An affective disorder in zebrafish with mutation of the glucocorticoid receptor.

Upon binding of cortisol, the glucocorticoid receptor (GR) regulates the transcription of specific target genes, including those that encode the stress hormones corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone. Dysregulation of the stress axis is a hallmark of major depression in human patients. However, it is still unclear how glucocorticoid signaling is linked to affective disorders. We identified an adult-viable zebrafish mutant in which the negative feedback on the stress response is disrupted, due to abolition of all transcriptional activity of GR. As a consequence, cortisol is elevated, but unable to signal through GR. When placed into an unfamiliar aquarium ('novel tank'), mutant fish become immobile ('freeze'), show reduced exploratory behavior and do not habituate to this stressor upon repeated exposure. Addition of the antidepressant fluoxetine to the holding water and social interactions restore normal behavior, followed by a delayed correction of cortisol levels. Fluoxetine does not affect the overall transcription of CRH, the mineralocorticoid receptor (MR), the serotonin transporter (Serta) or GR itself. Fluoxetine, however, suppresses the stress-induced upregulation of MR and Serta in both wild-type fish and mutants. Our studies show a conserved, protective function of glucocorticoid signaling in the regulation of emotional behavior and reveal novel molecular aspects of how chronic stress impacts vertebrate brain physiology and behavior. Importantly, the zebrafish model opens up the possibility of high-throughput drug screens in search of new classes of antidepressants.

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

[Epileptic seizures during treatment with antidepressants and neuroleptics]. / Epileptische Anfälle unter der Behandlung mit Antidepressiva und Neuroleptika.

Epileptic seizures are observed during treatment with antidepressants and neuroleptics more frequently than is the case for other neuroactive substances. Evidence from experimental and observational studies is mixed, suggesting an increased incidence of seizures for certain drugs, whilst other drugs such as SSRIs appear to have a protective effect. There is robust evidence for an elevated seizure incidence (up to 4.5 % of treated patients) associated with clozapine treatment, but with other neuroleptics the effect is moderate (2-fold). The evaluation of data from FDA approval reports reveals lower standardised incidence rates associated with antidepressants vs. placebo except for clomipramine and bupropione. Psychiatric disorders such as schizophrenia, depression, and obsessive-compulsive disorder are associated with a considerably increased incidence of seizures. Therefore, in clinical practice, taking into account ictogenic properties of substances is required only in patients with a history of seizures.

Axon guidance by gradients of a target-derived component.

Spatial gradients of axon guiding molecules have long been suspected to provide positional and directional cues for retinal ganglion cell axons growing within the optic tectum. With the identification of a guiding activity from tectal cell membranes, it has become possible to investigate the potential physiological significance of molecular gradients for retinal growth cone behavior in vitro. A subset of retinal growth cones, those from the temporal half, were highly sensitive to small concentration changes of the guiding component. The degree of response was correlated with the strength of the gradient. These findings demonstrate that the neural growth cone can read gradients of surface-associated information.

Retinal ganglion cell genesis requires lakritz, a Zebrafish atonal Homolog.

Mutation of the zebrafish lakritz (lak) locus completely eliminates the earliest-born retinal cells, the ganglion cells (RGCs). Instead, excess amacrine, bipolar, and Müller glial cells are generated in the mutant. The extra amacrines are found at ectopic locations in the ganglion cell layer. Cone photoreceptors appear unaffected by the mutation. Molecular analysis reveals that lak encodes Ath5, the zebrafish eye-specific ortholog of the Drosophila basic helix-loop-helix transcription factor Atonal. A combined birth-dating and cell marker analysis demonstrates that lak/ath5 is essential for RGC determination during the first wave of neurogenesis in the retina. Our results suggest that this wave is skipped in the mutant, leading to an accumulation of progenitors for inner nuclear layer cells.

Perception of Fourier and non-Fourier motion by larval zebrafish.

A moving grating elicits innate optomotor behavior in zebrafish larvae; they swim in the direction of perceived motion. We took advantage of this behavior, using computer-animated displays, to determine what attributes of motion are extracted by the fish visual system. As in humans, first-order (luminance-defined or Fourier) signals dominated motion perception in fish; edges or other features had little or no effect when presented with these signals. Humans can see complex movements that lack first-order cues, an ability that is usually ascribed to higher-level processing in the visual cortex. Here we show that second-order (non-Fourier) motion displays induced optomotor behavior in zebrafish larvae, which do not have a cortex. We suggest that second-order motion is extracted early in the lower vertebrate visual pathway.

