Monoaminergic Neurons Research Articles

Understanding How Physical Exercise Improves Alzheimer's Disease: Cholinergic and Monoaminergic Systems.

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder, characterized by the accumulation of proteinaceous aggregates and neurofibrillary lesions composed of β-amyloid (Aβ) peptide and hyperphosphorylated microtubule-associated protein tau, respectively. It has long been known that dysregulation of cholinergic and monoaminergic (i.e., dopaminergic, serotoninergic, and noradrenergic) systems is involved in the pathogenesis of AD. Abnormalities in neuronal activity, neurotransmitter signaling input, and receptor function exaggerate Aβ deposition and tau hyperphosphorylation. Maintenance of normal neurotransmission is essential to halt AD progression. Most neurotransmitters and neurotransmitter-related drugs modulate the pathology of AD and improve cognitive function through G protein-coupled receptors (GPCRs). Exercise therapies provide an important alternative or adjunctive intervention for AD. Cumulative evidence indicates that exercise can prevent multiple pathological features found in AD and improve cognitive function through delaying the degeneration of cholinergic and monoaminergic neurons; increasing levels of acetylcholine, norepinephrine, serotonin, and dopamine; and modulating the activity of certain neurotransmitter-related GPCRs. Emerging insights into the mechanistic links among exercise, the neurotransmitter system, and AD highlight the potential of this intervention as a therapeutic approach for AD.

Perturbation of monoamine metabolism and enhanced fear responses in mice defective in the regeneration of tetrahydrobiopterin.

Increasing evidence suggests the involvement of peripheral amino acid metabolism in the pathophysiology of neuropsychiatric disorders, whereas the molecular mechanisms are largely unknown. Tetrahydrobiopterin (BH4) is a cofactor for enzymes that catalyze phenylalanine metabolism, monoamine synthesis, nitric oxide production, and lipid metabolism. BH4 is synthesized from guanosine triphosphate and regenerated by quinonoid dihydropteridine reductase (QDPR), which catalyzes the reduction of quinonoid dihydrobiopterin. We analyzed Qdpr-/- mice to elucidate the physiological significance of the regeneration of BH4. We found that the Qdpr-/- mice exhibited mild hyperphenylalaninemia and monoamine deficiency in the brain, despite the presence of substantial amounts of BH4 in the liver and brain. Hyperphenylalaninemia was ameliorated by exogenously administered BH4, and dietary phenylalanine restriction was effective for restoring the decreased monoamine contents in the brain of the Qdpr-/- mice, suggesting that monoamine deficiency was caused by the secondary effect of hyperphenylalaninemia. Immunohistochemical analysis showed that QDPR was primarily distributed in oligodendrocytes but hardly detectable in monoaminergic neurons in the brain. Finally, we performed a behavioral assessment using a test battery. The Qdpr-/- mice exhibited enhanced fear responses after electrical foot shock. Taken together, our data suggest that the perturbation of BH4 metabolism should affect brain monoamine levels through alterations in peripheral amino acid metabolism, and might contribute to the development of anxiety-related psychiatric disorders. Cover Image for this issue:https://doi.org/10.1111/jnc.15398.

Cellular diversity and gene expression profiles in the male and female brain of Aedes aegypti

BackgroundAedes aegypti is a medically-important mosquito vector that transmits arboviruses including yellow fever, dengue, chikungunya, and Zika viruses to humans. The mosquito exhibits typical sexually dimorphic behaviors such as courtship, mating, host seeking, bloodfeeding, and oviposition. All these behaviors are mainly regulated by the brain; however, little is known about the function and neuron composition of the mosquito brain. In this study, we generated an initial atlas of the adult male and female brain of Ae. aegypti using 10xGenomics based single-nucleus RNA sequencing.ResultsWe identified 35 brain cell clusters in male and female brains, and 15 of those clusters were assigned to known cell types. Identified cell types include glia (astrocytes), Kenyon cells, (ventral) projection neurons, monoaminergic neurons, medulla neurons, and proximal medulla neurons. In addition, the cell type compositions of male and female brains were compared to each other showing that they were quantitatively distinct, as 17 out of 35 cell clusters varied significantly in their cell type proportions. Overall, the transcriptomes from each cell cluster looked very similar between the male and female brain as only up to 25 genes were differentially expressed in these clusters. The sex determination factor Nix was highly expressed in neurons and glia of the male brain, whereas doublesex (dsx) was expressed in all neuron and glia cell clusters of the male and female brain.ConclusionsAn initial cell atlas of the brain of the mosquito Ae. aegypti has been generated showing that the cellular compositions of the male and female brains of this hematophagous insect differ significantly from each other. Although some of the rare brain cell types have not been detected in our single biological replicate, this study provides an important basis for the further development of a complete brain cell atlas as well as a better understanding of the neurobiology of the brains of male and female mosquitoes and their sexually dimorphic behaviors.

