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Mental stress modifies both cholinergic neurotransmission and alternative splicing in the brain, via incompletely understood mechanisms. Here, we report that stress changes brain microRNA (miR) expression and that some of these stress-regulated miRs regulate alternative splicing. Acute and chronic immobilization stress differentially altered the expression of numerous miRs in two stress-responsive regions of the rat brain, the hippocampal CA1 region and the central nucleus of the amygdala. miR-134 and miR-183 levels both increased in the amygdala following acute stress, compared to unstressed controls. Chronic stress decreased miR-134 levels, whereas miR-183 remained unchanged in both the amygdala and CA1. Importantly, miR-134 and miR-183 share a common predicted mRNA target, encoding the splicing factor SC35. Stress was previously shown to upregulate SC35, which promotes the alternative splicing of acetylcholinesterase (AChE) from the synapse-associated isoform AChE-S to the, normally rare, soluble AChE-R protein. Knockdown of miR-183 expression increased SC35 protein levels in vitro, whereas overexpression of miR-183 reduced SC35 protein levels, suggesting a physiological role for miR-183 regulation under stress. We show stress-induced changes in miR-183 and miR-134 and suggest that, by regulating splicing factors and their targets, these changes modify both alternative splicing and cholinergic neurotransmission in the stressed brain.

Brain acetylcholinesterase activity was measured in 14 patients with Alzheimer disease and 14 age-matched control subjects by positron emission tomography with a radioactive acetylcholine analogue. Kinetic analysis was performed to calculate k3, an index of acetylcholinesterase activity. The k3 values were significantly reduced in the neocortex, hippocampus, and amygdala of all patients with Alzheimer disease, suggesting a loss of cholinergic innervation from the basal forebrain. Most profound reductions of k3 values were observed in the temporal (-30%) and parietal cortices (-31%), although reductions of k3 values were relatively uniform in the cerebral neocortex. This technique may be a powerful tool for early diagnosis of Alzheimer disease and also for therapeutic monitoring of acetylcholinesterase inhibitors in Alzheimer disease.

Development and maturation of the basolateral complex of the amygdaloid body were studied in fetal and postnatal rat brains. In cresyl violet-stained sections the basolateral complex was distinguishable at the 17th prenatal day. On about the 14th postnatal day, it showed all the features of the adult structure. The acetylcholinesterase activity appeared in neuropil of the basolateral complex on the seventh postnatal day; it increased slowly up to the 60th day. After that age, the acetylcholinesterase activity was stable in all parts of the basolateral complex and corresponded to its cytoarchitectonic differentiation. Our results suggest that the process of maturation of the amygdaloid basolateral complex, being related to the ingrowing of the cholinergic fibers from the basal forebrain, lasts at least up to the end of the second month of postnatal life.

To investigate the possible relationship between acetylcholinesterase (AChE)-containing fiber density and senile plaque density and between AChE-positive plaques and beta/A4 protein deposition, AChE histochemistry, the modified Bielschowsky's method and beta/A4 protein immunohistochemistry were performed on the amygdala of Alzheimer's disease (AD) and aged control cases. Abundant AChE-positive senile plaques were found in the amygdala and related structures in AD. These AChE-positive plaques were mainly of the primitive or diffuse type. In addition to senile plaques of typical morphologies a variety of AChE-positive structures were observed in the amygdala and related regions in AD. A comparison of serial sections stained alternatively with AChE histochemistry and beta/A4 protein immunohistochemistry has revealed that these AChE-positive structures with variable morphological appearances displayed beta/A4 protein immunoreactivity, indicating that AChE is localized in a variety of beta/A4 protein deposition including the diffuse plaque. Thus, it is suggested that AChE is present in some senile plaques at the earliest stage. However, there was no apparent correlation between the numerical density of AChE-positive fibers and senile plaque density. These findings suggest that the degeneration of cholinergic neurons is not attributed to the occurrence of AChE activity in beta/A4 protein.

