Effects of combined acetylcholinesterase inhibition and serotonergic receptor blockade on age-associated memory impairments in rats.

We recently reported that post-training administration of serotonergic receptor antagonists attenuated the inhibitory-avoidance memory deficits normally exhibited by aged rats. In the present study, we determined whether a subeffective dose of the serotonergic type-2 receptor antagonist, ketanserin, would augment the facilitative effects produced by the acetylcholinesterase inhibitor, physostigmine, on memory in aged rats using the same task. The drugs were injected intraperitoneally alone, or in combination, immediately following training. Retention testing occurred 24 hours following training. A dose-dependent enhancement of memory was demonstrated as a result of the two treatment conditions (physostigmine 0.01-10.0 micrograms/kg, ketanserin 1.0 mg/kg + physostigmine 0.001-0.01 micrograms/kg). The facilitation of memory produced by the combined treatment was observed at doses well below those required to produce a similar effect when each drug was administered alone. The results provide additional evidence for an interaction between the cholinergic and serotonergic neurotransmitter systems in learning and memory, and may have important implications in the treatment of age-related memory impairments.

What is the nature of the role of the serotonergic nervous system in learning and memory: prospects for development of an effective treatment strategy for senile dementia.

The serotonergic nervous system has long been suspected of playing an important role in the processes underlying learning and memory. However, owing frequently to inconsistent and divergent results, its precise role is difficult to define. Recently, there has been a renewed interest in this neurotransmitter's role in the processes underlying learning and memory. This has, in part, been due to the observations that this system appears to undergo significant deterioration as a result of the pathology associated with certain age-related cognitive disorders as well as the discovery of multiple receptor subtypes. Reviewed here are the results of a number of studies designed to gain further insight into the role the serotonergic nervous system plays in learning and memory. A variety of methods are used to manipulate this system, and the effects of these manipulations on performance in a variety of behavioral tasks are summarized. Consistent with past observations, it is difficult to incorporate the results of the present series of studies into a single unified theory of serotonin's role in learning and memory. However, it is clear that not all of the inconsistencies can be attributed to differences in the methods used to manipulate the system or in the types of tasks used to assess learning and memory. Some of the inconsistencies clearly belie a selective role of this neurotransmitter in the processes underlying learning and memory and further underscore the complex nature of this system's role in the processing of information by the brain.

Enhanced passive avoidance retention following posttrain serotonergic receptor antagonist administration in middle-aged and aged rats.

The experiments examined the ability of posttrain administration of serotonergic receptor antagonists to attenuate the age-related deficits in avoidance retention normally exhibited by middle-aged (12 months) and aged (22 months) rats. Ketanserin (0, 1.0, 10.0 mg/kg) produced a significant dose-dependent increase in test step-through latencies in both age groups. The suppression of responding did not appear to be due to generalized learned aversion as nonshocked rats, injected with the highest dose of ketanserin, did not exhibit similar elevations in test latencies. In order to determine whether the ketanserin-induced effect could be generalized to other serotonergic antagonists, middle-aged and aged animals were injected with a single dose of mianserin (10.0 mg/kg). This antagonist also significantly increased step-through latencies in both age groups, while not affecting the performance of nonshocked animals. The results provide additional evidence for a role of the serotonergic nervous system in memory, and may have important implications in the development of effective treatment strategies for geriatric-related cognitive disorders.

Age-related changes in regional cerebral blood flow and behavior in Sprague-Dawley rats.

Studies of regional cerebral blood flow (rCBF) were carried out in male, Sprague-Dawley rats at ages 6, 12 and 24 months. These animals were characterized behaviorally for their ability to learn a complex 14-choice sequential T-maze (i.e., Stone maze) for food reward. Old animals (i.e., 24 month) demonstrated a clear and consistent impairment in maze performance which correlated significantly with decreased cortical blood flow. The results are discussed in relation to cerebral blood flow studies in aging humans and as they relate to the use of rats, the Sprague-Dawley strain in particular, to study brain aging.

