RESUMEN
The circadian system comprises multiple clocks, including central and peripheral clocks. The central clock generally governs peripheral clocks to synchronize circadian rhythms throughout the animal body. However, whether the peripheral clock influences the central clock is unclear. This issue can be addressed through a system comprising a peripheral clock (compound eye clock [CE clock]) and central clock (the optic lobe [OL] clock) in the cricket Gryllus bimaculatus. We previously found that the compound eye regulates the free-running period (τ) and the stability of locomotor rhythms driven by the OL clock, as measured by the daily deviation of τ at 30°C. However, the role of the CE clock in this regulation remains unexplored. In this study, we investigated the importance of the CE clock in this regulation using RNA interference (RNAi) of the period (per) gene localized to the compound eye (perCE-RNAi). The perCE-RNAi abolished the compound eye rhythms of the electroretinogram (ERG) amplitude and clock gene expression but the locomotor rhythm driven by the OL clock was maintained. The locomotor rhythm of the tested crickets showed a significantly longer τ and greater daily variation of τ than those of control crickets treated with dsDsRed2. The variation of τ was comparable with that of crickets with the optic nerve severed. The τ was considerably longer but was comparable with that of crickets with the optic nerve severed. These results suggest that the CE clock regulates the OL clock to maintain and stabilize τ.
Asunto(s)
Relojes Circadianos , Gryllidae , Lóbulo Óptico de Animales no Mamíferos , Animales , Gryllidae/fisiología , Relojes Circadianos/fisiología , Lóbulo Óptico de Animales no Mamíferos/fisiología , Ojo Compuesto de los Artrópodos/fisiología , Regulación de la Expresión Génica , Locomoción/fisiología , Ritmo Circadiano/fisiologíaRESUMEN
The incidence of cerebral infarction triggered by abnormal glucose tolerance has increased; however, the relationship between glucose concentration in the brain and the detailed mechanism of post ischemic cell death remains unclear. Nicotinamide phosphoribosyltransferase (NAMPT), an adipocytokine, is the rate-limiting enzyme for NAD+ synthesis in the salvage pathway. Although NAMPT activation prevents neuronal injury, the relationship between NAMPT activity, glucose metabolism disorders, and cerebral ischemia-induced neuronal cell death is unknown. In this study, we determined changes in NAMPT on cerebral ischemic injuries with diabetes using a db/db mouse model of type 2 diabetes and then identified the underlying mechanisms using Neuro2a cells. The expression of inflammatory cytokine mRNAs was increased in db/db and db/+ middle cerebral artery occlusion and reperfusion (MCAO/R) mice. Although NeuN-positive cells were decreased after MCAO/R, the number of NAMPT and NeuN double-positive cells in NeuN-positive neuronal cells increased in db/db MCAO/R mice. Next, the role of NAMPT in Neuro2a cells under conditions of high glucose (HGC) and oxygen-glucose deprivation (OGD), which mimics diabetes-complicated cerebral infarction, was examined. Treatment with P7C3-A20, a NAMPT activator, suppressed the decrease in cell viability caused by HGC/OGD; however, there were no significant differences in the levels of cleaved caspase-3 and Bax proteins. Moreover, increased FoxO3a and LC3-II levels after HGC/OGD were inhibited by P7C3-A20 treatment. Our findings indicate that NAMPT activation is associated with neuronal survival under ischemic conditions with abnormal glucose tolerance through the regulation of FoxO3a/LC3.
