Nkx2.9 Contributes to Mid-Hindbrain Patterning by Regulation of mdDA Neuronal Cell-Fate and Repression of a Hindbrain-Specific Cell-Fate.

Nkx2.9 is a member of the NK homeobox family and resembles Nkx2.2 both in homology and expression pattern. However, while Nkx2.2 is required for development of serotonergic neurons, the role of Nkx2.9 in the mid-hindbrain region is still ill-defined. We have previously shown that Nkx2.9 expression is downregulated upon loss of En1 during development. Here, we determined whether mdDA neurons require Nkx2.9 during their development. We show that Nkx2.9 is strongly expressed in the IsO and in the VZ and SVZ of the embryonic midbrain, and the majority of mdDA neurons expressed Nkx2.9 during their development. Although the expression of Dat and Cck are slightly affected during development, the overall development and cytoarchitecture of TH-expressing neurons is not affected in the adult Nkx2.9-depleted midbrain. Transcriptome analysis at E14.5 indicated that genes involved in mid- and hindbrain development are affected by Nkx2.9-ablation, such as Wnt8b and Tph2. Although the expression of Tph2 extends more rostral into the isthmic area in the Nkx2.9 mutants, the establishment of the IsO is not affected. Taken together, these data point to a minor role for Nkx2.9 in mid-hindbrain patterning by repressing a hindbrain-specific cell-fate in the IsO and by subtle regulation of mdDA neuronal subset specification.

Context-dependent representations of movement in Drosophila dopaminergic reinforcement pathways.

Dopamine plays a central role in motivating and modifying behavior, serving to invigorate current behavioral performance and guide future actions through learning. Here we examine how this single neuromodulator can contribute to such diverse forms of behavioral modulation. By recording from the dopaminergic reinforcement pathways of the Drosophila mushroom body during active odor navigation, we reveal how their ongoing motor-associated activity relates to goal-directed behavior. We found that dopaminergic neurons correlate with different behavioral variables depending on the specific navigational strategy of an animal, such that the activity of these neurons preferentially reflects the actions most relevant to odor pursuit. Furthermore, we show that these motor correlates are translated to ongoing dopamine release, and acutely perturbing dopaminergic signaling alters the strength of odor tracking. Context-dependent representations of movement and reinforcement cues are thus multiplexed within the mushroom body dopaminergic pathways, enabling them to coordinately influence both ongoing and future behavior.

Pain induces adaptations in ventral tegmental area dopamine neurons to drive anhedonia-like behavior.

The persistence of negative affect in pain leads to co-morbid symptoms such as anhedonia and depression-major health issues in the United States. The neuronal circuitry and contribution of specific cellular populations underlying these behavioral adaptations remains unknown. A common characteristic of negative affect is a decrease in motivation to initiate and complete goal-directed behavior, known as anhedonia. We report that in rodents, inflammatory pain decreased the activity of ventral tegmental area (VTA) dopamine (DA) neurons, which are critical mediators of motivational states. Pain increased rostromedial tegmental nucleus inhibitory tone onto VTA DA neurons, making them less excitable. Furthermore, the decreased activity of DA neurons was associated with reduced motivation for natural rewards, consistent with anhedonia-like behavior. Selective activation of VTA DA neurons was sufficient to restore baseline motivation and hedonic responses to natural rewards. These findings reveal pain-induced adaptations within VTA DA neurons that underlie anhedonia-like behavior.

Proteomic characterization of secretory granules in dopaminergic neurons indicates chromogranin/secretogranin-mediated protein processing impairment in Parkinson's disease.

Parkinson's disease (PD) is an aging disorder related to vesicle transport dysfunctions and neurotransmitter secretion. Secretory granules (SGs) are large dense-core vesicles for the biosynthesis of neuropeptides and hormones. At present, the involvement of SGs impairment in PD remains unclear. In the current study, we found that the number of SGs in tyrosine hydroxylase-positive neurons and the marker proteins secretogranin III (Scg3) significantly decreased in the substantia nigra and striatum regions of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) exposed mice. Proteomic study of SGs purified from the dopaminergic SH-sy5Y cells under 1-methyl-4-phenylpyridinium (MPP+) treatments (ProteomeXchange PXD023937) identified 536 significantly differentially expressed proteins. The result indicated that disabled lysosome and peroxisome, lipid and energy metabolism disorders are three characteristic features. Protein-protein interaction analysis of 56 secretory proteins and 140 secreted proteins suggested that the peptide processing mediated by chromogranin/secretogranin in SGs was remarkably compromised, accompanied by decreased candidate proteins and peptides neurosecretory protein (VGF), neuropeptide Y, apolipoprotein E, and an increased level of proenkephalin. The current study provided an extensive proteinogram of SGs in PD. It is helpful to understand the molecular mechanisms in the disease.

