An Optogenetic Approach for Investigation of Excitatory and Inhibitory Network GABA Actions in Mice Expressing Channelrhodopsin-2 in GABAergic Neurons

Abstract

To investigate excitatory and inhibitory GABA actions in cortical neuronal networks, we present a novel optogenetic approach using a mouse knock-in line with conditional expression of channelrhodopsin-2 (ChR2) in GABAergic interneurons. During whole-cell recordings from hippocampal and neocortical slices from postnatal day (P) 2-P15 mice, photostimulation caused depolarization and excitation of interneurons and evoked barrages of postsynaptic GABAergic currents. Excitatory/inhibitory GABA actions on pyramidal cells were assessed by monitoring the alteration in the frequency of EPSCs during photostimulation of interneurons. We found that in slices from P2-P8 mice, photostimulation evoked an increase in EPSC frequency, whereas in P9-P15 mice the response switched to a reduction in EPSC frequency, indicating a developmental excitatory-to-inhibitory switch in GABA actions on glutamatergic neurons. Using a similar approach in urethane-anesthetized animals in vivo, we found that photostimulation of interneurons reduces EPSC frequency at ages P3-P9. Thus, expression of ChR2 in GABAergic interneurons of mice enables selective photostimulation of interneurons during the early postnatal period, and these mice display a developmental excitatory-to-inhibitory switch in GABA action in cortical slices in vitro, but so far show mainly inhibitory GABA actions on spontaneous EPSCs in the immature hippocampus and neocortex in vivo

Significance statement: We report a novel optogenetic approach for investigating excitatory and inhibitory GABA actions in mice with conditional expression of channelrhodopsin-2 in GABAergic interneurons. This approach shows a developmental excitatory-to-inhibitory switch in the actions of GABA on glutamatergic neurons in neocortical and hippocampal slices from neonatal mouse pups in vitro, but also reveals inhibitory GABA actions in the neonatal mouse neocortex and hippocampus in vivo.

Keywords: ChR2; GABA; cortex; hippocampus; inhibition; interneurons; optogenetics; patch clamp.

Figure 1.

EGFP-ChR2 expression in interneurons of neonatal mice. A, Gad2::CreER-R26ChR2-EGFP mice were treated with tamoxifen during in utero development [embryonic days (E) 10–E14] and the first days (P1–P3) after birth to induce EGFP-ChR2 expression in a Cre-dependent manner by P2–P15. B, Left, Distribution of EGFP-ChR2-expressing GAD neurons on coronal sections of the P6 mouse forebrain. SS Cx, Somatosensory cortex; H, hippocampus; St, striatum. Scale bar, 1 mm. Right, Enlargements of framed areas on the left panel (1–4 from the top). EGFP labeling (arrows) shows the soma and dendrites of numerous neurons in somatosensory cortex (1), hilus (2), stratum pyramidale of CA3 (3), and stratum oriens of CA1 (4) regions. Scale bars, 50 μm.

Figure 2.

Photoactivation of EGFP-ChR2-expressing interneurons in mouse cortical slices. A, Example traces of cell-attached recording (at −60 mV) of a P11 neocortical interneuron firing in response to the light pulse (blue bar above the traces) illuminating slice area around the tip of the patch pipette (as illustrated in Fig. 3A). B, The current-clamp recording of an interneuron spiking during the light flash. A, B, Traces, AP frequency plot averaged over data obtained during cell-attached (A, n = 6) and current-clamp (B, n = 6) recordings from the given interneuron and hippocampal stratum oriens interneurons, individually illuminated by a 10–20 μm light spot using a two-photon pulsed laser-scanning system coupled to a microscope. Data were fitted with basis spline function. The shaded region around the curve shows SE bands. C, Firing pattern of the same neuron as in A and B during a depolarizing current step.

Figure 3.

