Mechanisms of neuromodulatory volume transmission.

A wealth of neuromodulatory transmitters regulate synaptic circuits in the brain. Their mode of signaling, often called volume transmission, differs from classical synaptic transmission in important ways. In synaptic transmission, vesicles rapidly fuse in response to action potentials and release their transmitter content. The transmitters are then sensed by nearby receptors on select target cells with minimal delay. Signal transmission is restricted to synaptic contacts and typically occurs within ~1 ms. Volume transmission doesn't rely on synaptic contact sites and is the main mode of monoamines and neuropeptides, important neuromodulators in the brain. It is less precise than synaptic transmission, and the underlying molecular mechanisms and spatiotemporal scales are often not well understood. Here, we review literature on mechanisms of volume transmission and raise scientific questions that should be addressed in the years ahead. We define five domains by which volume transmission systems can differ from synaptic transmission and from one another. These domains are (1) innervation patterns and firing properties, (2) transmitter synthesis and loading into different types of vesicles, (3) architecture and distribution of release sites, (4) transmitter diffusion, degradation, and reuptake, and (5) receptor types and their positioning on target cells. We discuss these five domains for dopamine, a well-studied monoamine, and then compare the literature on dopamine with that on norepinephrine and serotonin. We include assessments of neuropeptide signaling and of central acetylcholine transmission. Through this review, we provide a molecular and cellular framework for volume transmission. This mechanistic knowledge is essential to define how neuromodulatory systems control behavior in health and disease and to understand how they are modulated by medical treatments and by drugs of abuse.

Molecular definition of distinct active zone protein machineries for Ca2+ channel clustering and synaptic vesicle priming.

Action potentials trigger neurotransmitter release with minimal delay. Active zones mediate this temporal precision by co-organizing primed vesicles with CaV2 Ca2+ channels. The presumed model is that scaffolding proteins directly tether primed vesicles to CaV2s. We find that CaV2 clustering and vesicle priming are executed by separate machineries. At hippocampal synapses, CaV2 nanoclusters are positioned at variable distances from those of the priming protein Munc13. The active zone organizer RIM anchors both proteins, but distinct interaction motifs independently execute these functions. In heterologous cells, Liprin-α and RIM from co-assemblies that are separate from CaV2-organizing complexes upon co-transfection. At synapses, Liprin-α1-4 knockout impairs vesicle priming, but not CaV2 clustering. The cell adhesion protein PTPσ recruits Liprin-α, RIM and Munc13 into priming complexes without co-clustering of CaV2s. We conclude that active zones consist of distinct complexes to organize CaV2s and vesicle priming, and Liprin-α and PTPσ specifically support priming site assembly.

Synaptotagmin-1 is a Ca2+ sensor for somatodendritic dopamine release.

Modes of somatodendritic transmission range from rapid synaptic signaling to protracted regulation over distance. Somatodendritic dopamine secretion in the midbrain leads to D2 receptor-induced modulation of dopamine neurons on the timescale of seconds. Temporally imprecise release mechanisms are often presumed to be at play, and previous work indeed suggested roles for slow Ca2+ sensors. We here use mouse genetics and whole-cell electrophysiology to establish that the fast Ca2+ sensor synaptotagmin-1 (Syt-1) is important for somatodendritic dopamine release. Syt-1 ablation from dopamine neurons strongly reduces stimulus-evoked D2 receptor-mediated inhibitory postsynaptic currents (D2-IPSCs) in the midbrain. D2-IPSCs evoked by paired stimuli exhibit less depression, and high-frequency trains restore dopamine release. Spontaneous somatodendritic dopamine secretion is independent of Syt-1, supporting that its exocytotic mechanisms differ from evoked release. We conclude that somatodendritic dopamine transmission relies on the fast Ca2+ sensor Syt-1, leading to synchronous release in response to the initial stimulus.

The intracellular C-terminus confers compartment-specific targeting of voltage-gated Ca2+ channels.

