Olfactory function in apoE knockout mice

Olfactory function in apoE knockout mice

Behavioural Brain Research 150 (2004) 1–7 Research report Olfactory function in apoE knockout mice Britto P. Nathan a,∗,1 , Johnathan Yost a,1 , Mel...

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Behavioural Brain Research 150 (2004) 1–7

Research report

Olfactory function in apoE knockout mice Britto P. Nathan a,∗,1 , Johnathan Yost a,1 , Melissa T. Litherland a , Robert G. Struble b , Paul V. Switzer a b

a Department of Biological Sciences, Eastern Illinois University, 600 Lincoln Avenue, Charleston, IL 61920, USA Center for Alzheimer’s Disease and Related Disorders, P.O. Box 19628, Southern Illinois School of Medicine, Springfield, IL 62794-9626, USA

Received 10 February 2003; received in revised form 30 April 2003; accepted 24 June 2003

Abstract Apolipoprotein E (apoE), a lipid transporting protein, has been shown to play a vital role in nerve repair and remodeling. Since the olfactory system is in a continuous state of remodeling, the present study tested the hypothesis that apoE is required for normal functioning of the olfactory system. Olfactory behavior of wild-type (WT) and apoE-deficient (apoE KO) mice was assessed by using three standard olfactory tests: (1) the buried food pellet (BFP) test; (2) the odor choice (OC) test; and (3) the odor cued taste avoidance (OCTA) test. ApoE KO mice performed poorly in all the three tests as compared to WT mice, although they learned the tasks at a rate comparable to WT mice. ApoE KO mice had a significantly longer latency to find the buried pellet than WT mice. In the OC experiment, apoE KO mice did not differentiate water from an odorant solution. Furthermore, in the OCTA test the apoE KO mice were significantly less successful than WT mice at avoiding water containing an odorant and a bad tastant. These data demonstrate that apoE deficiency in apoE KO mice leads to a deficit in olfactory function, suggesting an important role for apoE in the olfactory system. © 2003 Elsevier B.V. All rights reserved. Keywords: Apolipoprotein E; apoE knockout; Lipoproteins; Alzheimer’s disease olfaction; Olfactory behavior

1. Introduction ApoE is a 34-kDa protein component of lipoproteins that functions in the redistribution of lipids among cells of various organs [4,26]. It is primarily synthesized in the liver but is also expressed by astrocytes and microglia in significant amounts in the brain [15]. Humans have three major isoforms of apoE (apoE2, apoE3, and apoE4) that are produced by three alleles (ε2, ε3, and ε4) at a single gene locus on chromosome 19 [9,26,51,54]. ApoE genotype has major effects on the development and expression of several neurological diseases. The ε4 allele is a dose-dependent risk factor for Alzheimer’s disease (AD) [16]. Furthermore, AD patients with the ε4 allele usually showed an earlier age of onset and a more rapid progression of the disease [6,7,41]. ApoE occurs in neurofibrillary tangles and amyloid plaques, the two Abbreviations: AD, Alzheimer’s disease; apoE, apolipoprotein E; CNS, central nervous system; WT, wild-type; LRP, low-density lipoprotein receptor related protein; KO, knockout; RAP, receptor associated protein; BFP, buried food pellet; OC, odor choice; OCTA, odor cued taste avoidance ∗ Corresponding author. Tel.: +1-217-581-6891; fax: +1-217-581-7141. E-mail address: [email protected] (B.P. Nathan). 1 They contributed equally to this study. 0166-4328/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0166-4328(03)00219-5

characteristic lesions in AD brain [31,45]. In addition, apoE genotype can effect the development of dementia in several neurological conditions such as dementia pugilistica [22]. ApoE probably plays an important role in neuronal repair and plasticity. ApoE levels increase in both the central and peripheral nervous system following crush injury, and have been proposed to scavenge lipids from the degenerating neurons for recycling to growth cones of sprouting axons [40]. We have reported that apoE is associated with the terminal distribution of the olfactory nerve and apoE is significantly elevated following injury to the olfactory nerve [33,48]. The olfactory system is actually in a continuing state of repair and plasticity and shows very early changes in AD [5,12,28,30,35,46]. In fact, complex olfactory deficits occur early in the course of the disease, and other studies have proposed that olfactory dysfunction is a predictor of the disease [2,10,19,27,29,34]. Furthermore, AD patients show neuropathological changes in components of the brain involved in olfactory processing [36,47]. Recent studies have demonstrated that apoE4 individuals have a significant decline in odor threshold and odor identification, and have delays in processing of olfactory information [2,29,50,52]. Taken together, these data suggest that apoE plays an important role in olfactory function.

