Distributional coding of associative learning in discrete populations of midbrain dopamine neurons.

Midbrain dopamine neurons are thought to play key roles in learning by conveying the difference between expected and actual outcomes. Recent evidence suggests diversity in dopamine signaling, yet it remains poorly understood how heterogeneous signals might be organized to facilitate the role of downstream circuits mediating distinct aspects of behavior. Here, we investigated the organizational logic of dopaminergic signaling by recording and labeling individual midbrain dopamine neurons during associative behavior. Our findings show that reward information and behavioral parameters are not only heterogeneously encoded but also differentially distributed across populations of dopamine neurons. Retrograde tracing and fiber photometry suggest that populations of dopamine neurons projecting to different striatal regions convey distinct signals. These data, supported by computational modeling, indicate that such distributional coding can maximize dynamic range and tailor dopamine signals to facilitate specialized roles of different striatal regions.

A transcriptomic taxonomy of mouse brain-wide spinal projecting neurons.

The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.

The molecular cytoarchitecture of the adult mouse brain.

The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq1,2-a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource ( www.BrainCellData.org ), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases.

Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

Dopamine subsystems that track internal states.

Food and water are rewarding in part because they satisfy our internal needs1,2. Dopaminergic neurons in the ventral tegmental area (VTA) are activated by gustatory rewards3-5, but how animals learn to associate these oral cues with the delayed physiological effects of ingestion is unknown. Here we show that individual dopaminergic neurons in the VTA respond to detection of nutrients or water at specific stages of ingestion. A major subset of dopaminergic neurons tracks changes in systemic hydration that occur tens of minutes after thirsty mice drink water, whereas different dopaminergic neurons respond to nutrients in the gastrointestinal tract. We show that information about fluid balance is transmitted to the VTA by a hypothalamic pathway and then re-routed to downstream circuits that track the oral, gastrointestinal and post-absorptive stages of ingestion. To investigate the function of these signals, we used a paradigm in which a fluid's oral and post-absorptive effects can be independently manipulated and temporally separated. We show that mice rapidly learn to prefer one fluid over another based solely on its rehydrating ability and that this post-ingestive learning is prevented if dopaminergic neurons in the VTA are selectively silenced after consumption. These findings reveal that the midbrain dopamine system contains subsystems that track different modalities and stages of ingestion, on timescales from seconds to tens of minutes, and that this information is used to drive learning about the consequences of ingestion.

Involvement of Denervated Midbrain-Derived Factors in the Formation of Ectopic Cortico-Mesencephalic Projection after Hemispherectomy.

Neuronal remodeling after brain injury is essential for functional recovery. After unilateral cortical lesion, axons from the intact cortex ectopically project to the denervated midbrain, but the molecular mechanisms remain largely unknown. To address this issue, we examined gene expression profiles in denervated and intact mouse midbrains after hemispherectomy at early developmental stages using mice of either sex, when ectopic contralateral projection occurs robustly. The analysis showed that various axon growth-related genes were upregulated in the denervated midbrain, and most of these genes are reportedly expressed by glial cells. To identify the underlying molecules, the receptors for candidate upregulated molecules were knocked out in layer 5 projection neurons in the intact cortex, using the CRISPR/Cas9-mediated method, and axonal projection from the knocked-out cortical neurons was examined after hemispherectomy. We found that the ectopic projection was significantly reduced when integrin subunit ß three or neurotrophic receptor tyrosine kinase 2 (also known as TrkB) was knocked out. Overall, the present study suggests that denervated midbrain-derived glial factors contribute to lesion-induced remodeling of the cortico-mesencephalic projection via these receptors.SIGNIFICANCE STATEMENT After brain injury, compensatory neural circuits are established that contribute to functional recovery. However, little is known about the intrinsic mechanism that underlies the injury-induced remodeling. We found that after unilateral cortical ablation expression of axon-growth promoting factors is elevated in the denervated midbrain and is involved in the formation of ectopic axonal projection from the intact cortex. Evidence further demonstrated that these factors are expressed by astrocytes and microglia, which are activated in the denervated midbrain. Thus, our present study provides a new insight into the mechanism of lesion-induced axonal remodeling and further therapeutic strategies after brain injury.

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.

