This is the accepted version of the journal article: Rojic-Becker, Divka; Portero Tresserra, Marta; Martí Nicolovius, Margarita; [et al.]. «Effects of caloric restriction on monoaminergic neurotransmission, peripheral hormones, and olfactory memory in aged rats». Behavioural Brain Research, Vol. 409 (2021), p. 113328. 9 pàg. DOI 10.1016/j.bbr.2021.113328 This version is available at https://ddd.uab.cat/record/257988 under the terms of the license Manuscript File Click here to view linked References 1 Effects of caloric restriction on monoaminergic neurotransmission, 2 peripheral hormones, and olfactory memory in aged rats 3 Divka Rojic-Becker#, Marta Portero-Tresserra#, Margarita Martí-Nicolovius, Anna 4 Vale-Martínez, Gemma Guillazo-Blanch* 5 Affiliation of all the authors: Departament de Psicobiologia i Metodologia de les 6 Ciències de la Salut, Institut de Neurociències, Universitat Autònoma de Barcelona, 7 Barcelona (Spain). 8 Short title: Caloric restriction and olfactory memory in aged rats 9 Number of text pages: 32 10 Number of Figures: 8 11 Number of Tables: 0 # 12 These authors contributed equally to this work. 13 14 * Corresponding autor: Dr Gemma Guillazo-Blanch, Departament de Psicobiologia i 15 Metodologia de les Ciències de la Salut, Institut de Neurociències, Universitat 16 Autònoma de Barcelona, Barcelona (Spain). 17 Email: gemma.guillazo@uab.cat Telephone: 34 93 581 1173 18 1 19 Abstract 20 Aging is associated with a reduced ability to identify and discriminate scents, and 21 olfactory dysfunction has been linked to preclinical stages of neurodegenerative 22 diseases in humans. Moreover, emerging evidence suggests that smell-driven behaviors 23 are regulated by hormones like insulin or leptin, and by metabolic parameters like 24 glucose, which in turn may influence monoaminergic neurotransmission in brain areas 25 related to cognition. Several studies have suggested that dietary interventions like 26 caloric restriction (CR) can mitigate the age-induced decline in memory by modifying 27 metabolic parameters and brain monoaminergic levels. The present study explored the 28 effects of CR on age-dependent olfactory memory deficits, as well as their relationship 29 with peripheral leptin, insulin and glucose levels, and brain monoamines. To this end, 30 aged rats (24-months-old) fed on a CR diet or with ad libitum access to food, and adult 31 rats (3-4 months), were trained in an odor discrimination task (ODT). The peripheral 32 plasma levels of insulin, leptin, and glucose, and of monoamines and 33 metabolites/precursors in brain areas related to olfactory learning and memory 34 processes, such as the striatum and frontal cortex (FC), were determined. The data 35 obtained indicated that CR attenuated the age-dependent decline in olfactory sensitivity 36 in old animals fed ad libitum, which was correlated with the performance in ODT 37 retention trial, as well as with leptin plasma levels. CR enhanced dopamine levels in the 38 striatum, while it attenuated the age-related decline in serotonin levels in the striatum 39 and FC. Such findings support a positive effect of CR on age-dependent olfactory 40 sensitivity decline and dysfunctions in brain monoamine levels. 41 42 Keywords: Aging, striatum, frontal cortex, leptin, IGF-1, odor-reward association 2 43 1. Introduction 44 Olfactory dysfunction is a common feature in older individuals, its prevalence and 45 severity increasing substantially with age [1]. Olfactory impairment is frequently 46 associated with some neurodegenerative diseases and it is regarded as a clinical 47 correlate in both Alzheimer’s (AD) and Parkinson’s disease (PD) [2–6]. Behavioral 48 studies of olfactory function in both old rodents and aged humans, have indicated a 49 decreased sensitivity and ability in terms of discriminating [7–9] and associating odors 50 [10–13], as well as a disruption in performing odor-based learning tasks [14–16]. 51 Multiple factors may contribute to such age-related functional impairment. For example, 52 olfactory dysfunction and memory loss have been related to anatomical and biochemical 53 alterations within the olfactory system [3,8], and also in specific brain networks in the 54 frontal cortex (FC) [17–19] and striatum [20,21]. As the olfactory system (considered 55 an indicator of the integrity of the aging brain) influences physical well-being and daily- 56 life activities, preventing age-related functional olfactory decline is relevant to promote 57 healthier aging. 58 A major tool available to prevent some negative consequences of aging is caloric 59 restriction (CR) which consists of restricting food intake by organisms without causing 60 malnutrition [22]. CR is the only dietary intervention shown to significantly increase 61 longevity and improve health during the aging process in several species [23–26]. Such 62 dietary interventions may regulate the neurotransmitter and neuromodulatory systems 63 affected by aging [27,28]. In this regard, CR may also regulate peripheral hormones 64 [29–31], such as insulin or leptin, and metabolic parameters like glucose [32,33], as 65 well as influence monoaminergic neurotransmission in brain areas related to olfactory- 66 driven behaviors in rodents [33–38]. Nevertheless, while the beneficial effects of CR on 67 life expectancy and health are well known [23,39,40], it is less clear whether CR slows 3 68 age-dependent cognitive decline [36,41–43]. Hence, further studies are necessary to 69 investigate whether CR can attenuate age-related olfactory memory decline. 