- Email: [email protected]
Memory enhancement by the angiotensinergic system in the crab Chasmagnathus is mediated by endogenous angiotensin II
Neuroscience Letters 266 (1999) 1±4
Memory enhancement by the angiotensinergic system in the crab Chasmagnathus is mediated by endogenous angiotensin II Alejandro Delorenzi*, HeÂctor Maldonado Laboratorio de Neurobiologia de la Memoria, Facultad de Ciencias Exactas y Naturales, Depto BiologõÂa, Universidad de Buenos Aires (1428) Buenos Aires, Argentina Received 8 January 1999; received in revised form 22 February 1999; accepted 22 February 1999
Abstract In previous work with the crab Chasmagnathus, it was reported that either exogenous angiotensin II (ANGII) or angiotensin IV (ANGIV), have an enhancing effect on long-term memory, which involves an association between context and an iterative danger stimulus (context-signal memory, CSM). Present results indicate that Dival, an ANGIV antagonist, reverts the facilitatory effect of ANGIV but not that of ANGII, whereas saralasin but not Dival, disrupts CSM. These ®ndings suggest that ANGII is the endogenous angiotensin that plays a signi®cant role in long-term memory, while the ANGIV receptor would not be encompassed in the cascade of events related to crab's CSM. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angiotensin II; Angiotensin IV; Dival; Neuropeptides; Crab; Memory
Several roles have been ascribed to angiotensins, including physiological actions on the cardiovascular, renal and neuroendocrine systems [12,14]. Besides, manipulation of brain angiotensin levels appears to in¯uence acquisition and recall of a newly learned task. A disrupting effect on memory was initially attributed to centrally applied ANGII acting on AT1 receptor [14]. In contrast, further reports indicated that either ANGII or angiotensin II (3± 8), called ANGIV, have a facilitatory effect [1,14,15], in spite of acting on different receptor subtypes. Although the angiotensinergic system emerged early en evolution [5,6,9], research on the role of angiotensins in memory has been con®ned to vertebrates, However, it has been recently extended to an invertebrate, the crab Chasmagnathus granulatus, which presents an instance of robust associative memory, intensively investigated from mechanistic and behavioral standpoint [7]. The repeated presentation of a danger stimulus (a screen passing overhead) elicits in the crab Chasmagnathus an escape reaction that declines after few trials, and such decrement persists for a long period. This long-term memory is mediated by an association between the danger stimulus and the environmental cues [10], being called context-signal * Corresponding author. Tel.: 154-11-4576-3384; fax: 154-114576-3348. E-mail address: [email protected] (A. Delorenzi)
memory (CSM). Retention of CSM for at least 5 days is ensured by solely 15 9-s screen presentations, separated by 3 min, but not by 10 or less training trials [3]. However, post-training administration of ANG II (50 r mol) or ANG IV (5 r mol) allows an insuf®cient amount of training to induce CSM in Chasmagnathus [3,4]. Such facilitatory effect is reverted by the ANGII antagonist saralasin (5 r mol) which shows, in turn, an amnestic effect on CSM acquired after 30 training trials [3]. In contrast, two nonpeptide antagonists for ANGII, i.e. DUP-753 and PD 124177, are unable to suppress the facilitatory effect of ANGII in Chasmagnathus; neither do they show to have amnestic effects [4]. Both ANGII-like immunoreactivity and angiotensin-converting enzyme-like activity are described in gills, thoracic ganglia and brain of Chasmagnathus granulatus [3]. In attempt to give a comprehensive explanation of the foregoing results with Chasmagnathus, two hypotheses were offered [4]. According to a ®rst view, either ANGII or ANGIV induce memory enhancement in crabs through different speci®c receptor subtypes; while saralasin antagonizes ANGII receptors producing an amnestic effect that, in turn, masks the facilitatory effect of ANGIV when both compounds are coinjected. The second view considers that the effects obtained with ANGII are a consequence of its cleavage to ANGIV, namely, that ANGIV is the actual agent of the angiotensin enhancing effect on the CSM. In
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 23 2- 3
2
A. Delorenzi, H. Maldonado / Neuroscience Letters 266 (1999) 1±4
Fig. 1. Dival blocks the facilitatory effect of angiotensin IV, 24 h after a seven-trial training session. Ordinate: average accumulative scores for the two testing trials (two-trial block). VEH, groups injected with vehicle; AIV, with angiotensin IV; AIV 1 DIV, injected with angiotensin IV plus Dival. Planned comparisons between each group and the control group injected with vehicle: *P , 0:05. The height of each bar is the mean ^ SEM.
