Rendered images of Lobule I Purkinje cells with VGluT2/climbing fiber innervation. Images were produced using Imaris software. A: Rendered image of the calbindin-D28k immunostaining demonstrating cerebellar Purkinje cells. Note that the soma layer is outlined in blue. The background squares are 20 microns per side. B: Rendered image of the VGluT2 immunostaining. This represents the VGluT2 positive portion of the climbing fibers from the inferior olive. C: Combined red and green channels showing the interactions of the Purkinje cells with the climbing fibers.

Purkinje cell (PC) numbers in Lobule I of PN14 pups following ethanol exposure at two dose levels (3.6 g/kg and 4.5 g/kg) during the early postnatal period. Analyses identified a significant main effect of treatment (F_(6,70)=4.593, p=0.0005). Custom contrasts at the p<0.05 level identified significant differences within the 3.6 g/kg cohort for E4 and E4-6 vs E7-9 (†) and within the 4.5 g/kg cohort for E4 vs C4 (*) as well as E4-6 vs C4-6 (*). No significant impact of sex was identified (F_(1,70)=0.080). MRC14 = mother reared control group; C4, C4-6, C7-9 = Isocaloric vehicle control groups for PN4, PN4-6, and PN7-9, respectively; E4, E4-6, E7-9 = Ethanol exposure on PN4, PN4-6, and PN7-9, respectively.

Body weights of PN14 pups following ethanol exposure at two dose levels (3.6 g/kg and 4.5 g/kg) during the early postnatal period. Analyses identified a significant main effect of treatment (F_(6,99)=6.524, p<0.001) and dose (F_(1,99)=4.91, p<0.03). Custom contrasts at the p<0.05 level identified significant differences within the 3.6 g/kg dose cohort for E7-9 vs C7-9 (*), and E4 (‡); and within the 4.5 g/kg dose cohort for E7-9 vs E4 (‡). No significant impact of sex was identified (F_(1,99)=0.002). MRC14 = mother reared control group; C4, C4-6, C7-9 = Isocaloric vehicle control groups for PN4, PN4-6, and PN7-9, respectively; E4, E4-6, E7-9 = Ethanol exposure on PN4, PN4-6, and PN7-9, respectively.

Alterations in Climbing Fiber endings in the Molecular Layer of Lobule I on PN14 following ethanol exposure at two dose levels (3.6 g/kg and 4.5 g/kg) during the early postnatal period. MRC14 = mother reared control group; C4, C4-6, C7-9 = Isocaloric vehicle control groups for PN4, PN4-6, and PN7-9, respectively; E4, E4-6, E7-9 = Ethanol exposure on PN4, PN4-6, and PN7-9, respectively. Staining and confocal image acquisition was completed as two cohorts by ethanol dose.
A. VGlut2 expression intensity relative to total PC tissue volume (VG2 Intensity-Ratio). There were no significant control group differences (F_{(3, 62)}=0.75) that impact the conclusions. Treatment analyses identified a significant treatment x dose interaction (F_{(6, 99)}=4.772; p<0.0003). Custom contrasts at the p<0.05 level identified significant differences only within the 4.5 g/kg dose cohort for E4 vs C4 (*) and E7-9 (†) and E4-6 vs C4-6 (*) and E7-9 (†). No significant impact of sex was identified (F_(1,99)=0.003).
B. Co-localized expression intensity relative to total PC tissue volume (Co-localized Intensity-Ratio). There were no significant control group differences (F_{(3, 62)}=1.30) that impact the conclusions. Treatment analyses identified a significant treatment x dose interaction (F_{(6, 99)}=3.343; p<0.005). Custom contrasts at the p<0.05 level identified significant differences only within the 4.5 g/kg dose cohort for E4 vs C4 (*) and E7-9 (†)and for E4-6 vs C4 -6(*) and E7-9 (†). No significant impact of sex was identified (F_(1,99)=0.259).
C. VGlut2 expression volume relative to total PC tissue volume (VG2 Volume-Ratio).There was a significant difference within the control groups (F_{(3, 62)}=3.92; p<0.02). This difference is between the lowest group value C7-9 (0.144±0.017) compared to the MRC14 (0.211±0.015) and C4 (0.214±0.017) values. These differences do not impact the conclusions or interpretation of the effects of ethanol treatment in this study. Treatment analyses identified a significant treatment x dose interaction (F_{(6, 99)}=6.313; p<0.001). Custom contrasts at the p<0.05 level identified significant differences only within the 4.5 g/kg dose cohort for E4 vs C4 (*) and E7-9 (†), and E4-6 vs C4-6 (*) and E7-9 (†). No significant impact of sex was identified (F_(1,99)=3.08; p=0.082).
D. Co-localized expression volume relative to total PC tissue volume (Co-localized Volume-Ratio). There were no significant control group differences (F_{(3, 62)}=1.27) that impact the conclusions. Treatment analyses identified a significant treatment x dose interaction (F_{(6, 99)}=2.238; p<0.05). Custom contrasts at the p<0.05 level identified significant differences only within the 4.5 g/kg dose level for E4 vs E7-9 (†), E4-6 vs E7-9 (†), and E7-9 vs C7-9 (*). No significant impact of sex was identified (F_(1,99)=2.174; p=0.14).

