- Email: [email protected]
Effects of repeated cocaine administration on the thyrotropin-releasing hormone level and receptors in the rat brain
Neuropeptides (1997) 31 (3), 253-258 © PearsonProfessionalLtd 1997
Effects of r e p e a t e d c o c a i n e a d m i n i s t r a t i o n on the thyrotropin. releasing h o r m o n e level and r e c e p t o r s in the rat brain L. Jaworska-Feil, B. Budziszewska, W. Lasofi Department of Endocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krak6w, Poland
Summary The effects of single and repeated administration of cocaine on the thyrotropin-releasing hormone (TRH) level and receptors in discrete rat brain structures were evaluated. Male Wistar rats received saline or cocaine (15 mg/kg i.p., once an hour within 3 h, for 8 days). The animals were killed by decapitation at 45 min and 72 h (chronic group only) after the last injection. A radioimmunoassay (RIA) study showed that a single dose of cocaine increased the TRH level in the striatum by 68%, but had no significant effect on the peptide content in the nucleus accumbens, hippocampus, amygdala, septum, hypothalamus, frontal and prefrontal cortex at 45 min after the drug injection. Repeated administration of cocaine increased the TRH level in the striatum by 89% at 45 min, and in the hippocampus by 26% at 72 h after the last dose. No changes in the TRH level were found in other brain structures. In vitro cocaine (10-6--10-4 M) inhibited the K+-stimulated release in a concentration-dependent manner, but had no effect on the basal release of TRH from the striatum and nucleus accumbens of naive rats. Acute cocaine decreased the Br,ax of TRH receptors in the striatum, but had no effect on the density and affinity of TRH receptors in other brain regions. Repeated administration of cocaine evoked a long-lasting decrease in the Bm~x of TRH receptors in the striatum (by c. 30%), whereas an increase in that parameter was observed in the frontal cortex. The Binsx and affinity of TRH receptors following repeated cocaine remained unchanged in the nucleus accumbens. The results obtained indicate that cocaine affects the TRH system mainly in the striatum, and to a lesser extent in the nucleus accumbens, cortex and hippocampus. Furthermore, the above changes do not resemble those induced by amphetamine, which points to certain differences in adaptation of the TRH neuronal system to these psychostimulants. On the other hand, the increase in the hippocampal TRH level during both chronic cocaine and morphine withdrawal is a common feature of the mechanism of dependence on these drugs.
INTRODUCTION
It has been well recognized that apart from its hypophysiotropic activity, thyrotropin-releasing hormone (TRH), has a neurotransmitter- or a neuromodulator-like function.l,2 TRH and its receptors are widely distributed in the central nervous system, including dopamine-innervated extrahypothalamic structures. B TRH increases the dopamine release and metabolism and evokes a number
Received 3 December 1996 Accepted 25 March 1997 Correspondence to: WCadysiaw Lasofi, PhD, Department of Endocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Cracow, Poland. Tel: +48 12 37 40 22. Fax: +48 12 37 45 00.
of behavioural changes resembling those induced by psychomotor stimulants. 4-9 TRH may also be involved in an adaptive response to long-term treatment with psychomotor stimulants. Chronic amphetamine enhances the activity of the endogenous TRH system in the rat nucleus accumbens and striatum, 10 whereas chronic cocaine has recently been reported to decrease the prepro-TRH mRNA level in the former brain region.11 In order to better understand the role of TRH in the mechanism of cocaine action, the effects of single and repeated administration of cocaine on the TRH level and receptors in discrete rat brain structures were evaluated. Additionally, an in-vitro effect of cocaine on the TRH release from nucleus accumbens and striatum of naive rats was studied. 253
254
Jaworska-Fefl et al
MATERIALS A N D METHODS
TRH extraction procedures
Animals
Frozen brain structures were weighed and homogenized in chilled ethanol with an Ultra-Turrax homogenizer. 13 Dried ethanol extracts of the striatum, nucleus accumbens, hippocampus, septum, amygdala, frontal cortex, prefrontal cortex and hypothalamus, collected from individual rats, were used to determine the TRH content by a radioimmunoassay (RIA) method. The media from the release procedure were treated with ice-cold ethanol (2.4 ml) for TRH extraction. After centrffugation (3000 rpm, 0°C, 30 min), the supernatant was evaporated to dryness (VR Vacuum Concentrator, Heto Lab Equipment, Denmark). The residue was reextracted with l ml of ice-cold ethanol (-15°C, overnight), and was then centrifuged (3000 rpm, 0°C, 30min). The resultant supernatant was dried (VR Vacuum Concentrator), and the residue was used to determine the TRH content. The media extracted by that method did not shift the RIA standard curve. With [3HPro]-TRH as a tracer, the average recovery of the whole procedure was found to be 82 + 2% (n = 16). The results were corrected for that recovery.
