Anatomical identification of a corticocortical top-down recipient inhibitory circuitry by enhancer-restricted transsynaptic tracing.

Despite the importance of postsynaptic inhibitory circuitry targeted by mid/long-range projections (e.g., top-down projections) in cognitive functions, its anatomical properties, such as laminar profile and neuron type, are poorly understood owing to the lack of efficient tracing methods. To this end, we developed a method that combines conventional adeno-associated virus (AAV)-mediated transsynaptic tracing with a distal-less homeobox (Dlx) enhancer-restricted expression system to label postsynaptic inhibitory neurons. We called this method "Dlx enhancer-restricted Interneuron-SpECific transsynaptic Tracing" (DISECT). We applied DISECT to a top-down corticocortical circuit from the secondary motor cortex (M2) to the primary somatosensory cortex (S1) in wild-type mice. First, we injected AAV1-Cre into the M2, which enabled Cre recombinase expression in M2-input recipient S1 neurons. Second, we injected AAV1-hDlx-flex-green fluorescent protein (GFP) into the S1 to transduce GFP into the postsynaptic inhibitory neurons in a Cre-dependent manner. We succeeded in exclusively labeling the recipient inhibitory neurons in the S1. Laminar profile analysis of the neurons labeled via DISECT indicated that the M2-input recipient inhibitory neurons were distributed in the superficial and deep layers of the S1. This laminar distribution was aligned with the laminar density of axons projecting from the M2. We further classified the labeled neuron types using immunohistochemistry and in situ hybridization. This post hoc classification revealed that the dominant top-down M2-input recipient neuron types were somatostatin-expressing neurons in the superficial layers and parvalbumin-expressing neurons in the deep layers. These results demonstrate that DISECT enables the investigation of multiple anatomical properties of the postsynaptic inhibitory circuitry.

Fluorescence quenching by high-power LEDs for highly sensitive fluorescence in situ hybridization.

Recent technical advances have made fluorescent in situ hybridization (ISH) a pivotal method to analyze neural tissue. In a highly sensitive ISH, it is important to reduce tissue autofluorescence. We developed a photobleaching device using a light-emitting diode (LED) illuminator to quench autofluorescence in neural tissue. This device was equipped with 12 high-power LEDs (30 W per single LED) and an evaporative cooling system, and these features achieved highly efficient bleaching of autofluorescence and minimized tissue damage. Even after 60 min of photobleaching with evaporative cooling, the temperature gain of the tissue slide was suppressed almost completely. The autofluorescence of lipofuscin-like granules completely disappeared after 60 min of photobleaching, as did other background autofluorescence observed in the mouse cortex and hippocampus. In combination with the recently developed fluorescent ISH method using the hybridization chain reaction (HCR), high signal/noise ratio imaging was achieved without reduction of ISH sensitivity to visualize rare mRNA at single copy resolution by quenching autofluorescence. Photobleaching by the LED illuminator was also effective in quenching the fluorescent staining of ISH-HCR. We performed multiround ISH by repeating the cycle of HCR staining, confocal imaging, and photobleaching. In addition to the two-round ISH, fluorescent immunohistochemistry or fluorescent Nissl staining was conducted on the same tissue. This LED illuminator provides a quick and simple way to reduce autofluorescence and quench fluorescent dyes for multiround ISH with minimum tissue degradation.

Different Photoperiodic Responses in Four Lines of Japanese Quail.

Organisms measure day length to better adapt to seasonal changes in the environment; this phenomenon is called photoperiodism. The Japanese quail has a highly sophisticated photoperiodic mechanism and is an excellent model for the study of photoperiodism. Various lines of quail have been established during the domestication process. In the present study, we examined the effect of long day (LD) followed by short day (SD) on testicular weight in four lines of quail (L, AMRP, NIES-Br, and WE). When the quail were raised under SD conditions, testicular development was suppressed in all examined lines. The speed of the LD-induced testicular development of NIES-Br line was faster than that of AMRP line, while the speed of the SD-induced testicular regression of L line was significantly faster than that of WE line. These quail lines provide excellent model to uncover the underlying mechanism of seasonal testicular regression.

Lactic acid is a sperm motility inactivation factor in the sperm storage tubules.

Although successful fertilization depends on timely encounters between sperm and egg, the decoupling of mating and fertilization often confers reproductive advantages to internally fertilizing animals. In several vertebrate groups, postcopulatory sperm viability is prolonged by storage in specialized organs within the female reproductive tract. In birds, ejaculated sperm can be stored in a quiescent state within oviductal sperm storage tubules (SSTs), thereby retaining fertilizability for up to 15 weeks at body temperature (41°C); however, the mechanism by which motile sperm become quiescent within SSTs is unknown. Here, we show that low oxygen and high lactic acid concentrations are established in quail SSTs. Flagellar quiescence was induced by lactic acid in the concentration range found in SSTs through flagellar dynein ATPase inactivation following cytoplasmic acidification (

Low temperature-induced circulating triiodothyronine accelerates seasonal testicular regression.

