Synaptic pruning of murine adult-born neurons by microglia depends on phosphatidylserine.

New neurons, continuously added in the adult olfactory bulb (OB) and hippocampus, are involved in information processing in neural circuits. Here, we show that synaptic pruning of adult-born neurons by microglia depends on phosphatidylserine (PS), whose exposure on dendritic spines is inversely correlated with their input activity. To study the role of PS in spine pruning by microglia in vivo, we developed an inducible transgenic mouse line, in which the exposed PS is masked by a dominant-negative form of milk fat globule-EGF-factor 8 (MFG-E8), MFG-E8D89E. In this transgenic mouse, the spine pruning of adult-born neurons by microglia is impaired in the OB and hippocampus. Furthermore, the electrophysiological properties of these adult-born neurons are altered in MFG-E8D89E mice. These data suggest that PS is involved in the microglial spine pruning and the functional maturation of adult-born neurons. The MFG-E8D89E-based genetic approach shown in this study has broad applications for understanding the biology of PS-mediated phagocytosis in vivo.

Visually cued fear conditioning test for memory impairment related to cortical function.

AIM: Fear conditioning tests are intended to elucidate a subject's ability to associate a conditioned stimulus with an aversive, unconditioned stimulus, such as footshock. Among these tests, a paradigm related to precise cortical functions would be increasingly important in drug screening for disorders such as schizophrenia and dementia. Therefore, we established a new fear conditioning paradigm using a visual cue in mice. In addition, the validity of the test was evaluated using a genetically engineered mouse, heterozygous deficient in Mdga1 (Mdga1+/-), which is related to schizophrenia. RESULTS: Mice were given footshocks associated with a visual cue of moving gratings at training in 25-minute sessions. The mice showed the conditioned response of freezing behavior to the visual stimulus at testing 24 hours after the footshocks. In the test for validation, the Mdga1+/- deficient mice showed significantly less freezing than wild-type mice. CONCLUSION: The visually cued fear conditioning paradigm with moving gratings has been established, which is experimentally useful to evaluate animal cortical functions. The validity of the test was confirmed for Mdga1-deficient mice with possible deficiency in cortical functions.

Traceable stimulus-dependent rapid molecular changes in dendritic spines in the brain.

Dendritic spines function as microcompartments that can modify the efficiency of their associated synapses. Here, we analyzed stimulus-dependent molecular changes in spines. The F-actin capping protein CapZ accumulates in parts of dendritic spines within regions where long-term potentiation has been induced. We produced a transgenic mouse line, AiCE-Tg, in which CapZ tagged with enhanced green fluorescence protein (EGFP-CapZ) is expressed. Twenty minutes after unilateral visual or somatosensory stimulation in AiCE-Tg mice, relative EGFP-CapZ signal intensification was seen in a subset of dendritic spines selectively in stimulated-side cortices; this right-left difference was abolished by NMDA receptor blockade. Immunolabeling of α-actinin, a PSD-95 binding protein that can recruit AMPA receptors, showed that the α-actinin signals colocalized more frequently in spines with the brightest EGFP-CapZ signals (top 100) than in spines with more typical EGFP-CapZ signal strength (top 1,000). This stimulus-dependent in vivo redistribution of EGFP-CapZ represents a novel molecular event with plasticity-like characteristics, and bright EGFP-CapZ in AiCE-Tg mice make high-CapZ spines traceable in vivo and ex vivo. This mouse line has the potential to be used to reveal sequential molecular events, including synaptic tagging, and to relate multiple types of plasticity in these spines, extending knowledge related to memory mechanisms.

A link between vascular damage and cognitive deficits after whole-brain radiation therapy for cancer: A clue to other types of dementia?

Whole brain radiation therapy for the treatment of tumors can sometimes cause cognitive impairment. Memory deficits were noted in up to 50% of treated patients over a short period of several months. In addition, an increased rate of dementia in young patients has been noted over the longer term, i.e. years. A deficit in neurogenesis after irradiation has been postulated to be the main cause of cognitive decline in patients, but recent data on irradiation therapy for limited parts of the brain appear to indicate other possibilities. Irradiation can directly damage various types of cells other than neuronal stem cells. However, this paper will focus on injury to brain vasculature leading to cognitive decline since vessels represent a better therapeutic target for drug development than other cells in the brain because of the blood-brain barrier.

Angiogenesis in refractory depression: A possible phenotypic target to avoid the blood brain barrier.

