A comparative analysis of preprocessing techniques of cardiac event series for the study of heart rhythm variability using simulated signals

In the present study, using noise-free simulated signals, we performed a comparative examination of several preprocessing techniques that are used to transform the cardiac event series in a regularly sampled time series, appropriate for spectral analysis of heart rhythm variability (HRV). First, a group of noise-free simulated point event series, which represents a time series of heartbeats, was generated by an integral pulse frequency modulation model. In order to evaluate the performance of the preprocessing methods, the differences between the spectra of the preprocessed simulated signals and the true spectrum (spectrum of the model input modulating signals) were surveyed by visual analysis and by contrasting merit indices. It is desired that estimated spectra match the true spectrum as close as possible, showing a minimum of harmonic components and other artifacts. The merit indices proposed to quantify these mismatches were the leakage rate, defined as a measure of leakage components (located outside some narrow windows centered at frequencies of model input modulating signals) with respect to the whole spectral components, and the numbers of leakage components with amplitudes greater than 1%, 5% and 10% of the total spectral components. Our data, obtained from a noise-free simulation, indicate that the utilization of heart rate values instead of heart period values in the derivation of signals representative of heart rhythm results in more accurate spectra. Furthermore, our data support the efficiency of the widely used preprocessing technique based on the convolution of inverse interval function values with a rectangular window, and suggest the preprocessing technique based on a cubic polynomial interpolation of inverse interval function values and succeeding spectral analysis as another efficient and fast method for the analysis of HRV signals

Electrolocation-communication discharges of the fish Gymnotus carapo L. (Gymnotidae: Gymnotiformes) during behavioral sleep

Technical problems have hampered the study of sleep in teleosts. The electrical discharges of Gymnotus carapo L. (Gymnotidae: Gymnotiformes) were monitored to evaluate their ease and reliability as parameters to study sleep. The discharges were detected by electrodes immersed in a glass aquarium and were recorded on a conventional polygraph. G. carapo showed conspicuous signs of behavioral sleep. During these periods, opercular beat rates were counted, electric discharges recorded, and the "sharp discharge increase" (SDI) of the orienting reflex was investigated. All 20 animals monitored maintained electrical discharges during behavioral sleep. The discharge frequencies during sleep (50.3 ± 10.4 Hz) were not significantly different from those observed when the fish was awake and inactive (57.2 ± 12.1 Hz) (Wilcoxon matched-pairs signed-ranks test, P>0.05). However, the SDI, which was prevalent in the awake fish, was not observed during periods of behavioral sleep. Additional observations showed that the species had cannibalistic habits. When presented with electrical discharges from a conspecific, the sleeping fish showed an initial decrease or pause in discharge frequency, while the awake fish did not have this response. We conclude that the electrical discharges of G. carapo were not conspicuous indicators of behavioral sleep. Discharges may have been maintained during sleep for sensory purposes, i.e., conspecific detection and avoidance of cannibalistic attacks.

Natural preference of zebrafish (Danio rerio) for a dark environment

The zebrafish (Danio rerio) has been used as a model in neuroscience but knowledge about its behavior is limited. The aim of this study was to determine the preference of this fish species for a dark or light environment. Initially we used a place preference test and in a second experiment we applied an exit latency test. A two-chamber aquarium was used for the preference test. The aquarium consisted of a black chamber and a white chamber. In the first experiment the animal was placed in the aquarium and the time spent in the two compartments was recorded for 10 min. More time was spent in the black compartment (Wilcoxon matched-pairs signed-rank test, T = 7, N1 = N2 = 18, P = 0.0001). In the second experiment the animal was placed in the black or white compartment and the time it took to go from the initial compartment to the opposite one was recorded. The test lasted a maximum of 10 min. The results showed that the animal spent more time to go from the black to the white compartment (Mann-Whitney rank sum test, T = 48, N1 = 9, N2 = 8, P<0.0230). These data suggest that this fish species has a natural preference for a dark environment and this characteristic can be very useful for the development of new behavioral paradigms for fish.