Measuring the iron content of dopaminergic neurons in substantia nigra with MRI relaxometry.

In Parkinson's disease, the depletion of iron-rich dopaminergic neurons in nigrosome 1 of the substantia nigra precedes motor symptoms by two decades. Methods capable of monitoring this neuronal depletion, at an early disease stage, are needed for early diagnosis and treatment monitoring. Magnetic resonance imaging (MRI) is particularly suitable for this task due to its sensitivity to tissue microstructure and in particular, to iron. However, the exact mechanisms of MRI contrast in the substantia nigra are not well understood, hindering the development of powerful biomarkers. In the present report, we illuminate the contrast mechanisms in gradient and spin echo MR images in human nigrosome 1 by combining quantitative 3D iron histology and biophysical modeling with quantitative MRI on post mortem human brain tissue. We show that the dominant contribution to the effective transverse relaxation rate (R2*) in nigrosome 1 originates from iron accumulated in the neuromelanin of dopaminergic neurons. This contribution is appropriately described by a static dephasing approximation of the MRI signal. We demonstrate that the R2* contribution from dopaminergic neurons reflects the product of cell density and cellular iron concentration. These results demonstrate that the in vivo monitoring of neuronal density and iron in nigrosome 1 may be feasible with MRI and provide directions for the development of biomarkers for an early detection of dopaminergic neuron depletion in Parkinson's disease.

Dorsal Raphe Dopamine Neurons Signal Motivational Salience Dependent on Internal State, Expectation, and Behavioral Context.

The ability to recognize motivationally salient events and adaptively respond to them is critical for survival. Here, we tested whether dopamine (DA) neurons in the dorsal raphe nucleus (DRN) contribute to this process in both male and female mice. Population recordings of DRNDA neurons during associative learning tasks showed that their activity dynamically tracks the motivational salience, developing excitation to both reward-paired and shock-paired cues. The DRNDA response to reward-predicting cues was diminished after satiety, suggesting modulation by internal states. DRNDA activity was also greater for unexpected outcomes than for expected outcomes. Two-photon imaging of DRNDA neurons demonstrated that the majority of individual neurons developed activation to reward-predicting cues and reward but not to shock-predicting cues, which was surprising and qualitatively distinct from the population results. Performing the same fear learning procedures in freely-moving and head-fixed groups revealed that head-fixation itself abolished the neural response to aversive cues, indicating its modulation by behavioral context. Overall, these results suggest that DRNDA neurons encode motivational salience, dependent on internal and external factors.SIGNIFICANCE STATEMENT Dopamine (DA) contributes to motivational control, composed of at least two functional cell types, one signaling for motivational value and another for motivational salience. Here, we demonstrate that DA neurons in the dorsal raphe nucleus (DRN) encode the motivational salience in associative learning tasks. Neural responses were dynamic and modulated by the animal's internal state. The majority of single-cells developed responses to reward or paired cues, but not to shock-predicting cues. Additional experiments with freely-moving and head-fixed mice showed that head-fixation abolished the development of cue responses during fear learning. This work provides further characterization on the functional roles of overlooked DRNDA populations and an example that neural responses can be altered by head-fixation, which is commonly used in neuroscience.

Activation of dopaminergic VTA inputs to the mPFC ameliorates chronic stress-induced breast tumor progression.

