Proc. Natl. Acad. Sci. USA Vol. 93, pp. 11202-11207, October 1996 Neurobiology

Chronic morphine induces visible changes in the morphology of mesolimbic neurons (opiate /brain-derived neurotrophic factor//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 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 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 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 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 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 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 dopamine neurons as being 40-,um section throughout the rostral-caudal extent of the located medial to the medial terminal nucleus of the accessory ventral midbrain. An immunoreactive area was counted as a optic tract (Fig. 1). The VTA cells injected with Lucifer yellow cell if a region of central pallor, consistent with the absence of were located at -5.3 + 0.2 mm Bregma. staining of a nucleus, could be visualized. The slides were The dopaminergic phenotype of the Lucifer yellow-injected counted in a blind fashion. Regional boundaries of the VTA neurons was determined by TH immunohistochemistry. The were determined by use of neuroanatomical landmarks ac- fixed slices were washed with PBS and permeabilized by 2-3 cording to published criteria (20, 23). The major landmark cycles of freezing (at -70°C) and thawing. The slices were defining the lateral boundary of the VTA was the medial incubated for 18 hr with a mouse monoclonal anti-TH antibody lemniscus. Other major landmarks used to establish level and (1:1500; Incstar, Stillwater, MN) in PBS plus 0.3% Triton orientation included the , the red X-100 at 4°C. The slices were washed three times in PBS at nucleus, and the fasciculus retroflexus. Gross regions of TH room temperature, after which time they were incubated for immunoreactivity were compared and aligned with previously 2.5 hr with Texas Red-conjugated anti-mouse IgG made in published studies of the protein in the rat (23, 24). horse (1:200; Vector Laboratories) in PBS plus 0.3% Triton X-100 and 10% normal horse serum at room temperature. RESULTS Slices were washed three times in PBS, mounted on gelatin- coated slides, dried, dehydrated, cleared in methyl salicilate, Regulation of VTA Neuronal Morphology by Chronic Mor- and coverslipped with DPX (Merck). phine Administration. In the present study, we assessed di- Slices were examined under fluorescence microscopy (Ni- rectly the changes in cellular morphology in the VTA produced kon) to visualize Lucifer yellow-filled neurons and to deter- by chronic morphine administration. We used an approach mine whether the filled neurons were TH+ or TH-. The filled based on previous work (see refs. 16-19), which involved filling neurons were recorded with a Xabion charged coupled device- individual neurons with the fluorescent dye Lucifer yellow in paraformaldehyde-fixed brain slices. The injected cells were then identified as dopaminergic or nondopaminergic by im- munohistochemically labeling the slices for TH. This technique resulted in the reliable labeling of individual neurons and the proximal extents of their processes as visu- alized under fluorescence microscopy. In control rats (Fig. 2A), Lucifer yellow-filled TH' neurons exhibited considerable variability in their shape, although most cells were elliptical with several large caliber proximal processes, consistent with previous descriptions ofthe morphology of VTA dopaminergic neurons (see refs. 16, 17, and 25). In contrast, in morphine- treated rats (Fig. 2B), Lucifer yellow-filled TH' neurons exhibited smaller cell bodies and tended to show a more spherical shape with narrowed proximal processes. Analysis of 41 neurons from seven control and from seven morphine- treated rats revealed a 20-25% reduction in mean cell body area and perimeter elicited by chronic morphine exposure FIG. 1. Schematic illustration of the location of VTA neurons (Table 1). An equivalent (-25%) reduction in the area and sampled in the present study (box). The dotted lines indicate the perimeter of VTA dopamine neurons was observed after positions of oblique brain slices, 100- to 150-,um thick, obtained in a chronic morphine administration when the neurons were plane perpendicular to the printed page. The medial terminal nucleus with Lucifer via electrodes in of the accessory optic tract (mt) was used as the lateral boundary of injected yellow patch-recording living brain slices (data not shown). the VTA. VTA neurons were sampled at -5.3 ± 0.2 mm Bregma, the location of the coronal section shown in the figure. SNR, substantia In addition to a reduction in mean cell body size, there was nigra reticulata; and SNC, substantia nigra compacta. This figure is a dramatic shift in the distribution of cell body sizes between modified from Paxinos and Watson (20). the control and chronic morphine-treated conditions. This is Downloaded by guest on September 25, 2021 11204 Neurobiology: Sklair-Tavron et al. Proc. Natl. Acad. Sci. USA 93 (1996)

