Highlights d Fibrinogen is a blood-derived inducer of spine elimination d Fibrinogen promotes synapse loss in AD mice d 3D volume imaging of BBB disruption and Ab in cleared human AD brain d Genetic inhibition of fibrinogen-CD11b binding improves cognition in AD mice

Immunocytochemical and electrophysiological techniques were used to localize TTX-sensitive sodium channels (NaChs) over the soma-dendritic axis of basilar and nonbasilar pyramidal cells of the electrosensory lateral line lobe (ELL) of weakly electric fish (Apteronotus leptorhynchus). Dense NaCh-like immunolabel was detected on the membranes of basilar and nonbasilar pyramidal cell somata. Punctate regions of immunolabel (approximately 15 microns) were separated by nonlabeled expanses of membrane over the entire extent of basal dendrites. Similar punctate immunolabel was observed over the apical dendrites, and frequently on membranes of afferent parallel fiber boutons in the distal apical dendritic region. Intracellular recordings from pyramidal cell somata or proximal apical dendrites (75–200 microns) were obtained using an in vitro ELL slice preparation. TTX- sensitive potentials were identified by focal pressure ejection of TTX. Somatic recordings demonstrated both TTX-sensitive fast spike discharge and a slow prepotential; similar but lower amplitude potentials were recorded in apical dendrites. Dendritic spikes were composed of at least two active components triggered by a fast prepotential (FPP) generated by the somatic spike. TTX-sensitive spikes propagated in a retrograde fashion over at least the proximal 200 microns of the apical dendrites, as determined by the conduction of an antidromic population spike and focal TTX ejections. Somatic spikes were followed by a depolarizing afterpotential (DAP) that was similar in duration and refractory period to that of proximal dendritic spikes. During repetitive spike discharge, the DAP could increase in amplitude and attain somatic spike threshold, generating a high-frequency spike doublet and a subsequent hyperpolarization that terminated spike discharge. Repetition of this process gave rise to an oscillatory burst discharge (2–6 spikes/burst) with a frequency of 40–80 Hz. Both the DAP and oscillatory discharge were selectively blocked by TTX ejections restricted to the proximal apical dendritic region. The present study demonstrates an immunolocalization of NaChs over somatic and dendritic membranes of a vertebrate sensory neuron that correlates with the distribution of TTX-sensitive potentials. The interaction of somatic and dendritic action potentials is further shown to underlie an oscillatory discharge believed to be important in electrosensory processing.

Serial block face scanning electron microscopy (SBFSEM) is a powerful technique originally introduced by Leighton [1], substantially improved by Denk [2] and subsequently commercialized (Gatan Inc., Pleasanton, CA.). SBFSEM allows for the automated image acquisition of relatively large volumes of tissue at near nanometer-scale resolution, using a dry cutting ultramicrotome fitted into an SEM. In an automated process, a low voltage backscatter electron (BSE) image is obtained from the surface of an epoxy embedded tissue block face. The ultramicrotome then removes an ultra-thin section of tissue with a specially designed oscillating diamond knife (Diatome AG, Switzerland), and a block face image from the corresponding region is again obtained. This sequence is repeated over and over until the desired volume of tissue has been imaged. Although SBFSEM overcomes many obstacles routinely encountered with serial section TEM reconstruction, until recently there was a significant limitation to the resolution obtainable by this method compared to conventional TEM. This was due primarily to difficulties encountered using BSE imaging at low accelerating voltages. To overcome this we have developed a protocol for vastly increasing the heavy metal staining of specimens to improve BSE yield. This is accomplished by combining a variety of preexisting heavy metal staining methodologies not normally used together, including ferrocyanide-reduced osmium tetroxide, thiocarbohydrazide-osmium tetroxide (OTO), prolonged uranyl acetate treatment and en bloc lead aspartate staining. Using this approach, we demonstrate a dramatic improvement in image contrast and resolution from existing methods in a variety of specimens (Fig. 1).We have also combined this approach with a number of selective labeling methods such as Golgi impregnation to allow the reconstruction of whole cells in the nervous system (Fig. 2), as well as fluorescence photoconversion to label specifically targeted proteins [3,4]. Additionally, a powerful application of SBFSEM is its use in conjunction with a newly developed genetically encoded fluorescent reporter termed miniSOG (for mini singlet oxygen generator). MiniSOG is a small (106-residue) singlet oxygen generating protein engineered from a flavin-binding, blue light phototropin from Arabidopsis thaliana. MiniSOG has quantum yields for fluorescence and singlet oxygen of 0.30 and 0.47 respectively. It can be genetically fused to the target protein of interest for both fluorescence imaging and efficient photooxidation of diaminobenzidine into an osmiophilic polymer for subsequent electron microscopy [3]. Since miniSOG is genetically encoded and all other reactants (O 2 , diaminobenzidine, OsO 4 ) are permeant small molecules, there is no need to compromise chemical fixation to preserve protein epitopes or to permeabilize with detergents, which further degrade cellular ultrastructure. We have combined this approach with the intense heavy metal staining procedure outlined above for SBFSEM to enable 3D localization of gen...

