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Dopamine-dependent facilitation of LTP induction in hippocampal CA1 by exposure to spatial novelty

2003 Nature Publishing Group http://www.nature.com/natureneuroscience

Shaomin Li13, William K. Cullen1,2, Roger Anwyl2,4 and Michael J. Rowan1,2

1 Department of Pharmacology and Therapeutics, Biotechnology Building, Trinity College, Dublin 2, Ireland 2 Trinity College Institute of Neuroscience, Trinity College, Dublin 2, Ireland 3 Present address: Laboratory of Neurophysiology, Department of Psychiatry, Harvard Medical School, 74 Fenwood Road,

Boston, Massachusetts 02115, USA

4 Department of Physiology, Trinity College, Dublin 2, Ireland

Correspondence should be addressed to M.J.R. (mrowan@tcd.ie)

Published online 21 April 2003; doi:10.1038/nn1049 In addition to its role in memory formation, the hippocampus may act as a novelty detector. Here we investigated whether attention to novel events can promote the associative synaptic plasticity mechanisms believed to be necessary for storing those events in memory. We therefore examined whether exposure to a novel spatial environment promoted the induction of activity-dependent persistent increases in glutamatergic transmission (long-term potentiation, LTP) at CA1 synapses in the rat hippocampus. We found that brief exposure to a novel environment lowered the threshold for the induction of LTP. This facilitatory effect was present for a short period following novelty exposure but was absent in animals that explored a familiar environment. Furthermore, the facilitation was dependent on activation of D1/D5 receptors. These findings support an important role for dopamine-regulated synaptic plasticity in the storage of unpredicted information in the CA1 area.

There is growing evidence that, in addition to its role in memory formation13, the hippocampus may act as a novelty detector identifying mismatches between incoming and stored information411. Unpredicted information that triggers exploration may be of future adaptive significance to an animal and may thus gain preferential status for memory encoding. The question arises as to whether attention to novel events can promote the associative synaptic plasticity mechanisms believed to be necessary for their storage12. Long-term potentiation (LTP), an activity-dependent persistent increase in synaptic strength13, is the prevailing model of learning-related synaptic plasticity. LTP-like synaptic plasticity is believed to be necessary for information storage in brain areas such as the hippocampus, but there is still considerable debate concerning its exact role in memory formation12. Many forms of LTP have been reported, some of which may mimic naturally occurring mechanisms more closely than others 14 . The hippocampus receives input from a wide range of other brain regions that are active during learning, indicating that learningrelated synaptic plasticity is regulated by such input. It is important to determine whether, how and when such factors affect LTP in freely behaving animals as they acquire information in hippocampus-dependent tasks8,15. As the rodent hippocampus is particularly tuned to spatial information storage1,3, we reasoned that if LTP-like mechanisms are involved in memory encoding, then exposure to a novel environment should facilitate LTP induction. Here we
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show that freely behaving animals that explored a novel environment had a reduced threshold for LTP induction in a narrow time window. Furthermore, the facilitation of LTP induction by the novel environment was dependent on endogenous dopaminergic tone. Thus, block of D1/D5 receptors with the antagonist SCH23390 prevented the facilitation of LTP by novelty in awake animals. Activation of D1/D5 receptors with the agonist SKF38393 in anesthetized animals was sufficient to facilitate LTP induction. These results strongly implicate a requirement for dopamine-dependent LTP-like synaptic plasticity in hippocampal memory formation.

RESULTS
Novelty exposure facilitates LTP induction We investigated the effect of exposure to a novel environment on LTP induction threshold in freely behaving animals (Methods). When animals crossed from the familiar to the novel environment, they explored continuously for at least 5 minutes. The ability of a weak conditioning stimulation to induce LTP was then studied 5 minutes after the animals had returned to the familiar environment, a time when the animals were in a quiescent but alert state. This allowed us to compare the effects of conditioning stimulation with and without novelty exposure under an equivalent behavioral state, thereby minimizing any potential confounding effect of concurrent transient activity-related changes in excitability1619 on LTP induction (Methods). The application of weak conditioning stimulation to animals in a still
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Fig. 1. Exposure to a novel environment facilitates the induction of LTP at hippocampal CA1 synapses in freely behaving rats. (a) Application of weak high-frequency conditioning stimulation (arrow) did not induce LTP if the animal remained in a familiar environment throughout the recording period (n = 7). Two graphs show a typical example (top) and mean s.e.m (bottom). (b) The application of the same conditioning protocol 5 min after exploration of a novel environment for 5 min (vertical bar) now induced LTP (n = 7). (c) This protocol did not induce LTP if the animal had been habituated to the novel environment on three previous days (n = 5). (d) Exposure to the novel environment even in the absence of the objects (vertical bar) also facilitated the induction of LTP (n = 7). Insets show representative field EPSP traces at the time points indicated by the numbers. Horizontal calibration line, 5 ms; vertical line, 1 mV.