Male Wistar rats, weighing 250-290 g, were housed in groups of eight to a cage on a constant light-dark cycle (the light on between 08.00 and 20.00 h), with free access to food and water. In the chronic experiment, the weight of rats was controlled every second day. Experimental procedure
The rats were treated with cocaine hydrochloride (Polfa), dissolved in sterile 0.9% saline. The drug was administered intraperitonealy (i.p.) in a dose of 15 mg/kg, once an hour within 3 h, for 8 days. 12 Control rats were injected with saline by the same schedule. The animals were killed by rapid decapitation at 45 rain (single and chronic cocaine) or 72 h (chronic cocaine) after the last dose of cocaine. Their brains were quickly removed and dissected. Brain structures (the striatum, nucleus accumbens, hippocampus, septum, amygdala, frontal cortex, prefrontal cortex and hypothalamus) were immediately frozen on dry ice and stored at -70°C for determination of the TRH level and for quantification of the TRH receptor binding. Release experiments
Slices 300 gm thick, made from 70-90 mg of the striatal tissue, or fragments obtained from 30-35 mg of the nucleus accumbens were deposited in a basket with a nylon mesh bottom. The basket containing tissue was transferred into a vial containing 800 gl of pre-gassed Locke medium (154 mM NaC1, 5.6 mM KC1, 1 mM MgC12, 2.2 mM CaC12, 6 mM NaHCO3) , pH 7.2, enriched with 2 mM HEPES, 10 mM glucose and 20 gM bacitracin (to prevent the enzymatic degradation of TRH). A calciumfree solution was prepared by omission of CaC12 from the Locke medium. When 56 mM of KC1 were used for stimulation, the molarity of Na + was reduced to 104 mM to preserve the osmolarity equal to the control values. After a washing for 10 rain at a room temperature in calciumfree Locke medium, the tissues were transferred into fresh Locke medium, and incubated at 37°C under genfie, continuous agitation in a 95% O 2 and 5% CO 2 atmosphere. Afterwards, the tissue was incubated for 20 min to allow the slices to stabilize. TRH released between 21 and 40 rain of incubation was accepted as a basal release of TRH; the following 20 min of incubation constituted the TRH release effected by K+-stimulation and/or cocaine. During an experiment lasting 60 min (20 min of stabilization plus 20 rain of basal release plus 20 min of K~-stimu lation), the tissue was transferred to fresh medium every 10 min. Neuropeptides (1997) 31(3), 253-258
Determination of TRH
The TRH content was determined in dried ethanol extracts of tissues and incubation media by a RIA method as described previously, TM with commercially available specific anti-TRH antibodies (Peninsula Laboratories Europe Ltd), raised in sheep which were immunized with TRH bisdiozatised benzidine conjugated to 13 BSA. The assays were performed in duplicate in 0.04 M phosphate-buffered saline, pH 7.40, containing 0.1% bovine serum albumin. The RIA incubation mixture (consisting of an anti-TRH antibody, a control standard or sample and [125I]TRH) was incubated at 2°C for 20 h; afterwards, the antibody-bound peptide was precipitated with ice-cold absolute ethanol and separated by centrifugation. The supernatant was decanted, and samples were counted in a 7-counter (LKB). The average sensitivity of the RIA was 4 pg/tube. The inter-assay variation was 10%; the intra-assay variation was 4.2%.
TRH receptor assay
The TRH radioreceptor assay (RRA) was performed following principally a method of Sharff and Burt. 15,16Briefly, pooled tissue samples were homogenized in a sodium phosphate buffer (20 raM, pH 7.4) and centrifuged at 30 000 g for 30 rain. The resultant pellets were washed twice by resuspension and centrifugation. The washed membranes were dispersed in fresh buffer, and were used for the TRH receptor binding assay. 15,j~ The © Pearson Professional Ltd 1997
Effects of cocaine on TRH level and receptors
STRIATUM
[3H]MeTRH was expressed as fmoles/mg of protein. The resultant data were subjected to the 6-point Scatchard analysis. The receptor density (Bronx)and apparent dissociation constant (Ka) values were determined.
NUCLEUS ACCUMBENS D
20-
255
==15"
=
.B
Protein determination
o,10" E
Protein was determined by the method of Lowry et aP z with bovine serum albumin as a standard.
\
O.
115) 161 1121 1131 I---] CONTROL F~J C0£ single;/,S rain
O'
Statistical analysis
(15) i 6 ) . o ) . ~ ) COC repeated; 45rain COC repeated; 72 hr
All the data are expressed as the mean + SEM. Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by Dunnett's test. The differences were considered significant when P < 0.05.
Fig. 1 The TRH level in the rat striatum and nucleus accumbens after single (15 mg/kg i.p.) and chronic (15 mg/kg i.p. once an hour within 3 h for 8 days) treatments with cocaine (COC). The values (pg/mg wet tissue) are presented as mean _+SEM The number of estimations is given in parentheses. *P < 0.05, **P < 0.001, Dunnett's test following ANOVA.
RESULTS
membranes of the examined structures were incubated in 250~d of the total volume with 0.5-8nM of [3H]MeTRH (NEN, specific activity 62.8 Ci/mmol), in the presence or absence of 10 BM of TRH, for 5-6 h at 0°C (in a water-ice bath). The receptor-bound and free pH]MeTRH was separated by rapid filtration through a glass fibre filter GF/B (Whatman) under reduced pressure (Harvester-Brandel, USA). The trapped receptor-bound radioactivity was determined by liquid scintillation spectrometry (Beckman). The amount of specifically bound
A radioimmunological study showed that acute cocaine increased the TRH level in the striatum by 680, but had no significant effect on the peptide content in the nucleus accumbens, hippocampus, amygdala, septum, frontal and prefrontal cortex at 45 min after the drug injection (Fig. 1, Table 1). Repeated cocaine increased the TRH level in the striatum and nucleus accumbens by 89 and 44%, respectively, at 45 min, and in the hippocampus by 26% at 72 h after the last dose. No changes in the TRH level were found in other brain structures (Fig. 1, Table 1)~ Addition of cocaine to the incubation medium had no effect on the basal release of TRH from the nucleus
Table 1 The effect of single (15 mg/kg i.p.) and repeated (15 mg/kg i.p. once an hour within 3 h for 8 days) treatments with cocaine (COC) on the TRH level in the striatum and nucleus accumbens of the rat.
