Neurotox Res DOI 10.1007/s12640-016-9669-6

ORIGINAL ARTICLE

Modulation of Postnatal Neurogenesis by Perinatal Asphyxia: Effect of D1 and D2 Dopamine Receptor Agonists A. Tapia-Bustos1 • R. Perez-Lobos1 • V. Vı´o1 • C. Lespay-Rebolledo1 • E. Palacios1 • A. Chiti-Morales1 • D. Bustamante1 • M. Herrera-Marschitz1,2 P. Morales1,2

•

Received: 4 August 2016 / Revised: 6 September 2016 / Accepted: 8 September 2016 Ó Springer Science+Business Media New York 2016

Abstract Perinatal asphyxia (PA) is associated to delayed cell death, affecting neurocircuitries of basal ganglia and hippocampus, and long-term neuropsychiatric disabilities. Several compensatory mechanisms have been suggested to take place, including cell proliferation and neurogenesis. There is evidence that PA can increase postnatal neurogenesis in hippocampus and subventricular zone (SVZ), modulated by dopamine, by still unclear mechanisms. We have studied here the effect of selective dopamine receptor agonists on cell death, cell proliferation and neurogenesis in organotypic cultures from control and asphyxia-exposed rats. Hippocampus and SVZ sampled at 1–3 postnatal days were cultured for 20–21 days. At day in vitro (DIV) 19, cultures were treated either with SKF38393 (10 and 100 lM, a D1 agonist), quinpirole (10 lM, a D2 agonist) or sulpiride (10 lM, a D2 antagonist) ? quinpirole (10 lM) and BrdU (10 lM, a mitosis marker) for 24 h. At DIV 20–21, cultures were processed for immunocytochemistry for microtubule-associated protein-2 (MAP-2, a neuronal marker), and BrdU, evaluated by confocal microscopy.

Some cultures were analysed for cell viability at DIV 20–21 (LIVE/DEAD kit). PA increased cell death, cell proliferation and neurogenesis in hippocampus and SVZ cultures. The increase in cell death, but not in cell proliferation, was inhibited by both SKF38393 and quinpirole treatment. Neurogenesis was increased by quinpirole, but only in hippocampus, in cultures from both asphyxia-exposed and control-animals, effect that was antagonised by sulpiride, leading to the conclusion that dopamine modulates neurogenesis in hippocampus, mainly via D2 receptors. Keywords Neonatal hypoxia
Neurogenesis
Dopamine receptor
Subventricular zone
Hippocampus
Rat

Introduction Perinatal asphyxia (PA) implies a deregulation of gas exchange resulting in hypoxemia, hypercapnia and metabolic acidosis in vital organs, including the brain (Low

& P. Morales [email protected]

A. Chiti-Morales [email protected]

A. Tapia-Bustos [email protected]

D. Bustamante [email protected]

R. Perez-Lobos [email protected]

M. Herrera-Marschitz [email protected]

V. Vı´o [email protected] C. Lespay-Rebolledo [email protected] E. Palacios [email protected]

1

Programme of Molecular & Clinical Pharmacology, ICBM, Medical Faculty, University of Chile, Av. Independencia 1027, PO Box 8389100, Santiago, Chile

2

Biomedical Neuroscience Institute, BNI, ICBM, Medical Faculty, University of Chile, Santiago, Chile

123

Neurotox Res

1997). Despite important advances in perinatal care, PA remains a severe condition, with high prevalence (1–10/ 1000 live births) worldwide, leading to mortality or longlasting neuropsychiatric dysfunctions in newborns (see Morales et al. 2011; Douglas-Escobar and Weiss 2015; Marriott et al. 2016; Farı´as et al. 2016). The interruption of oxygen supply causes energy failure, triggering a biochemical cascade leading to cell dysfunction and ultimately to cell death, affecting in particular neurocircuitries of basal ganglia and hippocampus (Klawitter et al. 2007; Morales et al. 2008; Neira-Pena et al. 2015). Several compensatory mechanisms have been reported to take place after PA including increased neurogenesis, observed in cornu Amonnis CA1 and dentate gyrus (DG) of hippocampus (Bartley et al. 2005; Morales et al. 2008), subventricular zone (SVZ, located in the lateral wall of the lateral ventricles), (Plane et al. 2004; Ong et al. 2005), striatum (Str) and neocortex (Inta et al. 2015). The effect of PA on cell death, cell proliferation and neurogenesis probably implies long-term deficits in dopamine (DA) systems, which have been shown to be vulnerable to PA (Andersson et al. 1995; Chen et al. 1997a, b; Morales et al. 2011; Lo´pez-Pe´rez et al. 2015). There is a rich network of dopaminergic fibres impinging onto neurogenic niches in DG and SVZ (Verney et al. 1985; Ho¨glinger et al. 2014). In rodents, experimental depletion of DA leads to a decrease of cell proliferation in DG and SVZ (Ho¨glinger et al. 2004), and there is a decrease in the number of neuronal progenitors in DG of patients suffering from Parkinson disease (Ho¨glinger et al. 2004). Also, apomorphine, a non-selective DA agonist, increases neuronal proliferation in DG, together with an increase in the expression of bFGF, growth factor associated to neurogenesis (O’Keeffe et al. 2009; Reuss and Unsicker 2000). DA antagonism has also been shown to induce neural stem/ progenitor cell division (Dawirs et al. 1998; Kippin et al. 2005; Yang et al. 2008; Aponso et al. 2008; Park and Enikolopov 2010), making evident that this issue has to be further investigated. Five dopaminergic receptors have been cloned, coupled to G-proteins, defining two DA receptor families, D1 and D2, based on different intracellular second messengers signalling pathways. The D1 family (D1 and D5 receptors) activates the enzyme adenylate cyclase via a Gs coupled protein, increasing intracellular cAMP levels, while the D2 family (including D2, D3 and D4 receptors) inhibits the same enzyme via a Gi and/or Go coupled protein (Giros et al.1989). A heterogeneous distribution of DA receptors has also been described (see Goldsmith and Joyce 1994; see Bentivoglio and Morelli 2005). A dense-specific amount of D5 has been not only described in DG, but also D2 and D3 receptors, as well as in medial Str, linked to

123

the SVZ (Sokoloff et al.1990; Ohtani et al. 2003; Voorn et al. 1988). In proliferative germinal zones of the rat brain, DA receptors can already be observed at early embryonic periods (Lidow and Rakic 1995; Dı´az et al. 1997). Electron microscopy studies showed that neuroblasts (A cells) and transit-amplifying progenitor cells (C cells) express both D1 and D2 receptor subtypes, but the expression of D2 is several times stronger than that of D1 receptors (Ho¨glinger et al. 2004). No DA receptors have been observed on stem cells (B cells; Ho¨glinger et al. 2004). C cells also express the D3 receptor subtype (Dı´az et al.1997). Studies of our laboratory have showed that there is an increase of D2, but a decrease of D1 receptor levels in hippocampus 7 days after PA, although no changes have been observed in D1 or D2 receptor levels in SVZ under the same conditions. Thus, we have studied here the effects of selective DA receptor agonists on hippocampal and SVZ cell death, cell proliferation and neurogenesis using immunohistochemistry for BrdU and MAP-2 (proliferation and neuronal markers) in organotypic cultures from control and asphyxia-exposed rats. We found that dopamine agonists decrease cell death, and that the D2 receptor agonist quinpirole promotes neuronal proliferation, but only in hippocampus, providing a target for therapeutic strategies preventing neurologic and neuropsychiatric sequelae induced by PA.

