The Ca2+/calmodulin signaling system in the neural response to excitability. Involvement of neuronal and glial cells.

Ca2+ plays a critical role in the normal function of the central nervous system. However, it can also be involved in the development of different neuropathological and neurotoxicological processes. The processing of a Ca2+ signal requires its union with specific intracellular proteins. Calmodulin is a major Ca(2+)-binding protein in the brain, where it modulates numerous Ca(2+)-dependent enzymes and participates in relevant cellular functions. Among the different calmodulin-binding proteins, the Ca2+/calmodulin-dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. We present an overview on different works aimed at the study of the Ca2+/calmodulin signalling system in the neural response to convulsant agents. Ca2+ and calmodulin antagonists inhibit the seizures induced by different convulsant agents, showing that the Ca2+/calmodulin signalling system plays a role in the development of the seizures induced by these agents. Processes occurring in association with seizures, such as activation of c-fos, are not always sensitive to calmodulin, but depend on the convulsant agent considered. We characterized the pattern of expression of the three calmodulin genes in the brain of control mice and detected alterations in specific areas after inducing seizures. The results obtained are in favour of a differential regulation of these genes. We also observed alterations in the expression of the Ca2+/calmodulin-dependent protein kinase II and calcineurin after inducing seizures. In addition, we found that reactive microglial cells increase the expression of calmodulin and Ca2+/calmodulin-dependent protein kinase II in the brain after seizures.

Effects of epidural anaesthesia-analgesia on intravenous anaesthesia with propofol.

The main purpose of this study was to demonstrate that the use of epidural anaesthesia-analgesia reduces the amount of propofol necessary to maintain surgical anaesthesia in dogs during ovariohysterectomy. The study was carried out on 28 bitches undergoing ovariohysterectomy with general anaesthesia using an intravenous infusion of propofol. Dogs were allocated to one of two groups. Group 1 received systemic buprenorphine together with epidural analgesia using lidocaine and buprenophine, and in Group 2 systemic buprenorphine only was given. The mean propofol infusion rate was significantly lower in dogs with epidural analgesia (P < 0.0001). In addition, the mean endotracheal extubation time was significantly longer in dogs with epidural analgesia (P < 0.0001). No significant differences were detected in any of the physiological parameters measured.

The Ca2+/calmodulin system in neuronal hyperexcitability.

Calmodulin (CaM) is a major Ca2+-binding protein in the brain, where it plays an important role in the neuronal response to changes in the intracellular Ca2+ concentration. Calmodulin modulates numerous Ca2+-dependent enzymes and participates in relevant cellular functions. Among the different CaM-binding proteins, the Ca2+/CaM dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. Therefore, the role of the Ca2+/CaM signalling system in different neurotoxicological or neuropathological conditions associated to alterations in the intracellular Ca2+ concentration is a subject of interest. We here report different evidences showing the involvement of CaM and the CaM-binding proteins above mentioned in situations of neuronal hyperexcitability induced by convulsant agents. Signal transduction pathways mediated by specific CaM binding proteins warrant future study as potential targets in the development of new drugs to inhibit convulsant responses or to prevent or attenuate the alterations in neuronal function associated to the deleterious increases in the intracellular Ca2+ levels described in different pathological situations.

Regional concentrations of GABA, serotonin and noradrenaline in brain at onset of seizures induced by lindane (gamma-hexachlorocyclohexane).

