Spaced training facilitates long-term retention of place navigation in adult but not in adolescent rats

Young and adult Long Evans rats were tested in the water maze according to two different procedures: half of the subjects were given one session of four trials a day for 6 days, whereas the other subjects had the same amount of training massed in 1 day. For both conditions, a 14-day retention interval was then introduced to test long-term memory. This was followed by a four-trial reversal session. All groups showed a significant learning curve, but escape latencies were shorter for the adult than for the young rats, without differential effect of the training procedure. A first probe trial (PT1) confirmed similar accurate short-term retention in all the groups. But unimpaired long-term memory was only seen in the adult rats trained with the spaced procedure. The young rats trained over 1 day also showed some retention of the platform location after 14 days, but not the other two groups. Reversal acquisition of the new platform location was rapid in the four groups. These results indicate that although accurate short-term spatial memory in the water maze is seen after a 1-day massed training in both age groups, unimpaired long-term retention is only observed in adult rats trained with 24-h inter-session intervals.

The PPARbeta/delta agonist GW0742 relaxes pulmonary vessels and limits right heart hypertrophy in rats with hypoxia-induced pulmonary hypertension.

BACKGROUND: Pulmonary vascular diseases are increasingly recognised as important clinical conditions. Pulmonary hypertension associated with a range of aetiologies is difficult to treat and associated with progressive morbidity and mortality. Current therapies for pulmonary hypertension include phosphodiesterase type 5 inhibitors, endothelin receptor antagonists, or prostacyclin mimetics. However, none of these provide a cure and the clinical benefits of these drugs individually decline over time. There is, therefore, an urgent need to identify new treatment strategies for pulmonary hypertension. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the PPARbeta/delta agonist GW0742 induces vasorelaxation in systemic and pulmonary vessels. Using tissue from genetically modified mice, we show that the dilator effects of GW0742 are independent of the target receptor PPARbeta/delta or cell surface prostacyclin (IP) receptors. In aortic tissue, vascular relaxant effects of GW0742 were not associated with increases in cGMP, cAMP or hyperpolarisation, but were attributed to inhibition of RhoA activity. In a rat model of hypoxia-induced pulmonary hypertension, daily oral dosing of animals with GW0742 (30 mg/kg) for 3 weeks significantly reduced the associated right heart hypertrophy and right ventricular systolic pressure. GW0742 had no effect on vascular remodelling induced by hypoxia in this model. CONCLUSIONS/SIGNIFICANCE: These observations are the first to show a therapeutic benefit of 'PPARbeta/delta' agonists in experimental pulmonary arterial hypertension and provide pre-clinical evidence to favour clinical trials in man.

Intrahippocampal cholinergic grafts in aged rats compensate impairments in a radial maze and in a place learning task

Age-related cognitive impairments were studied in rats kept in semi-enriched conditions during their whole life, and tested during ontogeny and adult life in various classical spatial tasks. In addition, the effect of intrahippocampal grafts of fetal septal-diagonal band tissue, rich in cholinergic neurons, was studied in some of these subjects. The rats received bilateral cell suspensions when aged 23-24 months. Starting 4 weeks after grafting, they were trained during 5 weeks in an 8-arm maze made of connected plexiglass tunnels. No age-related impairment was detected during the first eight trials, when the maze shape was that of a classical radial maze in which the rats had already been trained when young. The older rats were impaired when the task was made more difficult by rendering two arms parallel to each other. They developed an important neglect of one of the parallel tunnels resulting in a high amount of errors before completion of the task. In addition, the old rats developed a systematic response pattern of visits to adjacent arms in a sequence, which was not observed in the younger subjects. None of these behaviours were observed in the old rats with a septal transplant. Sixteen weeks after grafting, another experiment was conducted in a homing hole board task. Rats were allowed to escape from a large circular arena through one hole out of many, and to reach home via a flexible tube under the table. The escape hole was at a fixed position according to distant room cues, and olfactory cues were made irrelevant by rotating the table between the trials. An additional cue was placed on the escape position. No age-related difference in escape was observed during training. During a probe trial with no hole connected and no proximal cue present, the old untreated rats were less clearly focussed on the training sector than were either the younger or the grafted old subjects. Taken together, these experiments indicate that enriched housing conditions and spatial training during adult life do not protect against all age-related deterioration in spatial ability. However, it might be that the considerable improvement observed in the grafted subjects results from an interaction between the graft treatment and the housing conditions.

