Chronic Administrations of Guanfacine on Mesocortical Catecholaminergic and Thalamocortical Glutamatergic Transmissions

International Journal of Molecular Sciences

Article Chronic Administrations of Guanfacine on Mesocortical Catecholaminergic and Thalamocortical Glutamatergic Transmissions

Kouji Fukuyama, Tomosuke Nakano, Takashi Shiroyama and Motohiro Okada *

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

Abstract: It has been established that the selective α2A adrenoceptor agonist guanfacine reduces hyperactivity and improves cognitive impairment in patients with attention-deﬁcit/hyperactivity disorder (ADHD). The major mechanisms of guanfacine are considered to involve the activation of the postsynaptic α2A adrenoceptor of glutamatergic pyramidal neurons in the frontal cortex, but the effects of chronic guanfacine administration on catecholaminergic and glutamatergic transmissions associated with the orbitofrontal cortex (OFC) are yet to be clariﬁed. The actions of guanfacine on catecholaminergic transmission, the effects of acutely local and systemically chronic (for 7 days) administrations of guanfacine on catecholamine release in pathways from the locus coeruleus (LC) to OFC, the ventral tegmental area (VTA) and reticular thalamic-nucleus (RTN), from VTA to OFC, from RTN to the mediodorsal thalamic-nucleus (MDTN), and from MDTN to OFC were determined using multi-probe microdialysis with ultra-high performance liquid chromatography. Additionally, the Citation: Fukuyama, K.; Nakano, T.; effects of chronic guanfacine administration on the expression of the α2A adrenoceptor in the plasma Shiroyama, T.; Okada, M. Chronic membrane fraction of OFC, VTA and LC were examined using a capillary immunoblotting system. Administrations of Guanfacine on The acute local administration of therapeutically relevant concentrations of guanfacine into the LC Mesocortical Catecholaminergic and Thalamocortical Glutamatergic decreased norepinephrine release in the OFC, VTA and RTN without affecting dopamine release Transmissions. Int. J. Mol. Sci. 2021, in the OFC. Systemically, chronic administration of therapeutically relevant doses of guanfacine 22, 4122. https://doi.org/10.3390/ for 14 days increased the basal release of norepinephrine in the OFC, VTA, RTN, and dopamine ijms22084122 release in the OFC via the downregulation of the α2A adrenoceptor in the LC, OFC and VTA. Fur- thermore, systemically, chronic guanfacine administration did not affect intrathalamic GABAergic Academic Editor: Maurizio Memo transmission, but it phasically enhanced thalamocortical glutamatergic transmission. The present study demonstrated the dual actions of guanfacine on catecholaminergic transmission—acute attenu- Received: 2 March 2021 ation of noradrenergic transmission and chronic enhancement of noradrenergic transmission and Accepted: 14 April 2021 thalamocortical glutamatergic transmission. These dual actions of guanfacine probably contribute to Published: 16 April 2021 the clinical effects of guanfacine against ADHD.

Publisher’s Note: MDPI stays neutral Keywords: attention-deﬁcit/hyperactivity disorder; guanfacine; dopamine; norepinephrine; L- with regard to jurisdictional claims in glutamate; GABA; α2A adrenoceptor published maps and institutional afﬁl- iations.

1. Introduction Attention deﬁcit hyperactivity disorder (ADHD) is a common neurodevelopmental Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and neuropsychiatric disorder in children and adolescents (estimated prevalence of ADHD This article is an open access article in adults is 5%) [1,2]. Usually, ADHD onsets in childhood and persists to adulthood. distributed under the terms and Sometimes onset occurs during adolescence [2]. Adult-onset ADHD is characterized by conditions of the Creative Commons dysregulations of emotion, attention, executive function, impulsivity, and restlessness, Attribution (CC BY) license (https:// which result in several socio-psychological disturbances [2]. creativecommons.org/licenses/by/ The treatment of ADHD usually combines pharmacotherapy and behavioural therapy [2]. 4.0/). In pharmacotherapy, it has been established that stimulant class agents (methylphenidate

Int. J. Mol. Sci. 2021, 22, 4122. https://doi.org/10.3390/ijms22084122 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 4122 2 of 22

and amphetamines) are the first-line agents, since these show the most effectiveness [3,4]. Unfortunately, stimulant class agents are probably not effective in up to 30% of patients with ADHD [5], and they are possibly associated with several adverse responses, including dependency or diversion [6]. Therefore, non-stimulant class medications need to be used as an adjunctive therapy or as a monotherapy alternative in patients with suboptimal responses to or intolerable side-effects from stimulants [6,7]. The availability of different mechanisms of medications provides opportunities for additional/augmentation medication strategies when ADHD symptoms cannot be adequately improved by prior therapies, such as stimulant class agents [7]. Guanfacine is an approved agent for the treatment of ADHD as a non-stimulant class agent in Japan, the USA and Europe [1–4]. Clinical trials indicate that guanfacine is effective for the treatment of ADHD in children and adolescents [7]. Notably, guanfacine improves several symptoms of ADHD, including oppositional symptoms [8]. Compared to atomoxetine, guanfacine may contribute to greater control of ADHD and rapid onset of efficacy [7]. Furthermore, a meta-analysis study reported a higher incidence of events of suicidal ideation induced by atomoxetine within one month of starting atomoxetine [9]; however, there is no reported risk of suicidal ideation with guanfacine [7,10]. Guanfacine is a selective agonist of the α2A adrenoceptor [11–14]. The α2A adrenoceptor is mainly expressed in the dendritic spines of frontal glutamatergic pyramidal neurones [13]. Based on the findings, the major mechanisms of action of guanfacine are proposed as two hypotheses [7,14,15]. The first hypothesis is that guanfacine activates frontal pyramidal neurones associated with working memory due to blockades of the hyperpolarisation-activated cyclic nucleotide-gated channel (HCN) [16], induced by the activation of the postsynaptic α2A adrenoceptor in superficial layers (HCN hypothesis) [14]. The second hypothesis is that guanfacine suppresses the hyper-function of pyramidal neurones of ADHD due to an enhanced inhibitory postsynaptic α2A adrenoceptor (excitatory postsynaptic current hypothesis) [7,15]. These two hypotheses emphasise the importance of the intra-frontal glutamatergic system. Both hypotheses were supported by a number of experiments. In particular, both guanfacine and clonidine improve attention/cognition performance and the regulation of impulsivity in rat ADHD models [17], but do not improve behaviour in the α2A adrenoceptor knockout model [18]. The behavioural effects of both guanfacine and clonidine were attenuated by α2A adrenoceptor antagonists but were unaffected by antagonists of α2B or α2C adrenoceptors [17]. These preclinical findings suggest that the modulation of noradrenergic transmission via the activation of the α2A adrenoceptor probably plays fundamental roles in the pathophysiology of ADHD. Clinical imaging studies revealed that the functional disturbance associated with the orbitofrontal cortex (OFC) [19], ventral tegmental area (VTA) [20] and mediodorsal thalamic nucleus (MDTN) [21] contributed to cognitive dysfunction in patients with ADHD. The locus coeruleus (LC), the most dominant noradrenergic nucleus, regulates cognition/perception via the projection of noradrenergic terminals to various brain regions, including the OFC and reticular thalamic nucleus (RTN) [22,23]. Therefore, to further understand the pathophysiology of ADHD, the effects of acute and chronic administrations of guanfacine on noradrenergic transmission in the frontal cortex and outside of the cortex should be clarified. Acute guanfacine administration in particular decreased releases of norepinephrine in the LC and OFC, and dopamine in the OFC, whereas the subchronic administration of a therapeutically relevant dose of guanfacine (for 7 days) suppressed the inhibitory effects of the α2A adrenoceptor but decreased basal releases of norepinephrine and dopamine in the OFC [24]. The mechanisms of suppression of the inhibitory effects of the α2A adrenoceptor induced by subchronic administration of guanfacine are speculated to cause the desensitisation or downregulation of the α2A adrenoceptor. However, neither the kinetics of desensitisation nor the downregulation of the α2A adrenoceptor induced by guanfacine has been clarified [24]. The OFC receives catecholaminergic projections, including the selective dopaminergic pathway from the VTA and selective noradrenergic and dopamine/norepinephrine co- Int. J. Mol. Sci. 2021, 22, 4122 3 of 22

releasing pathways from the LC [25–28]. The selective dopaminergic and noradrenergic terminals receive GABAergic inhibitory inputs in the deeper layers of the OFC, whereas dopamine/norepinephrine co-releasing terminals from the LC to the superﬁcial layers of the OFC receive stimulatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/glutamatergic regulation from the MDTN [27,28]. Glutamatergic neurones in the MDTN are also regulated by GABAergic inhibition from the RTN, which receives excitatory mesothalamic noradrenergic inputs via the excitatory α1 adrenoceptor [29]. Based on these anatomical ﬁndings regarding catecholaminergic pathways, to explore the mechanisms of chronic administration of guanfacine, the present study determined the effects of acute and chronic (for 14 days) administration of therapeutically relevant doses of guanfacine on the catecholaminergic transmission in LC-OFC, LC-VTA-OFC, and glutamatergic transmission in LC-RTN-MDTN-OFC using multiprobe microdialysis, as well as α2A adrenoceptor expression in the plasma membranes of LC, VTA and OFC using capillary immunoblotting systems.

2. Results 2.1. Effects of Acute and Chronic Administration of Therapeutic-Relevant Dose of Guanfacine on Extracellular Levels of Norepinephrine and Dopamine in the OFC and VTA (Study 1) To determine the effects of local administrations of guanfacine (200 nM) into the LC [24] on extracellular levels of norepinephrine and dopamine in the OFC, after the systemically chronic administration of therapeutic-relevant dose of guanfacine (0 or 0.12 mg/kg/day) [17,24,30] for 14 days using osmotic pump (2002, Alzet, Cupertino, CA, USA) (N = 48), microdialysis probes were inserted into the LC, VTA, RTN, MDTN and OFC [27,28,31–33]. After the chronic guanfacine-free administration for 14 days, the perfusate in the LC was switched from MRS to MRS containing guanfacine (200 nM) (acute effects). After the systemically chronic administration of guanfacine (0.12 mg/kg/day) for 14 days, the perfusate in LC was switched from MRS to MRS containing guanfacine (200 nM) (chronic effects).

