Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGLalpha) and beta (DGLbeta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGLalpha knockout mice, whereas the retrograde suppression was intact in DGLbeta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGLalpha knockout brains, whereas the 2-AG content was normal in DGLbeta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGLalpha, but not by DGLbeta, mediates retrograde suppression at central synapses.

The analytical technique of nuclear magnetic resonance (NMR) is based on coherent quantum mechanical superposition of nuclear spin states. Recently, NMR has received considerable renewed interest in the context of quantum computation and information processing, which require controlled coherent qubit operations. However, standard NMR is not suitable for the implementation of realistic scalable devices, which would require all-electrical control and the means to detect microscopic quantities of coherent nuclear spins. Here we present a self-contained NMR semiconductor device that can control nuclear spins in a nanometre-scale region. Our approach enables the direct detection of (otherwise invisible) multiple quantum coherences between levels separated by more than one quantum of spin angular momentum. This microscopic high sensitivity NMR technique is especially suitable for probing materials whose nuclei contain multiple spin levels, and may form the basis of a versatile multiple qubit device.

Transcription of hypoxia-inducible genes is regulated by hypoxia response elements (HREs) located in either the promoter or enhancer regions. Analysis of these elements reveals the presence of one or more binding sites for hypoxia-inducible factor 1 (HIF-1). Hypoxia-inducible genes include vascular endothelial growth factor (VEGF), erythropoietin, and glycolytic enzyme genes. Site-directed mutational analysis of the VEGF gene promoter revealed that an HIF-1 binding site (HBS) and its downstream HIF-1 ancillary sequence (HAS) within the HRE are required as cis-elements for the transcriptional activation of VEGF by either hypoxia or nitric oxide (NO). The core sequences of the HBS and the HAS were determined as TACGTG and CAGGT, respectively. These elements form an imperfect inverted repeat, and the spacing between these motifs is crucial for activity of the promoter. Gel shift assays demonstrate that as yet unknown protein complexes constitutively bind to the HAS regardless of the presence of these stimuli in several cell lines, in contrast with hypoxia-or NO-induced activation of HIF-1 binding to the HBS. A common structure of the HRE, which consists of the HBS and the HAS, is seen among several hypoxia-inducible genes, suggesting the presence of a novel mechanism mediated by the HAS for the regulation of these genes.

The glutamate receptor delta2 subunit (GluRdelta2) is specifically expressed in cerebellar Purkinje cells (PCs) from early developmental stages and is selectively localized at dendritic spines forming synapses with parallel fibers (PFs). Targeted disruption of the GluRdelta2 gene leads to a significant reduction of PF-->PC synapses. To address its role in the synaptogenesis, the morphology and electrophysiology of PF-->PC synapses were comparatively examined in developing GluRdelta2 mutant and wild-type cerebella. PCs in GluRdelta2 mutant mice were normally produced, migrated, and formed spines, as did those in wild-type mice. At the end of the first postnatal week, 74-78% of PC spines in both mice formed immature synapses, which were characterized by small synaptic contact, few synaptic vesicles, and incomplete surrounding by astroglial processes, eliciting little electrophysiological response. During the second and third postnatal weeks when spines and terminals are actively generated, the percentage of PC spines forming synapses attained 98-99% in wild type but remained as low as 55-60% in mutants, and the rest were unattached to any nerve terminals. As a result, the number of PF synapses per single-mutant PCs was reduced to nearly a half-level of wild-type PCs. Parallelly, PF stimulation less effectively elicited EPSCs in mutant PCs than in wild-type PCs during and after the second postnatal week. These results suggest that the GluRdelta2 is involved in the stabilization and strengthening of synaptic connectivity between PFs and PCs, leading to the association of all PC spines with PF terminals to form functionally mature synapses.

