Effects of cytotoxic hippocampal lesions in mice on a cognitive test battery

Mice received cytotoxic lesions which selectively removed all of the hippocampus and dentate gyrus except the most ventral portions. They were impaired on both spontaneous and rewarded discrete-trial alternation in T-mazes. Acquisition of reference memory for the location of a hidden platform in the Morris water maze was impaired in lesioned mice. On an elevated Y-maze reference memory task, in which only one arm was rewarded, lesioned mice showed no evidence of learning. In a Lashley III maze task, however, where maze rotation demonstrated that control performance was independent of distal spatial cues, acquisition in the lesioned mice was unimpaired. Control levels of continuous spontaneous alternation in a Y-maze were too low to reveal a hippocampal deficit. A small impairment in acquisition of a multiple-trial passive avoidance task was seen in lesioned mice, despite a small but significant increase in reactivity to the footshock. These results are largely consistent with findings in hippocampal lesioned rats on the same or similar tasks, and reflect a major impairment of spatial cognition, with relative sparing of non-spatial task performance.     

A study of hippocampal structure-function relations along the septo-temporal axis

This study examined structural-functional differences along the septo-temporal axis of hippocampus using radial-maze tasks that involved two different memory processes [reference memory (RM) and working memory (WM)], and the use of two kinds of information (spatial vs. nonspatial cue learning). In addition, retention of the nonspatial cue task was tested nine weeks following completion of acquisition, and the rats then underwent discrimination reversal training. Ibotenic acid lesions limited to either the dorsal pole, intermediate area, or ventral pole had minimal effects on acquisition of the complex place and cue discrimination tasks. The one exception was that rats with lesions confined to the dorsal third of hippocampus made more WM errors on the spatial task (but not the cue task) early in training. Selective lesions of the three hippocampal regions had no effects on either long-term retention or reversal of the nonspatial cue discrimination task. In contrast, rats that had all of the hippocampus removed were severely impaired in learning the spatial task, making many RM and WM errors, whereas on the nonspatial cue task, the impairment was limited to WM errors. Further analysis of the WM errors made in acquisition showed that rats with complete lesions were significantly more likely on both the spatial and nonspatial cue tasks to reenter arms that had been baited and visited on that trial compared to arms that had not been baited. A similar pattern of errors emerged for complete hippocampal lesioned rats during reversal discrimination. This pattern of errors suggests that in addition to an impairment in handling spatial information, complete removal of hippocampus also interferes with the ability to inhibit responding to cues that signal reward under some conditions but not under others. The finding that selective lesions limited to the intermediate zone of the hippocampus produce no impairment in either WM ("rapid place learning") or RM in our radial maze tasks serve to limit the generality of the conclusion of Bast et al. (Bast et al. (2009) PLos Biol 7:730-746) that the intermediate area is needed for behavioral performance based on rapid learning about spatial cues.     

Effects of enriched postoperative housing conditions on spatial memory deficits in rats with selective lesions of either the hippocampus,subiculum or entorhinal cortex

Long-Evans male, adult rats received selective and bilateral lesions of either the hippocampus, subiculum or lateral entorhinal cortex, and were then housed for 30 days in either enriched or standard conditions. Rats were then tested in the eight-arm radial maze to assess spatial working memory and the strategies that were employed (i.e. pattern of arms visited). Lesions of the hippocampus induced both a working-memory impairment and a loss in the use of allocentric strategies to perform the task. Rats with lesions of the subiculum were also impaired but less than hippocampectomized rats and showed a similar pattern of arm visits as control rats. In contrast with other lesioned rats, rats with lateral entorhinal cortex lesions performed the task like control rats. Postoperative enriched housing conditions (EHC) globally enhanced performance of rats, but did not affect the strategies selected by the rats to solve the task. The beneficial effect of EHC was particularly obvious in rats with lesions of the subiculum. In enriched rats with such lesions, performance was not significantly different from that of control rats housed in standard conditions. The present results indicate that 1) the structures within the hippocampal formation are not similarly involved in spatial learning and memory processes and in the management of navigational demands of the radial maze, and 2) enriched conditions may enhance the spared spatial abilities of some lesioned rats thus promoting functional recovery.     