The main objective of this study was to determine the modifications induced by the pesticide lindane (gamma-hexachlorocyclohexane) in the regional concentration of neurotransmitters in brain, taking the tonic-clonic seizure as the main sign of its neurotoxic action. The animals were given lindane (150 mg/kg p.o. in olive oil) and killed at the onset of the first seizure (mean latency time: 18.3 +/- 5.5 min, n = 16). The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and the biogenic amines noradrenaline (NA) and serotonin (5-HT), as well as its corresponding acidic metabolite, were determined by high performance liquid chromatography with fluorimetric or electrochemical detection in different areas of the brain: hippocampus, mesencephalon, colliculi, frontal cortex, parietal cortex, striatum and thalamus. The concentration of GABA in whole tissue was only significantly decreased in the colliculi. The concentration of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were decreased in the colliculi, striatum and frontal cortex, suggesting a decreased synthesis and release of 5-HT, but increased in the parietal cortex. The concentration of NA was significantly decreased in the parietal cortex. Thus, the results indicate that lindane induces some modifications of the concentration of neurotransmitters in cortical structures, basal nuclei, midbrain and colliculi. These changes may be consequent upon the proposed interaction of lindane with the GABAergic system, although a direct action of lindane on other neurotransmitter systems cannot be excluded.

Differential response of calmodulin genes in the mouse brain after systemic kainate administration.

In the central nervous system, many of the effects resulting from an increase in the intracellular levels of calcium are mediated by calmodulin, a major calcium-binding protein in the mammalian brain. Calmodulin is expressed by three different genes, namely CaM I, CaM II and CaM III, all of which encode an identical protein. We studied the expression of calmodulin in the mouse brain at different times after the administration of a convulsant dose of kainate, a potent neuroexcitotoxic agent. We detected the presence of the different calmodulin messenger RNAs and of the protein itself in brain sections by in situ hybridization histochemistry and immunocytochemistry respectively. In addition, we determined the calmodulin content in brain regions by radioimmunoassay. Kainate-treated animals did not show areas of neuronal death at the different times following administration considered. An increase in the hybridization signal for CaM I messenger RNAs was observed from 5 h after kainate administration in the different brain regions tested. In contrast, the CaM II messenger RNA signal decreased gradually to a minimum 24 h after treatment in the hippocampus, while the CaM III messenger RNA signal was mostly unaffected. Calmodulin immunoreactivity also increased in the hippocampus. Nevertheless, we did not detect any significant difference in calmodulin content between brain regions of control and treated animals by radioimmunoassay. Kainate treatment induced modifications in the expression of calmodulin at the level of both messenger RNAs and protein. The results suggest a differential regulation of the three calmodulin genes in the adult mouse brain and a post-transcriptional or a post-translational regulation of calmodulin expression.

Calmodulin is expressed by reactive microglia in the hippocampus of kainic acid-treated mice.

Calmodulin is a calcium-binding protein that is highly abundant in the brain, where it is involved in many essential functions. The protein is mainly expressed by neuronal cells. Calmodulin is encoded by three different genes in mammals, all of them producing an identical protein. Alterations in the expression of either calmodulin genes or protein have been reported in the rodent brain by several authors in different experimental situations. However, no mention has been made to date of possible alterations in calmodulin expression in glial cells in response to certain stimuli. In the present study, we found an increase in the expression of calmodulin in reactive microglial cells in the mouse hippocampus 24 h after an intraperitoneal administration of a convulsant dose of kainic acid. The results show that a high expression of calmodulin can be added to the list of changes described to occur in microglial cells when they become reactive microglia in response to certain kinds of stimuli, in contrast to the non-detectable level of expression of this protein observed in the resting microglial cells. It is difficult to explain such an increase due to the great number of processes in which calmodulin is involved, but the great level of calmodulin observed in the reactive microglial cells shows that calmodulin immunolabelling can be used to reveal these kinds of cells.

Comparative study of the pattern of expression of calmodulin messenger RNAs in the mouse brain.

