SP6 controls human cytotrophoblast fate decisions and trophoblast stem cell establishment by targeting MSX2 regulatory elements.

The commitment and differentiation of human placental progenitor cytotrophoblast (CT) cells are crucial for a successful pregnancy, but the underlying mechanism remains poorly understood. Here, we identified the transcription factor (TF), specificity protein 6 (SP6), as a human species-specific trophoblast lineage TF expressed in human placental CT cells. Using pluripotent stem cells as a model, we demonstrated that SP6 controls CT generation and the establishment of trophoblast stem cells (TSCs) and identified msh homeobox 2 (MSX2) as the downstream effector in these events. Mechanistically, we showed that SP6 interacts with histone acetyltransferase P300 to alter the landscape of H3K27ac at targeted regulatory elements, thereby favoring transcriptional activation and facilitating CT cell fate decisions and TSC maintenance. Our results established SP6 as a regulator of the human trophoblast lineage and implied its role in placental development and the pathogenies of placental diseases.

Angiotensin-(1-7) protects against sepsis-associated left ventricular dysfunction induced by lipopolysaccharide.

Sepsis-induced myocardial dysfunction is a major cause of death. The present study explored whether angiotensin (Ang)-(1-7), an important biologically active peptide of the renin-angiotensin system, could improve cardiac dysfunction and attenuate inflammation and apoptosis. Experiments were carried out in mice and in neonatal rat cardiomyocytes (NRCMs) treated with lipopolysaccharide (LPS) or Ang-(1-7). Angiotensin converting enzyme 2 (ACE2), Ang-(1-7) and Mas receptor (MasR) expressions were reduced in the mouse left ventricular and NRCM treated with LPS. Ang-(1-7) increased the ejection fraction and fractional shortening of left ventricular, which were reduced upon LPS injection in mice. Ang-(1-7) pre-treatment reversed LPS-induced decreases of α-myosin heavy chain (MHC) and ß-MHC, and increases of S100 calcium binding protein A8 (S100A8) and S100A9 in the mouse left ventricular. The LPS-induced increases of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in the mouse left ventricular and NRCMs were inhibited by Ang-(1-7) administration. Ang-(1-7) treatment reversed the increases of cleaved-caspase 3, cleaved-caspase 8 and Bax, and the decrease of Bcl2 induced by LPS in the mouse left ventricular and NRCMs. The increases of MAPKs pathway induced by LPS in NRCMs were inhibited by Ang-(1-7). These results indicate that Ang-(1-7) protects against sepsis-associated left ventricular dysfunction induced by LPS, and increases cardiac contractility via attenuating inflammation and apoptosis.

Differential contributions of voltage-gated potassium channel subunits in enhancing temporal coding in the bushy cells of the ventral cochlear nucleus.

Temporal coding precision of bushy cells in the ventral cochlear nucleus (VCN), critical for sound localization and communication, depends on the generation of rapid and temporally precise action potentials (APs). Voltage-gated potassium (Kv) channels are critically involved in this. The bushy cells in rat VCN express Kv1.1, 1.2, 1.3, 1.6, 3.1, 4.2, and 4.3 subunits. The Kv1.1 subunit contributes to the generation of a temporally precise single AP. However, the understanding of the functions of other Kv subunits expressed in the bushy cells is limited. Here, we investigated the functional diversity of Kv subunits concerning their contributions to temporal coding. We characterized the electrophysiological properties of the Kv channels with different subunits using whole cell patch-clamp recording and pharmacological methods. The neuronal firing pattern changed from single to multiple APs only when the Kv1.1 subunit was blocked. The Kv subunits, including the Kv1.1, 1.2, 1.6, or 3.1, were involved in enhancing temporal coding by lowering membrane excitability, shortening AP latencies, reducing jitter, and regulating AP kinetics. Meanwhile, all the Kv subunits contributed to rapid repolarization and sharpening peaks by narrowing half-width and accelerating fall rate, and the Kv1.1 subunit also affected the depolarization of AP. The Kv1.1, 1.2, and 1.6 subunits endowed bushy cells with a rapid time constant and a low input resistance of membrane for enhancing spike timing precision. The present results indicate that the Kv channels differentially affect intrinsic membrane properties to optimize the generation of rapid and reliable APs for temporal coding.NEW & NOTEWORTHY This study investigates the roles of Kv channels in effecting precision using electrophysiological and pharmacological methods in bushy cells. Different Kv channels have varying electrophysiological characteristics, which contribute to the interplay between changes in the membrane properties and regulation of neuronal excitability which then improve temporal coding. We conclude that the Kv channels are specialized to promote the precise and rapid coding of acoustic input by optimizing the generation of reliable APs.