The spontaneous recessive mutant mouse stargazer (stg) begins to show ataxia around postnatal day 14 and display a severe impairment in the acquisition of classical eyeblink conditioning in adulthood. These abnormalities have been attributed to the specific reduction in brain-derived neurotrophic factor (BDNF) and the subsequent defect in TrkB receptor signaling in cerebellar granule cells (GCs). In the stg mutant cerebellum, we found that EPSCs at mossy fiber (MF) to GC synapses are devoid of the fast component mediated by AMPA-type glutamate receptors despite the normal slow component mediated by NMDA receptors. The sensitivity of stg mutant GCs to exogenously applied AMPA was greatly reduced, whereas that to NMDA was unchanged. Glutamate release from MF terminals during synaptic transmission to GCs appeared normal. By contrast, AMPA receptor-mediated EPSCs were normal in CA1 pyramidal cells of the stg mutant hippocampus. Thus, postsynaptic AMPA receptor function was selectively impaired in stg mutant GCs, although the transcription of four AMPA receptor subunit genes in the stg GC was comparable to the wild-type GC. We also examined the cerebellum of BDNF knockout mice and found that their MF-GC synapses had a normal AMPA receptor-mediated EPSC component. Thus, the impaired AMPA receptor function in the stg mutant GC is not likely to result from the reduced BDNF-TrkB signaling. These results suggest that the defect in MF to GC synaptic transmission is a major factor that causes the cerebellar dysfunction in the stg mutant mouse.

Functional neural circuits are formed by eliminating early-formed redundant synapses and strengthening necessary connections during development. In newborn mouse cerebellum, each Purkinje cell (PC) is innervated by multiple climbing fibers (CFs) with similar strengths. Subsequently, a single CF is selectively strengthened by postnatal day 7 (P7). We find that this competition among multiple CFs occurs on the soma before CFs form synapses along dendrites. Notably, in most PCs, the single CF that has been functionally strengthened (the "winner" CF) undergoes translocation to dendrites while keeping its synapses on the soma. Synapses of the weaker CFs (the "loser" CFs) remain around the soma and form "pericellular nests" with synapses of the winner CFs. Then most perisomatic synapses are eliminated nonselectively by P15. Thus, our results suggest that the selective translocation of the winner CF to dendrites in each PC determines the single CF that survives subsequent synapse elimination and persistently innervates the PC.

We investigate interactions between electrons and nuclear spins by using the resistance (R xx ) peak which develops near filling factor ν = 2/3 as a probe. By temporarily tuning ν to a different value, ν temp, with a gate, the R xx peak is shown to relax quickly on both sides of ν temp = 1. This is due to enhanced nuclear spin relaxation by Skyrmions, and demonstrates the dominant role of nuclear spin in the transport anomaly near ν = 2/3. We also observe an additional enhancement in the nuclear spin relaxation around ν = 1/2 and 3/2, which suggests a Fermi sea of partially-polarized composite fermions.

Climbing fiber (CF) synapse formation onto cerebellar Purkinje cells (PCs) is critically dependent on the synaptogenesis from parallel fibers (PFs), the other input to PCs. Previous studies revealed that deletion of the glutamate receptor delta2 subunit (GluRdelta2) gene results in persistent multiple CF innervation of PCs with impaired PF synaptogenesis, whereas mutation of the metabotropic glutamate receptor subtype 1 (mGluR1) gene causes multiple CF innervation with normal PF synaptogenesis. We demonstrate that atypical CF-mediated EPSCs (CF-EPSCs) with slow rise times and small amplitudes coexisted with typical CF-EPSCs with fast rise times and large amplitudes in PCs from GluRdelta2 mutant cerebellar slices. CF-EPSCs in mGluR1 mutant and wild-type PCs had fast rise times. Atypical slow CF responses of GluRdelta2 mutant PCs were associated with voltage-dependent Ca(2+) signals that were confined to PC distal dendrites. In the wild-type and mGluR1 mutant PCs, CF-induced Ca(2+) signals involved both proximal and distal dendrites. Morphologically, CFs of GluRdelta2 mutant mice extended to the superficial regions of the molecular layer, whereas those of wild-type and mGluR1 mutant mice did not innervate the superficial one-fifth of the molecular layer. It is therefore likely that surplus CFs of GluRdelta2 mutant mice form ectopic synapses onto distal dendrites, whereas those of wild-type and mGluR1 mutant mice innervate proximal dendrites. These findings suggest that GluRdelta2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendrites, whereas the mGluR1-signaling pathway does not affect PF synaptogenesis but is involved in eliminating surplus CF synapses at the proximal dendrites.