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Intracellular accumulation of tau protein is a hallmark of Alzheimer's Disease and Progressive Supranuclear Palsy, as well as other neurodegenerative disorders collectively known as tauopathies. Despite our increasing understanding of the mechanisms leading to the initiation and progression of tau pathology, the field still lacks appropriate disease models to facilitate drug discovery. Here, we established a novel and modulatable seeding-based neuronal model of full-length 4R tau accumulation using humanized mouse cortical neurons and seeds from P301S human tau transgenic animals. The model shows specific and consistent formation of intraneuronal insoluble full-length 4R tau inclusions, which are positive for known markers of tau pathology (AT8, PHF-1, MC-1), and creates seeding competent tau. The formation of new inclusions can be prevented by treatment with tau siRNA, providing a robust internal control for use in qualifying the assessment of potential therapeutic candidates aimed at reducing the intracellular pool of tau. In addition, the experimental set up and data analysis techniques used provide consistent results in larger-scale designs that required multiple rounds of independent experiments, making this is a versatile and valuable cellular model for fundamental and early pre-clinical research of tau-targeted therapies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Amorim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Trace Amine-Associated Receptor 1 (TAAR1) is a potential target for the treatment of depression and other CNS disorders. However, the precise functional roles of TAAR1 to the actions of clinically used antidepressants remains unclear. Herein, we addressed these issues employing the TAAR1 agonist, o-phenyl-iodotyramine (o-PIT), together with TAAR1-knockout (KO) mice. Irrespective of genotype, systemic administration of o-PIT led to a similar increase in mouse brain concentrations. Consistent with the observation of a high density of TAAR1 in the medial preoptic area, o-PIT-induced hypothermia was significantly reduced in TAAR1-KO mice. Furthermore, the inhibition of a prepulse inhibition response by o-PIT, as well as its induction of striatal tyrosine hydroxylase phosphorylation and elevation of extracellular DA in prefrontal cortex, were all reduced in TAAR1-KO compared to wildtype mice. O-PIT was active in both forced-swim and marble-burying tests, and its effects were significantly blunted in TAAR1-KO mice. Conversely, the actions on behaviour and prefrontal cortex dialysis of a broad suite of clinically used antidepressants were unaffected in TAAR1-KO mice. In conclusion, o-PIT is a useful tool for exploring the hypothermic and other functional antidepressant roles of TAAR1. By contrast, clinically used antidepressants do not require TAAR1 for expression of their antidepressant properties.

The novel benzopyranopyrrolidine, S33138 [N-[4-[2-[(3aS,9bR)-8-cyano-1,3a,4,9b-tetrahydro[1]benzopyrano[3,4-c]pyrrol-2(3H)-yl)-ethyl]phenylacetamide], is a preferential antagonist of cloned human D(3) versus D(2L) and D(2S) receptors. In mice, S33138 (0.04-2.5 mg/kg i.p.) increased levels of mRNA encoding c-fos in D(3) receptor-rich Isles of Calleja and nucleus accumbens more potently than in D(2) receptor-rich striatum. Furthermore, chronic (3 weeks) administration of S33138 to rats reduced the number of spontaneously active dopaminergic neurones in the ventral tegmental area (0.16-10.0 p.o.) more potently than in the substantia nigra (10.0). In primates treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, antiparkinson actions of the D(3)/D(2) agonist, ropinirole, were potentiated by low doses of S33138 (0.01-0.16 p.o.) but diminished by a high dose (2.5). Consistent with antagonism of postsynaptic D(3)/D(2) sites, S33138 attenuated hypothermia and yawns elicited by the D(3)/D(2) agonist 7-OH-DPAT [(+)-7-dihydroxy-2-(di-n-propylamino)-tetralin] in rats, and it blocked (0.01-0.63, s.c.) discriminative properties of PD128,907 [(+)-(4aR,10bR)-3,4, 4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol; trans-N-[4-[2-(6-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-4-quinolininecarboxamide]. Suggesting antagonist properties at D(3)/D(2) autoreceptors, S33138 prevented (0.16-2.5 s.c.) the inhibitory influence of PD128,907 upon dopamine release in frontal cortex, nucleus accumbens, and striatum and abolished (0.004-0.25 i.v.) its inhibition of ventral tegmental dopaminergic neuron firing. At higher doses, antagonist actions of S33138 (0.5-4.0 i.v.) at alpha(2C)-adrenoceptors were revealed by an increased firing rate of adrenergic perikarya. Finally, antagonism of 5-hydroxytryptamine (5-HT(2A) and 5-HT(7)) receptors was shown by blockade of 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane-induced head twitches (0.63-10.0 s.c.) and 5-carboxytryptamine-induced hypothermia (2.5-20.0 i.p.), respectively. In conclusion, S33138 displays modest antagonist properties at central alpha(2C)-adrenoceptors, 5-HT(2A) and 5-HT(7) receptors. Furthermore, in line with its in vitro actions, it more potently blocks cerebral populations of D(3) versus D(2) receptors.