Deep Learning in Automatic Sleep Staging With a Single Channel Electroencephalography.

This study centers on automatic sleep staging with a single channel electroencephalography (EEG), with some significant findings for sleep staging. In this study, we proposed a deep learning-based network by integrating attention mechanism and bidirectional long short-term memory neural network (AT-BiLSTM) to classify wakefulness, rapid eye movement (REM) sleep and non-REM (NREM) sleep stages N1, N2 and N3. The AT-BiLSTM network outperformed five other networks and achieved an accuracy of 83.78%, a Cohen's kappa coefficient of 0.766 and a macro F1-score of 82.14% on the PhysioNet Sleep-EDF Expanded dataset, and an accuracy of 81.72%, a Cohen's kappa coefficient of 0.751 and a macro F1-score of 80.74% on the DREAMS Subjects dataset. The proposed AT-BiLSTM network even achieved a higher accuracy than the existing methods based on traditional feature extraction. Moreover, better performance was obtained by the AT-BiLSTM network with the frontal EEG derivations than with EEG channels located at the central, occipital or parietal lobe. As EEG signal can be easily acquired using dry electrodes on the forehead, our findings might provide a promising solution for automatic sleep scoring without feature extraction and may prove very useful for the screening of sleep disorders.

Difference in serum miRNA expression between immunoglobulin-sensitive and -insensitive incomplete Kawasaki disease patients.

The present study aimed to investigate the expression of microRNAs (miRNAs/miRs) and inflammatory factors in patients with immunoglobulin-sensitive and IVIG-insensitive incomplete Kawasaki disease (KD). One hundred and eighty-five patients with incomplete KD were included as the study group (KD group), and 182 patients with respiratory infection as the control group. Neutrophil to lymphocyte ratio (NLR), C-reactive protein (CRP) levels, alanine aminotransferase (ALT), aspartate aminotransferase (AST), white blood cell count (WBC), hemoglobin level (Hb), platelet count (PLT) and T cell subsets (CD3+, CD3+ CD4+) were compared. Patients in the KD group received aspirin (30 mg/kg orally daily) and gamma globulin (IVIG, 1 g/kg intravenously daily). According to the sensitivity to IVIG, patients were divided into IVIG-sensitive group and IVIG-insensitive KD group. The relative expression levels of miRNA-21, miRNA-145, miRNA-155 and miRNA-199b-5p in the serum were detected by RT-qPCR. Serum TNF-α, IL-6 and IL-1ß levels were assessed using ELISA. Before treatment, the neutrophil to lymphocyte ratio (NLR), levels C-reactive protein, and leukocytes in the KD group were significantly higher compared with the control group (P<0.05). After medical intervention, the relative expression of miRNA-21, miRNA-145 and miRNA-155 in the serum of patients in IVIG-sensitive and IVIG-insensitive KD groups were increased when compared with these levels in the control group (P<0.05). Meanwhile, the relative expression of miRNA-199b-5p was decreased (P<0.05). Compared with the IVIG-sensitive KD group, the relative expression levels of miRNA-145 and miRNA-155 were increased in the serum of patients in the IVIG-insensitive KD group (P<0.05). Compared with the control group, the levels of TNF-α, IL-6 and IL-1ß were increased in the serum of patients in the IVIG-sensitive and IVIG-insensitive KD groups (P<0.05). Compared with the IVIG-sensitive KD group, the serum levels of TNF-α and IL-6 were increased in patients of the IVIG-insensitive KD group (P<0.05). Except for NLR and CRP, there were differences in the expression of peripheral blood miRNA-145, miRNA-155 and serum TNF-α and IL-6 in patients with immunoglobulin-sensitive and -insensitive incomplete KD.

Reliability-based robust dynamic positioning for a turret-moored floating production storage and offloading vessel with unknown time-varying disturbances and input saturation.

