Dopamine-mediated calcium channel regulation in synaptic suppression in L. stagnalis interneurons

ABSTRACT D2 dopamine receptor-mediated suppression of synaptic transmission from interneurons plays a key role in neurobiological functions across species, ranging from respiration to memory formation. In this study, we investigated the mechanisms of D2 receptor-dependent suppression using soma-soma synapse between respiratory interneuron VD4 and LPeD1 in the mollusk Lymnaea stagnalis (L. stagnalis). We studied the effects of dopamine on voltage-dependent Ca2+ current and synaptic vesicle release from the VD4. We report that dopamine inhibits voltage-dependent Ca2+ current in the VD4 by both voltage-dependent and -independent mechanisms. Dopamine also suppresses synaptic vesicle release downstream of activity-dependent Ca2+ influx. Our study demonstrated that dopamine acts through D2 receptors to inhibit interneuron synaptic transmission through both voltage-dependent Ca2+ channel-dependent and -independent pathways. Taken together, these findings expand our understanding of dopamine function and fundamental mechanisms that shape the dynamics of neural circuit.


Introduction
Dopamine (DA) regulation of neural circuit function is ubiquitously observed across species. Acting through D1 and D2 classes of G protein-coupled receptors (GPCRs), DA broadly regulates synaptic transmission in the central nervous system (CNS) through modulation of presynaptic neurotransmitter release and postsynaptic response [92]. In particular, DAergic modulation of synaptic output from interneurons, through the activation of D2 receptors, modulates circuit function in a wide range of neurobiological functions, from fundamental homeostatic processes to higher order neural function. For example, in the freshwater pond snail Lymnaea stagnalis, rhythmic DAergic inhibition of respiratory interneuron output is key to mediating aerial respiration behaviour [88,75]. In the mammalian brain, DAergic D2 receptor-dependent inhibition of neurotransmitter release from interneurons in the striatum [21,23,53] and amygdala [10,19,72] are critically involved in motor control and associative learning. Therefore, understanding the mechanisms underlying D2 receptor-mediated DAergic inhibition of interneuron synaptic output is of particular physiological significance.
Activation of D2 receptors inhibited voltage-gated Ca 2+ current (I Ca ) in both L. stagnalis [4] and mammalian [99,16,74] interneurons. As activity-induced Ca 2+ influx is a key determinant of presynaptic vesicle release [104], this likely contributes to DAergic inhibition of interneuron synaptic output. D2 receptor-mediated inhibition of voltage-gated Ca 2+ channels occurs through both voltage-dependent and -independent mechanisms [15,15,60]. The voltage-dependent mechanism involves direct binding between the G-protein bg subunit and the a1 subunit of voltage-gated Ca 2+ channels [20,37,79,101], which results in reduction of the peak current amplitude and slowing of current activation kinetics [71,2,5,31]. The voltage-dependence stems from the ability of a strong depolarization to induce unbinding of the Gbg subunit and relieve part of the inhibition [5,13,30,42,57]. In contrast, the slower and longer-lasting voltage-independent inhibition can be mediated through a variety of mechanisms, including regulation of voltage-gated Ca 2+ channel surface expression levels through direct interaction with GPCRs [60], involvement of the Ga subunit [51], soluble second messenger cascades [49,38] and kinase activation [80,15,25]. However, whether D2 receptormediated inhibition of voltage-gated Ca 2+ channels leads to synaptic suppression in interneurons remains unclear.
The central pattern generator network that gives rise to the aerial respiration behaviour in the mollusk Lymnaea stagnalis provides a well-characterized and highly tractable model for examining DA-mediated regulation of voltage-gated Ca 2+ channels and synaptic output in interneurons. This circuit consists of the DAergic pacemaker neuron RPeD1 and the follower interneurons VD4 and IP3i [96]. DAergic input from the RPeD1 to the follower cells is necessary for rhythmic network activity of the CPG, as when VD4 and IP3i are cultured together in the absence of the RPeD1, pulsatile application of DA elicited a series of alternating bursts similar to that exhibited by the intact three-interneuron network both in vivo and in vitro [88]. In a well-established in vitro soma-soma synapse model [34], VD4 forms an excitatory cholinergic synapse with its postsynaptic target LPeD1 [45,97,98]. The soma-soma synapse between VD4 and LPeD1 allows direct access to both pre-and post-synaptic compartments for electrophysiological, pharmacological, and imaging studies. Furthermore, DA receptors, G protein subunits and Cav2 channels have been well-characterized in the L. stagnalis CNS. Both D1-and D2-like receptors have been identified in L. stagnalis central neurons [26,48]. The effects of DA on the VD4 and IP3i are shown to be mediated through D2-like receptors, as the effects can be blocked only by the D2 receptor antagonist, sulpiride [68,69].
In this study, we employed the VD4 to LPeD1 synapse model to study the endogenous mechanisms underlying DAergic modulation of voltage-gated Ca 2+ channels and synaptic output in interneurons. We found that DA not only inhibits I Ca in the VD4 through both voltage-dependent and independent pathways, but also suppresses synaptic vesicle release downstream of voltage-gated Ca 2+ channel-conducted Ca 2+ influx.
Protein sequences of vertebrate orthologs of D2 subfamily receptors, G protein subunits and Cav2 channels were retrieved from UniProt [91] (Table 1). For each protein, sequences of the orthologs were aligned via MUSCLE [29] to create a profile HMM (HMMER v3.1b1) [28] that was then used to query the predicted protein-coding sequences of the L. stagnalis CNS transcriptome (cut-off E-value:1e-5). The top hit was searched against the Nr database using BLAST [1] to confirm protein identity. The L. stagnalis sequences were aligned with their respective vertebrate orthologs using MUSCLE and analyzed in MEGA7 (Kumar et al., 2016) to find the best-fit model and construct the phylogenetic tree with 500 bootstrap replicates. Functional protein domains were identified according to UniProt annotations.

