The Journal of Neuroscience, February 1995, f5(2): 1162-i 171 Variants of Olfactory Memory and Their Dependencies on the Hippocampal Formation Ursula Sttiubli,’ To-Tam Le,2 and Gary Lynch* ‘Center for Neural Science, New York University, New York, New York 10003 and *Center for the Neurobiology of Learning and Memory, University of California, Irvine, California 92717 Olfactory memory in control rats and in animals with entor- pal pyramidal cells (e.g., Hjorth-Simonsen, 1972; Witter, 1993). hinal cortex lesions was tested in four paradigms: (1) a known Moreover, physiological activity in rat hippocampus becomes correct odor was present in a group of familiar but nonre- synchronized with that in the olfactory bulb and cortex during warded odors, (2) six known correct odors were simulta- odor sampling(Macrides et al., 1982). Theseobservations have neously present in a maze, (3) correct responses required prompted speculationand experimentation concerning the pos- the learning of associations between odors and objects, and sible contributions of the several stagesof the olfactory-hip- (4) six odors, each associated with a choice between two pocampal circuit to the encoding and use of memory. Lesions objects, were presented simultaneously. Control rats had no to the lateral entorhinal cortex, which separatethe hippocampus difficulty with the first problem and avoided repeating se- from its primary source of olfactory input, did not detectably lections in the second; this latter behavior resembles that affect the ability of rats to perform odor discriminations learned reported for spatial mazes but, in the present experiments, prior to surgery although they did disrupt the learning of new was not dependent upon memory for the configuration of discriminations (Staubli et al., 1984, 1986).This result suggested pertinent cues. Control animals varied considerably in their that the olfactory cortex and its connections with telencephalic acquisition of odor-object associations with only a subgroup regionsother than hippocampusare the sitesin which odors are learning every set of pairings. These latter animals also per- recognized and assignedsignificance. In accord with this idea, formed well in the fourth task and, as indicated by post hoc long-term potentiation was found to develop in the piriform analyses, developed complex strategies in dealing with the cortex when patterned stimulation of the lateral olfactory tract problem of serial odor-object pairs. Lesioned animals had was used as a discriminative cue (Roman et al., 1987). no difficulty in selecting correct odors learned prior to sur- Hypotheses about which aspectsof olfactory memory might gery (problem one) but repeated their choices in problem be encoded in the hippocampus have, for the most part, been two. This latter result suggests that hippocampus contrib- borrowed from studies using nonolfactory cues, for example, utes to the transient memory of prior choices for odors as the capacity of rats to rememberchoices made earlier in a spatial it does for prior choices in spatial mazes. Entorhinal rats maze (Olton et al., 1979) and the increasedfiring of field CA1 were able to form odor-object associations (problem three), neuronswhen an improper sequenceof visual cuesoccurs (Ranck, and a subgroup of the animals periodically succeeded in 1973). The extent to which these results hold for odor cues is doing a long series of such choices (problem four), though unknown and indeed there is controversy about their proper with less frequency than controls. These results indicate that interpretation with regard to memory of visual stimuli (seebe- rats use both long-term memory and transient memory in low). Accordingly, the present experiments tested olfactory dealing with olfactory problems and suggest that the second memory acrossseveral behavioral paradigms with the goal of of these is dependent upon a hippocampal process that providing information needed for evaluation and further de- encodes a type of information other than the relationship velopment ofhypotheses regardingpossible encoding operations between cues. by the different stagesof the olfactory-hippocampal circuit. The [Key words: o/factory memory, paired-associates, cross- first problem involved long-term memory and required rats to modal learning, olfactory-hippocampal circuit, hippocam- selecta known correct cue from a group of simultaneously pres- pus, entorhinal cortex, working memory, cognitive map, spa- ent familiar odors. Lesionsof the lateral entorhinal cortex were tial memory] used to test if hippocampus contributes to recognition memory in a situation in which the animal is required to reject several Anatomical and physiological studiesindicate that the olfactory possiblechoices. The secondparadigm examined the possibility system provides substantial and direct input to the hippocam- that rats have a transient or “working” memory which allows pus. The lateral entorhinal cortex, the posterior extension of the them to identify odors respondedto