Effects of housing on consummatory successive negative contrast in rats: wirebottom cages versus polycarbonate tubs

Effects of housing on consummatory successive negative contrast in rats: wirebottom cages versus polycarbonate tubs Michael Wood, MS 1, Alan M. Daniel, MS 1, Egeenee Daniels, DVM 2 & Mauricio R. Papini, PhD 1 In consummatory successive negative contrast, rats that have had experience drinking 32% sucrose solution drink significantly less 4% sucrose solution than rats that have drunk only 4% solution. This contrast effect occurs reliably when rats are housed in wire-bottom cages, but it occurs significantly less frequently when rats are housed in polycarbonate tubs. Although it is unclear what causes these differences among housing conditions, the present study underscores the impact that housing conditions outside the domain of the training environment can have on behavioral outcomes. Consummatory successive negative contrast (csnc) is an incentive contrast situation for inducing stress as a result of reward loss 1. The training protocol occurs in two phases. In a preshift phase, independent groups of rats have exposure to either a high or a low concentration of sucrose solution (typically, 32% versus 4% sucrose). In a postshift phase all the animals receive the low sucrose concentration. As a result of this incentive downshift (32 4), the consummatory response (i.e., drinking the solution) decreases sharply below the level of the unshifted, low-sucrose control (4 4; ref. 2). Extensive research supports the conclusion that the suppression of consummatory behavior is accompanied by an aversive emotional reaction leading to conflict 1,3. For example, increased corticosterone levels 4, suppression of aggressive behavior 5, and disruption of sexual behavior 6 follow incentive downshift. In addition, anxiolytics reduce csnc, including benzodiazepines 7,8, and opioid peptides modulate it 9 11. Evidence reported here suggests that csnc is sensitive to the caging conditions. Researchers usually focus on the conditions prevailing at the time of testing, failing to consider that the conditions in which a laboratory animal is raised and housed may play a significant role in the behavior under analysis even when housing and testing are carried out under different conditions. In recent years there has been an increasing shift away from the traditional wire-bottom cages to polycarbonate tubs for housing rats under laboratory conditions. A variety of results support this change. For example, Manser et al. 12 observed that rats showed preference for solid surfaces over grated flooring. Rats spent 55% of their time on the solid surface during the awake portion of the day and 80% of their time during the sleep portion of the day. Other studies showed that polycarbonate tubs may be less harmful than wire-mesh housing. Mizisin et al. 13 compared withdrawal time following filament stimulation of the hindpaw in animals housed with sawdust flooring and wire-grate flooring. Data revealed significantly faster withdrawal time from animals housed with sawdust flooring. Histology of the hindpaw revealed signs of plantar nerve injury (e.g., Renaut bodies, demyelination, Wallerian degeneration) after 9 and 12 weeks of exposure to wire-grate flooring. Ortman et al. 14 also found Renaut bodies (i.e., loosely textured, amorphous, fibrous material and collagen) directly between the plantar nerve and the dorsal surface of highest compression. Whereas the significance of Renaut bodies is still under debate, the authors concluded that these formations are probably due to mechanical stress. There are also reports that suggest potential problems with the polycarbonate tubs. Raynor et al. 15 exposed rats to the microenvironment of a polycarbonate caging system and measured particle counts under two different conditions: wire-mesh flooring inserts over the bedding and bedding without wire inserts. Results showed 1 Department of Psychology, Texas Christian University, Box 298920, Fort Worth, TX 76129. 2 Laboratory Animal Medicine, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. Correspondence should be addressed to M.R.P. (m.papini@tcu.edu). 34 Volume 35, No. 3 MARCH 2006

