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RIM is essential for stimulated but not spontaneous somatodendritic dopamine release in the midbrain

  1. Brooks G Robinson  Is a corresponding author
  2. Xintong Cai
  3. Jiexin Wang
  4. James R Bunzow
  5. John T Williams
  6. Pascal S Kaeser  Is a corresponding author
  1. Oregon Health & Science University, United States
  2. Harvard Medical School, United States
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Cite this article as: eLife 2019;8:e47972 doi: 10.7554/eLife.47972

Abstract

Action potentials trigger neurotransmitter release at active zones, specialized release sites in axons. Many neurons also secrete neurotransmitters or neuromodulators from their somata and dendrites. However, it is unclear whether somatodendritic release employs specialized sites for release, and the molecular machinery for somatodendritic release is not understood. Here, we identify an essential role for the active zone protein RIM in stimulated somatodendritic dopamine release in the midbrain. In mice in which RIMs are selectively removed from dopamine neurons, action potentials failed to evoke significant somatodendritic release detected via D2 receptor-mediated currents. Compellingly, spontaneous dopamine release was normal upon RIM knockout. Dopamine neuron morphology, excitability, and dopamine release evoked by amphetamine, which reverses dopamine transporters, were also unaffected. We conclude that somatodendritic release employs molecular scaffolds to establish secretory sites for rapid dopamine signaling during firing. In contrast, basal release that is independent of action potential firing does not require RIM.

https://doi.org/10.7554/eLife.47972.001

Introduction

In addition to secretion from axonal nerve terminals, many neurons release neurotransmitters or neuromodulators from their somata and dendrites (Ludwig et al., 2016). Important examples include neuropeptides, monoamines and neurotrophins, and signaling through these pathways is essential for brain function. However, the somatodendritic secretory machinery is not well understood.

A prominent example for somatodendritic secretion is the release of dopamine in the ventral midbrain. Subsequent activation of dopamine receptors is important for regulating neuronal excitability, for the response to drugs of abuse, and for the control of motor function (Bjijou et al., 1996; Crocker, 1997; Ford, 2014; Ludwig et al., 2016; Vezina, 1996). Somatodendritic dopamine release is mediated by vesicular exocytosis, as it is sensitive to clostridial toxins (Bergquist et al., 2002; Fortin et al., 2006). Despite years of study, essential molecular machinery for somatodendritic dopamine release has not been identified, but specific SNARE requirements and high calcium sensitivity have been proposed (Chen et al., 2011; Mendez et al., 2011; Witkovsky et al., 2009).

Electrophysiological recordings from midbrain dopamine neurons revealed that somatodendritic dopamine release evokes a D2 receptor mediated inhibitory postsynaptic current (D2-IPSC) that is mediated by GIRK channels and rises in 200 ms and decays in 500 ms (Beckstead et al., 2004). The D2-IPSC is produced by a high concentration of dopamine (100 µM; Courtney and Ford, 2014), and the duration is defined by efficient dopamine re-uptake through the dopamine transporter (DAT) (Ford et al., 2009). In addition, spontaneous dopamine release occurs and produces D2-IPSCs (Gantz et al., 2013). These studies indicate that somatodendritic release of dopamine can lead to activation of nearby receptors. Hence, mechanisms for targeting somatodendritic secretion towards specific membrane domains close to receptor clusters on target cells must be present.

The goal of this study was to identify molecular machinery that could provide for precise targeting of somatodendritic secretion of dopamine, focusing on the active zone organizer RIM. RIM localization within neurons is thought to be restricted to axons, where it is present in small clusters within active zones and organizes these release sites (de Jong et al., 2018; Kaeser et al., 2011; Tang et al., 2016; Wang et al., 2016; Wang et al., 1997). Recent reports suggest that specialized RIM isoforms may localize and function in dendrites (Alvarez-Baron et al., 2013), and postsynaptic roles for RIM1α in LTP have been proposed (Wang et al., 2018). In dopamine neurons, RIM was recently identified to be localized to active-zone like secretory sites in striatal dopamine axons, and RIM is essential for dopamine secretion in the striatum (Liu et al., 2018). It is not known whether RIM is present in dopamine neuron somata and dendrites.

Results and discussion

We generated RIM cKODA mice, in which RIM1 and RIM2 were specifically removed in dopamine neurons by crossing conditional RIM ‘floxed’ alleles to DATIRES-cre mice (Bäckman et al., 2006; Liu et al., 2018). Mice heterozygous for the RIM floxed and DATIRES-cre alleles were used as controls (RIM control). We prepared acute brain slices from these mice and recorded from individual substantia nigra dopamine neurons. There were no differences in cell capacitance, resistance, spontaneous firing rates, Ih current, or the current measured immediately following break-in between RIM cKODA and RIM control mice (Figure 1—figure supplement 1). Electrical stimulation (5 pulses at 40 Hz) was used to elicit dopamine release measured as D2-IPSCs. If an IPSC was not initially present, the stimulation intensity was increased and/or the electrode was relocated. In RIM controls, IPSCs of 10 to 50 pA were reliably induced (Figure 1A and B), and the time course of dopamine transmission was dependent on reuptake rather than diffusion of dopamine in these control animals (Figure 1—figure supplement 2). Compellingly, D2-IPSCs were difficult or impossible to evoke in RIM cKODA slices (Figure 1A and B). The currents in most recordings were indistinguishable from baseline, and only one stimulated response was over 10 pA. The stimulating electrode was relocated 21 times in RIM cKODA, while only six relocations were necessary in RIM control, illustrating the strong impairment in RIM cKODA mice. We conclude that somatodendritic dopamine release strongly depends on RIM, suggesting that it is controlled by active zone-like scaffolds.

Figure 1 with 3 supplements see all
RIM is essential for stimulated somatodendritic dopamine release.

Somatodendritic release was characterized in substantia nigra dopamine neurons. Release was induced using a monopolar electrode and measured by recording D2 receptor IPSCs in control mice (RIM control) and in mice with conditional knockout of RIM specifically in dopamine neurons (RIM cKODA). (A, B) Example traces (A) and quantification (B) of IPSCs in RIM control and RIM cKODA mice with vs. without the presence of the D2 receptor antagonist sulpiride, n = 23 cells/6 mice in RIM control, and n = 23/6 in RIM cKODA, significance was calculated by two-way ANOVA and is reported in panel B (RIM control vs. RIM cKODA: F(1) = 57.63, p < 0.001; stimulated response vs. + sulpiride: F(1) = 60.71, p < 0.001), and was followed by Bonferroni post-hoc analysis (RIM control stimulated response vs. RIM control + sulpiride t = 9.70, p < 0.05; RIM cKODA stimulated response vs. RIM cKODA + sulpiride: t = 1.33, p > 0.05; RIM control stimulated response vs. RIM cKODA stimulated response: t = 9.62, p < 0.05). (C, D) Example traces (top) and quantification (bottom) of IPSCs stimulated in RIM control slices (C) or RIM cKODA slices (D) before and after treatment with L-DOPA (10 µM) and subsequent application of sumatriptan (1 µM, to inhibit dopamine release from serotonin terminals), n = 6 cells/6 mice in each group, significance was calculated by repeated measures ANOVA and is reported in panels C and D, (C: F = 10.44, p = 0.01 D: F = 22.75, p < 0.005), and was followed by Tukey’s multiple comparison test (C: baseline vs. L-DOPA p < 0.05, L-DOPA vs. sumatriptan p < 0.05; D: baseline vs. L-DOPA p < 0.05, L-DOPA vs. sumatriptan p < 0.05). Data in B-D are shown as mean ± standard error of mean (SEM) and small circles represent individual cells.