Environmental blue light prevents stress in the fish Nile tilapia

The present study aimed to test the effects of blue, green or white light on the stress response of the Nile tilapia, Oreochromis niloticus (L.). Each color was tested on two groups of isolated adult Nile tilapia (8 replicates each): one being subjected to confinement stress, and the other not (control). A different environmental color was imposed on each compartment by covering the light source with cellophane of the respective color (green or blue; no cellophane was used for white light). The intensity of green, white and blue lights was 250, 590 and 250 lux, respectively. Basal plasma cortisol levels were determined for each fish prior to the experimental procedures. The fish were confined by being displaced toward one side of the aquarium using an opaque partition for 1 h both in the morning and the afternoon of the two consecutive days of the test. At the end of this 48-h period, plasma cortisol levels were measured again. Basal cortisol levels (ng/ml) were similar for each group (ANOVA, F(2;42) = 0.77, P = 0.47). Thus, plasma cortisol levels were analyzed in terms of variation from their respective basal level. After confinement, plasma cortisol levels were not increased in fish submitted to a blue light environment. Thus, blue light prevents the confinement-induced cortisol response, an effect not necessarily related to light intensity.

Sex-specific compensatory growth in food-deprived Nile tilapia

Female Nile tilapia incubate fertilized eggs in their mouth until they are released as alevins. Consequently, the female may not eat during this period. Thus, it would be expected that female Nile tilapia are more adapted to recovering from fasting than males, which do not display this behavior. To test this hypothesis we conducted an experiment with two groups of fish consisting of 7 males and 7 females each, with one fish per aquarium. The experiment was divided into three phases involving adjustment of the animals to experimental aquaria (0-15th day), fasting (16th-27th day), and refeeding (27th-42nd day). Compensatory growth performance was assessed by specific growth rate, weight, food conversion efficiency and food intake. Food conversion efficiency increased after fasting with a similar rate for both sexes. However, specific growth rate, food intake and weight gain (%) were significantly higher in males than in females in the refeeding phase. Thus, we conclude that male Nile tilapia can compensate for a fasting period more efficiently than females, refuting our hypothesis. A possible mechanism involved in the greater male compensation is that they presented greater hyperphagia than females, concomitantly with a similar rate of food conversion efficiency for both sexes during refeeding, which would probably be provoking greater growth in males.

Modulation of hormone secretion by functional electrical stimulation of the intact and incompletely dysfunctional dog pancreas

The purpose of the present study was to modulate the secretion of insulin and glucagon in Beagle dogs by stimulation of nerves innervating the intact and partly dysfunctional pancreas. Three 33-electrode spiral cuffs were implanted on the vagus, splanchnic and pancreatic nerves in each of two animals. Partial dysfunction of the pancreas was induced with alloxan. The nerves were stimulated using rectangular, charge-balanced, biphasic, and constant current pulses (200 µs, 1 mA, 20 Hz, with a 100-µs delay between biphasic phases). Blood samples from the femoral artery were drawn before the experiment, at the beginning of stimulation, after 5 min of stimulation, and 5 min after the end of stimulation. Radioimmunoassay data showed that in the intact pancreas stimulation of the vagal nerve increased insulin (+99.2 µU/ml) and glucagon (+18.7 pg/ml) secretion and decreased C-peptide secretion (-0.15 ng/ml). Splanchnic nerve stimulation increased insulin (+1.7 µU/ml), C-peptide (+0.01 ng/ml), and glucagon (+50 pg/ml) secretion, whereas pancreatic nerve stimulation did not cause a marked change in any of the three hormones. In the partly dysfunctional pancreas, vagus nerve stimulation increased insulin (+15.5 µU/ml), glucagon (+11 pg/ml), and C-peptide (+0.03 ng/ml) secretion. Splanchnic nerve stimulation reduced insulin secretion (-2.5 µU/ml) and increased glucagon (+58.7 pg/ml) and C-peptide (+0.39 ng/ml) secretion, and pancreatic nerve stimulation increased insulin (+0.2 µU/ml), glucagon (+5.2 pg/ml), and C-peptide (+0.08 ng/ml) secretion. It was concluded that vagal nerve stimulation can significantly increase insulin secretion for a prolonged period of time in intact and in partly dysfunctional pancreas.

