A Search for Discrete Cholinergic Nuclei in the Human Ventral Forebrain

Abstract: Slices cut from five frozen human brains were dissected into 2-mm cubes and assayed for choline acetyltransferase (ChAT) activity and protein content. A pattern of enrichment of ChAT activity was found ventral to the anterior commissure; this finding is consistent with the location of the enzyme in the cells of the nucleus basalis of Meynert. The region beneath the anterior commissure was the only place a discrete enrichment of activity could be found, and the precise topography of the enrichment was somewhat variable from brain to brain. The results are discussed in the light of recent knowledge concerning the source of the cortical cholinergic innervation.     

Vascular supply of the anterior interventricular epicardial nerves and ventricular Purkinje fibers in the porcine hearts

The aim of this study was to perform a pilot histological and quantitative analysis of the blood vessels accompanying the epicardial nerves (vasa nervorum) in the porcine hearts. Twenty healthy porcine hearts were used in this study. The blood vessels were analyzed by light microscopy using four different staining techniques in transverse sections taken from the upper, middle, and lower segments of the anterior part of the interventricular region and the adjacent parts of the right and left ventricles containing epicardial nerves and the endocardial peripheral parts of the Purkinje fibers. In total, 317 epicardial nerves were detected. The vasa nervorum were present in 75.7% of these nerves. The vasa nervorum resembled arterioles and postcapillary and collecting venules. One hundred and forty nine epicardial nerves were perivascular, located in the adventitia of the anterior interventricular artery and vein. The remaining 168 nerves ran freely through the epicardial interstitium. The presence of the vasa nervorum was not related to topographical location or nerve diameter. Additionally, from a total of 33 analyzed ventricular complexes of Purkinje fibers small blood vessels located in their proximity were identified in only two cases. It can be concluded that the majority of the anterior epicardial nerves of porcine heart possess well-developed vasa nervorum. In contrast, similar blood vessels are rarely present in the vicinity of the Purkinje fibers. The data obtained contribute to a better understanding of the nutrition of the cardiac nerves.     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Adrenergic nerves and 5-hydroxytryptamine-containing cells in the pulmonary vasculature of the aquatic file snake Acrochordus granulatus

Summary The adrenergic innervation of the pulmonary vasculature of the file snake Acrochordus granulatus was examined by use of glyoxylic acid-induced fluorescence. Perivascular plexuses of blue-green fluorescent nerves are observed around the common pulmonary artery, the anterior and posterior pulmonary arteries, the arterioles leading to the gas exchange capillaries of the lung, the venules draining the lung, and the anterior and posterior pulmonary veins. Adrenergic nerves are also associated with the visceral smooth muscle of the lung septa and other tissues. Thus, adrenergic control of pulmonary blood flow may occur either at the common pulmonary artery or more regionally within the lung. Regional control of blood flow in the elongate lung of this snake may be important in matching pulmonary perfusion with the distribution of respiratory gas. Glyoxylic acid-histochemistry and immunohistochemistry revealed that populations of cells located in the common pulmonary artery contain the indoleamine 5-hydroxy-tryptamine. Many of the cells are intimately associated with varicose blue-green fluorescent nerves. It is proposed that the 5-hydroxytryptamine-containing cells may be involved in intravascular chemoreception.     

Galanin-immunoreactive nerves in the female rat paracervical ganglion and uterine cervix: distribution and reaction to capsaicin

Summary The presence and distribution of galanin-immunoreactivity was examined in the uterine cervix and paracervical autonomic ganglia of the female rat. Some animals were treated with capsaicin to determine if galanin-immunoreactivity was present in small-diameter primary afferent nerves. Other animals were treated with the noradrenergic neurotoxin 6-hydroxydopamine to ascertain if galanin-immunoreactivity was present in sympathetic noradrenergic nerves. Galanin-immunoreactive nerve fibers were sparse in the cervical myometrium and vasculature, but numerous in the paracervical ganglion where they appeared to innervate principal neurons. Immunoreactivity was also present in dorsal root ganglia, dorsal horn of spinal cord, and inferior mesenteric ganglia. Capsaicin treatment resulted in a marked reduction of galanin-immunoreactivity in the spinal cord dorsal horn, but not in the dorsal root ganglia, paracervical ganglia, or cervix (although there was a substantial reduction of substance P-, neurokinin A-, and calcitonin gene-related peptide-immunoreactivity in the dorsal horn, dorsal root ganglia, and uterine cervix). 6-Hydroxydopamine treatment did not cause any appreciable change in the galanin-immunoreactivity in any tissues. We conclude that galanin-like immunoreactivity is expressed in nerve fibers innervating the paracervical ganglia and uterine cervix of the female rat. This immunoreactivity is probably present in afferent nerves and could play a role in neuroendocrine reflexes and in reproductive function.     

Neuronal organization of the ascidian (Urochordata) branchial basket revealed by cholinesterase activity

Summary Innervation of the ascidian branchial basket and other structures is demonstrated by staining for cholinesterase. Cholinesterase activity is not restricted to synaptic sites but is present throughout the neurons. Primary and secondary axonal bundles form a bilaterally symmetric innervation pattern around the large dorsal visceral nerve. These bundles continue to split into progressively smaller bundles as they course throughout the basket. Axons are suspended in a fibrous matrix and run within the blood sinuses on the atrial side of the basket. Stigmatal ciliated cells of the branchial basket are innervated by highly branched distal portions of neurons, whose cell bodies are located in the ganglion. Synaptic boutons, containing electron-lucent vesicles, are found at nearly all stigmatal ciliated cells. NiCl₂backfills of the visceral nerve reveal a distinct population of central neurons, some of which presumably control ciliary arrest.     

