Role of Dopamine D2/D3 Receptors in Development,Plasticity, and Neuroprotection in Human iPSC-Derived Midbrain Dopaminergic Neurons

The role of dopamine D2 and D3 receptors (D2R/D3R), located on midbrain dopaminergic (DA) neurons, in the regulation of DA synthesis and release and in DA neuron homeostasis has been extensively investigated in rodent animal models. By contrast, the properties of D2R/D3R in human DA neurons have not been elucidated yet. On this line, the use of human-induced pluripotent stem cells (hiPSCs) for producing any types of cells has offered the innovative opportunity for investigating the human neuronal phenotypes at the molecular levels. In the present study, hiPSCs generated from human dermal fibroblasts were used to produce midbrain DA (mDA) neurons, expressing the proper set of genes and proteins typical of authentic, terminally differentiated DA neurons. In this model, the expression and the functional properties of the human D2R/D3R were investigated with a combination of biochemical and functional techniques. We observed that in hiPSC-derived mDA neurons, the activation of D2R/D3R promotes the proliferation of neuronal progenitor cells. In addition, we found that D2R/D3R activation inhibits nicotine-stimulated DA release and exerts neurotrophic effects on mDA neurons that likely occur via the activation of PI3K-dependent mechanisms. Furthermore, D2R/D3R stimulation counteracts both the aggregation of alpha-synuclein induced by glucose deprivation and the associated neuronal damage affecting both the soma and the dendrites of mDA neurons. Taken together, these data point to the D2R/D3R-related signaling events as a biochemical pathway crucial for supporting both neuronal development and survival and protection of human DA neurons.     

Nicotinic Acetylcholine Receptors Control Encoding and Retrieval of Associative Recognition Memory through Plasticity in the Medial Prefrontal Cortex

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Contribution of Noradrenergic Neurons to the Regulation of Dopaminergic (D1) Receptor Denervation Supersensitivity in Rat Prefrontal Cortex

3,4-Dihydroxyphenylethylamine (DA, dopamine) levels in the rat prefrontal cortex were selectively decreased by 52%, leaving noradrenaline (NA) levels unaffected, 4 weeks following restricted bilateral electrolytic lesions of the ventral mesencephalic tegmentum (VMT). These lesions also induced a significant increase in DA-sensitive, but not isoproterenol-sensitive, adenylate cyclase activity in tissue homogenates (+38%). We had shown previously that chemical (6-hydroxydopamine, 6-OHDA) lesions of the VMT destroy both ascending DA and NA fibers but do not alter the D1-receptor density in the prefrontal cortex. In this study, electrolytic lesions of the VMT were combined with bilateral injections of 6-OHDA made laterally in the pedunculus cerebellaris superior to assess the role of NA fibers in the development of D1-receptor supersensitivity. This combined treatment produces a large decrease of cortical NA levels (-95%), an increase in beta-adrenergic-sensitive adenylate cyclase activity (+110%), and a decrease in DA levels (-60%), but does not alter D1-receptor density in the prefrontal cortex. These results indicate that the development of D1-receptor supersensitivity in the prefrontal cortex following electrolytic lesion of the VMT depends on the presence of an intact NA innervation.     

Pharmacological Characterization of 5-HT1A Autoreceptor-Coupled GIRK Channels in Rat Dorsal Raphe 5-HT Neurons

G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT_1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltage-clamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT_1A autoreceptor-activated inwardly rectifying K⁺ conductance in rat dorsal raphe 5-HT neurons. 5-HT_1A autoreceptor-activated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba²⁺ (EC₅₀ = 9.4 μM, full block with 100 μM) and by SCH23390 (EC₅₀ = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT_1A autoreceptor-activated GIRK conductance with high potency (EC₅₀ = 33.6 nM), but incompletely, i.e. ~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC₅₀s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K⁺ channels coupled to 5-HT_1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.     

