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The effects of water deprivation on conditioned freezing to contextual cues and to a tone in rats
Behavioural Brain Research 119 (2001) 49 – 59 www.elsevier.com/locate/bbr
Research report
The effects of water deprivation on conditioned freezing to contextual cues and to a tone in rats Bruno Pouzet a, Wei-Ning Zhang a, Mark A. Richmond b, J. Nicholas P. Rawlins b, Joram Feldon a,* a
Beha6ioural Neurobiology Laboratory, Swiss Federal Institute of Technology, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland b Department of Experimental Psychology, South Parks Road, Oxford OX1 3UD, UK Received 28 April 2000; received in revised form 19 September 2000; accepted 19 September 2000
Abstract In two experiments we used an automated system for quantifying freezing responses in rats to replicate and extend Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645–9; Maren S, DeCola JP, Swain RA, Fanselow MS, Thompson RF. Parallel augmentation of hippocampal long-term potentiation, theta rhythm and contextual fear conditioning in water deprived rats. Behav Neurosci 1994;108:44–57) who found that water deprivation in rats produced a selective enhancement in conditioning to context, as opposed to conditioning to a tone. In experiment 1 we gave water deprived and non-deprived rats either three or ten pairings of a tone and foot shock. During conditioning water deprivation decreased overall freezing only in rats that received ten pairings. On 2 subsequent days we assessed conditioned freezing (1) to the contextual cues of the conditioning chamber and (2) to the tone when presented in a distinctive, novel environment. We found, in direct contrast to Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645–9), that (a) water deprived rats did not differ from non-deprived rats in levels of conditioned contextual freezing and that (b) water deprived rats did show reduced levels of freezing to the tone stimulus. In the same experiment we found that the number of tone–shock pairings did not affect levels of conditioned contextual freezing but that rats that had received three pairings did show reduced levels of freezing to the tone stimulus compared with rats that had received ten pairings, thereby demonstrating that the behavioural procedure and analysis system that we used was appropriately sensitive to differences in conditioning. In experiment 2, therefore, we sought to replicate Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645– 9) using, as far as possible, exactly the same procedural parameters. Here we found that water deprivation produced no effects on conditioned freezing to the contextual cues or to the tone. We conclude that there is sufficient reason to doubt the generality of the previously reported findings. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Water deprivation; Freezing; Conditioning; CS; Context; Fear; Hippocampus; LTP; Rat
1. Introduction It is well established that rats who have received pairings of a tone and electric foot shock in a conditioning chamber will come to show freezing behaviour, * Corresponding author. Tel.: +41-1-6557448; fax: 6557203. E-mail address: [email protected] (J. Feldon).
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in the absence of the shock, when placed back into that chamber and/or when re-presented with the tone [1,2]. This change in the behavioural response to a stimulus presentation reflects learning on the part of the animal, since freezing typically does not occur as a response to either conditioning chambers or tones before they are paired with shock [16]. Thus, the freezing response appears to be a suitable index of learning a Pavlovian stimulus–shock relationship. Recently the freezing re-
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sponse has become a focus of attention among neuroscientists who are interested in the anatomical and physiological correlates of behaviour. For instance, there are now many published demonstrations of hippocampal lesion-induced impairments in conditioned freezing to contextual cues, such as the conditioning chamber in which conditioning took place, but not in conditioned freezing to the tone with which shock was directly paired (e.g. [6,7,12,16 – 18], but see [22]). Meanwhile, lesions of the amygdala have been found to impair conditioned freezing to both contextual and discrete cues [16] (see also McNish et al. [13]). Such findings, and their interpretation in terms of impaired contextual processing, are generally believed to be consistent with several theories of hippocampal and amygdaloid function. For instance, both spatial theory [14,15] and configural association theory [25,27] of hippocampal function treat contextual and spatial cues as similar in kind, albeit for different reasons. Proponents of these theories note the physical similarities between typical contextual cues and typical spatial cues: both are large, diffuse, multi-cue and poly-modal. They contrast such cues with both auditory and visual discrete CSs: these are often single cues in a single modality. Thus there is some agreement that spatial and contextual learning require an intact hippocampus because of the relatively complex nature of the stimuli involved (see also [8]). Nonetheless, there is an alternative and simpler interpretation of the results obtained in all these freezing studies. This notes that lesions of the hippocampus produce increases in general locomotor activity and an increased tendency to explore environments [4,5]. According to this view, the impairments in conditioned freezing to contextual cues result from the rat’s tendency to be hyperactive or more vigorously to explore its environment, rather than from a spatial or configural learning impairment. Because in the majority of studies, conditioned tone freezing has been at higher levels than conditioned context freezing, Good and Honey [4] have suggested that a given level of activity is more likely to interact with a stimulus that elicits little freezing (a context) than one that elicits more substantial freezing (a tone) (p. 487). For this reason alone hippocampal lesions might induce an increase in activity that alters conditioned freezing to contextual cues at the same time as leaving conditioned freezing to a tone intact [22]. Moreover, it has long been understood that putative lesion-induced or drug-induced selective impairments in behaviour could be a consequence of different initial response rates rather than of selective action on memory. For instance, Rawlins et al. [20] found that chlordiazepoxide HCl produced a selective impairment in instrumental aversive conditioning, as opposed to classical aversive conditioning, only when the initial response rates were not matched. Thus, previ-
ous reports might have observed what appear to be selective effects on contextual conditioning simply because of the higher levels of freezing seen to the tone than to the context. The primary advantage of the freezing response, over and above (say) suppression of licking, is that it is a measure of suppression of an unlearned spontaneous locomotor activity. Alternative measures have typically assessed the suppression of stable learned behaviours, such as lever pressing or licking. In these experiments rats would typically be maintained on food or water deprivation. In studies of animal learning it is becoming increasingly clear that motivational factors play a major part, at least in the operant learning process (e.g. [3]). Furthermore, there is evidence emerging from the neurosciences to suggest that food and water deprivation might act at brain regions such as the hippocampus or nucleus accumbens to produce changes in conditioned behaviour [9,31]. In particular, Maren et al. [9,10] found that water deprivation in non-lesioned rats caused an enhancement in conditioned contextual freezing but no changes in conditioned tone freezing. This pattern suggests that water deprivation produced a selective increase in conditioning to context, as opposed to conditioning to a tone. Maren et al. further found that, as well as showing an enhancement in conditioned freezing to context, water deprived rats also showed an enhancement of perforant path long-term potentiation (LTP) and of hippocampal theta. These authors note the compatibility between this correlation and the commonly observed impairment in conditioned contextual freezing after hippocampal lesions (see above). They suggest that because hippocampal LTP has been (previously) implicated in the mnemonic encoding of contextual and spatial information, the greater LTP induced in waterdeprived subjects is a putative neural mechanism for the superior representation of contextual stimuli in these rats ([10] p. 53). They conclude that their results provide evidence for a role of the hippocampus in the augmentation of contextual fear conditioning by water deprivation, and more generally, in the mediation of contextual learning ([9] p. 648). A similar rationale has been applied to sex differences in LTP and contextual conditioning (as opposed to discrete CS conditioning), with the argument that male rats show more LTP and contextual conditioning compared with female rats [11], but see the results and discussion in Pryce et al. [19]. One strong implication of this conclusion is that interpretation of studies that have used water or food deprived rats to investigate the involvement of the hippocampus in behaviour might be compromised. For instance, Selden et al. [26] found impaired conditioned context preference in hippocampal lesioned rats that had been water deprived. It is possible that Selden et al. observed an enhancement in conditioned place prefer-
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ence in their control rats that had been water deprived. Because according to Maren et al., water deprivation acts at the hippocampus to produce such changes, rats with hippocampal lesions would not have shown such an enhancement. Therefore, in this case rats with hippocampal lesions could have shown less conditioning to context than control rats without suffering from any impairments whatsoever. Thus, Selden et al.’s results can not be interpreted unambiguously as demonstrating a hippocampal lesion-induced impairment in conditioning to context. This ambiguity has serious implications since Winocur et al. ([30], see also [29]), in the first study to assess the effects of hippocampal lesions on conditioning to context, found that conditioned avoidance of a black conditioning chamber in which tone–shock pairings had been presented was enhanced in the (non-deprived) hippocampal lesion group when rats were given a choice between the black chamber and an adjacent larger white chamber. Thus, there appear to be potential problems associated both with the use of freezing in hippocampal lesioned rats — i.e. that hippocampal lesions cause hyperactivity — and with the use of some alternative response measures that require such rats to be water deprived — i.e. that water deprivation itself acts at the hippocampus to produce changes in contextual conditioning. A principal point of interest in our laboratory is to assess the involvement of the hippocampus in conditioning using response measures that avoid interpretations in terms of lesion-induced hyperactivity. But given the potential problems associated with the use of water deprived hippocampal lesioned rats we sought, as a first step, to assess the generality of Maren et al.’s findings. Furthermore, there are at least two reasons to believe that Maren et al.’s claim for a selective enhancement of contextual fear in water deprived rats, and by implication the claim for hippocampal involvement, may be premature. First of all, we have found, using a new computer controlled analysis of freezing behaviour that enables one to measure the development of freezing over time, that group differences can emerge as a significant interaction with repeated measures over time, in the absence of any significant main effects (see [21]). Maren et al.’s method of data collection gave an overall score and so could have failed to detect a pattern of freezing developing over time. Secondly, their behavioural testing procedure could have differentially contributed to this problem of measurement: Maren et al.’s [9] tone test had a duration of only 64 s and the context test had a duration of 8 min. Thus an effect of water deprivation on the tone test might have been obscured simply by its relatively short duration; the test may have ended before a differential pattern of freezing had developed. In our study context and tone were tested under equivalent conditions.