FIGURE 1 Animals from two experiments run separately. In one experiment (left bars), rats housing was in wirebottom cages, whereas in another (right bars) rats resided in polycarbonate tubs. In both experiments, one group received a 32 4 downshift and the other was a 4 4 unshifted control. Plot includes means and standard errors. *P = 0.012 (other comparisons were not significant). that tubs with no wire-mesh flooring have higher particle counts than those with inserts, which may result in infection or disease due to urease and/or increased bacteria 16. Although bedding helps absorb urea, the authors indicated that increased ammonia concentration directly correlates with the presentation of murine respiratory mycoplasmosis 17. Hirsjarvi and Valiaho 18 compared temperature, humidity, and ammonia concentration of a polycarbonate tub with wood shavings versus wire-bottom cages with paperboard in the bedding tray. Temperature and humidity showed no differences between housing conditions, but ammonia concentration was significantly higher in the vicinity of the wire-mesh cages after 3 d (as much as 25 p.p.m., considered a threshold limit for humans; ref. 19). However, this study is not conclusive because each caging condition had a different bedding material. Raynor et al. 15 found that paperboard bedding allowed for significantly higher ammonia concentrations than wood-shaving bedding. Therefore, it is possible that the differences that Hirsjarvi and Viliaho 18 reported were due to the type of bedding and not the type of cage. In the process of converting our facility from wirebottom caging to polycarbonate tubs, we experienced problems during experiments involving csnc. There had been no study of the possible effects of housing in polycarbonate tub environments on behavioral tests such as csnc. The starting point of the research reported here was the discrepant results between the last experiment on csnc carried out with rats housed in wire-bottom cages, and the first experiment with rats housed in polycarbonate tubs. These experiments were similar in design, in that they used rats bred at the Texas Christian University (TCU) animal research facility from similar stocks of Long-Evans rats purchased from Harlan (Indianapolis, IN), were based on exactly the same behavioral testing procedure, and were separated by ~2 months. Figure 1 illustrates the data. For clarity we present only trial 10 (last preshift trial) and trial 11 (first postshift trial) and place the emphasis on the degree of consummatory suppression that accompanies the 32 4 incentive downshift. In these experiments caging apparently affected the degree of consummatory suppression in the downshifted groups. We qualify this effect as an apparent effect, because the experiments were not simultaneous. Rats housed in wire-bottom cages (32/W) showed a significant decrease in goal tracking times from trial 10 to 11 (z = 2.52, N = 8, P = 0.012), whereas rats housed in polycarbonate tubs (32/T) exhibited a nonsignificant reduction in consummatory behavior (z = 1.86, N = 8, P = 0.063). Caging conditions had no appreciable effect on the performance of unshifted, 4 4 groups (z < 1.13, N = 8, P > 0.25). To determine whether caging affects consummatory behavior after incentive downshift, the two main conditions of the previous experiments, we replicated this scenario with behavioral testing carried out simultaneously. METHODS Subjects In this experiment we used 12 Long-Evans rats that had been bred, raised, and housed in the TCU laboratory animal facility under a 12 h light/12 h dark cycle (lights on at 07:00 h) with free access to water at all times. Of the two housing designs, one consisted of stainless-steel wire-bottom cages measuring 24.5 18 18 cm (length width height) and the other consisted of polycarbonate tubs measuring 20.5 23 20.5 cm (all cages from Lab Products, Inc., Seaford, DE). In both cases we provided corncob bedding, placed either in a tray below the wire-bottom cages or directly into the tubs, and replaced it weekly. Animals matured in individual polycarbonate tubs, from postweaning day 21 until ~80 d of age. At 80 d of age we placed half of the animals in wire-mesh cages, whereas the remainder stayed in the polycarbonate cages. Both groups received no food until they reached 81 85% of their ad libitum body weight. Behavioral testing began when rats were ~90 d old. The TCU Institutional Animal Care and Use Committee reviewed and approved all animal work. Apparatus Training took place in four conditioning boxes (Coulbourn Instruments, Inc., Allentown, PA) constructed of aluminum and Plexiglas, and measuring 29.3 21.3 26.8 cm. The floor consisted of steel rods, 0.4 cm in diameter and 1.6 cm apart, running parallel to the front wall. A bedding tray filled with corncob bedding was below the floor to collect fecal pellets and urine. Against the front wall was an elliptical hole 1 cm wide, 2 cm high, and 4 cm from the floor. We inserted a sipper tube, 1 cm in diameter, to extend no more than 1.0 cm into the box. A house light (GE 1820) located in the LAB ANIMAL Volume 35, No. 3 MARCH 2006 35

