Brain Research, 289 (1983) 223-234 223 Elsevier Chronic Naltrexone Increases Opiate Binding in Brain and Produces Supersensitivity to Morphine in the Locus Coeruleus of the Rat M. T. BARDO*, R. K. BHATNAGAR and G. F. GEBHART** Department of Pharmacology, University of lowa, Iowa City, IA 52242 (U.S.A.) (Accepted May 3rd, 1983) Key words: naltrexone -- morphine -- opiate receptor -- a-adrenergic receptor -- locus coeruleus -- receptor supersensitivity Rats were implanted subcutaneously for 2-4 weeks with slow-release pellets of naltrexone (10 rag) or placebo and then the pellets were removed. One day after removal of the pellet, animals were either (1) sacrificed and various CNS regions examined for specific binding of [3H]naloxone, [3H]etorphine or [3H]rauwolscine or (2) they were anesthetized and prepared acutely for assessing mor- phine-induced changes in the spontaneous activity of neurons in the locus coeruleus (LC). Naltrexone treatment significantly in- creased the number of specific binding sites for opiates, but not for a2-adrenergic antagonists, in spinal cord, hypothalamus, striatum and cortex. Specific binding of [3H]naloxone was also increased in the LC. The spontaneous activity of neurons in the LC was reduced by the chronic naltrexone treatment, suggesting that these neurons became supersensitive to the tonic inhibitory effect of endogenous opioid peptides. Moreover, neurons in the LC of chronic naltrexone-treated rats exhibited an enhanced response to the inhibitory ef- fects of morphine administered systemically. These results demonstrate that chronic opiate receptor blockade increases the number of receptor sites for morphine and that this increase in receptors is accompanied by a neuronal supersensitivity in the LC to morphine which can be assessed electrophysiologically. INTRODUCTION The chronic administration of drugs which block the action of a neurotransmitter may produce an in- crease in the number of postsynaptic receptor sites for that neurotransmitter. This increase in receptors is often accompanied by an enhanced response of the postsynaptic neuron to the neurotransmitter (i.e. su- persensitivity). Increase in the number of receptor sites following the chronic administration of receptor antagonists have been demonstrated for a variety of classical neurotransmitter systems, including dop- aminergic 11,34.46, noradrenergic63 and choliner- gic8,32, 48 systems. In general, the increase in these neurotransmitter receptor sites is accompanied by a supersensitivity to the neurotransmitter which is as- sessable electrophysiologicallylO. Recent evidence indicates that opioid neuronal systems also may adapt similarly to the chronic ad- ministration of opiate antagonists. The chronic ad- ministration of naloxone produces an increase in the number of opiate receptors which preferentially bind morphine and naloxone (i.e./t-type opiate receptors) in the brains of infantS, 7 and adult29,45, 66 animals. However, it is not known presently whether this in- crease in opiate receptors is also accompanied by a neuronal supersensitivity to opiate agonists. The brainstem locus coeruleus (LC) offers a poten- tially useful neuronal system for assessing the effects of chronic opiate blockade on subsequent electro- physiologic responses to opiates. The LC is involved functionally in a wide range of physiologic and be- havioral processes 2 and is thought to have a modula- tory influence on brainstem neurons which are in- volved directly in nociceptive and antinociceptive mechanisms20. There is a dense population of opiate receptors located in the LC 3.6,39,40 and the norepi- nephrine-containing cell bodies and fibers within this nucleus are innervated by enkephalinergic and endo- phinergic terminals44.49, 53.59. Although the response * Present Address: Department of Psychology, University of Kentucky, Lexington, KY 40506, U.S.A. ** To whom requests for reprints should be addressed. 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
