Development of multi-electrode array screening for anticonvulsants in acute rat brain slices

The acute hippocampal brain slice preparation is an important in vitro screening tool for potential anticonvulsants. Application of 4-aminopyridine (4-AP) or removal of external Mg2+ ions induces epileptiform bursting in slices which is analogous to electrical brain activity seen in status epilepticus states. We have developed these epileptiform models for use with multi-electrode arrays (MEAs), allowing recording across the hippocampal slice surface from 59 points. We present validation of this novel approach and analyses using two anticonvulsants, felbamate and phenobarbital, the effects of which have already been assessed in these models using conventional extracellular recordings. In addition to assessing drug effects on commonly described parameters (duration, amplitude and frequency), we describe novel methods using the MEA to assess burst propagation speeds and the underlying frequencies that contribute to the epileptiform activity seen. Contour plots are also used as a method of illustrating burst activity. Finally, we describe hitherto unreported properties of epileptiform bursting induced by 100M4-AP or removal of external Mg2+ ions. Specifically, we observed decreases over time in burst amplitude and increase over time in burst frequency in the absence of additional pharmacological interventions. These MEA methods enhance the depth, quality and range of data that can be derived from the hippocampal slice preparation compared to conventional extracellular recordings. It may also uncover additional modes of action that contribute to anti-epileptiform drug effects

Induction of cytochrome P-450 activity in individual Chironomus riparius Meigen larvae exposed to xenobiotics

Cytochrome P450 activity in individual Chironomus riparius larvae was measured using a microtiter plate adaptation of the ethoxyresorufin-O-deethylase (EROD) assay. The sensitivity of this biomarker was tested by exposing larvae to phenobarbital (0.5 and 1.0 mM) and permethrin (1 and 10 mug/g). Both chemicals induced EROD activity in C. riparius larvae by up to 1.58-fold with PB and 2.47-fold with permethrin. EROD induction was more pronounced after 48 h. The initially high EROD activity in the controls suggested that P450s are induced by stress. Feeding levels prior to exposure also had a significant effect on EROD activity. EROD activity compared to the control was highest when larvae were fed double the normal ration. These results indicate that EROD activity in individual C. riparius may be a useful biomarker to add to a suite of biomarkers for the detection of freshwater pollution. (C) 2002 Elsevier Science (USA). All rights reserved.

Intraclonal variability in Daphnia acetylcholinesterase activity: The implications for its applicability as a biomarker

The relationship between individual growth and acetylcholinesterase (AChE).activity was evaluated for Daphnia magna. Analysis on the influence of two different culture media on baseline AChE activity was performed with Daphnia similis. The results indicated an inverse relationship between D. magna body length and AChE activity. An increase in total protein, which was not proportional to an increase in the rate of the substrate hydrolysis (Delta absorbance/min), seems to be the reason for this inverse size versus AChE activity relationship. Therefore, toxicants such as phenobarbital, which affect protein and size but not AChE activity directly, have an overall affect on AChE activity. In contrast, the AChE inhibitor parathion altered AChE activity but not protein. Culture medium also had a significant affect on AChE activity in D. similis. Changes in total protein seem to be the main reason for the variations in baseline AChE activity in Daphnia observed in the different evaluations performed in this work. Therefore, AChE activity in Daphnia must be interpreted carefully, and variations related to changes in total protein must be taken into account when applying this enzyme as a biomarker in biological monitoring.

Development of multi-electrode array screening for anticonvulsants in acute rat brain slices

The acute hippocampal brain slice preparation is an important in vitro screening tool for potential anticonvulsants. Application of 4-aminopyridine (4-AP) or removal of external Mg2+ ions induces epileptiform bursting in slices which is analogous to electrical brain activity seen in status epilepticus states. We have developed these epileptiform models for use with multi-electrode arrays (MEAs), allowing recording across the hippocampal slice surface from 59 points. We present validation of this novel approach and analyses using two anticonvulsants, felbamate and phenobarbital, the effects of which have already been assessed in these models using conventional extracellular recordings. In addition to assessing drug effects on commonly described parameters (duration, amplitude and frequency), we describe novel methods using the MEA to assess burst propagation speeds and the underlying frequencies that contribute to the epileptiform activity seen. Contour plots are also used as a method of illustrating burst activity. Finally, we describe hitherto unreported properties of epileptiform, bursting induced by 100 mu M 4-AP or removal of external Mg2+ ions. Specifically, we observed decreases over time in burst amplitude and increase over time in burst frequency in the absence of additional pharmacological interventions. These MEA methods enhance the depth, quality and range of data that can be derived from the hippocampal slice preparation compared to conventional extracellular recordings. it may also uncover additional modes of action that contribute to anti-epileptiform drug effects. (C) 2009 Elsevier B.V. All rights reserved.

Cannabidivarin is anticonvulsant in mouse and rat in vitro and in seizure models

Summary Background and purpose: Phytocannabinoids in Cannabis sativa have diverse pharmacological targets extending beyond cannabinoid receptors and several exert notable anticonvulsant effects. For the first time, we investigated the anticonvulsant profile of the phytocannabinoid cannabidivarin (CBDV) in vitro and in in vivo seizure models. Experimental approach: The effect of CBDV (1-100μM) on epileptiform local field potentials (LFPs) induced in rat hippocampal brain slices by 4-AP application or Mg2+-free conditions was assessed by in vitro multi-electrode array recordings. Additionally, the anticonvulsant profile of CBDV (50-200 mg kg-1) in vivo was investigated in four rodent seizure models: maximal electroshock (mES) and audiogenic seizures in mice, and pentylenetetrazole (PTZ) and pilocarpine-induced seizures in rat. CBDV effects in combination with commonly-used antiepileptic drugs were investigated in rat seizures. Finally, the motor side effect profile of CBDV was investigated using static beam and gripstrength assays. Key results: CDBV significantly attenuated status epilepticus-like epileptiform LFPs induced by 4-AP and Mg2+-free conditions. CBDV had significant anticonvulsant effects in mES (≥100 mg kg-1), audiogenic (≥50 mg kg-1) and PTZ-induced seizures (≥100 mg kg-1). CBDV alone had no effect against pilocarpine-induced seizures, but significantly attenuated these seizures when administered with valproate or phenobarbital at 200 mg kg-1 CBDV. CBDV had no effect on motor function. Conclusions and Implications: These results indicate that CBDV is an effective anticonvulsant across a broad range of seizure models, does not significantly affect normal motor function and therefore merits further investigation in chronic epilepsy models to justify human trials.