Asunto(s)
Isquemia Encefálica , Supervivencia Celular , Proteína Forkhead Box O3 , Glucosa , Neuronas , Nicotinamida Fosforribosiltransferasa , Transducción de Señal , Animales , Nicotinamida Fosforribosiltransferasa/metabolismo , Proteína Forkhead Box O3/metabolismo , Glucosa/metabolismo , Glucosa/deficiencia , Neuronas/metabolismo , Neuronas/patología , Neuronas/efectos de los fármacos , Masculino , Ratones , Supervivencia Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Citocinas/metabolismo , Ratones Endogámicos C57BL , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/metabolismo , Línea Celular Tumoral , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/complicacionesRESUMEN
Ischemic stroke in patients with abnormal glucose tolerance results in poor outcomes. Nicotinamide phosphoribosyltransferase (NAMPT), an adipocytokine, exerts neuroprotective effects. However, the pathophysiological role of NAMPT after ischemic stroke with diabetes and the relationship of NAMPT with cerebrovascular lesions are unclear. The purpose of this study was to clarify the pathophysiological role of NAMPT in cerebral ischemia with diabetes, using db/db mice as a type 2 diabetes animal model. The number of degenerating neurons increased after middle cerebral artery occlusion and reperfusion (MCAO/R) in db/db mice compared with the degenerating neurons in db/+ mice. Extracellular NAMPT (eNAMPT) levels, especially monomeric eNAMPT, increased significantly in db/db MCAO/R mice but not db/+ mice in isolated brain microvessels. The increased eNAMPT levels were associated with increased expression of inflammatory cytokine mRNA. Immunohistochemical analysis demonstrated that NAMPT colocalized with GFAP-positive cells after MCAO/R. In addition, both dimeric and monomeric eNAMPT levels increased in the conditioned medium of primary cortical astrocytes under high glucose conditions subsequent oxygen/glucose deprivation. Our findings are the first to demonstrate the ability of increased monomeric eNAMPT to induce inflammatory responses in brain microvessels, which may be located near astrocyte foot processes.
Asunto(s)
Diabetes Mellitus Tipo 2 , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Animales , Humanos , Ratones , Citocinas , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Nicotinamida Fosforribosiltransferasa/metabolismo , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/patologíaRESUMEN
Most insects show circadian rhythms of which the free-running period changes in a light-dependent manner and is generally longer under constant light (LL) than under constant dark conditions in nocturnal animals. However, the mechanism underlying this LL-dependent period change remains unclear. Here, using the cricket Gryllus bimaculatus, we examined the effects of long-term LL exposure on the free-running period of locomotor rhythms. Initially, the free-running period was considerably longer than 24 h but it gradually became shorter during long-term exposure to LL. The initial lengthening and ensuing gradual shortening under long-term LL exposure were observed even after unilateral removal of the optic lobe. Thus, these changes in the free-running period could be attributable to a single optic lobe clock. RNA interference (RNAi)-mediated silencing of the clock genes Par domain protein 1 (Pdp1) and timeless (tim) revealed that the treatments eliminated the initial period lengthening by LL without reducing circadian photoreceptor gene expression. However, they did not affect the period shortening during long-term LL exposure. The slopes of the regression line for the period change during long-term LL for Pdp1RNAi-treated and timRNAi-treated crickets were not different from that of the dsDsRed2-treated control. These results suggest that the initial period lengthening after transfer to LL requires tim and Pdp1, while the ensuing period shortening during long-term LL exposure is caused by a mechanism independent of tim and Pdp1.
Asunto(s)
Gryllidae , Animales , Ritmo Circadiano , Gryllidae/genética , Gryllidae/metabolismo , Luz , Interferencia de ARNRESUMEN
The light cycle is the most powerful Zeitgeber entraining the circadian clock in most organisms. Insects use CRYPTOCHROMEs (CRYs) and/or the compound eye for the light perception necessary for photic entrainment. The molecular mechanism underlying CRY-dependent entrainment is well understood, while that of the compound eye-dependent entrainment remains to be elucidated. Using molecular and behavioral experiments, we investigated the role of timeless (tim) in the photic entrainment mechanism in the cricket Gryllus bimaculatus. RNA interference of tim (timRNAi) disrupted the entrainment or prolonged the transients for resynchronization to phase-delayed light-dark cycles. The treatment reduced the magnitude of phase delay caused by delayed light-off, but augmented advance shifts caused by light exposure at late night. TIM protein levels showed daily cycling with an increase during the night and reduction by light exposure at both early and late night. These results suggest that tim plays a critical role in the entrainment to delayed light cycles.
Asunto(s)
Relojes Circadianos , Gryllidae , Animales , Relojes Circadianos/genética , Ritmo Circadiano , Gryllidae/genética , Luz , Fotoperiodo , Interferencia de ARNRESUMEN
Many insects show daily and circadian changes in morphology and physiology in their compound eye. In this study, we investigated whether the compound eye had an intrinsic circadian rhythm in the cricket Gryllus bimaculatus. We found that clock genes period (per), timeless (tim), cryptochrome 2 (cry2), and cycle (cyc) were rhythmically expressed in the compound eye under 12-h light/12-h dark cycles (LD 12:12) and constant darkness (DD) at a constant temperature. After the optic nerves were severed (ONX), a weak but significant rhythmic expression persisted for per and tim under LD 12:12, while under DD, tim and cyc showed rhythmic expression. We also found that more than half of the ONX compound eyes exhibited weak but significant circadian electroretinographic rhythms. These results clearly demonstrate that the cricket compound eye possesses an intrinsic circadian oscillator which can drive the circadian light sensitivity rhythm in the eye, and that the circadian clock in the optic lobe exerts its influence on the oscillator in the eye.
