Pacemaker neurons spontaneously generate rhythmic electrical activity. Because the somatodendritic membrane of neurons is heterogeneous not only in morphological structure but also in excitability [1,2], the pacemaking mechanisms of neurons are quite complex. The midbrain dopamine (DA) neuron is a slow pacemaker that intrinsically generates action potentials (APs) at a regular rhythm [3,4]. Notably, these DA neurons have a soma that extends three to six primary dendrites [5,6], and an axon usually emerges from one of their proximal regions [7,8]. In addition, spontaneous APs (sAPs) and co-occurring Ca²⁺ oscillations in DA neurons take place in both the soma and dendrites [9], indicating that the dendrites, as an excitable element, participate in pacemaking. The survival of rhythmic Ca²⁺ oscillations in the soma and dendrites after complete suppression of sAPs with tetrodotoxin (TTX) further supports that both the soma and dendrites are active elements [10,11].

Multipolar midbrain DA neurons are known to initiate sAPs in the axon-bearing dendrite (ABD) due to the low AP threshold of the axon initial segment (AIS) [7,8,12,13]. Once the slowly depolarizing membrane potential reaches the threshold of the AIS, sAPs are generated and then rapidly propagated into the soma and non-axon-bearing dendrites (nABDs). However, the slow development of pacemaker potentials is not exclusively controlled by the soma, but multiple dendrites are also involved in the process [9,14]. In neurons, Ca²⁺ influx and efflux pathways that accompany endogenous membrane potential oscillations, such as pacemaking, lead to Ca²⁺ oscillations in each compartment having a various surface area to volume ratio (SVR). Because SVR greatly affects Ca²⁺ removal time, it is theoretically very likely that dendritic compartments could be oscillating at faster frequencies than the soma [9,15]. Despite this, sAPs and Ca²⁺ spikes occur synchronously throughout the somatodendritic compartment [9,11], making DA neuron behave like a single compartment. Thus, a coupled oscillator model has been proposed, which suggests the tight electrical coupling between the slow-oscillating soma and fast-oscillating dendrites in DA neurons [9].

Maintaining normal brain functions requires a baseline level of ambient DA, which is regulated by the rate of spontaneous firing, also known as tonic firing [16-18]. Burst discharges or phasic firing, can trigger additional surges of DA from this baseline [19-21]. Therefore, any abnormality in the pacemaker system of DA neurons could lead to various neuropsychiatric and neurodegenerative diseases [22,23]. Consequently, it is crucial to regulate pacemaker activity tightly in these DA neurons. The coupled oscillator model suggests that the slow-oscillating soma and fast-oscillating dendrites determine the spontaneous firing rate of individual DA neurons [9]. However, midbrain DA neurons are morphologically diverse, not uniform [14,24]. Nevertheless, spontaneous firing occurs within a limited range of 2–6 Hz, and the spontaneous firing rate is determined by the balance between the soma and the proximal dendritic compartments (PDCs), implying that the PDCs, not the whole dendritic compartment, might play a major role in pacemaking [14]. Very recently, we find two essential pacemaker channels, TRPC3 and NALCN in DA neurons [25] and NALCN is localized only in the PDCs [26], explaining why PDCs are highly excitable. However, the morphological aspects of pacemaking in central neurons including midbrain DA neurons still remain largely elusive.

In this study, we describe the functional somatodendritic organization of pacemaker activity in multipolar DA neurons. Multiple PDCs of the primary dendrites in DA neurons are a stochastically-fluctuating highly-excitable compartment, but the large, centrally-connected, less-excitable, and inertial soma counteracts and balances their high and stochastic excitabilities. This complementary dynamic interaction between the soma and multiple PDCs could explain how DA neurons generate both slow regular pacemaker activities as well as various and flexible types of evoked firings.

Preparation of midbrain slices and single DA neurons

All experiments on animals were carried out in accordance with the approved animal care and use guidelines of the Laboratory Animal Research Center in Sungkyunkwan University School of Medicine and all experimental protocols were approved by the Laboratory Animal Research Center in Sungkyunkwan University School of Medicine (SKKUIACUC2018-05-10-5). For midbrain slices recording we used the STOCK Tg (TH-eGFP) DJ76Gsat/Mmnc line (NIH Mutant Mouse Regional Resource Centers), which was maintained as heterozygous mice by breeding with ICR (CrljOri: CD1) inbred mice [14]. Horizontal midbrain slices which contain the substantia nigra pars compacta (SNc) area, were prepared in postnatal day 21–28 transgenic mice expressing enhanced green fluorescent protein (eGFP) driven by the tyrosine hydroxylase (TH) promoter. For acutely dissociated DA neurons, postnatal day 9–12 Sprague Dawley rats used. The mouse and rat brains were removed rapidly after decapitation and then sliced into 300–400 µm thicknesses in ice-cold, oxygenated, artificial cerebrospinal fluid (ACSF in mM: 125 NaCl, 2.5 KCl, 10 glucose, 1.25 NaH₂PO₄, 25 NaHCO₃, 1 MgCl₂, 2 CaCl₂, pH 7.4) or ice-cold high-glucose solution (in mM: 135 NaCl, 5 KCl, 10 HEPES, 1 CaCl₂, 1 MgCl₂, 25 D-glucose, pH 7.3) using a vibratome (VT 1000S; Leica Microsystems, or Series 1000; Technical Products International). The acute mice brain slices were incubated in ACSF for 30 min at 33°C in dark chamber during the recovery period. However, for dissociated DA neurons, the SNc regions were cut out from the midbrain slices without recovery time, and then the DA neuron-containing tissues were incubated in oxygenated high-glucose solution with 8 U/ml papain (Worthington Biochemical Corporation) for 30 min at 36°C–37°C. After enzymatic digestion, the tissue was washed three times with 36°C high-glucose solution. After that, the dissociated DA neurons were obtained from the tissue with gentle serial agitation with Pasture pipettes of varying pore sizes. Some dissociated DA neurons were also obtained using the same dissociation protocol from postnatal day 9–12 Sprague Dawley rats [14]. The acutely dissociated DA neurons were attached to a poly-D-lysine (0.01%)-coated cover slip for 30 min at room temperature (20°C–25°C).

Two-photon laser confocal imaging and axon laser cutting

Two-photon confocal imaging was performed using a Zeiss LSM 510 Meta confocal/two-photon microscope system in the Research Core Facility of the Samsung Biomedical Research Institute (SBRI). Neuron morphologies were visualized by Alexa Fluor 594 (30 µM, Invitrogen) and eGFP with a tunable (690–1,020 nm) Ti::sapphire laser (Mai Tai; Spectra Physics). Intracellular Ca²⁺ signals were measured with a Ca²⁺ indicator, Oregon Green BAPTA-1 (OGB-1, 200 µM, Invitrogen) and 800 nm wavelength before and after removing AIS from the DA neurons (800 nm for GFP or OGB and 720 nm for axon laser cutting).

To remove an AIS from the DA neuron after visualizing the thin axon, the fluorescence dyes were fully loaded into recording neurons via low resistance recording electrodes (2–3.5 MΩ) in resting state for ≥ 15 min. The axon was identified by a characteristic axon retraction ball, a non-spiny membrane, and non-tapering diameters. The 720 nm wavelength of laser was used for axon laser cutting with the line scanning mode or minimized region of interest (ROI) on target regions. The origination site of an axon close to the dendrite, where is potentially including AIS, was exposed gradually increased laser power. Laser power was increased from 5% to 50% until the axon was cut from the dendrite. Axons were usually cut at around 40% of the laser output power, and then a sudden increase in spontaneous firing frequency occurred with structural changes including bleb formation. To allow the neurons to stabilize and recover after axon removal, we injected a small amount of negative currents to keep the membrane potentials in a resting state for 15–20 min.

