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Enhancers function as DNA logic gates and may control specialized functions of billions of neurons. Here we show a tailored program of noncoding genome elements active in situ in physiologically distinct dopamine neurons of the human brain. We found 71,022 transcribed noncoding elements, many of which were consistent with active enhancers and with regulatory mechanisms in zebrafish and mouse brains. Genetic variants associated with schizophrenia, addiction, and Parkinson’s dis- ease were enriched in these elements. Expression quantitative trait locus analysis revealed that Parkinson’s disease-associated variants on chromosome 17q21 cis-regulate the expression of an enhancer RNA in dopamine neurons. This study shows that enhancers in dopamine neurons link genetic variation to neuropsychiatric traits.

Background The fimbria/fornix fiber system is an essential part of the hippocampal-VTA loop, and therefore activities that are propagated through this fiber system control the activity of the mesolimbic dopamine system. Objectives/hypothesis We hypothesized that stimulation of the fimbria/fornix with an increasing number of electrical pulses would cause increasing activity of the mesolimbic dopamine system, which coincides with concurrent changes in neuronal activities in target regions of the mesolimbic dopaminergic system. Methods Right fimbria/fornix fibers were electrically stimulated with different pulse protocols. Stimulus-induced changes in neuronal activities were visualized with BOLD-fMRI, whereas stimulus-induced release of dopamine, as measured for the activity of the mesolimbic dopamine system, was determined in the nucleus accumbens with in vivo fast-scan cyclic voltammetry. Results Dependent on the protocol, electrical fimbria/fornix stimulation caused BOLD responses in various targets of the mesolimbic dopamine system. Stimulation in the low theta frequency range (5 Hz) triggered significant BOLD responses mainly in the hippocampal formation, infralimbic cortex, and septum. Stimulation in the beta frequency range (20 Hz) caused additional activation in the medial prefrontal cortex (mPFC), nucleus accumbens, striatum, and VTA. Stimulation in the high-gamma frequency range (100 Hz) caused further activation in the hippocampus proper and mPFC. The strong activation in the mPFC during 100 Hz stimulations depended not only on the number of pulses but also on the frequency. Thus, short bursts of 5 or 20 high-frequency pulses caused stronger activation in the mPFC than continuous 5 or 20 Hz pulses. In contrast, high-frequency burst fimbria/fornix stimulation did not further activate the mesolimbic dopamine system when compared to continuous 5 or 20 Hz pulse stimulation. Conclusions There exists a frequency-dependent dissociation between BOLD responses and activation of the dopaminergic system. Low frequencies were more efficient to activate the mesolimbic dopamine system, whereas high frequencies were more efficient to trigger BOLD responses in target regions of the mesolimbic dopamine system, particularly the mPFC.

HIGHLIGHTS: eOPN3 is a mosquito-derived rhodopsin that inhibits neurotransmission in neurons. Activation of eOPN3 activates the G(i/o) pathway and reduces Ca(2+) channel activity; eOPN3 can suppress neurotransmission in a variety of cell types in vitro and in vivo. Activation of eOPN3 in nigrostriatal dopamine axons modulates locomotor behavior. SUMMARY: Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity. However, existing inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time. Here, we show that a targeting-enhanced mosquito homolog of the vertebrate encephalopsin (eOPN3) can effectively suppress synaptic transmission through the G(i/o) signaling pathway. Brief illumination of presynaptic terminals expressing eOPN3 triggers a lasting suppression of synaptic output that recovers spontaneously within minutes in vitro and in vivo. In freely moving mice, eOPN3-mediated suppression of dopaminergic nigrostriatal afferents induces a reversible ipsiversive rotational bias. We conclude that eOPN3 can be used to selectively suppress neurotransmitter release at presynaptic terminals with high spatiotemporal precision, opening new avenues for functional interrogation of long-range neuronal circuits in vivo.

