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Blockade of pro-cognitive effects of angiotensin IV and physostigmine in mice by oxytocin antagonism
European Journal of Pharmacology 683 (2012) 155–160
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural Pharmacology
Blockade of pro-cognitive effects of angiotensin IV and physostigmine in mice by oxytocin antagonism Paul R. Gard ⁎, Cathy Naylor, Sofiya Ali, Clare Partington School of Pharmacy & Biomolecular Sciences, University of Brighton, Moulsecoomb, BRIGHTON, BN2 4GJ, UK
a r t i c l e
i n f o
Article history: Received 18 October 2011 Received in revised form 13 February 2012 Accepted 26 February 2012 Available online 10 March 2012 Keywords: Oxytocin Angiotensin IV Anticholinesterase Learning Memory
a b s t r a c t Low doses of oxytocin enhance learning and memory in animal models. Angiotensin IV inhibits cysteine aminopeptidase, also known as insulin-regulated aminopeptidase and oxytocinase, and enhances memory in animals. The mechanism of this effect of angiotensin IV is unknown. This study explored the role of oxytocin in the cognitive effects of angiotensin IV with physostigmine as a control and used isolated smooth muscle to assess the pharmacological selectivity of the observed antagonism. Using novel object recognition in male mice, the effects of angiotensin IV (4.7 μg/kg), oxytocin (0.1 ng/kg) or physostigmine (200 μg/kg) administered subcutaneously immediately after the second training trial, were assessed in the presence and absence of 10 μg/kg β-mercapto-β–β-cyclopenta-methylenepropionyl; O-Me-Tyr 2, Orn8-oxytocin, an oxytocin antagonist; n = 8 in all cases. The effects of the antagonist on angiotensin IV, oxytocin and acetylcholine-induced contractions of rat isolated uterus were also determined. Oxytocin, angiotensin IV and physostigmine significantly enhanced consolidation of learning (P = 0.04, 0.004 and 0.008 respectively), and there were no significant effects on locomotor activity. The oxytocin antagonist similarly not only significantly improved novel object recognition (P = 0.03) but also significantly increased locomotor activity (P = 0.04). In the learning paradigm the oxytocin antagonist prevented the effects of oxytocin, angiotensin IV and physostigmine but in the uterus, contractions induced by angiotensin IV and acetylcholine were unaffected whilst effects of oxytocin were significantly reduced. These results suggest that the pro-cognitive effects of angiotensin IV may be mediated by accumulation of endogenous oxytocin although the mechanisms underlying the observed interaction between the oxytocin antagonist and physostigmine are unclear. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In rats, subcutaneous and intraperitoneal oxytocin in the pg–ng/kg dose range improves social memory i.e. recognition of conspecific animals (Arletti et al., 1995; Popik et al., 1992, 1996). That oxytocin is inherently involved in this form of memory is demonstrated by the fact that oxytocin-knockout mice display a deficit in social memory which is restored by intracerebroventricular (i.c.v.) oxytocin (1 pg) (Ferguson et al., 2001). In normal animals, however, higher doses of oxytocin, i.e. peripheral doses in the μg–mg/kg range and i.c.v. doses in the ng range, disrupt memory. This is true not only for social memory (Benelli et al., 1995; Popik and Vetulani, 1991; Popik et al., 1996); but also for spatial memory (Wu and Yu, 2004), conditioned avoidance (Boccia et al., 1998), passive avoidance (De Wied et al., 1987, 1991); and learned helplessness (Arletti and Bertolini, 1987; Nowakowska et al., 2002). Angiotensin IV is a component of the renin–angiotensin system, a metabolic product of angiotensin II (see Fig. 1). It has been shown to have positive effects on learning and memory in a range of rodent
⁎ Corresponding author. Tel.: + 44 1273 642084; fax: + 44 1273 642674. E-mail address: [email protected] (P.R. Gard). 0014-2999/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2012.02.048
models such as the Morris water maze in rats (Braszko et al., 1988; Wright et al., 1999) and novel object recognition in mice (Golding et al., 2010).The mechanism of this action is uncertain although the receptor for angiotensin IV has been identified as insulin regulated amino peptidase (IRAP) (Albiston et al., 2001), a membrane-spanning protein of the M1 family of zinc-dependent metallopeptidases with the accepted name of cysteine aminopeptidase (EC 3.4.11.3) but also labelled placental leucine aminopeptidase and oxytocinase. The enzyme is co-located with the insulin-dependent glucose transporter GLUT4 in intracellular vesicles which slowly translocate to the cell surface. Insulin and oxytocin accelerate the translocation (Demaegdt et al., 2008; Nakamura et al., 2000). The action of angiotensin IV on cognition may depend on inhibition of IRAP, and thereby prolongation of activity of substrates such as oxytocin, somatostatin and met-enkephalin, or on trafficking of IRAP and GLUT4 to the cell membrane and increased cellular uptake of glucose (Vanderheyden, 2009). That the effects of angiotensin IV on cognition involve increased glucose uptake is suggested by the facts that angiotensin IV and analogues enhance glucose uptake into rat hippocampal cells in vitro (Albiston et al., 2008; Fernando et al., 2008), and that glucose enhances memory in rodents (Lee et al., 1988; Ragozzino et al., 1998). The recent demonstration that angiotensin IV elevates brain oxytocin
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Angiotensinogen Renin Angiotensin converting enzyme 2
Angiotensin 1-9
Angiotensin I Angiotensin converting enzyme Angiotensin converting enzyme 2
Angiotensin II
Angiotensin 1-7
Angiotensin III
Angiotensin IV
AT1 receptor
AT2 receptor
AT4 receptor (IRAP)
Fig. 1. Schematic illustration of the brain renin–angiotensin system showing synthetic pathways for the angiotensins, the enzymes involved and the target receptors.
