Astroglial glutamate transporters are essential for maintenance of respiratory activity in the rhythmic slice preparation

Abstract

Astrocytes are the major type of glial cells in the brain and were already described in the 19th century by Rudolf Virchow. The physiological investigation of astrocytes has long been hampered by difficulties to identify astrocytes during physiological experiments. Apart from using transgenic mice with astrocytes expressing fluorescent proteins, the method of selectively labeling astrocytes with the fluorescent dye Sulforhodamine 101 (SR101) emerged in recent years. SR101 was reported to label astrocytes in the hippocampus and the neocortex of rodents. However, here it was shown that SR101 labeling was not sufficient to reliably identify astrocytes in the brainstem. Investigations of the cause of this discrepancy revealed that passive diffusion of SR101 via gap junction or pannexin hemichannels does not account for the labeling differences. Since the inhibitor of ATP-binding cassette transporters, MK-571 blocked astroglial SR101 labeling in the hippocampus but did not improve SR101 labeling of brainstem astrocytes, active transport of SR101 into hippocampal astrocytes via a not yet identified, MK-571 sensitive transporter is more probable. During rhythmic bursting activity of respiratory neurons in the pre-Bötzinger Complex (preBötC), extracellular concentrations of K+ and neurotransmitters increase rhythmically. It was found in this study, that around 10 % of the patched astrocytes detected these rhythmic changes of the extracellular milieu and showed respiratory-related current fluctuations that based on Kir4.1 channels and glutamate transporters. It was further tested under different conditions for Ca2+-dependent coupling of astrocytes to neurons in the preBötC. Astrocyte-to-neuron communication is mostly dependent on intracellular Ca2+ signals in astrocytes. Though, during normal respiratory activity and in conditions of increased activity after blockade of inhibitory transmission, we found no correlation between respiratory bursts and [Ca2+]in in preBötC astrocytes. Only after blocking the glial glutamate uptake and subsequent increase of extracellular glutamate levels, astroglial Ca2+ oscillations were induced. Furthermore, depolarization of single astrocytes during normal respiratory activity did not affect respiratory bursts. Thus, we conclude that during normal respiratory activity, coupling between astrocytes and neurons in the preBötC is low. However, the astroglial glutamate uptake is essential for maintenance of respiratory network activity and adds further data for the importance of astrocytes in the preBötC.