Convulsive Response

Electroshock induces a continuum of motor convulsions that are dependent on the intensity of the electrical stimulation current. The convulsive continuum as it occurs in rodents is indicated in Table 1.1 and is based on corneal stimulation (also known as transcorneal stimulation) using a 0.2 s duration, 60 Hz stimulus. Lower currents (below 20 mA in rats and 5 mA in mice) activate the forebrain selectively and produce only clonic convulsions. The clonic convulsions are useful

FIGURE 1.2

Tonic extension in the rat. Note that the animal has been rolled onto the side so that the convulsive phases may be observed. Depicted in panel A is a rat in tonic extension with tonic hindlimb extension (THE). This is the most severe electroshock response and may last up to 15 s. The rat depicted in part B is in tonic extension without THE. Abolition of THE is an indication of antiepileptic drug activity in generalized tonic-clonic (grand mal) seizures. Inhibition of THE is considered to have occurred when the legs do not extend beyond a 90° angle to the torso.29,30

FIGURE 1.2

Tonic extension in the rat. Note that the animal has been rolled onto the side so that the convulsive phases may be observed. Depicted in panel A is a rat in tonic extension with tonic hindlimb extension (THE). This is the most severe electroshock response and may last up to 15 s. The rat depicted in part B is in tonic extension without THE. Abolition of THE is an indication of antiepileptic drug activity in generalized tonic-clonic (grand mal) seizures. Inhibition of THE is considered to have occurred when the legs do not extend beyond a 90° angle to the torso.29,30

for detecting anticonvulsant activity of drugs effective against absence (petit mal) seizures. Repetition of such low current electroshock stimuli on a daily basis induces limbic kindling, as described below. Higher corneal electroshock currents activate brainstem mechanisms that produce tonic-clonic convulsions. The minimal electrical currents that are just sufficient to induce tonic-clonic convulsions are called threshold currents. The threshold currents induce tonic-clonic convulsions that are predictive of general anticonvulsant drug activity. Supramaximal currents that are five to seven times threshold induce tonic-clonic convulsions that are predictive of anticonvulsant drug activity against generalized tonic-clonic (grand mal) seizures. Thus, depending on the intensity of the stimulus a number of preclinical models of epilepsy are possible using corneal electroshock.

The convulsive continuum associated with increasing current strength of corneal electroshock, as outlined in Table 1.1, is detailed in the following sections. It should be noted that the electrical stimulation current strengths (in milliamps) shown to evoke each convulsive response in rats and mice as depicted in Table 1.1 are approximate and should be expected to vary, sometimes greatly, depending on the strain of animal.1781415 When initiating studies that require use of convulsive thresholds, pilot studies should always be performed to determine the electrical current thresholds for a given strain of animal in a particular laboratory setting.

1. Subconvulsive Response

Corneal electroshock in rats at currents below approximately 18 mA induces no convulsive activity (Table 1.1). Instead the animals appear irritated and display hyperactivity as well as escape and pain behaviors.1 The animals may exhibit a catatonic or "stun" response.1

2. Face and Forelimb Clonus

Slightly higher currents, in the 18 to 20 mA range in the rat, induce face and forelimb clonic activity11415 that may include a 5 s period of clonic spasms which involve facial clonus as well as clonic activity of all four limbs (Table 1.1).815 These convulsions are also termed minimal clonic seizures or minimal electroshock.115 The currents as administered by corneal electrodes are thought to activate forebrain areas and induce limbic seizure activity.12 The face and forelimb clonus induced by the initial stimulations only last for a few seconds.816 However, minimal electroshock repeated once or twice daily with corneal electrodes induces limbic kindling, with the forebrain convulsions becoming progressively longer in duration and greater in severity.816 The duration of the stimulus for this kindling effect is 4.0 s rather than the 0.2 s duration used in most electroshock procedures.8 This means that the investigator applying the stimulus must hold the corneal electrodes tightly to the animal for the entire 4 s period. Such corneal kindling induces convulsions that are similar to those observed in amygdala kindling17 and have a similar anticonvulsant profile.18

