Genetic Analysis of the Predisposition to Audiogenic Seizure Fits in Krushinsky-Molodkina Rat Strain

The expression of audiogenic seizure fits has been studied in F₁ hybrids between audiogenic seizure-prone Krushinsky-Molodkina rat strain and Wistar rats not prone to audiogenic seizures, as well as in two backcross generations. Only 10% of F₁ hybrids exhibit audiogenic seizure fits, whereas the frequency of this character in two generations of their backcrosses with Krushinsky-Molodkina rats is about 50%. A digenic model with incomplete penetrance has been put forward to explain the control of audiogenic seizure fits. This model fits the data obtained: the theoretically expected distributions of the character in offsprings of different crosses do not differ significantly from those observed in experiments. The model explains why the distribution of the character is the same in the first and second backcross offsprings.     

Laboratory rat selection for the trait "the absence of audiogenic seizure proneness"

The hybrids between Krushinsky-Molodkina (KM) inbred strain, selected for high predisposition to audiogenic epilepsy (AE), and Wistar rats non-prone to audiogenic seizure were the initial population for selection. Rats were selected for the trait "the absence of audiogenic seizure proneness". The creation of such strain in which the significant proportion of animals develop no AE in response to sound and share partly the genetic background of the KM strain is very important for the correct use of RV strain as the laboratory model of seizure states. As alleles which determine the AE proneness are recessive the selection for the "opposite" trait proceeds necessarily slow.     

Laboratory rat selection for the trait “the absence of audiogenic seizure proneness”

The hybrids between Krushinsky-Molodkina (KM) inbred strain, selected for high predisposition to audiogenic epilepsy (AE), and Wistar rats non-prone to audiogenic seizure were the initial population for selection. Rats were selected for the trait ??the absence of audiogenic seizure proneness??. The creation of such strain in which the significant proportion of animals develop no AE in response to sound and share partly the genetic background of the KM strain is very important for the correct use of KM strain as the laboratory model of seizure states. As alleles which determine the AE proneness are recessive the selection for the ??opposite?? trait proceeds necessarily slow.     

Energetic,oxidative and ionic exchange in rat brain and liver mitochondria at experimental audiogenic epilepsy (Krushinsky–Molodkina model)

The role of brain and liver mitochondria at epileptic seizure was studied on Krushinsky-Molodkina (KM) rats which respond to sound with an intensive epileptic seizure (audiogenic epilepsy). We didn't find significant changes in respiration rats of brain and liver mitochondria of KM and control rats; however the efficiency of АТР synthesis in the KM rat mitochondria was 10% lower. In rats with audiogenic epilepsy the concentration of oxidative stress marker malondialdehyde in mitochondria of the brain (but not liver) was 2-fold higher than that in the control rats. The rate of H₂O₂ generation in brain mitochondria of КМ rats was twofold higher than in the control animals when using NAD-dependent substrates. This difference was less pronounced in liver mitochondria. In KM rats, the activity of mitochondrial ATP-dependent potassium channel was lower than in liver mitochondria of control rats. The comparative study of the mitochondria ability to retain calcium ions revealed that in the case of using the complex I and complex II substrates, permeability transition pore is easier to trigger in brain and liver mitochondria of KM and КМs rats than in the control ones. The role of the changes in the energetic, oxidative, and ionic exchange in the mechanism of audiogenic epilepsy generation in rats and the possible correction of the epilepsy seizures are discussed.     

Effect of seizure activity on subunit composition of Ca2+/calmodulin-dependent protein kinase II in hippocampus of Krushinskii-Molodkina rats

Using a Western blot method it was been shown that a aontent of beta-CaMKII subunit was decreased on 40 % in hippocampus of Krushinskii-Molodkina rats (rats, genetically prone to audiogenic seizures) in comparison with normal Wistar rats. Additionally, we have investigate the temporal modifications of alpha/beta-CaMKII subunits in dependent from number audiogenic fits (n = 1 or n = 5). The level of beta-CaMKII in hippocampus of naive KM rats and KM rats in 24 h after single audiogenic seizure was not different, but it was increased on 56 % through 72 h after convulsion fit. In contrast, both 5-fold seizures and 20-fold seizures (audiogenic kindling) reduced the beta-CaMKII levels in comparison with naive group. The levels of alpha-subunit protein kinase in hippocampus these experimental groups of animals were not different. We suggest a hypothesis, that modifications alpha/beta ratio can be viewed as specific "homeostatic mechanism", which provide for neuronal function in hippocampus, thereby changing the rules governing synaptic plasticity in dependence from preliminary neuronal activity.     

