Acquisition Deficit and Time-Dependent Retrograde

Beha¨ioural Pharmacology 2000; 11:93᎐97 SHORT REPORT

Acquisition de®cit and time-dependent retrograde amnesia for contextual fear conditioning in agmatine-treated rats

L.S. Stewart and B.E. McKay

Neurochemistry and Neuroanatomy Laboratory, Department of Biology, Laurentian University, Sudbury, Ontario, Canada

Correspondence to Lee S. Stewart, Departments of Physiology and Clinical Neurological Sciences, University of Western Ontario, London, Ontario, Canada N6A 5A5. E-mail: ly᎐ [email protected]

Received 25 October 1999; accepted as revised 29 November 1999

Cumulative evidence indicates that the hippocampus plays a time-limited role in contextual learning paradigms. Pharmacological studies have indicated that acquisition of background contextual cues during Pavlovian fear conditioning is dependent upon hippocampal function, whereas early inactivation of the hippocampus after training produces retrograde amnesia. When administered prior to contextual fear conditioning, agmatine() 5 and 10 mgrkg, i.p. , an endogenous polyamine and N-methyl-D-aspartate() NMDA receptor ligand found at excitatory synapses in the hippocampus, impaired the acquisition of contextual fear() measured as defensive freezing 26 hours later without a reduction in baseline motor activity during training. Furthermore, ascending doses of agmatine were found not to exert analgesic effects on response thresholds to peripheral shock. This negated the possibility that the observed learning deﬁcit resulted from a difference in perceived shock intensity. Post-training agmatine treatment produced a time-dependent impairment of consolidation, with subjects approaching a level of fear equivalent to that of a reference group as the delay of treatment increased() up to 6 hours . Since physiologically high levels of agmatine are able to inhibit NMDA receptor activity, these results suggest that polyamine modulation of NMDA receptors, most likely within the hippocampus, is required for the acquisition and consolidation of contextual fear stimuli. ᮊ 2000 Lippincott Williams & Wilkins.

Keywords: agmatine; fear conditioning; hippocampus; learning; NMDA receptor; rat

INTRODUCTION deﬁcits in CF if made shortly after trainingŽ Anag- nostaras et al., 1999. and data obtained from addi- Pavlovian fear conditioning is a rapid form of learn- tional experiments suggest that NMDA receptor ac- ing whereby the polymodal, or contextual, stimulus tivity within the hippocampal formation is critical for elements of a novel environment are placed into the consolidation of emotional learning tasks that association with an aversive engram following a stressful eventŽ. i.e. footshock . Cumulative evidence possess a strong contextual element, although such implicating the hippocampal formation in this form activity appears to have a limited, or time-depen- Ž of learningŽ. Maren et al., 1998 also points to a role dent, role in post-training memory processing Fer- for hippocampal N-methyl-D-aspartateŽ. NMDA - reira et al., 1992; Izquierdo et al., 1997; Roesler et . dependent long-term potentiationŽ. LTP , a cellular al., 1998 . analog of behavioral memoryŽ Bliss and Collingridge, The polyamine agmatine is an upstream precursor 1993. , in the acquisition of contextual information for the synthesis of spermine and is localized to Ž.Maren et al., 1994 . The inhibitory effects of sys- numerous cortical and subcortical regionsŽ Otake et temic NMDA receptor antagonists on contextual al., 1998. . Endogenous agmatine interacts with a fear conditioningŽ. CF suggests that activity at these number of receptor subtypes, including imidazoline, receptors is a necessary component of associative ␣ 2-adrenergic and NMDAŽ Reis and Regunatham, plasticityŽ. acquisition required for the subsequent 1999. . Available data suggest that agmatine is found expression of punishment-suppressed behavior in hippocampal pyramidal cellsŽ. Reis et al., 1998 Ž.Young et al., 1994 . Lesions of the hippocampal and functions as a modulator of the NMDA recep- formation have been shown to produce retrograde tor channel pore, but high systemic levels display a

