Enaminone Modulators of Extrasynaptic α4β3δ γ-Aminobutyric AcidA Receptors Reverse Electrographic Status Epilepticus in the Rat After Acute Organophosphorus Poisoning

Seizures induced by organophosphorus nerve agent exposure become refractory to treatment with benzodiazepines because these drugs engage synaptic γ-aminobutyric acid-A receptors (GABAARs) that rapidly internalize during status epilepticus (SE). Extrasynaptic GABAARs, such as those containing α4β3δ subunits, are a putative pharmacological target to comprehensively manage nerve agent-induced seizures since they do not internalize during SE and are continuously available for activation. Neurosteroids related to allopregnanolone have been tested as a possible replacement for benzodiazepines because they target both synaptic and extrasynaptic GABAARs receptors. A longer effective treatment window, extended treatment efficacy, and enhanced neuroprotection represent significant advantages of neurosteroids over benzodiazepines. However, neurosteroid use is limited by poor physicochemical properties arising from the intrinsic requirement of the pregnane steroid core structure for efficacy rendering drug formulation problematic. We tested a non-steroidal enaminone GABAAR modulator that interacts with both synaptic and extrasynaptic GABAARs on a binding site distinct from neurosteroids or benzodiazepines for efficacy to control electrographic SE induced by diisopropyl fluorophosphate or soman intoxication in rats. Animals were treated with standard antidotes, and experimental therapeutic treatment was given following 1 h (diisopropyl fluorophosphate model) or 20 min (soman model) after SE onset. We found that the enaminone 2-261 had an extended duration of seizure termination (>10 h) in the diisopropyl fluorophosphate intoxication model in the presence or absence of midazolam (MDZ). 2-261 also moderately potentiated MDZ in the soman-induced seizure model but had limited efficacy as a stand-alone anticonvulsant treatment due to slow onset of action. 2-261 significantly reduced neuronal death in brain areas associated with either diisopropyl fluorophosphate- or soman-induced SE. 2-261 represents an alternate chemical template from neurosteroids for enhancing extrasynaptic α4β3δ GABAAR activity to reverse SE from organophosphorous intoxication

Spike timing of lacunosom-moleculare targeting interneurons and CA3 pyramidal cells during high-frequency network oscillations in vitro

Network activity in the 200- to 600-Hz range termed high-frequency oscillations (HFOs) has been detected in epileptic tissue from both humans and rodents and may underlie the mechanism of epileptogenesis in experimental rodent models. Slower network oscillations including theta and gamma oscillations as well as ripples are generated by the complex spike timing and interactions between interneurons and pyramidal cells of the hippocampus. We determined the activity of CA3 pyramidal cells, stratum oriens lacunosum-moleculare (O-LM) and s. radiatum lacunosum-moleculare (R-LM) interneurons during HFO in the in vitro low-Mg2+ model of epileptiform activity in GIN mice. In these animals, interneurons can be identified prior to cell-attached recordings by the expression of green-fluorescent protein (GFP). Simultaneous local field potential recordings from s. pyramidale and on-cell recordings of individual interneurons and principal cells revealed three primary firing behaviors of the active cells: 36% of O-LM interneurons and 60% of pyramidal cells fired action potentials at high frequencies during the HFO. R-LM interneurons were biphasic in that they fired at high frequency at the beginning of the HFO but stopped firing before its end. When considering only the highest frequency component of the oscillations most pyramidal cells fired on the rising phase of the oscillation. These data provide evidence for functional distinction during HFOs within otherwise homogeneous groups of O-LM interneurons and pyramidal cells

Increased neuronal firing in computer simulations of sodium channel mutations that cause generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+) is an autosomal dominant familial syndrome with a complex seizure phenotype. It is caused by mutations in one of 3 voltage-gated sodium channel subunit genes (SCN1B, SCN1A, and SCN2A) and the GABAA receptor {gamma}2 subunit gene (GBRG2). The biophysical characterization of 3 mutations (T875M, W1204R, and R1648H) in SCN1A, the gene encoding the CNS voltage-gated sodium channel {alpha} subunit Nav1.1, demonstrated a variety of functional effects. The T875M mutation enhanced slow inactivation, the W1204R mutation shifted the voltage dependency of activation and inactivation in the negative direction, and the R1648H mutation accelerated recovery from inactivation. To determine how these changes affect neuronal firing, we used the NEURON simulation software to design a computational model based on the experimentally determined properties of each GEFS+ mutant sodium channel and a delayed rectifier potassium channel. The model predicted that W1204R decreased the threshold, T875M increased the threshold, and R1648H did not affect the threshold for firing a single action potential. Despite the different effects on the threshold for firing a single action potential, all of the mutations resulted in an increased propensity to fire repetitive action potentials. In addition, each mutation was capable of driving repetitive firing in a mixed population of mutant and wild-type channels, consistent with the dominant nature of these mutations. These results suggest a common physiological mechanism for epileptogenesis resulting from sodium channel mutations that cause GEFS+

Development and physiology of GABAergic feedback excitation in parvalbumin expressing interneurons of the mouse basolateral amygdala

