Valproate release from polycaprolactone implants prepared by 3D-bioplotting.

In this study we examined the release kinetics of valproate from polycaprolactone (PCL) implants constructed for local antiepileptic therapy. The PCL implants were produced with a novel 3D-Bioplotting technology. Release kinetics were determined by superfusion of these implants. Valproate was measured in the superfusate fractions with high pressure liquid chromatography (HPLC). The HPLC measurements were linear over a concentration range of 10-500 microg/mL for valproate and the limit of quantification was found to be 9 microg/mL. The HPLC method used is simple, accurate and sensitive. Within the first day, valproate (10% w/w)-PCL implants released already 77% of the maximum possible liberated amount whereas (5% w/w)-PCL implants released only 53%. After four days, 88% of valproate was released from (10% w/w)-PCL implants and 94% valproate from (5% w/w)-PCL implants. When valproate was ground before the 3D-Bioplotting process, only 63% from (10% w/w)-PCL implants was released within the first day. This released amount of ground valproate was significantly lower compared to that which was not ground from the (10% w/w)-PCL implants. After three days of superfusion a total amount of 89% of ground valproate within the implants was released, corresponding to 88% of non-ground valproate after four days. The fast releasing PCL implants can be used to study acute effects of locally applied valproate on epileptogenesis in vivo after initiation of an epileptic focus in an animal model. The corresponding biocompatibility may also be analysed.

Effects of antiepileptic drugs on GABA release from rat and human neocortical synaptosomes.

In epilepsy, allegedly, a neurotransmitter imbalance between the inhibitory GABA and the excitatory glutamate prevails. Therefore, some antiepileptic drugs (AEDs) are thought to increase GABA release. Because little is known about corresponding presynaptic effects of AEDs in the human brain, this study investigated the effects of carbamazepine, lamotrigine, phenytoin, gabapentin, pregabalin, levetiracetam, and valproate on (3)H-GABA release from human neocortical synaptosomes preincubated with (3)H-GABA. To obtain information on possible species differences, rat neocortical synaptosomes were investigated concomitantly. Release was evoked by either veratridine (1, 3.2, or 10 µM), which prevents activated voltage-dependent Na(+) channels from closing, or elevation of extracellular [K(+)] from 3 to 15 mM. The exocytosis inhibitor tetanus toxin (TeT) or withdrawal of buffer Ca(2+) (Ca (e) (2+) ) reduced K(+)-evoked release in both species, while blockade of Na(+) channels with tetrodotoxin had no effect. K(+)-evoked release was characterized as predominant, Ca(2+)-dependent and Na(+)-independent, exocytosis. Carbamazepine and phenytoin in the rat and carbamazepine, phenytoin, lamotrigine, and valproate in human tissue reduced K(+)-evoked (3)H-GABA release. With respect to veratridine-evoked release, Ca (e) (2+) withdrawal did not reduce release in the rat; it even increased the release in human tissue. TeT was slightly inhibitory in the rat. Blockade of GABA transport diminished veratridine-evoked (3)H-GABA release in either species. This release was characterized as mediated mainly by transporter reversal. Carbamazepine, lamotrigine, and phenytoin in rat tissue and carbamazepine and phenytoin in human decreased veratridine-induced (3)H-GABA release. Interestingly, no AED increased (3)H-GABA release. The reduction by AEDs of veratridine-evoked release was more intense than that of K(+)-evoked release. In conclusion, reduction of GABA release by AEDs may be the actual objective in a pathologically altered neuronal network where GABA acts in a depolarizing fashion.

Differential inhibitory effects of drugs acting at the noradrenaline and 5-hydroxytryptamine transporters in rat and human neocortical synaptosomes.

