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Differential expression of voltage-sensitive K⁺ and Ca²⁺ currents in neurons of the honeybee olfactory pathway

Bernd Grünewald

Institut für Biologie, Neurobiologie, Freie Universität Berlin, Königin-Luise-Strasse 28/30, D-14195 Berlin, Germany

View larger version (152K): [in a new window]   Fig. 1. Confocal micrograph of the honeybee antennal lobe with labelled projection neurons. Injections of dextran-coupled rhodamine into the mushroom bodies stained projection neurons (PN); their somata are clustered around the antennal lobe. In this specimen only the median cluster was labelled, but in other preparations the other clusters around the antennal lobe were also labelled. The roots of the antennocerebralis tracts (ACT) are also stained (arrows). Within the protocerebrum the somata of mushroom body feedback neurons within a lateral cluster are densely stained (asterisk), because they innervate all calycal regions. Frontal view of a whole-mount preparation; lateral (l) and dorsal (d) directions are indicated. Scale bar, 50 µm.

View larger version (109K): [in a new window]   Fig. 2. Outgrowth of Kenyon cells (A,A') and projection neurons (B,B') after 2 (A,B) and 6 (A',B') days in primary cell culture. The neurons start to grow processes after 1 day in vitro. These neurites continue growing and branching throughout the period of 1 week. The somata diameters of projection neurons are larger and they grow longer neurites with more branches. Phase contrast; primary magnification 32x. Scale bars, 20µm.

View larger version (30K): [in a new window]   Fig. 3. Different levels of transient K+ currents. Typical examples of voltage-sensitive currents of a Kenyon cell (left) and a projection neuron (right) are shown. (A) In the presence of tetrodotoxin (TTX) and Cd2+ in the external saline to block currents through voltage-sensitive Na+ and Ca2+ channels, voltage-sensitive outward currents were isolated. Activation protocols for the experiments: cells were held at -80 mV. To remove channel inactivation, a long conditioning pulse to -120 mV (1 s) (cf. Pelz et al., 1999) preceded depolarizing voltage commands (potentials from -100 to +120 mV, 10 mV increments, duration 100 ms). Under these conditions Kenyon cells expressed a prominent inactivating K+ current (IPeak), which was less pronounced in projection neurons, where a sustained K+ current (ISust) dominated. Arrows indicate time points where currents were measured in the other figures and they point to the peak current (IPeak) and to the sustained current (ISust) at the end of the voltage pulse. (B) Following an inactivating prepulse to -20 mV (1 s), the transient K+ current of Kenyon cells was completely inactivated during depolarising pulses to various command potentials, whereas the outward currents of projection neurons remained relatively unaffected. (C) Subtracting trace B from trace A gave the amount of inactivating K+ current in the two neurons. Note the different scale bars for Kenyon cells and projection neurons.

View larger version (32K): [in a new window]   Fig. 5. Voltage- and Ca2+ -sensitive outward K+ currents of a typical Kenyon cell and a projection neuron. (A) Currents were recorded in the standard external saline (ES std., see Materials and methods) with the Na+ channel blocker tetrodotoxin (TTX, 100 nmol l-1) added. As in Fig. 3, projection neurons showed higher current amplitudes (note the different scale bars) and Kenyon cells expressed prominent transient K+ currents. Furthermore, the current amplitudes of Kenyon cells are increasing continuously with increasing depolarisations. By contrast, outward currents of projection neurons show an apparent non-linearity with a decrease of current amplitude increases between command potentials of 70-110 mV (see N-shaped I-V curve in Fig. 5C). The activation protocol for all experiments consisted of a hyperpolarising conditioning prepulse to -120 mV (1 s) and subsequent depolarizing voltage commands to various potentials (-100 to +120 mV, 10 mV increments, duration 100 ms; holding potential -80 mV). (B) Addition of the irreversible Ca2+ channel blocker 50 µmol l-1 CdCl2 (Cd2+) to the bath solution had little effect on K+ currents of Kenyon cells (left), but removed non-linearity of current activation in projection neurons (right). (C) Corresponding I-V curves of the two cells showing the Cd2+ block of the N-shaped I-V curve for the projection neuron (right), but no significant change in the I-V curve of voltage-sensitive K+ currents of the Kenyon cell (left). The peak currents were measured at the time points indicated by the arrows in A and B.

