Developmental origin dictates interneuron AMPA and NMDA receptor subunit composition and plasticity

Abstract

Disrupted excitatory synapse maturation in GABAergic interneurons may promote neuropsychiatric disorders such as schizophrenia. However, establishing developmental programs for nascent synapses in GABAergic cells is confounded by their sparsity, heterogeneity and late acquisition of subtype-defining characteristics. We investigated synaptic development in mouse interneurons targeting cells by lineage from medial ganglionic eminence (MGE) or caudal ganglionic eminence (CGE) progenitors. MGE-derived interneuron synapses were dominated by GluA2-lacking AMPA-type glutamate receptors (AMPARs), with little contribution from NMDA-type receptors (NMDARs) throughout development. In contrast, CGE-derived cell synapses had large NMDAR components and used GluA2-containing AMPARs. In neonates, both MGE- and CGE-derived interneurons expressed primarily GluN2B subunit-containing NMDARs, which most CGE-derived interneurons retained into adulthood. However, MGE-derived interneuron NMDARs underwent a GluN2B-to-GluN2A switch that could be triggered acutely with repetitive synaptic activity. Our findings establish ganglionic eminence-dependent rules for early synaptic integration programs of distinct interneuron cohorts, including parvalbumin- and cholecystokinin-expressing basket cells.

Figure 1. MGE- and CGE-dependent expression of synaptic glutamate receptors

(a,b) MGE- and CGE-derived cohorts of inhibitory interneurons were targeted using hippocampal slices derived from the Nkx2-1-cre:RCE GFP and Htr3a-GFP reporter mouse lines, respectively. Scale bars, 100 µm). (c,d) Top, representative total glutamate receptor (AMPAR and NMDAR)-mediated EPSCs evoked between −60 mV and +40 mV in 20-mV increments triggered by Schaffer collateral stimulation in MGE-derived (c) and CGE-derived (d) interneurons located in CA1 stratum radiatum. Bottom, I–V relationships of the AMPAR-mediated component measured at the time point of the EPSC peak obtained at −60 mV (indicated by dotted lines). Lines are the extrapolated linear fit of the data between −60 mV and 0 mV to reveal deviations from linearity at positive potentials. (e,f) AMPAR-mediated EPSCs before (black) and after (red) application of 2 µM philanthotoxin (PhTx) for representative recordings from MGE-derived (e) and CGE-derived (f) interneurons. EPSCs were evoked as pairs (with a 50-ms interstimulus interval), and PhTx did not alter the PPRs. (g,h) Representative EPSC traces from an MGE-derived (g) and CGE-derived (h) interneuron measured at −60 mV and +40 mV to extract the NMDAR-to-AMPAR amplitude ratio. (i–k) Summary plots of the AMPAR rectification index (RI) (i; MGE, 79 cells from 79 slices from 57 mice; CGE, 59 cells from 59 slices from 51 mice), philanthotoxin sensitivity (j; MGE, 8 cells from 8 slices from 5 mice; CGE, 8 cells from 8 slices from 6 mice) and NMDAR-to-AMPAR ratios (k; MGE, 85 cells from 85 slices from 57 mice; CGE, 88 cells from 88 slices from 51 mice) measured from all MGE- and CGE-derived interneurons regardless of their developmental age or anatomical identity. MGE-derived interneurons typically had AMPARs with significantly lower rectification indices (P = 2.73 × 10⁻²⁵, degrees of freedom (d.f.) = 136, t = −12.9), higher philanthotoxin sensitivity (P = 7.9 × 10⁻⁵, d.f. = 14, t = −5.5) and lower NMDA-to-AMPA ratios (P = 1.87 × 10⁻²⁰, d.f. = 171, t = −10.6) than their CGE-derived counterparts (P values were determined by unpaired t tests). Group data are presented as mean ± s.e.m., with results from individual experiments represented by open circles.

Figure 2. MGE and CGE specification of synaptic glutamate receptor expression is maintained through early development

