Article Figures & Data
Figures
- Figure 1.
Experimental timeline and the influence of prenatal ethanol exposure on early postnatal sensorimotor development. A, Pregnant dams were exposed to 5% (w/w) ethanol in a liquid diet, or a control (lab chow) diet from embryonic day (E) 13.5–16.5, a period of significant migration of early-born striosomal striatal projection neurons (SPNs) and striatal GABAergic interneurons (GINs), to the developing striatum. After birth at postnatal day (P) 0, mice were maintained to postnatal time points, P2, P4, P6, P8, P10, or P14, assessed for the development of a set of nine sensorimotor behaviors, and then killed for whole-cell patch-clamp recordings and morphological analysis of biocytin dye-filled cells. B, A brief binge prenatal ethanol exposure does not alter the postnatal growth: body weight (g) of neonatal mice (3-way ANOVA: exposure: F(1,197) = 0.387, p = 0.535, sex: F(1,197) = 2.686, p = 0.103, postnatal day: F(5,197) = 223.659, p < 0.001, exposure × sex × postnatal day: F(5,197) = 1.416, p = 0.220, exposure × sex: F(1,197) = 2.847, p = 0.093, exposure × postnatal day: F(5,197) = 0.355, p = 0.879, sex × postnatal day: F(5,197) = 0.222, p = 0.953). C, Prenatal ethanol exposure resulted in decreased total motor score (TMS) in ethanol-exposed M mice, which significantly differed from control M mice at P8 (Kruskal–Wallis test, P2: H(3) = 3.602, p = 0.308, P4: H(3) = 4.883, p = 0.181, P6: H(3) = 2.778, p = 0.427, P8: H(3) = 11.343, p = 0.010, Dunn's post hoc test: ethanol M vs ethanol F: p = 0.009, P10: H(3) = 0.000, p = 1.000, P14: H(3) = 2.893, p = 0.408). D, Prenatal ethanol exposure delayed the transition from forelimb-driven pivoting behavior, to crawling, and eventually running in ethanol-exposed M mice resulting in decreased quadruped walking scores (Kruskal–Wallis tests, P2: H(3) = 4.509, p = 0.211, P4: H(3) = 3.647, p = 0.301, P6: H(3) = 1.407, p = 0.704, P8: H(3) = 0.937, p = 0.817, P10: H(3) = 3.568, p = 0.312, P14: H(3) = 8.728, p = 0.033). E, Prenatal ethanol exposure delayed the development of mature vertical screen task behavior in M mice (Kruskal–Wallis tests, P2: H(3) = 0.731, p = 0.866, P4: H(3) = 6.436, p = 0.096, P6: H(3) = 7.248, p = 0.064, P8: H(3) = 4.311, p = 0.230, P10: H(3) = 0.602, p = 0.896, P14: H(3) = 1.422, p = 0.700). F, Prenatal ethanol exposure altered surface righting times in a sex-dependent manner (2-way ANOVA, group: F(3,200) = 6.307, p = 0.0004, postnatal day: F(5,200) = 81.71, p < 0.0001, group × postnatal day: F(15,200) = 3.502, p < 0.001, Bonferroni’s post hoc tests: control M vs ethanol M: P2: t = 3.856, p < 0.001, ethanol M vs ethanol F: P6: t = 3.400, p < 0.01, control F vs control M: P2: t = 2.956, p < 0.05, P4: t = 5.498, p < 0.001, control M vs ethanol F: P4: t = 3.443, p < 0.01, P6: t = 3.226, p < 0.01, control F vs ethanol M: P4: t = 3.969, p < 0.001). G, Prenatal ethanol exposure altered negative geotaxis time (2-way ANOVA, group: F(3,200) = 3.502, p = 0.0164, postnatal day: F(5,200) = 131.3, p < 0.001, group × postnatal day: F(15,200) 0.7252, p = 0.7253, Bonferroni’s post hoc tests: P8: ethanol M vs control M, t = 3.447, p < 0.01). Data are presented as mean score or time, error bars are standard error of the mean (SEM), **p < 0.01, ***p < 0.001, control male versus ethanol male; ##p < 0.01, control female versus ethanol +p < 0.05; +++p < 0.001, control male versus control female; @@@p < 0.001: control female versus ethanol. Data supported by Extended Data Figure 1-1.
- Figure 2.
Identifying striatal GABAergic interneurons (GINs) and striatal projection neurons (SPNs) in acute cortical slices from Nkx2.1Cre × TdTomato mice. A, Striatal GINs and SPNs were identified during whole-cell patch-clamp recordings from 250 µm acute coronal slices from Nkx2.1Cre × Tdtomato mice based on tdTomato (red) expression in MGE-derived GABAergic interneurons. Top, Schematic of whole-cell patch-clamp recordings from striatal neurons; middle, 40× magnification Hoffman modulated contrast image of acute slice during recording from a striatal GIN and a neighboring SPN (arrow); bottom, fluorescent tdTomato + striatal GIN (white) and a neighboring SPN (arrow). B, A SPN-specific nuclear immunomarker CTIP2 (green) does not colabel td-Tomato + GINs (red) in the dorsal striatum of a P6 Nkx2.1Cre mice, with a DAPI-counterstain (blue). C, Image of a neurobiotin-filled P14 striatal GIN after recording, pseudocolored (red). D, Image of a neurobiotin-filled P14 SPN after recording (green). Scale bars, 20 µm.
- Figure 3.
