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The therapeutic benefits of photobiomodulation (PBM) in pain management, although well documented, are accompanied by concerns about potential risks, including pain, particularly at higher laser intensities. This study investigated the effects of laser intensity on pain perception using behavioral and electrophysiological evaluations in rats. Our results show that direct laser irradiation of 1000 mW/cm2 to the sciatic nerve transiently increases the frequency of spontaneous firing in the superficial layer without affecting the deep layer of the spinal dorsal horn, and this effect reverses to pre-irradiation levels after irradiation. Interestingly, laser irradiation at 1000 mW/cm2, which led to an increase in spontaneous firing, did not prompt escape behavior. Furthermore, a significant reduction in the time to initiate escape behavior was observed only at 9500 mW/cm2 compared to 15, 510, 1000, and 4300 mW/cm2. This suggests that 1000 mW/cm2, the laser intensity at which an increase in spontaneous firing was observed, corresponds to a stimulus that did not cause pain. It is expected that a detailed understanding of the risks and mechanisms of PBM from a neurophysiological perspective will lead to safer and more effective use of PBM.
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Introduction: Atopic dermatitis (AD) is one of the most prevalent intractable chronic itch diseases worldwide. In recent years, new molecular-targeted drugs have emerged, but side effects and economic challenges remain. Therefore, since it is important for AD patients to have a wider range of treatment options, it is important to explore new therapeutic agents. Gabapentinoids, gabapentin and pregabalin, have been shown to be effective for the clinical treatment of several chronic itch. Recently, mirogabalin (MGB) was developed as a novel gabapentinoid. MGB is a drug for neuropathic pain and has a margin of safety between its side effects and the analgesic effect for animal experiments. Herein, we showed that MGB exhibited an antipruritic effect in a mouse model of AD using NC/Nga mice. Methods and results: The oral administration of MGB (10 mg/kg) inhibited spontaneous scratching behavior in AD mice and its effect was dose dependently. Then, when MGB (10 mg/kg) was orally administrated to healthy mice, it did not affect motor function, including locomotor activity, wheel activity, and coordinated movement. Moreover, gabapentin (100 mg/kg) and pregabalin (30 mg/kg), inhibited spontaneous scratching behavior in AD mice and decreased motor function in healthy mice. Furthermore, intracisternal injection of MGB (10 µg/site) significantly suppressed spontaneous scratching behavior in AD mice. Discussion: In summary, our results suggest that MGB exerts an antipruritic effect via the spinal dorsal horn using NC/Nga mice. We hope that MGB is a candidate for a novel therapeutic agent for AD with relatively few side effects.
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Genetic factors significantly affect the pathogenesis of psychiatric disorders. However, the specific pathogenic mechanisms underlying these effects are not fully understood. Recent extensive genomic studies have implicated the protocadherin-related 15 (PCDH15) gene in the onset of psychiatric disorders, such as bipolar disorder (BD). To further investigate the pathogenesis of these psychiatric disorders, we developed a mouse model lacking Pcdh15. Notably, although PCDH15 is primarily identified as the causative gene of Usher syndrome, which presents with visual and auditory impairments, our mice with Pcdh15 homozygous deletion (Pcdh15-null) did not exhibit observable structural abnormalities in either the retina or the inner ear. The Pcdh15-null mice showed very high levels of spontaneous motor activity which was too disturbed to perform standard behavioral testing. However, the Pcdh15 heterozygous deletion mice (Pcdh15-het) exhibited enhanced spontaneous locomotor activity, reduced prepulse inhibition, and diminished cliff avoidance behavior. These observations agreed with the symptoms observed in patients with various psychiatric disorders and several mouse models of psychiatric diseases. Specifically, the hyperactivity may mirror the manic episodes in BD. To obtain a more physiological, long-term quantification of the hyperactive phenotype, we implanted nano tag® sensor chips in the animals, to enable the continuous monitoring of both activity and body temperature. During the light-off period, Pcdh15-null exhibited elevated activity and body temperature compared with wild-type (WT) mice. However, we observed a decreased body temperature during the light-on period. Comprehensive brain activity was visualized using c-Fos mapping, which was assessed during the activity and temperature peak and trough. There was a stark contrast between the distribution of c-Fos expression in Pcdh15-null and WT brains during both the light-on and light-off periods. These results provide valuable insights into the neural basis of the behavioral and thermal characteristics of Pcdh15-deletion mice. Therefore, Pcdh15-deletion mice can be a novel model for BD with mania and other psychiatric disorders, with a strong genetic component that satisfies both construct and surface validity.
