Characterization of Fetal Antigen 1/Delta-Like 1 Homologue Expressing Cells in the Rat Nigrostriatal System: Effects of a Unilateral 6-Hydroxydopamine Lesion

Fetal antigen 1/delta-like 1 homologue (FA1/dlk1) belongs to the epidermal growth factor superfamily and is considered to be a non-canonical ligand for the Notch receptor. Interactions between Notch and its ligands are crucial for the development of various tissues. Moreover, FA1/dlk1 has been suggested as a potential supplementary marker of dopaminergic neurons. The present study aimed at investigating the distribution of FA1/dlk1-immunoreactive (-ir) cells in the early postnatal and adult midbrain as well as in the nigrostriatal system of 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian adult rats. FA1/dlk1-ir cells were predominantly distributed in the substantia nigra (SN) pars compacta (SNc) and in the ventral tegmental area. Interestingly, the expression of FA1/dlk1 significantly increased in tyrosine hydroxylase (TH)-ir cells during early postnatal development. Co-localization and tracing studies demonstrated that FA1/dlk1-ir cells in the SNc were nigrostriatal dopaminergic neurons, and unilateral 6-OHDA lesions resulted in loss of both FA1/dlk1-ir and TH-ir cells in the SNc. Surprisingly, increased numbers of FA1/dlk1-ir cells (by 70%) were detected in dopamine-depleted striata as compared to unlesioned controls. The higher number of FA1/dlk1-ir cells was likely not due to neurogenesis as colocalization studies for proliferation markers were negative. This suggests that FA1/dlk1 was up-regulated in intrinsic cells in response to the 6-OHDA-mediated loss of FA1/dlk1-expressing SNc dopaminergic neurons and/or due to the stab wound. Our findings hint to a significant role of FA1/dlk1 in the SNc during early postnatal development. The differential expression of FA1/dlk1 in the SNc and the striatum of dopamine-depleted rats could indicate a potential involvement of FA1/dlk1 in the cellular response to the degenerative processes.     

Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons

Striatal delivery of dopamine (DA) by midbrain substantia nigra pars compacta (SNc) neurons is vital for motor control and its depletion causes the motor symptoms of Parkinson's disease. While membrane potential changes or neuronal activity regulates tyrosine hydroxylase (TH, the rate limiting enzyme in catecholamine synthesis) expression in other catecholaminergic cells, it is not known whether the same occurs in adult SNc neurons. We administered drugs known to alter neuronal activity to mouse SNc DAergic neurons in various experimental preparations and measured changes in their TH expression. In cultured midbrain neurons, blockade of action potentials with 1?μM tetrodotoxin decreased TH expression beginning around 20?h later (as measured in real time by green fluorescent protein (GFP) expression driven off TH promoter activity). By contrast, partial blockade of small-conductance, Ca(2+) -activated potassium channels with 300?nM apamin increased TH mRNA and protein between 12 and 24?h later in slices of adult midbrain. Two-week infusions of 300?nM apamin directly to the adult mouse midbrain in vivo also increased TH expression in SNc neurons, measured immunohistochemically. Paradoxically, the number of TH immunoreactive (TH+) SNc neurons decreased in these animals. Similar in vivo infusions of drugs affecting other ion-channels and receptors (L-type voltage-activated Ca(2+) channels, GABA(A) receptors, high K(+) , DA receptors) also increased or decreased cellular TH immunoreactivity but decreased or increased, respectively, the number of TH+ cells in SNc. We conclude that in adult SNc neurons: (i) TH expression is activity-dependent and begins to change ～20?h following sustained changes in neuronal activity; (ii) ion-channels and receptors mediating cell-autonomous activity or synaptic input are equally potent in altering TH expression; and (iii) activity-dependent changes in TH expression are balanced by opposing changes in the number of TH+ SNc cells.     