Prenatal ethanol exposure alters the functional development of striatal GABAergic interneurons (GINs) in early postnatal mice in an age- and sex-dependent manner. A, Prenatal ethanol exposure modifies the firing rate (Hz) of striatal GIN in an age- and sex-dependent manner. At P2, striatal GINs from ethanol-exposed F mice had higher firing rates relative to those from ethanol-exposed and control-fed M mice (2-way ANOVA, group: F(3,450) = 10.56, p < 0.0001, current: F(8,450) = 24.52, p < 0.0001, group × current F(24,450) = 1.282, p = 0.1691, Bonferroni’s post hoc tests: ethanol F vs ethanol M: 60 pA: t = 2.981, p < 0.05, 70 pA: t = 3.474, p < 0.01, 80 pA: t = 3.620, p < 0.01; ethanol F vs control M: 70 pA: t = 3.242, p < 0.05, 80 pA: t = 3.574, p < 0.01). At P4–6, firing rate significantly differed between groups with striatal GINs: control-fed M mice demonstrated higher firing rates relative to those from ethanol-exposed M and F and control-fed M mice (2-way ANOVA, group: F(3,468) = 4.934, p = 0.0022, current: F(8,468) = 14.26, p < 0.001, group × current: F(24,468) = 0.4322, p = 0.9922). At P8–10 and P14, firing rate again significantly differed between groups with striatal GINs from ethanol-fed mice demonstrating higher firing rates relative to those from control-fed mice (2-way ANOVAs, P8–10: group: F(3,621) = 11.15, p < 0.0001, current: F(8,621) = 28.31, p < 0.0001, group × current: F(24,621) = 0.4459, p = 0.9904; P14: group: F(3,726) = 4.871, p = 0.0023, current: F(10,726) = 56.96, p < 0.0001, group × current: F(30,726) = 0.3229, p = 0.9998). B, Example traces of voltage responses of striatal GINs following depolarizing current steps from control-fed female mice at P2 and P14. C, Prenatal ethanol exposure significantly depolarized AP threshold in GINs from F mice relative to those from control-fed F mice at P2, hyperpolarized AP threshold in GINs from F and M mice relative to control-fed F and M mice at P14, and control-fed F relative to control-fed M mice but did not alter AP threshold from P4–10 (1-way ANOVAs, P2: F(3,50) = 4.298, p = 0.005, Bonferroni’s post hoc tests: ethanol F vs control F, p = 0.005, P4–6: F(3,50) = 0.949, p = 0.424, P8–10: F(3,69) = 2.859, p = 0.043, P14: F(3,69) = 5.352, p = 0.002 Bonferroni’s post hoc tests: ethanol F vs control F, p = 0.003, ethanol M vs control M: p = 0.027, control F vs control M: p = 0.023). D, Prenatal ethanol exposure significantly increased GIN AP half-width in M mice relative to those from control-fed M or F, or ethanol-exposed F mice at P2, but did not affect GIN AP half-width P4–14 (1-way ANOVAs, P2: group: F(3,50) = 5.639, p = 0.002, Bonferroni’s post hoc tests: ethanol M vs control M, p = 0.002, ethanol M vs ethanol F, p = 0.014, ethanol M vs control F, p = 0.014; P4–6 F(3,50) = 1.335, p = 0.273; P8–10 F(3,69) = 0.699, p = 0.556; P14: F(3,61) = 1.086, p = 0.362). Data are presented as means (bars), error bars are standard error of the mean (SEM), dots are individual neurons from at least three animals per group. *p < 0.05, control male versus ethanol male; ##p < 0.01, control female versus ethanol female; +p < 0.05, control male versus control female; @@p < 0.01, control female versus ethanol male; $p < 0.05, $$p < 0.01, control male versus ethanol female; xp < 0.05, xxp < 0.01, ethanol male versus ethanol female. Data supported by Extended Data Figures 3-1, 3-2.
- Figure 4.
Prenatal ethanol exposure alters the functional development of striatal spiny projection neurons (SPNs) in early postnatal mice in an age- and sex-dependent manner. A, Prenatal ethanol exposure modifies the firing rate of SPN in an age- and sex-dependent manner. While no significant differences were observed between groups: control F, ethanol F, control M, and ethanol M at P2, group-dependent differences were observed at P4–6, P8–10, and P14, with group differences varying based on current input at P14 (2-way ANOVAs, P2: F(3,531) = 0.6138, p = 0.6063, current: F(8,531) = 44.82 p < 0.0001, group × current: F(8,531) = 0.7447, p = 0.8059; P4–6: group: 2-way ANOVA, F(3,567) = 6.923, p = 0.0001, current: F(8,567) = 81.60, p < 0.001, group × current: F(24,567) = 0.7128, p = 0.8409; P8–10: group: F(3,729) = 12.97, p < 0.001, current: F(8,729) = 99.70, p < 0.001, group × current: F(24,729) = 0.6896, p = 0.8647; P14: group: F(3,803) = 7.874, p < 0.001, current: F(8,803) = 104.4, p < 0.0001, group × current: F(24,803) = 1.676, p = 0.0136). At P8, prenatal ethanol exposure significantly increased SPN firing rate in M mice relative to ethanol-exposed F mice. At P14, prenatal ethanol exposure significantly decreased SPN firing rate in M mice relative to control-fed M mice, while control-fed M mice demonstrated higher firing rates than both control-fed and ethanol-exposed F mice (Bonferroni’s post hoc tests, ethanol M vs control M: 500 pA: t = 2.864, p < 0.05, control M vs control F: 450 pA: t = 2.854, p < 0.05, 500 pA: t = 3.856, p < 0.01, control M vs ethanol F: 500 pA: t = 4.050, p < 0.001). B, Example traces of voltage responses of striatal GINs following hyperpolarizing and depolarizing current steps from control-fed female mice at P2 and P14. C, Prenatal ethanol exposure significantly depolarized AP threshold at P2 and hyperpolarized at P14 in SPNs from M mice, relative to those from control-fed M mice, and in depolarized AP threshold in SPNs from F mice relative to control-fed M mice at P14 but did not alter AP threshold between P4 and 10 (1-way ANOVAs, P2: F(3,59) = 3.862, p = 0.014, Bonferroni’s post hoc test: ethanol M vs control M: p 0.009; P4–6: F(3,66) = 1.039, p = 0.381; P8–10: F(3,81) = 1.180, p = 0.323; P14: F(3,73) = 5.055, p = 0.003, Bonferroni’s post hoc tests: ethanol M vs control M: p 0.020, ethanol F vs control M: p = 0.010). D, Prenatal ethanol exposure did not alter the SPN AP half-width (1-way ANOVAs, P2: F(3,59) = 1.384, p = 0.257, P4–6: F(3,66) = 1.979, p = 0.126, P8–10: F(3,81) = 1.017, p = 0.389, P14: F(3,73) = 0.419, p = 0.740). Data are presented as means (bars), error bars are standard error of the mean (SEM), dots are individual neurons from at least three animals per group. *p < 0.05, control male versus ethanol male; +p < 0.05, ++p < 0.01, control male versus control female; $p < 0.05,$$$p < 0.001, control male versus ethanol female; xp < 0.05, ethanol male versus ethanol female. Data supported by Extended Data Figures 4-1, 4-2.
- Figure 5.