Brain region
Control
COC single (45 min)
COC chronic (45 min)
COC chronic (72 h)
5.09 _+0.26
4.81 + 0.65
4.29 + 0.19
6.41 + 0.41"
Percentage of control Hippocampus
(15) 100 21.54 _+2.68 (15) 100 102.5 + 3.11 (17) 100 2.01 + 0.15 (11) 100 0.99 + 0.11 (11) 100 215.1 + 7.0
(6) 94 18.70 + 3.57 (6) 87 122.0 + 10.3 (5) 119 1.95 _+0.20 (10) 97 1.48 _+0.27 (10) 149 197.6 + 9.6
(12) 84 21.49 + 3.76 (7) 100 98.37 + 7.05 (12) 96 1.84 _+0.18 (12) 92 1.30 + 0.18 (12) 131 231.4 + 10.5
(15) 126 21.68 + 1.80 (12) 101 110.5 _+6.37 (14) 108 2.31 + 0.33 (11) 115 1.31 + 0.21 (11) 132 206.31 _ 5.8
Percentage of control
(20) 100
(6) 92
(12) 108
(16) 96
Hippocampus Percentage of control Amygdala Percentage of control Septum Percentage of control Frontal cortex Percentage of control Prefrontal cortex
Values (pg/mg wet tissue) are presented as mean _+SEM. The number of estimations is given in parentheses. * P < 0.05, Dunnett's test following ANOVA. © Pearson Professional Ltd 1997
Neuropeptides (1997) 31(3), 253-258
Jaworska-Feil et al
256
STRIATUM
NUCLEUS ACCUMBEN$
300 ¸
;"7 W/ ,/
r~
~ 200
I%
o
O
coc
o
10-6M 10"5M lO'"M
0
lO'(iM IO'~M IO-"M
[--'I basal retease ~ ] 56 mM KEI sfimu[ated release Fig. 2 The in-vitro effect of cocaine on TRH release (basal and stimulated by 56 mM KCI) from the rat striatum and nucleus accumbens. The results are presented as mean _+SEM of the percentage of basal release obtained in 3-5 independent incubations. *P < 0.05, **P < 0.0001, Dunnett's test following ANOVA.
accumbens and striatal slices, whereas the stimulated (KC1, 56 mM) release was significantly attenuated in both those structures (Fig. 2).
As shown in Table 2, acute cocaine decreased the Bmax of TRH receptors in the striatum, amygdala and septum by 27, 35 and 27%, respectively. In the latter structure a decrease in the TRH receptor Ka value of 32% was also observed. Acute cocaine had no effect on the density or affinity of TRH receptors in other brain regions. Repeated cocaine evoked a decrease in the Bm~ of TRH receptors in the striatum after 45 rain and 72 h by 34 and 31%, respectively, whereas in the frontal cortex an increase in that parameter was observed at both the time points studied (by 40% after 45 min, and by 29% after 72 h). The Kd of TRH receptors was decreased by 38% only in the striatum at 72 h. The Bmax and Kd of TRH receptors remained unchanged in the hippocampus, amygdala, septum and prefrontal cortex after repeated cocaine (Table 2). DISCUSSION
The data indicate that the striatum is the most sensitive brain structure to cocaine effects, where both acute and repeated drug administration increased the TRH tissue
Table 2 The effect of single (15 mg/kg i.p.) and repeated (15 mg/kg i.p. once an hour within 3 h for 8 days) treatments with cocaine (COC) on the affinity (Kd) and density (Bmax)of TRH receptors in the striatum, nucleus accumbens, hippocampus, amygdala, septum, frontal cortex and prefrontal cortex of the rat Control
COC single (45 min)
COO chronic (45 min)
COC chronic (72 h)
Striatum B K max Nucleus a c c u m b e n s B max Kd Hippocampus Bma× Kd Amygdala B K~ a× Septum B K dmax Frontal cortex Bma× K~ Prefrontal cortex Brnax Ko
25.57 + 4.85 _ 4.01 + 0.35 (7)
18.71 + 1.38** 3.14 +_0.36 (7)
16.78 _+ 0.79** 3.54 + 0.39 (8)
17.55 + 1.16"* + ** 2.47 _ 0.21 (8)
59.17 + 4.19 3.63 -+ 0.29 (6)
56.47 + 2.49 4.65 + 0.50 (6)
66.73 _+3.38 4.04 -+ 0.45 (8)
65.41 + 5.09 3.69 + 0.43 (8)
58.63 -+ 3.70 2.17 + 0.19 (9)
54.62 -+ 1.67 2.50 + 0.12 (8)
58.31 + 2.58 2.30 + 0.15 (7)
58.40 + 2.99 2.53 + 0.24 (8)
52.50 + 3.29 2.32 -+ 0.22 (8)
34.16 _4 - 1 .83 * * 2.15 + 0.20 (6)
- - 0.95 41.89 + 2.00 + 0.12 (7)
__ 6,11 46.42 + 2.66 + 0,24 (6)
56.90 + 2.50 3.61 _+ 0.22 (9)
41.53 + 1,51"* 2.46 +_ 0,24 ** (8)
55.33 _+2.17 3.57 + 0.22 (8)
52.08 +_ 3.81 4.07 + 0.35 (8)
29.46 -+ 1.31 3.18 + 0.16 (6)
33.54 +-3.12 3.54 + 0.41 (6)
41.18 -+ 2.49* 3.93 "4"0.27 (7)
38.10 + 1.43* 3.18 --- 0.24 (7)
29.38 + 1.90 2.57 + 0.37 (8)
34.03 + 2.04 2.78 + 0.26 (9)
36.07 _+2.87 2.76 _+0.35 (6)
33.29 + 1.84 2.84 + 0.26 (7)
The B a x and K d values are presented as m e a n + SEM of 6-point Scatchard plots. The n u m b e r of estimations is given in parentheses. *P < 0.05, **P <0.001, Dunnett's test following ANOVA.
