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Copper (Cu) is a naturally occurring micronutrient of eco-toxicological concern in aquatic ecosystems. Current knowledge of Cu-speciation and bioavailability in natural saline environments is insufficient to adequately inform environmental protection policy for estuarine systems. We assessed the combined effect of two of the main drivers of metal bioavailability, salinity and natural dissolved organic carbon (DOC), on Cu-speciation and associated Cu-toxicity to blue mussel (Mytilus galloprovincialis) embryos in a standard 48-h bioassay. We placed special emphasis on measurement of Cu-speciation rather than modelling. Cu-toxicity was found to be a function of DOC and salinity. The varying protective effect of different DOC-types suggests that estuarine DOC is more protective against Cu-toxicity than oceanic DOC. Salinity was negatively correlated with [Cu48-h-EC50], indicating a salinity-induced alteration in the physiology of the exposed mussel embryos and/or Cu-DOC-reactivity. These two assumptions were supported by (1) the relative uniformity of bioavailable copper ([Cu']) across similar salinity treatments despite considerable variation in [Cu48-h-EC50] and DOC-concentrations, and (2) the fact that Cu-toxicity and [Cu'] were slightly higher in the 35 salinity treatment compared to the 25 salinity treatment. Stripping voltammetry studies determined the presence of only one strong Cu-binding ligand class (i.e., L1), either actively or passively released by the exposed embryos. [L1] was found to be proportional to the total dissolved Cu-concentration ([CuT]), suggesting a protective effect of Cu-binding-ligands, in addition to the protective effect of DOC. There was also a strong positive correlation between [L1] and [Cu48-h-EC50], implying that electrochemically defined ligand concentrations along with measurements of [Cu'], DOC-quality, and salinity can be used as proxies for 48-h-EC50 Cu-values in estuarine waters, which may result in a significant improvement to risk assessments of Cu in estuarine systems.

Alzheimer's disease (AD) is a neurodegenerative disorder which accounts for 60-70% of the 50 million worldwide cases of dementia and is characterised by cognitive impairments, many of which have long been associated with dysfunction of the cholinergic system. Although the M muscarinic acetylcholine receptor (mAChR) is considered a promising drug target for AD, ligands targeting this receptor have so far been unsuccessful in clinical trials. As modulatory receptors to cholinergic transmission, the endocannabinoid system may be a promising drug target to allow fine tuning of the cholinergic system. Furthermore, disease-related changes have been found in the endocannabinoid system during AD progression and indeed targeting the endocannabinoid system at specific disease stages alleviates cognitive symptoms in numerous mouse models of AD. Here we review the role of the endocannabinoid system in AD, and its crosstalk with mAChRs as a potential drug target for cholinergic dysfunction. [Abstract copyright: Copyright © 2019. Published by Elsevier Inc.]

Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-N-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di in vivo can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for in vivo studies where metabolic conversion of CNO to clozapine is a concern.

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer’s disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that “pure” positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.

Grasshoppers produce an extraordinary oviposition behavior that is associated with multiple specializations of the skeletal and neuromuscular systems in the posterior abdomen, including a central pattern generator (CPG) in the female’s terminal abdominal ganglion. Two pairs of shovel-shaped appendages, the ovipositor valves on the abdomen tip, excavate the soil for deposition of eggs. By contrast, the sexually monomorphic pregenital region of the abdomen is without appendages. Morphological homologues of ovipositor muscles and efferent neurons in the eighth abdominal segment are nevertheless present in pregenital segments of males and females. In both sexes, a robust rhythmic motor program was induced in pregenital segments by the same experimental methods used to elicit oviposition digging. The activity, recorded extracellularly, was oviposition-like in burst period (5–6 s) and homologous muscle phase relationships, and it persisted after sensory inputs were removed, indicating the presence of pregenital CPGs. The abdomen exhibited posterior-going waves of activity with an intersegmental phase delay of approximately 1 s. These results indicate that serially homologous motor systems, including functional CPGs, provided the foundation for the evolution of oviposition behavior.

The acute toxicity of carbosulfan and chlorpyrifos in formulated pesticides to glochidia (larvae) of the freshwater mussel (Hyriopsis bialata Simpson, 1900) was evaluated under static conditions in moderately hard dechlorinated tap water. Measured pesticide concentrations were 26 to 34% lower than nominal concentrations; therefore, all results are expressed in terms of measured active ingredient. Carbosulfan was relatively non-toxic to the mussel larvae with median effective concentrations (EC50) of carbosulfan at 24 and 48 h greater than 0.10 mg/L. The EC50s of chlorpyrifos at 24 and 48 h were 0.083 and 0.078 mg/L, respectively (measured concentrations). The 48-h EC50 of a combined exposure to a mixture of chlorpyrifos and carbosulfan at a constant ratio of 2.9:1 was 0.0142:0.049 mg CP:CB/L. In a separate experiment, the effect of water hardness on carbosulfan, chlorpyrifos, or a combined exposure was assessed using glochidia exposed to either soft, moderately hard, or hard reconstituted water. There was no effect of water hardness on the survival of glochidia after 24- or 48-h exposure to carbosulfan. The chlorpyrifos 48-h EC50s in soft water, moderately hard water, and hard water were 0.18, 0.11, and 0.16 mg/L, respectively. The data indicate that the lowest water hardness resulted in the highest survival of glochidia, whereas an increase to moderate water hardness resulted in significantly decreased survival of glochidia (F = 15.5, P

Alzheimer's disease (AD) is a neurodegenerative disorder which accounts for 60–70% of the 50 million worldwide cases of dementia and is characterised by cognitive impairments, many of which have long been associated with dysfunction of the cholinergic system. Although the M1 muscarinic acetylcholine receptor (mAChR) is considered a promising drug target for AD, ligands targeting this receptor have so far been unsuccessful in clinical trials. As modulatory receptors to cholinergic transmission, the endocannabinoid system may be a promising drug target to allow fine tuning of the cholinergic system. Furthermore, disease-related changes have been found in the endocannabinoid system during AD progression and indeed targeting the endocannabinoid system at specific disease stages alleviates cognitive symptoms in numerous mouse models of AD. Here we review the role of the endocannabinoid system in AD, and its crosstalk with mAChRs as a potential drug target for cholinergic dysfunction.