70 Therefore, the objective of the current study was to explore whether a life-long CR diet 71 was able to prevent the age-dependent deficits in an odor-association task [44], the odor 72 discrimination task (ODT) performed by aged Wistar rats. In these animals, we also 73 analyzed IGF-1 as this serves as a marker of cognitive deterioration in normal aging 74 [45]. In the aged and adult animals tested, we examined plasma levels of insulin, leptin 75 and glucose, and the levels of the monoamines noradrenaline (NA), dopamine (DA), 76 and serotonin (5-HT) in the striatum and FC, as well as those of their metabolites and 77 precursors 3,4-Dihydroxyphenylacetic (DOPAC), 3-Methoxytyramine (3MT), and 5- 78 Hydroxyindoleacetic acid (5-HIAA). Accordingly, in the light of this study, life-long 79 CR diets can attenuate age-related deterioration in olfactory sensitivity, probably by 80 reducing high levels of leptin, and improve dysfunctions in brain monoamines. More 81 studies are needed to see if these effects can help reduce olfactory memory deficits. 82 83 2. Materials and Methods 84 2.1. Subjects 85 In this experiment, 45 naive male Wistar rats from our laboratory breeding stock 86 (Prolabor, Charles River Laboratories, Arbresle, France) were paired-housed in 50 x 22 87 x 14 cm transparent plastic cages with sawdust bedding, and they were maintained in a 88 controlled environment of 60-70% humidity, at a temperature of 20 to 22 ºC and on a 89 12-hour light-dark cycle. Rats were fed on standard laboratory food (dry pellets 90 obtained from Harlan Laboratories Inc., Madison, USA), containing wheat, maize, 91 maize gluten, calcium carbonate, soybean oil, mineral dicalcium phosphate, corn gluten 4 92 feed, sodium chloride, magnesium oxide and additives (per kg): vitamin A (E672) 6000 93 I.U, vitamin D3 (E671) 600 I.U, Fe (E1) 50 mg, Mn (E5) 44 mg, Zn (E6) 31 mg, Cu 94 (E4) 7mg, I (E2) 6.2mg, Co (3b302) 0.5mg and with the following analytical 95 constituents: Moisture 12%, Crude protein 14.5%, Crude oils and fats 4%, Crude fiber 96 4.5%, Crude ash 4.7%. Food was distributed inside the cage to avoid competition 97 among individuals. Animals were assigned to three experimental groups: a CR group (n 98 = 19; age = 24-27 months; weight = 469.38 g, SEM = 6.85 g) that followed a 25-30% 99 reduction in food intake from 4 months of age; an Ad libitum group (n = 11; age = 24-27 100 months; weight = 641.46 g, SEM = 29.72 g) composed by old animals that had 101 unrestricted access to food and water; and the Adult group (n =15; age =3-4 months; 102 weight = 433.66 g, SEM = 9.54 g). The amount of food consumed AL was 103 approximately 25 grams/day by adult rats and 32 grams/day by old rats. Food was 104 available once daily at 8 am. All groups had free access to water. 105 All the behavioral experiments were performed during the light cycle (lights on at 7 106 am). Moreover, all the procedures were performed following the EU Directive on the 107 protection of animals used for experimental and other scientific purposes (2010/63/EU), 108 and with the authorization of the Generalitat de Catalunya (DOGC 2450 7/8/1997, 109 DARP number 3866). 110 2.2. Behavioral studies 111 2.2.1. Odor Discrimination Task 112 The ODT requires the discrimination of three odors, one of which is associated with an 113 edible reward [44]. The odor-reward associative task procedure is based on foraging for 114 palatable food in a single, four-trial session, without the element of stress and fear [46] 115 and it has been described in detail elsewhere [47]. The test was performed in a black 116 square box (60 x 60 x 40 cm) that contained three yellow sponges (8.5 x 6.5 x 5.5 cm) 5 117 placed in a glass slide staining rack, with a 3 cm diameter and 2 cm deep hole in the 118 center. The food reinforcement placed in the hole of the sponge was chocolate rice 119 crispy breakfast food (Kellogg’s, Spain), such that the rat had to put its head into the 120 sponge (nose poke) to obtain the reward. Each sponge was infused with an odor, vanilla 121 (0.3 ml), orange (0.6 ml) and anise (0.2 ml: Vahiné, Ducros S.A. Sabadell, Spain). In 122 each trial, some cereal pieces were crumbled and scattered all over the box floor to 123 avoid the animal being guided by the chocolate rice odor and ensure that was guided by 124 the odor of the sponge. The odors used in the experiment were reinforced in a balanced 125 way between the different experimental groups throughout the study. All groups were 126 food deprived (12 g/day, to maintain body weight at 85% of freely feeding weight) 5 127 days prior to the ODT procedure, to ensure motivation to perform the task. The ODT 128 procedure was conducted after the three habituation sessions over three consecutive 129 days in which the animals were given free access to the food reinforcement in a plastic 130 bottomed cage (50 x 22 x 14 cm) and the time to eat 10 pieces of cereal was recorded. 131 After consuming the cereal, they were placed in the training box without the 132 reinforcement and allowed to explore it for 15 minutes to get habituated to the training- 133 test conditions. 