Inhalation Of Convulsant Substances

Inhalation is a less usual route of delivery for a convul-sant drug. For inhalation, the drug must have some special features, such as easy evaporation at room temperature. This is true for all ether-like substances. Indeed, ethers have excitatory features, reflected in the original classification of ether-induced anesthesia as stage II. However, in most of these drugs, stage III of surgical anesthesia develops quickly and covers the excitatory stage II. Although delivery of inhalatory convulsants requires some special equipment, it also has significant advantages over administration by injection. Minimum requirements for the equipment are an airtight glass cylinder, which serves as the inhalation chamber, and a fume hood for draining away excessive fumes of the convulsant. More sophisticated systems (and less hazardous for the laboratory worker) consist of an airtight chamber with sufficient inside volume (Figure 11). This chamber is attached to the vacuum exhaust system through the system of valves, which allows the chamber to be flushed after each experiment. Therefore the convulsant fumes do not escape to the surrounding atmosphere. The chamber is placed in the fume hood for additional protection. The convulsant drug is delivered using a constant infusion rate pump, and it evaporates at the filter paper with a standard size placed at the top of the chamber. Latency to onset of seizures from the beginning of the convulsant drug delivery is recorded. The major advantage of inhalation delivery is that no potentially painful injection and stressful restraint of the animal is involved, which results in minimal interference of stressors with the seizure model.

Flurothyl (Indoklon)

Methods of Generation

Flurothyl (bis-2,2,2-trifluoroethyl ether) seizures have been described in mice, rats, gerbils, and humans. The procedure consists of placing the animal (rat or mouse) in an airtight cylinder of a sufficient volume and injecting a specific volume of flurothyl, which then evaporates and forms convulsant fumes (Prichard et al., 1969; Truitt et al., 1960). Our laboratory uses 9.34 l airtight chamber for the rat exper-

To vacuum pump

To vacuum pump

Inhalation Chamber

FIGURE 11 Scheme of the chamber for the induction of flurothyl seizures. Flurothyl is delivered by precision microinfusion pump onto a filter paper, where it evaporates. After the experiment, the chamber is flushed with vacuum. The entire chamber, including the microinfusion pump, is placed in the fume hood for increased protection of the crew because flurothyl vapors have the capability of inducing seizures in humans.

FIGURE 11 Scheme of the chamber for the induction of flurothyl seizures. Flurothyl is delivered by precision microinfusion pump onto a filter paper, where it evaporates. After the experiment, the chamber is flushed with vacuum. The entire chamber, including the microinfusion pump, is placed in the fume hood for increased protection of the crew because flurothyl vapors have the capability of inducing seizures in humans.

iments. Flurothyl is delivered using a microinfusion pump at a constant rate; for this size chamber, the usual rate is 20 ml/min. Latency to onset of seizures from the application of the convulsant is measured. In our paradigm, the exact amount of flurothyl required to produce a certain seizure type can be calculated (flurothyl seizure threshold). It is imperative to run the tests in control and experimental conditions back to back because atmospheric factors may affect evaporation of flurothyl. Mortality after acute flurothyl exposure is low. However, if the adult rats are subjected to long-term flurothyl exposure to induce SE, they regularly die, in contrast to immature PN14 rats (Huang et al., 1999; Sogawa et al., 2001; Sperber et al., 1999a).

Defining Features

Seizures induced by flurothyl are age specific (Sperber and Moshe, 1988). Flurothyl induces initial agitation and increased exploratory activity in rats throughout development (Gatt et al., 1993). In rats during the first postnatal week, swimming movement occurs, followed by a tonus. Clonic seizures develop after PN10. After this age, rats usually display several myoclonic twitches followed by a clonic seizure, which at between PN10 and 20 usually evolves into a tonic-clonic seizure with loss of posture. After

PN20, several separated episodes of clonic seizures appear before the development of tonic-clonic seizures. It should be noted that the sequence and seizure phenomena after flurothyl exposure are very similar to the seizures occurring after systemic administration of GABA-related drugs (especially PTZ and bicuculline). However, in contrast to these drugs, flurothyl induces seizures that are more violent: Clonic seizures often involve tonic components of axial muscles, which may cause a temporary loss of posture. Clonic seizure may involve all four limbs simultaneously, which also interferes with the upright posture. However, in all these cases, the rat struggles to get up. This is in contrast to the loss of posture in tonic-clonic seizure, in which no active effort is made to regain posture. During the tonic phase, cyanosis and acidosis may occur, with fast correction within 5 minutes after the end of the seizure (McCabe et al., 2001). EEG shows rhythmic discharges (Figure 12) associated with a motionless stare from the third postnatal week onward. Additionally, individual spikes (or sharp waves in immature animals) are recorded. In young rats, these discharges very loosely correlate with myoclonic twitches; the correlation improves with age. Bilateral forelimb clonus is associated with long periods of spikes or sharp waves (Figure 13). In adult rats, discharges are found in the cortex, whereas in immature rats the discharges occur in both the cortex and the hippocampus (Sperber et al., 1999a). Metabolic studies using 2DG in adult rats demonstrate complex involvement of basal ganglia structures, including the substantia nigra reticulata. The pattern of metabolic involvement depends on the seizure type and whether in ictal or postictal state (Veliskova et al., 2005, in press). In immature rats, mild seizures induce decreases in 2DG uptake while severe seizures produce increases in the brain-stem and decreases in the cortex (Szot et al., 2001). In adult rats, c-fos immunoreactivity after flurothyl seizures was found throughout the cortex; PN10 rats had immuno-positivity only in the deep neocortical layers (Jensen et al., 1993).