concentration in vivo (Beyer et al., 2010), however, raises the possibility that the procognitive effect of angiotensin IV involves oxytocin. The current study explores the consequences of oxytocin antagonism on the effects of angiotensin IV on learning and memory in mice. The cholinesterase inhibitor physostigmine was used as a control nootrope to ascertain the behavioural selectivity of any observed antagonism and the pharmacological selectivity of any antagonism was assessed in isolated rat uterine smooth muscle which possesses oxytocin receptors of similar characteristics to those of the mouse hippocampus (Elands et al., 1987; Lukas et al., 2011).
was calculated as (A− B) / (A+ B) where A was the time spent exploring the novel object and B was the time spent exploring the familiar object. The effects of oxytocin (0.1 ng/kg), angiotensin IV (4.7 μg/kg) and physostigmine (200 μg/kg), administered subcutaneously immediately after the second training session, were assessed in the presence and absence of 10 μg/kg OTA. Drug effects were compared with those of a parallel saline control for each combination, and all experimental group sizes were 8. All behavioural experiments were licenced under the UK Animals (Scientific Procedures) Act, 1986.
2. Materials and methods 2.3. Isolated uterine smooth muscle contractility 2.1. Protocol Memory consolidation was assessed using novel object recognition in the mouse. The effects of oxytocin were determined as were the effects of angiotensin IV and the acetylcholinesterase inhibitor physostigmine in the absence and presence of the selective oxytocin receptor antagonist β-mercapto-β–β-cyclopenta-methylenepropionyl, O-Me-Tyr 2, Orn8-oxytocin (OTA). Selectivity of OTA was assessed by determining its antagonistic effects on the contractile effects of oxytocin, angiotensin IV and acetylcholine in rat isolated uterus. 2.2. Novel object recognition Mice were placed into an open field (34 × 60 cm), the floor of which was marked with a 3 × 6 grid and in which two identical solid, impermeable objects were positioned at one end of the field, each 10 cm from the adjacent walls. Mice were allowed to explore the area for 3 min. One hour later they were again exposed to the same open field and objects, which had been cleaned thoroughly with 50% ethanol, for 3 min. After 24 h the mice were placed again into the cleaned field containing one of the original objects and a novel (different size, shape and colour) object. Time spent exploring each of the objects was recorded over 3 min, as was locomotor activity (line crossing). Exploration was defined as the mouse facing the object with the nose within 5 mm of the object, any time spent on the object, facing outward was thus excluded. Learning (Discrimination, ‘D’, score) was quantified as the proportion of time spent exploring the novel object relative to the familiar object, and
Rat uteri were removed immediately post-mortem and the myometrium was separated immediately distal of the oviduct and proximal of the cervix. The tissue was suspended in De Jalon's solution, normally at 32 °C, gassed with 95%O2/5%CO2 and under a resting tension of 1 g. Isotonic contractile responses to oxytocin, angiotensin IV and acetylcholine, all at 10 − 9–10 − 5 M., were recorded using an eight minute dose cycle and a 30 second contact time. For each tissue, results were expressed as a percentage of the contractile response to 10 − 6 M acetylcholine. The effects of the oxytocin antagonist OTA (10 − 8 and 10 − 7 M) were determined by its addition 60 s prior to the agonist. The experiment was repeated for each drug combination a minimum of six times. In the small number of cases where the tissue was not quiescent at 32 °C, the temperature was reduced to 28 or 30 °C; the effects of any temperature change on contractility were overcome by expression of responses as a percentage of the response obtained to acetylcholine in the same tissue under the same conditions.
2.4. Animal husbandry Male C57BL/6 mice, mean weight 20 g, were bred and reared inhouse under identical conditions. Female Sprague–Dawley rats (mean 240 g) were obtained from Charles River. Animals were housed at a temperature of 21± 1 °C and relative humidity of 55 ± 5% and fed SDS expanded rat and mouse diet ad libitum. All behavioural determinations took place between 1100 and 1600 h.
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2.5. Drugs and chemicals Oxytocin, physostigmine and β-mercapto-β–β-cyclopentamethylenepropionyl; O-Me-Tyr 2, Orn 8-oxytocin were purchased from Sigma-Aldrich; angiotensin IV and acetylcholine were purchased from Bachem. De Jalon's solution is comprised of NaCl (154 mM), KCl (5.63 mM), CaCl2 (0.648 mM), NaHCO3 (5.95 mM) and glucose (2.77 mM). 2.6. Data analysis Behavioural data for the separate saline-treated control groups were tested for normality using the Kolmogorov–Smirnoff test, neither D scores nor locomotor activity (line crossing) significantly differed from normal, and data are thus presented as mean D scores or mean number of lines crossed plus and minus the standard error of the mean. Group means were compared with those of the appropriate parallel saline control value using Student's two-tailed independent ttest, and comparison of multiple groups used one-way ANOVA. Probability of less than 0.05 is taken as indicating a significant difference between the means. Contractile responses of the uterine smooth muscle are expressed as a percentage of the mean response elicited by acetylcholine (10 − 6 M), and are presented as means plus and minus the standard error of the mean. Log concentration–response curves were fitted using a four-variable equation with variable Hill slope (GraphPad Prism™). 3. Results 3.1. Novel object recognition Data from the saline-treated animals showed some natural variation in D score and locomotor activity. Whilst ANOVA demonstrated that there were significant group differences in locomotor activity (F = 38.543; P b 0.01), there were no significant differences in D score between the 4 groups (F = 2.100; P = 0.12); demonstrating the comparability of the litter groups, experimenters, etc. The overall mean D score for the saline groups was 0.03 ± 0.13 (95% C.I.). Despite
Fig. 3. The effects of a combination of oxytocin and β-mercapto-β–β-cyclopentamethylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA) on discrimination (D) scores in the novel object recognition test in male C57BL/6 mice. There were no significant drug effects in comparison to parallel saline-control, P > 0.05. Sample size equals 8 in all cases.
