Time-specific effects of ethanol exposure on cranial nerve nuclei: gastrulation and neuronogenesis

Abstract

During the development of the central nervous system, neurons pass through critical periods or periods of vulnerability. We explored periods of vulnerability for cranial nerve nuclei by determining the effects of acute exposure to ethanol during development on the number of neurons in mature brainstem. Long-Evans rats were injected with 2.9 g ethanol/kg body weight on one day between gestational day (G) 7 and G13, inclusive. Two hours later, animals received a second injection of 1.45 g/kg. Controls were injected with equivalent volumes of saline. Brainstems of 31-day-old offspring were cryosectioned and stained with cresyl violet. Stereological methods were used to determine the volume and numerical density of neurons in three trigeminal sensory nuclei (the principal sensory nucleus of the trigeminal nerve, and the oral and interpolar subnuclei of the spinal trigeminal nuclear complex) and three motor nuclei (the trigeminal, facial, and hypoglossal nuclei). The numbers of neurons in most nuclei were lower following early (on G7 and/or G8) or later (on G12 and/or G13) exposure. Only the trigeminal interpolar nucleus was affected by neither early nor late ethanol exposure. Thus, prenatal exposure to ethanol affects the number of neurons in brainstem nuclei in a time-dependent manner. Windows of vulnerability coincide with gastrulation (G7/G8) and the period of neuronal generation (G12/G13).

Figure 1

Blood ethanol concentration (BEC). The BEC rose precipitously to reach 233 mg/dl at 1.0 hour after the first injection. Peak BEC was reached two hours later. By eight hours post-injection, the BEC was 0. Arrows show the two injection times. Symbols represent the means of four animals (± standard errors of the means).

Figure 2

Appearance of the sensory cranial nerve nuclei Three sensory trigeminal nuclei were identifiable in horizontal sections stained with cresyl violet. Samples representing the animals treated with saline (controls) or ethanol were taken from cohorts dosed on gestational day 8. No gross differences were apparent between the groups. Rostral is oriented to the left and lateral to the top. Scale bars are 500 μm.

Figure 3

Quantitative measures of the effects of ethanol on sensory nuclei Stereological methods were used to estimate the volume (top) and the neuronal density of each nucleus (middle). Total neuronal number was estimated as the product of volume and density. Bars represent the means and T-bars signify the standard errors of the means. Each mean is based on five or six animals per group. Asterisks identify differences relative to the controls that were statistically significant (p<0.05).

Figure 4

Appearance of the motor cranial nerve nuclei Three motor nuclei (the motor nuclei of the trigeminal (MoV), facial (MoVII), and hypoglossal (MoXII) nerves were evident in horizontal sections. Images are oriented so that rostral is to the left and lateral to the top. Scale bars are 500 μm.

Figure 5

Quantitative measures of the effects of ethanol on motor nuclei Notations as in Figure 3.