https://doi.org/10.7554/eLife.47972.002

It is possible that the secretory defect in RIM cKODA slices is misjudged because RIM cKODA may lead to altered expression or function of D2 receptors. Application of the dopamine precursor L-DOPA increases D2-IPSCs (Beckstead et al., 2004; Gantz et al., 2015), in part due to higher levels of vesicular dopamine in dopamine neurons. A significant component, however, results from the metabolism of L-DOPA in serotonin terminals, which then release dopamine and enhance D2-IPSCs. This component is blocked by the serotonin autoreceptor agonist sumatriptan (Gantz et al., 2015). This phenomenon was leveraged here. In RIM control slices, L-DOPA increased D2-IPSCs, and sumatriptan partially reversed it (Figure 1C). In RIM cKODA slices, initial stimulation did not evoke significant D2-IPSCs, but application of L-DOPA resulted in sizeable IPSCs that were blocked by sumatriptan (Figure 1D). Hence, D2 receptors are present and functional in RIM cKODA mice, and D2-IPSCs are produced when dopamine is artificially released from sources other than the dopamine neurons. An alternative approach to assess D2 receptors was to superfuse dopamine onto the slices (1 and 100 µM) and to determine the current density. There was no significant difference between RIM control and RIM cKODA animals (Figure 1—figure supplement 3). We conclude that D2 receptor levels and function were not strongly altered upon RIM knockout.

RIM cKODA may have resulted in altered dopamine neuron structure or excitability, or in a loss of dopamine vesicles in the somatodendritic compartment. To assess these alternative explanations for the loss of dopamine release, we first characterized the morphology of dopamine neurons that were dye-filled through a patch pipette, fixed, and imaged by confocal microscopy. RIM control and RIM cKODA neurons were indistinguishable in shape as assessed by Sholl analyses (Figure 2A and B). We next characterized excitability of the neurons in acute brain slices. We injected depolarizing currents of increasing size into individual neurons, and measured the resulting number of action potentials in those neurons. On average, RIM cKODA neurons fired the same number of action potentials in response to these currents (Figure 2C and D). Finally, amphetamine was used to reverse the plasma membrane and vesicular dopamine transporters (DAT and vMAT2, respectively). The resulting increase in extracellular dopamine triggered D2-GIRK currents that were similar in RIM cKODA and RIM control slices (Figure 2E and F), indicating that the somatodendritic level of dopamine containing vesicles is similar between RIM cKODA and RIM control mice. Hence, RIM removal did not strongly alter the size and development of dopamine neurons.

Figure 2 with 1 supplement see all
Dopamine neuron shape, excitability and amphetamine induced dopamine release are unaffected by RIM knockout.

(A, B) Example images (A) and Sholl analysis (B) of individual neurobiotin-filled dopamine neurons in RIM control (n = 21 cells/6 animals) and RIM cKODA (n = 24/6) slices. RIM control and RIM cKODA neurons in B were compared using two-way ANOVA (F (1, 43) = 0.53, p = 0.47). (C, D) Neuron excitability was tested by hyperpolarizing cells to − 70 mV and applying progressively larger 500 ms long positive current steps. Example traces (C) and quantification (D) of action potential firing recorded in current clamp are shown. The number of action potentials during each step was quantified in RIM control (n = 27/6) and RIM cKODA (n = 27/6), and then compared using two-way ANOVA (current step size effect F(4, 240) = 71.50, p < 0.0001; RIM control vs. RIM cKODA F(1, 240) = 0.85, p = 0.36). (E, F) Example traces (E) and quantification of current density (F) from RIM control and RIM cKODA mice of D2 receptor currents produced by bath application of amphetamine (10 or 30 µM, which causes the reverse transport of dopamine into the extracellular space); n = 5/5 in RIM control, 4/4 in RIM cKODA, compared by Student’s t-test (t = 1.12, p = 0.30). Data in B, D and F are shown as mean ± SEM and small circles in F represent individual cells.

https://doi.org/10.7554/eLife.47972.006

Basal or spontaneous release of dopamine occurs in vivo and in slices, and its detection as D2-IPSCs is facilitated in a low concentration of cocaine and forskolin (Gantz et al., 2013). Spontaneous D2-IPSCs were readily detected in RIM control and RIM cKODA slices (Figure 3A and B) and were blocked by the D2 receptor antagonist sulpiride. They were identical in amplitude and 20% peak width in both conditions, and had a non-significant trend towards increased frequency in RIM cKODA mice (Figure 3C-E). We conclude that, while removal of RIM abolishes stimulated IPSCs, spontaneous release does not necessitate RIM. The dichotomy between evoked and spontaneous somatodendritic dopamine release is surprising, because at typical synapses, for example glutamatergic or GABAergic synapses in the hippocampus, RIM is important for both forms of release (Deng et al., 2011; Kaeser et al., 2011). The normal amplitudes and kinetics of spontaneous dopamine release further strengthen the point that D2 receptor function and localization are not altered in RIM cKODA slices.

Spontaneous dopamine release is unaffected by RIM knockout.

(A, B) Recording of spontaneous D2 receptor IPSCs (sIPSC) in RIM control and RIM cKODA mice. Example traces (A) and aligned events (B, averages in bold) of D2 receptor IPSCs produced by spontaneous dopamine release from RIM control and RIM cKODA slices in the presence of cocaine (300 nM) and forskolin (1 µM) followed by addition of the D2 receptor antagonist sulpiride (RIM control n = 161 events/16 cells/6 mice, RIM cKODA n = 185/16/6). (C) The frequency of spontaneous IPSCs was quantified per cell in RIM control (n = 16 cells/6 mice) and RIM cKODA (n = 16/6) slices, and the groups were compared by Student’s t-test (t = 1.79, p = 0.09). (D, E) The amplitude (D) and 20% peak width (E) of the spontaneous IPSCs were analyzed and compared between all events of RIM control and RIM cKODA mice using Student’s t-test (n as in B; amplitude: t = 1.32, p = 0.19; half peak width: t = 1.46, p = 0.14). Data in C-E are shown as mean ± SEM and small circles in C represent individual cells.

