Innervation by a GABAergic neuron depresses spontaneous release in glutamatergic neurons and unveils the clamping phenotype of synaptotagmin-1

Abstract

The role of spontaneously occurring release events in glutamatergic and GABAergic neurons and their regulation is intensely debated. To study the interdependence of glutamatergic and GABAergic spontaneous release, we compared reciprocally connected "mixed" glutamatergic/GABAergic neuronal pairs from mice cultured on astrocyte islands with "homotypic" glutamatergic or GABAergic pairs and autaptic neurons. We measured mEPSC and mIPSC frequencies simultaneously from both neurons. Neuronal pairs formed both interneuronal synaptic and autaptic connections indiscriminately. We find that whereas mEPSC and mIPSC frequencies did not deviate between autaptic and synaptic connections, the frequency of mEPSCs in mixed pairs was strongly depressed compared with either autaptic neurons or glutamatergic pairs. Simultaneous imaging of synapses, or comparison to evoked release amplitudes, showed that this decrease was not caused by fewer active synapses. The mEPSC frequency was negatively correlated with the mIPSC frequency, indicating interdependence. Moreover, the reduction in mEPSC frequency was abolished when established pairs were exposed to bicuculline for 3 d, but not by long-term incubation with tetrodotoxin, indicating that spontaneous GABA release downregulates mEPSC frequency. Further investigations showed that knockout of synaptotagmin-1 did not affect mEPSC frequencies in either glutamatergic autaptic neurons or in glutamatergic pairs. However, in mixed glutamatergic/GABAergic pairs, mEPSC frequencies were increased by a factor of four in the synaptotagmin-1-null neurons, which is in line with data obtained from mixed cultures. The effect persisted after incubation with BAPTA-AM. We conclude that spontaneous GABA release exerts control over mEPSC release, and GABAergic innervation of glutamatergic neurons unveils the unclamping phenotype of the synaptotagmin-1-null neurons.

Keywords: autaptic neuron; pHluorin imaging; spontaneous GABA release; spontaneous glutamate release; synaptotagmin-1.

Figure 1.

Reciprocal connectivity within minimal neuronal networks. A, B, Bright-field and fluorescence images of an isolated neuron (A) and a pair consisting of two connected neurons (B, DIV14). Alexa Fluor-467 (red) or lucifer yellow (yellow) was used to demonstrate neurite arborization (A2, B2; scale bars, 25 μm). C, Illustrations summarizing the different experimental groups. D, Whole-cell recording of isolated neuron and pairs containing two reciprocally connected cells. An action potential induced evoked autaptic release in isolated glutamatergic (eEPSC; left, green) and GABAergic (eIPSC; middle, magenta) neurons. An action potential induced both autaptic and synaptic eEPSCs (left illustration of mixed pair, green traces) and eIPSCs (right illustration of mixed pair, magenta traces) in mixed pairs. E, F, Evoked amplitudes were comparable between autaptic and synaptic connections in glutamatergic (E) and GABAergic (F) isolated neurons and minimal networks. G, H, No differences were found in decay time (90–10%) between autaptic and synaptic connections in glutamatergic (G) and GABAergic (H) isolated neurons and pairs. Also, evoked charge was comparable between the different groups (glutamatergic evoked charge: isolated, 0.042 ± 0.0085 pC; homotypic autaptic, 0.029 ± 0.0067 pC; homotypic synaptic, 0.0399 ± 0.010 pC; mixed autaptic, 0.028 ± 0.0071 pC; mixed synaptic = 0.033 ± 0.0066 pC; GABAergic evoked charge: isolated, 0.27 ± 0.032 pC; homotypic autaptic, 0.36 ± 0.085 pC; homotypic synaptic, 0.26 ± 0.089 pC; mixed autaptic, 0.28 ± 0.049 pC; mixed synaptic, 0.38 ± 0.058 pC). Differences in amplitude and decay time of eEPSCs and eIPSCs between different groups were tested using one-way ANOVA (p > 0.05).

Figure 2.