tracking time, was the dependent variable. All statistical tests compared goal tracking times on trial 10 versus trial 11 with a Wilcoxon test for paired samples, twotailed. An α level of 0.05 was used in all tests. FIGURE 2 Animals housed in polycarbonate tubs (T) or wirebottom cages (W) received access to 32% sucrose solution for 10 trials, followed by access to 4% sucrose solution for an additional 5 trials. Means and standard errors of each group on trials 10 and 11 appear in this figure. *P = 0.028 (other comparisons were not significant). center of the box s ceiling provided diffuse light. A computer located in an adjacent room controlled the presentation and retraction of the sipper tube. The computer also detected contact with the sipper tube by way of a circuit involving the steel rods in the floor. We placed each conditioning box in a sound-attenuating chamber that contained a speaker to deliver white noise and a fan for ventilation. Together, the speaker and fan produced noise with an intensity of 80.1 db (SPL, scale C). Procedure Rats were in one of two groups (n = 6) by random assignment: group 32/W (housed in wire-bottom cages) and group 32/T (housed in polycarbonate tubs). All rats received 10 daily trials of access to a 32% sucrose solution, followed by 5 trials of access to a 4% sucrose solution. Each trial started with a mean 30-s pretrial interval (range: 15 45 s), followed by insertion of the sipper tube. The 5-min trial began after the first contact with the sipper tube. Trials ended with a mean posttrial interval of 30 s (range: 15 45 s). We shifted the rats to a transport rack and moved them to a holding room in squads of four. Then we moved squads to the training room, where we placed the rats in the conditioning boxes. At the end of the trial we returned the rats to their cages, cleaned the conditioning boxes with a damp paper towel, and moved the transport rack to the holding room. Each rat was always part of the same squad and always trained in the same conditioning box. Randomization of squad training order occurred daily. Assignment to conditioning boxes was counterbalanced with caging condition. Preparation of sucrose solutions consisted of mixing 32 g (or 4 g) of commercial sugar for every 78 g (or 96 g) of distilled water (w/w). Automatic recording of contacts with the sipper tube accumulated in 0.05-s units. This measure, called goal RESULTS The results of interest are the performance of rats on trial 10, the last trial with exposure to the 32% sucrose solution, and trial 11, the first trial after the downshift to 4% sucrose. Figure 2 shows the performance of these groups in these two critical trials. There was suppression of consummatory behavior in both groups, but whereas group 32/T dropped 20.0% from trial 10 to trial 11, group 32/W dropped 34.4% on the same trials. Statistical tests revealed that, whereas the change in performance was not significant in group 32/T (z = 1.57, N = 6, P = 0.116), the rats from group 32/W showed a significant level of consummatory suppression on trial 11, relative to trial 10 (z = 2.52, N = 6, P = 0.012). DISCUSSION Housing rats in the polycarbonate tubs reduced the effect of a 32 4 incentive downshift in a consummatory situation. These results confirm previous results from experiments run under the same conditions but carried out at different times. These data do not indicate, however, the reason for the effect of caging condition on incentive downshift. Several variables differ across these two caging conditions, in addition to those mentioned in the introduction. First, the amount of effort necessary to procure food is clearly different. In the wire-bottom cages the food hopper rests against the front wall of the cage, and