Ciliogenesis defects after neurulation impact brain development and neuronal activity in larval zebrafish.

Cilia are slender, hair-like structures extending from cell surfaces and playing essential roles in diverse physiological processes. Within the nervous system, primary cilia contribute to signaling and sensory perception, while motile cilia facilitate cerebrospinal fluid flow. Here, we investigated the impact of ciliary loss on neural circuit development using a zebrafish line displaying ciliogenesis defects. We found that cilia defects after neurulation affect neurogenesis and brain morphology, especially in the cerebellum, and lead to altered gene expression profiles. Using whole brain calcium imaging, we measured reduced light-evoked and spontaneous neuronal activity in all brain regions. By shedding light on the intricate role of cilia in neural circuit formation and function in the zebrafish, our work highlights their evolutionary conserved role in the brain and sets the stage for future analysis of ciliopathy models.

The forkhead transcription factor Foxj1 controls vertebrate olfactory cilia biogenesis and sensory neuron differentiation.

In vertebrates, olfactory receptors localize on multiple cilia elaborated on dendritic knobs of olfactory sensory neurons (OSNs). Although olfactory cilia dysfunction can cause anosmia, how their differentiation is programmed at the transcriptional level has remained largely unexplored. We discovered in zebrafish and mice that Foxj1, a forkhead domain-containing transcription factor traditionally linked with motile cilia biogenesis, is expressed in OSNs and required for olfactory epithelium (OE) formation. In keeping with the immotile nature of olfactory cilia, we observed that ciliary motility genes are repressed in zebrafish, mouse, and human OSNs. Strikingly, we also found that besides ciliogenesis, Foxj1 controls the differentiation of the OSNs themselves by regulating their cell type-specific gene expression, such as that of olfactory marker protein (omp) involved in odor-evoked signal transduction. In line with this, response to bile acids, odors detected by OMP-positive OSNs, was significantly diminished in foxj1 mutant zebrafish. Taken together, our findings establish how the canonical Foxj1-mediated motile ciliogenic transcriptional program has been repurposed for the biogenesis of immotile olfactory cilia, as well as for the development of the OSNs.

A-kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii.

Cilia are microtubule-based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A-kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central-pair microtubules of Chlamydomonas reinhardtii cilia uses two C-terminal amphipathic helices to bind to its partner FAP174, an RIIα-like protein with a D/D domain at the N-terminus. Co-immunoprecipitation using anti-FAP174 antibody with an enriched central-pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub-ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central-pair microtubules.

Expression of taste sentinels, T1R, T2R, and PLCß2, on the passageway for olfactory signals in zebrafish.

The senses of taste and smell detect overlapping sets of chemical compounds in fish, e.g. amino acids are detected by both senses. However, so far taste and smell organs appeared morphologically to be very distinct, with a specialized olfactory epithelium for detection of odors and taste buds located in the oral cavity and lip for detection of tastants. Here, we report dense clusters of cells expressing T1R and T2R receptors as well as their signal transduction molecule PLCß2 in nostrils of zebrafish, i.e. on the entrance funnel through which odor molecules must pass to be detected by olfactory sensory neurons. Quantitative evaluation shows the density of these chemosensory cells in the nostrils to be as high or higher than that in the established taste organs oral cavity and lower lip. Hydrodynamic flow is maximal at the nostril rim enabling high throughput chemosensation in this organ. Taken together, our results suggest a sentinel function for these chemosensory cells in the nostril.

Methods to study motile ciliated cell types in the zebrafish brain.

Cilia are well conserved hair-like structures that have diverse sensory and motile functions. In the brain, motile ciliated cells, known as ependymal cells, line the cerebrospinal fluid (CSF) filled ventricles, where their beating contribute to fluid movement. Ependymal cells have gathered increasing interest since they are associated with hydrocephalus, a neurological condition with ventricular enlargement. In this article, we highlight methods to identify and characterize motile ciliated ependymal lineage in the brain of zebrafish using histological staining and transgenic reporter lines.

Elevated photic response is followed by a rapid decay and depressed state in ictogenic networks.