Chronic nicotine increases midbrain dopamine neuron activity and biases individual strategies towards reduced exploration in mice.

Long-term exposure to nicotine alters brain circuits and induces profound changes in decision-making strategies, affecting behaviors both related and unrelated to drug seeking and consumption. Using an intracranial self-stimulation reward-based foraging task, we investigated in mice the impact of chronic nicotine on midbrain dopamine neuron activity and its consequence on the trade-off between exploitation and exploration. Model-based and archetypal analysis revealed substantial inter-individual variability in decision-making strategies, with mice passively exposed to nicotine shifting toward a more exploitative profile compared to non-exposed animals. We then mimicked the effect of chronic nicotine on the tonic activity of dopamine neurons using optogenetics, and found that photo-stimulated mice adopted a behavioral phenotype similar to that of mice exposed to chronic nicotine. Our results reveal a key role of tonic midbrain dopamine in the exploration/exploitation trade-off and highlight a potential mechanism by which nicotine affects the exploration/exploitation balance and decision-making.

Adult Neural Stem Cells from Midbrain Periventricular Regions Show Limited Neurogenic Potential after Transplantation into the Hippocampal Neurogenic Niche.

The regulation of adult neural stem or progenitor cell (aNSC) proliferation and differentiation as an interplay of cell-intrinsic and local environmental cues remains in part unclear, impeding their role in putative regenerative therapies. aNSCs with all major properties of NSCs in vitro have been identified in a variety of brain regions beyond the classic neurogenic niches, including the caudal periventricular regions (PVRs) of the midbrain, though active neurogenesis is either limited or merely absent in these regions. To elucidate cell-intrinsic properties of aNSCs from various PVRs, we here examined the proliferation and early differentiation capacity of murine aNSCs from non-neurogenic midbrain PVRs (PVRMB) compared to aNSCs from the neurogenic ventricular-subventricular zone (PVRV-SVZ) 7 days after transplantation into the permissive pro-neurogenic niche of the dentate gyrus (DG) of the hippocampus in mice. An initial in vitro characterization of the transplants displayed very similar characteristics of both aNSC grafts after in vitro expansion with equal capacities of terminal differentiation into astrocytes and Tuj1+ neurons. Upon the allogenic transplantation of the respective aNSCs into the DG, PVRMB grafts showed a significantly lower graft survival and proliferative capacity compared to PVRV-SVZ transplants, whereby the latter are exclusively capable of generating new neurons. Although these differences might be-in part-related to the transplantation procedure and the short-term study design, our data strongly imply important cell-intrinsic differences between aNSCs from neurogenic compared to non-neurogenic PVRs with respect to their neurogenic potential and/or their sensitivity to neurogenic cues.

Diverse processing underlying frequency integration in midbrain neurons of barn owls.

Emergent response properties of sensory neurons depend on circuit connectivity and somatodendritic processing. Neurons of the barn owl's external nucleus of the inferior colliculus (ICx) display emergence of spatial selectivity. These neurons use interaural time difference (ITD) as a cue for the horizontal direction of sound sources. ITD is detected by upstream brainstem neurons with narrow frequency tuning, resulting in spatially ambiguous responses. This spatial ambiguity is resolved by ICx neurons integrating inputs over frequency, a relevant processing in sound localization across species. Previous models have predicted that ICx neurons function as point neurons that linearly integrate inputs across frequency. However, the complex dendritic trees and spines of ICx neurons raises the question of whether this prediction is accurate. Data from in vivo intracellular recordings of ICx neurons were used to address this question. Results revealed diverse frequency integration properties, where some ICx neurons showed responses consistent with the point neuron hypothesis and others with nonlinear dendritic integration. Modeling showed that varied connectivity patterns and forms of dendritic processing may underlie observed ICx neurons' frequency integration processing. These results corroborate the ability of neurons with complex dendritic trees to implement diverse linear and nonlinear integration of synaptic inputs, of relevance for adaptive coding and learning, and supporting a fundamental mechanism in sound localization.

In situ and transcriptomic identification of microglia in synapse-rich regions of the developing zebrafish brain.

