Introduction

Primary neuronal cultures from rodent hippocampus have become a standard preparation for studying synaptic physiology, offering in vitro mechanical stability and ready access to the extracellular space. The FM® styryl dyes, which have a high membrane affinity, allow direct monitoring of vesicle release and uptake in presynaptic terminals in dissociated neuronal cultures, yielding valuable insights into transmitter release (for review see Reference 1). Neuronal cultures have met significant criticism, though, for abnormal neuronal behavior that can result from formation of synapses and neuronal networks under artificial conditions, leading many to favor acute brain slice preparations or in vivo recording. For example, properties of inhibitory synapses in dissociated neuronal cultures exhibit abnormal maturation from postnatal days 6–15, continuing to show properties of early (P1–5) postnatal synapses even after 13–21 days in vitro (2). Cortical oscillatory behavior in vivo does not require γ-aminobutyric acid (GABA)_A-ergic transmission and disappears at postnatal days 6–7 (3), but develops much more slowly in cortical cultures only after days 9–15 in vitro, and this oscillatory activity now requires GABA_A-receptor activation (4). In brain slices, electro-physiology and histology typically supply, with fewer technical challenges, data that show excellent agreement with in vivo findings. Brain slices offer, over in vivo preparations, additional experimental windows of opportunity, but FM-imaging has been fatally hampered by nonspecific binding of dye.In this study, we demonstrate that, in brain slices, two-photon laser-scanning microscopy (TPLSM) of FM 1-43-labeled presynaptic terminals is a superior method for visualizing dynamics of total and readily releasable vesicle pools (TP and RRP). This method avoids many of the technical problems associated with microdi-alysis, quantal analysis, or presynaptic patch clamp recording. TPLSM gives superior signal over background as compared with quenching background fluorescence with a high concentration of sulforhodamine 101 (S-Rhd101; 50 µM) or using an even higher concentration of the β-cyclodextrin ADVASEP-7 (1 mM) as a carrier to facilitate wash-out of nonspecifically bound FM 1-43 (5,6,7,8), and TPLSM allows direct monitoring not only of vesicle release, but also of uptake.

Materials and Methods

Slice Preparation

Wistar rats 15–22 days of age, were decapitated under deep ether anesthesia, the hippocampi were dissected free, and 300-µm-thick transverse slices were cut on a DTK-1000 vibratome (Dosaka, Kyoto, Japan) or a Vibracut 3 (FTB, Weinheim, Germany). Slices were placed in an interface holding chamber at 25°C for at least 1 h before being transferred to a submerged chamber on the microscope stage, also at 25°C. Slices were perfused with artificial cerebrospinal fluid (ACSF; 2 mL/min) containing 126 mM NaCl, 5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM MgCl₂, 2 mM CaCl₂,26 mM NaHCO₃, and 10 mM glucose; saturated with 95% O₂ and 5% CO₂, pH 7.4. All drugs were bath applied. Schaffer collateral/ commissural axons in stratum radiatum were stimulated every 30 s for 1–5 s at either 10 or 50 Hz, with baseline stimulus intensities chosen to evoke half-maximal field excitatory postsyn-aptic potentials (EPSPs) in stratum radiatum of field CA1.

Labeling of the Total and Readily Releasable Vesicle Pools

Brain slices were labeled with FM 1 -43 by perfusing the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 µM) for 10 min to reduce spontaneous activity, followed by CNQX + FM 1-43 (5 µM; Molecular Probes, Eugene, OR, USA) for 5 min, and then CNQX + FM 1-43 in high [K⁺]_o (45 mM) for 15 min to stimulate uptake of FM 1-43 via endocytosis of vesicles. After loading, high [K⁺]_o and FM 1-43 were washed out for 30 min prior to imaging. To selectively release and load the RRP, we performed a rapid volume replacement of chamber saline with hyperosmotic ACSF solution (800 mOsm/L: ACSF plus 500 mOsm sucrose) for either 25 or 120 s, followed by rapid solution exchange back to control ACSF + CNQX for the 30-min FM 1-43 washout period. Stimulus-induced staining for measuring kinetics of vesicle endocytosis was recorded in the continued presence of CNQX and FM 1-43 by stimulating with 10 Hz for 2 s every 30 s. Stimulus-induced destaining was measured after 30 min in dye-free ACSF, evoked by 5-s bursts of 10 Hz or 1-s bursts of 50 Hz stimulation, applied once every 30 s. These short, discontinuous bursts produced a much slower time course of release than continuous stimulation (7,8,9,10,11). In this way, tissue movement artifacts were minimized and washout of released FM 1-43 from the vicinity of the synapse was facilitated.