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`224 of neurons to opiates may be characterized as hetero- genous 20.z7, neurons within the LC respond consis- tently to opiates and opioid peptides by decreasing their spontaneous firing rate 1.9,26,28,65. Thus, opiate- induced depression of neuronal activity in the LC might be potentiated following the chronic adminis- tration of opiate antagonists. In the present report, we examined the effect of chronic naltrexone treatment on the specific binding of opiates to various CNS regions and on the re- sponse of neurons in the LC to systemically adminis- tered morphine. The results presented here demon- strate that an increase in the number of opiate recep- tors (#-type) following chronic naltrexone blockade is accompanied also by a neuronal supersensitivity in the LC to morphine assessed electrophysiologically. METHODS Animals The animals were adult male Sprague-Dawley rats (Harlan, Madison, WI) weighing between 200 and 250 g at the start of the experiments. Food and water were available continuously under group housing conditions. Naltrexone pellets Long-acting naltrexone pellets were manufactured by the method of Misra and Pontani33. Briefly, nal- trexone free base (620 mg) was mixed with cholester- ol (2230 mg), glyceryltristerate (248 mg) and CHCI 3 (62 ml) in a round-bottom flask. The mixture was evaporated to dryness in a flash evaporator and pow- dered with mortar and pestle. Portions (50 mg) of the powder were then pressed into fiat-face pellets (7 mm diameter, 1 mm thick) using a Carver press. Thus, each 50 mg pellet contained 10 mg of naltrex- one as base. Placebo pellets were manufactured as above, except that naltrexone was omitted from the mixture. Each animal was anesthetized lightly with ether and a single pellet of either naltrexone or placebo was implanted subcutaneously in the mid-scapular region. At 2 or 4 weeks after implantation, the pellet was removed and, one day later, each animal was prepared for assessing either opiate binding or super- sensitivity to morphine electrophysiologically. A pre- liminary experiment confirmed the results of Misra and Pontani33; we found that the naltrexone pellets antagonized completely the analgetic effect of mor- phine (10 mg/kg) in the hot plate test for at least 20 days and that this antagonist action was dissipated completely within 24 h after removal of the pellet. Opiate and a 2 receptor assays One day after pellet removal, some animals were decapitated and their brains and spinal cords remov- ed rapidly. Brains were dissected on a cold plate (4 °C) into hypothalamus, striatum and frontal cor- tex according to the method of Glowinski and Iver- sen zl. Tissue was frozen immediately on dry ice and stored at --80 °C for 1-3 months. Following storage, the tissue was prepared for assaying the specific bind- ing of either the opiate antagonist [3H]naloxone (New England Nuclear, 50 Ci/mmol), the opiate ag- onist [3H]etorphine (Amersham, 51 Ci/mmol) or the az-noradrenergic antagonist [3H]rauwolscine (New England Nuclear, 88 Ci/mmol). For determining the specific binding of [3H]naloxone, tissue was homoge- nized (Polytron, setting 7, 10 s) in 200 vols. of ice- cold 50 mM Tris-HCl buffer (pH 7,4) containing 100 mM NaC1. Portions (0.95 ml) of the tissue homoge- nate were incubated at 0 °C for 180 min with 1 nM [3H]naloxone in the presence or absence of 100 nM levallorphan tartrate (Hoffman-La Roche). The final incubation volume was 1 ml. For Scatchard analyses, tissue was pooled from 3-4 animals and incubated with varying concentrations of [3H]naloxone (0.1-4.0 nM). Incubation was terminated by filtra- tion under vacuum pressure over Whatman glass fi- ber circles (GF/B) and washed