GABA release from mouse axonal growth cones.

Using developing hypothalamic neurons from transgenic mice that express high levels of green fluorescent protein in growing axons, and an outside-out patch from mature neuronal membranes that contain neurotransmitter receptors as a sensitive detector, we found that GABA is released by a vesicular mechanism from the growth cones of developing axons prior to synapse formation. A low level of GABA release occurs spontaneously from the growth cone, and this is substantially increased by evoked action potentials. Neurotransmitters such as acetylcholine can enhance protein kinase C (PKC) activity even prior to synapse formation; PKC activation caused a substantial increase in spontaneous GABA release from the growth cone, probably acting at the axon terminal. These data indicate that GABA is secreted from axons during a stage of neuronal development when GABA is excitatory, and that neuromodulators could alter GABA release from the growing axon, potentially enabling other developing neurons of different transmitter phenotype to modulate the early actions of GABA. One of the central questions in brain development is how axons find and initiate communication with their correct neuronal partners among millions or billions of cells. A crucial element of this scenario is the axonal growth cone, the advancing hand- or torpedo-like structure that crawls through the brain searching for its correct targets. The axonal growth cone is critical for axonal extension, pathfinding and initial contact with potential synaptic partners (Lectourneau et al. 1991). On the working assumption that some form of cellular communication can modulate and facilitate neuronal development, our study tested the hypothesis that GABA, the primary inhibitory transmitter throughout the mature brain, is released by growing axons and can serve as a local excitatory intercellular signal prior to synaptic contact. To detect small amounts of transmitter that might be released from the axonal growth cone, we used an outside-out membrane detector patch bearing transmitter receptors. This approach has been used with muscle patches to show that acetylcholine (ACh) is released from peripheral axons of avian or amphibian motoneurons (Hume et al. 1983; Young & Poo, 1983). Parallel studies have not previously examined amino acid transmitter release from mammalian CNS neurons. Work with cell fractionation has suggested that GABA may be released from a fraction enriched in growth cones, but proof that putative growth cone fractions are not contaminated with presynaptic axons is difficult to achieve (Gordon-Weeks et al. 1984; Lockerbie, 1990). Microscopic studies on plasmalemma recycling have suggested membrane turnover in the axon and its growth cone (Kraszewski et al. 1995; Diefenbach et al. 1999), consistent with the possibility that transmitter release occurs in developing neurons through an exocytotic mechanism. We focused on GABA because it is the primary inhibitory transmitter throughout the mature brain, and because a number of studies have demonstrated the importance of GABA during early neuronal development. In developing neurons, GABA alters mitosis of neuronal precursors, enhances synaptic thickening, alters the expression of subsets of GABA receptors, increases neuritic outgrowth, enhances synthesis of specific proteins and increases the density of cytoplasmic organelles (Ben-Ari et al. 1994; LoTurco et al. 1995; Belhage et al. 1998). Hypothalamic neurons were used because at least half of all synapses here use GABA (Decavel & van den Pol, 1990), axonal growth cones of hypothalamic embryonic neurons contain GABA (van den Pol, 1997), most hypothalamic neurons show excitatory responses to GABA during development (Obrietan & van den Pol, 1995; Chen et al. 1996), and GABA receptors are expressed at the earliest stage of development and prior to glutamate receptors (Chen et al. 1995). Neurons from other regions of the brain also use GABA and show depolarizing responses to GABA in early development (Ben-Ari, 1994; Reichling et al. 1994).