Localization and axonal transport of immunoreactive cholinergic organelles in rat motor neurons—An immunofluorescent study

Antisera produced in rabbits against pure fractions of cholinergic vesicles from Narcine brasiliensis were used to study cholinergic organelles in rat motor neurons. The indirect immunofluorescence method was used on perfusion-fixed material. The rats were surgically sympathectomized to remove sympathetic adrenergic and cholinergic nerves from the sciatic nerve. In the intact animal immunoreactive material, likely to represent cholinergic vesicles, was observed in motor endplates, identified by labelling with rhodamine-conjugated α-bungarotoxin or with subsequent acetylcholinesterase staining. The motor perikarya contained very little immunoreactive material. Non-terminal axons were virtually devoid of immunofluorescence in the intact animal. After crushing the sciatic nerve, immunoreactive material (likely to represent axonal cholinergic organelles) accumulated rapidly on both sides of the crush, indicating a rapid bidirectional transport. The transport was sensitive to local application of mitotic inhibitors.The axons which accumulated immunoreactive organelles were motor axons, as demonstrated by various procedures: (1) Cutting of ventral roots prevented accumulation of immunoreactive material in the nerve. (2) Deafferentation did not notably influence accumulations of immunoreactive material. (3) Ligated axons with immunoreactive material were acetylcholinesterase positive when identification was made on the same section; the intra-axonal distribution of immunoreactive material and acetylcholinesterase was not identical, however, and the Narcine antisera did not cross-react with bovine acetylcholinesterase in a solid phase immunoassay. (4) Most axons in ventral roots, but not in dorsal roots, accumulated strongly fluorescent immunoreactive material, while axons in dorsal roots contained weakly fluorescent material. On the other hand, substance P-like immune reactivity was present in many dorsal root axons, but only very rarely in ventral roots.It is suggested that the antisera against Narcine cholinergic vesicles can be used as a marker for cholinergic organelles in the motor neuron, and may be an important tool for studying the axonal cholinergic vesicles. It cannot, however, be used to identify cholinergic structures in unknown locations because it recognizes common antigenic determinants in transmitter organelles of other nerves e.g. adrenergic nerves. The axonal cholinergic organelles may carry important molecules, other than acetylcholine to the nerve endings.     

Age and neurochemical correlates of radial maze performance in rats

Young adult (8 months) and aged (26 months) female Wister rats were tested in a 12-arm radial maze in which the optimal strategy was to enter all arms without a repetition. In order to determine if possible age-associated alterations in behavior were correlated with defects in cholinergic. GABAergic and adrenergic neurons in the hippocampus and cerebral cortex, the activities of choline acetyltransferase (CAT), glutamic acid decarboxylase (GAD) and tyrosine hydroxylase (TH) were assayed in these regions of all animals after testing in the radial maze. In the maze, the aged rats continued to perform at the chance level after 15 trials, whereas the young rats had virtually mastered the task. The only significant neurochemical age effect was an increase in hippocampal TH. However, analysis of individual differences among rats revealed positive correlations between maze performance and hippocampal CAT in the aged group and cortical GAD in both the young and aged groups.     

Differential distribution of growth associated protein (GAP-43) in the motor nuclei of the adult rat conus medullaris

GAP-43 is normally produced by neurons during developmental growth and axonal regeneration, but it is also expressed in specific regions of the normal adult nervous system. We studied the protein expression of GAP-43 within the conus medullaris portion of the spinal cord in adult male rats. Immunohistochemistry for choline acetyltransferase (ChAT) was first performed to identify specific efferent autonomic and motor nuclei in lumbosacral segments of the spinal cord. Adjacent sections were then processed for GAP-43 immunoreactivity (IR). We show GAP-43 IR in the superficial portion of the dorsal horn, the intermediolateral nucleus, and the dorsal commissural tract. We also demonstrate a differential distribution of GAP-43 IR between different motor nuclei of the conus medullaris. Using densitometry, the most prominent GAP-43 IR was detected in the dorsolateral and dorsomedial motor nuclei, which represent the human Onufs nucleus homologue. Confocal microscopy of double immunofluorescent labeling for ChAT and GAP-43 demonstrate GAP-43 IR in the neuropil of the autonomic and motor nuclei, and many of the GAP-43 IR arbors are in close apposition with the efferent cholinergic neurons. We note that the efferent neurons of both the autonomic and somatic nuclei, which are ultimately responsible for the integrated normal control of the lower urinary tract, bowel and sexual functions, are heavily innervated by GAP-43 enriched projections. We speculate that these functionally related neurons retain a physiological GAP-43-associated synaptic plasticity throughout adult life.     