In this paper, we derived a mathematical model for a floating production storage and offloading (FPSO) vessel and its buoy mooring system and developed a new robust positioning controller to keep vessels in a desired region in the presence of unknown time-varying disturbances with uncertainties and input saturation. Different materials (chain and polyester) and buoys are considered in the model of mooring system to make the developed model more realistic. We employed a disturbance observer to estimate the disturbances and designed an auxiliary dynamic system integrated with the structural reliability's derivative to quantify the input saturation's influence, and its states are used to the control design. Our proposed controller can keep the structural reliability and heading at desired values with arbitrarily small errors while guaranteeing the uniform ultimate boundedness of all signals in the closed-loop control system. It is easier for the control design because disturbances and input saturation are handled simultaneously and so is the stability analysis because only one Lyapunov function is needed. Simulations are conducted to demonstrate our proposed controller's effectiveness and a comparison with a robust controller based on hyperbolic tangent functions shows our proposed controller can avoid steady errors with desired control goals.

Attenuated DHEA and DHEA-S response to acute psychosocial stress in individuals with depressive disorders.

BACKGROUND: In recent years, a relationship between depression and basal dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S) levels has frequently been suggested, but responses of these adrenal steroids to psychosocial stress have not been examined in individuals with depressive disorders. In this study, we examined salivary DHEA, DHEA-S, and cortisol/DHEA response to the Trier Social Stress Test (TSST) in individuals with depressive disorders and in healthy controls to discover whether the responses of DHEA and DHEA-S to acute psychosocial stress could be a more sensitive marker of HPA dysfunction in depressive disorders. METHODS: We compared salivary cortisol, DHEA, DHEA-S, and cortisol/DHEA levels to the TSST tests between 38 individuals with depression and 43 healthy controls aged 18.4-25.9 years. Depression severity was assessed by the self-reported Beck Depression Inventory-II (BDI-II). Salivary samples were evaluated at four time points: the baseline (-10 time point), before the TSST started (0 time point), the end of the TSST (+20 time point), and the recovery (+50 time points). RESULTS: No significant differences existed in the basal adrenal hormonal levels between subjects with depressive disorders and controls; however, at the end of TSST, attenuated DHEA and DHEA-S response was identified in subjects with depressive disorders compared to that found in healthy subjects. The differences in the DHEA and DHEA-S levels at the +20 time point, as well as the differences in the cortisol/DHEA at the +50 time point, exhibited negative correlations with depression severity. CONCLUSION: Attenuated DHEA and DHEA-S response to acute psychosocial stress was identified in subjects with depressive disorders. These findings help us to discover the bi-directional relationship between depression and the hypothalamic-pituitary-adrenal (HPA) axis function, hence furthering our understanding of whether altered DHEA and DHEA-S response to psychosocial stress may be a more sensitive method than basal adrenal steroid analysis for detecting HPA axis dysfunction in depressive disorders. LIMITATIONS: As this is a case control study, we could only draw the conclusion of the bi-directional relationship between the depression and the altered DHEA (S) response to stress, and could not identify whether depression was due to the HPA dysfunction, or vice versa. Prospective studies such as such as cohort studies or epidemiology experiments are needed to further test the cause of depression or HPA dysfunction; and the mechanisms responsible for altered DHEA and DHEA-S in response to acute psychosocial stress in individuals with depressive disorders are also needed to be clarified.

Cardiac insufficiency in infants.

Cardiac failures in young children have shown an enormous trudge in recent past. The reason being medical community has laid more stress on cardiac insufficiency management on adult patients and has conducted a large amount of research on the management of heart failure in adults, which has given rise to significant changes in management in the last decade. However, there are far fewer studies in children and those which do exist are often small, retrospective and use a diverse range of measures to assess efficacy. Current research is being focused worldwide to deal with this life threatening problem of young patients. The present review shall enlighten the above focus of the research and will discuss latest developments in therapeutic advances like paediatric use of ace inhibitors, beta blockers in young patients for the efficient management of cardiac insufficiency.

Pediatric cardiovascular risk factors.

