Figure 1.
GABA receptors are located at axon bleb and trunk.
A, Left, schematic diagram of bleb recording and GABA iontophoresis in a pyramidal neuron. Positive (but not negative) pulses could induce current responses. Vhold = –50 mV; iontophoresis pulses: 200 nA, 5 ms; retention current: –10 nA. Right, whole-cell recording from an axon bleb (top, fluorescence image; bottom, DIC image). Scale bar: 20 µm. The sharp electrode was used for GABA iontophoresis. Alexa Fluor 488 was added to the patch pipette solution so that the recording pipette was visible. B, Plot of the normalized GABA response as a function of the distance between the bleb and the tip of the iontophoresis electrode. Different symbols indicate different cells. The measurement of distance L is shown in the schematic diagram in panel A (indicated by arrows). C, GABA-induced responses could be observed when GABA was applied to the bleb (site a) or the main axon trunk (site c). The distance between sites a and c was approximately 50 µm, whereas that between a and b was approximately 25 µm. Vhold = –80 mV; iontophoresis pulses: 200 nA, 5 ms.
Figure 2.
The presence of GABAA (but likely not GABAB) receptors in the axon.
A, Reversal potential of GABA responses (IGABA) in the axon bleb. Left, representative currents induced by GABA application at different holding potentials (from –100 to –40 mV). At –60 mV (near reversal potential), GABA application induced no obvious change in baseline current (gray). Right, I-V curve of the GABA-induced responses shown on the left. B, IGABA could be blocked by GABAA receptor blocker PTX. Left, example traces before (black), during (gray) and after (Wash, dashed line) the bath application of PTX (25 µM). Vhold = –50 mV, GABA was applied via iontophoresis. Middle, time course of the effect of PTX. Right, group data showing the change of IGABA during (n = 6) and after (n = 3) PTX application. The dashed line indicates 100% of control. C, Left, currents evoked by puffing baclofen (200 µM), a GABAB receptor agonist, to the soma (16 psi, 15 ms). Right, no response was observed when baclofen was applied to the axon trunk (16 psi, 20 ms). D, Group data showing that GABA-induced currents at the axon blebs could not be blocked by the GABAB receptor antagonist CGP 35348 (100 µM); however, PTX could diminish these responses. Different symbols indicate different cells.
Figure 3.
Properties of axonal GABAA receptors.
A, DAB staining of an isolated axon bleb. The axon bleb was mechanically isolated from the main axon trunk before recording (see the Methods section). Arrow indicates the direction of the pia. Dashed line indicates the cut. Scale bar: 20 µm. B, Left, an example trace showing an increase in outward holding current after bath application of 5 µM muscimol (Vhold = 10 mV) and the blockade of this increase by 100 µM PTX. The dashed line indicates the baseline holding current. The mean values of Ihold for “Ctrl”, “Musci” and “Musci + PTX” group were 19.8, 41.4 and 16.9 pA, respectively. Right, histograms of the membrane currents shown on the left. The best-fit curves (dashed lines) were single Gaussian distributions. Axon blebs were recorded with patch pipettes filled with a low-Cl− ICS (7 mM [Cl−]i). C, Left, an example trace showing a decrease in inward holding current after bath application of 100 µM PTX (Vhold = –70 mV). The mean values of Ihold for “Ctrl” and “PTX” group were –22.8 and –12.5 pA, respectively. Right, histograms of the membrane currents shown on the left. Patch pipettes were filled with CsCl-based ICS (149 mM [Cl−]i). D, Current traces recorded in an isolated bleb. Vhold = –60 mV, CsCl-based ICS was used. Top, the actual current response; bottom, current trace with DC Remove. E, Power spectral density plots of membrane current fluctuations in control and muscimol-treated conditions (same data as in D). F, Subtraction of the power spectral density in the control from that with muscimol treatment (same data from D and E). The red line was the fitting curve of double Lorentzian functions. Cut-off frequencies () of the two components (arrowheads) were 3.5 and 18.1 Hz.
Figure 4.
Reversal potential of GABA responses (EGABA) is more negative than the local RMP.
A, Gramicidin perforated patch recording from an axon bleb. Arrow indicates the recorded bleb. Top, DIC image of the recording; middle, fluorescence image (unlabeled bleb); bottom, fluorescence image (labeled bleb, indicating rupture of patch membrane). Scale bar: 50 µm. B, Example traces showing GABA responses at different holding potentials (from –90 to –50 mV) before (black) and after the break-in (membrane rupture, gray). C, Comparison of EGABA and RMP. Note that EGABA at both the soma and the distal axon bleb were more hyperpolarized than their local RMP. *, P<0.05, paired t-test.