Microglia are brain resident macrophages that play vital roles in central nervous system (CNS) development, homeostasis, and pathology. Microglia both remodel synapses and engulf apoptotic cell corpses during development, but whether unique molecular programs regulate these distinct phagocytic functions is unknown. Here we identify a molecularly distinct microglial subset in the synapse rich regions of the zebrafish (Danio rerio) brain. We found that ramified microglia increased in synaptic regions of the midbrain and hindbrain between 7 and 28 days post fertilization. In contrast, microglia in the optic tectum were ameboid and clustered around neurogenic zones. Using single-cell mRNA sequencing combined with metadata from regional bulk sequencing, we identified synaptic-region associated microglia (SAMs) that were highly enriched in the hindbrain and expressed multiple candidate synapse modulating genes, including genes in the complement pathway. In contrast, neurogenic associated microglia (NAMs) were enriched in the optic tectum, had active cathepsin activity, and preferentially engulfed neuronal corpses. These data reveal that molecularly distinct phagocytic programs mediate synaptic remodeling and cell engulfment, and establish the zebrafish hindbrain as a model for investigating microglial-synapse interactions.

Inactivation mode of sodium channels defines the different maximal firing rates of conventional versus atypical midbrain dopamine neurons.

Two subpopulations of midbrain dopamine (DA) neurons are known to have different dynamic firing ranges in vitro that correspond to distinct projection targets: the originally identified conventional DA neurons project to the dorsal striatum and the lateral shell of the nucleus accumbens, whereas an atypical DA population with higher maximum firing frequencies projects to prefrontal regions and other limbic regions including the medial shell of nucleus accumbens. Using a computational model, we show that previously identified differences in biophysical properties do not fully account for the larger dynamic range of the atypical population and predict that the major difference is that originally identified conventional cells have larger occupancy of voltage-gated sodium channels in a long-term inactivated state that recovers slowly; stronger sodium and potassium conductances during action potential firing are also predicted for the conventional compared to the atypical DA population. These differences in sodium channel gating imply that longer intervals between spikes are required in the conventional population for full recovery from long-term inactivation induced by the preceding spike, hence the lower maximum frequency. These same differences can also change the bifurcation structure to account for distinct modes of entry into depolarization block: abrupt versus gradual. The model predicted that in cells that have entered depolarization block, it is much more likely that an additional depolarization can evoke an action potential in conventional DA population. New experiments comparing lateral to medial shell projecting neurons confirmed this model prediction, with implications for differential synaptic integration in the two populations.

Fibrillar α-synuclein induces neurotoxic astrocyte activation via RIP kinase signaling and NF-κB.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the death of midbrain dopamine neurons. The pathogenesis of PD is poorly understood, though misfolded and/or aggregated forms of the protein α-synuclein have been implicated in several neurodegenerative disease processes, including neuroinflammation and astrocyte activation. Astrocytes in the midbrain play complex roles during PD, initiating both harmful and protective processes that vary over the course of the disease. However, despite their significant regulatory roles during neurodegeneration, the cellular and molecular mechanisms that promote pathogenic astrocyte activity remain mysterious. Here, we show that α-synuclein preformed fibrils (PFFs) induce pathogenic activation of human midbrain astrocytes, marked by inflammatory transcriptional responses, downregulation of phagocytic function, and conferral of neurotoxic activity. These effects required the necroptotic kinases RIPK1 and RIPK3, but were independent of MLKL and necroptosis. Instead, both transcriptional and functional markers of astrocyte activation occurred via RIPK-dependent activation of NF-κB signaling. Our study identifies a previously unknown function for α-synuclein in promoting neurotoxic astrocyte activation, as well as new cell death-independent roles for RIP kinase signaling in the regulation of glial cell biology and neuroinflammation. Together, these findings highlight previously unappreciated molecular mechanisms of pathologic astrocyte activation and neuronal cell death with implications for Parkinsonian neurodegeneration.

Protocol for tissue clearing and 3D analysis of dopamine neurons in the developing mouse midbrain.

Advances in tissue clearing enable analysis of complex migratory patterns of developing neurons in whole intact tissue. Here, we implemented a modified version of 3DISCO to study migration of midbrain dopamine (DA) neurons. We provide a detailed protocol starting from whole-brain immunostaining, tissue clearing, and ultramicroscopic imaging to post-acquisition quantification and analysis. This protocol enables precise quantification of DA neuron migration but can also be applied more generally for analyzing neuron migration throughout the nervous system. For complete details on the use and execution of this protocol, please refer to Brignani et al. (2020).