Photolysis of caged compounds

To stimulate glutamate receptors or GABA receptors on the soma or dendrites, 100 µM 4-methoxy-7-nitroindolinyl-caged L-glutamate (MNI-glutamate) (Tocris Bioscience) with 10 µM glycine or 100 µM γ-Aminobutyric Acid, α-Carboxy-2-Nitrobenzyl Ester, Trifluoroacetic Acid Salt (O-(CNB-Caged) GABA) (Invitrogen) was added to the bath solution. Since caged-compounds are very sensitive of lights, whole procedures were avoided light exposure and we made fresh working solutions for every experiment to keep high efficiency of caged-compounds. In the local caged Ca²⁺ photolysis, we used an internal solution with 50 µM o-nitrophenyl EGTA (NP-EGTA) (Tocris Bioscience). Photolysis was performed using a Zeiss 510 confocal microscope with a UV laser (wavelengths 351 and 364 nm) and an oil-immersion objective lens (40× with a NA of 1.3, Olympus). Detailed procedures were described previously [14].

Measuring cytosolic Ca²⁺ oscillations and a calibration

For cytosolic Ca²⁺ measurements in tissue slices, DA neurons were loaded with Alexa-594 (red dye, 30 µM, Invitrogen) and Oregon Green Bapta-1 (green dye, OGB-1, 200 µM, Invitrogen) together. Optical signals were acquired at 800 nm of two-photon excitation beam to simultaneously excite both dyes. ROI images were acquired during frame scanning (512 × 512 pixels) with 10–20 ms time intervals and Ca²⁺ levels from the ROIs were quantified as changes in green Ca²⁺ fluorescence from OGB-1 divided by morphological red fluorescence of Alexa-594 (G/R). Ratio between OGB-1 and Alexa-594 was used to minimize interference of the fluorescence photobleaching.

Acutely dissociated DA neurons were incubated with 3–5 µM Fluo 4-AM in high-glucose solution at room temperature (20°C–25°C) for 30 min. The fluorescence intensities of the neurons were measured using a Zeiss 510 confocal microscope (40× oil immersion objective lens or 60× water immersion objective lens). Fluo 4-AM Ca²⁺ indicators were excited at 488 nm (argon laser) and cytosolic Ca²⁺ signals were collected through 550 nm long-pass filter. Ca²⁺ level changes represented delta fluorescence intensity devided by basal level of fluorescence (ΔF/F₀). To measure cytosolic Ca²⁺ concentration in some cases, we used a calibration kit (Calcium Calibration Buffer Kit #1; Invitrogen).

Electrophysiology

We used the patch clamp systems (EPC-9 with Patchmaster 2.73; HEKA Elektronik) to measure the electrical activity of DA neurons in single or two-photon confocal microscopes. Low-resistance (2.0–3.5 M_Ω) patch electrodes were prepared from borosilicate glass capillaries with a 1.5 mm outer diameter (World Precision Instruments) using a Narishige puller (MODEL PP-830). Spontaneous firing was recorded in the current-clamp mode at a sampling rate of 10 kHz filtered at 1 kHz under the cell-attached, nystatin-perforated, or whole-cell patch-clamp configurations. For nystatin-perforated patch-clamp recordings the patch pipettes were filled with the solution containing (in mM) 140 K-gluconate, 5 KCl, 10 HEPES, 5 MgCl₂ and Nystatin (200 µg/ml, MP biomedical), and pH 7.2 was adjusted with KOH. For whole-cell patch-clamp recordings, the patch pipettes were filled with (in mM) 130 K-gluconate, 1 KCl, 10 HEPES, 2 Mg-ATP, 0.3 Na-GTP, 4 Na-Vit C, 10 Na-phosphocreatine, at pH 7.2 adjusted with KOH. The normal bath solution for acutely dissociated DA neurons contained (in mM) 140 NaCl, 5 KCl, 10 HEPES, 10 D-glucose, 1 CaCl₂, and 1 MgCl₂, at pH 7.4 adjusted with NaOH.

Immunocytochemistry

The midbrain slices and the dissociated DA neurons were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 20 min and 15 min, respectively. After that, the slices or dissociated neurons were washed in PBS four times for 15 min or three times for 10 min, respectively. Slices were incubated for 15 min in permeabilization buffer (PBS containing 2% normal goat serum [NGS] and 1% Triton X-100) at 4°C. After permeabilization of midbrain slices, we incubated slices in blocking buffer (PBS containing 2% NGS) for 20 min with gentle shaking. For permeabilization of the dissociated neurons, we used PBS containing 1% NGS and 0.2% Triton X-100. After 10 min at 4°C incubation, neurons were rinsed in PBS and blocked for 20 min in blocking buffer (PBS containing 1% NGS). For TH and AIS staining, we used mouse anti-TH antibody (1:500 or 1:300, Abcam Inc.) and rabbit anti-ankyrin G antibody (1:300 or 1:200, Abcam Inc.). For primary antibody reactions in brain slices and dissociated neurons, they were incubated overnight in the primary antibodies in permiabilization buffer at 4°C. Primary antibody-loaded brain slices and neurons were rinsed four times with blocking buffer and incubated in blocking buffer with the secondary antibodies: Alexa 488-conjugated goat anti-mouse IgG (1:100, Invitrogen) and Alexa 647-conjugated goat anti-rabbit IgG (1:100, Invitrogen). After incubation for 2 h at room temperature fluorescence images were obtained using a confocal or two photon laser microscope.

Statistics

Statistical analyses were performed with Origin 8.1 (Origin Lab Corporation). All presented numeric values and graphic representations represent mean ± standard error of the mean, and statistical analyses used one-way ANOVA tests followed by Tukey’s multiple-comparison posttest. p-values < 0.05 were regarded as significant.

Changes in sAP initiation sites after removing the AIS from midbrain DA neurons

In midbrain DA neurons, both the soma and multiple dendrites participate in pacemaking [9,14]. But, it is not clear whether the AIS also contributes to pacemaking. Therefore, we examined what happens to the spontaneous firing of DA neurons after removal of an axon with the two-photon laser-cutting technique in mouse midbrain slices. DA neurons were identified with GFP driven by the promoters of TH [27]. A DA neuron in the SNc extended several long primary dendrites in various directions (left image, Fig. 1A) and a non-tapering thin axon was identified with a terminal retraction ball formed during slice preparation (middle upper image, Fig. 1A), as previously described [8,12]. After spontaneous firing was stabilized in the midbrain slices (right, cell-attached recording, Fig. 1A), the axon was cut using strong two-photon laser illumination (720 nm) at the origination site near the dendrite (the red line in the middle upper image of Fig. 1A). A scar was left as a bleb in this case (middle lower image, Fig. 1A), but very often the neuron ruptured and became invisible because of dye leakage. In this typical surviving DA neuron, the spontaneous firing rate temporally increased as soon as the axon was detached from the primary dendrite, but the resting spontaneous firing rate was restored after 10–20 min (Fig. 1A). After restabilization, spontaneous firing rate did not differ significantly from that before the axon-cutting (spontaneous firing rate _{before axon-cut} = 3.45 ± 0.01 Hz, spontaneous firing rate _{after axon-cut} = 3.54 ± 0.01 Hz, p = 0.356, n = 3, Fig. 1B), suggesting that the AIS does not significantly contribute to pacemaking. Nevertheless, after AIS removal, the variance in the interspike interval (ISI) did statistically increase (variance _{before axon-cut} = 0.0034 ± 0.00048, variance _{after axon-cut} = 0.0256 ± 0.0065, p < 0.05, n = 3, Fig. 1C), suggesting that the AIS lowers the irregularity of spontaneous firing.

