Chemical transmission between dopaminergic neuron pairs

Abstract

Midbrain dopaminergic (DAergic) neurons play a major regulatory role in in goal-directed behavior and reinforcement learning. DAergic neuron activity, and therefore spatiotemporal properties of dopamine release, precisely encodes reward signals. Neuronal activity is shaped both by external afferences and local interactions (chemical and electrical transmissions). Numerous hints suggest the existence of chemical interactions between DAergic neurons, but direct evidence and characterization are still lacking. Here, we show, using dual patch-clamp recordings in rat brain slices, a widespread bidirectional chemical transmission between DAergic neuron pairs. Hyperpolarizing postsynaptic potentials were partially mediated by D2-like receptors, and entirely resulted from the inhibition of the hyperpolarization-activated depolarizing current (Ih). These results constitute the first evidence in paired recordings of a chemical transmission relying on conductance decrease in mammals. In addition, we show that chemical transmission and electrical synapses frequently coexist within the same neuron pair and dynamically interact to shape DAergic neuron activity.

The substantia nigra pars compacta (SNc) is the main modulatory nucleus of basal ganglia, a network of subcortical nuclei involved in procedural learning and habit formation (1, 2). Dopaminergic (DAergic) neurons composing SNc mainly project to the dorsal striatum, the major input nucleus of basal ganglia. In striatum, dopamine (DA) potently modulates the processing of corticostriatal information (3–5), contributing to the formation of sensory-motor linkages allowing selection of adapted motor behavior in response to environmental cues.

Nigrostriatal DAergic neurons display two modes of discharge: a tonic firing associated with a low but constant DA release supporting a permanent tune-up of the striatal network, and a phasic firing leading to peaks of DA release, coding for a predictive reward value and attention to salient environmental events (6–9). These modes of activity are controlled by intrinsic electrophysiological properties, external inputs, and local interactions (chemical and electrical synapses) between DAergic neurons. SNc DAergic neurons are connected by gap junctions (10), and display electrical coupling able to control their spontaneous tonic activity (11). Yet, electrical coupling might not be their sole mode of communication. Numerous hints strongly support the existence of chemical transmission. DAergic neurons are known to release DA from dendrites (12), bear autoreceptors, and display a characteristic hyperpolarization in response to DA (13, 14) or DA agonist application (15, 16), and to electrical stimulation of the SNc (17) or the subthalamus (18). Moreover, ultrastructural studies revealed the presence of dendrodendritic synaptic contacts between DAergic cells in the SNc (19, 20). However, a direct demonstration of neuron-to-neuron communication allowing the characterization of the synaptic properties at the single cell level was still lacking.

Results

Pairs of DAergic neurons (n = 161 pairs) were recorded in the SNc from coronal rat brain slices maintained at 34°C. DAergic neurons were identified by their typical electrophysiological characteristics (16, 21) (Fig. 1A) including a prominent sag [hyperpolarization-induced depolarizing current (Ih)] (−20.5 ± 3.7 mV for a −90-pA hyperpolarizing stimulus), a depolarized action potential (AP) threshold (−37.0 ± 0.7 mV), a long AP duration (4.2 ± 0.1 ms), a large after-hyperpolarization amplitude (−23.0 ± 1.3 mV), and a slow regular spontaneous spiking activity (2.5 ± 0.3Hz) (n = 25). In a subset of experiments, recorded neurons (n = 28) were filled with biocytin. Immunohistochemical revelation of biocytin and tyrosine hydroxylase (TH), the DA synthesis enzyme, confirmed in all cases the DAergic phenotype of recorded neurons (Fig. 1B). Biocytin staining of recorded neuron pairs showed that the proximal part of their dendritic fields always overlapped and displayed putative synaptic contacts.

Fig. 1.

Chemical transmission in identified DAergic neuron pairs. (A) Electrophysiological identification of DAergic neurons. Typical responses of a DAergic neuron to current injections (−90 to +60 pA relative to holding current). (B) Immunohistochemical identification of DAergic neurons. The identities of recorded cells were confirmed by double immunoreactivity for tyrosine hydroxylase (red) and biocytin (green). (C) A 250 pA current injection (I1) evokes an AP train in cell1 (V1), whereas cell2 membrane potential is maintained at −80 mV. Time-locked averaging (using the peak of cell1 AP as reference) of 600 cell1 APs and corresponding 600 cell2 responses across six pairs (100 spike-response from each cell pair) reveals a hyperpolarizing response in cell2.

