Parkinson's LRRK2-G2019S risk gene mutation drives sex-specific behavioral and cellular adaptations to chronic variable stress.

Anxiety is a psychiatric non-motor symptom of Parkinson's that can appear in the prodromal period, prior to significant loss of brainstem dopamine neurons and motor symptoms. Parkinson's-related anxiety affects females more than males, despite the greater prevalence of Parkinson's in males. How stress, anxiety and Parkinson's are related and the basis for a sex-specific impact of stress in Parkinson's are not clear. We addressed this using young adult male and female mice carrying a G2019S knockin mutation of leucine-rich repeat kinase 2 ( Lrrk2 G2019S ) and Lrrk2 WT control mice. In humans, LRRK2 G2019S significantly elevates the risk of late-onset Parkinson's. To assess within-sex differences between Lrrk2 G2019S and control mice in stress-induced anxiety-like behaviors in young adulthood, we used a within-subject design whereby Lrrk2 G2019S and Lrrk2 WT control mice underwent tests of anxiety-like behaviors before (baseline) and following a 28 day (d) variable stress paradigm. There were no differences in behavioral measures between genotypes in males or females at baseline, indicating that the mutation alone does not produce anxiety-like responses. Following chronic stress, male Lrrk2 G2019S mice were affected similarly to male wildtypes except for novelty-suppressed feeding, where stress had no impact on Lrrk2 G2019S mice while significantly increasing latency to feed in Lrrk2 WT control mice. Female Lrrk2 G2019S mice were impacted by chronic stress similarly to wildtype females across all behavioral measures. Subsequent post-stress analyses compared cFos immunolabeling-based cellular activity patterns across several stress-relevant brain regions. The density of cFos-activated neurons across brain regions in both male and female Lrrk2 G2019S mice was generally lower compared to stressed Lrrk2 WT mice, except for the nucleus accumbens of male Lrrk2 G2019S mice, where cFos-labeled cell density was significantly higher than all other groups. Together, these data suggest that the Lrrk2 G2019S mutation differentially impacts anxiety-like behavioral responses to chronic stress in males and females that may reflect sex-specific adaptations observed in circuit activation patterns in stress-related brain regions.

Bidirectional Neuronal Actuation by Uncaging with Violet and Green Light.

We have developed a photochemical protecting group that enables wavelength selective uncaging using green versus violet light. Change of the exocyclic oxygen of the laser dye coumarin-102 to sulfur, gave thio-coumarin-102, a new chromophore with an absorption ratio at 503/402 nm of 37. Photolysis of thio-coumarin-102 caged γ-aminobutyric acid was found to be highly wavelength selective on neurons, with normalized electrical responses >100-fold higher in the green versus violet channel. When partnered with coumarin-102 caged glutamate, we could use whole cell violet and green irradiation to fire and block neuronal action potentials with complete orthogonality. Localized irradiation of different dendritic segments, each connected to a neuronal cell body, in concert with 3-dimenional Ca2+ imaging, revealed that such inputs could function independently. Chemical signaling in living cells always involves a complex balance of multiple pathways, use of (thio)-coumarin-102 caged compounds will enable arbitrarily timed flashes of green and violet light to interrogate two independent pathways simultaneously.

Development and cadherin-mediated control of prefrontal corticostriatal projections in mice.

Action-outcome associations depend on prefrontal cortex (PFC) projections to the dorsal striatum. To assess how these projections form, we measured PFC axon patterning, synapse formation, and functional maturation in the postnatally developing mouse striatum. Using Hotspot analysis, we show that PFC axons form an adult-like pattern of clustered terminations in the first postnatal week that remains largely stable thereafter. PFC-striatal synaptic strength is adult-like by P21, while excitatory synapse density increases until adulthood. We then tested how the targeted deletion of a candidate adhesion/guidance protein, Cadherin-8 (Cdh8), from corticostriatal neurons regulates pathway development. Mutant mice showed diminished PFC axon targeting and reduced spontaneous glutamatergic synaptic activity in the dorsal striatum. They also exhibited impaired behavioral performance in action-outcome learning. The data show that PFC-striatal axons form striatal territories through an early, directed growth model and they highlight essential contributions of Cdh8 to the anatomical and functional features critical for the formation of action-outcome associations.

