The 5-HT(1A) receptor active compounds (R)-8-OH-DPAT and (S)-UH-301 modulate auditory evoked EEG responses in rats.

Schizophrenics commonly demonstrate abnormalities in central filtering capability following repetitive sensory stimuli. Such sensory inhibition deficits can be mirrored in rodents following administration of psycho-stimulatory drugs. In the present study, male Sprague-Dawley rats were implanted with brain surface electrodes to record auditory evoked EEG potentials in a paired-stimulus paradigm, using 87 dB clicks delivered 0.5 s apart. Amphetamine (1.83 mg/kg, i.p.) produced the expected loss of sensory inhibition, as defined by an increase in the ratio between test (T) and conditioning (C) amplitudes at N40, a mid-latency peak of the evoked potentials. Also, the 5-HT(1A) agonist (R)-8-OH-DPAT caused a significant increase in the TC ratio at the highest dose studied (0.5 mg/kg s.c.), while the 5-HT(1A) antagonist (S)-UH-301 did not significantly affect the TC ratio at any dose studied (0.1-5 mg/kg s.c.). When administered with amphetamine, a lower dose of 8-OH-DPAT (0.1 mg/kg) and the highest dose of UH-301 tested (5 mg/kg, s.c.) were able to reverse the amphetamine-induced increase in TC ratio. The findings suggest that 5-HT(1A) signaling is involved in sensory inhibition and support the evaluation of 5-HT(1A) receptor active compounds in conditions with central filtering deficits, such as schizophrenia.

Effects of perinatal asphyxia on cell survival, neuronal phenotype and neurite growth evaluated with organotypic triple cultures.

The effect of perinatal asphyxia on brain development was studied with organotypic cultures from substantia nigra, neostriatum and neocortex. Asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Following asphyxia, the pups were nursed by a surrogate dam and sacrificed after three days for preparing organotypic cultures. Non-asphyxiated caesarean-delivered pups were used as controls. Morphological features and cell viability were recorded during in vitro development. At day in vitro (DIV) 24, the cultures were treated for immunocytochemistry using antibodies against the N-methyl-D-aspartate receptor subunit 1 (NR1) and tyrosine hydroxylase (TH). While in vitro survival was similar in cultures from both asphyxiated and control animals, differences were observed when the neuronal phenotype was assessed. Compared to controls, the total number of NR1-positive neurons in substantia nigra, as well as the number of secondary to higher level branching of TH-positive neurites from asphyxiated pups were decreased, illustrating the vulnerability of the dopaminergic systems to perinatal asphyxia.

Efficacy of trimethoprim-sulfadoxine against Escherichia coli in a tissue cage model in calves.

Tissue cages implanted subcutaneously in calves were infected with Escherichia coli. Twenty-four hours later, the calves were treated either with single doses of 2.5 + 12.5 or 5 + 25 mg/kg trimethoprim (TMP) + sulfadoxine (SDX) or with five doses of 7.5 + 37.5 mg/kg TMP + SDX at 12-h intervals. In addition, one cage in each of three calves in the highest dose group was infected 3 h after initiation of treatment. Untreated calves were kept as controls. Concentrations of TMP and SDX in plasma and tissue cage fluid (TCF) and counts of viable bacteria in TCF were determined. In the highest dose group, concentrations of TMP in TCF remained above the minimum inhibitory concentration of the test strain for 94-101 h and peak to minimum inhibitory concentration (MIC) ratio was close to 10. In spite of this, an effect of treatment was noted only in cages infected after initiation of treatment. In vitro studies and analysis of thymidine content in serum and TCF from calves suggest that levels of thymidine in TCF are high enough to antagonize the antibacterial effect of TMP. The results indicate that soft tissue infections in secluded infection sites of calves are refractory to treatment with TMP + SDX.

Insulin sensitivity of heifers on different diets.

The hyperinsulinemic euglycemic clamp technique was used to investigate the effect on insulin sensitivity of 2 different diets used in practical cattle feeding in calves. Ten 4 to 5-month-old heifer calves were allocated to 2 feeding groups, LO or HI, to obtain growth rates of 400 g/day or 900 g/day. The heifers were fed and housed individually for 5 weeks. Growth rates close to calculated rates were obtained with the diets used. Weekly blood samples were collected from the jugular vein for analysis of glucose, insulin, cortisol, total serum protein, urea, cholesterol and non-esterified fatty acids. During week 5, insulin sensitivity was estimated using the hyperinsulinemic euglycemic clamp technique. Insulin sensitivity did not differ between the groups, but the plasma glucose levels were higher during weeks 3 and 4 for the HI group compared to the LO group. It may be concluded that the amount of concentrate in the diet was too low to induce changes in either the basal plasma insulin levels or the insulin sensitivity in the HI group.

