Genetic mapping of the rat mutation creeping and evaluation of its positional candidate gene reelin

We have previously described a rat autosomal recessive mutation, creeping (cre), causing severe ataxia and disarrangement of neuronal cells in the central nervous system. The mutant strain has recently been successfully inbred, named Komeda Zucker creeping (KZC) rat. In the present study, we have performed a genetic analysis of the creeping mutation, and mapped it to rat Chromosome (Chr) 4. Comparative mapping, together with the similarity of the phenotype, suggested that the creeping mutation is homologous to the mouse reeler mutation. In fact, reelin expression was markedly reduced in the homozygous mutant (cre/cre) animals compared with the normal littermates. Thus, the KZC rat should become a useful biological model with a novel mutation in the reelin gene. Received: 25 June 1999 / Accepted: 19 October 1999     

Effects of physical training on IL-1beta, IL-6 and IL-1ra concentrations in various brain areas of the rat

There is increasing evidence that voluntary physical activity and exercise training have beneficial effects on brain function by facilitating neurovegetative, neuroadaptative and neuroprotective processes. Cytokines are chronically expressed at elevated levels within the CNS in many neurological disorders and may contribute to the histopathological, pathophysiological, and cognitive deficits associated with such disorders. In the present study, we examined the influence of seven weeks of physical training on IL-1b, IL-6 and IL-1ra concentrations in hypothalamus, pituitary, hippocampus, cerebellum and frontal cortex in rats. We determined circulating concentrations of cytokines, corticosterone, prolactin and leptin. Two groups of 10 rats were investigated: one group (trained rats) was progressively trained (5 days/week); the other group (sedentary rats) was used as a sedentary group. The training program induced a decrease of (i) IL-1b concentration in the hippocampus (0.7 +/- 0.16 versus 0.99 +/- 0.14 pg/mg protein; p < 0.05), (ii) IL-6 concentration in the cerebellum (10.7 +/- 1.00 in trained rats versus 14.8 +/- 1.34 pg/mg protein in sedentary rats; p < 0.05), (iii) IL-1ra concentration in the pituitary (245 +/- 14.31 versus 328 +/- 17.73 pg/mg protein; p < 0.01). We also found positive correlations between (i) serum prolactin and the concentration of IL-6 in the cerebellum, (ii) serum leptin and the concentration of IL-1ra in the pituitary. There was no effect of physical training on IL-1b, IL-6, and IL-1ra serum levels. These findings suggest that the decrease in particular pro-inflammatory, central cytokines such as IL-1b and IL-6 induced by the training program may play a role in the positive effects of regular physical activity on the central nervous system.     

Expression of Tropomyosin-Encoding Gene in the Kidney Depends on Functioning of Vasopressin Gene

Brattleboro diabetes insipidus mutant rats and normal WAG rats were subjected to water loading or thirst during 3 days. It was found that tropomyosin-encoding gene expression has a tissue-specific pattern in the kidney. Northern blot and western blot analysis had shown that the main expression of Tpm3(3) takes place in the renal medulla, and its intensity differs in normal and mutant rats. The differences between mutants with an ineffective vasopressin synthesizing system and the rats having an intact vasopressin gene were more distinct under long-lasting dehydration. The ratio between renal medullary tropomyosin of Brattleboro and WAG lines of rats was 39.76 +/- 0.90 versus 18.29 +/- 0.86 under water loading, and 46.12 +/- 2.14 versus 13.83 +/- 0.66 in thirst.     

GABAA receptor-related chloride flux of rats receiving continuous intracerebroventricular infusion of pentobarbital

The in vitro receptor-stimulated synaptoneurosomal chloride uptake induced by pentobarbital or muscimol was studied in the brains of rats which had been rendered tolerant to or dependent on pentobarbital. In the chronic study, rats received continuous administration of sodium pentobarbital by intracerebroventricular (i.c.v.) infusion. In rats continuously infused with pentobarbital for 6 days or in rats which had the infusion terminated for 24 h, the basal synaptoneurosomal chloride uptake was not altered in either the cerebral cortex or the cerebellum. On the other hand, pentobarbital (500 µM)-stimulated chloride uptake was significantly decreased in both cerebral cortices and cerebella of rats which received pentobarbital for 6 days as compared with the saline control groups. Twenty-four hours after termination of pentobarbital infusion, this high concentration of pentobarbital-stimulated chloride uptake remained low in both cerebral cortices and cerebella of rats which had been infused with pentobarbital for 6 days. In addition, similar results were also obtained in muscimol (2.5 µM)-stimulated chloride uptake in the cerebral cortices of pentobarbital-infused animals. However, pentobarbital infusion failed to alter muscimol-stimulated chloride uptake in the cerebellum. These results suggest that the GABA_A receptor regulated chloride uptake is downregulated after chronic pentobarbital administration. The results also suggest that down-regulation of the GABA_A receptor chloride channel complex takes place at different recognition sites on the complex. It further substantiates the allosteric effects of GABA and barbiturate recognition sites.     

