Agents that affect cAMP levels or protein kinase A activity modulate memory consolidation when injected into rat hippocampus but not amygdala

Male Wistar rats were trained in one-trial step-down inhibitory avoidance using a 0.4-mA footshock. At various times after training (0, 1.5, 3, 6 and 9 h for the animals implanted into the CA1 region of the hippocampus; 0 and 3 h for those implanted into the amygdala), these animals received microinfusions of SKF38393 (7.5 µg/side), SCH23390 (0.5 µg/side), norepinephrine (0.3 µg/side), timolol (0.3 µg/side), 8-OH-DPAT (2.5 µg/side), NAN-190 (2.5 µg/side), forskolin (0.5 µg/side), KT5720 (0.5 µg/side) or 8-Br-cAMP (1.25 µg/side). Rats were tested for retention 24 h after training. When given into the hippocampus 0 h post-training, norepinephrine enhanced memory whereas KT5720 was amnestic. When given 1.5 h after training, all treatments were ineffective. When given 3 or 6 h post-training, 8-Br-cAMP, forskolin, SKF38393, norepinephrine and NAN-190 caused memory facilitation, while KT5720, SCH23390, timolol and 8-OH-DPAT caused retrograde amnesia. Again, at 9 h after training, all treatments were ineffective. When given into the amygdala, norepinephrine caused retrograde facilitation at 0 h after training. The other drugs infused into the amygdala did not cause any significant effect. These data suggest that in the hippocampus, but not in the amygdala, a cAMP/protein kinase A pathway is involved in memory consolidation at 3 and 6 h after training, which is regulated by D1, ß, and 5HT1A receptors. This correlates with data on increased post-training cAMP levels and a dual peak of protein kinase A activity and CREB-P levels (at 0 and 3-6 h) in rat hippocampus after training in this task. These results suggest that the hippocampus, but not the amygdala, is involved in long-term storage of step-down inhibitory avoidance in the rat.

Conditioned medium from activated spleen cells supports the survival of rat retinal cells in vitro

Cytokines are a heterogeneous group of molecules that have been associated with several functions in the nervous system, such as survival and differentiation of neuronal and glial cells. In the present study, we demonstrated that conditioned medium from spleen cells activated with concanavalin A increased neuritogenesis and survival of retinal cells, as measured by biochemical and morphological criteria. Our data showed that conditioned medium induced a five-fold increase in the amount of protein after 120 h in vitro. This effect was not inhibited by the blockade of voltage-dependent L-type calcium channels with 5.0 µM nifedipine. However, the use of an intracellular calcium chelator (15.0 µM BAPTA-AM) inhibited this effect. Our results support the idea that factors secreted by activated lymphocytes, such as cytokines, can modulate the maintenance and the differentiation of rat retinal cells in vitro, indicating a possible role of these molecules in the development of retinal cells, as well as in its protection against pathological conditions

Hippocampal histamine receptors and conflictive exploration in the rat: studies using the elevated asymmetric plus-maze

The possible role of histamine receptors in the hippocampal formation on the exploratory motivation and emotionality of the rat was studied. An elevated asymmetric plus-maze composed of 4 different arms (no walls, single high wall, high and low walls and two high walls) arranged at 90o angles was used. The exploration score, considered to be an index of exploratory motivation, and the permanency score, considered to be an index of emotionality (anxiety), were determined. Histamine was administered locally into the ventral hippocampus at three different doses (9, 45 and 90 nmol). Another group of rats was also microinjected with 45 nmol of pyrilamine (a histamine H1 receptor antagonist) or ranitidine (a histamine H2 receptor antagonist) in addition to 9 nmol of histamine in order to identify the possible type of histamine receptor involved. Histamine administration significantly inhibited the exploration score and increased the permanency score at the doses of 9 and 45 nmol in two of four arms. These effects were completely blocked by the administration of either histamine receptor antagonist. The present results suggest that in the hippocampal formation histamine inhibits exploratory motivation and decreases emotionality by activating both types of histamine receptors. Also, the elevated asymmetric plus-maze appears to be a suitable technique to quantify exploration and possibly" anxiety"

