Enhancement of parvalbumin interneuron-mediated neurotransmission in the retrosplenial cortex of adolescent mice following third trimester-equivalent ethanol exposure

Abstract

Prenatal ethanol exposure causes a variety of cognitive deficits that have a persistent impact on quality of life, some of which may be explained by ethanol-induced alterations in interneuron function. Studies from several laboratories, including our own, have demonstrated that a single binge-like ethanol exposure during the equivalent to the third trimester of human pregnancy leads to acute apoptosis and long-term loss of interneurons in the rodent retrosplenial cortex (RSC). The RSC is interconnected with the hippocampus, thalamus, and other neocortical regions and plays distinct roles in visuospatial processing and storage, as well as retrieval of hippocampal-dependent episodic memories. Here we used slice electrophysiology to characterize the acute effects of ethanol on GABAergic neurotransmission in the RSC of neonatal mice, as well as the long-term effects of neonatal ethanol exposure on parvalbumin-interneuron mediated neurotransmission in adolescent mice. Mice were exposed to ethanol using vapor inhalation chambers. In postnatal day (P) 7 mouse pups, ethanol unexpectedly failed to potentiate GABA_A receptor-mediated synaptic transmission. Binge-like ethanol exposure of P7 mice expressing channel rhodopsin in parvalbumin-positive interneurons enhanced the peak amplitudes, asynchronous activity and total charge, while decreasing the rise-times of optically-evoked GABA_A receptor-mediated inhibitory postsynaptic currents in adolescent animals. These effects could partially explain the learning and memory deficits that have been documented in adolescent and young adult mice exposed to ethanol during the third trimester-equivalent developmental period.

Figure 1

Overview of electrode placement and timeline for the two experiments performed. (a) Electrode placement and timeline for Experiment 1. The acute effects of ethanol and flunitrazepam on GABA_A receptor mediated PSCs were measured in layer V pyramidal neurons and INs of the RSC. To electrically evoke GABA_A-PSCs, a bipolar stimulating electrode was placed on the border of layers I/II. (b) Electrode placement, laser stimulation location, and timeline for Experiment 2. The long-term effects of P7 ethanol vapor chamber exposure were measured at P40–60. PV-INs expressing channel rhodopsin-2/tdTomato were stimulated with a 473 nm laser using a 40 × objective lens in layer V of the RSC. Optically evoked IPSCs were recorded in layer V pyramidal neurons. Atlas images were adapted from the Allen Brain Coronal Atlas (©2015 Allen Institute for Brain Science. Allen Brain Atlas API. Available from: htttp://brain-map.org/api/index.html). Image credit: Allen Institute.

Figure 2

Effect of acute ethanol application on evoked GABA_A receptor-mediated postsynaptic current (GABA_A-ePSC) amplitude and decay in layer V pyramidal neurons and INs. (a) Average ePSC traces from layer V pyramidal neurons during the baseline (black trace) and acute 90 mM ethanol application (blue trace) phases. Scale bars 40 ms, 50 pA. (b) Average ePSC traces from layer V INs during the baseline and acute 90 mM ethanol application phases. Scale bars 40 ms, 20 pA. (c) Normalized GABA_A-ePSC amplitudes for both pyramidal neurons (black circles) and INs (red squares) in layer V during the baseline, 90 mM ethanol application, washout, and gabazine (25 µM) application phases. (d) Normalized decay constants (tau) for both pyramidal neurons and INs during the baseline, 90 mM ethanol application and washout phases. Data are presented as mean ± SEM.

Figure 3

Effect of acute flunitrazepam application on evoked GABA_A receptor-mediated postsynaptic current (GABA_A-ePSC) amplitude and decay in layer V pyramidal neurons and INs. (a) Average ePSC traces from layer V pyramidal neurons during the baseline (black trace) and acute 1 µM flunitrazepam application (green trace) phases. Scale bars 40 ms, 50 pA. (b) Average GABA_A-ePSC traces from layer V INs during the baseline and acute 1 µM flunitrazepam application phases. Scale bars 40 ms, 20 pA. (c) Normalized GABA_A-ePSC amplitudes for both pyramidal neurons (black circles) and INs (red squares) in layer V during the baseline, 1 µM flunitrazepam, washout, and gabazine application phases. (d) Normalized decay constants (tau) for both pyramidal neurons and INs during the baseline, 1 µM flunitrazepam, and washout phases. Data are presented as mean ± SEM.

