Interaction Between Mesocortical and Mesothalamic Catecholaminergic Transmissions Associated with NMDA Receptor in the Locus Coeruleus

biomolecules

Article Interaction between Mesocortical and Mesothalamic Catecholaminergic Transmissions Associated with NMDA Receptor in the Locus Coeruleus

Motohiro Okada * and Kouji Fukuyama

Department of Neuropsychiatry, Division of Neuroscience, Graduate School of Medicine, Mie University, Tsu 514-8507, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-59-231-5018

Received: 22 May 2020; Accepted: 30 June 2020; Published: 1 July 2020

Abstract: Noncompetitive N-methyl-D-aspartate/glutamate receptor (NMDAR) antagonists contribute to the pathophysiology of schizophrenia and mood disorders but improve monoaminergic antidepressant-resistant mood disorder and suicidal ideation. The mechanisms of the double-edged sword clinical action of NMDAR antagonists remained to be clariﬁed. The present study determined the interaction between the NMDAR antagonist (MK801), α1 adrenoceptor antagonist (prazosin), and α2A adrenoceptor agonist (guanfacine) on mesocortical and mesothalamic catecholaminergic transmission, and thalamocortical glutamatergic transmission using multiprobe microdialysis. The inhibition of NMDAR in the locus coeruleus (LC) by local MK801 administration enhanced both the mesocortical noradrenergic and catecholaminergic coreleasing (norepinephrine and dopamine) transmissions. The mesothalamic noradrenergic transmission was also enhanced by local MK801 administration in the LC. These mesocortical and mesothalamic transmissions were activated by intra-LC disinhibition of transmission of γ-aminobutyric acid (GABA) via NMDAR inhibition. Contrastingly, activated mesothalamic noradrenergic transmission by MK801 enhanced intrathalamic GABAergic inhibition via the α1 adrenoceptor, resulting in the suppression of thalamocortical glutamatergic transmission. The thalamocortical glutamatergic terminal stimulated the presynaptically mesocortical catecholaminergic coreleasing terminal in the superﬁcial cortical layers, but did not have contact with the mesocortical selective noradrenergic terminal (which projected terminals to deeper cortical layers). Furthermore, the α2A adrenoceptor suppressed the mesocortical and mesothalamic noradrenergic transmissions somatodendritically in the LC and presynaptically/somatodendritically in the reticular thalamic nucleus (RTN). These discrepancies between the noradrenergic and catecholaminergic transmissions in the mesocortical and mesothalamic pathways probably constitute the double-edged sword clinical action of noncompetitive NMDAR antagonists.

Keywords: N-methyl-D-aspartate; schizophrenia; mood disorder; L-glutamate; GABA; catecholamine

1. Introduction Recently, the impact of N-methyl-D-aspartate/glutamate receptor (NMDAR) modulation has been a fundamental psychopharmacological discussion, since (S)-ketamine was approved by the Food and Drug Administration (FDA) in early 2019 as a rapid-acting antidepressant for the treatment of major depressive disorders [1]. In particular, monoaminergic antidepressants exhibit limited eﬀectiveness against the suicidal ideation of mood disorders, whereas ketamine has also been shown to have distinct and independent anti-suicidal eﬀects in patients with mood disorders [2,3]. Therefore, ketamine can improve monoaminergic antidepressant-resistant cognitive dysfunction in mood disorders. Contrary

Biomolecules 2020, 10, 990; doi:10.3390/biom10070990 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 990 2 of 19 to these clinical advantages of ketamine, numerous clinical and preclinical studies have reported that noncompetitive NMDAR antagonists, such as phencyclidine, ketamine, and dizocilpine (MK801), contribute to the pathophysiology of schizophrenia, as NMDAR antagonists generate schizophrenia-like positive–negative symptoms and cognitive impairments in healthy individuals and experimental animal models, as well the exacerbation of psychotic symptoms of patients with schizophrenia [4–13]. Acute ketamine administration rapidly improves several symptoms in mood disorders, but reduces attention [14,15]. It has been considered that noradrenergic transmission associated with the locus coeruleus (LC) is one of the major fundamental neural circuits for the regulation of attention [16]. It has been well known that the major pharmacological mechanism of ketamine is the inhibition of NMDAR, whereas various other target systems have been explored in order to understand the paradoxical clinical actions of ketamine, including monoaminergic transmission [17–19]. The inhibition of the transporters of serotonin and norepinephrine provides an anti-depressive action and the improvement of dysfunctions of cognition and emotional perception in patients with major depression [20,21]. Noradrenergic signalling is also considered to be a candidate target of the mechanism of action of ketamine. Ketamine dose-dependently increases norepinephrine release in the medial prefrontal cortex, but clonidine inhibits ketamine-induced norepinephrine release via the activation of the presynaptic α2 adrenoceptor [22,23]. These findings suggest that ketamine enhances noradrenergic transmission similar to serotonin/norepinephrine transporter inhibitors. Contrary to microdialysis studies, norepinephrine transporter inhibiting antidepressants and ketamine suppress and enhance neuronal activities in the LC, respectively [24,25]. These contradictive demonstrations between electrophysiological and microdialysis studies suggest that the LC is probably one of the major target regions of the clinical action of ketamine, but the norepinephrine transporter inhibition probably cannot play an important role in the stimulatory effects of ketamine on noradrenergic transmission. Recently, we reported that the enhanced glutamatergic transmission in the thalamocortical pathway is one of the candidate responsible pathways of systemic NMDAR antagonist induced L-glutamate release in the frontal cortex [26–31]. The major thalamocortical glutamatergic pathways [32] composed of projection form the mediodorsal thalamic nucleus (MDTN) to the medial prefrontal cortex, insula, and orbitofrontal cortex (OFC) [26,27,30,31,33–39]. These thalamocortical glutamatergic pathways contribute to the constitution of cognition, and to stability and flexibility against environmental changes [26–31,33,34,40,41]. In particular, mesothalamic noradrenergic transmission plays important roles in the appropriate response of thalamocortical glutamatergic transmission to various inputs [36]. The activation of the α2A adrenoceptor in the mesothalamic noradrenergic pathway enhances intrathalamic inhibition of γ-aminobutyric acid (GABA) [36], resulting in the attenuation of thalamocortical glutamatergic transmission [26,27,30,31,36]. A clinical study using functional magnetic resonance imaging reported that a subanaesthetic dose of ketamine injection reduced connectivity between the LC and MDTN [42], consistent with the hypothesis that ketamine augments noradrenergic transmission from the LC to various target regions. LC projects selective noradrenergic and catecholamine coreleasing (norepinephrine and dopamine) terminals to the frontal cortex (mesocortical pathways) and noradrenergic terminal to the reticular thalamic nucleus (RTN) (mesothalamic pathway) [36–38,43]. The thalamocortical glutamatergic pathway receives GABAergic inhibition from the RTN, and projects terminals to superficial layers in the frontal cortex [27,29,31]. The thalamocortical glutamatergic terminal stimulates mesocortical catecholaminergic coreleasing terminals in the superficial layers of the frontal cortex via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/glutamate receptor (AMPAR), but does not make contact with the mesocortical selective noradrenergic terminals [30,31,37,38]. These clinical and preclinical findings indicate that the interaction between the mesocortical and mesothalamic noradrenergic/catecholaminergic transmissions on thalamocortical glutamatergic transmission contributes to the double-edged sword clinical action of the NMDAR antagonist on mood and cognition. Therefore, according to our hypothesis, the present study determines the effects of MK801, α1 adrenoceptor antagonist (prazosin), and α2A adrenoceptor agonist (guanfacine) on Biomolecules 2020, 10, 990 3 of 19 mesocortical and mesothalamic noradrenergic/catecholaminergic transmissions and thalamocortical glutamatergic transmission using multiprobe microdialysis.