Environmental color affects Nile tilapia reproduction

We investigated the effects of environmental color on the reproductive behavior of Nile tilapia, Oreochromis niloticus. Two environmental colors were tested by covering the aquarium (60 x 60 x 40 cm) with white (12 groups) or blue (13 groups) cellophane and observing reproductive behavior in groups of 2 males (10.27 ± 0.45 cm) and 3 females (10.78 ± 0.45 cm) each. After assignment to the respective environmental color (similar luminosity = 100 to 120 Lux), the animals were observed until reproduction (identified by eggs in the female's mouth) or up to 10 days after the first nest building. Photoperiod was from 6:00 h to 18:00 h every day. Food was offered in excess once a day and water quality was similar among aquaria. Daily observations were made at 8:00, 11:00, 14:00 and 17:00 h regarding: a) latency to the first nest, b) number of nests, c) gravel weight removed (the male excavates the nest in the bottom of the aquarium), d) nest area, and e) mouthbrooding incubation (indication of reproduction). The proportion of reproducing fish was significantly higher (6 of 13) in the group exposed to the blue color compared the group exposed to the white color (1 of 12; Goodman's test of proportions). Moreover, males under blue light removed significantly larger masses of gravel (blue = 310.70 ± 343.50 g > white = 130.38 ± 102.70 g; P = 0.01) and constructed wider nests (blue = 207.93 ± 207.80 cm² > white = 97.68 ± 70.64 cm²; P = 0.03) than the control (white). The other parameters did not differ significantly between light conditions. We concluded that reproduction in the presence of blue light was more frequent and intense than in the presence of white light.

Attraction to amino acids by Lymnaea acuminata, the snail host of Fasciola species

Adult Lymnaea acuminata (average length 20-22 mm) were collected locally from lakes and low-lying submerged fields from Gorakhpur. The chemoattraction studies were made in round glass aquaria measuring 30 cm in diameter and filled to a depth of 10 mm with 500 ml dechlorinated tap water. Each aquarium was divided into four concentric zones. At the starting time of the assay 10 snails were placed on the circumference of outermost zone 0. Snail attractant pellets (SAP) were added simultaneously in the center of central zone 3. SAP of different amino acids were prepared at concentrations of 10, 20, 50, 80 and 100 mM/2% agar solution and, subsequently, spread to a uniform thickness of 5 mm. After cooling, SAP were cut in small pieces of 5 mm in diameter. Lymnaea acuminata's attraction to amino acids was studied using different amino acid concentrations in SAP. Pellets containing amino acids with non-polar R groups (proline and tryptophan), a charged polar group (arginine) and uncharged polar R groups (serine, citrulline and asparagine) were tested. The snails were more attracted to the uncharged polar R group amino acid serine than to other groups of amino acids. The preferred amino acid concentration was 80 mM. The attraction of snails to different amino acids was concentration dependent. Snails could discriminate amongst the different amino acids at > or = 50 mM.

Stress responses of the fish Nile tilapia subjected to electroshock and social stressors

Plasma cortisol and glucose levels were measured in 36 adult Nile tilapia males, Oreochromis niloticus (standard length, mean ± SD, 14.38 ± 1.31 cm), subjected to electroshock and social stressors. Pre-stressor levels were determined 5 days after the adjustment of the fish to the experimental aquaria (1 fish/aquarium). Five days later, the effects of stressors on both cortisol and glucose levels were assessed. The following stressors were imposed for 60 min: pairing with a larger resident animal (social stressor), or a gentle electroshock (AC, 20 V, 15 mA, 100 Hz for 1 min every 4 min). Each stressor was tested in two independent groups, one in which stress was quantified immediately after the end of the 60-min stressor imposition (T60) and the other in which stress was quantified 30 min later (T90). Pre-stressor values for cortisol and glucose were not statistically different between groups. Plasma cortisol levels increased significantly and were of similar magnitude for both electroshock and the social stressor (mean ± SD for basal and final samples were: electroshock T60 = 65.47 ± 15.3, 177.0 ± 30.3; T90 = 54.8 ± 16.0, 196.2 ± 57.8; social stress T60 = 47.1 ± 9.0, 187.6 ± 61.7; T90 = 41.6 ± 8.1, 112.3 ± 26.8, respectively). Plasma glucose levels increased significantly for electroshock at both time points (T60 and T90), but only at T90 for the social stressor. Initial and final mean (± SD) values are: electroshock T60 = 52.5 ± 9.2, 115.0 ± 15.7; T90 = 35.5 ± 1.1, 146.3 ± 13.3; social stress T60 = 54.8 ± 8.8, 84.4 ± 15.0; T90 = 34.5 ± 5.6, 116.3 ± 13.6, respectively. Therefore, electroshock induced an increase in glucose more rapidly than did the social stressor. Furthermore, a significant positive correlation between cortisol and glucose was detected only at T90 for the social stressor. These results indicate that a fish species responds differently to different stressors, thus suggesting specificity of fish stress response to a stressor.

