Restoring lost nigrostriatal fibers in Parkinson's disease based on clinically-inspired design criteria.

Parkinson's disease is a neurodegenerative disease affecting around 10 million people worldwide. The death of dopaminergic neurons in the substantia nigra and the axonal fibers that constitute the nigrostriatal pathway leads to a loss of dopamine in the striatum that causes the motor symptoms of this disease. Traditional treatments have focused on reducing symptoms, while therapies with human fetal or stem cell-derived neurons have centered on implanting these cells in the striatum to restore its innervation. An alternative approach is pathway reconstruction, which aims to rebuild the entire structure of neurons and axonal fibers of the nigrostriatal pathway in a way that matches its anatomy and physiology. This type of repair could be more capable of reestablishing the signaling mechanisms that ensure proper dopamine release in the striatum and regulation of other motor circuit regions in the brain. In this manuscript, we conduct a review of the literature related to pathway reconstruction as a treatment for Parkinson's disease, delve into the limitations of these studies, and propose the requisite design criteria to achieve this goal at a human scale. We then present our tissue engineering-based platform to fabricate hydrogel-encased dopaminergic axon tracts in vitro for later implantation into the brain to replace and reconstruct the pathway. These tissue-engineered nigrostriatal pathways (TE-NSPs) can be characterized and optimized for cell number and phenotype, axon growth lengths and rates, and the capacity for synaptic connectivity and dopamine release. We then show original data of advances in creating these constructs matching clinical design criteria using human iPSC-derived dopaminergic neurons and a hyaluronic acid hydrogel. We conclude with a discussion of future steps that are needed to further optimize human-scale TE-NSPs and translate them into clinical products.

Reduced complement of dopaminergic neurons in the substantia nigra pars compacta of mice with a constitutive "low footprint" genetic knockout of alpha-synuclein.

Previous studies of the alpha-synuclein null mutant mice on the C57Bl6 genetic background have revealed reduced number of dopaminergic neurons in their substantia nigra pars compacta (SNpc). However, the presence in genomes of the studied mouse lines of additional genetic modifications that affect expression of genes located in a close proximity to the alpha-synuclein-encoding Snca gene makes these data open to various interpretations. To unambiguously demonstrate that the absence of alpha-synuclein is the primary cause of the observed deficit of dopaminergic neurons, we employed a recently produced constituent alpha-synuclein knockout mouse line B6(Cg)-Sncatm1.2Vlb/J. The only modification introduced to the genome of these mice is a substitution of the first coding exon and adjusted short intronic fragments of the Snca gene by a single loxP site. We compared the number of dopaminergic neurons in the SNpc of this line, previously studied B6(Cg)-Sncatm1Rosl/J line and wild type littermate mice. A similar decrease was observed in both knockout lines when compared with wild type mice. In a recently published study we revealed no loss of dopaminergic neurons following conditional inactivation of the Snca gene in neurons of adult mice. Taken together, these results strongly suggest that alpha-synuclein is required for efficient survival or maturation of dopaminergic neurons in the developing SNpc but is dispensable for survival of mature SNpc dopaminergic neurons.

Does Developmental Variability in the Number of Midbrain Dopamine Neurons Affect Individual Risk for Sporadic Parkinson's Disease?

Parkinson's disease (PD) is a slowly progressing neurodegenerative disorder that is coupled to both widespread protein aggregation and to loss of substantia nigra dopamine (DA) neurons, resulting in a wide variety of motor and non-motor signs and symptoms. Recent findings suggest that the PD process is triggered several years before there is sufficient degeneration of DA neurons to cause onset of overt motor symptoms. According to this concept, the number of DA neurons present in the substantia nigra at birth could influence the time from the molecular triggering event until the clinical diagnosis with lower number of neurons at birth increasing the risk to develop the disease. Conversely, the risk for diagnosis would be reduced if the number of DA neurons is high at birth. In this commentary, we discuss the genetic and epigenetic factors that might influence the number of nigral DA neurons that each individual is born with and how these may be linked to PD risk.

