Makoto Itakura

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC β-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The β construct beta-AID dominant negative, which disrupts the RIM1-β association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with β in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

Left-right asymmetry of the hippocampal synapses with differential subunit allocation of glutamate receptors

Left-right asymmetry of the brain has been studied mostly through psychological examination and functional imaging in primates, leaving its molecular and synaptic aspects largely unaddressed. Here, we show that hippocampal CA1 pyramidal cell synapses differ in size, shape, and glutamate receptor expression depending on the laterality of presynaptic origin. CA1 synapses receiving neuronal input from the right CA3 pyramidal cells are larger and have more perforated PSD and a GluR1 expression level twice as high as those receiving input from the left CA3. The synaptic density of GluR1 increases as the size of a synapse increases, whereas that of NR2B decreases because of the relatively constant NR2B expression in CA1 regardless of synapse size. Densities of other major glutamate receptor subunits show no correlation with synapse size, thus resulting in higher net expression in synapses having right input. Our study demonstrates universal left-right asymmetry of hippocampal synapses with a fundamental relationship between synaptic area and the expression of glutamate receptor subunits.

TARPs γ-2 and γ-7 are essential for AMPA receptor expression in the cerebellum

The α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, γ‐2 and γ‐7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, γ‐2 and γ‐7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in γ‐2‐knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in γ‐7‐KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in γ‐2/γ‐7 double‐KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in γ‐2‐KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber–Purkinje cell synapses, and AMPA receptor‐mediated currents at climbing fiber–Purkinje cell synapses were all reduced to approximately 10% of the wild‐type levels in DKO mice. On the other hand, the reduction in GluA4 in γ‐7‐KO granular layer reflected its loss at mossy fiber–granule cell synapses, whereas that of GluA1 and GluA4 in γ‐7‐KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, γ‐2 and γ‐7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.

Numbers, Densities, and Colocalization of AMPA- and NMDA-Type Glutamate Receptors at Individual Synapses in the Superficial Spinal Dorsal Horn of Rats

Ionotropic glutamate receptors play important roles in spinal processing of nociceptive sensory signals and induction of central sensitization in chronic pain. Here we applied highly sensitive freeze-fracture replica labeling to laminae I–II of the spinal dorsal horn of rats and investigated the numbers, densities, and colocalization of AMPA- and NMDA-type glutamate receptors at individual postsynaptic membrane specializations with a high resolution. All glutamatergic postsynaptic membranes in laminae I–II expressed AMPA receptors, and most of them (96%) were also immunoreactive for the NR1 subunit of NMDA receptors. The numbers of gold particles for AMPA and NMDA receptors at individual postsynaptic membranes showed a linear correlation with the size of postsynaptic membrane specializations and varied in the range of 8–214 and 5–232 with median values of 37 and 28, whereas their densities varied in the range of 325–3365/μm2 and 102–2263/μm2 with median values of 1115/μm2 and 777/μm2, respectively. Virtually all (99%) glutamatergic postsynaptic membranes expressed GluR2, and most of them (87%) were also immunoreactive for GluR1. The numbers of gold particles for pan-AMPA, NR1, and GluR2 subunits showed a linear correlation with the size of postsynaptic surface areas. Concerning GluR1, there may be two populations of synapses with high and low GluR1 densities. In synapses larger than 0.1 μm2, GluR1 subunits were recovered in very low numbers. Differential expression of GluR1 and GluR2 subunits suggests regulation of AMPA receptor subunit composition by presynaptic mechanism.

TARPs gamma-2 and gamma-7 are essential for AMPA receptor expression in the cerebellum.