Sleep, Narcolepsy, and Sodium Oxybate.

Sodium oxybate (SO) has been in use for many decades to treat narcolepsy with cataplexy. It functions as a weak GABAB agonist but also as an energy source for the brain as a result of its metabolism to succinate and as a powerful antioxidant because of its capacity to induce the formation of NADPH. Its actions at thalamic GABAB receptors can induce slow-wave activity, while its actions at GABAB receptors on monoaminergic neurons can induce or delay REM sleep. By altering the balance between monoaminergic and cholinergic neuronal activity, SO uniquely can induce and prevent cataplexy. The formation of NADPH may enhance sleep’s restorative process by accelerating the removal of the reactive oxygen species (ROS), which accumulate during wakefulness. SO improves alertness in normal subjects and in patients with narcolepsy. SO may allay severe psychological stress - an inflammatory state triggered by increased levels of ROS and characterized by cholinergic supersensitivity and monoaminergic deficiency. SO may be able to eliminate the inflammatory state and correct the cholinergic/ monoaminergic imbalance.

Electrophysiological Quality Control of Human Dopaminergic Neurons: Are We Doing Enough?

The breakthrough of induced pluripotent stem (iPS) cell technology (Takahashi and Yamanaka, 2006) has allowed scientists to design protocols allowing the differentiation of these cells into the main cell types of our organism. This possibility is particularly exciting in the field of Parkinson's disease (PD), since early experiments using heterologous dopaminergic (DA) or chromaffin cells have demonstrated the ability of such cells, when injected in adequate sites, to improve symptoms of the disease in a sustained manner (Kefalopoulou et al., 2014). The possibility of reproducing this effect using autologous cells, thereby free of ethical concerns, is of course fascinating (Barker et al., 2017; Parmar et al., 2020). It has clearly been demonstrated that symptoms of experimental parkinsonism can be relieved in several species by such grafts (Kikuchi et al., 2017; Song et al., 2020), with a first human case report study yielding encouraging results (Schweitzer et al., 2020). Protocols are being refined to improve the yield of DA neurons and minimize the survival of undifferentiated cells (e.g., Piao et al., 2021). Immunohistochemical methods allow to clearly define the presence of DA neurons, the absence of other monoaminergic neurons (e.g., serotoninergic neurons) and the molecular subtype of these DA neurons (by examining the expression of a variety of transcription factors such as NURR1, PITX3, etc…). Many experiments have confirmed the possibility of obtaining preparations devoid of oncogenic potential. In the published papers, some efforts have been made to characterize the physiological properties of the DA neurons, but I argue that, in many cases, the data is not completely convincing and, more importantly, that the reader has no idea of the percentage of neurons that display the specific properties of these neurons. I believe that more standardization of these experiments is important to assess the functional quality of the future grafts. Although I reckon that safety is of paramount importance, I also believe that suboptimal functionality may impair the clinical effectiveness of future procedures.

Effect of Psychostimulants on the Subunit Stoichiometry of the Dopamine and Serotonin Transporter