A novel mutation vf causing abnormal vacuoles in the central nervous system maps on rat chromosome 8.

Body-tremorous rats were found in a colony of WTC-tm rats and a new coisogenic mutant strain void of the tm mutation was established. Histological analysis revealed that these rat mutants had abnormal vacuoles in the red nucleus of the midbrain, the reticular formation in the brain stem, and the white matter of the cerebellum and spinal cord. Electron microscopic observation showed many irregular myelin-bound vacuoles and degenerated oligodendroglia. Genetic analysis indicated that the presence of the abnormal vacuoles in the central nervous system (CNS) is controlled by a recessive gene named "vacuole formation (vf)" on chromosome (Chr) 8, and that this gene is also involved in the appearance of body tremors. Comparative maps suggested that the mouse and human orthologs would be located on Chr 9 (43-48 cM) and Chr 6 (328-370 cR3000), respectively. Since similar mutations have not been mapped yet around these regions, the authors believe this novel rat mutation will allow the discovery of a new function of these particular genes that is involved in the development and maintenance of the CNS.     

Acetylcholinesterase level and molecular isoforms are altered in brain of Reelin Orleans mutant mice

In this study we examined changes in acetylcholinesterase (AChE) pattern in the brain of adult Reelin Orleans (RelnOrl) homozygous mutant mice. The AChE histochemistry firstly revealed an abnormal distribution of AChE-positive cells in several areas of the reeler brain, including cortices; the strongest labelling was observed in cerebellum and hippocampus when compared with controls. Biochemical determinations demonstrated an increase of 80-90% in AChE specific activity from cerebellar and hippocampal extracts. We also report that the AChE tetrameric form (G4) was selectively increased in the RelnOrl brain. The relationship between AChE and Reelin and suggested morphogenetic functions are also discussed.     

Two proteins associated with cerebellar hypoplasia in jaundiced Gunn rats

Two cerebellar proteins with apparent molecular weights of 250,000 (GR-250) and 50,000 (GR-50) are closely associated with cerebellar hypoplasia in jaundiced homozygous Gunn rats. These proteins, found in Gunn rat cerebellum (4–60 days of age) and cerebrum as well as staggerer mouse cerebellum, were studied with electrophoretic techniques. After 8 days of life, GR-250 decreased and GR-50 increased in the homozygous Gunn rat cerebellum. The pI's of GR-250 and GT-50 were 4.7–5.8 and 4.6–4.9, respectively, and the former protein was shown to bind to Concanavalin A. A comparative study between cerebella of Gunn rats and staggerer mice revealed that GR-250 and P₄₀₀, a protein generally thought to be characteristic of the Purkije cells, were identical. Evidence was also obtained showing that GR-250 was present in the Gunn rat cerebrum. GR-50 was not detectable in the staggerer mouse cerebellum but instead, a protein (MW 47,000) was found to be increased in the mutant mouse cerebellum.     

A new neurological mutant rat with symmetrical calcification of Purkinje cells in cerebellum.

A new neurological mutant has been found in the inbred F344 strain of rats. The mutation is inherited as an autosomal recessive trait and is manifest clinically by a hesitant and wobbling gait with asynergic limbs and slight tremor. These symptoms begin at 16-18 days of age and remain essentially constant thereafter. Histologic examination revealed severe degeneration of the Purkinje cells and symmetrical calcification in these and in their dendritic branches in the cerebellar cortex. Such calcified Purkinje cells were intensely stained with the periodic acid-Schiff (PAS) method. PAS-positive substances in the Purkinje cells and extending diffusely over the lesioned sites in the molecular layer were also evident before calcification took place. We have named this neurological mutant the Cerebellar Calcification (CC) rat with the gene symbol cc. This offers a new animal model for the study of the Purkinje cell degeneration and intracranial calcification.     

