Search

Seizures have sleep–wake and circadian patterns in various epilepsies and, in turn, disrupt sleep and circadian rhythms. The resultant sleep deprivation (SD) is an exacerbating factor for seizures that sets up a vicious cycle that can potentially lead to disease progression and even to epilepsy-related mortality. A variety of cellular or network electrophysiological changes and changes in expression of clock-controlled genes or other transcription factors underlie sleep–wake and circadian distribution of seizures, as well as the disruptions seen in both. A broad understanding of these mechanisms may help in designing better treatments to prevent SD-induced seizure exacerbation, disrupt the vicious cycle of disease progression, and reduce epilepsy-related mortality.

Summary Objective Manual detection of spike‐wave discharges (SWDs) from electroencephalography (EEG) records is time intensive, costly, and subject to inconsistencies/biases. In addition, manual scoring often omits information on SWD confidence/intensity, which may be important for the investigation of mechanistic‐based research questions. Our objective is to develop an automated method for the detection of SWDs in a mouse model of absence epilepsy that is focused on the characteristics of human scoring of preselected events to establish a confidence‐based, continuous‐valued scoring. Methods We develop a support vector machine (SVM)–based algorithm for the automated detection of SWDs in the γ2R43Q mouse model of absence epilepsy. The algorithm first identifies putative SWD events using frequency‐ and amplitude‐based peak detection. Four humans scored a set of 2500 putative events identified by the algorithm. Then, using predictors calculated from the wavelet transform of each event and the labels from human scoring, we trained an SVM to classify (SWD/nonSWD) and assign confidence scores to each event identified from 60, 24‐hour EEG records. We provide a detailed assessment of intra‐ and interrater scoring that demonstrates advantages of automated scoring. Results The algorithm scored SWDs along a continuum that is highly correlated with human confidence and that allows us to more effectively characterize ambiguous events. We demonstrate that events along our scoring continuum are temporally and proportionately correlated with abrupt changes in spectral power bands relevant to normal behavioral states including sleep. Significance Although there are automated and semi‐automated methods for the detection of SWDs in humans and rats, we contribute to the need for continued development of SWD detection in mice. Our results demonstrate the value of viewing detection of SWDs as a continuous classification problem to better understand “ground truth” in SWD detection (ie, the most reliable features agreed upon by humans that also correlate with objective physiologic measures).

Granule cells (GCs) of the dentate gyrus use sparse encoding to perform redundancy reduction, pattern separation, and novelty detection. One likely candidate mechanism to enforce low spiking activity is feedforward inhibition, in which the cortical excitatory drive from the perforant path (PP) recruits GABAergic interneurons that then inhibit GCs. Little is known, however, about how PP drive is balanced between GCs versus inhibitory neurons. In simultaneous recordings of GCs and fast-spiking (FS) interneurons from C57BL/6 mice, we find that focal PP stimulation preferentially recruits spiking in FS interneurons over GCs, because GCs require a larger excitatory synaptic current density to reach spike threshold. Blocking inhibition reversed this relationship, revealing a stronger intrinsic coupling between the PP and GCs versus FS interneurons and showing that inhibition can sparsify the output of the dentate gyrus by tightly regulating GC spike probability. Moreover, this regulation is dynamic, because the spiking profile of FS interneurons was frequency tuned, displaying bursting behavior in response to PP stimulation near theta rhythm frequency (∼10 Hz). The later spikes in the bursts were part of the feedback inhibitory pathway because they were driven by late EPSCs, were blocked by an inhibitor of synaptic output from GCs, and shared the same frequency dependence as GC spiking. Therefore, the temporal content of signals arriving via the PP determines whether a FS interneuron participates in only feedforward (one spike) or both feedforward and feedback (burst) inhibition.

