Differential localization of NADPH-diaphorase and calbindin-D28k within the cholinergic neurons of the basal forebrain, striatum and brainstem in the rat, monkey, baboon and human.

The localization of Calbindin-D28k and NADPH-diaphorase in the cholinergic neurons of the basal forebrain, striatum and brainstem was investigated in the rat, monkey, baboon and human using calbindin and choline acetyltransferase immunohistochemistry and NADPH-diaphorase histochemistry. Considerable regional and species-specific variations were observed. Double-stained sections demonstrated that NADPH-diaphorase activity occurred in as much as 20-30% of basal forebrain cholinergic neurons in the rat but in virtually none of those neurons in the monkey, baboon or human. In all of the species studied, virtually every cholinergic neuron within the pedunculopontine and laterodorsal tegmental nuclei contained NADPH-diaphorase activity, while none of the cholinergic neurons of the striatum did so. In the rat brain, calbindin immunoreactivity was not present in any of the cholinergic neurons of the basal forebrain, while in the primate brain virtually all of the basal forebrain cholinergic neurons were also calbindin-positive. None of the cholinergic neurons of the striatum, pedunculopontine nucleus or laterodorsal tegmental nucleus were found to be calbindin-positive in any of the species examined. These results demonstrate major species-specific differences in the cytochemical signatures of the basal forebrain cholinergic neurons, in contrast to the cholinergic neurons of the striatum and brainstem, which displayed little interspecies variation with respect to the markers that were used in this study. Our findings also suggest that caution must be exercised in using results from studies of rodent basal forebrain cholinergic systems to infer the role of this system in the primate brain.

High-resolution localization of acetylcholinesterase at the rat neuromuscular junction.

To overcome the limited ultrastructural resolution of conventional acetylcholinesterase (AChE) ultrahistochemistry, acetylcholine (ATCh) was used to reduce the rate of enzymic thiocholine liberation. The conventionally limited resolution is mainly due to the high focal activity of the enzyme in neural structures, because cleavage of substrate is faster than histochemical trapping reactions. Therefore, using the copper-thiocholine method, we investigated the reduction of thiocholine liberation by acetylcholine (ACh). As examined biochemically, the apparent Ki for ACh was close to the Km for ATCh. The ACh/ATCh ratio, therefore, determined the reduction of thiocholine production in histochemical experiments. In addition, the morphological appearance of the precipitated reaction product after its changes during the histochemical procedure was monitored using electric eel AChE immobilized on Sepharose 4B. The improved fine structural resolution at 40- to 100-fold excess of ACh over ATCh is demonstrated at the neuromuscular junction of rat lumbricalis muscle. The highest focal enzyme activity is found at the presynaptic membrane and in the secondary cleft, but not on top of the junctional folds, indicating the separation of esterase and nicotinic receptors. The physiological events during neuromuscular transmission are discussed on the basis of the new "gradient switch hypothesis" suggested in this report.

A one-step cobalt-ferrocyanide method for histochemical demonstration of acetylcholinesterase activity in central nervous system tissue.

We introduce a one-step histochemical method with cobalt as the precipitating agent for ferrocyanide for the light microscopic demonstration of acetylcholinesterase activity. This method was used to demonstrate acetylcholinesterase in normal cortical fibers and neurons, as well as pathological elements such as plaques and tangles. This procedure can also be easily combined with immunohistochemical methods that use diaminobenzidine as a chromogen.

Competitive substrate inhibition in the histochemistry of cholinesterase activity in Alzheimer's disease.

We used acetylcholine and butyrylcholine to competitively inhibit the cleavage of acetylthiocholine or butyrylthiocholine in plaques and tangles of Alzheimer's disease. Butyrylcholine was much more effective than acetylcholine in reducing the histochemical reaction for acetylcholinesterase not only in neuronal fibers, but also in plaques and tangles. This is in keeping with biochemical data on acetylcholinesterase and supports the existence of true acetylcholinesterase activity within plaques and tangles. However, 2-4 times higher acetylcholine and buturylcholine concentrations were necessary to inhibit the plaque and tangle bound enzyme. Together with the previously reported different pH optima, this suggests that the plaque- and tangle-bound acetylcholinesterase may represent an altered form of this enzyme.

Stereotactic management of lesions of the pineal region.

OBJECTIVE: The relevance of the computed tomography-guided stereotactic approach for the management of lesions of the pineal region is analyzed. METHODS: In a retrospective analysis conducted between 1985 and 1993, the risk profile, the diagnostic accuracy, and the therapeutic relevance of the stereotactic approach in 106 patients was studied. Survival analysis was used to assess the reliability of the stereotactically obtained diagnosis in terms of follow-up observation. RESULTS: A histological diagnosis was obtained in 103 of the 106 patients. In three patients, a conclusive diagnosis could not be established because of intraoperative complications. One lesion was misdiagnosed as a pineocytoma instead of a pineoblastoma. Two of the 106 patients died; 9 patients experienced perioperative morbidity. In 38 patients, the stereotactic approach was also useful for therapy. Cyst aspiration and/or internal drainage was performed in 18 patients with symptomatic cystic lesions, and radiosurgical treatment with use of interstitial 125iodine was performed in 16 patients with low-grade tumors and in 4 patients with solitary metastases. In 12 patients, the obtained tissue diagnosis was the basis for deferring additional therapy. In 43 patients with germ-cell tumors, pineoblastomas, or malignant gliomas, a stereotactic biopsy was the starting point for additional radiotherapy/chemotherapy. Open tumor resection played a minor role (five patients). CONCLUSION: The stereotactic approach to the pineal region is a relatively safe procedure in experienced hands. The diagnosis obtained by computed tomography-guided stereotactic biopsy is a valid basis for treatment decisions. Long-term follow-up observation of the benign lesions is necessary for a definite confirmation of diagnostic accuracy.

Interstitial 125-iodine radiosurgery of low-grade gliomas of the insula of Reil.

Between 1979 and 1991 67 patients with low-grade gliomas of the insula (of Reil) were treated with 125-iodine interstitial radiosurgery. Retrospective analysis with a median follow-up of 55 months demonstrated a 5- and 10-year survival rate of 54% and 47%, respectively, for all low-grade gliomas treated and a 5- and 10-year survival rate of 57% for 49 patients with astrocytomas WHO grade II analysed separately. The median Karnofsky performance status of survivors was 90%. Malignant change was the cause of death in 85%, failure to control tumour growth in the remaining cases. Multivariate analysis with a Cox proportional hazard model identified solely the pre-operative Karnofsky performance score of 70-80 vs. 90% as a prognostic factor for outcome (p = 0.001, risk ratio 3.62), but not age, gender, tumour volume, length of disease before treatment, mode of implantation, or major vs. moderate or no shrinkage of tumour volume after interstitial radiosurgery. Thus, 125-iodine radiosurgery yielded survival rates in these deep-seated insular gliomas comparable to those reported after surgery and radiation therapy of lobar tumours. This was achieved with a low peri-operative mortality and morbidity and at low costs.