Intranasal immunization with the recombinant measles virus encoding the spike protein of SARS-CoV-2 confers protective immunity against COVID-19 in hamsters.

BACKGROUND: As the nasal mucosa is the initial site of infection for COVID-19, intranasal vaccines are more favorable than conventional vaccines. In recent clinical studies, intranasal immunization has been shown to generate higher neutralizing antibodies; however, there is a lack of evidence on sterilizing immunity in the upper airway. Previously, we developed a recombinant measles virus encoding the spike protein of SARS-CoV-2 (rMeV-S), eliciting humoral and cellular immune responses against SARS-CoV-2. OBJECTIVES: In this study, we aim to provide an experiment on nasal vaccines focusing on a measles virus platform as well as injection routes. STUDY DESIGN: Recombinant measles viruses expressing rMeV-S were prepared, and 5 × 105 PFUs of rMeV-S were administered to Syrian golden hamsters via intramuscular or intranasal injection. Subsequently, the hamsters were challenged with inoculations of 1 × 105 PFUs of SARS-CoV-2 and euthanized 4 days post-infection. Neutralizing antibodies and RBD-specific IgG in the serum and RBD-specific IgA in the bronchoalveolar lavage fluid (BALF) were measured, and SARS-CoV-2 clearance capacity was determined via quantitative reverse-transcription PCR (qRT-PCR) analysis and viral titer measurement in the upper respiratory tract and lungs. Immunohistochemistry and histopathological examinations of lung samples from experimental hamsters were conducted. RESULTS: The intranasal immunization of rMeV-S elicits protective immune responses and alleviates virus-induced pathophysiology, such as body weight reduction and lung weight increase in hamsters. Furthermore, lung immunohistochemistry demonstrated that intranasal rMeV-S immunization induces effective SARS-CoV-2 clearance that correlates with viral RNA content, as determined by qRT-PCR, in the lung and nasal wash samples, SARS-CoV-2 viral titers in lung, nasal wash, BALF samples, serum RBD-specific IgG concentration, and RBD-specific IgA concentration in the BALF. CONCLUSION: An intranasal vaccine based on the measles virus platform is a promising strategy owing to the typical route of infection of the virus, the ease of administration of the vaccine, and the strong immune response it elicits.

Mechanism of Bisphenol F Affecting Motor System and Motor Activity in Zebrafish.

Bisphenol F (BPF; 4,4'-dihydroxydiphenylmethane) is one of the most frequently used compounds in the manufacture of plastics and epoxy resins. Previous studies have demonstrated that BPF affects locomotor behavior, oxidative stress, and neurodevelopment in zebrafish. However, its neurotoxic effects are controversial, and the underlying mechanisms are unclear. In order to determine whether BPF affects the motor system, we exposed zebrafish embryos to BPF and assessed behavioral, histological, and neurochemical changes. Spontaneous locomotor behavior and startle response were significantly decreased in BPF-treated zebrafish larvae compared with control larvae. BPF induced motor degeneration and myelination defects in zebrafish larvae. In addition, embryonic exposure to BPF resulted in altered metabolic profiles of neurochemicals, including neurotransmitters and neurosteroids, which may impact locomotion and motor function. In conclusion, exposure to BPF has the potential to affect survival, motor axon length, locomotor activity, myelination, and neurochemical levels of zebrafish larvae.

Graphene/III-V Quantum Dot Mixed-Dimensional Heterostructure for Enhanced Radiative Recombinations via Hole Carrier Transfer.

Fabrication of high quantum efficiency nanoscale device is challenging due to increased carrier loss at surface. Low dimensional materials such 0D quantum dots and 2D materials have been widely studied to mitigate the loss. Here, we demonstrate a strong photoluminescence enhancement from graphene/III-V quantum dot mixed-dimensional heterostructures. The distance between graphene and quantum dots in the 2D/0D hybrid structure determines the degree of radiative carrier recombination enhancement from 80% to 800% compared to the quantum dot only structure. Time-resolved photoluminescence decay also shows increased carrier lifetimes when the distance decreases from 50 to 10 nm. We propose that the optical enhancement is due to energy band bending and hole carrier transfer, which repair the imbalance of electron and hole carrier densities in quantum dots. This 2D graphene/0D quantum dot heterostructure shows promise for high performance nanoscale optoelectronic devices.

