Diazepam prevents post-training drug effects related to state dependency, but not post-training memory facilitation by epinephrine

Rats were trained and tested in a step-down inhibitory avoidance task (0.3-mA footshock). Training-test interval was 6 h. In Experiment 1, animals received, 1 h before training, an ip injection of vehicle or diazepam (2.0 mg/kg) and, 30 s after training and/or 30 min prior to testing, ip saline, epinephrine (6.25 micrograms/kg or 125.0 micrograms/kg), naloxone (0.5 mg/kg), or beta-endorphin (1 micrograms/kg). In the vehicle-pretreated animals, post-training epinephrine (6.25 micrograms/kg) and naloxone enhanced, and post-training beta-endorphin and epinephrine (125.0 micrograms/kg) reduced, retention test performance; and pretest beta-endorphin and epinephrine (125.0 micrograms/kg) reversed the latter effect and enhanced retention on their own. Diazepam lowered memory scores on its own and prevented all other drug effects with the exception of post-training facilitation by epinephrine (6.25 micrograms/kg). In previous papers it was shown that post-training facilitation by epinephrine is due to an influence on storage processes, whereas all the other drug effects described above result from the post-training establishment of state dependency to either beta-endorphin or epinephrine, and therefore to a process involving further acquisition and storage. The present findings suggest that diazepam selectively hindered the acquisition and/or storage processes involved in state dependency. This conclusion is strengthened by the findings from Experiment 2, which showed, using a classic 2 x 2 design, that diazepam itself did not induce state dependency but, rather, depressed acquisition and/or storage of the avoidance task.     

Reversal of retrieval impairment caused by retroactive interference on a two-way active avoidance task in rats

Rats were exposed to an open field with flashing light (OFL; 60-W lamp, 30 Hz, for 7 min) 2 h after training and/or 2 h before testing in a two-way active avoidance task (30 trials, 0.5-mA footshock). Post-training OFL presentation caused retroactive interference, i.e., a retrieval impairment/amnesia for the avoidance task. Pretest OFL exposure reversed the post-training OFL-induced retrieval deficit. Diazepam (2.0 mg/kg), atropine (2.0 mg/kg), and methylatropine (0.1 mg/kg) administered before post-training OFL presentation blocked the OFL amnesic effect. However, these drugs did not counteract the pretest OFL-induced recovery of retrieval. Atropine and methylatropine administered 2 h before testing to rats receiving only post-training OFL presentation canceled the OFL-interfering effect. These results suggest: (1) that the amnesic effect of post-training OFL is due to failure of retrieval of the avoidance task, (2) that the reversal of this retrieval impairment by pretest OFL exposure may involve either priming or state-dependent mechanisms, and (3) that there are different modulatory mechanisms involved in post-training and pretest OFL effects.     

Dual action of post-training naloxone on memory

Naloxone administered at a dose of 0.2 mg/kg post-training antagonizes the deleterious effect of post-training beta-endorphin administration and prevents the enhancing effect of pretest beta-endorphin administration, on retention of a step-down inhibitory avoidance task in rats. Given at a higher dose (0.4 mg/kg), naloxone caused, in addition to these effects, a pronounced retrograde facilitation which was additive with that caused by post-training ACTH or epinephrine administration. These findings show that post-training naloxone has two different effects or sets of effects, each with a different dose threshold: An interference with beta-endorphin induced state dependency and a true modulatory effect.     

Memory-modulatory effects of centrally acting noradrenergic drugs: possible involvement of brain cholinergic mechanisms.

