Modifications to the gene encoding human alpha-synuclein have been linked to development of Parkinson’s disease. The highly conserved structure of alpha-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human alpha-synuclein including new genetic tags developed for correlated LM and EM (the tetracysteine-biarsenical labeling system or the new fluorescent protein for EM, MiniSOG), we determined the distribution of alpha-synuclein when over-expressed in primary neurons at supramolecular and cellular scales, in three dimensions (3D). We observed specific association of alpha-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, alpha-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice over-expressing human alpha-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. 3D electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulo-vesicular structures similar to what observed in vitro. We propose that alpha-synuclein over-expression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that alpha- synuclein is involved in processes associated with the sorting, channeling, packaging and transport of synaptic material destined for degradation.

No abstract

No abstract

Recent advances in high-throughput technology facilitate massive data collection and sharing, enabling neuroscientists to explore the brain across a large range of spatial scales. One such form of high-throughput data collection is the construction of large-scale mosaic volumes using light microscopy (Chow et al., 2006; Price et al., 2006). With this technology, researchers can collect and analyze high-resolution digitized volumes of whole brain sections down to 0.2 μm. However, until recently, scientists lacked the tools to easily handle these large high-resolution datasets. Furthermore, artifacts resulting from specimen preparation limited volume reconstruction using this technique to only a single tissue section. In this paper, we carefully describe the steps we used to digitally reconstruct a series of consecutive mouse brain sections labeled with three stains, a stain for blood vessels (DiI), a nuclear stain (TO-PRO-3), and a myelin stain (FluoroMyelin). These stains label important neuroanatomical landmarks that are used for stacking the serial sections during reconstruction. In addition, we show that the use of two software applications, ir-Tweak and Mogrifier, in conjunction with a volume flattening procedure enable scientists to adeptly work with digitized volumes despite tears and thickness variations within tissue sections. These applications make processing large-scale brain mosaics more efficient. When used in combination with new database resources, these brain maps should allow researchers to extend the lifetime of their specimens indefinitely by preserving them in digital form, making them available for future analyses as our knowledge in the field of neuroscience continues to expand.

The study of dopaminergic influences on acetylcholine release is especially useful for the understanding of a wide range of brain functions and neurological disorders, including schizophrenia, Parkinson's disease, Alzheimer's disease, and drug addiction. These disorders are characterized by a neurochemical imbalance of a variety of neurotransmitter systems, including the dopamine and acetylcholine systems. Dopamine modulates acetylcholine levels in the brain by binding to dopamine receptors located directly on cholinergic cells. The dopamine D5 receptor, a D1-class receptor subtype, potentiates acetylcholine release and has been investigated as a possible substrate underlying a variety of brain functions and clinical disorders. This receptor subtype, therefore, may prove to be a putative target for pharmacotherapeutic strategies and cognitive-behavioral treatments aimed at treating a variety of neurological disorders. The present study investigated whether cholinergic cells in the dopamine targeted areas of the cerebral cortex, striatum, basal forebrain, and diencephalon express the dopamine D5 receptor. These receptors were localized on cholinergic neurons with dual labeling immunoperoxidase or immunofluorescence procedures using antibodies directed against choline acetyltransferase (ChAT) and the dopamine D5 receptor. Results from this study support previous findings indicating that striatal cholinergic interneurons express the dopamine D5 receptor. In addition, cholinergic neurons in other critical brain areas also show dopamine D5 receptor expression. Dopamine D5 receptors were localized on the somata, dendrites, and axons of cholinergic cells in each of the brain areas examined. These findings support the functional importance of the dopamine D5 receptor in the modulation of acetylcholine release throughout the brain.