Research report
Facilitation of olfactory recognition by lateral entorhinal cortex lesion in rats

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Abstract

An original olfactory recognition task was developed in order to examine the effect of lateral entorhinal cortex (LEC) lesion on olfactory mnesic processes. The task was based on the spontaneous exploratory behavior of rats toward odor sources. It consisted of a learning phase during which an odor was presented twice and in a recognition test, during which the same odor plus a new one was presented. The time rats spent sniffing the odor sources was measured. Olfactory recognition was identified by a short investigatory duration for the familiar odor as compared to a normal investigatory duration for the new odor during the test. The first three experiments aimed to validate the procedure. Experiment 1 was designed to show the decay of investigatory behavior caused by repeated exposure of the rats to one odor. Experiment 2 showed that normal rats display recognition when a short (5 or 40 min) pre-test delay was used, but not when a long pre-test delay (120 min) was used. Experiment 3 showed that FG7142, a well-known promnesic drug, enhanced the performance of the rats in this test as it allowed recognition at longer pre-test delays. The last experiment aimed at testing the effects of aspirative lesion of the LEC. Therefore, LEC-lesioned and sham-lesioned rats were submitted to variable pre-test delays. The experiment showed that an entorhinal lesion did not produce an impairment, but on the contrary facilitated olfactory recognition, as lesioned rats displayed recognition for delays at which sham-operated rats did not. These results show that LEC lesion apparently prolongs the duration of the olfactory mnesic trace. This effect might result from a modification of the functioning of structures innervated by the LEC. In this regard, it is noteworthy that LEC lesion produced a sprouting of septo-hippocampal fibers in the dentate gyrus of the hippocampus as assessed by acetylcholinesterase staining. Although the functional significance of this regrowth is not fully understood, the possible role of this sprouting should be considered.

Introduction

Numerous procedures have been designed to evaluate time dependent memory. Many of these tests derive from the principle of delayed conditioned response or discrimination based on stored information rather than on immediate stimulation. The tests evaluate the ability of an animal to perform the correct response controlled only by events that are no longer present at the time of the execution of the response. The most documented test for assessing this time dependent memory in a variety of species is delayed-non-matching-to-sample (DNMS) 1, 7, 9, 35, 37, 38. Short-term memory is assessed in this test by the use of a delay between the sample and choice trials and it shows that the number of correct responses decreases with increasing delays. In contrast, little attention has been paid to non-operant memory tasks such as spontaneous recognition tests. These procedures are based upon the spontaneous exploratory behavior of cues: rats display an intense exploratory behavior towards new stimuli that tends to disappear after repeated exposures to this stimulus; the decrease in investigatory behavior reflecting recognition [3]. Short-term memory can be evaluated in these tests by inserting a delay between two presentations of a stimulus. Thus, recognition memory can be defined as the time during which the animal keeps on considering a stimulus to be familiar. In this regard, the social recognition test 20, 12or the object recognition task [13]have proved very useful for investigating short-term memory.