Materials and Methods Animals Wistar albino rats from the animal station of the Molecular & Clinical Pharmacology Programme, ICBM, Faculty of Medicine, University of Chile, Santiago, Chile, were used along the experiments. The animals were kept on a temperature- and humidity-controlled environment with a 12/12 h light/dark cycle and fed ad libitum, when not used for the experiments, monitoring permanently the well being of the animals by qualified personnel. Ethic Statement All procedures were conducted in accordance with the animal care and use protocol established by a Local Ethics Committee for experimentation with laboratory animals at the Medical Faculty, University of Chile (Protocol CBA# 0389 FMUCH) and by the ad hoc commission of the Chilean Council for Science and Technology Research, (CONICYT), endorsing the principles of laboratory animal care (NIH; N° 86–23; revised 1985).

Neurotox Res

Perinatal Asphyxia Pregnant Wistar rats within the last day of gestation (G22) were euthanized by neck dislocation and hysterectomized. One or two pups per rat dam were removed immediately and used as non-asphyxiated caesarean-delivered controls (CS). The remaining foetuses-containing uterine horns were immersed in a water bath at 37 °C for 21 min (asphyxia-exposed rats, AS). Following asphyxia, the uterine horns were incised and the pups excised, stimulated to breathe and after an approximately 60-min observation period on a warming pad, evaluated with a Apgar scale for rats, according to Dell’Anna et al. (1997). Organotypic Culture Different series of rat neonates of both sexes were used for preparing organotypic cultures, one to three days after birth according to Morales et al. (2005). The brain was rapidly removed under sterile conditions and stored in a Petri dish containing Dulbecco’s modified Eagle medium (DMEM; GIBCO BRL, Life Technologies AB, Ta¨by, Sweden). Coronal Sects. (300 lm) were cut by a microslicer (DTK2000, Dosaka CO, Japan; 300 lm thick) and stored in cold DMEM. The SVZ was dissected from a frontal slices. A 0.5–1-mm strip of SVZ tissue apposed to the striatum was dissected under the corpus callosum down to the ventral tip of the lateral ventricle (Lois and Alvarez-Buylla, 1993). Samples from hippocampus and SVZ were placed on a coverslip (NuncThermanox Coverslips; Nunc, Naperville, IL, USA), containing a spread layer of chicken plasma (25 lL), coagulated by bovine thrombin (20/350 lL DMEM, Sigma, St. Louis, MO, USA) (the standard thrombin solution contained 1000 NIH units in 750 lL of H2O). The coverslips were then transferred into sterile Nunc flat CT-tubes containing 750 lL of culture medium, and grown at 35 °C, and 10 % CO2 in a Cell Incubator using a roller device exposing the cultures to gaseous or water phases every minute (Morales et al. 2005; Klawitter et al. 2007). At DIV 3, the medium was changed to a serum-free medium (Neurobasal-A medium supplemented with B27 2 %, GIBCO BRL; glucose 5 mM; L-glutamine 2.5 mM, Sigma). The medium was changed every 3–4 days. In Vitro Monitoring and Cell Viability The progression of the cultures was periodically monitored with an inverted microscope equipped with Hoffmann optics (Nikon T100), taking representative pictures. Some cultures were analysed for cell viability at DIV 20–21 by the LIVE/DEADÒ Viability/Cytotoxicity kit L3224 (Molecular Probes, Eugene, OR, USA), using ethidium-

homodimer (EthD-1) and calcein-acetoxymethyl ester (AM) for labelling dead and alive cells, respectively. Alive cells are distinguished by a green fluorescence produced by the action of intracellular esterases that cleave the AM group, retaining the calcein dye within the alive cells. Dying cells are identified by a red fluorescence produced by EthD-1 entering the cells with damaged membranes and binding to nucleic acids. For quantification, six samples of 1.13 mm2 (*0.02 mm3) per culture were counted, according to Morales et al. (2005). Pharmacological Treatments At DIV 19–20, different groups of cultures (n = 4–5) were treated either with vehicle (0.1 M phosphate-buffered saline, PBS), SKF38393 (10 or 100 lM, D1 agonist, Smith Kline and French Labs, Philadelphia, PA), or quinpirole (10 lM, D2 agonist, LY 171555; Eli Lilly Co, Indianapolis, IN) and BrdU for 24 h. Another group of cultures was treated with sulpiride (Seeman 1980) (10 lM, D2 antagonist, racemic, Laboratoire Etudes et developpment Chimiques, France; dissolved in a drop of ethanol 99 % and diluted in PBS) for 2 h, then treated with quinpirole (10 lM) or vehicle, and BrdU (10 lM) (Sigma, USA) for 24 h, before formalin fixation (paraformaldehyde 4 %, PF; Sigma, in 0.1 M PBS, pH 7.4) for 45 min at 4 °C, rinsed and stored in 0.1 M PBS at 4 °C pending further experiments. Immunohistochemistry Fixed tissue was washed with 0.1 M PBS (3 9 5 min). DNA was denatured using 1 N HCl for 40 min. After washing cycles, the sections were permeabilised with 1 % Triton X-100 in 0.1 M PBS for 10 min, and pre-incubated in a blocking solution (6.5 % normal goat serum, NGS; 2 % bovine serum albumin, BSA; 0.5 % Triton X-100 in 0.1 M PBS) for 1 h at room temperature. A rat anti-BrdU antibody (Abcam ab6326, 1:750 dilutions in blocking solution) was applied overnight at 4 °C. Following extensively washings with 1 % NGS in 0.1 M PBS, cultures were incubated with a goat anti-rat secondary antibody (Alexafluor 488, 1:500 dilution in 1 % NGS in PBS 0.1 M) for 2 h. Then, tissue was washed with 0.5 % Triton X-100 in 0.1 M PBS, for 10 min. For MAP-2 immunohistochemistry, cultures were post-fixed in 4 % PF for 15 min at 4 °C, washed and pre-incubated in blocking solution (2 % horse serum, NHS); 2 % BSA; 0.5 % Triton X-100 in 0.1 M PBS) for 1 h. A chicken anti-MAP-2 antibody (Abcam ab5392, 1:750 dilution in blocking solution) was applied overnight at 4 °C. Following extensively washing with 0.5 % horse serum in 0.1 M PBS, cultures were incubated with goat anti-chicken secondary antibody

123

Neurotox Res

(Alexafluor 594, 1:400) in 0.5 % horse serum in 0.1 M PBS, for 2 h. The tissue was washed, coverslipped with hydrophilic resin (Fluoromount) and examined with confocal microscopy (Olympus-fv10i). Image Processing and Sterologic Analysis Two parameters were quantified: (1) number of BrdU? cells/mm3, and (2) number of BrdU?/MAP-2? cells/mm3, as described by Morales et al. (2005). An investigator blinded to the treatment counted MAP-2? or BrdU? cells. Microphotographs (5–6) were taken from hippocampus or SVZ areas in the field of a confocal-inverted Olympusfv10i microscope with a 609 objective lens (NA 1,2). The area inspected was 0.04 mm2. The thickness (Z axis) was measured for each case. The number of cell proliferation was estimated by counting BrdU? cells, and neurogenesis by counting double labelled MAP-2?/BrdU? cells, expressed as cells/mm3, in cultures from CS and AS (n = 4–5), using ImageJ software. Cells were considered double-labelled when MAP-2 and BrdU immunoreactivity overlapped at four levels through a section (Z-step 1 lm). Statistical Analysis Values are expressed as mean ± SEM. Data were analysed by a proper Student’s t test, using GraphPad Prism software. *p \ 0.05 level, critical for statistically significant differences.