Dissection
All animals were dissected under sterile conditions. Snails were anaesthetized for 10 minutes in »10% (v/v) Listerine in water. The anaesthetized snails were then de-shelled with fine curved forceps and pinned to a black silicone rubber-based dissection dish containing normal snail saline (NSS) (mM: 51.3 NaCl, 1.7 KCl, 4.1 CaCl2, and 1.5 MgCl2, 5.0 HEPES, pH adjusted to 7.9 with NaOH). A dorsal midline incision was made to expose the CNS, including the central ring and the buccal ganglia from under the buccal mass. The CNS was subsequently removed from the animal using fine dissection scissors and immediately placed in the antibiotic-containing solution (ACS): NSS containing 50 mg/mL gentamycin (Sigma, ON, Canada).

Cell culture
Conditioning medium (CM) was prepared by incubating 8-12 acutely isolated central ring ganglia in 6 ml defined medium (DM) (mM: serum-free 50% (v/v) Liebowitz L-15 medium (GIBCO, NY, USA) containing 40.0 NaCl, 1.7 KCl, 4.1 CaCl2, 1.5 MgCl2, 5.0 HEPES, 10 glucose, 1.0 L-glutamine, 20-mg/mL gentamycin, pH adjusted to 7.9 with NaOH) in glass Petri dishes pre-coated with Sigmacote (Sigma) for 72 hrs, as described previously [81,34,89]. The resulting 1X CM was frozen in polypropylene tubes at ¡20 C until required. Subsequently, the brains were then rewashed in ACS and then re-incubated in DM for another 3-5 days to produce 2X CM. A further session of washing and re-incubating for another week produced 3X CM. For cell isolation, the central ring ganglia were treated with trypsin (Type III, 3 mg/mL, Sigma) dissolved in NSS for 20 min to weaken the outer glial sheath surrounding the central ganglia. The brains were then treated with trypsin inhibitor (3 mg/mL, Sigma) dissolved in NSS for 18 min. Upon completion of digestion, the ganglia were then placed in CM on a black silicone rubber based dissection dish. The connective tissue sheath surrounding the neuronal ganglia was removed using fine forceps. The VD4 and LPeD1 neurons were identified by colour and size in the visceral ganglia and left pedal dorsal ganglia, respectively. Gentle suction via a 2mm fire-polished pipette (World Precision Instruments, FL, USA) that was coated with Sigmacote (Sigma) was used to isolate the identified neurons. The neurons were subsequently plated onto a poly-L-lysine coated glass bottom culture dish filled with pre-prepared CM.
For single cell configuration, acutely isolated VD4 neurons were plated in culture dishes independently from one another for 24 hrs prior to testing. To culture synaptic pairs, acutely isolated VD4 and LPeD1 neurons were plated in culture dishes in close proximity to each other to allow for direct contact between respective neurites. Neurons were kept in culture for 24 hrs to allow for synaptic formation prior to experiments. The formation of the synapse was confirmed by electrical analysis through noting the existence of a postsynaptic response correlated to presynaptic electrical stimulation.