The dominant cause of mortality, morbidity and hospitalization worldwide is cardiac complications, and are the major public health problem in adult populations. The worldwide research is being focused on the idea that cardiovascular disease is an early life pathological state. Early unfavorable exposures, acting in different periods of fetal and early postnatal life, have been observed to be responsible for permanent editions in the cardiac system. This idea has been confirmed by preclinical experimental studies confirming the early life growth restriction leading to developmental adaptations in cardiovascular form and system. All these editions results in elevated susceptibility to cardiovascular disease. The present review article will put emphasis on these risk factors, which lead to the deadly cardiac pathology state in young infants.

Emergency treatment and nursing of children with severe pneumonia complicated by heart failure and respiratory failure: 10 case reports.

Pneumonia refers to lung inflammation caused by different pathogens or other factors, and is a common pediatric disease occurring in infants and young children. It is closely related to the anatomical and physiological characteristics of infants and young children and is more frequent during winter and spring, or sudden changes in temperature. Pneumonia is a serious disease that poses a threat to children's health and its morbidity and mortality rank first, accounting for 24.5-65.2% of pediatric inpatients. Due to juvenile age, severe illness and rapid changes, children often suffer acute heart failure, respiratory failure and even toxic encephalopathy at the same time. The concurrence in different stages of the process of emergency treatment tends to relapse, which directly places the lives of these children at risk. Severe pneumonia constitutes one of the main causes of infant mortality. In the process of nursing children with severe pneumonia, intensive care was provided, including condition assessment and diagnosis, close observation of disease, keeping the airway unblocked, rational oxygen therapy, prevention and treatment of respiratory and circulatory failure, support of vital organs, complications, and health education. The inflammatory response was proactively controlled, to prevent suffocation and reduce mortality. In summary, positive and effective nursing can promote the rehabilitation of children patients, which can be reinforced with adequate communication with the parents and/or caretakers.

Significance of serum 25-hydroxyvitamin D3 and interleukin-6 levels in immunoglobulin treatment of Kawasaki disease in children.

The aim of the study was to investigate the significance of the level of serum 25-hydroxyvitamin D3 [25-(OH)D3] and interleukin (IL)-6 in serum prior to and after immunoglobulin treatment in children suffering from Kawasaki disease in order to provide a reference for the successful treatment of Kawasaki disease in children. From February, 2013 to February, 2015, 45 patients with Kawasaki disease were enrolled in the observation group. The normal control group comprised 43 healthy volunteers and the feverish control group 46 patients with respiratory infection and fever. Venous blood was collected from each case before and after immunoglobulin treatment and the level of 25-(OH)D3 and IL-6 in the serum were measured using fluorescent quantitative PCR, enzyme-linked immunosorbent assay and western blotting. Before treatment, the level of 25-(OH)D3 in the feverish control group was significantly lower than that of the normal control group, while the level of 25-(OH)D3 in the observation group was significantly higher than that of the normal control group. The level of 25-(OH)D3 in the feverish control group was lower than the IL-6 level in the normal children, but the difference was not statistically significant (P>0.05). The level 25-(OH)D3 in the observation group was significantly higher than the IL-6 level in the normal control group. The serum content of 25-(OH)D3 was significantly higher after the treatment compared to before treatment levels and after treatment IL-6 level was only slightly lower. It was observed that the 25-(OH)D3 level in the observation group was significantly increased after immunoglobulin treatment and this was positively correlated with the effects of the treatment. The IL-6 level had no significant changes after treatment and had little correlation with the treatment effect. The results suggested that 25-(OH)D3 may be involved in the occurrence of Kawasaki disease in children and in the aggravation of the disease to some extent.

Bioinspired Coordinated Path Following for Vessels with Speed Saturation Based on Virtual Leader.

This paper investigates the coordinated path following of multiple marine vessels with speed saturation. Based on virtual leader strategy, the authors show how the neural dynamic model and passivity-based techniques are brought together to yield a distributed control strategy. The desired path following is achieved by means of a virtual dynamic leader, whose controller is designed based on the biological neural shunting model. Utilizing the characteristic of bounded and smooth output of neural dynamic model, the tracking error jump is avoided and speed saturation problem is solved in straight path. Meanwhile, the coordinated path following of multiple vessels with a desired spatial formation is achieved through defining the formation reference point. The consensus of formation reference point is realized by using the synchronization controller based on passivity. Finally, simulation results validate the effectiveness of the proposed coordinated algorithm.

The Effects of Urethane on Rat Outer Hair Cells.