Post-training administration of the centrally acting muscarinic agonist oxotremorine (50.0 microgram/kg, ip) facilitated 48-hr retention, in mice, of a one-trial step-through inhibitory avoidance response. Oxotremorine-induced memory facilitation was not prevented by the simultaneous post-training administration of the central beta-adrenoceptor antagonist propranolol (2.0 mg/kg, ip). In contrast, post-training administration of atropine (0.5 mg/kg, ip), but not methylatropine (0.5 mg/kg, ip), completely prevented the facilitatory effects of the central beta-adrenoceptor agonist clenbuterol (30.0 micrograms/kg, ip) on retention. Low subeffective doses of clenbuterol (3.0 micrograms/kg, ip) and oxotremorine (6.25 or 12.5 micrograms/kg, ip) potentiated their effects and facilitated retention when given simultaneously immediately post-training. These results suggest that clenbuterol may induce memory facilitation through an increase of the release of acetylcholine in the brain. Post-training administration of a high dose of clenbuterol (1.0 mg/kg, ip) significantly impaired retention. Clenbuterol (1.0 mg/kg, ip)-induced impairment of retention was completely prevented by simultaneous post-training administration of oxotremorine (6.25, 12.5, or 50.0 micrograms/kg, ip). The centrally acting anticholinesterase physostigmine (21.5 or 68.0 micrograms/kg, ip) partially prevented clenbuterol-induced impairment of memory. The peripherally acting anticholinesterase neostigmine (68.0 micrograms/kg, ip) modified neither retention nor the amnestic effects of clenbuterol. Considered together, these findings are consistent with the view that brain muscarinic cholinergic mechanisms are involved in both the facilitatory and impairing effect of post-training clenbuterol on the modulation of memory storage.     

The enhancement of retention induced by vasopressin in mice may be mediated by an activation of central nicotinic cholinergic mechanisms.

Immediate post-training subcutaneous administration of lysine vasopressin (LVP, 0.003-1.00 microgram/kg) enhanced retention, whereas the vasopressin antagonist AAVP (0.01-0.30 microgram/kg) impaired it, in male Swiss mice tested 48 h after training in an inhibitory avoidance task. Both effects were dose-dependent. Neither LVP nor AAVP affected response latencies in mice not given the footshock on the training trial. The simultaneous administration of AAVP at a dose (0.01 microgram/kg) which had no effect on retention shifted the dose-response curve of LVP to the right. Nicotine (1.0-30.0 micrograms/kg, sc), a central nicotinic cholinergic agonist, also facilitated retention in a dose-related manner without affecting the retention performance of unshocked mice. The effect of nicotine was prevented by the central acting nicotinic cholinergic receptor antagonist mecamylamine (5 mg/kg, sc.). In contrast, neither hexamethonium (5 mg/kg, sc), a peripheral acting nicotinic receptor blocker, nor atropine (0.5 mg/kg, sc) or methylatropine (0.5 mg/kg, sc), two anticholinergic drugs which are known to act on muscarinic cholinergic receptors, prevented the effect of post-training nicotine. The effects of LVP and nicotine were time-dependent, suggesting that both treatments enhanced retention by influencing post-training processes involved in memory storage. Low doses of nicotine (1.50 microgram/kg, sc) or the central anticholinesterase physostigmine (35 micrograms/kg, sc) and LVP (0.003 microgram/kg, sc), which had no effect on retention when administered alone, produced a synergistic interaction when given together following training. The influence of LVP (0.03 microgram/kg, sc) on retention was prevented not only by AAVP (0.01 microgram/kg, sc) but also by mecamylamine (5 mg/kg, sc), whereas the effects of nicotine (10.0 micrograms/kg, sc) were prevented only by mecamylamine. These results suggest that the enhancement of retention induced by vasopressin is probably due to an activation of central nicotinic cholinergic mechanisms which are critical for memory formation.     

Glucose effects on memory: behavioral and pharmacological characteristics

Recent findings indicate that post-training glucose injections can modulate memory storage for inhibitory (passive) avoidance training. Experiment I extended these findings to determine whether glucose, like other memory modulating treatments, enhances memory storage when administered after training with low footshock and impairs memory storage after high footshock training. In Experiment I, male Sprague-Dawley rats were trained in a one-trial inhibitory avoidance task using either a brief footshock (0.5 mA, 0.7 s) or slightly more intense footshock kept on until escape (0.7 mA, mean escape latency = 3.4 s). Immediately after training, each rat received a subcutaneous injection of glucose (100 mg/kg). When tested for retention performance 24 h later, the glucose-injected animals exhibited enhanced retention performance for low footshock training and impaired retention for high footshock training. Experiment II determined whether pretreatment with adrenergic antagonists blocked the effects of glucose on memory. Pretreatment with the alpha- or beta-adrenergic receptor antagonists, phenoxybenzamine, or propranolol, respectively, had no effect on acquisition or retention in animals trained with the brief footshock and did not affect glucose facilitation of that memory. In animals trained to escape footshock, phenoxybenzamine did not attenuate the amnesia produced by glucose. Propranolol-pretreated animals had impaired retention whether or not they received post-training amnestic injections of glucose; glucose had no effect on retention in these amnestic animals. These findings add further support to the view that glucose release after training and treatment may represent a physiological response subsequent to epinephrine release in modulating memory storage processing.     

