Discussion

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nicotine enters cerebral circulation and binds to neuronal nicotinic acetylcholine receptors (nAChR). nAChR are ligand-gated ion channels which are permeable to sodium, potassium, and calcium ions. Most nAChR in the CNS are presynaptically located and modulate the release of acetylcholine (Ach), DA, serotonin, glutamate, gama-aminobutyric acid (GABA), and norepinephrine (Dani and Bertrand, 2007). nAChR can also be postsynaptically located, such as on the DA neurons in the ventral tegmental area (VTA).

The two most commonly expressed nAChR in the brain are α4β2 or α7nAChR. Stimulation of the α4β2 nAChR located on the DA cell bodies in the VTA shifts these cells from tonic to phasic firing mode, which results in increased DA release in both the NAC and the prefrontal cortex to upregulate the dopamine expression. As with other drugs of abuse, this increased DA release in the NAC mediates the rewarding and pleasurable effects of nicotine and is presumed to be a critical mechanism for initiation and maintenance of nicotine addiction. Increased DA release in the prefrontal cortex is thought to be critical in mediating the cognitive-enhancing effects of nicotine (Herman et al., 2014). Altogether, dopamine projection from diverse region could upregulate the dopamine D1 and D2 receptor expression.

Furthermore, blockade of both DA D1R and DA D2R prevents nicotine generates addiction (Dani, 2003; Takahashi et al., 2008; Bergen et al., 2009). Consistent with our results (Fig. 16D), D1R antagonist, SCH23390 (Nisell et al., 1997; Jonkman and Markou, 2006; Markou, 2008; Hall et al., 2015b; General, 2017) and D2R antagonist, eticlopride (Araki et al., 2004; Laviolette et al., 2008; Liu et al., 2010) potent to relieve nicotine-induced dependence. Infusion SCH23390 into accumbens shell (AcS), parietal association cortex (PtA) and granular insula on the rats reduced nicotine self-administration 50% up to 75% (Kutlu et al., 2013; Hall et al., 2015a). Nicotine generates the formation of new

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dendritic branches and spine, completely blocked by SCH23390 on rats MSN (Ehlinger et al., 2017). Eticlopride reduced the ability of nicotine to enhance the responding for conditioned reinforcement effect (Guy and Fletcher, 2014), eticlopride inhibited luciferase activation by nicotine/ethanol on culture cell VTA and NAc of 17 old day embryonic Sprague-Dawley rats (Guy and Fletcher, 2014). These results showed the evidence that blocks of D1 receptor or D2 receptor alone by selective dopamine antagonist prevents nicotine dependence. Taken together, these pharmacological approaches demonstrated pivotal roles of D1R and D2R in the nicotine dependence.

Moreover, through nicotine’s interactions with the mesocorticolimbic dopamine system believed to mediate nicotine’s reinforcing effects leading to dependence (Sutherland et al., 2012; Jasinska et al., 2014), but this can be attenuated by D2R antagonsit. In Sprague Dawley rats administration of variety doses of eticlopride rapidly attenuated the magnitude of cue-elicited reinstatement of nicotine-seeking responding (Liu et al., 2010). Likewise, nicotine enhances dopamine release in striatal cholinergic interneurons in mice, as measured by fast-scan cyclic voltammetry, and this is correlated with nicotine dependence (Mclaughlin, Dani and Biasi, 2015). We here confirmed that D2RKO prevents dependence via modification of synaptic signal assessed by CPP method.

The projection of dopamine from VTA to NAc is crucial involved in behavioral sensitization, the rewarding/reinforcing effect of nicotine and also drug abuse (Risinger and Oakes, 1995). The D2RKO mice exhibited the slow response for acquisition of place-learning task on spatial task training for prediction of reward is associated with the place (Tran et al., 2003). In the D2RKO mice, the disruption of D2R in the mesolimbocortical dopaminergic pathway cause the pre-reward inhibitory response was abolished, whereas the inhibitory responses recorded during and after reward were unchanged due to changes in

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neural responses to incentive stimuli. The motoric activity of the mice enhanced by nicotine in WT mice but not in D2RKO mice (Fig. 17B), D2R gene in a mouse confirmed influence on specific central components of locomotion such as initiation of movement, time spent in motion, and horizontal distance traveled (Kelly et al., 1998; Picciotto and Corrigall, 2002).

Also, Nigrostriatal dopamine neurons participate in motor acts, locomotion impairment observed on lack D2R mice Proposed reflecting changes on the D2R system location in the nigrostriatal area (Wang et al., 2000).

