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Cortistatin modulates memory processes in rats
Brain Research 858 Ž2000. 78–83 www.elsevier.comrlocaterbres
Research report
Cortistatin modulates memory processes in rats a a Manuel Sanchez-Alavez , Margarita Gomez-Chavarın , ´ ´ ´ a , Luz Navarro a , Anabel Jimenez-Anguiano ´ a a a,b Eric Murillo-Rodrıguez , Roberto A. Prado-Alcala´ , Rene Drucker-Colin , ´ Oscar Prospero-Garcıa ´ ´ a, ) a
Grupo de Neurociencias, Depto. de Fisiologıa, de Mexico, Apdo. Postal 70-250, Mexico, D.F. ´ Fac. de Medicina, UniÕersidad Nacional Autonoma ´ ´ 04510, Mexico b Depto. de Neurociencias, IFC, UniÕersidad Nacional Autonoma de Mexico, Apdo. Postal 70-250, Mexico, D.F. 04510, Mexico ´ ´ Accepted 9 November 1999
Abstract Cortistatin ŽCST. is a recently described neuropeptide with high structural homology with somatostatin. Its mRNA is restricted to gamma amino butyric acid ŽGABA.-containing cells in the cerebral cortex and hippocampus. CST modulates the electrophysiology of the hippocampus and cerebral cortex of rats; hence, it may be modulating mnemonic processes. In this study, we have evaluated the effect of CST and somatostatin ŽSS. on short- and long-term memory ŽSTM and LTM, respectively., as well as on the extinction of the behavior by using the footshock passive avoidance behavioral test. In addition, we tested the ability of both neuropeptides to affect the generation of cAMP in hippocampal neurons in culture. Results showed that the administration of either CST or SS into the hippocampal CA1 deteriorates memory consolidation in a dose–response fashion and facilitates the extinction of the learned behavior. CST was more potent than SS. Likewise, CST increases cAMP while SS decreases it. These results strongly support a modulatory role for CST in memory processes. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Somatostatin; Short-term memory; Long-term memory; Memory extinction; Passive avoidance test; cAMP
1. Introduction Memory is a series of cognitive processes whose neurochemical control is highly complex w26x. For example, in the hippocampus, a cerebral structure crucially involved in the memory network w25,28x, at least three neurotransmitters are regulating its activity, i.e., glutamate ŽGlu. w15,21x, acetylcholine ŽACh. w11x and gamma amino butyric acid ŽGABA. w2,27x. Glu and ACh enhance hippocampal excitability and exert a facilitating effect on memory w11,24x; whereas GABA depresses hippocampal excitability w27x and facilitates memory extinction w2,6x. In addition, a series of neuropeptides are also involved in this regulation, i.e., somatostatin ŽSS. and vasoactive intestinal polypeptide ŽVIP.. Both SS and VIP seem to modulate cholinergic activity, and memory processes w10,16,17x. On the other hand, cAMP has been associated with learning and memory in several animal preparations w19x, and its disregulation causes learning and memory deterioration. ) C orresponding author. [email protected]
Fax:
q52-5-623-2241;
e-m ail:
Cortistatin ŽCST. is a neuropeptide recently described in rat w7x and human brains w8,14x. Its mRNA was detected in the brain of rats but not in other organs such as liver or testis w7x. This peptide exhibits an exclusive cortical and hippocampal distribution. Due to the fact that CST mRNA cohybridizes with GAD67 mRNA in the hippocampus, we believe it is synthesized, stored and released by GABAergic cells w9x. CST’s active form is conformed by 14 amino acids Žaa.. Its aa sequence shows a high structural homology with SS-14. Both molecules share 11 aa. CST binds all five SS receptors with high affinity ŽpM range. w23x; therefore, we expect it to induce effects similar to those produced by SS. Accordingly, it produces similar hyperpolarizing effects as SS on hippocampal pyramidal cells in in vitro preparations; however, this effect lasts longer w7x. In contrast, there are several other effects caused by CST that seem to go in an opposite direction. For example, CST reduces the ACh excitatory action in the hippocampus while SS facilitates it w7x. In this context, CST seems to be a neuropeptide with potential anticholinergic antagonizing effects. This CST effect may be mediated by SS receptors, or, alternatively, by its own receptors Žto be described. or
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 3 6 - 7
M. Sanchez-AlaÕez et al.r Brain Research 858 (2000) 78–83 ´
by facilitating GABA activity. In addition, a recent report has indicated that CST may be inhibiting locus coeruleus ŽLC. neurons, which are mainly noradrenergic cells, by facilitating Kq conductance w5x. Consistent with CST’s inhibitory effects on hippocampal activity, a recently published study has suggested that CST may be regulating memory processes in mice w12x. In this context, the goal of this study is to assess if CST actually plays a role in memory regulation. In order to reach this goal, we decided to investigate whether or not CST administration directly into the hippocampus affects short- and long-term memory ŽSTM and LTM, respectively. as well as the process of extinction, evaluated by a footshock passive avoidance test and additionally if CST affects cAMP production in hippocampal cells.