To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify the mechanisms that target voltage-gated Ca2+ channels (CaVs) to distinct subcellular compartments. In hippocampal neurons, CaV2s trigger neurotransmitter release at the presynaptic active zone, and CaV1s localize somatodendritically. After knockout of all three CaV2s, expression of CaV2.1, but not of CaV1.3, restores neurotransmitter release. Chimeric CaV1.3 channels with CaV2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release fully sensitive to blockade of CaV1 channels. This dominant targeting function of the CaV2.1 C-terminus requires an EF hand in its proximal segment, and replacement of the CaV2.1 C-terminus with that of CaV1.3 abolishes CaV2.1 active zone localization. We conclude that the intracellular C-termini mediate compartment-specific CaV targeting.

Protein composition of axonal dopamine release sites in the striatum.

Dopamine is an important modulator of cognition and movement. We recently found that evoked dopamine secretion is fast and relies on active zone-like release sites. Here, we used in vivo biotin identification (iBioID) proximity proteomics in mouse striatum to assess which proteins are present at these sites. Using three release site baits, we identified proteins that are enriched over the general dopamine axonal protein content, and they fell into several categories, including active zone, Ca2+ regulatory, and synaptic vesicle proteins. We also detected many proteins not previously associated with vesicular exocytosis. Knockout of the presynaptic organizer protein RIM strongly decreased the hit number obtained with iBioID, while Synaptotagmin-1 knockout did not. α-Synuclein, a protein linked to Parkinson's disease, was enriched at release sites, and its enrichment was lost in both tested mutants. We conclude that RIM organizes scaffolded dopamine release sites and provide a proteomic assessment of the composition of these sites.

Munc13 supports fusogenicity of non-docked vesicles at synapses with disrupted active zones.

Active zones consist of protein scaffolds that are tightly attached to the presynaptic plasma membrane. They dock and prime synaptic vesicles, couple them to voltage-gated Ca2+ channels, and direct neurotransmitter release toward postsynaptic receptor domains. Simultaneous RIM + ELKS ablation disrupts these scaffolds, abolishes vesicle docking, and removes active zone-targeted Munc13, but some vesicles remain releasable. To assess whether this enduring vesicular fusogenicity is mediated by non-active zone-anchored Munc13 or is Munc13-independent, we ablated Munc13-1 and Munc13-2 in addition to RIM + ELKS in mouse hippocampal neurons. The hextuple knockout synapses lacked docked vesicles, but other ultrastructural features were near-normal despite the strong genetic manipulation. Removing Munc13 in addition to RIM + ELKS impaired action potential-evoked vesicle fusion more strongly than RIM + ELKS knockout by further decreasing the releasable vesicle pool. Hence, Munc13 can support some fusogenicity without RIM and ELKS, and presynaptic recruitment of Munc13, even without active zone anchoring, suffices to generate some fusion-competent vesicles.

An action potential initiation mechanism in distal axons for the control of dopamine release.

Information flow in neurons proceeds by integrating inputs in dendrites, generating action potentials near the soma, and releasing neurotransmitters from nerve terminals in the axon. We found that in the striatum, acetylcholine-releasing neurons induce action potential firing in distal dopamine axons. Spontaneous activity of cholinergic neurons produced dopamine release that extended beyond acetylcholine-signaling domains, and traveling action potentials were readily recorded from dopamine axons in response to cholinergic activation. In freely moving mice, dopamine and acetylcholine covaried with movement direction. Local inhibition of nicotinic acetylcholine receptors impaired dopamine dynamics and affected movement. Our findings uncover an endogenous mechanism for action potential initiation independent of somatodendritic integration and establish that this mechanism segregates the control of dopamine signaling between axons and somata.

Rebuilding essential active zone functions within a synapse.