the rat must grab pieces of pellet by biting through the metallic grid. Additionally, pieces of food that fall on the floor of the cage are likely to drop below, into the unreachable tray. In contrast, procuring food in the polycarbonate tubs is a simpler task. Food rests in a hopper located inside the tub and is reached through relatively wide openings created by vertical bars in the front of the hopper. Moreover, if a piece of food is dropped, it is still inside the tub. Available evidence demonstrates that the amount of effort to procure food in the cage can affect behavioral performance under the same testing conditions. Eisenberger et al. 20 trained rats to run in a straight alley for food reinforcement in a goal box and measured the speed of running. After performance became stable, they shifted the conditions to extinction, in which food was withdrawn from the goal box. Although all rats were kept in wire-bottom cages, the high-effort group had to procure food through the metallic grid (as in our wirebottom cages), whereas the low-effort group received food in a Petri dish inside the cage. High-effort rats persisted longer during extinction than low-effort rats. This experiment demonstrated that effort in procuring food can affect behavior in a different situation. 36 Volume 35, No. 3 MARCH 2006

FIGURE 3 Rats (group 32/T/effort) housed in polycarbonate tubs had exposure to a food hopper with a metallic grid similar to that used in wire-bottom cages and then received training in the csnc as described in the present experiment for group 32/T. Plot also includes the performance of the conventional group 32/T/pooled on the side for comparison (average of the two 32/T groups shown in Figs. 1 and 2). Plot includes means and standard errors. *P = 0.012 (other comparisons were not significant). To test the effect of effort in procuring food on consummatory suppression, a group of rats housed in the polycarbonate tubs received food through a hopper that required a greater effort. The front of this food hopper had vertical bars crossed by horizontal bars in a grid fashion similar to the hopper used in wire-bottom cages. The rats housed in this way then received training under conditions identical to those described in the Methods section. In Figure 3 is a plot of the response to incentive downshift along with the data from the two 32/T groups plotted in Figures 1 and 2 for the purpose of comparison. Thus these rats all had polycarbonate tubs for housing, although they received training at different times. There was a significant degree of consummatory suppression in the group trained with this high-effort feeder (z = 2.52, N = 8, P = 0.012), suggesting that this may be one reason for the difference in behavioral performance between rats housed in wirebottom cages versus polycarbonate tubs. A second difference between these two caging conditions is the availability of social cues from other rats housed in adjacent cages and racks. The lateral walls and the back wall of the wire-bottom cages are solid metallic plates. Although the front wall is a metallic grid, the hopper and the cage card usually placed there cause a considerable blocking of visual cues. Olfactory and auditory cues may be present, but rats have virtually no visual contact with other rats. In contrast, rats housed in polycarbonate tubs can observe each other through the translucent lateral walls of the tubs, although olfactory and auditory cues may be reduced due to a more isolated environment relative to the wire cages. Furthermore, direct contact with bedding material may block olfactory cues released by other rats either by providing competing cues or by a more effective absorption of odoriferous compounds present in urine. There is no available information on the potential effects of these housing peculiarities, but one study shows that social interactions during housing play a significant role in behavioral testing. Shanab and Ralph 21 trained four groups of 126- d-old rats in the same running response situation as did Eisenberger et al. 20, with either continuous reinforcement (food in every trial) or 50% partial reinforcement (food in a random half of the trials). They then shifted all rats to extinction, withholding food in all trials. The rats in two of the groups (one continuous and one partial) had had individual housing since