T-type calcium channel enhancer SAK3 promotes dopamine and serotonin releases in the hippocampus in naive and amyloid precursor protein knock-in mice

T-type calcium channels in the brain mediate the pathophysiology of epilepsy, pain, and sleep. Recently, we developed a novel therapeutic candidate, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo[1,2-a] pyridine]-2-ene-3-carboxylate), for Alzheimer’s disease (AD). The cognitive improvement by SAK3 is closely associated with enhanced acetylcholine (ACh) release in the hippocampus. Since monoamines such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT) are also involved in hippocampus-dependent learning and psychomotor behaviors in mice, we investigated the effects of SAK3 on these monoamine releases in the mouse brain. Oral administration of SAK3 (0.5 mg/kg, p.o.) significantly promoted DA and 5-HT releases in the naive mouse hippocampal CA1 region but not in the medial prefrontal cortex (mPFC), while SAK3 did not affect NA release in either brain region. The T-type calcium channel-specific inhibitor, NNC 55–0396 (1 μM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampus. Interestingly, the α7 nicotinic ACh receptor (nAChR) antagonist, methyllycaconitine (1 nM) significantly inhibited DA release, and the α4 nAChR antagonist, dihydro-β-erythroidine (100 μM) significantly blocked both DA and 5-HT releases following SAK3 (0.5 mg/kg, p.o.) administration in the hippocampus. SAK3 did not alter basal monoamine contents both in the mPFC and hippocampus. SAK3 (0.5 mg/kg, p.o.) administration also significantly elevated DA and 5-HT releases in the hippocampal CA1 region of amyloid-precursor protein (APP)NL-GF knock-in (KI) mice. Moreover, hippocampal DA and 5-HT contents were significantly decreased in APPNL-GF KI mice. Taken together, our data suggest that SAK3 promotes monoamine DA and 5-HT releases by enhancing the T-type calcium channel and nAChR in the mouse hippocampus.


Introduction
Monoamines including dopamine (DA), serotonin (5-HT), and noradrenaline (NA) mediate various central nerve system functions such as motivation, motor function, and cognition [1,2]. Dysregulation of monoamine systems is associated with various psychiatric and neurodegenerative disorders [3]. In patients with schizophrenia, mesocorticolimbic DA dysfunction accounts for both psychotic and cognitive disturbances. Anti-psychotics with DA receptor blockers, such as risperidone, are generally used for therapy [4,5]. In addition, blockade of 5-HT and NA reuptake is the most common target of therapeutics for depression and behavioral and psychological symptoms of dementia (BPSD) in patients with Alzheimer's disease (AD) [6,7]. Furthermore, 5-HT levels are markedly reduced in the cerebral limbic and basal ganglia areas in patients with AD compared to healthy subjects [8,9]. These reports indicated that dysregulation of monoamine levels has a critical role in psychomotor disturbance in both psychiatry diseases and AD.
In this context, we investigated the effects of SAK3 on monoamine release in the mouse medial prefrontal cortex (mPFC) and hippocampal CA1 region. We also evaluated the effects of SAK3 (0.5 mg/kg, p.o.) on monoamine release in the hippocampus in amyloid precursor protein (APP) NL-GF knock-in (KI) mice as an animal model of AD [20]. Our results provide evidence that T-type calcium channel stimulation can increase monoamine release in both physiological and pathological conditions.

Animals
Male 6-week-old ddY mice were purchased from Clea Japan, Inc. (Tokyo, Japan). APP NL-GF KI mice were obtained from Dr. Takashi Saito and Dr. Takaomi C Saido (Riken, Saitama, Japan). Cav3.1 knock-out (KO) mice were generated by Dr. Kenji Sakimura [21]. Wild-type (WT) C57BL/6J mice were also purchased from Clea Japan, Inc. (Tokyo, Japan). Animals were housed under conditions of constant temperature (23 ± 2˚C) and humidity (55 ± 5%) on a 12-h light-dark cycle (light from 9 am-9 pm) and fed with standard forage. Animals were euthanized by isoflurane overdose or cervical dislocation after experiments. All animal procedures were approved by the Committee on Animal Experiments of Tohoku University.