Factors that influence test session performance measured 0, 3, or 6 h after inhibitory avoidance training

Rats were trained in a step-down inhibitory avoidance task using a 0.3-mA, 60-Hz footshock, and were tested at 0, 3, and 6 h from training. Retrieval scores (test session minus training session step-down latencies) were higher in control groups at 0 than at 3 or 6 h. Test session performance at 0 h was unaffected by the pretraining ip injection of ACTH1-24 (0.2 microgram/kg), epinephrine-HCl (5.0 micrograms/kg), human beta-endorphin (1.0 microgram/kg), or naloxone-HCl (0.4 mg/kg); or by a pretreatment with dexamethasone phosphate (2.0 mg/kg in divided doses 24 and 12 h before training); or by anterior or posterior hypothalamic deafferentation. Test session performance at 0 h was depressed by prior bilateral transection of the fornix, which suggests it depends on hippocampal function. The effect of the fornix lesion on test session performance at 0 h was not counteracted by ACTH, epinephrine, or beta-endorphin administration. When animals were tested 3 h after training, the post-training administration of ACTH and epinephrine caused an enhancement of test session performance; neither post-training beta-endorphin or naloxone, nor pretest ACTH, epinephrine, or beta-endorphin administration, had any effect in these animals. At 6 h from training, the post-training facilitatory action of ACTH and epinephrine was still present and the post-training depressant effect of beta-endorphin and the post-training facilitatory effect of naloxone became manifest, and so did the naloxone-reversible pretest facilitation induced by ACTH, epinephrine, or beta-endorphin. The influence of post-training naloxone or pretest beta-endorphin on retrieval scores at 6 h was not observed in the fornix-lesioned animals. In conclusion: Test session performance of this task at 0 h from training is regulated by different mechanisms than those which regulate test session performance at 3 or 6 h; in particular, it is less susceptible to modulation by the drugs used in the present study and it depends on the fornix; At least two major classes of modulatory factors influence retrieval scores at later times: consolidation-enhancing effects of ACTH and epinephrine, which become manifest at 3 h, and mechanisms related to beta-endorphin, which involve a form of state dependency and only become manifest at 6 h from training.     

Retention enhancement with post-training picrotoxin: lack of state dependency

Immediate post-training intraperitoneal administration of the GABA-antagonist picrotoxin (0.5 or 1.0 mg/kg) significantly enhanced retention of CD1 mice tested 24 h after training in an inhibitory avoidance task. Administration of picrotoxin prior to the retention test did not affect the retention performance of mice given post-training injections of either saline or picrotoxin. These findings indicate that the memory-enhancing effects of post-training administration of picrotoxin are not state-dependent.     

Benzodiazepine receptor ligand influences on acquisition: suggestion of an endogenous modulatory mechanism mediated by benzodiazepine receptors

In rats, pretraining ip administration of the central benzodiazepine receptor antagonist, flumazenil (5.0 mg/kg), or of the inverse agonist, n-butyl-beta-carboline-3-carboxylate (BCCB) (0.2 or 0.5 mg/kg), facilitated retention of a step-down inhibitory avoidance task; the central agonists, clonazepam and diazepam (0.4 or 1.0 mg/kg), had an opposite effect, and the peripheral agonist, 4'-chlordiazepam (1.25 or 6.25 mg/kg), was without effect. Pre- but not post-training flumazenil (2.0 mg/kg) blocked the effect of BCCB (0.5 mg/kg), clonazepam (1.0 mg/kg), or diazepam (1.0 mg/kg) given also pretraining. The post-training administration of all of these drugs had no effect on retention of the avoidance task. Flumazenil (5.0 mg/kg) and BCCB (0.5 mg/kg), given before training, enhanced retention test performance of habituation to a buzzer but not of habituation to an open field. In the three tasks studied, none of the drugs used had any appreciable effect on training session parameters. These results suggest that there is an endogenous mechanism mediated by benzodiazepine agonists, sensitive to inverse agonists, that normally down-regulates acquisition of certain behaviors; this mechanism becomes activated only when the tasks involve or occur with a certain degree of stress or anxiety (i.e., inhibitory avoidance or habituation to the buzzer) and not in less stressful or anxiogenic tasks (i.e., habituation to an open field).     

