The next generation of cholinesterase inhibitors

Clinical trials with tacrine (THA) have resulted in elevations of liver enzymes in Alzheimer patients that showed improvement. In an effort to minimize these side effects several THA analogues were synthesized. These analogues were compared to THA in biochemical as well as behavioural studies. The biochemical effects of these drugs on plasma cholinesterase activity and cholinergic receptors as well as the effect of these drugs on spatial learning in adult rats were examined. It is possible that some of these analogues with more potent cholinergic effect than THA might be the next generation of cholinesterase inhibitors which can be useful in the treatment of Alzheimer's disease.     

Anatomy of cholinesterase inhibition in Alzheimer's disease: effect of physostigmine and tetrahydroaminoacridine on plaques and tangles

The histochemical distribution of cholinesterases in the cerebral cortex and their response to cholinesterase inhibitors such as physostigmine and tetrahydroaminoacridine (THA) were investigated in brains from patients with Alzheimer's disease and control subjects. In the temporal neocortex of the control subjects, most of the cholinesterase activity was located within axons and cell bodies belonging to cholinergic pathways. In keeping with their well-known cholinomimetic effects, physostigmine and THA effectively inhibited this cholinesterase activity. Cholinesterase-containing normal axons (and in some cases cells) were severely depleted in the cerebral cortex of patients with Alzheimer's disease. Although the cerebral cortex of these patients continued to display abundant cholinesterase activity, the location of this enzyme was largely shifted to the neuritic plaques and neurofibrillary tangles. In fact, the majority of these pathological structures demonstrated intense acetylcholinesterase and butyrylcholinesterase activities. Physostigmine and THA were potent inhibitors of these plaque- and tangle-bound cholinesterases as well. In patients with Alzheimer's disease, cholinesterase inhibitors would therefore appear to have a major and widespread effect directly upon the enzymatic activity of plaques and tangles. Consequently, the clinical effects of anticholinesterases in Alzheimer's disease may be based on mechanisms that are different from those that apply to the normal brain.     

Interaction between the cholinergic system and CRH in the modulation of spatial discrimination learning in mice

Both cholinergic and CRH systems have been linked to cognitive processes such as learning and memory, and neuroanatomical as well as neurochemical evidence suggests important interactions between these two systems. Moreover, recent reports of pro-mnestic effects of CRH open the possibility that CRH could have beneficial effects in animals with cholinergic dysfunction. In a first experiment, spatial discrimination of C57BL/6 mice treated with various doses of scopolamine (0.5--2.0 mg/kg IP) was tested in a two-choice water maze task. Scopolamine, but not methylscopolamine, impaired accuracy and decreased responsivity. In contrast, similar doses of the nicotinic antagonist mecamylamine had no effect on choice accuracy but altered responsivity, as indicated by increased errors of omission and a reduction in swim speed during early experimental stages. ICV CRH (0.5--1.0 microg) also failed to significantly affect accuracy, but a strong tendency was observed to impair percentage correct responses. Measures of responsivity, such as errors of omission, choice latency and distance traveled, and of thigmotaxis were not significantly affected by CRH. However, initial swim speed was reduced by the peptide. Combined treatment with scopolamine (0.5 mg/kg IP) and CRH (0.5 microg ICV) had only mild, and primarily independent, effects, but overall suggested that concomitant blockade of muscarinic receptors and activation of the CRH system would rather act synergistically to disrupt spatial discrimination learning. Synergistic effects were also observed when animals receiving a combination of mecamylamine (2.0 mg/kg IP) and CRH (0.5 microg ICV) were tested, both in terms of responsivity and thigmotaxis, and there was limited evidence that part of these effects were potentiating. Thus, the cholinergic and CRH systems interact in the modulation of learning, but CRH, contrary to prediction, worsens the impairment caused by cholinergic blockade.     

