Memory enhancement in mice: role of drug dose and training-testing interval.

Pharmacologic probes are useful for studying memory mechanisms. For eight drug treatments affecting a variety of transmitter systems [arecoline, piribedil, clonidine, fluoxetine, naloxone, ACTH (4-10)], we determined how long memory retention would remain improved with a dose sufficient to improve 3-hour retention. While all 6 treatments enhanced 3-hour retention test performance at p less than 0.05, only 5 treatments significantly enhanced retention 24 hour after training and none of the treatments significantly affected retention at 168 hours. A detailed analysis of the dose and retention interval interaction for arecoline indicated that at low doses retention decreased as the retention interval increased while higher doses improved retention up to 3 hours and only the highest dose tested enhanced retention at 3 and 24 hours. Drug doses that enhance short-term retention (3 hours) were not adequate to enhance long term retention (168 hours). The 6 drug treatments had no significant or systematic effect on activity or on acquisition. We conclude that short-term retention performance was better because of enhanced memory processing or recall and not because of performance effects per se.

Effect of acute arecoline, tacrine and arecoline + tacrine post-training administration on retention in old mice.

The amnesias characteristic of Alzheimer's disease and other age-related dementias are refractory to conventional pharmacotherapy. A recent treatment strategy is to combine drugs to improve their memory enhancing effect. We previously reported that in young weakly trained mice, the combination of arecoline and tacrine was more effective on a mg/kg basis than either drug administered alone. This was true whether the route of administration was intracerebroventricular, subcutaneous or oral. We now report that 24 month old mice trained to avoid footshock in a T-maze show poor retention when tested one week later. Subcutaneous administration of arecoline, tacrine (also referred to as tetrahydroaminoacridine, THA) and arecoline plus tacrine administered immediately after T-maze footshock avoidance training enhanced retention of 24 month old mice compared to the saline-injected control. Since the combination was as effective as the single drug treatments even though 96% less arecoline and 99.7% less tacrine was administered, the combination showed marked potentiation of drug action of memory processing.

Two-drug combinations of memory enhancers: effect of dose ratio upon potency and therapeutic window, in mice.

Two-drug combinations have been reported to enhance retention more effectively than when either drug was administered alone at the same dose. Some combinations of cholinergic drugs enhance retention even though the total drug dosage is reduced by as much as 97% compared to the dose needed to improve retention when the same drugs are administered singly. The choice of dose ratio is usually arbitrary or based on empirical results. The present study systematically varied the ratio of two drugs in a combination and at the same time varied the dosage of each drug. The drug combinations were administered to mice immediately after training on T-maze footshock avoidance task. Retention was tested one week later. Three two-drug combinations were selected for presentation because they differed considerably as to (a) the lowest effective total dose that improved memory-retention, (b) the optimal ratio that improved retention and (c) the width of the therapeutic window. The effect of a drug combination on retention was found to be dependent on the particular drugs in the combination, the ratio and the dose administered.

Antagonism of endogenous opioids modulates memory processing.

The studies reported here demonstrate that opioid antagonism enhances memory in two classes of animals viz. Aves and Mammalia. In mice, immediate posttraining administration of naloxone produces a time-dependent improvement in retention tested one week later. This effect is stereospecific. As naloxone was approximately 1000-fold more potent when administered intracerebroventricularly compared to subcutaneously, it appears that it produces its effect within the central nervous system. Pretest administration of naloxone, at a dose that failed to alter acquisition, also improved test performance, suggesting that naloxone also improved recall. Similar improvement in retention was demonstrated with the more potent opioid antagonist, nalmefene, at a 500-fold lower dose. The dose response to naloxone in both the mouse and the chick and to nalmefene in the mouse had the characteristics of an inverted U, with high doses either being ineffective or suppressing memory retention. In mice, naloxone demonstrated anti-amnestic properties against both anisomycin, a protein synthesis inhibitor, and scopolamine, an acetylcholine receptor blocker. Administration of beta-funaltrexamine (B-FNA) 72 h prior to training did not alter acquisition but did enhance retention. In studies where the mu-opioid receptor was blocked with B-FNA, naloxone was unable to enhance retention. B-FNA failed to impair the memory enhancing properties of arecoline, fluoxetine or clonidine. This demonstrates specificity of the B-FNA ability to prevent naloxone from enhancing memory and suggests that the opioid antagonist effects on memory are mediated by the mu-receptor.

