Antiepileptic drugs: detection, quantification, and evaluation

The anticonvulsant potential of chemical substances can be identified with test procedures that act at various biological levels, ranging from subcellular elements to the normal or modified intact animal. All of these procedures modify either some minimal overt threshold electrochemical or neurochemical event or a suprathreshold manifestation such as seizure spread. This suggests that laboratory tests for the detection, quantification, and evaluation of antiepileptic drugs should be designed to identify substances that elevate seizure threshold and/or prevent seizure spread. The s.c. Metrazol (pentylenetetrazol) seizure threshold test and the supramaximal electroshock seizure test are commonly used to achieve this objective. Additional chemoshock tests may be used to delineate further the mechanisms of anticonvulsant action. Numerous variables, such as experimental animals, electroshock apparatus, parameters of electrical and chemical stimulus, and routes of drug administration, must be controlled to ensure accurate, reliable, and reproducible results. The in vivo procedures described are reliable and reproducible, and predict clinical utility of the drugs tested. New models for testing anticonvulsant activity are evaluated against clinically effective antiepileptic drugs originally identified by these same procedures.     

Age related increased threshold for electroshock seizure in BDF1 mice

The thresholds for inducing the minimal and maximal electroshock seizures were examined in relation to age in BDF1 mice of both sexes. The 50 percent effective intensities for the maximal electroshock seizure (tonic hindlimb extensor component) were lowest in the youngest age groups (6-month-old) for both male (10.68 mA) and female (9.18 mA) animals. The threshold increased with age and became significantly higher at 24 months (14.00 mA, 12.70 mA for male and female mice respectively). There was also a further increase in threshold at 30 months for both sexes. Similarly, the threshold for inducing the minimal seizure also increased with age but the differences in mean threshold levels between the youngest and oldest groups were much smaller in comparison to the maximal seizure. It was concluded that the threshold for inducing electroshock seizures significantly increases with age in mice of both sexes.     

Stimulus- and cell-type-specific release of purines in cultured rat forebrain astrocytes and neurons

Adenosine is formed during conditions that deplete ATP, such as ischemia. Adenosine deaminase converts adenosine into inosine, and both adenosine and inosine can be beneficial for postischemic recovery. This study investigated adenosine and inosine release from astrocytes and neurons during chemical hypoxia or oxygen-glucose deprivation. In both cell types, 2-deoxyglucose was the most effective stimulus for depleting cellular ATP and for evoking inosine release; in contrast, oxygen-glucose deprivation evoked the greatest adenosine release. alpha,beta-Methylene ADP, an inhibitor of ecto-5'nucleotidase, significantly reduced adenosine release from astrocytes but not neurons. Dipyridamole, an inhibitor of equilibrative nucleoside transporters, inhibited both adenosine and inosine release from neurons. Erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase, reduced neuronal inosine release evoked by oxygen-glucose deprivation but not by 2-deoxyglucose treatment. These data indicate that (1). astrocytes release adenine nucleotides that are hydrolyzed extracellularly to adenosine, whereas neurons release adenosine per se, (2). inosine is formed intracellularly and released via nucleoside transporters, and (3). inosine is formed by an adenosine deaminase-dependent pathway during oxygen-glucose deprivation but not during 2-deoxyglucose treatment. In summary, the metabolic pathways for adenosine formation and release were cell-type dependent whereas the pathways for inosine formation were stimulus dependent.     