Vestibular loss promotes procedural response during a spatial task in rats

Declarative memory refers to a spatial strategy using numerous sources of sensory input information in which visual and vestibular inputs are assimilated in the hippocampus. In contrast, procedural memory refers to a response strategy based on motor skills and familiar gestures and involves the striatum. Even if vestibular loss impairs hippocampal activity and spatial memory, vestibular‐lesioned rats remain able to find food rewards during complex spatial memory task. Since hippocampal lesions induce a switch from declarative memory to procedural memory, we hypothesize that vestibular‐lesioned rats use a strategy other than that of hippocampal spatial response to complete the task and to counterbalance the loss of vestibular information. We test, in a reverse T‐maze paradigm, the types of strategy vestibular‐lesioned rats preferentially uses in a spatial task. We clearly demonstrate that all vestibular‐lesioned rats shift to a response strategy to solve the spatial task, while control rats use spatial and response strategies equally. We conclude that the loss of vestibular informations leading to spatial learning impairments is not offset at the hippocampus level by integration process of other sense mainly visual informations; but favors a response strategy through procedural memory most likely involving the striatum, cerebellum, and motor learning. © 2014 Wiley Periodicals, Inc.     

Preserved configural learning and spatial learning impairment in rats with hippocampal damage.

This study was undertaken to compare the effect of hippocampal neurotoxic lesions in rats on two behavioral tasks, one a test of spatial learning, and the other an operant discrimination task that is acquired by forming nonspatial configural associations. Lesions of the hippocampus were made with microinjections of ibotenic acid. After postoperative recovery, rats were trained initially to locate a camouflaged escape platform in a water maze using distal spatial cues. Rats also were trained in the maze apparatus with a visible escape platform under conditions in which spatial information was made irrelevant to performance, i.e., cue learning. In an operant task, the same rats were then trained on a discrimination that included simultaneous feature positive and feature negative components (trial types XA+, A-, XB-, B+). After completion of this nonspatial configural learning task, rats received additional training in the water maze using a new platform location for spatial learning. To the extent that proficient performance in both the maze and operant tasks depends on a common function of the hippocampus, i.e., configural learning, the expectation was that hippocampal lesions would prove equally detrimental to performance in both tasks. Contrary to this expectation, lesioned rats were severely impaired in spatial learning but readily acquired the operant discrimination, even exhibiting some evidence of enhanced performance on this nonspatial configural learning task. Performance of the lesioned rats during cue training in the water maze was also enhanced relative to the control group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rats with lesions of the vestibular system require a visual landmark for spatial navigation.

The role of the vestibular system in acquisition and performance of a spatial navigation task was examined in rats. Male Long-Evans rats received sham or bilateral sodium arsanilate-induced vestibular lesions. After postoperative recovery, under partial water-deprivation, rats were trained (16 trials/day) to find a water reward in one corner of a black square enclosure. A cue card fixed to one wall of the enclosure served as a stable landmark cue. The orientation of the rat at the start of each trial was pseudo-randomized such that the task could not be solved by an egocentric response strategy. Rats with vestibular lesions acquired the task in fewer trials than the sham treated control rats. Vestibular lesions did not influence the motivation or motor function necessary to perform the task. Performance of sham rats was maintained during probe trials in which the cue card was removed from the enclosure, while lesioned rats were markedly impaired. Rotation of the cue card (+/-90 degrees ) caused an equivalent shift in corner choice behavior of the lesioned rats. However, sham rats often disregarded the rotated cue card and made place responses. These results suggest that the vestibular lesions disrupt idiothetic navigation or path integration and render navigational behavior critically dependent upon external landmarks. These results are consistent with the navigational abilities of humans with bilateral vestibular dysfunction.     

Involvement of the hippocampus and associative parietal cortex in the use of proximal and distal landmarks for navigation

Rats with dorsal hippocampus or associative parietal cortex (APC) lesions and sham-operated controls were trained on variants of the Morris water maze navigation task. In the 'proximal landmark condition', the rats had to localize the hidden platform solely on the basis of three salient object landmarks placed directly in the swimming pool. In the 'distal landmark condition', rats could rely only on distal landmarks (room cues) to locate the platform. In the 'beacon condition', the platform location was signaled by a salient cue directly attached to it. Rats with hippocampal lesions were impaired in the distal and to a less extent in the proximal landmark condition whereas rats with parietal lesions were impaired only in the proximal landmark condition. None of the lesioned groups was impaired in the beacon condition. These results suggest that the processing of information related to proximal, distal landmarks or associated beacon are mediated by different neural systems. The hippocampus would contribute to both proximal and distal landmark processing whereas the APC would be involved in the processing of proximal landmarks only. Navigation relying on a cued-platform would not require participation of the hippocampus nor the APC. Assuming that the processing of proximal landmarks heavily depends on the integration of visuospatial and idiothetic information, these results are consistent with the hypothesis that the APC plays a role in the combination of multiple sensory information and contributes to the formation of an allocentric spatial representation.     

