Characteristic Features

Behavioral and Electrographic Features Flathead Mutant Rat

In the first postnatal week, flathead pups display severe generalized seizures and motor deficits. During ambulation, astatic episodes can be observed and are characterized by the splaying of limbs. Mutant rats also often fall to one side during walking but are able to right themselves quickly. The falling and astatic episodes in flathead mutants are likely related to general muscle weakness, since flathead mutants cannot grip a small metal bar and are unable to rear onto their hind limbs. Seizures begin reliably toward the end of the first postnatal week and increase in duration and frequency until the third postnatal week (Figure 1). Seizures subsequently become more rare, but more severe, as identified by behavioral observations and electrophysiologic recordings.

As early as postnatal day 8 (P8), flathead mutants display intermittent episodes of tail flexion and extension (strub tail) and tremor of the limbs and head. These observations are the earliest signs of spontaneous generalized seizures of any of the models described here. Alternating forelimb clonus becomes more prevalent between P15 and P19. During clonic episodes, animals frequently ambulate around the home cage, propelling themselves by their forelimbs. Bursts of forelimb movements often result in forward lunges. Clonus of the neck musculature produces rhythmic head movements. Flathead rats display episodes of tonus that can involve one or more limbs or the entire body; tonus persists for approximately 1 minute and is sometimes preceded by loud vocalizations. Behavioral seizures are rare after P21, although single episodes of loud vocalization, jumping, and tonus have been observed in animals as late as P25—near the time of premature death, which is thought to be caused by a lethal seizure.

Tish Mutant Rat

Convulsive seizures become apparent in the tish mutant approximately 1 month after birth (K. Lee, personal communication). Otherwise the behavior of tish mutants, assessed by routine observation, is indistinguishable from unaffected littermates or wildtype Sprague-Dawley rats. Behavioral seizures occur simultaneously with high amplitude voltage oscillations in the EEG. The rat's behavior during seizures is more variable than the electrographic seizure activity and is characterized by facial and forelimb twitching that can progress to convulsive tonic-clonic seizures. The average frequency of such seizures is 48 seconds, and the frequency of the events (assessed in 4-6 months of monitoring) is between 1.5 to 15 events/week (Chen et al., 2000).

Seizures in Otx-/- mice range from short episodes of approximately 30 seconds, to longer lasting, 60-second episodes. The shorter episodes are characterized by automatisms, including head bobbing and teeth chattering. The longer seizures are characterized by upper limb clonus, rearing, and falling. Similarly to the flathead mutant, seizure frequency in Otx-/- mice is reduced in older animals, although there is no indication of an overall cessation or remission of seizures (Acampora et al., 1996). During the shorter episodes, electrographic recordings indicate large amplitude oscillations in hippocampus with occasional and asynchronous spikes in the neocortex. This pattern is consistent with focal generation of epileptiform events. The longer convulsive seizures, in contrast, are characterized by synchronized activity in both the neocortex and hippocampus (Avanzini et al., 2000).

FIGURE 1 Flathead seizures are generalized and increase in duration with age. A: Representative six-channel, electrographic recordings of a P16 flathead rat. Electrodes were placed across the rostrocaudal extent of the cortical surface (top to bottom: anterior left hemisphere; anterior right hemisphere; middle left hemisphere; middle right hemisphere; posterior left hemisphere; posterior right hemisphere). B: The average duration of seizures increases with age in flathead. C: There were no significant differences in average interval between seizures across age. D: Representative in vitro extracellular recording from a flathead hippocampal slice (electrode positioned in CA1 pyramidal layer). Ictal and interictal periods are identifiable in the upper trace (10-minute recording). Burst-type spiking can be seen in the expanded (lower) trace. Scale bars: A, 1000ms/200 ||V; D, upper trace, 2min/70 ||V; lower trace, 8sec/70 ||V.

FIGURE 1 Flathead seizures are generalized and increase in duration with age. A: Representative six-channel, electrographic recordings of a P16 flathead rat. Electrodes were placed across the rostrocaudal extent of the cortical surface (top to bottom: anterior left hemisphere; anterior right hemisphere; middle left hemisphere; middle right hemisphere; posterior left hemisphere; posterior right hemisphere). B: The average duration of seizures increases with age in flathead. C: There were no significant differences in average interval between seizures across age. D: Representative in vitro extracellular recording from a flathead hippocampal slice (electrode positioned in CA1 pyramidal layer). Ictal and interictal periods are identifiable in the upper trace (10-minute recording). Burst-type spiking can be seen in the expanded (lower) trace. Scale bars: A, 1000ms/200 ||V; D, upper trace, 2min/70 ||V; lower trace, 8sec/70 ||V.

