Regulation of trophic factor expression by innervating target regions in intraocular double transplants.

Trophic factors have been found to play a significant role both in long-term survival processes and in more rapid and dynamic processes in the brain and spinal cord. However, little is known regarding the regulation of expression of growth factors, and how these proteins interact on a cell-to-cell basis. We have studied protein levels of one growth factor known to affect the noradrenergic innervation of the hippocampal formation, namely brain-derived neurotrophic factor (BDNF). The purpose of the present study was to determine if appropriate innervation or contact between the LC noradrenergic neurons and their target, the hippocampus, affects expression of this growth factor in either brain region. Fetal brain stem tissue, containing the LC, and hippocampal formation were dissected from embryonic day 17 rat fetuses and transplanted together or alone into the anterior chamber of the eye of adult Fisher 344 rats. The tissue was grown together for 6 weeks, after which the animals were sacrificed and ELISAs for BDNF were undertaken. Transplantation to the anterior chamber of the eye increased the expression of BDNF in the hippocampal but not the brain stem tissue, compared with levels observed in fetal and adult rats in vivo. In addition, double grafting with hippocampal tissue more than tripled BDNF levels in brain stem grafts and doubled BDNF levels in the hippocampal portion of double grafts compared with hippocampal single grafts. Triple grafts containing basal forebrain, hippocampus, and brain stem LC tissue increased brain stem and hippocampal BDNF levels even further. Colchicine treatment of LC-hippocampal double grafts gave rise to a significant decrease in hippocampal BDNF levels to levels seen in single hippocampal grafts, while only a partial reduction of BDNF levels was seen in the brain stem portion of the same double grafts treated with colchicine. The findings suggest that an appropriate hippocampal innervation or contact with its target tissues is essential for regulation of BDNF expression in the brain stem, and that retrograde transport of BDNF can occur between double grafted fetal tissues in oculo.

Regulation of Trophic Factor Expression by Innervating Target Regions in Intraocular Double Transplants.

Trophic factors have been found to play a significant role both in long-term survival processes and in more rapid and dynamic processes in the brain and spinal cord. However, little is known regarding the regulation of expression of growth factors, and how these proteins interact on a cell-to-cell basis. We have studied protein levels of one growth factor known to affect the noradrenergic innervation of the hippocampal formation, namely brain-derived neurotrophic factor (BDNF). The purpose of the present study was to determine if appropriate innervation or contact between the LC noradrenergic neurons and their target, the hippocampus, affects expression of this growth factor in either brain region. Fetal brain stem tissue, containing the LC, and hippocampal formation were dissected from embryonic day 17 rat fetuses and transplanted together or alone into the anterior chamber of the eye of adult Fisher 344 rats. The tissue was grown together for 6 weeks, after which the animals were sacrificed and ELISAs for BDNF were undertaken. Transplantation to the anterior chamber of the eye increased the expression of BDNF in the hippocampal but not the brain stem tissue, compared with levels observed in fetal and adult rats in vivo. In addition, double grafting with hippocampal tissue more than tripled BDNF levels in brain stem grafts and doubled BDNF levels in the hippocampal portion of double grafts compared with hippocampal single grafts. Triple grafts containing basal forebrain, hippocampus, and brain stem LC tissue increased brain stem and hippocampal BDNF levels even further. Colchicine treatment of LC-hippocampal double grafts gave rise to a significant decrease in hippocampal BDNF levels to levels seen in single hippocampal grafts, while only a partial reduction of BDNF levels was seen in the brain stem portion of the same double grafts treated with colchicine. The findings suggest that an appropriate hippocampal innervation or contact with its target tissues is essential for regulation of BDNF expression in the brain stem, and that retrograde transport of BDNF can occur between double grafted fetal tissues in oculo.

Glial cell line-derived neurotrophic factor is essential for neuronal survival in the locus coeruleus-hippocampal noradrenergic pathway.