A proteomic approach to investigate enhanced responsiveness in rechallenged adult rats prenatally exposed to lindane.

Proteomic analysis was carried out in substantia nigra (SNi) and hippocampus (Hi) isolated from rat offspring born to mothers exposed to lindane (orally; 0.25 mg/kg) from gestation day 5 (GD5) to GD 21 and subsequently rechallenged (orally; 2.5 mg/kg X 21 days) at adulthood (12 weeks). 2D gel electrophoresis revealed no significant differences in the expression of proteins in brain regions isolated from prenatally exposed offspring at adulthood. Significantly greater magnitude of alterations was observed in the expression of proteins related to mitochondrial and energy metabolism, ubiquitin-proteasome pathway, structural and axonal growth leading to increased oxidative stress in Hi and SNi isolated from rechallenged offspring when compared to control offspring treated postnatally with lindane. Western blotting and DNA laddering showed a greater magnitude of increase in apoptosis in the Hi and SNi of rechallenged offspring. Ultrastructural analysis demonstrated disrupted mitochondrial integrity, synaptic disruption and necrotic structures in the brain region of rechallenged offspring. Neurobehavioral studies also demonstrated a greater magnitude of alterations in cognitive and motor functions in rechallenged rats. The data suggest that prenatal exposure of lindane induces persistent molecular changes in the nervous system of offspring which are unmasked leading to neurodegeneration following rechallenge at adulthood.

Effect of Wnt1 and Wnt5a on the development of dopaminergic neurons, and toxicity induced by combined exposure to paraquat and maneb during gestation and lactation.

Paraquat (PQ) and maneb (MB) are widely used herbicides. Wingless (Wnt) proteins serve a role in the development and differentiation of dopaminergic neurons. Previous studies demonstrated that combined exposure to PQ and MB damages dopaminergic neurons in the midbrain. Effects of PQ and MB exposure on midbrain Wnt proteins have also been previously reported. In the present study, from the 5th day of gestation to weaning of the offspring, pregnant Sprague­Dawley rats were administrated saline, or PQ and MB at two different doses: high, 15 mg/kg body weight PQ + 45 mg/kg body weight MB; or low, 10 mg/kg body weight PQ + 30 mg/kg body weight MB. Dopamine content in the striatum was examined by high performance liquid chromatography with a fluorescence detector and mRNA and protein expression of Wnt1, Wnt5a, nuclear receptor related factor 1 (Nurr1) and tyrosine hydroxylase (TH) in the midbrain was examined by reverse transcription­quantitative polymerase chain reaction and western blotting. Combined exposure to PQ and MB during development decreased mRNA and protein expression of Wnt1, TH and Nurr1 and increased expression of Wnt5a in the offspring.

Neonatal rotenone lesions cause onset of hyperactivity during juvenile and adulthood in the rat.

Attention deficit hyperactivity disorder (ADHD) is characterized by behavioral and cognitive symptoms. Longitudinal studies demonstrated that the symptoms remains clinically significant for the majority of ADHD children into adulthood. Furthermore, a population-based birth cohort provided the initial evidence of adult ADHD that lacks a history of childhood ADHD. We previously demonstrated that neonatal exposure to bisphenol A, an environmental chemical caused hyperactivity in the juvenile. Here, we extend to examine other chemical such as rotenone, a dopaminergic toxins. Oral administration of rotenone (3mg/kg) into 5-day-old male Wistar rats significantly caused hyperactivity at adulthood (8∼11 weeks old; p<0.05). It was about 1.3∼1.4-fold more active in the nocturnal phase after administration of rotenone than control rats. Higher dose (16mg/kg) or repeated lower dose of rotenone (1mg/kg/day for 4days) caused hyperactivity in the juvenile. Furthermore, DNA array analyses showed that neonatal exposure to rotenone altered the levels of gene expression of several molecules related to apoptosis/cell cycle, ATPase, skeletal molecule, and glioma. Bivariate normal distribution analysis indicates no correlation in gene expression between a hyperactivity disorder model and a Parkinson's disease model by rotenone. Thus, we demonstrate a rotenone models of ADHD whose onset varies during juvenile and adulthood.