2.1.1. Effects of Acute and Chronic Administration of Therapeutic-Relevant Dose of Guanfacine on Extracellular Norepinephrine Level in the OFC Perfusion with guanfacine into the LC, after systemically chronic administration (0 or 0.12 mg/kg/day guanfacine for 14 days), affected extracellular norepinephrine level in the OFC [Ftime(5.6,113.0) = 7.0 (p < 0.01), Fadministration(1,20) = 33.4 (p < 0.01), Fguanfacine(1,20) = 4.8 (p < 0.05), Fguanfacine*time(5.6,113.0) = 11.1 (p < 0.01), Fguanfacine*adminisdtration(1,20) = 2.6 (p > 0.1), Fadminisdtration*time(5.6,113.0) = 7.9 (p < 0.01), Fguanfacine*administration*time(5.6,113.0) = 5.7 (p < 0.01)] (Figure1). Acute administration of guanfacine (perfusion with 200 nM guanfacine into the LC) decreased extracellular norepinephrine in the OFC (Figure1A), whereas after chronic guanfacine administration, perfusion with 200 nM guanfacine into the LC did not have an effect (Figure1B). Further- more, chronic administration of guanfacine increased basal extracellular norepinephrine level in the OFC (Figure1C). Therefore, chronic administration of therapeutic-relevant does of guanfacine (0.12 mg/kg/day for 14 days), prevented the acute inhibitory effects of guanfacine on the mesocortical (LC-OFC) noradrenergic transmission and enhances the mesocortical noradrenergic transmission. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 22 Int. J. Mol. Sci. 2021, 22, 4122 4 of 22

Figure 1. 1. EffectsEffects of of acutely acutely local local administration administration (perfusion (perfusion with) with) 200 200 nM nM guanfacine guanfacine into into the thelocus locus coeruleus coeruleus (LC) (LC) on ex- on tracellularextracellular norepinephrine norepinephrine level level in inthe the orbitofrontal orbitofrontal cortex cortex (OFC), (OFC), after after the the systemically systemically chronic chronic administration of guanfacine (0 (0 or or 0.12 0.12 mg/kg/day mg/kg/day for for 14 14days). days). Panel Panel (A) ( Aindicates) indicates the the effects effects of perfusion of perfusion with with 200 200nM nMguanfacine guanfacine into intothe LC after chronic administration of guanfacine (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 200 the LC after chronic administration of guanfacine (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects nM guanfacine after systemically chronic administration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Or- dinates:of 200 nM mean guanfacine ± SD (n = after6) of extracellular systemically norepinephrine chronic administration level (nM), of guanfacineabscissa: time (0.12 after mg/kg/day) administration for of 14 perfusion days (chronic with ± guanfacineeffects). Ordinates: into the meanLC (min).SD Blue (n = bars: 6) of perf extracellularusion of 200 norepinephrine nM guanfacine level into (nM), LC. abscissa: Panel (C time) indicates after administration the AUC20–180 min of valuesperfusion of extracellular with guanfacine norepinephrine into the LC le (min).vel during Blue perfusion bars: perfusion with guanfacine of 200 nM guanfacine(from 20 to 180 into min). LC. Panel Ordinates: (C) indicates mean ± theSD (nAUC = 6)20–180 of AUC min values20–180 min of values extracellular of extracellular norepinephrine norepinephrine level during level perfusion (pmol). with Black guanfacine and blue (from columns 20 to 180indicate min). respective Ordinates: controlmean ± andSD perfusion (n = 6) of AUCof guanfacine20–180 min valuesof AUC of values extracellular of norepine norepinephrinephrine levels. level When (pmol). the effects Black andof guanfacine blue columns were indicate statis- ticallyrespective significant control compared and perfusion using of multivariate guanfacine analysis of AUC valuesof variance of norepinephrine (MANOVA), the levels. data When were theanalysed effects using of guanfacine Tukey’s postwere hoc statistically test. ** p signiﬁcant< 0.01, compared compared with using control. multivariate @@ p < 0.01: analysis compared of variance with acute (MANOVA), effects of guanfacine. the data were analysed using Tukey’s post hoc test. ** p < 0.01, compared with control. @@ p < 0.01: compared with acute effects of guanfacine. 2.1.2. Effects of Acute and Chronic Administration of Therapeutic-Relevant Dose of Guanfacine2.1.2. Effects on of Extracellular Acute and Chronic Dopamine Administration Level in the ofOFC Therapeutic-Relevant Dose of GuanfacinePerfusion on with Extracellular guanfacine Dopamine into the LevelLC, after in the systemically OFC chronic administration (0 or 0.12Perfusion mg/kg/day with guanfacine guanfacine for into 14 thedays), LC, affected after systemically extracellular chronic dopamine administration level in the (0 OFCor 0.12 [Ftime mg/kg/day(9,180) = 1.3 guanfacine (p > 0.1), F foradministration 14 days),(1,20) affected = 36.3 extracellular (p < 0.01), Fguanfacine dopamine(1,20) level = 0.4 in (p the > 0.1),OFC F [Fguanfacine*timetime(9,180)(9,180) = 1.3 = (p 1.4> 0.1), (p > F administration0.1), Fguanfacine*adminisdtration(1,20) = 36.3(1,20) (p < 0.01), = 0.3 F guanfacine(p > 0.1),(1,20) Fadminisdtra- = 0.4 tion*time(p > 0.1),(9,180) Fguanfacine*time = 1.3 (p > 0.1),(9,180) Fguanfacine*administration*time = 1.4 (p > 0.1), Fguanfacine*adminisdtration(9,180) = 0.5 (p > 0.1)](1,20) (Figure = 0.3 2). (p Acute> 0.1), administrationFadminisdtration*time of guanfacine(9,180) = 1.3 (perfusion (p > 0.1), with Fguanfacine*administration*time 200 nM guanfacine into(9,180) the LC) = 0.5 did (p not> 0.1)] affect(Figure extracellular2 ). Acute dopamine administration in the ofOFC guanfacine (Figure 2A), (perfusion and after with chronic 200 nM guanfacine guanfacine admin- into istration,the LC) did perfusion not affect with extracellular 200 nM guanfacine dopamine into in thethe OFCLC also (Figure had2 A),no effect and after (Figure chronic 2B). However,guanfacine the administration, chronic administration perfusion of with guan 200facine nM increased guanfacine the into basal the extracellular LC also had do- no pamineeffect (Figure level in2B). the However, OFC (Figure the chronic2C). These administration results indicate of guanfacine the difference increased mechanisms the basal of theextracellular effects of chronic dopamine administration level in the OFCof guan (Figurefacine2C). on Thesemesocortical results indicatenoradrenergic the difference and do- paminergicmechanisms transmissions, of the effects ofsince, chronic similar administration to norepinephrine, of guanfacine chronic on administration mesocortical no-of therapeuticallyradrenergic and relevant dopaminergic does of transmissions, guanfacine (0.12 since, mg/kg/day similar tofor norepinephrine, 14 days) enhances chronic the mesocorticaladministration dopaminergic of therapeutically transmission relevant with doesout of guanfacineacutely affecting (0.12 mg/kg/dayextracellular for dopamine 14 days) levelenhances in the the OFC. mesocortical dopaminergic transmission without acutely affecting extracellular dopamine level in the OFC.

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FigureFigure 2. 2. EffectsEffects of of acute acute local local administration administration (perfusion (perfusion with) with) 200 200 nM nM guanfacine guanfacine into into the the LC LC on on extracellular extracellular dopamine dopamine levellevel in in the the OFC, OFC, after after the the syst systemicallyemically chronic chronic administration administration of ofguanfacine guanfacine (0 (0or or0.12 0.12 mg/kg/day mg/kg/day for 14 for days). 14 days). Panel Panel (A) indicates(A) indicates the effects the effects of perfusion of perfusion with with 200 200nM nMguanfacine guanfacine into intothe theLC LCafter after chronic chronic administration administration of guanfacine of guanfacine (0 mg/kg/day)(0 mg/kg/day) for 14 for days 14 days(acute (acute effects). effects). Panel ( PanelB) indicates (B) indicates effects effectsof 200 nM of 200 guanfacine nM guanfacine after systemically after systemically chronic admin- chronic istration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean±SD (n = 6) of extracellular dopamine administration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean±SD (n = 6) of extracellular level (nM), abscissa: time after administration of perfusion with guanfacine into the LC (min). Blue bars: perfusion of 200 dopamine level (nM), abscissa: time after administration of perfusion with guanfacine into the LC (min). Blue bars: nM guanfacine into LC. Panel (C) indicates the AUC20–180 min values of extracellular dopamine level during perfusion with C guanfacineperfusion of (from 200 nM 20 to guanfacine 180 min). intoOrdinates: LC. Panel mean ( ) ± indicates SD (n = 6) the of AUCAUC20–18020–180 min min valuesvalues of of extracellular extracellular dopamine dopamine level level (pmol). during Blackperfusion and blue with columns guanfacine indicate (from respective 20 to 180 control min). Ordinates:and perfusion mean of guanfacine± SD (n = 6)of ofAUC AUC values20–180 of min dopaminevalues of levels. extracellular When thedopamine effects of level guanfacine (pmol). Blackwere andstatistically blue columns significant indicate compared respective using control MANOVA, and perfusion the data of were guanfacine analysed of using AUC valuesTukey’s of postdopamine hoc test. levels. @@ p When< 0.01: thecompared effects ofwith guanfacine acute effects were of statistically guanfacine. signiﬁcant compared using MANOVA, the data were analysed using Tukey’s post hoc test. @@ p < 0.01: compared with acute effects of guanfacine. 2.1.3. Effects of Acute and Chronic Administration of Therapeutic-Relevant Dose of Guanfacine2.1.3. Effects on of Extracellular Acute and Chronic Norepinephrine Administration Level in of the Therapeutic-Relevant VTA Dose of GuanfacinePerfusion on with Extracellular guanfacine Norepinephrine into the LC, after Level systemically in the VTA chronic administration (0 or 0.12Perfusion mg/kg/day with guanfacine guanfacine for into 14 thedays), LC, affected after systemically extracellular chronic norepinephrine administration level (0 in or the0.12 VTA mg/kg/day [Ftime(9,180) guanfacine = 5.2 (p < for 0.01), 14 days),Fadministration affected(1,20) extracellular = 69.4 (p < 0.01), norepinephrine Fguanfacine(1,20) level = 1.7 in the(p >VTA 0.1), [FFtimeguanfacine*time(9,180)(9,180) = 5.2 ( p=< 6.8 0.01), (p < F 0.01),administration Fguanfacine*adminisdtration(1,20) = 69.4 ((1,20)p < 0.01), = 1.3 F guanfacine(p > 0.1),(1,20) Fadminisdtra- = 1.7 tion*time(p > 0.1),(9,180) Fguanfacine*time = 1.5 (p > 0.05),(9,180) Fguanfacine*administration*time = 6.8 (p < 0.01), Fguanfacine*adminisdtration(9,180) = 1.8 (p > 0.05)](1,20) (Figure = 1.3 3). (p Acute> 0.1), administrationFadminisdtration*time of guanfacine(9,180) = 1.5 (perfusion (p > 0.05), with Fguanfacine*administration*time 200 nM guanfacine into(9,180) the =LC) 1.8 decreased (p > 0.05)] extracellular(Figure3). Acute norepinephrine administration in the of VTA guanfacine (Figure (perfusion 3A), whereas with after 200 nMchronic guanfacine guanfacine into administration,the LC) decreased perfusion extracellular with 200 norepinephrine nM guanfacine in into the the VTA LC (Figure had no3 A),effect whereas (Figure after 3B). Furthermore,chronic guanfacine the chronic administration, administration perfusion of guanfacine with 200 increased nM guanfacine basal extracellular into the LC nore- had pinephrineno effect (Figure level in3B). the Furthermore, VTA (Figure 3C). the chronicTherefore, administration similar to the of LC-OFC guanfacine noradrenergic increased pathway,basal extracellular the chronic norepinephrine administration level of therap in theeutically VTA (Figure relevant3C). does Therefore, of guanfacine similar to (0.12 the mg/kg/dayLC-OFC noradrenergic for 14 days) prevented pathway, thethe chronicacute inhi administrationbitory effects ofof therapeuticallyguanfacine on relevantthe LC- VTAdoes noradrenergic of guanfacine transmission, (0.12 mg/kg/day but enhanced for 14 days) the preventedLC-VTA noradrenergic the acute inhibitory transmission. effects of guanfacine on the LC-VTA noradrenergic transmission, but enhanced the LC-VTA noradrenergic transmission.