Subconjunctival Injection of XG-102, a c-Jun N-Terminal Kinase Inhibitor Peptide, in the Treatment of Endotoxin-Induced Uveitis in Rats.

Abstract Purpose: XG-102, a TAT-coupled dextrogyre peptide inhibiting the c-Jun N-terminal kinase, was shown efficient in the treatment of experimental uveitis. Preclinical studies are now performed to determine optimal XG-102 dose and route of administration in endotoxin-induced uveitis (EIU) in rats with the purpose of clinical study design. METHODS: EIU was induced in Lewis rats by lipopolysaccharides (LPS) injection. XG-102 was administered at the time of LPS challenge by intravenous (IV; 3.2, 35 or 355 μg/injection), intravitreal (IVT; 0.08, 0.2 or 2.2 μg/eye), or subconjunctival (SCJ; 0.2, 1.8 or 22 μg/eye) routes. Controls received either the vehicle (saline) or dexamethasone phosphate injections. Efficacy was assessed by clinical scoring, infiltrating cells count, and expression of inflammatory mediators [inducible nitric oxide synthase (iNOS), cytokine-induced neutrophil chemoattractant-1 (CINC-1)]. The effect of XG-102 on phosphorylation of c-Jun was evaluated by Western blot. RESULTS: XG-102 demonstrated a dose-dependent anti-inflammatory effect in EIU after IV and SCJ administrations. Respective doses of 35 and 1.8 μg were efficient as compared with the vehicle-injected controls, but only the highest doses, respectively 355 and 22 μg, were as efficient as dexamethasone phosphate. After IVT injections, the anti-inflammatory effect of XG-102 was clinically evaluated similar to the corticoid's effect with all the tested doses. Regardless of the administration route, the lowest efficient doses of XG-102 significantly decreased the ration of phospho c-Jun/total c-Jun, reduced cells infiltration in the treated eyes, and significantly downregulated iNOS and CINC-1 expression in the retina. CONCLUSION: These results confirm that XG-102 peptide has potential for treating intraocular inflammation. SCJ injection appears as a good compromise to provide a therapeutic effect while limiting side effects.

Effect of ganglion blockade with pentolinium on circulating neuropeptide Y levels in conscious rats.

The vasoconstrictor peptide, neuropeptide Y (NPY), is present in perivascular noradrenergic neurons of all mammals studied and may be important in the regulation of blood pressure. High plasma levels of NPY have been measured in the rat. To investigate partially the source and factors controlling the release of the circulating peptide, the effect of pentolinium tartrate administration has been studied in conscious rats. Pentolinium given as a bolus (5 mg/kg) followed by an infusion of a further 5 mg/kg/30 min produced a highly significant reduction in blood pressure of more than 40 mm Hg, when compared to either basal values or control animals treated with saline. Pentolinium treatment resulted in significantly lower plasma neuropeptide Y levels (31.0 +/- 6.7 fmol/ml) compared with those of control animals (78.6 +/- 8.2 fmol/ml). Circulating catecholamines were also significantly reduced in those animals receiving pentolinium. These results are compatible with circulating NPY arising from the sympathetic nervous system, with release being controlled by the mechanisms already established for catecholamines.

Calcium-sensing receptors modulate renin release in vivo and in vitro in the rat.