The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation.

Several pathogenic Alzheimer's disease (AD) mutations have been described, all of which cause increased amyloid beta-protein (Abeta) levels. Here we present studies of a pathogenic amyloid precursor protein (APP) mutation, located within the Abeta sequence at codon 693 (E693G), that causes AD in a Swedish family. Carriers of this 'Arctic' mutation showed decreased Abeta42 and Abeta40 levels in plasma. Additionally, low levels of Abeta42 were detected in conditioned media from cells transfected with APPE693G. Fibrillization studies demonstrated no difference in fibrillization rate, but Abeta with the Arctic mutation formed protofibrils at a much higher rate and in larger quantities than wild-type (wt) Abeta. The finding of increased protofibril formation and decreased Abeta plasma levels in the Arctic AD may reflect an alternative pathogenic mechanism for AD involving rapid Abeta protofibril formation leading to accelerated buildup of insoluble Abeta intra- and/or extracellularly.

Anticonvulsant effects of the 3-hydroxyanthranilic acid dioxygenase inhibitor NCR-631.

The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the pro-convulsive excitotoxin quinolinic acid. In the present study, the anticonvulsant effect of the 3-HAO inhibitor NCR-631 was investigated in models of chemically- and sound-induced seizures. Administration of NCR-631 i.c.v. at a dose of 300nmol in Sprague-Dawley rats was found to prolong the latency of occurrence of pentylenetetrazole (PTZ)-induced seizures. Also systemic pre-treatment with NCR-631 s.c. in N.M.R.I. mice subjected to PTZ-induced seizures provided an increase in the latency until onset of seizures, concomitant with a reduction in the severity of the seizures. However, the anticonvulsant effect of NCR-631 was short lasting (15-30min), and only observed at a dose of 250 mg/kg. A similar dose- and time-dependent anticonvulsant effect of NCR-631 was found in seizure-prone DBA/2J mice following sound-induced convulsions. Hence, the findings show that NCR-631 has anticonvulsant properties against generalized tonic-clonic seizures of different origin, suggesting that it may constitute a useful tool to study the role of kynurenines in various convulsive states.

3-Hydroxyanthranilic acid accumulation following administration of the 3-hydroxyanthranilic acid 3,4-dioxygenase inhibitor NCR-631.

In the kynurenine pathway of tryptophan metabolism, 3-hydroxyanthranilic acid is the substrate for formation of the excitotoxin quinolinic acid by 3-hydroxyanthranilic acid 3, 4-dioxygenase. This study was designed to characterize the effects on 3-hydroxyanthranilic acid after treatment with the 3-hydroxyanthranilic acid 3,4-dioxygenase inhibitor 4, 6-di-bromo-3-hydroxyanthranilic acid (NCR-631) in Sprague-Dawley rats. The blood plasma and brain concentrations of 3-hydroxyanthranilic acid were found to increase rapidly in a dose-dependent manner after gavage administration of NCR-631. However, the effect was relatively transient, with a decline in 3-hydroxyanthranilic acid levels already at 1h after NCR-631 treatment. Similar increases in plasma levels of 3-hydroxyanthranilic acid were observed following either gavage or parenteral (i.v. or s.c.) administration of NCR-631 (25 mg/kg). Only a minor enhancement of the NCR-631-induced increase in plasma 3-hydroxyanthranilic acid levels was found after sub-chronic treatment (25 mg/kg by gavage; 7 days, b.i.d.), suggesting a low propensity for altered 3-hydroxyanthranilic acid 3,4-dioxygenase activity following repeated inhibition. Administration of [14C]NCR-631 suggested 20 min initial plasma half life and an oral absorption around 50%. A dose of 250 mg/kg [14C]NCR-631 given by gavage provided plasma levels of almost 2 micromol/ml and a brain concentration of approximately 16 nmol/g, when analyzed 15 min after administration. Neither acute nor sub-chronic administration of NCR-631 caused any substantial effects on quinolinic acid levels in plasma or brain. Also, the plasma levels of kynurenic acid, another neuroactive kynurenine pathway metabolite, were unaffected by acute NCR-631 treatment. Moreover, the brain levels of the major cerebral tryptophan metabolites 5-hydroxytryptamine and 5-hydroxyindoleacetic acid remained unchanged following administration of NCR-631. Although reversible inhibition of 3-hydroxyanthranilic acid 3, 4-dioxygenase with NCR-631 in normal rats is insufficient to cause substantial changes in the levels of quinolinic acid or other important tryptophan metabolites, it causes a major accumulation of the substrate 3-hydroxyanthranilic acid.

