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.     

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.     

Acute and Chronic Acetazolamide Administration in DBA and C57 Mice: Effects of Age

The clinical utility of the carbonic anhydrase (CA) inhibitor acetazolamide (ACTZ) is limited because of rapid development of tolerance to its effects. Tolerance is thought to develop as a result of glial cell proliferation and/or increased CA synthesis. DBA mice, susceptible to audiogenic seizures (AGSs) in an age-dependent manner, have increased CA activity as compared with C57 (non-audiogenic seizure susceptible) mice at 21 and 110 days of age. The present work utilized ACTZ to help determine the relationship between increased CA activity in brain and AGSs in DBA mice. Also, minimal electroshock seizure threshold (EST) was measured at various ages in DBA and C57 mice to determine age-related changes in CNS excitability. EST was significantly lower in DBA as compared with C57 mice at 18 days and between 40 and 115 days of age, suggesting that DBA mice remain hyperexcitable to electrical stimulation after they develop resistance to AGSs. ACTZ ED50s against maximal electroshock seizures (MES) were significantly higher in DBA as compared with C57 mice at 26,36, and 115 days of age. This finding correlates with higher CA activity in this strain at 110 days of age, noted previously. However, at 21 days of age, when CA activity is also higher in DBA versus C57 mice, there were no significant differences in ACTZ ED50s against MES between the strains. ACTZ ED50s against AGSs in DBA mice were considerably lower than ACTZ ED50s against MES in either strain, suggesting that a particular fraction of CA is intimately involved in the production of AGSs.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-HT2 receptor binding and 5-HT uptake in mouse brain: developmental changes and the relationship to audiogenic seizure susceptibility in DBA/2J mice

Abnormal function of serotonergic neurones may be involved in the age-related susceptibility of DBA/2J mice to generalised convulsions induced by auditory stimulation. We have measured 5-HT2 receptor binding sites and synaptosomal 5-HT uptake in 5 brain regions of DBA/2J mice at ages before, during and after their maximal susceptibility to audiogenic seizures and in age-matched C57 B1/6 mice, a strain resistant to audiogenic seizures at all ages. The number of 5-HT2 binding sites was 20% higher in the cerebral cortex of DBA/2J than C57 B1/6 mice at the time of maximal susceptibility of DBA/2J mice to audiogenic seizures but did not differ at other ages. The number of 5-HT2 binding sites did not differ between the two strains at the ages studied in forebrain, mid-brain, hippocampus and pons-medulla. A marked reduction in the number of 5-HT2 binding sites was apparent in the mid-brain, hippocampus and pons-medulla of both strains of mice between 13-15 days of age and 21-23 days of age. Synaptosomal 5-HT uptake did not differ significantly between DBA/2J and C57 B1/6 in any of the brain regions at the ages studied. The higher density of cortical 5-HT2 binding sites in DBA/2J mice may contribute to their susceptibility to sound-induced seizures.     

Effects of age and caloric intake on glutathione redox state in different brain regions of C57BL/6 and DBA/2 mice

The main purpose of the present study was to determine whether specific regions of the mouse brain exhibit different age-related changes in oxidative stress, as indicated by glutathione redox state and the level of protein-glutathionyl mixed disulfides. Comparison of 3- and 21-month-old mice indicated an age-related decrease in the ratio of reduced to oxidized glutathione (GSH/GSSG) as well as a pro-oxidizing shift in the calculated redox potential (ranging from 6 to 15 mV) in the cortex, hippocampus, striatum and cerebellum, whereas there was little change in the brainstem. This pro-oxidizing shift in redox state was due to a modest decrease in GSH content occurring in all the brain regions examined, and elevations in GSSG amount that were most pronounced in the striatum and cerebellum. The regional changes in glutathione redox state were paralleled by increases in the amounts of protein-mixed disulfides. A reduction of caloric intake by 40% for a short period (7 weeks), implemented in relatively old mice (17 months), increased the GSH/GSSG ratio and redox potential at 19 months in the same brain regions that exhibited age-related decreases. The effects of age and caloric restriction were qualitatively similar in C57BL/6 and DBA/2 mice. However, young DBA/2 mice, which do not show extension of life span in response to long-term caloric restriction, had lower GSH/GSSG ratios and higher protein-mixed disulfides than age-matched C57BL/6 mice. The current findings demonstrate that oxidative stress, as reflected by glutathione redox state, increases in the aging brain in regions linked to age-associated losses of function and neurodegenerative diseases.     

