Dinara Shakiryanova

Activity-dependent liberation of synaptic neuropeptide vesicles

Despite the importance of neuropeptide release, which is evoked by long bouts of action potential activity and which regulates behavior, peptidergic vesicle movement has not been examined in living nerve terminals. Previous in vitro studies have found that secretory vesicle motion at many sites of release is constitutive: Ca2+ does not affect the movement of small synaptic vesicles in nerve terminals or the movement of large dense core vesicles in growth cones and endocrine cells. However, in vivo imaging of a neuropeptide, atrial natriuretic factor, tagged with green fluorescent protein in larval Drosophila melanogaster neuromuscular junctions shows that peptidergic vesicle behavior in nerve terminals is sensitive to activity-induced Ca2+ influx. Specifically, peptidergic vesicles are immobile in resting synaptic boutons but become mobile after seconds of stimulation. Vesicle movement is undirected, occurs without the use of axonal transport motors or F-actin, and aids in the depletion of undocked neuropeptide vesicles. Peptidergic vesicle mobilization and post-tetanic potentiation of neuropeptide release are sustained for minutes.

Neuropeptide delivery to synapses by long-range vesicle circulation and sporadic capture.

Neurotransmission requires anterograde axonal transport of dense core vesicles (DCVs) containing neuropeptides and active zone components from the soma to nerve terminals. However, it is puzzling how one-way traffic could uniformly supply sequential release sites called en passant boutons. Here, Drosophila neuropeptide-containing DCVs are tracked in vivo for minutes with a new method called simultaneous photobleaching and imaging (SPAIM). Surprisingly, anterograde DCVs typically bypass proximal boutons to accumulate initially in the most distal bouton. Then, excess distal DCVs undergo dynactin-dependent retrograde transport back through proximal boutons into the axon. Just before re-entering the soma, DCVs again reverse for another round of anterograde axonal transport. While circulating over long distances, both anterograde and retrograde DCVs are captured sporadically in en passant boutons. Therefore, vesicle circulation, which includes long-range retrograde transport and inefficient bidirectional capture, overcomes the limitations of one-way anterograde transport to uniformly supply release sites with DCVs.

Activity-dependent synaptic capture of transiting peptidergic vesicles.

Synapses require resources synthesized in the neuronal soma, but there are no known mechanisms to overcome delays associated with the synthesis and axonal transport of new proteins generated in response to activity, or to direct resources specifically to active synapses. Here, in vivo imaging of the Drosophila melanogaster neuromuscular junction reveals a cell-biological strategy that addresses these constraints. Peptidergic vesicles continually transit through resting terminals, but retrograde peptidergic vesicle flux is accessed following activity to rapidly boost neuropeptide content in synaptic boutons. The presence of excess transiting vesicles implies that synaptic neuropeptide stores are limited by the capture of peptidergic vesicles at the terminal, rather than by synthesis in the soma or delivery via the axon. Furthermore, activity-dependent capture from a pool of transiting vesicles provides a nerve terminal–based mechanism for directing distally and slowly generated resources quickly to active synapses. Finally, retrograde transport in the nerve terminal is regulated by activity.

Presynaptic Ryanodine Receptor-Activated Calmodulin Kinase II Increases Vesicle Mobility and Potentiates Neuropeptide Release

Although it has been postulated that vesicle mobility is increased to enhance release of transmitters and neuropeptides, the mechanism responsible for increasing vesicle motion in nerve terminals and the effect of perturbing this mobilization on synaptic plasticity are unknown. Here, green fluorescent protein-tagged dense-core vesicles (DCVs) are imaged in Drosophila motor neuron terminals, where DCV mobility is increased for minutes after seconds of activity. Ca2+-induced Ca2+ release from presynaptic endoplasmic reticulum (ER) is shown to be necessary and sufficient for sustained DCV mobilization. However, this ryanodine receptor (RyR)-mediated effect is short-lived and only initiates signaling. Calmodulin kinase II (CaMKII), which is not activated directly by external Ca2+ influx, then acts as a downstream effector of released ER Ca2+. RyR and CaMKII are essential for post-tetanic potentiation of neuropeptide secretion. Therefore, the presynaptic signaling pathway for increasing DCV mobility is identified and shown to be required for synaptic plasticity.

In vivo imaging of vesicle motion and release at the Drosophila neuromuscular junction.

Recently, it has become possible to directly detect changes in neuropeptide vesicle dynamics in nerve terminals in vivo and to measure the release of neuropeptides induced experimentally or evoked by normal behavior. These results were obtained with the use of transgenic fruit flies that express a neuropeptide tagged with green fluorescent protein. Here, we describe how vesicle movement and neuropeptide release can be studied in the larval Drosophila neuromuscular junction using fluorescence microscopy. Analysis methods are described for quantifying movement based on time lapse and fluorescence recovery after photobleaching data. Specific approaches that can be applied to nerve terminals include single particle tracking, correlation and Fourier analysis. Utilization of these methods led to the first detection of vesicle mobilization in nerve terminals and the discoveries of activity-dependent capture of transiting vesicles and post-tetanic potentiation of neuropeptide release. Overall, this protocol can be carried out in an hour with ready Drosophila.

Presynaptic ryanodine receptor-CamKII signaling is required for activity-dependent capture of transiting vesicles.