Correlation of behavioral and cerebrovascular functions in the aging rat.

Regional cerebral blood flow (rCBF), maze performance and the influence of environmental enrichment on these parameters were studied in Sprague-Dawley rats aged 6, 12 and 24 months. Learning ability in a complex sequential T-maze (Stone maze) progressively declined with increasing age in rats which were normally housed in standard caging. Environmental enrichment significantly improved maze performance but did not prevent the age-dependent impairment. Following completion of the learning studies, rCBF was measured in each of 13 brain regions in conscious, unrestrained, resting animals. In the absence of any significant change in cardiac output over the entire age range, rCBF was lower in all brain regions by an average of 16% in 12-14 month old rats and 8% in aged rats (24-26 months old); the occipital cortex, inferior and superior colliculi and hypothalamus were particularly affected regions in both age groups. The sharp reduction of rCBF that occurred between 6 and 12 months of age did not reflect, and probably preceded the progressive decline in maze performance. Such highly significant age-related changes in rCBF were not affected, however, by environmental enrichment procedures. This contrasts with the substantial influence of enrichment on maze performance. Finally, mean brain blood flow and mean cortical blood flow correlated inversely and significantly with average daily numbers of errors made by 24 month old rats during Stone maze acquisition.

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

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

Cerebrovascular permeability and cognition in the aging rat.

Regional cerebrovascular permeability-capillary surface area products (rPS) and brain vascular space (BVS) were measured in aging, conscious, unrestrained Sprague-Dawley rats. Three groups of animals were examined: young-mature (6 months), middle-aged (12-14 months), and old (24-26 months) rats. Complex maze learning had been previously characterized in these same animals. Maze learning declined with age. Brain vascular space did not differ significantly with age in any brain region. However, small, but significant age-dependent decreases in rPS (25-33%) were observed. These decreases occurred mainly in the old animals in the basal ganglia and parietal cortex, and in the middle-aged and old rats in the olfactory bulbs. Significant and unexpected positive average correlations between brain permeability-capillary surface area products (PS) and learning errors occurred primarily in young rats and were attributable mainly to changes in 5 of 14 brain regions; hypothalamus, hippocampus, parietal cortex, septal area and superior colliculus. The higher correlations between maze learning errors and PS in young animals may indicate dynamic regulation of this cerebrovascular parameter which is lessened with aging. Average correlations between PS and cerebral blood flow also were determined and found to be generally small and not significant for most brain regions and age groups.

Plasma concentrations of inorganic sulfate in Alzheimer's disease.

Recent reports have suggested that plasma concentrations of inorganic sulfate may be lower in patients with Alzheimer's disease (AD). We measured sulfate concentrations in 10 patients with AD and found an average concentration of 0.28 mM, which was not significantly different from the mean concentration in age-matched controls (0.32 mM) or young healthy controls (0.27 mM). These results indicate that plasma sulfate concentrations are not altered in AD and that previous reports suggesting altered metabolism of sulfur-containing xenobiotics in neurodegenerative diseases should be reevaluated.

Association of sleep parameters and memory in intact old rats and young rats with lesions in the nucleus basalis magnocellularis.

Relations between sleep and memory were examined as a function of aging in rats. Sleep (24 hr), passive avoidance retention, and choline acetyltransferase (CAT) activity were assessed in 3 age-groups (6, 15, and 24 months old). Age-related alterations were evident in sleep, memory, and cortical and striatal CAT activity. Retention deficits in old rats were significantly correlated with several measures of paradoxical sleep. Similar analyses in 6- and 15-month-old rats with ibotenic acid-induced lesions of the nucleus basalis magnocellularis (NBM) showed several alterations in sleep, memory, and cortical CAT activity comparable to those seen in the old rats. One measure of paradoxical sleep, bout duration, correlated significantly with retention scores in rats with lesions. Thus, fragmented paradoxical sleep accompanies memory impairments in old rats and in young rats with NBM lesions.