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Trastorno Bipolar , Temperatura Corporal , Cadherinas , Modelos Animales de Enfermedad , Locomoción , Ratones Noqueados , Animales , Masculino , Ratones , Conducta Animal , Trastorno Bipolar/genética , Trastorno Bipolar/fisiopatología , Cadherinas/genética , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Locomoción/genética , Ratones Endogámicos C57BL , Inhibición Prepulso/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , ProtocadherinasRESUMEN
Photobiomodulation (PBM) has attracted attention as a treatment for chronic pain. Previous studies have reported that PBM of the sciatic nerve inhibits neuronal firing in the superficial layers (lamina I-II) of the spinal dorsal horn of rats, which is evoked by mechanical stimulation that corresponds to noxious stimuli. However, the effects of PBM on the deep layers (lamina III-IV) of the spinal dorsal horn, which receive inputs from innocuous stimuli, remain poorly understood. In this study, we examined the effect of PBM of the sciatic nerve on firing in the deep layers of the spinal dorsal horn evoked by mechanical stimulation. Before and after PBM, mechanical stimulation was administered to the cutaneous receptive field using 0.6-26.0 g von Frey filaments (vFFs), and vFF-evoked firing in the deep layers of the spinal dorsal horn was recorded. The vFF-evoked firing frequencies were not altered after the PBM for any of the vFFs. The inhibition rate for 26.0 g vFF-evoked firing was approximately 13 % in the deep layers and 70 % in the superficial layers. This suggests that PBM selectively inhibits the transmission of pain information without affecting the sense of touch. PBM has the potential to alleviate pain while preserving the sense of touch.
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Terapia por Luz de Baja Intensidad , Ratas , Animales , Ratas Sprague-Dawley , Asta Dorsal de la Médula Espinal , Neuronas , Nervio Ciático , Dolor , Médula Espinal/fisiologíaRESUMEN
Whole genome analysis has identified rare copy number variations (CNV) that are strongly involved in the pathogenesis of psychiatric disorders, and 3q29 deletion has been found to have the largest effect size. The 3q29 deletion mice model (3q29-del mice) has been established as a good pathological model for schizophrenia based on phenotypic analysis; however, circadian rhythm and sleep, which are also closely related to neuropsychiatric disorders, have not been investigated. In this study, our aims were to reevaluate the pathogenesis of 3q29-del by recreating model mice and analyzing their behavior and to identify novel new insights into the temporal activity and temperature fluctuations of the mouse model using a recently developed small implantable accelerometer chip, Nano-tag. We generated 3q29-del mice using genome editing technology and reevaluated common behavioral phenotypes. We next implanted Nano-tag in the abdominal cavity of mice for continuous measurements of long-time activity and body temperature. Our model mice exhibited weight loss similar to that of other mice reported previously. A general behavioral battery test in the model mice revealed phenotypes similar to those observed in mouse models of schizophrenia, including increased rearing frequency. Intraperitoneal implantation of Nano-tag, a miniature acceleration sensor, resulted in hypersensitive and rapid increases in the activity and body temperature of 3q29-del mice upon switching to lights-off condition. Similar to the 3q29-del mice reported previously, these mice are a promising model animals for schizophrenia. Successive quantitative analysis may provide results that could help in treating sleep disorders closely associated with neuropsychiatric disorders.
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Discapacidades del Desarrollo , Discapacidad Intelectual , Humanos , Niño , Ratones , Animales , Discapacidades del Desarrollo/genética , Deleción Cromosómica , Variaciones en el Número de Copia de ADN , Temperatura Corporal , Discapacidad Intelectual/genética , Modelos Animales de Enfermedad , FenotipoRESUMEN
Photobiomodulation is an effective treatment for pain. We previously reported that the direct laser irradiation of the exposed sciatic nerve inhibited firing in the rat spinal dorsal horn evoked by mechanical stimulation, corresponding to the noxious stimulus. However, percutaneous laser irradiation is used in clinical practice, and it is unclear whether it can inhibit the firing of the dorsal horn. In this study, we investigated whether the percutaneous laser irradiation of the sciatic nerve inhibits firing. Electrodes were inserted into the lamina II of the dorsal horn, and mechanical stimulation was applied using von Frey filaments (vFFs) with both pre and post laser irradiation. Our findings show that percutaneous laser irradiation inhibited 26.0 g vFF-evoked firing, which corresponded to the noxious stimulus, but did not inhibit 0.6 g and 8.0 g vFF-evoked firing. The post- (15 min after) and pre-irradiation firing ratios were almost the same as those for direct and percutaneous irradiation. A photodiode sensor implanted in the sciatic nerve showed that the power density reaching the sciatic nerve percutaneously was attenuated to approximately 10% of that on the skin. The relationship between the laser intensity reaching the nerve and its effect could be potentially useful for a more appropriate setting of laser conditions in clinical practice.