Calmodulin is a major calcium-binding protein in the mammalian brain, playing an important role in neuronal cell function. Its amino acid sequence is highly conserved and the protein is encoded by multiple genes. In the mouse brain, as well as in the rat and the human brain, three different genes have been detected for calmodulin, CaM I, CaM II and CaM III, all of which encode an identical protein. We studied the pattern of expression of the three calmodulin genes and the pattern of calmodulin distribution in the mouse brain by in situ hybridization histochemistry and immunohistochemistry. We found that calmodulin messenger RNAs from the three calmodulin genes were widely expressed in the mouse brain. Nevertheless, there were differences in their patterns of distribution. In general, all calmodulin messenger RNAs were preferentially distributed in hippocampus, cerebral cortex and cerebellar cortex, and CaM II messenger RNA also in caudate-putamen. However, all messenger RNAs showed clearly differentiated patterns of distribution in the hippocampus and the cerebellar cortex. Calmodulin immunoreactivity was present in all cells so far examined. Immunostaining was observed both in the cell nucleus, where it was especially strong, and in the cytoplasm. Our results suggest that the three calmodulin genes are differentially regulated in the mouse brain and also that, although all calmodulin genes have a basal expression, precise regulation of calmodulin levels might be attained through the different contribution of the three calmodulin genes.

Effect of hexachlorocyclohexane isomers on calmodulin mRNA expression in the central nervous system.

Three different calmodulin genes that encode the same protein have been found in the brain of all mammalian species so far examined. Little is known about the factors involved in regulating the expression of this gene family in the central nervous system. We have investigated the possibility of differential expression of two calmodulin genes, CaM I and CaM II, which are expressed strongly in neuronal cells in the adult rat brain, after treatment with the gamma (lindane) and the delta isomers of the hexachlorocyclohexane (HCH). In this study a decrease of CaM I mRNA (mainly in the 4.0 kb transcript) was found in the cortex of the rats after 24 h of isomer administration. CaM I expression seemed to be more sensitive to delta isomer action, whereas the gamma isomer acted mainly at CaM II level. The levels of mRNA of calmodulin CaM II gene were also found to decrease after lindane administration; delta-HCH produced an increase of this transcript. These results were obtained by Northern blot analysis and confirmed by means of in situ hybridization. Our results suggest that levels of neuronal calmodulin mRNA species are modified in response to changes in neuronal activity.

Effect of different convulsants on calmodulin levels and proto-oncogene c-fos expression in the central nervous system.

In the present study, a relationship between convulsant activity and two cellular events, changes in calmodulin (CaM) concentration and proto-oncogene c-fos expression has been considered. c-fos has been found activated after the administration of the organochlorine insecticide lindane, the Ca2+ channel agonist Bay K, and N-methyl-D-aspartate (NMDA). The administration of the voltage-dependent Ca2+ channel antagonist nifedipine was able to block the expression elicited by lindane. The effect of lindane on c-fos expression could not be blocked by prior administration of MK-801, a non-competitive antagonist of the NMDA receptor. These results suggest a possible role for the voltage-dependent Ca2+ channels in the mechanism of action of lindane. By means of in situ hybridization, the different patterns of c-fos expression after the administration of the mentioned compounds have been described. A possible modification of the levels of CaM has also been investigated. Among all the subcellular fractions considered, only levels of nuclear CaM appeared to be affected after the different treatments. The changes observed seemed to follow a similar pattern to that described for c-fos induction. Calcium entry through these voltage-dependent calcium channels would be the link between membrane depolarizing events and expression of c-fos and/or increase in nuclear CaM.

Anticonvulsant activity of delta-HCH, calcium channel blockers and calmodulin antagonists in seizures induced by lindane and other convulsant drugs.