Glutamatergic synaptic activity in the developing striatum is disrupted by prenatal ethanol exposure depending on sex and neuronal subtype: striatal GABAergic interneurons (GINs) and striatal projection neurons (SPNs). A, Example whole-cell voltage-clamp recordings of spontaneous glutamatergic postsynaptic current (sPSC) recordings from striatal GINs in control-fed female mice at P2 and P14. B, Prenatal ethanol exposure differentially effects the frequency of glutamatergic sPSCs recorded in striatal GIN from female and male mice depending upon their postnatal age. At P2, control-fed male mice demonstrate a higher glutamatergic sPSC frequency than control-fed female mice, while frequency does not differ in GINs from ethanol-exposed male versus female mice (1-way ANOVA, F(3,49) = 3.884, p = 0.014; Bonferroni’s post hoc tests: control M vs control F: p = 0.017). At P14, prenatal ethanol exposure results in a decreased frequency of glutamatergic sPSCs in female mice relative to control-fed female mice (1-way ANOVA, F(3,47) = 4.007, p = 0.025, Bonferroni’s post hoc tests: ethanol F vs control F: p = 0.037). Prenatal ethanol exposure does not affect glutamatergic sPSC frequency in striatal GINs from P4–6 or P8–10 mice (1-way ANOVAs, P4–6: F(3,47) = 0.530, p = 0.664; P8–10: F(3,47) = 0.092, p = 0.964). C, Prenatal ethanol exposure decreases glutamatergic sPSC amplitude in striatal GINs from P14 female mice relative to control-fed female mice but does not alter glutamatergic sPSC frequency in striatal GINs from female between P2 and P10 or from male mice (1-way ANOVAs, P2: F(3,49) = 2.283, p = 0.091; P4–6: F(3,47) = 0.666, p = 0.577; P8–10: F(3,45) = 1.266, p = 0.298; P14: F(3,47) = 0.666, p = 0.012, Bonferroni’s post hoc tests: ethanol F vs control: p = 0.012). D, Example glutamatergic sPSC recordings from SPNs in control-fed female mice at P2 and P14. E, Prenatal ethanol exposure results in an early postnatal (P2) decrease in glutamatergic sPSC frequency in SPNs from female mice, relative to control-fed male mice (1-way ANOVA, F(3,41) = 4.852, p = 0.006; Bonferroni’s post hoc tests: control M vs ethanol F: p = 0.005). At P14, prenatal ethanol exposure resulted in significantly decreased glutamatergic sPSC frequency in SPNs from male mice relative to control-fed female mice (1-way ANOVA, F(3,47) = 4.853, p = 0.005, Bonferroni’s post hoc tests: control F vs ethanol M: p = 0.005). Prenatal ethanol exposure did not affect glutamatergic sPSC frequency in SPNs from male and female mice at P4–6 or P8–10 (1-way ANOVAs, P4–6: F(3,56) = 0.537, p = 0.659; P8–10: F(3,52) = 0.094, p = 0.963). F, Prenatal ethanol exposure did not alter amplitude of glutamatergic sPSCs recorded from SPN during the first two postnatal weeks (one-way ANOVAs, P2: F(3,41) = 0.861, p = 0.469; P4–6: F(3,56) = 1.332, p = 0.273; P8–10: F(3,52) = 1.341, p = 0.271; P14: F(3,47) = 1.093, p = 0.362). Data are presented as means (bars), error bars are standard error of the mean (SEM), dots are individual neurons from at least three animals per group. For all: +p < 0.05, control M versus control F; #p < 0.05, control F versus ethanol F; @@p < 0.01, control F versus ethanol M; $$p < 0.01, control M versus ethanol F.
- Figure 6.
GABAergic synaptic activity in the developing striatum is disrupted by prenatal ethanol exposure depending on sex and neuronal subtype: striatal GABAergic interneurons (GINs) and striatal projection neurons (SPNs). A, Example whole-cell voltage-clamp recordings of spontaneous GABAergic postsynaptic current (sPSC) recordings from striatal GINs in control-fed female mice at P2 and P14. B, Prenatal ethanol exposure increases the frequency of GABAergic sPSC recorded from striatal GINs in female mice relative to control-fed F mice at P4–6 but does not alter GABAergic sPSC frequency in F mice at P2, P8–10, or P14 and does not affect GABAergic sPSC frequency in male mice (1-way ANOVAs, P2: F(3,48) = 2.919, p 0.043; P4–6: F(3,52) = 3.307, p = 0.028; Bonferroni’s post hoc tests: control F vs ethanol F: p = 0.023; P8–10: F(3,41) = 0.699, p 0.558; P14: F(3,47) = 2.005, p 0.127). C, Striatal GINs from control-fed F mice had significantly increased GABAergic sPSC amplitude relative to those from control-fed M and ethanol-exposed M P14 mice (1-way ANOVA, F(3,47) = 3.150, p = 0.034, Bonferroni’s post hoc tests: control M vs control F: p = 0.028, control F vs ethanol M: p = 0.028). Prenatal ethanol exposure did not alter GABAergic sPSC amplitude between P2 and P10 (one-way ANOVAs, P2: F(3,40) = 1.461, p = 0.240; P4–6: F(3,52) = 2.253, p = 0.094; P8–10: F(3,41) = 0.160, p = 0.923, P14: F(3,44) = 3.150, p = 0.034, Bonferroni’s post hoc tests: ethanol M versus control F: p = 0.028. D, Example whole-cell voltage-clamp recordings of spontaneous GABAergic sPSC recordings from SPNs in control-fed female mice at P2 and P14. E, Prenatal ethanol exposure significantly decreased the frequency of GABAergic sPSC in SPNs from M mice at P2, relative to control-fed M and F mice, and ethanol-exposed F mice (1-way ANOVA, F(3,42) = 6.383, p = 0.001; Bonferroni’s post hoc test: control M vs ethanol M: p = 0.008, control M vs control F: p = 0.004, control M vs ethanol F: p = 0.004). Prenatal ethanol exposure did not alter GABAergic sIPSC frequency in SPNs P4–14 (1-way ANOVAs, P4–6: F(3,57) = 0.921, p = 0.437; P8–10: F(3,54) = 0.921, p = 0.437; P14: F(3,46) = 1.344, p = 0.272). F, At P8–10, prenatal ethanol exposure increases the amplitude of GABAergic SPCs in SPNs from M mice relative to control-fed M (1-way ANOVA, F(3,54) = 3.623, p = 0.019, Bonferroni’s post hoc: control M vs ethanol M: p = 0.027, control F vs ethanol M: p = 0.052). Prenatal ethanol exposure does not affect GABAergic sPSC amplitude at P2, P4–6, or P14 (1-way ANOVAs, P2: F(3,38) = 0.587, p = 0.627; P4–6: F(3,57) = 1.920, p = 0.137, P14: F(3,46) = 1.865, p = 0.149). Data are presented as means (bars), error bars are standard error of the mean (SEM), and dots are individual neurons from at least three animals per group. For all: *p < 0.05, **p < 0.01, control male versus ethanol male, +p < 0.05, control M versus control F; #p < 0.05, control F versus ethanol F; @p < 0.05, control F versus ethanol M; $$p < 0.01 control M versus ethanol F.
- Figure 7.
