Electrical Kindling in Developing Rats

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30 Electrical Kindling in Developing Rats ARISTEA S. GALANOPOULOU AND SOLOMON L. MOSHÉ

METHODS OF GENERATION

Procedures Electrode Implantation

Animal Issues

Electrode implantation is done at least 1 day prior to kindling in P7–8 Sprague-Dawley rats (Baram et al., 1993; Baram et al., 1998) and 2 days prior to testing in P9 or older rats (Baram et al., 1993; Baram et al., 1998; Moshé, 1981; Moshé et al., 1981). The recovery period needs to be shorter in infant rats than in adults, to avoid misplacement of the electrode tip due to the ongoing head growth. P6–10 rats are anesthetized under halothane anesthesia (Baram et al., 1993; Baram et al., 1998), whereas P12 or older rats are injected with a mixture of ketamine (70 mg/kg IP) and xylazine (6 mg/kg IP) (Moshé 1981; Moshé et al., 1981). When deeply anesthetized, rats are placed on an infantile rat stereotaxic apparatus. The tooth bar is set at 3.5 mm. Bipolar twisted wire electrodes are targeted through a burr hole, to the basolateral nucleus of the amygdala unilaterally. In P6–10 pups, bipolar electrodes with a wire diameter of 0.1–0.15 mm and vertical inter-tip distance of 0.5–1 mm have been used (Baram et al., 1993; Baram et al., 1998). In older pups, insulated electrode with wire diameters 0.23–0.35 mm (MS 303/1 or MS 303/2, Plastic One, Roanoke, VA, USA) can be used (Moshé 1981; Haas et al., 1998). The coordinates used to target each structure are based on the stereotaxic coordinates included in the atlas by Paxinos (Paxinos et al., 1994; Paxinos and Watson, 1997) but they need to be adjusted according to the age, strain, source, or weight of the rats. Table 1 presents published coordinates that can be used as starting points when targeting the amygdala or other sites.

Because of the rapid growth of infant rats, kindling stimulations must be delivered within a finite period of time, usually 48 hours. The first protocol of amygdala kindling in infantile rats (P15–18) was published by Moshé in 1981 using 1 hour interstimulus intervals (ISI) with 60 Hz alternating current stimulations. However, even shorter (15 minute) ISI were able to effect successful kindling, due to the lack of a refractory period (Moshé, 1981; Moshé et al., 1981; Moshé and Albala, 1983; Haas et al., 1990). With this paradigm, kindling can occur within 1 day. This is not the case with adult rats. Irrespective of the ISI, kindling once induced is permanent and persists into adulthood either in the original kindling site or the contralateral site (Moshé and Albala, 1982). Subsequently, several other studies adopted the 15 minute ISI kindling protocol and succeeded in kindling rats as young as P7 (Baram et al., 1993; Baram et al., 1998), and covering all the stages of development, i.e., the infantile (P8–P20), juvenile (P21–P34), and pubertal period (P35–P40) (Dahl et al., 1988; Ojeda et al., 1986). These studies revealed several age-related differences in kindling parameters, expression, and consequences. Briefly, age-related differences have been found in the presence of a refractory period, the afterdischarge (AD) threshold (ADT), kindling rate, behavioral manifestations of kindled seizures, the interaction between 2 foci kindled concurrently, and neuropathologic and metabolic sequelae.

Models of Seizures and Epilepsy

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In this lab, electrodes are usually inserted at a 0° angle in reference to the vertical plane (Table 1A). If additional electrodes or cannulae need to be inserted, the electrodes can be placed at a different angle, with appropriate adjustment of the coordinates (Table 1B). Electrodes are then fixed to the skull with 2 or 3 screws and dental acrylic. The pups are then returned to the litter. We prefer to keep the mother separated from the infants till all the pups are operated. We then cover the pups with the shavings already present in the cage to cover up any obnoxious smells acquired during the procedure. When pups are ready to kindle, they are placed in a test box and are connected to the stimulating and recording apparatus via a connector cable (Plastics One; MS 303 series). Recording of the EEG is done before and after each stimulation. Kindling The first step is to determine the ADT. A 60 Hz sinusoidal current is delivered for 1 second. Stimulations can be delivered as frequently as every 2 minutes, until an AD is elicited.