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In experiment 1 we assessed the effects of water deprivation and number of tone–shock pairings on conditioned freezing using parameters we have used previously in our laboratories. On day 1 all rats received either three or ten pairings of a tone and foot shock in a distinctive chamber. Twenty-four hours later they were placed back into the conditioning chamber to measure conditioned freezing to contextual cues. Fortyeight hours following conditioning they were placed into a novel chamber. Here we measured conditioned freezing to the tone during an 8-min presentation. To avoid ‘state dependency’ effects, which might mask transfer of information from the conditioning process to its expression in contextual and discrete tone induced freezing, the rats in experiment 1 were maintained on water deprivation throughout the experimental process. Because in this procedure we failed to observe the pattern of findings reported by Maren et al. [9] we conducted experiment 2 in which we sought to replicate Maren et al. [9] using, as far as possible, exactly the same procedural parameters. Rats were either water deprived or sated and, on day 1, received conditioning with three tone–shock pairings in an 8-min session. Immediately after conditioning, all rats were allowed free access to water for the remainder of the experiment. On day 2 all rats were placed into a novel chamber and conditioned tone freezing was measured. On day 3 all rats were placed back into the conditioning chamber to measure conditioned freezing to contextual cues.
2. Materials and methods
2.1. Subjects Subjects were 28 male hooded Lister rats (Harlan CPB, the Netherlands) that weighed between 300 and 350 g during behavioural testing. Rats were housed in groups of four in a temperature (2291°C) and humidity (559 5%) controlled room under a 12:12 h reversed light–dark cycle with lights on from 19:00 to 07:00 h. All behavioural testing was carried out in the dark phase of the cycle.
2.2. Apparatus Eight Coulbourn Habitest operant test chambers (Coulbourn Instruments, Allentown, PA) were used for behavioural testing. Four identical chambers were used for conditioning and assessment of contextual learning (model no. E10-10RF). These had two aluminium walls and two Perspex walls. The house light remained off throughout the experimental sessions in this chamber. The chamber floor consisted of 16 stainless steel bars with a diameter of 5 mm, spaced 1.3 cm apart, through
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which shocks could be delivered. An additional four chambers were used for assessing conditioned freezing to the tone stimulus. These had three black walls and one Perspex wall. A house light (1.12 W) positioned 1.8 cm from the ceiling on one wall and directed upwards, was lit during all the testing in this box. The floor of these chambers was a cross grid. Presentation of stimuli was controlled by a Coulbourn Universal Environmental Interface (model no. E91-12) and a Coulbourn Universal Environmental Port (model no. L91-12). Shocks were delivered, where appropriate, via a Coulbourn Precision Regulated Animal Shocker (model no. E13-12). We assessed immobility in two ways. The first used an automated analysis system developed in our laboratory (see [19,21,22]): Attached to the centre of each chamber’s ceiling was a wide-angle (100°) 2.5-mm lens (VPC-465B; CES AG, Zurich, Switzerland), through which a black and white video image of the experimental session was recorded (Sony video recorder, SVT1000). For the purposes of recording, each chamber was illuminated by four infrared LEDs (Hewlett Packard; model no. HSDL-4220; wavelength 875 nm), which were attached to the ceiling. Data analysis was carried out simultaneously using a Power Macintosh (7600/120) with a permanent video input, and a script derived from the ‘NIH IMAGE’ programme (available from web-site http://rsb.info.nih.gov/nih-image). Our script works by comparing every two adjacent seconds of video tape which are presented on separate screens, each containing 100 000 pixels. Each pixel is given a ‘grey value’ from 0 to 255 where 0 refers to black and 255 to white. Then the value of each pixel on screen 1 (the image of second 1) is compared with the value of the corresponding pixel on screen 2 (the image of the following second). A ‘grey value’ difference score for each pair of pixels is calculated and this data is used to prepare a third screen which represents the difference between screens 1 and 2. In the experiments reported here our criteria were as follows: a difference score of 5 or less resulted in a ‘no-movement’ score for the corresponding pixel on screen 3, which was therefore left white. A difference score of greater than 5 resulted in a ‘movement’ score for the corresponding pixel on screen 3, which was, therefore, coloured black. After minor adjustments for random noise, the analysis was thus able to provide, for each second of the experimental session, a score which represents the percentage change between that second and the following one. We scored immobility for each second if and only if the number of black pixels on screen 3 was less than 0.05% of the total. This percentage had been calibrated visually to ensure that breathing movements alone were not sufficient to produce a movement score. Furthermore, unpublished pilot observations revealed a 90% correspondence between the overall scores from this
automated system and those scores obtained using visual criteria similar to those commonly employed (e.g. see [4,6,12,24]). Thus, our automated measures of immobility can be validly used as an index of freezing behaviour. The second method of immobility assessment was used in experiment 1 only: A trained observer, who was blind to the experimental conditions of each rat, measured freezing from a video (cf. [9]). A score was taken every 8 s to yield 60 measurements over the 8-min test periods. This was in order to compare directly the results obtained with Maren’s method and those obtained using our own automated system.
2.3. Procedures 2.3.1. Experiment 1 For 7 days prior to conditioning, and throughout the entire duration of the experiment, one group of rats (water deprived WD, N=8) were maintained on a restricted-fluid schedule of 1 h access to water per day. The remaining group of rats (non-deprived ND, N=8) remained on ad-lib water throughout. During this period all rats were handled daily (3 min per rat per day). On day 1 of the experiment all rats were placed in the conditioning chambers for 30 min. During this session, half the rats in each group received three pairings (group 3-SH) of a tone (30 s duration, 85 dB) and foot shock (1 s, 0.5 mA), and the other half (group 10-SH) received ten pairings of a tone (30 s, 85 dB) and foot shock (1 s, 0.5 mA). Group 3-SH received the first tone–shock pairing 5 min after placement in the chamber and the subsequent intertrial interval was fixed at 9 min. They were removed from the chamber 5 min after the final shock. Group 10-SH received the first tone– shock pairing 90 s after placement in the chamber and the subsequent intertrial interval was fixed at 150 s. Rats in this group were removed from the chamber 90 s after the final shock. We assessed freezing during acquisition of conditioning using the automated system. Twenty-four hours after conditioning all rats were placed back into the conditioning chamber for 8 min and conditioned freezing to the contextual cues was assessed. Freezing was scored by means of the automated system and also using a visual scorer (see above). Twenty-four hours after the context test we assessed conditioned freezing to the tone stimulus. Rats were placed in a novel chamber, which differed from the conditioning chamber as described above, for 11 min. After 3 min the tone was presented for the remainder of the test session. Freezing behaviour was assessed in the same manner as described for the context test. 2.3.2. Experiment 2 For 7 days prior to conditioning one group of rats (deprived, N=6) were maintained on a restricted-fluid schedule of 1 h access to water per day. The remaining
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group of rats (non-deprived, N =6) remained on ad-lib water throughout. During this period all rats were handled daily (3 min per rat per day). On day 1 of the experiment all rats were placed in the conditioning chambers. During this session, rats in each group received three pairings of a tone (64 s, 85 dB) and foot shock (1 s, 0.5 mA). Rats received the first tone –shock pairing 3 min after placement in the chamber and the subsequent intertrial interval was 94 s. They were removed from the chamber 30 s after the final shock, returned to their home cages and all rats were allowed ad-lib water for the remainder of the experiment. Twenty-four hours after conditioning rats were placed in a novel chamber, that differed from the conditioning chamber as described above, for 11 min in order to assess conditioned freezing to the tone. After 3 min the tone was presented for the remainder of the test session. Freezing behaviour was assessed using the automated system (see above). Twenty-four hours later all rats were placed back into the conditioning chamber for 8 min and conditioned freezing to the contextual cues was assessed. Freezing was assessed using the automated system.
2.4. Statistical analyses Results were calculated as the average percentage of time spent freezing in a specified time period. Specifically we calculated the total number of seconds spent freezing divided by the maximum possible number of seconds spent freezing. This value was then multiplied by 100. Thus a rat that was immobile for 15 s during a block of 30 s would receive a freezing score of 50%. For experiment 1 we conducted two three-way splitplot ANOVAs on the acquisition of conditioned freezing, using automated measurement data obtained during the conditioning session: (1) for rats receiving ten tone–shock pairings, (2) for rats receiving three tone –shock pairings. These ANOVAs had variables of water deprivation schedule (two levels: water deprived vs. non-deprived), trial (either ten or three levels) and period (three levels: pre-CS, CS and post-CS). The pre-CS, CS and post-CS periods refer to the 30 s before each tone presentation, the 30 s of each tone presentation and the 30 s following each tone presentation, respectively. We conducted three further ANOVAs of automated measurement data obtained during the extinction test sessions: (1) of the 8-min context test, (2) of the first minute of the context test, (3) of the 8 min of tone presentation during test, (4) of the first minute of tone presentation during test. For these ANOVAs data were combined into blocks of 30 s. The between subjects variables were water deprivation schedule (two levels: water deprived vs. non-deprived) and number of tone –shock pairings (two levels: 3-SH vs. 10-SH). In four additional ANOVAs, we compared directly the
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measurements taken from the automated system and those taken by the visual scorer. The first assessed the 8-min context extinction test; the second assessed the 8-min tone extinction test itself; and the third assessed the initial 64 s of the tone extinction test (exactly as Maren et al. [9] had done). The analyses all had between subjects variables of water deprivation schedule (two levels) and number of tone–shock pairings (two levels), and a within subjects variable of measurement (two levels: automated vs. visual). For experiment 2 we conducted two split-plot ANOVAs: (1) of the 8-min context test, (2) of the 8-min tone presentation during test. For these ANOVAs data were combined into blocks of 30 s. The between subjects variable was water deprivation schedule (two levels).