Effects of estradiol benzoate on 5'-iodothyronine deiodinase activities in female rat anterior pituitary gland, liver and thyroid gland

There is little information on the possible effects of estrogen on the activity of 5'-deiodinase (5'-ID), an enzyme responsible for the generation of T3, the biologically active thyroid hormone. In the present study, anterior pituitary sonicates or hepatic and thyroid microsomes from ovariectomized (OVX) rats treated or not with estradiol benzoate (EB, 0.7 or 14 µg/100 g body weight, sc, for 10 days) were assayed for type I 5'-ID (5'-ID-I) and type II 5'-ID (5'-ID-II, only in pituitary) activities. The 5'-ID activity was evaluated by the release of 125I from deiodinated 125I rT3, using specific assay conditions for type I or type II. Serum TSH and free T3 and free T4 were measured by radioimmunoassay. OVX alone induced a reduction in pituitary 5'-ID-I (control = 723.7 ± 67.9 vs OVX = 413.9 ± 26.9; P<0.05), while the EB-treated OVX group showed activity similar to that of the normal group. Thyroid 5'-ID-I showed the same pattern of changes, but these changes were not statistically significant. Pituitary and hepatic 5'-ID-II did not show major alterations. The treatment with the higher EB dose (14 µg), contrary to the results obtained with the lower dose, had no effect on the reduced pituitary 5'-ID-I of OVX rats. However, it induced an important increment of 5'-ID-I in the thyroid gland (0.8 times higher than that of the normal group: control = 131.9 ± 23.7 vs ovx + EB 14 µg = 248.0 ± 31.2; P<0.05), which is associated with increased serum TSH (0.6-fold vs OVX, P<0.05) but normal serum free T3 and free T4. The data suggest that estrogen is a physiological stimulator of anterior pituitary 5'-ID-I and a potent stimulator of the thyroid enzyme when employed at high doses

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine

We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo) and 200 µM carbamylcholine (Cch). No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively). High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A more marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.

Expression of Fos protein in the rat central nervous system in response to noxious stimulation: effects of chronic inflammation of the superior cervical ganglion

The aim of this study was to investigate the possible interactions between the nociceptive system, the sympathetic system and the inflammatory process. Thus, the superior cervical ganglion of rats was submitted to chronic inflammation and Fos expression was used as a marker for neuronal activity throughout central neurons following painful peripheral stimulation. The painful stimulus consisted of subcutaneously injected formalin applied to the supra-ocular region. Fos-positive neurons were identified by conventional immunohistochemical techniques, and analyzed from the obex through the cervical levels of the spinal cord. In the caudal sub-nucleus of the spinal trigeminal nuclear complex, the number of Fos-positive neurons was much higher in rats with inflammation of the superior cervical ganglion than in control rats, either sham-operated or with saline applied to the ganglion. There was a highly significant difference in the density of Fos-positive neurons between the inflamed and control groups. No significant difference was found between control groups. These results suggest that the inflammation of the superior cervical ganglion generated an increased responsiveness to painful stimuli, which may have been due to a diminished sympathetic influence upon the sensory peripheral innervation.

Study of the effects of ß-myrcene on rat fertility and general reproductive performance

