Lateral hypothalamic orexin neurons are critically involved in learning to associate an environment with morphine reward

Abstract

Previously, we reported that lateral hypothalamic (LH) orexin neurons are stimulated in proportion to the preference shown for reward-associated cues during conditioned place preference (CPP) testing. Here, we examine for the first time the role of these neurons in the acquisition of morphine CPP. Results show that LH orexin neurons, but not those in the perifornical area (PFA), are stimulated during conditioning when morphine is given in a novel drug-paired environment (CPP compartment) but not when given in the home cage, nor when saline was given in the CPP environment. Furthermore, bilateral excitotoxic lesions of the LH orexin area completely blocked the acquisition of morphine CPP. Lesions that spared LH orexin neurons had no effect. Orexin neurons in the LH project to the ventral tegmental area (VTA), an area important in the acquisition of morphine CPP. Therefore, we investigated the importance of the LH orexin connection to the VTA in the acquisition of a morphine CPP using a disconnection technique involving a unilateral excitotoxic lesion of LH orexin neurons and contralateral blockade of VTA orexin receptors. Results indicated that a unilateral LH orexin lesion together with a microinjection of the orexin A antagonist (SB 334867) into the contralateral VTA prior to each morphine-pairing session was sufficient to block the development of a morphine CPP. Either of these treatments by themselves was not sufficient to block CPP development. These results demonstrate the importance of LH orexin neurons and their projections to the VTA in the formation of associations between environmental cues and drug reward.

Figure 1

Activation (as indicated by Fos immunoreactivity) of orexin neurons in the lateral hypothalamus (LH) (a) and perifornical area (PFA) (b) following morphine administration in the home cage or conditioned place preference box (CPP), or following saline in the CPP box, on the first and third conditioning days. All animals in each group received a single injection of morphine (10 mg/kg) or saline and were exposed to the CPP environment, or returned to their home cage, for 30 min. Animals were perfused 2 hours after drug or saline conditioning, and brains were processed for orexin and Fos immunohistochemistry. Standard daily alternating injections of morphine and saline were given to all Day 3 animals on Days 1 and 2 of the procedure. *significantly different from other groups P<.01. (c) Photomicrographs of the lateral hypothalamus taken from representative animals on Day 1 of drug or saline exposure. Arrows indicate orexin positive neurons that also were Fos positive. In all cases medial is on the left. f=fornix, LH=lateral hypothalamus.

Figure 2

Conditioned place preference scores (a) and orexin-positive neuronal cell counts (b) for animals given bilateral ibotenic acid microinjections in the lateral hypothalamus (LH). (a) Preference scores were calculated by subtracting the time spent in the morphine-paired chamber on the preconditioning day from the time spent in that chamber on the postconditioning (test) day. Scores represent group means ± standard errors. (b) Counts of orexin-positive neurons in LH or perifornical area (PFA) from 6 adjacent 40 um-thick sections in each animal at the level of the neurotoxin injection. Control animals were given similar ibotenic acid injections that were either dorsal or caudal to the orexin cell field. Sections from control animals were chosen to match the same anterior/posterior levels as the animals with orexin neuron lesions (*P<.01, lesioned group n=7, control n=9).

Figure 3

Photomicrographs (a) and diagram (b) illustrating the ibotenic acid lesions of the lateral hypothalamus. (a) Photomicrographs of an effective ibotenic acid lesion from an animal that failed to learn morphine CPP (top photograph) and an ineffective ventral control lesion from an animal that developed a morphine CPP (bottom photograph). (b) Diagram showing the extent of the effective ibotenic acid LH orexin lesions. The gray shaded area indicates the largest extent of the lesions and the black area indicates the smallest. Drawings were adapted from Paxinos and Watson 1998. ZI=zona incerta, DMH=dorsomedial hypothalamus, LH=lateral hypothalamus, PFA perifornical area, Me= medial amygdala nuc., VMH=ventomedial hypothalamus, Arc=arcuate nuc., f=fornix.

Figure 4

Conditioned place preference (CPP) scores (a) and orexin neuronal cell counts (b) for animals given unilateral NMDA injections in the LH and microinjections of SB 334867 in the contralateral VTA during CPP training. (a) Preference scores were calculated by subtracting the time spent in the morphine-paired chamber during the preconditioning day from the time spent in that chamber on the test day (i.e., post-conditioning). Control animals received vehicle instead of NMDA in the LH and received the same SB injections in the contralateral VTA. Scores represent group means ± standard errors. (b) Neuronal cell counts of surviving orexin neurons from 6 adjacent 40 um-thick sections at the level of the neurotoxin injection from animals with effective lesions. Control refers to the number of orexin neurons found on the non-lesioned side in the same slices (*P<.01, n=9). (c) Photomicrographs of an effective NMDA lesion from one experimental animal. f=fornix, PFA=perifornical area, LH=lateral hypothalamus (d) Diagram of VTA injection sites. The grey stars indicate effective sites of SB 334867 injection when combined with effective LH orexin lesion, black circles indicate SB injection sites in animals without LH lesions, and the white squares indicate ineffective SB injection sites in animals with LH orexin lesions. SN=substantia nigra, RN=red nucleus. Drawings were adapted from Paxinos and Watson 1998.

Figure 5

Diagram showing the extent of the effective NMDA LH orexin lesions. The black shaded area indicates the largest extent of the lesions. Drawings were adapted from Paxinos and Watson 1998. ZI=zona incerta, DMH=dorsomedial hypothalamus, LH=lateral hypothalamus, PFA perifornical area, Me= medial amygdala nuc., VMH=ventomedial hypothalamus, Arc=arcuate nucleus, f=fornix.