2. Materials and Methods

2.1. Experimental Animals The animal care and experimental procedures in this report were performed in compliance with the ethical guidelines established by the Institutional Animal Care and Use Committee at Mie University (no. 29-22-R2). All of the studies involving animals were reported in accordance with the relevant ARRIVE guidelines [44]. A total of 92 male Sprague-Dawley rats (approximately 250 g, 7 8 weeks − old, SLC, Shizuoka, Japan) were used in this study, and were maintained in a controlled environment (22 C 1 C) with a 12-h light/12-h dark cycle. ◦ ± ◦ 2.2. Chemical Agents

Dizocilpine (MK801; noncompetitive NMDAR antagonist), muscimol (MUS; GABAA receptor agonist), and prazosin (PRZ; α1 adrenoceptor antagonist) was obtained from Fujifilm-Wako (Osaka, Japan). Guanfacine (GUNA; α2A adrenoceptor agonist) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; AMPAR agonist) were obtained from Cosmo Bio (Tokyo, Japan). Each compound was prepared on the day of the experiments. These drugs were perfused in modified Ringer’s solution (MRS) containing 145 mM Na+, 2.7 mM K+, 2+ 2+ 1.2 mM Ca , 1.0 mM Mg , and 154.4 mM Cl−, which was adjusted to pH 7.4 using 2 mM phosphate buffer and 1.1 mM Tris buffer [30,31]. MK801, GUNA, MUS, and AMPA were dissolved in MRS directly. PRZ was initially dissolved at a concentration of 25 mM in dimethyl sulfoxide. According to previous reports, in the present study, to study the effects of MK801, MUS, PRZ, and GUNA, each rat was administered with MK801 (1 and 5 µM) [26,27,29–31], MUS (1 µM) [37,38], AMPA (100 µM) [30,31,33,36], PRZ (100 µM) [43], and GUNA (0.2 µM) [36] into the LC, MDTN or RTN.

2.3. Microdialysis System The rats were anesthetized with 1.8% isoflurane and were then placed in a stereotaxic frame for the implantation of dialysis probes [38,45]. Concentric direct insertion type dialysis probes (0.22 mm diameter; Eicom, Kyoto, Japan) were implanted into the orbitofrontal cortex (OFC; A = +3.2 mm, L = +2.4 mm, V = 6.5 mm, relative to bregma; 0.22 mm diameter, 2 mm exposed membrane; Eicom), − mediodorsal thalamic nucleus (MDTN; A = 3.0 mm, L = +0.6 mm, V = 6.4 mm, relative to bregma; − − 0.22 mm diameter, 2 mm exposed membrane; Eicom) at a lateral angle of 30◦, reticular thalamic nucleus (RTN: A = 1.4 mm, L = +1.2 mm, V = 7.2 mm, relative to bregma) at a lateral angle of 30 , or locus − − ◦ coeruleus (LC: A = 9.7 mm, L = +1.3 mm, V = 7.6 mm, relative to bregma; 0.22 mm diameter, 1 mm − − exposed membrane; Eicom) [31,34,36,40,41,46]. During recovery and experimentation, the rats were housed individually in cages and were provided food and water ad libitum. The perfusion experiments were initiated 18-h after recovery from isoflurane anaesthesia [47–49]. During the experiments, single rats were placed in an in vivo microdialysis system for freely moving rats (Eicom, Kyoto, Japan), equipped with a two-channel swivel (TCS2-23; ALS, Tokyo, Japan). The perfusion rate was set at 2 µL/min in all of the experiments, using modified Ringer’s solution (MRS; composition described above) [47,50], and dialysates were collected over 20 min sampling epochs. The extracellular levels of norepinephrine, dopamine, L-glutamate, and GABA were measured 8 h after the start of the perfusions. After baseline recording, the perfusion medium was replaced with MRS containing MK801, PRZ, GUNA, or AMPA, as indicated. The dialysate samples were then injected into the ultra-high-performance liquid chromatography (UHPLC) apparatus. All of the samples were taken from freely moving animals. After the microdialysis studies, the rat brains were removed following isoflurane overdose anesthesia. The locations of the dialysis probes were checked in each animal using histological Biomolecules 2020, 10, 990 4 of 19 examinations of 200 µm thick brain slices with a Vibratome 1000 (Technical Products International Inc., St. Louis, MO, USA).

2.4. Determination of Extracellular Levels of L-Glutamate, GABA, Dopamine and Norepinephrine The extracellular levels of L-glutamate and GABA in the perfusate were analysed by UHPLC (xLC3185PU; Jasco, Tokyo, Japan) with fluorescence resonance energy transfer detection (xLC3120FP; Jasco) after dual derivatization with isobutyryl-l-cysteine and o-phthalaldehyde. The derivative reagent solutions were prepared by dissolving isobutyryl-l-cysteine (2 mg) or o-phthalaldehyde (1 mg) in 0.1 mL ethanol, followed by the addition of 0.9 mL of sodium borate buffer (0.2 M, pH 9.0) [30,31,47,50]. The automated precolumn derivation was performed by mixing 5 µL of sample, standard, or blank solutions with 5 µL of derivative reagent solution in reaction vials for 5 min before injection (xLC3059AS; Jasco). Derivative samples (5 µL) were injected using an autosampler (xLC3059AS; Jasco). The flow rate was set at 0.5 mL/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 [30,31,47,50]. The excitation and emission wavelengths of the fluorescence detector were set at 280 and 455 nm, respectively. The analytical column (YMC Triart C18, particle 1.8 µm, 50 2.1 mm; × YMC, Kyoto, Japan) was maintained at 45 ◦C. The concentrations of dopamine and norepinephrine were determined using UHPLC (xLC3185PU; Jasco) with an electrochemical detector (ECD-300; Eicom) by a graphite carbon electrode set at +450 mV (vs. an Ag/AgCl reference electrode) [30,31,47,50]. 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 ethylenediaminetetraacetic acid (EDTA)-2Na (final pH 6.0) [51].

2.5. Data Analysis To determine the extracellular transmitter levels, the sample order was set on the autosampler according to a random number table. The drug levels and sample sizes of each experimental group were selected according to previous studies [26–31,36]. All of the experiments in this study were designed with equivalent sized groups (n = 6), without carrying out a formal power analysis [26–31,36], and all of the data were expressed as mean standard deviation (SD), and p < 0.05 (two-tailed) was ± considered statistically signiﬁcant. The drug concentration of the acute local administration (perfusion) was selected based on previous studies. Levels of the transmitter were analysed by Mauchly’s sphericity test, followed by multivariate analysis of variance (MANOVA) (BellCurve for Excel v. 3.2, Social Survey Research Information Co., Ltd., Tokyo, Japan). If data did not violate the assumption of sphericity (p > 0.05), the F-value of MANOVA was analysed by sphericity assumed degrees of freedom. Contrary, if the assumption of sphericity was violated (p < 0.05), the F-value was analysed by Chi–Muller’s corrected degrees of freedom. When the F-value for the drug*time factors of MANOVA was signiﬁcant, the data was analysed by Tukey’s post hoc test.

3. Results

3.1. Effects of NMDAR in the LC on Transmitter Release in the OFC, RTN and MDTN (Study_1) To explore the threshold level of local administration (perfusion with) of MK801 into the LC, Study_1 was designed to determine the effects of perfusion with 1 µM and 5 µM MK801 into the LC on the extracellular levels of GABA in the LC and MDTN, and extracellular levels of norepinephrine and dopamine in the OFC and RTN (MK801-induced release). Additionally, to clarify the mechanisms of MK801-induced releases of norepinephrine, dopamine and GABA, the effects of perfusion with 100 µM PRZ (α1 adrenoceptor antagonist) or 0.2 µM GUNA (α2A adrenoceptor agonist) into the LC Biomolecules 2020, 10, 990 5 of 19 on MK801-induced releases of norepinephrine and dopamine in the OFC and RTN, and GABA release in the LC and MDTN were determined. BiomoleculesPerfusion 2020 mediums, 10, 990 in the LC was commenced with MRS containing without (control)5 orof with19 100 µM PRZ or 0.2 µM GUNA. After the stabilisation of transmitter level in perfusate, perfusion mediumPerfusion in the LC mediums was switched in the LC to was the samecommenced MRS containing with MRS containing with MK801 without (1 or 5(control)µM) for or 180 with min. Perfusion100 μM mediumPRZ or 0.2 in theμM OFC,GUNA. RTN After and the MDTN stabilisation were maintained of transmitter with level MRS in alone perfusate, during perfusion Study_1. medium in the LC was switched to the same MRS containing with MK801 (1 or 5 μM) for 180 min. 3.1.1.Perfusion Effects medium of Local in Administration the OFC, RTN and of PRZ MDTN and were GUNA maintained into the LCwith on MRS MK801-Induced alone during Study_1. Releases of Norepinephrine and Dopamine in the OFC 3.1.1. Effects of Local Administration of PRZ and GUNA into the LC on MK801-Induced Releases of µ NorepinephrinePerfusions with and MK801 Dopamine (1 and in 5 theM) OFC into the LC concentration-dependently increased extracellular norepinephrine level in the OFC [FMK801 (2,15) = 29.7 (p < 0.01), FTime (4.0, 60.4) = 95.9 (p < 0.01), Perfusions with MK801 (1 and 5 μM) into the LC concentration-dependently increased FMK801*Time (8.1, 60.4) = 33.6 (p < 0.01)] (Figure1A,C). Perfusion with 100 µM PRZ (α1 adrenoceptor antagonist)extracellular into nor theepinephrine LC affected level neitherin the OFC basal [FMK801 nor (2,15) MK801-induced = 29.7 (p < 0.01) (5, FµTimeM (4.0, MK801 60.4) into = 95.9 the (p < LC) 0.01), FMK801*Time (8.1, 60.4) = 33.6 (p < 0.01)] (Figure 1A,C). Perfusion with 100 μM PRZ (α1 adrenoceptor norepinephrine releases in the (Figure1A,C). Perfusion with 0.2 µM GUNA (α2A adrenoceptor antagonist) into the LC affected neither basal nor MK801-induced (5 μM MK801 into the LC) agonist) into the LC decreased both basal and MK801-induced norepinephrine release in the OFC norepinephrine releases in the (Figure 1A,C). Perfusion with 0.2 μM GUNA (α2A adrenoceptor [F (2,15) = 29.3 (p < 0.01), F (5.0, 75.0) = 104.1 (p < 0.01), F (10.0, 75.0) = 33.3 (p < 0.01)] GUNAagonist) into the LC decreasedTime both basal and MK801-induced GUNA*Timenorepinephrine release in the OFC (Figure1A,C). Perfusions with MK801 (1 and 5 µM) into the LC concentration-dependently increased [FGUNA (2,15) = 29.3 (p < 0.01), FTime (5.0, 75.0) = 104.1 (p < 0.01), FGUNA*Time (10.0, 75.0) = 33.3 (p < 0.01)] extracellular(Figure 1A, dopamineC). Perfusions level with in theMK801 OFC (1 [F andMK801 5 μM)(2,15) into= the9.0 LC (p

