Protocol to isolate a large amount of functional oligodendrocyte precursor cells from the cerebral cortex of adult mice and humans.

During development, oligodendrocytes are generated from oligodendrocyte precursor cells (OPCs), a cell type that is a significant proportion of the total cells (3-8%) in the adult central nervous system (CNS) of both rodents and humans. Adult OPCs are responsible for the spontaneous remyelination that occurs in demyelinating diseases like Multiple Sclerosis (MS) and they constitute an interesting source of cells for regenerative therapy in such conditions. However, there is little data regarding the neurobiology of adult OPCs isolated from mice since an efficient method to isolate them has yet to be established. We have designed a protocol to obtain viable adult OPCs from the cerebral cortex of different mouse strains and we have compared its efficiency with other well-known methods. In addition, we show that this protocol is also useful to isolate functional OPCs from human brain biopsies. Using this method we can isolate primary cortical OPCs in sufficient quantities so as to be able to study their survival, maturation and function, and to facilitate an evaluation of their utility in myelin repair.

FGF-2 and Anosmin-1 are selectively expressed in different types of multiple sclerosis lesions.

Multiple sclerosis is a demyelinating disease that affects ≈ 2,000,000 people worldwide. In the advanced stages of the disease, endogenous oligodendrocyte precursors cannot colonize the lesions or differentiate into myelinating oligodendrocytes. During development, both FGF-2 and Anosmin-1 participate in oligodendrocyte precursor cell migration, acting via the FGF receptor 1 (FGFR1). Hence, we performed a histopathological and molecular analysis of these developmental modulators in postmortem tissue blocks from multiple sclerosis patients. Accordingly, we demonstrate that the distribution of FGF-2 and Anosmin-1 varies between the different types of multiple sclerosis lesions: FGF-2 is expressed only within active lesions and in the periplaque of chronic lesions, whereas Anosmin-1 is upregulated within chronic lesions and is totally absent in active lesions. We show that the endogenous oligodendrocyte precursor cells recruited toward chronic-active lesions express FGFR1, possibly in response to the FGF-2 produced by microglial cells in the periplaque. Also in human tissue, FGF-2 is upregulated in perivascular astrocytes in regions of the normal-appearing gray matter, where the integrity of the blood-brain barrier is compromised. In culture, FGF-2 and Anosmin-1 influence adult mouse oligodendrocyte precursor cell migration in the same manner as at embryonic stages, providing an explanation for the histopathological observations: FGF-2 attracts/enhances its migration, which is hindered by Anosmin-1. We propose that FGF-2 and Anosmin-1 are markers for the histopathological type and the level of inflammation of multiple sclerosis lesions, and that they may serve as novel pharmacogenetic targets to design future therapies that favor effective remyelination and protect the blood-brain barrier.

Development of the cholinergic system in the brain and retina of the zebrafish.

We have analyzed the distribution pattern of choline acetyltransferase (ChAT) in the zebrafish brain and retina during ontogeny. ChAT-immunoreactive (ChAT-ir) neurons are observed in the prosencephalon from 60 h postfertilization (hpf) onwards, exclusively in the preoptic area (basal plate of p6) derived from the secondary prosencephalon. In the mesencephalon, ChAT-ir cells are observed in both the optic tectum and the tegmentum. Stained cells in the tegmentum are observed from 60 hpf onwards, while in the optic tectum they appear after hatching. In the rhombencephalon, ChAT-ir cells are first observed in the isthmic region (rh1) and in the medulla oblongata (rh5-rh7) at the end of embryonic life. The rhombencephalic cholinergic cell groups develop in a gradual caudorostral sequence. Motoneurons of the spinal cord are ChAT-ir from 48 hpf onwards. The retina displays ChAT-ir neuropil in both the inner and outer plexiform layers from embryonic life, whereas stained amacrine cells are only observed after hatching. The staining in the outer plexiform layer gradually decreases during juvenile development. The optic nerve axons show a transient expression of ChAT at the end of embryonic development. The early presence of ChAT immunolabeling suggests an important neuromodulator role for acetylcholine in the first developmental stages.

Comparative analysis of the distribution of choline acetyltransferase in the central nervous system of cyprinids.

The general organization of the cholinergic system in the central nervous system is similar among vertebrates, though fish show higher variability. Thus, in zebrafish, cholinergic cells are absent from the habenula and the rhombencephalic reticular formation, where such neurons are present in most vertebrate species analyzed. In this work, we compared the distribution of choline acetyltransferase in the central nervous system of both zebrafish and tench, in order to investigate whether these divergences in the distribution of cholinergic cells in zebrafish are species-specific, or a feature shared by members of the cyprinid family. Our data show that these two cyprinid possess in common some peculiarities in their cholinergic system that are not present in the rest of fish analyzed (e.g. absence of cholinergic cells in the habenula and their presence in the descendent octaval nucleus). Nonetheless, some cholinergic cells were observed in the dorsal thalamus and rhombencephalic reticular nuclei of the tench, which were absent in the same regions in zebrafish. The comparative analysis suggests a divergent evolution of the cholinergic system among close-related cyprinid species.

Cholinergic elements in the zebrafish central nervous system: Histochemical and immunohistochemical analysis.

Recently, the zebrafish has been extensively used for studying the development of the central nervous system (CNS). However, the zebrafish CNS has been poorly analyzed in the adult. The cholinergic/cholinoceptive system of the zebrafish CNS was analyzed by using choline acetyltransferase (ChAT) immunohistochemistry and acetylcholinesterase (AChE) histochemistry in the brain, retina, and spinal cord. AChE labeling was more abundant and more widely distributed than ChAT immunoreactivity. In the telencephalon, ChAT-immunoreactive (ChAT-ir) cells were absent, whereas AChE-positive neurons were observed in both the olfactory bulb and the telencephalic hemispheres. The diencephalon was the region with the lowest density of AChE-positive cells, mainly located in the pretectum, whereas ChAT-ir cells were exclusively located in the preoptic region. ChAT-ir cells were restricted to the periventricular stratum of the optic tectum, but AChE-positive neurons were observed throughout the whole extension of the lamination except in the marginal stratum. Although ChAT immunoreactivity was restricted to the rostral tegmental, oculomotor, and trochlear nuclei within the mesencephalic tegmentum, a widespread distribution of AChE reactivity was observed in this region. The isthmic region showed abundant AChE-positive and ChAT-ir cells in the isthmic, secondary gustatory and superior reticular nucleus and in the nucleus lateralis valvulae. ChAT immunoreactivity was absent in the cerebellum, although AChE staining was observed in Purkinje and granule cells. The medulla oblongata showed a widespread distribution of AChE-positive cells in all main subdivisions, including the octavolateral area, reticular formation, and motor nuclei of the cranial nerves. ChAT-ir elements in this area were restricted to the descending octaval nucleus, the octaval efferent nucleus and the motor nuclei of the cranial nerves. Additionally, spinal cord motoneurons appeared positive to both markers. Substantial differences in the ChAT and AChE distribution between zebrafish and other fish species were observed, which could be important because zebrafish is widely used as a genetic or developmental animal model.