A-40-- B40 3-

A 2- 32-

_ _ median 22 I ,

I2 * 12

6-

4-

control morphine control morphine FIG. 3. Distribution of the perimeter of VTA dopamine and nondopamine neurons under control and morphine-treated condi- tions. (A) Dopaminergic (i.e., TH+) neurons; and (B) nondopamin- ergic (TH-) neurons. Bar and cross-hatch show mean ± SEM. *, P < 0.01 by Student's t test. (Table 1). These findings indicate that the effects of morphine on neuronal morphology represent specific effects achieved via the activation of opioid receptors. Interestingly, the chronic morphine-induced decrease in neuronal size was selective for the dopaminergic neurons in the VTA. Analysis of Lucifer yellow-filled TH- neurons, in the FIG. 2. Fluorescence photomicrographs of Lucifer yellow-filled same brain slices that were analyzed for TH+ neurons, re- neurons in the rat VTA. All neurons shown in the figure are dopa- vealed no difference in cell body area or perimeter between minergic (i.e., TH+). (A) Neurons from control rats; (B) neurons from control and morphine-treated rats [area, 38.1 ± 1.5 jim2 (n = morphine-treated rats; (C) neuron from BDNF-treated rat; and (D) 32) after morphine treatment versus 32.9 ± 1.8 (n = 28) neuron from BDNF- and morphine-treated rat. (Bar = 8 ,um.) tLm2 for control; perimeter, 24.2 + 0.7 ,Am (n = 32) after morphine treatment versus 23.4 ± 0.8 ,um (n = 28) for control]. There in which shows that the of all but illustrated Fig. 3A, perimeter also was no difference in the distribution of cell sizes under one of the VTA dopamine neurons of morphine-treated rats these treatment conditions, as illustrated in Fig. 3B. was below the median value exhibited by the neurons from Influence of BDNF on Morphine-Induced Morphological control rats. A similar shift in distribution was observed for cell Changes in the VTA. Based on the ability of BDNF infused body area (data not shown). directly into the VTA to prevent many of the biochemical Chronic morphine exposure also was found to result in a adaptations elicited in the VTA by systemic morphine admin- significant (=30%) reduction in the mean length of dopami- istration (15), we tested whether such BDNF infusions would nergic neuronal processes. The average length of Lucifer also prevent the morphine-induced structural changes in VTA yellow-filled processes in TH+ neurons from control rats was dopamine neurons. As shown in Fig. 2C, intra-VTA infusions 80 ± 10 ,um (n = 25) compared with 56 ± 6 ,um (n = 20) in of BDNF for 10 days did not alter the size of VTA dopamine TH+ neurons from morphine-treated rats (P < 0.03 by Stu- neurons, nor did they appear to influence the shape or dent's t test). arborizations of these cells (see also Table 1). However, 5 days The reduction in cell body area and perimeter elicited by of pretreatment with BDNF, followed by concomitant admin- chronic morphine administration was blocked by concomitant istration of BDNF and morphine for another 5 days, com- treatment of rats with the opioid receptor antagonist naltrex- pletely prevented the morphine-induced reductions in the size one, which by itself had no effect on the size of the neurons of VTA dopamine neurons (Fig. 2D and Table 1). BDNF also appeared to prevent the changes in cell body shape seen with Table 1. Effect of drug treatments on the size of VTA chronic morphine, in that most cells were elliptical. dopamine neurons Lack of Effect of Chronic Morphine Administration on the Treatment* Perimeter, ,um Area, j.m2 Number of VTA Dopamine Neurons. An immunohistochemi- cal analysis of TH was carried out to obtain a more complete ± ± Control 27.5 0.6 (41) 43.5 1.7 (41) assessment of dopaminergic neurons in the VTA. As shown in Morphine 22.1 ± 0.5 (41)t 33.2 ± 1.4 (41)t Fig. 4, TH immunohistochemical staining under low magnifi- ± ± Naltrexone 26.8 2.8 (9) 40.2 8.0 (9) cation revealed no gross abnormalities within the ventral Morphine plus naltrexone 26.4 ± 1.5 (15) 41.6 ± 4.6 (15) midbrain after chronic morphine administration. There also 26.2 ± 2.1 43.3 ± 2.9 Vehicle: (9) (9) were no gross differences evident between the dopaminergic BDNF 27.9 ± 0.8 49.2 ± 2.4 (28) (28) neurons in the VTA under control and morphine-treated ± ± Morphine plus BDNF 27.4 1.1 (19) 48.1 3.5 (19) conditions at this magnification. Data are expressed in means ± SEM (n). Under higher magnification (Fig. 4 B and E), the number of *All animals that were not treated with morphine received subcuta- dopaminergic neurons present throughout the rostral-caudal neous implantation of placebo pellets. Number of animals used were extent of the VTA was counted to determine whether chronic as follows: mor- control, seven; morphine, seven; naltrexone, three; was loss. No phine plus naltrexone, four; vehicle, three; BDNF, five; and morphine morphine treatment associated with neuronal plus BDNF, four. difference was found between the number of VTA dopami- tP < 0.01 by Student's t test. nergic neurons in control and morphine-treated rats, which tAnimals received intra-VTA infusions of vehicle solution (see ref. 15) were, respectively, 7128 ± 120 and 6983 ± 169 (mean ± SEM; as well as subcutaneous implantation of placebo pellets. n = 6). These numbers are consistent with previous reports of Downloaded by guest on September 25, 2021 Neurobiology: Sklair-Tavron et al. Proc. Natl. Acad. Sci. USA 93 (1996) 11205