Ncx3-Induced Mitochondrial Dysfunction in Midbrain Leads to Neuroinflammation in Striatum of A53t-α-Synuclein Transgenic Old Mice.

The exact mechanism underlying selective dopaminergic neurodegeneration is not completely understood. The complex interplay among toxic alpha-synuclein aggregates, oxidative stress, altered intracellular Ca2+-homeostasis, mitochondrial dysfunction and disruption of mitochondrial integrity is considered among the pathogenic mechanisms leading to dopaminergic neuronal loss. We herein investigated the molecular mechanisms leading to mitochondrial dysfunction and its relationship with activation of the neuroinflammatory process occurring in Parkinson's disease. To address these issues, experiments were performed in vitro and in vivo in mice carrying the human mutation of α-synuclein A53T under the prion murine promoter. In these models, the expression and activity of NCX isoforms, a family of important transporters regulating ionic homeostasis in mammalian cells working in a bidirectional way, were evaluated in neurons and glial cells. Mitochondrial function was monitored with confocal microscopy and fluorescent dyes to measure mitochondrial calcium content and mitochondrial membrane potential. Parallel experiments were performed in 4 and 16-month-old A53T-α-synuclein Tg mice to correlate the functional data obtained in vitro with mitochondrial dysfunction and neuroinflammation through biochemical analysis. The results obtained demonstrated: 1. in A53T mice mitochondrial dysfunction occurs early in midbrain and later in striatum; 2. mitochondrial dysfunction occurring in the midbrain is mediated by the impairment of NCX3 protein expression in neurons and astrocytes; 3. mitochondrial dysfunction occurring early in midbrain triggers neuroinflammation later into the striatum, thus contributing to PD progression during mice aging.

Thyroid hormone deficiency during zebrafish development impairs central nervous system myelination.

Thyroid hormones are messengers that bind to specific nuclear receptors and regulate a wide range of physiological processes in the early stages of vertebrate embryonic development, including neurodevelopment and myelogenesis. We here tested the effects of reduced T3 availability upon the myelination process by treating zebrafish embryos with low concentrations of iopanoic acid (IOP) to block T4 to T3 conversion. Black Gold II staining showed that T3 deficiency reduced the myelin density in the forebrain, midbrain, hindbrain and the spinal cord at 3 and 7 dpf. These observations were confirmed in 3 dpf mbp:egfp transgenic zebrafish, showing that the administration of IOP reduced the fluorescent signal in the brain. T3 rescue treatment restored brain myelination and reversed the changes in myelin-related gene expression induced by IOP exposure. NG2 immunostaining revealed that T3 deficiency reduced the amount of oligodendrocyte precursor cells in 3 dpf IOP-treated larvae. Altogether, the present results show that inhibition of T4 to T3 conversion results in hypomyelination, suggesting that THs are part of the key signaling molecules that control the timing of oligodendrocyte differentiation and myelin synthesis from very early stages of brain development.

Transcriptomic encoding of sensorimotor transformation in the midbrain.

Sensorimotor transformation, a process that converts sensory stimuli into motor actions, is critical for the brain to initiate behaviors. Although the circuitry involved in sensorimotor transformation has been well delineated, the molecular logic behind this process remains poorly understood. Here, we performed high-throughput and circuit-specific single-cell transcriptomic analyses of neurons in the superior colliculus (SC), a midbrain structure implicated in early sensorimotor transformation. We found that SC neurons in distinct laminae expressed discrete marker genes. Of particular interest, Cbln2 and Pitx2 were key markers that define glutamatergic projection neurons in the optic nerve (Op) and intermediate gray (InG) layers, respectively. The Cbln2+ neurons responded to visual stimuli mimicking cruising predators, while the Pitx2+ neurons encoded prey-derived vibrissal tactile cues. By forming distinct input and output connections with other brain areas, these neuronal subtypes independently mediated behaviors of predator avoidance and prey capture. Our results reveal that, in the midbrain, sensorimotor transformation for different behaviors may be performed by separate circuit modules that are molecularly defined by distinct transcriptomic codes.