BACKGROUND AND PURPOSE: Although the amino acid sequences of rat and human 5-hydroxytryptamine (5-HT) and noradrenaline (NA) transporters (i.e. SERT and NET) are highly homologous, species differences exist in the inhibitory effects of drugs acting at these transporters. Therefore, comparison of the potencies of drugs acting at SERT and NET in native human and rat neocortex may serve to more accurately predict their clinical profile. EXPERIMENTAL APPROACH: Synaptosomes prepared from fresh human and rat neocortical tissues were used for [(3)H]-5-HT and [(3)H]-NA saturation and competition uptake experiments. The drugs tested included NA reuptake inhibitors (desipramine, atomoxetine and (S,S)-reboxetine), 5-HT reuptake blockers (citalopram, fluoxetine and fluvoxamine) and dual 5-HT/NA reuptake inhibitors (duloxetine and milnacipran). KEY RESULTS: In saturation experiments on synaptosomal [(3)H]-5-HT and [(3)H]-NA uptake, the dissociation constants did not indicate species differences although a smaller density of both SERT and NET was observed in human tissues. In competition experiments with the various drugs, marked species differences in their potencies were observed, especially at SERT. The rank order of selectivity ratios (SERT/NET) in human neocortex was as follows: citalopram >or= duloxetine = fluvoxamine >or= fluoxetine > milnacipran > desipramine = atomoxetine > (S,S)-reboxetine. Significant species differences in these ratios were observed for duloxetine, atomoxetine and desipramine. CONCLUSIONS AND IMPLICATIONS: This study provides the first compilation of drug potency at native human neocortical SERT and NET. The significant species differences (viz., human vs. rat) in drug potency suggest that the general use of rodent data should be limited to predict clinical efficacy or profile.

Influence of narcotics on luminance and frequency modulated visual evoked potentials in rats.

Quantification of visual function is essential for the impact of disease models and their treatment. Recently, we introduced a chronic implant model to record visual evoked potentials (VEP) in awake Brown-Norway rats. Here, we investigated the hemispheric distribution of VEP after monocular stimulation, the chronic electrode implantation and the influence of commonly used anesthetics. Potentials were recorded by electrodes, implanted epidurally over the superior colliculus. The entire visual field of the rat was stimulated. Flicker stimuli were modulated in luminance with a modulation depth from 5 to 80% at 7.5 Hz and flashes with a modulation depth of >95% in a frequency range of 2.9-38 Hz. Recordings were constant over 9 days. When one eye was blinded, the potentials recorded from the contralateral side were not affected, while the potentials of the ipsilateral side were markedly reduced. Further, potentials of awake animals were compared with those receiving general anesthetics. For one group of rats (n = 8), we administered isoflurane by inhalation in five concentrations. Four different groups (n = 7-11) were given choralhydrate (200 and 400 mg/kg) and the combination of ketamine/xyaline (65/7 or 130/14 mg/kg, respectively) intraperitoneally. Isoflurane depressed the VEP in a concentration-dependent manner. Treatment with chloralhydrate and ketamine/xyaline increased the VEP at low concentrations and depressed it in high concentrations. The new VEP paradigm assesses distinct qualities of contrast vision in rats. All tested narcotics alter VEP amplitudes and can be avoided.

Effects of gabapentin and pregabalin on K+-evoked 3H-GABA and 3H-glutamate release from human neocortical synaptosomes.

One site of action of the anticonvulsant, analgesic, and anxiolytic drugs gabapentin and pregabalin is the alpha(2)delta-subunit of voltage-sensitive Ca(2+) channels (VSCC). We therefore analyzed the effects of gabapentin and pregabalin on K(+)-evoked release of (3)H-gamma-aminobutyric acid (GABA) and (3)H-glutamate from superfused human neocortical synaptosomes. These neurotransmitters are released by Ca(2+)-dependent exocytosis and by Ca(2+)-independent uptake reversal. When a GABA transport inhibitor was present throughout superfusion to isolate exocytotic conditions, gabapentin and pregabalin (100 microM each) reduced K(+)-evoked (3)H-GABA release by 39% and 47%, respectively. These effects were antagonized by the alpha(2)delta-ligand L: -isoleucine (1 microM) suggesting the alpha(2)delta-subunit of terminal VSCC to mediate the reduction of exocytosis. Both drugs had no effect on exocytotic (3)H-glutamate release and also failed to modulate the release of (3)H-GABA and (3)H-glutamate caused by reversed uptake in the absence of external Ca(2+). Thus, an inhibition of glutamate release by gabapentin and pregabalin as main anticonvulsant principle is not supported by our experiments. An anticonvulsant mode of action of both drugs may be the reduction of a proconvulsant exocytotic GABA release.