Figure 1. Eﬀects of local administration of prazosin (PRZ) and guanfacine (GUNA) into the locus Figure 1. Effects of local administration of prazosin (PRZ) and guanfacine (GUNA) into the locus coeruleus (LC) on dizocilpine (MK801)-induced releases of norepinephrine (A) and dopamine (B) in coeruleus (LC) on dizocilpine (MK801)-induced releases of norepinephrine (A) and dopamine (B) in the orbitofrontal cortex (OFC). Ordinates: mean standard deviation (SD) (n = 6) of extracellular the orbitofrontal cortex (OFC). Ordinates: mean ± standard deviation (SD) (n = 6) of extracellular catecholaminecatecholamine levels levels (nM); (nM); abscissa: abscissa: time time after after MK801 administration (min). (min). Black Black bars bars indicate indicate perfusionperfusion with with 100 100µ MμM prazosin prazosin (PRZ) (PRZ) oror 0.20.2 µμMM guanfacine (GUNA) (GUNA) into into the the LC. LC. Gray Gray bars bars indicate indicate perfusionperfusion with with 1 µ1M μM or or 5 µ5 MμM MK801 MK801 into into the the LC. LC. (C (C,D,D)) indicate indicate the the area area underunder curvecurve (AUC)(AUC) valuesvalues of extracellularof extracellular levels levels of norepinephrine of norepinephrine and and dopamine dopamine (pmol) (pmol) after after perfusion perfusion with with MK801MK801 (from(from 20 to 180to min), 180 min),based based on (A ) on and (A ()B and), respectively. (B), respectively. Gray columns Gray columns represent represent the AUC the values AUC before values perfusion before withperfusion MK801 with (basal MK801 release). (basal * p release).< 0.05, ** * pp < 0.05,0.01; ** relative p < 0.01; to relative control, to @ control,p < 0.05, @ @@p < p0.05,< 0.01; @@ p relative < 0.01; to MK801relative (5 toµM) MK801 by MANOVA (5 μM) by withMANOVA Tukey’s with post Tukey’s hoc test. post hoc test.

3.1.2. Effects of Local Administration of PRZ and GUNA into the LC on MK801-Induced Releases of Norepinephrine and Dopamine in the RTN Perfusions with MK801 (1 and 5 μM) into the LC concentration-dependently increased extracellular norepinephrine level in the RTN [FMK801 (2,15) = 17.7 (p < 0.01), FTime (3.9, 58.7) = 16.4 (p < Biomolecules 2020, 10, 990 6 of 19

3.1.2. Effects of Local Administration of PRZ and GUNA into the LC on MK801-Induced Releases of Norepinephrine and Dopamine in the RTN Perfusions with MK801 (1 and 5 µM) into the LC concentration-dependently increased extracellular Biomolecules 2020, 10, 990 6 of 19 norepinephrine level in the RTN [FMK801 (2,15) = 17.7 (p < 0.01), FTime (3.9, 58.7) = 16.4 (p < 0.01), F (7.8, 58.7) = 14.2 (p < 0.01)] (Figure2A,C). Perfusion with 5 µM MK801 into the LC increased 0.01),MK801*Time FMK801*Time (7.8, 58.7) = 14.2 (p < 0.01)] (Figure 2A,C). Perfusion with 5 μM MK801 into the LC extracellularincreased extracellular norepinephrine norepinephrine level in the level RTN, in but the 1 RTN,µM MK801 but 1 μM did MK801 not aff ectdid (Figure not affect2A,C). (Figure Perfusion 2A,C). withPerfusion 100 µ Mwith PRZ 100 into μM the PRZ LC into affected the neitherLC affected basal neither nor MK801-induced basal nor MK801 norepinephrine-induced norepinephrine release in the RTNrelease (Figure in the2 A,C).RTN Perfusion(Figure 2A,C). with 0.2PerfusiµMon GUNA with into0.2 μM the GUNA LC decreased into the both LC basaldecreased and MK801-induced both basal and norepinephrine release in the RTN [F (2,15) = 16.4 (p < 0.01), F (4.2, 62.9) = 43.8 (p < 0.01), MK801-induced norepinephrine releaseGUNA in the RTN [FGUNA (2,15) = 16.4Time (p < 0.01), FTime (4.2, 62.9) = 43.8 F (8.4, 62.9) = 13.4 (p < 0.01)] (Figure2A,C). Perfusions with MK801 (1 and 5 µM) into the LC (pGUNA*Time < 0.01), FGUNA*Time (8.4, 62.9) = 13.4 (p < 0.01)] (Figure 2A,C). Perfusions with MK801 (1 and 5 μM) didinto not the a ff LCect the didextracellular not affect the dopamine extracellular level in dopamine the RTN (Figure level 2 inB,D). the Neither RTN (Figure perfusion 2B,D). with 100 NeitherµM PRZperfusion nor 0.2 withµM 100 GUNA μM PRZ into thenor LC 0.2 aμMffected GUNA the extracellularinto the LC affected dopamine the level extracellular in the RTN dopamine (Figure2 B,D).level Therefore,in the RTN the (Figure threshold 2B,D). concentration Therefore, ofthe local threshold administration concentration of MK801 of local into administration the LC on norepinephrine of MK801 releaseinto the in LC the on RTN norepinephrine was lower than release 5 µM, in but the catecholaminergic RTN was lower neurones than 5 μM, in the but LC catecholaminergic does not project toneurones RTN. in the LC does not project to RTN.

Figure 2. Eﬀects of local administration of PRZ and GUNA into the LC on MK801-induced releases ofFigure norepinephrine 2. Effects of (Alocal) and administration dopamine (B of) in PRZ the and reticular GUNA thalamic into the nucleus LC on (RTN).MK801- Ordinates:induced release means ofSD norepinephrine (n = 6) of extracellular (A) and dopamine catecholamine (B) in the levels reticular (nM); thalamic abscissa: nucleus time after (RTN) MK801. Ordinates: administration mean ± ± (min).SD (n = Black 6) of barsextracellular indicate perfusioncatecholamine with levels 100 µM (nM); PRZ abscissa: or 0.2 µM time GUNA after into MK801 the LC. administration Gray bars indicate (min). perfusionBlack bars with indicate 1 µM perfusion and 5 µM with MK801 100 μM into PRZ the LC. or 0.2 (C, DμM) indicate GUNA theinto AUC the LC. values Gray of bars extracellular indicate levelsperfusion of norepinephrine with 1 μM and and 5 μM dopamine MK801 (pmol) into the after LC. perfusion (C,D) indicate with MK801 the AUC (from values 20 to 180of extracellular min), based onlevels (A) of and norepinephrine (B), respectively. and Gray dopamine columns (pmol) represent after theperfusion AUC values with beforeMK801 perfusion (from 20 withto 180 MK801 min), (basalbased release).on (A) and ** p (

