6.2.1 Function of Accumbal Shati/Nat8l in Nicotinic Effects
We previously reported that overexpression of Shati/Nat8l in the nucleus accumbens (NAc) of mice depressed the pharmacological effects of methamphetamine, especially addiction-related behaviors, hyperactivity, and place preference. In an in vivo microdialysis experiment, the extracellular dopamine (DA) level was increased by 200–300% of the base line by peripheral methamphetamine injection. In the NAc of these mice, NAA and NAAG levels were significantly reduced (Miyamoto et al. 2014). The mGluR3 antagonist, LY341495, cancelled the Shati-Nat8l-indeuced reduction of hyperlocomotion and conditioned place preference after methamphetamine treatment. Furthermore, LY341495 also cancelled the Shati/Nat8l-associated increase in extracellular DA levels in the NAc of methamphetamine-treated mice. These results indicate that the overexpression of Shati/Nat8l in the NAc suppresses the increase in dopamine release caused by methamphetamine via mGluR3 (Miyamoto et al. 2014).
We also investigated the effects of Shati/Nat8l on nicotine preference using a three-bottle paradigm. Experiments were performed as follows: I. Both NAc-Mock and NAc-Shati/Nat8l overexpressing mice were habituated to an experimental chamber which contained three water bottles on days 1–3. II. The tap water in all three bottles was changed to 2% saccharin to habituate animals to saccharin on days 4–6. III. The contents of all three bottles were replaced with a mixture of 75 μg/mL and 2% saccharin on days 8–14 (Fig. 6.2). During the subsequent test phase, three bottles containing either 0 μg/mL nicotine +2% saccharin, 75 μg/mL nicotine +2% saccharin or 150 μg/mL nicotine +2% saccharin, were presented to each mouse, and the total intake of nicotine was measured in each mouse on days 15–21 (Fig. 6.2). The daily amounts of nicotine intake are shown in Fig. 6.3a. On the first day of the test phase, an average of 300 μg of nicotine was consumed by both groups. In the NAc-Mock mice, the amount of nicotine consumed increased each day during days 15–17 (Fig. 6.3a). In contrast, the Shati/Nat8l mice showed a lower intake of nicotine enriched solution during this period (Fig. 6.3a). These results suggest that overexpression of Shati/Nat8l in NAc lowers nicotine intake and preference. The 2% of saccharin were essential for the experimental protocol, since nicotine also has aversive effects due to its bitter taste. While overexpression of Shati/Nat8l in NAc lowered nicotine preference during days 16–17 (Fig. 6.3a), total intake (water and nicotine solution) was not changed (Fig. 6.3c, d).
Next, we performed in vivo microdialysis experiments to measure the amount of extracellular DA induced by nicotine in the system (Fig. 6.4a). Basal levels of extracellular DA in the NAc of Shati/Nat8l-overexpressing mice were significantly lower than those of NAc-Mock mice (Fig. 6.4b). In the NAc-Mock mice group, the extracellular DA level was significantly increased 60–120 min after nicotine injection, up to a level of approximately 170% of that of the saline injection. Nicotine-injected NAc-Shati/Nat8l mice showed low levels of extracellular DA at the 60–120 min time point similar to saline-injected groups (Fig. 6.5). Furthermore, the suppressive effects of Shati/Nat8l on nicotine-induced DA elevation were partially reversed by LY341495, an mGluR2/3 antagonist (Fig. 6.6). These results indicate that the function of Shati/Nat8l to suppress the effect of nicotine-induced extracellular DA in the NAc is partially dependent on mGluR3. These results are similar to those showing the action of Shati/Nat8l against the pharmacological effects of methamphetamine. One difference is the complete or partial contribution of mGluR3 in methamphetamine and nicotine use, respectively. Both nicotine and methamphetamine regulate the DA reward system in the brain. Methamphetamine directly alters dopamine uptake and release, while nicotine first binds to the nicotinic acetylcholine receptor (nAChR) and, following signal transduction contributes to the potentiation of DA release. The suppressive effects of Shati/Nat8l on the DA release might happen downstream of the mGluR3 pathway. Further studies are required to discern whether there is a mechanistic crossover between the pharmacological actions of nicotine and methamphetamine. We attempted to investigate Shati/Nat8l mRNA changes in the NAc, the hippocampus and the frontal cortex, related to reward pathways following single or repeated treatments with nicotine. Unfortunately, we could not reproduce the results of the mRNA measurements, potentially because the levels of Shati/Nat8l or NAAG do not change based on the activation of nAChR. In relation to Shati/Nat8l production, methamphetamine and nicotine most likely act through different pathways.