0955-8810 ᮊ 2000 Lippincott Williams & Wilkins Behavioural Pharmacology 2000, Vol 11 No 1 93 STEWART AND MCKAY pharmacological profile similar to that of systemi- PlexiglassŽ. front, back and hinged ceiling door cally administered NMDA receptor antagonists chamber with a raised floor composed of 18 steel Ž.Kolesnikov et al., 1996; Gilad et al., 1995 . Further- rods spaced 1.5 cm apart. The intensity of electric more, high concentrations of agmatine Ž.)100 nM current was controlled by an A-615-C Master have been shown to displace binding of spermine, a ShockerŽ Lafayette Instruments, Lafayette, Indiana, local NMDA polyamine site agonist, to the receptor USA. . After the subject had been placed in the channel in hippocampal slicesŽ Rock and McDonald, chamber, the intensity of electric current was slowly 1995. . Interestingly, in-vivo experiments have impli- increased by increments of 0.01 mA until the subject cated spermine in the formation of LTP in the displayed a qualitatively distinct flinch. After 30 s the dentate gyrusŽ. Chida et al., 1992 . amount of current required to evoke the initial To examine the possible effects of agmatine on flinch response was increased by 0.05 mA and then CF, we administered agmatine prior to, or at specific quickly decreased until no flinch was observed. The intervals following, trainingŽ acquisition versus con- threshold was then recorded in milliamperes. solidation. . Peripherally, the effects of agmatine in- clude the ability to decrease blood pressure and Experiment 2a: Acquisition Ž sympathetic neuronal activity Forsang and Kapocsi, Both conditioning and CF testing were performed . ␣ 1999 . Agonists of imidazoline and 2-adrenergic using the footshock apparatus described above. The receptors have been reported to decrease spinal chamber was cleaned with a 10% acetic acid solu- nociceptive processing and induce thermal antinoci- tion between conditioning trials. On the day of con- Ž ception, respectively Diaz et al., 1997; Graham et ditioning, subjects were randomly allocated to treat- . al., 1997 . To discern possible non-specific drug ef- ment groups Ž.ns6 each and received one of the fects on learning related to either decreases in three doses of agmatineŽ. intraperitoneally 20 min arousal or differences in perceived shock intensity, prior to conditioning. An additional six animals re- additional measures were employed that monitored ceived a saline vehicle and were assigned to a refer- the exploratory activity of subjects in the presence of ence group. Ž. contextual conditioned stimuli CSs prior to uncon- CF was accomplished by placing each subject in Ž. ditional stimulus UCS delivery, and response the chamber for 3 min, after which three unsignaled thresholds to peripheral shock. We hypothesized footshocksŽ. 2-s, 0.5 mA, 60-s interstimulus interval that if the pharmacological effects of systemic agma- were delivered. Sixty seconds following the last foot- tine are comparable to that of traditional NMDA shock, the subjects were returned to their home antagonists, then an impairment in acquisition and cages. BaselineŽ. pre-shock locomotor activity was early consolidation would be expected. This hy- measured by conditioning chamber crossovers and pothesis was consistent with the results of previous was defined as forward movement over the chamber Ž work Ferreira et al., 1992; Young et al., 1994; midline. Subjects were then returned to the original . Izquierdo et al., 1997; Roesler et al., 1998 . conditioning chamber 26 hours later and scored for defensive freezing, a behavioral index of fearŽ Bolles, Methods 1970.Ž. , by an experimenter B.E.M. blind to treat- Subjects ment conditions. Freezing was assessed every 8 s In total, 68 male Wistar strain ratsŽ Charles River, during an 8-min extinction test, for a total of 60 Quebec, Canada. , approximately 80᎐90 days of age observations per subject. To satisfy parametric re- and weighing between 310 and 360 g served as sub- quirements, freezing measures were converted to a jects. All animals were group housed in plastic percentage of total observations. shoebox cages and maintained under a 20 " 1ЊC ambient temperature and 12 : 12 light᎐dark cycle Experiment 2b: Consolidation Ž.lights on at 07.30 hours . Food and water were A second experiment was performed using the same freely available throughout the experiment. conditioning parameters outlined above. Subjects were administered 10 mgrkg agmatineŽ. i.p. at Experiment 1: Effects of agmatine on response thresh- 30 min, 2 h, or 6 h Ž.ns6 each following condition- olds to peripheral shock ing. This single dose was chosen following the results Subjects were randomly assigned to receive one of of experiment 1 but has been shown to produce the three ascending doses of agmatineŽ 1, 5, or 10 mgrkg, behavioral effects of systemically administered i.p..Ž. 20 min prior to testing ns5 each . Response NMDA receptor antagonistsŽ Kolesnikov et al., thresholds to peripheralŽ. foot shock were assessed 1996. . An additional six animals served as a refer- using a 28 = 20 = 20 cm aluminumŽ. side walls and ence group. Context testing was performed 26 h later.

