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. Author manuscript; available in PMC: 2023 May 15.
Published in final edited form as: Physiol Behav. 2022 Mar 2;249:113769. doi: 10.1016/j.physbeh.2022.113769

Effects of Nucleus Accumbens Insulin Inactivation on Microstructure of Licking for Glucose and Saccharin in Male and Female Rats

Kenneth D Carr 1,2,*, Sydney P Weiner 1
PMCID: PMC8969111  NIHMSID: NIHMS1786083  PMID: 35247443

Abstract

Insulin of pancreatic origin enters the brain where several regions express a high density of insulin receptors. Functional studies of brain insulin signaling have focused predominantly on hypothalamic regulation of appetite and hippocampal regulation of learning. Recent studies point to involvement of nucleus accumbens (NAc) insulin signaling in a diet-sensitive response to glucose intake and reinforcement of flavor-nutrient learning. The present study used NAc shell microinjection of an insulin inactivating antibody (InsAb) to evaluate effects on the microstructure of licking for flavored 6.1% glucose. In both male and female rats, InsAb had no effect on the number of lick bursts emitted (a measure of motivation and/or satiety), but decreased the size of lick bursts (a measure of reward magnitude) in a series of five 30-minute test sessions. This effect persisted beyond microinjection test sessions and was shown to depend on previous flavored glucose consumption under InsAb treatment rather than InsAb treatment alone. This suggests learning of diminished reward value and aligns with the previous finding that InsAb blocks flavor-nutrient learning. Specificity of the InsAb effect for nutrient reward was indicated by failure to affect any parameter of licking for flavored 0.25% saccharin solution. Finally, maintenance of rats on a ‘Western’ diet for twelve weeks produced a decrease in lick burst size for glucose in male rats, but an increase in lick burst size in females. Possible implications of these results for flavor-nutrient learning, maladaptive consequences of NAc insulin receptor subsensitivity, and the plausible involvement of distinct insulin-regulated mechanisms in NAc are discussed.

Keywords: insulin, nucleus accumbens, reward, lick microstructure, flavor-nutrient learning

1. Introduction

CNS exposure to pancreatic insulin follows delivery through the choroid plexus into the CSF and trans-endothelial transport via the brain microvasculature (1–7). Insulin receptors are expressed in neurons and glia (8,9) with particularly high concentrations of protein and mRNA in olfactory bulb, hippocampus, hypothalamus, cerebellum, and ventral striatum (10–13). To date, studies aimed at determining the behavioral and physiological functions regulated by brain regional insulin receptor populations have focused predominantly on hypothalamic control of energy metabolism and appetite (14–17), and hippocampal mechanisms of neuroplasticity and learning (18,19).

The insulin receptor population in nucleus accumbens (NAc) is of interest based on the involvement of this structure in reward-related learning, incentive motivation, and hedonic reactivity, and the regulation of these behavioral functions by energy balance and nutritional feedback signals (for several representative reviews see: 20–22). Moreover, the fact that over forty percent of the US adult population is affected by insulin subsensitivity (U.S. CDC, National Diabetes Statistics Report, 2014) raises the possibility that depressed insulin signaling in NAc contributes to the increased prevalence of maladaptive eating behavior in this population (e.g., 23–25). Several recent basic science studies have begun to illuminate the local circuit and behavioral effects of NAc insulin signaling. For example, NAc insulin receptors and their downstream Akt signaling pathway are activated by oral and intra-gastric glucose delivery, indicating a short latency response to the associated insulin surge (26). In striatal slices, insulin facilitates evoked dopamine release by increasing excitability of cholinergic interneurons (27), and increases excitation of postsynaptic medium spiny neurons (MSNs) via an opioid receptor-dependent disinhibition of glutamate release (28). All of these effects are diet-sensitive. Subjects maintained on a high energy diet (HED) fail to display glucose-induced activation of NAc insulin receptors, insulin facilitation of evoked DA (26,27), and insulin facilitation of excitatory transmission in MSNs (28). Insight into the behavioral functions regulated by NAc insulin signaling is provided by the finding that inactivation of NAc insulin inhibits both glucose-induced insulin receptor activation and learning to prefer a flavor associated with oral intake of 6.1% glucose versus a flavor associated with 1% glucose/0.125% saccharin. Interestingly, rats maintained on HED also fail to acquire a flavor preference in this protocol. In a second behavioral protocol, where two different flavored solutions with equal glucose concentration are provided, rats maintained on a standard laboratory diet display a marked preference for a flavor repeatedly preceded by NAc microinjection of immunoglobulin G (IgG) as a control, compared to a flavor preceded by an insulin-inactivating antibody (InsAb) (26). These coordinated ex-vivo and in-vivo findings point to a role of NAc insulin signaling in flavor-nutrient learning and, specifically, in reinforcing preference for a flavor to the degree that it predicts glucose delivery. The disabling of this mechanism by maintenance on HED raises questions of whether, and in what manner, impaired ability to discriminate between foods based on nutritive yield may contribute to maladaptive eating behavior.

