Anticonvulsant and neuroprotective effect of (S)-3,4-dicarboxyphenylglycine against seizures induced in immature rats by homocysteic acid.

The present study has examined the anticonvulsant and neuroprotective effect of (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG), a highly selective agonist for subtype 8 of group III metabotropic glutamate receptors (mGluRs), against seizures induced in immature 12-day-old rats by bilateral icv infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side). For biochemical analyses, rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45-50 min after infusion. Comparable time intervals were used for sacrificing the animals which had received (S)-3,4-DCPG (0.25 nmol/each side, 15-20 min prior to infusion of DL-HCA or saline). This agonist provided a pronounced anticonvulsant effect, generalized clonic-tonic seizures were completely suppressed and cortical energy metabolite changes which normally accompany these seizures were either normalized (decrease of glucose and glycogen) or markedly reduced (an accumulation of lactate). Anticonvulsant effect of (S)-3,4-DCPG was also evident from the EEG recordings, nevertheless, it was not complete. In spite of the absence of obvious motor phenomena, sporadic ictal activity could be seen in some animals. Isolated spikes could also be observed in some animals after administration of (S)-3,4-DCPG alone. The neuroprotective effect of (S)-3,4-DCPG was evaluated after 24 h and 6 days of survival following DL-HCA-induced seizures. Massive neuronal degeneration was observed in a number of brain regions following infusion of DL-HCA alone (seizure group), whereas pretreatment with (S)-3,4-DCPG provided substantial neuroprotection. The present findings suggest that receptor subtype 8 of group III mGluRs may be considered a promising target for drug therapy in childhood epilepsies in the future.

Synergistic effect of CART (cocaine- and amphetamine-regulated transcript) peptide and cholecystokinin on food intake regulation in lean mice.

BACKGROUND: CART (cocaine- and amphetamine-regulated transcript) peptide and cholecystokinin (CCK) are neuromodulators involved in feeding behavior. This study is based on previously found synergistic effect of leptin and CCK on food intake and our hypothesis on a co-operation of the CART peptide and CCK in food intake regulation and Fos activation in their common targets, the nucleus tractus solitarii of the brainstem (NTS), the paraventricular nucleus (PVN), and the dorsomedial nucleus (DMH) of the hypothalamus. RESULTS: In fasted C57BL/6 mice, the anorexigenic effect of CART(61-102) in the doses of 0.1 or 0.5 microg/mouse was significantly enhanced by low doses of CCK-8 of 0.4 or 4 microg/kg, while 1 mg/kg dose of CCK-A receptor antagonist devazepide blocked the effect of CART(61-102) on food intake. After simultaneous administration of 0.1 microg/mouse CART(61-102) and of 4 microg/kg of CCK-8, the number of Fos-positive neurons in NTS, PVN, and DMH was significantly higher than after administration of each particular peptide. Besides, CART(61-102) and CCK-8 showed an additive effect on inhibition of the locomotor activity of mice in an open field test. CONCLUSION: The synergistic and long-lasting effect of the CART peptide and CCK on food intake and their additive effect on Fos immunoreactivity in their common targets suggest a co-operative action of CART peptide and CCK which could be related to synergistic effect of leptin on CCK satiety.

Cocaine- and amphetamine-regulated transcript (CART) peptide specific binding in pheochromocytoma cells PC12.

CART (cocaine- and amphetamine-regulated transcript) peptides have been studied for ten years. We report specific binding of 125I-CART(61-102) to the rat adrenal pheochromocytoma PC12 cell line, both intact cells and cell membranes. Saturation binding to intact plated cells resulted in Kd of 0.48+/-0.16 nM and Bmax of 2228+/-529 binding sites/cell. 125I-CART(61-102) was also bound to PC12 cells differentiated using nerve growth factor to the neuronal phenotype with non-specific binding below 20%, and Kd of 1.90+/-0.27 nM and Bmax of 11,194+/-261 binding sites/cell. In competitive binding experiments, CART(61-102), CART(55-102) and di-iodinated CART(61-102) were bound to PC12 cell membranes with Ki in low nM range; their affinity to intact non-differentiated and differentiated cells was in low 10(-8) M range. In order to prove that iodination did not eliminate the pharmacological properties of CART, we tested the biological activity of di-iodinated CART(61-102). It decreased food intake in in vivo feeding experiment on fasted mice in a dose of 1 microg/mouse to the same extent as CART(61-102) in a dose of 0.5 microg/mouse.

Opposing changes of trimeric G protein levels during ontogenetic development of rat brain.

