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.     

Cholinergic synapses of the associative temporal area of the neocortex in the realization of cognitive functions

The activity of choline acetyltransferase in the subsynaptic fractions of light and heavy synaptosomes of the associative temporal areas of the neocortex of cats with varying capacities for the formation of preverbal concepts was investigated. With respect to the majority of the subfractions, differences were detected between animals with normal and decreased intellect. Some theoretical conclusions were drawn relative to the origin of the individual subfractions; the role of cholinergic synapses of the area in question in the realization of the function of generalization and abstraction is discussed. Translated from Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 79, No. 9, pp. 18–25, September, 1993.     

Effects of long-term hormone replacement and of tibolone on choline acetyltransferase and acetylcholinesterase activities in the brains of ovariectomized,cynomologus monkeys

We examined long-term effects of low and high doses of tibolone, conjugated equine estrogens, and conjugated equine estrogens plus medroxyprogesterone acetate on choline acetyltransferase and acetylcholinesterase activities within different regions of the brain in cynomologus monkeys. All treatments were administered for 2 years. None of the treatments produced significant increases in either choline acetyltransferase or acetylcholinesterase in any of eight brain regions analyzed. In contrast, treatment with conjugated equine estrogens plus medroxyprogesterone acetate, but not conjugated equine estrogens alone, produced significant reductions in both choline acetyltransferase and acetylcholinesterase in the medial septum/diagonal band of Broca compared with untreated controls. Treatment with tibolone also resulted in significant reductions in both choline acetyltransferase and acetylcholinesterase in the medial septum/diagonal band of Broca, and this effect was dose-related. These findings are the first to report the effects of long-term therapies used by postmenopausal women on cholinergic measures in the primate brain. The findings are consistent with recent reports in rats, and suggest that any positive effects of long-term estrogen or hormone replacement therapy on cognitive processes are probably not due to significant effects on choline acetyltransferase or acetylcholinesterase activities.     

Inverter mechanism of the influence exerted by insulin on the plasma membranes of cardiac myocytes in rats of different age

In the plasma membranes of cardiac myocytes from old rats, Na, K-ATPase activity and phosphatidylinositol levels were lower and cardiolipin levels higher than in those from younger (adult) animals. Insulin injected into adult rats elevated Na, K-ATPase activity and phosphatidylethanolarnine levels and caused a sharp fall in phosphatidylinositol levels. In old rats, insulin had no effect on Na, K-ATPase activity, but lowered phosphatidylethanolamine levels. In experiments with cellular hybrids (cytosol+plasma membranes), cytosol from adult rats activated Na, K-ATPase in both adult and old rats, whereas cytosol from old rats failed to activate the enzyme both in old and in adult rats. Actinomycin D prevented the stimulatory effect of insulin on Na,K-ATPase activity. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 8, pp. 122–125, August, 1995     

Transplantation of embryonic ventral forebrain neurons to the neocortex of rats with lesions of nucleus basalis magnocellularis--I. Biochemical and anatomical observations

Unilateral ibotenic acid lesions of the rat nucleus basalis magnocellularis produce approximately 60% depletion of choline acetyltransferase activity in ipsilateral frontal and frontoparietal neocortex. This depletion, which represents the loss of most of the extrinsic neocortical cholinergic input, is stable for at least 6 months. Embryonic ventral forebrain neurons survive transplantation to such cholinergically denervated neocortex. Cholinergic cells abound within these transplants and appear able to reinnervate the cholinergically depleted host cortex, as assessed histochemically and by measurement of choline acetyltransferase activity. Outgrowing fibres may extend beyond 2 mm from the grafts and often appear to be organized in an appropriate laminar pattern within the host cortex. Peptidergic neurons are sparse within the grafts and their fibres frequently appear unable to grow into the host tissue. Control grafts of non-cholinergic embryonic hippocampal cells survive well but have no effect on cortical depletions of acetylcholinesterase or choline acetyltransferase activity. Reconstruction of the extrinsic cholinergic input to the cortex by transplantation provides a useful tool for understanding the functions of this pathway.     

