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Role for purinergic receptors in memory processing in young chicks
Behavioural Brain Research 223 (2011) 417–420
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Role for purinergic receptors in memory processing in young chicks Catherine Cronin a,∗,2 , Thomas M. Edwards a,3 , Marie E. Gibbs b,1 a b
School of Psychology & Psychiatry, Monash University, Clayton, 3800 Victoria, Australia Department of Anatomy & Developmental Biology, School of Biomedical Sciences, Monash University, Clayton, 3800 Victoria, Australia
a r t i c l e
i n f o
Article history: Received 16 December 2010 Received in revised form 1 May 2011 Accepted 3 May 2011 Available online 10 May 2011 Keywords: ATP Suramin PPADS Discriminative avoidance learning Memory Chick
a b s t r a c t The current study used a single trial bead discrimination task for the young chick to ascertain if inhibitors of P2 purinergic receptors would impair memory retention. Suramin and PPADS provided similar retention profiles. Loss of memory retention was evident by 60 min post-training. Both drugs caused persistent memory loss which was still evident 24 h post-training. These findings suggest that P2 receptors have a role in memory processing. © 2011 Elsevier B.V. All rights reserved.
Purinergic receptors have important roles in many systems in the peripheral and central nervous system [3,7]. They are found in many brain regions, including those associated with learning and memory, e.g. the hippocampus, cerebral cortex, basal ganglia, locus coeruleus, and it is likely that they are important in information processing [1]. In the chick, purinergic receptors are also found in memory-associated brain regions [22] and on both neurons and glial cells [7,21]. The activation of purinergic receptors is associated with adenosine-triphosphate (ATP), which acts as a co-transmitter in the central nervous system, being released from neurons along with glutamate, acetylcholine, GABA, dopamine and noradrenaline [4]. Purinergic receptors were categorised, based upon ligand affinity, into two families, the P1 and the P2 receptors [1,2]. ATP can activate P1 and P2 receptors either directly or indirectly, through hydrolysation. Specifically, P1 receptors are activated by adeno-
Abbreviations: APV, dl-2-amino-5-phosphonopentanoic acid; ATP, adenosine triphosphate; ADP, adenosine diphosphate; DR, discrimination ratio; IMM, intermediate medial mesopallium; NMDA, N-methyl d-aspartate; PPADS, pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonate; P1 and P2, purinergic receptors; s.e.m., standard error of mean. ∗ Corresponding author. Tel.: +61 3 0421461688. E-mail addresses: [email protected], catherine [email protected] (C. Cronin). 1 Current address: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052 Victoria, Australia. 2 Current address: Swinburne University of Technology, John Street, Hawthorn, VIC 3122, Australia. 3 Current address: Tabor Victoria, 9/44-60 Jacksons Rd, Mulgrave, VIC 3170, Australia. 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.05.002
sine, while P2 receptors are activated by nucleotides, such as ATP, and also by the hydrolyzed products of ATP–ADP and adenosine [3,17]. The P2 receptor family is further subdivided into ionotropic P2X receptors, which are ligand-gated ion channels permeable to Ca2+ and Na+ ions [3], and metabotropic P2Y G-protein-coupled receptors (GPCR), whose function varies depending on the specific G-protein. Currently eight P2Y receptors and seven P2X receptors have been identified. Although there are some studies on the role of purines in longterm potentiation (LTP) in the mammalian hippocampus (e.g. Ref. [3]), there are few studies using in vivo animal models, which directly investigate memory processing [1,2,20]. It was the intention of this study to use a pharmaco-behavioural methodology to identify a role for P2 receptors in memory processing. Young chicks were used in place of rodents given that they are amenable to a temporally precise and well defined single trial bead discrimination task. Moreover, any retention loss could be compared to the detailed Gibbs and Ng model of memory formation [11] and inferences made about potential direct, or indirect [5,13], actions on memory processing. To investigate the involvement of purinergic receptors in memory processing suramin sodium salt (suramin) and peridoxal phosphate-6-azo benzene-2,4,-disulfonic acid (PPADS); SigmaAldrich, NSW, Australia were used. Suramin is a broad spectrum P2 receptor antagonist while PPADS is a more selective P2 receptor antagonist. An inhibitory effect of drug administration on memory retention is suggestive of a role for P2 receptors in memory processing. One- and two-day-old chicks from an egg-laying hybrid strain (New Hampshire, Rhode Island Red, White Leghorn and Black Aus-
