Yo Shinoda

Repetition of mGluR-dependent LTd causes slowly developing persistent reduction in synaptic strength accompanied by synapse elimination

Synaptic plasticity, the cellular basis of memory, operates in a bidirectional manner. LTP (long-term potentiation) is followed by structural changes that may lead to the formation of new synapses. However, little is known whether LTD (long-term depression) is followed by morphological changes. Here we show that the repetitive induction of metabotropic glutamate receptor (mGluR)-dependent LTD in stable cultures of rat hippocampal slices led to a slowly developing persistent (ranging over weeks) reduction in synaptic strength that was accompanied by the loss of synaptic structures. LTD was induced pharmacologically 1-3 times at 24-h intervals by applying aseptically ACPD (1-aminocyclopentane-1,3-dicarboxylic acid), an agonist of group I/II mGluR, and APV (2-amino-5-phosphonovalerate), an antagonist of the NMDA (N-methyl-D-aspartate) receptor. One ACPD/APV application induced LTD that lasted less than 24 h. After three LTD inductions, however, a gradual attenuation of the fEPSP (field excitatory postsynaptic potential) amplitude and a decrease in the number of pre- and postsynaptic structures were observed. The blockade of LTD by an mGluR antagonist or a protein phosphatase 2B inhibitor abolished the development of the synaptic attenuation. In contrast to our previous finding that the repetitive LTP induction triggered a slowly developing persistent synaptic enhancement, the incremental and decremental forms of synaptic plasticity appeared to occur symmetrically not only on the minutes-hours time order but also on the days-weeks time order.

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.

2008 Special Issue: Cerebellar development transcriptome database (CDT-DB): Profiling of spatio-temporal gene expression during the postnatal development of mouse cerebellum

A large amount of genetic information is devoted to brain development and functioning. The neural circuit of the mouse cerebellum develops through a series of cellular and morphological events (including neuronal proliferation and migration, axogenesis, dendritogenesis, synaptogenesis and myelination) all within three weeks of birth. All of these events are controlled by specific gene groups, whose temporal and spatial expression profiles must be encoded in the genome. To understand the genetic basis underlying cerebellar circuit development, we analyzed gene expression (transcriptome) during the developmental stages on a genome-wide basis. Spatio-temporal gene expression data were collected using in situ hybridization for spatial (cellular and regional) resolution and fluorescence differential display, GeneChip, microarray and RT-PCR for temporal (developmental time series) resolution, and were annotated using Gene Ontology (controlled terminology for genes and gene products) and anatomical context (cerebellar cell types and circuit structures). The annotated experimental data were integrated into a knowledge resource database, the Cerebellar Development Transcriptome Database (CDT-DB http://www.cdtdb.brain.riken.jp), with seamless links to the relevant information at various bioinformatics database websites. The CDT-DB not only provides a unique informatics tool for mining both spatial and temporal pattern information on gene expression in developing mouse brains, but also opens up opportunities to elucidate the transcriptome for cerebellar development.

Long-lasting synaptic loss after repeated induction of LTD: independence to the means of LTD induction.

Short‐ and long‐lasting synaptic plasticity is assumed to be the cellular basis of short‐ and long‐lasting memory, respectively. However, the cellular consequences leading to the long‐lasting synaptic plasticity, assumed to include the processes of synapse formation and elimination, remain unknown. Using hippocampal slices maintained stably in culture, we found previously that the repeated induction of long‐term potentiation (LTP) triggered a slowly developing long‐lasting enhancement in synaptic transmission strength accompanied by synapse formation, which was separate from LTP itself. We recently reported a phenomenon apparently of a mirror‐image effect. The repeated activations of metabotropic glutamate receptor (mGluR), which induces long‐term depression (LTD), triggered a long‐lasting reduction in synaptic strength accompanied by synapse elimination. To clarify whether the reported long‐lasting effect was specific to the drugs used previously and whether the effect was specific to mGluR‐mediated LTD, we exposed the cultured slices repeatedly to another Group I metabotropic glutamate receptor (mGluR) agonist, an N‐methyl‐d‐aspartate receptor agonist, and a Na+/K+‐pump inhibitor. All these treatments resulted in an equivalent long‐lasting synaptic reduction/elimination when repeated three times, indicating that the repeated LTD induction leads to synapse elimination. The independence of synapse elimination to the means of LTD induction suggests that the signals leading to short‐term plasticity and long‐term plasticity are independent. Detailed inspections in the representative case of mGluR activation revealed that the reduction in synaptic strength developed with a ∼1‐week delay from the decrease in the number of synaptic structures. This synapse elimination should be unique as it is activity‐dependent rather than inactivity‐dependent.

Reduced axonal localization of a Caps2 splice variant impairs axonal release of BDNF and causes autistic-like behavior in mice

Ca2+-dependent activator protein for secretion 2 (CAPS2 or CADPS2) potently promotes the release of brain-derived neurotrophic factor (BDNF). A rare splicing form of CAPS2 with deletion of exon3 (dex3) was identified to be overrepresented in some patients with autism. Here, we generated Caps2-dex3 mice and verified a severe impairment in axonal Caps2-dex3 localization, contributing to a reduction in BDNF release from axons. In addition, circuit connectivity, measured by spine and interneuron density, was diminished globally. The collective effect of reduced axonal BDNF release during development was a striking and selective repertoire of deficits in social- and anxiety-related behaviors. Together, these findings represent a unique mouse model of a molecular mechanism linking BDNF-mediated coordination of brain development to autism-related behaviors and patient genotype.

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

Background Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)4-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. Methodology/Principal Findings PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. Conclusions/Significance Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.

Persistent synapse loss induced by repetitive LTD in developing rat hippocampal neurons.