The effects of adrenergic, opioid and pancreatic polypeptidergic compounds on feeding and other behaviors in neonatal leghorn chicks.

The present study examined the effects of intracerebral (IC) administration of pancreatic polypeptide (PP), neuropeptide Y (NPY), norepinephrine (NE), dynorphin and naloxone on food intake in 2-day-old Leghorn chicks. Of the compounds studied, only PP (20 micrograms) and naloxone (10 and 20 micrograms) elevated food intake significantly as compared to saline injections. NPY, a potent orexigenic agent in mammals, did not elevate consumption significantly in a dose-related fashion. This latter finding was attributed to the occurrence of tonic-clonic convulsions following NPY administration. However, for those chicks which did not exhibit behavioral convulsions, food intake appeared to be elevated by 1, 5 and 10 micrograms of NPY. Similarly, NE did not elevate food intake but instead induced sedation and narcolepsy, a behavioral response which could be distinguished from the convulsions observed after NPY. In a separate group of chicks, the effect of NPY on cortical activity was examined. Bipolar electrodes were used to record EEG activity before and after IC injections of saline, NPY or NE. The behavioral convulsions induced by NPY corresponded with an increase in high amplitude sharp-wave activity, which persisted for up to 30 min post-injection. Collectively, these results suggest that the neurochemical substrates for feeding in 2-day-old Leghorn chicks are distinct from those underlying food intake in adult mammals.

Fluoxetine enhances memory processing in mice.

Fluoxetine (FLU) increases brain concentrations of serotonin by blocking its uptake, without appreciably affecting the dopamine or norepinephrine systems. The present experiments provide evidence that a subcutaneous injection of FLU enhanced post-memory processing ("consolidation") and retrieval, but not acquisition in young adult mice. FLU (15 mg/kg) enhanced 1-week memory retention when injected 2 min post-training. Similar enhancement was obtained with intracerebroventricular injection (20 micrograms per mouse). FLU enhanced retention when administered prior to training (1-5 mg/kg). FLU (2.5 mg/kg) enhanced recall scores when injected 1 h before the 1-week retention test, indicating an enhancing effect on memory retrieval. Neither the pre-training nor pre-testing effects depended on improved acquisition, since FLU did not improve acquisition of T-maze foot-shock avoidance over the dose range 0.5-35 mg/kg. The sensitive period for post-training enhancement by FLU (15 mg/kg) was less than 90 min, as shown by the temporal gradient typical of memory-enhancing drugs. The amnesia induced by a protein synthesis inhibitor anisomycin, or by an anticholinergic drug scopolamine, was blocked by FLU (15 mg/kg) injected post-training. Finally, FLU (15 mg/kg) injected after one-trial passive avoidance training enhanced 1-week retention, demonstrating effectiveness in this task as well as in the active avoidance task.

Scopolamine effects on memory retention in mice: a model of dementia?

Scopolamine-treated normal young human subjects exhibit memory dysfunctions analogous to those observed in demented patients. The dysfunctions are reversible by physostigmine but not by d-amphetamine which suggests that the memory impairment is specifically related to reduced cholinergic transmission caused by scopolamine. Scopolamine-induced amnesia has been proposed as a model for dementia where reduced cholinergic function is the suspected cause. We report seven experiments in young adult mice which examine scopolamine's effects on memory retention and whether its amnestic effects are specifically blocked by cholinergic agonists or cholinomimetics. Young adult mice were trained to avoid footshock in a T maze and their retention tested 1 week after training. Pretraining subcutaneous injection of scopolamine improved retention scores of "undertrained" mice at a dose of 0.01 mg/kg but impaired at a dose of 0.1 mg/kg. Post-training injection showed no effect at 0.01 mg/kg, enhanced retention scores at 0.1 mg/kg, and impaired at 1.0 mg/kg. The impairment by 1.0 mg/kg was blocked by injection 45 min post-training of each of two cholinergic drugs but was also counteracted by six drugs which act upon five other neural systems (catecholamine, serotonin, glycine, GABA, and hormonal). When scopolamine was injected 40 min pretraining, and each of eight drugs was injected immediately after training, the amnestic effect of scopolamine was only partially counteracted. This suggests that scopolamine impaired acquisition, in addition to some impairment of memory processing. This was confirmed by a direct study of acquisition rates of the avoidance response; 0.1 mg/kg of scopolamine impaired acquisition. The overall results indicate that pretraining administration of scopolamine impairs learning and to some degree memory processing. Counteracting scopolamine-induced amnesia, by either pretraining or post-training drug administration, is not specific to the cholinergic system.