Developmental changes in the distribution of guanine nucleotide-binding regulatory proteins (G proteins) were investigated in the rat brain during postnatal development. Using a standard or high-resolution urea-SDS-PAGE and specific polyclonal antipeptide antibodies oriented against G(i)alpha1/G(i)alpha2, G(i)alpha3, G(s)alpha, G(o)alpha1/G(o)alpha2, G(q)alpha/G(11)alpha and Gbeta subunit, all these proteins were determined by quantitative immunoblotting in homogenates prepared from cortex, thalamus, hippocampus and pituitary of 1-, 7-, 12-, 18-, 25- and 90-day-old animals. The levels of the majority of G protein alpha subunits, namely G(i)alpha1, G(i)alpha2, G(i)alpha3, G(o)alpha1, G(o)alpha2, G(q)alpha, G(11)alpha and Gbeta, were high already at birth. Whereas the short variant of G(s)alpha, G(s)alphaS, rose sharply in all tested brain regions between postnatal day (PD) 1 and 90, the long variant of G(s)alpha, G(s)alphaL, was unchanged in cortex and thalamus and slightly increased in hippocampus. An increase was observed also in expression of G(i)alpha1/G(i)alpha2 and G(o)alpha1 protein, while G(o)alpha2 remained constant. Minority protein G(o)alpha* dramatically increased in cortex and thalamus, was unchanged in hippocampus and not detectable in pituitary. By contrast, the highest levels of G(i)alpha3 and G(q)alpha/G(11)alpha were detected as early as at PD 1. During the next 90 days, the immunological signal of G(i)alpha3 almost disappeared and G(q)alpha/G(11)alpha continuously declined to the levels corresponding to 50% of the levels determined at birth. Expression of Gbeta subunit was basically unchanged during postnatal development. Our present analysis indicates that G(s)alpha, G(i)alpha/G(o)alpha and G(q)alpha/G(11)alpha proteins are differently expressed in the course of brain development. Differential expression of the individual alpha subunits of trimeric G proteins during postnatal development suggests their different roles in maturation of the brain tissue.

Ontogenetic development of the G protein-mediated adenylyl cyclase signalling in rat brain.

Maturation of the brain adenylyl cyclase (AC) signalling system was investigated in the developing rat cortex, thalamus and hippocampus. Expression of AC type II, IV and VI measured by Western blot dramatically increased in all tested brain regions during the first 3 weeks after birth and these levels were maintained in adulthood. AC type I did not change during ontogenesis. In parallel, AC enzyme activities were determined in order to obtain the functional correlates to the preceding structural (immunoblot) analyses of trimeric G proteins [Ihnatovych et al., Dev. Brain Res. (2002) in press]. Surprisingly, basal, manganese-, fluoride-, forskolin- and GTP-stimulated adenylyl cyclase developed similarly. The relatively low enzyme activities, which were determined at birth, progressively increased (about four times) to a clear maximum around postnatal day PD 12. This was followed by a progressive regression to adulthood so that activity of AC at PD 90 was comparable with the low neonatal level. The peak of AC activities at PD 12 was detected in all tested brain regions. Stimulatory (isoproterenol) effect on basal AC activity as well as inhibitory (baclofen) effect on forskolin-stimulated AC activity were unchanged between PD 12 and PD 90. Thus, comparison of results of the structural and functional analyses of adenylyl cyclase signalling system revealed a clear dissociation between the increase in the amount protein of various AC isoforms and the decrease of total G-protein mediated enzyme activities between PD 12 and adulthood. As none of the complex changes in trimeric G protein levels can explain this difference, the future research has to be oriented to identification of potential negative regulators of AC in the course of brain development. Among these, the newly discovered group of GTPase activating proteins, RGS, appears to be of primary importance because these proteins represent potent negative regulators of any G protein-mediated signalling in brain.

Unequal development of thresholds for various phenomena induced by cortical stimulation in rats.

Electrical stimulation of sensorimotor cortical area was performed in 9-, 12-, 18-, 25-, 35- and 90-day-old rats with implanted electrodes to establish threshold intensities of currents necessary to elicit four different motor or EEG phenomena. Two different stimulation frequencies (8 and 50 Hz) were used. Development of thresholds for stimulation-bound movements, spike-and-wave afterdischarges and clonic seizures accompanying these afterdischarges was similar: the lowest threshold was found in 18-, respectively, in 18- and 25-day-old rats with the 8 and 50 Hz frequencies. Younger as well as older animals exhibited higher threshold intensities. The fourth phenomenon, transition into another, 'limbic' type of afterdischarges appeared only exceptionally in the youngest rats and its incidence increased whereas the threshold decreased with age. Higher frequency was more efficient in elicitation of limbic afterdischarges than the 8 Hz stimulation in rats aged 18 and more days. Our data represent a background for pharmacological studies and indicate the development of cortical excitability and of connections between the thalamocortical system and limbic structures.