Distinct cellular and molecular environments support aging-related DNA methylation changes in the substantia nigra.

AIM: We aimed to couple brain region-specific changes in global DNA methylation over aging to underlying cellular and molecular environments. MATERIALS & METHODS: We measured two major forms of DNA methylation and analyzed Dnmt, Tet and metabolite levels in the striatum and substantia nigra (SN) over aging in healthy male mice. RESULTS: The ratio of 5-hydroxymethylcytosine to 5-methylcytosine increases over aging in the SN, and 5-hydroxymethylcytosine increases preferentially in dopaminergic neurons. Additionally, this age-dependent alteration in methylation correlates with a reduction in the ratio of α-ketoglutarate to succinate in the SN. CONCLUSION: Distinct cellular and molecular environments correlate with aging-associated methylation changes in the SN, implicating this epigenetic mechanism in the susceptibility of this brain region to age-related cell loss.

Early prenatal exposure to MPTP does not affect nigrostrial neurons in macaque monkey.

The discovery of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a toxin that induces parkinsonism in both human and primate, has prompted the search for environmental toxins potentially responsible for idiopathic Parkinson's disease (PD). The present study reports the ultimate effects of MPTP intoxication of a female macaque monkey, which unraveled to be pregnant after parkinsonism had developed, upon its fetus. Detailed examination of the offpsring nigrostriatal pathway showed that tyrosine hydroxylase immunoreactivity in caudate-putamen nuclei and substantia nigra compacta (SNc) was not different from an age-matched control. Biochemical analysis of the tissue content of dopaminergic markers further suggested modification of metabolism in the MPTP-exposed monkey. These data suggest that early prenatal intoxication does not destroy nigrostriatal neurons, most likely because dopamine neurons had not developed yet when exposed to MPTP.

Sonic Hedgehog Controls the Phenotypic Fate and Therapeutic Efficacy of Grafted Neural Precursor Cells in a Model of Nigrostriatal Neurodegeneration.

The expression of soluble growth and survival promoting factors by neural precursor cells (NPCs) is suggested to be a prominent mechanism underlying the protective and regenerative effects of these cells after transplantation. Nevertheless, how and to what extent specific NPC-expressed factors contribute to therapeutic effects is not well understood. Using RNA silencing, the current study investigated the roles of two donor NPC molecules, namely glial cell-line derived neurotrophic factor (GDNF) and sonic hedgehog (SHH), in the protection of substantia nigra dopamine neurons in rats treated with 6-hydroxydopamine (6-OHDA). Analyses indicate that as opposed to the knock-down of GDNF, SHH inhibition caused a profound decline in nigrostriatal neuroprotection. Further, SHH silencing also curbed endogenous neurogenesis and the migration of host brdU+/dcx+ neural precursors into the striatum, which was present in the animals receiving control or GDNF silenced NPCs. A change in graft phenotype, mainly reflected by a reduced proportion of undifferentiated nestin+ cells, as well as a significantly greater host microglial activity, suggested an important role for these processes in the attenuation of neuroprotection and neurogenesis upon SHH silencing. Overall these studies reveal core mechanisms fundamental to grafted NPC-based therapeutic effects, and delineate the particular contributions of two graft-expressed molecules, SHH and GDNF, in mediating midbrain dopamine neuron protection, and host plasticity after NPC transplantation.

Ebf2 is required for development of dopamine neurons in the midbrain periaqueductal gray matter of mouse.