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Depression is a common psychiatric disorder affecting around 300 million people worldwide. Serum cortisol and glucocorticoid levels in humans are reportedly higher in patients with depression compared to controls. Furthermore, rodents repeatedly treated with exogenous corticosterone (CORT), a glucocorticoid in rodents, exhibit deficits in emotional behaviors. To confirm the availability of mice with chronic CORT treatment as an animal model of depression, we investigated the effect of chronic CORT treatment on depression-like behavioral and neuropathological phenotypes in C57BL/6N male mice. Behavioral studies showed depression- and anxiety-like behaviors in mice treated with CORT compared with control mice in the forced-swim and elevated-plus maze tests. Additionally, treated mice represented anhedonia and social behavior impairments in the sucrose preference and social interaction tests, respectively. Brains of depression patients have altered expression of reelin, an extracellular matrix protein involved in neuronal development and function. Likewise, in the present study, mice with chronic CORT treatment also exhibited reelin downregulation in cells of the hippocampus. Hence, we investigated therapeutic effects of reelin supplementation on CORT-induced behavioral abnormalities in mice. Microinjections of recombinant reelin protein into the hippocampus did not rescue behavioral deficits in mice with chronic CORT treatment. These results suggest that C57BL/6N male mice chronically treated with CORT are a suitable animal depression model, in which depressive behaviors may occur independently of the alternation of hippocampal Reelin expression.
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Corticosterona , Glucocorticoides , Humanos , Masculino , Ratones , Animales , Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Ratones Endogámicos C57BL , Hipocampo/metabolismo , Emociones , Depresión/metabolismo , Ratones Endogámicos , Conducta Animal , Modelos Animales de EnfermedadRESUMEN
Schizophrenia (SCZ) is a severe psychiatric disorder characterized by positive symptoms, negative symptoms, and cognitive deficits. Current antipsychotic treatment in SCZ improves positive symptoms but has major side effects and little impact on negative symptoms and cognitive impairment. The pathoetiology of SCZ remains unclear, but is known to involve small GTPase signaling. Rho kinase, an effector of small GTPase Rho, is highly expressed in the brain and plays a major role in neurite elongation and neuronal architecture. This study used a touchscreen-based visual discrimination (VD) task to investigate the effects of Rho kinase inhibitors on cognitive impairment in a methamphetamine (METH)-treated male mouse model of SCZ. Systemic injection of the Rho kinase inhibitor fasudil dose-dependently ameliorated METH-induced VD impairment. Fasudil also significantly suppressed the increase in the number of c-Fos-positive cells in the infralimbic medial prefrontal cortex (infralimbic mPFC) and dorsomedial striatum (DMS) following METH treatment. Bilateral microinjections of Y-27632, another Rho kinase inhibitor, into the infralimbic mPFC or DMS significantly ameliorated METH-induced VD impairment. Two proteins downstream of Rho kinase, myosin phosphatase-targeting subunit 1 (MYPT1; Thr696) and myosin light chain kinase 2 (MLC2; Thr18/Ser19), exhibited increased phosphorylation in the infralimbic mPFC and DMS, respectively, after METH treatment, and fasudil inhibited these increases. Oral administration of haloperidol and fasudil ameliorated METH-induced VD impairment, while clozapine had little effect. Oral administration of haloperidol and clozapine suppressed METH-induced hyperactivity, but fasudil had no effect. These results suggest that METH activates Rho kinase in the infralimbic mPFC and DMS, which leads to cognitive impairment in male mice. Rho kinase inhibitors ameliorate METH-induced cognitive impairment, perhaps via the cortico-striatal circuit.
