David L. Wokosin

Selective elimination of glutamatergic synapses on striatopallidal neurons in Parkinson disease models

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 Ca2+ 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.

Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability

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 H2O2 may account, in part, for this inhibition. Thus, two-photon 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.

Oxidant stress evoked by pacemaking in dopaminergic neurons is attenuated by DJ-1

Parkinson’s disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson’s disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), 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 Parkinson’s disease.

Differential excitability and modulation of striatal medium spiny neuron dendrites.

The loss of striatal dopamine (DA) in Parkinsons 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 backpropagating action potentials (bAPs) 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. After DA depletion, bAP-evoked Ca2+ transients were enhanced in distal dendrites and spines in D2 MSNs. 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.

In situ characterization of CD4 T cell behavior in mucosal and systemic lymphoid tissues during the induction of oral priming and tolerance

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.

Convergent cortical innervation of striatal projection neurons

Anatomical studies have led to the assertion that intratelencephalic and pyramidal tract cortical neurons innervate different striatal projection neurons. To test this hypothesis, we measured the responses of mouse striatal neurons to optogenetic activation of intratelencephalic and pyramidal tract axons. Contrary to expectation, direct and indirect pathway striatal spiny projection neurons responded to both intratelencephalic and pyramidal tract activation, arguing that these cortical networks innervate both striatal projection neurons.

MEF-2 regulates activity-dependent spine loss in striatopallidal medium spiny neurons.

Striatal dopamine depletion profoundly reduces the density of spines and corticostriatal glutamatergic synapses formed on D(2) dopamine receptor expressing striatopallidal medium spiny neurons, leaving D(1) receptor expressing striatonigral medium spiny neurons relatively intact. Because D(2) dopamine receptors diminish the excitability of striatopallidal MSNs, the pruning of synapses could be a form of homeostatic plasticity aimed at restoring activity into a preferred range. To characterize the homeostatic mechanisms controlling synapse density in striatal medium spiny neurons, striatum from transgenic mice expressing a D(2) receptor reporter construct was co-cultured with wild-type cerebral cortex. Sustained depolarization of these co-cultures induced a profound pruning of glutamatergic synapses and spines in striatopallidal medium spiny neurons. This pruning was dependent upon Ca(2+) entry through Cav1.2 L-type Ca(2+) channels, activation of the Ca(2+)-dependent protein phosphatase calcineurin and up-regulation of myocyte enhancer factor 2 (MEF2) transcriptional activity. Depolarization and MEF2 up-regulation increased the expression of two genes linked to synaptic remodeling-Nur77 and Arc. Taken together, these studies establish a translational framework within which striatal adaptations linked to the symptoms of Parkinsons disease can be explored.

Imaging Mitochondrial Organization in Living Primate Oocytes and Embryos using Multiphoton Microscopy

We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage-in the same embryo-thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

Cellular Mechanisms Underlying Burst Firing in Substantia Nigra Dopamine Neurons

Burst firing of substantia nigra dopamine (SN DA) neurons is believed to represent an important teaching signal that instructs synaptic plasticity and associative learning. However, the mechanisms through which synaptic excitation overcomes the limiting effects of somatic Ca2+-dependent K+ current to generate burst firing are controversial. Modeling studies suggest that synaptic excitation sufficiently amplifies oscillatory dendritic Ca2+ and Na+ channel currents to lead to the initiation of high-frequency firing in SN DA neuron dendrites. To test this model, visually guided compartment-specific patch-clamp recording and ion channel manipulation were applied to rodent SN DA neurons in vitro. As suggested previously, the axon of SN DA neurons was typically found to originate from a large-diameter dendrite that was proximal to the soma. However, in contrast to the predictions of the model, (1) somatic current injection generated firing that was similar in frequency and form to burst firing in vivo, (2) the efficacy of glutamatergic excitation was inversely related to the distance of excitation from the axon, (3) pharmacological blockade or genetic deletion of Ca2+ channels did not prevent high-frequency firing, (4) action potential bursts were invariably detected first at sites that were proximal to the axon, and (5) pharmacological blockade of Na+ channels in the vicinity of the axon/soma but not dendritic excitation impaired burst firing. Together, these data suggest that SN DA neurons integrate their synaptic input in a more conventional manner than was hypothesized previously.

Characterization of a Range of Fura Dyes with Two-Photon Excitation

Two-photon excitation (TPE) spectra of Fura-2, -4F, -6F, -FF, and Furaptra were characterized using a tunable (750-850 nM) ultra-short pulse laser. Two-photon fluorescence of these dyes was studied in free solution and in the cytosol of isolated rabbit ventricular cardiomyocytes. The TPE spectra of the Ca(2+)-free and Ca(2+)-bound forms of the dyes were measured in free solution and expressed in terms of the two-photon fluorescence cross section (Goppert-Meyer units). The Fura dyes displayed the same Ca(2+)-free TPE spectrum in the intracellular volume of permeabilized and intact cardiomyocytes. Fluorescence measurements over a range of laser powers confirmed the TPE of both Ca(2+)-free and Ca(2+)-bound forms of the dyes. Single-wavelength excitation at 810 nM was used to determine the effective dissociation constants (K(eff)) and dynamic ranges (R(f)) of Fura-2, -4F, -6F, -FF, and Furaptra dyes (K(eff) = 181 +/- 52 nM, 1.16 +/- 0.016 micro M, 5.18 +/- 0.3 micro M, 19.2 +/- 1 micro M, and 58.5 +/- 2 micro M; and R(f) = 22.4 +/- 3.8, 12.2 +/- 0.34, 6.3 +/- 0.17, 16.1 +/- 2.8, and 25.4 +/- 4, respectively). Single-wavelength excitation of intracellular Fura-4F resolved diastolic and peak [Ca(2+)] in isolated stimulated cardiomyocytes after calibration of the intracellular signal using reversible exposure to low (100 micro M) extracellular [Ca(2+)]. Furthermore, TPE of Fura-4F allowed continuous, long-term (5-10 min) Ca(2+) imaging in ventricular cardiomyocytes using laser-scanning microscopy without significant cellular photodamage or photobleaching of the dye.