Acupuncture on experimental epilepsies

The therapeutic effect of acupuncture on epilepsies was evaluated in 4 experimental models. 24 acupuncture points were tried. In electroconvulsive threshold model, square wave electrical stimulus of 0.2 msec and 6 Hz was applied through a pair of cotton electrodes at the cornea of mice for 3 sec. The stimulus intensity to induce stun reaction of the mouse was compared. In 86 control animals, the stimulus threshold was 0.70 +/- 0.22 mA. In the acupuncture treated group (N = 80), the threshold was 0.75 +/- 0.14 mA. In maximal electroshock model, the stimulus parameters were 60 Hz, 0.4 sec and 75 mA. The tonic extensor response of the hindlimbs of the mice was observed. 75.7% of the 115 control mice and 77.5% of the 80 acupuncture treated mice were observed to have tonic extensor response. In the focal cortical penicillin model, penicillin was applied at the subpial space over the exposed cortex of 24 cats. After the appearance of repeated spikes in ECoG, acupuncture was performed. In 175 trials the interictal spikes were decreased in 16 times, increased in 82 times. In 99 trials during seizures, the ictal activity was decreased in 4 times, increased in 79 times. In the intravenous penicillin model, high dose penicillin (1,000,000-1,500,000 U/kg) was given to 20 cats. It induced repetitive spikes and frequently even seizure discharges in EEG. Acupuncture was then tried. In 192 instances, acupuncture reduced the spikes in 13, increased the spikes in 103 times. In 74 trials during seizures, the ictal activity was suppressed in 4 times and aggravated in 66 times.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in threshold and pattern of electroshock-induced seizures in rats depending on site of stimulation

Although most laboratories employ transcorneal stimulation as a means of producing electroshock seizures, transauricular stimulation is also used by many investigators. The present study shows that seizures produced with transcorneal electroshock differ from those produced by transauricular electroshock in several ways: (1) transauricular stimulation is more effective at eliciting tonic convulsions; (2) the threshold for clonus is lower when transcorneal electrodes are used; and (3) the face and forelimb clonus produced by transcorneal stimulation cannot be produced with transauricular stimulation at any current. The present findings are consistent with the hypothesis that tonic seizures are more easily triggered with transauricular stimulation because they originate in the brainstem and because this brain region is preferentially activated when ear-clip electrodes are used.     

Change in the signal properties of a conditioned stimulus during two-way avoidance conditioning in rats

Acquisition of two components of conditioned active avoidance behavior by rats was studied. First presentations of electroshock evoked a number of different behavioral reactions. However, after five trials many rats learned to escape punishment running away to another part of a shuttle-box. The efficiency of the avoidance reaction conditioning significantly depended on the ability of an animal to learn the correct escape reaction to the unconditioned stimulus. However, some animals were incapable for acquisition of the conditioned reaction despite their high level of successful escapes. Increase in the number of negative reactions to the conditioned stimulus (light) at the next stage of learning suggests that the conditioned stimulus becomes the signal of forthcoming punishment. The ability of an animal to identify the conditioned stimulus as a signal significantly affected the efficiency of conditioned avoidance acquisition.     

Anticonvulsant effect of cytoskeletal depolymerizers in combination with potassium channel opener and adenylate cyclase activator; a causative link with nerve growth factor?

Anticonvulsant effect of cytoskeletal depolymerizing drugs in combination with potassium channel (KATP) opener and adenylate cyclase activator was evaluated in animal models of epilepsy. Seizures were induced in the animals by subjecting them to maximal electroshock (MES) or by injecting a chemical convulsant, pentylenetetrazole (PTZ). Moreover a correlation with the nerve growth factor (NGF) was also investigated. The anticonvulsant effect of minoxidil (1200 micrograms/kg i.p.) and Deacetylforskolin (600 micrograms/kg i.p.) was significantly enhanced in the mice pre-treated with cytoskeletal depolymerizing drugs. On the other hand nerve growth factor potentiated the convulsive phenomenon and decreased the seizure threshold in both the electroshock and chemically induced convulsions. Another interesting feature was the interaction of cytochalasin B, a microfilament disrupter in preventing the action of mNGF and PTZ. This study demonstrates the importance of interaction between cytoskeletal structures and signalling molecules in determining the convulsive threshold. This study clearly points to the importance of the nerve growth factor in convulsive phenomenon.     

