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Neurobiology of Learning and Memory 86 (2006) 66–71 www.elsevier.com/locate/ynlme

Enhancement of long-term memory retention by Colostrinin in one-day-old chicks trained on a weak passive avoidance learning paradigm Michael G. Stewart ¤, Duncan Banks Department of Biological Science, The Open University, Milton Keynes MK7 6AA, UK Received 16 October 2005; revised 5 December 2005; accepted 31 December 2005 Available online 13 February 2006

Abstract Colostrinin (CLN) is a biologically active proline-rich polypeptide which has therapeutic potential for the alleviation of memory deWcits in age-related dementias in a number of human conditions, particularly Alzheimer’s disease. To examine the eYcacy of CLN in other species, day-old domestic chicks were used as a model system to study its eVects on retention of memory for a single one-trial learning paradigm—avoidance of a bitter-tasting substance (methylanthranilate, MeA). Birds were presented with a bead coated with either a dilute (10%) solution of MeA or a bead coated with 100% MeA. Those trained on 100% MeA avoided pecking at a similar but dry bead 24 h later, thereby demonstrating long-term memory whereas chicks trained on the 10% solution pecked the bead at 24 h, indicating lack of long term memory for the task. However, when CLN was injected (i.c.) into a region known to be important in memory formation, the mesopallium intermediomediale (IMM), prior to training with 10% MeA, chicks exhibited strong memory retention at 24 h, similar to those trained on 100% MeA. Control chicks trained on 10% MeA but injected i.c. with a 10% saline solution did not show improvement in memory retention. Intraperitoneal (i.p.) injections of CLN were as eVective as the i.c. route. These data extend the known eYcacy of CLN from mammals demonstrating its widespread eYcacy as a cognitive enhancer. © 2006 Elsevier Inc. All rights reserved. Keywords: Chick; Passive avoidance learning; Memory enhancement; Colostrinin

1. Introduction The young chick is a powerful model system in which to study the biochemical and morphological processes underlying memory formation, in particular using the paradigms of passive avoidance learning (Dermon et al., 2002; Gibbs & Summers, 2002; Mileusnic, Lancashire, & Rose, 2004, 2005; Rose & Stewart, 1999;Stewart, 1991; Stewart & Rusakov, 1995; Stewart, Rusakov, Davies, & Harrison, 1995); single trial discrimination learning (Gibbs & Hertz, 2005), and Wlial imprinting (Horn, 2004; Meredith, McCabe, Kendrick, & Horn, 2004).

Passive avoidance learning provides an inexpensive and reliable model, in which a single speciWc learning paradigm—avoidance of a bitter-tasting substance, methyl anthranilate (MeA),1 can be utilized with precise timing. Moreover, because the training is given in day old birds, it does not suVer from the problem of “noise” of other learning systems which occur during normal behavioural development. In addition, because the thin skull of the chick is unossiWed, sites of memory formation can be accessed easily and rapidly by direct injection through the thin skull. Training of day old chicks on a one trial passive avoidance task results in a cascade of cellular events (paralleling

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Corresponding author. Fax: +44 1908 654167. E-mail addresses: [email protected] (M.G. Stewart), D.Banks @open.ac.uk (D. Banks). 1074-7427/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nlm.2005.12.011

1 Abbreviations used: IMM, mesopallium intermediomediale; CLNO, ovine Colostrinin; CLNB, bovine Colostrinin; MeA, methyl anthranilate.

