Experimental anxiety and the reinforcing effects of ethanol in rats

In order to examine the relationship between anxiety and reinforcing effects of alcohol, drug-naive male Wistar rats weighing 250-300 g were classified as "anxious" and "non-anxious" in the elevated plus-maze test. A conditioned place preference test was then used to investigate the reinforcing effects of ethanol (EtOH) on these animals. On 2 alternate days, groups of "anxious", "non-anxious" and "normal" rats received intraperitoneal (ip) injections of EtOH (0.5, 1.0 or 1.5 g/kg) immediately before a 15-min confinement to the white compartment. On the 2 intervening days the same rats received ip injections of saline before confinement to the opposite compartment. On day 5, a 15-min free-choice test was carried out with no injections. Rats classified as "anxious" showed a significant, though not dose-dependent preference for all doses of ethanol compared to saline-treated animals. These data demonstrate that rats regarded as "anxious" are more sensitive to the reinforcing effects of EtOH than "non-anxious" and "normal" Wistar rats and emphasize the relevance of the basal levels of anxiety of rats when trying to detect the reinforcing effects of EtOH.

DL-methionine supplementation of rice-and-bean diets affects gamma-glutamyltranspeptidase activity and glutathione content in livers of growing rats

Gamma-glutamyltranspeptidase (GGT-EC 2.3.2.2) activity and glutathione (GSH) content were measured in livers of female weanling Wistar rats (N = 5-18), submitted to rice-and-bean diets (13 and 6% w/w protein), both supplemented or not with DL-methionine (0.5 and 0.23 g/100 g dry diet, respectively). After 28 days, the rats on the rice-and-bean diets showed significantly higher levels (four times higher) of liver GGT activity and a concomitant 50% lower concentration of liver GSH in comparison with control groups feeding on casein. The addition of DL-methionine to rice-and-bean diets significantly increased the liver GSH content, which reached levels 50% higher than those found in animals on casein diets. The increase in GSH was accompanied by a decrease in liver GGT activity, which did not reach levels as low as those observed in the control groups. No significant correlation could be established between GGT and GSH changes under the present experimental conditions. Linear correlation analysis only revealed that in animals submitted to unsupplemented rice-and-bean diets GSH concentration was positively associated (P<0.05) with weight gain, food intake and food efficiency. GGT, however, was negatively correlated (P<0.05) with food intake only, and exclusively for supplemented rice-and-bean diets. The high levels of GGT activity observed in the present study for rats receiving a rice-and-bean mixture could be a result of the poor quality of these diets associated with their deficiency in sulfur amino acids. The results also suggest that diet supplementation with methionine could be important in the reduction of the deleterious effects of GSH depletion by restoring the intracellular concentration of this tripeptide.

Ethological analysis of mother-pup interactions and other behavioral reactions in rats: effects of malnutrition and tactile stimulation of the pups

Mother-pup interaction, as well as other behavioral reactions were studied during the lactation period in 24 litters of Wistar rats and their dams fed either a 16% (control - C; 12 litters) or a 6% (malnourished - M; 12 litters) protein diet. The diets were isocaloric. Throughout lactation there was a 36.4% weight loss of M dams and a 63% body weight deficit in the M pups when compared to control pups. During this period, half of the litters were exposed daily to additional tactile stimulation (CS or MS), while the other half were submitted to normal rearing conditions (CN or MN). The tactile stimulation of pups (handling) consisted of holding the animal in one hand and gently touching the dorsal part of the animal's body with the fingers for 3 min. A special camera and a time-lapse video were used to record litter behavior in their home cages. Starting at 6 p.m. and ending at 6 a.m., on days 3, 6, 12, 15, 18 and 21 of lactation, photos were taken at 4-s intervals. An increase in the frequency (154.88 ± 16.19) and duration (455.86 ± 18.05 min) of suckling was observed throughout the lactation period in all groups compared to birth day (frequency 24.88 ± 2.37 and duration 376.76 ± 21.01 min), but the frequency was higher in the C (84.96 ± 8.52) than in the M group (43.13 ± 4.37); however, the M group (470.2 ± 11.87 min) spent more time suckling as compared with the C group (393.67 ± 13.09 min). The M dams showed a decreased frequency of resting position throughout the lactation period (6.5 ± 2.48) compared to birth day (25.42 ± 7.74). Pups from the C group were more frequently observed separated (73.02 ± 4.38) and interacting (258.99 ± 20.61) more with their mothers than the M pups (separated 66.94 ± 5.5 and interacting 165.72 ± 12.05). Tactile stimulation did not interact with diet condition, showing that the kind of stimulation used in the present study did not lead to recovery from the changes induced by protein malnutrition. The changes in mother-pup interaction produced by protein malnutrition of both may represent retardation in neuromotor development and a higher dependence of the pups on their mothers. These changes may represent an important means of energy saving and heat maintenance in malnourished pups.

