Dendritic SNAREs add a new twist to the oldneuron theorySaak V. Ovsepian1 and J. Oliver Dolly1

International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, Ireland

Edited by Thomas C. Südhof, Stanford University School of Medicine, Palo Alto, CA, and approved October 4, 2011 (received for review November 22, 2010)

Dendritic exocytosis underpins a broad range of integrative and homeostatic synaptic functions. Emerging data highlight the essentialrole of SNAREs in trafficking and fusion of secretory organelles with release of peptides and neurotransmitters from dendrites. ThisPerspective analyzes recent evidence inferring axo-dendritic polarization of vesicular release machinery and pinpoints progress made withexisting challenges in this rapidly progressing field of dendritic research. Interpreting the relation of new molecular data to physiologicalresults on secretion from dendrites would greatly advance our understanding of this facet of neuronal mechanisms.

dendritic release | neuronal polarization | retrograde transmission

Classically, a single neuronreceives multimodal informa-tion as local electro-chemicalsignals through synaptic inputs

converging onto its soma and dendrites.After integration, these confined eventsare translated into action potentials at theaxon initial segment, which rapidly prop-agate to nerve terminals and trigger therequantal release of neurotransmitters. Re-cent electrophysiological and imaging data,however, dispute this rather naïve view ofneurons, highlighting unforeseen traitsand dynamics of dendritic integration andcommunication mechanisms betweennerve cells. It emerges that neurons, inaddition to transferring signals throughcanonical chemical synapses at the axonterminals, also interact via mixed electro-chemical and electrical synapses at so-matic and dendritic juxtapositions, as wellas through diffuse nonsynaptic volumetransmission signaling (1–4). Significantevidence has also accrued suggesting es-sential roles for dendritic exocytosis inthese and numerous other integrative andhomeostatic processes. These include re-lease of transmitters, neurotrophins, andmodulatory peptides from dendrites, alongwith activity-dependent remodeling ofsynaptic connections via regulated ex-pression of receptors and ion channels(5–7). Although a great body of dataadvocate the involvement of SNAREs intrafficking and vesicular fusion at den-drites, nonvesicular release of retrogrademessengers (e.g., endocannabinoids,arachidonic acid, nitric oxide, and carbonmonoxide) from this location with theirautocrine and paracrine effects on targetand precursor neurons has additionallybeen documented (8–10). This Perspectivereviews and analyzes key research streamson SNARE-dependent retrosynapticsignaling in mammalian central neuronsand highlights the outstanding issuesand challenges in this rapidly progressingtopic. Despite the well-defined signifi-cance of SNAREs for trafficking and

exocytosis at dendrites in compliance withthe view of highly conserved fusion ma-chinery among different secretory path-ways, new findings indicate their notablespecialization, supporting the premise ofmolecular asymmetry as one of the keyaffiliates of neuronal polarization.

SNARE Hypothesis and SecretoryVesicle Fusion at SynapsesMembrane fusion constitutes one of thefundamental biological processes governingtargeting and merging of intracellularorganelles. Analysis of its mechanisms ledto the discovery of SNAREs as highlyconserved key nanoengines of variousintracellular trafficking steps (11, 12).According to the contemporary model ofmembrane fusion, SNAREs, along withseveral regulatory proteins, which are lo-calized in opposing membranes, attach andpropel the merger of membranes, using thefree energy that is released during theformation of a four-helix SNARE bundle.Although not undisputed, during the last2 decades this model attracted scientificscrutiny from numerous angles and gainedoverwhelming support from functional,molecular, and genetic studies (13–15).In eukaryotic cells, the SNARE protein

family is represented by nine subfamilies,with a total of 36 members identified inhumans (16). Originally, a strict separationwas assumed between these proteins re-siding on the “donor” and “acceptor”membrane compartments, which led totheir classification as vesicle membraneor target membrane SNAREs (v- andt-SNAREs) (17). This categorization,however, proved to be inept when homo-typic fusion events were considered andespecially in cases in which certainSNAREs function in several transportsteps, with varying partners. The more re-cent and widely accepted classification is,therefore, based on reactive motifs con-tributed by each partner toward the for-mation of ultrastable coiled-coil SNAREcomplexes during membrane fusion, with