AIMS: Chronic stress plays an important role in promoting the progression and migration of cancers. However, little is known of any direct impact on tumor progression related to the regulation of emotion-related circuitry. The aim of this study was to explore the neural-circuit mechanisms underlying stress-induced progression of cancers and the impact of emotion-related regulation of circuitry on tumor growth. METHODS: Optogenetic manipulation was applied to unpredictable chronic mild stress (UCMS)-treated mice bearing breast tumor cell. The stress-related hormones, tumor-related cytokines, the tyrosine hydroxylase (TH)-positive neurons and their fibers, dopamine receptor-positive cells, and anxiety level were measured using ELISA, immunohistochemical staining, fluorescence in situ hybridization, and behavioral test, respectively. RESULTS: By investigating breast cancer mouse models with a chronic mild stress model, optogenetic stimulation, and behavioral analysis, we show that chronic stress induced anxiety-like behavior in mice and increased serum concentration of norepinephrine and corticosterone, hormones closely related to stress and anxiety. Optogenetic activation of VTA TH terminals in the mPFC rescued anxiety-like behavior induced by chronic stress. Chronic stress resulted in marked progression of breast tumors, and repetitive optogenetic activation of VTA TH terminals in the mPFC significantly attenuated stress-induced progression of breast cancers and reduced serum concentration of norepinephrine and corticosterone. Furthermore, there was a positive correlation between serum norepinephrine or corticosterone concentration and tumor size. CONCLUSIONS: These findings indicate a positive role of an emotion regulation circuit on the progression of breast cancer and reveal a link between stress, emotion regulation, and the progression of breast cancers. Our findings provide new insights pertinent to therapeutic interventions in the treatment of breast cancers.

Whole-brain efferent and afferent connectivity of mouse ventral tegmental area melanocortin-3 receptor neurons.

The mesolimbic dopamine (DA) system is involved in the regulation of multiple behaviors, including feeding, and evidence demonstrates that the melanocortin system can act on the mesolimbic DA system to control feeding and other behaviors. The melanocortin-3 receptor (MC3R) is an important component of the melanocortin system, but its overall role is poorly understood. Because MC3Rs are highly expressed in the ventral tegmental area (VTA) and are likely to be the key interaction point between the melanocortin and mesolimbic DA systems, we set out to identify both the efferent projection patterns of VTA MC3R neurons and the location of the neurons providing afferent input to them. VTA MC3R neurons were broadly connected to neurons across the brain but were strongly connected to a discrete set of brain regions involved in the regulation of feeding, reward, and aversion. Surprisingly, experiments using monosynaptic rabies virus showed that proopiomelanocortin (POMC) and agouti-related protein (AgRP) neurons in the arcuate nucleus made few direct synapses onto VTA MC3R neurons or any of the other major neuronal subtypes in the VTA, despite being extensively labeled by general retrograde tracers injected into the VTA. These results greatly contribute to our understanding of the anatomical interactions between the melanocortin and mesolimbic systems and provide a foundation for future studies of VTA MC3R neurons and the circuits containing them in the control of feeding and other behaviors.

Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations.

Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after non-cytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 µg.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 µM) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62 % of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labeled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and for the resulting neurotoxic effects.

Noncoding RNAs and Midbrain DA Neurons: Novel Molecular Mechanisms and Therapeutic Targets in Health and Disease.

Midbrain dopamine neurons have crucial functions in motor and emotional control and their degeneration leads to several neurological dysfunctions such as Parkinson's disease, addiction, depression, schizophrenia, and others. Despite advances in the understanding of specific altered proteins and coding genes, little is known about cumulative changes in the transcriptional landscape of noncoding genes in midbrain dopamine neurons. Noncoding RNAs-specifically microRNAs and long noncoding RNAs-are emerging as crucial post-transcriptional regulators of gene expression in the brain. The identification of noncoding RNA networks underlying all stages of dopamine neuron development and plasticity is an essential step to deeply understand their physiological role and also their involvement in the etiology of dopaminergic diseases. Here, we provide an update about noncoding RNAs involved in dopaminergic development and metabolism, and the related evidence of these biomolecules for applications in potential treatments for dopaminergic neurodegeneration.

Morphine selectively disinhibits glutamatergic input from mPFC onto dopamine neurons of VTA, inducing reward.

Ventral tegmental area (VTA) dopamine (DA) neurons presynaptic glutamate release plays a very important role in the mechanism of morphine. Previously, a study from our lab found that morphine disinhibited glutamatergic input onto the VTA-DA neurons, which was an important mechanism for the morphine-induced increase in the VTA-DA neuron firing and related behaviors (Chen et al., 2015). However, what source of glutamatergic inputs is disinhibited by morphine remains to be elucidated. Using optogenetic strategy combined with whole-cell patch-clamp, qRT-PCR, immunofluorescence and chemical genetic approach combined with behavioral methods, our results show that: 1) morphine promotes glutamate release from glutamatergic terminals of medial prefrontal cortex (mPFC) neurons projecting to VTA-DA neurons but does not on those from glutamatergic terminals of the lateral hypothalamus (LH) neurons projecting to VTA-DA neurons; 2) different response of glutamatergic neurons projecting to VTA-DA neurons from the mPFC or the LH to morphine is related to the expression of GABAB receptors at terminals of these neurons; 3) inhibition of projection neurons from the mPFC to the VTA significantly reduces morphine-induced locomotor activity increase and conditioned place preference but inhibition of projection neurons from the LH to the VTA does not. These results suggest that morphine selectively promotes glutamate release of the glutamatergic input from mPFC onto VTA-DA neurons by removing the inhibition of the GABAB receptors in this glutamatergic input from mPFC.