Presynaptic active zones are molecular machines that control neurotransmitter secretion. They form sites for vesicle docking and priming and couple vesicles to Ca2+ entry for release triggering. The complexity of active zone machinery has made it challenging to determine its mechanisms in release. Simultaneous knockout of the active zone proteins RIM and ELKS disrupts active zone assembly, abolishes vesicle docking, and impairs release. We here rebuild docking, priming, and Ca2+ secretion coupling in these mutants without reinstating active zone networks. Re-expression of RIM zinc fingers recruited Munc13 to undocked vesicles and rendered the vesicles release competent. Action potential triggering of release was reconstituted by docking these primed vesicles to Ca2+ channels through attaching RIM zinc fingers to CaVß4-subunits. Our work identifies an 80-kDa ß4-Zn protein that bypasses the need for megadalton-sized secretory machines, establishes that fusion competence and docking are mechanistically separable, and defines RIM zinc finger-Munc13 complexes as hubs for active zone function.

Molecular and functional architecture of striatal dopamine release sites.

Despite the importance of dopamine for striatal circuit function, mechanistic understanding of dopamine transmission remains incomplete. We recently showed that dopamine secretion relies on the presynaptic scaffolding protein RIM, indicating that it occurs at active zone-like sites similar to classical synaptic vesicle exocytosis. Here, we establish using a systematic gene knockout approach that Munc13 and Liprin-α, active zone proteins for vesicle priming and release site organization, are important for dopamine secretion. Furthermore, RIM zinc finger and C2B domains, which bind to Munc13 and Liprin-α, respectively, are needed to restore dopamine release after RIM ablation. In contrast, and different from typical synapses, the active zone scaffolds RIM-BP and ELKS, and RIM domains that bind to them, are expendable. Hence, dopamine release necessitates priming and release site scaffolding by RIM, Munc13, and Liprin-α, but other active zone proteins are dispensable. Our work establishes that efficient release site architecture mediates fast dopamine exocytosis.

Presynaptic Short-Term Plasticity Persists in the Absence of PKC Phosphorylation of Munc18-1.

Post-tetanic potentiation (PTP) is a form of short-term plasticity that lasts for tens of seconds following a burst of presynaptic activity. It has been proposed that PTP arises from protein kinase C (PKC) phosphorylation of Munc18-1, an SM (Sec1/Munc-18 like) family protein that is essential for release. To test this model, we made a knock-in mouse in which all Munc18-1 PKC phosphorylation sites were eliminated through serine-to-alanine point mutations (Munc18-1SA mice), and we studied mice of either sex. The expression of Munc18-1 was not altered in Munc18-1SA mice, and there were no obvious behavioral phenotypes. At the hippocampal CA3-to-CA1 synapse and the granule cell parallel fiber (PF)-to-Purkinje cell (PC) synapse, basal transmission was largely normal except for small decreases in paired-pulse facilitation that are consistent with a slight elevation in release probability. Phorbol esters that mimic the activation of PKC by diacylglycerol still increased synaptic transmission in Munc18-1SA mice. In Munc18-1SA mice, 70% of PTP remained at CA3-to-CA1 synapses, and the amplitude of PTP was not reduced at PF-to-PC synapses. These findings indicate that at both CA3-to-CA1 and PF-to-PC synapses, phorbol esters and PTP enhance synaptic transmission primarily by mechanisms that are independent of PKC phosphorylation of Munc18-1.SIGNIFICANCE STATEMENT A leading mechanism for a prevalent form of short-term plasticity, post-tetanic potentiation (PTP), involves protein kinase C (PKC) phosphorylation of Munc18-1. This study tests this mechanism by creating a knock-in mouse in which Munc18-1 is replaced by a mutated form of Munc18-1 that cannot be phosphorylated. The main finding is that most PTP at hippocampal CA3-to-CA1 synapses or at cerebellar granule cell-to-Purkinje cell synapses does not rely on PKC phosphorylation of Munc18-1. Thus, mechanisms independent of PKC phosphorylation of Munc18-1 are important mediators of PTP.

PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure.

The active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid-liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function.

Spatial and temporal scales of dopamine transmission.