2. Methods 2.1. Animals Male Wistar rats Ž250–350 g. were used in this study. All rats were implanted with a pair of guide cannulae aimed bilaterally to the hippocampal CA1 regions ŽP s 4.0, L s 2.5, V s 2.0.. Surgeries were performed under halothane anesthesia Ž2–3%.. Rats were individually housed 2 days before surgery and throughout the experiment. They were maintained under a controlled light–dark cycle Ž12:12, lights on at 0800 h., with food and water ad libitum. Antibiotics and analgesics were administered immediately after the surgery and during the following 2 days. 2.2. Training One week after surgery, rats were trained in the footshock passive avoidance behavioral test, in order to evaluate STM and LTM. The general method was as follows: the training apparatus was a trough-shaped alley Ž94 cm long, 25 cm wide and 30 cm deep. that was separated into two compartments by a guillotine door that retracted into the floor. The starting compartment Ž32 cm long. was illuminated by a 10-W lamp and the floor of this compartment was a grid made of aluminum bars; while the shock compartment Ž62 cm. was dark and V-shaped stainless-steel plates formed the floor and walls. This apparatus is equipped with 10 photocells distributed as follows: four in the starting compartment and six in the shock compartment. The apparatus is controlled by a PC computer and is placed in a sound-attenuated, non-illuminated room. Photocells relay information to the computer about the rat’s position within the chamber. The experimenter can view the rat directly as well as on-line through a cartoon representing the rat, displayed on the computer screen. Once the rat was placed in the starting compartment, the rat explored for 10 s, then the guillotine door was opened.
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Immediately after the rat entered the shock compartment with all four paws, the door was closed and a footshock Ž0.8 mA. was delivered through the stainless-steel plates. Five seconds after the initiation of the footshock delivery, the door was opened and the rat was allowed to escape. Rats stayed in the starting compartment for 30 s, then removed and a microinjector was inserted through the guide cannulae into the CA1 region. With the aid of an injection pump, one of the following compounds was injected: vehicle Žsaline., CST, or SS ŽRBI.. 2.3. Test Retention of the inhibitory avoidance training was tested 30 min and 24 h after training. Each rat was placed in the starting compartment and 30 s later, the door was opened. The latency to enter into the shock compartment with all four paws was recorded. Shock was not delivered. The rat was left in the starting compartment with the door open for a maximum of 600 s. 2.4. STM In order to evaluated the effect of SS and CST on STM, three groups Ž10 rats each group. were trained in the passive avoidance test. Immediately after training, rats of each group received either: the vehicle Ž1 ml., CST Ž100 ng., or SS Ž100 ng.. All rats were tested 30 min after trainingq administration. 2.5. LTM Nine groups of rats Ž n s 10 each group. were trained as described and then injected with either: the vehicle, CST Ž10, 50, 100, 1000 ng. or SS Ž10, 50, 100, 1000 ng.. Rats were tested 24 h later to evaluate memory retention. They were also tested at 48, 72, 96 and 120 h after trainingq administration to evaluate memory extinction. 2.6. Extinction of the behaÕior The next part of this study was performed in three groups of rats Ž10 rats each group.. Animals were trained and 24 h later tested. All of them exhibited the avoidance behavior, then they were tested until they showed extinction of the behavior, which took place by the fifth day after training. The sixth day, rats were injected with either: the vehicle, CST Ž100 ng. or SS Ž100 ng.. Half an hour, 24 and 48 h after the injection rats were tested. 2.7. cAMP production In the last part of this study, we evaluated the generation of cAMP in dissociated hippocampal neurons obtained from P16 male Wistar rats. These cells were seeded in a 24-well plate with DMEM added with L-glutamine Ž2
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M. Sanchez-AlaÕez et al.r Brain Research 858 (2000) 78–83 ´
mM., fetal bovine serum Ž10%., insulin Ž4.5 mgrml., citosinerabinocide Ž5 mgrml. and antibiotics and maintained for 6 days, at which time, glial cells were removed. On the sixth day, neurons were washed with a balanced salt solution at 378C, then incubated with DMEM added with either CST or SS Ž10 or 100 ng. for different periods of time, i.e., 15, 30 min, 1, 3, 6, 12, 24 h. Upon the completion of each incubation period, neurons were collected and prepared for cAMP determination by enzymeimmunoassay ŽEIA..
cal tests due to the ceiling latency imposed by the experimenters Ž600 s., which result in a non-normal distribution of the sample. As for the cAMP production evaluation, an ANOVA and a post-hoc Sheffe´ tests were used to determine statistical significance.
2.8. Statistics
3.1. STM
Statistical significance was obtained by using Kruskal– Wallis and Mann–Whitney U tests. We used these statisti-
Results indicated that SS Ž100 ng. and CST Ž100 ng. were unable to modify STM.