Concurrent muscarinic and beta-adrenergic blockade in rats impairs place-learning in a water maze and retention of inhibitory avoidance

These experiments examined the effects of separate and concurrent muscarinic cholinergic and beta-adrenergic blockade on inhibitory (passive) avoidance performance and spatial learning in the Morris water maze. Pretraining systemic administration of either scopolamine (0.3 or 1.0 mg/kg) or propranolol (3.0 or 10.0 mg/kg) had no significant effect on one-day retention of step-through inhibitory avoidance training. Similarly, pretraining administration of either 0.3 mg/kg scopolamine or 10 mg/kg propranolol did not affect spatial learning in the Morris water maze. However, combined administration of scopolamine and 10.0 mg/kg of propranolol impaired performance on these tasks. These findings further support a role for interactions between norepinephrine and acetylcholine in the modulation of learning and memory and implicate the participation of beta-adrenergic mechanisms in this interaction. Because cholinergic and noradrenergic deterioration is found in aging and Alzheimer's disease, these results also have implications regarding the role of age-related noradrenergic and cholinergic dysfunction in cognitive decline.     

Hebb-Williams performance and scopolamine challenge in rats with partial immunotoxic hippocampal cholinergic deafferentation

Recent studies suggested that the cholinergic innervation of the hippocampus is not crucial for spatial learning, but it might be important for other forms of learning. This study assessed the effects of partial immunotoxic cholinergic lesions in the medial septum and concurrent scopolamine challenge in a complex learning task, the Hebb-Williams maze. Long-Evans rats were given intraseptal injections of 192 IgG-saporin (SAPO). Rats injected with phosphate-buffered saline (PBS) served as controls. Starting 25 days after surgery, behavioural performance was assessed in the Hebb-Williams maze test without prior or after injection of scopolamine (0.17 or 0.5 mg/kg, i.p.). In SAPO rats, histochemical analysis showed a 40-45% decrease in the density of hippocampal AChE staining. The number of ChAT-positive cell bodies in the medial septum was also significantly decreased (-56%) and there was a non-significant reduction of the number of parvalbumine-positive neurons. The behavioural results demonstrated that the lesions induced small but significant learning deficits. At 0.17 mg/kg, scopolamine produced more impairments in SAPO rats than in PBS-injected rats, suggesting an additive effect between the partial lesion and the drug. These observations indicate that the Hebb-Williams test may be more sensitive to alterations of septohippocampal cholinergic function, than radial- or water-maze tasks. They also show that subtle learning deficits can be detected after partial lesions of the cholinergic septohippocampal pathways. Finally, the data from the scopolamine challenge are in keeping with clinical results showing higher sensitivity to muscarinic blockade in aged subjects in whom weaker cholinergic functions can be presumed.     

Tetrahydroaminoacridine improves the spatial acquisition deficit produced by nucleus basalis lesions in rats

We administered tetrahydroaminoacridine (THA), a cholinesterase inhibitor, to rats with bilateral nucleus basalis magnocellularis lesions and measured their performance in a spatial learning task. The subjects, 34 male Fischer-344 rats, received bilateral excitotoxic NBM lesions; 10 other rats served as unlesioned controls. Two weeks later the animals were tested in a circular water maze for time and distance swum to find a submerged platform. We tested three different doses (5.0, 2.5, and 1.25 mg/kg) of daily subcutaneous THA against a lesioned control group receiving saline and a fifth group of untreated unlesioned controls. The saline-treated lesioned group showed a significant impairment of acquisition. The 1.25 mg/kg group performed significantly better than the lesioned controls with respect to latency. Analysis of swim speed data showed slowing in the 2.5 and 5.0 mg/kg groups. Analysis of the distance swum to find the platform, an untimed task that corrects for the difference in swim speeds, showed statistically significant improvement in all three treated groups. Additionally, spatial memory for the platform location was improved by two of the three doses of THA tested. Passive avoidance retention was not impaired by our lesion. All lesioned groups had comparable reductions of cortical choline acetyltransferase. Our data show significantly improved spatial learning with THA. These data provide an additional rationale for further clinical testing of THA and other centrally active cholinergic agents in diseases with cholinergic loss.     

N-tert-butyl-alpha-phenylnitrone, a free radical scavenger with anticholinesterase activity does not improve the cognitive performance of scopolamine-challenged rats.

