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. 2000 Mar 15;523(Pt 3):629–637. doi: 10.1111/j.1469-7793.2000.t01-1-00629.x

GABA release from mouse axonal growth cones

Xiao-Bing Gao 1, Anthony N van den Pol 1
PMCID: PMC2269824  PMID: 10718743

Abstract

  1. 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.

  2. A low level of GABA release occurs spontaneously from the growth cone, and this is substantially increased by evoked action potentials.

  3. 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.

  4. 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).

METHODS

Culture

Pregnant mice were given an i.p. overdose of Nembutal (100 mg kg−1), embryos were harvested, and then the pregnant mouse was given a second lethal i.p. dose of Nembutal (150 mg kg−1). Experiments were approved by the university committee on animal use. Two groups of hypothalamic neurons were prepared from the brains of embryonic mice. One group was cultured on polylysine-coated coverslips for 1–2 weeks as donors for outside-out patches. A second group of neurons from green fluorescent protein (GFP)-expressing transgenic mice (Okabe et al. 1997; van den Pol & Ghosh, 1998) was cultured for 3–7 days for growth cone study. Both groups of hypothalamic neurons were dissociated from embryonic day (E)15-E18 mouse embryos and cultured as described previously (Gao et al. 1998).

Electrophysiology

The recording chamber was perfused (1.5-2 ml min−1) with a bath solution containing (mM): NaCl 150, KCl 2.5, CaCl2 2, Hepes 10 and glucose 10; pH 7.3 with NaOH. The patch pipette was made of borosilicate glass (World Precision Instruments) with a Narishige puller (pp-83) and coated with Sylgard (Dow Corning Corp., Midland, MI, USA) to minimize pipette capacitance. Although harder glass may generate crisper channel recordings, we used this softer glass because it worked better for whole-cell recordings, also needed in many of the same experiments. The tip resistance of the recording pipettes was 4–6 MΩ after filling with pipette solution containing (mM): KCl 145, MgCl2 1, Hepes 10, EGTA 1.1, Mg-ATP 2 and Na2-GTP 0.5; pH 7.3 with KOH. Outside-out membrane patches were taken from mature donor neurons after 1–2 weeks in culture. The detector patch was held at -75 mV and recorded with an L/M EPC-7 amplifier. Unless otherwise noted, the detector patch was held within 0.5-1 μm of the growth cone tested, and bath perfusion was stopped during recordings to reduce diffusion of the GABA. To stimulate action potentials in the neurons forming axonal growth cones, a loose seal was formed with a micropipette on the soma of the parent neuron by slight suction. A square pulse of 0.5-1.0 ms in duration and 4–20 V in amplitude was applied through the micropipette to generate a spike.

All data were sampled at 3–10 kHz and filtered at 1 kHz using AxoData 1.2.2 (Axon Instruments). Data were analysed with Axograph 3.5 (Axon Instruments) and Igor Pro software (WaveMetrics, Lake Oswego, OR, USA). Data are reported as means ±s.e.m. Student's t test was used to compare two groups of data. ANOVA test was used with three groups of data. 2-Amino-5-phosphonopentanoic acid (AP5), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), bicuculline and the PKC activator phorbol 12,13-dibutyrate (PDBu) were obtained from RBI, and α-latrotoxin was from Alomone Labs Ltd (Jerusalem, Israel).

RESULTS

All detector patches respond to GABA

To demonstrate that detector patches are a viable approach to detect small amounts of GABA that might be released from growing CNS axons (Fig. 1), we used outside-out patches from 1- to 2-week-old cultured hypothalamic neurons, and tested them with a series of flow-pipe GABA applications. All patches tested (n = 6) showed robust concentration-dependent GABA responses, suggesting a relatively high level of functional GABA receptors on the detector patch (Fig. 2A).

Figure 1. Growth cones are visualized by GFP expression.

