Symbiont genotype influences holobiont response to increased temperature.

As coral reefs face warming oceans and increased coral bleaching, a whitening of the coral due to loss of microalgal endosymbionts, the possibility of evolutionary rescue offers some hope for reef persistence. In tightly linked mutualisms, evolutionary rescue may occur through evolution of the host and/or endosymbionts. Many obligate mutualisms are composed of relatively small, fast-growing symbionts with greater potential to evolve on ecologically relevant time scales than their relatively large, slower growing hosts. Numerous jellyfish species harbor closely related endosymbiont taxa to other cnidarian species such as coral, and are commonly used as a model system for investigating cnidarian mutualisms. We examined the potential for adaptation of the upside-down jellyfish Cassiopea xamachana to increased temperature via evolution of its microalgal endosymbiont, Symbiodinium microadriaticum. We quantified trait variation among five algal genotypes in response to three temperatures (26 °C, 30 °C, and 32 °C) and fitness of hosts infected with each genotype. All genotypes showed positive growth rates at each temperature, but rates of respiration and photosynthesis decreased with increased temperature. Responses varied among genotypes but were unrelated to genetic similarity. The effect of temperature on asexual reproduction and the timing of development in the host also depended on the genotype of the symbiont. Natural selection could favor different algal genotypes at different temperatures, affecting host fitness. This eco-evolutionary interaction may be a critical component of understanding species resilience in increasingly stressful environments.

Dynamic species interactions associated with the range-shifting marine gastropod Mexacanthina lugubris.

Globally, species are undergoing range shifts in response to climate change. However, the potential impacts of climate-driven range shifts are not well understood. In southern California, the predatory whelk Mexacanthina lugubris has undergone a northward range shift of more than 100 km in the past four decades. We traced the history of the whelk's range shift and assessed potential effects using an integrated approach, consisting of field surveys, as well as feeding and thermotolerance experiments. We found that at sites where Mexacanthina and native species co-occurred, native whelks distributions peaked lower in the intertidal. In laboratory experiments, we found that the presence of Mexacanthina led to reduced growth in native whelks (Acanthinucella spirata). Additionally, the range-shifting whelk was able to tolerate higher temperatures than common native species (A. spirata and Nucella emarginata), suggesting further impacts as a result of climate warming. Many species are likely to undergo range shifts as a coping mechanism for changing climatic conditions. However, communities are unlikely to shift as a whole due to species-specific responses. By studying the impacts of range-shifting species, like Mexacanthina, we can better understand how climate change will alter existing community structure and composition.

Thirty-Day Mortality for Proximal Femoral Fractures Treated at a U.K. Elective Center with a Site-Streaming Policy During the COVID-19 Pandemic.

COVID-19 led to the reconfiguration of U.K. orthopaedic trauma services because surgical capacity was threatened in acute centers. We report the 30-day mortality of proximal femoral fractures in older adults treated at an elective orthopaedic center. METHODS: Patients >60 years old who presented with a proximal femoral fracture to any of 4 sites in the regional trauma network were transferred to our elective center for emergency surgery. Care was modeled according to the National Institute for Health and Care Excellence guidelines, and efforts were made to treat all patients within 36 hours. Data were collected prospectively, and mortality outcomes were recorded. RESULTS: Of the 192 patients who presented to the elective orthopaedic center, 167 were treated there. The median age of the latter patients was 88 years (interquartile range, 83 to 79 years). The median Charlson Comorbidity Index was 4 (interquartile range, 4 to 6). The median time from emergency department admission to surgical treatment was 24.5 hours (interquartile range, 18.8 to 34.7 hours). The 30-day rate of mortality was 10.2%. A total of 29 (17.4%) tested positive for COVID-19 during their admission, of whom 10 died, for a case-fatality rate of 34.5%. There were no significant differences in age (p = 0.33) or Charlson Comorbidity Index (p = 0.13) between patients who tested positive and those who did not. There was no significant difference in age between those who tested positive and died and those who tested positive and did not die (p = 0.13), but there was a significant difference in Charlson Comorbidity Index between those subgroups (p = 0.03). CONCLUSIONS: During a pandemic, an elective orthopaedic center can be reconfigured to a surgical center for older patients with proximal femoral fractures with acceptable health-care quality outcomes. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Ancient deep-sea sponge grounds on the Flemish Cap and Grand Bank, northwest Atlantic.