The anticonvulsant activity of delta-HCH and of a calmodulin antagonist, W-7 were investigated on convulsions induced in mice by lindane (ED100 100 mg/kg), by GABAergic antagonists PTZ (ED100 60 mg/kg) and PTX(ED100 4 mg/kg), by calcium channel agonist BAY-K-8644 (ED100 5 mg/kg), by two agonists of excitatory amino acid receptors, kainic acid (ED100 80 mg/kg) and NMDA (ED100 160 mg/kg and by the atypical benzodiazepine Ro 5-4864 (ED100 40 mg/kg). The anticonvulsant activity of a voltage-dependent calcium channel antagonist, nifedipine was also investigated on convulsions induced by Ro 5-4864, BAY-K-8644, kainic acid and NMDA. delta-HCH antagonized lindane- and BAY-K-8644-induced convulsions (ED50 231 (172-309) mg/kg and 148 (142-154) mg/kg, respectively) and at concentrations up to 300 mg/kg failed to antagonize Ro 5-4864, kainic acid and NMDA convulsions. In contrast delta-HCH potentiated PTX-induced seizures. Nifedipine antagonized BAY-K-8644- and kainic acid-induced convulsions (ED50 6.5 (4.3-9.7) mg/kg and 30 (13-70) mg/kg but at concentrations up to 20 mg/kg failed to antagonize Ro 5-4864 and 25% of protection was observed on NMDA-induced convulsions at the highest dose (20 mg/kg). The ED50 of W-7 to antagonize convulsions induced by lindane and BAY-K-8644 were 12 (8-19) mg/kg and 49 (29-85) mg/kg, respectively. Some anticonvulsant effect was observed against PTZ and NMDA but without any dose-dependent anticonvulsant activity. W-7 did not protect against PTX and kainic acid convulsions and 30% of protection was observed against convulsions at the highest dose of W-7 (75 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Role of calmodulin in the differentiation/activation of microglial cells.

In the present work the role of calmodulin (CaM) in regulating lipopolysaccharide (LPS)-induced microglial activation and in the spontaneous microglial differentiation has been investigated. We used pure rat microglial cell cultures to examine the effects of W13, a specific inhibitor of CaM, on microglial activation produced by LPS and the effect of CaM inhibition on microglial proliferation induced by the macrophage colony-stimulating factor (M-CSF). Microglial morphological transformation, inducible nitric oxide synthase (iNOS) activity and proliferating cell nuclear antigen (PCNA) immunostaining were determinate. Results show that CaM does not participate in the microglial increase of iNOS produced by LPS. In contrast, it is involved in spontaneous microglial ramification and in the activation of proliferation from quiescence. Multiple second-messenger pathways are involved in the transduction of signals initiated by LPS. The study of these mechanisms may allow us to extend our knowledge of the signals controlling the expression of these mediators.

Lindane-induced convulsions in NMRI and OF1 mice: antagonism with (+)MK-801 and voltage-dependent calcium channel blockers.

The convulsant profile of lindane was investigated in OF1 and NMRI mice lines in relation to other convulsants acting at the GABAA and NMDA receptor complexes. Thus, a specific GABA-gated chloride channel blocker, PTX, a GABAA receptor antagonist, PTZ, and an excitatory amino acid receptor agonist, NMDA, were used. Antagonism of the convulsant effects of each of these drugs was investigated with (+)MK-801, a blocker of the NMDA-operated cation channel, and with nifedipine, a voltage-dependent calcium channel antagonist. While no differences in potency for PTX or PTZ to induce seizures were observed between OF1 and NMRI mice, lindane was approximately 80 and 90% more potent in its ability to induce seizures and lethality, respectively, in OF1 than in NMRI mice. Brain lindane concentrations at the moment of convulsion, measured after ED100 doses of lindane (400 and 200 mg/kg for NMRI and OF1 mice, respectively), did not differ between OF1 and NMRI mice, suggesting that the different potency of lindane between these mouse lines is a consequence of pharmacokinetic factors. Furthermore, (+)MK-801 antagonized seizures induced by either lindane, PTX or PTZ with similar potencies in both mouse lines. These results, coupled with the different pharmacokinetics of lindane in OF1 and NMRI mice, suggest that the distinct effects of lindane in these mice are not mediated by different activities at either NMDA or GABAA receptor complexes. Nonetheless, nifedipine antagonized lindane-induced seizures with a three-fold higher potency in NMRI than in OF1 mice. In contrast, nifedipine failed to antagonize PTX and PTZ convulsions in both OF1 and NMRI mice. These results suggest that besides the GABAA receptor complex other mechanisms related to calcium mobilization may be involved in the convulsant action of lindane.

