Sequential generation of two distinct synapse-driven network patterns in developing neocortex

Abstract

Developing cortical networks generate a variety of coherent activity patterns that participate in circuit refinement. Early network oscillations (ENOs) are the dominant network pattern in the rodent neocortex for a short period after birth. These large-scale calcium waves were shown to be largely driven by glutamatergic synapses albeit GABA is a major excitatory neurotransmitter in the cortex at such early stages, mediating synapse-driven giant depolarizing potentials (GDPs) in the hippocampus. Using functional multineuron calcium imaging together with single-cell and field potential recordings to clarify distinct network dynamics in rat cortical slices, we now report that the developing somatosensory cortex generates first ENOs then GDPs, both patterns coexisting for a restricted time period. These patterns markedly differ by their developmental profile, dynamics, and mechanisms: ENOs are generated before cortical GDPs (cGDPs) by the activation of glutamatergic synapses mostly through NMDARs; cENOs are low-frequency oscillations (approximately 0.01 Hz) displaying slow kinetics and gradually involving the entire network. At the end of the first postnatal week, GABA-driven cortical GDPs can be reliably monitored; cGDPs are recurrent oscillations (approximately 0.1 Hz) that repetitively synchronize localized neuronal assemblies. Contrary to cGDPs, cENOs were unexpectedly facilitated by short anoxic conditions suggesting a contribution of glutamate accumulation to their generation. In keeping with this, alterations of extracellular glutamate levels significantly affected cENOs, which are blocked by an enzymatic glutamate scavenger. Moreover, we show that a tonic glutamate current contributes to the neuronal membrane excitability when cENOs dominate network patterns. Therefore, cENOs and cGDPs are two separate aspects of neocortical network maturation that may be differentially engaged in physiological and pathological processes.

Figure 1.

Multibeam two-photon imaging of the four maturation steps of spontaneous neuronal activity in somatosensory cortical slices from embryonic stages to first postnatal days. A, Two-photon calcium fluorescence images of rat somatosensory cortical slices of the four types of spontaneous activity: calcium spikes (left), cortical synchronous plateau assemblies (cSPAs), cortical early network oscillations (cENOs), and cortical giant depolarizing potentials (cGDPs, right) recorded at E20, P0 (cortical plate), P3 (cortical plate, horizontal slice), and P7 (deeper layers), respectively. White arrows indicate direction of pial surface. B, Automatically detected contours of the cells from the fluorescence images: open contours indicate silent cells, black filled contours indicate cells producing calcium spikes, red filled contours are cSPAs-cells, green filled contours are cENO cells, and blue filled contours are cGDPs-cells; scale bar: 100 μm. C, Raster plots of the activity from the four slices illustrated in A in control ACSF. Each row represents a single cell and each horizontal line the duration of detected calcium transients. Four populations of events can be distinguished as shown by representative fluorescence traces below the raster plots (black: calcium spikes; red: calcium plateaus i.e., cSPA-events; green: cENO-events; blue: cGDP-events). D, Current-clamp recordings (V_rest of approximately −60 mV) in four representative neurons displaying the four types of calcium activities described above. D1, A calcium spike recorded in a neuron at E20. D2, Red: cSPA recorded in a neuron at P0. Note that calcium plateaus are associated to rhythmic membrane potential oscillations as SPAs described in the hippocampus. D3, Green: a cortical ENO. D4, Blue: three successive cortical GDPs.

Figure 2.

Cortical ENOs and GDPs display two distinct spatiotemporal dynamics. A1, A2, Contour maps of seven successive movie frames taken from a P3 (A1) and a P8 (A2) horizontal somatosensory slice to illustrate the slower dynamics of cENOs (A1) compared with cGDPs (A2). Black filled contours indicate cells active in frames where network synchronization reaches significance threshold (see Materials and Methods). One frame every 150 ms; scale bar: 100 μm B1, Histogram indicating the fraction of imaged cells detected as being active for each movie frame in a P1 horizontal somatosensory cortical slice. Each peak of the histogram represents a cENO. Calcium fluorescence traces of four cells implicated in the two cENOs illustrated in the above histogram on an expanded time scale. B2, Simultaneous field potential recording (FP) and calcium imaging (raster plot) during a cENO occurring in a P3 horizontal cortical slice. Raster plot indicates the onset of each calcium event in all imaged cells as a function of time. Note the strong correlation between field potential oscillations and multineuron calcium activity. B3, Spectrogram of the FP oscillation associated to the cENO illustrated in B2. a.u.: arbitrary units. C1, C2, Same as A1 and A2 but in a P6 somatosensory horizontal slice where cGDPs could be recorded (small peaks of synchrony). Note that peaks associated with cGDPs are much smaller and more frequent than those associated with cENOs (B2). They involve fewer cells as shown in the C2 raster plot. C3, Cortical GDPs are not associated with any remarkable oscillatory pattern but correspond to a significant increase in MUA as shown by the frequency histogram of MUA as a function of time and by the MUA recording trace below.

Figure 3.

Single-cell electrophysiological and calcium events associated with cortical ENOs and GDPs. A, B, Current-clamp recordings at resting membrane potential and corresponding calcium fluorescence traces (bottom traces) of cells implicated in cENOs (A) and cGDPs (B). Recording periods indicated in i are illustrated on an expanded time scale. C1, Plots of the duration versus rise time of individual membrane potential oscillations associated with cGDPs (blue squares, n = 4 cells, 75 events) and cENOs (green, n = 5 cells, 65 events). C2, C3, Normalized distribution of the decay (2) and rise (3) times of single calcium events associated with cGDPs (blue, n = 1000) and cENOs (green, n = 1000). Distribution of the duration of the calcium plateaus associated with cSPAs (red, n = 500, see Materials and Methods) is also plotted in C2. D1, Comparison of three representative normalized calcium fluorescence traces recorded in single cells during cGDPs, cENOs, and cSPAs clearly illustrates the kinetics difference between these events. D2, Graph indicates the fraction of calcium spike-, cSPA-, cENO-, and cGDP-cells relative to the number of active cells at four successive age groups between embryonic to first postnatal stages. Error bars indicate SEMs.

