Sequestration of Holotrich Protozoa in the Reticulo-Rumen of Cattle

Studies were carried out to determine the means by which holotrich protozoa can maintain their numbers within the rumen against the washout effect associated with the flow of ingesta. When a diet composed of 2 kg of concentrate and 1.5 kg of rice straw was fed to Holstein cows, about a fourfold increase in holotrich numbers per ml of rumen fluid was observed within 1 h after the commencement of feeding, and an abrupt decrease followed. This fluctuation in numbers was not related to the time of feeding. A sole feeding of 2 kg of concentrate had almost the same effect on the holotrichs as a sole feeding of 1.5 kg of rice straw. Administration of either 2 kg of concentrate or 1.5 kg of rice straw through the rumen fistula caused similar changes, though the extent of response to the former was greater than that to the latter. The administration of either 0.7 kg of starch or 0.2 kg of glucose through the fistula had a relatively minor effect on the holotrich population. Addition of rice straw to 0.5 kg of concentrate increased the change in numbers, but its addition had little, if any, effect when 1 kg of concentrate was fed. These results suggested that the fluctuation in holotrich numbers was related not only to the nature or component of feed but also to other factors such as the quantity or volume of a diet and the act of ingesting feed. Increasing the number of feedings up to eight times per day at 3-h intervals caused a decrease in the peak heights of holotrich numbers per milliliter of rumen fluid. A thick protozoal mass which primarily consisted of holotrichs was found on the wall of the reticulum of Holstein steers slaughtered after overnight starvation. These findings suggest that holotrichs would usually sequester on the reticulum wall and migrate into the rumen only for a few hours after feeding, and that this mode of behavior would be essential for holotrichs to maintain their population within the rumen of cattle. Possible mechanisms of the migration are also discussed.     

Carbohydrate and lipid metabolism during the last larval moult of the tobacco hornworm, Manduca sexta

Abstract. At 25°C and with a light regime of 17 h light and 7h dark, the last larval moult of the tobacco hornworm, Manduca sexta , lasts approximately 32 h, during which profound changes of metabolism were observed. At the onset of the moult, which coincides with the cessation of feeding, the proportion of active fat body glycogen phosphorylase increased from 10 (-2h) to 25–30% (Oh). A biphasic pattern with peak activities of 45–50% after t – 12 h and again just prior to the shedding of the cuticle (32 h) was subsequently observed. Haemolymph trehalose concentration decreased significantly from c. 35 (Oh) to 20mM (8h), but then recovered to an intermediate level (30mM; 12h). After completion of the moult, the trehalose concentration was 35–40 mM. The haemolymph glucose level in feeding fourth instar larvae was 4–5 mM, but decreased sharply before the onset of the moult to c. 1 mM, followed by a slow 6-fold increase over the next 20h. Prior to the shedding of the cuticle, the glucose level dropped again dramatically. The haemolymph lipid level increased slowly from an initial level of 1.2–1.4mg/ml during the early part of the moult, reaching a maximum of 1.8mg/ml after /= 16 h. Afterwards, a decrease of c. 50% was observed until ecdysis occurred. Oxygen consumption per animal decreased steadily from 30–35 μl/min pre-moult by approximately 70% to c. 10 μl/min but started to increase about 5 h before the animals resumed feeding.     

Hydrogen Production by Rumen Holotrich Protozoa: Effects of Oxygen and Implications for Metabolic Control by In Situ Conditions

Experiments with washed suspensions of holotrich protozoa (Isotricha spp. and Dasytricha ruminantium ) showed that both organisms have an efficient 0,-scavenging capability (apparent K_m values 2.3 and 0.3 μM, respectively). Reversible inhibition of H₂, production increased almost linearly with increasing O₂ up to 1.5 μM; higher levels of O₂ gave irreversible inhibition. In situ determinations of H, CH₄, O₂, and CO₂, in ovine rumen liquor, using a membrane inlet mass spectrometer probe, indicated that O₂, was present before feeding at 1-1.5 μM and decreased to undetectable levels (<0.25 μM) within 25 min after feeding. A transient increase in O₂. concentration after feeding occurred only in defaunated animals and resulted in suppression of CH₄ and CO₂ production. The presence of washed holotrich protozoa decreases the O₂ sensitivity of CH₄ production by suspensions of a cultured methanogenic bacterium Methanosarcina barkeri . It is concluded that holotrich protozoa play a role in ruminal O₂ utilization as well as in the production of fermentation end products (especially short-chain volatile fatty acids) utilized by the ruminant and H, utilized by methanogenic bacteria. These hydrogenosome-containing protozoa thus both control patterns of fermentation by influencing O₂ levels, and are themselves regulated by the low ambient O₂ concentrations they experience in the rumen.     

