Juvenile social isolation immediately affects the synaptic activity and firing property of fast-spiking parvalbumin-expressing interneuron subtype in mouse medial prefrontal cortex.

A lack of juvenile social experience causes various behavioral impairments and brain dysfunction, especially in the medial prefrontal cortex (mPFC). Our previous studies revealed that juvenile social isolation for 2 weeks immediately after weaning affects the synaptic inputs and intrinsic excitability of fast-spiking parvalbumin-expressing (FSPV) interneurons as well as a specific type of layer 5 (L5) pyramidal cells, which we termed prominent h-current (PH) cells, in the mPFC. However, since these changes were observed at the adult age of postnatal day 65 (P65), the primary cause of these changes to neurons immediately after juvenile social isolation (postnatal day 35) remains unknown. Here, we investigated the immediate effects of juvenile social isolation on the excitability and synaptic inputs of PH pyramidal cells and FSPV interneurons at P35 using whole-cell patch-clamp recording. We observed that excitatory inputs to FSPV interneurons increased immediately after juvenile social isolation. We also found that juvenile social isolation increases the firing reactivity of a subtype of FSPV interneurons, whereas only a fractional effect was detected in PH pyramidal cells. These findings suggest that juvenile social isolation primarily disturbs the developmental rebuilding of circuits involving FSPV interneurons and eventually affects the circuits involving PH pyramidal cells in adulthood.

Ischemic Postconditioning Reduces NMDA Receptor Currents Through the Opening of the Mitochondrial Permeability Transition Pore and KATP Channel in Mouse Neurons.

Ischemic postconditioning (PostC) is known to reduce cerebral ischemia/reperfusion (I/R) injury; however, whether the opening of mitochondrial ATP-dependent potassium (mito-KATP) channels and mitochondrial permeability transition pore (mPTP) cause the depolarization of the mitochondrial membrane that remains unknown. We examined the involvement of the mito-KATP channel and the mPTP in the PostC mechanism. Ischemic PostC consisted of three cycles of 15 s reperfusion and 15 s re-ischemia, and was started 30 s after the 7.5 min ischemic load. We recorded N-methyl-D-aspartate receptors (NMDAR)-mediated currents and measured cytosolic Ca2+ concentrations, and mitochondrial membrane potentials in mouse hippocampal pyramidal neurons. Both ischemic PostC and the application of a mito-KATP channel opener, diazoxide, reduced NMDAR-mediated currents, and suppressed cytosolic Ca2+ elevations during the early reperfusion period. An mPTP blocker, cyclosporine A, abolished the reducing effect of PostC on NMDAR currents. Furthermore, both ischemic PostC and the application of diazoxide potentiated the depolarization of the mitochondrial membrane potential. These results indicate that ischemic PostC suppresses Ca2+ influx into the cytoplasm by reducing NMDAR-mediated currents through mPTP opening. The present study suggests that depolarization of the mitochondrial membrane potential by opening of the mito-KATP channel is essential to the mechanism of PostC in neuroprotection against anoxic injury.

Developmental dysregulation of excitatory-to-inhibitory GABA-polarity switch may underlie schizophrenia pathology: A monozygotic-twin discordant case analysis in human iPS cell-derived neurons.

Schizophrenia is a major psychiatric disorder, but the molecular mechanisms leading to its initiation or progression remain unclear. To elucidate the pathophysiology of schizophrenia, we used an in vitro neuronal cell culture model involving human induced pluripotent stem cells (hiPSCs) derived from a monozygotic-twin discordant schizophrenia pair. The cultured neurons differentiated from hiPSCs were composed of a mixture of glutamatergic excitatory neurons and gamma aminobutyric acid (GABA)ergic inhibitory neurons. In the electrophysiological analysis, a different pattern of spontaneous neuronal activity was observed under the condition without any stimulants. The frequency of spontaneous excitatory post-synaptic currents (sEPSCs) was significantly higher in the hiPSC-derived neurons of the patient with schizophrenia than in the control sibling at day-in-vitro 30. However, the synaptic formation was not different between the patient with schizophrenia and the control sibling during the same culture period. To explain underlying mechanisms of higher excitability of presynaptic cells, we focused on the potassium-chloride co-transporter KCC2, which contributes to excitatory-to-inhibitory GABA polarity switch in developing neurons. We also revealed the altered expression pattern of KCC2 in hiPSC-derived neurons from the patient with schizophrenia, which could contribute to understanding the pathology of schizophrenia in the developing nervous system.

Maternal Immune Activation Affects Hippocampal Excitatory and Inhibitory Synaptic Transmission in Offspring From an Early Developmental Period to Adulthood.

