Gabriela B. Acosta

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.

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 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.

Area-dependent changes in GABAergic function after acute and chronic cold stress.

(1) The function of the gamma-aminobutyric acid (GABA)ergic system in certain areas of the rat brain was investigated after acute and chronic cold stress. (2) GABA concentration, [3H]GABA uptake and the activity of the synthesis enzyme glutamate decarboxylase (GAD) were measured. (3) Acute stress: (a) reduced GABA concentration in the corpus striatum (29%); (b) decreased GAD activity (under non-saturating substrate concentration) in the olfactory bulbs (24%); (c) diminished neuronal uptake of [3H]GABA in the frontal cerebral cortex (65%), hypothalamus (86%) and olfactory bulbs (82%). (4) Chronic stress: (a) reduced the endogenous levels of GABA in the frontal cerebral cortex (51%), hypothalamus (26%) and olfactory bulbs (15%); (b) decreased GAD activity in the corpus striatum (32%) and olfactory bulbs (34%); (c) decreased neuronal uptake of [3H]GABA in the hypothalamus (83%). (5) These findings suggest that compensatory changes may develop in the GABAergic system after chronic stress.

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.

GABAA receptors mediate the changes produced by stress on GABA function and locomotor activity

(1) This study shows the effects of bicuculline pretreatment on the GABAergic system in certain areas of the rat brain after acute ether and cold stress. (2) The spontaneous locomotor activity was diminished by either ether stress or cold stress or bicuculline. (3) Acute ether stress enhanced GABA concentration in the hypothalamus (69%) and in the frontal cerebral cortex (26%). Bicuculline prevented the increase in GABA levels induced by ether stress in both areas. (4) Acute cold stress decreased GABA concentration in the corpus striatum (29%). Bicuculline prevented the decrease in GABA levels induced by cold stress. (5) It is concluded that there is a GABAA receptor involved in stress induced changes on endogenous GABA levels as well as on locomotor activity.

Diazepam fails to potentiate GABA-induced chloride uptake and to produce anxiolytic-like action in aged rats.

The pharmacological response to benzodiazepines has been demonstrated to be different in aged individuals in comparison to adults. We studied the age-dependent changes in some of the in vitro and behavioral effects of diazepam in aged (24 months old) rats, comparing them to adults (3 months old). We evaluated the in vitro gamma-aminobutyric acid (GABA)-induced 36Cl- uptake and the diazepam potentiation of GABA-stimulated 36Cl- uptake in microsacs from cerebral cortex of both groups of animals. We found no differences in the GABA-stimulated 36Cl- uptake between adult and aged animals, and diazepam failed to potentiate GABA-induced 36Cl- flux in the aged cortical microsacs. We also examined the effect of 0.03-10 mg of diazepam on locomotor activity in an open-field test and the anxiolytic-like action of diazepam in doses ranging from 0.03 to 1 in a dark-light transition test. We observed no anxiolytic-like action of the drug in the dark-light transition test in the aged rats, while there was a shift to the left in the diminution of locomotor activity evaluated by the open-field test. We conclude that the pharmacodynamic changes observed in cortical GABA(A) receptors in aged rats could partially explain the lack of anxiolytic-like action but not the oversedation evidenced in this group of animals.

Atropine, an anticholinergic drug, impairs memory retrieval of a high consolidated avoidance response in mice

Immediate post-training intraperitoneal administration of the centrally acting anticholinesterase physostigmine (70.0, or 150.0 microg/kg) enhanced retention of male CF-1 mice tested 48 h after training in a one-trial step-through inhibitory avoidance task (0.8 mA, 50 Hz, 1 s footshock). The effect was observed in mice that received saline 30 min before the retention test; on the contrary, the pre-test administration of the centrally active muscarinic cholinergic antagonist, atropine (1.0 mg/kg, i.p.), but not methylatropine (1.0 mg/kg, i.p.), instead of saline, prevents the enhancement of retention induced by both doses of the anticholinesterase when given immediately after training. The high retention performance caused by post-training physostigmine was recovered following a second administration of the same doses of the drug, 10 min after the pre-test injections of atropine. Since, physostigmine do not influence memory retrieval when given prior to the retention test, and its post-training effects are not due to the induction of state-dependency, the recover of the high retention performance was probably due to a classical interaction between a muscarinic competitive antagonist and an indirect cholinergic agonist. Further, atropine probably does not modify the memory trace by erasing it, but by producing a poor retrieval.

Reactivated memory of an inhibitory avoidance response in mice is sensitive to a nitric oxide synthase inhibitor.

It is accepted that once consolidation is completed memory becomes permanent. However, it has also been suggested that reactivation (retrieval) of the original memory, again, makes it sensitive to the same treatments that affect memory consolidation when given after training. Previous results demonstrated that the immediate post-training intraperitoneal administration of N(omega)-nitro-l-arginine methyl ester (L-NAME), a non-specific inhibitor of nitric oxide synthase (NOS), impairs retention test performance of a one-trial step-through inhibitory avoidance response in adult mice. The effect of L-NAME on retention was attributed to an action on memory consolidation of the original learning. For the first time, we report that the administration of L-NAME after the first retention test (memory reactivation) of the inhibitory avoidance response impairs retention performance over six consecutive days. This impairment effect is dose-and-time dependent and could not be attributed to a retrieval deficit since a mild footshock did not reinstate the original avoidance response and no spontaneous recovery was observed at least 21 days after training. Further support for a storage deficit interpretation as opposed to a retrieval deficit was obtained from the fact that L-NAMEs effects after retrieval were not due to state-dependency. The impairment effect of L-NAME was dependent on the age of the original memory. That is, there was an inverse correlation between the susceptibility of the memory trace when reactivated and the time elapsed between training and the first retrieval session. We suggest an action of L-NAME on memory reactivation-induced processes that are different from memory extinction of the original learning extending the biological significance of nitric oxide on memory.

Tolerance to the sedative effect of lorazepam correlates with a diminution in cortical release and affinity for glutamate.

Benzodiazepines are anxiolytic, anticonvulsant, sedative and hypnotic compounds usually prescribed on a long-term basis. Chronic treatment with these compounds induces tolerance, which has been extensively attributed to modifications in the GABAergic neurotransmission. However, a compensatory increase in the excitatory response, named as an oppositional response, has also been put forward as a means for explaining such tolerance. Changes in the excitatory neurotransmission have been found in withdrawn rats after a long treatment with benzodiazepines but these modifications have not been conclusively studied during tolerance. In this work we studied several parameters of the glutamatergic neurotransmission in rats made tolerant to the sedative effect of 3 mg/kg (i.p.) of lorazepam (LZ). We found a decrease in the affinity of cortical NMDA receptors for (3)H-glutamate (K(D): 124.4 +/- 13.3 nM in tolerant rats, 71.6 +/- 10.4 nM in controls, P<0.05) together with a decrease in the in vitro 60 mM K(+)-stimulated cortical glutamate release (59+/- 12% vs. 153 +/- 38%, tolerant rats vs. controls, P<0.05). We conclude that tolerance to the sedative effect of LZ correlates with a decreased sensitivity for glutamate that may in turn diminish the cortical response to a chemical stimulus. Our findings constitute an evidence against the oppositional model of pharmacodynamic tolerance in this experimental condition.