Differential effects of cholinergic blockade on performance of rats in the water tank navigation task and in a radial water maze

The disruptive effect of cholinergic blockade was tested under conditions in which either the working memory or the spatial mapping requirements of the behavioral task were emphasized. In Experiment 1, 13 rats were trained in an eight-arm radial water maze to asymptotic performance. When delays of 5, 10, 20, and 40 min were inserted between Choice 4 and Choice 5, incidence of errors in Choices 5-8 increased after pretrial (20 min) scopolamine injection (0.2 mg/kg, ip) faster than under control conditions and approached chance level with the 40-min delay. Scopolamine after Choice 4 or pretrial methylscopolamine was ineffective. In Experiment 2, 30 rats were trained in a Morris water tank. Acquisition was impaired by pretrial injection (20 min) of 0.1 and 0.2 mg/kg scopolamine, but a higher dose (1.0 mg/kg) was required to impair overtrained performance. In a working memory version of the navigation task, scopolamine administered 20 min before the first trial deteriorated retention tested 40 min later at a dose of 1.0 but not at 0.4 and 0.2 mg/kg. It is concluded that the disruptive effect of scopolamine is proportional to the demands on the working memory component of the task whereas the use of an overtrained mapping strategy is relatively resistant to cholinergic blockade.     

Scopolamine treatment and fimbria-fornix lesions: mimetic effects on radial maze performance

Long-Evans female rats were "trained" in an 8-arm radial maze and subsequently tested under systemic treatment with physostigmine (0.05 mg/kg, IP), scopolamine methylbromide (MBr) and scopolamine hydrobromide (HBr; 0.5 mg/kg, IP), whose effects were compared to those of aspirative lesions of the fimbria-fornix pathways. During the predrug trials, rats with lesions showed impaired performances compared to those of intact rats. Whereas physostigmine had no significant effect in either group, scopolamine HBr impaired performances of intact rats in a manner closely parallel to all measured behavioral effects of the lesions (errors, "correct arms" and strategies). The scopolamine HBr-induced deficits were not correlated with the percentage of "spatial" strategies. Under scopolamine HBr treatment the performances of rats showing preferences for "spatial" strategies did not differ significantly from those of rats showing preferences for "orientation" strategies. These results provide further support for the involvement of cholinergic processes in working memory and suggest that scopolamine-induced central cholinergic disruption may mimic the effects of fimbria-fornix lesions in an 8-arm radial maze. They also somewhat qualify previous reports on 1) the poor sensitivity of an uninterrupted radial maze testing procedure to pharmacological treatment and 2) the abilities of rats to resist muscarinic blockade depending on the strategies they use in the maze.     

Cholinergic and GABAergic modulation of medial septal area: effect on working memory

The role of the septohippocampal pathway in working memory was investigated by direct microinfusion of compounds into the medial septal area (MSA). Behavior was measured by performance in a continuous spatial alteration task in a T maze, and hippocampal theta rhythm was also recorded. Intraseptal saline had no effect on choice accuracy or hippocampal theta rhythm. Tetracaine decreased choice accuracy and theta rhythm 10 min, but not 90 min, after infusion. Likewise, muscimol and scopolamine produced a transient, dose-dependent suppression of hippocampal theta rhythm and a simultaneous dose-dependent impairment in choice accuracy. A significant correlation (r = .78) emerged between a compound's influence on theta rhythm and its effect on choice accuracy. The data support a role for the septohippocampal projection in working memory and suggest that gamma-aminobutyric acid and acetylcholine may have opposing influences on neurons in the MSA.     

Effects of cholinergic-rich neural grafts on radial maze performance of rats after excitotoxic lesions of the forebrain cholinergic projection system--II. Cholinergic drugs as probes to investigate lesion-induced deficits and transplant-induced functional recovery.