Memory enhancement: supra-additive effect of subcutaneous cholinergic drug combinations in mice.

The amnesias characteristic of Alzheimer's disease and other age-related dementias are refractory to conventional pharmacotherapy. A recent treatment strategy is to combine present drugs to improve their memory enhancing effect. We used mice weakly trained on active avoidance in a T-maze to compare the effect of cholinergic drugs, given alone and in two-drug combinations, on retention test performance. All drugs were injected SC immediately after training. Memory retention was tested 1 week later. A dose-response curve was determined for each of four drugs (arecoline, edrophonium, oxotremorine, tacrine) and for each of the six possible two-drug combinations. Each drug and each combination improved retention test performance up to an optimal dose; the improvement decreased with further increases in dose. A striking reduction (66.2%-95.7%) in the optimal dose for enhanced retention was observed with these two-drug combinations.

Hydergine enhances memory in mice.

Moderate improvement of cognitive function has been reported in many clinical studies of Hydergine (HYD) but in only a few controlled dose-response studies in animals. We therefore evaluated the effects of HYD upon one-week retention of training by T-maze active avoidance and by one-trial passive avoidance in a stepthrough apparatus. HYD satisfied seven criteria for a memory enhancing drug, as follows. Post-training subcutaneous injection improved memory recall scores; the dose-response curve was the inverted-U typical of many memory enhancers. Post-training intracerebral injection showed a similar effect, indicating a central mechanism of action. Pre-testing subcutaneous injection had a similar effect, indicating enhancement of retrieval processes. Pre-training subcutaneous injection had a similar effect, at much lower doses than post-training injection. HYD did not improve acquisition of T-maze active avoidance, indicating that its effects were on memory processing rather than learning per se. Enhancement by post-training injection declined as the training-to-injection interval increased; this temporal gradient is typical of memory-enhancing drugs. Subcutaneous HYD reversed the amnestic effects of anisomycin (a protein synthesis inhibitor) and of scopolamine (an anticholinergic drug). The results characterize HYD as an effective enhancer of memory processing and of memory retrieval, in the mouse paradigms of active avoidance and passive avoidance.

Memory retention test performance in mice: improvement by chronic oral cholinergic drug treatment.

Mice consumed solutions containing 0, 0.025, 0.050 or 0.075 mg/ml of arecoline hydrobromide (ARE) one week prior to training (T-maze, footshock, active avoidance) and a total of two weeks prior to testing memory retention. The mean daily doses of ARE were estimated to be 0, 157, 302, or 500 micrograms per mouse, respectively. An inverted-U dose-response curve was obtained; the best retention test performance was by the group receiving 0.050 mg/ml of ARE. Measures of activity and weight taken over the experiment indicated no significant differences between ARE groups and the control group; thus no apparent toxicity. Separate groups of mice consumed 0 or 0.050 mg/ml of ARE for one week, then were trained to a criterion of 5 avoidances in 6 training trials. There were no significant differences in trials to first avoidance response or to criterion. Thus the enhanced retention test performance of the 0.050 mg/ml ARE group reflected improved memory processing rather than better learning.

Memory retention: effect of prolonged cholinergic stimulation in mice.

The fundamental hypothesis that drugs may affect memory processing by prolonging transmitter action was tested by extending the time of drug action, using repeated administrations of the cholinergic agonist, arecoline hydrobromide (ARE). The ARE was injected intracerebroventricularly into mice immediately after training (T-maze footshock avoidance) and at 90-min intervals thereafter, for a total of 1, 2, or 3 injections. The results indicate that 1 injection had no effect whereas 3 successive injections significantly improved memory retention test performance. The results confirm the hypothesis being tested; six control groups ruled out other plausible interpretations of the results.

Brain glutamate inhibition and amnesia: evidence provided by proline analog action.