Results Perinatal Asphyxia An Apgar scale was applied approximately 60 min after delivery. Following 21 min of PA, the rate of survival was *70 %. Surviving pups showed a decreased respiratory frequency supported by gasping, decreased vocalisation, cyanotic skin, akinesia and rigidity (see Table 1). Organotypic Cultures In Vitro Evaluation and Cell Viability Organotypic cultures obtained from CS (n = 30) and AS (n = 30) neonates were monitored, assessing development on the coverslip (pictures were taken at DIV 1, 3, 11, 18 and 20–21 with a Nikon digital camera). At DIV 1, thickness of organotypic cultures was 300 lm, decreasing to *45 lm at DIV 20–21, measured at the stage of the confocal microscope (Z- axis). Hippocampus cultures (Fig. 1a, b) preserved the typical layering and the tissue structure, whereas SVZ cultures (Fig. 1c, d) became fused

123

and integrated, adopting a circular shape at DIV 7. No differences in coverslip adhesion or development were observed in hippocampus or SVZ organotypic cultures from CS or AS animals, as previously reported (Morales et al. 2005). At DIV 20–21, a viability test was applied, one sample for each culture series. Many alive (green fluorescence, calcein-AM), but only some few dead (red fluorescence, EThD-1?) cells were observed in hippocampus (Fig. 2a) and SVZ (Fig. 2b) organotypic cultures from CS animals. The amount of dead cells/mm3 increased in cultures from AS, compared to that observed in vehicle-treated CS animals, both in hippocampus (*1.5 X; CS: 7418 ± 494.3 (n = 4); AS: 12395 ± 1341 cells/mm3 (n = 4); t = 3.482 df = 6, p \ 0.007) (Fig. 2c) and SVZ ([2 X; CS: 11195 ± 1419 (n = 3); AS: 27808 ± 5801 cells/mm3 (n = 5); t = 2.129 df = 6, p \ 0.0387) (Fig. 2d). Cell Death (EThD-11) Compared to the effect by the vehicle, SKF38393 (10 lM, SKF) treatment did not significantly affect the number of dying (EThD-1?) cells in hippocampus or SVZ from CS animals, but SKF38393 decreased the number of dying cells in hippocampus (by 70 % AS: 12395 ± 1341 (N = 4); ASKF: 3791 ± 1518 cells/mm3 (n = 4); t = 4.248 df = 6, p \ 0.0027) and SVZ (by 60 %, AS: 27808 ± 5801 (n = 5); ASKF: 11597 ± 2252 cells/mm3 (n = 5); t = 2.605 df = 8, p \ 0.0157) from AS animals compared to the corresponding vehicle condition (Fig. 2c, d). The number of EThD-1?cells/mm3 in hippocampus was decreased by quinpirole (10 lM, Q), compared to that produced by the vehicle, in cultures from both CS (CS: 7418 ± 494.3 (N = 4); CQ: 4396 ± 872.5 cells/mm3 (n = 4); t = 3.013 df = 6, p \ 0.0118) and AS animals (AS: 12395 ± 1341 (N = 4); AQ: 7051 ± 952.2 (n = 4); t = 3.249 df = 6, p \ 0.0087) (Fig. 2c). The effect of quinpirole in SVZ was only observed in cultures from AS animals (AS: 27808 ± 5801(n = 5); AQ: 11597 ± 2252 cells/mm3 (n = 5); t = 1.822 df = 8, p \ 0.05) (Fig. 2d). Cell Proliferation (BrdU1- Cells) At DIV 19, cultures were treated with vehicle, SKF38393 (10–100 lM), and/or quinpirole (10 lM) for 24 h, formalin fixed, and processed for immunocytochemistry using antibodies for MAP-2 and BrdU, evaluated with confocal microscopy. Representative microphotographs obtained by confocal microscopy of hippocampus (Fig. 3a, CS; Fig. 3b, AS) and SVZ (Fig. 4a, CS; Fig. 4b, AS) cultures, treated with vehicle, SKF38393 (10 lM), or quinpirole (10 lM), labelled for BrdU? (green) and MAP-2? (red) cells are shown. Arrows show double BrdU/MAP-2 labelled cells,

Neurotox Res Table 1 An Apgar scale for rodents Parameters

Control (Caesarean-delivered pups; 0 min asphyxia) (m = 26, n = 30)

Asphyxia-exposed pups (21 min asphyxia) (m = 26, n = 30)

5.97 ± 0.70

5.87 ± 0.73

Respiratory frequency (events x min )

78 ± 6

39 ± 22

Presence of gasping* (yes; %)

0%

33 %

Pink–Blue

100 %

0%

Blue–Pink–Blue

0%

100 %

Presence of vocalisations (yes, %) Spontaneous movements** (4, 3, 2, 1, 0)

100 % 4

27 % 0

Body weight (g) -1

Skin colour

An Apgar scale for evaluating the consequences of perinatal asphyxia 60 min after delivery. Data expressed as the mean ± SEM (m number of dams; n number of pups) * Gasping forced reflex inspirations. In control rats gasping is absent after 1 min ** 4 indicates coordinated and spontaneous movements, 0 indicates no movement

Fig. 1 Overview of organotypic cultures from hippocampus (a, b) and subventricular zone, SVZ (c, d). The progression of hippocampus (a, b) and SVZ (c, d) cultures is shown at days in vitro (DIV) 3 and DIV18. Observe that the thickness of organotypic cultures from Hippocampus b and SVZ d was decreased at DIV 18. Hippocampus cultures (b) preserved the typical layering and the tissue, whereas SVZ culture (d) became fused and integrated, adopting a circular shape. (Scale bar: 1000 lm)

123

Neurotox Res

Fig. 2 Viability test of hippocampus and subventricular zone organotypic cultures from control and asphyxia-exposed rats: effect of dopamine agonists. At DIV 20–22, cell viability was evaluated in hippocampus (a) and subventricular zone (SVZ) (b) cultures. ab Representative microphotographs showing alive (Calcein-AM?, green fluorescence) and dead (EThD-1?, red fluorescence, arrows) cells in cultures from control and asphyxia-exposed rats, treated with vehicle, SKF38393 (10 lM) or quinpirole (10 lM), added directly into the culture medium for 24 h, before the viability test. Scale bar:

50 lm. Quantification of EThD-1? cells/mm3 in c hippocampus and d SVZ cultures treated with vehicle, SKF38393 or quinpirole, from control (open bars; N = 3–5) and asphyxia–exposed (filled bars; N = 4–5) rats. *p \ 0.05, **p \ 0.005, Student’s t test. aComparison between vehicle-treated cultures from asphyxia-exposed and control animals; bComparison between treatment and vehicle in cultures from control animals; cComparison between treatment and vehicle in cultures from asphyxia-exposed animals

indicating neuronal proliferation. Differences in size and shape of BrdU? nuclei (round, elongated, small and large nuclei) were observed, probably indicating different cell phenotypes. Stereological analysis revealed that PA induced a significant increase in cell proliferation (BrdU? cells/mm3) in both hippocampus (*1.5X; CS: 38441 ± 2766 (n = 5); AS: 57806 ± 5961 cells/mm3 (n = 5); t = 2.947 df = 8, p \ 0.009) (Fig. 3c) and SVZ (*1.5X, CS: 58117 ± 6389 (n = 5); AS: 77620 ± 6625 cells/mm3 (n = 6); t = 2.092 df = 9, p \ 0.033) (Fig. 4c), compared to the corresponding vehicle-treated CS controls. SKF38393 (10 lM) did not produce any effect on the number of BrdU?-cells in cultures from CS animals, but it appeared to reverse the effect of PA on cell proliferation. In order to verify this effect, the experiment was repeated with 100 lM of SKF38393, at which dose SKF38393

