We performed experiments on wild-type or TH-GFP C57BL/6 mice (Matsushita et al., 2002) maintained in our institutional animal facility. Female mice were examined for the presence of a vaginal plug the morning after mating. The day of plug discovery was designated as embryonic day 0 (E0). Experiments were performed on mice of either sex. We removed E16 and E18 mice from deeply anesthetized dams [subcutaneous injection of a mixture of xylazine (Rompun 2%; used at 0.05%) and ketamine (Imalgene 1000 used at 50 g/L) volume injected: 0.1 mL/10 g]. The embryos were kept in an ice-cold oxygenated solution containing (in mM): 110 choline, 2.5 KCl, 1.25 NaH₂PO₄, 7 MgCl₂, 0.5 CaCl₂, 25 NaHCO₃, 7 glucose. Postnatal mice (P0–P7) were killed by decapitation under isofluorane anaesthesia. Coronal and parasagittal slices (400 μ m thick) were cut in the ice-cold oxygenated choline solution using a vibratome (VT1200 Leica Microsystems Germany). During the recovery period, slices were placed at room temperature with standard artificial cerebrospinal fluid (ACSF) saturated with 95%O₂/5%CO₂ and containing the following (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH₂PO₄, 1.3 MgCl₂, 2 CaCl₂, 25 NaHCO₃, 11 glucose.

Slices were incubated in the dark with 25 μ L of a fura-2 AM solution (1 mM in DMSO + 0.8% pluronic acid; Molecular Probes). We performed imaging studies with a multibeam two-photon laser scanning system (Trimscope-LaVision Biotec) coupled to an Olympus microscope. Slices were imaged using a high numerical aperture objective (20×, NA 0.95, Olympus). Images (4 × 4 binning) were acquired via a CCD camera (La Vision Imager 3QE) with a time resolution of 115–147 ms per frame. Size of the scan field (444 × 336 μ m) and duration of the movies (1000 frames) were unchanged. We first took images of the GFP-expressing neurons located in the mesencephalon (laser at 910 nm) before acquiring spontaneous fura-2 fluorescence changes (laser at 780 nm). To verify the location of the recorded field, at the end of the imaging session we bleached the fura-2 fluorescence from the field and observed its corresponding location on the GFP image. During the analysis, GFP-expressing fura 2-loaded neurons were identified by superposing the two fields. We performed analysis of the calcium activity with custom-made software written in Matlab (MathWorks) (Bonifazi et al., 2009). Active cells were neurons exhibiting any Ca²⁺ event of at least 5% DF/F deflection within the period of recording. Ca²⁺ spike or Ca²⁺ plateau cells were neurons exhibiting at least one Ca²⁺ spike or one Ca²⁺ plateau within the period of recording. A calcium plateau sustained a calcium level for at least 30 frames as opposed to a calcium spike which started decaying at the peak. We computed the activity correlation of cell pairs as previously described (Crepel et al., 2007; Dehorter et al., 2011).

We performed all recordings at 32°C. Cells were visualized with infrared–differential interference optics (Axioskop2; Zeiss). For whole-cell current clamp recordings the pipette (6–10 MΩ) contained the following (in mM): 128.5 K-gluconate, 11.5 KCl, 1 CaCl₂, 10 HEPES, 10 EGTA, 2.5 MgATP and 0.3 NaGFP, pH 7.2–7.4 (275–285 mOsm). We determined input membrane resistance (R_m) by on-line fitting analysis of the transient currents in response to a 5–10 mV pulse at V_H = –60 mV. Criteria for considering a recording included R_m > 100 MΩ. The input resistance (R_m) of mDA neurons decreased significantly from 355 ± 39 MΩ before birth (E18, n = 8) to 203 ± 31 MΩ at P5–P7 (n = 5, p < 0.05, Mann–Whitney test). In parallel, the percentage of mDA neurons displaying the hyperpolarization-activated cationic current Ih increased from 61% at E18 to 100% at P7. Amplitude of action potentials was measured from peak to after spike hyperpolarization (AHP) potential and their duration half-way between threshold and peak (half-width duration).