One of the risk factors for schizophrenia is maternal infection. We have previously shown that Polyriboinosinic-polyribocytidylic acid (poly I:C) induced maternal immune activation in mice caused histological changes in the hippocampal CA1 area of offspring during the developmental period and impaired sensorimotor gating in offspring during adulthood, resulting in behavioral changes. However, it remains unclear how maternal immune activation functionally impacts the hippocampal neuronal activity of offspring. We studied the effect of prenatal poly I:C treatment on synaptic transmission of hippocampal CA1 pyramidal cells in postnatal and adult offspring. Treatment with poly I:C diminished excitatory and enhanced inhibitory (GABAergic) synaptic transmission on pyramidal cells in adult offspring. During the early developmental period, we still observed that treatment with poly I:C decreased excitatory synaptic transmission and potentially increased GABAergic synaptic transmission, which was uncovered under a condition of high extracellular potassium-activated neurons. In conclusion, we demonstrate that maternal immune activation decreased excitatory and increased inhibitory synaptic transmission on hippocampal pyramidal cells from an early developmental period to adulthood, which could result in net inhibition in conjunction with poor functional organization and integration of hippocampal circuits.

Juvenile Social Isolation Enhances the Activity of Inhibitory Neuronal Circuits in the Medial Prefrontal Cortex.

During brain development, the design of primary neural networks is primarily determined by environmental stimuli after their formation. In particular, the juvenile period is critical, during which neuronal circuits that consist of both excitatory and inhibitory neurons are remodeled by experience. Social isolation during the juvenile period profoundly affects brain development and contributes to the development of psychiatric disorders. We previously reported that 2 weeks of social isolation after weaning reduced excitatory synaptic inputs and intrinsic excitability in a subtype of layer 5 pyramidal cells, which we defined as prominent h-current (PH) cells, in the medial prefrontal cortex (mPFC) in mice. However, it remains unclear how juvenile social isolation affects inhibitory neuronal circuits that consist of pyramidal cells and interneurons. We found that 2 weeks of social isolation after weaning increased inhibitory synaptic inputs exclusively onto PH cells with a concomitant deterioration of action potential properties. Although social isolation did not alter the inhibitory synaptic release mechanisms or the number of inhibitory functional synapses on PH cells, we found that it increased the intrinsic excitability of fast-spiking (FS) interneurons with less excitatory synaptic inputs and more h-current. Our findings indicate that juvenile social isolation enhances the activity of inhibitory neuronal circuits in the mPFC.

Ischemic postconditioning prevents surge of presynaptic glutamate release by activating mitochondrial ATP-dependent potassium channels in the mouse hippocampus.

A mild ischemic load applied after a lethal ischemic insult reduces the subsequent ischemia-reperfusion injury, and is called ischemic postconditioning (PostC). We studied the effect of ischemic PostC on synaptic glutamate release using a whole-cell patch-clamp technique. We recorded spontaneous excitatory post-synaptic currents (sEPSCs) from CA1 pyramidal cells in mouse hippocampal slices. The ischemic load was perfusion of artificial cerebrospinal fluid (ACSF) equilibrated with mixed gas (95% N2 and 5% CO2). The ischemic load was applied for 7.5 min, followed by ischemic PostC 30 s later, consisting of three cycles of 15 s of reperfusion and 15 s of re-ischemia. We found that a surging increase in sEPSCs frequency occurred during the immediate-early reperfusion period after the ischemic insult. We found a significant positive correlation between cumulative sEPSCs and the number of dead CA1 neurons (r = 0.70; p = 0.02). Ischemic PostC significantly suppressed this surge of sEPSCs. The mitochondrial KATP (mito-KATP) channel opener, diazoxide, also suppressed the surge of sEPSCs when applied for 15 min immediately after the ischemic load. The mito-KATP channel blocker, 5-hydroxydecanoate (5-HD), significantly attenuated the suppressive effect of both ischemic PostC and diazoxide application on the surge of sEPSCs. These results suggest that the opening of mito-KATP channels is involved in the suppressive effect of ischemic PostC on synaptic glutamate release and protection against neuronal death. We hypothesize that activation of mito-KATP channels prevents mitochondrial malfunction and breaks mutual facilitatory coupling between glutamate release and Ca2+ entry at presynaptic sites.

Social Isolation During the Critical Period Reduces Synaptic and Intrinsic Excitability of a Subtype of Pyramidal Cell in Mouse Prefrontal Cortex.