Zinc and water intake in rats: investigation of adrenergic and opiatergic central mechanisms

We have demonstrated that central administration of zinc in minute amounts induces a significant antidipsogenic action in dehydrated rats as well as in rats under central cholinergic and angiotensinergic stimulation. Here we show that acute third ventricle injections of zinc also block water intake induced by central ß-adrenergic stimulation in Wistar rats (190-250 g). Central inhibition of opioid pathways by naloxone reverses the zinc-induced antidipsogenic effect in dehydrated rats. After 120 min, rats receiving third ventricle injections of isoproterenol (160 nmol/rat) exhibited a significant increase in water intake (5.78 ± 0.54 ml/100 g body weight) compared to saline-treated controls (0.15 ± 0.07 ml/100 g body weight). Pretreatment with zinc (3.0, 30.0 and 300.0 pmol/rat, 45 min before isoproterenol injection) blocked water intake in a dose-dependent way. At the highest dose employed a complete blockade was demonstrable (0.54 ± 0.2 ml/100 g body weight). After 120 min, control (NaAc-treated) dehydrated rats, as expected, exhibited a high water intake (7.36 ± 0.39 ml/100 g body weight). Central administration of zinc blocked this response (2.5 ± 0.77 ml/100 g body weight). Naloxone pretreatment (82.5 nmol/rat, 30 min before zinc administration) reverted the water intake to the high levels observed in zinc-free dehydrated animals (7.04 ± 0.56 ml/100 g body weight). These data indicate that zinc is able to block water intake induced by central ß-adrenergic stimulation and that zinc-induced blockade of water intake in dehydrated rats may be, at least in part, due to stimulation of central opioid peptides.

Central lead administration inhibits water intake and sodium appetite in rats

We have demonstrated that acute third ventricle injections of lead acetate (PbAc) exert a powerful antidipsogenic effect and induce a significant increase in renal sodium excretion. In the present study we confirm the antidipsogenic effect of lead and demonstrate that central administration of this metal, in minute amounts, significantly reduces salt intake both during dehydration and after central angiotensinergic stimulation. Adult male Wistar rats had the third ventricle cannulated seven days before the experiments. During this period they had free access to distilled water and hypertonic saline solution (1.5%). After a 24-h period of fluid deprivation, experimental animals received third ventricle injections of PbAc (0.3, N = 8 and 3.0 nmol/rat, N = 14) while controls received sodium acetate (NaAc; 3.0 nmol/rat, N = 10). Rats treated with PbAc at the highest dose showed a significant reduction (P<0.05) both in water and hypertonic saline intake when compared to controls. When the effect of lead administration on angiotensin II-induced water and salt intake was studied, normohydrated animals received third ventricle injections of angiotensin II (9.6 nmol/rat) after pretreatment with 3.0 nmol/rat of PbAc (experimental group, N = 10) or NaAc (controls, N = 8). The group pretreated with PbAc presented a significant reduction (P<0.05) in both water and salt intake compared to controls. Thus, this study confirms the antidipsogenic effect of central lead injections and demonstrates that the presence of lead in the brain exerts a significant inhibition of sodium appetite.

Defense reaction induced by a metabotropic glutamate receptor agonist microinjected into the dorsal periaqueductal gray of rats

The behavioral effects of trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), a metabotropic glutamate receptor (mGluR) agonist, or 0.9% (w/v) saline, injected into the dorsal periaqueductal gray (DPAG), was investigated. Male Wistar rats showed defense reactions characterized by jumps toward the top edges of the cages (saline = 0 vs t-ACPD = 6.0, medians P<0.05) and gallops (saline = 0 vs t-ACPD = 10.0, medians P<0.05) during the 60-s period after the beginning of the injection. In another experiment animals were placed inside an open arena for 5 min immediately after injection. Their behavior was recorded by a video camera and a computer program analyzed the videotapes. Eleven of fifteen rats injected with t-ACPD showed a short-lasting (about 1 min) flight reaction. No saline-treated animal showed this reaction (P<0.0005, chi-square test). The drug induced an increase in turning behavior (P = 0.002, MANOVA) and a decrease in the number of rearings (P<0.001, MANOVA) and grooming episodes (P<0.001, MANOVA). These results suggest that mGluRs play a role in the control of defense reactions in the DPAG.

