How To Repair Dopamine Receptors Domain_10

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Dopamine Receptor Antagonists Heighten Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors

Eva Hedlund,^{a, 1, 2, *} Laure Belnoue,^{b, three, *} Spyridon Theofilopoulos,⁴ Carmen Salto,⁴ Chris Bye,⁵ Clare Parish,⁵ Qiaolin Deng,^{ane, 3} Banafsheh Kadkhodaei,¹ Johan Ericson,ⁱⁱⁱ Ernest Arenas,^{4, †} Thomas Perlmann,^{1, 3, †} and András Simon^{three, †}

Eva Hedlund

^oneLudwig Found for Cancer Research, Stockholm Branch, Nobels five iii, 171 77 Stockholm, Sweden

²Section of Neuroscience, Karolinska Institutet, Retzius v. 8, 171 77 Stockholm, Sweden

Laure Belnoue

³Section of Cell and Molecular Biology, Karolinska Institutet, von Eulers v. iii, 171 77 Stockholm, Sweden

Spyridon Theofilopoulos

⁴Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Retzius v. viii, 171 77 Stockholm, Sweden

Carmen Salto

⁴Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Retzius v. 8, 171 77 Stockholm, Sweden

Chris Bye

^vFlorey Establish for Neuroscience and Mental Health, Academy of Melbourne, Parkville, Victoria, 3010 Australia

Clare Parish

⁵Florey Plant for Neuroscience and Mental Wellness, University of Melbourne, Parkville, Victoria, 3010 Australia

Qiaolin Deng

^oneLudwig Institute for Cancer Inquiry, Stockholm Branch, Nobels v 3, 171 77 Stockholm, Sweden

³Department of Cell and Molecular Biological science, Karolinska Institutet, von Eulers v. 3, 171 77 Stockholm, Sweden

Banafsheh Kadkhodaei

¹Ludwig Constitute for Cancer Research, Stockholm Branch, Nobels 5 three, 171 77 Stockholm, Sweden

Johan Ericson

³Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers v. 3, 171 77 Stockholm, Sweden

Ernest Arenas

⁴Laboratory of Molecular Neurobiology, Section of Medical Biochemistry and Biophysics, Karolinska Institutet, Retzius v. eight, 171 77 Stockholm, Sweden

Thomas Perlmann

ⁱLudwig Institute for Cancer Enquiry, Stockholm Branch, Nobels v 3, 171 77 Stockholm, Sweden

^threeDepartment of Prison cell and Molecular Biology, Karolinska Institutet, von Eulers five. iii, 171 77 Stockholm, Sweden

András Simon

³Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers v. 3, 171 77 Stockholm, Sweden

Received 2022 Dec 21; Accepted 2022 Apr 25.

Abstruse

Degeneration of dopamine neurons in the midbrain causes symptoms of the movement disorder, Parkinson illness. Dopamine neurons are generated from proliferating progenitor cells localized in the embryonic ventral midbrain. Nonetheless, it remains unclear for how long cells with dopamine progenitor character are retained and if there is any potential for reactivation of such cells later on cessation of normal dopamine neurogenesis. We evidence here that cells expressing Lmx1a and other progenitor markers remain in the midbrain aqueductal zone across the major dopamine neurogenic menstruation. These cells express dopamine receptors, are located in regions heavily innervated by midbrain dopamine fibres and their proliferation can exist stimulated by antagonizing dopamine receptors, ultimately leading to increased neurogenesis in vivo. Furthermore, treatment with dopamine receptor antagonists enhances neurogenesis in vitro, both from embryonic midbrain progenitors every bit well as from embryonic stem cells. Altogether our results indicate a potential for reactivation of resident midbrain cells with dopamine progenitor potential beyond the normal period of dopamine neurogenesis.

Midbrain dopamine neurons play key roles in neurotransmission controlling locomotion, knowledge and reward. Major motor symptoms in Parkinson's affliction are caused by degeneration of dopamine neurons and an of import inquiry aim has therefore been to develop methodology for technology dopamine neurons from stem cells in vitro for cell replacement therapy in Parkinson'south patients^{1 ,ii ,three}, reviewed in ref. ⁴. Detailed understanding of the normal procedure of dopamine neurogenesis has been essential in these efforts, reviewed in ref. ⁵. During development, spatially organized signalling events lead to the expression of transcription factors, including the LIM-homeodomain protein Lmx1a in early proliferating neural progenitors localized close to the ventricular wall of the midbrain channel^{6 ,seven}. Lmx1a, together with the related transcription factor Lmx1b, specify neural progenitors and is essential for the initiation of a molecular program for dopamine neurogenesis^{8 ,9}. Equally Lmx1a-specified progenitor cells get out the cell cycle additional transcription factors are induced, including Nurr1 and Pitx3⁵. These factors promote dopamine neuron differentiation and the acquisition of dopaminergic characteristics equally cells migrate kickoff radially and then tangentially towards the prospective ventral tegmental area and substantia nigra^{10 ,xi}.

The bulk of dopamine neurogenesis occurs normally between embryonic days (East) 10–14 in mice¹². An heady possibility would exist if substantial dopamine neurogenesis could be induced after the chief menstruation of embryonic dopamine neurogenesis. The potential for late dopamine neurogenesis has previously been investigated. Some studies point that loss of dopamine neurons in the adult brain evokes responses that could lead to de novo generation of dopamine neurons, while others discover no evidence for such events^{13 ,xiv ,15 ,xvi}. However, both the identity of potential progenitor cells as well as the mechanisms regulating their fate have remained elusive.

Here nosotros addressed the duration of Lmx1a expression in midbrain ventricular cells, and whether Lmx1a-expressing cells could constitute a cell population with progenitor potential for reactivation at later stages of embryogenesis. We also asked if neurogenesis in the developing ventral midbrain could exist regulated past the neurotransmitter dopamine itself since previous findings revealed that in aquatic salamanders, dopamine negatively controls the product of dopamine neurons both during homeostasis and regeneration¹⁷. Salamanders are the only vertebrates known to engagement with the ability to fully restore the dopaminergic system past a process that is driven past reactivation of dopamine neurogenesis^{18 ,xix}. Together our analyses indicate a potential for neurogenesis from persisting Lmx1a-expressing cells.

Results

Ventral midbrain ventricular cells maintained expression of Lmx1a and other progenitor markers

To investigate if Lmx1a expression was temporally restricted to the catamenia of dopamine neurogenesis (E10-E14)¹² nosotros first performed immunostaining and in situ hybridizations at various time points during development. We plant that Lmx1a expression was maintained in ventricular cells of the ventral midbrain at E15.five. In situ hybridization also demonstrated persistent Lmx1a mRNA expression at E15.5 and E18.5 (Fig. 1A–D). All the same, while Lmx1a protein was expressed at E15.5, no poly peptide expression could be detected by immunohistochemistry at E18.5 and at postnatal stages. Nosotros confirmed these findings by examining heterozygous Lmx1a ^eGFP/+ reporter mouse embryos^eight, which in accordance with immunostaining and in situ hybridization showed declining expression, but persistent presence of eGFP⁺ cells throughout development and as well in the adult brute at three and eight months of age (Fig. 1E–H).