M.G. Stewart, D. Banks / Neurobiology of Learning and Memory 86 (2006) 66–71

that in mammalian brain), from immediate early gene expression, through biochemical and pharmacological to morphological changes, in speciWc brain regions, initially in mesopallium intermediomediale (IMM) (Rose & Stewart, 1999). The IMM was formerly known as the intermediate and medial hyperstriatum ventrale (IMHV) until the adoption of the new avian brain nomenclature (Reiner et al., 2004); the recent nomenclature will be adopted throughout this paper. There are also increases in cell proliferation following avoidance learning in chicks with a signiWcant MeA-training induced increase in labelled cells in the dorsal ventricular zone surface bordering the IMM at 1 day post-training and later in the striatum mediale (StM, Dermon et al., 2002). The chick avoidance learning model has also proven a useful paradigm in which to examine the eVects of compounds which either inhibit or enhance memory. Steele, Dermon, and Stewart (1996) demonstrated that D-cycloserine, a partial agonist of the glycine site on the NMDA receptor, enhanced memory retention after training on weak aversive stimuli (10% MeA). The eYcacy of peptides as cognitive enhancers has been examined recently by Mileusnic et al. (2004, 2005). An amino acid sequence containing the palindromic peptide RER (which matches amino acids 328–330 of the amyloid precursor protein APP) can protect against memory loss induced by -amyloid (A) when injected into chick brain just before or shortly after training. Moreover, RER acts as a cognitive enhancer in a variant of the avoidance training model in which the strongly aversive bitter-tasting substance methyl anthranilate is presented to chick in a 10% solution. Normally with 100% MeA coated on a bead (strong training), memory is retained for at least 48 h. However, in weak training, when the bead is coated with 10% MeA, memory is retained only for about 6–8 h. A cognitive enhancer such as RER will increase memory retention toward that seen with 100% MeA. Colostrinin, a complex of polypeptides derived from sheep or bovine colostrum retards the progress of Alzheimer’s disease (Bilikiewicz & Gaus, 2004; Leszek et al., 1999). The mechanism of this process is unclear but CLN is known to attenuate the cytotoxic eVect of A1–40 on SHSY5Y neuroblastoma cells by reducing the aggregation of A Wbrils (Schuster et al., 2005). In a cell diVerentiation model system, pheochromocytoma (PC12) cells, CLN has the ability to increase neurite outgrowth in a dose dependent manner (Bacsi, Stanton, Hughes, Kruzel, & Boldogh, 2005). Colostrinin also facilitates acquisition and retrieval of spatial memory (Popik, Bobula, Janusz, Lisowski, & Vetulani, 1999) in aged rats and a CLN-derived non-apeptide may delay the extinction of long-term memories in these older animals (Popik, Galoch, Janusz, Lisowski, & Vetulani, 2001). In the present study, we have investigated the ability of ovine (CLNO) or bovine (CLNB) Colostrinin to enhance memory retention for the weak aversive stimulus of 10% MeA in day-old domestic chicks. Our data demonstrate

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that CLNO injected prior to training either i.c. or i.p. at very low concentrations has a remarkable memory enhancing eVect, and CLNB, tested i.c. only, exerted similar eVects on memory. 2. Materials and methods 2.1. Subjects: Chicks Commercially obtained Ross Chunky eggs (domestic chicks, Gallus domesticus) of both sexes were incubated and hatched in our own brooders and held until 16 § 6 h old. Chicks were then placed in pairs in small aluminium pens. Each pen was illuminated and heated with a 25 W red bulb and the temperature maintained at 25–28 °C. All experimental procedures were carried out under a UK Home OYce Project Licence and care was taken to minimize any suVering of the chicks.

2.2. Training Chicks were pre-trained and trained essentially as described earlier (Dermon et al., 2002; Lössner & Rose, 1983; Mileusnic, Lancashire, Johnson, & Rose, 2000, 2004). BrieXy, for pre-training, after 1 h of equilibration in pens, chicks were given two 10 s presentations of a dry white bead (2 mm in diameter). Only chicks that pecked the bead twice (normally more than 80%) were included in the subsequent experiment.