Coronal slices were placed in a chamber and perfused with O₂ saturated ACSF at 32°C. We measured the evoked and not the spontaneous release of dopamine as performed in P9–14 primary cultures of mDA neurons (Kim et al., 2008) or 30–40 days organotypic slices of the striatum (Cragg et al., 1998) because the high perfusion rate of ACSF needed to keep slices healthy prevents such a measure. Stimulation was performed with a bipolar tungsten electrode with a tip separation of 100 μ m (World Precision Instruments TST33C05KT, stereo tungsten electrode, in vitro impedance of 1 MΩ) inserted into the striatum. We did not study DA overflow in response to median forebrain bundle (MFB) stimulation because medial sagittal slices containing the MFB cannot be reliably obtained at embryonic stages. To evoke a reproducible DA release, we used a train of four 100 Hz square pulses of 50 V amplitude and 100 μ s duration. To monitor the electrically evoked dopamine release, we used continuous amperometry with carbon fiber electrodes because it gives similar results as cyclic voltammetry in the striatum (Schmitz et al., 2001). The carbon fiber electrode (active surface 10 μ m in diameter and 500 μ m long; World Precision Instruments, CF10) was implanted into the striatum at an angle of 60° from vertical so that the entire length of the active surface was inside the slice at a depth of about 50 μ m from the surface. This was done in the ventrolateral and dorsomedial striatum where the evoked DA release was maximal and minimal respectively. The carbon fiber electrode was connected to a potentiostat (MicroC, World Precision instruments) to apply voltage and measure current. To measure DA release, the imposed voltage between the carbon fiber electrode and the Ag/AgCl pellet was 0.5 V. In response to the stimulus train, the current generated by oxidation of evoked dopamine released was recorded. To separate the evoked current from an artefact, the same stimulus protocol was done with 0V applied between the carbon electrode and the Ag/AgCl reference. At this voltage, no oxidation of DA should occur. Signals were digitized using a Digidata data acquisition system (Digidata 1440A) coupled to a PC running the clampex nine program responding to the Multiclamp700A amplifier. Results are presented as maximum response obtained per brain hemisphere. To measure DA release during the blockade of dopamine reuptake, we incubated the slices in nomifensine (10 μ M) for a minimum of 20 min. To test the calcium-dependence of dopamine release, we used a modified ACSF containing the following (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH₂PO₄, 3.3 MgCl₂, 25 NaHCO₃, 11 glucose.

To visualize the TH-positive fibers in the striatum we performed immunocytochemistry of TH in embryonic and early postnatal slices, and to identify the recorded cells we revealed the neurobiotin injected during whole-cell recordings in recorded slices, as previously described (Dehorter et al., 2009). Dendritic and axonal fields were reconstructed for morphological analysis using the Neurolucida system (MicroBrightField Inc., Colchester, VT).

Drugs were prepared as concentrated stock solutions and diluted in ACSF for bath application. Gabazine, D-amino pyruvate (D-APV), 6-cyano-7-nitroquinoxaline 2,3-dione (CNQX), Tetrodotoxin (TTX), Nifedipine, Thapsigargin and Nomifensine maleate were purchased from Sigma (St. Louis, MO, USA). ω–conotoxin GVIA was purchased from Alomone Labs (Jerusalem, Israel).

Statistical results are given as means ± SEM. We performed statistical analysis using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA): one-way ANOVA (Tukey's Test as post hoc test), Mann–Whitney test (non-parametric t-test), and paired t-tests as indicated in the results section. Differences were considered significant at p ≤ 0.05 (^*** for p ≤ 0.001, ^** for p ≤ 0.01 and ^* for p ≤ 0.05). We grouped the P5 and P7 sets of data since they did not present a statistical difference. In the box plots of Figures 1A and 5B the bottom and top of the boxes represent the 25^th and 75^th, the band inside the box is the 50^th percentile (median) and the top and bottom vertical bars (whiskers) denote the maximum and minimum values.