F:. _ 611F:i. ~ _. , '.. /l

.f.. .4 s. 4 0 .:

..

X

-J.

00040j;0.0;;.00r...... t D:itt '"~~~~~~~~~~~~~~~~.0: .:30.,: i;; ...... ~~~~~~~~... ,Ui4...... ,5, v )g+

...... ,._...

~~~~~~~~~~~L

FIG. 4. Immunohistochemical analysis of TH in the rat VTA. (A and D) Low power (X25) bright field photomicrographs of coronal sections of the VTA and portions of adjacent substantia nigra. Photomicrographs of sections through the VTA at (B and E) X400 and (C and F) high power (x 1000). A, B, and C were derived from control rats; and D, E, and F were derived from morphine-treated rats. The photomicrographs shown correspond to stereotaxic coordinate -5.4 mm Bregma and are representative of the analysis of numerous sections from six control and six morphine-treated rats. [Bar = 94 ,tm (A and D), 13 ,tm (B and E), and 6 ,um (C and F).] VTA dopamine neuronal counts in various rodent strains (see 6-hydroxydopamine) administered in vivo (e.g., see refs. 27- refs. 22 and 24). 35). The present study shows that BDNF can in addition exert At the higher magnification, there did appear to be a small a more subtle effect on these neurons: it cannot only rescue the decrease in the size of dopamine neurons under morphine- cells and prevent cell death, it can also prevent neuroplastic treated conditions (Fig. 4 C and F). However, this was difficult changes that are not associated with cell death in response to to quantify due to the high density of TH+ immunoreactivity chronic drug exposure. in cell bodies and processes in the tissue sections and to the The results of the present study are also of interest in light difficulty in identifying complete (i.e., unsectioned) neurons. of recent work, which suggests that some of the morphine- Indeed, this limitation in the quantitative capability of immu- induced biochemical adaptations in the VTA may be achieved nohistochemistry necessitated the use of Lucifer yellow injec- via morphine perturbation of BDNF signaling cascades. For tions originally. example, chronic morphine administration has been shown to increase the phosphorylation state and activity of extracellular signal regulated kinase (ERK), a major effector of BDNF and DISCUSSION related neurotrophins, selectively in this brain region (36). The results of the present study provide the first direct Direct evidence has been obtained to implicate such regulation demonstration that dopaminergic neurons in the VTA exhibit of ERK in chronic morphine induction of TH in the VTA. In structural changes after chronic morphine administration. The this sense, the ability of BDNF to prevent some of morphine's observed reduction in cell size is consistent with earlier effects may represent not only a pharmacological intervention, speculation (12, 13), which was based on adaptations in but also an intervention at or near the pathophysiological cytoskeletal proteins (9, 10) and on the impairment in axo- mechanisms that mediate some of the long-term actions of plasmic transport (14) in this brain region associated with morphine in this brain region. In addition, these studies suggest chronic morphine exposure. In the present study, we show that possible approaches to the development of novel pharmaco- this reduction in cell size is specific to dopaminergic neurons logical treatments of opiate addiction; they raise the possibility within this brain region and requires the specific action of that agents that activate the BDNF signaling cascade may be morphine at opioid receptors. At the