Epileptic activity triggers rapid ROCK1-dependent astrocyte morphology changes.

Long-term modifications of astrocyte function and morphology are well known to occur in epilepsy. They are implicated in the development and manifestation of the disease, but the relevant mechanisms and their pathophysiological role are not firmly established. For instance, it is unclear how quickly the onset of epileptic activity triggers astrocyte morphology changes and what the relevant molecular signals are. We therefore used two-photon excitation fluorescence microscopy to monitor astrocyte morphology in parallel to the induction of epileptiform activity. We uncovered astrocyte morphology changes within 10-20 min under various experimental conditions in acute hippocampal slices. In vivo, induction of status epilepticus resulted in similarly altered astrocyte morphology within 30 min. Further analysis in vitro revealed a persistent volume reduction of peripheral astrocyte processes triggered by induction of epileptiform activity. In addition, an impaired diffusion within astrocytes and within the astrocyte network was observed, which most likely is a direct consequence of the astrocyte remodeling. These astrocyte morphology changes were prevented by inhibition of the Rho GTPase RhoA and of the Rho-associated kinase (ROCK). Selective deletion of ROCK1 but not ROCK2 from astrocytes also prevented the morphology change after induction of epileptiform activity and reduced epileptiform activity. Together these observations reveal that epileptic activity triggers a rapid ROCK1-dependent astrocyte morphology change, which is mechanistically linked to the strength of epileptiform activity. This suggests that astrocytic ROCK1 signaling is a maladaptive response of astrocytes to the onset of epileptic activity.

Dysfunction of NG2 glial cells affects neuronal plasticity and behavior.

NG2 glia represents a distinct type of macroglial cells in the CNS and is unique among glia because they receive synaptic input from neurons. They are abundantly present in white and gray matter. While the majority of white matter NG2 glia differentiates into oligodendrocytes, the physiological impact of gray matter NG2 glia and their synaptic input are still ill defined. Here, we asked whether dysfunctional NG2 glia affect neuronal signaling and behavior. We generated mice with inducible deletion of the K+ channel Kir4.1 in NG2 glia and performed comparative electrophysiological, immunohistochemical, molecular and behavioral analyses. Kir4.1 was deleted at postnatal day 23-26 (recombination efficiency about 75%) and mice were investigated 3-8 weeks later. Notably, these mice with dysfunctional NG2 glia demonstrated improved spatial memory as revealed by testing new object location recognition while working and social memory remained unaffected. Focussing on the hippocampus, we found that loss of Kir4.1 potentiated synaptic depolarizations of NG2 glia and stimulated the expression of myelin basic protein while proliferation and differentiation of hippocampal NG2 glia remained largely unaffected. Mice with targeted deletion of the K+ channel in NG2 glia showed impaired long-term potentiation at CA3-CA1 synapses, which could be fully rescued by extracellular application of a TrkB receptor agonist. Our data demonstrate that proper NG2 glia function is important for normal brain function and behavior.

Limited contribution of astroglial gap junction coupling to buffering of extracellular K+ in CA1 stratum radiatum.

Astrocytes form large networks, in which individual cells are connected via gap junctions. It is thought that this astroglial gap junction coupling contributes to the buffering of extracellular K+ increases. However, it is largely unknown how the control of extracellular K+ by astroglial gap junction coupling depends on the underlying activity patterns and on the magnitude of extracellular K+ increases. We explored this dependency in acute hippocampal slices (CA1, stratum radiatum) by direct K+ -sensitive microelectrode recordings and acute pharmacological inhibition of gap junctions. K+ transients evoked by synaptic and axonal activity were largely unaffected by acute astroglial uncoupling in slices obtained from young and adult rats. Iontophoretic K+ -application enabled us to generate K+ gradients with defined spatial properties and magnitude. By varying the K+ -iontophoresis position and protocol, we found that acute pharmacological uncoupling increases the amplitude of K+ transients once their initial amplitude exceeded ~10 mM. Our experiments demonstrate that the contribution of gap junction coupling to buffering of extracellular K+ gradients is limited to large and localized K+ increases.

Hierarchical spike clustering analysis for investigation of interneuron heterogeneity.

Action potentials represent the output of a neuron. Especially interneurons display a variety of discharge patterns ranging from regular action potential firing to prominent spike clustering or stuttering. The mechanisms underlying this heterogeneity remain incompletely understood. We established hierarchical cluster analysis of spike trains as a measure of spike clustering. A clustering index was calculated from action potential trains recorded in the whole-cell patch clamp configuration from hippocampal (CA1, stratum radiatum) and entorhinal (medial entorhinal cortex, layer 2) interneurons in acute slices and simulated data. Prominent, region-dependent, but also variable spike clustering was detected using this measure. Further analysis revealed a strong positive correlation between spike clustering and membrane potentials oscillations but an inverse correlation with neuronal resonance. Furthermore, clustering was more pronounced when the balance between fast-activating K(+) currents, assessed by the spike repolarisation time, and hyperpolarization-activated currents, gauged by the size of the sag potential, was shifted in favour of fast K(+) currents. Simulations of spike clustering confirmed that variable ratios of fast K(+) and hyperpolarization-activated currents could underlie different degrees of spike clustering and could thus be crucial for temporally structuring interneuron spike output.