weaning, whereas the rats in two other groups (one continuous and one partial) had had housing in groups of nine animals since weaning. Shanab and Ralph 21 found greater resistance to extinction in the partial than in the continuous reinforcement group for animals housed individually but no differences in extinction for animals housed in groups. Thus social interaction among rats playing a greater role in the polycarbonate tubs (e.g., visual cues) or in the wire-bottom cages (e.g., auditory or chemical communication) may be responsible for the differences found in the present situation. Wire cages are also darker than polycarbonate cages, because the walls are not transparent. Lockard 22 established that levels of illumination in the home cage can produce differences in a behavioral test involving selfexposure to light. It is unclear how levels of illumination may affect unrelated behaviors, such as consumption of sucrose, but the potential for such effects exists. A third potential influence that may differentiate these housing procedures concerns stress level. Some of the differences pointed out previously include levels of ammonia and the potential consequences for respiratory problems in polycarbonate tubs, potential chronic pain derived from tissue damage in the feet in wirebottom cages, differences in food-procuring effort, and relative social isolation. Although we noted no differences with respect to temperature under similar conditions 18, the particular cages used in these studies and the specific conditions prevailing in the colony room may have been associated with different temperatures. Different temperatures may impose different homeostatic demands and thus induce different levels of stress. Moreover, the rats used in these experiments had matured in polycarbonate cages before being subsequently shifted to wire-bottom cages 10 d before the start of training. It is possible that this change in housing, rather than the housing conditions themselves, contributed to the group differences. Any of these influences could introduce differential levels of stress and thus affect consummatory performance in the csnc situation. As indicated earlier, csnc involves aversive emotionality, increased hypothalamic-pituitary-adrenal activation, and conflict 1,3. There is substantial evidence that chronic levels of stress induced by a variety LAB ANIMAL Volume 35, No. 3 MARCH 2006 37

of procedures can affect behavior in a wide range of testing situations (e.g., refs. 23,24). Although there has been no study of the issue of chronic stress in relation to the csnc effect, there is a well-documented connection between stress and csnc. Recent evidence indicates, for example, that opioid agonists reduce csnc 11, opioid antagonists increase it 9, copulatory experience before incentive downshift (a known trigger of opioid activity 6 ) reduces it, and post-trial 11 administration of corticosterone (a stress hormone known to potentiate aversive memories) increases it 25. Although the interaction between physical pain and csnc has not yet been studied in detail, data demonstrating that exposure to incentive downshift raises the threshold for peripheral pain in the hot-plate test apparatus 26, as well as the attenuating effects of both opioids 11 and cannabinoid agonists 27 on csnc, suggest the connection. This evidence indicates a connection between chronic stress and the csnc effect, even if the direction of this potential relationship is difficult to predict. Does chronic stress derived from housing conditions sensitize rats to any stress-inducing event, thus increasing the size of the csnc effect, as it happens in wire-bottom cages? Or does chronic stress induced by tub housing provide an aversive psychological framework that reduces the emotional impact of incentive downshift in the conditioning box? The connection between pain and the csnc (e.g., ref. 26) suggests an affirmative answer to the first question, whereas the relativistic nature of the csnc effect (e.g., ref. 28) suggests an affirmative answer to the second. ACKNOWLEDGMENTS This research was supported with funds from the Department of Psychology at TCU. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 18 November 2005; accepted 13 January 2006. Published online at http:// 1. Flaherty, C.F. Incentive Relativity (Cambridge University Press, New York, 1996). 