Effect of GABAmimetics on electrocorticographic spike discharges induced by guanidinoethanesulfonic acid (amidino-taurine) in the rat

The effect of guanidinoethanesulfonic acid (GES) on rat electrocorticograms (ECoG) and the effects of -aminobutyric acid (GABA) and GABA-agonists on the ECoG changes induced by GES were studied. Sporadic spike discharges began 2–5 min after 1 mol GES/10 l on filter paper was applied to the pia mater of the left sensorimotor cortex; spike discharges extended to the opposite cerebral hemisphere 60 min after the onset of the ipsilateral spike discharges. The spike discharges with a frequency of 5–10 spikes/min lasted until the end of the 4 hour recording. The induced spike discharges were suppressed when the original GES soaked filter paper was replaced by one containing GES (1 mol) supplement combined with taurine (1 mol/10 l). GABA (1 mol) and its receptor agonist, muscimol (10nmol) and (3R)-(–)-4-amino-3-hydroxybutyric acid (1 mol) also suppressed the GES-induced spike discharges when applied topically. Diazepam (DZP) (10 mg/kg) suppressed the GES-induced spike discharges 10 min after i.p. injection, but phenobarbital (20 mg/kg) increased the frequency and voltage of spike discharges 100 min following subcutaneous administration. Intraperitoneal injection of either valproate (200 mg/kg) or phenytoin (25 mg/kg), after the completion of the spike discharges, showed no effect. These findings suggest that neurotransmission or neuromodulatory effects of taurine participate in GES-induced seizure activity, and that GABA_A and DZP receptors may play a role in the mechanism that suppresses GES-induced seizures.     

The modeling of rat EEG signals in absence epilepsy in the analysis of brain connectivity

Simple models that describe some features of the electrical brain activity in rats with genetic absence epilepsy recorded before and after an epileptic seizure have been proposed in this study. These models can help to analyze the efficiency of the Granger causality analysis of the directional connectivity determination. The comparison of the results of the experimental and modeled signal analysis, on one hand, reveals a number of artifacts of this method, and on the other hand, proves its effectiveness in the research on absence epilepsy mechanisms.     

Cholinergic mechanisms in the pathogenesis of genetically-caused absence epilepsy

Frontoparietal cortex and the thalamocortical circuit comprising reticular thalamic nucleus (RTN) and relay nuclei of the ventrolateral thalamus (VLT) are critical structures in the generation of spike-wave discharges (SWD) during absence seizures. The activity of these nuclei is under the control of the ascending cholinergic projections of nucleus basalis of Meynert. The aim of our study is to make an attempt to change the pattern of SWD in WAG/Rij rats by injecting of cholinotoxine AF64A to the area of RTN. Spontaneous SWD were registered in cortex of WAG/Rij rats with genetically determined absences. The spectral content of SWD was analyzed by means of the Fast Fourier Transformation (FFT) procedure. Unilateral injections of AF64A (1 nmol) to RTN led the decrease in duration and number of SWD comparing to the basal EEG recordings 2 days after the lesion. The FFT analysis showed the disappearance of 17-18 Hz spike on the side of the lesion compared with the intact side. The immunohistochemical study for acetylcholinetransferase (ChaT)-containing neurons showed the loss of ChaT-positive cells in the nucleus basalis area on the side of the lesion. The removal of cholinergic afferentation of RTN and cortex from nucleus basalis inhibits the SWD developing most likely due to the decrease of cortical excitability. Moreover, possibly cholinergic transmission is involved in the transforation of the synchronized phenomena (SWD) to another with close mechanism of generation.     

Specificity of gene di (diabetes insipidus) expression in homologous inbred rat strains

The diabetes insipidus mutation is displayed in homozygotes in the form of diabetes insipidus with water consumption from 30 to 100% of body weight per day. We developed two inbred sublines of the di/di Brattleboro rats as well as the recombinant inbred subline by integrating genes of August rats into the di/di mutant genome. Changes in the genetic background proved to have no effect on the quantitative parameters of the diabetes insipidus. The intensity of the secondary immune response and the content of tropomyosin in the medulla of the rat kidney can serve as additional marker traits of the di/di genotype.     

Establishment of a set of combined immunodeficient DA/Slc-Foxn1(rnu) Lyst(bg) congenic rat strains.

