Synaptosomal high-affinity noradrenaline uptake does not differ between mice susceptible (DBA/2J) and resistant (C57 BL/6) to audiogenic seizures

Abnormalities in noradrenaline-mediated neurotransmission have been advocated as a basis of the age-related susceptibility of DBA/2J mice to generalised convulsions induced by auditory stimulation. We have measured the kinetics of synaptosomal high-affinity noradrenaline uptake in 5 brain regions of DBA/2J mice at ages before, during and after their maximal susceptibility to audiogenic seizures, and age-matched C57 BL/6 mice, a strain resistant to audiogenic seizures at all ages. No differences were found between the two strains of mice in any of the brain regions studied. Abnormalities of high-affinity noradrenaline uptake do not contribute to audiogenic seizure susceptibility of DBA/2J mice.     

GABA(A) receptor alpha4 subunit in DBA/2J and C57BL/6J mice

GABA(A) receptors are chloride channels in the brain activated by binding of gamma-aminobutyric acid (GABA). Several important classes of drugs, including alcohol and certain antiepileptic drugs, modulate the actions of GABA. We report the sequence and expression of alpha4 subunits of GABA(A) receptors in two inbred strains of mice, DBA/2J and C57BL/6J, which differ in susceptibility to seizures and to behavioral effects of alcohol. We find no differences between the two strains in cDNA sequence, or in levels of alpha4 mRNA in whole brains of the two strains at 21 days of age, when DBA/2J are most susceptible to audiogenic seizures. We also describe the pattern of developmental expression and brain regional distribution of this subunit in mice, finding the highest developmental expression at about 14 days of age in whole brains, and the highest regional levels in hippocampus and basal forebrain (including thalamus) in adults.     

GABA-gated chloride ion influx and receptor binding studies in C57BL6J and DBA2J mice

GABA-gated chloride ion influx was measured in brain 'microsac' preparations of young (20-22-day-old) and older (40-42-day-old) C57BL6J and DBA2J mice. The young DBA2J mice are susceptible to audiogenic seizures. GABA sensitivity was reduced in young DBA2J mice as compared to age-matched C57BL6J mice or older mice of either strain. Age and strain differences in ligand binding to GABA/benzodiazepine receptor complex and glutamate receptor could not account for this finding. These results provide evidence for a defect in GABA-gated chloride ion influx in audiogenic seizure-susceptible DBA2J mice.     

Strain comparisons and developmental profile of the delta subunit of the murine GABAA receptor.

GABAA receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of gamma-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABAA receptor delta subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunit's postnatal developmental profile. We did not detect any unique features in the delta subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the delta subunit's first putative transmembrane region.     

Strain comparisons and developmental profile of the delta subunit of the murine GABAA Receptor

GABA_A receptors are multisubunit inhibitory chloride channels in the brain which open in response to binding of -γ-aminobutyric acid (GABA) and are thought to be involved in some forms of seizures. We compare the sequence and expression of the GABA_A receptor δ subunit in audiogenic seizure prone (DBA/2J) and seizure resistant (C57BL/6J) inbred strains of mice and also report this subunit's postnatal developmental profile. We did not detect any unique features in the 6 subunits of DBA/2J mice which might explain their seizure susceptibility, but did detect in some clones from both DBA/2J mice and C57BL/6J mice an unusual substitution of His for a conserved Tyr in the δ subunit's first putative transmembrane region.     

Influence of Audiogenic Seizures on Synaptic Facilitation in Mouse Hippocampal Slices Is Mediated by N-Methyl-d-Aspartate Receptor

The influence of audiogenic seizures (AGS) on synaptic facilitation was studied in DBA/2J (D2) and C57BL/6J (B6) mice. AGS susceptibility is inherited in D2 mice, but can be acquired in AGS-resistant B6 mice through acoustic priming. The experiments were per formed on hippocampal slices obtained from D2 and B6 mice both with and without seizures. Long-term potenti ation (LTP) and low-Mg^{2 +}-induced synaptic facilitation (LMISF) were evaluated after stimulation of Schaffer col laterals. The magnitude of LTP and LMISF was signifi cantly greater in slices obtained from mice with seizures than from mice without seizures in both strains. Seizure induced enhancement of LTP and LMISF was markedly reduced by the JV-methyl-D-aspartate (NMDA) receptor antagonist AP-5. The noncompetitive NMDA receptor antagonist MK 801 reduced the efficiency of priming in B6 mice and abolished AGS in D2 mice and primed B6 mice, the data suggest that audiogenic seizures can en hance synaptic facilitation through activation of the NMDA receptor.     

