Further pharmacological investigation

So far we can explain the observed differences of the two cell types by the temper-ature. To confirm that there are no other chemical side effects Antje Heidemann performed several pharmacological experiments. Their main results are listed below and we refer the interested reader to [198].

NO is involved in modulating oscillatory Ca²⁺ signalling in astrocytes:

NO is a potential mediator of the observed Ca²⁺ responses in astrocytes, since NO is implicated in astrocytic Ca²⁺ signalling [119]. To block the production of NO, the nitric oxide synthase inhibitor Nω-nitro-L-arginine (L-NNA; 2 mM) was applied for 12-20 minutes. The number of spontaneously active cells was significantly reduced when compared to control (39 ± 30% with the number of cells active at control conditions set to 100%, p <0,005; n = 16, averaged cell number/slice = 32± 16).

2min 0.4

∆F

28^oC 5min 20^oC

0 20 40 60

spike width (s)

31^oC

24^oC 37^oC 24^oC

** ** **

slice

0 30 60 90

16 20 24 28 32 36 40

spike width (s)

T (^oC) 0

20 40 60 80

spike width (s)

30^oC

20^oC 20^oC 28^oC 20^oC

** ** **

culture A

B D

C

Figure 5.3: The spike width of calcium oscillations is temperature dependent for both, cultured astrocytes and cells within acute brain slicesA: ∆F traces from three different cultured astrocytes at 28^◦C and 20^◦C. Note that the transient fluorescence changes have a smaller spike width and occur less frequently at higher temperature.

The recordings were separated by 5 min. B: The spike widths of the fluorescence transients were determined from recordings as depicted in A. The histogram shows the average values for all experiments obtained from cultured astrocytes determined at different temperatures as indicated (error bars indicate s.e.). C: Similar values as shown in B but for astrocytes in brain slices. D: From data shown in B and Cthe duration of the spike width is plotted as a function of temperature for astrocytes from culture (black dots) and brain slices (circles). The data points were fitted by f(T) = 620 s exp(−0.109T/^◦C), where we computed the weighted average for the two lowest temperatures from the data shown in B and C(**, p < 0.005).

The NO-donor S-nitrosoglutathione (SNOG; 100µM) was applied to investigate whether NO could influence the oscillatory astrocytic Ca²⁺ signalling in astrocytes in acute cortical brain slices. SNOG induced reproducible oscillatory Ca²⁺responses in cortical astrocytes at 31^◦C.

The observed Ca²⁺ signalling is mainly driven by release from inter-nal stores: The stopped flow paradigm was used to further study the properties of spontaneous Ca²⁺ activity in astrocytes. Cells were maintained in a perfusion chamber with the inflowing buffer heated to 30-33^◦C. Subsequently, we activated spontaneous Ca²⁺ activity by lowering the temperature via stopping the perfusion system.

First, it was tested whether spontaneous Ca²⁺ activity was due to Ca²⁺ influx from the extracellular space or due to Ca²⁺released from intracellular stores. There-fore, Ca²⁺ was omitted in the perfusion buffer. The total number of responsive cells was slightly, but not significantly, reduced to 78 ± 34% (n = 13, averaged cell number/slice = 31 ± 15). Interestingly, only cells, which had reacted in control conditions, reacted under Ca²⁺-free conditions. For assessing the contribution of intracellular Ca²⁺ stores, a first control recording was compared to a second one after perfusion with buffer containing 1µM thapsigargin for approximately 15 min.

After that treatment, in the presence of thapsigargin, astrocytes exhibited a largely reduced oscillatory activity .

We conclude that intracellular Ca²⁺ increase due to spontaneous activity are to a large extent controlled by Ca²⁺ release from intracellular Ca²⁺ stores and that NO is an important factor in controlling spontaneous Ca²⁺ activity also via Ca²⁺

release from internal stores.