134 One day after the last habituation session, ODT acquisition was assessed in a four-trial 135 session. Reinforcement (unconditioned stimulus) was associated with the same odor 136 (conditioned stimulus) in each of the trials for each rat, and the target odor was assigned 137 randomly to the different animals. At the start of the trial, the rats were placed in the 138 training box facing the corner without the sponge. An inter-trial interval of 1 min was 139 maintained, and there was a 3 min limit for the rats to find and consume the 140 reinforcement. After the rats had found and eaten the cereal, they remained in the box 141 for a few seconds before being removed and placed in the intertrial cage. The rats were 6 142 tested for retention (first trial) and relearning (three trials) 72 hours after acquisition, 143 using the same procedure as that used for acquisition except the first test trial was not 144 reinforced to directly measure long-term olfactory memory. 145 The learning and memory variables scored were the latency to make a correct response 146 (nose-poking into the target sponge) and the combination of the two kinds of errors: 147 omission error (sniffing the rewarded sponge with no subsequent nose-poking) and 148 commission error (nose-poking into a non-rewarding sponge [48]. The sessions were 149 recorded with a video camera fixed above the apparatus using the SMART video 150 tracking system (SMART v3.0, Panlab, Spain). 151 2.2.2. Buried Food Test 152 An additional olfactory test was carried out 24 hours after finishing the ODT test. The 153 buried food test measures how quickly a rat can find a small piece of familiar palatable 154 food, such as a cookie, to discard deficits in olfactory abilities. The test was carried out 155 as described previously [47,48], having fed the rats with a butter-flavored cookie 24 156 hours before (Brambly Hedge, Denmark) to habituate them to the new flavor. The 157 following day a clean plastic cage was set-up and filled with 2 cm of sawdust, and a 158 piece of the cookie was buried in one of its corners. The rats were then placed in the 159 cage, and the latency to find the buried cookie and start eating it was measured. 160 2.3. Biochemical procedures 161 2.3.1. Blood collection and sample acquisition 162 After completing the behavioral tests, the animal’s food was removed the day before 163 sacrifice [36] in order to minimize the possible differences due to the amount of food 164 consumed immediately before sacrifice [49] or to lipemia [50], in a subset of old and 165 adult rats (Old Ad Libitum, n= 6; Old CR, n= 10; Adult, n= 8). Blood samples were 7 166 collected in Heparin tubs (Sodium Heparin, 5000 USP/mL: Chiesi Spain, SA, Spain), 167 centrifuged for 15 min at 4000 rpm and 4 ºC (RCF 2361 xg), and the supernatant was 168 recovered and frozen. Subsequently, the rat’s brain was removed rapidly, and the whole 169 striatum and the whole FC were dissected out on ice. Both these brain regions were 170 weighed, frozen and stored individually at -80 ºC in 1 ml Eppendorf tubes, to then be 171 analyzed by HPLC-ED and in Western Blots. 172 2.3.2. Blood plasma analysis 173 Insulin, leptin and IGF-1 levels were measured by competitive ELISA on an EMS 174 Reader MF V.2.9-0 using the following kit for each protein: Rat Insulin ELISA 175 (Mercodia AB, Sweden; Quantikine®), Mouse/Rat leptin ELISA (R&D Systems, Inc. 176 USA; Quantikine®); and Mouse/Rat IGF-1 ELISA (R&D Systems, Inc. USA). Glucose 177 levels were detected using the Hexokinase method. All of the tests described above 178 were carried out using the Olympus AU400 (Germany) analyzer and the reactive OSR 179 (Olympus System Reagent, Beckman Coulter®, Ireland). 180 2.3.2. Monoamine quantification by High Performance Liquid chromatography 181 (HPLC-ED) 182 Brain samples were homogenized in buffer (perchloric acid 60% w/w 0.25M, sodium 183 metabisulphite 100 µM, EDTA Na2 2H2O 250 µM) at a 9/1 ratio (p/v; ml/mg). The 184 tissue was rapidly disrupted in a polytron homogenizer, the homogenate was 185 centrifuged (10 min at 4000 rpm, 4 ºC: RCF 1485 xg) and the supernatant filtered from 186 the purified supernatant (50 µL aliquots) were analyzed by HPLC on a reverse phase 187 column (Cromolith Performance 4.6 mm internal diameter x 10 cm length) coupled to a 188 pre-column (4.6 mm x 5 cm). The mobile phase consisted of 0.1M citric acid, 0.05M 189 EDTA, 1.2M SOS, 10% acetonitrile (v/v) adjusted to pH 2.75 with tetraethylammonium 8 190 (TEA). The column was eluted at a flow rate of 0.8 mL/min and the HPLC apparatus 191 (LaChrom Elite) was coupled to an electrochemical detector (ED: ESA Coulochem 192 5100A) with an ESA analytical dual electrode cell 5011A (detection potential for 193 electrodes 1 and 2 was set at 70.05 and +0.4 V). NA, DOPAC, DA, 3MT, 5-HIAA and 194 5-HT levels were detected electrochemically, and the current produced was monitored 195 by an interface connected to a computer. The concentrations (ng/g) of the compounds in 196 a given sample were calculated by interpolating the peak height into a standard 197 calibration curve using the EZChrom Elite Software. 198 2.3.4. Western Blotting 199 The striatum and the FC tissue were collected separately in lysis buffer (0.15M NaCl, 200 1% Triton X-100, 10% Glycerol, 0.001M EDTA, 0.05M Tris pH 7.4) with phosphatase 201 and protease inhibitor tablets (Roche, France). The lysates were homogenized and 202 centrifuged, and the total amount of protein was quantified using a PierceTM BCA assay 203 kit (Pierce Chemical Co., Thermo Fisher Scientific, USA). Equal amounts of protein 204 (30 μg/well) were resolved on 8–12% polyacrylamide SDS-PAGE gels, then transferred 205 to nitrocellulose membranes (Whatman, Dassel, Germany) for 90 min at 120 V on ice to 206 avoid possible overheating, and for 70 min at 100 V. The membranes were then blocked 207 for 1 hour at room temperature in 5 % non-fat milk in TBS-T buffer (75 mM NaCl, 1.5 208 mM KCl, 12.4 mM Tris-HCl pH 7.4 and 