Light and electron microscopic imaging of synaptic vesicle endocytosis at mouse hippocampal cultures

Abstract

Following the release of neurotransmitters at synaptic vesicles via exocytosis, endocytosis is initiated to retrieve vesicles that have fused with the plasma membrane of nerve terminals and recycle them, thus sustaining synaptic transmission. Here, we describe imaging-based protocols for quantitative measurements of endocytosis at cultured synapses. These protocols include (1) primary culture of mouse hippocampal neurons, (2) studying endocytosis at neurons transfected with a pH-sensitive synaptophysin-pHluorin2× using fluorescent microscopy, and (3) imaging endocytosis at fixed neurons with electron microscopy. For complete details on the use and execution of this protocol, please refer to Wu et al. (2016) and Wu et al. (2021).

Keywords: Cell Biology; Microscopy; Neuroscience.

Graphical abstract

Figure 1

Dissection of hippocampus from newborn mouse (A) A set of surgical tools, including an angled scissors, curved tweezers, blunt tweezers, fine tweezers, and fine scissors. (B) A P0−1 pup. Scale bar: 1 cm. (C) An isolated head in the dish containing ice-cold Dissection Solution. Scale bar (2 mm) also applies to panels (D–J). (D) A head after exposing brain. (E) An isolated brain. (F) A dissection microscope. The arrow shows a frozen-cold iron plate under a dish. (G) The location of the hippocampus in the ventromedial region of cerebral hemisphere. (H) Hippocampus marked with a dotted line, a “C”-shaped structure. (I) Isolated hippocampal tissue. (J) Chopped hippocampal tissue.

Figure 2

Trituration of hippocampal tissue (A) Before trituration, the digested hippocampal tissue in 2 mL of 1/1 Solution settled to the bottom of the tube. (B) Using a 1-mL tip to triturate the tissue. (C) After one trituration, hippocampal tissue becomes smaller pieces. (D) After trituration two more times, the suspension appears cloudy without any large pieces left.

Figure 3

Culture and development of hippocampal neurons (A) Under a light microscope (10×), the cells within a square chamber (marked with a black dash box) of the hemocytometer will be counted before cell plating. Bright dots are cells. (B) In a 37°C, 5% CO₂ incubator, 6-well plates with coverslip-plated neurons are placed on the top of empty 6-well plates. (C) Under a light microscope (10×), cultured neurons were imaged at 1 h after cell plating. Scale bar (100 μm) also applies to panels (D and E). (D) Cultured neurons at DIV7 are ready for transfection. (E) Cultured neurons at DIV14 are mature and ready for imaging experiments. (F–H) Zoomed-in views of neurons shown in (C, D, and E). Arrows in (F) show neurons with neurites. Scale bar (50 μm) applies to panels (F, G, and H).

Figure 4

Lipofectamine transfection procedures

Figure 5

Preparation of the coverslip-chamber unit (A) Bottom view of the imaging chamber. Two parallel platinum electrodes are used for field stimulation. The bath volume is 260 μL. Smear the grease on the bottom of the chamber. (B) Smear the grease on the top of the platform. (C) Mount a coverslip (colored with red to recognize it easily) on the top of the platform. (D) Put the chamber on the coverslip, with the bottom of the chamber with the grease facing the coverslip. The coverslip forms the floor of the chamber. (E) Put a clamp on the top of the chamber and tighten the screws to fix the chamber on the platform. (F) Coverslip-chamber unit was built. Add 250 μL of Saline Solution to bath.

Figure 6

Fluorescence images (A) Diagram of SypH tracking exocytosis and endocytosis. 1: In resting condition, SypH is quenched by low pH (pH_vesicle = 5.5) in the vesicle (see Figure 6B, left). 2: When the vesicle fuses with the plasma membrane, pH_vesicle increases to 7.2 because the protons move out of the vesicle, which activates SypH to produce the fluorescence (see Figure 6B, center). 3: Vesicle endocytosis. 4: After endocytosis, vesicles are re-acidified, which quenches SypH again (see Figure 6B, right). Therefore, exocytosis and endocytosis can be tracked in real-time by monitoring changes in SypH signals (see Figure 6C). (B) Snapshots at 10 s (left), 40 s (center) and 170 s (right) in a 180-s imaging video. ROI squares (2 μm × 2 μm) marked with different colors represent five boutons, which were visualized by dim fluorescence puncta. The intensity of the fluorescence signal reached its maximum at 40 s (center) at the end of a 10-s train stimulation (200 1-ms pulse at 20 Hz). Scale bar (10 μm) applies to all photos in panel B. (C) In Time Measurement window, fluorescence intensity traces were collected from 5 corresponding ROIs in (B). Their baselines, from 0 to 30 s, have different intensity levels. During a train stimulation (200 1-ms pulse at 20 Hz), from 30 s to 40 s, the intensity of signals increases, representing the net outcome of exocytosis and endocytosis. After 40 s, signals decay slowly, reflecting endocytosis. (D) The mean trace is averaged from the 5 traces shown in panel (C).