Rumen microbial changes in cattle fed diets with or without salinomycin

Four rumen-fistulated steers, randomly assigned to two groups (control and salinomycin fed) were used to monitor the changes in rumen microbial populations and volatile fatty acids (VFA) concentrations associated with feeding salinomycin (0.22 mg X kg-1 X day-1). Steers were adapted to an alfalfa hay and grain (80:20) diet before supplementing the diet with salinomycin, and then the diet was changed to 50:50 and 20:80 ratios of alfalfa hay to grain at 2-week intervals. Rumen samples for total and selective enumeration of anaerobic bacteria. VFA analysis, and enumeration of protozoa were collected during the 80:20 alfalfa hay-to-grain diet before salinomycin feeding, and during the 80:20, 50:50, and 20:80 hay-to-grain diets with salinomycin. At each sampling period, rumen samples were collected at 3 h after feeding on three consecutive days. Salinomycin feeding had no effect on rumen pH and total VFA concentration. The acetate-to-propionate ratio was significantly lower in salinomycin-fed steers than in the control. The molar proportion of butyrate increased in both control and salinomycin-fed steers. Total anaerobic bacterial counts were lower in salinomycin-fed steers than in the control steers after 8 weeks of salinomycin feeding. Salinomycin-resistant bacteria increased from 7.6 to 15.6% in salinomycin-fed steers but remained unchanged in control steers. Salinomycin had no effect on cellulolytic and lactate-utilizing bacteria, but the proportion of amylolytic bacteria was higher in salinomycin-fed steers than in control steers. The total number of protozoa decreased initially in salinomycin-fed steers. The initial reduction was due to reduced numbers of Entodinium species. Holotrichs were unaffected by salinomycin feeding.     

Rumen microbial changes in cattle fed diets with or without salinomycin.

Four rumen-fistulated steers, randomly assigned to two groups (control and salinomycin fed) were used to monitor the changes in rumen microbial populations and volatile fatty acids (VFA) concentrations associated with feeding salinomycin (0.22 mg X kg-1 X day-1). Steers were adapted to an alfalfa hay and grain (80:20) diet before supplementing the diet with salinomycin, and then the diet was changed to 50:50 and 20:80 ratios of alfalfa hay to grain at 2-week intervals. Rumen samples for total and selective enumeration of anaerobic bacteria. VFA analysis, and enumeration of protozoa were collected during the 80:20 alfalfa hay-to-grain diet before salinomycin feeding, and during the 80:20, 50:50, and 20:80 hay-to-grain diets with salinomycin. At each sampling period, rumen samples were collected at 3 h after feeding on three consecutive days. Salinomycin feeding had no effect on rumen pH and total VFA concentration. The acetate-to-propionate ratio was significantly lower in salinomycin-fed steers than in the control. The molar proportion of butyrate increased in both control and salinomycin-fed steers. Total anaerobic bacterial counts were lower in salinomycin-fed steers than in the control steers after 8 weeks of salinomycin feeding. Salinomycin-resistant bacteria increased from 7.6 to 15.6% in salinomycin-fed steers but remained unchanged in control steers. Salinomycin had no effect on cellulolytic and lactate-utilizing bacteria, but the proportion of amylolytic bacteria was higher in salinomycin-fed steers than in control steers. The total number of protozoa decreased initially in salinomycin-fed steers. The initial reduction was due to reduced numbers of Entodinium species. Holotrichs were unaffected by salinomycin feeding.     