Invertor-mediated modulation of Na,K-ATPase activity in myocardial plasma membranes in the emergency stage of compensatory cardiac hyperfunction in mature and senescent rats

Myocardial hyperfunction is experimentally modeled by coarctation of the aorta in mature (6–8-month-old) and senescent (26–28-month-old) Wistar rats. During the emergency phase of cardiac hyperfunction Na,K-ATPase activity is shown to rise reliably in mature animals, while in old rats it remains unchanged. Cardiomyocyte cytosol and blood plasma from mature (but not old) rats with experimental coarctation of the aorta activate Na,K-ATPase in membranes from the myocardium of both the mature and old rats. It is assumed that the activation of Na,K-ATPase during the emergency stage of cardiac hyperfunction is mediated through synthesis of specific invertors. In senescent animals the synthesis of invertors probably becomes insufficient, while membrane sensitivity to them is preserved. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 6, pp. 631–633, June, 1996     

Role of cholinergic regulation of the heart in the protective antiarrhythmic effect of adaptation to continuous moderate stress

Rats were adapted to the continuous action of moderate immobilization stress for 1, 5, and 15 days. Thereafter the threshold of ventricular fibrillation and the heart rate were compared with biochemical indexes of adrenergic and cholinergic regulation of the heart, namely, catecholamine, cAMP, and cGMP content, acetylcholinesterase and choline acetyltransferase activity, the number and affinity of cardiac muscarinic receptors, and the catecholamine content in the adrenals. The threshold of ventricular fibrillation fell on the 1st day due to a predominance of the adrenergic regulatory effect over the cholinergic. Adaptation for 5 days is attended by a rise of the threshold of ventricular fibrillation to the norm and by marked bradycardia, both these shifts being abolished by atropine. Elevation of the heart's resistance to arrhythmias stems from the prevalence of cholinergic regulation. Equilibrium between the cholinergic and adrenergic effects on the heart was found as a results of 15-day adaptation. The normal threshold of ventricular fibrillation and the increased cardiac resistance to arrhythmia were preserved and dictated largely by adaptive changes at the cardiomycyte level. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120. N ^o 7, pp. 36–39, July, 1995 Presented by G. N. Kryzhanovskii, Member of the Russian Academy of Medical Sciences     

GM1 ganglioside enhances cholinergic parameters in the brain of senescent rats.

GM1 ganglioside and nerve growth factor both promote the recovery of injured central cholinergic neurons in young animals. Brain cholinergic activity declines with aging and nerve growth factor has been shown to correct cholinergic deficits in senescent animals. We have administered GM1, to young (three months old) or senescent (22-24 months old) rats and evaluated acetylcholine and choline content, choline acetyltransferase and acetylcholinesterase activity as well as choline uptake in striatum, hippocampus and frontal cortex. For some studies, nerve growth factor was administered alone or together with GM1. Our results indicate that cholinergic neurochemical parameters are decreased in some brain areas of senescent animals and that both GM1 and nerve growth factor can enhance their recovery.     

The cholinergic innervation of normal and transplanted superior colliculus in the rat: an immunohistochemical study

The distribution of choline acetyltransferase was determined in normal and transplanted rat superior colliculus with choline acetyltransferase immunohistochemistry. This distribution was compared to the pattern of histochemically detected acetylcholinesterase activity. To determine cholinergic input to the superficial superior colliculus, double labelling experiments combining retrograde tracing methods and choline acetyltransferase immunohistochemistry were carried out. No choline acetyltransferase-containing neurons were observed in the rat superior colliculus. A dense network of choline acetyltransferase-immunoreactive fibres and terminals was seen in the intermediate layers of the normal superior colliculus. The distribution was patchy and very similar to the pattern of acetylcholinesterase activity. Occasional fibres and terminals were seen in the deep tectal laminae. The superficial layers contained a low number of choline acetyltransferase-stained fibres and terminals but a comparatively high level of acetylcholinesterase activity. Following a unilateral injection of a tracer into the superficial superior colliculus, retrogradely labelled choline acetyltransferase-immunoreactive neurons were found in the dorsal and ventral subnuclei of the ipsilateral parabigeminal nucleus. As in the normal superior colliculus, choline acetyltransferase-positive neurons were not found in tectal transplants. However, choline acetyltransferase-immunoreactive fibres and terminals were seen in grafts but only in those which had extensive connections with the host midbrain. The pattern of staining most closely resembled that seen in the intermediate layers of the normal superior colliculus. The similar arrangement of choline acetyltransferase and acetylcholinesterase activity in the intermediate layers of normal rat superior colliculus provides further evidence for cholinergic innervation to these layers, probably originating in the dorsal and pedunculopontine tegmental nuclei. The data from the double labelling experiments indicate that the choline acetyltransferase-immunoreactive terminals observed in the superficial layers represent the terminal field of an ipsilateral cholinergic projection from the parabigeminal nucleus. Tectal grafts receive cholinergic innervation from the host. The evidence suggests that much of this input derives from the cholinergic nuclei in the brainstem tegmentum which normally project to the intermediate tectal layers.     