Figure 7

Analysis of endocytosis rate (A) In Igor software, a sampled trace shows how the initial rate of endocytosis (Rate_decay) was calculated. Exocytosis and endocytosis were induced by a train stimulation (black bar, 200 1-ms pulse at 20 Hz). The same stimulation also applies to (B and C). (B and C) Fluorescence response (mean + SEM) induced by stimulation in WT (B, n=17) and Kv3.3 KO (C, n=15) boutons at 34–37°C. Kv3.3: a voltage-dependent potassium channel. (D) Summary of Rate_decay (mean + SEM) collected from B and C. ∗∗: p < 0.01; t test (compared to WT). Note: Data in Figure B‒D indicate that Kv3.3 enhances the vesicle endocytosis in hippocampal synapses. Data in Figure B‒D have been modified from Wu et al. (2021).

Figure 8

Four experimental conditions before fixation in the EM protocol (A) HRP staining with or without application of high K⁺ solution to the samples before fixation. (B) Diagram of HRP staining a vesicle. 1: Vesicle in resting condition. 2: Vesicle exocytosis. 3: HRP enters the vesicle during endocytosis. 4: HRP in endocytosed vesicle. DAB and H₂O₂ penetrate the plasma membrane into the cytosol. 5: DAB and H₂O₂ enter the vesicle. 6: Vesicle stained by the oxidized DAB.

Figure 9

Determination of the regions of interest (ROIs) and preparation of the thin sections (A) The cells (grey spots) embedded in the resin are viewed under an inverted light microscope (10×). Cells in Circle 1 and Circle 2 show a difference in their densities. Two circles have the same size (diameter = 300 μm). Scale bar: 300 μm. (B) Zoomed-in view of Circle 1, showing low density of neuronal somas and with many interconnecting neurites. The areas like Circle 1 will be the ROIs for further thin section cutting. Scale bar: 100 μm. (C) Zoomed-in view of Circle 2, showing high density of neuronal somas. This kind of area may yield fewer synapses. Scale bar: 100 μm. (D) Serial thin sections were cut by the ultramicrotome (Leica Ultracut S).

Figure 10

Workflow for preparing EM sample

Figure 11

Ultrastructure of hippocampal neuron imaged by transmission electron microscopy (A) The neurites of hippocampal neurons were imaged immediately after washout of 90 mM KCl and 5 mg/mL HRP (Mag: 20000×) (condition 2). A synapse is shown in the center of the picture, including the presynaptic bouton (yellow dash line), synaptic cleft, and postsynaptic compartment (empty triangle). Scale bar: 400 nm. (B) The bouton is zoomed in for detail. Arrows: HRP (-) vesicles; arrowhead: HRP (+) vesicles (filled with the oxidized DAB); ∗: HRP (+) endosome. Scale bar: 100 nm.

Figure 12

Ultrastructural changes in β-actin KO hippocampal boutons (A) Electron microscopy images of wild-type and β-actin KO hippocampal boutons fixed at rest (R) and at 0 (K⁺), 3, and 10 min after the end of 1.5 min 90 mM KCl application. For R, HRP was applied for 1.5 min before fixation; for other three conditions, HRP and KCl were co-applied for 1.5 min. Scale bar (200 nm) applies to all photos in panel A. (B and C) The number of HRP(+) vesicles (B) and bulk endosome area (C) per bouton (μm²) is plotted versus the time before (R) and at 0 (K⁺), 3, and 10 min after the end of KCl application in control (black bars) and β-actin KO (red bars) hippocampal cultures (mean + SEM; each group was from 40–100 synaptic profiles). ^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05 (ANOVA). Note: These results suggest that β-actin enhanced both vesicle endocytosis and bulk endocytosis in hippocampal neurons. Data in Figure 12 have been modified from Wu et al. (2016).