Recent studies on deep-sea sponges have focused on mapping contemporary distributions while little work has been done to map historical distributions; historical distributions can provide valuable information on the time frame over which species have co-evolved and may provide insight into the reasons for their persistence or decline. Members of the sponge family Geodiidae are dominant members of deep-sea sponge assemblages in the northwestern Atlantic. They possess unique spicules called sterrasters, which undergo little transport in sediment and can therefore indicate the Geodiidae sponge historical presence when found in sediment cores. This study focuses on the slopes of Flemish Cap and Grand Bank, important fishing grounds off the coast of Newfoundland, Canada, in international waters. Sediment cores collected in 2009 and 2010 were visually inspected for sponge spicules. Cores containing spicules were sub-sampled and examined under a light microscope for the presence of sterrasters. These cores were also dated using X-radiographs and grouped into five time categories based on known sediment horizons, ranging from 17,000 years BP to the present. Chronological groupings identified Geodiidae sponges in four persistent sponge grounds. The oldest sterrasters were concentrated in the eastern region of the Flemish Cap and on the southeastern slope of the Grand Bank. Opportunistic sampling of a long core in the southeastern region of the Flemish Cap showed the continuous presence of sponge spicules to more than 130 ka BP. Our results indicate that the geodiids underwent a significant range expansion following deglaciation, and support a contemporary distribution that is not shaped by recent fishing activity.

Assignment of Empirical Mode Decomposition Components and Its Application to Biomedical Signals.

OBJECTIVES: Empirical mode decomposition (EMD) is a frequently used signal processing approach which adaptively decomposes a signal into a set of narrow-band components known as intrinsic mode functions (IMFs). For multi-trial, multivariate (multiple simultaneous recordings), and multi-subject analyses the number and signal properties of the IMFs can deviate from each other between trials, channels and subjects. A further processing of IMFs, e.g. a simple ensemble averaging, should determine which IMFs of one signal correspond to IMFs from another signal. When the signal properties have similar characteristics, the IMFs are assigned to each other. This problem is known as correspondence problem. METHODS: From the mathematical point of view, in some cases the correspondence problem can be transformed into an assignment problem which can be solved e.g. by the Kuhn-Munkres algorithm (KMA) by which a minimal cost matching can be found. We use the KMA for solving classic assignment problems, i.e. the pairwise correspondence between two sets of IMFs of equal cardinalities, and for pairwise correspondences between two sets of IMFs with different cardinalities representing an unbalanced assignment problem which is a special case of the k-cardinality assignment problem. RESULTS: A KMA-based approach to solve the correspondence problem was tested by using simulated, heart rate variability (HRV), and EEG data. The KMA-based results of HRV decomposition are compared with those obtained from a hierarchical cluster analysis (state-of-the-art). The major difference between the two approaches is that there is a more consistent assignment pattern using KMA. Integrating KMA into complex analysis concepts enables a comprehensive exploitation of the key advantages of the EMD. This can be demonstrated by non-linear analysis of HRV-related IMFs and by an EMD-based cross-frequency coupling analysis of the EEG data. CONCLUSIONS: The successful application to HRV and EEG analysis demonstrates that our solutions can be used for automated EMD-based processing concepts for biomedical signals.

The challenge for innovation in direct restorative materials.

