Parkinson's disease (PD) is a pervasive, aging-related neurodegenerative disease whose cardinal motor symptoms reflect the loss of a small group of neurons – dopaminergic neurons in the substantia nigra pars compacta (SNc)1. Mitochondrial oxidant stress is widely viewed as responsible for this loss2, but why these particular neurons should be stressed is a mystery. Using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, it was discovered that the unusual engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. This stress engaged defenses that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, antagonists of cloned uncoupling proteins. Knocking out DJ-1, a gene associated with an early onset form of PD, down-regulated the expression of two uncoupling proteins (UCP4, 5), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of PD.

Parkinson disease is a common neurodegenerative disorder that leads to difficulty in effectively translating thought into action. Although it is known that dopaminergic neurons that innervate the striatum die in Parkinson disease, it is not clear how this loss leads to symptoms. Recent work has implicated striatopallidal medium spiny neurons (MSNs) in this process, but how and precisely why these neurons change is not clear. Using multiphoton imaging, we show that dopamine depletion leads to a rapid and profound loss of spines and glutamatergic synapses on striatopallidal MSNs but not on neighboring striatonigral MSNs. This loss of connectivity is triggered by a new mechanism-dysregulation of intraspine Cav1.3 L-type Ca(2+) channels. The disconnection of striatopallidal neurons from motor command structures is likely to be a key step in the emergence of pathological activity that is responsible for symptoms in Parkinson disease.

A major challenge for fluorescence imaging of living mammalian cells is maintaining viability following prolonged exposure to excitation illumination. We have monitored the dynamics of mitochondrial distribution in hamster embryos at frequent intervals over 24 h using two-photon microscopy (1,047 nm) while maintaining blastocyst, and even fetal, developmental competence. In contrast, confocal imaging for only 8 h inhibits development, even without fluorophore excitation. Photo-induced production of H 2 O 2 may account, in part, for this inhibition. Thus, twophoton microscopy, but not confocal microscopy, has permitted long-term fluorescence observations of the dynamics of three-dimensional cytoarchitecture in highly photosensitive specimens such as mammalian embryos.Keywords two-photon microscopy; laser scanning confocal microscopy; live cell fluorescence imaging; embryo; mitochondrial dynamics; mammal; hamster The detection of specific cellular components by imaging techniques such as wide-field epifluorescence or laser scanning confocal microscopy (LSCM) requires exposure to high intensity light that can cause cellular damage 1 . Consequently, the quantity or quality of images that can be collected is limited or, even worse, the reliability of the images may be compromised. This is a particular problem when imaging events that occur over periods of time ranging from hours to days, such as embryonic development. For this reason, much of our current understanding of subcellular morphological changes during mammalian embryonic development is based on images of fixed or static specimens at different developmental stages [2][3][4][5][6] . Thus, it can be difficult to interpret dynamic processes accurately, because the continuity of events must be inferred. The establishment of long-term fluorescence imaging methods that maintain the viability of live specimens is critical for advancing our understanding of cell biology and embryonic development in areas such as ion dynamics 7 , cytoplasmic reorganization, compaction and blastocoel formation, embryonic development in exotic species (where specimens are heterogeneous and difficult * Corresponding author (jsquirre@students.wisc.edu). to obtain), use of fluorescent tags for the preselection of embryos for subsequent embryo transfer 8 , and studies of protein expression in living cells using green fluorescent protein 9 . HHS Public AccessEmbryos of some mammals, particular hamsters, are very sensitive to culture conditions 10 . Furthermore, studies suggest that mammalian oocytes and embryos are adversely affected by exposure to visible light [11][12][13] . Because of this sensitivity, mammalian embryos are ideal to test live-cell imaging techniques. In addition, there are obvious morphological changes associated with differentiation, namely compaction and blastocoel formation, which can be used to assess viability. The embryo must undergo cell division during and after imaging as well as maintain a level of developmental competence that allows it to initiate dif...