Chemical Transmission Between Dopaminergic Neuron Pairs.

Local interactions between DAergic neurons were investigated by using dual whole-cell recordings, allowing not only the control of the receiving neuron but also a specific stimulation of a single DAergic neuron. When AP trains were triggered in a DAergic neuron (cell1), averaging of cell2 membrane potential (time-locked on the peak of each cell1 APs, n = 600 spikes in 6 neuron pairs) revealed that cell1 APs evoked an hyperpolarization in a 20 μV range (Fig. 1C). Such spike-evoked hyperpolarization (SEH) did not result from electrical coupling. Indeed, SEHs were found in electrically and nonelectrically coupled DAergic neuron pairs. Furthermore, the latency [17 ms; supporting information (SI) Fig. 6A] of the cell2 SEH was incompatible with the ultrafast kinetics of electrical transmission (22). Accordingly, SEHs sign the functional existence of an hyperpolarizing chemical transmission between DAergic neurons. The subsequent characterization of chemical transmission was performed exclusively on nonelectrically coupled DAergic neuron pairs.

Intracellular K⁺ was replaced by an impermeant cation (Cs⁺) to obtain larger unitary SEHs by enhancing the spike duration. In this condition, the I–V relationship of DAergic neurons was not significantly modified (SI Fig. 7A), and AP duration was increased between 25 and 160 ms (Fig. 2A). These large APs systematically evoked a SEH in the receiving neuron (Fig. 2A). This transmission was observed in almost all pairs of neighboring DAergic neurons (97%, n = 116), was nearly always bidirectional (96%, n = 72 pairs tested in both directions) (Fig. 2B) and SEH amplitudes were stable overtime (SI Fig. 8A).

Fig. 2.

Chemical transmission between DAergic neuron pairs recorded with CsGlu intracellular solution. (A) SEHs (V2) evoked by single long-duration APs (V1). The black line represents the average of 20 consecutive SEH raw traces (gray lines). Histograms show the distributions of the AP durations, SEH amplitudes, and SEH latencies in this cell pair. (B) Chemical transmission is bidirectional: Within the same pair, an AP evoked in cell1 induces a SEH in cell2, and, reciprocally, an AP in cell2 induces a SEH in cell1 (raw traces). (C) SEH amplitude increases with AP duration. Normalized SEH amplitude was plotted as a function of the AP duration. Different markers indicate different cell pairs. For each cell pair, the SEH amplitude was normalized by its predicted value (obtained by linear regression) at 87.5 ms. Linear fit of the four cell pair values after normalization is indicated.

The SEHs were strictly dependent on the presence of APs in the stimulated cell, because subthreshold depolarizations of cell1 never evoked a SEH in cell2 (SI Fig. 8B). When single APs were evoked in the presynaptic cell (AP duration: 82.6 ± 4.0 ms, range: 27.5–155.5 ms, n = 69 measurements, n = 4 cells), SEH amplitude was 4.5 ± 0.2 mV (range: 0.8–7.3 mV), with a latency of 20.4 ± 1.0 ms (range: 6.2–44.7 ms, SD: 7.2 ms). The SEH amplitude displayed a significant positive correlation with the AP duration (n = 69 measurements on n = 4 pairs, r = 0.935, P < 0.0001) (Fig. 2C), whereas SEH latency was not affected by the AP duration (n = 60 measurements on n = 3 pairs, r = 0.3377, P = 0.0083). This suggests that wider APs evoke a more important neurotransmitter release and consequently larger SEHs. Successive APs in cell1 induced the summation of cell2 SEHs but the responses evoked by a train of APs in cell1 reached a plateau of fixed amplitude value (SI Fig. 8B), specific for each cell pair, suggesting a saturation phenomenon.