Prenatal ethanol exposure result in early postnatal increases in spiny projection neuron (SPN) dendritic morphology. A, Representative reconstructions (right) and images (left) of neurobiotin-filled SPNs from female (F) control, male (M) control (top), F ethanol-exposed, and M ethanol-exposed (bottom) postnatal day (P) 2 neonates. B, Representative reconstructions (right) and images (left) of neurobiotin-filled SPNs from F control, M control (top), F ethanol-exposed, and M ethanol-exposed (bottom) P8 neonates. C, Sholl analysis indicates that prenatal ethanol exposure increases the dendritic complexity (number of intersections/increasing 10 µm radius) in a radius-dependent manner of SPNs from P2 F and M + mice relative to control-fed F mice, P4–6 M mice relative control-fed F mice, and decreased in P8–10 F mice relative to ethanol-exposed M and control-fed M, while differences have resolved by P14 (two-way repeated measures ANOVA, P2: group: F(3,1320) = 1.559, p = 0.2097, radius: F(24,1320) = 188.9, p < 0.001, group × radius: F(72,1320) = 1.241, p = 0.0878, Bonferroni’s post hoc tests: ethanol F vs control F: radius = 50 µm, t = 3.348, p < 0.05, radius = 60 µm, t = 3.866, p < 0.01); ethanol M vs control F: radius = 40 µm, t = 3.837, p < 0.01, radius = 50 µm, t = 3.189, p < 0.05, radius = 60 µm, t = 3.241, p < 0.05; P4–6: group: F(3,1596) = 1.701, p = 0.1770, radius: F(28,1596) = 228.5, p < 0.001, group × radius: F(84,1596) = 1.093, p = 0.2684, Bonferroni’s post hoc tests: ethanol M vs control F: radius = 20 µm, t = 4.540, p < 0.0001, 40 µm, t = 3.692, p < 0.01, 50 µm, t = 3.412, p < 0.05. P8–10: group: F(3,1782) = 0.6876, p = 0.5635, radius: F(33,1782) = 475.5, p < 0.0001, group × radius: F(99,1782) = 0.8764, Bonferroni’s post hoc tests: ethanol F vs ethanol M: radius = 80 µm, t = 4.061, ethanol F vs control M: radius = 60 µM, t = 3.694, p < 0.01, 70 µm, t = 3.207, p < 0.05. P14: group: F(3,2112) = 0.4037, p = 0.7508, radius F(33,2112) = 437.2, p < 0.0001, group × radius: F(99,2112) = 0.6128, p = 0.9990). For all images, scale bar, 20 µm. Data are presented as means (bars), error bars are standard error of the mean (SEM), and dots are individual neurons from at least three animals per group. For all: +p < 0.05, ++p < 0.01, control M versus control F; #p < 0.05, ##p < 0.01, control M versus ethanol M, @p < 0.05; @@@p < 0.001, control F versus ethanol M, $$p < 0.01 control M versus ethanol F; xp < 0.05, xxp < 0.01, ethanol F versus ethanol M. Extended Data Figure 7-1.
- Figure 8.
Prenatal ethanol exposure results in sex-dependent differences in early motor deficits coinciding with altered functional, synaptic, and morphological development of striatal neurons that vary with postnatal age. At P2, prenatal ethanol exposure results in a male-specific increase in surface righting time coinciding with significant sex differences in the effects of prenatal ethanol exposure on synaptic inputs to and functional properties of striatal GINs. In male mice exposed prenatally to ethanol, GIN demonstrate less increased AP half-width suggestive of a maturational delay, while SPNs receive fewer or weaker GABAergic synaptic inputs. In female mice, GINs show no differences in membrane properties but fire action potentials (APs) at a higher rate, while SPNs demonstrate no differences in GABAergic synaptic inputs and develop an increased dendritic complexity. However, SPNs from both male and female mice tended to have decreased glutamatergic synaptic innervation and had less mature/more excitable functional properties. At P4–6, prenatal ethanol exposure results in increased surface righting time only in female mice, while only male mice exposed prenatally to ethanol demonstrate increased negative geotaxis time and increased total motor score (TMS) at P8. Unlike female mice, male mice show fewer morphological differences early on (P2) but develop a longer lasting increase in dendritic complexity (P4–10), associated with longer, more highly branched dendrites. Differences in dendritic complexity coincided with changes in SPN AP firing rate that differ between female and male mice. Though both female and male mice demonstrate transient increases in SPN AP firing rate between P4 and P10, the increase observed at P4–6 in SPNs from female mice exposed prenatally to ethanol precedes a similar elevation in firing rate at P8–10 in SPNs from male mice with the same prenatal ethanol exposure (Fig. 4A). These changes in SPN firing properties occur simultaneously with increases in GABAergic activity: while at P4–6 the firing rate of striatal GINs from ethanol-exposed female mice does not differ from that of control-fed females, striatal GINs do display a higher GABAergic sPSC frequency (Figs. 4A, 6B). Alternatively, at P8–10, an increase in the amplitude of GABAergic sPSC frequency is increased in SPNs recorded from male mice exposed prenatally to ethanol (Fig. 6C). Data highlighted in blue represents a phenotype present in striatal GIN, while those highlighted in green represent a phenotype present in SPN. ↑ indicates a significant increase, ↓ indicates a significant decrease, - indicates no significant differences, * represents trend versus significant result.
Tables