Failure of hypoxic pulmonary vasoconstriction in the canine asthma model. Effect of prostaglandin inhibitors.

Measurements of respiratory mechanics, arterial blood gases, and pulmonary vascular resistance were made before and 15 min after inhalation challenge with Ascaris suum extract in dogs with natural sensitivity to this antigen. 25 of 47 dogs were treated before inhalation challenge with a prostaglandin inhibitor (90 mg/kg of aspirin or 2 mg/kg of indomethacin by intravenous infusion). In response to the challenge, bronchospasm developed in approximately half (responders) of each group reflected by decreases in mean specific respiratory system conductance and arterial oxygen tension. While the dogs were breathing room air, pulmonary vascular resistance remained unchanged after antigen challenge in the responders not given aspirin or indomethacin, but increased significantly and was associated with a lesser degree of arterial hypoxemia in the responders pretreated with either of the prostaglandin inhibitors. Prevention of arterial hypoxemia by oxygen breathing blocked an increase in pulmonary vascular resistance in four pretreated responders. No changes in respiratory mechanics, pulmonary hemodynamics, or arterial blood gases were noted in the 21 dogs who did not develop bronchospasm regardless of whether or not they were pretreated. 12 additional dogs in whom arterial hypoxemia was produced by 10% oxygen breathing, showed an increase in pulmonary vascular resistance that was not potentiated by pretreatment with aspirin in 6. We conclude that in acute experimental canine asthma, vasodilator prostaglandins appear to blunt the hypoxic pulmonary vasoconstrictor response, thereby further compromising gas exchange but preventing the development of pulmonary hypertension.

Surfactant deficiency in rats without a decreased amount of extracellular surfactant.

Low volume ventilation without periodic large inflations leads to diminished alveolar stability and to the accumulation of increased amounts of airway disaturated phosphatidylcholine (DSPC) in large aggregates that sediment at 1,000 g; surfactant in this form lowers surface tension less rapidly than surfactant present in the 1,000-g supernatant fraction. These observations led to the present work in which we tested the notion that alveolar instability may develop in the presence of an undiminished quantity of total airway surfactant, if the amount of surfactant found in the 1,000-g supernatant fraction is diminished. Pulmonary compliance fell and the alveolar-arterial O2 gradient widened in normothermic rats during constant ventilation in the resting tidal volume range, and, in hyperthermic rats (approximately 39 degrees C) similarly ventilated but with the addition of periodic sighs. The total amount of airway DSPC was undiminished in each group, but in each less DSPC was present in the 1,000-g supernatant fraction compared with controls. Alveolar instability and hypoxemia also developed in hyperthermic rats during low volume ventilation without periodic sighs. Although the total amount of airway DSPC was decreased in these rats, enough remained to theoretically form a continuous monomolecular film over the entire alveolar surface at functional residual capacity; however, there was insufficient surfactant in the 1,000-g supernatant fraction to form such a continuous film. These findings demonstrate that the mode of ventilation, and moderate hyperthermia, may lead to decreased alveolar stability despite the presence of normal amounts of airway surfactant, and, by inference, indicate the extracellular form or state of surfactant has an important effect on alveolar stability.

Zebrafish on the move: towards a behavior-genetic analysis of vertebrate vision.

Behavioral screens have uncovered dozens of zebrafish mutants with striking visual defects. In parallel with mutant studies, recent psychophysical experiments indicate that zebrafish are capable of high-level motion processing, previously thought to be restricted to animals with a visual cortex. It should be possible now to devise assays to screen for mutations in visual perception.

Transcutaneous Vagus Nerve Stimulation (tVNS) for Treatment of Drug-Resistant Epilepsy: A Randomized, Double-Blind Clinical Trial (cMPsE02).

