Name: measuring synaptic vesicle endocytosis in cultured hippocampal neurons
Uploaded: 2017-09-04T12:30:00
Description: Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

We thank Dr. Yong-Ling Zhu for providing synaptophysin-pHluorin2x construct, and Dr. James E. Rothman for providing VAMP2-phluorin. We thank Dr. Susan Cheng and Virginia Crocker of NINDS Electron Microscopy Facility for their technical support and help. This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program in USA and a grant from the KRIBB Research Initiative Program (Korean Biomedical Scientist Fellowship Program), Korea Research Institute of Bioscience and Biotechnology, Republic of Korea.

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
<ol>
	<li>Hippocampal neuron culture
	<ol>
		<li>Prepare Hippoc...

<ol>
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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...

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 cells1,2,3. The pHluorin protein is typically attached to the lumenal side of synaptic vesicle proteins, such as synaptophysin or VAMP2/synaptobrevin. At rest, pHluorin is quenched in the 5.5 pH lumen of synaptic vesicles. Vesicle fusion to the plasma membrane exposes the vesicular lumen to the extracellular solution where the pH is ~7.3, resulting in an increase in pHluorin fluorescence. After exocytosis, the increased fluorescence decays, due to endocytosis of synaptic vesicle proteins followed by vesicle re-acidification within those recovered vesicles. Although the decay reflects both endocytosis and vesicular re-acidification, it mostly reflects endocytosis, because re-acidification is faster than endocytosis in most conditions1,4. The time constant of re-acidification is 3-4 s or less5,6, which is generally faster than the 10 s or more required for vesicle endocytosis4,5. If experiments are needed to distinguish endocytosis from re-acidification, acid quenching experiments using the 4-Morpholineethanesulfonic acid (MES) solution (25 mM) with a pH of 5.5 can be used to determine whether synaptic vesicle proteins are retrieved from the plasma membrane via endocytosis1,3,4. Thus, the pHluorin fluorescence intensity increase reflects a balance of exo- and endocytosis, and the decrease after nerve stimulation specifically reflects endocytosis.
pHluorin imaging may be used not only to measure the time course of endocytosis, but also the size of synaptic vesicle pools7,8, and the probability of evoked release and spontaneous release9. Many factors and proteins involved in regulating endocytosis, such as calcium, soluble NSF-attachment protein receptor (SNARE) proteins, brain-derived neurotrophic factor(BDNF), and calcineurin have been identified using pHluorin imaging1,2,10,11,12,13,14,15,16. Moreover, release of neurotransmitter could be detected in not only primary neurons but in neuroblastoma cells with TIRFM17. Recently, pHluorin variants, dsRed, mOrange and pHTomato were developed for monitoring simultaneous recordings of multiple factors in a single synapse18,19. For example, pHTomato has been fused with synaptophysin and used with a genetically encoded calcium indicator (GCaMP5K) to monitor presynaptic vesicle fusion and Ca2+ influx in the postsynaptic compartment20. Therefore, pHluorin attached to synaptic proteins provides a useful method to analyze the relationship between endocytosis and exocytosis.
EM is another method commonly used to study endocytosis, due to the high spatial resolution that shows ultrastructural changes during endocytosis. Two general areas are the ability to visualize pathological changes within neuronal cells21 and track vesicle proteins22. In particular, the observation of synaptic vesicle uptake, membrane curvature coated by clathrin in the periactive zone, and endosomal structures are possible with EM3,23,24,25,26,27,28. While EM involves potential artifacts, such as fixative-induced malformations, that may affect endocytosis, and data analysis is labor intensive, the resolution provides an attractive opportunity to visualize cellular structure. Potential fixative problems and the limitation in EM temporal resolution can be overcome by high pressure freezing, providing a fast and non-chemical method of stabilizing the delicate structures present during endocytosis27.