The cochlea converts sound vibration into electrical impulses and amplifies the low-level sound signal. Urethane, a widely used anesthetic in animal research, has been shown to reduce the neural responses to auditory stimuli. However, the effects of urethane on cochlea, especially on the function of outer hair cells, remain largely unknown. In the present study, we compared the cochlear microphonic responses between awake and urethane-anesthetized rats. The results revealed that the amplitude of the cochlear microphonic was decreased by urethane, resulting in an increase in the threshold at all of the sound frequencies examined. To deduce the possible mechanism underlying the urethane-induced decrease in cochlear sensitivity, we examined the electrical response properties of isolated outer hair cells using whole-cell patch-clamp recording. We found that urethane hyperpolarizes the outer hair cell membrane potential in a dose-dependent manner and elicits larger outward current. This urethane-induced outward current was blocked by strychnine, an antagonist of the α9 subunit of the nicotinic acetylcholine receptor. Meanwhile, the function of the outer hair cell motor protein, prestin, was not affected. These results suggest that urethane anesthesia is expected to decrease the responses of outer hair cells, whereas the frequency selectivity of cochlea remains unchanged.

Synchronized Progression of Prestin Expression and Auditory Brainstem Response during Postnatal Development in Rats.

Prestin is the motor protein expressed in the cochlear outer hair cells (OHCs) of mammalian inner ear. The electromotility of OHCs driven by prestin is responsible for the cochlear amplification which is required for normal hearing in adult animals. Postnatal expression of prestin and activity of OHCs may contribute to the maturation of hearing in rodents. However, the temporal and spatial expression of prestin in cochlea during the development is not well characterized. In the present study, we examined the expression and function of prestin from the OHCs in apical, middle, and basal turns of the cochleae of postnatal rats. Prestin first appeared at postnatal day 6 (P6) for basal turn, P7 in middle turn, and P9 for apical turn of cochlea. The expression level increased progressively over the next few days and by P14 reached the mature level for all three segments. By comparison with the time course of the development of auditory brainstem response for different frequencies, our data reveal that prestin expression synchronized with the hearing development. The present study suggests that the onset time of hearing may require the expression of prestin and is determined by the mature function of OHCs.

Promising effects of Chinese traditional treatment for child typhoid complicated by myocarditis.

The clinical effects were compared and analyzed of traditional Chinese medicine (TCM) 'Ling Gui Long Mu soup' combined with the conventional Western medications in treating child typhoid complicated by myocarditis. From July, 2010 to May, 2014, 54 children suffering from typhoid complicated by myocarditis were enrolled in the present study. The patients were divided into the observation and control groups (n=27 cases per group) according to the random number table. Patients in the observation group were treated with basic Western medicine combined with TCM 'Ling Gui Long Mu soup' while patients in the control group were treated only with Western medicine. We analyzed the final curative effects in the two groups. The total effective rate in the observation group was significantly higher than that of the control group and the difference was statistically significant (P<0.05). The improvement rate of the syndrome in the TCM observation group was significantly higher than that in the control group and the difference was statistically significant (P<0.05). Although the C-reactive protein (CRP) and creatinine kinase-MB (CK-MB) levels in the two groups were decreased following the treatment, CRP and CK-MB levels in the observation group were further reduced, and the difference was statistically significant (P<0.05). In conclusion, for child typhoid complicated by myocarditis, TCM 'Ling Gui Long Mu soup' significantly improved the clinical efficiency of the treatment and improved the syndrome. Therefore, 'Ling Gui Long Mu soup' is useful in clinical practice.

The association between HERG gene expression and cardiac arrhythmia disease in children.

We explored the possible link between the expression of HERG gene and cardiomyopathy in children. From April 2013 to April 2015, 73 children with cardiac arrhythmia who were treated were enrolled in the present study to serve as the observation group. At the same time, 76 normal individuals were also enrolled as the control group. HERG expression level in the observation group was compared with the control group. To determine the level of HERG gene expression we used fluorescent directional PCR, enzyme immunoassay and western blot analysis. The results showed that HERG mRNA level in the observation group was significantly higher than that of the control group. The level of HERG protein in the observation group was significantly higher as well. In the observation group, HERG expression gradually increased with time during the course of the disease. This result suggested that HERG gene expression was associated with the severity of cardiac arrhythmia in children. HERG expression may be the cause of deterioration in cardiomyopathy. The results have provided a theoretical and practical basis for the diagnosis and treatment of children cardiomyopathy. Thus, we established a correlation between HERG expression and cardiac arrhythmia in children.