Chronic effect of antidopaminergic drugs or estrogen on male Wistar rat lactotrophs and somatotrophs

The aim of the present study was to evaluate the effect of antidopaminergic agents on the somatotrophs in the presence of hyperprolactinemia. Adult male Wistar rats were divided into 6 groups: a control group and five groups chronically treated (60 days) with haloperidol, fluphenazine, sulpiride, metoclopramide or estrogen. Somatotrophs and lactotrophs were identified by immunohistochemistry and the data are reported as percent of total anterior pituitary cells counted. The drugs significantly increased the percentage of lactotrophs: control (mean ± SD) 21.3 ± 4.4, haloperidol 27.8 ± 2.2, fluphenazine 34.5 ± 3.6, sulpiride 32.7 ± 3.5, metoclopramide 33.4 ± 5.5 and estrogen 42.4 ± 2.8. A significant reduction in somatotrophs was observed in animals treated with haloperidol (23.1 ± 3.0), fluphenazine (22.1 ± 1.1) and metoclopramide (24.2 ± 3.0) compared to control (27.3 ± 3.8), whereas no difference was observed in the groups treated with sulpiride (25.0 ± 2.2) and estrogen (27.1 ± 2.8). In the groups in which a reduction occurred, this may have simply been due to dilution, secondary to lactotroph hyperplasia. In view of the duplication of the percentage of prolactin-secreting cells, when estrogen was applied, the absence of a reduction in the percent of somatotrophs suggests a replication effect on this cell population. These data provide additional information about the direct or indirect effect of drugs which, in addition to interfering with the dopaminergic system, may act on other pituitary cells as well as on the lactotrophs.

Effect of inhibitory avoidance training on [3H]-glutamate binding in the hippocampus and parietal cortex of rats

Glutamate receptors have been implicated in memory formation. The aim of the present study was to determine the effect of inhibitory avoidance training on specific [3H]-glutamate binding to membranes obtained from the hippocampus or parietal cortex of rats. Adult male Wistar rats were trained (0.5-mA footshock) in a step-down inhibitory avoidance task and were sacrificed 0, 5, 15 or 60 min after training. Hippocampus and parietal cortex were dissected and membranes were prepared and incubated with 350 nM [3H]-glutamate (N = 4-6 per group). Inhibitory avoidance training induced a 29% increase in glutamate binding in hippocampal membranes obtained from rats sacrificed at 5 min (P<0.01), but not at 0, 15, or 60 min after training, and did not affect glutamate binding in membranes obtained from the parietal cortex. These results are consistent with previous evidence for the involvement of glutamatergic synaptic modification in the hippocampus in the early steps of memory formation.

Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats

The role of sympathetic nerve activity in the changes in arterial blood pressure and renal function caused by the chronic administration of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was examined in sham and bilaterally renal denervated rats. Several studies have demonstrated that sympathetic nerve activity is elevated acutely after L-NAME administration. To evaluate the role of renal nerve activity in L-NAME-induced hypertension, we compared the blood pressure response in four groups (N = 10 each) of male Wistar-Hannover rats weighing 200 to 250 g: 1) sham-operated vehicle-treated, 2) sham-operated L-NAME-treated, 3) denervated vehicle-treated, and 4) denervated L-NAME-treated rats. After renal denervation or sham surgery, one control week was followed by three weeks of oral administration of L-NAME by gavage. Arterial pressure was measured weekly in conscious rats by a tail-cuff method and renal function tests were performed in individual metabolic cages 0, 7, 14 and 21 days after the beginning of L-NAME administration. L-NAME (60 mg kg-1 day-1) progressively increased arterial pressure from 108 ± 6.0 to 149 ± 12 mmHg (P<0.05) in the sham-operated group by the third week of treatment which was accompanied by a fall in creatinine clearance from 336 ± 18 to 222 ± 59 µl min-1 100 g body weight-1 (P<0.05) and a rise in fractional urinary sodium excretion from 0.2 ± 0.04 to 1.62 ± 0.35% (P<0.05) and in sodium post-proximal fractional excretion from 0.54 ± 0.09 to 4.7 ± 0.86% (P<0.05). The development of hypertension was significantly delayed and attenuated in denervated L-NAME-treated rats. This was accompanied by a striking additional increase in fractional renal sodium and potassium excretion from 0.2 ± 0.04 to 4.5 ± 1.6% and from 0.1 ± 0.015 to 1.21 ± 0.37%, respectively, and an enhanced post-proximal sodium excretion compared to the sham-operated group. These differences occurred despite an unchanged creatinine clearance and Na+ filtered load. These results suggest that bilateral renal denervation delayed and attenuated the L-NAME-induced hypertension by promoting an additional decrease in tubule sodium reabsorption in the post-proximal segments of nephrons. Much of the hypertension caused by chronic NO synthesis inhibition is thus dependent on renal nerve activity.