Objective and hypothesis Monoamine transporters control extracellular monoamine levels in the brain by mediating their reuptake from the synaptic cleft, thereby terminating signal transduction in monoaminergic neurons. Previous studies of our lab have revealed that these transporters co-exist as monomers and oligomers at the plasma membrane. Transporter oligomerization has been suggested to play a role in transporter trafficking, drug-induced monoamine efflux, and drug tolerance development. However, many aspects regarding monoamine transporter oligomerization remain poorly understood. The objective of the present study was to investigate how psychostimulants affect monoamine transporter oligomerization for the first time on a single molecule level. Based on findings of other studies, the hypothesis was that substrate-type psychostimulants dissociate monoamine transporter oligomers whereas transporter inhibitors have no effect. Methods Transporter oligomerization was measured using single molecule microscopy in live transporter-transfected Chinese hamster ovary cells. The oligomeric state of fluorescently-labeled dopamine and serotonin transporter (DAT and SERT, respectively) was assessed in total internal reflection mode one hour after exposure to various transporter inhibitors and substrates. Results The substrates 3,4-methylenedioxymethamphetamine and mephedrone dissociated higher-order SERT oligomers, resulting in a statistically significant decrease in average oligomeric state (Student's t-test, P<0.05). Furthermore, both substrates reduced the kinetic trapping of SERT oligomers by phosphatidylinositol 4,5-bisphosphate (PIP2). In contrast, none of the tested transporter inhibitors altered SERT oligomerization. Furthermore, none of the tested transporter substrates or inhibitors altered the oligomeric state of DAT. Conclusion The effect of psychostimulants on DAT and SERT oligomerization was for the first time investigated on a single molecule level in live cells. Substrate-type stimulants dissociated SERT higher-order oligomers whereas transporter inhibitors had no effect. None of the psychostimulants affected DAT oligomers. The effect of substrate-type psychostimulants therefore resembles that of direct phospholipase C agonists.

Trace Amine‐Associated Receptor Intracellular Localization and Trafficking

Trace amine-associated receptor 1 (TAAR1) is a G-protein coupled receptor (GPCR) found in monoaminergic neurons of the central nervous system. Amphetamine-like psychostimulants activate TAAR1, which couples to Gαs and Gα13 downstream signaling cascades, and the latter regulates the RhoA-mediated internalization of neurotransmitter transporters. TAAR1 stimulation is crucial to methamphetamine (MA) mechanism of action, and a common variant of TAAR1 is associated with increased MA craving in MA users. Although the impact of TAAR1 on behavior has been characterized, the molecular mechanisms underlying TAAR1 pharmacology are incompletely understood. TAAR1 is a unique GPCR as it resides inside the cell rather than at the cell surface, though the precise location of TAAR1 within the cell is unknown. Understanding the intracellular localization, orientation, and trafficking of TAAR1 will further inform the molecular mechanisms of TAAR1 signaling. However, available antibodies and fluorescent fusion proteins for imaging TAAR1 are inadequate for such studies. Therefore, we have developed a novel method for imaging human TAAR1 (hTAAR1) through both light and electron microscopy using the miniVIPER peptide labeling system for multiscale microscopy. The hTAAR1-miniVIPER system has been applied to in vitro systems and has been used in conjunction with light microscopy to explore subcellular localization of the recombinant receptor in Chinese hamster ovary cells. Further investigation with electron microscopy will be carried out to determine the orientation of hTAAR1 on internal membranes and will indicate how TAAR1 mobilizes spatially distinct signaling cascades within the cell. In order to study the receptor in native tissue, AAV constructs will be employed to express the hTAAR1-miniVIPER construct in the dopamine neurons of mice. Tissue from these animals will be imaged through correlative light and electron microscopy to determine the subcellular localization and orientation of TAAR1. Animals expressing hTAAR1-miniR will be subjected to acute and chronic drug treatments to determine how TAAR1 agonism affects receptor localization and trafficking. Together these studies will expand our current understanding of molecular mechanisms of TAAR1 signaling and will inform future work concerning the role TAAR1 plays in the modulation of neural circuity relevant to addiction and methamphetamine use disorder.

Neuropathology of the Brainstem to Mechanistically Understand and to Treat Alzheimer's Disease.

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder as yet without effective therapy. Symptoms of this disorder typically reflect cortical malfunction with local neurohistopathology, which biased investigators to search for focal triggers and molecular mechanisms. Cortex, however, receives massive afferents from caudal brain structures, which do not only convey specific information but powerfully tune ensemble activity. Moreover, there is evidence that the start of AD is subcortical. The brainstem harbors monoamine systems, which establish a dense innervation in both allo- and neocortex. Monoaminergic synapses can co-release neuropeptides either by precisely terminating on cortical neurons or, when being “en passant”, can instigate local volume transmission. Especially due to its early damage, malfunction of the ascending monoaminergic system emerges as an early sign and possible trigger of AD. This review summarizes the involvement and cascaded impairment of brainstem monoaminergic neurons in AD and discusses cellular mechanisms that lead to their dysfunction. We highlight the significance and therapeutic challenges of transmitter co-release in ascending activating system, describe the role and changes of local connections and distant afferents of brainstem nuclei in AD, and summon the rapidly increasing diagnostic window during the last few years.