Figure 4.

GABAergic transmission is not involved in the generation of cENOs but is crucial for cGDPs. A, B, Representative histograms indicating the fraction of imaged cells detected as being active for each movie frame as a function of time in a P3 (A) and a P8 (B) somatosensory horizontal slice. The occurrence of cENOs (peaks of synchrony in A) was not significantly affected in the presence of the GABA_AR antagonist (bicuculline, 10 μm) compared with control conditions. In contrast, cGDPs (peaks of synchrony in B) were blocked in the presence of bicuculline. Below, Calcium fluorescence traces of three representative cells implicated in cENOs (A) and cGDPs (B) in control and after adding bicuculline.

Figure 5.

Differential role of glutamate in the generation of cortical ENOs and GDPs. A1, Histograms indicating the fraction of imaged cells detected as being active for each movie frame as a function of time in a P0 somatosensory horizontal slice. The occurrence of cENOs (peaks of synchrony, left histogram) was strongly reduced when the NMDAR antagonist (d-APV, 40 μm) was added to the saline (right histogram). Dashed horizontal line indicates statistical significance threshold. A2, Same type of histograms as in A1 showing that the occurrence of cENOs was fully blocked in the presence of both NMDAR and AMPA/KAR antagonists (d-APV, 40 μm and NBQX, 10 μm, right histogram). A3, Left, Average current–voltage relationship of cENO-associated postsynaptic currents (PSCs, 5 cENOs-PSCs averaged for each point) obtained in a representative cortical neuron. I/V curve displays a negative slope at negative membrane potential values and reverses around 0 mV, indicating a strong contribution of NMDARs. Error bars indicate SEM. Right, Representative traces of PSCs associated with cENOs at different holding potentials from the same recorded neuron. A4, Same type of histograms as in A1 in a P1 somatosensory horizontal slice, showing that perfusion with the enzymatic glutamate scavenger (GPT 5 U/ml with pyruvate 2 mm) significantly reduces the frequency of cENOs (peaks of synchrony). The effect of GPT is reversible upon wash out of the drug (right histogram). B, Same as A but in P6 somatosensory horizontal slices where cGDPs could be recorded (small peaks of synchrony). B1, d-APV had a significantly smaller effect on the occurrence of cGDPs compared with cENOs (see A1). Dashed horizontal line indicates significance threshold; B2, Blockade of ionotropic glutamatergic transmission almost completely prevented the occurrence of cGDPs. B3, Same as A3, but current–voltage relationship of cGDP-PSCs is linear and reverses close to −50 mV, indicating a strong contribution of GABA_ARs. B4, Same experiments as in A4 but in a P8 slice, showing that GPT (5 U/ml) does not affect cGDPs as much as cENOs (see A4).

Figure 6.

Perfusion rate and anoxic/aglycemic episodes differentially affect cENOs and cGDPs. A1, Histograms indicating the fraction of imaged cells detected as being active for each movie frame as a function of time in a P2 somatosensory horizontal slice. The frequency of cENOs (peaks of synchrony indicated by *) was significantly increased when decreasing the rate of ACSF perfusion from 4 to 1 ml/min. Dashed line indicates the time when perfusion rate was modified. A2, Same histograms as in A1 in a P3 horizontal cortical slice. The frequency of cENOs (peaks of synchrony in the histogram) was significantly increased compared with control (left histogram) after 5 min of anoxia/aglycemia (right). B1, B2, Same as A but in a P7 somatosensory horizontal slice where cGDPs could be recorded (small peaks of synchrony indicated by *). In contrast to cENOs (A), the frequency of cGDPs was dramatically decreased in low perfusion conditions (B1, right) as well as after 5 min of anoxia/aglycemia (B2, right).

Figure 7.

Differential modulation of cENOs and cGDPs, simultaneously recorded in a neocortical slice. A1, Histograms indicating the fraction of imaged cells detected as being active for each movie frame as a function of time in a P5 somatosensory horizontal slice in control (left) and during anoxic/aglycemic conditions (right, 164 ms per frame). Two types of synchronous network events can be distinguished: cGDPs (blue) are smaller amplitude highly recurrent synchronizations associated to fast and small amplitude calcium transients and cENOs (green) are less frequent large peaks of synchrony associated to slower and larger calcium transients. Perfusion with anoxic/aglycemic ACSF increases the frequency of cENOs but reduces that of cGDPs. A2, Same histograms as in A1 but on an expanded time scale for the time period indicated in A1 by a horizontal bar. Representative calcium fluorescence traces from four imaged cells illustrating the amplitude and kinetics difference between cENO and cGDP-associated calcium events. A3, Comparison of all the digitally averaged calcium fluorescence events associated to cENOs (green) and cGDPs (blue) from the entire duration of the recording clearly indicates the amplitude difference between the two network patterns. Comparison of the scaled digital averages shows that the rise and decay time constants of cENOs-associated calcium transients are significantly slower than those associated to cGDPs (rise time: 1.0 vs 0.6 s; decay: 5.7 vs 1.6 s). B, Same as A, but comparing control and perfusion with the enzymatic glutamate scavenger (GPT 5 U/ml with pyruvate 2 mm) Perfusion with GPT (5 U/ml) selectively blocks the occurrence of cENOs (green) without significantly affecting cGDPs (blue).