Axons of E16 neurobiotin-filled mDA neurons project rostrally toward the striatum even when their somas do not have dendrites yet (Figure 1A). The dendritic length and number of dendritic ends of mDA neurons significantly increase from E16 (90 ± 31 μ m; 1.5 ± 0.4 ends; n = 13) to E18 (245 ± 66 μ m; 3.9 ± 0.6 ends; n = 7; p < 0.05, Mann–Whitney test), and from E18 to P0 (1031 ± 287 μ m, 12 ± 3 ends; n = 6; p < 0.05, Mann–Whitney test) (Figure 1A). Accordingly, a substantial diffuse innervation of the striatum by TH⁺ fibers is already present at E14–E16 in the ventro-lateral part of the striatum (Ohtani et al., 2003) (Figure 1B). Later at E18–P0, TH⁺ fibers invade the more dorsal regions of the striatum. Therefore, mDA neurons extend long axons that reach the striatum already at E16 before developing their dendritic tree.

From the 1052 fura-2-loaded/GFP-positive (mDA) imaged neurons recorded in a total of 45 movies, 412 spontaneously generated calcium events (Figures 2A,B). mDA neurons were already active at E16, the youngest age tested (Figures 2C,D). Embryonic (E16–E18) and early postnatal (P0–P7) mDA neurons generated two patterns of activity (Crepel et al., 2007): Ca²⁺ spikes that were sporadic brief Ca²⁺ events (1.41 ± 0.08 s duration, n = 264 neurons in 45 fields referred as 264/45) and long lasting Ca²⁺ plateaus (9 ± 0.3 s duration, 106/45; Figure 2C). These two patterns of activity significantly differed in duration (p < 0.05, Mann–Whitney test). The percent of mDA neurons generating Ca²⁺ spikes was stable between E16 (28 ± 4% of fura-2 AM-loaded GFP neurons, 131/11), and P0 (32 ± 4%, 87/13), significantly decreased at P3 (11 ± 5%, 30/4, p < 0.001, Mann–Whitney test, data not shown) and then remained stable until P7 (13 ± 3%, 11/12; Figure 2D). Ca²⁺ spikes had a low frequency at E16–E18 (0.05 ± 0.01 Hz, 166/16) that increased at P0 (0.09 ± 0.01 Hz) and P7 (0.16 ± 0.04 Hz, data not shown). The percent of mDA neurons generating Ca²⁺ plateaus was low at E16 (11 ± 3%, 37/11) and remained stable until P0 (10 ± 2%, 43/13) before decreasing at P7 (4 ± 2%, 5/12; Figure 2D). Ca²⁺ plateaus had a similar mean frequency (0.040 ± 0.004 Hz vs. 0.08 ± 0.04 Hz; p = 0.28 Mann–Whitney test) and a similar mean duration (8.9 ± 1.7 s vs. 8.6 ± 3.7 s; p = 0.67 Mann–Whitney test) at E16 and P7 (data not shown). Overall, the percent of spontaneously active mDA neurons generating at least one Ca²⁺ spike and/or one Ca²⁺ plateau did not change significantly between E16, E18, and P0 (39.5 ± 3.6%, 50.6 ± 10.5%, and 45 ± 4% of imaged mDA neurons, respectively, p = 0.4 between E16 and E18, and p = 0.7 between E18 and P0), and then significantly declined from P0 to P7 (17 ± 5% at P7; p < 0.01, Mann–Whitney test). Ca²⁺ spikes and Ca²⁺ plateaus were poorly correlated between neurons (0.2% cell pairs significantly correlated, see materials and methods) at all ages tested.