this comparability between groups, results from drug treatment groups were compared with results from parallel saline control animals drawn from the same litter groups, in the same experimental session, by the same experimenter. The results presented in Fig. 2 illustrate that, in comparison to parallel saline control, oxytocin was effective in enhancing novel object recognition in male mice (P= 0.04). Angiotensin IV and physostigmine had similarly significant effects (P = 0.004 and 0.008 respectively). The oxytocin antagonist alone also significantly improved novel object recognition (P= 0.03), but the size of the effect was much smaller than that seen for oxytocin, angiotensin IV and physostigmine. In comparison to parallel saline control, oxytocin in the presence of OTA had no significant effect on D score (P = 0.23, Fig. 3). Similarly, in the presence of OTA, angiotensin IV and physostigmine were unable to produce any significant increase in discrimination between the novel and familiar objects either in comparison to parallel saline control (P = 0.35 and 0.51 respectively) nor in comparison to OTA alone (P = 0.4 and 0.7, Fig. 4). In comparison to saline-control neither angiotensin IV, oxytocin nor physostigmine had any significant effect on locomotor activity, however OTA alone caused a significant increase in locomotor activity
Fig. 2. The effects of oxytocin, angiotensin IV, physostigmine and β-mercapto-β–β-cyclopenta-methylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA) on discrimination (D) scores in the novel object recognition test in male C57BL/6 mice. All drug effects were significantly different from parallel saline control, P b 0.05. Sample size equals 8 in all cases.
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Fig. 4. The effects of angiotensin IV and physostigmine on discrimination (D) scores in the novel object recognition test in male C57BL/6 mice in the presence of β-mercapto-β–βcyclopenta-methylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA). There were no significant drug effects in comparison to OTA alone, P > 0.05. Sample size equals 8 in all cases.
(P = 0.04), which did not persist when the antagonist was combined with angiotensin IV or physostigmine (P = 0.06 and 0.9 respectively), illustrative data are presented in Fig. 5. 3.2. Selectivity of antagonism Log concentration response curves of the contractile effects of oxytocin, angiotensin IV and acetylcholine on isolated rat uterus are presented in Fig. 6, and oxytocin was the most potent and angiotensin IV the least potent of the three agonists. Estimated EC50 values for oxytocin, angiotensin IV and acetylcholine were 6.62 × 10 − 8, 7.87 × 10 − 7 and 3.87 × 10 − 7 M respectively. None of the Hill slopes differed significantly from 1. Contractions induced by oxytocin 10 − 7 M were significantly reduced by the antagonist at 10 − 7 M (P = 0.002, Fig. 7). Contractions induced by angiotensin IV and acetylcholine at 10 − 6 M were not significantly affected by OTA at 10 − 8 M nor 10 − 7 M although in both cases an apparent trend suggests that higher concentrations of OTA may reduce responses to both angiotensin IV and acetylcholine. 4. Discussion The results of this study are the first demonstration that oxytocin can enhance learning and memory in the novel object recognition paradigm in mice; our previous research failed to find a significant effect of oxytocin in BKW strain mice at a much higher, possibly amnesic, dose (Gard et al., 2007). That angiotensin IV has a positive effect in this test has been reported before (Golding et al., 2010). The primary focus of this study was to explore the possible involvement of oxytocin with the effects of angiotensin IV on learning and memory.
Fig. 5. The effects of β-mercapto-β–β-cyclopenta-methylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA) in the presence and absence of angiotensin IV and physostigmine on locomotor activity (line crossing) in the novel object recognition test. OTA alone, but not in combination, caused a significant increase in locomotors activity, P b 0.05. Sample size equals 8 in all cases.