Considering the latency of the SEH, we investigated whether chemical transmission between DAergic neurons was mono- or polysynaptic. Should the latter be true, bath application of tetrodotoxin (TTX) would block the release of the neurotransmitter by an intermediate neuron (with little impact on Cs⁺-enhanced APs in the stimulated cell) and therefore abolish the SEHs. Conversely, we observed reliable SEHs under TTX application (n = 3) (SI Fig. 9), indicating a monosynaptic chemical connection between DAergic neurons.

Chemical Transmission Partially Involves Dopamine.

Chemical transmission was calcium-dependent. Indeed, blockade of voltage-dependent calcium channels by external cadmium application (300 μM) significantly inhibited SEH amplitude by −63 ± 9% (n = 3 pairs) (Fig. 3). This suggests that chemical transmission relies on a vesicular release of neurotransmitter. We have investigated the involvement of DA in the chemical transmission between DAergic neurons. D2-like receptors are expressed by DAergic neuron somata and dendrites (23). Blockade of D2-like receptors by raclopride (1 μM) or sulpiride (20–30 μM) induced a significant decrease of the evoked SEHs (mean decrease was −40% with raclopride, and −27% with sulpiride, n = 5) (Fig. 3). Therefore, the SEHs resulted at least partially from a DA release acting on D2-like receptors. Involvement of other neurotransmitters releasable by DAergic neurons (24) was tested. No significant inhibition of SEH was observed after GABA_A, type-1 cannabinoid, type-2 serotoninergic and type-1 metabotropic glutamatergic receptor antagonist applications (bicuculline, 20 μM, n = 2; AM251, 1 μM, n = 3; ketanserin, 20 μM, n = 4; LY314495, 100 μM, n = 2).

Fig. 3.

Chemical transmission between DAergic neuron pairs involves DA. A partial inhibition (gray traces) of the SEH (average of 10 traces) was observed in presence of cadmium, a voltage-sensitive calcium channel blocker (300 μM) (Left) or raclopride, a D2 antagonist (1 μM) (Right), when compared with control (black traces).

Decrease of Membrane Conductance During Chemical Transmission.

Classically, chemical transmission acts through the activation of membrane conductances. Here, we report the first evidence in paired recordings of a decrease of membrane conductance during chemical transmission, in mammals. Indeed, when the R_i of the receiving cell was monitored before and during SEHs, a significant decrease (−17 ± 2%) of membrane conductance was observed in 70% of DAergic neuron pairs (n = 10) (Fig. 4A). This observation suggests that chemical transmission between DAergic neurons involves closure of already opened channels.

Fig. 4.

SEHs are mediated by temporary Ih inhibition. (A) Decrease of membrane conductance during chemical transmission. (A1) Cell2 R_i was measured by injection of hyperpolarizing currents (−10 pA, 100 ms), before and during evoked-spiking discharge in cell1. Superimposition of traces before (black trace) and during (gray trace) SEH are shown below at a higher magnification. (A2) R_i variations before and during SEH in 10 neuron pairs. * indicates significant (P < 0.05) R_i increases (n = 50 measurements per cell pair). (B) Voltage-dependency of the SEH. (Left) averaged SEHs (10 traces) for different cell2 membrane potentials. (Right) normalized SEH amplitude as a function of cell2 membrane potential in six cell pairs (symbols stand for different pairs). The dashed line indicates linear regression performed on the non-null points. (C) ZD7288, Ih voltage-dependent inhibitor, abolished the SEH. After 5 min of ZD7288 50 μM, SEH amplitude was unchanged. Once cell2 had been depolarized at −60 mV, ZD7288 became effective and abolished the SEH. (D) ZD7288 effect on DAergic neuron I–V relationships, monitored in the same cells as in C, shows that SEH inhibition is concomitant with the suppression of the Ih-induced sag. ZD7288 efficiently inhibits Ih because the sag was abolished after ZD7288. Accordingly, R_i is significantly increased specifically at potentials in the activation range of Ih: 129 ± 26 in control versus 415 ± 50MΩ with ZD7288 for −110 pA injected current, n = 7 neurons (while Ri is similar in control and ZD7288 for the −10-pA injected current). Raw traces current injections: −150, −120, −90, −60, −40, −20, and 0 pA, from a holding potential of −60 mV. APs are truncated at −40 mV. I/V relationships (n = 7 DAergic neurons) measured at sag peak (open markers) and steady state (closed markers).