OBJECTIVES: Calcium-sensing receptors (CaSRs) have been localized in the juxtaglomerular apparatus where they may contribute to the regulation of renin release. In the present study, we investigated the in-vitro and in-vivo effects of the calcimimetic R-568 on renin release. METHODS: In vitro, the effect of calcimimetics on renin release was assessed by incubating freshly isolated rat juxtaglomerular cells with or without R-568 (1 and 10 mumol/l) in serum-free medium in the presence or absence of forskolin or CaCl2. In vivo, we measured the impact of R-568 (20 ng/min intravenously) on the acute changes in plasma renin activity (PRA) induced by either a 90 min infusion of the angiotensin-converting enzyme inhibitor captopril, or the beta-receptor agonist isoproterenol, or of a vehicle in or after a furosemide challenge in conscious Wistar rats. RESULTS: In vitro, R-568 dose-dependently blunted renin release, but also reduced the increase in renin due to forskolin (P < 0.01). Both isoproterenol and enalapril increased in vivo PRA to 3.1 +/- 0.3 and 3.7 +/- 0.5 ng Ang I/ml per h, respectively (P < 0.01), compared with vehicle (1.5 +/- 0.2 ng Ang I/ml per h). R-568 significantly reduced PRA to 2.1 +/- 0.1 ng/ml per h in isoproterenol-treated rats and to 1.6 +/- 0.2 ng/ml per h in enalapril-treated rats (P < 0.05). In low-salt treated animals, acute infusion of furosemide increased PRA from 8.7 +/- 3.2 to 18.6 +/- 2.3, whereas R-568 partially blunted this rise to 11.2 +/- 1.5 (P = 0.02). In vivo, R-568 significantly lowered serum calcium and PTH1-84, but the drug-induced changes in PRA were independent of the changes in calcium and parathyroid hormone. CONCLUSION: After the recent discovery of CaSRs in juxtaglomerular cells of mice, our results confirm the presence of such receptors in rats and demonstrate that these receptors modulate renin release both in vitro and in vivo. This suggests that CaSRs play a role as a regulatory pathway of renin release.

Endotoxin-induced hypotension in rats is not mediated by prekallikrein activation.

To test whether endotoxin decreases blood pressure acutely in rats by activating the plasma kinin-forming system, plasma kallikrein activity was determined in different experimental settings of endotoxemia. Conscious normotensive rats were infused for 45 min with endotoxin (LPS E. coli 0111:B4) at a dose (0.01 mg/min) which had no effect on blood pressure. Additional rats were infused with the vehicle of endotoxin. Plasma prekallikrein activity was measured at the end of the 45 min infusions. In other rats, a bolus intravenous injection of endotoxin (2 mg) was administered following the 45 min infusion of endotoxin or its vehicle. In these two latter groups of rats, plasma prekallikrein activity was determined 15 min after administration of the bolus dose of endotoxin. In rats pretreated with the endotoxin infusion, the bolus dose of endotoxin had no significant effect on blood pressure, whereas rats infused with the vehicle became and remained hypotensive up to the end of the experiment. There was however no significant difference in plasma prekallikrein activity within the different groups of rats. In another group of rats, dextran sulfate (0.25 mg i.v.), which activates factor XII and thereby the conversion of prekallikrein to kallikrein, induced a short-lasting fall in blood pressure. 15 min after administration of dextran sulfate, plasma prekallikrein activity was almost completely suppressed. These results obtained in unanesthetized rats strongly suggest that the blood pressure fall induced by E. coli endotoxin is not due to activation of prekallikrein and consequently of the kinin-forming system.

Endothelin-1-induced spreading depression in rats is associated with a microarea of selective neuronal necrosis.