<table>
	<tbody>
		<tr>
			<td>Lipofectamine LTX with Plus</td>
			<td>Thermo Fisher</td>
			<td>15338-100</td>
			<td>Transfection of plasmid DNA including synaptophysin or VAMP2-pHluorin</td>
		</tr>
		<tr>
			<td>neurobasal medium</td>
			<td>Thermo Fisher</td>
			<td>21103-049</td>
			<td>Growth medium for neuron, Warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Thermo Fisher</td>
			<td>17504-044</td>
			<td>Gradient for neuronal differentiation</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Thermo Fisher</td>
			<td>35050-061</td>
			<td>Gradient for neuronal culture</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine coated coverslip</td>
			<td>Neuvitro</td>
			<td>GG-25-pdl</td>
			<td>Substrate for neuronal growth and imaging of pHluorin</td>
		</tr>
		<tr>
			<td>Trypsin XI from bovine pancrease</td>
			<td>Sigma</td>
			<td>T1005</td>
			<td>Neuronal culture-digest hippocampal tissues</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma</td>
			<td>D5025</td>
			<td>Neuronal culture-inhibits viscous cell suspension</td>
		</tr>
		<tr>
			<td>pulse stimulator</td>
			<td>A-M systems</td>
			<td>model 2100</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>Slotted bath with field stimulation</td>
			<td>Warner Instruments</td>
			<td>RC-21BRFS</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>stimulus isolation unit</td>
			<td>Warner Instruments</td>
			<td>SIU102</td>
			<td>Apply electrical stimulation</td>
		</tr>
		<tr>
			<td>lubricant</td>
			<td>Dow corning</td>
			<td>111</td>
			<td>pHluorin imaging-seal with coverslip and imaging chamber, avoid leak from chamber</td>
		</tr>
		<tr>
			<td>AP5</td>
			<td>Tocris</td>
			<td>3693</td>
			<td>Gradient for normal saline, selective NMDA receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>190</td>
			<td>Gradient for normal saline, competitive AMPA/kainate receptor antagonist, inhibit postsynaptic activity which have potential for recurrent activity</td>
		</tr>
		<tr>
			<td>Illuminator</td>
			<td>Nikon</td>
			<td>C-HGFI</td>
			<td>Metal halide light source for pHluorin</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon3</td>
			<td>pHluorin imaging, detect pHluorin fluorescence intensity</td>
		</tr>
		<tr>
			<td>Inverted microscopy</td>
			<td>Nikon</td>
			<td>Ti-E</td>
			<td>Imaging for synaptophysin or VAMP2 pHluorin transfected cells</td>
		</tr>
		<tr>
			<td>NIS-Elements AR</td>
			<td>Nikon</td>
			<td>NIS-Elements Advanced Research</td>
			<td>Software for imaging acquisition and analysis</td>
		</tr>
		<tr>
			<td>Igor Pro</td>
			<td>WaveMetrics</td>
			<td>Igor pro</td>
			<td>Software for imaging analysis and data presentation</td>
		</tr>
		<tr>
			<td>imaging chamber</td>
			<td>Warner Instruments</td>
			<td>RC21B</td>
			<td>pHluorin imaging, apply field stimulation on living cells</td>
		</tr>
		<tr>
			<td>poly-l-lysine</td>
			<td>Sigma</td>
			<td>P4832</td>
			<td>Electron microscopy, substrate for neuronal growth, apply on multiwell plate for 1 h at room temperature then wash with sterilized water 3 times</td>
		</tr>
		<tr>
			<td>Horseradish peroxidase(HRP)</td>
			<td>Sigma</td>
			<td>P6782</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Na cacodylate</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td>Electron microscopy, buffer for fixatives and washing, final concentration is 0.1 N</td>
		</tr>
		<tr>
			<td>3,3&#8242;-Diaminobenzidine(DAB)</td>
			<td>Sigma</td>
			<td>D8001</td>
			<td>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</td>
		</tr>
		<tr>
			<td>Hydrogen peroxide solution</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Electron microscopy, labeling of endocytosed synaptic vesicles by inducing HRP-DAB reaction, final concentration is 0.3% in DDW, make fresh before use</td>
		</tr>
		<tr>
			<td>glutaraldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>16365</td>
			<td>Electron microscopy, fixatives, final concentration is 4% in Na-cacodylate buffer, make fresh before use, shake well before to use</td>
		</tr>
		<tr>
			<td>TEM</td>
			<td>JEOL</td>
			<td>200CX</td>
			<td>Electron microscopy, imaging of endocytosed vesicles and ultrastructural changes</td>
		</tr>
		<tr>
			<td>CCD digital camera</td>
			<td>AMT</td>
			<td>XR-100</td>
			<td>Electron microscopy, capturing images</td>
		</tr>
		<tr>
			<td>Lead citrate</td>
			<td>Leica microsystems</td>
			<td>16707235</td>
			<td>Electron microscopy, grid staining</td>
		</tr>
	</tbody>
</table>

measuring synaptic vesicle endocytosis in cultured hippocampal neurons

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.

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 (&#916;F) 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-pHl...

Watch this Scientific Journal Video about Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons at JoVE.com

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

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

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Research

JoVE Journal

Neuroscience

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Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

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Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...