Figure 1

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A, a typical neuron after 3 days in culture, with short dendrites arising from the soma and a long axon with a growth cone at the end (arrow). Fluorescence is due to GFP expression. Scale bar, 16 μm. B and C, axonal growth cones that were recorded generally had active filopodia, as shown here in a time-lapse sequence from a typical motile growth cone. The two images are separated by 10 min. Arrows (a, b and c) indicate filopodia with altered morphology. Scale bar, 5 μm. D-G, morphology of some typical GFP-expressing growth cones that were recorded. Scale bar, 6 μm. H, an axonal growth cone (arrow) after 3 days in vitro shows blue immunoreactivity for GABA after immunoperoxidase staining with GABA antiserum viewed with differential interference contrast and digital inversion, as described in detail elsewhere (van den Pol, 1997). Absorption of the antiserum with GABA conjugated to a carrier protein eliminated staining. Scale bar, 4 μm.

Figure 2. Spontaneous release of GABA from an axonal growth cone.

Figure 2

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A, outside-out patches taken from 1- to 2-week-old cultured neurons were placed near an array of flow pipes containing increasing concentrations of GABA. The traces presented here are the responses of such a patch to 0, 1, 5, 10 and 50 μM GABA under voltage clamp at -75 mV in the presence of the antagonists of ionotropic glutamate receptors AP5 (50 μM) and CNQX (10 μM). B, traces presented here were from a typical experiment. In the presence of AP5 and CNQX, activity resembling channel opening was recorded by the outside-out patch under voltage clamp at -75 mV when it was placed near an axon growth cone. The activity recorded by the outside-out patch was reversibly blocked by 30 μM bicuculline (BIC). Ca, traces presented here were recorded from a single growth cone with an outside-out patch placed less than 1 μm, or 2, 10, 50 or 200 μm away from the growth cone. These raw traces were recorded under voltage clamp at -75 mV in the presence of AP5 and CNQX. Cb, plots of the data from all four neurons. The frequency of events recorded by the outside-out patch at each distance was normalized to that recorded at the distance of less than 1 μm.

Spontaneous release of GABA from the axonal growth cone

To facilitate detection and tracking of axonal growth cones, we used mice that were genetically altered to express the jellyfish gene coding for GFP. GFP expression was sufficiently strong that the entire neuronal arbor, including cell body, dendrites and axon, showed green fluorescence. Motile axon growth cones could be clearly identified with fluorescent microscopy (Fig. 1) 3 days after hypothalamic neurons from GFP-expressing transgenic mice were cultured at low density. As previously demonstrated, 3 days after plating the longest process had axonal attributes, and the shorter ones had dendritic characteristics (Bartlett & Banker, 1984). After fixation some long processes and their growth cones typical of the ones recorded showed strong immunostaining with GABA antiserum (Fig. 1H), suggesting the presence of GABA in the growth cone. When an outside-out patch was placed near (< 1 μm) a developing axon growth cone, an increase in electrical activity was recorded (Fig. 2B). The events recorded generally had brief observed open times (from less than 1 ms to 10 ms) although some with longer open times were also found. The mean (±s.e.m.) open time for events (6 neurons) was 3.8 ± 0.8 ms before addition of the GABAA receptor antagonist bicuculline; this decreased to 0.1 ± 0 ms in bicuculline (30 μM), and recovered after bicuculline washout. The amplitude of the recorded events ranged from 1.08 to 2.94 pA with a mean of 1.90 ± 0.04 pA (8 neurons). The conductance was 25.6 ± 0.5 pS. GABA release was shown by 32 of 53 neurons; in all positive cases, responses were completely blocked by bicuculline (30 μM) and recovered after bicuculline washout (Fig. 2B), suggesting that they were mediated by spontaneous GABA release. In the present experiment, and in all other experiments described below, bicuculline was added as a first step to block channel activity in order to ensure that the channel openings were due to GABA; bicuculline was then washed out, leading to full recovery. All experiments were done in the presence of the glutamate receptor antagonists AP5 (50 μM) and CNQX (10 μM) to eliminate events mediated by glutamate at ionotropic receptors. Even when action potentials were blocked by tetrodotoxin (1 μM), spontaneous release of GABA was still found (n = 4), indicating transmitter release independent of spikes, similar to the spontaneous release of GABA found at mature presynaptic hypothalamic axon terminals (Chen & van den Pol, 1996).