Electrophysiology
As previously described electrophysiological recordings were performed under current clamp mode [34,33,39], or voltage-clamp mode [33,52,65], using an Axopatch 700A amplifier (Axon Instruments, CA, USA) connected to an analog-to-digital interface Digidata 1322 that was linked to a personal computer running pClamp9 (Axon Instruments) [39,65]. Recording pipettes were pulled using a Flaming-Brown micropipette puller (Model P-87, Sutter Instruments, Novato, CA, USA. The data were filtered at 1 kHz (-3 dB) using a 4-pole Bessel filter and digitized at a sampling frequency of 2 kHz. Data was analyzed using Clampfit version 9.2 (Axon Instruments). All curve fittings were carried out using Origin 7 (OriginLab, MA, USA).
For voltage step experiments, currents were elicited by stepping from the holding potential to the test potential of +30mV for 50 ms. A prepulse protocol was applied consisting of a holding potential of ¡70 mV which first stepped to a depolarization of +80 mV for 10 ms, then to test voltage of +30mV for 50 ms separated by an inter-pulse voltage of ¡70 mV for 5 ms ( Figure 7A). To generate the current-voltage (IV) curve, the holding potential was set to ¡70 mV and 50 ms depolarizing steps from ¡40 mV to +100mV with a +5mV interval was used. The IV relation was generated from peak inward current using a Boltzmann fit (I=G Ã (V-Vrev) Ã (1-(1/(1+exp((V-Vh)/ S))))).
To generate the AP waveform (APW), single VD4 neurons were current clamped via a sharp intracellular pipette in NSS bath solution to evoke and record APs endogenous to those neurons. Positive current was injected at levels to induce and maintain an AP train at 2Hz. This frequency was chosen because it represents a sustainable firing rate composed of single APs. The averaged AP profile showed a threshold voltage of ¡29 mV, an overshoot voltage of +35mV, and half width of 4.5 ms ( Figure 6A). Experiments conducted using the APW was controlled using pClamp9. The APW was flanked with the holding potential of ¡70 mV. Data acquisition was filtered at 1 kHz (-3 dB) using a 4-pole Bessel filter and digitized at a sampling frequency of 2 kHz. Leak currents were subtracted via P/N leak subtraction protocol using 4 positive subsweeps from a holding level of ¡70 mV. Data was analyzed using Clampfit version 9.2. All curve fittings were carried out using Origin 7.
Simultaneous pre-and post-synaptic monitoring of VD4-LPeD1 synaptic transmission To study synaptic transmission between the VD4 and LPeD1 neurons, a dual clamp protocol was used ( Figure 4B). The presynaptic cell was current clamped via the intracellular sharp electrode and the postsynaptic cell was simultaneously voltage clamped via the patch electrode. Prior to testing, the presynaptic neuron was kept hyperpolarized to ¡70 mV by negative current injection to prevent any unwanted firing of APs. During testing, APs were induced by positive current injection to depolarize the neuron just past its firing threshold. A 5s long 1 Hz current pulse train was used to elicit five APs. Postsynaptic responses were detected as excitatory postsynaptic currents (EPSCs). The existence of a functional synapse was confirmed when an EPSC was correlated with a presynaptic AP. All recordings were carried out in NSS bath solution.
Ratiometric FURA-2 Ca 2+ imaging Intracellular Ca 2+ ([Ca 2+ ] i ) was measured using a FURA-2 ratiometric Ca 2+ imaging system, as described previously [33]. For intracellular sharp recording experiments, the cell-permeant AM-FURA-2, was used. The cells were incubated with 10 mM AM-FURA-2 (Molecular Probes, OR, USA) for 30 minutes and washed out with recording solution three times prior to imaging. The experiments were carried out in the dark to prevent photobleaching of the dye. The cells were stimulated with simultaneous intracellular recording, and the corresponding FURA-2 fluorescence signal was excited at 340 and 380 nm wavelengths generated by UV light from a 100W Hg/Xe-arc lamp, alternately passed through 340 and 380 nm excitation filters using a high speed random monochromator controlled by Image Pro 5 (Photon Technology International). The fluorescence signal, generated after the excitation light, was reflected via a 430 nm dichroic mirror, passed through a 530 nm long-pass emission filter, and detected and digitized by an intensified charged-coupled device (ICCD) camera (Photon Technology International) in Image Pro 5. Signal acquisition was done in hispeed acquisition mode using a 10 ms exposure time per excitation wavelength at 8x8 binning. Intensity of fluorescent signals detected at 340 and 380 nm were then processed to floating point images before being used to generate Poenie-Tsien ratio intensity images. These images were used for all FURA-2 analyses.
For voltage clamp experiments, the cell impermeant FURA-2 penta-potassium salt was loaded into neurons through the patch pipette by inclusion in the pipette solution. A final concentration of 100 mM FURA-2 penta-potassium salt was used and allowed to dialyze into the neuron after establishing a seal and rupture.

