Effects of monomethylarsonic and monomethylarsonous acid on evoked synaptic potentials in hippocampal slices of adult and young rats.

Arsenite and its metabolites, dimethylarsinic or dimethylarsinous acid, have previously been shown to disturb synaptic transmission in hippocampal slices of rats (Krüger, K., Gruner, J., Madeja, M., Hartmann, L.M., Hirner, A.V., Binding, N., Mubetahoff, U., 2006a. Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals. Arch. Toxicol. 80, 492-501, Krüger, K., Straub, H., Binding, N., Mubetahoff, U., 2006b. Effects of arsenite on long-term potentiation in hippocampal slices from adult and young rats. Toxicol. Lett. 165, 167-173, Krüger, K., Repges, H., Hippler, J., Hartmann, L.M., Hirner, A.V., Straub, H., Binding, N., Mubetahoff, U., 2007. Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats. Toxicol. Appl. Pharmacol. 225, 40-46). The present experiments investigate, whether the important arsenic metabolites monomethylarsonic acid (MMA(V)) and monomethylarsonous acid (MMA(III)) also influence the synaptic functions of the hippocampus. In hippocampal slices of young (14-21 days-old) and adult (2-4 months-old) rats, evoked synaptic field potentials from the Schaffer collateral-CA1 synapse were measured under control conditions and during and after 30 and 60 min of application of the arsenic compounds. MMA(V) had no effect on the synapse functions neither in slices of adult nor in those from young rats. However, MMA(III) strongly influenced the synaptic transmission: it totally depressed the amplitudes of fEPSPs at concentrations of 50 micromol/l (adult rats) and 25 micromol/l (young rats) and LTP amplitudes at concentrations of 25 micromol/l (adult rats) and 10 micromol/l (young rats), respectively. In contrast, application of 1 micromol/l MMA(III) led to an enhancement of the LTP amplitude in young rats, which is interpretable by an enhancing effect on NMDA receptors and a lack of the blocking effect on AMPA receptors at this concentration (Krüger, K., Gruner, J., Madeja, M., Hartmann, L.M., Hirner, A.V., Binding, N., Mubetahoff, U., 2006a. Blockade and enhancement of glutamate receptor responses in Xenopus oocytes by methylated arsenicals. Arch. Toxicol. 80, 492-501). These effects are probably not mediated by changes in cell excitability or in presynaptic glutamate release rates, since antidromically induced population spikes and paired-pulse facilitation failed to show any MMA(III) effect. The impairment of the excitatory CA1 synapse is more likely caused by the action of MMA(III) on postsynaptic glutamatergic receptors and may be jointly responsible for dysfunctions of cognitive effects in arsenic toxicity.

Effects of arsenite on long-term potentiation in hippocampal slices from young and adult rats.

The effects of trivalent arsenite were tested at the Schaffer collateral-CA1 synapse of adult (2-4 month) and young (14-21 days) rats. Exposure of 100micromol/l arsenite led to a slight and reversible reduction of the amplitudes of evoked excitatory postsynaptic field potentials in adult and young rats, while exposure of 0.1 and 1micromol/l arsenite had no effects. The long-term potentiation (LTP) was significantly inhibited by arsenite in adult but not in young rats. Exposure of 0.1, 1 and 100micromol/l arsenite to slices of adult rats before and during the LTP stimulus led to a significant reduction in the potentiated amplitudes amounting to a maximum of 50% of the control values. In young animals, however, exposure of 1micromol/l arsenite showed no effect on the LTP potentiated amplitudes, while exposure of 100micromol/l arsenite led initially to a significant reduction in the amplitudes, compared to the control level, which was completely reversible 20min after washout. Exposure of 100micromol/l arsenite did not affect the paired-pulse facilitation, indicating that arsenite does not exert its effects by influencing presynaptic transmitter release mechanisms.

Reduction of human neocortical and guinea pig CA1-neuron A-type currents by organic calcium channel blockers.