Partial agonism at the human alpha(2A)-autoreceptor: role of binding duration.

Temperature-induced changes of affinity and efficacy of the alpha2-adrenoceptor full agonist UK14,304 and the partial agonists clonidine and guanfacine were investigated to elucidate the mechanism of partial agonism at the terminal alpha2-autoreceptor. The effect of temperature on the efficacy of the substances was tested in 3H-noradrenaline release experiments at 37 degrees C and at room temperature. Human neocortical slices were prelabeled with 3H-noradrenaline, superfused, and stimulated electrically under autoinhibition-free conditions. Furthermore, saturation binding experiments with human neocortical synaptosomes were performed at 37 degrees C and 17 degrees C to evaluate the influence of temperature on the affinity of 3H-clonidine and 3H-UK14,304. Temperature-induced changes of the association and dissociation rate constants of 3H-UK14,304 and 3H-clonidine were assessed in corresponding kinetic binding experiments. Our experiments reveal that clonidine and guanfacine lose efficacy when the temperature is lowered, whereas no change was noted for the full agonist UK14,304. Moreover, the affinity of clonidine and guanfacine was shown to decrease at lower temperature. Kinetic experiments indicated that the loss of affinity observed for 3H-clonidine at 17 degrees C is due to a marked reduction of the association rate. The loss of efficacy of the partial agonists is most likely related to the short binding duration; partial agonists do not bind long enough to the receptor to mediate a maximum response. The discrepancy between the time required to elicit a maximum response and the actual binding time may be greater for partial agonists at lower temperatures, thus, causing the intrinsic activity to decline.

Differential modulation of K(+)-evoked (3)H-neurotransmitter release from human neocortex by gabapentin and pregabalin.

Anticonvulsant, analgesic, and anxiolytic effects have been observed both in preclinical and clinical studies with gabapentin (GBP) and pregabalin (PGB). These drugs appear to act by binding to the alpha(2)delta subunit of voltage-sensitive Ca(2+) channels (VSCC), resulting in the inhibition of neurotransmitter release. In this study, we examined the effects of GBP and PGB (mostly 100 microM, corresponding to relatively high preclinical/clinical plasma levels) on the release of neurotransmitters in human neocortical slices. These slices were prelabeled with (3)H-dopamine ((3)H-DA), (3)H-choline (to release (3)H-acetylcholine ((3)H-ACh)), (3)H-noradrenaline ((3)H-NA), and (3)H-serotonin ((3)H-5-HT), and stimulated twice in superfusion experiments by elevation of extracellular K(+) in the presence and absence of GBP and PGB. The alpha(2)delta ligands produced significant inhibitions of K(+)-evoked (3)H-ACh, (3)H-NA, and (3)H-5-HT release between 22% and 56% without affecting (3)H-DA release. Neither drug reduced (3)H-NA release in the presence of L: -isoleucine, a putative alpha(2)delta antagonist. Interestingly, this antagonism did not occur using the enantiomer, D: -isoleucine. These results suggest that GBP and PGB are not general inhibitors of VSCC and neurotransmitter release. Such alpha(2)delta ligands appear to be selective modulators of the release of certain, but not all, neurotransmitters. This differential modulation of neurotransmission presumably contributes to their clinical profile.

Why do cannabinoids not show consistent effects as analgetic drugs in multiple sclerosis?

The effectiveness of cannabinoids (CB) in the treatment of pain in patients with multiple sclerosis (MS) varies. The pathogenesis of pain in MS is diverse as are the possible effects of CB at different sites of CB receptors in the peripheral and central nervous system, this may explain the variable impact on individual patients. The aim of this review is to summarize pre-clinical and clinical studies to explain this variability from a neuropharmacological point of view. Future studies are needed to examine specific effects on distinct symptoms in homogenous groups of patients.