SLC35D3 increases autophagic activity in midbrain dopaminergic neurons by enhancing BECN1-ATG14-PIK3C3 complex formation

Searching for new regulators of autophagy involved in selective dopaminergic (DA) neuron loss is a hallmark in the pathogenesis of Parkinson disease (PD). We here report that an endoplasmic reticulum (ER)-associated transmembrane protein SLC35D3 is selectively expressed in subsets of midbrain DA neurons in about 10% TH (tyrosine hydroxylase)-positive neurons in the substantia nigra pars compacta (SNc) and in about 22% TH-positive neurons in the ventral tegmental area (VTA). Loss of SLC35D3 in ros (roswell mutant) mice showed a reduction of 11.9% DA neurons in the SNc and 15.5% DA neuron loss in the VTA with impaired autophagy. We determined that SLC35D3 enhanced the formation of the BECN1-ATG14-PIK3C3 complex to induce autophagy. These results suggest that SLC35D3 is a new regulator of tissue-specific autophagy and plays an important role in the increased autophagic activity required for the survival of subsets of DA neurons.     

Galanin inhibits tyrosine hydroxylase expression in midbrain dopaminergic neurons

Galanin (GAL) inhibits midbrain dopamine (DA) activity in several experimental paradigms, yet the mechanism underlying this inhibition is unclear. We examined the effects of GAL on the expression of tyrosine hydroxylase (TH) in primary cultures of rat embryonic (E14) ventral mesencephalon (VM). One micromolar GAL had no effect on the number of TH-immunoreactive (ir) neurons in VM cultures. However, 1 micro m GAL reduced an approximately 100% increase in TH-ir neurons in 1 mm dibutyryl cAMP (dbcAMP)-treated cultures by approximately 50%. TH-ir neuron number in dbcAMP-treated VM cultures was dose-responsive to GAL and the GAL receptor antagonist M40 blocked GAL effects. Semi-quantitative RT-PCR and quantitative immunoblotting experiments revealed that GAL had no effect on TH mRNA levels in VM cultures but reduced TH protein. VM cultures expressed GALR1, GALR2, and GALR3 receptor mRNA. However, dbcAMP treatment resulted in a specific approximately 200% increase in GALR1 mRNA. GALR1 activity is linked to a pertussis toxin (PTX)-sensitive opening of G protein-gated K+ channels (GIRKs). GAL reduction of TH-ir neuron number in dbcAMP + GAL-treated cultures was sensitive to both PTX and tertiapin, a GIRK inhibitor. GAL inhibition of midbrain DA activity may involve a GALR1- mediated reduction of TH in midbrain dopaminergic neurons.     

Fibroblast growth factor receptor signaling affects development and function of dopamine neurons - inhibition results in a schizophrenia-like syndrome in transgenic mice

Developing and mature midbrain dopamine (DA) neurons express fibroblast growth factor (FGF) receptor-1 (FGFR1). To determine the role of FGFR1 signaling in the development of DA neurons, we generated transgenic mice expressing a dominant negative mutant [FGFR1(TK-)] from the catecholaminergic, neuron-specific tyrosine hydroxylase (TH) gene promoter. In homozygous th(tk-)/th(tk-) mice, significant reductions in the size of TH-immunoreactive neurons were found in the substantia nigra compacta (SNc) and the ventral tegmental area (VTA) at postnatal days 0 and 360. Newborn th(tk-)/th(tk-) mice had a reduced density of DA neurons in both SNc and VTA, and the changes in SNc were maintained into adulthood. The reduced density of DA transporter in the striatum further demonstrated an impaired development of the nigro-striatal DA system. Paradoxically, the th(tk-)/th(tk-) mice had increased levels of DA, homovanilic acid and 3-methoxytyramine in the striatum, indicative of excessive DA transmission. These structural and biochemical changes in DA neurons are similar to those reported in human patients with schizophrenia and, furthermore, these th(tk-)/th(tk-) mice displayed an impaired prepulse inhibition that was reversed by a DA receptor antagonist. Thus, this study establishes a new developmental model for a schizophrenia-like disorder in which the inhibition of FGF signaling leads to alterations in DA neurons and DA-mediated behavior.     