3. Results
3.1. Experiment 1 3.1.1. Acquisition of freezing during conditioning Results are depicted in Figs. 1 and 2 which show conditioning data for rats in groups 10-SH and 3-SH respectively. In group 10-SH rats showed an increase in pre-CS and CS freezing during the first three trials followed by no further increases thereafter (Fig. 1A and B). Freezing during the post-CS periods did not increase over trials (Fig. 1C). Furthermore, water deprived rats showed lower levels of freezing during pre-CS, CS and post-CS periods than did non-deprived rats. ANOVA supported these conclusions as follows. There was a significant main effect of trial, F(9,108)= 7.27, PB 0.001, and a significant main effect of period, F(2,12)= 37.14, PB 0.001. The effect of period reflected higher levels of freezing during pre-CS and CS periods (which did not differ from each other) than during post-CS periods, PB 0.05 in both cases. There was also a significant interaction between these two variables, F(18,108)=4.63, PB 0.001. Post-hoc t-tests conducted on the interaction revealed a significant increase in freezing over trial only during pre-CS and CS periods. There was a main effect of water deprivation that approached conventional levels of significance, F(1,6)= 5.89, P= 0.051. Water deprived animals tended to be more active overall, and therefore had lower freezing scores. None of the remaining interactions was significant at the 0.1 level. In group 3-SH rats showed an increase in pre-CS (Fig. 2A) and CS (Fig. 2B) freezing during the three trials. Freezing during the post-CS periods decreased slightly over trials (Fig. 2C). ANOVA revealed a significant main effect of trial, F(2,24)= 9.03, PB 0.01, and a significant main effect of period, F(2,12)= 14.59, PB 0.001. The effect of period reflected a significant in-
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crease in freezing at the onset of the CS as well as a significant decrease in freezing at the offset of the CS, P B0.05 in both cases. There was no significant effect of water deprivation either as a main effect or as an interaction with any other variable, P \ 0.1 in all cases.
Fig. 2. (A – C) Show the development of conditioned freezing as a function of trials (1 – 3) for rats in group 3-SH. The figure shows rats split into groups water deprived (WD) and non-deprived (ND). (A) Shows the pre-CS period, (B) shows the CS period, (C) shows the post-CS period. Data are presented as percentage of freezing. Error bars denote S.E.M. The inter-trial interval was 300 s.
Fig. 1. (A – C) Show the development of conditioned freezing as a function of trials (1 – 10) for rats in group 10-SH. The figure shows rats split into groups water deprived (WD) and non-deprived (ND). (A) Shows the pre-CS period, (B) shows the CS period, (C) shows the post-CS period. Data are presented as percentage of freezing. Error bars denote S.E.M. The inter-trial interval was 90 s.
3.1.2. Freezing during extinction sessions Overall results of the context test (shown in Fig. 3A and B) were as follows: group WD/10-SH, 38.79 4.4%. Group ND/10-SH, 33.19 3.9%. Group WD/3-SH, 21.59 3.6%; Group ND/3-SH, 27.39 3.4%. ANOVA revealed no statistically significant differences between
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the groups or any significant interactions, except for an overall significant effect of block, F(15,180) = 4.21, PB 0.001. Overall freezing increased from 8.1% during the first block of testing to 46.6% during the fourth minute of testing. Thereafter freezing declined steadily to 25.1% during the final block of testing. In the ANOVA of the first minute of the context test there was an identical pattern of statistical findings: No effects of any between subjects variable, nor any significant interactions, P\ 0.1, except for a significant main effect of block, F(1,12) =5.78, P B0.05, this time reflecting an overall increase in freezing from 8.1% during the first block of testing to 17.6% during the second block. Results from the tone test are shown in Fig. 4A and B. Tone-induced freezing was much more pronounced than context-induced freezing (overall mean: tone= 71.0%, context=30.1%). In the ANOVA of the entire 8
Fig. 4. (A and B) Show conditioned freezing during the tone test of experiment 1. (A) Shows rats split into groups water deprived (WD) and non-deprived (ND). (B) Shows rats split into groups that had received three or ten conditioning trials. Data are presented as percentage of freezing for each 30 s of the 8-min tone presentation. Error bars denote S.E.M.
Fig. 3. (A and B) Show conditioned freezing during the context test of experiment 1. (A) Shows rats split into groups water deprived (WD) and non-deprived (ND). (B) Shows rats split into groups that had received three or ten conditioning trials. Data are presented as percentage of freezing for each 30 s of the 8-min context test. Error bars denote S.E.M.
min of tone presentation we found a significant effect of water deprivation, F(1,12)= 6.78, PB 0.05. As in the 10-SH acquisition study, this reflected the fact that water deprived rats showed decreased conditioned freezing to the tone compared with non-deprived rats (Fig. 4A). We also found a significant effect of pairings, F(1,12)=6.03, P B0.05, reflecting the fact that rats that had received three pairings showed decreased conditioned freezing to the tone compared with rats that had received ten pairings (Fig. 4B). There was a significant main effect of block, F(15,180)= 5.28, P B0.01, reflecting an initial increase followed by extinction of conditioned freezing during the test. None of the interactions was significant at the 0.05 level. Overall mean and standard error values for the four individual groups were as follows. Group WD/10-SH, 75.39 3.3%; group ND/10-SH, 88.291.7%; group WD/3-SH, 56.49 3.1%; group ND/3-SH, 76.29 2.4%.
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In the ANOVA of the first minute of tone presentation we found only a significant main effect of block, F(1,12)=17.45, P B0.01, reflecting the fact that freezing increased over the first minute of testing. The main effect of water deprivation was not statistically significant, F(1,12)= 1.31, P \0.1. All other main effects or interactions, F B1.
3.1.3. Freezing using the automated system compared with 6isual scoring We found that, on both context test and tone test, the overall pattern of between group differences obtained using the automated system was identical to that obtained using visual scoring of freezing. There were no effects of either water deprivation or number of pairings during the context test, P \ 0.1, but there were significant effects of both during the tone test presentation (see Fig. 5: selected because the main effect of measurement and its interactions reached their highest values in this analysis). Here, water deprived rats froze significantly less than non-deprived rats, F(1,12) = 9.01, P B0.02); rats in group 3-SH froze significantly less than rats in group 10-SH, F(1,12) =5.15, P B 0.05). Furthermore, in both cases, there was a significant effect of measurement: During the context test the visual scoring revealed higher overall levels of freezing than the automated system, F(1,12) = 9.96, P B 0.01. Means and standard error values were as follows: visual, 37.496.7%; automated, 30.1 95.0%. Meanwhile during the tone test presentation the visual scoring revealed lower overall levels of freezing than the automated system, F(1,12) =12.83, P B0.01. Mean and standard error values were as follows: visual, 66.69 3.9%; automated, 74.094.1%. Importantly for our purposes there were no significant interactions between measurement and any other variable(s) on either test, (maximum interaction F(1,12) = 1.10, P \0.3 in all
Fig. 5. Shows overall freezing scores during the tone test of experiment 1. The rats are split into water deprived (WD) and non-deprived (ND) groups; and into three conditioning trials (3-SH) and ten conditioning trials (10-SH) groups; the scoring methods are by visual assessment (observer) or by automated measurement (computer).
Fig. 6. Shows conditioned freezing during the context test of experiment 2. The figure shows rats split into groups water deprived (WD) and non-deprived (ND). Data are presented as percentage of freezing for each 30 s of the 8-min context test. Error bars denote S.E.M.
cases). This pattern suggests that, although the overall means might have differed as a function of automated versus visual measurement, the particular between group differences obtained were the same in both cases. Accordingly, the analysis of the initial 64 s of the tone test (parallelling Maren et al. [9]) revealed no significant effects of water deprivation F(1,12)= 1.63, n.s., nor of pairings, nor any significant interactions with measurement (all FB 1.0). The sole significant finding was the main effect of measurement, F(1,12)= 12.26, PB0.01): visual score, 80.595.3%; automated score 6893.7%. Thus, our interpretation of results from the automated system is identical to that derived from visual scoring methods; all the treatment effects and interactions of interest remained essentially unchanged, despite changes in absolute scores with the different assessment methods (see also [21]).
3.2. Experiment 2 As in experiment 1, there was much less freezing in response to contextual cues than in response to tone presentation. Water deprivation had no statistical effects on freezing during the context test either as a main effect or as an interaction with block, FB 1 in both cases (see Fig. 6). Furthermore, there was no main effect of block, F(15,150)=1.34, P\ 0.05. Mean and standard error values were as follows: group WD, 23.49 6.3%; group ND, 17.297.6%. Water deprivation also had no statistical effects on freezing during the tone test either as a main effect or as an interaction with block, FB 1 in both cases (see Fig. 7). There was, however, a significant main effect of block, F(15,150)= 2.64, PB 0.01, reflecting the extinction of conditioned freezing during the test. Mean and standard error values were as follows: group WD, 81.996.3%; group ND, 85.39 3.7%.