Reversible inhibitors of acetylcholinesterase improve spatial learning and memory in animal models of cognitive impairment. Here we investigate if the beneficial effects of free radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN) on cognitive performance could be explained by its recently discovered anticholinesterase activity. Morris water maze experiment was performed to examine the effect of PBN on the impairment of spatial learning and memory induced by the antagonist of cholinergic muscarinic transmission scopolamine. In situ hybridization histochemistry experiment was performed to study its effects on the induction of immediate early gene expression (c-fos, c-jun) by dopamine D1 receptor agonist SKF-82958 and on the augmentation of the SKF-82958-induced expression of these genes by scopolamine. In both experiments, the effects of PBN were compared to the effects of reversible anticholinesterase physostigmine. We found that physostigmine but not PBN significantly reversed the cognitive impairment in scopolamine-challenged rats, prevented the induction of c-fos and c-jun mRNAs by SKF-82958 and attenuated the augmentation of the SKF-82958-induced expression of these genes by scopolamine. The present experiments did not reveal a significant in vivo anticholinesterase activity of PBN.     

Medial septal galanin and acetylcholine: influence on hippocampal acetylcholine and spatial learning

Neurochemical and behavioral studies in the rat have provided evidence for the view that galanin impairs learning via an inhibitory modulation of cholinergic neurons in the septohippocampal projection, believed to be important for learning and memory. To test this hypothesis, galanin was microinjected via a unilateral chronic cannula located in MS/dBB of rats. Infusion of galanin in the MS/dBB, which contains a high number of 125I-galanin binding sites, did not impair spatial acquisition or memory. On the contrary, spatial acquisition tended to be facilitated by 1 and 3 nmoles of galanin, while the 0.3 nmol dose had no effect. Intraseptal injections of scopolamine (10 microg/rat), a non-specific muscarinic antagonist, also failed to alter learning performance. In contrast, co-injections of galanin (3 nmol) and scopolamine (10 microg) resulted in a marked impairment of spatial acquisition. The effect of intraseptal galanin on basal acetylcholine release in the ventral hippocampus was examined by in vivo microdialysis and high-performance liquid chromatography. Both galanin (3 nmol/rat) and scopolamine (10 microg/rat) infused into the MS/dBB increased basal acetylcholine release in the ventral hippocampus. The combined injections of galanin and scopolamine resulted in an excessive increase in acetylcholine release. These results indicate, that galanin activates septohippocampal cholinergic neurons, suggesting that septal galanin may have a facilitatory role in spatial learning. Moreover, the level of muscarinic activity within the septal area appears to be critical for the effects of galanin on cognitive functions, since the combination of galanin and scopolamine produced a marked impairment in spatial learning, despite a marked increase in hippocampal acetylcholine release. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.     

The Role of the Central Cholinergic Projections in Cognition: Implications of the Effects of Scopolamine on Discrimination Learning by Monkeys

In humans, administration of the cholinergic antagonist scopolamine impairs the encoding of information into long-term memory and has effects on other cognitive processes. It has been supposed that it is inhibition of the rising cholinergic projections from the basal forebrain, specifically from the basal nucleus of Meynert (NBM) to the neocortex and from the medial septum/vertical limb of the diagonal band of Broca (MS/VDB) to the hippocampus, that results in these cognitive impairments. In this paper, we describe the effects of scopolamine treatment in monkeys on learning different sorts of visual discrimination and visuospatial conditional tasks and compare these results to the effects of lesions of the rising cholinergic projections. Experiments in rodents in which these projections have been selectively destroyed have failed to produce a consensus view of the functions of these two areas. In particular, highly specific immunotoxic lesions of the NBM have largely failed to produce changes in task performance that can be interpreted as resulting from a cognitive impairment. In monkeys, lesions of the NBM produce modest or short-lasting, impairments in visual discrimination learning, retention, and reversal, whereas lesions of the MS/VDB produce large and permanent impairments of certain types of conditional learning. Similar impairments produced by scopolamine in monkeys and additive effects of lesions of the NBM or MS/VDB with scopolamine suggest that scopolamine has these effects by acting on the rising cholinergic pathways rather than on other cholinergic systems in the brain. It is argued that the rising cholinergic projections sustain the functions of the target areas; in the case of the hippocampus in humans, the function is usually regarded as being the analysis of information in a way that is pertinent to the formation of episodic memories and in the case of the neocortex, is the analysis of information in a manner that is relevant to the cognitive processing of on-going events and the acquisition of semantic knowledge.     