0.1% Tween-20), and they were subsequently 209 incubated overnight with the primary antibodies (Sigma, USA) at 4 °C diluted in 5% 210 (w/v) bovine serum albumin (BSA): mouse anti-tyrosine hydroxylase (TH, 1:1000, 211 T2928) and mouse anti-tryptophan hydroxylase (TPH, 1:500, T-06781). A mouse anti- 212 GAPDH antibody (1:10,000G8795) was also used to control for loading control and to 213 normalize the levels of protein detected. The secondary antibody was anti-mouse-HRP 214 (1:2,000: Dako Denmark, Glostrup, Denmark), which was visualized with enhanced 9 215 chemiluminescence. The chemiluminescence signals of the bands obtained were all 216 within the linear range of the imaging system and were not saturated (ChemiDoc XRS+ 217 System, Bio-Rad Laboratories). Densitometry and quantification were carried out using 218 a ChemiDoc MP Imaging System, Image Lab program (Bio-Rad) and Microsoft Excel 219 was used to determine the levels of the proteins, and the monoamine precursors TPH 220 and TH. 221 2.3.5 Statistical analysis 222 The behavioral and biochemical data were analyzed using the Statistical Package for the 223 Social Sciences (SPSS) v20 software and plotted as the mean ± SEM. A test of 224 normality and of the homoscedasticity of variances (Levene’s test) were applied to each 225 data set before all the variables were compared among the three experimental groups. 226 To analyze the ODT acquisition, retention and relearning trials, a repeated measures 227 ANOVA was carried out followed by post hoc contrast tests when necessary, and 228 multiple comparisons were performed with the Bonferroni correction. A one-way 229 analysis of variance (ANOVA) was carried out to determine the between-group 230 differences in the retention trial, olfactory test, and for the monoamine, glucose and 231 hormone levels. Moreover, Spearman Rank correlations were assessed to examine the 232 relationship between behavioral performance and blood plasma parameters. A p-value 233 of 5% or lower was considered to be statistically significant. 234 235 3. Results 236 3.1. Behavioral data 237 3.1.1. Odor discrimination task 10 238 In the ODT habituation, no significant between-group differences were detected in the 239 latency to eat the ten pieces of cereal during the first (F(2,42) = 2.269, p = 0.116) and the 240 second (F(2,42) = 1.915, p = 0.160) habituation sessions, but significant between-group 241 differences were detected in the third session (F(2,42) = 3.877, p = 0.028), specifically 242 between the CR and Adult groups (p=0.027), indicating that CR rats took longer to eat 243 the cereal. An ANOVA of the latencies to make the correct response in the acquisition, 244 retention and relearning ODT trials failed to identify significant Group factor (F(2,42) = 245 2.184, p = 0.125) or Group x Trial (F(4,84) = 1.552, p = 0.195) interactions (Fig. 1A) . 246 However, the Trial factor was significantly affected (F(2,84) = 4.759, p = 0.011), 247 indicating that the rats’ performance generally differed across trials, with shorter 248 latencies for CR and adult rats in the retention trials. Interestingly, the latency scores of 249 a high percentage of CR aged rats were comparable those of Adult animals in the ODT 250 retention trial (Fig. 2), while some extended beyond that range, indicating a dispersion 251 effect of the measure that was more noticeable in the Ad Libitum group. Hence, CR 252 might reduce the behavioral variability between aged animals, making their 253 performance more like that of control animals. The statistical dispersion could explain 254 the lack of significant between-group differences in the retention session, as observed in 255 other studies analyzing rats’ performance in probe trials of a spatial learning task 256 [51,52]. In fact, when an ANOVA was applied to the retention trial, significant 257 between-group differences were observed (F(2,44) = 4.616, p= 0.015). Specifically, the 258 Ad libitum group had a longer latency than the Adult group (p = 0.014), whose 259 performance did not differ from that of the CR group (p = 0.179). An ANOVA analysis 260 of the mean number of total errors (Fig. 1B) failed to detect significant differences for 261 the three factors: Group (F(2,42) = 1322, p = 0.278), Trial (F(2,84) = 2.856, p = 0.057), and 262 Group x Trial (F(4,84) = 0.970, p = 0.428). 11 263 3.1.2. Buried Food Test 264 265 An ANOVA of the latencies to find the buried cookie in the olfactory test (Fig. 3) 266 indicated between-group differences (F(2,42) = 10.866, p < 0.001), and a contrast analysis 267 showed that the Ad Libitum group performed significantly worse than both the Adult (p 268 < 0.001) and CR (p = 0.001) rats. A significant positive relationship was evident 269 through the Spearman correlation between the latency to find the reinforcement during 270 the ODT retention trial and the latency to find the buried cookie (r = 0.445, p = 0.001: 271 Fig. 4), suggesting that the rats that took the longest to find the buried cookie were those 272 with the highest latencies in the ODT retention test. 273 3.2. Biochemical data 274 3.2.1. Blood plasma analysis 275 The glucose levels obtained differed significantly between the groups (F(2,21) = 19.215, p 276 < 0.001: Fig. 5A) and a contrast analysis showed that Adult rats had lower glucose 277 levels than CR (p< 0.001) and Ad Libitum rats (p= 0.002). In terms of leptin (Fig. 5B), 278 ANOVA also demonstrated between-group differences (F(2,21) = 21.536, p < 0.001), 279 with the contrast analysis indicating there was more leptin in the Ad Libitum rats than in 280 the Adult (p < 0.001) and CR (p < 0.001) animals. Between-group differences in plasma 281 IGF-1 were also detected (F(2,21) = 8.630, p = 0.002: Fig. 5D) and specifically, there 282 were higher plasma IGF-1 concentrations in Adult rats than in CR (p = 0.005) and Ad 283 Libitum (p = 0.005) rats. However, and somewhat unexpectedly, no between-group 284 differences in the insulin levels were evident (F(2,21) = 0.153, p = 0.859: Fig. 5C). 