https://doi.org/10.7554/eLife.47972.008

RIM is important for rapid and precise exocytosis of synaptic vesicles (Kaeser et al., 2011; Koushika et al., 2001; Müller et al., 2012; Wang et al., 2016). Dopamine has long been considered a neuromodulator that impacts circuits in a paracrine fashion. However, it was recently found that axonal dopamine release in the striatum is fast and depends on active zone-like release sites (Liu et al., 2018). Here, we establish that RIM is essential for stimulated release from dopamine neuron somata and dendrites that is detected via D2-IPSCs. Three points suggest that RIM organizes or generates secretory sites to mediate somatodendritic release. First, it is likely that RIM function is similar at somatodendritic and axonal release sites. At axonal sites, RIM controls the docking and priming of vesicles (Deng et al., 2011), the close by tethering of Ca2+ channels (Han et al., 2011; Kaeser et al., 2011), and the coupling of these functions to PI(4,5)P2 (de Jong et al., 2018), a target membrane phospholipid that is important for synaptic vesicle release (Milosevic et al., 2005; Di Paolo et al., 2004). Second, somatodendritic dopamine release has a high initial release probability (Beckstead et al., 2007), indicating that scaffolding mechanisms to tether dopamine-laden vesicles to Ca2+ channels and other secretory machinery are essential. Third, although the overall time course of the dopamine IPSC is dependent on GPCR signaling, IPSC activation requires a rapid rise in dopamine concentration (Courtney and Ford, 2014; Ford et al., 2009). This is best achieved by synchronous release of dopamine from nearby point sources. Together, these points suggest that RIM operates as a molecular scaffold to establish somatodendritic release sites that direct release towards D2 receptors on target cells. While it remains uncertain whether the time scales of dopamine coding require molecular machinery for rapid dopamine transmission, recent findings suggest that dopamine mediates effects on relatively fast time scales (Howe and Dombeck, 2016; Menegas et al., 2018; da Silva et al., 2018; Yagishita et al., 2014), and the machinery we identify here and in a previous study (Liu et al., 2018) could serve such roles. Regardless of the timing of dopamine coding, this machinery is well suited to provide the high dopamine concentrations that are necessary to activate D2 receptors and for the regulation of dopamine signaling.

Compellingly, spontaneous dopamine release in the midbrain remains intact upon RIM knockout, indicating that molecular scaffolding mediated by RIM is dispensable for spontaneous dopamine release. Consistent with this point, spontaneous dopamine release is less dependent on Ca2+-entry compared to stimulated release (Gantz et al., 2013). Together, these findings suggest that at least two modes of dopamine transmission exist. A large, fast, and directed mode, likely dependent on excitatory inputs and depolarization-induced Ca2+ entry, requires RIM. A basal mode, in contrast, does not rely on RIM. It is unclear whether these two modes occur at the same or different locations. It is further possible that not all dopamine release is reported by D2-IPSCs, and that additional transmission modes exist.

In dopamine axons, RIM was only present in ~1/3 of the varicosities, and this fraction is likely responsible for stimulated dopamine release (Liu et al., 2018; Pereira et al., 2016). Whether a similar pattern is present in the somatodendritic compartment is unclear, but our findings raise the possibility that stimulated and spontaneous release occur at different locations in axonal and somatodendritic compartments. Given that the spontaneous dopamine IPSCs scale to stimulated IPSCs (Gantz et al., 2013), it is likely that the pre- and postsynaptic elements of somatodendritic transmission are in close proximity for both release modes. RIMs’ scaffolding functions may allow for the synchronization of release from multiple vesicles at an individual site, from multiple sites, or from multiple neurons when activated simultaneously, allowing for more powerful activation of D2 receptors than spontaneous events.

Finally, our results establish important roles for RIM mediating rapid and efficient secretion from somata and dendrites, suggesting that active zone scaffolding is employed for fast exocytosis beyond that of synaptic vesicles in axonal boutons.

Materials and methods

Key resources table
Reagent
type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent (mouse)B6.SJL-Slc6a3tm1.1(cre)Bkmn/JBäckman et al., 2006RRID:IMSR_JAX:006660
Genetic reagent (mouse)Rims1tm3Sud/JKaeser et al., 2008RRID:IMSR_JAX:015832
Genetic reagent (mouse)Rims2tm1.1Sud/JKaeser et al., 2011RRID:IMSR_JAX:015833

Animals

RIM cKODA and RIM control mice were generated as described in Liu et al. (2018), crossing mice with essential exons flanked by loxp sites in the Rims1 (‘floxed RIM1’) and Rims2 (‘floxed RIM2’) genes (Kaeser et al., 2008; Kaeser et al., 2011) with DATIRES-cre mice (Bäckman et al., 2006). RIM cKODA mice are homozogyote floxed for RIM1 and RIM2 and are heterozygous for DATIRES-cre, and RIM control mice are heterozygous for floxed RIM1, RIM2 and DATIRES-cre. Male and female adult mice (100–160 days old) were used for all experiments, and mice were either littermates from the same litter or age-matched controls from the same intercrosses. All procedures and experiments were approved by and done in accordance with the policies of the IACUC at Oregon Health and Science University and at Harvard University.

Slice preparation and electrophysiological recordings

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Mice were deeply anesthetized with isoflurane and decapitated. Brains were rapidly removed and slices (222 µM thick) containing the substantia nigra were taken in the horizontal plane using a vibrating blade microtome and placed in a recovery chamber for > 30 min prior to experimentation. All preparation was done in warm (32–34° C) Krebs buffer containing (in mM) 126 NaCl, 1.2 MgCl2, 2.4 CaCl2, 1.4 NaH2PO4, 25 NaHCO3, 11 D-glucose, along with 10 µM MK-801 and continuous bubbling with 95%/5% O2/CO2. Following recovery, slices were placed in a recording chamber and perfused with Krebs buffer at a rate of 3 ml/min and maintained at 34–36° C. For all experiments DNQX (10 µM), picrotoxin (100 µM), and CGP55845 (300 nM) were included in the solution to block AMPA, GABA-A, and GABA-B receptors. In all experiments except those involving L-DOPA, sumatriptan (1 µM) was also included in the bath. In all experiments, a gigaohm seal was made on a dopamine neuron in the subatantia nigra with a glass electrode (1.3–1.8 megaohm resistance) filled with an internal solution containing (in mM) 10 BAPTA (4 k), 90 K-methanesulphonate, 20 NaCl, 1.5 MgCl2, 10 HEPES (K), 2 ATP, 0.2 GTP, and 10 phosphocreatine. In this configuration, the firing rate of the cell was measured. All cells were firing in a pacemaker fashion between 0.75 and 4 Hz and the firing frequencies were recorded. Then the seal was broken and whole-cell voltage clamp recordings (held at − 60 mV with an axopatch-1D amplifier) were achieved. The cell capacitance, input resistance, and series resistance were documented. Ih currents were measured using a 60 mV hyperpolarization. D2-IPSC measurements were conducted in voltage clamp and continuously recorded and monitored using Chart 7 (AD Instruments, Colorado Springs, CO). To test for D2 receptor IPSCs, a glass electrode filled with Krebs buffer was lowered into the slice 20–50 µM away from the cell of interest. To begin, five 0.5 ms pulses at 40 Hz were applied with a stimulus isolator (World Precision Instruments) once every minute and recorded in an episodic manner with AxoGraph software (Berkeley, CA). The stimulation intensity was started at 1 µA. If no IPSC was produced, the stimulation was gradually increased and the stimulation electrode was repositioned. The stimulation intensity of 12 µA was never exceeded, as this would often cause loss of recording or unclamped depolarization. In some experiments, the stimulation was reduced to a single pulse. To measure cell excitability, recordings were done in current clamp. A current was injected such that the membrane potential was − 70 mV to quiet pacemaker firing, and current steps were applied in + 50 pA intervals (from + 50 – + 250 pA for 500 ms each) over the injected holding current, and the number of action potentials during each step was recorded. The application of drugs was done using bath superfusion in all experiments. For the L-DOPA experiments, 10 µM L-DOPA was applied for 10 min, and then removed for 10 min before applying sumatriptan (1 µM). For testing D2 receptor sensitivity, 1 µM dopamine was applied until a peak current was achieved and then removed. Once the current returned to a steady baseline, 100 µM dopamine was applied until a new peak was achieved. For amphetamine experiments, 10 or 30 µM were applied for 5 min (Figure 2C and D), followed by the application of sulpiride, or applied until the rise in current had nearly stopped and then quinpirole was added (Figure 2—figure supplement 1). All electrophysiological experiments were performed and analyzed by an experimenter blind to the genotype.

