At chemical synapses, signals are transmitted via secretion of neurotransmitter. The neurotransmitter is stored in synaptic vesicles, which fuse with the plasma membrane at specialized release sites, active zones. After fusion, vesicles are recycled by endocytosis, refilled with neurotransmitter and used in subsequent rounds of release.

Our laboratory studies the molecular basis of presynaptic membrane trafficking. Our main model is the lamprey giant reticulospinal axon, which permits acute perturbation of protein-protein interactions by presynaptic microinjection.
 
Molecular mechanisms in neurotransmitter release: analysis of intact nerve terminals

Lennart Brodin, Peter Löw

To address the molecular basis of synaptic vesicle cycling, a giant synapse in a lower vertebrate (lamprey) is used. The function of specific proteins is perturbed in the living synapse by microinjection of fluorescence-labeled proteins, toxins and antibodies under inspection with a CCD camera. The effects are tested by intracellular recording of the transmitter released onto a target cell, by fluorescence imaging and by electron microscopic analysis.

In vitro studies have revealed an increasingly complex pattern of protein-protein interactions among presynaptic proteins. Many presynaptic proteins also interact with actin/actin-regulating proteins, and with membrane phospholipids.

 

An example of a protein which has been studied in our laboratory is endophilin. Endophilin is a BAR domain proteins with an SH3 domain, which interacts with dynamin and synaptojanin. Our initial studies showed that microinjection of endophilin antibodies blocks clathrin-mediated endocytosis at an early stage, before the shallow coated pit starts to invaginate (Ringstad et al. 1999). Subsequent work showed that compounds which disrupt interactions of the SH3 domain of endophilin with dynamin and synaptojanin impair synaptic vesicle endocytosis (Gad et al., 2000). Two distinct types of endocytic intermediate was found to accumulate. Free clathrin-coated vesicles were induced by a peptide blocking endophilin´s SH3 domain, and by antibodies to the proline-rich domain of synaptojanin. Invaginated clathrin-coated pits were induced by the same peptide and by the SH3 domain of endophilin. Together these studies suggest that endophilin participates at multiple steps of endocytosis, including invagination, in fission and uncoating. Current work is aimed a defining the mechanisms by which endophilin exerts its different actions.

Other ongoing projects include

-       analysis of the role of actin at distinct steps of the synaptic vesicle cycle

-       analysis of presynaptic actin-regulating pathways

-       analysis of SNARE protein trafficking and its link with endocytosis

-       analysis of the role of small GTPases (e.g. ral) in vesicle traffic

 The project provides new insight into the functions of proteins mediating synaptic vesicle trafficking. This group of proteins has been pointed out as candidate genes for schizophrenia and neurological disorders.
Reviews:

Brodin L, Shupliakov O. Giant reticulospinal synapse in lamprey: molecular links between active and periactive zones. Cell Tissue Res. 2006 Jun 20

Brodin, L., Löw, P. and Shupliakov, O. Sequential steps in clathrin-mediated synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10 (2000) 312-320.

De Camilli, P., Slepnev, V, Shupliakov, O. and Brodin, L. Synaptic vesicle endocytosis and recycling. In "Synapses", M. Cowan, C.F. Stevens, T.C. Sudhof (eds.) Johns Hopkins University Press (2000).

Shupliakov, O., and Brodin, L. A model glutamate synapse I: the lamprey giant reticulospinal axon. In: Handbook of Chemical Neuroanatomy: Glutamate, Elsevier, Amsterdam (2000).

Brodin, L., Löw, P., Gad, H., Gustafsson, J., Pieribone, V. and Shupliakov, O. Sustained neurotransmitter release: new molecular clues. Eur. J. Neurosci. 9 (1997) 2503-2511.

Original papers:

Evergren E, Zotova E, Brodin L, Shupliakov O. Differential efficiency of the endocytic machinery in tonic and phasic synapses. Neuroscience. 2006 Aug 7;141(1):123-31.

Owe-Larsson B, Chaves-Olarte E, Chauhan A, Kjaerulff O, Brask J, Thelestam M, Brodin L, Löw P. Inhibition of hippocampal synaptic transmission by impairment of Ral function. Neuroreport. 2005 Nov 7;16(16):1805-8.