Behavioral task Trials Scoring Description Surface righting 3 0 = absent, 1 = present; time to complete task (s) Pup held on back for 5 s then given 30 s to right itself Auditory startle 3 0 = absent, 1 = present Pup presented with loud tone, observed for startle Tactile startle 3 0 = absent, 1 = present Pup presented with air puff, observed for startle Grasp reflex 3 0 = absent, 1 = present Pup stimulated on forepaw or hindpaw with dull side of metal blade, observed for grasp reflex Horizontal screen test 3 0 = absent, 1 = grasp screen, 2 = lift screen Pup pulled against horizontal wire mesh Vertical screen test 3 0 = absent, 1 = grasp screen, 2 = climb screen Pup pulled against vertical wire mesh Negative geotaxis 3 0 = absent, 1 = orients to horizontal, 2 = orients to vertical, time to complete task (s) Pup placed on 45° wire mesh head facing down, given 45 s to reverse direction and climb Cliff avoidance 3 0 = absent, 1 = present Mouse placed with snout and forepaw digits over a ledge (box), given up to 30 s to remiove Quadruped walking 2 0 = no forward movement, 1 = crawling, asymmetric movements, 2 = crawling, symmetric movements, 3 = fast crawling/walking Mouse recorded for 2 or 3 min in an empty cage Postnatal age Control female Control male Ethanol female Ethanol male P2 8 12 5 13 P4 6 9 5 5 P6 9 10 4 9 P8 18 16 7 11 P10 10 9 7 5 P14 14 21 14 13 Effects Total motor score Quadruped walking score Vertical screen score Surface righting score Surface righting time Negative geotaxis score Negative geotaxis time Tactile startle score Auditory startle score Forepaw grasp score Hindpaw grasp score Horizontal screen score Cliff avoidance score Exposure (E) Wald χ2(1) = 13.975, ***p < 0.001 Wald χ2(1) = 12.266, ***p < 0.001 Wald χ2(1) = 0.000, p = 0.994 Wald χ2(1) = 0.000, p = 1.000 F(1,200) = 1.245, p = 0.266 Wald χ2(1) = 0.000, p = 0.998 F(1,200) = 6.660, *p = 0.011 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 0.999 Sex (S) Wald χ2(1) = 0.418, p = 0.518 Wald χ2(1) = 0.140, p = 0.708 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 0.321 F(1,200) = 9.424, p = 0.002 Wald χ2(1) = 0.000, p = 0.999 F(1,200) = 0.016, p = 0.899 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 0.999 Postnatal day (P) Wald χ2(6) = 313.444, ***p < 0.001 Wald χ2(6) = 145.427, ***p < 0.001 Wald χ2(6) = 19.103, **p = 0.004 Wald χ2(6) = 6.998, p = 1.000 F(5/2000) = 81.703, **p < 0.001 Wald χ2(6) = 109.019, ***p < 0.001 F(5,200) = 131.345, p < 0.0001 Wald χ2(6) = 0.000, p = 1.000 Wald χ2(6) = 0.000, p = 1.000 Wald χ2(6) = 0.000, p = 1.000 Wald χ2(6) = 0.000, p = 1.000 Wald χ2(6) = 73.755, p < 0.001 Wald χ2(6) = 79.290, p < 0.001 E × S Wald χ2(1) = 7.355, **p = 0.007 Wald χ2(1) = 0.713, p = 0.399 Wald χ2(1) = 6.381, *p = 0.012 Wald χ2(1) = 2.050, p = 0.152 F(1,200) = 4,242, *p = 0.041 Wald χ2(1) = 3.625, p = 0.057 F(1,200) = 2.537, p = 0.113 Wald χ2(1) = 0.000, p = 1.000 Wald χ2(1) = 0.000, p = 0.997 Wald χ2(1) = 0.000, p = 0.997 Wald χ2(1) = 1.486, p = 0.223 Wald χ2(1) = 0.909, p = 0.340 Wald χ2(1) = 0.192, p = 0.661 E × P Wald χ2(5) = 2.204, p = 0.820 Wald χ2(5) = 4.098, p = 0.535 Wald χ2(5) = 2.994, p = 0.701 Wald χ2(5) = 1.882, p = 0.865 F(5,200) = 2.628 *p = 0.025 Wald χ2(5) = 6.054, p = 0.301 F(5,200) = 0.964, p = 0.441 Wald χ2(5) = 0.000, p = 1.000 Wald χ2(5) = 0.000, p = 1.000 Wald χ2(5) = 0.000, p = 1.000 Wald χ2(5) = 1.589, p = 0.903 Wald χ2(5) = 1.662, p = 0.894 Wald χ2(5) = 3.515, p = 0.621 P × S Wald χ2(5) = 5.999, p = 0.306 Wald χ2(5) = 4.022, p = 0.546 Wald χ2(5) = 2.238, p = 0.815 Wald χ2(5) = 9.778, p = 0.082 F(5,200) = 3.791, *p = 0.003 Wald χ2(5) = 3.674, p = 0.597 F(5,200) = 0.204, p = 0.961 Wald χ2(5) = 0.000, p = 1.000 Wald χ2(5) = 0.000, p = 1.000 Wald χ2(5) = 0.000, p = 1.000 Wald χ2(5) = 0.000, p = 1.000 Wald χ2(5) = 1.501, p = 0.913 Wald χ2(5) = 2.915, p = 0.713 E × S × P Wald χ2(24) = 323.717, ***p < 0.001 Wald χ2(24) = 158.526, ***p < 0.001 Wald χ2(24) = 97.034, ***p < 0.001 Wald χ2(24) = 50.548, **p = 0.001 F(5,200) = 4.128, p = 0.001 Wald χ2(24) = 154.322, ***p < 0.001 F(5,200) = 1.046, p = 0.392 Wald χ2(24) = 0.000, p = 1.000 Wald χ2(24) = 0.958, p = 1.000 Wald χ2(24) = 25.564, p = 0.376 Wald χ2(24) = 0.072, p = 1.000 Wald χ2(24) = 280.082, ***p < 0.001 Wald χ2(24) = 97.404, ***p < 0.001 Statistical differences in performance on motor behavioral tasks completed by neonatal mice were determined with ordinal logistic regressions (scored behavioral tasks) or three-way ANOVAs (timed behavioral tasks). Major effects: exposure (control chow vs prenatal ethanol exposure embryonic day 13.5–16.5), sex (male vs female), and postnatal day (P2, P4, P6, P8, P10, and P14), with interactions: E × S (exposure × sex), E × P (exposure × postnatal day), P × S (postnatal day × sex), and E × S × P (exposure × sex postnatal day). Statistics with p value ≤0.05 are bolded.
Effects GIN AP threshold GIN AP half-width GIN AP peak amplitude SPN AP threshold SPN AP half-width SPN AP peak amplitude Exposure (E) F(1,231) = 6.504, *p = 0.011 F(1,230) = 7.020, *p = 0.019 F(1,231) = 0.646, p = 0.422 F(1,271) = 0.034, p = 0.853 F(1,271) = 13.193, ***p < 0.001 F(1,271) = 0.006, p = 0.939 Sex (S) F(1,231) = 10.088, **p = 0.002 F(1,230) = 7.268, *p = 0.017 F(1,231) = 0.542, p = 0.463 F(1,271) = 0.016, p = 0.899 F(1,271) = 0.035, p = 0.852 F(1,271) = 0.397, p = 0.529 Postnatal day (P) F(3,231) = 12.869, **p < 0.001 F(3,230) = 81.193, ***p < 0.001 F(3,231) = 2.121, p = 0.098 F(3,271) = 3.173 *p = 0.025 F(3,271) = 13.813, ***p < 0.001 F(3,271) = 13.990, ***p < 0.001 E × S F(1,231) = 0.976, p = 0.324 F(1,230) = 12.062, ***p < 0.001 F(1,231) = 0.021, p = 0.884 F(1,271) = 1.698, p = 0.194 F(1,271) = 1.165, p = 0.281 F(1,271) = 0.980, p = 0.323 E × P F(3,231) = 3.178, *p = 0.025 F(3,230) = 1.977, p = 0.118 F(3,231) = 1.405, p = 0.242 F(3,271) = 7.306, ***p < 0.001 F(3,271) = 2.193, p = 0.089 F(3,271) = 1.544, p = 0.203 P × S F(3,231) = 5.538, **p = 0.001 F(3,230) = 5.826, ***p < 0.001 F(3,231) = 0.300, p = 0.826 F(3,271) = 0.454, p = 0.715 F(3,271) = 0.326, p = 0.807 F(3,271) = 1.424, p = 0.236 E × S × P F(3,231) = 3.316, *p = 0.021 F(3,230) = 7.781, ***p < 0.001 F(3,231) = 1.197, p = 0.312 F(3,271) = 2.620, p = 0.051 F(3,230) = 0.020, p = 0.996 F(3,230) = 0.349, p = 0.790 Statistical differences in AP threshold, half-width, and peak amplitude were determined with three-way ANOVAs. Major effects: exposure (control chow vs prenatal ethanol exposure embryonic day 13.5–16.5), sex (male vs female), and postnatal day (P2, P4, P6, P8, P10, and P14), with interactions: E × S (exposure × sex), E × P (exposure × postnatal day), P × S (postnatal day × sex), and E × S × P (exposure × sex postnatal day). Statistics with p value ≤0.05 are bolded.