Though dopaminergic mechanisms modulate cholinergic transmission and cognitive function, the significance of specific receptor subtypes remains uncertain. Here, we examined the roles of dopamine D(3) versus D(2) receptors. By analogy with tacrine (0.16-2.5 mg/kg, s.c.), the selective D(3) receptor antagonists, S33084 (0.01-0.63) and SB277,011 (0.63-40.0), elicited dose-dependent, pronounced and sustained elevations in dialysis levels of acetylcholine (ACh) in the frontal cortex, but not the hippocampus, of freely-moving rats. The actions of these antagonists were stereospecifically mimicked by (+)S14297 (1.25), whereas its inactive distomer, (-)S17777, was ineffective. The preferential D(2) receptor antagonist, L741,626 (10.0), failed to modify levels of ACh. S33084 (0.01-0.63) and SB277,011 (0.16-2.5) also mimicked tacrine (0.04-0.63) by dose-dependently attenuating the deleterious influence of scopolamine (1.25) upon social memory (recognition by an adult rat of a juvenile conspecific). Further, (+)S14297 (1.25) versus (-)S17777 stereospecifically blocked the action of scopolamine. Using an intersession interval of 120 min (spontaneous loss of recognition), S33084 (0.04-0.63), SB277,011 (0.16-10.0) and (+)S14297 (0.63-10.0) likewise mimicked tacrine (0.16-2.5) in enhancing social memory. In contrast, L741,626 (0.16-10.0) displayed amnesic properties. In conclusion, selective blockade of D(3) receptors facilitates frontocortical cholinergic transmission and improves social memory in rats. These data support the pertinence of D(3) receptors as a target for treatment of disorders in which cognitive function is compromised.

Information concerning striatal levels of noradrenaline (NA) remains inconsistent. Here we have addressed this issue using a sensitive method of HPLC coupled to amperometric detection. The NA reuptake-inhibitor, reboxetine, selectively elevated levels of NA versus dopamine (DA), and NA levels were also selectively elevated by the alpha2-adrenoceptor (AR) antagonist, atipamezole. The actions of atipamezole were mimicked by the preferential alpha2A-AR antagonist, BRL44408, while JO-1 and prazosin, preferential antagonists at alpha2C-ARs, caused less marked elevations in NA levels. In contrast to antagonists, the alpha2-AR agonist, S18616, decreased NA levels and likewise suppressed those of DA. Unilateral lesions of the substantia nigra with 6-hydroxydopamine depleted DA levels without affecting those of NA. Further, the D3/D2 receptor agonist, quinelorane, decreased levels of DA without modifying those of NA. However, the D3/D2 receptor antagonists, haloperidol and raclopride, and the DA reuptake-inhibitor, GBR12935, elevated levels of both DA and NA. Levels of 5-HT (but not of NA or DA) were increased only by the 5-HT reuptake-inhibitor, citalopram. They were decreased by S18616 and prazosin, reflecting the inhibitory and excitatory influence of alpha2- and alpha1-ARs, respectively, upon serotonergic pathways. In conclusion, NA in the striatum is derived from adrenergic terminals. Its release is subject to tonic, inhibitory control by alpha2-ARs, possibly involving both alpha2A- and alpha2C-AR subtypes, though their respective contribution requires clarification. A role of dopaminergic terminals in the reuptake of NA likely explains the elevation in its levels elicited by DA reuptake-inhibitors and D3/D2 receptor antagonists.