Baroreceptors are a promising new drug target for treating high blood pressure (hypertension). Hypertension is the world’s commonest killer disease because it is the biggest underlying risk factor for cardiovascular disease, stroke and kidney failure (WHO). These account for over 7 m deaths per year. The WHO regards current treatments as inadequate. Even in the developed world most patients need multiple drugs, both for the initial condition and combatting the side-effects the primary medication produces. Even with multi-drug therapies,>10% of patients are unresponsive. No more effective drugs have been introduced for over 20 years. We hypothesise this is because of the focus on old targets. Therefore, the WHO has asked urgently for new therapeutic targets. Baroreceptors are emerging as such a novel, and highly effective target. Electrical stimulation of baroreceptor nerves reduces long-term blood pressure by >20 mmHg even in the most drug-resistant patients. In some cases, it can even replace medication. However, only a small number of patients get this treatment, as it is highly invasive, expensive and requires rare surgical expertise. Thus, only resistant hypertensives are treated. Even then, this is a high-risk patient group, and only 50% are deemed suitable due to the anatomical condition of the stimulation site, and the difficulties of post-operative stimulation optimisation, gagging reflex activation, equipment maintenance, etc. We propose a pharmaceutical targeting of baroreceptors could be just as effective, and would be suitable for all patients, plus avoid the side-effects associated with current medications. We have discovered a modulatory system controlling mechanosensory terminal sensitivity that involves an unusual glutamate receptor with highly atypical pharmacology. We are therefore pharmacologically characterising the receptor in the aorta-aortic depressor nerve (ADN) system, as a translational step towards the clinic. Isolated aorta-ADN or working-heart brainstem (WHBS) preparations were humanely prepared (ASPA, 1986) from adult Sprague-Dawley rats. Baroreceptor immunolabelling for the atypical glutamate receptor (PLD-GluR) showed it to be present. In physiological experiments, pressure pulses were delivered periodically to the isolated or WHBS aorta. The effects of glutamate receptor or transducer channel ligands on ADN firing was recorded, as well as effects on sympathetic output and heart rate. PLD-GluR agonists robustly increased baroreceptor firing. Concanavalin A increased kainate-mediated enhancement by blocking receptor desensitisation. Blocking TRP or DEG/ENaC channels with 2-APB or amiloride inhibited pressure-evoked firing. Glutamatergic stimulation of baroreceptors also inhibited sympathetic output, indicating decreased sympathetic tone. Selective PLD-GluR antagonist PCCG-13 inhibited pressure-evoked firing and changes in sympathetic tone. These data show baroreceptor firing can be pharmacologically controlled. And, this influences baroreflex, inhibiting sympathetic output. Thus, glutamate receptor activation on baroreceptors offer a novel target, increasing baroreceptor stretch-sensitivity and the baroreflex response which may have important clinical implications. Further experiments are planned to fully characterise this response and the GluR involved.

The production of accurate and reliable data on metal toxicity during ecotoxicological bioassays is important for credible environmental risk assessments and management in aquatic environments. Actual measurements and reporting of contaminant concentrations in bioassays are, however, often disregarded; and potential contaminant loss attributable to adsorption processes (e.g., wall adsorption) in bioassays is widely omitted, which can have detrimental effects on calculated metal toxicity thresholds. In the present we assessed copper (Cu) mass balance during a standard 48-h bioassay test with blue mussel (Mytilus galloprovincialis) embryos to evaluate effects on calculated toxicity endpoints. We demonstrated that measured Cu concentrations at the test conclusion need to be used to quantify the risk of Cu toxicity because nominal Cu and initial Cu concentrations underestimate overall Cu toxicity by up to 1.5-fold, owing to Cu loss in solution attributable to adsorption and bioaccumulation processes. For the first time we provide evidence that extracellular adsorption to the biological surface of the embryos is the most important sink for total dissolved Cu in a bioassay. We also established that adsorbed extracellular Cu accumulation reduces Cu toxicity to embryos, potentially by inhibiting Cu from entering the cell of the mussel embryo. Environmental factors (e.g., salinity and dissolved organic carbon) did not influence the partitioning of Cu within the laboratory-based bioassay. The present results 1) demonstrate the importance of differentiating extra- and intracellular Cu pools to improve our understanding of Cu toxicity and associated processes, 2) reveal the potential for bias with respect to calculated Cu toxicity thresholds when results are based on nominal and initial Cu concentrations, and 3) point out the need to follow current guidelines for the testing of chemicals to standardize toxicity tests and data reporting. Environ Toxicol Chem 2019;38:561-574. © 2019 SETAC.

Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine-N-oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5H-dibenzo[b,e][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di in vivo can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for in vivo studies where metabolic conversion of CNO to clozapine is a concern.

Copper (Cu) is a naturally occurring micronutrient of eco-toxicological concern in aquatic ecosystems. Current knowledge of Cu-speciation and bioavailability in natural saline environments is insufficient to adequately inform environmental protection policy for estuarine systems. We assessed the combined effect of two of the main drivers of metal bioavailability, salinity and natural dissolved organic carbon (DOC), on Cu-speciation and associated Cu-toxicity to blue mussel (Mytilus galloprovincialis) embryos in a standard 48-h bioassay. We placed special emphasis on measurement of Cu-speciation rather than modelling. Cu-toxicity was found to be a function of DOC and salinity. The varying protective effect of different DOC-types suggests that estuarine DOC is more protective against Cu-toxicity than oceanic DOC. Salinity was negatively correlated with [Cu

The realization of the therapeutic potential of targeting the M1 muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer’s disease has prompted the discovery of M1 mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1H-benzimidazol-2-one), described previously as a potent M1 receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M1 mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that “pure” positive allosteric modulators showing selectivity for the M1 mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.