Figure 4

Effect of acute ethanol application on spontaneous GABA_A receptor-mediated PSC (GABA_A-sPSC) characteristics from layer V pyramidal neurons and INs. (a) Average GABA_A-sPSC current traces from pyramidal neurons during baseline (black trace), during 90 mM ethanol bath application (red trace), and during washout (blue trace). (b) Average GABA_A-sPSC current traces from INs during baseline, 90 mM ethanol application, and washout. Scale bars 50 ms, 10 pA. (c–j) Cumulative probability plots and average data from each cell (insets) showing effect of 90 mM ethanol application and washout on (c) pyramidal neuron inter-event interval (frequency in inset), (d) IN inter-event interval (frequency in inset), (e) pyramidal neuron amplitude, (f) IN amplitude, (g) pyramidal neuron rise time, (h) IN rise time, (i) pyramidal neuron decay tau, and (j) IN decay tau. Baseline = black line/black squares, 90 mM ethanol application = red line/ red circles, washout = blue line/blue triangles. Asterisks (*, **) denote a p-value of p < 0.05 and p < 0.01, respectively. Please see Supplemental Table S1 for exact p-values. Two-Way ANOVA effects: C = cell type effect, P = exposure phase effect, CxP = cell type by exposure phase interaction. Male pyramidal n = 4 cells from 4 animals from 4 litters; female pyramidal = 4 cells from 4 animals from 3 litters; male IN = 4 cells from 4 animals from 4 litters, female IN = 3 cells from 3 animals from 2 litters. Data are presented as mean ± SEM in all cases.

Figure 5

Representative immunohistochemistry (IHC) images from B6 PV^cre-Ai27D mice and analysis of both penetrance and specificity of transgene expression. (a–d) Representative IHC images showing colocalization of PV and ChR2- tdTomato. (a) IHC demonstrating PV expression. (b) Expression of endogenous ChR2–tdTomato transgene expression. (c) DAPI nuclear stain. (d) Merged image. Scale bar 25 µm. (e) Penetrance analysis of B6 PV^cre-Ai27D transgene expression. Penetrance of transgene expression was measured as the number of PV expressing cells colocalized with tdTomato divided by the number of PV expressing cells. (f) Specificity analysis of B6 PV^cre-Ai27D transgene expression. Specificity of transgene expression was measured as the number of cells expressing only tdTomato divided by the number of cells expressing both PV and tdTomato. Air male n = 3 animals from 3 litters, air female n = 2 animals from 2 litters; ethanol male n = 3 animals from 3 litters, ethanol female n = 2 animals from 2 litters. Data are presented as mean ± SEM.

Figure 6

Effect of P7 ethanol exposure on optically-evoked inhibitory postsynaptic currents (oIPSCs) in layer V RSC pyramidal neurons at P40–60. (a) Average oIPSC current traces from air-exposed animals using 0.5 ms (black trace), 1 ms (red trace), 2 ms (green trace), 4 ms (purple trace), and 8 ms (orange trace) laser pulse durations. (b) Average oIPSC current traces from ethanol-exposed animals using 0.5, 1, 2, 4, and 8 ms laser pulse durations. Scale bars 20 ms, 200 pA. Blue arrows indicate onset of laser pulse. (c–h) Collected peak amplitudes (c), current densities (d), GABAergic total charge (e), half widths at half-maximum amplitude (f), rise times (g), and # of oIPSC events (h) for all cells from air exposed (black circles) and ethanol exposed (red squares) animals presented for each laser pulse duration. Data are presented collapsed across sex due to a lack of sex effects from LMM analyses. Dagger (^†) denotes a p-value of < 0.06, and asterisks (*, **, ***, ****) denote p-values of p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Please see Supplemental Tables S2 and S3 for exact p-values from LMMs and post hoc tests, respectively. LMM effects: E = exposure effect, L = laser pulse duration effect, ExL = exposure by laser pulse duration interaction. Significance indicators next to x-axis laser pulse duration labels of panel (e) and panel (h) indicate a Mann–Whitney U post hoc effect of exposure within laser pulse duration. Female air n = 32 cells from 8 animals from 7 litters, male air n = 46 cells from 9 animals from 8 litters; female ethanol n = 35 cells from 8 animals from 8 litters, male ethanol n = 40 cells from 8 animals from 7 litters. Data are presented as mean ± SEM.

Figure 7

Effect of P7 ethanol exposure on optically-evoked inhibitory postsynaptic currents (oIPSCs) paired-pulse ratios in layer V RSC pyramidal neurons at P40–60. (a) Average oIPSC PPR current traces from air-exposed (black trace) and ethanol-exposed (red trace) animals, collapsed across sex. Amplitudes of traces from air- and ethanol-exposed animals are normalized to the amplitude of the first peak to illustrate PPRs. Scale bar 40 ms. (b) Collected amplitude PPRs for all cells from air-exposed (black circles) and ethanol-exposed (red squares) animals. (c) Collected total charge PPRs for all cells from air-exposed and ethanol-exposed animals. Asterisk (*) denotes a p-value of p < 0.05. Please see Supplemental Table S2 for exact p-values. S = sex effect. Air female n = 19 cells from 5 animals from 4 litters, air male n = 22 cells from 6 animals from 5 litters; ethanol female n = 22 cells from 6 animals from 5 litters; ethanol male n = 20 cells from 5 animals from 5 litters. Data are presented as mean ± SEM.