its central hydrophilic layer containingthree conserved glutamines (Q) and onearginine (R) (18). Accordingly, the con-tributing molecules are grouped into Qa-,Qb-, Qc-, Qbc-, and R-SNAREs (19, 20)(Fig. 1A). In neurons, SNAREs drivingsecretion are represented by a vesicle-associated membrane protein (VAMP,known also as synaptobrevin, R-SNARE),plasma membrane anchored-attached syn-taxin (Qa-SNARE), and SNAP (compris-ing two SNARE motifs, Qbc-SNARE)variants (21). Advantageously for research,these proteins constitute natural targets forClostridial neurotoxins (CNTs, both botu-linum and tetanus toxins), which recognizeand proteolytically inactivate SNAREswith astounding efficiency and specificity,causing blockade of synaptic transmission(22–24) (Fig. 1B). Unstructured in mono-meric state, SNAREs when associatedform highly stable ternary helical com-plexes (syntaxin and VAMP contributingone, whereas SNAP-25 provides twoα-helices) before vesicular release (13, 18).This multireaction process, which was ex-plicitly demonstrated at both the ensembleand single molecular levels, is initiatedby the opening of syntaxin and binding ofMunc18 to the syntaxin Habc N-terminaldomain (25, 26), a step that promotesloose and reversible interaction betweenSNARE motifs of syntaxin with SNAP-25and VAMP, leading to anchoring of thesynaptic vesicle at the release site (Fig.1C). Upon binding of complexin, anothercytosolic regulatory protein, the transientSNARE core scaffold gets primed forrapid activation by the Ca2+ sensor syn-aptotagmin (27); the latter enforces thedissociation of complexin from theSNARE complex and puts into effect the

Author contributions: S.V.O. and J.O.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.1To whom correspondence may be addressed. E-mail: saak.ovsepian@dcu.ie or oliver.dolly@dcu.ie.

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zippering of four α-helical parallel bundles,resulting in vesicle fusion with the plasmamembrane and neurosecretion (28)(Fig. 1 C and D). The bulk of recent dataemerging from research on dendritic exo-cytosis, in general consistent with themodel outlined above, suggests subtle var-iations in the machinery and mechanismsregulating vesicular release.

Dendritic Secretory OrganellesIn somato-dendrites and axon terminals ofnerve cells, secretory organelles are rep-resented primarily by small synaptic vesi-cles (SSVs), filled with low molecularweight transmitters, and large dense corevesicles (LDCVs), containing peptides,modulators, biological amines, or neuro-trophins (29, 30) (Fig. 2A). Although bothLDCVs and SSVs are generated from theGolgi, they exhibit significant differencesin their life cycles and destiny. In LDCVs

the cargo proteins and peptides form ag-gregates, resulting in condensation of thegranule matrix and membrane buddingwith the formation of early secretorygranules, which mature by fusion withother granules while being transportedthrough the trans-Golgi networks towardtheir release sites. After fusion with theplasmalema, vesicle membrane compo-nents of LDCVs are retrieved and re-cycled to the Golgi, where they get refilledwith new cargo, thereby generating vesi-cles de novo in each cycle. SSVs also budfrom Golgi as immature vesicles but havemorphology and protein composition dif-ferent from those of mature SSVs. Thesealso reach release sites through activetransport and there undergo maturation.However, in contrast to LDCVs, SSVs arerefilled and extensively recycled locallybefore their degradation (31, 32).

Release of both “classic” transmittersand peptides from dendrites seems to beprimarily mediated via regulated exo-cytosis, except under basal conditions theirconstitutive release has been documented(33–35) (Fig. 2B). Tannic acid fixation ofdendrites within the supraoptic nucleusrevealed the morphology of LDCVs, withevidence of “omega” fusion profiles at theplasma membrane, clearly indicating thevesicular secretion of peptides from den-drites (36) (Fig. 2A). In several otherneuron types, smaller dendritic vesicularelements have been observed, which maycorrespond to SSVs (31, 36). Despite theexistence of discrete populations of se-cretory organelles (rapid releasable andreserve pools) in dendrites and their ac-tivity-dependent segregation have beenproposed long ago (37), mechanismsof their targeting to the various poolsand plasma membrane are essentially