Figure 1. Axon removal reveals dendritic origin of sAPs in DA neurons. (A) Fluorescence images of a DA neuron in a midbrain slice and pacemaking and spontaneous firing diagram. The yellow rectangular area containing an axon is expanded into the middle images before and after axon removal at the red line. Right, spontaneous firing traces recorded in the cell-attached mode before and after axon removal. (B) Mean spontaneous firing frequencies before and after axon removal. (C) ISI variances before and after axon removal. (D) Ca²⁺ spikes were measured in two opposite proximal primary dendrites, P1 (ABD, blue circles) and P2 (nABD, red circles). (E) Before axon-cutting, Ca²⁺ spikes always developed immediately after sAPs from the ABD (P1, blue) and later at the nABD (P2, red). Black line represents AP appearance timing. AP propagation direction was marked with arrow in an insert. (F) Time-lags between APs and rising-points of Ca²⁺ spikes (mean time lag: P1 = 0.473 ± 0.046, P2 = 10.39 ± 1.04, n = 3). (G) Variances in the time-lags before and after axon removal. (H) Ca²⁺ spikes were measured in two opposite proximal primary dendrites (P1 and P2) after axon removal. The order of the two dendritic Ca²⁺ spikes varied. The case in which the Ca²⁺ spike occurred first at the nABD, immediately after sAP, is presented. (I) The order of Ca²⁺ spikes between two primary dendrites with the time-lags (mean time lag: P1 = 10.86 ± 2.69, P2 = 6.85 ± 2.19, n = 3). (J) Incidence of AP initiation in the ABD (P1) and nABD (P2) changed dramatically after axon removal (from P1 = 100% and P2 = 0% to P1 = 55.56%, and P2 = 44.44%, n = 3). Values are presented as mean ± SE. DA, dopamine; ISI, interspike interval; ABD, axon-bearing dendrite; nABD, non-axon-bearing dendrite; AP, action potential; sAPs, spontaneous action potentials; AIS, axon initial segment. **p < 0.001.

In multipolar DA neurons, it is well known that sAP always initiates from ABD and then propagates to the soma and other dendritic trees [7,8]. So, after removing axons, we wonder if sAP still initiates from the same ABD. First, we recorded spontaneous firing at the soma with a whole-cell patch-pipette and simultaneously compared corresponding Ca²⁺ spikes between the ABD and opposite nABD to detect the sAP initiation site [9,11,28]. After sAP onset (P1, blue lines and circles), Ca²⁺ spikes always started immediately from the ABD and later on the contralateral nABD (P2, red lines and circles) (Fig. 1D–F), as previously reported [7,8]. But, interestingly, after AIS removal with a two-photon laser cut (the red line in Fig. 1D), Ca²⁺ spikes from the ABD did not always start first (time-lag _{before axon-cut} = 0.52 ± 0.10 ms, time-lag _{after axon-cut} = 5.36 ± 2.11 ms, p < 0.05, n = 15 and 39 spikes from 3 neurons). Sometimes, Ca²⁺ spikes in the opposite nABD occurred before those in the previous ABD (Fig. 1H–J). In addition, after AIS removal, some paired Ca²⁺ spikes measured in the two dendrites (P1 and P2) did not match exactly with sAP onset times, with concurrent delays in both spikes (Ca²⁺ spikes 1, 2, 4, 6, and 8 in Fig. 1I), indicating that some sAPs developed in another primary dendrite. These data suggest that, if the AIS is removed, sAPs could be generated variably from many primary dendrites, including the previous ABD.

Regarding this aspect, it is interesting to see the changes in variances of the time-lags of dendritic Ca²⁺ spikes against sAPs which were measured with patch pipettes at the soma (Fig. 1F). The variances in the time-lags between the Ca²⁺ spikes in ABDs and the sAPs were much smaller than those in the nABDs (Fig. 1G), but after AIS removal, the variance in the ABDs increased to the level of the nABDs (p < 0.005, n = 3). All these data indicate that the slowly depolarizing pacemaker potentials of multiple primary dendrites are not equal and strictly coupled to each other. The pacemaker potentials of each primary dendrite appear to develop with some variation, but one of them reaches the AP threshold more quickly than the soma (see diagram in Fig. 1A). Thus, AIS appears to lower the ISI variability in the fluctuating ABD by triggering earlier sAPs therein. Consequently, AIS removal appears to remove the dominance of the ABD over the nABDs, thereby revealing the presence of variable sAP initiation sites scattered among many primary dendrites (Fig. 1H–J).

Stochastic initiation of sAPs from multiple primary PDCs in AIS-disabled dissociated DA neurons

Acutely dissociated DA neurons attached with multiple primary dendrites from the rat SNc are a good model for studying pacemaker mechanisms, due to the large cell bodies attached with long dendrites compared with those of mice, preservation of intrinsic regular firing properties, easy spatial Ca²⁺ measurements from the dendrites on cover-glasses, and clear environmental conditions [14,28,29]. In the dissociated DA neurons, we rarely found axon-like structures, unlike the DA neurons in midbrain slices (Fig. 2A). They appeared to be lost during the isolation procedures. When we stained AIS in these neurons with ankyrin G, a marker for AIS, axon was not found in more than half of the cells, and even in stained cells, significantly reduced in length (Fig. 2B–D). Consequently, as seen below, the AIS in dissociated DA neurons seems to be disabled or non-functional. Therefore, we are able to investigate the endogenous dendritic properties of pacemaking without any interference from the AIS in dissociated DA neurons.

Figure 2. Comparison of the AIS between midbrain slices and dissociated DA neurons. (A) AISs were identified in the DA neurons in the midbrain slices using two-photon confocal imaging and a terminal retraction ball formed during slice preparation. Bottom, the simplified structure of a DA neuron that includes a thin axon and multiple dendrites is reconstructed from the upper fluorescence image. (B) Immunocytochemistry of TH (red; Alexa 647) and ankyrin G (green; Qdot 488) in a SNc brain slice and dissociated DA neurons. The AISs of one DA neuron marked as ʻaʼ in the slice and a dissociated neuron are marked by white triangles. (C) Significant loss of AIS in dissociated DA neurons. AIS loss in 62.5% (5/8 cells) and decreased AIS in 37.5% (3/8 cells). (D) Average AIS length in the dissociated DA neurons decreased significantly after dissociation procedures, as shown by comparing them with DA neurons in the slices (AIS lengths of DA neurons in slices, 35.20 ± 2.26 µm, n = 15; in dissociated DA neurons, 11.70 ± 3.64 µm, n = 8). AIS, axon initial segment; DA, dopamine; TH, tyrosine hydroxylase; SNc, substantia nigra pars compacta.

