Orexin signaling is necessary for hypoglycemia-induced prevention of conditioned place preference

Abstract

While the neural control of glucoregulatory responses to insulin-induced hypoglycemia is beginning to be elucidated, brain sites responsible for behavioral responses to hypoglycemia are relatively poorly understood. To help elucidate central control mechanisms associated with hypoglycemia unawareness, we first evaluated the effect of recurrent hypoglycemia on a simple behavioral measure, the robust feeding response to hypoglycemia, in rats. First, food intake was significantly, and similarly, increased above baseline saline-induced intake (1.1 ± 0.2 g; n = 8) in rats experiencing a first (4.4 ± 0.3; n = 8) or third daily episode of recurrent insulin-induced hypoglycemia (IIH, 3.7 ± 0.3 g; n = 9; P < 0.05). Because food intake was not impaired as a result of prior IIH, we next developed an alternative animal model of hypoglycemia-induced behavioral arousal using a conditioned place preference (CPP) model. We found that hypoglycemia severely blunted previously acquired CPP in rats and that recurrent hypoglycemia prevented this blunting. Pretreatment with a brain penetrant, selective orexin receptor-1 antagonist, SB-334867A, blocked hypoglycemia-induced blunting of CPP. Recurrently hypoglycemic rats also showed decreased preproorexin expression in the perifornical hypothalamus (50%) but not in the adjacent lateral hypothalamus. Pretreatment with sertraline, previously shown to prevent hypoglycemia-associated glucoregulatory failure, did not prevent blunting of hypoglycemia-induced CPP prevention by recurrent hypoglycemia. This work describes the first behavioral model of hypoglycemia unawareness and suggests a role for orexin neurons in mediating behavioral responses to hypoglycemia.

Keywords: awareness; diabetes; perifornical hypothalamus; serotonin.

Fig. 1.

Timeline of conditioned place preference (CPP) protocol for single (A) and recurrent (B) hypoglycemia studies. A: intrinsic place preference was assessed on day 1 (pretest), CPP training was done on days 2-7, testing for CPP acquisition was done on day 8, rats underwent insulin-induced hypoglycemia (IIH) in the CPP apparatus on day 9, and CPP was again assessed on day 10. B: protocol for days 1-8 was identical to above, but on each of days 9-11 rats underwent single daily 2-h home cage bouts of IIH or saline injections followed by hypoglycemia in the CPP apparatus on day 12 and CPP assessment on day 13 (see materials and methods for details).

Fig. 2.

Day 1 and 2 blood glucose values during IIH. A: day 1 blood glucose values in animals exposed to 3 saline injections in a 2-day protocol (S/S/S). B: day 1 blood glucose values in animals exposed to 2 saline injections on day 1 followed by IIH on day 2 (S/S/H). C: day 1 blood glucose values in animals exposed to 3 insulin injections in a 2-day protocol (H/H/H). D: day 2 blood glucose values in animals exposed to 3 saline injections (S/S/S), 2 saline injections followed by a single bout of IIH (S/S/H), or 3 bouts of IIH (H/H/H) in a 2-day protocol. C: *P < 0.05, day 1 vs. day 2. D: *P < 0.05 S/S/S vs. S/S/H and S/S/S vs. H/H/H. Error bars indicate ± SE throughout.

Fig. 3.

Day 2 cumulative food intake during IIH. Day 2 cumulative (3-h) food intake in animals exposed to S/S/S, S/S/H, or H/H/H in a 2-day protocol. Letters denote significant differences in treatment × time by 2-way repeated-measures ANOVA. Error bars indicate ± SE. F(2, 17) = 80.3 (S/S/S vs. S/S/H vs. H/H/H).

Fig. 4.

CPP in rats before (day 1) and after (day 8) training with a palatable food reward and 1 day after IIH (day 10). CPP is expressed as minutes spent on either side of a 2-sided CPP apparatus. Data are means ± SE; *P ≤ 0.05 between groups. F(3,3) = 2.7 (day 10, side 1 vs. side 2).

Fig. 5.

CPP in rats before (day 1) and after training with a palatable food reward (day 8) and after IIH (day 13) preceded by 3 previous bouts of home cage hypoglycemia (RH) or saline injections (SH). A: CPP is expressed as the difference in time spent on either side of a 2-sided CPP apparatus. Change in the sign of the CPP value reflects a change in preference relative to the previous time point. B: preproorexin mRNA relative abundance in the PFH and LHA in recurrent hypoglycemic or single hypoglycemic rats. Data are means ± SE; *P ≤ 0.05 between groups. Letters denote significant differences in treatment × time by 2-way repeated-measures ANOVA. F(5,4) = 5.3 (day 13, SH vs. RH).

Fig. 6.

CPP in rats before (day 1) and after (day 8) training with a palatable food reward and after IIH (day 10) or saline paired with SB-334867A (20 mg/kg ip) or vehicle. CPP is expressed as the difference in time spent on either side of a 2-sided CPP apparatus. Change in the sign of the CPP value reflects a change in preference relative to the previous time point. Data are means ± SE. Letters denote significant differences in treatment × time by 2-way repeated-measures ANOVA. F(3,3) = 2.8 (day 13, Hypo-SB vs. Sal-SB/Sal-DMSO); F(3,2) = 6.6 (day 13, Hypo-DMSO vs. Sal-SB/Sal-DMSO).

Fig. 7.

CPP with (RH) and without (SH) 3 bouts of prior home cage hypoglycemia in rats pretreated with 21 days of 7.5 mg·kg⁻¹·day⁻¹ sertraline and assessed for CPP on day 13. CPP is expressed as the difference in time spent on either side of a 2-sided CPP apparatus. Change in the sign of the CPP value reflects a change in preference relative to the previous time point. Data are means ± SE. Letters denote significant differences in treatment × time by 2-way repeated-measures ANOVA. F(3,5) = 3.1 (day 13, SH vs. RH).