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Fig. 1. SNAREs and vesicular secretion from neurons. (A) Types of SNAREs with their gross structures schematized. The C-terminal of SNAREs corresponds tothe transmembrane (TM) domains; the central portion contains the SNARE motif, and the N-terminal of Qa-SNAREs (syntaxins in neurons) possesses antiparallelthree-helix bundles. In Qbc SNAREs (SNAP-23 and SNAP-25 in neurons), duplicated SNARE motifs are connected by a linker that is frequently palmitoylated (zig-zag lines) and anchors the protein to the membrane. During vesicular fusion at nerve terminals Qbc SNAREs (SNAP-23/25 variants) contribute two α-helices tothe four-helical core complex, with the other two provided by Qa (syntaxin) and R (VAMP) SNAREs. Dashed borders highlight domains that are missing in somesubfamily members. Modified with permission from ref. 18. (B) SNAREs constitute molecular targets of Clostridial neurotoxins, which recognize and cleavethese fusogenic proteins at specific scissile bonds. Neuronal SNAREs serve as substrate for the protease light chains of tetanus toxin and botulinum neurotoxins:arrows indicate the cleavage sites of individual SNAREs by the different neurotoxins (BoNT, A-G serotypes of botulinum neurotoxin; TeNT, tetanus toxin).Plasma and vesicle membranes are indicated in gray (Left and Right, respectively). (C) Schematic of the SNARE-dependent synaptic vesicle cycle. Conforma-tional shift from a closed to an open state of the N-terminal motif of syntaxin (Qa SNARE, red) conditioned by SM protein Munc18 initiates the loose in-teraction of syntaxin with SNAP-25 (Qbc SNARE, green) and VAMP (R-SNARE, blue), leading to ATP-dependent docking of SVs at the active zone (1, 2). Thiscomplex gets primed by complexin and Munc18 for activation by synaptotagmin and Ca2+ (3). Upon Ca2+ influx, synaptotagmin dissociates the complexin fromthe fusion complex and reacts with membrane phospholipids and the SNAREs, driving membrane fusion (4) and release of vesicular contents into the synapticcleft. This step is followed by dissociation of the SNARE complex by NSF using the hydrolysis of ATP as energy source, liberating SNAREs for further reuse. (D) Acrystal structure of SNARE core complex with four parallel α-helices associated in the ternary complex. The C-terminal of the helices, which points toward thevesicle and surface membranes, are oriented to the right. Modified with permission from ref. 136.

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unknown. Unlike axon terminals, whichrely on a somatic supply of LDCVs, sub-stantial data suggest local mRNA trans-lation and peptides synthesis in dendrites,with neurotransmitter secretion endo-plasmic reticular (ER) sites visualizedthroughout the dendritic shafts of hippo-campal and hypothalamic neurons (31,38, 39). The lack of functional data onthe principles and organization of dendriticendomembranes renders current un-derstanding of postsynaptic membrane dy-namics descriptive and incomplete (40).The significance of locally made vs. so-matically produced neurotransmitters andpeptides for dendritic secretion is currentlyalso a matter of debate and intense re-search. Equally, it remains unclear whyonly a fraction of dendrites contain Golgisecretory outspots, raising the fundamentalquestion of the significance of local trans-lation in the absence of secretory pathwaystherein. Interestingly, the presence ofsmooth ER-derived organelles associatedwith small vesicular structures raises thepossibility that some exocytotic vesicles can

be developed directly from dendritic ERelements, bypassing the Golgi apparatus(38, 41). Although this straight route ofsecretory organelles to plasma membranein dendrites has so far received littlefunctional evidence, with targeting proteinsremaining essentially unknown, if true itwould represent a unique, noncanonicalintracellular vesicle flow pathway thatcould explain how membrane proteins andpeptides get locally trafficked in dendriteslacking Golgi outposts (42). In addition tosecretory vesicles, several other exocytoticorganelles, including early endosomes, re-cycling endosomes, late endosomes, andlysosomes, have been identified in den-drites of various neuron types, extensivelyreviewed recently (42, 43).

Secretion from Dendrites of CentralNeurons Relies on SNAREsThere is considerable evidence for the in-volvement of SNAREs in secretion ofboth SSVs and LDCVs from dendrites ofcentral neurons. The bulk of the data onSNARE-dependence of LDCV release

