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. In addition to secretion from axonal nerve terminals, many neurons release neurotransmitters or neuromodulators from their somata and dendrites . ImportaA 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 . SomatodElectrophysiological 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 . The D2-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 . Recent DA mice, in which RIM1 and RIM2 were specifically removed in dopamine neurons by crossing conditional RIM ‘floxed’ alleles to DATIRES-cre mice . 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  and to determine the current density. There was no significant difference between RIM control and RIM cKODA animals  and Rims2 (‘floxed RIM2’) genes  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.RIM cKO’) genes  with DATcre mice . RIM cKO2, 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 . 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 . 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 (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 MgClor 5 min , followeor 5 min . All eleDA 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  + 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 . Cell filling, image acquisition and quantification were performed by experimenters blind to the genotype.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  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 . SpontanDrugs 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. 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.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).Thank you for submitting your article "RIM is essential for stimulated but not spontaneous somatodendritic dopamine release in the midbrain" for consideration by 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 ?- 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 ?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 . 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 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? 1) Validation of DA neurons:- What midbrain dopamine neurons were analyzed in this study ?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.DA neurons . 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 , 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.We have performed the requested experiments. First, we have quantified spontaneous firing rates and there was no difference between RIM control and RIM cKODA.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  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 . 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 cKO- Is the global density of dopaminergic dendrites was altered in these KO mice (suggested experiment:TH immunostaining in the substantia nigra)?DA 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.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 slices by confocal microscopy, and performed Sholl-analyses  to analyze their branch structure. RIM knockout dopamine neurons appear identical to the control neurons and electrophysiological properties of the neurons were unchanged .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 cKOThese 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.DA 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  and amphetamine  are unchanged, and most importantly the amplitude of spontaneous D2 IPSCs is unaffected by RIM cKODA .We thank the reviewers for bringing this up and have performed this experiment as instructed . 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 cKOThe 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.DA 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 . 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.We have performed new experiments and analyses of the frequency and kinetics of spontaneous events in RIM control and RIM cKOThe kinetics of spontaneous and evoked D2-IPSCs and how they relate to one another have been extensively studied before . 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  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 ?We thank the reviewers to raise the point of RIM localization and we now address it . 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 , as best assessed by STED microscopy . Two reports, however, proposed that RIM also may have dendritic functions. Specifically, it was proposed that RIM1α may support postsynaptic trafficking in dendrites , and RIM3 and 4, small RIM version, localize in dendrites and control their growth .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 . 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  of these RIM clusters are within somata and dendrites . 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  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 .DA 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.We also agree that it would be great to have an electron microscopic assessment of the vesicular compartment for dopamine release in the RIM cKO- 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 . 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 , 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 , 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  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 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.DA 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.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 animals was a gratifying confirmation of the severity of loss of dopamine exocytosis in these animals!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 . 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 cKO5) 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 . 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.