418
C. Cronin et al. / Behavioural Brain Research 223 (2011) 417–420
tralorp) were used. Chicks were collected from a local hatchery. In a purpose-built laboratory they were housed in random pairs in wooden pens (18 cm × 25 cm × 20 cm), with the measures from 20 chicks representing one data-point. Chicks were kept in pairs to avoid isolation stress, which can affect retention [23]. Crushed poultry food was provided ad libitum. The bead discrimination task consisted of various pre-training, training and testing trials [10]. During successive pretraining trials the chicks were presented with chrome, red and blue beads dipped in water. Red and blue bead trials lasted 10 s and all pretraining trials were designed to encourage pecking at novel objects. A single training trial used a similar red bead to that used in pretraining but now dipped in a bitter-tasting non-toxic taste aversant, methylanthranilate (MeA; Sigma-Aldrich Inc., NSW, Australia). This bead was presented to each pair of chicks for 10 s. For each group of 20 chicks retention testing occurred at a time related to the purpose of the experiment. Testing consisted of sequential presentation of a dry red bead and a dry blue bead, similar to those used in pretraining, for 10 s. Chicks that did not train or peck the non-aversive blue bead at test were excluded from the data analysis. Exclusions were few and equally scattered amongst all groups. The numbers of pecks from each chick in each trial was recorded. The level of memory retention for each chick was calculated as a discrimination ratio (DR), this being the number of pecks to the blue bead at test compared to the total number of peaks to the red and blue beads at test. A DR of 1.0 indicated perfect retention while a DR of 0.5 indicated complete amnesia. A mean DR was then calculated for each group of ≤20 chicks representing a single data-point. Suramin, PPADS or physiological saline (154 mM NaCl) were injected bi-laterally and intracranially into the intermediate medial mesopallium (IMM), an area important for memory processing [14,16]. Injections were administered freehand by a trained researcher using a Hamilton repeating dispenser syringe. The syringe was fitted with a 27-gauge needle of which only the final 3.5 mm was exposed. 10 l of solution was injected per hemisphere. Dose response studies were carried out to determine if suramin and/or PPADS impaired retention and to find the lowest concentration that produced maximal impairment. Each concentration tested was administered 2.5 min after training. Retention was measured 120 min post-training, which is well within the long-term memory stage. A one-way analysis of variance (ANOVA) revealed a significant effect of suramin [F(5,106) = 7.73, p < .001]. Post hoc analysis using Dunnett’s test showed that all doses greater than 5 pmol resulted in significant retention loss when compared to the saline control group. 300 pmol/hemisphere of suramin (p < .05) was the lowest dose to maximally inhibit retention and was used in subsequent experiments (Fig. 1A). The dose response function for PPADS (Fig. 1B) also revealed a significant retention loss [F(3,72) = 9.81, p < .001]. Using a Dunnett’s post hoc analysis doses of 30 (p < .05) and 100 pmol/hemisphere (p < .001) produced significant retention losses when compared with the saline control group. In subsequent experiments 100 pmol/hemisphere of PPADS was used as it maximally impaired retention. Experiments were conducted to determine the optimal time of administration for suramin and PPADS. Drugs were injected at predetermined intervals after training. Chicks were administered either 300 pmol suramin or 100 pmol PPADS, with saline administered at 2.5 min after training as the control in both experiments. Retention was tested at 120 min post-training. When suramin was administrated at 2.5 and 5 min post-training or 25 and 35 min post-training, there was a significant loss of retention – but no loss of retention was observed in the interim. A one-way ANOVA revealed a significant effect [F(10,192) = 7.419, p < .001]). Dunnett’s post hoc analysis showed that injection times of +2.5, +5, +25, +30
Fig. 1. Effect of increasing concentrations of (A) suramin and (B) PPADS on retention tested 120 min after training. All drugs were administered to the chicks 2.5 min after training. All groups were tested 120 min post-training. The mean discrimination ratio ± s.e.m. were calculated for each group of chicks (*p < .05).
