Satellite NG2 Progenitor Cells Share Common Glutamatergic Inputs with Associated Interneurons in the Mouse Dentate Gyrus

A subpopulation of hilar sNG2⁺ cells receives a glutamatergic input and exhibits distinct membrane properties. A, B, Representative responses in voltage-clamp (A) and current-clamp (B) mode of a sNG2⁺EGFP⁺ cell to voltage steps (A) (−100 to −10 mV; step size, 10 mV) and current injection (B) (−100–350 pA; step size, 50 pA). C, Single-plane confocal merged image (left) showing an example of a bright EGFP⁺ cell expressing the proteoglycan NG2 (bottom right) and filled with biocytin (top right) during patch-clamp recording. Scale bar, 20 μm. D, Representative examples of current-clamp (top traces) and voltage-clamp (V_h = −80 mV) (bottom traces) recordings from a sNG2⁺EGFP⁺ cell exhibiting spontaneous synaptic glutamatergic activity. E, Average traces of sEPSPs (top trace) and sEPSCs (bottom trace) recorded from the cell shown in D. Average sEPSPs and sEPSCs exhibit a fast-rising phase (0.67 and 0.27 ms, respectively) and a decay time (2.79 and 0.79 ms, respectively) typical of synaptic events mediated by AMPA/kainate receptors. F, Average of sEPSCs recorded under control conditions (black trace) and in the presence of 100 μm CTZ (gray trace). Sensitivity to CTZ indicates that these sEPSCs are mediated by AMPA receptors (V_h = −80 mV). G, Plots showing the individual membrane resistance (left plot) and capacitance (right plot) values of NG2⁺ cells recorded between P9 and P13 in which no sEPSCs were observed (black circles, silent cells) and NG2⁺ cells exhibiting sEPSCs (white circles, active cells). Average values (red circles) and SDs (red bars) are indicated for each cell population. All recordings were performed in the presence of 100 μm picrotoxin using a K-gluconate internal solution. ***p <0.001.

Hilar sNG2⁺ cells are progressively integrated into the network during postnatal development and their inputs exhibit functional maturation. A, Examples of 15 individual EPSCs (gray traces) and the corresponding averaged trace (red traces) recorded in three representative sNG2⁺ cells at P4 (left), P11 (middle), and P20 (right). B, Scatter plot of the sEPSC frequency (y-axis) in sNG2⁺ cells as a function of the age of the animal (x-axis) showing a positive correlation (linear regression; Pearson's product-moment correlation, r = +0.44; p < 0.01). C, Scatter plot of the sEPSC average amplitude (y-axis) in sNG2⁺ cells as a function of the age of the animal (x-axis) showing a positive correlation (linear regression; Pearson's product-moment correlation, r = 0.55; p < 0.01). D, Scatter plot of the sEPSC 20–80% rise time (y-axis) in sNG2⁺ cells as a function of the age of the animal (x-axis) showing a negative correlation (linear regression; Pearson's product-moment correlation, r = −0.43; p < 0.01). E, Scatter plot of the sEPSC decay time constant (y-axis) in sNG2⁺ cells as a function of the age of the animal (x-axis) showing a negative correlation (linear regression; Pearson's product-moment correlation, r = −0.65; p < 0.001). All recordings were performed in the presence of 100 μm CTZ using a K-gluconate internal solution.

sNG2⁺ cells and EGFP⁺ hilar interneurons receive glutamatergic inputs both from granule cells and CA3 pyramidal neurons. A, B, Evoked responses to granule cell layer stimulation in a hilar EGFP⁺ interneuron (A) (V_h = −60 mV) and its sNG2⁺ cell (B) (V_h = −80 mV) under control conditions (black trace) and in the presence of 1 μm DCG-IV (gray trace). Traces are averaged from 25 consecutive responses recorded from the same cell in the voltage-clamp mode (stimulus: 100 μs, 200 μA). The significant reduction of the evoked response after application of DCG-IV indicates that both cell types are contacted by mossy fiber synapses. Responses were evoked in an isolated dentate gyrus slice preparation. C, D, Facilitation of evoked responses by paired minimal stimulation of the granule cell layer (interstimulus interval, 50 ms) in a hilar EGFP⁺ interneuron (C) (V_h = −60 mV) and its sNG2⁺ cell (D) (V_h = −80 mV) (stimulus: 100 μs, 10 and 50 μA). The traces are averaged from 25 consecutive responses recorded from the same cell in the voltage-clamp mode. E, F, Superposition of consecutive responses evoked by CA3 layer stimulation in a hilar EGFP⁺ interneuron (E) (V_h = −60 mV) and its sNG2⁺ cell (F) (V_h = −80 mV) (stimulus: 100 μs, 200 μA). The right traces show examples of single responses evoked by CA3 layer stimulation in both cell types. All recordings were performed in the presence of 100 μm CTZ and 100 μm picrotoxin using a K-gluconate internal solution. stim, Stimulus; Rec, recording.