In this article, an innovative approach for magnetic data communication is presented. For this purpose, the receiver coil of a conventional magneto-inductive communication system is replaced by a high-sensitivity wideband magnetic field sensor. The results show decisive advantages offered by sensitive magnetic field sensors, including a higher communication range for small receiver units. This approach supports numerous mobile applications where receiver size is limited, possibly in conjunction with multiple detectors. Numerical results are supported by a prototype implementation employing an anisotropic magneto-resistive sensor. Magneto-inductive (MI) communication is of growing interest in communication environments, where radio, optical wireless, acoustical, and molecular communications are challenging or not possible . ExampleIn this publication, an innovative MI communication system approach is introduced: the receiver coil of a conventional MI communication system is replaced by a high-sensitivity magnetic field detector, in our prototype an anisotropic magneto-resistive (AMR) detector b. These The paper is structured as follows. First, characteristics of magnetic field detectors are assessed in the context of MI communication. Afterwards, the sensitivity of coil-to-coil and coil-to-AMR communication is analyzed and compared. Finally, the results are summarized, and an outlook is given.SQUIDs are ultra-sensitive magnetic field sensors . The senThe optical pumping resonance method exhibits very high sensitivity at low frequencies, but the sensor is large and is only able to detect the scalar  value of a magnetic field. The major problem regarding communication purposes is that the sensitivity of these magnetometers decreases rapidly for frequencies above 10 Hz . This prHall effect sensors are among the most popular magnetic field sensors. The sensors are very cheap and are mainly used as proximity indicators. They cannot be used for navigation purposes due to their low sensitivity . Most prFluxgate sensors can detect magnetic fields on the order of pT. Due to the small size and battery power supply, fluxgate magnetometers are qualified for mobile applications. The achievable data rate is limited by the upper frequency of a fluxgate sensor, which is about 10 kHz .Another well-known type of detector is magneto-resistive sensors. GMR and AMR sensors are implemented today in almost every smartphone, e.g. for compass functionality. Both sensor types require ultra-low power consumption and are cheap. The main advantage is the high bandwidth of up to 1 MHz and beyond. Due to the lower detection limit of AMR sensors, this method is favorable for communication systems.Presently, various magnetic sensors are commercially available. Most of the sensors measure the magnetic flux density of the magnetic field based on different sensing principles. For certain measurement tasks certain types of magnetic field sensors have been established. For example, giant magneto-resistive (GMR) sensors are used in reading heads of hard disks, whereas superconducting quantum interference devices (SQUIDs) are applied for measuring weak biomagnetic fields. In this section, the most common sensors are characterized regarding their suitability as a wideband receiver in a magnetic communication system. The crucial characteristics with respect to mobile communication purposes are summarized in Beside the features mentioned in In the following, the receiver coil of a conventional MI communication system a and theB as weak as Coil-based magnetometers employ the principle of MI. This implies that a change of the magnetic flux density through a coil leads to a voltage difference between the clamps of the coil. The voltage difference is proportional to the alternating magnitude of the magnetic flux density. The magnetic flux density through a coil changes if it is exposed to a time-varying magnetic field. Based on the sensitivity analysis made in , the dynmeasured .The AMR effect has been discovered by William Thomsen in Glasgow in 1857 . He founIn our prototype implementation, a Sensitec AFF755 low noise AMR sensor in a SO8-housing is used  14]. Th. Th14]. A flip coil is implemented between pin 1 and 8 for offset correction and detection of weak magnetic fields . If a suIn this section, theoretical investigations and the corresponding numerical results are presented, based on measurements of our prototype implementation. For this purpose, the near-field far-field boundary is elaborated and the equation for transmission range is derived for coil-to-coil and coil-to-AMR communication. Then, the receiver circuit is characterized. Subsequently, coil-to-coil and coil-to-AMR communications are compared regarding different aspects.d at which the near-field passes into the far-field is conventionally defined by the frequency f of the transmitted signal and the speed of light in the medium In antenna engineering, the near-field and the far-field are spatial regions with different characteristics. In the region close to an antenna, i.e., in the near field, there is no electro-magnetic (EM) radiation. In the far-field region, a combination of electrical and magnetic waves, known as EM wave, is radiated. For a coil system, the distance  cmedium :(1)d=cmeThis equation is valid for all setups where the size of the antenna is much smaller than the wavelength. The maximum transmission range of a near-field MI communication system is limited by the corresponding near-field far-field boundary. For example, the near-field far-field boundary in air for An essential property for communication systems is the maximal transmission distance. In this section, the influence of different crucial parameters on the communication