Novel anticonvulsant drugs.

Principles of complex mechanisms of action of anticonvulsants including latest reports concerning new antiepileptic drugs (AED) are considered. Different aspects of new anticonvulsant drugs (2nd generation) from preclinical and clinical testing, pharmacokinetics, and mono or combination therapy in children and adults are summarized. In the following condensed synopsis pharmacological and clinical characteristics of gabapentin (GBP), lamotrigine (LTG), levetiracetam (LEV), oxcarbazepine (OXC), pregabalin (PGB) and tiagabine (TGB) as well as topiramate (TPM) and zonisamide (ZNS) are discussed. In addition to the mechanisms of action, pharmacokinetics, interactions, indications and dosages as well as side effects are considered. Important data concerning the effect and tolerability of anticonvulsant drugs can be obtained from controlled studies. In comparison to drugs of the first generation (phenobarbital [PB], primidon [PRD], phenytoin [PHT], carbamazepine [CBZ] and valproic acid [VPA]) the potential for interactions and side effects due to enzyme induction or inhibition is reduced by most of the anticonvulsant drugs of the second generation. New anticonvulsant drugs increase the spectrum of treatment and represent further steps with regard to the optimization of an individual therapy of the epilepsies.

Neuronal electrical high frequency stimulation enhances GABA outflow from human neocortical slices.

Electrical high frequency stimulation of the globus pallidus internus or the subthalamic nucleus has beneficial motor effects in advanced Parkinson's disease. The mechanisms underlying these clinical results remain, however, unclear. From previous studies it is proposed that the gamma-aminobutyric acid (GABA) system is involved in the effectiveness of electrical high frequency stimulation. In these experiments, human neocortical slices were stimulated electrically (130 Hz) in vitro, and GABA outflow was measured after o-phthaldialdehyde sulphite derivatization using HPLC with electrochemical detection. Our results could demonstrate that high frequency stimulation (HFS) significantly increased basal GABA outflow in the presence of submaximal concentrations of the voltage-gated sodium channel opener veratridine. This effect could be abolished by the GABA antagonists bicuculline or picrotoxin. These results suggest that HFS has an activating effect on GABAergic neuronal terminals in human neocortical slices, depending on sodium and chloride influx. Since GABA plays a role in CNS disorders of basal ganglia, anxiety and epilepsy, its neocortical modulation by HFS may be (patho)physiologically relevant.

Cannabinoid CB1 receptor-mediated modulation of evoked dopamine release and of adenylyl cyclase activity in the human neocortex.

1. The present study investigated the binding characteristics of various ligands to cannabinoid CB(1) receptors in human neocortex and amygdala. In addition, the functionality of CB(1) receptors in the human neocortex was assessed by examining the effects of CB(1) receptor ligands on evoked [(3)H]-dopamine (DA) release in superfused brain slices and on synaptosomal cAMP accumulation. 2. Saturation-binding assays in human neocortical and amygdala synaptosomes using a radiolabelled cannabinoid receptor agonist ([(3)H]-CP55.940) revealed pK(d) values of 8.96 and 8.63, respectively. The numbers of binding sites (B(max)) were 3.99 and 2.67 pmol (mg protein)(-1), respectively. 3. Various cannabinoid receptor ligands inhibited [(3)H]-CP55.940 binding with rank order potencies corresponding to those of previous studies in animal tissues. 4. Electrically evoked [(3)H]-DA release from human neocortical slices was inhibited by CP55.940 (IC(50) 6.76 nm, I(max) 65%) and strongly enhanced by the cannabinoid receptor antagonist AM251. However, [(3)H]-DA release was not influenced in rat neocortex. In human tissue, the estimated endocannabinoid concentration in the biophase of the release-modulating CB(1) receptors was 1.07 nm, expressed in CP55.940 units. 5. K(+)-evoked [(3)H]-DA release in the presence of tetrodotoxin (TTX) was strongly inhibited by CP55.940 in humans, but not in rats. 6. In human tissue, CP55.940 inhibited forskolin-stimulated cAMP accumulation (IC(50) 20.89 nm, I(max) 35%). AM251 blocked this effect and per se increased forskolin-stimulated cAMP accumulation by approximately 20%. 7. In conclusion, cannabinoids modulate [(3)H]-DA release and adenylyl cyclase activity in the human neocortex. CB(1) receptors are located on dopaminergic nerve terminals and seem to be tonically activated by endocannabinoids.