twice with 5 ml vols. of ice-cold Tris buffer, The filters with washed tissue fragments were soaked in 8 ml Aquasol-2 (New-En- gland Nuclear) and the radioactivity was determined by liquid scintillation spectrometry. All samples were assayed in duplicate. The specific binding of [3HI na- loxone was calculated as the radioactivity obtained in the absence of levallorphan (total binding) minus the radioactivity obtained in the presence of levatlor- phan (non-specific binding). The receptor binding as- says for [3H]etorphine and [3H]rauwolscine were similar to that described for [3H]naloxone. In deter- mining the specific binding of [3H]etorphine, the Tris buffer did not contain NaCi and the opiate agonist le- vorphanol tartrate (Hoffman-LaRoche) was substi- tuted for the antagonist levallorphan: for determin-
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`ing the specific binding of [3H]rauwolscine, 50 mM Na,K-phosphate buffer (pH 7.4) was substituted for Tris buffer and phentolamine hydrochloride (Ciba- Geigy) was substituted for levallorphan. In another experiment, the specific binding of 1 nM [3H]naloxone in the LC was determined in tissue microdissected according to the method of Palko- vits35, 36. The brainstem was frozen, mounted and sliced in a freezer-microtome (American Optical, Model 840C). Each brain slice (300/~m thickness) was mounted on a cold glass slide and microdissected at --10 °C using a stainless steel needle (800/xm inter- nal diameter) which was kept on dry ice. Bilateral punches were made on each of 3 serial slices through the LC. The tissue was pooled from two animals, ho- mogenized by sonication in 4.5 ml of ice-cold Tris buffer, and assayed for [3H]naloxone binding. With the microdissection procedure, the tissue was pri- marily, but not solely, from within the anatomical borders of the LC. The glass fiber filters used in the assay procedure (Whatman GF/C) were prewashed in water saturated with amyl alcohol in order to re- duce the binding of [3H]naloxone to the filter 6. Pro- tein concentrations were determined by the method of Lowry 31. Electrophysiologic recording One day after pellet removal, some animals were prepared acutely for assessing the effect of opiates on extracellular single-unit activity within the LC. Each animal was anesthetized with chloral hydrate (400 mg/kg, i.p.) and the femoral vein was cannulated with polyethylene tubing (PE-10) attached to a two- way valve which allowed for delivery of drugs intra- venously. A glass microelectrode (1-2/xm tip, 1-5 Mff2 impedance at 1000 Hz) filled with 3 M NaC1/Fast Green dye was placed stereotaxically in the LC with the incisor bar at 0 mm. The coordinates were 1.1-1.3 mm lateral to lambda, 1.2-1.6 mm posterior to lambda and 5.8-6.8 mm below the dura. We used the caudal electrode approach described by Guye- net 25 in order to spare the transverse sinus dorsal to the LC, as severe blood loss can alter the firing rate of neurons in the LC 55. A large portion of the skull was removed so that the dura posterior to the sinus could be slit open laterally with a small knife. The electrode was lowered 4.5 mm through the dural opening and then slowly moved anterior to the ap- 225 propriate AP coordinate. With this caudal approach, we found that all electrode tracts in the AP plane were confined to the cerebellum and that, with expe- rience, the transverse sinus was not damaged. After the electrode was in place, neuronal activity was am- plified, monitored oscillographically, and discrimi- nated using conventional techniques. The output of the window discriminator was counted and the data preserved on a chart recorder. The spontaneous fir- ing rate was recorded, counted and averaged for 5 min. Morphine sulfate (Merck) was subsequently ad- ministered in incremental doses (0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mg/kg, i.v.), followed