NHLRC1 repeat expansion in two beagles with Lafora disease.

Lafora disease is a fatal genetic disorder characterised by neurotoxic deposits of malformed insoluble glycogen. In humans it is caused by mutation in the EPM2A or NHLRC1 genes. There is a known mutation in miniature wirehaired dachshunds which has not been documented in other dog breeds, including beagles, in which the disease is relatively commonly reported. This case report describes the causative defect in two affected beagles, namely the same massive expansion as in miniature wirehaired dachshunds of a 12-nucleotide repeat sequence that is unique to the canine NHLRC1 gene. This is the first mutation described in beagles with Lafora disease, and so far the only Lafora disease genetic variant in dogs.

Addisonian crisis in a dog treated with phenobarbitone.

BACKGROUND: A 2-year-old intact female Irish Setter was presented with a 1-week history of anorexia, lethargy, vomiting and diarrhoea. Previous medical therapy included a 3-week treatment with phenobarbitone for suspected idiopathic epilepsy. In humans, phenobarbitone accelerates metabolism of both exogenous and endogenous steroids. CASE REPORT: Based on history, the physical examination showing abnormal mentation and laboratory abnormalities including azotaemia, hyponatraemia and hyperkalaemia, Addisonian crisis was suspected. An adrenocorticotropic hormone stimulation test was performed and confirmed the diagnosis. Treatment with intravenous fluid therapy, glucocorticoids and mineralocorticoids led to a resolution of clinical signs in 3 days. CONCLUSION: To the authors' current knowledge, this is the first reported case of Addisonian crisis in a dog most probably related to phenobarbitone administration. As Addisonian crisis can be life-threatening, clinicians should be aware of this adverse effect of phenobarbitone and use it cautiously in dogs with borderline hypoadrenocorticism.

[Sleep disorders and the 24-hour profile of melatonin and cortisol]. / Poruchy spánku a 24 hodinový profil melatoninu a kortizolu.

The aim of our study was to assess the prevalence of the specific disorders of the circadian rhythm of cortisol and melatonin in the patients with sleep disorders. The group of our patients consisted of 93 persons (25 men, 68 women), from 4-72 years (mean age 38.3 years) with a sleep disorders. These patients were studied on Department of Neurology of 1st Medical Faculty of Charles University in Prague during years 1997-1999. Patients were divided by the clinical diagnosis into many subgroups: idiopathic hypersomnia (IH) 24 patients and other hypersomnias 8, narcolepsy 22, degenerative disorders 9, delay sleep phase syndrome (DSPS) 7, periodic leg movements syndrome (PLMS) 6, insomnia 7 and Parkinson's disease 10 patients. Twelve salivary samples were taken from each patient during a period of 24 hours in order to investigate the circadian secretion pattern of melatonin and cortisol. Salivary melatonin and cortisol levels were estimated by radioimmunoassay in the Department of Physiology of the Academy of Science, Czech Republic. Significant differences were found between our patients and the control group in idiopathic hypersomnia--acrophase of melatonin was delayed and secretion was prolonged. Patients suffered from narcolepsy often displayed multiple peak of melatonin secretion. The peak of the melatonin concentration occurred later in DSPS (non significantly). Low level of melatonin and prolonged signal of melatonin was in Parkinson's disease patients.

Adjustment of the human circadian system to changes of the sleep schedule under dim light at home.

In a field study at home, eight volunteers experienced a sleep period from 01:00 to 09:00 h encompassed by dim light for 6 days, then for another 6 days they were subjected to a 3-h advance of the sleep period, and for the last 6 days they were subjected to a 3-h delay of the sleep period, i.e. they experienced again the original sleep schedule. Following the 3-h advance of the sleep period, the circadian salivary melatonin and cortisol rhythms phase advanced by about 1 h within 6 days as compared with the original rhythm profiles, following the subsequent 3-h delay of the sleep period, both rhythms phase delayed by about 1 h within 6 days and returned roughly to their original phase. The data indicate that shifting of the sleep time under dim light at home, which may occur commonly in everyday life, phase shifts the human circadian system.