It has been shown that the noradrenergic (NE) locus coeruleus (LC)-hippocampal pathway plays an important role in learning and memory processing, and that the development of this transmitter pathway is influenced by neurotrophic factors. Although some of these factors have been discovered, the regulatory mechanisms for this developmental event have not been fully elucidated. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor influencing LC-NE neurons. We have utilized a GDNF knockout animal model to explore its function on the LC-NE transmitter system during development, particularly with respect to target innervation. By transplanting various combinations of brainstem (including LC) and hippocampal tissues from wildtype or GDNF knockout fetuses into the brains of adult wildtype mice, we demonstrate that normal postnatal development of brainstem LC-NE neurons is disrupted as a result of the GDNF null mutation. Tyrosine hydroxylase immunohistochemistry revealed that brainstem grafts had markedly reduced number and size of LC neurons in transplants from knockout fetuses. NE fiber innervation into the hippocampal co-transplant from an adjacent brainstem graft was also influenced by the presence of GDNF, with a significantly more robust innervation observed in transplants from wildtype fetuses. The most successful LC/hippocampal co-grafts were generated from fetuses expressing the wildtype GDNF background, whereas the most severely affected transplants were derived from double transplants from null-mutated fetuses. Our data suggest that development of the NE LC-hippocampal pathway is dependent on the presence of GDNF, most likely through a target-derived neurotrophic function.

Degeneration of neurons and glia in the Niemann-Pick C mouse is unrelated to the low-density lipoprotein receptor.

The BALB/c mouse model of Niemann-Pick type C disease exhibits similar neuropathological features to the human condition, including cerebral atrophy, demyelination of the corpus callosum, and degeneration of cerebellar Purkinje cells. The gene defect in Niemann-Pick C disease causes cholesterol to accumulate within the lysosomal compartment of neurons and glial cells. In order to determine whether cholesterol accumulation through the low-density lipoprotein receptor pathway plays an important role in the degenerative process, Niemann-Pick C mice were crossed with low-density lipoprotein receptor knockout mice. The purpose of the present study was to determine whether degeneration of neurons and glial cells is reduced in Niemann-Pick C animals lacking the low-density lipoprotein receptor. Using stereological counting methods, Purkinje cells were counted in the cerebellum and glial cell bodies were counted in the corpus callosum in mice at 3, 7.5 and 11 weeks of age. In the Niemann-Pick C animals, compared to wild-type control mice, there were 48% fewer glial cells at 3 weeks of age, and by 11 weeks of age there were 63% fewer glial cells. Purkinje cells were decreased in number by 13% at 3 weeks of age, and by 11 weeks of age there was a 96% loss. In the Niemann-Pick C animals lacking low-density lipoprotein receptors, there was no difference in the magnitude of glial cell or Purkinje cell loss compared to the Niemann-Pick C animals. These data indicate that both neurons and glia are vulnerable to degeneration in the Niemann-Pick C mouse, but that blocking the accumulation of cholesterol through the low-density lipoprotein receptor pathway does not alter the degenerative phenotype of Niemann-Pick C disease.

Selective neurodegeneration, without neurofibrillary tangles, in a mouse model of Niemann-Pick C disease.

The BALB/c mouse model of Niemann-Pick type C (NPC) disease exhibits neuropathological similarities to the human condition. There is an age-related cerebral atrophy, demyelination of the corpus callosum, and degeneration of cerebellar Purkinje cells in the NPC mouse. In human NPC, many cortical and subcortical neurons contain neurofibrillary tangles, which are thought by some investigators to play an important role in the neurodegenerative process. The purpose of the present study was to determine whether neurodegeneration occurs in the NPC mouse, in brain regions other than the cerebellum and whether the degeneration is related to the presence of neurofibrillary tangles. Using light microscopic methods with immunohistochemistry, electron microscopy, and cell counting methods, 11-week-old NPC(+/+) and NPC(-/-) animals were examined. In the NPC(-/-) mice, there were 96% fewer Purkinje cells, 28% fewer neurons in the prefrontal cortex, 20% fewer neurons in the thalamus, and 63% fewer glial cells in the corpus callosum. On the other hand, previous studies indicate normal numbers of neurons and glial cells in these same neuroanatomical regions in young NPC(-/-) mice. There were normal numbers of cholinergic neurons in sections assessed in the striatum and basal forebrain in the 11-week-old animals and no evidence of neurofibrillary tangles within cells. The present data indicate that both neurons and glial cells die in the NPC mouse but that all cells are not equally vulnerable. There was no evidence for neurofibrillary tangles in the NPC mouse, and therefore the degenerative process in the mouse is unrelated to the neurofibrillary tangle.