range is theoretically analyzed for coil-to-coil and coil-to-AMR communication. The system parameters used in the numerical results throughout this paper are listed in In conventional MI communication systems, the properties of transmitter and receiver coil and the coupling between the coils have an influence on the transmission range. The basis for the calculation of the communication characteristics is the Agbinya-Masihour model . The MI dcc61+rT2If the communication distance is large compared to the transmitter coil radius, the following approximation holds:This simplifies the link budget, so that the transmission range B at the receiver side [N the number of turns of the transmitter coil, and I the electrical current through the transmitter coil. Accordingly, the transmission range If an AMR detector is used, the maximum transmission distance depends on the magnetic flux density ver side :(7)B=μ0μB is replaced by the detection threshold of the magnetic sensor, If Equation  proves td = 10.1 cm in boresight direction regarding the transmitter coil. Then, the output voltage of the receiver is measured for different magnetic flux densities. The magnetic flux density, which is present at the AMR sensor, is a function of the coil properties, the current through the transmitter coil, and the distance between transmitter and receiver. To verify the given AMR sensitivity, the receiver is placed at a distance of To get the AMR sensitivity, the AMR output voltage before signal amplifying must be calculated and normalized to 1 V input voltage. Therefore, the voltage at the receiver output is divided by the amplifier gain (G = 2000) and the AMR sensor input voltage. The second important parameter that affects the detection threshold, is the noise level of the receiver. The magnetic field sensor in our prototype system can work at frequencies from 0 Hz up to 1 MHz. The main components that contribute to the total noise are the AMR sensor with 10 d 100 Hz ,18. For Based on the measured receiver sensitivity and the noise voltages for different bandwidths the corresponding detection thresholds can be determined. The results are compared with the detection thresholds calculated with the theoretical values given in the datasheets. For the bandwidth Equations  and 8)  8) show In the following, the influence of the transmitter and receiver coil radii on the coil-to-coil and the transmitter radius on the coil-to-AMR communication is investigated. Let us start with air coils, i.e., ion term  is of neIn the next investigation, the receiver side of both communication approaches is examined. For this purpose, the receiver coil radius for coil-to-coil transmission is varied. The results indicate that the range for coil-to-coil communication is severely restricted when small receiver coil radii are required. This is necessary for mobile applications such as AUVs and sensor networks. For these requirements, coil-to-AMR communication can serve much longer distances.Equation  shows thSubsequently, the influence of the permeability of the coil core material on the transmission range is examined for both communication techniques. For coil-to-coil communication, the transmitter and receiver coil core permeability are adapted simultaneously. For coil-to-AMR communication, only the transmitter coil core permeability can be changed.L according to L of a coil has a limiting effect on the resonant frequencyC of the resonant circuit is lower bounded by parasitic capacitances.However, the enhancement of the possible communication distance by means of increasing the permeability of the core material is limited by a few parameters. Primarily, there are no materials available that exhibit arbitrary high permeabilities. Additionally, the permeability of a material depends on the frequency, so that extremely high permeabilities for high frequencies usually cannot be achieved. Moreover, the saturation flux density nstraint . This reNow, the influence of the sensor’s detection limit, In this contribution, an innovative approach for magnetic communication is considered. The key idea is to employ a high-sensitivity wideband magnetic field sensor at the receiver side. Several advantages have been indicated compared to conventional MI coil-to-coil communication, because a different measurement principle is used compared to a receiver coil. In most of our numerical results an AMR sensor is assumed. AMR receivers are extremely small, so that they can be considered to be a point sink. Given the same form factor, they are much more sensitive than coils. This enables longer transmitting distances for mobile applications, where small receiver sizes are necessary. The frequency characteristic of AMR sensors is almost flat below the cut-off frequency. This feature together with the small size is of great interest for multiple-input single-output (MISO) system approaches. Vice versa, single-input multiple-output (SIMO) processing can be used for sensitivity improvements. Some AMR detectors include 3D functionality. This aspect is useful for joint communication and localization purposes. Possibly, the data rate can be enhanced by 3D processing. Moreover, a high permeability transmitter core material can be used for coil-to-AMR communication to extend the possible range drastically. Our investigation demonstrates that magnetic field communication with an AMR receiver is a promising technique for mobile applications in challenging environments. Since the effective transmission distance is a function of the sensitivity of the detectors, the fundamental observations of this paper can be exported to other measurement principles. For example, the AMR sensor used in our prototype receiver could be replaced by a fluxgate sensor.