The α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐type glutamate receptors require auxiliary subunits termed transmembrane AMPA receptor regulatory proteins (TARPs), which promote receptor trafficking to the cell surface and synapses and modulate channel pharmacology and gating. Of six TARPs, γ‐2 and γ‐7 are the two major TARPs expressed in the cerebellum. In the present study, we pursued their roles in synaptic expression of cerebellar AMPA receptors. In the cerebellar cortex, γ‐2 and γ‐7 were preferentially localized at various asymmetrical synapses. Using quantitative Western blot and immunofluorescence, we found severe reductions in GluA2 and GluA3 and mild reduction in GluA4 in γ‐2‐knockout (KO) cerebellum, whereas GluA1 and GluA4 were moderately reduced in γ‐7‐KO cerebellum. GluA2, GluA3 and GluA4 were further reduced in γ‐2/γ‐7 double‐KO (DKO) cerebellum. The large losses of GluA2 and GluA3 in γ‐2‐KO mice and further reductions in DKO mice were confirmed at all asymmetrical synapses examined with postembedding immunogold. Most notably, the GluA2 level in the postsynaptic density fraction, GluA2 labeling density at parallel fiber–Purkinje cell synapses, and AMPA receptor‐mediated currents at climbing fiber–Purkinje cell synapses were all reduced to approximately 10% of the wild‐type levels in DKO mice. On the other hand, the reduction in GluA4 in γ‐7‐KO granular layer reflected its loss at mossy fiber–granule cell synapses, whereas that of GluA1 and GluA4 in γ‐7‐KO molecular layer was caused, at least partly, by their loss in Bergmann glia. Therefore, γ‐2 and γ‐7 cooperatively promote synaptic expression of cerebellar AMPA receptors, and the latter also promotes glial expression.

Hippocampal CA3 and CA2 have distinct bilateral innervation patterns to CA1 in rodents

Ipsilateral and contralateral hippocampal CA3–CA1 and CA2–CA1 projections were investigated in adult male Long–Evans rats by retrograde tracing. Injection of the retrograde tracer cholera toxin subunit B in the strata oriens and radiatum of dorsal CA1 resulted in labeling of predominantly pyramidal cells in ipsilateral and contralateral CA3 and CA2. The contralateral and ipsilateral anterior–posterior extents of CA3 innervation to CA1 were similar. Fifteen to twenty per cent of the hippocampus proper cells that give rise to CA1 stratum oriens innervation were CA2 pyramidal cells, whereas CA2 cells were a mere 3% for CA1 stratum radiatum innervation. The preferred projection of CA2 pyramidal cells to the CA1 stratum oriens was also manifested in transgenic mice that express GFP under the control of the CACNG5 promoter, in which CA2 cells express high amounts of GFP. The ratios of ipsilateral to contralateral projections were compared. For the CA3–CA1 connection, we found that dorsal CA1 stratum radiatum received more ipsilateral projections whereas CA1 stratum oriens received more contralateral innervation. Interestingly, ipsilateral connections dominated for both CA2–CA1 stratum oriens and CA2–CA1 stratum radiatum. These results demonstrate that the primary intrahippocampal target of CA2 pyramidal cells is the ipsilateral CA1 stratum oriens, in contrast to CA3 cells which project more diversely to bilateral CA1 regions. Such innervation patterns may suggest differential dendritic information processing in apical and basal dendrites of CA1 pyramidal cells.

Differential distributions of the Ca2+-dependent activator protein for secretion family proteins (CAPS2 and CAPS1) in the mouse brain

The Ca2+‐dependent activator protein for secretion (CAPS/Cadps) family consists of two members, CAPS1 and CAPS2, and plays an important role in secretory granule exocytosis. It has been shown that CAPS1 regulates catecholamine release from neuroendocrine cells, whereas CAPS2 is involved in the release of two neurotrophins, brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3), from parallel fibers of cerebellar granule cells. Although both CAPS proteins are expressed predominantly in the brain, their cellular and regional distributions in the brain are largely unknown. In this study we analyzed the immunohistochemical distributions of the CAPS family proteins in the mouse brain. In most areas of the embryonic nervous system CAPS1 and CAPS2 proteins were complementarily expressed. In the postnatal brain, CAPS1 was widespread at different levels. On the other hand, CAPS2 was localized to distinct cell types and fibers of various brain regions, including the olfactory bulb, cerebrum, hippocampal formation, thalamus, mesencephalic tegmentum, cerebellum, medulla, and spinal cord, except for some regions that overlapped with CAPS1. These CAPS2 cellular distribution patterns had the marked feature of coinciding with those of BDNF in various brain regions. Immunolabels for CAPS2 were also colocalized with those for some proteins related to exocytosis (VAMP and SNAP‐25) and endocytosis (Dynamin I) in the cell soma and processes of the mesencephalic tegmentum and cerebellum, suggesting that these proteins might be involved in the dynamics of CAPS2‐associated vesicles, although their colocalization on vesicles remains elusive. These results demonstrate that the CAPS family proteins are involved in the secretion of different secretory substances in developing and postnatal brains, and that CAPS2 is probably involved in BDNF secretion in many brain areas. J. Comp. Neurol. 495:735–753, 2006.

