Saturday, August 18, 2007

Mind The Gap Junctions

Gap junctions in the locus coeruleus??

Rash JE, Olson CO, Davidson KG, Yasumura T, Kamasawa N, Nagy JI. (2007). Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus. Neuroscience 147:938-56.

Locus coeruleus neurons are strongly coupled during early postnatal development, and it has been proposed that these neurons are linked by extraordinarily abundant gap junctions consisting of connexin32 (Cx32) and connexin26 (Cx26), and that those same connexins abundantly link neurons to astrocytes. Based on the controversial nature of those claims, immunofluorescence imaging and freeze-fracture replica immunogold labeling were used to re-investigate the abundance and connexin composition of neuronal and glial gap junctions in developing and adult rat and mouse locus coeruleus. In early postnatal development, connexin36 (Cx36) and connexin43 (Cx43) immunofluorescent puncta were densely distributed in the locus coeruleus, whereas Cx32 and Cx26 were not detected. By freeze-fracture replica immunogold labeling, Cx36 was found in ultrastructurally-defined neuronal gap junctions, whereas Cx32 and Cx26 were not detected in neurons and only rarely detected in glia. In 28-day postnatal (adult) rat locus coeruleus, immunofluorescence labeling for Cx26 was always co-localized with the glial gap junction marker Cx43; Cx32 was associated with the oligodendrocyte marker 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase); and Cx36 was never co-localized with Cx26, Cx32 or Cx43. Ultrastructurally, Cx36 was localized to gap junctions between neurons, whereas Cx32 was detected only in oligodendrocyte gap junctions; and Cx26 was found only rarely in astrocyte junctions but abundantly in pia mater. Thus, in developing and adult locus coeruleus, neuronal gap junctions contain Cx36 but do not contain detectable Cx32 or Cx26, suggesting that the locus coeruleus has the same cell-type specificity of connexin expression as observed ultrastructurally in other regions of the CNS. Moreover, in both developing and adult locus coeruleus, no evidence was found for gap junctions or connexins linking neurons with astrocytes or oligodendrocytes, indicating that neurons in this nucleus are not linked to the pan-glial syncytium by Cx32- or Cx26-containing gap junctions or by abundant free connexons composed of those connexins.

Fig. 3 (adapted from Rash et al., 2007). Immunofluorescence localization of connexins in rat LC. (A) Double immunofluorescence of the same field from 7 day postnatal rat showing absence of labeling for Cx26 in the LC (A1), and moderate labeling for Cx43 in the LC and surrounding regions, including ependyma (A2, arrow). Arrows indicate location of the fourth ventricle, asterisks mark the center of the LC, and arrowheads (A1) show positive labeling for Cx26 within leptomeninges (arrowheads, shown at higher magnification in inset). (B) Micrograph showing absence of labeling for Cx32 within the LC (asterisk).

Fig. 8 (adapted from Rash et al., 2007). Cx36-immunogold-labeled gap junctions in P28 (adult) rat LC. (A) At low magnification, a few myelinated axons (Ax) are seen to pass through the nucleus of the LC. This sample was triple-labeled for Cx36 (two 18-nm gold beads) and for Cx32 and Cx47 (6-nm and 12-nm gold beads; none present on neuronal gap junctions). However, Cx32 and Cx47 were abundant in oligodendrocyte gap junctions. Inscribed area in A is enlarged as B. Extremely low background (A) allowed detection of gap junctions consisting of ca. 46 and 48 connexons labeled by two (B) and three (C) 18-nm gold beads (labeling efficiency (LE)≈1:20).

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