Carlos M. Baratti

Memory facilitation with posttrial injection of oxotremorine and physostigmine in mice.

The immediate posttrial injection of oxotremorine (0.125, 0.250 and 0.500 μMol/kg i.p.) and equimolecular doses of physostigmine can facilitate the retention of a passive avoidance response in mice. Injections given 10 min after training also significantly facilitate retention, but injections given 30 or 120 min after training do not affect retention. These findings suggest an action of oxotremorine and physostigmine on mechanisms involved in memory storage. The enhanced retention produced by oxotremorine and physostigmine was blocked by pretreatment with atropine (2 μMol/kg, 20 min, i.p.) but was not affected by methylatropine (2 μMol/kg, 20min, i.p.). The retention was not modified by posttrial injection of metoxotremorine (0.250 μMol/kg i.p.) or neostigmine (0.250 μMol/kg i.p., quaternary analogs of oxotremorine and physostigmine, respectively. The results suggest a central action of both cholinergic agents attributable to an activation of muscarinic brain receptors.

NF-κB transcription factor is required for inhibitory avoidance long-term memory in mice

Although it is generally accepted that memory consolidation requires regulation of gene expression, only a few transcription factors (TFs) have been clearly demonstrated to be specifically involved in this process. Increasing research data point to the participation of the Rel/nuclear factor‐κB (NF‐κB) family of TFs in memory and neural plasticity. Here we found that two independent inhibitors of NF‐κB induced memory impairment in the one‐trial step‐through inhibitory avoidance paradigm in mice: post‐training administration of the drug sulfasalazine and 2 h pretraining administration of a double‐stranded DNA oligonucleotide containing the NF‐κB consensus sequence (κB decoy). Conversely, one base mutation of the κB decoy (mut‐κB decoy) injection did not affect long‐term memory. Accordingly, the κB decoy inhibited NF‐κB in hippocampus 2 h after injection but no inhibition was found with mut‐κB decoy administration. A temporal course of hippocampal NF‐κB activity after training was determined. Unexpectedly, an inhibition of NF‐κB was found 15 min after training in shocked and unshocked groups when compared with the naïve group. Hippocampal NF‐κB was activated 45 min after training in both shocked and unshocked groups, decreasing 1 h after training and returning to basal levels 2 and 4 h after training. On the basis of the latter results, we propose that activation of NF‐κB in hippocampus is part of the molecular mechanism involved in the storage of contextual features that constitute the conditioned stimulus representation. The results presented here provide the first evidence to support NF‐κB activity being regulated in hippocampus during consolidation, stressing the role of this TF as a conserved molecular mechanism for memory storage.

Possible interaction between central cholinergic muscarinic and opioid peptidergic systems during memory consolidation in mice.

Naloxone (0.01-1.00 mg/kg, ip) facilitated retention of a one-trial inhibitory avoidance task, when administered to male Rockland mice immediately after training, as indicated by performance on a retention test 48 hr later. The dose-response curve was an inverted U in this range of dose. In these conditions naloxone did not lengthen latencies to step-through during the retest of unshocked mice. Higher doses of naloxone (3.00 and 10.00 mg/kg, ip) tended to increase latencies to step-through of both shocked and unshocked mice. These facts rule out an aversive effect of naloxone for low and moderate doses but not for high doses. The influence of naloxone (0.10 mg/kg, ip) on retention was time dependent, which suggests that naloxone facilitated memory consolidation processes. The effects of naloxone were prevented by morphine in both an amnesic and a nonamnesic dose (1.0 and 0.5 mg/kg, ip, respectively). Therefore, naloxone probably facilitated retention as a function of its opiate antagonist properties. The memory facilitation induced by naloxone (0.10 mg/kg, ip) was antagonized by atropine (0.5 mg/kg, ip) but not by methylatropine (0.5 mg/kg, ip), mecamilamine (5 mg/kg, ip), or hexametonium (5 mg/kg, ip). Further, there was a mutual potentiation for both naloxone (0.01 mg/kg, ip) and the muscarinic agonist oxotremorine (6.25 and 12.5 micrograms/kg, ip) administered simultaneously, in doses which had no effect on their own. Moreover, an amnesic dose of atropine (10.00 mg/kg, ip) prevented the enhancement of retention induced by naloxone, while an amnesic dose of morphine (1.00 mg/kg, ip) did not modify the facilitatory effect of oxotremorine (50 micrograms/kg, ip) on retention. An inhibitory modulatory role for endogenous opioid systems on the activity of central cholinergic muscarinic systems during memory consolidation is suggested.