Grasshoppers produce an extraordinary oviposition behavior that is associated with multiple specializations of the skeletal and neuromuscular systems in the posterior abdomen, including a central pattern generator (CPG) in the female's terminal abdominal ganglion. Two pairs of shovel-shaped appendages, the ovipositor valves on the abdomen tip, excavate the soil for deposition of eggs. By contrast, the sexually monomorphic pregenital region of the abdomen is without appendages. Morphological homologues of ovipositor muscles and efferent neurons in the eighth abdominal segment are nevertheless present in pregenital segments of males and females. In both sexes, a robust rhythmic motor program was induced in pregenital segments by the same experimental methods used to elicit oviposition digging. The activity, recorded extracellularly, was oviposition-like in burst period (5-6 s) and homologous muscle phase relationships, and it persisted after sensory inputs were removed, indicating the presence of pregenital CPGs. The abdomen exhibited posterior-going waves of activity with an intersegmental phase delay of approximately 1 s. These results indicate that serially homologous motor systems, including functional CPGs, provided the foundation for the evolution of oviposition behavior.

The phosphorus (P)-inactivation agents alum or modified zeolite (Aqual-P™) are used in eutrophic lake remediation. Lake managers must evaluate the benefits of P-removal against potential adverse effects on lake biota. Laboratory mesocosms were used to determine whether a 2 month exposure to alum or Aqual-P had lethal or sublethal effects on native benthic-dwelling macroinvertebrates or fish. The P-inactivation agents were applied while the organisms were present to evaluate both acute- and longer-term effects. A gradient of doses up to 344 g alum m−2 (>7 mm capping layer thickness) and a single 200 g Aqual-P m−2 dose were applied with no detectable acute effects on survival or behaviour. After 2 months, there was no significant effect of alum or Aqual-P on the survival or growth of the crayfish, mussels or fish, but aluminium accumulation was measurable in some treatments. Fingernail clams were held in a sub-mesocosm to prevent predation, which resulted in exposure to intact capping layers. The highest alum dose significantly decreased fingernail clam survival and reburial rates, while 200 g Aqual-P m−2 caused highly variable survival. Our findings can be used by lake managers to assist the selection of site-specific application rates for these P-inactivation agents.

In female grasshoppers, oviposition is a highly specialized behavior involving a rhythm-generating neural circuit, the oviposition central pattern generator, unusual abdominal appendages, and dedicated muscles. This study of Schistocerca americana (Drury) grasshoppers was undertaken to determine whether the simpler pregenital abdominal segments, which do not contain ovipositor appendages, share common features with the genital segment, suggesting a roadmap for the genesis of oviposition behavior. Our study revealed that although 5 of the standard pregenital body wall muscles were missing in the female genital segment, homologous lateral nerves were, indeed, present and served 4 ovipositor muscles. Retrograde labeling of the corresponding pregenital nerve branches in male and female grasshoppers revealed motor neurons, dorsal unpaired median neurons, and common inhibitor neurons which appear to be structural homologues of those filled from ovipositor muscles. Some pregenital motor neurons displayed pronounced contralateral neurites; in contrast, some ovipositor motor neurons were exclusively ipsilateral. Strong evidence of structural homology was also obtained for pregenital and ovipositor skeletal muscles supplied by the identified neurons and of the pregenital and ovipositor skeletons. For example, transient embryonic segmental appendages were maintained in the female genital segments, giving rise to ovipositor valves, but were lost in pregenital abdominal segments. Significant proportional differences in sternal apodemes and plates were observed, which partially obscure the similarities between the pregenital and genital skeletons. Other changes in reorganization included genital muscles that displayed adult hypertrophy, 1 genital muscle that appeared to represent 2 fused pregenital muscles, and the insertion points of 2 ovipositor muscles that appeared to have been relocated. Together, the comparisons support the idea that the oviposition behavior of genital segments is built upon a homologous, segmentally iterated motor infrastructure located in the pregenital abdomen of male and female grasshoppers.

Adult New Zealand freshwater mussel Echyridella menziesii were collected from three locations in the North Island of New Zealand. In a series of tests that followed standard test guidance, glochidia were exposed to either dissolved copper (Cu), zinc (Zn), or total ammonia nitrogen (TAN) for 6, 24, or 48 h (20 °C, pH 7.8, water hardness 30 mg L(-1) as CaCO3, dissolved organic carbon [DOC] 2.0-2.9 mg L(-1)). Of the three contaminants and tests that met control survival criteria, mussel larvae (glochidia) were most sensitive to Cu exposure (48-h EC50 = 1.7-3.4 μg L(-1), 48-h no observed effect concentrations (NOEC) of 1.3-2.6 μg L(-1)). The Zn 48-h EC50 concentrations were 229-337 μg L(-1) and the 48-h NOEC values were 128-240 μg L(-1). Compared with other native New Zealand species, glochidia were also relatively sensitive to TAN exposure (48-h EC50 12-15 mg TAN L(-1) [pH 7.8], 48-h NOEC 8-10 mg TAN L(-1)). Comparison of our data with those of previous studies on North American freshwater mussels indicates that (1) E. menziesii are among those aquatic species most sensitive to acute Cu or TAN exposure; and (2) E. menziesii juveniles would not be adequately protected by current ANZECC water quality guidelines for TAN or Cu. Inclusion of North American juvenile mussel data in a revision of the current ANZECC water-quality guideline (95th percentile) for chronic ammonia exposure results in a decrease from 0.9 mg to 0.2 mg TAN L(-1) (pH 8).