The purpose of the present study was to use lickometry to further explore the behavioral function(s) regulated by NAc insulin signaling and how it is affected by sex and maintenance diet. An advantage of lick microstructure analysis is the ability to differentiate lick parameters that are indicative of reward magnitude, motivation to consume, and satiety, and to monitor changes that may develop as a function of repeated experience with a particular fluid (29–32). Consequently, in Experiment 1 we measured lick parameters for a flavored 6.1% glucose solution in male and female rats over a series of five 30-min sessions, with sessions preceded by NAc microinjection of either InsAb or control IgG. Based on the observation that InsAb induced changes in lick behavior that persisted beyond the five microinjection sessions, Experiment 2 tested the effect of five microinjections of InsAb versus IgG, in the absence of consumption, on future lick microstructure in order to assess whether enduring effects of InsAb are due to InsAb treatment alone or InsAb combined with the experience of consumption. As a further test of the hypothesis that NAc insulin signaling mediates flavor-nutrient learning, Experiment 3 tested the effect of InsAb versus IgG microinjection on lick microstructure for a non-nutritive saccharin solution. Finally, Experiment 4 tested the prediction that subjects maintained on HED would develop changes in lick microstructure similar to those seen in subjects that had been microinjected with InsAb.

2. Materials and Methods

2.1. Rats

Subjects were male and female Sprague–Dawley rats purchased from Taconic Farms (Germantown, NY) at 10-12 weeks of age. Rats were housed individually in plastic cages with bedding and free access to water. They were maintained on a 12-h light/dark cycle (lights on at 6 am), and allowed at least three days acclimation to vivarium housing prior to initiation of any experimentation.

All experimental procedures were approved by the New York University Grossman School of Medicine Institutional Animal Care and Use Committee and were performed in accordance with the “Principles of Laboratory Animal Care” (NIH publication number 85-23). All efforts were made to minimize animal suffering and to reduce the number of animals used.

2.2. Diets

Rats in Experiment 1 (effect of NAc InsAb on glucose lickometry), Experiment 2 (persistence of NAc InsAb effect) and Experiment 3 (effect of NAc InsAb on saccharin lickometry) had free access to standard lab pellets (Rodent Diet #5001, Lab Diet, St. Louis, MO) in the home cage. Rats in Experiment 4 (effect of ‘Western’ diet on glucose lickometry) were maintained for twelve weeks on either a ‘Western’ (D12079Bi, Research Diets Inc., New Brunswick NJ) or control (D14042701i) diet. The ‘Western’ diet is 35% sucrose and 20% butter fat by weight. The control diet is matched for protein, vitamin, and mineral content but contains 58% cornstarch and 3.5% milk fat by weight.

2.3. Surgery

Rats were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), and stereotaxically implanted with chronically indwelling guide cannulas (P1 Technologies, Roanoke, VA). Two guide cannulas (26 ga) were placed bilaterally 2.0 mm dorsal to the injection sites in the NAc shell (1.6 mm anterior to bregma; 2.1 mm lateral to the sagittal suture, tips angled 8° toward the midline, 5.8 mm ventral to skull surface). Cannula patency was maintained with occlusion stylets. The cannulas and four mounting screws were then permanently secured to the skull by flowing dental acrylic around them. Postsurgical analgesia was achieved by administration of Carprofen (5.0 mg/kg, s.c.) at the time of removal from the stereotaxic frame and twenty four hours later.