Arenicola marina. To study effects of arenicin on complement activation in human blood serum, we used hemolytic assays of two types, with antibody sensitized sheep erythrocytes and rabbit erythrocytes. Complement activation was also assessed, by the level of C3a production that was measured by ELISA. We found that the effect of arenicin depends on its concentration. At relatively low concentrations the peptide stimulates complement activation and lysis of target erythrocytes, whereas at higher concentrations arenicin acts as a complement inhibitor. A hypothetical mechanism of peptide action is proposed, suggesting its interaction with two complement proteins, C1q and C3. The results lead to the possibility of the development of new approaches for therapy of diseases connected with complement dysregulation, using peptide regulators derived from natural antimicrobial peptides of invertebrates.Antimicrobial peptides from marine invertebrates are known not only to act like cytotoxic agents, but they also can display some additional activities in mammalian organisms. In particular, these peptides can modulate the complement system as was described for tachyplesin, a peptide from the horseshoe crab. In this work, we investigated the influence on complement activation of the antimicrobial peptide arenicin-1 from the marine polychaete  Arenicola marina. Three isoforms of arenicins have been described: arenicin-1, -2 [A. marina [The biologically active compounds derived from marine organisms are known to be unique, and can be used as a pattern for the development of new, effective pharmacological agents for treatment of different diseases . Antimicin-1, -2 . These cin-1, -2 ,5 stabil. marina . At micr. marina ,7,8,9, a. marina . Several. marina ,12,13,14Tachypleus trindentatus, and polyphemusins from Limulus polyphemus, which are related to tachyplesins [Arenicins have similar amino acid sequence and spatial structure to another group of antimicrobial peptides, tachyplesins, which have been isolated earlier from the horseshoe crab yplesins ,16  ,21. CompAccording to the structural similarity between arenicins and tachyplesins, we assumed that arenicin could also interact with C1q and that this might influence the complement activation. In a previous paper, we demonstrated that arenicin-1 really forms a stable complex with human C1q .In this paper, we studied the effects of arenicin-1 on complement activation and target cell lysis in two hemolytic models in vitro. We also used an ELISA test for C3a to confirm the complement activation in these models. We found that the effect of arenicin on the complement system depends on the concentration of the peptide. At relatively low concentrations, arenicin stimulates complement activation and/or target erythrocyte lysis. However, at higher concentrations arenicin behaves as a complement system inhibitor. Though it was not demonstrated directly, our results provide strong evidence about the interaction of arenicin with the C3 complement protein, in addition to the previously established interaction with C1q.sh), which is the model for the classical pathway of complement activation, we observed more than twice the baseline level of Esh lysis by 1% normal human serum (NHS) in the presence of arenicin-1 at concentrations 10 and 20 μg/mL, compared with control (samples without peptide) (In the hemolytic assay with antibody sensitized sheep erythrocytes (Epeptide) A. Howevesh lysis level differences reflect different complement activation levels, we developed an ELISA system for human C3a, a derivative of the complement protein C3, which is produced on complement activation. The test system was suitable for detection of C3a in the range 25–500 ng/mL, with no cross-reactivity to the uncleaved C3 protein.To assess whether Esh lysis and C3a production is illustrated in p < 0.05.After hemolytic assay, the same samples were used for C3a determination B. Both irab) is widely used. We utilized this assay to study arenicin action on the complement system as well. Since we used 4% serum in this model, we took a broader concentration range of arenicin. We found that in this model, arenicin retained its inhibitory action at high concentrations (80 and 160 μg/mL), essentially decreasing hemolysis level (rab lysis itself in heat-inactivated serum (data not shown).As a model of complement activation via the alternative pathway, a hemolytic assay with rabbit erythrocytes  were significantly