Asunto(s)
Relojes Circadianos/genética , Ojo Compuesto de los Artrópodos/fisiología , Gryllidae/genética , Gryllidae/fisiología , Animales , Ritmo Circadiano/fisiología , Ojo Compuesto de los Artrópodos/inervación , Electrorretinografía , Masculino , FotoperiodoRESUMEN
BACKGROUND: Entrainment to the environmental light cycle is an essential property of the circadian clock. Although the compound eye is known to be the major photoreceptor necessary for entrainment in many insects, the molecular mechanisms of photic entrainment remain to be explored. RESULTS: We found that cryptochromes (crys) and c-fos mediate photic entrainment of the circadian clock in a hemimetabolous insect, the cricket Gryllus bimaculatus. We examined the effects of RNA interference (RNAi)-mediated knockdown of the cry genes, Gb'cry1 and Gb'cry2, on photic entrainment, and light-induced resetting of the circadian locomotor rhythm. Gb'cry2 RNAi accelerated entrainment for delay shifts, while Gb'cry1/ Gb'cry2 double RNAi resulted in significant lengthening of transient cycles in both advance and delay shifts, and even in entrainment failure in some crickets. Double RNAi also strongly suppressed light induced resetting. The Gb'cry-mediated phase shift or resetting of the rhythm was preceded by light-induced Gb'c-fosB expression. We also found that Gb'c-fosB, Gb'cry2 and Gb'period (Gb'per) were likely co-expressed in some optic lobe neurons. CONCLUSION: Based on these results, we propose a novel model for photic entrainment of the insect circadian clock, which relies on the light information perceived by the compound eye.
RESUMEN
Although functional disruption of the cerebrovasculature, which is called the "neurovascular unit (NVU)", may lead to amplification of ischemia-induced injury, changes in the gap junctional proteins within the NVU and their pathophysiological roles after brain injury remain controversial. We previously demonstrated that the intravenous injection of neural progenitor cells (NPCs) have therapeutic potential for improving the spatial learning dysfunction and depression-like behaviors observed after cerebral ischemia. In this study, we investigated whether severe cerebral ischemia would alter the expression of gap junctional proteins in isolated brain capillaries and examined the effect of intravenous injection of NPCs on the levels of these proteins. Cerebral ischemia induced a sustained decrease in the level of the gap junctional protein connexin 43 (Cx43) in the isolated brain capillaries, whereas the level of aquaporin 4 (AQP-4) was transiently increased. The injection of NPCs increased the level of Cx43 compared that of vehicle in the microsphere embolism (ME) rats, suggesting this decrease to be a possible mechanism for disruption of the astrocyte-endothelial cell interface within the NVU without causing any changes in the level of AQP-4 and N-cadherin. We also demonstrated that some of the intravenously injected NPCs migrated into the blood vessels in the peri-infarct area. These results suggest that the intravenous injection of the NPCs would remodel the NVU after severe cerebral ischemia, which remodeling might be associated with functional improvement following the NPC injection.
Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Capilares/metabolismo , Conexina 43/metabolismo , Células-Madre Neurales/trasplante , Animales , Acuaporina 4/farmacología , Astrocitos/metabolismo , Encéfalo/irrigación sanguínea , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Cadherinas/metabolismo , Movimiento Celular , Células Cultivadas , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Células-Madre Neurales/patología , Células-Madre Neurales/fisiología , Ratas , Ratas Transgénicas , Ratas WistarRESUMEN
Earlier we demonstrated that the injection of neural progenitor cells (NPCs) has therapeutic potential for the improvement of learning dysfunction after cerebral ischemia. However, it remained to be clarified how transplantation of NPCs can improve ischemia-induced dysfunction. In this study, we examined whether intravenous injection of NPCs after cerebral ischemia could enhance angiogenesis by affecting the expression of angiogenic factors. The injection of NPCs on day 7 after cerebral ischemia enhanced angiogenesis on day 28. Vascular endothelial growth factor (VEGF) and its receptor VEGFR2 were increased in expression by the NPC injection. The level of angiopoietin-1 (Ang-1), an angiogenic factor, but not that of Ang-2, which acts as an antagonist for the Ang-1 receptor, was also increased on day 28. In addition, the expression of Ang-1 receptor Tie2 was enhanced in brain capillaries. Furthermore, the amounts of tight junctional proteins, which are in the blood-brain barrier and whose expression occurs downstream of Ang-1/Tie2 signaling, were increased by the NPC injection. These results suggest that the NPC injection promoted angiogenesis through Ang-1/Tie2 and/or VEGF/VEGFR2 signaling in brain capillaries after cerebral ischemia. Such signaling might have the potential for causing vascular stabilization and maturation for a long period after cerebral ischemia.