FigureFigure 3. 3.Eﬀ Effectsects of of local local administration administration ofof PRZPRZ and GUNA into into the the LC LC on on MK801 MK801-induced-induced reduction reduction in γFigurein-aminobutyric γ-aminobutyric 3. Effects of acid acid local (GABA) (GABA) administration release release in inof the thePRZ LC.LC and. Ordinate:Ordinate: GUNA into mean the ± LCSDSD on(n (n =MK801 =6)6) of of extracellular-induced extracellular reduction GABA GABA ± levelinlevel γ (µ-aminobutyric M);(μM); abscissa: abscissa: acid time time (GABA) after after MK801 MK801release administration inadministration the LC. Ordinate: (min). (min). Blackmean Black bars± SDbars indicate(n indicate = 6) of perfusion extracellular perfusion with with GABA 100 100µ M PRZlevelμM or PRZ 0.2(μM); µorM 0.2abscissa: GUNA μM GUNA time into the afterinto LC. theMK801 GrayLC. Grayadministration bars bars indicate indicate perfusion (min). perfusion Black with bawith 1rsµ indicate1M μM and and 5perfusionµ 5M μM MK801 MK801 with into into100 the LC.μMthe (B LC.)PRZ indicates ( Bor) indicates0.2 theμM AUCGUNA the valuesAUC into values the of extracellularLC. of Gray extracellular bars GABA indicate GABA level perfusion level (nmol) (nmol) with after after1 perfusion μM perfusion and 5 withμM with MK801 MK801 MK801 into (from 20the(from to 180LC. 20 min),(B to) indicates180 based min), onthe based ( AAUC). Grayon values (A columns). Gray of extracellular columns represent represent GABA the AUC level the values (nmol)AUC values before after perfusionbefore perfusion perfusion with with MK801 MK801with (basal(fromMK801 release). 20 (basal to 180 * release).p min),< 0.05, based * **p p< <0.05,on0.01; (A **). relativepGray < 0.01; columns torelative control represent to bycontrol MANOVA the by AUC MANOVA with values Tukey’s withbefore Tukey’s post perfusion hoc post test. with hoc MK801test. (basal release). * p < 0.05, ** p < 0.01; relative to control by MANOVA with Tukey’s post hoc 3.1.4. Etest.ﬀects of Local Administration of PRZ and GUNA into the LC on MK801-Induced GABA Release3.1.4. Effects in the MDTNof Local Administration of PRZ and GUNA into the LC on MK801-Induced GABA 3.1.4.Release Effects in the of MDTN Local Administration of PRZ and GUNA into the LC on MK801-Induced GABA Perfusions with MK801 (1 and 5 µM) into the LC concentration-dependently increased extracellular Release in the MDTN GABA levelPerfusions in the withMDTN MK801 [FMK801 (1 (2,15) and 5= 6.7 μM) (p into< 0.01), the F LCTime concentration(4.7, 70.1) = 14.7-dependently (p < 0.01), increasedFMK801*Time (9.3,extracellular 70.1)Perfusions= 8.2 (GABAp < with0.01)] level MK801 (Figure in the 4 (1 A,B).MDTN and Perfusion 5[F μM)MK801 (2,15) into with the= 5 6.7µ M LC (p MK801

Figure 4. Eﬀects of local administration of PRZ and GUNA into the LC on MK801-induced GABA releaseFigure in 4. the Effects mediodorsal of local ad thalamicministration nucleus of PRZ (MDTN). and GUNA Ordinate: into the mean LC onSD MK801 (n = -6)induced of extracellular GABA ± GABAFigurerelease level 4.in Effects (theµM); mediodorsal abscissa:of local ad time thalamicministration after MK801nucleus of PRZ administration(MDTN) and GUNA. Ordinate (min).into: themean Black LC ± on barsSD MK801 (n indicate = 6)- inducedof perfusionextracellular GABA with 100releaseGABAµM PRZ levelin the or (μM 0.2mediodorsal);µ abscissa:M GUNA timethalamic into after the nucleusMK801 LC. Gray administration(MDTN) bars indicate. Ordinate (min). perfusion: mean Black ± bars withSD indicate(n 1 µ= M6) andof perfusion extracellular 5 µM MK801with intoGABA100 the μM LC.level PRZ (B (μM or) indicates 0.2); abscissa: μM GUNA the time AUC into after valuesthe MK801 LC. ofGray administration extracellular bars indicate GABA(min). perfusion Black level with bars (nmol) 1indicate μM after and perfusion perfusion5 μM MK801 with with MK801100into μM the (from PRZLC. 20 or(Bto )0.2 indicates 180 μM min), GUNA the based AUCinto onthe values ( ALC.). Gray Grayof extracellular columnsbars indicate represent GABA perfusion level the with AUC(nmol) 1 valuesμM after and perfusion before 5 μM perfusionMK801 with withinto MK801 the LC. (basal (B) indicates release). the * p AUC< 0.05, values ** p

MK801 (from 20 to 180 min), based on (A). Gray columns represent the AUC values before perfusion Biomolecules 2020, 10, 990 8 of 19 with MK801 (basal release). * p < 0.05, ** p < 0.01; relative to control, @@ p < 0.01; relative to MK801 (5 μM) by MANOVA with Tukey’s post hoc test. 3.2. Eﬀects of Local Administration of MUS into the LC on MK801-Induced Releaes of Norepinephrine and Doapmine3.2. Effects in of the Local OFC Administration and RTN (Study_2) of MUS into the LC on MK801-Induced Releaes of Norepinephrine and Doapmine in the OFC and RTN (Study_2) Study_1 suggests that MK801-induced releases of dopamine in the OFC and norepinephrine in the OFCStudy_1 and RTN suggests are possibly that MK801 generated-induced by intra-LC releases GABAergicof dopamine disinhibition, in the OFC similarand norepinephrine to serotonergic in andthe glutamatergic OFC and RTN transmission are possibly [29 ,31 generated,34]. Based by on intra the-LC previous GABAergic demonstration, disinhibition, to explore similar the to mechanismsserotonergic of and MK801-induced glutamatergic catecholamine transmission [29,31,34] release in. the Based OFC on and the RTN, previous the e ﬀ demonstration,ects of perfusion to withexplore 1 µM the MUS mechanisms (GABAA ofreceptor MK801 agonist)-induced into catecholamine the LC on MK801-induced release in the OFC releases and ofRTN, norepinephrine the effects of inperfusion the OFC, with RTN 1 and μM dopamine MUS (GABA in theA receptor OFC were agonist) determined into the [37 LC,38 , on48, 52 MK801]. Perfusion-induced mediums releases in of thenorepinephrine OFC and RTN in werethe OFC, maintained RTN and with dopamine MRS alone in the during OFC were Study_2. determined Perfusion [37,38,48,52] mediums. inPerfusion the LC wasmediums commenced in the OFC with and MRS RTN containing were maintained without (control) with MRS or alone with 1 duringµM MUS. Study_2. After Perfusion the stabilisation mediums of transmitterin the LC waslevel commenced in perfusate, with perfusion MRS medium containing in the without LC was (control) switched or to with the same1 μM MRS MUS. containing After the withstabi 5lisationµM MK801 of transmitter for 180 min. level in perfusate, perfusion medium in the LC was switched to the same MRSPerfusions containing with with 1 5µ μMM MUSMK801 into for the 180 LC min. decreased both basal and MK801-induced releases of Perfusions with 1 μM MUS into the LC decreased both basal and MK801-induced releases of norepinephrine in the OFC [FMUS (2,15) = 14.3 (p < 0.01), FTime (3.6, 35.6) = 106.4 (p < 0.01), FMUS*Time norepinephrine in the OFC [FMUS (2,15) = 14.3 (p < 0.01), FTime (3.6, 35.6) = 106.4 (p < 0.01), FMUS*Time (3.6, (3.6, 35.6) = 7.1 (p < 0.01)] and RTN [FMUS (2,15) = 12.2 (p < 0.01), FTime (3.5, 35.2) = 42.6 (p < 0.01), 35.6) = 7.1 (p < 0.01)] and RTN [FMUS (2,15) = 12.2 (p < 0.01), FTime (3.5, 35.2) = 42.6 (p < 0.01), FMUS*Time FMUS*Time (3.5, 35.2) = 2.4 (p > 0.05)] (Figure5A). Perfusions with 1 µM MUS into the LC also decreased (3.5, 35.2) = 2.4 (p > 0.05)] (Figure 5A). Perfusions with 1 μM MUS into the LC also decreased both both basal and MK801-induced releases of dopamine in the OFC [FMUS (2,15) = 6.0 (p < 0.01), FTime basal and MK801-induced releases of dopamine in the OFC [FMUS (2,15) = 6.0 (p < 0.01), FTime (4.9, 49.4) (4.9, 49.4) = 37.6 (p < 0.01), FMUS*Time (4.9, 49.4) = 1.1 (p > 0.05)] (Figure5B). Taken together with results in= 37.6 Study_1, (p < 0.01), the results FMUS*Time of (4.9, Study_2 49.4) indicate= 1.1 (p > that 0.05)] MK801 (Figure generates 5B). Taken intra-LC together GABAergic with results disinhibition in Study_1, resultingthe results in of the Study_2 increase indicate in catecholamine that MK801 release generates in the intra OFC-LC and GABAergic RTN. disinhibition resulting in the increase in catecholamine release in the OFC and RTN.