Arginine vasotocin activates phosphoinositide signal transduction system and potentiates N-acetyltransferase activity in the rat pineal gland.

The pineal gland is innervated by pinealopetal peptidergic fibers originating in the hypothalamic nuclei which release arginine vasopressin (AVP) and arginine vasotocin (AVT) from their endings. Since the mechanism of AVT action on the pineal signal transduction and melatonin synthesis has not been determined so far, we examined the effect of AVT on the phosphoinositide signalling system and the N-acetyltransferase (NAT) activity in the rat pineal gland. The effect of AVP 4-9 fragment and AVP analogue desmopressin was also tested. The phosphoinositide signalling system was studied by measuring 32P labelling of phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP) and phosphatidylinositol bisphosphate (PIP2) which reflects PI cycle activation. AVT (10(-5) and 10(-4) M) induced a significant increase in 32P labelling of PI, PIP and PIP2. The AVT mediated activation of the PI signal cascade was supressed by the vasopressin V1 receptor antagonist. The desmopressin and AVP 4-9 fragment were without the effect on PI signalling. To assess the AVT role in the melatonin synthesis we studied the daily pattern of the pineal NAT activity in rats treated by AVT (10 microg/100 g b.w). AVT application in the dark period of the day significantly increased nocturnal NAT activity. It can be summarized that AVT activates PI signalling system and potentiates NAT activity in the rat pineal gland.

Adjustment of the human melatonin and cortisol rhythms to shortening of the natural summer photoperiod.

Fifteen human subjects were exposed to natural outdoor summer light from 0415 h until 2000 h for 4 days and then from 0800 h until 1600 h for another 4 days. Following shortening of the natural summer photoperiod, times of the morning salivary melatonin decline and cortisol rise did not change whereas the time of the evening melatonin rise phase advanced by about 1.5 h within 1 day and further did not change significantly. Consequently, the melatonin signal duration extended markedly within 1 day. The data show that the compressed melatonin rhythm waveform in humans experiencing a long natural summer photoperiod from sunrise until sunset may change rapidly following a shortening of the photoperiod.

Exposure to long summer days affects the human melatonin and cortisol rhythms.

Exposure of 8 human subjects in summer to a natural 16 h bright light photoperiod phase advanced the morning salivary melatonin decline and cortisol rise and shortened the nocturnal melatonin signal by 2 h relative to the winter patterns of the same subjects followed under a combined artificial and natural light 16 h photoperiod. The data suggest that summer days experienced from sunrise till sunset and not winter days with a combined artificial and natural light long photoperiod evoke a true long day response of the human circadian system.

A fixed morning awakening coupled with a low intensity light maintains a phase advance of the human circadian system.

The next day after an early morning awakening of human subjects coupled with a single exposure to bright light from 0600 to 0900 h, the salivary melatonin rhythm was phase advanced by about 1 h as compared with the pre-treatment profile. The phase advance was maintained for 4 days when the subjects adhered to the 0600 h awakening coupled with just low intensity light, but vanished when they slept till 0900 h. Similarly, 3 days following a twice repeated exposure to bright light from 0600 to 0900 h coupled with an early morning awakening, the salivary melatonin rhythm was phase advanced relative to the pre-treatment profile only in the subjects who adhered to the 0600 h awakening but not in those who slept till 0900 h. The findings may be used for treatment of chronobiological disorders.

Acute and chronic effects of potassium and noradrenaline on Na+, K+-ATPase activity in cultured mouse neurons and astrocytes.