Major depressive syndrome (so-called depression) is a common but serious mental disease that causes low mood. Most patients are treatable, mainly because of high response rates for medicines such as selective serotonin-reuptake inhibitors (SSRIs). However, there are still a considerable number of patients with refractory or drug-resistant depression. On the other hand, recent findings suggest that angiogenesis, i.e., making new blood vessels, could have an important role in the recovery from depressive disorders, at least in part. It has been reported that the brain capillaries are physiologically capable of undergoing angiogenesis upon stimuli such as exercise and SSRIs seem to accelerate brain angiogenesis. Drugs targeting angiogenesis may possibly be another good concept. In addition, the blood brain barrier (BBB), which is a major obstacle for drug development for the central nervous system, would be circumvented. Here I summarize the reports that relate angiogenesis to a cure for major depression and discuss some of the potential molecular targets.

17α-estradiol is generated locally in the male rat brain and can regulate GAD65 expression and anxiety.

Increasing evidence suggests that 17ß-estradiol, a sex hormone, is synthesized by neurons. In addition, 17α-estradiol, the stereoisomer of 17ß-estradiol, is reported to be the dominant form in the male mouse brain. However, probably because the method to detect these isomers requires unusual and precise experimental design, the presence of this endogenous 17α-estradiol has not been reported subsequently and the actual role is therefore not well elucidated. We first quantified the estradiol level in hippocampal extracts using gas chromatography/mass spectrometry. As a result, 17α-estradiol was found in all of the male rats tested, while that of 17ß-estradiol was detected only in a certain subset. The estrogen-biosynthesis inhibitor letrozole decreased the expression of the major presynaptic GABA synthesizing enzyme GAD65 in cultured neurons and the effect was abrogated by exogenously supplied 17α-estradiol. Next, injection of the inhibitor into the brain reduced the 17α-estradiol level, indicating its biogenesis in the brain. Under the same conditions, immuno-staining of GAD65 was also decreased. Furthermore, the inhibitor treatment increased anxiety index of rats in the open field and this was ameliorated by the addition of 17α-estradiol. We showed that 17α-estradiol was generated in the brain and acted as a regulator of inhibitory neurotransmission as well as behavior. These results may have implications for a variety of diseases, such as the menopausal depression and Alzheimer's disease that have been reported to be related to estrogen levels.

Postsynaptic spiking homeostatically induces cell-autonomous regulation of inhibitory inputs via retrograde signaling.

Developing neural circuits face the dual challenge of growing in an activity-induced fashion and maintaining stability through homeostatic mechanisms. Compared to our understanding of homeostatic regulation of excitatory synapses, relatively little is known about the mechanism mediating homeostatic plasticity of inhibitory synapses, especially that following activity elevation. Here, we found that elevating neuronal activity in cultured hippocampal neurons for 4 h significantly increased the frequency and amplitude of mIPSCs, before detectable change at excitatory synapses. Consistently, we observed increases in presynaptic and postsynaptic proteins of GABAergic synapses, including GAD65, vGAT, and GABA(A)Rα1. By suppressing activity-induced increase of neuronal firing with expression of the inward rectifier potassium channel Kir2.1 in individual neurons, we showed that elevation in postsynaptic spiking activity is required for activity-dependent increase in the frequency and amplitude of mIPSCs. Importantly, directly elevating spiking in individual postsynaptic neurons, by capsaicin activation of overexpressed TRPV1 channels, was sufficient to induce increased mIPSC amplitude and frequency, mimicking the effect of elevated neuronal activity. Downregulating BDNF expression in the postsynaptic neuron or its extracellular scavenging prevented activity-induced increase in mIPSC frequency, consistent with a role of BDNF-dependent retrograde signaling in this process. Finally, elevating activity in vivo by kainate injection increased both mIPSC amplitude and frequency in CA1 pyramidal neurons. Thus, spiking-induced, cell-autonomous upregulation of GABAergic synaptic inputs, through retrograde BDNF signaling, represents an early adaptive response of neural circuits to elevated network activity.

Activity-dependent localization in spines of the F-actin capping protein CapZ screened in a rat model of dementia.

