The interaction of meal-related, rhythmic and homeostatic mechanisms and the generation of thirst and drinking

One of the primary goals of the study of thirst is to understand why drinking occurs under ad libitum or natural conditions. An appreciation of the experimental strategies applied by physiologists studying thirst from different perspectives can facilitate progress toward understanding the natural history of drinking behavior. Drinking research carried out using three separate perspectives - homeostatic, circadian rhythms, and food-associated - generates types of information about the mechanisms underlying drinking behavior. By combining research strategies and methods derived from each of these approaches, it has been possible to gain new information that increases our appreciation of the interactions between homeostatic mechanisms and circadian rhythms in the modulation of water intake and how these might be related to drinking associated with food intake under near natural conditions

Faster gastric emptying of a liquid meal in rats after hypothalamic dorsomedial nucleus lesion

The effects of dorsomedial hypothalamic (DMH) nucleus lesion on body weight, plasma glucose levels, and the gastric emptying of a liquid meal were investigated in male Wistar rats (170-250 g). DMH lesions were produced stereotaxically by delivering a 2.0-mA current for 20 s through nichrome electrodes (0.3-mm tip exposure). In a second set of experiments, the DMH and the ventromedial hypothalamic (VMH) nucleus were lesioned with a 1.0-mA current for 10 s (0.1-mm tip exposure). The medial hypothalamus (MH) was also lesioned separately using a nichrome electrode (0.3-mm tip exposure) with a 2.0-mA current for 20 s. Gastric emptying was measured following the orogastric infusion of a liquid test meal consisting of physiological saline (0.9% NaCl, w/v) plus phenol red dye (6 mg/dl) as a marker. Plasma glucose levels were determined after an 18-h fast before the lesion and on the 7th and 15th postoperative day. Body weight was determined before lesioning and before sacrificing the rats. The DMH-lesioned rats showed a significantly faster (P<0.05) gastric emptying (24.7% gastric retention, N = 11) than control (33.0% gastric retention, N = 8) and sham-lesioned (33.5% gastric retention, N = 12) rats, with a transient hypoglycemia on the 7th postoperative day which returned to normal by the 15th postoperative day. In all cases, weight gain was slower among lesioned rats. Additional experiments using a smaller current to induce lesions confirmed that DMH-lesioned rats had a faster gastric emptying (25.1% gastric retention, N = 7) than control (33.4% gastric retention, N = 17) and VMH-lesioned (34.6% gastric retention, N = 7) rats. MH lesions resulted in an even slower gastric emptying (43.7% gastric retention, N = 7) than in the latter two groups. We conclude that although DMH lesions reduce weight gain, they do not produce consistent changes in plasma glucose levels. These lesions also promote faster gastric emptying of an inert liquid meal, thus suggesting a role for the DMH in the regulation of gastric motility

The C-peptide response to a standard mixed meal in a group of Brazilian type 1 diabetic patients

In order to analyze the different parameters used in the interpretation of C-peptide response in a functional test, we compared a group of 26 type 1 diabetics aged 21.1 ± 8.2 years, with a diabetes duration of 7.9 ± 6.7 months, with a group of 24 non-diabetic subjects aged 25.0 ± 4.4 years. A standard mixed meal of 317 kcal was used as a stimulus. Blood sampling for C-peptide determinations was performed at regular intervals. Although all the studied C-peptide variables were significantly lower in the diabetic group (P<0.0001), some overlapping of parameters was observed between the two groups. The highest degree of overlapping was found for basal value (BV) (30.8%) and percent increase (42.31%), and the lowest for incremental area, absolute increase, peak value (PV) (3.8%), and total area (7.7%) (c2 = 31.6, P<0.0001). We did not observe a definite pattern in the time of maximum response among the 21 diabetics who showed an increase in C-peptide levels after the stimulus. In this group, however, there was a highly significant number of late responses (120 min) (c2 = 5.7, P<0.002). Although BV showed a significant correlation with PV (rS = 0.95, P<0.0001), the basal levels of C-peptide did not differentiate the groups with and without response to the stimulus. We conclude that the diabetic group studied showed delayed and reduced C-peptide responses, and that the functional test can be an important tool for the evaluation of residual ß cell function.

