Dopamine-induced oscillations of the pyloric pacemaker neuron rely on release of calcium from intracellular stores

Abstract

Endogenously bursting neurons play central roles in many aspects of nervous system function, ranging from motor control to perception. The properties and bursting patterns generated by these neurons are subject to neuromodulation, which can alter cycle frequency and amplitude by modifying the properties of the neuron's ionic currents. In the stomatogastric ganglion (STG) of the spiny lobster, Panulirus interruptus, the anterior burster (AB) neuron is a conditional oscillator in the presence of dopamine (DA) and other neuromodulators and serves as the pacemaker to drive rhythmic output from the pyloric network. We analyzed the mechanisms by which DA evokes bursting in the AB neuron. Previous work showed that DA-evoked bursting is critically dependent on external calcium (Harris-Warrick RM, Flamm RE. J Neurosci 7: 2113-2128, 1987). Using two-photon microscopy and calcium imaging, we show that DA evokes the release of calcium from intracellular stores well before the emergence of voltage oscillations. When this release from intracellular stores is blocked by antagonists of ryanodine or inositol trisphosphate (IP(3)) receptor channels, DA fails to evoke AB bursting. We further demonstrate that DA enhances the calcium-activated inward current, I(CAN), despite the fact that it significantly reduces voltage-activated calcium currents. This suggests that DA-induced release of calcium from intracellular stores activates I(CAN), which provides a depolarizing ramp current that underlies endogenous bursting in the AB neuron.

Fig. 1.

Release of calcium from intracellular stores is essential for dopamine (DA)-induced anterior burster (AB) oscillations. A: 30 μM BAPTA-AM (BAPTA) blocked 10⁻⁴ M DA-induced bursting in a synaptically isolated AB. In this experiment, the AB neuron was isolated from all synaptic and descending neuromodulatory inputs by photoablation of the pyloric dilator (PD) and ventricular dilator (VD) neurons and local application of tetrodotoxin (TTX) to a Vaseline pool around the stomatogastric nerve. B–D: 10 nM ryanodine (Ry), 10 μM xestospongin C (Xe), and 1 μM thapsigargin (Tg) blocked DA-induced oscillations in the AB. In these experiments, the AB neuron was isolated by application of TTX and picrotoxin (PTX) directly to the stomatogastric ganglion (STG).

Fig. 2.

Calcium imaging in the AB neuron filled with the calcium indicator Indo-1. A: Z stack of the AB neuron labeled with Indo-1, showing fluorescence from the 395-nm channel. Cell body with a bright nucleus is at bottom left. The primary and secondary neurites, along with an extensive dendritic tree, are clearly labeled. Yellow arrowheads mark the approximate regions of fine neuropil where calcium measurements were typically taken. Size bar, 100 μm. Region in yellow square is shown enlarged in B. B: the same region of fine neuropil, showing the ratios of the calcium emission signal detected at 395 nm and 495 nm. Size bar, 25 μm. Measurements are repeated after blockade of synaptic input with TTX, and again after addition of 10⁻⁴ M DA.

Fig. 3.

Onset of the DA-induced calcium rise precedes the onset of membrane potential (V_m) oscillations. A: time course of the voltage (top) and free Ca²⁺ concentration ([Ca²⁺]_in; bottom) change during DA application; 10⁻⁴ M DA has reached the bath solution at the 50 s time point. Each dendrite [region of interest (ROI)] is indicated by different color; the data points from one of the neurites are fit with a sigmoid function (plotted as a red solid line). Note that in the time period highlighted with the vertical light gray bar, [Ca²⁺] started rising before emergence of bursting. Voltage oscillations initially are slower and accelerate as the DA concentration reaches steady state. B1: complete time course of AB oscillations and intracellular calcium measurements as DA is added and washed out. As DA was washed out of the bath solution, the voltage oscillations and calcium signal returned back to their pre-DA levels within 10–15 min. B2: summary of the changes in the average calcium levels under different conditions: physiological saline (140 ± 8 nM), TTX (67 ± 3 nM), 10⁻⁴ M DA (193 ± 17 nM), and washout of DA. The neuron was oscillating in saline and in the presence of DA and quiescent in TTX and after DA (wash). C: preincubation with intracellular calcium channel blockers (Ry+Xe) abolishes the DA-evoked rise in [Ca²⁺]_in and oscillations in the AB neuron. C1: the AB neuron was preincubated with Ry+Xe for 30–60 min before DA application. Intracellular [Ca²⁺] did not change upon DA application; the neuron depolarized, because of DA's effects on other currents, but did not oscillate. C2: there was no significant difference in mean [Ca²⁺] under different conditions: 82 ± 11 nM before DA (in Ry+Xe) and 87 ± 17 nM in the presence of DA and Ry+Xe (P > 0.05, n = 10 ROIs).