OBJECTIVE: The switch between nonseizure and seizure states involves profound alterations in network excitability and synchrony. In this study, we aimed to identify and compare features of neural excitability and dynamics across multiple zebrafish seizure and epilepsy models. METHODS: Inspired by video-electroencephalographic recordings in patients, we developed a framework to study spontaneous and photically evoked neural and locomotor activity in zebrafish larvae, by combining high-throughput behavioral tracking and whole-brain in vivo two-photon calcium imaging. RESULTS: Our setup allowed us to dissect behavioral and physiological features that are divergent or convergent across multiple models. We observed that spontaneous locomotor and neural activity exhibit great diversity across models. Nonetheless, during photic stimulation, hyperexcitability and rapid response dynamics were well conserved across multiple models, highlighting the reliability of photically evoked activity for high-throughput assays. Intriguingly, in several models, we observed that the initial elevated photic response is often followed by rapid decay of neural activity and a prominent depressed state. Elevated photic response and following depressed state in seizure-prone networks are significantly reduced by the antiseizure medication valproic acid. Finally, rapid decay and depression of neural activity following photic stimulation temporally overlap with slow recruitment of astroglial calcium signals that are enhanced in seizure-prone networks. SIGNIFICANCE: We argue that fast decay of neural activity and depressed states following photic response are likely due to homeostatic mechanisms triggered by excessive neural activity. An improved understanding of the interplay between elevated and depressed excitability states might suggest tailored epilepsy therapies.

Diversity and function of motile ciliated cell types within ependymal lineages of the zebrafish brain.

Motile cilia defects impair cerebrospinal fluid (CSF) flow and can cause brain and spine disorders. The development of ciliated cells, their impact on CSF flow, and their function in brain and axial morphogenesis are not fully understood. We have characterized motile ciliated cells within the zebrafish brain ventricles. We show that the ventricles undergo restructuring through development, involving a transition from mono- to multiciliated cells (MCCs) driven by gmnc. MCCs co-exist with monociliated cells and generate directional flow patterns. These ciliated cells have different developmental origins and are genetically heterogenous with respect to expression of the Foxj1 family of ciliary master regulators. Finally, we show that cilia loss from the tela choroida and choroid plexus or global perturbation of multiciliation does not affect overall brain or spine morphogenesis but results in enlarged ventricles. Our findings establish that motile ciliated cells are generated by complementary and sequential transcriptional programs to support ventricular development.

Eating and rumination behaviour in Brahman grade cattle and crossbred water buffalo fed on high roughage diet.

The purpose of this study is to compare feeding and other behavior and nutrient digestibility of tropical grade Brahman (body weight (BW) = 231 kg ± 12.4; n = 3) and crossbred water buffalo (BW = 300 kg ± 13.9; n = 3). This experiment on digestibility and measures of muscles of mastication utilized one-way, and animal behavior two-way, analysis of variance, respectively. Two video camcorders were installed in each pair of buffalo and Brahman for 24 h period programmed on the 107th, 109th and 111th days of the digestion trials. Frequency and duration of feeding, meal intake, rumination, bolus, chews, drinking, defecating, standing and lying were recorded daily. Muscle diameter of Digastricus, Masseter and Pterygoid and different regions of the tongue were sampled and measured under light microscope using a standard micrometer. Buffalo obtained significantly higher intake of dry matter, roughage, crude protein, total digestible nutrient and metabolized energy than Brahman. This was supported by longer meal duration (P ≤ 0.05), and shorter meal breaks (P ≤ 0.05) of buffalo than Brahman. The diameter of the muscles for mastication was bigger (P ≤ 0.05) in buffalo than in Brahman, which is indicative of stronger chewing ability. Briefly, lesser and slower chewing action; higher intake of roughage and crude protein; and longer resting behavior of crossbred water buffalo than Brahman are all indicative of better digestive and metabolic performance of the buffalo under high roughage feeding conditions.

Force matching errors following eccentric exercise.

During eccentric exercise contracting muscles are forcibly lengthened, to act as a brake to control motion of the body. A consequence of eccentric exercise is damage to muscle fibres. It has been reported that following the damage there is disturbance to proprioception, in particular, the senses of force and limb position. Force sense was tested in an isometric force-matching task using the elbow flexor muscles of both arms before and after the muscles in one arm had performed 50 eccentric contractions at a strength of 30% of a maximum voluntary contraction (MVC). The exercise led to an immediate reduction of about 40%, in the force generated during an MVC followed by a slow recovery over the next four days, and to the development of delayed onset muscle soreness (DOMS) lasting about the same time. After the exercise, even though participants believed they were making an accurate match, they made large matching errors, in a direction where the exercised arm developed less force than the unexercised arm. This was true whichever arm was used to generate the reference forces, which were in a range of 5-30% of the reference arm's MVC, with visual feedback of the reference arm's force levels provided to the participant. The errors were correlated with the fall in MVC following the exercise, suggesting that participants were not matching force, but the subjective effort needed to generate the force: the same effort producing less force in a muscle weakened by eccentric exercise. The errors were, however, larger than predicted from the measured reduction in MVC, suggesting that factors other than effort might also be contributing. One factor may be DOMS. To test this idea, force matches were done in the presence of pain, induced in unexercised muscles by injection of hypertonic (5%) saline or by the application of noxious heat to the skin over the muscle. Both procedures led to errors in the same direction as those seen after eccentric exercise.

