Awake, long-term intranasal insulin treatment does not affect object memory, odor discrimination, or reversal learning in mice

Abstract

Intranasal insulin delivery is currently being used in clinical trials to test for improvement in human memory and cognition, and in particular, for lessening memory loss attributed to neurodegenerative diseases. Studies have reported the effects of short-term intranasal insulin treatment on various behaviors, but less have examined long-term effects. The olfactory bulb contains the highest density of insulin receptors in conjunction with the highest level of insulin transport within the brain. Previous research from our laboratory has demonstrated that acute insulin intranasal delivery (IND) enhanced both short- and long-term memory as well as increased two-odor discrimination in a two-choice paradigm. Herein, we investigated the behavioral and physiological effects of chronic insulin IND. Adult, male C57BL6/J mice were intranasally treated with 5μg/μl of insulin twice daily for 30 and 60days. Metabolic assessment indicated no change in body weight, caloric intake, or energy expenditure following chronic insulin IND, but an increase in the frequency of meal bouts selectively in the dark cycle. Unlike acute insulin IND, which has been shown to cause enhanced performance in odor habituation/dishabituation and two-odor discrimination tasks in mice, chronic insulin IND did not enhance olfactometry-based odorant discrimination or olfactory reversal learning. In an object memory recognition task, insulin IND-treated mice did not perform differently than controls, regardless of task duration. Biochemical analyses of the olfactory bulb revealed a modest 1.3 fold increase in IR kinase phosphorylation but no significant increase in Kv1.3 phosphorylation. Substrate phosphorylation of IR kinase downstream effectors (MAPK/ERK and Akt signaling) proved to be highly variable. These data indicate that chronic administration of insulin IND in mice fails to enhance olfactory ability, object memory recognition, or a majority of systems physiology metabolic factors - as reported to elicit a modulatory effect with acute administration. This leads to two alternative interpretations regarding long-term insulin IND in mice: 1) It causes an initial stage of insulin resistance to dampen the behaviors that would normally be modulated under acute insulin IND, but ability to clear a glucose challenge is still retained, or 2) There is a lack of behavioral modulation at high concentration of insulin attributed to the twice daily intervals of hyperinsulinemia caused by insulin IND administration without any insulin resistance, per se.

Keywords: IR kinase; Intranasal; Kv1.3; Meal frequency; Olfactometry; Olfactory.

Figure 1

Ingestive behavior and glucose clearance in mice following chronic insulin intranasal delivery (IND) along two time schedules. (A) Timeline of behavioral experiments for Animal Cohorts 1 and 2. (B,C) Scatter plot demonstrating the number of meal bouts per 12 h interval during the (B) light or (C) dark cycle for mice in Cohort 1 (Metabolic Chambers). Each data point represents cumulative number of meals for an individual mouse collected continuously during specified time interval. (D) Line graph of plasma glucose concentration over time for an intraperitoneal glucose tolerance test (IPGTT) for mice in Cohort 2 following final IND treatment (Sacrifice). Inset, bar graph of the mean integration of the area under the curve (iAUC). (E) Scatter plot of plasma insulin levels for 8 mice in Cohort 1 (open symbols) and 7 mice in Cohort 2 (closed symbols) following final IND treatment (Sacrifice). Squares = PBS (Control), Circles = insulin. B, C – Kolmogorov-Smirnov test, **p < 0.0025. D - Not-significantly different (NS) means, Student t-test, p > 0.05. Dark bars or circles = Control, Light bars or light squares = Insulin. E – Student’s t-test, p < 0.05. All values represent mean ± SEM in this and subsequent figures. N = Number of mice as indicated.

Figure 2

Long-term intranasal insulin administration does not alter olfactory ability. (A) Line graph of the correct responses for mice in Cohort 2 trained in a two-odor discrimination task (Olfactometry Testing; Figure 1) that were operant trained to discriminate between the odorants 5% ethyl acetate (EA) and 1% acetophenone (AP). NS, two-way RM ANOVA, using treatment and time as factors, p > 0.05. (B) Line graph of the correct responses for the same mice trained to perform an odor reversal-learning paradigm. S+/S− reversed at bar break. Two-way RM ANOVA using treatment (p > 0.05) and time (***p < 0.001) as factors. Control (dark circle) vs. Insulin (open square). Dashed line = 80% correct responses (criteria). N = Number of mice as indicated.

Figure 3

Long-term intranasal insulin does not enhance short- or long-term memory in a novel object recognition task. Bar graphs of the exploratory time to explore two objects during a familiarization phase (Object 1, Object 2) followed by a recognition phase (Object 1, Object 3) where a novel object is presented (Object 3). Time between familiarization and recognition phase is set for (A) short-term memory, or 5 min, or (B) long-term memory, or 24 h. Dashed line = 50% exploration of Object 1, or equal exploration across objects. Two-way, repeated-measure mixed ANOVA, Bonferroni’s post-hoc test, ****p < 0.0001. N = 5 animals/treatment group, Cohort 2 (Figure 1; Novel Object Recognition).

Figure 4

Long-term Intranasal insulin increases insulin receptor (IR) kinase phosphorylation within the olfactory bulb but does not uniformly phosphorylate downstream substrates. Immunoprecipitated (IP) tyrosine phosphorylated proteins (4G10) were separated by SDS-PAGE and blotted (Blot) with antisera against insulin (IRCT3) or Kv1.3 channel (Kv1.3). Western analysis and associated bar graph of normalized immunodensity values for (A,C) IR kinase phosphorylation and (B,D) Kv1.3 phosphorylation for animals pooled across Cohorts 1 and 2 (Figure 1, Sacrifice). Also shown is input lysate for A–D, blotted with the loading control, beta-tubulin III (Tubulin). (E–H) Same as (A–D) for animals from Cohort 1 (Figure 1, Sacrifice), but tyrosine phosphorylated proteins (4G10) were blotted for AKT, MAPK (Erk1/2). Input lysates were blotted with Akt and MAPK (Erk 1/2), respectively. M_r in kDa as specified. Dashed line = ratio 1.0 for insulin/control. C = Control, +I = Insulin. N = number of animals (both olfactory bulbs per sample). Student’s t-test, *p < 0.05.