Vibrational microspectroscopy of single proteins

Vibrational infrared absorption of a single protein molecule was detected at a few kelvins as infrared-induced recovery of visible fluorescence of a dye with which the protein was labeled. This sensitive method of detecting infrared absorption was demonstrated for a single bovine serum albumin (BSA) molecule labeled with Alexa Fluor 660 by determining the vibrational infrared absorption spectrum of the backbone vibrations of the α-helical structure in the wavelength region around 6 μm (1650 cm -1). In addition to measuring the vibrational infrared absorption spectrum, the visible fluorescence can be simultaneously used for imaging of the same dye-labeled single protein molecules

Calcium-dependent activator protein for secretion 2 (CAPS2) promotes BDNF secretion and is critical for the development of GABAergic interneuron network

Calcium-dependent activator protein for secretion 2 (CAPS2) is a dense-core vesicle-associated protein that is involved in the secretion of BDNF. BDNF has a pivotal role in neuronal survival and development, including the development of inhibitory neurons and their circuits. However, how CAPS2 affects BDNF secretion and its biological significance in inhibitory neurons are largely unknown. Here we reveal the role of CAPS2 in the regulated secretion of BDNF and show the effect of CAPS2 on the development of hippocampal GABAergic systems. We show that CAPS2 is colocalized with BDNF, both synaptically and extrasynaptically in axons of hippocampal neurons. Overexpression of exogenous CAPS2 in hippocampal neurons of CAPS2-KO mice enhanced depolarization-induced BDNF exocytosis events in terms of kinetics, frequency, and amplitude. We also show that in the CAPS2-KO hippocampus, BDNF secretion is reduced, and GABAergic systems are impaired, including a decreased number of GABAergic neurons and their synapses, a decreased number of synaptic vesicles in inhibitory synapses, and a reduced frequency and amplitude of miniature inhibitory postsynaptic currents. Conversely, excitatory neurons in the CAPS2-KO hippocampus were largely unaffected with respect to field excitatory postsynaptic potentials, miniature excitatory postsynaptic currents, and synapse number and morphology. Moreover, CAPS2-KO mice exhibited several GABA system-associated deficits, including reduced late-phase long-term potentiation at CA3–CA1 synapses, decreased hippocampal theta oscillation frequency, and increased anxiety-like behavior. Collectively, these results suggest that CAPS2 promotes activity-dependent BDNF secretion during the postnatal period that is critical for the development of hippocampal GABAergic networks

The brain-specific RasGEF very-KIND is required for normal dendritic growth in cerebellar granule cells and proper motor coordination

<div><p>Very-KIND/Kndc1/KIAA1768 (v-KIND) is a brain-specific Ras guanine nucleotide exchange factor carrying two sets of the kinase non-catalytic C-lobe domain (KIND), and is predominantly expressed in cerebellar granule cells. Here, we report the impact of v-KIND deficiency on dendritic and synaptic growth in cerebellar granule cells in v-KIND knockout (KO) mice. Furthermore, we evaluate motor function in these animals. The gross anatomy of the cerebellum, including the cerebellar lobules, layered cerebellar cortex and densely-packed granule cell layer, in KO mice appeared normal, and was similar to wild-type (WT) mice. However, KO mice displayed an overgrowth of cerebellar granule cell dendrites, compared with WT mice, resulting in an increased number of dendrites, dendritic branches and terminals. Immunoreactivity for vGluT2 (a marker for excitatory presynapses of mossy fiber terminals) was increased in the cerebellar glomeruli of KO mice, compared with WT mice. The postsynaptic density around the terminals of mossy fibers was also increased in KO mice. Although there were no significant differences in locomotor ability between KO and WT animals in their home cages or in the open field, young adult KO mice had an increased grip strength and a tendency to exhibit better motor performance in balance-related tests compared with WT animals. Taken together, our results suggest that v-KIND is required for compact dendritic growth and proper excitatory synaptic connections in cerebellar granule cells, which are necessary for normal motor coordination and balance.</p></div

Increased number of postsynaptic densities in mossy fiber–granule cell synapses in v-KIND KO cerebellum.

<p>(A) Representative electron microscopic images of cerebellar glomeruli in WT (<i>left</i>) and v-KIND KO (<i>right</i>) mice (8-week-old). Arrow heads indicate excitatory synapses between mossy fiber terminals (MFTs) and dendrites of cerebellar granule cells. High power views of each synaptic structure indicated by arrow heads are shown below. Scale bar, 1 μm. (B) Structural analysis of granule cell–mossy fiber synapses in the glomerulus. <i>Left</i>, the number of granule cell postsynaptic densities (PSDs) per MFT was increased in the KO compared with WT. <i>Right</i>, the perimeter of the MFT was not different between KO and WT animals. MFTs in two different electron microscopic images from 3 independent mice (<i>N</i> = 3) for each genotype were analyzed (number of MFTs analyzed: WT, <i>n</i> = 50; KO, <i>n</i> = 53). Data are shown as mean ± SEM. Two sample Student’s <i>t</i>-test assuming equal variances; **<i>p</i> < 0.01.</p

v-KIND KO mice show increased grip strength but no difference in locomotion or wire hanging ability compared to their WT littermates.