Important aspects of the current study are that the peptides are administered after the second training trial and therefore assess drug effects on consolidation rather than on acquisition or recall, and secondly the peptides are administered peripherally rather than by intracerebroventricular injection, suggesting that the peptides are able to cross the blood brain barrier or act at a site unprotected by that barrier. This approach differs from that used by other groups studying the effects of angiotensin IV and oxytocin on memory, for example Braszko and Harding and Wright, who typically administer angiotensin IV intracerebroventricularly to rats immediately before the recall trial and therefore study drug effects on recall (Braszko et al., 1988; Wright et al., 1999). The sites and mechanisms of the memory-enhancing effects of angiotensin IV and oxytocin remain unresolved. Features of one-trial novel object recognition have been reviewed by Dere et al. (2007) and it is reasonable to suggest that a two-trial novel object recognition has similar neuronal substrates. Dere et al. concluded that the hippocampus is involved in the one-trial form of the memory, but that other brain areas can substitute for the hippocampus following hippocampal damage. Angiotensin IV receptors are present in mouse hippocampus (Fernando et al., 2008), and therefore may be involved in object recognition but oxytocin receptor gene expression was not identified in this tissue (Gould and Zingg, 2003). Of the non-hippocampal brain areas, the perirhinal cortex is probably the most important, and indeed this area is probably critical for novel object recognition. The same review considered the neurochemical components and reported that both muscarinic and nicotinic cholinergic receptors; AMPA and NMDA glutamate receptors; dopaminergic; serotonergic and other receptors may all be involved with the process. Endogenous oxytocin and angiotensin IV and their receptors have not previously been implicated in novel object recognition although there is some evidence that oxytocin influences serotonin
Fig. 6. Contractile responses of rat isolated uterus to oxytocin, angiotensin IV (AngIV) and acetylcholine (ACh), all at 10− 9 to 10− 5 M.
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Fig. 7. The effects of β-mercapto-β–β-cyclopenta-methylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA) on contractions of rat isolated uterus induced by oxytocin, angiotensin IV (AngIV) and acetylcholine (Ach). The antagonist was without significant effect other than against oxytocin at 10− 7 M (P b 0.01). Sample size greater than 6 in all cases.
release in the Raphe nucleus (Yoshida et al., 2009), which may suggest a direction for future research. As outlined in the Introduction, the mechanisms underlying the effects of angiotensin IV on learning and memory remain to be elucidated, although there is a good theoretical basis to the suggestion that the effects may involve translocation of the GLUT4 transported to the cell surface and an increase uptake of glucose. The current demonstration, however, that an oxytocin antagonist prevents the cognition-enhancing effects of angiotensin IV at the dose tested, suggests that oxytocin may be involved in the pro-cognitive effects, potentially via an accumulation of endogenous oxytocin as a consequence of inhibition of IRAP/oxytocinase. The subcutaneous dose of OTA selected for use in vivo was approximately one tenth of that used intraperitoneally previously in prairie voles to demonstrate a behavioural effect (Bales et al., 2004) and thus hopefully was not so excessive as to become non-selective. Indeed the behavioural results suggest that OTA may actually be acting as a partial agonist although the significant change in D score in comparison to parallel saline control may be an anomaly, as may the observed effect on locomotor activity, which would be worthy of replication. Although it is difficult to draw conclusions about drug actions by comparing complex behaviour to contractions of smooth muscle because the two systems are so different and receptor characteristics may differ between different species and tissues (see Introduction), data from the isolated rat uterus suggests that the OTA is a selective antagonist at the oxytocin receptor at the concentrations tested, without any intrinsic activity and devoid of any effect on responses to angiotensin IV. Such data, however, require further consideration: oxytocin stimulates uterine contraction by interaction with the oxytocin receptor which is G-protein coupled, utilising inositol trisphosphate as the second messenger. The mechanism of action of angiotensin IV is less clear. AT1 and AT2 receptors have been clearly identified in the uterus (de Gasparo et al., 2000) but the nature of any uterine AT4 receptors is unknown. Angiotensin IV clearly induces uterine contractions, in a manner that is not fully blocked by the selective AT1-receptor antagonist losartan (Gard et al., 2007), however it is unlikely that interaction of angiotensin IV with IRAP would result in smooth muscle contraction. Investigation of the effects of angiotensin IV on vascular smooth muscle suggests that the actions are mediated by AT1 receptors (Yang et al., 2008), and the AT1 receptor has even been implicated in some central effects of angiotensin IV (De Bundel et al., 2010). The interplay of angiotensin IV, AT4 and AT1 receptors therefore remains to be clarified but the results of the current study clearly indicate that OTA does not block the effects of angiotensin IV in the uterus at the concentrations tested but whether this indicates that the antagonist does not block the AT4 receptor in vivo in those brain areas responsible for memory consolidation requires elucidation. The dose of physostigmine selected was in line with doses used by previous workers, albeit with conflicting results (Dong et al., 2005;
Ennaceur and Meliani, 1992); in this study there were positive effects on memory consolidation, presumably due to accumulation of brain acetylcholine subsequent to inhibition of cholinesterase. OTA was able to prevent the positive effects of physostigmine in the novel object recognition test. The studies in rat isolated uterus indicated that the oxytocin antagonist does not block the effects of acetylcholine in the uterus. Acetylcholine induces uterine contractions via the muscarinic M3 receptor (Varol et al., 1988), and these results therefore suggest that the prevention of the behavioural effect of physostigmine by the oxytocin antagonist was not due to antagonism of M3 receptors in the brain. Other possible explanations for the effects of the oxytocin antagonist on the effects of physostigmine would be prevention of the anticholinesterase activity; action as an antagonist at the muscarinic (non-M3) or nicotinic receptors or possible mediation of the effects of physostigmine by oxytocin. There is no previously published evidence to support the latter suggestions and thus the selectivity of antagonism elicited by OTA is worthy of further investigation. In conclusion, the results of the current study have demonstrated a positive effect of low dose oxytocin on memory consolidation in the two-trial novel object recognition test in male C57BL/6 mice. Oxytocin is also implicated in mediating the memory-enhancing effects of both angiotensin IV (and possibly physostigmine) by the demonstration that the memory-enhancing actions of both of these agents were prevented by a selective oxytocin antagonist.