Actin reorganization in dendritic spines is hypothesized to underlie neuronal plasticity. Actin-related proteins, therefore, might serve as useful markers of plastic changes in dendritic spines. Here, we utilized memory deficits induced by fimbria-fornix transection (FFT) in rats as a dementia model to screen candidate memory-associated molecules by using a two-dimensional gel method. Comparison of protein profiles between the transected and control sides of hippocampi after unilateral FFT revealed a reduction in the F-actin capping protein (CapZ) signal on the FFT side. Subsequent immunostaining of brain sections and cultured hippocampal neurons revealed that CapZ localized in dendritic spines and the signal intensity in each spine varied widely. The CapZ content decreased after suppression of neuronal firing by tetrodotoxin treatment in cultured neurons, indicating rapid and activity-dependent regulation of CapZ accumulation in spines. To test input specificity of CapZ accumulation in vivo, we delivered high-frequency stimuli to the medial perforant path unilaterally in awake rats. This path selectively inputs to the middle molecular layer of the dentate gyrus, where CapZ immunoreactivity increased. We conclude that activity-dependent, synapse-specific regulation of CapZ redistribution might be important in both maintenance and remodeling of synaptic connections in neurons receiving specific spatial and temporal patterns of inputs.

Influence of brain-derived neurotrophic factor on pathfinding of dentate granule cell axons, the hippocampal mossy fibers.

Mossy fibers, the dentate granule cell axons, are generated throughout an animal's lifetime. Mossy fiber paths and synapses are primarily restricted to the stratum lucidum within the CA3 region. Brain-derived neurotrophic factor (BDNF), a neurotrophin family protein that activates Trk neurotrophin receptors, is highly expressed in the stratum lucidum in an activity-dependent manner. The addition of a Trk neurotrophin receptor inhibitor, K252a, to cultured hippocampal slices induced aberrant extension of mossy fibers into ectopic regions. BDNF overexpression in granule cells ameliorated the mossy fiber pathway abnormalities caused by a submaximal dose of K252a. A similar rescue was observed when BDNF was expressed in CA3 pyramidal cells, most notably in mossy fibers distal to the expression site. These findings are the first to clarify the role of BDNF in mossy fiber pathfinding, not as an attractant cue but as a regulator, possibly acting in a paracrine manner. This effect of BDNF may be as a signal for new fibers to fasciculate and extend further to form synapses with neurons that are far from active BDNF-expressing synapses. This mechanism would ensure the emergence of new independent dentate gyrus-CA3 circuits by the axons of new-born granule cells.

Experience-dependent, rapid structural changes in hippocampal pyramidal cell spines.

Morphological changes in dendritic spines may contribute to the fine tuning of neural network connectivity. The relationship between spine morphology and experience-dependent neuronal activity, however, is largely unknown. In the present study, we combined 2 histological analyses to examine this relationship: 1) Measurement of spines of neurons whose morphology was visualized in brain sections of mice expressing membrane-targeted green fluorescent protein (Thy1-mGFP mice) and 2) Categorization of CA1 neurons by immunohistochemical monitoring of Arc expression as a putative marker of recent neuronal activity. After mice were exposed to a novel, enriched environment for 60 min, neurons that expressed Arc had fewer small spines and more large spines than Arc-negative cells. These differences were not observed when the exploration time was shortened to 15 min. This net-balanced structural change is consistent with both synapse-specific enhancement and suppression. These results provide the first evidence of rapid morphological changes in spines that were preferential to a subset of neurons in association with an animal's experiences.

A novel chalcone polyphenol inhibits the deacetylase activity of SIRT1 and cell growth in HEK293T cells.

SIRT1 is one of seven mammalian orthologs of yeast silent information regulator 2 (Sir2), and it functions as a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase. Recently, resveratrol and its analogues, which are polyphenols, have been reported to activate the deacetylase activity of SIRT1 in an in vitro assay and to expand the life-span of several species through Sir2 and the orthologs. To find activators or inhibitors to SIRT1, we examined thirty-six polyphenols, including stilbenes, chalcones, flavanones, and flavonols, with the SIRT1 deacetylase activity assay using the acetylated peptide of p53 as a substrate. The results showed that 3,2',3',4'-tetrahydroxychalcone, a newly synthesized compound, inhibited the SIRT1-mediated deacetylation of a p53 acetylated peptide and recombinant protein in vitro. In addition, this agent induced the hyperacetylation of endogenous p53, increased the endogenous p21CIP1/WAF1 in intact cells, and suppressed the cell growth. These results indicated that 3,2',3',4'-tetrahydroxychalcone had a stronger inhibitory effect on the SIRT1-pathway than sirtinol, a known SIRT1-inhibitor. Our results mean that 3,2',3',4'-tetrahydroxychalcone is a novel inhibitor of SIRT1 and produces physiological effects on organisms probably through inhibiting the deacetylation by SIRT1.