Recombinant measles virus encoding the spike protein of SARS-CoV-2 efficiently induces Th1 responses and neutralizing antibodies that block SARS-CoV-2 variants.

Owing to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, the development of effective and safe vaccines has become a priority. The measles virus (MeV) vaccine is an attractive vaccine platform as it has been administered to children for more than 40 years in over 100 countries. In this study, we developed a recombinant MeV expressing the full-length SARS-CoV-2 spike protein (rMeV-S) and tested its efficacy using mouse and hamster models. In hCD46Tg mice, two-dose rMeV-S vaccination induced higher Th1 secretion and humoral responses than one-dose vaccination. Interestingly, neutralizing antibodies induced by one-dose and two-dose rMeV-S immunization effectively blocked the entry of the α, ß, γ, and δ variants of SARS-CoV-2. Furthermore, two-dose rMeV-S immunization provided complete protection against SARS-CoV-2 in the hamster model. These results suggest the potential of rMeV-S as a vaccine candidate for targeting SARS-CoV-2 and its variants.

Enhanced Photoluminescence of 1.3 µm InAs Quantum Dots Grown on Ultrathin GaAs Buffer/Si Templates by Suppressing Interfacial Defect Emission.

We report on the photoluminescence enhancement of 1.3 µm InAs quantum dots (QDs) epitaxially grown on an ultrathin 250 nm GaAs buffer on a Si substrate. Decreasing the GaAs buffer thickness from 1000 to 250 nm was found to not only increase the coalesced QD density from 6.5 × 108 to 1.9 × 109 cm-2 but also decrease the QD photoluminescence emission intensity dramatically. Inserting an Al0.4Ga0.6As potential barrier layer maintained strong photoluminescence from the QDs by effectively suppressing carrier leakage to the GaAs/Si interfacial region even when the GaAs buffer was thinned to 250 nm. We then fabricated a light-emitting diode using the ultrathin 250 nm GaAs buffer on Si and confirmed strong electroluminescence peaking at 1.28 µm without interfacial defect emission at room temperature. We believe that this work is promising for monolithically integrated evanescent Si lasers using InAs/GaAs QDs.

A robust multivariate structure of interindividual covariation between psychosocial characteristics and arousal responses to visual narratives.

People experience the same event but do not feel the same way. Such individual differences in emotion response are believed to be far greater than those in any other mental functions. Thus, to understand what makes people individuals, it is important to identify the systematic structures of individual differences in emotion response and elucidate how such structures relate to what aspects of psychological characteristics. Reflecting this importance, many studies have attempted to relate emotions to psychological characteristics such as personality traits, psychosocial states, and pathological symptoms across individuals. However, systematic and global structures that govern the across-individual covariation between the domain of emotion responses and that of psychological characteristics have been rarely explored previously, which limits our understanding of the relationship between individual differences in emotion response and psychological characteristics. To overcome this limitation, we acquired high-dimensional data sets in both emotion-response (8 measures) and psychological-characteristic (68 measures) domains from the same pool of individuals (86 undergraduate or graduate students) and carried out the canonical correlation analysis in conjunction with the principal component analysis on those data sets. For each participant, the emotion-response measures were quantified by regressing affective-rating responses to visual narrative stimuli onto the across-participant average responses to those stimuli, while the psychological-characteristic measures were acquired from 19 different psychometric questionnaires grounded in personality, psychosocial-factor, and clinical-problem taxonomies. We found a single robust mode of population covariation, particularly between the 'accuracy' and 'sensitivity' measures of arousal responses in the emotion domain and many 'psychosocial' measures in the psychological-characteristics domain. This mode of covariation suggests that individuals characterized with positive social assets tend to show polarized arousal responses to life events.

Non-invasive administration of AAV to target lung parenchymal cells and develop SARS-CoV-2-susceptible mice.

