Ca2+ -independent transmission at the central synapse formed between dorsal root ganglion and dorsal horn neurons.

A central principle of synaptic transmission is that action potential-induced presynaptic neurotransmitter release occurs exclusively via Ca2+ -dependent secretion (CDS). The discovery and mechanistic investigations of Ca2+ -independent but voltage-dependent secretion (CiVDS) have demonstrated that the action potential per se is sufficient to trigger neurotransmission in the somata of primary sensory and sympathetic neurons in mammals. One key question remains, however, whether CiVDS contributes to central synaptic transmission. Here, we report, in the central transmission from presynaptic (dorsal root ganglion) to postsynaptic (spinal dorsal horn) neurons in vitro, (i) excitatory postsynaptic currents (EPSCs) are mediated by glutamate transmission through both CiVDS (up to 87%) and CDS; (ii) CiVDS-mediated EPSCs are independent of extracellular and intracellular Ca2+ ; (iii) CiVDS is faster than CDS in vesicle recycling with much less short-term depression; (iv) the fusion machinery of CiVDS includes Cav2.2 (voltage sensor) and SNARE (fusion pore). Together, an essential component of activity-induced EPSCs is mediated by CiVDS in a central synapse.

Tuning the Size of Large Dense-Core Vesicles and Quantal Neurotransmitter Release via Secretogranin II Liquid-Liquid Phase Separation.

Large dense-core vesicles (LDCVs) are larger in volume than synaptic vesicles, and are filled with multiple neuropeptides, hormones, and neurotransmitters that participate in various physiological processes. However, little is known about the mechanism determining the size of LDCVs. Here, it is reported that secretogranin II (SgII), a vesicle matrix protein, contributes to LDCV size regulation through its liquid-liquid phase separation in neuroendocrine cells. First, SgII undergoes pH-dependent polymerization and the polymerized SgII forms phase droplets with Ca2+ in vitro and in vivo. Further, the Ca2+ -induced SgII droplets recruit reconstituted bio-lipids, mimicking the LDCVs biogenesis. In addition, SgII knockdown leads to significant decrease of the quantal neurotransmitter release by affecting LDCV size, which is differently rescued by SgII truncations with different degrees of phase separation. In conclusion, it is shown that SgII is a unique intravesicular matrix protein undergoing liquid-liquid phase separation, and present novel insights into how SgII determines LDCV size and the quantal neurotransmitter release.

A method for recording the two phases of dopamine release in mammalian brain striatum slices.

Striatal dopamine (DA) release plays an essential role in many physiological functions including motor and non-motor behaviors (such as reward, motivation, and cognition). We have previously reported that, following a single electrical field stimulation, the amperometric recording of DA release from presynaptic terminals in striatal slices (both ventral and dorsal) contains two temporally separated phases. The first phase (direct DA transmission, direct DT) arises from DA terminal release following autologous action potentials (APs), while the second phase (cholinergic transmission-induced DA transmission, CTDT) arises from delayed DA release triggered by the activation of cholinergic interneurons to DA terminals (axon-axon transmission). The millisecond time-resolution of amperometry permits separation of an ∼7 ms latency difference from the single synapse (axon-axon) within the two-phase DA-release (2pDA) signal, and thus the 2pDA signal provides a novel method to study either direct DT, or CTDT, or both. Here, we describe the 2pDA method, including signal recording, processing, analysis, and troubleshooting (anti-artifact). Compared with other DA assays using different stimuli, recording methods, and preparations (such as high performance liquid chromatography or fast scan cyclic voltammetry), 2pDA recording is a novel and powerful physiological recording method for the study of DA transmissions in situ.