Phytoestrogens alter hypothalamic calbindin-D28k levels during prenatal development.

Calbindin-D28K (CALB) in the medial basal hypothalamic (MBH) and preoptic area (POA) of male and female fetuses from pregnant rats fed different phytoestrogens diets during gestation was examined by Western analysis. In animals fed a phytoestrogen containing diet (Phyto-200), males displayed significantly higher CALB levels compared to females. Whereas, in animals fed a phytoestrogen-free diet (Phyto-free), females exhibited significantly higher CALB levels compared to Phyto-200 female values. The present data have far reaching implications where phytoestrogen content in diets apparently influence MBH-POA CALB levels prenatally. The altered CALB levels may in turn modify sexually dimorphic brain structures during neuronal development by buffering Ca2+ that is associated with programmed cell death.

The neurotoxin MPTP increases calbindin-D28k levels in mouse midbrain dopaminergic neurons.

The calcium-binding protein calbindin-D28k (CALB) has been localized in high concentrations in several neuronal populations within the central nervous system (CNS) and is believed to act as an intracellular calcium (Ca2+) buffer. There has been much interest and speculation concerning its potential neuroprotective function. However, there is little direct evidence linking CALB content of individual neurons to Ca2+ buffering ability, resistance to Ca(2+)-mediated excitotoxicity, or vulnerability to Ca(2+)-mediated degeneration. It is necessary to demonstrate these relationships on a cellular level so that more definitive conclusions can be made. We have utilized immunocytochemical and Western blot techniques to determine whether cellular CALB content is altered in the nucleus A10 dopaminergic region of the midbrain following administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our data demonstrate a significant increase in the CALB content of nucleus A10 neurons (up to 227 +/- 23% above control) 3 and 6 h after MPTP treatment. CALB elevation demonstrated both time and dosage dependence as 6-h groups exhibited larger increases than 3-h groups, and a 60 mg/kg dosage induced a larger increase than a 20 mg/kg dosage. These data support the hypothesis that MPTP is neurotoxic by causing increases in free intracellular Ca2+ and that increased CALB in the midbrain dopaminergic neurons is a protective response to elevated intracellular free Ca2+.

The effect of glutaraldehyde pulpotomy treatment on pulpal enzymes.

The enzymes lactate dehydrogenase (LD), succinate dehydrogenase (SD), alkaline phosphatase (AP), and glucose-6-phosphate dehydrogenase (G-6-PD) were evaluated at specific locations in pulp tissue following glutaraldehyde pulpotomies. Pulpotomies were performed on maxillary first molars in 40 rats after sacrifice at times 0, 24 hr, seven days, or one month, and experimental teeth were treated with 6% buffered glutaraldehyde. Teeth were prepared for frozen sectioning and the sections were subjected to incubation media to detect each enzyme. Significant differences were found regarding the location of staining and the time intervals at which stain was present or absent. Staining for LD and AP was the most intense throughout the four observation time periods, though staining for these enzymes as well as for G-6-PD had begun to diminish by day seven in the coronal thirds. Staining was completely absent in the coronal thirds at one month but remained in the middle and apical thirds throughout one month. SD was the only enzyme that was totally absent in the coronal third at seven days and totally absent in the middle third at one month. These findings suggest that LD, AP, and the biochemical mechanisms that they represent remain active longer than SD and G-6-PD following exposure to glutaraldehyde. This information is important in identifying mechanisms that are important for repair, healing, and recovery following pulpal injury.