2.4. Intracerebral microinjection

Prior to behavioral test sessions described below, subjects received bilateral microinjection of either InsAb (low endotoxin, azide-free; AB46707, Abcam, Cambridge MA) or the control IgG1 (low endotoxin, azide-free; AB18437, Abcam, Cambridge MA). InsAb efficacy had been established in previous studies showing that it prevents insulin-enhanced DA uptake in striatal synaptosomes (27) and glucose-induced insulin receptor phosphorylation in NAc (26). For microinjections, two 30 cm lengths of PE-50 tubing were attached on one side to 5-μL Hamilton syringes filled with distilled water, and on the other side to 31-gauge injector cannulas extending 2.0 mm beyond the implanted guide cannulas. Rats were placed on a cushion and their stylets were removed. Injectors were inserted and microinjections were made at rate of 0.05 μL every 10 seconds over a period of 100 s. Injector cannulas were left in place for another minute before being removed, at which point stylets were replaced and the rat was placed in the test chamber where a lickometry test session was initiated. Alternatively, rats were returned to their home cage depending on the experimental protocol as described below. Mock microinjections included all aspects of the procedure with the exception of antibody delivery into brain tissue.

2.5. Methods of habituation and preliminary behavioral testing

After a 5-7 day period of postsurgical recovery, each rat was placed in the lickometry test chamber and given access to 6.1% unflavored glucose (Sigma-Aldrich). Sustained licking for a period of 60 sec was criterion for advancing to the next phase of experimental preparation and the subject was removed from the chamber. Otherwise, the session continued for up to 30 min. Any rat that did not meet criterion was re-tested on up to two additional occasions. All rats satisfied criterion, with most doing so in the first or second session. This criterion was a reliable indicator that rats had detected the sipper tube, its palatable content, and would engage in glucose licking during subsequent test sessions. Following satisfaction of the lick criterion, rats underwent several days habituation to mild restraint on a foam cushion and received a mock intracerebral microinjection. This was followed by a preliminary test session of 30 min duration, preceded by mock microinjection, in which rats had access to unflavored 6.1% glucose. Results of this test, using the measure of total number of licks/30 min, were used to assign subjects to matched groups for experimental testing.

2.6. Apparatus and behavioral measures

Testing was conducted in Med Associates (Georgia, VT) operant chambers with a contact lickometer using Med PC and custom software. The fluid available for consumption in test sessions was 6.1% glucose or 0.25% saccharin flavored with 0.05% grape Kool-Aid (Kraft Foods, Northfield, IL). Grape flavor was used in order to match, as far as possible, taste properties of the glucose and saccharin solutions. Each subject was tested in five 30-min sessions spaced 48-72 h apart. The lick parameters recorded included total number of licks per session, a proxy for total consumption, plus multiple measures of lick patterning. These included lick bursts, which are groups of licks separated by an inter-lick interval of > 1 sec. This criterion for lick bursts is based on analyses of several laboratories which have concluded that an interval of 1 second is most likely to represent a true pause rather than “missed licks” or lateral tongue protrusions (for review see: 32). The number of bursts emitted per unit time is considered a measure of incentive salience that is also subject to modulation by satiety. Burst size is the number of licks in a burst, where only bursts of > 3 licks are counted and is considered a measure of reward magnitude or hedonic impact. All behavioral data were automatically collected by computer.

2.7. Behavioral testing

2.7.1. Experiment 1

Male and female rats were tested as separate cohorts. Only those subjects whose microinjection cannulas remained patent for the entire series of test sessions and whose injection sites were histologically localized to the NAc shell or the shell/core or shell/olfactory tubercle border were included in the experimental analysis.

Each subject was tested in five 30-min duration sessions in which grape-flavored 6.1% glucose was available. Sessions were spaced 48 to 72 hours apart and each test was immediately preceded by intra-NAc microinjection. Eleven female subjects comprised the group microinjected with IgG and eleven comprised the group microinjected with InsAb. Eleven males were microinjected with IgG and eleven with InsAb. Following completion of the series of five test sessions, subjects were tested in three additional sessions in which lickometry was preceded by mock microinjection rather than delivery of antibody into tissue. Results for the five microinjection test sessions and three mock injection test sessions were analyzed separately for each sex. Separate analyses were performed on three measures, namely, total number of licks per session, total number of bursts per session, and average burst size (i.e., licks per burst) per session.