The effects of two doses of muscarinic (arecoline and scopolamine) and nicotinic (nicotine and mecamylamine) cholinergic receptor agonists and antagonists on the radial maze errors of rats, performing poorly after ibotenate lesions to the nucleus basalis and medial septal brain regions, were assessed before and after transplantation of cholinergic-rich and -poor fetal grafts, using tasks which measured short- (working) and long-term (reference) spatial and associative memory. Lesioned rats showed improvement with the agonists, and impairment with the antagonists, at low doses which did not affect the performance of controls; these effects were more marked for working than reference memory, especially in the spatial task. The peripherally acting antagonists N-methylscopolamine and hexamethonium did not affect the performance of control or lesioned rats. Effects of the cholinergic probes were re-examined 16 weeks after grafting, in groups with cholinergic-rich grafts to cortex and/or hippocampus which showed functional recovery, and groups with cholinergic-rich grafts to basal forebrain, or cholinergic-poor grafts to basal forebrain, cortex, and hippocampus, which showed no improvement. All lesioned rats, regardless of site, type, or efficacy of transplant, continued to show marked impairment with the antagonists. Poorly performing grafted animals also showed improvement with the agonists. In rats with behaviourally effective cholinergic-rich grafts, arecoline had no effect, but nicotine substantially increased working and reference memory errors, particularly spatial working memory errors. Rats with grafts in both cortex and hippocampus showed the largest increases in errors after nicotine. These results show that lesioned rats were more sensitive to the bi-directional effects of cholinergic receptor ligands than controls, consistent with a role for acetylcholine in the lesion-induced deficits. The predominant effect of drugs on working memory may also be consistent with disruption of acquisition rather than of storage or retrieval processes in memory, and may be related to impairment of attention. The results further show that, despite behavioural recovery, supersensitive responses to cholinergic drugs were not normalized in rats with cholinergic-rich grafts, and that an additive interaction between graft and host may have occurred in response to nicotine.     

Age-related changes in rostral basal forebrain cholinergic and GABAergic projection neurons: relationship with spatial impairment

Both cholinergic and GABAergic projections from the rostral basal forebrain contribute to hippocampal function and mnemonic abilities. While dysfunction of cholinergic neurons has been heavily implicated in age-related memory decline, significantly less is known regarding how age-related changes in codistributed GABAergic projection neurons contribute to a decline in hippocampal-dependent spatial learning. In the current study, confocal stereology was used to quantify cholinergic (choline acetyltransferase [ChAT] immunopositive) neurons, GABAergic projection (glutamic decarboxylase 67 [GAD67] immunopositive) neurons, and total (neuronal nuclei [NeuN] immunopositive) neurons in the rostral basal forebrain of young and aged rats that were first characterized on a spatial learning task. ChAT immunopositive neurons were significantly but modestly reduced in aged rats. Although ChAT immunopositive neuron number was strongly correlated with spatial learning abilities among young rats, the reduction of ChAT immunopositive neurons was not associated with impaired spatial learning in aged rats. In contrast, the number of GAD67 immunopositive neurons was robustly and selectively elevated in aged rats that exhibited impaired spatial learning. Interestingly, the total number of rostral basal forebrain neurons was comparable in young and aged rats, regardless of their cognitive status. These data demonstrate differential effects of age on phenotypically distinct rostral basal forebrain projection neurons, and implicate dysregulated cholinergic and GABAergic septohippocampal circuitry in age-related mnemonic decline.     

Orexin-saporin lesions of the medial septum impair spatial memory

The medial septum and diagonal band of Broca (MSDB) provide a major input to the hippocampus and are important for spatial learning and memory. Although electrolytic MSDB lesions have prominent memory impairing effects, selective lesions of either cholinergic or GABAergic MSDB neurons do not or only mildly impair spatial memory. MSDB neurons are targets of orexin-containing neurons from the hypothalamus. At present, the functional significance of orexin afferents to MSDB is unclear, and the present study investigated a possible involvement of orexin innervation of the MSDB in spatial memory. Orexin-saporin, a toxin that damages neurons containing the hypocretin-2 receptor, was administered into the MSDB of rats. Rats were subsequently tested on a water maze to assess spatial reference memory and a plus maze to assess spatial working memory. At 100 ng/microl, orexin-saporin destroyed primarily GABAergic septohippocampal neurons, sparing the majority of cholinergic neurons. At 200 ng/microl, orexin-saporin almost totally eliminated GABAergic septohippocampal neurons and destroyed many cholinergic neurons. Spatial reference memory was impaired at both concentrations of orexin-saporin with a dramatic impairment observed for 24-h retention. Short-term reference memory was also impaired at both concentrations. Rats treated with 200 ng/microl, but not 100 ng/microl, of orexin-saporin were also impaired on a spontaneous alternation task, showing a deficit in spatial working memory. Our results, together with previous studies, suggest that orexin innervation of the MSDB may modulate spatial memory by acting on both GABAergic and cholinergic septohippocampal neurons.     

