Postsynaptic synucleins mediate vesicular exocytosis of endocannabinoids

Two seemingly unrelated questions have long motivated studies in neuroscience: How are endocannabinoids, among the most powerful modulators of synaptic transmission, released from neurons? What are the physiological functions of synucleins, key contributors to Parkinson’s Disease? Here, we report an unexpected convergence of these two questions: Endocannabinoids are released via vesicular exocytosis from postsynaptic neurons by a synuclein-dependent mechanism. Specifically, we find that deletion of all synucleins selectively blocks all endocannabinoid-dependent synaptic plasticity; this block is reversed by postsynaptic expression of wildtype but not of mutant α-synuclein. Loading postsynaptic neurons with endocannabinoids via patch-pipette dialysis suppressed presynaptic neurotransmitter release in wildtype but not in synuclein-deficient neurons, suggesting that the synuclein deletion blocks endocannabinoid release. Direct optical monitoring of endocannabinoid release confirmed the requirement of synucleins. Given the role of synucleins in vesicular exocytosis, the requirement for synucleins in endocannabinoid release indicates that endocannabinoids are secreted via exocytosis. Consistent with this hypothesis, postsynaptic expression of tetanus-toxin light chain, which cleaves synaptobrevin SNAREs, also blocked endocannabinoid-dependent plasticity and release. The unexpected finding that endocannabinoids are released via synuclein-dependent exocytosis assigns a function to synucleins and resolves a longstanding puzzle of how neurons release endocannabinoids to induce synaptic plasticity.

Postsynaptic synucleins mediate vesicular exocytosis of endocannabinoids

Two seemingly unrelated questions have long motivated studies in neuroscience: How are endocannabinoids, among the most powerful modulators of synaptic transmission, released from neurons? What are the physiological functions of synucleins, key contributors to Parkinson's Disease? Here, we report an unexpected convergence of these two questions: Endocannabinoids are released via vesicular exocytosis from postsynaptic neurons by a synuclein-dependent mechanism. Specifically, we find that deletion of all synucleins selectively blocks all endocannabinoid-dependent synaptic plasticity; this block is reversed by postsynaptic expression of wildtype but not of mutant α-synuclein. Loading postsynaptic neurons with endocannabinoids via patch-pipette dialysis suppressed presynaptic neurotransmitter release in wildtype but not in synuclein-deficient neurons, suggesting that the synuclein deletion blocks endocannabinoid release. Direct optical monitoring of endocannabinoid release confirmed the requirement of synucleins. Given the role of synucleins in vesicular exocytosis, the requirement for synucleins in endocannabinoid release indicates that endocannabinoids are secreted via exocytosis. Consistent with this hypothesis, postsynaptic expression of tetanus-toxin light chain, which cleaves synaptobrevin SNAREs, also blocked endocannabinoid-dependent plasticity and release. The unexpected finding that endocannabinoids are released via synuclein-dependent exocytosis assigns a function to synucleins and resolves a longstanding puzzle of how neurons release endocannabinoids to induce synaptic plasticity.

Phosphatidylinositol-4,5-Biphosphate (PI(4,5)P2) Is Required for Rapid Endocytosis in Chromaffin Cells

Objective. Phosphoinositides play a regulatory role in clathrin-mediated endocytosis. However, their involvement in clathrin-independent endocytosis termed rapid endocytosis (RE), which is the mode of vesicle recycling during neurotransmitter release by transient fusion (known as kiss-and-run), has not been investigated. Here, we used patch-clamp recording of whole-cell membrane capacitance in adrenal chromaffin cells (ACC) to monitor changes of RE kinetics in response to pharmacological alteration of phosphatidylinositol-4,5-biphosphate (PI(4,5)P2) level by phenylarsine oxide (PAO) or antibody against phosphatidylinositol 4-kinase (AbPI4K). Results. We found that PAO and AbPI4K significantly abrogated RE kinetics. Infusion of PI(4,5)P2 through the patch pipette potentiated RE kinetics and reversed PAO- and AbPI4K-induced blockade of RE. Similarly, the application of the bifunctional thiol dithiothreitol (DTT) to PAO-treated cells completely prevented the inhibitory effect of PAO on RE. These findings indicate that PI(4,5)P2 is implicated in the signaling (mechanistic) process of RE in ACC.