RESUMEN
Reverse genetic studies have revealed that common clock genes, such as period (per), timeless (tim), cycle (cyc), and Clock (Clk), are involved in the circadian clock mechanism among a wide variety of insects. However, to what degree the molecular oscillatory mechanism is conserved is still to be elucidated. In this study, cDNA of the clock gene Clk was cloned in the cricket Gryllus bimaculatus, and its function was analyzed using RNA interference (RNAi). In adult optic lobes, the Clk mRNA level showed no significant rhythmic changes both under light-dark cycle (LD) and constant darkness (DD). A single injection of Clk double-stranded RNA (dsRNA) resulted in a knockdown of the mRNA level to about 25% of the peak level of control animals. The injected crickets lost their locomotor rhythms in DD. The arrhythmicity in locomotor activity persisted for up to 50 days after the Clk dsRNA injection. Control animals injected with DsRed2 dsRNA showed a clear locomotor rhythm like intact animals. Injection of Clk dsRNA not only suppressed the mRNA levels of both per and tim but also abolished their rhythmic expression. per RNAi down-regulates the Clk mRNA levels, suggesting that per is required for sufficient expression of Clk. These results suggest that Clk is an essential component and plays an important role in the cricket's circadian clock machinery like in Drosophila, but regulation of its expression is probably different from regulation in Drosophila.
Asunto(s)
Proteínas CLOCK/metabolismo , Relojes Circadianos , Gryllidae/metabolismo , Proteínas de Insectos/metabolismo , Lóbulo Óptico de Animales no Mamíferos/metabolismo , Animales , Clonación Molecular , Locomoción , Masculino , Proteínas Circadianas Period/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Activación TranscripcionalRESUMEN
In the cricket Gryllus bimaculatus, a hemimetabolous insect, the compound eyes begin to form in the embryo and increase 5-6 fold in size during the postembryonic development of the nymphal stage. Retinal stem cells in the anteroventral proliferation zone (AVPZ) of the nymphal eye proliferate to increase retinal progenitors, which then differentiate to form new ommatidia in the anterior region of the eye. However, mechanisms underlying this type of eye formation have not been well elucidated yet. Here, we found that the homologues of the retinal determination transcription factor genes of eyes absent (eya) and sine oculis (so) are expressed during the cricket embryonic eye formation. eya is also expressed intensely in the AVPZ of the nymphal eye. To explore their functions, we performed knockdown by RNA interference (RNAi). Knockdown of Gb'eya resulted in loss of the embryonic eye. In the nymphal eye, RNAi against Gb'eya or Gb'so impaired retinal morphology by apparently transforming cornea structures into head cuticle. These results imply that Gb'eya and Gb'so are essential for the differentiation of the retinal progenitor cells and maintaining retinal structures during eye development.