The distribution pattern of cholinesterase activity in the basal forebrain region was examined in five human brains without any history of neurologic or psychiatric disorders. Complete sets of serial sections allowed a three-dimensional reconstruction of this region. The intensity grading and the distribution pattern of the specific and non-specific cholinesterase activity was depicted diagramatically. The distribution pattern of cholinesterase activity in the supracommissural striatum demonstrated the well-known striosomal configuration, particularly in the head of the caudate nucleus. Within this nucleus caudatus the striosomes appeared connected with a subventricular zone of low acetylcholinesterase-activity. Bands of very high activity could be demonstrated from the dorsolateral and ventral areas of the caudate nucleus to the lateral border of the putamen and the commissural and subcommissural division of the ventral striatum. The distribution pattern of cholinesterase activity in the subcommissural region showed very close correlation to the cytomorphological subdivisions of the striatum as defined by Brockhaus (1942). In addition to his topographic description it was possible to define the tuberculum olfactorium and several subdivisions of the interstitial nucleus of the stria terminalis. The inhibition of non-specific esterase activity by ISO-OMPA in the globus pallidus allowed distinction between striatal and pallidal components. Three-dimensional reconstructions of the terminal islands revealed several types, which were named according to their topography as insulae substriatales, -subventriculares, -olfactoriae, -magnae, and -interstitiales. Characteristically, the core of these islands consisted of clusters of tightly packed, extremely high acetylcholinesterase-positive cells. Cholinesterase activity of the surrounding rim region ranged from negative to strongly positive depending on the position and type of the island. The findings suggest that the islands represent derivatives of the fundus striati region as defined by Brockhaus and are connected to the dorsal striatum by means of cellular bridges.

Histo- and immunohistochemical distribution of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in the amygdaloid nuclei of young adult (3 month old) and aged (26 month old) Wistar rats was compared. AChE staining and ChAT immunoreactivity showed the same regional variations in the amygdaloid nuclei of young adult rats. The density and staining of AChE- and ChAT-positive fibres, terminals, and nerve cells were reduced in aged rat amygdala. Moreover, heavily stained aberrant fibres and coarse terminals were located around the nerve cells, blood vessels, and occasionally in patches. In aged rats, atrophic AChE positive and ChAT immunoreactive nerve cells exhibited serpentine-like, thicker, and less extensively branched dendrites than those in young adult rats. These changes are similar to the age-related changes in the cholinergic enzymes in other brain regions which are targets to the basal forebrain.

The origin of the cholinergic innervation to the amygdaloid complex was investigated with the use of acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) assay of microdissected nuclei. Visualization of AChE-positive neurones in the ventral forebrain was facilitated by pretreatment of rats with 1.5 mg/kg di-isopropyl phosphofluoridate (DFP). The AChE-positive neurones in the ventral forebrain are distributed in a continuous system from the septum through the lateral preoptic area to the entopeduncular nucleus caudally. Knife cuts or kainic acid injections (1.5 microgram/l microliter) placed in the lateral preoptic area resulted in substantial depletion of the AChE and ChAT content of the amygdala nuclei. Kainic acid injections (1.5 microgram/l microliter) in the diagonal band area or cuts through the stria terminalis dorsally did not significantly modify the AChE staining or ChAT content of the amygdala (although diagonal band injections partially depleted the hippocampus of ChAT). Knife cuts severing both the so-called ventral pathway and the stria terminalis did not produce significantly greater ChAT depletion in the amygdala than those produced by the knife cuts or kainic acid injections in the lateral preoptic area. Parasagittal knife cuts undercutting the lateral pyriform cortex also failed to modify the AChE or ChAT content of the amygdala, but they depleted the undercut cortex of both ChAT and AChE; AChE-positive material accumulated ventrally and medially to the knife cut. It is suggested that the major source of the cholinergic innervation of the amygdala is the magnocellular AChE-positive neurones in the lateral preoptic area and adjacent regions of the ventral forebrain.

Orchidectomy plus estradiol administration decreased acetylcholinesterase activity in the rat cerebral cortex and mesencephalon, while in the amygdala it was increased. In the adenohypophysis, orchidectomy increased enzyme activity, but subsequent estradiol treatment decreased it. The hypothalamus did not respond to either manipulation.