Expression of presenilin 1 mRNA in rat peripheral organs and brain.

At least 50 different mutations in the presenilin 1 gene have been shown to cause early onset familial Alzheimer's disease. Although presenilin 1 has an obvious role in the pathogenesis of Alzheimer's disease, its function is still unknown. In the present study, the occurrence and distribution of presenilin 1 mRNA was examined in rat peripheral organs as well as in the brain by in situ hybridization histochemistry, using a radiolabelled oligonucleotide probe. In comparison to the brain, a high presenilin 1 mRNA expression was found in the testis, kidney, spleen, adrenal gland and thymus. It was also observed in skeletal muscle, liver, small intestine and lung, whereas no presenilin 1 could be detected in the heart, spinal cord and pancreas. Since presenilin 1 mRNA was found to be abundant in peripheral tissues which apparently are not affected in Alzheimer's disease, additional functions of presenilin 1 are suggested, unrelated to its role in the pathological processes of the disease.

Flumethasone-induced insulin resistance in calves.

The impact of one single therapeutic dose of flumethasone (0.5 mg/100 kg b.w.) on insulin sensitivity was studied in calves. Hyperinsulinemic, euglycemic clamp tests were performed before and after flumethasone treatment. At 24 h after treatment, insulin-dependent glucose utilization was reduced by 74% (P < 0.005). No significant changes occurred 72 h post-treatment.

Clenbuterol-induced insulin resistance in calves measured by hyperinsulinemic, euglycemic clamp technique.

Hyperinsulinemic, euglycemic clamp tests were performed on calves before and after clenbuterol treatment. Clenbuterol was given as 2 intramuscular injections with an interval of about 12 h. The dose used was 1 microgram/kg b.w. The treatment resulted in increased plasma levels of insulin and glucose. The results of the clamp tests showed that clenbuterol induced a transient decrease in insulin sensitivity. Both insulin mediated glucose disposal (M), expressed as mumol/kg live b.w./min. and the M/I-index (M divided by the average insulin concentration at steady state) were significantly reduced after treatment. The effect of clenbuterol on carbohydrate metabolism seemed to be rather short-lived, since significant changes occurred only in animals treated 5-6 h prior to the test. According to the literature, the metabolic effects of clenbuterol have been studied only after the high doses used for growth promoting purposes. The results from the present study showed that similar changes occur also after doses at the therapeutic level. The hyperinsulinemic, euglycemic clamp test was considered to be a valuable tool for the study of insulin sensitivity in cattle.

Effects of the 3-hydroxyanthranilic acid analogue NCR-631 on anoxia-, IL-1 beta- and LPS-induced hippocampal pyramidal cell loss in vitro.

The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the putative neuropathogen quinolinic acid (QUIN). In the present study, the neuroprotective effects of the 3-HANA analogue and 3-HAO inhibitor NCR-631 was investigated using organotypic cultures of rat hippocampus. An anoxic lesion was induced by exposing the cultures to 100% N2 for 150 min, resulting in a pronounced loss of pyramidal neurons, as identified using NMDA-R1 receptor subunit immunohistochemistry. NCR-631 provided a concentration-dependent protective effect against the anoxia. NCR-631 was also found to counteract the loss of pyramidal neurons in two models of neuroinflammatory-related damage; incubation with either LPS (10 ng/ml) or IL-1 beta (10 IU/ml). The findings suggest that NCR-631 has neuroprotective properties and that it may be a useful tool to study the role of kynurenines in neurodegeneration.

Pharmacokinetics of penicillin-G in ewes and cows in late pregnancy and in early lactation.

Concentrations of penicillin-G in serum were determined after single intravenous doses of potassium penicillin-G (10 mg/kg body wt) to ewes and cows in late pregnancy and in early lactation. Penicillin-G in serum was analysed by a microbiological method and pharmacokinetic parameters were calculated by model-independent methods. Serum concentrations were lower in early lactation than in late pregnancy in both ewes and cows. The differences were statistically significant at most sampling times in ewes but only during the first hour in cows. Weight corrected values of clearance and volume of distribution were significantly higher in early lactation than in late pregnancy in both ewes and cows. Mean residence time and elimination-half life were shorter in lactating than in pregnant ewes, however for the latter parameter the difference was not statistically significant. Neither of these parameters changed in cows. It is concluded that in both ewes and cows pharmacokinetic parameters of penicillin-G are altered from late pregnancy to early lactation but that these changes have little practical impact and do not call for a revised dosage regime of the studied drug.

Delayed neuronal death following perinatal asphyxia in rat.