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4. Discussion In two separate experiments we found no evidence that water deprived rats showed a selective enhancement in conditioning to context, as opposed to conditioning to a tone. In experiment 1 we found that, during conditioning, water deprivation produced an overall decrease in freezing, both to the context (pre-CS measure) and to the tone (CS measure), only in those rats that received ten tone – shock pairings. Rats that received three tone – shock pairings exhibited no changes in acquisition of conditioning on the basis of deprivation schedule. Furthermore, water deprivation did not produce any statistically reliable effects on conditioned freezing to contextual cues during an extinction test session. However, rats that were water deprived showed reduced levels of conditioned freezing during the tone test. Thus, in experiment 1 the results failed to conform to the pattern reported by Maren et al. [9] in two respects. Not only did we find that water deprivation produced no reliable effects on contextual conditioning, whereas Maren et al. reported that contextual conditioning was significantly enhanced; we also found that water deprivation decreased conditioning to the tone, whereas Maren et al. reported no effects on tone induced freezing. Furthermore, it can be seen from Fig. 4A, as well as from the ANOVA we conducted on the first minute of tone presentation during test, that this between group difference, although it was seen statistically as a main effect of water deprivation rather than as a water deprivation – block interaction, would not have been observed had we run a 64-s tone test, as Maren et al. did. In experiment 1 we also found that rats that had received three or ten conditioning trials showed similar levels of conditioned contextual freezing. However, rats in group 3-SH showed less conditioned freezing to the
Fig. 7. Shows conditioned freezing during the tone test of experiment 2. The figure shows rats split into groups water deprived (WD) and non-deprived (ND). Data are presented as percentage of freezing for each 30 s of the 8-min tone presentation. Error bars denote S.E.M.
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tone than rats in group 10-SH. This pattern — reduced conditioned tone freezing, but no change in conditioned contextual freezing, after fewer tone–shock pairings — has been reported previously using the same freezing analysis [21]. This consistency across experiments serves to support our contention that the present system enables us validly to compare groups using small numbers of subjects. Furthermore, none of these effects can be attributed to supposed peculiarities of our automated system: we obtained an identical pattern of results by visual scoring methods. The pattern of results we obtained is also noteworthy in the following respect. There is a strong indication that freezing measures taken during acquisition and during extinction tests are both not adequate indices of conditioning: The effect of water deprivation on conditioned contextual freezing was observed only during acquisition of conditioning to contextual cues (pre-CS measure) but not during subsequent context extinction sessions. On the other hand, the effect of water deprivation on conditioned tone freezing was observed during both the acquisition of conditioning (CS measure) and the tone extinction test. However, during acquisition of conditioning the effects of water deprivation on either context or tone conditioning were observed only in rats that received 10 pairings of tone and shock, whereas, during the extinction session both group 10-SH and group 3-SH showed water deprivation-induced decreases in tone freezing. Thus we have obtained a double dissociation of between group effects during acquisition of conditioning and during extinction sessions. Effects of water deprivation on contextual conditioning were observed during acquisition but not during extinction, whereas effects of water deprivation on tone conditioning in group 3-SH were observed during extinction but not during acquisition. In our view, this pattern is consistent with the notion that the foot shock produced a delayed unconditioned freezing response, displayed only during conditioning training (when shocks were presented) but not during extinction sessions (when shocks were not presented). That is to say, we propose that the two types of measure — freezing during acquisition and freezing during extinction — might be qualitatively different rather than merely differing in sensitivity. Note that this notion assumes that the freezing response during both pre-CS and CS periods in the acquisition phase largely reflects unconditioned freezing in response to the foot shock, rather than conditioned contextual freezing and conditioned tone freezing, respectively. This analysis argues strongly against using interchangeably the freezing scores gained during acquisition and during extinction sessions (e.g. [7,9,16,19,23]). Interestingly the non-selective effects of water deprivation during acquisition of conditioning were observed only in group 10-SH. Unconditioned or generalised
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freezing is likely to have been greater in this group, which received more shocks with shorter gaps between each shock, and hence presumably a greater carry-over from a US delivery to the next pre-CS measurement. Thus water deprivation induced decreases in freezing in conditions that generated relatively high levels of overall freezing (acquisition of both context and tone conditioning in group 10-SH and tone extinction sessions) but not in conditions that generated relatively lower levels of overall freezing (acquisition of both context and tone conditioning in group 3-SH and context extinction sessions). There is no need, on the basis of our findings, to suppose that water deprivation acts either to increase or to decrease levels of freezing only to a certain type of stimulus. In experiment 2 we sought to replicate the procedures used by Maren et al. as closely as possible, (1) by giving the tone test before the context test and (2) by giving rats ad-lib water after conditioning ensuring that deprivation levels at test were the same in both groups of rats. In this case, unlike Maren et al., we found no effects of water deprivation during the context test, but like them we found that water deprivation produced no effects on conditioned freezing to the tone. It is worth noting that the original study ([9] three-pairings-group), using visual assessment of a relatively small sample of data, was conducted on 14 rats as opposed to our 12. Thus, it is highly unlikely that our failure to observe group differences in experiment 2 can be explained in terms of group sizes or lack of experimental power generally. In conclusion our results serve to limit the generality of Maren et al.’s findings. They suggest that any general conclusions regarding the relationship between conditioning to context and water deprivation, whether by action through LTP or theta at the hippocampus or not, are likely to be incorrect, as is the case for gender differences, compare Maren et al. [11] with Pryce et al. [19]. Furthermore our results suggest that some minor procedural variation might account for the different pattern of results we obtained. Possibilities include: (1) The housing conditions — we housed rats in groups of four in plastic cages with a straw floor covering while Maren et al. housed rats individually or in pairs in metal cages without straw. Recently published results in our laboratory show that both group and individually housed rats are more active if raised on sawdust than if raised on a grid floor [28]. Such differences might indeed account for different patterns of freezing behaviour in different published reports since baseline response rates are likely to have been different in the two studies; (2) Time of testing — we tested rats during the dark phase of the cycle while Maren et al. tested during the light phase. Again while this difference might be unlikely to produce effects on learning, general activity levels across the light/dark cycle might well interact with deprivation condition.
Acknowledgements This work was supported by grants from the Swiss Federal Institute of Technology and The Swiss National Science Foundation (grant no. 31-42009.94). The authors thank the animal facility team for their care of the animals. References [1] Blanchard RJ, Blanchard DC. Crouching as an index of fear. J Comp Physiol Psychol 1969;67:370 – 5. [2] Bouton ME, Bolles RC. Conditioned fear assessed by freezing and by the suppression of three different baselines. Anim Learn Behav 1980;8:429 – 34. [3] Dickinson A. Instrumental conditioning. In: Mackintosh NJ, editor. Animal Learning and Cognition. London: Academic Press, 1994:45 – 79. [4] Good M, Honey RC. Dissociable effects of selective lesions to hippocampal subsystems on exploratory behaviour, contextual learning and spatial learning. Behav Neurosci 1997;111:487–93. [5] Jarrard LE. Behaviour of hippocampal lesioned rats in home cage and novel situations. Physiol Behav 1968;3:65 – 70. [6] Kim JJ, Fanselow MS. Modality-specific retrograde amnesia of fear. Science 1992;256:675 – 7. [7] Maren S, Aharonov G, Fanselow MS. Neurotoxic lesions of the dorsal hippocampus and Pavlovian fear conditioning in rats. Behav Brain Res 1997;88:261 – 74. [8] Maren S, Anagnostaras SG, Fanselow MS. The startled seahorse: is the hippocampus necessary for contextual fear conditioning. Trends Cognit Sci 1998;2:39 – 43. [9] Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645 – 9. [10] Maren S, DeCola JP, Swain RA, Fanselow MS, Thompson RF. Parallel augmentation of hippocampal long-term potentiation, theta rhythm and contextual fear conditioning in water deprived rats. Behav Neurosci 1994;108:44 – 57. [11] Maren S, De Oca B, Faneslow MS. Sex differences in hippocampal long-term potentiation (LTP) and Pavlovian fear conditioning in rats: Positive correlation between LTP and contextual learning. Brain Res 1994;661:25 – 34. [12] Maren S, Fanselow MS. Electrolytic lesions of the fimbria/ fornix, dorsal hippocampus, or entorhinal cortex produce anterograde deficits in contextual fear conditioning in rats. Neurobiol Learn Mem 1997;67:142 – 9. [13] McNish KA, Gewirtz JC, Davis M. Evidence of contextual fear after lesions of the hippocampus: a disruption of freezing but not fear potentiated startle. J Neurosci 1997;17:9353 – 60. [14] Nadel L, Willner J. Context and conditioning: a place for space. Physiol Psychol 1980;8:218 – 28. [15] O’Keefe J, Nadel L. The Hippocampus as a Cognitive Map. Oxford: Clarendon Press, 1978. [16] Phillips RG, LeDoux JE. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav Neurosci 1992;106:274 – 85. [17] Phillips RG, LeDoux JE. Lesions of the dorsal hippocampal formation interfere with background but not foreground contextual fear conditioning. Learn Mem 1994;1:34 – 44. [18] Phillips RG, LeDoux JE. Lesions of the fornix but not the entorhinal or perirhinal cortex interfere with contextual fear conditioning. J Neurosci 1995;15:5308 – 15. [19] Pryce CR, Lehmann J, Feldon J. Effect of sex on fear conditioning is similar for context and discrete CS in Wistar, Lewis, and Fischer rat strains. Pharmacol Biochem Behav 1999;64:753–9.