comes from nigral dopaminergic cells andhypothalamic neurons. Dopamine hasbeen observed in a variety of secretoryorganelles, including LDCV, SSVs, andtuberovesicular structures resemblingsmooth endoplasmic reticular stores (44).The advantageous anatomical arrange-ments of dopaminergic neurons with seg-regated dendrites, in substantia nigra parsreticulate, from somatic and axonal com-partments not only made it possible toperform selective quantitative analysis ofthe dendritic release but provides a modelfor real-time monitoring of this physiolog-ical process in vivo (45, 46). Nevertheless,identification of subcellular storage com-partments and precise molecular eventsleading to dendritic secretion of dopamineremained a subject of longstanding con-troversy (44, 47–49) until a dual, quantal-reverse carrier-mediated secretion hy-pothesis was proposed (45). According tothis model, dopamine can be releasedthrough both vesicular and carrier-medi-ated secretory mechanisms. Importantly,potent inhibition of dopamine secretionfrom somato-dendritic compartments ofthese neurons by CNTs in vitro and in vivoconfirmed the involvement of SNAREs(50, 51). These functional data receivedsupport from more recent immunocyto-chemical studies, which highlighted thepresence of the main CNTs-sensitiveSNARE variants, including syntaxin3,VAMP2, and SNAP-25, in dendrites ofnigral dopaminergic neurons (52).Similar experiments also provided con-

clusive evidence for dendritic vesicular se-cretion from hypothalamic neurons. Indendrites of magnocellular neurons, forinstance, a steep Ca2+-dependence of oxy-tocin release with clear structural attributesof vesicular storage and secretion fromLDCVs has been firmly established (36,53). The presence of SNAP-25 and syntaxinwere also verified at this location (54).Importantly, probing dendritic oxytocinrelease with nanomolar concentrations oftetanus toxin revealed an essential role forVAMP in the secretion of this peptide-hormone (55). With earlier studies dem-onstrating the presence of α-SNAP mRNAand possible local translation of SNAP-25in dendrites of hypothalamic neurons (56,57), blockade of oxytocin release by CNTssubstantiated the role of SNAREs as keymediators of dendritic peptide secretionhere. Moreover, the data from severalother brain areas, including hippocampus(58, 59), olfactory bulb (60), cerebellum(61), and neocortex (62, 63), are consistentwith the requirement of SNARE proteinsin dendritic neurosecretion and suggest thepervasion of this mechanism throughoutthe brain. It should be highlighted, how-ever, that most of the data on SNARE-dependent dendritic secretion rely exclu-sively on CNT-induced inhibition of

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Fig. 2. (A) Electron micrographs of rat hypothalamic area showing a dendritic shaft packed with LDCVs.The presence of synaptic contacts with axon terminal identifies the process as a dendrite. Arrowhead(Lower) indicates released dendritic secretory granule content into the interstitial space. Modified withpermission from refs. 36 and 137. (Scale bar, 1.0 μm.) (B) Tufted cell (TC) dendro-dendritic inhibitionmediated by periglomerular (PG) neurons. In the presence of tetrodotoxin, a voltage step is followed bya long-lasting barrage of inhibitory postsynaptic currents that were abolished by the GABAA receptorantagonist bicuculine (BMC). (C) L-type Ca2+ currents, which are normally not coupled to exocytosis, arerequired for TC–PG dendro-dendritic inhibition. Dendro-dendritic inhibition is reduced by nimodepine(L-type Ca2+ channel blocker) and recovered by activation of the same channel with Bay K. Modified withpermission from ref. 34.

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synaptic transmission, which is indirect ev-idence for SNARE involvement. In lightof significant functional overlap and re-dundancy between different SNARE var-iants (64, 65) and recently recognizedreliance of constitutive and regulated re-lease on different SNAREs (64, 66–68),the data described above should be in-terpreted with a great degree of cautionand warrant further in-depth research.

Evidence for Specialization of ExocytoticMachinery for Dendritic SecretionIn biological systems, membrane fusionmust be carefully controlled to excludevesicle delivery to incorrect acceptor com-partments that may prove detrimental.Therefore, intracellular pathways of secre-tory organelles follow highly regulatedroutes, to ensure targeted delivery to theirfusion end-points. These journeys seem tobe controlled primarily by Ras-relatedGTPases, Rab proteins instrumental inensuring the specificity of contacts betweenfusion partners (18, 20). Although special-ization of certain SNARE variants for in-dividual fusion reactions and for guidingvesicular traffic has been recognized, theextent to which SNAREs contribute tothese regulated flows of exocytotic organ-elles and their routing to different com-partments remains a subject of greatinterest and ongoing debate; this is pri-marily due to the capacity of someSNAREs to assemble promiscuouslywith different variants (69, 70). For in-stance, in yeast ER–Golgi traffic, Sec22and Ykt6 can substitute for each other asR-SNAREs. Similarly, in mammalian cells,SNAP-25 and SNAP-23, as well as VAMP2and cellubrevin, are capable of replacingeach other to varying degrees in regulatedexocytosis (64, 71). Nevertheless, specificSNAREs have been assigned to most ofthe fusion steps and organelle types (18),with illustrative examples of such speciali-zation in neuronal dendrites representedby syntaxin 4 and SNAP-23 (72, 73).Highly enriched in dendritic spines, theseSNAREs are known to mediate dendrite-specific fusion reactions, reviewed in detailrecently (42).Adaptation of biochemical and physio-

