17 research outputs found
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
Pyramidal cell activity levels affect the polarity of activity-induced gene transcription changes in interneurons
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 âactive probingâ 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 0.033â
Hz (periodâ=â30â
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 â 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<sub>V</sub>1.6 and Dual Na<sub>V</sub>1.6/Na<sub>V</sub>1.2 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