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We have previously reported that apoE is expressed at high levels in human and mouse olfactory bulbs, particularly in the olfactory nerve layer and around the glomeruli [48]. In a later study we showed that apoE deficiency in apoE gene-deficient/knockout (apoE KO) mice leads to substantial delay in olfactory nerve repair [32]. In the present study we hypothesized that apoE KO mice should have a deficit in olfactory function. We assessed the performance of apoE KO and wild-type (WT) mice in three commonly used tests of olfaction that evaluate qualitative differences in olfactory behavior. The results from this study revealed that apoE KO mice are defective in olfactory functioning.

back into its home cage. The bedding in the test chamber was changed between trials. The visible pellet test was identical to the BFP test except the pellet was randomly placed on the top of the bedding. Only a randomly selected subset of mice (n = 9 per genotype) from the BFP test was tested in the visible pellet test. 2.3. Odor choice experiment

ApoE KO (C57BL/6J-Apoe tm1Unc ) and wild-type (WT) C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in a sound attenuated room under constant temperature (22 ◦ C), light from 4 a.m. to 4 p.m. and access to water and food ad libitum. Mice were housed individually. Male mice, 2–4-month-old, were used in the experiment. All mice were acclimatized to the sound attenuated environment and 12 h, fluorescent light schedule for a period of 7 days prior to any testing. To avoid circadian variation, all experiments were started between 17:00 and 18:00 h. A single-blind procedure was used in all experiments so that the experimenter was unaware of the genotype of the animals used in the study. Different individuals were used for each experiment below.

The odor choice (OC) test used in this study was similar to that described for rats by Darling and Slotnick [8]. The test chamber consisted of a metal-floored box (20 cm × 30 cm × 12 cm) fitted with two 10 ml syringes each with a stainless steel drinking nozzle (0.7 cm outside diameter). One syringe contained tap water (S+), and the other syringe contained 0.1% isoamyl acetate (ICN Biomedicals Inc.) in tap water (S−). The nozzles of the syringes were connected to a Powerlab (ADI Instruments) by an electrical cable. The metal floor was also connected to the PowerLab via a cable. Thus, a touch circuit was established that was measured by a computer using Chart software (ADI Instruments). This computerized touch circuit precisely measured the latency to first contact, and the time spent in contact with the nozzle. Twenty-four hours before testing, the apoE KO (n = 12) and WT (n = 7) mice were deprived of water. After the deprivation period mice received one trial. The mouse was placed in the center of the testing chamber, facing the wall between the two nozzles, and allowed free access to the nozzles for 5 min. A positive contact score was awarded when a mouse made 0.5 s of sustained oral contact with a nozzle. The aluminum foil floor of the test cage was changed between the testing of each mouse.

2.2. Buried food pellet test

2.4. Odor cued taste avoidance

A modification of the buried food pellet (BFP) test was employed except a Purina mouse chow pellet (PMI Nutrition International, Inc.) was used instead of an exogenously scented pellet [13]. This modification eliminated the need for pre-training the mice to associate an exogenous odor to food. ApoE KO (n = 25) and WT (n = 28) mice were placed on a food-restricted diet (0.2 g chow per mouse/24 h) starting 2 days prior to testing and during the 5-day experimental period. On each of the 5 testing days mice received one trial per day. In each trial a single mouse was placed at random in a test cage (45 cm × 24 cm × 20 cm) to recover a 1-g food pellet. The food pellet was buried approximately 0.5 cm below the surface of a 3-cm deep layer of mouse bedding material. The location of the food pellet was changed daily in a random fashion. The latency to find the food pellet was defined as the time between when the mouse was placed in the cage and when the mouse uncovered the food pellet and grasped it in its forepaws and/or teeth. Animals were allowed to consume the pellet they found and were then returned to their home cage. An animal that did not find the food pellet within 5 min was removed and placed