logical mechanisms of secretion from axonterminals and dendrites has also beenproposed on the basis of data obtained fromother models adopted for studying thetargeting of secretory vesicles to these twopoles of neurons (74, 75). In hypothalamicneurons, for example, dendritic LDCVsseem to differ in their content from thosedestined for release from axon terminalsand have been proposed to rely on distinctexocytotic mechanisms (76). In the sameway, in hippocampal neurons the mem-brane-associated SNAP-25 explicitly shownin axons was hardly detectable in dendrites,even though its presence has been dem-

onstrated in the cytoplasm of both thesoma and large dendrites of these cells(77). Evidence from nigral dopaminergicneurons suggests functional polarizationof VAMP isoforms, with VAMP7 (TI-VAMP) [but not botulinum neurotoxintype B (BoNT/B)-sensitive VAMP variants]contributing to regulated vesicular secre-tion from dendrites (50, 53) despite theabundance of VAMP2 in both compart-ments. Intriguingly, the controversy overthe presence of nonfunctional SNAREs indendrites of hippocampal neurons hasbeen partially resolved by the identifica-tion of VAMP2 on the pool of vesiclesthat bypass this neuronal compartment onthe transcytotic journey to final functionaldestination at axon terminals (78). It re-mains to be established whether VAMP2redundant in dendrites of nigral dopami-nergic neurons (52) can be involvedwith LDCVs targeted for release ataxon terminals.

Ca2+ Triggering of Dendritic VesicularSecretionAt a canonical synapse, secretory vesicles inpreparation for fusion dock at release sitesand get primed for Ca2+-induced fusionwith the plasma membrane (65) (Fig. 1C).Upon arrival of action potentials, voltage-gated Ca2+ channels open and allow influxof a pulse of Ca2+, thereby raising the in-tracellular [Ca2+] so that synchronous ve-sicular release gets triggered. Althoughthere is convincing evidence to support the

existence of such regulated Ca2+-induceddendritic secretion (79, 80), both the sourceand routes of Ca2+ entry, as well as theCa2+ sensitivity of fusion machinery indendrites, seem to differ significantly fromthose at axon terminals. Unlike releaseat axon endings that relies on actionpotential-induced nondecremented de-polarization, Ca2+ elevations in dendritescan be graded and may be driven by backpropagating action potentials or prolongeddendritic Ca2+ spikes. Additionally, they canbe mediated through activation of Ca2+-permeable receptor-channel complexesor by release of Ca2+ from internal stores(37, 81, 82). Interestingly, a number of in-vestigations suggest that different voltage-gated Ca2+ channels operate at these twopoles of neurons, with L- and R-typesdominating in dendrites, in contrast to axonterminals that depend primarily on P/Q-and N-type channels to mediate Ca2+ in-flux (83, 84) (Fig. 2B and see below). Anillustrative example of such polarization isprovided by dentate gyrus granule cells,where blockade of L-type Ca2+ currentinhibits the dendritic release of dynorphinat the perforant pathway synapses but hasno effect on secretion of the same sub-stance from the axon terminals of theseneurons (85). Differences have also beendocumented in the kinetics of free cytosolic[Ca2+], with fast dendritic Ca2+ transientsdisplaying more diffuse and protractedcharacteristics. The latter seem to dependmore heavily on activation of store-

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Fig. 3. Molecular architecture of the active zone. (A) Schematic of a canonical synapse with presynapticrelease site enriched with secretory vesicles (SV, blue), active zone proteins (light brown), SNAREs/Ca2+

sensor (red), scaffolding/cytoskeletal proteins (orange), and voltage-gated Ca2+ channels (yellow). Theadjacent postsynaptic receptive element comprises PSD95, SAP-97 (pink), receptors (green), scaffolding/cytoskeletal proteins (orange), and SNAREs (red). (B) Protein interaction network targeting secretoryvesicles and driving their regulated fusion at the active zone. In cooperation with Rab3-GTPase andanchoring/cytoskeletal proteins [leucocyte common antigen-related protein (LAR), neurexins, actin],active zone proteins that include members of five protein families (RIMs, Bassoon and Piccolo, ELKS, andliprins-α) assist docking and priming of synaptic vesicles for SNARE-mediated fusion. VDCC, voltage-dependent Ca2+ channel.