2.3. Drugs and injection Colostrinin (either from ovine or bovine sources, see Kruzel et al., 2004 for details of puriWcation process) was dissolved in 0.9% sterile saline (SAL). The concentrations injected were based upon those used previously by Popik et al. (1999), with a maximum dose of 2 g/l (CLNB) or 1 g/l (CLNO). Bilateral intracranial injections of 2.5 l/hemisphere were made using a 5 l Hamilton syringe Wtted with a plastic sleeve to allow a penetration of 3 mm into the mesopallium intermediomediale (IMM), which is known to play a key role in the initial stages of memory formation. After injection the syringe was kept in place for 5 s. The head-holder with guide tracts for the syringe was custom built and has been described previously (Mileusnic et al., 2004; Steele et al., 1996). The injections were made 5 min after the second pre-training trial and t20 min before the actual training trial. Control birds where injected with 2.5 l/hemisphere of 0.9% sterile saline. Any chicks with abnormal behaviour (listless or lying on the Xoor of the pens) after i.c. injection were excluded from subsequent analyses. Intraperitoneal (i.p.) injections were made via a 1.0 ml syringe with the CLNO (100 g/chick) dissolved in a volume of 0.1 ml sterile SAL.

2.4. Training and testing All training and testing was done with an experimenter blind to the nature of the substance injected. Codes were only broken only at the end of the testing trial 24 h later, or in the experiments examining the eVect of diVerent concentrations of CLNO or CLNB, after discrimination testing. Twenty minutes after injection of CLN or SAL (whether i.c. or i.p.), chicks were presented with a chrome bead dipped in a bitter-tasting substance, methylanthranilate (MeA, Sigma, UK) either with 100% MeA or MeA diluted to 10% of its original volume with 41% ethyl alcohol. Chicks were recorded as peck and shake (the disgust reaction), or no peck (up to 20% of birds). At the end of training the birds were provided with water and chick crumb in their pens. The following day, t24 h later, chicks were tested for memory retention by presentation of a dry chrome bead. Birds were scored as avoid (indicating memory retention) or peck (amnestic). Recall was calculated as a percent avoidance score, i.e., the percentage of chicks in each group which avoided the chrome bead on test. Discrimination testing: In the two series of experiments in which ovine and bovine Colostrinin were injected i.c., to ensure that the chick behaviour on testing at 24 h after training was not due to adverse reactions to

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the drug, 10 min after the testing trial on the dry chrome bead chicks were oVered a white bead for 20 s. Chicks were considered to remember if they had avoided the chrome bead at test but pecked at the white bead (discrimination), and to have forgotten if they pecked at both beads. Only chicks that pecked the bitter bead on training and the white bead on test were included in the Wnal results from these experiments. Birds were used only once and after testing they were immediately killed humanely by decapitation. All experiments were carried out under a UK Home OYce Project Licence.

2.5. Statistical analysis The numbers of chicks avoiding or pecking were used to estimate percentage avoidance. The 2 test was used to examine the statistical signiWcance of diVerences in avoidance levels of individual CLN treated groups of chicks, in comparison to control birds injected only with SAL. SigniWcance was taken at p < .05.

3. Results 3.1. Experiment 1: The eVect of CLNO on avoidance learning with an aversive stimulus of 100% MeA; control birds injected with sterile saline To ensure that there was no adverse eVect of CLN on chick behaviour, an initial i.c. injection of a “maximum” dose (1 mg/ml, 2.5 l volume) of ovine-derived CLN was made bilaterally into the mesopallium intermediomediale (IMM), and chicks were trained on the one-trial passive avoidance task with 100% MeA. Control birds were injected with saline. Fig. 1 shows that there is no signiWcant diVerence ( D 3.09; p D .1) between CLNO and SAL injected birds in the percentage avoidance which was 89% when injected with SAL and 80% when injected with CLNO. Twenty birds were used in this experiment.

3.2. Experiment 2: Determination of a dose–response relationship for CLNO on retention of memory for a weak aversant (10% MeA) Six doses of CLNO at varying concentrations as below were injected i.c. into the IMM of chicks to be trained on a weak aversant (10% MeA). Control chicks were injected with sterile 0.9% SAL. A total of 301 birds were used in these experiments and each experimental group contained 44 birds except for the 1:1 CLNO concentration where a total of 81 birds were used because testing at this dilution was repeated several times. The CLNO concentrations used were: (i) (ii) (iii) (iv) (v) (vi)

1.0 g/l, 0.5 g/l, 0.1 g/l, 0.01 g/l, 0.005 g/l, 0.001 g/l.

