Quantitative analysis of synaptic vesicle release and readily releasable pool size in hippocampal neurons

( views:, downloads: )
XU Shu-Jun(Medical School, Ningbo University, Ningbo 315211, China)
WANG Ping(Medical School, Ningbo University, Ningbo 315211, China)
XIA Di(Institute of Neuroscience and Key Laboratory of Neurobiology,Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China)
Journal Title:
Volume 61, Issue 06, 2009
Key Word:
endocytosis; synaptic vesicles; whole cell recording; cultured cell; hippocampus

Abstract: In central nervous system only a limited number of vesicles exist in the presynaptic terminals. The size and fusion modes of the vesicles were particularly important because of their potential impact on neuronal communications. Efficient methods were needed to analyze the recycling kinetics of synaptic vesicle and the size of readily releasable pool (RRP). In this study, fluorescent dyes with different affinity for membranes (FM1-43 with high affinity and FM2-10 with low affinity) were used to stain the functional synaptic vesicles of cultured hippocampal neurons and the kinetics of vesicle recycling was measured. The results showed that the destaining proportion was larger for FM2-10 than that for FM1-43 during the first trial, while it was greater for FM1-43 than FM2-10 during the second and third trials (first round, 93.0%±5.9% versus 57.9%±3.5% for FM2-10 and FM1-43, respectively, P<0.0001; second round, 1.4%±3.8% versus 24.0%±2.3%, P<0.0001; third round, 2.3%±1.6% versus 8.6%±1.5%, P=0.005). The results indicated that rapid endocytosis existed not only in the first round but also occurred when the vesicles were reused. Moreover, Both high-frequency stimuli and hypertonic sucrose stimuli were used to estimate the RRP sizes in the mix cultured hippocampal inhibitory neurons at 13-14 days in vitro (DIV). We found that the RRP size estimated by hypertonic sucrose stimuli [(200±23.0) pC] was much larger than that estimated by high-frequency stimuli [(51.1±10.5) pC]. One possible reason for the discrepancies in RRP estimates is that in mix cultured conditions, one neuron may receive inputs from several neurons and hypertonic sucrose stimuli will cause RRP of all those neurons release, while using dual patch recording, only the connection between two neurons was analyzed. Thus, to exclude out the impacts of inputs numbers on RRP sizes, it is more reasonable to use high-frequency stimuli to estimate the RRP size in mix cultured neurons.