Neuroinflammation not associated with cholinergic degeneration in aged-impaired brain

Degeneration of the cholinergic neurons in the basal forebrain and elevation of inflammatory markers are well-established hallmarks of Alzheimer's disease; however, the interplay of these processes in normal aging is not extensively studied. Consequently, we conducted a neuroanatomical investigation to quantify cholinergic neurons and activated microglia in the medial septum/vertical diagonal band (MS/VDB) of young (6 months) and aged (28 months) Fisher 344 × Brown Norway F₁ rats. Aged rats in this study were impaired relative to the young animals in spatial learning ability as assessed in the Morris water maze. Stereological analysis revealed no difference between aged and young rats in the total numbers of cholinergic neurons, demonstrating that loss of cholinergic neurons is not a necessary condition to observe impaired spatial learning in aged rats. In this same region, the total number of activated microglia was substantially greater in aged rats relative to young rats. Jointly, these data demonstrate that aging is characterized by an increase in the basal inflammatory state within the MS/VDB, but this inflammation is not associated with cholinergic neuron death.     

Dissociation of cholinergic function in spatial and procedural learning in rats

The cholinergic system has long been known for its role in acquisition and retention of new information. Scopolamine, a muscarinic acetylcholine receptor antagonist impairs multiple memory systems, and this has promoted the notion that drug-induced side effects are responsible for diminished task execution rather than selective impairments on learning and memory per se. Here, we revisit this issue with the aim to dissociate the effects of scopolamine (0.2-1.0 mg/kg) on spatial learning in the water maze. Experiments 1 and 2 showed that acquisition of a reference memory paradigm with constant platform location is compromised by scopolamine independent of whether the animals are pre-trained or not. Deficits were paralleled by drug induced side-effects on sensorimotor parameters. Experiment 3 explored the role of muscarinic receptors in acquisition of an episodic-like spatial delayed matching to position (DMTP) protocol, and scopolamine still caused a learning deficit and side-effects on sensorimotor performance. Rats extensively pre-trained in the DMTP protocol with 30 s and 1 h delays over several months in experiment 4 and tested in a within-subject design under saline and scopolamine had no sensorimotor deficits, but spatial working memory remained compromised. Experiment 5 used the rising Atlantis platform in the DMTP paradigm. Intricate analysis of the amount of dwelling and its location revealed a clear deficit in spatial working memory induced by scopolamine, but there was no effect on sensorimotor or procedural task demands. Apart from the well-known contribution to sensorimotor and procedural learning, our findings provide compelling evidence for an important role of muscarinic acetylcholine receptor signaling in spatial episodic-like memory.     

Spatial working memory is independent of hippocampal CA1 long-term potentiation in rats

This study investigated the relationship between spatial working memory and hippocampal long-term potentiation (LTP) using the allocentric place discrimination task (APDT) in rats, in which the selection accuracy is a good index for spatial working memory. Either the selective M1 muscarinic receptor antagonist pirenzepine (50 microg) or the choline uptake inhibitor hemicholinium-3 (5 microg) impaired APDT selection accuracy, but neither affected the induction of LTP in the hippocampal CA1 region in anesthetized rats. In contrast, the selective N-methyl-D-aspartate receptor antagonist D-amino-5-phosphonopentanoate (200 nmol) did not impair APDT selection accuracy but completely blocked hippocampal CA1 LTP. These results suggest that spatial working memory is independent of hippocampal CA1 LTP and that the central cholinergic system is involved in spatial working memory, but not through the modulation of hippocampal CAI LTP.     

Recognition of novel objects and their location in rats with selective cholinergic lesion of the medial septum

The importance of cholinergic neurons projecting from the medial septum (MS) of the basal forebrain to the hippocampus in memory function has been controversial. The aim of this study was to determine whether loss of cholinergic neurons in the MS disrupts object and/or object location recognition in male Sprague-Dawley rats. Animals received intraseptal injections of either vehicle, or the selective cholinergic immunotoxin 192 IgG-saporin (SAP). 14 days later, rats were tested for novel object recognition (NOR). Twenty-four hours later, these same rats were tested for object location recognition (OLR) (recognition of a familiar object moved to a novel location). Intraseptal injections of SAP produced an 86% decrease in choline acetyltransferase (ChAT) activity in the hippocampus, and a 31% decrease in ChAT activity in the frontal cortex. SAP lesion had no significant effect on NOR, but produced a significant impairment in OLR in these same rats. The results support a role for septo-hippocampal cholinergic projections in memory for the location of objects, but not for novel object recognition.     