Adeno-associated virus (AAV)-mediated gene delivery holds great promise for gene therapy. However, the non-invasive delivery of AAV for lung tissues has not been adequately established. Here, we revealed that the intratracheal administration of an appropriate amount of AAV2/8 predominantly targets lung tissue. AAV-mediated gene delivery that we used in this study induced the expression of the desired protein in lung parenchymal cells, including alveolar type II cells. We harnessed the technique to develop severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-susceptible mice. Three kinds of immune function-relevant gene knockout (KO) mice were transduced with AAV encoding human angiotensin-converting enzyme 2 (hACE2) and then injected with SARS-CoV-2. Among these mice, type I interferon receptor (IFNAR) KO mice showed increased viral titer in the lungs compared to that in the other KO mice. Moreover, nucleocapsid protein of SARS-CoV-2 and multiple lesions in the trachea and lung were observed in AAV-hACE2-transduced, SARS-CoV-2-infected IFNAR KO mice, indicating the involvement of type I interferon signaling in the protection of SARS-CoV-2. In this study, we demonstrate the ease and rapidness of the intratracheal administration of AAV for targeting lung tissue in mice, and this can be used to study diverse pulmonary diseases.

Ultra- and micro-structural changes of respiratory tracts in SARS-CoV-2 infected Syrian hamsters.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.

Immunization with RBD-P2 and N protects against SARS-CoV-2 in nonhuman primates.

Since the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), various vaccines are being developed, with most vaccine candidates focusing on the viral spike protein. Here, we developed a previously unknown subunit vaccine comprising the receptor binding domain (RBD) of the spike protein fused with the tetanus toxoid epitope P2 (RBD-P2) and tested its efficacy in rodents and nonhuman primates (NHPs). We also investigated whether the SARS-CoV-2 nucleocapsid protein (N) could increase vaccine efficacy. Immunization with N and RBD-P2 (RBDP2/N) + alum increased T cell responses in mice and neutralizing antibody levels in rats compared with those obtained using RBD-P2 + alum. Furthermore, in NHPs, RBD-P2/N + alum induced slightly faster SARS-CoV-2 clearance than that induced by RBD-P2 + alum, albeit without statistical significance. Our study supports further development of RBD-P2 as a vaccine candidate against SARS-CoV-2. Also, it provides insights regarding the use of N in protein-based vaccines against SARS-CoV-2.

The Effect of Ulinastatin to the Learning and Memory in Zebrafish.

Previous study indicated that Ulinastatin (UTI) increased glutamine uptake by upregulation of glutamate transporters in astrocytes. These glutamate transporters have important role to improve cognitive function in hippocampus. In this study, we wanted to demonstrate whether UTI could improve learning and memory by using zebrafish behavior model and bio-markers. Zebrafish were 6-8 months of age and were 2.5-3.5 cm long. They were divided into four groups by control, 1X PBS-injected control, UTI 10,000, and 50,000 injected group. All PBS and UTI injected zebrafish were anesthetized by Tricainemethanesulphonate. We measured total time, distance moved, and frequency in each compartment of T-maze. We also measured the expression levels of glutamate transporter levels and cognitive bio-markers such as c-fos, c-jun, BDNF. UTI affected the learning and memory in zebrafish in a dose-dependent manner. In 50,000 unit/kg UTI-treated zebrafish, there were increases of time, distance, and frequency in target compartment. In 50,000 unit/kg UTI-treated zebrafish, there was an increase of time in target compartment. There was no difference among control, PBS-injected, and UTI 10,000 unit/kg-treated groups. EAAT4 glutamate transporter, c-fos and BDNF were significantly increased in 50,000 unit/kg UTI-treated group. UTI-enhanced learning and memory in zebrafish. The expressions of EAAT4 glutamate transporter, c- fos and BDNF in zebrafish were highly correlated may play a role.

Neuroprotective effects of magnesium L-threonate in a hypoxic zebrafish model.