Enhancement of the memory of a previously learned aversive habit following pre-test administration of a variety of serotonergic antagonists in mice.

The present study examined the effects of pre-test administration of a number of serotonergic receptor antagonists on the retrieval of a previously learned aversive habit in the mouse. All of the receptor antagonists (pirenperone, ketanserin, mianserin, methysergide and metergoline) produced a dose-dependent increase in the latency to complete 5 s of drinking 48 h after training. This suppression of drinking could not be attributed to nonspecific effects of the drugs on behavior (e.g., illness, reduced thirst, or activity), as non-contingently trained mice failed to exhibit similar elevations in their test scores. These results are, therefore, further support for an important role for serotonin in the processes underlying learning and memory.

Evidence for a possible interaction between noradrenergic and serotonergic neurotransmission in the retrieval of a previously learned aversive habit in mice.

The present series of experiments examined the effects of pretreating pirenperone-injected mice with a variety of non-serotonergic receptor antagonists on retrieval of a one-trial inhibitory (passive) avoidance task. Water-deprived mice were trained to avoid drinking from a water spout located in the avoidance chamber by pairing foot-shock with licks from the water spout. Retention was measured as the suppression of drinking (latency to drink) 48 h later. Pre-test administration of pirenperone (1.0 mg/kg) significantly enhanced retrieval (increased latencies). The suppression of drinking could not be attributed to the non-specific effects of pirenperone on behavior in general, as the performance of non-contigently shocked mice injected with the same dose of pirenperone did not exhibit a similar elevation in latencies. Of the seven pretreatment drugs examined, only phenoxybenzamine (1.0 mg/kg) completely blocked the pirenperone-induced response. Bicuculline (1.0 mg/kg) partially attenuated the enhanced performance resulting from pre-test pirenperone administration. The results suggest that the pirenperone-induced response may be partly due to activation of noradrenergic (alpha) neurotransmission.

Benzodiazepine anti-conflict effects in Maudsley reactive (MR/Har) and non-reactive (MNRA/Har) rats.

The Maudsley Reactive (MR/Har) and Non-Reactive (MNRA/Har) rat strains, bred originally by Broadhurst for differences in Open Field Defecation, also differ in their control (i.e., non-drug) behavior in the Conditioned Suppression of Drinking (CSD) conflict procedure, a second "model" behavior for the study of anxiety and/or emotionality in rats. The present studies compared the effects of diazepam and alprazolam on CSD behavior in these two strains of rats. In daily 10-min sessions, water-deprived rats were trained to drink from a tube that was occasionally electrified (0.2-0.5 mA), electrification being signaled by a tone. Both diazepam and alprazolam increased punished responding in a dose-related manner. The per cent increase in punished responding (for diazepam only) was comparable in the two strains; however, both statistical and empirical approaches indicated that the magnitude of the anti-conflict effect of benzodiazepines in MNRA/Har versus MR/Har rats was not related to differences in baseline (i.e., non-drug) punished responding. Based on the absolute change in shocks received, rats of the MNRA/Har strain exhibited a significantly greater anti-conflict effect following diazepam or alprazolam treatment than did rats of the MR/Har strain. These findings further the hypothesis that the behavioral differences exhibited by Maudsley MR/Har and MNRA/Har rat strains may constitute a genetically-based "animal model" for the study of emotionality and/or anxiety.

Role of serotonin in memory: facilitation by alaproclate and zimeldine.