Juvenile social experience is crucial for the functional development of forebrain regions, especially the prefrontal cortex (PFC). We previously reported that social isolation for 2 weeks after weaning induces prefrontal cortex dysfunction and hypomyelination. However, the effect of social isolation on physiological properties of PFC neuronal circuit remained unknown. Since hypomyelination due to isolation is prominent in deep-layer of medial PFC (mPFC), we focused on 2 types of Layer-5 pyramidal cells in the mPFC: prominent h-current (PH) cells and nonprominent h-current (non-PH) cells. We found that a 2-week social isolation after weaning leads to a specific deterioration in action potential properties and reduction in excitatory synaptic inputs in PH cells. The effects of social isolation on PH cells, which involve reduction in functional glutamatergic synapses and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/N-methyl-d-aspartate charge ratio, are specific to the 2 weeks after weaning and to the mPFC. We conclude that juvenile social experience plays crucial roles in the functional development in a subtype of Layer-5 pyramidal cells in the mPFC. Since these neurons project to subcortical structures, a deficit in social experience during the critical period may result in immature neural circuitry between mPFC and subcortical targets.

A Subtype of Olfactory Bulb Interneurons Is Required for Odor Detection and Discrimination Behaviors.

UNLABELLED: Neural circuits that undergo reorganization by newborn interneurons in the olfactory bulb (OB) are necessary for odor detection and discrimination, olfactory memory, and innate olfactory responses, including predator avoidance and sexual behaviors. The OB possesses many interneurons, including various types of granule cells (GCs); however, the contribution that each type of interneuron makes to olfactory behavioral control remains unknown. Here, we investigated the in vivo functional role of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic arborization of 5T4-expressing GCs (5T4 GCs), the level of which is reduced in the OB of 5T4 knock-out (KO) mice. Electrophysiological recordings with acute OB slices indicated that external tufted cells (ETCs) can be divided into two types, bursting and nonbursting. Optogenetic stimulation of 5T4 GCs revealed their connection to both bursting and nonbursting ETCs, as well as to mitral cells (MCs). Interestingly, nonbursting ETCs received fewer inhibitory inputs from GCs in 5T4 KO mice than from those in wild-type (WT) mice, whereas bursting ETCs and MCs received similar inputs in both mice. Furthermore, 5T4 GCs received significantly fewer excitatory inputs in 5T4 KO mice. Remarkably, in olfactory behavior tests, 5T4 KO mice had higher odor detection thresholds than the WT, as well as defects in odor discrimination learning. Therefore, the loss of 5T4 attenuates inhibitory inputs from 5T4 GCs to nonbursting ETCs and excitatory inputs to 5T4 GCs, contributing to disturbances in olfactory behavior. Our novel findings suggest that, among the various types of OB interneurons, the 5T4 GC subtype is required for odor detection and discrimination behaviors. SIGNIFICANCE STATEMENT: Neuronal circuits in the brain include glutamatergic principal neurons and GABAergic interneurons. Although the latter is a minority cell type, they are vital for normal brain function because they regulate the activity of principal neurons. If interneuron function is impaired, brain function may be damaged, leading to behavior disorder. The olfactory bulb (OB) possesses various types of interneurons, including granule cells (GCs); however, the contribution that each type of interneuron makes to the control of olfactory behavior remains unknown. Here, we analyzed electrophysiologically and behaviorally the function of oncofetal trophoblast glycoprotein 5T4, a regulator for dendritic branching in OB GCs. We found that, among the various types of OB interneuron, the 5T4 GC subtype is required for odor detection and odor discrimination behaviors.

Plant regeneration: cellular origins and molecular mechanisms.

Compared with animals, plants generally possess a high degree of developmental plasticity and display various types of tissue or organ regeneration. This regenerative capacity can be enhanced by exogenously supplied plant hormones in vitro, wherein the balance between auxin and cytokinin determines the developmental fate of regenerating organs. Accumulating evidence suggests that some forms of plant regeneration involve reprogramming of differentiated somatic cells, whereas others are induced through the activation of relatively undifferentiated cells in somatic tissues. We summarize the current understanding of how plants control various types of regeneration and discuss how developmental and environmental constraints influence these regulatory mechanisms.

A simple and reliable multi-gene transformation method for switchgrass.