Measurement of monoamine releases using in vivo microdialysis
Stereotaxic surgery for in vivo microdialysis in mice was performed as previously described [24]. Mice were anesthetized with pentobarbital Na (50 mg/kg, i.p.), and the head was placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, U.S.A.). A guide cannula (AG-4 for the hippocampus or AG-3 for the mPFC; Eicom, Kyoto, Japan) was inserted into the dorsal hippocampal CA1 region [2.9 mm posterior and 3.3 mm lateral to the bregma and 1.4 mm below the brain surface, according to [25]] or the mPFC [1.9 mm posterior and 0.3 mm lateral to the bregma and 1.8 mm below the brain surface, according to [25]], and the skull was covered by dental cement. The microdialysis probe (A-I-4-02 for the hippocampus or A-I-3-02 for the mPFC; Eicom) was inserted through the guide cannula. After recovery, Ringer's solution was perfused at 2 μL/min using a micro-syringe pump (ESP-64; Eicom) under free moving conditions. PP-ODS (Eicom) was used for measurement of hippocampal DA and 5-HT [24]. Hippocampal NA and monoamines in the mPFC were measured using CAX-EICOMPAK (Eicom). Perfused dialysates were collected every 6 min (PP-ODS) or 15 min (CAX-EICOMPAK) in the sample loop of an auto-injector (EAS-20; Eicom) connected to a high-performance liquid chromatography (HPLC)-electrochemical detector (ECD) system (HTEC-500; Eicom). When monoamine levels reached a steady state, mice were treated with SAK3 (0.5 mg/kg, p.o.). The T-type calcium channel specific blocker, NNC 55-0396 (1 μM), the α7 nAChR antagonist, MLA (1 nM), or the α4β2 nAChR antagonist, DhβE (100 μM) in Ringer's solution was infused to brain regions through a microdialysis probe before SAK3 administration. Monoamine levels were calculated in the chromatogram (Fig 1B and 1C). Monoamine release was assessed as a percentage of basal levels. Released monoamine levels were calculated after SAK3 treatment by comparison to the responses of vehicle-treated animals at the same time points.

Measurement of monoamine contents in the brain tissues
Fifteen minutes after SAK3 (0.5 mg/kg, p.o.) administration, monoamine contents were measured in ddY mice. Ten-month-old WT and APP NL-GF KI mice were used for the measurement. Animals were sacrificed by cervical dislocation for dissection of brain tissues. After decapitation, the mPFC and hippocampal CA1 region were dissected, frozen in liquid nitrogen, and stored at -80˚C until assayed. Analyses of monoamine contents were performed as previously described [24]. Each frozen tissue sample was weighed and homogenized in 200 μL of 0.2 M perchloric acid containing 100 ng/mL isoproterenol as an internal standard. The homogenate was placed on ice for 30 min and then centrifuged at 20,000 x g for 15 min at 4˚C. Monoamine contents in the supernatants were quantified with the HPLC-ECD system and expressed as ng/g tissue weight.

Statistical analysis
Significant differences were determined using Student's t-test for two-group comparison and by two-way analysis of variance (ANOVA) for multi-group comparisons to analyze in vivo microdialysis. Other comparison between multiple groups was performed using oneway ANOVA followed by Tukey's multiple comparisons test. Results are expressed as mean ± standard error of the mean (SEM).