In one dissociated DA neuron of the rat to that three primary dendrites were attached, we measured Ca²⁺ spikes along one long primary dendrite, together with sAPs using a patch-pipette at the soma (Fig. 3A). In this typical DA neuron, we found two different types of Ca²⁺ spike propagations: in one case, Ca²⁺ spikes started from the proximal dendritic region and propagated bidirectionally to the soma and the distal dendritic region (Fig. 3B). In the other case, Ca²⁺ spikes occurred unidirectionally from the soma to the proximal and then distal dendritic regions (Fig. 3C). When we analyzed the time-lags of the Ca²⁺ spikes between the soma and variable sites along a dendrite in this neuron, within the proximal dendritic regions < 80 µm from the soma, the dendritic Ca²⁺ spikes preceded the somatic Ca²⁺ spikes in 13 out of 24 sAPs (black open circles in Fig. 3D) and the reversed occurred in 11 out of 24 sAPs (red open circles in Fig. 3D). However, in the distal dendritic regions > 80 µm from the soma, no dendritic Ca²⁺ spike preceded the somatic Ca²⁺ spikes at all (gray circles in Fig. 3D). We obtained similar results from six different neurons. These data, together with the previous results, suggest that in AIS-disabled DA neurons, sAPs begin variably from one of the primary proximal dendritic regions < 80 µm from the soma and then propagate to other parts of the neuron. When the somatic Ca²⁺ spikes preceded both the proximal and distal dendritic Ca²⁺ spikes (Fig. 3C), the sAPs could probably be originated from one of the unmeasured PDCs. These results are compatible with our previous report [14] that PDCs < 80 µm from the soma are important for pacemaking and more excitable than the distal dendritic compartments. To examine this point further, in another dissociated DA neuron attached with four primary dendrites (Fig. 3E), we simultaneously measured Ca²⁺ spikes at opposite sites of two primary dendrites equally distanced from the central soma (~40.2 µm), together with the center of the soma (P1 as red dots, P2 as green dots, and the soma as black dots). In this case, there were variable combinations of Ca²⁺ spike initiation site orders among them (Fig. 3E–G). As expected, the initiation of the first Ca²⁺ spikes was observed evenly between the two contralateral primary dendrites (Fig. 3E, G), indicating that both primary dendrites could generate their own sAPs. However, most notably and importantly, in many cases (37.1% of 300 Ca²⁺ spikes in 13 cells, Fig. 3G) both the contralateral primary dendrites developed their own Ca²⁺ spikes before the soma initiated any Ca²⁺ spikes (Ca²⁺ spikes 2, 3, 4, 6, and 11 in Fig. 3E and the first trace in Fig. 3F), indicating that these two dendrites are able to generate their own sAPs independently. We also found some cases in which the somatic Ca²⁺ spikes preceded the two dendritic Ca²⁺ spikes (Fig. 3E–G), possibly due to the propagation from initiation sites of other primary dendrites elsewhere. Taken together, these data suggest that multiple primary PDCs in DA neurons drive pacemaking in DA neurons (Fig. 3H).

Figure 3. Variable initiation of sAPs from multiple PDCs in the primary dendrites in acutely dissociated, AIS-disabled DA neurons. (A) The red fluorescence image with differential interference contrast image of an acutely dissociated DA neuron loaded with Fluo 4-AM and Ca²⁺ measuring sites (S, soma; P, proximal dendrite; and D, distal dendrite). (B) After sAP, the Ca²⁺ spike started immediately from the PDC (red) and then propagated into the soma (black) and distal dendritic region (blue). Normalized traces expanded from the inset. (C) The Ca²⁺ spike started from the soma and propagated directionally to the proximal and distal dendritic regions. Normalized traces expanded from the inset. (D) Distribution of the time-lags of Ca²⁺ spike onsets between the dendritic regions and the soma. Black circles indicate Ca²⁺ propagation from the soma to the dendrite. Red circles indicate Ca²⁺ propagation from the proximal dendrite to the soma. Gray closed circles indicate time-lags in the distal dendritic region. Right, time lags of Ca²⁺ onset are represented by a bar graph (P→S: –6.06 ± 1.66 ms, 13/24, S→P: 6.75 ± 2.33 ms, 11/24). (E) Initiation order of Ca²⁺ spikes among three different parts, including two opposite proximal primary dendrites. Two individual PDCs, P1 (red closed circles) and P2 (green closed circles), alternatively generated APs, but in some cases (*), two proximal dendrites independently generated Ca²⁺ spikes earlier than the somatic one (black closed circles). (F, G) The order of generation of Ca²⁺ spikes between two opposite primary dendrites and their statistics (P1 and P2 > S; both P1 and P2 Ca²⁺ spikes preceded the soma Ca²⁺ spikes; P1 > S or P2 > S, either P1 or P2 generated the earliest Ca²⁺ spike; S, the soma Ca²⁺ spikes preceded both the P1 and P2 Ca²⁺ spikes; n = 13). (H) Schematic models of sAP generation in multipolar DA neurons. In normal DA neurons with an attached axon, sAPs always begin in the AIS and propagate into the soma and other primary dendrites. When the AIS is disabled or removed, the excitable PDCs of many primary dendrites depolarize independently, and the one that first reaches the AP threshold fires first. Values are presented as mean ± SE. sAPs, spontaneous action potentials; PDCs, proximal dendritic compartments; AIS, axon initial segment; DA, dopamine; AP, action potential.

Stochastic Ca²⁺ fluctuations in the highly-excitable PDCs and regular Ca²⁺ oscillations in the large less-excitable soma

In spontaneously and regularly firing dissociated DA neurons, the intracellular Ca²⁺ oscillations synchronized with the pacemaking cycle reflect depolarization-induced Ca²⁺ influxes [10,28]. Also, small-conductance Ca²⁺-activated K⁺ channels (SK) are abundantly expressed in DA neurons [30-33]. Therefore, the amount of Ca²⁺ influx is not only a good indicator of the depolarizing power of pacemaker activities, but also an important regulator of the pacemaking cycle [9,10]. Thus, we analyzed Ca²⁺ oscillations or spikes in regularly firing dissociated DA neurons (Fig. 4). When we measured intracellular Ca²⁺ spikes synchronized with sAPs in the soma and PDCs (Fig. 4A), the amplitude of Ca²⁺ spikes in the PDCs was higher and more variable ([Ca²⁺]_dendrites = 0.118 ± 0.007 ∆F/F₀, SD = 0.0448, n = 36) than that of the somatic Ca²⁺ spikes ([Ca²⁺]_soma = 0.051 ± 0.005 ∆F/F₀, SD = 0.0302, n = 31). Consequently, the areas under the curves (AUCs) of Ca²⁺ spikes in the PDCs were significantly larger than those in the soma (Fig. 4B), and the variations in the AUCs of Ca²⁺ spikes in the PDCs were also higher than those in the soma (CV_soma = 0.427 ± 0.012, CV_dendrites = 0.472 ± 0.016, p < 0.05, n = 21, Fig. 4C). It is also noteworthy that during pacemaking cycles, the lowest levels of Ca²⁺ oscillations or spikes in the PDCs were significantly higher than those in somatic Ca²⁺ concentrations ([Ca²⁺]_dendrites = 93.40 ± 0.94 µM, [Ca²⁺]_soma = 88.06 ± 1.04 µM, p < 0.001, n = 6, Fig. 4D). When spontaneous firing was blocked by TTX application, the Ca²⁺ level in the PDCs dropped to the same level in the soma ([Ca²⁺]_dendrites = 76.81 ± 0.41 µM, [Ca²⁺]_soma = 77.24 ± 0.88 µM, p = 0.667, n = 6), indicating that Ca²⁺ oscillations in the PDCs during pacemaking cycles are higher and more variable than those in the soma. All these data strongly indicate the higher excitability of the PDCs compared with the soma, and they are in line with our previous conclusion that multiple PDCs dominantly drive pacemaking in multipolar DA neurons. Therefore, if many primary PDCs in multipolar DA neurons generate variable and stochastically-fluctuating Ca²⁺ influxes by their own higher excitability, they could be responsible for minor firing irregularities observed in normal pacemaking cycles (Fig. 1A, 4A). Compatible with this idea, isradipine, which inhibits the Ca_v1.3 channel, the major voltage-dependent Ca²⁺ channel in DA neurons [11], decreased the ISI variance (73.2 ± 14.3%, p < 0.001, n = 7) by reducing variable Ca²⁺ influxes (Fig. 4E).