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.     

Genetic analysis of serum thyroxine content and audiogenic seizures in recombinant inbred and congenic strains of mice

We previously proposed that the audiogenic seizure (AGS) susceptibility of 21 +/- 1-day-old DBA/2 (D2) mice may result from an early postnatal elevation in serum thyroxine (T4) concentration. In the present study we used seven C57 X DBA (BXD) recombinant inbred strains and the D2.B6-Iasb congenic strain to study the association between serum T4 content and susceptibility to AGS. The D2.B6-Iasb congenic mice are genetically similar to the D2 mice except for the Iasb gene, which inhibits AGS susceptibility. The total and estimated free serum T4 concentrations in these strains at 14 +/- 1 days of age were compared with the previously determined AGS susceptibilities of these strains at 21 +/- 1 days of age. We found no significant correlations between serum T4 concentration and AGS susceptibility in these strains. It is unlikely, therefore, that inherited differences in neonatal serum T4 content are directly responsible for differences in susceptibility to AGS in 21 +/- 1-day-old mice. The mechanisms by which the experimental manipulation of serum T4 content influences AGS susceptibility are discussed.     

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.     

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.     

Differences in basal levels of CREB and NPY in nucleus accumbens regions between C57BL/6 and DBA/2 mice differing in inborn alcohol drinking behavior

An increasing body of evidence suggests that genetic factors play a role in alcohol drinking behaviors. C57BL/6J (C57) mice innately consume larger amounts of alcohol compared to that consumed by DBA/2J (DBA) mice. Furthermore, alterations in cAMP-responsive element binding (CREB) protein function in the brain have been implicated in alcohol drinking behaviors. The present investigation examined innate expression and phosphorylation of CREB in various brain structures of C57 and DBA mice. It was found that CREB expression and phosphorylation was lower, specifically in the shell structure of the nucleus accumbens, in C57 mice compared to that in DBA mice. CREB expression and phosphorylation were similar in other brain regions such as the nucleus accumbens core and the cortical, amygdaloid, hippocampal, and striatal structures of C57 and DBA mice. The expression of a cAMP-inducible gene, neuropeptide Y (NPY), was also investigated in the nucleus accumbens region of C57 and DBA mice. It was found that in C57 mice, NPY protein levels were lower in the shell but not in the core structure of the nucleus accumbens compared to that in DBA mice. It was also found that C57 mice are not innately anxious, but they consume larger amounts of alcohol than do DBA mice. Because the shell structure of the nucleus accumbens has been implicated in reward mechanisms of alcohol, it is possible that lower CREB function in this brain structure may be in part associated with the excessive alcohol drinking behavior of C57 mice.     

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.     

Strain differences in the development of susceptibility to audiogenic seizures after acoustic priming but not after hearing loss

Either exposure to an initial auditory stimulus (IAS) or external ear plugging (EEP) was used to produce susceptibility to audiogenic seizures in C57BL/6Bg and DBA/1Bg mice. After the IAS, increments in seizure susceptibility occurred by 5 h in C57BL/6Bg mice and by 24 h in DBA/1Bg-ras mice, whereas after EEP, increments in seizure susceptibility occurred by 48 h in C57BL/6Bg and by 24 h in DBA/1Bg-ras mice. Because both the IAS and EEP produce hearing loss, the strain differences in the effect of the IAS on the development of susceptibility and the strain similarities in the effect of the EEP on the development of susceptibility support the hypothesis that acoustic priming in the C57BL/6Bg at 19 days of age involves another mechanism in addition to that of hearing loss and disuse supersensitivity. It was suggested elsewhere that the other mechanism is mediated by a post-IAS decrease in the concentration of brain γ-aminobutyric acid and requires brain protein synthesis for a brief period post-IAS.     

N(2)O stimulates NOS enzyme activity in C57BL/6 but not DBA/2 mice

Exposure to 70% N(2)O produces a prominent antinociception in C57BL/6 mice but not DBA/2 mice. N(2)O exposure also increases conversion of [14C]L-arginine to [14C]L-citrulline in homogenates prepared from whole brains of C57BL/6 mice; there was no such increase in NOS activity in the DBA/2 whole brain. A differential N(2)O effect on brain NOS in these inbred strains might explain why the C57BL/6 but not DBA/2 mice are responsive to N(2)O antinociception.     

