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Objective: [¹⁸F]FE-PE2I (FE-PE2I) is a new radiotracer for dopamine transporter (DAT) imaging with PET. The aim of this study was to evaluate the visual interpretation of FE-PE2I images for the diagnosis of idiopathic Parkinsonian syndrome (IPS). The inter-rater variability, sensitivity, specificity, and diagnostic accuracy for visual interpretation of striatal FE-PE2I compared to [¹²³I]FP-CIT (FP-CIT) single-photon emission computed tomography (SPECT) was evaluated.

Methods: Thirty patients with newly onset parkinsonism and 32 healthy controls with both an FE-PE2I and FP-CIT were included in the study. Four patients had normal DAT imaging, of which three did not fulfil the IPS criteria at the clinical reassessment after 2 years. Six raters evaluated the DAT images blinded to the clinical diagnosis, interpreting the image as being ‘normal’ or ‘pathological’, and assessed the degree of DAT-reduction in the caudate and putamen. The inter-rater agreement was assessed with intra-class correlation and Cronbach’s α. For calculation of sensitivity and specificity, DAT images were defined as correctly classified if categorized as normal or pathological by ≥4/6 raters.

Results: The overall agreement in visual evaluation of the FE-PE2I- and FP-CIT images was high for the IPS patients (α = 0.960 and 0.898, respectively), but lower in healthy controls (FE-PE2I: α = 0.693, FP-CIT: α = 0.657). Visual interpretation gave high sensitivity (both 0.96) but lower specificity (FE-PE2I: 0.86, FP-CIT: 0.63) with an accuracy of 90% for FE-PE2I and 77% for FP-CIT.

Conclusion: Visual evaluation of FE-PE2I PET imaging demonstrates high reliability and diagnostic accuracy for IPS.

Glial cell line-derived neurotrophic factor (GDNF) is potent for survival and promotion of nerve fibers from midbrain dopamine neurons. It is also known to exert different effects on specific subpopulations of dopamine neurons. In organotypic tissue cultures, dopamine neurons form two diverse nerve fiber growth patterns, targeting the striatum differently. The aim of this study was to investigate the effect of GDNF on the formation of dopamine nerve fibers. Organotypic tissue cultures of ventral mesencephalon of gdnf gene-deleted mice were studied. The results revealed that dopamine neurons survive in the absence of GDNF. Tyrosine hydroxylase immunoreactivity demonstrated, in gdnf knockout and wildtype cultures, nerve fiber formation with two separate morphologies occurring either in the absence or the presence of astrocytes. The outgrowth that occurred in the absence of astrocytes was unaffected by gdnf deletion, whereas nerve fibers guided by the presence of astrocytes were affected in that they reached significantly shorter distances from the gdnf gene-deleted tissue slice, compared to those measured in wildtype cultures. Treatment with GDNF reversed this effect and increased nerve fiber density independent of genotype. Furthermore, migration of astrocytes reached significantly shorter distances from the tissue slice in GDNF knockout compared to wildtype cultures. Exogenous GDNF increased astrocytic migration in gdnf gene-deleted tissue cultures, comparable to lengths observed in wildtype tissue cultures. In conclusion, cultured midbrain dopamine neurons survive in the absence of GDNF, and the addition of GDNF improved dopamine nerve fiber formation - possibly as an indirect effect of astrocytic stimulation.

Tyrosine hydroxylase-positive nerve fiber formation occurs in two diverse morphological patterns in rat fetal ventral mesencephalic slice cultures; one is non-glial-associated and the other is glial-associated. The aim of this study was to characterize the non-glial-associated nerve fibers and its relation to migration of astrocytes. Organotypic slice cultures were prepared from embryonic days 12, 14, and 18 rat fetuses and maintained for 5, 7 or 14 days in vitro. Inhibition of cell proliferation using cytosine beta-D-arabinofuranoside was conducted in embryonic day 14 ventral mesencephalic cultures. The treatment impaired astrocytic migration at 7 and 14 days in vitro. The reduced migration of astrocytes exerted a negative effect on the glial-associated tyrosine hydroxylase-positive nerve fibers, reducing the outgrowth from the tissue slice. The non-glial-associated outgrowth was, however, positively affected by reduced astrocytic migration, reaching distances around 3mm in 2 weeks, and remained for longer time in culture. Co-cultures of fetal ventral mesencephalon and frontal cortex revealed the cortex as a target for the non-glial-associated tyrosine hydroxylase-positive outgrowth. The age of the fetal tissue at plating affected the astrocytes such that older tissue increased the length of astrocyte migration. Younger tissue at plating promoted the presence of non-glial-associated outgrowth and long radial-glia-like processes, while older tissue promoted migration of neurons instead of formation of nerve fiber network. In conclusion, inhibition of astrocytic proliferation promotes the persistence of long-distance growing tyrosine hydroxylase-positive nerve fibers in ventral mesencephalic slices cultures. Furthermore, the long-distance growing nerve fibers target the frontal cortex and are absent in cultures derived from older tissue.

