Early developmental electroencephalography abnormalities, neonatal seizures, and induced spasms in a mouse model of tuberous sclerosis complex.

OBJECTIVE: Tuberous sclerosis complex (TSC) is one of the most common genetic causes of epilepsy. Seizures in TSC typically first present in infancy or early childhood, including focal seizures and infantile spasms. Infantile spasms in TSC are particularly characteristic in its strong responsiveness to vigabatrin. Although a number of mouse models of epilepsy in TSC have been described, there are very limited electroencephalographic (EEG) or seizure data during the preweanling neonatal and infantile-equivalent mouse periods. Tsc1GFAP CKO mice are a well-characterized mouse model of epilepsy in TSC, but whether these mice have seizures during early development has not been documented. The objective of this study was to determine whether preweanling Tsc1GFAP CKO mice have developmental EEG abnormalities or seizures, including spasms. METHODS: Longitudinal video-EEG and electromyographic recordings were performed serially on Tsc1GFAP CKO and control mice from postnatal days 9-21 and analyzed for EEG background abnormalities, sleep-wake vigilance states, and spontaneous seizures. Spasms were also induced with varying doses of N-methyl-D-aspartate (NMDA). RESULTS: The interictal EEG of Tsc1GFAP CKO mice had excessive discontinuity and slowing, suggesting a delayed developmental progression compared with control mice. Tsc1GFAP CKO mice also had increased vigilance state transitions and fragmentation. Tsc1GFAP CKO mice had spontaneous focal seizures in the early neonatal period and a reduced threshold for NMDA-induced spasms, but no spontaneous spasms were observed. SIGNIFICANCE: Neonatal Tsc1GFAP CKO mice recapitulate early developmental aspects of EEG abnormalities, focal seizures, and an increased propensity for spasms. This mouse model may be useful for early mechanistic and therapeutic studies of epileptogenesis in TSC.

Transient use of a systemic adenosine kinase inhibitor attenuates epilepsy development in mice.

OBJECTIVE: Over one-third of all patients with epilepsy are refractory to treatment and there is an urgent need to develop new drugs that can prevent the development and progression of epilepsy. Epileptogenesis is characterized by distinct histopathologic and biochemical changes, which include astrogliosis and increased expression of the adenosine-metabolizing enzyme adenosine kinase (ADK; EC 2.7.1.20). Increased expression of ADK contributes to epileptogenesis and is therefore a target for therapeutic intervention. We tested the prediction that the transient use of an ADK inhibitor administered during the latent phase of epileptogenesis can mitigate the development of epilepsy. METHODS: We used the intrahippocampal kainic acid (KA) mouse model of temporal lobe epilepsy, which is characterized by ipsilateral hippocampal sclerosis with granule cell dispersion and the development of recurrent hippocampal paroxysmal discharges (HPDs). KA-injected mice were treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 1.6 mg/kg, b.i.d., i.p.) during the latent phase of epileptogenesis from day 3-8 after injury; the period when gradual increases in hippocampal ADK expression begin to manifest. HPDs were assessed at 6 and 9 weeks after KA administration followed by epilepsy histopathology including assessment of granule cell dispersion, astrogliosis, and ADK expression. RESULTS: 5-ITU significantly reduced the percent time in seizures by at least 80% in 56% of mice at 6 weeks post-KA. This reduction in seizure activity was maintained in 40% of 5-ITU-treated mice at 9 weeks. 5-ITU also suppressed granule cell dispersion and prevented maladaptive ADK increases in these protected mice. SIGNIFICANCE: Our results show that the transient use of a small-molecule ADK inhibitor, given during the early stages of epileptogenesis, has antiepileptogenic disease-modifying properties, which provides the rationale for further investigation into the development of a novel class of antiepileptogenic ADK inhibitors with increased efficacy for epilepsy prevention.

Longitudinal analysis of developmental changes in electroencephalography patterns and sleep-wake states of the neonatal mouse.

The neonatal brain undergoes rapid maturational changes that facilitate the normal development of the nervous system and also affect the pathological response to brain injury. Electroencephalography (EEG) and analysis of sleep-wake vigilance states provide important insights into the function of the normal and diseased immature brain. While developmental changes in EEG and vigilance states are well-described in people, less is known about the normal maturational properties of rodent EEG, including the emergence and evolution of sleep-awake vigilance states. In particular, a number of developmental EEG studies have been performed in rats, but there is limited comparable research in neonatal mice, especially as it pertains to longitudinal EEG studies performed within the same mouse. In this study, we have attempted to provide a relatively comprehensive assessment of developmental changes in EEG background activity and vigilance states in wild-type mice from postnatal days 9-21. A novel EEG and EMG method allowed serial recording from the same mouse pups. EEG continuity and power and vigilance states were analyzed by quantitative assessment and fast Fourier transforms. During this developmental period, we demonstrate the timing of maturational changes in EEG background continuity, frequencies, and power and the emergence of identifiable wake, NREM, and REM sleep states. These results should serve as important control data for physiological studies of mouse models of normal brain development and neurological disease.

The specificity and role of microglia in epileptogenesis in mouse models of tuberous sclerosis complex.

OBJECTIVE: Microglial abnormalities have been reported in pathologic specimens from patients with tuberous sclerosis complex (TSC), a genetic disorder characterized by epilepsy, intellectual disability, and autism. However, the pathogenic role of microglia in epilepsy in TSC is poorly understood, particularly whether microglia defects may be a primary contributor to epileptogenesis or are secondary to seizures or simply epiphenomena. In this study, we tested the hypothesis that Tsc1 gene inactivation in microglia is sufficient to cause epilepsy in mouse models of TSC. METHODS: Using a chemokine receptor, Cx3cr1, to target microglia, conventional Tsc1Cx3cr1-Cre CKO (conditional knockout) mice and postnatal-inducible Tsc1Cx3cr1-CreER CKO mice were generated and assessed for molecular and histopathologic evidence of microglial abnormalities, mechanistic target of rapamycin 1 (mTORC1) pathway activation, and epilepsy. RESULTS: Tsc1Cx3cr1-Cre CKO mice exhibited a high efficiency of microglia Tsc1 inactivation, mTORC1 activation, increased microglial size and number, and robust epilepsy, which were rapamycin-dependent. However, Cre reporter studies demonstrated that constitutive Cx3cr1 expression affected not only microglia, but also a large percentage of cortical neurons, confounding the role of microglia in epileptogenesis in Tsc1 Cx3cr1-Cre CKO mice. In contrast, postnatal inactivation of Tsc1 utilizing a tamoxifen-inducible Cx3cr1-CreER resulted in a more-selective microglia Tsc1 inactivation with high efficiency, mTORC1 activation, and increased microglial size and number, but no documented epilepsy. SIGNIFICANCE: Microglia abnormalities may contribute to epileptogenesis in the context of neuronal involvement in TSC mouse models, but selective Tsc1 gene inactivation in microglia alone may not be sufficient to cause epilepsy, suggesting that microglia have more supportive roles in the pathogenesis of seizures in TSC.