The Influence of Gender and Age on Neonatal Rat Hypothalamic 5-HT1A and 5-HT2A Receptors

1.Rat hypothalamic 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) concentrations are transiently sexually differentiated in the second week postpartum (pp), with higher levels in the female. In this report we investigate the possibility that 5-HT receptors may also exhibit sexual dimorphism in the neonatal period.2.5-HT1A and 5-HT2A receptors were quantitated by radioligand binding of [³H]ketanserin and [³H]8-OH DPAT, respectively, in hypothalamus and amygdala from male and female rats at days 8–16 pp.3.There was no sexual dimorphism or change in the density of 5-HT2A binding in hypothalamus or amygdala over days 8–16 pp. There was also no sexual dimorphism of 5-HT1A receptors.4.There was an increase in 5-HT1A receptor density in both the hypothalamus and the amygdala. In the hypothalamus, but not the amygdala, this increase was interrupted on day 14 by a decrease in 5-HT1A receptors, which we suggest may be of physiological significance in modifying the eventual pattern of adult agonistic activity.5.The results suggest that the sexual dimorphism in 5-HT turnover is predominantly presynaptic, relating to altered synthesis and/or release, and is not of sufficient magnitude or duration to produce adaptive responses in postsynaptic 5-HT1A or 5-HT2A receptors.     

Molecular Design of Novel Ligands for 5-HT1A Receptors

Abstract

Serotonin (5-HT) is a potent bioactive substance known to function through a number of different receptor types and subtypes. In our attempt to develop new agents that would interact selectively at certain 5-HT receptors, especially the 5-HT_1A subtype, 8-hydroxy-2-di-n-propylamino tetralin (8-OH-DPAT) served as a template for the design of novel agents sharing aspects of the pharmacophore of 8-OH-DPAT and 5-HT. 5-HT contains no center of asymmetry, and 8-OH-DPAT shows only very modest stereospecificity for 5-HT_1A receptors. To develop agents having enhanced potency and selectivity for the 5-HT_1A site, several ring systems offering enhanced conformational rigidity which approximate the oxygen to nitrogen interatomic distances of 8-OH-DPAT and (to a lesser extent) 5-HT were synthesized. Exemplary ring systems include the 8-alkoxy-hexahydroindeno[1,2-c]pyrrole, 5-alkoxy-hexahydro-1H-indeno-[2,1-c]pyridine, and 9-alkoxy-hexahydro-1H-benz[e]isoindole systems. These couformationally restricted molecules demonstrated moderate stereospecificity in their interaction with the 5-HT_1A binding site, which was enhanced in compounds with larger nitrogen substituents. Appropriate choice of such derivatives led to highly potent compounds selective for 5-HT_1A sites compared with their activity at other 5-HT and/or adrenergic receptors. The pharmacological profile of compounds which appear to act as agonists at 5-HT_1A receptors in the central nervous system to lower blood pressure in animal models of hypertension is presented     

Fluoxetine Increases Extracellular Dopamine in the Prefrontal Cortex by a Mechanism Not Dependent on Serotonin

Fluoxetine at 10 and 25 mg/kg increased (167 and 205%, respectively) the extracellular dopamine concentration in the prefrontal cortex, whereas 25 (but not 10) mg/kg citalopram raised (216%) dialysate dopamine. No compound modified dialysate dopamine in the nucleus accumbens. The effect of 25 mg/kg of both compounds on cortical extracellular dopamine was not significantly affected by 300 mg/kg p-chlorophenylalanine (PCPA) (fluoxetine, saline, 235%; PCPA, 230%; citalopram, saline, 179%; PCPA, 181%). PCPA depleted tissue and dialysate serotonin by approximately 90 and 50%, respectively, and prevented the effect of fluoxetine and citalopram on dialysate serotonin (fluoxetine, saline, 246%; PCPA, 110%; citalopram, saline, 155%; PCPA, 96%). Citalopram significantly raised extracellular serotonin from 0.1 to 100 microM (251-520%), whereas only 10 and 100 microM increased dialysate dopamine (143-231%). Fluoxetine similarly increased extracellular serotonin (98-336%) and dopamine (117-318%). PCPA significantly reduced basal serotonin and the effects of 100 microM fluoxetine (saline, 272%; PCPA, 203%) and citalopram (saline, 345%; PCPA, 258%) on dialysate serotonin but did not modify their effect on dopamine (fluoxetine, saline, 220%; PCPA, 202%; citalopram, saline, 191%; PCPA, 211%). The results clearly show that the effects of fluoxetine and of high concentrations of citalopram on extracellular dopamine do not depend on their effects on serotonin.     

