Date Published: March 30, 2017
Publisher: Public Library of Science
Author(s): Mackenzie E. Hofmann, Michael C. Andresen, Fabien Tell.
The recycling of vesicle membrane fused during exocytosis is essential to maintaining neurotransmission. The GTPase dynamin is involved in pinching off membrane to complete endocytosis and can be inhibited by dynasore resulting in activity-dependent depletion of release-competent synaptic vesicles. In rat brainstem slices, we examined the effects of dynasore on three different modes of glutamate release–spontaneous, evoked, and asynchronous release–at solitary tract (ST) inputs to neurons in the nucleus of the solitary tract (NTS). Intermittent bursts of stimuli to the ST interspersed with pauses in stimulation allowed examination of these three modes in each neuron continuously. Application of 100 μM dynasore rapidly increased the spontaneous EPSC (sEPSC) frequency which was followed by inhibition of both ST-evoked EPSCs (ST-EPSC) as well as asynchronous EPSCs. The onset of ST-EPSC failures was not accompanied by amplitude reduction–a pattern more consistent with conduction block than reduced probability of vesicle release. Neither result suggested that dynasore interrupted endocytosis. The dynasore response profile resembled intense presynaptic TRPV1 activation. The TRPV1 antagonist capsazepine failed to prevent dynasore increases in sEPSC frequency but did prevent the block of the ST-EPSC. In contrast, the TRPV1 antagonist JNJ 17203212 prevented both actions of dynasore in neurons with TRPV1-expressing ST inputs. In a neuron lacking TRPV1-expressing ST inputs, however, dynasore promptly increased sEPSC rate followed by block of ST-evoked EPSCs. Together our results suggest that dynasore actions on ST-NTS transmission are TRPV1-independent and changes in glutamatergic transmission are not consistent with changes in vesicle recycling and endocytosis.
To sustain synaptic transmission, exocytotic vesicle release must be balanced with restoration of the pool of ready-releasable vesicles. Regenerating vesicles requires an endocytotic step in which membrane is retrieved and recycled to generate new vesicles in a timely fashion. Key aspects of these processes are calcium dependent and different forms of transmission likely engage multiple pools of vesicles [1–4]. The small molecule, dynasore, selectively and reversibly interrupts membrane endocytosis by inhibition of dynamin and thus vesicle recycling [5, 6]. Block of endocytosis by dynasore leads to vesicle depletion and produces vesicle component accumulation at the surface membrane in an activity dependent manner . Dynasore reduces evoked response amplitudes independent from spontaneous release suggesting differential actions across release modes . Thus, dynasore discriminated between activity-dependent and activity-independent synaptic vesicle release.
All animal procedures were approved by the Institutional Animal Care and Use Committee at Oregon Health and Science University and conformed to animal welfare guidelines issued by the National Institutes of Health publication Guide for the Care and Use of Laboratory Animals.
Many neurons rapidly recycle vesicle membrane during bouts of synaptic fusions and vesicle release in synaptic transmission [8, 30, 31]. Dynasore can disrupt action potential dependent vesicular release through the inhibition of dynamin to block endocytosis. Here, we investigated how dynasore affected spontaneous, evoked, and asynchronous release from TRPV1 expressing ST axons in the NTS. Dynasore facilitated both spontaneous and miniature frequency, suggesting a presynaptic site of action to increase the probability of release while interrupting evoked ST-EPSC transmission in the same neurons. With no graded changes in evoked amplitudes, we found no evidence of the activity-dependent vesicle depletion that was expected and conclude that vesicle recycling in ST afferents likely has no dynamin sensitive step. The relative timing of the effects of dynasore and its form–increases in spontaneous release prior to ST-EPSC failures–were reminiscent of TRPV1 activation in these ST afferents. Prior application of TRPV1 antagonists brought mixed and puzzling results. CPZ prevented the increase in ST-EPSC failures but not the increase in sEPSC frequency, while JNJ prevented both actions of dynasore suggesting a link to TRPV1 receptors. However, dynasore blocked the ST-EPSC and increased the spontaneous rate at a neuron whose ST afferent input lacked TRPV1 suggesting that dynasore acts independent of TRPV1. Overall and across 13 neurons, our results consistently point to presynaptic actions with decreases in afferent action potential conduction into the ST terminal as well as a separate action which facilitates spontaneous glutamate release. These actions of dynasore at ST afferents do not fit with the canonical view of disruption of endocytosis.