FM1-43 imaging of vesicle release
Levels of vesicle release were monitored using the styryl dye N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide (FM1-43) (Molecular Probes). This water-soluble dye fluoresces when it binds to a lipid-rich membrane and has been commonly used to visualize synaptic vesicles following endocytotic processes. FM1-43 dye (4 mM) was added to culture dishes and allowed to equilibrate for 5 minutes. To load the dye, neurons were induced to fire APs by intracellular sharp electrode current injection. Two 5s current pulses separated by 30s were used, which caused endocytosis of the FM1-43 dye. Any dye that was not endocytosed was subsequently washed away by exchanging NSS bath solution once every 5 minutes for a total of three washes. The experiments were carried out in the dark to prevent photobleaching of the dye. Exocytosis of FM1-43 dye and subsequent destaining of dye was achieved by either electrical stimulation or ionomycin application. Electrical stimulation of vesicle release was performed using intracellular electrodes to inject current pulses into FM1-43 loaded neurons, to cause unloading of dye. The current injection protocol consisted of three current injection pulses of 5s each separated by 30s, which was controlled by pClamp9 software. Ionomycin stimulation of vesicle release was performed by rapid bath exchange of 2 mM ionomycin dissolved in NSS bath solution onto neurons.
Imaging of FM1-43 fluorescence was acquired simultaneously with the above mentioned stimulus protocols. FM1-43 fluorescence signal was excited at 488 nm wavelength generated by UV light from a 100 W Hg/Xe-arc lamp, alternately passed through a 488nm excitation filter, which was controlled by Image Pro 5. Emitted light was passed through a band pass filter 510 nm-560 nm and detected and digitized by an intensified chargedcoupled device (ICCD) camera in Image Pro 5. Signals were sampled at 0.5Hz using a 300 ms exposure time and 4£4 binning.
Fluorescent intensities in single neurons were analyzed using the averaged fluorescence from the entire soma. In synaptically paired neurons, fluorescence intensities were taken from three regions: (i) presynaptic contact site (CS), (ii) presynaptic non-contact site, (iii) postsynaptic non-contact site ( Figure 4E).

Data analysis and statistics
Unless otherwise stated, data are presented as the mean § s.e.m. Statistical analyses were carried out using SigmaStat 3.0 (Jandel Scientific, IL, USA). Differences between mean values from each experimental group were tested using a paired t-test for two groups or one-way analysis of variance (ANOVA, Holm-Sidak or Tukey method) followed by Holm-Sidak post-hoc test for multiple comparisons. Differences were considered significant if p < 0.05.

