Clock-driven vasopressin neurotransmission mediates anticipatory thirst prior to sleep.

Circadian rhythms have evolved to anticipate and adapt animals to the constraints of the earth's 24-hour light cycle. Although the molecular processes that establish periodicity in clock neurons of the suprachiasmatic nucleus (SCN) are well understood, the mechanisms by which axonal projections from the central clock drive behavioural rhythms are unknown. Here we show that the sleep period in mice (Zeitgeber time, ZT0-12) is preceded by an increase in water intake promoted entirely by the central clock, and not motivated by physiological need. Mice denied this surge experienced significant dehydration near the end of the sleep period, indicating that this water intake contributes to the maintenance of overnight hydromineral balance. Furthermore, this effect relies specifically on the activity of SCN vasopressin (VP) neurons that project to thirst neurons in the OVLT (organum vasculosum lamina terminalis), where VP is released as a neurotransmitter. SCN VP neurons become electrically active during the anticipatory period (ZT21.5-23.5), and depolarize and excite OVLT neurons through the activation of postsynaptic VP V1a receptors and downstream non-selective cation channels. Optogenetic induction of VP release before the anticipatory period (basal period; ZT19.5-21.5) excited OVLT neurons and prompted a surge in water intake. Conversely, optogenetic inhibition of VP release during the anticipatory period inhibited the firing of OVLT neurons and prevented the corresponding increase in water intake. Our findings reveal the existence of anticipatory thirst, and demonstrate this behaviour to be driven by excitatory peptidergic neurotransmission mediated by VP release from central clock neurons.

Molecular analysis and genotype-phenotype correlation of Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) features hypoplastic anemia and congenital malformations, largely caused by mutations in various ribosomal proteins. The aim of this study was to characterize the spectrum of genetic lesions causing DBA and identify genotypes that correlate with phenotypes of clinical significance. Seventy-four patients with DBA from across Canada were included. Nucleotide-level mutations or large deletions were identified in 10 ribosomal genes in 45 cases. The RPS19 mutation group was associated with higher requirement for chronic treatment for anemia than other DBA groups. Patients with RPS19 mutations, however, were more likely to maintain long-term corticosteroid response without requirement for further chronic transfusions. Conversely, patients with RPL11 mutations were less likely to need chronic treatment. Birth defects, including cardiac, skeletal, hand, cleft lip or palate and genitourinary malformations, also varied among the various genetic groups. Patients with RPS19 mutations had the fewest number of defects, while patients with RPL5 had the greatest number of birth defects. This is the first study to show differences between DBA genetic groups with regards to treatment. Previously unreported differences in the rate and types of birth defects were also identified. These data allow better patient counseling, a more personalized monitoring plan, and may also suggest differential functions of DBA genes on ribosome and extra-ribosomal functions.

A one-generation reproductive toxicity study of the mycotoxin ochratoxin A in Fischer rats.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium molds. Grain-based foods account for most human dietary exposures to OTA. OTA is a teratogen, but its reproductive and developmental effects are poorly understood. A one-generation reproductive toxicity study was conducted with groups of 16 male and 16 female Fischer rats exposed to 0, 0.026, 0.064, 0.16, 0.4 or 1.0 mg OTA/kg in diet. Dams exposed to 1.0 mg OTA/kg diet had statistically significant F1 pup losses between implantation and postnatal day (PND 4). Delays in preputial separation (PPS) and vaginal opening (VO) were indicative of delayed puberty in F1 rats. Mild renal lesions in nursing pups indicated that exposure prior to weaning impacted the kidneys. The developing kidney was more susceptible to OTA than the adult kidney. Significant increases in multi-oocyte follicles (MOFs) and proportional changes in resting and growing follicles were observed in F1 female ovaries. Plasma testosterone was reduced in F0 males, and there were negative effects on sperm quality in F0 and F1 male rats. The results confirm that continuous dietary exposure to OTA causes post-implantation fetotoxicity in dams, and renal and reproductive toxicity in their male and female offspring.

Simulated management effects on ammonia emissions from field applied manure.