Dopamine is a prototypical neuromodulator that controls circuit function through G protein-coupled receptor signalling. Neuromodulators are volume transmitters, with release followed by diffusion for widespread receptor activation on many target cells. Yet, we are only beginning to understand the specific organization of dopamine transmission in space and time. Although some roles of dopamine are mediated by slow and diffuse signalling, recent studies suggest that certain dopamine functions necessitate spatiotemporal precision. Here, we review the literature describing dopamine signalling in the striatum, including its release mechanisms and receptor organization. We then propose the domain-overlap model, in which release and receptors are arranged relative to one another in micrometre-scale structures. This architecture is different from both point-to-point synaptic transmission and the widespread organization that is often proposed for neuromodulation. It enables the activation of receptor subsets that are within micrometre-scale domains of release sites during baseline activity and broader receptor activation with domain overlap when firing is synchronized across dopamine neuron populations. This signalling structure, together with the properties of dopamine release, may explain how switches in firing modes support broad and dynamic roles for dopamine and may lead to distinct pathway modulation.

Intact synapse structure and function after combined knockout of PTPδ, PTPσ, and LAR.

It has long been proposed that leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cell-adhesion proteins that control synapse assembly. Their synaptic nanoscale localization, however, is not established, and synapse fine structure after knockout of the three vertebrate LAR-RPTPs (PTPδ, PTPσ, and LAR) has not been tested. Here, superresolution microscopy reveals that PTPδ localizes to the synaptic cleft precisely apposed to postsynaptic scaffolds of excitatory and inhibitory synapses. We next assessed synapse structure in newly generated triple-conditional-knockout mice for PTPδ, PTPσ, and LAR, complementing a recent independent study of synapse function after LAR-RPTP ablation (Sclip and Südhof, 2020). While mild effects on synaptic vesicle clustering and active zone architecture were detected, synapse numbers and their overall structure were unaffected, membrane anchoring of the active zone persisted, and vesicle docking and release were normal. Hence, despite their localization at synaptic appositions, LAR-RPTPs are dispensable for presynapse structure and function.

Synapse and Active Zone Assembly in the Absence of Presynaptic Ca2+ Channels and Ca2+ Entry.

Presynaptic CaV2 channels are essential for Ca2+-triggered exocytosis. In addition, there are two competing models for their roles in synapse structure. First, Ca2+ channels or Ca2+ entry may control synapse assembly. Second, active zone proteins may scaffold CaV2s to presynaptic release sites, and synapse structure is CaV2 independent. Here, we ablated all three CaV2s using conditional knockout in cultured hippocampal neurons or at the calyx of Held, which abolished evoked exocytosis. Compellingly, synapse and active zone structure, vesicle docking, and transsynaptic nano-organization were unimpaired. Similarly, long-term blockade of action potentials and Ca2+ entry did not disrupt active zone assembly. Although CaV2 knockout impaired the localization of ß subunits, α2δ-1 localized normally. Rescue with CaV2 restored exocytosis, and CaV2 active zone targeting depended on the intracellular C-terminus. We conclude that synapse assembly is independent of CaV2s or Ca2+ entry through them. Instead, active zone proteins recruit and anchor CaV2s via CaV2 C-termini.

Synaptotagmin-1 is the Ca2+ sensor for fast striatal dopamine release.

Dopamine powerfully controls neural circuits through neuromodulation. In the vertebrate striatum, dopamine adjusts cellular functions to regulate behaviors across broad time scales, but how the dopamine secretory system is built to support fast and slow neuromodulation is not known. Here, we set out to identify Ca2+-triggering mechanisms for dopamine release. We find that synchronous dopamine secretion is abolished in acute brain slices of conditional knockout mice in which Synaptotagmin-1 is removed from dopamine neurons. This indicates that Synaptotagmin-1 is the Ca2+ sensor for fast dopamine release. Remarkably, dopamine release induced by strong depolarization and asynchronous release during stimulus trains are unaffected by Synaptotagmin-1 knockout. Microdialysis further reveals that these modes and action potential-independent release provide significant amounts of extracellular dopamine in vivo. We propose that the molecular machinery for dopamine secretion has evolved to support fast and slow signaling modes, with fast release requiring the Ca2+ sensor Synaptotagmin-1.