Chlordiazepoxide improves the performance of septal lesioned but not hippocampal lesioned animals in a Morris maze

The effects of hippocampal and lateral septum lesions were compared in rats tested in a water maze spatial memory task, and the effect of chlordiazepoxide (CDP) was examined. There was a significant interaction for lesion and CDP in the septal lesioned subjects, with the lesioned animals performing worse than control animals, and CDP improving the performance of lesioned animals. CDP had no effect on impaired performance in hippocampal lesioned animals.     

Impaired remote spatial memory after hippocampal lesions despite extensive training beginning early in life

Damage to the hippocampus typically produces temporally graded retrograde amnesia, whereby memories acquired recently are impaired more than memories acquired remotely. This phenomenon has been demonstrated repeatedly in a variety of species and tasks, and it has figured prominently in theoretical treatments of memory and hippocampal function. A striking exception to the finding of temporally graded retrograde amnesia comes from studies with rodents using spatial tasks like the water maze. In these studies, recent and remote memory were similarly impaired following hippocampal lesions. In contrast to work with rodents, studies of patients with medial temporal lobe lesions, including complete hippocampal lesions, indicate that remote spatial memory can be intact. One difference between studies in humans and studies in rodents is that spatial memory in animal studies is acquired during a limited period of time when the animals are adults. In contrast, the spatial memory studied in humans was acquired beginning at an early age and learning continued for a considerable period of time. We initiated training in a standard water maze immediately after rats had been weaned at 21 days of age and continued training until the rats were young adults (90 days old). Large hippocampal lesions were made 100 days after the completion of training. After recovery from surgery, control rats exhibited good retention on the first retention probe trial, but rats with hippocampal lesions performed at chance. Thus, even after extended training beginning early in life, and with a prolonged training-surgery interval, hippocampal lesions impair performance in the water maze task. Possible reasons for these findings are discussed in the context of the specific performance requirements of the water maze task.     

Early rearing environment and dorsal hippocampal ibotenic acid lesions: long-term influences on spatial learning and alternation in the rat

Behavioural responses in a set of spatial and cue tasks were assessed in adult rats that had been given ibotenic acid lesions of the dorsal hippocampus at weaning. The lesions or sham operations were immediately followed by one month of differential rearing, either in enriched, social or isolated housing environments. The differential rearing was followed by standard (social) housing conditions until behavioural testing began at 4 months of age. Compared to sham-operated rats, the rats with early cytotoxic lesions showed substantial impairments on learning and efficient strategy formation in radial arm maze, retention of a spatial location, but not of a cue-marked location, in a + maze and spontaneous alternation. Differential rearing had some long-term effects depending on the task. Sham-operated rats which had been housed in isolation used a pattern of strategies in the radial arm maze that resembled the pattern used by rats with lesions. Early enrichment, on the other hand, alleviated lesion deficits only in a spontaneous alternation task in a T-maze where the variety and salience of proximal cues were maximised. Enrichment increased lesion deficits in the radial maze task, where distal cues only could guide performance. The results suggest that the hippocampus may play an important role in the use of contextual information and that behavioural recovery after early hippocampal damage--limited to situations in which featural information is highly salient--may be permanently induced by rearing in environments, as in enriched ones, where rats can attend to and manipulate environmental cues.     