and +35 min caused significant retention loss (p < .05), and further confirmed two periods of statistically significant retention loss (Fig. 2A). The time of administration study for PPADS showed a similar pattern of effective administration times (one-way ANOVA [F(8,145) = 10.56, p < .001]). Dunnett’s post hoc test showed that injection times of +0, +2.5 and +35 min caused significant retention loss (p < .001) – but not at times of administration between these (Fig. 2B). Retention function studies were conducted with both drugs to determine at what time retention loss first occurred and whether it was persistent or transient. Time of retention loss indicates the stage of memory formation a drug interferes with and when compared to past findings suggests potential biochemical relationships between mechanisms. Whether a retention loss profile is persistent or transient is important as it indicates whether a memory formation or retrieval mechanism is affected. For example an effect on retrieval mechanisms is suggested if a transient retention loss is apparent given that the trace is not extinguished but its expression only interrupted for a time. Suramin (300 pmol/hemisphere) or saline were injected 2.5 min post-training and retention was measured at times between10 min and 24 h after training in separate groups of chicks (Fig. 3A). A trend in memory loss became evident after 30 min post-training. A two-way ANOVA yielded a significant main effect for drug [F(1,342) = 66.14, p < .001], time of test [F(9,342) = 2.65, p < .01] and
C. Cronin et al. / Behavioural Brain Research 223 (2011) 417–420
Fig. 2. Time of administration study for (A) suramin and (B) PPADS. Suramin (300 pmol/hemisphere), PPADS (100 pmol/hemisphere) or saline injected at times post-training. Retention was tested 120 min post-training. The mean discrimination ratio ± s.e.m. were calculated for each group of chicks (*p < .05).
significant drug x time interaction effects [F(9,343) = 2.46, p < 005]. Simple main effects post hoc analysis identified a significant retention loss (p < .05) from 60 min post-training, and at the following times: +65, +85, +120, and +180 min, until the completion of the experiment 24 h post-training. A retention function study with PPADS (100 pmol/hemisphere) administered 2.5 min after training also demonstrated a trend in loss of memory after 30 min post-training (Fig. 3B). A one-way ANOVA revealed a significant difference in retention between saline and PPADS treated chicks [F(6.127) = 13.59, p < .001]. Using a Dunnett’s post hoc analysis chicks tested 60, 120 min and 24 h post-training had retention levels significantly less than controls (p < .001). Purinergic receptors represent a new and interesting target for behavioural neuroscientists. However, little is known about what role they may have in memory processing, the memory stage in which their action becomes important and the cellular processes impacting – or impacted by – P2 receptors. To overcome this problem in this study we first sought to determine if P2 receptors were required for memory processing using a well-established avian task. Given the temporal specificity of this task it was possible to identify the stage of memory in which P2 receptors were required. Given the wealth of literature [8,11,12] in which this task has been used, and the Gibbs and Ng model generated from such findings, it is possible to speculate upon those cellular processes related to P2 receptor activation. The results of this study show that P2 receptors are important for memory processing given that administration of both suramin
419
Fig. 3. Retention function study observed the effect of (A) Suramin (300 pmol/hemisphere) administered 2.5 min post-training. Separate groups of chicks were tested at times between 10 min and 24 h after training. (B) PPADS (100 pmol/hemisphere) 2.5 min after training and separate groups of chicks tested at times between 10 and 24 h min after training. The mean discrimination ratio +/s.e.m. were calculated for each group of chicks (*p < .05).
and PPADS resulted in retention loss. That two periods of effective administration times were found suggests an action of these drugs early in the short-term memory stage and at the interface of phase A and B of the intermediate-term memory stage as defined by the Gibbs and Ng model. This pattern is also seen with the NMDA receptor antagonist D-APV when administered into the hippocampus [9] and in the IMM (Gibbs unpublished). Moreover, memory loss following drug administration 2.5 min after training results in a progressive trend in retention loss after 30 min post-training which was significant by 60 min post-training and persistent until the end of the experiment 24 h post-training. It is interesting to note that 60 min represents the start of the long-term memory stage within the Gibbs and Ng model and that this retention loss profile is similar to that of APV injection into the IMM [18,19]. Suramin is not as selective for purinergic receptors as is PPADS [6,15] and memory is vulnerable to inhibition at more times than PPADS, which may reflect actions on other receptors as well. Nonetheless the pattern of effective drug administration times and retention loss profile is very similar for both drugs providing confirmatory support that P2 receptor inhibition was the cause of the retention losses observed. Whether there is an indirect or direct relationship between the P2 purinergic receptors and NMDA receptors and LTP is not revealed in these experiments, but they do suggest that there is endogenous release of ATP at the time of learning and memory consolidation because inhibition of P2 receptors will prevent the action of endogenously released ATP.