Impairment of radial-arm maze performance in rats following lesions involving the cholinergic medial pathway: reversal by arecoline and differential effects of muscarinic and nicotinic antagonists.

Pharmacologic studies have indicated that accurate performance on the radial-arm maze depends upon the integrity of both nicotinic and muscarinic cholinergic neurotransmitter systems and that these systems interact in a complex fashion. Although numerous studies have suggested that pathways deriving from the basal nuclear complex of the forebrain are critical for the cholinergic modulation of learning and memory, most have focussed on the septohippocampal projection, and none have specifically targeted the medial or lateral systems. In Experiment 1, cortical knife cuts interrupting the medial cholinergic pathway were made at the level of the caudate-putamen nucleus. Such transections produced a robust but temporary disruption of choice accuracy performance in the radial-arm maze. Recovery of this behavior occurred within 10 days and before cholinergic fiber regeneration, suggesting that compensatory changes could have taken place in non-ablated neuronal circuits. In Experiment 2, daily postsurgical administration of arecoline, an agonist with predominantly muscarinic actions, was found to virtually eliminate the adverse behavioral effects of medial pathway transections, indicating that the deficit could be attributable, in part, to disruption of cholinergic projections. In Experiment 3, the effects of scopolamine, a muscarinic antagonist, and mecamylamine, a nicotinic antagonist, were examined in rats with medial cholinergic pathway transections after behavior had returned postsurgically to control levels. Although both drugs attenuated radial-arm maze performance before knife cuts, only scopolamine reduced choice accuracy following surgery. We conclude that the medial cholinergic pathway, particularly its nicotinic actions, plays an important role in cognitive function, at least as exemplified by radial-arm maze performance. Muscarinic mechanisms associated with other telencephalically projecting cholinergic networks, as well as possibly with the medial pathway itself, appear to operate interactively with nicotinic influences.     

Central cholinergic involvement in working memory: effects of scopolamine on continuous nonmatching and discrimination performance in the rat

Rats were trained to stable baselines of lever pressing on a variable intertrial interval continuous nonmatching to sample schedule (CNM) or on an analogous discrimination schedule. Scopolamine reduced accuracy of CNM performance to a similar extent over the three intertrial (retention) intervals: 2.5, 5, and 10 s, results indicating that the drug did not affect the time-dependent process of retention in working memory. When baseline levels of performance accuracy were similar in the CNM and discrimination tasks (but stimulus discriminability was greater in the CNM task), scopolamine reduced accuracy equally in the two procedures. Effects of scopolamine on accuracy of noncorrection trial CNM performance were simulated by reducing stimulus discriminability; however, scopolamine disrupted CNM correction trial performance much more than did reductions in stimulus discriminability. It is concluded that scopolamine's effects on working memory are not limited to possible effects on stimulus discrimination: Scopolamine may also affect retrieval of response rules from reference memory.     

Tetrahydroaminoacridine alleviates medial septal lesion-induced and age-related spatial reference but not working memory deficits

This study examined the effects of tetrahydroaminoacridine (THA, an anticholinesterase) on water-maze (WM) spatial reference (stable platform location during training) and spatial working memory (reversal of platform location) learning in young intact/medial septal (MS)-lesioned and aged rats. THA (1 or 3 mg/kg, IP) had no effect on reference or working memory performance of young intact rats. MS lesions decreased cholineacetyltransferase activity in the hippocampus and also impaired spatial reference and working memory. THA at 3 mg/kg stabilized MS lesion-induced reference memory performance deficit (path length increase), but was ineffective at 1 mg/kg. THA had no effect on the working memory performance of MS-lesioned rats. Aged rats were impaired in spatial reference and working memory tasks. THA at 3 mg/kg partially stabilized the age-induced spatial reference memory deficits, but was ineffective at 1 mg/kg. THA at either 1 or 3 mg/kg did not alleviate the age-related deficit in the working memory version of WM. The present results suggest that some of the age-related WM deficits may be related to the degeneration of the MS-hippocampus cholinergic system and that THA may be effective in stabilizing the reference memory deficits induced by hippocampal cholinergic dysfunction.     

