Cannabidiol and epilepsy.

Cannabidiol (CBD) has been investigated as a pharmacological approach for treating a myriad of neurological and psychiatric disorders, the most successful of them being its use as an antiseizure drug (ASD). Indeed, CBD has reached the clinics for the treatment of certain epileptic syndromes. This chapter aims to overview the pharmacology of CBD and its potential mechanisms of action as an ASD. First, we give an outline of the concepts, mechanisms and pharmacology pertaining to the field of study of epilepsy and epileptic seizures. In the second section, we will summarize the effects of CBD as an ASD. Next, we will discuss its potential mechanisms of action to alleviate epileptic seizures, which seem to entail multiple neurotransmitters, receptors and intracellular pathways. Finally, we will conclude and present some limitations and perspectives for future studies.

Respiratory activity during seizures induced by pentylenetetrazole.

This study investigated the respiratory activity in adult Wistar rats across different behavioral seizure severity induced by pentylenetetrazole (PTZ). Animals underwent surgery for electrodes implantation, allowing simultaneous EEG and diaphragm EMG (DIAEMG) recordings and the respiratory frequency and DIAEMG amplitude were measured. Seizures were acutely induced through PTZ injection and classified based on a pre-established score, with absence-like seizures (spike wave discharge (SWD) events on EEG) representing the lowest score. The respiratory activity was grouped into the different seizure severities. During absence-like and myoclonic jerk seizures, the breathing frequency decreased significantly (∼50% decrease) compared to pre- and post-ictal periods. Pronounced changes occurred with more severe seizures (clonic and tonic) with periods of apnea, especially during tonic seizures. Apnea duration was significantly higher in tonic compared to clonic seizures. Notably, during PTZ-induced tonic seizures the apnea events were marked by tonic DIAEMG contraction (tonic-phase apnea). In the majority of animals (5 out of 7) this was a fatal event in which the seizure-induced respiratory arrest preceded the asystole. In conclusion, we provide an assessment of the respiratory activity in the PTZ-induced acute seizures and showed that breathing dysfunction is more pronounced in seizures with higher severity.

Different types of Status Epilepticus may lead to similar hippocampal epileptogenesis processes.

About 1-2% of people worldwide suffer from epilepsy, which is characterized by unpredictable and intermittent seizure occurrence. Despite the fact that the exact origin of temporal lobe epilepsy is frequently unknown, it is frequently linked to an early triggering insult like brain damage, tumors, or Status Epilepticus (SE). We used an experimental approach consisting of electrical stimulation of the amygdaloid complex to induce two behaviorally and structurally distinct SE states: Type I (fully convulsive), with more severe seizure behaviors and more extensive brain damage, and Type II (partial convulsive), with less severe seizure behaviors and brain damage. Our goal was to better understand how the various types of SE impact the hippocampus leading to the development of epilepsy. Despite clear variations between the two behaviors in terms of neurodegeneration, study of neurogenesis revealed a comparable rise in the number of Ki-67 + cells and an increase in Doublecortin (DCX) in both kinds of SE.

Divergent Effects of Systemic and Intracollicular CB Receptor Activation Against Forebrain and Hindbrain-Evoked Seizures in Rats.

Cannabinoid (CB) receptor agonists are of growing interest as targets for anti-seizure therapies. Here we examined the effect of systemic administration of the CB receptor agonist WIN 55,212-2 (WIN) against audiogenic seizures (AGSs) in the Genetically Epilepsy Prone Rat (GEPR)-3 strain, and against seizures evoked focally from the Area Tempestas (AT). We compared these results to the effect of focal administration of the CB1/2 receptor agonist CP 55940 into the deep layers of the superior colliculus (DLSC), a brain site expressing CB1 receptors. While systemic administration of WIN dose-dependently decreased AGS in GEPR-3s, it was without effect in the AT model. By contrast, intra-DLSC infusion of CP 55940 decreased seizures in both models. To determine if the effects of systemic WIN were dependent upon activation of CB1 receptors in the DSLC, we next microinjected the CB1 receptor antagonist SR141716, before WIN systemic treatment, and tested animals for AGS susceptibility. The pretreatment of the DLSC with SR141716 was without effect on its own and did not alter the anti-convulsant action of WIN systemic administration. Thus, while CB receptors in the DLSC are a potential site of anticonvulsant action, they are not necessary for the effects of systemically administered CB agonists.