Results
Identification and phylogenetic analyses of L. stagnalis orthologs of D2-like receptors, G protein subunits and Cav2 channels We first examined the L. stagnalis CNS transcriptome [82] to characterize the conservation of genes involved in DAergic regulation of Cav2 channels in central neurons. Whereas the D2-like subfamily of receptors in vertebrates is known to include three isoforms, D2, D3 and D4 receptors, we found that the L. stagnalis CNS expresses a single D2-like receptor (Figure 1) in which the two agonist bindings sites show partial sequence similarity with vertebrate D2-4 receptors. Consistent with previous reports [62,61], we observed that L. staganalis Gai ( Figure 2A) and Gao ( Figure 2B) subunits share high degrees of sequence identity with their vertebrate counterparts, with complete conservation of the four guanine nucleotide binding sites required for GTP binding. Similarly, the seven WD repeat domains in the single L. stagnalis Gb protein ( Figure 2C), which are implicated in protein-protein interaction [63], are nearly identical to those in vertebrate Gb 1 orthologs. Greater sequence divergence was observed in the single L. stagnalis Gg isoform as compared to vertebrate Gg 2 orthologs ( Figure 2D). Finally, sequence analyses of the L. stagnalis Ca V 2 (LCa V 2) channel ortholog Ca V 2, demonstrate that two of the three Gb 1 g 2 binding sites, one in the N-terminus cytoplasmic tail and another in the I-II linker region, on vertebrate Ca V 2.1 and Ca V 2.2 channels that are essential for G protein-mediated    voltage-dependent regulation of Cav2 channels [102] are conserved in considerable degrees in L Ca V 2. Taken together, these findings demonstrate L. stagnalis to be a conserved model for examining the fundamental mechanisms of G protein-mediated DAergic modulation of Ca V 2 channels in the CNS.

DA inhibits synaptic output of the VD4
VD4 and LPeD1 were acutely isolated and cultured for 24 hours to allow for soma-soma synapse formation prior to testing, as previously described by [34]. Only pairs adopting the soma-soma synapse morphology were selected for recordings ( Figure 4A). To test the effects of DA on synaptic transmission between the VD4 and LPeD1, postsynaptic responses to presynaptic APs were examined using dual-clamp recordings ( Figure 4B). As it has previously been shown that 10 mM DA nearly abolishes all evoked action potential firing activity in VD4 interneurons [4], we employed 1 mM DA here to ensure sufficient VD4 synaptic output and detectable LPeD1 post-synaptic response. Indicative of the formation of an excitatory synapse between VD4 and LPeD1, recordings showed a oneto-one correlation between presynaptic APs and EPSCs ( Figure 4C). As commonly observed, the first EPSC from the train of five APs had a larger amplitude than the remaining four APs. We noted that DA affected the properties of the AP train, however did not change the first AP of the train. We thus compared the first EPSC elicited by the first AP, before and after DA application. We found that DA resulted in a 37.8% reduction in peak amplitude of the first EPSC (no DA: ¡126.15 § 7.97 pA; DA: ¡78.46 § 10.38 pA, n = 4, p < 0.05) ( Figure 4D). Inhibition of synaptic transmission by DA was reversed by washing with NSS bath solution (¡101.33 § 9.67 pA, n = 4).
We next sought to determine whether the DAmediated reduction in EPSC amplitude was due to changes in vesicle release from the VD4, using the fluorescent styryl dye FM1-43 to visualize synaptic vesicle exocytosis ( Figure 4E). FM1-43 labelled synaptic pairs show intensified labelling at the  Figure 4F). Fewer vesicles situated at the pre-and postsynaptic non-contact sites (non-CSs) were released in response to evoked APs and their release was insensitive to DA ( Figure 4F). Taken together, these findings indicate that DA inhibited synaptic output of the VD4 through suppression of vesicle release.

