Acetylcholinesterase Reaction Product Research Articles

Protective Effects of a synthetic glycosaminoglycan mimetic (OTR4132) in a rat immunotoxic lesion model of septohippocampal cholinergic degeneration

Using a partial hippocampal cholinergic denervation model, we assessed the effects of the RGTA® named OTR4132, a synthetic heparan-mimetic biopolymer with neuroprotective/neurotrophic properties. Long-Evans male rats were injected with the cholinergic immunotoxin 192 IgG-saporin into the medial septum/diagonal band of Broca (0.37 µg); vehicle injections served as controls. Immediately after surgery, OTR4132 was injected into the lateral ventricles (0.25 µg/5 µl/rat) or intramuscularly (1.5 mg/kg). To determine whether OTR4132 reached the lesion site, some rats received intracerebroventricular (ICV) or intramuscular (I.M.) injections of fluorescent OTR4132. Rats were sacrificed at 4, 10, 20, or 60 days post-lesion (DPL). Fluorescein-labeled OTR4132 injected ICV or I.M. was found in the lesion from 4 to 20 DPL. Rats with partial hippocampal cholinergic denervation showed decreases in hippocampal acetylcholinesterase reaction products and in choline acetyltransferase-positive neurons in the medial septum. These lesions were the largest at 10 DPL and then remained stable until 60 DPL. Both hippocampal acetylcholinesterase reaction products and choline acetyltransferase-positive neurons in the medial septum effects were significantly attenuated in OTR4132-treated rats. These effects were not related to competition between OTR4132 and 192 IgG-saporin for the neurotrophin receptor P75 (p75NTR), as OTR4132 treatment did not alter the internalization of Cy3-labelled 192 IgG. OTR4132 was more efficient at reducing the acetylcholinesterase reaction products and choline acetyltransferase-positive neurons than a comparable heparin dose used as a comparator. Using the slice superfusion technique, we found that the lesion-induced decrease in muscarinic autoreceptor sensitivity was abolished by intramuscular OTR4132. After partial cholinergic damage, OTR4132 was able to concentrate at the brain lesion site possibly due to the disruption of the blood-brain barrier and to exert structural and functional effects that hold promises for neuroprotection/neurotrophism.

Involvement of the Basal Cholinergic Forebrain in the Mediation of General (Propofol) Anesthesia

Recent studies have pointed out the involvement of the basal forebrain gamma-aminobutyric acid-mediated system in mediating the effects of general anesthesia. In this study, the authors asked whether the basal forebrain cholinergic system is also involved in mediating the effects of general anesthetics such as propofol. Cholinergic lesions were produced by administration of the selective immunotoxin 192 immunoglobulin G-saporin into the lateral ventricles, the medial septum, or the nucleus basalis magnocellularis. The anesthetic potency of propofol was determined using an anesthetic score with a crossover counterbalanced design. Animals were given intraperitoneal propofol (25 or 50 mg/kg) repeatedly every 15 min to set up a subanesthetic (low-dose) or anesthetic (high-dose) state. The anesthetic score was assessed for each cumulative dose. Control of the cholinergic depletion was performed using histochemical acetylcholinesterase staining on brain slices. A shift from a subanesthetic state to an anesthetic state was observed mainly in the rats with the immunotoxin injected into the lateral ventricles or the medial septum and vertical diagonal band of Broca, compared with controls. In those rats, the density of acetylcholinesterase reaction products was normal in the striatum and the thalamus, but reduced in the cortex and the hippocampus. The anesthetic potency of propofol was increased in all rats with hippocampal lesions, whatever the injection sites, compared with controls. These results demonstrate that a cholinergic dysfunction in the basal forebrain potentiates the anesthetic effects of propofol.

Selective cholinergic lesions in the rat nucleus basalis magnocellularis with limited damage in the medial septum specifically alter attention performance in the five-choice serial reaction time task