Fig. 2 shows that control birds injected i.c. with SAL showed avoidance at only 37% one day after training on a 10% dilution of MeA as compared to 90% avoidance on the strong stimulus of 100% MeA (Fig. 1). When the eVect of CLNO dilutions on avoidance at 10% MeA is examined there is a marked enhancement in memory retention for the weak aversant. There is relatively little diVerence between 1.0 and 0.01 g/l CLNO, with signiWcant enhancement in retention from 67% avoidance at 1.0 g/l and 66% at 0.01 g/l CLNO. Maximal eVect is actually at 0.1 g/l CLNO at 81% avoidance. However at 0.005 g/l the 100

CLN

O

Percentage avoidance

n.s. 80

60

40

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100 ***

80

***

***

***

60

n.s.

40

n.s.

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0 SAL

1

0.5

0.1

0.01

0.005

0.001

Dose (µg/µl)

0 100% MeA 100% MeA SAL

CLNO 1µg/µl

Fig. 1. EVect of passive avoidance learning of bitter-tasting methyl anthranilate (MeA) in one-day-old chick of i.c. injections of either 0.9% saline (SAL) or 1 g/l ovine Colostrinin (CLNO). There is no signiWcant diVerence in the percentage avoidance ( D 3.09; p D .1) between CLNO and SAL injected birds.

Fig. 2. EVect on passive avoidance learning of bitter-tasting methyl anthranilate (MeA) in one-day-old chicks of i.c. injections of either Saline (SAL) or various ovine Colostrinin (CLNO) dilutions (dose–response curve dilutions from 1 to 0.0001 g/l). There is marked enhancement in memory retention for the weak aversant (10% MeA) when chicks are injected with all but the weakest CLNO dilutions. (¤¤¤: 1.0 g/l, D 20.14; p < .001; 0.5 g/l, D 32.64, p < .001; 0.1 g/l, D 40.02, p < .001; 0.01 g/l, D 15.51, p < .001; n.s. D not signiWcant for 0.005 g/ l, and 0.001g/l).


percentage avoidance is non-signiWcant and has fallen to 35%, a level it remains at with a concentration of 0.001 g/ l. These latter two percentage avoidance levels are similar to those seen in birds injected i.c. with SAL (Fig. 2). When birds were tested on the discriminating learning task by presentation of a white bead, on average across the six concentrations 90% of the birds who had avoided the chrome bead on test, pecked the white bead. 3.3. Experiment 3: Determination of a dose–response relationship for CLNB on retention of memory for a weak aversant (10% MeA) Six doses of CLNB at varying concentrations as below were injected i.c. into the IMM of chicks to be trained on a weak aversant (10% MeA). Control chicks were injected with sterile 0.9% SAL. A total of 209 birds were used in these experiments and each experimental group contained a minimum of 38 animals and no more than 58 animals. The CLNB concentrations used were: (i) (ii) (iii) (iv) (v)

2.0 g/l, 0.2 g/l, 0.02 g/l, 0.002 g/l, 0.0002 g/l.

Fig. 3 (SAL) shows that control birds injected i.c. with SAL showed avoidance at 27%. When the eVect of various CLNB dilutions (range 0.0002–1.0 g/l) on avoidance at 10% MeA is examined there is a marked enhancement in memory retention for the weak aversant. There is no signiW-