Cytokine-induced conversion of mesencephalic-derived progenitor cells into dopamine neurons

We have previously shown that a combination of the cytokines interleukin (IL)-1, IL-11, leukemia inhibitory factor (LIF), and glial cell line-derived neurotrophic factor (GDNF) can convert rat fetal (E14.5) mesencephalic progenitor cells into tyrosine hydroxylase (TH)-immunoreactive (ir) neurons in vitro. The experiments described here characterize the mesencephalic progenitor cells and their cytokine-induced conversion into dopamine (DA) neurons. For all experiments, we used bromodeoxyuridine (BrdU)-ir cultures of (E14.5) mesencephalic progenitor cells that had been expanded at least 21 days. We first demonstrated that IL-1 induced DA neuron conversion in mesencephalic progenitors, but not in striatal progenitors (P < 0.001). Thus, these cells should be classified as lineage-restricted progenitors, and not omnipotent stem cells. To further characterize cell populations in these cultures, we used monoclonal antibodies against Hu (an early marker for neurons), growth-associated protein (GAP)-43 (a marker for neuronal process extension), TH (a marker for DA neurons), and glial fibrillary acidic protein (GFAP, a marker for astrocytes). We assessed (E14.5) mesencephalic progenitor cell cultures (plated at 125,000 cells/cm2) incubated in the cytokine mixture (described above) or in complete media (CM, negative control). Following 7 days incubation, GFAP-positive cells formed a nearly confluent carpet in both types of cultures. However, numbers of Hu-ir and GAP-43-ir cells in the cytokine-incubated cultures far exceeded those in CM-incubated controls (P = 0.0003, P = 0.0001, respectively), while numbers of TH-ir cells were 58-fold greater in the cytokine-incubated cultures versus CM-incubated controls. The TH phenotype persisted for 7 days following withdrawal of the differentiation media. Numerous double-labeled cells that were BrdU-ir and also TH-ir, or Hu-ir and also TH-ir, were observed in the cytokine-incubated cultures. These data suggest that cytokines "drive" the conversion of progenitor cells into DA neurons.     

Heterogeneous Distribution of Kir3 Potassium Channel Proteins Within Dopaminergic Neurons in the Mesencephalon of the Rat Brain

1. Dopaminergic neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) of the ventral mesencephalon play an important role in the regulation of the parallel basal ganglia loops. 2. We have raised affinity-purified polyclonal rabbit antibodies specific for all four members of the Kir3 family of inwardly rectifying potassium channels (Kir3.1–Kir3.4) to investigate the distribution of the channel proteins in the dopaminergic neurons of the rat mesencephalon at light and electron microscopic level. In addition, immunocytochemical double labeling with tyrosine hydroxylase (TH), a marker of dopaminergic neurons, were performed. 3. All Kir3 channels were present in this region. However, the individual proteins showed differential cellular and subcellular distributions. 4. Kir3.1 immunoreactivity was found in SNc fibers and some neurons of the substantia nigra pars reticulata (SNr). Few Kir3.3-positive neurons were found in the SNc. However, a strong Kir3.3 signal was identified in the SNr neuropil. Weak Kir3.4 staining was detected in neuronal somata as well as in dendritic fibers of both parts of the SN. 5. In the VTA, Kir3.1, Kir3.3, and Kir3.4 showed only weak staining of neuropil structures. The distribution of the Kir3.2 channel protein was especially striking with strong labeling in the SNc and in the lateral but not central VTA. 6. Our results suggest that the heterogeneously distributed Kir3.2 channel proteins could help to discriminate the dopaminergic neurons of VTA and SNc.     