Selective immunotoxic cholinergic lesions in the nucleus basalis magnocellularis (NBM) impair visuospatial attention performance in a 5-choice serial reaction time task (5-CSRT task). The features of the reported deficits, however, do not perfectly match among studies, in which some lesions may have been too weak while others largely encroached onto the septal region. Using the 5-CSRT task, we therefore re-assessed the effects of NBM lesions that produced minimal septal damage. Long-Evans adult male rats were trained to stable 5-CSRT task performance (stimulus duration: 0.5 s) and subsequently subjected to intra-NBM injections of 192 IgG-saporin (200 ng/side). The lesions induced more than 90% loss of choline acetyltransferase–positive neurons in the NBM vs. only 28% in the medial septum. The decrease of the optical density of acetylcholinesterase reaction products was significant in the cortex (−91%), not in the hippocampus. In the 5-CSRT task, the lesions resulted in increased omissions (from 10% to 30%) and decreased correct responses (from 80% to 60%), with negligible or no effects on all other usually collected variables. This deficit disappeared with lengthened stimulus duration (i.e. 0.5–1 and then 5 s). Furthermore, overall performance levels decreased when the stimulus duration was shortened (i.e. 0.5–0.2 s) or its intensity attenuated, and rats with cholinergic lesions remained consistently impaired vs. controls. These results show that disruption of sustained visual attention functions by damage to the NBM cholinergic neurons can be evidenced despite weak or no effects on variables accounting for motivational, locomotion- or impulsivity-related biases. Discrepancies with previously reported results are discussed in terms of differences in lesion extent/specificity and training levels.

Optic nerve transection affects development and use-dependent plasticity in neocortex of the rat: Quantitative acetylcholinesterase imaging

We investigated the effects of neonatal optic nerve transection on cortical acetylcholinesterase (AChE) activity in hooded rats during postnatal development and following behavioral manipulation after weaning. AChE reaction product was quantified on digitized images of histochemically stained sections in layer IV of primary somatic sensory, primary visual and visual association cortex. Rats with optic nerve transection were compared to sham-operated littermates. In all cortical regions of both types of animal, AChE reaction product was increased to peak 2 weeks after birth and decreased thereafter, reaching adult levels at the end of the third postnatal week. During postnatal development, reaction product in primary visual cortex was lower in rats deprived of retinal input than in sham-operated littermates and the area delineated by reaction product was smaller. However, optic nerve transection did not modify the time course of postnatal development or statistically significantly diminish adult levels of AChE activity. Behavioral manipulations after weaning statistically significantly increased enzyme activity in sham-operated rats in all cortical areas examined. Compared with cage rearing, training in a discrimination task with food reward had a greater impact than environmental enrichment. By contrast, in the rats with optic nerve transection enrichment and training resulted in statistically significantly increased AChE activity only in lateral visual association cortex. Our findings provide evidence for intra- and supramodal influences of the neonatal removal of retinal input on neural activity- and use-dependent modifications of cortical AChE activity. The laminar distribution of the AChE reaction product suggests that the observed changes in AChE activity were mainly related to cholinergic basal forebrain afferents. These afferents may facilitate the stabilization of transient connections between the somatic sensory and the visual pathway.

Distribution of catecholaminergic afferent fibres in the rat globus pallidus and their relations with cholinergic neurons

The topographical distribution of catecholaminergic nerve fibres and their anatomical relationship to cholinergic elements in the rat globus pallidus were studied. Peroxidase–antiperoxidase and two-colour immunoperoxidase staining procedures were used to demonstrate tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) and choline acetyltransferase (ChAT) immunoreactivities, combined with acetylcholinesterase (AChE) pharmacohistochemistry. TH immunoreactive nerve fibres were seen to enter the globus pallidus from the medial forebrain bundle. The greatest density of such fibres was found in the ventral region of the globus pallidus, which was also characterized by the greatest density of ChAT immunoreactive neurons. TH immunoreactive nerve fibres showed varicose arborizations and sparse boutons, which were occasionally seen in close opposition to cholinergic structures. In all regions of the globus pallidus, there were also larger, smooth TH immunoreactive nerve fibres of passage to the caudate putamen. A smaller number of DBH immunoreactive nerve fibres and terminal arborizations were found in the substantia innominata, internal capsule and in the globus pallidus bordering these structures. A few PNMT immunoreactive nerve fibres in the substantia innominata and internal capsule did not enter the globus pallidus. Electron microscopy revealed TH immunoreactive synaptic profiles in the ventromedial area of the globus pallidus corresponding to the nucleus basalis magnocellularis of Meynert (nBM). These made mainly symmetrical and only a few asymmetrical synaptic contacts with dendrites containing AChE reaction product. The results indicate that cholinergic structures in the nBM are innervated by dopaminergic fibres and terminals, with only a very small input from noradrenergic fibres.

Ultrastructural localization of acetylcholinesterase and choline acetyltransferase in oligodendrocytes, glioblasts and vascular endothelial cells in the external cuneate nucleus of the gerbil.