Nicotine does not influence the D1R expression in D2RKO mice (fig. 22E), probably due to the affinity of the D1R is lower than D2R (Cristina Missale et al., 1998), in mouse neostriatal slices, nicotine 1µM decreased the level of phospho-Thr34 DARPP-32 as representative of D1R (Hamada et al., 2004), suspected for modulation of D1R on D2RKO mice the high concentration of nicotine is required. Direct activation of D1R and inactivation of D2R through the indirect pathway are a prominent feature for reward-based learning (Hikida et al., 2013). Probably, the projection pathway in dopaminergic neuron divided into 2 major population on the basis of their distinct projection through differential gene expression, a direct pathway from MSN, expressing dopamine D1 receptors, and an indirect pathway from MSN, expressing D2 receptor (Kreitzer and Malenka, 2008). This phenomena may be explained that expression of D1 and D2 receptor may have direct or indirect reciprocal interaction in the core of nucleus accumbens play important roles in drug dependence (Miyamoto et al., 2014). Additionally, D1 and D2 receptor co-activation generates a novel phospholipase C-mediated calcium signal, the pathway not activated by either receptor alone (Lee et al., 2004). For instance, cross-talk between Gi- and Gq- coupled receptors, mediated by Gβγ exchange has been observed to results in potentiation of Gq- mediated signaling due to Gi-Coupled receptor activation (Quitterer and Lohse, 1999).

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Inactivation dopamine receptor in I-aRNB mice by dopamine agonist quinpirole and aripiprazole produces selective impairments of aversive behavior (Hikida et al., 2013), that phenomena evoked by postsynaptic D2R serve as prerequisite determinant to modulates the pathway LTP-generating synaptic receptor (NMDA, A2a, and CB1 receptors) (Higley and Sabatini, 2010), this evidenced by administration of D2R agonist quinpirole and aripiprazole decrease the latency of memory aversion this represents the D2R is necessary for memory formation (Hikida et al., 2013). On direct pathway-specific manner, unstimulated of D1 receptor and inhibition of D2 receptor prevents cocaine evokes addiction reward learning (Hikida et al., 2013). Altogether, perturb of D1R and D2R respectively will change the downstream pathway on memory formation of nicotine dependence.

Lack of CaMKII in may correlate by LTP on memory formation by nicotine, in PD a dopamine deficiency condition, lacks CaMKII generates memory deterioration, the deficiency of CaMKII on D2RKO proposed to abolish the memory formation on remembering the nicotine-paired compartment and failed to evoke CPP(Picconi, 2004;

Silva et al., 2007). A recent study shows D2-like receptors, D4 strongly enhanced CaMKII activity through the stimulation of PLC pathway and elevation of intracellular Ca²⁺ via IP3Rs, the data obtained from westernblott analyses result of the additional D4R agonist PD168077 on suppressed neuronal activity of PFC by TTX (Tetrodotoxin), interestingly the effect of D4R agonist is disappeared since added 2APB a membrane-permeable IP3R antagonist (Gu and Yan, 2004). Fukunaga reported the collaboration D2R and CaM as a cellular Ca2+ sensor to activate ion channel to govern the CaMKII release (Fukunaga and Shioda, 2012). Visualization of pCaMKII by immunostaining to prove the prominent feature of pCaMKII on nicotine dependence (fig.19), nicotine injection not only increase the number of pCaMKII cells but also increase the fluorescence of pCaMKII and pERK

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cell revealed on extra magnification figure (fig. 19A, 20D). Nicotine increase the colocalization of CaMKII and Nicotine-induced fluo-4 [Ca²⁺] signaling along hippocampus axon (Zhong, Talmage and Role, 2015).

In addition to CaMKII signaling, the ERK1/2 phosphorylation has been increased in NAc and CA1 region nicotine dependence condition (Figure 18B and 21B) due to calcium influx was enhanced by nAChR stimulation (Nakayama et al., 2001). The intracellular Ca²⁺ elevation induces activation a tyrosine kinase, PYK2 and in turn the RAS is activated as upstream of ERK1/2 (Tahara et al., 2001). In the present study, D2R KO mice deficits the nicotine effect on ERK1/2 phosphorylation, therefore D2R is required for transactivating the tyrosine kinase to generate ERK modulation (Luttrell, Daaka and Lefkowitz, 1999; Tahara et al., 2001). We further demonstrated that these kinase activation promotes phosphorylation CREB in NAc and CA1 region. CREB(Ser 133/143) are phosphorylated by CaMKII (Matthews et al., 1994; Sun et al., 1994; Chiamulera et al., 2008; Yan et al., 2016) and CREB(Ser133) is also phosphorylate by ERK1/2 (Wu, Deisseroth and Tsien, 2001) and CREB activation in NAc is essential for nicotine-induced CPP (Walters et al., 2005; Brunzell et al., 2009; Pascual, Pastor and Bernabeu, 2009).

Genetic modulation of BDNF through TrkB receptor, modify the rewarding effect of drugs abuse such as alcohol and cocaine (Hall et al., 2003; Hensler, Ladenheim and Lyons, 2003;

Unterwald et al., 2013). Nicotine-stimulated increases in hippocampal BDNF expression may contribute to synaptic rearrangement involved in the development and maintenance of the nicotine addiction in humans (Kenny, File and Rattray, 2000). Notably, nicotine not alters the BDNF expression on D2RKO mice, suggesting a critical role in linkage between DR and BDNF production through calcium signaling and/or CaMKII activation (Hasbi et al., 2009). Additionally, α4β2nAChR in coordination with D2R controls BDNF expression

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after nicotine treatment in mouse NAc (Peterson et al., 2017), in where α4nAChR trans-activates tyrosine kinase B (TrkB) (Li et al., 2005; Beuten et al., 2007). BDNF-TrkB signaling mediates the neuronal structural changes by addictive drugs (Ohira and Hayashi, 2009). The main intracellular pathways activated by BDNF-TrkB signaling are MEK-ERK, PI3K-Akt-mTORC1 and PLC-PKC pathways(Kumar, 2005). Indeed, nicotine activates MEK-ERK and Akt-mTORC1 pathways in primary cultures dopamine neurons (Collo et al., 2013).