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alert state that had remained in the familiar environment throughout the recording period failed to produce a persistent change in synaptic strength (109.9 5.6, 111.1 6 and 110.1 9.6% of pre-conditioning baseline EPSP slope s.e.m. at 10 min, 1 h and 3 h, respectively; P > 0.05 compared to baseline, n = 7) (Fig. 1a). Remarkably, if the animals were allowed to explore the novel environment for a 5-minute period and then an identical weak conditioning stimulation was applied 5 minutes after returning to the familiar environment (while the animals were in a still alert state), a rapid and robust LTP was induced (176.1 19.1, 155.3 11.8 and 151.1 10.8% at 10 min, 1 h and 3 h, respectively; P < 0.05, n = 7; Fig. 1b). If the facilitation of LTP induction indeed resulted from the novel environment triggering a state in the hippocampus that favors the storage of new information, we reasoned that if the animals were habituated to the novel environment, then reexposure to such an environment should have little effect on LTP induction. A group of animals was familiarized with the novel environment by allowing exploration for a 30-minute period on three consecutive days. Subsequent application of the weak conditioning stimulation on the fourth or fifth day after the animals had visited the now-familiar environment for 5 minutes did not facilitate LTP induction (116.9 6.7, 120.9 9.4 and 116.6 11.5% at 10 min, 1 h and 3 h, respectively; P > 0.05, n = 5; Fig. 1c). These experiments show that the novelty aspect of the unfamiliar environment is crucial for the facilitation of LTP induction. We further investigated the nature of the novelty by removing all objects from the novel environment. The animals explored such a novel environment to the same extent as when the objects were present (that is, continuously during the 5-minute exposure period), and LTP was induced by the weak stimulation after they had returned to the familiar environment (144.3 10.7, 143.3 7 and 142.6 7.9% at 10 min, 1 h and 3 h, respectively; P < 0.05, n = 7; Fig. 1d). These experiments show that the presence of the novel objects was not essential and that relatively simple novelty in the environment was sufficient to trigger novelty exploration and to facilitate LTP induction.
Time window for LTP facilitation We examined the time window for the facilitation of LTP induction by novelty exposure to determine the temporal constraints
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on this form of information encoding by CA1 synapses. In one set of experiments, the animals remained in the novel environment for 25 minutes. The animals explored actively for 15 minutes and then became quiescent. Five minutes later, after returning to the familiar environment, application of the weak conditioning stimulation failed to facilitate LTP (124.5 10.5% at 1 h; P > 0.05, n = 5; Fig. 2a). A similar lack of LTP facilitation was observed when the animals explored the novel environment for 5 minutes, and then the weak tetanus was applied 25 minutes later in the familiar environment (98.9 6.1%; P > 0.05, n = 4; Fig. 2b). Thus, neither a prolonged (15 min) nor a brief (5 min) period of exploration facilitated LTP induction if there was a 30-minute delay between the start of exploration and the application of the conditioning stimulation. We also examined the effect of allowing the animals to explore the novel environment starting 5 minutes after applying the weak conditioning stimulation. Unlike in experiments where brief exploration took place 5 minutes before the weak tetanus, no LTP was induced (118.9 7.4%, P > 0.05, n = 5; Fig. 2c). Taken together, these experiments show that the time window for the facilitation of LTP by novelty exposure is relatively narrow.
Role of D1/D5 dopamine receptors Exposure to novelty activates mesolimbic dopaminergic neurons, which innervate several brain areas including the hippocampus2022. We therefore assessed the role of dopamine in facilitating the LTP induction seen in the present studies. As LTP at CA1 synapses can be regulated by D1/D5 dopamine receptor activation2325, we examined the ability of the selective antagonist SCH23390 to affect novelty-facilitated LTP induction. In animals pre-injected with SCH23390 (15 g in 5 l, i.c.v.) 50 minutes before introduction into the novel environment, the weak high-frequency conditioning protocol did not induce LTP even though the animal continuously explored the novel environment for the 5-minute exposure period (118.4 3.3% at 1 h; P > 0.05, n = 6; Fig. 3a). To quantify the exploratory activity in more detail, we carried out a separate behavioral experiment. The same dose (15 g in 5 l, i.c.v.) of SCH23390 that inhibited LTP facilitation did not significantly affect the time spent actively exploring the novel environment (121.1 11.1 s out of 150 s, n = 8 versus 131.8 11.6 s, n = 6 in controls; P > 0.05). There was, however, a difference between control and pretreated animals when they were allowed to explore the novel environment for a second time 1 h later. Control animals showed significant habituation (from 131.8 11.6 to 94.8 14.5 s, P < 0.05), but SCH23390 pretreated animals did not (from 121.1 11.1 to 103.3 14.5 s, P > 0.05). Most previous reports focus on the contribution of dopamine to LTP induced by relatively strong high-frequency stimulation in the CA1 area 14,2426. Here we examined how
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Fig. 2. Time window for facilitated induction of LTP by novelty exposure. (a) Exposure to the novel environment for 25 min (vertical bar) before application of the weak high-frequency conditioning stimulation (HFS, arrow) did not facilitate LTP (n = 5). Two graphs show a typical example (left) and mean s.e.m. (right). (b) Similarly, LTP was not induced if the animal explored the novel environment for 5 min and the weak HFS was applied 25 min later (n = 4). (c) Application of the weak HFS 5 min before exposure to the novel environment did not induce LTP (n = 5).