The odor cued taste avoidance (OCTA) test is a modification of previously published procedures [8,24]. The test chamber used in the OCTA test was similar to that described in the OC test except the box had a single opening through which the stainless steel metal drinking nozzle, fitted with the 10 ml syringe, could be placed at variable distances from the opening. Furthermore, a PVC tube was attached over the nozzle such that the nozzle slid freely in the PVC tube. The computerized touch circuit was similar to that described in the OC test. ApoE KO (n = 9) and WT (n = 8) mice were placed on a 0.4 ml per day water deprivation schedule that started 24 h before testing, and continued throughout the 6-day test schedule. Each trial consisted of two periods: (1) a sampling period of 30 s in which the nozzle end was recessed 1.5 cm into the PVC tube such that the mouse could sniff the odor from the nozzle, but could not make contact with the nozzle, and (2) a drinking period of 60 s in which the nozzle was moved forward so that the mouse could contact the drinking nozzle. A positive contact score was awarded upon any contact with the nozzle. Trials were separated by a 3-min

2. Materials and methods 2.1. Animals

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3. Results 3.1. Buried food pellet recovery A two factor, repeated measures ANOVA with day and mouse strain as factors and latency as the dependent variable was performed to investigate whether WT and apoE KO mice differed in their ability to find buried food pellets (Fig. 1). Both mouse genotypes found the pellets more rapidly over days [F2,102 = 42.0, P < 0.0001], but apoE KO mice were significantly slower at finding buried pellets than WT mice [F1,51 = 31.8, P < 0.0001]. Within genotypes, individuals did not differ consistently in their ability to find the pellets [F51,102 = 1.07, P = 0.37]. The interaction between genotype and day was not significant [F2,102 = 1.28, P = 0.28], indicating that mice from both genotypes improved similarly over time in their ability to find buried food pellets. These results of the BFP test indicate that WT mice were faster at recovering a buried pellet than were apoE KO mice. In contrast, the result from the visible pellet test was dramatically different than that from the BFP test (Fig. 1). In the visible pellet test, the apoE KO mice were actually faster than WT mice at finding the pellet (t = 2.88, P = 0.01, n = 9). 3.2. Odor choice experiment When given a choice between a bottle containing only water and a bottle containing water and 0.1% isoamyl actetate, WT mice strongly preferred the plain water; 7/7 WT individuals (100%) contacted water first (χ2 = 7.0, df = 1, P < 0.01). In contrast, 7/12 (58%) of apoE KO mice contacted water first, which is not significantly different than random (χ2 = 0.33, df = 1, P > 0.50). For those apoE KO individuals that contacted both bottles, latencies until con-

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rest period during which the mouse was placed in a separate cage, with no access to food or water. The aluminum foil flooring of the test chamber was changed between each mouse. At the end of the sixth trial, the mouse was returned to his home cage that contained 0.4 ml water and food ad libitum. Each mouse received 6 trials per day for 6 consecutive days. During days 1–3, mice were trained to drink tap water from the nozzle. During days 4 and 5, animals were offered three trials with S+ solution (water) and three trials with S− solution, 1% vanillin (Acros Organics) plus 0.05% quinine monohydrochloride dihydrate (QHCl, Acros Organics). The order of S+ and S− was randomized within a day such that each individual received the same random order within a day but the sequence of S+ and S− differed among days. On day 6, the randomly ordered six trials continued, however, the S− concentration of vanillin was reduced from 1 to 0.001%, while the concentration of QHCl remained the same.

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Fig. 1. Performance of WT (n = 28) and apoE KO (n = 25) on buried and visible pellet tests. On each of the first 3 consecutive days, the latency for the mice to recover a buried food pellet was recorded. Latency declined significantly across days in both apoE KO and WT mice. However, latency of apoE KO mice was significantly higher than WT on all the 3 days. In visible pellet test latency to find a visible pellet was recorded (WT, n = 9; apoE KO, n = 9). In contrast to buried pellet test, apoE KO mice had significantly shorter latency to find the visible pellet as compared to WT mice.

tact with water were not significantly shorter than those for isoamyl actetate (n = 11; water: 104.2 ± 18.2 s, isoamyl actetate: 105.4 ± 28.9 s; paired t = 0.05; P = 0.96). 3.3. Odor cued taste avoidance In order to investigate whether WT and apoE KO mice differed in their ability to perform the OCTA test (Fig. 2), we used the results from the initial 3-day (S+ only) training period and performed a repeated measures ANOVA with genotype and day as factors. WT and apoE KO mice did not differ in their S+ latencies during the training period [F1,15 = 0.20, P = 0.65]. Latencies decreased over days [F2,30 = 6.76, P = 0.004] and consistent differences existed among individual mice [F15,30 = 2.89, P = 0.006], but no significant interaction existed between genotype and day [F2,30 = 0.88, P = 0.42]. Thus, the latencies for both WT and apoE KO mice decreased at the same rate which suggests that WT and apoE KO mice did not differ in their ability to learn this task. In order to examine whether WT and apoE KO mice responded to the vanillin-cued quinine, within-individual comparisons for WT and apoE KO mice were conducted separately for days 4 and 5 (vanillin 1% + QHCl 0.05%) and day 6 (vanillin 0.001% + QHCl 0.05%) trials (Fig. 3). Wild-type mice had significantly longer latencies to S− than S+ for all 3 days (paired t-tests: day 4, t = 4.77, P = 0.002; day 5, t = 6.86, P = 0.0002; day 6, t = 10.6, P < 0.0001).