How Many Angels Can Dance on the Head of a Patch Pipette? Understanding Neuronal Hyperexcitability in Angelman Syndrome

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A critical assessment of single-cell transcriptomes sampled following patch-clamp electrophysiology

Patch-seq, combining patch-clamp electrophysiology with single-cell RNA-sequencing (scRNAseq), enables unprecedented single-cell access to a neuron’s transcriptomic, electrophysiological, and morphological features. Here, we present a systematic review and re-analysis of scRNAseq profiles from 4 recent patch-seq datasets, benchmarking these against analogous profiles from cellular-dissociation based scRNAseq. We found an increased likelihood for off-target cell-type mRNA contamination in patch-seq, likely due to the passage of the patch-pipette through the processes of adjacent cells. We also observed that patch-seq samples varied considerably in the amount of mRNA that could be extracted from each cell, strongly biasing the numbers of detectable genes. We present a straightforward marker gene-based approach for controlling for these artifacts and show that our method improves the correspondence between gene expression and electrophysiological features. Our analysis suggests that these technical confounds likely limit the interpretability of patch-seq based single-cell transcriptomes. However, we provide concrete recommendations for quality control steps that can be performed prior to costly RNA-sequencing to optimize the yield of high quality samples.

Suppression of ciliary movements by a hypertonic stress in the newt olfactory receptor neuron

Olfactory receptor neurons isolated from the newt maintain a high activity of the ciliary beat. A cilium of neuron is so unique that only little is known about regulatory factors for its beat frequency. We examined the olfactory receptor neuron immersed in various extracellular media under the video-enhanced differential interference contrast microscope. The activation of voltage-gated Ca2+ channels by K+ depolarization or by application of Ca2+ to membrane-permeabilized olfactory cells did not affect the ciliary movement, suggesting that Ca2+ influx through the cell membrane has no direct effect on the movement. However, when an extracellular medium contained NaCl or sucrose at concentrations only 30% higher than normal levels, ciliary movement was greatly and reversibly suppressed. In contrast, a hypotonic solution of such a solute did not change the ciliary movement. The hypertonic solutions had no effect when applied to permeabilized cells. Suction of the cell membrane with a patch pipette easily suppressed the ciliary movement in an isotonic medium. Application of positive pressure inside the cell through the same patch pipette eliminated the suppressive effect. From these findings, we concluded that the hypertonic stress suppressed the ciliary movement not by disabling the motor proteins, microtubules, or their associates in the cilia, but rather by modifying the chemical environment for the motor proteins. The ciliary motility of the olfactory receptor cell is directly sensitive to the external environment, namely, the air or water on the nasal epithelium, depending on lifestyle of the animal.

Hypotonicity-induced cell swelling activates TRPA1

Hypotonic solutions can cause painful sensations in nasal and ocular mucosa through molecular mechanisms that are not entirely understood. We clarified the ability of human TRPA1 (hTRPA1) to respond to physical stimulus, and evaluated the response of hTRPA1 to cell swelling under hypotonic conditions. Using a Ca2+-imaging method, we found that modulation of AITC-induced hTRPA1 activity occurred under hypotonic conditions. Moreover, cell swelling in hypotonic conditions evoked single-channel activation of hTRPA1 in a cell-attached mode when the patch pipette was attached after cell swelling under hypotonic conditions, but not before swelling. Single-channel currents activated by cell swelling were also inhibited by a known hTRPA1 blocker. Since pre-application of thapsigargin or pretreatment with the calcium chelator BAPTA did not affect the single-channel activation induced by cell swelling, changes in intracellular calcium concentrations are likely not related to hTRPA1 activation induced by physical stimuli.

High membrane permeability for melatonin

The pineal gland, an endocrine organ in the brain, synthesizes and secretes the circulating night hormone melatonin throughout the night. The literature states that this hormone is secreted by simple diffusion across the pinealocyte plasma membrane, but a direct quantitative measurement of membrane permeability has not been made. Experiments were designed to compare the cell membrane permeability to three indoleamines: melatonin and its precursors N-acetylserotonin (NAS) and serotonin (5-HT). The three experimental approaches were (1) to measure the concentration of effluxing indoleamines amperometrically in the bath while cells were being dialyzed internally by a patch pipette, (2) to measure the rise of intracellular indoleamine fluorescence as the compound was perfused in the bath, and (3) to measure the rate of quenching of intracellular fura-2 dye fluorescence as indoleamines were perfused in the bath. These measures showed that permeabilities of melatonin and NAS are high (both are uncharged molecules), whereas that for 5-HT (mostly charged) is much lower. Comparisons were made with predictions of solubility-diffusion theory and compounds of known permeability, and a diffusion model was made to simulate all of the measurements. In short, extracellular melatonin equilibrates with the cytoplasm in 3.5 s, has a membrane permeability of ∼1.7 µm/s, and could not be retained in secretory vesicles. Thus, it and NAS will be “secreted” from pineal cells by membrane diffusion. Circumstances are suggested when 5-HT and possibly catecholamines may also appear in the extracellular space passively by membrane diffusion.