In epilepsy models, organic calcium antagonists regularly induce a transient activity increase before suppression of epileptiform discharges. This action was speculated to be mediated by a modulation of potassium currents. Since A-type currents potently regulate neuronal excitability, their modulation by calcium channel blockers was investigated in acutely isolated human neocortical temporal lobe neurons and CA1 neurons of guinea pigs using the whole-cell voltage-clamp technique. In human neurons, 40 microM nifedipine caused an amplitude reduction by 28% at a command potential of -6 mV and produced a biexponential, markedly accelerated current inactivation with time constants of 8.4 +/- 1.1 ms (n = 6) and 62.9 +/- 6.4 ms (n = 5). The time constant under control conditions was 50.1 +/- 8.5 ms (n = 6). Verapamil (40 microM) did not affect the current amplitude, but accelerated the monoexponential current inactivation from 40.2 +/- 7.1 ms to 13.3 +/- 0.8 ms (n = 9). Accordingly, verapamil accelerated the inactivation from 42.3 +/- 5.9 ms to 15.0 +/- 1.3 ms (n = 11) in guinea pig CA1 neurons, without affecting the current amplitude. In this preparation, it was shown that the two enantiomers of verapamil do not differ in their actions. The results show that the A-type current in human neocortical and in guinea pig hippocampal neurons is reduced by organic calcium channel blockers.

Adenosine A1 but not A2a receptor agonist reduces hyperalgesia caused by a surgical incision in rats: a pertussis toxin-sensitive G protein-dependent process.

BACKGROUND: Activation of A1 adenosine receptors (A1Rs) causes antinociception after nerve injury and inflammation. However, the role of A2a adenosine receptors (A2aRs) for pain processing is less clear. In the current study, the authors investigated the role of spinal adenosine A1Rs and A2aRs for the maintenance of mechanical hyperalgesia in an animal model for postoperative pain. METHODS: Rats with intrathecal catheters were anesthetized and underwent plantar incision. Spontaneous pain behavior and withdrawal threshold to punctuate stimulation were measured before and after administration of intrathecal R-phenylisopropyl-adenosine (R-PIA; A1R agonist), 2-w p-2-carbonyl-ethyl-phenylethylaminox-5X-N-ethylcarboxami-doadenosine (CGS21680; A2aR agonist), or vehicle. In separate groups of animals, the effects of pertussis toxin, forskolin, glibenclamide, 4-aminopyridine, tetraethylammonium, apamin, charybdotoxin, or margatoxin on R-PIA-induced antinociception were examined. RESULTS: Intrathecal administration of 5 nmol R-PIA but not 10 nmol CGS21680 decreased nonevoked spontaneous pain behavior. Furthermore, intrathecal administration of R-PIA but not of CGS21680 increased withdrawal thresholds after incision. Pretreatment with pertussis toxin and administration of forskolin, glibenclamide, 4-aminopyridine, and tetraethylammonium inhibited R-PIA-induced antinociception. In addition, intrathecal administration of apamin, charybdotoxin, or margatoxin did not modify mechanical hypoalgesia mediated by R-PIA. CONCLUSIONS: Spinal A1Rs but not A2aRs play an important role in the maintenance of nonevoked and evoked pain behaviors after an incision. Furthermore, A1R-induced spinal antinociception is mediated by interactions with pertussis toxin-sensitive G proteins. In addition, the opening of adenosine triphosphate-sensitive K channels but not of calcium-activated potassium channels and voltage-gated Kv1.3 or Kv1.6 channels contribute to the antinociceptive effect of A1R agonists.

Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats.

In this study, the effects of pentavalent dimethylarsinic acid ((CH(3))(2)AsO(OH); DMA(V)) and trivalent dimethylarsinous acid ((CH(3))(2)As(OH); DMA(III)) on synaptic transmission generated by the excitatory Schaffer collateral-CA1 synapse were tested in hippocampal slices of young (14-21 day-old) and adult (2-4 month-old) rats. Both compounds were applied in concentrations of 1 to 100 micromol/l. DMA(V) had no effect on the amplitudes of evoked fEPSPs or the induction of LTP recorded from the CA1 dendritic region either in adult or in young rats. However, application of DMA(III) significantly reduced the amplitudes of evoked fEPSPs in a concentration-dependent manner with a total depression following application of 100 micromol/l DMA(III) in adult and 10 micromol/l DMA(III) in young rats. Moreover, DMA(III) significantly affected the LTP-induction. Application of 10 micromol/l DMA(III) resulted in a complete failure of the postsynaptic potentiation of the fEPSP amplitudes in slices taken both from adult and young rats. The depressant effect was not reversible after a 30-min washout of the DMA(III). In slices of young rats, the depressant effects of DMA(III) were more pronounced than in those taken from adult ones. Compared to the (absent) effect of DMA(V) on synaptic transmission, the trivalent compound possesses a considerably higher neurotoxic potential.

Comparison of brain extracellular fluid, brain tissue, cerebrospinal fluid, and serum concentrations of antiepileptic drugs measured intraoperatively in patients with intractable epilepsy.

