Effects of cycloheximide and puromycin on learning and retention in the cockroach,P. americana

A new one-session T-maze training procedure for cockroaches, in which animals were trained to turn right or left to avoid shock, is described. This paradigm was utilized to investigate effects of protein synthesis inhibiting drugs on learning and retention. Cycloheximide (CXM), which inhibited protein synthesis by over 90% during the training period, did not impair acquisition and did not produce retention deficits at any interval up to 1 day after training. Puromycin (PURO), which inhibited protein synthesis by about 70% during the training period, produced amnesia 5 hr after training, while acquisition was not affected. Thus invertebrates, as well as vertebrates, are susceptible to amnesic effects of puromycin. Although PURO-injected animals showed retention deficits as measured by the number of correct turns, no retention deficit occurred for the behavioral modification consisting of an increase in runway time during the training period. Therefore, PURO appears to show specificity for the different types of longer-term memories that are formed in a training situation.     

Injections of atropine into the caudate nucleus impair the acquisition and the maintenance of passive avoidance

Two experiments were performed to test the hypotheses that cholinergic activity of the caudate nucleus (CN) is involved in the acquisition and in the maintenance of passive avoidance behavior. Rats were trained, in one trial, to avoid one of two compartments of a conditioning box and retention of the task was measured 24 hours later. Several doses of atropine were injected into the CN of independent groups of animals. In Experiment 1 the atropine was injected 2 minutes after training and in Experiment 2 it was injected 6 minutes before retention testing. In both cases a dose-dependent retention deficit was found. These results indicate that striatal cholinergic activity is indeed involved in the processes that mediate passive avoidance conditioning.     

DDC-Induced amnesia and norepinephrine: A correlated behavioral-biochemical analysis

Diethyldithiocarbamic acid (DDC), a dopamine-B-hydroxylase inhibitor, when injected into rats 30 min to 6 h before training of a passive avoidance task, impaired formation of long-term memory as indicated by performance on a retention test 24 h later. Performance of the task was at its minimum when injection occurred 2 to 4 h prior to training; recovery was evident in animals trained 5 or 6 h after drug treatment. Catecholamine assay of brains of temporally yoked animals showed that norepinephrine depletion followed a time course paralleling that of the amnesia. These findings support the hypothesis that the degree of memory storage, as reflected in performance following training in a passive avoidance task, can be directly correlated with the level of norepinephrine existing at the time of training.     

A new method for studying working memory by using the three-panel runway apparatus in rats

We designed a new method for studying working memory, by using a repeated acquisition procedure in the three-panel runway apparatus. This apparatus is composed of a start box, a goal box and four consecutive choice points; each choice point consists of three panel gates. Male Wistar rats were trained with 6 consecutive trials (one session) per day. Each trial was performed every two minutes. In this apparatus, rats could pass through only one gate (correct gate) among three panel gates in the direction of the goal box and were given 100 mg food pellets as the positive reinforcement. The sequence of correct gate position in each rat was changed everyday, but not in each session. Error responses (pushing the incorrect gate) were gradually reduced as training was repeated, and the learning was established within 16 training sessions to achieve criterion performance. Intraperitoneal scopolamine and intrahippocampal ethylcholine aziridinium ion (AF64A) produced increases in both the number of errors and the latency in a dose-dependent manner. The increase in errors induced by AF64A did not return to the control level, though the prolonged latency returned to normal. As a conclusion, this experimental procedure using the three-panel runway apparatus would be a useful method for studying working memory, and its memory deficit is involved at least in the dysfunction of the cholinergic system in the hippocampus.     

Effects of acute alcohol administration on object recognition learning in C57BL/6J mice.

The goal of the present study was to investigate effects of alcohol intoxication on the object recognition learning task. Male C57BL/6J mice habituated to saline injections and exploratory arena received different doses of ethanol (0, 1.6 or 2.4 g/kg) before or after a 10-min training session. During training, animals were exposed to a small object (a marble or a die). On the next day, during a 10-min testing session, animals were exposed to two objects: the familiar object from the previous day and a novel object. Analysis of behavior during testing showed that mice injected with 0 and 1.6 g/kg of ethanol before training spent more time exploring a novel than a familiar object during testing. In contrast, mice injected with 2.4 g/kg ethanol spent equal amounts of time exploring the novel and the familiar object. Mice injected with this dose of ethanol after training did not show a decreased ratio of object exploration during testing. Analysis of behavior during training showed that mice injected with this dose of ethanol spent less time exploring the object, although their locomotor activity was not decreased. Our results show that in C57BL/6J mice, ethanol intoxication interferes with exploratory activity during object exploration, but not with consolidation of memory.     