Both partial and generalized seizures occur in p35 -/mice (Wenzel 2001). Approximately 75% of mutants display some sign of electrographic abnormality, and 25% display both behavioral and electrographic features of generalized seizure episodes (Wenzel et al., 2001). Seizures typically start from a state of quiet rest or sleep and initiate upon a high-amplitude spike in the EEG. Behaviorally, generalized seizures begin with back and forth head movements that progress to a period characterized by a rigidly up-turned head and extended forelimbs. In many animals this behav-

FIGURE 2 The flathead phenotype is associated with a decrease in brain size but preserved patterning. A: Saggital Nissl-stained sections from a P1 (left column) and P14 (right column) flathead brain (top row) and wildtype brain (bottom row). B: Coronal Nissl-stained section of a P21 flathead brain (left) and wildtype brain (right). Note lack of the infrapyramidal blade of the dentate gyrus but normal lamination of neocortex. Scalebars: A & B, 1 mm.

FIGURE 2 The flathead phenotype is associated with a decrease in brain size but preserved patterning. A: Saggital Nissl-stained sections from a P1 (left column) and P14 (right column) flathead brain (top row) and wildtype brain (bottom row). B: Coronal Nissl-stained section of a P21 flathead brain (left) and wildtype brain (right). Note lack of the infrapyramidal blade of the dentate gyrus but normal lamination of neocortex. Scalebars: A & B, 1 mm.

ioral pattern progresses to a period of vigorous hind limb thrusts. The behavioral manifestations last 1 to 1.5 minutes (Wenzel et al., 2001). During seizures, high-amplitude elec-trographic spiking is observed bilaterally in both hippocampus and neocortex. Epileptiform spiking can continue longer in the hippocampus than in the neocortex, and ictal periods are followed by a 3 to 5 minute postictal period characterized by a quiet EEG and behavioral inactivity (Wenzel et al., 2001).

Neuropathology

Flathead Mutant

Although the primary developmental disruptions in flat-head mutants are restricted to the CNS (Roberts et al., 2000), disruptions have been reported in testes and liver (Liu et al., 2003; Naim et al., 2004). The brain of flathead mutants is approximately 50% the size of unaffected littermates (Figure 2). This difference emerges in late embryogenesis and is not detectable at embryonic day 16 (E16). By E18, mutant brains weigh 26% less than unaffected littermates, and by

E19 weigh 51% less. Reduction in size involves most brain areas, although the tectum is least affected (Figure 2). In addition to general reduction in brain size, some neuronal populations are disproportionally reduced in number. The most affected cell populations include granule cells in cerebellum, dentate granule cells of the hippocampus, and layer 2/3 pyramidal neurons of neocortex. This pattern of loss is consistent with a model of progenitor depletion that would preferentially target the latest born populations of neurons.

In flathead mutants, the number of inhibitory interneu-rons—as a proportion of the total number of neurons in the cerebral cortex—is also greatly reduced (Figure 3). Quantitative analysis in both somatosensory cortex (SS) and entorhinal cortex (EC) indicates that the percentage of GABA-positive neurons is reduced in mutants relative to littermates. In upper layers of EC, 30% of neurons in normal littermates are GABA-positive, compared to only 9% of neurons in mutants; in deeper layers of EC, 15% of neurons in normal littermates are GABA-positive compared to only 6% of neurons in flathead rats. In deeper layers of SS cortex, 15% of neurons in unaffected littermates, compared to only 4% of neurons in mutants, are GABA-positive. In contrast, in upper layers of SS there is only a slight decrease in the percentage of GABA-positive neurons: ~11% of neurons in littermates compared to 9% of neurons in mutants. The calcium-binding proteins calretinin (CR), parvalbumin (PARV), and calbindin (CAL) are specifically expressed in largely nonoverlapping populations of GABA-positive interneurons in the cerebral cortex and correspond to different functional and morphological classes of inhibitory interneurons. The flathead mutation results in differential loss of these different classes (Alcantara et al., 1993; Alcantara et al., 1996); the CR-positive interneurons are not significantly decreased in number, but the PARV and CAL-positive cells are reduced by 70% (Sarkisian et al., 2001). Therefore, the flathead mutation causes a widespread and differential decrease in the relative number of inhibitory interneurons across neocortex.