B. Pouzet et al. / Beha6ioural Brain Research 119 (2001) 49–59 [20] Rawlins JNP, Feldon J, Salmon P, Garrud P, Gray JA. The effects of chlordiazepoxide HCl administration upon punishment and conditioned suppression in the rat. Psychopharmacology 1980;70:317 – 22. [21] Richmond MA, Murphy CA, Pouzet B, Schmid P, Rawlins JNP, Feldon J. A computer controlled analysis of freezing behaviour. J Neurosci Methods 1998;86:91–9. [22] Richmond MA, Yee BK, Pouzet B, Veenman L, Rawlins JNP, Feldon J, Bannerman DM. Dissociating context and space within the hippocampus: effects of complete, dorsal and ventral excitotoxic hippocampal lesions on conditioned freezing and spatial learning. Behav Neurosci 1999;113:1189–203. [23] Riedel G, Harrington NR, Hall G, Macphail EM. Nucleus accumbens lesions impair context, but not cue, conditioning in rats. Neuroreport 1997;8:2477–82. [24] Rudy JW, Morledge P. Ontogeny of contextual fear conditioning in rats: implications for consolidation, infantile amnesia, and hippocampal system function. Behav Neurosci 1994;108:227 – 34. [25] Rudy JW, Sutherland RJ. Configural association theory and the hippocampal formation: an appraisal and reconfiguration.
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Hippocampus 1995;5:375 – 89. [26] Selden NRW, Everitt BJ, Jarrard LE, Robbins TW. Complementary roles for the amygdala and hippocampus in aversive conditioning to explicit and contextual cues. Neuroscience 1991;42:335 – 50. [27] Sutherland RJ, Rudy JW. Configural association theory: the role of the hippocampal formation in learning, memory and amnesia. Psychobiology 1989;17:129 – 44. [28] Weiss IC, Feldon J, Domeney AM. Isolation rearing-induced disruption of prepulse inhibition: further evidence for fragility of the response. Behav Pharmacol 1999;10:139 – 49. [29] Winocur G. Hippocampal lesions alter conditioning to conditional and contextual stimuli. Behav Brain Res 1997;88:219–29. [30] Winocur G, Rawlins JNP, Gray JA. The hippocampus and conditioning to contextual cues. Behav Neurosci 1987;101:617– 27. [31] Young AMJ, Joseph MH, Gray JA. Increased dopamine release in vivo in nucleus accumbens and caudate nucleus of the rat during drinking: a microdialysis study. Neuroscience 1992;48:871 – 6.
Research report
The effects of water deprivation on conditioned freezing to contextual cues and to a tone in rats Bruno Pouzet a, Wei-Ning Zhang a, Mark A. Richmond b, J. Nicholas P. Rawlins b, Joram Feldon a,* a
Beha6ioural Neurobiology Laboratory, Swiss Federal Institute of Technology, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland b Department of Experimental Psychology, South Parks Road, Oxford OX1 3UD, UK Received 28 April 2000; received in revised form 19 September 2000; accepted 19 September 2000
Abstract In two experiments we used an automated system for quantifying freezing responses in rats to replicate and extend Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645–9; Maren S, DeCola JP, Swain RA, Fanselow MS, Thompson RF. Parallel augmentation of hippocampal long-term potentiation, theta rhythm and contextual fear conditioning in water deprived rats. Behav Neurosci 1994;108:44–57) who found that water deprivation in rats produced a selective enhancement in conditioning to context, as opposed to conditioning to a tone. In experiment 1 we gave water deprived and non-deprived rats either three or ten pairings of a tone and foot shock. During conditioning water deprivation decreased overall freezing only in rats that received ten pairings. On 2 subsequent days we assessed conditioned freezing (1) to the contextual cues of the conditioning chamber and (2) to the tone when presented in a distinctive, novel environment. We found, in direct contrast to Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645–9), that (a) water deprived rats did not differ from non-deprived rats in levels of conditioned contextual freezing and that (b) water deprived rats did show reduced levels of freezing to the tone stimulus. In the same experiment we found that the number of tone–shock pairings did not affect levels of conditioned contextual freezing but that rats that had received three pairings did show reduced levels of freezing to the tone stimulus compared with rats that had received ten pairings, thereby demonstrating that the behavioural procedure and analysis system that we used was appropriately sensitive to differences in conditioning. In experiment 2, therefore, we sought to replicate Maren et al. (Maren S, DeCola JP, Fanselow MS. Water deprivation enhances fear conditioning to contextual, but not discrete, conditional stimuli in rats. Behav Neurosci 1994;108:645– 9) using, as far as possible, exactly the same procedural parameters. Here we found that water deprivation produced no effects on conditioned freezing to the contextual cues or to the tone. We conclude that there is sufficient reason to doubt the generality of the previously reported findings. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Water deprivation; Freezing; Conditioning; CS; Context; Fear; Hippocampus; LTP; Rat
1. Introduction It is well established that rats who have received pairings of a tone and electric foot shock in a conditioning chamber will come to show freezing behaviour, * Corresponding author. Tel.: +41-1-6557448; fax: 6557203. E-mail address: [email protected] (J. Feldon).
+41-1-
in the absence of the shock, when placed back into that chamber and/or when re-presented with the tone [1,2]. This change in the behavioural response to a stimulus presentation reflects learning on the part of the animal, since freezing typically does not occur as a response to either conditioning chambers or tones before they are paired with shock [16]. Thus, the freezing response appears to be a suitable index of learning a Pavlovian stimulus–shock relationship. Recently the freezing re-
0166-4328/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 3 2 8 ( 0 0 ) 0 0 3 3 7 - 5
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sponse has become a focus of attention among neuroscientists who are interested in the anatomical and physiological correlates of behaviour. For instance, there are now many published demonstrations of hippocampal lesion-induced impairments in conditioned freezing to contextual cues, such as the conditioning chamber in which conditioning took place, but not in conditioned freezing to the tone with which shock was directly paired (e.g. [6,7,12,16 – 18], but see [22]). Meanwhile, lesions of the amygdala have been found to impair conditioned freezing to both contextual and discrete cues [16] (see also McNish et al. [13]). Such findings, and their interpretation in terms of impaired contextual processing, are generally believed to be consistent with several theories of hippocampal and amygdaloid function. For instance, both spatial theory [14,15] and configural association theory [25,27] of hippocampal function treat contextual and spatial cues as similar in kind, albeit for different reasons. Proponents of these theories note the physical similarities between typical contextual cues and typical spatial cues: both are large, diffuse, multi-cue and poly-modal. They contrast such cues with both auditory and visual discrete CSs: these are often single cues in a single modality. Thus there is some agreement that spatial and contextual learning require an intact hippocampus because of the relatively complex nature of the stimuli involved (see also [8]). Nonetheless, there is an alternative and simpler interpretation of the results obtained in all these freezing studies. This notes that lesions of the hippocampus produce increases in general locomotor activity and an increased tendency to explore environments [4,5]. According to this view, the impairments in conditioned freezing to contextual cues result from the rat’s tendency to be hyperactive or more vigorously to explore its environment, rather than from a spatial or configural learning impairment. Because in the majority of studies, conditioned tone freezing has been at higher levels than conditioned context freezing, Good and Honey [4] have suggested that a given level of activity is more likely to interact with a stimulus that elicits little freezing (a context) than one that elicits more substantial freezing (a tone) (p. 487). For this reason alone hippocampal lesions might induce an increase in activity that alters conditioned freezing to contextual cues at the same time as leaving conditioned freezing to a tone intact [22]. Moreover, it has long been understood that putative lesion-induced or drug-induced selective impairments in behaviour could be a consequence of different initial response rates rather than of selective action on memory. For instance, Rawlins et al. [20] found that chlordiazepoxide HCl produced a selective impairment in instrumental aversive conditioning, as opposed to classical aversive conditioning, only when the initial response rates were not matched. Thus, previ-
ous reports might have observed what appear to be selective effects on contextual conditioning simply because of the higher levels of freezing seen to the tone than to the context. The primary advantage of the freezing response, over and above (say) suppression of licking, is that it is a measure of suppression of an unlearned spontaneous locomotor activity. Alternative measures have typically assessed the suppression of stable learned behaviours, such as lever pressing or licking. In these experiments rats would typically be maintained on food or water deprivation. In studies of animal learning it is becoming increasingly clear that motivational factors play a major part, at least in the operant learning process (e.g. [3]). Furthermore, there is evidence emerging from the neurosciences to suggest that food and water deprivation might act at brain regions such as the hippocampus or nucleus accumbens to produce changes in conditioned behaviour [9,31]. In particular, Maren et al. [9,10] found that water deprivation in non-lesioned rats caused an enhancement in conditioned contextual freezing but no changes in conditioned tone freezing. This pattern suggests that water deprivation produced a selective increase in conditioning to context, as opposed to conditioning to a tone. Maren et al. further found that, as well as showing an enhancement in conditioned freezing to context, water deprived rats also showed an enhancement of perforant path long-term potentiation (LTP) and of hippocampal theta. These authors note the compatibility between this correlation and the commonly observed impairment in conditioned contextual freezing after hippocampal lesions (see above). They suggest that because hippocampal LTP has been (previously) implicated in the mnemonic encoding of contextual and spatial information, the greater LTP induced in waterdeprived subjects is a putative neural mechanism for the superior representation of contextual stimuli in these rats ([10] p. 53). They conclude that their results provide evidence for a role of the hippocampus in the augmentation of contextual fear conditioning by water deprivation, and more generally, in the mediation of contextual learning ([9] p. 648). A similar rationale has been applied to sex differences in LTP and contextual conditioning (as opposed to discrete CS conditioning), with the argument that male rats show more LTP and contextual conditioning compared with female rats [11], but see the results and discussion in Pryce et al. [19]. One strong implication of this conclusion is that interpretation of studies that have used water or food deprived rats to investigate the involvement of the hippocampus in behaviour might be compromised. For instance, Selden et al. [26] found impaired conditioned context preference in hippocampal lesioned rats that had been water deprived. It is possible that Selden et al. observed an enhancement in conditioned place prefer-