Remote effects of early postnatal pituitary hormone melatonin injection on audiogenic seizures in Krushinsky-Molodkina rats

Rats Krushinsky-Molodkina inbred strain (KM) genetically prone to audiogenic seizures were injected with pineal hormone melatonin (50 mg/kg, s.c.) within the period 7th to the 14th posnatal days (PND). The remote effects of this injection adult KM rats consisted in a decrease in the latency and increase in severity of myoclonic seizures produced by audiogenic kindling (20 sound stimuli, 100 dB and 12-15 kHz). As compared to the control, in the cortex and hippocampus of rats of melatonin group, we also found a significant reduction of both total and functional activity of Ca2+/calmodulin-dependent protein kinase II (CAMK II) after audiogenic kindling. On the contrary, melatonin administration within the 1st to 7th PND and the 14th to the 21st PND resulted in a decrease in seizure activity. In the first case, both the total (cortical) and functional (hippocampal) CAMK II activities in melatonin-injected rats were increased as compared to control, whereas in the second case, only a slight increase in Ca2+-independent CAMK II activity in the hippocampus of melatonin-injected rats was observed. Probably, the melatonin administration in the period of early postnatal development changes the features of expression and/or regulation of CAMK II activity, and this could be one of the mechanisms of audiogenic seizure modulation in KM rats.     

Rats of Krushinsky-Molodkina strain: studies of audiogenic epilepsy, vascular pathology, and behavior

Physiological, biochemical and genetic studies of audiogenic epilepsy in Krushinsky-Molodkina (KM) rat strain are analyzed. In brief, the history of KM strain selection and its current state are reviewed. Physiological mechanisms of the typical audiogenic seizure development and the audiogenic myoclonus (audiogenic kindling phenomena) are considered. The EEG correlates of these phenomena are presented. Biochemical features of the strain (neurotransmitter and neuronal membrane biochemistry) are described.     

Cottonwood hybrids gain fitness traits of both parents: a mechanism for theirlong-term persistence?

Using surveys of natural populations, experimental crosses, and common garden trials, we tested the hypothesis that hybrid cottonwoods (Populus fremontii × P. angustifolia) from the Weber River in northern Utah would produce as many viable offspring as their parental species. We found that both F(1) generations and backcross generations can be just as fit as the parent taxa. First, F(1) hybrids produced as many viable seed as P. angustifolia (but less than P. fremontii), and backcross genotypes produced as many viable seeds as both parent taxa. Second, hybrids produced nearly two times as many ramets from root sprouts as P. angustifolia and four times as many ramets as P. fremontii. Third, the high mortality of germinated seedlings of all tree types (i.e., >90%) and very low mortality of asexually derived ramets provide hybrids with equal sexual reproduction and enhanced asexual reproduction, especially since backcross hybrids exhibit transgressive segregation in ramet production. Our findings suggest that the introgression of P. fremontii seed traits into the hybrid genome is responsible for their equivalent performance (at least to one parent) in sexual reproduction, while the contributions of asexual traits from P. angustifola results in hybrids having equal or greater fitness.     

