Local Electrical Stimulation

Methods of Generation

Electroencephalograph^ afterdischarges (ADs) elicited by local electrical stimulation have different electrographic patterns and behavioral correlates according to the structure stimulated. The experiments can be aimed at two different phenomena: (1) measurement of threshold intensities of electrical current necessary for elicitation of ADs or (2) repeated stimulations with the same intensity but with short intervals not leading to kindling. This second type of arrangement can be used for studies of acute anticonvulsant effects.

Epileptic ADs represent a model of complex partial seizures (stimulation of limbic structures) or myoclonic seizures (stimulation of sensorimotor cortex). Animals used in these experiments are mostly rats; mice, rabbits, and cats are nowadays rarely used.

Procedures

The experiment starts with surgical preparation. Volatile anesthetics (diethylether, halothane, isoflurane) or barbiturates with a short to middle duration of action (e.g., pento-barbitone) are recommended. Dissociative anesthesia (ketamine 100mg/kg IP [administered intraperitoneally] and xylazine 20mg/kg IM [administered intramuscularly]) is also possible. Postsurgical recovery takes at least 1 week in adult animals with chronically implanted electrodes. Electrodes can be placed epidurally (flat or ball electrodes; higher stimulation intensities are then needed). If the electrodes are introduced into the cerebral cortex (the same type of electrodes as for subcortical stimulation), a stereotaxic apparatus must be used. Stimulation of subcortical structures can be performed using concentric electrodes or twisted twin electrodes. Care must be given to isolating the electrodes up to the tips.

Different frequencies of stimulation can be used similarly to the electroshock model: high frequency (60 or 50 Hz, less frequently higher frequencies) or low frequency (from 3 to 12 Hz). Rectangular pulses are more common, but sinusoid waves may also be used. Biphasic pulses (i.e., those with the same duration of both phases) are preferable to avoid excessive polarization of electrodes. Duration of stimulation series is in an inverse relation to frequency: High-frequency stimulation is usually applied only for 1 to 2 seconds, whereas low-frequency stimulation has to be applied for longer time (up to tens of seconds). Stimulation sessions might be repeated if very high intensities of stimulation are not used. It is necessary to avoid initial kindling because progressive lengthening of ADs (see Chapter 28) might interfere with the action of AEDs. Long-lasting stimulation can lead to status epilepticus (see Chapter 36). In the case of pharmacologic experiments, the half-life of the administered drug has to be taken into account.

Monitoring requires EEG apparatus; a sample rate of 200 Hz might not be sufficient, or the sharp EEG discharges can be distorted. Digitization at a rate of 500 Hz is recommended. The evaluation focuses on the presence or absence of epileptic ADs (a duration of 5 seconds is sometimes taken as a criterion), their pattern, and accompanying behavioral phenomena.

This method is not difficult to develop, but it is reliable only with experience in the field (anesthesia of animals, stereotaxic implantation of electrodes, and concomitant registration of EEG and behavior).

Characteristics

Behavioral Features

Behavior depends on the stimulated structure and age (see below). Spread of epileptic activity (especially if high stimulation intensities are used) modifies behavioral pattern of seizures (see Chapters 28 and 30).

Electroencephalographs Features

Afterdischarges may be of different types in relation to the stimulated structure: cortical or thalamic low-frequency stimulation elicits spike-and-wave rhythm, whereas limbic ADs are characterized by trains of fast spikes, large delta waves (sometimes with superimposed low-amplitude spikes), or sharp theta waves. Again the spread of epileptic activity can modify the EEG pattern of ADs (e.g., high-intensity neocortical stimulation; see later). The duration of ADs also depends on the stimulated structure: spike-and-wave type lasts usually only a few seconds; limbic ADs are substantially longer: from tens of seconds to few minutes.