285 Finally, the possible relationship between behavioral performance and blood plasma 286 parameters was evaluated by analyzing the blood parameters that show significant 287 between-group differences. Accordingly, only leptin levels showed a significant 12 288 correlation (r = 0.518, p < 0.005) with the latency to find the buried cookie in the 289 olfactory test, with more leptin associated with a worse performance in the test (Fig. 6). 290 3.2.2. Monoamine levels 291 3.2.2.1. Striatum 292 Regarding the concentrations of monoamines, precursors and metabolites in the striatum 293 (Fig. 7A), ANOVA showed significant between-group differences in DOPAC (F(2,21) = 294 19.764, p < 0.001), DA (F(2,19) = 65.948, p < 0.001), 3MT (F(2,21) = 30.595, p < 0.001) 295 and 5-HT (F(2,21) = 4.970, p = 0.017). Contrast analyses indicated there were higher 296 concentrations of DOPAC (p = 0.007, p = 0.002), DA (p < 0.001) and 3MT (p < 0.001) 297 in the CR group than in the Ad Libitum and Adult groups. Spearman correlation 298 demonstrated a significant relation between the levels of DA and the latency to find the 299 reinforcement during the ODT acquisition (r = 0.385, p = 0.039) and the latency to find 300 the buried cookie in the olfactory test (r = 0,504, p = 0.008), but not with the ODT 301 retention. In terms of 5HT, there were higher levels in the CR group than in Ad Libitum 302 animals (p < 0.026), while no between-group differences were found in NA and 5- 303 HIAA in striatum. 304 3.2.2.2. Frontal cortex 305 In the FC (Fig. 7B), ANOVA revealed between-group differences in DOPAC (F(2,21) = 306 10.909, p = 0.001), DA (F(2,21) = 27.537, p < 0.001) and 5-HT levels (F(2, 19) = 3.931, p = 307 0.037). The post hoc analysis indicated that there were higher levels of DOPAC (p = 308 0.004, p = 0.0014) and DA (p = 0.035, p < 0.001) in the Adult group than in the Ad 309 Libitum and CR groups. By contrast, there were higher 5HT levels in the CR group than 310 in Ad Libitum animals (p = 0.038). No between-group differences were found for NA 311 and 5-HIAA. 13 312 3.2.3. Monoamine precursor levels 313 There were significant between-group differences in monoamine precursors levels when 314 assessed by ANOVA, particularly of the TH levels in both the striatum (F(2,12) = 32.793, 315 p < 0.001) and FC (F(2,14) = 4.107, p = 0.040: Fig. 8). In the striatum, more TH was 316 detected in the CR than in the Adult (p < 0.001) and Ad Libitum (p < 0.001) rats, and 317 there was a tendency towards significantly higher TH levels in the FC of the Adult rats 318 than in the CR animals (p = 0.051). Regarding the TPH, no between-group differences 319 were evident in the FC (F(2,10) = 0.917, p = 0.431). 320 321 4. Discussion 322 The data obtained here indicate that a CR dietary intervention in rats from 4 months of 323 age attenuates age-related decline in olfactory sensitivity, observed in the performance 324 of the Ad Libitum old rats in the buried food test. Although the CR diet did not improve 325 ODT acquisition, when latencies were assessed in the 72-hour ODT retention trial, the 326 Ad Libitum group performed worse, which could be interpreted as if diet affected 327 somehow odor sensitivity and/or memory encoding, since the ODT test cannot measure 328 both processes separately. However, as such deficit was not observed in the number of 329 errors, it cannot be discarded that differences in latencies could be explained by other 330 factors, such as locomotor activity. Moreover, our results also confirm that the 331 nutritional state influences odor sensitivity [53,54] given that higher levels of leptin 332 were related to a worse performance in the buried food test, but not in the ODT. All 333 animals were food-deprived 5 days before the ODT procedure, so the results can hardly 334 be attributed to a differential acute effect of diet on motivation. Moreover, no significant 335 between-group effects were detected in the first and second habituation ODT sessions, 14 336 when all rats were habituated to the edible reward. However, old CR rats were slower in 337 consuming cereal compared to Adult rats in the last habituation session. This result also 338 does not seem to indicate that CR rats were less motivated to eat as they performed 339 similarly to the Adult animals in the ODT task and the buried food test. 340 In general, the data obtained are in agreement with previous experiments demonstrating 341 that old rodents can discriminate between different odors [14,15,55,56], even though 342 they experience mild olfactory dysfunction [8,56,57]. Furthermore, earlier experiments 343 [12,51] also suggested that there is wide variability among old rodents, with only some 344 individuals exhibiting significant olfactory learning deficits. Therefore, the greater 345 variability of the scores in both the aged groups, but especially in the Ad Libitum group, 346 could have prevented more statistically significant differences appearing in the data. In 347 addition, the absence of general differences between the groups in the task might be due 348 to the olfactory memory deficit