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cant eVect of the weakest CLNB concentration (0.0002 g/ l) with relatively little diVerence in avoidance level (31%) compared to SAL injected birds (27%). However, there is a signiWcant enhancement for the other four concentrations from 2.0 to 0.002 g/l CLNB, with the maximal eVect (78% avoidance) at a CLNB concentration of 0.2 g/l. When birds were tested on the discriminating learning task by presentation of a white bead, on average across the six concentrations 90% of the birds who had avoided the chrome bead on test, pecked the white bead, the same Wgure as when CLNO had been injected in Experiment 2. 3.4. Experiment 4: Determination of eYcacy of i.p. injected CLNO on retention of memory for a weak aversant (10% MeA): Comparison with the i.c. route described in Experiment 2 The maximally eVective i.c. dose of CLNO (adjusted for body weight) was injected i.p. in one-day-old chicks in 0.9% sterile SAL. The CLNO concentration was 1.0 g/l injected in 0.1 ml. Control chicks were injected i.p. with 0.1 ml of 0.9% SAL. A total of 66 one-day-old chicks were used; the results of this experiment are shown in Fig. 4. There is a signiWcant enhancement ( D 15.13; p < .001) of memory retention for 10% MeA, on testing, 71% of the CLNO injected chicks avoided the bead whereas one day later, only 22% of saline injected control animal avoided the bead, demonstrating that there was little memory retention in this group. These data demonstrate that for CLNO the i.p. injection route is as eYcacious as the i.c. route in the cognitive enhancement eVect on the passive avoidance-learning task.

80 CLN

B

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60

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*

40

n.s.

20



100

60

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2

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Dose (µg/µl)

Fig. 3. EVect on Passive Avoidance Learning of bitter-tasting methyl anthranilate (MeA) in one-day-old chicks of i.c. injections of either Saline (SAL) or various bovine Colostrinin (CLNB) dilutions (dose–response curve dilutions from 2 to 0.0002 g/l). There is signiWcant enhancement in memory retention for the weak (10%) aversant (MeA) at all but the weakest dilution tested (0.002 g/l). (¤¤¤: 2.0 g/l, D 15.12; p < .001; 0.2 g/l, D 49.51, p < .001; **0.02 g/l, D 7.03, p < .01; *0.002 g/l, D 0.025; 0.002 g/l, n.s., not signiWcant).

0 10% MeA SAL

10% MeA 1.0 µg/µl CLN O

Fig. 4. The eVect of i.p. injections of 0.1 ml 0.9% Saline (SAL) or 0.1 ml ovine Colostrinin (CLNO) (0.1 g/l) on the passive avoidance learning response in one-day-old domestic chicks: for CLNO the i.p. injection route is as eYcacious as the i.c. route in the cognitive enhancement eVect on training on the weak aversant, 10% MeA. D 48.26, ***p < .001.

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4. Discussion These experiments have demonstrated clearly that CLN derived from ovine or bovine sources exhibits potency as a cognitive enhancer of avoidance training on the weak aversive stimulus of 10% MeA in newly hatched chicks. It brings retention of memory up to the level normally seen with 100% MeA. CLN appears to be without adverse eVect on the chick’s ability to learn. Even when injected i.c. at the highest concentration employed, it has no adverse eVect on memory in chicks when trained on full strength 100% MeA, compared to control chicks injected with SAL. The enhancement of memory for the weak aversant (10% MeA) is comparable to that shown previously in the chick passive avoidance model by Sandi, Rose, Mileusnic, and Lancashire (1995) and also Loscertales, Rose, Daisley, and Sandi (1998) where memory was improved with compounds which stimulated corticosterone action. It is also similar to the eVect shown by Mileusnic et al. (2004), with the RER peptide using this weak learning paradigm. Here CLN eYcacy on improving memory for the weak aversant showed a dose–response curve with potency over a 100-fold range (Fig. 3). The route of CLN injections, whether i.c. or i.p., does not appear to aVect the cognitive enhancing eVect of the compound. The likely explanation for this Wnding is that in the newly hatched chick the blood brain barrier is incomplete, thus allowing rapid access of CLN to the brain. The mechanism of action of CLN has not been yet clariWed but it would not appear necessarily to be acting directly in unmodiWed form on CNS tissue. In mammals where there is a notable blood brain barrier CLN can exert a cognitive eVect even when injected via the i.p. route. In the rat, Popik et al. (1999), demonstrated that CLN injected i.p. in older animals (of 13 months of age) at a dose of 4 g/rat, facilitated the acquisition of spatial learning. At this concentration it also improved incidental learning. CLN also has eYcacy in treatment of humans suVering from mild or moderate Alzheimer’s disease (AD). In a clinical trial with CLN administered to patients in tablet form orally, a signiWcant improvement in cognitive performance was seen compared to a control group given placebo tablets (Bilikiewicz & Gaus, 2004). Studies in vitro have provided evidence of the possible mode of action of CLN. Colostrinin prevents the aggregation of -amyloid peptide A1–40 in a cell culture model (SHSY-5Y) (Schuster et al., 2005). A further clue to its mode of action may be found in studies of Boldogh et al. (2003) which demonstrated in PC12 cells that CLN has a variety of eVects including reduction in the abundance of 4-hydroxynonenal (4HNE)-protein adducts, and reduction in intracellular levels of reactive oxygen species (ROS). They concluded that CLN appeared to down-regulate 4HNE-mediated lipid peroxidation and its productinduced signalling that otherwise might lead to pathological changes at the cellular and organ level. In chicks, although the precise mechanism of CLN action in enhancing memory is unclear, one possibility is