Differential and spatial expression meta-analysis of genes identified in genome-wide association studies of depression

Major depressive disorder (MDD) is the most prevalent psychiatric disorder worldwide and affects individuals of all ages. It causes significant psychosocial impairments and is a major cause of disability. A recent consortium study identified 102 genetic variants and 269 genes associated with depression. To provide targets for future depression research, we prioritized these recently identified genes using expression data. We examined the differential expression of these genes in three studies that profiled gene expression of MDD cases and controls across multiple brain regions. In addition, we integrated anatomical expression information to determine which brain regions and transcriptomic cell types highly express the candidate genes. We highlight 12 of the 269 genes with the most consistent differential expression: MANEA, UBE2M, CKB, ITPR3, SPRY2, SAMD5, TMEM106B, ZC3H7B, LST1, ASXL3, ZNF184 and HSPA1A. The majority of these top genes were found to have sex-specific differential expression. We place greater emphasis on ZNF184 as it is the top gene in a more conservative analysis of the 269. Specifically, the differential expression of ZNF184 was strongest in subcortical regions in males and females. Anatomically, our results suggest the importance of the dorsal lateral geniculate nucleus, cholinergic, monoaminergic and enteric neurons. These findings provide a guide for targeted experiments to advance our understanding of the genetic underpinnings of depression.

Interaction between Orexin Neurons and Monoaminergic Systems.

Orexins have received a lot of attention as potent endogenous arousal-promoting peptides, and orexin receptor antagonists have shown clinical efficacy for the treatment of insomnia. Orexin neurons are thought to act primarily on monoaminergic neurons to maintain arousal and vigilance. In this chapter, we discuss the functional interaction between monoaminergic systems, including noradrenaline, serotonin and histamine, and orexin neurons, as well as interactions between the acetylcholine system and the orexin neurons, focusing, in particular, on their function in the regulation of sleep-wakefulness states. Orexin also has close interactions with the dopaminergic system, and many studies have suggested roles of orexin signaling in the reward system and roles for orexins in drug addiction.

Selective Knockout of the Vesicular Monoamine Transporter 2 (Vmat2) Gene in Calbindin2/Calretinin-Positive Neurons Results in Profound Changes in Behavior and Response to Drugs of Abuse.

The vesicular monoamine transporter 2 (VMAT2) has a range of functions in the central nervous system, from sequestering toxins to providing conditions for the quantal release of monoaminergic neurotransmitters. Monoamine signaling regulates diverse functions from arousal to mood, movement, and motivation, and dysregulation of VMAT2 function is implicated in various neuropsychiatric diseases. While all monoamine-releasing neurons express the Vmat2 gene, only a subset is positive for the calcium-binding protein Calbindin 2 (Calb2; aka Calretinin, 29 kDa Calbindin). We recently showed that about half of the dopamine neurons in the mouse midbrain are positive for Calb2 and that Calb2 is an early developmental marker of midbrain dopamine cells. Calb2-positive neurons have also been identified in other monoaminergic areas, yet the role of Calb2-positive monoaminergic neurons is poorly understood. To selectively address the impact of Calb2-positive monoaminergic neurons in behavioral regulation, we took advantage of the Cre-LoxP system to create a new conditional knockout (cKO) mouse line in which Vmat2 expression is deleted selectively in Calb2-Cre-positive neurons. In this Vmat2lox/lox;Calb2−Cre cKO mouse line, gene targeting of Vmat2 was observed in several distinct monoaminergic areas. By comparing control and cKO mice in a series of behavioral tests, specific dissimilarities were identified. In particular, cKO mice were smaller than control mice and showed heightened sensitivity to the stereotypy-inducing effects of amphetamine and slight reductions in preference toward sucrose and ethanol, as well as a blunted response in the elevated plus maze test. These data uncover new knowledge about the role of genetically defined subtypes of neurons in the brain’s monoaminergic systems.