KEY MESSAGE: A simple and reliable Agrobacterium -mediated transformation method was developed for switchgrass. Using this method, many transgenic plants carrying multiple genes-of-interest could be produced without untransformed escape. Switchgrass (Panicum virgatum L.) is a promising biomass crop for bioenergy. To obtain transgenic switchgrass plants carrying a multi-gene trait in a simple manner, an Agrobacterium-mediated transformation method was established by constructing a Gateway-based binary vector, optimizing transformation conditions and developing a novel selection method. A MultiRound Gateway-compatible destination binary vector carrying the bar selectable marker gene, pHKGB110, was constructed to introduce multiple genes of interest in a single transformation. Two reporter gene expression cassettes, GUSPlus and gfp, were constructed independently on two entry vectors and then introduced into a single T-DNA region of pHKGB110 via sequential LR reactions. Agrobacterium tumefaciens EHA101 carrying the resultant binary vector pHKGB112 and caryopsis-derived compact embryogenic calli were used for transformation experiments. Prolonged cocultivation for 7 days followed by cultivation on media containing meropenem improved transformation efficiency without overgrowth of Agrobacterium, which was, however, not inhibited by cefotaxime or Timentin. In addition, untransformed escape shoots were completely eliminated during the rooting stage by direct dipping the putatively transformed shoots into the herbicide Basta solution for a few seconds, designated as the 'herbicide dipping method'. It was also demonstrated that more than 90 % of the bar-positive transformants carried both reporters delivered from pHKGB112. This simple and reliable transformation method, which incorporates a new selection technique and the use of a MultiRound Gateway-based binary vector, would be suitable for producing a large number of transgenic lines carrying multiple genes.

High-throughput cryopreservation of plant cell cultures for functional genomics.

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell-LSP solution mixtures was kept at -30 °C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics.

A non-destructive screenable marker, OsFAST, for identifying transgenic rice seeds.

The production of transgenic plants has contributed greatly to plant research. Previously, an improved method for screening transgenic Arabidopsis thaliana seeds using the FAST (Fluorescence-Accumulating-Seed Technology) method and FAST marker was reported. Arabidopsis seeds containing the FAST marker may be visually screened using a fluorescence stereomicroscope or blue LED handy-type instrument. Although the FAST method was originally designed for Arabidopsis screens, this study endeavors to adapt this method for the screening of other plants. Here, an optimized technology, designated the OsFAST method, is presented as a useful tool for screening transgenic rice seeds. The OsFAST method is based on the expression of the OsFAST-G marker under the control of a seed-embryo-specific promoter, similar to the Arabidopsis FAST-G marker. The OsFAST method provides a simple and non-destructive method for identifying transgenic rice seeds. It is proposed that the FAST method is adaptable to various plant species and will enable a deeper analysis of the floral-dip method. 

Cryopreservation and metabolic profiling analysis of Arabidopsis T87 suspension-cultured cells.

We established a simple cryopreservation protocol for Arabidopsis T87 cells using an encapsulation-dehydration method. T87 cells were encapsulated into alginate beads containing 2 M glycerol and 0.4 M sucrose. Alginate beads containing T87 cells were dehydrated with silica gel for 2 h (to c. 0.7 g water per g dry weight followed by immersed in LN. After rewarming at 35 degree C for 3 min and 1-d incubation under continuous illumination at 22 degree C, cryopreserved T87 cells exhibited considerable regrowth. Exponentially-grown 7-d-old T87 cells regrew more vigorously (86% of control) than 14-d-old cells after cryopreservation without preculture in medium containing 0.3 M sucrose. Genetic stability of cryopreserved T87 cells was demonstrated by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) and principal component analysis (PCA). Transformed T87 cells were cryopreserved using established protocols, and GUS expression was maintained within a 2-fold variance. These results indicate that cryopreservation of T87 cells is useful for comprehensive metabolomics research and for the large scale collection of transformed cultured cell lines for functional genomics research.

Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics.

We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.

Voltage- and Ca2+-activated potassium channels in Ca2+ store control Ca2+ release.

Ca2+ release from Ca2+ stores is a 'quantal' process; it terminates after a rapid release of stored Ca2+. To explain the quantal nature, it has been supposed that a decrease in luminal Ca2+ acts as a 'brake' on store release. However, the mechanism for the attenuation of Ca2+ efflux remains unknown. We show that Ca2+ release is controlled by voltage- and Ca2+-activated potassium channels in the Ca2+ store. The potassium channel was identified as the big or maxi-K (BK)-type, and was activated by positive shifts in luminal potential and luminal Ca2+ increases, as revealed by patch-clamp recordings from an exposed nuclear envelope. The blockage or closure of the store BK channel due to Ca2+ efflux developed lumen-negative potentials, as revealed with an organelle-specific voltage-sensitive dye [DiOC5(3); 3,3'-dipentyloxacarbocyanine iodide], and suppressed Ca2+ release. The store BK channels are reactivated by Ca2+ uptake by Ca2+ pumps regeneratively with K+ entry to allow repetitive Ca2+ release. Indeed, the luminal potential oscillated bistably by approximately 45 mV in amplitude. Our study suggests that Ca2+ efflux-induced store BK channel closures attenuate Ca2+ release with decreases in counter-influx of K+.

