Lurasidone Sub-Chronically Activates Serotonergic Transmission via Desensitization of 5-HT1A and 5-HT7 Receptors in Dorsal Raphe Nucleus

Lurasidone is an atypical mood-stabilizing antipsychotic agent with unique receptor-binding profile, including 5-HT7 receptor (5-HT7R) antagonism. Effects of 5-HT7R antagonism on transmitter systems of schizophrenia and mood disorders, however, have not been well clarified. Thus, this study examined the mechanisms underlying the clinical effects of lurasidone by measuring mesocortical serotonergic transmission. Following systemic and local administrations of lurasidone, MK801 and 5-HT receptor modulators, we determined releases of 5-HT in dorsal raphe nucleus (DRN), mediodorsal thalamic nucleus (MDTN) and medial prefrontal cortex (mPFC) and γ-aminobutyric acid (GABA) in DRN using multiprobe microdialysis with ultra-high-performance liquid chromatography (UHPLC). Serotonergic and GABAergic neurons in the DRN are predominantly regulated by inhibitory 5-HT1A receptor (5-HT1AR) and excitatory 5-HT7R, respectively. Lurasidone acutely generates GABAergic disinhibition by 5-HT7R antagonism, but concomitant its 5-HT1AR agonism prevents serotonergic hyperactivation induced by 5-HT7R inhibition. During treatments with 5-HT1AR antagonist in DRN, lurasidone dose-dependently increased 5-HT release in the DRN, MDTN and mPFC. Contrary, lurasidone chronically enhanced serotonergic transmission and GABAergic disinhibition in the DRN by desensitizing both 5-HT1AR and 5-HT7R. These effects of lurasidone acutely prevented MK801-evoked 5-HT release by GABAergic disinhibition via N-methyl-D-aspartate (NMDA)/glutamate receptor (NMDA-R)-mediated inhibition of 5-HT1AR function, but enhanced MK801-induced 5-HT release by desensitizing 5-HT1AR and 5-HT7R. These results indicate that acutely lurasidone fails to affect 5-HT release, but chronically enhances serotonergic transmission by desensitizing both 5-HT1AR and 5-HT7R. These unique properties of lurasidone ameliorate the dysfunctions of NMDA-R and augment antidepressive effects.


Dose-Dependent Effects of Systemic Lurasidone Administration on Extracellular Levels of 5-HT in the DRN, MDTN,mPFC and GABA in the DRN Following Inhibition of 5-HT1AR in the DRN (Study_2)
In the DRN, 5-HT1AR is a major autoregulation inhibitory receptor against serotonergic neurons in the DRN [18,22]. Study_2 was designed to determine dose-dependent effects of systemic lurasidone administrations at 0, 0.3, or 1 mg/kg, and distinguish these from the effects of 5-HT1AR on extracellular levels of 5  In the DRN, 5-HT1AR is a major autoregulation inhibitory receptor against serotonergic neurons in the DRN [18,22]. Study_2 was designed to determine dose-dependent effects of systemic lurasidone administrations at 0, 0.3, or 1 mg/kg, and distinguish these from the effects of 5-HT1AR on extracellular levels of 5-HT in the DRN, MDTN, mPFC, and GABA in the DRN. Perfusate in the DRN was commenced with MRS containing 10 µM N- , and perfusions in the MDTN and the mPFC were commenced with MRS. After confirming stabilization of extracellular levels of 5-HT and GABA in perfusates, rats were administered lurasidone (0, 0.3, or 1 mg/kg) intraperitoneally ( Figure 2 and Table 1).

