In vivo and in vitro Flow Cytometry Comparative Analysis of Somatostatin-Positive Cells in the Pineal Gland of the Neonatal Rat

Abstract

Flow cytometry was used for comparative in vivo and in vitro analysis of cell populations staining positively for somatostatin. Experiments were carried out with pineals obtained from neonatal, 8- and 15-day-old rats. Pineal cells were obtained by dispersion with collagenase and then processed in a flow cytometer or maintained in culture for 1 or 2 weeks. Identification of somatostatin-immunopositive cell populations was performed using a polyclonal somatostatin antibody and confirmed by indirect immunostaining of cytospun smears with the avidin-biotin-peroxidase method. In vivo, the percentage of somatostatin-positive cells was 60.6 ± 4% in neonatal pineals and declined to 22.2 ± 11 % in 15-day-old animals (p < 0.04). The density of peptide immunostaining decreased in 8-day-old animals but recovered to the neonate levels in 15 day-old animals; homogeneity in the immunopositive population increased with age. Maintenance in culture for 1 week resulted in an increase in positive somatostatin staining in animals of 8 and 15 days with no changes in neonates; however, after 2 weeks of culture, the percent of immunopositive cells decreased from 53.3 ± 6 to 12.2 ± 4% in the older animals and remained unchanged in neonates. We conclude that somatostatin is found in pinealocytes and shows a declining pattern during the perinatal period; this probably implies that the peptide plays a paracrine role important for cell differentiation in these young animals, since maximal cellularity and a high mitotic index occur within the first 3 days of life, and pineal cell differentiation is completed before the end of the third week of extrauterine life.

Expression of the somatostatin gene and receptors in the rat Harderian gland

“…A role for somatostatin in cell maturation has similarly been suggested in the pineal gland (Viader et al, 1995). Further recent unpublished observations of our own also show that pineal somatostatin has a seasonal expression similar to the one described here in the Harderian gland, although expression in the pineal is positive up to 15 days of age and up to 30 days in the Harderian gland.…”

Developmental potency of cultured pineal cells: An approach to pineal developmental biology

“…Neuronal properties have been confirmed under culture conditions; synaptic membrane proteins are immunohistochemically localized in cultured rat pineal cells (Redecker et al, 1997) and expression of somatostatin, a neuromodulating substance, is also seen in culture cells (Mato et al, 1993;Viader et al, 1995). In a series of our culture studies on rat pineal tissues, we used undissociated, collagenase-treated pineal tissues as culture materials without dissociating to the single-cell level (Araki and Tokunaga, 1990;Araki et al, 1988Araki et al, , 1994.…”

Developmental potency of cultured pineal cells: An approach to pineal developmental biology

“…In previous studies on cultured rat pineal cells, several interesting observations have been reported, mainly aiming to reveal the molecular events in physiological function (Rowe et al, 1977;Karasek and Vollrath, 1982;Walker and Aloyo, 1985). Neuronal properties have been confirmed under culture conditions; synaptic membrane proteins are immunohistochemically localized in cultured rat pineal cells (Redecker et al, 1997) and expression of somatostatin, a neuromodulating substance, is also seen in culture cells (Mato et al, 1993;Viader et al, 1995). In a series of our culture studies on rat pineal tissues, we used undissociated, collagenase-treated pineal tissues as culture materials without dissociating to the single-cell level (Araki and Tokunaga, 1990;Araki et al, 1988Araki et al, , 1994.…”