Even a single AD results in plastic changes. Immediately after the end of the Ads, postictal depression (postictal refractoriness) appears (Dyer et al., 1979); its duration may change with stimulated structure and age (see later discussion). Postictal depression prevents elicitation of the second seizure for a maximum of few minutes, with a progressive return to the prestimulation level (i.e., there is an absolute and relative refractory phase). This refractoriness is probably due to prolonged activation of inhibitory systems responsible for termination of seizures. Depression is followed by potentiation; if stimulation is repeated with long intervals, kindling may be induced (see Chapters 28-30). Possible changes in the range of tens of minutes to a few hours were studied only exceptionally, and repeated poten-

tiation and suppression phases cannot be excluded Neuropathology was not studied after a single or a few seizures. Investigators should be aware that even the insertion of electrodes may lead to focal changes (focal hemorrhage, neuronal damage, activation of glia). Penetration of electrodes into the hippocampus (i.e., a mechanical insult) may induce a short-lasting seizure.

Response to AEDs was repeatedly studied. There are many studies, but they are far from being comparable and complete because of differences in stimulated structure, stimulation paradigm, methods of evaluation, and selection of AEDs to be studied.

Limitations

Electrically induced ADs require good equipment and operator experience to be reproducible. In addition, they are time consuming. On the other hand, they provide much more information than electroshock seizures do. Mortality of animals depends mostly on surgical technique and post-surgical care.

Afterdischarges can be elicited if a certain level of maturation is achieved. The exact age depends on the structure stimulated (see later discussion).

Standardization is necessary in evaluation of behavioral phenomena accompanying ADs. Most common is a scale published by Racine (1972), originally for limbic stimulation (see Table 1); but with slight modification it can be used for ADs elicited from various structures (e.g., cortical ADs) (Mares et al., 2002).

Afterdischarges Elicited by Stimulation of Individual Brain Structures

Neocortex

Two experimental arrangements using rats have been published ( Kubova et al., 1996; Voskuyl et al., 1989, 1992).

Voskuyl's method uses electrodes over right and left motor cortical areas; that is, stimulation of both hemispheres is performed. The intensity of current is progressively increased during the stimulation series. Originally they estimated only the threshold intensity of current necessary to induce clonic movements during stimulation (threshold for localized seizure activity); later this method was extended to the second parameter: spread of convulsions (threshold for generalized seizure activity) (Hoogerkamp et al., 1994; Krupp and Löscher, 1998). An advantage of this method is the possibility of performing repeated measurements. Using daily stimulation, the threshold values initially decreased, but after 10 days they stabilized and did not change further (Della Paschoa et al., 1998). Thus animals can be used for repeated measurements of drug effects. This has been done (with parallel measurement of pharmacokinetic parameters) with oxazepam, midazolam, phenytoin, valproate, lamotri-gine, tiagabine, and loreclezole (see Jonker et al., 2003).

The method developed in our laboratory applies both electrodes over the sensorimotor area of the same hemisphere; that is, only one hemisphere is stimulated, but the cortical area is more extensive than in the Voskuyl's model. Low-frequency stimulation (8 Hz for 15 seconds) is used; it allows evaluation of motor phenomena during stimulation (i.e., induced by direct excitation of the motor cortex) and epileptic ADs appearing after the end of stimulation. High-

frequency stimulation (50 Hz for 2 seconds) resulted in a similar pattern of ADs, but it cannot be used for studies of the direct effects of stimuli on the motor system (Mares et al., 2002). Repeated stimulations with a stepwise increase of current intensity make it possible to measure four different phenomena: (1) movements during stimulation, (2) spike-and-wave EEG ADs (Figure 2); (3) clonic seizures as a motor correlate of spike-and-wave ADs; (4) transition into

FIGURE 2 Electroencephalographic (EEG) recording of cortical aferdischarges (ADs) in an adult rat. Upper part: Spike-and-wave type of ADs accompanied by stage 4 with a transition into stage 3. Lower part: mixed type of ADs elicited in the same rat by higher stimulation intensity. The right sensorimotor cortical area was stimulated. Individual leads from top to bottom: LF, left sensorimotor (frontal); LP, left parietal; LO, left visual (occipital); and RO, right visual cortical regions. The last second of the 15-second stimulation series is in the frame at the beginning of the recordings. Full arrows mark the end of the spike-and-wave type of ADs; empty arrow denotes the end of the mixed type. Time mark is 2 seconds; amplitude calibration is 0.5mV.