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ence in their control rats that had been water deprived. Because according to Maren et al., water deprivation acts at the hippocampus to produce such changes, rats with hippocampal lesions would not have shown such an enhancement. Therefore, in this case rats with hippocampal lesions could have shown less conditioning to context than control rats without suffering from any impairments whatsoever. Thus, Selden et al.’s results can not be interpreted unambiguously as demonstrating a hippocampal lesion-induced impairment in conditioning to context. This ambiguity has serious implications since Winocur et al. ([30], see also [29]), in the first study to assess the effects of hippocampal lesions on conditioning to context, found that conditioned avoidance of a black conditioning chamber in which tone–shock pairings had been presented was enhanced in the (non-deprived) hippocampal lesion group when rats were given a choice between the black chamber and an adjacent larger white chamber. Thus, there appear to be potential problems associated both with the use of freezing in hippocampal lesioned rats — i.e. that hippocampal lesions cause hyperactivity — and with the use of some alternative response measures that require such rats to be water deprived — i.e. that water deprivation itself acts at the hippocampus to produce changes in contextual conditioning. A principal point of interest in our laboratory is to assess the involvement of the hippocampus in conditioning using response measures that avoid interpretations in terms of lesion-induced hyperactivity. But given the potential problems associated with the use of water deprived hippocampal lesioned rats we sought, as a first step, to assess the generality of Maren et al.’s findings. Furthermore, there are at least two reasons to believe that Maren et al.’s claim for a selective enhancement of contextual fear in water deprived rats, and by implication the claim for hippocampal involvement, may be premature. First of all, we have found, using a new computer controlled analysis of freezing behaviour that enables one to measure the development of freezing over time, that group differences can emerge as a significant interaction with repeated measures over time, in the absence of any significant main effects (see [21]). Maren et al.’s method of data collection gave an overall score and so could have failed to detect a pattern of freezing developing over time. Secondly, their behavioural testing procedure could have differentially contributed to this problem of measurement: Maren et al.’s [9] tone test had a duration of only 64 s and the context test had a duration of 8 min. Thus an effect of water deprivation on the tone test might have been obscured simply by its relatively short duration; the test may have ended before a differential pattern of freezing had developed. In our study context and tone were tested under equivalent conditions.
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In experiment 1 we assessed the effects of water deprivation and number of tone–shock pairings on conditioned freezing using parameters we have used previously in our laboratories. On day 1 all rats received either three or ten pairings of a tone and foot shock in a distinctive chamber. Twenty-four hours later they were placed back into the conditioning chamber to measure conditioned freezing to contextual cues. Fortyeight hours following conditioning they were placed into a novel chamber. Here we measured conditioned freezing to the tone during an 8-min presentation. To avoid ‘state dependency’ effects, which might mask transfer of information from the conditioning process to its expression in contextual and discrete tone induced freezing, the rats in experiment 1 were maintained on water deprivation throughout the experimental process. Because in this procedure we failed to observe the pattern of findings reported by Maren et al. [9] we conducted experiment 2 in which we sought to replicate Maren et al. [9] using, as far as possible, exactly the same procedural parameters. Rats were either water deprived or sated and, on day 1, received conditioning with three tone–shock pairings in an 8-min session. Immediately after conditioning, all rats were allowed free access to water for the remainder of the experiment. On day 2 all rats were placed into a novel chamber and conditioned tone freezing was measured. On day 3 all rats were placed back into the conditioning chamber to measure conditioned freezing to contextual cues.
2. Materials and methods
2.1. Subjects Subjects were 28 male hooded Lister rats (Harlan CPB, the Netherlands) that weighed between 300 and 350 g during behavioural testing. Rats were housed in groups of four in a temperature (2291°C) and humidity (559 5%) controlled room under a 12:12 h reversed light–dark cycle with lights on from 19:00 to 07:00 h. All behavioural testing was carried out in the dark phase of the cycle.
2.2. Apparatus Eight Coulbourn Habitest operant test chambers (Coulbourn Instruments, Allentown, PA) were used for behavioural testing. Four identical chambers were used for conditioning and assessment of contextual learning (model no. E10-10RF). These had two aluminium walls and two Perspex walls. The house light remained off throughout the experimental sessions in this chamber. The chamber floor consisted of 16 stainless steel bars with a diameter of 5 mm, spaced 1.3 cm apart, through
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which shocks could be delivered. An additional four chambers were used for assessing conditioned freezing to the tone stimulus. These had three black walls and one Perspex wall. A house light (1.12 W) positioned 1.8 cm from the ceiling on one wall and directed upwards, was lit during all the testing in this box. The floor of these chambers was a cross grid. Presentation of stimuli was controlled by a Coulbourn Universal Environmental Interface (model no. E91-12) and a Coulbourn Universal Environmental Port (model no. L91-12). Shocks were delivered, where appropriate, via a Coulbourn Precision Regulated Animal Shocker (model no. E13-12). We assessed immobility in two ways. The first used an automated analysis system developed in our laboratory (see [19,21,22]): Attached to the centre of each chamber’s ceiling was a wide-angle (100°) 2.5-mm lens (VPC-465B; CES AG, Zurich, Switzerland), through which a black and white video image of the experimental session was recorded (Sony video recorder, SVT1000). For the purposes of recording, each chamber was illuminated by four infrared LEDs (Hewlett Packard; model no. HSDL-4220; wavelength 875 nm), which were attached to the ceiling. Data analysis was carried out simultaneously using a Power Macintosh (7600/120) with a permanent video input, and a script derived from the ‘NIH IMAGE’ programme (available from web-site http://rsb.info.nih.gov/nih-image). Our script works by comparing every two adjacent seconds of video tape which are presented on separate screens, each containing 100 000 pixels. Each pixel is given a ‘grey value’ from 0 to 255 where 0 refers to black and 255 to white. Then the value of each pixel on screen 1 (the image of second 1) is compared with the value of the corresponding pixel on screen 2 (the image of the following second). A ‘grey value’ difference score for each pair of pixels is calculated and this data is used to prepare a third screen which represents the difference between screens 1 and 2. In the experiments reported here our criteria were as follows: a difference score of 5 or less resulted in a ‘no-movement’ score for the corresponding pixel on screen 3, which was therefore left white. A difference score of greater than 5 resulted in a ‘movement’ score for the corresponding pixel on screen 3, which was, therefore, coloured black. After minor adjustments for random noise, the analysis was thus able to provide, for each second of the experimental session, a score which represents the percentage change between that second and the following one. We scored immobility for each second if and only if the number of black pixels on screen 3 was less than 0.05% of the total. This percentage had been calibrated visually to ensure that breathing movements alone were not sufficient to produce a movement score. Furthermore, unpublished pilot observations revealed a 90% correspondence between the overall scores from this
automated system and those scores obtained using visual criteria similar to those commonly employed (e.g. see [4,6,12,24]). Thus, our automated measures of immobility can be validly used as an index of freezing behaviour. The second method of immobility assessment was used in experiment 1 only: A trained observer, who was blind to the experimental conditions of each rat, measured freezing from a video (cf. [9]). A score was taken every 8 s to yield 60 measurements over the 8-min test periods. This was in order to compare directly the results obtained with Maren’s method and those obtained using our own automated system.