This study reports the reactivities of acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) in some of the nonneuronal elements in the external cuneate nucleus (ECN) of gerbils. AChE reaction products were localized in some oligodendrocytes in their cisternae of rough endoplasmic reticulum, nuclear envelope and Golgi saccules. The basal lamina lining the capillary endothelia also displayed AChE reactivity. In ChAT immunocytochemistry, the reaction products were found to be associated with the vascular basal lamina as well as the endothelial plasma membrane facing the lumen. The most remarkable finding was the localization of ChAT immunoreactivity in some oligodendrocytes and occasional glioblasts (small glial precursor cells containing a thin rim of cytoplasm surrounding an irregular nucleus with homogeneous chromatin materials). The ChAT-positive oligodendrocytes consisted of two types, medium-dense and dark cells, either associated with blood vessels or ChAT-stained neuronal elements. It is suggested from these new findings that at least some of the oligodendrocytes and glioblasts in the ECN of gerbils may be involved in the synthesis, storage, release and degradation of acetylcholine.

Ultrastructural identification of cholinergic neurons in the external cuneate nucleus of the gerbil: acetylcholinesterase histochemistry and choline acetyltransferase immunocytochemistry.

Using acetylcholinesterase histochemical and choline acetyltransferase immunocytochemical localization methods, this study has provided conclusive evidence for the existence of cholinergic neurons in the external cuneate nucleus of gerbils. By light microscopy, both acetylcholinesterase and choline acetyltransferase labelling was confined to the rostral portion of the external cuneate nucleus. Ultrastructurally, acetylcholinesterase reaction products were found in the nuclear envelope, cisternae of rough endoplasmic reticulum and Golgi saccules of some somata and large dendrites as well as in the membranes of small dendrites, myelinated axons and axon terminals. These neuronal elements were also stained for choline acetyltransferase; immunoreactivity was associated with nuclear pores, nuclear envelope, perikaryal membrane and all the membranous structures within the cytoplasm. Of the total choline acetyltransferase-labelled neuronal profiles analysed, 79% were myelinated axons, 15% dendrites, 4% somata and 2% axon terminals. The immunostained axon terminals consisted of two types containing either round (Rd type; 62.5%) or pleomorphic (Pd type; 37.5%) vesicles. Both were associated directly with choline acetyltransferase-positive dendrites. In contrast to the paucity of choline acetyltransferase-labelled axon terminals, numerous choline acetyltransferase-positive myelinated axons were present. It may thus be hypothesized that most, if not all, of the external cuneate nucleus cholinergic neurons are projection cells; such cells may give rise to axonal collaterals which synapse onto their own dendrites for possible feedback control. Choline acetyltransferase-positive dendrites were contacted by numerous unlabelled presynaptic boutons, 60% of which contained round or spherical synaptic vesicles (Rd boutons) and 40% flattened vesicles (Fd boutons), suggesting that these neurons are under strong inhibitory control. The preferential concentration of cholinergic components in the rostral external cuneate nucleus may be significant in the light of the highly organized somatotopy in the external cuneate nucleus and its extensive efferent projections to medullary autonomic-related nuclei. Our results suggest that the cholinergic neurons may be involved in somatoautonomic integration.

Distribution of acetylcholinesterase activity in the rat embryonic heart with reference to HNK-1 immunoreactivity in the conduction tissue

Acetylcholinesterase (AChE) activity was topographically investigated in the presumptive cardiac conduction tissue regions visualized by HNK-1 immunoreactivity in rat embryos, and AChE-positive cells were examined with the electron microscope. On embryonic day (ED) 14.5, when HNK-1 was most intensely visualized, AChE activity could not be detected enzyme-histochemically in the conduction tissue regions, except in the ventricular trabeculae and part of the AV node. On ED 16.5, however, the AChE activity was clearly demonstrated in some parts of the developing conduction tissue. One exception was the AV node region, where an AChE-positive area was in close proximity to an area showing HNK-1 immunoreactivity but did not overlap. Furthermore, AChE activity was demonstrated predominantly in the ventricular trabeculae, including cardiac myocytes, but was rather weak in the atrium. With the electron microscope, AChE reaction products were observed predominantly intracellularly in both developing conduction tissue cells and developing ordinary myocytes, and no reactivity was found in neuronal components. From ED 18.5 until birth, both AChE activity and HNK-1 immunoreactivity faded away in the conduction tissue. Thus, transient AChE activity in the embryonic heart seems to be different from the developing adult form and may be related to a morphogenetic function in embryonic tissues, as proposed by other authors.