Parkinson's-linked LRRK2-G2019S derails AMPAR trafficking, mobility and composition in striatum with cell-type and subunit specificity.

Parkinson's (PD) is a multi-factorial disease that affects multiple brain systems and circuits. While defined by motor symptoms caused by degeneration of brainstem dopamine neurons, debilitating non-motor abnormalities in fronto-striatal based cognitive function are common, appear early and are initially independent of dopamine. Young adult mice expressing the PD-associated G2019S missense mutation in Lrrk2 also exhibit deficits in fronto-striatal-based cognitive tasks. In mice and humans, cognitive functions require dynamic adjustments in glutamatergic synapse strength through cell-surface trafficking of AMPA-type glutamate receptors (AMPARs), but it is unknown how LRRK2 mutation impacts dynamic features of AMPAR trafficking in striatal projection neurons (SPNs). Here, we used Lrrk2 G2019S knockin mice to show that surface AMPAR subunit stoichiometry is altered biochemically and functionally in mutant SPNs to favor incorporation of GluA1 over GluA2. GluA1-containing AMPARs were resistant to internalization from the cell surface, leaving an excessive accumulation of GluA1 on the surface within and outside synapses. This negatively impacted trafficking dynamics that normally support synapse strengthening, as GluA1-containing AMPARs failed to increase at synapses in response to a potentiating stimulus and showed significantly reduced surface mobility. Surface GluA2-containing AMPARs were expressed at normal levels in synapses, indicating subunit-selective impairment. Abnormal surface accumulation of GluA1 was independent of PKA activity and was limited to D 1 R SPNs. Since LRRK2 mutation is thought to be part of a common PD pathogenic pathway, our data suggest that sustained, striatal cell-type specific changes in AMPAR composition and trafficking contribute to cognitive or other impairments associated with PD. SIGNIFICANCE STATEMENT: Mutations in LRRK2 are common genetic risks for PD. Lrrk2 G2019S mice fail to exhibit long-term potentiation at corticostriatal synapses and show significant deficits in frontal-striatal based cognitive tasks. While LRRK2 has been implicated generally in protein trafficking, whether G2019S derails AMPAR trafficking at synapses on striatal neurons (SPNs) is unknown. We show that surface GluA1-AMPARs fail to internalize and instead accumulate excessively within and outside synapses. This effect is selective to D 1 R SPNs and negatively impacts synapse strengthening as GluA1-AMPARs fail to increase at the surface in response to potentiation and show limited surface mobility. Thus, LRRK2-G2019S narrows the effective range of plasticity mechanisms, supporting the idea that cognitive symptoms reflect an imbalance in AMPAR trafficking mechanisms within cell-type specific projections.

Development of prefrontal corticostriatal connectivity in mice.