2.7.2. Experiment 2

This experiment followed the observation, in Experiment 1, that effects of InsAb on lick parameters persisted in subjects that were later tested under mock injection conditions.

New male subjects, whose results were expected to illuminate the persistent effect of InsAb microinjection observed in Experiment 1, underwent preliminary testing and were assigned to groups matched for total number of licks per session as in Experiment 1. Five subjects then underwent a series of five intra-NAc microinjections of IgG and five underwent a series of five intra-NAc microinjections of InsAb at 48 to 72 h intervals but without fluid consumption. They each were then tested in a series of three mock microinjection sessions with access to grape-flavored 6.1% glucose as in Experiment 1. Lick parameters analyzed and compared between groups were the same as those analyzed in Experiment 1.

2.7.3. Experiment 3

Methods of Experiment 3 were as described for Experiment 1 with the exception that the fluid available for consumption was grape-flavored 0.25% saccharin. The fluid available was devoid of nutritive sweetener. Based on evidence that female rats are more avid and reliable consumers of saccharin than are males (33), subjects in this experiment were eight new females microinjected with IgG and eight injected with InsAb. Behavioral measures analyzed were as described in Experiment 1.

2.7.4. Experiment 4

Sixteen male and sixteen female rats underwent preliminary lickometric testing with unflavored 6.1% glucose as in Experiment 1. Based on total number of licks emitted during the session, half the subjects of each sex were assigned to a ‘Western’ diet and half to the control diet. Both diets were initiated immediately and were the only food available to these subjects. For the next twelve weeks, 24 h food intake and body weight were measured once per week and lickometric testing with unflavored 6.1% glucose was conducted bi-weekly. Behavioral measures analyzed were as described in Experiment 1.

2.8. Histology

Rats were briefly exposed to CO2 and decapitated by guillotine. Brains were removed and fixed in 10 % buffered formalin for 48 h. Frozen 40 μm coronal sections were cut on a Reichert-Jung 2800 cryostat, thaw-mounted on gelatin-coated slides and stained with cresyl violet. Microinjection sites were determined by visual inspection under an Olympus SZ40 microscope.

3. Results

3.1. Experiment 1

In the preliminary ‘mock’ microinjection test that preceded testing with antibody treatment, males assigned to the IgG group emitted 1170 (± 266) licks, and males assigned to the InsAb group emitted 1195 (± 255) licks. Females assigned to the IgG group emitted 2417 (± 377) licks, and females assigned to the InsAb group emitted 2202 (± 267) licks. The higher baseline lick rate of females is not surprising given past reports that females show greater preference and/or instrumental responding for sweet solutions than do males (e.g., 34). As displayed in Figure 1, both male (F1,100 = 80.6, p<.001) and female (F1,100 = 45.4, p<.001) rats displayed a marked decrease in total number of licks per session following InsAb microinjection with no differences between sessions (male: F(sessions)4,100 =0.97; F(interaction)4,100 = 0.68; female: F(sessions)4,100 =1.86; F(interaction)4,100 = 1.32). Both sexes showed an InsAb-induced decrease in the size of lick bursts (male: F1,100 = 38.1, p<.001); female: F1,100 = 16.1, p<.001) but no change in the number of lick bursts per session (male: F1,100 = 0.01); female: F1,100 = 0.53).

Figure 1. Effects of nucleus accumbens insulin antibody microinjection on microstructure of licking for flavored 6.1% glucose in male and female rats.

Figure 1.

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Left column: Lick microstructure parameters (mean ± SEM) for male rats consuming grape-flavored 6.1% glucose in a series of five 30-min sessions. Each session was preceded by microinjection of either insulin antibody (InsAb) or the control immunoglobulin G (IgG) in nucleus accumbens shell. N=11 per microinjection group. ***Main effect of microinjection treatment, p<.001. Right column: Lick microstructure parameters (mean ± SEM) for female rats consuming grape-flavored 6.1% glucose in a series of five 30-min sessions. Each session was preceded by microinjection of either insulin antibody (InsAb) or the control immunoglobulin G (IgG) in nucleus accumbens shell. N=11 per microinjection group. ***Main effect of microinjection treatment, p<.001.

In general, each subject’s burst number and average burst size accounted for 90% or more of the total licks recorded.