Enhanced activation of Ca2+/Calmodulin-dependent protein kinase II upon downregulation of cyclin-dependent kinase 5-p35

Cyclin‐dependent kinase 5 (Cdk5)‐p35 is downregulated in cultured neurons by N‐methyl‐d‐aspartate (NMDA) via the proteasomal degradation of p35. However, it is not known where in neurons this downregulation occurs or the physiologic meaning of the reaction. We show the enrichment of Cdk5 and p35 in the postsynaptic density and the NMDA‐induced degradation of postsynaptic p35 using brain slices and cultured neurons. To evaluate the role of this downregulation, we examined the relationship between Ca2+/calmodulin‐dependent protein kinase II (CaMKII) activation and Cdk5 downregulation, as events downstream from NMDA stimulation. Glutamate or NMDA stimulation induced CaMKII autophosphorylation over a time course that mirrored the time course of p35 degradation. To simulate the downregulation of postsynaptic Cdk5 in invitro experiments, we used the Cdk5 inhibitor roscovitine. The inhibition of Cdk5 activity by roscovitine enhanced CaMKII autophosphorylation and activation in cultured neurons, and in an isolated postsynaptic‐density‐enriched fraction. These results suggest that Cdk5 activity suppresses CaMKII activation, and that the downregulation of Cdk5 activity after treatment withNMDA facilitates CaMKII activation, leading to the easier induction of long‐term potentiation.

Differential expression of SNAP-25 family proteins in the mouse brain.

Soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP)‐25 is a neuronal SNARE protein essential for neurotransmitter release from presynaptic terminals. Three palmitoylated SNAP‐25 family proteins: SNAP‐25a, SNAP‐25b, and SNAP‐23, are expressed in the brain, but little is known about their distributions and functions. In the present study, we generated specific antibodies to distinguish these three homologous proteins. Immunoblot and immunohistochemical analyses revealed that SNAP‐25b was distributed in synapse‐enriched regions throughout almost the entire brain, whereas SNAP‐25a and SNAP‐23 were expressed in relatively specific brain regions with partially complementary expression patterns. SNAP‐25a and SNAP‐25b, but not SNAP‐23, were also present in the axoplasm of nerve fibers. The intracellular localization was also different, and although SNAP‐25b and SNAP‐23 were found primarily in membrane and lipid raft‐enriched fractions of mouse brain homogenates, a substantial amount of SNAP‐25a was recovered in soluble fractions. In PC12 cells, SNAP‐25b was localized to the plasma membrane, but SNAP‐25a and SNAP‐23 were distributed throughout the cytoplasm. The expression and distribution of these three proteins were also differentially regulated in the early postnatal period. These results indicate that the three SNAP‐25 family proteins display a differential distribution in the brain as well as in neuronal cells, and possibly play distinct roles. J. Comp. Neurol. 519:916–932, 2011.

Dual regulation of astrocyte gap junction hemichannels by growth factors and a pro-inflammatory cytokine via the mitogen-activated protein kinase cascade.

Evidence that glutamate and ATP release from astrocytes can occur via gap junction hemichannels (GJHCs) is accumulating. However, the GJHC is still only one possible release mechanism and has not been detected in some studies, although this may be because the levels were below those detectable by the system used. Because of these conflicting results, we hypothesized that release from astrocyte GJHCs might depend on different astrocyte states, and screened for factors affecting astrocyte GJHC activity by measuring fluorescent dye leakage via GJHCs using a conventional method for GJHC acivation, i.e. removal of extracellular divalent cations. Astrocytes cultured in Dulbeccos minimal essential medium containing 10% fetal calf serum, a medium widely used for astrocyte studies, did not show dye leakage, whereas those cultured in a defined medium showed substantial dye leakage, which was confirmed pharmacologically to be due to GJHCs and not to P2x7 receptors. EGF and bFGF inhibited the GJHC activity via the mitogen‐activated protein kinase cascade, and the effect of the growth factors was reversed by interleukin‐1β. These factors altered GJHC activity within 10 min, but did not affect connexin 43 expression. GJHC activity in hippocampal slice culture preparations was measured using the same methods and found to be regulated in a similar manner. These results indicate that astrocyte GJHC activity is regulated by brain environmental factors.