Once an AD is observed, the ISI is kept at 15 minutes or as otherwise stated in the selected protocol (Table 2). If determination of ADT is an endpoint in the experimental design, a protocol using small increments of stimulation amplitude may be used. For example, stimulations can be started at 30 mA and increased by 30 mA in each subsequent trial until an AD is obtained (Moshé et al., 1981). The current can then be decreased by 15 mA. If an AD is evoked, the current can further be increased by 5 mA increments until the lowest current that can generate an AD is reached (Moshé et al., 1981). In most cases, however, ADT determination simply serves the purpose of ensuring that all subsequent stimulations will be above ADT to achieve successful kindling. In such cases, the starting stimulus intensity can be set at 100 mA and progressive increments by 100 mA are applied till an AD is recorded. Generally, rats not responding to 500 mA stimulation with an AD are excluded, as they have been shown to be refractory to kindling (Moshé SL, unpublished data). Determination of the ADT is done the same day as kindling.

TABLE 1 Age- and Site-Specific Coordinates for Targeting Limbic Structures for Kindling A. Insertion at 00 angle Age (days)

Tooth bar (mm)

Antero-posterior in reference to bregma (mm)

Lateral (mm)

Depth (mm)

Reference

-1.5 to -1.8 -1.5

3.5 3.5

6.5–7.4 8.8

(Moshé, 1981) (Moshé, 1981)

Basolateral nucleus of the amygdala 7–14 -3.5 ≥32 -3.5 Dorsal hippocampus 13–14

-3.5

-3.2

2.7 to 3

2.8–3

(Haas et al., 1990)

Ventral hippocampus 7–21 28

0 5

-3 to -2.9 -3.6

3.7–4.9 4.9

3.7–4.9 4.9

(Michelson and Lothman, 1991)

Deep endopiriform cortex 13 -3.5

-1.2

3.1

5.5

(Sperber et al., 1998)

Piriform cortex 13

-1.2

3.7

6

(Sperber et al., 1998)

-3.7

B. Angled coordinates

Age (days)

Tooth bar (mm)

Basolateral nucleus of the amygdala 14 -3.5 Dorsal hippocampus 13–14

-3.5

Angle

Antero-posterior in reference to bregma (mm)

Lateral (mm)

Depth (mm)

Reference

150 forward

+5

3.5

7.4

(Haas et al., 1992)

150 backward

-4

3

2.9

(Haas et al., 1992)

Stereotaxic coordinates for targeting the basolateral nucleus of the amygdala, the dorsal or ventral hippocampus, the deep endopiriform cortex, and the piriform cortex in developing Sprague-Dawley rats. Insertion is routinely made at a 00 angle, in reference to the vertical plane (Table 1A). If additional electrodes need to be placed, angled coordinates can be used (Table 1B). Further adjustments may need to be made according to the specific strain, source, or weight of the rats.

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Methods of Generation

TABLE 2 Age and Site-Specific Protocols of Kindling in Developing Rats Site Amygdala (basolateral nucleus)

Age P7–12 P15–18 P30–35

Protocol

Reference

1 sec pulse, 60 Hz monophasic current, 400 mA, 15 min ISI 1 sec pulse, 60 Hz sinusoidal current, 400 mA, 15 min ISI 1st day: 20 stimulations; 2nd day: 10 stimulations 1 sec pulse, 60 Hz sinusoidal current, 400 mA, 60 min ISI 1st day: 20 stimulations; 2nd day: 10 stimulations

(Baram et al., 1993) (Moshé et al., 1983) (Moshé, 1981) (Moshé et al., 1981) (Holmes and Weber, 1983)