diminished . In both models, arenicin revealed both activating and inhibitory effects depending on its concentration.To sum up, complement activation stimulated by different types of erythrocytes was of an approximately equal level. Differences between C3a concentrations for control samples B are duePreviously, it has been demonstrated that the antimicrobial peptide tachyplesin interacts with C1q protein and activates the classical complement pathway. Generally, the classical pathway is initiated as a consequence of recognition by C1q of IgGs or IgMs in complex with their antigens. However, tachyplesin seems to activate the complement system independently of antibodies . Since ash stimulates the classical pathway of the complement system and activation via the alternative pathway is insignificant, whereas Erab hemolysis is a model for alternative pathway investigations. However, we observed a stimulating action of arenicin-1 in both hemolytic models, though it was expressed mainly in lysis level in the case of Esh, and in C3a production in the case of Erab. Unexpectedly, at high doses (80–160 µg/mL) arenicin displayed reverse effects, essentially diminishing lysis level and C3a generation, in both experimental systems. Apparently, high doses of arenicin inhibit both the classical and alternative pathways, and the latter cannot be explained by the interaction of arenicin with C1q. Instead, the inhibitory effect of arenicin must be related to the action on the common point of both pathways, i.e., C3 cleavage. Most likely, this is the interaction of arenicin with C3 protein leading to its protection from cleavage, although interactions of arenicin with C3-convertases are also possible.It is commonly accepted that antibody sensitized E2O)Bb generated at spontaneous ‘tick-over’ activation of the complement cascade by the alternative pathway; C3bBb, a convertase of the ‘amplification loop’ of the complement system, also usually considered as part of the alternative pathway. The tick-over C3-convertase is an exclusively fluid phase enzyme, while the other two could be covalently bound to the surface of a target cell [There are three types of C3-convertases: C4b2a, a common C3-convertase of classical and lectin pathways; C3(Hget cell . Our resrab (not treated with antibodies) and sensitized Esh, respectively. In the case of using Esh, arenicin interacts with C1q near the cell surface because C1q is bound to antibodies to surface antigens. On the other hand, C1q remains in the fluid phase in the Erab model, and most C3b generated by classical pathway C3-convertase will not be attached to the erythrocyte surface. Thus, both classical pathway and amplification loop C3-convertases locate mainly in a fluid phase where they are highly unstable [2+-free buffer for Erab hemolysis, which is not compatible with the classical pathway activation. Thus, stimulating effects of arenicin on the complement system remain enigmatic, at least for Erab hemolysis assay.It is more difficult to explain stimulating effects of arenicin on the complement system at relatively low concentrations. It could be assumed that interaction of arenicin with C1q can lead both to antibody-independent classical pathway activation (as it was described for tachyplesin ) and to unstable ,23, and While the antimicrobial peptide tachyplesin-1 was shown to activate the classical complement pathway , we descThe ability of arenicin-1 to act as a potent complement system inhibitor makes it a prospective molecular source for the design of novel therapeutics directed to the complement system. Currently, the almost complete lack of therapeutic agents that regulate the level of complement activation is a serious medical problem. When hyperactivated, the complement system is perhaps the most dangerous pro-inflammatory and cytotoxic machinery in the human body. The complement system plays a significant role in the pathogenesis of a number of diseases, including age-related macular degeneration, paroxysmal nocturnal hemoglobinuria, hereditary angioedema, and some kidney diseases such as atypical hemolytic uremic syndrome and membranoproliferative glomerulonephritis type II ,25,26,27Our results indicate that at low concentrations arenicin-1 acts differently on the complement system, depending on the presence of antibodies detecting target cells. Although we described this action as complement activation in both cases, fluid phase C3 conversion could be regarded as