Besides serving as a model of limbic kindling that does not involve the placement of intracranial electrodes,18 the forebrain convulsions induced by corneal electroshock are proposed to represent a threshold for minimum seizures.15 The 0.2 s duration electrical current required to induce these convulsions represents the threshold current or the minimal stimulus needed to induce seizure activity in 97% to 100% of the animals.15 Drugs that raise this threshold prevent the occurrence of face and forelimb clonus or clonic spasms and are active against absence (petit mal) seizures.15 For example, valproate and ethosuximide are active in both the minimal clonic seizures induced by electroshock and generalized absence seizures.15 The absolute change in threshold induced by anticonvulsant drugs can be determined by the method of Litchfield and Wilcoxon,19 in which multiple groups of animals are tested with various stimulation currents until a CC50 (convulsant current in 50% of the animals) is determined.1 Alternatively, fewer animals and time are required to determine changes in threshold using the "up-down" or "staircase" methods in which the stimulation current for each animal is determined by the response of the previous animal.120-22

Minimal clonic seizures are also produced by low doses of the chemical con-vulsants pentylenetetrazol, bicuculline, and picrotoxin,81215 which are presented in Chapter 2. The face and forelimb clonus produced by these chemical convulsants is analogous to that produced by corneal stimulation in that both identify drugs that raise seizure threshold and that are effective against absence seizures.6815

The animals are considered to be unconscious during the minimal clonic sei-zures.1 This is because the seizure activity generalizes to, or involves, the entire forebrain and disrupts conscious awareness. Since seizure generalization is a feature common to all electroshock-induced convulsions, it is likely that all animals are unconscious during electroshock-induced minimal clonic seizures, as well as during more severe convulsions induced by higher electroshock currents.1

3. Running-Bouncing Clonus; Tonic Flexion

The next two successive convulsive responses evoked by progressively higher corneal electroshock stimulating currents are running-bouncing clonus and tonic flexion (Table 1.1). These occur in relatively limited current ranges and are therefore difficult to observe in isolation using corneal electroshock. The running-bouncing clonic convulsions have also been referred to as wild running and represent a response to the initial, minimal activation of the brainstem nuclei that mediate tonic-clonic convulsions.12 The running-bouncing clonus or wild running convulsions induced by electroshock correspond to the convulsions that are reported as rank 1 seizures (audiogenic response score of 1) or minimal audiogenic seizures in genetically epilepsy-prone rats,23 as described in Chapter 6, and the initial convulsive response seen with seizures induced by localized electrical stimulation of the inferior collic-ulus.24

Tonic flexion represents the first stage of the flexion-extension sequence that is characteristic of tonic-clonic convulsions. This convulsion is the result of contraction of the flexor muscles with the back of the animal arched and the limbs directed forward in a flexed position. This convulsion corresponds to the clonic phase of the running-bouncing clonus described as ranks 2 and 3 seizures (audiogenic response scores of 2 and 3) in genetically epilepsy-prone rats23 and also has been described as opisthotonus.12 Tonic flexion represents slightly greater activation of the brainstem nuclei that mediate tonic-clonic convulsions than the activation that induces running-bouncing clonus.1 Although it is possible to induce flexion-only convulsions with electroshock, such convulsions typically are not used to test for antiepileptic drugs, in part because the narrow current range for inducing the convulsions makes for unreliable seizure responses.

4. Tonic-Clonic Convulsions

Further increases in corneal electroshock stimulation current elicit convulsions that include both tonic flexion and tonic extension. These are commonly known as tonic-clonic convulsions (Table 1.1). While tonic-clonic convulsions are classified as threshold tonic-clonic or maximal tonic-clonic (Table 1.1), the convulsions are qualitatively the same in overall appearance. The convulsions are observed in the rat as a 2 to 3 s period of tonic flexion followed by a tonic extension phase lasting a period of approximately 10 to 12 s. The durations of the flexion and extension phases are slightly shorter in mice. The tonic extension is observed as a "wave" of muscular contraction that passes down the body from the head to the tail (rostro-caudal). The forelimbs extend first and this occurs reliably.25 Tonic hindlimb extension (Figure 1.2) represents the maximal convulsive response, but may not always occur reliably as described below. The body remains in a rigid extension for most of the 10 to 15 s period before relaxing in reverse order from tail to head (caudorostral).