During the past 50 years, a series of key UN conferences have established a framework to minimize human health risks from environmental exposures to key chemicals. In January 2013, more than 140 countries agreed to the text of new treaty to minimize Hg effects on the environment (the Minamata Convention). Dental caries is omnipresent around the globe, affecting 60% to 90% of school children and most adults, and producing discomfort that affects quality of life. Dental amalgam is frequently used to treat carious lesions and its use releases mercury into the environment. The best way to avoid the use of dental amalgam is to emphasize caries prevention. Alternatives to amalgam are suitable in some applications, but no replacement for amalgam has been found for large posterior restorations. For any restorative material, safety and environmental impacts are part of clinical risk assessment. Safety is freedom from unacceptable risks. Risk is a combination of probability of exposure and severity of harm. Best management practices are crucial to manage dental amalgam, but these impose additional that are disproportionately more for developing countries. The Minamata Convention seeks a phase-out of all mercury-based products except dental amalgam, where a phase-down is the present goal. For dentistry, the most important focus is the promotion of caries prevention and research on new materials.

Geochemistry of bed and suspended sediment in the Mississippi river system: provenance versus weathering and winnowing.

Stream-bed sediment for the size fraction less than 150 microm, examined in 14,000 samples collected mostly from minor tributaries to the major rivers throughout the Mississippi River drainage system, is composed of 5 mineral fractions identified by factor analysis-Al-silicate minerals, quartz, calcite and dolomite, heavy minerals, and an Fe-Mn fraction. The Al-silicate fraction parallels its distribution in the regolith, emphasizing the local sediment source as a primary control to its distribution. Quartz and the heavy-mineral fraction, and associated trace elements, exhibit a complementary distribution to that of the Al-silicate fraction, with a level of enrichment in the bed sediment that is achieved through winnowing and sorting. The carbonate fraction has a distribution suggesting its dissolution during transport. Trace elements partitioned onto the Fe-Mn, possibly amorphous oxyhydride, fraction are introduced to the streams, in part, through human activity. Except for the heavy-mineral fraction, these fractions are identified in suspended sediment from the Mississippi River itself. Although comparison of the tributary bed sediment with the riverine suspended sediment is problematic, the geochemistry of the suspended sediment seems to corroborate the interpretation of the geochemistry of the bed sediment.

Evidence implicating a role for orexin-1 receptor modulation of paradoxical sleep in the rat.

The neuropeptide orexin-A modulates the sleep-wake cycle such that central administration to rats increases arousal, reduces slow-wave-sleep (SWS) and paradoxical sleep (PS) and delays PS onset. The contribution of orexin-1 and -2 receptor (OXR) activation to this orexin-A response is still unknown. Using the OX(1)R antagonist SB-334867-A we investigated the role of this receptor in orexin-A-induced PS alteration. Male rats prepared for frontal-occipital electroencephalograph, nuchal muscle electromyograph recording and lateral ventricle cannulae received vehicle or orexin-A (10 microg icv) at lights on in combination with vehicle or SB-334867-A (10 or 30 mg/kg ip) 30 min pre-icv injection. The amount of arousal, SWS 1, SWS 2 and PS was determined during the 1st h post icv administration along with the latency to onset of the first> or =10 s epoch of PS. Orexin-A administration reduced the amount and increased the latency to onset of PS. SB-334867-A reversed this effect of orexin-A. The present study demonstrates that the OX(1)R also has a role in orexinergic sleep modulation.

Effect of SB-243213, a selective 5-HT(2C) receptor antagonist, on the rat sleep profile: a comparison to paroxetine.

5-HT(2) receptor antagonists promote slow wave sleep (SWS) in humans and rats, conversely 5-HT(2) agonists inhibit SWS in rats. These alterations are thought to be predominantly mediated via the 5-HT(2C) receptor subtype. It is evident that 5-HT(2) receptor function also plays an important role in depression. Here, we examine the acute effect of the selective 5-HT(2C) receptor antagonist 5-methyl-1-[[-2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-triflouromethylindoline hydrochloride (SB-243213-A) on rat sleep in comparison to the selective serotonin reuptake inhibitor (SSRI) paroxetine. Both SB-243213-A (10 mg/kg po) and paroxetine (3 mg/kg po) significantly increased deep SWS (SWS2) quantity (27% and 24%, respectively) and reduced paradoxical sleep (PS) quantity (35%) during the sleep period. Following SB-243213-A, SWS2 occurrence frequency was reduced (24.1%); however, elevated quantity of SWS2 can be attributed to an increase in occurrence duration (81%). Reduced PS quantity results from a decrease in occurrence frequency (46%). In comparison, paroxetine increased SWS2 occurrence frequency (50%), with decreased frequency (27%) and duration (21%) of PS. The data for SB-243213-A in the present study is consistent with that following ritanserin supporting 5-HT(2C) receptor subtype mediation of this response. The similar effect of SB-243213-A to paroxetine with regard to PS quantity provides further evidence that 5-HT(2C) receptor antagonists maybe beneficial in the treatment of depression/anxiety.