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C. Cronin et al. / Behavioural Brain Research 223 (2011) 417–420
Acknowledgement We gratefully acknowledge the technical assistance of Ms E. Hartley. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbr.2011.05.002. References [1] Bonan CD, Dias MM, Battastini AM, Dias RD, Sarkis JJ. Inhibitory avoidance learning inhibits ectonucleotidases activities in hippocampal synaptosomes of adult rats. Neurochem Res 1998;23:977–82. [2] Bonan CD, Roesler R, Quevedo J, Battastini AM, Izquierdo I, Sarkis JJ. Effects of suramin on hippocampal apyrase activity and inhibitory avoidance learning of rats. Pharmacol Biochem Behav 1999;63:153–8. [3] Burnstock G. Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 2007;87:659–797. [4] Burnstock G. Purinergic signalling and disorders of the central nervous system. Nat Rev Drug Discov 2008;7:575–90. [5] Burnstock G. Vessel tone remodeling. Nat Med 2006;12:16–7. [6] Edwards FA. ATP receptors. Curr Opin Neurobiol 1994;4:347–52. [7] Fields RD, Burnstock G. Purinergic signalling in neuron-glia interactions. Nat Rev Neurosci 2006;7:423–36. [8] Gibbs ME. Memory systems in the chick: regional and temporal control by noradrenaline. Brain Res Bull 2008;76:170–82. [9] Gibbs ME, Bowser DN, Hutchinson DS, Loiacono RE, Summers RJ. Memory processing in the avian hippocampus involves interactions between beta-adrenoceptors, glutamate receptors, and metabolism. Neuropsychopharmacology 2008;33:2831–46.
[10] Gibbs ME, Johnston AN, Mileusnic R, Crowe SF. A comparison of protocols for passive and discriminative avoidance learning tasks in the domestic chick. Brain Res Bull 2008;76:198–207. [11] Gibbs ME, Ng KT. Psychobiology of memory: towards a model of memory formation. Neurosci Behav Rev 1977;1:113–36. [12] Gibbs ME, Summers RJ. Role of adrenoceptor subtypes in memory consolidation. Prog Neurobiol 2002;67:345–91. [13] Haydon PG, Carmignoto G. Astrocyte control of synaptic transmission and neurovascular coupling. Physiol Rev 2006;86:1009–31. [14] Kossut M, Rose SPR. Differential 2-deoxyglucose uptake into chick brain structures during passive avoidance training. Neuroscience 1984;12:971–7. [15] Lambrecht G. Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications. Naunyn-Schmiedeberg’s Arch Pharmacol 2000;362:340–50. [16] Sedman G, O’Dowd B, Rickard N, Gibbs ME, Ng KT. Brain metablic activity associated with long-term memory consolidation. Mol Neurobiol 1992;5:351–3. [17] Langer D, Hammer K, Koszalka P, Schrader J, Robson S, Zimmermann H. Distribution of ectonucleotidases in the rodent brain revisited. Cell Tissue Res 2008;334:199–217. [18] Ng KT, O’Dowd BS, Rickard NS, Robinson SR, Gibbs ME, Rainey C, et al. Complex roles of glutamate in the Gibbs–Ng model of one-trial aversive learning in the new-born chick. Neurosci Biobehav Rev 1997;21:45–54. [19] Rickard NS, Poot AC, Gibbs ME, Ng KT. Both non-NMDA and NMDA glutamate receptors are necessary for memory consolidation in the day-old chick. Behav Neural Biol 1994;62:33–40. [20] Yarbrough GG, McGuffin-Clineschmidt JC. In vivo behavioral assessment of central nervous system purinergic receptors. Eur J Pharmacol 1981;76:137–44. [21] Meyer MP, Clarke JD, Patel K, Townsend-Nicholson A, Burnstock G. Selective expression of purinoceptor cP2Y1 suggests a role for nucleotide signalling in development of the chick embryo. Dev Dyn 1999;214:152–8. [22] Webb TE, Simon J, Barnard EA. Regional distribution of [35S]2 -deoxy 5 -O-(1thio) ATP binding sites and the P2Y1 messenger RNA within the chick brain. Neuroscience 1998;84:825–37. [23] De Vaus JE, Gibbs ME, Ng KT. Effects of social isolation on memory formation. Behav Neural Biol 1980;29:473–80.