Two different theories of migraine aura exist: In the vascular theory of Wolff, intracerebral vasoconstriction causes migraine aura via energy deficiency, whereas in the neuronal theory of Leão and Morison, spreading depression (SD) initiates the aura. Recently, it has been shown that the cerebrovascular constrictor endothelin-1 (ET-1) elicits SD when applied to the cortical surface, a finding that could provide a bridge between the vascular and the neuronal theories of migraine aura. Several arguments support the notion that ET-1-induced SD results from local vasoconstriction, but definite proof is missing. If ET-1 induces SD via vasoconstriction/ischemia, then neuronal damage is likely to occur, contrasting with the fact that SD in the otherwise normal cortex is not associated with any lesion. To test this hypothesis, we have performed a comprehensive histologic study of the effects of ET-1 when applied topically to the cerebral cortex of halothane-anesthetized rats. Our assessment included histologic stainings and immunohistochemistry for glial fibrillary acidic protein, heat shock protein 70, and transferase dUTP nick-end labeling assay. During ET-1 application, we recorded (i) subarachnoid direct current (DC) electroencephalogram, (ii) local cerebral blood flow by laser-Doppler flowmetry, and (iii) changes of oxyhemoglobin and deoxyhemoglobin by spectroscopy. At an ET-1 concentration of 1 muM, at which only 6 of 12 animals generated SD, a microarea with selective neuronal death was found only in those animals demonstrating SD. In another five selected animals, which had not shown SD in response to ET-1, SD was triggered at a second cranial window by KCl and propagated from there to the window exposed to ET-1. This treatment also resulted in a microarea of neuronal damage. In contrast, SD invading from outside did not induce neuronal damage in the absence of ET-1 (n = 4) or in the presence of ET-1 if ET-1 was coapplied with BQ-123, an ET(A) receptor antagonist (n = 4). In conclusion, SD in presence of ET-1 induced a microarea of selective neuronal necrosis no matter where the SD originated. This effect of ET-1 appears to be mediated by the ET(A) receptor.

Evidence for a compulsive-like behavior in rats exposed to alternate access to highly preferred palatable food.

Converging evidence suggests that recurrent excessive calorie restriction causes binge eating by promoting behavioral disinhibition and overeating. This interpretation suggests that cognitive adaptations may surpass physiological regulations of metabolic needs after recurrent cycles of dieting and binging. Intermittent access to palatable food has long been studied in rats, but the consequences of such diet cycling procedures on the cognitive control of food seeking remain unclear. Female Wistar rats were divided in two groups matched for food intake and body weight. One group received standard chow pellets 7 days/week, whereas the second group was given chow pellets for 5 days and palatable food for 2 days over seven consecutive weeks. Rats were also trained for operant conditioning. Intermittent access to palatable food elicited binging behavior and reduced intake of normal food. Rats with intermittent access to palatable food failed to exhibit anxiety-like behaviors in the elevated plus maze, but displayed reduced locomotor activity in the open field and developed a blunted corticosterone response following an acute stress across the diet procedure. Trained under a progressive ratio schedule, both groups exhibited the same motivation for sweetened food pellets. However, in contrast to controls, rats with a history of dieting and binging exhibited a persistent compulsive-like behavior when access to preferred pellets was paired with mild electrical foot shock punishments. These results highlight the intricate development of anxiety-like disorders and cognitive deficits leading to a loss of control over preferred food intake after repetitive cycles of intermittent access to palatable food.

Bone marrow dosimetry in rats using direct tissue counting after injection of radio-iodinated intact monoclonal antibodies or F(ab')2 fragments.

Normal rats were injected intravenously with 131I- and 125I-labeled intact murine and chimeric mouse-human monoclonal antibodies directed against carcinoembryonic antigen or with the corresponding F(ab')2 fragments. At different times after injection, individual animals were killed and radioactivity of blood and major organs, including bones and bone marrow, was determined. Ratios comparing radioactivity concentration in different tissues with that of bone marrow were calculated and found to remain stable during several effective half-lives of the antibodies. Mean bone marrow radioactivity was 35% (range, 29%-40%) of that of blood and 126% (range, 108%-147%) of that of liver after injection of intact Mabs or F(ab')2 fragments. In nude rats bearing human colon carcinoma xenografts producing carcinoembryonic antigen, relative bone marrow radioactivity was slightly lower than that in normal rats.