Mapping of Histamine H1 Receptors in the Human Brain Using [11C]Pyrilamine and Positron Emission Tomography

We have studied the characteristics of carbon-11 labeled pyrilamine as a radioligand for investigating histamine H1 receptors in human brain with positron emission tomography (PET). [11C]Pyrilamine is distributed evenly in proportion to cerebral blood flow at initial PET images. Later (after 45-60 min), 11C radioactivity was observed at high concentrations in the frontal and temporal cortex, hippocampus, and thalamus, and at low concentrations in the cerebellum and pons. The regional distribution of the carbon-11 labeled compound in the brain corresponded well with that of the histamine H1 receptors determined in vitro in autopsied materials. In six controls, the frontal and temporal cortices/cerebellum ratio increased during the first 60 min to reach a value of 1.22 +/- 0.071. Intravenous administration of d-chlorpheniramine (5 mg) completely abolished the specific binding in vivo in the frontal cortex and temporal cortex (cortex/cerebellum ratio, 0.955 +/- 0.015). The availability of this method for measuring histamine H1 receptors in vivo in humans will facilitate studies on neurological and psychiatric disorders in which histamine H1 receptors are thought to be abnormal.     

Adrenomedullin and its receptor complexes in remnant kidneys of rats with renal mass ablation: decreased expression of calcitonin receptor-like receptor and receptor-activity modifying protein-3.

Adrenomedullin (AM) has vasodilator and diuretic actions, similarly to natriuretic peptides. AM receptor complexes are composed of calcitonin receptor-like receptor (CRLR) and receptor-activity modifying protein-2 (RAMP2), or CRLR and RAMP3. We aimed to know whether gene expression of AM and AM receptor complexes are regulated in kidneys under pathophysiological conditions. Expression of AM, RAMP2, RAMP3 and CRLR mRNA was studied in the remnant kidney of rats with renal mass ablation using competitive quantitative RT-PCR techniques. Partial cloning was performed to determine the rat RAMP3 nucleotide sequence. In normal rat kidneys, expression levels of RAMP2, RAMP3, CRLR and AM mRNAs were 26.5 +/- 1.9 mmol/mole of GAPDH, 7.7 +/- 0.9 mmol/mole of GAPDH, 3.6 +/- 0.2 mmol/mole of GAPDH and 0.57 +/- 0.03 mmol/mole of GAPDH (mean +/- SE, n = 6), respectively. RAMP3 mRNA levels decreased significantly to about 50% and about 70% of control (sham-operated rats) 4 days and 14 days after 5/6 nephrectomy, respectively. CRLR mRNA levels also decreased significantly to about 30% and about 43% of control. Sodium intake restriction had no significant effects on the RAMP3 and CRLR gene expression. On the other hand, RAMP2 mRNA expression in the kidney was suppressed by sodium intake restriction regardless of nephrectomy, while RAMP2 levels in the remnant kidney were not significantly changed by 5/6 nephrectomy. Neither 5/6 nephrectomy or sodium intake restriction had any significant effects on the AM gene expression in the kidney. The present study showed that expression of mRNAs encoding AM, RAMP2, RAMP3 and CRLR were differentially regulated in remnant kidneys of rats with renal mass ablation.     

Fixed feeding potentiates interdigestive gastric motor activity in rats: importance of eating habits for maintaining interdigestive MMC

Endogenous ghrelin regulates the occurrence of interdigestive gastric phase III-like contractions in rats. However, the fasted motor pattern is not as regular and potent in humans and dogs. We hypothesize that eating habits play an important role in maintaining a regular interdigestive gastric contractions. We studied the effect of fixed-feeding regimen on interdigestive gastric contractions and plasma acyl ghrelin levels. The fixed-fed rats were trained to the assigned meal feeding regimen, once daily at 12:00 PM to 4:00 PM for 14 days. Free-fed rats were maintained with free access to food. As ghrelin regulates gastric emptying as well, solid gastric emptying was also studied in fixed-fed rats and free-fed rats. In free-fed rats, two of six rats did not show interdigestive gastric phase III-like contractions. In contrast, phase III-like contractions were observed in all rats 14 days after starting the fixed-feeding regimen. The maximal amplitude of phase III-like contractions significantly increased from 8.4 +/- 0.6 to 16.3 +/- 1.8 g (n = 6, P < 0.05) 14 days after the start of the fixed feeding. Fasted and postprandial plasma ghrelin levels were significantly increased after 14 days of fixed feeding. Solid gastric emptying was significantly accelerated in fixed-fed rats (72.1 +/- 4.2%) compared with that of free-fed rats (58.7 +/- 2.7%, n = 6, P < 0.05). Our present findings suggest that fixed feeding increases plasma ghrelin levels, potent interdigestive contractions, and acceleration of gastric emptying.     