sNG2⁺ cells are not directly contacted by associated hilar interneurons. A, Example of spontaneous GABAergic IPSCs recorded in a sNG2⁺ cell (top traces) and blocked by 1 μm gabazine (bottom traces). GABAergic activity was recorded using a Cs-methanesulfonate solution (V_h = 0 mV; estimated E_Cl = −80 mV). B–D, Confocal images showing a hilar interneuron (red) and its sNG2⁺ cell (green) filled with biocytin (red in B and gray in C) and tetramethylrhodamine dextran (green in B and gray in D), respectively. Scale bar, 20 μm. E, Example of a hilar interneuron (top trace) recorded in current clamp and its sNG2⁺ cell (bottom trace) recorded in voltage clamp. The injection of a current step in the interneuron (200 pA; 600 ms) evoked a volley of action potentials, but did not evoke any response in the sNG2⁺ cell. The hilar neuron was recorded in K⁺-gluconate internal solution, whereas the sNG2⁺ cell was recorded in Cs-methanesulfonate solution and voltage clamped at 0 mV (E_Cl = −80 mV). Our ability to detect GABAergic activity is demonstrated by the occurrence of spontaneous GABAergic IPSCs in some sNG2⁺ cells recorded under the same conditions.

CA3 spontaneous bursts are synchronized between hilar interneurons and sNG2⁺ cells. A, Example of synchronized bursts observed in a hilar interneuron (V_h = −60 mV) (top trace) and its sNG2⁺ cell (V_h = −80 mV) (bottom trace) recorded in a whole hippocampal slice preparation in the presence of 100 μm picrotoxin. The black bar indicates the interval of the trace in A that is magnified in B. B, Example of synchronized recurrent EPSCs observed within each burst in a hilar interneuron (top trace) and its sNG2⁺ cell (bottom trace). The dotted lines indicate recurrent EPSCs, which are usually observed after the initial discharge of EPSCs indicated by a solid line. C, Cross-correlation histogram shows the high degree of temporal correlation of recurrent EPSCs occurring in the interneuron and sNG2⁺ cell shown in A and B. A total of 57 recurrent events from 14 synchronized bursts were analyzed. Initial discharges were excluded from the analysis. Bin width, 5 ms. D, Scatter plot of the amplitudes of the recurrent EPSCs in the interneuron recorded in A and B (x-axis) against the amplitudes of the corresponding EPSCs in the associated sNG2⁺ cell (y-axis). EPSCs amplitudes in the interneuron and in the sNG2⁺ cell exhibit a positive correlation (gray linear regression; Pearson's product-moment correlation, r = 0.71; p < 0.0001; 57 synchronized events analyzed) and fit a linear relationship obtained by linear regression. As in C, a total of 57 recurrent events from 14 synchronized bursts were analyzed. Initial discharges were excluded from the analysis.

sNG2⁺ cells and hilar interneurons are contacted by the same neuron. A, Example of spontaneous activity recorded in a hilar interneuron (V_h = −60 mV) (top trace) and its sNG2⁺ cell (V_h = −80 mV) (bottom trace) using a specific cesium-methanesulfonate internal solution to isolate EPSCs while preserving GABAergic activity (E_Cl = −80 mV; E_cation = 0 mV). The application of 20 μm carbachol to the hippocampal slice strongly increases the spontaneous EPSC activity in both cells. B, C, Cumulative plots of the amplitudes of EPSCs observed in the absence (black) and presence (gray) of 20 μm carbachol. EPSC amplitude distributions are not significantly affected by the application of carbachol, either in the interneuron (B) or in its sNG2⁺ cell (C). D, Example of synchronized spontaneous activity recorded in the hilar interneuron (top trace) and sNG2⁺ cell (bottom trace) shown in A. The bottom traces show two examples of synchronized EPSCs. E, Cross-correlation histogram shows that a significant fraction of EPSCs are synchronized between interneurons and their sNG2⁺ cells. The histograms are from data pooled from eight sNG2⁺ cell–interneuron pairs. The gray box highlights events that are in a time window of ±1 ms and above the noise level (corresponding to random correlations). F, Cross-correlation histogram shows that only a few EPSCs are synchronized when interneurons and sNG2⁺ cells are separated by >200 μm. The histograms show data pooled from seven sNG2⁺ cell–interneuron pairs. The gray box highlights events that are in a time window of ±1 ms and above the noise level (corresponding to random correlations). G, Plot of the percentage of synchronized events above the noise level in each recorded cell pair showing a significant difference (p < 0.01, Mann–Whitney U test) between pairs of anatomically associated cells (11.6 ± 4.2%; n = 7) versus pairs of distant cells (3.8 ± 1.8%; n = 6).