If GABA was released from a particular site, we reasoned that the intensity of the response should decrease as the detector patch was moved away from the source. The response to spontaneous GABA release recorded by the outside-out patch was distance sensitive (Fig. 2C), with decreasing responses when the patch was moved away from the axonal growth cone. The largest GABA response was recorded when the outside-out patch was placed within 1 μm of the growth cone, a small response was detected at a distance of 10 μm, and no response was found at a distance of 50 μm or greater away from the axon and its growth cone. In two cells we recorded both from the axonal growth cone and from the axon shaft of the same cell away from the growth cone. The detector patch showed a 90% decrease in activity when the patch was moved > 100 μm away from the growth cone or its parent cell body and placed within 1 μm of the axon shaft. These results suggest that GABA was released by the growth cone.

Evoked release of GABA from the axonal growth cone

To determine whether action potentials could increase GABA release from growth cones, a loose membrane seal was formed with a second micropipette on the soma of the parent neuron by slight suction to allow stimulation of this neuron. The evoked release of GABA was recorded at the axon growth cone with the outside-out patch on a different pipette coupled to a second amplifier, as shown in the schematic diagram in Fig. 3. For stimulation, a square pulse was applied through the micropipette to evoke an action potential. The evoked response had a mean amplitude of approximately 40 pA and a duration of several seconds. In part this long response duration was due to the absence of buffer movement during these experiments. The evoked response could be eliminated completely by application of the GABA antagonist bicuculline (30 μM; Fig. 3), and recovered after bicuculline washout. Similar to the study above examining spontaneous GABA release, when the outside-out patch was moved 50 μm or more away from the growth cone, an evoked GABA response could not be detected (Fig. 3). These data suggest that action potentials increase GABA release from the growth cone.

Figure 3. Action potential increases GABA release from the axon growth cone.

Figure 3

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Upper panel, schematic diagram of a typical dual-pipette recording experiment to study evoked GABA release. One pipette contains the outside-out patch detector used to record GABA release from the GFP-expressing axon growth cone, and the other is used to evoke action potentials. Lower panel, an evoked current was recorded by a patch near an axon growth cone when the target neuron was stimulated. The inset shows part of the evoked current on an expanded time scale. The evoked current was completely blocked by the antagonist bicuculline, and recovered after bicuculline washout. No evoked current could be recorded when the outside-out patch was moved 50 μm away from the growth cone. All traces were recorded under voltage clamp at -75 mV in the presence of AP5 (100 μM) and CNQX (10 μM).

Vesicular release

GABA could be released through the machinery of vesicular exocytosis, although there is no synapse at this stage of neuronal development, or via outward transporters. To test the hypothesis that GABA release may be based on an exocytotic mechanism, the effect of α-latrotoxin (α-LTx) on spontaneous GABA activity was examined. α-LTx has been demonstrated to evoke exocytosis and neurotransmitter release from synaptic vesicles in a wide variety of vertebrate central and peripheral synaptic junctions (Meldolesi et al. 1984; Valtorta et al. 1988). The action of α-LTx requires binding of the toxin to a high affinity receptor on the axon surface (Valtorta et al. 1988). After the application of 1 nM α-LTx, the frequency of spontaneous activity of GABA was increased dramatically (Fig. 4Aa and Ab). With controls defined as 100%, the enhancement of frequency varied from 179 to 467% with a mean of 274 ± 55% (n = 5) with respect to the control (Fig. 4Ac; paired t test, P < 0.05). These results support the hypothesis that GABA release is dependent, at least in part, on vesicular release from axonal growth cones.