addition, it proposes that saralasin is also cleaved to a fragment that antagonizes the AT4 receptor, disrupting CSM when injected after strong training or blocking the facilitatory effect of ANGIV when both compounds are coinjected after a weak training. The present paper has the purpose of testing these hypotheses, using Dival, a speci®c antagonist for ANGIV [13]. The experimental design and set-up were as those previously used [7]. Brie¯y, the experimental unit was the actometer: a bowl-shaped plastic container suspended by strings from an upper wooden framework and illuminated from above. A crab was placed in the container and an opaque screen was moved across the upper border of the framework by a motor, thus provoking a crab's running response and consequently container vibrations. These vibrations induced, through a piezoelectric transducer, elec-
Fig. 2. Dival fails to block the facilitatory effect of angiotensin II, 24 h after a seven-trial training session. VEH, groups injected with vehicle; AII, with angiotensin II; AII 1 DIV, injected with angiotensin II plus Dival. Ordinate, symbols and contrasts as in Fig. 1.
trical signals which were ampli®ed, integrated during the recording time (9 s), translated into numerical units ranging from 0 to 3000, and processed by computer. The experimental room had 40 actometers, isolated from each other by partitions. Animals were adult male crabs weighing 17 ^ 0:2 g, collected from water less than 1 m deep in narrow coastal inlets of San Clemente del TuyuÂ, Argentina. Trained groups (TR) included 34±36 crabs that underwent either 15 9-s trials separated by 3-min intervals (strong training protocol), or seven trials with the same intertrial interval (weak training protocol) [2]. Control groups (CT) with the same number of individuals, remained in the actometers during the entire training session but without being trained. Immediately after the training session, animals were given either 50 ml of vehicle (a crustacean saline solution) or drug solution, through the dorsal cephalothoracic-abdominal membrane, ensuring that the injected solutions were released roughly at the center of the pericardial sac. Human ANGII, ANGIV and saralasin (SAR) were purchased from Sigma and the ANGIV antagonist Dival (WSU1291) [13] from Hedral Therapeutics, USA. Twenty-four hours after training, crabs were given two test trials. In all previous experiments at our laboratory a signi®cant difference (t-test, a 0:05) at testing was invariably disclosed between vehicle injected CT and TR groups, when TR received strong training. In contrast, no difference was found when TR was given weak training. Consequently, testing results of this paper were analyzed by performing planned comparisons, weighted means ANOVA with P 0:5 [8], between each group and a vehicle injected CT group, using average accumulative scores for the two test trials. To determine if Dival can revert the enhancing effect of exogenous ANGIV in Chasmagnathus, we carried out the following experiment. Three groups of crabs were given a weak training protocol (7 trials) and then injected with either the vehicle or ANGIV (5 r mol) or ANGIV plus Dival (5 r mol each), and termed VEH-TR, ANGIV-TR, ANGIV 1 DIV-TR, respectively. In addition, a control group (VEH-CT) injected with the vehicle, was formed. All groups had 35 crabs. In previous work [4], a curve dose-response had shown an enhancing effect of ANGIV in the range from 5 to 500 r mol. Present results at testing are shown in Fig. 1. Planned comparisons between each trained group and VEH-CT revealed a signi®cant difference only for ANGIV-TR vs. VEH-CT (F 1; 132 5:44, P , 0:05). Thus, the enhancing effect of ANGIV is abolished when coinjected with the antagonist Dival. A second experiment was aimed at exploring the effect of Dival concerning ANGII. Should the hypothesis that memory facilitation by ANGII is mediated by ANGIV prove viable, an injection of Dival would be expected to block such facilitation. Three groups of crabs were given a weak training protocol (7 trials) and then injected
A. Delorenzi, H. Maldonado / Neuroscience Letters 266 (1999) 1±4
Fig. 3. Saralasin but not Dival, has amnestic effect, 24 h after a 30-trial training session. Planned comparisons: **P , 0:01. Ordinate, symbols and contrasts as in Fig. 1.