3.2. Experiment 2

We then tested the durability of insulin-dependent licking patterns in male subjects from Experiment 1. These animals were allowed 48 h after the final microinjection test session and then tested in a series of three mock microinjection sessions at 48-72 h intervals. The group that had been treated with InsAb microinjections continued to display a decrease in total number of licks per session (F1,60 = 14.94, p<.001) (Figure 2), with no differences between sessions (F(sessions)2,60 = 2.52, p >.05; F(interaction)2,60 = 0.33). Further, as was also observed in the microinjection test sessions, they displayed a decrease in burst size (F1,60 = 6.32, p <.02) but no change in number of lick bursts per session (F1,60 = 2.79, p >.05).

Figure 2. Effects of prior insulin antibody microinjection, with or without concomitant flavored glucose consumption, on lick microstructure in subsequent mock microinjection tests.

Figure 2.

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Left column: Lick microstructure parameters (mean ± SEM) for male rats consuming grape-flavored 6.1% glucose in a series of three 30-min sessions. Each session was preceded by ‘mock’ microinjection. These rats had a history (Experiment 1) of five sessions in which they consumed grape-flavored 6.1% glucose immediately following microinjection of insulin antibody (InsAb) or the control immunoglobulin G (IgG) (see Figure 1). N=11 per group. ***Main effect of microinjection treatment, p<.001. *Main effect of microinjection treatment p<.02. Right column: Lick microstructure parameters (mean ± SEM) for male rats consuming grape-flavored 6.1% glucose in a series of three 30-min sessions. Each session was preceded by ‘mock’ microinjection. These rats had a history (Experiment 2) of five microinjections of insulin antibody (InsAb) or the control immunoglobulin G (IgG), but without access to grape-flavored 6.1% glucose. N=5 per group. **Main effect of microinjection treatment, p<.005.

As follow-up to this observation two new groups of male rats were prepared to investigate the necessity of learning in these behaviors. In the preliminary ‘mock’ microinjection test that preceded testing under antibody treatment, those assigned to the IgG group emitted 915 (± 286) licks, and those assigned to the InsAb group emitted 969 (± 248) licks. When these subjects underwent a series of InsAb versus IgG microinjection treatments without fluid access and were then subject to three mock microinjection test sessions, the group that had been treated with InsAb displayed a total number of licks per session that tended to be greater than displayed by the IgG-treated group (F1,24 = 4.13, p =.053), in sharp contrast to those in which there was prior association with consumption (Figure 2). In another departure from the pattern of results seen in subjects that consumed flavored glucose following each microinjection, these subjects displayed a change in lick burst number (F1,24 = 11.37, p<.005) but not burst size (F1,24 = 0.66). Specifically, in line with their increased number of licks per session, InsAb-treated subjects displayed an increase in burst number.

3.3. Experiment 3

In the preliminary ‘mock’ microinjection test that preceded testing with antibody treatment, females assigned to the IgG group emitted 1047 (± 679) licks, and those assigned to the InsAb group emitted 1035 (± 794) licks. Female subjects that underwent the same experimental protocol of treatment and testing as in Experiment 1, except that the fluid consumed was flavored 0.25% saccharin rather than 6.1% glucose, displayed no effect of InsAb microinjection on total number of licks (F1,70 = 0.42), number of lick bursts (F1,70 = 0.005), or average size of lick bursts per session (F1,70 = 0.09) (Figure 3). They did, however, display an increase in total number of licks across test sessions, regardless of microinjection treatment (F4,70 = 3.82, p<.01), which was reflected in a trend toward greater lick burst size (F4,70 = 2.24, p=.074) but less clearly a trend toward greater number of lick bursts (F4,70 = 1.87, p=.124).

Figure 3. Effects of nucleus accumbens insulin antibody microinjection on microstructure of licking for flavored 0.25 % saccharin.

Figure 3.

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Lick microstructure parameters (mean ± SEM) for female rats consuming grape-flavored 0.25% saccharin in a series of five 30-min sessions. Each session was preceded by microinjection of insulin antibody (InsAb) or the control immunoglobulin G (IgG) in nucleus accumbens shell. N=8 per microinjection group.