Gustafsson JS, Birinyi A, Crum J, Ellisman M, Brodin L, Shupliakov O. Ultrastructural organization of lamprey reticulospinal synapses in three dimensions. J Comp Neurol. 450:167-82, 2002

Gad H, Ringstad N, Löw P, Kjaerulff O, Gustafsson J, Wenk M, Di Paolo G, Nemoto Y, Crum J, Ellisman M, De Camilli P, Shupliakov O, Brodin L. Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron, 27: 301-312, 2000.

Shayan, A.J., Brodin, L., Ottersen, O.P., Birinyi, A., Hill, C.E., Govind, C.K., Atwood, H.L. and Shupliakov, O. Neurotransmitter levels and synaptic strength at the Drosophila larval neuromuscular junction are not altered by mutation in the sluggish-A gene, which encodes proline oxidase and affects adult locomotion. J. Neurogenet. 14 (2000) 165-192.

Ringstad N, Gad H, Löw P, Di Paolo G, Brodin L, Shupliakov O, De Camilli P. Endophilin/SH3p4 is required for the transition from early to late stages in clathrin-mediated synaptic vesicle endocytosis. Neuron, 24:143-154, 1999.

Löw, P., Norlin, T., Risinger, C., Larhammar, D., Pieribone, V.A., Shupliakov, O. and Brodin, L. Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25. Eur. J. Cell Biol. 78 (1999) 787-793.

Owe-Larsson, B., Berglund, M.M., Kristensson, K., Garoff, H., Larhammar, D., Brodin, L. and Löw, P. Perturbation of the synaptic release machinery in hippocampal neurons by overexpression of SNAP-25 with the Semliki Forest virus vector. Eur. J. Neurosci. 11 (1999) 1981-1987.

Brodin, L., Bakeeva, L and Shupliakov, O. Presynaptic mitochondria and the temporal pattern of neurotransmitter release. Phil. Trans. Royal Soc. 354 (1999) 365-372.

Gad, H., Löw, P., Zotova, E., Brodin, L. & Shupliakov, O. Dissociation between Ca2+-evoked synaptic vesicle exocytosis and clathrin-mediated endocytosis in a central vertebrate synapse. (1998) Neuron 21:607-616.

Owe-Larsson, B., Kristensson, K., Hill, R.H. & Brodin, L. Distinct effects of Clostridial toxins on activity-dependent modulation of autaptic responses in cultured hippocampal neurons. Eur.J. Neurosci. 9 (1997) 1773-1777.

Shupliakov, O., Löw, P., Grabs, D., Gad, H., Chen, H., David, C., Takei, K., De Camilli, P.& Brodin, L. Synaptic vesicle endocytosis impaired by disruption of dynamin - SH3 domain interactions. Science 276 (1997) 259-263.

Shupliakov, O., Storm-Mathisen, J., Ottersen, O.P. & , Brodin, L. Glial and neuronal glutamine pools at glutamatergic synapses with distinct properties. Neuroscience 77(1997) 1201-1212.

Li, L., Chin, L.-S., Shupliakov, O., Brodin, L., Sihra, T.S., Hvalby, Ö.S., Jensen, V., Zheng, D., McNamara, J., Greengard, P. & Andersen, P. Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity in synapsin I-deficient mice. Proc. Natl. Acad. Sci. USA 92 (1995) 9235-9239.

Pieribone, V., Shupliakov, O., Brodin, L., Czernik, A.J., Hilfiker-Rotenfluh, S. & Greengard, P. Distinct pools of synaptic vesicles in neurotransmitter release. Nature 375(1995)493-497.

Shupliakov, O. Atwood, H.L., Storm-Mathisen, J., Ottersen, O.P. & Brodin, L. Presynaptic glutamate levels in tonic and phasic excitatory motor axons correlate with properties of synaptic release. J. Neurosci. 15(1995)7168-7180.

Shupliakov, O., Pieribone, V., Gad, H. & Brodin, L. Synaptic vesicle depletion in reticulospinal axons is reduced by 5-HT: direct evidence for presynaptic modulation of glutamatergic transmission. Eur. J. Neurosci. 7(1995)1111-1116