Acetylcholinesterase staining was examined in the substantia innominata of 3 normal human brains. Large intensely stained neurones were seen within the region of the basal nucleus of Meynert which is believed to be the origin of the cholinergic projection to the neocortex in animals. On the basis of the acetylcholinesterase staining pattern, the substantia innominata was dissected from post-mortem brain tissue of 19 cases of Alzheimer's disease (AD) and 16 controls so as to include the basal nucleus. Choline acetyltransferase (ChAT) activity was found to be reduced in the substantia innominata and amygdala in AD but not in the adjacent lentiform nucleus and hypothalamus.

The topographical distribution and morphological characteristics of neurons containing acetylcholinesterase (AChE, EC 3.1.1.7) in the basal forebrain of the cat were studied by means of the Butcher's pharmaco-histochemical technique involving staining for AChE at various times after the administration of di-isopropylfluorophosphate (DFP). This method allows a clear visualization of numerous AChE-producing neurons in olfactory tubercle, medial septal-diagonal band area, hypothalamus and basal ganglia. In olfactory tubercle the AChE neurons abound in the deep polymorph layer where they form several types of cell clusters topographically related to the islands of Calleja. The AChE neurons in medial septal nucleus appear morphologically similar to those in nucleus of the diagonal band. As a whole, the medial septal-diagonal band area stands out as one of the major AChE cell collections in basal forebrain. In hypothalamus, neurons staining intensely for AChE occur particularly in supraoptic and paraventricular nuclei and in supramammillary nucleus. The neurons in lateral hypothalamic area stain only moderately for the enzyme. On the other hand, numerous large-sized AChE cells are scattered throughout the neostriatum. However, the AChE cells in putamen appear morphologically different, more numerous per mm2, and larger than those in caudate nucleus suggesting that the feline neostriatum is not a homogeneous structure. The globus pallidus contains both small and large cells that stain lightly and intensely for AChE, respectively. The small pallidal cells are similar to entopeduncular cells and it is proposed that they form the typical pallidal elements. In contrast, the large pallidal AChE cells appear similar to AChE neurons lying in adjacent substantia innominata, and are continuous with the AChE cells in medial septal-diagonal band area. It is suggested that these magnocellular AChE neurons form a population of "limbic" elements within the feline basal ganglia.

(1) The activities of choline acetyltransferase (ChAc) (EC 2.3.1.6) and acetylcholinesterase (AChE) (EC 3.1.1.7) were determined in about one hundred regions and subregions of baboon brain. The activities and distributions of these enzymes were in comparable to those found previously in the brains of other species. (2) ChAc activity was highest in the interpeduncular nucleus, where it was about twice that in the putamen, the region previously thought to be the richest in this enzyme. The caudate nucleus, the substantia perforata, the nucleus basalis, the central part of the amygdala and the oculomotor nucleus also had high activities. The activities in the cerebral and cerebellar cortex were less than one twentieth of that in the interpeduncular nucleus. (3) The distribution of AChE activity was not entirely in parallel with that of ChAc.

The relationship between neuritic plaque formation in Alzheimer's disease and cholinergic innervation of brain regions is unclear. Many neuritic plaques are found in the amygdala, which also receives dense cholinergic innervation from the ventral forebrain, predominantly to the basolateral complex. To determine whether the regional distribution of neuritic plaques is related to the pattern of cholinergic innervation, we studied serial sections through the amygdala of four patients with Alzheimer's disease and four neurologically normal patients. We compared acetylcholinesterase reactivity, neuritic plaques stained with thioflavine S, and cytoarchitectural features in adjacent sections. Neuritic plaque counts were high in most amygdaloid nuclei but were significantly lower in the most acetylcholinesterase-positive region, the lateral portion of the basal nucleus of the amygdala. Acetylcholinesterase reactivity was reduced in the Alzheimer's cases, but the basal nucleus was easily recognized by the characteristic large neurons. The morphology of neuritic plaques also differed in the various regions. These results show that neuritic plaques occur to varying degrees in all nuclei of the amygdala, but are significantly less frequent in the region that receives the most prominent innervation from the cholinergic ventral forebrain.