Evaluation of 12 beta-lactam antibiotics for Agrobacterium-mediated transformation through in planta antibacterial activities and phytotoxicities.

The antibacterial activities of 12 beta-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 living in tobacco (Nicotiana tabacum L.) leaf tissues, and their phytotoxicities to tobacco leaf tissues were evaluated. All beta-lactams at minimum bactericidal concentration (MBC) or higher showed weak bactericidal activities against agrobacteria persisting in tobacco leaf tissues. The beta-lactams evaluated were classified into two major groups according to their inhibitory effect on shoot regeneration of tobacco leaf tissues: (1) highly phytotoxic drugs, and (2) moderately phytotoxic drugs. According to these results, suitable kind and concentration of beta-lactam antibiotics were evaluated for Agrobacterium-mediated transformation.

Screening for highly active beta-lactam antibiotics against Agrobacterium tumefaciens.

Quantitative in vitro antibacterial activities, i.e., minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs), of 12 beta-lactam antibiotics against Agrobacterium tumefaciens strains LBA4404 and EHA101 were examined, in order to identify antibiotics effective in eliminating the bacteria in Agrobacterium-mediated plant genetic transformation. The antibacterial activities of beta-lactams tested against strain EHA101 were equal to or less than those tested against strain LBA4404. Cefotaxime, cefbuperazone, and meropenem had high activities against strain LBA4404 (MBC <1 mg l(-1)). Against strain EHA101, however, only meropenem showed activity comparable to that against strain LBA4404. The production of beta-lactamase was observed only in strain EHA101.

Chemical preconditioning prevents paradoxical increase in glutamate release during ischemia by activating ATP-dependent potassium channels in gerbil hippocampus.

Ischemic tolerance induced by pretreatment with a low dose of 3-nitropropionic acid (3-NPA), called chemical preconditioning, prolongs the delay to hypoxic depolarization and improves the recovery of synaptic transmission (Exp. Neurol. 166 (2000), 385-391). We studied the effect of chemical preconditioning on the presynaptic site by analyzing spontaneous excitatory postsynaptic currents (sEPSCs) and miniature EPSCs (mEPSCs) with a whole cell patch-clamp technique in gerbil hippocampal slices. The frequency of sEPSCs decreased first and then dramatically increased during ischemia (10 min in duration, low pO(2), and deprivation of glucose) up to 200-300%. This increase was apparently a paradox, since synaptic transmission evoked by electrical stimulation diminished when the sEPSC frequency started to increase. The frequency of mEPSCs also increased in the same time course. Increases in sEPSC and mEPSC frequencies were prevented by chemical preconditioning with 3-NPA (4 mg/kg) administered intraperitoneally 3 h before the preparation of brain slices. These effects of chemical preconditioning were abolished by glibenclamide (5 microM), a blocker of ATP-dependent potassium (K(ATP)) channels, applied in vitro before the ischemic insult. The application of diazoxide (500 microM), an opener of K(ATP) channels, produced the same preventive effects on sEPSC and mEPSC frequencies. These results suggested that chemical preconditioning acted on presynaptic terminals to prevent the paradoxical increase in glutamate release during ischemia through the activation of K(ATP) channels.

High transmittance of X-rays in the utricular otolith of upside-down swimming catfish, Synodontis nigriventris.

The upside-down swimming catfish (Synodontis nigriventris) has unique behavior, i.e., it frequently shows a stable upside-down posture during swimming and resting. To examine whether the unique postural control in S. nigriventris results from the characteristics of the vestibular organ, we observed the morphological aspects of the otolith and the orientation of sensory hair cells in the utricle. Soft X-ray densitometry analysis showed that the transmittance of soft X-rays in the otolith of S. nigriventris was higher than that in a closely related species (Synodontis multipunctatus) belonging to Synodontis family, goldfish (Carassius auratus) or miniature catfish (Corydoras paleatus) which shows upside-up swimming. The higher transmittance of soft X-rays suggests that the density of the otolith in S. nigriventris is lower than that in S. multipunctatus, C. auratus or C. paleatus. It is possible that the low density of the otolith may have a relation to the control of the unique upside-down posture of S. nigriventris. The hair cells in S. nigriventris were present at the ventral to ventro-lateral site of the utricular epithelium, forming a single hair cell layer as in the other 3 species of fish. The orientation of the sensory hair cells does not appear to cause the unique postural control.