Kainate and AMPA receptor binding in seizure-prone and seizure-resistant inbred mouse strains

Glutamate and its receptors represent the major excitatory neurotransmission system in the mammalian brain and are considered important in the pathogenesis of many neurological diseases. The present study describes saturation binding experiments performed to measure the affinity (K_d) and density (B_max) of kainate and AMPA receptors in striatum, cortex and hippocampus from mature DBA/2J (DBA) and C57BL/6J (C57) mice. Previous studies have documented that these two strains differ significantly in seizure susceptibility, with DBA mice exhibiting greater sensitivity in various convulsant tests compared to C57 mice. Non-linear regression analysis of binding data together with Student's t-test and ANOVA revealed significantly higher densities of kainate receptors in striatum and of AMPA receptors in cortex of DBA mice. C57 mice exhibited higher striatal [³H]AMPA binding. There were no significant differences between the mouse strains in binding sites prepared from hippocampus and no differences in affinity for either receptor in any brain region studied. The results support a role for kainate and AMPA receptors in seizure sensitivity, possibly by influencing glutamate transmission in specific pathways. It is unlikely, however, that these receptors account for the generation of seizures alone but rather cooperate with other glutamatergic and non-glutamatergic neurotransmitter systems.     

Radioiodide uptake in brain, CSF, thyroid, and salivary glands of audiogenic seizure mice

DBA/2J (DBA) mice are susceptible to audiogenic seizures (ASs) in an age-dependent manner. Anion transport as measured by radioiodide uptake was determined in thyroid gland, salivary gland, skeletal muscle, cerebral cortex, cerebellum, brainstem, and CSF from these mice at various ages. Anion transport was also determined in C57BL/6J(C57) mice, an AS-resistant strain. In thyroid, DBA mice had an enhanced ability to concentrate iodide at 21 days of age when they have maximal AS susceptibility, as compared with the same-aged C57 mice. This difference in thyroid function was less marked at 40 days of age, when DBA mice are less AS susceptible, and was absent at 110 days of age, when DBA mice are AS resistant. In brain, differences in iodide uptake were also noted between these two strains of mice at 21 days of age. DBA mice had an increased concentration of iodide in CSF, an indication that they have a defect in the transport of iodide out of the CSF across the choroid plexus. In addition, DBA mice had a lower ratio of cerebral cortex to CSF iodide, which suggests that DBA mice have a defect in the transport of this anion into cerebral cortical cells from brain interstitial fluid. These differences in iodide transport in brain decreased with age as the AS susceptibility of DBA mice decreased. These results suggest a relation between anion transport in thyroid gland, cerebral cortex, and choroid plexus and AS susceptibility in DBA mice at 21 days of age.     

Brain 5-HT2 receptor binding sites in depressed suicide victims

5-HT2 receptor binding sites were measured (by saturation binding of [3H]ketanserin) in brain tissue obtained at postmortem from 19 suicide victims with definite evidence of depression and 19 sex and age-matched control subjects. Five of the suicide victims were receiving antidepressant drugs prior to death; 13 suicide victims had not been prescribed antidepressant or other psychoactive drugs recently and none were found in their blood at postmortem. The number of serotonin-2 (5-HT2) binding sites in frontal, temporal and occipital cortex and amygdala did not differ significantly between the depressed suicide victims and controls, either in the total suicide group or in the antidepressant drug-free suicides. The number of 5-HT2 binding sites in the hippocampus did not differ from controls in the total suicide group but was significantly lower (by 23%) in the antidepressant-free suicide group. The affinity of [3H]ketanserin binding did not differ from controls in the antidepressant-free suicides but was lower (increased Kd) in those subjects receiving antidepressant drugs. No correlation was found between the time of death and storage of tissue or the duration of tissue storage prior to assay and the number or affinity of 5-HT2 binding sites. A significant negative correlation was found between age of the subject and the number of 5-HT2 binding sites in the frontal and occipital cortex. The present study of suicide victims with definite evidence of depression do not confirm previous studies of increased numbers of 5-HT2 binding sites in suicide victims and suggest that these previous findings may be related to factors other than depression.     