Dopaminergic (DA) neurons in the midbrain ventral periaqueductal gray matter (PAG) play critical roles in various physiological and pathophysiological processes including sleep-wake rhyme, antinociception, and drug addiction. However, the molecular mechanisms underlying their development are poorly understood. Here, we showed that PAG DA neurons arose as early as E15.5 in mouse embryos. During the prenatal period, the majority of PAG DA neurons was distributed in the intermediate and caudal regions of the PAG. In the postnatal brain, ∼50% of PAG DA neurons were preferentially located in the caudal portion of the PAG. Moreover, transcription factor early B-cell factor 2 (Ebf2) was transiently expressed in a subset of DA neurons in embryonic ventral mesencephalon. Functional analysis revealed that loss of Ebf2 in vivo caused a marked reduction in the number of DA neurons in the midbrain PAG but not in the substantia nigra and ventral tegmental area. Thus, Ebf2 is identified as a novel and important regulator selectively required for midbrain PAG DA neuron development.

Mutant α-synuclein enhances firing frequencies in dopamine substantia nigra neurons by oxidative impairment of A-type potassium channels.

Parkinson disease (PD) is an α-synucleinopathy resulting in the preferential loss of highly vulnerable dopamine (DA) substantia nigra (SN) neurons. Mutations (e.g., A53T) in the α-synuclein gene (SNCA) are sufficient to cause PD, but the mechanism of their selective action on vulnerable DA SN neurons is unknown. In a mouse model overexpressing mutant α-synuclein (A53T-SNCA), we identified a SN-selective increase of in vivo firing frequencies in DA midbrain neurons, which was not observed in DA neurons in the ventral tegmental area. The selective and age-dependent gain-of-function phenotype of A53T-SCNA overexpressing DA SN neurons was in part mediated by an increase of their intrinsic pacemaker frequency caused by a redox-dependent impairment of A-type Kv4.3 potassium channels. This selective enhancement of "stressful pacemaking" of DA SN neurons in vivo defines a functional response to mutant α-synuclein that might be useful as a novel biomarker for the "DA system at risk" before the onset of neurodegeneration in PD.

Canonical BMP-Smad signalling promotes neurite growth in rat midbrain dopaminergic neurons.

Ventral midbrain (VM) dopaminergic (DA) neurons project to the dorsal striatum via the nigrostriatal pathway to regulate voluntary movements, and their loss leads to the motor dysfunction seen in Parkinson's disease (PD). Despite recent progress in the understanding of VM DA neurogenesis, the factors regulating nigrostriatal pathway development remain largely unknown. The bone morphogenetic protein (BMP) family regulates neurite growth in the developing nervous system and may contribute to nigrostriatal pathway development. Two related members of this family, BMP2 and growth differentiation factor (GDF)5, have neurotrophic effects, including promotion of neurite growth, on cultured VM DA neurons. However, the molecular mechanisms regulating their effects on DA neurons are unknown. By characterising the temporal expression profiles of endogenous BMP receptors (BMPRs) in the developing and adult rat VM and striatum, this study identified BMP2 and GDF5 as potential regulators of nigrostriatal pathway development. Furthermore, through the use of noggin, dorsomorphin and BMPR/Smad plasmids, this study demonstrated that GDF5- and BMP2-induced neurite outgrowth from cultured VM DA neurons is dependent on BMP type I receptor activation of the Smad 1/5/8 signalling pathway.

