Dynamic changes in interneuron morphophysiological properties mark the maturation of hippocampal network activity

Abstract

During early postnatal development, neuronal networks successively produce various forms of spontaneous patterned activity that provide key signals for circuit maturation. Initially, in both rodent hippocampus and neocortex, coordinated activity emerges in the form of synchronous plateau assemblies (SPAs) that are initiated by sparse groups of gap-junction-coupled oscillating neurons. Subsequently, SPAs are replaced by synapse-driven giant depolarizing potentials (GDPs). Whether these sequential changes in mechanistically distinct network activities correlate with modifications in single-cell properties is unknown. To determine this, we studied the morphophysiological fate of single SPA cells as a function of development. We focused on CA3 GABAergic interneurons, which are centrally involved in generating GDPs in the hippocampus. As the network matures, GABAergic neurons are engaged more in GDPs and less in SPAs. Using inducible genetic fate mapping, we show that the individual involvement of GABAergic neurons in SPAs is correlated to their temporal origin. In addition, we demonstrate that the SPA-to-GDP transition is paralleled by a remarkable maturation in the morphophysiological properties of GABAergic neurons. Compared with those involved in GDPs, interneurons participating in SPAs possess immature intrinsic properties, receive synaptic inputs spanning a wide amplitude range, and display large somata as well as membrane protrusions. Thus, a developmental switch in the morphophysiological properties of GABAergic interneurons as they progress from SPAs to GDPs marks the emergence of synapse-driven network oscillations.

Figure 1.

GABAergic neurons are preferentially involved in SPAs at early postnatal stages (P0–P5). A1, Two-photon calcium fluorescence image from the CA3 region of a P1 GAD67-KI-GFP mouse (left) and the GFP signal from the same field (right). A2, Automatically detected contours of the cells (left) from the calcium fluorescence image (A1) and overlap of this contour map with that of GFP-positive cells (right). Open contours, Silent cells; black-filled contours, cells producing calcium spikes; red-filled contours, SPA cells; green open contours, GFP-positive cells. Note that in this case, half of the SPA cells are GFP-positive. B1, Histogram indicating the fraction of SPA cells that are GFP-positive at three time points (P1, P3, P5). B2, Histogram indicating the fraction of interneurons (solid-colored bars) and pyramidal cells (striped bars) involved in SPAs (red) or in GDPs (blue) relative to the total interneuron or pyramidal cell population, respectively, as a function of time. C1, Two-photon calcium fluorescence image from the CA3 region of a P5 GAD67-KI-GFP mouse (left) and the GFP signal from the same field (right). C2, Contour map of the imaged cells (left) from the boxed areas in C1 and superimposed on the GFP-positive cell contour map (right). Open contours, Silent cells; red-filled contours, SPA cells; blue-filled contours, GDP cells; green open contours, GFP-positive cells. C3, High-magnification image of a neurobiotin-filled SPA interneuron (left, arrow) and the corresponding GFP confocal image (right) suggest that the targeted SPA interneuron is coupled with another interneuron by a gap junction. D1, Patch-clamp recording in current-clamp mode at resting membrane potential (0 pA, top red trace) of the targeted SPA interneuron in C, the corresponding calcium fluorescence trace (middle), and histogram (bottom) indicating the fraction of active cells as a function of time: each blue peak represents a GDP. Note that the occurrence of a GDP switches off the calcium plateau, as previously shown (Crépel et al., 2007). D2, Recurrent membrane potential oscillations characteristic of SPA cells from the same SPA interneuron that further supports gap-junction coupling by the presence of spikelets; shown on an expanded time scale below.

Figure 2.

SPA cells develop into GDP cells as the hippocampal network matures. A, Two-photon calcium fluorescence image (left) and contour map of the cells imaged (right) in the CA3 region of an organotypic hippocampal slice prepared from a P4 mouse. SR, Stratum radiatum; SP, Stratum pyramidale. B, Calcium fluorescence traces as a function of time of four pyramidal cells (Pyr. Cell #1–4) and four interneurons (In #5–8), circled in A and monitored after 2 d in culture (Day D) and 1 d later (D+1). Scale bars: x-axis, 10 s; y-axis, 20% DF/F. Note that pyramidal cells 1–3 and interneurons 5–7 first produce calcium plateaus characteristic of SPAs at D and display synchronous fast calcium transients associated with GDPs at D+1. C, Graph showing the fraction of SPA cells relative to all active cells (y-axis on the left) at D and the fraction of SPA and GDP cells relative to the number of SPA cells at D on D+1 (y-axis on the right; n = 4 slices, 520 neurons). Note that the proportion of SPA cells at D that become involved in GDPs the next day is greater than that still producing SPAs at D+1 (56 ± 5% vs 11 ± 10%).

Figure 3.

SPA and GDP interneurons are morphologically diverse, with SPA interneurons displaying larger somata and a higher density of membrane protrusions. Neurolucida reconstructions of 18 Neurobiotin-filled neurons, nine SPA interneurons (left) and nine GDP interneurons (right) from P4 GAD67-KI-GFP mice. Both groups are morphologically diverse (black, dendrites and soma; red, axons for SPA interneurons; blue, axons for GDP interneurons). SR, Stratum radiatum; SP, Stratum pyramidale; SO, Stratum oriens. Scale bar, 50 μm. B, Confocal fluorescence images and the corresponding neurolucida reconstructions of the three SPA (left) and three GDP interneurons (right) illustrated in A at a high magnification centered on the soma. Note that the soma of SPA interneurons is larger than that of GDP interneurons. Scale bar, 50 μm. C, Confocal fluorescence images of a fragment of second-order dendritic tree for three SPA (left) and three GDP interneurons (right) illustrated in A. Scale bar, 5 μm. Note that the dendrites of SPA interneurons are covered with numerous protrusions of various shapes, i.e., round and small (arrows) or elongated (arrowheads).