Stress induces divergent gene expression among lateral habenula efferent pathways

The lateral habenula (LHb) integrates critical information regarding aversive stimuli that shapes decision making and behavioral responses. The three major LHb outputs innervate dorsal raphe nucleus (DRN), ventral tegmental area (VTA), and the rostromedial tegmental nucleus (RMTg). LHb neurons that project to these targets are segregated and nonoverlapping, and this led us to consider whether they have distinct molecular phenotypes and adaptations to stress exposure. In order to capture a time-locked profile of gene expression after repeated forced swim stress, we used intersectional expression of RiboTag in rat LHb neurons and next-gen RNA sequencing to interrogate the RNAs actively undergoing translation from each of these pathways. The “translatome” in the neurons comprising these pathways was similar at baseline, but diverged after stress, especially in the neurons projecting to the RMTg. Using weighted gene co-expression network analysis, we found one module, which had an overrepresentation of genes associated with phosphoinositide 3 kinase (PI3K) signaling, comprising genes downregulated after stress in the RMTg-projecting LHb neurons. Reduced PI3K signaling in RMTg-projecting LHb neurons may be a compensatory adaptation that alters the functional balance of LHb outputs to GABAergic vs. monoaminergic neurons following repeated stress exposure.

Monoamine Mapping Using Mass Spectrometry Identified New Monoamine-rich Brain Nuclei

Monoamine neurotransmitters are released by specialized neurons that regulate behavioral and cognitive functions. Although localization of monoaminergic neurons in the brain is well known, the distribution, concentration, and kinetics of monoamines remain unclear. We used mass spectrometry imaging (MSI) for simultaneous and quantitative imaging of endogenous monoamines to generate a murine brain atlas of serotonin (5-HT), dopamine (DA), and norepinephrine (NE) levels. We observed several nuclei rich in both 5-HT and a catecholamine (DA or NE). Additionally, we analyzed de novo monoamine synthesis or fluctuations in those nuclei. We propose that MSI is a useful tool to gain deeper understanding of associations among the localization, levels, and turnover of monoamines in different brain areas and their role in inducing behavioral changes.

Ventrolateral Preoptic Nucleus (VLPO) Role in Turning the Ascending Arousal System Off During Sleep

Coordinated manner of action of the projections from monoaminergic cell groups, cholinergic and orexin neurons...

Enhancement of the rewarding effects of 3,4-methylenedioxymethamphetamine in orexin knockout mice

Orexinergic neurons, which are closely associated with narcolepsy, regulate arousal and reward circuits through the activation of monoaminergic neurons. Psychostimulants as well as 5-HT-related compounds have potential in the treatment of human narcolepsy. Previous studies have demonstrated that orexin receptor antagonists as well as orexin deficiencies affect the pharmacological effects of psychostimulants. However, little information is available on the consequences of psychostimulant use under orexin deficiency. Therefore, the present study was designed to investigate the abuse liability of psychostimulants in orexin knockout (KO) mice. In the present study, conditioned place preferences induced by methamphetamine and methylphenidate were not altered in orexin KO mice. Interestingly, we found that MDMA induced a conditioned place preference in orexin KO mice, but not in wild type (WT) mice. In addition, MDMA produced methylphenidate/methamphetamine-like discriminative stimulus effects in orexin KO mice, but not WT mice. Increases in 5-HT and dopamine release in the nucleus accumbens induced by MDMA were not altered by knockout of orexin; the steady-state level of G protein activation was higher in the limbic forebrain of orexin KO mice. In substitution tests using a drug discrimination procedure, substitution of 5-HT1A receptor agonist for the discriminative stimulus effects of methylphenidate was enhanced in orexin KO mice. These findings indicate that the orexinergic system is involved the rewarding effects of psychostimulants. However, there is a risk of establishing rewarding effects of psychostimulants even under orexin deficiency. On the other hand, deficiencies in orexin may enhance the abuse liability of MDMA by changing a postsynaptic signal transduction accompanied by changes in discriminative stimulus effects themselves.

3,4-Methylenedioxypyrovalerone: Neuropharmacological Impact of a Designer Stimulant of Abuse on Monoamine Transporters.