Reserpine potentiates NMDA-induced c-fos mRNA expression in the mouse brain.

The systemic administration of a non-convulsant dose of N-methyl-D-aspartate (NMDA; 75 mg/kg i.p.), which was associated with motor activation, induced a regional c-fos mRNA expression in the mouse brain. The NMDA-induced c-fos mRNA expression was predominant in the dentate gyrus and in the medial mammillary nucleus and less pronounced in other hippocampal areas, cortical areas, bed nucleus of the stria terminalis and posterior amygdaloid nuclei. It is suggested that the hippocampus and/or the extended amygdala might be involved in the previously hypothesized dopamine-independent NMDA-mediated motor activation mechanism. No increase in c-fos mRNA expression was observed 21 h after reserpine treatment (5 mg/kg s.c.). However, reserpinization induced a significant potentiation of the NMDA-induced c-fos mRNA expression. These results show the existence of a strong and selective amine-dependent modulation of NMDA neurotransmission in the brain.

Convulsant effect of lindane and regional brain concentration of GABA and dopamine.

Lindane (gamma-hexachlorocyclohexane) is an organochlorine insecticide with known neurotoxic effects. Its mechanism of action is not well understood although it has been proposed that lindane acts as a non-competitive antagonist at the gamma-aminobutyric acid (GABA)-A receptor. We studied the effect of lindane (150 mg/kg) on the GABAergic and dopaminergic systems by measuring the concentration of GABA, dopamine and its metabolites in 7 brain areas at the onset of seizures. All animals suffered tonic convulsions at 18.3 +/- 1.4 min after lindane administration. The concentration of GABA was only slightly but significantly decreased in the colliculi without modifications in the other areas. The concentration of dopamine was increased in the mesencephalon and that of its metabolite DOPAC was also increased in the mesencephalon and the striatum.

Effect of lindane at repeated low doses.

The relationship between brain concentration of lindane and its convulsant effect have been studied in rats after repeated low doses of lindane. The mean brain plateau concentration was achieved in 5-8 days. The doses administered (5, 12 and 20 mg/kg) and the brain concentration of lindane at the plateau were highly correlated. The incidence rate of response (percentage of rats with tonic convulsions) was also highly correlated with the log of concentration of lindane in brain for all doses and days studied. A decrease in brain concentration of lindane was observed after 12 days of daily administration at doses of 5 and 12 mg/kg but not at the highest dose (20 mg/kg).

Anticonvulsant activity of calmodulin antagonist W-7 in convulsions induced by lindane and BayK-8644: effects in c-fos expression.

The anticonvulsant activity of calmodulin antagonist W-7, was investigated on convulsions induced in mice by the insecticide lindane and by the calcium channel agonist BayK-8644. We also studied the inhibitory effect of W-7 on on c-fos mRNA expression induced by both convulsants. We observed a good correlation between doses and the acute convulsive effects of lindane and BayK-8644. The incidence rate and time to onset were clearly dose-dependent. W-7 antagonized the convulsive effects of lindane and BayK-8644 in all the parameters studied. A significant decrease in the incidence rate and time to onset were observed when they are compared with the values obtained with the ED100 of lindane- and BayK-8644 induced seizures. Both were able to activate the mRNA expression of the proto-oncogene. The pattern of this expression displayed by in situ hybridization was very similar. A dramatic increase was found in dentate gyrus and high levels of mRNA expression also occurring in hippocampal fields and cortical regions. In accordance with the behavioural results, W-7 antagonized also the c-fos expression induced by lindane and BayK-8644. Our results suggest that lindane as BayK-8644 may activate voltage-dependent calcium channels leading to calmodulin activation.

Effect of gamma-hexachlorocyclohexane and its isomers on proto-oncogene c-fos expression in brain.