Fig. 4.

The AB neuron calcium signal oscillates in phase with voltage oscillations but is delayed in onset and peak levels. Data were collected at 164 Hz by a fast line scan over a single dendrite. Simultaneous recordings of calcium signal and membrane: V_m (top) and relative change in fluorescence (dF/F; bottom). Time markers, in seconds, are below the voltage trace. A: spontaneously bursting AB neuron in physiological saline. B: 10⁻⁷ M TTX+ 5 × 10⁻⁵ M PTX blocked both voltage and calcium oscillations in <10 min. C: 10⁻⁴ M DA induced V_m and calcium signal oscillations. D: application of 10 μM Ry stopped both voltage and calcium oscillations. E: overlay of voltage and calcium traces. Calcium oscillations had similar delayed time course and amplitude both in a spontaneously bursting AB neuron with no blockers (red traces) and in a synaptically isolated AB (treated with TTX and PTX) in the presence of DA (black traces). Top: voltage trace. Bottom: calcium signal change (dF/F).

Fig. 5.

Calcium-activated inward current I_CAN is a likely target of DA modulation. A: flufenamic acid (FFA) abolishes DA oscillations in a synaptically isolated AB neuron. Top: the AB neuron was isolated by photoablation, and bursting was evoked with 10⁻⁴ M DA. Bottom: the AB neuron was isolated from modulatory and synaptic input by application of 10⁻⁷ M TTX + 5 × 10⁻⁵ M PTX, and oscillations were evoked with 10⁻⁴ M DA. In both cases, 3 μM FFA stopped oscillations within 30 min. B: properties of I_CAN in the AB neuron: the current is measured during voltage-clamp recordings as a slowly deactivating tail current evoked by an activating prestep to 0 mV and a series of hyperpolarizing steps (inset at right, voltage protocol). Arrowheads on the current traces mark the peak voltage-gated calcium current [I_Ca(V)] activated by the depolarizing prestep and the peak I_CAN tail currents measured after repolarization of the cell. C: dose-response curve for FFA block of I_CAN (IC₅₀ = 24.7 ± 5 μM; n = 4). The amplitude of the blocked current was normalized by the corresponding control current amplitude. D: DA enhances the amplitude of I_CAN while inhibiting I_Ca(V). Voltage-clamp current traces under control conditions (thin black trace), during application of 10⁻⁴ M DA (thick black trace), and after 30 min of washout (thin gray trace). DA inhibited I_Ca(V) while enhancing I_CAN amplitude (arrowheads). Current traces are not leak subtracted. The voltage protocol used to measure I_CAN is shown in the small inset. E: I_CAN current-voltage relationship under control conditions (C, open squares), in the presence of DA (filled circles), and after washout (W, gray triangles). All data points are means ± SE. Asterisks mark the voltage steps where the difference between control and DA was statistically significant (n = 8, P < 0.05, Student's t-test).

Fig. 6.

Multiple effects of DA on the ionic currents in AB neuron. A: schematics of the changes in the voltage and [Ca²⁺] during DA application, based on our results with indo-1 and Calcium Green-1 imaging. B: summary of DA effects on AB neuron. DA inhibits transient potassium current I_A, leak current (I_leak), and I_Ca(V), enhances I_K(V) and hyperpolarization-activated current I_h, and increases intracellular [Ca²⁺]. C: mechanism of the DA-induced oscillations in the AB neuron (see discussion for the description of the mechanism of action of DA). IP₃R, inositol trisphosphate receptor; RyR, Ry receptor.