甘蓝型油菜Ogura 雄性不育×白菜的回交杂种后代与亲本之间蕾期基因表达差异比较研究*

以甘蓝型油菜（Brassica napus L, AACC,2n＝38）Ogura细胞质雄性不育材料为母本,以不同白菜（B campestris ssp. chinensis Makino, AA, 2n＝20） 自交系‘新选一号’和‘矮脚黄’为父本进行杂交,获得了杂种F1、BC1、BC2代。利用cDNA-AFLP技术对两种材料的不同回交世代BC1、BC2代与其亲本在蕾期的基因表达进行分析。结果表明,两种白菜回交世代与其亲本的基因表达有明显差异,在质和量上都存在差异。基因表达模式有5类共7种：（1）单亲沉默型（2种）,（2）单亲一致型（2种）,（3）双亲共沉默型,（4）杂种特异型,（5）表达一致型。随着回交世代的增加,回交杂种和亲本的基因表达在单亲沉默型、双亲共沉默型呈增加趋势。而在母本一致型、父本一致型、杂交种特异型、表达一致型呈下降的趋势。两种白菜在F1、BC1、BC2 3个世代与回交亲本花蕾间的基因差异表达有15种类型,其中以在轮回亲本、F1、BC1、BC2中共同出现表达的带的比例最高。Abstract: Crosses between female parent of Ogura male sterility Brassica napus L. and male parents of B. campestris ssp. chinensis Makino were made and F1, BC1 and BC2 generations produced. Gene expression of two Chinese cabbage backcross hybrid BC1, BC2 and their parents at bud stage was analyzed by means of cDNA-AFLP technique. The results indicated that the patterns of gene expression differ significantly between BC1 and BC2 generations and their parents. There were many patterns of gene expression, including gene overexpression and gene silencing. Five patterns (seven kinds) of gene expression were observed, which include: (1) bands occurring in only one parent (two kinds); (2) bands observed in hybrids and one parent (two kinds); (3) bands occurring in only parents (one kind); (4) bands visualized in only hybrids (one kind); (5) bands observed in parents and hybrids (one kind). In accompany with the addition of backcross, the increase trend in backcross hybrids and their parents were described in the aspects of differential gene expression, bands expressed only in one parent and bands expressed only in both parents. The declined trend in backcross hybrids and their parents were observed in the aspects of bands expressed in both hybrids and one parent (two kinds), bands visualized in only hybrids and bands observed in parents and hybrid. Fifteen patterns of gene expression were observed in F1、BC1、BC2 and backcross parents. The percent of bands expressed in F1、BC1、BC2 and backcross was highest.     

Multifaceted,Cross-Generational Costs of Hybridization in Sibling Drosophila species

Maladaptive hybridization, as determined by the pattern and intensity of selection against hybrid individuals, is an important factor contributing to the evolution of prezygotic reproductive isolation. To identify the consequences of hybridization between Drosophila pseudoobscura and D. persimilis, we estimated multiple fitness components for F1 hybrids and backcross progeny and used these to compare the relative fitness of parental species and their hybrids across two generations. We document many sources of intrinsic (developmental) and extrinsic (ecological) selection that dramatically increase the fitness costs of hybridization beyond the well-documented F1 male sterility in this model system. Our results indicate that the cost of hybridization accrues over multiple generations and reinforcement in this system is driven by selection against hybridization above and beyond the cost of hybrid male sterility; we estimate a fitness loss of >95% relative to the parental species across two generations of hybridization. Our findings demonstrate the importance of estimating hybridization costs using multiple fitness measures from multiple generations in an ecologically relevant context; so doing can reveal intense postzygotic selection against hybridization and thus, an enhanced role for reinforcement in the evolution of populations and diversification of species.     

Gentics of urogenital abnormalities in ACI inbread rats.

The frequency of urogenital abnormalities in adult ACI rats was 18.8%. These abnormalities consisted of a spectrum of defects ranging from focal aplasia of the ureter to the complete absence of the ureter, kidney, and tubular genital tract on the affected side. When ACI rats with normal urogenital tracts tracts were crossed with F344 rats the frequency of the defects decreased to a constant level in the F1, F2, and F3 generations. There was no clearly significant decrease in the frequency of the defects in the backcross offspring of (ACI times F344)F1 times ACI matings, but there was a significant and dramatic decrease in the backcross to F344 rats. Attempts to select against these defects by brother-sister matings among normal ACI rats were not successful: 5 generations of inbreeding did not alter the frequency of the urogenital abnormalities. The frequency of the abnormalities was the same in the offspring of affected parents as in the offspring of normal parents, indicating homozygosity of the factors responsible for the defects. The results suggest that the transmission of these defects is polygenic.     

