Neurotrophin ligand-receptor systems in somatosensory cortex of adult rat are affected by repeated episodes of ethanol

Abstract

Ethanol exposure profoundly affects learning and memory and neural plasticity. Key players underlying these functions are neurotrophins. The present study explored the effects of ethanol on the distribution of neurotrophins in the cerebral cortex of the adult rat. Age- and weight-matched pairs of adult male, Long-Evans rats were fed a liquid, ethanol-containing (6.7% v/v) diet or pair-fed an isocaloric control diet three consecutive days per week for 6, 12, 18, or 24 weeks. Brains were processed immunohistochemically for nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression and for the expression of three neurotrophin receptors, p75, trkA, and trkB. Total numbers of immunolabeled neurons in specific layers of somatosensory cortex of ethanol- and control-fed animals were determined stereologically. Ethanol exposure induced an increase in the numbers of NGF- or BDNF-expressing neurons and in neurotrophin content per somata. These changes were (a) time and (b) laminar dependent. In contrast, the number of receptor-expressing neurons did not change due to ethanol exposure or to length of time on the ethanol diet. Thus, ethanol induces the recruitment of cortical neurons to express neurotrophins and an increase in the amount of neurotrophin expression per neuron.

Figure 1

Distribution of neurotrophin expression in somatosensory cortex. Pairs of animals were fed either the control (Ct) or ethanol (Et) liquid diet and processed for neurotrophin immunohistochemistry. The distribution of NeuN, NGF, and BDNF immunolabeling through the full depth of cortex (top) and in layer V (bottom) are shown. [n.b. The images provided were obtained from tissue prepared with matched incubations; they reflect the treatment-induced differences in immunolabeling. The specific labeling in the present study was like that described previously (Pitts and Miller, 2000).] Horizontal lines indicate laminar boundaries and Roman numerals identify the layers. Vertical scale bars in the lower right of each micrograph are 50 μm.

Figure 2

Layer volumes in somatosensory cortex of adult rat. Total volume of layer II/III (circles) and layer V (squares) was estimated by Cavalieri’s method in both control-fed (filled symbols and solid lines) and ethanol-fed (open symbols and dashed lines) rats. Each point represents the mean ± standard error of 5 (6 and 12 wk exposures) or 6 (18 and 24 wk exposures) rats per group.

Figure 3

Numbers of NeuN-, NGF-, and BDNF-immunopositive cells. Stereological methods were used to determine the total numbers of cells in layers II/III and V that were immunoreactive for NeuN, NGF, or BDNF. Rats were maintained on a control diet (Ct; filled circles) or an ethanol-containing diet (Et; open circles) for 6, 12, 18, or 24 weeks. Each symbol represents the mean of at least five rats (± the standard error of the mean). Linear regression lines were fit to the data: Ct (solid lines) and Et (dashed lines). Bolded lines indicate data that were significantly different.

Figure 4

Amount of cellular NGF and BDNF expression. Rats were exposed to ethanol episodically during six weeks. Quasi-quantitative microdensitometry was used to determine the relative intensity of NGF and BDNF immunoreactivity in both layer II/III (top) and layer V (bottom) on a per cell basis. Relative optical densities were determined in 50 cells per layer per animal using a total of five yoked pairs. Data are normalized against the amount detected in the controls. Asterisks identify statistically significant (p < 0.05) differences.

Figure 5

Distribution of neurotrophin and receptor expression in somatosensory cortex. Expression of receptors, i.e., p75, trkA, and trkB, in cortices of ethanol-treated and control rats is shown. Notations as for Figure 1.

Figure 6

Numbers of neurons expressing neurotrophin receptors. Stereological analysis results are illustrated here from animals maintained on the control or ethanol diet for 6, 12, 18, or 24 weeks. Data shown represent the means of at least five rats (± standard error of the means) per group. No significant differences were observed between treatment groups or with length of treatment.