increased BrdU?-cell proliferation in hippocampus (*1.5X, CS: 38441 ± 2766 (n = 5); CSSKF100lM: 57734 ± 5477 (n = 5); t = 3.144 df = 8, p \ 0.007) and SVZ (*1.5X, CS: 58117 ± 6624 (n = 5); CSSKF100lM: 90326 ± 14231 (n = 4); t:2.229 df = 7, p \ 0,03) but only in CS animals when compared to the vehicle (Figs. 3c, 4c). Quinpirole (10 lM) increased cell proliferation in hippocampus ([1.5X; CS: 38441 ± 2766 (n = 5); CQ: 60382 ± 2962 (n = 5); t = 5.415 df = 8, p \ 0.0003) and SVZ ([1.5X; CS: 58117 ± 6389 (n = 5); CQ: 94058 ± 3968 cells/mm3 (n = 5); t = 4.779 df = 8, p \ 0.0007) from CS, compared to vehicle-treated controls, but that effect was not evident in cultures from AS rats, since cell proliferation was already enhanced under the vehicle condition (Figs. 3d, 4d).

123

Neurotox Res

Fig. 3 Cell (BrdU?) and neuronal proliferation (BrdU?/MAP-2?) in hippocampus organotypic cultures from control and asphyxia-exposed rats: effect of dopamine agonists. Representative microphotographs obtained by confocal microscopy showing BrdU? (green) and MAP-2? (red)-cells in hippocampus organotypic culture from control (a) and asphyxia-exposed (b) rats. Arrows show BrdU/MAP-2 double labelling, indicating neuronal proliferation. Scale bar: 20 lm. Stereological quantification of BrdU?cells/mm3 in cultures from control (open bars) and asphyxia-exposed (filled bars) rats, treated with vehicle; SKF38393 (10 and 100 lM) (c); quinpirole (10 lM) and sulpiride ? quinpirole (10 lM each) (d), for 24 h (n = 4–5 for

each group). Stereological quantification of double-labelled (BrdU?/ MAP-2?) cells/mm3 in cultures from control (open bars) and asphyxia-exposed (filled bars) rats, treated for 24 h with vehicle; SKF38393 (10 and 100 lM) (e); quinpirole (10 lM), and sulpiride ? quinpirole (10 lM each) (f) (n = 4–5, for each group). *p \ 0.05, **p \ 0.005, Student’s t-test. aComparison between vehicle-treated cultures from asphyxia-exposed and control animals; b Comparison between treatment and vehicle in cultures from control animals; cComparison between treatment and vehicle in cultures from asphyxia-exposed animals; dComparison between sulpiride ? quinpirole versus quinpirole

123

Neurotox Res

Fig. 4 Cell (BrdU?) and neuronal proliferation (BrdU?/MAP-2?) in SVZ organotypic cultures from control and asphyxia-exposed rats: effect of dopamine agonists. Representative microphotographs obtained by confocal microscopy showing BrdU? (green) and MAP-2? (red)-cells in SVZ organotypic culture from control (a) and asphyxia-exposed (b) rats. Arrows show BrdU/MAP-2 double labelling, indicating neuronal proliferation. Scale bar: 20 lm. Stereological quantification of BrdU?cells/mm3 in cultures from control (open bars) and asphyxia-exposed (filled bars) rats, treated with vehicle; SKF38393 (10 and 100 lM) (c); quinpirole (10 lM) and sulpiride ? quinpirole (10 lM each) (d), for 24 h (n = 4–5 for each

123

group). Stereological quantification of double labelled (BrdU?/MAP2?) cells/mm3 in cultures from control (open bars) and asphyxiaexposed (filled bars) rats, treated for 24 h with vehicle; SKF38393 (10 and 100 lM) (e); quinpirole (10 lM), and sulpiride ? quinpirole (10 lM each) (f) (n = 4–5, for each group). *p \ 0.05, **p \ 0.005, Student’s t-test. aComparison between vehicle-treated cultures from asphyxia-exposed and control animals; bcomparison between treatment and vehicle in cultures from control animals; ccomparison between treatment and vehicle in cultures from asphyxia-exposed animals; dcomparison between sulpiride ? quinpirole versus quinpirole

Neurotox Res

Neurogenesis (BrdU1/MAP-21) Double labelling revealed that approximately 40 % of BrdU? cells were also positive for MAP-2 in cultures from CS and AS animals (c.f. Figs. 3c, d, 4c, d). The number of BrdU?/MAP-2?-cells was increased in cultures from AS, compared to that from CS animals, but only in hippocampus reached the significance level (CS: 15788 ± 1832 (n = 5); AS: 21855 ± 2236 cells/mm3 (n = 5); t = 2.099 df = 8, p \ 0.0345; Fig. 3e). 100 lM of SKF38393 decreased neurogenesis by *40 %, but only in SVZ from CS animals (CS: 23908 ± 3074 (n = 5); CSKF: 13438 ± 3536 cells/mm3 (n = 5) t = 2.233 df = 7, p \ 0.0304) (see Figs. 3e, 4e). Quinpirole (10 lM) increased neurogenesis in hippocampus from CS animals ([1.7X; CS: 15788 ± 1832 (n = 5); CQ: 28230 ± 3214 cells/mm3 (n = 5); t = 3.363 df = 8, p \ 0.0049), and AS ([1.6X, AS: 21855 ± 2236 (n = 5); AQ 35480 ± 5366 cells/mm3 (n = 5); t = 2.344 df = 8, p \ 0.0236) animals, compared to that observed under the vehicle-treated condition (Fig. 3f). No significant effects were observed in SVZ (see Fig. 4f). D2 Antagonism on the Effect of Quinpirole The effect of sulpiride (10 lM, S), a selective D2 antagonist, was studied on the increase of neurogenesis elicited by quinpirole (10 lM) in hippocampus. Stereological analysis revealed that sulpiride prevented the effect of quinpirole on neurogenesis (BrdU?/MAP-2?-cells) in hippocampus from CS (CQ: 28230 ± 3214 (n = 5); CQ?S: 16860 ± 4164 cells/mm3 (n = 5); t = 2.162 df = 8, p \ 0.0313) and AS animals (AQ: 35480 ± 5366, (n = 5); AQ?S:12608 ± 1463, cells/mm3 (n = 4); t = 3.161 df = 6, p \ 0.0098) (Fig. 3f). No effect was observed on cell proliferation (Fig. 3d).