Rational decision making is grounded in learning to associate actions with outcomes, a process that depends on projections from prefrontal cortex to dorsomedial striatum. Symptoms associated with a variety of human pathological conditions ranging from schizophrenia and autism to Huntington's and Parkinson's disease point toward functional deficits in this projection, but its development is not well understood, making it difficult to investigate how perturbations in development of this circuitry could contribute to pathophysiology. We applied a novel strategy based on Hotspot Analysis to assess the developmental progression of anatomical positioning of prefrontal cortex to striatal projections. Corticostriatal axonal territories established at P7 expand in concert with striatal growth but remain largely unchanged in positioning through adulthood, indicating they are generated by directed, targeted growth and not modified extensively by postnatal experience. Consistent with these findings, corticostriatal synaptogenesis increased steadily from P7 to P56, with no evidence for widescale pruning. As corticostriatal synapse density increased over late postnatal ages, the strength of evoked PFC input onto dorsomedial striatal projection neurons also increased, but spontaneous glutamatergic synaptic activity was stable. Based on its pattern of expression, we asked whether the adhesion protein, Cdh8, influenced this progression. In mice lacking Cdh8 in PFC corticostriatal projection neurons, axon terminal fields in dorsal striatum shifted ventrally. Corticostriatal synaptogenesis was unimpeded, but spontaneous EPSC frequency declined and mice failed to learn to associate an action with an outcome. Collectively these findings show that corticostriatal axons grow to their target zone and are restrained from an early age, do not undergo postnatal synapse pruning as the most dominant models predict, and that a relatively modest shift in terminal arbor positioning and synapse function has an outsized, negative impact on corticostriatal-dependent behavior.

Non-Motor Symptoms of Parkinson's Disease: The Neurobiology of Early Psychiatric and Cognitive Dysfunction.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that has been recognized for over 200 years by its clinically dominant motor system impairment. There are prominent non-motor symptoms as well, and among these, psychiatric symptoms of depression and anxiety and cognitive impairment are common and can appear earlier than motor symptoms. Although the neurobiology underlying these particular PD-associated non-motor symptoms is not completely understood, the identification of PARK genes that contribute to hereditary and sporadic PD has enabled genetic models in animals that, in turn, have fostered ever deepening analyses of cells, synapses, circuits, and behaviors relevant to non-motor psychiatric and cognitive symptoms of human PD. Moreover, while it has long been recognized that inflammation is a prominent component of PD, recent studies demonstrate that brain-immune signaling crosstalk has significant modulatory effects on brain cell and synaptic function in the context of psychiatric symptoms. This review provides a focused update on such progress in understanding the neurobiology of PD-related non-motor psychiatric and cognitive symptoms.

Cognitive deficits and altered cholinergic innervation in young adult male mice carrying a Parkinson's disease Lrrk2G2019S knockin mutation.

Impaired executive function is a common and debilitating non-motor symptom of idiopathic and hereditary Parkinson's disease (PD), but there is little understanding of the underlying pathophysiological mechanisms and circuits. The G2019S mutation in the kinase domain of leucine-rich repeat kinase 2 (LRRK2) greatly increases risk for late-onset PD, and non-manifesting LRRK2G2019S carriers can also exhibit early and significant cognitive impairment. Here, we subjected young adult male mice carrying a Lrrk2G2019S knockin mutation to touchscreen-based operant tasks that measure attention, goal-directed learning and cognitive flexibility, all of which rely on frontal-striatal connectivity and are strongly modulated by cholinergic innervation. In a visuospatial attention task, mutant mice exhibited significantly more omissions and longer response latencies than controls that could not be attributed to deficits in motivation, visual sensory perception per se or locomotion, thereby suggesting impairments in divided attention and/or action-selection as well as generally slower information processing speed. Pretreating mice with the acetylcholinesterase inhibitor donepezil normalized both higher omission rates and longer response latencies in the mutants, but did not affect any performance metric in controls. Strikingly, cholinergic fiber density in cortical areas PL/IL and DMS (dorsomedial striatum) was significantly sparser in mutants than in controls, while further behavioral interrogation of the mutants revealed significant impairments in action-outcome associations but preserved cognitive flexibility. These data suggest that the Lrrk2G2019S mutation negatively impacts cholinergic innervation anatomically and functionally by young adulthood, impairing corticostriatal network function in ways that may contribute to early PD-associated executive function deficits.

Mitochondrial localization and moderated activity are key to murine erythroid enucleation.