Chemical Transmission Is Mediated by the Temporary Inhibition of Ih.

To determine the nature of the ionic flux underlying the SEH, the voltage dependency of the SEH was investigated (Fig. 4B). The SEH amplitude increased with the hyperpolarization of the postsynaptic DAergic neuron, which is opposite to the expected effect for a classic inhibitory postsynaptic potential. Furthermore, beyond the cancellation of the SEH at −52 mV, no reversion was observed (explored up to +35 mV). This atypical I–V relationship suggested that SEHs were not due to the activation of a hyperpolarizing membrane current, but rather to the temporary closure of already opened channels maintaining a depolarization, accordingly with the observed membrane conductance decrease (Fig. 4A). Interestingly, DAergic neurons display a potent depolarizing current activated by hyperpolarization (Ih) below −45 mV (25), constitutively active at DAergic neuron resting membrane potential and responsible for the typical sag in membrane potential response to hyperpolarizing current pulses observed in these neurons. Similarly to the SEH (Fig. 4B), Ih reversion is not observable in the conditions of current-clamp recordings, because Ih reversal potential [approximately −35 mV (26)] is above the Ih activation range, and Ih amplitude increases with hyperpolarization (25). This suggests that SEHs could be mediated by a temporary inhibition of Ih. This hypothesis was tested by analyzing the effect of ZD7288, a specific voltage-dependent Ih inhibitor (27). Accordingly in our experiments, ZD7288 blocked chemical transmission between DAergic neuron pairs in a voltage-dependent manner. When the postsynaptic neuron (cell2) was maintained at a hyperpolarized potential (−80 mV), the SEH amplitude was unchanged by ZD7288 (Fig. 4 C and D). In contrast, when cell2 was briefly held at −60 mV and then hyperpolarized back at −80 mV (still under ZD7288), AP trains in cell1 never induced any SEH in cell2 (n = 4 pairs) (Fig. 4 C and D). This demonstrates clearly that the observed chemical transmission between DAergic neurons relies on a temporary inhibition of Ih.

The hyperpolarizing effect of DA is classically described as mediated through an activation of potassic conductances (13, 14, 17, 18). However, SEH I–V relationships were not significantly different whether receiving neurons were recorded by using CsGlu or KGlu intracellular solutions (SI Fig. 7B). We applied exogenous DA (30 μM) while recording DAergic neurons in KGlu intracellular solution (SI Fig. 10). In all neurons tested, bath application of DA induced an outward current (58 ± 15 pA, n = 5 neurons held at −60 mV) that was significantly inhibited (8 ± 3 pA, n = 5; −86%, P = 0.03 when compared with control) in presence of D2 antagonist sulpiride (20 μM). A significant decrease in the spontaneous firing activity (−73 ± 18%, n = 2), and in the Ri (−19 ± 5%, n = 3) was also observed upon exogenous DA application. This indicates that the differences between our results and previous studies (13, 14, 17, 18) is due to the stimulation procedure (stimulation of a single DAergic neuron versus exogenous DA application or DAergic neuron population stimulation).

Coexistence of Electrical and Chemical Transmissions.