Aromatic-Turmerone Analogs Protect Dopaminergic Neurons in Midbrain Slice Cultures through Their Neuroprotective Activities.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra. The inflammatory activation of microglia participates in dopaminergic neurodegeneration in PD. Therefore, chemicals that inhibit microglial activation are considered to have therapeutic potential for PD. Aromatic (ar)-turmerone is a main component of turmeric oil extracted from Curcuma longa and has anti-inflammatory activity in cultured microglia. The aims of the present study are (1) to investigate whether naturally occurring S-enantiomer of ar-turmerone (S-Tur) protects dopaminergic neurons in midbrain slice cultures and (2) to examine ar-turmerone analogs that have higher activities than S-Tur in inhibiting microglial activation and protecting dopaminergic neurons. R-enantiomer (R-Tur) and two analogs showed slightly higher anti-inflammatory effects in microglial BV2 cells. S- and R-Tur and these two analogs reversed dopaminergic neurodegeneration triggered by microglial activation in midbrain slice cultures. Unexpectedly, this neuroprotection was independent of the inhibition of microglial activation. Additionally, two analogs more potently inhibited dopaminergic neurodegeneration triggered by a neurotoxin, 1-methyl-4-phenylpyridinium, than S-Tur. Taken together, we identified two ar-turmerone analogs that directly and potently protected dopaminergic neurons. An investigation using dopaminergic neuronal precursor cells suggested the possible involvement of nuclear factor erythroid 2-related factor 2 in this neuroprotection.

IGF-1 receptor is involved in the regulatory effects of icariin and icaritin in astrocytes under basal conditions and after an inflammatory challenge.

Icariin and icaritin, the major active components of Epimedii Genus, are considered as promising drugs with anti-inflammatory, anti-aging and neuroprotective effects. Our previous studies have demonstrated that icariin and icaritin can protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridinium (MPP+)-induced neurotoxicity on dopaminergic neurons via insulin-like growth factor-1 receptor (IGF-1 receptor) signaling. In the present study, we aimed to evaluate the role of IGF-1 receptor signaling in mediating the anti-inflammatory effects of icariin and icaritin against lipopolysaccharide (LPS)-induced neuroinflammation as well as their biological regulation effects in midbrain primary astrocytes. Our results showed that both icariin and icaritin significantly inhibited LPS-induced mRNA expressions of tumor necrosis factor (TNF-α) and interleukin-1ß (IL-1ß). Pre-treatment with IGF-1 receptor antagonist JB-1 could significantly block the anti-inflammatory effects of icariin and icaritin on LPS-induced up-regulations of TNF-α, IL-1ß, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Under basal conditions of astrocytes, icariin and icaritin treatment alone increased the phosphorylation of ERK1/2 and AKT, which could be blocked by JB-1. Moreover, the mRNA expressions of glutamate transptor-1 (GLT-1) and glutamate-aspartate transporter (GLAST) could be up-regulated by icariin and icaritin in a time-dependent manner via IGF-1 receptor. Taken together, our results suggest for the first time that both icariin and icaritin exert regulatory effects in astrocytes under basal conditions and after an inflammatory challenge via IGF-1 receptor signaling pathway.

Efficient generation of dopaminergic induced neuronal cells with midbrain characteristics.

The differentiation of pluripotent stem cells can be accomplished by sequential activation of signaling pathways or through transcription factor programming. Multistep differentiation imitates embryonic development to obtain authentic cell types, but it suffers from asynchronous differentiation with variable efficiency. Transcription factor programming induces synchronous and efficient differentiation with higher reproducibility but may not always yield authentic cell types. We systematically explored the generation of dopaminergic induced neuronal cells from mouse and human pluripotent stem cells. We found that the proneural factor Ascl1 in combination with mesencephalic factors Lmx1a and Nurr1 induce peripheral dopaminergic neurons. Co-delivery of additional midbrain transcription factors En1, FoxA2, and Pitx3 resulted in facile and robust generation of functional dopaminergic neurons of midbrain character. Our results suggest that more complex combinations of transcription factors may be needed for proper regional specification of induced neuronal cells generated by direct lineage induction.