(a–c) Representative reconstructions of a juvenile CGE-derived perisomatic-targeting basket cell (a), a wide-range dendrite-targeting cell (b) and a Schaffer collateral–associated cell (c). Somata and dendrites are shown in black, and axons are shown in red. Approximate boundaries of strata oriens, pyramidale and radiatum (s.o., s.p., and s.r., respectively) are drawn. Scale bars, 100 µm. (d–f) Representative EPSC profiles for CGE-derived interneurons from neonates (traces were from a CGE-derived basket cell). (d) Superimposed averaged AMPAR-mediated EPSCs (V_h = −70 mV) and isolated NMDAR-mediated EPSCs (+40 mV; recorded in the presence of 5 µM NBQX). (e) AMPAR EPSC I–V relationship (V_h = −60 mV to +40 mV in 20-mV increments and in the presence of 100 µM AP5). (f) Plot of the I–V relationship for the AMPAR EPSC peak amplitude shown in e. (g–i) As in d–f but for a juvenile CGE-derived basket cell. (j–l) Reconstruction of a juvenile MGE-derived perisomatic basket cell (j), a bistratified cell (k) and an ivy cell (l). Scale bars, 100 µm. (m–r) Representative data from MGE-derived basket cells in neonates (m–o) and juveniles (p–r) as in d–i. (s) Summary histogram for the NMDAR-to-AMPAR ratios for anatomically confirmed neonate and juvenile MGE-and CGE-derived interneurons (MGE: basket neonate, 8 cells from 8 slices from 6 mice; basket juvenile, 11 cells from 11 slices from 9 mice; bistratified neonate, 9 cells from 9 slices from 6 mice; bistratified juvenile, 10 cells from 10 slices from 8 mice; ivy neonate, 4 cells from 4 slices from 4 mice; ivy juvenile, 5 cells from 5 slices from 5 mice; CGE: basket neonate, 8 cells from 8 slices from 7 mice; basket juvenile, 7 cells from 7 slices from 7 mice; dendrite-targeting neonate, 9 cells from 9 slices from 8 mice; dendrite-targeting juvenile, 14 cells from 14 slices from 12 mice; Schaffer collateral–associated (SC assoc) neonate, 14 cells from 14 slices from 12 mice; Schaffer collateral–associated juvenile, 19 cells from 19 slices from 19 mice). (t) Summary data for the rectification index of AMPAR-mediated EPSCs (MGE: basket neonate, 7 cells from 7 slices from 6 mice; basket juvenile, 7 cells from 7 slices from 7 mice; bistratified neonate, 8 cells from 8 slices from 6 mice; bistratified juvenile, 6 cells from 6 slices from 4 mice; ivy neonate, 4 cells from 4 slices from 4 mice; ivy juvenile, 5 cells from 5 slices from 5 mice; CGE: basket neonate, 7 cells from 7 slices from 5 mice; basket juvenile, 7 cells from 7 slices from 7 mice; dendrite-targeting neonate, 8 cells from 8 slices from 5 mice; dendrite-targeting juvenile, 7 cells from 7 slices from 7 mice; Schaffer collateral–associated neonate, 8 cells from 8 slices from 4 mice; Schaffer collateral–associated juvenile, 6 cells from 6 slices from 5 mice). One-way analysis of variance (s, d.f. = 11, F = 11.37; t, d.f. = 11, F = 18.7) and post hoc Tukey analysis were conducted to test for significant differences (P < 0.05) across means. Horizontal lines above the data indicate no significant difference. Group data are presented as mean ± s.e.m., with results from individual experiments represented by open circles.