In Parkinson's disease, degeneration of substantia nigra dopaminergic neurons is accompanied by damage on other neuronal systems. A severe denervation is for example seen in the locus coerulean noradrenergic system. Little is known about the relation between noradrenergic and dopaminergic degeneration, and the effects of noradrenergic denervation on the function of the dopaminergic neurons of substantia nigra are not fully understood. In this study, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) was injected in rats, whereafter behavior, striatal KCl-evoked dopamine and glutamate releases, and immunohistochemistry were monitored at 3 days, 3 months, and 6 months. Quantification of dopamine-beta-hydroxylase-immunoreactive nerve fiber density in the cortex revealed a tendency towards nerve fiber regeneration at 6 months. To sustain a stable noradrenergic denervation throughout the experimental timeline, the animals in the 6-month time point received an additional DSP4 injection (2 months after the first injection). Behavioral examinations utilizing rotarod revealed that DSP4 reduced the time spent on the rotarod at 3 but not at 6 months. KCl-evoked dopamine release was significantly increased at 3 days and 3 months, while the concentrations were normalized at 6 months. DSP4 treatment prolonged both time for onset and reuptake of dopamine release over time. The dopamine degeneration was confirmed by unbiased stereology, demonstrating significant loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra. Furthermore, striatal glutamate release was decreased after DSP4. In regards of neuroinflammation, reactive microglia were found over the substantia nigra after DSP4 treatment. In conclusion, long-term noradrenergic denervation reduces the number of dopaminergic neurons in the substantia nigra and affects the functionality of the nigrostriatal system. Thus, locus coeruleus is important for maintenance of nigral dopaminergic neurons.

Rat fetal ventral mesencephalic organotypic cultures have demonstrated two morphologically different dopamine nerve fiber growth patterns, in which the initial nerve fibers are formed in the absence of astrocytes and the second wave is guided by astrocytes. In this study, the presence of subpopulations of dopamine neurons, other neuronal populations, and glial cells was determined. We used "roller-drum" organotypic cultures, and the results revealed that beta-tubulin-positive/tyrosine hydroxylase (TH)-negative nerve fibers were present as early as 1 day in vitro (DIV). A similar growth pattern produced by TH-positive neurons was present from 2 DIV. These neurites grew to reach distances over 4 mm and over time appeared to be degenerating. Thin, vimentin-positive processes were found among these nerve fibers. As the first growth was retracted, a second outgrowth was initiated and formed on migrating astrocytes. TH- and aldehyde dehydrogenase-1 (ALDH1)-positive nerve fibers formed both the nonglia-associated and the glia-associated outgrowth. In cultures with membrane inserts, only the glia-associated outgrowth was found. Vimentin-positive cells preceded migration of NG2-positive oligodendrocytes and Iba-1-positive microglia. Oligodendrocytes appeared not to be involved in guiding neuritic growth, but microglia was absent over areas dense with TH-positive neurons. In conclusion, in "roller-drum" cultures, nerve fibers are generally formed in two sequences. The early-formed nerve fibers grow in the presence of thin, vimentin-positive processes. The second nerve fiber outgrowth is formed on astroglia, with no correlation to the presence of oligodendrocytes or microglia. ALDH1-positive nerve fibers, presumably derived from A9 dopamine neurons, participate in formation of both sequences of outgrowth.

Objectives: Current treatments for Parkinson’s disease using pharmacological approaches alleviate motor symptoms but do not prevent neuronal loss or dysregulation of dopamine neurotransmission. In this article, we have explored the molecular mechanisms underlying the neuroprotective effect of the antioxidant N-acetylcysteine (NAC) on the damaged dopamine system.

