Cleverson Rodrigues

Different hippocampal molecular requirements for short- and long-term retrieval of one-trial avoidance learning.

Rats were trained in one-trial step-down inhibitory avoidance and tested either 3 h or 31 days later. Ten minutes prior to the retention test, through indwelling cannulae placed in the CA1 region of the dorsal hippocampus, they received 0.5 microl infusions of: saline, a vehicle (2% dimethylsulfoxide in saline), the glutamate NMDA receptor blocker, aminophosphonopentanoic acid (AP5) (5.0 microg), the AMPA/kainate receptor blocker, cyanonitroquinoxaline dione (CNQX) (0.25 or 1.25 microg), the metabotropic receptor antagonist, methylcarboxyphenylglycine (MCPG) (0.5 or 2.5 microg), the inhibitor of calcium/calmodulin-dependent protein kinase II (KN62) (3.5 microg), the inhibitor of cAMP-dependent protein kinase (PKA), Rp-cAMPs (0.1 or 0.5 microg), the stimulant of the same enzyme, Sp-cAMPs (0.1 or 0.5 microg), or the inhibitor of the mitogen-activated protein kinase (MAPK) kinase, PD098059 (10 or 50 microM). CNQX, KN62 and PD098059 were dissolved in the vehicle; the other drugs were dissolved in saline. All these drugs, at the same doses, had been previously found to affect short- and long-term memory formation of this task. Retrieval measured 3 h after training (short-term memory) was blocked by CNQX and MCPG, and was unaffected by all the other drugs. In contrast, retrieval measured at 31 days was blocked by MCPG, Rp-cAMPs and PD098059, enhanced by Sp-cAMPs, and unaffected by CNQX, AP5 or KN62. The results indicate that, in CA1, glutamate metabotropic receptors are necessary for the retrieval of both short- and long-term memory; AMPA/kainate receptors are necessary for short-term but not long-term memory retrieval, and NMDA receptors are uninvolved in retrieval. Both the PKA and MAPK signalling pathways are required for the retrieval of long-term but not short-term memory.

Differential involvement of cortical receptor mechanisms in working, short-term and long-term memory

Rats received, through bilaterally implanted indwelling cannulae, 0.5 µl infusions of 6-cyano-7-nitroquinoxaline2,3-dione (CNQX) (0.5 µg), D-2-amino-5-phophono pentanoic acid (AP5) (5.0 µg), muscimol (0.5 µg), scopolamine (2.0 µg), SCH23390 (2.5 µg), saline or a vehicle into the CA1 region of the hippocampus, or into the antero-lateral prefrontal (PRE), posterior parietal (PP) and entorhinal cortex (EC). The infusions were given 6min prior to one-trial step-down inhibitory avoidance training in order to measure their effect on working memory (WM), or immediately post-training in order to measure their effect on short-term (STM) and long-term memory (LTM), 1.5 and 24 h later, respectively. WM was inhibited by CNQX or muscimol given into any of the cortical areas, by SCH23390 given into CA1, PRE or PP, and by scopolamine given into PRE or EC. STM was unaffected by any of the treatments given into PRE, and was inhibited by CNQX or muscimol given into CA1, PP and EC and by scopolamine given into PP, and enhanced by SCH given into CA1. LTM was inhibited by CNQX, muscimol, scopolamine or SCH23390 given into PRE, by scopolamine given into PP, by SCH23390 given into the entorhinal cortex, and by AP5, CNQX, muscimol or scopolamine given into CA1. The results indicate a differential involvement of the various neurotransmitter systems in the three types of memory in the various brain areas, and a separation of the mechanisms and of the regions involved in each. In addition, some of the findings suggested links between WM and LTM processing in PRE, between WM and STM processing in EC and PP, and between all three types of memory in CA1.