Post-training systemic and intra-amygdala administration of the GABA-B agonist baclofen impairs retention

The effects of the GABA-B receptor agonist baclofen on memory storage were studied in two series of experiments. In the first series, CD-1 mice were trained in two aversively motivated tasks: a one-trial inhibitory avoidance task and a classical conditioning task (conditional emotional response). Immediate post-training ip administration of (+/-)baclofen (10 and 30 mg/kg) impaired retention of animals in both tasks. The effect was time-dependent: Retention was not affected by baclofen administered 120 min after training. In the second series of experiments, which used Sprague-Dawley rats, post-training intra-amygdala administration of baclofen impaired retention of an inhibitory avoidance response. These results support the view that the GABAergic system is involved in the modulation of memory storage and that the amygdaloid complex may be a critical site for effects of drugs affecting the GABAergic system.     

Effects of post-training bicuculline and muscimol on retention: lack of state dependency

Immediate post-training intraperitoneal injections of the GABA antagonist bicuculline (0.25 or 0.5 mg/kg) or of the GABA agonist muscimol (1.0 or 2.0 mg/kg) improved and impaired, respectively, retention of CD1 mice tested 24 h after training in a one-trial inhibitory avoidance task. Administration of bicuculline or muscimol prior to the retention test did not modify retention latencies of mice that had received either saline or the same drug immediately after training. These findings indicate that the effects of post-training administration of bicuculline and muscimol on retention are not state dependent and, thus, argue against a general state-dependency interpretation of the effects of post-training treatments affecting retention. The findings are consistent with previous evidence indicating that GABAergic drugs affect retention through influences on memory storage processes.     

Mecamylamine prevents the enhancement of retention induced by lysine vasopressin in mice

Lysine vasopressin (0.03 microgram/kg, sc) enhanced retention of a one-trial, step-through inhibitory avoidance task when injected into male Swiss mice immediately post-training, as indicated by retention performance 48 h later. A low dose of the vasopressin antagonist, AAVP (0.01 microgram/kg, sc), did not significantly affect retention, whereas a higher dose (0.03 microgram/kg, sc) impaired retention. Neither lysine vasopressin nor AAVP modified latencies to step-through of mice that had not received a footshock during training. The simultaneous injection of AAVP (0.01 microgram/kg, sc) prevented the enhancement of retention induced by lysine vasopressin. The influence of lysine vasopressin on retention was antagonized by the simultaneous administration of mecamylamine (5 mg/kg, sc) but not by hexamethonium (5 mg/kg, sc), atropine (0.5 mg/kg, sc), or methylatropine (0.5 mg/kg, sc). A modulatory role of vasopressin on the activity of central cholinergic nicotinic mechanisms which participate in memory formation is suggested.     

Distinct mechanisms underlying memory modulation after the first and the second session of two avoidance tasks

Rats were subjected to three consecutive sessions, one session per day, of either a step-down inhibitory avoidance task using a 60-Hz. 0.3-mA footshock, or a two-way active avoidance task using 20 presentations of a 5-s, 1-kHz tone and a 0.3-mA footshock. After either the first or the second training session animals received an intraperitoneal injection of ACTH (0.2 microgram/kg), epinephrine-HCl (5.0 micrograms/kg), or naloxone-HCl (0.8 mg/kg). All these treatments caused memory facilitation on both tasks when administered after the first training session. When administered after the second training session only ACTH and adrenaline were effective, on both tasks. As previous physiological and pharmacological reports point to the activation of the brain beta-endorphin system after the first, but not the second, session of a task, we propose that (a) memory facilitation by naloxone depends on the previous activation of the brain beta-endorphin system; and (b) memory facilitation due to ACTH or epinephrine does not depend on the opioid activity, so their effects are expressed after both the first and the second training sessions. It was also observed that the enhancement of performance in the second training session due to post-training facilitatory treatments carried over to the test session. These results suggest that some form of consolidation occurs both after the first and after the second training session.     