Degradation of mimosine, 2,3-dihydroxy pyridine and 3-hydroxy-4(1H)-pyridine by bacteria from the rumen of sheep in Venezuela

Abstract The addition of Leucaena leucocephala herbage did not diet of sheep in Venezuela did not affect the concentration of volatile fatty acids (VFA) in the rumen, the degradation of rice straw incubated in sacco, or the numbers of rumen fungi or bacteria. However, feeding Leucaena increased the concentration of ammonia in the rumen. In addition, two products of the degradation of the toxic amino acid mimosine were detected in the rumen when Leucaena was fed. One of these products, 2,3-dihydroxy pyridine (2,3-DHP), was detected at concentrations of up to 1.1 μmol/ml. The other, 3-hydroxy-4(1H)-pyridone (3,4-DHP) was found at concentrations of up to 0.96 μmol/ml. The examination of bacterial cultures isolated from the rumen of the sheep under investigation showed that feeding Leucaena increased the relative proportions of short Gram-negative rods and decreased the proportion of long roads and coccobacilli present. Although the animals fed Leucaena showed a small loss in weight during the feeding trial, no evidence of Leucaena toxicity was seen. A total of 18 cultures capable of degrading 2,3-DHP or 3,4-DHP were isolated from the rumen of the sheep before Leucaena was fed. These included both Gram-positive and Gram-negative bacteria, and a Gram-positive sporeformer. It seems that 2,3-DHP and 3,4-DHP may be degraded by a much wider range of bacteria than has been recognised previously. The detection of these bacteria before Leucaena was fed suggests that they were indigenous members of the rumen microflora of sheep in Venezuela.     

Changes in haemolymph octopamine levels associated with food deprivation in the locust, Schistocerca gregaria

ABSTRACT. Levels of octopamine in the haemolymph and locomotor activity have been measured over 24 h in fed and food-deprived adult male locusts, Schistocerca gregaria (Forskål). Locusts are predominantly diurnal, the locomotor activity of individuals with continuous access to food was higher during the photophase. The basal level of octopamine in haemolymph sampled during the first quarter of the light cycle was 7.8±1.1 pg/μl (51 nm). Depriving locusts of food at the start of the photophase for 9 h caused an increase in both the speed of movement and total activity compared to fed individuals. There was a corresponding increase in the haemolymph octopamine concentration which continued for as long as the insects were unable to feed, reaching a concentration of 20.7 ± 3.7 pg/μl (135 nM). While declining, the levels of octopamine remained higher in fed locusts for up to 4 h after replacing the food. The results are discussed in relation to the regulation of feeding behaviour and a proposed neurohormonal role of octopamine in insects.     

Effect of protein supplementation on ruminal parameters and microbial community fingerprint of Nellore steers fed tropical forages

In tropical regions, protein supplementation is a common practice in dairy and beef farming. However, the effect of highly degradable protein in ruminal fermentation and microbial community composition has not yet been investigated in a systematic manner. In this work, we aimed to investigate the impact of casein supplementation on volatile fatty acids (VFA) production, specific activity of deamination (SAD), ammonia concentration and bacterial and archaeal community composition. The experimental design was a 4×4 Latin square balanced for residual effects, with four animals (average initial weight of 280±10 kg) and four experimental periods, each with duration of 29 days. The diet comprised Tifton 85 (Cynodon sp.) hay with an average CP content of 9.8%, on a dry matter basis. Animals received basal forage (control) or infusions of pure casein (230 g) administered direct into the rumen, abomasum or divided (50 : 50 ratio) in the rumen/abomasum. There was no differences (P>0.05) in ruminal pH and microbial protein concentration between supplemented v. non-supplemented animals. However, in steers receiving ruminal infusion of casein the SAD and ruminal ammonia concentration increased 33% and 76%, respectively, compared with the control. The total concentration of VFA increased (P<0.05) in steers receiving rumen infusion of casein. SAD and the microbial protein concentration did not vary significantly among treatments during the feeding cycle, but mean SAD values were greater in steers supplemented in the rumen and rumen/abomasum. Ruminal ammonia concentration was positively correlated with SAD in animals receiving ruminal infusion of casein. Polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE) analysis revealed low similarity between treatments, animals and time of sample collection. Richness analysis and determination of the Shannon–Wiener index indicated no differences (P>0.05) in species richness and diversity of γ-proteobacteria, firmicutes and archaea between non-supplemented Nellore steers and steers receiving casein supplementation in the rumen. However, species richness and the Shannon–Wiener index were lower (P<0.05) for the phylum bacteroidetes in steers supplemented with casein in the rumen compared with non-supplemented animals. Venn diagrams indicated that the number of unique bands varied considerably among individual animals and was usually higher in number for non-supplemented steers compared with supplemented animals. These results add new knowledge about the effects of ruminal and postruminal protein supplementation on metabolic activities of rumen microbes and the composition of bacterial and archaeal communities in the rumen of steers.     