Spatiotemporal expression of MANF in the developing rat brain.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved neurotrophic factor which exhibited neuroprotective properties. Recent studies suggested that MANF may play a role in the neural development of Drosophila and zebra fishes. In this study, we investigated the spatiotemporal expression of MANF in the brain of postnatal and adult rats. MANF expression appeared wide spread and mainly localized in neurons. In the cerebral cortex, neurons in layer IV and VI displayed particularly strong MANF immunoreactivity. In the hippocampus, intensive MANF expression was observed throughout the subfields of Cornu Amonis (CA1, CA2, and CA3) and the granular layer of the dentate gyrus (DG). In the substantia nigra, high MANF expression was shown in the substantia nigra pars compacta (SNpc). In the thalamus, the anterodorsal thalamic nucleus (ADTN) exhibited the highest MANF immunoreactivity. In the hypothalamus, intensive MANF immunoreactivity was shown in the supraoptic nucleus (SON) and tuberomammillary nucleus (TMN). In the cerebellum, MANF was localized in the external germinal layer (EGL), Purkinje cell layer (PCL), internal granule layer (IGL) and the deep cerebellar nuclei (DCN). We examined the developmental expression of MANF on postnatal day (PD) 3, 5, 7, 9, 15, 21, 30 and adulthood. In general, the levels of MANF were high in the early PDs (PD3 and PD5), and declined gradually as the brain matured; MANF expression in the adult brain was the lowest among all time points examined. However, in some structures, such as PCL, IGL, SON, TMN and locus coeruleus (LC), high expression of MANF sustained throughout the postnatal period and persisted into adulthood. Our results indicated that MANF was developmentally regulated and may play a role in the maturation of the central nervous system (CNS).

Altered cerebellum development and dopamine distribution in a rat genetic model with congenital hypothyroidism.

Thyroid hormones play crucial roles in the development and functional maintenance of the central nervous system. Despite extensive studies of the neural function of thyroid hormones, little is known about the effects of hypothyroidism on behavioural traits and the mechanisms underlying such effects. In the present study, we report an investigation of congenitally hypothyroid mutant rdw rats, revealing a novel function of thyroid hormones in the central nervous system. The rdw rats were subjected to behavioural analyses such as the rotarod test, open field test and circadian activity measurement. To determine the cause of behavioural disorders, cerebellar morphogenesis was examined by immunohistochemical analysis, and the axonal transport of dopamine in the nigrostriatal pathway was analysed by high-performance liquid chromatography and western blotting. The effects of thyroxine administration to the rdw rats were examined by behavioural analysis. The rdw rats showed severe impairment of motor coordination and balance. This could be explained by the fact that the rats showed severe retardation of cerebellar morphogenesis, which correlates with the small somata and poor dendritic arborisation of Purkinje cells and retarded migration of granule cells particularly during the first two postnatal weeks. Moreover, the rdw rats showed hypoactivity, characterised by decreased circadian locomotor activity. After weaning, thyroxine administration improved the dwarfism in rdw rats but had no effect on cerebellar function. In addition, the rdw rats showed anxiety and depression intrinsically to novel surroundings. Interestingly, the rdw rats showed high levels of dopamine in the substantia nigra and low levels in the striatum, an important centre for the coordination of behaviour. Furthermore, low levels of tubulin in the striatum were detected, indicating the aberrant axonal transport of dopamine in the nigrostriatal pathway as a result of the reduced delivery of microtubules. These findings indicate an important function of thyroid hormones in cerebellar formation and in the regulation of axonal transport of dopamine. Moreover, rdw rats will be useful for studies of brain function and behavioural disorders in congenital hypothyroidism.

Neonatal exposure to estradiol valerate increases dopamine content in nigrostriatal pathway during adulthood in the rat.

Research in programming has focused in the study of stimuli that affect sensitive periods of development such as prenatal and neonatal stage. We previously showed that exposure to estradiol valerate to female rats during the first 12 h of life increased catecholamine content in ventromedial-arcuatus hypothalamus of the adult rat. However, changes in others dopaminergic circuits have not been studied. The purpose of this work was to determine the neurotransmitters changes induced by neonatal estradiol valerate (0.1 mg/50 µl s. c. per rat) administration on nigrostriatal pathway of adult female rats. Sesame oil (50 µl s. c. per rat) was administered in a control parallel group. EV-1 adult rats presented effective markers of long-term estrogenization as decreased serum levels of progesterone and a reduction in the size of estrogen-sensitive organs. In the brain, neonatal estradiol valerate administration led to a significant increase in dopamine content in striatum, substantia nigra and ventral tegmental area. With respect to the contents of dopamine metabolites, only 3-methoxytyramine content increased in substantia nigra and ventral tegmental area. In addition, the content of noradrenaline increased only in striatum. Interestingly, estrogenized rats lacked locomotor activity induced by acute dose of amphetamine (1 mg/kg i. p.). Altogether, these results show that neonatal exposure to estradiol valerate permanently modified the content of monoamine neurotransmitters in nigrostriatal pathway and amphetamine-induced locomotor activity of adult female rats. This might imply that estrogenized rats could have changes in the expression of key proteins in dopaminergic regulation, as tyrosine hydroxylase and dopamine transporter.