2.3. Procedures 2.3.1. Experiment 1 For 7 days prior to conditioning, and throughout the entire duration of the experiment, one group of rats (water deprived WD, N=8) were maintained on a restricted-fluid schedule of 1 h access to water per day. The remaining group of rats (non-deprived ND, N=8) remained on ad-lib water throughout. During this period all rats were handled daily (3 min per rat per day). On day 1 of the experiment all rats were placed in the conditioning chambers for 30 min. During this session, half the rats in each group received three pairings (group 3-SH) of a tone (30 s duration, 85 dB) and foot shock (1 s, 0.5 mA), and the other half (group 10-SH) received ten pairings of a tone (30 s, 85 dB) and foot shock (1 s, 0.5 mA). Group 3-SH received the first tone–shock pairing 5 min after placement in the chamber and the subsequent intertrial interval was fixed at 9 min. They were removed from the chamber 5 min after the final shock. Group 10-SH received the first tone– shock pairing 90 s after placement in the chamber and the subsequent intertrial interval was fixed at 150 s. Rats in this group were removed from the chamber 90 s after the final shock. We assessed freezing during acquisition of conditioning using the automated system. Twenty-four hours after conditioning all rats were placed back into the conditioning chamber for 8 min and conditioned freezing to the contextual cues was assessed. Freezing was scored by means of the automated system and also using a visual scorer (see above). Twenty-four hours after the context test we assessed conditioned freezing to the tone stimulus. Rats were placed in a novel chamber, which differed from the conditioning chamber as described above, for 11 min. After 3 min the tone was presented for the remainder of the test session. Freezing behaviour was assessed in the same manner as described for the context test. 2.3.2. Experiment 2 For 7 days prior to conditioning one group of rats (deprived, N=6) were maintained on a restricted-fluid schedule of 1 h access to water per day. The remaining
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group of rats (non-deprived, N =6) remained on ad-lib water throughout. During this period all rats were handled daily (3 min per rat per day). On day 1 of the experiment all rats were placed in the conditioning chambers. During this session, rats in each group received three pairings of a tone (64 s, 85 dB) and foot shock (1 s, 0.5 mA). Rats received the first tone –shock pairing 3 min after placement in the chamber and the subsequent intertrial interval was 94 s. They were removed from the chamber 30 s after the final shock, returned to their home cages and all rats were allowed ad-lib water for the remainder of the experiment. Twenty-four hours after conditioning rats were placed in a novel chamber, that differed from the conditioning chamber as described above, for 11 min in order to assess conditioned freezing to the tone. After 3 min the tone was presented for the remainder of the test session. Freezing behaviour was assessed using the automated system (see above). Twenty-four hours later all rats were placed back into the conditioning chamber for 8 min and conditioned freezing to the contextual cues was assessed. Freezing was assessed using the automated system.
2.4. Statistical analyses Results were calculated as the average percentage of time spent freezing in a specified time period. Specifically we calculated the total number of seconds spent freezing divided by the maximum possible number of seconds spent freezing. This value was then multiplied by 100. Thus a rat that was immobile for 15 s during a block of 30 s would receive a freezing score of 50%. For experiment 1 we conducted two three-way splitplot ANOVAs on the acquisition of conditioned freezing, using automated measurement data obtained during the conditioning session: (1) for rats receiving ten tone–shock pairings, (2) for rats receiving three tone –shock pairings. These ANOVAs had variables of water deprivation schedule (two levels: water deprived vs. non-deprived), trial (either ten or three levels) and period (three levels: pre-CS, CS and post-CS). The pre-CS, CS and post-CS periods refer to the 30 s before each tone presentation, the 30 s of each tone presentation and the 30 s following each tone presentation, respectively. We conducted three further ANOVAs of automated measurement data obtained during the extinction test sessions: (1) of the 8-min context test, (2) of the first minute of the context test, (3) of the 8 min of tone presentation during test, (4) of the first minute of tone presentation during test. For these ANOVAs data were combined into blocks of 30 s. The between subjects variables were water deprivation schedule (two levels: water deprived vs. non-deprived) and number of tone –shock pairings (two levels: 3-SH vs. 10-SH). In four additional ANOVAs, we compared directly the
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measurements taken from the automated system and those taken by the visual scorer. The first assessed the 8-min context extinction test; the second assessed the 8-min tone extinction test itself; and the third assessed the initial 64 s of the tone extinction test (exactly as Maren et al. [9] had done). The analyses all had between subjects variables of water deprivation schedule (two levels) and number of tone–shock pairings (two levels), and a within subjects variable of measurement (two levels: automated vs. visual). For experiment 2 we conducted two split-plot ANOVAs: (1) of the 8-min context test, (2) of the 8-min tone presentation during test. For these ANOVAs data were combined into blocks of 30 s. The between subjects variable was water deprivation schedule (two levels).
3. Results
3.1. Experiment 1 3.1.1. Acquisition of freezing during conditioning Results are depicted in Figs. 1 and 2 which show conditioning data for rats in groups 10-SH and 3-SH respectively. In group 10-SH rats showed an increase in pre-CS and CS freezing during the first three trials followed by no further increases thereafter (Fig. 1A and B). Freezing during the post-CS periods did not increase over trials (Fig. 1C). Furthermore, water deprived rats showed lower levels of freezing during pre-CS, CS and post-CS periods than did non-deprived rats. ANOVA supported these conclusions as follows. There was a significant main effect of trial, F(9,108)= 7.27, PB 0.001, and a significant main effect of period, F(2,12)= 37.14, PB 0.001. The effect of period reflected higher levels of freezing during pre-CS and CS periods (which did not differ from each other) than during post-CS periods, PB 0.05 in both cases. There was also a significant interaction between these two variables, F(18,108)=4.63, PB 0.001. Post-hoc t-tests conducted on the interaction revealed a significant increase in freezing over trial only during pre-CS and CS periods. There was a main effect of water deprivation that approached conventional levels of significance, F(1,6)= 5.89, P= 0.051. Water deprived animals tended to be more active overall, and therefore had lower freezing scores. None of the remaining interactions was significant at the 0.1 level. In group 3-SH rats showed an increase in pre-CS (Fig. 2A) and CS (Fig. 2B) freezing during the three trials. Freezing during the post-CS periods decreased slightly over trials (Fig. 2C). ANOVA revealed a significant main effect of trial, F(2,24)= 9.03, PB 0.01, and a significant main effect of period, F(2,12)= 14.59, PB 0.001. The effect of period reflected a significant in-
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crease in freezing at the onset of the CS as well as a significant decrease in freezing at the offset of the CS, P B0.05 in both cases. There was no significant effect of water deprivation either as a main effect or as an interaction with any other variable, P \ 0.1 in all cases.
Fig. 2. (A – C) Show the development of conditioned freezing as a function of trials (1 – 3) for rats in group 3-SH. The figure shows rats split into groups water deprived (WD) and non-deprived (ND). (A) Shows the pre-CS period, (B) shows the CS period, (C) shows the post-CS period. Data are presented as percentage of freezing. Error bars denote S.E.M. The inter-trial interval was 300 s.
Fig. 1. (A – C) Show the development of conditioned freezing as a function of trials (1 – 10) for rats in group 10-SH. The figure shows rats split into groups water deprived (WD) and non-deprived (ND). (A) Shows the pre-CS period, (B) shows the CS period, (C) shows the post-CS period. Data are presented as percentage of freezing. Error bars denote S.E.M. The inter-trial interval was 90 s.
3.1.2. Freezing during extinction sessions Overall results of the context test (shown in Fig. 3A and B) were as follows: group WD/10-SH, 38.79 4.4%. Group ND/10-SH, 33.19 3.9%. Group WD/3-SH, 21.59 3.6%; Group ND/3-SH, 27.39 3.4%. ANOVA revealed no statistically significant differences between
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the groups or any significant interactions, except for an overall significant effect of block, F(15,180) = 4.21, PB 0.001. Overall freezing increased from 8.1% during the first block of testing to 46.6% during the fourth minute of testing. Thereafter freezing declined steadily to 25.1% during the final block of testing. In the ANOVA of the first minute of the context test there was an identical pattern of statistical findings: No effects of any between subjects variable, nor any significant interactions, P\ 0.1, except for a significant main effect of block, F(1,12) =5.78, P B0.05, this time reflecting an overall increase in freezing from 8.1% during the first block of testing to 17.6% during the second block. Results from the tone test are shown in Fig. 4A and B. Tone-induced freezing was much more pronounced than context-induced freezing (overall mean: tone= 71.0%, context=30.1%). In the ANOVA of the entire 8
Fig. 4. (A and B) Show conditioned freezing during the tone test of experiment 1. (A) Shows rats split into groups water deprived (WD) and non-deprived (ND). (B) Shows rats split into groups that had received three or ten conditioning trials. Data are presented as percentage of freezing for each 30 s of the 8-min tone presentation. Error bars denote S.E.M.
Fig. 3. (A and B) Show conditioned freezing during the context test of experiment 1. (A) Shows rats split into groups water deprived (WD) and non-deprived (ND). (B) Shows rats split into groups that had received three or ten conditioning trials. Data are presented as percentage of freezing for each 30 s of the 8-min context test. Error bars denote S.E.M.
min of tone presentation we found a significant effect of water deprivation, F(1,12)= 6.78, PB 0.05. As in the 10-SH acquisition study, this reflected the fact that water deprived rats showed decreased conditioned freezing to the tone compared with non-deprived rats (Fig. 4A). We also found a significant effect of pairings, F(1,12)=6.03, P B0.05, reflecting the fact that rats that had received three pairings showed decreased conditioned freezing to the tone compared with rats that had received ten pairings (Fig. 4B). There was a significant main effect of block, F(15,180)= 5.28, P B0.01, reflecting an initial increase followed by extinction of conditioned freezing during the test. None of the interactions was significant at the 0.05 level. Overall mean and standard error values for the four individual groups were as follows. Group WD/10-SH, 75.39 3.3%; group ND/10-SH, 88.291.7%; group WD/3-SH, 56.49 3.1%; group ND/3-SH, 76.29 2.4%.
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In the ANOVA of the first minute of tone presentation we found only a significant main effect of block, F(1,12)=17.45, P B0.01, reflecting the fact that freezing increased over the first minute of testing. The main effect of water deprivation was not statistically significant, F(1,12)= 1.31, P \0.1. All other main effects or interactions, F B1.