The action of proline and its analog as glutamate antagonists was investigated in CNS tissue of the neonatal chick. Avian brain slices were incubated in low concentrations of L-proline, D-proline, DL-3,4-dehydro-proline, L-prolyl-L-proline, or in avian physiological salt solution before depolarization was induced by application of 45 mM K+. Glutamate was determined in the efflux material collected both before and after tissue stimulation. The release of endogenous glutamate was inhibited significantly by exposure to L-proline, DL-3,4-dehydroproline and L-prolyl-L-proline. The degree of glutamate inhibition correlated with the amnestic potency of these substances. The manner in which these results strengthen the hypothesis of glutamate involvement in memory processes is discussed.

L-prolyl-L-arginyl-glycineamide induces memory enhancement in chicks.

Two-day-old chicks were injected either intraventricularly or intraperitoneally with saline or a L-prolyl-L-arginyl-glycineamide solution. This C-terminal tripeptide of arginine vasopressin produced dose dependent enhancement effects when injected centrally but not peripherally. Physical debilitation and/or aversive effects of the peptide were eliminated as the cause of the decreased responding noted in memory enhancement studies using this avoidance paradigm. Possible memory mechanisms are discussed in light of this peptide's relationship to vasopressin, vasotocin, and L-propyl-L-leucyl-glycineamide.

Dose-dependent and time-dependent action of oxytocin on chick memory.

Experiments were conducted to investigate the dose-related and time-dependent effects of oxytocin on memory for a one-trial conditioned taste aversion task using two-day old chicks. Oxytocin was administered intracerebrally 1 min posttraining to 5 groups of chicks in dose levels differing by a factor of 10 and ranging from 5.0 pg to 50 ng. A second experiment tested the time-dependent nature of the neuropeptide's action. In this experiment the oxytocin (5.0 ng) was administered at either 1 min, 9 min or 59 min posttraining. In both experiments saline-injected control groups were included. The taste aversion training for all experiments consisted of presenting an attractive lure, coated with an aversive liquid (EtOH), to each chick for a 10-s training trial. Most chicks pecked 1 or 2 times at the lure before inhibiting any further response. The retention testing took place 24 h after the training and consisted of presenting the dry, uncoated lure to each chick for an additional 10 s. Chicks that avoided pecking at the lure were considered to have exhibited enhanced retention. The groups of chicks receiving 50 pg to 50 ng of oxytocin exhibited enhancement of retention, as did the 1 min group of the time-dependent experiment. These results are compared to the effects on memory consolidation in chicks induced by vasopressin and L-prolyl-L-leucyl-glycineamide. The apparent conflict between these results and those obtained in mammalian studies with oxytocin are discussed.

Memory retention: potentiation of cholinergic drug combinations in mice.

The amnesias characteristic of Alzheimer's disease and other age-related dementias are refractory to conventional pharmacotherapy. A recent strategy is to combine present drugs, to improve their memory enhancing effect. We utilize mice weakly trained on active avoidance in a T-maze in order to compare the effect on retention test performance of cholinergic drugs given alone and in two-drug and three-drug combinations. All drugs were injected intraventricularly immediately after training. Memory retention was tested one week later. A dose-response curve was determined for each of four drugs (arecoline, edrophonium, oxotremorine, deanol) and for several of their fixed-ratio combinations. The results indicate that each drug and each combination improved retention test performance up to an optimal dose; the improvement decreased with further increases in dose. A striking reduction (as much as 95%) in the optimal dose for enhanced retention was observed with these two-drug combinations, and further reduction with a three-drug combination. The practical implications of planned drug interactions as an improved means of treating amnesias associated with aging are under investigation.

Memory enhancement induced in chicks by L-prolyl-L-leucyl-glycinamide.

Two-day-old chicks were injected either intraventricularly or intraperitoneally with saline or a L-prolyl-L-leucyl-glycinamide solution. This C-terminal tripeptide of oxytocin produced retrograde enhancement when injected centrally but not peripherally. Possible memory mechanisms are discussed in light of this peptide's relationship to oxytocin, MSH, and dopaminergic systems.

D-glutamine produces seizures and retrograde amnesia in the chick.

D-glutamine, 6 mumols/chick, injected intracranially one min after training, produces retrograde amnesia when tested 24 and 48 hr post training on an avoidance task. D-glutamine also produces ectostriatal seizures that correlate with amnestic activity.