Otoconia‐deficient mice show selective spatial deficits

The vestibular system contributes to the performance of various spatial memory tasks, but few studies have attempted to disambiguate the roles of the semicircular canals and otolith organs in this performance. This study tested the otolithic contribution to spatial working and reference memory by evaluating the performance of otoconia‐deficient tilted mice on a radial arm maze and a Barnes maze. One radial arm maze task provided both intramaze and extramaze cues, whereas the other task provided only extramaze cues. The Barnes maze task provided only extramaze cues. On the radial arm maze, tilted mice performed similar to control mice when intramaze cues were available, but committed more working and reference memory errors than control mice when only extramaze cues were available. On the Barnes maze task, control and tilted mice showed similar latency, distance, and errors during acquisition training. On the subsequent probe trial, both groups spent the greatest percentage of time in the goal quadrant, indicating they were able to use extramaze cues to guide their search. Overall, these results suggest signals originating in the otolith organs contribute to spatial memory, but are not necessary for all aspects of spatial performance. © 2014 Wiley Periodicals, Inc.     

Hippocampal lesioned rats are able to learn a spatial position using non-spatial strategies

In the last two decades, many experiments have demonstrated that the hippocampus plays a role in the learning and processing of spatial and contextual information. Despite these demonstrations, some recent publications have indicated that the hippocampus is not the only structure involved in spatial learning and that even after hippocampal lesions, rats can perform spatial tasks. However, it is not well established whether animals with hippocampal dysfunction still have some spatial learning capacities or develop non-spatial solutions; these may require lengthier acquisition training. We now report the effects of conventional, dorsal hippocampal ablation on rats' performance on the water maze. We tested rats using a short (4 days) versus a long (16 days) acquisition period. We demonstrated that animals with dorsal hippocampal lesions have some residual capacity for learning the localization of a hidden escape platform in a pool during both a reference memory task and a working memory task. The animals with dorsal hippocampal lesions learned to escape at a fixed location, but only with extended training. It is suggested that these animals used non-spatial strategies to compensate for a spatial memory impairment. The results are discussed with respect to the experimental procedure and the strategy applied by the lesioned rats.     

Hippocampus and remote spatial memory in rats

Damage to the hippocampus typically produces temporally graded retrograde amnesia, whereby memories acquired recently are impaired more than memories acquired remotely. This phenomenon has been demonstrated repeatedly in a variety of species and tasks. It has also figured prominently in theoretical treatments of memory and hippocampal function. Yet temporally graded retrograde amnesia has not been demonstrated following hippocampal damage in spatial tasks like the water maze. We have assessed recent and remote spatial memory following hippocampal lesions in three different tests of spatial memory: (1) the standard water maze; (2) the Oasis maze, a dry-land version of the water maze; and (3) the annular water maze, where training and testing occur within a circular corridor. Training protocols were developed for each task such that retention of spatial memory could be expressed after very long retention intervals. In addition, retention in each task was assessed with single probe trials so that the assessment of remote memory did not depend on the ability to relearn across multiple trials. The findings were consistent across the three tasks. In the standard water maze (Experiment 1), spatial memory was impaired after training-surgery intervals of 1 day, 8 weeks, or 14 weeks. Similarly, in the Oasis maze (Experiment 2), spatial memory was impaired after training-surgery intervals of 1 day and 9 weeks. Finally, in the annular water maze (Experiment 3), spatial memory was impaired after training-surgery intervals of 9 weeks and 14 weeks. Dorsal hippocampal lesions impaired performance to the same extent as complete lesions. The impairment in remote spatial memory could reflect disruption of previously acquired spatial information. Alternatively, it is possible that in these tasks hippocampal lesions might produce an impairment in performance that prevents the expression of an otherwise intact spatial memory.     

The effects of ibotenic acid lesions of the nucleus accumbens on spatial learning and extinction in the rat

Rats with ibotenic acid lesions of the nucleus accumbens (N. Acc) were studied in two spatial learning paradigms: a T-maze and a Morris water maze. Learning of a spatial discrimination task and its reversal in the T-maze were disrupted by the N. Acc lesions. As both original and reversal learning were impaired, there was no evidence of a specific lesion effect on reversal learning. The lesioned rats did not perseverate excessively in their choice of the previously reinforced arm. There was evidence of behavioural inflexibility during extinction when the lesioned rats failed to slow the pace at which they ran the maze in the absence of reward. Spontaneous alternation was not significantly affected by the lesion. Acquisition of the second spatial task, locating the hidden platform in the Morris water maze, was also impaired. The lesioned rats did eventually learn the task and successfully reached the platform with similar latencies and heading errors to controls. Thus, the N. Acc lesion impaired but did not abolish spatial learning in the T-maze and the water maze. The deficits observed in this study may reflect a role for the N. Acc in the reorganisation of behaviour in response to external change.     