Salt-sensitive hypertension and cardiac hypertrophy in mice deficient in the ubiquitin ligase Nedd4-2.

Nedd4-2 has been proposed to play a critical role in regulating epithelial Na+ channel (ENaC) activity. Biochemical and overexpression experiments suggest that Nedd4-2 binds to the PY motifs of ENaC subunits via its WW domains, ubiquitinates them, and decreases their expression on the apical membrane. Phosphorylation of Nedd4-2 (for example by Sgk1) may regulate its binding to ENaC, and thus ENaC ubiquitination. These results suggest that the interaction between Nedd4-2 and ENaC may play a crucial role in Na+ homeostasis and blood pressure (BP) regulation. To test these predictions in vivo, we generated Nedd4-2 null mice. The knockout mice had higher BP on a normal diet and a further increase in BP when on a high-salt diet. The hypertension was probably mediated by ENaC overactivity because 1) Nedd4-2 null mice had higher expression levels of all three ENaC subunits in kidney, but not of other Na+ transporters; 2) the downregulation of ENaC function in colon was impaired; and 3) NaCl-sensitive hypertension was substantially reduced in the presence of amiloride, a specific inhibitor of ENaC. Nedd4-2 null mice on a chronic high-salt diet showed cardiac hypertrophy and markedly depressed cardiac function. Overall, our results demonstrate that in vivo Nedd4-2 is a critical regulator of ENaC activity and BP. The absence of this gene is sufficient to produce salt-sensitive hypertension. This model provides an opportunity to further investigate mechanisms and consequences of this common disorder.

Absence of snapshot memory of the target view interferes with place navigation learning by rats in the water maze

Contribution of visual and nonvisual mechanisms to spatial behavior of rats in the Morris water maze was studied with a computerized infrared tracking system, which switched off the room lights when the subject entered the inner circular area of the pool with an escape platform. Naive rats trained under light-dark conditions (L-D) found the escape platform more slowly than rats trained in permanent light (L). After group members were swapped, the L-pretrained rats found under L-D conditions the same target faster and eventually approached latencies attained during L navigation. Performance of L-D-trained rats deteriorated in permanent darkness (D) but improved with continued D training. Thus L-D navigation improves gradually by procedural learning (extrapolation of the start-target azimuth into the zero-visibility zone) but remains impaired by lack of immediate visual feedback rather than by absence of the snapshot memory of the target view.

Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis.

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.

Markedly reduced blood pressure responsiveness in endotoxemic rats; reversal by neuropeptide Y.

This study in conscious normotensive rats was performed to assess the effect of the vasoconstrictor peptide, neuropeptide Y (NPY), on blood pressure responsiveness to exogenous norepinephrine in endotoxaemia. NPY and endotoxin were infused at doses which had no effect on blood pressure, whether given alone or in combination. Endotoxin markedly reduced the pressor responses to bolus injections of norepinephrine. However, blood pressure responsiveness could be enhanced by infusing NPY simultaneously with the endotoxin. It is suggested that low dose NPY infusions may be clinically useful in reversing the reduced vascular responsiveness to pressor amines in shock.

Feasibility of in vivo 15N MRS detection of hyperpolarized 15N labeled choline in rats.

The increase of total choline in tumors has become an important biomarker in cancer diagnosis. Choline and choline metabolites can be measured in vivo and in vitro using multinuclear MRS. Recent in vivo(13)C MRS studies using labeled substrates enhanced via dynamic nuclear polarization demonstrated the tremendous potential of hyperpolarization for real-time metabolic studies. The present study demonstrates the feasibility of detecting hyperpolarized (15)N labeled choline in vivo in a rat head at 9.4 T. We furthermore report the in vitro (172 +/- 16 s) and in vivo (126 +/- 15 s) longitudinal relaxation times. We conclude that with appropriate infusion protocols it is feasible to detect hyperpolarized (15)N labeled choline in live animals.