氯霉素乙酰基转移酶双抗体夹心ELISA检测方法的建立

目的 建立氯霉素乙酰基转移酶（chloramphenicol acetyltransferase,CAT)双抗体夹心ELISA的实验室检测方法。方法 从质粒pBLCAT6经PCR扩增CAT基因序列，插入到原核表达质粒pGEX-2T中，在大肠埃希菌DH5α中诱导表达；以纯化的融合蛋白GST-CAT为抗原免疫实验用兔，得到的CAT抗血清进行生物素标记，并在此基础上利用生物素链和亲和素放大系统建立双抗体夹心ELISA法，构建不同YY1结合位点突变的HPV16LCRCAT报道质粒，体外瞬时转染真核细胞，提取胞质蛋白，以建立的方法进行CAT表达检测。结果 SDS-PAGE显示表达的GST-CAT融合蛋白相对分子质量约为54000；制备的CAT抗血清可有效地识别原核及哺乳动物细胞表达的CAT蛋白；在不同启动子及调节序列的控制下，利用建立的CAT检测技术证明在瞬时转染的细胞中可诱导2-8倍的CAT表达增强。结论 建立的双抗体夹心ELISA方法可敏感地反映上游序列的启动子活性，以及启动子调节序列变化对启动子活性的影响，使CAT作为报道基因的研究更为简单化。     

Stimulation of chloramphenicol acetyltransferase synthesis by cyclic AMP in bacterial cell systems

The effect of cyclic 3,5-adenosine monophosphate (cAMP) on production of the enzyme chloramphenicol acetyltransferase (CAT) by whole bacterial cells was studied in strainsEscherichia coli CSH-2/R222 and WZ-78/R222 (cya₈₅₅). CAT synthesis in strainE. coli WZ-78/R222 was shown to have an intensity only half as great as that of strainE. coli CSH-2/R222. The production of CAT by strainE. coli CSH-2/R222 was increased only very slightly by cAMP, but its effect on the production of this enzyme in strain WZ-78/R222 was appreciable.Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 10, pp. 65–66, October, 1975.     

Cell cholesterol esters and high-density lipoprotein plasma levels during liver hyperplasia in choline-fed male and female rats

Sexual dimorphism exists in the response of rats to lead nitrate, liver hyperplasia occuring earlier and being more pronounced in males. Excess dietary choline in females shifted the growth pattern towards that of males. To determine whether phosphatidylcholine-induced growth modulations could be related to a derangement of cholesterol metabolism, liver accumulation of cholesterol esters and plasma lipoprotein patterns were investigated. In males, lead-induced liver hyperplasia was associated with increased total cholesterol hepatic content, accumulated cholesterol esters and reduced concentration of plasma High Density Lipoprotein (HDL) cholesterol. Females were less responsive to the liver mitogenic signal of lead nitrate; there was no elevation of cholesterol content nor any marked accumulation of cholesterol esters. This is consistent with the lack of change in the plasma levels of HDL cholesterol. Continuous choline feeding displaced the liver cholesterol ester pattern and plasma HDL cholesterol levels in females, and in parallel that of DNA synthesis, towards those of males. Choline was not observed to have any effect in males. These results suggest that the derangement of phosphatidylcholine metabolism induces growth-related changes in cholesterol turnover; they are consistent with the proposal that the intracellular content of cholesterol esters may have a role in regulating liver growth rates.     

IPSI- and contralateral changes in acetylcholinesterase and choline acetyltransferase activities in the hippocampus following unilateral septal lesions in the rat

Unilateral, left electrocoagulative septal lesions of the rat brain were performed. Acetylcholinesterase and choline acetyltransferase activities in the left and right hippocampi were measured 6–8 and 14–17 days postoperatively.Small lesions, damaging mainly the nucleus lateralis evoked a small (10–20%) decrease in the activity of both enzymes on the ipsilateral and side a tendency to an increase in the enzymatic activity on the contralateral side. No differences were observed between values obtained on the sixth-eighth and the fourteenth-seventeenth days after the operation. Larger lesions, including the nucleus fimbriatus and the nucleus of the diagonal band evoked on the sixth-eighth day a decrease (40–50%) in the activity of both enzymes on the ipsilateral side, but on the fourteenth-seventeenth day a significant recovery in the enzymatic activity was noted. Contralaterally, following larger lesions, an initial decrease on the sixth-eighth day in choline acetyltransferase activity was followed by an overshoot in comparison to controls on the fourteenth-seventeenth day; acetylcholinesterase activity remained unchanged on the sixth-eighth day, but also surpassed the control value on the fourteenth-seventeenth day.It is suggested that the post-lesion changes in enzyme activity in the ipsilateral hippocampus occur mainly within the cholinergic system, while in the contralateral hippocampus they may be the result of damage to non-cholinergic fibres followed by sprouting of cholinergic axons.     