Identification and characterization of neuronal precursors and their progeny from human fetal tissue.

We have examined primary human neuronal precursors (HNPs) from 18-22-week-old fetuses. We showed that E-NCAM/MAP2/beta-III tubulin-immunoreactive neuronal precursors divide in vitro and could be induced to differentiate into mature neurons in 2 weeks. HNPs did not express nestin and differentiated slowly compared to rodent neuronal restricted precursors (NRPs, 5 days). Immunocytochemical and physiological analyses showed that HNPs could generate a heterogeneous population of neurons that expressed neurofilament-associated protein and various neurotransmitters, neurotransmitter synthesizing enzymes, voltage-gated ion channels, and ligand-gated neurotransmitter receptors and could fire action potentials. Undifferentiated and differentiated HNPs did not coexpress glial markers. Only a subset of cells that expressed GFP under the control of the Talpha1 tubulin promoter was E-NCAM/beta-III tubulin-immunoreactive, indicating nonexclusive overlap between these two HNP cell populations. Overall, HNPs resemble NRPs isolated from rodent tissue and appear to be a neuronal precursor population.

Clozapine enhances breakpoint in common marmosets responding on a progressive ratio schedule.

RATIONALE: Motivational effects of psychotropic drugs may contribute to their therapeutic profile and progressive ratio (PR) schedules provide a method of measuring these effects in animals. OBJECTIVE: Determine effects of selected antipsychotic, psychotomimetic, anxiolytic and antidepressant drugs on PR performance in common marmosets. METHOD: Marmosets were trained to lever press for banana milkshake reinforcement using a PR schedule, in which the number of lever presses to achieve successive reinforcements increased by one until responding ceased (breakpoint). RESULTS: Clozapine administered intramuscularly (0.01-2 mg/kg IM; 30 min pretreatment time (ptt) or by oral gavage (0.1-4 mg/kg PO; 60 min ptt) significantly increased the breakpoint. Independent tests of fluid consumption failed to show enhanced fluid intake after clozapine pretreatment, suggesting this effect was not due to drug induced polydipsia. Neither haloperidol (0.005-0.1 mg/kg PO; 60 min ptt) nor risperidone (0.0025-0.05 mg/kg PO; 60 min ptt) altered breakpoint. Olanzapine (0.01-1 mg/kg PO; 60 min ptt) significantly enhanced the breakpoint at 0.05 mg/kg PO, but the effect was not robust. Amphetamine (0.2-2 mg/kg SC; 30 min ptt) significantly reduced the breakpoint at 2 mg/kg and fluoxetine (0.1-1 mg/kg PO; 60 min ptt) was without effect. Diazepam significantly increased the breakpoint at 0.5 mg/kg PO. Drug-induced polydipsia might play a role in this response as independent tests showed increased fluid consumption following diazepam. CONCLUSIONS: These results demonstrate that, unlike other antipsychotics, clozapine over a wide dose range increased the motivational state of marmosets to respond for banana milkshake. This motivational aspect of clozapine's actions may contribute to its unique clinical profile and the PR procedure may provide a method for detecting novel antipsychotics with a clozapine-like profile.

Implicit learning and implicit memory for odors: the influence of odor identification and retention time.