Ventral midbrain Lmx1a-expressing ventricular cells maintained progenitor backdrop.

Lmx1a is expressed in ventral midline cells that generate midbrain dopamine neurons at E12.5, shown past staining against Lmx1a poly peptide and using Lmx1a^GFP/+ reporter mice (A). The expression of Lmx1a was maintained in ventricular progenitors at E15.5 (B,C) and E18.5 (D) shown by immunofluorescence and in situ hybridization. Lmx1a^GFP/+ reporter mice revealed a persistent presence of eGFP⁺ ventricular cells during evolution and in the developed animals, depicted at E12.5 (E), E15.5 (F), 3 months (G) and 8 months of historic period (H). Nonetheless, the eGFP⁺ cells decreased in numbers with fourth dimension and changed in morphology with a gradual shortening of the processes (Due east–H). Ventral eGFP⁺ ventricular cells expressed nestin both in the embryo and the adult animal (I–G). At E15.five the majority of cells lining the aqueduct were nestin⁺ (I), while at E18.5, nestin was restricted mainly to the eGFP⁺ cells (J). eGFP⁺ cells were distinguished from other ventricular cells by their lack of Sox1 expression (L), but showed overlapping expression with Sox2 (Thou), Sox3 (N) and prominin (O,P). The number of proliferating eGFP⁺ ventricular cells steadily decreased during development, with 123.9 ± 12.5 Ki67⁺ cells at E12.five (Q,T, north = vi mice), 74.4 ± 9.1 Ki67⁺ cells at E15.5 (R,T, n = eight mice) and 2.5 ± ane.7 Ki67⁺ cells at E18.5 (Due south,T, n = 5 mice) (**P < 0.01, ***P < 0.001, ANOVA, Tukey's mail hoc test). Scale bars: 50 μM in (B) (applicative to A,K), 50 μM in (D) (applicable to C), 50 μM in (H) (applicable to E–1000, P, Q–S), 20 μM in (N) (applicable to I,J,50,M) and 150 μM in (O).

To characterize ventral eGFP⁺ ventricular cells we analysed the expression of stem and progenitor markers during late embryogenesis. We found that eGFP⁺ cells expressed nestin both in the embryo and the adult animal (Fig. 1I–G). At E15.5 nearly ventricular cells lining the channel were nestin⁺ (Fig. 1I), while at E18.five, the expression of nestin was restricted mainly to eGFP⁺ cells in the flooring plate (FP) (Fig. 1J) and the roof plate (RP) (information not shown). Although nestin was maintained in the adult brute, the morphology of the eGFP⁺ cells changed markedly with age, displaying gradual shortening of their processes, equally visualised by eGFP (Fig. 1G,H) and nestin expression (Fig. 1K). In dissimilarity to the neighboring ventricular cells, eGFP⁺ cells were devoid of Sox1 (Fig. 1L), but showed an overlapping expression with Sox2 (Fig. 1M) and Sox3 (Fig. 1N). The Sox1, Sox2 and Sox3 expression patterns were maintained at E18.5 (Supplementary Fig. 1A–F). Notably, the eGFP⁺ cells also expressed the stem cell marker prominin (Fig. 1O,P). Developed ventricular eGFP⁺ cells also displayed a robust expression of Sox2 (Supplementary Fig. 2A–E), indicative of their maintained progenitor potential. In addition, a subset of the adult eGFP⁺ cells expressed Sox3 and prominin (Supplementary Fig. 2F–M). Ki67 staining showed that the number of proliferating eGFP⁺ ventricular cells steadily decreased with developmental age (Fig. 1Q–T) indicating that these cells exited the cell cycle concomitant with the termination of dopamine neurogenesis. Collectively, our information demonstrate that cells of the Lmx1a-lineage maintain progenitor markers during late embryogenesis until E18.v. Thus, dopamine progenitor backdrop may extend beyond the principal period of dopamine neurogenesis.

eGFP⁺ cells expressed dopamine receptors and were localized within a domain innervated past midbrain dopamine neurons

We previously showed that mature dopamine neurons are in close contact with neural stem cells in the adult salamander, a highly regenerative species in which the dopaminergic system can be completely restored post-obit chemic ablation of dopamine neurons, and in which dopamine negatively regulates midbrain dopamine neurogenesis¹⁷. Nosotros set out to accost to what extent such a regulatory mechanism might exist evolutionary conserved. Start, we found that eGFP⁺ cells expressed dopamine D2 receptors (D2R) both during embryogenesis and in the adult brain (Fig. 2A–D; orthogonal view of E18.5 shown in Supplementary Fig. 3A,B). Next, immunofluorescent analyses revealed that tyrosine hydroxylase (TH⁺) fibers projected onto the ventral aqueduct in the midbrain (Fig. 2E). High magnification confocal images and orthogonal sections indicated that Thursday fibers partially surrounded a proportion of the eGFP⁺ progenitor cells (Supplementary Fig. 3C,D). To investigate the origin of the Th⁺ fibers projecting to the midbrain channel, we analysed DATCreYFP mice, in which YFP is exclusively expressed in dopamine transporter (DAT)-expressing midbrain dopamine neurons. We found that TH⁺ fibers surrounding the ventricular Lmx1a-expressing cells expressed YFP in these embryos, indicating their midbrain origin (Fig. 2F,G). Periaqueductal TH⁺ neurons, located just lateral to the ventral aqueduct, do non express DAT and for this reason did not express YFP in transgenic embryos. These periaqueductal TH⁺ neurons also extended processes in close proximity to the channel (Fig. 2F,1000). Histological assay of conditional Nurr1^DATCre (Nurr1CKO) mice, in which well-nigh dopamine neurons are lost^twenty, showed a 7.ix-fold reduction in TH⁺ fiber innervation (Fig. 2H–L) suggesting that a majority of fibers originated from midbrain dopamine neurons in the ventral midbrain surface area. Every bit periaqueductal Th⁺ neurons are not lost in Nurr1-ablated mice (Fig. 2H,K,50), it is possible that some of the remaining fibers originated from this neuronal subpopulation. Furthermore, we plant that eGFP⁺ cells lining the quaternary ventricle in the hindbrain were also in close proximity to TH⁺ fibers originating from the locus coeruleus (Supplementary Fig. 3E,F). Notably, analysis showed that TH⁺ fibers were in close proximity with the outer layers of the midbrain aqueductal cells also in the human encephalon (Fig. 2M,N). Together these data indicated a possibility for dopamine regulation of Lmx1a-expressing ventricular cells.

