Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

Summary

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Abstract

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Introduction

Neurotransmitters are stored in synaptic vesicles and released by exocytosis. The synaptic vesicle membrane and protein are then internalized by endocytosis, and reused in the next round of exocytosis. Endocytosis of synaptic vesicles is important for maintaining synaptic vesicle pools and removes protruding vesicles from the plasma membrane. The pH-sensitive green fluorescent protein pHluorin, which is quenched in acidic circumstances and dequenched in neutral pH, has been used to measure endocytosis time courses in live cells^1,2,3. The pHluorin protein is typically attached....

Protocol

NOTE: The following protocol describes the pHluorin imaging methods and EM methods used in cultured hippocampal neurons. pHluorin monitors synaptic vesicle protein uptake in living cells and EM detects uptake of synaptic vesicle and ultrastructural changes.

Animal care and procedure followed NIH guidelines and were approved by the NIH Animal Care and Use Committee.

1. pHluorin Imaging

1. Hippocampal neuron culture
   1. Prepare Hippocampus Buffer (HB) by combining 4 mM NaHCO₃ and 5 mM HEPES and adjust to pH 7.3 with 5 M NaOH. Make the culture medium by mixing neurobasal ....

Results

Using the lipid carrier method, SpH was expressed in hippocampal neurons, allowing for the identification of boutons (Figure 1a). Electrical stimulation of the cells induced exocytosis, and a corresponding increase in fluorescence intensity. The increase in fluorescence (ΔF) was stopped by ending the stimulus (Figure 1b). The increased fluorescence was followed by a slow decrease, due to endocytosis. In the case of VAMP2-pHluorin.......

Discussion

Here we demonstrate two methods for monitoring synaptic vesicle endocytosis. In the first method, we monitored pHluorin fused with a synaptic vesicle protein in transfected neurons and subsequently electrically stimulated. Secondly, we used EM imaging of HRP uptake as induced by KCl. We used different stimuli for two reasons. First, high potassium application induces depolarization of all neurons in the culture. This facilitates EM examination, given that our EM methodology could not distinguish between non-stimulated an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Lipofectamine LTX with Plus	Thermo Fisher	15338-100	Transfection of plasmid DNA including synaptophysin or VAMP2-pHluorin
neurobasal medium	Thermo Fisher	21103-049	Growth medium for neuron, Warm up to 37°C before use
B27	Thermo Fisher	17504-044	Gradient for neuronal differentiation
Glutamax	Thermo Fisher	35050-061	Gradient for neuronal culture
Poly-D-Lysine coated coverslip	Neuvitro	GG-25-pdl	Substrate for neuronal growth and imaging of pHluorin
Trypsin XI from bovine pancrease	Sigma	T1005	Neuronal culture-digest hippocampal tissues
Deoxyribonuclease I from bovine pancreas	Sigma	D5025	Neuronal culture-inhibits viscous cell suspension
pulse stimulator	A-M systems	model 2100	Apply electrical stimulation
Slotted bath with field stimulation	Warner Instruments	RC-21BRFS	Apply electrical stimulation
stimulus isolation unit	Warner Instruments	SIU102	Apply electrical stimulation
lubricant	Dow corning	111	pHluorin imaging-seal with coverslip and imaging chamber, avoid leak from chamber
AP5	Tocris	3693	Gradient for normal saline, selective NMDA receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity
CNQX	Tocris	190	Gradient for normal saline, competitive AMPA/kainate receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity
Illuminator	Nikon	C-HGFI	Metal halide light source for pHluorin
EMCCD camera	Andor	iXon3	pHluorin imaging, detect pHluorin fluorescence intensity
Inverted microscopy	Nikon	Ti-E	Imaging for synaptophysin or VAMP2 pHluorin transfected cells
NIS-Elements AR	Nikon	NIS-Elements Advanced Research	Software for imaging acquisition and analysis
Igor Pro	WaveMetrics	Igor pro	Software for imaging analysis and data presentation
imaging chamber	Warner Instruments	RC21B	pHluorin imaging, apply field stimulation on living cells
poly-l-lysine	Sigma	P4832	Electron microscopy, substrate for neuronal growth, apply on multiwell plate for 1 h at room temperature then wash with sterilized water 3 times
Horseradish peroxidase(HRP)	Sigma	P6782	Electron microscopy, labeling of endocytosed synaptic vesicles by catalyzing DAB in presence hydrogen peroxide, final concentration is 5 mg/mL in normal saline, make fresh before use
Na cacodylate	Electron Microscopy Sciences	12300	Electron microscopy, buffer for fixatives and washing, final concentration is 0.1 N
3,3′-Diaminobenzidine(DAB)	Sigma	D8001	Electron microscopy, labeling of endocytosed synaptic vesicles, substrate for HRP, final concentration is 0.5 mg/mL in DDW and filtered, make fresh before use
Hydrogen peroxide solution	Sigma	H1009	Electron microscopy, labeling of endocytosed synaptic vesicles by inducing HRP-DAB reaction, final concentration is 0.3% in DDW, make fresh before use
glutaraldehyde	Electron Microscopy Sciences	16365	Electron microscopy, fixatives, final concentration is 4% in Na-cacodylate buffer, make fresh before use, shake well before to use
TEM	JEOL	200CX	Electron microscopy, imaging of endocytosed vesicles and ultrastructural changes
CCD digital camera	AMT	XR-100	Electron microscopy, capturing images
Lead citrate	Leica microsystems	16707235	Electron microscopy, grid staining