The effect of electroshock on regional CNS energy reserves in mice

ATP, phosphocreatine, glycogen, glucose and lactate levels were measured in the cerebral cortex, thalamus, cerebellum, brain stem and spinal cord of mice following supramaximal electroshock. During the initial 17 s after the onset of a 2 s electrical stimulus high energy phosphate expenditure exceeded formation in all regions but was slower in spinal cord than in the other regions. In cerebral cortex high energy phosphate utilization continued to exceed formation for 32 s which was twice as long as in any other region studied. Altered levels of metabolites recovered most rapidly in spinal cord and least rapidly in cerebral cortex. Pretreatment with a non-anaesthetic dose of phenobarbitone influenced the effect of electroshock. Most of the clinical seizure was prevented, and increased high energy phosphate utilization was sustained for a much shorter period. Only in cerebral cortex did high energy phosphate expenditure exceed formation for as long as 15 s after the electrical stimulus; but even in this region the excess of expenditure over formation was much less than in untreated animals.     

Adenine derivatives as neurohumoral agents in the brain. The quantities liberated on excitation of superfused cerebral tissues

Adenine nucleotides of guinea-pig neocortical tissues were labelled by incubation with [(14)C]adenine and excess of adenine was then removed by superfusion with precursor-free medium. Adenine derivatives released from the tissue during continued superfusion, including a period of electrical stimulation of the tissue, were collected by adsorption and examined after elution and concentration. The stimulation greatly increased the (14)C output, and material collected during and just after stimulation had a u.v. spectrum which indicated adenosine to be a major component. The additional presence of inosine and hypoxanthine was shown by chromatography and adenosine was identified also by using adenosine deaminase. Total adenine derivatives released from the tissue during a 10min period of stimulation were obtained as hypoxanthine, after deamination and hydrolysis of adenosine and inosine, and amounted to 159nmol/g of tissue. This corresponded to the release of approx. 7pmol/g of tissue per applied stimulus. The hypoxanthine sample derived from superfusate hypoxanthine, inosine and adenosine was of similar specific radioactivity to the sample of inosine separated chromatographically, and each was of higher specific radioactivity than the adenine nucleotides obtained by cold-acid extraction of the tissue.     

On Blair's theory of excitation and the role of internal energy sources

It is shown that Blair's theory of excitation is independent of, and consequently valid for, any possible relationship between the threshold and the magnitude of the stimulus. It is pointed out that if a dependence of threshold on stimulus is assumed, the concept of rheobase becomes meaningless. Consequently contrary to Blair's impression, the disagreement between his theory in its original form and the experimental data on the time of incipient excitation with constant stimulus (response time) cannot be explained by assuming a dependence of the threshold on the magnitude of the stimulus. It is shown that a modification of Blair's interpretation, obtained by taking into account effects of internal energy sources released by the stimulus, eliminates the disagreement mentioned above between theory and experiment. The role of such modification in connection with propagation of excitation is discussed.     

New method for quantitative evaluation of esophageal sensibility

A method for quantitating esophagus sensibility by an electric stimulation test is described. Square stimulus waveform at different voltages and durations were transmitted to the esophagus, three series of electric stimuli being used in successive durations (0.5, 1, 2, 4, 8 and 16 ms); in each series the voltage discharge was increased progressively from 0 mV, until the subject noted the first sensation. This procedure was carried out at all esophageal levels. The following parameters were analyzed: sensitive threshold along the esophagus; the relation of threshold sensibility (mV) duration of stimulus (ms), and reobase and cronaxia for each esophageal level. At all esophageal levels, the sensitive threshold was regular and coherent; in the middle esophagus a zone was found having higher sensitive threshold than the proximal and distal esophageal zones. The relationship between sensitive threshold and inverse of the stimulus duration indicated that esophageal sensibility follows the basic law of excitation of WEISS, at least with this type of stimulus, reobase and cronaxia being representative of the sensibility threshold along the esophagus. Quantitative esophageal sensibility, therefore is concluded to be particularly suited to evaluation by electric stimulation.     