Dose- and time-dependent hippocampal cholinergic lesions induced by ethylcholine mustard aziridinium ion: Effects of nerve growth factor,GM1 ganglioside,and vitamin E

Ethylcholine mustard aziridinium ion (ECMA) was infused intracerebroventricularly (icv) to rats followed by measurement of two markers of presynaptic cholinergic neurons, choline acetyltransferase (ChAT) activity and high affinity choline transport (HAChT), in the hippocampus and cortex. Bilateral icv administration of 1, 2, or 3 nmol of ECMA per side produced dose-dependent reductions in each marker in the hippocampus, but not in the cortex, one week after treatment. Reductions of 52% and 46% for ChAT activity and HAChT, respectively, were produced in the hippocampus by 3 nmol ECMA. Measurement of these two markers at different times after icv infusion of 2 nmol ECMA/ventricle revealed that the activity of ChAT was reduced to a greater extent than was HAChT in the hippocampus 1 day and 1, 2, 4, and 6 weeks after treatment. The maximal reductions of ChAT activity and HAChT (61% and 53%, respectively) were reached between 1 and 2 weeks after ECMA administration. There was no evidence of regeneration of either marker at 4 or 6 weeks posttreatment. HAChT and ChAT activity in the cortex were not altered at any of the posttreatment times examined.ECMA-induced deficits in hippocampal ChAT activity and HAChT were not counteracted by the following treatments: (i) daily administration of GM₁ ganglioside (10 mg/kg, intraperitoneally (ip)) from the day prior to infusion of ECMA until 2 weeks later; (ii) daily administration of GM₁ ganglioside between 2 and 6 weeks after infusion of ECMA; and (iii) icv administration of nerve growth factor (NGF) twice per week for 2 weeks after ECMA treatment. Since similar treatments with NGF and GM₁ ganglioside ameliorate lesions induced by other methods, these results indicate that the mechanism of lesion formation and the surviving cellular components influence the functional effects of neurotrophic factors. In contrast to the above results, treatment with vitamin E significantly attenuated ECMA-induced deficits of ChAT activity and HAChT. Further studies of the effects of vitamin E on the development of ECMA-induced deficits may help to elucidate the mechanism action of ECMA.     

Antibodies to Cholinergic Neurons in Alzheimer''s Disease

Alzheimer's disease (AD) is associated with degenerative changes in nuclei of the basal forebrain which provide most of the cholinergic input to the cortex and hippocampus and with a reduction in presynaptic cholinergic parameters in these areas. Although the etiology and pathogenesis of AD are not known, several reports indicate the involvement of immunological mechanisms. In the present work we examined the existence of antibodies in sera of AD patients that bind specifically to cholinergic neurons. As antigens we employed the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogeneous and cross-react antigenically with human and other mammalian cholinergic neurons. Our findings show that immunoglobulins from sera of AD patients bind to a specific antigen (molecular mass 200 kilodaltons) in the cell bodies and axons of Torpedo electromotor neurons and that the levels of such antibodies are significantly higher in AD patients than in controls. The possible role of these antibodies in the cholinergic dysfunction in AD and their diagnostic potential are discussed.     

Ciliary Neuronotrophic Factor Stimulates Choline Acetyltransferase Activity in Cultured Chicken Retina Neurons

It has been demonstrated that cultured cholinergic retinal neurons from 8-day-old chicken embryos respond to a polypeptide factor present in retinal cell-conditioned medium (RCM) and in retinal extracts. Compared with control cultures, the activity of acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; ChAT) is enhanced more than twofold in neuronal retinal cultures grown for 7 days in the presence of RCM. The present study demonstrates that both ciliary neuronotrophic factor (CNTF), which is characterized by its trophic activity on parasympathetic ciliary neurons, and RCM exhibit identical stimulatory effects on ChAT activity in retinal monolayer cultures. Similarly, RCM supports the in vitro survival of ciliary neurons to the same extent as CNTF. The active species in RCM has a molecular weight (20,900 +/- 1,000) identical to that of CNTF, as determined by preparative sodium dodecyl sulfate gel electrophoresis. The results indicate that cholinergic retinal neurons represent a central neuronal target for CNTF or a closely related protein.     

Chicken retinospheroids as developmental and pharmacological in vitro models: acetylcholinesterase is regulated by its own and by butyrylcholinesterase activity

Summary The phylo- and ontogenetically related enzymes butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are expressed consecutively at the onset of avian neuronal differentiation. In order to investigate their possible co-regulation, we have studied the effect of highly selective inhibitors on each of the cholinesterases with respect to their expression in rotary cultures of the retina (retinospheroids) and stationary cultures of the embryonic chick tectum. Adding the irreversible BChE inhibitor iso-OMPA to reaggregating retinal cells has only slight morphological effects and fully inhibits BChE expression. Unexpectedly, iso-OMPA also suppresses the expression of AChE to 35%–60% of its control activity. Histochemically, this inhibition is most pronounced in fibrous regions. The release of AChE into the media of both types of cultures is inhibited by iso-OMPA by more than 85%. Control experiments show that AChE suppression by the BChE inhibitor is only partially explainable by direct cross-inhibition of iso-OMPA on AChE. In contrast, the treatment of retinospheroids with the reversible AChE inhibitor BW284C51 first accelerates the expression of AChE and then leads to a rapid decay of the spheroids. After injection of BW284C51 into living embryos, we find that AChE is expressed prematurely in cells that normally express BChE. We conclude that the cellular expression of AChE is regulated by the amount of both active BChE and active AChE within neuronal tissues. Thus, direct interaction with classical cholinergic systems is indicated for the seemingly redundant BChE.