Figure 4.

Figure 4

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Modulation of growth cone GABA release

A, vesicular release of GABA from the growth cone. Raw traces recorded before (Aa) and after (Ab) application of α-latrotoxin, a potent stimulator of exocytosis. All traces were recorded at -75 mV in the presence of AP5 and CNQX. The mean release from all five neurons is shown in the bar graph (Ac). Student's paired t test indicates a significant difference between these two groups (*P < 0.05). B, PKC modulation of GABA release. Voltage-clamp recordings were made at -75 mV in the presence of AP5 and CNQX. PDBu evoked an increase in the frequency of GABA events (Ba). The mean frequency of GABA events during and 10 min after the application of PDBu was normalized to that of the control (before application of PDBu) and is shown in the bar graph (Bb). An ANOVA statistical test suggests that the PDBu significantly (*P < 0.05) increased the frequency of GABA events. Events were reversibly blocked by bicuculline (30 μM).

PKC modulation of GABA release from the growth cone

During early development, a number of transmitters are synthesized and released; many of the receptors for these, including muscarinic ACh, some group I metabotropic glutamate, opiate, serotonin and cholecystokinin receptors function by activating PKC (van Hooff et al. 1989). Previous work has demonstrated that PKC can modulate transmitter release from mature presynaptic axons (Majewski & Iannazzo, 1998) and can phosphorylate growth cone proteins (van Hooff et al. 1989). To determine whether other transmitters that may act through PKC could modulate GABA release from axons prior to synapse formation, we first tested ACh and its agonist muscarine, and found increases in the frequency of spontaneous postsynaptic currents from hypothalamic neurons in response to flow-pipe application. ACh (100 μM) evoked a 170% increase in postsynaptic current frequency (n = 4). For four out of seven neurons that showed an increase in response to the ACh agonist muscarine (1 μM), the mean increase was 180%. This suggests that within 3 days of culture, at a time when neuritic outgrowth is occurring, the neurons expressed functional ACh receptors. To test the hypothesis that activation of the PKC second messenger pathway was capable of altering growth cone GABA release, we used the PKC activator PDBu, which acts on the diacylglycerol binding site of PKC isoforms to activate the enzyme. PDBu increases the release of glutamate, ACh, dopamine and noradrenaline from actions at the mature presynaptic bouton.

After recording a baseline level of axonal GABA release, PDBu (2 μM) was applied. The frequency of spontaneous GABA events increased from control levels (defined as 100%) to a range of 131–256% with a mean of 192.0 ± 23.5% (n = 6) following PDBu application. This increase declined to 114.5 ± 55.0% of control (n = 3) 10 min after washout of PDBu. An ANOVA test suggested that the enhancement in the frequency of recorded GABA spontaneous activity was very significant (P < 0.01) (Fig. 4Ba). All six cells tested showed a PKC-mediated increase in GABA release. For control purposes, in the absence of a growth cone, we compared the responses of excised patches to 5 μM GABA in the presence or absence of 2 μM PDBu: PDBu had no effect on GABA activity detected by the patch (n = 4, not shown). As the spontaneous release of GABA was independent of action potentials, the PKC activation was probably directly at the axonal growth cone.

DISCUSSION

In this paper we have shown that some motile axonal growth cones, identified by the expression of GFP, release GABA spontaneously. The release of GABA from the growth cone is substantially increased by action potentials. In previous work focusing on growth cones of motoneurons from birds and frogs, ACh release was detected (Hume et al. 1983; Young & Poo, 1983). Together with the work on mammalian CNS GABA neurons reported here, these data suggest that other neurotransmitters may also be released from their growing axons prior to synapse formation.