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Disfunción Cognitiva , Metanfetamina , Proteínas de Unión al GTP Monoméricas , Inhibidores de Proteínas Quinasas , Esquizofrenia , Animales , Masculino , Ratones , Clozapina , Disfunción Cognitiva/tratamiento farmacológico , Haloperidol/farmacología , Haloperidol/uso terapéutico , Proteínas de Unión al GTP Monoméricas/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Esquizofrenia/inducido químicamente , Esquizofrenia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Amyloid ß (Aß) plays a key role in the pathology of Alzheimer's disease (AD) and is toxic owing to its ability to aggregate into oligomers and fibrils. Aß has high aggregative ability and potent toxicity due to the "toxic turn" at positions 22 and 23. Furthermore, APP knock-in mice producing E22P-Aß with the toxic turn exhibited AD-related phenotypes such as cognitive impairment, Aß plaque accumulation, and tau hyperphosphorylation. In these mice, it is suggested that the activation of neuroinflammation and dysregulation of hypoxia-inducible factor (HIF) expression in the hippocampus contribute to the pathogenesis of AD-related phenotype. However, it remains unclear which cells are responsible for the dysregulation of HIF expression and the neuroinflammation which was induced by E22P-Aß with the toxic turn. Here, we investigated the effects of chronic treatment with E22P-Aß42 and lipopolysaccharides (LPS) on the inflammatory response in BV-2 microglia. Chronic treatment with E22P-Aß42 and LPS increased nitric oxide production and the expression of interleukin-6 (IL-6), whereas it reduced the expression of HIF-1α and HIF-3α in BV-2 microglia. The reduction of HIF-1α caused by E22P-Aß42 and LPS was milder than that caused by LPS. Furthermore, chronic treatment with E22P-Aß42 and LPS increased the nuclear translocation of nuclear factor-kappaB (NF-κB). E22P-Aß42 could enhance the inflammatory response of microglia with abnormal HIF signaling and contribute to the progression of AD pathology.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Ratones , Animales , Péptidos beta-Amiloides/metabolismo , Microglía , Lipopolisacáridos/toxicidad , Enfermedades Neuroinflamatorias , Enfermedad de Alzheimer/metabolismo , HipoxiaRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that requires further pathological elucidation to establish effective treatment strategies. We previously showed that amyloid ß (Aß) toxic conformer with a turn at positions 22-23 is essential for forming highly toxic oligomers. In the present study, we evaluated phenotypic changes with aging in AD model AppNL-P-F/NL-P-F (NL-P-F) mice with Swedish mutation (NL), Iberian mutation (F), and mutation (P) overproducing E22P-Aß, a mimic of toxic conformer utilizing the knock-in technique. Furthermore, the role of the toxic conformer in AD pathology was investigated. NL-P-F mice produced soluble toxic conformers from an early age. They showed impaired synaptic plasticity, glial cell activation, and cognitive decline, followed by the accumulation of Aß plaques and tau hyperphosphorylation. In addition, the protein expression of hypoxia-inducible factor (HIF)-1α was increased, and gene expression of HIF-3α was decreased in NL-P-F mice. HIF dysregulation due to the production of soluble toxic conformers may be involved in AD pathology in NL-P-F mice. This study could reveal the role of a highly toxic Aß on AD pathogenesis, thereby contributing to the development of a novel therapeutic strategy targeting the toxic conformer.
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Enfermedad de Alzheimer , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Factor 1 Inducible por Hipoxia , Animales , Ratones , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Ratones Transgénicos , Fenotipo , Placa Amiloide/metabolismo , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Current antipsychotics used to treat schizophrenia have associated problems, including serious side effects and treatment resistance. We recently identified a significant association of schizophrenia with exonic copy number variations in the Rho GTPase activating protein 10 (ARHGAP10) gene using genome-wide analysis. ARHGAP10 encodes a RhoGAP superfamily member that is involved in small GTPase signaling. In mice, Arhgap10 gene variations result in RhoA/Rho-kinase pathway activation. We evaluated the pharmacokinetics of fasudil and hydroxyfasudil using liquid chromatography-tandem mass spectrometry in mice. The antipsychotic effects of fasudil on hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits were also investigated in a MK-801-treated pharmacological mouse schizophrenia model. Fasudil and its major metabolite, hydroxyfasudil, were detected in the brain at concentrations above their respective Ki values for Rho-kinase after intraperitoneal injection of 10 mg kg-1 fasudil. Fasudil improved the hyperlocomotion, social interaction deficits, prepulse inhibition deficits, and novel object recognition deficits in MK-801-treated mice in a dose-dependent manner. Following oral administration of fasudil, brain hydroxyfasudil was detected at concentration above the Ki value for Rho-kinase whilst fasudil was undetectable. MK-801-induced hyperlocomotion was also improved by oral fasudil administration. These results suggest that fasudil has antipsychotic-like effects on the MK-801-treated pharmacological mouse schizophrenia model. There are two isoforms in Rho-kinase, and further investigation is needed to clarify the isoforms involved in the antipsychotic-like effects of fasudil in the MK-801-treated mouse schizophrenia model.