Neuropeptides (1997) 31(3), 253-258
© Pearson Professional Ltd 1997
Effects of cocaine on TRH level and receptors
level. Only chronic treatment elevated the TRH level in the nucleus accumbens. These effects are in contrast to the action of amphetamine which, when given acutely and/or chronically, decreases the TRH level in both these structures. ~° The reason for such a discrepancy may be different influence of cocaine and amphetamine on TRH release. Cocaine has no effect on the basal TRH release and attenuates stimulated TRH release in vitro from the striatum and nucleus accumbens, whereas amphetamine enhances the peptide release. ~°,~4 Inhibition of TRH release by cocaine possibly leads to accumulation of the peptide and may thus account for its elevated tissue level. Furthermore, as TRH enhances the dopamine release and pharmacological effects of dopaminomimetics, 5,z9 the above data suggest that this peptide participates in the psychomotor stimulatory action of amphetamine, but not that of cocaine. Apart from the changes in the striatum and nucleus accumbens, an elevated TRH level was found only in the hippocampus at 72 h after chronic cocaine withdrawal. Increases in the hippocampal TRH level and prepro-TRH mRNA level, 1~ indicate enhancement of the TRH biosynthesis in this tissue during chronic cocaine withdrawal. An increasing body of evidence points to a close relationship between tolerance/dependence and memory processes in which the hippocampus is considered to be the main anatomical snbstrate. TM TRH, a positive modulator of glutamatergic transmission, ~9 has been shown to improve memory in both animal amnesia models and humans. 2° Therefore it is not unlikely that the up-regulation of the hippocampal TRH biosynthesis participates in the maintenance of the state of dependence on cocaine. Interestingly, an earlier study demonstrated an increase in the hippocampal TRH level in the rat at 72 h after repeated morphine withdrawal. 21 This finding supports an assumption that the hippocampal TRH system may play some role in the mechanism of dependence on drugs of different classes.22,a3 The present binding study showed that cocaine had more profound effects on the TRH receptor than on the peptide level. The most sensitive structure was the striaturn, where a long-lasting decrease in the Bm~ of TRH receptors was found. Changes induced by cocaine and amphetamine in the Bm~ of stfiatal TRH receptors were generally negatively correlated with the tissue TRH leveI2 ° On the other hand, in the amygdala, frontal cortex and septum, cocaine affected only parameters of TRH receptors. As the TRH tissue levels remained unchanged, it seems that changes in TRH receptors are not a compensatory response to alterations in the availability of the endogenous agonist. It is noteworthy that another drug of abuse, morphine - when used chronically - induces changes in TRH receptor parameters also irrespective of its influence on the TRH tissue level. 2~ © Pearson Professional Ltd 1997
257
The results of the present study indicate that cocaine affects the TRH system mainly in the striatum, and to a lesser extent in the nucleus accumbens, cortex and hippocampus. In contrast to amphetamine, cocaine inhibits TRH release from the striatum and nucleus accumbens; consequently, the cocaine-induced changes in the striatal/mesolimbic TRH system do not resemble those induced by amphetamine. This observation points to certain differences in adaptation of the TRH neuronal system to these psychostimulants. On the other hand, the increase in the hippocampal TRH level during both chronic cocaine and chronic morphine withdrawal is a common feature of the mechanism of dependence on these drugs.
ACKNOWLEDGEMENT We wish to thank Mrs B. Korzeniak for her skilful technical assistance.
REFERENCES 1. BrownsteinM J, Palkovits M, SaavedraJ M, Bassiri R M, Utiger R D. Thyrotropin releasing hormone in specific nuclei of rat brain. Science 1974; 185: 267-269. 2. MorleyJE. Extrahypothalamic thyrotropin releasing hormone (TRH) - its distribution and its functions. LifeSci 1979; 25: 1539-1550. 3. Winokur A, Utiger R D. Thyrotropin releasing hormone: regional distribution in rat brain. Science 1974; 185: 265-266.
4. Sharp T, Bennett G W, Marsden C A. Thyrotropin-releasing hormone analogues increase dopamine release from slices of rat brain. J Neurochem 1982; 39:1763-1766. 5. KerwinR W, Pycock C J. Thyrotropin releasing hormone stimulates release of [3H]-dopaminefrom slices of rat nucleus accumbens in vitro. BrJ Pharmacol 1979; 67: 323-326. 6. Narumi S, Nagai Y, Saji Y, Nagawa Y.A possible mechanism of action of thyrotropin-releasing hormone (TRH)and its analog DN-1417 on the release of dopamine from the nucleus accumbens and striatum in rats. JpnJ Pharmacol 1985; 39: 425-435. Z KalivasP W, Stanley D, Prange A J Jr. Interaction between thyrotropin-releasing hormone and the mesolimbic dopamine system. Neuropharmacology 1987; 26: 33-38. 8. PlotnikoffN P, Breese G R, Prange AJ Jr. Thyrotropin-releasing hormone (TRH):DOPApotentiation and biogenic amine studies. Pharmacol BiochemBehav 1975; 3: 665-670. 9. Manberg P J, NemeroffC B, Prange AJ Jr. Thyrotropin-releasing hormone and amphetamine: a comparison of pharmacological profiles in animals. Prog Neuropsychopharmacol 1979; 3: 303-314.