The consequences of perinatal asphyxia on the rat brain were studied 80 min to 8 days after birth with hematoxylin-eosin and in situ DNA double-strand-breaks labeling histochemistry. Asphyxia was induced by immersing fetus-containing uterus horns, removed from ready-to-deliver Sprague-Dawley rats, in a water bath at 37 degrees C for various time periods (0-22 min). Spontaneous- and cesarean-delivered pups were used as controls. Perinatal asphyxia led to a decrease in the rate of survival, depending upon the length of the insult. No gross morphological changes could be seen in the brain of either control or asphyctic pups at any of the studied time points after delivery. However, in all groups, nuclear chromatin fragmentation, corresponding to in situ detection of DNA fragmentation, was observed at different stages. Nuclear fragmentation in control pups showed a specific distribution that appeared to be related to brain maturation, thus indicating programmed cell death. A progressive and delayed increase in nuclear fragmentation was found in asphyctic pups, which was dependent upon the length of the perinatal insult. The most evident effect was seen in frontal cortex, striatum, and cerebellum at postnatal day 8, although changes were also found in ventral-posterior thalamus, at days 1 and 2. Thus, nuclear chromatin fragmentation in asphyctic pups indicates a delayed post-asphyctic neuronal death. The absence of signs of inflammation or necrosis suggests that delayed neuronal cell death following perinatal asphyxia is an active, apoptosis-like phenomenon.

Low-level cadmium exposure of lactating rats causes alterations in brain serotonin levels in the offspring.

Effects on monoaminergic and cholinergic transmitter systems as well as neurotrophins were characterized in developing Sprague-Dawley rats directly exposed to 5 ppm cadmium in the drinking water or indirectly via exposed dams. Cadmium was given to dams during the lactation period, from parturition to postnatal day 17, and/or to the offspring until postnatal day 42. Cresyl violet staining and glial fibrillary acidic protein immunohistochemistry did not reveal any obvious neuropathology after cadmium exposure. Following high-power microwave fixation, concentrations of acetylcholine (ACh) and monoamines were determined in cerebral cortex, striatum, and hippocampus using HPLC with electro-chemical detection. ACh, dopamine, and noradrenaline levels were not significantly affected after the different cadmium exposures. Cortical levels of serotonin were significantly reduced in rats exposed to cadmium during lactation as well as in rats exposed to cadmium during both lactation and postweaning. A major decrease in 5-hydroxyindoleacetic acid was found in cortex and hippocampus in rats exposed to cadmium during lactation. The regional characteristics of cadmium toxicity as reflected in changes of neurotrophins were studied using in situ hybridization histochemistry with oligonucleotide probes and phosphoimaging evaluation. No significant changes in the mRNA expression of brain-derived neurotrophic factor (BDNF), neurotrophin-3, and the high-affinity tyrosine kinase receptor of BDNF, trkB, were detected. The present results demonstrate that exposure to levels of cadmium as low as 5 ppm in the drinking water leads to neurochemical disturbances of the serotonergic system in the offspring during the lactational period.

Trimethyltin exposure in the rat induces delayed changes in brain-derived neurotrophic factor, fos and heat shock protein 70.

Trimethyltin chloride (TMT) treatment in adult rats leads to limbic brain lesions that are detectable with classical neuropathological techniques 3 days after exposure. In particular, the hippocampal cells of the CA3c region are affected. The temporal and regional characteristics of TMT toxicity as reflected in changes of activity-dependent factors were studied in adult male Sprague-Dawley rats using quantitative in situ hybridization and immunohistochemistry. No significant alterations in the BDNF mRNA were detected in hippocampus and cerebral cortex 1 and 4 h after 8 mg TMT/kg. Three days after TMT, a significant increase in BDNF mRNA was detected in CA1, and increases in BDNF mRNA were also seen in cortical layers. An increase in BDNF hybridization signal was seen over scattered neurons within and outside CA3c at 3 days. Four h after 8 mg TMT/kg, BDNF immunoreactivity was reduced in the pyramidal cells of the CA3c and CA1 regions as well as in the dentate gyrus. No significant change in BDNF immunoreactivity was seen in hippocampus or cerebral cortex 3 days after TMT. BDNF interacts with the high-affinity receptor tyrosine kinase B (trkB). No immediate alteration in trkB mRNA was seen in hippocampus or cerebral cortex after 8 mg TMT/kg, while at 3 days trkB mRNA was significantly reduced in the CA3c pyramidal cell layer. No changes could be detected in neurotrophin-3 mRNA at either 1, 4 h or 3 days after TMT. Three days after 8 mg TMT/kg, a major induction of hsp70 mRNA occurred in a subset of neurons in the CA3c region, concomitant with an increased expression of c-fos mRNA as well as Fos protein in the hilar region of hippocampus. Hence, an early and transient decrease in BDNF appears to occur after TMT exposure, which is succeeded at 3 days by increases in BDNF, c-fos and hsp 70 mRNAs, concomitant with a decrease in trkB mRNA in regions known to be vulnerable to TMT. These results demonstrate that TMT causes a delayed, spatially restricted increase in activity-dependent gene expression, making TMT-induced disturbances an interesting model of neurodegenerative events.