The waters of the north Tasman Sea are adjacent to the arid Australian sub-continent and are north of the circumpolar Subtropical convergence. Nutrient and phytoplankton stocks in this region were investigated on two voyages during February 2005 and March 2006 to establish the spatial extent and magnitude of oligotrophy in the region. Surface nitrate, phosphate, ammonium and nitrite were all in the nanomolar concentration range north and south of the Tasman Front (∼33°S; 165–175°E). The location of the nitracline was found to be at or above the 1% light level. The distributions of pico-eukaryotic cells, Synechococcus and Prochlorococcus showed strong latitudinal and vertical gradients, with higher abundances south of the Tasman Front that decreased northward, but consistent with changes in nutrient concentration. A nitrite maximum was observed at and below the 1% light level and results from incomplete assimilatory nitrate reduction by phytoplankton. Mixed layer dissolved iron concentrations varied between 0.05 and 0.70 nmol L−1, and were dependent on the vertical supply rate of iron from below and on sporadic atmospheric dust deposition. Based on the rate of iron supply, phytoplankton located south of the Tasman Front were unlikely to be iron limited whereas phytoplankton located north of the Tasman Front were near the threshold for iron limitation. Deck-board incubation experiments involving the addition of macro- (ammonium, nitrate and phosphate) and micro- (iron, dust and zinc) nutrients confirm nitrogen availability to be the primary control on biological production, with the potential for secondary phosphate, silicate and dissolved organic carbon limitation, when nitrogen limitation was alleviated. The form of nitrogen required to stimulate the phytoplankton community also varied; ammonium stimulated Prochlorococcus growth whereas nitrate stimulated Synechococcus growth. Predator-free incubation experiments indicate that grazing was an important constraint on phytoplankton production. Water column observations and incubation results confirm that the supply of dissolved inorganic nitrogen into the euphotic zone was the primary factor controlling new primary production in the northern Tasman Sea region.

This article describes impromptu presentations, a new pedagogical approach for college classrooms that produces full engagement and rapid information processing by students. The technique offers an additional tool to the growing literature on active and collaborative learning. Given a short timeframe, students are required to investigate, analyze, or solve problems assigned by the instructor. This brief critical thinking phase is followed by group presentations in which the students must support their arguments and teach others about relevant course concepts. The traditional idea of learning by teaching serves as the foundational principle behind this approach. Various ways of employing impromptu presentations in the classroom are also described. Finally, the merits of the impromptu presentation approach are highlighted by drawing on the active learning, peer teaching, and levels of learning literature streams.

The staining of neurons with silver began in the 1800s, but until now the great resolving power of the laser scanning confocal microscope has not been utilized to capture the in-focus and three-dimensional cytoarchitecture of metal-impregnated cells. Here, we demonstrate how spectral confocal microscopy, typically reserved for fluorescent imaging, can be used to visualize metal-labeled tissues. This imaging does not involve the reflectance of metal particles, but rather the excitation of silver (or gold) nanoparticles and their putative surface plasmon resonance. To induce such resonance, silver or gold particles were excited with visible-wavelength laser lines (561 or 640 nm), and the maximal emission signal was collected at a shorter wavelength (i.e., higher energy state). Because the surface plasmon resonances of noble metal nanoparticles offer a superior optical signal and do not photobleach, our novel protocol holds enormous promise of a rebirth and further development of silver- and gold-based cell labeling protocols. DOI: http://dx.doi.org/10.7554/eLife.09388.001, eLife digest A fresh slice of brain tissue has a fairly uniform appearance, even when viewed under a microscope. To study the neurons and other cells in the brain, scientists must therefore first prepare tissue samples using methods that make it easier to see certain kinds of cells, or particular features of them. One method that has been available for over a century is to use metal particles to stain some of the cells. For example, when the Spanish anatomist Santiago Ramón y Cajal investigated how brain cells – or neurons – are organized in the brain in the late 1880s, he made the cells visible by staining them with silver. This silver staining technique, called the Golgi method, bears the name of Camillo Golgi who first discovered it. Both Golgi and Ramón y Cajal are considered to be the pioneers of neuroscience, and shared the Nobel Prize in Physiology or Medicine in 1906. Over the years, silver staining was superseded by the use of fluorescent probes. Light travels in the form of waves, and different colors of light have different wavelengths (the distance between the peaks of the wave). Shining light of one specific color onto a fluorescent probe causes it to emit light of a longer wavelength. By detecting this emitted light, it is possible to visualize structures that contain the probes. In the late 1980s, the invention of the laser-scanning confocal microscope allowed highly detailed three-dimensional reconstructions of individual neurons to be obtained using these fluorescent labels. Unfortunately, the lifespan of fluorescent probes is limited by the fact that their fluorescence decreases with repeated use, in a process called photobleaching. Traditional silver stains avoid this problem, but standard confocal microscopy cannot obtain good images from metal-stained cells. Now, Thompson, Harley et al. have overcome this problem by using the confocal microscope in a new way to detect emitted light with shorter wavelengths than the light that was initially absorbed (rather than the longer wavelength light normally detected). This protocol produced highly detailed three-dimensional images of individual metal-stained neurons that had been impregnated with silver or gold particles. The short wavelength light is thought to result from the activity of free electrons called plasmons that are present on the surface of small metal particles (nanoparticles) that are about one millionth of a centimeter in size. When plasmons absorb radiation of a specific wavelength, they vibrate rapidly and emit their excess energy in the form of light. Medieval craftsmen unknowingly exploited this same phenomenon when they added silver and gold particles to molten stained glass, producing windows with vivid red and yellow colors that are still vibrant today. A return to metal-based staining of brain tissue could produce similar longevity for today’s tissue samples. Equally, the procedure developed by Thompson, Harley et al. opens up the possibility of revisiting archived material with the tools of modern confocal microscopy. DOI: http://dx.doi.org/10.7554/eLife.09388.002

The S OLAS A ir–Sea G as E xchange (SAGE) experiment was conducted in Sub-Antarctic waters off the east coast of the South Island of New Zealand in the late summer of 2004. This mesoscale iron enrichment experiment was unique in that chlorophyll a (chl a ) and primary productivity were only 2× OUT stations values toward the end of the experiment and this enhancement was due to increased activity of non-diatomaceous species. In addition, this enhancement in activity appeared to occur without a significant build up of particulate organic carbon. Picoeukaryotes ( a , only accumulated when phytoplankton growth exceeded grazing and when net algal growth exceeded dilution rate. The SAGE results highlight the function of the smallest phytoplankton size fraction described by the ecumenical Iron Hypothesis. Thus, adding iron to HNLC-low silicic acid regions during certain times of the year may simply transfer more carbon through the microbial food web. A primary implication of this study is that any iron-mediated gain in fixed carbon with this set of environmental conditions has a high probability of being recycled in surface waters.