Influence of vestibular input on spatial and nonspatial memory and on hippocampal NMDA receptors

It has recently been shown that a lack of vestibular sensory information decreases spatial memory performance and induces biochemical changes in the hippocampus in rodents. After vestibular neurectomy, patients display spatial memory deficit and hippocampal atrophy. Our objectives were to explore: (a) spatial (Y maze, radial-arm maze), and non-spatial (object recognition) memory performance, (b) modulation of NMDA receptors within the hippocampus using radioligand binding, and (c) hippocampal atrophy, using MRI, in a rat model of bilateral labyrinthectomy realized in two operations. Chemical vestibular lesions (VLs) were induced in 24 animals by transtympanic injections of sodium arsanilate (30 mg/0.1 ml/ear), one side being lesioned 3 weeks after the other. The control group received transtympanic saline solution (0.1 ml/ear) (n = 24). Spatial memory performance (Y maze and radial maze) decreased after VL. Conversely, non-spatial memory performance (object recognition) was not affected by VL. No hippocampal atrophy was observed with MRI, but density of NMDA receptors were increased in the hippocampus after VL. These findings show that the lack of vestibular information induced specific deficits in spatial memory. Additionally, quantitative autoradiographic data suggest the involvement of the glutamatergic system in spatial memory processes related to vestibular information. When studying spatial memory performances in the presence of vestibular syndrome, two-step labyrinthectomy is a suitable procedure for distinguishing between the roles of the specific components of vestibular input loss and those of impaired locomotor activity.     

Egocentric spatial orientation in a water maze by rats subjected to transection of the fimbria-fornix and/or ablation of the prefrontal cortex

The acquisition of a water maze based task requiring egocentric spatial orientation in the absence of distal cues was studied in four groups of rats: animals in which the fimbria-fornix had been transected, rats that received bilateral ablations of the anteromedial prefrontal cortex, animals in which both of these structures had been lesioned, and a sham-operated control group. Isolated lesions of both the anteromedial prefrontal cortex and the hippocampus were associated with a significantly impaired task acquisition. Both of these individually lesioned groups did, however, eventually demonstrate full functional recovery by reaching the task proficiency of the sham-operated control group. In contrast, the group in which both of these structures had been lesioned failed to demonstrate full functional recovery and was severely and long-lastingly impaired when compared to all other groups. Behavioural challenges in the form of a no-platform session and two reversals of platform position demonstrated that while the sham-operated control group and the group subjected to fimbria-fornix transections in isolation utilized rather pure egocentric orientation strategies, the two prefrontally lesioned groups (and especially the combined lesion group) employed a different set of solution strategies which at least partly relied on a "circling" method. Even in the behaviour of the prefrontally lesioned groups, however, indications of a certain level of cognitive representations of the platform positions were seen. It is concluded that both the prefrontal cortex and the hippocampus contribute to the mediation of egocentric spatial orientation. Furthermore, the hippocampus is a significant and potentially irreplaceable part of the neural substrate of functional recovery of the presently studied task after prefrontal lesions--while the prefrontal cortex may play a similar role with respect to hippocampal lesions.     

Spatial correlates of hippocampal unit activity are altered by lesions of the fornix and entorhinal cortex

Behavioral and electrophysiological evidence supports the role of the hippocampus in the processing of spatial information. In the present study, neuronal activity recorded from chronically implanted hippocampal microelectrodes was correlated with a rat's spatial orientation while traversing a radial maze for food reward. Place units were found in all fields of the dorsal hippocampus and dentate gyrus. Rotation of the maze relative to extramaze cues failed to disrupt the intact animal's spatial task performance or the spatial correlates of the unit activity. Lesions of the fornix or entorhinal cortex disrupted performance of the task. Unit activity correlated to the animal's spatial orientation was also disrupted by either lesion. There was no correlation between the disruption of the unit activity and location of the unit within hippocampal fields. Unit activity from lesioned animals showed correlation to the physical properties of the maze rather than to the orientation of the maze in space. These results further support the role of the hippocampus in the processing of spatial information.     