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D1/D5 receptor activation by an agonist affects the ability of weak conditioning stimulation to induce LTP. We chose to study the effect of the agonist SKF38393 in anesthetized animals to avoid possible behavior-related changes. The same weak highfrequency stimulation protocol that we used for experiments in awake animals did not trigger a persistent change in baseline synaptic transmission in urethane-anesthetized animals (107.6 3.6%, n = 8) (Fig. 4a, open circles). However, the injection of the D1/D5 agonist SKF38393 at a dosage that had no effect on baseline transmission (2.5 mg/kg, i.p., 106.8 17.9% at 3 h after injection; P > 0.05, n = 6), facilitated LTP induction (138 6.6% at 1 h; n = 7, P < 0.05 compared to baseline and vehicle-injected controls) (Fig. 4a, closed circles). Thus, in the intact hippocampus, dopamine receptor activation is not only necessary for the facilitation of LTP by novelty exposure, but also may be sufficient to mediate it. The facilitation of LTP induction by D1/D5 receptor activation appeared to depend on activation of protein kinase A (PKA). We examined the effect of the membrane-permeable PKA inhibitor Rp-cAMPS, at a dosage (80 g in 5 l, i.c.v.) that did not affect baseline synaptic transmission (111.0 16.1% at 3 h, n = 4) and well below that having nonPKA related effects on LTP when applied intracellularly 27 . Injection of Rp-cAMPS at the same time as SKF38393 prevented the induction of LTP by weak high-frequency stimulation (102.7 7.5% at 1 h, n = 6, P > 0.05 compared to baseline, P < 0.05 compared to SKF38393 alone) (Fig. 4b).
Role of muscarinic receptors and -adrenoceptors Novelty exposure is also associated with increased cholinergic28 and noradrenergic29 drive to the hippocampus, and activation of muscarinic cholinoceptors and -adrenoceptors 30,31 can

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facilitate LTP induction. Therefore we also examined the effects of selective antagonists for these receptors. Injection of the -adrenoceptor antagonist propranolol (2 g in 5 l) 10 minutes before novelty exposure did not block the facilitation of LTP induction (139.6 17.2% at 1 h, P < 0.05, n = 5) (Fig. 3b). Similarly, in the presence of the muscarinic receptor antagonist scopolamine (5 g in 5 l), the weak conditioning stimulation induced a robust LTP after novelty exposure (217.4 17.2% at 1 h; P < 0.05, n = 4) (Fig. 3c).