FIGURE 2 Electroencephalographic (EEG) recording of cortical aferdischarges (ADs) in an adult rat. Upper part: Spike-and-wave type of ADs accompanied by stage 4 with a transition into stage 3. Lower part: mixed type of ADs elicited in the same rat by higher stimulation intensity. The right sensorimotor cortical area was stimulated. Individual leads from top to bottom: LF, left sensorimotor (frontal); LP, left parietal; LO, left visual (occipital); and RO, right visual cortical regions. The last second of the 15-second stimulation series is in the frame at the beginning of the recordings. Full arrows mark the end of the spike-and-wave type of ADs; empty arrow denotes the end of the mixed type. Time mark is 2 seconds; amplitude calibration is 0.5mV.

another type of AD (Figure 2) similar to that induced by stimulation of limbic structures accompanied by automatisms, mostly wet-dog shakes. Repeated elicitation of spike-and-wave ADs with unchanged stimulation intensity can be used for testing of antiepileptic drugs (Kubova et al., 1996).

Ontogeny Ontogenetic study demonstrated reliable elicitation of cortical ADs in rats from the age of 12 days and older if low-frequency stimulation is used or from postnatal day 9 and older if 50-Hz stimulation is applied. Twelve-day-old rat pups are not able to generate spike-and-wave rhythm; the AD is formed by rhythmic sharp delta waves (Mares et al., 2002). The maximal sensitivity to cortical stimulation is around the end of the third postnatal week of life in rats (Mares et al., 2002). In addition, repetition of stimulation in 12-day-old rats with 10- or 20-minute intervals leads to a progressive increase of duration of ADs; postictal depression, which is observed in adult rats, is absent in this age group.

Hippocampus

Electrodes can be introduced into the dorsal or ventral hippocampus. The pattern of ADs is variable: low-amplitude spikes, delta waves, and delta waves with superimposed low-amplitude spikes. The first AD is followed by a period of EEG depression, and then the secondary (recurrent) AD appears (Dyer et al., 1979; Leung, 1987). The most common behavioral pattern is wet-dog shakes (Le Gal la Salle et al., 1983; Velisek and Mares, 2004).

Ontogeny It is possible to evoke hippocampal ADs in rats at the age of 7 days (Velisek and Mares, 1991). In rat pups 12 to 15 days old, postictal depression is absent; 7-day-old rat pups exhibit postictal refractoriness, probably because of the fatigability of the system, not to a presence of active inhibition (Velisek and Mares, 1991).

Amygdala

There are differences among individual nuclei studied in detail in the kindling model. Epileptic automatisms form a behavioral pattern of these seizures: arrest at the beginning, followed by chewing movements that continue for several seconds after the end of stimulation (Goddard et al., 1969).

Ontogeny Ontogenetic studies demonstrated the possibility of eliciting these ADs at the end of the second postnatal week in rats (Moshe, 1981). In contrast to the results in older animals, amygdala ADs are not followed by a period of postictal depression in 2-week-old rats (Moshe and Albala, 1983). Baram et al. (1993) was able to elicit epileptic ADs by amygdala stimulation in 7-day-old rats.

Entorhinal Cortex

Electrodes can be introduced either into the entorhinal cortex or into the perforant path. It is possible to perform a preoperative electrophysiologic control using single stimuli and recording responses in the dentate gyrus. The pattern of ADs may be variable: low-amplitude spikes, large delta waves, and delta waves with superimposed low-amplitude spikes. Behavioral concomitants are represented by an intense orienting reaction in a cage where the animal spent tens of minutes and include locomotion, sniffing, and rearing. Such behavior is appropriate for the first 2 to 3 minutes in a new environment. This automatism appears toward the end of the AD and continues even after the EEG epileptic activity is over. Wet-dog shakes are observed less frequently.

Ontogeny Afterdischarges can be induced by stimulation of angular bundle in rat pups 10 to 11 days old, and adult characteristics are attained at the age of 21 days (Stringer and Lothman, 1992).

Piriform Cortex

Afterdischarges and their behavioral correlates elicited by stimulation of this structure do not differ from those induced by amygdala stimulation (Goddard et al., 1969; Honack et al., 1991). A disadvantage of piriform cortex is its anatomy: a very thin, rather long structure difficult for an exact placement of electrodes.

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