Morphological and behavioural effects of granule cell degeneration induced by intrahippocampal fluid injections in intact and fimbria-fornix lesioned rats

This study was aimed at determining whether granule cell degeneration induced by intragyral injections of a neutral fluid (0.9% NaCl with 0.6% glucose, pH 7.0, 2 sites per hippocampus, 2 μl/site, 1 μl/min) produced behavioural deficits in rats which, 2 weeks prior to the injections, had received either fimbria-fornix lesions or sham-operations. In both sham-operated and lesioned rats, we found such injections to induce a comparable, topographically-limited loss of granule cells in the dorsal leaf of the dentate gyrus and, in the close vicinity of the degeneration area, a severe shrinkage of the molecular layer with concomitant morphological reorganizations (e.g. acetylcholinesterase reaction products were distributed uniformly throughout the molecular layers of sham-operated rats). While the fimbria-fornix lesions produced classically reported behavioural deficits (hyperactivity in both a familiar and an unfamiliar environment, reduced T-maze alternation rates and impaired radial-maze performance), we could not detect adversive effects of the granule cell degeneration on either of these variables in sham-operated and lesioned rats. Our data suggest that limited granule cell degeneration induced by intragyral fluid injections has no effect on locomotor activity, spontaneous alternation and spatial learning. Therefore, we may also infer that the granule cell damage observed after an intragyral implantation of a fetal neural cell suspension does probably not account for the behavioural deficits which, in some experiments, have been found in fimbria-fornix lesioned rats bearing intragyral cell suspension grafts.

Ultrastructural cytochemistry of acetylcholinesterase enzyme activity in pancreatic tissue transplants in rats.

The distribution of acetylcholinesterase (AChE) at the ultrastructural level was investigated in normal and in pancreatic fragments transplanted for 56 days into the anterior eye chamber of heterologous rats using enzyme cytochemical methods. Acetylcholinesterase reaction products were seen on the basal surface of the acinar cells in normal pancreas. Acetylcholinesterase enzyme activity was also detected on the axolemma of the surviving nerve fibres. This enzyme reaction product forms alternating thick and thin bands on the axolemma. Some of these AChE-positive nerve fibres accompany blood vessels that also survive after transplantation. AChE were seen in cytoplasm adjacent to the surviving alpha and pancreatic polypeptide cells. We conclude that the ability of some neurons and cells to produce and or store acetylcholinesterase is still retained after transplantation of pancreatic tissue into the anterior eye chamber of rats.

Laminar organization of acetylcholinesterase and cytochrome oxidase in the lateral geniculate nucleus of prosimians

Hess and Rockland [Hess and Rockland (1983) Brain Res. 289, 322–325] proposed that the distribution of acetylcholinesterase within the lateral geniculate nucleus might correlate with the daily activity patterns shown by primates. In diurnal primates, the magnocellular laminae show a greater acetylcholinesterase reaction product. In nocturnal primates, the parvocellular laminae are more heavily stained. We have examined the laminar distribution of acetylcholinesterase and cytochrome oxidase in the lateral geniculate nucleus of a series of rare prosimian primates. In all prosimians examined, the most dense acetylcholinesterase reaction product is seen in the parvocellular layers of the lateral geniculate nucleus. Heavy cytochrome oxidase activity is seen in both the magnocellular and parvocellular layers, but not the koniocellular layers of the prosimian lateral geniculate nucleus. We have also employed a polyclonal antibody to choline acetyltransferase to examine the laminar organization or cholinergic activity in the Galago (Bushbaby) lateral geniculate nucleus. We report that choline acetyltransferase immunoreactivity does not correlate with acetylcholinesterase activity in the prosimian lateral geniculate nucleus. Although the lateral geniculate nucleus is more immunoreactive than most other thalamic structures and although the intercalated koniocellular laminae demonstrate somewhat lighter choline acetyltransferase immunoreactivity, no great difference in staining intensity is seen between the parvocellular and magnocellular laminae. In addition, we examined the phenotype of known inputs to assess the laminar specificity of cholinergic projections to the bushbaby lateral geniculate nucleus. Layer VI of primary visual cortex, which is known to be a source of acetylcholinesterase in the parvocellular layers, does not contain cholinergic cells, nor does the pretectal nucleus, which projects mainly to the parvocellular layers. The parabigeminal nucleus is cholinergic; however, this nucleus is known to project to the koniocellular layers, along with the noncholinergic superior colliculus. Finally, the pedunculopontine tegmental nucleus, which provides a strong input to many regions of the thalamus, including the lateral geniculate nucleus, is cholinergic. The laminar organization of its input to the lateral geniculate nucleus is not known. Increased acetylcholinesterase reaction product within the parvocellular layers of the lateral geniculate nucleus is common to all strepsirhine primates. The pattern is also seen in the only two nocturnal haplorhine primates, Tarsius and Aotus (owl monkey). The relation of this increased acetylcholinesterase activity to cholinergic function remains unclear. We note exceptions to the suggested correlation between nocturnality and increased density of acetylcholinesterase within the parvocellular layers. We hypothesize that rather than reflecting nocturnal/diurnal lifestyles, the acetylcholinesterase staining pattern may be a primitive feature of geniculate organization in primates.