Lmx1a-expressing ventricular cells expressed dopamine receptors and their domain was innervated past midbrain dopamine neurons.

In that location was a gradual increase in the fraction of eGFP⁺ cells expressing D2 receptor during embryonic evolution (A–C). eGFP⁺ cells expressed D2 receptors in adult animals (D). Immunofluorescent assay of 3-calendar month-onetime mice showed that the ventral aqueduct in the midbrain was innervated past tyrosine hydroxylase (Th⁺) fibers (E). Analysis of DATCrexROSA26YFP (DATCreYFP) mice demonstrated that Thursday⁺ fibers surrounding the ventricular eGFP⁺ cells were YFP⁺ and thus of a midbrain dopamine neuron origin (F, enlarged in G). Periaqueductal TH⁺ neurons, which lack DAT, too extended processes in close proximity to the aqueduct (F,G). Nurr1CKO mice, which lack SNc dopamine neurons, and have reduced numbers of VTA dopamine neurons, showed a 7.9-fold reduction in Th⁺ fiber innervation at 5 months of age (P = 0.0021, n = 3 + 4, unpaired Student's t test) (H–L). Human midbrain sections demonstrated that TH⁺ fibers were in contact with the outer layers of the aqueductal cells (M,N). Scale bars: 50 μM in (A) (applicative to B,C), 50 μM in (D), 200 μM in (F), l μM in (K), 200 μM in (G) (applicative to I), 50 μM in (L) (applicable to E,J).

Haloperidol increased eGFP⁺ jail cell proliferation and neurogenesis

To examine if eGFP⁺ cells may be regulated by dopamine, we administered the broad-spectrum dopamine receptor adversary, haloperidol, to significant Lmx1a ^eGFP/+ females. Animals received two daily injections of haloperidol or vehicle and were sacrificed the following day. We first carried out this series of experiments during E12.5–E14.5 and found that haloperidol administration led to an increased number of eGFP⁺ cells that were also positive for the mitotic marker pH3 (Fig. 3A,B). Interestingly, haloperidol induced a particularly strong increment in the number of pH3⁺ eGFP⁺ cells in the caudal portions of the midbrain (Supplementary Fig. 4A). This is in concordance with the more prominent Thursday innervation in caudal midbrain (Supplementary Fig. 5). We likewise administered the nucleotide analogue BrdU to animals via osmotic pumps, but positive cells could non be counted because virtually all cells had incorporated the analogue due to the intense proliferation during this agile neurogenic period (data non shown). In contrast, when haloperidol was administered after the decline of normal dopamine neurogenesis, we could evaluate BrdU-incorporation and plant a i.5-fold increment of BrdU⁺ eGFP⁺ cells both at E16.5 and E17.5 (Fig. 3C,D). Similarly to the pH3-index, we noted a marked shift towards the caudal portions of the midbrain harbouring more than BrdU⁺ eGFP⁺ cells (Supplementary Fig. 4B). We too found that haloperidol appeared to increment the proliferation of the eGFP-negative cells (Supplementary Fig. 4C). In agreement with these information we noted a marked increase in the number of cycling cells in the midbrain progenitor zone at E11.v in factor targeted mice lacking both alleles of the D2R gene (Fig. 3E).

Haloperidol treatment increases the proliferation of eGFP⁺ cells and neurogenesis in vivo.

Haloperidol treatment increased the percentage of pH3⁺ eGFP⁺ ventricular cells (A,B, Haloperidol during E12.5–13.five, northward = viii (Ctrl), n = ix (Hal); Haloperidol during E13.5–14.5, n = 11 (Ctrl), n = 9 (Hal); Haloperidol during E14.5–15.5, north = 13 (Ctrl), n = ten (Hal), 2-way-ANOVA, handling effect P = 0,003). Haloperidol treatment increased the per centum of BrdU⁺ eGFP⁺ cells at E16.5 in the ventral part of the third ventricle (C,D, Haloperidol during E15.v–16.five, due north = x (Ctrl), n = 11 (Hal); Haloperidol during E16.5–17.5, n = 12 (Ctrl), n = 12 (Hal), 2-mode-ANOVA, handling effect P = 0,016). The number of Ki67⁺ ventricular cells in the ventral midbrain was increased in D2R KO mice at E11.5 compared to wild-type littermates (Eastward, n = v (Wt), northward = 5 (KO), P = 0.04, unpaired Student's t examination). Haloperidol treatment at E15.5–E17.five increased the number of BrdU⁺ cells in the prospective substantia nigra and ventral tegmental area (F,1000, n = 6 (Ctrl), north = 7 (Hal), P = 0.0014, unpaired Student'south t exam). Haloperidol treatment at E15.5–E17.5 did not modify the percent of BrdU⁺ cells expressing TH in the prospective substantia nigra and ventral tegmental expanse (H, northward = 4 (Ctrl), northward = 4 (Hal), P = 0.32, unpaired Student'due south t examination). Orthogonal sections showing BrdU⁺ TH⁺ (acme panel, arrow) and BrdU⁺ Th ⁻ (bottom panel, arrow) (I). Haloperidol treatment at E15.5–E17.v increased the density of BrdU⁺ Thursday⁺ cells in the prospective substantia nigra and ventral tegmental area (P = 0.002, unpaired Student'southward t test). Calibration confined: 50 μM in (B,D,E), 100 μM in (Yard) and 20 μM in (H).

Next nosotros tested whether haloperidol handling could increase the proliferation of the eGFP⁺ cells and the production of new dopamine neurons after E14.5. We thus administered haloperidol and BrdU between E15.5 to E17.5, as described above, just this fourth dimension evaluated the number BrdU⁺ cells in the prospective substantia nigra and ventral tegmental surface area instead of the ventricular zone. A i.5-fold increase in the density of BrdU⁺ cells was detected in haloperidol treated animals (Fig. 3F,G). These BrdU⁺ cells were equally distributed betwixt VTA and SNc (Supplementary Fig. 4D). Next we analysed the identity of BrdU⁺ cells and found that the percentage of BrdU⁺ cells expressing Th was unaffected, indicating that dopamine neuron differentiation was similar in both groups (Fig. 3H). Hence, the total number of newborn Thursday⁺ neurons showed a 1.5-fold increase in haloperidol compared to vehicle treated animals (Fig. 3I). These data collectively testify that antagonizing dopamine signalling increases the proliferation of ventral aqueductal cells and ultimately the number of newborn Thursday⁺ neurons in the midbrain.