We have previously shown that in the basolateral amygdala (BLA), action potentials in one type of parvalbumin (PV)-expressing GABAergic interneuron can evoke a disynaptic feedback excitatory postsynaptic potential (fbEPSP) onto the same presynaptic interneuron. Here, using whole-cell recordings from PV-expressing interneurons in acute brain slices we expand on this finding to show that this response is first detectable at 2-week postnatal, and is most prevalent in animals beyond 3 weeks of age (>P21). This circuit has a very high fidelity, and single action potential evoked fbEPSPs display few failures. Reconstruction of filled neurons, and electron microscopy show that interneurons that receive feedback excitation make symmetrical synapses on both the axon initial segments (AIS), as well as the soma and proximal dendrites of local pyramidal neurons, suggesting fbEPSP interneurons are morphologically distinct from the highly specialized chandelier neurons that selectively target the axon initial segment of pyramidal neurons. Single PV interneurons could trigger very large (~ 1 nA) feedback excitatory postsynaptic currents (fbEPSCs) suggesting that these neurons are heavily reciprocally connected to local glutamatergic principal cells. We conclude that in the BLA, a subpopulation of PV interneurons forms a distinct neural circuit in which a single action potential can recruit multiple pyramidal neurons to discharge near simultaneously and feed back onto the presynaptic interneuron

Properties of doublecortin expressing neurons in the adult mouse dentate gyrus

The dentate gyrus is a neurogenic zone where neurons continue to be born throughout life, mature and integrate into the local circuitry. In adults, this generation of new neurons is thought to contribute to learning and memory formation. As newborn neurons mature, they undergo a developmental sequence in which different stages of development are marked by expression of different proteins. Doublecortin (DCX) is an early marker that is expressed in immature granule cells that are beginning migration and dendritic growth but is turned off before neurons reach maturity. In the present study, we use a mouse strain in which enhanced green fluorescent protein (EGFP) is expressed under the control of the DCX promoter. We show that these neurons have high input resistances and some cells can discharge trains of action potentials. In mature granule cells, action potentials are followed by a slow afterhyperpolarization that is absent in EGFP-positive neurons. EGFP-positive neurons had a lower spine density than mature neurons and stimulation of either the medial or lateral perforant pathway activated dual component glutamatergic synapses that had both AMPA and NMDA receptors. NMDA receptors present at these synapses had slow kinetics and were blocked by ifenprodil, indicative of high GluN2B subunit content. These results show that EGFP-positive neurons in the DCX-EGFP mice are functionally immature both in their firing properties and excitatory synapses

Comparison of electrotonic properties of EGFP-positive granule cells and mature dentate gyrus granule cells.

<p>Comparison of electrotonic properties of EGFP-positive granule cells and mature dentate gyrus granule cells.</p

Dendritic Calcium Signaling in EGFP-expressing Granule Cells.

<p>An intracellular calcium rise was elicited by a train of 4 APs at 100 Hz and measured at approximately 20–30 µM from the soma, before the first branch point of the main apical dendrite (white boxes). A. One group of immature EGFP-expressing granule cells, that were unable to fire APs (middle) had no detectable dendritic calcium rise (bottom, average and standard deviation, n = 5) in response to current injections that elicited a change in membrane potential similar in magnitude to an AP. B. More mature looking EGFP-expressing granule cells with sparsely spiny dendrites (top) were able to fire 4 APs (middle) that resulted in a detectable rise in intracellular calcium concentration in the apical dendrite (bottom, average and standard deviation, n = 5). C. Mature granule cells were densely spiny and 4 APs elicited a relatively large rise in dendritic calcium (bottom, average and standard deviation, n = 6). (Inset scale bars are 10 mV and 10 ms).</p

Intrinsic Properties of EGFP-expressing and Mature Granule Cells.

<p>Histograms of the whole-cell capacitance, resting membrane potential and input resistance demonstrate that EGFP-expressing granule cells (green lines) are typically smaller, more depolarized (though inaccurately measured, see text) and have a higher input resistance than mature dentate gyrus granule cells (DGGC, black lines).</p

Both Immature and Mature Granule Cells Have Slow NMDA Kinetics and Rectifying AMPA Receptors in Swiss Webster Mice.

<p>A. NMDA currents recorded at +40 mV in response to medial perforant path stimulation in a mature granule cell (left) and from and EGFP-positive newborn neurons (right). Recordings were fit with a double exponential equation and the fit is overlaid (red lines). B. The NMDA receptor mediated EPSC in newborn neurons is blocked by ifenprodil. Panel on left shows the normalized NMDA-receptor mediated EPSC amplitude for one cell recorded at +40 mV before and following application of 5 µM ifenprodil. The EPSCs recorded at the indicated time points are superimposed on the right. C. AMPA-receptor mediated synaptic currents recorded over a range of membrane potentials from −80 to +40 mV following blockade of NMDA receptor currents with d-AP5. Currents were normalized to the peak current recorded at −80 mV and plotted against voltage to demonstrate the slight rectification at depolarized potentials in both mature (black traces) and newborn (green traces) granule cells. The peak current-voltage relationship for the two cell types are shown on the right.</p