MATING BEHAVIOR OF THE CAT FLEA, CTENOCEPHALIDES FELZS BOUCHE(SIPHONAPTERA:PULICIDAE) AND MALE RESPONSE TO FEMALE EXTRACT ON AN ARTIFICIAL FEEDING SYSTEM

Abstract  The mating behavior of cat flea, Ctenocepholides felis (Bouche) was studied on an artificial feeding device. Male and female can mate repeatedly with same partner or Merent ones. In the situation of male: female ratio of 1 :5, each mating lasted an average of 6.6 min, with a mean interval between matings at 2.5 min., compared to 11. 1 min and 12.1 min respectively in a cell with 5 males and 1 female. As many as 48 mating events were observed for one male during an 8 h period. One female mated 27 times in 7 h with 5 males in the same cell. Newly emerged males and females can not mate before blood meal and about 24 h blood feeding is rewuired for successful mating. Newly emerged males can not mate with fed females (fed for 48 h), but fed males can mate with newly emerged females who are feeding the blood. Significantly more male contacts and male-male mating attempts were observed after the paper treated with female extract was introduced into the cell. The paper contacts and mating attempts were 16.75–32.25 times and 15.75–31.38 times, respectively, on average during a period of 20 min when different doses (FE) of extract were provided.     

Effect of intake on fasting heat production,respiratory quotient and plasma metabolites measured using the washed rumen technique

The objective was to investigate the effect of intake before fasting on concentrations of metabolites and hormones, respiratory quotient (RQ) and fasting heat production (HP) using the washed rumen technique and to compare these values with those from the fed state. Six Holstein steers (360±22 kg) were maintained at 21°C and fed three different energy intakes within a replicated 3×3 Latin square design with 21-day periods. Steers were fed alfalfa cubes to provide 1.0, 1.5 and 2.0×NE_m during 19 days of each experimental period. Steers were placed in individual metabolism stalls fitted with indirect calorimetry head-boxes on day 20 of each experimental period (FED steers) and fed their normal meal. On day 21 of each period the reticulorumen was emptied, washed and refilled with ruminal buffer (NaCl=96; NaHCO₃=24; KHCO₃=30; K₂HPO₄=2; CaCl₂=1.5; MgCl₂=1.5 mmol/kg of buffer) aerated with 75% N₂ and 25% CO₂ before introduction to the rumen (steers were not fed; WASHED steers). Each gas exchange was measured over 24 h. HP for 1.0, 1.5 and 2.0×NE_m were 479, 597 and 714 kJ/daykg^0.75 (s.e.m. =16), respectively. The plateau RQ was 0.756, 0.824 and 0.860 for the 1.0, 1.5 and 2.0×NE_m intakes for the FED steers, respectively. After rumen washing, fasting HP was 331, 359 and 400 kJ/daykg^0.75 (s.e.m.=13) for 1.0, 1.5, and 2.0×NE_m intakes before fasting, respectively. The RQ for WASHED rumen steers was 0.717, 0.710 and 0.719, respectively. Cortisol and β-hydroxybutyrate concentrations in WASHED rumen steers did not exceed threshold levels for severe energy deficit and stress as can be induced from prolonged fasting. This study demonstrates that a fasting state can be emulated using the washed rumen technique, minimizing the time required as opposed to traditional fasting methodologies, without causing a severe energy deficit and stress.     

Non-invasive corticosterone treatment changes foraging intensity in red-eyed vireos Vireo olivaceus

Corticosterone is thought to play an important role in food caching and foraging behaviour. However, the direct influence of increased plasma corticosterone on feeding behaviour is still unclear. In this study the effect of increased corticosterone on feeding behaviour in migratory active red-eyed vireos Vireo olivaceus was investigated. We hypothesized that if increased corticosterone levels facilitate foraging behaviour, an increased number of visits to the food bowl by corticosterone treated birds would be seen. In addition to ad lib food during the experiment, the vireos were fed every full hour between 09.00–13.00 h with one meal worm injected either with 4 μg corticosterone dissolved in 20 μl DMSO, or with DMSO only as a control treatment. The presence or absence of a bird in the food bowl was recorded by a motion detector between 09.00–15.00 h. The non-invasive corticosterone treatment increased plasma corticosterone levels and caused vireos to visit the food bowl more frequently compared to control treated individuals between 11.00–12.00 h and 13.00–15.00 h. Our data indicate that corticosterone has an effect on feeding behaviour in birds.     