To corroborate these findings we tested the effect of various neurotransmitters on primary cultures of embryonic midbrain cells from animals undergoing dopamine neurogenesis. Nosotros took advantage of the fact that these cultures independent a mixture of forerunner, including both dopamine and γ-aminobutyric acid (GABA) progenitors and used an array of neurotransmitter agonists and antagonists targeting dopamine and GABA receptors. In accordance with the in vivo observations we found that haloperidol handling increased the proliferation of primary embryonic midbrain cells one.5 fold as assayed by BrdU-incorporation (Fig. 4A). Moreover treatment with the dopamine two receptor (D2R) antagonist, sulpiride, but not the dopamine one receptor (D1R) antagonist SCH-23390, increased proliferation (Fig. 4A). In dissimilarity, neither dopamine itself nor the dopamine receptor agonists quinpirole or dihydrexidine increased proliferation. We also found that the GABA_A receptor agonist muscimol decreased proliferation, while the GABA_A receptor blocker, picrotoxin, increased proliferation (Fig. 4A, Supplementary Fig. 6). Consistently with these results nosotros observed an increased fraction of TH⁺ cells in the cultures upon haloperidol and sulpiride handling, while neither the dopamine receptor agonists nor SCH-23390 had this effect (Fig. 4B). Furthermore, sulpiride as well increased the fraction of BrdU⁺ cells expressing Thursday (Fig. 4C). Although we found that the GABA_A receptor blocker, picrotoxin, increased proliferation, the fraction of TH⁺ cells did not increment in the cultures (Fig. 4B), indicating a selective part for dopamine receptor signalling in dopamine neurogenesis. To farther validate these observations and to analyse if dopamine signalling could regulate the number of dopamine neurons generated from mouse embryonic stem cells (mESCs) we induced neurogenesis in mESC-derived midbrain cultures and plant that dopamine decreased the fraction of EdU⁺/Th⁺ cells whereas both haloperidol and sulpiride increased it (Fig. 4D,E). These in vitro information are consistent with the results obtained in vivo and point that dopamine receptor antagonists may serve as a tool to enhance dopamine neurogenesis.

Dopamine receptor antagonists increased cell proliferation and neurogenesis in embryonic midbrain and mESC cultures.

The fraction of principal midbrain cells increased significantly upon treatment with the dopamine receptor (DR) antagonists haloperidol and sulpiride, but non with SCH-23390. Dopamine and the DR agonist quinpirole did not increase prison cell proliferation. The GABA_A receptor agonist muscimol decreased proliferation, while the GABA_A receptor blocker, picrotoxin, increased proliferation (A). Only the DR antagonists haloperidol and sulpiride increased the fraction of TH⁺ cells in primary midbrain cultures (B). The DR adversary sulpiride increased the fraction of BrdU⁺ TH⁺ cells in primary midbrain cultures (C). Dopamine (DA) decreased, while haloperidol (Hal) and sulpiride (Sulpi) increased the fraction of EdU⁺ TH⁺/TH⁺ cells derived from mESCs (D,Eastward). (A,B,D, *P < 0.05; **P < 0.01; ***P < 0.001, i-mode ANOVA and Newman-Keuls mail service hoc examination, (C), unpaired Student's t examination). Calibration confined: xx μM.

Word

In this work we characterized ventral midbrain ventricular cells in the Lmx1a lineage through embryogenesis into adulthood in mice. These cells retain expression of nestin, Sox2, Sox3 and prominin across the menstruum of normal dopamine neurogenesis during embryonic development and even in the adult, suggesting that they might have sustained progenitor cell backdrop. We further showed that proliferation and subsequent neurogenic conversion of these cells was enhanced by dopamine receptor antagonist treatment, an result that extended beyond the bulk of normal embryonic dopamine neurogenesis.

Several previous studies have shown that neurotransmitters influence neurogenesis both during embryonic development and in the adult brain and spinal cord in several species, e.g. in mammals, salamanders and zebrafish^{21 ,22 ,23 ,24}. The office of dopamine signalling in neurogenesis in the mammalian brain was addressed in unlike experimental settings, which collectively indicated region- and cell-type specific effects. Dopamine depletion has been seen to subtract cell proliferation in the dentate gyrus and subventricular zone in some of these studies^{25 ,26 ,27 ,28 ,29 ,thirty}, while others documented increased jail cell proliferation^{13 ,31 ,32}. Furthermore, observations in gerbils and in rats showed increased jail cell proliferation in dentate gyrus³³, thus revealing that haloperidol treatment can increase the number of stem cells and the proliferation of transient-amplifying progenitors in the SVZ³⁴.

Importantly, the present work is the first to address how modulating dopamine signalling influences cell proliferation and neurogenesis in the developing mammalian midbrain after the acme of dopamine neurogenesis. Although the effects in mice, equally shown here, were rather modest, the results are in line with previous findings in aquatic salamanders, which demonstrated increased midbrain dopamine neurogenesis upon haloperidol handling¹⁷. This indicates an evolutionary conserved regulatory machinery by which dopamine suppresses the proliferation of dopamine progenitors and ultimately neurogenesis. Whether dopamine acts in a negative feedback-like fashion in the mammalian midbrain remains to exist proven. Notwithstanding, it is noteworthy that several reports place regionally committed stalk and progenitor cells that reply to varying levels of neurotransmitters. For case, the neurotransmitter (GABA) negatively controls proliferation of cells in the subventricular zone and in the rostral migratory stream^{35 ,36 ,37}, and quiescent neural stem cells respond tonically to GABA by means of γ2-subunit-containing GABA_A receptors in the adult dentate gyrus³⁸. Hence, a plausible hypothesis is that a function of dopaminergic innervation of Lmx1a-expressing cells could exist to maintain progenitors in quiescence, a state that can be transitioned into proliferation upon dopamine receptor antagonist treatment.

Dopamine neurogenesis in the adult salamander midbrain is critically dependent on reduced dopamine levels. Our observations heighten the possibility that neurogenesis could be activated from resident Lmx1a-expressing cells by modulation of dopamine receptor signalling also in the postnatal mammalian brain. However, several significant ontogenetic changes occur as the region harbouring these cells transition beyond embryonic neurogenesis. The number of Lmx1a-expressing cells decreases, the expression level of Lmx1a in these cells is decreased and their processes become smaller and do not extend far beyond the channel in the adult encephalon. As these processes probable back up cell migration during evolution their disappearance may contribute to the diminution of neurogenesis in the adult midbrain. Nevertheless our findings warrant further experiments targeting the neurogenic potential of Lmx1a-expressing population in the adult midbrain.