The loss of striatal dopamine (DA) in Parkinson's disease (PD) models triggers a cell-type specific reduction in the density of dendritic spines in D2 receptor-expressing striatopallidal medium spiny neurons (D2 MSNs). How the intrinsic properties of MSN dendrites, where the vast majority of DA receptors are found, contribute to this adaptation is not clear. To address this question, two-photon laser scanning microscopy (2PLSM) was performed in patch-clamped mouse MSNs identified in striatal slices by expression of green fluorescent protein (eGFP) controlled by DA receptor promoters. These studies revealed that single back-propagating action potentials (bAP) produced more reliable elevations in cytosolic Ca2+ concentration at distal dendritic locations in D2 MSNs than at similar locations in D1 receptor-expressing striatonigral MSNs (D1 MSNs). In both cell types, the dendritic Ca2+ entry elicited by bAPs was enhanced by pharmacological blockade of Kv4, but not Kv1 K+ channels. Local application of DA depressed dendritic bAP-evoked Ca2+ transients, whereas application of ACh increased these Ca2+ transients in D2 MSNs—but not in D1 MSNs. Following DA depletion, bAP-evoked Ca2+ transients were enhanced in distal dendrites and spines in D2 MSNs. Taken together, these results suggest that normally D2 MSN dendrites are more excitable than those of D1 MSNs and that DA depletion exaggerates this asymmetry, potentially contributing to adaptations in PD models.

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SUMMARY The two principal movement-suppressing pathways of the basal ganglia, the so-called hyperdirect and indirect pathways interact within the subthalamic nucleus (STN). An appropriate level and pattern of hyperdirect pathway cortical excitation and indirect pathway external globus pallidus (GPe) inhibition of the STN are critical for normal movement and greatly perturbed in Parkinson’s disease. Here, we demonstrate that motor cortical inputs to the STN heterosynaptically regulate through activation of postsynaptic NMDA receptors the number of functional GABAA receptor-mediated GPe-STN inputs. Thus, a homeostatic mechanism, intrinsic to the STN, balances cortical excitation by adjusting the strength of GPe inhibition. However, following loss of dopamine, excessive cortical activation of STN NMDA receptors triggers GPe-STN inputs to strengthen abnormally, contributing to the emergence of pathological, correlated activity.

Summary Loss of noradrenergic locus coeruleus (LC) neurons is a prominent feature of aging–related neurodegenerative diseases, like Parkinson’s disease (PD). The basis of this vulnerability is not understood. To explore possible physiological determinants, LC neurons were studied using electrophysiological and optical approaches in ex vivo mouse brain slices. These studies revealed that autonomous activity in LC neurons was accompanied by oscillations in dendritic Ca2+ concentration attributable to opening of L–type Ca2+ channels. This oscillation elevated mitochondrial oxidant stress and was attenuated by inhibition of nitric oxide synthase. The relationship between activity and stress was malleable, as arousal and carbon dioxide, each increased the spike rate, but differentially affected mitochondrial oxidant stress. Oxidant stress also was increased in an animal model of PD. Thus, our results point to activity–dependent Ca2+ entry and a resulting mitochondrial oxidant stress as factors contributing to the vulnerability of LC neurons.

The behavior of antigen-specific CD4+ T lymphocytes during initial exposure to antigen probably influences their decision to become primed or tolerized, but this has not been examined directly in vivo. We have therefore tracked such cells in real time, in situ during the induction of oral priming versus oral tolerance. There were marked contrasts with respect to rate and type of movement and clustering between naive T cells and those exposed to antigen in immunogenic or tolerogenic forms. However, the major difference when comparing tolerized and primed T cells was that the latter formed larger and longer-lived clusters within mucosal and peripheral lymph nodes. This is the first comparison of the behavior of antigen-specific CD4+ T cells in situ in mucosal and systemic lymphoid tissues during the induction of priming versus tolerance in a physiologically relevant model in vivo.