β-Endorphin and corticotropin immunoreactivity and specific binding in the neuromuscular system of obese-diabetic mice

Immunoreactivity for two derivatives of pro-opiomelanocortin, β-endorphin and α-melanocortin (or corticotropin), was demonstrated, using a conventional immunoperoxidase method, in some of the intramuscular nerves in muscle sections from obese diabetic (ob/ob) mice and homozygous lean (+/+) mice. The endplate regions were visualized in the sections by staining for acetylcholinesterase reaction product. The proportion of muscle endplates with β-endorphin-immunoreactive motor nerves was approximately 2.5-fold higher in soleus and extensor digitorum longus muscles and approximately 1.5-fold higher in the diaphragm of the obese (ob/ob) mice compared to the normal lean mice. The proportion of muscle endplates with α-melanotropin-immunoreactive motor nerves was between 30 and 53% lower, depending on the muscle type, in the ob/ob mice compared to the lean mice. The muscles of ob/ob and lean mice were investigated for the presence of specific binding sites for [ 125I]β-endorphin and for [ 125I]corticotropin, using autoradiography. Some muscle fibres in soleus, extensor digitorum longus and diaphragm in both the ob/ob and the lean mice exhibited specific binding sites for the radioactive ligands. The binding sites were distributed over the entire surface in these muscle fibres. In the ob/ob mice the number of muscle fibres with specific [ 125I]β-endorphin binding sites was six-fold higher in soleus and approximately 10-fold higher in extensor digitorum longus and diaphragm, than in the corresponding muscles of the lean mice. In contrast, the number of muscle fibres with specific [ 125I]corticotropin binding sites was similar in obese (ob/ob) and lean mice. The results suggest a disturbance in the expression of pro-opiomelanocortin peptides in motoneurones of obese-diabetic (ob/ob) mice, and of the peptide receptors in the skeletal muscles. As there is a selective increase in β-endorphin-immunoreactive nerves, the defect in peptide expression could be in the post-translational processing of the pro-opiomelanocortin precursor. The disturbance may be a manifestation of early neuropathic change.

Ultrastructural localization of acetylcholinesterase in neurofibrillary tangles, neuropil threads and senile plaques in aged and Alzheimer's brain

Acetylcholinesterase (AChE) histochemistry was used to evaluate the accumulation of this enzyme in senile plaques, neurofibrillary tangles and neuropil threads using light and electron microscopy in Alzheimer's disease as well as non-demented aged brains. Under the electron microscope, a crystalline-like AChE precipitate was localized over paired helical filaments and straight filaments in both neurofibrillary tangles and neuropil threads. AChE reaction product also decorated the amyloid fibrils in diffuse plaques as well as the halo and the heavy accumulation of amyloid which forms the core of classical plaques. In both diffuse plaques and the halo of classical plaques, we found AChE-positive structures resembling cell processes, which in some cases appeared to contain amyloid fibrils. The possible origin and significance of AChE localized over paired helical filaments, straight filaments and amyloid is discussed.