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Antipsicóticos , Esquizofrenia , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/uso terapéutico , Animales , Antipsicóticos/farmacología , Antipsicóticos/uso terapéutico , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Maleato de Dizocilpina/farmacología , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Esquizofrenia/tratamiento farmacológico , Quinasas Asociadas a rhoRESUMEN
The Reelin gene (RELN) encodes a large extracellular protein, which has multiple roles in brain development and adult brain function. It activates a series of neuronal signal transduction pathways in the adult brain that function in synaptic plasticity, dendritic morphology, and cognitive function. To further investigate the roles of Reln in brain function, we generated a mouse line using the C57BL/6 J strain with the specific Reln deletion identified from a Japanese patient with schizophrenia (Reln-del mice). These mice exhibited abnormal sociality, but the pathophysiological significance of the Reln deletion for higher brain functions, such as learning and behavioral flexibility remains unclear. In this study, cognitive function in Reln-del mice was assessed using touchscreen-based visual discrimination (VD) and reversal learning (RL) tasks. Reln-del mice showed normal learning in the simple VD task, but the learning was delayed in the complex VD task as compared to their wild-type (WT) littermates. In the RL task, sessions were divided into early perseverative phase (sessions with <50% correct) and later learning phase (sessions with ≥50% correct). Reln-del mice showed normal perseveration but impaired relearning ability in both simple RL and complex RL task as compared to WT mice. These results suggest that Reln-del mice have impaired learning ability, but the behavioral flexibility is unaffected. Overall, the observed behavioral abnormalities in Reln-del mice suggest that this mouse model is a useful preclinical tool for investigating the neurobiological mechanism underlying cognitive impairments in schizophrenia and a therapeutic strategy.
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Aprendizaje Discriminativo/fisiología , Proteína Reelina/genética , Aprendizaje Inverso/fisiología , Esquizofrenia/genética , Percepción Visual/genética , Animales , Cognición/fisiología , Modelos Animales de Enfermedad , Eliminación de Gen , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismoRESUMEN
Reelin, a large extracellular matrix protein, helps to regulate neuronal plasticity and cognitive function. Several studies have shown that Reelin dysfunction, resulting from factors such as mutations in gene RELN or low Reelin expression, is associated with schizophrenia (SCZ). We previously reported that microinjection of Reelin into cerebral ventricle prevents phencyclidine-induced cognitive and sensory-motor gating deficits. However, it remains unclear whether and how Reelin ameliorates behavioral abnormalities in the animal model of SCZ. In the present study, we evaluated the effect of recombinant Reelin microinjection into the medial prefrontal cortex (mPFC) on abnormal behaviors induced by MK-801, an N-methyl-D-aspartate receptor antagonist. Microinjection of Reelin into the mPFC prevented impairment of recognition memory of MK-801-treated mice in the novel object recognition test (NORT). On the other hand, the same treatment had no effect on deficits in sensory-motor gating and short-term memory in the pre-pulse inhibition and Y-maze tests, respectively. To establish the neural substrates that respond to Reelin, the number of c-Fos-positive cells in the mPFC was determined. A significant increase in c-Fos-positive cells in the mPFC of MK-801-treated mice was observed when compared with saline-treated mice, and this change was suppressed by microinjection of Reelin into the mPFC. A K2360/2467A Reelin that cannot bind to its receptor failed to ameliorate MK-801-induced cognitive deficits in NORT. These results suggest that Reelin prevents MK-801-induced recognition memory impairment by acting on its receptors to suppress neural activity in the mPFC of mice.
