Transient food insecurity during the juvenile-adolescent period affects adult weight, cognitive flexibility, and dopamine neurobiology.

A major challenge for neuroscience, public health, and evolutionary biology is to understand the effects of scarcity and uncertainty on the developing brain. Currently, a significant fraction of children and adolescents worldwide experience insecure access to food. The goal of our work was to test in mice whether the transient experience of insecure versus secure access to food during the juvenile-adolescent period produced lasting differences in learning, decision-making, and the dopamine system in adulthood. We manipulated feeding schedules in mice from postnatal day (P)21 to P40 as food insecure or ad libitum and found that when tested in adulthood (after P60), males with different developmental feeding history showed significant differences in multiple metrics of cognitive flexibility in learning and decision-making. Adult females with different developmental feeding history showed no differences in cognitive flexibility but did show significant differences in adult weight. We next applied reinforcement learning models to these behavioral data. The best fit models suggested that in males, developmental feeding history altered how mice updated their behavior after negative outcomes. This effect was sensitive to task context and reward contingencies. Consistent with these results, in males, we found that the two feeding history groups showed significant differences in the AMPAR/NMDAR ratio of excitatory synapses on nucleus-accumbens-projecting midbrain dopamine neurons and evoked dopamine release in dorsal striatal targets. Together, these data show in a rodent model that transient differences in feeding history in the juvenile-adolescent period can have significant impacts on adult weight, learning, decision-making, and dopamine neurobiology.

Dopamine neuron morphology and output are differentially controlled by mTORC1 and mTORC2.

The mTOR pathway is an essential regulator of cell growth and metabolism. Midbrain dopamine neurons are particularly sensitive to mTOR signaling status as activation or inhibition of mTOR alters their morphology and physiology. mTOR exists in two distinct multiprotein complexes termed mTORC1 and mTORC2. How each of these complexes affect dopamine neuron properties, and whether they have similar or distinct functions is unknown. Here, we investigated this in mice with dopamine neuron-specific deletion of Rptor or Rictor, which encode obligatory components of mTORC1 or mTORC2, respectively. We find that inhibition of mTORC1 strongly and broadly impacts dopamine neuron structure and function causing somatodendritic and axonal hypotrophy, increased intrinsic excitability, decreased dopamine production, and impaired dopamine release. In contrast, inhibition of mTORC2 has more subtle effects, with selective alterations to the output of ventral tegmental area dopamine neurons. Disruption of both mTOR complexes leads to pronounced deficits in dopamine release demonstrating the importance of balanced mTORC1 and mTORC2 signaling for dopaminergic function.

Dopaminergic Dysregulation in Syndromic Autism Spectrum Disorders: Insights From Genetic Mouse Models.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder defined by altered social interaction and communication, and repetitive, restricted, inflexible behaviors. Approximately 1.5-2% of the general population meet the diagnostic criteria for ASD and several brain regions including the cortex, amygdala, cerebellum and basal ganglia have been implicated in ASD pathophysiology. The midbrain dopamine system is an important modulator of cellular and synaptic function in multiple ASD-implicated brain regions via anatomically and functionally distinct dopaminergic projections. The dopamine hypothesis of ASD postulates that dysregulation of dopaminergic projection pathways could contribute to the behavioral manifestations of ASD, including altered reward value of social stimuli, changes in sensorimotor processing, and motor stereotypies. In this review, we examine the support for the idea that cell-autonomous changes in dopaminergic function are a core component of ASD pathophysiology. We discuss the human literature supporting the involvement of altered dopamine signaling in ASD including genetic, brain imaging and pharmacologic studies. We then focus on genetic mouse models of syndromic neurodevelopmental disorders in which single gene mutations lead to increased risk for ASD. We highlight studies that have directly examined dopamine neuron number, morphology, physiology, or output in these models. Overall, we find considerable support for the idea that the dopamine system may be dysregulated in syndromic ASDs; however, there does not appear to be a consistent signature and some models show increased dopaminergic function, while others have deficient dopamine signaling. We conclude that dopamine dysregulation is common in syndromic forms of ASD but that the specific changes may be unique to each genetic disorder and may not account for the full spectrum of ASD-related manifestations.

Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations.

Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.

Tsc1-mTORC1 signaling controls striatal dopamine release and cognitive flexibility.

Tuberous Sclerosis Complex (TSC) is a neurodevelopmental disorder caused by mutations in TSC1 or TSC2, which encode proteins that negatively regulate mTOR complex 1 (mTORC1). TSC is associated with significant cognitive, psychiatric, and behavioral problems, collectively termed TSC-Associated Neuropsychiatric Disorders (TAND), and the cell types responsible for these manifestations are largely unknown. Here we use cell type-specific Tsc1 deletion to test whether dopamine neurons, which modulate cognitive, motivational, and affective behaviors, are involved in TAND. We show that loss of Tsc1 and constitutive activation of mTORC1 in dopamine neurons causes somatodendritic hypertrophy, reduces intrinsic excitability, alters axon terminal structure, and impairs striatal dopamine release. These perturbations lead to a selective deficit in cognitive flexibility, preventable by genetic reduction of the mTOR-binding protein Raptor. Our results establish a critical role for Tsc1-mTORC1 signaling in setting the functional properties of dopamine neurons, and indicate that dopaminergic dysfunction may contribute to cognitive inflexibility in TSC.

Imaging striatal dopamine release using a nongenetically encoded near infrared fluorescent catecholamine nanosensor.

Neuromodulation plays a critical role in brain function in both health and disease, and new tools that capture neuromodulation with high spatial and temporal resolution are needed. Here, we introduce a synthetic catecholamine nanosensor with fluorescent emission in the near infrared range (1000-1300 nm), near infrared catecholamine nanosensor (nIRCat). We demonstrate that nIRCats can be used to measure electrically and optogenetically evoked dopamine release in brain tissue, revealing hotspots with a median size of 2 µm. We also demonstrated that nIRCats are compatible with dopamine pharmacology and show D2 autoreceptor modulation of evoked dopamine release, which varied as a function of initial release magnitude at different hotspots. Together, our data demonstrate that nIRCats and other nanosensors of this class can serve as versatile synthetic optical tools to monitor neuromodulatory neurotransmitter release with high spatial resolution.

Targeted Activation of Cholinergic Interneurons Accounts for the Modulation of Dopamine by Striatal Nicotinic Receptors.

Striatal dopamine (DA) is a major player in action selection and reinforcement. DA release is under strong local control by striatal ACh acting at axonal nicotinic ACh receptors (nAChRs) on DA axons. Striatal nAChRs have been shown to control how DA is released in response to ascending activity from DA neurons, and they also directly drive DA release following synchronized activity in a small local cholinergic network. The source of striatal ACh has been thought to arise solely from intrinsic cholinergic interneurons (ChIs), but recent findings have identified a source of cholinergic inputs to striatum from brainstem nuclei, the pedunculopontine nucleus (PPN) and laterodorsal tegmentum (LDT). Here, we used targeted optogenetic activation alongside DA detection with fast-scan cyclic voltammetry to test whether ChIs alone and/or brainstem afferents to the striatum can account for how ACh drives and modulates DA release in rat striatum. We demonstrate that targeted transient light activation of rat striatal ChIs drives striatal DA release, corroborating and extending previous observations in mouse to rat. However, the same light stimulation targeted to cholinergic brainstem afferents did not drive DA release, and nor did it modulate DA release activated subsequently by electrical stimulation, whereas targeted activation of ChIs did so. We were unable to obtain any evidence for DA modulation by PPN/LDT stimulation. By contrast, we could readily identify that striatal ChIs alone are sufficient to provide a source of ACh that powerfully regulates DA via nAChRs.

Combinatorial Expression of Grp and Neurod6 Defines Dopamine Neuron Populations with Distinct Projection Patterns and Disease Vulnerability.