Limitations

A more sophisticated airtight chamber is required to produce flurothyl seizures by inhalation of flurothyl vapors. Atmospheric factors such as barometric pressure, air humidity, and temperature may affect the evaporation rate of flurothyl. Therefore the experiments should be carefully planned. Sometimes it is difficult to compare corresponding data collected over different periods.

Insights into Human Disorders

The mechanisms of action of flurothyl are uncertain. Increased opening of sodium channels has been suggested (Woodbury, 1980a). Antagonism at the GABAa receptor may also contribute to the convulsant potency of flurothyl (Araki et al., 2002). Additionally, activation of cholinergic system has been proposed (Eger et al., 2002). Because flurothyl induces a motionless stare with accompanying

Freezing Behavior
FIGURE 12 Rhythmic, spindle-shaped discharge induced by inhalation of flurothyl in sensorimotor cortex of an adult Sprague-Dawley rat. This discharge was associated with freezing behavior. Bipolar recording from the sensorimotor cortex is shown.
Sensorimotor Cortex Rats

FIGURE 13 Spike-and-wave and polyspike discharges induced by inhalation of flurothyl in sensorimotor cortex of an adult Sprague-Dawley rat. Arrow indicates onset of clonic seizure and rearing. The onset of this discharge significantly preceded the onset of motor seizures. However, the occurrence of high-frequency polyspike discharges was associated with the onset of clonic seizure.

FIGURE 13 Spike-and-wave and polyspike discharges induced by inhalation of flurothyl in sensorimotor cortex of an adult Sprague-Dawley rat. Arrow indicates onset of clonic seizure and rearing. The onset of this discharge significantly preceded the onset of motor seizures. However, the occurrence of high-frequency polyspike discharges was associated with the onset of clonic seizure.

EEG spindles, this can be considered a model of generalized seizures—typical absence seizures (Engel, 2001). Clonic seizures can be a model of generalized myoclonic seizures. Finally, tonic-clonic flurothyl seizures are a model of generalized tonic-clonic seizures (Engel, 2001).

TOPICAL (FOCAL) APPLICATION OF SUBSTANCES

This approach is frequently used to create an acute or chronic seizure focus in a particular brain area, usually in the cortex. Additional structures may be injected with a con-vulsant substance, such as the hippocampus, amygdala, substantia nigra, area tempestas, etc. Many of the convulsant drugs can be injected in a solution into the brain ventricular system. Some of these ICV administrations have already been mentioned herein. The advantage of this approach is that it produces seizures very reliably and only micro-amounts of the drug are required. However, there is no exact control of the focus of the seizure onset. All structures surrounding the injected ventricle are affected by the drug; thus seizure onset can be multifocal. For precise localization of the focus, intraparenchymal administration of a convulsant substance is preferred. Many different convulsant substances are used for production of seizure focus, and its major representatives are briefly mentioned as follow.

Metals (Cobalt, Zinc, Antimony, Alumina Cream, Iron)

Methods of Generation

Cobalt can be implanted as a powder or rod, or it can be injected as a solution of CoCl2 (Cooper and Legare, 1997). ICV injection of cobalt chloride has the CD50 of 10 ml of 0.45 mM solution. Seizures develop very slowly in powdered or rod cobalt implants and vanish in about a month. After ICV injection of a soluble cobalt salt, seizures develop within an hour and last for about a week (Zhao et al., 1985). Other metals such as nickel and antimony have high epileptogenic potency; iron has a somewhat lower potency than cobalt (Colasanti and Craig, 1992; Craig and Colasanti, 1992; Dambinova et al., 1998; DeDeyn et al. 1992; Doi et al., 2000; Kabuto et al., 1998; Pei and Koyama, 1986; Ueda and Willmore, 2000a, b, c; see also Fisher, 1989). Additional technique of application includes positioning of a filter paper soaked with a soluble salt (e.g., saturated solution of NiCl26H2O) on the cortex (Cooper et al., 2001). Alumina cream (Kopeloff et al., 1942, 1954; Ward, 1969; Wyler et al., 1978), 4% AlOH (Spencer, 2000a), is a very potent and frequently used topical convulsant; however, its effects are limited to monkeys and rabbits (see Chapter 14). The latent period is several months and seizures may last for up to several years (Louis et al., 1987).

