树鼩中脑腹侧被盖区向尾状核的DOPA能和AChE阳性纤维投射研究

应用荧光逆行标记分别与荧光组化(FAGLU)和AChE-药理组化相结合技术,研究了树鼩中脑腹侧被盖区向尾状核头部的DOPA能和ACHE-阳性纤维投射。结果表明:树鼩中脑腹侧被盖区除分别向同侧和对侧尾状核头部发出DOPA能纤维投射外,还向同侧尾状核头部发出AChE阳性投村纤维;此外,腹侧被盖区存在着向双侧尾核头部发出分叉投射纤维的DOPA能细胞。     

The stimulating effect of ganglioside injections on the recovery of choline acetyltransferase and acetylcholinesterase activities in the hippocampus of the rat after septal lesions

The effect of intramuscular administration of a mixture of gangliosides (21% GM₁, 39.7% GD_1a,, 16% GD_1b, 19% GT₁ in a daily dose of 50 mg per kg upon the time course of changes in hippocampal acetylcholinesterase and choline acetyltransferase activities after extensive medioventral septal lesions in the rat was checked on days 3, 5, 18 and 50 after the operation. Following the early decrease in the enzyme activities to about 25% of control due to degeneration, a gradual recovery up to about 50% of control activity at the 50th day was found. When gangliosides were administered, the recovery in the activity of both enzymes was more pronounced. The ratio of the enzyme activities from the animals injected with gangliosides to that from uninjected animals was 1.45 and 1.48 on the 18th day and 1.62 and 1.50 on the 50th day after the operation, for choline acetyltransferase and acetylcholinesterase activity, respectively. Since no significant effect of ganglioside injection was seen at early postoperative times i.e. on days 3 and 5, the effects seen on days 18 and 50 seem to be specifically due to facilitation of the recovery processes and not to retardation of the degeneration processes.Assuming that the spontaneous recovery of cholinergic enzyme activity reflects reinnervation of the hippocampus through collateral sprouting, gangliosides would seem to facilitate the regrowth of new cholinergic nerve terminals.     

The isolation of pure cholinergic nerve terminal sacs (T-sacs) from the electric organ of juvenile Torpedo.

The problem of synaptosome formation in the electric organ of Torpedo has been re-investigated using tissue from juvenile fish. This tissue is softer than adult material and can be easily homogenized in an Aldridge-type homogenizer. Homogenates so prepared contain a significant number of synaptosome-like structures which can be purified by differential and density gradient centrifugation. The purified particles are enriched in acetylcholine and choline acetyltransferase; they also contain lactate dehydrogenase activity, most of which is in an occluded form. The structure of these particles as revealed by electron microscopy is unusual in that they have no post-synaptic adhesions, relatively few synaptic vesicles and no intraterminal mitochondria. Because of their unusual morphology we have named these particles nerve terminal sacs (T-sacs). A high-affinity, hemicholinium-3 sensitive choline uptake system with an apparent K_m of 1–3 μm is associated with the T-sacs.     

Guidance of acetylcholinesterase-containing fibres by target tissue in co-cultured brain slices

Slices of various brain regions were prepared from newborn and from 7-day old rats and co-cultured in different combinations. In the majority of co-cultures of septal and hippocampal slices, acetylcholinesterase-positive fibres originating in the septal nuclei invaded the adjacent hippocampal slice. A similar pattern of hippocampal ingrowth by acetylcholinesterase-positive fibres occurred with slices prepared from the nucleus basalis of Meynert and from spinal cord. Septal neurones also projected to cortical slices, an effect which even occurred in the presence of their natural target tissue. In contrast to these massive projections to brain areas which in situ receive cholinergic inputs, no significant acetylcholinesterase-positive fibre ingrowth was observed in tissues which lack major cholinergic afferents in situ (hypothalamus, substantia nigra and cerebellum). These results indicate that under our culture conditions, acetylcholinesterase-positive fibres selectively invade cholinergic target areas. This effect is independent of the brain area from which the cholinergic neurones were derived.     