Pharmacological demonstration of the differential involvement of protein kinase C isoforms in short- and long-term memory formation and retrieval of one-trial avoidance in rats

Abstract Rationale: The hippocampal protein kinase C (PKC) family is involved in the early events of consolidation of long-term potentiation (LTP) and long-term memory (LTM). Results so far are indecisive about which PKC isoform is involved and as to whether any of them plays a role in short-term memory (STM) processes, which have recently been shown to be separate from those of LTM in the hippocampus-dependent one-trial step-down inhibitory avoidance task. Objectives: To measure the effect of two PKC inhibitors, one (Gö 6976) selective to the calcium-dependent isoforms α and βI, and the other (Gö 7874) unspecific as to PKC isoforms on the formation and retrieval of STM and LTM of one-trial inhibitory avoidance. Methods: Rats bilaterally implanted with cannulae in the CA1 region of the dorsal hippocampus were trained in one-trial step-down inhibitory avoidance. The effect of these two drugs on STM and LTM formation was investigated as follows. Animals were infused 10 min before or 50, 110, or 170 min after inhibitory avoidance training with a vehicle (2% dimethylsulfoxide in saline), or with Gö 6976 (0.92 nM or 4.6 nM) or Gö 7874 (1.96 nM or 8 nM) dissolved in the vehicle. Infusion volume was 0.5 µl in all cases. Animals were tested 1.5 h and 3 h after training for STM and at 24 h for LTM. In order to study the effects of these compounds on retrieval, they were infused into the hippocampus 10 min prior to STM testing at 3 h (see above) or 10 min before LTM testing at 24 h. In addition, the effect of Gö 6976 and Gö 7874 was studied on general activity measured in an open field, and on performance in an elevated plus maze. Results: STM was suppressed by 4.6 nM Gö 6976 given 10 min before or 50 min after training. LTM was cancelled by the higher dose of the two compounds given 10 min before, or 50 min or 110 min after training. None of the two compounds infused 170 min post-training affected the retrieval of STM measured 10 min later. However, both compounds given 10 min before testing inhibited the retrieval of LTM measured at 24 h. This effect cannot be attributed to influences on locomotor activity or anxiety levels, since the drugs had no effect on performance in the open field but were mildly ”anxiogenic” (pro-conflict) and reduced the number of entries into open and closed arms and rearings. Conclusions: LTM consolidation requires in part α- and/or βI-PKC and in part other PKC isoforms. STM formation requires instead only α and/or βI-PKC and during a more limited period of time. In addition, PKC appears not to be necessary for the retrieval of STM, but is crucial for the retrieval of LTM. These findings further point to a biochemical separation of STM and LTM, as ascertained in numerous previous studies.

Late and prolonged post-training memory modulation in entorhinal and parietal cortex by drugs acting on the cAMP/protein kinase A signalling pathway.

Rats implanted bilaterally with cannulae in the entorhinal or posterior parietal cortex or in the amygdaloid nucleus were trained in one-trial step-down inhibitory (passive) avoidance using a 0.3 mA footshock. At 0, 3, 6 or 9 h after training, they received localized 0.5 µl infusions into these areas of a vehicle, or of 8-Br-cAMP, forskolin (adenylyl cyclase activator), KT5720 (protein kinase A inhibitor), SKF38393 (dopamine D, receptor agonist), SCH23390 (Dt antagonist), norepinephrine hydrochloride, timolol hydrochloride (βblocker), 8-HO-DPAT (5-HT1A receptor agonist) or NAN-190 (5-HT1A antagonist) dissolved in 20% dimethylsulfoxide (DMSO) in saline (vehicle). Rats were tested for retention 24 h after training. 8-Br-cAMP, forskolin, SKF 38393 and norepinephrine caused memory facilitation and KT5720, SCH23390, timolol and 8-HO-DPAT caused retrograde amnesia when given into the entorhinal cortex 0,3 or 6 h but not 9 h after training. When given into the posterior parietal cortex 0, 3 or 6 but not 9 h after training, KT5720 was amnestic. When given into this structure 3 or 6 h but not 0 or 9 h after training 8-Br-cAMP, forskolin and norepinephrine caused memory facilitation and KT5720, SCH23390 and timolol caused retrograde amnesia. All treatments given into the amygdala 0,3 or 6 h after training were ineffective except for norepinephrine given at 0 h, which caused facilitation. The data point to a role of cAMP/protein kinase A-dependent mechanisms in memory formation in the entorhinal and parietal cortex, but not the amygdala, from 0 to 6 h after training, and to a strong modulation of these mechanisms by dopaminergic D1, β-noradrenergic and 5-HT1A receptors. The lack of effect of NAN-190 but not 8-HO-DPAT in both cortical regions suggests that 5-HT1A receptors do not play a physiological role but can be activated pharmacologically. The fact that SCH23390 was amnestic but SKF38393 had no effect when given into the parietal cortex suggests that D1 receptors may play a maintenance rather than a stimulant role in this area.