BACKGROUND: Hypoxia inhibits the uptake of glutamate (a major neurotransmitter in the brain closely related to cognitive function) into brain cells, and the initial response of cells to cortical hypoxia depends on glutamate. Previous studies have suggested that magnesium may have protective effects against hypoxic injuries. In particular, magnesium L-threonate (MgT) may increase magnesium ion concentrations in the brain better than MgSO4 and improve cognitive function. METHODS: We evaluated cell viability under hypoxic conditions in the MgT- and MgSO4-treated human SH-SY5Y neurons, in vivo behavior using the T-maze test following hypoxia in MgT-treated zebrafish, activity of brain mitochondrial dehydrogenase by 2,3,5-triphenyltetrazolium chloride (TTC) staining, and protein expression of the excitatory amino acid transporter (EAAT) 4 glutamate transporter by western blotting. RESULTS: Among the groups treated with hypoxia, cell viability significantly increased when pre-treated with 1 or 10 mM MgT (p = 0.009 and 0.026, respectively). Despite hypoxic insult, MgT-treated zebrafish showed preferences for the red compartment (p = 0.025 for distance and p = 0.007 for frequency of entries), suggesting memory preservation. TTC staining showed reduced cerebral infarction and preserved absorbance in the MgT-treated zebrafish brain after hypoxia (p = 0.010 compared to the hypoxia group). In addition, western blot showed upregulation of EAAT4 protein in the MgT treated group. CONCLUSIONS: Pre-treatment with MgT attenuated cell death and cerebral infarction due to hypoxia and protected cognitive function in zebrafish. In addition, MgT appeared to modulate expression of the glutamate transporter, EAAT4.

Effects of hypoxic preconditioning on memory evaluated using the T-maze behavior test.

Perioperative brain ischemia and stroke are leading causes of morbidity and mortality. Brief hypoxic preconditioning is known to have protective effects against hypoxic-ischemic insult in the brain. Current studies on the neuroprotective effects of ischemic preconditioning are based on histologic findings and biomarker changes. However, studies regarding effects on memory are rare. To precondition zebrafish to hypoxia, they were exposed to a dissolved oxygen (DO) concentration of 1.0 ± 0.5 mg/L in water for 30 s. The hypoxic zebrafish were then exposed to 1.0 ± 0.5 mg/L DO until the third stage of hypoxia, for 10 min ± 30 s. Zebrafish were assessed for memory retention after the hypoxic event. Learning and memory were tested using the T-maze, which evaluates memory based on whether or not zebrafish moves to the correct target compartment. In the hypoxic preconditioning group, infarct size was reduced compared with the hypoxic-only treated zebrafish group; memory was maintained to a degree similar to that in the hypoxia-untreated group. The hypoxic-only group showed significant memory impairments. In this study, we used a hypoxic zebrafish model and assessed the effects of ischemic preconditioning not only on histological damages but also on brain function, especially memory. This study demonstrated that a brief hypoxic event has protective effects in hypoxic brain damage and helped maintain memory in zebrafish. In addition, our findings suggest that the zebrafish model is useful in rapidly assessing the effects of ischemic preconditioning on memory.

The protective effect of hydromorphone to ischemia in rat glial cells.

BACKGROUND: Ischemic insults during operation can cause ischemic-reperfusion injuries in brain as well as memory impairments. Total intravenous anesthesia (TIVA) is the preferred anesthetic method in brain surgery, as it utilizes motor evoked potential monitoring. And the use of opioids is common in TIVA. However there are few studies about ischemic protective effect of opioids to glial cells. METHODS: We used mixed cultures of rat glial cells, which were harvested from the brain of 1-day old rat. We divided the experimental groups according to their hydromorphone conditioning period: (a) pre-culture, (b) per-culture, or (c) pre- and per-culture. We measured the levels of the reactive oxygen species (ROS) induced by tert-butyl hydroperoxide (TBH) using flow cytometry. The ROS levels in the glial cells were also measured after the administration of 100 nM hydromorphone and selective opioid receptor antagonists. RESULTS: The ROS levels were reduced in the hydromorphone-treated group, as compared to the control group (only TBH treated). There were no differences between pre-conditioned and per-conditioned groups. However, the ROS levels were more reduced in pre- and per-conditioned group compared to pre-conditioned or per-conditioned only groups. Furthermore, selective antagonists for the delta, kappa, or mu opioid receptor partially negated the hydromorphone effect. CONCLUSION: This study demonstrated that hydromorphone can have additional protective effects on oxidative stress when pre- and per-conditioning is combined. Furthermore we proved that µ, δ, κ opioid receptors participate in protective mechanism of hydromorphone to glial cells.