FigureFigure 5.5. EEffectsﬀects ofof locallocal administrationadministration ofof muscimolmuscimol (MUS:(MUS: 11µ μM)M)into intothe theLC LC on on MK801-induced MK801-induced releasesreleases of of norepinephrine norepinephrine in in the the OFC OFC and and RTN RTN ( A(A)) and and dopamine dopamine in in the the OFC OFC (B (B).). Ordinate: Ordinate: mean mean ± ± SDSD (n(n= =6) 6) ofof extracellular extracellular levelslevels ofof norepinephrine norepinephrineand and dopamine dopamine (nM); (nM); abscissa: abscissa: timetime afterafter MK801MK801 administrationadministration (min). (min). ClosedClosed andand graygray barsbars indicateindicate perfusionperfusion with with 1 1µ μMM MUS MUS and and 5 5µ μMM MK801 MK801 into into thethe LC, LC, respectively. respectively. ** **p p< <0.01; 0.01; relativerelative toto controlcontrol by by MANOVA MANOVA with with Tukey’s Tukey’s post post hoc hoc test. test. 3.3. Interaction between NMDAR in the LC and Adrenoceptor in the RTN on Transmitter Release in the RTN, 3.3. Interaction between NMDAR in the LC and Adrenoceptor in the RTN on Transmitter Release in the and MDTN (Study_3) RTN, and MDTN (Study_3) To explore the mechanisms of MK801-induced releases of norepinephrine in the RTN and GABA To explore the mechanisms of MK801-induced releases of norepinephrine in the RTN and GABA in the MDTN, the eﬀects of perfusion with 100 µM PRZ (α1 adrenoceptor antagonist) and 0.2 µM in the MDTN, the effects of perfusion with 100 μM PRZ (α1 adrenoceptor antagonist) and 0.2 μM GUNA (α2A adrenoceptor agonist) into the RTN on MK801-induced releases of catecholamine in the GUNA (α2A adrenoceptor agonist) into the RTN on MK801-induced releases of catecholamine in the RTN and GABA in the MDTN were determined. Perfusion mediums in the MDTN were maintained RTN and GABA in the MDTN were determined. Perfusion mediums in the MDTN were maintained with MRS alone during Study_3. Perfusion mediums in the LC was commenced with MRS. Perfusion with MRS alone during Study_3. Perfusion mediums in the LC was commenced with MRS. Perfusion medium in the RTN was commenced with containing without (control) or with 100 µM PRZ or 0.2 µM medium in the RTN was commenced with containing without (control) or with 100 μM PRZ or 0.2 GUNA. After the stabilisation of transmitter level in perfusate, perfusion medium in the LC was μM GUNA. After the stabilisation of transmitter level in perfusate, perfusion medium in the LC was switched to MRS containing with 5 µM MK801 for 180 min. switched to MRS containing with 5 μM MK801 for 180 min. Biomolecules 2020, 10, 990 9 of 19

Biomolecules 2020, 10, 990 9 of 19 3.3.1. Effects of Local Administration of PRZ and GUNA into the RTN on MK801-Induced Catecholamine3.3.1. Effects of ReleaseLocal Administration in the RTN of PRZ and GUNA into the RTN on MK801-Induced Catecholamine Release in the RTN Perfusions with MK801 (5 µM) into the LC increased extracellular norepinephrine level in the RTNPerfusions (MK801-induced with MK801 release in(5 theμM) RTN) into (Figurethe LC6 increasedA,C). Perfusion extracellular with 100 norepinephrineµM PRZ (α1 adrenoceptor level in the antagonist)RTN (MK801 into-induced the RTN release affected in neither the RTN) basal (Figure nor MK801-induced 6A,C). Perfusion norepinephrine with 100 releases μM PRZ in the(α1 RTNadrenoceptor (Figure5A,C). antagonist) Perfusion into with the 0.2 RTNµM affected GUNA (neitherα2A adrenoceptor basal nor MK801 agonist)-induced into the norepinephrine RTN decreased releases in the RTN (Figure 5A,C). Perfusion with 0.2 μM GUNA (α2A adrenoceptor agonist) into the both basal and MK801-induced norepinephrine releases in the RTN [FGUNA (2,15) = 20.8 (p < 0.01), RTN decreased both basal and MK801-induced norepinephrine releases in the RTN [FGUNA (2,15) = FTime (5.2, 78.0) = 68.7 (p < 0.01), FGUNA*Time (10.4, 78.0) = 20.8 (p < 0.01)] (Figure6A,C). Perfusions with20.8 ( MK801p < 0.01) (5, µFTimeM) into(5.2, the78.0) LC = did68.7 not (p < a ff0.01),ect extracellular FGUNA*Time (10.4, dopamine 78.0) = level 20.8 in(p the< 0.01)] RTN (Figure (Figure 66AB,D).,C). NeitherPerfusions perfusion with MK801 with 100 (5 µμM)M PRZ into nor the 0.2 LCµ Mdid GUNA not affect into theextracellular RTN affected dopamine dopamine level releases in the in RTN the RTN(Figure (Figure 6B,D).6B,D). Neither perfusion with 100 μM PRZ nor 0.2 μM GUNA into the RTN affected dopamine releases in the RTN (Figure 6B,D).

Figure 6. Effects of local administration of PRZ and GUNA into the RTN on MK801-induced releases Figure 6. Effects of local administration of PRZ and GUNA into the RTN on MK801-induced releases of norepinephrine (A) and dopamine (B) in the RTN. Ordinates: mean SD (n = 6) of extracellular of norepinephrine (A) and dopamine (B) in the RTN. Ordinates: mean ±± SD (n = 6) of extracellular catecholamine levels (nM); abscissa: time after MK801 administration (min). Stripped bars indicate perfusioncatecholamine with levels 100 µM (nM); PRZ abscissa: or 0.2 µM time GUNA after into MK801 the RTN. administration Gray bars indicate(min). Stripped perfusion bars with indicate 5 µM MK801perfusion into with the LC.100 μM (C,D PRZ) indicate or 0.2 AUCμM GUNA values ofinto extracellular the RTN. Gray levels bars of norepinephrine indicate perfusion and wi dopamineth 5 μM (pmol)MK801 after into perfusion the LC. ( withC,D) MK801indicate (from AUC 20 values to 180 min), of extracellular based on (A levels) and of (B ), norepinephrine respectively. Gray and columnsdopamine represent (pmol) after the AUC perfusion values with before MK801 perfusion (from with20 to MK801180 min), (basal based release). on (A) **andp < (B0.01;), respectively. relative to control,Gray columns and @@ representp < 0.01; relativethe AUC to values MK801 before (5 µM) perfusion by MANOVA with withMK801 Tukey’s (basal post release). hoc test. ** p < 0.01; relative to control, and @@ p < 0.01; relative to MK801 (5 μM) by MANOVA with Tukey’s post hoc 3.3.2.test. Effects of Local Administration of PRZ and GUNA into the RTN on MK801-Induced GABA Release in the MDTN 3.3.2.Perfusions Effects of Local with MK801Administration (5 µM) intoof PRZ the and LC GUNA increased into extracellular the RTN on GABAMK801 level-Induced in the GABA MDTN (MK801-inducedRelease in the MDTN GABA release in the MDTN) (Figure7A,B). Perfusion with 100 µM PRZ into the RTN did notPerfusions affect basal with GABA MK801 release (5 μM) in theinto MDTN, the LC but increased decreased extracellular MK801-induced GABA GABA level in release the MDTN in the MDTN(MK801 [F-inducedPRZ (2,15) GABA= 8.2( prelease< 0.01), in F Timethe MDTN)(3.7, 55.4) (Figure= 34.9 (7Ap <,B).0.01), Perfusion FPRZ*Time with(7.4, 100 55.4) μM= 7.4PRZ (p

Figure 7. Eﬀects of local administration of PRZ and GUNA into the RTN on MK801-induced GABA releaseFigure (7.A )Effects in the MDTN.of local Ordinate:administration mean of SDPRZ (n and= 6) GUNA of extracellular into the GABARTN on level MK801 (µM);-induced abscissa: GABA time ± afterrelease MK801 (A) in administration the MDTN. Ordinate: (min). Stripped mean ± barsSD (n indicate = 6) of extracellular perfusion with GABA 100 µ levelM PRZ (μM or); 0.2 abscissa:µM GUNA time intoafter the MK801 RTN. Grayadministration bars indicate (min). perfusion Stripped with bars 5 µM indicate MK801 perfusioninto the LC. with (B) indicates 100 μM thePRZ AUC or 0.2 values μM ofGUNA extracellular into the GABA RTN. levelGray (nmol) bars indicate after perfusion perfusion with with MK801 5 μM (from MK801 20 to into 180 the min), LC. based (B) indicates on (A). Gray the columnsAUC values represent of extracellular the AUC values GABA before level perfusion(nmol) afte withr perfusion MK801 (basal with release).MK801 (from** p < 200.01 to; relative180 min), to control,based on @@ (Ap).