Sholl analysis of individual dopamine neurons

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Dopamine neurons for morphological analyses were first identified and characterized electrophysiologically. Whole cell recordings were made from one cell per slice using an internal solution that contained neurobiotin (0.05%). Cells were recorded for a minimum of 15 min prior to removal of the electrode, and capacitance and resistance were recorded (no differences were observed between RIM control and RIM cKODA cells, not shown). Slices were then incubated for 30–45 min in extracellular solution at 35 °C prior to fixation. Slices were fixed for 30 min in PBS (phosphate buffered saline) + 4% paraformaldehyde (PFA) at room temperature and washed 3 × 15 min in PBS. Slices were incubated in PBS + 0.5% fish skin gelatin (FSG) + 0.3% Tween 20 with streptavidin Alexa Fluor 568 (at 1:1000) and washed 3 × 15 min in PBS at room temperature. Sections were mounted on glass slides with coverglasses with a 1.5 refractive index using fluorescent mounting medium. Laser-scanning confocal images were acquired on a ZEISS LSM880 with airyscan, with laser excitation at 560 nm at 20 x magnification with Z steps of 1 µm. The images were acquired through the Z plane such that the whole cell and all processes in the slice were captured. The Z-stacks were collapsed with z-project using Fiji for analysis. Individual neurons in confocal images were then traced manually. Sholl analysis of each traced neuron was performed using the ImageJ Sholl analysis plug-in. The cell body was selected, and the number of neurite crossings of concentric circles around the center of the cell body was measured at increasing radii (from 20 µm to 180 µm in 10 µm intervals). Cell filling, image acquisition and quantification were performed by experimenters blind to the genotype.

Data analyses and statistics

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To analyze IPSC amplitudes, the maximum amplitude for a given IPSC was recorded between 200 and 400 ms following the stimulation onset. This range was used because the stereotyped peak of D2-IPSCs reliably falls within it and a measurement for very small or non-existent IPSCs would still be generated. Each reported value is the average of three consecutive stimulation events. For analysis of spontaneous IPSCs, AxoGraph software was used to continuously collect data (sampling at 10 kHz) following the application of cocaine (300 nM) and forskolin (1 µM). For analysis, recordings were filtered at 1 kHz and decimated (averaging 10 points). Spontaneous IPSCs were automatically detected using a sliding template procedure in AxoGraph. The template was generated by averaging multiple events that conformed to previously published kinetic analysis (Gantz et al., 2013). Spontaneous IPSCs were only detected with amplitudes greater than 2.1 x the standard deviation of baseline noise. Detected events were manually examined for quality assurance. Statistics were performed using GraphPad Prism. All data are shown as mean ± SEM, and individual cells are shown as small circles. Student’s t-test was used for comparison of two groups while ANOVA was used to compare more than two groups or if there were multiple variables. Tukey’s (for one-way) or Sidak’s (for two-way) multiple comparison tests were used if a repeated measures ANOVA reached significance and Bonferroni post-hoc comparisons were done if the one- or two-way ANOVA reached significance.

Drugs

Drugs were bath applied. MK-801, forskolin, CGP55845, and picrotoxin were acquired from Hello Bio Princeton, NJ. CNQX, sulpiride, L-DOPA, were acquired from Sigma-Aldrich. Amphetamine was acquired from NIH NIDA and sumatriptan was acquired from Glaxo Wellcome Inc.

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Decision letter

  1. Olivier J Manzoni
    Reviewing Editor; Aix-Marseille University, INSERM, INMED, France
  2. Eve Marder
    Senior Editor; Brandeis University, United States
  3. Nicolas Tritsch
    Reviewer
  4. Pablo E Castillo
    Reviewer; Albert Einstein College of Medicine, United States
  5. Louis-Eric Trudeau
    Reviewer; Université de Montréal, Canada

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "RIM is essential for stimulated but not spontaneous somatodendritic dopamine release in the midbrain" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Nicolas Tritsch (Reviewer #1); Pablo E Castillo (Reviewer #2); Louis-Eric Trudeau (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The study by Robinson et al. shows that RIM proteins are required for evoked somatodendritic release of dopamine from midbrain DA neurons in mice. Specifically, selective removal of RIM from dopamine midbrain DA neurons abolished D2 receptor mediated-IPSCs induced by electrical bulk stimulation, whereas spontaneous release of dopamine remained intact. Together the data indicate that the mechanisms underlying evoked and spontaneous release of dopamine differ.

Essential revisions:

1) Validation of DA neurons:

- What midbrain dopamine neurons were analyzed in this study (SN, VTA or both)?

- Are dopamine neurons in cKO mice just as likely to be stimulated electrically to release dopamine than control neurons? Please compare spontaneous firing rates and action potential waveforms, and then demonstrate that electrical stimulation equally recruits dopamine neurons in both genotypes.

- Is the global density of dopaminergic dendrites was altered in these KO mice (suggested experiment:TH immunostaining in the substantia nigra)?

2) D2-IPSCs: To more directly address the possibility of changes in D2 receptor responsiveness of dopamine neurons in the RIM KO mice, please measure membrane currents evoked in dopamine neurons by local application of a D2 agonist.

The kinetics of spontaneous D2-IPSCs and electrically evoked D2-IPSCs seem to differ as seen in Figure 3B. Please measure and analyze the frequency, rise-time and decay of evoked and spontaneous D2-IPSCs.

3) RIM-dependent synchronization:

Can action potentials triggered by direct depolarization of DA neurons induce DA-IPSCs? If so, the authors may want to use this single-cell approach to confirm that RIM-dependent synchronization is stimulation protocol-independent and can be observed with a more subtle manipulation.

4)Rim localization:

The authors need to state more explicitly what is known about the localization of RIM1-2 in neurons and cite the relevant work. Can the authors show the presence of RIM proteins in the somatodendritic domain of dopamine neurons? Can the authors detect RIM in dendrites (As: "In dopamine axons, RIM was only present in ~1/3 of the varicosities…")?

Reviewer #1:

This brief report makes a simple point, and does so clearly and well: it reveals that the presynaptic protein RIM is essential for electrically evoked, but not spontaneous dendritic release of dopamine in the mouse midbrain. This paper comes on the heels of a compelling report from the same last author, showing that the active zone proteins RIM 1 and 2 distribute to presynaptic terminals of midbrain dopaminergic neurons in striatum, and that mice in which these proteins are conditionally deleted (cKO) from dopaminergic neurons are unable to release dopamine when measured with amperometry (Liu et al., 2008). Interestingly, these mice did not appear to suffer from gross locomotor defects and microdialysis experiments revealed trace amounts of extracellular dopamine in striatum, suggesting that spontaneous release may be intact. Here, the authors build upon this work, comparing dendritic release of dopamine in of control and RIM cKO mice using whole cell recordings of D2 receptor-evoked GIRK currents in midbrain dopamine neurons. They report the absence of electrically-evoked GIRK currents in RIM cKO mice, and perform a series of control experiments to rule out insufficient stimulation, or changes in D2 receptor expression. Interestingly, spontaneously occurring D2 receptor mediated GIRK currents appear intact in these mice, harking back to their earlier speculation that spontaneous release may occur in a RIM-independent fashion.