Discussion In the present study, we investigated the effect of PA on postnatal cell death, cell proliferation and neurogenesis, and on the modulation by DA agonists, in hippocampus and SVZ organotypic cultures. It was found that PA increased cell death, cell proliferation and neurogenesis in hippocampus and SVZ organotypic cultures. Treatment with the DA receptor agonists, SKF38393 (D1) or Quinpirole (D2), decreased significantly cell death in cultures from AS animals, but in cultures from CS animals that effect was only observed after quinpirole, only in hippocampus. Quinpirole increased cell proliferation and neurogenesis in hippocampus from CS animals, but in cultures from AS animals the effect of quinpirole was only

observed on neurogenesis. In SVZ, the effect of quinpirole on cell proliferation was only observed in cultures from CS animals. No effect was observed by a low dose of SKF38393 (10 lM), but 100 lM of SKF38393 produced a decrease of neurogenesis in SVZ from AS animals. No synergism was observed by simultaneously treating with SKF38393 ? quinpirole (10 lM, each) (data not shown). In hippocampus, the effect of quinpirole (10 lM) on neurogenesis was prevented by the selective D2 antagonist sulpiride (10 lM), in cultures from both asphyxia-exposed and control animals. Thus, the present results provide evidence that D2 is the receptor promoting postnatal neurogenesis in hippocampus, but not in SVZ. PA remains a severe clinical condition leading to neonatal mortality, and short- and long-term sequelae in the surviving infants (see Herrera-Marschitz et al. 2014). The pathophysiology underlying these long-term consequences is not fully understood, and there is no, at present, consensus on a therapeutic strategy, apart of hypothermia, showing protection only if started soon after the insult (see Wassink et al. 2014; Sabir and Cowan 2015). Postnatal establishment of neurocircuitries can be disturbed by metabolic insults, including PA, with long-term consequences (Pastuzko 1994; Morales et al. 2003; Klawitter et al. 2007). The dopaminergic pathways are main targets of the present study, implied in striatal and mesolimbic functions (see Herrera-Marschitz et al. 2011), as well as in postnatal neurogenesis (Ho¨glinger et al. 2004; Morales et al. 2011). Previous studies from our laboratory showed that PA induces a decrease in tyrosine hydroxylase (TH)-labelled cells, together with a decrease in cell viability in substantia nigra, suggesting an increased vulnerability of DA cells to hypoxic insult (Neira-Pena et al. 2015; Klawitter et al. 2007; Decker et al. 2003; Morales et al. 2003; Gross et al. 2000, 2005; Chen et al. 1997a, b). Vaarmann et al. (2013) showed that a low concentration of both D1 and D2- receptor agonists had protective effect on glutamate-induced cell death. Particularly, D2 receptor agonists have been shown to be neuroprotective in animal models of global cerebral ischemia (Liu et al. 1995; O’Neill et al. 1998). D2 receptor stimulation is associated with adenylate cyclase inhibition, reducing intracellular Ca?2 levels (Sawada et al. 1998; Vallar et al. 1990), which tamper the role of glutamate-mediated toxicity. In addition, it has been shown that stimulation of DA autoreceptors by D2 agonists may provide neuroprotection by scavenging oxidative radicals (ROS) that are produced by increased DA metabolism (Sawada et al. 1998). D2 receptors modulate the activity of TH, by competing with glutamate on SER19 and SER 40 phosphorylation sites, activating or deactivating the enzyme. Glutamate acts via NMDA receptors, phosphorylating TH via Ser 19 and 40,

123

Neurotox Res

activating the enzyme, while DA inhibits the enzyme, dephosphorylating Ser 40, via D2 autoreceptors (Lindgren et al. 2001, 2003; Castro et al. 1999; Nisenbaum et al.1998; Zackheim and Abercrombie 2001). D2 agonists may also neuroprotect by a receptor independent manner, inducing the synthesis of ROS scavengers (Sawada et al. 1998), or acting themselves as scavengers (Nishibayashi et al. 1996). It has further been discussed that the neuroprotection produced by DA agonists is mediated by neurotrophic factors (Ohta et al. 2010; Fancellu et al. 2003). Another potential target is the vasculature of the CNS (Choi et al. 2006). A direct effect of DA upon the vasculature has been suggested, on the basis of increased cerebral blood flow measured after the administration of apomorphine, a non-selective dopamine receptor agonist (Ingvar et al. 1983). Indeed, DA has direct effects on brain microcirculation, inducing hemodynamic changes of many brain functions (Krimer et al. 1998), which can promote compensatory mechanisms after a hypoxic-ischemic insult. Thus, the neuroprotective effects of DA agonists could explain the decrease in the number of EThD-1? cells/mm3 observed in CS and AS cultures from hippocampus and SVZ (see Fig. 2). Several compensatory mechanisms have been proposed against the deleterious effects elicited by PA, including neurogenesis. Increased neurogenesis has been observed in DG, CA1 and SVZ after hypoxia/ischemia in rats (Jin et al. 2001; Nakatomi et al. 2002; Daval and Vert 2004; Morales et al. 2008; Bartley et al. 2005), as it is also shown here following PA. Neurogenesis was observed only in hippocampus following PA, but not in SVZ (Figs. 3e, 4e). Neurogenesis represented approximately 40 % of the total cell proliferation induced by the insult, supporting previous reports showing that PA induces cell proliferation with both glial and neuronal phenotypes (Morales et al. 2005, 2007, 2008). The involvement of DA on cell proliferation in rodents has been suggested by several studies (Baker et al. 2004; Ho¨glinger et al. 2004). DA regulates cell proliferation during embryonic development (Borta and Ho¨glinger 2007), and its receptors are expressed in the neurogenic regions of the brain (Dı´az et al. 1997). Ohtani et al. (2003) demonstrated that D1 receptor stimulation reduced the entry of progenitor cells to the S phase, whereas D2 receptors promote G1 to S phase entry, thereby driving proliferation. Furthermore, experimental depletion of DA (by DA antagonism, or 6-hydroxydopamine or MPTP lesions) results in decreased cell proliferation, both in hippocampus and SGZ, in rodents (Ho¨glinger et al. 2004; Yang et al. 2008; Khaindrava et al. 2011) and primates (O’Keeffe et al. 2009). D1 receptors might modulate neurogenesis, but indirectly, perhaps via GABAergic neurons (Goffin et al.

123

2008). Nevertheless, no effects were observed here by treating with a low dose of SKF38393 (10 lM). 100 lM of SKF38393 produced instead a decrease of neurogenesis in SVZ of CS animals. It has been reported that DA-mediated proliferation is dependent on ciliary neurotrophic factor (CNTF), which is known to promote proliferation in the hipocampus and SVZ (Emsley and Hagg 2003; Yang et al. 2008; Mori et al. 2008). A recent study proposed that DA induces proliferation through Akt and extracellular signal-regulated kinase 1/2 signalling, while other studies suggest that DA stimulates the release of epidermal growth factor (EGF), which is known to promote proliferation of neural stem cells (O’Keeffe et al. 2009; Lao et al. 2013). In agreement, it has been shown that D2 and D3 receptor stimulation promotes proliferation of neural progenitor cells in SVZ (van Kampen et al. 2004) and hippocampus (Hiramoto et al. 2007; Coronas et al. 2004; van Kampen and Robertson 2005). When treated with apomorphine or a combination of D1 and D2 agonists (O’Keeffe et al. 2009; Yang et al. 2008), the expression of growth factors, such as EGF, CNTF and FGF-2, increased, promoting cell proliferation (O’Keeffe et al. 2009). Bromocriptine has also been shown to increase cell proliferation, prevented by sulpiride (Ho¨glinger et al. 2004; Coronas et al. 2004). The D2 receptor agonists, quinpirole and ropinerole, have been shown to reverse a decreased neurogenesis observed after DA de-afferentation, suggesting that postsynaptic D2 receptors regulate CNTF (Ho¨glinger et al. 2004; Yang et al. 2008; Sesack et al. 1994). A cooperation between D1 and D2 receptors on the regulation of bFGF levels has also been suggested, involving neurotrophic cascades contributing to neuronal repair and cell survival, in agreement with a report showing that both apomorphine and quinpirole up-regulate striatal bFGF mRNA levels, which can be prevented by SCH23390 (D1) or haloperidol (D2 antagonists) (Roceri et al. 2001). It has been shown that alterations in dopaminergic neurotransmission differentially increase neuronal expression of c-Fos, an immediate early response gene involved in brain cell proliferation and differentiation (Robertson et al. 1991; Hughes and Dragunow 1995). Activation of D1 and blocking D2 receptors elevates fos mRNA and Fosimmunoreactivity (Robertson and Jian 1995; Robertson and Fibiger 1992; Davoodi et al. 2014). Also, pharmacological studies revealed synergistic c-fos responses to combined D1- and D2-stimulation (Keefe and Gerfen 1995). Recent studies have showed that DA cells located in VTA and SNc innervate differentially the hippocampus and SVZ (Ho¨glinger et al. 2014). Because of their diversity, it is possible to speculate that neurogenic areas are differentially affected by region-specific changes in DA signalling (Ho¨glinger et al. 2014). Furthermore, previous