Acknowledgements This research was funded by internal funds made available by the University of Brighton.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural Pharmacology
Blockade of pro-cognitive effects of angiotensin IV and physostigmine in mice by oxytocin antagonism Paul R. Gard ⁎, Cathy Naylor, Sofiya Ali, Clare Partington School of Pharmacy & Biomolecular Sciences, University of Brighton, Moulsecoomb, BRIGHTON, BN2 4GJ, UK
a r t i c l e
i n f o
Article history: Received 18 October 2011 Received in revised form 13 February 2012 Accepted 26 February 2012 Available online 10 March 2012 Keywords: Oxytocin Angiotensin IV Anticholinesterase Learning Memory
a b s t r a c t Low doses of oxytocin enhance learning and memory in animal models. Angiotensin IV inhibits cysteine aminopeptidase, also known as insulin-regulated aminopeptidase and oxytocinase, and enhances memory in animals. The mechanism of this effect of angiotensin IV is unknown. This study explored the role of oxytocin in the cognitive effects of angiotensin IV with physostigmine as a control and used isolated smooth muscle to assess the pharmacological selectivity of the observed antagonism. Using novel object recognition in male mice, the effects of angiotensin IV (4.7 μg/kg), oxytocin (0.1 ng/kg) or physostigmine (200 μg/kg) administered subcutaneously immediately after the second training trial, were assessed in the presence and absence of 10 μg/kg β-mercapto-β–β-cyclopenta-methylenepropionyl; O-Me-Tyr 2, Orn8-oxytocin, an oxytocin antagonist; n = 8 in all cases. The effects of the antagonist on angiotensin IV, oxytocin and acetylcholine-induced contractions of rat isolated uterus were also determined. Oxytocin, angiotensin IV and physostigmine significantly enhanced consolidation of learning (P = 0.04, 0.004 and 0.008 respectively), and there were no significant effects on locomotor activity. The oxytocin antagonist similarly not only significantly improved novel object recognition (P = 0.03) but also significantly increased locomotor activity (P = 0.04). In the learning paradigm the oxytocin antagonist prevented the effects of oxytocin, angiotensin IV and physostigmine but in the uterus, contractions induced by angiotensin IV and acetylcholine were unaffected whilst effects of oxytocin were significantly reduced. These results suggest that the pro-cognitive effects of angiotensin IV may be mediated by accumulation of endogenous oxytocin although the mechanisms underlying the observed interaction between the oxytocin antagonist and physostigmine are unclear. © 2012 Elsevier B.V. All rights reserved.
1. Introduction In rats, subcutaneous and intraperitoneal oxytocin in the pg–ng/kg dose range improves social memory i.e. recognition of conspecific animals (Arletti et al., 1995; Popik et al., 1992, 1996). That oxytocin is inherently involved in this form of memory is demonstrated by the fact that oxytocin-knockout mice display a deficit in social memory which is restored by intracerebroventricular (i.c.v.) oxytocin (1 pg) (Ferguson et al., 2001). In normal animals, however, higher doses of oxytocin, i.e. peripheral doses in the μg–mg/kg range and i.c.v. doses in the ng range, disrupt memory. This is true not only for social memory (Benelli et al., 1995; Popik and Vetulani, 1991; Popik et al., 1996); but also for spatial memory (Wu and Yu, 2004), conditioned avoidance (Boccia et al., 1998), passive avoidance (De Wied et al., 1987, 1991); and learned helplessness (Arletti and Bertolini, 1987; Nowakowska et al., 2002). Angiotensin IV is a component of the renin–angiotensin system, a metabolic product of angiotensin II (see Fig. 1). It has been shown to have positive effects on learning and memory in a range of rodent
⁎ Corresponding author. Tel.: + 44 1273 642084; fax: + 44 1273 642674. E-mail address: [email protected] (P.R. Gard). 0014-2999/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2012.02.048
models such as the Morris water maze in rats (Braszko et al., 1988; Wright et al., 1999) and novel object recognition in mice (Golding et al., 2010).The mechanism of this action is uncertain although the receptor for angiotensin IV has been identified as insulin regulated amino peptidase (IRAP) (Albiston et al., 2001), a membrane-spanning protein of the M1 family of zinc-dependent metallopeptidases with the accepted name of cysteine aminopeptidase (EC 3.4.11.3) but also labelled placental leucine aminopeptidase and oxytocinase. The enzyme is co-located with the insulin-dependent glucose transporter GLUT4 in intracellular vesicles which slowly translocate to the cell surface. Insulin and oxytocin accelerate the translocation (Demaegdt et al., 2008; Nakamura et al., 2000). The action of angiotensin IV on cognition may depend on inhibition of IRAP, and thereby prolongation of activity of substrates such as oxytocin, somatostatin and met-enkephalin, or on trafficking of IRAP and GLUT4 to the cell membrane and increased cellular uptake of glucose (Vanderheyden, 2009). That the effects of angiotensin IV on cognition involve increased glucose uptake is suggested by the facts that angiotensin IV and analogues enhance glucose uptake into rat hippocampal cells in vitro (Albiston et al., 2008; Fernando et al., 2008), and that glucose enhances memory in rodents (Lee et al., 1988; Ragozzino et al., 1998). The recent demonstration that angiotensin IV elevates brain oxytocin
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Angiotensinogen Renin Angiotensin converting enzyme 2
Angiotensin 1-9
Angiotensin I Angiotensin converting enzyme Angiotensin converting enzyme 2
Angiotensin II
Angiotensin 1-7
Angiotensin III
Angiotensin IV
AT1 receptor
AT2 receptor
AT4 receptor (IRAP)
Fig. 1. Schematic illustration of the brain renin–angiotensin system showing synthetic pathways for the angiotensins, the enzymes involved and the target receptors.