Deep endopiriform nucleus

P15–16

1 sec pulse, 60 Hz sinusoidal current, 400 mA, 15 min ISI, 1st day: 20 stimulations; 2nd day: 10 stimulations

(Sperber et al., 1998)

Piriform cortex

P15–16

1 sec pulse, 60 Hz sinusoidal current, 400 mA, 15 min ISI, 1st day: 20 stimulations; 2nd day: 10 stimulations

(Sperber et al., 1998)

Dorsal hippocampus

P15–17

(Haas et al., 1992)

P30

1-sec, 400 mA. 60 Hz, sinusoidal current, every 15 min 1st day: 20 stimulations; 2nd day: 10 stimulations 10-sec, 10 Hz or 60 Hz, 1000 mA, 5 min ISI

Ventral hippocampus

P7–28

10-sec, 20 Hz, every 30 min for 9 hours per day, 2 days

(Michelson and Lothman, 1991)

Perforant pathway

P14–60

10-sec, 60 Hz, bipolar square wave pulses, twice the ADT, every 30 min

(Trommer et al., 1994)

When the ADT is determined, the amplitude of the stimulation current used for kindling can be set at 100 mA higher than the ADT and kept the same throughout the kindling protocol. In most protocols, however, amplitude can be set at 400 mA, which is sufficient to kindle rats at all ages and in most sites. To kindle the amygdala, stimulations (1 second, 60 Hz sinusoidal, 400 mA) are delivered every 15–20 minutes, to a maximum of 20 stimulations the first day. Kindling continues until pups develop three consecutive bilateral/generalized seizures (i.e., stage 3.5 to stage 7; Table 3). A measure of how fast kindling proceeds is the kindling rate, i.e., the number of stimulations required to achieve the first of three consecutive bilateral seizures (stage 3.5 to stage 7, Table 3). Between stimulations, pups are returned to their dam. After 10 stimulations, pups are rested for 1 hour before resuming kindling. The following day, pups are again kindled up to 10 times, or until they develop three consecutive stage 3.5–7 seizures. A total of 30 stimulations over a 2-day period can elicit stage 6 or 7 repeated seizures, which last for 100–120 seconds (Sperber et al., 1992). Postictal refractoriness can be measured an hour after the last kindled seizure, delivering eight repetitive 1-second stimulations, 400 mA, 60 Hz pulses, every 2 minutes, and comparing the AD duration and the kindling stage (Moshé and Albala, 1983). When kindling permanence or the long-term effects of kindling are studied, it is necessary to remove the original electrodes to avoid misplacement of the tip due to continuing head growth as well as infectious/inflammatory complications. New electrodes may then be re-inserted at a later stage if needed in the experimental design (Moshé and Albala, 1982). The sensitivity of different brain regions to kindling is, however, different. Attempts to kindle the dorsal hippocam-

(Holmes and Thompson, 1987)

TABLE 3 Behavioral Manifestations of Kindling in Infantile Rats Stage

Behavior

0

Behavioral arrest

1

Mouth clonus

2

Head bobbing

3

Unilateral forelimb clonus

3.5

Alternating forelimb clonus

4

Bilateral forelimb clonus

5

Bilateral forelimb clonus with rearing and falling

6

Wild running and jumping with vocalizations

7

Tonus

The currently used seizure scales describing the progression of seizures during kindling of infantile pups are based on the modified infantile scale initially proposed by Moshé (Haas et al., 1990; Haas et al., 1998). Age- and site-related variations may be observed as discussed in section IIA. (Reproduced from Haas et al., 1990, with permission from Elsevier.)

pus, piriform cortex, deep endopiriform nucleus (“area tempestas”) and amygdala in P15–17 pups, using the same experimental protocol, showed that the deep endopiriform nucleus kindled the fastest (Sperber et al., 1998; Haas et al., 1998). The different sensitivity to kindling in certain cases necessitated the use of site-specific protocols, as summarized in Table 2. Modifications may thus need to be made in the duration and frequency of the stimulation as well as the ISI. In the study by Michelson and Lothman, kindling of the ventral hippocampus of P7–28 rats with 5 minute-ISI resulted in erratic kindling, whereas efficiency was improved with ISIs of 30 minutes (Michelson and Lothman,

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1991). These differences may suggest the existence of a relative refractory period in the ventral hippocampus at this age.