Concentration-Dependent Effects of Local Lurasidone Administrations into the DRN on Extracellular Levels of 5-HT in the DRN, MDTN, mPFC and GABA in the DRN (Study_3)
Both study_1 and study_2 indicate that systemic administration of lurasidone dose-dependently increased 5-HT release in the DRN without affecting those in the MDTN or mPFC; however, under the 5-HT1AR inhibition in the DRN, systemic administration of lurasidone increased 5-HT release in all three regions with reduced GABA release in the DRN. To clarify the mechanisms of these discrepant effects of systemic administration of lurasidone under the conditions between 5-HT1AR  and inhibition, the present study determined the DRN regional specific action of lurasidone, 5-HT1AR and 5-HT7R on 5-HT release and GABA in the DRN.
Our previous studies showed that local administration of 3 µM lurasidone into the insular cortex increased regional extracellular levels of dopamine and norepinephrine, whereas lower than 1 µM had no effects [11].
Therefore, study_3-1 was designed to determine the concentration-dependent effects of local lurasidone administrations in the DRN, and study_3-2 was designed to determine the effects of 5-HT1AR, 5-HT7R, and GABA A receptors (GABA A -R) in the DRN on extracellular levels of 5-HT in the DRN, MDTN, mPFC, and GABA in the DRN. In study_3, perfusate in the DRN, MDTN and mPFC were commenced with MRS. After confirming stabilization of extracellular 5-HT and GABA levels in perfusates, perfusates in the DRN were switched to MRS containing lurasidone (1 or 3 µM), 5-HT1AR agonist, 1-  Table 1). The perfusates in the MDTN and mPFC were remained MRS during experiment.
Perfusions with 1 µM AS19 into the DRN did not affect extracellular levels

Effects of Local Administrations of BP554, SB269970, and Muscimol in the DRN on Changes in Extracellular Levels of 5-HT in the DRN, MDTN, mPFC and GABA in the DRN (Study_5)
Study_5 was designed to determine the effects of local administrations of agents of 5-HT1AR, 5-HT7R, GABAA-R and lurasidone into the DRN on MK801-induced 5-HT release and MK801-induced GABA reductions ( Figure 6). After study_3 or Study_4, perfusion media in the DRN were switched to the same MRS containing with 5 μM MK801 for 180 min ( Figure 6 and Table 1

Effects of Local Administration of MK801, AS19 and BP554 in the DRN on 5-HT Release in the DRN, MDTN, mPFC, and GABA in the DRN After Sub-Chronic Administration of Lurasidone (Study_6)
Study_6 was designed to determine the effects of systemically sub-chronic administration of effective does of lurasidone (3 mg/kg/day for 7 days) [11] on 5 µM MK801-, 50 µM BP554-and 1 µM AS19-induced release of 5-HT and GABA (Figure 7 and Table 1). After sub-chronic systemic administration of effective doses of lurasidone (3 mg/kg/day) for 7 days using an osmotic pumps (2ML_1; Alzet, Cupertino, CA) implanted subcutaneously in the dorsal region (N = 24), microdialysis probes were implanted. Perfusion experiments were initiated at 18 h after recovery from isoflurane anesthesia (36 h after withdraw from sub-chronic lurasidone treatments). A previous study of pharmacokinetics in rats reported a half-life (T 1/2 ) of lurasidone of 6-9 h [23]. According to this evidence, we started perfusions at 36 h after withdraw from sub-chronic lurasidone treatments, and avoided the effects of residual lurasidone. Perfusions into the DRN, MDTN and mPFC were started with MRS. After confirming that 5-HT and GABA levels were stable in perfusates, perfusates in the DRN were switched to MRS containing 5 µM MK801, 50 µM BP554, or 1 µM AS19 for 180 min (Figure 7). The perfusates in the MDTN and mPFC were remained MRS during experiment.