One hundred and fifty-two subjects, divided into eight groups, were exposed to a room with a low concentration of either orange or lavender and to an odorless room. In a careful double-blind procedure, neither the subjects nor the experimenters were made aware of the presence of the odors in the experimental conditions. Later they were asked to indicate how well each of 12 odor stimuli, including the experimental and control odors, befitted each of 12 visual contexts, including the exposure rooms. At the end of this session they rated the pleasantness and the familiarity of the odors, and identified them by name. Finally they were debriefed and asked specifically whether they had perceived the experimental odors anywhere in the building. The results of four subjects who answered positively to the latter question were omitted. The results confirm the earlier finding that non-identifiers implicitly link odor and exposure room, whereas identifiers do not show such a link. It is suggested that episodic information is an essential constituent of olfactory memory and that its function is comparable to that of form and structure in visual and auditory memory systems.

SB-334867, a selective orexin-1 receptor antagonist, enhances behavioural satiety and blocks the hyperphagic effect of orexin-A in rats.

Intracerebroventricular (i.c.v.) administration of the novel hypothalamic neuropeptide orexin-A stimulates food intake in rats, and delays the onset of behavioural satiety (i.e. the natural transition from feeding to resting). Furthermore, preliminary findings with the selective orexin-1 receptor antagonist, SB-334867, suggest that orexin-A regulation of food intake is mediated via the orexin-1 receptor. At present, however, little is known about either the intrinsic effects of SB-334867 on the normal structure of feeding behaviour, or its effects upon orexin-A-induced behavioural change. In the present study, we have employed a continuous monitoring technique to characterize the effects of SB-334867 (3-30 mg/kg, i.p.) on the microstructure of rat behaviour during a 1-h test with palatable wet mash. Administered alone, SB-334867 (30 mg/kg, but not lower doses) significantly reduced food intake and most active behaviours (eating, grooming, sniffing, locomotion and rearing), while increasing resting. Although suggestive of a behaviourally nonselective (i.e. sedative) action, the structure of feeding behaviour was well-preserved at this dose level, with the reduction in behavioural output clearly attributable to an earlier onset of behavioural satiety. As previously reported, orexin-A (10 microg per rat i.c.v.) stimulated food intake, increased grooming and delayed the onset of behavioural satiety. Pretreatment with SB-334867 dose-dependently blocked these effects of orexin-A, with significant antagonism evident at dose levels (3-10 mg/kg) below those required to produce intrinsic behavioural effects under present test conditions. Together, these findings strongly support the view that orexin-A is involved in the regulation of feeding patterns and that this influence is mediated through the orexin-1 receptor.

Dose-response effects of orexin-A on food intake and the behavioural satiety sequence in rats.

Although intracerebroventricular (i.c.v.) administration of orexin-A has been reported to stimulate food intake and/or feeding behaviour in rats, mice and goldfish, little attention has thus far been paid to its effects on normal patterns of feeding. In the present study, a continuous monitoring technique was used to characterise the effects of this novel neuropeptide on the microstructure of rat behaviour during a 1-h test with palatable wet mash. Particular attention was devoted to the behavioural satiety sequence, in which feeding is followed by grooming and resting. Although results confirmed the hyperphagic effects of orexin-A (3.33-30.0 microg i.c. v.), gross behavioural analysis failed to reveal any reliable effects of peptide treatment on eating, drinking, sniffing, grooming, resting, locomotion or rearing. However, microstructural analysis revealed behavioural effects of orexin-A that are both dose- and time-dependent. At lower doses (3.33-10.0 microg), orexin-A primarily delayed behavioural satiety, i.e. the normal transition from eating to resting. In contrast, the 30 microg dose initially induced a sedative-like effect, significantly suppressing eating and other active behaviours for the first 15-20 min of the test period. This sedative-like effect resulted in a phase-shifting of the entire behavioural sequence with higher than control levels of eating, grooming, locomotion, rearing and sniffing observed over the second half of the test session. Present findings illustrate the advantages of microstructural behavioural analysis and suggest that the hyperphagic response to low doses of orexin-A results largely from a delay in behavioural satiety while that seen in response to high doses may occur in rebound to initial behavioural suppression. Further studies will be required to confirm the identity of the specific orexin receptors (i.e. OX(1) or OX(2)) involved in mediating the dose-dependent behavioural effects reported.