In accordance with the pioneering observation of dye-coupling between DAergic neurons (10), we have reported the existence of functional electrical synapses between subsets of DAergic neurons (11). Their strong low-pass filtering properties allow an efficient transmission of slow kinetic events that modulates spontaneous postsynaptic activity. Here, we observed the coexistence of chemical and electrical synapses in 27% of recorded DAergic neuron pairs (n = 69) (Fig. 5). Hyperpolarizing pulses in cell1 induced a hyperpolarization of cell2 membrane potential (Fig. 5B), demonstrating the existence of electrical coupling between these cells. Similarly, subthreshold sinusoidal stimulations of different frequencies in cell1 induced corresponding cell2 sinusoidal oscillations (assessed by fast Fourier transform analysis) (Fig. 5B). The mean value of the coupling coefficient was 2.4 ± 0.1%, corresponding to a junctional conductance value of 49 ± 4pS (n = 19). When a suprathreshold depolarization was applied to cell1 (Fig. 5B), a biphasic response was observed in cell2. The initial depolarization (arrow), occurring before cell1 AP, was due to the electrotonic transmission of cell1 depolarization. The following hyperpolarization corresponded to the SEH mediated by chemical transmission. Indeed, given that APs recorded in CsGlu intracellular solution did not display AHPs, the hyperpolarization observed in cell2 could not result from the electrotonic transmission of the AHP. Therefore, the depolarization of the stimulated neuron activated both type of transmissions that resulted in opposite effects on the membrane potential of the postsynaptic neuron. This biphasic shape of the response was efficiently modified by the membrane potential of the receiving cell (Fig. 5C). Indeed, at hyperpolarized membrane potentials, cell2 responses clearly displayed the two components (electrical and chemical), resulting in a net hyperpolarization. In contrast, when cell2 membrane potential was depolarized, chemical transmission was less efficient (accordingly to its voltage-dependency), and only the electrical component remained visible, leading to a net depolarization of the receiving cell. These results indicate that, when activated together, chemical and electrical transmissions dynamically interact in an antagonistic way.

Fig. 5.

Coexistence of electrical and chemical transmissions. (A) DAergic neuron pair connected by chemical interactions but not electrical synapses. A hyperpolarization (Left) or a sinusoidal stimulation (Right) in cell1 (V1) fails to evoke an electrotonic synaptic current in cell2 (V2), but a suprathreshold depolarization of cell1 (Center) evokes a SEH in cell2. (B) DAergic neuron pair connected by both electrical synapses and chemical interactions. A hyperpolarization (Left) or a sinusoidal stimulation (Right) of cell1 evoke a hyperpolarizing or sinusoidal response in cell2 mediated by electrical synapses. The mean value of the coupling coefficient was 3.3 ± 0.3% corresponding to a Gj value of 60pS. A suprathreshold depolarization of cell1 evokes a SEH in the receiving neuron (V2). Note the initial depolarization in cell2 (arrow), corresponding to the electrotonic transmission of the subthreshold component of cell1 stimulation. (C) In electrically and chemically coupled pairs, the response of cell2 (V2) to a train of APs in cell1 (V1) depends on the membrane potential of cell2. At hyperpolarized membrane potentials (−90 mV), the net effect is hyperpolarizing (chemical transmission is predominant), whereas at −70 mV, the net effect is depolarizing (electrical coupling is predominant).

Discussion

Chemical Transmission Between Dopaminergic Neurons.

Numerous studies have suggested an interaction between DAergic neurons, based on evidences of release and effects of DA in DAergic neurons (12–16, 19, 20). Two recent studies have brought functional evidences of hyperpolarization evoked by DA after extracellular stimulation in the SNc (17) or the subthalamus (18). Here, using double patch-clamp recordings, we provide direct functional demonstration of a DA-mediated chemical transmission between DAergic neurons. A crucial improvement of paired recordings is brought by the stimulation of a single and tightly controlled DAergic neuron. Consequently, the effect observed in the receiving cell can be specifically related to the strict output of the stimulated DAergic neuron, in contrast to extracellular stimulations recruiting heterogeneous cell populations. In these conditions, we observed in nearly all pairs of neighboring DAergic neurons an hyperpolarization of the receiving cell, strictly dependent on the occurrence of an AP in the presynaptic cell, involving DA and acting through the temporary inhibition of Ih.

Chemical Transmission Relies on an Inhibition of Ih Partially Mediated by Dopamine.

Our observation of SEHs mediated partially by DA and resulting from a temporary inhibition of the constitutively active Ih current is coherent with previous studies reporting that application of exogenous DA induced a D2-like receptor mediated inhibition of Ih on various cell populations (28–30). However, it should be noted that DA-mediated inhibition of Ih has been reported to be secondary to activation of potassic conductances (31).