with the vehicle, ANGII (50 r mol) or ANGII plus Dival (50 1 5 r mol), and termed VEH-TR, ANGII-TR, ANGII 1 DIV-TR, respectively. In addition, a control group (VEH-CT) injected with vehicle was formed. All groups had 36 crabs. In previous work [2], a curve doseresponse showed an enhancing effect of ANGII only at 50 r mol. Results at testing are shown in Fig. 2. Planned comparisons between each trained group and VEH-CT revealed a signi®cant difference either for ANGII vs. VEH-CT (F 1; 136 7:76, P , 0:05) or for ANGII 1 DIV-TR vs. VEH-CT (F 1; 136 5:67, P , 0:05), indicating that Dival fails to prevent the enhancing effect of ANGII, i.e. that ANGII induces memory enhancement through an angiotensin receptor subtype that is different from that of ANGIV. The foregoing result is at variance with the hypothesis that ANGIV is the only agent of the angiotensin enhancing effect on the crab's CSM. However, to conclude that the alternative hypothesis is right, i.e. that both angiotensins are modulating CSM, it is required to demonstrate that endogenous ANGII or ANGIV play a role in this mnemonic process. A favored technique for identifying the endogenous role of neuropeptides is the use of antagonists [16]. In the present case, a persuasive ®nding would be to demonstrate an amnestic effect of either the ANGII or ANGIV antagonist after a strong training. For testing this possibility, a third experiment was conducted. Three groups of crabs were given 30 trials (TR-groups) and other three were controls (CT-groups). One TR-CT pair of groups was injected with the vehicle (VEH), a second pair with 5 r mol of Dival, and a third pair with 50 r mol of saralasin [3], i.e. an ANGII analogous peptide with a wellknown antagonist effect on the whole current family of ANGII receptors [11] and that proved to have amnestic effect on crab when used in such dose. Thus, six groups were formed: VEH-CT, VEH-TR, DIV-CT, DIV-TR, SAR-CT and SAR-TR. Each group had 34 individuals. Results corresponding to test trials are presented in Fig. 3. Planned comparisons disclosed a signi®cant difference
3
for VEH-CT vs. VEH-TR (F 1; 128 11:26, P , 0:01) and between DIV-CT vs. DIV-TR (F 1; 128 9:44, P , 0:01) but neither for SAR-CT vs. SAR-TR nor between CT-groups. No amnestic effect of Dival was obtained with doses as high as 50 r mol or 5 h mol (data not shown). Thus, while saralasin showed a clear-cut disrupting effect on CSM, con®rming a previous result [4], no amnestic effect was produced by post-training injection of Dival. In brief, we may conclude that ANGII, but not ANGIV, would be the endogenous angiotensin that plays a signi®cant role within the cascade of events related to CSM acquisition and/or consolidation in Chasmagnathus. Such conclusion is supported by three lines of evidence based on both present and previously reported results [2±4]. First, ANGII has a memory facilitatory effect since it yields CSM retention when injected after a weak training protocol. Second, the antagonist saralasin abolishes memory enhancement when it was coinjected with ANGII after weak training and has a notable amnestic effect when it was given after strong training. Third, ANGIV has memory facilitatory effect reverted by Dival, a result similar to that obtained in mammals [1,15], but the endogenous activity of this peptide fails to be demonstrated in crab since amnestic effect of the antagonist can not be shown. Therefore, the role of ANGII as memory enhancer would have merged early in evolution; while the ANGIV receptor, though present at such an early evolutive stage, would not be encompassed in the cascade of