Calcium and ionophore A23187 stimulates deposition of extracellular matrix and acetylcholinesterase release in cultured myotubes

Calcium (Ca2+) and calcium-transporting ionophores stimulate protein secretion in many cellular systems. We demonstrate here than increases in intracellular calcium concentration induce a time- and concentration-dependent deposition of extracellular matrix and an increase in acetylcholinesterase secretion. Scanning and transmission electron-microscopy revealed that treatment with the calcium ionophore A23187, or high extracellular Ca2+ levels (5 mM to 15 mM) produce significant deposits of extracellular matrix around the myotubes, as well as a marked increase in the acetylcholinesterase reaction-product. Blocking muscle contraction was not necessary for the induction of AChE secretory activity. Sucrose density-gradients of media conditioned by muscle cells revealed 3 separate acetylcholinesterase molecular forms. However, incubation with A23187 increased only the 4.5 S and the 7.2 S molecular forms, whereas the 12.0 S form showed no significant differences from controls. Polyacrylamide gel electrophoresis, and autoradiography using [3H]diisopropyl fluorophosphate revealed a broad band at 65,000 daltons. This band was broader than for controls when medium was obtained from A23187-treated cells. Our results show that increasing intracellular Ca2+ concentration induces marked deposition of extracellular matrix and increased acetylcholinesterase secretion, with an apparent selectivity for the monomeric and dimeric acetylcholinesterase molecular forms.

Ultrastructural localization of acetylcholinesterase in the guinea pig pineal gland

The ultrastructural localization of acetylcholinesterase (AChE) activity in guinea pig pineal gland was studied using the copper-glycine procedure. A small number of pinealocytes and bundles of unmyelinated nerve fibers were labeled by the AChE reaction. The AChE-positive pinealocytes were located near blood vessels and distributed in small groups. The AChE reaction product was localized in the perinuclear cistern, in the cisternae of the endoplasmic reticulum (ER), and in the saccules of the Golgi apparatus. These findings suggest that the AChE-positive pinealocytes synthesize AChE. The AChE reaction product was also seen in the intercellular space between pinealocyte processes. Besides pinealocytes, AChE activity was localized on the axolemma of myelinated and unmyelinated nerve fibers and in the basement membrane surrounding unmyelinated nerve fibers. Pseudocholinesterase activity was confined to Schwann cells, which showed the reaction product in their perinuclear cistern, in the cisternae of the ER, and on the plasmalemma.

Effects of transient cerebral ischemia on the hippocampal dentate theta (θ) profile in the acute rat: a study 4–5 months following recirculation

This study mainly describes the long-term effects of 20 min of cerebral ischemia on the profile of the presumed cholinegic θ rhythm in the rat dorsal hippocampal formation during ether anesthesia and injection of the muscarinic agonist agent arecoline. The experimental data were collected 4–5 months after ischemia. They show that ischemia results in a statistically significant reduction in both superficial and deep θ recorded from the CA1 area of the hippocampus and the dentate gyrus, respectively. Amplitude reduction is similar for both rhythms and co-varies positively with the extent of CA1 stratum pyramidale damage which, from light microscope observation, appeared to be the major neuroanatomical consequence of ischemic insult in the dorsal hippocampal formation. The medial septal nucleus-diagonal band of Broca complex involved in θ generation did not suffer visible anatomical damage. Moreover, no significant alteration in the spatial distribution and the density of hippocampal dentate acetylcholinesterase reaction product was seen in ischemic animals. These histological data were statistically confirmed by computerized image analysis. Finally, this is the first investigation to show that transient interruption of cerebral blood flow results in a long-lasting alteration of θ rhythm which is probably the major aspect of the basic activity of the hippocampal formation. Thus, the present findings obtained in the acute rat at 4–5 months postischemia confirm and extend, in most respects, our previous results collected in the chronic animal 2–29 days following 4-vessel occlusion. Possible significance of these findings for the hypothesis of the dependant generation sites of superficial and deep θs in the hippocampus assumed to be crucial in learning and memory, is discussed.

Effects of nimodipine on medial septal area cells after section of the fimbria-fornix

In rats, sectioning the fimbria-fornix leads to a decrease in cells reactive for acetylcholinesterase and choline acetyltransferase, as well as other types of cells, in the medial septal area that project to the hippocampus. In this study cells in the medial septal area were stained for acetylcholinesterase reaction product or by a Nissl method two weeks after unilateral section of the fimbria-fornix. Counts were made of cells on each side of the midline by independent observers. The counts of the cells on the two sides of the brain were compared in rats that had been given nimodipine (70 μg/kg) just after the lesion and for 7 additional days and in rats that had been given only the vehicle for an equivalent amount of time. The number of cells reactive for acetylcholinesterase was reduced in the medial septal area on the side of the brain in which the forebrain fornix was sectioned, but this occurred to a significantly less extent in the nimodipine-treated rats. No differences were noted in the number of cells on the two sides of the medial septal area when counted in Nissl-stained sections.