A need exists to improve the utilization of manure nutrients by minimizing NH(3) emissions from land application of manure. Management strategies to reduce NH(3) emissions are available; however, few have been validated under Canadian conditions. A well tested and accurate simulation model, however, can help overcome this challenge by determining appropriate management strategies for a given set of field conditions. The Volt'Air simulation model was utilized to estimate NH(3) volatilization from manure spreading for various manure spreading considerations under a range of atmospheric conditions typically encountered in eastern Canada. Considerations included: (i) soil liming, (ii) time of day of manure spreading, (iii) rainfall (timing and amount) and (iv) manure incorporation (timing, depth and manure coverage). Results demonstrated that liming to increase soil pH, increased NH(3) emissions by 3.3 kg ha(-1) for each increment of 0.1 pH (up to a 1.5 total increase), over no liming at 34.6 kg ha(-1). For each hour delay in manure spreading past 0800 h, NH(3) losses were reduced by 1.5 kg ha(-1). Rainfall (10mm) at least 20 h after manure application reduced losses, with increased reductions at higher rainfall amounts. Incorporation soon (1h) after application was best for NH(3) mitigation. Increasing the depth of incorporation by 5c m reduced NH(3) emissions by 4.4 kg ha(-1); also increasing manure coverage by incorporation reduced losses by 2 kg ha(-1) for each 10% increase in coverage, compared to surface application at 34.6 kg ha(-1). This investigation using Volt'Air yielded valuable information about simulating manure management strategies and the magnitude of their effects on NH(3) emissions.

Gadolinium uncouples mechanical detection and osmoreceptor potential in supraoptic neurons.

Stretch-sensitive ion channels are ubiquitous, yet evidence of their role in mechanotransduction remains scarce. The presence of stretch-inactivated cation channels in supraoptic neurons is consistent with the osmoreceptor potentials regulating vasopressin release. However, whether osmosensitivity depends on mechanical gating and ion flux through stretch-inactivated channels is unknown. Here we report that changes in channel open probability associated either with modification of pipette pressure or with external osmolality selectivity result from variations in closed time. While channel mechanosensitivity and osmotically evoked changes in cell volume are not affected by gadolinium, similar concentrations of the lanthanide inhibit cation permeation through the single channels and macroscopic osmoreceptor potentials. Mechanotransduction through stretch-inactivated channels is therefore necessary for osmoreception in supraoptic neurons.

Coincident detection of CSF Na+ and osmotic pressure in osmoregulatory neurons of the supraoptic nucleus.

Behavioral and neuroendocrine responses underlying systemic osmoregulation are under the concerted control of centrally located osmoreceptors and cerebrospinal fluid (CSF) Na+ concentration ([Na+]) detectors. Although the process underlying osmoreception is understood, the mechanism by which [Na+] is detected and integrated with cellular information derived from osmoreceptors is unknown. Here, we show that shifts in extracellular [Na+] ([Na+]0) cause proportional changes in the relative Na+ permeability of mechanosensitive cation channels responsible for signal transduction in the osmosensory neurons of the supraoptic nucleus. This effect causes the generation of Na+ specific receptor potentials under isotonic conditions and modulates osmoreceptor potentials and electrical responsiveness during osmotic perturbation. These results provide a cellular basis for Na+-sensing and for the coordinated detection of CSF [Na+] and osmolality in central osmoregulatory neurons.

Contribution of TRPV channels to osmosensory transduction, thirst, and vasopressin release.

Systemic osmoregulation is an integrated physiological process through which water intake and excretion are continuously balanced against salt intake and excretion to maintain the osmolality of the extracellular fluid near an optimal 'set-point' value. The behaviors (that is, thirst and sodium appetite) and renal responses (diuresis and natriuresis) that are modulated to mediate osmoregulatory homeostasis are mainly controlled by the nervous system. Appropriate regulation of these parameters depends in large part on specialized osmosensitive neurons, termed osmoreceptors, which convert changes in plasma osmolality into electrical signals that ultimately modulate effector functions to achieve homeostasis. Previous work has shown that mechanosensitive cation channels expressed in osmoreceptor neurons play a key role in the process of osmosensory transduction. Although the molecular identity of these channels remains unknown, a growing body of evidence, reviewed here, indicates that members of the transient receptor potential vanilloid family of ion channels may contribute to osmosensory transduction and to homeostatic responses implicated in the control of water balance.