Asunto(s)
Ojo/embriología , Ojo/metabolismo , Proteínas de Insectos/metabolismo , Factores de Transcripción/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Gryllidae , Proteínas de Insectos/genética , Interferencia de ARN/fisiología , Factores de Transcripción/genéticaRESUMEN
The ability of stem cells to enhance neurological recovery seen after cerebral ischemia has been reported. However, it remains to be clarified whether neural progenitor cells (NPCs) improve cerebral ischemia-induced learning dysfunction. We found in an earlier study that the direct injection of NPCs into the hippocampus prevents spatial learning dysfunction after cerebral ischemia. As the intravascular injection of cells represents a minimally invasive therapeutic approach, we sought to determine whether the intravenous injection of NPCs also would improve ischemia-induced spatial learning dysfunction. Cerebral ischemia was produced by the injection of 700 microspheres into the right hemisphere of rats. The injection of NPCs via a femoral vein on day 7 after the induction of ischemia improved the modified neurological severity score and reduced the prolongation of the escape latency seen in the water maze task on days 12-28 after cerebral ischemia. The intravenous injection of NPCs on day 7 did not affect the viable area of the ipsilateral hemisphere on day 28 compared with that of the non-treated ischemic rats. Furthermore, the NPCs injected via the vein were detected in the ipsilateral hemisphere; and they expressed brain-derived neurotrophic factor (BDNF) on day 28. The decrease in the BDNF level in the ipsilateral hemisphere was also inhibited by the injection of NPCs. These results suggest that the NPCs injected via the vein after cerebral ischemia improved spatial learning dysfunction, but without having any restorative effect on the damaged areas, possibly by acting as a source of neurotrophic factors.
Asunto(s)
Isquemia Encefálica/complicaciones , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Trastornos del Conocimiento/tratamiento farmacológico , Aprendizaje por Laberinto , Células-Madre Neurales/trasplante , Animales , Isquemia Encefálica/metabolismo , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/fisiopatología , Inyecciones Intravenosas , Masculino , Microesferas , Ratas , Ratas Wistar , Tiempo de Reacción , Índice de Severidad de la EnfermedadRESUMEN
Pigment-dispersing factor (PDF) is a neuropeptide widely distributed in insect brains and plays important roles in the circadian system. In this study, we used RNA interference to study the role of the pigment-dispersing factor (pdf) gene in regulating circadian locomotor rhythms in the cricket, Gryllus bimaculatus. Injections of pdf double-stranded RNA (dspdf) effectively knocked down the pdf mRNA and PDF peptide levels. The treated crickets maintained the rhythm both under light-dark cycles (LD) and constant darkness (DD). However, they showed rhythms with reduced nocturnal activity with prominent peaks at lights-on and lights-off. Entrainability of dspdf-injected crickets was higher than control crickets as they required fewer cycles to resynchronize to the LD cycles shifted by 6 h. The free-running periods of the dspdf-injected crickets were shorter than those of control crickets in DD. These results suggest that PDF is not essential for the rhythm generation but involved in control of the nocturnality, photic entrainment, and fine tuning of the free-running period of the circadian clock.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de Insectos/metabolismo , Neuropéptidos/química , Receptores Acoplados a Proteínas G/metabolismo , Animales , Conducta Animal , Relojes Biológicos , Ritmo Circadiano , Gryllidae , Inmunohistoquímica , Luz , Masculino , Movimiento , Neuropéptidos/metabolismo , Interferencia de ARN , ARN Bicatenario/metabolismo , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Disruption of the blood-brain barrier (BBB) after cerebral ischemia is considered to be the initial step in the development of brain injuries, and an increase in the tyrosine phosphorylation of the tight junctional protein occludin has been shown to cause an increase in BBB permeability. Prostaglandin E2 (PGE2) appears to be associated with both toxic and protective effects on neuronal survival in vitro. However, it remains to be determined whether the prostanoid EP1 receptor is involved in the disruption of the BBB after cerebral ischemia. So we examined the effect of a prostanoid EP1 receptor antagonist, SC51089, on BBB leakage and tyrosine phosphorylation of occludin after cerebral ischemia. We demonstrated that SC51089 attenuated the increase in the tyrosine phosphorylation of occludin in isolated brain capillaries, which was coincident with a decrease in BBB leakage. These results suggest that the prostanoid EP1 receptor is involved in the tyrosine phosphorylation of occludin at tight junction, which may lead to disruption of the BBB and be linked to the development of cerebral infarctions.
Asunto(s)
Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Receptores de Prostaglandina E/antagonistas & inhibidores , Animales , Isquemia Encefálica/fisiopatología , Capilares/efectos de los fármacos , Capilares/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/metabolismo , Hidrazinas/farmacología , Masculino , Proteínas de la Membrana/metabolismo , Ocludina , Oxazepinas/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Ratas , Ratas Wistar , Subtipo EP1 de Receptores de Prostaglandina E , Albúmina Sérica/metabolismo , Tirosina/metabolismoRESUMEN
The circadian clock gene period (Gryllus bimaculatus period, Gb'per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb'per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb'per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb'per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb'per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb'per.
