Synergistic Positive Feedback Mechanisms Underlying Seizure Initiation

Investigations into seizure initiation, in recent years, have focused almost entirely upon alterations of interneuronal function, chloride homeostasis, and extracellular potassium levels. In contrast, little attention has been directed toward a possible role of dendritic plateau potentials in the actual ictogenic transition, despite a substantial literature dating back 40 years regarding its importance generally in epilepsy. Here, we argue that an increase in dendritic excitability, coordinated across the population of pyramidal cells, is a key stage in ictogenesis

Graphical user interface for simultaneous profiling of activity patterns in multiple neuronal subclasses

We provide notes on how to use a graphical user interface (GUI), implemented with MATLAB, for aligning imaging datasets of biological tissue. The original use was for matching two imaging data sets, where one set was taken of the living preparation and another was taken post-fixation and following immunohistochemical processing. This technique is described in detail in an accompanying paper (Parrish et al., [1], where we also include information about the experimental procedures, and examples of the usage of the GUI. Keywords: Pyramidal neurons, Interneurons, Glia, Astrocytes, MATLAB, Immunohistochemistry, Ca2+ imagin

Optogenetic stimulation reveals a latent tipping point in cortical networks during ictogenesis

Brain state transitions are readily apparent from changes in brain rhythms1, but are difficult to predict, suggestive that the underlying cause is latent to passive recording methods. Among the most important transitions, clinically, are the starts of seizures. We here show that an “active probing” approach may have several important benefits for epileptic management, including by helping predict these transitions. We used mice expressing the optogenetic actuator, channelrhodopsin, in pyramidal cells, allowing this population to be stimulated in isolation. Intermittent stimulation at frequencies as low as 0.033 Hz (period = 30 s) delayed the onset of seizure-like events in an acute brain slice model of ictogenesis, but the effect was lost if stimulation was delivered at even lower frequencies (1/min). Notably, active probing additionally provides advance indication of when seizure-like activity is imminent, revealed by monitoring the postsynaptic response to stimulation. The postsynaptic response, recorded extracellularly, showed an all-or-nothing change in both amplitude and duration, a few hundred seconds before seizure-like activity began – a sufficient length of time to provide a helpful warning of an impending seizure. The change in the post-synaptic response then persisted for the remainder of the recording, indicative of a state change from a pre-epileptic to a pro-epileptic network. This occurred in parallel with a large increase in the stimulation-triggered Ca2+ entry into pyramidal dendrites, and a step increase in the number of postsynaptic somatic action potentials, both consistent with a reduction in the threshold for dendritic action potentials. In 0 Mg2+ bathing media, the reduced threshold was not associated with changes in glutamatergic synaptic function, nor of GABAergic release from either parvalbumin or somatostatin interneurons, but simulations indicate that the step change in the optogenetic response can instead arise from incremental increases in intracellular [Cl-]. The change in the response to stimulation was replicated by artificially raising intracellular [Cl-], using the optogenetic chloride-pump, Halorhodopsin. By contrast, increases in extracellular [K+] cannot account for the firing patterns in the response to stimulation, although this, and other cellular changes, may contribute to ictal initiation in other circumstances. We describe how these various cellular changes form a synergistic network of positive feedback mechanisms, which may explain the precipitous nature of seizure onset. This model of seizure initiation draws together several major lines of epilepsy research and as well as providing an important proof-of-principle regarding the utility of open-loop brain stimulation for clinical management of the condition

Indirect effects of Halorhodopsin activation: potassium redistribution, non-specific inhibition and spreading depolarisation