Regulatory Mechanisms of Serotonergic Transmission Associated with the DRN
Serotonergic modulations, including 5-HT release, receptors, transporters, and enzymes, have well established roles in the pathophysiology of schizophrenia, mood disorders, and anxiety disorders [1][2][3][4]11,18,22,24,25]. The DRN is a major serotonergic nucleus that projects terminals to The inhibitory effects of perfusions with 50 µM BP554 into the DRN on extracellular 5-HT levels in the DRN, MDTN and mPFC ( Figure 4) were abolished by sub-chronic treatments with effective doses of lurasidone (3 mg/kg/day for 7 days; Figure 7A,C-E,G,H). Similar to BP554, the stimulatory effects of perfusion with 1 µM AS19 into the DRN on regional extracellular GABA levels ( Figure 4B,F) were abolished by sub-chronic treatments with effective doses of lurasidone ( Figure 7B,F). Thus, sub-chronic administration of effective doses of lurasidone may desensitize or downregulate 5-HT1AR and 5-HT7R. Contrary

Regulatory Mechanisms of Serotonergic Transmission Associated with the DRN
Serotonergic modulations, including 5-HT release, receptors, transporters, and enzymes, have well established roles in the pathophysiology of schizophrenia, mood disorders, and anxiety disorders [1][2][3][4]11,18,22,24,25]. The DRN is a major serotonergic nucleus that projects terminals to various brain regions, including the mPFC and MDTN [26,27] (Figure 8). Therefore, activation of serotonergic transmission from the DRN is considered fundamental to the pathophysiology of cognitive impairments and mood disturbances [1][2][3][4]11,18,22,24,25]. hypothesis for neural circuitry in the interaction between serotonergic and GABAergic transmissions in the DRN is represented in Figure 8. Herein, we demonstrate that 5-HT release in the DRN is regulated by inhibitory 5-HT1AR and GABAA-R predominantly in the DRN, as indicated by decreased regional 5-HT release following local perfusions with BP554 (5-HT1AR agonist) and muscimol (GABAA-R agonist) into the DRN ( Figure   Figure 8. Proposed hypothesis for the extended neural circuitry involved in the DRN, MDTN and mPFC. Our proposed hypothesis for the extended neural circuitry involved in the transmissions of DRN-MDTN and the DRN-mPFC pathways. Serotonergic neurons in the DRN (green circle), which receives intra-DRN GABAergic inhibition, project terminals to the MDTN and deeper layers in the mPFC. GABAergic neurons in the DRN are predominantly regulated by excitatory 5-HT7R and NMDA-R rather than inhibitory 5-HT1AR. In contrast, serotonergic neurons in the DRN are regulated by inhibitory 5-HT1AR rather than excitatory 5-HT7R and NMDA-R. MK801 inhibits NMDA-R on the GABAergic neurons resulting in increase in 5-HT releases in the DRN, MDTN and mPFC; however, lurasidone acutely also enhances GABAergic disinhibition via its 5-HT7R antagonism but inhibits serotonergic neuronal activity via its 5-HT1AR agonism in the DRN. Contrary to acute treatment, lurasidone chronically, enhances 5-HT release via desensitization of 5-HT1AR and 5-HT7R in the DRN. The DRN comprises heterogeneous serotonergic, GABAergic, and glutamatergic neurons [4,19], which may correspond with a variety of physiological and pathological serotonergic functions. This heterogeneous organization complicates the understanding of regulatory mechanisms of serotonergic transmission in the DRN. Therefore, understanding of such properties of GABAergic and serotonergic neuronal activities in the DRN may reveal various functions of the DRN. Our hypothesis for neural circuitry in the interaction between serotonergic and GABAergic transmissions in the DRN is represented in Figure 8.
Herein, we demonstrate that 5-HT release in the DRN is regulated by inhibitory 5-HT1AR and GABA A -R predominantly in the DRN, as indicated by decreased regional 5-HT release following local perfusions with BP554 (5-HT1AR agonist) and muscimol (GABA A -R agonist) into the DRN (Figure 4). Contrary to 5-HT, GABA release in the DRN is regulated more by excitatory 5-HT7R than 5-HT1AR in the DRN, reflecting decreased regional GABA release following local perfusions with SB269970 (5-HT7R antagonist) into the DRN (Figure 4).