3. Results
Fig. 1. This graph shows the performance of rats trained in the passive avoidance paradigm, under the effect of several doses of CST Ža. or SS Žb.. p - 0.05 compared to saline.
U
M. Sanchez-AlaÕez et al.r Brain Research 858 (2000) 78–83 ´
81
Fig. 2. This graph depicts the reversal of extinction induced by CST and in much less degree by SS. U p - 0.05 compared to saline.
3.2. LTM
3.3. Memory extinction
CST Ž100 and 1000 ng. as well as SS Ž1000 ng. strongly suppressed memory retrieval 24 h after training. CST Ž50 ng. does not modify memory retrieval but facilitates memory extinction Žsee Fig. 1a.. However, SS Ž100 ng. significantly impairs memory retrieval 24 h after training and facilitates memory extinction Žsee Fig. 1b..
In addition, a reversal of extinction was produced in all the animals that were treated with CST on day 6 Žsee Fig. 2.. 3.4. cAMP concentration EIA showed that the concentration of cAMP after incubating the neurons with CST significantly increased; while decreasing after incubating the cells with SS Žsee Fig. 3..
4. Discussion
Fig. 3. This graph illustrates the effect of CST and SS on the generation of cAMP in dissociated hippocampal cells. Both CST and SS produced significant differences from the second point in the graph Ž30 min. through the last point compared to the control. U p- 0.05.
In summary, these experiments show that CST is capable of deteriorating LTM but not STM. In addition, it reverts extinction of the behavior, mimicking the effects previously observed with anticholinergic drugs. In addition, CST increased the concentration of cAMP in dissociated hippocampal cells. These results suggest that CST modulates mnemonic processes, by using a putative disregulating cAMP production mechanism. This effect on memory may also be explained by its potential interaction with ACh or alternatively, by its hyperpolarizing effects on the LC neurons w5x that participate facilitating arousal and attention w1x. It is also interesting that rats exhibiting extinction of the inhibitory avoidance behavior, showed a reversal of extinction after CST administration. We have previously obtained this effect by using scopolamine, an anticholinergic
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drug w22x. Although the interpretation of this observation is controversial, we believe extinction may be viewed as Miller and Stevenson w18x put it forth ‘‘the acquisition of a new habit that opposes the original habit’’. It is possible that both anticholinergic drugs as well as CST, impair the establishment of the new habit: to go into the shock compartment; therefore, facilitating the reinstatement of the originally learned response: to avoid the shock compartment. This effect further supports the possibility of CST having an anticholinergic action and via this action may interfere with the retrieval of the on-going behavior. This possibility also postulates that the first behavior, the inhibitory avoidance, is stored somewhere in the brain and not forgotten, yet substituted by the re-learned behavior. In addition, these findings suggest that CST, like the anticholinergic drugs, interferes with the retrieval of the relearned behavior. These results altogether support the hypothesis suggesting that CST plays an important role in the modulation of memory processes. The fact indicating that CST is capable of preventing LTM but not STM implies that this peptide interferes with memory consolidation but not with learning expressed in the short term. As it has been amply documented, cAMP plays a critical role in memory consolidation in mollusks: Aplysia californica; insects: Drosophila melanogaster; and mammals: mice and rats w19x. In this context, our results indicating that CST disregulates cAMP formation is consistent with the deterioration of memory consolidation. For example, although there is an enormous difference between models, the mutants of the fruit fly D. melanogaster, that disregulate cAMP production, i.e., rutabaga mutant has cAMP production reduced and the dunce mutant has it increased, have an impairment in memory consolidation w13x. This disregulation that disrupts the consolidation of memory has also been observed in mice disregulating CaM KII w19x. As for SS effect, our results show that the action of this peptide on memory is weaker than that induced by CST, and in contrast with CST, SS decreases cAMP. This finding is easier to reconcile with the memory-impairing effect of this peptide. However, other systems might additionally be affected. For example, SS increases M-current, which is inhibited by muscarinic cholinergic activity w3x, in hippocampal pyramidal cells; thereby hyperpolarizing these neurons w20x. Likewise, SS seems to be capable of inhibiting excitatory transmission mediated by Glu in the hippocampus w4x. Our results indicate that CST is capable of deteriorating memory consolidation, potentially by disregulating cAMP formation in the neurons of the hippocampus. The physiological meaning of this activity is unknown and any effort to explain it becomes merely speculative. However, it is tempting to say that a system like this may be important to inhibit the retrieval of memory traces that are negligible for specific demands of the environment in a given mo-