Acute blood volume expansion delays the gastrointestinal transit of a charcoal meal in awake rats

The present study evaluates the effect of blood volume expansion on the gastrointestinal transit of a charchoal meal (2.5 ml of an aqueous suspension consisting of 5% charcoal and 5% gum arabic) in awake male Wistar rats (200-270 g). On the day before the experiments, the rats were anesthetized with ether, submitted to left jugular vein cannulation and fasted with water ad libitum until 2 h before the gastrointestinal transit measurement. Blood volume expansion by iv infusion of 1 ml/min Ringer bicarbonate in volumes of 3, 4 or 5% body weight delayed gastrointestinal transit at 10 min after test meal administration by 21.3-26.7% (P<0.05), but no effect was observed after 1 or 2% body weight expansion. The effect of blood volume expansion (up to 5% body weight) on gastrointestinal transit lasted for at least 60 min (P<0.05). Mean arterial pressure increased transiently and central venous pressure increased and hematocrit decreased (P<0.05). Subdiaphragmatic vagotomy and yohimbine (3 mg/kg) prevented the delay caused by expansion on gastrointestinal transit, while atropine (0.5 mg/kg), L-NAME (2 mg/kg), hexamethonium (10 mg/kg), prazosin (1 mg/kg) or propranolol (2 mg/kg) were ineffective. These data show that blood volume expansion delays the gastrointestinal transit of a charcoal meal and that vagal and yohimbine-sensitive pathways appear to be involved in this phenomenon. The delay in gastrointestinal transit observed here, taken together with the modifications of gastrointestinal permeability to salt and water reported by others, may be part of the mechanisms involved in liquid excess management.

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.

Meal-feeding scheme: twenty years of research in Brazil

Naomi Shinomiya Hell was the first researcher to investigate the physiological adaptations to a meal-feeding scheme (MFS) in Brazil. Over a period of 20 years, from 1979 to 1999, Naomi's group determined the physiological and metabolic adaptations induced by this feeding scheme in rats. The group showed the persistence of such adaptations even when MFS is associated with moderate exercise training and the performance to a session of intense physical effort. The metabolic changes induced by the feeding training were discriminated from those caused by the effective fasting period. Naomi made an important contribution to the understanding of the MFS but a lot still has to be done. One crucial question still remains to be satisfactorily answered: what is the ideal control for the MFS?

Influence of season and pollution on the antioxidant defenses of the cichlid fish acará (Geophagus brasiliensis)

The livers of Geophagus brasiliensis collected from both a non-polluted site and a polluted site were analyzed for different antioxidant defenses, O2 consumption, thiobarbituric acid-reactive substance (TBARS) levels, and histological damage. Compared to controls (116.6 ± 26.1 nmol g-1), TBARS levels were enhanced at the polluted site (284.2 ± 25.6 nmol g-1), as also was oxygen consumption (86.6 ± 11.3 and 128.5 ± 9.8 µmol O2 min-1 g-1, respectively). With respect to enzymatic antioxidants, increased catalase activities (8.7 ± 1.3 and 29.2 ± 2.4 mmol min-1 g-1, respectively), unchanged superoxide dismutase activities (767.2 ± 113.3 and 563.3 ± 70.2 U g-1, respectively), and diminished glutathione S-transferase activities (29.0 ± 3.2 and 14.9 ± 3.2 µmol min-1 g-1, respectively) were detected. Reduced glutathione (1.91 ± 0.17 and 1.37 ± 0.25 mM, respectively), oxidized glutathione (1.50 ± 0.20 and 0.73 ± 0.17 mM, respectively), and total glutathione (3.40 ± 0.26 and 2.07 ± 0.27 mM, respectively) concentrations were also below control values at the polluted site. Nevertheless, the observed ethoxyresorufin-O-deethylase activities (1.34 ± 0.11 and 16.7 ± 0.21 pmol min-1 mg-1, respectively) showed enhanced values at the polluted site. The main histological damage observed in the hepatocytes from fish collected at the polluted site was characterized by heavy lipid infiltration. Fish collected at the end of spring showed higher O2 consumption, higher superoxide dismutase and glutathione S-transferase activities, and higher total and oxidized glutathione concentrations compared to the beginning of autumn. No seasonal changes were observed in catalase activities, glutathione or TBARS levels. Fish chronically exposed to relatively high pollution levels seem to be unable to set up adequate antioxidant defenses, probably due to severe injury to their hepatocytes. The higher antioxidant defenses found at the end of spring are probably related to the enhanced activities during high temperature periods in thermoconforming organisms.