In a dialysis procedure not requiring perfusate addition of acetylcholinesterase inhibitors to "boost" basal levels of acetylcholine (ACh), the influence of the antiparkinson agent piribedil upon levels of ACh in frontal cortex and dorsal hippocampus of freely moving rats was compared with those of other antiparkinson drugs and selective ligands at alpha(2)-adrenoceptors (ARs). Suggesting a tonic, inhibitory influence of alpha(2A)-ARs upon cholinergic transmission, the alpha(2)-AR agonist 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline tartrate (UK14,304), and the preferential alpha(2A)-AR agonist guanabenz reduced levels of ACh. They were elevated by the antagonists 2(2-methoxy-1,4 benzodioxan-2-yl)-2-imidazoline HCl (RX821002) and atipamezole and by the preferential alpha(2A)-AR antagonist 2-(2H-(1-methyl-1,3-dihydroisoindole)methyl)-4,5-dihydroimidazole (BRL44008). In contrast, trans-2,3,9,13b-tetrahydro-1,2-dimethyl-1H-dibenz[c,f]imidazo[1,5-a]azepine (BRL41992) and prazosin, preferential alpha(2B/2C)-AR antagonists, were inactive. The dopaminergic agonist and antiparkinson agent piribedil, which behaves as an antagonist at alpha(2)-ARs, dose dependently increased extracellular levels of ACh. This action was absent upon pretreatment with a maximally effective dose of RX821002. On the other hand, a further dopaminergic agonist and antiparkinson agent, talipexole, which possesses agonist properties at alpha(2)-ARs, dose dependently reduced levels of ACh. This action was also blocked by RX821002. In contrast to piribedil and talipexole, quinelorane, which interacts with dopaminergic receptors but not alpha(2)-ARs, failed to affect ACh levels. Finally, in analogy to the frontal cortex, piribedil likewise elicited a dose-dependent increase in extracellular levels of ACh in the dorsal hippocampus. In conclusion, in distinction to talipexole and quinelorane, and reflecting its antagonist properties at alpha(2A)-ARs, piribedil reinforces cholinergic transmission in the frontal cortex and dorsal hippocampus of freely moving rats. These actions may be related to its facilitatory influence upon cognitive function.

Rationale: Although drug discrimination procedures have proven difficult to apply to antidepressant agents, we recently characterized discriminative stimulus properties of the selective serotonin (5-HT) reuptake inhibitor, citalopram, in rats. However, discriminative stimulus properties of selective norepinephrine (NE) reuptake inhibitors remain to be evaluated., Objective: We determined the potential discriminative stimulus properties of the highly selective NE reuptake inhibitor and antidepressant, reboxetine., Methods: Employing a two-lever discrimination procedure, rats were trained to discriminate reboxetine (2.5 mg/kg, IP) from saline. In parallel, the influence of reboxetine (2.5 mg/kg) upon dialysate levels of monoamines in frontal cortex and dorsal hippocampus of freely moving rats was determined., Results: After 54+/-10 training sessions, reboxetine elicited robust stimulus recognition, fully generalizing to itself with an ED50 of 1.2 mg/kg. Two further NE reuptake inhibitors, desipramine (5.3) and maprotiline (1.8), as well as the 5-HT/NE reuptake inhibitor, venlafaxine (1.0), likewise generalized. In contrast, the 5-HT reuptake inhibitors, paroxetine, citalopram and sertraline, and the DA reuptake inhibitors, GBR12935 and bupropion, did not show significant generalization. Reboxetine markedly increased dialysate levels of NE, but not 5-HT, in frontal cortex and hippocampus. Dopamine (DA) levels were also (though less markedly) enhanced in frontal cortex., Conclusion: In parallel with an elevation in extracellular levels of NE, the selective NE reuptake inhibitor, reboxetine, elicits a specific discriminative stimulus in rats.