GABA(A)-mediated postsynaptic response to photoactivation of local interneurons in the neonatal mouse cortex in vitro and in vivo. A, Schematic drawing of the experimental setup demonstrates patch-clamp recording from the principal neuron during light stimulation of EGFP-ChR2-expressing interneurons located close to the light source. B, Averaged plot of GABA-PSC frequency change in response to local interneurons photoactivation by the light pulse (highlighted area) in P2–P15 mouse cortical slices (on the top, n = 42) and in the P3–P9 mouse cortex in vivo (on the bottom, n = 13) fitted with basis spline function. The shaded region around the curve shows SE bands. C, D, The whole-cell voltage-clamp recording of GABA-PSCs with a low-chloride pipette solution at the reversal potential of Glu-EPSCs in a hippocampal slice (C) and the intact brain (D) of P4 mice. Five responses evoked by the light flash are shown with a blue bar above the traces indicating the onset and the length of the light pulse. Red bars indicate individual GABA-PSCs. Below, Corresponding GABA-PSC frequency histograms based on the results of 30–100 photostimulation trials (bin size, 10 ms). E, F, The time course of light-evoked GABA-mediated responses at different postnatal ages in vitro (E) and in vivo (F). Each pair of symbols shows the onset and the end of GABA response to the light flash (highlighted area) recorded from individual hippocampal (squares) or neocortical (triangles) neurons. Animal age is indicated on the right. G, H, Pooled data on GABA-PSC frequency during light-evoked responses normalized to the baseline frequency in hippocampal (squares) and neocortical (triangles) neurons at different postnatal ages in vitro (G) and in vivo (H). Closed circles show mean values with SE. Baseline GABA-PSC frequency was calculated in the 0.5 s preceding the light pulse. In case of the absence of GABA-PSCs detected during this time window, the number of GABA-PSCs for baseline frequency calculation was considered by convention to be equal to 1 (marked with a stroke near the symbol). GABA-PSC frequency during the response was calculated within an individual time window for each neuron (displayed in E and F).

Figure 4.

Inhibition of light-evoked GABA-mediated response by the GABA(A) receptor antagonist bicuculline. A, Representative traces of whole-cell voltage-clamp recording of the GABA(A)-mediated response to photostimulation of interneurons in a P6 neocortical slice in control (upper traces) and after bath application of 10 μm bicuculline (lower traces). Corresponding histograms of GABA-PSC frequency based on 100 stimulation sweeps are shown under the traces. B, The statistical plot of GABA-PSC frequency change in response to the light flash (highlighted in blue) in control and in the presence of bicuculline. Data were averaged over six recorded cells and fitted with basis spline function. Shaded area around the curve shows SE bands.

Figure 5.

Photoactivation of interneurons induces glutamate release from principal cells in neonatal but not in adolescent mice in vitro. A, B, Example traces of Glu-EPSCs recorded during photostimulation at the reversal potential of GABA-PSCs (−75 mV) in P6 hippocampal (A) and P15 neocortical (B) slices. Red bars indicate individual Glu-EPSCs. Below, Corresponding histograms of Glu-EPSC frequency based on results of 100–110 photostimulation trials. C, The time course of glutamatergic response evoked by the light pulse (highlighted area) at different postnatal ages (blue and red symbols correspond to a decrease and an increase in Glu-EPSC frequency during responses, respectively). Each pair of symbols shows the onset and the end of response recorded from individual hippocampal (squares) or neocortical (triangles) neurons. Animal age is indicated on the right. D, Pooled data on the change in Glu-EPSC frequency during the light pulse in relation to the baseline frequency in hippocampal (squares) and neocortical (triangles) neurons of P2–P15 mice. Color code of symbols is same as in C. The Glu-EPSC frequency during response was calculated within a time window unique to each neuron (displayed in C). Gray symbols correspond to the neurons where no significant change in Glu-EPSC frequency was detected during illumination. Response frequency in these neurons was calculated within the mean-response time window. Closed circles show mean values with SE. Note that photostimulation of interneurons elicits an increase in Glu-EPSC frequency during the first postnatal week and a decrease in Glu-EPSC frequency after P8, indicating a developmental excitatory-to-inhibitory switch in the action of GABA photoreleased from interneurons on glutamatergic neurons. The cumulative distribution of Glu-EPSC amplitudes during baseline activity (black trace) and throughout the photostimulation-evoked response (blue trace) is shown in the inset graph. The shaded area around the curves corresponds to SE bands.