Methylenedioxypyrovalerone (MDPV) is an abused synthetic cathinone, commonly referred to as a "bath salt." Because the dopamine (DA) transporter (DAT) and vesicular monoamine transporter-2 (VMAT-2) are key regulators of both the abuse and neurotoxic potential of structurally and behaviorally related agents, the impact of MDPV on these transporters was investigated. Results revealed that a single in vivo MDPV administration rapidly (within 1 hour) and reversibly increased both rat striatal DAT and VMAT-2 activity, as assessed via [3H]DA uptake in synaptosomes and synaptic vesicles, respectively, prepared from treated rats. There was no evidence of an MDPV-induced increase in plasmalemmal membrane DAT surface expression. Plasma concentrations of MDPV increased dose-dependently as assessed 1 hour after 2.5 and 5.0 mg/kg (s.c.) administration and returned to levels less than 10 ng/ml by 18 hours after 2.5 mg/kg (s.c.). Neither pretreatment with a D1 receptor (SCH23390), a D2 receptor (eticlopride), nor a nicotinic receptor (mecamylamine) antagonist attenuated the MDPV-induced increase in DAT activity. In contrast, eticlopride pretreatment attenuated both the MDPV-induced increase in VMAT-2-mediated DA uptake and an associated increase in cytoplasmic-associated vesicle VMAT-2 immunoreactivity. SCH23390 did not attenuate the MDPV-induced increase in VMAT-2 activity. Repeated MDPV injections did not cause persistent DAergic deficits, as assessed 7 to 8 days later. The impact of MDPV on striatal and hippocampal serotonergic assessments was minimal. Taken together, these data contribute to a growing pharmacological rubric for evaluating the ever-growing list of designer cathinone-related stimulants. The profile of MDPV compared with related psychostimulants is discussed. SIGNIFICANCE STATEMENT: Pharmacological characterization of the synthetic cathinone, 3,4-methylenedioxypyrovalerone (MDPV; commonly referred to as a "bath salt"), is critical for understanding the abuse liability and neurotoxic potential of this and related agents. Accordingly, the impact of MDPV on monoaminergic neurons is described and compared with that of related psychostimulants.

Genetic identification of cell types underlying brain complex traits yields insights into the etiology of Parkinson's disease.

Genome-wide association studies (GWAS) have discovered hundreds of loci associated with complex brain disorders but it remains unclear in which cell types these loci are active. Here we integrate GWAS results with single-cell transcriptomic data from the entire mouse nervous system to systematically identify cell types underlying brain complex traits. We show that psychiatric disorders are predominantly associated with projecting excitatory and inhibitory neurons. Neurological diseases were associated with different cell types, which is consistent with other lines of evidence. Notably, Parkinson’s disease was not only genetically associated with cholinergic and monoaminergic neurons (which include dopaminergic neurons) but also with enteric neurons and oligodendrocytes. Using post-mortem brain transcriptomic data, we confirmed alterations in these cells, even at the earliest stages of disease progression. Our study provides an important framework for understanding the cellular basis of complex brain maladies, and reveals an unexpected role of oligodendrocytes in Parkinson’s disease.

Teratogenic effects of 1.5% EtOH in Cranial Blood Vessel development and Neurogenesis in Zebrafish larvae at 72hpf

Fetal alcohol spectrum disorder, which is observed in children who have been exposed to alcohol in utero, is a leading cause of neurodevelopmental disability. Studies in rodents have shown that prenatal ethanol exposure results in decreased blood flow from the umbilical artery to the fetal brain, compromising neuron formation and angiogenesis in the maturing brain. Ethanol (EtOH) has shown to disrupt coronary artery formation in the embryonic zebrafish, but the effects of ethanol on the developing cerebral blood vessels and neurogenesis in zebrafish larvae remains poorly understood. We exposed zebrafish larvae to 1.5% ethanol at 4 hours post fertilization (hpf) for 24 hours and imaged the brain at 72 hpf. We used Tg(fli1:EGFP) to image cerebral blood vessels and Tg(ETvmat2:GFP) to visualize monoaminergic neurons using confocal microscopy. Morphological data collected at 72hpf depicted significantly higher rates of craniofacial deformities, pericardial edema, and severe axial malformations in 1.5% EtOH‐treated embryos in comparison to control. Our preliminary results suggest that EtOH at 1.5% leads to reduced number of cranial vessels and altered vessel branching, and decreased density of telencephalic and hindbrain neurons. These findings lead to questions regarding molecular and vascular disparities in affected regions of the developing brain.Support or Funding InformationI am currently seeking for funds (nonmember student registration fee) for our undergraduate students to attend and present this poster at the conference.