ment. With all, we can say that CST seems to play a critical role in memory modulation. Acknowledgements This study was partially supported by CONACyT, Grant no. 25488N, and a Grant from FUNSALUD to OPG. CST was generously provided by Dr. Luis de Lecea. The authors wish to thank Mr. Raul ´ Ramırez ´ for his technical support, Mr. Manuel Zarate for the care of the animals and MS Edith Monroy for the English translation. References w1x G. Aston-Jones, J. Rajkowski, P. Kubiak, Conditioned responses of monkey locus coeruleus neurons anticipate acquisition of discriminative behavior in vigilance task, Neuroscience 80 Ž1997. 697–715. w2x J.D. Brioni, J.L. McGaugh, Post-training administration of GABAergic antagonists enhances retention of aversively motivated tasks, Psychopharmacology 96 Ž1988. 505–510. w3x D. Brown, M-currents: an update, TINS 11 Ž1988. 294–299. w4x S. Boehm, H. Betz, SS inhibits excitatory transmission at rat hippocampal synapses via presynaptic receptors, J. Neurosci. 17 Ž1997. 4066–4075. w5x M. Connor, S.L. Ingram, M.J. Christie, Cortistatin increase of a potassium conductance in rat locus coeruleus in vitro, Br. J. Pharmacol. 122 Ž1997. 1567–1572. w6x S.E. Cruz-Morales, G.L. Quirarte, M.A. Dıaz ´ del Guante, R.A. Prado-Alcala, ´ Effects of GABA antagonists on inhibitory avoidance, Life Sciences 53 Ž1993. 1325–1330. w7x L. de Lecea, J.R. Criado, O. Prospero-Garcıa, ´ ´ K.M. Gautvik, P. Schweitzer, P.E. Danielson, C.L. Dunlop, G.R. Siggins, S.J. Henriksen, J.G. Sutcliffe, A cortical neuropeptide with neuronal depressant and sleep-modulating properties, Nature 381 Ž1996. 242–245. w8x L. de Lecea, P. Ruiz-Lozano, P.E. Danielson, J. Peelle-Kirley, P.E. Foye, W.N. Frankel, J.G. Sutcliffe, Cloning, mRNA expression, and chromosomal mapping of mouse and human preprocortistatin, Genomics 42 Ž1997. 499–506. w9x L. de Lecea, J.A. del Rio, J.R. Criado, S. Alcantara, M. Morales, P.E. Danielson, S.J. Henriksen, E. Soriano, J.G. Sutcliffe, Cortistatin is expressed in a distinct subset of cortical interneurons, J. Neurosci. 17 Ž1997. 5868–5880. w10x P. Dournaud, F. Jazat-Poindessous, A. Slama, Y. Lamour, J. Epelbaum, Correlations between water maze performance and cortical SS mRNA and high-affinity binding sites during ageing in rats, Eur. J. Neurosci. 8 Ž1996. 476–485. w11x B.J. Everitt, T.W. Robbins, Central cholinergic systems and cognition, Annu. Rev. Psychol. 48 Ž1997. 649–684. w12x J.F. Flood, K. Uezu, J.E. Morley, The cortical neuropeptide, cortistatin-14, impairs post-training memory processing, Brain Res. 775 Ž1997. 250–252. w13x D.A. Frank, M.E. Greenberg, CREB: a mediator of long-term memory from mollusks to mammals, Cell 79 Ž1994. 5–8. w14x S. Fukusumi, K. Chieko, S. Takekawa, H. Kizawa, J. Sakamoto, M. Miyamoto, S. Hinuma, Identification and characterization of a novel human cortistatin-like peptide, Biochem. Biophys. Res. Commun. 232 Ž1997. 157–163. w15x J.D.C. Lambert, R.S.G. Jones, M. Andreasen, M.S. Jensen, U. Heinemann, The role of excitatory amino acids in synaptic transmission in the hippocampus, Comp. Biochem. Physiol. 93A Ž1989. 195–201. w16x P.J. Magistretti, VIP neurons in the cerebral cortex, TIPS 11 Ž1990. 250–254.
M. Sanchez-AlaÕez et al.r Brain Research 858 (2000) 78–83 ´ w17x N. Matsuoka, S. Kanko, M. Satoh, Somatostatin augments long-term potentiation of the mossy fiber-CA3 system in guinea-pig hippocampal slices, Brain Res. 553 Ž1991. 188–194. w18x N.E. Miller, S.S. Stevenson, Agitated behavior of rats during experimental extinction and a curve of spontaneous recovery, J. Comp. Psychol. 21 Ž1936. 231–250. w19x B. Milner, L.R. Squire, E.R. Kandel, Cognitive neuroscience and the study of memory, Neuron 20 Ž1998. 445–468. w20x S.D. Moore, S.D. Madamba, M. Joels, G.R. Siggins, Somatostatin augments the M-current in hippocampal neurons, Science 239 Ž1988. 278–280. w21x R.A. Nicoll, R.C. Malenka, J.A. Kauer, Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system, Physiol. Rev. 70 Ž1990. 513–565. w22x R.A. Prado-Alcala, ´ M. Haiek, S. Rivas, G. Roldan-Roldan, G. Quirarte, Reversal of extinction by scopolamine, Physiol. Behav. 56 Ž1994. 27–30.