Nerve growth factor affects uninjured, adult rat septohippocampal cholinergic neurons

The effect of nerve growth factor on the intact versus injured septohippocampal cholinergic system of adult rats was studied. Nerve growth factor was continuously infused into the lateral ventricle of adult uninjured rats or rats that had received unilateral partial transection of the fimbria. Controls (operated and unoperated) received intraventricular infusion of cytochrome c. After 2 weeks of nerve growth factor or cytochrome c treatments, choline acetyltransferase and acetylcholinesterase activities were measured in the septal area and in the hippocampus (divided into dorsal, medial and ventral parts). The continuous infusion of nerve growth factor resulted in a marked dose-dependent increase of choline acetyltransferase activity in both septum and hippocampus of adult unlesioned rats. In lesioned rats the nerve growth factor treatment was capable of inducing choline acetyltransferase activity in the hippocampus of not only the lesioned but also the unlesioned side, as well as in the septal area. In addition, nerve growth factor affected choline acetyltransferase activity differently in the hippocampus of the operated side with respect to the contralateral side or in unoperated animals. The chronic infusion of nerve growth factor did not affect acetylcholinesterase activity in the septum or in the hippocampus of either lesioned or unlesioned rats. The present findings indicate that nerve growth factor is capable of modulating the function of not only damaged but also normal adult forebrain cholinergic neurons. This suggests that nerve growth factor may modulate the function of these neurons in adulthood.     

Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterase-positive neurons

Cholinergic neurons intrinsic to rat cortex were studied using a sensitive method for the localization of choline acetyltransferase immunoreactivity, acetylcholinesterase histochemistry, combined localization of choline acetyltransferase and acetylcholinesterase, and combined localization of choline acetyltransferase and retrogradely transported horseradish peroxidase-wheat germ agglutinin. Choline acetyltransferase immunoreactivity was localized predominantly in small bipolar cortical neurons within the upper layers of isocortex, while small multipolar neurons were the predominantly stained cell type in allocortical regions. Acetylcholinesterase histochemistry demonstrated mainly small polymorphic cells scattered throughout all cellular layers in all cortices. Combined staining for choline acetyltransferase and acetylcholinesterase resulted in localization of the markers in different cell populations; choline acetyltransferase-immunoreactive neurons did not contain detectable acetylcholinesterase and acetylcholinesterase-positive neurons did not contain detectable immunoreactivity to choline acetyl-transferase. Some possible connections of the cortical choline acetyltransferase-immunoreactive cells were studied in rats which had received injections of horseradish peroxidase-wheat germ agglutinin into either cortex or brainstem. The choline acetyltransferase-immunoreactive cells were frequently admixed with cells labeled with the retrograde marker; however, no double-labeled cells were observed.It was concluded that cortical cholinergic cells are not visualized by acetylcholinesterase histochemistry, and are likely to be involved in local circuitry.     