Adrenomedullin improves cardiac function and prevents renal damage in streptozotocin-induced diabetic rats

Adrenomedullin (AM) is a potent vasodilating peptide and is involved in cardiovascular and renal disease. In the present study, we investigated the role of AM in cardiac and renal function in streptozotocin (STZ)-induced diabetic rats. A single tail-vein injection of adenoviral vectors harboring the human AM gene (Ad.CMV-AM) was administered to the rats 1-wk post-STZ treatment (65 mg/kg iv). Immunoreactive human AM was detected in the plasma and urine of STZ-diabetic rats treated with Ad.CMV-AM. Morphological and chemical examination showed that AM gene delivery significantly reduced glycogen accumulation within the hearts of STZ-diabetic rats. AM gene delivery improved cardiac function compared with STZ-diabetic rats injected with control virus, as observed by decreased left ventricular end-diastolic pressure, increased cardiac output, cardiac index, and heart rate. AM gene transfer significantly increased left ventricular long axis (11.69 +/- 0.46 vs. 10.31 +/- 0.70 mm, n = 10, P < 0.05) and rate of pressure rise and fall (+6,090.1 +/- 597.3 vs. +4,648.5 +/- 807.1 mmHg/s), (-4,902.6 +/- 644.2 vs. -3,915.5 +/- 805.8 mmHg/s, n = 11, P < 0.05). AM also significantly attenuated renal glycogen accumulation and tubular damage in STZ-diabetic rats as well as increased urinary cAMP and cGMP levels, along with increased cardiac cAMP and Akt phosphorylation. We also observed that delivery of the AM gene caused an increase in body weight along with phospho-Akt and membrane-bound GLUT4 levels in skeletal muscle. These results suggest that AM plays a protective role in hyperglycemia-induced glycogen accumulation and cardiac and renal dysfunction via Akt signal transduction pathways.     

nNOS gene transfer to RVLM improves baroreflex function in rats with chronic heart failure

We hypothesized that gene transfer of neuronal nitric oxide synthase (nNOS) into the rostral ventrolateral medulla (RVLM) improves baroreflex function in rats with chronic heart failure (CHF). Six to eight weeks after coronary artery ligation, rats showed hemodynamic signs of CHF. A recombinant adenovirus, either Ad.nNOS or Ad.beta-Gal, was transfected into the RVLM. nNOS expression in the RVLM was confirmed by Western blot analysis, NADPH-diaphorase, and immunohistochemical staining. We studied baroreflex control of the heart rate (HR) and renal sympathetic nerve activity (RSNA) in the anesthetized state 3 days after gene transfer by intravenous injections of phenylephrine and nitroprusside. Baroreflex sensitivity was depressed for HR and RSNA regulation in CHF rats (2.0 +/- 0.3 vs. 0.8 +/- 0.2 beats.min-1.mmHg-1, P < 0.01 and 3.8 +/- 0.3 vs. 1.2 +/- 0.1% max/mmHg, P < 0.01, respectively). Ad.nNOS transfer into RVLM significantly increased the HR and RSNA ranges (152 +/- 19 vs. 94 +/- 12 beats/min, P < 0.05 and 130 +/- 16 vs. 106 +/- 5% max/mmHg, P < 0.05) compared with the Ad.beta-Gal in CHF rats. Ad.nNOS also improved the baroreflex gain for the control of HR and RSNA (1.8 +/- 0.2 vs. 0.8 +/- 0.2 beats.min-1.mmHg-1, P < 0.01 and 2.6 +/- 0.2 vs. 1.2 +/- 0.1% max/mmHg, P < 0.01). In sham-operated rats, we found that Ad.nNOS transfer enhanced the HR range compared with Ad.beta-Gal gene transfer (188 +/- 15 vs. 127 +/- 14 beats/min, P < 0.05) but did not alter any other parameter. This study represents the first demonstration of altered baroreflex function following increases in central nNOS in the CHF state. We conclude that delivery of Ad.nNOS into the RVLM improves baroreflex function in rats with CHF.     

A rice mutant displaying a heterochronically elongated internode carries a 100 kb deletion

We have isolated a recessive rice mutant,designated as indeterminate growth(ing),which displays creeping and apparent heterochronic phenotypes in the vegetative period with lanky and winding culms.Rough mapping and subsequent molecular characterization revealed that the ing mutant carries a large deletion,which corresponds to a 103 kb region in the Nipponbare genome,containing nine annotated genes on chromosome 3.Of these annotated genes,the SLRI gene encoding a DELLA protein is the only one that is well characterized in its function,and its null mutation,which is caused by a single base deletion in the middle of the intronless SLR1 gene,confers a slender phenotype that bears close resemblance to the ing mutant phenotype.The primary cause of the ing mutant phenotype is the deletion of the SLR1 gene,and the ing mutant appears to be the first characterized mutant having the entire SLRI sequence deleted.Our results also suggest that the deleted region of 103 kb does not contain an indispensable gene,whose dysfunction must result in a lethal phenotype.     