3H-1-glutamate binding with the synaptic membranes isolated from the cerebral cortex and hippocampus of Krushinski?-Molodkina strain rats

Specific binding of 3H-L-glutamate to synaptic membranes isolated from the cerebral cortex and hippocamp of Wistar and Krushinsky-Molodkina (KM) rats examined both in a quiet awake state and after audiogenic seizures was compared. The dissociation constant (KD) values and binding capacity (Bmax) for KM rats did not differ significantly from the corresponding parameters of binding determined for Wistar rats (KD--89.8 +/- 18.1 and 102.6 +/- 12.5 nm, Bmax--1.23 +/- +/- 0.08 and 1.30 +/- 0.15 pmol/mg for the cortex and hippocamp, respectively). After audiogenic seizures the binding capacity of the hippocamp of KM rats was reduced by 30%. It is suggested that hippocampal glutamate receptors of KM rats are involved in the mechanism of convulsive activity formation.     

High pressure neurologic syndrome type 2 seizure in mice

Progressive compression in helium/oxygen (heliox) atmospheres elicits in mice and many other vertebrates tested a complex series of effects known as the high pressure neurologic syndrome (HPNS). The most dramatic behavioral manifestations of the syndrome are two successive and distinct convulsive seizures. HPNS type 1 and type 2. In the present study, a maximum likelihood estimation procedure was applied to 11 models of inheritance of the difference in the time elapsed until manifestation of the type 2 seizure in heliox-compressed C57BL/6 and BALB/c mouse strains, their F1 hybrid, and the backcross generations. The "preferred" model specifying interaction between two major unlinked autosomal loci was confirmed indirectly by further breeding tests. The tests also showed that type 1 and type 2 seizure thresholds are uncorrelated but type 1 always preceded type 2. A challenge to the latter result involving mating backcross mice with high type 1 and type 2 seizure thresholds to mice with low type 1 and type 2 seizure thresholds produced no instance of alteration of the seizure order.     

Further studies on resistance to Leptosphaeria coniothyrium in the red raspberry and related species

Rubus pileatus and its F₁ hybrids with raspberry (R. idaeus) were resistant to cane blight (Leptosphaeriu conioth-yriurn), but little resistance was obtained in subsequent backcross generations apart from a hybrid identified in the second backcross. Two hybrids from backcrossing R. coreanus to raspberry also showed resistance. R. pileatus and its F, hybrids produced hard growth, unlike that of raspberries, which may have been associated with a form of resistance that could not easily be transferred to commercial raspberry cultivars. Some of the genotypes used as parents showed intermediate levels of resistance and it is possible that the highly resistant genotype identified in the second backcross arose from a recombination of genes for resistance. Plants with pubescent canes (gene H) were up to 20% more resistant to mycelial inoculation than those with non-pubescent canes (gene h), and the percentage of machine-harvester inflicted wounds with disease symptoms that resulted from natural infection was also less in genotypes with pubescent canes. Many genotypes with intermediate levels of resistance suffered only limited damage from mycelial inoculation and so there are good prospects for breeding cultivars with an effective resistance, despite the limited value of R. pileatus as a donor species.     