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FigureFigure 3. 3. EffectsEffects of of acutely acutely local local administration administration (perfusion (perfusion with) with) 200 200 nM nM guanfacine guanfacine into into the the LC LC on onextracellular extracellular norepi- nore- nephrinepinephrine level level in inthe the VTA, VTA, after after the the syst systemicallyemically chronic chronic administration administration of of guanfa guanfacinecine (0 (0 or or 0.12 0.12 mg/kg/day for for 14 14 days). days). PanelPanel ( (AA)) indicates indicates the the effects effects of of perfusion perfusion with with 200 200 nM nM guanfacine guanfacine into into the the LC LC after after chronic chronic ad administrationministration of of guanfacine guanfacine (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 200 nM guanfacine after systemically chronic ad- (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 200 nM guanfacine after systemically chronic ministration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean±SD (n = 6) of extracellular nore- administration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean±SD (n = 6) of extracellular pinephrine level (nM), abscissa: time after administration of perfusion with guanfacine into the LC (min). Blue bars: per- fusionnorepinephrine of 200 nM levelguanfacine (nM), abscissa:into LC. Panel time after (C) indicates administration the AUC20–180 of perfusion min with values guanfacine of extracellular into the norepinephrine LC (min). Blue level bars: duringperfusion perfusion of 200 nMwith guanfacine guanfacine into (from LC. 20 Panel to 180 (C min).) indicates Ordinates: the AUC20–180 mean±SD (n min = 6) valuesof AUC20–180 of extracellular min values norepinephrine of extracel- lularlevel norepinephrine during perfusion level with (pmol). guanfacine Black and (from blue 20 columns to 180 min). indicate Ordinates: respective mean control±SD and (n = perfusion 6) of AUC20–180 of guanfacine min values of AUC of valuesextracellular of norepinephrine norepinephrine levels. level When (pmol). the Blackeffects and of guanfacine blue columns were indicate statistically respective significant control compared and perfusion using of MANOVA, guanfacine theof AUCdata were values analysed of norepinephrine using Tukey’s levels. post Whenhoc test. the * effectsp < 0.01, of compared guanfacine with were control. statistically @@ p 0.05), [Ftime F(9,180)guanfacine*time = 11.8(9,180) (p < = 0.01), 16.3 F(padministration < 0.01), Fguanfacine*adminisdtration(1,20) = 20.5 (p <(1,20) 0.01), = F guanfacine2.5 (p > 0.1),(1,20) Fadmin- = 3.2 isdtration*time(p > 0.05),(9,180) Fguanfacine*time = 5.3 (p <(9,180) 0.01), F =guanfacine*administration*time 16.3 (p < 0.01), Fguanfacine*adminisdtration(9,180) = 6.8 (p < 0.01](1,20) (Figure = 2.5 4). (p Acute> 0.1), administrationFadminisdtration*time of guanfacine(9,180) = 5.3 (perfusion (p < 0.01), with Fguanfacine*administration*time 200 nM guanfacine into(9,180) the =LC) 6.8 decreased (p < 0.01)] extracellular(Figure4). Acute norepinephrine administration in the of guanfacineRTN (Figure (perfusion 4A), whereas with after 200 nMchronic guanfacine guanfacine into administration,the LC) decreased perfusion extracellular with 200 norepinephrine nM guanfacine in into the the RTN LC (Figuredid not4 A),affect whereas (Figure after 4B). Furthermore,chronic guanfacine chronic administration, administration perfusionof guanfacine with increased 200 nM guanfacinebasal extracellular into the norepi- LC did nephrinenot affect level (Figure in the4B). RTN Furthermore, (Figure 4C). chronic Therefore, administration similar to of LC-OFC guanfacine and increasedLC-VTA nora- basal drenergicextracellular pathways, norepinephrine chronic administration level in the RTN of (Figure therapeutic-relevant4C). Therefore, does similar of toguanfacine LC-OFC (0.12and mg/kg/day LC-VTA noradrenergic for 14 days), pathways,prevented the chronic acute administrationinhibitory effects of of therapeutic-relevant guanfacine on the LC-RTNdoes of guanfacinenoradrenergic (0.12 transmission, mg/kg/day but for enha 14 days),nces preventedthe LC-RTN the noradrenergic acute inhibitory transmis- effects sion.of guanfacine on the LC-RTN noradrenergic transmission, but enhances the LC-RTN noradrenergic transmission.

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FigureFigure 4. 4.Effects Effects ofof acutelyacutely locallocal administrationadministration (perfusion with) 200 200 nM nM guanfacine guanfacine in intoto the the LC LC on on extracellular extracellular norepi- nore- pinephrinenephrine level level in in the the RTN, RTN, after the systemicallysystemically chronicchronic administrationadministration of of guanfacine guanfacine (0 (0 or or 0.12 0.12 mg/kg/day mg/kg/day forfor 1414 days).days). Panel (A) indicates the effects of perfusion with 200 nM guanfacine into the LC after chronic administration of guanfacine Panel (A) indicates the effects of perfusion with 200 nM guanfacine into the LC after chronic administration of guanfacine (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 200 nM guanfacine after systemically chronic ad- (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 200 nM guanfacine after systemically chronic ministration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean ± SD (n = 6) of extracellular ± administrationnorepinephrine of level guanfacine (nM), abscissa: (0.12 mg/kg/day) time after foradministration 14 days (chronic of perfusion effects). Ordinates:with guanfacine mean intoSD the (n =LC 6) (min). of extracellular Blue bars: norepinephrineperfusion of 200 level nM (nM),guanfacine abscissa: into time LC. afterPanel administration (C) indicates the of perfusionAUC20–180 min with values guanfacine of extracellular into the LCnorepinephrine (min). Blue bars: level perfusionduring perfusion of 200 nM with guanfacine guanfacine into (from LC. Panel 20 to (180C) indicatesmin). Ordinates: the AUC mean±SD20–180 min values(n = 6) of AUC extracellular20–180 min values norepinephrine of extracellular level duringnorepinephrine perfusion withlevel guanfacine(pmol). Black (from and 20 toblue 180 columns min). Ordinates: indicate meanrespective±SD (ncontrol = 6) of and AUC perfusion20–180 min ofvalues guanfacine of extracellular of AUC norepinephrinevalues of norepinephrine level (pmol). levels. Black When and blue the columnseffects of indicate guanfacine respective were statistically control and significant perfusion of compared guanfacine using of AUC MANOVA, values ofthe norepinephrine data were analysed levels. using When Tukey’s the effects post of hoc guanfacine test. ** p were < 0.01, statistically compared signiﬁcant with control. compared @@ p < using0.01: compared MANOVA, with the dataacute wereeffects analysed of guanfacine. using Tukey’s post hoc test. ** p < 0.01, compared with control. @@ p < 0.01: compared with acute effects of guanfacine. 2.1.5. Effects of Acute and Chronic Administration of Therapeutically Relevant Dose of 2.1.5.Guanfacine Effects on of AcuteExtracellular and Chronic GABA Administration Level in the MDTN of Therapeutically Relevant Dose of GuanfacinePerfusion on Extracellularwith guanfacine GABA into Level the LC, in the after MDTN systemically chronic administration (0 or 0.12Perfusion mg/kg/day with guanfacine) guanfacine for into 14 thedays, LC, affected after systemically extracellular chronic GABA administrationlevel in the MDTN (0 or[Ftime 0.12(7.3,144.5) mg/kg/day = 6.6 ( guanfacine)p < 0.01), Fadministration for 14 days,(1,20) affected= 4.0 (p > extracellular0.05), Fguanfacine GABA(1,20) = level 1.9 (p in > the0.1), MDTNFguanfacine*time [Ftime(7.3,144.5)(7.3,144.5) = =7.7 6.6 ( (pp < 0.01), FFadministrationguanfacine*adminisdtration(1,20) =(1,20) 4.0 ( p= >0.8 0.05), (p > F guanfacine0.1), Fadminisdtra-(1,20) =tion*time 1.9 (p(7.3,144.5)> 0.1), Fguanfacine*time = 5.8 (p < 0.01),(7.3,144.5) Fguanfacine*administration*time = 7.7 (p < 0.01),(7.3,144.5) Fguanfacine*adminisdtration = 5.5 (p < 0.01)](1,20) (Figure = 0.8 5). (pAcute> 0.1 ),administration Fadminisdtration*time of guanfa(7.3,144.5)cine (perfusion = 5.8 (p < 0.01), with F200guanfacine*administration*time nM guanfacine into the(7.3,144.5) LC) de- =creased 5.5 (p < extracellular 0.01)] (Figure GABA5). Acute in the administration MDTN (Figure of 5A), guanfacine whereas (perfusion after chronic with guanfacine 200 nM guanfacineadministration, into the perfusion LC) decreased with 200 extracellular nM guanfacine GABA into in the MDTNLC had (Figureno effect5A), (Figure whereas 5B). afterContrary chronic to guanfacinecatecholamine, administration, the chronic perfusion administration with 200 of nMguanfacine guanfacine did into not theaffect LC basal had noextracellular effect (Figure GABA5B). Contrarylevel in the to catecholamine,MDTN (Figure the 5C). chronic The GABAergic administration neurons of guanfacine receive ex- didcitatory not affect noradrenergic basal extracellular inputs via GABA postsynaptic level in the α1 MDTN adrenoceptor (Figure5 [24,34,35].C). The GABAergic The reduction neu- ronsin GABA receive level excitatory in the MDTN noradrenergic by local administration inputs via postsynaptic of guanfacineα1 adrenoceptor into the LC is[ generated24,34,35]. Theby reduced reduction norepinephrine in GABA level release in the in MDTN the RTN. by Contrary local administration to acute response, of guanfacine the increased into theextracellular LC is generated norepinephrine by reduced level norepinephrine cannot produce release the enhancement in the RTN. of ContraryGABAergic to acuteinputs response,to the MDTN, the increased which suggests extracellular that excitatory norepinephrine noradrenergic level cannottransmission produce to the the RTN enhance- prob- mentably affects of GABAergic phasic activation inputs to [24,35]. the MDTN, which suggests that excitatory noradrenergic transmission to the RTN probably affects phasic activation [24,35].

