Binge-like ethanol exposure during the brain growth spurt disrupts the function of retrosplenial cortex-projecting anterior thalamic neurons in adolescent mice

Abstract

Ethanol (EtOH) exposure during late pregnancy leads to enduring impairments in learning and memory that may stem from damage to components of the posterior limbic memory system, including the retrosplenial cortex (RSC) and anterior thalamic nuclei (ATN). In rodents, binge-like EtOH exposure during the first week of life (equivalent to the third trimester of human pregnancy) triggers apoptosis in these brain regions. We hypothesized that this effect induces long-lasting alterations in the function of RSC-projecting ATN neurons. To test this hypothesis, vesicular GABA transporter-Venus mice (expressing fluorescently tagged GABAergic interneurons) were subjected to binge-like EtOH vapor exposure on postnatal day (P) 7. This paradigm activated caspase 3 in the anterodorsal (AD), anteroventral (AV), and reticular thalamic nuclei at P7 but did not reduce neuronal density in these areas at P60-70. At P40-60, we injected red retrobeads into the RSC and performed patch-clamp slice electrophysiological recordings from retrogradely labeled neurons in the AD and AV nuclei 3-4 days later. We found significant effects of treatment on instantaneous action potential (AP) frequency and AP overshoot, as well as sex × treatment interactions for AP threshold and overshoot in AD neurons. A sex × treatment interaction was detected for AP number in AV neurons. EtOH exposure also reduced the frequency and amplitude of spontaneous excitatory postsynaptic currents and increased the charge transfer of spontaneous inhibitory postsynaptic currents. These results highlight a novel cellular mechanism that could contribute to the lasting learning and memory deficits associated with developmental EtOH exposure.

Keywords: Apoptosis; Cortex; Development; Electrophysiology; Ethanol; Fetal; GABA; Glutamate; Neonatal; Retrosplenial; Thalamus.

Figure 1.. Ethanol (EtOH) exposure at postnatal day 7 activated apoptotic pathways in anterior thalamic neurons.

Images from VGAT-Venus mice from the Air (control) (A) and 8 hours post EtOH vapor conditions (B). Red: cleaved caspase 3 (CC3), blue: DAPI, green: Venus. AD, anterodorsal thalamic nucleus; AV, anteroventral thalamic nucleus; TRN, thalamic reticular nucleus. C) Panoramic image illustrating the AD, AV, and TRN location in a coronal brain slice of a P7 VGAT-Venus mouse. Blue: DAPI; green: Venus. D). Summary data of the effect of EtOH treatment on the percentage of DAPI-positive cells that were positive for cleaved caspase 3 (CC3) in the AD nucleus (p<0.0001), AV nucleus (p<0.0001) and TRN (p=0.032) (Šídák’s multiple comparisons test for control vs. EtOH). Control, n=4 pups (2 males and 2 females from 2 litters) and EtOH, n=5 pups (4 males and 1 female from 3 litters).

Figure 2.. Neonatal ethanol (EtOH) exposure did not significant affect neuronal densities in anterior thalamic nuclei at postnatal days 60–70.

A) Sample images of neurons stained with anti-NeuN antibodies (red) and Venus+ GABAergic interneurons (green) from the Air (control) and EtOH groups (scale bars 100 μm). B) Summary graph illustrating the effect of third trimester-equivalent EtOH exposure on neuronal densities in the anterodorsal (AD), anteroventral (AV), or thalamic reticular (TRN) nuclei. Data were analysed by three-way ANOVA of log-transformed data. Posthoc analysis with the Holm-Šídák’s test yielded treatment p values ranging from >0.75 to >0.9 for both males and females in the AD nucleus, AV nucleus, and TRN. Control female, n=8 pups from 6 litters; EtOH female, n=7 pups from 4 litters; control male, n=6 pups from 3 litters; EtOH male, n=8 pups from 6 litters.

Figure 3.. Schematic representation of retrobead injection into the retrosplenial cortex to label neurons in the anterodorsal (AD) and anteroventral (AV) thalamic nuclei.

A) Parasagittal image of a Nissl stained section (image #18) from the Allen Reference Atlas – Mouse Brain (postnatal day 56) illustrating the site of red retrobead injection in the retrosplenial cortex (RSC). B) Nissl stained image with anatomical annotations of a coronal section (image #75) from the same atlas also showing the site of injection in the RSC. C) Parasagittal image of a Nissl stained section (image #16) from the same atlas illustrating the site of electrophysiological recordings in the AD and AV thalamic nuclei. D) Nissl-stained image with anatomical annotations of a coronal section (image #62) from the same atlas also showing the site of electrophysiological recordings in the AD and AV thalamic nuclei. The inset shows an image of red retrobeads in the AD and AV nuclei from a mouse that received an injection in the RSC. Allen Mouse Brain Atlas, mouse.brain-map.org.

Figure 4.. Neonatal ethanol (EtOH) exposure disrupted action potential firing in retrosplenial cortex-projecting anterodorsal (AD) thalamic nucleus neurons at postnatal days 40–60.