10. Jaworska-Feil L, BudziszewskaB, LasofiW. The effects of repeated amphetamine administration on the thyrotropinreleasing hormone level. Its release and receptors in the rat brain. Neuropeptides 1995; 29:171-176. 11. SevarinoK A, Primus RJ. Cocaine regulation of brain preprothyrotropin-releasing hormone mRNA.J Neurochem 1993; 60: 1151-1154. Neuropeptides (1997) 31(3), 253-258
258
Jaworska-Feil et al
12. Spangler R, Unterwald E M, Kreek M J. "Binge" cocaine administration induces a sustained increase of prodynorphin mRNA in rat caudate-putamen. Mol Brain Res 1993; 19: 323-32Z 13. Przegaliflski E, Jaworska L. The effect of repeated administration of antidepressant drugs on the thyrotropin-releasing hormone (TRH) content of rat brain structures. Psychoneuroendocrinology 1990; 15: 147-153. 14. PrzegalifisM E, Jaworska L, Budziszewska B. The role of dopamine receptors in the release of thyrotropin-releasing hormone from the rat striatum and nucleus accumbens: an in vitro study. Neuropeptides 1993; 25: 277-282. 15. Sharif N A, Burt D R. Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects. Regul Pept 1983 7:399-411. 16. Przegalifiski E, Jaworska L, Konarska R. Repeated treatment with amitriptyline or electroconvulsise shock does not affect thyrotropin-releasing hormone (TRIg) receptors in discrete rat brain structures. Neurosci Lett 1990; 115:86-91. 1Z Lowry O H, Rosenbrough N J, Farr A L, Randall R J. Protein measurement with the Folin reagent. J Biol Chem. 1951; 193: 265-275.
Neuropeptides (1997) 31(3), 253-258
18. Trujillo K A, Akil H. Inhibition of opiate tolerance by noncompetitive N-methyl-D-aspartate receptor antagonists. Brain Res 1994; 633: 178-188. 19. Canonico P L, Bruno V, Fiore L, Nicoletti F, Scapagnini U. Thyrotropin-releasing hormone (TRH): a positive modulation of excitatory amino acid transmission in neuronal cultures. Pharmacol Res Commun 1988; 20 Suppl. II: 66. 20. Kasparov S A, Chizh B A. The NMDA-receptor antagonist dizocflpine (MK-801) suppresses the memory facflatory action of thyrotropin-releasing hormone. Neuropeptides 1992; 23: 87-92. 21. Jaworska-FeiI J, Budziszewska B, Lasofl W. The effects of single and repeated morphine administration on the level of thyrotropin-releasing hormone and its receptors in the rat brain. Neuropeptides 1995; 29: 343-349. 22. Bhargava H N. The effects of thyrotropin releasing hormone on the central nervous system responses to chronic morphine administration. Psychopharmacology 1980; 68: 185-189. 23. Bhargava H N. Modification of development of physical dependence on morphine by thyrotropin releasing hormone. In: Way EL, ed. Endogenous and Exogenous Opiate Agonists and Antagonists. New York: Pergamon. 1980: 549.
© Pearson Professional Ltd 1997
Effects of r e p e a t e d c o c a i n e a d m i n i s t r a t i o n on the thyrotropin. releasing h o r m o n e level and r e c e p t o r s in the rat brain L. Jaworska-Feil, B. Budziszewska, W. Lasofi Department of Endocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krak6w, Poland
Summary The effects of single and repeated administration of cocaine on the thyrotropin-releasing hormone (TRH) level and receptors in discrete rat brain structures were evaluated. Male Wistar rats received saline or cocaine (15 mg/kg i.p., once an hour within 3 h, for 8 days). The animals were killed by decapitation at 45 min and 72 h (chronic group only) after the last injection. A radioimmunoassay (RIA) study showed that a single dose of cocaine increased the TRH level in the striatum by 68%, but had no significant effect on the peptide content in the nucleus accumbens, hippocampus, amygdala, septum, hypothalamus, frontal and prefrontal cortex at 45 min after the drug injection. Repeated administration of cocaine increased the TRH level in the striatum by 89% at 45 min, and in the hippocampus by 26% at 72 h after the last dose. No changes in the TRH level were found in other brain structures. In vitro cocaine (10-6--10-4 M) inhibited the K+-stimulated release in a concentration-dependent manner, but had no effect on the basal release of TRH from the striatum and nucleus accumbens of naive rats. Acute cocaine decreased the Br,ax of TRH receptors in the striatum, but had no effect on the density and affinity of TRH receptors in other brain regions. Repeated administration of cocaine evoked a long-lasting decrease in the Bm~x of TRH receptors in the striatum (by c. 30%), whereas an increase in that parameter was observed in the frontal cortex. The Binsx and affinity of TRH receptors following repeated cocaine remained unchanged in the nucleus accumbens. The results obtained indicate that cocaine affects the TRH system mainly in the striatum, and to a lesser extent in the nucleus accumbens, cortex and hippocampus. Furthermore, the above changes do not resemble those induced by amphetamine, which points to certain differences in adaptation of the TRH neuronal system to these psychostimulants. On the other hand, the increase in the hippocampal TRH level during both chronic cocaine and morphine withdrawal is a common feature of the mechanism of dependence on these drugs.
INTRODUCTION
It has been well recognized that apart from its hypophysiotropic activity, thyrotropin-releasing hormone (TRH), has a neurotransmitter- or a neuromodulator-like function.l,2 TRH and its receptors are widely distributed in the central nervous system, including dopamine-innervated extrahypothalamic structures. B TRH increases the dopamine release and metabolism and evokes a number
Received 3 December 1996 Accepted 25 March 1997 Correspondence to: WCadysiaw Lasofi, PhD, Department of Endocrinology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Cracow, Poland. Tel: +48 12 37 40 22. Fax: +48 12 37 45 00.