Acute SAK3 administration promotes DA and 5-HT releases in the hippocampal CA1 region
We first investigated the effect of SAK3 on NA, DA, and 5-HT releases in the hippocampal CA1 region. NA, DA, and 5-HT levels were calculated by the area under the receiver operator characteristic curve (AUC: NA levels were calculated from time 0 to 160 min, DA and 5-HT levels were calculated from time 0 to 60 min). Acute SAK3 (0.5 mg/kg, p.o.) administration significantly promoted DA and 5-HT releases with a peak at 12 min in the hippocampal CA1 region (DA: p = 0.0011 vs. saline-treated mice; 5-HT: p = 0.0005 vs. saline-treated mice; Fig (Fig 2A). We also evaluated DA and 5-HT contents of hippocampal tissues dissected 15 min after SAK3 administration. However, SAK3 (0.5 mg/kg, p.o.) administration did not alter basal DA and 5-HT contents compared to saline-treated mice (DA: p = 0.2604 vs. saline treated mice; 5-HT: p = 0.7999 vs. saline treated mice; Table 1).

Acute SAK3 administration does not affect monoamine release in the mPFC
Since monoamine levels in the mPFC mediate cognition and psychic functions [26,27], we next tested whether SAK3 promotes monoamine release in the mPFC. In contrast to the hippocampal CA1 region, acute SAK3 (0.  Table 1).

T-type calcium channel inhibitor and nAChR antagonists prevent SAK3-promoted DA and 5-HT releases in the hippocampal CA1 region
We previously reported that SAK3 promotes ACh release through enhancing T-type calcium channels in the mouse hippocampus [18]. In addition, SAK3 shows neuroprotective effects against transient brain ischemia via activation of nAChR in the CA1 pyramidal neurons [19]. Thus, we tested whether the T-type calcium channel inhibitor or nAChR antagonists eliminate SAK3-promoted DA and 5-HT releases in the mouse hippocampus. Significant group effects were observed in DA [F (8, 48) = 7.384, p < 0.0001] and 5-HT [F (8, 48) = 6.452, p < 0.0001] levels (Fig 4B and 4D). T-type calcium channel specific inhibitor NNC 55-0396 (1 μM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampal CA1 region  Fig 4C). Taken together, SAK3 promotes DA and 5-HT releases by enhancing T-type calcium channels and activating nAChR in the hippocampal CA1 region.

Peak response of DA and 5-HT release followed by SAK3 administration is delayed in Cav3.1 KO hippocampal CA1 region
To reveal mechanism underlying SAK3-facilitated hippocampal DA and 5-HT releases, we assessed whether Cav3.1 gene deletion inhibits effects of SAK3 (Fig 5). SAK3 significantly promoted hippocampal DA but not 5-HT releases in Cav3.

Acute SAK3 administration enhances DA and 5-HT releases in the hippocampal CA1 region in APP NL-GF KI mice
Finally, we tested whether SAK3 can promote monoamine release in the hippocampus in an animal model of AD. For the analysis, we used 10-month-old APP NL-GF KI mice exhibiting memory impairments due to amyloid plaque formation in the hippocampus [20,28]. Importantly, SAK3 (0.5 mg/kg, p.o.) administration significantly elevated DA and 5-HT releases in the hippocampus of APP NL-GF KI mice (DA: p = 0.0136 vs. saline-treated mice; 5-HT: p = 0.0006 vs. saline-treated mice; Fig 6D and 6F). SAK3 did not affect NA release in APP NL-GF KI mice (p = 0.1799 vs. saline-treated mice; Fig 6B).  Fig 6E). We also measured basal monoamine contents in the hippocampal CA1 region and the mPFC of APP NL-GF KI mice. Whereas no differences were observed in monoamine content in the mPFC (NA: p = 0.7913 vs. WT mice; DA: p = 0.1159 vs. WT mice; 5-HT: p = 0.5970 vs. WT mice; Table 2), DA and 5-HT contents were markedly reduced in the hippocampal CA1 region of APP NL-GF KI mice than of WT mice of the same age (NA: p = 0.4602 vs. WT mice; DA: p = 0.0487 vs. WT mice; 5-HT: p = 0.0394 vs. WT mice; Table 2). Therefore, these results indicated that SAK3 can promote hippocampal DA and 5-HT releases under the AD-like condition.

