Electrical coupling regulates layer 1 interneuron microcircuit formation in the neocortex

Results

Development of electrical and GABAergic connections

Layer 1 interneurons expand their neurites horizontally26,27,38. To preserve neurites, we prepared whole-mounts of the somatosensory cortex from GAD67-green fluorescent protein (eGFP) transgenic mice aged postnatal days 1–5 (P1-5) and acute horizontal slices of the somatosensory cortex from mice aged P6-25 (Supplementary Fig. 1). We identified neocortical layer 1 on the basis of the sparsely distributed cells. Guided by infrared differential interference contrast (DIC) and epifluorescence illumination, we simultaneously recorded from four layer 1 interneurons whose cell bodies were between 30 and 150 μm apart (the distance between the centres of cell bodies) (Fig. 1a,d). Compared with parasagittal sections of the brain30,31, arborizations of layer 1 interneurons, filled with neurobiotin during the recording, covered a larger field in the horizontal slice preparations (Fig. 1b), indicating that the horizontal slice preparation preserved anatomical and functional connectivity in neocortical layer 1. Furthermore, we found that layer 1 interneurons exhibited tracer coupling (neurobiotin,Fig. 1c), as observed in interneurons of other brain regions39,40. As in our previous study26, we found the vast majority of layer 1 neurons were positive for interneuron markers (Supplementary Fig. 2).

Figure 1: Development of electrical and GABAergic connections between interneurons in neocortical layer 1.

(a) A schematic diagram of a quadruple whole-cell recording of four neurons in cortical layer 1. (b) An image of four layer 1 neurons filled with neurobiotin during recording and labelled with fluorescence-conjugated avidin. Scale bar, 100 μm. (c) A magnified image of b (dotted rectangular region). Red arrowheads indicate the four recorded neurons, and green arrowheads indicate dye-coupled neurons. Scale bar, 50 μm. (d) A DIC image of quadruple whole-cell recording of the four neurons in layer 1 shown in b and c. Scale bar, 50 μm. (e) Summary of the synaptic connections detected in this quadruple recording. The average traces of the postsynaptic responses are shown in the rectangle. Red traces indicate the existence of electrical synapses, and green traces indicate the existence of chemical synapses. Sample traces of AP and hyperpolarization potentials are shown to the left. Scale bar, 40 pA (green vertical scale bar), 20 ms (red horizontal scale bar), 200 mV (black vertical scale bar). ‘Pre-Stimu.’, presynaptic potential; ‘Pre’, presynaptic neuron; ‘Post’, postsynaptic neuron. (f) A schematic diagram showing connections between the four neurons in e. Wavy red arrowheads indicate electrical connections, and green arrowheads indicate chemical connections. (g) Summary of proportion of electrical connections (red bars) and unidirectional/bidirectional chemical connections (light blue bars for unidirectional chemical connections and dark blue bars for bidirectional chemical connections) between interneuron pairs in neocortical layer 1 at different postnatal stages.

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Once all four recordings were established, serial APs and hyperpolarization were sequentially triggered in one of the four neurons and the postsynaptic responses were then measured in the other three neurons to test chemical and electrical synapse formation between them. As shown in Fig. 1e and Supplementary Fig. 3, APs in the presynaptic neurons induced GABA-receptor-mediated inward currents in the postsynaptic neurons (green lines in Fig. 1e), while hyperpolarization induced electrical coupling-mediated outward currents (red lines in Fig. 1e). Although inward currents were induced by APs in both the chemically and electrically connected interneuron pairs, the GABA-receptor-mediated responses were distinguished based on their characteristic slow decay time course. To further confirm this, we examined the effects of bicuculline (BIC, 10 μM), a specific GABA-A receptor inhibitor. As expected, bicuculline treatment not only strongly reduced the inward current amplitudes but also completely eliminated the slow decay time responses (Supplementary Fig. 4a), suggesting that the latter are mediated by the GABA-A receptor. Furthermore, the treatment of carbenoxolone (CBX, 100 μM), a gap junction blocker, abolished hyperpolarization-induced outward currents (Supplementary Fig. 4b). Previous studies have shown that GABA-A and GABA-B receptors are both involved in mediating inhibitory synaptic response in neocortical layer 1 (refs 31, 35). However, single presynaptic APs barely induced GABA-B receptor-mediated postsynaptic response31,35, indicating that the inhibitory synaptic responses are mainly mediated by GABA-A receptors.

To further systematically study the development of electrical and GABAergic connections between layer 1 interneurons, we examined 947 pairs of layer 1 interneurons at different developmental stages (Fig. 1gand Supplementary Table 1). Our results showed that electrical and GABAergic connections (including unidirectional and bidirectional GABAergic connections) between layer 1 interneurons emerged at about the same developmental period (P5-6). The occurrence of electrical and GABAergic connections steadily increased during the second postnatal week, suggesting that the second postnatal week is a critical period for the development of synaptic connections between layer 1 interneurons. Together, these results suggest that the electrical and GABAergic connections between layer 1 interneurons have similar developmental time courses. Of note, we did not detect chemical or electrical connections between Cajal–Retzius cells, or between Cajal–Retzius cells and layer 1 interneurons at the early postnatal period.

Microcircuitry between layer 1 interneurons

On the basis of their electrophysiological properties, layer 1 interneurons were classified into two subtypes (Fig. 2a), burst spiking (BS) and late spiking (LS), as shown previously26,41,42. The key differences between them were a delay in the initial spike, spike firing pattern and after depolarization (the inset of Fig. 2a). LS neurons were identified by a delay with a steady ramp depolarization leading up to the initial spike at threshold current injections. BS neurons fired a burst of APs at the initial spike without any delay, and after depolarizations were only observed in BS neurons. Out of a total of 469 layer 1 interneurons obtained from P9-25 mice, the number of LS and BS interneurons was 374 (79.7%) and 95 (20.3%), respectively (Fig. 2b).

Figure 2: Electrical and GABAergic connections between layer 1 interneurons.

(a) Representative traces of voltage responses to 500 ms current pulse step injections recorded in the current-clamp mode. The red traces show the initial AP spike. Layer 1 interneurons were divided into two subtypes, LS and BS neurons, based on their AP firing patterns. The inset shows the ADP in the initial AP of a BS neuron. (b) Histogram showing that the majority of interneurons displayed LS firing pattern (LS, 79.7%, 374 cells; BS, 20.3%, 95 cells; n=63 mice). (c) Summary of the proportion of electrical coupling observed between layer 1 interneurons at P9–P25. The rate of electrical connections between LS interneurons is significantly higher than the rate between LS and BS interneurons and between BS interneurons. (d) The scatter plot of coupling coefficients revealed no significant difference between interneuron subtypes (LS–LS pairs, 1.8±0.12%, n=122; LS–BS pairs, 2.2±0.45%,n=22; BS–BS pairs, 1.1±0.15%, n=4). (e) Summary of the proportion of GABAergic connections between interneuron subtypes. (f) The unidirectional chemical synapses between LS and BS interneurons showed directional selectivity. (g) The amplitude of uIPSCs between LS interneuron pairs (37.98±2.13 pA, n=73) was significantly larger than those between LS–BS pairs (24.22±2.09 pA, n=39) and BS–BS pairs (21.44±1.73 pA, n=34) (not including failures). *P<0.05, **P<0.01, ***P<0.001, n.s., P>0.05, not significant. χ2-test, Fisher’s exact test and Mann–Whitney rank sum test. Error bars in d and g represent mean±s.e.m. ADP, after depolarization.