Asunto(s)
Gryllidae/fisiología , Proteínas de Insectos/metabolismo , Interferencia de ARN , Animales , Ritmo Circadiano , Gryllidae/genética , Gryllidae/crecimiento & desarrollo , Gryllidae/efectos de la radiación , Proteínas de Insectos/genética , Luz , Ninfa/genética , Ninfa/crecimiento & desarrollo , Ninfa/fisiología , Ninfa/efectos de la radiación , ARN Bicatenario/genéticaRESUMEN
The circadian clock gene period (Gryllus bimaculatus period, Gb'per) plays a core role in circadian rhythm generation in adults of the cricket Gryllus bimaculatus. We examined the role of Gb'per in nymphal crickets that show a diurnal rhythm rather than the nocturnal rhythm of the adults. As in the adult optic lobes, Gb'per mRNA levels in the head of the third instar nymphs showed daily cycling in light-dark cycles with a peak at mid night, and the rhythm persisted in constant darkness. Injection of Gb'per double-stranded RNA (dsRNA) into the abdomen of third instar nymphs knocked-down the mRNA levels to 25% of that in control animals. Most Gb'per dsRNA injected nymphs lost their circadian locomotor activity rhythm, while those injected with DsRed2 dsRNA as a negative control clearly maintained the rhythm. These results suggest that nymphs and adults share a common endogenous clock mechanism involving the clock gene Gb'per.
Asunto(s)
Ritmo Circadiano/genética , Gryllidae/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Interferencia de ARN , Animales , Conducta Animal/fisiología , Cartilla de ADN/genética , Gryllidae/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ninfa/metabolismo , Proteínas Circadianas Period , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.
Asunto(s)
Relojes Biológicos/genética , Ritmo Circadiano/genética , Gryllidae/metabolismo , Interferencia de ARN , Transactivadores/metabolismo , Animales , Proteínas CLOCK , Genes de Insecto , Gryllidae/genética , ARN Mensajero/análisis , Transactivadores/genéticaRESUMEN
Accumulating evidence indicates that stem cells have the ability to improve neurological deficits seen after cerebral ischemia. However, the effects of neural progenitor cells (NPCs) on cerebral ischemia-induced learning and memory dysfunction remain to be clarified. The purpose of the present study was to determine whether the injection of exogenous NPCs could prevent learning and memory dysfunction after cerebral ischemia. Sustained cerebral ischemia was produced by the injection of 700 microspheres into the right hemisphere of each rat. We demonstrated that injection of NPCs into the hippocampus at 10 min after the induction of cerebral ischemia reduced prolongation of the escape latency seen in acquisition and retention tests of the water maze task on Days 12-28 after cerebral ischemia. Injection of NPCs partially attenuated the decrease in viable areas of the ipsilateral hemisphere on Day 28 after the cerebral ischemia. We also demonstrated that injection of NPCs prevented the decrease in the level of BDNF seen at the early period after cerebral ischemia. These results suggest that the injection of exogenous NPCs into the hippocampus can prevent cerebral ischemia-induced learning and memory dysfunction, possibly through maintenance of the BDNF level.
Asunto(s)
Discapacidades para el Aprendizaje/terapia , Trastornos de la Memoria/terapia , Neuronas/fisiología , Trasplante de Células Madre/métodos , Células Madre/fisiología , Análisis de Varianza , Animales , Conducta Animal/fisiología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Infarto Cerebral/complicaciones , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Discapacidades para el Aprendizaje/etiología , Masculino , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/etiología , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Although stem cells are likely to improve neurological deficits seen after cerebral ischemia, the effects of neural progenitor cells (NPCs) on cerebral ischemia-induced learning dysfunction remain to be clarified. We tested whether the delayed injection of exogenous NPCs could prevent learning dysfunction after cerebral ischemia. Cerebral ischemia was produced by the injection of microspheres into the right hemisphere of each rat. Injection of NPCs obtained from green fluorescent protein transgenic rats into the hippocampus on Day 7 after the induction of cerebral ischemia improved the modified neurological severity score and reduced the prolongation of the escape latency seen in the water maze task. A few of the injected NPCs were positive for mature neuronal markers. In addition, the injected NPCs expressed BDNF on Day 28 after cerebral ischemia. Thus, the exogenous NPCs delivered by injection could act as a source of neurotrophic factors and prevent cerebral ischemia-induced learning dysfunction.