Modulation of electrically evoked acetylcholine release through cannabinoid CB1 receptors: evidence for an endocannabinoid tone in the human neocortex.

Cannabinoids are known to inhibit neurotransmitter release in the CNS through CB1 receptors. The present study compares the effects of synthetic cannabinoids on acetylcholine (ACh) release in human and mice neocortex. We further investigated a possible endocannabinoid tone on CB1 receptors in human neocortex caused by endogenous agonists like anandamide or 2-arachidonylglycerol. Brain slices, incubated with [3H]-choline, were superfused and stimulated electrically under autoinhibition-free conditions to evoke tritium overflow assumed to represent ACh release. The first series of experiments was performed with 26 pulses, 60 mA, at 0.1 Hz. In mice neocortical slices, the cannabinoid receptor agonist WIN55212-2 decreased ACh release (pIC50=6.68, I(max)=67%). In the human neocortex the concentration-response curve of WIN55212-2 was bell-shaped and flat (I(max observed) approximately 30%). The estimated maximum possible inhibition, however, was much larger: I(max derived)=79%. Lec, the negative logarithm (lg) of the biophase concentration of endocannabinoids in 'WIN55212-2 units,' was -6.52, the pKd of WIN55212-2 was 7.47. The CB1 receptor antagonist/inverse agonist SR141716 enhanced ACh release in the human neocortex (by 38%) and prevented the inhibitory effect of WIN55212-2. The concentration-response curve of WIN55212-2 was changed in its shape including a shift to the right due to the presence of SR141716. A pA2 of this antagonist between 11.60 and 11.18 was obtained. SR141716 alone had no effect in mice neocortical slices. A partial agonist without inverse agonistic activity, O-1184, enhanced ACh release in the human neocortex. The endocannabinoid uptake-inhibitor AM404 decreased ACh release in human, but not in mice, neocortical slices. Change of the stimulation parameters (eight trains of pseudo-one-pulse bursts (4 pulses, 76 mA, 100 Hz), spaced by 45 s intervals) led to a stronger inhibitory effect of WIN55212-2, and abolished the disinhibitory effect of SR141716 and O-1184. The results show that activation of CB1 cannabinoid receptors leads to inhibition of ACh release in the human and mouse neocortex. The endocannabinoid tone is high in the human, but not in the mouse neocortex and is dependent on neuronal activity. SR141716 acts as a competitive CB1 receptor antagonist.

Evidence for strychnine-sensitive glycine receptors in human amygdala.

Recent studies suggested the existence of strychnine-sensitive glycine-receptors in mammalian amygdala. In the present study, we investigated the amino acid concentrations as well as immunocytochemical and pharmacological properties of glycine-receptors in fresh human amygdala tissue obtained from epilepsy surgery. High pressure liquid chromatography revealed a considerable amount of glycine and its precursors and glycine-receptors agonists L-serine and taurine in this tissue. Immunohistochemistry using the monoclonal antibody mAb4a, recognizing an epitope common to all alpha-subunit variants of glycine receptors, displayed a specific labeling at the soma and on proximal dendrites of mostly tripolar, large-sized neurons of irregular distribution and arrangement. To elucidate the pharmacological properties of the glycine-receptors found slices of human amygdala were preloaded with [(3)H]-choline and superfused. Glycine induced an overflow of [(3)H]-acetylcholine, which was inhibited by strychnine in a concentration-dependent manner. Furthermore, the glycine-induced release of [(3)H]-acetylcholine was significantly inhibited by furosemide, indicating glycine-induced actions to be attributed to chloride channels. These actions of glycine were not influenced by MK-801, D-CP-Pene or bicuculline. Thus, the effects of glycine did not seem to be mediated through NMDA or GABA receptors. These observations indicate that strychnine-sensitive, chloride-conducting glycine receptors, which elicit the release of [(3)H]-acetylcholine, are present at the soma and on proximal dendrites of neurons in human amygdala. It is hypothesized that glycine may display a regulatory role in amygdaloid functions, probably via cholinergic interneurons.