Excitatory peptides and osmotic pressure modulate mechanosensitive cation channels in concert.

Behavioral and neuroendocrine responses underlying systemic osmoregulation are synergistically controlled by osmoreceptors and neuropeptides released within the hypothalamus. Although mechanisms underlying osmoreception are understood, the cellular basis for the integration of osmotic and peptidergic signals remains unknown. Here we show that the excitatory effects of angiotensin II, cholecystokinin and neurotensin on supraoptic neurosecretory neurons are due to the stimulation of the stretch-inactivated cation channels responsible for osmoreception. This molecular convergence underlies the facilitatory effects of neuropeptides on responses to osmotic stimulation and provides a basis for the gating effects of plasma osmolality on the responsiveness of osmoregulatory neurons to peptidergic stimulation.

A 90-day subchronic gavage toxicity study in Fischer 344 rats with 3-methylfuran.

A 90-day gavage study was conducted with 0.0, 0.02, 0.075, 0.25, 1.0 and 4.0 mg/kg bw/day dose groups of 3-methylfuran to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects including changes in gross anatomy, histopathology, clinical biochemistry and hematology. There were significant changes in the serum clinical biochemistry markers related to liver injury where males were more affected than the females for most parameters analysed. The serum liver injury marker γ-glutamyltransferase, alanine and aspartate aminotransferases were significantly increased in males in the 4.0 mg/kg dose group. Alkaline phosphatase was increased in females and males. There were increases in both gross and histological lesions in the liver of both sexes in addition to statistical differences in female liver weights at the 4.0 mg/kg bw/day dose. Significant increases in spleen weights were found in both genders. This was accompanied by a dose-dependent atrophy of both B- and T-cell regions in which the males were more affected. There were no significant changes in male kidney weights but there was microscopically decreased protein in the proximal tubules and crowding of their nuclei in the 4.0 mg/kg bw/day dose group. There were also significant changes in the kidney serum biomarkers including various electrolytes, blood urea nitrogen, creatinine and uric acid. A small, but significant increase in female kidney weights was observed and which increase was accompanied by changes in electrolytes, kidney specific markers and a dose-dependent increase in mineralization. In both genders, amylase decreased whereas lipase increased but these were not accompanied by any histological changes in the pancreas. Histopathological changes in the liver were observed consistently in male and female rats in the 0.25 mg/kg dose group and higher. Hence, a lowest observed adverse effect level (LOAEL) of 0.25 mg/kg bw/d and a no observed adverse effect level (NOAEL) of 0.075 mg/kg bw/day are proposed for 3-methylfuran-induced hepatic lesions in this study. Benchmark dose modelling based on a BMR of 10% change in lesion incidence, generated BMDLs10 of 0.08 mg/kg bw/day in male rats and 0.05-0.17 mg/kg bw/day in female rats for increased incidence of liver lesions.

Activity-dependent modulation of nerve terminal excitation in a mammalian peptidergic system.

Transmitter release at nerve terminals is triggered by voltage-gated calcium influx upon arrival of an action potential. Changes in coupling between axonal impulse discharge and depolarization of the nerve ending might therefore regulate exocytotic secretion. In the magnocellular neurosecretory system of the rat, the pattern of electrical activity can modify the duration of action potentials within individual nerve endings, and alter the spatial spread of excitation into the branching terminal field of neurohypophysial axons. The observed effects are consistent with the effects of firing rate and pattern on peptide release, indicating that activity-dependent changes in axon-terminal coupling may modulate secretion in this neuroendocrine system.

Osmoreception in magnocellular neurosecretory cells: from single channels to secretion.