A) Representative traces of action potential firing evoked by current injection (600 pA) in AD nucleus neurons from the Air (control) and EtOH groups. Whole trace scale bar = 50 ms X 10 mV; expanded trace scale bar = 5 ms X 10 mV. B) Summary graphs illustrating that EtOH treatment (red squares) reduced the instantaneous frequency of action potentials in females and males with respect to control (black circles) (data were analyzed by three-way ANOVA of aligned rank transformed data; Supplementary Data Tables). C) Mean effect of EtOH exposure on action potential amplitude in females and males (data were analyzed by three-way ANOVA of aligned rank transformed data; Supplementary Data Tables). Control female, n=9 pups from 6 litters; EtOH female, n=10 pups from 6 litters; control male, n=12 pups from 9 litters; EtOH male, n=12 pups from 7 litters.

Figure 5.. Neonatal ethanol (EtOH) exposure disrupted action potential firing in retrosplenial cortex-projecting anteroventral (AV) thalamic nucleus neurons at postnatal days 40–60.

A) Representative traces of action potential firing evoked by current injection (600 pA) in AV nucleus neurons from the Air (control; black circles) and EtOH (red squares) groups. Whole trace scale bar = 50 ms X 10 mV; expanded trace scale bar = 5 ms X 10 mV. B) Mean effect of EtOH treatment on the instantaneous frequency of action potentials in females and males (data were analyzed by three-way ANOVA of aligned rank transformed data; Supplementary Data Tables). C) Mean effect of EtOH exposure on action potential amplitude in females and males (data were analyzed by three-way ANOVA of aligned rank transformed data; Supplementary Data Tables). Control female, n=7 pups from 5 litters; EtOH female, n=9 pups from 6 litters; control male, n=10 pups from 8 litters; EtOH male, n=10 pups from 5 litters.

Figure 6.. Neonatal ethanol (EtOH) exposure reduced the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) in retrosplenial cortex-projecting anterodorsal (AD) thalamic nucleus neurons at postnatal days 40–60.

A) Representative sEPSC traces from the Air (control) (black trace) and EtOH groups (red trace). Scale bar is 0.4 s X 40 pA. B) Mean effect of EtOH exposure on sEPSC frequency (two-way ANOVA revealed the indicated significant effects; see Supplementary Data Tables for more details). C) Mean effect of EtOH exposure on sEPSC amplitude (two-way ANOVA revealed the indicated significant effect; see Supplementary Data Tables for more details). D) Mean effect of EtOH exposure on sEPSC total charge. E) Mean effect of EtOH exposure on sEPSC rise time 10–90%. F) Mean effect of EtOH exposure on sEPSC decay time. Control female, n=8 pups from 6 litters; EtOH female, n=10 pups from 7 litters; control male, n=11 pups from 8 litters; EtOH male, n=13 pups from 7 litters.

Figure 7.. Neonatal ethanol (EtOH) exposure did not affect spontaneous excitatory postsynaptic currents (sEPSCs) in retrosplenial cortex-projecting anteroventral (AV) thalamic nucleus neurons at postnatal days 40–60.

A) Representative sEPSC traces from the Air (control) (black trace) and EtOH groups (red trace). Scale bar is 0.4 s X 40 pA. B) Mean effect of EtOH exposure on sEPSC frequency. C) Mean effect of EtOH exposure on sEPSC amplitude. D) Mean effect of EtOH exposure on sEPSC total charge. E) Mean effect of EtOH exposure on sEPSC rise time 10–90%. F) Mean effect of EtOH exposure on sEPSC decay time. Control female, n=9 pups from 6 litters; EtOH female, n=9 pups from 6 litters; control male, n=8 pups from 7 litters; EtOH male, n=10 pups from 6 litters.

Figure 8.. Neonatal ethanol (EtOH) exposure increased the total charge of spontaneous inhibitory postsynaptic currents (sIPSCs) in retrosplenial cortex-projecting anterodorsal (AD) thalamic nucleus neurons at postnatal days 40–60.

A) Representative sIPSC traces from the Air (control) (black trace) and EtOH groups (red trace). Scale bar is 0.4 s X 40 pA. B) Mean effect of EtOH exposure on sIPSC frequency. C) Mean effect of EtOH exposure on sIPSC amplitude. D) Mean effect of EtOH exposure on sIPSC total charge (two-way ANOVA revealed the indicated significant effect; see Supplementary Data Tables for more details). E) Mean effect of EtOH exposure on sIPSC rise time 10–90%. F) Mean effect of EtOH exposure on sIPSC decay time. Control female, n=8 pups from 6 litters; EtOH female, n=9 pups from 6 litters; control male, n=11 pups from 8 litters; EtOH male, n=10 pups from 7 litters.

Figure 9.. Neonatal ethanol (EtOH) exposure increased the total charge of spontaneous inhibitory postsynaptic currents (sIPSCs) in retrosplenial cortex-projecting anterodorsal (AV) thalamic nucleus neurons at postnatal days 40–60.

A) Representative sIPSC traces from the Air (control) (black trace) and EtOH groups (red trace). Scale bar is 0.4 s X 40 pA. B) Mean effect of EtOH exposure on sIPSC frequency. C) Mean effect of EtOH exposure on sIPSC amplitude. D) Mean effect of EtOH exposure on sIPSC total charge. E) Mean effect of EtOH exposure on sIPSC rise time 10–90%. F) Mean effect of EtOH exposure on sIPSC decay time. Control female, n=8 pups from 6 litters; EtOH female, n=8 pups from 6 litters; control male, n=8 pups from 5 litters; EtOH male, n=7 pups from 6 litters.