by naloxone hydro- chloride (Dupont) given also in incremental doses (0.01, 0.02, 0.04 and 0.06 mg/kg, i.v.). Each dose was given at 2 min intervals and each was followed by 0.10 ml saline to insure that the drug was flushed en- tirely from the i.v. femoral cannula. Only one unit was recorded from each animal. At the end of each recording session, Fast Green dye was iontophoresed (40/~A, 10 min) at the recording site and the animals were perfused transcardially with 10% buffered for- malin. The brain was then removed, sectioned on a freezer-microtome and stained with cresyl violet, in order to verify electrode tip placement. Statistical analyses All data were analyzed with unbalanced factorial and split-plot analyses of variance, except for the data obtained from the receptor binding saturation curves 12. The saturation curves were subjected to Scatchard analyses, using linear regression plots to estimate receptor affinity (Kd), receptor number (Bmax) and strength of correlation (r2). RESULTS Opiate and a2-binding The specific binding of [3H]naloxone (1 nM) was increased significantly in each CNS region examined following 2 weeks of chronic naltrexone blockade (Fig. 1). The increase in opiate binding was 59% in spinal cord, 42% in hypothalamus, 39% in striatum and 51% in frontal cortex. There was no further in- crease in specific binding of [3H]naloxone when the naltrexone pellet was implanted for 4 weeks rather than 2 weeks. Although chronic naltrexone block- ade clearly increased specific opiate binding, spe-
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`226 35 30, ~' Z5 20 # I0 ] PLACEBO []NAL TREXONE SPINAL CORD HYPOTH,II~LAMUS iiiii~!iiii ii!i!iii!i!i :x: iiiiiiiiiiii iiiiiiiii! ! :::::: !ii~i~iiii ii~iiiiiiiii iiiiiiiii!i! ::::::::::: ;:::L::::: [:i:i:i:ili STRIAT UM FRONTAL CORTEX Fig. 1. Specific binding of [3H]naloxone in the CNS in animals implanted with either a placebo or a naltrexone pellet for 2 weeks, Values reflect binding 1 day after removal of the pel- lets. Each mean and S.E.M. is based on 8 rats. Asterisks rep- resent significant differences from the placebo control group, P < 0.001. cific binding of the a2-adrenergic antagonist [3H]- rauwolscine was not changed by the chronic naltrex- one in any of the CNS regions examined (data not shown). Scatchard analyses of the specific binding of [3H]naloxone (0.1-4.0 nM) revealed that the in- crease in ligand binding reflected largely an increase in the number of binding sites for opiates rather than a change in receptor affinity for opiates (Fig. 2). The Bma x values, which reflect the number of binding sites, were increased significantly by 64% in spinal cord, 41% in hypothalamus, 28% in striatum and 68% in frontal cortex (P values < 0.05). In addition to a change in Bmax, chronic naltrexone treatment also tended to produce a slight increase in Ka values in each CNS region examined (5% in spinal cord, 17% in hypothalamus, 12% in striatum and 6% in frontal cortex), indicating that receptor affinity was slightly tu t~ .40 .30 ,20 .10 \ o (3 .50 .20 .10 I SPINAL CORD I HYPOTHALAMUS 1 • NALTREXONE ] I o PLACEBO I KD=.74 , BMAX=33.4, R2=.96 b""'~/" K 6:5 B 23 7 R 2 96 2 " w~O / D=" ' MAX= " ' =" KD=.85, BMAX =lB.4, R =.99 ,,0 ~ / KD=.81, BMAX=II.2, R2=.9B ~0.,. x , , STRIATUM KD=.77, BMAX=54.3, R2=.99 ['~'~ ",,,,~/KD=.69, BMAX=42.3, R =.99 0 I0 20 30 40 0 FRONTAL CORTEX KD=.55, BMAX =29.4,R2=.99 jKD=.SZ, BMA×,~.5, R =.99 \./\ IO 20 30 40 3H-NALOXONE BOUND (pmol/g) Fig. 2. Scatchard plots of specific [3H]naloxone bound in the CNS of animals implanted with either a placebo or a naltrexone pellet for 2 weeks. Each plot was derived by pooling tissue from 3-4 animals. Values reflect binding 1 day after removal of the pellets. The K a values are expressed in nM and the Bma x values are expressed in pmol/g wet weight tissue.
