| Parkinson's disease (PD) is a progressive neurodegenerative disease that is pathologically characterized by preferential degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). As the number of older people is rapidly increasing, the incidence of PD is on the rise, becoming one of the major diseases that afflict aged population. Thus, it is a major task for neurobiological study to unravel the mechanisms underlying neurodegeneration and to find effective drugs to halt the degenerative process.The observation that the degree of terminal loss in the striatum appears to be more pronounced than the magnitude of SNc DA neuron loss, suggests that striatal DA nerve terminals are the primary targets of the degenerative process and that neuronal death in PD may result from a"dying back"process. Experimental support for the concept of"dying back"includes the observations that in MPTP-treated monkeys the destruction of striatal terminals precedes that of SNc cell bodies, and in MPTP-treated mice, protection of striatal terminals prevents the loss of SNc DA neurons.Recent studies from environmental toxicology and molecular genetics demonstrate that midbrain dopamine (DA) neurons are particularly vulnerable to microtubule-depolymerizing agents, indicating the involvement of microtubule dysfunction in the pathogenesis of Parkinson's disease (PD). So we want to know if microtubule disruption of axonal terminals of dopamine neuron may lead to the degeneration of dopamine neurons in SNc.So we used the intrastriatal injection method and applied one of the well-known microtubule disruptor, colchicine (COL), to the striatum where axon terminals of dopamine neuron are located. With the help of fluorescent tract tracing, immunofluorescence and immunohistochemistry, neurochemical analysis etc, we observed the series of pathological changes that occurred in both SNc and striatum, including neurodegeneration and glial reaction. In the mean time, we also analyzed the behavior changes of the COL treated mice so as to evaluate its pathological consequences.Major Results:1. Microtubule disruption caused by unilateral injection of COL blocked the retrograde axonal transport of fluorogold (FG) previously injected into striatum and induced substantial death of striatal neurons and dopamine neurons (DA) in SNc. It is possible that retrograde axonal transport dysfunction is responsible for the degeneration of dopamine neurons in SNc.2. Striatal neurons were sensitive to microtubule disruption and began to die three days after COL injection when the degeneration of dopamine neurons was still not observed. TUNEL analysis revealed strong signals in striatum one week after injection of COL while no signal in SNc. Surprisingly, strong TUNEL signal was found in medial geniculate body of the injection side, associated with obvious activation of microglia, indicating the degeneration of central auditory pathway.3. COL-induced pathological changes were associated with robust glial reaction, which may be conducive to the degeneration of striatonigral pathway. Microglia displayed graded reactive morphology through the days after injection of COL, peaking at seven days post injection while a unique morphology was observed for reactive astrocyte at this time. Three dimentional reconstruction of the immunofluorescent figure of microglia and astrocytes revealed their direct interaction, indicating that there may be direct contact between them which may mediate communication of important signals.4. Combined degeneration of both striatum and SNc represents a good pathological model for striatonigral degeneration (multiple system atrophy). Behavioral tests demonstrated that injured mice show ipsilateral rotation when stimulated by agonist of dopamine receptor, and spontaneous ipsilateral side bias in locomotion. The grid box we designed is a good device to quantify this side bias. Swim test not only cofirmed the side bias but found its shift toward contralateral side bias, reflecting plasticity of certain kind in the neural circuits controling swim direction. Major Conclusion:1. We demonstrated in vivo, that axonal microtubule injury can induce the death of dopamine neurons in SNc(not apoptosis), which may be caused by dysfunction of retrograde axonal transport.2. Striatal neurons is sensitive to microtubule injury, which began to degenerate three days after treatment of COL, though apoptosis appeared seven days after treatment of COL.3. COL can directily activated microglia, suggesting that it may activate microglia through a direct way in striatum,in addition to the very likely indirect way through injured neurons, triggering its graded activation through days, accompanied by unique activation of astrocytes.4. Three dimensional recontruction of confocal microscopic pictures of microglia and astrocytes revealed their intimate spatial relationship, which promote us to propose the concept of interglial synapse, which may be important for their communication and physiological function.5. The combined degeneration of striatal neurons and dopamine neurons in SNc could serve as a good model for striatonigral degeneration (multiple system atrophy). Unilateral degeneration of striatalnigral pathway could be tested by behaviroal analysis of injured mice. This could provide an experimental model for investigating potential therapeutic use of novel strategies of neuroprotection.6. Grid box may be a good device for investigation of brain lateralization.Together, our results provide in vivo data lending support to the concept that microtubule dysfunction may play a significant role in the demise of DA neurons, though glial reaction may be involved and contribute to the degenerative process. Combined injury of straitum and SNc makes the COL-induced degeneration paradigm a good model of striatonigral degeneration (multiple system atrophy). Morphological study on the direct contact of microglia and astrocytes promotes us to propose the concept of interglial synapse, which may inspire the studies on their interactions and biological consequences.
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