Immunofluorescence reveals unusual patterns of labelling for connexin43 localized to calbindin‐D28K‐positive interstitial cells in the pineal gland

Abstract

Gap junctions between cells in the pineal gland have been described ultrastructurally, but their connexin constituents have not been fully characterized. We used immunofluorescence in combination with markers of pineal cells to document the cellular localization of connexin43 (Cx43). Immunofluorescence labelling of Cx43 with several different antibodies was widely distributed throughout the pineal, whereas another connexin examined, connexin26, was not found in pineal but only in surrounding leptomeninges. Labelling apparently associated with plasma membranes was visualized either as fine Cx43-puncta (1-2 μm) or as unusually large pools of Cx43 ranging up to 4-7 μm in diameter or length. These puncta and pools were highly concentrated in perivascular spaces, where they were associated with numerous cells devoid of labelling for markers of pinealocytes (e.g. tryptophan hydroxylase and serotonin), and where they were minimally associated with blood vessels and lacked association with resident macrophages. Astrocytes labelled for glial fibrillary acidic protein were largely restricted to the anterior pole of the pineal gland, where they displayed only fine and sparse Cx43-puncta along their processes. Labelling for Cx43 was localized largely though not exclusively to the somata and long processes of a subpopulation of perivascular interstitial cells that were immunopositive for calbindin-D28K. These cells were often located among dense bundles or termination areas of sympathetic fibres labelled for tyrosine hydroxylase or serotonin. The results indicate that interstitial cells form abundant gap junctions composed of Cx43, and suggest that gap junction-mediated intracellular communication by these cells supports the activities of pinealocytes.

Connexin36 localization to pinealocytes in the pineal gland of mouse and rat

“…The presence of Cx36 also in the pineal stalk is consistent with the known distribution of pinealocytes in this structure (Calvo & Boya, ; Rath et al ., ). Although gap junctions linking pinealocytes to glial cells have been described (Cieciura & Krakowski, ), we found no association (not shown) between Cx36 localized to pinealocytes and Cx43 reported elsewhere to be localized to glial cells and interstitial cells (Tsao et al ., ), consistent with the nonpermissiveness of gap junction formation between these two connexins (Teubner et al ., ; Li et al ., ). FRIL double labelling for Cx36 and Cx43 showed no overlap of these connexins in any gap junctions, and no cells were labelled for both connexins.…”

“…The cellular localization of Cx36 in the pineal was examined by immunolabelling for Cx36 in combination with labelling either for TrypH or its downstream product, 5‐hydroxytryptamine (5‐HT). Labelling for TrypH is an adequate marker of pinealocytes, as tested in sections of adult rat pineal that were taken for fluorescence Nissl staining and simultaneous immunolabelling for TrypH (Tsao et al ., ). The vast majority of Nissl‐stained cells were TrypH‐positive, indicating that TrypH is likely expressed by all pinealocytes and can be readily detected in these cells by immunofluorescence.…”

“…However, in contrast to the high density of immunofluorescence labelling for Cx36 observed in this study, labelling of Cx26 previously reported in the pineal appeared to be relatively sparse (Saez et al ., ) in relation to the tightly packed nature of pinealocytes. In this regard, our recent work showed a total absence of Cx26 expression in the pineal despite robust labelling for this connexin in surrounding leptomeningeal tissues (Tsao et al ., ), as demosntrated using a set of well‐characterized anti‐Cx26 antibodies (Nagy et al ., , ). Further complicating this issue is that intercellular communication mediated by gap junctions between pinealocytes was demonstrated by observations of dye‐transfer following single cell injections of Lucifer Yellow (LY) (Saez et al ., ), which is poorly permeable across Cx36‐containing gap junction channels (Teubner et al ., ; Bukauskas, ; Ek‐Vitorin & Burt, ).…”

“…Further complicating this issue is that intercellular communication mediated by gap junctions between pinealocytes was demonstrated by observations of dye‐transfer following single cell injections of Lucifer Yellow (LY) (Saez et al ., ), which is poorly permeable across Cx36‐containing gap junction channels (Teubner et al ., ; Bukauskas, ; Ek‐Vitorin & Burt, ). Our localization of Cx36 to pinealocytes demonstrated here and lack of Cx26 detection in the pineal as reported elsewhere (Tsao et al ., ), together with demonstrations of LY‐coupling between these cells, raise inconsistences, the basis for which remain to be determined. However, we cannot exclude the possibilities that Cx36‐containing pinealocyte gap junctions are sufficiently dense to support some degree of LY‐coupling and/or that pineal cells express an additional connexin that was not examined here.…”

“…The pineal gland consists primarily of pinealocytes that produce melatonin (Pevet, , ; Vollrath, ; Moller & Baeres, ; Sapede & Cau, ) and which can be identified by immunolabelling for their markers tryptophan hydroxylase (TrypH) (Rath et al ., ), 5‐hydroxytryptamine (5‐HT) (Tsao et al ., ), or S‐antigen (aka, arrestin) (Schomerus et al ., ). Additional cells include a set of spatially restricted astrocytes identified by their labelling for glial fibrillary acidic protein (GFAP) and located largely in anterior regions of the gland (Moller et al ., ; Schachner et al ., ; Yamamoto et al ., ), and widely distributed perivascular interstitial cells that represent about 10–15% of the pineal cell population (Vollrath, ) and that can be identified, or at least a portion thereof, by their robust immunohistochemical labelling for calbindin‐D28K (Yamamoto et al ., ; Tsao et al ., ). Early studies described ultrastructurally identified gap junction in these cell types in the pineal of various species, including rat (Krstic, ; Taugner et al ., ; Cieciura & Krakowski, ), guinea‐pig (Huang & Taugner, ), rabbit (Romijn, ), monkey (Ichimura, ; Ichimura et al ., ) and humans (Moller, ).…”