Mammalian red blood cells (RBCs), which primarily contain hemoglobin, exemplify an elaborate maturation process, with the terminal steps of RBC generation involving extensive cellular remodeling. This encompasses alterations of cellular content through distinct stages of erythroblast maturation that result in the expulsion of the nucleus (enucleation) followed by the loss of mitochondria and all other organelles and a transition to anaerobic glycolysis. Whether there is any link between erythroid removal of the nucleus and the function of any other organelle, including mitochondria, remains unknown. Here we demonstrate that mitochondria are key to nuclear clearance. Using live and confocal microscopy and high-throughput single-cell imaging, we show that before nuclear polarization, mitochondria progressively move toward one side of maturing erythroblasts and aggregate near the nucleus as it extrudes from the cell, a prerequisite for enucleation to proceed. Although we found active mitochondrial respiration is required for nuclear expulsion, levels of mitochondrial activity identify distinct functional subpopulations, because terminally maturing erythroblasts with low relative to high mitochondrial membrane potential are at a later stage of maturation, contain greatly condensed nuclei with reduced open chromatin-associated acetylation histone marks, and exhibit higher enucleation rates. Lastly, to our surprise, we found that late-stage erythroblasts sustain mitochondrial metabolism and subsequent enucleation, primarily through pyruvate but independent of in situ glycolysis. These findings demonstrate the critical but unanticipated functions of mitochondria during the erythroblast enucleation process. They are also relevant to the in vitro production of RBCs as well as to disorders of the erythroid lineage.

Interclass GPCR heteromerization affects localization and trafficking.

Membrane trafficking processes regulate G protein-coupled receptor (GPCR) activity. Although class A GPCRs are capable of activating G proteins in a monomeric form, they can also potentially assemble into functional GPCR heteromers. Here, we showed that the class A serotonin 5-HT2A receptors (5-HT2ARs) affected the localization and trafficking of class C metabotropic glutamate receptor 2 (mGluR2) through a mechanism that required their assembly as heteromers in mammalian cells. In the absence of agonists, 5-HT2AR was primarily localized within intracellular compartments, and coexpression of 5-HT2AR with mGluR2 increased the intracellular distribution of the otherwise plasma membrane-localized mGluR2. Agonists for either 5-HT2AR or mGluR2 differentially affected trafficking through Rab5-positive endosomes in cells expressing each component of the 5-HT2AR-mGluR2 heterocomplex alone, or together. In addition, overnight pharmacological 5-HT2AR blockade with clozapine, but not with M100907, decreased mGluR2 density through a mechanism that involved heteromerization between 5-HT2AR and mGluR2. Using TAT-tagged peptides and chimeric constructs that are unable to form the interclass 5-HT2AR-mGluR2 complex, we demonstrated that heteromerization was necessary for the 5-HT2AR-dependent effects on mGluR2 subcellular distribution. The expression of 5-HT2AR also augmented intracellular localization of mGluR2 in mouse frontal cortex pyramidal neurons. Together, our data suggest that GPCR heteromerization may itself represent a mechanism of receptor trafficking and sorting.

LRRK2 mutation alters behavioral, synaptic, and nonsynaptic adaptations to acute social stress.

Parkinson's disease (PD) risk is increased by stress and certain gene mutations, including the most prevalent PD-linked mutation LRRK2-G2019S. Both PD and stress increase risk for psychiatric symptoms, yet it is unclear how PD-risk genes alter neural circuitry in response to stress that may promote psychopathology. Here we show significant differences between adult G2019S knockin and wild-type (wt) mice in stress-induced behaviors, with an unexpected uncoupling of depression-like and hedonia-like responses in G2019S mice. Moreover, mutant spiny projection neurons in nucleus accumbens (NAc) lack an adaptive, stress-induced change in excitability displayed by wt neurons, and instead show stress-induced changes in synaptic properties that wt neurons lack. Some synaptic alterations in NAc are already evident early in postnatal life. Thus G2019S alters the magnitude and direction of behavioral responses to stress that may reflect unique modifications of adaptive plasticity in cells and circuits implicated in psychopathology in humans.NEW & NOTEWORTHY Depression is associated with Parkinson's disease (PD), and environmental stress is a risk factor for both. We investigated how LRRK2-G2019S PD mutation affects depression-like behaviors, synaptic function, and intrinsic neuronal excitability following stress. In response to stress, the mutation drives abnormal synaptic changes, prevents adaptive changes in intrinsic excitability, and leads to aberrant behaviors, thus defining new ways in which PD mutations derail adaptive plasticity in response to stress that may contribute to disease onset.

