ACKNOWLEDGEMENTS

1. Innervation Of The Ventral Tegmental Area From Lateral Hypothalamic Orexin Neurons Is Necessary For Acquisition Of Morphine Conditioned Place Preference • M. Wimmer1, G.C. Harris1 and G. Aston-Jones2 • 1.Neuroscience Graduate Program, University of Pennsylvania, Philadelphia, PA • 2. Department of Neuroscience, Medical University of South Carolina, Charleston, SC INTRODUCTION LH Orexin Projection to VTA is Critical for Acquisition of Morphine CPP Contralateral disconnection experiment shows that orexin projections from LH to VTA are needed for acquisition of morphine CPP. Bilateral Lesions of LH Orexin Neurons Blocks Acquisition of Morphine CPP Bilateral lesions of LH orexin neurons blocked acquisition of morphine CPP while unilateral lesions and lesions outside of the LH did not affect morphine conditioning. LH Orexin Neurons are Stimulated During Drug Pairing LH orexin neurons display a significant increase in Fos activation when animals are injected with morphine in the CPP environment. There was no increase in activation in PFA orexin neurons. Previously, we found that the number of Fos+ neurons in the lateral hypothalamus (LH) was directly correlated to the amount of place preference shown for food- or morphine-associated cues during conditioned place preference (CPP) testing. The LH region is rich in orexin/hypocretin. Orexin has been implicated in feeding behavior, active waking and arousal states. Orexin neurons in the LH send projections to brain areas involved in reward related activity, including the nucleus accumbens and ventral tegmental area (VTA) (Peyron et al., 1998; Fadel and Deutch, 2002). Recently, we showed that orexin neurons in the LH are involved in reward processing. Chemical activation of these neurons reinstated a previously extinguished conditioned place preference for morphine (Harris et al., 2005). b a LH PFA DMH Conditioned place preference (CPP) scores (a) and orexin neuronal cell counts (b) for animals given a unilateral NMDA lesion of the LH (or vehicle control injection) and daily injections of SB 33487 in the contralateral VTA prior to morphine injections during CPP training. Control animals received vehicle instead of NMDA in the LH and received the same SB injections in the VTA. *P<.01 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 post-conditioning (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 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 Distribution of Orexin neurons in the Hypothalamus Photomicrographs of frontal sections of the LH showing neurons doubly labeled for Fos (black nuclei) and orexin (brown cytoplasm). Orexin neurons in the LH send excitatory projections to both dopamine (DA) and GABA neurons in the VTA, indicating a possible mechanism whereby LH stimulation could influence motivated behavior. 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). Fos induction in orexin neurons in the LH (a) or PFA (b) following morphine administration (8mg/kg, ip) in the home cage, CPP box , or following saline in the CPP box, on the first and third conditioning days. *significantly different from other groups P<.01. GOALS In the present experiments we investigated the role of the LH orexin neurons in the acquisition of morphine conditioned place preference. c CONTROL LESION f CONCLUSIONS Place Conditioning Procedure • These data indicate that LH orexin neurons play an important role in the conditioning of environmental cues to drug reward. • Our results suggest that orexinergic projections to the VTA are necessary for learning or memory of morphine place preference. 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. Place conditioning was done using a standard balanced design. Conditioning occurred on each of 3 days, and injections of morphine (8 mg/kg, ip) or saline alternated between morning and afternoon sessions. Male Sprague-Dawley rats 300-350g were used in all experiments. Photomicrographs of the lateral hypothalamus and perifonical area taken from representative control and lesioned animals. The sections were stained for orexin. ACKNOWLEDGEMENTS Research supported by PHS grant DA06214 and DA017289