Audiogenic kindling changes the subunit composition of BK-channels in dentate gyrus of Krushinskii-Molodkina rats

Subunit composition of voltage- and Ca²⁺-sensitive high-conductance K⁺ channels (BK channels) in dentate gyrus (DG) of Krushinskii-Molodkina (KM) rats, genetically prone to audiogenic seizures, was compared with that of normal Wistar rats, resistant to sound effects. Additionally, long-lasting changes in protein expression of α- and β4-subunits in DG of KM rats after audiogenic kindling (model of temporal lobe epilepsy) was investigated. Western blot analysis revealed no differences between the levels of the pore-forming α-subunit expression in DG of KM and Wistar rats. In contrast, the level of brain-specific auxiliary β4-subunit in DG of KM rats was increased twofold in comparison to that in Wistar rats. It is likely that the observed changes in the BK channel α/β4 subunits ratio can prevent the development of excessive neuronal exitability in DG of KM rats. The results obtained on the model of audiogenic kindling (20 convulsion fits) confirmed this assumption. Thus, α-subunit expression levels in DG of KM rats on day 3 and 14 after the last seizure were increased 2.5 times in comparison with intact KM rats. The expression level of β4 in DG of KM rats 3 days after kindling was reduced to 30% of the control level. On day 14 after finishing audiogenic kindling, a further reduction of β4 protein expression level occurred. We suggest that the changes in the subunit composition of BK channels in DG following chronic seizures can alter functional properties of DG as a physiological filter, which normally prevents the propagation of epileptiform activity in the hippocampus.     

The Wakayama epileptic rat (WER), a new mutant exhibiting tonic-clonic seizures and absence-like seizures.

A new mutant, the Wakayama epileptic rat (WER), exhibiting both spontaneous absence-like behavior and tonic-clonic convulsions, was identified in a colony of Wistar rats. To determine clear seizure characteristics of this mutant strain, we analyzed the mode of inheritance of the convulsion and observed patterns of electroencephalogram (EEG) during the seizures. F1 progeny were produced between the founder male and normal females of the same colony. Animals were monitored through the inbreeding course to analyze genetic control of epileptic behavior. EEGs were recorded using affected animals in the F3-4 and post F13 generations. After the F2 generation, affected rats spontaneously exhibited both absence-like immobile behavior and tonic-clonic convulsions. The absence-like seizures were characterized by motor arrest and head droop. The tonic-clonic convulsions began with neck and forelimb clonus, wild jumping/running, and opisthotonic posturing, and evolved to tonic, then clonic convulsions. Most convulsion onsets occurred between 25-70 days of age. Mating experiments revealed that 0%(0/18) of the animals in F1, 10%(3/26) in F2, 17%(1/6) in backcross progeny and 86% (100/116) in progeny of crosses between epileptic rats showed tonic-clonic convulsions. Ictal cortical EEGs were characterized by 4-6 (5.1 +/- 0.4, mean +/- SD) Hz spike-and-wave complexes in the absence-like seizures and by low-voltage fast waves in the tonic-clonic convulsions. This new mutant rat spontaneously exhibited both absence-like and tonic-clonic seizures. The tonic-clonic seizure was inherited as an autosomal recessive trait with 86% incidence. Thus, the new mutant rat may become a useful model for studying human inherited epilepsies.     

Genetic analysis for insulitis in NOD mice

Non-obese diabetic (NOD) mice spontaneously develop diabetic signs akin to those of Type I diabetes in man. Insulitis, i.e., lymphocytic infiltration around and into the pancreatic islets is one of the characteristics of such mice. It is also the etiologic pathological lesion in the development of diabetes mellitus in NOD mice. Thus, we chose insulitis as a marker for genetic analysis of the development of diabetes mellitus in NOD mice and clarified the mode of its inheritance. In breeding studies between NOD and C57BL/6J mice, insulitis was not observed in the F1 and (F1 X C57BL/6J) backcross generations, but was found with incidences of 3.9% in females and 1.4% in males in the F2 generation and 23.7% in females and 12.1% in males in the (F1 X NOD) backcross generation. These incidences in the F2 and (F1 X NOD) backcross females corresponded approximately to 1/16 and 1/4 of the incidences of 89.7% in the NOD females, respectively. A similar relationship was observed between the F2 and (F1 X NOD) backcross males and the NOD males. When the gene expressivity of both sexes for a double recessive homozygote was assumed to be the incidences of insulitis in 9-week-old NOD females and males, respectively, the expected numbers of both sexes with and without insulitis in the F2 and backcross generations agreed well with the observed ones. These observations suggest that two recessive genes on independent autosomal chromosomes are necessary for the development of insulitis in NOD mice.     