Neurotransmitter characteristics of brain grafts: striatal and septal tissues form the same laminated input to the hippocampus

In previous experiments we have studied the development of grafts of embryonic septal tissues implanted alongside the hippocampal formation of neonatal rats. In the present study we examined intracerebral implants of corpus striatum, a brain region that contains acetylcholinesterase-positive cells and does not normally project to the hippocampal formation, in order to evaluate the possibility that neurotransmitter identity may be involved in mechanisms guiding patterns of afferent growth and connectivity. Implant cavities were made in the entorhinal cortices of neonatal rat recipients and 3-6 days later embryonic striatal tissues were grafted to these preformed cavities. Implants were examined with acetylcholinesterase histochemistry one month after implantation. Grafts of embryonic striatal tissues did not survive implantation when the implant was introduced at the same time as the cavity was made. Grafts of corpora striata containing acetylcholinesterase-positive neurons were found in 7 of 11 rats in the delayed implant paradigm and, in all but one of these animals, acetylcholinesterase was present within those terminal laminae in the ipsilateral hippocampus and dentate gyrus that normally receive cholinergic input from the septal area. These findings suggest that cues underlying the development of specific connections between native (and implanted) septal efferents and hippocampal target neurons may be recognized by ingrowing acetylcholinesterase-reactive fibers from striatal implants.     

Membrane-bound choline acetyltransferase in Torpedo electric organ: A marker for synaptosomal plasma membranes?

The enzyme choline-O-acetyltransferase catalyses the biosynthesis of acetylcholine from acetyl coenzyme A and choline and is considered as one of the best markers for cholinergic nerve endings. The distribution of this enzymatic activity was analysed during the purification of plasma membranes of purely cholinergic nerve endings isolated from the electric organ of the fish Torpedo marmorata. This tissue, which receives a profuse and purely cholinergic innervation, can be considered as being a "giant" neuromuscular synapse. The isolated nerve endings (synaptosomes) were first osmotically disrupted and their plasma membranes isolated by equilibrium density centrifugation (discontinuous followed by continuous sucrose gradients). Choline acetyltransferase activity was found to exist in three forms: (1) a soluble form (the major one) present in the cytoplasm of the nerve endings, (2) a form which is ionically associated with membranes and which can be solubilized by washing exhaustively the membrane fraction with solutions of high ionic strength (0.5 M NaCl) and (iii) a form which is non-ionically bound to membranes and cannot be solubilized with high salt solution. The soluble and the non-ionically bound activities exhibited very similar affinities for choline (1.34 and 1.64 mM, respectively). The non-ionically membrane-associated form of choline acetyltransferase was found to "copurify" with the cholinergic synaptosomal plasma membranes of Torpedo, its specific activity being increased from 122 (crude fraction) to 475 (purified membrane fraction) nmol/h/mg protein. An enrichment was also observed for another cholinergic marker, the enzyme acetylcholinesterase, but not for the nicotinic receptor to acetylcholine, a marker for postsynaptic membranes. No choline acetyltransferase activity could be detected in preparations of synaptic vesicles that were highly purified from the electric organ. Also, the non-ionically associated form of choline acetyltransferase activity was hardly detectable (2.4 nmol/h/mg protein) in fractions enriched in axonal membranes prepared from the cholinergic electric nerves innervating the electric organ. The partition into soluble and membrane-bound activity was also analysed for choline acetyltransferase present in human placenta, a rich source for the enzyme but a non-innervated tissue. In this case the great majority of the enzyme appeared as soluble activity. Very low levels of non-ionically membrane-bound activity were found to be present in a crude membrane fraction from human placenta (2.8 nmol/h/mg protein).(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructural observations of the cholinergic neuron in the rat striatum as identified by acetylcholinesterase pharmacohistochemistry

In order to examine the ultrastructural features of the cholinergic neuron in the striatum (caudatoputamen) of the rat, cytochemistry for acetylcholinesterase was conducted 2–12 h after intramuscular injection of the irreversible acetylcholinesterase inhibitor diisopropylphosphorofluoridate. Light microscopic examination of Epon sections reacted for acetylcholinesterase showed that only large-sized cells in the striatum (25–35 μm in the long axis) were stained intensely. In the case of longer survival periods (10–12 h), some lightly stained cells (medium-sized) were seen dispersed amongst the large acetylcholinesterase-rich cells. Electron microscopic observations were made on ultrathin sections of selected large acetylcholinesterase-rich neurons that were first studied by light microscopy. The nucleus of these cells has an eccentric position and possesses several indentations of the nuclear envelope. The cytoplasm contains abundant organelles, many exhibiting features unique to this cell type. Many stacks of granular endoplasmic reticulum, arranged in a parallel manner and forming typical Nissl bodies, were observed in the periphery of the perikarya, and many distinct golgi complexes were seen in the perinuclear zone. At all post-diisopropylphosphorofluoridate survival times, heavy deposits of acetylcholinesterase reaction product were found within the perikarya of this cell type, for the most part within the cisternae of the granular endoplasmic reticulum. At the longer post-diisopropylphosphorofluoridate survival times, reaction product within the cytoplasm was very dense and appeared to have reached a maximum level. At these times reaction product also appeared in the secondary and tertiary dendritic branches of the large-sized neurons.

