Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11202-11207, October 1996 Neurobiology Chronic morphine induces visible changes in the morphology of mesolimbic dopamine neurons (opiate addiction/brain-derived neurotrophic factor/tyrosine hydroxylase/Lucifer yellow/mesolimbic dopamine system) LioRA SKLAIR TAVRON*, WEI-XING SHI, SARAH B. LANE, HERBERT W. HARRISt, BENJAMIN S. BUNNEY, AND ERIC J. NESTLERt Laboratory of Molecular Psychiatry, Departments of Psychiatry and Pharmacology, Yale University School of Medicine and Connecticut Mental Health Center, 34 Park Street, New Haven, CT 06508 Communicated by Paul Greengard, Rockefeller University, New York NY July 5, 1996 (received for review May 21, 1996) ABSTRACT The mesolimbic dopamine system, which structural changes within this brain region (12, 13). Indirect arises in the ventral tegmental area (VTA), is an important support for this possibility is provided by the observation that neural substrate for opiate reinforcement and addiction. chronic morphine treatment results in a 50% impairment in Chronic exposure to opiates is known to produce biochemical axoplasmic transport from the VTA to a major forebrain adaptations in this brain region. We now show that these target, with no change in transport observed in other neural adaptations are associated with structural changes in VTA pathways studied (14). Moreover, intra-VTA infusion of brain- dopamine neurons. Individual VTA neurons in paraformal- derived neurotrophic factor (BDNF) or related neurotrophins, dehyde-fixed brain sections from control or morphine-treated known to support the survival of dopaminergic neurons in cell rats were injected with the fluorescent dye Lucifer yellow. The culture (see Discussion), has been shown recently to prevent identity of the injected cells as dopaminergic or nondopam- the morphine-induced biochemical adaptations in this brain inergic was determined by immunohistochemical labeling of region (15). the sections for tyrosine hydroxylase. Chronic morphine The goal of the present study was to assess directly whether treatment resulted in a mean -25% reduction in the area and the various biochemical adaptations elicited in the VTA by perimeter of VTA dopamine neurons. This reduction in cell chronic morphine administration are associated with morpho- size was prevented by concomitant treatment of rats with logical changes in this brain region. We show here that chronic naltrexone, an opioid receptor antagonist, as well as by exposure to morphine elicits a selective reduction in the size of intra-VTA infusion of brain-derived neurotrophic factor. In VTA dopamine neurons, with no change observed in nondo- contrast, chronic morphine treatment did not alter the size of paminergic neurons in this brain region. Moreover, the reduc- nondopaminergic neurons in the VTA, nor did it affect the tion in size of VTA dopamine neurons is prevented by intra- total number of dopaminergic neurons in this brain region. VTA infusion of BDNF. The results of these studies provide direct evidence for struc- tural alterations in VTA dopamine neurons as a consequence of chronic opiate exposure, which could contribute to changes MATERIALS AND METHODS in mesolimbic dopamine function associated with addiction. In Vivo Drug Treatments. Sprague-Dawley rats (initial weight 100-120 g; Camm Research Institute, Wayne, NJ) were Dopaminergic neurons in the ventral tegmental area (VTA) used in these studies. Morphine was administered chronically, and their projections to the limbic forebrain are important as described (5), by daily subcutaneous implantation of mor- neural substrates for the acute reinforcing properties of opiates phine pellets (containing 75 mg of morphine base; National and other drugs of abuse (1-4). It has been proposed that Institute on Drug Abuse) for 5 days under light halothane chronic exposure to a drug of abuse elicits long-lasting adap- anesthesia. Rats were used 24 hr after the last pellet implan- tations within these same brain regions that underlie changes tation. This treatment paradigm produces morphine tolerance in drug reinforcement mechanisms, as well as drug craving, and dependence based on behavioral, electrophysiological, associated with drug addiction. and biochemical criteria (see ref. 5). Control rats received Among the most consistent drug-induced adaptations in the equivalent implantation of placebo pellets. VTA is induction of tyrosine hydroxylase (TH; refs. 5-8), the Naltrexone (Sigma) was administered daily [50 mg/ml i.p. in rate-limiting enzyme in the biosynthesis of dopamine. In saline and 50 mg/ml in an emulsion of saline and lipid] addition, chronic morphine administration has been shown to immediately before morphine or placebo pellet implantation decrease levels of the