94 Behavioural Pharmacology 2000, Vol 11 No 1 AGMATINE EFFECTS ON CONDITIONED FEAR

Results Footshock response() ¯inch thresholds Analysis of variance revealed no signiﬁcant difference in mean footshock ﬂinch thresholds in response to ascending doses of agmatine w FŽ.3, 19 s 0.63, NSx Ž. Table 1 . This observation was in agreement with previous workŽ. Kolesnikov et al., 1996 .

Locomotor activity and extinction test 2a During conditioning, no significant difference in baseline locomotor activityŽ. crossovers was observed between groups w FŽ.3, 23 s1.19, NSx Ž. Table 1 . An ANOVA revealed a significant difference in mean percent freezing among treatment groups 26 hours w Ž.s - x FIGURE 1. Mean percent ()"SEM freezing observed through- after training F 3, 23 6.95, P 0.05 . Although a out an 8 min extinction test for rats treated with agmatine prior to polynomial trend analysis indicated a significant lin- acquisition and during consolidation of contextual fear condition- ear relationship between treatments w FŽ.1, 23 s ing. Acquisition subjects exhibited a linear decrease in freezing - x behavior in response to ascending doses of agmatine. Adminis- 14.41, P 0.01 , which is suggestive of dose- tration of a single dose of agmatine() 10 mg/kg at three intervals dependency, only animals injected with 5 and during consolidation revealed a progressive increase in reten- 10 mgrkg agmatine exhibited significantly less Ž P- tion as the delay of the post-training treatment was extended. 0.05. freezing compared to the reference group Ž.Figure 1 . CFŽ. Fanselow et al., 1994; Young et al., 1994 , pre- Locomotor activity and extinction test 2b training administration of agmatine significantly im- No significant between-group or between-ex- paired acquisition. It is thought that the amnestic periment differences in chamber crossovers were effects of NMDA antagonists on learning and me- w s xw s observed FŽ.3, 23 1.50, NS and FŽ.7, 47 2.10, mory processes are due to an impairment of x NS , respectively. An ANOVA indicated a significant NMDA-dependent LTP in the hippocampus. Thus, difference in mean percent freezing among treat- inhibition of hippocampal NMDA receptor activity w Ž.s - x ment groups F 3, 23 9.29, P 0.01 . A post hoc by agmatine would account for the acquisition analysis of the treatment effect indicated that agma- deficits observed in experiment 1, although an addi- tine produced significant decreases in fear consoli- tional possibility emerges since NMDA receptor ac- dation when administered at 30 min and 2 h, but not tivity and associative LTP within the amygdala also 6 h after conditioningŽ. Figure 1 . appear to mediate acquisitionŽ. Maren et al., 1996 . Stimulation of afferent pathways to the amygdala Discussion produce a long-term enhancement of synaptic effi- Contextual fear, as demonstrated in the present cacy, and neurons of the lateral and basolateral study through an expression of freezing behavior, is nuclei show a subsequent increase in activity in mediated by a neural circuit in which the hippocam- response to a stimulus CS previously paired with a pal formation is thought to play an important role, shock UCSŽ. Maren and Fanselow, 1995 . This evi- by forming a configural representation of the train- dence suggests that the basolateral complex func- ing environmentŽ. Maren et al., 1998 . Consistent tions as a coincidence detector for CS᎐UCS pair- with the effects of NMDA receptor antagonism on ings, and the associative mechanism is thought to

TABLE 1. Effects of agmatine on pre-shock locomoter activity and response thresholds to peripheral shock