Expression of trefoil factor 1 in the developing and adult rat ventral mesencephalon.

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides with a characteristic tree-looped trefoil structure. TFFs are mainly expressed in the gastrointestinal tract where they play a critical role in the function of the mucosal barrier. TFF1 has been suggested as a neuropeptide, but not much is known about its expression and function in the central nervous system. We investigated the expression of TFF1 in the developing and adult rat midbrain. In the adult ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly found in the substantia nigra pars compacta (SNc), the ventral tegmental area (VTA) and in periaqueductal areas. While around 90% of the TFF1-ir cells in the SNc co-expressed tyrosine hydroxylase (TH), only a subpopulation of the TH-ir neurons expressed TFF1. Some TFF1-ir cells in the SNc co-expressed the calcium-binding proteins calbindin or calretinin and nearly all were NeuN-ir confirming a neuronal phenotype, which was supported by lack of co-localization with the astroglial marker glial fibrillary acidic protein (GFAP). Interestingly, at postnatal (P) day 7 and P14, a significantly higher proportion of TH-ir neurons in the SNc co-expressed TFF1 as compared to P21. In contrast, the proportion of TFF1-ir cells expressing TH remained unchanged during postnatal development. Furthermore, significantly more TH-ir neurons expressed TFF1 in the SNc, compared to the VTA at all four time-points investigated. Injection of the tracer fluorogold into the striatum of adult rats resulted in retrograde labeling of several TFF1 expressing cells in the SNc showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in SNc of 6-hydroxylase-lesioned hemiparkinsonian rats. In conclusion, we show for the first time that distinct subpopulations of midbrain dopaminergic neurons express TFF1, and that this expression pattern is altered in a rat model of Parkinson's disease.

Lack of vascular endothelial growth factor receptor-2/Flk1 signaling does not affect substantia nigra development.

Oxygen tension is critical for proliferation of human and murine midbrain-derived neural precursor cells (mNPCs). Lack of hypoxia-inducible factor-1α (HIF1α) impairs midbrain dopaminergic neurogenesis which could be rescued by vascular endothelial growth factor (VEGF) via VEGFR-2 signaling. Here, we conditionally inactivated the VEGFR-2, encoded by the fetal liver kinase 1 (Flk1) gene, in murine NPCs to determine its role in proliferation and survival in vitro as well as survival of dopaminergic neurons in vivo. Flk1 conditional knock-out (Flk1 CKO) mice showed no general brain phenotype. There was no midbrain-specific impairment of NPC proliferation as seen in HIF1α CKO mice. In the substantia nigra (SN) of adult Flk1 CKO mice, nonbiased stereological cell counts revealed no reduction of TH-positive neurons of Flk1 CKO mice compared with control Cre/wt mice (in which the wild-type Flk1 allele is expressed in parallel with the Cre recombinase allele). In conclusion, VEGF receptor signaling seems not to be relevant to the development and survival of substantia nigra dopaminergic neurons within the hypoxia-HIF1α signaling pathway.

Neonatal exposure to lipopolysaccharide enhances accumulation of α-synuclein aggregation and dopamine transporter protein expression in the substantia nigra in responses to rotenone challenge in later life.

