Ventral hippocampal lesions affect anxiety but not spatial learning

Abstract

Rats with cytotoxic ventral hippocampal lesions which removed approximately 50% of the hippocampus (including dentate gyrus) starting from the temporal pole, displayed a reduction in freezing behaviour following the delivery of an unsignalled footshock in an operant chamber. This was more plausibly a result of reduced susceptibility to fear than a result of a lesion-induced increase in general motor activity. There was no consistent difference between sham and lesioned animals in spontaneous locomotor activity, or locomotion following acute or chronic treatment with amphetamine. In contrast, ventral hippocampal lesioned animals were quicker to pass from the black to the white box during a modified version of the light/dark exploration test, and were quicker to begin eating during tests of hyponeophagia. Furthermore, rats with ventral hippocampal lesions defecated less than their sham counterparts both during open field testing and in extinction sessions following contextual conditioning. In contrast to these clear lesion effects, there were no signs of any spatial learning impairment either in the watermaze or on the elevated T-maze. Taken together these results suggest that the ventral hippocampus may play a role in a brain system (or systems) associated with fear and/or anxiety, and provide further evidence for a distinct specialisation of function along the septotemporal axis of the hippocampus.

Introduction

It is widely believed that the hippocampus (HPC) plays an important role in certain forms of learning and memory [15], [30], [56]. Humans with HPC damage are profoundly amnesic [50], [66]. Furthermore, rodents with HPC lesions display robust and reliable memory deficits on maze tasks such as the Morris watermaze, leading to the suggestion that, in rodents at least, the HPC is selectively involved in spatial learning and memory [40], [42], [44].

Recent studies have shown, however, that whereas selective cytotoxic lesions of the dorsal HPC impair spatial performance on tasks such as the watermaze or rewarded alternation on the elevated T-maze, selective ventral HPC lesions are without effect on these tasks [2], [25], [43]. These results suggest, therefore, that it is the dorsal component of the rodent HPC that supports spatial learning.

In contrast, less is known about the functions performed by the ventral HPC. In a previous study, selective ventral HPC lesions were found to mimic the effects of complete HPC lesions and reduce freezing behaviour in response to both an explicit conditioned stimulus (CS; a tone) which had been paired with a mild footshock, and to the context in which the conditioning event took place [49]. Indeed, the ventral lesioned animals (like rats with complete HPC lesions) also showed reduced levels of freezing immediately after the delivery of the very first footshock during the conditioning phase. In contrast, dorsal HPC lesions did not affect freezing behaviour.

Questions remain, however, regarding the exact nature of the freezing deficit observed in the ventral lesioned animals. One possible explanation is that the deficit in freezing behaviour simply reflects a general increase in locomotor activity in lesioned animals [16], [37]. It is well established that complete HPC lesions produce a marked increase in both spontaneous, and amphetamine-induced, locomotor activity [2], [14], [58], [62], [64]. Against this, however, the same ventral HPC lesion that was found to reduce freezing behaviour, had no effect on either spontaneous or amphetamine-induced locomotor activity as measured in photocell activity cages [2], even though it did result in an increase in swim speed during acquisition of the Morris watermaze [2].

An alternative explanation is that ventral HPC lesioned animals display increased levels of activity but only in anxiogenic situations of mild stress (e.g. after a footshock or during aversive watermaze training). Indeed, it has previously been suggested that the HPC (and the related structures which make up the septohippocampal system) may play an important role in the processing of, and/or response to anxiogenic stimuli [19], [20]. Rats with complete HPC lesions show reduced levels of anxiety in a number of different experimental situations (e.g. [12]). It is possible, therefore, that specifically ventral HPC cell loss impairs the processing of, or response to, fearful or anxiogenic stimuli. This is consistent with the anatomy of the ventral HPC. Projections to and from this region are intimately associated with subcortical structures, including areas that contribute to the hypothalamic–pituitary–adrenal (HPA) axis, and are therefore, likely to be involved in the mechanisms of stress and anxiety [23], [24], [27].