Intraruminal infusion of n-butyric acid induces an increase of ruminal papillae size independent of IGF-1 system in castrated bulls

Abstract

The objective of this study was to explore morphological alterations of rumen papillae induced by n-butyric acid in relation to the insulin-like growth factor (IGF) system in adult castrated bulls. Three animals fitted with rumen cannula were fed twice daily at a low and high nutritional level (LL and HL), i.e., at 1.1 × maintenance (M) and 1.6 × M, respectively. Diets contained artificial dried grass and concentrate (74:26 and 52:48). Bulls received no (B0) or daily intraruminal infusions of 500 g n-butyric acid (B500) over 14 d. The infusion started 1 h after the morning feeding (9:00) and lasted for 3.5 h. Thus, four treatments (B0LL, B500LL, B0HL, and B500HL) were compared. Blood and rumen mucosa samples from the atrium ruminis were taken at the last day of each period. Length, width and surface of rumen papillae were greater (p < 0.001) in B0HL than in B0LL. Treatment with n-butyric acid resulted in an increase of the papillae surface of 20 – 40% (p = 0.047) for both nutritional levels as compared to periods without n-butyric acid treatments. The higher nutritional level and intraruminal n-butyric acid infusion induced epithelial cell death. The percentage of proliferative cells was doubled by n-butyric acid treatment. The mRNA of IGF-1 and IGF type 1 receptor (IGF-1R), as well as IGF-1R binding capacity were unaffected by butyric acid treatments. The abundance of IGF-1 mRNA tended to be lower (p = 0.1) and IGF-1R abundance was lower (p = 0.03) in response to the HL. The plasma IGF-1 concentration was lower with butyric acid treatment (p < 0.01), but was unaffected by the nutritional level. In conclusion, under described experimental preconditions of daily short-time intraruminal n-butyric acid infusion alterations of rumen papillae morphology is not mediated by ruminal IGF type 1 receptor and by local IGF-1 expression in papillae in castrated bulls.     

Effects of dietary sulphur sources on concentrations of hydrogen sulphide in the rumen head-space gas of dairy cows

Three change-over design experiments investigated the origin of hydrogen sulphide in the rumen head-space gas of dairy cows, comparing the effects of single iso-S additions of methionine, cysteine and sodium sulphate, as well as the effects of single meals of fresh ryegrass or white clover. The concentration of hydrogen sulphide in rumen gas declined close to zero within 4 h after withdrawal of the previous feed. Sulphur sources were then given to cows and concentrations of hydrogen sulphide recorded in rumen head-space gas at 30-min intervals. Cysteine addition (8 g) led to a rapid (within 30 min) and a large (490 and 957 p.p.m. respectively in two experiments) increase in hydrogen sulphide concentration. Concentrations were significantly less following methionine addition. Increasing levels of cysteine addition led to significant increases in hydrogen sulphide concentrations ( P < 0.001 for the linear effect), although peak hydrogen sulphide was delayed and concentrations remained higher for longer with the highest (12 g) addition of cysteine ( P < 0.01 for the ‘cysteine level’ × ‘time’ interaction). The increase in concentration of hydrogen sulphide from sodium sulphate was smaller (230 p.p.m.) and slower (2 h) than for cysteine. Despite the much higher intake of cystine for white clover in comparison with perennial ryegrass ( P < 0.001), there was almost no increase in hydrogen sulphide concentration in rumen head-space gas from cows fed white clover. It seems likely that this is associated with the use of sulphur to produce thiocyanate to detoxify the hydrogen cyanide from cyanogenic white clover.     

Evidence for existence of lectins on the ruminal bacteria from steers fed roughage and concentrate diets

Mixed rumen bacteria, isolated by centrifugation from the rumen of steers fed a roughage (R) or concentrate (C) diet, were used to determine if lectins are present on rumen bacteria, based on hemagglutination (HA) and HA inhibition assays in vitro. Rumen bacteria from steers fed either diet agglutinated erythrocytes from cattle, sheep, pigs, and rats, suggested that lectins exist on rumen bacteria. Bacterial HA titers from steers receiving the R diet were much higher (p<0.001) than those from steers fed the C diet, depending on the erythrocyte source used. Centrifugation at 20,000xg for 30 min fractionated the rumen bacteria into upper (U) and lower (L) layers. The HA titers of the U bacterial fractions were significantly higher (p<0.001) than those of the L fractions. A remarkable reduction or complete disappearance of HA titers following treatment of rumen bacteria with protease, trypsin, sodium dodecyl sulfate (SDS) or sodium periodate indicates that rumen bacterial lectins are probably glycoproteins. Lectin specificity for saccharides (galactose, lactose, N-acetyl-D-galactosamine, methyl-alpha-D-galactopyranoside and methyl-beta-D-galactopyranoside) and glycoproteins (mucin, fetuin, and thyroglobulin) was found in the RU, RL, and CU bacterial fractions; no specific binding was determined in the CL fractions. The potential role of lectins in mediating the attachment of rumen bacteria to feed particles, rumen epithelia and other microorganisms is discussed.     