Immunocytochemical and physiological characterization of a population of cultured human neural precursors.

Human neural precursor cells (HNPC) have recently become commercially available. In an effort to determine the usefulness of these cells for in vitro studies, we have grown cultured HNPCs (cHNPCs) according to the supplier specifications. Here we report our characterization of cHNPCs under nondifferentiating and differentiating growth conditions and make a comparison to primary HNPCs (pHNPCs) obtained at the same developmental time point from a different commercial supplier. We found that under nondifferentiating conditions, cHNPCs expressed nestin, divided rapidly, expressed few markers of differentiated cells, and displayed both 4-aminopyridine (4-AP)-sensitive and delayed-rectifier type K(+) currents. No inward currents were observed. On changing to differentiating culture conditions, a majority of the cells expressed neuronal markers, did not divide, expressed inward and outward time- and voltage-dependent currents, and responded to the application of the neurotransmitters acetylcholine and glutamate. The outward current densities were indistinguishable from those in undifferentiated cells. The inward currents included TTX-sensitive and -resistant Na(+) currents, sustained Ca(2+) currents, and an inwardly rectifying K(+) current. Comparison of the properties of differentiated cells from cHNPCs with neurons obtained from primary fetal cultures (pHNPCs) revealed two major differences: the differentiated cHNPCs did not express embryonic neural cell adhesion molecule (E-NCAM) immunoreactivity but did co-express GFAP immunoreactivity. The co-expression of neuronal and glial markers was likely due to the growth of cells in serum containing medium as the pHNPCs that were never exposed to serum did express E-NCAM and did not co-express glial fibrillary acidic protein (GFAP). The relevance of these results is discussed and compared with results from other neuronal progenitor populations and cultured human neuronal cells.

Characterization of SB-269970-A, a selective 5-HT(7) receptor antagonist.

The novel 5-HT(7) receptor antagonist, SB-269970-A, potently displaced [(3)H]-5-CT from human 5-HT(7(a)) (pK(i) 8.9+/-0.1) and 5-HT(7) receptors in guinea-pig cortex (pK(i) 8.3+/-0.2). 5-CT stimulated adenylyl cyclase activity in 5-HT(7(a))/HEK293 membranes (pEC(50) 7.5+/-0.1) and SB-269970-A (0.03 - 1 microM) inhibited the 5-CT concentration-response with no significant alteration in the maximal response. The pA(2) (8.5+/-0.2) for SB-269970-A agreed well with the pK(i) determined from [(3)H]-5-CT binding studies. 5-CT-stimulated adenylyl cyclase activity in guinea-pig hippocampal membranes (pEC(50) of 8.4+/-0.2) was inhibited by SB-269970-A (0.3 microM) with a pK(B) (8.3+/-0.1) in good agreement with its antagonist potency at the human cloned 5-HT(7(a)) receptor and its binding affinity at guinea-pig cortical membranes. 5-HT(7) receptor mRNA was highly expressed in human hypothalamus, amygdala, thalamus, hippocampus and testis. SB-269970-A was CNS penetrant (steady-state brain : blood ratio of ca. 0.83 : 1 in rats) but was rapidly cleared from the blood (CLb=ca. 140 ml min(-1) kg(-1)). Following a single dose (3 mg kg(-1)) SB-269970 was detectable in rat brain at 30 (87 nM) and 60 min (58 nM). In guinea-pigs, brain levels averaged 31 and 51 nM respectively at 30 and 60 min after dosing, although the compound was undetectable in one of the three animals tested. 5-CT (0.3 mg kg(-1) i.p.) induced hypothermia in guinea-pigs was blocked by SB-269970-A (ED(50) 2.96 mg kg(-1) i.p.) and the non-selective 5-HT(7) receptor antagonist metergoline (0.3 - 3 mg kg(-1) s.c.), suggesting a role for 5-HT(7) receptor stimulation in 5-CT induced hypothermia in guinea-pigs. SB-269970-A (30 mg kg(-1)) administered at the start of the sleep period, significantly reduced time spent in Paradoxical Sleep (PS) during the first 3 h of EEG recording in conscious rats.