CHARACTERISTICS AND DEFINING FEATURES Monitoring ADT/Kindling Rates Age- and site-related differences in ADTs have been reported. In the amygdala, the highest ADT occurs at P15–18 and declines thereafter till adulthood (Moshé et al., 1981). Kindling rate is the lowest at P7–8 rats (Baram et al., 1998), highest at P35, and intermediate at the other ages (Moshé et al., 1981). The effects of age on ADTs and kindling rates may differ in other brain sites (Holmes et al., 1987; Trommer et al., 1994; Lee et al., 1989; Haas et al., 1998). For instance, although dorsal hippocampus kindles as fast as the amygdala and piriform cortex in 2-week-old pups, in adults hippocampal kindling proceeds at a slower rate than in the other brain sites (Haas et al., 1998; Lee et al., 1989; Moshé 1981; Moshé et al., 1981). Behavioral/Clinical Features Kindled seizures are semiologically different in infants than in adults. This prompted the adoption of a modified seizure scoring scale, based on the seizures observed in P15–18 rats undergoing amygdala kindling (Table 3) (Haas et al., 1990). The main differences from the adult scale include the appearance of alternating forelimb clonus in the same or consecutive seizures (stage 3.5), which is indicative of bilateral or generalized seizures. The incomplete myelination of the corpus callosum has been postulated to be a reason for the development of asynchronous bilateral motor manifestations (Haas et al., 1998; Gravel and Hawkes 1990; Kristensson et al., 1986). Another distinguishing feature of kindling of the immature rats is the early appearance of severe kindled seizures. Following the development of stage 5 seizures, P15 rats also develop more severe seizures, consisting of wild running with jumping and vocalizations (stage 6 seizures) and sometimes tonus (stage 7 seizures) (Haas et al., 1990). This may occur early on, after only less than 30 stimulations. In contrast, in adult rats more than 100 stimulations may be required to induce similarly severe seizures (Haas et al., 1998). The incidence of stage 6 or 7 seizures depends upon the stimulated site. Sixty to 80% of pups kindled at the amygdala, hippocampus, or area tempestas develop severe seizures, whereas none of those kindled at the piriform cortex do (Haas et al., 1990; Sperber et al., 1998). In P7–9 pups, alternating clonus and rearing were rarely observed (Baram et al., 1993). Furthermore,

P7–9 kindled rats rarely develop bilateral clonic seizures or rearing and progress from unilateral clonic seizures to tonic seizures (Baram et al., 1998). The kindled seizures in peripubertal rats are semiologically similar to those observed in adults, and therefore the adult scale can be used (Racine, 1972) (see also Chapter 28 on “The Kindling Phenomenon”). Similar scales have also been adopted in studies of kindling at other sites (Sperber et al., 1998; Haas et al., 1998; Michelson and Lothman, 1991; Holmes and Thompson, 1987; Lee et al., 1989) (Table 3). P15–17 pups are also more prone to develop recurrent kindled seizures and status epilepticus than adult rats (Moshé and Albala, 1983). The absence of a postictal refractory period in infant rats has been proposed to be a factor contributing to this increased propensity of the immature brain to develop status epilepticus or recurrent kindled seizures (Moshé and Albala, 1983). Kindling Antagonism In contrast to adult kindled rats, P15–17 pups do not exhibit kindling antagonism (Haas et al., 1998). Concurrent kindling of the amygdala with either the contralateral amygdala or the contralateral or ipsilateral hippocampus, using alternating stimulations at each site, accelerated the kindling at each site, with pups manifesting more severe, and occasional spontaneous, seizures (Haas et al., 1998; Haas et al., 1990; Haas et al., 1992). These findings suggest that the immature brain is not yet ready to suppress the development of multiple kindling foci, explaining therefore the higher incidence of multifocal seizures in the immature brain (Haas et al., 1992). Spontaneous Seizures Amygdala kindling may lead to the appearance of spontaneous seizures. In the study by Baram’s group, spontaneous seizures were defined as those observed at least 4 minutes after the last stimulation and typically after stage 3.5–5 seizures. Spontaneous seizures in that study persisted for 2–3 hours, till sacrifice. The investigators found that spontaneous seizures appeared in 25–50% of kindled P7–12 pups (Baram et al., 1998). Spontaneous seizures resembling stage 6 seizures (wild running, jumping, and vocalizations) have also been reported in P15–16 pups kindled with alternating stimuli at the ipsilateral amygdala and dorsal hippocampus (Haas et al., 1992). There are, however, no long–term monitoring studies to determine whether and how long spontaneous seizures continue to occur till adulthood. Persistence of Kindling Kindling of limbic structures during the infantile period appears to leave persistent alterations in the brain, through adulthood. P15–18 rats kindled to manifest either general-