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FigureFigure 5. 5.Effects Effects ofof acutely acutely local local administration administration (perfusion(perfusion with)with) 200200 nMnMguanfacine guanfacine intointo thetheLC LC onon extracellular extracellular GABAGABA levellevel in in the the MDTN, MDTN, after after the the systemically systemically chronic chronic administration administration of of guanfacine guanfacine (0 (0 or or 0.12 0.12 mg/kg/day mg/kg/day forfor 1414 days). days). PanelPanel (A(A)) indicatesindicates thethe effectseffects ofof perfusionperfusion with 200 nM nM guanfacine guanfacine in intoto the the LC LC after after chronic chronic administration administration of of guanfacine guanfacine (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 200 nM guanfacine after systemically chronic admin- (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 200 nM guanfacine after systemically chronic istration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean±SD (n = 6) of extracellular GABA administration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean±SD (n = 6) of extracellular level (μM), abscissa: time after administration of perfusion with guanfacine into the LC (min). Blue bars: perfusion of 200 GABAnM guanfacine level (µM), into abscissa: LC. Panel time (C after) indicates administration the AUC20–180 of perfusion min values with guanfacineof extracellular into theGABA LC (min).level during Blue bars: perfusion perfusion with ofguanfacine 200 nM guanfacine (from 20 intoto 180 LC. min). Panel Ordinates: (C) indicates mean the ± AUC20–180 SD (n = 6) minof AUC20–180 values of extracellular min values GABA of extracellular level during GABA perfusion level with(pmol). guanfacine Black and (from blue 20 columns to 180 min).indicate Ordinates: respective mean control± SD and (n perfusion = 6) of AUC20–180 of guanfacine min of values AUC ofvalues extracellular of GABA GABA levels. levelWhen (pmol). the effects Black of and guanfacine blue columns were indicatestatistically respective significant control compared and perfusion using MANOVA, of guanfacine the data of AUC were values analysed of GABA using levels.Tukey’s When post thehoc effectstest. * p of < guanfacine0.05, compared were with statistically control. signiﬁcant compared using MANOVA, the data were analysed using Tukey’s post hoc test. * p < 0.05, compared with control. 2.2. Effects of Local Administration of Prazosine into the VTA on Extracellular Levels of Dopa- 2.2.mine Effects in the of OFC Local of AdministrationChronic Guanfa ofcine Prazosine Administration into the VTA (Study on Extracellular2) Levels of Dopamine in theTo OFC determine of Chronic the Guanfacine effects of Administration local administrations (Study 2) of α1 adrenoceptor antagonist, 100 μM prazosinTo determine (PRZ) the [27,28,36,37] effects of local into administrations the VTA on extracellular of α1 adrenoceptor dopamine antagonist, level in the 100 OFC,µM prazosinduring the (PRZ) systemically [27,28,36 chronic,37] into administration the VTA on extracellular of guanfacine dopamine (0 or 0.12 levelmg/kg/day in the OFC,for 14 duringdays) using the systemically osmotic pump chronic (N = 24), administration microdialysis of probes guanfacine were (0inserted or 0.12 into mg/kg/day the VTA and for 14OFC. days) During using the osmotic chronic pump guanfacine-free (N = 24), microdialysis administration probes for 14 were days, inserted the perfusate into the in VTA the andVTA OFC. was Duringswitched the from chronic MRS guanfacine-free to MRS containing administration PRZ (100 μM) for (acute 14 days, effects). the perfusate During the in thesystemically VTA was switchedchronic administration from MRS to MRS of guanfacine containing (0.12 PRZ mg/kg/day) (100 µM) (acute for 14 effects). days, Duringthe per- thefusate systemically in VTA was chronic switched administration from MRS to of MRS guanfacine containing (0.12 PRZ mg/kg/day) (100 μM) (chronic for 14 days, effects). the perfusatePerfusion in VTA with was 100 switched μM PRZ from into MRS the VTA, to MRS during containing systemically PRZ (100 chronicµM) (chronic administration effects). (0 orPerfusion 0.12 mg/kg/day with100 guanfacine)µM PRZ for into 14 thedays, VTA, affected during extracellular systemically dopamine chronic level adminis- in the trationOFC [F (0time or(6.0,119.2) 0.12 mg/kg/day = 20.2 (p < guanfacine) 0.01), Fadministration for 14(1,20) days, = 26.9 affected (p < 0.01), extracellular Fguanfacine dopamine(1,20) = 7.3 level(p < 0.05), in the F OFCguanfacine*time [Ftime(6.0,119.2)(6.0,119.2) = =19.2 20.2 (p ( p< <0.01), 0.01), Fguanfacine*adminisdtration Fadministration(1,20)(1,20) = 26.9 = 0.2 (p (

0.01), 0.1), FFguanfacineadminisdtration*time(1,20)(6.0,119.2) = 7.3 (p = <3.5 0.05),(p < 0.01), Fguanfacine*time Fguanfacine*administration*time(6.0,119.2)(6.0,119.2) = 19.2 = 1.9 (p (

0.01),0.05)] F(Figureguanfacine*adminisdtration 6). Perfusion with(1,20) 100 = μ 0.2M PRZ (p > 0.1),into the Fadminisdtration*time VTA decreased(6.0,119.2) extracellular = 3.5 dopamine (p < 0.01), in Ftheguanfacine*administration*time OFC, and after chronic(6.0,119.2) guanfacine = 1.9 administration, (p > 0.05)] (Figure perfusion6). Perfusion with with 100 100μM PRZµM PRZ into intothe theVTA VTA also decreased decreased extracellular (Figure 6A,B). dopamine Furthermore, in the OFC, the and chronic after chronic administration guanfacine of administration,guanfacine increased perfusion basal with extracellular 100 µM PRZ dopa intomine the VTA level also in decreasedthe OFC (Figure 6 6C).A,B). These Fur- thermore,results suggest the chronic that the administration responses of of dopamine guanfacinergic increased neurons basal in the extracellular VTA to the dopamine excitatory levelnoradrenergic in the OFC inputs (Figure from6C). the These LC are results not af suggestfected by that chronic the responses guanfacine of dopaminergicadministration, neuronswhereas in the the elevation VTA to theof basal excitatory extracellular noradrenergic dopamine inputs level from in thethe LC OFC are was not affectedinduced byby chronicchronic guanfacineguanfacineadministration, administration. whereas the elevation of basal extracellular dopamine level in the OFC was induced by chronic guanfacine administration.

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FigureFigure 6. 6. EffectsEffects of of acutely acutely local local administration administration (perfusion (perfusion with) with) 100 100 μµMM prazosin prazosin (PRZ) (PRZ) into into the the VTA VTA on on extracellular dopaminedopamine level level in in the the OFC, after the system systemicallyically chronic administration of guanfacineguanfacine (0(0 oror 0.120.12 mg/kg/daymg/kg/day for for 14 14 days). days). μ PanelPanel ( (AA)) indicates indicates the the effects effects of of perfusion perfusion with with 100 100 MµM PRZ PRZ into into the the VTA VTA after after chronic chronic administration administration of guanfacine of guanfacine (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 100 μM PRZ after systemically chronic administration (0 mg/kg/day) for 14 days (acute effects). Panel (B) indicates effects of 100 µM PRZ after systemically chronic administration of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean ± SD (n = 6) of extracellular dopamine level of guanfacine (0.12 mg/kg/day) for 14 days (chronic effects). Ordinates: mean ± SD (n = 6) of extracellular dopamine level (nM), abscissa: time after administration of perfusion with PRZ into the VTA (min). Green bars: perfusion of 100 μM PRZ (nM), abscissa: time after administration of perfusion with PRZ into the VTA (min). Green bars: perfusion of 100 µM PRZ into VTA. Panel (C) indicates the AUC20–180 min values of extracellular dopamine level during perfusion with PRZ (from 20 tointo 180 VTA. min). Panel Ordinates: (C) indicates mean±SD the AUC (n =20–180 6) of minAUCvalues20–180 min of values extracellular of extracellular dopamine dopamine level during level perfusion (pmol). with Black PRZ and (from green 20 columnsto 180 min). indicate Ordinates: respective mean control±SD (n and = 6) perfusion of AUC20–180 of PRZ min valuesof AUC of values extracellular of dopamine dopamine levels. level When (pmol). the Black effects and of greenPRZ werecolumns statistically indicate significant respective controlcompared and using perfusion MANOVA, of PRZ th ofe AUC data valueswere analysed of dopamine using levels. Tukey’s When post the hoc effects test. of* pPRZ < 0.05, were ** pstatistically < 0.01, compared signiﬁcant with compared control. @@ using p < MANOVA,0.01: compared the data with were acute analysed effects of using PRZ. Tukey’s post hoc test. * p < 0.05, ** p < 0.01, compared with control. @@ p < 0.01: compared with acute effects of PRZ. 2.3. Effects of Local Administration of AMPA into the MDTN on Extracellular Levels of Norepi- nephrine,2.3. Effects Dopamine of Local Administrationand L-glutamate of in AMPA the OFC into of the Chronic MDTN Guanfacine on Extracellular Administration Levels of (Study 3)Norepinephrine, Dopamine and L-glutamate in the OFC of Chronic Guanfacine Administration (StudyTo 3)determine the effects of local administrations of AMPA/glutamate receptor agonist, 100To determineμM AMPA the into effects the MDTN of local administrationson extracellular oflevels AMPA/glutamate of norepinephrine, receptor dopamine agonist, and100 µL-glutamateM AMPA into in the the OFC, MDTN after on the extracellular systemically levels chronic of norepinephrine, administration dopamineof guanfacine and (0L-glutamate or 0.12 mg/kg/day) in the OFC, for 14 after days the using systemically osmotic chronicpump (N administration = 12), microdialysis of guanfacine probes were (0 or inserted0.12 mg/kg/day) into the MDTN for 14 and days OFC. using During osmotic the pumpchronic (N guanfacine-free = 12), microdialysis administration probes were for 14inserted days, the into perfusate the MDTN in the and MDTN OFC. was During switched the chronic from MRS guanfacine-free to MRS containing administration 100 μM AMPAfor 14 days,(acute theeffects). perfusate During in the the systemically MDTN was chronic switched administration from MRS toof guanfacine MRS containing (0.12 mg/kg/day)100 µM AMPA for (acute14 days, effects). the perfusate During thein MDTN systemically was switched chronic administration from MRS to MRS of guanfacine containing(0.12 AMPA mg/kg/day) (100 μM) for (chronic 14 days, effects). the perfusate in MDTN was switched from MRS to MRS containingPerfusion AMPA with (100 100 µμM)M AMPA (chronic into effects). the MDTN, during systemically chronic admin- istrationPerfusion (0 or 0.12 with mg/kg/day 100 µM AMPA guanfacine) into the for MDTN, 14 days, during affected systemically extracellular chronic norepineph- adminis- tration (0 or 0.12 mg/kg/day guanfacine) for 14 days, affected extracellular norepinephrine rine level in the OFC [Ftime(7.9,79.4) = 44.8 (p < 0.01), Fadministration(1,10) = 23.8 (p < 0.01), Fadmin- level in the OFC [F (7.9,79.4) = 44.8 (p < 0.01), F (1,10) = 23.8 (p < 0.01), isdtration*time(7.9,79.4) = 11.7time (p < 0.01)] (Figure 7A,B). Perfusionadministration with 100 μM AMPA into the µ MDTNFadminisdtration*time increased (7.9,79.4)extracellular = 11.7 norepinephrine (p < 0.01)] (Figure in7 A,B).the OFC Perfusion (Figure with 7A,B). 100 MChronic AMPA into the MDTN increased extracellular norepinephrine in the OFC (Figure7A,B). Chronic guanfacine administration enhanced 100 μM AMPA-evoked norepinephrine release in guanfacine administration enhanced 100 µM AMPA-evoked norepinephrine release in the the OFC (Figure 7A,C). Furthermore, chronic administration of guanfacine increased ba- OFC (Figure7A,C). Furthermore, chronic administration of guanfacine increased basal sal extracellular norepinephrine level in the OFC (Figure 7A,B). extracellular norepinephrine level in the OFC (Figure7A,B). Perfusion with 100 μM AMPA into the MDTN, during systemically chronic administration (0 or 0.12 mg/kg/day guanfacine) for 14 days, affected extracellular dopamine level in the OFC [Ftime(9,90) = 57.1 (p < 0.01), Fadministration(1,10) = 45.5 (p < 0.01), Fadminisdtra- tion*time(9,90) = 25.6 (p<0.01)] (Figure 7C,D). Perfusion with 100 μM AMPA into the MDTN increased extracellular dopamine in the OFC (Figure 7C,D). Chronic guanfacine administration enhanced 100 μM AMPA-evoked dopamine release in the OFC (Figure 7C,D). Furthermore, the chronic administration of guanfacine increased basal extracellular dopamine level in the OFC (Figure 7C,D).