earlier in a test session.It olfactory cortex, densely innervates the granule cells of the den- hasbeen proposedthat memory of this kind, in contrast to long- tate gyms and also projects monosynaptically to the hippocam- term recognition/significance memory, is dependent upon hip- pocampus, and in particular the mossy fiber synapsesbetween the dentate gyrus and field CA3 (Lynch and Staubli, 1991; Lynch Received Apr. 22, 1994; revised July 27, 1994; accepted July 27, 1994. et al., 1991; Staubli, 1992). It was also anticipated that the We thank Gene Go for excellent technical assistance. outcome of the experiment would be relevant to a controversy Correspondence should be addressed to Dr. Ursula Stiubli, New York Uni- versity, Center for Neural Science, 4 Washington Place, New York, NY 10003. concerning the nature of hippocampal contributions to memory Copyright 0 1995 Society for Neuroscience 0270-6474/95/l 5 1162-10$05.00/O involving spatial cues. Olton and coworkers (1979) showedthat The Journal of Neuroscience, February 1995, 15(2) 1163 rats with hippocampal damage make numerous reentries into B the arms of a radial maze during a given test session while control animals typically avoid earlier choices. They interpreted this as showing that the hippocampus encodes a transient mem- ory of prior selections. O’Keefe and others (e.g., O’Keefe and Nadel, 1978; Rasmussen et al., 1989) argue that reentry errors in hippocampal animals are secondary to the disruption of a spatial map, that is, a stable memory of the configuration of spatial cues. Tests with odor cues should be useful for deter- mining if memory of prior choices requires hippocampus in- dependent of any role it plays in encoding configurations of cues. The third paradigm involved the learning of associations be- C A--a vs b A*a vs d tween odors and objects. Youngentob and colleagues (1990, 1991) have recently shown that rats are adept at associating odors with visual cues in an open field environment. We were interested in testing if animals are able to acquire paired-asso- ciates involving odors and intra-maze objects and the extent to which this is dependent upon hippocampus. The hippocampus is the most obvious site for convergence of olfactory and visual information-as noted, it receives a very large input from the posterior extension of olfactory cortex (e.g., Hjorth-Simonsen, B--avsb LJ E--bvse 1972; Witter, 1993) and is known to be responsive to visual B--a vs b cues (e.g., O’Keefe and Nadel, 1978; O’Keefe and Conway, 1978). Figure I. Illustration ofthe various paradigms used in the study. Shape The dense interconnectivity within hippocampus would seem- and proportions of the 12 arm maze are schematized and do not exactly ingly provide a substrate for associating contiguous odors and correspond to the actual maze (dark arm = start alley). A, Long-term objects. However, O’Keefe and Nadel (1978) have made the recognition memory: choose correct odor (A) from a group of five fa- important point that hippocampus is not needed for stimulus- miliar odors; B, olfactory working memory: sample each of six odors only once within a trial, irrespective of odor rotations and delays in- response learning involving intra-maze cues and specific re- terposed after the third choice; C, Cross-modal paired-associates be- sponses, for example, maze learning in the absence of extramaze, tween odors and objects: associate odor A with object a and odor B spatial cues. From this perspective, it might well be the case with object b (phase l), then odor C with object c and odor D with that an extrahippocampal system encodes and uses intra-maze object d (phase 2), and last odor E with object e and odor F with object f (phase 3); D, serial odor-object associations: solve all six odor-object associations of odors with objects. The fourth paradigm com- associations within a single trial. bined problems two and three: rats were required to make a series of odor choices each of which was followed by an object choice. This provided (1) a test of the rat’s ability to integrate through an opening in the hurdle located at the entrance to each alley. transient memory (which odors were already selected) and long- A fan mounted 25 cm above the center platform served to exhaust the odors. term memory (which object is associated with the odor just Experiment 1: long-term memory for sign$cunce of simultaneously selected), and (2) an opportunity to determine if any deficits in present odors. Rats were trained to discriminate between five odors transient memory of choices caused by hippocampal denerva- ejected simultaneously from different arms of the radial maze (see Fig. tion would impair utilization of long-term memory. 1A). Two sets of four odors were used, the members of the first set [celery (X), rose (Y), garlic (Z), basil (W)] were present in each trial and always marked arms with empty food wells; the fifth odor was randomly Materials and Methods selected in each trial from the second odor set [coconut (A), banana (B), Subjects.