The congenitally athymic nude rat is used for studying cancer and transplantation owing to its hairlessness and T-cell defective function caused by the Foxn1(rnu) gene. However, NK cell activity of the nude rat is markedly increased. It is known that NK cells play a major role in rejection of xenografts and in cytotoxicity against tumor cells. Thus, the athymic nude rat with impaired NK cell activity should be a useful model for extensive studies. The DA-Lyst(bg)/Lyst(bg) rat, a model for human Chediak-Higashi syndrome (CHS) is characterized by diluted-coat color and impairment of NK cell activity. We planned to establish a combined immunodeficient double mutant rat introgressed with the Foxn1(rnu) and Lyst(bg) genes and a set of congenic strains having an identical genetic backgrounds simultaneously. Based on the phenotypic and genetic characteristics of the parental rat strains, the new strains were produced using continuous backcross and diagnosis with molecular genetic techniques. Each disease gene was diagnosed with PCR-RFLP or the long-nested PCR method. Furthermore, we used a marker-assisted congenic strategy based on scanning the genetic backgrounds of the parental rats with 461 rat microsatellite markers. We think that the newly established DA/Slc-Foxn1(rnu)/Foxn1(rnu) Lyst(bg)/Lyst(bg) double mutant will be useful as a severe disease model for human CHS, and the set of DA/Slc-Foxn1(rnu) Lyst(bg) congenic strains which have impaired NK cell activity and/or defective T cell function should be useful for studying in cancer research, xenotransplantation, immune function and other wide-ranging studies.     

GABAergic neurons and GABA(A)-receptors in temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE) is the most prevalent form of epilepsy, characterized by recurrent complex partial seizures and hippocampal sclerosis. The pathophysiology underlying this disorder remains unidentified. While a loss of benzodiazepine binding sites is a key diagnostic feature of MTLE, experimental studies have shown enhanced inhibitory transmission and increased expression of GABA(A)-receptors, suggesting that compensatory mechanisms are operative in epileptic hippocampus. In the present study, changes in the expression and cellular distribution of major GABA(A)-receptor subunits were investigated in the hippocampus of pilocarpine-treated rats during the phase of spontaneous recurrent seizures. A uniform decrease in GABA(A)-receptor subunit-immunoreactivity was observed in regions of extensive neuronal death (i.e. CA1, CA3, hilus). whereas a prominent increase occurred in the dentate gyrus (DG). Most strikingly, the increase was largest for the alpha3- and alpha5-subunits, which are expressed at very low levels in the DG of control rats, suggesting the formation of novel GABA(A)-receptor subtypes in epileptic tissue. Furthermore, an extensive loss of interneurons expressing the alpha1-subunit, representing presumptive basket cells, was seen in the DG. These changes were very similar to those reported in a novel mouse model of MTLE, based on the unilateral injection of kainic acid into the dorsal hippocampus (Bouilleret et al., 1999). This indicates that the regulation of GABA(A)-receptor expression is related to chronic recurrent seizures, and is not due to the extrahippocampal neuronal damage affecting pilocarpine-treated rats. These results allow causal relationships in the induction and maintenance of chronic recurrent seizures to be distinguished. The loss of a critical number of interneurons in the DG is a possible cause of seizure initiation, whereas the long-lasting upregulation of GABA(A)-receptors in granule cells represents a compensatory response to seizure activity.     

The rat pink-eyed dilution (p) mutation: An identical intragenic deletion in pink-eye dilute-coat strains and several Wistar-derived albino strains

We identified the rat pink-eyed dilution (p) and pink eye Mishima (p^m) mutations. The p^m mutation, which was isolated from a wild rat caught in Mishima Japan in 1961 and is carried in the NIG-III strain, is a splice donor site mutation in intron 5. The p mutation, which was first described in 1914 and is carried in several p/p rats including the RCS and BDV strains, is an intragenic deletion including exons 17 and 18. In addition to RCS and BDV strains, several albino strains, KHR, KMI and WNA, all descendants of albino stock of the Wistar Institute, are homozygous for the p allele. Analyses revealed that the colored p strains and the Wistar-derived albino p strains had the same marker haplotype spanning approximately 4 Mb around the P locus. This indicates that these p strains share a common ancestor and the p allele did not arise independently via recurrent mutations. The historical relationship among the p strains suggests that the p deletion had been maintained in stock heterogeneous for the C and P loci and then was inherited independently by the ancestor of the Wistar albino stock and the ancestor of the pink-eyed agouti rats in Europe.     

Activation of constitutive nitric oxide synthase(s) and absence of inducible isoform in aged rat brain

In this study, the effect of aging on nitric oxide synthases (NOS) was investigated in homogenates and cytosolic fractions from hippocampus, brain cortex and cerebellum of adult, old adult and old Wistar rats (3-4, 14, and 24 months old, respectively). Our results indicate the enhancement of Ca(2+) and calmoduline-dependent NOS activity in all investigated aged brain parts. Significantly higher NOS activity was found in the cerebellum.In the absence of Ca(2+) or in the presence of N-nitro-L-arginine (NNLA) the activity of NOS was absent. Inhibitor of constitutive NOS isoforms which preferentially inhibits neuronal NOS (nNOS), 7-nitroindazole, decreased NOS activity by 60 and 75% in adult and aged brain, respectively. However, using RT-PCR a significantly lower amount of mRNA for nNOS was detected in hippocampus. The ratio of NOS activity to nNOS mRNA was significantly higher in hippocampus and cerebellum of aged brain. No expression of the gene for inducible NOS was observed in adult and aged brain.These results indicate that probably nNOS is responsible for higher NOS activity in aged brain. Our data suggest that alteration of nNOS phosphorylation state may be responsible for the activation of NOS in aged brain. The down-regulation of nNOS mRNA expression may be an adaptive mechanism that protects the brain against excessive NO release.     