Cold seeps are widely distributed on active and passive margins and display considerable temporal variability in terms of gas and fluid expulsion rates and volume, over scales of hours to days. To constrain this variability, two cold seeps, the North and South Tower, located at the Wairarapa Seep site on Opouawe Bank, Cook Strait, New Zealand, were monitored in an integrated geological and biogeochemical study during eight surveys between 2005 and 2008. To ensure sampling of the water column within the flare overlying the seeps the vessel was manoeuvred in response to the backscatter data from a hull-mounted 38 kHz single-beam echo-sounder. High methane concentrations (> 100 nmol l− 1) were found within 400–450 m-high flares at both sites, and within an 825 m-high flare at a newly identified seep, southeast of Pahaua Bank. Elevated bottom water methane was also found over newly identified mud volcanoes on the Campbell Bank, Cook Strait. The locations of the seeps appear to be structurally controlled with active methane vents occurring on anticlinal ridges and associated with a 30 m-high normal fault scarp on Opouawe Bank. Fluid flow is facilitated by sedimentary layer permeability properties, structural focusing and dilational fracturing on the crest of a large anticlinal ridge, cored by a seaward-verging reverse fault. The South Tower cold seep did not significantly influence bulk sediment and water column particulates and nutrients, although high bacterial biomass, productivity and methane oxidation rates were associated with elevated methane in the flare. The seep flares exhibited depleted δ13C–CH4 values (− 63 to − 70‰), characteristic of shallow biogenic gas sources. Incorporation of the δ13C–CH4 and CH4 concentration data in a mixing model, and comparison of the water residence time with methane oxidation rates, indicated that the primary fate of methane in the flare was dilution. The dissolved methane originating from the seeps was retained in the water column at depths > 700 m, with minimal contribution to atmospheric emissions, as confirmed by surface methane mapping. Nevertheless, the South Tower seep was a significant source of methane with a column-integrated burden below 700 m of 5.6 mmol m− 2, and an estimated annual emission of 0.5–1 × 106 mol CH4. The structural and tectonic setting of the Wairarapa Seep provides a useful analogue model for other convergent margin cold seeps on the Hikurangi Margin and elsewhere. This unique, three-year integrated study provided valuable insights into the linkage between geological structure and overlying water column biogeochemistry, and confirmed the value of this cold seep site for long-term monitoring.

Designing an e-commerce site that will appeal to customers and convince them to purchase a product or service becomes difficult because of the variety of personalities and preferences that individual customers may have. One approach is to base the website's text, imagery, and navigation features on customers' personality types. The goal of this paper is to propose a conceptual model that links the Myers-Briggs Type Indicator's (MBTI) personality dimensions to business-to-consumer e-commerce website design. The model can offer guidance to those e-retailers who want to target their products at a particular audience.

PurposeThe purpose of this paper is to identify differences, if any, that exist in leadership style, behaviors, and competencies to drive performance between public/non‐profit and for‐profit organizational leaders.Design/methodology/approachThe study describes the results of in‐depth interviews with leaders in small to medium‐sized organizations in California. Approximately half of the leaders work in non‐profit and public organizations, while the other half work in for‐profit companies.FindingsThe findings reveal both similarities and differences between the two groups.Originality/valueThe results are a first step in examining the key leadership competencies required for success in each sector and serve as a springboard for future research.

Inspired by the educational benefits of project-based learning, a class project for leadership courses is described that takes interview assignments to a new level. Students use the findings from their interviews with managers to develop a book on leadership that they have had a hand in from start to finish. The project provides a comprehensive approach to linking the concepts and realities of leadership and engages students in a collaborative learning activity that challenges them as both individuals and group members. The project also provides numerous skill-building opportunities that maximize the value of the project. The six phases of the project are described in detail, along with suggestions for the project's timeline and assessment mechanisms. Options for altering the project's length are discussed as well as its potential for use in other management courses.

Stretch-activated afferent neurons, such as those of mammalian muscle spindles, are essential for proprioception and motor co-ordination, but the underlying mechanisms of mechanotransduction are poorly understood. The dorsal bipolar dendritic (dbd) sensory neurons are putative stretch receptors in the Drosophila larval body wall. We have developed an in vivo protocol to obtain receptor potential recordings from intact dbd neurons in response to stretch. Receptor potential changes in dbd neurons in response to stretch showed a complex, dynamic profile with similar characteristics to those previously observed for mammalian muscle spindles. These profiles were reproduced by a general in silico model of stretch-activated neurons. This in silico model predicts an essential role for a mechanosensory cation channel (MSC) in all aspects of receptor potential generation. Using pharmacological and genetic techniques, we identified the putative mechanosensory channel, DmPiezo, as a strong candidate for this functional role in dbd neurons, with TRPA1 playing a subsidiary role. We also show that rat muscle spindles exhibit a ruthenium red-sensitive current, but found no expression evidence this corresponds to Piezo activity. In summary, we show that the dbd neuron is a stretch receptor and demonstrate that this neuron is a tractable model for investigating mechanisms of mechanotransduction.

The authors examined the nature of e-business and non-profit alliances and their impact on brand equity, pricing, perceptions of product quality, trust in the company, consumer behavioral intentions towards the brand, and the consequences of the company's actions. E-businesses with strong reputations and e-businesses with weak reputations were paired with popular or not so popular causes. Results showed no significant differences in the dependent variables for e-businesses with strong reputations, whether they adopted causes with strong or weak reputations. However, for e-businesses with weak reputations, differences were found in perceptions of brand equity and consequences of the company's actions. These findings suggest that alliances with strong reputation causes may be beneficial for e-businesses with weak reputations. The results also showed that e-businesses with strong reputations adopting a non-profit cause received higher ratings for product quality and pricing than did weak reputation ...

