Co-release of glutamate and GABA from single, identified mossy fibre giant boutons

Abstract

Several laboratories have provided immunohistochemical, molecular biological and electrophysiological evidence that the glutamatergic granule cells of the dentate gyrus can transiently express a GABAergic phenotype during development. Electrophysiological recordings on hippocampal slices obtained during this period have shown that stimulation of the mossy fibres (MFs) provokes simultaneous monosynaptic GABA(A) and glutamate receptor-mediated responses in their target cells,which have the pharmacological and physiological characteristics of MF neurotransmission. This evidence, although strongly supporting the hypothesis that MFs co-release glutamate and GABA, is indirect, as the extracellular stimulation used in slice experiments could activate fibres other than MFs. In this study, we show that selective stimulation of single, identified MF boutons (MFBs) attached to the apical dendrites of dissociated pyramidal cells of developing rats produced synaptic currents mediated by either glutamate receptors only or by both glutamate and GABA(A) receptors. By contrast, stimulation of MFBs of adult rats produced exclusively glutamate receptor-mediated responses. All responses evoked by stimulation of MFBs underwent strong frequency-dependent potentiation and were depressed by the activation of presynaptic metabotropic glutamate receptors. On the other hand, synaptic responses evoked by stimulation of interneuronal boutons located on the soma or on the basal dendrites of the same pyramidal cells were exclusively mediated by GABA(A) receptors, underwent frequency-dependent depression and were unaffected by mGluR agonists.We here demonstrate that the simultaneous glutamatergic and GABAergic responses evoked by MF stimulation in pyramidal cells of CA3 during development have a common origin in the giant MFBs.

Figure 1. Dissociated pyramidal cells of CA3 with fluorescent giant MFBs attached to their apical dendrites

A, prior to dissociation, Zn²⁺-containing MFBs are labelled with the fluorescent sensor for Zn²⁺, Zinpyr-1, or (Ba and b) with dextranamine transported from the granule cell layer to the MFs terminals. C, enzyme-free mechanical dissociation is then conducted using a vibrating blunt pipette positioned over CA3 with a micromanipulator. MFBs can be identified attached to the apical dendrites of the dissociated pyramidal cells by their fluorescent label of either Zinpyr-1 (Da and b) or rhodamine-dextranamine (Ea and b). Other non-fluorescent bulks (putative boutons) can also be observed (arrowhead, IntB in Da and b), which can be electrophysiologically verified. F, pyramidal cells and G, interneurons are recognized by their characteristic shape and firing of action potentials on depolarization.

Figure 2. Spontaneous synaptic events and synaptic responses evoked by stimulation of attached, identified single MF and interneuronal boutons

A, viable dissociated pyramidal cells presented frequent spontaneous synaptic activity. B, schematic depiction of a pyramidal cell with a fluorescent MFB on the apical dendrite and an IntB on the basal dendrite, which were stimulated with a bipolar theta glass pipette. C, synaptic currents evoked in a pyramidal cell by stimulation of the fluorescent MFB and of an IntB. Both responses occur in an all-or-none fashion. D, intensity dependence of the synaptic responses evoked in pyramidal cells (n= 4) by MFB and IntB stimulation. Notice the all-or-none nature of their appearance and the higher amplitude of the responses to MFB stimulation. E, positioning of the stimulation pipette in the centre of the fluorescent bouton readily evoked a synaptic current (position 1), whereas a shift of the pipette (position 2) failed to evoke responses. F, synaptic currents evoked by stimulation of MFBs presented failures, consistent with previous reports using this preparation. In the cases where two MFBs on the same cell were found, their stimulation provoked a similar failure rate. Interestingly, the failure rate observed on IntB stimulation was lower than in the case of MFBs stimulation (n= 5 MFBs; n= 4 IntBs). G, the onset latency of the responses evoked by direct bouton stimulation was constant. Together, these results demonstrate that the synaptic responses originate from the activation of one bouton only.

Figure 3. The synaptic responses evoked by stimulation of MFBs have the physiological and pharmacological signature of transmission of MF origin

Aa and b, changing the stimulation frequency of MFBs from 0.05 to 1 Hz produced a robust frequency potentiation (>250%; n= 6; filled circles) and reduction of the failure rate (B; filled bars). By contrast, the same change in stimulation frequency of the IntBs produced a marked depression (60%, n= 6; open circles) and an increase in failure rate (B; open bars). C, perfusion of the mGluR agonist, DCG-IV (1 μm), produced a depression (Ca and b), as well as increase in the failure rate (D; filled bars) of the synaptic responses evoked by MFB stimulation. By contrast responses to IntB stimulation were unaffected (open circles and bars in Cb and D).

