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    • levels of Relative to the impact of moderate PAE on markers

    2022-03-03

    Relative to the impact of moderate PAE on markers of histaminergic neurotransmission, there were four salient observations from the studies reported here. First, it does not appear that moderate PAE affects the number of HDC-positive neurons in the tuberomammillary nucleus of the ventral hypothalamus. These results are in contrast to previous studies of the effects of PAE on brainstem raphe serotonergic neurons, where 5-hydroxytryptophan-positive neural cell numbers were reduced in PAE rodent offspring compared to controls (Sari and Zhou, 2004, Tajuddin and Druse, 1999, Zhou et al., 2002). A number of methodological differences between these studies and our study might be the basis for differential outcomes, including use of different rodent species or rat stocks, different ethanol exposure paradigms that result in different ethanol exposure levels, or measuring ethanol effects at different offspring ages. In particular, the peak maternal serum ethanol concentration achieved in the present study using a voluntary drinking paradigm (60 mg/dL) is about 20–70 mg/dL lower than the range of peak maternal ethanol concentrations reported previously using ethanol liquid diets in mice and rats (Tajuddin and Druse, 1999, Zhou et al., 2002). Thus, it is possible that higher peak maternal serum ethanol levels may have a greater impact on HDC-positive cell number in ventral hypothalamus. One limitation in these IHC studies is that stereological approaches were not employed that may have provided more assurance of the absolute numbers of HDC+ neurons. However, given that the analytical method used was identical for all rats, any sampling error inherent to the approach should be consistent for the two prenatal treatment groups. While the number of ventral hypothalamic HDC-positive cells does not appear to be different in PAE rats, ethanol may have disrupted histamine axonal navigation or innervation of target projections in a manner that could have reduced the distribution or density of histamine nerve terminals in various levels of regions. Attempts to use HDC immunohistochemistry coupled with quantitative microdensitometry to measure histamine nerve terminal density in brain regions of interest were frustrated by the relative sparseness of histaminergic projections, particularly in higher brain regions (Takagi et al., 1986). The signal to noise intensity ratio in most terminal field regions of interest was relatively low and too variable to reliably quantitate density in a reproducible manner. As a consequence of this technical limitation, we used quantitative Western blotting procedures. Given prior studies illustrating variable quantities of HDC in different rodent brain regions (Krusong et al., 2011), we first conducted tissue concentration studies in each of the five brain regions of interest to determine the optimal tissue concentration for use in each brain region of interest. As illustrated in Fig. 3, we were able to identify tissue concentrations that provided near optimal linearity of signal for both HDC isoforms (denoted by arrows on each graph) as well as the reference protein β-actin. The region-specific concentrations established in the characterization studies above were subsequently employed in the primary study of the effects of PAE on HDC isoform expression, as summarized in Fig. 4. Both HDC isoforms were present in all five brain regions of interest. The total of both isoforms was greatest in the hypothalamus, followed by the cerebellum, caudate nucleus, and smaller amounts present in the frontal cortex and dentate gyrus (Fig. 4B). The quantity of the 54-kDa isoform in the different brain regions was similar to levels reported previously (Krusong et al., 2011), with the exception of cerebellum, where we observed much higher levels. The relative expression of the 74-kDa HDC isoform was less than the 54-kDa isoform in all regions except the cerebellum, with the greatest differential expression in the hypothalamus. Moderate PAE reduced HDC expression in the frontal cortex, dentate gyrus, and the cerebellum, but not in the caudate nucleus or the hypothalamus (Fig. 4B). The expression of the 74-kDa isoform was significantly reduced in all three brain regions, whereas the effect on the 54-kDa isoform was only significant in the frontal cortex, but trended toward significance both in the dentate gyrus and cerebellum (p < 0.10).