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Trastornos de la Memoria/tratamiento farmacológico , Fármacos Neuroprotectores/administración & dosificación , Proteína Reelina/administración & dosificación , Animales , Conducta Animal/efectos de los fármacos , Células Cultivadas , Maleato de Dizocilpina , Masculino , Trastornos de la Memoria/inducido químicamente , Ratones Endogámicos C57BL , Microinyecciones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Corteza Prefrontal , Proteínas Proto-Oncogénicas c-fos/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Proteínas Recombinantes/administración & dosificación , Proteína Reelina/genéticaRESUMEN
We previously generated an ischemic stroke in a zebrafish model using N2 gas perfusion; however, this model was an unsuitable drug screening system due to low throughput. In this study, we examined a zebrafish ischemic stroke model using an oxygen absorber to assess drug effects. Hypoxic exposure more than 2 h using the oxygen absorber significantly induced cell death in the brain and damage to the neuronal cells. To confirm the utility of the ischemic model induced by the oxygen absorber, we treated zebrafish with neuroprotective agents. MK-801, an N-methyl-d-aspartate (NMDA) receptor antagonist, significantly suppressed cell death in the brain, and edaravone, a free radical scavenger, significantly reduced the number of dead cells. These results suggest that the activation of NMDA receptors and the production of reactive oxygen species induce neuronal cell damage in accordance with previous mammalian reports. We demonstrate the suitability of an ischemic stroke model in zebrafish larvae using the oxygen absorber, enabling a high throughput drug screening.
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Isquemia Encefálica/tratamiento farmacológico , Maleato de Dizocilpina/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Edaravona/uso terapéutico , Depuradores de Radicales Libres/uso terapéutico , Larva , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Pez Cebra , Animales , Encéfalo/patología , Isquemia Encefálica/etiología , Isquemia Encefálica/patología , Muerte Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Maleato de Dizocilpina/farmacología , Edaravona/farmacología , Depuradores de Radicales Libres/farmacología , Gases , Hipoxia/complicaciones , Hipoxia/patología , Neuronas/patología , NitrógenoRESUMEN
Cerebral infarct is caused by cerebrovascular occlusion and results in brain damage. Although many rodent models of cerebral infarct exist, there is none based on zebrafish. In this study, we developed a novel ischemia-reperfusion model induced by hypoxic treatment using zebrafish. We first examined the changes in blood flow under hypoxic conditions. Hypoxic treatment interrupted the blood flow in 4 dpf (days post fertilization) zebrafish larvae. To quantify the trunk and cerebral blood flow, we selected the middle mesencephalic central artery (MMCtA) as a cerebral blood vessel and the dorsal aorta (DA) as a blood vessel of the trunk. Interestingly, the interruption of blood flow in MMCtA preceded that in DA. Considering these results, we hypothesized that reoxygenation immediately after hypoxia-induced cerebral ischemia leads to reperfusion. As a result, hypoxia-reoxygenation (H/R) treatment induced ischemia-reperfusion in cerebral vessels. Furthermore, brain cell death was increased 24 h after H/R treatment. Transgenic zebrafish (HuC:kaede), with neuronal cells expressing the kaede fluorescent protein, was used to investigate the effect of H/R on neuronal cells. The H/R treatment reduced the fluorescence intensity of kaede. Besides, glial fibrillary acidic protein immunoreactivity in H/R-treated larvae was significantly increased. In conclusion, H/R-treated zebrafish larvae may provide a novel ischemia-reperfusion model.
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Isquemia Encefálica/fisiopatología , Muerte Celular/fisiología , Circulación Cerebrovascular/fisiología , Neuronas/fisiología , Daño por Reperfusión/fisiopatología , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Pez CebraRESUMEN
Reelin, an extracellular matrix protein, is secreted by Cajal-Retzius cells and plays crucial roles in the development of brain structures and neuronal functions. Reductions in Reelin cause the brain dysfunctions associated with mental disorders, such as schizophrenia. A recent genome-wide copy number variation analysis of Japanese schizophrenia patients identified a novel deletion in RELN encoding Reelin. To clarify the pathophysiological role of the RELN deletion, we developed transgenic mice carrying the RELN deletion (Reln-del) and found abnormalities in their brain structures and social behavior. In the present study, we performed an in vitro analysis of Reelin expression, intracellular Reelin signaling, and the morphology of primary cultured cortical neurons from wild-type (WT) and Reln-del mice. Reelin protein levels were lower in Reln-del neurons than in WT neurons. Dab1 expression levels were significantly higher in Reln-del neurons than in WT neurons, suggesting that Reelin signaling was decreased in Reln-del neurons. Reelin was mainly expressed in γ-aminobutyric acid (GABA)-ergic inhibitory neurons, but not in parvalbumin (PV)-positive neurons. A small proportion of Ca2+/calmodulin-dependent protein kinase II α subunit (CaMKIIα)-positive excitatory neurons also expressed Reelin. In comparisons with WT neurons, significant decreases were observed in neurite lengths and branch points as well as in the number of postsynaptic density protein 95 (PSD95) immunoreactive puncta in Reln-del neurons. A disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3) is a protease that inactivates Reelin by cleavage at the N-t site. The knockdown of ADAMTS-3 by short hairpin RNAs suppressed Reelin cleavage in conditioned medium and reduced Dab1 expression, indicating that Reelin signaling was enhanced in the primary cultured cortical neurons of WT and heterozygous Reln-del. Accordingly, the inhibition of ADAMTS-3 may be a potential candidate in the clinical treatment of schizophrenia by enhancing Reelin signaling in the brain.