Behavioral and EEGraphic Characterization of the Anticonvulsant Effects of the Predator Odor (TMT) in the Amygdala Rapid Kindling, a Model of Temporal Lobe Epilepsy.

Background: Clinical and experimental evidence indicates that olfactory stimulation modulates limbic seizures, either blocking or inducing ictal activity. Objective: We aim to evaluate the behavioral and electroencephalographic (EEGraphic) effects of dihydro-2,4,5-trimethylthiazoline (TMT) olfactory exposure on limbic seizures induced by amygdala rapid kindling (ARK). Materials and Methods: Wistar male rats (280-300 g) underwent stereotaxic surgery for electrode implantation in piriform cortex (PC), hippocampal formation (HIP), and amygdaloid complex (AMYG). Part of the animals was exposed to a saturated chamber with water or TMT, while others had ARK and olfactory exposure prior to the 21st stimulus. Behavioral responses were measured by traditional seizure severity scales (Racine and Pinel and Rovner) and/or by sequential analysis/neuroethology. The electrographic activity of epileptogenic limbic networks was quantified by the occurrence of the first and second EEG afterdischarges, comparing the 1st and 21st stimulus. The spectral analysis [Fast Fourier Transform (FFT)] of the first afterdischarge was performed at the 21st stimulus. Results: TMT olfactory exposure reduced the seizure severity in kindled rats, altering the displayed behavioral sequence. Moreover, TMT decreased the occurrence of first and second afterdischarges, at the 21st stimulus, and altered the spectral features. Conclusions: Both behavioral and EEGraphic evaluations indicated that TMT, a potent molecule with strong biological relevance, in fact, "predator odor," suppressed the epileptiform activity in limbic networks.

Impact of mTOR hyperactive neurons on the morphology and physiology of adjacent neurons: Do PTEN KO cells make bad neighbors?

Hyperactivation of the mechanistic target of rapamycin (mTOR) pathway is associated with epilepsy, autism and brain growth abnormalities in humans. mTOR hyperactivation often results from developmental somatic mutations, producing genetic lesions and associated dysfunction in relatively restricted populations of neurons. Disrupted brain regions, such as those observed in focal cortical dysplasia, can contain a mix of normal and mutant cells. Mutant cells exhibit robust anatomical and physiological changes. Less clear, however, is whether adjacent, initially normal cells are affected by the presence of abnormal cells. To explore this question, we used a conditional, inducible mouse model approach to delete the mTOR negative regulator phosphatase and tensin homolog (PTEN) from <1% to >30% of hippocampal dentate granule cells. We then examined the morphology of PTEN-expressing granule cells located in the same dentate gyri as the knockout (KO) cells. Despite the development of spontaneous seizures in higher KO animals, and disease worsening with increasing age, the morphology and physiology of PTEN-expressing cells was only modestly affected. PTEN-expressing cells had smaller somas than cells from control animals, but other parameters were largely unchanged. These findings contrast with the behavior of PTEN KO cells, which show increasing dendritic extent with greater KO cell load. Together, the findings indicate that genetically normal neurons can exhibit relatively stable morphology and intrinsic physiology in the presence of nearby pathological neurons and systemic disease.

Impact of strain, sex, and estrous cycle on gamma butyrolactone-evoked absence seizures in rats.