I only have 2 points that should be clarified, or excluded as potential confounds to confirm their conclusions:

- In Figure 2, the authors use amphetamine to test whether the cKO mice have a reduced number of vesicles (as opposed to a defect in release). They report no differences in current amplitude compared to WT mice, but a difference may only be revealed using this approach if GIRK conductances are not saturated. Can the authors rule this out? A direct measure of large dense core vesicles with EM would obviously be a better experiment, but probably not a resource available to either lab.

- In Figure 1—figure supplement 1, the authors make the case that the intrinsic properties of dopamine neurons are identical in both genotypes. But what the authors really need to show is that dopamine neurons in cKO mice are just as likely to be stimulated electrically to release dopamine than control neurons. The authors could start by comparing spontaneous firing rates and action potential waveforms, and then demonstrate that their electrical stimulation paradigm equally recruits dopamine neurons in both genotypes.

Reviewer #2:

In this study Robinson et al. report that RIM proteins are required for evoked somatodendritic release of dopamine from midbrain DA neurons in mice. The key finding is that selective removal of RIM from these neurons abolished D2-IPSCs induced by electrical bulk stimulation, whereas spontaneous release of dopamine remained intact. These observations strongly suggest that the mechanisms underlying evoked and spontaneous release of dopamine differ. The need for spatiotemporal precision of dopamine release and the associated secretory machinery, as seen for fast neurotransmitters, is unclear to this reviewer. In any event, the experiments are well designed and the results support the claims that are made. I only have a few suggestions that may strengthen the authors' conclusions.

1) "RIMs scaffolding functions may allow for the synchronization of release from multiple sites or multiple vesicles". Synchronization may also result from the stimulation method (extracellular bulk stimulation). Can action potentials triggered by direct depolarization of DA neurons induce DA-IPSCs? If so, the authors may want to use this single-cell approach to confirm that RIM-dependent synchronization is stimulation protocol-independent and can be observed with a more subtle manipulation.

2) The comparison between single stimulus and five stimuli IPSCs can only provide a very indirect estimation of Pr. Yet, the authors conclude that evoked somatodendritic release of DA has a high initial Pr and that a scaffolding mechanism to tether dopamine-laden vesicles to calcium channels and other secretory machinery are essential. Repetitive, bulk stimulation could release modulators that reduce transmitter release that can mistakenly interpreted as high initial Pr.

3) Why does somatodendritic DA release require the spatiotemporal precision that is typically observed in fast neurotransmission? Evoked DA-IPSCs are extremely slow -as compared to fast synaptic transmission- and do not seem to require (pre-postsynaptic) nanodomains. This issue should be discussed more thoroughly.

4) "In dopamine axons, RIM was only present in ~1/3 of the varicosities…". Can the authors detect RIM in dendrites?

5) The authors claim that spontaneous D2-IPSCs were kinetically similar to electrically evoked D2-IPSCs. However, consistent with Gantz et al., 2013 their kinetics seem to differ as seen in Figure 3B. Maybe the authors want to measure and analyze the rise-time and decay of evoked and spontaneous D2-IPSCs.

Reviewer #3:

In this manuscript by Robinson and colleagues, the authors explored the implication of the active zone proteins RIM1-2 in somatodendritic dopamine release in mice. The work is quite original as the molecular mechanisms of somatodendritic dopamine release are still incompletely characterized and whether the release machinery and scaffolding proteins involved are similar or different from the machinery in axon terminals in an important question. Globally, the work presented in this very short paper is quite convincing. The block of dendritic dopamine release is quite striking. However, a few issues need to be addressed.

1) The authors need to state more explicitly what is known about the localization of RIM1-2 in neurons and cite the relevant work. Obviously, it would add significantly to the paper to demonstrate the presence of RIM proteins in the somatodendritic domain of dopamine neurons. The authors should at least explain why this was not done in the present work (difficulty to distinguish between RIM protein directly in the dendrite as opposed to RIM in terminals contacting the dendrites?)

2) The Introduction should make it clearer that somatodendritic dopamine release is well established to be vesicular in origin and to be sensitive to cleavage of SNARE protein by clostridial toxins.

3) The data on the frequency-dependence of dendritic dopamine release presented in Figure 1—figure supplements 2 and 3, while interesting, are not really relevant to the main point of the paper. Also, the conclusions drawn from these results (that dendritic dopamine release employs scaffolds and that RIM heterozygosity did not strongly impair dendritic dopamine release) are not really supported by this data. I would suggest removing this data from the manuscript.

4) The experiments carried out with L-DOPA are presented as if they were performed to determine whether the loss of dendritic dopamine release in RIM KO mice is due to reduced levels of dopamine D2 receptors. In fact, the results do not really address this question, which is otherwise important. The fact that L-DOPA induces a D2R-dependent response which apparently comes from serotonin axon terminals is quite interesting in itself, but only supports the idea that some dopamine D2 receptors remain in the somatodendritic compartment of dopamine neurons in the KO mice. It does not allow saying whether there are less or more D2R. Also, in control mice, it is surprising to note that there was no difference between the baseline response and the response after L-DOPA and sumatriptan. This would tend to suggest that L-DOPA did not boost dendritic dopamine release at all. Also, the sumatriptan-sensitive component is bigger in RIM KO mice compared to the control mice. What this means is unclear. Was there some compensatory adaptation in the 5-HT terminals in response to abrogation of axonal and dendritic dopamine release in these mice? Globally, these experiments with L-DOPA raise more questions than anything else.

5) To more directly address the possibility of changes in D2 receptor responsiveness of dopamine neurons in the RIM KO mice, it seems to me that it would be better to simply measure membrane currents evoked in dopamine neurons by local application of a D2 agonist. This would nicely complement the experiments of Figure 2 with amphetamine.

6) The experiments of Figure 3 are quite interesting. The authors should also provide the frequency of these spontaneous events in control and KO neurons.

7) The absence of RIM starting from the late embryonic period could have perturbed the development of dopamine neurons, potentially leading to altered dendritic development. The capacitance results from Figure 1—figure supplement 1 argue that there were no major changes in the size of dopamine neurons in the KO mice. However, do the authors have the result of a TH immunostaining experiment in the substantia nigra to determine whether the global density of dopaminergic dendrites was altered in these KO mice?

https://doi.org/10.7554/eLife.47972.011

Author response

1) Validation of DA neurons:

- What midbrain dopamine neurons were analyzed in this study (SN, VTA or both)?

We apologize that we did not make this clear. All analyzed neurons were dopamine neurons in the pars compacta of the substantia nigra. This is now mentioned at various places including the results, the legend of Figure 1, and the Materials and methods section.

- Are dopamine neurons in cKO mice just as likely to be stimulated electrically to release dopamine than control neurons? Please compare spontaneous firing rates and action potential waveforms, and then demonstrate that electrical stimulation equally recruits dopamine neurons in both genotypes.