Defining Features

In the rat, unilateral cobalt implant produces seizures within 5 days. Simple partial seizures develop first and consist of contralateral clonus associated with EEG spikes above the implant. Although very frequent (more than 20 per day), these seizures disappear within 9 days. Generalization is characterized by a bilateral clonus, occurring about 10 times a day, most frequently 1 week after cobalt implantation. Behavioral arrest may also occur and is associated with gradual buildup of EEG activity (Chang et al., 2004). ICV administration of soluble cobalt salt produces seizures resulting from irritation of the hippocampus. Therefore these seizures are similar to KA-induced seizures (Zhao et al., 1985). Nickel salt applied on the filter paper on the cortex produces contralateral myoclonic twitches within an hour; these twitches last about 2 hours (Cooper et al., 2001). After cobalt implantation in the posterior cortex, dark patches of increased 2DG uptake appear around the implant site and in the lateral geniculate nucleus of the thalamus. This is, however, not true for anterior cortex cobalt implants and for implants of other convulsogenic metals such as nickel and antimony (Cooper and Legare, 1997). Metal implants cause focal necrosis.

Limitations

These models are labor intensive and expensive to prepare, especially for the monkey alumina cream model. The difference of clinical seizure foci may be the direct irritation of the brain tissue by the metal deposit. Cobalt-induced seizures are more sensitive to antiepileptic drugs used against absence seizures in humans and thus cannot be a predictive model of refractory focal epilepsy (Loscher, 1997). Except for the alumina cream model in monkeys, foci spontaneously deactivate after several weeks.

Insights into Human Disorders

Mechanisms of action of these metal deposits are largely unclear. Metal deposition models depend on the localization of the deposit; these are good models of focal motor seizures with elementary clonic motor signs or focal motor seizures with typical (temporal lobe) automatisms (Engel, 2001). Except for cobalt, metal deposit models may have good predictive value for screening anticonvulsant drugs effective against focal onset seizures.

Convulsant Drugs

Methods of Generation

All convulsant drugs mentioned previously in this section, plus cholinergics, anticholinergics, estrogens, and strychnine can act focally if applied on the cortex or in a particular seizure-prone structure, such as amygdala, hippocampus, substantia nigra, or area tempestas (Campbell and Holmes, 1984). Older studies usually used focal administration of the drug on the surface of the neocortex of paralyzed and ventilated or anesthetized animals (Veliskova et al., 1991). In that case, a filter paper of an exact geometry is used. Its area is either soaked with a water solution of a convulsant drug or a defined volume is delivered using a microsyringe to the filter paper positioned on the cortex. More recent studies use chronic cannulation with microinfusions of the drugs in freely moving animals (Gale, 1995; Halonen et al., 1994; Soukupova et al., 1993; Velisek et al., 1993).

Defining Features

Behavioral features depend on the following: (1) the drug used and (2) the exact placement of the microinfusion. For doses of commonly used convulsant drugs, see Table 5. Microinfusions of any drug in the limbic system will induce initial automatisms followed by generalization of seizure activity and clonic seizures (Sierra-Paredes and Sierra-Marcuno, 1996b). Application of the drugs on the motor cortex will produce myoclonic twitches and, later, clonic seizures (Soukupová et al., 1993). Finally, drugs with high affinity for limbic structures will produce automatisms and clonic seizures even when the injection site is remote to the limbic system (such as the substantia nigra pars reticulata (Sawamura et al., 2001). EEG usually displays individual discharges (Figure 14), which may cumulate over time and develop into ictal activity.

Limitations

These models are used less frequently because they are relatively labor intensive. Further, the use of NMDA as a focal convulsant drug may be surprising in some brain structures because NMDA has a very high propensity to

TABLE 5 Concentrations of Some Focally Applied Convulsant Drugs

Drug

Structure

Dose

Frequency

Reference

Bicuculline methiodide

Neocortex

5 ml of 1-2mM solution

1-2 times

Soukupová et al., 1993

Pentylenetetrazol

Neocortex

2.5 ml of 200mg/ml solution

Single

Velísek et al., 1993

Bicuculline methiodide

Neocortex

2.5 ml of 4mM solution

Single

Velísková et al., 1991

Picrotoxin

Hippocampus

100-500 mM

Continuous

Sierra-Paredes and Sierra-Marcuno, 1996a, b

Kainic acid

Amygdala

0.4-2.0 mg in 0.1-0.4 ml

Single

Ben-Ari et al., 198G

Kainic acid

Substantia nigra

1 mg in 1 ml

Single

Sawamura et al., 2GG1

Bicuculline

Area tempestas

118pmol

Single

(Wardas et al., 199G)

Carbachol

0 0

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