events related to crab's CSM. This work was supported by the University of Buenos Aires (grant No. 01/TY04), FONCYT (grant No. 01863) and CONICET (grant No. PID 3847/94). We thank Angel Vidal for technical assistance. Dival was provided by Hedral Therapeutics, USA. [1] Braszko, J.J., Kupryszewski, G., Witczuk, B. and Wisniewski, K., Angiotensin II (3±8)-hexapeptide affects motor activity, performance of passive avoidance and a conditioned avoidance response in rats. Neuroscience, 27 (1988) 777± 783. [2] Delorenzi, A., Pedreira, M.E., Romano, A., Pirola, C.J., Nahmod, V.E. and Maldonado, H., Acute administration of angiotensin II improves long-term habituation in the crab Chasmagnathus. Neurosci. Lett., 196 (1995) 193±196. [3] Delorenzi, A., Pedreira, M.E., Romano, A., Garcia, S.I., Pirola, C.J., Nahmod, V.E. and Maldonado, H., Angiotensin II enhances long-term memory in the crab Chasmagnathus. Brain Res. Bull., 41/4 (1996) 211±220. [4] Delorenzi, A., Locatelli, F., Romano, A., Nahmod, V. and Maldonado, H., Angiotensin II (3±8) induces long-term memory improvement in the crab Chasmagnathus. Neurosci. Lett., 226 (1997) 143±146. [5] Gonzalez, G.C., Roger, Y., Gonzalez, E., Martinez-Padron, M., Moore, G.J. and Lukowiak, K., Angiotensinogen-like epitopes are present in the CNS of Aplysia californica and co-localize with urotensin Y- and urotensin-like immunoreactivities in the cerebral ganglia. NeuroReport, 6 (1995) 541±544. [6] Laurent, V., Bulet, P. and Salzet, M., A comparison of the
4
A. Delorenzi, H. Maldonado / Neuroscience Letters 266 (1999) 1±4
[7]
[8] [9]
[10]
[11]
leech Theromyzon tessulatom angiotensin I-like molecule with forms of vertebrate angiotensinogens: a hormonal system conserved in the course of evolution. Neurosci. Lett., 190 (1995) 175±178. Maldonado, H., Romano, A. and Tomsic, D., Long-term habituation (LTH) in the crab Chasmagnathus: a model for behavioral and mechanistic studies of memory. Braz. J. Med. Biol. Res., 30 (1997) 813±826. Rosenthal, R. and Rosnow, R.L., Contrast Analysis Focused Comparisons in the Analysis of Variance, Cambridge University Press, Cambridge, 1985. Schoofs, L., Veelaert, D., De Loof, A., Hybrechts, R. and Isaac, E., Immunocytochemical distribution of angiotensin I-converting enzyme-like immunoreactivity in the brain and testis of insects. Brain Res., 785 (1998) 215±227. Tomsic, D., Pedreira, M.E., Romano, A. and Maldonado, H., Context-us association as a determinant of long-term habituation in the crab Chasmagnathus. Anim. Learn. Behav., 26 (1998) 196±206. Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T., Herblin,
[12] [13] [14] [15]
[16]
W.F., Ben®eld, P., Carini, D.J., Lee, R.J., Wexler, R.R., Saye, J.A.M. and Smith, R.D., Angiotensin II receptor and angiotensin II receptor antagonist. Pharmacol. Rev., 45 (1993) 206±251. Wright, J.W. and Harding, J.W., Brain angiotensin receptor subtypes in the control of physiological and behavioral responses. Neurosci. Behav. Rev., 18 (1994) 21±53. Wright, J.W., Koreas, L.T., Stab, J.W. and Harding, J.W., The angiotensin IV system: functional implications. Front. Neuroendocrinol., 16 (1995) 23±52. Wright, J.W. and Harding, J.W., Important role for angiotensin III and IV in the brain renin-angiotensin system. Brain Res. Rev., 25 (1997) 96±124. Wright, J.W., Miller-Wing, A.V., Shaffer, M.J., Higginson, C., Wright, D.E., Hanesworth, J.M. and Harding, J.W., Angiotensin II (3±8) hippocampal binding: potential role in the facilitation of memory. Brain Res. Bull., 32 (1993) 497± 502. De Wied, D., Neuropeptides in learning and memory processes. Behav. Brain Res., 83 (1997) 83±90.