Midbrain dopamine neurons project to numerous targets throughout the brain to modulate various behaviors and brain states. Within this small population of neurons exists significant heterogeneity based on physiology, circuitry, and disease susceptibility. Recent studies have shown that dopamine neurons can be subdivided based on gene expression; however, the extent to which genetic markers represent functionally relevant dopaminergic subpopulations has not been fully explored. Here we performed single-cell RNA-sequencing of mouse dopamine neurons and validated studies showing that Neurod6 and Grp are selective markers for dopaminergic subpopulations. Using a combination of multiplex fluorescent in situ hybridization, retrograde labeling, and electrophysiology in mice of both sexes, we defined the anatomy, projection targets, physiological properties, and disease vulnerability of dopamine neurons based on Grp and/or Neurod6 expression. We found that the combinatorial expression of Grp and Neurod6 defines dopaminergic subpopulations with unique features. Grp+/Neurod6+ dopamine neurons reside in the ventromedial VTA, send projections to the medial shell of the nucleus accumbens, and have noncanonical physiological properties. Grp+/Neurod6- dopamine neurons are found in the VTA as well as in the ventromedial portion of the SNc, where they project selectively to the dorsomedial striatum. Grp-/Neurod6+ dopamine neurons represent a smaller VTA subpopulation, which is preferentially spared in a 6-OHDA model of Parkinson's disease. Together, our work provides detailed characterization of Neurod6 and Grp expression in the midbrain and generates new insights into how these markers define functionally relevant dopaminergic subpopulations.

Cortical Control of Striatal Dopamine Transmission via Striatal Cholinergic Interneurons.

Corticostriatal regulation of striatal dopamine (DA) transmission has long been postulated, but ionotropic glutamate receptors have not been localized directly to DA axons. Striatal cholinergic interneurons (ChIs) are emerging as major players in striatal function, and can govern DA transmission by activating nicotinic receptors (nAChRs) on DA axons. Cortical inputs to ChIs have historically been perceived as sparse, but recent evidence indicates that they strongly activate ChIs. We explored whether activation of M1/M2 corticostriatal inputs can consequently gate DA transmission, via ChIs. We reveal that optogenetic activation of channelrhodopsin-expressing corticostriatal axons can drive striatal DA release detected with fast-scan cyclic voltammetry and requires activation of nAChRs on DA axons and AMPA receptors on ChIs that promote short-latency action potentials. By contrast, DA release driven by optogenetic activation of intralaminar thalamostriatal inputs involves additional activation of NMDA receptors on ChIs and action potential generation over longer timescales. Therefore, cortical and thalamic glutamate inputs can modulate DA transmission by regulating ChIs as gatekeepers, through ionotropic glutamate receptors. The different use of AMPA and NMDA receptors by cortical versus thalamic inputs might lead to distinct input integration strategies by ChIs and distinct modulation of the function of DA and striatum.

Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model.

The pathological end-state of Parkinson disease is well described from postmortem tissue, but there remains a pressing need to define early functional changes to susceptible neurons and circuits. In particular, mechanisms underlying the vulnerability of the dopamine neurons of the substantia nigra pars compacta (SNc) and the importance of protein aggregation in driving the disease process remain to be determined. To better understand the sequence of events occurring in familial and sporadic Parkinson disease, we generated bacterial artificial chromosome transgenic mice (SNCA-OVX) that express wild-type α-synuclein from the complete human SNCA locus at disease-relevant levels and display a transgene expression profile that recapitulates that of endogenous α-synuclein. SNCA-OVX mice display age-dependent loss of nigrostriatal dopamine neurons and motor impairments characteristic of Parkinson disease. This phenotype is preceded by early deficits in dopamine release from terminals in the dorsal, but not ventral, striatum. Such neurotransmission deficits are not seen at either noradrenergic or serotoninergic terminals. Dopamine release deficits are associated with an altered distribution of vesicles in dopaminergic axons in the dorsal striatum. Aged SNCA-OVX mice exhibit reduced firing of SNc dopamine neurons in vivo measured by juxtacellular recording of neurochemically identified neurons. These progressive changes in vulnerable SNc neurons were observed independently of overt protein aggregation, suggesting neurophysiological changes precede, and are not driven by, aggregate formation. This longitudinal phenotyping strategy in SNCA-OVX mice thus provides insights into the region-specific neuronal disturbances preceding and accompanying Parkinson disease.