PURPOSE: The mechanisms of drug resistance in epilepsy are only incompletely understood. According to a current concept, overexpression of drug efflux transporters at the blood-brain barrier may reduce levels of antiepileptic drugs (AEDs) in epileptogenic brain tissue. Increased expression of drug efflux transporters such as P-glycoprotein has been found in brain tissue surgically resected from patients with medically intractable epilepsy, but it is not known whether this leads to decreased extracellular (interstitial) AED concentrations in affected brain regions. This prompted us to measure concentrations of AEDs in the extracellular space of human neocortical tissue by using intraoperative microdialysis (IOMD) in those parts of the brain that had to be removed for therapeutic reasons. For comparison, AED levels were determined in brain tissue, subarachnoid CSF, and serum. METHODS: Concentrations of carbamazepine (CBZ), 10-hydroxy-carbazepine (10-OH-CZ, metabolite of oxcarbazepine), lamotrigine (LTG), levetiracetam (LEV), topiramate, or phenytoin were determined by using one to four catheters during IOMD in the medial temporal gyrus. Furthermore, to calculate the individual recovery of every catheter, an in vitro microdialysis was performed with ultrafiltrate of serum concurrently obtained from the respective patient. In addition, AED levels were determined in the resected brain tissue, CSF, and serum of the same patients. Altogether 22 pharmacoresistant epilepsy patients (nine male, 13 female patients; age 15-54 years) with complex partial seizures or secondarily generalized seizures were involved. In a first series, IOMD samples 40 min after beginning of the microdialysis (flow rate, 1 microl/min), and in a second series, continuous measurements 25, 30, 35, and 40 min from the beginning were evaluated (flow rate, 2 microl/min). With in vitro recovery data of the individual catheters, the concentration in the extracellular space (ECS) was estimated. RESULTS: AED concentrations in the ECS of the cortex measured by catheters located at a distance of 0.6 cm differed markedly in some patients, whereas concentrations in the ultrafiltrate of the serum of the respective patients measured with the same catheters varied only slightly. Furthermore, ECS concentrations related to the ultrafiltrate of serum showed considerable interindividual variations. The high intra- and interindividual variation of ECS concentrations is demonstrated by the low correlation between concentrations in ECS and the ultrafiltrate of serum (CBZ, r= 0.41; 10-OH-CZ, r= 0.42; LTG, r= 0.27) in contrast to the high correlation between brain tissue concentration and the ultrafiltrate of serum (CBZ, r= 0.97; 10-OH-CZ, r= 0.88; LTG, r= 0.98) in the same group of patients. When comparing AED concentrations in the ECS with those in the CSF, ECS concentrations were significantly lower for CBZ, 10-OH-CZ, LTG, and LEV. CONCLUSIONS: The data demonstrate that AED concentrations show a considerable intraindividual and interindividual variation in the ECS of cortical regions. Furthermore, the ECS concentration of several AEDs is significantly lower than their CSF concentration in patients with intractable epilepsy. However, in the absence of data from nonepileptic tissues, it is not possible to judge whether the present findings relate to overexpression of multidrug transporters in the brain. Instead, the present study illustrates the methodologic difficulties involved in performing IOMD studies in patients and may thus be helpful for future approaches aimed at elucidating the role of multidrug transporters in epilepsy.

Optical imaging of epileptiform activity in experimentally induced cortical malformations.

Electrophysiological studies of human cortical dysplasia and rodent models revealed widespread hyperexcitability in the malformation itself as well as in its vicinity. We here analyzed the initiation of paroxysmal epileptiform activity using optical imaging of neuronal activity in rats with cortical malformations induced by neonatal freeze lesions. Brain slice preparations were incubated with the voltage-sensitive dye RH795 and neuronal activity was monitored using a fast-imaging photodiode array combined with standard field potential recordings. Spontaneous paroxysmal epileptiform activity emerged in all slices from animals with cortical malformations and sham-operated controls 20-40 min after omission of extracellular Mg(2+). Following electrophysiological and optical recordings, slices were histochemically processed. Using this approach, the present study demonstrated that in animals with freeze-lesion-induced focal cortical malformations, paroxysmal epileptiform activity always emerged from the dysplastic cortex and then spread to adjacent areas through superficial layers. This distribution of initiation sites was significantly different to sham-operated controls in which epileptogenic foci were located in various cytoarchitectonic areas. The present study indicates that following global changes in excitability, the dysplastic cortex itself is the main initiation site of paroxysmal epileptiform activity in animals with focal cortical malformations.