Nonconvulsive Seizures Result in Behavioral but Not Electrophysiological Changes in Developing Rats.

It is not known if nonconvulsive seizures lead to functional or morphological changes in immature rats. Therefore we studied consequences of such seizures induced by kainic acid (KA) on Postnatal Day (PD) 12 (2 mg/kg ip). The animals were examined electrophysiologically (cortical epileptic afterdischarges (ADs) were elicited in rats with implanted electrodes on PD 14, 18 or 25) and behaviorally (open field was studied in another group of animals on PDs 18 and 25). Hippocampal and cortical morphology was checked by light microscopy (Nissl staining) on PDs 18 and/or 25. Another group of rats was injected with a 6 mg/kg dose of KA on PD 18 and examined on PD 25. The dose of KA used induced only nonconvulsive seizures characterized by automatisms (scratching on PD 12, wet dog shakes on PD 18). Cortical ADs in animals stimulated on PD 14, 18, or 25 did not differ from those in control rats. KA-Treated rats exposed to open field two times (on PDs 18 and 25) exhibited more exploratory activities during the second exposure than control animals. A similar difference was noted in PD 25 rats injected with KA on PD 18. Qualitative histology did not reveal any obvious neuronal damage in hippocampus and cortex. These results demonstrate that nonconvulsive seizures induced at early developmental stages that do not result in observable electrophysiological and morphological changes may have delayed behavioral consequences.

Candidatus Neoehrlichia mikurensis infection identified in 2 hematooncologic patients: benefit of molecular techniques for rare pathogen detection.

Hematooncologic patients often host rare or fastidious pathogens. Using 16S rDNA sequencing and transmission electron microscopy, we have identified 2 lymphoma patients infected with Candidatus Neoehrlichia mikurensis. In both individuals, the clinical presentation suggested ehrlichiosis-like syndrome. We believe that molecular techniques open new vistas in the field of pathogen detection.

A comprehensive study of TP53 mutations in chronic lymphocytic leukemia: Analysis of 1287 diagnostic and 1148 follow-up CLL samples.

TP53 plays a pivotal role in the process of DNA repair and apoptosis. In 10-20% of patients with chronic lymphocytic leukemia (CLL), the TP53 pathway is affected. In this study, we analyzed the TP53 mutation status in 2435 consecutive CLL samples, including 1287 diagnostic samples and 1148 samples during follow-up, using FASAY (Functional Analysis of Separated Alleles in Yeast) and direct sequencing. In a cohort of 1287 diagnostic CLL samples, we identified 237 cases with TP53 variants, including mutations, temperature-sensitive variants, deletions, insertions and aberrant splicing variants (18.4%). In 1148 follow-up samples, no TP53 clonal evolution was observed.

Sustained deficiency of mitochondrial complex I activity during long periods of survival after seizures induced in immature rats by homocysteic acid.

Our previous work demonstrated the marked decrease of mitochondrial complex I activity in the cerebral cortex of immature rats during the acute phase of seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side) and at short time following these seizures. The present study demonstrates that the marked decrease ( approximately 60%) of mitochondrial complex I activity persists during the long periods of survival, up to 5 weeks, following these seizures, i.e. periods corresponding to the development of spontaneous seizures (epileptogenesis) in this model of seizures. The decrease was selective for complex I and it was not associated with changes in the size of the assembled complex I or with changes in mitochondrial content of complex I. Inhibition of complex I was accompanied by a parallel, up to 5 weeks lasting significant increase (15-30%) of three independent mitochondrial markers of oxidative damage, 3-nitrotyrosine, 4-hydroxynonenal and protein carbonyls. This suggests that oxidative modification may be most likely responsible for the sustained deficiency of complex I activity although potential role of other factors cannot be excluded. Pronounced inhibition of complex I was not accompanied by impaired ATP production, apparently due to excess capacity of complex I documented by energy thresholds. The decrease of complex I activity was substantially reduced by treatment with selected free radical scavengers. It could also be attenuated by pretreatment with (S)-3,4-DCPG (an agonist for subtype 8 of group III metabotropic glutamate receptors) which had also a partial antiepileptogenic effect. It can be assumed that the persisting inhibition of complex I may lead to the enhanced production of reactive oxygen and/or nitrogen species, contributing not only to neuronal injury demonstrated in this model of seizures but also to epileptogenesis.