The effect of lesions of neural structures potentially involved in recognition memory have been examined both in DNMS and recognition test procedures. Lesions of the entorhinal cortex have been found to impair short-term retention in object recognition 36, 51or in olfactory DNMS [37]and olfactory discrimination [46]with increasing delays. Despite impairment in short-term memory, several studies have reported preserved learning abilities following entorhinal cortex lesions 37, 40, 47as EC-lesioned rats were able to acquire tasks based on olfactory cues. Importantly, the EC-lesion has produced deficits in olfactory discrimination learning when olfactory cues were presented simultaneously; however, the lesion produced a facilitation of acquisition when the cues were presented in a successive manner [40]. The EC-lesion was also found to facilitate an operant appetitive conditioning task in mice [19]. Taken together, these studies suggest that the EC-lesion may have different effects extending from impairment to facilitation, depending on the task demand. More recently, in our laboratory, lesion of the lateral entorhinal cortex (LEC) was found to produce a facilitation of conditioned odor aversion learning [14]. Conditioned odor aversion is a classical conditioning produced by the association between a tasteless odorized-water and a gastric distress induced by lithium chloride injection. This association is generally strictly obtained when the injection immediately following the consumption of the odorized water; that is when the inter-stimulus interval is short. The LEC-lesion was found to facilitate this acquisition, as LEC-lesions allowed the acquisition of a conditioned odor aversion with longer inter-stimulus intervals. These results led us to hypothesize that the LEC-lesion produces a prolongation of the duration of the mnesic trace. We therefore developed an original olfactory recognition test in order to test this hypothesis. The test is inspired from object recognition tests and social recognition tests in rats 13, 43, 49and in hamsters [25]. It is based on the spontaneous explorative behavior of rats elicited by sources of odors and basically consists of measuring the time the rats spend sniffing these sources. The first experiment was designed to show the decrease in investigatory behavior caused by repeated exposures of the rats to one odor. The second experiment was aimed at documenting the duration of the olfactory mnesic trace in intact animals by using variable pre-test delays. The third experiment explored the possibility to modulate the duration of the mnesic trace with pharmacological agents. Finally, the forth experiment tested whether LEC-lesions would modulate olfactory recognition memory.

Section snippets

Subjects

Male Long–Evans (275–300 g) obtained from CERJ rat farm (Janvier, France) served as subjects for all experiments. They were housed two per cage with food and water available ad libitum. They were maintained in a temperature controlled vivarium on a 12: 12 h light-dark cycle (light at 08:00 h). All testing took place during the light phase of the cycle. One week after their arrival to the laboratory, animals were handled for a short period each day in order to familiarize them to the researcher.

Apparatus

Experiment 1

The purpose of this experiment was to show the decay of investigatory behavior caused by repeated exposure of rats to one odor. Two groups were compared: the first one was given three successive presentations of one odor O1 (group 1), while the other group (group 2) was given two presentations of the odor O1 followed by the presentation of a novel odor (O2). The first two sessions were arbitrarily referred to as the learning phase and the third session as the recognition test.

Experiment 2

The aim of this experiment was to determine the time range over which rats recognize a previously encountered odor, that is to evaluate the duration of a short-term olfactory mnesic trace. To this end, the effects of various pre-test delays were examined. Moreover, as experiment 1 showed that the difference in time spent sniffing the familiar and the novel odor could be a good index of recognition, this test consisted of presenting both odors simultaneously.

Experiment 3

Experiments 1 and 2 indicated that our task is likely to assess the short-term olfactory memory of rats. The aim of experiment 3 was to evaluate whether it could be modulated by a drug known to have promnesic effects. To this end, we tested the effects of FG7142, a partial inverse agonist of the benzodiazepine receptor, the promnesic effects of which have been well documented in various tasks 43, 50, 24. The drug was injected before the experiment, and two pre-test delays were examined: 120 and

Experiment 4

The aim of this experiment was to evaluate the effects of LEC lesion on olfactory recognition and more precisely, to evaluate whether the lesion modifies the duration of the olfactory mnesic trace. Aspirative lesions of the LEC were performed. The effect of five pretest delays (5, 120, 240, 480 min and 24 h) were evaluated on the performance of subgroups of LEC- and sham-lesioned animals.

General discussion

The present study made use of an original olfactory recognition test in order to examine the effects of a lateral EC lesion. The first three experiments established that our test is valid and likely to assess short-term memory; the fourth experiment showed that the EC lesion enhanced the recognition performance of the rats in this test.

Experiment 1 showed that investigation (i.e. sniffing) of an odor source could be used as an index of recognition. As a matter of fact, sniffing duration

Acknowledgements

We are grateful to Karine Herbeaux for her expert technical assistance. This study was supported by a grant from IPSEN industry (Convention No. 96/912/067).

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