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.

Different biochemical strategies of two Neotropical fish to cope with the impairment of nitrogen excretion during air exposure

The exposure of fish to air is normally expected to interfere with the nitrogen excretion process. Hoplias malabaricus and Hoplerythrinus unitaeniatus, two teleost species, display distinct behaviors in response to decreases in natural reservoir water levels, although they may employ similar biochemical strategies. To investigate this point, plasma levels of ammonia, urea, uric acid, and the two urea cycle enzymes, ornithine carbamoyl transferase (OCT) and arginase (ARG), as well as glutamine synthetase (GS) were determined for both species after exposure to air. Plasma ammonia increased gradually during exposure to air, but only H. malabaricus showed increased concentrations of urea. Plasma uric acid remained very low in both fish. Enzymatic activities (mean ± SD, µmol min-1 g protein-1) of H. malabaricus showed significant increases (P<0.05, N = 6) in OCT from 0.84 ± 0.05 to 1.42 ± 0.03, in ARG from 8.07 ± 0.47 to 9.97 ± 0.53 and in GS from 1.15 ± 0.03 to 2.39 ± 0.04. The OCT and ARG enzymes remained constant in H. unitaeniatus (N = 6), but GS increased from 1.49 ± 0.02 to 2.06 ± 0.03. Although these species are very closely related and share the same environment, their biochemical strategies in response to exposure to air or to increased plasma ammonia are different.

Eye color as an indicator of social rank in the fish Nile tilapia

We investigated the association of eye color with the dominant-subordinate relationship in the fish Nile tilapia, Oreochromis niloticus. Eye color pattern was also examined in relation to the intensity of attacks. We paired 20 size-matched fish (intruder: 73.69 ± 11.49 g; resident: 75.42 ± 8.83 g) and evaluated eye color and fights. These fish were isolated in individual aquaria for 10 days and then their eye color was measured 5 min before pairing (basal values). Twenty minutes after pairing, eye color and fights were quantified for 10 min. Clear establishment of social hierarchy was observed in 7 of 10 pairs of fish. Number of attacks ranged from 1 to 168 among pairs. The quartile was calculated for these data and the pairs were then divided into two classes: low-attack (1 to 111 attacks - 2 lower quartiles) or high-attack (112 to 168 attacks - 2 higher quartiles). Dominance decreased the eye-darkening patterns of the fish after pairing, while subordinance increased darkening compared to dominance. Subordinate fish in low-attack confrontations presented a darker eye compared to dominant fish and to the basal condition. We also observed a paler eye pattern in dominants that shared low-attack interactions after pairing compared to the subordinates and within the group. However, we found no differences in the darkening pattern between dominants and subordinates from the high-attack groups. We conclude that eye color is associated with social rank in this species. Moreover, the association between eye color and social rank in the low-attack pairs may function to reduce aggression.

Effect of chronic fish oil supplementation on renal function of normal and cachectic rats