Effects GIN RMP GIN input resistance GIN membrane time constant GIN capacitance SPN RMP SPN input resistance SPN membrane time constant SPN capacitance Exposure (E) F(1,230) = 3.728, p = 0.055 F(1,232) = 1.254, p = 0.264 F(1,197) = 0.249, p = 0.619 F(1,197) = 0.642, p = 0.424 F(1,271) = 10.503, **p = 0.001 F(1,271) = 8.100, **p = 0.005 F(1,204) = 5.814, *p = 0.017 F(1,204) = 2.525, p = 0.114 Sex (S) F(1,230) = 4.940, *p = 0.027; F(1,232) = 0.530, p = 0.467 F(1,197) = 0.718, p = 0.398 F(1,197) = 0.187, p = 0.666 F(1,271) = 0.831, p = 0.363 F(1,271) = 0.009, p = 0.923 F(1,204) = 0.017, p = 0.897 F(1,204) = 0.020, p = 0.888 Postnatal day (P) F(3,230) = 22.167, ***p < 0.001 F(3,232) = 95.984 ***p < 0.001 F(3,197) = 11.167, ***p < 0.001 F(3,197) = 14.176, ***p < 0.001 F(3,271) = 4.910, **p = 0.002 F(3,271) = 216.643, ***p < 0.001 F(3,204) = 49.272, ***p < 0.001 F(3,204) = 85.567, **p < 0.001 E × S F(1,230) = 6.523, *p = 0.019 F(1,232) = 1.404, p = 0.237 F(1,197) = 0.257, p = 0.613, F(1,197) = 0.646, p = 0.422 F(1,271) = 1.418, p = 0.235 F(1,271) = 0.484, p = 0.487 F(1,204) = 0.086, p = 0.770 F(1,204) = 2.706, p = 0.102 E × P F(3,230) = 3.671, *p = 0.013 F(3,232) = 0.791, p = 0.500 F(3,197) = 0.362, p = 0.781 F(3,197) = 0.463, p = 0.708 F(3,271) = 1.295, p = 0.276 F(3,271) = 1.555, p = 0.201 F(3,204) = 0.937, p = 0.424 F(3,204) = 0.680, p = 0.565 P × S F(3,230) = 4.065, ***p < 0.001 F(3,232) = 1.378, p = 0.250 F(3,197) = 0.412, p = 0.745 F(3,197) = 1.950, p = 0.123 F(3,271) = 1.339, p = 0.262 F(3,271) = 0.369, p = 0.775 F(3,204) = 4.081, **p = 0.008 F(3,204) = 0.284, p = 0.837 E × S × P F(3,230) = 2.917, *p = 0.035 F(3,232) = 1.083, p = 0.357, F(3,197) = 2.120, p = 0.099 F(3,197) = 1.440, p = 0.232 F(3,271) = 0.670, p = 0.571 F(3,271) = 0.038, p = 0.990 F(3,204) = 0.815, p = 0.487 F(3,204) = 1.645, p = 0.180 Statistical differences in resting membrane potential (RMP), input resistance, membrane time constant, and capacitance were determined with three-way ANOVAs. Major effects: exposure (control chow vs prenatal ethanol exposure embryonic day 13.5–16.5), sex (male vs female), and postnatal day (P2, P4, P6, P8, P10 and P14), with interactions: E × S (exposure × sex), E × P (exposure × postnatal day), P × S (postnatal day × sex), and E × S × P (exposure × sex postnatal day). Statistics with p value ≤0.05 are bolded.
Effects GIN glutamatergic sPSC frequency GIN glutamatergic sPSC amplitude SPN glutamatergic sPSC frequency SPN glutamatergic sPSC amplitude GIN GABAergic sPSC frequency GIN GABAergic sPSC amplitude SPN GABAergic sPSC frequency SPN GABAergic sPSC amplitude Exposure (E) F(1,182) = 0.883, p = 0.349 F(1,182) = 0.164, p = 0.686 F(1,193) = 12.003, ***p < 0.001 F(1,193) = 0.253, p = 0.615 F(1,182) = 2.973, p = 0.476 F(1,172) = 0.751, p = 0.387 F(1,193) = 9.687, **p = 0.002 F(1,192) = 0.009, p = 0.924 Sex (S) F(1,182) = 0.883, p = 0.473 F(1,182) = 0.770, p = 0.381 F(1,193) = 0.305, p = 0.581 F(1,193) = 3.265, p = 0.072 F(1,182) = 6.748, *p = 0.010 F(1,172) = 0.851, p = 0.358 F(1,193) = 0.009, p = 0.923 F(1,192) = 7.388, **p = 0.007 Postnatal age (P) F(1,182) = 168.705, ***p < 0.001 F(1,182) = 6.263, ***p < 0.001 F(1,193) = 18.882, ***p < 0.001 F(1,193) = 12.194, ***p < 0.001 F(3,182) = 88.952, ***p < 0.001 F(1,172) = 9.632, ***p < 0.001 F(1,193) = 38.104, ***p < 0.001 F(1,192) = 9.302, ***p < 0.001 E × S F(1,182) = 0.909, p = 0.342 F(1,182) = 0.026, p = 0.872 F(1,193) = 2.621, p = 0.528 F(1,193) = 0.946, p = 0.332 F(1,182) = 0.064, p = 0.801 F(1,172) = 0.311, p = 0.578 F(1,193) = 0.105, p = 0.747 F(1,192) = 0.276, p = 0.600 E × P F(3,182) = 0.789, p = 0.501 F(3,182) = 3.885, *p = 0. 010 F(3,193) = 2.621, p = 0.052 F(3,193) = 1.199, p = 0.311 F(3,182) = 1.859, p = 0.138 F(3,172) = 4.467, **p = 0.005, F(3,193) = 0.365, p = 0.778 F(3,192) = 2.836, *p = 0.039, P × S F(3,182) = 0.807, p = 0.492 F(3,182) = 0.692, p = 0.058 F(3,193) = 3.487, p = 0.017 F(3,193) = 1.805, p = 0.148 F(3,182) = 2.581, p = 0.055 F(3,172) = 0.775, p = 0.509 F(3,193) = 2.107, p = 0.101 F(3,192) = 1.155, p = 0.328 E × S × P F(3,182) = 2.536, p = 0.058 F(3,182) = 0.603, p = 0.614 F(3,193) = 0.526, p = 0.665 F(3,193) = 0.706, p = 0.549 F(3,182) = 0.171, p = 0.916 F(3,172) = 1.638, p = 0.182 F(3,193) = 2.099, p = 0.102 F(3,192) = 0.324, p = 0.808 Statistical differences in glutamatergic and GABAergic postsynaptic current frequency and amplitude were determined with three-way ANOVAs. Major effects: exposure (control chow vs prenatal ethanol exposure embryonic day 13.5–16.5), sex (male vs female), and postnatal day (P2, P4, P6, P8, P10, and P14), with interactions: E × S (exposure × sex), E × P (exposure × postnatal day), P × S (postnatal day × sex), and E × S × P (exposure × sex postnatal day). Statistics with p value ≤0.05 are bolded.