Conductance decrease mediated SEHs is an unusual property of chemical transmission compared with the classical mechanism acting through membrane conductance increase. Up to now, transmitter-evoked inactivation of membrane conductances was induced mainly by exogenous application of neurotransmitters and involved longer time-scale (seconds to minutes, compared with milliseconds in our study) changes (32–37). Using paired recordings, we provide the first direct evidence of an hyperpolarizing chemical transmission through membrane conductance decrease, in mammals. Both this unusual mechanism of conductance-decrease mediated synaptic event and the metabotropic transduction pathway result in multiple steps between the DA release and the effect on membrane potential, which can account for the duration and variability of SEH latency. The partial inhibition of SEH by raclopride could be due to a combination of a lesser accessibility of synaptic receptors (compared with extrasynaptic receptors coupled to K⁺ channels activation) and the high amount of DA released during Cs-enhanced spikes that could displace the antagonists. Still, this does not explain the partial inhibition by sulpiride, therefore suggesting the existence of a cotransmitter that remains to be determined, or the local accumulation of extracellular cations.

SEHs observed between DAergic neurons pairs are not due to a DA-induced activation of K⁺ channels as reported in other studies (13, 14, 17, 18), yet we could observe the effects of DA application in our experiments. Therefore, the difference of D2-activation outcome results only from differences in the stimulation protocol, either from the difference in intensity of stimulation (larger DA amount and time of application with exogenous DA or population stimulation) or in its localization (more focalized with single neuron stimulation). The first hypothesis would mean that a larger DA amount or duration of activation of D2 receptors would be necessary to activate potassium channels than to inhibit Ih current, which is unlikely, considering that similar concentrations of DA are needed to inhibit Ih and to evoke an outward current in slices (30). The second hypothesis implies the existence of different pools of D2-like receptors: synaptic receptors coupled only to Ih channels, activated when a single neuron is stimulated, and para- or extrasynaptic receptors coupled to potassium channels (and possibly to Ih channels) that are activated by DA spillover [caused by exogenous DA application (13, 14) or electrical stimulation of a large population of neurons (17, 18)]. This hypothesis is supported by the >2-fold latency differences observed between SEHs and SNc stimulation induced outward currents (17), which could reflect the diffusion time of DA toward parasynaptic receptors. The differential G protein coupling and ion channel modulation among D2-like receptors could underlie the different outcome of the synaptic versus parasynaptic receptor activation. Indeed, the shorter isoform of D2 receptor, which is known to be responsible for the DA effects on potassium conductance in DAergic neurons, as shown by using KO mice for D3 or for the longer form of D2 (17, 38), could constitute the parasynaptic receptor pool, whereas D3 receptors, which are expressed by all DAergic neurons (39) and do not activate potassium channels (40), could constitute the synaptic receptor pool.

Functional Implications of Ih-Mediated Synaptic Transmission.

The outcome of chemical transmission between DAergic neurons being an hyperpolarization, this transmission would be considered as inhibitory. However, because the SEH relies on a membrane conductance decrease, the postsynaptic DAergic neuron is temporarily more electrotonically compact and might therefore be more excitable. Similarly, Ih blockade decreases the spontaneous firing rate in subpopulations of DAergic neurons (41, 42), but is also thought to promote bursting (30). These paradoxical effects of Ih suggest that the impact of chemical transmission on the activity of DAergic neurons might be more complex than a pure inhibitory transmission.

The small amplitude of SEHs when recorded in KGlu intracellular solution suggest that during tonic activity, a coactivation of numerous presynaptic DAergic neurons, might be needed to efficiently inhibit Ih in the postsynaptic neuron. During phasic activity however, chemical transmission might have a larger impact on the postsynaptic membrane potential. Indeed, the calcium influx induced by Cs-enhanced APs, critical for triggerring SEHs, might correspond to the calcium influxes occurring during bursting activity (43, 44). Therefore, DAergic neurons might have three “levels” of chemical transmission depending on the amount of DA released: (i) In tonic activity, little DA is released, and chemical transmission impact is weak. (ii) In phasic activity, a larger amount of DA is released and chemical transmission is able to influence the membrane potential and R_i of the postsynaptic cell by inhibiting Ih. This could also participate in stabilizing or spreading bursting activity across DAergic neurons. (iii) In case of a massive phasic activation of a population of DAergic neurons (or maybe also if DA recapture is affected), DA spillover reaches and activates para- or extrasynaptic receptors coupled to potassium conductances, which drastically hyperpolarizes DAergic neurons and decreases their activity.