BACKGROUND: Various brain stimulation techniques are in use to treat epilepsy. These methods usually require surgical implantation procedures. Transcutaneous vagus nerve stimulation (tVNS) is a non-invasive technique to stimulate the left auricular branch of the vagus nerve at the ear conch. OBJECTIVE: We performed a randomized, double-blind controlled trial (cMPsE02) to assess efficacy and safety of tVNS vs. control stimulation in patients with drug-resistant epilepsy. METHODS: Primary objective was to demonstrate superiority of add-on therapy with tVNS (stimulation frequency 25 Hz, n = 39) versus active control (1 Hz, n = 37) in reducing seizure frequency over 20 weeks. Secondary objectives comprised reduction in seizure frequency from baseline to end of treatment, subgroup analyses and safety evaluation. RESULTS: Treatment adherence was 84% in the 1 Hz group and 88% in the 25 Hz group, respectively. Stimulation intensity significantly differed between the 1 Hz group (1.02 ± 0.83 mA) and the 25 Hz group (0.50 ± 0.47 mA; p = 0.006). Mean seizure reduction per 28 days at end of treatment was -2.9% in the 1 Hz group and 23.4% in the 25 Hz group (p = 0.146). In contrast to controls, we found a significant reduction in seizure frequency in patients of the 25 Hz group who completed the full treatment period (20 weeks; n = 26, 34.2%, p = 0.034). Responder rates (25%, 50%) were similar in both groups. Subgroup analyses for seizure type and baseline seizure frequency revealed no significant differences. Adverse events were usually mild or moderate and comprised headache, ear pain, application site erythema, vertigo, fatigue, and nausea. Four serious adverse events were reported including one sudden unexplained death in epilepsy patients (SUDEP) in the 1 Hz group which was assessed as not treatment-related. CONCLUSIONS: tVNS had a high treatment adherence and was well tolerated. Superiority of 25 Hz tVNS over 1 Hz tVNS could not be proven in this relatively small study, which might be attributed to the higher stimulation intensity in the control group. Efficacy data revealed results that justify further trials with larger patient numbers and longer observation periods.

Genetic disorders of vision revealed by a behavioral screen of 400 essential loci in zebrafish.

We examined optokinetic and optomotor responses of 450 zebrafish mutants, which were isolated previously based on defects in organ formation, tissue patterning, pigmentation, axon guidance, or other visible phenotypes. These strains carry single point mutations in >400 essential loci. We asked which fraction of the mutants develop blindness or other types of impairments specific to the visual system. Twelve mutants failed to respond in either one or both of our assays. Subsequent histological and electroretinographic analysis revealed unique deficits at various stages of the visual pathway, including lens degeneration (bumper), melanin deficiency (sandy), lack of ganglion cells (lakritz), ipsilateral misrouting of axons (belladonna), optic-nerve disorganization (grumpy and sleepy), inner nuclear layer or outer plexiform layer malfunction (noir, dropje, and possibly steifftier), and disruption of retinotectal impulse activity (macho and blumenkohl). Surprisingly, mutants with abnormally large or small eyes or severe wiring defects frequently exhibit no discernible behavioral deficits. In addition, we identified 13 blind mutants that display outer-retina dystrophy, making this syndrome the single-most common cause of inherited blindness in zebrafish. Our screen showed that a significant fraction (approximately 5%) of the essential loci also participate in visual functions but did not reveal any systematic genetic linkage to particular morphological traits. The mutations uncovered by our behavioral assays provide distinct entry points for the study of visual pathways and set the stage for a genetic dissection of vertebrate vision.

Pentylenetetrazole induced changes in zebrafish behavior, neural activity and c-fos expression.

Rodent seizure models have significantly contributed to our basic understanding of epilepsy. However, medically intractable forms of epilepsy persist and the fundamental mechanisms underlying this disease remain unclear. Here we show that seizures can be elicited in a simple vertebrate system e.g. zebrafish larvae (Danio rerio). Exposure to a common convulsant agent (pentylenetetrazole, PTZ) induced a stereotyped and concentration-dependent sequence of behavioral changes culminating in clonus-like convulsions. Extracellular recordings from fish optic tectum revealed ictal and interictal-like electrographic discharges after application of PTZ, which could be blocked by tetrodotoxin or glutamate receptor antagonists. Epileptiform discharges were suppressed by commonly used antiepileptic drugs, valproate and diazepam, in a concentration-dependent manner. Up-regulation of c-fos expression was also observed in CNS structures of zebrafish exposed to PTZ. Taken together, these results demonstrate that chemically-induced seizures in zebrafish exhibit behavioral, electrographic, and molecular changes that would be expected from a rodent seizure model. Therefore, zebrafish larvae represent a powerful new system to study the underlying basis of seizure generation, epilepsy and epileptogenesis.

Cysticercotic cyst involving the pleura. An unusual case of an abnormal chest roentgenogram.

We describe a case of human cysticercosis appearing as an abnormal chest roentgenogram. The patient had a long history of ingestion of undercooked pork. The diagnosis was entertained only after surgical resection, but the history obtained from this patient gave an important clue toward a parasitic infestation.