Brain inflammation in early life may enhance adult susceptibility to develop neurodegenerative disorders triggered by environmental toxins. Our previous studies show that perinatal lipopolysaccharide (LPS) exposure enhances adult susceptibility to rotenone-induced injury to the dopaminergic system in the substantia nigra (SN) of the adult rat brain. To further investigate the enhanced adult susceptibility by neonatal LPS exposure to rotenone neurotoxicity, we used our neonatal rat model of LPS exposure (1mg/kg, intracerebral injection in postnatal day 5, P5, neonatal rats) to examine the protein levels of α-synuclein and dopamine transporters (DAT) in the adult rat. By P70, rats from the saline- or LPS-exposed group were challenged with rotenone, a commonly used pesticide, through subcutaneous mini-pump infusion at a dose of 1.25mg/kg/day for 14 days. The accumulation of α-synuclein aggregation and increment of DAT protein content were found in the SN of LPS-exposed rats. Neonatal LPS exposure enhanced rotenone-stimulated accumulation of α-synuclein aggregation and increment in DAT protein expression in the cytoplasmic compartment of the SN, and in the synaptosomal compartment of the striatum of adult rats. Rotenone treatment also resulted in reduction of [(3)H]dopamine uptake and mitochondrial complex I activity in the striatum of rats with neonatal LPS exposure, but not in those without this exposure. The current study suggests possible roles of α-synuclein aggregate and DAT distribution in the cytoplasm and synaptosome triggered by environmental toxins in later life in the development of neurodegenerative disorders. Our model may be useful in studying mechanisms involved in the pathogenesis of nonfamilial Parkinson's disease and for developing potential therapeutic treatments for this disease.

The vitamin D receptor in dopamine neurons; its presence in human substantia nigra and its ontogenesis in rat midbrain.

There is growing evidence that vitamin D is a neuroactive steroid capable of regulating multiple pathways important for both brain development and mature brain function. In particular, there is evidence from rodent models that prenatal vitamin D deficiency alters the development of dopaminergic pathways and this disruption is associated with altered behavior and neurochemistry in the adult brain. Although the presence of the vitamin D receptor (VDR) has been noted in the human substantia nigra, there is a lack of direct evidence showing that VDR is present in dopaminergic cells. Here we confirm that the VDR is present in the nucleus of tyrosine hydroxylase (TH)-positive neurons in both the human and rat substantia nigra, and it emerges early in development in the rat, between embryonic day 12 (E12) and E15. Consistent evidence based on immunohistochemistry, real-time PCR and western blot confirmed a pattern of increasing VDR expression in the rat midbrain until weaning. The nuclear expression of VDR in TH-positive neurons during critical periods of brain development suggests that alterations in early life vitamin D status may influence the orderly development of dopaminergic neurons.

Phosphodiesterase 2 inhibitors promote axonal outgrowth in organotypic slice co-cultures.

The development of appropriate models assessing the potential of substances for regeneration of neuronal circuits is of great importance. Here, we present procedures to analyze effects of substances on fiber outgrowth based on organotypic slice co-cultures of the nigrostriatal dopaminergic system in combination with biocytin tracing and tyrosine hydroxylase labeling and subsequent automated image quantification. Selected phosphodiesterase inhibitors (PDE-Is) were studied to identify their potential growth-promoting capacities. Immunohistochemical methods were used to visualize developing fibers in the border region between ventral tegmental area/substantia nigra co-cultivated with the striatum as well as the cellular expression of PDE2A and PDE10. The quantification shows a significant increase of fiber density in the border region induced by PDE2-Is (BAY60-7550; ND7001), comparable with the potential of the nerve growth factor and in contrast to PDE10-I (MP-10). Analysis of tyrosine hydroxylase-positive fibers indicated a significant increase after treatment with BAY60-7550 and nerve growth factor in relation to dimethyl sulfoxide. Additionally, a dose-dependent increase of intracellular cGMP levels in response to the applied PDE2-Is in PDE2-transfected HEK293 cells was found. In summary, our findings show that PDE2-Is are able to significantly promote axonal outgrowth in organotypic slice co-cultures, which are a suitable model to assess growth-related effects in neuro(re)generation.