Control of ion and neurotransmitter homeostasis by hippocampal astroglial networks

Abstract

The present study addressed the contribution of astrocytic gap junction channels to extracellular K⁺ clearance. Astrocyte form large networks through gap junction coupling that enables intercellular communication and trafficking of ions. The contribution of this network was proposed to facilitate extracellular K⁺ clearance by the spatial redistribution of K⁺ via the gap junction coupled network. However, the quantitative contribution of this mechanism to K⁺ clearance was not fully clear. In order to obtain further insights, the extracellular K⁺ concentration in the CA1 str. radiatum of hippocampal slices was recorded with K⁺-sensitive microelectrodes in parallel to the inhibition of intercellular molecule trafficking. For this purpose, a pharmacological approach to acutely uncouple gap junction channels was employed. This allowed the fast disruption of the gap junction coupled astrocyte network and prevented potential cellular alterations induced by a long-term absence of gap junction coupling. Next, different experimental approaches were used to evoke extracellular K⁺ transients before and after the acute inhibition of gap junction coupling. A commonly used stimulation paradigm to probe hippocampal circuit functions evoked neuronal activity and extracellular K⁺ transients with relatively small K⁺ peak amplitudes. However, acute gap junction uncoupling had no effect on these K⁺ transients. Also, extracellular K⁺ transients with larger K⁺ peak amplitudes evoked by more intense neuronal stimulation were largely unaffected by gap junction uncoupling. Next, the iontophoretic application of K⁺ was introduced in order to evoke extracellular K⁺ transients in the absence of neuronal activity. This allowed the application of K⁺ as a point source and the control of the K⁺ transient’s peak amplitudes by adjusting the amount of injected K⁺. Interestingly, iontophoretically evoked K⁺ transients with peak amplitudes in the low millimolar range were also unaffected, but large K⁺ transients with peak amplitudes that exceeded ~10 mM showed further augmentation after gap junction uncoupling. These results demonstrate that the contribution of gap junction coupling to buffering of extracellular K⁺ gradients is limited to large and localized K⁺ increases. Since such large extracellular K⁺ accumulations are commonly observed in association with pathophysiological conditions, astrocytic gap junction coupling might act as a rescue mechanism when other K⁺ clearance mechanisms are not able to cope with extensive extracellular K⁺ loads. Thus, the astrocyte network might not play a pivotal role for K⁺ clearance during physiological conditions but rather in pathophysiological scenarios. In particular the attenuation of spatially confined epileptic activity or the dampening of spreading depression could be an important function of astrocyte gap junction coupling since these conditions could provide a steep extracellular K⁺ gradient as observed in the present experiments. <br /> The second aspect addressed by this study was the impact of rapidly occurring astrocyte morphology changes in the CA1 str. radiatum that were triggered by epileptiform activity. Previous experiments have shown that this morphology changes were accompanied by an impaired intra- and intercellular diffusion and had a proepileptiform effect (Anders, 2016). However, the underlying mechanism that mediated this proepileptiform effect remained uncertain. Thus, in the present study several approaches investigated the link between astrocyte morphology and the increased epileptiform activity. First, it was tested if the induction of epileptiform activity and the concomitant morphology changes modulated inhibitory synaptic input on CA1 pyramidal neurons. This was quantified by recording spontaneous inhibitory currents from CA1 pyramidal neurons after epileptiform activity. We found that the frequency and amplitude of the spontaneous inhibitory currents were unaffected by epileptiform activity, but their decay was shorter compared to control conditions. This suggests that the astrocyte morphology changes decreased the inhibitory synaptic input onto CA1 pyramidale neurons and thereby might induced neuronal hyperexcitability. In order to validate this, further experiments that employ the acute inhibition of the astrocyte morphology changes will be performed. Second, it was analyzed if the extracellular space fraction was affected by the induction of epileptiform activity using a diffusion analysis of TMA⁺. This analysis revealed that the epileptiform activity and astrocyte morphology changes had no influence on the extracellular space fraction. Third, we employed K⁺-sensitive microelectrodes to measure extracellular K⁺ transients after epileptiform activity. The peak amplitude and decay of these K⁺ transients were not different compared to control conditions. This indicates that the astrocyte morphology changes did not impair extracellular K⁺ clearance and thus is unlikely to underlie the proepileptiform effect. Finally, the extracellular glutamate clearance was investigated using the fluorescent glutamate sensor iGluSnFR. Extracellular glutamate elevations were either evoked by an iontophoretic application or by synaptic activity. We found that also glutamate clearance was largely unaffected by the epileptiform activity and thus cannot explain the observed proepileptiform effect of the astrocyte morphology changes. However, there was a tendency toward an increased spatial spread of glutamate into the extracellular space. This could contribute to a facilitated activation of glutamate receptors and, in turn, to an increased neuronal activity. Again, additional experiments are required to validate this observation. In conclusion, this study provided further information about the functional consequences of rapid astrocyte morphology changes. Although, the mechanism underlying the proepileptiform effect of the morphology changes is still not finally clear, the conducted experiments helped to narrow down the list of possible candidates. Moreover, the experiments investigating the inhibitory synaptic transmission and glutamate clearance provided good starting points for further experiments.