Mapping the activity of the human mesolimbic dopamine system by BOLD-fMRI is a tempting approach to non-invasively study the action of the brain reward system during different experimental conditions. However, the contribution of dopamine release to the BOLD signal is disputed. To assign the actual contribution of dopaminergic and non-dopaminergic VTA neurons to the formation of BOLD responses in target regions of the mesolimbic system, we used two optogenetic approaches in rats. We either activated VTA dopaminergic neurons selectively, or dopaminergic and mainly glutamatergic projecting neurons together. We further used electrical stimulation to non-selectively activate neurons in the VTA. All three stimulation conditions effectively activated the mesolimbic dopaminergic system and triggered dopamine releases into the NAcc as measured by in vivo fast-scan cyclic voltammetry. Furthermore, both optogenetic stimulation paradigms led to indistinguishable self-stimulation behavior. In contrast to these similarities, however, the BOLD response pattern differed greatly between groups. In general, BOLD responses were weaker and sparser with increasing stimulation specificity for dopaminergic neurons. In addition, repetitive stimulation of the VTA caused a progressive decoupling of dopamine release and BOLD signal strength, and dopamine receptor antagonists were unable to block the BOLD signal elicited by VTA stimulation. To exclude that the sedation during fMRI is the cause of minimal mesolimbic BOLD in response to specific dopaminergic stimulation, we repeated our experiments using CBF SPECT in awake animals. Again, we found activations only for less-specific stimulation. Based on these results we conclude that canonical BOLD responses in the reward system represent mainly the activity of non-dopaminergic neurons. Thus, the minor effects of projecting dopaminergic neurons are concealed by non-dopaminergic activity, a finding which highlights the importance of a careful interpretation of reward-related human fMRI data.

We have developed a good manufacturing practice for long-term cultivation of fetal human midbrain-derived neural progenitor cells. The generation of human dopaminergic neurons may serve as a tool of either restorative cell therapies or cellular models, particularly as a reference for phenotyping region-specific human neural stem cell lines such as human embryonic stem cells and human inducible pluripotent stem cells. We cultivated 3 different midbrain neural progenitor lines at 10, 12, and 14 weeks of gestation for more than a year and characterized them in great detail, as well as in comparison with Lund mesencephalic cells. The whole cultivation process of tissue preparation, cultivation, and cryopreservation was developed using strict serum-free conditions and standardized operating protocols under clean-room conditions. Long-term-cultivated midbrain-derived neural progenitor cells retained stemness, midbrain fate specificity, and floorplate markers. The potential to differentiate into authentic A9-specific dopaminergic neurons was markedly elevated after prolonged expansion, resulting in large quantities of functional dopaminergic neurons without genetic modification. In restorative cell therapeutic approaches, midbrain-derived neural progenitor cells reversed impaired motor function in rodents, survived well, and did not exhibit tumor formation in immunodeficient nude mice in the short or long term (8 and 30 weeks, respectively). We conclude that midbrain-derived neural progenitor cells are a promising source for human dopaminergic neurons and suitable for long-term expansion under good manufacturing practice, thus opening the avenue for restorative clinical applications or robust cellular models such as high-content or high-throughput screening.

Olfactory dysfunction, often preceding the cardinal motor symptoms, such as bradykinesia, rigidity, tremor at rest and postural instability, is frequently reported in Parkinson's disease. This symptom appears to be related to an increased number of dopamine neurons in the periglomerular layer of the olfactory bulb. In animal models of Parkinson's disease, adult neural progenitor cells migrating from the subventricular zone of the lateral ventricle to the olfactory bulb are evidently altered in their survival and progeny. The modulation of neural progenitor cells contributing to the number of dopamine neurons in the periglomerular layer, however, is still poorly understood. In this study, we have investigated the survival and neuronal differentiation of newly generated cells in the olfactory bulb, following treatment with the dopamine precursor l-DOPA and the monoamine oxidase-B inhibitor selegiline in a unilateral, intranigral 6-hydroxydopamine lesion model in mice. Our data show that the number of neural progenitor cells in the subventricular zone is decreased after an intranigral 6-hydroxydopamine lesion, while there is no difference from control in lesioned mice with selegiline or l-DOPA treatment. Selegiline is able to normalize the number of dopamine neurons in the periglomerular layer, while l-DOPA treatment sustains the increased number observed in 6-hydroxydopamine lesioned animals. We conclude that there is a distinct modulation of newly generated dopamine neurons of the olfactory bulb after l-DOPA and selegiline treatment. The differential effects of the two drugs might also play a role in olfactory dysfunction in Parkinson's disease patients.