Previous electrophysiological studies show that inhibitory 5-HT1AR and excitatory 5-HT2AR responses in serotonergic neurons were more than 90% and lower than 20% in the DRN, respectively [19]. In contrast with serotonergic neurons, inhibitory and excitatory 5-HT responses in GABAergic neurons were 15% and 80% in the DRN, respectively, likely relating to 5-HT7R excitatory responses [19]. Taken with these reports, our data suggest that 5-HT1AR and 5-HT7R predominantly inhibit serotonergic neurons and excite GABAergic neurons, respectively.
Feedback inhibition via somatodendritic 5-HT1AR autoreceptor on serotonergic neurons in the DRN plays an important role in the regulation of 5-HT-mediated neuronal activity [18,22,28]. It has been established that GABAergic transmission in the DRN is also an important regulatory mechanism for controlling serotonergic neuronal activity [28]. Therefore, increased 5-HT release in the DRN enhances negative feedback for serotonergic transmission by activating 5-HT1AR, which directly inhibits serotonergic neuronal activity, and 5-HT7R, which generates GABAergic disinhibition. Furthermore, the negative feedback mechanisms between serotonergic and GABAergic interactions are phasic with each other.
5-HT1AR exists as functionally two sub-types, autoreceptor and heteroreceptor. Autoreceptors are expressed on the somatodendritic regions of serotonergic neurons in the DRN, which inhibits neuronal activity resulting in reduction of 5-HT release in terminal regions [35]. Contrary, heteroreceptors are expressed on postsynaptic regions in glutamatergic and GABAergic neurons in the cortex and limbic system [35]. The present study chronic lurasidone administration generated autoreceptor desensitization resulting in enhancement of serotonergic transmission from the DRN to mPFC and MDTN. The desensitization of heteroreceptor on the glutamatergic and GABAergic neurons in the mPFC probably activates excitatory glutamatergic and inhibitory GABAergic transmission. The present study demonstrated that the bottom-up regulation (from DRN to mPFC) of lurasidone on serotonergic transmission, whereas the detailed study to determine the top-down regulation (from mPFC to DRN) of lurasidone must be needed. 5-HT2AR antagonism is considered to be one of the major mechanisms of lurasidone (Ki = 2.0 nM) [31], similar to other atypical antipsychotics, such as risperidone, blonanserine, clozapine, zotepine and quetiapine [5,6,10,12]. Previous electrophysiological study demonstrated that 5-HT2AR probably activated both serotonergic and GABAergic neuronal activities in the DRN [19]. Therefore, the effects of 5-HT2AR antagonism on mesocortical and mesothalamic serotonergic transmission is probably more complex rather than those of 5-HT1AR and 5-HT7R, since 5-HT2AR blockade generates GABAergic disinhibition but at the same time prevention of serotonergic activities. We shall the effects of acute and chronic administration of lurasidone mesocortical and mesothalamic serotonergic transmission associated with 5-HT2AR.

5-HT7R-Assoicated Mechanisms of Lurasidone
Attenuation of 5-HT7R activities may induce and accelerate antidepressant actions. Recent preclinical studies show that inhibition of 5-HT7R leads to rapid, robust, and sustained antidepressant effects, representing a significant breakthrough in psychopharmacology. Behavioral studies also demonstrated that subeffective doses of SB269970 enhanced the antidepressive actions of several antidepressants [4,16]. Esketamine was approved by the FDA in early 2019 as a rapid-acting antidepressant for the treatment of major depressive disorders [36]. Monoaminergic antidepressants have delayed onset of antidepressive actions and take up to several weeks to exert their salutary effects. These types of antidepressants can improve depressive symptoms, but their ability to successfully reduce suicide ideation and behavior remains inconclusive [37]. In contrast, single doses of ketamine have superior response rates within hours to days of administration [38]. Similarly, single doses (0.5 mg/kg) of intravenous ketamine exert rapid and profound antidepressant effects with reduced suicidal ideation within hours to days of administration [38,39]. Although well-tolerated at sub-anesthetic (antidepressive) doses, ketamine retains its ability to promote abuse and accelerate acute psychotic episodes.