We have performed the requested experiments. First, we have quantified spontaneous firing rates and there was no difference between RIM control and RIM cKODA neurons (in Figure 1—figure supplement 1). We have then performed experiments to assess whether stimulation recruits the RIM knockout neurons efficiently. We have quantified the number of action potential spikes induced by current injections in dopamine neurons of RIM control and RIM cKODA mice (new Figures 2C and 2D), and found that stimulation equally recruits dopamine neurons in both genotypes. The observations that pacemaker firing, firing in response to depolarization, and input resistance are unchanged strongly indicate that excitability is normal in RIM cKODA neurons.

An additional way to assess this would be to determine the somatic action potential waveforms of dopamine neurons. We have not attempted this here because 1) all recordings were done with amplifiers that cannot truly follow rapid voltage changes and hence action potential shape may be distorted (Magistretti et al., 1996) and 2) the spike and afterhyperpolarization of these neurons include many conductances, some of which would only cause subtle changes in the action potential waveform if affected. A recent review on dopamine neurons points out this complexity (Gantz et al., 2018). We strongly feel that any change could easily be missed and would prefer not to reach conclusions about the many important conductances based on simply assessing somatic waveforms. Nevertheless, the observations that spontaneous firing, recruitment upon depolarization and input resistance are unchanged exclude that excitability defects account for the strong impairment in release in RIM cKODA.

- Is the global density of dopaminergic dendrites was altered in these KO mice (suggested experiment:TH immunostaining in the substantia nigra)?

We thank the reviewers for bringing this up and have performed experiments to address this point. First, we have stained TH neurons in RIM control and RIM cKODA mice in preliminary experiments. While the overall morphology and TH density appeared unaffected, dendrite and cell shapes could not be analyzed with certainty because the dendrites are dense and for many dendrites it is not possible to follow and assign them to a given cell.

Instead, we performed a full experiment in which we identify individual dopamine neurons electrophysiologically in acute brain slices and then dye-filled these neurons. We then fixed the slices and imaged individual dopamine neurons in RIM control and RIM cKODA slices by confocal microscopy, and performed Sholl-analyses (new Figures 2A and 2B) to analyze their branch structure. RIM knockout dopamine neurons appear identical to the control neurons and electrophysiological properties of the neurons were unchanged (data not shown as they would duplicate the data in Figure 1—figure supplement 1).

These data establish that there are no strong effects of RIM deletion on dopamine neuron cell size and shape.

2) D2-IPSCs: To more directly address the possibility of changes in D2 receptor responsiveness of dopamine neurons in the RIM KO mice, please measure membrane currents evoked in dopamine neurons by local application of a D2 agonist.

We thank the reviewers for bringing this up and have performed this experiment as instructed (Figure 1—figure supplement 3). We have measured D2 receptor currents in response to bath application of 1 µM followed by 100 µM dopamine. There is no significant difference in the currents induced by dopamine between RIM control and RIM cKODA mice. While a robust response is present in both genotypes upon application of 100 µM dopamine, there may be a non-significant trend towards a reduction in RIM cKODA mice in 1 µM dopamine. We note that these currents are small and unreliable. However, overall our data strongly support that D2 receptor localization and function is not significantly impaired, because the D2 responses to L-DOPA (metabolized and released by serotonin neurons, Figures 1C and 1D) and amphetamine (Figure 2—figure supplement 1) are unchanged, and most importantly the amplitude of spontaneous D2 IPSCs is unaffected by RIM cKODA (Figure 3).

The kinetics of spontaneous D2-IPSCs and electrically evoked D2-IPSCs seem to differ as seen in Figure 3B. Please measure and analyze the frequency, rise-time and decay of evoked and spontaneous D2-IPSCs.

We have performed new experiments and analyses of the frequency and kinetics of spontaneous events in RIM control and RIM cKODA animals. We note that these experiments are laborious given the low amplitude and overall frequency of the events and the difficulty of their detection. Our analysis reveals that the frequency of spontaneous events is not significantly changed in the RIM cKODA mice, but there is a sizable trend towards an increase in spontaneous event frequency (Figure 3C). We also provide a new analysis of the 20% peak width of the spontaneous events that establish that the kinetics of the spontaneous events are unchanged in RIM cKODA mice.

The kinetics of spontaneous and evoked D2-IPSCs and how they relate to one another have been extensively studied before (Courtney and Ford, 2014; Gantz et al., 2013). As presented by these studies and in the previous version of our manuscript, the tail of the evoked D2-IPSC is somewhat longer than that of the spontaneous D2-IPSC. It has also been shown in these previous studies that the spontaneous D2-IPSCs scale to evoked D2-IPSCs, and the decay is most likely dependent of downstream GPCR signaling with a decay time constant of 300-350 ms rather than the properties of dopamine release. While interesting, we strongly feel that a detailed analysis and discussion of these points is marginal to the manuscript and distracts from the main point. In the interest of keeping this brief report concise and focused on the key point (the mechanisms of somatodendritic dopamine release), we have removed the comparison of the spontaneous and evoked D2-IPSCs, but instead refer to the relevant literature.

3) RIM-dependent synchronization:

Can action potentials triggered by direct depolarization of DA neurons induce DA-IPSCs? If so, the authors may want to use this single-cell approach to confirm that RIM-dependent synchronization is stimulation protocol-independent and can be observed with a more subtle manipulation.

We have pursued experiments to test whether direct depolarization of a DA neurons triggers DA IPSCs in that neuron in previous experiments. Unfortunately, we have never succeeded in using a single neuron as a “sensor” for its dopamine release. Hence, this experiment is not possible. We have adjusted the text around the hypothesis of synchronization and have included the possibility suggested by reviewer 3 (synchronization of inputs across neurons, main text, eleventh paragraph) to better express the hypothetical nature of these models and their underlying mechanisms.

4)Rim localization:

The authors need to state more explicitly what is known about the localization of RIM1-2 in neurons and cite the relevant work. Can the authors show the presence of RIM proteins in the somatodendritic domain of dopamine neurons? Can the authors detect RIM in dendrites (As: "In dopamine axons, RIM was only present in ~1/3 of the varicosities…")?

We thank the reviewers to raise the point of RIM localization and we now address it (main text, fourth paragraph). RIM localization has been best characterized in hippocampal neurons. There, RIM is predominantly found in axons. Within axons, it is highly concentrated at active zones (Kaeser et al., 2011; Wang et al., 1997), as best assessed by STED microscopy (de Jong et al., 2018). Two reports, however, proposed that RIM also may have dendritic functions. Specifically, it was proposed that RIM1α may support postsynaptic trafficking in dendrites (Wang et al., 2018), and RIM3 and 4, small RIM version, localize in dendrites and control their growth (Alvarez-Baron et al., 2013).