Figure 4. Stochastic Ca²⁺ fluctuations in the PDCs and regular Ca²⁺ oscillations in the soma in DA neurons. (A) Fluorescence image of a dissociated DA neuron with cytosolic Ca²⁺ measuring sites. Ca²⁺ oscillations in the soma (S-black trace) and proximal dendritic region (P-red trace) were simultaneously measured with spontaneous firing. Right, mean amplitudes (red closed circle) and distributions of individual amplitudes (black open circle) of Ca²⁺ oscillations in the soma (0.051 ± 0.005 ∆F/F₀, n = 31) and PDC (0.118 ± 0.007 ∆F/F₀, n = 36). **p < 0.001. (B) Distribution of AUCs of Ca²⁺ oscillations in the soma (black) and PDCs (red). inset: Shaded zone indicates amount of Ca²⁺ influx from a single spike. (C) Coefficients of variation of Ca²⁺ oscillation AUCs in the proximal dendrite (SD = 0.04479, n = 36) and the soma (SD = 0.03015, n = 31). *p < 0.001. (D) Basal level of spontaneous Ca²⁺ oscillations in the soma and PDCs in spontaneous firing and silenced conditions by TTX treatment. Left, Ca²⁺ oscillation traces at the soma (black) and PDCs (red). Right, statistic results. Changes of Ca²⁺ levels in the soma and PDCs in the firing (proximal = 93.40 ± 1.04 µM, soma = 88.61 ± 1.96 µM, **p < 0.001, n = 6) and silenced conditions (TTX treatment, proximal = 76.98 ± 0.68 µM, soma = 78.95 ± 0.25 µM, p = 0.67, n = 6). (E) ISI variability during tonic firing was decreased by isradipine (50 µM, 0.27 ± 0.19, **p < 0.001, n = 8). (F) Dynamic interaction model between the soma and PDCs. Highly excitable and stochastically-fluctuating PDCs are electrically coupled with the regularly oscillating inertial soma. Values are presented as mean ± SE. PDCs, proximal dendritic compartments; DA, dopamine; AUCs, areas under the curves; TTX, tetrodotoxin; ISI, interspike interval; n.s., not significant.

If the soma and PDCs are basically different in intrinsic excitabilities (Fig. 4F), the firing responses of these two different compartments to external stimuli should be different. Therefore, we stimulated the soma and PDCs using the local glutamate uncaging technique by continuously increasing the uncaging area in these two compartments, respectively (Fig. 5A). The sequential increments of the uncaging area gradually increased the maximum frequencies of the evoked firings in both the soma and PDCs. But, as expected, the slope of firing frequency increase was higher than that of the soma (r2_{prox dendrite} = 0.91, p < 0.001; r2_soma = 0.83, n = 7, p < 0.01, Fig. 5B), indicating that the PDCs were more sensitive than the soma. In addition, when we performed local GABA uncaging on the soma and PDCs in spontaneously firing DA neurons using the same uncaging area (78.4 ± 0.82 µm²) (Fig. 5C, D), the firing pauses were induced immediately as soon as PDCs were exposed to GABA, whereas the pauses were induced only after some delay in the soma (latency_soma = 2.55 ± 0.28 s, latency_dendrite = 0.61 ± 0.29 s, p < 0.05, n = 6, Fig. 5E), indicating that the PDCs respond more quickly than the soma. All these data are consistent with the previous conclusions that the primary PDCs in DA neurons are a stochastically-fluctuating, highly-excitable energetic compartment, but the large soma is a slowly and stably-oscillating inertial compartment.

Figure 5. PDCs are more excitable and sensitive than the soma in DA neurons. (A) A transmitted image of a dissociated DA neuron with the locations of glutamate uncaging sites (red). To get the same effect from glutamate uncaging, the soma needed a larger uncaging area than the proximal dendritic regions. (B) Relationship between the uncaged areas and the maximum firing frequencies in the soma (red) and PDCs (black). (C) A transmitted image of a dissociated DA neuron with the locations of GABA-uncaging areas (red). GABA-uncaging on the soma or proximal dendritic region induced firing inhibitions with different latency times (∆t). Red triangles indicate the uncaging times. The uncaging areas are the same. (D) GABA uncaging on the proximal dendritic region immediately inhibited spontaneous firing, in contrast with the effects on the soma. Dots indicate spikes of action potentials in DA neurons. (E) Comparison of the latency of GABA-induced firing inhibitions between the soma and PDCs (**p < 0.001, n = 6). Values are presented as mean ± SE. PDCs, proximal dendritic compartments; DA, dopamine.

Dynamic coupling between the multiple, stochastically-fluctuating energetic PDCs and the large, stably-oscillating inertial soma

Next we examined how the large regularly-oscillating soma is coupled with the small, stochastically-fluctuating, and highly-excitable PDCs in DA neurons. Because cellular Ca²⁺ removal depends on the SVR of compartments, the high-SVR PDCs can remove elevated Ca²⁺ faster than the low-SVR soma [9,34]. Fig. 6A illustrates the case in which Ca²⁺ removal was dramatically faster in the PDCs than in the soma when we instantly raised free Ca²⁺ levels in the soma or PDCs using local Ca²⁺ uncaging (NP-EGTA) in dissociated DA neurons (half-maximal decay-time_dendrites = 0.59 ± 0.04 s, half-maximal decay-time_soma = 2.24 ± 0.13 s, p < 0.001, n = 7, Fig. 6B, C). Despite the significant difference in Ca²⁺ removal times between these two physically distinct compartments (Fig. 6C), Ca²⁺ spikes of the PDCs in pacemaking DA neurons were well synchronized with those in the soma (Fig. 4A, D), indicating that the stochastically-fluctuating PDCs are tightly coupled with the large regularly-oscillating soma electrically, as previously reported [9]. The tight coupling of membrane potentials appears to cause changes in Ca²⁺ influxes/effluxes in the coupled PDCs, resulting in synchronized Ca²⁺ oscillations.