High potassium-induced activation of choline-acetyltransferase in human neocortex: implications and species differences.

The role of electrical and potassium (K(+))-induced depolarisation on choline-acetyltransferase (ChAT) activity in human and mouse neocortical slices was studied. When [3H]-ACh release was evoked by two K(+) stimulations in human neocortex, the mean S(2)/S(1) ratio was significantly below unity. ChAT inhibitors, like bromo-acetylcholine and ocadaic acid, raised this ratio by 79 and 63%, respectively, suggesting that the diminished S(2)/S(1) value in the absence of ChAT inhibitors reflected an increased ChAT activity at S(2) following K(+) depolarisation at S(1). When stimulated electrically, however, the S(2)/S(1) ratio in human neocortex was near unity and ocadaic acid remained without effect. In parallel experiments on mouse neocortical slices, the S(2)/S(1) ratio was near unity in both electrically or K(+)-evoked [3H]-ACh release and was not altered by ChAT inhibition. ChAT activity following K(+) depolarisation was also determined directly. ChAT activation in human neocortical slices was highest at 10 and 20mM K(+). ChAT activity in mouse neocortical tissue was not altered by K(+) depolarisation. These results suggest that in human, but not in mouse, neocortex ChAT activity may be increased due to ongoing K(+) depolarisation. This increase of ChAT activity supports a cholinergic degeneration hypothesis which has been entitled "autocannibalism" by Wurtman [TINS 15 (1992) 177].

Characterization of muscarinic autoreceptors in the rabbit hippocampus and caudate nucleus.

Oxotremorine-induced inhibition of electrically evoked release of 3H-acetylcholine from brain slices preincubated with 3H-choline was used to characterize muscarinic autoreceptors in rabbit hippocampus and caudate nucleus. From the shifts to the right of the concentration-response curves of oxotremorine in the presence of muscarinic receptor antagonists, the following pKB values [95% C.I.] were determined in the hippocampus: tripinamide: 8.7 [8.5, 8.8]; himbacine: 8.4 [8.3, 8.5]; AQ-RA 741: 8.3 [8.2, 8.5]; 4-DAMP: 8.2 [8.0, 8.3]; hexahydrosiladifenidol: 7.4 [7.2, 7.5]; AF-DX 116: 7.3 [7.1, 7.4]; pirenzepine: 6.8 [6.6, 7.0]; and PD102807: 6.3 [6.0, 6.5]. In the caudate nucleus: tripinamide: 9.1 [8.9, 9.2]; 4-DAMP: 8.3 [8.2, 8.5]; himbacine: 8.1 [8.0, 8.2]; AQ-RA 741: 8.1 [8.0, 8.3]; hexahydrosiladifenidol: 7.3 [7.2, 7.4]; AF-DX 116: 7.1 [7.0, 7.2]; pirenzepine: 6.7 [6.6, 6.8]; and PD102807: 6.5 [6.2, 6.8]. These pKB values fit best to literature values for M2 receptors, suggesting that the muscarinic autoreceptor of the rabbit hippocampus and caudate nucleus is the m2 gene product.

Mice transgenic for exon 1 of Huntington's disease: properties of cholinergic and dopaminergic pre-synaptic function in the striatum.