Childhood absence epilepsy (CAE) is the most common pediatric epilepsy syndrome and is characterized by typical absence seizures (AS). AS are non-convulsive epileptic seizures characterized by a sudden loss of awareness and bilaterally generalized synchronous 2.5-4 Hz spike and slow-wave discharges (SWD). Gamma butyrolactone (GBL) is an acute pharmacological model of AS and induces bilaterally synchronous SWDs and behavioral arrest. Despite the long use of this model, little is known about its strain and sex-dependent features. We compared the dose-response profile of GBL-evoked SWDs in three rat strains (Long Evans, Sprague-Dawley, and Wistar), and examined the modulatory effects of estrous cycle on SWDs in female Wistar rats. We evaluated the number of seizures, the cumulative time seizing, and the average seizure duration as a function of dose, strain, and sex/estrous phase. Long Evans rats displayed the greatest sensitivity to GBL, followed by Wistar rats, and then by Sprague-Dawley rats. GBL-evoked SWDs were modulated by estrous cycle in female rats, with the lowest sensitivity to GBL occurring during metestrus. Wistar rats showed the greatest variability as a function of dose, and the least variability within dose; these features make this strain desirable for interventional studies. Moreover, our finding that the SWD response to GBL differs as a function of estrous cycle underscores the importance of cycle monitoring in studies examining female animals using this model. Together, these strain and sex-dependent findings provide guidance for future studies.

Kappa opioid receptors regulate hippocampal synaptic homeostasis and epileptogenesis.

OBJECTIVE: Homeostatic synaptic plasticity (HSP) serves as a gain control mechanism at central nervous system (CNS) synapses, including those between the dentate gyrus (DG) and CA3. Improper circuit control of DG-CA3 synapses is hypothesized to underlie epileptogenesis. Here, we sought to (1) identify compounds that preferentially modulate DG-CA3 synapses in primary neuronal culture and (2) determine if these compounds would delay or prevent epileptogenesis in vivo. METHODS: We previously developed and validated an in vitro assay to visualize the behavior of DG-CA3 synapses and predict functional changes. We used this "synapse-on-chip" assay (quantification of synapse size, number, and type using immunocytochemical markers) to dissect the mechanisms of HSP at DG-CA3 synapses. Using chemogenetic constructs and pharmacological agents we determined the signaling cascades necessary for gain control at DG-CA3 synapses. Finally, we tested the implicated cascades (using kappa opioid receptor (OR) agonists and antagonists) in two models of epileptogenesis: electrical amygdala kindling in the mouse and chemical (pentylenetetrazole) kindling in the rat. RESULTS: In vitro, synapses between DG mossy fibers (MFs) and CA3 neurons are the primary homeostatic responders during sustained periods of activity change. Kappa OR signaling is both necessary and sufficient for the homeostatic elaboration of DG-CA3 synapses, induced by presynaptic DG activity levels. Blocking kappa OR signaling in vivo attenuates the development of seizures in both mouse and rat models of epilepsy. SIGNIFICANCE: This study elucidates mechanisms by which synapses between DG granule cells and CA3 pyramidal neurons undergo activity-dependent homeostatic compensation, via OR signaling in vitro. Modulation of kappa OR signaling in vivo alters seizure progression, suggesting that breakdown of homeostatic closed-loop control at DG-CA3 synapses contributes to seizures, and that targeting endogenous homeostatic mechanisms at DG-CA3 synapses may prove useful in combating epileptogenesis.

PTEN deletion increases hippocampal granule cell excitability in male and female mice.