being more evident in complex learning tasks, such as 349 associative contextual and/or reversal learning [14,15]. In this regard, ODT could be 350 considered a robust memory task involving a survival-based behavior to find food in 351 conditions of deprivation [47]. As such, it might well be less sensitive to the effects of 352 aging or other factors. Similarly, senescent rats were not only seen to learn conditioned 353 taste aversion, which involved odor perception, but their performance was even better 354 than that of younger rats [7]. In fact, a CR diet in old Fischer-344 rats failed to reduce 355 age-related neophobic deficits to novel flavoring foods [58]. These data suggest that the 356 harmful effects of aging may differ depending on the type of learning considered and its 357 complexity or biological significance. 358 Our results suggest that nutritional status may influence odor sensitivity since higher 359 leptin levels correlated with poorer performance on the buried food test, consistent with 360 earlier studies suggesting that leptin and its receptors, present in the rat olfactory 15 361 mucosa, are modulated by the nutritional status [53] and modulate olfactory-mediated 362 pre-ingestive behavior [54]. Indeed, the olfactory system is more reactive to odors in 363 conditions of starvation and this activity diminishes after satiety [59]. In this regard, the 364 olfactory bulb is thought to be a metabolic or nutrient sensor, and metabolic hormones 365 like insulin and leptin can modulate olfactory-driven behaviors [38,60,61]. In general, 366 these hormones tend to increase with age [29,30,33,62] and dietary interventions like 367 CR could restore their levels to those of young animals [33]. Thus, as leptin modulates 368 odor sensitivity, high levels of this hormone in the Ad libitum rats could account, at least 369 in part, for the differences observed in the cookie test. Although our results confirm that 370 a lifelong hypocaloric diet may reduce leptin levels, which could be related to olfactory 371 sensitivity, insulin and glucose levels did not change here. These results do not fully 372 match with those previously observed in which CR reduced high insulin levels in aged 373 animals [33]. Therefore, further experiments are needed to confirm the capacity of a CR 374 diet to restore glucose and insulin blood levels in old rats. 375 The data presented also show that regardless of diet, older animals had less IGF-1, 376 considered a potential biomarker of cognitive decline in the aged brain [45,63,64]. 377 Plasma IGF-1 levels generally change with aging and here, CR did not reverse the 378 effects of aging on IGF-1 levels, consistent with our previous findings [33] but not with 379 those showing that this dietary intervention decreases [65] or may even increase [62] 380 IGF-1 levels. Therefore, more experiments should be carried out to assess the relevance 381 of this hormone to the age-induced impairment in olfactory perception and memory 382 decline, and how this may be affected by diet and other factors. 383 Regarding neurotransmitters, a 30% lifetime reduction in food intake enhanced DA 384 levels in the striatum, as well as its DOPAC and 3MT metabolites, when compared to 385 the Ad Libitum [33] and Adult rats. Comparably, more TH was also detected in the 16 386 striatum of CR rats. DA neurons in the midbrain provide rich innervation to the striatum 387 and FC, and they play a fundamental role in a multitude of cognitive processes, 388 including reward processing, learning, decision making and motivation to engage in 389 goal-orientated behaviors (e.g., eating and drinking) [66,67]. Moreover, the olfactory 390 bulb sends projections to the olfactory tubercle, that is considered a component of the 391 ventral striatum closely related to the mesolimbic dopaminergic pathway involved in 392 motivation and hedonic responses for foods (reviewed in [68]). In our experiment, DA 393 levels were correlated with performance in ODT acquisition and olfactory sensitivity 394 test, but not with ODT retention. Therefore, high levels of DA may increase motivation 395 to eat while learning the task, but not during later phases, once the smell has been 396 associated with the reward. 397 In addition, there was more 5-HT in the striatum of old CR rats, a neurotransmitter 398 related to cognitive processes [69,70]. Indeed, there is evidence that selective activation 399 of 5-HT4 receptors can improve memory performance in elderly rats in a task of 400 olfactory associative discrimination [10]. Such findings suggest that 5-HT may be 401 involved in olfactory memory and that its increase could enhance the olfactory 402 performance of old rats. As to the effects of aging on monoaminergic neurotransmission 403 in the striatum, we could not confirm that levels of DA decline with age, in agreement 404 with some previous studies [28,71–75] but see also [76–83]. Furthermore, in aged rats 405 there were no changes detected here in 5-HT [72,74,75,77,78] nor in NA [74,75,78– 406 81,83]. 407 The data regarding neurotransmission in the FC contrasts with that obtained in the 408 striatum. As reported previously [73,83–85], the levels of DA and its metabolite 409 decrease in aged rats irrespective of diet, as further supported by the low levels of TH. 410 However, no such effect [28,74,79,80,82,86] or even an increase [72] in DA and 17 411 DOPAC have also been described. The CR diet was unable to attenuate such age- 412 dependent decreases in the FC, unlike a previous report where a 60% reduction in the ad 413 libitum diet increased DA in the FC [37]. Our results confirmed that aged animals 414 experience an overall reduction of 5-HT in the FC [37,79,83,87] but see [28,72– 415 75,77,80,82], which in this case was reversed by a CR diet. However, neither aging nor 416 CR affected the levels of the TPH neurotransmitter precursor in the FC. Finally, no 417 differences were found between the groups in terms of NA, as seen previously in some 418 [28,37,74,79,84] but not all reports [73,75,80,82]. 