Moderate recurrent hypoglycemia during early development leads to persistent changes in affective behavior in the rat

Abstract

Recurrent hypoglycemia is a common problem among infants and children that is associated with several metabolic disorders and insulin-dependent diabetes mellitus. Although studies have reported a relationship between a history of juvenile hypoglycemia and psychological health problems, the direct effects of recurrent moderate hypoglycemia have not been fully determined. Thus, in this study, we used an animal model to examine the effects of recurrent hypoglycemia during the juvenile period on affective, social, and motor function (assessed under euglycemic conditions) across development. To model recurrent hypoglycemia, rats were administered 5 U/kg of insulin or saline twice per day from postnatal day (P)10 to P19. Body weight gain was retarded in insulin-treated rats during the treatment period, but recovered by the end of treatment. However, insulin-treated rats displayed increases in affective reactivity that emerged early during treatment and persisted after treatment into early adulthood. Specifically, insulin-treated pups showed increased maternal separation-induced vocalizations as infants, and an exaggerated acoustic startle reflex as juveniles and young adults. Moreover, young adult rats with a history of recurrent juvenile hypoglycemia exhibited increased fear-potentiated startle and increases in behavioral and hormonal responses to restraint stress. Some of these effects were sex-dependent. The changes in affective behavior in insulin-exposed pups were accompanied by decreases in adolescent social play behavior. These results provide evidence that recurrent, transient hypoglycemia during juvenile development can lead to increases in fear-related behavior and stress reactivity. Importantly, these phenotypes are not reversed with normalization of blood glucose and may persist into adulthood.

Fig. 1

Body growth for saline- (SAL) or insulin-treated (INS) pups prior to, during, and after the treatment period for cohorts of rats receiving twice daily treatments from P10 to P19. (A) Daily morning body weights beginning on P10 (prior to the first treatment), continuing through P19, then at 3 days post-treatment on P22. INS pups showed a significantly different growth rate as indicated by a significant “treatment × postnatal day” interaction (indicated by “#”; see text). By P22, however, body weights of the two groups were nearly identical. The arrow marks the age of eye opening, after which solid food consumption and rate of weight gain appeared to increase in both groups. (B and C) Linear growth functions (weight × day) regressed across the period from P10 to P15, prior to full eye opening in all animals, and P16 to P19, from eye opening until the end of treatment. (B) The linear functions; (C) shows a bar graph of the slopes (growth rates). INS pups showed a significantly slower rate of growth between P10 and P15, but a trend for a faster rate of growth from P16 to P19. ^#p < 0.001, treatment × postnatal day interaction (A); *p < 0.05, main effect of treatment (B and C); ^tp < 0.1, main effect of treatment (C).

Fig. 2

Ultrasonic vocalizations emitted by P12 and P14 pups during a 3 min period of separation from the dam. Baseline maternal separation-induced vocalizations assessed prior to the beginning of treatment on P10 were not different between groups (see text). The response to separation was assessed when all pups were euglycemic (see text). INS pups showed significant increases in separation-induced vocalizations. *p < 0.05 main effect of treatment; ^#p < 0.05 main effect of age.

Fig. 3

Habituation of the acoustic startle reflex in INS- and SAL-treated rats assessed at P22, 3 days following the final treatment. (A) A polynomial function for the acoustic startle reflex regressed across the 16 consecutive startle stimulus presentation for SAL (open symbols; dotted line) and INS (solid symbols and line) rats, showing persistent increase in the startle reflex in INS rats. The inset shows the habituation index, defined as the percent decrease in startle amplitude between the first two and last two trials of the series. (B) Habituation index values for a separate cohort tested as young adults (see text for minor differences in testing conditions). There was a significant interaction between treatment and sex on this measure in adults (see text). Contributing to this was a trend for decreased startle habituation in INS males (top panel) but the opposite trend in females (bottom panel). *p < 0.05, effect of treatment; ^tp < 0.1, effect of treatment.

Fig. 4

Baseline acoustic startle and prepulse inhibition of startle in SAL- or INS-treated pups tested as juveniles or young adults. Baseline startle was determined following full habituation of the response (see Section 1). (A) Baseline acoustic startle (inset) and prepulse inhibition of startle (as a function of the salience of a prepulse delivered 100 ms prior to the startle stimulus) in rats exposed to SAL or INS from P10 to P19, and tested on P22. (B) Baseline acoustic startle (inset) and prepulse inhibition of startle from a different cohort of rats tested as young adults. Consistent with previous reports (see Section 1) prepulse inhibition is more sensitive to prepulse salience in adults than in juveniles. An interaction between juvenile treatment and prepulse salience was found in the adult cohort, produced by a selective difference between groups at with the quietest prepulse. *p < 0.05, effect of treatment.

Fig. 5

Fear-potentiated startle (FPS) in young adult rats with a history of juvenile treatment with INS or SAL. FPS ratio calculated as the startle response on CS⁺ trials divided by the average startle response on CS⁻ trials. Shown is the average FPS across the first 3 test trials, prior to evidence for extinction. See Supplementary material for further explanation of the calculation of average FPS. *p < 0.05, effect of treatment.

Fig. 6

Behavioral and hormonal responses to restraint stress in young adult rats with a history of juvenile treatment with INS or SAL (A) Frequency of bouts of facial tremor during 30 min of restraint. (B) Plasma corticosterone measured following at the end of the restraint period. *p < 0.05, main effect of treatment; ^tp < 0.1, main effect of treatment; ^#p < 0.05, treatment × sex interaction.