ß-Myrcene (MYR) is a monoterpene found in the oils of a variety of aromatic plants including lemongrass, verbena, hop, bay, and others. MYR and essential oils containing this terpenoid compound are used in cosmetics, household products, and as flavoring food additives. This study was undertaken to investigate the effects of MYR on fertility and general reproductive performance in the rat. MYR (0, 100, 300 and 500 mg/kg) in peanut oil was given by gavage to male Wistar rats (15 per dose group) for 91 days prior to mating and during the mating period, as well as to females (45 per dose group) continuously for 21 days before mating, during mating and pregnancy, and throughout the period of lactation up to postnatal day 21. On day 21 of pregnancy one-third of the females of each group were submitted to cesarean section. Resorption, implantation, as well as dead and live fetuses were counted. All fetuses were examined for external malformations, weighed, and cleared and stained with Alizarin Red S for skeleton evaluation. The remaining dams were allowed to give birth to their offspring. The progeny was examined at birth and subsequently up to postnatal day 21. Mortality, weight gain and physical signs of postnatal development were evaluated. Except for an increase in liver and kidney weights, no other sign of toxicity was noted in male and female rats exposed to MYR. MYR did not affect the mating index (proportion of females impregnated by males) or the pregnancy index (ratio of pregnant to sperm-positive females). No sign of maternal toxicity and no increase in externally visible malformations were observed at any dose level. Only at the highest dose tested (500 mg/kg) did MYR induce an increase in the resorption rate and a higher frequency of fetal skeleton anomalies. No adverse effect of MYR on postnatal weight gain was noted but days of appearance of primary coat, incisor eruption and eye opening were slightly delayed in the exposed offspring. On the basis of the data presented in this paper the no-observed-adverse-effect level (NOAEL) for toxic effects on fertility and general reproductive performance can be set at 300 mg of ß-myrcene/kg body weight by the oral route.

Gender differences in the activities of aspirin-esterases in rat tissues

The activities of aspirin (acetylsalicylic acid)-esterases were measured in several tissues (liver, kidney, adrenal glands, brain and serum) from adult male and female Wistar rats. In males, both aspirin-esterase I (assayed at pH 5.5) and II (assayed at pH 7.4) activities were higher in liver homogenates when compared to females (aspirin-esterase I: males 48.9 ± 4.8 (N = 8) and females 29.3 ± 4.2 (N = 8) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 41.4 ± 4.1 (N = 8) and females 26.1 ± 4.5 (N = 8) nmol of salicylic acid formed min-1 mg protein-1, P<0.001). In serum, enzyme activity was higher in females than in males (aspirin-esterase I: males 0.85 ± 0.06 (N = 6) and females 1.18 ± 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1; aspirin-esterase II: males 1.03 ± 0.13 (N = 6) and females 1.34 ± 0.11 (N = 6) nmol of salicylic acid formed min-1 mg protein-1, P<0.001). In the other tissues assayed, no statistically significant difference between males and females was found. There were no statistically significant differences when the enzymes were assayed in different phases of the estrous cycle in liver and serum. These results show that the differences in aspirin-esterase activity observed between males and females are not due to the estrous cycle. The gender difference obtained in our study may indicate an involvement of gonadal hormones in the control of the hydrolysis of aspirin. This possibility is currently under investigation.

Histopathological analysis of rat mesentery as a method for evaluating neutrophil migration: differential effects of dexamethasone and pertussis toxin