View larger version (11K): [in a new window]   Fig. 4. Quantitative comparison of voltage-sensitive K+ currents of Kenyon cells and projection neurons. (A) Amplitudes of K+ currents (elicited by a depolarizing command pulse to +50 mV) measured at the peak during transient current component (IPeak) and during sustained current component (ISust; arrows in Fig. 3A) are higher in projection neuron (PN) than in Kenyon cells (KC; P<0.001). (B) The mean current density of the sustained (delayed rectifier type) current (ISust) is higher in projection neurons than those of Kenyon cells (P<0.01); the current densities measured at the peak currents do not differ significantly. (C) The ratio of peak (transient) versus sustained K+ current is higher in Kenyon cells (KC) than in projection neuron (PN, P<0.001). Levels of significance: *** P<0.001, ** P<0.01.

View larger version (16K): [in a new window]   Fig. 6. Comparison of current—voltage relationships of K+ currents of Kenyon cells (KC) and projection neurons (PN). Currents were recorded in external saline containing 100 nmoll-1 tetrodotoxin (TTX) and were elicited by command voltage pulses between -80 mV and +120 mV (increment 10 mV; preceded by a prepulse to -120 mV for 1 s; holding potential -80 mV; see pulse protocol in Fig. 5). Current amplitudes were measured at the peak of the transient current component (arrows in Fig. 5A) and averaged (values are means ± S.E.M.; the numbers of observations are indicated). Projection neurons express higher current amplitudes than Kenyon cells and show a pronounced N-shaped I-V curve at positive membrane potentials. The I-V curve of outward currents of Kenyon cells is rather linear between -20 and +120 mV.

View larger version (32K): [in a new window]   Fig. 7. Currents through voltage-sensitive Ca2+ channels. (A) Typical currents through Ca2+ channels of Kenyon cells (left) and projection neurons (right) show different amplitudes of inward currents. Currents were elicited by depolarizing voltage pulses (-90 to +60 mV, increment 10 mV; duration 100 ms; holding potential -80 mV); protocol (inset). Tetraethylammonium chloride (TEA, 10 mmoll-1) and 100 nmoll-1 TTX were added to the external standard saline (ES std., see Materials and methods). To block K+ outward currents further, the internal saline contained CsF (40 mmoll-1), Cs-gluconate (83 mmoll-1), Cs-EGTA (10 mmoll-1) and TEA-Cl instead of the corresponding K+ salts (cf. Materials and methods section). (B) Addition of 50 µmoll-1 CdCl2 (Cd2+) blocked the voltage-sensitive Ca2+ currents completely in Kenyon cells. A small residual Cd2+-insensitive current remained unblocked in projection neurons. The Cd2+ block was irreversible even after excessive periods of wash (up to 30 min). (C) Current—voltage relationships of the currents through voltage-sensitive Ca2+ currents of all measured neurons show different amplitudes of peak inward currents (measured at the time point indicated by the arrow in A) and diversity of Ca2+ currents between neurons of a given type. The amplitudes of the peak currents are higher in projection neurons (PN, right) than in those of Kenyon cells (KC, left). However, the voltage-dependencies of the currents and the variability of the current amplitudes at the various potentials (-90 to +60 mV) appear to be similar.

View larger version (12K): [in a new window]   Fig. 8. Comparison of Ca2+ currents of Kenyon cells and projection neurons. (A) The voltage-dependencies of Ca2+ currents are similar, but projection neurons (PN) show higher current amplitudes than Kenyon cells (KC). Values are means ± S.E.M. of the peak Ca2+ current amplitudes measured at the time point indicated by the arrows in Fig. 7A (numbers of observations are indicated) at the respective pulse potential (pulse protocol is given in Fig. 7). (B) Mean peak current amplitudes are higher in projection neurons than in Kenyon cells (P<0.001). (C) Ca2+ current densities (-pA/pF) do not differ significantly (NS). The cell capacitances were measured for each cell using the capacitance compensation of the patch amplifier. All values are means ± S.E.M.