a kind of complement inhibition, since the C3 pool becomes depleted (C3 consumption). However, the host cells could also be targets of the complement system, for example in the case of autoimmune disorders. Moreover, C3a, a moderate anaphylatoxin, is produced due to fluid phase C3 conversion, which can induce some proinflammatory effects ,29. If oArenicin-1 is known as a hemolytic peptide for human erythrocytes . It coulIn conclusion, we revealed that arenicin-1 is able to both activate and inhibit the complement system in utilized test models in vitro. Our results imply that arenicin can be considered as the basis for the development of a new therapeutic drug for complement system modulation. In addition, the ability of arenicin to interact with the complement system should be taken into account in attempts to create novel antibiotic drugs derived from its structure. The presented observations should be treated as initial results due to the application of relatively simple methods of analysis of the complement system activation. The assays were conducted only with diluted serum, which is not physiologically relevant. In vivo tests on an animal model were not conducted. Further investigations are required to clarify mechanisms of arenicin action on the complement system, and its feasibility under in vivo conditions.O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate/N,N-diisopropylethylamine (TBTU/DIEA) activation, using Wang-resin as the solid-phase and triphenylmethyl protecting groups for cysteines, as described previously [Arenicin-1 was synthesized based on the standard 9-fluorenylmethoxycarbonyl (Fmoc) protocol with the eviously . Human CNormal human serum (NHS) was collected by medical staff  from more than 20 healthy volunteers, pooled, aliquoted, and stored at −70 °C no longer than two months. Serum aliquots were thawed at 4 °C in the day of the experiment, kept in ice baths before introducing to test tubes, and were not used repetitively. To obtain serum with an inactivated complement system, it was incubated at 56 °C for an hour immediately before the experiment.++  for sheep erythrocytes (Esh), and GVB+  for rabbit erythrocytes (Erab). DGVB++ is a 2.5 mM sodium barbital buffer containing 71 mM NaCl, 150 mM glucose, 1 mM MgCl2, 0.15 mM CaCl2, 0.05% gelatin; pH 7.35. GVB+ is a 2.5 mM sodium barbital buffer containing 150 mM NaCl, 10 mM Mg-EGTA, 0.05% gelatin; pH 7.35. Before experiments, sheep erythrocytes were sensitized with antibodies ; we used a 1:1600 dilution of these antibodies and incubated sheep red blood cells for 30 min at 37 °C.Animal erythrocytes were purified from whole blood of rabbit and sheep. They were stored in Alsever’s solution at 4 °C for no more than 5 days. Before use, they were washed with an appropriate buffer: DGVBsh and Erab) as target cells as described elsewhere [Hemolytic functional assays were performed utilizing erythrocytes  in a microplate reader . After measurement, the samples were used for C3a determination by ELISA.The incubation was carried out for half an hour at 37 °C. After the incubation, the lysis of the erythrocytes was stopped by the addition of PBS  in a ratio of 1:7.5. To some test tubes, distilled water was added instead of buffer, and thus samples were obtained with 100% lysis. Samples were centrifuged at 500× For the immunization of F2-hybrid mice, 0.5 mg of C3a peptide in 1 mL of PBS was mixed with equal volume of Freund’s complete adjuvant (or Freund’s incomplete adjuvant for reimmunization). The reimmunization was carried out after 30 days; 12 days after that, the lymphocytes from the inguinal, peritoneal and axillary lymph nodes were collected for hybridization with Sp2/0 myeloma cells in the ratio 2:1. Cell hybridization was performed in PEG/DMSO (Sigma), using serial dilution with culture media . The hybClone screening was performed using ELISA in a 96-well microplate. Briefly, 50 µL of culture media was added to wells precoated with C3a antigen, and after incubation for 1 h, peroxidase-conjugated goat anti-mouse IgGs were added. Detection was performed with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution.For C3a ELISA development, two monoclonal murine antibodies to C3a, with different epitope specificities designated as CC3a-5 and CC3a-1, were selected. CC3a-5 was immobilized on a solid phase as a capture antibody, while CC3a-1 was conjugated with horseradish peroxidase and was used as a detection antibody.ELISA for