Function and developmental origin of a mesocortical inhibitory circuit.

Midbrain ventral tegmental neurons project to the prefrontal cortex and modulate cognitive functions. Using viral tracing, optogenetics and electrophysiology, we found that mesocortical neurons in the mouse ventrotegmental area provide fast glutamatergic excitation of GABAergic interneurons in the prefrontal cortex and inhibit prefrontal cortical pyramidal neurons in a robust and reliable manner. These mesocortical neurons were derived from a subset of dopaminergic progenitors, which were dependent on prolonged Sonic Hedgehog signaling for their induction. Loss of these progenitors resulted in the loss of the mesocortical inhibitory circuit and an increase in perseverative behavior, whereas mesolimbic and mesostriatal dopaminergic projections, as well as impulsivity and attentional function, were largely spared. Thus, we identified a previously uncharacterized mesocortical circuit contributing to perseverative behaviors and found that the diversity of dopaminergic neurons begins to be established during their progenitor phase.

Contribution of near-threshold currents to intrinsic oscillatory activity in rat medial entorhinal cortex layer II stellate cells.

The temporal lobe is well known for its oscillatory activity associated with exploration, navigation, and learning. Intrinsic membrane potential oscillations (MPOs) and resonance of stellate cells (SCs) in layer II of the entorhinal cortex are thought to contribute to network oscillations and thereby to the encoding of spatial information. Generation of both MPOs and resonance relies on the expression of specific voltage-dependent ion currents such as the hyperpolarization-activated cation current (I(H)), the persistent sodium current (I(NaP)), and the noninactivating muscarine-modulated potassium current (I(M)). However, the differential contributions of these currents remain a matter of debate. We therefore examined how they modify neuronal excitability near threshold and generation of near-threshold MPOs and resonance in vitro. We found that resonance mainly relied on I(H) and was reduced by I(H) blockers and modulated by cAMP and an I(M) enhancer but that neither of the currents exhibited full control over MPOs in these cells. As previously reported, I(H) controlled a theta-frequency component of MPOs such that blockade of I(H) resulted in fewer regular oscillations that retained low-frequency components and high peak amplitude. However, pharmacological inhibition and augmentation of I(M) also affected MPO frequencies and amplitudes. In contrast to other cell types, inhibition of I(NaP) did not result in suppression of MPOs but only in a moderation of their properties. We reproduced the experimentally observed effects in a single-compartment stochastic model of SCs, providing further insight into the interactions between different ionic conductances.

The range of intrinsic frequencies represented by medial entorhinal cortex stellate cells extends with age.

In both humans and rodents, the external environment is encoded in the form of cognitive maps. Neurons in the medial entorhinal cortex (mEC) represent spatial locations in a sequence of grid-like patterns scaled along the dorsal-ventral axis. The grid spacing correlates with the intrinsic resonance frequencies of stellate cells in layer II of mEC. We investigated the development of frequency preferences in these cells from weaning to adulthood using patch-clamp and sharp microelectrode recordings. We found that the dorsal-ventral gradient of stellate cell properties and frequency preferences exists before animals are able to actively explore their environment. In the transition to adulthood, cells respond faster and become less excitable, and the range of intrinsic resonance frequencies in the population expands in the dorsal direction. This is likely to reflect both the growth of the brain and the expansion of the internal representation caused by new exploratory experience.

Effects of XE991, retigabine, losigamone and ZD7288 on kainate-induced theta-like and gamma network oscillations in the rat hippocampus in vitro.

Ion currents such as M-currents (I(M)), persistent sodium currents (I(NaP)) and H-currents (I(h)) have been observed in a variety of brain regions, including the hippocampal formation, where storage and retrieval of information are facilitated by oscillatory network activities. They have been suggested to play an important role in neuronal excitability, synaptic transmission, membrane oscillatory activity, and in shaping resonance. Resonance and membrane potential oscillations have been implied in the generation of theta but not gamma oscillations. Here, we performed extracellular field potential recordings in hippocampal slices from adult rats and applied either the I(M) blocker XE991, the I(M) activator retigabine, the I(NaP) blocker losigamone or the I(h) inhibitor ZD7288 to test if these currents contribute to the generation of network oscillations. Kainate application induced network theta-like frequency oscillations in coronal slices as well as network gamma frequency oscillations in horizontal slices, and these remained stable for up to 3h. Power spectrum analysis revealed that all agents dose-dependently reduced the network oscillations in both frequency bands in areas CA3 and CA1. In contrast, the peak oscillation frequency was affected differentially. These results confirm that theta-like frequency oscillations are induced in longitudinal slices while gamma frequency oscillations dominate in horizontal slices. They also suggest that modifying neuronal excitability and transmitter release alters hippocampal network oscillations which are thought to be crucial for memory processing.