Characteristics and Defining Features

ized or focal seizures can be re-kindled faster during adulthood, whether stimulation is done at the ipsilateral or contralateral amygdala (Moshé and Albala, 1982; Moshé and Albala, 1983). For instance, rats that were fully or partially kindled at P18 required only 3–6 stimulations respectively to re-kindle in adulthood, as opposed to 10–13 stimulations needed in controls (Moshé and Albala, 1982). AD duration was also significantly longer in adult rats that were previously kindled in infancy and their seizures were more severe (Moshé and Albala, 1982).

Neuropathology Histologic examination of kindled rats has not demonstrated significant histopathologic differences compared to same-age rats implanted but not kindled (Moshé and Albala, 1983). Nissl staining of brain sections from rats kindled at P15–17 did not reveal significant cell loss at the CA3c or CA1 hippocampal regions, 2 weeks following the last kindled seizure (Haas et al., 2001). Similarly, there was no significant mossy fiber sprouting in Timm-stained hippocampal sections from these rats (Haas et al., 2001), in agreement with the studies supporting the relative resistance of the immature brain to seizure-induced pathologic changes. These studies demonstrate that kindling in pups is permanent in the absence of overt histologic changes, albeit only studied in amygdala and hippocampus. The development and neuropathologic consequences of kindling, however, are enhanced in pups that have a pre-existing neuronal migration disorder. Germano et al. showed that P15 pups with experimentally-induced neuronal migration disorder, produced by transplacental injection of methylazoxylmethanol acetate, had lower ADTs, faster kindling rates, and longer ADs the second day of hippocampal kindling. In addition, kindled-seizure induced damage was observed at the CA3 sectors bilaterally (Germano et al., 1998).

Neuroimaging Increased deoxyglucose uptake in rhinencephalic structures, but not in basal ganglia or neocortex, was observed in P15–16 albino rats with stage 6 and 7 kindled seizures (Ackermann et al., 1989). The increased deoxyglucose accumulation in the hippocampus was not associated with concomitant increase in glucose accumulation (Sperber et al., 1992). It has been proposed that the rapid propagation and increased severity of seizures at this age may reflect the immaturity of subcortical structures involved in seizure control (Haas et al., 1998).

Genetics and Molecular Changes Kindling has been described mainly in immature Sprague-Dawley rats (Moshé, 1981; Moshé et al., 1981;