Tish Mutant Rat

The tish mutant has large bilateral secondary cortices that form below the normal neocortical layers. The heterotopias are variable in size from animal to animal, but typically extend from the frontal to occipital poles (Lee et al., 1997). The normotopic cortex above the heterotopic cortex is thinner than normal, although all neocortical layers and major projection neurons are present. The heterotopic cortex, in contrast, is not laminated. Cellular birth-dating experiments with BrdU indicate that cells in normotopic cortex are born in a normal inside-to-outside pattern, while the cells in the nonlaminated heterotopic cortex do not show a clear birth-dating pattern (Lee et al., 1998). In spite of the lack of lamination, the heterotopic cortex (like the

FIGURE 3 The flathead phenotype is associated with cytokinesis failure of neocortical neurons and a reduction of GABAergic interneurons. A: Electron micrograph of a neocortical neuron where two nuclei are visible within a single cell membrane. B: An embryonic BrdU pulse results in labeling of multiple nuclei within single Nissl-stained neocortical neurons. C: Quantification of the relative number of GABAergic interneurons in the upper and deeper layers of entorhinal cortex and somatosensory cortex in flathead and wildtype brains at P0 and P14. D: Representative example of a calbindin-positive neocortical interneuron in flathead, containing two nuclei (arrows). E: Representative example of a parvalbumin-positive neocortical interneuron in the flathead rat, containing two nuclei (arrows). F: Relative percentage of GABAergic interneuron subtypes in the flathead (closed bars) and wildtype (open bars) neocortex (entorhi-nal and somatosensory). G: Percentage of GABAergic and DiI-labeled pyramidal neurons in flathead neocortex that contains multiple nuclei. Scale bars: A, 5 mm; B, 10 mm; D, 15 mm; E, 20 mm.

FIGURE 3 The flathead phenotype is associated with cytokinesis failure of neocortical neurons and a reduction of GABAergic interneurons. A: Electron micrograph of a neocortical neuron where two nuclei are visible within a single cell membrane. B: An embryonic BrdU pulse results in labeling of multiple nuclei within single Nissl-stained neocortical neurons. C: Quantification of the relative number of GABAergic interneurons in the upper and deeper layers of entorhinal cortex and somatosensory cortex in flathead and wildtype brains at P0 and P14. D: Representative example of a calbindin-positive neocortical interneuron in flathead, containing two nuclei (arrows). E: Representative example of a parvalbumin-positive neocortical interneuron in the flathead rat, containing two nuclei (arrows). F: Relative percentage of GABAergic interneuron subtypes in the flathead (closed bars) and wildtype (open bars) neocortex (entorhi-nal and somatosensory). G: Percentage of GABAergic and DiI-labeled pyramidal neurons in flathead neocortex that contains multiple nuclei. Scale bars: A, 5 mm; B, 10 mm; D, 15 mm; E, 20 mm.

normotopic cortex) contains neurons that project to appropi-ate subcortical and cortical targets. Similarly, the heterotopic cortex receives patterned thalamic input from somatosen-sory thalamus; cortical barrels form in both heterotopic and normotopic cortex (Schottler et al., 2001).

The brains of Otx-/- mice are significantly smaller than the brains of wildtype or heterozygous animals. The neo-cortex is most reduced in size, while the midbrain and hind brain are relatively unaffected. The neocortical lamina are reduced in thickness by approximately 25%, and there is a significant reduction in cellular density in neocortical lamina ranging from 4 to 35% (depending on the cortical area examined) (Cipelletti et al., 2002; Panto et al., 2004). This pattern of microencephaly is similar to, although less severe than, that in the flathead mutant.

There is also a decrease in the density of some interneu-ron populations within the neocortex of Otx1-/- mice. The density of GAD67-positive inhibitory interneurons in the Otx1 -/- neocortex, as well as parvalbumin- and calbindin-positve neurons, shows reductions. Parvalbumin interneurons are reduced by approximately 60% and calbindin-positive interneurons are reduced by approximately 52%, whereas the total decrease in neurons is approximately 30% (Cipelletti et al., 2002). Thus, similar to the flathead mutant cortex, the Otx-/- mutant cortex has an overall reduction in neurons and a disproportionate decrease in interneurons.

Both neocortex and hippocampus show developmental disruption in p35-/- mice. The lamination of neocortex is disrupted and the laminar position of pyramidal cell populations is generally inverted relative to control neo-cortex (Chae et al., 1997). There has been no report of significant/disproportionate interneuron loss. The normally compact dentate granule cell layer in hippocampus is more diffuse in the p35-/- mutants. In addition, the dentate granule cells exhibit ectopic sprouting. This sprouting has not been observed in the other three developmental models discussed here but is a common feature of many other models of epilepsy. Such axonal sprouting results in recurrent feedback excitation that may create hyperexcitable circuits in hippocampus (Patel et al., 2004).