Memory enhancement by the angiotensinergic system in the crab Chasmagnathus is mediated by endogenous angiotensin II Alejandro Delorenzi*, HeÂctor Maldonado Laboratorio de Neurobiologia de la Memoria, Facultad de Ciencias Exactas y Naturales, Depto BiologõÂa, Universidad de Buenos Aires (1428) Buenos Aires, Argentina Received 8 January 1999; received in revised form 22 February 1999; accepted 22 February 1999
Abstract In previous work with the crab Chasmagnathus, it was reported that either exogenous angiotensin II (ANGII) or angiotensin IV (ANGIV), have an enhancing effect on long-term memory, which involves an association between context and an iterative danger stimulus (context-signal memory, CSM). Present results indicate that Dival, an ANGIV antagonist, reverts the facilitatory effect of ANGIV but not that of ANGII, whereas saralasin but not Dival, disrupts CSM. These ®ndings suggest that ANGII is the endogenous angiotensin that plays a signi®cant role in long-term memory, while the ANGIV receptor would not be encompassed in the cascade of events related to crab's CSM. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Angiotensin II; Angiotensin IV; Dival; Neuropeptides; Crab; Memory
Several roles have been ascribed to angiotensins, including physiological actions on the cardiovascular, renal and neuroendocrine systems [12,14]. Besides, manipulation of brain angiotensin levels appears to in¯uence acquisition and recall of a newly learned task. A disrupting effect on memory was initially attributed to centrally applied ANGII acting on AT1 receptor [14]. In contrast, further reports indicated that either ANGII or angiotensin II (3± 8), called ANGIV, have a facilitatory effect [1,14,15], in spite of acting on different receptor subtypes. Although the angiotensinergic system emerged early en evolution [5,6,9], research on the role of angiotensins in memory has been con®ned to vertebrates, However, it has been recently extended to an invertebrate, the crab Chasmagnathus granulatus, which presents an instance of robust associative memory, intensively investigated from mechanistic and behavioral standpoint [7]. The repeated presentation of a danger stimulus (a screen passing overhead) elicits in the crab Chasmagnathus an escape reaction that declines after few trials, and such decrement persists for a long period. This long-term memory is mediated by an association between the danger stimulus and the environmental cues [10], being called context-signal * Corresponding author. Tel.: 154-11-4576-3384; fax: 154-114576-3348. E-mail address: [email protected] (A. Delorenzi)
memory (CSM). Retention of CSM for at least 5 days is ensured by solely 15 9-s screen presentations, separated by 3 min, but not by 10 or less training trials [3]. However, post-training administration of ANG II (50 r mol) or ANG IV (5 r mol) allows an insuf®cient amount of training to induce CSM in Chasmagnathus [3,4]. Such facilitatory effect is reverted by the ANGII antagonist saralasin (5 r mol) which shows, in turn, an amnestic effect on CSM acquired after 30 training trials [3]. In contrast, two nonpeptide antagonists for ANGII, i.e. DUP-753 and PD 124177, are unable to suppress the facilitatory effect of ANGII in Chasmagnathus; neither do they show to have amnestic effects [4]. Both ANGII-like immunoreactivity and angiotensin-converting enzyme-like activity are described in gills, thoracic ganglia and brain of Chasmagnathus granulatus [3]. In attempt to give a comprehensive explanation of the foregoing results with Chasmagnathus, two hypotheses were offered [4]. According to a ®rst view, either ANGII or ANGIV induce memory enhancement in crabs through different speci®c receptor subtypes; while saralasin antagonizes ANGII receptors producing an amnestic effect that, in turn, masks the facilitatory effect of ANGIV when both compounds are coinjected. The second view considers that the effects obtained with ANGII are a consequence of its cleavage to ANGIV, namely, that ANGIV is the actual agent of the angiotensin enhancing effect on the CSM. In
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 23 2- 3
2
A. Delorenzi, H. Maldonado / Neuroscience Letters 266 (1999) 1±4
Fig. 1. Dival blocks the facilitatory effect of angiotensin IV, 24 h after a seven-trial training session. Ordinate: average accumulative scores for the two testing trials (two-trial block). VEH, groups injected with vehicle; AIV, with angiotensin IV; AIV 1 DIV, injected with angiotensin IV plus Dival. Planned comparisons between each group and the control group injected with vehicle: *P , 0:05. The height of each bar is the mean ^ SEM.
addition, it proposes that saralasin is also cleaved to a fragment that antagonizes the AT4 receptor, disrupting CSM when injected after strong training or blocking the facilitatory effect of ANGIV when both compounds are coinjected after a weak training. The present paper has the purpose of testing these hypotheses, using Dival, a speci®c antagonist for ANGIV [13]. The experimental design and set-up were as those previously used [7]. Brie¯y, the experimental unit was the actometer: a bowl-shaped plastic container suspended by strings from an upper wooden framework and illuminated from above. A crab was placed in the container and an opaque screen was moved across the upper border of the framework by a motor, thus provoking a crab's running response and consequently container vibrations. These vibrations induced, through a piezoelectric transducer, elec-
Fig. 2. Dival fails to block the facilitatory effect of angiotensin II, 24 h after a seven-trial training session. VEH, groups injected with vehicle; AII, with angiotensin II; AII 1 DIV, injected with angiotensin II plus Dival. Ordinate, symbols and contrasts as in Fig. 1.