Origins of Parkinson's Disease in Brain Development: Insights From Early and Persistent Effects of LRRK2-G2019S on Striatal Circuits.

Late-onset Parkinson's disease (PD) is dominated clinically and experimentally by a focus on dopamine neuron degeneration and ensuing motor system abnormalities. There are, additionally, a number of non-motor symptoms - including cognitive and psychiatric - that can appear much earlier in the course of the disease and also significantly impair quality of life. The neurobiology of such cognitive and psychiatric non-motor symptoms is poorly understood. The recognition of genetic forms of late-onset PD, which are clinically similar to idiopathic forms in both motor and non-motor symptoms, raises the perspective that brain cells and circuits - and the behaviors they support - differ in significant ways from normal by virtue of the fact that these mutations are carried throughout life, including especially early developmental critical periods where circuit structure and function is particularly susceptible to the influence of experience-dependent activity. In this focused review, we support this central thesis by highlighting studies of LRRK2-G2019S mouse models. We describe work that shows that in G2019S mutants, corticostriatal activity and plasticity are abnormal by P21, the end of a period of excitatory synaptogenesis in striatum. Moreover, by young adulthood, impaired striatal synaptic and non-synaptic forms of plasticity likely underlie altered and variable performance by mutant mice in validated tasks that test for depression-like and anhedonia-like behaviors. Mechanistically, deficits in cellular, synaptic and behavioral plasticity may be unified by mutation-linked defects in trafficking of AMPAR subunits and other membrane channels, which in turn may reflect impairment in the function of the Rab family of GTPases, a major target of LRRK2 phosphorylation. These findings underscore the need to better understand how PD-related mutant proteins influence brain structure and function during an extended period of brain development, and offer new clues for future therapeutic strategies to target non-motor cognitive or psychiatric symptoms of PD.

Restraining Lysosomal Activity Preserves Hematopoietic Stem Cell Quiescence and Potency.

Quiescence is a fundamental property that maintains hematopoietic stem cell (HSC) potency throughout life. Quiescent HSCs are thought to rely on glycolysis for their energy, but the overall metabolic properties of HSCs remain elusive. Using combined approaches, including single-cell RNA sequencing (RNA-seq), we show that mitochondrial membrane potential (MMP) distinguishes quiescent from cycling-primed HSCs. We found that primed, but not quiescent, HSCs relied readily on glycolysis. Notably, in vivo inhibition of glycolysis enhanced the competitive repopulation ability of primed HSCs. We further show that HSC quiescence is maintained by an abundance of large lysosomes. Repression of lysosomal activation in HSCs led to further enlargement of lysosomes while suppressing glucose uptake. This also induced increased lysosomal sequestration of mitochondria and enhanced the competitive repopulation ability of primed HSCs by over 90-fold in vivo. These findings show that restraining lysosomal activity preserves HSC quiescence and potency and may be therapeutically relevant.

Cyfip1 Regulates SynGAP1 at Hippocampal Synapses.