Blockade of astrocyte metabolism causes delayed excitation as revealed by voltage-sensitive dyes in mouse brainstem slices.

Fluoroacetate is known to block cell metabolism and to change potassium conductances selectively in astrocytes. In a functional neuronal network with ongoing activity, we investigated the effects of such a blockade of the astrocytic metabolism by fluoroacetate on neuronal signal propagation. Transverse 400- microm slices were prepared from the caudal medulla of mice of postnatal day 3-8, which contained the hypoglossal nucleus receiving excitatory synaptic input from the ventral respiratory group. Propagation of excitation within this network was measured by optical imaging using the voltage-sensitive dye RH 795. A 464-element photodiode array allowed fast recordings of voltage changes within a small population of cells. The spatial and temporal resolution was advanced to 32 microm and 1.27 ms, respectively. Changes of cellular membrane potential levels were expressed as relative changes of fluorescence (DeltaI/I). Stimulus-evoked excitation of neurons propagating from the ventral respiratory group to the hypoglossal nucleus peaked after 7.2+/-0.6 ms ( n=6). The latency of this early excitatory response is consistent with the time course of stimulus-evoked EPSPs in whole-cell recordings. Mean changes of fluorescence in the hypoglossal nucleus were -2.1+/-0.5 x 10(-3) (DeltaI/I). After incubation in 1 mM fluoroacetate, the early depolarization was reduced to 69.1+/-9.8% of control ( n=6, p=0.034). Additionally, fluoroacetate induced a delayed excitatory response, such that fluorescence intensity did not return to baseline within 1s. Propagation velocity and spatial distribution of the voltage signal were not affected by fluoroacetate. Our results suggest that blockade of astrocyte metabolism impairs fast synaptic transmission and induces a delayed excitation, probably resulting from the combination of reduced repolarization of neurons and persistent depolarization of astrocytes.

A new neurophysiological/neuropathological ex vivo model localizes the origin of glioma-associated epileptogenesis in the invasion area.

Seizures commonly occur in glioma patients, but their pathogenesis is poorly understood, in part due to a lack of valid and versatile experimental models. We have established a new model that enables comprehensive neuropathological and neurophysiological analysis on identical tissue preparations. Rat C6 glioma cells stably transfected with a green fluorescence protein (GFP) gene are transplanted into rat neocortex, giving rise to diffusely invading gliomas histologically resembling human glioblastomas. After 2 weeks, 500- micro m-thick cerebral slices are prepared, stained with the voltage-sensitive dye RH795, and fluorescence changes associated with origin and spread of abnormal bioelectric activity upon washout of Mg(2+) are detected by a 464-element photodiode array at a rate of 785 frames/s. GFP fluorescence promotes identification of tumor cells during electrophysiological experiments and in neuropathological analyses using frozen and paraffin-embedded tissue sections. By performing subsequent histological analysis of the slices examined neurophysiologically, origin and spread of abnormal activity can be correlated with structural and molecular (immunohistochemical) features. Specifically, we found that ictaform activity was initiated in cortical areas diffusely invaded by single tumor cells. This model is useful for further elucidating the electrophysiological, molecular and structural basis of glioma-associated epileptogenesis.

Effect of levetiracetam on epileptiform discharges in human neocortical slices.

PURPOSE: The anticonvulsant effects of the novel antiepileptic drug (AED) levetiracetam (LEV) were tested in neocortical slice preparations from 23 patients who underwent surgery for the treatment of refractory epilepsy. METHODS: Slices were used to evaluate the effects of LEV on two different models of epilepsy: low-Mg2+-induced untriggered and bicuculline-evoked stimulus-triggered epileptiform burst discharges and spontaneously appearing rhythmic sharp waves. RESULTS: LEV (0.1-1 mM) did not influence spontaneously appearing rhythmic sharp waves or Mg2+-free aCSF-induced epileptiform field potentials. LEV affected neither the amplitudes or duration nor the repetition rates of burst discharges in these epilepsy models. However, LEV (100-500 microM) significantly suppressed the ictal-like discharges elicited by the gamma-aminobutyric acid subtype A (GABAA)-receptor antagonist bicuculline. A marked reduction of the amplitude and duration of bicuculline-evoked field response in the presence of LEV was observed. CONCLUSIONS: The results indicate the potential for LEV to inhibit epileptiform burst discharges in human neocortical tissue, which is consistent with its effects in animal models of epilepsy. These results also support the seizure reduction observed in clinical trials and support that this may, in part, be related to the ability of LEV to inhibit epileptiform discharges.