3.4. Experiment 4

As displayed in Figure 4, during the twelve weeks on experimental diet, both males and females consuming Western diet gained more weight than subjects consuming the control diet (males: F1,98 =30.6, p<.001; females: F1,98 =7.1, p<.01). Group average body weights of male subjects increased from 314 (± 1.7) to 516.3 (± 3.9) grams in the control group, and from 322 (± 1.3) to 584 (± 9.7) grams in the Western diet group. In females, averages increased from 178 (± 2.1) to 290 (± 4.9) grams in the control group, and from 177 (± 1.2) to 314 (± 3.2) g in the Western diet group. Consequently, males and females maintained on Western diet both gained approximately 16% more weight than controls. Twenty four hour intake measures in the final three weeks indicated that males and females consuming control diet consumed an average of 93.6 and 64.3 kcal, respectively, while those consuming Western diet consumed an average of 122.2 and 91.6 kcal, respectively.

Figure 4. Biweekly body weights of rats maintained on a Western or control diet.

Figure 4.

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Body weights (grams) recorded biweekly in male (left) and female (right) rats beginning with pre-diet (time-0) through diet week 12. Subjects were either maintained on a high fat-high sugar ‘Western’ diet or a Control diet matched for pelleted form, protein, vitamins and minerals. N=8 per diet/sex group. ***Main effect of diet from week 2 through 12, p<.001.

Lickometric testing for 6.1% glucose conducted bi-weekly revealed diet-dependent differences in both male and female subjects. As displayed in Figure 5, across the six test sessions, males consuming Western diet emitted fewer licks per session (F1,84=15.2, p<.001) and fewer licks per burst (F1,84=14.8, p<.001), with no difference in number of lick bursts (F1,84=0.01, p>.05), relative to males consuming the control diet. Females displayed a different pattern of change, with those consuming Western diet displaying a trend in the direction of greater number of licks per session (F1,84=3.3, p= .07) and significantly greater number of licks per burst (F1,84=14.8, p<.001), with no difference in number of lick bursts (F1,84=0.7, p>.05), relative to females consuming the control diet.

Figure 5. Effects of a Western diet on lick microstructure for 6.1% glucose.

Figure 5.

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Left column: Lick microstructure parameters (mean ± SEM) for male rats consuming 6.1% glucose in a series of five 30-min sessions as a function of number of weeks maintained on either a ‘Western’ or Control diet. N=8 per group. ***Main effect of diet, p<.001. Right column: Lick microstructure parameters (mean ± SEM) for female rats consuming 6.1% glucose in a series of five 30-min sessions as a function of number of weeks maintained on either a ‘Western’ or Control diet. N=8 per group. *Main effect of diet, p<.05.

4. Discussion

The antibody used to inactivate NAc insulin in the present study was previously shown to block exogenous insulin-enhanced DA uptake in striatal synaptosomes (27) and, when microinjected into NAc, blocked oral glucose-induced insulin receptor phosphorylation (26) and acquisition of preference for a glucose-paired flavor (26,27). In Experiment 1 of the present study, it markedly decreased licking for a flavored 6.1% glucose solution and did so by decreasing the average number of licks per burst while sparing the total number of bursts emitted during the 30-min test sessions. This effect was seen in both male and female subjects. The baseline level of licking was substantially higher in females than in males. This observation aligns with previous reports that although adult female rats drink less water than males (33), they display greater avidity for sweet solutions, reflected in measures of passive consumption (33–35), preference relative to water (34), and instrumental responding (34). The robustness of the InsAb effect on lick burst size is therefore supported both by its occurrence across sex and markedly different lick rates.

Previous studies have established that total number of licks per session is a close proxy for total volume consumed (36) and lick burst size is reflective of hedonic value or reward magnitude (for review see 32). For example, burst size increases in parallel with sugar concentration (29,30,36–38), is modulated by contrast with higher and lower available sugar concentrations (39), decreases with increasing quinine concentration (40,41), and appears to be dependent on the availability of D-2 DA but not D-1 or opioid receptors (42–45). On the other hand, the number of bursts emitted during a session is reflective of motivation or incentive salience (32). This parameter is increased by food deprivation (30,46) and sham feeding (29), and decreased by gastric filling (47) or treatment with the anorexigenic agents cholecystokinin (48) or d-fenfluramine (49). Further, unlike lick burst size, it is dependent upon availability of D-1 DA and opioid receptors (44,45).