The Mass1frings mutation underlies early onset hearing impairment in BUB/BnJ mice, a model for the auditory pathology of Usher syndrome IIC

The human ortholog of the gene responsible for audiogenic seizure susceptibility in Frings and BUB/BnJ mice (mouse gene symbol Mass1) recently was shown to underlie Usher syndrome type IIC (USH2C). Here we report that the Mass1frings mutation is responsible for the early onset hearing impairment of BUB/BnJ mice. We found highly significant linkage of Mass1 with ABR threshold variation among mice from two backcrosses involving BUB/BnJ mice with mice of strains CAST/EiJ and MOLD/RkJ. We also show an additive effect of the Cdh23 locus in modulating the progression of hearing loss in backcross mice. Together, these two loci account for more than 70% of the total ABR threshold variation among the backcross mice at all ages. The modifying effect of the strain-specific Cdh23ahl variant may account for the hearing and audiogenic seizure differences observed between Frings and BUB/BnJ mice, which share the Mass1frings mutation. During postnatal cochlear development in BUB/BnJ mice, stereocilia bundles develop abnormally and remain immature and splayed into adulthood, corresponding with the early onset hearing impairment associated with Mass1frings. Progressive base-apex hair cell degeneration occurs at older ages, corresponding with the age-related hearing loss associated with Cdh23ahl. The molecular basis and pathophysiology of hearing loss suggest BUB/BnJ and Frings mice as models to study cellular and molecular mechanisms underlying USH2C auditory pathology.     

Moderate body hypothermia alleviates behavioral and EEG manifestations of audiogenic seizures in metaphit-treated rats

We investigated the effects of hypothermia on the incidence and EEG signs of audiogenic seizures in rats treated with metaphit (1-[1(3isothiocyanatophenyl)-cyclohexyl] piperidine), an experimental model of generalized reflex epilepsy. After i.p. injection with metaphit (10 mg/kg) Wistar rats were exposed to audiogenic stimulation at hourly intervals during the time course of the experiment. After intermittent use of an ice pack 8 h after the metaphit treatment, when seizure was fully developed, the body temperature was reduced to 30 +/- 0.5 degrees C in one half of the rats, and maintained at 37 +/- 0.5 degrees C in the other half. Saline-injected rats served as a control group. In the hypothermia group, the incidence of audiogenic seizures induced by metaphit was completely suppressed during the 3 consecutive testing times, while no signs of epileptiform activity were noted in EEG tracings. The termination of hypothermic treatment resulted in the recovery of seizure susceptibility, and during audiogenic stimulation, bursts of spiking activity were recorded in the EEGs of metaphit-treated rats. These findings indicate that moderate body hypothermia is an effective anticonvulsant treatment for audiogenic seizures in metaphit-treated rats.     

Reproductive fitness of hybrids between Senecio jacobaea and S. aquaticus (Asteraceae)

Natural hybridization is increasingly recognized as an important process for the ecology and evolution of natural plant populations and species. There is a great need to initiate more studies based on natural populations in order to elucidate the possible role of hybrids in nature. The reproductive success of early generation hybrids can make or break hybrid lineages and may determine the genetic structure of hybrid swarms or the potential for gene flow through future generations, but studies of hybrid reproductive success are lacking. Here we measured components of male and female reproductive success in Senecio jacobaea and S. aquaticus (Asteraceae) species and F(1) hybrids between these species under laboratory conditions, and we measured reproductive output from crosses producing F(1), F(2), and backcross (BC) generation hybrids. F(1) hybrids were readily produced, and on average, the success of crosses producing subsequent generations (F(2), BC) decreased (though remained substantial), but the success of crosses was highly dependent on the genotypes involved. Also, F(1) hybrids were bigger, produced more flowers, and therefore produced more pollen than parental plants. Finally, crosses between parents were asymmetrical, such that S. aquaticus produced more and larger F(1) seeds than did S. jacobaea.