Imaging mass spectrometry technology and application on ganglioside study; visualization of age-dependent accumulation of C20-ganglioside molecular species in the mouse hippocampus.

Gangliosides are particularly abundant in the central nervous system (CNS) and thought to play important roles in memory formation, neuritogenesis, synaptic transmission, and other neural functions. Although several molecular species of gangliosides have been characterized and their individual functions elucidated, their differential distribution in the CNS are not well understood. In particular, whether the different molecular species show different distribution patterns in the brain remains unclear. We report the distinct and characteristic distributions of ganglioside molecular species, as revealed by imaging mass spectrometry (IMS). This technique can discriminate the molecular species, raised from both oligosaccharide and ceramide structure by determining the difference of the mass-to-charge ratio, and structural analysis by tandem mass spectrometry. Gangliosides in the CNS are characterized by the structure of the long-chain base (LCB) in the ceramide moiety. The LCB of the main ganglioside species has either 18 or 20 carbons (i.e., C18- or C20-sphingosine); we found that these 2 types of gangliosides are differentially distributed in the mouse brain. While the C18-species was widely distributed throughout the frontal brain, the C20-species selectively localized along the entorhinal-hippocampus projections, especially in the molecular layer (ML) of the dentate gyrus (DG). We revealed development- and aging-related accumulation of the C-20 species in the ML-DG. Thus it is possible to consider that this brain-region specific regulation of LCB chain length is particularly important for the distinct function in cells of CNS.

Enhancement of Trk signaling pathways by the cholestane amide conjugate MCC-257.

The cholestane amide conjugate MCC-257 has been shown to augment the effects of nerve growth factor (NGF) on cell survival and on tyrosine phosphorylation of the TrkA receptor in PC12 cells. Recent findings suggest that signaling pathways downstream of Trk are regulated independently. We describe here our finding that the NGF-induced phosphorylation of both ERK and Akt are accelerated by MCC-257. Analysis of the common features of the augmented pathways suggests that TrkA is most likely to be the primary target of MCC-257 and that both ERK and Akt may be involved in the cellular effects of this compound.

Contextual learning induces an increase in the number of hippocampal CA1 neurons expressing high levels of BDNF.

We examined behaviorally induced expression of brain-derived neurotrophic factor (BDNF) in area CA1 of the hippocampus. Sprague-Dawley rats were trained in a contextual fear conditioning (CFC) task, sacrificed 4h later, and their brains were processed for immunohistochemistry. We found distinctively high levels of BDNF immunoreactivity in a small number ( approximately 1%) of CA1 neurons in untrained animals. The number of these exceptional neurons, which are identified as BDNF(++) in this study, was increased by up to approximately 3% after CFC. This increase was blocked in the presence of a memory-impairing dose of a NMDA receptor antagonist (MK801 0.3 mg/kg, i.p.) given 30 min prior to training. The BDNF signal intensity in BDNF(++) neurons correlated with that of surrounding glutamic acid decarboxylase (GAD) 65. This correlation between GAD65 and BDNF signal intensities suggests that BDNF upregulation was associated with increased signaling via inhibitory GABAergic synapses that would lessen further intervening neuronal activity. Our observation that neurons which upregulate BDNF expression following a learning experience are rich in GAD65-enriched afferent synapses suggests that these neurons may have distinct roles in memory consolidation.

A low-cost method for brain slice cultures.

Low-cost, simple procedures for organotypic tissue cultures are desirable for high-throughput biological experiments such as large-scale medical/drug screening. We present a practical and economical method to cultivate brain slices using hydrophilic filtration membranes. With a cost reduction of more than 90%, this technique allows us to prepare hippocampal slice cultures that are morphologically and functionally indistinguishable from those obtained by the widely used Millicell-CM method.

Conserved role of brain-derived neurotrophic factor in Val66Met: target-selective reinforcement of GABAergic synapses.