The novel brain neuropeptide, orexin-A, modulates the sleep-wake cycle of rats.

Orexin-A is a novel neuropeptide initially isolated from hypothalamic extracts but now known to be present in fibres distributed throughout the rat CNS including many regions associated with sleep-wake regulation. The recognition of a particularly dense innervation of orexinergic nerves in the locus coeruleus, together with the observed increase in firing rate of locus coeruleus neurons following application of orexin-A in vitro, further highlighted a potential involvement of the peptide in modulating the arousal state. The present study was undertaken to determine the effects of intracerebroventricularly (ICV) administered orexin-A on the sleep-wake cycle of conscious rats using electroencephalographic and electromyographic recordings. When administered at the onset of the normal sleep period, orexin-A (1, 10 or 30 microg/rat ICV) produced a dose-dependent increase in the time rats spent awake during the second and third hours after dosing. The enhancement of arousal was accompanied by a marked reduction in paradoxical sleep and deep slow wave sleep at the highest dose. The latency to the first occurrence of paradoxical sleep was also prolonged. This overall profile of increased arousal and decreased paradoxical sleep is consistent with a high rate of firing of locus coeruleus neurons as would be expected to occur following ICV administration of orexin-A. It is concluded that orexin-A may play an important physiological role in regulating the sleep-wake state, a hypothesis considerably strengthened by the recently reported narcoleptic phenotype of prepro-orexin (the precursor for orexin-A) knockout mice.

Calcium signalling in squid olfactory receptor neurons.

Isolated squid olfactory receptor neurons respond to dopamine and betaine with hyperpolarizing conductances. We used Ca(2+) imaging techniques to determine if changes in intracellular Ca(2+) were involved in transducing the hyperpolarizing odor responses. We found that dopamine activated release of Ca(2+) from intracellular stores while betaine did not change internal Ca(2+) concentrations. Application of 10 mM caffeine also released Ca(2+) from intracellular stores, suggesting the presence of ryanodine-like receptors. Depletion of intracellular stores with 100 microM thapsigargin revealed the presence of a Ca(2+) store depletion-activated Ca(2+) influx. The influx of Ca(2+) through the store-operated channel was reversibly blocked by 10 mM Cd(2+). Taken together, these data suggest a novel odor transduction system in squid olfactory receptor neurons involving Ca(2+) release from intracellular stores.

Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells.

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted, self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani, K. Hobson, and M. S. Rao, 1997, Dev. Biol. 186, 202-223; M. S. Rao and M. Mayer-Proschel, 1997, Dev. Biol. 188, 48-63; M. Mayer-Proschel, A. J. Kalyani, T. Mujtaba, and M. S. Rao, 1997, Neuron 19, 773-785). We now show that cells identical to rat NEPs, NRPs, and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs, respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive, undergo self-renewal in defined medium, and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus, lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells.

Orexin A activates locus coeruleus cell firing and increases arousal in the rat.

The localization of orexin neuropeptides in the lateral hypothalamus has focused interest on their role in ingestion. The orexigenic neurones in the lateral hypothalamus, however, project widely in the brain, and thus the physiological role of orexins is likely to be complex. Here we describe an investigation of the action of orexin A in modulating the arousal state of rats by using a combination of tissue localization and electrophysiological and behavioral techniques. We show that the brain region receiving the densest innervation from orexinergic nerves is the locus coeruleus, a key modulator of attentional state, where application of orexin A increases cell firing of intrinsic noradrenergic neurones. Orexin A increases arousal and locomotor activity and modulates neuroendocrine function. The data suggest that orexin A plays an important role in orchestrating the sleep-wake cycle.