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Perfusion with 100 μM AMPA into the MDTN, during systemically chronic administration (0 or 0.12 mg/kg/day guanfacine) for 14 days, affected extracellular L-glutamate level in the OFC [Ftime(9,90) = 53.7 (p < 0.01), Fadministration(1,10) = 7.4 (p < 0.01), Fadminisdtra- tion*time(9,90) = 12.5 (p < 0.01)] (Figure 7E,F). Perfusion with 100 μM AMPA into the MDTN increased extracellular L-glutamate level in the OFC (Figure 7E,F). Chronic guanfacine administration enhanced 100 μM AMPA-evoked dopamine release in the OFC (Figure Int. J. Mol. Sci. 2021, 22, 4122 7E,F). Contrary to catecholamine, chronic administration of guanfacine did not affect10 basal of 22 L-glutamate level in the OFC (Figure 7E,F).

FigureFigure 7. 7. EffectsEffects of acutely local administrationadministration (perfusion(perfusion with) with) 100 100µ MμMα -amino-3-hydroxy-5-methyl-4-isoxazolepropionicα-amino-3-hydroxy-5-methyl-4-isoxazolepropi- onicacid acid (AMPA) (AMPA) into into the MDTNthe MDTN on extracellular on extracellular levels levels of norepinephrine, of norepinephrine, dopamine dopamine and and L-glutamate L-glutamate in the in the OFC, OFC, after after the thesystemically systemically chronic chronic administration administration of guanfacine of guanfacine (0 or (00.12 or 0.12 mg/kg/day mg/kg/day for for 14 14 days). days). Panels Panels (A (,AC,,CE),E indicate) indicate the the effects effects of ofperfusion perfusion with with 100 100µM μ AMPAM AMPA into into the the MDTN MDTN after after chronic chronic administration administrati ofon guanfacine of guanfacine (0 or 0.12(0 or mg/kg/day)0.12 mg/kg/day) for 14 for days 14 dayson extracellular on extracellular levels levels of norepinephrine, of norepinephrine, dopamine dopamine and L-glutamate and L-glutamate in the in OFC, the respectively.OFC, respectively. Ordinates: Ordinates: mean± mean±SDSD (n = 6) (nof = extracellular 6) of extracellular levels oflevels norepinephrine of norepinephrine (nM), dopamine(nM), dopamine (nM) or (nM) L-glutamate or L-glutamate (µM), abscissa:(μM), abscissa: time after time administration after administration of perfusion with AMPA into the MDTN (min). Red bars: perfusion of 100 μM AMPA into the MDTN. Panels of perfusion with AMPA into the MDTN (min). Red bars: perfusion of 100 µM AMPA into the MDTN. Panels (B,D,F) (B,D,F) indicate the AUC20–180 min values of extracellular levels of norepinephrine, dopamine and L-glutamate during indicate the AUC20–180 min values of extracellular levels of norepinephrine, dopamine and L-glutamate during perfusion perfusion with AMPA (from 20 to 180 min). Ordinates: mean ± SD (n = 6) of AUC20–180 min values of extracellular levels ofwith norepinephrine AMPA (from (pmol), 20 to 180 dopamine min). Ordinates:(pmol) and mean L-glutamate± SD (n (nmol), = 6) of respective AUC20–180ly. Opened, min values black of and extracellular red columns levels indi- of catenorepinephrine AUC vales during (pmol), pre-AMPA dopamine (pmol)and AMPA-evoked and L-glutamate releases (nmol), control respectively. and chronic Opened, guanfacine black andadministrations, red columns indicaterespec- tively.AUC valesWhen during the effects pre-AMPA of AMPA and were AMPA-evoked statistically releasessignificant control compared and chronic using MANOVA, guanfacine the administrations, data were analysed respectively. using Tukey’sWhen the post effects hoc test. of AMPA ** p < were0.01, statisticallycompared with signiﬁcant control. compared using MANOVA, the data were analysed using Tukey’s post hoc test. ** p < 0.01, compared with control. 2.4. Effects of Subacute and Chronic Guanfacine Administration on α2A Adrenoceptor ExpressionPerfusion in the with Plasma 100 MembraneµM AMPA of into LC, theOFC MDTN, and VTA during systemically chronic administration (0 or 0.12 mg/kg/day guanfacine) for 14 days, affected extracellular dopamine level in After the systemically subchronic (for 7 days) and chronic (for 14 days) administra- the OFC [F (9,90) = 57.1 (p < 0.01), F (1,10) = 45.5 (p < 0.01), tion of guanfacinetime (0.12 mg/kg/days) using osmoticadministration pump (N = 12), the total plasma mem- F (9,90) = 25.6 (p<0.01)] (Figure7C,D). Perfusion with 100 µM AMPA into braneadminisdtration*time proteins were extracted using Minute Plasma Membrane Protein Isolation Kit (In- the MDTN increased extracellular dopamine in the OFC (Figure7C,D). Chronic guan- vent Biotechnologies, Plymouth, MN, USA). The control was treated with vehicle (0.9% facine administration enhanced 100 µM AMPA-evoked dopamine release in the OFC (Figure7C,D ). Furthermore, the chronic administration of guanfacine increased basal extracellular dopamine level in the OFC (Figure7C,D). Perfusion with 100 µM AMPA into the MDTN, during systemically chronic administration (0 or 0.12 mg/kg/day guanfacine) for 14 days, affected extracellular L- glutamate level in the OFC [Ftime(9,90) = 53.7 (p < 0.01), Fadministration(1,10) = 7.4 (p < 0.01), Fadminisdtration*time(9,90) = 12.5 (p < 0.01)] (Figure7E,F). Perfusion with 100 µM AMPA into the MDTN increased extracellular L-glutamate level in the OFC (Figure7E,F). Chronic guanfacine administration enhanced 100 µM AMPA-evoked dopamine release in the OFC Int. J. Mol. Sci. 2021, 22, 4122 11 of 22

(Figure7E,F). Contrary to catecholamine, chronic administration of guanfacine did not affect basal L-glutamate level in the OFC (Figure7E,F).

2.4. Effects of Subacute and Chronic Guanfacine Administration on α2A Adrenoceptor Expression in the Plasma Membrane of LC, OFC and VTA After the systemically subchronic (for 7 days) and chronic (for 14 days) administra- Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 22 tion of guanfacine (0.12 mg/kg/days) using osmotic pump (N = 12), the total plasma membrane proteins were extracted using Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA). The control was treated with vehicle (0.9% saline)saline) for for 14 14 days days using using osmotic osmotic pump. pump. Subchronic Subchronic (for (for 7 7 days) days) and and chronic chronic (for (for 14 14 days) days) guanfacineguanfacine administration administrationss time-dependently time-dependently decreased α2A2A adrenoceptor adrenoceptor expressions expressions in in thethe plasma plasma membrane membrane of LC [F(2,15) = 9.2, 9.2, p << 0.01], 0.01], OFC OFC [F(2,15) [F(2,15) = = 20.6, 20.6, pp << 0.01] 0.01] and and VTA VTA [F(2,15)[F(2,15) = = 13.6, 13.6, pp << 0.01], 0.01], respectively respectively (Figure (Figure 88).). ExpressionExpression ofof α2A2A adrenoceptor adrenoceptor in in the plasmaplasma membrane ofof LCLC waswas decreased decreased by by chronic chronic (for (for 14 14 days) days) guanfacine guanfacine administration, administration,but notbut affectednot affected by subchronic by subchronic (for 7(for days) 7 days) administration administrati (Figureon (Figure8A). 8A). Contrary Contrary to LC, to LC,both both subchronic subchronic (for (for 7 days) 7 days) and and chronic chronic (for (for 14 days) 14 days) decreased decreasedα2A α2A adrenoceptor adrenoceptor in the in theplasma plasma membrane membrane of OFC of OFC and and VTA VTA (Figure (Figures8B,C). 8B,C).