Regional decreases of cortical choline acetyltransferase after lesions of the septal area and in the area of nucleus basalis magnocellularis

Choline acetyltransferase and [³H]choline uptake have been measured in neocortical regions and hippocampus one week after lesions which destroyed the septum bilaterally, and after unilateral lesions in the area of nucleus basalis magnocellularis. Lesions of the septal area, which severely decreased choline acetyltransferase in hippocampus, only moderately decreased choline acetyltransferase in a posterior cortical region and had no effect in frontal and parietal regions. In contrast, lesions which included nucleus basalis magnocellularis decreased choline acetyltransferase markedly in frontal and parietal regions and had less of an effect in the posterior cortical regions. Lesion-induced decreases of [³H]choline uptake paralleled those of choline acetyltransferase. Lesions which included nucleus basalis magnocellularis had no effect on choline acetyltransferase in hippocampus, nucleus accumbens, olfactory tubercle, midbrain or pons-medulla.These results suggest the existence of topographically distinct cholinergic inputs to neocortex. In agreement with previous studies, cholinergic projections from the peripallidal region of nucleus basalis magnocellularis are predominantly to frontal and parietal neocortex. In contrast to previous suggestions, cholinergic projections to neocortex from the septal area are limited to the posterior regions of neocortex.     

Further characterizations of the nature of the behavioral and neurochemical effects of lesions to the nucleus basalis of Meynert in the rat

Converging lines of evidence indicate an important role for the basal forebrain cholinergic system in memory processes. The principal origin of the cholinergic projection to the neocortex appears to be the magnocellular neurons in the region of the nucleus basalis of Meynert (NbM). We examined the effects of bilateral lesions of the NbM on retention of shock avoidance training by stereotaxically injecting rats with 0.5 microliter of ibotenic acid (14 micrograms/microliter) into the NbM. Two weeks later rats were given passive avoidance training and tested for retention of the original avoidance habit 5 min, 30 min, or 24 hr later. Rats with lesions of the NbM showed significantly impaired shock avoidance performance compared to non-operated controls at both 30 min and 24 hr, but not at 5 min after training. Lesioned animals also showed a significant decrease in cortical choline acetyltransferase (CAT) and acetylcholinesterase (AChE) activities. No differences in muscarinic receptor binding or plasma cholinesterase activity was observed. The results demonstrate the usefulness of NbM lesions as a model for studying the role of the basal forebrain cholinergic system in memory processes.     

Transmitter phenotype is a major determinant in the specificity of synapses formed by cholinergic neurons transplanted to the hippocampus

Embryonic habenular or striatal cholinergic tissues were transplanted to the hippocampal formation of adult rats. The connectivity of these grafts with the host hippocampal formation was analysed using acetylcholinesterase histochemistry and immunocytochemistry with a monoclonal antibody to choline acetyltransferase. Both graft types produced laminar arrangements of acetylcholinesterase-positive fibers in the hippocampal formation that closely resembled the native pattern of cholinergic innervation. In addition, graft-derived choline acetyltransferase-immunoreactive synapses were found in the host hippocampal formation. These synapses were formed on non-immunoreactive dendritic structures and were similar to the types of cholinergic synapses found in the hippocampal formation of normal animals. These data indicate that the cholinergic transmitter phenotype is a major determinant of whether a neuron will form typical cholinergic synapses with hippocampal targets.     

Concurrent visualization of choline acetyltransferase-like immunoreactivity and retrograde transport of neocortically injected markers in basal forebrain neurons of cat and rat

Magnocellular neurons in the basal forebrain of rats and cats were retrogradely labeled with Fast Blue or horseradish peroxidase injected into the neocortex. Using antisera against choline acetyltransferase (ChAT) a direct double-labeling technique was carried out and it was demonstrated that retrogradely transported markers and ChAT-like immunoreactivity occur within the same neurons. These findings strongly support the cholinergic nature of basal forebrain projection to the neocortex.     

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)     

A comparison of the localization of choline acetyltransferase and glutamate decarboxylase immunoreactivity in rat cerebral cortex