Recognizing that osmotic pressure is a principal factor controlling antidiuresis, Verney introduced the term 'osmoreceptor' to designate the mysterious cerebral structures that regulate vasopressin release from the posterior pituitary. While hormone secretion from the neurohypophysis is influenced by synaptic inputs from other osmoresponsive neurons, magnocellular neurosecretory cells currently provide our most comprehensive model of signal detection in an osmoreceptor.

Calcium-channel subtypes in the somata and axon terminals of magnocellular neurosecretory cells.

To understand the specific functions of Ca(2+)-channel types it is necessary to know how they are distributed within neurons. The unique structure of the magnocellular neurosecretory cells of the rat supraoptic nucleus has made it possible to obtain whole-cell recordings from individual somata and axon terminals acutely isolated from adult rats. Characterization of elicited Ca2+ currents in these cells has demonstrated that certain types are segregated in somata or axon terminals, and that current types defined pharmacologically can display different kinetic properties in the two loci. Observed biophysical properties correlate with functional requirements in the two compartments and have implications for the roles of specific Ca(2+)-channel subtypes.

Effects of Salt Loading on the Regulation of Rat Hypothalamic Magnocellular Neurosecretory Cells by Ionotropic GABA and Glycine Receptors.

Synaptic and extrasynaptic transmission mediated by ionotropic GABA and glycine receptors plays a critical role in shaping the action potential firing (spiking) activity of hypothalamic magnocellular neurosecretory cells and therefore determines the rate at which vasopressin and oxytocin are released from the neurohypophysis. The inhibitory effect of these transmitters relies on the maintenance of a low concentration of intracellular chloride ions such that, when activated by GABA or glycine, a hyperpolarisation of the neuronal membrane potential results. In this review, we highlight the various ways by which the two types of inhibitory receptors contribute to homeostasis by fine-tuning the spiking rate of vasopressin-releasing magnocellular neurosecretory cells in a manner dependent on the hydration state of the animal. In addition, we review the currently available evidence on how the strength of these inhibitory pathways can be regulated during chronic hypernatraemia via a form of activity-dependent depolarisation of the chloride reversal potential, leading to an abolition of these inhibitory pathways potentially causing sodium-dependent elevations in blood pressure.

Effects of Salt Loading on the Morphology of Astrocytes in the Ventral Glia Limitans of the Rat Supraoptic Nucleus.

In the ventral glial limitans (VGL) of the supraoptic nucleus (SON) of the rat, a unique astrocyte type is found with an ability to undergo striking morphological plasticity in response to a wide range of physiological stimulations such as chronic hypernatraemia. This includes a thinning of the VGL, which contains the somata and proximal processes of these astrocytes, as well as an almost complete withdrawal of their vertically-oriented distal processes. Currently, there is little information available on the types of astrocytes that reside in the SON-VGL and which of these exhibit state-dependent structural plasticity. To address this, we enabled the visualisation of single SON-VGL glia using two novel cell labelling techniques with fluorescence microscopy. First, we used an inducible genetic reporter mouse line that allowed the specific labelling of a low density of astrocytes expressing glutamate and aspartate transporter (GLAST)/excitatory amino acid transporter 1. This approach revealed a high degree of variability in the morphology of mouse SON-VGL astrocytes, in contrast to what has been reported for cortical astrocytes. Next, we used the DiOlistlic labelling approach to label single glial cells with DiI in the SON-VGL of rats. Astrocytes observed using this approach shared the morphological features of GLAST-expressing astrocytes in the mouse SON-VGL. Specific structural aspects of these cells were modified by chronic hypernatraemia achieved by 7-day salt loading. Notably, the average area of cells exhibiting protoplasmic features was significantly reduced in the horizontal plane, and the size of varicosities present on fibrous projections was significantly enlarged. These observations indicate that novel cell labelling methods can significantly advance our understanding of SON-VGL cells and reveal specific forms of morphological plasticity that can be driven by chronic hypernatraemia.

Excitatory role of the hyperpolarization-activated inward current in phasic and tonic firing of rat supraoptic neurons.