Survival of patients with cancer in a medical critical care unit.

The records of 40 patients with cancer and 684 patients without cancer admitted to a medical critical care unit were reviewed for the purpose of comparing survival. Patients with cancer had a higher mortality (55%, 22/40) than the patients without cancer (17%, 118/864). Patients with cancer and respiratory failure had a higher mortality (75%, 18/24) than patients without cancer but with respiratory failure (25%, 66/273) and a higher mortality than patients with cancer but without respiratory failure (25%, 4/16). In patients with cancer, the manifestation of respiratory failure as the adult respiratory distress syndrome (ARDS) was associated with a high mortality (86%, 12/14), which was not statistically higher than in patients with cancer with non-ARDS respiratory failure (60%, 6/10) or in patients without cancer with ARDS (65%, 28/43). Patients with cancer but without respiratory failure had a mortality of 25% (4/16). All eight patients with cancer admitted to the hospital because of life-threatening metabolic disturbances survived. In deciding whether to apply critical care techniques to a patient with cancer, the physician should consider the specific nature of the life-threatening illness.

Left ventricular surface tissue oxygen pressures determined by oxygen sensitive multiwire electrodes in pigs.

STUDY OBJECTIVE: Polarographic oxygen sensitive electrodes can be used to measure tissue oxygen pressures on the surface of the beating heart. The purpose of the study was to clarify the significance of these PO2 determinations. DESIGN: Changes in left ventricular surface oxygen pressures, subendocardial or subepicardial wall functions (ultrasonic dimension technique), and blood flow (radioactive microsphere technique) were correlated during different degrees of acute coronary artery stenoses in pigs. EXPERIMENTAL MATERIAL: 19 anaesthetised open chest pigs, 28-40 kg body weight, were studied during different degrees of constriction of the left anterior descending artery which did not influence overall left ventricular function or irreversibly damage the myocardium. MEASUREMENTS AND MAIN RESULTS: Highly significant (p less than 0.001) correlations (each % delta) were obtained for surface tissue oxygen pressures (y) with subepicardial (y = 0.002 e 0.10x; r = 0.89) and subendocardial (y = 1.44 e 0.04x; r = 0.98) blood flow values, as well as with subendocardial function (y = 82.4 + 0.22x; r = 0.89); a significant correlation was also obtained for subendocardial function (y) with subendocardial blood flow (y = 66.0 + 0.28x; r = 0.69). However, no significant correlation was obtained for polarographic tissue PO2 with subepicardial segment function, indicating that regional function was maintained when tissue PO2 recordings were not much above 0 kPa and when blood flow was reduced by 25 to 30%. CONCLUSIONS: Oxygen pressures of the superficial layers of the left ventricle are relatively high at normal values of oxygen consumption but decrease rapidly if blood supply is reduced. Regional wall function is preserved at low oxygen pressures. Polarographic surface PO2 electrodes hence can be used to study influences of experimental interventions on oxygenation of the normally perfused and of the moderately ischaemic myocardium.

Food intake, hunger, and satiety after preloads in women with eating disorders.

Food intake, food selection, macronutrient intake, sensory-specific satiety, and ratings of hunger and satiety were measured after high- and low-energy salad preloads (2414 kJ, or 172 kJ) or no preload to determine whether patients with eating disorders compensate appropriately for different energy intakes. Subjects were female patients with a DSM-III-R diagnosis of anorexia nervosa with bulimic features or bulimia nervosa, or non patient, normal-weight, nondieters (n = 9/group). At a self-selected lunch 30 min after the preloads, all of the groups reduced intake after the high-energy preload, with the bulimics showing the best compensation. The anorexics chose low-energy foods and in some conditions ate a smaller proportion of fat than did the other groups. The bulimics ate more high-energy foods than did the anorexics. The anorexics demonstrated sensory-specific satiety only after the high-energy salad and the bulimics only after the low-energy salad. Overall, these data suggest that while many of their responses to food are abnormal, patients with eating disorders have some capacity to respond to physiological hunger and satiety cues.

Generalized epilepsy with febrile seizures plus: further heterogeneity in a large family.