In temperate zones, animals restrict breeding to specific seasons to maximize the survival of their offspring. Birds have evolved highly sophisticated mechanisms of seasonal regulation, and their testicular mass can change 100-fold within a few weeks. Recent studies on Japanese quail revealed that seasonal gonadal development is regulated by central thyroid hormone activation within the hypothalamus, depending on the photoperiodic changes. By contrast, the mechanisms underlying seasonal testicular regression remain unclear. Here we show the effects of short day and low temperature on testicular regression in quail. Low temperature stimulus accelerated short day-induced testicular regression by shutting down the hypothalamus-pituitary-gonadal axis and inducing meiotic arrest and germ cell apoptosis. Induction of T3 coincided with the climax of testicular regression. Temporal gene expression analysis over the course of apoptosis revealed the suppression of LH response genes and activation of T3 response genes involved in amphibian metamorphosis within the testis. Daily ip administration of T3 mimicked the effects of low temperature stimulus on germ cell apoptosis and testicular mass. Although type 2 deiodinase, a thyroid hormone-activating enzyme, in the brown adipose tissue generates circulating T3 under low-temperature conditions in mammals, there is no distinct brown adipose tissue in birds. In birds, type 2 deiodinase is induced by low temperature exclusively in the liver, which appears to be caused by increased food consumption. We conclude that birds use low temperature-induced circulating T3 not only for adaptive thermoregulation but also to trigger apoptosis to accelerate seasonal testicular regression.

Application of IgY to sandwich enzyme-linked immunosorbent assays, lateral flow devices, and immunopillar chips for detecting staphylococcal enterotoxins in milk and dairy products.

Staphylococcal enterotoxins (SEs), produced by Staphylococcus aureus, are a major cause of staphylococcal food poisoning. Traditionally, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse passive latex agglutination with rabbit antibody IgG have been used to detect SEs. However, most of these kits require a long processing time and there is a risk of false-positive results since IgG reacts nonspecifically with protein A produced by S. aureus. In this study, we prepared antienterotoxin chicken IgY antibodies specific for each SE (SEA to SEE) without reaction to protein A, which enabled a drastic reduction in nonspecific reactions. ELISAs, lateral flow device (LFDs), and IgY-based immunopillar chips were developed for SE detection. All the ELISAs developed were as sensitive as commercially available kits. The SEs in milk were successfully detected by the ELISAs, LFDs, and immunopillar chips without any sample pretreatment. The LFD could detect SEA even at the low concentration of 0.2 ng/ml within 15 min in milk. The detection limit of the immunopillar chips for the SEs ranged from 0.01 to 0.1 ng/ml in milk; the SEs were detected within 12 min and specialized skills were not required. The ELISA and LFD detected SEA in dairy products artificially contaminated with S. aureus, including ice cream, yogurt, and café au lait, in a dose-dependent manner. In conclusion, IgY allows highly specific detection of SEs, and ELISAs, LFDs, and immunopillar chips should be useful tools for screening SEs in milk and dairy products.

Novel method of gene transfer in birds: intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms.

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

Phospholipase Czeta mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor.

This study was conducted to investigate the role of a sperm-borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Czeta (PLCzeta) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCzeta cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca(2+) chelator) before SE injection. On the other hand, when the oocytes were injected with PLCzeta cRNA at 60 microg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCzeta cRNA-induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCzeta cRNA can induce development. In addition, RT-PCR revealed that PLCzeta mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCzeta is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis.

Increased proportion of donor primordial germ cells in chimeric gonads by sterilisation of recipient embryos using busulfan sustained-release emulsion in chickens.

The aim of the present study was to improve the efficiency of endogenous primordial germ cell (PGC) depletion and to increase the ratio of donor PGCs in the gonads of recipient chicken embryos. A sustained-release emulsion was prepared by emulsifying equal amounts of Ca(2+)- and Mg(2+)-free phosphate-buffered saline containing 10% busulfan solubilised in N,N-dimethylformamide and sesame oil, using a filter. Then, 75 microg per 50 microL busulfan sustained-release emulsion was injected into the yolk. To determine the depletion and repopulation of PGCs in the gonads after 6 days incubation, whole-mount immunostaining was performed. The busulfan sustained-release emulsion significantly reduced the number of endogenous PGCs compared with control (P < 0.05). Moreover, the busulfan sustained-release emulsion significantly depleted endogenous PGCs compared with other previously reported busulfan delivery systems (P < 0.05), but with less variation, suggesting that the sustained-release emulsion delivered a consistent amount of busulfan to the developing chicken embryos. The PGC transfer study showed that the proportion of donor PGCs in the gonads of busulfan sustained-release emulsion-treated embryos after 6 days incubation increased 28-fold compared with control. In conclusion, the results demonstrate that exogenous PGCs are capable of migrating and settling in gonads from which endogenous PGCs have been removed using a busulfan sustained-release emulsion.

Possible role of calcium on oocyte development after intracytoplasmic sperm injection in quail (Coturnix japonica).

Although a rise in intracellular calcium concentration of vertebrate oocytes plays a pivotal role for the initiation of fertilization or oocyte activation, no study on this subject has been reported in birds. This study was conducted to study the role of intracellular calcium in relation to fertilization in avian oocytes. First, immediately after a quail oocyte was injected with a sperm, it was treated with strontium chloride as an inducer for intracellular calcium rise at doses of 0, 2.5, 5, 7.5, 10 mM for 4 hr in the culture medium and was followed by 20-hr culture. Treatment with 5 mM of strontium chloride induced blastodermal development in 24.2% of injected eggs, although no oocytes developed without strontium treatment. Second, quail oocytes were injected with a sperm and 0.1 M calcium chloride or a sperm and saline solution, cultured without calcium for 4 hr and was followed by 20-hr culture without strontium. The calcium solution induced blastodermal development in 20.5% of the oocytes, although no oocytes developed without calcium treatment. Third, quail oocytes were injected with 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) as a calcium chelator, cultured with strontium (5 mM) for 4 hr followed by 20-hr culture without strontium. Only one oocyte developed after BAPTA and strontium treatment of 36 oocytes examined. Developmental stages of all the oocytes ranged from IV to VII. These results suggest that intracellular calcium rise may participate in quail oocyte activation and allow fertilization and blastodermal development.