Various targets have been reported for ketamine, although inhibition of NMDA-R is considered the main pharmacological target [40,41]. Inhibition of NMDA-R activates glutamatergic transmission via GABAergic disinhibition in the cortex and in sub-cortical regions [5,6,[9][10][11][12]42,43]. In our hands, MK801 (NMDA-R antagonist) drastically increased 5-HT release via GABAergic disinhibition in the DRN. Conversely, the 5-HT7R antagonists lurasidone and SB269970 induced GABAergic disinhibition, but did not increase 5-HT release in the presence of functional 5-HT1AR. Following inhibition of 5-HT1AR in the DRN, both SB269970 and lurasidone increased 5-HT release in the DRN, MDTN and mPFC. Although both MK801 and 5-HT7R antagonists exhibit rapid-onset antidepressive effects, their actions on 5-HT release differ. These differences between the effects of MK801 and lurasidone on serotonergic transmission suggest that GABAergic disinhibition is a candidate mechanism for rapid-acting antidepressive actions rather than enhancement of serotonergic transmission.
The pharmacological properties of 5-HT7R have been described [44], but whereas SB269970 acts as a 5-HT7R antagonist, chronic administration of SB269970 desensitizes 5-HT7R and fails to downregulate its expression [44]. We also demonstrate that sub-chronic administration of effective doses of lurasidone desensitized 5-HT7R as shown for SB269970 [44]. Therefore, we suggest that lurasidone is an inverse agonist of 5-HT7R. In agreement, desensitization of 5-HT1AR and 5-HT7R following sub-chronic treatments with lurasidone inhibited their direct and indirect inhibitory (modulated by GABAergic disinhibition) actions on 5-HT release in the DRN, respectively. Accordingly, sub-chronic lurasidone administration enhanced MK801-induced 5-HT release in the DRN, MDTN and mPFC.
In recent reports, hyperactivation of thalamocortical glutamatergic transmission contributed to the cognitive impairments of several psychotic disorders [9,11,12,42,43,45]. Under physiological conditions, the MDTN regulates various cognitive processes, and enhanced sensitivity and reliability of MDTN signaling contributed to changes in flexibility and stability against environmental changes [45]. Hence, severe GABAergic disinhibition from the reticular thalamic nucleus following attenuation of NMDA-R in the MDTN probably sustains hyperactivity of MDTN glutamatergic activities, resulting in relative deterioration of sensitivity to signals from other regions and leading to functionally similar conditions to those following disruption of MDTN activity [46]. Inhibition of 5-HT7R in the MDTN normalized the hyperactivation of glutamatergic neurons in the MDTN. Although this is a reasonable outcome of antipsychotics, the effects of lurasidone on serotonergic transmission from the DRN to the MDTN remain unknown [11]. We also show that neither subeffective nor effective doses of lurasidone acutely affected 5-HT release in the MDTN. Therefore, taken with previous findings [11], this study suggests that lurasidone normalizes hyperactivation of glutamatergic neuronal hyperactivities in the MDTN by directly inhibiting 5-HT7R in the MDTN without affecting DRN-MDTN serotonergic transmission. Contrary to the known acute effects of lurasidone, chronic administration of lurasidone may activate DRN-MDTN serotonergic transmission, but may also lead to excitatory 5-HT7R desensitization. Alternatively, both acute and chronic treatments with lurasidone could normalize hyperactivated thalamocortical glutamatergic transmission via inverse 5-HT7R agonistic actions. To clarify this hypothesis, further studies of the chronic effects of lurasidone on MK801-induced thalamocortical glutamatergic transmission are awaited.