Ca²⁺ spikes and Ca²⁺ plateaus were sensitive to blockers of Na⁺/Ca²⁺ voltage-gated channels since TTX (1 μ M) + nifedipin (10 μ M) dramatically decreased the percent of active mDA neurons from 30.2 ± 7.7% to 4.3 ± 1.3% at E16 (p < 0.05, paired t-test, n = 4 slices), i.e., it decreased the activity of 94 ± 4% of the previously active E16 mDA cells (Figure 3A). Nifedipin alone at a concentration that specifically blocks L-type Ca²⁺ channels (3 μ M) decreased the calcium activity of 44 ± 14% of the previously active P1 mDA neurons (n = 8 slices, Figure 3B). Furthermore, ω-conotoxin GVIA (1 μ M), a specific blocker of N-type Ca²⁺ channels, decreased the calcium activity of 57 ± 7% of the previously active P1 mDA neurons (n = 4 slices, Figure 3C). In contrast, Ca²⁺ events recorded at P0 were totally insensitive to ionotropic GABA and glutamate receptor antagonists (44.9 ± 2.3% active vs. 37.2 ± 3.7% at P0, p = 0.7, paired t-test, i.e., 76.5 ± 6% of the previously active cells remained unaffected, Figure 3D). A few days later, at P3 the same synaptic blockers decreased the calcium activity of 69 ± 8% of the previously active mDA neurons (n = 4 slices, Figure 3E). In contrast, depletion of intracellular Ca²⁺ stores with thapsigargin (10 μ M) did not affect the number of cells generating Ca²⁺ spikes and Ca²⁺ plateaus, nor the frequency of these events (24.5 ± 4.1% control active vs. 24.6 ± 4.3% at P1, p = 0.9, paired t-test, i.e., 76 ± 10% of the active GFP-positive cells identified before thapsigargin treatment had their activity unaffected by the treatment, data not shown). These results showed that E16-P1 mDA neurons spontaneously generate intrinsically driven, L- and N-type mediated Ca²⁺ events. Only starting from P3, are these Ca²⁺ events sensitive to synaptic blockers suggesting that synapse-driven inputs to mDA neurons operate during the first postnatal week but not before.

In contrast to their capacity to generate Ca²⁺ events (Figures 2B and 3), E16 embryonic mDA neurons (n = 16) did not generate spontaneous (Figure 4A) or evoked (Figure 4B) Na⁺ spikes. They started to generate spontaneous Na⁺ spikes around birth since 28% and 50% of the recorded mDA neurons at E18 and P0, respectively, (n = 18; n = 12), spontaneously fired action potentials with a mean amplitude of 44.8 ± 5.4 mV and 52.6 ± 3.3 mV (Figures 4A–C). At P7, 100% of the recorded mDA neurons were spontaneously active (amplitude: 73.6 ± 4.5 mV; n = 10). Spike half-width duration significantly decreased from E18–P0 (3.8 ± 0.6 ms, n = 5; 4.2 ± 0.6, n = 6) to P7 (2.2 ± 0.2 ms, n = 10; p < 0.05, Mann–Whitney test; Figure 4D) but mean spontaneous firing frequency did not significantly increase from E18–P0 to P7 (0.2 ± 0.1 Hz at E18; 0.4 ± 0.2 Hz at P0; 0.6 ± 0.2 Hz at P7; Figure 4E). Therefore, half of the mDA neurons are already capable of generating spikes at birth (see Figure 2).

In agreement with the presence of TH⁺ fibers in the embryonic striatum, we detected from E18 DA release in the striatum in response to local stimulation (19.7 ± 1.5 pA, n = 13; Figures 5A,B). This evoked DA release significantly increased at birth (P0) to 43.1 ± 4.3 pA (p < 0.001, One-way ANOVA; n = 25) and stayed stable during the first postnatal week. The evoked DA release observed at E18 and P0 was entirely dependent on external Ca²⁺ ions, since the response disappeared in the absence of Ca²⁺ ions, and was rescued in the presence of Ca²⁺ ions (Figures 5A,B) To further support the view that the changes in oxidation current evoked by striatal stimulation actually correspond to an evoked dopamine overflow (Benoit-Marand et al., 2000), nomifensine (10 μ M) was added to the perfusion medium to inhibit dopamine reuptake. This did not alter the rising phase of dopamine overflow which corresponds to dopamine release, but slowed the kinetics of the decreasing phase which depends on dopamine reuptake (Figure 5A). Dopamine half-decay was significantly increased by nomifensine treatment (20 min) at P7 (0.4 ± 0.1 to 3.7 ± 0.3 s, p < 0.001 paired t-test, n = 5), and P25–40 (0.5 ± 0.2 to 3.7 ± 1 s, p < 0.05 paired t-test, n = 5; Figure 5B). These results confirmed the perinatal expression of the dopamine transporter in rodents (Galineau et al., 2004). At E18 and P0, the decay phase in the presence of nomifensine was too long and precluded its measure. This could be due to the fact that at these young ages the competitive inhibitor nomifensine, at the dose used, could not be rapidly displaced from its binding sites on the dopamine transporter (Tuomisto, 1977; Jones et al., 1995; Katz et al., 2000) by the small amount of evoked dopamine overflow.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.