5.3 Discussion

We have seen that a decrease in temperatures below the normal body core temper-ature increases the frequency of spontaneous Ca²⁺ transients in mouse astrocytes and prolongs the duration of single Ca²⁺ transients. The consistency of our findings between cultured astrocytes and astrocytes in acute brain slices, suggests that the temperature-dependence of spontaneous Ca²⁺ activity is an intrinsic property of as-trocytes and is not affected by the substantial differences in astrocyte morphology in the two experimental settings.

Spontaneous increases in the cytosolic Ca²⁺ concentration of cells are generally known to play a role in development, differentiation and maturation of tissues. Es-pecially in the nervous system it is described that spontaneous oscillatory electrical activity accompanied by Ca²⁺ oscillations underlie the establishment of connections in the CNS [274, 117, 80, 281, 280], and the astrocytic activity was hypothesized to drive those neuronal oscillations [46, 171].

All previous studies on spontaneous astrocytic Ca²⁺ signalling in culture or in acute brain slices have not been performed at the precise core temperature of 37^◦C.

Either the temperature was not reported as for the hippocampus [156] or, when the temperature was stated, it was commonly between 20^◦C and 25^◦C, as for the stud-ies in acute slices of the ventrobasal thalamus (20-24^◦C) [173] and of the neocortex (22-25^◦C) [3]. In epileptic tissue, the rate of spontaneous activity increased as com-pared to control slices in control conditions, but also in this study the temperature during the recording was not stated [240]. These results indicate that spontaneous astrocytic Ca²⁺ signalling at a high frequency could be a temperature artefact and astrocytesin vivo might not exhibit such frequent spontaneous activity. Indeed, the first Ca²⁺ recordings from astrocytes in vivo support this view [93]. In this study, it is reported that the astrocytes were either quiescent or responded with one or few events during a 10 min observation time. A similar observation was recently made in vivo in the barrel cortex: only two out of 93 astrocytes showed a somatic Ca²⁺

increase during a 10 min observation time [265]. Taken together, the findings indi-cate that the effect of temperature might explain the low frequency of spontaneous Ca²⁺ signalling in vivo.

The impact of temperature on Ca²⁺ signalling is most likely not an astrocyte-specific phenomenon. In cardiac muscle the incidence and frequency of Ca²⁺ spikes decreased dramatically at 37^◦C compared with 22^◦C. In other cell types as e.g. in rabbit renal tubules, hepatocytes, parenchymal, endothelial, and Kupffer cells of the liver acute hypothermia affects the intracellular Ca²⁺ homeostasis and goes along with a rise in cytosolic Ca²⁺ levels [140, 104, 87].

We found that temperature not only affected the frequency of spontaneous events, but also the time course of a single event. At physiological temperature, single events were significantly shorter as compared to lower temperature. A mech-anistic explanation could be a faster inhibition of the IP₃R channels yielding in shorter opening times at higher temperatures or by the temperature dependent ac-tivity of the SERCA pumps [50, 112]. Since acac-tivity of SERCAs increases with temperature, the communication between IP₃R clusters decreases and events do not occur in synchrony within a cell, thus ceasing the global oscillations.

In Bergmann glial cells, NO triggers a transient Ca²⁺ increase, and the Ca²⁺

transient triggered by parallel fibre stimulation is mediated by NO [139]. Here we demonstrated that SNOG, an NO-donor, triggered Ca²⁺ signalling in cortical astro-cytes in acute brain slices. We show a strong overlap of the cell populations exhibit-ing Ca²⁺ signalling upon SNOG application and at low temperatures. Whereas the NO-induced response is completely dependent on Ca²⁺ release from internal stores, the signalling induced by a drop in temperature is not completely abolished after emptying internal Ca²⁺ stores. Thus, we assume that NO is not the initial trigger of the drop-in-temperature-induced Ca²⁺ signalling but has a secondary role in its amplification. NO has already been suggested to function as an autocrine factor for

astrocytes [204, 200] and even to be an endogenous Ca²⁺ influx factor, responsible for the refill of internal stores [119]. This, together with the fact that NO-synthase is mainly activated by Ca²⁺ [106, 28], supports the possibility of an amplifying role for NO.