Effects of exercise training on autonomic and myocardial dysfunction in streptozotocin-diabetic rats

Several investigators have demonstrated that diabetes is associated with autonomic and myocardial dysfunction. Exercise training is an efficient non-pharmacological treatment for cardiac and metabolic diseases. The aim of the present study was to investigate the effects of exercise training on hemodynamic and autonomic diabetic dysfunction. After 1 week of diabetes induction (streptozotocin, 50 mg/kg, iv), male Wistar rats (222 ± 5 g, N = 18) were submitted to exercise training for 10 weeks on a treadmill. Arterial pressure signals were obtained and processed with a data acquisition system. Autonomic function and intrinsic heart rate were studied by injecting methylatropine and propranolol. Left ventricular function was assessed in hearts perfused in vitro by the Langendorff technique. Diabetes (D) bradycardia and hypotension (D: 279 ± 9 bpm and 91 ± 4 mmHg vs 315 ± 11 bpm and 111 ± 4 mmHg in controls, C) were attenuated by training (TD: 305 ± 7 bpm and 100 ± 4 mmHg). Vagal tonus was decreased in the diabetic groups and sympathetic tonus was similar in all animals. Intrinsic heart rate was lower in D (284 ± 11 bpm) compared to C and TD (390 ± 8 and 342 ± 14 bpm, respectively). Peak systolic pressure developed at different pressures was similar for all groups, but +dP/dt max was decreased and -dP/dt max was increased in D. In conclusion, exercise training reversed hypotension and bradycardia and improved myocardial function in diabetic rats. These changes represent an adaptive response to the demands of training, supporting a positive role of physical activity in the management of diabetes.

Abnormal proliferative response of the carotid artery of spontaneously hypertensive rats after angioplasty may be related to the depolarized state of its smooth muscle cells

Hypertension is one of the major precursors of atherosclerotic vascular disease, and vascular smooth muscle abnormal cell replication is a key feature of plaque formation. The present study was conducted to examine the relationship between hypertension and smooth muscle cell proliferation after balloon injury and to correlate neointima formation with resting membrane potential of uninjured smooth muscle cells, since it has been suggested that altered vascular function in hypertension may be related to the resetting of the resting membrane potential in spontaneously hypertensive rats (SHR). Neointima formation was induced by balloon injury to the carotid arteries of SHR and renovascular hypertensive rats (1K-1C), as well as in their normotensive controls, i.e., Wistar Kyoto (WKY) and normal Wistar (NWR) rats. After 14 days the animals were killed and the carotid arteries were submitted to histomorphometric and immunohistochemical analyses. Resting membrane potential measurements showed that uninjured carotid arteries from SHR smooth muscle cells were significantly depolarized (-46.5 ± 1.9 mV) compared to NWR (-69 ± 1.4 mV), NWR 1K-1C (-60.8 ± 1.6 mV), WKY (-67.1 ± 3.2 mV) and WKY 1K-1C (-56.9 ± 1.2 mV). The SHR arteries responded to balloon injury with an enhanced neointima formation (neo/media = 3.97 ± 0.86) when compared to arteries of all the other groups (NWR 0.93 ± 0.65, NWR 1K-1C 1.24 ± 0.45, WKY 1.22 ± 0.32, WKY 1K-1C 1.15 ± 0.74). Our results indicate that the increased fibroproliferative response observed in SHR is not related to the hypertensive state but could be associated with the resetting of the carotid smooth muscle cell resting membrane potential to a more depolarized state.

Effects of adrenalectomy and adrenal enucleation on liquid gastric emptying in rats

The effects of adrenalectomy and adrenal enucleation on liquid gastric emptying were studied in male Wistar rats that were adrenalectomized, adrenal enucleated (AE) or sham operated (SH). The animals in the first group had free access to a 1% NaCl solution (ADS), while the animals in the second and third groups were divided into two subgroups, which ingested either tap water (AEW, SHW) or 1% NaCl solution (AES, SHS). The gastric emptying study was performed on the 16th post-operative day. Three test meals labeled with phenol red (6 mg/dl) were used (0.9% NaCl, 1.8% NaCl and 5% glucose). Percent gastric retention was determined 10 min after orogastric infusion of the NaCl test meals and 15 min after the glucose meal. Gastric retention of the ADS subgroup was significantly lower (P<0.01) (median = 19.8% vs 25.5% for SHW, vs 31.9% for SHS, vs 25.7% for AEW, and vs 27.1% for AES) for the 0.9% NaCl test meal and for the 1.8% NaCl test meal (33.5% for ADS vs 47.5% for AEW and 50.6% for AES). When 5% glucose was used as a test meal, gastric retention was similar for all subgroups. These results suggest that ablation of the adrenal cortex results in increased gastric emptying of an isosmolar NaCl meal.