Interestingly, the effect of InsAb on licking for flavored 6.1% glucose persisted in a series of follow-up tests that were preceded by mock microinjection and no further antibody exposure. This may indicate that subjects acquired an association between grape flavor and low reward magnitude, aligning with the previous finding that NAc microinjection of InsAb blocked acquisition of preference for a glucose-paired flavor in a two-bottle choice test (26,27). Nevertheless, one alternative explanation is that repeated InsAb microinjection, itself, induces enduring cellular or local circuit-based changes that impact subsequent consummatory behavior. Consequently, in Experiment 2 subjects received a series of InsAb or IgG microinjections that were not followed by flavored glucose consumption, and follow-up behavioral tests were conducted as before. Rather than reproducing the effect seen in subjects of Experiment 1, subjects with a history of InsAb microinjection in Experiment 2 showed a trend toward increased number of total licks (p<.09) and a significant increase in number of lick bursts with no change in lick burst size. This facilitatory effect of repeated insulin inactivation may be a rebound response but interestingly, expresses in number of lick bursts rather than burst size. Several tentative conclusions can be drawn from this observation. First, the effects in Experiment 1 that persist beyond microinjection sessions are not a result of repeated treatment with InsAb itself, but are dependent upon consumption of flavored glucose following each microinjection; this supports the hypothesis that subjects have learned the diminished reward value of grape-flavored fluid. Second, the effects in Experiment 2 seem to unmask a second insulin mechanism that regulates motivation or incentive salience, and is apparently upregulated in response to repeated insulin-blocking treatments. If this is the case, one would expect to see evidence of its expression in the subjects of Experiment 1 during post-microinjection follow-up testing. Inspection of Figure 2 suggests that this is the case. While total number of licks and lick burst size are decreased, the number of lick bursts emitted is increased, though not significantly so, in the InsAb treatment group. Given that no changes were seen in number of lick bursts when subjects consumed flavored glucose with InsAb treatment, acute blockade of this proposed second mechanism either has no effect on consumption when in its normosensitive state, or is superseded by the effect of InsAb on burst size/reward magnitude. Mechanistically, it is at least plausible that different insulin receptor-bearing elements mediate the acute effect and after-effect of InsAb treatment on lick microstructure, given that three distinct insulin-modulated mechanisms have been identified in NAc (27,28,50,51). Speculation, based on the pharmacology of lick microstructure (42–45) would point to opioid-dependent facilitation of glutamate release (28) as the mechanism expressing rebound upregulation, and the cholinergic facilitation of DA release (27) as the mechanism underlying the acute and ‘learned’ decrease in reward magnitude.

In order to test the hypothesis that insulin signaling in NAc selectively modulates the reward value of a nutritive sugar, the protocol of Experiment 1 was repeated in Experiment 3 using the non-caloric sweetener, saccharin. Though devoid of caloric content, oral intake of saccharin may trigger a cephalic phase insulin response, albeit of shorter duration and lower amplitude than a nutritive sugar, but does not induce post-absorptive insulin release (52). At the level of NAc, saccharin consumption can induce DA signaling, but this effect is lost after first exposure, unlike a nutritive sugar which repeatedly induces DA signaling (53). Results of Experiment 3 indicate that InsAb had no effect on total number of licks, total number of lick bursts, or average lick burst size. Consequently, the inhibitory effect of InsAb on flavored glucose consumption is specific and selective. Irrespective of microinjection treatment, rats in Experiment 3 displayed a progressive increase in licking for saccharin across sessions, which may reflect increasing accommodation to the bitter off-taste.

The perceived reward value of flavored solutions is determined by a combination of orosensation and postingestive nutrient signaling (54). The specific contribution of nutrient signaling has been demonstrated in studies that combine oral consumption of non-nutritive solutions with intragastric infusion of nutrients (e.g., 55–58). The reward value added by post-oral nutrient delivery has been shown in assays of conditioned flavor preference (e.g.,56,58), taste reactivity (57) and, most germane to the present study, a change in lick microstructure expressing as a marked increase in lick burst size (55). Consequently, the present results demonstrating a selective decrease in lick burst size for a nutritive sugar, but not a non-nutritive sweetener, suggest that NAc insulin signaling plays a significant role in establishing the net rewarding effect of nutritive foods.