Posttreatment with group II metabotropic glutamate receptor agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate is only weakly effective on seizures in immature rats.

The present study has examined the anticonvulsant and neuroprotective effect of 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a selective agonist for group II metabotropic glutamate receptors (mGluRs) when given 10-15 min after the onset of seizures induced in 12-day-old rats by bilateral icv infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side). For biochemical analyses, rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45-50 min after infusion of DL-HCA. Comparable time intervals were used for sacrificing the animals which received 2R,4R-APDC (0.05 nmol/side) or saline. The severity of seizures was influenced only slightly when the agonist was given after the onset of seizures, as evaluated both from the behavioral symptoms and from EEG recordings. A tendency to lower number and a shorter duration of seizures was outlined in animals posttreated with 2R,4R-APDC, but the differences did not reach the level of statistical significance. Cortical energy metabolite changes which normally accompany seizures in immature rats (large decrease of glucose and glycogen and a marked rise of lactate) were ameliorated only partially. The neuroprotective effect of 2R,4R-APDC was evaluated after 24 h and 6 days of survival following DL-HCA-induced seizures. Massive neuronal degeneration in many brain regions, mainly in the hippocampus and thalamus, following infusion of DL-HCA alone was only partially attenuated after 2R,4R-APDC posttreatment. The present findings clearly indicate that both anticonvulsant and neuroprotective effect of 2R,4R-APDC against DL-HCA-induced seizures is substantially diminished when the agonist is given after the onset of seizures as compared with its efficacy after the pretreatment (Exp. Neurol.192, 420-436, 2005).

Mitochondrial complex I inhibition in cerebral cortex of immature rats following homocysteic acid-induced seizures.

The major finding of the present study concerns the marked decrease of respiratory chain complex I activity in the cerebral cortex of immature rats following seizures induced by bilateral intracerebroventricular infusion of dl-homocysteic acid (600 nmol/side). This decrease was already evident during the acute phase of seizures (60-90 min after infusion) and persisted for at least 20 h after the seizures. It was selective for complex I since activities of complex II and IV and citrate synthase remained unaffected. Inhibition of complex I activity was not associated with changes in complex I content. Based on enhanced lipoperoxidation and decreased aconitase activity, it can be postulated that oxidative modification is most likely responsible for the observed inhibition. Mitochondrial respiration, as well as cortical ATP levels remained in the control range, apparently due to excess capacity of the complex I documented by energy thresholds. On the other hand, the enhanced production of reactive oxygen species by inhibited complex I was observed in mitochondria from HCA-treated animals. The decrease of complex I activity was substantially attenuated when animals were treated with substances providing an anticonvulsant effect and also with selected free radical scavengers. We can assume that inhibition of complex I may elicit enhanced formation of reactive oxygen species and contribute thus to neuronal injury demonstrated in this model.

Effect of free radical spin trap N-tert-butyl-alpha-phenylnitrone (PBN) on seizures induced in immature rats by homocysteic acid.

The present study has examined the effect of free radical spin trap N-tert-butyl-alpha-phenylnitrone (PBN) in the model of seizures induced in immature 12-day-old rats by bilateral intracerebroventricular infusion of dl-homocysteic acid (dl-HCA, 600 nmol/side). PBN was given i.p. in two doses (100 mg/kg each), 30 min prior and 30 min after dl-HCA infusion. PBN did not significantly influence the severity of seizures, evident both from the behavioral symptoms and EEG recordings. PBN normalized decreased ATP levels in the hippocampus, occurring during the acute phase of seizures ( approximately 45-50 min after infusion) and persisting until the end of the 24-h recovery period. PBN also led to normalization of decreased glucose levels and to a significant reduction of lactate accumulation in the cerebral cortex and hippocampus. The neuroprotective effect of PBN was evaluated after 24 h and 6 days of survival following dl-HCA-induced seizures (Nissl and Fluoro-Jade B staining). The administration of PBN resulted in a partial amelioration of severe damage observed in many brain regions following infusion of dl-HCA alone. The data suggest that increased free radical production is apparently occurring during seizures induced in immature rats by homocysteic acid. Free radical scavenger PBN had a clear-cut protective effect, evident as the improved recovery of brain energy status and as a partial, but significant, attenuation of neuronal degeneration associated with this model of seizures.

Seizures induced in immature rats by homocysteic acid and the associated brain damage are prevented by group II metabotropic glutamate receptor agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate.