Identifying athletes at risk of hamstring strains and how to protect them.

1. One common soft-tissue injury in sports involving sprinting and kicking a ball is the hamstring strain. Strain injuries often occur while the contracting muscle is lengthened, an eccentric contraction. We have proposed that the microscopic damage to muscle fibres that routinely occurs after a period of unaccustomed eccentric exercise can lead to a more severe strain injury. 2. An indicator of susceptibility for the damage from eccentric exercise is the optimum angle for torque. When this is at a short muscle length, the muscle is more prone to eccentric damage. It is known that subjects most at risk of a hamstring strain have a previous history of hamstring strains. By means of isokinetic dynamometry, we have measured the optimum angle for torque for nine athletes with a history of unilateral hamstring strains. We also measured optimum angles for 18 athletes with no previous history of strain injuries. It was found that mean optimum angle in the previously injured muscles was at a significantly shorter length than for the uninjured muscles of the other leg and for muscles of both legs in the uninjured group. This result suggests that previously injured muscles are more prone to eccentric damage and, therefore, according to our hypothesis, more prone to strain injuries than uninjured muscles. 3. After a period of unaccustomed eccentric exercise, if the exercise is repeated 1 week later, there is much less evidence of damage because the muscle has undergone an adaptation process that protects it against further damage. We propose that for athletes considered at risk of a hamstring strain, as indicated by the optimum angle for torque, a regular programme of mild eccentric exercise should be undertaken. This approach seems to work because evidence from a group of athletes who have implemented such a programme shows a significant reduction in the incidence of hamstring strains.

Effects of local pressure and vibration on muscle pain from eccentric exercise and hypertonic saline.

In human subjects the triceps surae of one leg was exercised eccentrically by asking subjects to walk backwards on an inclined treadmill. Before the exercise controlled local pressure, applied to the muscle with an electromagnet, produced mild soreness, which was reduced when the pressure was combined with vibration. When delayed-onset muscle soreness (DOMS) had set in, 24-48 h after the exercise, vibration increased pain from local pressure. Vibrating at different frequencies suggested 80 Hz as the optimal frequency. During 2-h testing post-exercise, evidence of a change in character of the effects of vibration was first detected at 6 h. It persisted up to 72 h post-exercise. When muscle pain was generated in an unexercised triceps by injection of hypertonic (5%) saline, controlled local pressure applied to the sore area increased pain levels by 32% while pressure plus vibration reduced this to 11%. In a subject with DOMS, local pressure again increased pain from saline by 32% but combining it with vibration increased pain further by an additional 20%. The effect of vibration on DOMS could be abolished with a large nerve fibre block applied to the sciatic nerve. It is concluded that the vibration effects are the result of stimulation of large-diameter mechanoreceptive afferents in the muscle which, it is speculated, play a role in generating DOMS.

Force-matching errors after eccentric exercise attributed to muscle soreness.

1. Experiments were performed to test the ability of human subjects to match forces in their elbow flexor muscles following eccentric exercise of one arm and, in a second series, after biceps brachii of one arm had been made sore by injection of hypertonic saline. 2. In the force-matching task, the elbow flexors of one arm, the reference arm, generated 30% of maximum voluntary contraction (MVC) under visual control. Subjects matched that level with their other arm, the indicator arm, without visual feedback. 3. After eccentric exercise of elbow flexors of the indicator arm, subjects felt they had achieved a satisfactory match while indicating forces that were significantly lower, by approximately 5%, than the reference level. Errors were in the opposite direction (i.e. forces were overestimated) when the reference arm was exercised. 4. Errors were reduced when matching forces were expressed as fractions of the sessional MVC rather than the pre-exercise MVC. Residual errors from 24 h postexercise onwards were attributed to muscle soreness from the exercise. 5. In support of this view, a similar pattern of matching errors was observed when an unexercised arm was made sore by injection of hypertonic saline into the biceps. 6. It is concluded that muscle soreness can interfere with a subject's ability to match forces, perhaps as a result of a reduced excitability of motor cortex. It implies that muscle soreness may contribute to the weakness experienced after a period of unaccustomed eccentric exercise.