Dexmedetomidine Activation of Dopamine Neurons in the Ventral Tegmental Area Attenuates the Depth of Sedation in Mice.

BACKGROUND: Dexmedetomidine induces a sedative response that is associated with rapid arousal. To elucidate the underlying mechanisms, the authors hypothesized that dexmedetomidine increases the activity of dopaminergic neurons in the ventral tegmental area, and that this action contributes to the unique sedative properties of dexmedetomidine. METHODS: Only male mice were used. The activity of ventral tegmental area dopamine neurons was measured by a genetically encoded Ca indicator and patch-clamp recording. Dopamine neurotransmitter dynamics in the medial prefrontal cortex and nucleus accumbens were measured by a genetically encoded dopamine sensor. Ventral tegmental area dopamine neurons were inhibited or activated by a chemogenetic approach, and the depth of sedation was estimated by electroencephalography. RESULTS: Ca signals in dopamine neurons in the ventral tegmental area increased after intraperitoneal injection of dexmedetomidine (40 µg/kg; dexmedetomidine, 16.917 [14.882; 21.748], median [25%; 75%], vs. saline, -0.745 [-1.547; 0.359], normalized data, P = 0.001; n = 6 mice). Dopamine transmission increased in the medial prefrontal cortex after intraperitoneal injection of dexmedetomidine (40 µg/kg; dexmedetomidine, 10.812 [9.713; 15.104], median [25%; 75%], vs. saline, -0.498 [-0.664; -0.355], normalized data, P = 0.001; n = 6 mice) and in the nucleus accumbens (dexmedetomidine, 8.543 [7.135; 11.828], median [25%; 75%], vs. saline, -0.329 [-1.220; -0.047], normalized data, P = 0.001; n = 6 mice). Chemogenetic inhibition or activation of ventral tegmental area dopamine neurons increased or decreased slow waves, respectively, after intraperitoneal injection of dexmedetomidine (40 µg/kg; delta wave: two-way repeated measures ANOVA, F[2, 33] = 8.016, P = 0.002; n = 12 mice; theta wave: two-way repeated measures ANOVA, F[2, 33] = 22.800, P < 0.0001; n = 12 mice). CONCLUSIONS: Dexmedetomidine activates dopamine neurons in the ventral tegmental area and increases dopamine concentrations in the related forebrain projection areas. This mechanism may explain rapid arousability upon dexmedetomidine sedation.

CNTNAP4 deficiency in dopaminergic neurons initiates parkinsonian phenotypes.

Rationale: Contactin-associated protein-like 4 (CNTNAP4) belongs to the neurexin superfamily and has critical functions in neurological development and synaptic function. Loss of CNTNAP4 in interneurons has been linked to autism, schizophrenia, and epilepsy. CNTNAP4 is also highly enriched in dopaminergic (DA) neurons in the substantia nigra (SN), however, few studies have investigated the role of CNTNAP4 in DA neurons, and whether CNTNAP4 deficiency in DA neurons contributes to Parkinson's disease (PD) remains unclear. Methods: Effects of CNTNAP4 knockdown or overexpression on the DA MN9D cell line were assessed via Western blotting, immunocytochemistry, and RNA sequencing. An in vivo animal model, including CNTNAP4 knockout mice and stereotaxic injections of adeno-associated viral short-hairpin RNA with the tyrosine-hydroxylase promotor to silence CNTNAP4 in the SN, as well as the resulting physiological/behavioral effects, were evaluated via behavioral tests, Western blotting, immunohistochemistry, and transmission electron microscopy. Enzyme-linked immunosorbent assays (ELISAs) were performed to examine the cerebrospinal fluid (CSF) and plasma CNTNAP4 concentrations in PD patients. Results: We demonstrated that CNTNAP4 knockdown induced mitophagy and increased α-synuclein expression in MN9D cells. CNTNAP4 knockdown in the SN induced PD-like increases in SN-specific α-synuclein expression, DA neuronal degeneration, and motor dysfunction in mice. In addition, CNTNAP4 knockdown in SN-DA neurons increased autophagosomes and reduced synaptic vesicles in the SN. Furthermore, CNTNAP4 knockout mice showed movement deficits, nigral DA degeneration, and increased autophagy, which were consistent with the SN-specific knockdown model. We also found that CSF and plasma CNTNAP4 expression was increased in PD patients; in particular, plasma CNTNAP4 was increased in male PD patients compared with controls or female PD patients. Conclusion: Our findings suggest that CNTNAP4 deficiency may initiate phenotypes relevant to PD, of which we elucidated some of the underlying mechanisms.