<p>Male mice (8–12 weeks of age) were tested. (A) Home cage activity (indicated by the number of crossings of the beam per 15 min) during the dark period for 6 days. WT: <i>n</i> = 9; KO: <i>n</i> = 8. There was no statistically significant difference between the two genotypes under dark (Fig 5A) or light (data not shown) conditions. (B) Distance (cm) traveled in an open field, illuminated at 50 lux, in 3 5-min bins. WT: n = 10; KO: n = 10. (C) Number of rearings in a period of 5 min. WT: <i>n</i> = 9; KO: <i>n</i> = 8. (D) Grip strength (in Newton [N]). WT: <i>n</i> = 6; KO: <i>n</i> = 10. (E) Latency to fall (s) in three trials of wire hanging is shown. WT: <i>n</i> = 6; KO: <i>n</i> = 10. Data are shown as mean ± SEM. Two sample Student’s <i>t</i>-test assuming equal variances; **<i>p</i> < 0.01 in (D).</p

No apparent gross abnormality in the cerebellar lobules or layers, or in the densely-packed granule cell layer in Nissl-stained sections of v-KIND KO cerebella.

<p>Sagittal sections of KO and WT mice cerebella (8-week-old) were analyzed by Nissl staining. (A) The lobular structure of the whole cerebellum. Scale bar, 1 mm. (B) The layer structure of the cerebellar cortex. Scale bar, 100 μm. (C) Magnified view of the granular layer indicated by the red square in (B). Cerebellar lobules II–X. ML, molecular layer; PCL, Purkinje cell layer; GL, granular layer; WM, white matter. (D) Number of Nissl-stained puncta/1,000 μm² in the granule cell layer of WT and KO mice. 5~10 random areas per section, 2 sections per animal, and three mice for each genotype (<i>N</i> = 3) was statistically analyzed. Data are shown as mean ± SEM. Two sample Student’s <i>t</i>-test assuming equal variances showed no statistical significance (<i>p</i> > 0.5).</p

Expression of presynaptic markers for mossy fibers and Golgi cell axons in cerebellar glomeruli of v-KIND KO mice.

<p>(A) Immunohistochemistry of the presynaptic GAD65/67 protein (red) in Golgi cells and the presynaptic vGluT2 (green) in mossy fibers in the granule cell layers of WT and KO mice at 5 weeks of age. Panels a1 and b1 show representative images of co-immunostaining patterns in lobules IV–V of WT and KO mice, respectively. Panels a2 and b2 are magnified images of the cerebellar layer indicated by the white square in a1 and b1, respectively. Panels a3 and b3 are magnified images of the granular layer indicated by the red square in a2 and b2, respectively. Bars: 100 μm in a1 and b1, 10 μm in a2, a3, b2 and b3. Images of vGluT2 immunostaining of whole cerebellar sections are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173175#pone.0173175.s001" target="_blank">S1 Fig</a>. (B) Number of GAD65/67-immunopositive puncta (/1,000 μm²) was calculated by analyzing data from fifteen areas (7,182 μm²/area) (<i>n</i> = 15) of three different animals (<i>N</i> = 3) for each genotype. (C) Number of vGluT2-immunopositive puncta (/1,000 μm²) was calculated by analyzing 26 and 24 areas (2,500 μm²/area) in WT (<i>n</i> = 26) and KO (<i>n</i> = 24) mice, respectively, from five different animals (<i>N</i> = 5) for each genotype. Data are shown as mean ± SEM. Two sample Student’s <i>t</i>-test assuming equal variances (GAD65/67 <i>N</i> = 3; vGluT2 <i>N</i> = 5); ** <i>p</i> < 0.01 in (C).</p

v-KIND KO mice have a tendency to display better motor performance than WT mice.

<p>Balance beam test of WT and v-KIND KO mice. <i>Left</i>, number of slips while the mouse remained on the 9-mm or 6-mm beam. <i>Right</i>, time (s) the mouse crossed the 9-mm or 6-mm beam. Data are shown as mean ± SEM (WT: <i>n</i> = 6; KO: <i>n</i> = 10). *<i>p</i> < 0.05, **<i>p</i> < 0.01 (two-tailed, unequal variances Student’s <i>t</i>-test).</p

Increased branches and terminals of cerebellar granule cell dendrites in v-KIND KO cerebella.

<p>(A) Branched arborization pattern of granule cell dendrites in the granular layer of WT (<i>left</i>) and v-KIND KO (<i>right</i>) mouse cerebella at 8 weeks of age. Granule cells were visualized by DiI staining, and images were obtained by confocal microscopy. Scale bar, 10 μm. (B) Statistical analysis of the number of dendrites per cell (<i>top left</i>), number of branches per dendrite (<i>top right</i>), number of terminals per cell (<i>bottom left</i>) and average length of dendrites (<i>bottom right</i>) of cerebellar granule cells by Student’s <i>t</i>-test. Cells from four animals were analyzed for each genotype (<i>N</i> = 4) (WT: <i>n</i> = 13 cells; KO: <i>n</i> = 12 cells). Data are shown as mean ± SEM. Two sample Student’s <i>t</i>-test assuming equal variances; *<i>p</i> < 0.05, ***<i>p</i> < 0.001.</p