The movement of ions in and out of neurons can exert significant effects on neighboring cells. Here we report several experimentally important consequences of activation of the optogenetic chloride pump, Halorhodopsin. We recorded extracellular K+ concentration, [K+]extra, in neocortical brain slices prepared from young adult mice (both sexes) which express Halorhodopsin in pyramidal cells. Strong Halorhodopsin activation induced a pronounced drop in [K+]extra, that persisted for the duration of illumination. Pharmacological blockade of K+ channels reduced the amplitude of this drop, indicating that it represents K+ redistribution into cells during the period of hyperpolarization. Halorhodopsin thus drives the inward movement of both Cl- directly, and K+ secondarily. When the illumination period ended, a rebound surge in extracellular [K+] developed over tens of seconds, partly reflecting the previous inward redistribution of K+, but additionally driven by clearance of Cl- coupled to K+ by the potassium-chloride co-transporter, KCC2. The drop in [K+]extra during light activation leads to a small (2-3mV) hyperpolarization also of other cells that do not express Halorhodopsin. Its activation therefore has both direct and indirect inhibitory effects. Finally, we show that persistent strong activation of Halorhodopsin causes cortical spreading depolarisations (CSDs), both in vitro and in vivo. This novel means of triggering CSDs is unusual, in that the events can arise during the actual period of illumination, when neurons are being hyperpolarized and [K+]extra is low. We suggest that this fundamentally different experimental model of CSDs will open up new avenues of research to explain how they occur naturally

Targeting firing rate neuronal homeostasis can prevent seizures

Manipulating firing-rate neuronal homeostasis, which enables neurons to regulate their intrinsic excitability, offers an attractive opportunity to prevent seizures. However, to date, no drug-based interventions have been reported that manipulate this type of neuronal homeostatic mechanism. Here, we use a combination of Drosophila and mouse and, in the latter, both a pentylenetetrazole (PTZ) induced seizure model, and an electrically induced seizure model for refractory seizures to evaluate anticonvulsant efficacy of a novel class of anticonvulsant compounds, based on 4-tert-butyl-benzaldehyde (4-TBB). The mode-of-action includes increased expression of the firing rate homeostatic regulator PUMILIO (PUM). Knock-down of PUM expression, in Drosophila, blocks anticonvulsive effects of 4-TBB, whilst analysis of validated PUM targets in mouse brain show significant reductions following exposure to this compound. A structure-activity study identifies the active parts of the molecule and, further, shows the pyrazole analogue demonstrates highest efficacy, being active against both PTZ-, and electrically-induced, seizures. This study provides a proof-of-principle that anticonvulsant effects can be achieved through regulation of firing rate neuronal homeostasis and identifies a possible chemical compound for future development

Molecular Pharmacology of Selective Na_V1.6 and Dual Na_V1.6/Na_V1.2 Channel Inhibitors that Suppress Excitatory Neuronal Activity Ex Vivo

Voltage-gated sodium channel (NaV) inhibitors are used to treat neurological disorders of hyperexcitability such as epilepsy. These drugs act by attenuating neuronal action potential firing to reduce excitability in the brain. However, all currently available NaV-targeting antiseizure medications nonselectively inhibit the brain channels NaV1.1, NaV1.2, and NaV1.6, which potentially limits the efficacy and therapeutic safety margins of these drugs. Here, we report on XPC-7724 and XPC-5462, which represent a new class of small molecule NaV-targeting compounds. These compounds specifically target inhibition of the NaV1.6 and NaV1.2 channels, which are abundantly expressed in excitatory pyramidal neurons. They have a > 100-fold molecular selectivity against NaV1.1 channels, which are predominantly expressed in inhibitory neurons. Sparing NaV1.1 preserves the inhibitory activity in the brain. These compounds bind to and stabilize the inactivated state of the channels thereby reducing the activity of excitatory neurons. They have higher potency, with longer residency times and slower off-rates, than the clinically used antiseizure medications carbamazepine and phenytoin. The neuronal selectivity of these compounds is demonstrated in brain slices by inhibition of firing in cortical excitatory pyramidal neurons, without impacting fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model, whereas XPC-7224 does not, suggesting a possible requirement of Nav1.2 inhibition in 0-Mg2+- or 4-AP-induced brain slice seizure models. The profiles of these compounds will facilitate pharmacological dissection of the physiological roles of NaV1.2 and NaV1.6 in neurons and help define the role of specific channels in disease states. This unique selectivity profile provides a new approach to potentially treat disorders of neuronal hyperexcitability by selectively downregulating excitatory circuits