Taken together, our results show that Shati/Nat8l in the NAc has a protective effect against the deleterious physiological changes associated with nicotine or methamphetamine administration.
6.2.2 Striatal Shati/Nat8l and the Reward System
We produced Shati/Nat8l transgenic mice (Shati/Nat8l-Tg) to investigate global overexpression in the brain. A targeting vector was used to produce the Shati
/
Nat8l-Tg mice, which ubiquitously expressed his-tagged Shati
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Nat8l gene. A transgene cassette including the CAG promoter followed by the his-Shati
/
Nat8l sequence was obtained from a CAG promoter the his-Shati/Nat8l expression plasmid (Fig. 6.7a). The transgene cassette was microinjected into fertilized eggs from C57BL/6 J females mated with males. Genotyping confirmation of Shati
/
Nat8l-Tg mice using a wild type mouse as a control is shown in Fig. 6.7b. Shati/Nat8l mRNA levels were measured by quantitative real-time reverse transcriptase (RT)-PCR in the brain and are presented relative to the expression of the housekeeping gene, 36B4. Shati/Nat8l mRNA was highly expressed in the whole brain of 8-week-old transgenic mice (Fig. 6.8). However, Shati/Nat8l mRNA levels were increased in relation to wild type expression in the striatum only, not in other brain regions, such as the olfactory bulb, the prefrontal cortex or the NAc (Fig. 6.9). These Shati/Nat8l-Tg mice were therefore used for striatal Shati/Nat8l-overexpression mice. The mice showed no differences in basal locomotor activity compared to wild-type mice when observed in a novel environment (Fig. 6.10). These mice also performed a Y maze task as well as novel object recognition tests, to assess their learning abilities (Fig. 6.11). In the Y maze task, neither the number of total entries nor spontaneous alternation behaviors were different between Shati/Nat8l-Tg and wild type mice (Fig. 6.12). In the novel recognition test, both time and trial exploratory preference (in %) were similar between wild type and Shati/Nat8l-Tg mice (Fig. 6.12). Next, anxiety-like emotional behaviors in Shati/Nat8l-Tg mice were investigated using the light/dark box test, and no difference in the time spent on the light side was observed between Shati/Nat8l-Tg mice and wild type mice (Fig. 6.13). Results from both the light/dark box and elevated plus maze tests indicated no effect of increased levels of striatal Shati/Nat8l on anxiety-like behaviors. We also investigated the social abilities of the Shati/Nat8l-Tg mice. The experimental schedule of the three-chamber social interaction test is shown in Fig. 6.13a. In trial 1, the test mouse was placed in the center of the chamber, while the other side of the chamber remained empty and no wire cage was placed. Both Shati/Nat8l-Tg and wild type mice were more interested in the novel object (Fig. 6.13b, left). In trial 2, another novel object was placed in the wire cage on one side, and an unfamiliar mouse (C57BL/6J) was placed in a wire cage on the opposite side. The Shati/Nat8l-Tg mice and wild type mice showed a similar level of interest in the unfamiliar mouse (Fig. 6.13b, right). In a prepulse inhibition (PPI) test, auditory startle response (Fig. 6.14a) and sensory motor control function (Fig. 6.14b) were not changed in Shati/Nat8l-Tg mice. Furthermore, in forced swimming and tail suspension tests, the immobility times of Shati/Nat8l-Tg mice were the same as those found for wild type mice (Figs. 6.15 and 6.16). These results indicate Shati/Nat8l-Tg mice do not demonstrate schizophrenia or depression-like behaviors.
Shati/Nat8l-Tg mice were not stable for the overexpression of Shati/Nat8l in the striatum, depending on their generation. In general, mouse lines generated with transgenes are often not stable, since transecting genes is a process that does not happen naturally. Therefore, to measure the effect of increased striatal Shati/Nat8l on the reward system, we injected an EGFP-tagged AAV containing Shati/Nat8l into the striatum of mice (Str-Shati/Nat8l) and confirmed localized overexpression by in situ hybridization and EGFP immunohistochemistry in the dorsal striatum area. Str-Shati/Nat8l mice showed no difference in methamphetamine-induced hyperactivity compared to mock-injected mice (Fig. 6.17a). Methamphetamine-induced conditioned place preference was also not changed in Str-Shati/Nat8l mice (Fig. 6.17b). Therefore, we conclude that Shati/Nat8l in the striatum does not contribute to reward effects in mice.