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Fig. 2. Performance of WT (n = 8) and apoE KO (n = 9) mice on the odor cued taste avoidance task. In the initial 3-day training period, with only S+ (water) used, there were no significant differences between apoE KO and WT in the daily latency value. However, latency of both WT and apoE KO mice significantly decreased across days. Also, during the 3-day testing period, the latencies to S+ of both WT and apoE KO mice decreased across days. In both the training and testing period, there were no significant differences between apoE KO and WT mice in their latencies to S+. However, apoE KO mice had significantly shorter latency to S− than WT mice. VN: vanillin; QH: quinine monohydrochloride.

ApoE KO mice only had significantly longer latencies on day 5 (paired t-tests: day 4, t = 0.603, P = 0.56; day 5, t = 6.82, P = 0.0001). When vanillin concentrations were lowered on day 6, 7/9 apoE KO mice had longer average latencies to S− than to S+, but the latency differences were not great (Fig. 3) and the trend was non-significant (day 6, t = 1.92, P = 0.09). The significant effect on day 5, however, suggests that apoE KO mice were capable of making the association between the odor and the taste stimuli. Several lines of evidence suggest that WT and apoE KO mice differed in their response to the S+ and S− treatments. First, WT mice had significantly longer mean S− times than apoE KO on all 3 days (day 4: WT = 38.9 ± 4.7, apoE KO 14.0 ± 3.7; t = 5.3, P < 0.0001; day 5: WT = 44.9 ± 4.1, apoE KO = 19.5 ± 4.6; t = 4.79, P = 0.0002; day 6: WT = 38.7 ± 4.6, apoE KO = 9.9 ± 2.0; t = 8.9, P < 0.0001). Second, the mean treatment difference (i.e. S− minus S+) between WT and apoE KO mice indicates that a larger difference existed for WT mice on all 3 test days (day 4, t = 3.47, P = 0.003; day 5, t = 3.62, P = 0.002; day 6, t = 7.6, P < 0.0001), with the largest difference occurring on day 6, the day with the lowest vanillin concentration (Fig. 3). Third, 7/8 WT versus 0/9 apoE KO mice did not drink during at least one of their three S− trials on day

Fig. 3. Mean treatment differences [(S− latency) − (S+ latency)] between WT (n = 8) and apoE KO (n = 9) mice in the odor cued taste avoidance task. Mean treatment differences were significantly lower in the apoE KO mice than WT on all the 3 testing days. Within genotypes, WT had significantly longer latencies to S− vs. S+ on all days. In contrast, for apoE KO, latencies to S− were significantly greater than those to S+ only on day 5. VN: vanillin; QH: quinine monohydrochloride.

6 (Fisher’s exact test, P = 0.0004). Fourth, on the first comparison test day (i.e. day 4), the first trial was S+ and the second trial was S−. Therefore, we could examine these two trials to compare WT and apoE KO in their response to S− at their first exposure. Five of eight WT mice had longer latencies with S− than with S+, while all nine apoE KO mice had longer latencies with S+ (Fisher’s exact test, P = 0.009). This longer latency for WT with S− was unlikely to be due to them satiating their thirst; on the previous day (day 3, with all trials S+), 0/8 WT individuals had longer latencies with their second trial than their first. Finally, WT, but not apoE KO mice, exhibited a significant difference between S+ and S− when tested the first day (i.e. day 4) and when vanillin concentration was lowered (Fig. 3). The results of the OCTA test suggest that both the WT and apoE KO mice are able to associate the quinine taste with the vanillin odor. However, the WT mice are significantly more likely to avoid the vanillin odor than are the apoE KO mice, likely indicating that the WT mice are better able to detect the vanillin odor, at these concentrations, than are the apoE KO mice.

