Expression of L-type calcium channel alpha(1)-1.2 and alpha(1)-1.3 subunits on rat sacral motoneurons following chronic spinal cord injury.

In the presence of the monoamines serotonin and norepinephrine, motoneurons readily generate large persistent inward currents (PICs). The resulting plateau potentials amplify and sustain motor output. Monoaminergic input to the cord originates in the brainstem and the sharp reduction in monoamine levels that occurs following acute spinal cord injury greatly decreases motoneuron excitability. However, recent studies in the adult sacral cord of the rat have shown that motoneurons reacquire the ability to generate PICs and plateau potentials within 1-2 months following spinal transection. Ca(v)1.3 L-type calcium channels are involved in generating PICs in both healthy and injured animals. Additionally, expression of Ca(v)1.2 and Ca(v)1.3 L-type calcium channels is altered in several pathological conditions. Therefore, in this paper we analyzed the expression of L-type calcium channel alpha(1) subunits within the motoneuron pool following a complete transection of the spinal cord at the level of the sacral vertebra (S)2 segment. The analysis was done both caudally (S4 segment) and rostrally [thoracic vertebra (T)6 segment] from the injury site. The S4 segment was significantly reduced in diameter when compared with control animals, and this reduction was more evident in the white matter. Ca(v)1.2 alpha(1) subunit expression significantly increased (26%) in the motoneuron pool located caudally but not rostrally from the injury site. In contrast, the expression of Ca(v)1.3 alpha(1) subunit remained unchanged in both S4 and T6 segments. The differential expression of the two alpha(1) subunits in spinal injury suggests that Ca(v)1.2 and Ca(v)1.3 channels have different functions in neuronal adaptation following spinal cord injury.

Enrichment of unipolar brush cell-like neurons in primary rat cerebellar cultures.

Unipolar brush cells are a distinct class of excitatory interneurons situated in the granular layer of the cerebellar cortex, where they form giant synapses with individual mossy fiber terminals. We have previously shown that primary cerebellar cell cultures from embryonic and postnatal rodents contain neurons displaying morphological and chemical phenotypes characteristic of unipolar brush cells in situ, including intense staining with calretinin antiserum. In cultures from both embryonic and postnatal rats, the large majority of calretinin-positive neurons are unipolar brush cells, while granule cells are usually calretinin-negative. A small percentage of putative Golgi/Lugaro cells also express calretinin. We demonstrate here that the developmental stage of the source tissue, the concentration of potassium in the medium, and treatment with glutamate after differentiation have substantial effects on the density of putative unipolar brush cells in the cultures. In dissociated cultures obtained from embryos at gestation day E18 and E20 and from pups at postnatal day P0, P2, P5, P8, and P10 grown in 25 mM KCl, the percentage calretinin-positive cells progressively decreases from 24% to 0.1% of total cells. In cultures from E20 embryos grown in physiological potassium (5 mM KCl), calretinin-positive cells are enriched to approximately 60% of total cells, while the majority of calretinin-negative cells die. In embryonic cultures exposed to high concentrations of glutamate after 12 days in vitro, calretinin-positive neurons have a survival advantage over calretinin-negative cells and represent up to 83% of total cells.

Unipolar brush cells develop a set of characteristic features in primary cerebellar cultures.