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`.30 FRONTAL CORTEX ~e NALTREXONE: K D='42' EL i .20 • ~Y B MAX = 24"6' R?" ="91 PLACEBO: KD=.40, :, \ - \ ;'e \ "e \. \\\ II ~ , "\'i "° \~\\ • \\xl I I I o io 20 3o 5H-ETORPHINE BOUND (pmol/g) Fig. 3. Scatchard plots of specific [3H]etorphine bound in fron- tal cortex of animals implanted with a placebo or a naltrexone pellet for 2 weeks. Values reflect binding 1 day after removal of the pellets. Each plot was derived by pooling tissue from 3-4 animals. The K d values are expressed in nM and the Bma x val- ues are expressed in pmol/g wet weight tissue. reduced. However, these differences in Kd between naltrexone- and placebo-treated groups were not sta- tistically significant (P values > 0.05). As was ob- tained with [3H]naloxone, the specific binding of the opiate agonist [3H]etorphine (0.1-4.0 nM) was in- creased significantly following 2 weeks of chronic opiate blockade (Fig. 3). This increase in [3H]etor- phine binding also reflected an increase in Bmax, rath- er than a change in Kd. Chronic naltrexone treatment for 2 weeks also in- creased the specific binding of [3H]naloxone in locus coeruleus micropunches (Table I). Although there was a 45% mean increase in DPMs per assay tube produced by naltrexone, this effect was not statistic- 227 ally significant because the variances within each treatment group were relatively large. However, when the data were expressed as frnol [3H]naloxone bound/rag protein, the increase reached statistical significance. There were no significant differences in protein concentration in LC micropunches from nal- trexone- and placebo-treated animals. Electrophysiologic recording All data reported are from recording sites (n = 23 units) subsequently determined histologically to have been within the LC (e.g. Fig. 4). In placebo-treated animals anesthetized with chloral hydrate, single units in the LC were of relatively large amplitude, with a steady, spontaneous firing rate. A noxious pinch to the contralateral hindpaw typically in- creased their firing rate and this pinch-induced excit- ation was usually, but not always, followed by a peri- od of quiescence (Fig. 5A). The units were unaf- fected following the administration of dextrorphan, the d-isomer of levorphanol which is relatively de- void of analgetic action (Fig. 5B). In contrast to dex- trorphan, morphine consistently produced a dose-de- pendent inhibition of unit activity in the LC which was rapidly and reliably reversed by naloxone (Fig. 5C). Chronic naltrexone treatment (4 weeks) signifi- cantly lowered the spontaneous firing rate of units in the LC. One day after removal of the naltrexone pel- let, the mean spontaneous firing rate was reduced by 44%, from 2.36 to 1.32 spikes/s (Table II). There was no significant difference in the spontaneous unit ac- tivities in the LC between control (placebo) animals and animals whose naltrexone pellet was implanted for 1 week and left intact during the recording ses- sion. Chronic naltrexone treatment also increased the TABLE I Specific binding of [3H]naloxone in the locus coeruleus following 2 weeks of chronic naltrexone blockade Values reflect opiate binding 24 h after removal of pellets. Treatment Tissue Mean + S. E. M. pools (n) #g protein~tube dpm/tube fmol bound~rag protein Placebo 7 27.0 + 1.8 225 + 42 74.6 + 12.0 Naltrexone 7 24.6 + 2.1 326 + 22 129.2 + 21.4" * Significantly different from placebo control group, P < 0.05.
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`228 Fig. 4. Stained coronal section through the brainstem demonstrating the placement of a recording electrode tip in the LC (arrow). Ab- breviations: NMT, nucleus of the mesencephalic tract of the trigeminal nerve; LC, locus coeruleus; IV, fourth ventricle; PVG, peri- ventricular gray substance; and CBL, cerebellum. efficacy of morphine in inhibiting the firing rate of units in the LC. In placebo-treated animals, mor- phine administered in incremental doses (0.1-1.0 mg/kg i.v.) produced a dose-dependent in'hibition of unit activity in the LC. In 4 week naltrexone-treated animals which had their pellets removed one day be- fore the recording session, the morphine-induced in- hibition of unit activity was significantly potentiated at each dose of morphine relative to placebo-treated animals (Fig. 6, left portion). Naloxone administered