same time, this reduction useful in treating certain sequelae of long-term opiate exposure. in cell size was not associated with gross abnormalities in the The morphine-induced reduction in the size of VTA dopa- VTA, nor was it associated with death of VTA dopamine mine neurons did not appear to be due to a different rate of neurons. Since other drugs of abuse are known to produce diffusion of the Lucifer yellow in the neurons between control some similar biochemical adaptations in the VTA compared and morphine-treated conditions. This is because the dye was with morphine (see Introduction), it would be interesting in injected into individual neurons visualized under fluorescent future studies to determine whether the drugs also produce microscopy, and was observed to completely fill all of the similar effects on the morphology of VTA dopamine neurons. neurons sampled during the injection process. However, we We also show in the present study that the morphine- cannot exclude the possibility that a different rate of diffusion induced changes in the size of VTA dopamine neurons can be influenced the morphine-induced reduction in the mean prevented by intra-VTA infusion of BDNF. VTA dopamine length of VTA neuronal processes observed in these studies. neurons are known to express TrkB, the protein tyrosine A major question raised by the present findings is: What are kinase receptor that exhibits high affinity for BDNF (26). the functional consequences of the morphine-induced struc- Moreover, BDNF has been shown to enhance the survival of tural changes observed in VTA dopamine neurons? We know midbrain dopamine and other neurons in cell culture in vitro that opiates acutely activate VTA dopamine neurons, an effect and to protect the neurons from neurotoxins (e.g., MPTP or mediated, at least in brain slices, via inhibition of inhibitory Downloaded by guest on September 25, 2021 11206 Neurobiology: Sklair-Tavron et al. Proc. Natl. Acad. Sci. USA 93 (1996) GABAergic interneurons (37). However, the electrophysio- 2. Kuhar, M. J., Ritz, M. C. & Boja, J. W. (1991) Trends Neurosci. logical effects of chronic opiate exposure on these neurons 14, 299-302. have received virtually no attention to date. In addition, we do 3. Koob, G. F. (1992) Trends Pharmacol. Sci. 13, 177-184. not know whether the structural changes in VTA dopamine 4. Self, D. W. & Nestler, E. J. (1995) Annu. Rev. Neurosci. 18, 463-495. neurons seen after chronic morphine administration are me- 5. Beitner-Johnson, D. & Nestler, E. J. (1991) J. Neurochem. 57, diated via transsynaptic effects of morphine through the 344-347. GABAergic neurons or via direct effects of morphine on the 6. Hurd, Y. L., Linefors, N., Brodin, E., Brene, S., Persson, H., dopaminergic neurons. Evidence for such direct effects is the Ungerstedt, U. & Hokfelt, T. (1992) Brain Res. 578, 317-326. apparent expression of opioid receptors by VTA dopamine 7. Sorg, B. A., Chen, S.-Y. & Kalivas, P. W. (1993) J. Pharmacol. neurons (38) as well as the synaptic specializations between Exp. Ther. 266, 424-430. these neurons and enkephalin-containing nerve terminals 8. Vrana, S. L., Vrana, K. E., Koves, T. R., Smith, J. E. & Dworkin, observed by electron microscopy (39). S. I. (1993) J. Neurochem. 61, 2262-2268. One possible model of opiate regulation of the VTA is that 9. Beitner-Johnson, D., Guitart, X. & Nestler, E. J. (1992) J. Neu- rosci. 