4. Discussion ApoE KO mice performed poorly in all three olfactorybased tasks as compared to WT mice. These data suggest that apoE deficiency leads to deficit in olfactory-mediated behavior. Three alternative explanations, a simple deficit in locomotor ability, appetitive behavior or learning ability, do not explain the pattern of results. First, previous studies of

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locomotor ability have not found any significant differences between apoE KO and WT [11,25,37]. In our study, for the visible pellet test, apoE KO were faster than WT mice in finding the pellet. Also, the latency to drink water in the first 3 days (training period) for apoE KO mice and WT did not significantly differ in our OCTA test. Thus, these data suggest that apoE KO mice have normal locomotor function. Second, appetitive behaviors do not appear to be significantly impaired. Raber et al. have shown that 6-month-old apoE KO mice did not differ from WT in food or water intake [43]. Results from our study show that apoE KO animals were motivated to seek food and water. In the OC and the OCTA tests, the latency to drink water of apoE KO and WT were approximately equal. Also, in the visible pellet test apoE KO mice performed better than WT in finding the pellet. Taken together these data strongly argue against a simple lack of motivation or differences in hunger/thirst as the primary cause for the apparent deficit in apoE KO mice. Third, differences either in task learning ability or in the aversion to the negative stimulus are not a viable explanation for the differences between WT and apoE KO mice. Learning rates in both WT and apoE KO mice were similar. In the BFP test, both genotypes displayed similar learning curves in their ability to find the food pellet. In addition, in the initial 3-day training period of OCTA test with S+ alone (water), the latencies of both apoE KO and WT significantly decreased at the same rate, suggesting that mice from both genotypes did not differ in their ability to learn this task. Furthermore, we have no evidence that the stimulus in the OCTA test (QHCL) was less aversive to the apoE KO individuals than WT mice. If the results of the OCTA test for apoE KO were due to their response to QHCL, one would not expect their performance to decrease as we saw between S− and S+ between days 5 and 6 (Fig. 3). Rather, because the QHCL concentration remained the same and only the odorant (vanillin) concentration had decreased between days 5 and 6, we would have expected either no change in performance (because the QHCL concentration was the same) or an increase in performance due to learning (e.g. see Fig. 3 for WT). Thus, the most parsimonious explanation for the combined results of our study is that apoE KO mice have some deficit in olfactory functioning. The mechanism whereby apoE deficiency leads to olfactory dysfunction in mice is unknown. A series of observations suggest that apoE may play a vital role in the olfactory epithelium and olfactory bulb. Immunohistochemical studies have localized apoE in the Schwann cells of the olfactory nerves, and in a subpopulation of olfactory receptor neurons (ORN) in the human olfactory epithelium [53]. Grehan et al. have demonstrated exceptionally high levels of apoE in the olfactory bulb [20]. ApoE mRNA was particularly prominent in astrocytes in the glomerular layer of the bulb. We have previously demonstrated that apoE is found in the mouse and human olfactory bulbs, specifically in the olfactory nerve layer and around the glomeruli [48]. Furthermore, apoE level in the olfactory bulb increased following olfac-

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tory nerve lesion in mice [33]. Double labeling immunocytochemical studies revealed that both reactive astroglia and microglia contributed to the increase of apoE. Preliminary data from a recent study that examined the effects of apoE deficiency on olfactory nerve recovery post olfactory nerve lesioning revealed that the olfactory nerve regeneration is delayed in apoE KO mice as compared to WT mice [32]. Thus one potential role for apoE in the olfactory system is to support the continuous regeneration of neuronal terminals that occur in the olfactory bulb as a result of the continuous turnover of the receptor neurons in the olfactory epithelium. In addition to the olfactory epithelium and olfactory bulb, apoE is also expressed in brain regions such as the hippocampus and amygdala that are important for higher-level odor tasks [3,20,38,39]. In particular, these regions are important for odor identification, odor memory, and odor-induced behavior [14,34]. Cortical areas that process olfactory information, including the orbital frontal cortex, anterior olfactory nucleus, and the entorhinal cortex also produce apoE [3,20]. Hence, the complex deficits we observe could also be mediated by an apoE deficit in these other areas. To our knowledge this is the first study to document an olfactory dysfunction related to the absence of apoE in mice. Several previous studies have found cognitive deficit in apoE KO mice; however, these findings are controversial [1,17,18,21,23,25,37,42,44,49,55]. Given our result that apoE KO mice have an olfactory deficit, these previous studies that infer a learning or memory deficit have to be carefully interpreted. For example, poor performance of apoE KO mice in a cognitive test where odor-emitting rewards (e.g. food) are used could be misinterpreted as memory deficit, when the effect could be due to a complex olfactory deficit.

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