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`A (J Q °n n cn O.-a PP B e "O. 0 10 Sec Dextrorphan 2.5 ~-PP1 4 1 Min 229 C e e Q. Morphine Naloxone .2 .4 .8 .04 pp I, ,I, ,I, ,I, ,I, _ L __ J~m;,imlmigilililnml~inmr~;:ll 1 Min Fig. 5. Representative profile of spontaneous activity of neurons in the LC of placebo control animals. A: response to a paw-pinch (PP) of the contralateral hindpaw. B: dextrorphan (2.5 mg/kg, i.v.) was ineffective in altering the spontaneous firing rate. C: morphine in incremental doses (0.2-0.8 mg/kg, i.v., cumulative dose 1.4 mg/kg) inhibited the spontaneous firing rate; this inhibition was re- versed by naloxone (0.04 mg/kg). B and C are from the same experiment. in incremental doses (0.01-0.06 mg/kg, i.v.) reversed the morphine-induced inhibition of LC activity in both placebo-treated and naltrexone-treated animals which had their pellet removed (Fig. 6, right por- tion). However, we did not determine whether the naltrexone- and placebo-treated animals displayed a differential sensitivity to the naloxone-induced re- versal of the inhibitory effect of morphine, as not enough units were held for a sufficient length of time to allow for a valid statistical analysis. In contrast to animals which had their naltrexone pellet removed for I day, animals assessed with their naltrexone pel- TABLE II Spontaneous firing rates of single units in the LC Values reflect unit activity in 4-week placebo- or naltrexone-treated animals which had their pellets removed 1 day before the record- ing session (placebo and naltrexone removed groups, respectively) and in 1-week naltrexone-treated animals in which the pellet was not removed (naltrexone intact group). Treatment group Units Unit activity (spikes~s) (n) Mean S.E.M. Range " Placebo 8 2.36 0.31 1.38-3.92 Naltrexone removed 12 1.32" 0.21 0.40-2.74 Naltrexone intact 3 2.01 0.07 1.90-2.14 * Significantly different from placebo control group, P < 0.05.
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`230 40 } 2! ..,,..o. Pallet Intact -±-~,-T ,~ -'" "-----, - .Xl,/T ~, zo I\, "-.d, / I / 1 ~ ',T 60 Noltraxone ~ ~ ~'d Pallet I \ o R.moved IX T ' 8o ,A" 4 I 1 I I I Y • I .2 .4 .6 .8 1.0 B ,,,,,o"'; T ,1. / '-/ ,,,, i / - / / i / I I -- I 0 I I 1 I .01 .02 .04 .06 Noloxone Morphine CONSECUTIVE I.V. DOSES (mg/kg) Fig. 6. Percent change from the baseline spontaneous firing rate of neurons in the LC produced by morphine and naloxonein animals which were either placebo-treated, implanted for 4 weeks with a naltrexone pellet which was then removed 1 day before recording (naltrexone pellet removed), or were implanted for t week with a naltrexone pellet which was not removed before recording (naltrex- one pellet intact). Morphine was given in incremental consecutive doses 2 rain apart (0.1-1.0 mg/kg, i.v.; total cumulative dose, 3.1 mg/kg), followed 2 min later by incremental consecutive doses of naloxone given 2 min apart (0.01-0.06 mg/kg, i.v.; total cumulative dose, 0.13 mg/kg). The number next to each data point indicates the number of animals (1 unit/animal) used in calculating the mean and S.E.M. ; if no number is present, then the data point represents 1 animal. Asterisks represent significant differences from placebo group, P < 0.05. let intact (1 week) displayed no morphine-induced in- hibition of LC activity within the range of morphine doses tested. DISCUSSION Chronic administration of antagonist drugs which block the action of classical neurotransmitters such as dopamine, norepinephrine or acetylcholine general- ly produces an increase in the number of postsynaptic sites for the neurotransmitter s,n,32,34,46`48`63, as well as a supersensitivity of the postsynaptic neuron to the neurotransmitterlO. The results of the present experi- ments indicate that opioid-receptor systems also adapt to chronic receptor blockade in a manner which is similar to that observed in classical neurotransmitter systems. Animals implanted with slow-release pellets of naltrexone for 2--4 weeks ex- hibited an increase in the number of binding sites for opiates in spinal cord, hypothalamus, striatum and frontal cortex. This increase in receptors was specific for opiates, as binding of the a2-adrenergic antago- nist [3H]rauwolscine was unaffected by the chronic naltrexone treatment. In addition, there was an in- crease in opiate binding in the LC which was accom- panied by a supersensitivity of neurons in the LC to the inhibitory effects of morphine. Previous evidence indicates that a single injection of naloxone produces a transient increase in the num- ber of opiate receptors in whole brain41,42. The in-