The tonic-clonic convulsion may or may not include a terminal clonic phase.26 The terminal clonus does not occur reliably and is rarely used in seizure analysis.

a. Tonic Extension

Tonic extension is a manifestation of tonic contraction of the musculature. Although both the flexor and extensor muscles contract during the extension phase, extension dominates because the antigravity extensor muscles are stronger than the corresponding flexor muscles. Support for this hypothesis includes the observation that in the sloth, which hangs upside down and uses the flexors as antigravity muscles, tonic flexion is displayed after electroshock.27

b. Tonic Hindlimb Extension

By far the most frequently used endpoint in the quantification of tonic-clonic convulsions is tonic hindlimb extension (THE). Other methods for quantifying tonic-clonic convulsions (described below) are typically used only when THE cannot be reliably induced. THE is the last event to occur in the wave of contraction that progresses down the body and is observed as the hindlimbs projecting straight back behind the body (Figure 1.2). THE may be induced by electroshock or high doses of chemical convulsants that induce tonic-clonic convulsions.15 28 It is proposed that electroshock or the high-dose chemical convulsants act directly on the neural substrates in the brainstem that mediate tonic-clonic convulsions and THE.12 Drugs that inhibit THE in rats are also effective against generalized tonic-clonic (grand mal) seizures.68 Animals treated with effective antiepileptic drugs actually still display tonic extension but without the THE component (Figure 1.2). The endpoint for anticonvulsant protection is usually considered to be the failure of the hindlimbs to extend beyond a 90° angle to the torso.62930 Mice reliably respond to electroshock with THE.1 Unfortunately, as indicated in Table 1.2, not all Sprague-Dawley rats reliably respond with THE and the failureratemaybeashighas 500/o.14,31"35 This is discussed below in Section II.G, althoughitshouldbenotedthat 1.2 provides sources of rats that respond reliably with THE. It is recommended that rats be tested (screened) for THE before use in experimental studies involving the THE component of maximal electroshock (MES) and animals that do not respond with THE should not be used.836 In the event that a large percentage of the rats screened do not display THE, the flexion/extension (F/E) ratio may be used to quantify the seizure response. Alternatively, the duration of the tonic extension phase may be measured and a decrease in duration considered a decrease in seizure severity.35 37 38 These other methods for quantifying the tonic-clonic convulsive response are described next.

c. Flexion/Extension Ratio and Duration of Extension

The flexion-extension (tonic-clonic) convulsions represent a greater electroshock activation of the brainstem mechanisms that mediate generalized tonic-clonic convulsions than occurs when running-bouncing clonus or tonic flexion is induced.12 At electroshock currents just above threshold for tonic-clonic convulsions, the flexion phase is still prominent. With increasing stimulation current the seizure response becomes more severe as reflected by a decrease in duration of the tonic flexion phase

TABLE 1.2

The Tonic Hindlimb Extension (THE) Response to Electroshock as Reported in Various Rat Strains

TABLE 1.2

The Tonic Hindlimb Extension (THE) Response to Electroshock as Reported in Various Rat Strains

% with

Rat strain and source

THE

Stimulus parameters

Ref.

Sprague-Dawley albino (male), (source unknown)

90

Transcorneal, 150 mA, 0.2 s

29, 33

Sprague-Dawley albino (male), (source unknown)

100

Transcorneal, 150 mA, 0.2 s

8, 31

Sprague-Dawley albino (male), (source unknown)

50

Transcorneal, 150 mA, 0.2 s

14

Sprague-Dawley albino (male), (source unknown)

90

Transauricular, 150 mA, 0.2 s

14

Sprague-Dawley (male), Zivic-Miller, Allison Park, PA

50

Transcorneal, 50 mA, 0.2 s

35

Wistar (female), Harlan Windelmann, Borchen, F.R.G.