Coexistence of Chemical and Electrical Synapses.

Subsets of DAergic neurons are electrically coupled (refs. 10 and 11, but see ref. 45). Here, we report the coexistence of chemical and electrical transmissions. In case of cell firing, these transmissions have opposite effects on the membrane potential of the receiving neuron. Because of the voltage dependency of the chemical transmission, the net outcome in the receiving DAergic neuron is tuned by its membrane potential value. A coexistence of chemical and electrical transmissions has been described in GABAergic interneuron networks (22, 46–48), and promotes highly precise synchronization of spiking activity. However, tonic spontaneous activity recorded in DAergic neuron pairs is not synchronized in vitro (11), which may be essential for a homogenous low-level DA release in the striatum in the absence of relevant stimuli. The weaker precision of DAergic compared with GABAergic transmission (milliseconds compared with sub-milliseconds) could explain why the coexistence of chemical and electrical transmissions in DAergic neurons does not lead to the synchronization of their tonic activity. Moreover, modeling studies show that synchronization processes emerging from combination of electrical coupling and chemical transmissions differ between neuronal populations. Depending on the coupling strength, the membrane properties and the discharge rate, these two types of transmission can have synergistic or opposite effects on synchronization (49, 50). Given the differences in membrane properties and in electrical synapse characteristics between DAergic neurons and GABAergic interneuron (51), a different outcome of local interactions in terms of synchronization of tonic activity is therefore expected.

Concerning phasic activity, a modeling study shows that, in in vivo-like conditions (presence of NMDA receptor-mediated excitatory inputs), electrical coupling between DAergic neurons should promote the emergence of synchronous bursting activity (52). This prediction is in accordance with an in vivo study, reporting a synchronization of activity in subsets of DAergic neurons (10, 53). Phasic activity is characterized by bursts of APs followed by a prolonged hyperpolarization, involving different membrane conductances when compared with tonic firing (54, 55). These differences might induce a change in the single and combined impact of electrical and chemical transmissions on synchronization of DAergic neuron activity (49, 50). Furthermore, considering the voltage-dependency of the chemical transmission reported here, the important fluctuations of membrane potential during phasic firing should induce similar fluctuations of the weight of chemical transmission. An important step in the understanding of the impact of local interactions in DAergic neuron activity is the mapping of these connections in the whole DAergic nuclei. Indeed, if nearly all neighboring DAergic neurons are connected by chemical synapses (and one-fourth by electrical synapses), the medium- and long-range interaction map is yet unknown. Incidence of chemical (and electrical) synapses could decrease in distant pairs (in particular because putative contacts are seen in the proximal part of the dendritic field), confining the integration of information (and possible synchronization) in DAergic projecting to or receiving from the same striatal territories. Conversely, a constantly dense network of local interactions throughout the DAergic nuclei could result in synchronous acute variations of DA levels in distinct striatal territories and might therefore underlie a linkage of reward-related information concerning different behavioral aspects in which the nigrostriatal pathway is involved.

Methods

Whole-cell recordings were performed in coronal brain slices from Sprague–Dawley rats (postnatal days 15–18) at 34°C, using the same extra- and intracellular solutions as described in ref. 11, except when CsGlu intracellular solution was used [105 mM Cs-gluconate, 30 mM CsCl, 10 mM Hepes, 10 mM phosphocreatine, 4 mM ATP-Mg, 0.3 mM GTP-Na, and 0.3 mM EGTA (adjusted to pH 7.35 with CsOH)]. All experiments were done in accordance with European Union guidelines (directive 86/609/EEC). Liquid junction potentials were estimated as 13.6 mV and 14.2 mV for KGlu and CsGlu intracellular solutions, respectively. Signals were amplified by using an EPC10–2 amplifier (HEKA Elektronik), and analyzed by using IgorPro (Wavemetrics). All results are expressed as mean ± standard error of mean, and statistical significance was assessed by using the student t test or the nonparametric Mann–Whitney test. Effects of antagonists were measured after a 5-min bath application after 5–15 min of recording in control conditions. Detailed experimental and analysis methods are available in SI Methods.