Preparation of the Microdialysis System
All animal care and experimental procedures were performed in compliance with the ethical guidelines established by the Institutional Animal Care and Use Committee at Mie University (No. [29][30]. All studies involving animals are reported in accordance with the relevant ARRIVE guidelines [47] and European Union Council (2010/63/EU). A total of 132 rats were used in experiments.
All rats were weighed before the study. Rats were anesthetized with 1.8% isoflurane and were then placed in a stereotaxic frame for implantation of dialysis probes [5,7]. Concentric direct insertion-type dialysis probes (0.22 mm diameter; Eicom, Kyoto, Japan) were implanted in the mPFC (3 mm exposed membrane; A = +3.2 mm, L = +0.8 mm, V = −5.2 mm, relative to the bregma), MDTN (2 mm exposed membrane; A = −3.0 mm, L = +0.9 mm, V = −6.2 mm, relative to the bregma at a lateral angle of 30 • ) and DRN (1 mm exposed membrane; A = −8.2 mm, L = 0.2 mm, V = −6.8 mm, relative to the bregma at a lateral angle of 15 • ) [48]. During recovery and experimentation, rats were housed individually in cages and were provided food and water ad libitum. Perfusion experiments were initiated at 18 h after recovery from isoflurane anesthesia [8,49,50]. During experiments, single rats were placed in an in vivo dialysis system for freely moving animals (Eicom) equipped with a two-channel swivel (TCS2-23; ALS, Tokyo, Japan). The perfusion rate was set at 2 µL/min in all experiments using modified Ringer's solution (MRS, composition described below) [7,50], and dialysates were collected over 20 min sampling epochs. Extracellular l-glutamate levels were measured at 8 h after the start of perfusions. After baseline recording, perfusion medium was switched to MRS containing MK801, WAY100635, BP554, AS19, muscimol, SB269970 or lurasidone as indicated. Dialysate samples were then injected into the UHPLC apparatus. All samples were taken from freely moving animals.
After microdialysis experiments, brains were removed following cervical dislocation and overdose isoflurane anesthesia. The locations of the dialysis probes were verified in each animal using histological examinations of 200 µm thick brain tissue slices, which were prepared using a Vibratome 1000 (Technical Products International Inc., St. Louis, MO).

Determination of Extracellular GABA and 5-HT Levels
GABA concentrations in the perfusate (µM) were determined using UHPLC (xLC3185PU; Jasco) with fluorescence resonance energy transfer detection (xLC3120FP; Jasco, Tokyo, Japan) after dual derivatization with isobutyryl-l-cysteine and o-phthalaldehyde. Derivative reagent solutions were prepared by dissolving isobutyryl-l-cysteine (2 mg) or o-phthalaldehyde (1 mg) in 0.1 mL aliquots of ethanol, followed by addition of 0.9 mL of sodium borate buffer (0.2 M, pH 9.0) [50]. Automated precolumn derivation was conducted by mixing 5 µL sample, standard, or blank solutions with 5 µL of derivative reagent solution in reaction vials for 5 min before injection. Derivative samples (5 µL) were injected using an autosampler (xLC3059AS; Jasco). The analytical column (YMC Triart C18, particle 1.8 µm, 50 × 2.1 mm; YMC, Kyoto, Japan) was maintained at 45 • C. The flow rate was set at 500 µL/min, and elution was performed using a linear gradient of mobile phases A (0.05 M acetate buffer, pH 5.0) and B (0.05 M acetate buffer containing 60% acetonitrile, pH 3.5) over 10 min [51]. Excitation and emission wavelengths of the fluorescence detector were set at 280 and 455 nm, respectively.