Activity elicits capture of dense-core vesicles (DCVs) that transit through resting Drosophila synaptic boutons to produce a rebound in presynaptic neuropeptide content following release. The onset of capture overlaps with an increase in the mobility of DCVs already present in synaptic boutons. Vesicle mobilization requires Ca2+-induced Ca2+ release by presynaptic endoplasmic reticulum (ER) ryanodine receptors (RyRs) that in turn stimulates Ca2+/calmodulin-dependent kinase II (CamKII). Here we show that the same signaling is required for activity-dependent capture of transiting DCVs. Specifically, the CamKII inhibitor KN-93, but not its inactive analog KN-92, eliminated the rebound replacement of neuropeptidergic DCVs in synaptic boutons. Furthermore, pharmacologically or genetically inhibiting neuronal sarco-endoplasmic reticulum calcium ATPase to deplete presynaptic ER Ca2+ stores or directly inhibiting RyRs prevented the capture response. These results show that the presynaptic RyR–CamKII pathway, which triggers mobilization of resident synaptic DCVs to facilitate exocytosis, also mediates activity-dependent capture of transiting DCVs to replenish neuropeptide stores.

Synaptic neuropeptide release induced by octopamine without Ca2+ entry into the nerve terminal

Synaptic release of neurotransmitters is evoked by activity-dependent Ca2+ entry into the nerve terminal. However, here it is shown that robust synaptic neuropeptide release from Drosophila motoneurons is evoked in the absence of extracellular Ca2+ by octopamine, the arthropod homolog to norepinephrine. Genetic and pharmacology experiments demonstrate that this surprising peptidergic transmission requires cAMP-dependent protein kinase, with only a minor contribution of exchange protein activated by cAMP (epac). Octopamine-evoked neuropeptide release also requires endoplasmic reticulum Ca2+ mobilization by the ryanodine receptor and the inositol trisphosphate receptor. Hence, rather than relying exclusively on activity-dependent Ca2+ entry into the nerve terminal, a behaviorally important neuromodulator uses synergistic cAMP-dependent protein kinase and endoplasmic reticulum Ca2+ signaling to induce synaptic neuropeptide release.

Differential Control of Presynaptic CamKII Activation and Translocation to Active Zones

The release of neurotransmitters, neurotrophins, and neuropeptides is modulated by Ca2+ mobilization from the endoplasmic reticulum (ER) and activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). Furthermore, when neuronal cultures are subjected to prolonged depolarization, presynaptic CaMKII redistributes from the cytoplasm to accumulate near active zones (AZs), a process that is reminiscent of CaMKII translocation to the postsynaptic side of the synapse. However, it is not known how presynaptic CaMKII activation and translocation depend on neuronal activity and ER Ca2+ release. Here these issues are addressed in Drosophila motoneuron terminals by imaging a fluorescent reporter of CaMKII activity and subcellular distribution. We report that neuronal excitation acts with ER Ca2+ stores to induce CaMKII activation and translocation to a subset of AZs. Surprisingly, activation is slow, reflecting T286 autophosphorylation and the function of presynaptic ER ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs). Furthermore, translocation is not simply proportional to CaMKII activity, as T286 autophosphorylation promotes activation, but does not affect translocation. In contrast, RNA interference-induced knockdown of the AZ scaffold protein Bruchpilot disrupts CaMKII translocation without affecting activation. Finally, RyRs comparably stimulate both activation and translocation, but IP3Rs preferentially promote translocation. Thus, Ca2+ provided by different presynaptic ER Ca2+ release channels is not equivalent. These results suggest that presynaptic CaMKII activation depends on autophosphorylation and global Ca2+ in the terminal, while translocation to AZs requires Ca2+ microdomains generated by IP3Rs.

Prolonged presynaptic posttetanic cyclic GMP signaling in Drosophila motoneurons

Ca2+ can stimulate cyclic nucleotide synthesis, but it is not known whether this signaling occurs in nerve terminals in response to activity. Here, in vivo imaging of Drosophila motoneuron terminals shows that activity rapidly induces a long-lasting signal from a transgenically expressed optical indicator based on the epac1 (exchange protein directly activated by cAMP 1) cAMP-binding domain. The epac1-cAMP sensor (camps) response in synaptic boutons depends on extracellular Ca2+ and ryanodine receptor-mediated Ca2+-induced Ca2+ release from the endoplasmic reticulum. However, mutations that inhibit rutabaga Ca2+-stimulated adenylyl cyclase and dunce cAMP-specific phosphodiesterase (PDE) have no effect. Instead, the activity-dependent presynaptic epac1-camps signal reflects elevation of cGMP in response to nitric oxide-activated guanylyl cyclase. Posttetanic presynaptic cGMP is long-lived because of limited PDE activity. Thus, nerve terminal biochemical signaling induced by brief bouts of activity temporally summates on a time scale orders of magnitude longer than fast transmission.

Imaging Neuropeptide Release in the Drosophila Neuromuscular Junction (NMJ)

Electrophysiological studies of synaptic function cannot directly reveal the internal workings of the nerve terminal and do not robustly report release of neuropeptides and neurotrophins. These limitations can now be overcome with the presynaptic expression of green fluorescent protein (GFP) indicators of vesicle motion, release, and signaling. This protocol describes how to image single wavelength and ratiometric fluorescence resonance energy transfer (FRET)-based GFP indicators with fluorescence microscopy in living synaptic boutons of the Drosophila neuromuscular junction (NMJ). The steps for setting up the imaging equipment for epifluorescence microscopy are given, followed by special considerations for preparing the larval NMJ for peptide release studies.