DISCUSSION
In previous experiments, we found that exposure to a novel environment does not significantly affect the induction of LTP by strong high-frequency conditioning stimulation32. In the present study, we used a weak conditioning protocol to assess the ability of brief novelty exposure to modulate the induction of LTP. We discovered that novelty had a marked facilitatory effect on the induction of LTP by weak high-frequency stimulation. Moreover, re-exposure of animals to such an environment after habituation did not affect LTP induction. This indicates that when environmental novelty triggers a state in the hippocampus favoring the storage of new information, it also facilitates the induction of LTP. The finding that LTP induction was enhanced during a period of quiescence after novelty exposure has important implications for state-dependent models of hippocampal mnemonic function8,15. According to one model15, the induction of naturally occurring LTP-like potentiation in selected pyramidal cells takes place preferentially after exploration is complete, a time of

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Fig. 3. Dopamine-dependence of the facilitation of the induction of LTP by novelty exposure. (a) Application of the weak conditioning stimulation (arrow) after exploration of the novel environment (vertical bar) did not induce LTP in freely behaving rats in which D1/D5 receptors were blocked with the selective antagonist SCH23390 (15 g i.c.v., asterisk, n = 6). Graphs show a typical example (left) and mean s.e.m. (right). (b) Block of -adrenoceptors with the selective antagonist propranolol (2 g i.c.v., asterisk) in freely behaving rats did not prevent the facilitation of LTP induction by novelty exposure (n = 5). (c) Similarly, robust LTP was induced by the conditioning stimulation when applied after exploration of the novel environment if muscarinic cholinoceptors were blocked with the selective antagonist scopolamine (5 g i.c.v., asterisk, n = 4). Horizontal calibration line, 5 ms; vertical line, 1 mV. nature neuroscience volume 6 no 5 may 2003

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Fig. 4. Pharmacological activation of D1/D5 dopamine receptors mimics the effect of novelty exploration on LTP induction. (a) Injection of anesthetized animals with the selective agonist SKF38393 (2.5 mg/kg, i.p., asterisk) 30 min before weak high-frequency conditioning stimulation (arrow) facilitated the induction of LTP (n = 7). Application of the same weak conditioning protocol in vehicle-injected controls did not induce LTP (n = 8). (b) The PKA inhibitor Rp-cAMPS (80 g) injected i.c.v. at the same time as the i.p. injection of SKF38393 blocked the induction of LTP by the weak conditioning protocol. Values are mean s.e.m.

