Summary The basis for selective death of specific neuronal populations in neurodegenerative diseases remains unclear. Parkinson's disease (PD) is a synucleinopathy characterized by a preferential loss of dopaminergic neurons in the substantia nigra (SN), whereas neurons of the ventral tegmental area (VTA) are spared. Using intracellular patch electrochemistry to directly measure cytosolic dopamine (DAcyt) in cultured midbrain neurons, we confirm that elevated DAcyt and its metabolites are neurotoxic and that genetic and pharmacological interventions that decrease DAcyt provide neuroprotection. L-DOPA increased DAcyt in SN neurons to levels 2-3-fold higher than in VTA neurons, a response dependent on dihydropyridine-sensitive Ca2+ channels, resulting in greater susceptibility of SN neurons to L-DOPA-induced neurotoxicity. DAcyt was not altered by α-synuclein deletion, although dopaminergic neurons lacking α-synuclein were resistant to L-DOPA-induced cell death. Thus, an interaction between Ca2+, DAcyt and α-synuclein may underlie the susceptibility of SN neurons in PD, suggesting multiple therapeutic targets.

Within glomeruli, the initial sites of synaptic integration in the olfactory pathway, olfactory sensory axons terminate on dendrites of projection and juxtaglomerular (JG) neurons. JG cells form at least two major circuits: the classic intraglomerular circuit consisting of external tufted (ET) and periglomerular (PG) cells and an interglomerular circuit comprised of the long-range connections of short axon (SA) cells. We examined the projections and the synaptic inputs of identified JG cell chemotypes using mice expressing green fluorescent protein (GFP) driven by the promoter for glutamic acid decarboxylase (GAD) 65 kDa, 67 kDa, or tyrosine hydroxylase (TH). Virtually all (97%) TH+ cells are also GAD67+ and are thus DAergic–GABAergic neurons. Using a combination of retrograde tracing, whole-cell patch-clamp recording, and single-cell three-dimensional reconstruction, we show that different JG cell chemotypes contribute to distinct microcircuits within or between glomeruli. GAD65 + GABAergic PG cells ramify principally within one glomerulus and participate in uniglomerular circuits. DAergic–GABAergic cells have extensive interglomerular projections. DAergic–GABAergic SA cells comprise two subgroups. One subpopulation contacts 5–12 glomeruli and is referred to as “oligoglomerular.” Approximately one-third of these oligoglomerular DAergic SA cells receive direct olfactory nerve (ON) synaptic input, and the remaining two-thirds receive input via a disynaptic ON→ET→SA circuit. The second population of DAergic–GABAergic SA cells also disynaptic ON input and connect tens to hundreds of glomeruli in an extensive “polyglomerular” network. Although DAergic JG cells have traditionally been considered PG cells, their interglomerular connections argue that they are more appropriately classified as SA cells.

The thermoreversible gelation of methylcellulose in aqueous solution has been studied by static and dynamic light scattering (DLS), small-angle neutron scattering (SANS), and rheology. At 20 °C, dilute solution light scattering establishes the molecular weight, second virial coefficient, radius of gyration, and hydrodynamic radius of the polymer. Semidilute solutions exhibit two relaxation modes in DLS, one reflecting cooperative diffusion and the other attributable to pregel clusters. Rheological measurements in this regime also suggest a weak supermolecular association. The gelation of semidilute solutions proceeds in two stages with increasing temperature above 20 °C, consistent with previous reports. The first stage is attributable to clustering of chains, driven by hydrophobic association, and extends up to approximately 50 °C. This process is accompanied by an increase in the low-frequency dynamic elastic modulus, G‘, and an increase in both light and neutron scattered intensity. The DLS properties of these solutions, and the angular dependence of the scattered intensity, is not greatly affected by this association. The second stage of gelation occurs rather abruptly above ca. 50 °C and is attributed to phase separation accompanied by gelation. The elastic modulus increases rapidly with temperature, the samples become visibly turbid, and the scattered intensity increases markedly over a wide range of scattering wavevector, q. In contrast, dilute solutions do not gel but give clear evidence of aggregation in the high-temperature regime, consistent with crossing a phase boundary. The SANS structure factor S(q) in the gel state is well described by a sum of two terms, corresponding to two power-law regimes. The lower q regime follows S(q) ∼ q -1.8 in both the pregel and gel states, consistent with chains intermediate between good and ϑ solvent conditions. At higher q the exponent evolves from ca. −2.5 to −4 upon gelation. The latter exponent indicates a sharp boundary between the gel structure and the intervening fluid, consistent with liquid−liquid phase separation that is arrested by gelation.

