The connexin family of proteins (Cx) that form intercellular gap junctions in vertebrates is well represented in the mammalian central nervous system. Among these, Cx30 and Cx43 are present in gap junctions of astrocytes. Cx32 is expressed by oligodendrocytes and is present in heterologous gap junctions between oligodendrocytes and astrocytes as well as at autologous gap junctions between successive myelin layers. Cx36 mRNA has been identified in neurons, and Cx36 protein has been localized at ultrastructurally defined interneuronal gap junctions. Cx26 is also expressed in the CNS, primarily in the leptomeningeal linings, but is also reported in astrocytes and in neurons of developing brain and spinal cord. To establish further the regional, cellular, and subcellular localization of Cx26 in neural tissue, we investigated this connexin in adult mouse brain and in rat brain and spinal cord using biochemical and immunocytochemical methods. Northern blotting, western blotting, and immunofluorescence studies indicated widespread and heterogeneous Cx26 expression in numerous subcortical areas of both species. By confocal microscopy, Cx26 was colocalized with both Cx30 and Cx43 in leptomeninges as well as along blood vessels in cortical and subcortical structures. It was also localized at the surface of oligodendrocyte cell bodies, where it was coassociated with Cx32. Freeze-fracture replica immunogold labeling (FRIL) demonstrated Cx26 in most gap junctions between cells of the pia mater by postnatal day 4. By postnatal day 18 and thereafter, Cx26 was present at gap junctions between astrocytes and in the astrocyte side of most gap junctions between astrocytes and oligodendrocytes. In perinatal spinal cord and in five regions of adult brain and spinal cord examined by FRIL, no evidence was obtained for the presence of Cx26 in neuronal gap junctions. In addition to its established localization in leptomeningeal gap junctions, these results identify Cx26 as a third connexin (together with Cx30 and Cx43) within astrocytic gap junctions and suggest a further level of complexity to the heterotypic connexin channel combinations formed at these junctions.

Intrinsic membrane properties are important in the regulation of motoneuronal output during such behaviours as locomotion. A conductance through L-type calcium channels has been implicated as an essential component in the transduction of motoneuronal input to output during locomotion. Given the developmental changes in calcium currents occurring postnatally in some neurons, and the increasing interest in the study of spinal locomotor output in neonatal preparations, experiments were conducted to investigate the postnatal development of L-type calcium channels in mouse motoneurons. This was assessed both physiologically, using a chemically induced rhythmic motor output, and anatomically, using immunohistochemical methods. The electrophysiological data were obtained during rhythmic bursting produced by application of N-methyl-D-aspartate (NMDA) and strychnine to the isolated spinal cord at various postnatal ages. The L-type calcium channel blocker nifedipine has no effect on this ventral root bursting in postnatal day (P) P2-P5 animals, but reversibly reduced the amplitude and/or burst duration of this activity in animals greater than P7. The immunohistochemical evidence demonstrates a dramatic change in the cellular profile of both the alpha1C and alpha1D subunits of L-type calcium channels during postnatal development; the labelling of both subunits increases with age, approximating the adult pattern by P18. These results demonstrate that in the spinal cord, the L-type calcium channel profile develops both physiologically and anatomically in the early postnatal period. This development parallels the development of the mature functional behaviours of weight bearing and walking, and may be necessary for the production of complex motor behaviour in the mature mammal.

Following birth, when mammals are relatively immobile, significant development of the motor system facilitates weight bearing and locomotion. Prominent cholinergic C-terminals develop on somata and proximal dendrites of spinal motoneurons during this time period. It is hypothesized that these terminals are essential in regulating motoneuron excitability and thus their development contributes to motor system maturation. Therefore, the development of pre- and postsynaptic components of the C-terminal synapse on motoneurons in mice during the early postnatal period was investigated. Fluorescence immunohistochemical studies revealed that developmental increases in punctate labeling of presynaptic cholinergic terminals, as visualized by vesicular acetylcholine transporter immunoreactivity (VAChT-IR) corresponded to the progressive expression and spatial restriction of immunoreactivity for the calcium channel subunit alpha(1)2.2 (N-type) located presynaptically and the muscarinic type 2 acetylcholine receptor situated postsynaptically. In addition, clustering of immunoreactivity for the potassium channel subunit K(V)2.1 occurred within the early postnatal period in concert and colocalized with the maturation of the C-terminals. The time course of development of these components of the C-terminal synapse corresponds to the maturation of the motor system that enables the animal to locomote in an adult-like fashion.

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