Inhibition of gastric emptying and intestinal transit in anesthetized rats by a Tityus serrulatus scorpion toxin

The effects of a fraction (T1) of Tityus serrulatus scorpion venom prepared by gel filtration on gastric emptying and small intestinal transit were investigated in male Wistar rats. Fasted animals were anesthetized with urethane, submitted to tracheal intubation and right jugular vein cannulation. Scorpion toxin (250 µg/kg) or saline was injected iv and 1 h later a bolus of saline (1.0 ml/100 g) labeled with 99m technetium-phytate (10 MBq) was administered by gavage. After 15 min, animals were sacrificed and the radioactivity remaining in the stomach was determined. Intestinal transit was evaluated by instillation of a technetium-labeled saline bolus (1.0 ml) through a cannula previously implanted in the duodenum. After 60 min, the progression of the marker throughout 7 consecutive gut segments was estimated by the geometric center method. Gastric retention of the liquid test meal in rats injected with scorpion toxin (median: 88%; range: 52-95%) was significantly higher (P<0.02) than in controls (54%; 21-76%), an effect which was not modified by gastric secretion blockade with ranitidine. The progression of the isotope marker throughout the small intestine was significantly slower (P<0.05) in rats treated with toxin (1.2; 1.0-2.5) than in control animals (2.3; 1.0-3.2). Inhibition of both gastric emptying and intestinal transit in rats injected with scorpion toxin suggests an increased resistance to aboral flow, which might be caused by abnormal neurotransmitter release or by the local effects of venom on smooth muscle cells.

Sexual behavior and fertility of male rats submitted to prolonged immobilization-induced stress

In order to investigate whether prolonged stress interferes with the onset of sexual behavior at puberty and with fertility at adulthood, prepubertal male Wistar rats (40 days of age) were immobilized 6 h a day for 15 days (up to early puberty) or for 60 days (until sexual maturity). Pubertal stressed rats showed a two-fold increase in the latency for the first mount (probably due to repeated aversive experience in which a change of environment was always followed by immobilization) and a 2.5-fold increase in the frequency of thrusting (indicative of enhanced sexual performance). The apparently stimulatory effect of prolonged stress on the onset of sexual behavior is discussed in terms of increased testosterone level and interference with the complex interchanges between the neurotransmitters/neuropeptides involved in the central control of male sexual activity. Adult stressed animals were mated with normal females, which became pregnant but exhibited a more than two-fold increase in both pre-implantation and post-implantation loss, probably due to a smaller rate of fertilization and/or fertilization with damaged spermatozoa.

Interaction between repeated restraint stress and concomitant midazolam administration on sweet food ingestion in rats

Emotional changes can influence feeding behavior. Previous studies have shown that chronically stressed animals present increased ingestion of sweet food, an effect reversed by a single dose of diazepam administered before testing the animals. The aim of the present study was to evaluate the response of animals chronically treated with midazolam and/or submitted to repeated restraint stress upon the ingestion of sweet food. Male adult Wistar rats were divided into two groups: controls and exposed to restraint 1 h/day, 5 days/week for 40 days. Both groups were subdivided into two other groups treated or not with midazolam (0.06 mg/ml in their drinking water during the 40-day treatment). The animals were placed in a lighted area in the presence of 10 pellets of sweet food (Froot loops®). The number of ingested pellets was measured during a period of 3 min, in the presence or absence of fasting. The group chronically treated with midazolam alone presented increased ingestion when compared to control animals (control group: 2.0 ± 0.44 pellets and midazolam group: 3.60 ± 0.57 pellets). The group submitted to restraint stress presented an increased ingestion compared to controls (control group: 2.0 ± 0.44 pellets and stressed group: 4.18 ± 0.58 pellets). Chronically administered midazolam reduced the ingestion in stressed animals (stressed/water group: 4.18 ± 0.58 pellets; stressed/midazolam group: 3.2 ± 0.49 pellets). Thus, repeated stress increases appetite for sweet food independently of hunger and chronic administration of midazolam can decrease this behavioral effect.