The nucleus basalis magnocellularis: The origin of a cholinergic projection to the neocortex of the rat

The cells of origin of a neocortical cholinergic afferent projection have been identified by anterograde and retrograde methods in the rat. Horseradish peroxidase injected into neocortex labelled large, acetylcholinesterase-rich neurons in the ventromedial extremity of the globus pallidus. This same group of neurons underwent retrograde degeneration following cortical ablations. The region in which cell depletion occurred also showed significant decreases in the activities of choline acetyltransferase and acetylcholinesterase. Discrete electrolytic and kainic acid lesions restricted to the medial part of the globus pallidus each resulted in significant depletions of neocortical choline acetyltransferase and acetylcholinesterase. Hemitransections caudal to this cell group did not result in such depletions. Taken together these observations suggest that the acetylcholinesterase-rich neurons lying in the ventromedial extremity of the globus pallidus, as mapped in this study, constitute the origin of a major subcortical cholinergic projection to the neocortex. The utility of acetylcholinesterase histochemistry in animals pretreated with di-isopropylphosphorofluoridate in identifying cholinergic neurons is discussed in the light of this example; specifically, it is proposed that high acetylcholinesterase activity 4–8 h after this pretreatment is a necessary, but not sufficient, criterion for the identification of cholinergic perikarya.The neurons in question appear to be homologous to the nucleus basalis of the substantia innominata of primates, and are thus termed ‘nucleus basalis magnocellularis’ in the rat. No evidence was obtained to support the hypothesis that nucleus of the diagonal band projects to neocortex. However, striking similarities in size and acetylcholinesterase activity were observed among the putative cholinergic perikarya of the nucleus basalis magnocellularis, the nucleus of the diagonal band, and the medial septal nucleus.Kainic acid lesions of the neocortex produced uniform and complete destruction of neuronal perikarya. These lesions decreased neocortical glutamic acid decar?ylase activity, suggesting that there are GABAergic perikarya in the neocortex. However, the same lesions did not affect neocortical choline acetyltransferase. This observation suggests that there are no cholinergic perikarya in the neocortex, a conclusion that is consistent with the absence of intensely acetylcholinesterase-reactive neurons in the neocortex.     

Cortical projections arising from the basal forebrain: a study of cholinergic and noncholinergic components employing combined retrograde tracing and immunohistochemical localization of choline acetyltransferase

The neurochemical identity of ascending putative cholinergic pathways from the rat basal forebrain was investigated employing a method for simultaneously visualizing choline acetyltransferase immunoreactivity and retrogradely transported horseradish peroxidase-conjugated wheatgerm agglutinin. This histochemical procedure revealed three distinct populations of neurons: (1) cells which stained only for choline acetyltransferase immunoreactivity; (2) cells which stained only for retrograde tracer and (3) cells which stained simultaneously for choline acetyltransferase immunoreactivity and retrograde tracer. The results demonstrated that this projection is topographically organized and consists of both cholinergic and noncholinergic components. The relative contribution of each component varied with the telencephalic target area as follows: the olfactory bulb receives a projection from cells of the horizontal limb nucleus, 10-20% of which are cholinergic (Ch3); the hippocampal formation receives afferents from cells of the medial septal and vertical limb nuclei, 35-45% of which are cholinergic (Ch1 and Ch2); and the cortical mantle receives afferents primarily from cells within the substantia innominata-nucleus basalis complex, 80-90% of which are cholinergic (Ch4). The topographical organization of Ch4 projections is not as completely differentiated as we have previously observed in the primate.     

A lectin horseradish peroxidase study of the origin of ascending fibers in the medial forebrain bundle of the rat. The lower brainstem

The origins of projections within the medial forebrain bundle from the lower brainstem were examined with the horseradish peroxidase technique. Labeled cells were found in at least 15 lower brainstem nuclei following injections of a conjugate or horseradish peroxidase and wheat germ agglutinin at various levels of the medial forebrain bundle. Dense labeling was observed in the following cell groups (from caudal to rostral): A1 (above the lateral reticular nucleus); A2 (mainly within the nucleus of the solitary tract); a distinct group of cell trailing ventrolaterally from the medial longitudinal fasciculus at the level of the rostral pole of the inferior olive; raphe magnus; nucleus incertus; dorsolateral tegmental nucleus (of Castaldi); locus coeruleus; nucleus subcoeruleus; caudal part of the dorsal (lateral) parabrachial nucleus; and raphe pontis. Distinct but light labeling was seen in raphe pallidus and obscurus, nucleus prepositus hypoglossi, nucleus gigantocellularis pars ventralis, and the ventral (medial) parabrachial nucleus. Sparse labeling was observed throughout the medullary and caudal pontine reticular formation. Several lower brainstem nuclei were found to send strong projections along the medial forebrain bundle to very anterior levels of the forebrain. They were: A1, A2, raphe magnus (rostral part), nucleus incertus, dorsolateral tegmental nucleus, raphe pontis and locus coeruleus. With the exception of the locus coeruleus, attention has only recently been directed to the ascending projections of most of the nuclei mentioned above. Evidence was reviewed indicating that fibers from lower brainstem nuclei with ascending medial forebrain bundle projections distribute to widespread regions of the forebrain.It is concluded from the present findings that several medullary cell groups are capable of exerting a direct effect on the forebrain and that the medial forebrain bundle is the major ascending link between the lower brainstem and the forebrain.