C3a was performed in 96-well microplates. Wells of a microplate were coated with capture antibodies and blocked by 1% BSA in PBS, pH 7.2. The same BSA solution was used as diluent on subsequent steps. Ten-fold diluted experimental samples and calibration samples were introduced to the wells, after which the plates were incubated for 1 h at 37 °C. Then, 100 µL of peroxidase-conjugated detection antibodies were added to each well. After incubation for 1 h at 37 °C, the TMB substrate solution  was added. After 15–20 min, the reaction was stopped by adding sulfuric acid, and the absorbance at 450 nm was measured in a microplate reader .t-test using the software package STATISTICA . All of the experiments were repeated five times. All data are presented as mean ± standard deviation (SD). Pearson coefficients were calculated and used for evaluation of the correlation level between C3a concentration and percentage of lysed cells. For both tests, p-values of less than 0.05 were considered statistically significant.Statistical significance of the values was evaluated by paired


To investigate the effects of exenatide on renal injury instreptozotocin-induced diabetic rats.  Fifty SD rats were randomly divided into normal control, model, exenatide-1,exenatide-2 and exenatide-3 groups, 10 rats in each group. The diabeticnephropathy model was constructed in later 4 groups. Then, the later 3groups were treated with 2, 4 and 8 μg/kg exenatide for 8 weeks,respectively. The serum and urine biochemical indexes and oxidative stressand inflammatory indexes in renal tissue were determined. Compared to the model group, in exenatide-3 group the serum fasting plasmaglucose and hemoglobin A1c levels were significantly decreased, the fastinginsulin level was significantly increased, the renal index and blood ureanitrogen, serum creatinine and 24 h urine protein levels were significantlydecreased, the renal tissue superoxide dismutase and glutathione peroxidaselevels were significantly increased, the malondialdehyde level wassignificantly decreased, and the renal tissue tumor necrosis factor alpha,interleukin 6, hypersensitive C-reactive protein and chemokine (C-C motif)ligand 5 levels were significantly decreased P<0.05). Exenatide can mitigate the renal injury in diabetic rats. The mechanisms maybe related to its resistance of oxidative stress and inflammatory responsein renal tissue.  With the improvement of living standards and the aging of the population,the diabetic nephropathy has gradually become the primary cause of chronic kidneydisease, surpassing the chronic glomerulonephritis. Diabetic nephropathy is one ofthe most common microvascular complications of diabetes mellitus, and is one of themain causes of chronic renal failure and death of diabetes mellitus This study was performed in strict accordance with the recommendations in the Guidefor the Care and Use of Laboratory Animals of the National Institutes of Health. Theanimal use protocol has been reviewed and approved by the Institutional Animal Careand Use Committee of Tai’an Central Hospital.  Fifty male Sprague Dawley rats (200±30 g) were adaptively fed for 1 week. Then,the rats were divided into normal control, model, low-dose exenatide(exenatide-1), middle-dose exenatide (exenatide-2) and high-dose exenatide(exenatide-3) groups according to random number table, with 10 rats in eachgroup. The rats in model and 3 exenatide groups were fasted for 12h, followed bysingle sterile intraperitoneal injection of streptozotocin with dose of 60mg/kg. After 72h, the tail vein blood was sampled, and the fasting blood glucose(FBG) level higher than 16.7 mmol/L indicated the diabetes. After 3 weeks, the24-hours urine protein (24h UP) was detected. The 24h UP higher than 30 mgindicated the diabetic nephropathyoC for later determination. After establishment of diabetic nephropathy model, the rats in exenatide-1,exenatide-2 and exenatide-3 groups were subcutaneously injected with exenatide, with dose of and 2, 4 and 8μg/kg, respectively. The normal control and model groups were subcutaneouslyinjected with equal volume of solvent. The injection was performed once per day,and was lasted for 8 weeks. During the treatment, the general conditions of ratswere observed. After treatment, the 24-hours urine samples were collected. Thebody weight of rats was measured. After fasting for 12 h, the rats wereanesthetized with chloral hydrate. A 10 ml blood was collected from theabdominal aorta, and