trical signals which were ampli®ed, integrated during the recording time (9 s), translated into numerical units ranging from 0 to 3000, and processed by computer. The experimental room had 40 actometers, isolated from each other by partitions. Animals were adult male crabs weighing 17 ^ 0:2 g, collected from water less than 1 m deep in narrow coastal inlets of San Clemente del TuyuÂ, Argentina. Trained groups (TR) included 34±36 crabs that underwent either 15 9-s trials separated by 3-min intervals (strong training protocol), or seven trials with the same intertrial interval (weak training protocol) [2]. Control groups (CT) with the same number of individuals, remained in the actometers during the entire training session but without being trained. Immediately after the training session, animals were given either 50 ml of vehicle (a crustacean saline solution) or drug solution, through the dorsal cephalothoracic-abdominal membrane, ensuring that the injected solutions were released roughly at the center of the pericardial sac. Human ANGII, ANGIV and saralasin (SAR) were purchased from Sigma and the ANGIV antagonist Dival (WSU1291) [13] from Hedral Therapeutics, USA. Twenty-four hours after training, crabs were given two test trials. In all previous experiments at our laboratory a signi®cant difference (t-test, a 0:05) at testing was invariably disclosed between vehicle injected CT and TR groups, when TR received strong training. In contrast, no difference was found when TR was given weak training. Consequently, testing results of this paper were analyzed by performing planned comparisons, weighted means ANOVA with P 0:5 [8], between each group and a vehicle injected CT group, using average accumulative scores for the two test trials. To determine if Dival can revert the enhancing effect of exogenous ANGIV in Chasmagnathus, we carried out the following experiment. Three groups of crabs were given a weak training protocol (7 trials) and then injected with either the vehicle or ANGIV (5 r mol) or ANGIV plus Dival (5 r mol each), and termed VEH-TR, ANGIV-TR, ANGIV 1 DIV-TR, respectively. In addition, a control group (VEH-CT) injected with the vehicle, was formed. All groups had 35 crabs. In previous work [4], a curve dose-response had shown an enhancing effect of ANGIV in the range from 5 to 500 r mol. Present results at testing are shown in Fig. 1. Planned comparisons between each trained group and VEH-CT revealed a signi®cant difference only for ANGIV-TR vs. VEH-CT (F 1; 132 5:44, P , 0:05). Thus, the enhancing effect of ANGIV is abolished when coinjected with the antagonist Dival. A second experiment was aimed at exploring the effect of Dival concerning ANGII. Should the hypothesis that memory facilitation by ANGII is mediated by ANGIV prove viable, an injection of Dival would be expected to block such facilitation. Three groups of crabs were given a weak training protocol (7 trials) and then injected
A. Delorenzi, H. Maldonado / Neuroscience Letters 266 (1999) 1±4
Fig. 3. Saralasin but not Dival, has amnestic effect, 24 h after a 30-trial training session. Planned comparisons: **P , 0:01. Ordinate, symbols and contrasts as in Fig. 1.