Methods

Ethics statement

All the work involving animals or human subjects/tissues was carried out in accordance with the Lawmaking of Ideals of the World Medical Association (Annunciation of Helsinki) and with national legislation and institutional guidelines. Fauna procedures were approved by the regional animal ideals review board (Stockholms Norra Djurförsöksetiska nämnd) and the Florey Institute of Neuroscience and Mental Wellness brute ethics committee. Ethical approval for the use of man post mortem samples was obtained from the regional upstanding review board in Stockholm, Sweden (Regionala Etikprövningsnämnden, Stockholm, EPN), ethical approval number 2022/111-31/1. All post mortem human tissues were obtained from the National Affliction Research Interchange (NDRI, world wide web.ndriresource.org) with the written informed consent from the donors or the adjacent of kin.

Animals

All mouse lines were maintained on a C57Bl/6 background. The post-obit transgenic mouse lines were utilized: Lmx1a^eGFP/+ mice⁸; floxNurr1DATCre (Nurr1CKO) mice, generated by crossing floxed Nurr1 mice^twenty with mice carrying Cre inserted into the DAT gene locus³⁹; and DATYFPxROSA26 mice, which were generated past crossing B6.129X1-Gt(ROSA)26Sor^tm1(EYFP)cos/J mice (Jackson laboratory #006148) with DAT-CreERT2 mice; D2R knock-out (D2R KO) mice and wild-type littermates⁴⁰ (Jackson laboratory #003190).

BrdU delivery through subcutaneous pumps

The subcutaneous pump (Alzet, Model 2001) was filled with BrdU solution (0,08 mg/mL, Sigma), diluted in 60% DMSO and 40% dH₂O.

Haloperidol in vivo administration

Haloperidol (Sigma) solution was prepared in acetic acid and saline and neutralized by addition of NaOH. For the proliferation experiments, pregnant dams received two daily injections of haloperidol (three mg/kg) or vehicle at E12.5, E13.5, E14.5, E15.5 or E16.five, and were sacrificed the day after. For chase experiment performed from E15.5 to E18.5, animals were daily injected with vehicle or haloperidol (1.5 mg/kg).

Mouse tissue processing

Developed mice were anesthetized with avertin (tribromoethanol, Sigma) at 0.5–0.6 mg/g torso weight i.p. and perfused intracardially with phosphate buffered saline (PBS) (Invitrogen) and later 4% paraformaldehyde (Sigma). Brains were dissected, postfixed for ii–half dozen h in 4% PFA, cryoprotected in 20% then 30% sucrose, sectioned (30–xl μm), serially collected and stored in antifreeze solution (30% glycerol, 30% ethoxyethanol, 40% PBS) at −xx °C until used. Significant dams were anesthetised past CO₂ and sacrificed by cervical dislocation and embryos nerveless. For immunohistochemistry and in situ hybridization, collected embryos were postfixed in iv% PFA (1–iii h depending on the age of the embryo), cryoprotected in 20% and later in 30% sucrose, embedded in Oct (TissueTek), sectioned at 12–20 μm, serially collected and stored at −eighty °C.

Human tissue processing

The tissues were fixed in 4% PFA, sequentially placed through sucrose gradients (2%, x%, xx% and 30%) for cryoprotection and sectioned on a freezing microtome (Microme HM430) at 40 μm thickness and serially collected. Tissues were subjected to antigen retrieval (0.01 K citric acrid buffer, pH six.0 for 20 min at 95 °C) and blocking of endogenous peroxidases (iii% H₂O_ii in fifty% methanol in PBS) prior to staining⁴¹.

Histological procedures for mouse and human being tissues

For immunofluorescent staining of mouse tissues, sections were rinsed with PBS and incubated with blocking buffer (PBS, five–x% normal ass serum; NDS or normal goat serum; NGS (Jackson Laboratories), 0.1% Triton-X100) for i h prior to incubation with primary antibodies (Supplementary Table one) in blocking buffer overnight at four °C. The tissue sections were afterward incubated in Alexafluor secondary antibodies (1:500) for 1 h, and rinsed in PBS. Hoechst 33342 (four μg/ml) was used for counterstaining and tissue sections were mounted onto slides in Mowiol 4–88 (Calbiochem). Confocal assay was performed using a Zeiss LSM510/Meta Station (Thornwood, NY, http://www.zeiss.com). For identification of signal co-localization within a cell, optical thickness was kept to a minimum, and orthogonal reconstructions were obtained. Each experiment was performed in triplicate and all counts were done in a double-bullheaded fashion.

Sections from human postmortem tissues were incubated with blocking buffer for 1 h. Later on, sections were incubated overnight at 4 °C using caprine animal anti-tyrosine hydroxylase antibody (1:300, PelFreez). Sections were washed in PBS and incubated with a biotinylated secondary antibody (1:200; Vector Laboratories) for ane h at room temperature, followed by incubation in streptavidin-biotin complex (Vectastain ABC kit Elite, Vector laboratories) for 1 h and visualized by incubation in 3,three′-diaminobenzidine solution (DAB, Vector Laboratories). Nuclei were counterstained using Myers hematoxylin (Histolab) for 1–2 min, rinsed in ddH₂0, speedily immersed in 70% ethanol containing 36 mM HCl to remove background staining, followed by another rinse in ddH₂0. Sections were subsequently dehydrated past sequential steps in increasing ethanol concentration accordingly; 25% ethanol (2 min), 70% ethanol (2 min), differentiator (75% ethanol containing 96 mM acerb acid) (1 min), 95% ethanol (2 min), 100% ethanol (2 min), xylene (2 × 2 min) and coverslipped using Mountex (Histolab)⁴¹. Brightfield images were captured using a Zeiss Axio Imager M1 Upright microscope. Control experiments were performed for all stainings by omitting either primary or secondary antibodies.

Quantification of cell numbers and intensity measurements in vivo

The signal intensity of tyrosine hydroxylase fiber innervation of the midbrain aqueduct was quantified using Image J (http://rsb.info.nih.gov/ij/). Values are displayed as relative intensity of signal in controls. Quantification of the number of Ki67⁺ eGFP⁺ cells, using immunofluorescent staining coupled with confocal analysis, was performed at E12.5 (1/5 sections counted), E15.5 (1/6 sections counted) and E18.5 (1/7.5 sections counted) (Fig. 1Q–T). The total number of Ki67⁺ cells in the ventral midbrain regions were retrieved by using the counts and taken into account the total number of sections, the average diameter of the cells (ten μm), by performing the Abercrombie correction, co-ordinate to P = A • (Thousand/L + G) • n, where P is the average number of nuclear points, A the number of nuclei identified/department, M is the thickness of the department, 50 the average length of the jail cell (in μm) and due north the number of sections. TH innervation of the midbrain aqueduct in Nurr1CKO and wild-type littermate mice was conducted on confocal projection images derived from z-stacks of identical depth using the Image J software (https://imagej.nih.gov/ij/).

