCC). Efforts are underway to identify alternate reference regions for (18)F-nifene microPET studies in rodents.

Figure 1

Chemical structure of ¹⁸F-nifene.

Figure 2

In vivo microPET rat brain test-retest study. (A) Horizontal, (B) sagittal, (C) coronal of ¹⁸F-nifene. The thalamus (TH) shows the highest binding followed by the cortex (COR) and the cerebellum (CB). Test-retest study showing consistency in binding of ¹⁸F-nifene to the thalamus with respect to the cerebellum. BP_ND for the test study was 1.69 while the retest study was 1.64.

Figure 3

Blood and brain metabolite analysis in rats postadministration of intravenous ¹⁸F-nifene. (A) Blood plasma collected at different time points (5, 15, 60, and 90 min) and compared to ¹⁸F-nifene standard on TLC. A polar metabolite is seen, but the predominant radioactive species is ¹⁸F-nifene. (B) Analysis of TLC in (A) indicates 42% of ¹⁸F-nifene (blue) remaining at 90 min with little polar metabolites (red) remaining in the plasma. (C) Ex vivo rat brain was dissected into two hemispheres--the left hemisphere was cut into 40-μm thick sagittal brain sections and were scanned to reveal brain areas. (D) Binding of ¹⁸F-nifene in the thalamus (TH), cortex (COR), and least binding in the cerebellum (CB) was observed. (E) RadioTLC of ¹⁸F-nifene standard with 9:1 CH₂Cl₂:CH₃OH. (F) RadioTLC of brain extracts with 9:1 CH₂Cl₂:CH₃OH showing the presence of ¹⁸F-nifene.

Figure 4

Ex vivo microPET and autoradiographic brain images of a rat. MicroPET images ((A) horizontal, (B) coronal, and (C) sagittal) validate maximum binding in the thalamus (TH) followed by the cortical regions (COR). An autoradiograph of the brain in (A) showing 10-μm horizontal sections (D) and an anatomical view (E) of the slice in (D). ¹⁸F-nifene binding followed the order TH > subiculum (SUB) > cortex (COR) > striatum (STR) > cerebellum (CE).

Figure 5

In vivo displacement of ¹⁸F-nifene by nicotine. In vivo rat microPET brain slices of ¹⁸F-nifene before (A) and after (B) nicotine challenge. (C) Time-activity curve of ¹⁸F-nifene specific binding (thalamus-cerebellum) with nicotine (0.3 mg/kg) administered at 30 min pi, displacing ¹⁸F-nifene binding in the thalamus (inset shows dissociation rate, k_off of ¹⁸F-nifene was 0.06 min^-1).

Figure 6

Dose effects of nicotine on thalamus time-activity curves. Time-activity curves of ¹⁸F-nifene uptake in the thalamus of rats injected with different doses of nicotine.