DA inhibits I Ca in the VD4
Activity-induced Ca 2+ influx is a key determinant of presynaptic vesicle release. We first confirmed and expanded upon earlier findings by [4]. that 10 mM DA indeed inhibited the amplitude of I Ca in the VD4 ( Figure 5A and B), without affecting its activation properties ( Figure 5C). However, as a non-physiological square wave (SW) protocol was used to evoke I Ca , the physiological relevance of the observed inhibition by DA was unclear. Therefore, as shown in Figure 6A, we employed prerecorded VD4 AP waveforms (APW) as the voltage clamp command to examine the effect of the same concentration of DA on intracellular Ca 2+ dynamics during neuronal activity. We then compared the I Ca peak amplitude induced by either APW ( Figure 6B) or SW ( Figure 6C) in response to 10 mM DA. We found that DA reduced the average peak amplitude of APW-evoked I Ca by 36.9 § 9.01% (n = 4) ( Figure 6B), which was comparable with the extent of DA-mediated reduction observed in I Ca elicited by a SW voltage step to peak APW amplitude (+30 mV) (36.10 § 5.03%, n = 6; p>0.05) ( Figure 6C). The 10 mM DA-induced reduction of I Ca evoked by SW or APW DA was consistent, as summarized in Figure 6D. Clearly, DA inhibited Ca 2+ influx evoked by neuronal activity in the VD4.
Prepulse depolarization facilitates I Ca in the presence of DA Next, we sought to investigate the mechanisms underlying DAergic inhibition of I Ca in the VD4.
GPCR-mediated inhibition of presynaptic voltagegated Ca 2+ channels, via the liberated Gbg subunit, can be detected using a prepulse depolarization that causes unbinding of the Gbg subunit from the channel and disinhibition of I Ca [47,54] ( Figure 7A). We found that DAergic inhibition of I Ca (36.10 § 5.03%, n=5) in the VD4 was significantly reduced by prepulse depolarization (26.95 § 3.41%, n=5, p < 0.05) ( Figure 7B and C). The prepulse had no effect on I Ca in the absence of DA ( Figure 7B and C). These findings suggest that DAergic inhibition of I Ca in the VD4 involved Gbg-dependent signalling.

DA inhibits ionomycin-induced synaptic vesicle release from the VD4
Several studies have shown that neuromodulators can also inhibit presynaptic vesicle release in neurons independently of their effects on Ca 2+ dynamics, such as through direct G protein-dependent interaction with the exocytic machinery [70,11,12,40,41,78,93,100]. To investigate the possibility that DA also modulates VD4 synaptic release downstream of Ca 2+ influx, we employed the ionophore ionomycin to mediate Ca 2+ entry into the cell independently of voltage-gated Ca 2+ channels [18]. Upon addition of ionomycin, the linear relationship between extracellular Ca 2+ concentration and FURA-2 imaging of intracellular Ca 2+ concentration indicated that the source of the Ca 2+ influx is from the extracellular bath ( Figure 8A). First, we confirmed that ionomycin-induced Ca 2+ influx was sufficient to trigger vesicle release by monitoring intracellular Ca 2+ concentration and vesicle release using FURA-2 fluorescence and FM1-43 destaining, respectively ( Figure 8B). We observed that ionomycin caused a rapid influx of Ca 2+ that then reached an equilibrium level and remained constant. In parallel, FM1-43 fluorescence traces showed two components: a rapid exponential decrease in fluorescence directly after ionomycin application, followed by a slow linear decay in fluorescence. Finally, we examined the effects of DA on ionomycin-induced vesicle release. Interestingly, exogenous DA was still able to attenuate ionomycin-induced vesicle release in the VD4 by 66.9% (no DA: 221.2 § 35.6 a.u., n = 6; DA: 73.3 § 11.5 a.u., n = 4; p < 0.05) ( Figure 8C and D). Taken together, these results indicate that DA also inhibited VD4 synaptic output via voltage-gated Ca 2+ channel-independent mechanisms.

Discussion
In this study, we examined the effects of DA on I Ca and vesicle release in a well-characterized molluscan interneuron synapse model. We demonstrated for the first time that DAergic inhibition of synaptic output is mediated through both voltage-dependent and -independent inhibition of voltage-gated Ca 2+ channels, and voltage-gated Ca 2+ channel-independent suppression of vesicle release.