The neurotransmitter-synthesizing enzymes choline acetyltransferase and glutamate decarboxylase were localized immunocytochemically at the light microscopic level. Their respective laminar distributions were compared in 17 different cytoarchitectural areas, comprising limbic and neocortical regions of rat cerebral cortex. The immunoreactive intensities within these areas were measured with an image analysis system and dark-field optics. Choline acetyltransferase and glutamate decarboxylase immunoreactivity displayed distinctive distribution patterns throughout the cerebrum. In general, limbic cortex showed greater intensity of both choline acetyltransferase and glutamate decarboxylase immunoreactivity than neocortex. For example, choline acetyltransferase immunoreactivity in pyriform and retrosplenial cortex was 54% and 29% greater, respectively, than in neocortex, and glutamate decarboxylase immunoreactivity in the same cortical areas was 5% and 17% greater, respectively. In addition to these regional differences, the marked variations of choline acetyltransferase and glutamate decarboxylase immunostaining were characterized as either coincidental or complementary when comparing their laminar distributions. The laminar pattern and relative intensities of choline acetyltransferase and glutamate decarboxylase immunostaining were coincident in some layers of all cortical regions. For example, both choline acetyltransferase and glutamate decarboxylase immunoreactive intensities were high in cellular layers II and IV of the entorhinal cortex. In contrast, examples of complementary choline acetyltransferase and glutamate decarboxylase immunoreactive patterns were observed in retrosplenial cortex and neocortex. In neocortex, layers III and part of V were intensely glutamate decarboxylase-positive, whereas these same layers were less intensely choline acetyltransferase immunoreactive than the intervening layer IV and upper part of V. Quantitatively, choline acetyltransferase immunoreactivity in layers IV and upper V was 27-37% greater than adjacent layers II and deep V. The glutamate decarboxylase immunostaining pattern was complementary in that layer IV was 19-23% less intensely stained than adjacent layers III and V. Our results demonstrate that terminals immunoreactive for choline acetyltransferase and glutamate decarboxylase, and presumably the synaptic terminals that respectively use acetylcholine or gamma-aminobutyric acid as their neurotransmitters, are distributed in distinct laminar patterns that are strategically situated for modulating either afferent information in the case of cholinergic terminals or efferent transmission for GABAergic endings.(ABSTRACT TRUNCATED AT 400 WORDS)     

Basal forebrain neurons provide major cholinergic innervation of primate neocortex

In 3 monkeys, lesions were made in the basal forebrain by microinjections of ibotenic acid into the nucleus basalis. Bilateral samples of multiple neocortical gyri were assayed for the activity of choline acetyltransferase. Compared to control hemispheres, enzyme activity was reduced up to 69% in the neocortex ipsilateral to the lesion; in addition, acetylcholinesterase staining was decreased at the lesioned site and in the ipsilateral cortex. These results support the concept that the principal cholinergic innervation of the primate neocortex is derived from axons and nerve terminals of neurons whose perikarya are located in the basal forebrain, particularly the nucleus basalis.     

Neuroprotective effect of developmental docosahexaenoic acid supplement against excitotoxic brain damage in infant rats

Long-chain polyunsaturated fatty acid (LC-PUFA) composition of neural membranes is a key factor for brain development, in chemical communication of neurons and probably also their survival in response to injury. Viability of cholinergic neurons was tested during brain development following dietary supplementation of fish oil LC-PUFAs (docosahexaenoic acid [DHA], eicosapentaenoic acid, arachidonic acid) in the food of mother rats. Excitotoxic injury was introduced by N-methyl-D,L-aspartate (NMDA) injection into the cholinergic nucleus basalis magnocellularis of 14-day-old rats. The degree of loss of cholinergic cell bodies, and the extend of axonal and dendritic disintegration were measured following immunocytochemical staining of cell bodies and dendrites for choline acetyltransferase and p75 low-affinity neurotrophin receptor and by histochemical staining of acetylcholinesterase-positive fibres in the parietal neocortex. The impact of different feeding regimens on fatty acid composition of neural membrane phospholipids was also assayed at 12 days of age. Supplementation of LC-PUFAs resulted in a resistance against NMDA-induced excitotoxic degeneration of cholinergic neurones in the infant rats. More cholinergic cells survived, the dendritic involution of surviving neurons in the penumbra region decreased, and the degeneration of axons at the superficial layers of parietal neocortex also attenuated after supplementing LC-PUFAs. A marked increment in DHA content in all types of phospholipids was obtained in the forebrain neuronal membrane fraction of supplemented rats. It is concluded that fish oil LC-PUFAs, first of all DHA, is responsible for the neuroprotective action on developing cholinergic neurons against glutamate cytotoxicity.