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder with a prevalence of about 1% in a population older than 70 years and about 3% in individuals older than 80 years (Strickland and Bertoni 2004). Pathological hallmarks include, but are not confined to, the preferential loss of dopaminergic (DAergic) neurons within the substantia nigra pars compacta and the presence of intracytoplasmic inclusions containing α-synuclein and ubiquitin, so-called Lewy bodies (Braak et al. 2003). Next to the generation of free radicals in the course of dopamine metabolism, the increased presence of iron in the midbrain of PD patients has been assumed as a contributing pathogenic factor (Berg and Hochstrasser 2006). Iron participates in metabolic processes by undergoing oxidation–reduction reactions, a common property among transition metals, which allows this metal to undergo interconversion between the divalent cationic or ferrous (Fe2+), and trivalent cationic or ferric (Fe3+) states. These electron exchange processes can also lead to significant oxidative damage via free radical production within the brain when excess iron is present. In DAergic neurons during the dopamine catabolism, hydrogen peroxide is produced that in presence of iron favors Fenton reactions and oxidative stress [reviewed in (Papanikolaou and Pantopoulos 2005)]. Other transition metals such as Mn or Zn have been suggested to contribute to the pathogenesis of PD. For example, exposure of workers to high concentrations of manganese is an established risk factor for the development of a Parkinsonian syndrome, which clinically greatly mimics idiopathic Parkinson's disease [reviewed in (Guilarte 2010)]. Similar symptoms have been observed in psychostimulant drug abusers after repetitive intravenous injection of manganese-containing substances (Sikk 2011). Excessive levels of brain manganese have been linked to the loss of dopamine in the striatum, death of non-DAergic neurons in the globus pallidus, and damage of other neuronal pathways such as glutamatergic and GABAergic projections, all of which contribute to altered behavior, motor dysfunction, and cognition deficit (Erikson 2002). Manganese elevates intracellular H2O2 and related peroxides (HaMai et al. 2001) and reduces tyrosine hydroxylase activity and intracellular antioxidant levels (GSH, thiols, catalase) in DAergic neurons (Migheli 1999). Iron and manganese are known to pass across the blood–brain barrier [reviewed in (Yokel 2009; Moos 2007)]. Because of their chemical similarity, both metals share and compete for transport proteins in organisms ranging from bacteria to mammals. As such, during conditions of low iron, abnormal manganese accumulation occurs. Conversely, when manganese concentrations are altered, the homeostasis and deposition of iron and other transition metals are disrupted. However, the knowledge on the subcellular allocation of trace elements upon exposure to manganese or iron remains incomplete. Indirect methods, like radioactive 54Mn tracing after cellular extraction, are able to quantify manganese in cell extracts, but provide only limited spatial information (Kalia et al. 2008). The principle of X-ray fluorescence (XRF) is based on the irradiation of sample atoms with X-rays with energy sufficient to eject an electron from one of the atom's inner shells. The relaxation process is accompanied by the emission of a fluorescence photon. The energy of the emitted X-rays is characteristic of the excited element enabling the identification of the composition of the atoms constitutive of the sample. Beside element composition, X-ray spectromicroscopy performed using an X-ray nanoprobe beam is a unique method to determine the oxidation state of elements in cells and tissues (Qin 2011). X-rays can penetrate thick cells and tissues, eliminating the need of invasive preparation and sectioning of the specimen. Several studies have thus recently demonstrated the use of X-rays for trace element mapping of different cell types (Ortega 2007; Bacquart 2007; Ducic 2011). This study was designed to investigate the subcellular distribution of manganese and iron in primary midbrain neurons and to compare this distribution in the DAergic and non-DAergic subpopulations by X-ray fluorescence microscopy. Previous studies have been limited to cell lines, post-mortem tissue, or did not take into account changes in the oxidative state during sample processing (Bacquart 2007; Szczerbowska-Boruchowska 2007). In this study, we visualized the transition metal distribution upon exposure in primary midbrain neurons and used the cryo-preserving (so-called cryo-embedding) technique to preserve oxidative states of the metal ions, which, to the best of our knowledge, is the first description exploiting primary midbrain neurons culture model with X-ray imaging techniques.