Dance and Music in "Gangnam Style": How Dance Observation Affects Meter Perception.

Dance and music often co-occur as evidenced when viewing choreographed dances or singers moving while performing. This study investigated how the viewing of dance motions shapes sound perception. Previous research has shown that dance reflects the temporal structure of its accompanying music, communicating musical meter (i.e. a hierarchical organization of beats) via coordinated movement patterns that indicate where strong and weak beats occur. Experiments here investigated the effects of dance cues on meter perception, hypothesizing that dance could embody the musical meter, thereby shaping participant reaction times (RTs) to sound targets occurring at different metrical positions.In experiment 1, participants viewed a video with dance choreography indicating 4/4 meter (dance condition) or a series of color changes repeated in sequences of four to indicate 4/4 meter (picture condition). A sound track accompanied these videos and participants reacted to timbre targets at different metrical positions. Participants had the slowest RT's at the strongest beats in the dance condition only. In experiment 2, participants viewed the choreography of the horse-riding dance from Psy's "Gangnam Style" in order to examine how a familiar dance might affect meter perception. Moreover, participants in this experiment were divided into a group with experience dancing this choreography and a group without experience. Results again showed slower RTs to stronger metrical positions and the group with experience demonstrated a more refined perception of metrical hierarchy. Results likely stem from the temporally selective division of attention between auditory and visual domains. This study has implications for understanding: 1) the impact of splitting attention among different sensory modalities, and 2) the impact of embodiment, on perception of musical meter. Viewing dance may interfere with sound processing, particularly at critical metrical positions, but embodied familiarity with dance choreography may facilitate meter awareness. Results shed light on the processing of multimedia environments.

Effects of dietary corticosterone on yolk colors and eggshell quality in laying hens.

The objective of this study was to investigate the effects of dietary corticosterone on egg quality. For 2 weeks hens received either control or experimental diet containing corticosterone at 30 mg/kg diet. Feed intake and egg production were monitored daily, and body weight measured weekly. Egg weights and egg quality were measured daily. Corticosterone treatment resulted in a remarkable increase in feed intake and sharp decrease in egg production compared with control (p<0.05) whereas body weight remained unchanged. Decreased albumen height, but no changes in egg weight, led to decreased Haugh unit (p<0.05). Corticosterone caused elevated eggshell thickness (p<0.05) without altering weight and strength, suggesting possible changes in shell structure. Yolk color and redness were increased by corticosterone (p<0.05) but lightness and yellowness were either not changed or inconsistent over the time period of measurements. Increased concentrations in plasma were also found for corticosterone, glucose, cholesterol, creatinine, uric acid, albumin, aspartate aminotransferase, creatine kinase, lactate dehydrogenase, total protein, and amylase (p<0.05), suggesting that corticosterone increased protein breakdown, renal dysfunctions and pancreatitis. Together, the current results imply that dietary corticosterone affects egg quality such as yolk colors and shell thickness, in addition to its effects on feed intake and egg production.

Valproic acid decreases the proliferation of telencephalic cells in zebrafish larvae.

Valproic acid (VPA) is used as an antiepileptic drug (AED) or mood stabilizer. Recent studies have shown that exposure to VPA during embryonic development alters neural progenitor cell (NPC) proliferation, and can also lead to behavioral impairment in adult animals. The main goal of this investigation was to evaluate the effects of treatment with 2 mM VPA for 3h on cell proliferation in the telencephalic area of zebrafish larvae of 5 days post-fertilization (dpf) using immunohistochemistry (IHC). It was also determined whether VPA exposure affects the learning ability and anxiety levels of zebrafish during adulthood using bottom-dwelling behavior and passive avoidance tests. Result of the study demonstrated that VPA exposure during development transiently decreased neuronal cell proliferation without inducing apoptosis. Additionally, quantitative real-time PCR (qRT-PCR) data indicated that mRNA expression levels of wnt signaling pathway-related factors such as ß-catenin, lef1, and gsk3ß were altered in the zebrafish treated with VPA. Interestingly, these effects were reversed over time after VPA treatment had ceased. Alterations of passive avoidance learning and bottom dwelling behavior were not observed during adulthood after developmental VPA exposure. These results may be due to the restoration of cell proliferation during the recovery period after VPA treatment.

