Nicotine is very toxic and addictive substance that humans are exposed to by daily smoking, but it is a preventable risk to human health. Nicotine exerts its effects through the activation of nicotinic acetylcholine receptors (nAChRs) in multiple areas of the brain such as the hippocampus, amygdala, and prefrontal cortex.¹ Varenicline is used as a smoking cessation aid and is a partial agonist acting at the α4β2 nAChRs. Varenicline was developed based on the hypothesis that a partial agonist would help smokers quit smoking by blocking the effect of nicotine.² Thus, it would be interesting to compare the in vivo effects of nicotine and varenicline that contribute to the reward system. However, the influence of nicotine and varenicline on hippocampal neurochemical changes has not been experimentally investigated yet. We therefore studied the effects of repeated nicotine exposure and varenicline administration on neurochemical changes by using in vivo proton magnetic resonance spectroscopy (¹H MRS) at 9.4T.Eight-week-old male Wistar rats (n = 11; mean body weight, 304.9 ± 9.9 g; range, 290.1–323.21 g) were divided into 3 groups: control rats (control, saline injection, n = 3); nicotine-induced rats (nicotine, subcutaneous injection of nicotine, 0.4 mg/kg/day free base, n = 4); and nicotine- and varenicline-induced rats (varenicline, subcutaneous injection of nicotine, 0.4 mg/kg/day free base, intraperitoneal injection of varenicline 0.3 mg/kg/day free base, n = 4). The 8 rats in the nicotine and varenicline groups received a dose of 0.4 mg/kg nicotine subcutaneously once a day for 5 days and the varenicline group received a dose of 0.3 mg/kg varenicline intraperitoneally 30 min before the last nicotine injection on day 5.³ The 3 rats in control group received an equivalent volume of saline. On day 5, the rats were anaesthetized by inhalation of mixture of air and O2 containing 1–2% isoflurane after induction in a pre-anesthesia box with mixture of air and O2 containing 5% isoflurane. Then, ¹H MRS was performed on 9.4T Agilent MR scanner approximately 1 h after the last injection. A axial and coronal T2 weighted images (T2WI) was acquired with the following parameters: repetition time (TR) = 4000 ms, echo time (TE) eff = 32.95ms, field of view (FOV) = 3 cm × 3 cm, flip angle of 90º, slice thickness = 1.5 mm, matrix: 256 × 256. After the image acquisition, in vivo ¹H MRS were acquired in the voxel (1.5 × 2.5 × 3 mm³) mainly containing hippocampus using point-resolved spectroscopy (PRESS) sequence and T2WI for anatomical guidance with the following parameters : TR = 5000 ms, TE = 13.4 ms, 256 averages. The linear combination of model spectra (LCModel, version 6.3, Stephen W. Provencher) fitting software was used to quantify the metabolites in the frequency domain by using the basis metabolites.Fig 1 shows (A) axial and (B) coronal view of the rat brain and voxel plan. Fig 1(C-E) shows representative spectra obtained from the hippocampus regions of the 11 rats from the three groups (C: control, n = 3; D: nicotine, n = 4; E: varenicline, n = 4) respectively. Fig 2 illustrates the cerebral metabolite levels quantified from the 11 rats acquired in vivo spectra from the hippocampus region. The vertical lines on each of the bars indicate the (+) standard deviation of the mean values. Table 1 shows the mean values of the in vivo metabolite concentrations (μmol/g ± standard deviation [SD]) in the hippocampus of the rat brain with the Cramer-Raw low bound (CRLB) values.In summary, the present study conducted in vivo ¹H MRS in a rat model to determine the influence of repeated nicotine treatment on neurochemical in the rat hippocampus and the efficacy of varenicline on repeated nicotine-induced rats. In this study, the results show the tendency of increased glutamate (Glu) level in nicotine group than in the control and varenicline groups. Moreover, glutathione (GSH) and N-acetylaspartate (NAA) levels tended to decrease in the nicotine group in comparison with those in the control and varenicline groups. These findings indicate that the hippocampus is integrally linked to the brain reward sensitization involved in addiction and Glu release through mobilization of intracellular calcium stores.⁴ Further, oxidative stress and toxicity of nicotine on brain would cause the decline tendency of GSH and NAA.⁵ It satisfies that high resolution and short TE component adequately spilt the overlapped metabolite spectra and quantify the cerebral neurochemicals. In conclusion, we found that varenicline effectively inhibits the reward cycle.