Dopaminergic modulation of olfactory-evoked motor output in sea lampreys (Petromyzon marinus L.).

Detection of chemical cues is important to guide locomotion in association with feeding and sexual behavior. Two neural pathways responsible for odor-evoked locomotion have been characterized in the sea lamprey (Petromyzon marinus L.), a basal vertebrate. There is a medial pathway originating in the medial olfactory bulb (OB) and a lateral pathway originating from the rest of the OB. These olfactomotor pathways are present throughout the life cycle of lampreys, but olfactory-driven behaviors differ according to the developmental stage. Among possible mechanisms, dopaminergic (DA) modulation in the OB might explain the behavioral changes. Here, we examined DA modulation of olfactory transmission in lampreys. Immunofluorescence against DA revealed immunoreactivity in the OB that was denser in the medial part (medOB), where processes were observed close to primary olfactory afferents and projection neurons. Dopaminergic neurons labeled by tracer injections in the medOB were located in the OB, the posterior tuberculum, and the dorsal hypothalamic nucleus, suggesting the presence of both intrinsic and extrinsic DA innervation. Electrical stimulation of the olfactory nerve in an in vitro whole-brain preparation elicited synaptic responses in reticulospinal cells that were modulated by DA. Local injection of DA agonists in the medOB decreased the reticulospinal cell responses whereas the D2 receptor antagonist raclopride increased the response amplitude. These observations suggest that DA in the medOB could modulate odor-evoked locomotion. Altogether, these results show the presence of a DA innervation within the medOB that may play a role in modulating olfactory inputs to the motor command system of lampreys.

Sonic hedgehog expression in zebrafish forebrain identifies the teleostean pallidal signaling center and shows preglomerular complex and posterior tubercular dopamine cells to arise from shh cells.

Ventralization, a major patterning process in the developing vertebrate neural tube (central nervous system, CNS), depends on Sonic hedgehog (SHH) as a main signaling morphogen. We studied the CNS of late larval and young adult zebrafish in a transgenic shh-GFP line revealing increased neuroanatomical detail due to the progressed differentiation state compared to earlier stages. Some major findings emerge from the present study. (a) shh -GFP is still expressed along the adult zebrafish CNS neuraxis in most locations seen in larvae. (b) We newly identify a ventroposterior shh pallidal domain representing the basal telencephalic signaling center important for basal ganglia development known in other vertebrates (i.e., the anterior entopeduncular area-basal medial ganglionic eminence of mammals). (c) We further show late-emerging shh-GFP positive radial glia cells in the medial zone of the dorsal telencephalon (i.e., the teleostan pallial amygdala). (d) Immunostains for tyrosine hydroxylase demonstrate that there is selective colocalization in adult dopamine cells with shh-GFP in the posterior tuberculum, including in projection cells to striatum, which represents a striking parallel to amniote mesodiencephalic dopamine cell origin from shh expressing floor plate cells. (e) There is no colocalization of shh and islet1 as shown by respective shh-GFP and islet1-GFP lines. (f) The only radially far migrated shh-GFP cells are located in the preglomerular area. (g) There are no adult cerebellar and tectal shh-GFP cells confirming their exclusive role during early development as previously reported by our laboratory.

Dopaminergic and Prefrontal Basis of Learning from Sensory Confidence and Reward Value.

Deciding between stimuli requires combining their learned value with one's sensory confidence. We trained mice in a visual task that probes this combination. Mouse choices reflected not only present confidence and past rewards but also past confidence. Their behavior conformed to a model that combines signal detection with reinforcement learning. In the model, the predicted value of the chosen option is the product of sensory confidence and learned value. We found precise correlates of this variable in the pre-outcome activity of midbrain dopamine neurons and of medial prefrontal cortical neurons. However, only the latter played a causal role: inactivating medial prefrontal cortex before outcome strengthened learning from the outcome. Dopamine neurons played a causal role only after outcome, when they encoded reward prediction errors graded by confidence, influencing subsequent choices. These results reveal neural signals that combine reward value with sensory confidence and guide subsequent learning.