The effects of alaproclate and zimeldine on memory retrieval were examined in male Swiss-Webster mice using a one-trial inhibitory avoidance task. All drugs were administered IP prior to the retention test 24 h after training. Both drugs were found to facilitate memory retrieval significantly in a dose- and time-dependent fashion that could not be explained in terms of non-specific effects of the drug (illness, lack of motility, etc.) at the time of the test. The temporal effects of alaproclate and zimeldine on memory closely followed their course of concentration of the drug within the blood stream. The facilitation of retrieval induced by alaproclate and zimeldine was blocked by the putative serotonergic receptor agonist quipazine but not blocked by the antagonist cyproheptadine. Pretreatment with quipazine alone in a group of animals trained to a shock level which normally results in high levels of suppression was not sufficient to produce memory impairment, suggesting that quipazine was probably antagonizing the facilitative effects of alaproclate and zimeldine directly, rather than overriding the facilitation through an indirect action on retrieval in general. The present results lend further support to the suggestion that serotonin plays a significant role in memory.

Potentiation of oxotremorine-induced hypothermia by alaproclate in PCA lesioned and non-lesioned rats.

Stimulation of muscarinic cholinergic receptors with the highly potent and selective receptor agonist oxotremorine produced hypothermia in rats. Alaproclate, a purported selective serotonergic reuptake inhibitor, potentiated this response. Destruction of central presynaptic serotonergic terminals with the potent cytotoxin p-chloroamphetamine (PCA) failed to attenuate the hypothermic response to oxotremorine in alaproclate-pretreated animals. These results could be taken to suggest that alaproclate may act, at least in part, via a non-serotonergic mechanism to potentiate the oxotremorine-induced hypothermic response.

Antagonism of the anti-conflict effects of phenobarbital, but not diazepam, by the A-1 adenosine agonist l-PIA.

The present study examined the effects of the anxiolytics diazepam and phenobarbital, the A-1 adenosine agonist N6-R-phenylisopropyladenosine (l-PIA), and the A-2 adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) on conflict behavior. Water-restricted rats were trained to drink from a tube that was electrified (0.5 mA intensity) on a FI-29s schedule, electrification being signaled by a tone. After 3 weeks of daily 10-min sessions, the animals accepted a stable number of shocks (punished responding) and consumed a consistent volume of water (unpunished responding) per session. Different doses of l-PIA and NECA were then tested separately at weekly intervals. In addition, the effects of diazepam and phenobarbital were determined in animals pretreated with saline, l-PIA, or NECA. Neither l-PIA (15-250 nmole/kg) nor NECA (2.5-20 nmole/kg) produced a significant anti-conflict effect when administered alone. Diazepam (1.25-10 mg/kg) or phenobarbital (10-40 mg/kg) administration to saline-pretreated rats resulted in a dose-dependent increase in punished responding (shocks received) with minimal effects on unpunished responding (water intake). Neither l-PIA nor NECA pretreatment reliably altered the effects of diazepam on conflict behavior. Pretreatment with l-PIA, but not NECA, significantly reduced the anti-conflict effects of phenobarbital on conflict behavior. These data suggest that phenobarbital, but not diazepam, anti-conflict responses may involve interactions with A-1 adenosine receptors.

Facilitation of memory retrieval by centrally administered catecholamine stimulating agents.

Amnesia for inhibitory avoidance learning induced in mice by a post-training injection of the protein synthesis inhibitor anisomycin was alleviated in a dose-dependent fashion by introcerebroventricular injections of D-amphetamine (20 micrograms), dopamine (10, 25 and 50 micrograms) and the dopamine agonist lisuride (0.5 and 1.0 micrograms), given 5 min before the retention test. Animals which received avoidance training in a different apparatus did not show increased test latencies following drug treatment thus eliminating non-specific behavioral suppression as an explanation for the findings. Neither norepinephrine nor the norepinephrine agonist clonidine was able to reverse the amnesia. These findings indicate that central dopamine systems may play a role in the retrieval of inhibitory avoidance learning.

Effects of combined serotonin depletion and lesions of the nucleus basalis magnocellularis on acquisition of a complex spatial discrimination task in the rat.