FigureFigure 8. 8. EffectsEffects of of systemically systemically subchronic subchronic (for (for 7 days: 7 days: light light blue blue columns) columns) and and chronic chronic (for (for 14 days: 14 days: blue blue columns) columns) ad- α ministrationsadministrations of ofguanfacine guanfacine on on expression expression of of2Aα2A adrenoceptor adrenoceptor in in the the plasma plasma membrane membrane of of LC LC (Panel (Panel ( (AA)),)), OFC OFC (Panel (Panel (B)) and VTA (Panel (C)), using capillary immunoblotting system. Ordinate: mean ± SD (n = 6) of the relative protein level (B)) and VTA (Panel (C)), using capillary immunoblotting system. Ordinate: mean ± SD (n = 6) of the relative protein level of α2A adrenoceptor (ADRA2A) in the plasma membrane fraction. Effects of guanfacine on α2A adrenoceptor expression of α2A adrenoceptor (ADRA2A) in the plasma membrane fraction. Effects of guanfacine on α2A adrenoceptor expression were analysed by one-way ANOVA with Tukey’s post hoc test (* p < 0.05, ** p < 0.01 vs. Control). The pseudo-gel images, usingwere analysedcapillary byimmunoblotting one-way ANOVA were with represented Tukey’s postin the hoc lower test panels. (* p < 0.05, ** p < 0.01 vs. Control). The pseudo-gel images, using capillary immunoblotting were represented in the lower panels. 3. Discussion 3. Discussion Catecholaminergic neurons in the LC project catecholaminergic terminals to various brain Catecholaminergicregions, including neuronsthe OFC, in VTA the LC and project RTN catecholaminergic[24–28,34,35,38]. Interestingly, terminals to variousthe LC projectsbrain regions, selective including noradrenergic the OFC, terminals VTA and to the RTN deeper [24–28 layers,34,35 of,38 the]. Interestingly,OFC (the mesocorti- the LC calprojects noradrenergic selective noradrenergic pathway) and terminals co-releasing to the (dopamine deeper layers and of norepinephrine) the OFC (the mesocortical terminals to the superficial layers of the OFC (the mesocortical co-releasing catecholaminergic pathway) [24–29,36]. For the other noradrenergic projections, the LC-VTA noradrenergic pathway activates dopaminergic transmission in the mesocortical (VTA-OFC) dopaminergic pathway via the excitatory postsynaptic α1 adrenoceptor [38]. Similar to the LC-VTA noradrenergic pathway, the LC-RTN noradrenergic pathway also activates intrathalamic (RTN-MDTN) GABAergic transmission via postsynaptic α1 adrenoceptor [24,29,34,35]. Therefore, the reduction in3 mesothalamic noradrenergic transmission enhances thalamocortical (MDTN-OFC) glutamatergic transmission due to intrathalamic GABAer-

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noradrenergic pathway) and co-releasing (dopamine and norepinephrine) terminals to the superficial layers of the OFC (the mesocortical co-releasing catecholaminergic pathway) [24–29,36]. For the other noradrenergic projections, the LC-VTA noradrenergic pathway activates dopaminergic transmission in the mesocortical (VTA-OFC) dopaminergic α Int. J. Mol. Sci. 2021, 22, x FOR PEERpathway REVIEW via the excitatory postsynaptic 1 adrenoceptor [38]. Similar to the LC-VTA12 of 22 noradrenergic pathway, the LC-RTN noradrenergic pathway also activates intrathalamic (RTN-MDTN) GABAergic transmission via postsynaptic α1 adrenoceptor [24,29,34,35]. Therefore, the reduction in3 mesothalamic noradrenergic transmission enhances thalamo- corticalgic disinhibition. (MDTN-OFC) Interestingly, glutamatergic thalamocortica transmissionl glutamatergic due to intrathalamic transmission GABAergic selectively disinhi- ac- bition.tivates Interestingly, the mesocortical thalamocortical co-releasing glutamatergic catecholaminergic transmission terminal selectively in superficial activates layers the of mesocorticalthe frontalco-releasing cortex but catecholaminergic does not affect terminal the mesocortical in superficial layersnoradrenergic of the frontal pathway cortex[24,27,28,31,35,39]. but does not affect Based the mesocortical on these previous noradrenergic findings, pathway to explore [24,27 the,28 ,31clinical,35,39 ].action Based of onguanfacine, these previous the present findings, study to explore determined the clinical the chronic action ofadministration guanfacine, the of presenttherapeutically study determinedrelevant doses the chronicof guanfacine administration on catecholam of therapeuticallyinergic transmission relevant in doses the LC-OFC of guanfacine and LC- onVTA, catecholaminergic GABAergic inhibition transmission in LC-RTN-MDTN, in the LC-OFC and and thalamocortical LC-VTA, GABAergic glutamatergic inhibition trans- inmission LC-RTN-MDTN, in MDTN-OFC. and thalamocortical The catecholaminergic glutamatergic transmission transmission pathway in MDTN-OFC.associated with The the catecholaminergicLC is represented transmission in Figure 9. pathway associated with the LC is represented in Figure9.

FigureFigure 9. 9.The The proposed proposed hypothesis hypothesis for for the the catecholaminergic, catecholaminergic, noradrenergic,noradrenergic, andand co-releasingco-releasing catecholaminergic (norepi- (nore- nephrine/dopamine) LC-OFC pathways, the intermediate glutamatergic LC-OFC (LC-RTN-MDTN-OFC) pathway, and pinephrine/dopamine) LC-OFC pathways, the intermediate glutamatergic LC-OFC (LC-RTN-MDTN-OFC) pathway, and the LC-VTA (LC-VTA-OFC) dopaminergic pathway. LC projects three afferents, including the selective norepinephrine the LC-VTA (LC-VTA-OFC) dopaminergic pathway. LC projects three afferents, including the selective norepinephrine releasing terminal, to deeper OFC (the direct noradrenergic pathway), RTN (a part of the intermediate glutamatergic releasingpathway), terminal, VTA (a to part deeper of the OFC intermediate (the direct dopaminergic noradrenergic path pathway),way), and RTN superficial (a part of OFC the layers intermediate (the direct glutamatergic co-releasing pathway),catecholaminergic VTA (a part pathway). of the intermediate The co-releasi dopaminergicng terminal in pathway), the OFC receives and superﬁcial excitatory OFC glutamatergic layers (the input direct from co-releasing MDTN (a catecholaminergicpart of the intermediate pathway). glutamatergic The co-releasing pathway). terminal The inselect theive OFC norepinephrine receives excitatory releasing glutamatergic terminal in input RTN from activates MDTN the (aGABAergic part of the intermediateneuron in RTN, glutamatergic resulting in pathway). the inhibition The selectiveof the MDTN norepinephrine glutamatergic releasing neuron. terminal The selective in RTN no activatesrepinephrine the GABAergicreleasing terminal neuron in in RTN, the VTA resulting activates in the th inhibitione dopaminergic of the MDTNneuron glutamatergicin the VTA via neuron. the α1 The adrenoceptor, selective norepinephrine resulting in in- releasingcreased terminaldopamine in therelease VTA in activates the OFC. the The dopaminergic acute activation neuron of the in the postsy VTAnaptic/somatodendritic via the α1 adrenoceptor, α2A resulting adrenoceptor in increased in the dopamineLC by guanfacine release in reduces the OFC. noradrenergi The acute activationc transmission of the in postsynaptic/somatodendritic noradrenergic LC-OFC and LC-VTAα2A adrenoceptor pathways and in the catechola- LC by minergic transmission in the co-releasing catecholaminergic LC-OFC pathway. Contrary to the acute effect of guanfacine, guanfacine reduces noradrenergic transmission in noradrenergic LC-OFC and LC-VTA pathways and catecholaminergic chronic guanfacine administration enhances catecholaminergic transmission in both noradrenergic LC-OFC and LC-VTA transmission in the co-releasing catecholaminergic LC-OFC pathway. Contrary to the acute effect of guanfacine, chronic pathways and catecholaminergic LC-OFC pathways via the downregulation of inhibitory α2A adrenoceptors. guanfacine administration enhances catecholaminergic transmission in both noradrenergic LC-OFC and LC-VTA pathways and catecholaminergic LC-OFC pathwaysIn our viaprevious the downregulation study, acute of local inhibitory administα2Aration adrenoceptors. into the LC and subchronic (0.12 mg/kg/day for 7 days) administrations of guanfacine decreased basal extracellular levels In our previous study, acute local administration into the LC and subchronic of dopamine and norepinephrine in the OFC [24,35], whereas the present study demon- (0.12 mg/kg/day for 7 days) administrations of guanfacine decreased basal extracellular strated that chronic administration of the therapeutically relevant dose [17,24,30] of levels of dopamine and norepinephrine in the OFC [24,35], whereas the present study guanfacine (0.12 mg/kg/day for 14 days) increased basal extracellular levels of dopamine demonstrated that chronic administration of the therapeutically relevant dose [17,24,30] of and norepinephrine in the OFC and prevented the inhibitory effects of the local administration of guanfacine into the LC. The mechanisms of the time-dependent discrepant effects between acute, subchronic (for 7 days), and chronic (for 14 days) guanfacine administrations are probably induced by a combination of desensitization and downregulation of α2A adrenoceptor in the LC because the α2A adrenoceptor in the LC was downregulated by chronic (for 14 days) guanfacine administration but not by subchronic (for 7 days) administration (Figure 8). The α2A adrenoceptor in the LC is expressed in both presynaptic and postsynaptic/somatodendritic regions, which operates as an autoreceptor [24,35,40]. The α2A adrenoceptor is an inhibitory receptor that acts via coupling to the Gi