In the present study we determined the effect of chronic diet supplementation with n-3 PUFA on renal function of healthy and cachectic subjects by providing fish oil (1 g/kg body weight) to female rats throughout pregnancy and lactation and then to their offspring post-weaning and examined its effect on renal function parameters during their adulthood. The animals were divided into four groups of 5-10 rats in each group: control, control supplemented with fish oil (P), cachectic Walker 256 tumor-bearing (W), and W supplemented with fish oil (WP). Food intake was significantly lower in the W group compared to control (12.66 ± 4.24 vs 25.30 ± 1.07 g/day). Treatment with fish oil significantly reversed this reduction (22.70 ± 2.94 g/day). Tumor growth rate was markedly reduced in the P group (16.41 ± 2.09 for WP vs 24.06 ± 2.64 g for W). WP group showed a significant increase in mean glomerular filtration rate compared to P and control (1.520 ± 0.214 ml min-1 kg body weight-1; P < 0.05). Tumor-bearing groups had low urine osmolality compared to control rats. The fractional sodium excretion decreased in the W group compared to control (0.43 ± 0.16 vs 2.99 ± 0.87%; P < 0.05), and partially recovered in the WP group (0.90 ± 0.20%). In summary, the chronic supplementation with fish oil used in this study increased the amount of fat in the diet by only 0.1%, but caused remarkable changes in tumor growth rate and cachexia, also showing a renoprotective function.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 µg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 ± 393 cells/mm²) compared to control (1886 ± 892 cells/mm²; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g) and 845 ± 82 cells/mm² (6 µg/g), also lower than control (1312 ± 31 cells/mm²; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Agonistic-like responses from the torus semicircularis dorsalis elicited by GABA A blockade in the weakly electric fish Gymnotus carapo

Findings by our group have shown that the dorsolateral telencephalon of Gymnotus carapo sends efferents to the mesencephalic torus semicircularis dorsalis (TSd) and that presumably this connection is involved in the changes in electric organ discharge (EOD) and in skeletomotor responses observed following microinjections of GABA A antagonist bicuculline into this telencephalic region. Other studies have implicated the TSd or its mammalian homologue, the inferior colliculus, in defensive responses. In the present study, we explore the possible involvement of the TSd and of the GABA-ergic system in the modulation of the electric and skeletomotor displays. For this purpose, different doses of bicuculline (0.98, 0.49, 0.245, and 0.015 mM) and muscimol (15.35 mM) were microinjected (0.1 µL) in the TSd of the awake G. carapo. Microinjection of bicuculline induced dose-dependent interruptions of EOD and increased skeletomotor activity resembling defense displays. The effects of the two highest doses showed maximum values at 5 min (4.3 ± 2.7 and 3.8 ± 2.0 Hz, P < 0.05) and persisted until 10 min (11 ± 5.7 and 8.7 ± 5.2 Hz, P < 0.05). Microinjections of muscimol were ineffective. During the interruptions of EOD, the novelty response (increased frequency in response to sensory novelties) induced by an electric stimulus delivered by a pair of electrodes placed in the water of the experimental cuvette was reduced or abolished. These data suggest that the GABA-ergic mechanisms of the TSd inhibit the neural substrate of the defense reaction at this midbrain level.

Effects of mercury intoxication on the response of horizontal cells of the retina of thraira fish (Hoplias malabaricus)

Methyl mercury (MeHg) is highly neurotoxic, affecting visual function in addition to other central nervous system functions. The effect of mercury intoxication on the amplitude of horizontal cell responses to light was studied in the retina of the fish Hoplias malabaricus. Intracellular responses were recorded from horizontal cells of fish previously intoxicated with MeHg by intraperitoneal injection (IP group) or by trophic exposure (T group). Only one retina per fish was used. The doses of MeHg chloride administered to the IP group were 0.01, 0.05, 0.1, 1.0, 2.0, and 6.0 mg/kg. The amplitudes of the horizontal cell responses were lower than control in individuals exposed to 0.01 (N = 4 retinas), 0.05 (N = 2 retinas) and 0.1 mg/kg (N = 1 retina), whereas no responses were recorded in the 1.0, 2.0, and 6.0 mg/kg groups. T group individuals were fed young specimens of Astyanax sp previously injected with MeHg corresponding to 0.75 (N = 1 retina), 0.075 (N = 8 retinas) or 0.0075 (N = 4 retinas) mg/kg fish body weight. After 14 doses, one every 5 days, the amplitude of the horizontal cell response was higher than control in individuals exposed to 0.075 and 0.0075 mg/kg, and lower in individuals exposed to 0.75 mg/kg. We conclude that intoxication with MeHg affects the electrophysiological response of the horizontal cells in the retina, either reducing or increasing its amplitude compared to control, and that these effects are related to the dose and/or to the mode of administration.

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.