Akari Hagiwara

Non-redundant odor coding by sister mitral cells revealed by light addressable glomeruli in the mouse

Sensory inputs frequently converge on the brain in a spatially organized manner, often with overlapping inputs to multiple target neurons. Whether the responses of target neurons with common inputs become decorrelated depends on the contribution of local circuit interactions. We addressed this issue in the olfactory system using newly generated transgenic mice that express channelrhodopsin-2 in all of the olfactory sensory neurons. By selectively stimulating individual glomeruli with light, we identified mitral/tufted cells that receive common input (sister cells). Sister cells had highly correlated responses to odors, as measured by average spike rates, but their spike timing in relation to respiration was differentially altered. In contrast, non-sister cells correlated poorly on both of these measures. We suggest that sister mitral/tufted cells carry two different channels of information: average activity representing shared glomerular input and phase-specific information that refines odor representations and is substantially independent for sister cells.

Serotonergic modulation of odor input to the mammalian olfactory bulb

Centrifugal serotonergic fibers innervate the olfactory bulb, but the importance of these projections for olfactory processing is unclear. We examined serotonergic modulation of sensory input to olfactory glomeruli using mice that express synaptopHluorin in olfactory receptor neurons (ORN). Odor-evoked synaptic input to glomeruli was attenuated by increased serotonin signaling through serotonin 2C (5-HT2C) receptors and amplified by decreased serotonergic activity. Intravital multiphoton calcium imaging revealed that 5-HT2C receptor activation amplified odor-evoked activity in a subset of juxtaglomerular cells and attenuated glutamate release from ORN terminals via GABAB receptors. Endogenous serotonin released by electrical stimulation of the dorsal raphe nucleus attenuated odor-evoked responses without detectable bias in glomerular position or odor identity. Weaker glomerular responses, however, were less sensitive to raphe stimulation than strong responses. Our data indicate that the serotonergic system regulates odor inputs in the olfactory bulb and suggest that behavioral states may alter odor processing at the earliest stages.

Fibulin-5/DANCE has an elastogenic organizer activity that is abrogated by proteolytic cleavage in vivo

Elastic fibers are required for the elasticity and integrity of various organs. We and others previously showed that fibulin-5 (also called developing arteries and neural crest EGF-like [DANCE] or embryonic vascular EGF-like repeat–containing protein [EVEC]) is indispensable for elastogenesis by studying fibulin-5–deficient mice, which recapitulate human aging phenotypes caused by disorganized elastic fibers (Nakamura, T., P.R. Lozano, Y. Ikeda, Y. Iwanaga, A. Hinek, S. Minamisawa, C.F. Cheng, K. Kobuke, N. Dalton, Y. Takada, et al. 2002. Nature. 415:171–175; Yanagisawa, H., E.C. Davis, B.C. Starcher, T. Ouchi, M. Yanagisawa, J.A. Richardson, and E.N. Olson. 2002. Nature. 415:168–171). However, the molecular mechanism by which fiblin-5 contributes to elastogenesis remains unknown. We report that fibulin-5 protein potently induces elastic fiber assembly and maturation by organizing tropoelastin and cross-linking enzymes onto microfibrils. Deposition of fibulin-5 on microfibrils promotes coacervation and alignment of tropoelastins on microfibrils, and also facilitates cross-linking of tropoelastin by tethering lysyl oxidase-like 1, 2, and 4 enzymes. Notably, recombinant fibulin-5 protein induced elastogenesis even in serum-free conditions, although elastogenesis in cell culture has been believed to be serum-dependent. Moreover, the amount of full-length fibulin-5 diminishes with age, while truncated fibulin-5, which cannot promote elastogenesis, increases. These data suggest that fibulin-5 could be a novel therapeutic target for elastic fiber regeneration.

Immunocytochemical localization of the α1A subunit of the P/Q-type calcium channel in the rat cerebellum

Among various types of low‐ and high‐threshold calcium channels, the high voltage‐activated P/Q‐type channel is the most abundant in the cerebellum. These P/Q‐type channels are involved in the regulation of neurotransmitter release and in the integration of dendritic inputs. We used an antibody specific for the α1A subunit of the P/Q‐type channel in quantitative pre‐embedding immunogold labelling combined with three‐dimensional reconstruction to reveal the subcellular distribution of pre‐ and postsynaptic P/Q‐type channels in the rat cerebellum. At the light microscopic level, immunoreactivity for the α1A protein was prevalent in the molecular layer, whereas immunostaining was moderate in the somata of Purkinje cells and weak in the granule cell layer. At the electron microscopic level, the most intense immunoreactivity for the α1A subunit was found in the presynaptic active zone of parallel fibre varicosities. The dendritic spines of Purkinje cells were also strongly labelled with the highest density of immunoparticles detected within 180 nm from the edge of the asymmetrical parallel fibre–Purkinje cell synapses. By contrast, the immunolabelling was sparse in climbing fibre varicosities and axon terminals of GABAergic cells, and weak and diffuse in dendritic shafts of Purkinje cells. The association of the α1A subunit with the glutamatergic parallel fibre–Purkinje cell synapses suggests that presynaptic channels have a major role in the mediation of excitatory neurotransmission, whereas postsynaptic channels are likely to be involved in depolarization‐induced generation of local calcium transients in Purkinje cells.

Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling

Synaptic vesicle release occurs at a specialized membrane domain known as the presynaptic active zone (AZ). Several membrane proteins are involved in the vesicle release processes such as docking, priming, and exocytotic fusion. Cytomatrix at the active zone (CAZ) proteins are structural components of the AZ and are highly concentrated in it. Localization of other release‐related proteins including target soluble N‐ethylmaleimide‐sensitive‐factor attachment protein receptor (t‐SNARE) proteins, however, has not been well demonstrated in the AZ. Here, we used sodium dodecyl sulfate‐digested freeze‐fracture replica labeling (SDS‐FRL) to analyze quantitatively the distribution of CAZ and t‐SNARE proteins in the hippocampal CA3 area. The AZ in replicated membrane was identified by immunolabeling for CAZ proteins (CAZ‐associated structural protein [CAST] and Bassoon). Clusters of immunogold particles for these proteins were found on the P‐face of presynaptic terminals of the mossy fiber and associational/commissural (A/C) fiber. Co‐labeling with CAST revealed distribution of the t‐SNARE proteins syntaxin and synaptosomal‐associated protein of 25 kDa (SNAP‐25) in the AZ as well as in the extrasynaptic membrane surrounding the AZ (SZ). Quantitative analysis demonstrated that the density of immunoparticles for CAST in the AZ was more than 100 times higher than in the SZ, whereas that for syntaxin and SNAP‐25 was not significantly different between the AZ and SZ in both the A/C and mossy fiber terminals. These results support the involvement of the t‐SNARE proteins in exocytotic fusion in the AZ and the role of CAST in specialization of the membrane domain for the AZ. J. Comp. Neurol. 489:195–216, 2005.

Deletion of the Presynaptic Scaffold CAST Reduces Active Zone Size in Rod Photoreceptors and Impairs Visual Processing

How size and shape of presynaptic active zones are regulated at the molecular level has remained elusive. Here we provide insight from studying rod photoreceptor ribbon-type active zones after disruption of CAST/ERC2, one of the cytomatrix of the active zone (CAZ) proteins. Rod photoreceptors were present in normal numbers, and the a-wave of the electroretinogram (ERG)—reflecting their physiological population response—was unchanged in CAST knock-out (CAST−/−) mice. Using immunofluorescence and electron microscopy, we found that the size of the rod presynaptic active zones, their Ca2+ channel complement, and the extension of the outer plexiform layer were diminished. Moreover, we observed sprouting of horizontal and bipolar cells toward the outer nuclear layer indicating impaired rod transmitter release. However, rod synapses of CAST−/− mice, unlike in mouse mutants for the CAZ protein Bassoon, displayed anchored ribbons, normal vesicle densities, clustered Ca2+ channels, and essentially normal molecular organization. The reduction of the rod active zone size went along with diminished amplitudes of the b-wave in scotopic ERGs. Assuming, based on the otherwise intact synaptic structure, an unaltered function of the remaining release apparatus, we take our finding to suggest a scaling of release rate with the size of the active zone. Multielectrode-array recordings of retinal ganglion cells showed decreased contrast sensitivity. This was also observed by optometry, which, moreover, revealed reduced visual acuity. We conclude that CAST supports large active zone size and high rates of transmission at rod ribbon synapses, which are required for normal vision.

Optophysiological analysis of associational circuits in the olfactory cortex

Primary olfactory cortical areas receive direct input from the olfactory bulb, but also have extensive associational connections that have been mainly studied with classical anatomical methods. Here, we shed light on the functional properties of associational connections in the anterior and posterior piriform cortices (aPC and pPC) using optophysiological methods. We found that the aPC receives dense functional connections from the anterior olfactory nucleus (AON), a major hub in olfactory cortical circuits. The local recurrent connectivity within the aPC, long invoked in cortical autoassociative models, is sparse and weak. By contrast, the pPC receives negligible input from the AON, but has dense connections from the aPC as well as more local recurrent connections than the aPC. Finally, there are negligible functional connections from the pPC to aPC. Our study provides a circuit basis for a more sensory role for the aPC in odor processing and an associative role for the pPC.

Prickle2 is localized in the postsynaptic density and interacts with PSD-95 and NMDA receptors in the brain

The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.

Vangl2, the planner cell polarity protein, is complexed with postsynaptic density protein PSD-95

Vangl is a component of the non‐canonical Wnt/planar cell polarity pathway, which is implicated in various cell polarity functions. However, little is known about its synaptic localization in neurons. Here, we show that Vangl1 and Vangl2 are expressed in adult rat neurons, where they are tightly associated with the postsynaptic density (PSD) fraction. Vangl2 forms a complex with PSD‐95 through direct binding. Furthermore, the C‐terminal PDZ‐binding motif of Vangl2 is required for localization to dendritic spines. These results suggest that Vangl2 is a new component of the PSD that forms a complex with PSD‐95 in the adult brain.

Distribution of serine/threonine kinase SAD-B in mouse peripheral nerve synapse.

The serine/threonine kinase SAD regulates neural functions such as axon/dendrite polarization and neurotransmitter release. In the vertebrate central nervous system, SAD-B, a homolog of Caenorhabditis elegans SAD-1, is associated with synaptic vesicles and the active zone cytomatrix in nerve terminals. However, the distribution of SAD-B in the peripheral nervous system remains elusive. Here, we show that SAD-B is specifically localized to neuromuscular junctions. Although the active zone protein bassoon showed a punctated signal indicating its localization to motor end plates, SAD-B shows relatively diffuse localization indicating its association with both the active zone and synaptic vesicles. Therefore, SAD kinase may regulate neurotransmitter release from motor end plates in a similar manner to its regulation of neurotransmitter release in the central nervous system.