Discussion
In the present study, we demonstrated that SAK3 promotes DA and 5-HT releases in the naive mouse hippocampus but not in the mPFC. In addition, we also made the following novel observations: (1) T-type calcium channel inhibitor, NNC 55-0396, antagonized SAK3-induced DA and 5-HT releases in the hippocampal CA1 region; (2) α4 nAChR antagonist, DhβE, and/ or α7 nAChR antagonist, MLA, blocked DA and 5-HT releases by SAK3 administration in the hippocampus; (3) SAK3 significantly promoted DA and 5-HT releases in the hippocampal CA1 region of APP NL-GF KI mice. Previous reports have indicated that T-type calcium channel activities play a key role in DA release [29,30]. Blockade of T-type calcium channels by Ni 2+ (100 μM) decrease single pulseevoked DA release in the Hartley guinea pig striatum [29]. High K + levels (60 mM) increase the frequency of DA release in rat dopaminergic neurons in the substantia nigra pars compacta using carbon fiber microelectrodes, and this action is abolished by T-type calcium channel blocker, mibefradil (10 mM) [30]. On the other hand, several voltage-gated calcium channels are involved in 5-HT release. P/Q-type calcium channel inhibitor, ω-agatoxin IVA (0.1-1 μM) and N-type calcium channel inhibitor, ω-conotoxin GVIA (3-10 μM), but not the L-type calcium channel inhibitor, nifedipine (3 μM) antagonized high K + (50 mM)-induced 5-HT release in rat hippocampal slices [31]. We demonstrated here that SAK3 promotes DA and 5-HT releases in the hippocampus, an effect blocked by NNC 55-0396 (1 μM) administration. We previously defined that polymethoxyflavone nobiletin enhances hippocampal DA release via enhancing the T-type calcium channel in naive and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice [24]. Moreover, an in situ hybridization study indicated that T-type calcium channel mRNAs expressed in the rat substantia nigra and raphe nuclei [16], which are the origin of dopaminergic and serotonergic neurons respectively, and T-type calcium channels likely mediate each neuronal activity and development [32][33][34][35]. These observations suggest that SAK3 promotes hippocampal DA and 5-HT releases via enhancing T-type calcium channels.
On the other hand, the cholinergic system is known to mediate DA and 5-HT releases in the brain. For example, nicotine (0.3 mg/kg, s.c.) treatment increased DA release in the rat hippocampus, an effect blocked by the α7 nAChR selective antagonist, MLA (500 μM) and non- SAK3 promotes dopamine and serotonin releases in the hippocampus specific nAChR antagonist, mecamylamine (MEC: 100 μM) used in microdialysis analysis [36]. Administration of low dose nicotine (1 μM) also enhanced hippocampal DA release in rats [37]. Treatment with α7 nAChR agonist, EVP-6124 (0.1 mg/kg, i.p.) also increased DA release in the rat mPFC and nucleus accumbens (NAc) [38]. Electrostimulation-evoked DA release was inhibited by nAChRs inhibitor, hexamethonium (200 μM) administration in the mouse NAc [39]. Likewise, nicotine (50-500 μM) treatment promoted [ 3 H]-5-HT release in rat hippocampal slices in a concentration-dependent manner, and MEC (0.5 μM) treatment significantly antagonized it [40]. On the other hand, nicotine treatment decreases 5-HT release in the rat hippocampus using in vivo microdialysis [36,41]. Since nicotine stimulation promoted both monoamine and gamma-aminobutyric acid (GABA) release [42], GABA inhibitory neurotransmission may contribute to these discrepancies between in vivo and ex vivo conditions. Furthermore, nicotine stimulation increased the frequency of excitatory postsynaptic currents in serotonergic neurons in the dorsal raphe nucleus through the α4β2 nAChRs [43], suggesting that the nAChR may mediate serotonergic neuronal activity. Previously, we indicated that SAK3 promoted ACh release in the mouse hippocampus, an effect inhibited by T-type calcium channel inhibitor treatment and/or by deficiency of the Cav3.1 gene [18]. Moreover, SAK3 activates nAChR signaling in hippocampal CA1 pyramidal neurons [18]. Therefore, SAK3 possibly enhances