100

Transcorneal and transauricular, 150 mA, 0.2 s

7

Wistar (male), Harlan Sprague-Dawley Inc.

100

Transcorneal, 150 mA, 0.2 s

30

Wistar (male), Charles River, Sulzfeld

100

Transcorneal, 150 mA, 0.2 s

71

Note: The last three listings provide sources for rats that reliably respond with THE. However, it is recommended that all rats be seizure tested (screened) and the nonextenders discarded prior to use.

Note: The last three listings provide sources for rats that reliably respond with THE. However, it is recommended that all rats be seizure tested (screened) and the nonextenders discarded prior to use.

and an increase in duration of the extension phase. This may be quantified by the flexion/extension or F/E ratio.13139 The duration of the tonic flexion and tonic extension phases can be measured in a number of ways,14,31-34,40-43 but typically involve flexion being considered as the period from the electroshock stimulus to the fullest extent of the tonic extension and extension being the period from the fullest extent of the tonic extension until some point in the relaxation of the tonic phase. Whatever convulsion endpoints are chosen for the phases they must be applied consistently. Tonic-clonic convulsions evoked by low currents are less severe, as reflected by a short extension phase or a high F/E ratio. Antiepileptic drugs that inhibit generalized tonic-clonic seizures also reduce the severity of flexion-extension seizures which is observed as an increased F/E ratio as compared to untreated control animals.13139 For convenience the reciprocal of the F/E ratio, the extension/flexion (E/F) ratio, may be used because it allows the use of numbers greater than unity and thus an increase in the E/F ratio is associated with an increase in the seizure severity.41

It should be reemphasized that the critical standard for quantifying generalized tonic-clonic convulsions is the occurrence of THE. The F/E ratio has not been systematically evaluated as a parameter for screening anticonvulsant drug activity, since it was originally shown to correlate with the effectiveness of phenytoin and phenobarbital.26 Although a reduced duration of the tonic extension phase is correlated with anticonvulsant activity,35 37 44 the F/E ratio has not been systematically tested against all clinically effective antiepileptic drugs. Perhaps future studies might evaluate these convulsive parameters in addition to THE and determine their relative utility in detecting anticonvulsant drug activity.

d. Seizure Spread

It has been hypothesized that tonic-clonic convulsions represent the spread of seizure activity through the brain and drugs that inhibit tonic-clonic convulsions inhibit seizure spread.1545 The effectiveness of antiepileptic drugs is reflected by a change in seizure pattern, such as an increase in the duration of tonic flexion and a decrease in tonic extension or as an inhibition of THE.8 15 2631 Therefore, drugs that alter the threshold or maximal electroshock seizure pattern are proposed to inhibit seizure spread.145 This concept was originally based on the theory of an "oscillator" or a minimal collection of neurons that must discharge in an epileptiform manner to produce the seizure.45 The theoretical "oscillator" projects or spreads the aberrant epileptiform neuronal activity to other parts of the brain, resulting in the observed convulsions.45 The maximal tonic-clonic convulsions involve spread of seizure activity to the entire brain, resulting in the tonic flexion and tonic extension response.45 The greater the stimulating current, the greater the seizure spread, which results in a shorter period of tonic flexion and a greater period of tonic extension.31 Antiepi-leptic drugs are proposed to suppress the seizure spread and thereby alter the seizure patterns.1 The inhibition of THE is the alteration of seizure pattern that is most commonly used to evaluate anticonvulsant drug activity in electroshock7,8 and is thought to represent a reduction in seizure spread.15 2631 The increased F/E ratio is also a change in seizure pattern that is interpreted as anticonvulsant drug activity,1 26 31 as is the reduction in duration of tonic extension phase that is induced by anticon-vulsant drugs.35 37 3844