Effects of combined methysergide and mecamylamine/scopolamine treatment on spatial navigation.

In the present study, we investigated the effects of a 5-HT2 receptor antagonist, methysergide (2.5, 7.5 and 20 mg/kg), on spatial learning in saline, mecamylamine (10 mg/kg) and scopolamine (0.8 mg/kg) treated rats. Methysergide had no effect on water-maze (WM) spatial learning in rats subjected to saline or mecamylamine pretreatments. However, scopolamine-induced WM learning deficit was augmented by methysergide at doses of 7.5 and 20 mg/kg. These results further suggest (A) that cholinergic and serotonergic systems may interact in the regulation of spatial learning, and (B) that the cholinergic component of this interaction with serotonin2 receptors is mediated by muscarinic receptors, but not by nicotinic receptors.     

N-tert-butyl-alpha-phenylnitrone, a free radical scavenger with anticholinesterase activity does not improve the cognitive performance of scopolamine-challenged rats

Reversible inhibitors of acetylcholinesterase improve spatial learning and memory in animal models of cognitive impairment. Here we investigate if the beneficial effects of free radical scavenger N-tert-butyl-alpha-phenylnitrone (PBN) on cognitive performance could be explained by its recently discovered anticholinesterase activity. Morris water maze experiment was performed to examine the effect of PBN on the impairment of spatial learning and memory induced by the antagonist of cholinergic muscarinic transmission scopolamine. In situ hybridization histochemistry experiment was performed to study its effects on the induction of immediate early gene expression (c-fos, c-jun) by dopamine D1 receptor agonist SKF-82958 and on the augmentation of the SKF-82958-induced expression of these genes by scopolamine. In both experiments, the effects of PBN were compared to the effects of reversible anticholinesterase physostigmine. We found that physostigmine but not PBN significantly reversed the cognitive impairment in scopolamine-challenged rats, prevented the induction of c-fos and c-jun mRNAs by SKF-82958 and attenuated the augmentation of the SKF-82958-induced expression of these genes by scopolamine. The present experiments did not reveal a significant in vivo anticholinesterase activity of PBN.     

Cholinergic influence on memory facilitation induced by angiotensin II in rats

The influence of the cholinesterase inhibitor galanthamine (Nivalin), of the cholinergic agonist oxotremorine, and of the muscarinic cholinergic antagonist scopolamine on the retention-improving effect of angiotensin II (AT II) was studied in male Wistar rats trained and tested for retention (24h later) using two paradigms: two-way active avoidance (shuttle-box) and passive (step-through) avoidance. AT II and the cholinergic agonists, administered together potentiated their retention-improving effects, while scopolamine abolished the memory effect of AT II. It is suggested that brain cholinergic neurotransmission participates in the mechanisms of the memory-facilitating effect of AT II.     

Learning deficits in aged rats pretreated chronically with barbital and tested late in abstinence: alleviation by tetrahydroaminoacridine

Summary Physostigmine and tetrahydroaminoacridine (THA) have been reported to improve cognitive function in patients with Alzheimer's disease. Two experiments were conducted to examine the effects of these anticholinesterase agents on learning in aged rats pretreated chronically with barbital. In the first experiment animals received barbital in their drinking water for 46 weeks. Controls were given only water. On days 100–104 of abstinence, when the animals were 20 months old, acquisition of the Morris maze task was initiated after treatment with physostigmine. It was found that physostigmine improved learning of the maze task in control but not barbital treated rats. In the second experiment animals received barbital solution or water as in experiment one. On days 100–103 of abstinence they were injected with THA before being tested in the Morris water maze. It was found that THA improved learning in both barbital treated and control rats. These results corroborate clinical findings of improved cognitive function following treatment with THA, and suggest that the therapeutic effects of THA may be mediated by mechanisms distinct from cholinesterase inhibition. Furthermore chronic barbital treatment could be used as a model to study cognitive disturbances in experimental animals.     