Protective effect of Bacopa monniera on methyl mercury-induced oxidative stress in cerebellum of rats

Methyl mercury (MeHg) is a ubiquitous environmental pollutant leading to neurological and developmental deficits in animals and human beings. Bacopa monniera (BM) is a perennial herb and is used as a nerve tonic in Ayurveda, a traditional medicine system in India. The objective of the present study was to investigate whether Bacopa monniera extract (BME) could potentially inhibit MeHg-induced toxicity in the cerebellum of rat brain. Male Wistar rats were administered with MeHg orally at a dose of 5 mg/kg b.w. for 21 days. Experimental rats were given MeHg and also administered with BME (40 mg/kg, orally) for 21 days. After the treatment period, we observed that MeHg exposure significantly inhibited the activities of superoxide dismutase, catalase, glutathione peroxidase, and increased the glutathione reductase activity in cerebellum. It was also found that the level of thiobarbituric acid-reactive substances was increased with the concomitant decrease in the glutathione level in MeHg-induced rats. These alterations were prevented by the administration of BME. Behavioral interference in the MeHg-exposed animals was evident through a marked deficit in the motor performance in the rotarod task, which was completely recovered to control the levels by BME administration. The total mercury content in the cerebellum of MeHg-induced rats was also increased which was measured by atomic absorption spectrometry. The levels of NO(2) (-) and NO(3) (-) in the serum were found to be significantly increased in the MeHg-induced rats, whereas treatment with BME significantly decreased their levels in serum to near normal when compared to MeHg-induced rats. These findings strongly implicate that BM has potential to protect brain from oxidative damage resulting from MeHg-induced neurotoxicity in rat.     

Altered cerebellar function in mice lacking CaV2.3 Ca2+ channel

Voltage-dependent Ca(2+) channels play important roles in cerebellar functions including motor coordination and learning. Since abundant expression of Ca(V)2.3 Ca(2+) channel gene in the cerebellum was detected, we searched for possible deficits in the cerebellar functions in the Ca(V)2.3 mutant mice. Behavioral analysis detected in delayed motor learning in rotarod tests in mice heterozygous and homozygous for the Ca(V)2.3 gene disruption (Ca(V)2.3+/- and Ca(V)2.3-/-, respectively). Electrophysiological analysis of mutant mice revealed perplexing results: deficit in long-term depression (LTD) at the parallel fiber Purkinje cell synapse in Ca(V)2.3+/- mice but apparently normal LTD in Ca(V)2.3-/- mice. On the other hand, the number of spikes evoked by current injection in Purkinje cells under the current-clamp mode decreased in Ca(V)2.3 mutant mice in a gene dosage-dependent manner, suggesting that Ca(V)2.3 channel contributed to spike generation in Purkinje cells. Thus, Ca(V)2.3 channel seems to play some roles in cerebellar functions.     

Adiponectin mRNA levels in parametrial adipose tissue and serum adiponectin levels are reduced in mice during late pregnancy.

Adiponectin, a fat-derived factor, is downregulated in insulin resistance and obesity; insulin resistance has been demonstrated during late pregnancy in both humans and in rodents. The present study examines the physiological change of adiponectin gene expression as well as the circulating levels of adiponectin during pregnancy. We examined the relative quantity of adiponectin mRNA produced in the adipose tissues of pregnant compared to virgin mice. We also measured serum adiponectin levels and parametrial adipocyte size in mice throughout pregnancy. Adiponectin mRNA was significantly reduced by 74 +/- 8 % and 63 +/- 4 % at days 15 and 18 of pregnancy, respectively, compared to virgin mice. Serum adiponectin concentration decreased on days 15 (30.7 +/- 8.5 microg/ml) and 18 (27.9 +/- 8.7 microg/ml) of pregnancy, and the values were significantly lower than that of virgin mice (56.8 +/- 6.6 microg/ml). Parametrial adipocytes from mice on days 15 and 18 of pregnancy were significantly larger than in virgin mice or during early pregnancy. Fat-cell size was closely correlated to degradation of adiponectin gene expression and serum adiponectin levels. These results suggest that changes of adiponectin expression affect metabolic status in pregnant mice.