The purpose of the present experiment was to determine the effects of lesions of cholinergic neurons originating from the nucleus basalis magnocellularis (NBM), alone or in combination with central serotonin depletion, on learning and memory in rats trained in the Stone 14-unit T-maze--a complex, positively-reinforced spatial discrimination task. Lesion of cholinergic neurons within the NBM was accomplished by bilateral infusion of ibotenic acid. Serotonin depletion was accomplished by the systemic administration of p-chloroamphetamine (PCA). The results show that PCA-induced serotonin depletion enhanced learning. This effect was completely prevented by NBM lesions, despite the fact that NBM lesions alone did not affect the performance of rats in this task. The results of this study support the view that the cholinergic and serotonergic systems may functionally interact in learning and memory processes. The significance of this interaction in the etiology and treatment of dementia should be further investigated.

Enhanced spatial discrimination learning in rats following 5,7-DHT-induced serotonergic deafferentation of the hippocampus.

Learning in rats trained in the Stone 14-unit T-maze, a complex, positively reinforced spatial discrimination task was assessed following cytotoxic (5,7-dihydroxytryptamine; 5,7-DHT) deafferentation of the serotonergic inputs to the hippocampus. Serotonergic deafferentation was accomplished by infusing the cytotoxin in to the fornix-fimbria/cingulum bundle. Lesioned rats reached criterion (i.e. learned) in significantly fewer trials and made significantly fewer errors throughout training than either vehicle-injected or sham-operated controls. This represents the first time that the effects of selective chronic destruction of serotonergic inputs to the hippocampus have been investigated. The present results provide, therefore, evidence in support of a neuromodulatory role for serotonin (5-HT) within the rat hippocampus in the mediation of the processes underlying learning and memory for this task. Other studies are, therefore, warranted in order to determine whether hippocampal 5-HT also plays a role in the mediation of the processes underlying learning and memory in other types of tasks.

Central effects of adenosine analogs on locomotor activity in mice and antagonism of caffeine.

Mice implanted with chronic indwelling cannulas were injected in the lateral cerebral ventricle with a series of adenosine analogs and the effects on spontaneous locomotor activity were recorded. All analogs produced dose-related decreases in locomotor activity. The relative order of potency for locomotor depression was: NECA much greater than L-PIA greater than CADO greater than D-PIA. Caffeine at the lowest dose produced a significant decrease in locomotor activity. At higher doses caffeine had no effect on locomotor activity but it did antagonize the depressant effects of NECA, a finding consistent with the notion that the central stimulant action of methylxanthines is due to their antagonism of central adenosine receptors.

Prenatal exposure to alcohol in adult rats: relationships between sleep and memory deficits, and effects of glucose administration on memory.

Previous studies show that prenatal exposure to alcohol results in sleep deficits in rats, including reductions in paradoxical sleep. Little is known, however, about the extent or duration of sleep impairments beyond the neonatal period. The present experiment examined effects of prenatal exposure on sleep in young adulthood. Three-hour, daytime sleep EEGs were obtained in 6-month-old female rats prenatally exposed to alcohol. Compared to isocaloric pair-fed and ad libitum control groups, the alcohol-exposed group showed reduced paradoxical sleep. Non-paradoxical sleep did not differ between groups. Concurrent deficits were obtained in radial arm maze, but not inhibitory (passive) avoidance, performance. One year later, at the age of 18 months, alcohol-exposed rats showed deficits in spontaneous alternation behavior which were reversed by administration of glucose (100 mg/kg). Deficits in paradoxical sleep at 6 months of age were highly correlated with deficits in spontaneous alternation behavior at 18 months of age, in individual, alcohol-exposed animals. These results provide the first evidence that prenatal exposure to alcohol results in selective and persistent deficits in sleep. They also show that measures of paradoxical sleep can predict impaired memory over a large portion of the life span, and suggest that glucose can attenuate memory deficits in this population.