Strength of scopolamine-induced amnesia as a function of time between training and testing

Variations in the strength of scopolamine-induced amnesia as a function of age of the habit were studied in Swiss Webster mice. Animals were trained in an active avoidance task to a criterion of 9/10 avoidances and immediately following training injected with scopolamine hydrochloride (1.0 mg/kg) or saline. Retention of the avoidance learning was evaluated by testing different groups of animals 1, 3, 7, 10, 14, and 28 days following training. The retention test consisted of five trials in which the CS but not the UCS was presented. Results indicated that saline-treated mice exhibited near-perfect retention up to 14 days post-training with forgetting beginning to be apparent at 28 days. Scopolamine treatment produced strong amnesia in animals tested 1 and 3 days post-training but normal retention in animals tested 7 and 10 days after learning. The amnesia abruptly reappeared at 14 days after which time it remained stable. The marked similarity of the scopolamine retention curve to changes in the strength of memory of discrimination learning in undertrained rats reported by Deutsch suggested that scopolamine resulted in the storage of a weak memory of the avoidance response. To explore this idea further we trained mice to a criterion (4/5) which would result in a weak avoidance response and tested different groups 1, 3, 10, 14, and 28 days following learning. Results showed that strength of the memory of avoidance learning increased up to 10 days and then decreased abruptly at 14 days thus replicating the general shape of the retention curve produced by injecting scopolamine following strong training. These data suggest that scopolamine disrupts processes essential for the formation of durable memories.     

Honeybees (Apis mellifera) holding on to memories: response competition causes retroactive interference effects

Five experiments on honeybees examined how the learning of a second task interferes with what was previously learned. Free flying bees were tested for landmark-based memory in variations on a paradigm of retroactive interference. Bees first learned Task 1, were tested on Task 1 (Test 1), then learned Task 2, and were tested again on Task 1 (Test 2). A 60-min delay (waiting in a box) before Test 2 caused no performance decrements. If the two tasks had conflicting response requirements, (e.g., target right of a green landmark in Task 1 and left of a blue landmark in Task 2), then a strong decrement on Test 2 was found (retroactive interference effect). When response competition was minimised during training or testing, however, the decrement on Test 2 was small or nonexistent. The results implicate response competition as a major contributor to the retroactive interference effect. The honeybee seems to hold on to memories; new memories do not wipe out old ones.     

Task-dependent role for dorsal striatum metabotropic glutamate receptors in memory

The effect of post-training intradorsal striatal infusion of metabotropic glutamate receptor (mGluR) drugs on memory consolidation processes in an inhibitory avoidance (IA) task and visible/hidden platform water maze tasks was examined. In the IA task, adult male Long-Evans rats received post-training intracaudate infusions of the broad spectrum mGluR antagonist α-methyl-4-carboxyphenylglycine (MCPG; 1.0, 2.0 mM/0.5 μL), the group I/II mGluR agonist 1-aminocyclopentane-1,3-carboxylic acid (ACPD; 0.5 or 1.0 μM/0.5 μL), or saline immediately following footshock training, and retention was tested 24 h later. In the visible- and hidden-platform water maze tasks, rats received post-training intracaudate infusions of ACPD (1.0 μM), MCPG (2.0 mM), or saline immediately following an eight-trial training session, followed by a retention test 24 h later. In the IA task, post-training infusion of ACPD (0.5 and 1.0 μM) or MCPG (1.0 and 2.0 mM) impaired retention. In the IA and visible-platform water maze tasks, post-training infusion of ACPD (1.0 μM), or MCPG (2.0 mM) impaired retention. In contrast, neither drug affected retention when administered post-training in the hidden-platform task, consistent with the hypothesized role of the dorsal striatum in stimulus-response habit formation. When intradorsal striatal injections were delayed 2 h post-training in the visible-platform water maze task, neither drug affected retention, indicating a time-dependent effect of the immediate post-training injections on memory consolidation. It is hypothesized that MCPG impaired memory via a blockade of postsynaptic dorsal striatal mGluR's, while the impairing effect of ACPD may have been caused by an influence of this agonist on presynaptic “autoreceptor” striatal mGluR populations.     