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`crease in opiate receptors following an acute injec- tion of naloxone differs, however, from the increase in opiate receptors following chronic exposure to nal- trexone. With a single injection of naloxone, an in- crease in opiate receptors is evident only during the approximately 2-h period during which naloxone is present in vivo, and the brain tissue must be washed of free naloxone in order to prevent interference with the in vitro radioreceptor assay41. 42. In contrast, with chronic naloxone treatment, an increase in opiate re- ceptors is evident for at least 7 days after the period during which the antagonist is active in vivo 29, and the brain tissue does not require washing before per- forming an in vitro radioreceptor assay. In addition, while an increase in opiate receptors following a sin- gle injection of naloxone is not obtained when the in vitro assay is performed in the presence of sodi- um 7.42, an increase in opiate receptors following chronic treatment may be obtained in the presence of sodium. Taken together, these results indicate that the increase in opiate receptors obtained following either acute or chronic opiate blockade involves dif- ferent, perhaps independent, adaptive neuronal mechanisms. It is not known from the present study whether the increase in opiate receptors following chronic nal- trexone treatment involves p-type receptors exclu- sively, or perhaps other opiate receptor subtypes as well. Extensive evidence indicates that both p and 6 receptors exist in mammalian brain, with p receptors having preferential affinity for morphine and nalox- one, and b receptors having preferential affinity for Leu-enkephalin 14.30.~0.52. Although p and 6 receptors may coexist on the same neuron16, 62 and may be cou- pled functionally in the expression of analgesia4. 57, these two receptor subtypes are thought generally to be distinct binding sites which have unique structural requirements for their activationS7. Since we used a low concentration (1 nM) of the p antagonist [3H]na- loxone in the in vitro radioreceptor assay, it appears that the increase in opiate binding produced by chronic naltrexone blockade reflects primarily, if not solely, an increase in the number of p receptors. However, further investigation using drugs which bind with greater selectivity to either p or 6 recep- tors, such as the p agonist morphiceptin~5 and the 6 agonist metkephamid 19, may be necessary to answer this question definitely. Further, we cannot rule out 231 the possibility of the involvement of additional opiate receptor subtypes, including those recently charac- terized43, 64 which specifically bind either ethylketo- cyclazocine (u receptor) or SKF 10,047 (tr receptor). Indeed, a recent report indicates that these various receptor subtypes are altered differentially by chron- ic naltrexone treatment 56. Regardless whether chronic naltrexone increases the specific binding of opiates to receptor subtypes other than the p receptor, we have demonstrated that the increase in opiate receptors is accompanied by a neuronal supersensitivity to morphine in the LC. Pre- vious evidence indicates that the effect of opiates on neurons in the LC is dependent upon whether the opiates are administered acutely or chronically. Al- though the acute administration of morphine clearly inhibits the firing of neurons in the LC9, 2s by hyper- polarizing the neuronal membrane37, the chronic ad- ministration of morphine produces tolerance to mor- phine's inhibitory effect 1. Following chronic mor- phine administration, the administration of naloxone produces an hyperactivity in the LC 1 which corre- sponds to the well-characterized behavioral symp- toms of precipitated opiate withdrawal in ro- dents60. 61. In contrast to the excitatory effect of na- loxone in morphine-dependent animals, opiate an- tagonists are without effect on the spontaneous activ- ity of LC neurons in drug-naive animals, regardless whether the antagonists are administered acutely 9 or chronically, as in the present report (i.e. naltrexone intact group, Table II). One day after cessation of chronic naltrexone treatment, however, we found a significant depression in the spontaneous neuronal activity in the LC, which probably reflects a super- sensitivity to the tonic inhibitory effect of endoge- nous opioids which innervate the LC. In addition to the depressed spontaneous activity of neurons in the LC, chronic naltrexone treatment enhanced the in- hibitory effect of systemically administered mor- phine on spontaneous neuronal activity in the LC. These results support the notion that the efficacy of opiates and opioid peptides in inhibiting the activity of neurons in the LC is directly dependent upon the number of postsynaptic binding sites for opiates in this CNS region. In addition to the inhibitory effect of opiates, a2- adrenergic agonists such as clonidine are potent in in- hibiting the spontaneous activity of LC neurons 13,54.