Neurotox Res

studies have shown that there is heterogeneity in the DA systems, in term of trophic factors and neuropeptides, as well as electrophysiological and behavioural properties (Roeper 2013). Therefore, it is expected that neurogenesis induced by DA agonists is different in both neurogenic niches, which is consistent with our results. The present study provides evidence that quinpirole, a D2 agonist, increases neurogenesis in hippocampus following control and asphyxia-exposed conditions, while in the SVZ, the effect of quinpirole was only observed in cultures from CS animals (Fig. 4f), suggesting that the effect of PA abolishes the modulation of neurogenesis by DA, impairing the plasticity of the system in that area. That is a relevant observation, because the effect of PA on delayed cell death can be due to the interruption of postnatal plasticity rather than to a neurotoxic cascade. Modulating neurogenesis by D2 agonists provides perhaps a strategy for restoration of damaged neurocircuitries, which is interesting because there are clinical trials combining indirect and direct DA agonists for reducing longterm deficits associated to perinatal metabolic insults. Acknowledgments Contract Grant sponsors: FONDECYT-Chile 1110263; 1120079; 1170146; 1170074; Millenium Initiative-2010 BNI (P09-015-F; Chile). ATB (#21151232), RPL (#1110263), CLR (#21140281) are CONICYT PhD Fellows; VVM is a MECESUP (UCH0714) fellow. The skillful technical support by Mr Juan Santibanez, Ms Carmen Almeyda is acknowledged. Compliance with Ethical Standards Conflict of Interest All authors declare no conflict of interest on any section of the manuscript.

References Andersson K, Blum M, Chen Y, Eneroth P, Gross J, HerreraMarschitz M, Bjelke B, Bolme P, Diaz R, Jamison L, Loidl F, ˚ stro¨m G, O ¨ gren SO ¨ (1995) Perinatal asphyxia Ungethu¨m U, A increases bFGF mRNA levels and DA cell body number in mesencephalon of rats. NeuroReport 6(2):375–378 Aponso PM, Faull RL, Connor B (2008) Increased progenitor cell proliferation and astrogenesis in the partial progressive 6-hydroxydopamine model of Parkinson’s disease. Neuroscience 151(4):1142–1153 Baker S, Baker KA, Hagg T (2004) Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone. Eur J Neurosci 20(2):575–579 Bartley J, Soltau T, Wimborne H, Kin S, Martin-Studdard A, Hess D, Hill W, Waller J, Carroll J (2005) BrdU-positive cells in the neonatal mouse hippocampus following hypoxic-ischemic brain injury. BCM Neurosci 6:15 Bentivoglio M, Morelli M (2005) The organization and circuits of mesencephalic dopaminergic neurons and the distribution of dopamine receptors in the brain. Handbook of Chemical Neuroanatomy 21:1–107 Borta A, Ho¨glinger GU (2007) Dopamine and adult neurogenesis. J Neurochem 100(3):587–595

Castro NG, de Mello MC, de Mello FG, Aracava Y (1999) Direct inhibition of the N-methyl-D-aspartate receptor channel by dopamine and (?)-SKF38393. Br J Pharmacol 126(8):1847–1855 Chen Y, Hillefors-Berglund M, Herrera-Marschitz M, Bjelke B, Gross J, Andersson K, von Euler G (1997a) Perinatal asphyxia induces long-term changes in dopamine D1, D2, and D3 receptor binding in the rat brain. Exp Neurol 146(1):74–80 Chen Y, Engindawork E, Loidl F, Dell’Anna E, Goiny M, Lubec G, Andersson K, Herrera-Marschitz M (1997b) Short- and longterm effects of perinatal asphyxia on monoamine, amino acid and glycolysis product levels measured in the basal ganglia of the rat. Brain Res Dev Brain Res 104(1–2):19–30 Choi Ji, Chen Y, Hamel E, Bruce G (2006) Brain hemodynamic changes mediated by dopamine receptors: role of the cerebral microvasculature in dopamine-mediated neurovascular coupling. NeuroImage 30(3):700–712 Coronas V, Bantubungi K, Fombonne J, Krantic S, Schiffmann SN, Roger M (2004) Dopamine D3 receptor stimulation promotes the proliferation of cells derived from the postnatal subventricular zone. J Neurochem 91(6):1292–1301 Daval JL, Vert P (2004) Apoptosis and neurogenesis after transient hypoxia in the developing rat brain. Semin Perinatol 28(4):257–263 Davoodi N, te Riele P, Langlois X (2014) Examining dopamine D3 receptor occupancy by antipsychotic drugs via [3H]7-OH-DPAT ex vivo autoradiography and its cross-validation via c-fos immunohistochemistry in the rat brain. Eur J Pharmacol 740:669–675 Dawirs RR, Hildebrandt K, Teuchert-Noodt G (1998) Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus. J Neural Transm 105(2–3):317–327 Decker MJ, Hue GE, Caudle WM, Miller GW, Keating GL, Rye DB (2003) Episodic neonatal hypoxia evokes executive dysfunction and regionally specific alterations in markers of dopamine signaling. Neuroscience 117(2):417–425 Dell’Anna E, Chen Y, Engidawork E, Andersson K, Lubec G, Luthman J, Herrera-Marschitz M (1997) Delayed neuronal death following perinatal asphyxia in rat. Exp Brain Res 115(1):105–115 Dı´az J, Ridray S, Mignon V, Griffon N, Schawartz JC, Sokoloff P (1997) Selective expression of dopamine D3 receptor mRNA in proliferative zones during embryonic development of the rat brain. J Neurosci 17(11):4282–4292 Douglas-Escobar M, Weiss MD (2015) Hypoxic-ischemic encephalopathy: a review for the clinician. JAMA Pediatr 169(4):397–403 Emsley J, Hagg T (2003) Endogenous and exogenous ciliary neurotrophic factor enhances forebrain neurogenesis in adult mice. Exp Neurol 183(2):298–310 Fancellu R, Armentero MT, Nappi G, Blandini F (2003) Neuroprotective effects mediated by dopamine receptor agonists against malonate-induced lesion in the rat striatum. Neurol Sci 24(3):180–181 Farı´as JG, Herrera EA, Carrasco-Pozo C, Sotomayor-Za´rate R, Cruz G, Morales P, Castillo RL (2016) Pharmacological models and approaches for pathophysiological conditions associated with hypoxia and oxidative stress. Pharmacol Ther 158:1–23 Giros B, Sokoloff P, Martres MP, Riou JF, Emorine LJ, Schwartz JC (1989) Alternative splicing directs the expression of two D2 dopamine receptor isoforms. Nature 342(6252):923–926 Goffin D, Aarum J, Schroeder J, Jovanovic J, Chuang T (2008) D1like dopamine receptors regulate GABAA receptor function to modulate hippocampal neural progenitor cell proliferation. J Neurochem 107(4):964–975