Adenosine Triphosphate in the Bovine Rumen During Maximum Nutrient Supply and Starvation

The concentration of adenosine triphosphate (ATP) in rumen fluid from heifers fed high levels of nutrients continuously and after 12–24 h starvation has been determined in order to examine the influence of different physiological conditions. The conventional parameters for characterizing the rumen fermentation have been applied to control the effect of a different nutrient supply. The mean concentration of ATP, determined by the luciferin-luciferase assay, was 55.3 mg ATP/l rumen fluid in a heifer fed continuously, twice as much as found in a starved heifer. The mean ratio of biomass/ATP changed from 683 at the high level of energy intake to 1208 in the period of starvation. Consequently, the concentration of ATP in the rumen micro-organisms is markedly influenced by nutritional conditions. Finally, the applicability of ATP determinations in rumen microbiology is discussed.     

Uptake and metabolism of benzyladenine in the early stage of flower bud development in vitro in tobacco

Benzyladenine (BA) was found to regulate the number of flower buds regenerated in vitro from pedicel tissue of tobacco. Flower bud induction was particularly sensitive to BA levels in the range of 0.45 to 1.0 μ M , where a two-fold increase in concentration caused a threefold rise in the number of buds. When tissues were fed radioactive BA for 24h, only 9–12% of the counts were recovered in the original compound. The rest was present in metabolites, tentatively identified as the mono-, di- and triribotides, 7- and 9-glucosides and 9-riboside of BA. The amount of growth regulator taken up and the quantities of BA and its metabolites in the explants were all linearly related to the concentration of the medium. The internal BA concentration was ca 60% of the level in the medium after 24 h. When the concentration in the medium was raised, relatively more BA remained in the non-conjugated form. However, this change in the equilibrium between BA and the conjugates is too small to account for the steep rise in the curve representing concentration vs effect between 0.45 and 1.0 μ M .     

A Comparison of the Effects of Ethanol and the Competitive Glycine Antagonist 7-Chlorokynurenic Acid on N-Methyl-d-Aspartic Acid-Induced Neurotransmitter Release from Rat Hippocampal Slices

Abstract: N -Methyl- d -aspartate (NMDA; 500 μ M ) stimulated the net release of preloaded tritiated norepinephrine from rat hippocampal slices. Both ethanol and the competitive glycine antagonist 7-chlorokynurenic acid (7-CK) dose-dependently inhibited NMDA-stimulated release without affecting basal, nonstimulated efflux. These inhibitory effects were readily reversed upon washout of the drugs. Over the concentration range tested (25–200 m M ), ethanol inhibited ∼65% of NMDA-stimulated release with an estimated IC₅₀ of ∼70 m M . In contrast, 7-CK fully inhibited release (>95%) at a concentration of 30 μ M with half-maximal inhibition occurring at ∼2 μ M . The combination of 7-CK (1–30 μ M ) and ethanol (25–100 m M ) had an additive inhibitory effect on NMDA-stimulated release but did not alter the inhibitory potency of 7-CK. Calculated IC₅₀values for 7-CK in the presence of 25, 50, or 100 m M ethanol were (mean × SEM; μ M ) 2.33 (0.11), 2.38 (0.23), and 1.99 (0.30), respectively. 7-CK (3 μ M ) inhibited NMDA-stimulated [³H]norepinephrine release by ∼50%. This inhibition was fully attenuated by the addition of the glycine agonistserine with complete reversal occurring at 30 μ M d -serine. Increasing the 7-CK concentration to 10 μ M shifted the d -serine dose-effect curve to the right in a parallel fashion as expected for a competitive antagonist. In contrast, the inhibitory effects of ethanol or the combination of 7-CK (3 μ M ) and ethanol (25 or 50 m M ) were not reversed by the addition of d -serine (0.1–1,000 μ M ). Together, these results suggest that ethanol's inhibition of NMDA-stimulated [³H]norepinephrine release from hippocampal slices is not due to a simple competitive interaction with the glycine site on the NMDA receptor.     

