Activity-induced calcium transients in dendritic regions of cerebellar Purkinje neurones are well known to be larger than those in the soma. The plasma membrane calcium ATPase (PMCA), present at high density in Purkinje cell dendritic regions (Hillman et al. 1996), may be important in extruding the activity-induced calcium load. Counter-transport of protons by the PMCA means that this calcium extrusion is likely to be accompanied by an intracellular acidification. We report here dendritic pH shifts and compare them with pH changes seen at the soma.

Whole-cell patch-clamp recordings were made from Purkinje neurones in 200 µm thick sagittal cerebellar brain slices from 11- to 21-day-old Wistar rats killed by cervical dislocation. Slices were superfused with 95 % O₂ and 5 % CO₂ saturated saline at ~24°C. Neurones were loaded with the fluorescent pH indicator 8-hydroxy-pyrene-1,3,6-trisulphonic acid (HPTS) via the patch pipette (3-5 M¢) and imaged using a Zeiss LSM 510 confocal microscope. HPTS was excited at 458 nm and emitted fluorescence collected at > 505 nm (Schwiening & Willoughby, 2000).Substantial acid shifts were seen in Purkinje dendritic regions during bursts of action potentials (APs) with little pH change in the soma. Figure 1 shows two acid shifts estimated to be ~0.3 pH unit in the fine dendritic regions (ROI 3) during a burst of ~50 APs. The relatively large pH changes in dendritic regions are consistent with the large calcium transients seen in these regions during electrical activity. Similar relative distributions in pH changes were also seen when Purkinje neurones were held under voltage clamp and depolarized to +20 mV for 1 s (n = 3 in CO₂/HCO₃^–-buffered saline, n = 4 in Hepes-buffered saline). The large dendritic calcium changes in Purkinje neurones play an important role in synaptic plasticity (Konnerth et al. 1992). Since many cellular proteins are highly pH sensitive, it is possible that the accompanying acid shifts are also involved in modulating synaptic function.We are grateful to the MRC for funding.

Figure 1. A, confocal image shows fluorescence intensity of HPTS with dark areas representing areas of high intensity. B, three regions of interest (ROI) selected from the neurone for analysis of pH (ROI 1, soma; ROI 2, main dendritic branch; ROI 3, fine dendrites). C, upper trace shows membrane potential of the neurone under current clamp with bursts of 54 then 43 APs during depolarizing current injection; lower trace shows F/F0 (relative fluorescence shift) values for the three ROIs.

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