DA and 5-HT releases in the hippocampus via indirect stimulation of nAChR. However, SAK3 could not alter DA and 5-HT releases in the mPFC. Whereas cholinergic innervation in the mPFC is received from the nucleus basalis of Meynert, where it may show low expression levels of T-type calcium channel mRNAs [16,44], cholinergic neurons in medial septum input to the hippocampus [44]. Since T-type calcium channels are highly expressed in the medial septum [16,19], the fact that SAK3 did not affect the monoamines in the mPFC may be due to differences in cholinergic innervation. Further studies are required to define the action mechanism of SAK3 on DA and 5-HT releases in the brain.
Previous studies have indicated that NA release is mediated only by N-type calcium channels and not by any other type of voltage-gated calcium channels [45][46][47]. In rat hippocampal slices, nAChR agonist dimethylphenylpiperazinium-induced [ 3 H]-NA release is significantly blocked by N-type calcium channel inhibitors and not by other voltage-gated calcium channel blockers [47]. By contrast, functional L-type and T-type calcium channels are expressed and regulate neuronal pacemaking in the locus ceruleus located in noradrenergic neurons [48]. While the combined application of the L-type calcium channel inhibitor, isradipine (120 nM) and the T-type calcium channel inhibitor, mibefradil (2 μM) increases firing frequency and decreases afterhyperpolarization amplitude in noradrenergic neurons, mibefradil (2 μM) alone could not affect pacemaking, suggesting that both channel functions may be essential for neuronal activity in the locus ceruleus [48]. Thus, we concluded here that the T-type calcium channel enhancer SAK3 could not affect NA release in the mouse brain. Several postmortem studies have reported decreased density of dopamine receptors in the AD brain including the hippocampus [49][50][51]. In addition, D1-like receptor agonists significantly improve memory deficits seen in amyloid β-injected mice [52,53]. On the other hand, 5-HT concentration was significantly decreased in the platelets in patients with AD [54] and selective serotonin reuptake inhibitors improve decreased cognitive performance and BPSD seen in patients with AD [55,56]. Likewise, in the AD brain, reduction of DA and 5-HT contents was observed in the hippocampus of APP NL-GF KI mice, suggesting that decrease in dopaminergic and serotonergic pathways in the hippocampus may be associated with the cognitive impairments seen in APP NL-GF KI mice [20]. In addition, SAK3 could enhance DA and 5-HT release in the hippocampus in APP NL-GF KI mice. These observations suggest that SAK3 may have potential for improvement of cognitive impairments seen in APP NL-GF KI mice. Supported this idea, we reported that chronic SAK3 (0.5 mg/kg, p.o.) administration significantly antagonizes cognitive impairments seen in APP NL-F KI mice [22]. Therefore, SAK3 may be able to become an attractive therapeutic for both cognitive impairment and BPSD observed in patients with AD.
Here, we observed that SAK3-promoted DA and 5-HT releases reach a peak at 12 min in the hippocampal CA1 region. We previously reported that the brain concentration of SAK3 reaches approximately 0.2 nM within 15 min after SAK3 (0.5 mg/kg, p.o.) administration [22]. Since patch-clamp experiments indicate that 0.1 nM SAK3 can enhance Cav3.1 and 3.3 currents maximally in Cav3.1 and Cav3.3 over-expressed neuro2A cells [18], SAK3 (0.5 mg/kg, p. o.) administration in the present study may be enough to act in the brain.
In summary, the T-type calcium channel enhancer SAK3 promoted DA and 5-HT, but not NA, release in the mouse hippocampus under naive and AD-like conditions. SAK3 may promote DA and 5-HT releases through activation of the nAChR by enhancing ACh release in the hippocampus. Therefore, SAK3 can facilitate both ACh and DA and 5-HT releases in the hippocampus through enhancing T-type calcium channels. This evidence is particularly important for SAK3-induced improvement of memory deficits and BPSD seen in patients with AD.