419 5. Conclusions 420 The results of our study suggest that in general, a lifetime CR diet may attenuate the 421 age-related decline in olfactory sensitivity, observed in the Ad Libitum old rats. The 422 specific mechanism through which CR may exert such beneficial effect is not 423 completely determined, as a better locomotor activity could partially mediate the 424 performance in the tests. However, it could also be linked to a reduction in the age- 425 related increase in leptin, and enhancements in DA and 5-HT in the striatum, a region 426 related to olfaction. Our study will serve as a foundation for a follow-up study that can 427 address more causal contribution. Future research into the effects of CR on the aged 428 brain will be of great interest to fully understand the effects of such dietary intervention 429 on olfactory memory, and to favor behavioral habits that ensure a long and healthy life 430 in humans. 431 Figure legends 432 Figure 1: (A) Latency (s) to make the correct response (± SEM) and (B) mean number 433 of total errors (± SEM) in the acquisition, retention, and relearning trials of the ODT 434 (Latency: Group F(2,42) = 2.184, p = 0.125, Trial F(2,84) = 4.759, p = 0.01, Group x Trial 18 435 F(4,84) = 1.552, p = 0.195; Error: Group F(2,42) = 1322, p = 0.278, Trial F(2,84) = 2.856, p = 436 0.057, Group x Trial F(4,84) 0.970, p = 0.428). 437 Figure 2: Dispersion cloud of the ODT latency in the retention trial. Each dot represents 438 a specific subject and the black line across the dispersion cloud depicts the mean latency 439 to find the reinforcement for each group (*p < 0.05). (F(2,44) = 4.616, p = 0.015, Ad 440 Libitum > Adult group (p = 0.014). 441 Figure 3: Latency (s) to find the buried cookie (± SEM) for each group of animals. 442 (***p < 0.001). (F(2,42) = 10.866, p < 0.001; Ad Libitum > Adult (p < 0.001) and CR (p = 443 0.001)). 444 Figure 4: Spearman’s correlation between the latency to find the reinforcement in the 445 ODT retention trial and the latency to find the buried cookie in the olfactory test (r = 446 0.445, p = 0.001). 447 Figure 5. Blood plasma concentration of (A) Glucose, (B) Leptin, (C) Insulin, and (D) 448 IGF-1 in each group of animals (mean ± SEM). (** p < 0.01, *** p < 0.001). Glucose 449 (F(2,21) = 19.215, p < 0.001; Adult < CR (p < 0.001) and Ad Libitum (p = 0.002). Leptin 450 (F(2,21) = 21.536, p < 0.001); Ad Libitum > Adult (p < 0.001) and CR (p < 0.001). IGF-1 451 (F(2,21) = 8.630, p = 0.002); Adult > CR (p = 0.005) and Ad Libitum (p = 0.005). Insulin 452 (F(2,21) = 0.153, p = 0.859). 453 Figure 6. Spearman’s correlation between the latency to find the buried cookie in the 454 olfactory test and the plasma leptin levels (r = 0.518 p < 0.005). The dispersion cloud is 455 milder for the CR and adult animals than the Ad Libitum subjects. 456 Figure 7. Concentration of monoamines, precursors, and metabolites, in the striatum (A) 457 and FC (B), as analyzed by HPLC-ED for each group (mean ± SEM) (*p < 0.05, ** p < 458 0.01, *** p < 0.001). DOPAC (F(2,21) = 19.764, p < 0.001, DA F(2,19) = 65.948, p < 19 459 0.001, 3MT F(2,21) = 30.595, p < 0.001); CR > Ad Libitum and Adult. DOPAC (p = 460 0.007, p = 0.002), DA (p < 0.001) and 3MT (p < 0.001) and 5-HT (F(2,21) = 4.970, p = 461 0.017, CR > Ad Libitum (p < 0.026)). FC (DOPAC F(2,21) = 10.909, p = 0.001), DA 462 (F(2,21) = 27.537, p < 0.001; Adult > Ad Libitum and CR DOPAC (p = 0.004, p = 463 0.0014)) and DA (p = 0.035, p < 0.001). 5-HT (F(2, 19) = 3.931, p = 0.037, CR > Ad 464 Libitum (p = 0.038)). 465 Fig. 8: Representative Western Blots and percentage change (mean ± SEM) in the 466 intensity of the TPH and TH monoamine precursors in the striatum (A) and FC (B) of 467 Ad Libitum, CR and Adult rats, taking the intensity in Adults rats as 100% (*p < 0.05, 468 **p <0.01, *** p < 0.001); (C, D) Representative images of the integrated density bands 469 of proteins for each group and brain region. TH striatum (F(2,12) = 32.793, p < 0.001 CR 470 > Adult (p < 0.001) and Ad Libitum (p < 0.001) and FC F(2,14) = 4.107, p = 0.040), Adult 471 > CR (p = 0.051)). 472 Funding 473 This work was supported by funds from the Ministerio de Economía y Competitividad, 474 Spain (PSI2017-84290-R). 475 Acknowledgements 476 The authors thank Dr Mark Sefton for his support with English language editing. 477 Declaration of Competing Interest 478 The authors have no financial or potential conflict of interest to declare. 479 480 References 20 481 [1] D.P. Devanand, Olfactory Identification Deficits, Cognitive Decline, and 482 Dementia in Older Adults, Am. J. Geriatr. Psychiatry. 24 (2016) 1151–1157. 483 https://doi.org/10.1016/j.jagp.2016.08.010. 484 [2] M.W. Albers, M.H. Tabert, D.P. Devanand, Olfactory dysfunction as a predictor 485 of neurodegenerative disease, Curr. Neurol. Neurosci. Rep. 6 (2006) 379–386. 486 https://doi.org/10.1007/s11910-996-0018-7. 487 [3] J. Attems, L. Walker, K.A. 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Manag. 5 (2015) 425–443. https://doi.org/10.2217/nmt.15.34. 