The relationships between neurons containing dopamine and nitric oxide synthase in the ventral tegmental area

Ventral tegmental area (VTA) is a heterogeneous group of dopaminergic cells which contains interfascicular (IF), parabrachial (PBP) and rostral linear (RLi) nuclei. Neurons of this area are involved in the regulation of motor and motivational aspects of behavior and reveal high neuronal plasticity. Among many various neurotransmitters and neuromodulators, nitric oxide (NO) is localized in this region. In the present study, we investigated morphology and distribution of nitric oxide synthase (NOS)-positive neurons in VTA and their colocalization with dopaminergic neurons. The study was performed on six adult Wistar rats. After perfusional fixation, the brains were cut, immunostained for tyrosine hydroxylase (TH) and NOS and studied by confocal laser microscopy. In each of the three studied nuclei of VTA we investigated three different neuronal populations. Numerous TH-immunoreactive (TH-ir) and NOS-immunoreactive (NOS-ir) neurons are present in the studied region. Among them, a considerable number showed coexistence of both neurotransmitters. The populations of TH-ir and NOS-ir neurons interact with each other as manifested by the presence of NOS-ir endings on TH-ir neurons and vice versa. Taking the above into account, it may be suspected that NO is involved in the modulation of dopaminergic transmission.     

Long term osmotic stress exposure outcomes on rat dopaminergic innervations and the associated motor behavior

The osmotic stress is a powerful stimulus that elicits profound peripheral and central disturbances. In the mammalian brain, osmotic stress has been associated to several glial and neuronal changes. The lack of data regarding the impact on the dopaminergic system and locomotion led us to investigate the effect of prolonged water deprivation in rat on the midbrain dopaminergic system and locomotor performance by dehydrating rats for one and two weeks. Locomotor activity and tyrosine hydroxylase (TH) expression were assessed using the open field test and immunohistochemistry respectively. Water deprivation was accompanied with a significant increment of TH expression within substantia nigra compacta (SNc) and ventral tegmental area (VTA) gradually as the duration of dehydration increases. While locomotor activity showed the inverse tendency manifested by a drop of crossed boxes number following one and two weeks of water deprivation. Our data suggest a substantial implication of midbrain dopaminergic system in the central response to the osmotic stimuli accompanied with locomotor deficiencies.     

The hTH-GFP Reporter Rat Model for the Study of Parkinson's Disease

Parkinson disease (PD) is the second leading neurodegenerative disease in the US. As there is no known cause or cure for PD, researchers continue to investigate disease mechanisms and potential new therapies in cell culture and in animal models of PD. In PD, one of the most profoundly affected neuronal populations is the tyrosine hydroxylase (TH)-expressing dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). These DA-producing neurons undergo degeneration while neighboring DA-producing cells of the ventral tegmental area (VTA) are largely spared. To aid in these studies, The Michael J. Fox Foundation (MJFF) partnered with Thomas Jefferson University and Taconic Inc. to generate new transgenic rat lines carrying the human TH gene promoter driving EGFP using a 11 kb construct used previously to create a hTH-GFP mouse reporter line. Of the five rat founder lines that were generated, three exhibited high level specific GFP fluorescence in DA brain structures (ie. SN, VTA, striatum, olfactory bulb, hypothalamus). As with the hTH-GFP mouse, none of the rat lines exhibit reporter expression in adrenergic structures like the adrenal gland. Line 12141, with its high levels of GFP in adult DA brain structures and minimal ectopic GFP expression in non-DA structures, was characterized in detail. We show here that this line allows for anatomical visualization and microdissection of the rat midbrain into SNpc and/or VTA, enabling detailed analysis of midbrain DA neurons and axonal projections after toxin treatment in vivo. Moreover, we further show that embryonic SNpc and/or VTA neurons, enriched by microdissection or FACS, can be used in culture or transplant studies of PD. Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson''s disease research.     