The unipolar brush cells (UBCs) are a class of excitatory interneurons recently discovered in the cerebellar granular layer. UBCs differ morphologically and biochemically from granule cells, although they share the same mossy fiber and Golgi cell inputs. To elucidate development of the UBCs, we sought to ascertain their presence in primary cerebellar cultures and the class-specific properties they develop in vitro outside of the context of the tissue. By light and electron microscopy, we demonstrate that primary cultures from embryonic and postnatal mouse and rat cerebella contain UBC-like neurons that are highly polarized and can be distinguished from granule cells on several grounds. Granule cells are more numerous in dissociated postnatal cultures than in embryonic cultures; express little, if any, calretinin immunoreactivity; and develop dendritic processes devoid of typical claw-like endings, but provided with small synaptic junctions. By contrast, UBC-like neurons occur more frequently in embryonic cultures than in postnatal cultures, are intensely calretinin-positive, and develop characteristic cell organelles, dendrites, and large synapses. In embryonic cultures, the UBC-like neurons have a clear nucleus and contain a special cytoplasmic array of ringlet subunits, resembling the botrysome. At 12-28 days in vitro, the UBC dendrites contain abundant mitochondria, are provided with clusters of non-synaptic appendages, and engage in glomerular arrays together with large and small axon terminals. The large terminals contain round synaptic vesicles, form extensive, asymmetric synapses with the cell bodies and the dendrites of the UBC-like neurons, and resemble mossy terminals, while the small terminals contain pleomorphic vesicles, form symmetric synaptic junctions, and resemble Golgi terminals. In postnatal cultures grown for 12 days, UBC-like neurons are rare and resemble in most aspects the cells observed in embryonic cultures, although they rarely develop elaborate dendritic brushes.

Characterization of an ecto-phosphorylated protein of cultured cerebellar granule neurons.

Previous work identified the phosphorylation by extracellular ATP of an endogenous 45-kDa protein substrate and established the presence of ecto-protein kinase activity associated with cultured cerebellar granule neurons (Volonté et al.: J Neurochem 63:2028-2037, 1994). In this work, we characterize such ecto-phosphorylated 45-kDa protein substrate and its association with the cellular membrane. The total radioactive content of the 45-kDa protein is stable for the first 15 min after phosphorylation, and decreases about 70% in 30 min and 90% in approximately 2 hr. Rinsing the cells after the phosphorylating reaction causes a 50% removal of the incorporated radioactivity. Glycosidic residues are present on the 45-kDa ecto-protein, which is held in position on the cellular membrane through a specific glycosyl-phosphatidylinositol anchor. The extracellular incorporation of phosphate on the 45-kDa protein is not modulated by agents interfering with cytoskeleton stability, such as colchicine and taxol, or by gangliosides. The extracellular phosphorylation occurs mostly on serine residues, since the phosphate ester linkage is unstable at high pH and only antibodies raised against phosphoserine are capable of recognizing the 45-kDa ecto-protein.

Lithium induces apoptosis in immature cerebellar granule cells but promotes survival of mature neurons.

Lithium (Li+) has been used in the treatment of manic-depressive disorders for several decades. More recently, Li+ has been shown to affect the signaling pathway of various neurotransmitters and growth/neurotrophic factors. We examined the effect of Li+ on the survival of cerebellar granule neurons in culture. Treatment of immature granule cells with Li+ resulted in programmed cell death (apoptosis). The death process is accompanied by DNA fragmentation, a hallmark of apoptosis. Following maturation in vitro, granule neurons are dependent on elevated concentrations of extracellular potassium ([K+]o) for survival. Lowering of [K+]o to physiological levels induces apoptosis. Surprisingly, Li+ prevents death of mature neurons caused by low [K+]o. Moreover, the concentration range at which Li+ exerts its protective effect is the same as that at which it induces apoptosis in immature neurons. Thus, a single agent under similar extracellular conditions has opposing effects on survival, depending on the developmental status of the neuron.

Cytotoxic activity of human lymphocytes against differentiated intestinal tumour cell lines.

The natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic activity of human peripheral blood lymphocytes (PBL) against various human tumour cell lines from intestinal origin (WIDR, HT29, Caco-2) has been investigated. The differentiated Caco-2 cells were then used as a model to investigate the cytotoxic activity against enterocyte-like target cells. Caco-2 were seeded on polycarbonate filters and maintained in culture for at least 15 days to allow the differentiation and formation of tight junctions. The integrity of tight junctions was assayed by measuring [3H]mannitol flux from apical to basolateral compartment. Cytotoxic analysis showed that both differentiated and undifferentiated Caco-2 cells were similarly susceptible to NK and LAK activity. The capacity of cytotoxic lymphocytes to kill enterocyte-like cells with intact junctional complex may suggest a direct role of cytotoxic lymphocytes in causing intestinal lesions under inflammatory conditions.