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guanfacine (0.12 mg/kg/day for 14 days) increased basal extracellular levels of dopamine and norepinephrine in the OFC and prevented the inhibitory effects of the local administration of guanfacine into the LC. The mechanisms of the time-dependent discrepant effects between acute, subchronic (for 7 days), and chronic (for 14 days) guanfacine administrations are probably induced by a combination of desensitization and downregulation of α2A adrenoceptor in the LC because the α2A adrenoceptor in the LC was downregulated by chronic (for 14 days) guanfacine administration but not by subchronic (for 7 days) administration (Figure8). The α2A adrenoceptor in the LC is expressed in both presynaptic and postsynaptic/somatodendritic regions, which operates as an autoreceptor [24,35,40]. The α2A adrenoceptor is an inhibitory receptor that acts via coupling to the Gi protein, similar to the serotonin 5-HT1A receptor [41–43]. The chronic administration of selective serotonin reuptake inhibitors enhances serotonergic transmission via the downregulation of the serotonin 5-HT1A receptor [42,43]. Therefore, in the present results, the downregulation of the somatodendritic α2A adrenoceptor in the LC induced by chronic guanfacine administration generates the enhancement of catecholaminergic transmission via the attenuation of the function of the inhibitory α2A adrenoceptor. Contrary to chronic guanfacine administration, the subchronic administration of therapeutically relevant doses of guanfacine (for 7 days) generated weak desensitisation without the downregulation of the α2A adrenoceptor in the LC, resulting in the attenuation of basal extracellular catecholamine levels in the OFC. In other words, the function of the α2A adrenoceptor in the LC is attenuated but possibly remains functional during subchronic guanfacine administration [24]. Contrary to the LC, both subchronic and chronic guanfacine administration time- dependently downregulated the α2A adrenoceptor in the VTA. In the VTA, the α2A adrenoceptor is mainly expressed on the presynaptic region and suppresses norepinephrine release [38], whereas in our previous study, subchronic guanfacine administration weakly decreased basal extracellular dopamine levels in the OFC [24]. Released norepinephrine from noradrenergic terminals in the VTA enhances dopaminergic neurones in the VTA via the activation of the postsynaptic α1 adrenoceptor. The downregulation of the presynaptic α2A adrenoceptor in the VTA activates the postsynaptic α1 adrenoceptor, leading to the activation of dopaminergic neurones due to an enhanced release of norepinephrine induced by the disinhibition associated with the presynaptic α2A adrenoceptor. However, the affinity of the α1 adrenoceptor is more than 10 times lower in terms of sensitivity compared to the α2A adrenoceptor [13]. Therefore, enhanced basal dopamine release in the OFC possibly requires the higher elevation of norepinephrine release than the threshold level for the α1 adrenoceptor, induced by downregulations of both the somatodendritic α2A adrenoceptor in the LC and presynaptic α2A adrenoceptor. A clinical study using a reinforcement-learning task with fMRI revealed that stimulants improved task performance in patients with ADHD (enhancement of reward-learning rates and the ability to differ- entiate optimal from non-optimal novel choices) via reduced VTA responses to novelty compared to the control [20]. Taken together with the clinical findings, the present results suggest that enhanced tonic dopaminergic transmission due to the rapid downregulation of the presynaptic α2A adrenoceptor induced by guanfacine contributes to the mechanisms of clinical action of guanfacine as an improvement in novelty processing. Thalamocortical glutamatergic transmission in the MDTN-OFC pathway plays important roles in cognitive/perception functions, including sensory integration and executive function [23,42,44]. MDTN glutamatergic neurones receive inputs from the amygdala, cortical, and other subcortical regions associated with learning, memory, emotion, and perceptual integration [29,45,46]. Indeed, the model of MDTN lesions continued to respond to stimuli after being satiated with food rewards [47]. Therefore, the thalamocortical (MDTN-OFC) pathway plays important roles in maintaining flexible stimulus–reward associations and emotional processing [48]. In other words, the MDTN regulates the integration of emotional and sensory information to the OFC. Anatomical/functional have also emphasised that functional abnormalities associated with the MDTN are involved in the cognitive dysfunction of ADHD [21]. Glutamatergic neurones in the MDTN output Int. J. Mol. Sci. 2021, 22, 4122 14 of 22

glutamatergic signalling to the OFC through select/integrate various sensory/emotional inputs to the MDTN [24,33,35,39,49–54]. The impairment of sensory/emotional integration in the MDTN is generated by two types of functional abnormalities—the elevation of the threshold for input signalling (less sensitivity to signalling inputs) and the desensitisation of phasic output signalling due to tonic hyperactivation. N-methyl-D-aspartate (NMDA)/glutamate receptor impairment schizophrenia models exhibit hyperactivation of thalamocortical glutamatergic transmission, which is compensated by several atypical antipsychotics, clozapine, aripiprazole and lurasidone [33,49–54]. In contrast, hypo- glutamatergic transmission in the thalamocortical pathway was observed in the genetic rodent model of autism [39,55–58]. A clinical study using fMRI reported that ketamine (an NMDA/glutamate receptor antagonist) suppressed functional LC-MDTN connectivity [59] through intrathalamic GABAergic disinhibition [23]. Although further studies address- ing how the LC-RTN-MDTN pathway is involved in the pathophysiology and cognitive function of various neuropsychiatric disorders are needed, these clinical and preclinical findings emphasise that the LC-RTN-MDTN pathway plays important roles in the efficient switching between hippocampus-dependent and hippocampus-independent learning processes [60] leading to the regulation of attention and memory consolidation/reconsolidation processes [23,24,42–44,61,62]. Both acute guanfacine administration and chronic clonidine administration suppressed locomotion [17,63]. Therefore, both acute and chronic activations of the α2 adrenoceptor at least partially improve hyperactivity. In our previous study, the subchronic administration of therapeutically relevant doses of guanfacine (0.12 mg/kg/day for 7 days) enhanced thalamocortical (MDTN-OFC) glutamatergic transmission [24], and in the present study, chronic guanfacine administration (0.12 mg/kg/day for 14 days) also enhanced thalamocortical glutamatergic transmission; however, the mechanisms of enhanced thalamocortical glutamatergic transmission between acute, subchronic and chronic guanfacine administrations are not identical. Acute and subchronic guanfacine administrations improve phasic thalamocortical glutamatergic transmission, since both acute and subchronic guanfacine administrations lead to intrathalamic (RTN-MDTN) GABAergic disinhibition via reductions in mesothalamic (LC-RTN) noradrenergic transmission [24,35]. In contrast, chronic guanfacine administration also phasically enhanced thalamocortical glutamatergic transmission without affecting intrathalamic GABAergic transmission. Voltage-dependent sodium-channel-inhibiting antiepileptic drugs did not aggravate symptoms, whereas the AMPA/glutamate receptor inhibitor perampanel led to a high incidence of hostile and aggressive reactions [64]. Therefore, the acute and chronic administration of guanfacine improves the integration of sensory/emotional inputs in the MDTN, resulting in the enhancement of phasic glutamatergic output in the thalamocortical pathway. The present study could not explain the detailed mechanisms of these incomprehensible effects of chronic guanfacine administration on thalamocortical glutamatergic transmission. Both GABAergic neurones in the RTN and glutamatergic neurones in the MDTN receive a number of signals associated with GABA, L-glutamate, acetylcholine, noradrenaline, dopamine and serotonin [23,24,33,35,39,42,44,51–53,61,62]. The stimulatory effects of chronic guanfacine administration on thalamocortical glutamatergic transmission are probably mediated by combination with various transmission systems. To clarify this process, further studies are needed. The consistent enhancement of thalamocortical glutamatergic transmission by guanfacine, even if the mechanisms among acute, subchronic and chronic administrations are not identical, can reasonably explain the rapid onset of the clinical effects of guanfacine. Both atomoxetine (a selective norepinephrine transporter inhibitor) and guanfacine (a selective α2A adrenoceptor agonist) are approved for the treatment of ADHD as non- stimulant class medications in Japan [7,24]. Recent meta-analyses and systematic reviews revealed the efficacies of both stimulant and non-stimulant class medications on the management of ADHD symptoms [7,65–67]; however, guanfacine displays a faster onset of improvement in ADHD symptoms compared to atomoxetine [7]. Indeed, one double- blind/placebo-controlled clinical trial demonstrated that guanfacine and atomoxetine Int. J. Mol. Sci. 2021, 22, 4122 15 of 22

displayed effectiveness onsets within 1 and 3 weeks, respectively [7]. Notably, another meta-analysis study reported that atomoxetine exhibited incidences associated with suicidal ideation within one month of starting treatment [9], whereas there are no reports of the risk of suicide-related events with guanfacine [7,10]. Therefore, the long-term effectiveness of atomoxetine and guanfacine are considered to be almost equal in the treatment of ADHD [1–4], whereas during the initiation of treatment with non-stimulant class medication, guanfacine is held in high esteem due to its rapid onset and fewer reports of serious adverse reactions compared to atomoxetine [7,9,10]. Furthermore, compared with atomoxetine, guanfacine probably provides greater control of certain ADHD symptoms, such as hypermotor and oppositional symptoms [7,8]. Noradrenergic transmission in the OFC is considered to be involved in fostering stability and flexibility against environmental changes [68]. The persistent reduced noradrenergic transmission in the OFC generates robust stability and remains in an initial state, but does not respond well to critical changes, whereas conversely, the persistent enhanced noradrenergic transmission found in the OFC generates quite unstable and cycling concentrations in response to a sequence of environmental changes [68]. It has also been established that mesocortical dopaminergic transmission is critically involved in modulating the neural circuits responsible for motivation associated with adaptive responses to rewarding and aversive events [69,70]. Therefore, mesocortical dopaminergic transmission also contributes to stability and flexibility against escape/avoidance events via a combination of suppressing inputs, which does not require immediate action with amplifying inputs associated with escape/avoidance events [71]. Atomoxetine acutely and chronically (for 21 days) enhanced frontal norepinephrine release, whereas atomoxetine-induced norepinephrine release was reduced by chronic atomoxetine administration [72]. Considering the clinical findings that three weeks is needed for the onset of the efficacy of atomoxetine, the downregulation of adrenoceptor probably contributes to the clinical action of atomoxetine. Therefore, the drastic enhanced noradrenergic transmission taking place until the downregulation of the adrenoceptor is induced by atomoxetine may provide unstable and cycling concentrations in response to a sequence of environmental changes. In contrast, during the initial stage, guanfacine selectively activates the α2A adrenoceptor, since guanfacine decreases norepinephrine release until the adrenoceptor is downregulated. Additionally, the enhancement of thalamocortical glutamatergic transmission during the initiation and chronic continuation of medication with guanfacine plays an important role in the rapid onset of the clinical efficacy of guanfacine. Taken together with previous clinical and preclinical findings, the present results regarding the effects of guanfacine on basal extracellular catecholamine levels in the OFC suggest that a combination of the direct activation of the α2A adrenoceptor with a reduction in basal releases of norepinephrine and dopamine in the OFC induced by guanfacine play important roles in the mechanisms of the rapid onset of improvement in hypermotor and oppositional symptoms. Additionally, the enhanced basal extracellular levels of norepinephrine and dopamine in the OFC induced by chronic guanfacine administration also contribute to improvement in the attention deficits of ADHD due to the activation of noradrenergic regulation associated with flexibility and dopaminergic regulation associated with amplifying important information. The present study extensively explored the effects of acute and chronic administration of guanfacine on noradrenergic transmission associated with LC. Other noradrenergic nuclei, nucleus tractus solitarii, and the parabrachial nucleus also project noradrenergic terminals to various subcortical regions, such as the amygdala and thalamic nuclei, affecting cognition and emotions via interactions with monoaminergic transmissions [73–76]. Guan- facine is a selective α2A agonist, but the mechanism of clinical action via the modulation of noradrenergic transmission is probably more complicated than we expect. Therefore, further studies are required to elucidate the detailed pathophysiology of ADHD. Int. J. Mol. Sci. 2021, 22, 4122 16 of 22