In Huntington's disease (HD), neuronal loss is most prominent in the striatum leading to emotional, cognitive and progressive motor dysfunction. The R6/2 mice, transgenic for exon 1 of the HD gene, develop a neurological phenotype with similarities to these features of HD. In striatal tissue, electrically evoked release of tritiated acetylcholine (ACh) and dopamine (DA) were compared in wild-type (WT) and R6/2 mice. In R6/2 mice, the evoked release of ACh, its M2 autoreceptor-mediated maximum inhibition and its dopamine D2 heteroreceptor-mediated maximum inhibition was diminished to 51%, 74% and 87% of controls, respectively. Also, the activities of choline acetyltransferase and of synaptosomal high-affinity choline uptake decreased progressively with age in these mice. In the DA release model, however, electrical stimulation elicited equal amounts of [3H]-DA both in WT and R6/2 mice. Moreover, high-affinity DA uptake into striatal slices was similar in WT and R6/2 mice. In order to confirm these findings in vivo, intrastriatal levels of extracellular DA were measured by intracerebral microdialysis in freely moving mice: striatal DA levels were found to be equal in WT and R6/2 mice. In conclusion, in the transgenic R6/2 mice changes occur mainly in striatal cholinergic neurones and their pre-synaptic modulation, but not in the dopaminergic afferent terminals. Whether similar events also contribute to the pathogenesis of HD in humans has to be established.

Up-regulation of D3 dopaminergic receptor mRNA in the core of the nucleus accumbens accompanies the development of seizures in a genetic model of absence-epilepsy in the rat.

The basal ganglia system is thought to play a key role in the control of absence-seizures and there is ample evidence that epileptic seizures modify brain dopamine function. We recently reported that local injections of dopamine D1 or D2 agonists in the core of the nucleus accumbens suppressed absence-seizures in a spontaneous, genetic rodent model of absence-epilepsy whereas injections of D1 or D2 antagonists had aggravating effects. These findings raised the possibility that the dopaminergic system may be altered in absence-epilepsy prone rats. Therefore, we studied by in situ hybridization histochemistry the expression of pre- and postsynaptic components of the dopaminergic system in this strain of rats. When compared to non-epileptic control rats, epileptic rats displayed no change in the expression of mRNAs coding for the neuronal dopaminergic markers (tyrosine hydroxylase, membraneous and vesicular dopamine transporters). In addition, there was no difference between the two strains concerning the expression of the dopamine receptor transcripts D1, D2 and D5. In adult absence-epilepsy prone rat with an overt epileptic phenotype, however, an elevated level of D3 mRNA expression was observed in neurons of the core of the nucleus accumbens (+23% increase in silver grain density compared to non-epileptic control rats). D3 transcripts were not increased in juvenile epileptic rats without seizures. These findings suggests that up-regulation of D3 receptor mRNA is part of the epileptic phenotype in absence-epilepsy prone rats. Its localization in the core of the nucleus accumbens bears close resemblance to the dopamine-sensitive antiepileptic sites in ventral striatum and further support the involvement of ventral structures of the basal ganglia system in the control of absence-seizures.

The effects of continuous and single-dose radiation on choline uptake in organotypic tissue slice cultures of rabbit hippocampus.

The objective of the present study was to determine the time-dependent course of choline uptake in mature organotypic slice cultures of rabbit hippocampal formation and to assess the effects of continuous and single high-dose irradiation on choline uptake in cultivated slices in vitro. Transverse slices of hippocampus were dynamically incubated in a cerebrospinal fluid-like culture medium for 72 h. To study the changes in choline uptake longitudinally, the slice cultures were processed with 0.1 microM [3H]-choline, and tritium accumulation was counted. Two different gamma irradiation sources (125I seeds and a clinical 60Co source) were used as representative models of interstitial radiosurgery and other radiosurgical techniques. A total dose of approximately 6000 cGy was delivered to the brain slices in one session or in a continuous, relatively low-dose rate fashion, and their effects on high-affinity choline uptake were examined. In another set of experiments with 125I, 5 microM hemicholinium-3 was used in choline uptake procedures as a competitive high-affinity choline uptake inhibitor. The results can be summarized as follows: (1) in the control group of the hippocampal tissue culture, there was a significant increase in tritium accumulation values from 0 to 48 h and a decrease thereafter; (2) continuous 125I irradiation caused a highly significant depression of the accumulation of tritium compared to that observed in the control group throughout its application for 72 h; (3) there was no significant change in the accumulation of tritium in the slices after single high-dose rate irradiation with a 60Co source; and (4) 5 microM hemicholinium significantly depressed the accumulation of tritium in both the control and the 125I-irradiated groups, and there was no longer a difference between 125I-irradiated and control groups when both groups were treated with hemicholinium. These results demonstrate that the delivery of continuous but relatively low-dose rate gamma irradiation is more efficacious than single high-dose external irradiation on high-affinity choline uptake in hippocampal nervous tissue. The results also indicate that continuous irradiation specifically affected the high-affinity energy-dependent choline uptake mechanism, whereas nonspecific choline uptake did not seem to be disturbed.