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Moléculas de Adhesión Celular Neuronal/deficiencia , Corteza Cerebral/metabolismo , Proteínas de la Matriz Extracelular/deficiencia , Eliminación de Gen , Proteínas del Tejido Nervioso/deficiencia , Neuronas/metabolismo , Esquizofrenia/metabolismo , Serina Endopeptidasas/deficiencia , Animales , Moléculas de Adhesión Celular Neuronal/biosíntesis , Moléculas de Adhesión Celular Neuronal/genética , Células Cultivadas , Corteza Cerebral/citología , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteína Reelina , Esquizofrenia/genética , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/genética , Transducción de Señal/fisiologíaRESUMEN
We recently found a significant association between exonic copy-number variations in the Rho GTPase activating protein 10 (Arhgap10) gene and schizophrenia in Japanese patients. Special attention was paid to one patient carrying a missense variant (p.S490P) in exon 17, which overlapped with an exonic deletion in the other allele. Accordingly, we generated a mouse model (Arhgap10 S490P/NHEJ mice) carrying a missense variant and a coexisting frameshift mutation. We examined the spatiotemporal expression of Arhgap10 mRNA in the brain and found the highest expression levels in the cerebellum, striatum, and nucleus accumbens (NAc), followed by the frontal cortex in adolescent mice. The expression levels of phosphorylated myosin phosphatase-targeting subunit 1 and phosphorylated p21-activated kinases in the striatum and NAc were significantly increased in Arhgap10 S490P/NHEJ mice compared with wild-type littermates. Arhgap10 S490P/NHEJ mice exhibited a significant increase in neuronal complexity and spine density in the striatum and NAc. There was no difference in touchscreen-based visual discrimination learning between Arhgap10 S490P/NHEJ and wild-type mice, but a significant impairment of visual discrimination was evident in Arhgap10 S490P/NHEJ mice but not wild-type mice when they were treated with methamphetamine. The number of c-Fos-positive cells was significantly increased after methamphetamine treatment in the dorsomedial striatum and NAc core of Arhgap10 S490P/NHEJ mice. Taken together, these results suggested that schizophrenia-associated Arhgap10 gene mutations result in morphological abnormality of neurons in the striatum and NAc, which may be associated with vulnerability of cognition to methamphetamine treatment.
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Cognición/efectos de los fármacos , Cuerpo Estriado/patología , Proteínas Activadoras de GTPasa/genética , Metanfetamina/farmacología , Mutación/genética , Neuronas/patología , Esquizofrenia/genética , Esquizofrenia/fisiopatología , Proteína de Unión al GTP rhoA/genética , Animales , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Discriminación en Psicología , Proteínas Activadoras de GTPasa/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Ratones Endogámicos C57BL , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Núcleo Accumbens/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
In the majority of schizophrenia patients, chronic atypical antipsychotic administration produces a significant reduction in or even complete remission of psychotic symptoms such as hallucinations and delusions. However, these drugs are not effective in improving cognitive and emotional deficits in patients with schizophrenia. Atypical antipsychotic drugs have a high affinity for the dopamine D2 receptor, and a modest affinity for the serotonin 5-HT2A receptor. The cognitive and emotional deficits in schizophrenia are thought to involve neural networks beyond the classical dopaminergic mesolimbic pathway, however, including serotonergic systems. For example, mutations in the RELN gene, which encodes Reelin, an extracellular matrix protein involved in neural development and synaptic plasticity, are associated with neurodevelopmental disorders such as schizophrenia and autism spectrum disorder. Furthermore, hippocampal Reelin levels are down-regulated in the brains of both schizophrenic patients and in rodent models of schizophrenia. In the present study, we investigated the effect of Reelin microinjection into the mouse hippocampus on behavioral phenotypes to evaluate the role of Reelin in neurodevelopmental disorders and to test a therapeutic approach that extends beyond classical monoamine targets. To model the cognitive and emotional deficits, as well as histological decreases in Reelin-positive cell numbers and hippocampal synaptoporin distribution, a synaptic vesicle protein, offspring that were prenatally exposed to maternal immune activation were used. Microinjections of recombinant Reelin protein into the hippocampus rescued impairments in object memory and anxiety-like behavior and recruited synaptoporin in the hippocampus in offspring exposed to antenatal inflammation. These results suggest that Reelin supplementation has the potential to treat cognitive and emotional impairments, as well as synaptic disturbances, in patients with neurodevelopmental disorders such as schizophrenia.