Figure 4.

Different SPA interneurons display similar intrinsic electrophysiological properties; firing diversity only emerges in GDP interneurons. A, Electrophysiological recordings in current-clamp mode at resting membrane potential (I = 0 pA) in the presence of blockers of synaptic transmission (10 μm bicuculline, 10 μm NBQX, 40 μm APV) from the three SPA interneurons (SPA#1–3) and the three GDP interneurons (GDP#1–3) illustrated in Figure 3A from P4 GAD67-KI-GFP mice. Scale bars: x-axis, 1 s; y-axis, 20 mV. B, Firing patterns in response to a depolarizing current step (+40 pA). Scale bars: x-axis, 5 s; y-axis, 10 mV. Note that at the same developmental stage (P4),a basket-like SPA interneuron (SPA#3; Fig. 3A) presents a strongly delayed and adapting firing pattern while a basket-like GDP interneuron (GDP#3; Fig. 3A) displays a fast-spiking pattern. C, Diagram representing the distribution of different firing patterns recorded in SPA interneurons (n = 11). NA, Non-adaptating; IS, irregular-spiking; Stut, stuttering. Note that SPA interneurons display similar intrinsic electrophysiological properties with an adapting firing pattern and recurrent membrane potential oscillations at resting membrane potential; GDP interneurons display a diversity of firing patterns.

Figure 5.

Large amplitude miniature postsynaptic currents are only recorded in SPA cells. A1, GABA_AR-mediated mIPSCs recorded at +10 mV and AMPA/KAR-mediated mEPSCs recorded at −60 mV in the CA3 hippocampal region of P4 GAD67-KI-GFP mice in four SPA interneurons. A2, Same as A1, but in four GDP interneurons. Note the occurrence of high amplitude mIPSC and mEPSCs only in SPA interneurons. B, Histograms plotting the pooled distribution of mIPSCs and mEPSCs amplitudes of SPA (red) and GDP (blue) interneurons. Note the strong right skew of both mIPSCs and mEPSCs distributions for SPA interneurons, indicating a large variety of event amplitudes with a significant occurrence of larger events.

Figure 6.

Morphophysiological specificities of SPA interneurons are age-independent. A, Confocal images of three neurobiotin-filled CA3 interneurons targeted at P4 and P7 in GAD67-KI-GFP mice: SPA-interneuron at P4 (left; inset is a higher magnification of the boxed area), SPA interneuron at P7 (middle), and GDP interneuron at P7 (right). Scale bar, 10 μm. Note the presence, at both ages, of somatic (stars) and dendritic (arrowheads) protrusions on SPA interneurons, whereas the GDP interneuron is free of protrusions and presents a smaller cell body than SPA interneurons. Note also the global increase in cell body size between P4 and P7. B, Electrophysiological recordings in current-clamp mode, at resting membrane potential (I = 0 pA) in the presence of blockers of synaptic transmission (10 μm bicuculline, 10 μm NBQX, 40 μm APV) for the three interneurons illustrated in A. Scale bars: x-axis, 2 s; y-axis, 20 mV. C, Firing patterns in response to a depolarizing current step (+40 pA). Scale bars: x-axis, 1 s; y-axis, 20 mV. D, Diagram representing the distribution of different firing patterns recorded in the three interneurons illustrated in A (left, n = 11; middle, n = 5; right, n = 5) and showing that the adapting pattern is the most represented one in the SPA interneuron population. NA, Non-adaptating; IS, irregular-spiking; Stut, stuttering. Note that in addition to membrane protrusions (A), both SPA interneurons display similar intrinsic electrophysiological characteristics, i.e., adapting firing patterns and recurrent membrane potential oscillations, even at late postnatal stages (P7) where only few SPA interneurons can be found.

Figure 7.

Interneuron involvement in SPAs or GDPs depends on birth date. A1, Current-clamp recording at resting membrane potential (0 pA) of an EG-In of the CA3 region of a P3 Mash1^CreERTM;RCE:LoxP mouse treated with tamoxifen at E11.5 (see Materials and Methods). Note the presence of GDPs. A2, Same as A1 but in voltage-clamp mode recorded at +10 mV (top trace) and −60 mV (bottom trace) in another EG-In. Note the presence of characteristic GABAergic (top trace) and glutamatergic (bottom trace) bursts of postsynaptic currents associated with GDPs. A3, Bar histogram indicating the fraction, at P3, of SPA (red) and GDP (blue) interneurons of the total EG-Ins population (left) and in the overall GFP-positive cell population from GAD67-KI mice (right). The percentage of either immature spiking or inactive interneurons is shown in white. Note that all EG-Ins are already involved in GDPs at a developmental stage (P3) dominated by immature activities. B1, GFP-positive LG-In (red contour and white-filled soma) imaged in the CA1 hippocampal region of a P7 Mash1^CreERTM;RCE:LoxP mouse treated with tamoxifen at E18.5 (see Materials and Methods) and superimposed with the contour map of all imaged cells. White contours, Silent cells; blue contours, GDP-cells; red contours, SPA-cells. B2, Traces of calcium fluorescence changes as a function of time for two representative GDP cells and for the SPA LG-In indicated in B1. B3, Bar histograms indicating the percentage, at P7, of SPA (red) and GDP (blue) interneurons of the total population of LG-Ins from Mash1^CreERTM;RCE:LoxP mice treated with tamoxifen at E18.5 (left) and out the total population of GAD-GFP-positive cells from GAD67 KI mice (right). Note that a large fraction of LG-Ins are still involved in SPA at a development stage (P7) dominated by GDPs.