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operating Ca2+ channels, with a metabo-tropic mechanism contributing significantlyto the process (82, 86). In concert withslower kinetics of dendritic Ca2+ spikes,a greater component of diffuse efflux of[Ca2+] from internal stores would contrib-ute appreciably toward global [Ca2+] ele-vations, which in turn should favor diffuserelease of mediators or secretion of peptideand hormone-like substances from den-drites (87–89). Interestingly, although therelation between [Ca2+] and secretion ofboth classic transmitters and peptides (62,90) seems to be steeper at axon terminals,neurosecretion from dendrites exhibitsgreater sensitivity to smaller elevations in[Ca2+] and exhibits higher tolerance to re-duction of extracellular [Ca2+] (84, 91, 92).Hence, in contrast to micromolar [Ca2+]being required for release of classic trans-mitters at axon terminals (93, 94), the ab-solute minimum [Ca2+] elevations requiredfor dendritic release range between 50 and200 nM (62, 95). These concentrationsmatch closely the intracellular [Ca2+] re-quired for spontaneous exocytosis of neu-rotransmitters at nerve endings withoutnotable effects on evoked release proper-ties (67, 96). The higher efficiency of Ca2+

in dendrites has been speculated to be dueto distinct synaptotagmin variants or con-tribution of an additional high-affinity Ca2+

sensor linking voltage-activated Ca2+ influxto the dendritic secretory machinery (92).This notion received support from experi-ments showing that despite the fact that theNMDA receptor-channel complex at den-drites in olfactory bulb granule cells is notdirectly coupled to vesicular release, smallCa2+ elevations mediated through NMDAreceptor activation can be sufficient totrigger dendritic secretion of GABA (79).It is noteworthy that synaptotagmin 12 (97)and double C2 domain 2b (doc2b) (98)seem to ensure greater [Ca2+] sensitivity ofthe fusion machinery mediating spontane-ous release at nerve terminals and, perhaps,could contribute toward dendritic secre-tion. Intriguingly, the direct contribution ofsynaptotagmin 10 to insulin-like growthfactor 1 (IGF-1) release from somato-den-drites of olfactory bulb neurons has beenidentified recently (99). Localized on IGF-1containing vesicles, Ca2+ binding by syn-aptotagmin 10 causes these vesicles to un-dergo exocytosis with spatial and temporalcharacteristics distinct from Ca2+-de-pendent exocytosis controlled by synapto-tagmin 1 (99). These and perhaps otheryet-unidentified sensor proteins also con-tribute toward greater responsiveness ofdendritic secretory machinery to small ele-vations of cytoplasmic [Ca

2+]. Thus, the

higher responsiveness of dendritic fusionmachinery to local and global Ca2+ ele-vations and the activation of graded vesic-ular release of transmitters or peptides areconsistent with the specialization of den-

drites for diffuse neurosecretion with ret-rograde modulator signaling. A range ofdiffuse metabotropic effects caused bydendritic release provides direct experi-mental support for this inference.

Sites for Dendritic Secretion: ActiveZone vs. Ectopic Release?Requirement of active zone-like special-izations for exocytosis from dendrites is oneof the long-standing research queries ofneurobiology. These restricted protein-richareas of presynaptic plasma membraneprovide a molecular scaffold for dockingand priming of synaptic vesicles, with theircontrolled fusion and release of neuro-transmitters triggered by action potential-induced Ca2+ elevations (Fig. 3 A and B).In nerve cells, vesicular secretion at activezones and distant from membrane releasespecializations (ectopic release) is welldocumented (100). Although the dendro-dendritic juxtapositions, with closely asso-ciated secretory organelles, were amongthe first synaptic elements recognized in thebrain at an ultrastructural level (101), thereis so far no evidence for the presence ofelectron-dense active zone tufts in den-drites. Notably, together with the presenceof dense uniform vesicular pools, reminis-cent of specialized release sites at canonicalsynapses (101–105), diffusely distributedsecretory organelles have also been dem-onstrated in dendrites of central neurons,without preference for aggregations atmembrane juxtapositions (36, 106, 107).Manifestly, vesicular clustering at axonterminal is typically associated with anelectron-dense matrix, referred to as pre-synaptic grids (108), which have not beenidentified at dendrites of central neurons(31, 36, 106). Likewise, the molecularcomposition and architecture of dendro-dendritic synapses remains poorly studied(42), despite the latter being well recog-nized anatomically and physiologically. Thepresence of postsynaptic scaffolding mole-cules, such as PSD-93 and PSD-95, atdendro-dendritic juxtapositions of granule/mitral cells suggests their homology withconventional postsynaptic elements (109).However, no data are available on Rab3ainteracting molecules (RIMs), proteins richin E, L, K, and S (ELKS), Velis, Liprin-α orMunc13, Munc18, membrane-associatedSNAREs, and other hallmark active zoneproteins here and at other potential den-dro-dendritic junctions. Likewise, in mag-nocellular hypothalamic neurons, Ca2+-dependent relocation of LDCVs from thedeeper cytoplasm to the submembranecompartments has been demonstrated, yetwith no ultrastructural evidence for releasesite specializations nearby (107). In theabsence of active zones, the sensitivity ofdendritic LDCV neurosecretion to CNTs(107, 110) indicates ectopic SNARE-de-pendent (v-SNAREs) release of peptides