The central aim of the present study was to determine whether the previous effects that we have observed with selective ventral HPC lesions (e.g. the reduced freezing response, increased swim speed in the watermaze) were due to a general increase in locomotor activity levels per se, or whether they reflected a role for the ventral HPC in brain systems associated with the response to stressful and/or anxiogenic stimuli. Sham and ventral HPC (including dentate gyrus) lesioned animals were therefore tested on a range of behavioural paradigms assessing responses to fear and/or anxiety-inducing stimuli in both conditioned and unconditioned paradigms. The freezing response to mild footshock was examined using a contextual fear conditioning paradigm with no explicit conditioned stimulus (CS). Freezing was measured both immediately after a series of unsignalled footshocks and also in subsequent extinction sessions during which the animals were returned to the context in which they had previously received footshock. In addition to measuring freezing, levels of defecation were also recorded.

The response to anxiogenic stimuli was also assessed in several different unconditioned paradigms. Sham and ventral lesioned animals were compared on a test of hyponeophagia during which the latency to begin eating in a novel (and hence potentially anxiogenic), test situation was recorded. Under such circumstances, rats with complete HPC lesions have been found to eat much more readily than controls, suggesting that the lesioned animals were less anxious [1], [12], [28], [33], [38]. We wished to determine whether damage confined to the ventral portion alone could reduce hyponeophagia. In addition, latency to eat a novel foodstuff in a familiar environment was also examined. In this situation it is the novelty of the foodstuff, rather than the experimental context, that suppresses feeding.

A quite different measure of susceptibility to fear or anxiety can be derived from the light/dark exploration test. This has been widely used to assess anxiety levels in rodents, and is sensitive to anxiolytic drugs but not to other psychoactive drugs [9], [10]. As the bright, white box is potentially more anxiogenic than the darker box, the latency to cross into the white compartment is regarded as a measure of anxiety.

We additionally assessed anxiety levels in an open field test. The open field consists of a large white open arena which is brightly illuminated from above. Measures of locomotor activity, rearing, thigmotaxis and defecation can provide an index of anxiety levels in this apparatus. Open field behaviour has been widely studied with conventional HPC lesioned rats (see [20] for a review).

General locomotor activity was assessed in photocell activity cages. Both spontaneous (at various time points during the course of these experiments) and amphetamine-induced locomotor activity were measured. Previous studies have shown that although ventral HPC lesions increase activity both after a mild footshock, and in terms of increased swim speeds during watermaze training, they have no effect on either spontaneous or amphetamine-induced locomotor activity as measured in photocell activity cages [2].

It is well established that locomotor activity is associated with dopamine release in the nucleus accumbens [52]. Furthermore, both mild stressors (e.g. footshock) and a single, acute injection of amphetamine are known to induce dopamine release in the accumbens shell region [13], [65]. It is not immediately clear therefore why ventral HPC lesioned animals might display increased activity levels after the delivery of a mild footshock, but not after an injection of amphetamine. One possible explanation is that, unlike the naturally-elicited dopamine release that follows an event such as mild footshock, the dopamine release following a single injection of amphetamine is not impulse dependent [26]. It may be that impulse-dependent dopamine release is more sensitive to ventral HPC cell loss. To test this hypothesis, we examined the effects of ventral HPC lesions on the development of sensitisation to repeated administration of systemic amphetamine. Sensitisation results in accumbens dopamine release becoming impulse-dependent [61]. Consequently, the locomotor response to repeated doses of amphetamine was examined. If ventral HPC cell loss potentiates impulse-dependent, but not impulse-independent, accumbens dopamine release then one would predict that there would be no difference in the response to a single injection of amphetamine between sham and lesioned animals, but that the lesioned animals should demonstrate a bigger increase in activity following repeated exposure to the drug.

Finally spatial learning was assessed in two separate paradigms. The aim of these tests was to determine whether or not the dorsal HPC was functioning normally in the ventral lesioned animals. First, spatial reference memory was assessed in the Morris watermaze and second, spatial working memory was examined using a non-matching to position (rewarded alternation) task on the elevated T-maze. On the basis of previous work, both in this laboratory [2] and elsewhere (e.g. [43]), we predicted that our ventral HPC lesions should be without effect on these spatial learning tasks. Clear lesion effects in tests of anxiety, coupled with a lack of any such effects in tests of spatial learning and memory, would strengthen the case for regional specialisation of functions within the HPC.