Involvement of central cholinergic mechanisms in the effects of oxytocin and an oxytocin receptor antagonist on retention performance in mice

Oxytocin (OT, 0.10 microg/kg, sc) impaired retention of a one-trial step-through inhibitory avoidance task when injected into male Swiss mice 10 min after training, as indicated by retention performance 48 h later. In contrast, the immediate post-training administration of the putative oxytocin receptor antagonist d(CH(2))(5)[Tyr(Me)(2), Thr(4), Thy-NH(9)(2)] OVT (AOT, 0.30 microg/kg, sc) significantly enhanced retention performance. Neither OT nor AOT affected response latencies in mice not given footshock on the training trial, and neither the impairing effects of OT nor the enhancing effects of AOT were seen when the training-treatment interval was 180 min, suggesting that both treatments influenced memory storage. The effects of OT (0.10 microg/kg, sc) on retention were prevented by AOT (0.03 microg/kg, sc) given immediately after training, but 10 min prior to OT treatment. The central acting anticholinesterase physostigmine (35, 70, or 150 microg/kg, i.p.), but not its quaternary analogue neostigmine (150 microg/kg, i.p.), reversed the impairment of retention performance induced by OT, whereas low subeffective doses of the centrally active muscarinic cholinergic antagonist atropine (0.5 mg/kg, i.p.) or the central acting nicotinic cholinergic antagonist mecamylamine (5 mg/kg, i.p.), but not methylatropine (0.5 mg/kg, i.p.) or hexamethonium (5 mg/kg, i.p.) prevented the enhancement of retention performance caused by AOT. We suggest that oxytocin negatively modulates the activity of central cholinergic mechanisms during the posttraining period that follows an aversively motivated learning experience, leading to an impairment of retention performance of the inhibitory avoidance response.     

Epinephrine modulates long-term retention of an aversively motivated discrimination

These experiments examined the effects of post-training epinephrine (Epi) on retention of an aversively motivated discrimination task. Male CFW mice were trained to escape from footshock by entering one of two alleys of a Y-maze. On a 24-h retention test (six trials) the correct alley was reversed. The findings of Experiment 1 indicate that errors on the discrimination reversal varied directly with number of trials (criterion of 0, 3, or 6 successive correct choices) on the original training. These findings indicate that errors on discrimination reversal training provide a sensitive index of retention of the original training. In Experiment 2, mice were trained to a criterion of three successive correct choices and were given post-training injections of saline or Epi (0.1, 0.3, or 1.0 mg/kg ip). On a 24-h discrimination reversal test mice given the low doses of Epi made more errors than did saline controls while mice given the high dose made fewer errors. In Experiment 3, mice trained as in Exp 2 received post-training saline or Epi (0.3 or 1.0 mg/kg) and were tested for retention either 1 week or 1 month later. At each retention interval, performance was comparable to that found with a 24-h retention interval. The findings provide additional evidence that post-training Epi produces long-lasting dose-dependent modulating effects on memory storage.     

Beta-endorphin enhancement of retrieval in a three-session paradigm

Rats were submitted to three consecutive sessions, one session per day, of step-down inhibitory avoidance task (60-Hz, 0.3-mA footshock) or of two-way active avoidance task (25 trials of a 5-s, 1-kHz tone and a 0.4-mA footshock). Animals received intraperitoneal (ip) injections of saline or beta-endorphin (2.0 micrograms/kg) before or after the second session and before the third session. beta-Endorphin given before either the second or the third session improved retention of both tasks, while its administration after the second session had no effect upon performance on both tasks. In Experiment 2, it was shown that rats receiving naloxone (0.2 mg/kg) ip after the first session did not exhibit the enhancement of retrieval by beta-endorphin administration before the second session, so a non-state-dependent improvement of retrieval by the opioid seems to be unlikely. In view of these results we can propose that the presession retrieval enhancing effect of beta-endorphin is due to an endogenous state dependency on the opioid that can be expressed either in the second or in the third session of aversive tasks.     

Post-training brain catecholamine levels: lack of response to water-motivated training

Rats were trained in a one-trial appetitive task using water motivation. Brain catecholamine and metabolite levels were assessed in samples collected 10 min after training. There was no evidence that brain NE levels were modified by training, although catecholamine levels increased when the animals were placed in a novel environment. These results differ from those obtained after avoidance training where the extent of a post-training decrease in brain norepinephrine predicts later retention performance.