The properties and functional roles of the hyperpolarization-activated inward current (I(H)) in magnocellular neurosecretory cells (MNCs) were investigated during sharp microelectrode recordings from supraoptic neurons in superfused explants of rat hypothalamus. Under current clamp, voltage responses to hyperpolarizing current pulses featured depolarizing sags that were abolished by the I(H) blocker ZD 7288. Under voltage clamp, subtraction of current responses to hyperpolarizing steps recorded in the absence and presence of ZD 7288 was used to investigate the properties of I(H). Current-voltage analysis revealed that steady-state I(H) amplitude increases with hyperpolarization, with half-maximal activation of the underlying conductance occurring at -78 mV. The time course of activation of I(H) during hyperpolarizing steps was monoexponential with time constants (100-800 msec) decreasing with hyperpolarization. The effects of ZD 7288 on I(H) were slow (tau, approximately 15 min), irreversible, and half-maximal at 1.8 micrometer. When tested on continuously active MNCs, application of 30-60 micrometer ZD 7288 caused a significant reduction in firing rate. In phasically active MNCs, the drug decreased burst duration and intraburst firing frequency and caused an increase in the duration of interburst intervals. These effects were accompanied with a small hyperpolarization of the membrane potential. In contrast, ZD 7288 had no effect on spike duration, on the amplitude of calcium-dependent afterpotentials, or on the frequencies and amplitudes of spontaneous synaptic potentials. These results confirm the presence of I(H) in MNCs of the rat supraoptic nucleus and suggest that the presence of this conductance provides an excitatory drive that contributes to phasic and tonic firing.

The function of Ca(2+) channel subtypes in exocytotic secretion: new perspectives from synaptic and non-synaptic release.

By mediating the Ca(2+) influx that triggers exocytotic fusion, Ca(2+) channels play a central role in a wide range of secretory processes. Ca(2+) channels consist of a complex of protein subunits, including an alpha(1) subunit that constitutes the voltage-dependent Ca(2+)-selective membrane pore, and a group of auxiliary subunits, including beta, gamma, and alpha(2)-delta subunits, which modulate channel properties such as inactivation and channel targeting. Subtypes of Ca(2+) channels are constituted by different combinations of alpha(1) subunits (of which 10 have been identified) and auxiliary subunits, particularly beta (of which 4 have been identified). Activity-secretion coupling is determined not only by the biophysical properties of the channels involved, but also by the relationship between channels and the exocytotic apparatus, which may differ between fast and slow types of secretion. Colocalization of Ca(2+) channels at sites of fast release may depend on biochemical interactions between channels and exocytotic proteins. The aim of this article is to review recent work on Ca(2+) channel structure and function in exocytotic secretion. We discuss Ca(2+) channel involvement in selected types of secretion, including central neurotransmission, endocrine and neuroendocrine secretion, and transmission at graded potential synapses. Several different Ca(2+) channel subtypes are involved in these types of secretion, and their function is likely to involve a variety of relationships with the exocytotic apparatus. Elucidating the relationship between Ca(2+) channel structure and function is central to our understanding of the fundamental process of exocytotic secretion.

Cholera-like diarrhea in Canada. Report of a case associated with enterotoxigenic Escherichia coli and a toxin-producing Aeromonas hydrophila.

A 67-year-old Indian patient was admitted with an acute cholera-like illness. Toxigenic Escherichia coli producing both heat-stable and heat-labile enterotoxins grew from cultures of feces. In addition, an Aeromonas hydrophila producing a cytotoxic toxin was also isolated from this patient's feces. The unusual severity of this patient's illness may have resulted from coinfection with these two toxigenic organisms, although any role of the toxin produced by a hydrophila is speculative.

Mechanical basis of osmosensory transduction in magnocellular neurosecretory neurones of the rat supraoptic nucleus.