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Fig. 4. Polarized exocytotic machinery of centralneurons: a schematic. With both LDCVs and SSVs(light brown and white circles, respectively) iden-tified in dendrites and axon terminals, Ca2+ ele-vations that triggers dendritic secretion aremediated by activation of L- and R-type voltage-dependent Ca2+ channels (VDCC), NMDA receptor-channel complex, and store-operating Ca2+ chan-nels (SOCC). This contrasts with axon terminals,where Ca2+ elevations are mediated by P/Q-, N-type VDCC as well as SOCC mechanisms. PrincipalSNAREs identified at dendrites of neurons areVAMP7, SNAP-23, syntaxin3 and -4, and SNAP-25;at axon terminals SNAREs are represented byVAMP1 and -2, SNAP-25, and syntaxin1 and 2.PSD, postsynaptic density. DDS, dendro-dendriticsynapse; ADS, axo-dendritic synapse. Note thatneurosecretion at axon terminal can be caused byreversible hemifusion (“kiss-and-stay,” “kiss-and-run”) (1) or complete collapse of synaptic vesicleswith the plasmalemma (2). Recycling of synapticvesicles after their complete fusion involves cla-thrin-dependent endocytosis (coated vesicles).

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from the dendrites of these neurons, similarto that described in adrenal chromaffincells (111, 112). Data obtained from nigraldopaminergic cells indicate that althoughtheir axon terminals in the striatum areenriched with vesicular aggregates and ac-tive zone type specializations with closelypositioned vesicular pools (44, 113), theirdendrites in the substantia nigra are devoidof such aggregations or any release spe-cializations (114, 115). Note that as in hy-pothalamic neurons, dopamine secretion atboth corpus striatum and substantia nigra isstrongly inhibited by CNTs, which is con-sistent with its reliance on SNAREs (50).Interestingly, the ectopic dopamine releasefrom dendrites of nigral neurons agreeswith diffuse immunoreactivity of SNAP-25therein (52, 54), contrasting with thepunctuated presence of this SNARE vari-ant at presynaptic axon terminals of othercentral neurons (116, 117). Finally, analysisof the distribution of vesicular glutamatetransporters-3 (VGluT3), an SSV proteinexpressed preferentially in dendrites ofcortical neurons (118, 119), also revealedits dispersed immunoreactivity at bothsubsynaptic areas as well as along the pri-mary and secondary apical and basal den-drites (119), with no indication of activezone-like specializations therein. Thus, al-though the distribution of secretory organ-elles in dendrites of some central neuronssuggests exocytotic specializations, the bulkof the data are consistent with their diffusedistribution with fusion outside of activezones, posing the fundamental question ofregulatory mechanisms and the physiologi-cal significance of extrasynaptic retrogradesignaling. Unquestionably, recent advancesin optical imaging combined with computermodels simulating transmission betweenneurons, which made possible analysis ofpresynaptic dynamics at the level of theindividual vesicle (100, 120), should greatlyassist future studies in these and numerousother fascinating questions raised bydendritic research.