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Role for purinergic receptors in memory processing in young chicks Catherine Cronin a,∗,2 , Thomas M. Edwards a,3 , Marie E. Gibbs b,1 a b
School of Psychology & Psychiatry, Monash University, Clayton, 3800 Victoria, Australia Department of Anatomy & Developmental Biology, School of Biomedical Sciences, Monash University, Clayton, 3800 Victoria, Australia
a r t i c l e
i n f o
Article history: Received 16 December 2010 Received in revised form 1 May 2011 Accepted 3 May 2011 Available online 10 May 2011 Keywords: ATP Suramin PPADS Discriminative avoidance learning Memory Chick
a b s t r a c t The current study used a single trial bead discrimination task for the young chick to ascertain if inhibitors of P2 purinergic receptors would impair memory retention. Suramin and PPADS provided similar retention profiles. Loss of memory retention was evident by 60 min post-training. Both drugs caused persistent memory loss which was still evident 24 h post-training. These findings suggest that P2 receptors have a role in memory processing. © 2011 Elsevier B.V. All rights reserved.
Purinergic receptors have important roles in many systems in the peripheral and central nervous system [3,7]. They are found in many brain regions, including those associated with learning and memory, e.g. the hippocampus, cerebral cortex, basal ganglia, locus coeruleus, and it is likely that they are important in information processing [1]. In the chick, purinergic receptors are also found in memory-associated brain regions [22] and on both neurons and glial cells [7,21]. The activation of purinergic receptors is associated with adenosine-triphosphate (ATP), which acts as a co-transmitter in the central nervous system, being released from neurons along with glutamate, acetylcholine, GABA, dopamine and noradrenaline [4]. Purinergic receptors were categorised, based upon ligand affinity, into two families, the P1 and the P2 receptors [1,2]. ATP can activate P1 and P2 receptors either directly or indirectly, through hydrolysation. Specifically, P1 receptors are activated by adeno-
Abbreviations: APV, dl-2-amino-5-phosphonopentanoic acid; ATP, adenosine triphosphate; ADP, adenosine diphosphate; DR, discrimination ratio; IMM, intermediate medial mesopallium; NMDA, N-methyl d-aspartate; PPADS, pyridoxalphosphate-6-azophenyl-2 ,4 -disulfonate; P1 and P2, purinergic receptors; s.e.m., standard error of mean. ∗ Corresponding author. Tel.: +61 3 0421461688. E-mail addresses: [email protected], catherine [email protected] (C. Cronin). 1 Current address: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052 Victoria, Australia. 2 Current address: Swinburne University of Technology, John Street, Hawthorn, VIC 3122, Australia. 3 Current address: Tabor Victoria, 9/44-60 Jacksons Rd, Mulgrave, VIC 3170, Australia. 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.05.002
sine, while P2 receptors are activated by nucleotides, such as ATP, and also by the hydrolyzed products of ATP–ADP and adenosine [3,17]. The P2 receptor family is further subdivided into ionotropic P2X receptors, which are ligand-gated ion channels permeable to Ca2+ and Na+ ions [3], and metabotropic P2Y G-protein-coupled receptors (GPCR), whose function varies depending on the specific G-protein. Currently eight P2Y receptors and seven P2X receptors have been identified. Although there are some studies on the role of purines in longterm potentiation (LTP) in the mammalian hippocampus (e.g. Ref. [3]), there are few studies using in vivo animal models, which directly investigate memory processing [1,2,20]. It was the intention of this study to use a pharmaco-behavioural methodology to identify a role for P2 receptors in memory processing. Young chicks were used in place of rodents given that they are amenable to a temporally precise and well defined single trial bead discrimination task. Moreover, any retention loss could be compared to the detailed Gibbs and Ng model of memory formation [11] and inferences made about potential direct, or indirect [5,13], actions on memory processing. To investigate the involvement of purinergic receptors in memory processing suramin sodium salt (suramin) and peridoxal phosphate-6-azo benzene-2,4,-disulfonic acid (PPADS); SigmaAldrich, NSW, Australia were used. Suramin is a broad spectrum P2 receptor antagonist while PPADS is a more selective P2 receptor antagonist. An inhibitory effect of drug administration on memory retention is suggestive of a role for P2 receptors in memory processing. One- and two-day-old chicks from an egg-laying hybrid strain (New Hampshire, Rhode Island Red, White Leghorn and Black Aus-