An animal model of human-type memory loss based on aging, lesion, forebrain ischemia, and drug studies with the rat

The goals of this research were to develop a within-subject test of spatial working memory and performance for the rat in a T-maze, based on a delayed alternation, or "win-shift" foraging strategy. Using this model, specific aims were to compare the effects of: (1) age, (2) basal forebrain, medial septal, and amygdala lesions, (3) four vessel occlusion (4-VO), forebrain ischemia, and (4) physostigmine, scopolamine, arecoline, piracetam, and clonidine on memory and performance of young middle-aged, and old rats. Aging significantly impaired working memory and performance of Long-Evans rats. Memory of septal and basal forebrain, but not of amygdala lesioned rats was significantly impaired without effects on performance. Transient, 4-VO forebrain ischemia produced significant memory impairment, without effects on performance, and highly selective CA1 cell loss in the hippocampus. Physostigmine enhanced working memory in middle-aged and old rats. Scopolamine impaired memory in young, middle-aged, and old rats. Physostigmine reversed the scopolamine impairments of working memory. Arecoline enhanced memory in old rats without effects on performance. Piracetam and clonidine had no direct effects on memory, but piracetam increased and clonidine decreased speed of performance. From the aging, lesion, ischemia, and drug studies it was concluded that there was a convergence of evidence from 4 different approaches for a critical role for the hippocampus, particularly the CA1 fields, in spatial working memory.     

Short-term memory: facilitation and disruption with cholinergic agents

Male mice (C57BL/6 strain) trained in a T-maze to develop a successive reversal learning set were delayed-response tested at several intervals. When treated with scopolamine, behavior was impaired at all intervals; however, physostigmine facilitated performance at the longer intervals. The impairment and facilitation were most probably due to the actions of the cholinergic agents upon the expression of short-term memory, and it was concluded that the cholinergic synapse was necessary for short-term memory.     

Neurotransmitters and memory: role of cholinergic, serotonergic, and noradrenergic systems

In Alzheimer's disease (AD), pathological changes are found in the basal forebrain cholinergic system (BFCS), serotonergic raphe (RA), and noradrenergic locus coeruleus (LC) systems. The present study was designed to determine the extent to which selective damage in each of these systems individually could produce an impairment of memory, one of the clinical symptoms of AD. Rats were given selective lesions by injecting ibotenic acid into the nucleus basalis magnocellularis and medial septal area (i.e., BFCS); 5,7-dihydroxytryptamine into the medial and dorsal RA; and 6-hydroxydopamine (6-OHDA) into the LC or by ip injections of (2-chloroethyl)N-ethyl-2-bromobenzylamine HCl (DSP4). Levels of choline acetyltransferase (ChAT), norepinephrine, and serotonin verified lesion effectiveness and selectivity. Chronic changes in serotonergic-2 and beta-adrenergic receptors were also determined. Rats were tested in a delayed spatial alternation in a T-maze. BFCS lesions impaired choice accuracy with intertrial delays of 5, 30, and 60 s. RA lesions or DSP4 injections impaired choice accuracy only when the intertrial delay was 60 s. LC lesions (by 6-OHDA) did not impair choice accuracy at any delay. The results suggest that the pathological changes in the BFCS and RA are sufficient to produce the types of memory impairments associated with dementia, but the quantitative effects of pathology in these two systems are different.     

Neonatal lesion of forebrain cholinergic neurons: further characterization of behavioral effects and permanency

Intraventricular injections of 192 IgG-saporin in the neonatal rat caused severe loss of basal forebrain cholinergic neurons and ectopic hippocampal ingrowths. These were evident at 24 months of age and thus, were lifelong consequences of the 192 IgG-saporin treatment. When tested as young adults on a novel water-escape radial arm maze, the rats with this lesion were slower to learn the task, committing significantly more working and reference memory errors before they achieved control level of performance. It is unlikely that this was a result of attentional impairment as the lesioned rats performed as vigilantly as controls in a five choice serial reaction time task. When tested in the Morris water maze at 22 months of age, they were slower at learning the hidden platform location. This contrasts with previous studies which have repeatedly shown that they normally acquire this task as young adults. It was concluded that this neonatal cholinergic lesion has modest but discernable effects on problem solving in young adulthood that are consistent with the reported effects of the lesion on cortical pyramidal neurons. The cognitive effects of the lesion may become more severe in aging, perhaps as a result of the added effects of aging on these neurons.     