12, 2165-2176. opiates acutely activate VTA dopamine neurons with an 10. Beitner-Johnson, D., Guitart, X. & Nestler, E. J. (1993) J. Neu- intensity and persistence not seen under normal conditions. rochem. 61, 1766-1773. After chronic exposure, compensatory adaptations occur in 11. Self, D. W., McClenahan, A. W., Beitner-Johnson, D., Terwill- the VTA to oppose this activation. In the absence of drug, iger, R. Z. & Nestler, E. J. (1995) Synapse 21, 312-318. these changes would contribute to deficient VTA neuronal 12. Nestler, E. J. (1992) J. Neurosci. 12, 2439-2450. function and possibly to an aversive state postulated to occur 13. Nestler, E. J., Hope, B. T. & Widnell, K. L. (1993) Neuron 11, during (3, 4, 40-42). According to this model, 995-1006. the structural alterations in VTA neurons could reflect an 14. Beitner-Johnson, D. & Nestler, E. J. (1993) NeuroReport 5, impairment in the functional capacity of these cells, which 57-60. 15. Berhow, M. T., Russell, D. S., Terwilliger, R. Z., Beitner-John- would thereby contribute to motivational dependence. son, D., Self, D. W., Lindsay, R. M. & Nestler, E. J. (1995) It is also possible that the changes observed in the morphol- Neuroscience 68, 969-979. ogy of VTA dopamine neurons after chronic morphine expo- 16. Grace, A. A. & Bunney, B. S. (1983) Neuroscience 10, 317-331. sure represent a form of neural injury. Indirect support for this 17. Grace, A. A. & Onn, S.-P. (1989) J. Neurosci. 9, 3463-3481. possibility comes from prior observations that chronic mor- 18. Onn, S.-P. & Grace, A. A. (1994) J. Neurophysiol. 71, 1917-1934. phine administration, as well as heroin self-administration, 19. Shi, W. X. & Rayport, S. (1994) J. Neurosci. 14, 4548-4560. results in reduced levels of neurofilaments and elevated levels 20. Paxinos, G. & Watson, C. (1986) The Rat Brain in Stereotaxic of glial fibrillary acidic protein selectively within the VTA (see Coordinates (Academic, Sydney), 2nd Ed.. Introduction). Glial fibrillary acidic protein is an astrocyte- 21. Shi, W. X. & Bunney, B. S. (1991) Synapse 9, 157-164. in concert 22. Harris, H. W. & Nestler, E. J. (1996) Brain Res. 706, 1-12. enriched protein often induced with astrocyte 23. Hokfelt, T., Mortensson, R., Bjorkland, A., Kleinau, S. & Gold- activation or proliferation. While this would represent the first stein, M. (1984) in Handbook of Chemical Neuroanatomy, eds. evidence for neural injury in response to chronic opiate Bjorkland, A. & Hokfelt, T. (Elsevier Science, Amsterdam), pp. administration, damage to midbrain dopamine neurons and 277-379. concomitant induction of glial fibrillary acidic protein in select 24. German, D. C. & Manaye, K. F. (1993) J. Comp. Neurol. 331, brain regions have been well-documented in response to 297-309. chronic exposure to , particularly and 25. Oades, R. D. & Halliday, G. M. (1987) Brain Res. Rev. 12, its derivatives (for example, see refs. 43 and 44). However, it 117-165. is important to emphasize that the altered levels of neurofila- 26. Seroogy, K. B. & Gall, C. M. (1993) Exp. Neurol. 124, 119-128. ments and fibrillary acidic protein and the changes in 27. Altar, C. A., Boylan, C. B., Fritsche, M., Jones, B. E., Jackson, C., glial Wiegand, S. J., Lindsay, R. M. & Hyman, C. (1994) J. Neuro- VTA neuronal morphology seen after morphine administra- chem. 63, 1021-1032. tion, while perhaps suggestive of neural injury, could instead 28. Beck, K. D., Knusel, B. & Hefti, F. (1993) Neuroscience 52, reflect neuroplastic changes associated with increased or 855-866. decreased functioning of these cells. Indeed, one must be very 29. Fadda, E., Negro, A., Facci, L. & Skaper, S. D. (1993) Neurosci. cautious in interpreting the observed changes as reflecting Lett. 159, 147-150. neural injury, given the current and successful practice of 30. Hyman, C., Juhasz, M., Jackson, C., Wright, P., Ip, N. Y. & treating opiate addiction with opiate agonists, such as meth- Lindsay, R. M. (1994) J. Neurosci. 