hippocampal sharp-wave electroencephalogram (EEG) activity. This would allow encoding of memories of important recent events15. In a second model8, learning-related NMDA receptordependent LTP occurs primarily during (not after) periods when novel stimuli appear, and is associated with hippocampal theta/gamma EEG oscillations. Our results show clearly that brief exposure to new information could prime the CA1 area to enable the triggering of persistent increases in synaptic strength at a time after the novelty had been actively explored and replaced by familiar information. Quiescent periods soon after investigative behavior may provide favorable conditions for naturally occurring LTP-like synaptic strengthening that is believed to be necessary for memory formation in the hippocampus12. Such a mechanism provides a means for the preferential processing for storage of new information and events that occur in a novel context together and thereby enable subsequent retrieval in a context-dependent manner. The present data also elucidate how novelty exposure can control learning by activation of mesolimbic dopaminergic neurons22,33. Indeed, the model8 that posits a key role of increased dopaminergic drive to the hippocampus in the novelty-driven promotion of information encoding at CA3-CA1 synapses is consistent with our findings. The present results show that a salient environmental change can trigger a transient dopaminedependent state in the hippocampus that favors the synaptic storage of recently encountered events. This provides an explanation for the known ability of hippocampal dopamine depletion to impair34 (and dopamine D1 receptor agonists to promote) certain types of hippocampus-dependent learning35,36. The present finding that the dopamine D1 receptor antagonist SCH23390 inhibited behavioral habituation to the same novel environment that facilitated LTP induction is consistent with such a mechanism. Dopamine D1/D5 receptor activation was sufficient for novelty facilitation of LTP induction in the intact hippocampus without needing either muscarinic cholinoceptor or -adrenoceptor activation. The ability of a selective D1/D5 receptor agonist to mimic the effect of novelty in anesthetized animals will allow a detailed evaluation of the cellular actions of dopamine that may be responsible for the reduction of the threshold of LTP induction in vivo. The present studies point to a key requirement for activation of PKA. It will be of interest to determine the role of further downstream effectors, such as cyclic AMP response element (CRE)-mediated transcription, that are known to be essential for hippocampus-dependent memory formation14,37. In contrast to the present experiments where exposure to a novel environment was found to facilitate the induction of LTP, previous studies32,38,39 report that novelty exploration promotes
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the reversal of previously established LTP (depotentiation). The two phenomena clearly complement one another, with the prevailing experimental conditions determining which effect will predominate. The factors controlling the dominance of either the facilitation of LTP induction or the reversal of LTP expression include the sequence of novelty exposure/conditioning stimulation, the strength of the conditioning stimulation32 and the duration 40 of novelty exposure (see Supplementary Table 1 online). The facilitation of LTP induction described here required that brief (5 min) novelty be introduced before (10 min) the conditioning stimulation that was sub-threshold (weak HFS, 10 trains of 10 pulses at 100 Hz). The reversal of LTP expression described in our previous reports32,40 requires that prolonged (20 min) novelty be introduced after (1 h) the conditioning stimulation that is at or near maximal (strong HFS, 10 trains of 20 pulses at 200 Hz). On the other hand, LTP induction was not facilitated if the novelty exposure was prolonged (25 min) or introduced after (5 min) the conditioning stimulation (present results) or if strong HFS is used 32. LTP is not reversed if the novelty is brief (10 min)40 or if it is introduced before (5 min) the HFS32. Thus the temporal constraints of these complementary phenomena are different, indicating different underlying mechanisms. This is consistent with the idea that although the acquisition of novel information by the hippocampus may lead to a persistent widespread weakening of recently strengthened synaptic contacts, it occurs in tandem with the selective activity-dependent strengthening of sparsely distributed synaptic networks32. Such interplay would favor continuous updating of important information for transfer to other brain regions, such as the cortex, for long-term storage. The time window for the facilitatory effect of novelty on LTP induction would presumably change rapidly as the behavioral relevance of the new information for learning fluctuates. The CA1 area seems to act as both an information store and a mismatch detector8, suggesting that the interaction between novelty detectiontriggered dopamine release and the subsequent promotion of information encoding should be critical for the normal function of the intact hippocampus.