three major neurofilament proteins, as described (10). This treatment paradigm has been shown to NF200, NF160, and NF68 (9), and to increase levels of glial completely block the development of morphine tolerance and fibrillary acidic protein (10) in this brain region. Each of these dependence based on behavioral, electrophysiological, and biochemical adaptations occurs specifically in the VTA and is biochemical criteria (see ref. 10). not elicited by chronic exposure to other psychotropic drugs BDNF was provided by Ronald Lindsay (Regeneron Phar- that lack reinforcing properties. In addition, similar adapta- maceuticals, Tarrytown, NY). It was infused at a dose of 2 tions in the VTA are observed in rats after chronic heroin ,ug/day (in 1QmM sodium phosphate, pH 7.4/0.9% NaCl/1% self-administration, indicating that they are induced in re- bovine serum albumin) directly into the VTA by osmotic sponse to self-regulated drug intake (11). minipumps via midline cannulae exactly as described (15). This The lower levels of neurofilament proteins and the recip- rocal increase in glial fibrillary acidic protein present in the Abbreviations: VTA, ventral tegmental area; TH, tyrosine hydroxy- VTA after chronic opiate administration raises the possibility lase; BDNF, brain-derived neurotrophic factor. that chronic exposure to opiates may result in prominent *Present address: Technion Institute of Technology, Bat-Galim, Haifa 31096, Israel. tPresent address: National Institute on Aging, National Institutes of The publication costs of this article were defrayed in part by page charge Health, Bethesda, MD 20892. payment. This article must therefore be hereby marked "advertisement" in ITo whom reprint requests should be addressed. e-mail: eric_nestler@ accordance with 18 U.S.C. §1734 solely to indicate this fact. qm.yale.edu. 11202 Downloaded by guest on September 25, 2021 Neurobiology: Sklair-Tavron et al. Proc. Natl. Acad. Sci. USA 93 (1996) 11203 BDNF dose has been shown to prevent many of the biochem- gated video camera. The morphological parameters of the ical effects elicited in the VTA by chronic morphine administra- neurons were measured by National Institutes of Health IMAGE tion (15). Control rats received intra-VTA infusions of vehicle. version 1.57 software; this was done with the experimenter Measurements of VTA Neuronal Morphology. The mor- blinded to the identity of the neurons as TH+ or TH- and of phology of VTA neurons was assessed by filling individual cells the slices as control or morphine-treated. Cell perimeter and with Lucifer yellow by use of a procedure based on previous area were then calculated. studies (16-19). Rats were deeply anesthetized with chloral In pilot experiments, 400-gm thick oblique midbrain slices hydrate (400 mg/kg i.p.) and then perfused transcardially with containing the VTA from control and morphine-treated rats 50 ml of ice-cold phosphate-buffered saline (PBS), followed by were prepared as described (21). Dopamine neurons, identi- 200 ml of 4% paraformaldehyde in PBS at a rate of 15 ml/min. fied by well-established electrophysiological criteria (for ex- Brains were removed and 100- to 150-,um thick oblique slices ample, see refs. 16 and 21), were recorded in whole cell mode (described below) were obtained through the VTA by use of with electrodes filled with an intracellular solution containing a vibratome. Neurons in the fixed slices were injected with 1% Lucifer yellow. The electrodes were withdrawn after 15 Lucifer yellow under an upright fluorescence microscope. min in cell-attached mode. One to two neurons were injected Individual neurons were visualized with Nomarski optics, per slice. The slices were then fixed in 4% paraformaldehyde, impaled with a glass microelectrode filled with 0.2% Lucifer cut into 50- to 100-gm thick sections, and mounted for analysis yellow in PBS, and injected by applying negative current pulses of cell morphology as described above. (2 Hz, 400 msec, 1 nA) for 5-10 min. After filling 4-8 cells per Counting VTA Dopamine Neurons. Control and morphine- slice, the slices were fixed in 4% paraformaldehyde in PBS treated rats were perfused with paraformaldehyde, as de- overnight at 4°C. scribed above. TH immunohistochemistry was then conducted Oblique brain slices, illustrated in Fig. 1, were used in these on fixed 40-gm thick coronal brain sections, and the number studies according to published procedures (21). In these of TH+ neurons was counted at x400 magnification, exactly as oblique slices, VTA dopamine neurons can be readily distin- described (22). Briefly, TH+ neurons were counted in every guished from substantia nigra dopamine neurons as being 40-,um section throughout the rostral-caudal extent of the located medial to the medial terminal nucleus of the