Based on a previous observation that as little as five weeks of liquid high fat-high sugar diet (chocolate Ensure) induced NAc insulin receptor insensitivity in male rats (26,27), it was predicted that maintenance on a high fat-high sugar diet would induce the same alterations in glucose lick microstructure as InsAb microinjection induced in Experiment 1. For the present study a pelleted ‘Western’ diet was used to enable matching of the experimental and control diets for form, protein, vitamin, and mineral composition. Another factor recommending the pelleted diet over chocolate Ensure was the potential confounding effect of licking sweet fluid both as maintenance diet and as acute probe of lick microstructure. In the twelve weeks of diet access both male and female rats on the ‘Western’ diet gained more weight than subjects maintained on the control diet. Diet group differences in microstructure of licking for unflavored 6.1% glucose were also seen but, unexpectedly, with marked sex differences. Males on the Western diet displayed a consistent decrease in total number of licks after just three weeks on the diet, while females displayed an increase in total number of licks that emerged after seven weeks on the Western diet. Matching the effect of InsAb microinjection in Experiment 1, Western diet had no effect on the number of lick bursts emitted by either sex but did alter burst size. Males displayed a decrease in burst size, and females displayed an increase in burst size.

The similar effect of InsAb microinjection and Western diet on lick microstructure in male rats preserves the hypothesis that NAc insulin receptor subsensitivity, previously demonstrated in male rats maintained on HED, is a determinant of the diet-induced decrease in reward magnitude of glucose. In addition, the previous findings that NAc insulin inactivation and HED both decreased flavor-nutrient (glucose) learning in male rats suggests homology between the insulin-modulated function revealed in the lickometric analysis and the insulin-dependent reinforcement of flavor-nutrient learning. The translational implication of diet-induced uncoupling of taste and nutrient-based reward/reinforcement for ‘free-range’ eating behavior was not explored here but could express as more avid consumption of sweets in search of the ‘predicted’ nutrient signal and/or a shift toward preference of exceptionally high sugar concentrations. If so, this would have a perpetuating or exacerbating effect on the overweight and insulin-resistant state, and align with the mechanistic hypothesis invoked to explain paradoxical health effects of chronic non-caloric sweetener consumption (59).

In seeming contradiction of the foregoing hypothesis, Myers and coworkers have shown that maintenance on HED for thirty weeks enhances flavor-nutrient learning (60). Interestingly, that result was obtained in female rats which, in the present study, displayed a HED-induced increase in reward magnitude, opposing the effect seen in males. While the apparent consonance between results of the two behavioral assays in females continues to suggest homology between reward magnitude inferred from lick burst size and reinforcement of a flavor-glucose association, the status and involvement of NAc insulin signaling in females on HED is currently unknown; effects of NAc insulin inactivation on flavor-nutrient learning, and effects of HED on glucose-induced insulin receptor phosphorylation and signaling have yet to be tested in female rats. Consequently, results of the present study add another dimension to the question of whether reward magnitude, as reflected in lick burst size, and reinforcement of flavor-nutrient learning are modulated, and in what manner, by a common diet-sensitive NAc insulin receptor-dependent mechanism. Finally, to the extent that NAc insulin signaling has a role in the development and perpetuation of maladaptive eating behavior, it remains to be determined which among the several local insulin-regulated elements are involved; namely, DAT surface expression and function (50,51), cholinergic facilitation of DA release (27), and/or opioid-dependent facilitation of glutamate release (28).

Highlights.

  • Analysis of lick microstructure indicates that microinjection of insulin antibody in nucleus accumbens decreases the reward magnitude of flavored glucose in male and female rats

  • Repeated consumption of flavored glucose in conjunction with insulin antibody treatment leads to a persistent decrease in reward magnitude, reflecting a learning process

  • Repeated insulin inactivation in nucleus accumbens induces a rebound increase in motivation to consume flavored glucose, unmasking a second insulin-regulated mechanism

  • Insulin antibody microinjection in nucleus accumbens has no effect on microstructure of licking for flavored non-nutritive saccharin

  • Maintenance on a ‘Western’ diet leads to a decrease in reward magnitude of glucose in male rats, but an increase in females

Acknowledgements

This research was supported by R01 DA050165 from NIDA/NIH. We would like to thank Rhonda Kolaric for technical assistance and support.

Footnotes

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