was kept for test. The left and right kidneys were takenout quickly. After removing the capsules, the kidneys were weighed. The renalindex  was calculated. The kidneys were storedat -80 The blood samples were centrifuged at 2000 r/min for 10 min to obtain the serum.The FGB and fasting insulin (FINS) levels were detected according to the kitinstructions. The hemoglobin A1c (HbA1c) was detected by enzyme linkedimmunosorbent assay. The blood urea nitrogen (BUN), serum creatinine (Scr) and24h UP levels were measured by automatic biochemical analyzer. The kits wereprovided by Sigma-Aldrich Corp. . The kidneys of rats were taken, and the 10% renal tissue homogenate was madefrom 100 mg renal tissue using 5 ml Tris-HCl solution (pH 7.4). Aftercentrifugation at 2000 r/min for 10 min, the supernatant was obtained. Thesuperoxide dismutase (SOD) level was determined using WST-1 method The data were analyzed using SPSS 22.0 software . The datawere presented as mean±standard deviation. The differences among differentgroups were analyzed using one-way analysis of variance, followed by pairwisecomparison using SNK-q test. A P < 0.05 was accepted as statisticallysignificant. During the treatment period, the rats in normal control group were in goodcondition, with glossy fur, free movement and sensitive reaction. In modelgroup, the rats had obviously poor mental state. The fur gradually lost luster.The movement and response were slow. Compared with model group, the symptoms ofrats in 3 exenatide groups were mild, especially in exenatide-2 and exenatide-3groups. There was no rat dying in each group during the experiments. At the end of treatment, the body weight of rats in normal control group was442.34±58.12 g. The body weight in model, exenatide-1, exenatide-2 andexenatide-3 groups were 277.62±42.33 g, 288.14±36.29 g, 311.58±45.48 g and334.04±57.46 g, respectively, which was significantly lower than normal controlgroup, respectively (P<0.05). There was no significant difference of bodyweight among model group and 3 exenatide groups (P>0.05) .  After treatment, compared with the normal control group, in model group and 3exenatide groups the FBG and HbA1 levels were significantly increased,respectively (P < 0.05), and the FINS level was significantly decreased,respectively (P < 0.05). Compared with the model group, the FBG and HbA1levels in exenatide-2 and exenatide-3 groups were significantly decreased,respectively (P < 0.05), and the FINS level in exenatide-3 group wassignificantly increased (P < 0.05) . After treatment, compared with the normal control group, in mode, exenatide-1and exenatide-2 groups the renal tissue SOD and GSH-Px levels were significantlydecreased, respectively (P < 0.05), and the renal tissue MDA level wassignificantly increased, respectively (P < 0.05). Compared with model group,the SOD level in exenatide-2 and exenatide-3 groups was significantly increased,respectively (P < 0.05), the GSH-Px level in exenatide-3 group wassignificantly increased (P < 0.05), and the MDA level in exenatide-2 andexenatide-3 groups was significantly decreased, respectively (P < 0.05).  As shown in , GLP-1 is one of the important intestinal regulatory hormones in the homeostasis ofblood glucose. On the one hand, GLP-1 can enhance the glucose-dependent insulinsecretion response. On the other hand, it can promote the satiety and reduce foodintake by acting on the central nervous system, thereby reducing the burden of isletbeta cells and lowering the body weight. At present, GLP-1 is one of the hotspots inthe research of hypoglycemic drugs Oxidative stress plays an important role in the development of diabeticnephropathy TNF-α is a pro-inflammatory factor with negative inotropic action, and is theinitiation factor of the inflammatory cascade reaction. It can induce glomerularvascular endothelial cells to secrete adhesion factors, promote the proliferation ofglomerular mesangial cells, and induce the glomerular lesions The exenatide can mitigate the renal injury in diabetic rats. The mechanisms may berelated to its resistance of oxidative stress and inflammatory response in renaltissue. This study has provided an experimental basis for clinical application ofexenatide to prevention and treatment of diabetic nephropathy. There are still somelimitations in this study. Firstly, the correlations among different indexes are notinvestigated. Secondly, maybe there are other mechanisms of exenatide in alleviatingrenal injury.