Acute and chronic effects of elevated extracellular concentrations of potassium ions ([K+]0) and/or noradrenaline were studied in homogenates of primary cultures of mouse astrocytes, from the cerebral cortex or the spinal cord, and of primary cultures of mouse cerebral cortical neurons. NA+, K+-ATPase activity in cerebral cortical astrocytes showed a Km value of 1.9 mM with confidence limits of 1.3-2.9 mM and a Vmax of 5.4 mumol/h/mg protein with confidence limits of 3.3-8.1 mumol/h/mg protein. Due to the high Km value, the activity of the enzyme was significantly increased by an increase in [K+]0 in the interval 5-12 mM. In cerebral cortical neurons, Vmax was lower (1.77 +/- 0.06 mumol/h/mg protein) but the affinity was higher (Km 0.43 +/- 0.8 mM). With these kinetics, there is no stimulation of enzyme activity when [K+]0 is increased beyond control levels. In spinal cord astrocytes, the relative effect of increasing [K+]0 above 6 mM was larger than in cerebral astrocytes but the absolute activity of the enzyme was lower. Na+, K+-ATPase activity in both types of astrocyte was stimulated by noradrenaline and its beta-adrenergic subtype agonist isoproterenol but mainly or exclusively at 6 mM [K+]0. Noradrenaline also caused a stimulation in cortical neurons, but at non-physiological K+ concentrations this stimulation was converted to an inhibition, and isoproterenol had no stimulatory effect. Chronic exposure of cerebral cortical astrocytes to elevated [K+]0 caused a decrease in Na+, K+-ATPase activity when enzyme activity in the cells was subsequently measured at normal [K+]0. During exposure to 30 mM [K+]0 this "down-regulation" took place within 10 min. Conversely, chronic exposure to reduced [K+]0 led to an increase in Na+, K+-ATPase activity. Chronic exposure to noradrenaline had no significant effect but there was a tendency towards an increase.

Astrocyte-neuron interaction during one-trial aversive learning in the neonate chick.

During two specific stages of the Gibbs-Ng model of one-trial aversive learning in the neonate chick, we have recently found unequivocal evidence for a crucial involvement of astrocytes. This evidence is metabolic (utilization of the astrocyte-specific energy store, glycogen, during normal learning and inhibition of memory formation by the astrocyte specific metabolic inhibitors, fluoroacetate and methionine sulfoximine) as well as physiological (abolition of memory formation in the presence of ethacrynic acid, an astrocyte-specific inhibitor of cellular reaccumulation of potassium ions). These findings are discussed in the present review in the framework of a more comprehensive description of metabolic and physiological neuronal-astrocytic interactions across an interstitial (extracellular) space bounded by minute processes from either cell type.

Signalling effect of elevated potassium concentrations and monoamines on brain energy metabolism at the cellular level.

The effects of elevated K+ concentrations and monoamine transmitters on different cell types in the CNS and on different subcellular structures in these cells are reviewed. Pronounced differences exist in the metabolic processes that are stimulated by excess K+ and by adrenergic agonists, e.g., noradrenaline. An elevation in the extracellular K+ concentration appears to enhance neuronal-astrocytic interaction by stimulating metabolic processes involved in (1) the promotion of supply of precursors for transmitter glutamate, and (2) reestablishment of resting ion distribution following neuronal excitation. The monoamine transmitters stimulate energy production and Na+,K(+)-ATPase activity in astrocytes in a complex manner and, in so doing, facilitate their role in ion regulation. However, in contrast to excess K+, they do not enhance the production of astrocytic precursors for neuronal glutamate production. Emphasis is placed on possible profound differences in metabolic effects on excitatory and inhibitory neurotransmission and on the importance of stimulation of glycolytic metabolism in astrocytes versus oxidative metabolism in neurons.

Na+,K(+)-ATPase activity in young chicks after taste stimulation.

The activity of specific ouabain-sensitive Na+,K(+)-ATPase was studied in crude membrane fraction of the brain of 1- to 3-day-old chicks after the administration of a chemical aversant methylanthranilate (MeA), shown in previous behavioral studies to induce avoidance of pecking of an otherwise attractive stimulus. Enzyme activity was dramatically decreased (by 40-50%) in the time interval between 10 min-2 h after MeA administration onto the tongue of awake chicks. It was possible to localize these changes in Na+,K(+)-ATPase activity into forebrain structures contained within the dorsal ventricular ridge comprising the hyperstriatum accessorium (HA), hyperstriatum ventrale (HV), hyperstriatum dorsale (HD), and parts of neostriatum (N). In contrast, Na+,K(+)-ATPase activity in the ectostriatum (E), the medial neostriatum (NM), and the paleostriatal complex were unaffected. Results from experiments involving preincubation of membrane fractions and with partial purification using detergents, suggest that some substances with inhibitory effects were produced under the effect of MeA and bound to membrane fractions in their respective areas. A similar decrease of Na+,K(+)-ATPase activity as after MeA administration in vivo was observed when inhibitory mediators (GABA, glycine) were added to membrane fractions in vitro. These findings may have implications for memory processing in chicks following aversive learning using MeA as the aversant.