Glutamate excitotoxicity leads to fragmented mitochondria in neurodegenerative diseases, mediated by nitric oxide and S-nitrosylation of dynamin-related protein 1, a mitochondrial outer membrane fission protein. Optic atrophy gene 1 (OPA1) is an inner membrane protein important for mitochondrial fusion. Autosomal dominant optic atrophy (ADOA), caused by mutations in OPA1, is a neurodegenerative disease affecting mainly retinal ganglion cells (RGCs). Here, we showed that OPA1 deficiency in an ADOA model influences N-methyl-D-aspartate (NMDA) receptor expression, which is involved in glutamate excitotoxicity and oxidative stress. Opa1enu/+ mice show a slow progressive loss of RGCs, activation of astroglia and microglia, and pronounced mitochondrial fission in optic nerve heads as found by electron tomography. Expression of NMDA receptors (NR1, 2A, and 2B) in the retina of Opa1enu/+ mice was significantly increased as determined by western blot and immunohistochemistry. Superoxide dismutase 2 (SOD2) expression was significantly decreased, the apoptotic pathway was activated as Bax was increased, and phosphorylated Bad and BcL-xL were decreased. Our results conclusively demonstrate that not only glutamate excitotoxicity and/or oxidative stress alters mitochondrial fission/fusion, but that an imbalance in mitochondrial fission/fusion in turn leads to NMDA receptor upregulation and oxidative stress. Therefore, we propose a new vicious cycle involved in neurodegeneration that includes glutamate excitotoxicity, oxidative stress, and mitochondrial dynamics.

Glaucoma is the leading cause of irreversible blindness and is characterized by slow and progressive degeneration of the optic nerve head axons and retinal ganglion cell (RGC), leading to loss of visual function. Although oxidative stress and/or alteration of mitochondrial (mt) dynamics induced by elevated intraocular pressure (IOP) are associated with this neurodegenerative disease, the mechanisms that regulate mt dysfunction-mediated glaucomatous neurodegeneration are poorly understood. Using a mouse model of glaucoma, DBA/2J (D2), which spontaneously develops elevated IOP, as well as an in vitro RGC culture system, we show here that oxidative stress, as evidenced by increasing superoxide dismutase 2 (SOD2) and mt transcription factor A (Tfam) protein expression, triggers mt fission and loss by increasing dynamin-related protein 1 (DRP1) in the retina of glaucomatous D2 mice as well as in cultured RGCs exposed to elevated hydrostatic pressure in vitro. DRP1 inhibition by overexpressing DRP1 K38A mutant blocks mt fission and triggers a subsequent reduction of oxidative stress, as evidenced by decreasing SOD2 and Tfam protein expression. DRP1 inhibition promotes RGC survival by increasing phosphorylation of Bad at serine 112 in the retina and preserves RGC axons by maintaining mt integrity in the glial lamina of glaucomatous D2 mice. These findings demonstrate an important vicious cycle involved in glaucomatous neurodegeneration that starts with elevated IOP producing oxidative stress; the oxidative stress then leads to mt fission and a specific form of mt dysfunction that generates further oxidative stress, thus perpetuating the cycle. Our findings suggest that DRP1 is a potential therapeutic target for ameliorating oxidative stress-mediated mt fission and dysfunction in RGC and its axons during glaucomatous neurodegeneration. Thus, DRP1 inhibition may provide a new therapeutic strategy for protecting both RGCs and their axons in glaucoma and other optic neuropathies.