RESUMEN
Schizophrenia (SCZ) is known to be a heritable disorder; however, its multifactorial nature has significantly hampered attempts to establish its pathogenesis. Therefore, in this study, we performed genome-wide copy-number variation (CNV) analysis of 2940 patients with SCZ and 2402 control subjects and identified a statistically significant association between SCZ and exonic CNVs in the ARHGAP10 gene. ARHGAP10 encodes a member of the RhoGAP superfamily of proteins that is involved in small GTPase signaling. This signaling pathway is one of the SCZ-associated pathways and may contribute to neural development and function. However, the ARHGAP10 gene is often confused with ARHGAP21, thus, the significance of ARHGAP10 in the molecular pathology of SCZ, including the expression profile of the ARHGAP10 protein, remains poorly understood. To address this issue, we focused on one patient identified to have both an exonic deletion and a missense variant (p.S490P) in ARHGAP10. The missense variant was found to be located in the RhoGAP domain and was determined to be relevant to the association between ARHGAP10 and the active form of RhoA. We evaluated ARHGAP10 protein expression in the brains of reporter mice and generated a mouse model to mimic the patient case. The model exhibited abnormal emotional behaviors, along with reduced spine density in the medial prefrontal cortex (mPFC). In addition, primary cultured neurons prepared from the mouse model brain exhibited immature neurites in vitro. Furthermore, we established induced pluripotent stem cells (iPSCs) from this patient, and differentiated them into tyrosine hydroxylase (TH)-positive neurons in order to analyze their morphological phenotypes. TH-positive neurons differentiated from the patient-derived iPSCs exhibited severe defects in both neurite length and branch number; these defects were restored by the addition of the Rho-kinase inhibitor, Y-27632. Collectively, our findings suggest that rare ARHGAP10 variants may be genetically and biologically associated with SCZ and indicate that Rho signaling represents a promising drug discovery target for SCZ treatment.
Asunto(s)
Esquizofrenia , Animales , Variaciones en el Número de Copia de ADN , Proteínas Activadoras de GTPasa/genética , Humanos , Ratones , Esquizofrenia/genética , Transducción de Señal , Proteína de Unión al GTP rhoARESUMEN
AIM: A Japanese individual with schizophrenia harboring a novel exonic deletion in RELN was recently identified by genome-wide copy-number variation analysis. Thus, the present study aimed to generate and analyze a model mouse to clarify whether Reln deficiency is associated with the pathogenesis of schizophrenia. METHODS: A mouse line with a novel RELN exonic deletion (Reln-del) was established using the CRISPR/Cas9 method to elucidate the underlying molecular mechanism. Subsequently, general behavioral tests and histopathological examinations of the model mice were conducted and phenotypic analysis of the cerebellar granule cell migration was performed. RESULTS: The phenotype of homozygous Reln-del mice was similar to that of reeler mice with cerebellar atrophy, dysplasia of the cerebral layers, and abrogated protein levels of cerebral reelin. The expression of reelin in heterozygous Reln-del mice was approximately half of that in wild-type mice. Conversely, behavioral analyses in heterozygous Reln-del mice without cerebellar atrophy or dysplasia showed abnormal social novelty in the three-chamber social interaction test. In vitro reaggregation formation and neuronal migration were severely altered in the cerebellar cultures of homozygous Reln-del mice. CONCLUSION: The present results in novel Reln-del mice modeled after our patient with a novel exonic deletion in RELN are expected to contribute to the development of reelin-based therapies for schizophrenia.