Effect of vasopressin on brain swelling at the cellular level: do astrocytes exhibit a furosemide--vasopressin-sensitive mechanism for volume regulation?

Two sets of new observations are reported: (i) astrocytes in primary cultures show an increased potassium-induced swelling in the presence of 1-100 x 10(-12) M vasopressin, whereas no similar phenomenon is found in primary cultures of neurons, and (ii) the furosemide-sensitive cotransport system for uptake of K+, Na+, and Cl-, which is known to exist in astrocytes, is absent in neurons. On the basis of these findings and observations by other investigators on transport of ions and water in the brain in vivo, a novel mechanism is suggested, according to which all boundaries of brain parenchymal tissue (perivascular astrocytic end-feet, glia limitans, and ependyma) in the absence of vasopressin are capable of performing a net uptake of K+, Na+, and Cl- without uptake of water, and that the resulting hyperosmolarity in the presence of vasopressin leads to water uptake (cell swelling), which causes a reduction in the amount of water in the interstitial fluid and thus an increase in extracellular concentrations of ions.

Opioid receptors in the rat spinal cord after longlasting deafferentation.

In vitro binding of specific opioid ligands to their respective sites in membrane fractions and the contribution of individual receptor classes (mu, delta, kappa) was studied in rats after longlasting (up to 22 months) section of spinal dorsal roots at the cervical (C5-8) or thoracic (Th1-4) level. This procedure leads to autotomy or scratching of the skin on the operated side. The total number of receptors in the cervical and thoracic spinal cord was more than doubled in both operated and contralateral part of the cord in comparison with intact controls of the same age. In the cervical region, this increase mainly represented a rise in the number of free receptors, whilst in the thoracic region both free and saturated receptors were increased. On the deafferented side, receptor selectivity, especially in the delta and kappa types was decreased.

Na(+), K(+) ATPase activity in membrane fractions of rat central and peripheral nervous tissues modified by opioids.

The inhibition by ouabain of Na(+), K(+) ATPase activity in lumbar spinal cord membranes followed a sigmoid curve and in sciatic nerve membranes an exponential curve. The degree of inhibition in spinal cord depended on the presence of opioid ligands; fractions became more sensitive to ouabain after removal of bound opioids. The saturation of receptors by specific ligands reversed the effect of preincubation. Opioids had no effect on Na(+), K(+) ATPase in sciatic nerve. Membranes isolated from spinal cord were highly sensitive to oubain inhibition and their Na(+), K(+) ATPase resembled that containing the ? (+) subunit; membranes from peripheral nerve were less sensitive to ouabain and their enzyme reacted as that containing the ? subunit.

Increase in extracellular potassium level in rat spinal dorsal horn induced by noxious stimulation and peripheral injury.

Changes in extracellular K+ concentration ([K+]e) in dorsal horn were studied by means of double-barrel K+-sensitive microelectrodes in rats anesthetized with Nembutal. Acute nociceptive stimuli (pinch, press, heat) applied to the hind paw induced a transient increase in [K+]e of about 0.1-0.5 mM which persisted for 5-30 s. Regional variations in [K+]e were found in unstimulated rat spinal cords. The K+ level in the lower dorsal horn (laminae III-V) was by about 0.4 +/- 0.06 mM higher than that in more superficial laminae and in the ventral horn. Chemical or thermal injury was evoked by: s.c. injection of 0.1-0.5 ml formalin or turpentine into the hind paw, by application of mustard oil onto the skin, or by thermal injury of the hind paw. These produced a long-term increase in [K+]e in the lower dorsal horn by 0.3-3.0 mM in 75% of animals (n = 27). The increase in [K+]e began 5-15 min after injury and persisted for more than 2 h. In the rest of the animals, occasional elevation in [K+]e of 0.1-0.3 mM were observed. The K+ increases evoked by acute nociceptive stimuli as well as by injury were blocked by preinjecting the hind paw with 1% procaine. However, when procaine was applied 20 min or later after injury, the evoked long-term rise in [K+]e was not affected. This shows that the long-term K+ accumulation results from self-sustained abnormal neuronal firing induced in the dorsal horn by injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of carbon dioxide on extracellular potassium accumulation and volume in isolated frog spinal cord.