Alterations in Climbing Fiber endings in the Soma region of Lobule I on PN14 following ethanol exposure at two dose levels (3.6 g/kg and 4.5 g/kg) during the early postnatal period. MRC14 = mother reared control group; C4, C4-6, C7-9 = Isocaloric vehicle control groups for PN4, PN4-6, and PN7-9, respectively; E4, E4-6, E7-9 = Ethanol exposure on PN4, PN4-6, and PN7-9, respectively. Staining and confocal image acquisition was completed as two cohorts by ethanol dose.
A. VGlut2 expression intensity relative to total PC tissue volume (VG2 Intensity-Ratio). There were no significant control group differences (F_{(3, 62)}=1.618; p=0.19) that impact the conclusions. Treatment analyses identified a significant main effect of treatment (F_{(6, 99)}=2.971; p=0.0104). Custom contrasts at the p<0.05 level identified significant differences only within the 4.5 g/kg dose cohort for E4 vs C4 (*). No significant impact of sex was identified (F_(1,99)=0.0011).
B. Alterations in the Colocalized expression intensity relative to total PC tissue volume (Colocalized Intensity-Ratio). There were no significant control group differences (F_{(3, 62)}=0.7354) that impact the conclusions. Treatment analyses identified a significant treatment x dose interaction (F_{(6, 99)}=2.650; p=0.020). Custom contrasts at the p<0.05 level identified significant differences within the 4.5 g/kg dose cohort for E4 vs C4 (*) and E7-9 (†), as well as E4-6 vs E7-9 (†), and E7-9 vs C7-9 (*). No significant impact of sex was identified (F_(1,99)=0.8718).
C. Alterations in the VGlut2 expression volume relative to total PC tissue volume (VG2 Volume-Ratio). There was a significant difference within the control groups (F_{(3, 62)}=4.714; p=0.005). This difference is between the C7-9 group (0.162±0.021) compared to the C4 group (0.246±0.020) or the MRC14 group (0.254±0.018. These differences do not impact the conclusions or interpretation of the effects of ethanol treatment in this study. Treatment analyses identified a significant treatment x dose interaction (F_{(6, 99)}=3.952; p=0.002). Custom contrasts at the p<0.05 level identified significant differences only within the 4.5 g/kg dose level for E4 vs C4 (*) and E7-9 (†) and for E4-6 vs the E7-9 group (†). No significant impact of sex was identified (F_(1,99)=1.308; p=0.255).
D. Alterations in relative Colocalized expression volume relative to total PC tissue volume (Colocalized Volume-Ratio). There were no significant control group differences (F_{(3, 62)}=0.597) that impact the conclusions. Treatment analyses identified significant treatment x dose interaction (F_{(6, 99)}=2.487; p=0.028). Custom contrasts at the p<0.05 level identified significant differences only within the 4.5 g/kg dose level for E4 and E4-6 vs E7-9 (†), as well as E7-9 vs C7-9 (*). No significant impact of sex was identified (F_(1,99)=2.380; p=0.126).