The distribution of acetylcholinesterase enzyme was studied in the amygdala of some rodents, subprimates and several primates. The cytoarchitecture of the amygdala has presented various problems to anatomists, including the question as to how many nuclear groups and subgroups should be identified. Among the mammals examined, the arrangement of the amygdaloid nuclei is remarkably uniform and no clear phylogenetic trend can be recognised. Although there are minor differences, there seems to be a general similarity between most mammals examined in so far as the distribution of cholinesterase is concerned. The staining is less intense in the brains of the monkeys examined. The sole exception to the rule, that cholinesterase distribution is slightly different from nucleus to nucleus in different animals, is the magnocellular part of the basal nucleus. This amygdaloid nucleus stains quite strongly in all animals examined. From these findings, and those of others studying the distribution of choline acetyltransferase, it was concluded that the basal amygdaloid nucleus is cholinergic and possible cholinoceptive. The ultrastructural investigations appear to confirm this point. This is particularly applicable to the magnocellular part of the basal amygdaloid nucleus.

The localization of acetylcholinesterase (AChE) activity in the nuclei of the amygdaloid body of man was studied by Gerebtzoff's modification of Koelle's acetylthiocholine method on 10 human brains. To compare the histochemical results with the cytoarchitectionics, additionally sections from 10 human brains were stained with cresyl violet. The intensity of AChE activity in the different amygdaloid nuclei varied widely. The high AChE activity occurred in the basal lateral nucleus and in the lateral part of the central nucleus. Very little activity was found in the medial nucleus and in the intermediate part of the central nucleus. All remaining nuclei showed AChE activity of moderate intensity. In almost each amygdaloid nucleus, differences of AChE activity among various areas were observed. In the amygdala of man all nuclei described in animals were found except nucleus of the lateral olfactory tract. Most of the human amygdaloid nuclei show more differentiated structure than in lower mammals in terms of not only cytoarchitectonic subdivision, but also in the intensity of AChE activity.

The cellular structure and distribution of histochemically demonstrated acetylcholinesterase (AChE) activity were studied in the amygdaloid body of 9 dogs. Cytoarchitectonic observations were made in series of paraffin and celloidin sections stained with cresyl violet. For the demonstration of the acetylcholinesterase activity, modifications of Koelle method were used. The general pattern of morphological structure of the dog's amygdaloid body is similar to that in other mammalian species. The corticomedial group of the nuclei was characterized generally by cytoarchitectonic uniformity of small, lightly stained cells and low intensity of the AChE reaction, except for the nucleus of the lateral olfactory tract and the lateral part of the central nucleus. The latter showed further differentiation in both cellular arrangement and distribution of AChE activity and may be divided into three subdivisions. The basolateral group of nuclei was characterized by higher differentiation of the cellular arrangement and distribution of the AChE activity. The highest enzyme activity was observed in the basal magnocellular nucleus. These findings support the homology of particular amygdaloid nuclei in various mammalian species.

Kainic acid (KA) was injected into the amygdala (AM) complex of the rat and its effect on the cholinergic enzymes, choline acetyltransferase (CAT) and acetylcholinesterase (AChE), and the binding of the muscarinic ligand, [3H]quinuclidinyl-benzilate (QNB) and the nicotinic ligand [125I]alpha-bungarotoxin (aBuTX) was investigated. Ka produced a loss of approximately 35% of the CAT activity in the AM. However, no effect on AChE activity was observed. A 30--50% decrease in the number of muscarinic and nicotinic receptor sites was also found. CAT, AChE and QNB binding in the AM contralateral to the injection did not change. However, the binding of aBuTX was found to decrease by approximately 40%. The present results suggest that the loss of CAT activity in the AM after treatment with KA is due to the destruction of cholinergic neurons within the AM. The lack of effect on AChE suggests that the major cholinergic input to the AM is not affected by KA. In addition, the loss of nicotinic receptors in the contralateral AM may reflect anterograde degeneration of terminals that have nicotinic sites located on them, or may be secondary to the elicitation of intense seizure activity evoked by the KA.