The DAergic system in L. stagnalis
In mammalian neurons, DA broadly regulates neurotransmission through activation of a family of GPCRs, which are divided into two major classes based on their structural, pharmacological, and signalling properties: D1 and D5 receptors belong to the subfamily of D1-like receptors, whereas D2, D3, and D4 receptors are grouped into the D2-like receptor class. Both D1/ 3-and D2/4-like receptors have been identified in L. stagnalis based on their pharmacological properties, albeit with differing sensitivities to various agonists and antagonists as compared to their vertebrate counterparts [86,3,48,69,94]. The D1-and D2-like receptors in L. stagnalis exhibit differential expression patterns, with the former present on growth cones and the latter on the soma [26]. The effects of DA on the VD4 are shown to be mediated by D2-like receptors, as it has been shown that the effets of both synaptically released DA from the RPeD1 and exogenously applied dopamine on the VD4 can be blocked only by sulpiride, a D2 receptor antagonist [68]. Indeed, DAergic inhibition of I Ca in the VD4 [4] is consistent with observations of D2 receptor effects in mammalian CNS [74,16,50,73]. While the downstream targets and signaling mechanisms of D2-like receptors in L. stagnalis remain unclear, previous studies [62,61]; Knol et al., 1995) and our sequence analyses of G protein subunits (Figure 2) in the L. stagnalis CNS have demonstrated that they are highly homologous to their vertebrate counterparts. Taken together, L. stagnalis provides a conserved model for studying DAergic regulation of interneuron function.
There are several potential limitations to the cultured VD4 interneuron electrophysiological experimental model employed in studies thus far, including our current report, in recapitulating the physiological conditions of DAergic modulation of interneurons in L. stagnalis. As the VD4 likely receives pulsatile DAergic input from the presynaptic rhythmically active pacemaker neuron RPeD1 [85,88], the bath-applied tonic DA treatment may not fully mimick the effects of phasically released DA on VD4 membrane and synaptic properties in vivo. While the sensitivity of cultured VD4 interneurons to bath-applied DA has been characterized [4], it is yet unclear what range of DA concentrations act on the VD4 interneuron in vivo.

Voltage-dependent and independent inhibition of I Ca by DA
Activity-induced Ca 2+ influx is a key determinant of presynaptic vesicle release [104]. A reduction in the amount of Ca 2+ influx through voltage-gated Ca 2+ channels at the active zone would efficiently reduce the probability of synaptic vesicle fusion and therefore neurotransmission [58,46]. Previous studies have reported that DAergic inhibition of synaptic output in interneurons is associated with reductions in I Ca in mammalian interneurons [99,16]. In this study, we confirmed and expanded upon the findings of [4]. by demonstrating that DA inhibits voltage-dependent Ca 2+ influx elicited by physiologically relevant stimuli, i.e. action potential waveforms, in the VD4. These findings suggest that DA likely inhibits activityinduced Ca 2+ influx in the presynaptic terminal that is required for neurotransmitter release.
Prepulse facilitation of VD4 I Ca in the presence of DA indicates that this inhibition is mediated in part through voltage-dependent G-protein-mediated inhibition, which is shown to be mediated through direct binding of membrane-delimited Gbg to cytoplasmic regions of the a1 subunit of Ca V 2 family of voltagegated Ca 2+ channels, resulting in delay of the activation kinetics and a depolarizing shift in the activation voltage of the channel [27,5,31,36,47,54,103]. In this study, we observed only a reduction in I Ca amplitude without changes in its activation kinetics. As the effects of Gbg-mediated inhibition are shown to be dependent on the Ca V b subunit subtype [17,32], this may in part be due to differences in the L. stagnalis and mammalian Ca v b subunit properties. A previous study reported that the L. stagnalis Ca V 2 ortholog, LCa V 2, does not exhibit prepulse facilitation when coexpressed in HEK-293T cells with rat orthologs of the Ca V 2 channel accessory subunits a2d1 and b1b and G protein subunits b1 and g2 [51]. As protein-protein interaction between LCa V 2 and rat Gb1g2 was not directly confirmed in the study, and given the sequence divergence we observed between L. stagnalis and vertebrate Gb ( Figure 2C), Gg ( Figure 2D), and Ca V 2 channel Gbg binding sites (Figure 3), the discrepancies between our findings may be attributable in part to non-canonical associations between LCa V 2 and mammalian Gbg protein. In addition, endogenous factors present in L. stagnalis neurons but absent in HEK-293T cells may also be required for the G protein-mediated voltage-dependent regulation of LCa V 2 channels observed in this study.
The depolarizing prepulse relieved only »30% of DAergic inhibition of I Ca in the VD4 in our study, suggesting that DA also inhibited I Ca through a voltage-independent mechanism. G protein-mediated voltage-independent inhibition of LCa V 2 in the VD4 has been reported to be mediated through the Ga subunit and increases in cAMP levels, with a slow time course on the scale of minutes [51]. Slow but persistent voltage-independent G protein-mediated inhibition of voltage-gated Ca 2+ channels has also been well-documented in mammalian neurons [9,6,7,22,56,66,83,87,102] and shown to be mediated by a variety of mechanisms, including regulation of Ca v surface expression levels through direct interaction with GPCRs [59,60] and phosphatidylinositol 4,5-bisphosphate (PIP 2 ) depletion [49,38] and kinase activation [80,25].
Parallel activation of voltage-dependent and independent inhibition of voltage-gated Ca 2+ channels has been hypothesized to allow for fine-tuning of intracellular Ca 2+ levels and activity-dependent plasticity [66,14,51,76,95]. The relative ratio between voltagedependent and -independent modes of inhibition in chick sensory neurons is shown to be critically dependent on intracellular Gbg concentration [64]. Activation of D2 receptors induces both voltage-dependent and -independent inhibition of the N-type voltagegated Ca 2+ channels in HEK-293T cells [60] and differentiated NG108-15 cells [15]. Consistent with the previous reports, our findings demonstrated DA effects are mediated by both voltage-dependent and -independent inhibition. It is likely that the synaptically released DA level onto interneurons varies, and thus can differentially modify interneuron intracellular Ca 2+ dynamics and synaptic output, thereby inducing cellular and circuit-wide plasticity.