Sequence and Functional Analysis of Cloned Guinea Pig and Rat Serotonin 5-HT1D Receptors: Common Pharmacological Features Within the 5-HT1D Receptor Subfamily

Abstract: This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)_1D receptor sites. Guinea pig, rat, and mouse 5-HT_1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT_1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13–27%) in the N-terminal extracellular region of these 5-HT_1D receptors. Guinea pig and rat 5-HT_1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT_1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT_1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT_1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT_1D receptor-mediated antagonist potency for methiothepin and the 5-HT₂ receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT_1D receptor subtype seems, unlike the 5-HT_1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

Dopaminergic Inhibition of Catecholamine Secretion from Chromaffin Cells: Evidence that Inhibition Is Mediated by D4 and D5 Dopamine Receptors

Abstract: Previous studies have suggested that activation of D2-like dopamine receptors inhibits catecholamine secretion from adrenal chromaffin cells. The purpose of this study was to determine whether the activation of D1-like receptors on chromaffin cells affects either catecholamine release from the cells or the inhibition of secretion by D2-like dopamine receptors. Both D1- and D2-selective agonists inhibited secretion elicited by dimethylphenylpiperazinium (DMPP), veratridine, and high K⁺ levels. The D1-selective agonists 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (Cl-APB) and SKF-38393 inhibited DMPP-stimulated catecholamine secretion in a concentration-dependent manner; 50% inhibition was obtained with ～10 µM Cl-APB and ～100 µM SKF-38393. Of the D2-selective agonists, bromocriptine was a more potent inhibitor of DMPP-stimulated catecholamine release than was quinpirole. The inhibition of secretion caused by Cl-APB or SKF-38393 was additive with the inhibition caused by bromocriptine. Pertussis toxin treatment (50 ng/ml, 18 h) attenuated the inhibitory effect of D2-selective, but not D1-selective, dopamine agonists. In addition, forskolin-stimulated adenylyl cyclase activity was inhibited by D2-selective, but not D1-selective, agonists. Neither D1- nor D2-selective agonists stimulated adenylyl cyclase activity in the cells, although cyclase activity was stimulated by forskolin, carbachol, and vasoactive intestinal peptide. DMPP-stimulated Ca²⁺ uptake was inhibited by both D1- and D2-selective dopamine agonists. PCR analysis was used to determine which of the dopamine receptor subtypes within the D1-like and D2-like subfamilies was responsible for the observed inhibition. PCR analysis indicated that mRNA for only D4 and D5 dopamine receptor subtypes was present in chromaffin cells. These combined data suggest that D1- and D2-selective agonists inhibit Ca²⁺ uptake and catecholamine secretion by activating D4 and D5 dopamine receptors on chromaffin cells.     

Effect of chronic paroxetine treatment on 5-HT1B and 5-HT1D autoreceptors in rat dorsal raphe nucleus

This study reports the effect of chronic paroxetine (10 mg/kg p.o., 21 days) on 5-HT1B and 5-HT1D autoreceptors controlling stimulated 5-HT efflux in slices of rat dorsal raphe nucleus. Electrically evoked 5-HT (10 pulses, 200 Hz, 0.1 ms, 10 mA) was measured using fast cyclic voltammetry. 5-HT efflux was inhibited by CP 93129 (10 nM-10 microM) and by sumatriptan (1 nM-1 microM) agonists at 5-HT1B and 5-HT1D receptors, respectively. Chronic paroxetine did not, initially, appear to alter the sensitivity of the 5-HT1B autoreceptors to CP 93129. However, when constructed in the presence of WAY 100635 (10 nM) the selective and silent 5-HT1A antagonist, there was a significant (P < 0.001) rightward shift of the CP 93129 concentration-response curve in the paroxetine-treated rats but not in the controls, implying a desensitisation of the 5-HT1B autoreceptor by paroxetine. Chronic paroxetine did not affect the sumatriptan concentration-response curve, even with WAY 100635 present, implying that there was no (de)sensitisation of the 5-HT1D autoreceptor. These data suggest that chronic paroxetine treatment may desensitise 5-HT1B autoreceptors in the dorsal raphe nucleus but that this effect is unmasked only when the dominant 5-HT1A autoreceptor control is antagonised.     

Repeated Haloperidol Administration has No Effect on Vitamin D Signaling but Increase Retinoid X Receptors and Nur77 Expression in Rat Prefrontal Cortex

Both vitamin D (VD) signaling and Nur77 are implicated in dopaminergic neurotransmission and dopamine-related neuropsychiatric disorders, such as schizophrenia and Parkinson’s disease. Developmental vitamin D (DVD) deficiency rats exhibit schizophrenia-like behaviors and disturbance of dopamine system, which could be partly normalized by haloperidol treatment. By blocking dopamine D₂ receptor, haloperidol induces Nur77 expression, suggesting a modulatory role of Nur77 in brain dopamine system. Rxr is the heterodimeric partner of both Nur77 and vitamin D receptor and also participates in homeostatic regulation of central dopamine neurotransmission. Although D₂ antagonist-induced Nur77 expression has been reported by several studies, the change of its active partner Rxr remains elusive. Here, we studied the impact of 2 weeks administration of haloperidol on VD signaling and Nur77/Rxr expression in rat prefrontal cortex. It was found that haloperidol has no effect on local VD signaling, but could significantly increase Nur77, Rxr_β, and Rxr_γ expression, which indicated that Nur77/Rxr, but not vdr/Rxr, was implicated in dopamine-related neuroadaptation. Given that VD deficiency is commonly observed in schizophrenia patients, the renal metabolism of VD was also examined.     