In the present study, histopathological analysis of rat mesentery was used to quantify the effect of two anti-inflammatory agents, dexamethasone (Dex) and pertussis toxin (Ptx), on leukocyte migration. The intravenous injection of Dex (1 mg/kg) and Ptx (1,200 ng) 1 h prior to the intraperitoneal injection of the inflammatory stimuli lipopolysaccharide (LPS) or formyl-methionyl-leucyl-phenylalanine (fMLP) significantly reduced the neutrophil diapedesis (LPS: Ptx = 0.86 ± 0.19 and Dex = 0.35 ± 0.13 vs saline (S) = 2.85 ± 0.59; fMLP: Ptx = 0.43 ± 0.09 and Dex 0.01 ± 0.01 vs S = 1.08 ± 0.15 neutrophil diapedesis/field) and infiltration (LPS: Ptx = 6.29 ± 1.4 and Dex = 3.06 ± 0.76 vs S = 15.94 ± 3.97; fMLP: Ptx = 3.85 ± 0.56 and Dex = 0.40 ± 0.16 vs S = 7.15 ± 1.17 neutrophils/field) induced by the two agonists in the rat mesentery. The inhibitory effect of Dex and Ptx was clearly visible in the fields nearest the venule (up to 200 µm), demonstrating that these anti-inflammatory agents act preferentially in the transmigration of neutrophils from the vascular lumen into the interstitial space, but not in cell movement in response to a haptotactic gradient. The mesentery of rats pretreated with Dex showed a decreased number of neutrophils within the venules (LPS: Dex = 1.50 ± 0.38 vs S = 4.20 ± 1.01; fMLP: Dex = 0.25 ± 0.11 vs S = 2.20 ± 0.34 neutrophils in the lumen/field), suggesting that this inhibitor may be acting at a step that precedes neutrophil arrival in the inflamed tissue. In contrast to that observed with Dex treatment, the number of neutrophils found in mesenteric venules was significantly elevated in animals pretreated with Ptx (LPS: Ptx = 9.85 ± 2.25 vs S = 4.20 ± 1.01; fMLP: Ptx = 4.66 ± 1.24 vs S = 2.20 ± 0.34 neutrophils in the lumen/field). This discrepancy shows that Ptx and Dex act via different mechanisms and suggests that Ptx prevents locomotion of neutrophils from the vascular lumen to the interstitial space. In conclusion, the method described here is useful for quantifying the inflammatory and anti-inflammatory effect of different substances. The advantage of this histopathological approach is that it provides additional information about the steps involved in leucocyte migration.

Respiratory effects of kynurenic acid microinjected into the ventromedullary surface of the rat

Several studies demonstrate that, within the ventral medullary surface (VMS), excitatory amino acids are necessary components of the neural circuits involved in the tonic and reflex control of respiration and circulation. In the present study we investigated the cardiorespiratory effects of unilateral microinjections of the broad spectrum glutamate antagonist kynurenic acid (2 nmol/200 nl) along the VMS of urethane-anesthetized rats. Within the VMS only one region was responsive to this drug. This area includes most of the intermediate respiratory area, partially overlapping the rostral ventrolateral medulla (IA/RVL). When microinjected into the IA/RVL, kynurenic acid produced a respiratory depression, without changes in mean arterial pressure or heart rate. The respiratory depression observed was characterized by a decrease in ventilation, tidal volume and mean inspiratory flow and an increase in respiratory frequency. Therefore, the observed respiratory depression was entirely due to a reduction in the inspiratory drive. Microinjections of vehicle (200 nl of saline) into this area produced no significant changes in breathing pattern, blood pressure or heart rate. Respiratory depression in response to the blockade of glutamatergic receptors inside the rostral VMS suggests that neurons at this site have an endogenous glutamatergic input controlling the respiratory cycle duration and the inspiratory drive transmission.

The left ventricular contractility of the rat heart is modulated by changes in flow and a1-adrenoceptor stimulation

Myocardial contractility depends on several mechanisms such as coronary perfusion pressure (CPP) and flow as well as on a1-adrenoceptor stimulation. Both effects occur during the sympathetic stimulation mediated by norepinephrine. Norepinephrine increases force development in the heart and produces vasoconstriction increasing arterial pressure and, in turn, CPP. The contribution of each of these factors to the increase in myocardial performance needs to be clarified. Thus, in the present study we used two protocols: in the first we measured mean arterial pressure, left ventricular pressure and rate of rise of left ventricular pressure development in anesthetized rats (N = 10) submitted to phenylephrine (PE) stimulation before and after propranolol plus atropine treatment. These observations showed that in vivo a1-adrenergic stimulation increases left ventricular-developed pressure (P<0.05) together with arterial blood pressure (P<0.05). In the second protocol, we measured left ventricular isovolumic systolic pressure (ISP) and CPP in Langendorff constant flow-perfused hearts. The hearts (N = 7) were perfused with increasing flow rates under control conditions and PE or PE + nitroprusside (NP). Both CPP and ISP increased (P<0.01) as a function of flow. CPP changes were not affected by drug treatment but ISP increased (P<0.01). The largest ISP increase was obtained with PE + NP treatment (P<0.01). The results suggest that both mechanisms, i.e., direct stimulation of myocardial a1-adrenoceptors and increased flow, increased cardiac performance acting simultaneously and synergistically.