GABAergic input attenuates mEPSC frequency in mixed pairs. A, Example traces of spontaneous glutamatergic (mEPSCs, green) and GABAergic (mIPSCs, magenta) release events in all experimental groups. Thick traces represent the averaged spontaneous release event in each condition. Internal recording solution contained high [Cl⁻], thus both mEPSCs and mIPSCs were detected as inward currents. B, A threshold of 5 ms was used to segregate mEPSCs and mIPSCs in mixed pairs (histogram, bin size = 0.1 ms). Inset, mEPSC (green arrowheads) and mIPSC (magenta arrowheads) events in mixed pairs. C, No network-induced changes were found in spontaneous decay time kinetics using threshold segregation between mEPSCs and mIPSCs in mixed pairs. D, Network association did not affect mIPSC or mEPSC amplitude. E, Heterosynaptic connectivity in mixed pairs attenuates mEPSC release frequency (p < 0.01, Student's t test comparing average mEPSC frequency, averaged from both cells within a pair, in homotypic pairs with average mEPSC frequency in mixed pairs). F, Acute application of bicuculline (40 μm) blocked both mIPSCs and eIPSCs in mixed pairs (magenta), but mEPSCs and eEPSCs release was unaffected (green). Consecutive application of bicuculline (40 μm) and CNQX (10 μm) blocked all spontaneous release. Insets show eIPSCs and eEPSCs before and during bicuculline or bicuculline/CNQX application. Note that bicuculline only blocks mIPSCs, indicating that the segregation based on decay time accurately distinguishes between mEPSCs and mIPSCs. The remaining events during bicuculline represent mis-sorted mEPSCs, which are blocked during bicuculline/CNQX application.

Figure 3.

Spontaneous GABA release regulates spontaneous glutamatergic release in mixed pairs. A, Acute application of bicuculline-containing medium that was incubated for 3 d at 37°C completely blocked eIPSCs, demonstrating that bicuculline is stable and active during this period. B, Long-term pretreatment with bicuculline (3 d, +Bic_pre) exposed an increase in mEPSC frequency in mixed pairs (p < 0.001, Student's t test comparing average mEPSC frequency between control and bicuculline pretreated mixed pairs). Bicuculline had no effect on spontaneous release rate in glutamatergic pairs or GABAergic spontaneous release rate in mixed pairs. Note that bicuculline was not present during the whole-cell recordings. C, Acute application of TTX-containing medium that was incubated for 3 d at 37°C completely blocked both eEPSCs and eIPSCs, demonstrating that TTX is stable and active during this period. D, Long-term TTX treatment (3 d, +TTX_pre) did not affect mEPSC frequency in mixed pairs, suggesting spontaneous GABA release control mEPSC release in mixed pairs (p > 0.05; Student's t test comparing average mEPSC frequency between control and TTX pretreated mixed pairs). TTX was not present during the whole-cell recordings. E, Spontaneous glutamatergic release is regulated by GABAergic input (left). Long-term silencing of spontaneous GABAergic input (using bicuculline) blocks the GABAergic control of mEPSC frequency in mixed pairs (E, middle). This GABAergic control of spontaneous glutamatergic release does not depend on network activity, since long-term TTX treatment did not affect mEPSC frequency (E, right).

Figure 4.

Counting synapses in isolated (autaptic) neurons and interconnected neuronal pairs. A, B, The total number of synaptic connections formed by isolated (A1) or pairs (B1) of neurons was determined using superfusion of NH₄Cl, herewith unquenching all SypHy-positive synaptic contacts (DIV8; A2, B2). MPTS (red) or Alexa Fluor-568 (blue) was infused during whole-cell recordings to visualize dendritic arborization. Recording electrodes were retracted before imaging, exposing the complete arborization. A3, B3, Manual tracking of the neuronal arborization resulted in dendritic masks of the isolated autaptic neurons and of neuronal pairs. Filled circles represent SypHy-positive puncta that were automatically identified and colocalized with the dendritic mask (Schmitz et al., 2011). A3, SypHy-positive puncta representing autaptic connections (orange dots) based on colocalization with a red dendritic mask of an isolated autaptic neuron. B3, SypHy-positive spots in a pair, representing either autapses or interneuronal synapses on the red neuron (orange dots) or the blue neuron (light blue dots). A small fraction of synapses were allocated to both neurons due to dendritic overlap (purple dots). A4, B4, High-frequency trains (40 Hz, 300 stimulations) were used to determine the number of active GABAergic or glutamatergic synapses. C, The total number of SypHy-positive spots that appear during superfusion with NH₄Cl is comparable between glutamatergic and GABAergic isolated neurons. Pairs of neurons establish twice as many synaptic connections, suggesting the number of synaptic connections made per neuron is constant (One-way ANOVA (p < 0.05), followed by Tukey's test for subsequent pairwise comparisons). D, Evoked amplitude is comparable between autaptic and synaptic connections in glutamatergic neurons in isolation and minimal networks (DIV8–9), indicating no strong preference in formation of functional autaptic or synaptic glutamatergic connections (Kruskal–Wallis test, p > 0.05). E, Similarly, evoked amplitude is comparable between autaptic and synaptic connections in GABAergic neurons in isolation and minimal networks (Kruskal–Wallis test, p > 0.05). F, G, Correlation plot between the total number of active synapses (assessed using 40 Hz trains) and the total mEPSC/mIPSC release frequency in all groups (frequencies summed up from both neurons in the case of a pair). F, G, The total mIPSC frequency and GABAergic synapse number correlate in mixed pairs, but the total mEPSC frequency in mixed pairs was lower than expected based on number of glutamatergic synapses. H, I, Correlation plot between the summed eEPSC/eIPSC amplitudes and the total mEPSC/mIPSC release frequency in all groups (frequencies summed up from both neurons in the case of a pair). The mEPSC frequency was much lower than expected based on the summed eEPSC amplitude in mixed pairs, while the mIPSC frequency was more consistent with the eIPSC amplitude found in mixed pairs. Long-term treatment with bicuculline blocks the GABAergic inhibition of mEPSC frequency (Fig. 3B), herewith shifting the mEPSC frequency toward the predicted value (H, gray arrow marked ‘Bic’ and square).