Rat magnocellular neurosecretory cells (MNCs) release vasopressin and oxytocin to promote antidiuresis and natriuresis at the kidney. The osmotic control of oxytocin and vasopressin release at the neurohypophysis is required for osmoregulation in these animals, and this release is mediated by a modulation of the action potential firing rate by the MNCs. Under basal (isotonic) conditions, MNCs fire action potentials at a slow rate, and this activity is inhibited by hypo-osmotic conditions and enhanced by hypertonicity. The effects of changes in osmolality on MNCs are mediated by a number of different factors, including the involvement of synaptic inputs, the release of taurine by local glial cells and regulation of ion channels expressed within the neurosecretory neurones themselves. We review recent findings that have clarified our understanding of how osmotic stimuli modulate the activity of nonselective cation channels in MNCs. Previous studies have shown that osmotically-evoked changes in membrane potential and action potential firing rate in acutely isolated MNCs are provoked mainly by a modulation of nonselective cation channels. Notably, the excitation of isolated MNCs during hypertonicity is mediated by the activation of a capsaicin-insensitive cation channel that MNCs express as an N-terminal variant of the transient receptor potential vanilloid 1 (Trpv1) channel. The activation of this channel during hypertonicity is a mechanical process associated with cell shrinking. The effectiveness of this mechanical process depends on the presence of a thin layer of actin filaments (F-actin) beneath the plasma membrane, as well as a densely interweaved network of microtubules (MTs) occupying the bulk of the cytoplasm of MNCs. Although the mechanism by which F-actin contributes to Trpv1 activation remains unknown, recent data have shown that MTs interact with Trpv1 channels via binding sites on the C-terminus, and that the force mediated through this complex is required for channel gating during osmosensory transduction. Indeed, displacement of this interaction prevents channel activation during shrinking, whereas increasing the density of these interaction sites potentiates shrinking-induced activation of Trpv1. Therefore, the gain of the osmosensory transduction process can be regulated bi-directionally through changes in the organisation of F-actin and MTs.

An investigation of the mechanisms of action of 5-hydroxytryptamine in the suppression of ethanol intake.

The effect of blockage of 5-hydroxytryptamine and norepinephrine uptake on voluntary ethanol consumption in rats was investigated. It was demonstrated that attenuation of ethanol intake occurred only as a result of treatment with specific 5-hydroxytryptamine uptake inhibitors. These results suggested that increasing the availability of central 5-hydroxytryptamine may in some way interfere with the positive reinforcing properties of ethanol. The second phase was designed to determine whether the attenuation of ethanol intake following blockade of 5-hydroxytryptamine uptake may be due to increased post-synaptic activity. Ethanol-preferring animals were pretreated with methergoline, a post-synaptic receptor blocker, followed by treatment with zimelidine, a 5-hydroxytryptamine uptake inhibitor. The results indicate that treatment with methergoline did not alter the zimelidine-induced attenuation of ethanol intake. Based on these results it is suggested that blockade of 5-hydroxytryptamine uptake produces an attenuation of ethanol intake but not as a result of increased post-synaptic activity.

Serial reconstruction of Lucifer yellow-labeled supraoptic nucleus neurons in perfused rat hypothalamic explants.

Intracellular recordings from supraoptic nucleus neurons in perfused explants of rat hypothalamus were followed by intracellular injections of the fluorescent dye, Lucifer yellow. Following fixation, 40 microns sections were processed for whole cell light-microscopic reconstruction in the horizontal or coronal plane. The somata of most supraoptic neurons were elongated (mean 25 X 13 microns) with 1-3 sparsely branched dendrites (length 30-725 microns) that displayed numerous spines. Most (95%) dendrites turned in the ventral direction to end in the glial lamina along the base of the nucleus. Each neuron had one axon: in 60% of cells, the axon arose from a dendritic profile and immediately assumed a varicose appearance; in the other 40% of cells, the axon appeared to arise directly from the soma and demonstrated in its initial 80-200 microns numerous spines and few varicosities, i.e. the morphological features of a dendrite. All axons coursed in a dorsomedial direction over the optic tract. At this point, most axons revealed smaller secondary processes 2-15 microns in length. Axons then turned ventrally towards the basal hypothalamus; some could be followed for up to 2100 microns from the cell somata. This approach to the light microscope morphology of supraoptic neurons provides a surprising array of detail on soma, dendrite and axon characteristics, while retaining the overall relationship between individual neurons and neighboring structures, including the boundaries of the nucleus itself.