Interaction between perfusion with 5 μM MK801 into the LC and 0.2 μM GUNA into the RTN

(onp < AMPA-induced0.01), FGUNA*Time releases(11.9, 89.4)of norepinephrine= 8.0 (p < 0.01)], [FGUNA dopamine (2,15) [F= 31.1GUNA (p(2,15) < 0.01),= 18.4FTime ((6.0,p < 0.01), 89.4) F=Time 95.6(4.9, (p < 74.1)0.01),= FGUNA*Time65.3 (p < (11.9,0.01), 89.4) FGUNA*Time = 8.0 (p (9.9,< 0.01)], 74.4) dopamine= 13.2 (p [F< GUNA0.01)] (2,15) and L-glutamate= 18.4 (p < 0.01), [FGUNA FTime (2,15)(4.9, 74.1)= 5.2 = (65.3p < 0.05),(p < 0.01), FTime FGUNA*Time(5.4, 80.9) (9.9,= 159.2 74.4) (=p 13.2< 0.01), (p < F0.01)]GUNA*Time and L-glutamate(10.8, 80.9) =[F8.5GUNA (p (2,15)< 0.01)] = 5.2 in (p the < 0.05), OFC wasFTime detected(5.4, 80.9) (Figure = 159.28 A–F).(p < 0.01), Similar FGUNA*Time to PRZ, (10.8, perfusion 80.9) = with 8.5 (p GUNA < 0.01)] into in the the OFC RTN was prevented detected the (Figure inhibitory 8A– eF).ﬀects Similar of perfusion to PRZ, withperfusion MK801 with into GUNA the LC into on AMPA-inducedthe RTN prevented releases the inhibitory of norepinephrine, effects of dopamineperfusion andwith L-glutamate MK801 into inthe the LC OFC on AMPA-induced (Figure8A–F). releases of norepinephrine, dopamine and L-glutamate

Figure 8. Eﬀects of local administration of MK801 into the LC, PRZ and GUNA into the RTN on αFigure-amino-3-hydroxy-5-methyl-4-isoxazolepropionic 8. Effects of local administration of MK801 acidinto (AMPA)-inducedthe LC, PRZ and GUNA releases into of norepinephrine the RTN on α- (amino-3-hydroxy-5-methyl-4-isoxazolepropionicA,D), dopamine (B,E) and L-glutamate (C,F) in the acid OFC. (AMPA)-induced Ordinate: mean releasesSD (n = of6) norepinephrine of extracellular ± levels(A,D), ofdopamine norepinephrine (B,E) and (nM), L-glutamate dopamine (C, (nM)F) in the and OFC. L-glutamate Ordinate: (µ meanM); abscissa: ± SD (n = time6) of afterextracellular AMPA administrationlevels of norepinephrine (min). Closed (nM), and dopamine stripped bars(nM) indicate and L-glutamate perfusion with (μM); 5 µ abscissa:M MK801 time into after the LC, AMPA and 100administrationµM PRZ or 0.2(min).µM Closed GUNA and into stripped the RTN, bars respectively. indicate perfusion Gray bars with indicate 5 μM MK801 perfusion into with the 100LC, µandM AMPA100 μM into PRZ the or MDTN. 0.2 μM (GUNAD–F) indicate into the the RTN, AUC respectively. values of extracellular Gray bars levelsindicate of norepinephrineperfusion with 100 (pmol), μM dopamineAMPA into (pmol) the MDTN. and L-glutamate (D–F) indicate (nmol) the after AUC perfusion values ofwith extracellular AMPA (from levels 20 to of 180 norepinephrine min), based on(pmol), (A–C dopamine), respectively. (pmol) Gray and columns L-glutamate represent (nmol) the after AUC perfusion values before with perfusionAMPA (from with 20 AMPA to 180 (basal min), release).based on **(Ap–C<),0.01; respectively. relative toGray control, columns @@ prepresent< 0.01; relativethe AUC to values MK801 before (5 µM) perfusion by MANOVA with AMPA with Tukey’s(basal release). post hoc ** test.p < 0.01; relative to control, @@ p < 0.01; relative to MK801 (5 μM) by MANOVA with Tukey’s post hoc test. 4. Discussion 4. DiscussionThe present study demonstrated the presence of the regulatory systems of the transmissionThe present in thalamocortical study demonstrated (MDTN-OFC) the presence glutamatergic, of the regulatory intrathalamic systems (RTN-MDTN) of the transmission GABAergic, in mesothalamicthalamocortical (LC-RTN) (MDTN-OFC) noradrenergic, glutamatergic, mesocortical (LC-OFC) intrathalamic noradrenergic (RTN-MDTN) and catecholaminergic GABAergic, (norepinephrinemesothalamic /dopamine (LC-RTN) coreleasing) noradrenergic, pathways, mesocortical using multiprobes (LC-OFC) microdialysis. noradrenergic According and to thecatecholaminergic results in this study (norepinephrine/dopamine and the published neural coreleasing) circuits [26, 27,30 pathways,,31,36–38 ,54 using–56], our multiprobes proposed hypothesismicrodialysis. regarding According the to neural the results networks in this from study the and LC the associated published with neural NMDAR circuits are [26,27,30,31,36– indicated in Figure38,54–56],9. our proposed hypothesis regarding the neural networks from the LC associated with NMDAR are indicated in Figure 9. Biomolecules 2020, 10, 990 12 of 19 Biomolecules 2020, 10, 990 12 of 19

Figure 9. Proposed hypothesis for the extended neural circuitry involved in thalamocortical (RTN–MDTN–OFC)Figure 9. Proposed hypothesis glutamatergic, for the mesothalamic extended neural (LC-MDTN) circuitry involved noradrenergic, in thalamocortical and mesocortical (RTN– (LC-OFC)MDTN–OFC catecholaminergic) glutamatergic, connectivitymesothalamic and (LC their-MDTN) regulation noradrenergic, systems. and GABAergic mesocortical neurones (LC-OFC) in thecatecholaminergic LC receive excitatory connectivity glutamatergic and their inputs regulation via NMDAR. systems. Both GABAergic noradrenergic neurones and catecholaminergic in the LC receive