Figure 5.

Enhanced spontaneous release in synaptotagmin-1-null mutants depends on ‘mixed’ connectivity. A1, Example traces of spontaneous release in wild-type homotypic (Syt-1^+/+, top trace) and mixed pairs (bottom trace). Note the discrete difference in kinetics between mEPSCs and mIPSCs in the mixed pair. A2, Example traces of spontaneous release in synaptotagmin-1-null (Syt-1^−/−) mutant homotypic (top trace) and mixed pairs (bottom trace). B1, B2, No difference was found in mEPSC frequency between isolated and homotypic pairs from wild-type and synaptotagmin-1-null neurons. Note that the GABAergic inhibition of mEPSC frequency is also present in mixed pairs from wild-type (Syt-1^+/+) C57BL/6 mice (B1, p < 0.01, Student's t test comparing average mEPSC frequency in homotypic pairs with average mEPSC frequency in mixed pairs). B2, The increased spontaneous release typical for synaptotagmin-1-null mutants is exposed only in mixed pairs (p < 0.01; Student's t test comparing average mEPSC and mIPSC frequencies between control and Syt-1^−/− mixed pairs). C, D, mEPSC and mIPSC frequencies were inversely correlated in mixed pairs from wild-type NMRI mice (C, Spearman's rank correlation = −0.455, p = 0.0001) and in mixed pairs form wild-type C57BL/6 mice (D, Spearman rank correlation = −0.405, p = 0.001). E, Correlation between mEPSC and mIPSC frequencies was absent in synaptotagmin-1-null mutant mixed pairs (Spearman's rank correlation: r = 0.228, p = 0.145).

Figure 6.

Synaptotagmin-1-null-induced disinhibition of spontaneous release is independent of GABAergic control of mEPSCs and partly calcium dependent. A1, Long-term bicuculline treatment (+ Bic _pre) increases mEPSC frequency in wild-type (C57BL/6; Syt-1^+/+) mixed pairs (p < 0.001, Student's t test comparing average mEPSC frequency between control and bicuculline pretreated mixed pairs). A2, Long-term bicuculline treatment showed an additive effect on mEPSC frequency in Syt-1^−/− mutant mixed pairs (right, p < 0.05, Student's t test comparing average mEPSC frequency between control and bicuculline pretreated Syt-1^−/− mixed pairs). Data from Figure 5B are included in the “−Bic_pre” group. B, Summarizing effect of long-term bicuculline treatment on mEPSC frequency in NMRI (magenta), Syt-1^+/+ (green), and Syt-1^−/− (blue) mixed pairs. Note the increase in both mIPSC and mEPSC release rate in Syt-1^−/− mixed pairs, while additional long-term bicuculline treatment affects only the mEPSC release rate in these pairs. C1, BAPTA-AM (20 μm, 20 min at 37°C) strongly reduced mEPSC frequency in homotypic pairs (∼95% of events are calcium dependent), and both mEPSC and mIPSC frequency in mixed pairs (∼80% are calcium dependent). C2, BAPTA-AM pretreatment induced a similar decrease in mEPSC release in Syt-1^−/− homotypic pairs. In contrast, mEPSC and mIPSC frequency remained elevated in Syt-1^−/− mixed pairs compared with control, suggesting an increase in spontaneous release willingness (p < 0.01, Student's t test, comparing average mEPSC and mIPSC frequency between control and BAPTA-AM pretreated Syt-1^−/− mixed pairs. n.s.: not significant).