Effects of Ethanol and NAP on Cerebellar Expression of the Neural Cell Adhesion Molecule L1

The neural cell adhesion molecule L1 is critical for brain development and plays a role in learning and memory in the adult. Ethanol inhibits L1-mediated cell adhesion and neurite outgrowth in cerebellar granule neurons (CGNs), and these actions might underlie the cerebellar dysmorphology of fetal alcohol spectrum disorders. The peptide NAP potently blocks ethanol inhibition of L1 adhesion and prevents ethanol teratogenesis. We used quantitative RT-PCR and Western blotting of extracts of cerebellar slices, CGNs, and astrocytes from postnatal day 7 (PD7) rats to investigate whether ethanol and NAP act in part by regulating the expression of L1. Treatment of cerebellar slices with 20 mM ethanol, 10−12 M NAP, or both for 4 hours, 24 hours, and 10 days did not significantly affect L1 mRNA and protein levels. Similar treatment for 4 or 24 hours did not regulate L1 expression in primary cultures of CGNs and astrocytes, the predominant cerebellar cell types. Because ethanol also damages the adult cerebellum, we studied the effects of chronic ethanol exposure in adult rats. One year of binge drinking did not alter L1 gene and protein expression in extracts from whole cerebellum. Thus, ethanol does not alter L1 expression in the developing or adult cerebellum; more likely, ethanol disrupts L1 function by modifying its conformation and signaling. Likewise, NAP antagonizes the actions of ethanol without altering L1 expression

Opiate sensitization induces FosB/ΔFosB expression in prefrontal cortical, striatal and amygdala brain regions.

Sensitization to the effects of drugs of abuse and associated stimuli contributes to drug craving, compulsive drug use, and relapse in addiction. Repeated opiate exposure produces behavioral sensitization that is hypothesized to result from neural plasticity in specific limbic, striatal and cortical systems. ΔFosB and FosB are members of the Fos family of transcription factors that are implicated in neural plasticity in addiction. This study examined the effects of intermittent morphine treatment, associated with motor sensitization, on FosB/ΔFosB levels using quantitative immunohistochemistry. Motor sensitization was tested in C57BL/6 mice that received six intermittent pre-treatments (on days 1, 3, 5, 8, 10, 12) with either subcutaneous morphine (10 mg/kg) or saline followed by a challenge injection of morphine or saline on day 16. Mice receiving repeated morphine injections demonstrated significant increases in locomotor activity on days 8, 10, and 12 of treatment (vs. day 1), consistent with development of locomotor sensitization. A morphine challenge on day 16 significantly increased locomotor activity of saline pre-treated mice and produced even larger increases in motor activity in the morphine pre-treated mice, consistent with the expression of opiate sensitization. Intermittent morphine pre-treatment on these six pre-treatment days produced a significant induction of FosB/ΔFosB, measured on day 16, in multiple brain regions including prelimbic (PL) and infralimbic (IL) cortex, nucleus accumbens (NAc) core, dorsomedial caudate-putamen (CPU), basolateral amygdala (BLA) and central nucleus of the amygdala (CNA) but not in a motor cortex control region. Opiate induced sensitization may develop via Fos/ΔFosB plasticity in motivational pathways (NAc), motor outputs (CPU), and associative learning (PL, IL, BLA) and stress pathways (CNA)

L1 expression in adult cerebellum after chronic binge drinking.

<p>Total RNA and tissue lysates were collected from cerebella of adult rats following one year of self-administration of ethanol (2% sucrose/10% ethanol) or sucrose (2% sucrose). L1 mRNA expression was measured by qRT-PCR and normalized to 18S. L1 protein levels were measured by Western blot and normalized to β-tubulin. The inset shows representative Western blot images above corresponding bars. Bars represent normalized mean + SEM (ethanol, n = 6; sucrose, n = 7).</p

Summary of ethanol and NAP effects on L1 mRNA and protein expression in three cerebellar culture systems.

<p>Summary of ethanol and NAP effects on L1 mRNA and protein expression in three cerebellar culture systems.</p

RNA quality, assay reliability, and validation of endogenous controls.

<p>Total RNA quality was assessed using an Agilent Bioanalyzer 2100 and endogenous controls were validated for RT-qPCR and Western blot. A) Generated gel images show representative slice, CGN, and astrocyte samples with and without 24-hr ethanol treatment. B) A standard curve was performed using a dilution series of purified L1 template. Increases in template concentration result in decreasing Cq values, as expected for a well-functioning qPCR assay (n = 4). C) 18S Cq values are shown for all tissue/cell types with and without ethanol treatment. D) Representative Western blots for β-tubulin are shown for each sample type with and without 24-hr ethanol treatment. All bars (C, D) show the mean + SEM of 4 independent experiments.</p

Primers used for PCR amplification of target genes.

<p>Primers used for PCR amplification of target genes.</p

Intermittent morphine administration results in the development of locomotor sensitization in C57BL/6 mice.

<p>Mice were given either a saline (10 mg/ml) or morphine (10 mg/kg) injection on days 1, 3, 5, 8, 10, 12 and a locomotor activity test was conducted and recorded immediately after each injection for 180 min. Data are recorded as total distance traveled for 180 min and shown as total distance traveled (cm) ± S.E.M in each treatment group (n = 8–16 mice per group). *<i>P</i><0.05 in comparison to the morphine treated group's first day of testing.</p

Quantitative analyses of FosB/ΔFosB immunoreactivity from subjects from the vehicle and morphine pre-treatment groups following vehicle injection and motor activity testing on day 16.

<p>The quantification of the ΔFosB/FosB expression was determined by immunohistochemistry from multiple sections in each of the following brain regions: PL, IL, M1/2, NAcC, NAcS, CPU, CNA and BLA. Data are shown as mean ± S.E.M of n = 4–8 mice from each treatment group. *<i>P</i><0.05, **<i>P</i><0.01, and ***<i>P</i><0.001 vs. vehicle pre-treatment group.</p