of behavioural changes resembling those induced by psychomotor stimulants. 4-9 TRH may also be involved in an adaptive response to long-term treatment with psychomotor stimulants. Chronic amphetamine enhances the activity of the endogenous TRH system in the rat nucleus accumbens and striatum, 10 whereas chronic cocaine has recently been reported to decrease the prepro-TRH mRNA level in the former brain region.11 In order to better understand the role of TRH in the mechanism of cocaine action, the effects of single and repeated administration of cocaine on the TRH level and receptors in discrete rat brain structures were evaluated. Additionally, an in-vitro effect of cocaine on the TRH release from nucleus accumbens and striatum of naive rats was studied. 253
254
Jaworska-Fefl et al
MATERIALS A N D METHODS
TRH extraction procedures
Animals
Frozen brain structures were weighed and homogenized in chilled ethanol with an Ultra-Turrax homogenizer. 13 Dried ethanol extracts of the striatum, nucleus accumbens, hippocampus, septum, amygdala, frontal cortex, prefrontal cortex and hypothalamus, collected from individual rats, were used to determine the TRH content by a radioimmunoassay (RIA) method. The media from the release procedure were treated with ice-cold ethanol (2.4 ml) for TRH extraction. After centrffugation (3000 rpm, 0°C, 30 min), the supernatant was evaporated to dryness (VR Vacuum Concentrator, Heto Lab Equipment, Denmark). The residue was reextracted with l ml of ice-cold ethanol (-15°C, overnight), and was then centrifuged (3000 rpm, 0°C, 30min). The resultant supernatant was dried (VR Vacuum Concentrator), and the residue was used to determine the TRH content. The media extracted by that method did not shift the RIA standard curve. With [3HPro]-TRH as a tracer, the average recovery of the whole procedure was found to be 82 + 2% (n = 16). The results were corrected for that recovery.
Male Wistar rats, weighing 250-290 g, were housed in groups of eight to a cage on a constant light-dark cycle (the light on between 08.00 and 20.00 h), with free access to food and water. In the chronic experiment, the weight of rats was controlled every second day. Experimental procedure
The rats were treated with cocaine hydrochloride (Polfa), dissolved in sterile 0.9% saline. The drug was administered intraperitonealy (i.p.) in a dose of 15 mg/kg, once an hour within 3 h, for 8 days. 12 Control rats were injected with saline by the same schedule. The animals were killed by rapid decapitation at 45 rain (single and chronic cocaine) or 72 h (chronic cocaine) after the last dose of cocaine. Their brains were quickly removed and dissected. Brain structures (the striatum, nucleus accumbens, hippocampus, septum, amygdala, frontal cortex, prefrontal cortex and hypothalamus) were immediately frozen on dry ice and stored at -70°C for determination of the TRH level and for quantification of the TRH receptor binding. Release experiments
Slices 300 gm thick, made from 70-90 mg of the striatal tissue, or fragments obtained from 30-35 mg of the nucleus accumbens were deposited in a basket with a nylon mesh bottom. The basket containing tissue was transferred into a vial containing 800 gl of pre-gassed Locke medium (154 mM NaC1, 5.6 mM KC1, 1 mM MgC12, 2.2 mM CaC12, 6 mM NaHCO3) , pH 7.2, enriched with 2 mM HEPES, 10 mM glucose and 20 gM bacitracin (to prevent the enzymatic degradation of TRH). A calciumfree solution was prepared by omission of CaC12 from the Locke medium. When 56 mM of KC1 were used for stimulation, the molarity of Na + was reduced to 104 mM to preserve the osmolarity equal to the control values. After a washing for 10 rain at a room temperature in calciumfree Locke medium, the tissues were transferred into fresh Locke medium, and incubated at 37°C under genfie, continuous agitation in a 95% O 2 and 5% CO 2 atmosphere. Afterwards, the tissue was incubated for 20 min to allow the slices to stabilize. TRH released between 21 and 40 rain of incubation was accepted as a basal release of TRH; the following 20 min of incubation constituted the TRH release effected by K+-stimulation and/or cocaine. During an experiment lasting 60 min (20 min of stabilization plus 20 rain of basal release plus 20 min of K~-stimu lation), the tissue was transferred to fresh medium every 10 min. Neuropeptides (1997) 31(3), 253-258
Determination of TRH
The TRH content was determined in dried ethanol extracts of tissues and incubation media by a RIA method as described previously, TM with commercially available specific anti-TRH antibodies (Peninsula Laboratories Europe Ltd), raised in sheep which were immunized with TRH bisdiozatised benzidine conjugated to 13 BSA. The assays were performed in duplicate in 0.04 M phosphate-buffered saline, pH 7.40, containing 0.1% bovine serum albumin. The RIA incubation mixture (consisting of an anti-TRH antibody, a control standard or sample and [125I]TRH) was incubated at 2°C for 20 h; afterwards, the antibody-bound peptide was precipitated with ice-cold absolute ethanol and separated by centrifugation. The supernatant was decanted, and samples were counted in a 7-counter (LKB). The average sensitivity of the RIA was 4 pg/tube. The inter-assay variation was 10%; the intra-assay variation was 4.2%.
TRH receptor assay
The TRH radioreceptor assay (RRA) was performed following principally a method of Sharff and Burt. 15,16Briefly, pooled tissue samples were homogenized in a sodium phosphate buffer (20 raM, pH 7.4) and centrifuged at 30 000 g for 30 rain. The resultant pellets were washed twice by resuspension and centrifugation. The washed membranes were dispersed in fresh buffer, and were used for the TRH receptor binding assay. 15,j~ The © Pearson Professional Ltd 1997
Effects of cocaine on TRH level and receptors
STRIATUM
[3H]MeTRH was expressed as fmoles/mg of protein. The resultant data were subjected to the 6-point Scatchard analysis. The receptor density (Bronx)and apparent dissociation constant (Ka) values were determined.
NUCLEUS ACCUMBENS D
20-
255
==15"
=
.B
Protein determination
o,10" E
Protein was determined by the method of Lowry et aP z with bovine serum albumin as a standard.
\
O.
115) 161 1121 1131 I---] CONTROL F~J C0£ single;/,S rain
O'
Statistical analysis
(15) i 6 ) . o ) . ~ ) COC repeated; 45rain COC repeated; 72 hr
All the data are expressed as the mean + SEM. Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by Dunnett's test. The differences were considered significant when P < 0.05.