that this may involve the second of a two-stage memory process (Rose & Stewart, 1999). The Wrst does not require de novo synthetic processes and decays at some 6–8 h posttraining, whereas the second stage requires a cascade of synthetic events which ensure that memory is formed as a long term store. The weak (10% MeA) avoidance training task fails to stimulate the second wave of events and thus memory is lost after some 6–8 h. CLN may exert an eVect by acting upon the transmitter/receptor systems which are known to be rapidly up-regulated on weak avoidance learning in the chick, inXuenced by NMDA agonists (Steele et al., 1996). If so, then the cascade of processes involved in long term memory formation are initiated, including protein synthesis, and hence the second phase of memory formation occurs and at 24 h testing memory retention for 10% MeA is almost similar to that when birds are trained on 100% MeA. However, only by use of e.g., protein synthesis inhibitors such as Anisomycin which inhibits the second stage of memory formation in chicks when given at appropriate time in relation to training (Sojka, Davies, Harrison, & Stewart, 1995), could we be conWdent that this is the case. In summary, our data show a clear enhancing eVect of CLN on training for a weak aversive task, and demonstrate its widespread eYcacy as a cognitive enhancer in other animal species. They also demonstrate the versatility of the chick as a model system to test cognitive enhancing drugs, with potential for treatment of degenerative diseases such as Alzheimer’s, adding to the recent study by Carrodeguas et al. (2005) which suggested that the chick embryo appears as a suitable natural model to study cell biology and developmental function of A precursor protein and a potential assay system for drugs that regulate A precursor protein processing. The results in this paper have previously been published in abstract form at the Federation of European Neuroscience Societies meeting (Banks & Stewart, 2004). Acknowledgments This research was supported in part by a grant from ReGen Therapeutics Plc, London, UK. We are grateful for helpful advice from Dr. Radmila Mileusnic on aspects this research and to S. Walters, K. Evans, and D. Sadler for help with chicks. References Bacsi, A., Stanton, G. J., Hughes, T. K., Kruzel, M., & Boldogh, I. (2005). Colostrinin-driven neurite outgrowth requires p53 activation in PC12 cells. Cellular and Molecular Neurobiology, 25, 1123–1139. Banks, D., & Stewart, M.G. (2004). The eVects of Colostrinin™ (CLN) on one-trial passive-avoidance learning in day-old chicks (Gallus domesticus). FENS Abstracts, 2, A078.3. Bilikiewicz, A., & Gaus, W. (2004). Colostrinin (a naturally occurring, proline-rich, polypeptide mixture) in the treatment of Alzheimer’s disease. Journal of Alzheimers Disease, 6, 17–26. Boldogh, I., Liebenthal, D., Hughes, T. K., Juelich, T. L., Georgiades, J. A., Kruzel, M. L., et al. (2003). Modulation of 4HNE-mediated signaling by proline-rich peptides from ovine colostrum. Journal of Molecular Neuroscience, 20, 125–134.

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