Conditioning chamber crossoversa Flinch thresholds() mA b

Group n Mean SEM Group n Mean SEM

Reference 6 7.8 0.8 Reference 5 0.12 0.01 1 mg/kg 6 8.2 0.66 1 mg/kg 5 0.1 0.03 5 mg/kg 6 9.6 0.6 5 mg/kg 5 0.11 0.01 10 mg/kg 6 8.6 0.74 10 mg/kg 5 0.13 0.02 a[(F 3, 23 ) =1.19, NS ] . b[(F 3, 19 ) =0.63, NS ] . Behavioural Pharmacology 2000, Vol 11 No 1 95 STEWART AND MCKAY rely on NMDA receptor activity, since pretraining The possibility that physiological concentrations inactivation of NMDA receptors in the basolateral of agmatine might approach a level sufficient to amygdala also impairs acquisitionŽ Fanselow and block NMDA receptor activation, and non-specific Kim, 1994; Maren et al., 1996. . interactions with central-type imidazoline and ␣ 2- Temporally graded post-training agmatine treat- adrenergic receptors resulting from pharmacological ment produced a time-dependent impairment of doses, merits additional consideration. Yang and consolidation when delivered at 30 min and 2 h, but ReisŽ. 1999 have reported that, like other neurotrans- not 6 h following training. Although there is general mitters, agmatine does not display a uniform dis- agreement that the hippocampal formation is critical tribution among postsynaptic sites. Consequently, for acquiring contextual CSs, prior work has demon- agmatine levels at some sites may approach the strated that this structure may contribute to post- necessary concentration required to inhibit NMDA training memory consolidation for contextual learn- receptor activity upon release. The available data ing tasks. Lesions of the hippocampal formation pertaining to the role of imidazoline and ␣ 2 recep- made 1 day after CF impair the later expression of tors in learning processes suggest that these receptor Ž. freezing Anagnostaras et al., 1999 . Infusion of the subtypes may participate in certain learning tasks. NMDA receptor antagonist, AP5, immediately and Stimulation of imidazoline and ␣ 2 receptors have 3 h following inhibitory avoidance training, to hip- been reported to facilitate spatial working memory pocampal tissue and the adjacent entorhinal cortex, in primatesŽ. Arnsten and Jentsch, 1997 , whereas respectively, where performance is also dependant ␣ 2 antagonism improves acquisition of a spatial upon the acquisition of contextual CSs, impairs task maze task in rodentsŽ. Haapalinna et al., 1998 . These retentionŽ. Ferreira et al., 1992; Roesler et al., 1998 . inconsistent results may reflect a difference in func- Other evidence suggests that the basolateral amyg- tional anatomy, since working memory generally in- dala also modulates post-training memory consolida- volves prefrontal activity and spatial tasks rely heav- tion for inhibitory avoidanceŽ Vazdarjanova and Mc- ily on the hippocampus. Although CF does possess a Gaugh, 1999.Ž but not for CF Maren et al., 1996 . . strong hippocampal component, it is unlikely that an Although procedurally similarŽ i.e. both tasks involve ␣ 2-adrenergic substrate underlies the present obser- aversive stimulation in a novel context. , the discrep- vations, given thatŽ. 1 agmatine stimulates ␣ 2 activ- ancy following post-training inactivation is believed ity, andŽ. 2 deficits in both acquisition and consolida- to result from a differential involvement of the basolateral amygdala based on response contin- tion were observed following agmatine treatment. gency. In inhibitory avoidance paradigms, the UCS Furthermore, if the present effects were localized to ␣ is contingent on the animal’s response pattern, while 2 activity within prefrontal cortex, we would antici- in CF the UCS is inescapable and is delivered re- pate an effect if agmatine were administered prior gardless of the animal’s behaviorŽ Vazdarjanova and to testing, as the prefrontal region has been impli- McGaugh, 1999. . Thus, deficits in fear consolidation cated in neural processes fundamental to extinction Ž. observed in the 30 min and 2 h treatment groups LeDoux, 1998 . Recent work has indicated that ␣ ␤ support the view that the hippocampal formation an enhancement of both 1- and 1-adrenergic alone, or in association with the entorhinal cortexŽ a transmission between the amygdala and hippocam- major source of sensory input to the hippocampal pus enhances retention of emotional learning by formation. , modulates the consolidation of contex- augmenting NMDA receptor activity and associative tual stimuli following aversive training through plasticity that is critical for the formation and storage NMDA-dependent processes. of an aversive contextual representationŽ Katsuki et The results of experiment 2 may also reflect dif- al., 1997; Ferry et al., 1999. . ferential effects on the induction and maintenance The present study has shown that systemic admin- of NMDA-dependent LTP. Associative LTP occurs istration of agmatine, a putative neurotransmitter when sensory inputŽ. i.e. contextual stimuli is weak and endogenous NMDA receptor ligand, disrupts and sufficient postsynaptic depolarization occurs with both the acquisitionŽ without affecting normal ex- a concomitant strong input such as aversive shock ploratory behavior or nociceptive responding. and Ž.Bliss and Collingridge, 1993 . The resulting progres- early consolidation of aversively conditioned contex- sive increase in synaptic efficacy is estimated at tual stimuli. Our data provide further support for 10 min to 1᎐2 h in duration, thereby presenting a the view that NMDA receptor activity mediates con- window for exploring NMDA ligand effects, since textual fear conditioning, although a precise neu- late-LTP is dependent on structural changes in- roanatomical substrate for the observed deficits can- duced by previously activated intracellular calcium not be determined. 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