Extracellular (36C1) Space, Electrolyte, Protein, and DNA Content in Brain of DBA and C57 Mice: Effects of Age

DBA/2J (DBA) mice are susceptible to audiogenic seizures (AGSs) in an age-dependent manner, susceptibility being maximal at 21 days and absent at 110 days of age. Previous studies have demonstrated that there is a decrease in anion transport and an increase in carbonic anhydrase (CA) activity in brain from DBA mice as compared with C57BL/6J (C57, non-AGS) mice at 21 days. Since these results suggest that there are alterations in cellular and electrolyte composition of brain from DBA mice, the present work was directed toward determining electrolyte content, extracellular space, and DNA content of brain from DBA and C57 mice at 21 and 110 days of age. There was a decrease in intracellular chloride and sodium content, and an increase in intracellular potassium content in cerebral cortex, cerebellum, and brainstem from DBA mice at both 21 and 110 days. Also, extracellular space was larger in these three brain tissues from DBA mice at both ages. DNA content, not different between the strains at 21 days, was significantly lower in cerebellum from DBA mice at 110 days. These findings give further evidence of alterations in the transport of ions in brain from DBA mice. In addition, they demonstrate that there are alterations in the intracellular and extracellular space values, an indication of changes in cellular composition of brain in these mice. Such changes may contribute to AGS susceptibility by disrupting the balance that normally exists in the neuronal microenvironment of the central nervous system.     

Brain cationic ATPase activities in epileptic (El) mice

Total, Mg2+-, Na+,K+-, and Ca2+-ATPase activities were studied in fresh brain membrane preparations from adult epileptic (El) mice and nonepileptic C57BL/6J (B6) mice. The El mice have an inherited type of temporal lobe epilepsy. No significant differences were observed between the El and B6 mice for any of the ATPase activities in the hippocampus, brain stem, or cerebellum. These findings indicate that seizure susceptibility in El mice is not associated with differences in the activities of these cationic ATPases and that seizure susceptibility in El mice and audiogenic DBA/2 mice may involve different biochemical mechanisms.     

Voltage-Sensitive Calcium Channel Development in Epileptic DBA/2J Mice Suggests Altered Presynaptic Function

Summary: Aberrant synapse formation has been implicated in development and propagation of epileptic potential. Litzinger et al. (1993a) showed that ω-GVIA conotoxin may be used as a marker for synapse formation in nonepileptic mice. We conducted ω-GVIA binding in synaptosomal preparations from epileptic DBA/2J mice at different developmental ages. Binding in DBA/2J mice was compared with ω-GVIA binding in synaptosomal preparations from nonepileptic C57/B1, Swiss Webster, and AJ mice. Striking differences between these strains of mice are evident in the developmental sequence and pattern of N-type voltage-sensitive calcium channels (VSCC). In contrast to nonepileptic mice, the DBA/2J mice show a slow increase in ω-GVIA binding between postnatal days 2 and 8. This increase corresponds to onset of susceptibility to seizure in this strain. In addition to the difference in developmental sequence, DBA/2J mice have fewer binding sites for ω-GVIA throughout development, suggesting changes in channel structure or number. These data show that in DBAI2J mice development of the VSCC in brain is different from that in nonepileptic mice. This difference in development in presynaptic membranes responsible for neurotransmitter release may represent a change in synaptic activity that plays a role in epileptogenesis.     

Audiogenic seizure proneness requires the contribution of two susceptibility loci in mice

Juvenile mice of the DBA/2J strain undergo generalised seizures when exposed to a high-intensity auditory stimulus. Genetic analysis identified three different loci underlying this audiogenic seizure proneness (ASP)—Asp1, Asp2 and Asp3 on chromosomes 12, 4 and 7, respectively. Asp1 is thought to have the strongest influence, and mice with only Asp1 derived from the DBA/2J strain are reported to exhibit ASP. The aim of this study was to characterise more accurately the contributions of the Asp1 and Asp3 loci in ASP using congenic strains. Each congenic strain contains a DBA/2J-derived interval encompassing either Asp1 or Asp3 on a C57BL/6J genetic background. A double congenic C57BL/6J strain containing both Asp loci derived from DBA/2J was also generated. Here, we report that DBA/2J alleles at both of these Asp loci are required to confer ASP because congenic C57BL/6 mice harbouring DBA/2J alleles at only Asp1 or Asp3 do not exhibit ASP, whereas DBA/2J alleles at both loci resulted in increased susceptibility for audiogenic seizure in double congenic C57BL/6 mice.     

