FM dyes have been used to label and then monitor synaptic vesicles, secretory granules and other endocytic structures in a variety of preparations. Here, we describe the general procedure for using FM dyes to study endosomal trafficking in general, and synaptic vesicle recycling in particular. The dye, dissolved in normal saline solution, is added to a chamber containing the preparation to be labeled. Stimulation evokes exocytosis, and compensatory endocytosis that follows traps FM dye inside the retrieved vesicles. The extracellular dye is then washed from the chamber, and labeled endocytic structures are examined with a fluorescence microscope. Fluorescence intensity provides a direct measure of the labeled vesicle number, a good measure of the amount of exocytosis. If the preparation is stimulated again, without dye in the chamber, dimming of the preparation provides a measure of exocytosis of labeled vesicles. With a synaptic preparation on hand, this protocol requires 1 day.

Synchronous neurotransmitter release is a highly regulated process that takes place at specializations at the presynaptic membrane called active zones (AZs). The relationships between AZs, quantal release, and vesicle replenishment are not well understood in a mature synapse. We have measured the number, distribution, and other properties of AZs in mouse motor nerve terminals and combined these observations with electrophysiological estimates of the size of the readily releasable pool (RRP) of synaptic vesicles. On average, we counted 850 AZs per terminal. Assuming two primary docked vesicles per AZ, we predict a total of ϳ1700 vesicles optimally positioned for exocytosis. Electrophysiological estimates of the size of the RRP, using a simple kinetic model that assumes exponential depletion of the initial pool and refilling by recruitment, gave an average value of 1730 quanta during 100 Hz stimulation, in satisfying agreement with the morphology. At lower stimulus frequencies, however, the model revealed that the estimated RRP size is smaller, suggesting that not all AZs participate in release at low stimulation frequencies.

We used fluorescence recovery after photobleaching (FRAP) to measure the mobility of synaptic vesicles in frog motor nerve terminals. Vesicles belonging to the recycling pool or to the reserve pool were selectively labeled with FM1-43. In resting terminals, vesicles in the reserve pool were immobile, while vesicles in the recycling pool were mobile. Nerve stimulation increased the mobility of reserve pool vesicles. Treatment with latrunculin A, which destroyed actin filaments, had no significant effect on mobility, and reducing the temperature likewise had little effect, suggesting that recycling pool vesicles move by simple diffusion. Application of okadaic acid caused vesicle mobility in both pools to increase to the same level. We could model these and others' results quantitatively by taking into account the relative numbers of mobile and immobile vesicles in each pool, and vesicle packing density, which has a large effect on mobility.

In addition to cognitive impairments, neurodevelopmental disorders (NDDs) often result in sensory processing deficits. However, the biological mechanisms that underlie impaired sensory processing associated with NDDs are generally understudied and poorly understood. We found that SYNGAP1 haploinsufficiency in humans, which causes a sporadic neurodevelopmental disorder defined by cognitive impairment, autistic features, and epilepsy, also leads to deficits in tactile-related sensory processing. In vivo neurophysiological analysis in Syngap1 mouse models revealed that upper-lamina neurons in somatosensory cortex (SSC) weakly encode information related to touch. This was caused by reduced synaptic connectivity and impaired intrinsic excitability within upper-lamina SSC neurons. These results were unexpected given that Syngap1 heterozygosity is known to cause circuit hyperexcitability in brain areas more directly linked to cognitive functions. Thus, Syngap1 heterozygosity causes a range of circuit-specific pathologies, including reduced activity within cortical neurons required for touch processing, which may contribute to sensory phenotypes observed in patients.

We measured synaptic vesicle mobility using fluorescence recovery after photobleaching of FM 1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] stained mouse motor nerve terminals obtained from wild-type (WT) and synapsin triple knock-out (TKO) mice at room temperature and physiological temperature. Vesicles were mobile in resting terminals at physiological temperature but virtually immobile at room temperature. Mobility was increased at both temperatures by blocking phosphatases with okadaic acid, decreased at physiological temperature by blocking kinases with staurosporine, and unaffected by disrupting actin filaments with latrunculin A or reducing intracellular calcium concentration with BAPTA-AM. Synapsin TKO mice showed reduced numbers of synaptic vesicles and reduced FM 1-43 staining intensity. Synaptic transmission, however, was indistinguishable from WT, as was synaptic vesicle mobility under all conditions tested. Thus, in TKO mice, and perhaps WT mice, a phospho-protein different from synapsin but otherwise of unknown identity is the primary regulator of synaptic vesicle mobility.

Inhibition from molecular layer interneurons (MLIs) is thought to play an important role in cerebellar function by sharpening the precision of Purkinje cell spike output. Yet the coding features of MLIs during behavior are poorly understood. To study MLI activity, we used in vivo Ca 2ϩ imaging in head-fixed mice during the performance of a rhythmic motor behavior, licking during water consumption. MLIs were robustly active during lick-related movement across a lobule-specific region of the cerebellum showing high temporal correspondence within their population. Average MLI Ca 2ϩ activity strongly correlated with movement rate but not to the intentional, or unexpected, adjustment of lick position or to sensory feedback that varied with task condition. Chemogenetic suppression of MLI output reduced lick rate and altered tongue movements, indicating that activity of these interneurons not only encodes temporal aspects of movement kinematics but also influences motor outcome pointing to an integral role in online control of rhythmic behavior.

We monitored presynaptic exocytosis and vesicle recycling at neuromuscular junctions of transgenic mice expressing synaptopHluorin (spH), using simultaneous optical and electrophysiological recordings. Synaptic transmission was indistinguishable from that in wildtype controls. Fluorescence rose during and decayed monotonically after stimulus trains to the nerve, with amplitudes and decay times increasing with the amount of stimulation. The relatively large size of synaptic terminals allowed us to examine the spatial profile of fluorescence changes. We identified hot spots of exocytosis, which were stable with repeated trains. Photobleach experiments showed that spH freshly exposed by nerve stimulation was not preferentially retrieved by compensatory endocytosis; instead, most retrieved spH preexisted in the surface membrane. Finally, we compared fluorescence and electrical [summed end-plate potentials (EPPs)] estimates of exocytosis, which diverged during repeated trains, as fluorescence exceeded summed EPPs, although the average amplitude of miniature EPPs was unchanged. This might reflect exocytosis of spH-containing, acetylcholine-free ("empty") vesicles or other organelles during intense stimulation.

Motor learning involves neural circuit modifications in the cerebellar cortex, likely through re-weighting of parallel fiber inputs onto Purkinje cells (PCs). Climbing fibers instruct these synaptic modifications when they excite PCs in conjunction with parallel fiber activity, a pairing that enhances climbing fiber-evoked Ca2+ signaling in PC dendrites. In vivo, climbing fibers spike continuously, including during movements when parallel fibers are simultaneously conveying sensorimotor information to PCs. Whether parallel fiber activity enhances climbing fiber Ca2+ signaling during motor behaviors is unknown. In mice, we found that inhibitory molecular layer interneurons (MLIs), activated by parallel fibers during practiced movements, suppressed parallel fiber enhancement of climbing fiber Ca2+ signaling in PCs. Similar results were obtained in acute slices for brief parallel fiber stimuli. Interestingly, more prolonged parallel fiber excitation revealed latent supralinear Ca2+ signaling. Therefore, the balance of parallel fiber and MLI input onto PCs regulates concomitant climbing fiber Ca2+ signaling.