Neurophysiology

Flathead Mutant

Flathead electrographic seizures occur at a high enough frequency, and with sufficient reliability, to allow for quan titative measurements of seizure duration and interval across postnatal development. Figure 1 shows the changes in seizure frequency and duration from animals from P7 to P18. The interval between seizures does not change significantly; however, the seizure duration increases with age. By P20, electrographic and behavioral seizures are rare; however, severe lethal seizures have been noted in several animals from P21 to P27. Long-duration seizures increase in number from P7 to P18, and this increase accounts for the increase in mean seizure duration.

Simultaneous multi-site surface recordings, as well as recordings from brain slices, demonstrate that epileptiform activity in the flathead can be generated from multiple sites within cerebral cortex. Following callosal transection, both hemispheres can show independent and isolated electro-graphic seizure activity. Similarly, spontaneous 20 to 60 second self-regenerating epileptiform discharges are present in brain slices prepared from neocortex or hippocampus.

Tish Mutant

In vivo depth electrode recordings and slice recordings from tish rats indicate that epileptiform activity is present in both the normotopic and heterotopic cortices (Chen et al.,

2000). Epileptiform activity in normotopic cortex appears to precede activity in the heterotopic cortex. In experiments on brain slices, when normotopic cortex was isolated from heterotopic cortex (either pharmacologically or surgically), epileptiform activity persisted in normotopic but not in heterotopic cortex (Chen et al., 2000). Thus, the driver of epileptiform activity in the tish rat appears to be normotopic, not heterotopic, cortex.

A thorough electrophysiologic analysis has been carried out in brain slices prepared from Otx-/- mice. Stimulation of white matter can initiate polysynaptic bursts in the Otx-/- cortex at stimuli strengths that fail to elicit bursts in normal controls (Sancini et al., 2001). These polysynaptic bursts are characterized by prominent slow inhibitory post-synaptic potentials (IPSPs) and delayed large excitatory PSPs (EPSPs). The polysynaptic burst activity is blocked by NMDA receptor antagonists but not by AMPA receptor antagonists. Intrinsic excitability of pyramidal cells is not altered in the mutant. However, a redistribution of electro-physiologic cell types is apparent in layer V, apparently resulting from a loss of a subpopulation of layer V pyramidal neurons. It is likely that the combination of increased synchrony of synaptic inhibition, along with increases in NMDA-mediated excitation, results in the aberrant poly-synaptic activity described in Otx-/- cortex (Sancini et al.,

2001). The enhanced synchrony of inhibition may seem paradoxical considering the relative loss of inhibitory interneu-

rons. However, it is possible that a decrease in interneuron number may result in increased synchrony because fewer inhibitory neuronal elements contribute to the IPSPs.

Both in vivo recordings and in vitro brain slice experiments have been carried out in p35 -/- mutant mice. In vivo depth electrode recordings from hippocampus and neocor-tex show that intermittent epileptiform spikes typically occur in the hippocampus, and sustained ictal discharges occur synchronously in hippocampus and neocortex (Wenzel et al., 2001). Field EPSP recordings from the dentate gyrus further indicate an aberrant response in the granule cell layer that is likely due to a dispersed "somal" current sink (Patel et al., 2004). Intrinsic properties of dentate granule cells are similar between mutants and controls, but hyperexcitability of dentate granule cell responses, relative to controls, is observed in low concentrations of the GABA receptor antagonist bicuculline methiodide (BMI) (Patel et al., 2004). In addition, consistent with an increase in granule cell sprouting, antidromic activation of granule cells in the p35 -/- hippocampus results in enhanced excitatory synaptic responses in granule cells (Patel et al., 2004).

Response to AEDs

To date, only the flathead rat has been used in published studies to test the effectiveness of different antiepileptic drugs (AEDs) (Sarkisian et al., 1999). Three AEDs have been evaluated for their effects on the duration and frequency of electrographic seizures in flathead mutants. Phenobarbital (PB), ethosuximide (ESM), and valproate (VPA) induce sedation for at least 2 hours in neonatal rats (PB, 40mg/kg; ESM, 600mg/kg; VPA, 400mg/kg), and suppress behavioral manifestations of the seizures. However, electrographic seizures are still observed after AED treatment, and occur at rates of at least 2 per hour. Seizure occurrence is differently affected by the different AEDs. PB significantly increases the interval between seizures in mutants but does not significantly change the duration of seizures. VPA, in contrast, does not significantly alter the interval between seizures but shortens the duration of seizures in the flathead mutant. ESM has no effect on seizure interval or duration in the flathead mutant (Sarkisian et al., 1999).

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