Wine marketers realise that to increase the overall size of the wine consuming population they must make wine more approachable and easier to understand. As it now stands, many consumers lack confidence in their ability to select a wine for either their own consumption or to share with others. Therefore, understanding the role played by consumer self‐confidence is especially relevant to marketers of wine, and the need to accurately measure the construct is important to scholarly research. Recently, the development of a scale to measure consumer self‐confidence has appeared in the consumer behaviour literature (Bearden, Hardesty and Rose, 2001). This study first adapts this consumer self‐confidence scale for use in wine‐related research. Next, the impact of six distinct dimensions of consumer self‐confidence on three different wine purchase situations is demonstrated. Results show the scale has the potential to inform both researchers and marketers about consumers' self‐confidence related to wine purchases.

The four brown midrib (bm) mutants of maize have a reduced content and altered subunit composition of the cell wall polymer lignin. The bm mutations have traditionally been considered completely recessive, because the brown midrib phenotype is only apparent in plants homozygous for the mutation. In addition to an effect on cell wall composition, some bm mutations have been shown to affect flowering time. We had preliminary evidence for a dosage effect of the Bm1 locus on flowering time, which prompted this detailed study on the Bm1 locus. In this study, near-isogenic lines (in an A619 background) with zero, one or two bm1 mutant alleles were compared. The bm1 heterozygotes flowered significantly earlier than both the wild-type plants and bm1 mutants. This difference can at least be partly attributed to an accelerated growth rate in the later stages of plant development. Furthermore, Fourier transform infrared spectroscopy revealed that the cell wall composition of the bm1 heterozygous plants is distinct from both the bm1 and wild-type homozygotes. The combination of the data on flowering time and the data on cell wall composition provide evidence for a dosage effect at the Bm1 locus.

A specialized behavior, oviposition, is produced by the eighth and ninth abdominal segments of female grasshoppers. To begin to understand how these segments produce the behavior, which is not displayed by males or pregenital regions of the abdomen in females, the structure and function of efferent neurons in abdominal ganglia of both sexes were examined. In females, the eighth and ninth segments are specialized differently for oviposition: 20 ovipositor motor neurons were found in the eighth segment, and 26 were found in the ninth segment. Males had fewer motor neurons in their eighth segment, but the same number in the ninth segment, which is the only genital segment in males. However, the axons of several of the ninth segmental male motor neurons traveled to the periphery in the genital nerve, which is only found in males. In both sexes, pregenital ganglia had the most motor neurons, but these neurons, for the most part, had morphologies that strongly resembled those of genital segments. Efferent modulatory neuron numbers were not sexually dimorphic in the segments examined, except that males had a greater number in their ninth segment. Experimental methods that activate oviposition were found to also activate a rhythmical motor pattern in pregenital abdominal segments of both sexes. In females, the pattern was phase-coupled to oviposition, but persisted after the connections with the terminal abdominal ganglion were severed. The preponderance of similarities among efferent neurons and elicited motor activity suggests a common pattern of neural circuitry in the behaviorally diverse abdominal segments of grasshoppers.

A uniquely female behavior in grasshoppers, oviposition, is driven by neural circuitry in the terminal abdominal segments of the female's central nervous system. Because it is known that the embryonic pattern of neuroblasts is sexually monomorphic in these animals, we were interested to know how the central nervous system of adults is organized to support the obvious behavioral dimorphism. Here, we compare three classes of identifiable adult neurons: ovipositor motor neurons, efferent dorsal unpaired median (DUM) neurons, and DUM interneurons in the eighth abdominal neuromere. Cobalt backfills of the eighth tergal nerves revealed identical complements of motor neurons in males and females. Included among these neurons in the male were putative homologues of two sets of ovipositor muscle motor neurons. Whereas these motor neurons supply two ovipositor muscles in the female, they are divided to supply three muscles in males. The eighth abdominal neuromere of both sexes contained seven efferent DUM neurons, but peripheral axon projections varied between males and females in accordance with gender-specific targets. In the eighth neuromere of females, some 22 small cell bodies of DUM interneurons were stained with Toluidine blue, whereas only three male DUM interneurons were found. Male muscle homologues were induced to express a rhythmical motor pattern by experimental methods that activate the oviposition pattern in females. The induced pattern in males is of unknown behavioral significance. Although oviposition normally occurs only after sexual maturity, the motor pattern could be activated at all life stages in females, including embryos, as early as 90% of embryonic development.

Objective: To discover if the measure 'stops walking to talk' is a rrseful indicator of falls risk in the frail elderly in residential care. Method: Twelve frail elderly residents of care facilities were assessed for falls likelihood with the Timed Upand-Go (TUG) and the Berg Balance Scale (BBS) tests, and then observed in walking to determine if they needed to stop walking to talk. These results were compared with the number of falls subjects had experienced in the previous six months. Results: Eight subjects were deemed at risk of falling by the TUG and seven by the BBS. Four of these subjects had fallen in the previous six months. Three subjects needed to stop walking to talk on at least one occasion and were deemed at risk of falling by the TUG and BBS. One of these had fallen in the previous six months. Conclusion: This study demonstrated in this group of frail older people that falling in the previou; six months was associated with high TUG and low scores on the BBS. Only three observations of 'stops walking to talk ' were made; this limited the comparisons as only one of these subjects had fallen but all were predicted 0s fallers by the TUG and BBS. A future study with a larger sample size and incorporating memory recall in the conversation should confirm these results.