For quantification of Ki67+ cells in the D2R KO mice analysis was conducted at E11.5 (1/v sections counted) (Fig. 3E). To evaluate the number ventral eGFP⁺ pH3⁺ cells or eGFP⁺ BrdU⁺ cells, a Zeiss upright microscope was used. The number of cells was quantified under 63X magnification with the optical fractionator method on a systematic random sampling of every fifth sections forth the rostro-caudal axis. To evaluate the number of BrdU⁺ cells in the prospective VTA and SN, 20x images encompassing the entire midbrain were generated and cell numbers inside the TH⁺ area counted (number of BrdU⁺ cells/Thursday⁺ surface area measured in μm³) in 3 or four sections. To analyse the percent of BrdU⁺Th⁺/BrdU⁺ cells, 40x confocal images forth the entire Z-axis (12 μm) using 1 μm intervals were counted. The co-labeled cells were analyzed with Imarys software.

In situ hybridization

In situ hybridization on sections was carried out essentially as previously described⁴² using a probe for Lmx1a^vi.

Mouse main midbrain cultures and immunocytochemistry

Brains from E11.5 mice were obtained and the midbrain region was dissected, mechanically dissociated, plated on poly-d-lysine (150,000 cells/cm^two), and grown in serum-complimentary N2 media consisting of F12/DMEM (i:1) with insulin (10 ng/ml), apo-transferrin (100 μg/ml), putrescine (100 μM), progesterone (twenty nM), selenium (30 nM), glucose (6 mg/ml), BSA (1 mg/ml) and FGF2 (10 ng/ml). Cells were treated for 3 days in vitro with either dopamine (10 μM, Sigma), haloperidol (one μM, Sigma), quinpirole (10μM, Tocris Bioscience), dihydrexidine (10 μM, Tocris Bioscience), muscimol (30 μM, Sigma) sulpiride (10 μM, Tocris Bioscience), SCH-23390 (x μM, Santa Cruz Biotechnology), picrotoxin (thirty μM, Tocris Bioscience) or media but and and then processed for immunocytochemistry. For BrdU analysis, cells were treated with BrdU 1 hour afterward plating. After a further 2 days in culture, cells were treated for 30 minutes with 2N HCl, and processed for immunocytochemistry.

Differentiation of mouse embryonic stem cells (mESCs)

R1 mESCs (Nagy's lab, MSH, Toronto, Canada) were cultured and differentiated on PA6 stromal feeders (RCB1127, Riken BRC Jail cell Bank, Japan)⁴³. Specifically, mESCs were plated at depression density (100 cells/cm²) on a confluent layer of PA6 cells and were grown in Serum Replacement Medium with Noggin (300 ng/ml; R&D Systems) equally previously described⁴⁴. At day 5, 200 ng/ml Shh (R&D Systems) and 25 ng/mL Fgf8b (R&D Systems) were added to the medium. At day 8, the medium was switched to N2 medium containing Shh, Fgf8b, and Fgf2 (10 ng/ml, R&D Systems). At mean solar day x of differentiation the cells were pulsed with EdU (10 μM, Life Technologies). From 24-hour interval xi of differentiation, Shh, Fgf8b and Fgf2 were removed from the N2 medium and replaced by BDNF (20 ng/ml, R&D Systems), GDNF (20 ng/ml, R&D Systems), and ascorbic acid (0.ii mM). Betwixt mean solar day 8–xv, cells were treated daily with either dopamine (10 μM), haloperidol (1 μM), quinpirole (ten μM), sulpiride (10 μM) or media simply. At day 15, cells were fixed in 4% PFA and processed for immunocytochemistry as described beneath.

Immunohistochemistry of mESC and master cultures and in vitro quantifications

Cells were incubated for 1 h in blocking buffer prior to overnight incubation with the primary antibodies (Supplementary Table 1) at iv °C. The coverslips were subsequently incubated in secondary antibodies (ane:500, Life Technologies or Jackson Immunoresearch) for ane h, and rinsed in PBS. EdU determinations were done according to Clik-it EdU kit (Life Technologies). DAPI or Hoechst (Sigma-Aldrich) was used for counterstaining. Each condition was analyzed in duplicates for three independent experiments (n = three). Specifically, for the primary cultures, cells positive for each marker were counted in 8 consecutive fields/well. For the mESC-derived cultures, the number of Th⁺ and Th⁺ EdU⁺ cell in each well were quantified in xv fields with an Olympus FV1000 confocal microscope.

Statistical analysis

Prism (GraphPad software inc) and Statistica eight.0 (Statsoft) software were used for the statistical analyses. Experimental data was analysed using either student's t test, 1-style ANOVA or two-way ANOVA. All data are presented as mean ± SEM.

Additional Information

How to cite this article: Hedlund, Eastward. et al. Dopamine Receptor Antagonists Enhance Proliferation and Neurogenesis of Midbrain Lmx1a-expressing Progenitors. Sci. Rep. 6, 26448; doi: ten.1038/srep26448 (2016).

Supplementary Fabric

Supplementary Information:

Acknowledgments

Nosotros would like to give thanks D Berg for exploratory experiments in the initial phase of this study. This work was funded past grants from the Swedish Inquiry Council, AFA Insurances, Cancerfonden and the European Research Quango to A.S. The European Matrimony, Seventh Framework Program (mdDANeurodev), Swedish Strategic Research Foundation and Knut and Alice Wallenberg Foundation to T.P. The Swedish Research Quango (VR projects: DBRM, 2008–2811, 2022–3116 and 2022–3318), Swedish Strategic Research Foundation (SRL plan), European Commission (NeuroStemcell) and Karolinska Institutet (SFO Thematic Middle in Stem cells and Regenerative Medicine) to E.A. Swedish Medical Research Council (2011–2651) and NEURO Sweden to East.H. Fifty.B. was supported by a postdoc fellowship from Wenner-Gren Foundation, Sweden. C.P. was supported past a senior medical enquiry fellowship from the Viertel charitable foundation, Commonwealth of australia. C.B. was supported by a Peter Doherty Fellowship from the National Wellness and Medical Research Quango, Commonwealth of australia. Human mail mortem tissues were kindly received from the National Disease Research Interchange (NDRI; world wide web.ndriresource.org). We would similar to thank Shanzheng Yang for help with the microphotographs of the midbrain cultures (E.A.).