Epilepsy, with a lifetime prevalence rate of 3%, is a common and serious neurological disorder. It is characterized by recurrent paroxysms resulting from hypersynchronous discharges in the brain. Mutations in more than a dozen ion channel genes have been associated with familial epilepsy syndromes providing a strong foundation on which to build an understanding of epileptogenesis. Ion channels have an acute role setting the real-time excitability of neurons and networks, and a developmental role in the coupling of neurons in networks. When mutations occur, they may influence either role to varying degrees to cause the clinical phenotype. Distinguishing acute from developmental roles has important therapeutic implications. Reversing the consequences of developmental dysfunction may not be a simple matter of compensating for the acute receptor deficit because the developmental consequences may be inherent in neural networks of patients with epilepsy. There is no evidence that familial disease causing ion channel mutations impacts neural development. Mouse models harboring human epilepsy-causing mutations that recapitulate aspects of the human disease are particularly valuable models to test the link between gene mutation and seizures. Manipulation of the temporal expression of a single mutation may provide a means to resolving the impact of a mutation on the developing and adult brain. To this end, we engineered a mouse model in which we can manipulate the temporal expression of a GABAA γ2 mutation, previously reported in a large Australian family with febrile seizures and generalized epilepsy.1 GABAA receptors are ligand-gated chloride channels and arbiters of fast inhibitory neurotransmission in the adult brain. Perturbation of fast inhibition has been linked to seizure genesis. During brain development, GABAA receptors also play an important role in regulating neuronal differentiation, proliferation, and synaptogenesis2 that can also influence seizure genesis. Here, we generate and characterize a conditional mouse model that allows a forebrain-specific switch of a human epilepsy GABAA γ2 (R43Q) mutation at specific times during development. Using this mouse, we demonstrate that expression of GABAA γ2 (R43Q) in the developing brain increases seizure susceptibility in adulthood.

Genetic background plays a significant role in producing variable seizure outcomes in patients and animal models. The neurobiological mechanism underlying this heterogeneity is not clear. Here we compare GABAergic synaptic properties within the thalamocortical circuit of two commonly used inbred mice strains, the C57B/6 and spike-and-wave discharge (SWD) prone DBA/2J. Differences between the strains occur in amplitude, kinetics, and frequency of miniature inhibitory postsynaptic currents (mIPSCs) in a region-specific manner. The biggest difference in synaptic inhibition was seen in the thalamus where DBA/2J mice showed a doubling of mIPSC frequency when compared to C57B/6. EEG analysis revealed higher power in the 6-12 Hz band during non-rapid eye movement sleep in DBA/2J mice. Increased susceptibility of the DBA2/J strain to develop SWDs and increase in the 6-12 Hz EEG power may be due to a hypersynchronous ventrobasal thalamus as a consequence of increased GABAergic input.

We investigated the role of Na(+)-K(+)-Cl(-) co-transporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchanger (NCX(rev)) in glutamate-mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6-8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na(+)/Ca(2+) exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1-mediated Na(+) influx. In the presence of a specific AMPA receptor inhibitor 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ-induced sustained rise led to mitochondrial Ca(2+) accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ-elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCX(rev). These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1-mediated Na(+) influx. The latter triggers NCX(rev) and NCX(rev)-mediated overload and compromises mitochondrial function and cellular viability.

GABAAreceptor function can be conceptually divided into interactions between ligand and receptor (binding) and the opening and closing of the ligand-bound channel (gating). The relationship between binding, gating, and receptor structure remains unclear. Studies of mutations have identified many amino acid residues that contribute to the GABAbinding site. Most of these studies assayed changes in GABA dose–response curves, which are macroscopic measures that depend on the interplay of many processes and cannot resolve individual microscopic transitions. Understanding the microscopic basis of binding and gating is critical, because kinetic transition rates predict how receptors will behave at synapses. Furthermore, microscopic rates are directly related to the molecular interactions underlying receptor function.Here, we focused on a residue (β2-R207) previously identified as lining the GABA-binding site that, when mutated to cysteine, greatly reduces apparent GABA affinity and was predicted to affect both binding and gating. To better understand the role of β2-R207, we expressed α1β2and α1β2-R207C receptors in human embryonic kidney 293 cells and studied receptor kinetics using fast solution applications. The mutation accelerated deactivation by 10-fold, without altering desensitization in the presence of saturating GABA. Maximum open probability and single-channel open times were also unaltered by the mutation, but the GABA-binding rate was reduced eightfold. Therefore, the effects of this mutation in a predicted binding site residue are solely attributable to changes in GABA-binding and unbinding kinetics, with no changes in channel gating. Because β2-R207 stabilizes GABA in the binding pocket, it may directly contact the GABA molecule.