Pharmacological consequences of cholinergic plus serotonergic manipulations

The present study investigated pharmacological consequences of combined cholinergic and serotonergic blockade. Raphe medianus (RM) lesions (5,7-DHT) had no effect on spatial learning, but augmented scopolamine 0.8 mg/kg induced learning deficit. Pilocarpine (4 mg/kg) could reverse scopolamine (0.8 mg/kg), but not scopolamine (0.8 mg/kg) + RM lesion induced spatial learning impairment. However, a higher dose of pilocarpine could restore spatial learning deficit induced by scopolamine (0.8 mg/kg) and RM lesions. These findings support the important role of cholinergic-serotonergic interaction in the regulation of spatial learning and suggests that the combined cholinergic-serotonergic deficit in patient's with Alzheimer's disease may have an impact on therapeutic approaches which seek to normalize AD related cognitive impairments.     

Clinical experiences and biochemical findings with tacrine (THA)

A clinical comparison of tacrine (THA) and placebo was performed in 15 Alzheimer patients using a double blind crossover technique over 4 plus 4 weeks with one drug-free week in between. Treatment results, as evaluated by clinical rating scales and neuropsychological tests, were mostly negative. Side effects were few, except for elevation liver enzymes which occured in one third of the patients. CSF levels of the monoamine metabolites HVA and 5-HIAA increased on tacrine as evidence for activation of dopamine and serotonin pathways through cholinergic receptors. Pharmacokinetic investigations showed that the oral bioavailability of tacrine was low and greatly varying between subjects. Patients with high bioavailability of the drug tended to improve more, and also to have more liver enzyme elevations, than those with low bioavailability. A gel preparation for rectal administration was manufactured for comparison of plasma levels attained during one week's treatment with levels attained with oral capsules. Preliminary results indicate that the dose of tacrine can be reduced to 50 per cent when administered rectally, probably as by this route the rapid first-pass metabolism of the drug in the liver is diminished. A clinical trial of tacrine via the rectal route would be justified as this could decrease the number of patients with liver side effects and increase the number of patients improving on the treatment.     

The systemic administration of tacrine or selegiline facilitate spatial learning in aged fisher 344 rats

Summary When compared to young Fisher 344 rats, aged Fisher 344 rats were impaired in their acquisition of the water maze task as indicated by longer escape latencies and distances to find a hidden platform. In a free swim trial which was performed after the training period, young rats had a better spatial bias, since they spent more time swimming in the previous training quadrant. Tacrine 3mg/kg, an anticholinesterase, and selegiline 0.25mg/kg, a MAO-B inhibitor, partially reversed the acquisition deficit in aged rats when administered on their own, and drug-treated aged rats swam more in the previous training quadrant than vehicle-treated aged rats during the free swim trial. Aged rats also swam slower than young rats. Tacrine, but not selegiline, increased swimming speed in aged rats. Taken as a whole, these data support the proposal that tacrine may be effective at alleviating age-related learning impairment and confirm the role of cholinergic dysfunction in the spatial learning deficit in aged rats.     

Effects of bipolar affective disorder and lithium administration on the cholinergic system in human blood

Several processes relating to the cholinergic system that are present in human blood were measured in samples obtained from patients with bipolar affective disorder, both before and during treatment with lithium, and from controls. The biochemical measurements include RBC and plasma choline concentrations, the kinetics of RBC choline uptake, plasma non-specific cholinesterase and RBC and plasma acetylcholinesterase. The RBC choline level is increased and the Vmax of the RBC choline uptake system is decreased in samples from lithium-free bipolar patients during manic episodes. There are no differences from control in the plasma choline levels or in the acetylcholinesterase enzyme activities in blood samples from the lithium-free or lithium-treated patients. Plasma non-specific cholinesterase is below control levels in all patients. Lithium treatment increases the RBC choline concentration to more than ten-times control levels and reduces the Vmax and the affinity for choline of the RBC choline transport system. In vitro addition of lithium does not replicate the effects of in vivo administration of lithium. Possible mechanisms for these effects of lithium are discussed.     