Adenine nucleotide degradation in the thoroughbred horse with increasing exercise duration

Adenine nucleotide (AN) degradation has been shown to occur during intense exercise in the horse and in man, at or close to the point of fatigue. The aim of the study was to compare the concentrations of muscle inosine 5'-monophosphate (IMP) and plasma ammonia (NH3) during intense exercise with the concentrations of muscle and blood lactate. Seven trained thoroughbred horses were used in the study. Each exercised on a treadmill for periods of between 30 s and 150 s, at 11 and/or 12 m.s-1. Blood and muscle samples were taken and analysed for lactate and NH3 and adenosine 5'-triphosphate (ATP), phosphorylcreatine (PCr), IMP, creatine, lactate and glycerol-3-phosphate respectively. Horses showed varying degrees of AN degradation as indicated by plasma [NH3] and muscle [ATP] and [IMP]. Comparisons of [IMP] with muscle [lactate], and plasma [NH3] with that of blood [lactate] indicated a threshold to the start of AN degradation. This threshold corresponded to a lactate content of around 80 mmol.kg-1 dry muscle and 15 mmol.l-1 in blood. We discuss the mechanisms which have been proposed to account for AN degradation and suggest that IMP formation occurs as a result of a sudden rise in the concentration of adenosine 5'-diphosphate (ADP) and consequently the concentration of adenosine 5'-monophosphate. The data suggest a critical pH below which there may be a substantial reduction in the kinetics of ADP rephosphorylation provided by PCr resulting in an increase in [ADP], which is the stimulus to AN degradation during intense exercise.     

Memory retention of appetitive and aversive conditioning in the damselfish Stegastes fuscus

We compared the memory of damselfish Stegastes fuscus in an aversive and appetitive conditioning task. Fish were trained to associate the sides of the tank that corresponded to the presence of a positive (conspecific presence) or negative (electroshock) stimulus. After two conditioning sessions, they were tested for learning. The fish conditioned to the stimulus were then re-tested for memory retention after 5, 10 or 15 days. Both the positive and negative rewards were associated with a specific side of the tank, indicating learning ability. Additionally, in both contexts, S. fuscus stored the information learned and showed similar behavioural patterns after 5, 10 and 15 days, suggesting long-lasting memory. For the ecological context, long lasting memories of social encounters outcomes and negative experiences of threatening situations may confer advantages that ultimately affect fishes’ fitness.     

Energy model for contrast detection: spatiotemporal characteristics of threshold vision

A model for contrast detection of spatiotemporal stimuli is proposed which consists of a spatiotemporal linear filter, an energy device and a threshold device. Assuming the existence of independent intrinsic noise, the probability of stimulus detection was approximated by a Weibull function of the response energy. With this assumption, the stimulus energy is a constant at fixed detection probability. This energy model for contrast detection satisfactorily accounted for the elliptical threshold contours of line pairs at stimulus separations within the range 2–30 min and at stimulus onset asynchronies within the range 20–140 ms. The threshold contour at a large stimulus onset asynchrony (300 ms) was in the form of a rounded square. This finding was explained by assuming that the probability of seeing the line pair was determined by the joint probability that at least one stimulus had been detected. With the energy model, the temporal and spatial autocorrelation functions of the response to a flashed line were evaluated. The autocorrelation functions thus determined were used to predict the temporal contrast sensitivity function to a flickering line stimulus and the spatial contrast sensitivity function to flashed gratings, which were in agreement with the experimental data. The data obtained were fitted adequately by an impulse response approximated by a spatiotemporal Gabor-like function. Received: 08 December 1997 / Accepted in revised form: 26 January 1999     