Of the other cell types in the striatum, two types of medium-sized cells displayed a light deposit of reaction product in their perikarya, but this was observed only at longer recovery times (8–12 h). The majority of cells in the striatum lacked reaction particles. Throughout the early post-diisopropylphosphorofluoridate period, the recovery of enzyme activity in the neuropil was moderate compared to that seen within cell bodies.

These findings indicate that the large-sized neuron is the only striatal structure that shows rapid regeneration of acetylcholinesterase activity during the early recovery phase after diisopropylphosphorofluoridate administration. Previous studies have indicated that this type of neuron represents the cholinergic interneuron of the striatum. The present results indicate that under appropriate conditions acetylcholinesterase pharmacohistochemistry can be utilized to determine the ultrastructural features of central cholinergic neurons.     

Catecholaminergic neurites in senile plaques in prefrontal cortex of aged nonhuman primates

Immunocytochemical studies, using a polyclonal antibody directed against tyrosine hydroxylase, identified catecholaminergic axons in prefrontal cortex of young and aged nonhuman primates. Aged monkeys, who showed cortical senile plaques in silver stains, had swollen tyrosine hydroxylase-immunoreactive axons in neocortex. Some of these abnormal processes were associated with deposits of amyloid (visualized by thioflavin-T fluorescence) and were similar in appearance to neurites demonstrated by silver impregnation methods. This study provides evidence for structural abnormalities in catecholaminergic axons/nerve terminals in the neocortices of aged primates.     

乙酰胆碱酯酶阳性神经元在鼠脑的分布和形态特征

本文用乙酰胆碱酯酶(AChE)再生技术,研究AChE阳性神经元在鼠脑的分布和形态特征。按其染色程度,可见强度、中度和轻度三种染色细胞。强染色细胞多数是较大的多极细胞,主要分布于纹状体、基底前脑、下丘脑、黑质、红核、蓝斑,腹侧被盖区、臂旁核、桥被盖核和脑神经运动核。本文将AChE染色结果和胆碱乙酰转移酶(ChAT)免疫组织化学资料进行了比较,对AChE和胆碱能神经元的关系,以及AChE阳性神经元的性质和意义,进行了讨论。     

Organotypic culture of embryonic electromotor system tissues from Torpedo marmorata

An explant culture system has been used to study the electric organ and electric lobe tissues of Torpedo marmorata at different stages during the development of the electromotor system.The myotubes in tissue expiants, taken from the electric organ primordia of 33–38 mm body-length embryos prior to electrocyte differentiation, contract spontaneously on explantation and have electrogenic membranes. The myotubes subsequently lose these properties in vitro and can differentiate in the absence of neural tissue into immature electrocytes which have morphologically characteristic postsynaptic membranes.Isolated expiants of differentiated electric organ tissue from 60–100 mm body-length embryos can be maintained for 3 to 4 weeks in vitro but cellular outgrowth is minimal. In contrast, a rapid, dense outgrowth of cells and a subsequent regeneration of myotubes occurs when differentiated electric organ explants are co-cultured with electric lobe tissue from embryos of the same stage. Cellular outgrowth from differentiated electric-organ tissue expiants can be stimulated by spinal cord, medulla, cerebellum and heart tissues but a subsequent regeneration of myotubes has not been observed. Myotube regeneration in the presence of electric lobe tissue is maximal with tissue from 60–80 mm body-length embryos. The myotubes that regenerate from differentiated electric organ expiants have not been observed to differentiate into electrocytes.Neuritic outgrowth in vitro occurs with electric lobe tissue taken at two different embryonic stages. The first stage corresponds to a period when most of the neuroepithelial cells in the lobe anlagen are withdrawing from the mitotic cycle and projecting axons into the branchial arches. The second, later stage is when the electromotorneurones are normally generating axon collaterals that are invading the interelectrocyte space of electrocyte columns. Maximum neuritic outgrowth at this second, later stage is obtained with tissue from 60–80 mm body-length embryos. Although neuritic invasion of electrocyte column expiants can be obtained in electric organ—electric lobe co-cultures at this later stage, synapses similar to those observed during the early stages of synaptogenesis in the electric organs in vivo have not been observed in vitro.