Figure 3. Developmental expression of synaptic GluN2 is cell-type specific

(a–d) Evoked Schaffer collateral NMDAR-mediated EPSCs in the absence or presence of ifenprodil (ifen; 5 µM) for representative MGE-derived basket cell (a,b) and a CGE-derived basket cell (c,d) from a neonate (a,c) and a juvenile (b,d). (e) Summary plot for the NMDAR EPSC decay kinetics weighted time constant (π_w) for both neonate and juvenile MGE- and CGE-derived interneurons (MGE, basket neonate, 8 cells from 8 slices from 6 mice; basket juvenile, 7 cells from 7 slices from 7 mice; bistratified neonate, 6 cells from 6 slices from 5 mice; bistratified juvenile, 7 cells from 7 slices from 5 mice; ivy neonate, 7 cells from 7 slices from 7 mice; ivy juvenile, 5 cells from 5 slices from 5 mice; CGE: basket neonate, 6 cells from 6 slices from 5 mice; basket juvenile, 7 cells from 7 slices from 7 mice; dendrite-targeting neonate, 8 cells from 8 slices from 5 mice; dendrite-targeting juvenile, 13 cells from 13 slices from 8 mice; Schaffer collateral–associated neonate, 12 cells from 12 slices from 8 mice; Schaffer collateral–associated juvenile, 13 cells from 13 slices from 8 mice). (f) Summary graph of the developmental regulation of ifenprodil sensitivity expressed as the ratio of the NMDAR EPSC peak amplitude measured in the presence of ifenprodil divided by the control NMDA EPSC peak amplitude (MGE, basket neonate, 6 cells from 6 slices from 5 mice; basket juvenile, 6 cells from 6 slices from 4 mice; bistratified neonate, 6 cells from 6 slices from 5 mice; bistratified juvenile, 6 cells from 6 slices from 5 mice; ivy neonate, 6 cells from 6 slices from 6 mice; ivy juvenile, 5 cells from 5 slices from 5 mice; CGE: basket neonate, 6 cells from 6 slices from 5 mice; basket juvenile, 6 cells from 6 slices from 6 mice; dendrite-targeting neonate, 6 cells from 6 slices from 5 mice; dendrite-targeting juvenile, 9 cells from 9 slices from 8 mice; Schaffer collateral–associated neonate, 6 cells from 6 slices from 5 mice; Schaffer collateral–associated juvenile, 8 cells from 8 slices from 8 mice). Unpaired t test was used for the comparisons between neonates and juveniles for each cell type (e, MGE: basket, *P = 0.002, d.f. = 13, t = 3.9; bistratified, *P = 0.0001, d.f. = 11, t = 5.9; ivy, *P = 0.0005, d.f. = 10, t = 4.9; CGE: basket, P = 0.92, d.f = 11, t = −0.107; dendrite targeting, P = 0.49, d.f. = 19, t = −0.7; Schaffer collateral associated, *P = 9.9 × 10⁻⁶, d.f. = 23, t = 5.6; f, MGE: basket, *P = 0.001, d.f. = 10, t = −4.34; bistratified, *P = 0.004, d.f. = 10, t = −3.65; ivy, *P = 0.006, d.f. = 9, t = −4.55; CGE: basket, P = 0.91, d.f = 10, t = 0.118; dendrite targeting, P = 0.99, d.f. = 13, t = −0.01; Schaffer collateral associated, *P = 0.005, d.f. = 12, t = −3.39). Group data are presented as mean ± s.e.m., with results from individual experiments represented by open circles.

Figure 4. Afferent specificity of glutamatergic transmission maturation in MGE-derived cells

(a) AMPAR EPSC rectification index for Schaffer collateral inputs (black) and ALV inputs (red) onto identified MGE-derived basket and bistratified interneurons (pooled data). The histogram bars represent the mean data set, and open circles represent tethered Schaffer collateral and ALV individual data points from the same recording (neonate, 7 cells from 7 slices from 4 mice; juvenile: 7 cells from 7 slices from 3 mice). The upper traces show representative AMPAR I–V relationships of Schaffer collateral and ALV inputs onto a single MGE-derived basket cell (neonate, P = 0.94, d.f. = 6, t = 0.08; juvenile, P = 0.44, d.f. = 6, t = 0.83; paired t test). Traces are on the same time scale, and the vertical bars represent 50 pA. (b) NMDAR-to-AMPAR ratio for Schaffer collateral and ALV inputs across four developmental time points. The upper traces show representative mean NMDAR-to-AMPAR currents evoked by Schaffer collateral and ALV inputs onto a single MGE-derived basket cell (P6–P9, 5 cells from 5 slices from 2 mice; P10–P13, 8 cells from 8 slices from 4 mice; P14–P16, 5 cells from 5 slices from 3 mice; P17–P21, 6 cells from 6 slices from 3 mice). Traces are on the same time scale, and the vertical bars represent 100 pA. (c,d) The developmental loss of ifenprodil sensitivity in NMDAR EPSCs at Schaffer collateral (black) and ALV (red) inputs in neonate and juvenile MGE-derived basket cells. Traces in c and d are on the same time scale, and the vertical bars represent 25 pA. (e) Summary plot of the developmental profile of NMDAR EPSC decay kinetics from Schaffer collateral and ALV inputs (*P = 0.0002, d.f. = 24, t = 4.4 for the Schaffer collateral input and *P = 0.0152, d.f. = 14, t = 2.77 for the ALV input for the comparison of the P17–P21 and P6–P9 data sets). (f) Summary plot for the developmental profiles of NMDA EPSC ifenprodil sensitivity from Schaffer collateral and ALV inputs (*P = 0.0002, d.f. = 26, t = 4.26 for the Schaffer input and *P = 0.0352, d.f. = 18, t = −2.28 for the ALV input for the comparison of the P17–P21 and P6–P9 data sets). (e, Schaffer collateral P6–P9, 19 cells from 19 slices from 12 mice; ALV P6–P9, 9 cells from 9 slices from 5 mice; Schaffer collateral and ALV P10–P13, 8 cells from 8 slices from 4 mice; Schaffer collateral and ALV P14–P16, 5 cells from 5 slices from 3 mice; Schaffer collateral P17–P21, 7 cells from 7 slices from 3 mice; ALV P17–P21, 7 cells from 7 slices from 4 mice; f, Schaffer collateral P6–P9, 19 cells from 19 slices from 12 mice; ALV P6–P9, 11 cells from 11 slices from 5 mice; Schaffer collateral and ALV P10–P13, 8 cells from 8 slices from 4 mice; Schaffer collateral and ALV P14–P16, 5 cells from 5 slices from 3 mice; Schaffer collateral P17–P21, 9 cells from 9 slices from 4 mice; ALV P17–P21, 9 cells from 9 slices from 4 mice.) Group data are presented as mean ± s.e.m., with results from individual experiments represented by open circles.

