The Significance of Neuroglia for the Formation, Function and Plasticity of Synapses: Synaptically induced Sodium Transients in Glial Cells
1. ABSTRACT
Biologisch-Medizinischer Hintergrund:
Regulation of the sodium concentration in glial cells is of vital importance for synaptic function, because many cell functions like intracellular Ca2+ or pH homeostasis are directly dependent on the inwardly directed sodium gradient across the plasma membrane. In addition, reuptake of glutamate, which shapes the time course of synaptic conductance and limits spillover of glutamate out of the synaptic cleft, is driven by concomitant inward transport of sodium. It is well established that decreasing the electrochemical gradient for sodium directly decreases glutamate uptake and thereby can influence synaptic transmission. However, knowledge about intracellular sodium signals in glial cells that accompany synaptic transmission is extremely limited. Recently, we could demonstrate that synaptic activity induces sodium transients in the mM range in fine processes of Bergmann glial cells close to activated synapses. These sodium transients, in concert with a membrane depolarization and changes in the concentrations of other transported ions, result in a long-lasting reduction of the driving force for glial glutamate uptake at active synapses in the cerebellum.
Another well documented consequence of sodium accumulations, described from classical astrocytes, is increased ATP hydrolysis by the Na+/K+-ATPase, which has been suggested to promote glycogen breakdown. We have, therefore, analyzed if classical astrocytes are subject to sodium accumulation in response to synaptic activity by performing quantitative widefield and 2-photon sodium imaging combined with whole-cell patch-clamp in hippocampal astrocytes in acute tissue slices. These investigations were aimed to elucidate if synaptically-induced sodium transients in astrocytes might be suited to provide the observed close link between increased neuronal activity and glial metabolism.
Results
:We analysed sodium transients evoked by synaptic activity in SR101-positive, passive astrocytes and pyramidal neurons in acute hippocampal slices using whole-cell patch-clamp and quantitative sodium imaging. Cells were identified by labelling with the fluorescent dye SR101, which selectively stains classical astrocytes in the hippocampus throughout postnatal development. We found that both application of glutamate, as well as short bursts of Schaffer collateral stimulation evoke sodium transients in the mM range in the somata of neurons and astrocytes. Pharmacological analysis revealed that neuronal sodium signals were mainly attributable to sodium influx through ionotropic glutamate receptors. Activation of ionotropic receptors also contributed to glial sodium transients, while TBOA-sensitive glutamate uptake was the major pathway responsible for sodium influx into astrocytes following synaptic activation. Upon selective activation of glutamate uptake by D-aspartate as well as following synaptic stimulation, we found that glial sodium transients were defined to 1-2 primary branches and adjacent fine processes and only weakly invaded the soma with low stimulation intensities. More intense stimulation, in contrast, elicited global sodium transients throughout the entire cell. Our results establish that glutamatergic synaptic transmission in the hippocampus results in either local or global sodium transients in astrocytes that are mainly mediated by activation of glutamate uptake. They also suggest the existence of microdomains for sodium signalling, in which sodium-dependent processes could be modulated independently.
Fig. 1. Glutamate-induced sodium transients in astrocytes as revealed by 2-photon imaging. (A) Right: Image of a hippocampal astrocyte loaded with the fluorescent sodium indicator SBFI. In addition to the astrocyte, a blood vessel is visible. On the left, the application pipette is schematically indicated. Right: Sodium transients induced by puff application of glutamate (1 mM) for 500 ms. (B): Right Image of an astrocyte as in A. Left: Influence of CNQX on the glutamate-induced sodium transient. Sodium transients were neither altered by TTX, nor by CNQX. In the experiment depicted in (B), the recording was interrupted during the long decay phase to protect cells from dye bleaching.
| Fig. 2. Synaptically-induced sodium signals in different astrocytic regions. A, Image of the SR101-staining (top) and the SFBI-fluorescence (centre) of a patch-clamped astrocyte. Bottom: Inversed picture of the SBFI fluorescence. Coloured dashed lines indicate the regions of interest (r1-r7) analyzed for the experiment depicted in B. The stimulation pipette was positioned at the lower right hand side at a distance of about 40 µm from the cell body. B, Somatic currents (upper traces) and sodium signals in the different cellular regions r1-r7 (bottom) in response to synaptic stimulation at 20 V and 50 V (200 ms/50 Hz). Note that low intensity stimulation causes a significant sodium signal in region r1 only. Higher intensity stimulation induces sodium transients in all cellular regions, which differ in both rise times and amplitudes (see arrows). |
Goals:
Continuation of the described project was granted for another two years within the DFG Program 1172 (The Significance of Neuroglia for the Formation, Function and Plasticity of Synapses (Ro 2327/4-3)).
In this granting period, we will analyse sodium transients in hippocampal astrocytes mainly by using high-resolution, 2-photon microscopy. More specifically, we will study the developmental profile and underlying mechanisms of activity-induced sodium signals by investigating astrocytes at different stages of postnatal development (P3, P7, P14-16, P25). In addition, we will perform astrocytes such as fine processes and endfeet on blood vessels using sodium imaging techniques. These experiments will thus further elucidate the properties of glial sodium signalling and shed light on a possible functional specialization of different astrocyte processes close to synapses or on blood vessels.
2. Publikations:
Bennay M, Kafitz KW, Meier SD, Rose CR: Imaging glial glutamate uptake in the cerebellum. Proc. Glial Cells in Health and Disease, 2007; Medimont.
Bennay M., Meier SD, Kafitz KW, Langer J, Rose CR: Sodium signals in cerebellar Purkinje neurons and Bergmann glial cells evoked by glutamatergic synaptic transmission. Glia 2008; 56: 1138-49.
Kafitz KW, Meier SD, Stephan J, Rose, CR: Developmental profile and properties of sulforhodamine (SR101)-positive astrocytes in acute brain slices of rat hippocampus. J Neurosci Methods 2008; 169: 84-92.
Meier SD, Kafitz KW, Rose CR: Developmental profile and mechanisms of GABA-induced calcium signaling in hippocampal astrocytes. Glia 2008; 56: 1127-37.
Kelly T, Kafitz KW, Roderigo C, Rose CR: Ammonium-evoked alterations in intracellular sodium and pH reduce glial glutamate transport activity. Glia (in press).
Kelly, T, & Rose C.R.: Sodium signals in axons. Invited Review. In: Axons: Advances in Morphology, Physiology, Development, Computation and Pathophysiology. D. Feldmeyer and J. Lübke (Hrsg) Springer Verlag (in press).
Rose, C.R. Two-photon Na+ imaging in dendritic spines. In: Imaging in Neuroscience and Development: A Laboratory Manual. Invited Contribution. R. Yuste, F. Helmchen, A. Konnerth (Editors) CSH Laboratory Press, Cold Spring Harbor (in press).
Deitmer JW, Rose CR: Ion changes in perisynaptic glia. Invited Review. Brain Research Reviews (in preparation).