A 6-10-fold increase in pCO2 in the superfusing Ringer solution increased the volume of the extracellular space (ECS) and changed the spatial distribution and amplitude of the extracellular K+ accumulation which resulted from dorsal root stimulation. Using the increase in tetraethylammonium concentration [( TEA+]) resulting from iontophoretic injection of that ion in the extracellular fluid as an indication of the volume of the ECS, it was found that in high pCO2 the ECS volume in spinal dorsal horn increased by more than 60%. In addition, in the presence of raised pCO2 we also observed the following: (1) The rate of diffusion of TEA+ into the dorsal horn increased. (2) The accumulation of K+ evoked by single or tetanic stimulation of the dorsal root was less. (3) The clearance of K+ was slowed down. (4) The regions where K+ accumulated were more restricted. (5) The K+ evoked depolarization of the primary afferent fibres decreased. (6) In contrast to TEA+, the rate of diffusion of K+ into the dorsal horn decreased. The effects of an increase in pCO2 on K+ accumulation and clearance appear to result from an increase in ECS volume and a possible decrease in glial electrical coupling which interferes with glial spatial buffering of K+.

Changes in 3H-leucine enkephalin binding in spinal cord of frog after dorsal rhizotomy, cordotomy, transcutaneous stimulation and temperature variations: correlation with nociceptive sensitivity.

In the frog spinal cord about 50% of the 3H-leucine enkephalin (3H-LE) binding sites (b.s.) were blocked by an endogenous ligand. Three days after deafferentation and cordotomy the number of free b.s. increased by 44 and 56%, respectively. In spinal frogs the threshold of the flexor reflex responses evoked by nociceptive stimuli decreased. More than 7 days after deafferentation and cordotomy the number of both total and free 3H-LE b.s. decreased, while the threshold of the flexor reflex responses returned to that before spinalization. Transcutaneous electrical stimulation (TES) of the hind limbs (30 Hz, 5 minutes) in frogs spinalized 3 hours earlier increased 3H-LE binding at low intensities of stimulation (0.2 mA) and decreased the threshold of the flexor reflex responses. TES at higher intensities (1.0 mA) decreased 3H-LE binding and increased the threshold. Three days after spinalization TES even at low intensity diminished 3H-LE binding and raised flexor reflex threshold. A decrease in the number of free 3H-LE b.s. was found when the frog body temperature was elevated (from 15 to 24 degrees C) or lowered (from 15 to 1 degrees C) for 14 days and was accompanied by an increase in flexor reflex threshold. The data suggest the existence of an endogenous opioidergic system in the frog spinal cord which has a high degree of tonic activity.

The effect of opioids and of naloxone on Na+,K+-adenosine triphosphatase activity in frog spinal cord membrane fractions.

The effects of opioids and of naloxone on ouabain-sensitive Na+,K+-adenosine triphosphatase (ATPase) activity were studied in vitro on membrane fractions from frog spinal cords. The addition of morphine and of the stable enkephalin analogue, D-Ala2,D-Leu5-enkephalin, in concentrations from 10(-7) to 10(-4) M significantly increased Na+,K+-ATPase activity. No effect was found with methionine enkephalin (Met-Enk). However, the addition of two peptidase inhibitors, captopril and phosphoramidon (10(-5) M each), significantly increased Na+,K+-ATPase activity. A further increase in enzyme activity was found when Met-Enk (10(-4) or 10(-7) M) was added simultaneously with peptidase inhibitors. On the other hand, the addition of the opiate antagonist, naloxone, at low concentration (10(-7) M) decreased the activity of Na+,K+-ATPase. These results are discussed with respect to the effect of synthetic and endogenous opioids on the activity of Na+,K+-ATPase.