Figure 5. GluN2B-containing NMDARs participate in summation and spike timing of young MGE-derived interneurons

(a) Raster plot of a single representative cell-attached recording from a P6 MGE-derived interneuron demonstrating evoked spikes elicited by three stimuli given at a frequency of 40 Hz to the alvear path as indicated by the arrows (stim). The intersweep frequency was 0.1 Hz, and the numbers of sweeps were 30, 90 and 30 for the baseline, 5 µM ifenprodil and 5 µM ifenprodil plus 10 µM DNQX conditions, respectively. The conditions are delineated by shaded areas. (b) Raw traces of the same experiment as in a showing spiking of the neonate MGE-derived interneuron for 12 consecutive sweeps in each condition as indicated. Shaded red regions indicate the s.d. of the spike peak latency. Arrows at the bottom show the time of each stimulus (stim) in the 40-Hz train. (c) Effect of ifenprodil on the jitter of the spike in an MGE-derived neonate interneuron (measured as the s.d. of the spike peak latency) evoked by the first stimulation in the 40-Hz train. (d) As in c but for juvenile MGE-derived interneurons. (e) Pooled data for the effects of blocking GluN2B by ifenprodil on spike jitter in both neonate and juvenile MGE-derived interneurons (neonate, *P = 0.036, d.f. = 9, t = −2.28; juvenile, P = 0.18 (not significant (NS)), d.f. = 7, t = −1.17). (f) Effect of ifenprodil on the combined number of spikes after the second and third stimulation of the 40-Hz train in MGE-derived neonate interneurons. (g) As in f but for juvenile MGE-derived interneurons. (h) Pooled data for the effects of blocking GluN2B by ifenprodil on spike probability after the second and third spikes in the 40-Hz train in both neonate and juvenile MGE-derived interneurons (neonate, *P = 0.024, d.f. = 9, t = −2.57; juvenile, P = 0.36 (NS), d.f. = 7, t = −0.73). In c, d, f and g, each pair of data points joined by a line depicts an individual experiment. Solid and dotted lines indicate synaptic-evoked spikes in MGE-derived interneurons after alvear and Schaffer collateral stimulation, respectively. For the pooled graphs in e and h, data from alvear and Schaffer collateral stimulation were combined (neonate, n = 10 cells recorded in 10 slices from 5 mice; juvenile, n = 8 cells recorded in 8 slices from 2 mice). Group data are presented as mean ± s.e.m.

Figure 6. An activity-dependent change in GluN2 subunit expression in neonatal MGE-derived cells