Neurospecific S-100 protein content in brains of different mouse strains

Total whole brain concentrations of S-100 protein and of its water-soluble fraction were determined in 11 inbred mouse straine: DBA/2J, AKR/J, CBA/Lac, C57BL/6J, C57BL/6J-Ay, C3H/He, C3H/f, DD, A/He, BALB/cLac, CC57BR/Mv, and in cerebral cortex, cerebellum and hippocampus in DBA/2J, AKR/J and CBA/Lac strains. Highly significant differences in the concentrations of the water-soluble S-100 protein were found between some strains. Slight differences were found in total S-100 protein content in whole brains between the strains (0.01 less that P less than 0.05). The DBA/2J mice had the highest brain S-100 protein content, and were characterized by a higher learning rate in shuttle-box as compared to CBA/Lac and AKR/J mice, who had a low content of this neurospecific protein.     

Differential prostaglandin formation induced by convulsions in the brain of mice susceptible (DBA/2J) and resistant (CFLP) to acoustic stimulation

Endogenous cerebral prostanoids possess anticonvulsant properties. This study investigates possible age-dependent anomalies of prostanoid synthesis in the brain of seizure-prone DBA/2J (DBA2) mice as compared to sound stimulus-resistant CFLP mice. Irrespective of the age of the animals, a large increase of prostaglandin (PG) D2 and E2 in the brain of CFLP mice was observed in response to pentylenetetrazol (PTZ)-, or electroshock (ES)-induced seizures. Significantly less PGD2 and PGE2 was formed in the brain of DBA2 mice at day 21 after birth when subjected to PTZ or ES convulsions. At 42 days of age, however, this deficit of cerebral PGD2 synthesis in DBA2 mice disappeared concomitantly with the age-related decrease in audiogenic seizure (AS) susceptibility, whereas the deficit of PGE2 formation persisted. These results suggest that a deficiency of cerebral PGD2 synthesis may be one of the factors responsible for the AS susceptibility of the DBA2 mice. In contrast to PTZ or ES convulsions, acoustically induced seizures of the DBA2 mice were not accompanied by cerebral prostanoid synthesis. This supports the view that the pathways involved in AS are different from those involved in PTZ or ES models of epilepsy.     

Total RNA content of brain cells of inbred mice reared under different conditions.

AB/Jena, DBA/2Jena, C57B1/6 Jena inbred mice and ABD2F1- and B6D2F4-hybrid mice either were reared communally and weaned at day 30 or were reared by isolated mothers and weaned at day 21 (I21). On day 50 the total RNA content of pyramidal brain cells of male offspring was determined by cytophotometry. The studied brain areas were the frontal pole of cerebral cortex (FCC) and the CA1 region of the dorsal hippocampus (DHI). The DBA, C57 and ABD2 mice had a reduced FCC and DHI RNA-content under I21-rearing. Under these conditions the AB strain has the highest RNA-content in both substrates. The results call for the awareness of possible genetic influences when studying the effects of environmental enrichment/impoverishing.     

Social conflict-induced changes in nociception and β-endorphin-like immunoreactivity in pituitary and discrete brain areas of C57BL/6 and DBA/2 mice

The present study characterizes the time course of social conflict analgesia and its reversibility by opioid antagonist drugs in the C57BL/6 and DBA/2 inbred strains of mice and examines the relationship between alterations in brain and pituitary levels of β-endorphin-like immunoreactivity (β-ELIR) and the antinociception elicited by social stress. Data revealed statistically strain differences in regard to β-ELIR in control animals. The pituitary content of β-ELIR was higher in DBA/2, while the values in the periaqueductal grey (PAG) and in the amygdala were higher in C57BL/6 mice. No interstrain differences were found in the hypothalamus. Exposure to 50 attack bites resulted in a 6-fold higher analgesia in DBA/2 mice and in a strain-dependent fall of ELIR in pituitary (27%) and PAG (23%). PAG but not pituitary β-ELIR levels in C57BL/6 mice correlated positively with the increase in tail-flick latency after attack. Mere confrontation with a non-aggressive opponent failed to induce analgesia and was associated in C57BL/6 mice with a significant reduction in the β-ELIR content of both the pituitary and the PAG. The data are discussed in terms of genotype-dependent sensitivity of the β-endorphin system to stress and its relation to analgesia.     

