MEMBRANE AND CELLULAR BIOPHYSICS AND BIOCHEMISTRY NEUROREPORT 0959-4965 & Lippincott Williams & Wilkins Vol 12 No 17 4 December 2001 3649 Activity-dependent accumulation of Ca 2 in axon and dendrites of the leech Leydig neuron Christian Lohr, CA Andreas Beck and Joachim W. Deitmer Abteilung fu Èr Allgemeine Zoologie, Universita Èt Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany CA Corresponding Author Received 17 August 2001; accepted 12 September 2001 We have investigated Ca 2 changes evoked by single action potentials (APs) in axon and dendrites of leech Leydig neurons. Dendritic Ca 2 transients induced by an AP were twice as large as in the axon, and Ca 2 recovery was signi®cantly faster in the dendrites as compared to the axon. The AP-induced Ca 2 transients were blocked by Co 2 and suppressed in Ca 2 -free saline, indicating Ca 2 in¯ux through voltage-activated channels. During a train of APs, Ca 2 accumulated signi®cantly more in the axon than in the dendrites. Suppression of the Ca 2 in¯ux changed the shape of the action potential and increased the ®ring frequency. The results suggest a functional role of Ca 2 in¯ux and Ca 2 accumulation during electrical activity in different neuronal subcompartments. NeuroReport 12:3649± 3653 & 2001 Lippincott Williams & Wilkins. Key words: Action potential; Confocal microscopy; Hirudo; Voltage-activated calcium channel INTRODUCTION Action potential (AP)-induced Ca 2 in¯ux through voltage- activated Ca 2 channels (VACCs) can contribute to the inward current during the AP or modulate the shape of the AP by the activation of a Ca 2 -dependent K  conduc- tance [1,2]. During trains of APs, Ca 2 accumulates and can lead to cell responses that require high Ca 2 concentra- tions, e.g. the modulation of gene expression, presynaptic facilitation or facilitation of APs backpropagated into dendrites [3±5]. Studies of the spatial distribution of the Ca 2 signals induced by single APs revealed differences in the amplitude and/or the kinetics of the Ca 2 transients in different cell compartments [6,7]. Few studies, however, investigated how these differences in the kinetics of the Ca 2 transients affect the accumulation of Ca 2 in cellular subcompartments [8], and it is not known whether Ca 2 accumulation during trains of APs differs between axon and dendrites. In the present study, we have measured Ca 2 transients in the Leydig neuron in the leech CNS using confocal microscopy. This neuron has been characterized as a modulatory interneuron, affecting various neural circuits such as those underlying the heart beat and the local bending re¯ex [9,10]. In each segmental ganglion, two Leydig neurons are present which are synchronized by electrical coupling [11]. MATERIALS AND METHODS Dissection procedure and solutions: Leeches ( Hirudo med- icinalis L.) were obtained from Zaug (Biebertal, Germany) and kept in fresh water at 16±188C. For 2±7 days before an experiment the animals were kept at room temperature (20±258C). Animals were anaesthetized in ice-cold water before dissection. The dissection and experimental proce- dures were performed as described before [7,12]. A single midbody segmental ganglion was pinned ventral side upwards into a Sylgard-lined experimental chamber con- taining modi®ed Leibovitz-15 medium [13] and the ventral ganglion capsule was removed. After enzyme treatment (collagenase/dispase, 2 mg/ml for 15 min) both ventro- medial cell packets were sucked off by a small ®re- polished pipette (tip diameter 40 ìm) to enable optical access to the neuropil. The Leydig neurons, located in the posterio-lateral cell packets, were identi®ed according to Keyser et al. [11]. During an experiment, the ganglia were continuously superfused with physiological saline contain- ing (in mM): NaCl, 85; KCl, 4; CaCl 2 , 2; MgCl 2 , 1; HEPES, 10; the pH was adjusted to 7.4 with NaOH. In Ca 2 -free saline, Ca 2 was substituted by Mg 2 , and 0.5 mM EGTA was added. Co 2 (2 mM) was directly added to the saline in some experiments. Electrophysiology: Double-barrelled microelectrodes were pulled from è-type borosilicate glass capillaries (TST150-6, World Precision Instruments, USA) for simultaneous mem- brane potential recording and dye injection. The recording channel was ®lled with 3 M potassium acetate, while the second channel was ®lled with 5 mM of the Ca 2 -sensitive dye Oregon Green 488 BAPTA-1 (OGB) dissolved in deio- nized water. The microelectrodes were bevelled on a rotat-