Insulin receptor has tyrosine kinase activity toward Shc in rat liver

Insulin induces tyrosine phosphorylation of Shc in cell cultures and in insulin-sensitive tissues of the intact rat. However, the ability of insulin receptor (IR) tyrosine kinase to phosphorylate Shc has not been previously demonstrated. In the present study, we investigated insulin-induced IR tyrosine kinase activity towards Shc. Insulin receptor was immunoprecipitated from liver extracts, before and after a very low dose of insulin into the portal vein, and incubated with immunopurified Shc from liver of untreated rats. The kinase assay was performed in vitro in the presence of exogenous ATP and the phosphorylation level was quantified by immunoblotting with antiphosphotyrosine antibody. The results demonstrate that Shc interacted with insulin receptor after infusion of insulin, and, more important, there was insulin receptor kinase activity towards immunopurified Shc. The description of this pathway in animal tissue may have an important role in insulin receptor tyrosine kinase activity toward mitogenic transduction pathways.

Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM) for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group) in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC). These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

Catabolism of Ap4A and Ap5A by rat brain synaptosomes

Adenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) and adenosine 5',5'''-P1,P5-pentaphosphate (Ap5A) are stored in and released from rat brain synaptic terminals. In the present study we investigated the hydrolysis of dinucleotides (Ap4A and Ap5A) in synaptosomes from the cerebral cortex of adult rats. Ap4A and Ap5A, but not Ap3A, were hydrolyzed at pH 7.5 in the presence of 20 mM Tris/HCl, 2.0 mM MgCl2, 10 mM glucose and 225 mM sucrose at 37oC. The disappearance of the substrates measured by FPLC on a mono-Q HR column was both time and protein dependent. Since synaptosome integrity was at least 90% at the end of the assay, hydrolysis probably occurred by the action of an ecto-enzyme. Extracellular actions of adenine dinucleotides at central nervous system terminate due to the existence of ecto-nucleotidases which specifically cleave these dinucleotides. These enzymes in association with an ATP diphosphohydrolase and a 5'-nucleotidase are able to promote the complete hydrolysis of dinucleotides to adenosine in the synaptic cleft.

The carotid body of the spontaneous insulin-dependent diabetic rat

The carotid bodies from adult spontaneous insulin-dependent diabetic rats (strain BB/S) were perfusion-fixed at normal arterial blood pressure with 3% phosphate-buffered glutaraldehyde and compared with the organs from control rats (strain BB/Sc) prepared in the same way. Serial 5-µm sections were cut, stained, and using an interactive image analysis system, were analysed to determine the volumes of the carotid body and its vascular and extravascular compartments. There was no evidence of systemic arterial disease in the carotid stem arteries in either group of animals, and the microvasculature of the organs appeared normal by light microscopy. The volume of the carotid body was unchanged 3 months after the onset of diabetes but was increased at 6 months. The total vascular volume of the organ was unchanged, but the volume of the small vessels (5-12 µm) was increased. In the control group the small vessels comprised 5% of the total volume of the carotid body, or about 44% of the vascular compartment. The percentage of small vessels increased at 3 months in the diabetic group, but had returned to normal at 6 months. The extravascular volume followed the same pattern as the total carotid body volume and so did not change appreciably when expressed as a percentage of the total volume of the organ. The increase in size of the carotid body in diabetic rats is due, therefore, to an augmented extravascular volume. In one diabetic specimen the carotid sinus nerve showed signs of diabetic neuropathy, axonal swelling and intramyelinic oedema. The clinical implications of these results are discussed.