  • [1]Rizzoli SO,Betz WJ.Synaptic vesicle pools.Nat Rev Neurosci 2005; 6(1):57-69.
  • [2]Harata NC,Choi S,Pyle JL,Aravanis AM,Tsien RW.Frequency-dependent kinetics and prevalence of kiss-and-run and reuse at hippocampal synapses studied with novel quenching methods.Neuron 2006; 49(2):243-256.
  • [3]Zhang Q,Li Y,Tsien RW.The dynamic control of kiss-and-run and vesicular reuse probed with single nanoparticles.Science 2009;323(5920):1448-1453.
  • [4]Richards DA,Bai J,Chapman ER.Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles.J Cell Biol 2005; 168(6):929-939.
  • [5]Gandhi SP,Stevens CF.Three modes of synaptic vesicular recycling revealed by single-vesicle imaging.Nature 2003; 423 (6940):607-613.
  • [6]Harata N,Pyle JL,Aravanis AM,Mozhayeva M,Kavalali ET,Tsien RW.Limited numbers of recycling vesicles in small CNS nerve terminals:implications for neural signaling and vesicular cycling.Trends Neurosci 2001; 24(11):637-643.
  • [7]Priller C,Mitteregger G,Paluch S,Vassallo N,Staufenbiel M,Kretzschmar HA,Jucker M,Herms J.Excitatory synaptic transmission is depressed in cultured hippocampal neurons of APP/ PS1 mice.Neurobiol Aging 2009; 30(8):1227-1237.
  • [8]Chen XW,Feng YQ,Hao CJ,Guo XL,He X,Zhou ZY,Guo N,Huang HP,Xiong W,Zheng H,Zuo PL,Zhang CX,Li W,Zhou Z.DTNBP1,a schizophrenia susceptibility gene,affects kinetics of transmitter release.J Cell Biol 2008; 181(5):791-801.
  • [9]Zhang W,Buckmaster PS.Dysfunction of the dentate basket cell circuit in a rat model of temporal lobe epilepsy.J Neurosci 2009; 29(24):7846-7856.
  • [10]Cho JH,Askwith CC.Presynaptic release probability is increased in hippocampal neurons from ASIC1 knockout mice.J Neurophysiol 2008; 99(2):426-441.
  • [11]Otsu Y,Murphy TH.Optical postsynaptic measurement of Acta Physiologica Sinica,December 25,2009,61 (6):505-510vesicle release rates for hippocampal synapses undergoing asynchronous release during train stimulation.J Neurosci 2004; 24 (41):9076-9086.
  • [12]Chiappalone M,Casagrande S,Tedesco M,Valtorta F,Baldelli P,Martinoia S,Benfenati F.Opposite changes in glutamatergic and GABAergic transmission underlie the diffuse hyperexcit-ability of synapsin I-deficient cortical networks.Cereb Cortex 2009; 19(6):1422-1439.
  • [13]Liu H,Dean C,Arthur CP,Dong M,Chapman ER.Autapses and networks of hippocampal neurons exhibit distinct synaptic transmission phenotypes in the absence of synaptotagmin I.J Neurosci 2009; 29(23):7395-7403.
  • [14]Luo JH,Fu ZY,Losi G,Kim BG,Prybylowski K,Vissel B,Vicini S.Functional expression of distinct NMDA channel subunits tagged with green fluorescent protein in hippocampal neurons in culture.Neuropharmacology 2002; 42(3):306-318.
  • [15]Xu S,Ning W,Xu Z,Zhou S,Chiang H,Luo J.Chronic exposure to GSM 1800-MHz microwaves reduces excitatory synaptic activity in cultured hippocampal neurons.Neurosci Lett 2006; 398(3):253-257.
  • [16]Shen W,Wu B,Zhang Z,Dou Y,Rao ZR,Chen YR,Duan S.Activity-induced rapid synaptic maturation mediated by presynaptic cdc42 signaling.Neuron 2006; 50(3):401-414.
  • [17]Klingauf J,Kavalali ET,Tsien RW.Kinetics and regulation of fast endocytosis at hippocampal synapses.Nature 1998; 394(6693):581-585.
  • [18]Moulder KL,Mennerick S.Reluctant vesicles contribute to the total readily releasable pool in glutamatergic hippocampal neurons.J Neurosci 2005; 25(15):3842-3850.
  • [19]Stevens CF,Williams JH.Discharge of the readily releasable pool with action potentials at hippocampal synapses.J Neurophysiol 2007; 98(6):3221-3229.
  • [20]Kashani,AH,Chen BM,Grinnell AD.Hypertonic enhancement of transmitter release from frog motor nerve terminals:Ca~(2+) independence and role of integrins.J Physiol 2001; 530(pt 2):243-252.
  • [21]Smith SM,Renden R,von Gersdorff H.Synaptic vesicle endocytosis:fast and slow modes of membrane retrieval.Trends Neurosci 2008; 31(11):559-568.
  • [22]Stevens CF,Williams JH."Kiss and run" exocytosis at hippocampal synapses.Proc Natl Acad Sci USA 2000; 97(23):12828-12833.
WanfangData CO.,Ltd All Rights Reserved
About WanfangData | Contact US
Healthcare Department, Fuxing Road NO.15, Haidian District Beijing, 100038 P.R.China
Tel:+86-010-58882616 Fax:+86-010-58882615 Email:yiyao@wanfangdata.com.cn