ELKS1 Captures Rab6-Marked Vesicular Cargo in Presynaptic Nerve Terminals.

Neurons face unique transport challenges. They need to deliver cargo over long axonal distances and to many presynaptic nerve terminals. Rab GTPases are master regulators of vesicular traffic, but essential presynaptic Rabs have not been identified. Here, we find that Rab6, a Golgi-derived GTPase for constitutive secretion, associates with mobile axonal cargo and localizes to nerve terminals. ELKS1 is a stationary presynaptic protein with Golgin homology that binds to Rab6. Knockout and rescue experiments for ELKS1 and Rab6 establish that ELKS1 captures Rab6 cargo. The ELKS1-Rab6-capturing mechanism can be transferred to mitochondria by mistargeting ELKS1 or Rab6 to them. We conclude that nerve terminals have repurposed mechanisms from constitutive exocytosis for their highly regulated secretion. By employing Golgin-like mechanisms with anchored ELKS extending its coiled-coils to capture Rab6 cargo, they have spatially separated cargo capture from fusion. ELKS complexes connect to active zones and may mediate vesicle progression toward release sites.

Assembly of the presynaptic active zone.

In a presynaptic nerve terminal, the active zone is composed of sophisticated protein machinery that enables secretion on a submillisecond time scale and precisely targets it toward postsynaptic receptors. The past two decades have provided deep insight into the roles of active zone proteins in exocytosis, but we are only beginning to understand how a neuron assembles active zone protein complexes into effective molecular machines. In this review, we outline the fundamental processes that are necessary for active zone assembly and discuss recent advances in understanding assembly mechanisms that arise from genetic, morphological and biochemical studies. We further outline the challenges ahead for understanding this important problem.

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

Firing Rate Homeostasis Can Occur in the Absence of Neuronal Activity-Regulated Transcription.

Despite dynamic inputs, neuronal circuits maintain relatively stable firing rates over long periods. This maintenance of firing rate, or firing rate homeostasis, is likely mediated by homeostatic mechanisms such as synaptic scaling and regulation of intrinsic excitability. Because some of these homeostatic mechanisms depend on transcription of activity-regulated genes, including Arc and Homer1a, we hypothesized that activity-regulated transcription would be required for firing rate homeostasis. Surprisingly, however, we found that cultured mouse cortical neurons from both sexes grown on multi-electrode arrays homeostatically adapt their firing rates to persistent pharmacological stimulation even when activity-regulated transcription is disrupted. Specifically, we observed firing rate homeostasis in Arc knock-out neurons, as well as knock-out neurons lacking the activity-regulated transcription factors AP1 and SRF. Firing rate homeostasis also occurred normally during acute pharmacological blockade of transcription. Thus, firing rate homeostasis in response to increased neuronal activity can occur in the absence of neuronal-activity-regulated transcription.SIGNIFICANCE STATEMENT Neuronal circuits maintain relatively stable firing rates even in the face of dynamic circuit inputs. Understanding the molecular mechanisms that enable this firing rate homeostasis could potentially provide insight into neuronal diseases that present with an imbalance of excitation and inhibition. It has long been proposed that activity-regulated transcription could underlie firing rate homeostasis because activity-regulated genes turn on when neurons are above their target firing rates and include many genes that could regulate firing rate. Surprisingly, despite this prediction, we found that cortical neurons can undergo firing rate homeostasis in the absence of activity-regulated transcription, indicating that firing rate homeostasis can be controlled by non-transcriptional mechanisms.

Nanoscale Location Matters: Emerging Principles of Ca2+ Channel Organization at the Presynaptic Active Zone.

How does diversity in the organization of secretory machines determine properties of neurotransmitter release? In this issue of Neuron, Rebola et al. (2019) found that distinct nanoscale assemblies of Ca2+ channels and Munc13, not overall channel abundance, mediate differing release characteristics of two cerebellar synapses.