Footnotes

Author Contributions E.H.: Conceived the study; performed the characterization of the Lmx1a-GFP animals, conducted the D2R expression and Th innervation experiments in mouse and man, analysed all data, and wrote the paper. L.B.: Conceived the study; performed the in vivo pharmacological studies, analysed all data, and wrote the paper. South.T.: Designed, performed, analysed and interpreted the primary midbrain culture experiments. C.S.: Designed, performed, analysed and interpreted the ESCs experiments. C.B. and C.P.: Performed and analysed in vivo D2R KO mouse experiments. Q.D.: Initiated the analysis of Lmx1a-GFP mice and performed some characterization. B.K.: Contributed to Nurr1CKO and DATCreYFP mouse experiments. J.E.: Conceived, analysed and, supervised characterization of the Lmx1a-GFP animals. E.A.: Conceived, analysed and supervised the mESC and midbrain progenitor culture experiments and contributed to writing the paper. T.P.: Conceived the written report, analysed all data, supervised characterization of the Lmx1a-GFP animals, and wrote the paper. A.S.: Conceived the study, analysed all data, supervised the in vivo pharmacological studies, and wrote the paper. All authors reviewed and edited the manuscript.

References

• Grealish South. et al. Human ESC-derived dopamine neurons show similar preclinical efficacy and potency to fetal neurons when grafted in a rat model of Parkinson's disease. Cell stem cell xv, 653–665, doi: ten.1016/j.stem.2014.09.017 (2014). [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
• Kriks South. et al. Dopamine neurons derived from human ES cells efficiently engraft in brute models of Parkinson's disease. Nature 480, 547–551, doi: x.1038/nature10648 (2011). [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
• Hedlund E. et al. Embryonic stem cell-derived Pitx3-enhanced green fluorescent protein midbrain dopamine neurons survive enrichment past fluorescence-activated cell sorting and part in an brute model of Parkinson'southward illness. Stem cells 26, 1526–1536, doi: x.1634/stemcells.2007-0996 (2008). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Steinbeck J. A. & Studer Fifty. Moving stem cells to the clinic: potential and limitations for brain repair. Neuron 86, 187–206, doi: 10.1016/j.neuron.2015.03.002 (2015). [PMC gratis article] [PubMed] [CrossRef] [Google Scholar]
• Arenas Due east., Denham Chiliad. & Villaescusa J. C. How to make a midbrain dopaminergic neuron. Development 142, 1918–1936, doi: 10.1242/dev.097394 (2015). [PubMed] [CrossRef] [Google Scholar]
• Andersson E. et al. Identification of intrinsic determinants of midbrain dopamine neurons. Cell 124, 393–405, doi: ten.1016/j.jail cell.2005.ten.037 (2006). [PubMed] [CrossRef] [Google Scholar]
• Ono Y. et al. Differences in neurogenic potential in flooring plate cells along an anteroposterior location: midbrain dopaminergic neurons originate from mesencephalic floor plate cells. Evolution 134, 3213–3225, doi: ten.1242/dev.02879 (2007). [PubMed] [CrossRef] [Google Scholar]
• Deng Q. et al. Specific and integrated roles of Lmx1a, Lmx1b and Phox2a in ventral midbrain development. Development 138, 3399–3408, doi: x.1242/dev.065482 (2011). [PubMed] [CrossRef] [Google Scholar]
• Yan C. H., Levesque M., Claxton S., Johnson R. 50. & Ang Southward. L. Lmx1a and lmx1b role cooperatively to regulate proliferation, specification, and differentiation of midbrain dopaminergic progenitors. The Journal of neuroscience: the official periodical of the Lodge for Neuroscience 31, 12413–12425, doi: 10.1523/JNEUROSCI.1077-xi.2011 (2011). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Vitalis T., Cases O. & Parnavelas J. G. Development of the dopaminergic neurons in the rodent brainstem. Experimental neurology 191 Suppl one, S104–112, doi: 10.1016/j.expneurol.2004.05.044 (2005). [PubMed] [CrossRef] [Google Scholar]
• Yang S. et al. Cxcl12/Cxcr4 signaling controls the migration and procedure orientation of A9-A10 dopaminergic neurons. Development 140, 4554–4564, doi: 10.1242/dev.098145 (2013). [PubMed] [CrossRef] [Google Scholar]
• Bayer S. A., Wills One thousand. V., Triarhou 50. C. & Ghetti B. Time of neuron origin and gradients of neurogenesis in midbrain dopaminergic neurons in the mouse. Experimental brain research 105, 191–199 (1995). [PubMed] [Google Scholar]
• Aponso P. M., Faull R. L. & Connor B. Increased progenitor prison cell proliferation and astrogenesis in the partial progressive 6-hydroxydopamine model of Parkinson'southward disease. Neuroscience 151, 1142–1153, doi: 10.1016/j.neuroscience.2007.11.036 (2008). [PubMed] [CrossRef] [Google Scholar]
• Kay J. North. & Blum Grand. Differential response of ventral midbrain and striatal progenitor cells to lesions of the nigrostriatal dopaminergic project. Developmental neuroscience 22, 56–67, doi: 17427 (2000). [PubMed] [Google Scholar]
• Zhao K. et al. Evidence for neurogenesis in the adult mammalian substantia nigra. Proceedings of the National Academy of Sciences of the United States of America 100, 7925–7930, doi: 10.1073/pnas.1131955100 (2003). [PMC complimentary article] [PubMed] [CrossRef] [Google Scholar]
• Frielingsdorf H., Schwarz Chiliad., Brundin P. & Mohapel P. No evidence for new dopaminergic neurons in the adult mammalian substantia nigra. Proceedings of the National University of Sciences of the The states 101, 10177–10182, doi: x.1073/pnas.0401229101 (2004). [PMC complimentary article] [PubMed] [CrossRef] [Google Scholar]
• Berg D. A., Kirkham One thousand., Wang H., Frisen J. & Simon A. Dopamine controls neurogenesis in the developed salamander midbrain in homeostasis and during regeneration of dopamine neurons. Jail cell stalk cell viii, 426–433, doi: ten.1016/j.stalk.2011.02.001 (2011). [PubMed] [CrossRef] [Google Scholar]
• Parish C. Fifty., Beljajeva A., Arenas E. & Simon A. Midbrain dopaminergic neurogenesis and behavioural recovery in a salamander lesion-induced regeneration model. Development 134, 2881–2887, doi: ten.1242/dev.002329 (2007). [PubMed] [CrossRef] [Google Scholar]
• Berg D. A. et al. Efficient regeneration by activation of neurogenesis in homeostatically quiescent regions of the developed vertebrate brain. Evolution 137, 4127–4134, doi: 10.1242/dev.055541 (2010). [PubMed] [CrossRef] [Google Scholar]
• Kadkhodaei B. et al. Nurr1 is required for maintenance of maturing and developed midbrain dopamine neurons. The Periodical of neuroscience: the official journal of the Gild for Neuroscience 29, 15923–15932, doi: 10.1523/JNEUROSCI.3910-09.2009 (2009). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Berg D. A., Belnoue L., Song H. & Simon A. Neurotransmitter-mediated control of neurogenesis in the adult vertebrate encephalon. Development 140, 2548–2561, doi: 10.1242/dev.088005 (2013). [PMC complimentary commodity] [PubMed] [CrossRef] [Google Scholar]