In order to study the distribution of acetylcholinesterase (AChE) in the primate hippocampal formation, we have stained serial sections through the brains of nine macaque monkeys for AChE by two variants of the Koelle acetylthiocholine method. We have found a distinctive pattern of staining in the hippocampal formation which varies in intensity both from region to region, and along rostrocaudal and radial gradients within each region. In the dentate gyrus, there is intense staining of the inner one-third of the molecular layer with much lighter staining in the rest of the molecular layer except for a moderately stained band at its outer edge. In the caudal half of the dentate gyrus, the inner portion of the molecular layer is less intensely stained though there is a distinctly denser band of staining just above, and partly within, the superficial margin of the granule cell layer. The granule cells are unstained but there are AChE-positive fibers which run through the granule cell layer to the molecular layer. The hilar region of the dentate gyrus has a narrow band of heavy staining (which corresponds to an acellular layer in Nissl-stained sections) just subjacent to the granule cell layer; the remainder of the hilus, where most of the hilar cells reside, is less intensely stained and at caudal levels is almost entirely unstained. In the regio inferior of the hippocampus, there is intense staining of the stratum oriens which extends into the pyramidal cell layer; the stratum radiatum and the stratum lacunosum- moleculare are also stained and here the staining pattern shows some degree of stratification. By contrast, most of the alveus, the pyramidal cell somata, and the layer of mossy fibers (stratum lucidum) are unstained. The border region between regio inferior and regio superior of the hippocampus (field CA2 of Lorente de No, '34) is especially heavily stained. This contrasts markedly with regio superior, which is more lightly stained than regio inferior. Stratum oriens and stratum radiatum of regio superior have a more evenly distributed pattern of staining, though the intensity of staining increases sharply at the border with the subiculum. Stratum lacunosum- moleculare is only lightly stained throughout much of the transverse extent of regio superior but there is also a conspicuous and constant patch of heavier staining at the border with the subiculum.(ABSTRACT TRUNCATED AT 400 WORDS)

This paper describes in detail the acetylcholinesterase (AChE) staining characteristics of the normal human amygdaloid complex using fixed post-mortem brain tissue. It was shown that the differential AChE staining of the amygdala could be used to accurately delineate amygdaloid subdivisions macroscopically. Using the same staining method on the amygdala from brains previously frozen and stored at -70 degrees C resulted in a staining pattern similar to that seen using fixed tissue, despite numerous ice artifacts. It was concluded that this stain may be useful to researchers working on post-mortem human amygdala, particularly those involved in microdissecting this region for neurochemical analysis.

The distribution of "marker" enzymes for cholinergic neurons has been studied in 10 subdivisions of the amygdaloid complex of the rat brain. Choline acetyltransferase activity was measured using a radiochemical method in samples dissected from fresh serial sections. Acetylcholinesterase was studied using a histochemical procedure. Both enzymes had similar patterns of distribution within the amygdaloid complex and were most concentrated in the posterior lateral and basolateral nuclei and in the nucleus of the lateral olfactory tract. These enzymes were much less concentrated in the cortical, medial, central, and basomedial nuclei. Large differences in acetylcholinesterase staining were found within the lateral posterior and the basolateral nuclei and within the pyriform cortex. Biochemical studies showed a parallel distribution of choline acetyltransferase within these nuclei. The results indicate that cholinergic neural elements in the amygdala are concentrated primarily in the basolateral complex and suggest that this region may be innervated by cholinergic fibers traveling in the ventral amygdalo-fugal pathway.

The aim of the study is to present a detailed and thorough description of the acetylcholinesterase enzyme (AChE) content of the limbic structures of the rabbit, now commonly used in our laboratory for investigating the electrographic-behavioral correlations of the cholinergic limbic system. We use 'chemitrodes' for intracerebral mi croinjections of cholinomimetic drugs; a detailed histochemical study (using Koelle's method) of the distributions of AChE enzyme in the limbic system of the rabbit was deemed necessary. Such areas as the septum and the amygdala are particularly poorly described in the rabbit and an attempt was made to clarify the confusion that still exists in the literature on the comparative anatomy and exact localization of these structures. On the whole, it could be inferred from this study that AChE-bearing neurons are generally better developed in phylogenetically older parts of the brain. Practically all the relays in Papez's 'cortical circuits of emotion' have a fairly AChE activity. As noted by us in other phyogenetically lower macrosmatic mammals, the reaction of AChE in the rabbit brain is more intense than in primates.