Methods: SH-SY5Y cells were differentiated towards a dopaminergic phenotype and exposed to 6-hydroxydopamine (6-OHDA) to establish an in vitro model of Parkinson’s disease. We examined the potential of NAC to restore the pathological effects of 6-OHDA on cell survival, dopamine synthesis as well as on key proteins regulating dopamine metabolism. Specifically, we evaluated gene- and protein expression of tyrosine hydroxylase (TH), vesicle monoamine transporter 2 (VMAT2), and α-synuclein, by using qPCR and Western blot techniques. Moreover, we quantified the effect of NAC on total dopamine levels using a dopamine ELISA assay.

Results: Our results indicate that NAC has a neuroprotective role in SH-SY5Y cells exposed to 6-OHDA by maintaining cell proliferation and decreasing apoptosis. Additionally, we demonstrated that NAC treatment increases dopamine release and protects SH-SY5Y cells against 6-OHDA dysregulations on the proteins TH, VMAT2, and α-synuclein.

Conclusions: Our findings contribute to the validation of compounds capable to restore dopamine homeostasis and shed light on the metabolic pathways that could be targeted to normalize dopamine turnover. Furthermore, our results highlight the effectiveness of the antioxidant NAC in the prevention of dopaminergic neurodegeneration in the present model.

Chronic implantation of neurotransmitter measuring devices is essential for awake, behavioral studies occurring over multiple days. Little is known regarding the effects of long term implantation on surrounding brain parenchyma and the resulting alterations in the functional properties of this tissue. We examined the extent of tissue damage produced by chronic implantation of either ceramic microelectrode arrays (MEAs) or microdialysis probes. Histological studies were carried out on fixed tissues using stains for neurons (cresyl violet), astrocytes (GFAP), microglia (Iba1), glutamatergic nerve fibers (VGLUT1), and the blood-brain barrier (SMI-71). Nissl staining showed pronounced tissue body loss with microdialysis implants compared to MEAs. The MEAs produced mild gliosis extending 50-100 microm from the tracks, with a significant change in the affected areas starting at 3 days. By contrast, the microdialysis probes produced gliosis extending 200-300 microm from the track, which was significant at 3 and 7 days. Markers for microglia and glutamatergic fibers supported that the MEAs produce minimal damage with significant changes occurring only at 3 and 7 days that return to control levels by 1 month. SMI-71 staining supported the integrity of the blood-brain barrier out to 1 week for both the microdialysis probes and the MEAs. This data support that the ceramic MEA's small size and biocompatibility are necessary to accurately measure neurotransmitter levels in the intact brain. The minimal invasiveness of the MEAs reduce tissue loss, allowing for long term (>6 month) electrochemical and electrophysiological monitoring of brain activity.

Chondroitin sulfate proteoglycans (CSPGs) promote nerve growth during development, and inhibit axonal growth in the adult CNS after injury. Chondroitinase ABC (ChABC) and methyl-umbelliferyl-β-d-xyloside (β-xyloside), two enzymes that degrade CSPGs, promote regeneration after injury, however, they demonstrate opposing results in tissue culture. To elucidate the effect of the two enzymes, organotypic tissue cultures, treated with ChABC or β-xyloside, were employed to monitor nerve fiber outgrowth and astrocytic migration. Rat ventral mesencephalon (VM) and spinal cord (SC) from embryonic day (E) 14 and E18 were treated early, from the plating day for 14 days in vitro, or late where treatment was initiated after being cultured for 14 days. In the early treatment of E14 VM and SC cultures, astrocytic migration and nerve fiber outgrowth were hampered using both enzymes. Early treatment of E18 cultures reduced the astrocytic migration, while nerve growth was promoted by β-xyloside, but not by ChABC. In the late treated cultures of both E14 and E18 cultures, no differences in distances that astrocytes migrated or nerve fiber growth were observed. However, in β-xyloside-treated cultures, the confluency of astrocytic monolayer was disrupted. In E18 cultures both early and late treatments, neuronal migration was present in control cultures, which was preserved using ChABC but not β-xyloside. In conclusion, ChABC and β-xyloside had similar effects and hampered nerve fiber growth and astrocytic migration in E14 cultures. In E18 cultures nerve fiber growth was stimulated and neuronal migration was hampered after β-xyloside treatment while ChABC treatment did not exert these effects.