Research ArticleNEUROPHYSIOLOGY

GABAergic interneurons excite neonatal hippocampus in vivo

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Science Advances  12 Jun 2020:
Vol. 6, no. 24, eaba1430
DOI: 10.1126/sciadv.aba1430
  • Fig. 1 GABAergic interneurons are excitatory in 3-day-old hippocampus in vivo.

    (A) Experimental design. (B) Colabeling of AAV-dF-KORD-IRES-mCitrine expressed in GAD2-Cre mouse with anti-GABA. Scale bars, 100 and 20 μm. (C) Percentage of mCitrine(KORD)–expressing neurons coexpressing GABA and percentage of GABA-expressing neurons coexpressing mCitrine(KORD) (mean ± 95% CI, n = 3, 3). (D) Change in membrane potential in hippocampal slices at P3 and P11. SalB hyperpolarized KORD–expressing neurons and CNO depolarized hM3Dq–expressing neurons at both ages (n = 6, 6 , 5, and 7; ANOVA, P < 0.001). (E) Representative localization of electrode and viral expression in P3 animal. (F) Representative recording for P3 reduction of GABAergic neuron excitability. MUA of spontaneous activity in CA1 hippocampus, along with associated stratum radiatum LFP and thoracic movement detection and electromyography. Activity is dominated by early sharp waves (eSPW) whose spike density is reduced following subcutaneous SalB (KORD agonist) injection. (G) Quantification of KORD-induced suppression of GABAergic neuron excitability and control conditions. [Pyramidal cell layer firing rate (n = 10, 6, and 8; ANOVA, P < 0.001), eSPW LFP amplitude (n = 10, 6, and 8; P = 0.002), and normalized (to mean of 1- to 100-Hz baseline) spectral power for stratum radiatum LFP, n = 10]. (H) Quantification of hM3Dq-induced increase in GABAergic excitability (n = 7, 6, and 6; P = 0.005; P = 0.33; n = 7). All values and statistics are listed in table S1.

  • Fig. 2 Hippocampal GABAergic neurons are inhibitory by P7.

    (A) Experimental design. (B) Representative recording for GABAergic neuron suppression in P7 hippocampus. (C and D) Quantification of suppression (C) and enhancement (D) of GABAergic neuron excitability at P7 [(C): CA1 firing rate: KORD-SalB: 1.14 ± 0.62 (n = 5), KORD-saline: 0.04 ± 0.35 (n = 4), GFP-SalB: −0.04 ± 0.43 (n = 4), P = 0.001; LFP spectra: P < 0.05 at 6.9 to 14.7 Hz (n = 5); (D): CA1 firing rate: hM3Dq-CNO: −2.37 ± 2.02 (n = 5), hM3Dq-saline: 0.17 ± 0.21 (n = 4), GFP-CNO: 0.13 ± 0.43 (n = 5), P = 0.003; LFP spectra: P < 0.05 at 7.7 to 93.3 Hz (n = 5)]. (E and F) Similar quantification at P11 [(E): CA1 firing rate: KORD-SalB: 1.11 ± 0.57 (n = 6), KORD-saline: 0.12 ± 0.22 (n = 8), GFP-SalB: −0.03 ± 0.28 (n = 4), P < 0.001; LFP spectra: not significant (n.s.) (n = 6); (F): CA1 firing rate, hM3Dq-CNO: −2.09 ± 1.29 (n = 7), hM3Dq-saline: 0.18 ± 0.22 (n = 5), GFP-CNO: −0.07 ± 0.37 (n = 5), P = 0.001; LFP spectra, P < 0.05 at 2.3 to 93.3 Hz (n = 7)].

  • Fig. 3 GABAergic neurons in visual cortex have a net inhibitory action as early as P3.

    (A) Experimental design. (B and C) Colocalization of KORD expression with GABA [93.7 ± 6.7% (n = 3)]. Scale bars, 50 and 10 μm. (D) Change in membrane potential of GAD2+ neurons by activation of KORD and hM3Dq at P3 and P11 in visual cortical slices [P3 KORD: −6.62 ± 1.88 (n = 5), P11 KORD: −8.03 ± 2.14 (n = 7), P3 hM3Dq: 7.18 ± 2.23 (n = 6), P11 hM3Dq: 7.23 ± 2.06 (n = 8), P < 0.001]. (E) Representative recording of visual cortex at P3 and the effect of GABAergic neuron suppression. LFP spectrogram is from the presumptive input layer. (F) Quantification of change in superficial layer firing rate [KORD-SalB: 1.25 ± 0.5 (n = 6), KORD-saline: 0.22 ± 0.4 (n = 4), GFP-SalB: −0.03 ± 0.35 (n = 5), P = 0.001] and LFP spectral power [n.s. (n = 6)] following suppression of GABAergic neuron excitability by KORD activation. (G) Firing rate change at P3, P7, and P11 to KORD activation [P3: 1.25 ± 0.5 (n = 6), P7: 1.11 ± 0.71 (n = 6), P11: 1.17 ± 0.43 (n = 5); P = 0.89]. (H and I) As (F) and (G) but for GABAergic neuron enhancement by hM3Dq activation [(H): VC firing rate: hM3Dq-CNO: −1.51 ± 1.18 (n = 6), hM3Dq-saline: 0.11 ± 0.46 (n = 4), GFP-CNO: 0.02 ± 0.32 (n = 5), P = 0.007; LFP spectra: n.s. (n = 6); (I): P3: −1.51 ± 1.18 (n = 6), P7: −1.43 ± 1.02 (n = 5), P11: −1.94 ± 1.35 (n = 6), P = 0.71].

  • Fig. 4 Changing role of anion conductance likely underlies the regional and age heterogeneity of GABA function.

    (A to C) stGtACR2, an anion-conducting channelrhodopsin with a soma-targeting motif, is virally expressed in non-GABAergic neurons in hippocampus or cortex of EMX1-Cre mice [(C): hippocampus: 8.5 ± 8.5% (n = 3); visual cortex: 10.7 ± 7.6% (n = 3)]. Scale bars, 50 and 10 μm. (D) Photostimulation of stGtACR2 in hippocampal glutamatergic neurons (470 nm LED, 1 s) increased CA1 firing at P3 but decreases it at P7 [P3 stGtACR2: 0.74 ± 0.38 (n = 5), P3 GFP: −0.07 ± −0.35 (n = 5), P = 0.001; P7 stGtACR2: −3.51 ± −4.25 (n = 7), P7 GFP: 0.04 ± −0.01 (n = 5), P = 0.001]. (E) In visual cortex, photostimulation of stGtACR2 in glutamatergic neurons decreased MUA at both P3 and P7 [P3 stGtACR2: −2.65 ± −4.68 (n = 4), P3 GFP: 0.08 ± −0.29 (n = 5), P = 0.002; P7 stGtACR2: −2.43 ± −3.27 (n = 4), P7 GFP: 0.18 ± 0.04 (n = 3), P = 0.001].

Supplementary Materials

  • Supplementary Materials

    GABAergic interneurons excite neonatal hippocampus in vivo

    Yasunobu Murata and Matthew T. Colonnese

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