Role of calcium in neurotensin-evoked enhancement in firing in mesencephalic dopamine neurons

Abstract

Neurotensin (NT) increases neurotransmission within the mesolimbic dopamine system by enhancing the firing rate of dopaminergic (DAergic) neurons and by acting at the nerve terminal level. The signal transduction pathways involved in these effects have not been characterized, but NT receptors are coupled to the phospholipase C pathway and Ca(2+) mobilization. However, an enhancement of intracellular Ca(2+) concentration ([Ca(2+)](i)) evoked by NT in DAergic neurons has yet to be demonstrated. Furthermore, the hypothesis that the excitatory effects of NT in DAergic neurons are Ca(2+) dependent is currently untested. In whole-cell recording experiments, DAergic neurons in culture were identified by their selective ability to express a cell-specific green fluorescent protein reporter construct. These experiments confirmed that NT increases firing rate in cultured DAergic neurons. This effect was Ca(2+) dependent because it was blocked by intracellular dialysis with BAPTA. Using Ca(2+) imaging, we showed that NT caused a rapid increase in [Ca(2+)](i) in DAergic neurons. Most of the Ca(2+) originated from the extracellular medium. NT-induced excitation and Ca(2+) influx were blocked by SR48692, an antagonist of the type 1 NT receptor. Blocking IP(3) receptors using heparin prevented the excitatory effect of NT. Moreover, Zn(2+) and SKF96365 both blocked the excitatory effect of NT, suggesting that nonselective cationic conductances are involved. Finally, although NT can also induce a rise in [Ca(2+)](i) in astrocytes, we find that NT-evoked excitation of DAergic neurons can occur independently of astrocyte activation.

Figure 1.

Neurotensin induces a Ca²⁺-dependent increase in firing rate in DAergic neurons. A, DAergic neurons were identified before patch-clamp recording using a selective transfection strategy allowing the expression of GFP under the control of the dopamine transporter promoter (green neuron; left image). The phenotype of recorded neurons was always confirmed after patch-clamp recording or Ca²⁺ imaging by detecting TH immunoreactivity (red signal; right image). Note that the field contained three DAergic neurons, but that only one was transfected with GFP. Scale bar, 15 μm. B, Whole-cell current-clamp recording of spontaneous action potentials in a DAergic neuron. Perfusion of 100 nm NT for 1 min (black bar) caused a delayed increase in firing rate. Basal firing rate was, on average, 1.05 ± 0.05 Hz in control condition and 1.6 ± 0.2 Hz in the presence of neurotensin. C, Summary data showing the change in action potential firing rate caused by bath-applied NT in DAergic neurons under control conditions (black circles) or when the patch pipette contained BAPTA (black squares). The change in firing rate over time is expressed as a difference score (delta) representing the variation in average firing rate within 15 sec bins compared with the average firing rate within the eight initial bins acquired before peptide application. Basal firing rate was, on average, 1.5 ± 0.1 Hz in the presence of BAPTA. For statistical comparisons, the average number of action potentials during the control period was compared with the average number of action potentials during the minute after the application of NT (in which the maximal effect was detected in experiments without BAPTA). There was a significant increase in firing in control experiments (t test; t = 4.21; p < 0.05) but not in experiments in which neurons were loaded with BAPTA (t test; t = 0.20; p > 0.05).

Figure 2.

Neurotensin induces calcium influx in DAergic neurons. A, Typical time course of the rise in [Ca²⁺]_i in a DAergic neuron during a 1 min exposure to NT (100 nm). Intracellular Ca²⁺ concentration was estimated using Calcium Green-1 AM. Data are represented as the mean fluorescence ratio as a function of time (ΔF/F_o). Images were acquired at a rate of 0.5 Hz. B, Dose-response effect of NT on [Ca²⁺]_i for NT concentrations ranging from 0.01 to 100 nm (0.01 nm, n = 3; 0.1 nm, n = 12; 1 nm, n = 11; 10 nm, n = 9; 100 nm, n = 13). Each bar represents the mean ± SEM value. C, The effect of NT on [Ca²⁺]_i persists in the presence of TTX. D, NT-evoked intracellular Ca²⁺ elevation in DAergic neurons is mostly blocked in the absence of extracellular Ca²⁺ (0 Ca²⁺) but is maintained after treatment with thapsigargin (Thap) to deplete intracellular Ca²⁺ stores or after blockade of IP₃ receptors with heparin (Hep). E, Thapsigargin and heparin cause a delay in the time-to-peak (T_max) of NT-evoked intracellular Ca²⁺ elevation. Ctrl, Control.

Figure 3.

The effect of NT on firing rate is mediated by the NTS1 receptor. A, SR48692 (1 μm), an NTS1 receptor antagonist, blocked the effect of NT (100 nm) on the firing rate of DAergic neurons (black circles). The response of the control group (CTRL) (from Fig. 1C) is shown for comparison (gray circles). Basal firing rate in the presence of SR48692 was, on average, 0.6 ± 0.1 Hz. B, The elevation in [Ca²⁺]_i induced by NT was also blocked by SR48692 and by SR142948A, a broader-spectrum NT receptor antagonist. The number of neurons responding to NT as a function of the total number of neurons tested is indicated below each column (Responsive neurons).

Figure 4.

Neurotensin-evoked excitation of DAergic neurons occurs independently of astrocyte activation. A, Graph representing the proportion of astrocytes that demonstrate an increase in [Ca²⁺]_i in response to NT (100 nm). Whereas a response was obtained in a significant proportion of primary mesencephalic astrocytes in culture, this was almost never observed in purified astrocyte cultures initially grown in flasks. B, Comparison of the NT-evoked enhancement in firing rate in DAergic neurons when grown together with responsive (open circles) (n = 7) or nonresponsive (dark squares) astrocytes (n = 10). The time course of the change in firing rate over time is expressed as a difference score (delta) representing the variation in average firing rate within 15 sec bins compared with the average firing rate within the eight initial bins acquired before peptide application. Basal firing rate was, on average, 0.40 ± 0.05 Hz in neurons grown on responsive astrocytes and 1.05 ± 0.05 in neurons grown on nonresponsive astrocytes. NT was used at 100 nm.

Figure 5.

NT-evoked increase in firing rate requires IP₃ receptors and cationic channels. A, Lack of effect of the PKC inhibitor bisindolylmaleimide on NT-evoked change in firing rate over time. Basal firing rate was, on average, 1.2 ± 0.1 Hz. B, Intra-pipette dialysis of the IP₃ receptor antagonist heparin blocked the ability of NT (100 nm) to enhance firing rate. Basal firing rate was, on average, 0.75 ± 0.05 Hz. C, Extracellular Zn²⁺ (10 μm), a blocker of nonselective cationic channels, prevented NT(8-13)-evoked enhancement in firing rate in DAergic neurons. Basal firing rate was, on average, 0.7 ± 0.1 Hz. D, Extracellular SKF96365 (1 μm), a different blocker of nonselective cationic channels, also prevented NT(8-13)-evoked enhancement in firing rate in DAergic neurons. Basal firing rate was, on average, 0.6 ± 0.2 Hz.