Novelty enhances retrieval: molecular mechanisms involved in rat hippocampus

Rats exposed to a novel environment just prior to or 1–2 h, but not 4 or 6 h, before retention testing exhibited an enhanced retrieval of a one‐trial inhibitory avoidance training. The bilateral intrahippocampal infusion of PD098059, an inhibitor of mitogen‐activated protein kinase (MAPK), the specific upstream activator of p42 and p44 MAPKs, given 10 min before the exposure to the novel environment, blocked the enhancing effect of novelty on memory retrieval. In addition, prenovelty infusion of dl‐2‐amino‐5‐phosphonovalerate (APV), an antagonist of glutamate NMDA receptors, produced similar effects. The exposure to the novel environment is associated with an activation of p42 and p44 MAPKs and an increase in the phosphorylation state of the transcription factor cAMP response element binding protein (CREB). No changes were observed in cAMP‐dependent protein kinase (PKA) activity or in α‐CAMKII activation. Taken together, our results indicate that novelty activates hippocampal MAPKs, which are necessary, along with glutamate NMDA receptors, for the enhancing effect of novelty on retrieval.

Differential Role of Hippocampal cAMP-Dependent Protein Kinase in Short- and Long-Term Memory

One-trial step-down inhibitory (passive) avoidance training is followed by two peaks of cAMP-dependent protein kinase (PKA) activity in rat CA1: one immediately after training and the other 3 h later. The second peak relies on the first: Immediate posttraining infusion into CA1 of the inhibitor of the regulatory subunit of PKA, Rp-cAMPS, at a dose that reduces PKA activity during less than 90 min, cancelled both peaks. Long-term memory (LTM) of this task measured at 24 h depends on the two peaks: Rp-cAMPS given into CA1 0 or 175 min posttraining, but not between those times, blocked LTM. However, the effect of immediate posttraining Rp-cAMPS on LTM could not be reversed by the activator of the regulatory subunit of PKA, Sp-cAMPS, given at 180 min, which suggests that, for LTM, the first peak may be more important than the second. When given at 0, 22, 45, or 90, but not at 175 min from training, Rp-cAMPS blocked short-term memory (STM) measured at 90 or 180 min. This effect of immediate posttraining Rp-cAMPS infusion on STM but not that on LTM was readily reversed by Sp-cAMPS infused 22 min later. On its own, Sp-cAMPS had effects exactly opposite to those of the inhibitor. It enhanced LTM when given at 0 or 175 min from training, and it enhanced STM when given at 0, 22, 45, or 90 min from training. These findings show that STM and LTM formation require separate PKA-dependent processes in CA1. STM relies on the continued activity of the enzyme during the first 90 min. LTM relies on the two peaks of PKA activity that occur immediately and 180 min posttraining.

Pharmacological differences between memory consolidation of habituation to an open field and inhibitory avoidance learning.

Rats implanted bilaterally with cannulae in the CA1 region of the dorsal hippocampus or the entorhinal cortex were submitted to either a one-trial inhibitory avoidance task, or to 5 min of habituation to an open field. Immediately after training, they received intrahippocampal or intraentorhinal 0.5-microl infusions of saline, of a vehicle (2% dimethylsulfoxide in saline), of the glutamatergic N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphono pentanoic acid (AP5), of the protein kinase A inhibitor Rp-cAMPs (0.5 microg/side), of the calcium-calmodulin protein kinase II inhibitor KN-62, of the dopaminergic D1 antagonist SCH23390, or of the mitogen-activated protein kinase kinase inhibitor PD098059. Animals were tested in each task 24 h after training. Intrahippocampal KN-62 was amnestic for habituation; none of the other treatments had any effect on the retention of this task. In contrast, all of them strongly affected memory of the avoidance task. Intrahippocampal Rp-cAMPs, KN-62 and AP5, and intraentorhinal Rp-cAMPs, KN-62, PD098059 and SCH23390 caused retrograde amnesia. In view of the known actions of the treatments used, the present findings point to important biochemical differences in memory consolidation processes of the two tasks.

Involvement of the medial precentral prefrontal cortex in memory consolidation for inhibitory avoidance learning in rats.