Subcellular distribution of carbonic anhydrase and Na+, K+- and HCO3--ATPases in brains of DBA and C57 mice

DBA/2J mice are susceptible to audiogenic seizures (ASs) in an age-dependent manner, susceptibility being maximal at 21 days of age and declining thereafter. DBA, as compared with AS-resistant C57BL/6J (C57) mice, had higher carbonic anhydrase (CA) activity in cerebral cortex, brainstem, and cerebellum homogenates at 21 days of age. CA activity was also increased in cytosolic (82%), microsomal (167%), and myelin (68%) subcellular fractions from cerebral cortex, and in cytosolic (51%) and mitochondrial (102%) fractions from brainstem of DBA mice at 21 days of age. In addition, DBA mice had a higher Na+, K+-ATPase activity in myelin from cerebral cortex, and a lower HCO3--ATPase activity in mitochondria from brainstem. The differences in CA activity in the cerebral cortex and in HCO3--ATPase were not present at 110 days of age, when DBA mice are no longer susceptible to ASs. Because CA and HCO3--ATPase are involved in maintaining a proper ionic environment for neuronal function, these data suggest that alterations in activity of these enzymes are related to the age-dependent changes in AS susceptibility in DBA mice.     

Audiogenic seizures and brain serotonin after l-tryptophan and p-chlorophenylalanine

Despite intense investigation, the role of serotonergic neurons in audiogenic seizures in mice remains uncertain. In the work reported here, audiogenic seizure susceptibility and brain tryptophan and serotonin concentrations were measured in DBA/2J mice after administration of three doses of L-tryptophan or p-chlorophenylalanine. p-Chlorophenylalanine reduced brain serotonin and significantly prolonged the latency to appearance of all seizure phases. L-tryptophan was largely ineffective in protecting against seizures and in elevating brain serotonin content, despite the fact that it caused a marked increase in brain tryptophan content. Thus, it appears that DBA/2J mice have an impaired ability to synthesize serotonin from tryptophan.     

Effect of Milacemide on Audiogenic Seizures and Cortical (Na+, K+)-ATPase of DBA/2J Mice

Milacemide (MLM, CP 1552 S, 2-N-pentylaminoacetamide), a glycinamide derivative, is currently being evaluated clinically for antiepileptic activity. Anticonvulsant properties have been shown in various animal models, but the mechanism of action of MLM is unclear. We studied its activity in audiogenic seizures of DBA/2J mice. MLM was effective in inhibiting the convulsions induced by sound with a biphasic dose-effect relation. The ED50 was 109 mg/kg orally against tonic extension. Higher doses were necessary to abolish clonic convulsion and running response. Because impaired cerebral (Na+, K+)-ATPase activity is supposed to play a role in epileptogenesis, we tested MLM on in vitro cortical enzymatic activity of DBA/2J mice. Basal (Na+, K+)-ATPase activity was unchanged by several concentrations of MLM in normal C57BL/6J and audiogenic DBA/2J mice. K+ activation (from 3 to 18 mM) of (Na+, K+)-ATPase is abolished in DBA/2J mice as compared with C57BL/6J mice, suggesting impaired glial (Na+, K+)-ATPase. In the presence of MLM (from 30 to 1000 mg/L), cortical (Na+, K+)-ATPase of DBA/2J mice is activated by high concentrations of K+, as in C57BL/6J mice. Results suggest that the antiepileptic activity of MLM in audiogenic mice may be secondary to an activation of a deficient glial (Na+, K+)-ATPase.     

Transcranial imaging of audiogenic epileptic foci in the cortex of DBA/2J mice

Exposure to intense sound stimuli induces audiogenic seizures in DBA/2J mice. We investigated cortical activities during sound stimulation using flavoprotein fluorescence imaging. Most DBA/2J mice had seizures during intense sound stimulation, with more than half surviving after seizures. Surviving mice were anesthetized with urethane (1.6 g/kg, intraperitoneal), and the skull was exposed and then covered with clear resin. More than 3 days after surgery, the mice were lightly anesthetized with urethane (0.8 g/kg) and cortical activities during intense sound stimulation were visualized. Focal responses appeared near the somatosensory cortex together with spike activities localized in the response area. These findings indicate that epileptic foci of audiogenic seizure are formed in the cortex of DBA/2J mice.     