The central nervous systems of many insects contain segmentally arranged groups of unpaired midline neurones. These have been called DUM (dorsal unpaired median) neurones because of the position of their cell bodies. The larger DUM neurones, with bilaterally symmetrical peripheral axons, have been most extensively studied in grasshoppers and locusts. DUM cells in the metathoracic ganglion modulate tension of skeletal muscle, potentiate synaptic transmission at neuromuscular junctions and slow a myogenic rhythm (Evans and O’Shea, 1977, 1978; Whim and Evans, 1989), whereas those in the abdominal ganglia alter tension in visceral muscle (Lange and Orchard, 1984). In fireflies, DUM neurones of the terminal abdominal ganglia control flashing by activating the photocytes of the lantern (Christensen and Carlson, 1981). The effects are mediated by the biogenic amine octopamine (Carlson and Jalenak, 1986; Evans and O’Shea, 1977, 1978; Morton and Evans, 1984; Orchard and Lange, 1985), which is also implicated in other modulatory actions (see Ramirez and Orchard, 1990, for references). In grasshoppers, in addition to the octopamine-containing efferent DUM neurones, there are a substantial number of local and intersegmental DUM interneurones with small cell bodies (Thompson and Siegler, 1989, 1991).

The range of anatomical and physiological properties in the adult progeny of an identified neuroblast was investigated. Some 80-90 adult neurons constitute the dorsal unpaired median (DUM) group of the grasshopper metathoracic ganglion. Within the group are efferent, octopaminergic neurons with large cell bodies and overshooting action potentials. Our objective was to determine the properties of the neurons with small cell bodies that make up the majority of the clone, some 60-70 neurons, about which scant information was available. The small DUM neurons have cell body diameters of 10-20 microns and stain with antibodies to GABA (Thompson and Siegler, '89: Proc. Soc. Neurosci. 15:1296 (abstr.); Witten and Truman, '89: Proc. Soc. Neurosci. 15:365 (abstr.)). By employing intracellular electrophysiological and morphological techniques, we have established that the small DUM neurons are spiking interneurons, expressing passively conducted action potentials in the cell body. They fall into two basic classes: local interneurons with bilateral branches in the auditory neuropiles, and intersegmental interneurons with bilateral branches widespread in the methathoracic ganglion and axons traveling in both anterior connectives. The local interneurons typically respond to sound, whereas the intersegmental interneurons selectively respond to wind on the head or to generalized movements by the animal. Primary neurites of small and large DUM neurons enter the neuropil in a bundle, but the neurites of DUM interneurons are more posterior and have a separate trajectory from those of the efferent DUM neurons once in the ganglion core. A model is presented for the sequential development of efferent, local, and intersegmental DUM neurons from the median neuroblast.

Background With the recent advent of percutaneous valve therapy, an increased need for the evaluation of outcomes after open aortic valve replacement (AVR) in elderly patients is warranted. This study compares the short- and long-term survival outcomes of octogenarians after AVR with younger age groups in the modern surgical era. Methods A retrospective review was performed on patients who underwent isolated, primary AVR from 1996 to 2006 at the Emory Healthcare Hospitals. Five-hundred fifteen patients were divided into three age groups: 60 to 69 (n = 206), 70 to 79 (n = 221), and 80 to 89 years of age (n = 88). Outcomes were compared among the age groups using logistic regression and analysis of variance techniques. Long-term survival between age groups was compared using the Cox proportional hazards model. Kaplan-Meier plots were used to determine survival rates. Results The groups were similar with respect to in-hospital mortality ( p = 0.66) and hospital length of stay ( p = 0.08). Preoperative predictors of in-hospital mortality included stroke (odds ratio [OR] 5.36), chronic lung disease (OR 4.51), and renal failure (OR 1.39). As expected, age significantly impacted long-term survival (hazard ratio [HR] 1.06). Other predictors of long-term survival included stroke (HR 2.15), current smoker (HR 2.03), diabetes (HR 1.53), and renal failure (HR 1.4). The Kaplan-Meier estimate of median survival for octogenarians was 7.4 years. Conclusions In the modern era, octogenarians have acceptable short- and long-term results after open AVR. Comparisons of less invasive techniques for AVR should rely on outcomes based in the modern era and decisions regarding surgical intervention in patients requiring AVR should not be based on age alone.

Followership has been an understudied topic in the academic literature and an underappreciated topic among practitioners. Although it has always been important, the study of followership has become even more crucial with the advent of the information age and dramatic changes in the workplace. This paper provides a fresh look at followership by providing a synthesis of the literature and presents a new model for matching followership styles to leadership styles. The model’s practical value lies in its usefulness for describing how leaders can best work with followers, and how followers can best work with leaders.

Issues of justice are integral to social, political, and economic change. And so are matters of leadership. While justice, on the one hand, and leadership, on the other, have both received massive research attention by scholars over the years, few efforts have focused explicitly on how these two broad issues commingle, either during times of change or under more placid circumstances. Is there a social psychology which links them? What are the dynamics involved? Surely such questions are reasonable ones for justice researchers to ask. After all, post-industrial nations are increasingly described as “societies of organizations”, in which economic and political power rests less with individuals, and more with formal organizations and organized interest groups in both the public and private sectors (e.g., Drucker, 1988; Zucker, 1983). This being the case, an understanding of justice, particularly during times of turbulence and revolutionary change, cannot be complete without being informed by those sciences which are dedicated to understanding and improving human organizations. Organizational science has a number of priorities, often conflicting. But in the pantheon of organizational concepts, leadership is seen as central (Hollander, 1986), with great relevance and importance for organizational forms, processes, and outcomes.

A group of lineally related neurons in the grasshopper was studied to determine the number of efferent neurons in the group and their morphological types. The neurons arise from the median neuroblast of the third thoracic neuromere and comprise what is commonly known as the DUM or dorsal unpaired median group. Of some 92 neurons in the group, about 20 are efferent neurons, the remainder being local or intersegmental interneurons. As part of our continuing developmental studies, we wished to identify the efferent neurons within the lineage and to determine their number. Ten efferent DUM neurons had been described in earlier studies, where neurons were stained individually through microelectrodes. The remaining unidentified neurons might be novel types, multiples of known types, or both, possibilities that would not be readily distinguished through further staining of neurons individually. Rather, we used methods of retrograde staining and axon tracing that allowed us to examine the entire group of efferent DUM neurons. Nineteen efferent neurons were identified, comprising two DUM1s, five DUM3s, six DUM3,4s, three DUM3,4,5s, and three DUM5s, neurons were named according to the lateral nerves containing their axons. The efferent neurons were further divided by type according to the distribution of axonal branches in lateral nerves, the course of the primary neurite within the deep or superficial DUM tract, and the diameter of the cell body. © 1995 Wiley-Liss, Inc.