Activation of Hippocampal Nuclear Factor-κB by Retrieval Is Required for Memory Reconsolidation

Initially, memory is labile and requires consolidation to become stable. However, several studies support that consolidated memories can undergo a new period of lability after retrieval. The mechanistic differences of this process, termed reconsolidation, with the consolidation process are under debate, including the participation of hippocampus. Up to this point, few reports describe molecular changes and, in particular, transcription factor (TF) involvement in memory restabilization. Increasing evidence supports the participation of the TF nuclear factor-κB (NF-κB) in memory consolidation. Here, we demonstrate that the inhibition of NF-κB after memory reactivation impairs retention of a hippocampal-dependent inhibitory avoidance task in mice. We used two independent disruptive strategies to reach this conclusion. First, we administered intracerebroventricular or intrahippocampal sulfasalazine, an inhibitor of IKK (IκB kinase), the kinase that activates NF-κB. Second, we infused intracerebroventricular or intrahippocampal κB decoy, a direct inhibitor of NF-κB consisting of a double-stranded DNA oligonucleotide that contains the κB consensus sequence. When injected immediately after memory retrieval, sulfasalazine or κB decoy (Decoy) impaired long-term retention. In contrast, a one base mutated κB decoy (mDecoy) had no effect. Furthermore, we also found NF-κB activation in the hippocampus, with a peak 15 min after memory retrieval. This activation was earlier than that found during consolidation. Together, these results indicate that NF-κB is an important transcriptional regulator in memory consolidation and reconsolidation in hippocampus, although the temporal kinetics of activation differs between the two processes.

Memory-improving actions of glucose: Involvement of a central cholinergic muscarinic mechanism

Post-training intraperitoneal administration of alpha-D[+]-glucose (10-300 mg/kg) facilitated 24-h retention, in male Swiss mice, of a one-trial step-through inhibitory avoidance task. The dose-response curve was an inverted U. Glucose did not increase the retention latencies of mice that had not received a footshock during training. The effect of glucose (30 mg/kg, ip) on retention was time-dependent, which suggests that the drug facilitated memory storage. The memory facilitation induced by glucose (30 mg/kg, ip) was prevented by atropine (0.5 mg/kg, ip) administered after training, but 10 min prior to glucose treatment. In contrast, neither methylatropine (0.5 mg/kg, ip), a peripherally acting muscarinic receptor blocker, nor mecamylamine (5 mg/kg, ip) or hexamethonium (5 mg/kg, ip), two cholinergic nicotinic receptor antagonists, prevented the effects of post-training glucose on retention. Low subeffective doses of the central acting anticholinesterase physostigmine (35 micrograms/kg, ip), administered immediately after training, and glucose (10 mg/kg, ip), given 10 min after training, acted synergistically to improve retention. The effects of glucose (10 mg/kg, ip) were not influenced by the peripherally acting anticholinesterase neostigmine (35 micrograms/kg, ip). Considered together, these findings suggest that the memory facilitation induced by post-training administration of glucose could result from an enhancement of brain acetylcholine synthesis and/or its release that, in turn, might modulate the activity of muscarinic cholinergic mechanisms that are critically involved in memory storage.

Memory consolidation and reconsolidation of an inhibitory avoidance response in mice: effects of i.c.v. injections of hemicholinium-3

The immediate post-training i.c.v. administration of hemicholinium-3 (HC-3) (1 microg), a specific inhibitor of the high-affinity choline uptake (HACU) in brain cholinergic neurons, impaired retention test performance of a one-trial step-through inhibitory avoidance response in adult male CF-1 mice. The effect was observed in mice that received a footshock (0.8 mA, 50 Hz, 1 s) on the learning trial, and not only 48 h after training, but also 7 days after it. After the completion of the retention test at each of the training-test interval that were studied, the HACU in the hippocampus of HC-3-treated mice was not significantly different from that of saline-injected (1 microl) control groups. Mice that were over-reinforced (1.2 mA, 50 Hz, 1 s) on the learning trial, exhibited a high retention performance 48 h after training. The immediate i.c.v. injection of HC-3 (1 microg) after the retention test, that is, after memory reactivation, significantly impaired retention performance over 4 consecutive days, whereas the saline-injected control group shown a slight, but significant performance decrease only at the last retention test. Retention performance was unchanged in HC-3-treated mice not undergoing memory reactivation session. These results, taken together, indicate that HC-3, not only impaired consolidation, but also reconsolidation of an inhibitory avoidance task in mice, suggesting a critical participation of central cholinergic mechanisms in both memory processes.

The impairment of retention induced by β-endorphin in mice may be mediated by a reduction of central cholinergic activity