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w23x S. Siehler, K. Seuwen, D. Hoyer, w125 Ix tyr 10 -cortistatin 14 labels all five somatostatin receptors, Arch. Pharmacol. 357 Ž1998. 483–489. w24x T. Steckler, A.B. Keith, R.G. Wiley, A. Sahgal, Cholinergic lesions by 192 IgG-saporin and short-term recognition memory: role of the septohippocampal projection, Neuroscience 66 Ž1995. 101–114. w25x T. Steckler, H.I.M. Drinkenburg, A. Sahgal, J.P. Aggleton, Recognition memory in rats — II. Neuroanatomical substrates, Prog. Neurobiol. 54 Ž1998. 313–332. w26x T. Steckler, A. Sahgal, J.P. Aggleton, H.I.M. Drinkenburg, Recognition memory in rats — III. Neurochemical substrates, Prog. Neurobiol. 54 Ž1998. 333–348. w27x S.C. Steffensen, S.J. Henriksen, Effects of baclofen and bicuculline on inhibition in the fascia dentata and hippocampus regio superior, Brain Res. 538 Ž1991. 46–53. w28x N.J. Woolf, A structural basis for memory storage in mammals, Prog. Neurobiol. 55 Ž1998. 59–77.
Research report
Cortistatin modulates memory processes in rats a a Manuel Sanchez-Alavez , Margarita Gomez-Chavarın , ´ ´ ´ a , Luz Navarro a , Anabel Jimenez-Anguiano ´ a a a,b Eric Murillo-Rodrıguez , Roberto A. Prado-Alcala´ , Rene Drucker-Colin , ´ Oscar Prospero-Garcıa ´ ´ a, ) a
Grupo de Neurociencias, Depto. de Fisiologıa, de Mexico, Apdo. Postal 70-250, Mexico, D.F. ´ Fac. de Medicina, UniÕersidad Nacional Autonoma ´ ´ 04510, Mexico b Depto. de Neurociencias, IFC, UniÕersidad Nacional Autonoma de Mexico, Apdo. Postal 70-250, Mexico, D.F. 04510, Mexico ´ ´ Accepted 9 November 1999
Abstract Cortistatin ŽCST. is a recently described neuropeptide with high structural homology with somatostatin. Its mRNA is restricted to gamma amino butyric acid ŽGABA.-containing cells in the cerebral cortex and hippocampus. CST modulates the electrophysiology of the hippocampus and cerebral cortex of rats; hence, it may be modulating mnemonic processes. In this study, we have evaluated the effect of CST and somatostatin ŽSS. on short- and long-term memory ŽSTM and LTM, respectively., as well as on the extinction of the behavior by using the footshock passive avoidance behavioral test. In addition, we tested the ability of both neuropeptides to affect the generation of cAMP in hippocampal neurons in culture. Results showed that the administration of either CST or SS into the hippocampal CA1 deteriorates memory consolidation in a dose–response fashion and facilitates the extinction of the learned behavior. CST was more potent than SS. Likewise, CST increases cAMP while SS decreases it. These results strongly support a modulatory role for CST in memory processes. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Somatostatin; Short-term memory; Long-term memory; Memory extinction; Passive avoidance test; cAMP
1. Introduction Memory is a series of cognitive processes whose neurochemical control is highly complex w26x. For example, in the hippocampus, a cerebral structure crucially involved in the memory network w25,28x, at least three neurotransmitters are regulating its activity, i.e., glutamate ŽGlu. w15,21x, acetylcholine ŽACh. w11x and gamma amino butyric acid ŽGABA. w2,27x. Glu and ACh enhance hippocampal excitability and exert a facilitating effect on memory w11,24x; whereas GABA depresses hippocampal excitability w27x and facilitates memory extinction w2,6x. In addition, a series of neuropeptides are also involved in this regulation, i.e., somatostatin ŽSS. and vasoactive intestinal polypeptide ŽVIP.. Both SS and VIP seem to modulate cholinergic activity, and memory processes w10,16,17x. On the other hand, cAMP has been associated with learning and memory in several animal preparations w19x, and its disregulation causes learning and memory deterioration. ) C orresponding author. [email protected]
Fax:
q52-5-623-2241;
e-m ail:
Cortistatin ŽCST. is a neuropeptide recently described in rat w7x and human brains w8,14x. Its mRNA was detected in the brain of rats but not in other organs such as liver or testis w7x. This peptide exhibits an exclusive cortical and hippocampal distribution. Due to the fact that CST mRNA cohybridizes with GAD67 mRNA in the hippocampus, we believe it is synthesized, stored and released by GABAergic cells w9x. CST’s active form is conformed by 14 amino acids Žaa.. Its aa sequence shows a high structural homology with SS-14. Both molecules share 11 aa. CST binds all five SS receptors with high affinity ŽpM range. w23x; therefore, we expect it to induce effects similar to those produced by SS. Accordingly, it produces similar hyperpolarizing effects as SS on hippocampal pyramidal cells in in vitro preparations; however, this effect lasts longer w7x. In contrast, there are several other effects caused by CST that seem to go in an opposite direction. For example, CST reduces the ACh excitatory action in the hippocampus while SS facilitates it w7x. In this context, CST seems to be a neuropeptide with potential anticholinergic antagonizing effects. This CST effect may be mediated by SS receptors, or, alternatively, by its own receptors Žto be described. or
0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 2 3 3 6 - 7
M. Sanchez-AlaÕez et al.r Brain Research 858 (2000) 78–83 ´
by facilitating GABA activity. In addition, a recent report has indicated that CST may be inhibiting locus coeruleus ŽLC. neurons, which are mainly noradrenergic cells, by facilitating Kq conductance w5x. Consistent with CST’s inhibitory effects on hippocampal activity, a recently published study has suggested that CST may be regulating memory processes in mice w12x. In this context, the goal of this study is to assess if CST actually plays a role in memory regulation. In order to reach this goal, we decided to investigate whether or not CST administration directly into the hippocampus affects short- and long-term memory ŽSTM and LTM, respectively. as well as the process of extinction, evaluated by a footshock passive avoidance test and additionally if CST affects cAMP production in hippocampal cells.