concentration in vivo (Beyer et al., 2010), however, raises the possibility that the procognitive effect of angiotensin IV involves oxytocin. The current study explores the consequences of oxytocin antagonism on the effects of angiotensin IV on learning and memory in mice. The cholinesterase inhibitor physostigmine was used as a control nootrope to ascertain the behavioural selectivity of any observed antagonism and the pharmacological selectivity of any antagonism was assessed in isolated rat uterine smooth muscle which possesses oxytocin receptors of similar characteristics to those of the mouse hippocampus (Elands et al., 1987; Lukas et al., 2011).
was calculated as (A− B) / (A+ B) where A was the time spent exploring the novel object and B was the time spent exploring the familiar object. The effects of oxytocin (0.1 ng/kg), angiotensin IV (4.7 μg/kg) and physostigmine (200 μg/kg), administered subcutaneously immediately after the second training session, were assessed in the presence and absence of 10 μg/kg OTA. Drug effects were compared with those of a parallel saline control for each combination, and all experimental group sizes were 8. All behavioural experiments were licenced under the UK Animals (Scientific Procedures) Act, 1986.
2. Materials and methods 2.3. Isolated uterine smooth muscle contractility 2.1. Protocol Memory consolidation was assessed using novel object recognition in the mouse. The effects of oxytocin were determined as were the effects of angiotensin IV and the acetylcholinesterase inhibitor physostigmine in the absence and presence of the selective oxytocin receptor antagonist β-mercapto-β–β-cyclopenta-methylenepropionyl, O-Me-Tyr 2, Orn8-oxytocin (OTA). Selectivity of OTA was assessed by determining its antagonistic effects on the contractile effects of oxytocin, angiotensin IV and acetylcholine in rat isolated uterus. 2.2. Novel object recognition Mice were placed into an open field (34 × 60 cm), the floor of which was marked with a 3 × 6 grid and in which two identical solid, impermeable objects were positioned at one end of the field, each 10 cm from the adjacent walls. Mice were allowed to explore the area for 3 min. One hour later they were again exposed to the same open field and objects, which had been cleaned thoroughly with 50% ethanol, for 3 min. After 24 h the mice were placed again into the cleaned field containing one of the original objects and a novel (different size, shape and colour) object. Time spent exploring each of the objects was recorded over 3 min, as was locomotor activity (line crossing). Exploration was defined as the mouse facing the object with the nose within 5 mm of the object, any time spent on the object, facing outward was thus excluded. Learning (Discrimination, ‘D’, score) was quantified as the proportion of time spent exploring the novel object relative to the familiar object, and
Rat uteri were removed immediately post-mortem and the myometrium was separated immediately distal of the oviduct and proximal of the cervix. The tissue was suspended in De Jalon's solution, normally at 32 °C, gassed with 95%O2/5%CO2 and under a resting tension of 1 g. Isotonic contractile responses to oxytocin, angiotensin IV and acetylcholine, all at 10 − 9–10 − 5 M., were recorded using an eight minute dose cycle and a 30 second contact time. For each tissue, results were expressed as a percentage of the contractile response to 10 − 6 M acetylcholine. The effects of the oxytocin antagonist OTA (10 − 8 and 10 − 7 M) were determined by its addition 60 s prior to the agonist. The experiment was repeated for each drug combination a minimum of six times. In the small number of cases where the tissue was not quiescent at 32 °C, the temperature was reduced to 28 or 30 °C; the effects of any temperature change on contractility were overcome by expression of responses as a percentage of the response obtained to acetylcholine in the same tissue under the same conditions.
2.4. Animal husbandry Male C57BL/6 mice, mean weight 20 g, were bred and reared inhouse under identical conditions. Female Sprague–Dawley rats (mean 240 g) were obtained from Charles River. Animals were housed at a temperature of 21± 1 °C and relative humidity of 55 ± 5% and fed SDS expanded rat and mouse diet ad libitum. All behavioural determinations took place between 1100 and 1600 h.
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2.5. Drugs and chemicals Oxytocin, physostigmine and β-mercapto-β–β-cyclopentamethylenepropionyl; O-Me-Tyr 2, Orn 8-oxytocin were purchased from Sigma-Aldrich; angiotensin IV and acetylcholine were purchased from Bachem. De Jalon's solution is comprised of NaCl (154 mM), KCl (5.63 mM), CaCl2 (0.648 mM), NaHCO3 (5.95 mM) and glucose (2.77 mM). 2.6. Data analysis Behavioural data for the separate saline-treated control groups were tested for normality using the Kolmogorov–Smirnoff test, neither D scores nor locomotor activity (line crossing) significantly differed from normal, and data are thus presented as mean D scores or mean number of lines crossed plus and minus the standard error of the mean. Group means were compared with those of the appropriate parallel saline control value using Student's two-tailed independent ttest, and comparison of multiple groups used one-way ANOVA. Probability of less than 0.05 is taken as indicating a significant difference between the means. Contractile responses of the uterine smooth muscle are expressed as a percentage of the mean response elicited by acetylcholine (10 − 6 M), and are presented as means plus and minus the standard error of the mean. Log concentration–response curves were fitted using a four-variable equation with variable Hill slope (GraphPad Prism™). 3. Results 3.1. Novel object recognition Data from the saline-treated animals showed some natural variation in D score and locomotor activity. Whilst ANOVA demonstrated that there were significant group differences in locomotor activity (F = 38.543; P b 0.01), there were no significant differences in D score between the 4 groups (F = 2.100; P = 0.12); demonstrating the comparability of the litter groups, experimenters, etc. The overall mean D score for the saline groups was 0.03 ± 0.13 (95% C.I.). Despite
Fig. 3. The effects of a combination of oxytocin and β-mercapto-β–β-cyclopentamethylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA) on discrimination (D) scores in the novel object recognition test in male C57BL/6 mice. There were no significant drug effects in comparison to parallel saline-control, P > 0.05. Sample size equals 8 in all cases.