Figure 6. Dynamic coupling between the inertial soma and the excitable, stochastically-fluctuating PDCs. (A) In a spontaneously firing DA neuron, Ca²⁺ uncaging was performed in the soma (S) and PDCs (P). Localized Ca²⁺ rises were seen in the subtracted images, together with firing pauses and different Ca²⁺ removal kinetics between the soma and dendrite. (B) Normalized Ca²⁺ decays in the soma (black) and PDCs (red). (C) Half-maximal Ca²⁺ decay times in the soma (2.02 ± 0.15 s, n = 3) and PDCs (0.79 ± 0.003 s, n = 3). **p < 0.001. (D) Fluorescence-overlapped image of a DA neuron and a magnified dendrite image with cytosolic Ca²⁺ measuring sites (yellow boxes). Local Ca²⁺ oscillations are shown at the marked sites of a dendrite. (E) The CV of local dendritic Ca²⁺ oscillations increased as a function of distance from the soma. (F) Correlation coefficient of the peaks of Ca²⁺ oscillations (R² = 0.67, Pearsonʼs product moment correlation coefficient, n = 5). (G) Left, A DA neuron loaded with Fluo 4-AM and Ca²⁺ measuring sites. Ca²⁺ measuring site: black circle in the soma (S), red circle in the proximal dendrite (P). Right, raw traces of Ca²⁺ oscillations and averaged spikes of Ca²⁺ oscillations in the soma and PDCs. (H) Normalized Ca²⁺ decay curve of soma (black) and PDCs (red) during spontaneous firing. (I) Half-maximal Ca²⁺ decay times in the soma (0.11 ± 0.01 s, n = 9) and PDCs (0.09 ± 0.01 s, n = 9) during spontaneous firing. Values are presented as mean ± SE. PDCs, proximal dendritic compartments; DA, dopamine; CV, coefficients of variance; n.s., not significant.

Therefore, we examined how strongly the PDCs are coupled with the soma in regularly firing DA neurons by observing the correlations in Ca²⁺ oscillations between them. When we analyzed the peaks of Ca²⁺ oscillations between the soma and different sites of a dendrite (Fig. 6D), the coefficients of variance (CV) increased according to the distance from the soma (Fig. 6E), and the correlation coefficients decreased (Fig. 6F). Thus, the coupling of Ca²⁺ oscillations between the regularly oscillating soma and irregularly fluctuating PDCs is distance-dependent. When we examined Ca²⁺ removal in the soma and PDCs during normal pacemaking cycles (Fig. 6G), we found no significant difference between them in the half-maximal decay times of Ca²⁺ spikes (dendrites = 0.09 ± 0.01 s, soma = 0.11 ± 0.01 s, n = 9, p = 0.53, Fig. 6H, I), despite the different amplitudes in their Ca²⁺ spikes (amplitude_soma = 0.12 ± 0.009 ∆F/F₀, amplitude_{dendritic compartment} = 0.24 ± 0.011 ∆F/F₀, p < 0.05, n = 9). It may suggest that the tight electrical coupling of PDCs with the large soma might restrict the vulnerable and fluctuating Ca²⁺ oscillations of PDCs, thereby subjugating dynamic dendritic Ca²⁺ changes during slow pacemaking cycles. Taken together, all of these data strongly suggest that the dynamic interaction between the large, stably-oscillating, inertial soma and the multiple, stochastically-fluctuating, energetic PDCs creates the slow pacemaker activity of DA neurons.

Dissociation of glutamate-evoked two distinct firing responses within the PDCs of a DA neuron

Since the dynamic interaction between two functionally different compartments produces regular spontaneous firing, this interaction model may underlie or affect other types of firing, such as evoked firing, other than slow pacemaking. Therefore, we next examined how DA neurons respond to fast external stimuli. Midbrain DA neurons can produce burst firing in respond to sufficient excitatory glutamatergic synaptic inputs [17,35]. It is well known that glutamatergic action on DA neurons is dualistic [36,37]: glutamate can activate several inotropic glutamate receptors that depolarize the membrane potential, generating burst discharges (fast excitatory components), but glutamate can also evoke a following Ca²⁺ transient due to the Ca²⁺ influxes and Ca²⁺ release from the endoplasmic reticulum store, consequentially suppressing spontaneous firing via activation of SK channels (slow inhibitory components) (Fig. 7A) [28,37,38]. Thus, a brief strong stimulation with glutamate mostly evokes two distinct firing responses in DA neurons: the initial burst discharge and subsequent postfiring pause (Fig. 7A). To see how glutamate works in the dynamic interaction model, we stimulated a different part of a dendrite in a dissociated DA neuron using the local glutamate uncaging technique (the numbered black circles in Fig. 7B). Usually, responsibility of DA neurons to local glutamate uncaging was strong in the proximal dendritic region and disappeared in the distal dendritic regions [14,26]. Accordingly, within the PDCs the typical burst-pause firing pattern was clearly observed (Fig. 7C). Interestingly, we found that the burst firing (the excitatory component, marked as red in Fig. 7C, D) was well preserved within the PDCs, but that the burst firing abruptly declined around 80 µm from the soma, and no more responses in the distal dendritic regions, indicating the higher excitability of the PDCs than distal dendritic compartments. This result is compatible with our previous report [14,26] that PDCs < 80 µm from the soma in rat DA neurons are more excitable than the distal dendritic compartments without a significant difference in glutamate receptor expressions. However, the postfiring pause (the inhibitory component, marked as blue in Fig. 7C, D) decreased exponentially with the distance from the soma in the same PDCs. While the excitatory response was well-preserved within the PDCs, the inhibitory response in the same region was exponentially decaying like a passive electrotonic dissipation of local membrane potentials. There was a clear dissociation between the excitatory and inhibitory responses to glutamate.

Figure 7. Dissociation between the excitatory and inhibitory responses of spontaneous firing to glutamate in the PDCs of DA neurons. (A) Schematic drawing of glutamate signals in the DA neuron. Glutamate evoked two distinct changes in spontaneous firing and cytosolic Ca²⁺ levels. (B, C) In a spontaneously firing dissociated DA neuron, local glutamate uncaging was serially performed along a dendrite. Firing traces are presented according to the numbers of the marked photolysis sites and their order. Glutamate induced-firing enhancements are marked as red areas, and firing inhibitions are marked as blue areas. Bottom, expansion of firing traces in the red areas. (D) Glutamate-induced excitatory (maximum frequency, red) and inhibitory firing responses (duration of firing pause, blue) are plotted against the distance from the soma (n = 8). PDCs, proximal dendritic compartments; DA, dopamine.

Dualistic firing responses to glutamate: the PDCs responsible for the excitatory response and the soma responsible for the inhibitory response