Time-dependent retention deficits induced by post-training injections of atropine into the caudate nucleus

Rats were trained on a one-trial passive avoidance task, and retention of the task was measured 24 hr later. Atropine was injected bilaterally into the anterior caudate nuclei (ACN) of rats from independent groups at one of several intervals after training. Application of atropine 2 min after training produced a lack of retention of passive avoidance. An intermediate degree of impairment was seen when the treatment was given 3 min 45 sec after the learning experience, and interference with retention was still noted when an interval of 7 min 30 sec was studied. In contrast, no deficits were observed in groups of animals injected with atropine 15 or 30 min after training. Rats injected with atropine into the parietal cortex 2 min after training showed only a minimal reduction of retention, and a group injected with saline solution into the ACN performed as well as non-treated animals. These results suggest that there is a time-dependent process that mediates the retention of passive avoidance, and that this process requires the activation of cholinergic synapses within the anterior caudate nucleus.     

Effects of nimodipine, felodipine and amlodipine on electroconvulsive shock-induced amnesia in the rat

The effects of various doses (0.03, 0.1, 0.3 or 1.0 mg/kg) of the Ca²⁺ channel blockers nimodipine, felodipine and amlodipine on the learning ability of rats exposed to electroconvulsive shock were examined. The animals were trained in a passive avoidance procedure. The drugs tested were injected 30 min before the learning trial started. The electroconvulsive shock was given immediately after the learning trial response had been acquired. A passive avoidance retention test was performed 24 h later. It was found that electroconvulsive shock strongly impaired the retention of the passive avoidance response. Nimodipine, felodipine and amlodipine did not influence the passive avoidance behavior in the sham electroconvulsive shock group, but significantly improved the retention deficits in the animals exposed to electroconvulsive shock. These findings support the hypothesis that perturbations in Ca²⁺ homeostasis can contribute to the memory deficits associated with electroconvulsive shock. The antiamnestic effects of the substances tested make them interesting candidates for clinical trials in patients with cognitive impairment caused by electroconvulsive shock therapy.     

Chronic administration of cholinergic agents: effects on behavior and calmodulin

Rats were implanted subcutaneously with Alza pumps and 0.9% saline, physostigmine, or scopolamine were continuously infused for 15 days. Twenty-four hours after removal of the pumps all animals were trained on a single trail passive avoidance task. Twenty-four hours after training they were tested for retention. Following behavioral testing, animals were sacrificed, brain regions dissected, frozen and stored (-20 degrees C) for calmodulin determinations. Animals which had previously received chronic infusions of scopolamine performed significantly better than controls, while those which previously received chronic infusions of physostigmine performed significantly worse during the retention test. No significant differences in calmodulin levels (soluble or particulate) were detected across brain regions or drug groups. These results indicate that continuous chronic infusion of drugs which can facilitate or inhibit CNS cholinergic activity can induce performance changes on a learning task opposite to those resulting following the acute administration of these same drugs.     

Differential expression of Arc in the mesocorticolimbic system is involved in drug and natural rewarding behavior in rats

Aim:

To investigate the different effects of heroin and milk in activating the corticostriatal system that plays a critical role in reward reinforcement learning.

Methods:

Male SD rats were trained daily for 15 d to self-administer heroin or milk tablets in a classic runway drug self-administration model. Immunohistochemical assay was used to quantify Arc protein expression in the medial prefrontal cortex (mPFC), the nucleus accumbens (NAc), the dorsomedial striatum (DMS) and the ventrolateral striatum (VLS) in response to chronic self-administration of heroin or milk tablets. NMDA receptor antagonist MK801 (0.1 mg/kg) or dopamine D1 receptor antagonist {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 (0.03 mg/kg) were intravenously injected at the same time as heroin was infused intravenously.

Results:

Runway training with heroin resulted in robust enhancement of Arc expression in the mPFC, the NAc and the DMS on d 1, 7, and 15, and in the VLS on d 1 and d 7. However, runway training with milk led to increased Arc expression in the mPFC, the NAc and the DMS only on d 7 and/or d 15 but not on d 1. Moreover, runway training with milk failed to induce increased Arc protein in the VLS. Both heroin-seeking behavior and Arc protein expression were blocked by MK801 or {"type":"entrez-protein","attrs":{"text":"SCH23390","term_id":"1052733334","term_text":"SCH23390"}}SCH23390 administration.