Force matching at the elbow joint is disturbed by muscle soreness.

These experiments are concerned with the ability of human subjects to match isometric torque in their elbow flexor muscles when biceps of one arm is made sore. Pain was induced by injection of hypertonic saline. Subjects were asked to generate a level of torque, 30% of maximum, with one arm, the reference arm. To achieve the required torque, subjects were given visual feedback. Subjects were then asked to match this torque with their other arm, the indicator arm. In control measurements, subjects were consistent in their matching ability and often were quite accurate. However, when biceps of one arm was made sore, subjects consistently and significantly underestimated the level of torque being generated by the sore arm. Painful heat applied to the skin over biceps produced a similar pattern of errors. Heating skin remote from elbow flexors had no significant effect. One interpretation of these findings is that the nociceptive input from the sore region of skin or muscle leads to reduced excitability of the motor cortex. That, in turn, disturbs the relationship between the centrally generated effort and motor output, leading to matching errors.

Matching different levels of isometric torque in elbow flexor muscles after eccentric exercise.

Human subjects generated a specified level of isometric torque with elbow flexor muscles of one arm, the reference arm, under visual feedback. They were then asked to generate what they perceived to be the same level, with the other arm, the indicator, but with no visual feedback. A number of torque levels, between 2% and 30% of maximum were used in the matching trials. Elbow flexors of one arm were then exercised eccentrically on a dynamometer. Immediately after the exercise, there was a large (40%) drop in maximum voluntary torque, as well as some soreness and swelling 24 h later, indicative of muscle damage. When the torque-matching experiment was repeated after the indicator arm had been exercised, the indicator signalled torque levels significantly below the reference level (P<0.05). When the reference arm was exercised, errors were in the opposite direction. Over the 4 days of testing post-exercise, errors became less as torque levels returned to normal. When errors were expressed in terms of maximum torque post-exercise, they were significantly reduced. This suggested that subjects were using as a matching cue the perceived effort required to generate a given level of torque rather than the level of torque itself. Persisting matching errors, from 24 h onwards after the eccentric contractions, were proposed to include a component attributable to the muscle soreness. Changes in electromyogram recorded after eccentric exercise were consistent with the effort-matching hypothesis. The muscle's torque-angle relationship was used to estimate matching ability in the absence of fatigue. One forearm was placed at various angles and its reference torque was matched by the other, the indicator, always at 90 degrees. Again, matching errors were consistent with an interpretation based on a match of effort rather than torque.

Ongoing relationships with a personal focus: mothers' perceptions of birth centre versus hospital care.

OBJECTIVE: to describe women's perceptions of care in Western Australian birth centres following a previous hospital birth. DESIGN, SETTING AND PARTICIPANTS: an exploratory study was undertaken to examine the care experiences of women from three Western Australian birth centres. Data were obtained from 17 women whose interviews were audio-recorded and transcribed. The research focused upon women's perceptions of their recent birth centre care as compared to previous hospital care during childbirth. FINDINGS: four key themes emerged from the analysis: 'beliefs about pregnancy and birth', 'nature of the care relationship', 'care interactions' and 'care structures'. The themes of 'care interactions' and 'care structures' will be presented in this paper. Care interactions refer to women's opportunities to develop rapport with their carers. Care structures involved the organisational framework in which care was delivered. The first two themes of 'beliefs about pregnancy and birth' and the 'nature of the care relationship' were discussed in a previous paper. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: differences in opportunities for care interactions and care structures were revealed between birth centre and hospital settings. Ongoing, cumulative contacts with midwives in the birth-centre setting were strongly supported by women as encouraging the development of rapport and perception of 'being known' as an individual. Additionally, care structures tailored to women were advocated over the systematised, fragmented care found in hospital settings.

Normality and collaboration: mothers' perceptions of birth centre versus hospital care.