Our data demonstrate that GABA is released from growth cones of hypothalamic neurons both spontaneously and after stimulation. Spontaneous release of ACh was also found in Xenopus motoneuron growth cones (Young & Poo, 1983). The frog motoneuron growth cone has been used in several studies of transmitter release from cholinergic cells (Young & Poo, 1983; Xie & Poo, 1986; Sun & Poo, 1987). Only a low level of spontaneous activity was recorded in our experiments on GABA cells, whereas ‘staircase’ patterns of channel openings, indicative of multiple simultaneous channel openings, were recorded in the frog. This could be due to several factors. The amount of GABA released from hypothalamic growth cones may be small compared to the amount of ACh released from the larger Xenopus motoneuron growth cones, which could in part relate to different levels of activity of the two cell types. Membrane patches from frog myoballs may contain relatively greater numbers of neurotransmitter receptors, which would increase the probability of detecting ACh release. Finally, the conductance of the ACh channels is larger (about 40 pS; Young & Poo, 1983) than the conductance (about 25 pS) of GABA-gated channels, also favouring the detection of ACh release.

A number of transmitters found in early development can stimulate PKC; we found that activation of PKC increases GABA release. As the PKC-mediated increase in activity was independent of action potentials, PKC probably acted at the axon terminal to enhance transmitter release from the growth cone. PKC may be involved in the phosphorylation of proteins associated with neurotransmitter release from mature axons (Majewski & Iannazzo, 1998). Several phosphoproteins can modulate transmitter release in mature axons. Some of these, for instance GAP-43 and MARCKS (myristoylated alanine-rich PKC substrate), are also found in the growth cone, and biochemical and cytochemical studies have shown them to be phosphorylated by PKC (Fukura et al. 1996; Dent & Meiri, 1998). Our physiological data are consistent with the hypothesis that PKC may facilitate phosphorylation of proteins leading to increased GABA release not only in mature axons as shown previously (Majewski & Iannazzo, 1998), but also in the developing axonal growth cone.

In mature neurons, glutamate is the primary excitatory neurotransmitter, and GABA the primary inhibitory transmitter. In contrast, in early hypothalamic development, where GABA receptors develop earlier than glutamate receptors, currents evoked by GABA are larger and more frequent than glutamate currents (Chen et al. 1995). In some neuron perikarya (Obrietan & van den Pol, 1995; Chen et al. 1996), and neurites and growth cones (Obrietan & van den Pol, 1996), GABA evokes a greater calcium rise than glutamate, suggesting that at this early developmental stage, GABA can be a more robust excitatory transmitter than glutamate. This may generalize to developing neurons from other brain or spinal cord regions where GABA excitation has also been reported (Ben-Ari et al. 1994; Reichling et al. 1994; LoTurco et al. 1995). Although in the present paper we focused on hypothalamic neurons, given the similarities with other CNS neurons, we suspect that neurons throughout the brain may release GABA from growing axons.

Cytosolic calcium can influence neuritic growth and extension (Kater et al. 1988). The data presented here suggest that GABA is released from growth cones and may participate in communication to other developing neurites, potentially by a mechanism involving GABA-mediated increases in calcium. Hypothetically this could alter the rate or direction of growth, modulate the affinity to calcium-sensitive adhesion molecules, and alter the calcium-sensitive cytoskeleton leading to changes in the microstructure of nearby cells. GABA release from growing axons could thus alter development during the course of the axon's growth, thereby modulating cell division and migration, cell survival, neurite growth and synaptogenesis (LoTurco et al. 1995; Barker et al. 1998). As the developing GABA neuron both releases transmitter and is sensitive to transmitter actions, the release of GABA from the growth cone could be further modulated by other transmitter molecules during the course of the axon's journey to its postsynaptic partners.

Acknowledgments

We thank Drs Y. Yang for excellent technical assistance, M. Okabe for providing some of the transgenic mice and P. Patrylo for suggestions on the manuscript. Support was provided by NIH grants NS10174, NS34887 and NS31573, and the National Science Foundation.

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