e. Seizure Threshold

Tonic-clonic convulsions induced by low corneal stimulation currents represent a convulsive threshold; in other words, the minimal current required to initiate the flexion-extension convulsion in 97% to 100% of the animals.15 For this reason these low current or submaximal threshold seizures are known as the threshold tonic extension (TTE) test,6 maximal threshold seizures,15 or threshold for maximal electroshock seizures (MEST) (Table 1.1).7 The quantal all or none response of THE or minimal clonic seizures can be used to establish the current threshold for tonic-clonic convulsions,1715 either by the Litchfield and Wilcoxon19 procedure or the "up-down" or "staircase" method.1 20-22 Although threshold currents are proposed to activate the "oscillator" sufficiently to initiate seizure activity and the seizure activity spreads to other areas of the brain, the spread is considered minimal compared to that induced by higher (supramaximal) currents.15 26 Although considered minimal, the seizure spread is still sufficient to induce a tonic-clonic convulsion (Table 1.1).1526 Thus, the maximal threshold seizures or MEST represent seizure mechanisms associated with both the threshold for seizure induction and the spread of the seizure activity to other brain areas.15 Further, MEST convulsions respond to antiepileptic drugs that raise seizure threshold, inhibit seizure spread, or both.15 In a classic experiment, Piredda et al.15 demonstrated this relationship by showing that ethosux-imide (which increases convulsive thresholds), phenobarbital (which inhibits seizure spread), and valproate (which increases threshold and inhibits spread) all are effective in MEST. Those authors concluded that MEST is capable of detecting anticonvulsant activity, but is nonspecific in differentiating spread and threshold mechanisms of action. However, Löscher et al.7 have shown that MEST is capable of detecting specific anticonvulsant drug activity where electroshock stimulation using higher currents (maximal electroshock) does not. In studies with mice, Löscher demonstrated that both primidone and clonazepam are effective anticonvulsants in MEST but not maximal electroshock.7 The Anticonvulsant Screening Project of the Epilepsy Branch of the National Institute of Neurological Disorders and Stroke has used MEST to identify compounds that are effective only in MEST convulsions; however, the clinical utility of such agents is debatable.8

5. Maximal Electroshock

The maximal tonic-clonic convulsion is the most severe electroshock convulsive response and it is induced by supramaximal electrical stimulation currents,15 more commonly known as maximal electroshock (MES) (Table 1.1). MES is typically induced in rats and mice using supramaximal corneal electroshock currents of 150 mA and 50 mA, respectively.18 Such currents are approximately five to seven times the threshold current and are considered supramaximal or suprathreshold. At the supramaximal currents the tonic-clonic seizures are more severe than those induced by threshold currents in that the tonic flexion phase is reduced in duration while the tonic extension phase is increased.31 This change in phase duration is reflected in a minimal F/E ratio (Table 1.1). In a rat population that does not uniformly respond to MES with THE, the percentage of animals responding with THE increases as the current is increased from threshold to supramaximal.31

The most common endpoint for anticonvulsant drug activity in MES is the inhibition of THE (Figure 1.2).6-8 Drugs that inhibit the THE component of MES are effective antiepileptics against generalized tonic-clonic (grand mal) seizures.6-8 Due to the suprathreshold current used to induce MES it is not possible to determine if the drugs raise convulsive threshold,15 but drugs that inhibit THE do alter the seizure pattern and therefore are proposed to reduce the spread of seizure activity.15 2645 Anti-epileptic drugs also reduce the severity of seizures, as observed by an increase in the duration of tonic flexion and a decrease in the duration of tonic extension resulting in an increased F/E ratio.2631 39 The reduction in the duration of the tonic extension phase has been used as a measure of anticonvulsant drug activity.35 373844

Electroshock stimulation currents greater than supramaximal (150 mA in rats, 50 mA in mice) do not produce significantly greater convulsive responses. Currents up to 10 times suprathreshold result in no significant change in the F/E ratio, but may increase the incidence of hindlimb paralysis after the seizure.31 Hindlimb paralysis is the result of vertebral fractures that occur during the course of the tonic-clonic convulsions. The animals do not recover from the paralysis and it is recommended that the animal be euthanized as quickly as possible after evidence of a vertebral fracture is recognized.

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