4. Materials and Methods 4.1. Chemical Agents and Drug Administration Guanfacine (selective α2A adrenoceptor agonist) was obtained from Cosmo Bio (Tokyo, Japan). Prazosin (PRZ, selective α1 adrenoceptor antagonist) and α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, selective AMPA/glutamate receptor agonist) were obtained from Fujifilm-Wako (Osaka, Japan). All compounds were prepared on the day of experiments. All drugs were perfused in modified Ringer’s solution (MRS), 145 mM Na+, 2.7 mM K+, 1.2 mM Ca2+, 1.0 mM Mg2+, and 154.4 mM Cl− adjusted to pH 7.4 using 2 mM phosphate buffer and 1.1 mM Tris buffer. For microdialysis study, guanfacine and AMPA were dissolved directly in MRS. PRZ was initially dissolved in dimethyl sulfoxide at 25 mM [77,78]. The final dimethyl sulfoxide concentration was lower than 0.1% (vol/vol). To study the effects of systemically subchronic and chronic administrations of therapeutically relevant doses of guanfacine, male Sprague Dawley rats (7 weeks old; SLC, Shizuoka, Japan) (total N = 84) were treated for 7 or 14 days with or without guanfacine (0.12 mg/kg/day), using osmotic pumps (2002; Alzet, Cupertino, CA, USA) implanted subcutaneously in the dorsal region, under 1.8% isoflurane anaesthesia. All rats were weighed prior to initiation of the study [43,50,51,55]. Guanfacine was dissolved in saline at 10 mg/mL/kg body weight. After surgery, rats were housed individually in cages, with food and water ad libitum in a controlled environment (22 ± 1 ◦C) under a 12 h dark/12 h light cycle.

4.2. Preparation of Microdialysis Animal care, experimental procedures, and protocols for the animal experiments were approved by the Animal Research Ethics Committee of the Mie University School of Medicine (No. 29–21, 7 April 2018). To determine the effects of guanfacine on catecholaminergic transmission, all rats were chronically (for 14 days) treated with 0.12 mg/kg/day guanfacine, since the continuous systemic administration of 0.12 mg/kg/day guanfacine for 7 days desensitizes the α2 adrenoceptor. Three days after osmotic pump removal (after systemically chronic administration for 14 days), rats were anesthetized with 1.8% isoflurane and then placed in a stereotaxic frame. Concentric direct insertion-type dialysis probes were implanted in the 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, Kyoto, Japan), 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◦, VTA (A = −5.6 mm, L = +2.1 mm, V = −9.0 mm, relative to bregma; 0.22-mm diameter; 1.5 mm of exposed membrane; Eicom) or 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) according to the atlas of Paxinos and Watson [35,49–52,54,55,79]. Following surgery, rats were individually housed in cages during recovery and the experiment, with food and water provided ad libitum. Perfusion experiments commenced 18 h after recovery from isoflurane anaesthesia. Rats were placed individually into the 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 MRS. Dialysate was collected every 20 min. Extracellular levels of norepinephrine, dopamine and L-glutamate were measured 8 h after the commencement of perfusion [80,81]. The microdialysis experiments were carried out on awake and freely moving rats. To determine the effects of guanfacine, PRZ and AMPA, the perfusion medium was then switched from MRS to MRS containing the target agent. Each dialysate was injected into an ultra-high- performance liquid chromatography (UHPLC) system. After the microdialysis experiments, the brain was removed after cervical dislocation during isoflurane overdose anaesthesia. The locations of the dialysis probes were verified using brain tissue slices by stereoscopic microscope (SZ60, Olympus, Tokyo, Japan). Int. J. Mol. Sci. 2021, 22, 4122 17 of 22

4.3. Determination of Levels of Dopamine, Norepinephrine and L-Glutamate The levels of L-glutamate were determined by UHPLC (PU-4285; Jasco, Tokyo, Japan) with fluorescence detection (FP-4025; Jasco) after dual derivatization with isobutyryl-L- cysteine and o-phthalaldehyde. Derivative reagent solutions were prepared by dissolving isobutyryl-L-cysteine (2 mg) and o-phthalaldehyde (2 mg) in 100 µL of ethanol followed by the addition of 900 µL of sodium borate buffer (0.2 M, pH 9.0). Automated pre-column derivatization was carried out by drawing up a 5 µL aliquot sample, standard or blank solution and 5 µL of derivative reagent solution and holding them in reaction vials for 5 min before injection. The derivatized samples (5 µL) were injected using an autosampler (AS-4150; Jasco). The analytical column (YMC Triart C18, particle 1.8 µm, 50 × 2.1 mm; YMC, Kyoto, Japan) was maintained at 45 ◦C and the flow rate was set at 500 µL/min. A linear gradient elution program was performed over 10 min with mobile phases A (0.05 M citrate buffer, pH 7.0) and B (0.05 M citrate buffer containing 30% acetonitrile and 30% methanol, pH 2.0). The excitation/emission wavelengths of the fluorescence detector were set at 280/455 nm [82–86]. The levels of norepinephrine and dopamine were determined by UHPLC (xLC3185PU; Jasco) and electrochemical detection (ECD-300; Eicom), with a graphite carbon electrode set at +450 mV (vs. Ag/AgCl reference electrode). The analytical column (Triart C18, particle 1.8 µm, 30 × 2.1 mm; YMC) was maintained at 25 ◦C and the flow rate of the mobile phase was set at 500 µL/min. The mobile phase was composed of 0.1 M acetate buffer containing 1% methanol and 50 mg/L EDTA-2Na; the final pH was 6.0 [35,37,87].

4.4. Capillary Immunoblotting Analysis Total plasma membrane proteins of LC, OFC and VTA were extracted using Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies, Plymouth, MN, USA). The capillary immunoblotting analyses were performed using Wes (ProteinSimple, Santa Clara, CA, USA) according to the mainly ProteinSimple user manual. The lysates of plasma membrane fraction were mixed with a master mix (ProteinSimple) to a final concentration of 1 × sample buffer, 1 × fluorescent molecular weight marker, and 40 mM dithiothreitol and then heated at 95 ◦C for 5 min. The samples, blocking reagents, primary antibodies, HRP- conjugated secondary antibodies, chemiluminescent substrate (SuperSignal West Femto: Thermo Fisher Scientific, Waltham, MA, USA), and separation and stacking matrices, were also dispensed to the designated wells in a 25-well plate. After plate loading, the separation electrophoresis and immunodetection steps took place in the capillary system and were fully automated. A capillary immunoblotting analysis was carried out at room tempera- ture, and the instrument’s default settings were used. Capillaries were first filled with a separation matrix followed by a stacking matrix, with about 40 nL of the sample used for loading. During electrophoresis, the proteins were separated by molecular weight through the stacking and separation matrices at 250 volts for 40–50 min and then immobilized on the capillary wall using proprietary photo-activated capture chemistry. The matrices were then washed out. The capillaries were next incubated with a blocking reagent for 15 min, and the target proteins were immunoprobed with primary antibodies followed by HRP-conjugated secondary antibodies (Anti-Rabbit IgG HRP, A00098, 10 µg/mL, GenScript, Piscataway, NJ, USA). The antibodies of ADRA2A (14266-1-AP, 1:100, Proteintech, Rosemont, IL, USA) and GAPDH (NB300-322, 1:100, Novus Biologicals, Littleton, CO, USA) were diluted in an antibody diluent (ProteinSimple) [50,55,82,83].

4.5. Data Analysis Where possible, we randomized and blinded sample data. To determine extracellular transmitter levels and α2A adrenoceptor expression, 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. All experiments in this study were designed with equally sized animal groups (N = 6) and all values were expressed as means ± standard deviation (SD). Differences were considered signiﬁcant when p < 0.05 (two-tailed). Int. J. Mol. Sci. 2021, 22, 4122 18 of 22

Regional transmitter concentrations were analysed 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 were 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 analysed using sphericity-assumed degrees of freedom. When the assumption of sphericity was violated (p < 0.05), F-values were analysed 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 analysed 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 agent administration. All statistical analyses complied with the recommendations for experimental design and analysis in pharmacology. The protein expression of α2A adrenoceptor in the LC, OFC and VTA in the plasma membrane fraction was analysed by a one-way ANOVA with Tukey’s multiple comparison using BellCurve for Excel.

5. Conclusions The present study demonstrated the time-dependent mechanisms of action of guanfacine. During acute guanfacine administration, guanfacine suppresses noradrenergic transmission from LC to OFC, VTA, and RTN due to the activation of the inhibitory postsynaptic/somatodendritic α2A adrenoceptor in the LC and presynaptic α2A receptor in the OFC, VTA and RTN. The co-releasing (norepinephrine with dopamine) pathway (from the LC to OFC) is also weakly suppressed by guanfacine, whereas thalamocortical glutamatergic transmission (MDTN-OFC) is enhanced via intrathalamic (RTN-MDTN) GABAergic disinhibition. In contrast with early-stage administration, chronic guanfacine administration (probably more than 2 weeks in duration) enhances noradrenergic transmission from the LC to OFC, VTA, and RTN due to the downregulation of the postsynaptic/somatodendritic and presynaptic α2A adrenoceptors. Furthermore, the attenuation of the inhibitory α2A adrenoceptor also produces an enhancement of the dopaminergic transmission in the LC-OFC co-releasing pathway and mesocortical (VTA-OFC) pathway. Unexpectedly, intrathalamic GABAergic transmission was not affected by acute guanfacine administration after chronic guanfacine administration, whereas thalamocortical glutamatergic transmission was enhanced via amplifying the integration of sensory/emotional inputs in the MDTN. These time-dependent actions of guanfacine, from the tonic-inhibition to tonic-activation of mesocortical catecholaminergic transmission, and phasic-activation associated with thalamocortical glutamatergic transmission support rapid onset and continuous efﬁcacy in the treatment of ADHD.

Author Contributions: Conceptualization, M.O.; Data curation, K.F., T.N. and M.O.; Formal analysis, K.F., T.N. and M.O.; Funding acquisition, M.O.; Methodology, M.O.; Project administration; M.O., Validation, T.S. and T.N.; Writing—original 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 (19K08073). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki. Animal care, the experimental procedures, and protocols for animal experiments were approved by the Animal Research Ethics Committee of the Mie University School of Medicine (No. 29–21, 7 April 2018). All studies involving animals have been reported in accordance with the ARRIVE guidelines for reporting experiments involving animals. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to equipment dependent data. Conﬂicts of Interest: The authors state no conﬂict of interest. Int. J. Mol. Sci. 2021, 22, 4122 19 of 22

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