2. Methods 2.1. Animals Male Wistar rats Ž250–350 g. were used in this study. All rats were implanted with a pair of guide cannulae aimed bilaterally to the hippocampal CA1 regions ŽP s 4.0, L s 2.5, V s 2.0.. Surgeries were performed under halothane anesthesia Ž2–3%.. Rats were individually housed 2 days before surgery and throughout the experiment. They were maintained under a controlled light–dark cycle Ž12:12, lights on at 0800 h., with food and water ad libitum. Antibiotics and analgesics were administered immediately after the surgery and during the following 2 days. 2.2. Training One week after surgery, rats were trained in the footshock passive avoidance behavioral test, in order to evaluate STM and LTM. The general method was as follows: the training apparatus was a trough-shaped alley Ž94 cm long, 25 cm wide and 30 cm deep. that was separated into two compartments by a guillotine door that retracted into the floor. The starting compartment Ž32 cm long. was illuminated by a 10-W lamp and the floor of this compartment was a grid made of aluminum bars; while the shock compartment Ž62 cm. was dark and V-shaped stainless-steel plates formed the floor and walls. This apparatus is equipped with 10 photocells distributed as follows: four in the starting compartment and six in the shock compartment. The apparatus is controlled by a PC computer and is placed in a sound-attenuated, non-illuminated room. Photocells relay information to the computer about the rat’s position within the chamber. The experimenter can view the rat directly as well as on-line through a cartoon representing the rat, displayed on the computer screen. Once the rat was placed in the starting compartment, the rat explored for 10 s, then the guillotine door was opened.
79
Immediately after the rat entered the shock compartment with all four paws, the door was closed and a footshock Ž0.8 mA. was delivered through the stainless-steel plates. Five seconds after the initiation of the footshock delivery, the door was opened and the rat was allowed to escape. Rats stayed in the starting compartment for 30 s, then removed and a microinjector was inserted through the guide cannulae into the CA1 region. With the aid of an injection pump, one of the following compounds was injected: vehicle Žsaline., CST, or SS ŽRBI.. 2.3. Test Retention of the inhibitory avoidance training was tested 30 min and 24 h after training. Each rat was placed in the starting compartment and 30 s later, the door was opened. The latency to enter into the shock compartment with all four paws was recorded. Shock was not delivered. The rat was left in the starting compartment with the door open for a maximum of 600 s. 2.4. STM In order to evaluated the effect of SS and CST on STM, three groups Ž10 rats each group. were trained in the passive avoidance test. Immediately after training, rats of each group received either: the vehicle Ž1 ml., CST Ž100 ng., or SS Ž100 ng.. All rats were tested 30 min after trainingq administration. 2.5. LTM Nine groups of rats Ž n s 10 each group. were trained as described and then injected with either: the vehicle, CST Ž10, 50, 100, 1000 ng. or SS Ž10, 50, 100, 1000 ng.. Rats were tested 24 h later to evaluate memory retention. They were also tested at 48, 72, 96 and 120 h after trainingq administration to evaluate memory extinction. 2.6. Extinction of the behaÕior The next part of this study was performed in three groups of rats Ž10 rats each group.. Animals were trained and 24 h later tested. All of them exhibited the avoidance behavior, then they were tested until they showed extinction of the behavior, which took place by the fifth day after training. The sixth day, rats were injected with either: the vehicle, CST Ž100 ng. or SS Ž100 ng.. Half an hour, 24 and 48 h after the injection rats were tested. 2.7. cAMP production In the last part of this study, we evaluated the generation of cAMP in dissociated hippocampal neurons obtained from P16 male Wistar rats. These cells were seeded in a 24-well plate with DMEM added with L-glutamine Ž2
80
M. Sanchez-AlaÕez et al.r Brain Research 858 (2000) 78–83 ´
mM., fetal bovine serum Ž10%., insulin Ž4.5 mgrml., citosinerabinocide Ž5 mgrml. and antibiotics and maintained for 6 days, at which time, glial cells were removed. On the sixth day, neurons were washed with a balanced salt solution at 378C, then incubated with DMEM added with either CST or SS Ž10 or 100 ng. for different periods of time, i.e., 15, 30 min, 1, 3, 6, 12, 24 h. Upon the completion of each incubation period, neurons were collected and prepared for cAMP determination by enzymeimmunoassay ŽEIA..