The another evidence provide, the knock in of BDNF Val66Met polymorphism gene on mice reduces nicotine-mediated anxiety-like behavior following withdrawal of nicotine and abolish the anxiolytic effect of chronic nicotine (Lee et al., 2015). The BDNF Val66Met polymorphism is related by deterioration of hippocampal function in humans assessed by assay of hippocampal physiology using the blood oxygenation level dependent (BOLD) fMRI technique (Egan et al., 2003). Genotype interaction of BDNF and smoking status on serum BDNF, which suggests that BDNF Val66Met polymorphism did not govern the association between smoking and serum BDNF in humans (Jamal et al., 2015).

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Figure 23. The Scheme of D1R and D2R actions on BDNF expression in nicotine dependence on WT-mice.

Activation of α4β2nAChRs and α7nAChRs in the dopaminergic terminals shifts these cells from tonic to phasic firing mode, which results in enhances DA release to upregulate the dopamine expression. Dopamine binds and activates D2R for phosphorylating of MEK and the upstream activity of ERK1/2. Additionally, D2R activation elevates intracellular calcium to enhanced CaMKII. Nuclear translocation of ERK1/2 and CaMKII induces phosphorylation the diverse of nuclear protein such as cAMP Response Element Binding (CREB) in core of the cells. Furthermore, phosphorylation of CREB is required to transformation of pre-Pro-BDNf to proBDNF by influencing axon

CREB p DA

α4β2nAChR/

α7nAChR

Nicotine, ACh

D2R

β αi ϒGDP

MEK p

ERK1/2

p CaMKII

p Ca²⁺

Dopamine terminal neuron

BDNF

Gi

D1R

cAMP PKA

Gs/olf AC

CREB p

BDNF

Activation

Neuron in Nucleus Accumbens

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no IV in transcription of pro-BDNF. pro-BDNF is a precursor of BDNF and is catalyzed by proteolytic enzyme change to become mature BDNF. On the other hand, D1R stimulation possibly causes synaptic rearrangement through the PKA pathway in the different cell types.

Figure 24. The Pattern pathway of nicotine action on D1R and D2R in dopaminergic neuron in D2KO mice

CREB p DA

α4β2nAChR/

α7nAChR Nicotine

ACh

D2R

β αi ϒGDP

MEK p

ERK1/2 p

CaMKII p Ca²⁺

Dopamine terminal neuron

BDNF

Gi

D1R

cAMP PKA

Gs/olf AC

CREB p

BDNF

Unactivation

Neuron in Nucleus Accumbens Unstimulation

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In contrast with WT mice in D2KO mice, stimulation of dopamine release by nicotinic acetylcholine receptor on dopaminergic neuron ((Anderson et al., 2009) unaffected the dopamine receptor. Deletion of dopamine D2 receptor on D2KO mice decrease the basal condition of ERK1/2 due to ERK activation induced by dopamine D2R stimulation in mesencephalic dopaminergic neurons related by nuclear ophan receptor Nurr1(Kim, 2006), Besides that absence of D2R reduces the CaMKII activity in D2KO mice, CaMKII binds with the D2R in intracellular loop 3 (IL3) domain (S. Zhang et al., 2014). ERK1/2 and CaMKII activity influenced phosphorylation of CREB in nucleolus (Impey et al., 1998; Yan et al., 2016) and the further action influence BDNF through maturation of proBDNF.

Interestingly, knock out of D2 receptor prevents the activation of D1 receptor by dopamine in D2KO mice. Inactivation D1 receptor by removing D2 receptor in KO mice to have an impact on unstimulating the PKC pathway and stabilized the BDNF expression via preCREB and proBDNF cycles.

All together nicotine evokes dependence through the α4β2nAChR and α7nAChR on dopaminergic neuron (Klink et al., 2001; Wooltorton et al., 2003; Faure et al., 2014), in where both activation D1R and D2R are crucial for the dependence formation. Activation CaMKII, ERK1/2, pCREB, proBDNF, and BDNF through D2R are the vital steps to generate nicotine dependence though D1 and D2 receptors (Fig. 23 and 24). In conclusion, activation of CaMKII and ERK1/2 through D2R are associated with nicotine-induced CPP behavior in a apart a BDNF as terminal signal. BDNF is very important in LTP memory formation during nicotine dependence acquisition. Our results shed new light on the detailed mechanism of nicotine-induced addiction through D2R activity. These findings provide new perspectives on the nicotine dependence is correlated with dopamine D2 transmission signal

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may explain the role of the D2R on nicotine addiction acquisition on wild-type mice and D2RKO mice particularly through BDNF expression.

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