Deletion of the mTOR pathway inhibitor PTEN from postnatally-generated hippocampal dentate granule cells causes epilepsy. Here, we conducted field potential, whole cell recording and single cell morphology studies to begin to elucidate the mechanisms by which granule cell-specific PTEN-loss produces disease. Cells from both male and female mice were recorded to identify sex-specific effects. PTEN knockout granule cells showed altered intrinsic excitability, evident as a tendency to fire in bursts. PTEN knockout granule cells also exhibited increased frequency of spontaneous excitatory synaptic currents (sEPSCs) and decreased frequency of inhibitory currents (sIPSCs), further indicative of a shift towards hyperexcitability. Morphological studies of PTEN knockout granule cells revealed larger dendritic trees, more dendritic branches and an impairment of dendrite self-avoidance. Finally, cells from both female control and female knockout mice received more sEPSCs and more sIPSCs than corresponding male cells. Despite the difference, the net effect produced statistically equivalent EPSC/IPSC ratios. Consistent with this latter observation, extracellularly evoked responses in hippocampal slices were similar between male and female knockouts. Both groups of knockouts were abnormal relative to controls. Together, these studies reveal a host of physiological and morphological changes among PTEN knockout cells likely to underlie epileptogenic activity. SIGNIFICANCE STATEMENT: Hyperactivation of the mTOR pathway is associated with numerous neurological diseases, including autism and epilepsy. Here, we demonstrate that deletion of the mTOR negative regulator, PTEN, from a subset of hippocampal dentate granule impairs dendritic patterning, increases excitatory input and decreases inhibitory input. We further demonstrate that while granule cells from female mice receive more excitatory and inhibitory input than males, PTEN deletion produces mostly similar changes in both sexes. Together, these studies provide new insights into how the relatively small number (≈200,000) of PTEN knockout granule cells instigates the development of the profound epilepsy syndrome evident in both male and female animals in this model.

Impact of rapamycin on status epilepticus induced hippocampal pathology and weight gain.

Growing evidence implicates the dentate gyrus in temporal lobe epilepsy (TLE). Dentate granule cells limit the amount of excitatory signaling through the hippocampus and exhibit striking neuroplastic changes that may impair this function during epileptogenesis. Furthermore, aberrant integration of newly-generated granule cells underlies the majority of dentate restructuring. Recently, attention has focused on the mammalian target of rapamycin (mTOR) signaling pathway as a potential mediator of epileptogenic change. Systemic administration of the mTOR inhibitor rapamycin has promising therapeutic potential, as it has been shown to reduce seizure frequency and seizure severity in rodent models. Here, we tested whether mTOR signaling facilitates abnormal development of granule cells during epileptogenesis. We also examined dentate inflammation and mossy cell death in the dentate hilus. To determine if mTOR activation is necessary for abnormal granule cell development, transgenic mice that harbored fluorescently-labeled adult-born granule cells were treated with rapamycin following pilocarpine-induced status epilepticus. Systemic rapamycin effectively blocked phosphorylation of S6 protein (a readout of mTOR activity) and reduced granule cell mossy fiber axon sprouting. However, the accumulation of ectopic granule cells and granule cells with aberrant basal dendrites was not significantly reduced. Mossy cell death and reactive astrocytosis were also unaffected. These data suggest that anti-epileptogenic effects of mTOR inhibition may be mediated by mechanisms other than inhibition of these common dentate pathologies. Consistent with this conclusion, rapamycin prevented pathological weight gain in epileptic mice, suggesting that rapamycin might act on central circuits or even peripheral tissues controlling weight gain in epilepsy.

PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure.

Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offsets the reduction in boutons per axon length. These morphological changes predict a net increase in granule cell innervation of CA3. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell communication with CA3. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy.

Impact of corticosterone treatment on spontaneous seizure frequency and epileptiform activity in mice with chronic epilepsy.

Stress is the most commonly reported precipitating factor for seizures in patients with epilepsy. Despite compelling anecdotal evidence for stress-induced seizures, animal models of the phenomena are sparse and possible mechanisms are unclear. Here, we tested the hypothesis that increased levels of the stress-associated hormone corticosterone (CORT) would increase epileptiform activity and spontaneous seizure frequency in mice rendered epileptic following pilocarpine-induced status epilepticus. We monitored video-EEG activity in pilocarpine-treated mice 24/7 for a period of four or more weeks, during which animals were serially treated with CORT or vehicle. CORT increased the frequency and duration of epileptiform events within the first 24 hours of treatment, and this effect persisted for up to two weeks following termination of CORT injections. Interestingly, vehicle injection produced a transient spike in CORT levels - presumably due to the stress of injection - and a modest but significant increase in epileptiform activity. Neither CORT nor vehicle treatment significantly altered seizure frequency; although a small subset of animals did appear responsive. Taken together, our findings indicate that treatment of epileptic animals with exogenous CORT designed to mimic chronic stress can induce a persistent increase in interictal epileptiform activity.