Long-lasting neurotoxic effects of exposure to methylmercury during development.

Amongst environmental chemical contaminants, methylmercury (MeHg) remains a major concern because of its detrimental effects on developing organisms, which appear to be particularly susceptible to its toxicity. Here, we investigated the effects of low MeHg levels on the development of the nervous system using both in vitro and in vivo experimental models. In neural stem cells (NSCs), MeHg decreased proliferation and neuronal differentiation and induced cellular senescence associated with impairment in mitochondrial function and a concomitant decrease in global DNA methylation. Interestingly, the effects were heritable and could be observed in daughter NSCs never directly exposed to MeHg. By chronically exposing pregnant/lactating mice to MeHg, we found persistent behavioural changes in the male offspring, which exhibited depression-like behaviour that could be reversed by chronic treatment with the antidepressant fluoxetine. The behavioural alterations were associated with a decreased number of proliferating cells and lower expression of brain-derived neurotrophic factor (Bdnf) mRNA in the hippocampal dentate gyrus. MeHg exposure also induced long-lasting DNA hypermethylation, increased histone H3-K27 tri-methylation and decreased H3 acetylation at the Bdnf promoter IV, indicating that epigenetic mechanisms play a critical role in mediating the long-lasting effects of perinatal exposure to MeHg. Fluoxetine treatment restored the Bdnf mRNA expression levels, as well as the number of proliferating cells in the granule cell layer of the dentate gyrus, which further supports the hypothesis that links depression to impaired neurogenesis. Altogether, our findings have shown that low concentrations of MeHg induce long-lasting effects in NSCs that can potentially predispose individuals to depression, which we have reported earlier to occur in experimental animals exposed to MeHg during prenatal and early postnatal development.

Neuropeptide effects on rat chondrocytes and perichondrial cells in vitro.

This study examines if cultured chondrocytes and perichondrial cells change the level of cAMP and/or cGMP in response to application of the neuropeptide calcitonin gene-related peptide (CGRP). Cells collected from the knee region of 4-8 days old rat pups were cultured in vitro. Cultures were exposed to 10(-10)-10(-6) M CGRP during 10 minutes. The levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the cultures and in controls were determined by radioimmunoassay. The results show that application of CGRP causes a distinctly increased level of cAMP, that was absent when CGRP was applied together with the CGRP(1) receptor antagonist. The level of cGMP was not obviously altered. Hence, it is possible that terminals of primary sensory neurones present in developing cartilage influence chondrocytes and perichondrial cells via local release of CGRP.

Neuropeptide content and physiological properties of rat cartilage-projecting sensory neurones co-cultured with perichondrial cells.

In young rats the cartilaginous epiphyses forming the knee joint are supplied with blood vessels and peptidergic sensory nerve fibres through the perichondrium and cartilage canals. In the present study we show that cartilage-related dorsal root ganglion neurones co-cultured with perichondrial cells develop extensive neurite trees and express calcitonin gene-related peptide (CGRP) and substance P (SP) in in vivo-like proportions using retrograde tracing and immunohistochemistry. Moreover, whole cell patch clamp recordings from these cells showed that the majority is depolarised by application of H(+)-ions. These results are compatible with the hypothesis that a local imbalance of blood flow and metabolism during normal skeletal maturation may cause tissue acidosis eliciting release of CGRP/SP from sensory nerve endings.

Retrograde tracing and neuropeptide immunohistochemistry of sensory neurones projecting to the cartilaginous distal femoral epiphysis of young rats.

Although cartilage is considered to be devoid of innervation, axons occur in the perichondrium and during development in cartilage canals, thereby having a relatively close spatial relationship to chondroblasts and chondrocytes. The present study locates the source of the sensory innervation of the femoral cartilaginous epiphyses of young rats and investigates whether the neuropeptide calcitonin gene-related peptide (CGRP) can influence chondrocytes. Retrograde tracing from the distal femoral epiphysis of young rats with Fast Blue (FB) showed labelled neuronal profiles in the L2-L5 dorsal root ganglia. Sample countings indicated that 50% of the FB-labelled neuronal profiles were located at the L3 level and 25% at the L4 level. The labelled neurones had diameters of 15-40 microm, with a peak at 25-30 microm. Immunohistochemistry showed that about 50% of the FB-labelled profiles contained CGRP. Together with the finding that CGRP influences bone cells to generate the second messenger cAMP, this result suggested the hypothesis that chondrocytes might be similarly influenced by CGRP. However, stimulation of cartilage slices with CGRP in vitro followed by an assay of the cAMP content did not provide support for this hypothesis. We conclude that primary sensory neurones containing CGRP project to the perichondrium and to cartilage canals of growing cartilage, and that exogenous CGRP does not elevate the cAMP content of cartilage slices in vitro.

The developmental skeletal growth in the rat foot is reduced after denervation.

It has long been known that bone is innervated. In recent years it has been suggested that the local nerves may influence the growth and metabolism of bone by way of neuropeptides. The transient local presence of nerve-containing cartilage canals just before formation of secondary ossification centres in rat knee epiphyses seems to support that view. The purpose of the present study was to see if denervation affects the developmental growth of metatarsal bones in the rat hindfoot. We made sciatic and femoral neurectomies in 7-day-old rat pups and examined the hindfeet at various times after surgery. Immunohistochemical analysis showed that denervation was complete. Radiographic examination revealed that the metatarsal bones were significantly shorter in denervated hindfeet 30 days after denervation (average relative shortening 9.9 +/- 2.3%). Measurements of total foot length showed that denervated feet were subnormally sized already five days postoperatively, before the onset of secondary ossification. The timing of the latter was not affected by denervation. Control rats subjected to tenotomies exhibited normal metatarsal bone lengths. On the basis of these results we suggest that the local nerves may influence the growth of immature bones but do not affect secondary ossification.