neuronesexcitatory in glutamatergic the LC are regulated inputs byvia theNMDAR inhibitory. Both postsynaptic noradrenergic GABA andA catecholaminergicreceptor and somatodendritic neurones in αthe2A adrenoceptor, LC are regulated but are by not the a ff inhibitoryected by the postsynapticα1 adrenoceptor GABA [36A, 57receptor,58]. Noradrenergic and somatodendritic neurons in α2A the LCadrenoceptor, project terminals but are to thenot OFCaffected (deeper by the layers) α1 adrenoceptor [37,38,56,59] and[36,57,58] RTN [35. Noradrenergic], whereas catecholaminergic neurons in the neuronesLC project in the terminals LC project to coreleasing the OFC terminal (deeper to layers) the OFC [37,38,56,59] (superficial and layers) RTN [37 ,[35]38,56, ,59 whereas], but notcatecholaminergic to RTN [35]. GABAergic neurones in neurones the LC project in the coreleasing RTN receive terminal excitatory to the noradrenergic OFC (superficial input layers) from the[37,38,56,59] LC via α, 1but adrenoceptor not to RTN [35] [36].. GABAergic The intrathalamic neurones GABAergicin the RTN receive pathway excitatory is regulated noradrenergic by the presynapticinput from/ somatodendriticthe LC via α1 adrenoceptorα2A adrenoceptor [36]. The [36 ].intrathalamic Activated intrathalamic GABAergic GABAergic pathway istransmission regulated by suppressesthe presynaptic/somatodendritic glutamatergic neurones α2A inthe adrenoceptor MDTN, resulting [36]. Activatedin the inhibition intrathalamic of thalamocortical GABAergic (MDTN-OFC)transmission glutamatergic suppresses glutamatergic transmission [27 neurones,29,35]. The in thalamocorticalthe MDTN, resulting (MDTN-OFC) in the glutamatergic inhibition of pathwaythalamocortical activates (MDTN catecholaminergic-OFC) glutamatergic coreleasing transmission terminals via[27,29,35] AMPAR,. The but tha doeslamocortical not make (MDTN contact- withOFC) the glutamatergic noradrenergic pathway terminals activates in the OFCcatecholaminergic [37,38,56,59]. coreleasing terminals via AMPAR, but does not make contact with the noradrenergic terminals in the OFC [37,38,56,59]. 4.1. Catecholaminergic Transmission Regulating System Associated with NMDAR 4.1. TheCatecholaminergic inhibition of Transmission NMDAR in Regulating the medial System prefrontal Associated cortex, with insula, NMDAR and dorsal raphe nucleus (DRN)The increased inhibition regional of NMDAR monoamine in the release medial via prefrontal GABAergic cortex,disinhibition insula, and [30 dorsal,31,34, 37 raphe,38,43 nucleus,48,52]. The(DRN) noradrenergic increased regional and monoamine catecholaminergic release via neurones GABAergic in disinhibition the LC project [30,31,34,37,38,43,48,52] three pathways;. namely,The noradrenergic the mesocortical and catecholaminergic noradrenergic neurones and catecholaminergic in the LC project coreleasing three pathways; (dopamine namely, with the norepinephrine)mesocortical noradrenergic pathways, as and well catecholaminergic as the mesothalamic coreleasing (LC-RTN) (dopamine noradrenergic with norepinephrine) pathway [36] (Figurepathways,9). The as well present as the study mesothalamic also demonstrated (LC-RTN) that noradrenergic the noradrenergic pathway neurones [36] (Figure in the 9). LC The project present a selectivestudy also noradrenergic demonstrated terminal that the to bothnoradrenergic OFC and RTN, neurones but catecholaminergic in the LC project neurones a selective in thenoradrenergic LC project coreleasingterminal to terminal both OFC to the and OFC RTN, but not but to catecholaminergic the RTN. Contrary to neurones catecholamine, in the the LC local project administration coreleasing ofterminal MK801 to into the OFC theLC but decreasednot to the RTN. regional Contrary GABA to catecholamine, release. Therefore, the local these administration increases in of releases MK801 ofinto norepinephrine the LC decreased and regional dopamine GABA induced release. by Therefore, the localadministration these increases ofin releases MK801 (MK801-inducedof norepinephrine release)and dopamine were generated induced by by intra-LC the local GABAergic administration disinhibition, of MK801 as the (MK801 local administration-induced release) of MUS were (GABAgeneratedA receptor by intra agonist)-LC GABAergic prevented disinhibition, the MK801-induced as the loca releasesl administration of norepinephrine of MUS (GABA and dopamineA receptor inagonist) the OFC prevented and RTN. the Therefore, MK801-induced MK801 inhibitsreleases the of GABAergicnorepinephrine neuronal and dopamine activity without in the aOFCffecting and theRTN. noradrenergic Therefore, and MK801 catecholaminergic inhibits the neurones GABAergic in the neuronal LC. Similar activity to the without LC, we have affecting already the demonstratednoradrenergic the and MK801-induced catecholaminergic enhancement neurones in of the monoaminergic LC. Similar to transmission the LC, we in have the frontalalready cortexdemonstrated [37,38,48 the,52], MK801 serotonergic-induced transmission enhancement in the of monoaminergic DRN [30,31,34], transmission and glutamatergic in the transmissionfrontal cortex in[37,38,48,52] the thalamus, serotonergic [27–29,33,40 transmission] via regional in GABAergic the DRN [30,31,34] disinhibition., and glutamatergic transmission in the 2+ 2+ thalamusDuring [27 the–29,33,40] resting via stage, regional cation GABAergic channels indisinhibition. NMDAR are inhibited by Mg and Zn caps 2+ 2+ via specificDuring binding the resting sites; stage, however, cation depolarizationchannels in NMDAR (higher are than inhibited20 mV) by repelsMg2+ and Mg Zn2+and caps Zn via − + 2+ fromspecific the binding channel sites; pore, however, leading depolarization to the voltage-dependent (higher than inflow −20 mV) of repels Na and Mg2+ Ca and, andZn2+ outflowfrom the + Kchannel[60]. Therefore,pore, leading NMDAR to the voltage is a ligand-gated-dependent cation inflow channel of Na+ and with Ca accompanying2+, and outflow voltage-sensitive K+ [60]. Therefore, NMDAR is a ligand-gated cation channel with accompanying voltage-sensitive features. Additionally, the resting membrane potential of GABAergic interneurons is usually positive (−50 ~ Biomolecules 2020, 10, 990 13 of 19 features. Additionally, the resting membrane potential of GABAergic interneurons is usually positive ( 50 ~ 60 mV) compared with that of monoaminergic and glutamatergic neurons [61,62]. Therefore, − − the unique profiles of the selective inhibitory effects of MK801 on GABAergic transmission rather than monoaminergic or glutamatergic transmissions are induced by the differences of the resting membrane potential between the GABA neurones and other neurones. The threshold concentration of the local administration of MK801 on the release of norepinephrine and dopamine in the OFC were lower than 1 µM and 5 µM, respectively. Therefore, the selective noradrenergic projection is predominantly sensitive to NMDAR, rather than that of catecholaminergic projection. Furthermore, the noradrenergic projection to the OFC is more sensitive to NMDAR rather than that to the RTN, as the threshold concentration of MK801 on norepinephrine release in the RTN was lower than 5 µM. These distinct sensitivities between noradrenergic and catecholaminergic projections constitute the unique regulation system of catecholaminergic transmission in the OFC. In LC-OFC pathways, noradrenergic and catecholaminergic neurones project deeper and superficial layers in the OFC, respectively [36–38,56,59]. The thalamocortical glutamatergic pathway projects to the superficial layers in the frontal cortex [35,37,38]. Therefore, the thalamocortical glutamatergic pathway makes contact with the catecholaminergic coreleasing terminal, but not with noradrenergic terminal in the OFC [36–38]. The MK801-induced norepinephrine release in the RTN inhibited thalamocortical (MDTN-OFC) glutamatergic transmission via the activation of intrathalamic GABAergic inhibition, as local administration in the LC unexpectedly increased GABA release in the MDTN. These results suggest that the functional abnormalities induced by NMDAR inhibition in the LC affect various transmission systems, and may be more complex than our expectation. Indeed, intra-LC GABAergic disinhibition by NMDAR inhibition in the LC increased the release of norepinephrine and dopamine in the OFC via the activation of both noradrenergic and catecholaminergic neurones in the LC, but enhanced noradrenergic transmission in the RTN leads to the suppression of the thalamocortical catecholaminergic coreleasing terminal via the inactivation of the thalamocortical glutamatergic transmission in the OFC.

4.2. Catecholaminergic Transmission Regulating System Associated with Adrenoceptor Cortical α2A adrenoceptor is predominantly expressed in the postsynaptic spine, whereas the α2A adrenoceptor in the LC are expressed in both the presynaptic and somatodendritic regions, and function as autoreceptors on both non-catecholaminergic and catecholaminergic terminals [57]. Contrary to the α2A adrenoceptor, the α1 adrenoceptor has restricted expression regions compared with the α2 receptors, and is expressed in the thalamus rather than that in the LC [58] (Figure9). Noradrenergic neurons in the LC directly project to the RTN and enhance GABAergic transmission via the activation of postsynaptic α1 adrenoceptor [36,63]. In the present study, local administrations of PRZ (α1 adrenoceptor antagonist) into the LC and RTN did not aﬀect basal or MK801-induced norepinephrine release in the OFC and RTN; however, GABA release in the MDTN induced by the local administration of MK801 in the LC was inhibited by the local administration of PRZ into the RTN, but not by that into the LC. Therefore, elevation of norepinephrine release into the RTN enhances GABAergic neurones in the RTN via the activation of the postsynaptic α1 adrenoceptor. Contrary to the α1 adrenoceptor, local administrations of GUNA (α2A adrenoceptor agonist) into the LC decreased the basal and MK801-induced norepinephrine release in the OFC and RTN. Furthermore, local administrations of GUNA into the RTN also decreased basal and MK801-induced norepinephrine release in the RTN. Therefore, the activation of somatodendritic α2A in the LC and presynaptic α2A adrenoceptor in the RTN inhibits mesothalamic noradrenergic transmission. The paradoxical activation of intrathalamic GABAergic inhibition induced by local administration in the LC was prevented by the activation of the somatodendritic α2A adrenoceptor in the LC, presynaptic α2A adrenoceptor in the RTN, and/or inhibition of postsynaptic α1 adrenoceptor in the RTN. In other words, the modulation of these adrenoceptor subtypes can compensate the Biomolecules 2020, 10, 990 14 of 19 suppression of the thalamocortical glutamatergic transmission induced by the inhibition of NMDAR in the LC.