Polymer dots as a novel probe for fluorescence sensing of dopamine and imaging in single living cell using droplet microfluidic platform.

We report here simple synthetic method for preparing polymer dots (Pdots) via hydrothermal treatment of organic dye (neutral red), urea and trisodium citrate. The prepared Pdots with enhanced quantum yield (quantum yield: 30.2%) was used as a selective and sensitive probe for fluorescent sensing of dopamine (DA) with high selectivity and sensitivity. The as-synthesized Pdots exhibited strong fluorescence intensity at 435 nm, which DA can trigger remarkable fluorescence quenching of such luminescent Pdots on the basis of inner filter effect (IFE) and static quenching effect (SQE). A wide linearity range (0.001 µM-900 µM) for DA detection was obtained with lower DL (3 S/N) of 0.28 nM, and no interference from other molecules such as ascorbic acid, urine acid, glutathione, glucose, epinephrine, arginine, cysteine, proline, creatinine, serine; alanine, L-therionine, Hg2+, Mg2+, K+, Ca2+ and Na+. The designed sensor was successfully applied in the imaging of DA in single living PC12 cells using droplet microfluidic approach, indicating its acceptable practicability of the proposed assay for DA detection with ultrahigh sensitivity in biological samples.

Whole-genome DNA hyper-methylation in iPSC-derived dopaminergic neurons from Parkinson's disease patients.

BACKGROUND: Parkinson's disease (PD) is characterized by the loss of midbrain dopaminergic neurons (DAn). Previously, we described the presence of DNA hyper- and hypo-methylation alterations in induced pluripotent stem cells (iPSC)-derived DAn from PD patients using the Illumina 450K array which prominently covers gene regulatory regions. METHODS: To expand and contextualize previous findings, we performed the first whole-genome DNA bisulfite sequencing (WGBS) using iPSC-derived DAn from representative PD subjects: one sporadic PD (sPD) patient, one monogenic LRRK2-associated PD patient (L2PD), and one control. RESULTS: At the whole-genome level, we detected global DNA hyper-methylation in the PD which was similarly spread across the genome in both sPD and L2PD and mostly affected intergenic regions. CONCLUSION: This study implements previous epigenetic knowledge in PD at a whole genome level providing the first comprehensive and unbiased CpG DNA methylation data using iPSC-derived DAn from PD patients. Our results indicate that DAn from monogenic or sporadic PD exhibit global DNA hyper-methylation changes. Findings from this exploratory study are to be validated in further studies analyzing other PD cell models and patient tissues.

Voltammetry and monoamines determination: an old acquaintance revisited.

First steps in brain research progress were made during the early 19th century, whose swift progress was accompanied by the discovery of monoamines and their localization in the brain. Since the discovery of polarography in 1924, several variations of electrochemical techniques for in vitro and in vivo determination of monoamines have been developed, with the most prevalent being microdialysis and voltammetry. Voltammetry takes advantage of the chemical property of certain species to oxidize, videlicet to produce a current that can be measured and subsequently interpreted to concentration gradient. Voltammetric techniques require a three-electrode system and operate under the application of a potential at the working electrode, responsible to evoke the oxidation processes. Methodological variations include, among others, amperometry, cyclic voltammetry, differential pulse voltammetry, etc. In the present work we attempted to review the available knowledge on voltammetry, its uses and future endeavors since voltammetry is a promising method towards the investigation of brain and central nervous system physiology and pathophysiology.

Dopamine Neurons Reflect the Uncertainty in Fear Generalization.

Generalized fear is a maladaptive behavior in which non-threatening stimuli elicit a fearful response. Here, we demonstrate that discrimination between predictive and non-predictive threat stimuli is highly sensitive to probabilistic discounting and increasing threat intensity in mice. We find that dopamine neurons of the ventral tegmental area (VTA) encode both the negative valence of threat-predictive cues and the certainty of threat prediction. As fear generalization emerges, the dopamine neurons that are activated by a threat predictive cue (CS+) decrease the amplitude of activation and an equivalent signal emerges to a non-predictive cue (CS-). Temporally precise enhancement of dopamine neurons during threat conditioning to high threat levels or uncertain threats can prevent generalization. Moreover, phasic enhancement of genetically captured dopamine neurons activated by threat cues can reverse fear generalization. These findings demonstrate the dopamine neurons reflect the certainty of threat prediction that can be used to inform and update the fear engram.