Differences between seizure-prone and non-seizure-prone mice with regard to glutamate and GABA receptor binding in the hippocampus and other regions of the brain

Quisqualate-preferring glutamate receptors were determined in membranes from frontal cortex, occipital cortex, hippocampus and cerebellum, from seizure-prone DBA/2J BOM and seizure-resistant C57/BL mice. The animals were studied 21, 27 and 40 days postnatally, i.e., before, during and after the age at which DBA mice are most susceptible to seizures. Radio-binding assays were performed using [3H]AMPA in the presence of 100 nM glutamate. Except for the occipital cortex, where no significant differences between the two strains were observed, all areas of the brain of DBA mice exhibited significantly (P less than 0.001, t test) higher AMPA binding than the corresponding areas of C57/BL mice at 27 days of age. At pre- and post-susceptible ages, the two strains showed no significant differences in the hippocampus and occipital cortex. A significant difference was observed, however, in the frontal cortex and cerebellum at the ages of 21 and 40 days, respectively, although this difference was considerably less than at 27 days. In addition to determination of glutamate receptors, GABA-receptor binding was also studied in membranes from the same cerebral areas and at the above-mentioned ages. Binding characteristics, using [3H]GABA as the ligand, were essentially identical in the two strains at all ages investigated, i.e., both low and high affinity GABA receptors could be identified with KD values of 6-16 nM and 100-800 nM, respectively.     

Development of muscarinic cholinergic receptors in inbred strains of mice: Identification of receptor heterogeneity and relation to audiogenic seizure susceptibility

The concentrations and biochemical properties of muscarinic acetylcholine receptors in the brains of two highly inbred strains of mice, DBA/2J and C57BL/6J, have been studied using [³H]quinuclidinyl benzilate (QNB), a potent and specific receptor antagonist. As is the case with rat brain, murine muscarinic receptors exist in at least two forms, which differ in their affinities for receptor agonists but which have the same high affinity for receptor antagonists. Carbamylcholine binding to mouse neural membranes can be resolved into two components with K_Ds of 5.2 × 10⁻⁷ and 7.9 × 10⁻⁵ M. There is a regional heterogeneity of brain receptors with respect to their distribution between these high and low agonist affinity forms. Brain stem and hypothalamus receptors display binding properties that would be expected if over 60% of their receptors were in the high affinity state, while only 30–40% of cortex, striatum and thalamus receptors appear to be in the high affinity form. Hippocampal receptors display the least amount of high agonist affinity character.Saturation curves and Scatchard plots of QNB binding at 2, 14, 21 and 42 days postnatal age in both strains indicate no differences or changes in the affinity or nature of the binding with age. Significant increases in QNB binding per mg membrane protein were observed between 14 and 42 days in the cortex, hippocampus, striatum, thalamus and hypothalamus, but not in the midbrain-pons-medulla region. In the hippocampus the DBA mice had significantly more QNB bindin. In the hypothalamus decreases with age in total binding were noted in DBA, while slight increases were noted in C57. Compared to C57, hippocampal receptors in DBA displayed lower agonist affinity at 14 and 21 days, a trait which was not apparent when DBA had outgrown their audiogenic seizure sensitivity at 42 days. The differences in receptor density and agonist state distribution between the two strains may be related to audiogenic seizure sensitivity.     

Excitability of auditory neurons in the dorsal and ventral cochlear nuclei of DBA2 and C57BL6 mice

Extracellular response properties were studied in neurons of the dorsal and ventral divisions of the cochlear nucleus (DCN and VCN, respectively) of $\text{DBA}\text{2}$ (DBA) and $\text{C57BL}\text{6}$ (C57) mice. Mice of the former inbred strain show susceptibility to audiogenic seizures and have severe high frequency hearing loss when young; mice of the latter strain do not. Whereas afterdischarges had been readily observed in the inferior colliculus of DBA mice in a previous study, they were never observed in the cochlear nucleus. The incidence of nonmonotonic intensity functions, the slopes of intensity functions, and the incidence of inhibition in response areas indicated that inhibition was diminished in the DCN of DBA mice. However, in the VCN, these response properties did not differ between the two strains. There appeared to be an “amplification” of excitability (i.e., attenuation of inhibition) from VCN to DCN to inferior colliculus in DBA mice.