375

Baram et al., 1993; Baram et al., 1998), but also in weanling (3-week old) Wistar pups (Kawahara et al., 1989). It is the experience of this lab (Moshé SL, unpublished observations) that the development of kindling in Sprague-Dawley pups obtained from different sources may vary, suggesting that exogenous or genetic factors influence this process. The existence of strain differences in the development of kindling has been well documented in adult Fast and Slow rats, a crossbreeding of Wistar and Long-Evans rats (Xu et al., 2004; McIntyre et al., 1999). Similar differences also exist in immature pups. During amygdala kindling, P15–16 Fast and Slow rats exhibit an elevated ADT and require longer ISIs (at least 45 minutes) compared to Sprague-Dawley pups (Veliskova et al., 2004) . Fast P15–16 pups kindle faster than Slow pups (Veliskova et al., 2004; Moshé et al., 1981). There are no studies about the effects of specific genetic or molecular alterations on the development of kindling or describing molecular changes resulting from kindling in developing rats. Furthermore, there are no definitive studies describing sex differences in kindling in developing rats, as all of the published data have been obtained from mixed litters, not analyzed according to sex.

Response to Antiepileptic Drugs/Usefulness in Screening Drugs Kindling has been used as a model to test the effects of a variety of conditions and drugs on the susceptibility of the developing brain to seizures and the development of the kindled state. To test whether a drug may alter sensitivity to kindling, drugs can be administered prior to the first daily kindling stimulus. It is also possible to study whether a drug may alter the establishment of the kindled state, by administering it once the rat is fully kindled. In these studies, the effects of drugs or pathologic states, such as hypoxia/ischemia, can be tested in regard to their ability to alter ADT, kindling rate, AD duration, and seizure severity. Only few antiepileptic drugs have so far been tested as to their ability to alter the development of kindling in immature pups. Diazepam (Albertson et al., 1982) and gabapentin (Lado et al., 2001) inhibited the development of kindling in immature rats. Acute and chronic ACTH treatment inhibited the development of the kindled state (Holmes and Weber, 1986), but had no effect once the kindled state was already established (Thompson and Holmes, 1987). Furthermore, other drugs and compounds not included among the conventional antiepileptics have also been tested. For instance, progesterone-like substances (Holmes and Weber, 1984), NMDA receptor antagonists (Trommer and Pasternak, 1990; Holmes et al., 1990), the GABAB receptor agonist baclofen (Wurpel, 1994), and the calcium channel blockers verapamil and nimodipine (Wurpel and Iyer, 1994) also inhibited kindling in developing rats. Estrogens had no effect on the development of kindling (Schultz-Krohn et al., 1986).

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Baclofen also suppressed the severity and duration of established kindled seizures and increased postictal refractoriness (Wurpel, 1994).

LIMITATIONS Ease of Development/Reliability Despite a higher ADT, amygdala kindling is produced faster in infantile rats compared to adults. The short ISI kindling protocol reliably kindles rats of most ages, although the success rate may differ according to the specific age and site. Amygdala kindling of P15–18 rats with 15 minute ISI kindled 100% of the pups (Moshé et al., 1983). In younger pups (P7–8) the success of inducing AD with this protocol falls to 50% (Baram et al., 1993).

Mortality Mortality during the kindling process is insignificant. If rats are allowed to grow to adulthood, a higher than expected mortality has been observed in kindled (40%) versus implanted but not kindled rats (20%) (Moshé and Albala, 1982).

WHAT’S IT GOOD FOR? Kindling of immature rats has been shown to be a reliable method of assessing susceptibility to the development of seizures and establishing a state of increased seizure susceptibility that persists till adulthood. Although spontaneous seizures, the principal feature of the epileptic state, have been reported, there are no studies documenting the persistence of these spontaneous seizures till adulthood. This may be due to excessive animal costs and the difficulty of maintaining intracerebral electrodes in growing animals. Therefore, kindling in developing rats can probably be used as a model of epileptogenesis but not as a model of spontaneous epilepsy. It is a useful method to assess the effect of a variety of drugs or conditions on several aspects of seizure susceptibility, i.e., ADT, kindling rates, recurrent triggered bilateral seizures, or postictal refractoriness. Kindling can also be used to study the consequences of repetitive seizures in the developing brain and persistence of any seizure-induced deficits.

Acknowledgements This work was supported by NIH NINDS grants NS20253 and NS048856 (SLM), and NS45243 (ASG).

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