4.3. Clinical Implication of NMDAR Antagonist and Adrenoceptor Agents The functional magnetic resonance study demonstrated that the subanaesthetic dose of ketamine generated the reduced functional connectivity between the LC and MDTN [42]. This study provided the mechanisms of ketamine-induced reduced connectivity between the LC and MDTN. Enhanced intrathalamic (RTN-MDTN) GABAergic inhibition induced by the hyperactivation of mesothalamic (LC-RTN) noradrenergic transmission inhibits the neuronal activity of glutamate neurons in the MDTN. Therefore, the reduced functional connectivity between LC and MDTN induced by ketamine is possibly due to the observed suppression of MDTN neuronal activity via the hyperactivation of intrathalamic GABAergic inhibition. Regarding the attention mechanism, the physiological enhancement of the thalamic activity generates a reduction in distraction in the attentional performance [64]; however, the pathological enhancement of the thalamocortical glutamatergic transmission induced by phencyclidine generates a working memory deficit, but is compensated by a therapeutic-relevant dose of GUNA [65]. Therefore, the imbalance between the neuronal hyperactivation in the LC and the hypofunction in the MDTN are related to a change in the regulation of arousal in terms of a disruption of the alerting function, a promotion of non-selective sensory signal detection, and an enhancement of behavioural flexibility [42]. Contrary to thalamocortical glutamatergic transmission, the enhancement of the postsynaptic α2A adrenoceptor in the frontal cortex improves cognitive deficits, including working memory, but activations both frontal α1 and β adrenoceptors, which impair working memory [66]. Indeed, the relatively lower extracellular norepinephrine level during pre-event periods with event-related transient increased norepinephrine release in OFC contributes to generating stability and flexibility against contingency and uncontrolled severe stresses [36]. Therefore, the persistent relatively lower noradrenergic tone in OFC generates stable and sticks in an initial state, but discards a critical change, whereas the continuous hyper-noradrenergic tone in the OFC reliably leads unstable and cycles concentrations to a sequence of environmental changes [36,67]. Therefore, an appropriate dose (relatively low dose) NMDAR antagonist possibly has the potential for improving working memory/cognition via the selective enhancement of mesocortical noradrenergic transmission without affecting mesothalamic noradrenergic transmission, as the threshold concentration of the local administration of MK801 into the LC on mesocortical selective noradrenergic transmission was lower than 1 µM, but those on the mesothalamic noradrenergic transmissions were lower than 5 µM. Further complex considerations are required regarding the relationship between mesocortical catecholaminergic coreleasing transmission and cognition. In our previous studies, we demonstrated that both local (into the RTN) and systemic administrations of MK801 enhanced the thalamocortical glutamatergic transmission concentration dose-dependently [28,29,35]; however, the MK801-induced activation of thalamocortical glutamatergic transmission is compensated by antipsychotics, clozapine (activation of group III metabotropic glutamate receptor), aripiprazole (activation of group II metabotropic glutamate receptor), and lurasidone (inhibition of serotonin 5-HT7 receptor) [26,27,30,31]. The incomprehensible results regarding the suppression of thalamocortical glutamatergic transmission induced by the local administration of the NMDAR antagonist, which aggravates or conducts psychosis and cognitive deficits, were similar to the actions of antipsychotics. We speculate that the paradoxical mechanisms that direct the inhibition of NMDAR are possibly predominant compared with the postsynaptic α1 adrenoceptor secondarily activation in the RTN GABAergic neurones, based on the systemic MK801-induced dose-dependent activation of thalamocortical glutamatergic transmission [26,28,29]. Therefore, the systemic administration of the NMDAR antagonist double-burstly enhances mesocortical catecholaminergic coreleasing transmission. Although this hypothesis needs to be clarified in further study, a relatively low dose NMDAR antagonist can selectively enhance mesocortical noradrenergic transmission, without affecting mesocortical catecholaminergic Biomolecules 2020, 10, 990 15 of 19 coreleasing transmission, whereas conversely, a relatively high-dose NMDAR antagonist enhances both mesocortical selective noradrenergic and catecholaminergic coreleasing transmissions in the OFC. GUNA, which is approved for the treatment of ADHD, enhances thalamocortical glutamatergic transmission via the inhibition of mesothalamic (LC-RTN) noradrenergic transmission [36]. In the frontal cortex, including the medial prefrontal cortex, insula, and OFC, the thalamocortical glutamatergic terminal enhances the mesocortical catecholaminergic coreleasing terminal from the LC [37,38]. Therefore, the impacts of the thalamocortical glutamatergic and mesocortical catecholaminergic transmission on cognitive function might be synonymous in the neuronal networks from the LC. Contrary to noradrenergic transmission, the inhibition of NMDAR in the DRN activates thalamocortical glutamatergic transmission via the activation of the serotonin 5-HT7 receptor in the MDTN [30,31,34]. The contradictive actions between the serotonergic and noradrenergic transmissions on the thalamocortical glutamatergic transmission possibly, at least partially, can explain the clinical advantage of selective serotonin transporter inhibitors to major depression, compared with selective norepinephrine transporter inhibitors. Both serotonergic and noradrenergic transmissions are recognized as fundamental transmission systems in the pathophysiology of major depression, whereas noradrenergic compounds have been less extensively utilized in clinical medication against major depression compared with selective serotonin transporter inhibitors. The development of the selective norepinephrine transporter inhibitors, reboxetine and atomoxetine, have not substantially improved the state of the art against mood disorders medication [68]. Additionally, the antidepressive mechanisms of atypical antipsychotics, quetiapine/norquetiapine, approved for the treatment of bipolar depression, are mediated by their α1 adrenoceptor inhibition [38,69]. Taken together with clinical evidence, the present results suggest that noradrenergic transmission is likely not sufficient to obtain a full antidepressive action, but the suppression of mesothalamic noradrenergic transmission via α1 adrenoceptor inhibition and/or α2A adrenoceptor activation represents an important element for effectiveness in the treatment of depressive or bipolar disorder with anxious distress/mixed features.

5. Conclusions The present study, to clarify the effects of NMDAR in the LC on thalamocortical (RTN-MDTN-OFC) GABAergic/glutamatergic, mesothalamic (LC-RTN) noradrenergic, and mesocortical (LC-OFC) selective noradrenergic and coreleasing catecholaminergic transmissions, determined the effects of the local administration of noncompetitive NMDAR antagonist, MK801, into the LC on releases of L-glutamate, GABA, norepinephrine, and dopamine in the OFC, RTN, MDTN, and LC, using multiprobe microdialysis. The inhibition of NMDAR in the LC generates intra-LC GABAergic disinhibition, resulting in enhancement in mesocortical selective noradrenergic and catecholaminergic coreleasing (norepinephrine and dopamine) transmissions and mesothalamic noradrenergic transmission. Interestingly, thalamocortical glutamatergic transmission is suppressed by the enhanced intrathalamic (RTN-MDTN) GABAergic inhibition, due to the MK801-induced activation of mesothalamic noradrenergic transmission. Intrathalamic (RTN-MDTN) GABAergic transmission was inhibited by the inhibition of the postsynaptic α1 adrenoceptor in the RTN, and the activations of the somatodendritic α2A adrenoceptor in the LC or presynaptic α2A adrenoceptor in the RTN. Remarkably, the sensitivity of mesocortical selective noradrenergic transmission to MK801 is relatively higher than those of mesothalamic noradrenergic and mesocortical catecholaminergic transmissions. Therefore, a relatively low dose of NMDAR antagonist possibly enhances mesocortical noradrenergic transmission without affecting mesocortical catecholaminergic or mesothalamic noradrenergic transmissions; however, a relatively high dose of NMDAR antagonist enhances both mesothalamic noradrenergic, mesocortical noradrenergic, and catecholaminergic transmissions. These distinct sensitivities of the three pathways associated with the LC probably contribute to the pathophysiology of cognitive deficits and mood disorders associated with NMDAR.

Author Contributions: Conceptualization, M.O.; Data curation, K.F. and M.O.; Formal analysis, K.F. and M.O.; Funding acquisition, M.O.; Methodology, M.O.; Project administration; M.O., Validation, M.O.; Writing original Biomolecules 2020, 10, 990 16 of 19 draft, M.O.; Writing review and editing, M.O. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by Japan Society for the Promotion of Science (15H04892 and 19K08073). Conﬂicts of Interest: The authors state no conﬂict of interest.

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