Interactive effects of morphine and scopolamine,MK-801, propanolol on spatial working memory in rhesus monkeys

Opiate, cholinergic, glutamatergic and beta-adrenergic neurotransmitters play key roles in learning and memory in humans and animals. Dysfunction of the interactions between these neurotransmitters may induce human diseases. In the present study, the interactions of morphine and acetylcholine (ACh), NMDA, and beta-adrenergic receptor antagonist (scopolamine, MK-801, and propanolol) were evaluated in a single-blind design by co-administrations of morphine and these drugs in a delayed response in rhesus monkeys. The results indicated that: (1) Co-administration of morphine and scopolamine deteriorated spatial working memory. (2) Co-treatment of morphine and MK-801 restored impairment caused by morphine and MK-801 in a dose-depending pattern. (3) Morphine plus propranolol impaired spatial working memory. High dose of morphine (0.01 mg/kg) reversed impaired spatial working memory induced by single propranolol and morphine treatment. These data suggested that the interactions of morphine and AChergic, NMDAergic and beta-adrenergic compounds were involved in spatial working memory in rhesus monkeys.     

Selective immunolesions of CH4 cholinergic neurons do not disrupt spatial memory in rats

Adult male Long-Evans rats were subjected to bilateral lesions of the cholinergic neurons in the nucleus basalis magnocellularis (NBM) by injection of 0.2 or 0.4 microg 192-IgG-saporin in 0.4 microl phosphate-buffered saline. Control rats received an equivalent amount of phosphate-buffered saline. Starting 2 weeks after surgery, all rats were tested for locomotor activity in their home cage, beam-walking performance, T-maze alternation rates (working memory), reference and working memory performance in a water-maze task, and memory capabilities in the eight-arm radial maze task using uninterrupted and interrupted (delay of 2 min, 2 h and 6 h after four arms had been visited) testing procedures. Histochemical analysis showed a significant decrease of acetylcholinesterase (AChE)-positive reaction products (30-66%) in various cortical regions at the 0.2-microg dose. At the dose of 0.4 microg, there was an additional, although weak, damage to the hippocampus (17-30%) and the cingulate cortex (34%). The behavioral results showed only minor impairments in spatial memory tasks, and only during initial phases of the tests (reference memory in the water maze, working memory in the radial maze). The behavioral effects of the dramatic cholinergic lesions do not support the idea of a substantial implication of cholinergic projections from the NBM to the cortex in the memory processes assessed in this study, but they remain congruent with an involvement of these projections in attentional functions.     

N-methyl-D-aspartate antagonists and working memory performance: comparison with the effects of scopolamine, propranolol, diazepam, and phenylisopropyladenosine.

The effects of the competitive N-methyl-D-aspartate (NMDA) antagonists CPP (5 & 10 mg/kg) and NPC 12626 (25 & 40 mg/kg) and the noncompetitive NMDA antagonists phencyclidine (1, 3, & 6.25 mg/kg) and MK 801 (0.1 & 0.2 mg/kg) on performance of rats on a nonspatial delayed matching-to-sample working memory task were evaluated. At the highest dose, each NMDA antagonist reduced choice accuracy at all retention intervals. In contrast, the reference anticholinergic agent scopolamine selectively reduced accuracy at long retention intervals, suggesting that scopolamine but not the NMDA antagonists directly interfered with time-dependent working memory retention. Propranolol, diazepam, and phenylisopropyladenosine had little or no effect on choice accuracy, suggesting that noradrenergic, gamma-aminobutyric acid-diazepam, and adenosine receptors may be relatively unimportant for working memory performance as assessed in this task. The NMDA antagonists also differed from scopolamine in that doses of NMDA antagonists that reduced response accuracy also reduced response probability, altered bias (competitive antagonists only), and increased intertrial interval responding (noncompetitive antagonists only). It was concluded that NMDA antagonists disrupt cognitive functions including, but not limited to, those required for accurate working memory performance.