A cholinergic model synapse to elucidate protein function at presynatic terminals

Abstract

A large number of proteins have been identified at nerve terminals and a cascade of protein–protein interactions has been suggested to be involved in cycling of synaptic vesicle states. To explore protein function in presynaptic terminals, only a few unique synapses such as the squid giant synapse, the calyx of Held synapse and the hippocampal neuron autapse have been used. The squid giant synapse and the calyx of Held are useful to introduce reagents into their large presynaptic terminals and the hippocampal neuron autapse is a good system to modify a protein level by exogenous DNA or RNA. The cholinergic synapse formed between superior cervical ganglion (SCG) neurons in long-term culture is a useful model for a fast synapse. The axon of the large cell body contacts with soma of neighboring neurons. The architecture of synaptic connections makes it possible to introduce reagents into the presynaptic terminals by diffusion from a cell body within a short time. Introduction of exogenous cDNA or siRNA performed by microinjection into a SCG neuron allows us to modulate the level of the protein of interest or to express mutant proteins in the neuron. Here, we describe use of the model SCG neuronal synapse to elucidate function of presynaptic proteins in mediating synaptic transmission.

Introduction

A large number of proteins play a role in mediating neurotransmitter release in response to arrival of each action potential at the nerve terminal. A cascade of protein–protein interactions has been proposed for cycling of synaptic vesicles from one state to the next along the maturation pathway (Mochida, 2000, Sudhof, 1995, Sudhof, 2004). Among these presynaptic proteins, the SNARE proteins, synaptobrevin/VAMP (a synapse vesicle-associated protein), syntaxin and SNAP-25 (membrane-associated proteins) have been extensively studied, these proteins are suggested to form a stable SNARE complex which underlies the synaptic vesicle fusion machinery (Jahn and Scheller, 2006, Rothman and Warren, 1994). A synaptic vesicle Ca²⁺-binding protein, synaptotagmin I, that interacts with the SNARE complex, Ca²⁺channels and other proteins and phospholipids, has also been well studied and suggested to act as a Ca²⁺-sensor to trigger the fusion of synaptic vesicles with the plasma membrane (Sudhof, 1995, Sudhof, 2004).

To explore protein function in the presynaptic terminals, few unique synapses, such as the squid giant synapse (McGuinness et al., 1989), the hippocampal neuron autapse (Yamaguchi et al., 2002) and the calyx of Held synapse (Xu et al., 1998) have been often used. Reagents that impair protein function can be easily introduced into the large presynaptic terminal of the squid giant synapse or the calyx of Held synapse, whilst simultaneously recording synaptic transmission. In contrast, protein levels can be controlled by exogenous cDNA expression or by RNA interference in autapses of hippocampal neurons in culture (Inoue et al., 2006). The cholinergic synapse formed between SCG neurons in long-term culture is a model for a fast synapse. This is a useful synapse for exploring the function of proteins in presynaptic terminals; reagents that impair protein function can be introduced into the large presynaptic neuron by microinjection, and protein levels can be controlled by microinjection of DNAs or small interference RNAs (siRNAs) into a presynaptic neuron.

The SCG is located at the rostral end of the paravertebral sympathetic chain and selectively innervates different organs located throughout the upper body (Gibbins, 2004, Li and Horn, 2006). SCG neurons control specialized vascular beds in the brain, muscle, skin, and glands as well as piloerector hairs, salivary glands, the iris, and pineal gland (Arbab et al., 1986, Bowers et al., 1984, Gibbins, 1991, Gibbins et al., 1996, Morris et al., 1999, Reuss and Moore, 1989, Uddman et al., 1989, Voyvodic, 1989). SCG neurons from rat and rabbit has been extensively studied to reveal the physiological significance of synaptic transmission mechanisms (Brown and Adams, 1980, Eccles, 1935, Feldberg and Gaddum, 1934, Higashida et al., 2003, Libet, 1970, Mochida et al., 1987, Nishi et al., 1965) as the location of the SCG benefits dissection from the animal body.

Cultures of isolated SCG neurons taken from neonatal rats extend processes to neighboring neurons and form synapses (O’Lague et al., 1974). Immunofluorescence staining using an antibody for presynaptic proteins, such as synaptophysin, showed small fluorescent spots both around the soma and on the hillocks of processes, suggesting that axo-somatic and axo-dendritic synapses are formed in long-term culture (Mochida et al., 1994b). The transmitter of the SCG neuron synapses changes from noradrenaline (Koh and Hille, 1997) to acetylcholine (ACh) in long-term culture (O’Lague et al., 1974, Wakshull et al., 1979, Mochida et al., 1994b). The cholinergic synapse of SCG neurons is favorable for investigation of protein function in neurotransmitter release. Firstly, synthetic proteins, peptides or antibodies that perturbs function of the protein of interest, can be introduced into the relatively large (30–40 μm) presynaptic cell body by microinjection, and the injected reagents can rapidly diffuse to nerve terminals forming synapses with adjacent neurons (Mochida et al., 1994a). Secondly, the expression level of specific proteins in a presynaptic neuron may be selectively modulated by siRNA or cDNA injection (Baba et al., 2005, Krapivinsky et al., 2006, Mochida et al., 2003a). Thirdly, the function of the protein of interest may be selectively impaired by expression of the dominant-negative mutant proteins transfected using synthetic DNAs (Baba et al., 2005, Krapivinsky et al., 2006, Mochida et al., 2003a). The effects of introduced reagents or those of gene-targeted treatments on the stimulated release of ACh can be accurately monitored by recording the excitatory postsynaptic potentials (EPSPs) evoked by action potentials in the presynaptic neurons. Here we describe an experimental system, SCG neuron synapses in long-term culture, and also demonstrate functional studies of presynaptic proteins employing the unique model synapse.