[Expression and functional assessment of solute carrier 26A transporter family in HEK-293 cells].

OBJECTIVE: To express solute carrier 26A proteins in HEK-293 cells and explore their functions. METHODS: SLC26A-eGFP plasmids were transiently transfected into HEK-293 cells, and the nonlinear capacitance of the cells expressing SLC26A proteins was measured by whole-cell patch recording. RESULTS: All the SLC26A transporters were expressed on the membrane of HEK-293 cells. Each member of the SLC26A transporter family showed robust nonlinear capacitance, which represented their binding capability with anions. CONCLUSION: The SLC26A transporters expressed on HEK cells show similar functions as expected in tissue environment. The plasmids we constructed facilitate structural and functional study of SLC26A transporters.

Polyphenols in Regulation of Redox Signaling and Inflammation During Cardiovascular Diseases.

Cardiovascular diseases remain one of the major health problems worldwide. The worldwide research against cardiovascular diseases as well as genome wide association studies were successful in indentifying the loci associated with this prominent life-threatening disease but still a substantial amount of casualty remains unexplained. Over the last decade, the thorough understanding of molecular and biochemical mechanisms of cardiac disorders lead to the knowledge of various mechanisms of action of polyphenols to target inflammation during cardiac disorders. The present review article summarizes major mechanisms of polyphenols against cardiovascular diseases.

[Effects of astragalus and its active ingredients on ischemia reperfusion injury in isolated guinea-pig heart].

OBJECTIVE: To explore the effects of astragalus (AST) , total flavone of astragalus (TFA), total saponins of astragalus (TSA) and astragalus polysaccharides (APS) on ischemia/reperfusion (40 min/60 min) injury in isolated guinea-pig heart. METHODS: Isolated guinea-pig hearts underwent ischemia, then followed by K-H perfusion (I/R group), AST (60 mg/L),AST (60 mg/L), TFA (60 mg/L), TSA (60 mg/L) and APS (60 mg/L) perfusion (n = 6 each).Isolated hearts without ischemia serve as control group (n = 6). Activity of lactate dehydrogenas (LDH) and creatine kinase (CK) in effluent were measured.Infarct size, myocardial superoxide dismutase (SOD) activity and malondiadehyde (MDA) contents were also determined. RESULTS: Compared to control hearts, heart rate, coronary flow and myocardial superoxide dismutase (SOD) activity were significantly reduced, while LDH and CK in effluent as well as myocardial MDA were significantly increased in the I/R hearts during reperfusion (all P < 0.05), these changes could be partly reversed by AST and TFA perfusion.Infarct size was also significantly reduced in AST (11.9 ± 2.03) % and TFA (13.31 ± 1.17) % treated hearts compared to that in I/R group (18.9 ± 2.27) % (all P < 0.01). CONCLUSIONS: The findings indicate that AST and TFA could attenuate I/R injury in isolated guinea-pig heart possibly through enhancing the activity of SOD and reducing lipid peroxidation.

[Functional refinement of bushy cells in the anterior ventral cochlear nucleus precedes hearing onset in neonatal rats].

OBJECTIVE: To investigate the development of the electrophysiological property of bushy cells in the anterior ventral cochlear nucleus (AVCN) of neonatal Sprague Dawley (SD) rats. METHODS: The development of action potential and spontaneous miniature excitatory postsynaptic currents (mEPSCs) in AVCN bushy cells were investigated by whole-cell patch-clamp technique in SD rats during the postnatal days 5-21 (P5-21). The half band width of the action potential (AP), 10%-90% risetime and decay tau of the mEPSCs were analyzed. RESULTS: The AP of the bushy cells became faster with age from P5 to P21 and stopped changing around the period of hearing onset, as evidenced by the alteration of half band width of the AP. The time accuracy of mEPSCs of the bushy cells also increased with age and stabilized around hearing onset as shown by briefer 10%-90% rise time and decay tau of mEPSCs in P14/P21 than in P7. CONCLUSION: The functional refinement of the bushy cells in the AVCN precedes hearing onset in neonatal rats.