Reverse controlled antegrade and retrograde subintimal tracking in chronic total occlusion of right coronary artery.

Passage failure of guidewire is still remained most common reason for percutaneous coronary intervention (PCI) failure in chronic total occlusion (CTO). Intravascular ultrasound study (IVUS) and cardiac CT angiography can help identify features that most influence current success rates of PCI. We report our experience using the reverse controlled antegrade and retrograde subintimal tracking technique under the aid of IVUS, cardiac CT angiography for an ambiguous CTO of proximal right coronary artery.

Improvement of pentylenetetrazol-induced learning deficits by valproic acid in the adult zebrafish.

Pentylenetetrazol (PTZ) has been shown to induce seizure-like behavior, learning deficits in passive avoidance response test, and an increase in hsp70 (heat shock protein 70) mRNA expression in the adult zebrafish; PTZ has been increasingly appreciated as an excellent model system for the study of seizures. In this study, we demonstrate that valproic acid (VPA), an antiepileptic drug, suppresses seizure-like behavior and improves learning ability in adult zebrafish treated with PTZ. Pretreatment with VPA significantly reduces rapid involuntary movement and abrupt changes in moving direction in the PTZ-treated zebrafish. PTZ-induced learning impairments were also improved in the zebrafish pretreated with 200 or 500 microM VPA. However, the scopolamine-induced impairments of learning ability were not improved by VPA pretreatment. It is worth noting that while the zebrafish treated with 500 microM VPA for 1-3 weeks learned the passive avoidance response, those treated with 1 or 2mM VPA for 3h didn't. Furthermore, the increased level of hsp70 expression induced by PTZ, a stress marker protein, was significantly reduced in the VPA-pretreated zebrafish brains. Collectively, our data show the antiepileptic effects of VPA in the adult zebrafish, which coincides with reduced hsp70 mRNA expression, rescued learning impairment under PTZ-treated conditions.

Reduced neuronal proliferation by proconvulsant drugs in the developing zebrafish brain.

Seizures have been reported to modify neural development in the immature brain. In this study, we attempted to determine whether pentylenetetrazol (a GABAergic receptor antagonist, PTZ)-induced seizures influence cell proliferation in zebrafish larvae (5 and 15 days of post-fertilization), using bromodeoxyuridine (BrdU) to label dividing cells. In the brains of 5 dpf larvae, PTZ treatment significantly reduced the number of BrdU-labeled cells in the telencephalic area (pallium and subpallium), diencephalic area (thalamus and preoptic area), medial tectal proliferation zone, and medial cerebellar proliferation zone to 52.4%, 62.9%, 47.2%, and 54.0% of the controls, respectively. In contrast, we noted no reductions in the number of BrdU-labeled cells in the brains of the 15 dpf larvae. The double-label of BrdU and Hu, a neuronal marker, demonstrated that the majority of newborn cells showed the neuronal phenotype. Similarly, kainic acid (200 microM), a glutamatergic receptor agonist, significantly reduced the number of BrdU-labeled cells in the telencephalic area, diencephalic area, and medial tectal proliferation zone to 51.4%, 61.9%, and 40.4% of the controls, respectively. Physostigmine (500 microM), an acetylcholinesterase inhibitor, also reduced the number of BrdU-labeled cells in the telencephalic area, diencephalic area, medial tectal proliferation zone, and medial cerebellar proliferation zone to 52.8%, 35.9%, 30.5%, and 39.8% of the controls, respectively. All of these drugs resulted in electrographic seizures in the larval brain when perfused directly through artificial cerebrospinal fluid. These results indicated that seizures result in a massive reduction in cell proliferation in wide-ranging areas of the developing brain.