Neuroactive steroids and GABAA receptor plasticity in the brain of the WAG/Rij rat, a model of absence epilepsy

The role of neuroactive steroids and GABA(A) receptors in the generation of spontaneous spike-and-wave discharges (SWDs) was investigated in the WAG/Rij rat model of absence epilepsy. The plasma, cerebrocortical, and thalamic concentrations of the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) were increased in the WAG/Rij rat at 2 months of age compared with those in control (Wistar) rats. In contrast, the brain and peripheral levels of 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC) did not differ between the two rat strains at this age. At 6 months of age, when absence epilepsy worsens in WAG/Rij rats, the plasma concentration of 3alpha,5alpha-TH PROG remained high whereas that of 3alpha,5alpha-TH DOC had increased, the cerebrocortical levels of both 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC had increased, and the thalamic concentrations of these metabolites had decreased. At 6 months of age the expression of the alpha(4) and delta subunits of the GABA(A) receptor in relay nuclei was increased. Finally, chronic stress induced by social isolation elicited a reduction in the amount of 3alpha,5alpha-TH PROG in the thalamus of 2-month-old WAG/Rij rats that was associated with a reduction in the number and overall duration of SWDs at 6 months of age. Absence epilepsy in the WAG/Rij rat is thus associated with changes in the abundance of neuroactive steroids and in the expression of specific GABA(A) receptor subunits in the thalamus, a brain area key to the pathophysiology of this condition.     

Non-linear analysis of epileptic seizures I. Correlation-dimension measurements for absence epilepsy and near-periodic signals

The study of six absence seizures from two patients confirmed the efficacy, in the search for low correlation dimensions, of using scaled-structure analysis, combined with the appropriate checking procedures. The analysis is directed towards characterizing an attractor not only by its correlation dimension, but also by its “quality” and by the probability for genuine identification. For near-periodic dynamics, we warn against: (1) artefacts that appear at high values of the correlation integral, in the form of apparent Grassberger-Procaccia scaling at very low values of the dimension (near-periodicity artefact); (2) erroneous interpretation of phase-randomization data, owing to destruction of the artefact by randomization rather than any evidence for low-dimensional dynamics. In single-channel analyses of two patients and six seizures altogether, high-quality attractors were found only for one seizure in two channels, at correlation dimensions 4.7 and 6, respectively. Furthermore, no attractor of measurable dimension was found from multichannel space reconstructions over durations approaching those of typical seizures. Both these results show that in an absence seizure, spatial extension of low-dimensional dynamics must be lost over such durations. Received: 27 April 1998 / Accepted in revised form: 10 November 1998     

Morphological aberrations in therapy-resistant partial epilepsy (TRPE)

Epileptic temporal and parietal cortices, removed from 6 patients with therapy-resistant (intractable) partial epilepsy (TRPE) during neurosurgery, were studied. Neurons (40–50 in each slice) in laminae I–VI and white matter were injected with Lucifer Yellow (LY). Samples were examined in a confocal laser scanning microscope (BioRad [Richmond, CA] MRC 600), and individual cells were scanned at 0.1–2 μm incremental levels. 2D maximal linear projection was used for overview. Frames (50–60) of scanned neurons were transformed into 3D volumes, using VoxelView software on a Silicone Graphics workstation, and rotated. All samples contained pyramidal neurons with duplicated apical dendrites, additional basal dendrites, or were misplaced in a horizontal position in the white matter. Rarely were such cells observed in normal cases. The relation between the observations and the disease is discussed. The attempt to simultaneously apply immunofluorescence was successful concerning synaptic vesicle antigens. This approach will be used for a detailed study of the synaptology of this disease.     

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.     

A set of highly informative rat simple sequence length polymorphism (SSLP) markers and genetically defined rat strains

Background

The National Bio Resource Project for the Rat in Japan (NBRP-Rat) is focusing on collecting, preserving and distributing various rat strains, including spontaneous mutant, transgenic, congenic, and recombinant inbred (RI) strains. To evaluate their value as models of human diseases, we are characterizing them using 109 phenotypic parameters, such as clinical measurements, internal anatomy, metabolic parameters, and behavioral tests, as part of the Rat Phenome Project. Here, we report on a set of 357 simple sequence length polymorphism (SSLP) markers and 122 rat strains, which were genotyped by the marker set.

Results

The SSLP markers were selected according to their distribution patterns throughout the whole rat genome with an average spacing of 7.59 Mb. The average number of informative markers between all possible pairs of strains was 259 (72.5% of 357 markers), showing their high degree of polymorphism. From the genetic profile of these rat inbred strains, we constructed a rat family tree to clarify their genetic background.

Conclusion

These highly informative SSLP markers as well as genetically and phenotypically defined rat strains are useful for designing experiments for quantitative trait loci (QTL) analysis and to choose strategies for developing new genetic resources. The data and resources are freely available at the NBRP-Rat web site [1].