Testing the importance of the retrosplenial guidance system: effects of different sized retrosplenial cortex lesions on heading direction and spatial working memory

The present study: (1) tested the importance of the retrosplenial cortex for learning a specific heading direction and distance and, (2) determined if lesion size could explain apparent inconsistencies in the results of different research groups. Dark agouti rats received either 'complete' cytotoxic retrosplenial cortex lesions or 'standard' lesions, the latter sparing the caudal retrosplenial cortex. Animals were first tested on two versions of a "landmark" task in a water maze. In condition 1 animals could use both heading direction and allocentric position, while in condition 2 only heading direction was effective. In condition 1, animals with complete retrosplenial lesions were impaired by the end of training, their profile of performance being consistent with a failure to use allocentric position information. When the water maze task changed (condition 2) so that allocentric cues became redundant, the animals with complete retrosplenial lesions were able to head in the appropriate direction although they showed longer swim paths. Subsequent testing in the radial-arm maze provided more evidence that retrosplenial lesions can disrupt the use of distal (allocentric) room cues. The impairments seen with retrosplenial lesions were often mild but throughout the study performance of rats with 'complete' lesions was more disrupted than those with 'standard' lesions, who often did not differ from the controls. These findings show that lesion size is a critical factor and may explain why some studies have failed to find comparable deficits after retrosplenial cortex lesions.     

Effects of post-training lesions in the hippocampus and the parietal cortex on idiothetic information processing in the rat

Dead reckoning can be defined as the ability to navigate using idiothetic information based on self-movement cues without using allothetic information such as environmental cues. In the present study, we investigated the effects of hippocampal and parietal cortex lesions on homing behavior using dead reckoning in rats. Experimentally naive Wistar rats were trained with a homing task in which rats were required to take a food pellet from a cup in the arena and to return home with the pellet. After training, rats were divided into a control (CONT) group (n = 16), hippocampal lesioned (HIPP) group (n = 16), and parietal cortex lesioned (PARC) group (n = 16), and rats in the lesioned groups underwent surgery. After surgery, Test 1 (with four cups) and Test 2 (with one cup but the outgoing path was diverted by a barrier) were conducted. The HIPP group showed severe impairment in homing, but the performance of the PARC group did not differ from that of the CONT group. HIPP rats either approached wrong doors or ate the pellet in the arena. Circular statistics showed that homing directions of CONT and PARC rats showed concentration towards home, whereas those of HIPP rats did not. Our results exhibiting HIPP rats' failure in homing agree with many previous studies, but the results obtained from PARC rats were different from previous studies. These results indicate that the intact hippocampus is important for dead reckoning, but the role of the parietal cortex in dead reckoning is still not clear.     

Selective immunolesions of hippocampal cholinergic input fail to impair spatial working memory

The septo-hippocampal cholinergic pathway has traditionally been thought of as essential for spatial memory. Recent studies have demonstrated intact spatial learning following removal of this pathway with an immunotoxin selective for cholinergic neurons. In the present experiment, rats with selective removal of hippocampal cholinergic input were tested in a delayed nonmatching-to-position task in a water version of the radial arm maze. This allowed us to increase and parametrically vary the memory load compared with the standard Morris water maze (by varying the delay between the initial four choices and the final four choices) to determine if this would reveal a deficit in rats with lesions of septo-hippocampal cholinergic projections. Male Long-Evans rats were given injections of 192 lgG-saporin, a selective immunotoxin for cholinergic neurons, into the medial septum/vertical limb of the diagonal band (MS/VDB) to remove cholinergic projections to the hippocampus, or a control surgery. The rats were trained on the radial maze task following surgery. An escape platform was located at the end of each arm of the maze and was removed after an arm was utilized for escape. After initial training, a delay was interposed between the first four trials and the second four trials. Errors during the second four-trial component were scored in two categories: retroactive (reentering an arm chosen before the delay) and proactive (reentering an arm chosen after the delay). Retroactive errors increased as delay increased (from 60 s to 6 h) but were equivalent in control and MS/VDB-lesion groups. Proactive errors did not vary with delay and were also unaffected by the lesion. Radioenzymatic assays for choline acetyltransferase activity in the hippocampus of lesioned rats confirmed a significant loss of cholinergic input from the MS/VDB. These results indicate that normal spatial working memory is possible after substantial loss of septo-hippocampal cholinergic projections. Hippocampus 7:130–136, 1997. © 1997 Wiley-Liss, Inc.