In humans, copy number variations in CYFIP1 appear to have sweeping physiological and structural consequences in the brain, either producing or altering the severity of intellectual disability, autism, and schizophrenia. Independently, SynGAP1 haploinsufficiency produces intellectual disability and, frequently, autism. Cyfip1 inhibits protein translation and promotes actin polymerization, and SynGAP1 is a synaptically localized Ras/Rap GAP. While these proteins are clearly distinct, studies investigating their functions in mice have shown that each regulates the maturation of synapses in the hippocampus and haploinsufficiency for either produces an exaggerated form of mGluR-dependent long-term depression, suggesting that some signaling pathways converge. In this study, we examined how Cyfip1 haploinsufficiency impacts SynGAP1 levels and localization, as well as potential sites for mechanistic interaction in mouse hippocampus. The data show that synaptic, but not total, levels of SynGAP1 in Cyfip1 +/- mice were abnormally low during early postnatal development and in adults. This may be in response to a shift in the balance of kinases that activate SynGAP1 as levels of Cdk5 were reduced and those of activated CaMKII were maintained in Cyfip1 +/- mice compared to wild-type mice. Alternatively, this could reflect altered actin dynamics as Rac1 activity in Cyfip1 +/- hippocampus was boosted significantly compared to wild-type mice, and levels of synaptic F-actin were generally enhanced due in part to an increase in the activity of the WAVE regulatory complex. Decreased synaptic SynGAP1 coupled with a CaMKII-mediated bias toward Rap1 inactivation at synapses is also consistent with increased levels of synaptic GluA2, increased AMPA receptor-mediated responses to stimulation, and increased levels of synaptic mGluR1/5 compared to wild-type mice. Collectively, our data suggest that Cyfip1 regulates SynGAP1 and the two proteins work coordinately at synapses to appropriately direct actin polymerization and GAP activity.

Of Molecules and Mechanisms.

Without question, molecular biology drives modern neuroscience. The past 50 years has been nothing short of revolutionary as key findings have moved the field from correlation toward causation. Most obvious are the discoveries and strategies that have been used to build tools for visualizing circuits, measuring activity, and regulating behavior. Less flashy, but arguably as important are the myriad investigations uncovering the actions of single molecules, macromolecular structures, and integrated machines that serve as the basis for constructing cellular and signaling pathways identified in wide-scale gene or RNA studies and for feeding data into informational networks used in systems biology. This review follows the pathways that were opened in neuroscience by major discoveries and set the stage for the next 50 years.

Are we listening to everything the PARK genes are telling us?

The cardinal motor symptoms that define Parkinson's disease (PD) clinically have been recognized for over 200 years. That these symptoms arise following the loss of dopamine neurons in the substantia nigra has been known for the last 50. These long-established facts have fueled a broadly held expectation that degenerating dopaminergic neurons alone hold the key to understanding and curing PD. This prevalent expectation is at odds with the observation that many nonmotor symptoms, including depression and cognitive inflexibility among others, can appear years earlier than the overt dopaminergic neuron degeneration that drives motor abnormalities and are not improved by levodopa treatment. Thus, preserving or rescuing dopamine neuron health and function is of paramount importance, but this alone fails to capture the underlying neurobiology of earlier-appearing nonmotor symptoms. Insight into the complete landscape of disease-related abnormalities and the context in which they arise can be gleaned from a more comprehensive consideration of the PARK genes that are known to cause PD. Here, we make the case that a full incorporation of research showing when and where PARK genes are expressed as well as the impact of gene mutation on function throughout life, in tandem with research studying how dopaminergic neuron degeneration begins, is essential for a full understanding of the multi-dimensional etiology of PD. A broad view may also reveal something about long-term adjustments cells and systems make in response to gene mutation and help to identify mechanisms conferring the resilience or susceptibility of some cells and systems over others.

Functional and behavioral consequences of Parkinson's disease-associated LRRK2-G2019S mutation.