418
C. Cronin et al. / Behavioural Brain Research 223 (2011) 417–420
tralorp) were used. Chicks were collected from a local hatchery. In a purpose-built laboratory they were housed in random pairs in wooden pens (18 cm × 25 cm × 20 cm), with the measures from 20 chicks representing one data-point. Chicks were kept in pairs to avoid isolation stress, which can affect retention [23]. Crushed poultry food was provided ad libitum. The bead discrimination task consisted of various pre-training, training and testing trials [10]. During successive pretraining trials the chicks were presented with chrome, red and blue beads dipped in water. Red and blue bead trials lasted 10 s and all pretraining trials were designed to encourage pecking at novel objects. A single training trial used a similar red bead to that used in pretraining but now dipped in a bitter-tasting non-toxic taste aversant, methylanthranilate (MeA; Sigma-Aldrich Inc., NSW, Australia). This bead was presented to each pair of chicks for 10 s. For each group of 20 chicks retention testing occurred at a time related to the purpose of the experiment. Testing consisted of sequential presentation of a dry red bead and a dry blue bead, similar to those used in pretraining, for 10 s. Chicks that did not train or peck the non-aversive blue bead at test were excluded from the data analysis. Exclusions were few and equally scattered amongst all groups. The numbers of pecks from each chick in each trial was recorded. The level of memory retention for each chick was calculated as a discrimination ratio (DR), this being the number of pecks to the blue bead at test compared to the total number of peaks to the red and blue beads at test. A DR of 1.0 indicated perfect retention while a DR of 0.5 indicated complete amnesia. A mean DR was then calculated for each group of ≤20 chicks representing a single data-point. Suramin, PPADS or physiological saline (154 mM NaCl) were injected bi-laterally and intracranially into the intermediate medial mesopallium (IMM), an area important for memory processing [14,16]. Injections were administered freehand by a trained researcher using a Hamilton repeating dispenser syringe. The syringe was fitted with a 27-gauge needle of which only the final 3.5 mm was exposed. 10 l of solution was injected per hemisphere. Dose response studies were carried out to determine if suramin and/or PPADS impaired retention and to find the lowest concentration that produced maximal impairment. Each concentration tested was administered 2.5 min after training. Retention was measured 120 min post-training, which is well within the long-term memory stage. A one-way analysis of variance (ANOVA) revealed a significant effect of suramin [F(5,106) = 7.73, p < .001]. Post hoc analysis using Dunnett’s test showed that all doses greater than 5 pmol resulted in significant retention loss when compared to the saline control group. 300 pmol/hemisphere of suramin (p < .05) was the lowest dose to maximally inhibit retention and was used in subsequent experiments (Fig. 1A). The dose response function for PPADS (Fig. 1B) also revealed a significant retention loss [F(3,72) = 9.81, p < .001]. Using a Dunnett’s post hoc analysis doses of 30 (p < .05) and 100 pmol/hemisphere (p < .001) produced significant retention losses when compared with the saline control group. In subsequent experiments 100 pmol/hemisphere of PPADS was used as it maximally impaired retention. Experiments were conducted to determine the optimal time of administration for suramin and PPADS. Drugs were injected at predetermined intervals after training. Chicks were administered either 300 pmol suramin or 100 pmol PPADS, with saline administered at 2.5 min after training as the control in both experiments. Retention was tested at 120 min post-training. When suramin was administrated at 2.5 and 5 min post-training or 25 and 35 min post-training, there was a significant loss of retention – but no loss of retention was observed in the interim. A one-way ANOVA revealed a significant effect [F(10,192) = 7.419, p < .001]). Dunnett’s post hoc analysis showed that injection times of +2.5, +5, +25, +30
Fig. 1. Effect of increasing concentrations of (A) suramin and (B) PPADS on retention tested 120 min after training. All drugs were administered to the chicks 2.5 min after training. All groups were tested 120 min post-training. The mean discrimination ratio ± s.e.m. were calculated for each group of chicks (*p < .05).