To investigate why the inhibitory response decreases with the distance from the soma within the PDCs, we performed local stimulation experiments on the soma and PDCs. Because Ca²⁺ is the main cause for glutamate-induced firing pauses [36,37], local glutamate uncaging on different sites of a dendrite might evoke different levels of Ca²⁺ rise. But it was not the case. Although the same local glutamate uncaging on different sites of a dendrite evoked similar Ca²⁺ spikes (Fig. 8A), the duration of the postfiring pause decreased with the distance from the soma (Fig. 8A, C), whereas the excitatory responses of firing to glutamate (the maximum frequencies of the evoked firings) were preserved within PDCs (Fig. 8A, B). When SK channels were suppressed by a specific blocker apamin (100 µM), or intracellular Ca²⁺ was buffered with a high concentration of BAPTA (20 µM), the postfiring pause was completely abolished (control = 2.44 ± 0.72 s, n = 6; apamin = 0.57 ± 0.03 s, n = 6, p < 0.05; BAPTA = 0.57 ± 0.01 s, n = 3, p < 0.05, Fig. 8D), indicating that the firing pauses are entirely mediated by intracellular Ca²⁺ rises. However, the excitatory response was not significantly affected (control = 13.01 ± 0.97 Hz, n = 9; apamin = 12.88 ± 1.55 Hz, n = 6, p = 0.997; BAPTA = 15.03 ± 2.29 Hz, n = 3, p = 0.649, Fig. 8E). Therefore, to understand why local Ca²⁺ rises at a different part of PDCs differently suppress spontaneous firing, we directly performed local Ca²⁺ uncaging (NP-EGTA) in a dendrite (Fig. 9A-C). When local Ca²⁺ spikes were serially evoked along a dendrite (Fig. 9A), the duration of the postfiring pause decreased exponentially with the distance from the soma (Fig. 9A, C), which is very similar to the result with glutamate uncaging (Fig. 7C-D). Interestingly, according to the distance from the soma we found an exponential decrease of the hyperpolarization of the soma (Fig. 9A, B). In addition, when higher Ca²⁺ spikes were evoked with stronger Ca²⁺ uncaging on the same site of a dendrite, stronger hyperpolarization of the soma occurred, together with correspondingly longer suppression of spontaneous firing (Fig. 9D). The magnitudes of the Ca²⁺ rises correlated with the degree of hyperpolarization in the soma (r = 0.76, n = 5, Fig. 9E). All these data strongly suggest that the extent of hyperpolarization of the central large soma determines the duration of the postfiring pause. Thus, the closer the dendritic stimulation site is to the soma, the larger its impact on the soma (Fig. 9A–C). Unless the large, centrally-connected soma is affected, stimulation of a small part of the dendritic regions cannot suppress the spontaneous firing of multipolar DA neurons (Fig. 9A). Therefore, we can now conclude that the key factor determining the inhibitory responses to glutamate in multipolar DA neurons is the hyperpolarization of the large inertial soma.

Figure 8. Glutamate induced-firing inhibition is mediated by intracellular Ca²⁺ rise. (A) Simultaneous measurements of Ca²⁺ rises and firing inhibition upon glutamate uncaging in a dissociated dopamine neuron. Glutamate uncaging sites and Ca²⁺ measuring sites are marked as white circles (ø < 5 µM). Red triangles and blue boxes in the lower traces indicate uncaging points and firing pauses, respectively. (B) Local glutamate uncaging at different sites on proximal dendrites evoked Ca²⁺ spikes of similar amplitudes, and the evoked maximum firing frequencies did not differ significantly within the proximal dendritic regions (n = 7, *p < 0.05). (C) Postfiring pauses in spontaneous firing by glutamate uncaging in the proximal dendritic region decreased exponentially as a function of distance from the soma (n = 7). (D) Glutamate-induced firing inhibitions were blocked by apamin application (100 µM) and by dialysis with BAPTA (20 mM) using patch pipettes. The upper traces are firing changes (black), and the lower traces are dendritic Ca²⁺ changes (red). (E) Statistics from (D): glutamate uncaging, 13.01 ± 0.97 Hz, n = 9; apamin, 12.88 ± 1.55 Hz, n = 6; BAPTA, 15.03 ± 2.29 Hz, n = 3. Dendritic Ca²⁺ increases were completely inhibited by BAPTA dialysis. Values are presented as mean ± SE. *p < 0.05.

Figure 9. The soma, responsible for inhibitory responses of spontaneous firing to glutamate, works together with the PDCs, to produce the typical burst-pause firing patterns in DA neurons. (A) A dissociated DA neuron was loaded with NP-EGTA and Fluo 4-AM. Serial photolysis of a small area along a dendrite led to localized cytosolic Ca²⁺ rises. Serial fluorescence images from the white box are magnified and overlapped. Dendritic Ca²⁺ changes in the uncaging regions are presented according to the distance from the soma, with the corresponding firing patterns recorded in the soma. Photolysis points are indicated by blue dotted lines. (B) Relative changes in the normalized hyperpolarization of membrane potential according to the distance from the soma. (C) The duration of postfiring pauses is plotted versus distance from the soma (n = 4). (D, E) When Ca²⁺ uncaging on the same site of a dendrite was repeated with different degrees, higher [Ca²⁺] rises caused longer pauses in spontaneous firing and stronger hyperpolarization of the membrane potential. Right, the relationship between the hyperpolarization of membrane potential and dendrite Ca²⁺ changes (n = 5, r = 0.76). (F) Strong glutamate uncaging in the local dendritic region induced different types of Ca²⁺ increases between the stimulated dendritic region (green) and the soma (blue). Spontaneous firing and evoked bursts were measured with a patch pipette at the soma (gray trace). (G) The recoveries of the elevated Ca²⁺ levels in the soma and stimulated dendritic region when the first spontaneous firing reappeared after glutamate uncaging (soma = 60.07 ± 7.35%, proximal = 2.77 ± 1.07%, n = 7). (H) Functional somatodendritic organization of the pacemaking and firing system in multipolar DA neurons. Highly excitable PDCs not only drive pacemaking but also serve as an integration site for evoked firings. PDCs, proximal dendritic compartments; DA, dopamine.

Generation of evoked firing patterns by the complementary interaction between the inertial soma and the excitable PDCs

It seems now clear that the functionally distinct soma and PDCs are independently responsible for the two different components of burst-pause firing to sudden glutamate stimulation in DA neurons. In actual conditions, if the summed effects of glutamatergic synaptic activities throughout all the dendrites are sufficient to affect one of the highly-excitable PDCs, high-frequency burst firing would occur in that PDC and propagate rapidly into the other somatodendritic trees, causing sequential Ca²⁺ elevations in the propagated areas and then activating regional SK channels. If the large inertial soma is sufficiently hyperpolarized, the postfiring pause would then occur. To confirm this hypothesis, we strongly stimulated one region of a dendrite in a spontaneously firing DA neuron and observed both the spontaneous firing patterns and Ca²⁺ changes in the soma and stimulated dendritic region simultaneously. Upon glutamate uncaging (the green rectangular area in Fig. 9F), the Ca²⁺ level (green line) went up abruptly in the uncaged dendritic region with an immediate burst discharge. However, somatic Ca²⁺ (blue line) increased relatively slowly, together with a mirrored change in the hyperpolarization of the soma. Consequently, after the immediate burst firing (the excitatory component), the postfiring pause (the inhibitory component) followed with a slow somatic Ca²⁺ rise. The dendritic Ca²⁺ level, which increased immediately with the glutamate uncaging, was restored to its previous level more quickly than the somatic Ca²⁺ level. Notably, the reappearance of spontaneous firing depended on the recovery of the somatic Ca²⁺ levels, but not dendritic Ca²⁺ levels. When the somatic Ca²⁺ levels dropped by 60.07 ± 7.35% (n = 7), spontaneous firing reappeared. In that condition, the increased dendritic Ca²⁺ levels had already returned to their unstimulated level (Fig. 9G). Thus, it is clear that the large inertial soma is responsible for the inhibitory components of firing to glutamate, whereas the PDCs are responsible for the excitatory firing responses to glutamate. This complementary interaction between the two compartments appears to determine the final firing output patterns in multipolar pacemaker DA neurons (Fig. 9H).