3.1.3. Freezing using the automated system compared with 6isual scoring We found that, on both context test and tone test, the overall pattern of between group differences obtained using the automated system was identical to that obtained using visual scoring of freezing. There were no effects of either water deprivation or number of pairings during the context test, P \ 0.1, but there were significant effects of both during the tone test presentation (see Fig. 5: selected because the main effect of measurement and its interactions reached their highest values in this analysis). Here, water deprived rats froze significantly less than non-deprived rats, F(1,12) = 9.01, P B0.02); rats in group 3-SH froze significantly less than rats in group 10-SH, F(1,12) =5.15, P B 0.05). Furthermore, in both cases, there was a significant effect of measurement: During the context test the visual scoring revealed higher overall levels of freezing than the automated system, F(1,12) = 9.96, P B 0.01. Means and standard error values were as follows: visual, 37.496.7%; automated, 30.1 95.0%. Meanwhile during the tone test presentation the visual scoring revealed lower overall levels of freezing than the automated system, F(1,12) =12.83, P B0.01. Mean and standard error values were as follows: visual, 66.69 3.9%; automated, 74.094.1%. Importantly for our purposes there were no significant interactions between measurement and any other variable(s) on either test, (maximum interaction F(1,12) = 1.10, P \0.3 in all
Fig. 5. Shows overall freezing scores during the tone test of experiment 1. The rats are split into water deprived (WD) and non-deprived (ND) groups; and into three conditioning trials (3-SH) and ten conditioning trials (10-SH) groups; the scoring methods are by visual assessment (observer) or by automated measurement (computer).
Fig. 6. Shows conditioned freezing during the context test of experiment 2. The figure shows rats split into groups water deprived (WD) and non-deprived (ND). Data are presented as percentage of freezing for each 30 s of the 8-min context test. Error bars denote S.E.M.
cases). This pattern suggests that, although the overall means might have differed as a function of automated versus visual measurement, the particular between group differences obtained were the same in both cases. Accordingly, the analysis of the initial 64 s of the tone test (parallelling Maren et al. [9]) revealed no significant effects of water deprivation F(1,12)= 1.63, n.s., nor of pairings, nor any significant interactions with measurement (all FB 1.0). The sole significant finding was the main effect of measurement, F(1,12)= 12.26, PB0.01): visual score, 80.595.3%; automated score 6893.7%. Thus, our interpretation of results from the automated system is identical to that derived from visual scoring methods; all the treatment effects and interactions of interest remained essentially unchanged, despite changes in absolute scores with the different assessment methods (see also [21]).
3.2. Experiment 2 As in experiment 1, there was much less freezing in response to contextual cues than in response to tone presentation. Water deprivation had no statistical effects on freezing during the context test either as a main effect or as an interaction with block, FB 1 in both cases (see Fig. 6). Furthermore, there was no main effect of block, F(15,150)=1.34, P\ 0.05. Mean and standard error values were as follows: group WD, 23.49 6.3%; group ND, 17.297.6%. Water deprivation also had no statistical effects on freezing during the tone test either as a main effect or as an interaction with block, FB 1 in both cases (see Fig. 7). There was, however, a significant main effect of block, F(15,150)= 2.64, PB 0.01, reflecting the extinction of conditioned freezing during the test. Mean and standard error values were as follows: group WD, 81.996.3%; group ND, 85.39 3.7%.
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4. Discussion In two separate experiments we found no evidence that water deprived rats showed a selective enhancement in conditioning to context, as opposed to conditioning to a tone. In experiment 1 we found that, during conditioning, water deprivation produced an overall decrease in freezing, both to the context (pre-CS measure) and to the tone (CS measure), only in those rats that received ten tone – shock pairings. Rats that received three tone – shock pairings exhibited no changes in acquisition of conditioning on the basis of deprivation schedule. Furthermore, water deprivation did not produce any statistically reliable effects on conditioned freezing to contextual cues during an extinction test session. However, rats that were water deprived showed reduced levels of conditioned freezing during the tone test. Thus, in experiment 1 the results failed to conform to the pattern reported by Maren et al. [9] in two respects. Not only did we find that water deprivation produced no reliable effects on contextual conditioning, whereas Maren et al. reported that contextual conditioning was significantly enhanced; we also found that water deprivation decreased conditioning to the tone, whereas Maren et al. reported no effects on tone induced freezing. Furthermore, it can be seen from Fig. 4A, as well as from the ANOVA we conducted on the first minute of tone presentation during test, that this between group difference, although it was seen statistically as a main effect of water deprivation rather than as a water deprivation – block interaction, would not have been observed had we run a 64-s tone test, as Maren et al. did. In experiment 1 we also found that rats that had received three or ten conditioning trials showed similar levels of conditioned contextual freezing. However, rats in group 3-SH showed less conditioned freezing to the
Fig. 7. Shows conditioned freezing during the tone test of experiment 2. The figure shows rats split into groups water deprived (WD) and non-deprived (ND). Data are presented as percentage of freezing for each 30 s of the 8-min tone presentation. Error bars denote S.E.M.
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tone than rats in group 10-SH. This pattern — reduced conditioned tone freezing, but no change in conditioned contextual freezing, after fewer tone–shock pairings — has been reported previously using the same freezing analysis [21]. This consistency across experiments serves to support our contention that the present system enables us validly to compare groups using small numbers of subjects. Furthermore, none of these effects can be attributed to supposed peculiarities of our automated system: we obtained an identical pattern of results by visual scoring methods. The pattern of results we obtained is also noteworthy in the following respect. There is a strong indication that freezing measures taken during acquisition and during extinction tests are both not adequate indices of conditioning: The effect of water deprivation on conditioned contextual freezing was observed only during acquisition of conditioning to contextual cues (pre-CS measure) but not during subsequent context extinction sessions. On the other hand, the effect of water deprivation on conditioned tone freezing was observed during both the acquisition of conditioning (CS measure) and the tone extinction test. However, during acquisition of conditioning the effects of water deprivation on either context or tone conditioning were observed only in rats that received 10 pairings of tone and shock, whereas, during the extinction session both group 10-SH and group 3-SH showed water deprivation-induced decreases in tone freezing. Thus we have obtained a double dissociation of between group effects during acquisition of conditioning and during extinction sessions. Effects of water deprivation on contextual conditioning were observed during acquisition but not during extinction, whereas effects of water deprivation on tone conditioning in group 3-SH were observed during extinction but not during acquisition. In our view, this pattern is consistent with the notion that the foot shock produced a delayed unconditioned freezing response, displayed only during conditioning training (when shocks were presented) but not during extinction sessions (when shocks were not presented). That is to say, we propose that the two types of measure — freezing during acquisition and freezing during extinction — might be qualitatively different rather than merely differing in sensitivity. Note that this notion assumes that the freezing response during both pre-CS and CS periods in the acquisition phase largely reflects unconditioned freezing in response to the foot shock, rather than conditioned contextual freezing and conditioned tone freezing, respectively. This analysis argues strongly against using interchangeably the freezing scores gained during acquisition and during extinction sessions (e.g. [7,9,16,19,23]). Interestingly the non-selective effects of water deprivation during acquisition of conditioning were observed only in group 10-SH. Unconditioned or generalised
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freezing is likely to have been greater in this group, which received more shocks with shorter gaps between each shock, and hence presumably a greater carry-over from a US delivery to the next pre-CS measurement. Thus water deprivation induced decreases in freezing in conditions that generated relatively high levels of overall freezing (acquisition of both context and tone conditioning in group 10-SH and tone extinction sessions) but not in conditions that generated relatively lower levels of overall freezing (acquisition of both context and tone conditioning in group 3-SH and context extinction sessions). There is no need, on the basis of our findings, to suppose that water deprivation acts either to increase or to decrease levels of freezing only to a certain type of stimulus. In experiment 2 we sought to replicate the procedures used by Maren et al. as closely as possible, (1) by giving the tone test before the context test and (2) by giving rats ad-lib water after conditioning ensuring that deprivation levels at test were the same in both groups of rats. In this case, unlike Maren et al., we found no effects of water deprivation during the context test, but like them we found that water deprivation produced no effects on conditioned freezing to the tone. It is worth noting that the original study ([9] three-pairings-group), using visual assessment of a relatively small sample of data, was conducted on 14 rats as opposed to our 12. Thus, it is highly unlikely that our failure to observe group differences in experiment 2 can be explained in terms of group sizes or lack of experimental power generally. In conclusion our results serve to limit the generality of Maren et al.’s findings. They suggest that any general conclusions regarding the relationship between conditioning to context and water deprivation, whether by action through LTP or theta at the hippocampus or not, are likely to be incorrect, as is the case for gender differences, compare Maren et al. [11] with Pryce et al. [19]. Furthermore our results suggest that some minor procedural variation might account for the different pattern of results we obtained. Possibilities include: (1) The housing conditions — we housed rats in groups of four in plastic cages with a straw floor covering while Maren et al. housed rats individually or in pairs in metal cages without straw. Recently published results in our laboratory show that both group and individually housed rats are more active if raised on sawdust than if raised on a grid floor [28]. Such differences might indeed account for different patterns of freezing behaviour in different published reports since baseline response rates are likely to have been different in the two studies; (2) Time of testing — we tested rats during the dark phase of the cycle while Maren et al. tested during the light phase. Again while this difference might be unlikely to produce effects on learning, general activity levels across the light/dark cycle might well interact with deprivation condition.
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