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`232 Clonidine reduces the hyperactivity assessed electro- physiologically which accompanies opiate withdra- waP, TM and is also efficacious in suppressing opiate withdrawal symptoms in rats 51,58 and man 22,23,24. Since naloxone does not reverse these effects of clo- nidine, it is thought that a2-adrenergic agonists re- duce opiate withdrawal symptoms by activating re- ceptors which are independent of opiate receptors. Consistent with this, chronic morphine treatment has been shown to be without effect on a 2 adrenergic re- ceptors in rat brain and spinal cord 47. In addition, we have shown in the present report that chronic nal- trexone treatment increases opiate binding without altering the specific binding of [3H]rauwolscine, a specific antagonist which radiolabels a2-adrenergic receptors in brain 38. From these receptor binding re- sults, it appears that a2 agonists and opiates inhibit the activity of neurons in the LC by independent mechanisms, and it seems unlikely that chronic nal- trexone treatment would affect the clonidine-in- duced inhibition of neurons in the LC. However, this hypothesis requires further experimentation. ACKNOWLEDGEMENTS We thank Bhavna Chatterjee and Elaine Herink for assisting in the radioreceptor andprotein assays, and we thank Razia Khan for the histological work. Naloxone hydrochloride was graciously provided by Endo Laboratories. Supported by USPHS Grants NS 12121 and DA 02879 and Fellowship DA 05195. REFERENCES 1 Aghajanian, G. K., Tolerance of locus coeruleus neurones to morphine and suppression of withdrawal response by clonidine, Nature (Lond.), 276 (1978) 186--188. 2 Amaral, D. G. and Sinnamon, H. M., The locus coeruleus: neurobiology of a central noradrenergic nucleus, Progr. Neurobiol., 9 (1977) 147-196. 3 Atweh, S. F. and Kuhar, M. J., Autoradiographic localiza- tion of opiate receptors in rat brain. II. The brain stem, Brain Research, 129 (1977) 1-12. 4 Audigier, Y., Maraguil, H., Gout, R. and Cros, J., Struc- ture-activity relationships of enkephalin analogs at opiate and enkephalin receptors: correlation with analgesia, Eu- rop. J. Pharmacol., 63 (1980) 35-46. 5 Bardo, M. T., Bhatnagar, R. K. and Gebhart, G. F., Dif- ferential effects of chronic morphine and naloxone on opiate receptors, monoamines, and morphine:induced be- haviors in preweanling rats, Develop. Brain Res., 4 (1982) 139-147. 6 Bardo, M. T., Bhatnagar, R. K. and Gebhart, G. F., An improved filtration procedure for measuring opiate recep- tors in small regions of rat brain, J. Neurochem., 39 (1982) 175t-1754. 7 Bardo, M. T., Bhatnagar, R. K. and Gebhart, G. F., Age- related differences in the effect of chronic naloxone on opiate binding in rat brain, Neuropharmacology, 22 (1983) 453-461. 8 Ben-Barak, J. and Dudai, Y., Scopolamine induces an in- crease in muscarinic receptor level in rat hippocampus, Brain Research, 193 (1980) 309--313. 9 Bird, S. J. and Kuhar, M. J., Iontophoretic application of opiates to the locus coeruleus, Brain Research, 122 (1977) 523-533. 10 Bloom, F. E., Siggins, G. R. and Henriksen, S. J., Electro- physiologic assessment of receptor changes following chronic drug treatment, Fed. Proc., 40 (1981) 166-172. 11 Burt, D. R., Creese, I. and Snyder, S. H., Antischizoph- renic drugs: chronic treatment elevates dopamine receptor binding in brain, Science, 196 (1977) 326--328. 12 Bylund, D. B., Analys