In this study, we explore the importance of the PDCs in the coordinated generation of pacemaker rhythm and evoked firing in multipolar midbrain DA neurons. In DA neurons, sAPs consistently initiate from the ABD and propagate to the soma and other nABDs without significant attenuation [7,8]. However, after AIS removal or in AIS-disabled dissociated DA neurons, sAPs were variably initiated from multiple primary PDCs, revealing the presence of endogenous excitable sites and/or primary pacemaker loci scattered in multiple primary dendrites. Observations of sAP initiation sites in AIS-disabled DA neurons using Ca²⁺ measurements, reveal that contralateral PDCs are able to independently generate their own sAPs without somatic trans-propagation, underscoring the role of multiple PDCs as primary pacemaker loci in DA neurons. Consistent with these findings, the soma and PDCs display distinct Ca²⁺ oscillation patterns, functioning as two distinct compartments. Low-amplitude, regular Ca²⁺ oscillations occur in the large soma compartment with lower SVR, while small PDC compartments with higher SVR exhibit high-amplitude, stochastic Ca²⁺ oscillations. Additionally, despite the higher Ca²⁺ removal rate in dendritic compartments compared to the soma, the lowest dendritic Ca²⁺ concentration of PDCs during pacemaking cycles remains consistently higher than the somatic Ca²⁺ concentration. These results suggest that primary PDCs are a highly excitable and stochastically fluctuating compartment, while the centrally connected large soma behaves as an inertial and regularly oscillating compartment. It is widely acknowledged that the sinoatrial node pacemaker cells' sAP interval is neither strictly stationary nor entirely random, regulated by stochastic mechanisms within ion channels and complex Ca²⁺ toolkits [39-41]. In computational models, stochasticity depends on the model size, reducing quickly with increasing system size but persisting even in large models [42]. In the case of DA neurons, the higher irregularity of Ca²⁺ oscillations in PDCs compared to the soma can be attributed partly to the smaller size of PDCs. However, as reported previously [14], the higher intrinsic excitabilities of PDCs in DA neurons compared to the soma seem to contribute to the increased PDCs' stochasticity.

Recently, two pacemaker channels, TRPC3 and NALCN, were identified in DA neurons [25], with NALCN channels highly expressed in PDCs [26]. This provides molecular evidence for the greater excitability of PDCs compared to the soma in DA neurons. Moreover, simulation models of DA neurons [9,14] suggest that the frequency of Ca²⁺ oscillations theoretically depends on compartment size. Therefore, interpreting the functional role of PDCs requires consideration of both the various physical properties of compartments, such as SVR, and the higher intrinsic excitability of PDCs in DA neurons.

Despite substantial differences in the physical and functional attributes outlined earlier for the soma and PDCs, these two dynamic compartments seemingly behave as a single compartment during pacemaking cycles, as shown in the synchronized spontaneous firing and Ca²⁺ oscillations between them. It is for this reason that tight electrical coupling throughout the somatodendritic architecture was previously proposed [9] and has become a basis for many simulation studies [15,34,43,44]. Our experimental results also support the strong electrical coupling between the soma and dendrites. Nevertheless, the entire dendritic compartment does not uniformly contribute to pacemaking. Instead, the specialized PDCs located within 80 μm from the soma, possessing higher excitability than other neuron regions, could be important as the primary pacemaking contributors [14,26]. Hence, we can infer that the pacemaker activities in DA neurons result from the interplay between two principal dynamic compartments: the highly excitable and stochastically fluctuating PDCs and the stable and regularly oscillating soma. Consequently, in an electrically coupled state, the large, centrally-connected, inertial soma counteracts and balances the excitable and vulnerable activities of the PDCs.

For midbrain DA neurons with a multipolar structure to function as a slow pacemaker, the heightened intrinsic excitabilities of multiple PDCs, arising unavoidably from physical constraints (i.e., a small compartment with a high SVR) and the localization of NALCN channels [26], must be counterbalanced by the reduced excitability of the large soma. This might lead the soma in DA neurons to become significantly less excitable and more inertial, potentially resulting in the axon not directly arising from the soma, deviating from the pattern observed in many typical neurons [45,46]. Utilizing the dynamic-clamp technique, Tucker et al. [47] also noted that the somatodendritic distribution of Na⁺ channels plays a pivotal role in determining repetitive spiking frequency. They observed that selectively reducing Na⁺ channel activity in the soma was adequate to decrease pacemaker frequency and enhance susceptibility to depolarization block, underscoring the significance of differing excitabilities between the soma and dendrites in the context of slow pacemaking. Despite the apparent synchronization of spontaneous firing and Ca²⁺ spikes between the soma and dendrites during slow pacemaking cycles [9], the electrical coupling of each dendritic compartment with the soma appears imperfect and distance-dependent. This is likely due to the very low axial resistance of dendrites and their limited Ca²⁺ diffusion [48,49]. Our observed firing responses to normal Ca²⁺ spikes, local stimuli or sudden perturbations of the soma or dendritic compartments, through stimulation or inhibition using glutamate or GABA uncaging, align with our model depicting two distinct compartments and their relationships. The excitable PDCs exhibit a sensitive and immediate response to excitatory or inhibitory stimuli, while the inertial soma responds less sensitively and with a delay. Consequently, in this dynamic interaction model, both dependencies and independencies between these compartments manifest in physiological conditions, depending on the time scale.

The slow time scale reveals dependencies, as observed in synchronized Ca²⁺ dynamics and spontaneous firings between compartments during the pacemaking cycle. Conversely, fast time scale independencies are evident in evoked firing responses to glutamate and GABA uncaging or sudden changes in Ca²⁺. Given the physical and functional disparities between the soma and PDCs, the pacemaking activities of DA neurons defy description as static coupling based on surface areas with specific physical parameters. Instead, they represent the dynamic interplay of the stably-oscillating inertial soma and stochastically-fluctuating energetic PDCs. This interaction model holds implications beyond pacemaker mechanisms, extending to diverse firing phenomena such as ISI variability and patterns in response to various spatiotemporal stimuli [16,19,50].

Using this dynamic interaction model, we can now interpret the diverse firing patterns evoked by local stimulations or perturbations, such as glutamate, GABA, and Ca²⁺ uncaging, and a wide range of firing diversities. In regularly firing DA neurons, glutamate typically induces two distinct firing responses: initial burst firing and a subsequent postfiring pause [36,37,51]. Notably, these firing patterns are not uniform across the entire dendrite but show a dissociation between excitatory and inhibitory responses within proximal dendritic regions < 80 μm from the soma, a key region for pacemaking [14]. The burst-pause patterns can now be explained by the distinct properties of the large, less-excitable, inertial soma and the highly-excitable PDCs. Quick-responding PDCs contribute to excitatory responses to glutamate, while the large soma is responsible for inhibitory responses (due to slow recovery upon hyperpolarization). Stronger inhibitory action occurs as the glutamate uncaging site approaches the soma, leading to more substantial somatic hyperpolarization. The higher intrinsic excitabilities of proximal excitatory dendritic regions within 80 μm from the soma, together with NALCN expression [26], suggest that PDCs act as integration sites for diverse synaptic activities across the entire dendritic arbor. In multipolar DA neurons, PDCs, as highly excitable elements, play a crucial role in both pacemaking and dendritic integration, while the soma, as a slowly responding element, counteracts and balances their energetic actions. This novel dynamic interaction model for DA neurons seems to serve as a common basis for generating both pacemaking and evoked firing patterns appropriately.

None.

This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI13C1793) and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2017R1A2B3005656 and 2022R1A2C2009159).

The authors declare no conflicts of interest.