Time course and action spectrum of vibrotactile adaptation

In a series of experiments designed to explore the processes underlying adaptation of the sense of flutter-vibration, vibrotactile threshold was measured on the pad of the index finger, using Békésy tracking. Unadapted thresholds were first measured, for a number of frequencies (4-90 Hz) and contactor sizes (1-8 mm diameter). As expected, these measurements indicated the presence of (1) a Pacinian system possessing spatial summation and increasing in sensitivity, as frequency was raised, at the rate of 12 dB/octave; and (2) a non-Pacinian system showing little spatial summation, and with a frequency characteristic matching that of the NP I mechanism of Bolanowski et al. (1988). These baseline data of Experiment 1 guided the selection of stimulus parameters for subsequent experiments, in which threshold for a test stimulus was measured before, during, and after periods of vibrotactile adaptation. In Experiment 2, test stimuli of 10 Hz and 50 Hz were combined factorially with 30-dB SL adapting stimuli of the same two frequencies. When the test stimulus was 10 Hz, the two adapting frequencies were equally effective in raising threshold; however, when the 50-Hz test stimulus was used, the 50-Hz adapting stimulus raised threshold by a greater amount than did the 10-Hz adapter. These results confirm on the finger the independence of adaptation in Pacinian and non-Pacinian channels, a result previously established on the thenar by other workers. For all four frequency combinations, threshold rose exponentially with a time constant of 1.5-2 min. In Experiment 3, an action spectrum was determined, showing the adapting amplitude needed at each of a series of frequencies to raise the threshold of a 10-Hz stimulus by 10 dB; this spectrum was essentially flat from 30 to 90 Hz. The results, taken in conjunction with what is known about rapidly adapting cutaneous mechanoreceptors, imply that the effectiveness of an adapting stimulus is not determined solely by the amount of activity it generates in first-order afferents.     

Effect of stimulus volume on taste detection threshold for NaCl

An ascending method of limits with a three stimulus forced choiceresponse procedure was used to determine detection thresholdsfor NaCl at stimulus volumes of 0.05, 0.5 and 0.9 ml, with andwithout an intertrial 0.9-ml water rinse. Results suggestedan inverse relationship between stimulus volume and taste threshold.The rinse condition produced a small non-significant improvementin detection for all volumes. Thresholds, varying from 1.8 to5.6 mM, were similar to those determined by other investigatorsin which much larger stimulus volumes were used. These resultsdemonstrate that a rapid, small volume, no-rinse method of thetype commonly used in clinical practice to measure taste detectionthresholds can be used to yield valid estimates of taste threshold.     

Time Course and Action Spectrum of Vibrotactile Adaptation

In a series of experiments designed to explore the processes underlying adaptation of the sense of flutter-vibration, vibrotactile threshold was measured on the pad of the index finger, using Békésy tracking. Unadapted thresholds were first measured, for a number of frequencies (4-90 Hz) and contactor sizes (1-8 mm diameter). As expected, these measurements indicated the presence of (1) a Pacinian system possessing spatial summation and increasing in sensitivity, as frequency was raised, at the rate of 12 dB/octave; and (2) a non-Pacinian system showing little spatial summation, and with a frequency characteristic matching that of the NP I mechanism of Bolanowski et al. (1988). These baseline data of Experiment 1 guided the selection of stimulus parameters for subsequent experiments, in which threshold for a test stimulus was measured before, during, and after periods of vibrotactile adaptation.

In Experiment 2, test stimuli of 10 H_z and 50 H_z were combined factorially with 30-dB SL adapting stimuli of the same two frequencies. When the test stimulus was 10 Hz, the two adapting frequencies were equally effective in raising threshold; however, when the 50-Hz test stimulus was used, the 50-Hz adapting stimulus raised threshold by a greater amount than did the 10-Hz adapter. These results confirm on the finger the independence of adaptation in Pacinian and non-Pacinian channels, a result previously established on the thenar by other workers. For all four frequency combinations, threshold rose exponentially with a time constant of 1.5-2 min.

In Experiment 3, an action spectrum was determined, showing the adapting amplitude needed at each of a series of frequencies to raise the threshold of a 10-Hz stimulus by 10 dB; this spectrum was essentially flat from 30 to 90 Hz. The results, taken in conjunction with what is known about rapidly adapting cutaneous mechanoreceptors, imply that the effectiveness of an adapting stimulus is not determined solely by the amount of activity it generates in first-order afferents.     