123

Neurotox Res Goldsmith SK, Joyce JN (1994) D2 receptor expression in hippocampus and parahippocampal cortex of rat, cat, and human in relation to tyrosine hydroxylase-immunoreactive fibers. Hippocampus 4(3):354–373 Gross J, Mu¨ller I, Chen Y, Elizalde M, Leclere N, Herrera-Marschitz M, Andersson K (2000) Perinatal asphyxia induces regionspecific long-term changes in mRNA levels of tyrosine hydroxylase and dopamine D(1) and D(2) receptors in rat brain. Brain Res Mol Brain Res 79(1–2):110–117 Gross J, Andersson K, Chen Y, Muller I, Andreeva N, HerreraMarschitz M (2005) Effect of perinatal asphyxia on tyrosine hydroxylase and D2 and D1 dopamine receptor mRNA levels expressed during early postnatal development in rat brain. Brain Res Mol Brain Res 134(2):275–281 Herrera-Marschitz M, Morales P, Leyton L, Bustamante D, Klawitter V, Espina-Marchant P, Allende C, Lisboa F, Cunich G, JaraCavieres A, Neira T, Gutierrez-Hernandez MA, Gonzalez-Lira V, Simola N, Schmitt A, Morelli M, Tasker RA, GebickeHaerter PJ (2011) Perinatal asphyxia: current status and approaches towards neuroprotective strategies, with focus on sentinel proteins. Neurotox Res 19(4):603–627 Herrera-Marschitz M, Neira-Pen˜a T, Rojas-Mancilla E, EspinaMarchant P, Esmar D, Pe´rez R, Mun˜oz V, Gutierrez-Herna´ndez M, Rivera B, Simola N, Bustamante D, Morales P, GebickeHaerter P (2014) Perinatal asphyxia: CNS development and deficits with delayed onset. Front Neurosci 26:8–47 Hiramoto T, Kanda Y, Satoh Y, Takishima K, Watanabe Y (2007) Dopamine D2 receptor stimulation promotes the proliferation of neural progenitor cells in adult mouse hippocampus. NeuroReport 18(7):659–664 Ho¨glinger GU, Rizk P, Muriel MP, Duyckaerts C, Oertel WH, Caill I, Hirsch EC (2004) Dopamine depletion impairs precursor cell proliferation in Parkinson disease. Nat Neurosci 7(7):726–735 Ho¨glinger GU, Arias-Carrio´n O, Ipach B, Oertel WH (2014) Origin of the dopaminergic innervation of adult neurogenic areas. J Comp Neurol 522(10):2336–2348 Hughes P, Dragunow M (1995) Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system. Pharmacol Rev 47(1):133–178 Ingvar M, Lindvall O, Stenevi U (1983) Apomorphine-induced changes in local cerebral blood flow in normal rats and after lesions of the dopaminergic nigrostriatal bundle. Brain Res 262(2):259–265 Inta D, Cameron HA, Gass P (2015) New neurons in the adult striatum: from rodents to humans. Trends Neurosci 38(9):517–523 Jin K, Minami M, Lan JQ, Mao XO, Batteur S, Simon RP, Greenberg DA (2001) Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. ProcNatlAcad Sci USA 98(8):4710–4715 Keefe KA, Gerfen CR (1995) D1-D2 dopamine receptor synergy in striatum: effects of intrastriatal infusions of dopamine agonists and antagonists on immediate early gene expression. Neuroscience 66(4):903–913 Khaindrava V, Salin P, Melon C, Ugrumov M, Kerkerian-Le-Goff L, Daszuta A (2011) High frequency stimulation of the subthalamic nucleus impact adult neurogenesis in a rat model of Parkinson’s disease. Neurobiol Dis 42(3):284–291 Kippin TE, Kapur S, van der Kooy D (2005) Dopamine specifically inhibits forebrain neural stem cell proliferation, suggesting a novel effect of antipsychotic drugs. J Neurosci 25(24): 5815–5823 Klawitter V, Morales P, Bustamante D, Gomez-Urquijo S, Ho¨kfelt T, Herrera-Marschitz M (2007) Plasticity of basal ganglia following perinatal asphyxia: neuroprotection by nicotinamide. Exp Brain Res 180(1):139–152

123

Krimer L, Muly E, Williams G, Goldman-Rakic P (1998) Dopaminergic regulation of cerebral cortical microcirculation. Nat Neurosci 1(4):286–289 Lao C, Lu C, Chen J (2013) Dopamine receptor activation promotes neural stem/progenitor cell proliferation through AKT and ERK1/2 pathways and expands type-B and -C cells in adult subventricular zone. Glia 61(4):475–489 Lidow MS, Rakic P (1995) Neurotransmitter receptors in the proliferative zones of the developing primate occipital lobe. J Comp Neurol 360(3):393–402 Lindgren N, Xu ZQ, Herrera-Marschitz M, Haycock J, Ho¨kfelt T, Fisone G (2001) Dopamine D(2) receptors regulate tyrosine hydroxylase activity and phosphorylation at Ser40 in rat striatum. Eur J Neurosci 13(4):773–780 Lindgren N, Usiello A, Goiny M, Haycock J, Erbs E, Greengard P, Hokfelt T, Borrelli E, Fisone G (2003) Distinct roles of dopamine D2L and D2S receptor isoforms in the regulation of protein phosphorylation at presynaptic and postsynaptic sites. Proc Natl Acad Sci USA 100(7):4305–4309 Liu XH, Kato H, Chen T, Kato K, Itoyama Y (1995) Bromocriptine protects against delayed neuronal death of hippocampal neurons following cerebral ischemia in the gerbil. J Neurol Sci 129(1):9–14 Lois C, Alvarez-Buylla A (1993) Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glı´a. Proc Natl Acad Sci USA 90:2074–2077 Lo´pez-Pe´rez SJ, Morales-Villagra´n A, Medina-Ceja L (2015) Effect of perinatal asphyxia and carbamazepine treatment on cortical dopamine and DOPAC levels. J Biomed Sci 22:14 Low JA (1997) Intrapartum fetal asphyxia: definition, diagnosis, and classification. Am J Obset Gynecol 176(5):957–959 Marriott AL, Rojas-Mancilla E, Morales P, Herrera-Marschitz M, Tasker RA (2016) Models of progressive neurological dysfunction originating early in life. Prog Neurobiol. doi:10.1016/j. pneurobio.2015.10.001 Morales P, Klawitter V, Johansson S, Huaiquin P, Barros VG, Avalos AM, Fiedler J, Bustamante D, Gomez-Urquijo S, Goiny M, Herrera-Marschitz M (2003) Perinatal asphyxia impairs connectivity and dopamine neurite branching in organotypic triple culture from rat substantia nigra, neostriatum and neocortex. Neurosci Lett 348(3):175–179 Morales P, Reyes P, Klawitter V, Huaiquı´n P, Bustamante D, Fiedler J, Herrera-Marschitz M (2005) Effects of perinatal asphyxia on cell proliferation and neuronal phenotype evaluated with organotypic hippocampal cultures. Neuroscience 135(2):421–431 Morales P, Huaiquı´n P, Bustamante D, Fiedler JL, Herrera-Marschitz M (2007) Perinatal asphyxia induces neurogenesis in hippocampus: an organotypic culture study. Neurotox Res 12(1):81–84 Morales P, Fiedler JL, Andre´s S, Berrios C, Huaiquı´n P, Bustamante D, Cardenas S, Parra E, Herrera-Marschitz M (2008) Plasticity of hippocampus following perinatal asphyxia: effects on markers for apoptosis and neurogenesis evaluated in vivo seven days after birth. J Neurosci Res 86:2650–2662 Morales P, Bustamante D, Espina-Marchant P, Neira-Pen˜a T, Gutie´rrez-Herna´ndez M, Allende-Castro C, Rojas-Mancilla E (2011) Pathophysiology of perinatal asphyxia: can we predict and improve individual outcomes? EPMA 2(2):211–230 Mori M, Jefferson JJ, Hummel M, Garbe DS (2008) CNTF: a putative link between dopamine D2 receptors and neurogenesis. J Neurosci 28(23):5867–5869 Nakatomi H, Kuriu T, Okabe S, Yamamoto S, Hatano O, Kawahara N, Tamura A, Kirino T, Nakafuku M (2002) Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110(4): 429–441