Conclusion:

The VLS is likely to be critically involved in drug-seeking behavior. The NMDA- and D1 receptor-dependent Arc expression is important in drug-seeking behavior.     

Impairment of working memory by neuronal degeneration with NMDA in rat hippocampal CA-1

Rats were trained on a three-panel runway task prior to injections of N-methyl-D-aspartate (NMDA; 40nmoles/μl/side) into the dorsal hippocampus. One week after the treatment, animals did not show any change in the number of errors on the first runway trial (reference memory), with one correct white and two incorrect black panels at each choice point, whereas they showed a marked increase in the number of errors on the following (2nd-6th) trials (working memory) with all black panels. The memory deficit persisted at least for 10 days. After the experiments, histological studies showed neuronal degeneration of hippocampal CA-1 pyramidal cells in all NMDA-treated rats but not in vehicle-treated rats. Pretreatment with the NMDA receptor antagonist MK-801 (3 or 10mg/kg, i.p) before the NMDA injections protected both the neuronal degeneration and the memory deficit. These findings suggest that selective neuronal degeneration induced by excessive stimulation of NMDA receptors in hippocampal CA-1 impaired working memory, but not reference memory.     

Amphetamine and the overtraining reversal effect

Rats were trained in a Y-maze on a two choice simultaneous brightness discrimination with light as S+ and dark as S- (position irrelevant). Animals in the Mastery group were trained until they reached criterion and were then switched to reversal, where the reinforcement contingencies of the original training were reversed. Animals in the Overtraining group received a further 110 trials before being switched to reversal. The administration of 1 mg/kg d-amphetamine facilitated dramatically reversal learning in Mastery group. Overtraining improved reversal in saline injected animals and slowed down reversal in amphetamine-treated animals. The drug also facilitated the acquisition of the initial brightness discrimination.     

Drug discrimination training with a single choice trial per session

All drug discrimination procedures share in common the practice of providing for multiple choice opportunities per training session. This practice allows nondrug cues (presence or absence of reinforcement) to mediate choice behavior during that portion of the session following the initial choice. Investigators who have acknowledged this problem typically use only first-trial choice performance to evaluate discriminative control by the drug cue, and consider additional choice training following the delivery or nondelivery of the first reinforcer to be facilitatory in establishing drug-mediated discriminative control. In this experiment, rats were trained to discriminate 4.0 mg/kg morphine from saline in a novel procedure that employed a single choice trial per training session. Choice performance was characterized during discrimination acquisition and in subsequent stimulus generalization testing. The results indicated that when all reinforcers are made contingent on correct performance during a single choice trial, rapid and stable control of drug-mediated choice behavior, is observed. In addition, the results demonstrated that additional choice training following the delivery or nondelivery of the first reinforcer is not a necessary antecedent toward establishing drug-mediated discriminative control.     

Protective effects of saffron extract and its active constituent crocin against oxidative stress and spatial learning and memory deficits induced by chronic stress in rats

Although it is well established that chronic stress impairs spatial learning and memory, few studies have investigated possible ways to prevent its deleterious effects. Here, we investigated the effects of Crocus sativus L., commonly known as saffron, and its active constituent crocin on learning and memory loss and the induction of oxidative stress in the hippocampus by chronic stress. Rats were injected with saffron extract, crocin or vehicle over a period of 21 days while being exposed to chronic restraint stress (6 h/day). After this, they were trained and tested on a water-maze spatial memory task. They performed four trials per day for 5 consecutive days, and this was followed by a probe trial two days later. At the end of the behavioral testing, several parameters of oxidative stress in the hippocampus were measured. Treatment with saffron extract or crocin blocked the ability of chronic stress to impair spatial learning and memory retention. Relative to controls that received vehicle, stressed animals that received saffron extract or crocin had significantly higher levels of lipid peroxidation products, significantly higher activities of antioxidant enzymes including glutathione peroxidase, glutathione reductase and superoxide dismutase and significantly lower total antioxidant reactivity capacity. Finally, crocin significantly decreased plasma levels of corticosterone, as measured after the end of stress. These observations indicate that saffron and its active constituent crocin can prevent the impairment of learning and memory as well as the oxidative stress damage to the hippocampus induced by chronic stress. Thus, using these substances may be useful in pharmacological alleviation of cognitive deficits.     