cal tests due to the ceiling latency imposed by the experimenters Ž600 s., which result in a non-normal distribution of the sample. As for the cAMP production evaluation, an ANOVA and a post-hoc Sheffe´ tests were used to determine statistical significance.
2.8. Statistics
3.1. STM
Statistical significance was obtained by using Kruskal– Wallis and Mann–Whitney U tests. We used these statisti-
Results indicated that SS Ž100 ng. and CST Ž100 ng. were unable to modify STM.
3. Results
Fig. 1. This graph shows the performance of rats trained in the passive avoidance paradigm, under the effect of several doses of CST Ža. or SS Žb.. p - 0.05 compared to saline.
U
M. Sanchez-AlaÕez et al.r Brain Research 858 (2000) 78–83 ´
81
Fig. 2. This graph depicts the reversal of extinction induced by CST and in much less degree by SS. U p - 0.05 compared to saline.
3.2. LTM
3.3. Memory extinction
CST Ž100 and 1000 ng. as well as SS Ž1000 ng. strongly suppressed memory retrieval 24 h after training. CST Ž50 ng. does not modify memory retrieval but facilitates memory extinction Žsee Fig. 1a.. However, SS Ž100 ng. significantly impairs memory retrieval 24 h after training and facilitates memory extinction Žsee Fig. 1b..
In addition, a reversal of extinction was produced in all the animals that were treated with CST on day 6 Žsee Fig. 2.. 3.4. cAMP concentration EIA showed that the concentration of cAMP after incubating the neurons with CST significantly increased; while decreasing after incubating the cells with SS Žsee Fig. 3..
4. Discussion
Fig. 3. This graph illustrates the effect of CST and SS on the generation of cAMP in dissociated hippocampal cells. Both CST and SS produced significant differences from the second point in the graph Ž30 min. through the last point compared to the control. U p- 0.05.
In summary, these experiments show that CST is capable of deteriorating LTM but not STM. In addition, it reverts extinction of the behavior, mimicking the effects previously observed with anticholinergic drugs. In addition, CST increased the concentration of cAMP in dissociated hippocampal cells. These results suggest that CST modulates mnemonic processes, by using a putative disregulating cAMP production mechanism. This effect on memory may also be explained by its potential interaction with ACh or alternatively, by its hyperpolarizing effects on the LC neurons w5x that participate facilitating arousal and attention w1x. It is also interesting that rats exhibiting extinction of the inhibitory avoidance behavior, showed a reversal of extinction after CST administration. We have previously obtained this effect by using scopolamine, an anticholinergic
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drug w22x. Although the interpretation of this observation is controversial, we believe extinction may be viewed as Miller and Stevenson w18x put it forth ‘‘the acquisition of a new habit that opposes the original habit’’. It is possible that both anticholinergic drugs as well as CST, impair the establishment of the new habit: to go into the shock compartment; therefore, facilitating the reinstatement of the originally learned response: to avoid the shock compartment. This effect further supports the possibility of CST having an anticholinergic action and via this action may interfere with the retrieval of the on-going behavior. This possibility also postulates that the first behavior, the inhibitory avoidance, is stored somewhere in the brain and not forgotten, yet substituted by the re-learned behavior. In addition, these findings suggest that CST, like the anticholinergic drugs, interferes with the retrieval of the relearned behavior. These results altogether support the hypothesis suggesting that CST plays an important role in the modulation of memory processes. The fact indicating that CST is capable of preventing LTM but not STM implies that this peptide interferes with memory consolidation but not with learning expressed in the short term. As it has been amply documented, cAMP plays a critical role in memory consolidation in mollusks: Aplysia californica; insects: Drosophila melanogaster; and mammals: mice and rats w19x. In this context, our results indicating that CST disregulates cAMP formation is consistent with the deterioration of memory consolidation. For example, although there is an enormous difference between models, the mutants of the fruit fly D. melanogaster, that disregulate cAMP production, i.e., rutabaga mutant has cAMP production