Neurotox Res Neira-Pena T, Rojas-Mancilla E, Munoz-Vio V, Perez R, Gutie´rrezHerna´ndez M, Bustamante D, Morales P, Hermoso MA, Gebicke-Haerter P, Herrera-Marschitz M (2015) Perinatal asphyxia leads to PARP-1 overactivity, p65 translocation, IL1b, and TNF-a overexpression, and apoptotic-like cell death in mesencephalon of neonatal rats: prevention by systemic neonatal nicotinamide administration. Neurotox Res 27(4):453–465 Nisenbaum ES, Mermelstein PG, Wilson CJ, Surmeier DJ (1998) Selective blockade of a slowly inactivating potassium current in striatal neurons by (±) 6-chloro-APB hydrobromide (SKF82958). Synapse 29(3):213–224 Nishibayashi S, Asanuma M, Kohno M, Go´mez-Vargas M, Ogawa N (1996) Scavenging effects of dopamine agonists on nitric oxide radicals. J Neurochem 67(5):2208–2211 O’Neill MJ, Hicks CA, Ward MA, Cardwell GP, Reymann JM, Allain H, Bentue´-Ferrer D (1998) Dopamine D2 receptor agonists protect against ischaemia-induced hippocampal neurodegeneration in global cerebral ischaemia. Eur J Pharmacol 352(1):37–46 Ohta K, Kuno S, Inoue S, Ikeda E, Fujinami A, Ohta M (2010) The effect of dopamine agonists: the expression of GDNF, NGF, and BDNF in cultured mouse astrocytes. J Neurol Sci 291(1–2):12–16 Ohtani N, Goto T, Waeber C, Bhide PG (2003) Dopamine modulates cell cycle in the lateral ganglionic eminence. J Neurosci 23(7):2840–2850 O’Keeffe GC, Barker RA, Caldwell MA (2009) Dopaminergic modulation of neurogenesis in the subventricular zone of the adult brain. Cell Cycle 8(18):2888–2894 Ong J, Plane JM, Parent JM, Silverstein FS (2005) Hypoxic-ischemic injury stimulates subventricular zone proliferation and neurogenesis in the neonatal rat. Pediatr Res 58(3):600–606 Park JH, Enikolopov G (2010) Transient elevation of adult hippocampal neurogenesis after dopamine depletion. ExpNeurol 222(2):267–276 Pastuzko A (1994) Metabolic responses of the dopaminergic system during hypoxia in newborn brain. Biochem Med Metab Biol 51(1):1–15 Plane JM, Liu R, Wang TW, Silverstein FS, Parent JM (2004) Neonatal hypoxic-ischemic injury increases forebrain subventricular zone neurogenesis in the mouse. Neurobiol Dis 16(3):585–595 Reuss B, Unsicker K (2000) Survival and differentiation of dopaminergic mesencephalic neurons are promoted by dopamine-mediated induction of FGF-2 in striatal astroglial cells. Mol Cell Neurosci 16(6):781–792 Robertson GS, Fibiger HC (1992) Neuroleptics increase c-fos expression in the forebrain: contrasting effects of haloperidol and clozapine. Neuroscience 46(2):315–328 Robertson GS, Jian M (1995) D1 and D2 dopamine receptors differentially increase fos-like immunoreactivity in accumbal projections to the ventral pallidum and midbrain. Neuroscience 64(4):1019–1034 Robertson HA, Paul ML, Moratalla R, Graybiel AM (1991) Expression of the immediate early gene c-fos in basal ganglia: induction by dopaminergic drugs. Can J Neurol Sci 18:380–383

Roceri M, Molteni R, Fumagalli F, Racagni G, Gennarelli M, Corsini G, Maggio R, Riva M (2001) Stimulatory role of dopamine on fibroblast growth factor-2 expression in rat striatum. J Neurochem 76(4):990–997 Roeper J (2013) Dissecting the diversity of midbrain dopamine neurons. Trends Neurosci 36(6):336–342 Sabir H, Cowan FM (2015) Prediction of outcome methods assessing short- and long-term outcome after therapeutic hypothermia. SemFetal Neonatal Med 20(2):115–121 Sawada H, Ibi M, Kihara T, Urushitani M, Akaike A, Kimura J, Shimohama S (1998) Dopamine D2-type agonists protect mesencephalic neurons from glutamate neurotoxicity: mechanisms of neuroprotective treatment against oxidative stress. Ann Neurol 44(1):110–119 Seeman P (1980) Brain dopamine receptors. Pharmacol Rev 32(3):229–313 Sesack SR, Aoki C, Pickel VM (1994) Ultrastructural localization of D2 receptor-like immunoreactivity in midbrain dopaminergic neurons and their striatal targets. J Neurosci 14(1):88–106 Sokoloff P, Giros B, Martres MP, Bouthenet ML, Schwartz JC (1990) Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347(6289):146–151 Vaarmann A, Kovac S, Holmstro¨m KM, Gandhi S, Abramov AY (2013) Dopamine protects neurons against glutamate-induced excitotoxicity. Cell Death Dis 4:e455 Vallar L, Muca C, Magni M, Albert P, Bunzow J, Meldolesi J, Civelli O (1990) Differential coupling of dopaminergic D-2 receptors expressed in different cell types. J Biol Chem 265:10320–10326 van Kampen JM, Robertson HA (2005) A possible role for dopamine D3 receptor stimulation in the induction of neurogenesis in the adult rat substantia nigra. Neuroscience 136(2):381–386 van Kampen JM, Hagg T, Robertson HA (2004) Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D receptor stimulation. Eur J Neurosci 19(9):2377–2387 Verney C, Baulac M, Berger B, Alvarez C, Vigny A, Helle KB (1985) Morphological evidence for a dopaminergic terminal field in hippocampal formation of young and adult rat. Neuroscience 14(4):1039–1052 Voorn P, Kalsbeck A, Jorritsma-Byham B, Groenewegen HJ (1988) The pre- and postnatal development of the dopaminergic cell groups in the ventral mesencephalon and the dopaminergic innervation of the striatum of the rat. Neuroscience 25(3):857–887 Wassink G, Gunn ER, Drury PP, Bennet L, Gunn AJ (2014) The mechanisms and treatment of asphyxial encephalopathy. Front Neurosci 8:40 Yang P, Arnold SA, Habas A, Hetman M, Hagg T (2008) Ciliary neurotrophic factor mediates dopamine D2 receptor-induced CNS neurogenesis in adult mice. J Neurosci 28(9):2231–2241 Zackheim JA, Abercrombie ED (2001) Decreased striatal dopamine efflux after intrastriatal application of benzazepine-class D1 agonists is not mediated via dopamine receptors. Brain Res Bull 54(6):603–607

123