OBJECTIVE: to describe women's perceptions of care in Western Australian birth centres following a previous hospital birth. DESIGN, SETTING AND PARTICIPANTS: an exploratory design was used to study the care experiences of 17 women recruited from three Western Australian birth centres. Data were obtained from in-depth interviews that explored women's perceptions of their care in both the birth centre and hospital context. FINDINGS: four key themes emerged from the analysis: 'beliefs about pregnancy and birth', 'nature of the care relationship', 'care interactions', and 'care structures'. The themes of 'beliefs about pregnancy and birth' and 'nature of the care relationship' are discussed in this paper. Beliefs about pregnancy and birth refer to the philosophical underpinnings of pregnancy and birth held by women and their carers. Nature of the care relationship identifies women's perceptions of their relationship with health professionals. Care interactions and care structures will be described in a subsequent paper. KEY CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: The women's comments suggested differences in philosophy between hospital and birth-centre settings. The philosophy and beliefs of caregivers was an important component of the care experience. Women valued the normality of the birth-centre approach and the opportunity to experience the birth of their child with collaborative support from a midwife.

Pyrene is metabolized to bound residues by Penicillium janthinellum SFU403.

We have previously shown that the filamentous fungus, Penicillium janthinellum SFU403 (SFU403) oxidizes pyrene to pyrene 1,6- and 1,8-quinones and that the level of pyrenequinones (PQs) subsequently declines suggesting that PQs are not terminal metabolites. The purpose of this study was to determine the fate of PQs in SFU403. First, we compared the fate of 14C-pyrene in SFU403 and a non-pyrene-oxidizing fungus, a Paecilomyces sp.. After 7 days of incubation, more than 80% of the radioactivity was cell-associated in both fungi; however, while 90% of the 14C could be extracted from the Paecilomyces sp. as unmetabolized pyrene, 65-80% of the bound radioactivity remained inextractable from SFU403. Further evidence that pyrene oxidation to PQs was required for irreversible binding was obtained by comparing the extent of 14C bound to SFU403 when it was grown for 21 days under conditions that resulted in differing amounts of 14C-pyrene oxidation. The results showed that approximately 40% of the inextractable products were bound residues derived from pyrene metabolites. The balance (60%) could be attributed to strong sorption of unreacted pyrene. We used electron paramagnetic resonance spectroscopy and oxygen consumption studies to demonstrate that both NADPH and glutathione can reduce PQs by one electron to their corresponding semiquinone anion radicals in vitro. These studies demonstrate that PQs are metabolized by SFU403 to bound residues, possibly via semiquinone intermediates.

Effect of nappy liners on temperature stability in very preterm infants.

OBJECTIVE: To determine whether absorbent liners used in posturally supportive cloth nappies influence temperature stability in infants < 31 weeks gestation. METHODOLOGY: Randomized cross-over trial conducted at King Edward Memorial Hospital for Women, Perth, Western Australia. Twenty-three infants nursed in incubators on Infant Servo Control were randomly assigned to wear cloth postural support nappies alternately with or without absorbent liners for 24-h periods over 4 days. Measurements of skin and incubator temperatures were recorded hourly. Times of all nappy changes and infant handling procedures were also recorded. RESULTS: There was no change in any temperature measurement over time, between days, or between day/night periods. Infants nursed with the liner demonstrated a higher skin temperature (0.04 degrees C), and a lower incubator temperature (1.05 degrees C). A drop in skin temperature of 0.02 degrees C and an increase in incubator temperature of 0.28 degrees C occurred following handling of infants. There was no effect due to sex, gestational age, or actual age of the infants. CONCLUSION: Use of an absorbent liner within a cloth postural support nappy promotes better temperature regulation in infants < 31 weeks gestation, by reducing incubator temperature and increasing skin temperature.

Vaccine know-how. Kimberley immunisation study: community nurses immunisation education, knowledge and practice.

In the remote Kimberley Region of Western Australia, community nurses administer almost all childhood vaccines. This paper discusses the immunisation education, knowledge and practice of this group of nurses. This research was part of a larger Kimberley immunisation study. The first phase investigated the immunisation cover and timing of vaccine administration to children in the 0-18 month age group in the Kimberley Region of Western Australia. Phase two investigated immunisation education, knowledge and practice. The study findings suggest that community nurses are knowledgeable about vaccine administration, and administer vaccines appropriately to children with multiple infections, weight loss and failure to thrive. They are also 'active' in following up children due and overdue for vaccines. However, a lack of on-going nursing immunisation education was reported.