Fig. 1 The TRH level in the rat striatum and nucleus accumbens after single (15 mg/kg i.p.) and chronic (15 mg/kg i.p. once an hour within 3 h for 8 days) treatments with cocaine (COC). The values (pg/mg wet tissue) are presented as mean _+SEM The number of estimations is given in parentheses. *P < 0.05, **P < 0.001, Dunnett's test following ANOVA.
RESULTS
membranes of the examined structures were incubated in 250~d of the total volume with 0.5-8nM of [3H]MeTRH (NEN, specific activity 62.8 Ci/mmol), in the presence or absence of 10 BM of TRH, for 5-6 h at 0°C (in a water-ice bath). The receptor-bound and free pH]MeTRH was separated by rapid filtration through a glass fibre filter GF/B (Whatman) under reduced pressure (Harvester-Brandel, USA). The trapped receptor-bound radioactivity was determined by liquid scintillation spectrometry (Beckman). The amount of specifically bound
A radioimmunological study showed that acute cocaine increased the TRH level in the striatum by 680, but had no significant effect on the peptide content in the nucleus accumbens, hippocampus, amygdala, septum, frontal and prefrontal cortex at 45 min after the drug injection (Fig. 1, Table 1). Repeated cocaine increased the TRH level in the striatum and nucleus accumbens by 89 and 44%, respectively, at 45 min, and in the hippocampus by 26% at 72 h after the last dose. No changes in the TRH level were found in other brain structures (Fig. 1, Table 1)~ Addition of cocaine to the incubation medium had no effect on the basal release of TRH from the nucleus
Table 1 The effect of single (15 mg/kg i.p.) and repeated (15 mg/kg i.p. once an hour within 3 h for 8 days) treatments with cocaine (COC) on the TRH level in the striatum and nucleus accumbens of the rat.
Brain region
Control
COC single (45 min)
COC chronic (45 min)
COC chronic (72 h)
5.09 _+0.26
4.81 + 0.65
4.29 + 0.19
6.41 + 0.41"
Percentage of control Hippocampus
(15) 100 21.54 _+2.68 (15) 100 102.5 + 3.11 (17) 100 2.01 + 0.15 (11) 100 0.99 + 0.11 (11) 100 215.1 + 7.0
(6) 94 18.70 + 3.57 (6) 87 122.0 + 10.3 (5) 119 1.95 _+0.20 (10) 97 1.48 _+0.27 (10) 149 197.6 + 9.6
(12) 84 21.49 + 3.76 (7) 100 98.37 + 7.05 (12) 96 1.84 _+0.18 (12) 92 1.30 + 0.18 (12) 131 231.4 + 10.5
(15) 126 21.68 + 1.80 (12) 101 110.5 _+6.37 (14) 108 2.31 + 0.33 (11) 115 1.31 + 0.21 (11) 132 206.31 _ 5.8
Percentage of control
(20) 100
(6) 92
(12) 108
(16) 96
Hippocampus Percentage of control Amygdala Percentage of control Septum Percentage of control Frontal cortex Percentage of control Prefrontal cortex
Values (pg/mg wet tissue) are presented as mean _+SEM. The number of estimations is given in parentheses. * P < 0.05, Dunnett's test following ANOVA. © Pearson Professional Ltd 1997
Neuropeptides (1997) 31(3), 253-258
Jaworska-Feil et al
256
STRIATUM
NUCLEUS ACCUMBEN$
300 ¸
;"7 W/ ,/
r~
~ 200
I%
o
O
coc
o
10-6M 10"5M lO'"M
0
lO'(iM IO'~M IO-"M
[--'I basal retease ~ ] 56 mM KEI sfimu[ated release Fig. 2 The in-vitro effect of cocaine on TRH release (basal and stimulated by 56 mM KCI) from the rat striatum and nucleus accumbens. The results are presented as mean _+SEM of the percentage of basal release obtained in 3-5 independent incubations. *P < 0.05, **P < 0.0001, Dunnett's test following ANOVA.
accumbens and striatal slices, whereas the stimulated (KC1, 56 mM) release was significantly attenuated in both those structures (Fig. 2).