and +35 min caused significant retention loss (p < .05), and further confirmed two periods of statistically significant retention loss (Fig. 2A). The time of administration study for PPADS showed a similar pattern of effective administration times (one-way ANOVA [F(8,145) = 10.56, p < .001]). Dunnett’s post hoc test showed that injection times of +0, +2.5 and +35 min caused significant retention loss (p < .001) – but not at times of administration between these (Fig. 2B). Retention function studies were conducted with both drugs to determine at what time retention loss first occurred and whether it was persistent or transient. Time of retention loss indicates the stage of memory formation a drug interferes with and when compared to past findings suggests potential biochemical relationships between mechanisms. Whether a retention loss profile is persistent or transient is important as it indicates whether a memory formation or retrieval mechanism is affected. For example an effect on retrieval mechanisms is suggested if a transient retention loss is apparent given that the trace is not extinguished but its expression only interrupted for a time. Suramin (300 pmol/hemisphere) or saline were injected 2.5 min post-training and retention was measured at times between10 min and 24 h after training in separate groups of chicks (Fig. 3A). A trend in memory loss became evident after 30 min post-training. A two-way ANOVA yielded a significant main effect for drug [F(1,342) = 66.14, p < .001], time of test [F(9,342) = 2.65, p < .01] and
C. Cronin et al. / Behavioural Brain Research 223 (2011) 417–420
Fig. 2. Time of administration study for (A) suramin and (B) PPADS. Suramin (300 pmol/hemisphere), PPADS (100 pmol/hemisphere) or saline injected at times post-training. Retention was tested 120 min post-training. The mean discrimination ratio ± s.e.m. were calculated for each group of chicks (*p < .05).
significant drug x time interaction effects [F(9,343) = 2.46, p < 005]. Simple main effects post hoc analysis identified a significant retention loss (p < .05) from 60 min post-training, and at the following times: +65, +85, +120, and +180 min, until the completion of the experiment 24 h post-training. A retention function study with PPADS (100 pmol/hemisphere) administered 2.5 min after training also demonstrated a trend in loss of memory after 30 min post-training (Fig. 3B). A one-way ANOVA revealed a significant difference in retention between saline and PPADS treated chicks [F(6.127) = 13.59, p < .001]. Using a Dunnett’s post hoc analysis chicks tested 60, 120 min and 24 h post-training had retention levels significantly less than controls (p < .001). Purinergic receptors represent a new and interesting target for behavioural neuroscientists. However, little is known about what role they may have in memory processing, the memory stage in which their action becomes important and the cellular processes impacting – or impacted by – P2 receptors. To overcome this problem in this study we first sought to determine if P2 receptors were required for memory processing using a well-established avian task. Given the temporal specificity of this task it was possible to identify the stage of memory in which P2 receptors were required. Given the wealth of literature [8,11,12] in which this task has been used, and the Gibbs and Ng model generated from such findings, it is possible to speculate upon those cellular processes related to P2 receptor activation. The results of this study show that P2 receptors are important for memory processing given that administration of both suramin
419
Fig. 3. Retention function study observed the effect of (A) Suramin (300 pmol/hemisphere) administered 2.5 min post-training. Separate groups of chicks were tested at times between 10 min and 24 h after training. (B) PPADS (100 pmol/hemisphere) 2.5 min after training and separate groups of chicks tested at times between 10 and 24 h min after training. The mean discrimination ratio +/s.e.m. were calculated for each group of chicks (*p < .05).
and PPADS resulted in retention loss. That two periods of effective administration times were found suggests an action of these drugs early in the short-term memory stage and at the interface of phase A and B of the intermediate-term memory stage as defined by the Gibbs and Ng model. This pattern is also seen with the NMDA receptor antagonist D-APV when administered into the hippocampus [9] and in the IMM (Gibbs unpublished). Moreover, memory loss following drug administration 2.5 min after training results in a progressive trend in retention loss after 30 min post-training which was significant by 60 min post-training and persistent until the end of the experiment 24 h post-training. It is interesting to note that 60 min represents the start of the long-term memory stage within the Gibbs and Ng model and that this retention loss profile is similar to that of APV injection into the IMM [18,19]. Suramin is not as selective for purinergic receptors as is PPADS [6,15] and memory is vulnerable to inhibition at more times than PPADS, which may reflect actions on other receptors as well. Nonetheless the pattern of effective drug administration times and retention loss profile is very similar for both drugs providing confirmatory support that P2 receptor inhibition was the cause of the retention losses observed. Whether there is an indirect or direct relationship between the P2 purinergic receptors and NMDA receptors and LTP is not revealed in these experiments, but they do suggest that there is endogenous release of ATP at the time of learning and memory consolidation because inhibition of P2 receptors will prevent the action of endogenously released ATP.
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