A missense mutation (R43Q) in the γ2 subunit of the γ-aminobutyric acid (GABA)(A) receptor is associated with generalized (genetic) epilepsy with febrile seizures plus (GEFS+). Heterozygous GABA(A) γ2(R43Q) mice displayed a lower temperature threshold for thermal seizures as compared to wild-type littermates. Temperature-dependent internalization of GABA(A) γ2(R43Q)-containing receptors has been proposed as a mechanism underlying febrile seizure genesis in patients with this mutation. We tested this idea using the GABA(A) γ2(R43Q) knockin mouse model and analyzed GABAergic miniature postsynaptic inhibitory currents (mIPSCs) in acute brain slices after exposure to varying temperatures. Incubation of slices at an elevated temperature increased mIPSC amplitude in neurons from heterozygous mice, with no change seen in wild-type controls. [³H]Flumazenil binding measured in whole-brain homogenates from mutant and control mice following elevation of body temperature showed no temperature-dependent differences in γ2-containing receptor density. Therefore, in vivo mouse data do not support earlier in vitro observations that proposed temperature-dependent internalization of γ2 R43Q containing GABA(A) receptors as the cellular mechanism underlying febrile seizure genesis in patients with the GABA(A) γ2(R43Q) mutation.

1. The effects of the volatile anesthetics enflurane, halothane, and isoflurane on gamma-aminobutyric acid (GABA) receptor-mediated inhibitory postsynaptic currents (IPSCs) were studied in cultured rat hippocampal neurons. The experimental concentrations of anesthetics were measured directly using gas chromatography. All three anesthetics increased the overall duration of IPSCs, measured as the time to half-decay (T1/2). Clinically effective concentrations of anesthetics [between 0.5 and 1.5 times MAC (minimum alveolar concentration)] produced between 100 and 400% increases in T1/2. These effects were fully reversible, and did not involve alterations in the reversal potential for the IPSC (EIPSC). 2. The decay of the IPSC was fitted as a sum of two exponential functions, yielding a fast component (tau fast = 20 ms), and a slow component (tau slow = 77 ms), such that the fast component accounted for 79% of the IPSC amplitude and 52% of the total charge transfer. All three anesthetics produced concentration-related increases in the amplitude and charge transfer of the slow component, while simultaneously decreasing the amplitude and charge transfer of the fast component. Thus T1/2 approximated tau fast under control conditions, but approximated tau slow in the presence of the anesthetics. 3. Varying the calcium chelating agents in the recording pipettes had no effect on the quality or magnitude of alterations in IPSC kinetics produced by halothane, suggesting that variations in intracellular calcium levels are not required for the effect of halothane on the time course of the IPSC. 4. The (+)-stereoisomer of isoflurane produced greater increases in the duration of the IPSC than the (-)-isomer when applied at approximately equal concentrations, suggesting that there is a structurally selective site of interaction for isoflurane that modulates the GABAA receptor. 5. These results suggest that the previously shown abilities of volatile anesthetics to potentiate responses to exogenously applied GABA and to prolong the duration of GABA-mediated synaptic inhibition may be due to an alteration in the gating kinetics of the GABAA receptor/channel complex. Prolongation of synaptic inhibition in the CNS is consistent with the physiological effects that accompany anesthesia and may contribute to the mechanism of anesthetic action.