In the dopamine neurons, RIM localization has previously only been assessed in striatal axons of substnatia nigra and VTA dopamine neurons, where it is clustered in sparse, active zone-like release sites (Liu et al., 2018). Recently, we have put significant effort into attempting to localize RIM in somata and dendrites of dopamine neurons with several super-resolution microscopic methods. Unfortunately, we have remained uncertain about RIM localization in these compartments. The key challenge is that dopamine somata and dendrites receive dense presynaptic inputs from other neurons, and hence somata and dendrites are densely decorated with RIM, but it is impossible to tell whether some (likely a very small fraction) of these RIM clusters are within somata and dendrites (as opposed to in active zones of nerve terminals onto somata and dendrites, only separated by a synaptic cleft of ~20 nm width from the inside of the dopaminergic cell). Hence, despite significant effort, we have not been able to establish or exclude somatic and dendritic localization of RIM at release-site like hotspots.

This point also relates to the broader question of whether there are defined release sites (for example generated or marked by RIM) in dopamine neuron somata and dendrites. While our data suggest that secretory hotspots in dopamine neurons are close to dopamine receptors on receiving cells, it has remained uncertain how this secretory pathway is organized. For example, fundamental aspects such as the nature of the membranous compartment that mediates somatodendritic release (small clear vesicles vs. tubulovesicle vs. other vesicles) has remained uncertain. In respect to RIM, it is possible that it is clustered at release sites, decorates the target membrane broadly, is instead associated with the vesicular compartment that contains dopamine, or is a soluble, cytosolic release factor to support release. While all data support the model that RIM is sparse in somata and dendrites, it is currently impossible to distinguish between these different models.

We hope that the better description of what is known about RIM localization in the text and the explanations provided here are sufficient to clarify this point. We will continue to study the structures that underlie somatodendritic dopamine exocytosis. We also hope that our findings motivate other laboratories to join the important effort to understand the structure and organization of the somatodendritic secretory pathway in dopamine neurons.

Reviewer #1:

[…] I only have 2 points that should be clarified, or excluded as potential confounds to confirm their conclusions:

- In Figure 2, the authors use amphetamine to test whether the cKO mice have a reduced number of vesicles (as opposed to a defect in release). They report no differences in current amplitude compared to WT mice, but a difference may only be revealed using this approach if GIRK conductances are not saturated. Can the authors rule this out? A direct measure of large dense core vesicles with EM would obviously be a better experiment, but probably not a resource available to either lab.

As outlined above in the response to the editorial decision, we have ruled out that D2 GIRK conductances are saturated (Figure 2—figure supplement 1).

We also agree that it would be great to have an electron microscopic assessment of the vesicular compartment for dopamine release in the RIM cKODA mice, but we note that despite many years of research it has not been established what vesicle dopamine is released from in wild type animals. We hope that the reviewer understands that we cannot solve this problem in the context of a revision.

- In Figure 1—figure supplement 1, the authors make the case that the intrinsic properties of dopamine neurons are identical in both genotypes. But what the authors really need to show is that dopamine neurons in cKO mice are just as likely to be stimulated electrically to release dopamine than control neurons. The authors could start by comparing spontaneous firing rates and action potential waveforms, and then demonstrate that their electrical stimulation paradigm equally recruits dopamine neurons in both genotypes.

We have now included these experiments as outlined in the response to the editorial decision letter (major point 1) in Figure 1—figure supplement 1 and in Figure 2.

Reviewer #2:

[…] I only have a few suggestions that may strengthen the authors' conclusions.

1) "RIMs scaffolding functions may allow for the synchronization of release from multiple sites or multiple vesicles". Synchronization may also result from the stimulation method (extracellular bulk stimulation). Can action potentials triggered by direct depolarization of DA neurons induce DA-IPSCs? If so, the authors may want to use this single-cell approach to confirm that RIM-dependent synchronization is stimulation protocol-independent and can be observed with a more subtle manipulation.

Unfortunately, the single cell approach has not been used successfully. In the past, this experiment has been taken on by many researchers in each of the monoamine nuclei, locus coeruleus, dorsal raphe, substantia nigra and VTA. Pharmacological experiments did further not find any evidence of autoreceptor dependent inhibition. Similarly, paired cell recordings have been the source of failed experiments in both the locus coeruleus and VTA. At this point, the site(s) of dendritic connections are not known. A similar experiment can be done in autaptic culture, but in this preparation the release is most likely axonal rather than dendritic. Finally, experiments using the intracellular application of reserpic acid, a membrane impermeant version of reserpine, did not block spontaneous IPSCs suggesting that at least the spontaneous events were not true autoreceptor dependent processes (Gantz et al., 2015). We hope that given these explanations, the reviewer agrees that this experiment is currently not possible.

Instead, we have toned down the Discussion and also express that synchronization may arise from synchronization of different neurons. We note that the RIM-dependent release machinery would also be well suited to do this as it time-locks release with firing. Hence, if dopamine neurons receive timed depolarizing inputs, the fast secretory machinery may serve to convey these signals as well.

2) The comparison between single stimulus and five stimuli IPSCs can only provide a very indirect estimation of Pr. Yet, the authors conclude that evoked somatodendritic release of DA has a high initial Pr and that a scaffolding mechanism to tether dopamine-laden vesicles to calcium channels and other secretory machinery are essential. Repetitive, bulk stimulation could release modulators that reduce transmitter release that can mistakenly interpreted as high initial Pr.

We agree to this point and thank the reviewer for pointing this out. Given this comment and the comments of the other reviewers, we have removed this figure. We further note that previous literature has established a high Pr (Beckstead et al., 2007), and we simply refer to this previous literature (which contains a much more extensive characterization than the one we provided in the previous version of the paper) in the interpretation of our data.

3) Why does somatodendritic DA release require the spatiotemporal precision that is typically observed in fast neurotransmission? Evoked DA-IPSCs are extremely slow -as compared to fast synaptic transmission- and do not seem to require (pre-postsynaptic) nanodomains. This issue should be discussed more thoroughly.

It has remained uncertain what the true time scales of dopamine coding are and why such fast machinery has been selected through evolution to mediate its release. Independent of the rapidity, it is important that D2 receptors require a relatively high concentration of dopamine for activation (Courtney and Ford, 2014; Ford et al., 2009), and the machinery we describe here likely accounts for this. Finally, we would note that secretory machinery like RIM may also be well suited for regulation. We now better discuss this in the manuscript with a specific statement referring to the need for fast signaling.

4) "In dopamine axons, RIM was only present in ~1/3 of the varicosities…". Can the authors detect RIM in dendrites?

We have addressed this question in the response to the editorial decision major point 4, we hope that this clarifies what is known at this point.

5) The authors claim that spontaneous D2-IPSCs were kinetically similar to electrically evoked D2-IPSCs. However, consistent with Gantz et al., 2013 their kinetics seem to differ as seen in Figure 3B. Maybe the authors want to measure and analyze the rise-time and decay of evoked and spontaneous D2-IPSCs.

We thank the reviewer for this comment and have addressed it by removing the claim of kinetics. As explained above and as noted by the reviewer, this point has been addressed in previous literature, and we refer to this body of literature now.

More generally, the differences between evoked and spontaneous transmitter release are subject of considerable discussion (see Kaeser and Regehr, 2014 for an in depth discussion of the topic) and the dopamine neurons are not alone. The Gantz et al., 2013 paper did find a difference in the time course of the IPSCs in that the evoked IPSCs had a somewhat longer tail. This was observed in experiments with minimal stimulation, and stimulation was most often done near the cell soma. However, the location of the site(s) of spontaneous or evoked release are not known and could account for the difference, but in the end the reason is not known.

Reviewer #3:

[…] A few issues need to be addressed.