Figure 5.
Activation of axonal GABAA receptors shapes the AP waveform.
A, Example traces showing the change in AP waveform after GABA iontophoresis to the axon bleb. Vm change was –3.9 mV in this bleb. The amplitude and the half-width of APs decreased to 95.6% and 86.8% of the control, respectively. B, Group data showing that activation of axonal GABAA receptors shaped AP waveforms by regulating the amplitude and the half-width. Note that the recordings were performed under current-clamp and that the Vm could be manipulated by DC current injection. Black, GABA responses were hyperpolarizing; gray, depolarizing. The blebs were recorded with low-Cl− ICS (7 mM [Cl−]i). C, Increasing [Cl−]i depolarized the Vm but still showed a shunting effect on AP waveforms. Amplitude and half-width were significantly reduced. Modified ICS (20 mM [Cl−]i) was used for these recordings. ***, P<0.001, paired t-test. D, Bath application of PTX could block the GABA-induced Vm depolarization and its shunting effect on AP waveform. Modified ICS was used. E, Example traces showing that GABA application caused a shunting effect on APs evoked by electric shocks (asterisks), although GABA itself could evoke an AP (arrow). High-Cl− ICS (75 mM [Cl−]i) was used here.
Figure 6.
Propagation of GABA-induced hyperpolarization at the axon regulates AP generation.
A, DAB staining of recorded neurons. Simultaneous recording from the soma and axon bleb were performed in a pyramidal neuron (left), and GABA was applied to the axon trunk (right). The axon length was 239 µm in this case. The distance between the iontophoresis site and the soma was 117 µm. Scale bar: 100 µm (left); 50 µm (right). B, The sign of the effect of GABA (hyperpolarization or depolarization) depended on the Vm. Top, traces were taken from the bleb. Bottom, traces were the corresponding responses at the soma. The Vm was clamped through somatic DC current injection. Asterisk indicates application of GABA to the main trunk. C, Left, application of GABA to the axon increased the amplitude but decreased the half-width of propagating APs. GABA iontophoresis hyperpolarized the Vm by 2.3±0.4 mV (n = 7). Right, similar results were obtained when Vm was hyperpolarized by 2.8±0.3 mV (n = 5) through DC current injection. *, P<0.05; **, P<0.01, paired t-test. D, Example traces showing activation of axonal GABAA receptors reduced firing probability and frequency. The distances between the iontophoresis site and the soma were 100 µm (distal axon) and 18 µm (AIS). E, Left, repetitive firing recorded at an axon bleb induced by 400 pA DC current injection at the soma before (black) and after (red) GABA application to the axon trunk. The arrow indicates GABA iontophoresis. Middle, instantaneous firing frequency of APs decreased after GABA application (same data as shown in the left). Right, group data showing a decrease in the mean frequency of APs after GABA iontophoresis at the axon trunk. **, P<0.01, paired t-test. Low-Cl− ICS was used in these experiments.
Figure 7.
Activation of axonal GABAA receptors decreases AP-induced Ca2+ transients.
A, Left, projection of 2-photon fluorescence images of a recorded pyramidal neuron filled with Alexa Fluor 594 (50 µM) and OGB-1 (200 µM). The dashed arrow indicates the direction of bath flow. Right, iontophoresis of GABA at the axon trunk (labeled by “GABA” in the left image) decreased Ca2+ transients evoked by single APs at nearby locations (ROI 1 and 2) but showed no effect on somatic transients. B, Group data showing the effect of GABA on single-AP-triggered Ca2+ transients. The results are presented as the ratio of the Ca2+ signal amplitude for “GABA” to “Ctrl.” Top, data collected from the axon trunk (n = 13 axons). Bottom, data collected from the soma. The site for GABA iontophoresis was defined as 0. If the ROI was located downstream from the iontophoresis site, the sign of the distance was positive; otherwise it was assigned a negative sign. *, P<0.05; **, P<0.01; ***, P<0.001, paired t-test. C, Group data showing the effect of GABA on Ca2+ transients induced by 3 APs (100 Hz). Data were collected from the axon trunk (n = 9 axons). D, PTX blocked GABA-induced decrease in Ca2+ transients. Gray, individual cells; black, average data.