Figure 6.

Photoactivation of interneurons suppresses glutamate release from principal cells in neonatal and adolescent mice in vivo. A, B, Example traces of Glu-EPSCs recorded during photostimulation at the reversal potential of GABA-PSCs (−75 mV) in P4 hippocampal (A) and P15 neocortical (B) neurons in vivo. Red bars indicate individual Glu-EPSCs. Below, Corresponding histograms of Glu-EPSC frequency based on 100–230 stimulation sweeps. C, The time course of glutamatergic responses evoked by the light pulse (highlighted area) at different postnatal ages. Each pair of symbols shows the onset and the end of response recorded from individual hippocampal (squares) or neocortical (triangles) neurons. The animal age is indicated on the right. D, Pooled data on the change in Glu-EPSC frequency during the light pulse in relation to the baseline frequency in hippocampal (squares) and neocortical (triangles) neurons of P3–P9 mice. Glu-EPSC frequency during the response was calculated within an individual time window for each neuron (displayed in C). Gray symbols correspond to the neurons where no significant change in Glu-EPSC frequency was detected during illumination. The response frequency in these neurons was calculated within the mean-response time window. Closed circles show mean values with SE. The cumulative distribution of Glu-EPSC amplitudes during baseline activity (black trace) and throughout the photostimulation-evoked response (blue trace) is shown in the inset graph. The shaded area around the curves corresponds to SE bands.

Figure 7.

Characteristics of the glutamatergic response evoked by photostimulation in cortical neurons of Gad2::CreER-R26ChR2-EGFP neonatal mice in vitro and in vivo. A, The time course of the Glu-EPSC frequency change in response to photoactivation of local interneurons by the light flash (highlighted in blue) recorded during the first postnatal week from cortical slices (P2–P8, n = 18 cells, upper plot) and the intact cortex in vivo (P3–P8, n = 12 cells, lower plot). Data were averaged for the neurons where photostimulation evoked a significant change in Glu-EPSC frequency (displayed in Figs. 5C, 6C; neurons shown by gray symbols in Figs. 5D and 6D are not included) and fitted with basis spline function. The shaded area around the curve indicates SE bands. B, Statistical plot of Glu-EPSC frequency observed during baseline activity and throughout the light-evoked response in different age groups in vitro and in vivo. Each pair of connected circles corresponds to an individual neuron. The medians of boxplots are shown by black lines; mean values are shown by black circles. Glu-EPSC frequency data in B include all cells recorded in vitro (n = 25 cells at P2–P8; n = 7 cells at P9–P15) and in vivo (n = 21 cells) and displayed in Figures 5D and 6D, respectively. *p < 0.05; ***p < 0.001.

Figure 8.

Effects of various experimental conditions on the excitatory GABA actions in vitro. A–C, Example traces of responses evoked by local GABA puff application (arrow) under control conditions, in the presence of bath-applied urethane (10 mm), and during coapplication of urethane and 10 μm NKCC1 antagonist bumetanide (A), in the presence of the mACSF (B), and after heating the ACSF in the recording chamber to 36°C (C). Averaged plots of AP frequency change in response to GABA puff application in P2–P6 wild-type mouse cortical slices fitted with basis spline function are shown below the traces. The shaded area around each curve shows SE bands. D–F, Pooled data on AP frequency during responses to puff application normalized to the baseline frequency obtained from P2–P6 wild-type mouse cortical slices under different experimental conditions. The control recordings were made in bath-applied ACSF from slices prepared using ACSF as slicing medium (A, D), in bath-applied ACSF from slices prepared using the choline-based solution as slicing medium (B, E), and in bath-applied mACSF from slices prepared using the choline-based solution as slicing medium (C, F). ***p < 0.001; n.s., p ≥ 0.05.