Laura M. Prolo

Circadian Rhythm Generation and Entrainment in Astrocytes

In mammals, the master circadian pacemaker is considered the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN consists of a heterogeneous population of neurons and relatively understudied glia. We investigated whether glia, like neurons, rhythmically express circadian genes. We generated pure cultures of cortical astrocytes from Period2::luciferase (Per2::luc) knock-in mice and Period1::luciferase (Per1::luc) transgenic rats and recorded bioluminescence as a real-time reporter of gene activity. We found that rat Per1::luc and mouse Per2::luc astroglia express circadian rhythms with a genetically determined period. These rhythms damped out after several days but were reinitiated by a variety of treatments, including a full volume exchange of the medium. If cultures were treated before damping out, the phase of Per1::luc rhythmicity was shifted, depending on the time of the pulse relative to the peak of Per1 expression. Glial rhythms entrained to daily 1.5°C temperature cycles and were significantly sustained when cocultured with explants of the adult SCN but not with cortical explants. Thus, multiple signals, including a diffusible factor(s) from the SCN, are sufficient to either entrain or restart circadian oscillations in cortical glia.

The Suprachiasmatic Nucleus Entrains, But Does Not Sustain, Circadian Rhythmicity in the Olfactory Bulb

The suprachiasmatic nucleus (SCN) of the hypothalamus has been termed the master circadian pacemaker of mammals. Recent discoveries of damped circadian oscillators in other tissues have led to the hypothesis that the SCN synchronizes and sustains daily rhythms in these tissues. We studied the effects of constant lighting (LL) and of SCN lesions on behavioral rhythmicity and Period 1 (Per1) gene activity in the SCN and olfactory bulb (OB). We found that LL had similar effects on cyclic locomotor and feeding behaviors and Per1 expression in the SCN but had no effect on rhythmic Period 1 expression in the OB. LL lengthened the period of locomotor and SCN rhythms by ∼1.6 hr. After 2 weeks in LL, nearly 35% of rats lost behavioral rhythmicity. Of these, 90% showed no rhythm in Per1-driven expression in their SCN. Returning the animals to constant darkness rapidly restored their daily cycles of running wheel activity and gene expression in the SCN. In contrast, the OB remained rhythmic with no significant change in period, even when cultured from animals that had been behaviorally arrhythmic for 1 month. Similarly, we found that lesions of the SCN abolished circadian rhythms in behavior but not in the OB. Together, these results suggest that LL causes the SCN to lose circadian rhythmicity and its ability to coordinate daily locomotor and feeding rhythms. The SCN, however, is not required to sustain all rhythms because the OB continues to oscillate in vivo when the SCN is arrhythmic or ablated.

Olfactory bulb neurons express functional, entrainable circadian rhythms

Circadian pacemakers drive many daily molecular, physiological and behavioural rhythms. We investigated whether the main olfactory bulb is a functional circadian pacemaker in rats. Long‐term, multielectrode recordings revealed that individual, cultured bulb neurons expressed near 24‐h oscillations in firing rate. Real‐time recordings of Period1 gene activity showed that a population of cells within the bulb expressed synchronized rhythmicity starting on embryonic day 19. This rhythmicity was intrinsic to the mitral, and not the granule, cell layer, entrainable to physiological temperature cycles and temperature compensated in its period. However, removal of the olfactory bulbs had no effect on running wheel behaviour. These results indicate that individual mitral/tufted cells are competent circadian pacemakers which normally synchronize to each other. The daily rhythms in gene expression and firing rate intrinsic to the olfactory bulb are not required for circadian patterns of locomotion, indicating that they are involved in rhythms outside the canonical circadian system.

The Lysosomal Sialic Acid Transporter Sialin Is Required for Normal CNS Myelination

Salla disease and infantile sialic acid storage disease are autosomal recessive lysosomal storage disorders caused by mutations in the gene encoding sialin, a membrane protein that transports free sialic acid out of the lysosome after it is cleaved from sialoglycoconjugates undergoing degradation. Accumulation of sialic acid in lysosomes defines these disorders, and the clinical phenotype is characterized by neurodevelopmental defects, including severe CNS hypomyelination. In this study, we used a sialin-deficient mouse to address how loss of sialin leads to the defect in myelination. Behavioral analysis of the sialin−/− mouse demonstrates poor coordination, seizures, and premature death. Analysis by histology, electron microscopy, and Western blotting reveals a decrease in myelination of the CNS but normal neuronal cytoarchitecture and normal myelination of the PNS. To investigate potential mechanisms underlying CNS hypomyelination, we studied myelination and oligodendrocyte development in optic nerves. We found reduced numbers of myelinated axons in optic nerves from sialin−/− mice, but the myelin that was present appeared grossly normal. Migration and density of oligodendrocyte precursor cells were normal; however, a marked decrease in the number of postmitotic oligodendrocytes and an associated increase in the number of apoptotic cells during the later stages of myelinogenesis were observed. These findings suggest that a defect in maturation of cells in the oligodendrocyte lineage leads to increased apoptosis and underlies the myelination defect associated with sialin loss.

Vesicular uptake and exocytosis of l-aspartate is independent of sialin

The mechanism of release and the role of l‐aspartate as a central neurotransmitter are controversial. A vesicular release mechanism for l‐aspartate has been difficult to prove, as no vesicular l‐aspartate transporter was identified until it was found that sialin could transport l‐aspartate and l‐glutamate when reconstituted into liposomes. We sought to clarify the release mechanism of l‐aspartate and the role of sialin in this process by combining l‐aspartate uptake studies in isolated synaptic vesicles with immunocyotchemical investigations of hippocampal slices. We found that radiolabeled l‐aspartate was taken up into synaptic vesicles. The vesicular l‐aspartate uptake, relative to the l‐glutamate uptake, was twice as high in the hippocampus as in the whole brain, the striatum, and the entorhinal and frontal cortices and was not inhibited by l‐glutamate. We further show that sialin is not essential for exocytosis of l‐aspartate, as there was no difference in ATP‐dependent l‐aspartate uptake in synaptic vesicles from sialin‐knockout and wild‐type mice. In addition, expression of sialin in PC12 cells did not result in significant vesicle uptake of l‐aspartate, and depolarization‐induced depletion of l‐aspartate from hippocampal nerve terminals was similar in hippocampal slices from sialin‐knockout and wild‐type mice. Further, there was no evidence for nonvesicular release of l‐aspartate via volume‐regulated anion channels or plasma membrane excitatory amino acid transporters. This suggests that l‐aspartate is exocytotically released from nerve terminals after vesicular accumulation by a transporter other than sialin.—Morland, C., Nordengen, K., Larsson, M., Prolo, L. M., Farzampour, Z., Reimer, R. J., Gundersen, V. Vesicular uptake and exocytosis of L‐aspartate is independent of sialin. FASEB J. 27, 1264–1274 (2013). www.fasebj.org

Anterolateral approach to the upper cervical spine: Case report and operative technique

Transcervical approaches to the upper cervical spine are challenging because several upper anterior neurovascular structures need to be displaced to provide access. Although various techniques have been described, the anterolateral approach is one of the safest and most effective methods available to access the anterior C2–C3 disc space. Despite the approachs efficacy, however, it can cause postoperative complications because of, at least partly, the inter‐surgeon differences in the methods by which the larynx and hypopharynx are displaced medially.