this comparability between groups, results from drug treatment groups were compared with results from parallel saline control animals drawn from the same litter groups, in the same experimental session, by the same experimenter. The results presented in Fig. 2 illustrate that, in comparison to parallel saline control, oxytocin was effective in enhancing novel object recognition in male mice (P= 0.04). Angiotensin IV and physostigmine had similarly significant effects (P = 0.004 and 0.008 respectively). The oxytocin antagonist alone also significantly improved novel object recognition (P= 0.03), but the size of the effect was much smaller than that seen for oxytocin, angiotensin IV and physostigmine. In comparison to parallel saline control, oxytocin in the presence of OTA had no significant effect on D score (P = 0.23, Fig. 3). Similarly, in the presence of OTA, angiotensin IV and physostigmine were unable to produce any significant increase in discrimination between the novel and familiar objects either in comparison to parallel saline control (P = 0.35 and 0.51 respectively) nor in comparison to OTA alone (P = 0.4 and 0.7, Fig. 4). In comparison to saline-control neither angiotensin IV, oxytocin nor physostigmine had any significant effect on locomotor activity, however OTA alone caused a significant increase in locomotor activity
Fig. 2. The effects of oxytocin, angiotensin IV, physostigmine and β-mercapto-β–β-cyclopenta-methylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA) on discrimination (D) scores in the novel object recognition test in male C57BL/6 mice. All drug effects were significantly different from parallel saline control, P b 0.05. Sample size equals 8 in all cases.
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Fig. 4. The effects of angiotensin IV and physostigmine on discrimination (D) scores in the novel object recognition test in male C57BL/6 mice in the presence of β-mercapto-β–βcyclopenta-methylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA). There were no significant drug effects in comparison to OTA alone, P > 0.05. Sample size equals 8 in all cases.
(P = 0.04), which did not persist when the antagonist was combined with angiotensin IV or physostigmine (P = 0.06 and 0.9 respectively), illustrative data are presented in Fig. 5. 3.2. Selectivity of antagonism Log concentration response curves of the contractile effects of oxytocin, angiotensin IV and acetylcholine on isolated rat uterus are presented in Fig. 6, and oxytocin was the most potent and angiotensin IV the least potent of the three agonists. Estimated EC50 values for oxytocin, angiotensin IV and acetylcholine were 6.62 × 10 − 8, 7.87 × 10 − 7 and 3.87 × 10 − 7 M respectively. None of the Hill slopes differed significantly from 1. Contractions induced by oxytocin 10 − 7 M were significantly reduced by the antagonist at 10 − 7 M (P = 0.002, Fig. 7). Contractions induced by angiotensin IV and acetylcholine at 10 − 6 M were not significantly affected by OTA at 10 − 8 M nor 10 − 7 M although in both cases an apparent trend suggests that higher concentrations of OTA may reduce responses to both angiotensin IV and acetylcholine. 4. Discussion The results of this study are the first demonstration that oxytocin can enhance learning and memory in the novel object recognition paradigm in mice; our previous research failed to find a significant effect of oxytocin in BKW strain mice at a much higher, possibly amnesic, dose (Gard et al., 2007). That angiotensin IV has a positive effect in this test has been reported before (Golding et al., 2010). The primary focus of this study was to explore the possible involvement of oxytocin with the effects of angiotensin IV on learning and memory.
Fig. 5. The effects of β-mercapto-β–β-cyclopenta-methylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA) in the presence and absence of angiotensin IV and physostigmine on locomotor activity (line crossing) in the novel object recognition test. OTA alone, but not in combination, caused a significant increase in locomotors activity, P b 0.05. Sample size equals 8 in all cases.
Important aspects of the current study are that the peptides are administered after the second training trial and therefore assess drug effects on consolidation rather than on acquisition or recall, and secondly the peptides are administered peripherally rather than by intracerebroventricular injection, suggesting that the peptides are able to cross the blood brain barrier or act at a site unprotected by that barrier. This approach differs from that used by other groups studying the effects of angiotensin IV and oxytocin on memory, for example Braszko and Harding and Wright, who typically administer angiotensin IV intracerebroventricularly to rats immediately before the recall trial and therefore study drug effects on recall (Braszko et al., 1988; Wright et al., 1999). The sites and mechanisms of the memory-enhancing effects of angiotensin IV and oxytocin remain unresolved. Features of one-trial novel object recognition have been reviewed by Dere et al. (2007) and it is reasonable to suggest that a two-trial novel object recognition has similar neuronal substrates. Dere et al. concluded that the hippocampus is involved in the one-trial form of the memory, but that other brain areas can substitute for the hippocampus following hippocampal damage. Angiotensin IV receptors are present in mouse hippocampus (Fernando et al., 2008), and therefore may be involved in object recognition but oxytocin receptor gene expression was not identified in this tissue (Gould and Zingg, 2003). Of the non-hippocampal brain areas, the perirhinal cortex is probably the most important, and indeed this area is probably critical for novel object recognition. The same review considered the neurochemical components and reported that both muscarinic and nicotinic cholinergic receptors; AMPA and NMDA glutamate receptors; dopaminergic; serotonergic and other receptors may all be involved with the process. Endogenous oxytocin and angiotensin IV and their receptors have not previously been implicated in novel object recognition although there is some evidence that oxytocin influences serotonin
Fig. 6. Contractile responses of rat isolated uterus to oxytocin, angiotensin IV (AngIV) and acetylcholine (ACh), all at 10− 9 to 10− 5 M.