The present study evaluated, via a combined electrophysiological and dialysis approach, the potential influence of serotonin (5-HT)(2C) as compared to 5-HT(2A) and 5-HT(2B) receptors on dopaminergic, adrenergic, and serotonergic transmission in frontal cortex (FCX). Whereas the selective 5-HT(2A) antagonist MDL100,907 failed to modify extracellular levels of dopamine (DA), noradrenaline (NA) or 5-HT simultaneously quantified in single dialysate samples of freely-moving rats, the 5-HT(2B)/5-HT(2C) antagonist SB206,553 dose-dependently increased levels of DA and NA without affecting those of 5-HT. This action was attributable to 5-HT(2C) receptor blockade inasmuch as the selective 5-HT(2C) antagonist SB242,084 likewise increased FCX levels of DA and NA, whereas the selective 5-HT(2B) antagonist SB204,741 was ineffective. Further, the preferential 5-HT(2C) receptor agonist Ro60-0175 dose-dependently depressed FCX levels of DA. The suppressive influence of 5-HT(2C) receptors on DA release was also expressed on mesolimbic and nigrostriatal dopaminergic pathways, in that levels of DA in nucleus accumbens and striatum were likewise reduced by Ro60-0175 and elevated, though less markedly, by SB206,553. In line with the above findings, Ro60-0175 dose-dependently decreased the firing rate of ventrotegmental dopaminergic and locus coeruleus (LC) adrenergic perikarya, whereas their activity was dose-dependently enhanced by SB206,553. Furthermore, SB206,553 transformed the firing pattern of ventrotegmental dopaminergic neurons into a burst mode. In contrast to SB206,553, MDL100,907 had little affect on the firing rate of dopaminergic or adrenergic neurons. In conclusion, as compared to 5-HT(2A) and 5-HT(2B) receptors, 5-HT(2C) receptors exert a tonic, suppressive influence on the activity of mesocortical - as well as mesolimbic and nigrostriatal - dopaminergic pathways, likely via indirect actions expressed at the level of their cell bodies. Frontocortical adrenergic, but not serotonergic, transmission is also tonically suppressed by 5-HT(2C) receptors., (Copyright 2000 Wiley-Liss, Inc.)

The mixed serotonin and noradrenaline reuptake inhibitor, duloxetine, (5.0 mg/kg, s.c.), increased levels of serotonin (220%), dopamine (180%) and noradrenaline (470%) in individual dialysates of frontal cortex of freely moving rats. Its influence on serotonin, but not dopamine or noradrenaline, levels was enhanced by the 5-HT1A receptor antagonist, WAY 100,635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclo-hexanecarboxamide 3HCl) (0.16 mg kg(-1), s.c). In the forced swimming test, although duloxetine was inactive alone, it dose dependently reduced immobility in the presence of WAY 100,635. Thus, blockade of 5-HT1A (auto)receptors selectively facilitates the influence of duloxetine on serotonin levels in the frontal cortex in rats and, in the forced swimming model, enhances its 'antidepressant' properties in parallel.

Evidence exists that a reinforcement in monoaminergic transmission in the frontal cortex (FCX) is associated with antidepressant (AD) properties. Herein, we examined whether blockade of alpha2-adrenergic receptors modified the influence of monoamine reuptake inhibitors on FCX levels of serotonin (5-HT), noradrenaline (NAD), and dopamine (DA). The selective alpha2-adrenergic receptor agonist S 18616 (0.16 mg/kg, s.c.) suppressed extracellular levels of NAD, DA, and 5-HT (by 100, 51, and 63%, respectively) in single dialysates of FCX of freely moving rats. In contrast, the selective alpha2-adrenergic receptor antagonists atipamezole (0.16 mg/kg, s.c.) and 1-(2-pyrimidinyl)piperazine (1-PP; 2.5 mg/kg, s.c.) increased levels of NAD (by 180 and 185%, respectively) and DA (by 130 and 90%, respectively), without affecting 5-HT levels. Duloxetine (5.0 mg/kg, s.c.), a mixed inhibitor of 5-HT and NAD reuptake, and fluoxetine (10.0 mg/kg, s.c.), a selective 5-HT reuptake inhibitor, both increased levels of 5-HT (by 150 and 120%, respectively), NAD (by 400 and 100%, respectively), and DA (by 115 and 55%, respectively). Atipamezole (0.16 mg/kg, s.c.) markedly potentiated the influence of duloxetine and fluoxetine on levels of 5-HT (by 250 and 330%, respectively), NAD (by 1,030 and 215%, respectively), and DA (by 370 and 170%, respectively). 1-PP similarly potentiated the influence of duloxetine on 5-HT, NAD, and DA levels (by 290, 1,320, and 600%, respectively). These data demonstrate that alpha2-adrenergic receptors tonically inhibit NAD and DA and phasically inhibit 5-HT release in the FCX and that blockade of alpha2-adrenergic receptors strikingly potentiates the increase in FCX levels of 5-HT, NAD, and DA elicited by reuptake inhibitors. Concomitant alpha2-adrenergic receptor antagonism and inhibition of monoamine uptake may thus provide a mechanism allowing for a marked increase in the efficacy of AD agents.