beta-Endorphin (0.03 to 1.00 microgram/kg, ip) impaired retention of a one-trial inhibitory avoidance task in a dose-dependent manner when injected into male Swiss mice immediately post-training, as indicated by retention performance 48 h later. The doses of 0.03 and 0.10 microgram/kg significantly impaired retention while the two higher doses (0.30 and 1.00 microgram/kg) did not significantly affect retention as compared with the control group, but tended to increase retention as compared with the dose of 0.10 microgram/kg. Thus, the dose-response curve shows an U-shaped form. The simultaneous injection of naloxone (0.1 mg/kg, ip) not only shifted the dose-response curve to the right but also prevented the tendency to increase retention latencies of the two higher doses. The two lower doses of beta-endorphin did not lengthen latencies to step-through of mice that had not received a footshock during the training while, under these conditions, the two higher doses of the peptide significantly increased latencies to step-through. This effect was prevented by naloxone (0.1 mg/kg). Taken together these results suggest that the effects of beta-endorphin on retention are the consequence of an interaction with opioid receptors and indicate that the right ascending arm of the dose-response curve would probably be due to a punitive effect of beta-endorphin which was also prevented by naloxone. The impairing effect of post-training administration of beta-endorphin (0.10 microgram/kg) on memory was time-dependent, since it was decreased as the training-treatment interval was increased. These results rule out a pharmacological proactive effect of beta-endorphin on retention performance and suggest that beta-endorphin affects memory consolidation. The simultaneous administration of beta-endorphin (0.10 microgram/kg) with the central muscarinic agonist oxotremorine (12.5 or 50.0 micrograms/kg) completely prevented the impairment of retention induced by beta-endorphin, while the simultaneous administration of the central-acting anticholinesterase physostigmine (17 or 68 micrograms/kg) only partially but significantly attenuated the effect of beta-endorphin on retention. Further, the peripheral-acting anticholinesterase neostigmine (68 micrograms/kg) and the nicotinic blocker hexamethonium (5 mg/kg) modified neither retention nor the behavioral effects of beta-endorphin. These results suggest that the impairment of retention induced by beta-endorphin is probably due to an inhibition of acetylcholine release at central cholinergic synapses which are critical for memory formation.

Opioid peptidergic systems modulate the activity of β-adrenergic mechanisms during memory consolidation processes

Post-training administration of the opioid receptor antagonist naloxone (0.1 mg/kg) facilitated 48-hr retention, in mice, of a one-trial step-through inhibitory avoidance response. The naloxone-induced memory facilitation was blocked in animals given the selective brain-noradrenergic neurotoxin DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) (50.0 mg/kg, ip) 7 days before training. Pretreatment with the norepinephrine-uptake inhibitor desmethylimipramine (10.0 mg/kg, ip, 30 min), but not with the serotonin-uptake inhibitor fluoxetine (5.0 mg/kg, ip, 30 min), prevented this antagonism. The simultaneous administration of the central beta-adrenoceptor blocker l-propranolol (2.0 mg/kg, ip), also blocked the effects of naloxone on memory. The effects of naloxone were not blocked by d-propranolol (2.0 mg/kg, ip), the peripheral beta-adrenoceptor blocker sotalol (2.0 mg/kg, ip), the alpha-adrenoceptor blocker phenoxybenzamine (10.0 mg/kg, ip), or the predominantly peripheral alpha-adrenoceptor blocker phentolamine (10.0 mg/kg, ip). These findings suggest that central beta-adrenergic mechanisms are involved in the effects of naloxone on memory. Naloxone (0.1 mg/kg, ip) potentiated the effects of the central beta-adrenoceptor agonist clenbuterol (0.001-1.00 mg/kg, ip), which, when administered alone, facilitates or impairs retention as a function of the dose injected. The simultaneous administration of beta-endorphin (0.1 micrograms/kg, ip) exerted effects opposite to those elicited by naloxone, that is, shifted the dose-response curve of clenbuterol to the right. Considered together, these findings are consistent with the view that the facilitatory action of naloxone on memory results from the release of central beta-adrenergic mechanisms from an inhibition induced by opioid peptides released during or immediately after training.

Dynorphin induces task-specific impairment of memory

Immediate posttraining administration of the opioid peptide dynorphin(1–13) (0.1, 0.3, and 1.0 μg/kg i.p.) significantly impaired 24-h retention of a one-trial inhibitory avoidance task in mice. In contrast, posttraining dynorphin did not modify retention of either a Y-maze discrimination (0.1, 1.0, or 10.0 μg/kg i.p.) or habituation of exploration (0.1, 0.3, 1.0, or 2.0 μg/kg i.p.). The administration of dynorphin (0.1, 1.0, and 10.0 μg/kg i.p.) 2 min prior to the inhibitory avoidance retention test did not modify retention latencies of mice injected with either saline or dynorphin (0.1 μg/kg i.p.) immediately after training. In mice, dynorphin appears to impair retention by interfering with memory storage processes, and this effect seems to be task specific.

Memory consolidation and reconsolidation of an inhibitory avoidance task in mice: Effects of a new different learning task

CF-1 male mice were trained in an inhibitory avoidance task using a high footshock (1.2mA, 50Hz, 1 s) in order to reduce the influence of extinction on retention performance. A single session of 5 min exposure to a hole-board (nose-poke behavior), either immediately after training or the first retention test (memory reactivation) impaired retention performance over two consecutive days. The effects were time-dependent since they were not observed when the exposure to the hole-board was delayed 3 h. When mice were habituated to the hole-board (5 min/day, 5 days), and then trained in an inhibitory avoidance task, the immediately post-training or memory reactivation exposure to the hole-board did not modify retention performance over two consecutive days. The effects of the post-reactivation acute exposure to the hole-board were long-lasting (21 days). Reinstatement was not observed in our experimental conditions. The non-spontaneous recovery of retention performance over 21-days and the lack of reinstatement, suggest that the impairment of retention performance observed was not probably due to a deficit in memory retrieval. These findings suggest that the exposure to a potential new learning situation impairs not only memory consolidation but also memory reconsolidation of the original learning task.