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra

We describe a strategy for developing hydrophilic chemical cocktails for tissue delipidation, decoloring, refractive index (RI) matching, and decalcification, based on comprehensive chemical profiling. More than 1,600 chemicals were screened by a high-throughput evaluation system for each chemical process. The chemical profiling revealed important chemical factors: salt-free amine with high octanol/water partition-coefficient (logP) for delipidation, N-alkylimidazole for decoloring, aromatic amide for RI matching, and protonation of phosphate ion for decalcification. The strategic integration of optimal chemical cocktails provided a series of CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis) protocols, which efficiently clear mouse organs, mouse body including bone, and even large primate and human tissues. The updated CUBIC protocols are scalable and reproducible, and they enable three-dimensional imaging of the mammalian body and large primate and human tissues. This strategy represents a future paradigm for the rational design of hydrophilic clearing cocktails that can be used for large tissues.

This study aimed to generate a mouse model of acquired glomerular sclerosis. A model system that allows induction of podocyte injury in a manner in which onset and severity can be controlled was designed. A transgenic mouse strain (NEP25) that expresses human CD25 selectively in podocytes was first generated. Injection of anti-Tac (Fv)-PE38 (LMB2), an immunotoxin with specific binding to human CD25, induced progressive nonselective proteinuria, ascites, and edema in NEP25 mice. Podocytes showed foot process effacement, vacuolar degeneration, detachment and downregulation of synaptopodin, WT-1, nephrin, and podocalyxin. Mesangial cells showed matrix expansion, increased collagen, mesangiolysis, and, later, sclerosis. Parietal epithelial cells showed vacuolar degeneration and proliferation, whereas endothelial cells were swollen. The severity of the glomerular injury was LMB2 dose dependent. With 1.25 ng/g body wt or more, NEP25 mice developed progressive glomerular damage and died within 2 wk. With 0.625 ng/g body wt of LMB2, NEP25 mice survived >4 wk and developed focal segmental glomerular sclerosis. Thus, the study has established a mouse model of acquired progressive glomerular sclerosis in which onset and severity can be preprogrammed by experimental maneuvers.

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Dopamine (DA)-producing neurons in the ventral midbrain are generated from a specified neuronal lineage and form selective axonal pathways that mediate multiple CNS functions. Expression of the gene encoding tyrosine hydroxylase (TH), which is a key enzyme of catecholamine biosynthesis, is regulated during the development of midbrain DA neurons. In the present study, we report the developmental regulation and cell type specificity of TH gene promoter in the ventral midbrain by using a green fluorescent protein (GFP) reporter system. Transgenic mice were generated that express GFP in the majority of midbrain DA neurons under the control of the 9-kb upstream region of the rat TH gene. At an early embryonic stage, GFP expression was induced in the developing DA neurons, and the expression was then markedly down-regulated at later embryonic stages. However, the expression was reactivated and approached the adult levels during early postnatal development. These developmental changes in GFP expression patterns suggest the presence of multistep regulatory mechanisms for TH gene expression during DA neuron development. The TH promoter appears to possess transcriptional elements at least necessary for the induction of TH expression at the early embryonic stage and its reactivation during the post-natal development.