The novel, potential anxiolytic, S 15535 (4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine), is an agonist and antagonist (weak partial agonist) at pre- and postsynaptic serotonin (5-HT)1A receptors, respectively. Herein, we characterized its influence on dialysate levels of 5-HT, dopamine (DA) and NAD simultaneously determined in single samples of the frontal cortex (FCX) of freely moving rats, and compared its activity in several other models of potential antidepressant (AD) properties with those of the 5-HT reuptake inhibitor (SSRI), fluoxetine. S 15535 displayed high affinity at cloned human (h) 5-HT1A receptors (Ki = 0.7 nM) and >250-fold lower affinity at cloned hD2 (400 nM), hD3 (248 nM) and h alpha2A-adrenergic (AR) (190 nM) receptors. S 15535 (0.08-5.0 mg/kg s.c.) markedly and dose-dependently suppressed dialysate levels of 5-HT in the FCX, nucleus accumbens and striatum of freely moving rats, whereas fluoxetine (10.0 mg/kg s.c.) elevated levels of 5-HT in each structure. In contrast to 5-HT, dialysate levels of DA and NAD in the FCX were dose-dependently increased by S 15535, and this effect was mimicked by fluoxetine. The influence of S 15535 and fluoxetine on FCX levels of DA was regionally specific inasmuch as dialysate levels of DA in the accumbens and striatum were not modified. The selective 5-HT1A antagonist, WAY 100,635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide (0.16) transiently elicited a slight increase in cortical levels of 5-HT, an action opposite to that of S 15535. Further, in the presence of WAY 100,635 (0.16), the influence of S 15535 (0.63) on cortical levels of 5-HT, DA and NAD was markedly attenuated. Upon chronic administration of S 15535 or fluoxetine (10.0 mg/kg s.c. daily for 14 days, in each case), there was no significant alteration in the density of beta-AR receptors in the FCX. However, in contrast to fluoxetine, S 15535 elicited a significant (25%) decrease in the density (Bmax) of 5-HT2A receptors labeled by [3H]ketanserin in the cortex; there was no alteration in Kd. In a learned helplessness paradigm in rats, S 15535 (0.63-40.0 mg/kg p.o.) markedly reduced escape deficits on each of three consecutive days of testing. Fluoxetine (2.0-8.0 mg/kg i.p.) was also active in each session, but presented a biphasic dose-response curve. Finally, under the conditions used, neither S 15535 (0.63-10.0) nor fluoxetine (0.63-10.0) decreased immobility time in the forced swim test. In conclusion, S 15535 is a selective ligand of cloned, h5-HT1A receptors. Its agonist actions at 5-HT1A autoreceptors underlie its ability to decrease extracellular levels of 5-HT in the FCX, and likely contribute to the increase in extracellular levels of DA and NAD evoked by S 15535 in this structure. Further, S 15535 is active in several other, although not all, models of potential AD activity. Thus, although S 15535 is under development as an anxiolytic agent, a further characterization of its putative AD actions would be of interest.

In freely moving rats, the novel, selective dopamine (DA) D3 receptor agonist PD 128,907 dose-dependently [effective dose (ED25) = 0.07 mg/kg, s.c.] reduced dialysate levels of DA in the frontal cortex, a structure innervated by the ventral tegmental area (VTA). This action of PD 128,907 (0.16 mg/kg, s.c.) was abolished by a selective DA D3 receptor antagonist S 14297 (1.25 mg/kg, s.c.), which alone did not modify levels of DA. In contrast to S 14297, its inactive distomer, S 17777, did not modify the actions of PD 128,907. In addition, PD 128,907 dose-dependently and potently inhibited the firing rate of VTA-localized neurons in anesthetized rats (ED50 = 0.001 mg/kg, i.v.). S 14297, but not S 17777, completely reversed the actions of PD 128,907 (0.005 mg/kg, i.v.) with a 50% inhibitory dose of 0.03 mg/kg, i.v. and did not itself significantly modify the firing rate. In conclusion, these data provide the first direct evidence that DA D3 (auto) receptors modulate (inhibit) the release of DA in the frontal cortex.