Further evidence for the cholinergic hypothesis of aging and dementia from the canine model of aging

Memory decline in human aging and dementia is linked to dysfunction of the cholinergic system. Aging dogs demonstrate cognitive impairments and neuropathology that models human aging and dementia. This paper reviews recent evidence suggesting cholinergic involvement in canine cognitive aging based on studies with the anti-cholinergic drug, scopolamine, and a novel acetylcholinesterase inhibitor, phenserine. In particular, we examine: (1) the cognitive specificity of scopolamine's impairment in dogs, (2) the effect of age on scopolamine impairment and (3) the effect of phenserine on cognitive performance in dogs. Our findings indicate that working memory performance is disrupted by scopolamine at doses that do not disrupt non-cognitive behavior or long-term, semantic-like, memory, as indicated by performance of previously learned discriminations. This pattern of deficits is also seen in human and canine aging. We demonstrate that aged dogs are more sensitive to the impairing effects of scopolamine than young dogs, suggesting a decrease in cholinergic tone with increasing age. Dogs receiving phenserine demonstrate improved learning and memory compared to placebo controls. Our findings suggest that cholinergic decline could result in memory impairment, but that the memory impairment may be secondary to deficits in attention and/or encoding of new information. Together, these results suggest that the canine cholinergic system declines with age and that the aged dog is a unique model for screening therapeutics and for examining the relationship between amyloid pathology and cholinergic dysfunction in age-dependent cognitive decline.     

Different effects of scopolamine on the retrieval of spatial memory and fear memory

Retrieval of memory is fundamental for our life as individuals. The participation of cholinergic system in memory consolidation process has been extensively studied, but there are few data concerning the function of this system in memory retrieval process. In the current study, we inject non-selective muscarinic antagonist scopolamine peripherally 20 min before training or testing to see whether cholinergic modulation has effects on the acquisition or retrieval of spatial memory by water maze task and fear memory by inhibitory avoidance task. We find that the cholinergic system is essential for the acquisition of both spatial memory and fear memory. As for the memory retrieval, the cholinergic system has a positive role in the retrieval of spatial memory, because mice injected with scopolamine 20 min before the testing in the water maze show impaired spatial memory retrieval. Whereas injection of scopolamine 20 min before the testing in the inhibitory avoidance task does not cause memory retrieval deficits. That indicates the cholinergic system is not essential for the retrieval of fear memory.     

Effects of complete immunotoxin lesions of the cholinergic basal forebrain on fear conditioning and spatial learning

Administration of muscarinic cholinergic antagonists such as scopolamine impairs the acquisition of contextual fear conditioning, but the role of the basal forebrain (BF) cholinergic system in consolidation is unclear. To test the hypothesis that BF cholinergic neurons are critical for acquisition and consolidation of fear conditioning, male Sprague-Dawley rats with 192 IgG-saporin lesions of the entire cholinergic BF made either before or after fear conditioning were tested for conditioned fear to context and tone by assessing freezing and 22 kHz ultrasonic vocalization (USV) responses. Spatial learning in a 1-day water maze task provided a comparison for effects of the BF lesions on fear conditioning. In the test phase, neither pre-training nor posttraining BF lesions affected freezing to the context or tone. During both training and testing, pre-lesioned rats were impaired in production of USVs associated with fear. Postlesioned rats emitted fewer USVs only during testing. Acquisition of a spatial water maze task was mildly impaired in lesioned rats, although probe trial and cued performance was unimpaired. Nevertheless, these data suggest that conditioned fear-induced USVs are more sensitive to the loss of BF cholinergic neurons than is conditioned fear-induced freezing. The failure of BF cholinergic lesions to impair contextual fear conditioning indicates that scopolamine-induced impairments in fear conditioning may not be mediated by affecting cholinergic input to the hippocampus and neocortex.