Functional Polarization of Neurons andSNAREsThe highly asymmetric morphology ofnerve cells led Ramon-y-Cajal to put for-ward the principle of functional polariza-tion of neurons, which postulatedunidirectional flow of information fromdendrites to axons. Together with the an-atomical, physiological, and biochemicaldiscreteness of nerve cells, the polarizationprinciple rests as one of the cornerstones ofneuronal doctrine, affirming nerve cellsas basic structural and functional modulesof the brain (121). He explained that

“for us, the cause of. . .polarization. . .isuniquely in the relationship which existsbetween the neurons. . .where the seat isthe entry of excitation” (122). Indeed, asestablished later, these entry sites corre-spond to chemical synapses, specializedfor vectorial transmission of signals viavesicular release of neurotransmitters(123, 124), a process blocked by CNTs.Accumulating data indicate congruity ofSNARE-dependent exocytotic mecha-nisms and machinery at axon terminalsand dendrites. However, as in other po-larized cells (125), some SNARE in-hibitors have revealed differential effectsat these two neuronal poles, implying axo-dendritic specialization of membrane fu-sion nanoengines and release mechanisms(Fig. 4). Interestingly, although fusigenicasymmetry in epithelial cells seems to beestablished primarily by differential dis-tribution of individual t-SNAREs (126,127) as well as their regulation by differentvariants of Sec/Munc SM proteins (128,129), in neurons both t- and v-SNAREsseem to contribute to such polarization(50, 73). Though explicit, these originaldata require further experimental valida-tion with the use of advanced optical toolsfor single-molecule studies combined withgenetic approaches and electrophysiology.Ad interim, it is tempting to speculate thatpolarization of secretory machinery andmechanisms facilitated the remarkableextension of dendritic functions duringevolution far beyond mere informationtransmission and wiring of individualneurons into functional networks. Thiswould provide dendrites with astoundingflexibility and the capacity for integratingand processing tremendous volumes ofinformation (130, 131). Indeed, alongwith the significant retrograde influenceon synaptic inputs and dendritic cableproperties through regulation of voltage-gated K+ and other currents, dendriticsecretion endows nerve cells with thecapacity to support a broad array of in-tegrative and homeostatic functions(132–134). Thus, in addition to sharedtraits and functions with those recognizedfor axon terminals, SNARE-dependentdendritic secretion seems to be specializedfor functions unique to this neuronalcompartment.

Concluding Remarks and FutureDirectionsDisentangling the precise means used bynerve cells for information processing andtransmission would afford important cluesto one of the most enduring mysteries ofour times—the human brain. It also is

likely to unveil recipes for potential rem-edies to normalize brain functions thathave been disrupted by numerous dis-orders and diseases. Emerging data sug-gest that release of mediators and peptidesfrom dendrites exhibiting instructive andhomeostatic effects offers neurons enor-mous integrative power and diversity. In-deed, with the capacity for graded tuningof afferent inputs through release ofmodulators and transmitters, dendriteseffectively filter and sort inputs and adjusttheir strength. Although recent researchgreatly advanced our understanding ofdendritic physiology, it has also raiseda number of fundamental questions withimportant implications for the SNAREhypothesis and neuronal doctrine. Recog-nition of SNAREs and other regulatoryproteins specialized for retrograde signal-ing at dendrites queries the canonicalsynapse-based view of redundancy of someSNARE proteins and predicts decoroussects for these molecules in the “functionalcatalog” of neurons. Likewise, vesicularsecretion from dendrites in the absence ofactive zones along with apparently higher[Ca2+] sensitivity of dendritic exocytosiscall for further refinement of the currentcanonical synapse-based neurotransmis-sion hypothesis. Ironically, the emergingview on asymmetric secretory mechanismsat axo-dendritic poles of neurons, thoughcomplementing Ramon-y-Cajal’s doctrineof dynamic polarization of neurons,also revitalizes the reticularist view ofneuronal assemblies, led by Ramon-y-Cajal’s key opponent Camillo Golgi,who championed the intimate reciprocalinteraction between nerve cells (135).Clearly, along with the deep physiologicaldivision between dendrites and axons,the data outlined above strongly suggesttheir common functional and evolutionarymissions—channeling and modulation ofinformation flow between neurons. Not-withstanding all successes and failures,the stakes remain high for defining themeaning and significance of axo-dendriticpolarization of release mechanisms forinformation processing at the level of thesingle neuron and overall integrativebrain functions.

ACKNOWLEDGMENTS. We thank Drs. GaryLawrence, Valerie B. O’Leary, and Neal Lemon forinsightful discussions and comments onthis manuscript. We apologize for not citing allrelevant articles on the topic because of spaceconstraints. This work was supported by theProgram for Research in Third Level Institu-tions Cycle 4 grant from the Irish HigherEducational Authority for the Neurosciencesection of “Targeted-driven therapeutics andtheranostics.”

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