BACKGROUND: Generalized epilepsy with febrile seizures plus (GEFS(+)) is a recently described benign childhood-onset epileptic syndrome with autosomal dominant inheritance. The most common phenotypes are febrile seizures (FS) often with accessory afebrile generalized tonic-clonic seizures (GTCS, FS(+)). In about one third, additional seizure types occur, such as absences, myoclonic, or atonic seizures. So far, three mutations within genes encoding subunits of neuronal voltage-gated Na(+) channels have been found in GEFS(+) families, one in SCN1B (beta(1)-subunit) and two in SCN1A (alpha-subunit). METHODS: The authors examined the phenotypic variability of GEFS(+) in a five-generation German family with 18 affected individuals. Genetic linkage analysis was performed to exclude candidate loci. RESULTS: Inheritance was autosomal dominant with a penetrance of about 80%. A variety of epilepsy phenotypes occurred predominantly during childhood. Only four individuals showed the FS or FS(+) phenotype. The others presented with different combinations of GTCS, tonic seizures, atonic seizures, and absences, only in part associated with fever. The age at onset was 2.8 +/- 1.3 years. Interictal EEG recordings showed rare, 1- to 2-second-long generalized, irregular spike-and-wave discharges of 2.5 to 5 Hz in eight cases and additional focal parietal discharges in one case. Linkage analysis excluded the previously described loci on chromosomes 2q21-33 and 19q13. All other chromosomal regions containing known genes encoding neuronal Na(+) channel subunits on chromosomes 3p21-24, 11q23, and 12q13 and described loci for febrile convulsions on chromosomes 5q14-15, 8q13-21, and 19p13.3 were also excluded. CONCLUSION: These results indicate further clinical and genetic heterogeneity in GEFS(+).

Evidence for increased permeability in reexpansion pulmonary edema.

Concurrent analyses of the colloid osmotic pressure of pulmonary edema fluid and serum were performed in two patients with reexpansion pulmonary edema. In addition, pulmonary artery wedge pressure was measured during pulmonary edema in one patient. The colloid osmotic pressure of the pulmonary edema fluid was 73 and 81 percent of the serum value. The pulmonary arterial wedge pressure in one patient was within normal limits (8 mm Hg). Reexpansion pulmonary edema appears to be due to increased pulmonary capillary permeability rather than to hemodynamic mechanisms.

Ventilatory drive in normal man during semistarvation.

The effects of reduced caloric intake on ventilatory drive were investigated in normal volunteers. During a ten-day semistarvation period, six subjects (group 1) received parenterally an amino acid solution providing 550 kcal/d sufficient to prevent a negative nitrogen balance. Six subjects (group 2) received in addition a safflower oil solution providing a total caloric intake of 1,100 kcal/d. Hypoxic ventilatory drive was estimated by an index (parameter A) of the relation between minute ventilation (VE) and hypoxia. In group 1, mean values (+/- SEM) of A decreased significantly from 161.5 (+/- 42.0) to 48.9 (+/- 12.0) by day 10 (p less than 0.05), indicating a severe depression of hypoxic drive despite a positive nitrogen balance. In group 2, A did not change significantly (p greater than 0.05) from control values indicating a preserved hypoxic ventilatory drive. In both groups, the slopes of the line relating VE to arterial PCO2 (delta VE/delta PaCO2) did not change significantly during the ten-day semistarvation period consistent with preservation of the hypercapnic ventilatory drive. These data indicate that during periods of starvation, parenteral administration of aminoacids in an amount sufficient to maintain nitrogen balance is inadequate to prevent depression of respiratory control mechanisms unless a minimum daily caloric intake is achieved.

In vivo estimation of tracheal wall water: effects of histamine and carbachol.

Based on double-indicator-dilution principles we developed and validated a new method of measuring water content in the tracheal wall by inscribing time-concentration curves with the inert gas, helium and the water-soluble gas, dimethylether. In the trachea of conscious adult sheep the measurable water volume of the wall over a 15-cm length ranged between 0.50 and 0.70 ml. The tracheal radius calculated from helium volume and known tracheal length ranged between 7.4 and 9.3 mm. The corresponding measurable "water depth" ranged between 63 and 82 microns. Changes in bronchial blood flow had no consistent influence on tracheal water volume. The intravenous injection of histamine (4 micrograms/kg) increased mean water depth to between 122 and 134% of base line (P less than 0.001), whereas the intravenous injection of carbachol (4 micrograms/kg) had no effect on water depth. The production of large amounts of airway secretions produced by carbachol were removed prior to inscription of the indicator-dilution curves. These findings indicate that the double-gas-indicator technique is capable of measuring in vivo the volume of a water compartment in the tracheal wall and that this volume is increased by histamine. This method may be applicable in human subjects.