with the vehicle, ANGII (50 r mol) or ANGII plus Dival (50 1 5 r mol), and termed VEH-TR, ANGII-TR, ANGII 1 DIV-TR, respectively. In addition, a control group (VEH-CT) injected with vehicle was formed. All groups had 36 crabs. In previous work [2], a curve doseresponse showed an enhancing effect of ANGII only at 50 r mol. Results at testing are shown in Fig. 2. Planned comparisons between each trained group and VEH-CT revealed a signi®cant difference either for ANGII vs. VEH-CT (F 1; 136 7:76, P , 0:05) or for ANGII 1 DIV-TR vs. VEH-CT (F 1; 136 5:67, P , 0:05), indicating that Dival fails to prevent the enhancing effect of ANGII, i.e. that ANGII induces memory enhancement through an angiotensin receptor subtype that is different from that of ANGIV. The foregoing result is at variance with the hypothesis that ANGIV is the only agent of the angiotensin enhancing effect on the crab's CSM. However, to conclude that the alternative hypothesis is right, i.e. that both angiotensins are modulating CSM, it is required to demonstrate that endogenous ANGII or ANGIV play a role in this mnemonic process. A favored technique for identifying the endogenous role of neuropeptides is the use of antagonists [16]. In the present case, a persuasive ®nding would be to demonstrate an amnestic effect of either the ANGII or ANGIV antagonist after a strong training. For testing this possibility, a third experiment was conducted. Three groups of crabs were given 30 trials (TR-groups) and other three were controls (CT-groups). One TR-CT pair of groups was injected with the vehicle (VEH), a second pair with 5 r mol of Dival, and a third pair with 50 r mol of saralasin [3], i.e. an ANGII analogous peptide with a wellknown antagonist effect on the whole current family of ANGII receptors [11] and that proved to have amnestic effect on crab when used in such dose. Thus, six groups were formed: VEH-CT, VEH-TR, DIV-CT, DIV-TR, SAR-CT and SAR-TR. Each group had 34 individuals. Results corresponding to test trials are presented in Fig. 3. Planned comparisons disclosed a signi®cant difference
3
for VEH-CT vs. VEH-TR (F 1; 128 11:26, P , 0:01) and between DIV-CT vs. DIV-TR (F 1; 128 9:44, P , 0:01) but neither for SAR-CT vs. SAR-TR nor between CT-groups. No amnestic effect of Dival was obtained with doses as high as 50 r mol or 5 h mol (data not shown). Thus, while saralasin showed a clear-cut disrupting effect on CSM, con®rming a previous result [4], no amnestic effect was produced by post-training injection of Dival. In brief, we may conclude that ANGII, but not ANGIV, would be the endogenous angiotensin that plays a signi®cant role within the cascade of events related to CSM acquisition and/or consolidation in Chasmagnathus. Such conclusion is supported by three lines of evidence based on both present and previously reported results [2±4]. First, ANGII has a memory facilitatory effect since it yields CSM retention when injected after a weak training protocol. Second, the antagonist saralasin abolishes memory enhancement when it was coinjected with ANGII after weak training and has a notable amnestic effect when it was given after strong training. Third, ANGIV has memory facilitatory effect reverted by Dival, a result similar to that obtained in mammals [1,15], but the endogenous activity of this peptide fails to be demonstrated in crab since amnestic effect of the antagonist can not be shown. Therefore, the role of ANGII as memory enhancer would have merged early in evolution; while the ANGIV receptor, though present at such an early evolutive stage, would not be encompassed in the cascade of events related to crab's CSM. This work was supported by the University of Buenos Aires (grant No. 01/TY04), FONCYT (grant No. 01863) and CONICET (grant No. PID 3847/94). We thank Angel Vidal for technical assistance. Dival was provided by Hedral Therapeutics, USA. [1] Braszko, J.J., Kupryszewski, G., Witczuk, B. and Wisniewski, K., Angiotensin II (3±8)-hexapeptide affects motor activity, performance of passive avoidance and a conditioned avoidance response in rats. Neuroscience, 27 (1988) 777± 783. [2] Delorenzi, A., Pedreira, M.E., Romano, A., Pirola, C.J., Nahmod, V.E. and Maldonado, H., Acute administration of angiotensin II improves long-term habituation in the crab Chasmagnathus. Neurosci. Lett., 196 (1995) 193±196. [3] Delorenzi, A., Pedreira, M.E., Romano, A., Garcia, S.I., Pirola, C.J., Nahmod, V.E. and Maldonado, H., Angiotensin II enhances long-term memory in the crab Chasmagnathus. Brain Res. Bull., 41/4 (1996) 211±220. [4] Delorenzi, A., Locatelli, F., Romano, A., Nahmod, V. and Maldonado, H., Angiotensin II (3±8) induces long-term memory improvement in the crab Chasmagnathus. Neurosci. Lett., 226 (1997) 143±146. [5] Gonzalez, G.C., Roger, Y., Gonzalez, E., Martinez-Padron, M., Moore, G.J. and Lukowiak, K., Angiotensinogen-like epitopes are present in the CNS of Aplysia californica and co-localize with urotensin Y- and urotensin-like immunoreactivities in the cerebral ganglia. NeuroReport, 6 (1995) 541±544. [6] Laurent, V., Bulet, P. and Salzet, M., A comparison of the
4
A. Delorenzi, H. Maldonado / Neuroscience Letters 266 (1999) 1±4
[7]
[8] [9]
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