Differential distribution of acetylcholinesterase activity among vasopressin- and oxytocin-containing supraoptic magnocellular neurons

Acetylcholinesterase activity was demonstrated histochemically at light- and electron-microscopic levels, in Vibratome sections of the supraoptic nucleus of fixed hypothalami derived from osmotically stimulated and unstimulated Long-Evans rats, from homozygous Brattleboro rats with hypothalamic diabetes insipidus, from lactating rats, from normal adult male house mice ( Mus musculus) and from mice with hereditary nephrogenic diabetes insipidus ( di/di). Reaction product was located in supraoptic magnocellular neurons; in dorsal and rostral aspects of the supraoptic nuclei lightly stained cells predominate, whereas in ventral and caudal regions densely staining perikarya predominate. Pre- and post-embedding immunocytochemical detection of oxytocin-neurophysin or vasopressin-neurophysin, combined with acetylcholinesterase histochemistry, showed that the lightly staining cells are oxytocinergic, and the densely staining cells vasopressinergic. Osmotic stimulation of the animals, either by substitution of drinking water for 3 days with 2.5% saline or by reason of genetic defects which result in diabetes insipidus, enhanced the acetylcholinesterase activity of the vasopressin neurons but had little effect on the weakly acetylcholinesterase-reactive oxytocin cells. Acetylcholinesterase activity was particularly marked in the hypertrophied abnormal magnocellular neurons of homozygous Brattleboro rats which do not release significant amounts of vasopressin. The increased acetylcholinesterase activity in osmotically stimulated animals cannot, therefore, be a function of vasopressin. Acetylcholinesterase activity was also detected in large multipolar neurons lying dorsolateral to the supraoptic nucleus, and in their fine axonal processes which project towards the supraoptic nucleus. A very few synaptic boutons surrounded by acetylcholinesterase reaction product were found in contact with magnocellular neuron basal dendrites. However, much of the punctate acetylcholinesterase reactivity observed at the light microscopic level and previously interpreted as representing the loci of cholinergic synaptic boutons was shown to be intracellular, and probably caused by acetylcholinesterase activity in some large, secondary lysosomes.

Distribution of acetylcholinesterase in the granular layer of the cerebellum of the rhesus monkey (Macaca mulatta).

A nonuniform distribution of acetylcholinesterase (AChE) activity was identified in the granular layer of the cerebellum in rhesus monkeys. The distribution of darkly AChE-stained clumps in the granular layer was determined for each lobule of the vermis and the lateral cortex. The vermis contained a greater density of AChE reaction product than the lateral cortex. In the vermis, lobules IX and X had significantly the highest level of activity, followed by lobules VII and VIII, which were significantly higher than lobules II-VI. In the lateral cortex, the flocculus had the highest level of the AChE activity, followed by crus I and the dorsal paraflocculus, which had significantly higher levels than the remaining lobules. The high levels of AChE activity in the flocculonodular lobe and lobule IX may correspond to cholinergic mossy fiber transmission, but the high levels of AChE activity in other cerebellar regions probably involve noncholinergic functions. The significance of the nonuniform AChE distribution is not yet known, but may correspond to regional differences in neuronal or metabolic activity in the cerebellum that occur in conjunction with specific behaviors.

Histochemical Localization of Acetylcholinesterase in the Cerebral Ganglia of Fasciola hepatica, a Parasitic Flatworm

Acetylcholinesterase activity was found in the cell bodies and extracellularly in the neuropile of the cerebral ganglia of the adult trematode parasite, Fasciola hepatica. Within neuronal cell bodies of the cerebral ganglion, acetylcholinesterase reaction product was found in the endoplasmic reticulum, in the cisternae of the Golgi apparatus, and in secretory vesicles near the inner (releasing face) cisternae. Acetylcholinesterase reaction product was not seen intracellularly within any nerve processes. The reaction product was found around the somatic cell membranes and in the extracellular space between closely apposed nerve processes in the neuropile. Acetylcholinesterase reaction product was associated with synaptic endings that contained clear spheroidal synaptic vesicles, and the reaction product was localized at the site of synaptic contact between the zone of apposition of the pre- and postsynaptic terminals. This intracellular and extracellular distribution of the enzyme is consistent with its function as the degrading enzyme in cholinergic transmission.