Adult male Wistar rats were trained in a step-down inhibitory avoidance learning task (3.0-s, 0.4-mA foot shock), received a 0.5-microl infusion of muscimol (0.02, 0.1, or 0.5 microg), AP5 (0.16, 0.34, 0. 5, 1.6, or 5.0 microg), SCH 23390 (0.05, 0.34, 0.5, or 1.75 microg), saline, or vehicle (DMSO 20%) into the anterior medial precentral area (Fr2) (CI) immediately after training, and were tested 24 h later. Muscimol (0.02, 0.1, or 0.5 microg), AP5 (0.34 or 0.5 microg), or SCH (0.5 or 1.75 microg) were amnesic. Then, animals were infused with muscimol (0.1 or 0.5 microg), AP5 (0.34, 0.5, or 5.0 microg), or SCH (0.5 microg) at other posttraining times and/or into the junction of Fr1-Fr2 (CII). Muscimol (0.1 and 0.5 microg) or SCH into CI were amnesic when given 90 or 180 min after training, but not when given 270 min after training. Muscimol (0.5 microg, but not 0.1 microg) or SCH into CII were amnesic when given 90 min after training, but not when given 0 or 180 min after training. AP5 (0.5, but not 5.0 microg) was amnesic when given into CI, but not into CII, at 0 or 180 min posttraining, and a trend toward an amnesic effect was seen at 90 min posttraining. The results suggest that 1) the glutamatergic, GABAergic, and dopaminergic systems in Fr2 are involved in the consolidation of memory for inhibitory avoidance learning, either directly or as parts of modulatory systems; and 2) timing of involvement of anterior Fr2 (CI) is different from that of posterior Fr2 (CII).

NMDA receptor antagonism in the basolateral amygdala blocks enhancement of inhibitory avoidance learning in previously trained rats

Extensive evidence suggests that N-methyl-D-aspartate (NMDA) glutamate receptor channels in the amygdala are involved in fear-motivated learning, and infusion of NMDA receptor antagonists into the amygdala blocks memory of fear-motivated tasks. Recent studies have shown that previous training can prevent the amnestic effects of NMDA receptor antagonists on spatial learning. In the present study, we evaluated whether infusion of the NMDA antagonist D,L-2-amino-5-phosphonopentanoic acid (AP5) into the basolateral nucleus of the amygdala (BLA) impairs reinforcement of inhibitory avoidance learning in rats given previous training. Adult male Wistar rats (220-310 g) were bilaterally implanted under thionembutal anesthesia (30 mg/kg, i.p.) with 9.0-mm guide cannulae aimed 1.0 mm above the BLA. Infusion of AP5 (5.0 microg) 10 min prior to training in a step-down inhibitory avoidance task (0.4 mA footshock) blocked retention measured 24 h after training. When infused 10 min prior to a second training session in animals given previous training (0.2 mA footshock), AP5 blocked the enhancement of retention induced by the second training. Control experiments showed that the effects were not due to alterations in motor activity or footshock sensitivity. The results suggest that NMDA receptors in the basolateral amygdala are involved in both formation of memory for inhibitory avoidance and enhancement of retention in rats given previous training.

Involvement of the serotonergic type 1A (5-HT1A) receptor in the agranular insular cortex in the consolidation of memory for inhibitory avoidance in rats.

Adult male Wistar rats were bilaterally implanted with indwelling cannulae in the agranular insular cortex of the prefrontal cortex. After recovery, animals were trained in a step‐down inhibitory avoidance task (3.0‐s, 0.4‐mA footshock) and received, immediately after training, a 0.5‐μl infusion of the serotonergic type 1A (5‐HT 1A ) receptor agonist dipropylamino‐8‐hydroxy‐1,2,3,4‐tetrahydronaphthalene hydrobromide (8‐OH‐DPAT) or of the 5‐ HT 1A receptor antagonist 1‐(2‐methoxyphenyl)‐4‐[4‐(2‐phthalimido)butyl] piperazine hydrobromide (NAN‐190), or of vehicle alone (20% DMSO). Retention testing was carried out 24 h after training. 8‐OH‐DPAT (1.25 and 6.25 μg but not 0.0125 or 0.125 μg) was amnesic. NAN‐190 was not effective at 0.125 or 1.25 μg any dose but reversed amnesia when given at 1.250 μg simultaneously with both effective doses of 8‐OH‐DPAT. These results show that an overactivation of 5‐HT 1A receptors in the agranular insular cortex impairs memory consolidation of inhibitory avoidance, in rats, immediately after training. This suggests that these receptors of the insular cortex may modulate memory consolidation.