Brain-derived neurotrophic factor has been implicated in higher cognitive functions, and several neurological and psychiatric disorders. Recently, a variant brain-derived neurotrophic factor (BDNFMet), having a substitution referred to as Val66Met, was reported as a product of a bdnf allele with a common single nucleotide polymorphism. It has been reported that BDNFMet is impaired in its potential for activity-dependent release. We sparsely transfected cultured hippocampal neurons with BDNFMet or wild-type BDNFVal cDNAs and examined the amount of GABA-synthetic enzyme glutamic acid decarboxylase 65 (GAD65) in the adjacent region, probably in the GABAergic synapses. BDNFMet transfection increased the GAD65 level to the same extent as transfection with BDNFVal. Our findings suggest that the activity-independent secretion of brain-derived neurotrophic factor may be sufficient to induce inhibitory regulation.

Estrogen produced in cultured hippocampal neurons is a functional regulator of a GABAergic machinery.

Accumulating evidence suggests that estrogen is produced locally by the neurons in the brain. We observed that a 48-hr treatment with the estrogen receptor antagonists ICI 182780 and tamoxifen decreased the level of glutamate decarboxylase (GAD)-65, a rate-limiting gamma-aminobutyric acid (GABA)-synthesizing enzyme, in a dissociated hippocampal neuronal culture. Aromatase is an essential enzyme for estrogen biosynthesis. Treatment with an aromatase inhibitor decreased the GAD 65 level, indicating that estrogen biogenesis functions to maintain the level of this enzyme for GABAergic neurotransmission. Furthermore, insofar as the effect of ICI 182780 was observed equivalently in the presence of either brain-derived neurotrophic factor (BDNF) or BDNF-receptor inhibitor K252a, estrogen probably regulates GAD level independently of brain-derived neurotrophic factor (BDNF). Thus, estrogen produced by neurons is considered to be an intrinsic regulatory factor for neuronal networks that maintain GABAergic neurotransmission.

K252a, an inhibitor of Trk, disturbs pathfinding of hippocampal mossy fibers.

Hippocampal mossy fibers, which are the axons of dentate granule cells, are continuously generated owing to adult neurogenesis of granule cells. They extend exclusively into the stratum lucidum, a proximal layer of the CA3 pyramidal cells. We visualized the mossy fiber tracts by Timm histochemical staining and DiI labeling in the cultured hippocampal slices from newborn rats. The fibers were abnormally expanded when the slices were cultured in the presence of K252a, an inhibitor of the neurotrophin receptor Trk. Similar defasciculation was observed with an inhibitor of MEK, which is one of the signaling molecules downstream of Trk. This study suggests for the first time that Trk and the MEK pathway are required for mossy fiber pathfinding.

IP3 receptor types 2 and 3 mediate exocrine secretion underlying energy metabolism.

Type 2 and type 3 inositol 1,4,5-trisphosphate receptors (IP3R2 and IP3R3) are intracellular calcium-release channels whose physiological roles are unknown. We show exocrine dysfunction in IP3R2 and IP3R3 double knock-out mice, which caused difficulties in nutrient digestion. Severely impaired calcium signaling in acinar cells of the salivary glands and the pancreas in the double mutants ascribed the secretion deficits to a lack of intracellular calcium release. Despite a normal caloric intake, the double mutants were hypoglycemic and lean. These results reveal IP3R2 and IP3R3 as key molecules in exocrine physiology underlying energy metabolism and animal growth.

BDNF locally potentiates GABAergic presynaptic machineries: target-selective circuit inhibition.

Inhibitory neurotransmission is critical for neuronal circuit formation. To examine whether inhibitory neurotransmission receives target-selective modulation in the long term, we expressed the cDNA of brain-derived neurotrophic factor (BDNF), which has been shown to induce the augmentation of GABAergic synapses in vivo and in vitro, in a small population of cultured hippocampal neurons. At 48 h after transfection, the expression level of glutamic acid decarboxylase 65 (GAD65), a GABA synthetic enzyme that resides mainly in GABAergic terminals, was selectively enhanced around the BDNF-expressing neurons, in comparison with the neighboring control neurons interposed between the BDNF-expressing neurons and inhibitory neurons. Exogenous BDNF application for 48 h also increased the GAD level and enhanced the GABA release probability. These potentiating effects were attenuated in inhibitory synapses on neurons expressing a dominant negative form of the BDNF receptor (tTrkB). This suggests that postsynaptic BDNF-TrkB signaling contributes to the target-selective potentiation of inhibitory presynaptic machineries. Since BDNF is expressed in an activity-dependent manner in vivo, this selectivity may be one of the key mechanisms by which the independence of functional neuronal circuits is maintained.