The purpose of this study was to determine the factors underlying differences in population size and composition between segmentally homologous neuronal lineages. The segmental median neuroblasts (MNBs) of grasshoppers are identified stem cells that each produce a midline group of neurons. We traced the embryonic development of the group in two disparate segments, counting MNB progeny and profiles of dying cells in fixed and stained preparations of staged embryos. In the metathoracic segment (T3), about 95 MNB progeny survive embryonic development, whereas in the next posterior segment, the first abdominal (A1), only about 60 survive. In T3, the MNB arises at 29% of embryogenesis and dies at 78%, whereas in A1 the MNB arises at 30% and dies at 73%. In T3, the number of MNB progeny initially increases at a steady rate, 10 cells being added per 5% of embryogenesis. Between 70% and 78% growth tapers off; although the T3 MNB continues to divide, cells die at the same time, specifically removing last-born progeny. By contrast, in A1 the MNB progeny increase in two phases, one from 30% to 45% and the other from 60% to 73%, again at the rate of 10 cells per 5%. Between the two phases, the number of A1 progeny is stable. The A1 MNB continues to divide, but cells die at the same time, specifically removing earlier-born progeny. The episodes of cell death in A1 and T3 coincide with embryonic molts, and thus may be hormonally triggered. Cell death is greater in A1 than T3, accounting for most of the difference in population size. The difference in MNB longevity makes a lesser contribution. The present data, together with corollary anatomical data (Thompson and Siegler, 1991), support the hypothesis that progeny fated to become certain neuronal types are selectively removed from the two MNB lineages: intersegmental interneurons from T3 and efferent neurons and local interneurons from A1.

Three antifouling (AF) sesterterpenes have been isolated from the New Zealand marine sponge Semitaspongia bactriana with toxicity against the diatom Nitzschia closterium and bryozoan Bugula neritina. The three metabolites have been characterised by spectroscopic techniques as 7 E, 12 E, 20 Z-variabilin (1), cavernosolide (2) and lintenolide A (3) (also called spongianolide C) and have low micromolar activity against our two test species. The γ-hydroxybutenolide containing sesterterpenes (2 and 3) show the most promise, with relative stability and suitable lipophilicity for incorporation of either these metabolites, or synthetic analogues, as biocides to produce paints or plastics with AF properties.

Extracellular stimulation over the dorsal funiculus in the spinal cord of lampreys was found to selectively activate prolonged episodes of fictive arousal respiration (Figs. 1, 3). The induced episodes showed comparable increases in cycle frequency and motoneuron burst duration to the spontaneous arousal pattern observed in isolated brain preparations (Fig. 2). Intracellular stimulation of primary sensory neurons with axons in the dorsal funiculus, called ‘dorsal cells’, also elicited the arousal pattern (Fig. 4). Mechanoreceptive dorsal cells respond to cutaneous stimulation. When mechanical stimuli were applied to the skin of intact lampreys (Fig. 6) or to lampreys with ipsilateral vagotomy, arousal respiration was induced (Figs. 7, 8). Bilateral, but not unilateral, trigeminal lesion blocked dorsal cell induction of the arousal response (Fig. 5). Spontaneous arousal respiration was recorded from intact, unrestrained lampreys (Fig. 9). These results suggest that fictive arousal respiration is the in vitro correlate of natural arousal respiration in lampreys, and that one mechanism leading to arousal respiration may be the activity of sensory dorsal cells. A model for respiratory motor pattern switching in lamprey is proposed. The model suggests that the normal and arousal patterns are produced by separately engaging rostral or caudal pattern generators in the medulla, rather than by modifying one pattern generator (Fig. 10).

Background Factors that affect flowering vary among different plant species, and in the grasses in particular the exact mechanism behind this transition is not fully understood. The brown midrib (bm) mutants of maize (Zea mays L.), which have altered cell wall composition, have different flowering dynamics compared to their wild-type counterparts. This is indicative of a link between cell wall biogenesis and flowering. In order to test whether this relationship also exists in other grasses, the flowering dynamics in sorghum (Sorghum bicolor (L.) Moench) were investigated. Sorghum is evolutionarily closely related to maize, and a set of brown midrib (bmr) mutants similar to the maize bm mutants is available, making sorghum a suitable choice for study in this context.

The intracellular activity of motoneurons during 'fictive' respiration in the isolated lamprey brain was investigated. In association with each respiratory cycle three distinct PSP phases were observed: an early, low amplitude EPSP phase; a large, brief EPSP phase that drove action potentials; and a subsequent IPSP phase. Selective midline and trigeminal lesions, and trigeminal stimulation, demonstrated that the large excitatory and inhibitory phases were generated by a previously described pair of central pattern generators located in the trigeminal region of the medulla. Lesion studies further showed that the low amplitude excitatory input could be produced independently of the trigeminal pacemakers, near the region of the medulla that contains the respiratory motoneurons. In addition to 'normal' fictive respiration, the isolated brain was found to produce several variations of the respiratory pattern. These motor programs, 'coughs', 'arousal breathing', and 'weak breathing', were distinguished from the normal respiratory pattern by their much longer burst durations, distinctive underlying synaptic input, and separate coordinating mechanism. Activity similar to these motor programs could be independently produced by the caudal medulla after both trigeminal central pattern generators had been removed. Lesion studies, and the observation that respiratory-related neural activity ceased in the trigeminal region during the production of these long-duration programs, suggest that the caudal medulla also contains paired central pattern generators involved in lamprey respiration.