Possible role of brain dopaminergic systems in the memory effects of central stimulants

In experiments on male Wistar rats trained and tested for memory retention on an active avoidance task in a maze, it was found that haloperidol at doses of 0.2 and 2 mg/kg injected intraperitoneally one hour prior to or immediately after training impaired learning and/or memory. The central stimulants caffeine (20 mg/kg), strychinine (1 mg/kg) and amphetamine (1 mg/kg) injected intraperitoneally immediately after training improved long-term retention; the central stimulants administered at the same doses but in combination with haloperidol (2 mg/kg) after training did not manifest their retention-facilitating effect. It is assumed that dopaminergic mechanisms underlie learning and memory processes in active avoidance conditioning and that an optimum activity of these mechanisms is necessary for the memory-facilitating effect of the central stimulants to appear.     

Cycloheximide produces adult-like retention deficits of prior learning in infant mice

Utilizing a dosage of cycloheximide which was found to inhibit cerebral protein synthesis by almost 90% after injection, separate groups of 13-day-old mice received either cycloheximide or saline followed by 0 (control), 15, or 25 training trials in a discriminated shock-escape T-maze. Twenty-four hr later, each mouse was treated with cycloheximide or saline and tested for retention by an additional 25 trails in the T-maze. As reflected by correct choice-point turns, the results suggest that whereas salinetreated mice demonstrated reliable retention of prior learning, cycloheximide treated mice exhibited memory impairment; cycloheximide per se had no effect on performance during either original training or retest. A final experiment indicated that this memory impairment was not due to cycloheximide's general debilitating side effects at the time of retention testing. Taken together, these data suggest that protein synthesis inhibition during training impaired consolidation and/or retrieval processes involved in memory. The biochemical and behavioral effects following cycloheximide injection in 13-14-day-old mice in the present study parallel those reported with adult animals and lend indirect support to the hypothesis that the 24-hr memory capacity exhibited by these young mice reflects the early functioning of those processes involved in adult long-term memory.     

Anxiolytic effects of oxytocin in cue-induced cocaine seeking behavior in rats

Rationale

Oxytocin (OT) is a neuropeptide previously related to reward, learning, memory, and stress, events associated with cocaine addiction. OT has shown anxiolytic properties in different animal models of anxiety. Moreover, previous data have demonstrated an increase in mRNA OT levels within the nucleus accumbens (NAc) following acute and chronic cocaine exposure in rats. Therefore, OT might play a modulatory role in the rewarding properties of cocaine.

Objectives

The present set of experiments aims to examine the role of OT on environmentally elicited cocaine-seeking behavior and whether OT could reduce anxiety associated with this behavior.

Methods

Separate groups of rats were trained in a cue-elicited cocaine-seeking behavior paradigm. Prior to the reinstatement phase, animals received microinfusions of artificial cerebrospinal fluid (aCSF), OT, OT agonist (TgOT), or OT antagonist (OTA) within the intracerebral ventricular intracerebroventricular (ICV) system. To test OT anxiolytic effects in reinstatement behavior, separate groups of animals were trained in a cue-elicited cocaine-seeking behavior protocol or in a cocaine-conditioning paradigm. At the end of each behavioral training, all animals were ICV pretreated with aCSF or OT, and then exposed to an elevated plus maze.

Results

Results showed that OT and TgOT pretreatment significantly reduced reinstatement of cocaine-seeking behavior. Most significantly, OT treatment reduced the anxiety triggered by cue-induced reinstatement conditions and cocaine-paired conditioned locomotion.

Conclusions

The present study demonstrates for the first time that OT actions within the brain mediate the anxiety response triggered by cues previously paired with cocaine intake.     

Effects of pre-training administration of scopolamine on learning and retention in the cockroach,P. americana

The muscarinic cholinergic blocker, scopolamine, has no effect on acquisition or retention of the choice behavior of the cockroach, P. americana trained to turn right or left to avoid shoch in a T-maze. Scopolamine does, however, prevent runaway habituation during training, suggesting that such habituation may be dependent on central cholinergic synapses as there are no known peripheral cholinergic receptors in cockroaches. Previous work demonstrating that no effect of scopolamine on such retention, suggests that puromycin does not produce its amnesic effect on choice behavior by interferring with central cholinergic synaptic processes.     