Effects Mean length/dendrite (µm) Mean nodes/dendrite (µm) Soma area (µm2) Exposure (E) F(1,232) = 1.030, p = 0.311 F(1,232) = 0.007, p = 0.933 F(1,232) = 1.030, p = 0.412 Sex (S) F(1,232) = 3.724, p = 0.055 F(1,232) = 1.362, p = 0.244 F(1,232) = 0.141, p = 0.707 Postnatal age (P) F(1,232) = 29.118, ***p < 0.001 F(1,232) = 7.700, ***p < 0.001 F(3,232) = 3.699,*p = 0.012 E × S F(1,232) = 1.162, p = 0.282 F(1,232) = 7.159, **p = 0.008 F(1,232) = 10.014,** p = 0.002 E × P F(3,232) = 1.503, p = 0.214 F(3,232) = 0.901, p = 0.442 F(3,232) = 1.503, p = 0.214 P × S F(3,232) = 11.774, ***p < 0.001 F(3,232) = 2.335, p = 0.075 F(3,232) = 2.890, *p = 0.036 E × S × P F(3,232) = 5.453, **p = 0.001 F(3,232) = 1.037, p = 0.377 F(3,232) = 1.540, p = 0.205 Statistical differences in mean length/dendrite, mean nodes/dendrite, and soma area were determined with three-way ANOVAs. Major effects: exposure (control chow vs prenatal ethanol exposure embryonic day 13.5–16.5), sex (male vs female), and postnatal day (P2, P4, P6, P8, P10, and P14), with interactions: E × S (exposure × sex), E × P (exposure × postnatal day), P × S (postnatal day × sex), and E × S × P (exposure × sex postnatal day). Statistics with p value ≤0.05 are bolded.
Figure 1-1
Prenatal ethanol exposure alters the development complex but not reflexive behaviors. Prenatal ethanol exposure results in significant differences in (A) surface righting score (B) negative geotaxis score (C) horizontal screen score, (D) cliff avoidance score, that depend on both sex and postnatal age (Kruskall-Wallis tests, surface righting score: P2: H(3) = 6.102, p = 0.107, P4: H(3) = 8.660, p = 0.034, Dunn’s post-hoc tests: ethanol F vs. control F: p=0.042, P6: H(3) = 3.176, p = 0.365, P8: H(3) = 0.796, p = 0.850, P10: H(3) = 0.000, p = 1.000, P14: H(3) = 0.000, p = 1.000; negative geotaxis score: P2: H(3) = 1.319, p = 0.725, P4: H(3) = 1.566, p = 0.667, P6: H(3) = 1.633, p = 0.652, P8: H(3) = 12.926, p = 0.005, Dunn’s post-hoc tests: ethanol M vs. ethanol F: P8: p= 0.007, P10: H(3) = 3.064, p = 0.382, P14: H(3) = 6.392, p = 0.094; horizonal screen score: P2: H(3) = 0.000, p = 1.000, P4: H(3) = 0.000, p = 1.000, P6: H(3) = 5.348, p = 0.148, P8: H(3) = 2.145, p = 0.543, P10: H(3) = 1.534, p = 0.674, P14: H(3) = 1.012, p = 0.798; cliff avoidance score: P2: H(3) = 9.160, p 0.027, Dunn’s post-hoc, control F vs. control M: P2: p=0.017, P4: H(3) = 3.190, p = 0.363, P6: H(3) = 5.861, p = 0.119, P8: H(3) = 0.159, p = 0.984, P10: H(3) = 1.250, p = 0.741, P14: H(3) = 5.279, p = 0.152). No differences were observed between groups in (E) tactile startle score, (F) auditory startle score, (G) forepaw grasp score, (H) hindpaw grasp score. Data are presented as mean score or time, error bars are standard error of the mean (SEM), **p<0.01, control male vs. ethanol male; +p<0.05, control male vs. control female; xp<0.05, ethanol male vs. ethanol female. Download Figure 1-1, TIF file.
Figure 3-1
Prenatal ethanol exposure differentially effects the intrinsic properties of striatal GINs from female and male mice, depending on the postnatal day. (A) IV curves for responses to hyperpolarizing current steps during whole-cell current clamp recordings of striatal GINs during the first postnatal week (2-way ANOVAs, P2: group: F(3,450)= 7.015, p=0.=0001, current: F(8,450) = 63.35, p<0.001, group x current: F(24,450)=0.2834, p=0.9996; P4-6: group: F(3,468)= 11.35, p<0.001, current: F(8,468) = 38.60, p<0.001, group x current: F(24,468)=0.2114, p=1.000; P8-10: group: F(3,621)= 25.22, p<0.001, current: F(8,621) = 74.29, p<0.001, group x current: F(24,621)=0.2189, p=0.9890; P14: group: F(3,671)= 15.53, p<0.001, current: F(10,671)= 83.66, p<0.001, group x current: F(30,671)=0.08655, p=1.000. (B) Prenatal ethanol exposure resulted in sex-dependent differences in resting membrane potential (RMP) that varied based on the postnatal day, though no significant differences were observed between groups on individual postnatal days (1 way ANOVAs, P2: F(3,50)= 2.008, p=0.125, P4-6: F(3,50)= 1.293, p=0.287, P8-10: F(3,69)= 2.244, p=0.091, P14: F(3,61)=0.642, p=0.591). (C) Input resistance (IR) (mΩ) and (D) membrane time constant (ms) were unaffected in striatal GINs were unaffected by prenatal ethanol exposure, sex or postnatal day (1-way ANOVAs, IR: P2: F(3,50) = 0.611, p =0.649 P4-6: F(3,50) = 0.219, p =0.883; P8-10: F(3,69) = 0.557, p =0.302; P14: F(3,61) = 0.611, p =0.610; membrane time constant: P2: F(3,50) = 1.318, p=0.279; P4-6: F(3,61) = 0.689, p =0.563; P8-10: F(3,69) = 1.239, p =0.302; P14: F(3,61) = 1.277, p =0.294). Data are presented as means (bars), error bars are standard error of the mean (SEM), dots are individual neurons from at least 3 animals per group. $p<0.05, control male vs. ethanol female. Download Figure 3-1, TIF file.
Figure 3-2
Prenatal ethanol exposure does not alter the action potential (AP) amplitude of developing striatal GABAergic interneurons (GINs) or striatal projection neurons (SPNs). (A) The peak amplitude of APs (pA) did not differ in striatal GINs or (B) SPNs following prenatal ethanol exposure. Data are presented as means (bars), error bars are standard error of the mean (SEM), dots are individual neurons from at least 3 animals per group. Download Figure 3-2, TIF file.