LRRK2 mutation is the most common inherited, autosomal dominant cause of Parkinson's disease (PD) and has also been observed in sporadic cases. Most mutations result in increased LRRK2 kinase activity. LRRK2 is highly expressed in brain regions that receive dense, convergent innervation by dopaminergic and glutamatergic axons, and its levels rise developmentally coincident with glutamatergic synapse formation. The onset and timing of expression suggests strongly that LRRK2 regulates the development, maturation and function of synapses. Several lines of data in mice show that LRRK2-G2019S, the most common LRRK2 mutation, produces an abnormal gain of pathological function that affects synaptic activity, spine morphology, persistent forms of synapse plasticity and behavioral responses to social stress. Effects of the mutation can be detected as early as the second week of postnatal development and can last or have consequences that extend into adulthood and occur in the absence of dopamine loss. These data suggest that the generation of neural circuits that support complex behaviors is modified by LRRK2-G2019S. Whether such alterations impart vulnerability to neurons directly or indirectly, they bring to the forefront the idea that neural circuits within which dopamine neurons eventually degenerate are assembled and utilized in ways that are distinct from circuits that lack this mutation and may contribute to non-motor symptoms observed in humans with PD.

Parkinson's Disease-Linked LRRK2-G2019S Mutation Alters Synaptic Plasticity and Promotes Resilience to Chronic Social Stress in Young Adulthood.

The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is a prevalent cause of late-onset Parkinson's disease, producing psychiatric and motor symptoms, including depression, that are indistinguishable from sporadic cases. Here we tested how this mutation impacts depression-related behaviors and associated synaptic responses and plasticity in mice expressing a Lrrk2-G2019S knock-in mutation. Young adult male G2019S knock-in and wild-type mice were subjected to chronic social defeat stress (CSDS), a validated depression model, and other tests of anhedonia, anxiety, and motor learning. We found that G2019S mice were highly resilient to CSDS, failing to exhibit social avoidance compared to wild-type mice, many of which exhibited prominent social avoidance and were thus susceptible to CSDS. In the absence of CSDS, no behavioral differences between genotypes were found. Whole-cell recordings of spiny projection neurons (SPNs) in the nucleus accumbens revealed that glutamatergic synapses in G2019S mice lacked functional calcium-permeable AMPARs, and following CSDS, failed to accumulate inwardly rectifying AMPAR responses characteristic of susceptible mice. Based on this abnormal AMPAR response profile, we asked whether long-term potentiation (LTP) of corticostriatal synaptic strength was affected. We found that both D1 receptor (D1R)- and D2R-SPNs in G2019S mutants were unable to express LTP, with D2R-SPNs abnormally expressing long-term depression following an LTP-induction protocol. Thus, G2019S promotes resilience to chronic social stress in young adulthood, likely reflecting synapses constrained in their ability to undergo experience-dependent plasticity. These unexpected findings may indicate early adaptive coping mechanisms imparted by the G2019S mutation.SIGNIFICANCE STATEMENT The G2019S mutation in LRRK2 causes late-onset Parkinson's disease (PD). LRRK2 is highly expressed in striatal neurons throughout life, but it is unclear how mutant LRRK2 affects striatal neuron function and behaviors in young adulthood. We addressed this question using Lrrk2-G2019S knock-in mice. The data show that young adult G2019S mice were unusually resilient to a depression-like syndrome resulting from chronic social stress. Further, mutant striatal synapses were incapable of forms of synaptic plasticity normally accompanying depression-like behavior and important for supporting the full range of cognitive function. These data suggest that in humans, LRRK2 mutation may affect striatal circuit function in ways that alter normal responses to stress and could be relevant for treatment strategies for non-motor PD symptoms.

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos.

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis. This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Visualizing and Characterizing Semaphorin Endocytic Events Using Quantum Dot-Conjugated Proteins.

Neurons can endocytose soluble semaphorins to either initiate or interrupt signaling at the cell membrane. Depending on the cell type and even on the specific subcellular domain, the endocytic process will differ in intensity, speed, and modality, and will subsequently facilitate diverse actions of semaphorin molecules. Therefore, in order to understand the physiology of guidance cues like semaphorins it is important to visualize endocytic events with good spatial and temporal resolution. Here, we describe methods to visualize endocytosed Semaphorin3A (Sema3A) molecules and to characterize the rate and pathway of internalization in primary rat neuronal cultures using semiconductor quantum dot nanoparticles (Q-dots).