• Young S. Z., Taylor M. G. & Bordey A. Neurotransmitters couple brain activeness to subventricular zone neurogenesis. The European periodical of neuroscience 33, 1123–1132, doi: x.1111/j.1460-9568.2011.07611.x (2011). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Corns L. F. et al. Cholinergic Enhancement of Prison cell Proliferation in the Postnatal Neurogenic Niche of the Mammalian Spinal Cord. Stem cells 33, 2864–2876, doi: 10.1002/stem.2077 (2015). [PMC gratuitous commodity] [PubMed] [CrossRef] [Google Scholar]
• Perez M. R. et al. Relationships between radial glial progenitors and 5-HT neurons in the paraventricular organ of developed zebrafish - potential effects of serotonin on adult neurogenesis. The European journal of neuroscience 38, 3292–3301, doi: 10.1111/ejn.12348 (2013). [PubMed] [CrossRef] [Google Scholar]
• Freundlieb Northward. et al. Dopaminergic substantia nigra neurons projection topographically organized to the subventricular zone and stimulate precursor jail cell proliferation in aged primates. The Journal of neuroscience: the official journal of the Society for Neuroscience 26, 2321–2325, doi: 10.1523/JNEUROSCI.4859-05.2006 (2006). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Winner B. et al. Striatal deafferentation increases dopaminergic neurogenesis in the developed olfactory bulb. Experimental neurology 197, 113–121, doi: 10.1016/j.expneurol.2005.08.028 (2006). [PubMed] [CrossRef] [Google Scholar]
• Sui Y., Horne M. K. & Stanic D. Reduced proliferation in the adult mouse subventricular zone increases survival of olfactory seedling interneurons. Plos ane 7, e31549, doi: 10.1371/journal.pone.0031549 (2012). [PMC complimentary article] [PubMed] [CrossRef] [Google Scholar]
• Suzuki G. et al. Destruction of dopaminergic neurons in the midbrain by 6-hydroxydopamine decreases hippocampal cell proliferation in rats: reversal by fluoxetine. Plos 1 5, e9260, doi: 10.1371/journal.pone.0009260 (2010). [PMC gratis article] [PubMed] [CrossRef] [Google Scholar]
• Hoglinger One thousand. U. et al. Dopamine depletion impairs precursor prison cell proliferation in Parkinson disease. Nature neuroscience 7, 726–735, doi: 10.1038/nn1265 (2004). [PubMed] [CrossRef] [Google Scholar]
• Baker S. A., Baker K. A. & Hagg T. Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the developed mouse subventricular zone. The European periodical of neuroscience 20, 575–579, doi: ten.1111/j.1460-9568.2004.03486.x (2004). [PubMed] [CrossRef] [Google Scholar]
• Peng J., Xie 50., Jin One thousand., Greenberg D. A. & Andersen J. Grand. Fibroblast growth gene two enhances striatal and nigral neurogenesis in the astute 1-methyl-4-phenyl-1,2,3,half dozen-tetrahydropyridine model of Parkinson'south disease. Neuroscience 153, 664–670, doi: 10.1016/j.neuroscience.2008.02.063 (2008). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Park J. H. & Enikolopov Yard. Transient tiptop of developed hippocampal neurogenesis after dopamine depletion. Experimental neurology 222, 267–276, doi: x.1016/j.expneurol.2010.01.004 (2010). [PMC gratis article] [PubMed] [CrossRef] [Google Scholar]
• Dawirs R. R., Hildebrandt Thou. & Teuchert-Noodt K. Adult handling with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus. Journal of neural transmission 105, 317–327 (1998). [PubMed] [Google Scholar]
• Kippin T. E., Kapur S. & van der Kooy D. Dopamine specifically inhibits forebrain neural stalk cell proliferation, suggesting a novel effect of antipsychotic drugs. The Journal of neuroscience: the official periodical of the Society for Neuroscience 25, 5815–5823, doi: 10.1523/JNEUROSCI.1120-05.2005 (2005). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Liu X., Wang Q., Haydar T. F. & Bordey A. Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAP-expressing progenitors. Nature neuroscience 8, 1179–1187, doi: x.1038/nn1522 (2005). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Nguyen Fifty. et al. Autocrine/paracrine activation of the GABA(A) receptor inhibits the proliferation of neurogenic polysialylated neural cell adhesion molecule-positive (PSA-NCAM+) precursor cells from postnatal striatum. The Journal of neuroscience: the official journal of the Social club for Neuroscience 23, 3278–3294 (2003). [PMC free article] [PubMed] [Google Scholar]
• Fernando R. N. et al. Cell bicycle brake by histone H2AX limits proliferation of developed neural stalk cells. Proceedings of the National Academy of Sciences of the United states of america of America 108, 5837–5842, doi: 10.1073/pnas.1014993108 (2011). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Song J. et al. Neuronal circuitry mechanism regulating adult quiescent neural stalk-prison cell fate conclusion. Nature 489, 150–154, doi: 10.1038/nature11306 (2012). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Ekstrand M. I. et al. Progressive parkinsonism in mice with respiratory-chain-scarce dopamine neurons. Proceedings of the National Academy of Sciences of the United States of America 104, 1325–1330, doi: 10.1073/pnas.0605208103 (2007). [PMC free article] [PubMed] [CrossRef] [Google Scholar]
• Kelly M. A. et al. Pituitary lactotroph hyperplasia and chronic hyperprolactinemia in dopamine D2 receptor-deficient mice. Neuron 19, 103–113 (1997). [PubMed] [Google Scholar]
• Comley L. et al. Motor neurons with differential vulnerability to degeneration show distinct protein signatures in health and ALS. Neuroscience 291, 216–229, doi: ten.1016/j.neuroscience.2015.02.013 (2015). [PubMed] [CrossRef] [Google Scholar]
• Anderson T. R., Hedlund E. & Carpenter E. One thousand. Differential Pax6 promoter activity and transcript expression during forebrain development. Mech Dev 114, 171–175 (2002). [PubMed] [Google Scholar]
• Perrier A. L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proceedings of the National Academy of Sciences of the Us 101, 12543–12548, doi: 10.1073/pnas.0404700101 (2004). [PMC costless commodity] [PubMed] [CrossRef] [Google Scholar]
• Andersson Due east. R. et al. Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stalk cells. Proceedings of the National Academy of Sciences of the Usa of America 110, E602–610, doi: 10.1073/pnas.1208524110 (2013). [PMC free article] [PubMed] [CrossRef] [Google Scholar]

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4887985/

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