The influence of rate of temperature change and peak stimulus duration on pain intensity and quality

An important aspect of experimental pain research is that the assessment methods can investigate the different aspects of pain perception. The aim of the present study was to investigate the influence of rate of temperature change and peak stimulus duration on heat evoked pain intensity and quality. All stimuli were applied within the medial aspect of the anterior forearm. The rate of temperature change was varied from 1 to 16 C/s without any effect on the pain threshold. The pain threshold decreased with an increasing peak stimulus duration from 0.1 to 2 s, but not from 2 to 3 s. The pain intensity for suprathreshold stimuli (46 C, 48 C, 50 C) increased for decreasing rates and increasing duration. The pain intensity was highly correlated with the energy of the stimulus. When the rates of temperature change (1-16 C/s) are varied, no differences between pricking and burning pain were present at either low stimulus intensity (46 C) or high stimulus intensity (50 C). At low stimulus intensity (46 C), the pricking pain was not influenced by the duration (0.1-3 s), but the burning pain was intensified when the duration was increased from 1.5 to 3 s. At high intensity stimuli (50 C), the pricking pain intensified with an increased duration, whereas burning pain did not. The heat pain threshold is influenced by the peak stimulus duration, and not by the rate of temperature change. If suprathreshold stimuli are used, both the rate of temperature change and the peak stimulus duration can strongly affect the pain intensity and the pain quality. Therefore, the same stimulus modality can be used to assess the modulation of different pain intensities and of the pricking and burning pain qualities simply by varying the stimulus configuration.     

Inosine-stimulated insulin release and metabolism of inosine in isolated mouse pancreatic islets.

Inosine is a potent primary stimulus of insulin secretion from isolated mouse islets. The inosine-induced insulin secretion was totally depressed during starvation, but was completely restored by the addition of 5 mM-caffeine to the medium and partially restored by the addition of 5 mM-glucose. Mannoheptulose (3 mg/ml) potentiated the effect of 10 mM-inosine in islets from fed mice. The mechanism of the stimulatory effect of inosine was further investigated, and it was demonstrated that pancreatic islets contain a nucleoside phosphorylase capable of converting inosine into hypoxanthine and ribose 1-phosphate. Inosine at 10 mM concentration increased the lactate production and the content of ATP, glucose 6-phosphate (fructose 1,6-diphosphate + triose phosphates) and cyclic AMP in islets from fed mice. In islets from starved mice inosine-induced lactate production was decreased and no change in the concentration of cyclic AMP could be demonstrated, whereas the concentration of ATP and glucose 6-phosphate rose. Inosine (10 mM) induced a higher concentration of (fructose 1,6-diphosphate + triose phosphates) in islets from starved mice than in islets from fed mice suggesting that in starvation the activities of glyceraldehyde 3-phosphate dehydrogenase or other enzymes below this step in glycolysis are decreased. Formation of glucose from inosine was negligible. Inosine had no direct effect on adenylate cyclase activity in islet homogenates. The observed changes in insulin secretion and islet metabolism mimic what is seen when glucose and glyceraldehyde stimulate insulin secretion, and as neither ribose nor hypoxanthine-stimulated insulin release, the results are interpreted as supporting the substrate-site hypothesis for glucose-induced insulin secretion according to which glucose has to be metabolized in the beta-cells before secretion is initiated.     

Effect of drugs modifying central serotonergic function on the response of extensor and nonextensor rats to maximal electroshock

Influences of reduction or enhancement of serotonergic function on convulsive responses and thresholds to maximal electroshock stimulation (MES) were studied in rats classified by MES as extensors or nonextensors. In extensors, serotonin reduction decreased the tonic convulsive threshold coincident with an increased incidence of hindlimb extension (HLE). Enhancement of serotonergic function with fluoxetine, pCA, 5-HTP, fenfluramine or 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) prevented HLE. This action was antagonized by pCA, strychnine or digitoxigenin and was not related to overt behavioral disruption or an elevated tonic threshold. In nonextensors, serotonin reduction restored HLE. HLE initiated by spinal cord stimulation was not remarkably effected by any treatment. Catecholamine reduction had no effect on any part of the tonic response in either group of rats. The results suggest the activation of central serotonergic inhibitory influences during the MES tonic convulsion. The possibility of graded-intensity serotonergic attenuation of seizure spread is discussed suggesting the action of serotonergic-enhancing drugs to prevent HLE may include a mechanism similar to a naturally-occurring “mechanism” to prevent HLE in nonextensor rats.