Focal stimulation of single GABAergic presynaptic boutons on the rat hippocampal neuron
Introduction
γ-Amino butyric acid (GABA) is a predominant fast inhibitory neurotransmitter in the central nervous system (CNS) (Bormann et al., 1987). Major advances in our knowledge of GABAergic synaptic transmission have been achieved using brain slice preparations (Edwards et al., 1990). However, complicated connections among CNS neurons often make multiple synaptic contacts on a single postsynaptic neuron, thus the electrical stimulation of presynaptic nerve bundle simultaneously activates not only multiple presynaptic axons but also the collateral pathways. As a result, the mechanisms of GABA release from a single presynaptic nerve terminal in the CNS are difficult to precisely elucidate using only brain slice preparations. Recently, a more simplified preparation of cultured neurons has been developed which enables the electrical stimulation of a single presynaptic terminal and the properties of transmitter release were clearly studied (Liu and Tsien, 1995, Fedulova et al., 1999, Kirischuk et al., 1999a, Kirischuk et al., 1999b). However, little information exists as to whether the release properties and presynaptic modulation of single native GABAergic terminals behave similarly to terminals formed in culture preparations. We have developed a mechanical acute dissociation technique of single CNS neurons attached with functional native excitatory and inhibitory synaptic terminals (boutons), namely a ‘synaptic bouton’ preparation, without using any enzymes. These dissociated neurons exhibit spontaneous postsynaptic currents, and their presynaptic modulation has been extensively studied (Koyama et al., 1999, Koyama et al., 2000, Rhee et al., 2000, Kishimoto et al., 2000, Katsurabayashi et al., 2001, Wang et al., 2001, Jang et al., 2001a, Jang et al., 2001b, Jang et al., 2001c, Kiyosawa et al., 2001). In the present study, we visualized single presynaptic boutons on a dissociated single hippocampal neuron and electrical stimulation was selectively given to one of the boutons. We herein show how such an electrophysiological approach allows us to understand the release of GABA in a single presynaptic element without any contamination of the collateral pathways. Furthermore, the present research approach given by combination of the focal stimulation and the synaptic bouton preparation could become a powerful method for understanding the transmitter release property based on an extremely small single native presynaptic terminal in the CNS.
Section snippets
Mechanical dissociation
The hippocampal CA1 pyramidal neurons were dissociated from 2-week-old Wistar rats as previously described (Harata et al., 1997). Briefly, slices cut at a thickness of 400 μm were kept for about 1 h in an incubation medium (in mM), 124 NaCl, 5 KCl, 1.2 KH2PO4, 1.3 MgSO4, 2.4 CaCl2, 24 NaHCO3 and 10 glucose, saturated with 95% O2 and 5% CO2 at room temperature (22–25 °C). Next, the slices were transferred into a 35 mm dish (Primaria 3801; Becton Dickinson, Franklin Lakes, NJ) filled with a
Identification of single presynaptic boutons
In order to see the distribution of functional single presynaptic boutons on a mechanically dissociated hippocampal CA1 pyramidal neuron, these boutons were stained with FM 1-43, which is generally used for labeling functional synaptic vesicles, before performing patch recordings. Fig. 1(Aa) shows a dissociated hippocampal CA1 pyramidal neuron stained with FM 1-43. To confirm whether or not these fluorescent spots were indeed presynaptic boutons, the external solution containing 15 mM K+ was
Single bouton stimulation
The all or none like relationship between the stimulus current intensity and the eIPSC amplitude, and the critical location of the stimulation pipette upon a bouton are considered to be electrophysiological evidences suggesting the selective and/or direct activation of a single bouton. The absolute distance of a stimulus glass pipette which could elicit eIPSCs slightly varied across individual boutons. Such differences might be due to the size and shape of individual bouton, or are more or less
Acknowledgements
The authors would like to thank Dr M. C. Andresen for his helpful comments and advice. This study was supported by the Japan Health Science Foundation (Research on Brain Science) to N. Akaike.
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