The present study has examined the anticonvulsant and neuroprotective effect of group II metabotropic glutamate receptor (mGluR) agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) in the model of seizures induced in immature 12-day-old rats by bilateral intracerebroventricular infusion of dl-homocysteic acid (DL-HCA, 600 nmol/side). For biochemical analyses, rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45-50 min after infusion. Comparable time intervals were used for sacrificing the pups which had received 2R,4R-APDC. Low doses of 2R,4R-APDC (0.05 nmol/side) provided a pronounced anticonvulsant effect which was abolished by pretreatment with a selective group II mGluR antagonist LY341495. Generalized clonic-tonic seizures were completely suppressed and cortical energy metabolite changes which normally accompany these seizures were either normalized (decrease of glucose and glycogen) or markedly reduced (an accumulation of lactate). EEG recordings support the marked anticonvulsant effect of 2R,4R-APDC, nevertheless, this was only partial. In spite of the absence of obvious motor phenomena, isolated spikes or even short periods of partial ictal activity could be observed. Isolated spikes could also be seen in some animals after application of 2R,4R-APDC alone, reflecting most likely subclinical proconvulsant activity of this agonist. The neuroprotective effect of 2R,4R-APDC was evaluated after 24 h and 6 days of survival following DL-HCA-induced seizures. Massive neuronal degeneration, as revealed by Fluoro-Jade B staining, was observed in a number of brain regions following infusion of DL-HCA alone (seizure group), whereas 2R,4R-APDC pretreatment provided substantial neuroprotection. The present findings support the possibility that group II mGluRs are a promising target for a novel approach to treating epilepsy.

Seizures induced by homocysteic acid in immature rats are prevented by group III metabotropic glutamate receptoragonist (R,S)-4-phosphonophenylglycine.

The potential anticonvulsant effect of group III metabotropic glutamate receptor (mGluR) agonist (R,S)-4-phosphonophenylglycine ((R,S)-PPG) against seizures induced in immature 12-day-old rats by bilateral intracerebroventricular (icv) infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side) was examined in the present study. Rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45 to 50 min after infusion. Comparable time intervals were used for sacrificing the pups which had received (R,S)-PPG. Low doses of (R,S)-PPG (10 nmol, icv) provided a pronounced anticonvulsant effect which was abolished by pretreatment with a selective group III mGluR antagonist (R,S)-alpha-methylserine-O-phosphate. Generalized clonic-tonic seizures were completely suppressed and cortical energy metabolite changes which normally accompany these seizures were either normalized (glucose and glycogen decreases) or markedly ameliorated (an accumulation of lactate). Despite the absence of obvious motor phenomena, EEG recordings revealed sporadic ictal activity, mostly in the dorsal hippocampus. Spreading of this activity into the frontal cortex was rather exceptional. The latency of ictal EEG in pretreated rats was significantly prolonged. Our data suggest that the predominant effect of (R,S)-PPG might concern seizure spread. The administration of (R,S)-PPG alone did not cause any overt behavioral side effects; it did not change the EEG pattern and did not influence cortical metabolite levels, with the exception of increased concentrations of glucose. The present findings suggest that group III mGlu receptor agonists may be of therapeutic significance for treating childhood epilepsies.

Neuronal cell death in hippocampus induced by homocysteic acid in immature rats.

PURPOSE: To examine the morphologic alterations in the cerebral cortex and hippocampus of immature rats 6 days after the generalized clonic-tonic seizures induced by homocysteic acid (HCA). METHODS: Seizures were induced by bilateral intracerebroventricular infusion of HCA (600 nmol per each side) in 12-day-old rats. After 6 days, rat pups were transcardially perfused under deep ether anesthesia with heparinized normal saline and subsequently with the fixation solution (4% paraformaldehyde in phosphate buffer, pH 7.4, for light microscopy) or with Karnovsky's solution (4% paraformaldehyde and 2% glutaraldehyde in phosphate buffer, pH 7.4, for electron microscopic analysis). Nissl stain and the DNA-specific dye bis-benzimide (Hoechst 33342) were used. RESULTS: No pathologic changes were found in the cerebral cortex, whereas serious alterations occurred in the hippocampus. A total loss of CA3 pyramidal cells was observed, with marked changes in the CA1 region and dentate gyrus. A prominent glial reaction was seen in many regions of the hippocampal formation. A slight dilatation of the cerebral ventricles was noticed in some experimental as well as control animals. In the granule cell layer of the dentate gyrus, neurons with segmented or fragmented nuclei in various stages of degeneration were detected, displaying the features of apoptotic death. CONCLUSIONS: These findings demonstrate the vulnerability of the immature rat brain, which most likely reflects both the direct neurotoxic effect of HCA and prolonged seizure activity. The relative contribution of these two factors still remains to be assessed.