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Fig. 7. The effects of β-mercapto-β–β-cyclopenta-methylenepropionyl; O-Me-Tyr2, Orn8-oxytocin (oxytocin antagonist, OTA) on contractions of rat isolated uterus induced by oxytocin, angiotensin IV (AngIV) and acetylcholine (Ach). The antagonist was without significant effect other than against oxytocin at 10− 7 M (P b 0.01). Sample size greater than 6 in all cases.
release in the Raphe nucleus (Yoshida et al., 2009), which may suggest a direction for future research. As outlined in the Introduction, the mechanisms underlying the effects of angiotensin IV on learning and memory remain to be elucidated, although there is a good theoretical basis to the suggestion that the effects may involve translocation of the GLUT4 transported to the cell surface and an increase uptake of glucose. The current demonstration, however, that an oxytocin antagonist prevents the cognition-enhancing effects of angiotensin IV at the dose tested, suggests that oxytocin may be involved in the pro-cognitive effects, potentially via an accumulation of endogenous oxytocin as a consequence of inhibition of IRAP/oxytocinase. The subcutaneous dose of OTA selected for use in vivo was approximately one tenth of that used intraperitoneally previously in prairie voles to demonstrate a behavioural effect (Bales et al., 2004) and thus hopefully was not so excessive as to become non-selective. Indeed the behavioural results suggest that OTA may actually be acting as a partial agonist although the significant change in D score in comparison to parallel saline control may be an anomaly, as may the observed effect on locomotor activity, which would be worthy of replication. Although it is difficult to draw conclusions about drug actions by comparing complex behaviour to contractions of smooth muscle because the two systems are so different and receptor characteristics may differ between different species and tissues (see Introduction), data from the isolated rat uterus suggests that the OTA is a selective antagonist at the oxytocin receptor at the concentrations tested, without any intrinsic activity and devoid of any effect on responses to angiotensin IV. Such data, however, require further consideration: oxytocin stimulates uterine contraction by interaction with the oxytocin receptor which is G-protein coupled, utilising inositol trisphosphate as the second messenger. The mechanism of action of angiotensin IV is less clear. AT1 and AT2 receptors have been clearly identified in the uterus (de Gasparo et al., 2000) but the nature of any uterine AT4 receptors is unknown. Angiotensin IV clearly induces uterine contractions, in a manner that is not fully blocked by the selective AT1-receptor antagonist losartan (Gard et al., 2007), however it is unlikely that interaction of angiotensin IV with IRAP would result in smooth muscle contraction. Investigation of the effects of angiotensin IV on vascular smooth muscle suggests that the actions are mediated by AT1 receptors (Yang et al., 2008), and the AT1 receptor has even been implicated in some central effects of angiotensin IV (De Bundel et al., 2010). The interplay of angiotensin IV, AT4 and AT1 receptors therefore remains to be clarified but the results of the current study clearly indicate that OTA does not block the effects of angiotensin IV in the uterus at the concentrations tested but whether this indicates that the antagonist does not block the AT4 receptor in vivo in those brain areas responsible for memory consolidation requires elucidation. The dose of physostigmine selected was in line with doses used by previous workers, albeit with conflicting results (Dong et al., 2005;
Ennaceur and Meliani, 1992); in this study there were positive effects on memory consolidation, presumably due to accumulation of brain acetylcholine subsequent to inhibition of cholinesterase. OTA was able to prevent the positive effects of physostigmine in the novel object recognition test. The studies in rat isolated uterus indicated that the oxytocin antagonist does not block the effects of acetylcholine in the uterus. Acetylcholine induces uterine contractions via the muscarinic M3 receptor (Varol et al., 1988), and these results therefore suggest that the prevention of the behavioural effect of physostigmine by the oxytocin antagonist was not due to antagonism of M3 receptors in the brain. Other possible explanations for the effects of the oxytocin antagonist on the effects of physostigmine would be prevention of the anticholinesterase activity; action as an antagonist at the muscarinic (non-M3) or nicotinic receptors or possible mediation of the effects of physostigmine by oxytocin. There is no previously published evidence to support the latter suggestions and thus the selectivity of antagonism elicited by OTA is worthy of further investigation. In conclusion, the results of the current study have demonstrated a positive effect of low dose oxytocin on memory consolidation in the two-trial novel object recognition test in male C57BL/6 mice. Oxytocin is also implicated in mediating the memory-enhancing effects of both angiotensin IV (and possibly physostigmine) by the demonstration that the memory-enhancing actions of both of these agents were prevented by a selective oxytocin antagonist.
Acknowledgements This research was funded by internal funds made available by the University of Brighton.
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