Figure 4. Stimulation of MFBs evokes mixed glutamatergic–GABAergic, glutamatergic-only and GABAergic-only currents

A, the pharmacologically isolated GABA_A-R-mediated current was evoked in an all-or-none fashion (open circles) and had a similar intensity threshold to the total current, evoked prior to perfusion of antagonists (filled circles). B, synaptic currents evoked by stimulation of some MFB were partially blocked by the iGluR antagonists NBQX and APV. The GABA_A-R antagonist, bicuculline, blocked the remaining current. C, stimulation of some MFBs evoked synaptic currents that were glutamatergic only because they were completely blocked by the iGluR antagonists. D, stimulation of some MFBs evoked synaptic currents that were insensitive to iGluR antagonists, but completely blocked by the GABA_A-R antagonist, thus GABAergic only. E, stimulation of all IntBs evoked a synaptic current that was blocked by the GABA_A-R antagonist, thus GABAergic only. F, the synaptic current evoked by stimulation of MFBs in normal ACSF reversed at −27 mV and, on blockage of iGluRs, the reversal potential shifted to −60 mV, consistent with currents mediated by GABA_A receptors. The trace in red, expanded below, shows a compound inward/outward current evoked at Vh −30 mV. G, by contrast, the synaptic currents evoked by stimulation of IntBs in normal ACSF and during blockage of iGluRs reversed at −64 mV, both consistent with currents mediated by GABA_A receptors. H, the synaptic currents evoked by stimulation of the MFBs in the absence (ACSF) and presence of iGluR antagonists (NBQX+APV) had the same onset latency. I, the length of the shift of the stimulation electrode away from the centre of the synaptic bouton, at which responses were no longer evoked, was similar in the absence (ACSF) and presence of iGluR antagonists (NBQX+APV).

Figure 5. The synaptic currents evoked by stimulation of MFBs have distinct kinetics

A, the synaptic currents evoked by stimulation of the MFBs had either a fast and a slow decay component (mixed), a fast component (fast), or a slow component (slow). Perfusion of iGluR antagonists isolated the current with the slow component. Subtracting the pharmacologically isolaged GABAergic (slow) component from the mixed response yielded a current with a fast component (B). C, in agreement with this observation, perfusion of bicuculline blocked the slow component and isolated a current with a fast component. D, the synaptic current evoked by IntBs stimulation under normal ACSF had a slow decay, similar to that obtained by MFB stimulation in the presence of iGluR antagonists. E, stimulation of MFBs of preparations older than 23 days of age evoked synaptic currents with a fast decay constant that were blocked by iGluR antagonists. F, values of the decay constant and rise time of each synaptic current evoked by MFB (n= 6) and IntB (n= 6). G, percentage of each type of current (mixed, slow – GABAergic, or fast – glutamatergic) evoked and failures on MFB and IntB stimulation in the different pharmacological conditions. Notice that iGluR blockage prevented currents with a fast decay constant and blockage of GABA_A-R prevented currents with a slow decay constant, while failure rates were not modified. Ha, spontaneous currents in 15-day-old rats could have either a fast and a slow decay component (mixed), a fast component (fast), or a slow component (slow), as the evoked ones, depicted in panel A. Hb, in 25-day-old rats, the spontaneous currents had either a slow or a fast decay component.

Figure 6. The pharmacologically isolated GABAergic synaptic currents evoked by stimulation of MFBs have the signature of transmission of MF origin

A, changing the stimulation frequency from 0.05 to 1 Hz produced a strong potentiation (>200%) of the GABAergic synaptic responses (filled circles) and reduction of the failure rate (B; filled bars; n= 6). By contrast stimulation of IntBs (A, open circles) produced frequency-dependent depression and increase of the failure rate (B; open bars; n= 6). C, group synaptic responses to MFB (filled circles; n= 6) and IntB stimulation (open circles; n= 6). Responses to MFB stimulation were partially blocked by iGluR blockers. The remaining responses were strongly and reversibly depressed by the activation of mGluRs with l-AP4, and completely blocked by bicuculline. Responses to IntB were not affected by the perfusion of iGluR blockers, but completely blocked by bicuculline. D, the effect of l-AP4 on the pharmacologically isolated GABAergic current evoked by MFB stimulation is presynaptic, as evidenced by the paired-pulse protocol. By contrast, GABAergic responses evoked by IntB were unaffected. E, further evidence for the presynaptic action of l-AP4 is the increase in the failure rate of MFB responses. Responses to IntB were unaffected.