METHODS
Electrode implantation and electrophysiology. Experiments were performed on freely behaving male Wistar rats, weighing 250350 g (711 weeks old) under license from the Department of Health and Children, Ireland. The method of implantation of the electrodes in the stratum radiatum of the CA1 region under pentobarbitone sodium (60 mg/kg, i.p.) anesthesia was similar to that described previously32. Experiments commenced after the animals had fully recovered from surgery and were used to being handled (usually about two weeks after surgery). After surgery, the rats were housed individually in their home cage between recording sessions. In the acute experiments, the animals were anesthetized by intraperitoneal (i.p.) injection of urethane (1.5 g/kg), and body temperature was maintained at 37 C. In all experiments, Schaffer collaterals/commissural fibers were stimulated at a frequency of 0.033 Hz and at an intensity evoking half-maximum population field EPSPs. On the
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basis of pilot studies in which the number of pulses and stimulus intensity were systematically adjusted, the weak high-frequency stimulation conditioning protocol (HFS) was set at a level subthreshold for LTP induction in the familiar recording environment (10 trains of 10 stimuli at the test pulse intensity, inter-stimulus interval 10 ms, i.e 100 Hz, inter-train interval 2 s). The magnitude of LTP is expressed as the mean percentage of preHFS baseline EPSP slope standard error of the mean (s.e.m.). Analyses of variance (ANOVAs) and Students t-tests were used for statistical analysis. In some animals, a cannula was implanted in the ipsilateral lateral cerebral ventricle to allow intracerebroventricular (i.c.v.) injection of drugs41. (D,L) Propranolol HCl, () scopolamine HBr, R-(+)-SCH23390 (7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine) HCl and Rp-cAMPS (adenosine 3,5-cyclic monophosphothioate) triethylammonium salt were purchased from Sigma-RBI, Ireland. SKF38393 (1-phenyl-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-3-benzazepine) HBr was obtained from Tocris (U.K.). Drugs were injected i.c.v. rather than intra-hippocampally to avoid loss of stability of intrahippocampal electrophysiological recording and stimulation. The doses chosen were based on previous studies using similar methods4247. Electrophysiological data from a total of 68 rats are presented. Four of the rats in Fig. 1c (exposure to novel environment after repeated habituation, no LTP elicited) had been studied 56 d previously in the experiments presented in Fig. 2b (first-time exposure to novel environment 25 minutes before conditioning stimulation, no LTP elicited). One of the rats in Fig. 1b (exposure to novel environment before weak conditioning stimulation, LTP elicited) had also been studied previously (Fig. 2c, exposure to novel environment after weak HFS, no LTP elicited). In the pharmacological experiments, only one drug protocol was tested in each animal. Experiments under the different conditions in which weak conditioning stimulation induced or did not induce LTP were carried out over the same time period. Recording apparatus and novelty exposure. Experiments were carried out in a well-lit (750 lux, fluorescent lighting) room. The Perspex plastic recording box consisted of two equally sized compartments (each 27 24 24 cm) with different colored walls (filter factor 3) and contents, separated by an opaque barrier. All animals were first habituated to the recording procedure in one compartment (red translucent walls with wood shaving bedding) over the post-surgery recovery period. The novel compartment had yellow translucent walls and 13 small novel objects instead of bedding. Upon removal of the opaque barrier separating the two compartments, animals were gently shepherded into the novel environment if they hadnt entered it spontaneously within 60 s. The barrier was removed temporarily again 5 min later to return animals to the familiar compartment, unless otherwise stated. The small size of the recording box, together with the short duration of exploration and application of high frequency stimulation under the same behaviorally quiescent state, reduced possible interference from motor activity effects such as increases in brain temperature1618,48. Rather than test the effect of novelty exposure on LTP induction during or immediately after exploration, we used a 5-min delay between re-entering the familiar environment and applying the conditioning stimulation to further minimize any possible confounding effects of exposure to the novel environment48. Thus EEG analysis, using the same intrahippocampal electrode that recorded EPSPs, showed that theta activity in the 46 Hz range increased by a factor of 2.87 when rats first entered and explored the novel environment, but returned to baseline 5 min after return to the familiar environment, a time when they were behaviorally quiescent (n = 4). Similarly, when brain temperature was monitored in animals that entered the novel environment, using a fine (tip diameter 0.5 mm) type-T thermocouple wire placed either in the contralateral hippocampus or cortex, the increase was relatively small (<1 C at 510 min, accuracy 0.05 C, n = 4)48. Furthermore, there was no difference in the serum corticosterone levels measured with high-performance liquid chromatography (HPLC) between animals in the familiar and novel environments (3 0.8 versus 5.2 1.2 g/dl respectively, n = 4 per group)48. None of the animals showed behavioral signs of stress such as freezing or piloerection48. Exploratory activity. A total of 14 animals were studied in a separate behavioral experiment to quantify the effect of SCH23390 on exploratory 530

activity in the novel environment. A cannula was chronically implanted in the lateral cerebral ventricle at least one week before testing, as described above for the electrophysiology experiments. The animals were pre-exposed to the familiar compartment of the two-compartment box for 10 min per day for 5. On the test day, the animals were injected i.c.v. with either SCH23390 (15 g in 5 l) or water vehicle (5 l) 1 h before they were placed in the familiar compartment for 150 s. The barrier was temporarily removed to allow the animal enter the novel compartment. After 300 s, the animal was allowed return to the familiar compartment for a further 150 s, and then it was returned to its home cage. A recall trial took place 1 h later to determine if the animal had habituated to the novel environment. The animal was replaced in the familiar compartment for 150 s before the barrier was again temporarily removed. The animal was allowed to re-explore the novel compartment again for 300 s. Time spent actively investigating the compartments was determined in 150-s epochs. Active investigation was operationally defined to include horizontal and vertical (rearing) locomotor activity as well as gross head movement, such as contact with the novel objects. The measure excluded time when the animal was immobile, in a still and alert state, or grooming. The experimenter rating the behavioral activity was blind as to treatment group.
Note: Supplementary information is available on the Nature Neuroscience website.

Acknowledgments
The authors wish to thank D. Balschun for help with the i.c.v. injection method. This research was funded by the Wellcome Trust, the Health Research Board of Ireland, Enterprise Ireland, the Irish Higher Education Authority (Programme for Research in Third-Level Institutions), European Union and Science Foundation Ireland.

Competing interests statement

The authors declare that they have no competing financial interests.

RECEIVED 5 FEBRUARY; ACCEPTED 27 MARCH 2003

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