Synthesis of Dopamine by Non-Dopaminergic Neurons Containing Aromatic Amino Acid Decarboxylase in the Suprachiasmatic Nucleus of Rats in Ontogeny

Abstract—The suprachiasmatic nucleus (SCN) regulates functions in accordance with circadian rhythms thanks to the pacemaker neurons present in it and afferents modulating their activity. One type of afferent is nerve fibers containing only tyrosine hydroxylase (TH) and secreting (L-dihydroxyphenylalanine) L-DOPA. The aim of this work was to test the hypothesis that L-DOPA comes from TH-containing fibers into neurons containing only aromatic L-amino acid decarboxylase (AAAD), where dopamine is synthesized. The synthesis of dopamine was evaluated by its content in SCN in intact rats on the 10th postnatal day (P10) and on P60, as well as by the total dopamine content in SCN and in the incubation medium after incubation of this nucleus in the medium with or without L-DOPA. It was shown that in intact rats SCN contains dopamine at the same concentration at P10 and P60. Some of the experiments with incubation of SCN slices were performed with rats injected with neurotoxins of monoaminergic neurons to exclude the synthesis of dopamine from L-DOPA in monoaminergic structures. At P10, during incubation of SCN in L-DOPA-containing medium, an increase in dopamine synthesis was observed equally in animals in control (normal) and after exposure to neurotoxins, which indicates synthesis of dopamine only in monoenzymatic AAAD-containing neurons. In a similar experiment at P60, the addition of L-DOPA into the incubation medium also led to an increase in the synthesis of dopamine, however, in animals exposed to neurotoxins to a lesser extent than in the control. This means that in adult animals, dopamine is synthesized in SCN mainly in monoenzymatic AAAD-containing neurons and to a lesser extent in monoaminergic structures. Thus, it was shown that dopamine in SCN is synthesized in monoenzymatic AAAD-containing neurons from L-DOPA secreted by monoenzymatic TH-containing fibers.

The neurotoxicity of psychoactive phenethylamines “2C series” in cultured monoaminergic neuronal cell lines

The aim of this study was to evaluate the neurotoxicity of psychoactive abused 2,5-dimethoxy-substituted phenethylamines “2C series” in monoaminergic neurons. After the exposure to “2C series”, 2,5-dimethoxy-4-propylthiophenethylamine (2C-T-7), 2,5-dimethoxy-4-isopropylthiophenethylamine (2C-T-4), 2,5-dimethoxy-4-ethylthiophenthylamine (2C-T-2), 2,5-dimethoxy-4-iodophenethylamine (2C-I) or 2,5-dimethoxy-4-chlorophenethylamine (2C-C), we examined their neurotoxicity, morphological changes, and effects of concomitant exposure to 3,4-methylenedioxymethamphetamine (MDMA) or methamphetamine (METH), using cultured neuronal dopaminergic CATH.a cells and serotonin-containing B65 cells. Single dose exposure to “2C series” for 24 h showed significant cytotoxicity as increase in lactate dehydrogenase (LDH) release from both monoaminergic neurons: 2C-T-7, 2C-C (EC50; 100 µM) > 2C-T-2 (150 µM), 2C-T-4 (200 µM) > 2C-I (250 µM) in CATH.a cells and 2C-T-7, 2C-I (150 µM) > 2C-T-2 (250 µM) > 2C-C, 2C-T-4 (300 µM) in B65 cells. The “2C series”-induced neurotoxicity in both cells was higher than that of MDMA or METH (EC50: ≥ 1–2 mM). In addition, apoptotic morphological changes were observed at relatively lower concentrations of “2C series”. The concomitant exposure to non-toxic dose of MDMA or METH synergistically enhanced 2C series drugs-induced LDH release and apoptotic changes in B65 cells, but to a lesser extent in CATH.a cells. In addition, the lower dose of 2C-T-7, 2C-T-2 or 2C-I promoted reactive oxygen species production in the mitochondria of B65 cells, even at the early stages (3 h) without apparent morphological changes. The 2,5-dimethoxy-substitution of “2C series” induced severe neurotoxicity in both dopaminergic and serotonin-containing neurons. The non-toxic dose of MDMA or METH synergistically enhanced its neurotoxicity in serotonergic neurons.