Effects of N-methyl-d-aspartate antagonism on spatial learning in mice

C57BL/6Ibg mice were treated with the N-methyl-d-aspartate (NMDA) receptor antagonist 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) and tested for selective deficits in spatial learning ability in the Morris water task. Two types of training protocols were used during the initial exposure to the training environment. In protocol 1, animals were given four massed trials before being returned to their home cages. In protocol 2, animals were returned to their home cages after each of the first four trials. Following the initial four trials, both sets of animals were given massed trials in blocks of four. CPP had minor effects on nonspatial learning, with greater impairment seen in animals trained according to protocol 1 than in animals trained according to protocol 2. The drug increased latency to find the platform in the spatial learning form of the task, with no effect of training protocol on latency. When spatial learning ability was measured in terms of the search behavior exhibited by the animals after the platform was removed from the pool, animals trained according to protocol 1 showed a severe CPP-induced impairment in search accuracy. Animals trained according to protocol 2 showed no effect of drug treatment. The results suggest that CPP does not have a reliable effect on place learning and that factors other than the type of learning being tested may contribute to performance deficits following CPP treatment.     

The effect of the CS-UCS interval and extinction on place conditioning and analgesic tolerance with morphine

In experiment 1, a CS-UCS interval study of place conditioning and analgesic tolerance with morphine was conducted. Morphine (10 mg/kg i.p.) was administered to separate groups of rats either 2 h prior to, 1 h prior to, immediately prior to, immediately after or 2 h after 30-min confinement in one end compartment of a place conditioning apparatus. A total of three choice tests was given, one after every six morphine injections. A preference for the end compartment contingent upon morphine injection was shown in groups that received morphine prior to end compartment placement. Groups that received morphine after end compartment placement were not different in their preference behavior from groups that received only saline during place conditioning training. A hot-plate test for tolerance to the analgesic effect of morphine was given at the end of all choice testing. All groups that had received morphine during place conditioning training were equally tolerant. These results indicate a dissociation between the analgesic effect of morphine and the effect that produces place preference, since the former was not affected by temporal parameters that did affect the latter. In the second experiment, the effect of extinction on a morphine-induced place preference was studied using extinction procedures that, in contrast to previous studies, equated exposure to both end compartments. Whereas the morphine-induced place preference was undiminished by a 10-day retention period in which animals received saline injections in the home cage, extinction trials during the same period eliminated the place preference. These results provide evidence that morphine-induced place preferences involve associative processes.     

Tacrolimus (FK506) reduces hippocampal damage but fails to prevent learning and memory deficits after transient, global cerebral ischemia in rats

Transient, global cerebral ischemia (TGCI) leads to hippocampal damage and disruption of spatial learning and memory. The immunosuppressant, tacrolimus (FK506), prevents TGCI-induced hippocampal neurodegeneration, but its effectiveness in promoting the recovery of learning and memory performance after TGCI has been little investigated. Here, we use a confined version of the aversive, non-food rewarded radial maze to evaluate further the effects of FK506 on TGCI-induced learning and memory deficits. In the first experiment, rats were rendered ischemic (15 min 4-VO) and 20 days later were tested for acquisition of the radial maze task over 15 consecutive days (post-operative training). In the second experiment, naive rats were trained for 10 days and subjected to TGCI (pre-operative training); retention of task performance was assessed on days 31, 35 and 39 post-ischemia. Acquisition and retention performances were expressed as a) latency to find a goal box, b) number of reference memory errors, and c) number of working memory errors. Data are presented both across daily training sessions (15 days, 3-day blocks) and as a total value (summed over the 15 days). Histological examination was performed on the day after behavioral testing. In both experiments, FK506 (1.0 mg/kg) was given i.v. at the beginning of reperfusion, followed by doses applied intraperitoneally (i.p.) 6, 24, 48 and 72 h post-ischemia. TGCI markedly disrupted both acquisition and retention performance (p<0.0001-0.05). Treatment with FK506 did not prevent the TGCI-induced acquisition and retention deficits, independently of whether performances were quantified 'daily' or as a 'total' value. In contrast, FK506 reduced hippocampal damage significantly compared to the vehicle alone (p<0.001-0.05). We conclude that the present study did not confirm our earlier behavioral data, and suggest that FK506 is not effective in treating the behavioral outcomes of TGCI, despite its efficacy in reducing CA1, hippocampal damage. However, further studies including other behavioral tasks and more extensive neurohistological analysis, are needed to better elucidate the effectiveness of FK506 in promoting functional recovery in models of transient, global cerebral ischemia.