14, 335-347. adone (see ref. 45). 31. Skaper, S. D., Negro, A., Facci, L. & Dal Toso, R. (1993) While more work is needed to the of J. Neurosci. Res. 34, 478-487. clearly study validity 32. Spenger, C., Hyman, C., Studer, L., Egli, M., Evtouchenko, L., these and alternative interpretations, this study highlights the Jackson, C., Dahl-Jorgensen, A., Lindsay, R. M. & Seiler, R. W. complex types of adaptations that can be elicited in the brain (1995) Exp. Neurol. 133, 50-63. following a chronic drug treatment. It also adds to the growing 33. Spina, M. B., Squinto, S. P., Miller, J., Lindsay, R. M. & Hyman, evidence that many examples of plasticity in the functioning of C. (1992) J. Neurochem. 59, 99-106. neurons in the fully differentiated adult brain are associated 34. Studer, L., Spenger, C., Seiler, R. W., Altar, C. A., Lindsay, R. M. with plasticity that can be visualized at the anatomical level & Hyman, C. (1995) Eur. J. Neurosci. 7, 223-233. (for example, see refs. 46-51). As the array of molecular and 35. Zhou, J., Bradford, H. F. & Stern, G. M. (1994) Brain Res. 656, cellular adaptations induced in the VTA and other brain 147-156. chronic exposure is these 36. Berhow, M. T., Hiroi, N. & Nestler, E. J. (1996) J. Neurosci. 16, regions by opiate delineated, changes 4707-4715. can be related to specific behavioral features of addiction and 37. Johnson, S. W. & North, R. A. (1992) J. Neurosci. 12, 483-488. may direct the development of fundamentally new approaches 38. Mansour, A., Watson, S. J. & Akil, H. (1995) Trends Neurosci. 18, to the treatment of addictive disorders. 69-70. 39. Sesack, S. R. & Pickel, V. M. (1995) Brain Res. 672, 261-275. This work was supported by U.S. Public Health Service Grants 40. Harris, G. C. & Aston-Jones, G. (1994) Nature (London) 371, DA08227, DA10160, and DA00203 and by the Abraham Ribicoff 155-157. Research Facilities of the Connecticut Mental Health Center, State of 41. Koob, G. F. (1996) Neuron 16, 893-896. Connecticut Department of Mental Health and Addiction Services. 42. Nestler, E. J. (1996) Neuron 16, 897-900. 43. Switzer, R. C. (1993) Ann. N.Y Acad. Sci. 679, 341-348. 1. Wise, R. A. (1990) in Psychotropic Drugs of Abuse, ed. Balfour, 44. O'Callaghan, J. P. & Miller, D. B. (1994)J. Pharmacol. Exp. Ther. D. J. K. (Pergamon, Oxford), pp. 23-57. 270, 741-751. Downloaded by guest on September 25, 2021 Neurobiology: Sklair-Tavron et al. Proc. Natl. Acad. Sci. USA 93 (1996) 11207

45. Novick, D. M., Richman, B. L., Friedman, J. M., Friedman, J. E., 48. Rocha, M. & Sur, M. (1995) Proc. Natl. Acad. Sci. USA 92, Fried, C., Wilson, J. P., Townley, A. & Kreek, M. J. (1993) Drug 8026-8030. Depend. 33, 235-245. 49. Segal, M. (1995) Neurosci. Lett. 193, 73-76. 46. Hosokawa, T., Rusakov, D. A., Bliss, T. V. P. & Fine, A. (1995) 50. Stein-Behrens, B., Mattson, M. P., Chang, I., Yeh, M. & Sapol- J. Neurosci. 15, 5560-5573. sky, R. (1994) J. Neurosci. 14, 5373-5380. 47. Marty, S. & Peschanski, M. (1995) J. Comp. Neurol. 356, 523-536. 51. Woolley, C. S. & McEwen, B. S. (1994)J. Neurosci. 14, 7680-7687. Downloaded by guest on September 25, 2021