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The observer was blinded as to which treatment group the subject belonged to during the stereological analysis. The average height of the tissue was The optical fractionator method Gundersen, Chakraborty et al. The neuronal number estimates did not depend on a direct measurement of the volume of reference of the region considered and therefore tissue shrinkage during histological processing would not influence the neuronal number estimates.

Coefficient of error CE values for the cell number estimates were determined using the Gundersen method incorporated in the StereoInvestigator software and then averaged for each group. The volumes of the MRN and DRN were measured by drawing the contour plot of each region at 10X magnification and then multiplying the contour area by the total thickness of the interslice distance number of sections between each contour multiplied by slice thickness i.

Volume estimates were calculated based on postprocessing tissues that have shrunk in all three directions. No attempts were made to correct for shrinkage because it probably differs in the z and x-y directions. The aqueduct and the medial longitudinal fasiculus were used as anatomical borders to demarcate the DRN Paxinos and Franklin, The observer was blinded to the treatment of each group during cell imaging and counting. M of n animals and were subject to a two-tailed t -test to analyse the differences between vehicle treated control, and cis -RA treated, mouse brains.

The n number for each region investigated varies because of variability in the tissue quality of some of the sections. Qualitative examination revealed that no gross anatomical or histochemical abnormalities were observed in cis -RA treated brains compared with vehicle treated controls. For the DRN, the average number of cells counted per mouse was For the MRN, the average number of cells counted per mouse was Immunohistochemical analysis of serotoninergic neurons in the brain of vehicle control and cis -RA treated young adult mice.

The volume of the MRN was 0. Similarly, in the larger DRN, the volume of the nucleus in control mice was 0. Quantitative analysis of the volume of the raphe nuclei in vehicle control and cis -RA treated young adult mice. Chronic cis -RA treatment did not alter the estimated volume A or the estimated density of serotoninergic neurons B in either the median MRN or dorsal DRN raphe nuclei compared with vehicle treated controls.

To exclude the possibility that subregional differences in the number of serotoninergic neurons could be obscured in the stereological analysis, we also examined the numbers of serotoninergic cells within subregions of the DRN using TPH-2 immunoreactivity Fig. Analysis of TPH-2 immunolabelling in the dorsal raphe nucleus from vehicle control and cis -RA treated adult mice. Stereological analysis of 5-HT immunolabelled neurons revealed that the volume, number of serotoninergic neurons and neuronal density of the MRN and DRN was not affected by cis -RA treatment.

To our knowledge, this study represents the first quantitative estimate of the number of serotoninergic neurons in the DRN and MRN of the adult mouse obtained by unbiased stereological methods. Stereological estimates of serotoninergic neuronal number in the adult DRN range from 15, in rats Casu et al. This variability in the number of serotoninergic neurons likely reflects the interspecies variation in size. The serotoninergic neurons of the DRN project to most forebrain areas, including those crucial for emotional behaviours, such as the amygdala and paraventricular nucleus of the hypothalamus, as well as limbic structures, such as the hippocampus Azmitia and Segal, Imai et al.

An increase in cell death in the DRN in response to retinoid treatment could therefore lead to a reduction in serotoninergic input to these forebrain regions involved in mediating responses to stress. Indeed, the chronic retinoid treatment regime we used here gives rise to plasma retinoid levels of 1. Although the total number of serotoninergic cells arising in the raphe nuclei remain unchanged, it is still possible that the serotoninergic input to the hippocampus is impaired by chronic cis -RA treatment.

It is known that the dendritic and cellular morphology of cultured cells is affected by high levels of cis -RA Ishikawa et al. The firing rate of serotoninergic neurons from the DRN can be regulated by 5-HT 1A autoreceptors, where a decrease in receptor number leads to increased basal firing activity Richer et al. The authors acknowledge Dr. Abstract The synthetic retinoid cis -retinoic acid cis -RA, prescribed for the treatment of severe nodular acne, has been linked to an increased incidence of depression.

Keywords: Retinoids, dorsal raphe nucleus, depression, stereology, 5-hydroxytryptamine, tryptophan hydroxylase.

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The synthetic retinoid cis -retinoic acid cis -RA, prescribed for the treatment of severe nodular acne, has been linked to an increased incidence of depression. Given that retinoids have apoptotic effects, we hypothesised that a decrease in the number of serotoninergic neurons in the DRN or roaccutane price pbs raphe nucleus MRN would lead to decreased serotoninergic tone and in turn to the behavioural changes seen with cis -RA administration. Here, we used immunolabelling and unbiased stereological methods to estimate the number of serotonin 5-hydroxytryptamine, 5-HT neurons in the MRN and DRN of vehicle control and cis -RA-treated adult mice. The potential pro-depressant behavioural and molecular effects associated with chronic administration of cis -RA may result from changes in serotoninergic activity rather than changes in the number of serotoninergic neurons.

Authored by Alex Grant Brann, PA-C, MMSC

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