33 788 34 Figure 1 Click here to access/download;Figure;FIGURE 1_Rojic_Becker_etal_2021.pptx A B 120 1.6 1.6 Mean number of total errors in the ODT Latency (s) to find the reinforcement 1.4 1.4 100 1.2 1.2 80 1.0 1.0 60 0.8 0.8 0.6 0.6 40 0.4 0.4 20 0.2 0.2 0 0.0 0.0 Acquisition Acquisition Test Retention Reacquisition Relearning Acquisition Acquisition Retention Test Relearning Reacquisition Ad Libitum CR Adult Ad Libitum CR Adult Figure 1: (A) Latency (s) to make the correct response (± SEM) and (B) mean number of total errors (± SEM) in the acquisition, retention, and relearning trials of the ODT (Latency: Group F(2,42) = 2.184, p = 0.125, Trial F(2,84) = 4.759, p = 0.01, Group x Trial F(4,84) = 1.552, p = 0.195; Error: Group F(2,42) = 1322, p = 0.278, Trial F(2,84) = 2.856, p = 0.057, Group x Trial F(4,84) 0.970, p = 0.428). Figure 2 Click here to access/download;Figure;FIGURE 2_Rojic_Becker_etal_2021.pptx 200 * Latency to find the reinforcement (s) 180 160 140 120 100 80 60 40 20 0 0 1 Ad Libitum 2 CR 3 Adult 4 Figure 2: Dispersion cloud of the ODT latency in the retention trial. Each dot represents a specific subject and the black line across the dispersion cloud depicts the mean latency to find the reinforcement for each group (*p < 0.05) (F(2,44) = 4.616, p = 0.015, Ad Libitum > Adult group (p = 0.014). Figure 3 Click here to access/download;Figure;FIGURE 3_Rojic_Becker_etal_2021.pptx 140 *** Latency (s) to find the buried cookie 120 *** 100 80 60 40 20 0 Ad Libitum CR Adult Figure 3: Latency (s) to find the buried cookie (± SEM) for each group of animals. (***p < 0.001). (F(2,42) = 10.866, p < 0.001; Ad Libitum > Adult (p < 0.001) and CR (p = 0.001)). Figure 4 Click here to access/download;Figure;FIGURE 4_Rojic_Becker_etal_2021.pptx 300 Latency (s) to find the buried cookie 250 200 150 100 50 0 0 50 100 150 200 Latency (s) to find the reinforcement at the test trial in the ODT AdAL Libitum CR Adult Figure 4: Spearman’s correlation between the latency to find the reinforcement in the ODT retention trial and the latency to find the buried cookie in the olfactory test (r = 0.445, p = 0.001) Figure 5 Click here to access/download;Figure;FIGURE 5_Rojic_Becker_etal_2021.pptx A B 180 ** 16000 *** 160 *** 14000 *** 140 Figure 5. Blood plasma concentration of (A) Glucose, (B) Leptin, 12000 (C) Insulin, and (D) IGF-1 in each group of animals (mean ± SEM). Glucose (mg/dL) Leptin (pg/mL) 120 (** p < 0.01, *** p < 0.001). Glucose (F(2,21) = 19.215, p < 0.001; 10000 100 Adult < CR (p < 0.001) and Ad Libitum (p = 0.002). Leptin (F(2,21) = 8000 80 21.536, p < 0.001); Ad Libitum > Adult (p < 0.001) and CR (p < 6000 0.001). IGF-1 (F(2,21) = 8.630, p = 0.002); Adult > CR (p = 0.005) 60 4000 and Ad Libitum (p = 0.005). Insulin (F(2,21) = 0.153, p = 0.859). 40 20 2000 0 0 Ad Libitum CR Adult Ad Libitum CR Adult C D 1.2 1200000 ** 1.0 ** 1000000 Insulin (µg/mL) IGF-1 (pg/mL) 0.8 800000 0.6 600000 0.4 400000 0.2 0.2 200000 0.0 0.0 0 Ad Libitum CR Adult Ad Libitum CR Adult Figure 6 Click here to access/download;Figure;FIGURE 6_Rojic_Becker_etal_2021.pptx 300 Time (s) to find the buried cookie 250 200 150 100 50 0 0 5000 10000 15000 20000 Plasma levels of leptin Ad Libitum CR Adult Figure 6. Spearman’s correlation between the latency to find the buried cookie in the olfactory test and the plasma leptin levels (r = 0.518 p < 0.005). The dispersion cloud is milder for the CR and adult animals than the Ad Libitum subjects. Figure 7 Click here to access/download;Figure;FIGURE 7_Rojic_Becker_etal_2021.pptx Striatum B Frontal Cortex A 11000 5500 10000 *** *** 5000 * 4500 *** Concentration (ng/g) 9000 Concentration (ng/g) 8000 4000 7000 3500 6000 3000 5000 2500 ** 4000 ** ** 2000 *** 3000 1500 * 2000 1000 1000 *** *** * 500 0 0 NA DOPAC DA 3MT 5-HIAA 5-HT NA DOPAC DA 5-HIAA 5-HT Ad Libitum CR Adult Ad Libitum CR Adult Figure 7. Concentration of monoamines, precursors, and metabolites, in the striatum (A) and FC (B), as analyzed by HPLC-ED for each group (mean ± SEM) (*p < 0.05, ** p < 0.01, *** p < 0.001). DOPAC (F(2,21) = 19.764, p < 0.001, DA F(2,19) = 65.948, p < 0.001, 3MT F(2,21) = 30.595, p < 0.001); CR > Ad Libitum and Adult. DOPAC (p = 0.007, p = 0.002), DA (p < 0.001) and 3MT (p < 0.001) and 5-HT (F(2,21) = 4.970, p = 0.017, CR > Ad Libitum (p < 0.026)). FC (DOPAC F(2,21) = 10.909, p = 0.001), DA (F(2,21) = 27.537, p < 0.001; Adult > Ad Libitum and CR DOPAC (p = 0.004, p = 0.0014)) and DA (p = 0.035, p < 0.001). 5-HT (F(2, 19) = 3.931, p = 0.037, CR > Ad Libitum (p = 0.038)). Figure 8 Click here to access/download;Figure;FIGURE 8_Rojic_Becker_etal_2021.pptx Fig. 8: Representative Western Blots and percentage change (mean ± SEM) in the intensity of the TPH and TH monoamine precursors in the striatum (A) and FC (B) of Ad Libitum, CR and Adult rats, taking the intensity in Adults rats as 100% (*p < 0.05, **p <0.01, *** p < 0.001); (C, D) Representative images of the integrated density bands of proteins for each group and C D brain region. TH striatum (F(2,12) = 32.793, p < 0.001 CR > Adult (p < 0.001) and Ad Libitum (p < 0.001) and FC F(2,14) = 4.107, p = 0.040), Adult > CR (p = 0.051)) Author Statement Author Agreement Statement We the undersigned declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We understand that the Corresponding Author is the sole contact for the Editorial process. She is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs Signed by all authors as follows: Divka Rojic-Becker,, Marta Portero-Tresserra, Margarita Martí-Nicolovius, Anna Vale- Martínez, Gemma Guillazo-Blanch