In vivo electrochemical measurements of serotonin clearance in rat striatum: effects of neonatal 6-hydroxydopamine-induced serotonin hyperinnervation and serotonin uptake inhibitors.

Diffusion and clearance of extracellular serotonin (5-HT) was examined using in vivo chronoamperometry with "delayed-pulse" recordings after pressure ejections of 1 to 60 picomoles 5-HT into rat striatum at a fixed distance from a Nafion-coated carbon fiber electrode. Signals obtained were identified based on the signal characteristics to consist of 5-HT. Clearance times of 5-HT decreased, while amplitudes and rise times increased with serotonergic hyperinnervation induced by neonatal 6-hydroxydopamine (6-OHDA) lesions of dopamine (DA) neurons. Local applications of the 5-HT uptake inhibitors zimelidine or fluoxetine, in conjunction with 5-HT ejections, produced increased clearance times in both normal and 6-OHDA-treated animals. Thus, direct in vivo evidence was obtained for the importance of high affinity nerve terminal uptake as a key mechanism for clearance of 5-HT from the extracellular space. Inhibitors of 5-HT uptake appear to prolong the extracellular presence of 5-HT by increasing its clearance time.

Functional changes induced by neonatal cerebral 6-hydroxydopamine treatment: effects of dose levels on behavioral parameters.

Male Sprague-Dawley rats were treated neonatally with either of three different doses of 6-hydroxydopamine (6-OHDA): 50 micrograms i.c., 75 micrograms i.c., or 2 x 100 micrograms i.c.v., 30 min after a subcutaneous injection of desipramine (DMI, 25 mg/kg), in order to obtain selective lesions of mesencephalic dopamine (DA) neurons to different extents. From juvenile ages onwards, rats in each dose condition were tested for spontaneous motor activity and exploration in an openfield/holeboard setting measuring ambulation, rearing and head-dips. Between 77 and 78 days, the animals were tested in a modified, enclosed radial arm maze, followed 1 week later by tests in the circular swim maze. Finally, motor activity was tested in automated activity test chambers. In the openfield/holeboard setting, hyperactivity was seen for both rearing and ambulation in rats administered 50 micrograms 6-OHDA, whereas the 75 micrograms and 2 x 100 micrograms groups showed hyperactivity for ambulation, but hypoactivity for rearing and head-dips. All three dose groups demonstrated a retardation of learning in the radial arm maze. The 75 and 2 x 100 micrograms groups, but not the 50 micrograms group, showed impairments of acquisition in the swim maze. In the activity test chambers locomotion and rearing behavior varied as a function of 6-OHDA dose, being negatively and positively, respectively, related to DA concentration in striatum. These results show that the extent of the neonatal DA lesion determines both changes in motor- and exploratory activity as well as the occurrence and severity of acquisition impairment in spatial learning tasks.

Pentylenetetrazol kindling decreases N-methyl-D-aspartate and kainate but increases gamma-aminobutyric acid-A receptor binding in discrete rat brain areas.

Pentylenetetrazol is a convulsive drug acting on gamma-aminobutyric acid-A (GABA[A]) gated-chloride receptors. In this study we used a subconvulsive dose (30 mg/kg) of pentylenetetrazol to induce a fully kindled state in rats. Glutamate receptors were evaluated using [3H]-[1(2-thienylcyclohexyl)]-piperidin (TCP) and [3H]kainate receptor autoradiography and [3H]muscimol autoradiography was used to study GABA(A) receptors. In fully kindled rats decreased N-methyl-D-aspartate receptor binding was found in parietal cortex, area CA2 of hippocampus and piriform cortex. Decreased kainate receptor binding was observed in all areas of the hippocampus, the medial amygdala and in the piriform cortex in the kindled rats. In contrast, GABA(A) receptor binding increased in the dentate gyrus. It is concluded that modulatory neuronal plasticity events are induced in fully pentylenetetrazol kindled rats, which appears to lead to decreased glutamatergic excitation and increased GABAergic inhibition in brain regions implicated in the development of seizure activity.