2. Vogel, J.R., Mikulka, P.J. & Spean, N.E. Effects of shifts in sucrose and saccharin concentrations on licking behavior in the rat. J. Comp. Physiol. Psychol. 66(3), 661 666 (1968). 3. Papini, M.R. Comparative psychology of surprising nonreward. Brain Behav. Evol. 62(2), 83 95 (2003). 4. Flaherty, C.F., Becker, H.C., & Pohorecky, L. Correlation of corticosterone elevation and negative contrast varies as a function of postshift day. Anim. Learn. Behav. 13, 309 314 (1985). 5. Mustaca, A.E., Martinez, C. & Papini, M.R. Surprising nonreward reduces aggressive behavior in rats. Int. J. Comp. Psychol. 13(1 2), 91 100 (2000). 6. Freidin, E. & Mustaca, A.E. Frustration and sexual behavior in male rats. Learn. Behav. 32(3), 311 320 (2004). 7. Flaherty, C.F., Grigson, P.S. & Rowan, G.A. Chlordiazepoxide and the determinants of contrast. Anim. Learn. Behav. 14, 315 321 (1986). 8. Mustaca, A.E., Bentosela, M. & Papini, M.R. Consummatory successive negative contrast in mice. Learn. Motiv. 31(3), 272 282 (2000). 9. Pellegrini, S., Wood, M.D., Daniel, A.M. & Papini, M.R. Opioid receptors modulate recovery from consummatory successive negative contrast. Behav. Brain Res. 164(2), 239 249 (2005). 10. Rowan, G.A. & Flaherty, C.F. Effect of morphine on negative contrast in consummatory behavior. Psychopharmacology (Berl) 93(1), 51 58 (1987). 11. Wood, M.D., Daniel, A.M. & Papini, M.R. Selective effects of the delta-opioid receptor agonist DPDPE on consummatory successive negative contrast. Behav. Neurosci. 119(2), 446 454 (2004). 12. Manser, C.E., Morris, T.H. & Broom, D.M. An investigation into the effects of solid or grid cage flooring on the welfare of laboratory rats. Lab. Anim. 29(4), 353 363 (1995). 13. Mizisin, A.P., Kalichman, M.W., Garrett, R.S. & Dines, K.C. Tactile hyperesthesia, altered epidermal innervation and plantar nerve injury in the hindfeet of rats housed on wire grates. Brain Res. 788(1 2), 13 19 (1998). 14. Ortman, J.A., Sahenk, Z. & Mendell, J.R. The experimental production of Renaut bodies. J. Neurol. Sci. 62(1 3), 233 241 (1983). 15. Raynor, T.H., Steinhagen, W.H. & Hamm, T.E. Differences in the microenvironment of a polycarbonate caging system: bedding vs raised wire floors. Lab. Anim. 17(2), 85 89 (1983). 16. Briel, J.E., Kruckenberg, S.M. & Besch, E.L. Observations of Ammonia Generation in Laboratory Animal Quarters. Kansas State University NIH Contract, 71 2511 (1972). 17. Broderson, J.R., Lindsey, J.R. & Crawford, J.E. The role of environmental ammonia in respiratory mycoplasmosis of rats. Am. J. Pathol. 85(1), 115 130 (1976). 18. Hirsjarvi, P.A. & Valiaho, T.U. Microclimate in two types of rat cages. Lab. Anim. 21(2), 95 98 (1987). 19. American Conference of Government Industrial Hygienics. Documentation of threshold limit values for substances in workroom air. Cincinatti, OH: ACGIH (1973). 20. Eisenberger, R., Masterson, F.A. & Over, S. Maintenancefeeding effort affects instrumental performance. Q. J. Exp. Psychol. A 34B, 141 148 (1982). 21. Shanab, M.E. & Ralph, L. Negative contrast and partial reinforcement effect as a function of crowded rearing conditions in the rat. J. Gen. Psychol. 100, 13 26 (1979). 22. Lockard, R.B. Some effects of maintenance luminance and strain differences upon self-exposure to light by rats. J. Comp. Physiol. Psychol. 55, 1118 1123 (1962). 23. Fontella, F.U. et al. Taste modulation of nociception differently affects chronically stressed rats. Physiol. Behav. 80(4), 557 561 (2004). 24 Vyas, A. & Chattarji, S. Modulation of different states of anxiety-like behavior by chronic stress. Behav. Neurosci. 118(6), 1450 1454 (2004). 25. Bentosela, M., Ruetti, E., Muzio, R.N., Mustaca, A.E. & Papini, M.R. Administration of corticosterone after the first downshift trial enhances consummatory successive negative contrast. Behav. Neurosci. (in press). 26. Mustaca, A.E. & Papini, M.R. Consummatory successive negative contrast induces hypoalgesia. Int. J. Comp. Psychol. 18, 255 262 (2005). 27. Genn, R.F., Tucci, S., Parikh, S. & File, S.E. Effects of nicotine and a cannabinoid receptor agonist on negative contrast: Distinction between anxiety and disappointment? Psychopharmacology (Berl) 177(1 2), 93 99 (2004). 28. Papini, M.R. & Pellegrini, S. Scaling relative incentive value in consummatory behavior. Learn. Motiv. (in press). 38 Volume 35, No. 3 MARCH 2006