Figure 4-1
Prenatal ethanol exposure differentially effects the intrinsic properties of striatal SPNs from female and male mice, depending on the postnatal day. (A) The effects of prenatal ethanol exposure on IV curves for responses to hyperpolarizing current steps during whole-cell current clamp recordings of SPNs during the first postnatal week vary by group (2-way ANOVAs, P2: group: F(3,531)= 3.047, p=0.0284, current: F(8,531) = 151.8, p<0.001, group x current: F(24, 531)=0.2490, p=0.9999; P4-6: group: F(3,540)= 13.10, p<0.001, current: F(8,540) = 169.0, p<0.001, group x current: F(24, 540)=0.4369, p=0.9999; P8-10: group: F(3,765)= 14.67, p<0.001, current: F(8,765) = 164.9, p<0.001, group x current: F(24,765)=0.4551, p=0.9890; P14: group: F(3,770)= 3.537, p=0.0145, current: F(10,770) = 243, p<0.001, group x current: F(30,770)=0.1753, p=1.000). (B) Prenatal ethanol exposure resulted in sex-dependent differences in SPN RMP that varied based on the postnatal day: At P4-6: prenatal ethanol exposure resulted in significantly depolarized RMP in male mice relative to control-fed male and female mice (1-way ANOVA: F(3,66) = 4.632, p=0.005, Bonferroni post-hoc tests: ethanol M vs. control F: p=0.023, ethanol M vs control F: p=0.004). SPN RMP was unaltered by prenatal ethanol exposure at P2, 8-10 or P14 (1-way ANOVAs, P2: F(3,59) = 1.790, p =0.159; P8-10: F(3,81) = 0.796, p =0.499; P14: F(3,73) = 1.042, p = 0.379). (C) Prenatal ethanol exposure did not alter the IR of SPNs (1-way ANOVAs, P2: F(3,59) = 0.946, p =0.424; P4-6: F(3,66) = 1.039, p =0.202; P8-10: F(3,81) = 1.182, p =0.322; P14: F(3,73) = 1.034, p=0.383). (D) Prenatal ethanol exposure results in decreased membrane time constant in SPNs from ethanol-exposed M mice at P8-10, relative to control-fed F mice, but did not alter membrane time constant in F mice. (1-way ANOVAs, P2: F(3,59) = 1.175, p =0.327; P4-6: F(3,66) =0.934, p =0.430; P8-10: F(3,81) = 3.405, p =0.024; P14: F(3,73) = 2.283, p = 0.093. Data are presented as means (bars), error bars are standard error of the mean (SEM), dots are individual neurons from at least 3 animals per group. *p<0.05, control male vs. ethanol male; @@p<0.01, control female vs. ethanol male. Download Figure 4-1, TIF file.
Figure 4-2
Prenatal ethanol exposure does not alter membrane capacitance of developing striatal GABAergic interneurons (GINs) or striatal projection neurons (SPNs) (A) Membrane capacitance was unaffected by prenatal ethanol exposure in striatal GINs or (B) SPNs. Data are presented as means (bars), error bars are standard error of the mean (SEM), dots are individual neurons from at least 3 animals per group. Download Figure 4-2, TIF file.
Figure 7-1
Prenatal ethanol exposure result in early postnatal increases in spiny projection neuron (SPN) dendritic morphology: length, number, branching, and soma area. (A) Prenatal ethanol exposure decreased the mean length/dendrite (µM) in SPN F mice relative to control-fed F while control-male mice also displayed significantly decreased mean length/dendrite relative to control fed F at P2, while prenatal ethanol exposure resulted in no significant differences at P4-6. At P8-10 prenatal ethanol exposure increased the mean length/dendrite in SPNs from M mice relative to ethanol-exposed and control-fed F mice, control-fed male mice also displayed significantly increased mean length/dendrite relative to control-fed F mice. At P14, SPNs from ethanol-exposed M mice relative displayed a decreased mean length/dendrite relative to ethanol-exposed and control-fed F mice. (P2: (Kruskall-Wallis test, H(3) = 13.49, p=0.0039, Dunn’s post-hoc tests: ethanol F vs. control F: p<0.05, ethanol F vs. control M, p>0.05; P4-6: Kruskal-Wallis test, H(3) = 6.905, p=0.0750; P8-10: one-way ANOVA, F(3,59) = 6.276, p = 0.001, Bonferroni post-hoc tests: ethanol M vs. ethanol F, t= 3.427, p<0.01, ethanol M vs. control F, t=3.710, p<0.01; P14: one-way ANOVA, F(3,64) = 3.962, p = 0.0118, Bonferroni post-hoc tests: ethanol M vs. ethanol F, t= 3.308, p<0.01). (B) Prenatal ethanol exposure results in a transient increase in the number of dendrites in SPNs from P2 and P4-6 F mice relative to those from control-fed F and ethanol-exposed M mice of the same ages, that resolves by P8-10 (Kruskal-Wallis tests, P2: H(3)= 12.832, p=0.005, Dunn’s post-hoc tests: ethanol F vs. control F: p< 0.05, ethanol F vs. ethanol M, p<0.05; P4-6: H(3) = 14.116, p=0.003, Dunn’s post-hoc tests: ethanol F vs. control F: p<0.01, ethanol F vs. ethanol M, p<0.01; P8-10: H(3)= 0.897, p=0.826; P14: H(3)= 0.747, p=0.862). (C) Prenatal ethanol exposure resulted in trend towards a decreased mean number of nodes/dendrite in SPNs from P4-6 F mice relative to control-fed F mice, and ethanol-exposed M mice. Control-fed F mice had increased mean nodes/dendrite relative to control-fed M mice at P2. No differences in mean nodes/dendrite mean nodes per dendrite were observed in SPNs at P8-10, or P14 (P2: one-way ANOVA, F(3,58)=4.596, p= 0.061, Bonferroni post-hoc tests, control F vs. control M, p<0.05; P4-6: Kruskal-Wallis test, H(3) = 8.619. p=0.0348; P8-10: one-way ANOVA: F(3,59) = 1.340, P14: F(3,67) = 0.6633). (D) Prenatal ethanol exposure increased the soma area (µM2) if SPNs from F mice, relative to those from control-fed F mice at P4. SPNs from control-fed F mice also had decreased soma area relative to those from control-fed male mice at P4. No differences in soma area were observed at P2, P8-10 or P14. P2: one way ANOVA, F(3,58) = 0.4028, p = 0.7515; P4: one-way ANOVA, F(3,60) = 5.002, p<0.0038, Bonferroni post-hoc tests: ethanol F vs. control F, t=3.121, p<0.05, control F vs. control M, t = 3.591, p<0.01; P8-10: one-way ANOVA, F(3,59) = 0.7238, p= 0.5420; P14: Kruskall-Wallis test, H(3) = 6.232. Data are presented as means (bars), error bars are standard error of the mean (SEM), dots are individual neurons from at least 3 animals per group. For all: +p<0.05, ++p<0.01, control M vs. control F; #p<0.05, ##p<0.01, control M vs. ethanol M, @p<0.05; @@@p<0.001, control F vs. ethanol M, $$ p<0.01 control M vs. ethanol F; xp<0.05, xxp<0.01, ethanol F vs. ethanol M. Download Figure 7-1, TIF file.
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