(a) A representative control (−plasticity) isolated NMDAR-mediated EPSC in the absence or presence of ifenprodil from a neonate MGE-derived interneuron (V_h = +40 mV). (b) A representative NMDAR EPSC in the absence or presence of ifenprodil measured after repetitive synaptic activity (+plasticity = 2 Hz, 200 stimuli delivered at a holding potential of −70 mV). (c) Scaled NMDAR EPSCs from a and b before ifenprodil administration to emphasize the differences in decay kinetics in cells receiving the plasticity induction paradigm. (d–f) The same synaptic induction paradigm evoked changes in NMDAR EPSC decay kinetics and ifenprodil sensitivity in the presence of AP5 (100 µM) but did not evoke GluN2 subunit plasticity when delivered in the presence of intracellular BAPTA (10 mM) or bath-applied NBQX (5 µM). (g) Summary graph of NMDAR EPSC decay kinetics for basal control conditions (P4–P6, 9 cells from 9 slices from 6 mice; P8–P10, 22 cells from 22 slices from 8 mice; P14–P16, 17 cells from 17 slices from 5 mice) and after repetitive synaptic activity (plasticity, P4–P6, 13 cells from 13 slices, from 7 mice; P8–P10, 13 cells from 13 slices from 6 mice; P14–P16, 10 cells from 10 slices from 6 mice) at the specified developmental periods (*P = 3.5 × 10⁻⁵, d.f. = 20, t = 5.3 for P4–P6; *P = 0.002, d.f. = 33, t = 3.4 for P8–P10; P = 0.51, d.f. = 25, t = −0.66 for P14–P16). (h) Summary graph of NMDAR EPSC ifenprodil sensitivity for basal control conditions (P4–P6, 6 cells from 6 slices from 6 mice; P8–P10, 22 cells from 22 slices from 8 mice; P14–P16, 16 cells from 16 slices from 5 mice) and after repetitive synaptic activity (P4–P6, 11 cells from 11 slices from 7 mice; P8–P10, 15 cells from 15 slices from 6 mice; P14–P16, 9 cells from 9 slices from 6 mice) at the specified developmental periods (*P = 0.02, d.f. = 15, t = −2.6 for P4–P6; *P = 0.03, d.f. = 35, t = −2.2 for P8–P10; P = 0.48, d.f. = 123, t = 0.72 for P14–P16). (i) Summary graph of NMDAR EPSC decay kinetics for basal control conditions and after repetitive synaptic activity in the absence (control, 11 cells from 11 slices from 8 mice; plasticity, 20 cells from 20 slices from 13 mice; *P = 1.3 × 10⁻⁵, d.f. = 29, t = 5.23) or presence of the NMDAR antagonist AP5 (100 µM; 10 cells from 10 slices from 4 mice; *P = 0.002, d.f. = 19, t = −3.66), the mGluR5 antagonist MTEP (10 µM; 10 cells from 10 slices from 3 mice; *P = 5.1 × 10⁻⁴, d.f. = 19, t = 4.18), BAPTA (intracellular 10 mM; 8 cells from 8 slices from 3 mice; P = 0.37, d.f. = 17, t = −0.92) or NBQX (5 µM; 8 cells from 8 slices from 3 mice; P = 0.46, d.f. = 17, t = −0.75). (j) Summary graph of NMDAR EPSC ifenprodil sensitivity under the same conditions as in i in the absence of inhibitors (control, 9 cells from 9 slices from 8 mice; plasticity, 15 cells from 15 slices from 13 mice; *P = 3.2 × 10⁻⁴, d.f. = 22, t = −4.26), or in the presence of AP5 (100 µM; 7 cells from 7 slices from 4 mice; *P = 0.005, d.f. = 14, t = 3.31), MTEP (10 µM; 9 cells from 9 slices from 3 mice; *P = 0.001, d.f. = 16, t = −3.97), BAPTA (intracellular 10 mM; 6 cells from 6 slices from 3 mice; P = 0.94, d.f. = 13, t = 0.072) or NBQX (5 µM; 8 cells from 8 slices from 3 mice; P = 0.57, d.f. = 15, t = −0.58). Traces in a,b and d–f are all on the same time scale, and the vertical bar in each panel represents 20 pA, as illustrated in a and d. Unpaired t test was used for the comparisons of the various conditions to the basal control NMDA EPSCs. Group data are presented as mean ± s.e.m., with the results from individual experiments represented by open circles.

Figure 7. Principal-cell bursting activity in CA3 evokes a rapid GluN2 subunit switch in MGE-derived neonatal basket and bistratified cells

(a) A confocal image (×20) of a biocytin-filled CA3 pyramidal cell and a CA1 basket cell to illustrate the typical experimental recording configuration. (The confocal image was converted to grayscale and the color was inverted; scale bar, 60 µm.) (b) Whole-cell recording from a CA3 pyramidal cell to establish repeated robust bursting activity in the presence of elevated amounts of extracellular potassium (5 mM). After 5–8 min of monitoring CA3 bursting activity, the whole-cell configuration is established in an MGE-derived interneuron. Scale bar, 20 mV/3 s. (c,d) Representative NMDA EPSCs and their ifenprodil sensitivity for neonate MGE-derived basket cells in control slices (c, black) and slices treated with a high-potassium (K⁺) solution to promote CA3 pyramidal neuron burst firing (d, red). Traces in c and d are on the same time scale, and the vertical bars represent 50 pA, as indicated in c. (e,f) Summary plots for MGE-derived basket and bistratified cell NMDA EPSC decay kinetics (e; control, 19 cells from 19 slices from 14 mice; K⁺, 17 cells from 17 slices from 8 mice; *P = 5.3 × 10⁻⁶, d.f. = 34, t = 5.39) and ifenprodil sensitivity (f; control, 19 cells from 19 slices from 14 mice; K⁺, 14 cells from 14 slices from 8 mice; *P = 7.8 × 10⁻⁴, d.f. = 31, t = −3.73) recorded in control and K⁺-treated slices. Group data are presented as mean ± s.e.m., with results from individual experiments represented by open circles.