Synaptic pruning is a physiological event that eliminates excessive or inappropriate synapses to form proper synaptic connections during development of neurons. Appropriate synaptic pruning is required for normal neural development. However, the mechanism of synaptic pruning is not fully understood. Strength of synaptic activity under competitive circumstances is thought to act as a selective force for synaptic pruning. Long-term depression (LTD) is a synaptic plasticity showing persistent decreased synaptic efficacy, which is accompanied by morphological changes of dendritic spines including transient retraction. Repetitive induction of LTD has been shown to cause persistent loss of synapses in mature neurons. Here, we show that multiple, but not single, induction of LTD caused a persistent reduction in the number of dendritic synapses in cultured rat developing hippocampal neurons. When LTD was induced in 14 days in vitro cultures by application of (RS)-3,5-dihydroxyphenylglycine (DHPG), a group I metabotropic glutamate receptor (mGluR) agonist, and repeated three times with a one day interval, there was a significant decrease in the number of dendritic synapses. This effect continued up to at least two weeks after the triple LTD induction. The persistent reduction in synapse number occurred in the proximal dendrites, but not the distal dendrites, and was prevented by simultaneous application of the group I/II mGluR antagonist (S)-a-methyl-4-carboxyphenylglycine (MCPG). In conclusion, we found that repetitive LTD induction in developing neurons elicits synaptic pruning and contributes to activity-dependent regulation of synapse number in rat hippocampal neurons.

Expression of the IP3R1 promoter‐driven nls‐lacZ transgene in Purkinje cell parasagittal arrays of developing mouse cerebellum

The cerebellar Purkinje cell monolayer is organized into heterogeneous Purkinje cell compartments that have different molecular compositions. Here we describe a transgenic mouse line, 1NM13, that shows heterogeneous transgene expression in parasagittal Purkinje cell arrays. The transgene consists of a nuclear localization signal (nls) fused to the β‐galactosidase (lacZ) composite gene driven by the type 1 inositol 1,4,5‐trisphosphate receptor (IP3R1) gene promoter. IP3R1‐nls‐lacZ transgene expression was detected at a single Purkinje cell level over the surface of a whole‐mount X‐gal‐stained cerebellum because of nuclear accumulation of the nls‐lacZ activity. Developing cerebella of 1NM13 mice showed stripe‐like X‐gal staining patterns of parasagittal Purkinje cell subsets. The X‐gal stripe pattern was likely determined by an intrinsic property as early as E15 and showed increasing complexity with cerebellar development. The X‐gal stripe pattern was reminiscent of, but not identical to, the stripe pattern of zebrin II immunoreactivity. We designated the symmetrical X‐gal‐positive (transgene‐positive, Tg+) Purkinje cell stripes about the midline as vermal Tg1+, Tg2(a, b)+ and Tg3(a, b)+ stripes and hemispheric Tg4(a, b)+, Tg5(a, b)+, Tg6(a, b, c)+, and Tg7(a, b)+ stripes, where a, b, and c indicate substripes. We also assigned three parafloccular substripes Tg8(a, b, c)+. The boundaries of X‐gal stripes at P5 were consistent with raphes in the Purkinje cell layer through which granule cells migrate, suggesting a possible association of the X‐gal stripes with raphe formation. Our results indicate that 1NM13 is a good mouse model with a reproducible and clear marker for the compartmentalization of Purkinje cell arrays.

Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans-Golgi Network

Ca2+-dependent activator protein for secretion (CAPS) regulates exocytosis of catecholamine- or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites, such as nerve terminals. However, large amounts of CAPS protein are localized in the cell soma, and the role of somal CAPS protein remains unclear. The present study shows that somal CAPS1 plays an important role in DCV trafficking in the trans-Golgi network. The anti-CAPS1 antibody appeared to pull down membrane fractions, including many Golgi-associated proteins, such as ADP-ribosylation factor (ARF) small GTPases. Biochemical analyses of the protein-protein interaction showed that CAPS1 interacted specifically with the class II ARF4/ARF5, but not with other classes of ARFs, via the pleckstrin homology domain in a GDP-bound ARF form-specific manner. The pleckstrin homology domain of CAPS1 showed high affinity for the Golgi membrane, thereby recruiting ARF4/ARF5 to the Golgi complex. Knockdown of either CAPS1 or ARF4/ARF5 expression caused accumulation of chromogranin, a DCV marker protein, in the Golgi, thereby reducing its DCV secretion. In addition, the overexpression of CAPS1 binding-deficient ARF5 mutants induced aberrant chromogranin accumulation in the Golgi and consequently reduced its DCV secretion. These findings implicate a functional role for CAPS1 protein in the soma, a major subcellular localization site of CAPS1 in many cell types, in regulating DCV trafficking in the trans-Golgi network; this activity occurs via protein-protein interaction with ARF4/ARF5 in a GDP-dependent manner.

Possible involvement of BDNF release in long-lasting synapse formation induced by repetitive PKA activation.

For the analysis of the cellular mechanism underlying long-term synaptic plasticity, a model system that allows long-lasting pursuit is required. Previously we reported that, in hippocampal neurons under dissociated cell culture conditions, repeated (but not a single) transient activation of protein kinase A (PKA) led to an increase in the number of synapses that lasted >3 weeks, and hence we proposed that this phenomenon should serve as an appropriate model system. Here we report that repeated pulsatile application of brain-derived neurotrophic factor (BDNF) leads to persistent synapse formation equivalent to that after the repeated transient activation of PKA. A BDNF-scavenging substance applied concomitantly with PKA activation abolished the synapse formation. The release of BDNF upon PKA activation was confirmed by phosphorylation of TrkB. These results indicate that the release of BDNF is involved in the putative signaling cascade connecting PKA activation and synapse formation.