By means of in situ hybridization, the induction of proto-oncogene c-fos in rat brain after administration of several convulsants has been studied. The organochlorine insecticide gamma-hexachlorocyclohexane (lindane) has been shown to induce c-fos expression in a dose dependent manner. 30 mg/kg of lindane increased c-fos expression in cortical and hippocampal areas. The two non convulsant isomers of lindane, alpha- and delta-HCH, were not able to induce the expression of the proto-oncogene, but blocked that elicited by lindane. Pentylenetetrazole (PTZ) and picrotoxin (PTX), a known GABAA-receptor antagonist, have also been considered. Both of them were able to induce c-fos, although the pattern of expression was not the same in each case. alpha- and delta-HCH, were administered prior to the mentioned toxicants, affecting the proto-oncogene expression in different ways. We propose here that the distribution of c-fos mRNA after different treatments can be used as a marker of neurotoxic action.

Evidence for acute tolerance to the behavioral effects of lindane: concomitant changes in regional monoamine status.

The effects of the organochlorine insecticide lindane on plus-maze ((+)-maze) behavior of rats and on regional monoamine status were studied at two time points, 30 min and 24 hr post-dosing. Animals were given lindane, 20 mg/kg, 30 min before (L1/2 group), or 40 mg/kg, 24 hr before (L24 group), experimental time; these schedules allowed the study of animals with equivalent brain concentration of lindane at two different time points after administration. The (+)-maze results indicated a reduction in the number of entries into the arms of the maze 30 min after administration of lindane that was not present 24 hr later, suggesting the development of an acute tolerance to the behavioral effects of the chemical. In a parallel group of animals, concentrations of noradrenaline (NA), serotonin (5HT), 5-hydroxyindoleacetic acid (5HIAA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined by HPLC in seven brain regions. Significant decreases in NA concentrations were found in L1/2 animals in hippocampus and cerebral cortex and also in the L24 group in the latter region. 5HT/5HIAA ratios increased in several brain regions in the L24 rats but not in the L1/2 rats, thus showing an inverse relationship with behavioral effects. DOPAC/DA and HVA/DA ratios in hypothalamus and cerebral cortex were, by contrast, increased in both groups of treated animals. In conclusion, it appears that an acute tolerance can develop to the behavioral effects of lindane, and that there are modifications produced in central monoaminergic systems by lindane that show brain region and and treatment schedule specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Repeated lindane exposure in the rat results in changes in spontaneous motor activity at 2 weeks post-exposure.

Male Wistar rats were given 25 doses of lindane, 10 mg/kg per day, 1 ml/kg p.o. in olive oil, or control vehicle. Two weeks after the last dose, the animals were assessed for modifications in spontaneous motor activity, plus-maze behavior, shuttle-box active avoidance acquisition, brain regional concentrations of biogenic amines and metabolites, and regional [35S]TBPS binding. Rats treated with lindane showed an increase in spontaneous motor activity. Although no additional behavioral or neurochemical modifications were found, the changes in activity observed at 2 weeks post-exposure further demonstrate the need to assess for long lasting neurobehavioral sequelae of repeated lindane exposure.

Microcomputer adaptation of the wheel-shaped activity monitor: effects of lindane.

The development of microcomputers with increased power and memory capacity may allow for the spreading of the techniques of microanalysis of animal behavior in neurotoxicology. The present work describes the adaptation of the wheel-shaped activity monitor to a personal computer system (IBM-PC/XT/AT-compatible). The system has been used to study the effects elicited by a single 30 mg/kg dose of the organochlorine insecticide gamma-hexachlorocyclohexane (lindane) on the spontaneous behavior of rats. Lindane induced complex changes in behavior, the most prominent being a disruption of the temporal pattern of activity and changes in the local activity/locomotor activity ratios and in place preferences in the monitor. Effects on body weight and number of fecal boluses were also observed.