Prefrontal Serotonergic Denervation Induces Increase in the Density of 5-HT<Subscript>2A</Subscript> Receptors in Adult Rat Prefrontal Cortex

The 5-HTergic system and particularly 5-HT_2A receptors have been involved in prefrontal cognitive functions, but the underlying mechanisms by which the serotonin (5-HT) system modulates these processes are still unclear. In this work, the effects of prefrontal 5-HTergic denervation on the density and expression levels of 5-HT_2A receptors were evaluated by immunohistochemical and molecular biology studies in the prefrontal cortex (PFC). The [³H]-Ketanserin binding study revealed an increase in the B_max, along with no change in the binding affinity (K_D) for 5-HT_2A receptors. The increase in PFC of 5-HT_2A receptor density in response to denervation was accompanied by increase in 5-HT_2A receptor mRNA and protein levels. This increase in the number of 5-HT_2A receptors may be interpreted as an adaptive plastic change, i.e., hypersensitivity; resulting from the selective pharmacological lesion of the raphe-proceeding 5-HTergic fibers to the PFC. Based on previous evidence, this could be strongly related to the abnormal expression of short-term memory.     

Visualization of D1 Dopamine Receptors on Living Nucleus Accumbens Neurons and Their Colocalization with D2 Receptors

Abstract: To examine the substrate for dopamine (DA) synaptic action in the nucleus accumbens (nAcc), we visualized the cellular and subcellular distribution of DA receptors on postnatal nAcc neurons in culture using fluoroprobe derivatives of DA receptor ligands. Previously, we have shown that rhodamine- N -( p -aminophenethyl)-spiperone (NAPS) (10 n M ), a derivative of the D2 antagonist spiperone, labels D2-like receptors on living nAcc neurons. We now show that rhodamine-Sch-23390 (30 n M ), a derivative of the D1 antagonist, labels D1-like receptors. Putative specific membrane labeling reached a plateau after about 20 min. Labeling was stereospecific, as it was unaffected by competition with (−)-butaclamol, but blocked with (+)-butaclamol. We found that 52 ± 7% of nAcc medium-sized neurons showed D1 labeling, which extended onto the dendrites. Labeling was also seen on presynaptic terminals, often abutting D1-positive and D1-negative cell bodies, consistent with a presynaptic modulatory role for D1 receptors. Larger neurons, which may be GABAergic or cholinergic interneurons, were also labeled. By sequential labeling first with rhodamine-Sch-23390 and then rhodamine-NAPS, we found that 38 ± 6% of medium-sized neurons express both D1- and D2-like receptors, indicating that D1–D2 interactions may occur at the level of single postsynaptic neurons.     

Plasticity of 5-HT 1A receptor-mediated signaling during early postnatal brain development

The presence of serotonin 1A receptor (5-HT(1A)-R) in the hippocampus, amygdala, and most regions of the frontal cortex is essential between postnatal day-5-21 (P5-21) for the expression of normal anxiety levels in adult mice. Thus, the 5-HT(1A)-R plays a crucial role in this time window of brain development. We show that the 5-HT(1A)-R-mediated stimulation of extracellular signal-regulated kinases 1 and 2 (Erk1/2) in the hippocampus undergoes a transition between P6 and P15. At P6, a protein kinase C (PKC) isozyme is required for the 5-HT(1A)-R -->Erk1/2 cascade, which causes increased cell division in the dentate gyrus. By contrast, at P15, PKC alpha participates downstream of Erk1/2 to augment synaptic transmission through the Schaffer Collateral pathway but does not cause increased cell division. Our data demonstrate that the 5-HT(1A)-R -->Erk1/2 cascade uses PKC isozymes differentially, first boosting the cell division to form new hippocampal neurons at P6 and then undergoing a plastic change in mechanism to strengthen synaptic connections in the hippocampus at P15.