Physical activity enhances spatial learning performance with an associated alteration in hippocampal protein kinase C activity in C57BL/6 and DBA/2 mice

The effects of physical activity on spatial learning performance and associated hippocampal functioning were examined in C57BL/6Ibg (C57) and DBA/2Ibg (DBA) mice. C57 and DBA mice, 3 months of age, were subjected to 8 weeks of a physical activity regime (consisting of moderate-pace treadmill running 5 days/week, 60 min/day, 0% grade, 12 m/min) or remained sedentary in their cages. Mice were then tested on the Morris water maze task for 6 days followed by 12 days of testing on the place learning-set task (8 trials/day with each task). Both C57 and DBA run mice showed no difference in swim speed compared to controls. Hippocampal protein kinase C (PKC) activity was measured in cytosolic, loosely bound, and membrane-bound homogenate fractions. Mice subjected to the physical activity protocol were compared to sedentary controls from the same set of litters. Physical activity produced a 2- to 12-fold enhancement in spatial learning performance on both the Morris (P < 0.0001) and place learning-set (P < 0.02) probe trials in both C57 and DBA mice. DBA mice, which characteristically perform poorly in comparison to C57 mice, were enhanced to perform similarly to C57 control mice. This physical activity-induced enhancement in spatial learning performance was accompanied by alterations in hippocampal bound PKC activity (P < 0.05). These data provide further support for our previous hypothesis of a PKC activity involvement in spatial learning and enhancement of spatial learning performance in rodents by physical activity. In addition, these data indicate that hippocampal PKC activity may be involved in the physical activity-induced enhancement of spatial learning performance.     

The GABAA receptor γ1-subunit in seizure prone (DBA/2) and resistant (C57BL/6) mice

γ-Aminobutyric acid (GABA)_A receptors are the sites of action for many antiepileptic drugs such as benzodiazepines and barbiturates. We report the results of molecular cloning of the γ₁-subunit from seizure prone DBA/2J and resistant C57BL/6J inbred mice, and analyses of nucleotide sequences and expression of the γ₁-subunit messenger RNA (mRNA) in DBA/2 and C57BL/6 inbred mice. The mouse γ₁-subunit complementary DNA (cDNA) shares 98% similarity with that of the rat at the level of amino acid sequence. Northern blot hybridization indicates that the γ₁-subunit mRNA is expressed predominantly in areas other than the cerebral cortex and cerebellum and shows little change with postnatal development. No differences have been found for the subunit between DBA/2 and C57BL/6 mice either for nucleotide sequence or for level of expression of the subunit’s mRNA in whole brain by Northern blots at 3 weeks of age.     

Basal levels and alcohol-induced changes in nociceptin/orphanin FQ, dynorphin, and enkephalin levels in C57BL/6J mice

In order to investigate the involvement of the opioid and nociceptin/orphanin FQ (N/OFQ) system in alcohol drinking behaviour, N/OFQ and the opioid peptides dynorphin B (DYNB) and Met-enkephalin-Arg(6) Phe(7) (MEAP) were examined in the alcohol-preferring C57BL/6J mice. Basal peptide levels were compared in the brain and the pituitary gland with basal levels in the alcohol-avoiding DBA/2J mice. Furthermore, the effects of chronic alcohol self-administration on peptides were studied in the C57BL/6J mice. Compared to the DBA/2J mice, C57BL/6J mice had low immunoreactive (ir) levels of DYNB and MEAP in the nucleus accumbens, the hippocampus, and the substantia nigra, low ir-DYNB levels in the striatum and low ir-MEAP levels in the frontal cortex. Higher ir-DYNB levels in the pituitary gland and in the periaqueductal gray (PAG) and higher ir-N/OFQ levels in the frontal cortex and in the hippocampus were detected in C57BL/6J mice compared to the DBA/2J mice. After 4 weeks of voluntary alcohol consumption, only minor changes in steady-state peptide levels were identified. However, 5 days after the alcohol-drinking period, lower levels of all peptides were detected in the ventral tegmental area and ir-DYNB levels were also lower in the amygdala and in the substantia nigra. Twenty-one days after cessation of alcohol self-administration, the opioid peptides in alcohol-consuming C57BL/6J mice were lower in the PAG, the N/OFQ was lower in the frontal cortex and DYNB was higher in the amygdala and substantia nigra as compared to control C57BL/6J mice. This study demonstrates strain differences between C57BL/6J mice and DBA/2J mice that could contribute to divergent drug-taking behaviour, and it also demonstrates time- and structure-specific changes in neuropeptide levels after alcohol self-administration in the C57BL/6J mice.