Extracellular Somatostatin Measured by Microdialysis in the Hippocampus of Freely Moving Rats: Evidence for Neuronal Release

Abstract: Intracerebral microdialysis combined with a sensitive and specific radioimmunoassay was used to monitor the neuronal release of somatostatin (somatostatin-like immunoreactivity, SLI) in the dorsal hippocampus of freely moving rats. The sensitivity of the radioimmunoassay was optimized to detect <1 fmol/ml. The basal concentration of SLI in 20-min dialysate fractions (5 μl/min) collected 24 h after probe implantation was stable over at least 200 min. The spontaneous efflux dropped by 54 ± 6.4% ( p < 0.05) when Ca²⁺ was omitted and 1 m M EGTA added to the Krebs-Ringer solution and by 65.5 ± 3.2% ( p < 0.05) in the presence of 1 μ M tetrodotoxin. Depolarizing concentrations of the Na⁺ channel opener veratridine (6.25, 25, 100 μ M ) induced 11 ± 2 ( p < 0.05), 17 ± 2 ( p < 0.05), and 21 ± 5 ( p < 0.01) fold increase in SLI concentration, respectively, during the first 20 min of perfusion. The effect of 100 μ M veratridine was blocked by coperfusion with 5 μ M tetrodotoxin ( p < 0.01) and reduced by 79% ( p < 0.01) in the virtual absence of Ca²⁺. Neuronal depolarization by 20 min of perfusion with Krebs-Ringer solution containing 25 and 50 m M KCl and proportionally lowered Na⁺ increased the dialysate SLI 4.4 ± 1 ( p < 0.05) and 17 ± 3 ( p < 0.01) fold baseline, respectively. Ten micromolar ouabain, a blocker of Na⁺,K⁺-ATPase, increased the dialysate SLI 15-fold baseline, on average ( p < 0.05), during 80 min of perfusion. The results demonstrate the suitability of brain microdialysis for monitoring the neuronal release of SLI and for studying its role in synaptic transmission.     

DEVELOPMENT AND CHARACTERISTICS OF THE HISTAMINE-INDUCED ACCUMULATION OF CYCLIC AMP IN THE RABBIT CEREBRAL CORTEX

Abstract— In slices of adult rabbit cerebral cortex histamine at 5 μM produced a detectable rise in adenosine 3',5'-monophosphate (cyclic AMP). A maximum (20-fold) increase was observed in response to 0–5 mM histamine, with higher concentrations being less effective. The antihistaminic agent, tripelennamine, inhibited the response to 50 μM histamine in a dose-related manner. No effect on basal levels of cyclic AMP was noted with the highest dose of tripelennamine. The cyclic AMP response to 50 μM histamine was sustained for up to 1 h of incubation whether the slices and included medium were assayed together or the slices were assayed separately, although after 60 min of incubation cyclic AMP levels were higher when the medium was included in the assay. During development of the rabbit cerebral cortex, the first detectable increase of cyclic AMP in response to histamine occurred at fetal day 25, and from day 28 to birth the response was a 4-to 5-fold increase. A maximal (10-fold) response was observed at 4–8 days postpartum and by 20 days of postnatal age the response had decreased to the adult levels.     

Availability, uptake and turnover of glycerol in hypersaline environments

Abstract A sensitive assay for glycerol and other polyols was developed, based on periodate oxidation to formaldehyde, followed by a colorimetric assay with 3-methyl-2-benzothiazolone hydrazone. Apparent glycerol concentrations thus measured in saltern crystallizer ponds were around 20–36 μM, while in the Dead Sea, during a Dunaliella bloom, values were up to 27 μM. However, these values probably overestimate the glycerol concentrations present, as shown by labeled glycerol uptake experiments. Values of [K + S_n] (natural concentration + affinity constant) in saltern ponds were as low as 0.76–1.4 μM, with V_max values of 193–303 nmol 1⁻¹h⁻¹, and turnover times between 2.6–7.2 h at 35°C. Similar measurements in the Dead Sea were: [K + S_n] 0.07–1.41 μM, V_max values 160–426 nmol 1⁻¹h⁻¹, and turnover times in the range of 0.45–3.3 h.