1) The authors need to state more explicitly what is known about the localization of RIM1-2 in neurons and cite the relevant work. Obviously, it would add significantly to the paper to demonstrate the presence of RIM proteins in the somatodendritic domain of dopamine neurons. The authors should at least explain why this was not done in the present work (difficulty to distinguish between RIM protein directly in the dendrite as opposed to RIM in terminals contacting the dendrites?)

We have addressed this point in detail in the response to the editorial letter, major point 4.

2) The Introduction should make it clearer that somatodendritic dopamine release is well established to be vesicular in origin and to be sensitive to cleavage of SNARE protein by clostridial toxins.

We have addressed this in the Introduction and cite the relevant literature.

3) The data on the frequency-dependence of dendritic dopamine release presented in Figure 1—figure supplements 2 and 3, while interesting, are not really relevant to the main point of the paper. Also, the conclusions drawn from these results (that dendritic dopamine release employs scaffolds and that RIM heterozygosity did not strongly impair dendritic dopamine release) are not really supported by this data. I would suggest removing this data from the manuscript.

We thank the reviewer for bringing this up and fully agree, we have removed these data as also explained in the response to the editorial letter.

4) The experiments carried out with L-DOPA are presented as if they were performed to determine whether the loss of dendritic dopamine release in RIM KO mice is due to reduced levels of dopamine D2 receptors. In fact, the results do not really address this question, which is otherwise important. The fact that L-DOPA induces a D2R-dependent response which apparently comes from serotonin axon terminals is quite interesting in itself, but only supports the idea that some dopamine D2 receptors remain in the somatodendritic compartment of dopamine neurons in the KO mice. It does not allow saying whether there are less or more D2R. Also, in control mice, it is surprising to note that there was no difference between the baseline response and the response after L-DOPA and sumatriptan. This would tend to suggest that L-DOPA did not boost dendritic dopamine release at all. Also, the sumatriptan-sensitive component is bigger in RIM KO mice compared to the control mice. What this means is unclear. Was there some compensatory adaptation in the 5-HT terminals in response to abrogation of axonal and dendritic dopamine release in these mice? Globally, these experiments with L-DOPA raise more questions than anything else.

We fully agree that the L-DOPA experiments do not indicate that the D2 receptor expression is identical in the two genotypes, and we currently do not have an approach to count D2 receptor numbers. We think that the data are important and would prefer to leave them in the manuscript because they show that RIM cKODA neurons have D2 dopamine receptors to sense dopamine. The striking absence of an IPSC cannot be explained by the complete or near complete loss of receptors.

The release of dopamine from 5-HT terminals and the augmentation of dopamine release from dopamine neurons has been studied in wild type animals before (Beckstead et al., 2004; Gantz et al., 2015). Those results show that the major increase in the IPSC results from the 5-HT terminals. There is also an increase in dendritic dopamine release that is relatively small compared to that from the 5-HT terminals. The new results in the control animals (which are heterozygous for RIM) mirror experiments in these wild type animals, and we hope that the reviewer concurs that there is value in having this comparison in the paper given that it was not possible to have wild type littermates as controls included. To us, the observation that sumatriptan completely blocked the IPSCs in the RIM cKODA animals was a gratifying confirmation of the severity of loss of dopamine exocytosis in these animals!

5) To more directly address the possibility of changes in D2 receptor responsiveness of dopamine neurons in the RIM KO mice, it seems to me that it would be better to simply measure membrane currents evoked in dopamine neurons by local application of a D2 agonist. This would nicely complement the experiments of Figure 2 with amphetamine.

We thank the reviewer for suggesting this experiment. We have performed it and included it in the revised manuscript (Figure 1—figure supplement 3). We have addressed the point in detail in the response to the editorial decision (major point 2) and also note that the spontaneous event amplitude does not change. Together, these observations make a strong effect on dopamine receptor localization or function very unlikely.

6) The experiments of Figure 3 are quite interesting. The authors should also provide the frequency of these spontaneous events in control and KO neurons.

We thank the reviewer for proposing this experiment. It is a laborious experiment given the low frequency and difficult detection, but we think that the new data added in response to this suggestion make the manuscript significantly stronger. This point is also addressed in the response to the editorial decision, major point 2.

7) The absence of RIM starting from the late embryonic period could have perturbed the development of dopamine neurons, potentially leading to altered dendritic development. The capacitance results from Figure 1—figure supplement 1 argue that there were no major changes in the size of dopamine neurons in the KO mice. However, do the authors have the result of a TH immunostaining experiment in the substantia nigra to determine whether the global density of dopaminergic dendrites was altered in these KO mice?

We have addressed this point with new experiments and described in the response to the editorial decision letter major point 1, and the new data are shown in Figures 2A and 2B. There is no detectable effect on dendritic development.

https://doi.org/10.7554/eLife.47972.012

Article and author information

Author details

  1. Brooks G Robinson

    The Vollum Institute, Oregon Health & Science University, Portland, United States
    Contribution
    Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Writing—original draft
    For correspondence
    robinbro@ohsu.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5020-531X
  2. Xintong Cai

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Formal analysis, Investigation, Methodology, Writing—review and editing
    Competing interests
    No competing interests declared
  3. Jiexin Wang

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Resources, Investigation
    Competing interests
    No competing interests declared
  4. James R Bunzow

    The Vollum Institute, Oregon Health & Science University, Portland, United States
    Contribution
    Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  5. John T Williams

    The Vollum Institute, Oregon Health & Science University, Portland, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing—original draft, Project administration
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0647-6144
  6. Pascal S Kaeser

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration
    For correspondence
    kaeser@hms.harvard.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1558-1958

Funding

National Institute of Neurological Disorders and Stroke (R01NS083898)

  • Pascal S Kaeser

National Institute on Drug Abuse (R01DA04523)

  • John T Williams

National Institute of Neurological Disorders and Stroke (R01NS103484)

  • Pascal S Kaeser

National Institute on Drug Abuse (K99DA044287)

  • Brooks G Robinson

Harvard Medical School

  • Pascal S Kaeser

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by the National Institutes of Health (R01NS083898 and R01NS103484 to PSK, R01DA04523 to JTW, K99DA044287 to BGR), by the Dean’s Initiative Award for Innovation (to PSK), and by a Harvard-MIT Joint Research Grant (to PSK). We thank Dr. Changliang Liu for insightful discussions.

Ethics

Animal experimentation: All animal experiments were performed according to institutional guidelines of Harvard University and of Oregon Health & Science University, and were in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animals were handled according to protocols (protocol numbers Harvard IS00000049, OHSU IP00000160) approved by the institutional animal care and use committee (IACUC).

Senior Editor

  1. Eve Marder, Brandeis University, United States

Reviewing Editor

  1. Olivier J Manzoni, Aix-Marseille University, INSERM, INMED, France

Reviewers

  1. Nicolas Tritsch
  2. Pablo E Castillo, Albert Einstein College of Medicine, United States
  3. Louis-Eric Trudeau, Université de Montréal, Canada

Publication history

  1. Received: April 26, 2019
  2. Accepted: September 4, 2019
  3. Accepted Manuscript published: September 5, 2019 (version 1)
  4. Version of Record published: September 20, 2019 (version 2)

Copyright

© 2019, Robinson et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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