Myoclonus in epilepsy patients with anticonvulsive add-on therapy with pregabalin.

PURPOSE: To report on the occurrence of myoclonus in patients receiving pregabalin (PGB) for the treatment of focal epilepsy. METHODS: Clinic records of 19 patients who were consecutively enrolled at a tertial referral epilepsy center in a randomized, double-blind and/or open add-on study with PGB were reviewed. RESULTS: In four patients treated with PGB, focal myoclonus newly developed. The side effect appeared with PGB doses of 50-600 mg/day; the intensity showed some dose dependency. All patients had medically refractory focal epilepsy and received other antiepileptic drugs (AEDs) besides the study medication. One patient showed focal myoclonic jerks of the left arm, whereas the other patients developed multifocal myoclonus. Polygraphic studies including electromyogram (EMG)-triggered back-averaging of the EEG in the patient with the highest frequency of myoclonic jerks showed no visible correlate of the myoclonus. In this patient, frequency and intensity of myoclonic jerks significantly decreased after dose reduction of PGB. In the other cases, myoclonus was only subtle and did not significantly interfere with daily activities, so that a dose reduction of PGB was not considered necessary. CONCLUSIONS: These data indicate a relatively high incidence (four of 19) of myoclonus associated with PGB therapy. The rate seems to be at least as high as reported in patients receiving the structurally similar anticonvulsant gabapentin.

Electrically evoked release of glutamate in rat hippocampal slices: effects of various drugs and fimbria-fornix lesions.

A model of electrically evoked release of glutamate from rat hippocampus was developed and used to detect possible changes induced by lesions of hippocampal afferences. Neuronal glutamate in hippocampal slices was labelled by preincubation with [3H]glutamine. The slices were then superfused with physiological medium in the presence of the glutamate uptake inhibitor L-transpyrrolidine-2,4-dicarboxylic acid (100 microM or 3 microM) and stimulated twice electrically (S1, S2: 240 pulses, 3 Hz, 2 ms, 26-30 mA); various drugs were added before S2. In order to determine the basal and evoked outflow of [3H]glutamate only, the mixture of 3H-labelled compounds (glutamine, glutamate and GABA) was separated by ion exchange chromatography in superfusate fractions and slices. The electrically evoked overflow of [3H]glutamate was largely Ca2+-dependent and tetrodotoxin-sensitive and hence represented action potential-induced exocytotic release of [3H]glutamate. Evoked [3H]glutamate release was significantly increased by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM), suggesting the presence of endogenous inhibitory adenosine, and reduced by the A1 receptor agonist N6-cyclopentyladenosine (1 microM, antagonized by DPCPX, 0.1 microM). There was no evidence for a cholinergic, serotonergic, or adrenergic modulation of the evoked release of [3H]glutamate: the corresponding selective agonists (or antagonists) were ineffective. After aspirative lesions of the septohippocampal pathways the hippocampal noradrenaline content was markedly increased, whereas cholinergic and serotonergic markers were reduced. The evoked release of [3H]glutamate in hippocampal slices of lesioned rats was significantly increased by a mechanism which still has to be determined, but which is not related to alterations in A1 receptor function. It is concluded that the present model was able to detect lesion-induced differences in electrically evoked release of [3H]glutamate, but the relationship of these differences to changes of noradrenergic, cholinergic or serotonergic hippocampal innervations remains to be established.