Reliability in the identification of midbrain dopamine neurons

Brain regions typically contain intermixed subpopulations of neurons with different connectivity and neurotransmitters. This complicates identification of neuronal phenotypes in electrophysiological experiments without using direct detection of unique molecular markers. A prime example of this difficulty is the identification of dopamine (DA) neurons in the midbrain ventral tegmental area (VTA). Although immunocytochemistry (ICC) against tyrosine hydroxylase (TH) is widely used to identify DA neurons, a high false negative rate for TH ICC following ex vivo electrophysiology experiments was recently reported, calling into question the validity of comparing DA and non-DA VTA neurons based on post-hoc ICC. However, in whole cell recordings from randomly selected rat VTA neurons we have found that TH labeling is consistently detected in ∼55% of neurons even after long recording durations (range: 2.5–150 min). This is consistent with our prior anatomical finding that 55% of VTA neurons are TH(+). To directly estimate a false negative rate for our ICC method we recorded VTA neurons from mice in which EGFP production is driven by the TH promoter. All 12 EGFP(+) neurons recorded with a K-gluconate internal solution (as used in our rat recordings) were strongly labeled by TH ICC (recording duration 16.6±1.8 min). However, using recording electrodes with an internal solution with high Cl⁻ concentration reduced the intensity of TH co-labeling, in some cases to background (recording duration 16.7±0.9 min; n = 10). Thus TH is a highly reliable molecular marker for DA neurons in VTA patch clamp recordings provided compatible microelectrode solutions are used.     

Delta-like 1 participates in the specification of ventral midbrain progenitor derived dopaminergic neurons

Delta-like 1 (Dlk1), a member of the Delta/Notch protein family, is expressed in the mouse ventral midbrain (VM) as early as embryonic day 11.5 (E11.5) followed by exclusive expression in tyrosine 3-monooxygenase (TH) positive neurons from E12.5 onwards. To further elucidate the yet unknown function of Dlk1 in VM neuron development, we investigated the effect of soluble Dlk1 protein as well as the intrinsic Dlk1 function in the course of VM progenitor expansion and dopaminergic (DA) neuron differentiation in vitro . Dlk1 treatment during expansion increased DA progenitor proliferation and the proportion of NR4A2+ neurons expressing TH after differentiation, whereas Dlk1 treatment during the course of DA precursor differentiation did not alter TH+ neuron counts. In contrast, silencing of endogenously expressed Dlk1 prior to DA precursor differentiation partially prevented the expression of DA neuron markers, which was not accompanied with alteration of overall or local proliferation. Due to the latter finding in combination with the absence of Dlk1 negative DA neurons in differentiated cultures, we suggest that Dlk1 expression might have a permissive effect on DA neuron differentiation in vitro . The study presented here is the first publication identifying Dlk1 effects on ventral midbrain-derived DA precursor differentiation.     

Estrogen regulates tyrosine hydroxylase expression in the neonate mouse midbrain

Estrogen plays an important role during differentiation of midbrain dopaminergic neurons. This is indicated by the presence of estrogen receptors and the transient expression of the estrogen-forming enzyme aromatase within the dopaminergic cell groups. We have previously shown that estrogen regulates the plasticity of dopamine cells through the stimulation of neurite growth/arborization. In this study, we have analyzed the capability of estrogen to influence the activity of developing mouse dopamine neurons. The expression of tyrosine hydroxylase (TH) was assessed by competitive RT-PCR and Western blotting. The developmental expression of TH in the ventral midbrain was studied from embryonic day 15 until postnatal day 15 and revealed highest TH levels early postnatally. This profile coincides with the transient aromatase expression in this brain area. Using cultured midbrain cells, we found that estrogen increased TH mRNA/protein levels. The application of the estrogen receptor antagonist ICI 182,780 resulted in a complete inhibition of estrogen effects. To verify these data in vivo, fetuses were exposed in utero from E15 until birth to the aromatase inhibitor CGS 16949A or to CGS supplemented with estrogen. CGS caused a robust reduction in TH mRNA/protein levels in the midbrain, which could be restored by estrogen substitution. Taken together, our data strongly suggest that estrogen controls dopamine synthesis in the developing nigrostriatal dopaminergic system and support the concept that estrogen is implicated in the regulation of ontogenetic steps but also in the function of midbrain dopamine neurons.