L-histidine enhances learning in stressed zebrafish

The aim of the present study was to determine the effect of the histaminergic precursor L-histidine and the H3 receptor antagonist thioperamide on the learning process of zebrafish submitted or not to confinement stress. On each of the 5 consecutive days of experiment (D1, D2, D3, D4, D5), animals had to associate an interruption of the aquarium air supply with food offering. Non-stressed zebrafish received an intraperitoneal injection of 100 mg/kg L-histidine, 10 mg/kg thioperamide or saline after training. Stressed animals received drug treatment and then were submitted to confinement stress for 1 h before the learning procedure. Time to approach the feeder was measured (in seconds) and was considered to be indicative of learning. A decrease in time to approach the feeder was observed in the saline-treated group (D1 = 141.92 ± 13.57; D3 = 55 ± 13.54), indicating learning. A delay in learning of stressed animals treated with saline was observed (D1 = 217.5 ± 25.66). L-histidine facilitated learning in stressed (D1 = 118.68 ± 13.9; D2 = 45.88 ± 8.2) and non-stressed (D1 = 151.11 ± 19.20; D5 = 62 ± 14.68) animals. Thioperamide inhibited learning in non-stressed (D1 = 110.38 ± 9.49; D4 = 58.79 ± 16.83) and stressed animals (D1 = 167.3 ± 26.39; D5 = 172.15 ± 27.35). L-histidine prevented the increase in blood glucose after one session of confinement (L-histidine = 65.88 ± 4.50; control = 53 ± 3.50 mg/dL). These results suggest that the histaminergic system enhances learning and modulates stress responses in zebrafish.

Participation of the GABAergic system in the anesthetic effect of Lippia alba (Mill.) N.E. Brown essential oil

The objective of this study was to identify the possible involvement of the GABAergic system in the anesthetic effect of Lippia alba essential oil (EO). We propose a new animal model using silver catfish (Rhamdia quelen) exposed to an anesthetic bath to study the mechanism of action of EO. To observe the induction and potentiation of the anesthetic effect of EO, juvenile silver catfish (9.30 ± 1.85 g; 10.15 ± 0.95 cm; N = 6) were exposed to various concentrations of L. alba EO in the presence or absence of diazepam [an agonist of high-affinity binding sites for benzodiazepinic (BDZ) sites coupled to the GABA A receptor complex]. In another experiment, fish (N = 6) were initially anesthetized with the EO and then transferred to an anesthetic-free aquarium containing flumazenil (a selective antagonist of binding sites for BDZ coupled to the GABA A receptor complex) or water to assess recovery time from the anesthesia. In this case, flumazenil was used to observe the involvement of the GABA-BDZ receptor in the EO mechanism of action. The results showed that diazepam potentiates the anesthetic effect of EO at all concentrations tested. Fish exposed to diazepam and EO showed faster recovery from anesthesia when flumazenil was added to the recovery bath (12.0 ± 0.3 and 7.2 ± 0.7, respectively) than those exposed to water (9.2 ± 0.2 and 3.5 ± 0.3, respectively). In conclusion, the results demonstrated the involvement of the GABAergic system in the anesthetic effect of L. alba EO on silver catfish.

Effect of (+)-dehydrofukinone on GABAA receptors and stress response in fish model

(+)-Dehydrofukinone (DHF) is a major component of the essential oil of Nectandra grandiflora (Lauraceae), and exerts a depressant effect on the central nervous system of fish. However, the neuronal mechanism underlying DHF action remains unknown. This study aimed to investigate the action of DHF on GABAA receptors using a silver catfish (Rhamdia quelen) model. Additionally, we investigated the effect of DHF exposure on stress-induced cortisol modulation. Chemical identification was performed using gas chromatography-mass spectrometry and purity was evaluated using gas chromatography with a flame ionization detector. To an aquarium, we applied between 2.5 and 50 mg/L DHF diluted in ethanol, in combination with 42.7 mg/L diazepam. DHF within the range of 10-20 mg/L acted collaboratively in combination with diazepam, but the sedative action of DHF was reversed by 3 mg/L flumazenil. Additionally, fish exposed for 24 h to 2.5-20 mg/L DHF showed no side effects and there was sustained sedation during the first 12 h of drug exposure with 10-20 mg/L DHF. DHF pretreatment did not increase plasma cortisol levels in fish subjected to a stress protocol. Moreover, the stress-induced cortisol peak was absent following pretreatment with 20 mg/L DHF. DHF proved to be a relatively safe sedative or anesthetic, which interacts with GABAergic and cortisol pathways in fish.