Concentrations of 5-HT in the perfusate (nM) were determined using UHPLC (xLC3185PU; Jasco) with electrochemical detection (ECD-300; Eicom, Kyoto, Japan) by a graphite carbon electrode set at +450 mV (vs. a Ag/AgCl reference electrode [49,52]. The analytical column (Triart C18, particle 1.8 µm, 30 × 2.1 mm; YMC) was maintained at 40 • C and the flow rate of the mobile phase was set at 400 µL/min. The mobile phase contained 0.1 M acetate buffer, 1% methanol, and 50 mg/L EDTA-2Na (final pH 6.0) [53,54]. Where possible, we randomized and blinded sample data. In particular, for determinations of extracellular transmitter levels, the sample order was dictated by the autosampler according to a random number table.

Data Analysis
Where possible, we randomized and blinded sample data. To determine extracellular transmitter levels, the sample order was set on the autosampler according to a random number table. Drug doses and sample sizes were selected according to previous studies [11,42,45]. All experiments in this study were designed with equally sized animal groups (N = 6) [9,11,42,43,45] and all values were expressed as means ± standard deviations (SD). Differences were considered significant when P < 0.05 (two-tailed).
Regional transmitter concentrations were analyzed using Mauchly's sphericity test followed by multivariate analysis of variance (MANOVA) using BellCurve for Excel ver. 3.20 (Social Survey Research Information Co., Ltd., Tokyo, Japan). The data composed of average of pre-treatment period (1 point) and each time point during target agent administration (9 points). When the data did not violate the assumption of sphericity (P > 0.05), the F-value of MANOVA was analyzed using sphericity-assumed degrees of freedom. When the assumption of sphericity was violated (P < 0.05), F-values were analyzed using Chi-Muller's corrected degrees of freedom by BellCurve for Excel. When F-values for drug factors were significant in MANOVA, the data were finally analyzed using Tukey's post hoc test with BellCurve for Excel. Transmitter levels were expressed as the area under the curve between 20 and 180 min (AUC20-180 min) after target agents administration. All statistical analyses complied with the recommendations for experimental design and analysis in pharmacology [55].
All compounds were prepared on the day of experiments. All drugs were perfused in MRS containing 145 mM Na + , 2.7 mM K + , 1.2 mM Ca 2+ , 1.0 mM Mg 2+ , and 154.4 mM Cl − , which was adjusted to pH 7.4 using 2 mM phosphate buffer and 1.1 mM Tris buffer [7,50]. Muscimol, MK-801, WAY100635, and SB269970 were dissolved in MRS directly, whereas BP554 and AS19 were initially dissolved in dimethyl sulfoxide at 50 mM. Lurasidone was initially dissolved in dimethyl sulfoxide at 1 mg/mL. The final dimethyl sulfoxide concentration was lower than 0.1% (vol/vol).

Conclusions
In the present study, we demonstrated interactions between 5-HT1AR and 5-HT7R that are relevant to serotonergic and GABAergic transmission and explored the mechanisms behind the antipsychotic and mood stabilizing actions of lurasidone. Serotonergic and GABAergic neuronal activities in the DRN were predominantly regulated by inhibitory 5-HT1AR and excitatory 5-HT7R responses, respectively. 5-HT7R antagonism of lurasidone generates GABAergic disinhibition, but partial agonism of 5-HT1AR by lurasidone prevents 5-HT release, resulting in no net changes in basal 5-HT release. Failure to increase 5-HT release in the MDTN and the mPFC reflects the interaction between 5-HT1AR partial agonism and 5-HT7R antagonism by lurasidone. Furthermore, 5-HT7R antagonism inhibits NMDA-R-antagonist induced thalamocortical glutamatergic transmission, but enhances serotonergic transmission via GABAergic disinhibition in the DRN. Finally, lurasidone acutely inhibits 5-HT release following NMDA-R inhibition by acting as a 5-HT1AR agonist, but chronically activates NMDA-R antagonist induced serotonergic transmission by desensitizing 5-HT1AR and 5-HT7R. These interactions between 5-HT1AR agonism and 5-HT7R antagonism of lurasidone probably contribute to the preclinically observed rapid-action and augmentation of antidepressive actions by lurasidone.