DAergic inhibition of vesicle release downstream of Ca 2+ influx
In neurons and secretory cells, GPCRs can inhibit vesicle release in parallel with changes in I Ca [70,18,24,77,84]. This inhibition can occur through direct binding of the Gbg subunit to the C-terminus of the SNARE protein SNAP-25, thereby displacing synaptotagmin and preventing vesicle exocytosis [40,93,105]. Gbg is also known to interact with several other proteins involved in synaptic vesicle release, including syntaxin, VAMP, CSP and synaptotagmin [55]. Binding of Gbg to these proteins may alter their function and/or interaction with other proteins in the exocytic machinery, thereby inhibiting vesicle release downstream of Ca 2+ influx [90]. While no direct interaction between G protein and presynaptic proteins have been reported in L. stagnalis, they are shown to be homologous in structure and function to their vertebrate counterparts (Figure 2) [62,61]; Knol et al., 1995;[35,43]. Here we employed the ionophore ionomycin to induce Ca 2+ -dependent synaptic release independently of voltage-gated Ca 2+ channel activation, as it has been shown in hippocampal neurons that ionomycin-induced synaptic release is unaffected by the voltage-gated Ca 2+ channel blocker Cd 2+ [18]. We demonstrated that DA inhibits vesicle release from VD4 interneurons downstream of activityinduced Ca 2+ influx. Our finding suggests that L. stagnalis may serve as an easily tractable model in which to elucidate the molecular mechanisms of G proteinmediated modulation of synaptic release.

Potential functional significance of redundant mechanisms of DAergic inhibition of synaptic transmission in interneurons
In this study, we provide the first demonstration that inhibition of I Ca and direct inhibition of synaptic vesicle release all contribute to DAergic presynaptic inhibition in interneurons. It may be hypothesized that whereas the Gbg subunit-mediated voltage-dependent inhibition allows for rapid activity-dependent regulation of synaptic transmission, the voltage-independent inhibition of I Ca and direct inhibition of vesicle release machinery may allow for more persistent and nuanced inhibition of interneuron synaptic output [66,14,51,76,95]. Indeed, strong but modifiable inhibition of the VD4 activity may be integral to respiratory control in L. stagnalis. In the context of the respiratory CPG network, the VD4 controls the muscles that close the pneumostome [89]. Increased firing of the RPeD1 neuron, which results in increased DA release onto VD4, coincides with opening of the pneumostome and initiation of the inspiration phase [88]. Therefore, complete inhibition of VD4 output during this phase is likely crucial to prevent interruption of inspiration and thus generating normal rhythmic respiration behaviour. In addition, the capacity for activity-dependent plasticity in the VD4 response to DA, endowed by voltage-dependent inhibition of voltage-gated Ca 2+ channels, may be important for the well-documented hypoxia and learning-induced modulation of CPG activity and aerial respiration behaviour in L. stagnalis [67,8].

Conclusion
In this study, we provide the first demonstration that DA suppresses synaptic output of a molluscan respiratory interneuron through both voltage-dependent and -independent inhibition of I Ca , and voltage-gated Ca 2+ channel-independent inhibition of synaptic vesicle release. In future studies, the relative sensitivity and function of each pathway and any potential interactions between them will be further investigated to expand our understanding of the fundamental mechanisms that shape interneuron function and neuronal network dynamics.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.