reduced and the dunce mutant has it increased, have an impairment in memory consolidation w13x. This disregulation that disrupts the consolidation of memory has also been observed in mice disregulating CaM KII w19x. As for SS effect, our results show that the action of this peptide on memory is weaker than that induced by CST, and in contrast with CST, SS decreases cAMP. This finding is easier to reconcile with the memory-impairing effect of this peptide. However, other systems might additionally be affected. For example, SS increases M-current, which is inhibited by muscarinic cholinergic activity w3x, in hippocampal pyramidal cells; thereby hyperpolarizing these neurons w20x. Likewise, SS seems to be capable of inhibiting excitatory transmission mediated by Glu in the hippocampus w4x. Our results indicate that CST is capable of deteriorating memory consolidation, potentially by disregulating cAMP formation in the neurons of the hippocampus. The physiological meaning of this activity is unknown and any effort to explain it becomes merely speculative. However, it is tempting to say that a system like this may be important to inhibit the retrieval of memory traces that are negligible for specific demands of the environment in a given mo-
ment. With all, we can say that CST seems to play a critical role in memory modulation. Acknowledgements This study was partially supported by CONACyT, Grant no. 25488N, and a Grant from FUNSALUD to OPG. CST was generously provided by Dr. Luis de Lecea. The authors wish to thank Mr. Raul ´ Ramırez ´ for his technical support, Mr. Manuel Zarate for the care of the animals and MS Edith Monroy for the English translation. References w1x G. Aston-Jones, J. Rajkowski, P. Kubiak, Conditioned responses of monkey locus coeruleus neurons anticipate acquisition of discriminative behavior in vigilance task, Neuroscience 80 Ž1997. 697–715. w2x J.D. Brioni, J.L. McGaugh, Post-training administration of GABAergic antagonists enhances retention of aversively motivated tasks, Psychopharmacology 96 Ž1988. 505–510. w3x D. Brown, M-currents: an update, TINS 11 Ž1988. 294–299. w4x S. Boehm, H. Betz, SS inhibits excitatory transmission at rat hippocampal synapses via presynaptic receptors, J. Neurosci. 17 Ž1997. 4066–4075. w5x M. Connor, S.L. Ingram, M.J. Christie, Cortistatin increase of a potassium conductance in rat locus coeruleus in vitro, Br. J. Pharmacol. 122 Ž1997. 1567–1572. w6x S.E. Cruz-Morales, G.L. Quirarte, M.A. Dıaz ´ del Guante, R.A. Prado-Alcala, ´ Effects of GABA antagonists on inhibitory avoidance, Life Sciences 53 Ž1993. 1325–1330. w7x L. de Lecea, J.R. Criado, O. Prospero-Garcıa, ´ ´ K.M. Gautvik, P. Schweitzer, P.E. Danielson, C.L. Dunlop, G.R. Siggins, S.J. Henriksen, J.G. Sutcliffe, A cortical neuropeptide with neuronal depressant and sleep-modulating properties, Nature 381 Ž1996. 242–245. w8x L. de Lecea, P. Ruiz-Lozano, P.E. Danielson, J. Peelle-Kirley, P.E. Foye, W.N. Frankel, J.G. Sutcliffe, Cloning, mRNA expression, and chromosomal mapping of mouse and human preprocortistatin, Genomics 42 Ž1997. 499–506. w9x L. de Lecea, J.A. del Rio, J.R. Criado, S. Alcantara, M. Morales, P.E. Danielson, S.J. Henriksen, E. Soriano, J.G. Sutcliffe, Cortistatin is expressed in a distinct subset of cortical interneurons, J. Neurosci. 17 Ž1997. 5868–5880. w10x P. Dournaud, F. Jazat-Poindessous, A. Slama, Y. Lamour, J. Epelbaum, Correlations between water maze performance and cortical SS mRNA and high-affinity binding sites during ageing in rats, Eur. J. Neurosci. 8 Ž1996. 476–485. w11x B.J. Everitt, T.W. Robbins, Central cholinergic systems and cognition, Annu. Rev. Psychol. 48 Ž1997. 649–684. w12x J.F. Flood, K. Uezu, J.E. Morley, The cortical neuropeptide, cortistatin-14, impairs post-training memory processing, Brain Res. 775 Ž1997. 250–252. w13x D.A. Frank, M.E. Greenberg, CREB: a mediator of long-term memory from mollusks to mammals, Cell 79 Ž1994. 5–8. w14x S. Fukusumi, K. Chieko, S. Takekawa, H. Kizawa, J. Sakamoto, M. Miyamoto, S. Hinuma, Identification and characterization of a novel human cortistatin-like peptide, Biochem. Biophys. Res. Commun. 232 Ž1997. 157–163. w15x J.D.C. Lambert, R.S.G. Jones, M. Andreasen, M.S. Jensen, U. Heinemann, The role of excitatory amino acids in synaptic transmission in the hippocampus, Comp. Biochem. Physiol. 93A Ž1989. 195–201. w16x P.J. Magistretti, VIP neurons in the cerebral cortex, TIPS 11 Ž1990. 250–254.
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