As shown in Table 2, acute cocaine decreased the Bmax of TRH receptors in the striatum, amygdala and septum by 27, 35 and 27%, respectively. In the latter structure a decrease in the TRH receptor Ka value of 32% was also observed. Acute cocaine had no effect on the density or affinity of TRH receptors in other brain regions. Repeated cocaine evoked a decrease in the Bm~ of TRH receptors in the striatum after 45 rain and 72 h by 34 and 31%, respectively, whereas in the frontal cortex an increase in that parameter was observed at both the time points studied (by 40% after 45 min, and by 29% after 72 h). The Kd of TRH receptors was decreased by 38% only in the striatum at 72 h. The Bmax and Kd of TRH receptors remained unchanged in the hippocampus, amygdala, septum and prefrontal cortex after repeated cocaine (Table 2). DISCUSSION
The data indicate that the striatum is the most sensitive brain structure to cocaine effects, where both acute and repeated drug administration increased the TRH tissue
Table 2 The effect of single (15 mg/kg i.p.) and repeated (15 mg/kg i.p. once an hour within 3 h for 8 days) treatments with cocaine (COC) on the affinity (Kd) and density (Bmax)of TRH receptors in the striatum, nucleus accumbens, hippocampus, amygdala, septum, frontal cortex and prefrontal cortex of the rat Control
COC single (45 min)
COO chronic (45 min)
COC chronic (72 h)
Striatum B K max Nucleus a c c u m b e n s B max Kd Hippocampus Bma× Kd Amygdala B K~ a× Septum B K dmax Frontal cortex Bma× K~ Prefrontal cortex Brnax Ko
25.57 + 4.85 _ 4.01 + 0.35 (7)
18.71 + 1.38** 3.14 +_0.36 (7)
16.78 _+ 0.79** 3.54 + 0.39 (8)
17.55 + 1.16"* + ** 2.47 _ 0.21 (8)
59.17 + 4.19 3.63 -+ 0.29 (6)
56.47 + 2.49 4.65 + 0.50 (6)
66.73 _+3.38 4.04 -+ 0.45 (8)
65.41 + 5.09 3.69 + 0.43 (8)
58.63 -+ 3.70 2.17 + 0.19 (9)
54.62 -+ 1.67 2.50 + 0.12 (8)
58.31 + 2.58 2.30 + 0.15 (7)
58.40 + 2.99 2.53 + 0.24 (8)
52.50 + 3.29 2.32 -+ 0.22 (8)
34.16 _4 - 1 .83 * * 2.15 + 0.20 (6)
- - 0.95 41.89 + 2.00 + 0.12 (7)
__ 6,11 46.42 + 2.66 + 0,24 (6)
56.90 + 2.50 3.61 _+ 0.22 (9)
41.53 + 1,51"* 2.46 +_ 0,24 ** (8)
55.33 _+2.17 3.57 + 0.22 (8)
52.08 +_ 3.81 4.07 + 0.35 (8)
29.46 -+ 1.31 3.18 + 0.16 (6)
33.54 +-3.12 3.54 + 0.41 (6)
41.18 -+ 2.49* 3.93 "4"0.27 (7)
38.10 + 1.43* 3.18 --- 0.24 (7)
29.38 + 1.90 2.57 + 0.37 (8)
34.03 + 2.04 2.78 + 0.26 (9)
36.07 _+2.87 2.76 _+0.35 (6)
33.29 + 1.84 2.84 + 0.26 (7)
The B a x and K d values are presented as m e a n + SEM of 6-point Scatchard plots. The n u m b e r of estimations is given in parentheses. *P < 0.05, **P <0.001, Dunnett's test following ANOVA.
Neuropeptides (1997) 31(3), 253-258
© Pearson Professional Ltd 1997
Effects of cocaine on TRH level and receptors
level. Only chronic treatment elevated the TRH level in the nucleus accumbens. These effects are in contrast to the action of amphetamine which, when given acutely and/or chronically, decreases the TRH level in both these structures. ~° The reason for such a discrepancy may be different influence of cocaine and amphetamine on TRH release. Cocaine has no effect on the basal TRH release and attenuates stimulated TRH release in vitro from the striatum and nucleus accumbens, whereas amphetamine enhances the peptide release. ~°,~4 Inhibition of TRH release by cocaine possibly leads to accumulation of the peptide and may thus account for its elevated tissue level. Furthermore, as TRH enhances the dopamine release and pharmacological effects of dopaminomimetics, 5,z9 the above data suggest that this peptide participates in the psychomotor stimulatory action of amphetamine, but not that of cocaine. Apart from the changes in the striatum and nucleus accumbens, an elevated TRH level was found only in the hippocampus at 72 h after chronic cocaine withdrawal. Increases in the hippocampal TRH level and prepro-TRH mRNA level, 1~ indicate enhancement of the TRH biosynthesis in this tissue during chronic cocaine withdrawal. An increasing body of evidence points to a close relationship between tolerance/dependence and memory processes in which the hippocampus is considered to be the main anatomical snbstrate. TM TRH, a positive modulator of glutamatergic transmission, ~9 has been shown to improve memory in both animal amnesia models and humans. 2° Therefore it is not unlikely that the up-regulation of the hippocampal TRH biosynthesis participates in the maintenance of the state of dependence on cocaine. Interestingly, an earlier study demonstrated an increase in the hippocampal TRH level in the rat at 72 h after repeated morphine withdrawal. 21 This finding supports an assumption that the hippocampal TRH system may play some role in the mechanism of dependence on drugs of different classes.22,a3 The present binding study showed that cocaine had more profound effects on the TRH receptor than on the peptide level. The most sensitive structure was the striaturn, where a long-lasting decrease in the Bm~ of TRH receptors was found. Changes induced by cocaine and amphetamine in the Bm~ of stfiatal TRH receptors were generally negatively correlated with the tissue TRH leveI2 ° On the other hand, in the amygdala, frontal cortex and septum, cocaine affected only parameters of TRH receptors. As the TRH tissue levels remained unchanged, it seems that changes in TRH receptors are not a compensatory response to alterations in the availability of the endogenous agonist. It is noteworthy that another drug of abuse, morphine - when used chronically - induces changes in TRH receptor parameters also irrespective of its influence on the TRH tissue level. 2~ © Pearson Professional Ltd 1997
257
The results of the present study indicate that cocaine affects the TRH system mainly in the striatum, and to a lesser extent in the nucleus accumbens, cortex and hippocampus. In contrast to amphetamine, cocaine inhibits TRH release from the striatum and nucleus accumbens; consequently, the cocaine-induced changes in the striatal/mesolimbic TRH system do not resemble those induced by amphetamine. This observation points to certain differences in adaptation of the TRH neuronal system to these psychostimulants. On the other hand, the increase in the hippocampal TRH level during both chronic cocaine and chronic morphine withdrawal is a common feature of the mechanism of dependence on these drugs.
ACKNOWLEDGEMENT We wish to thank Mrs B. Korzeniak for her skilful technical assistance.
REFERENCES 1. BrownsteinM J, Palkovits M, SaavedraJ M, Bassiri R M, Utiger R D. Thyrotropin releasing hormone in specific nuclei of rat brain. Science 1974; 185: 267-269. 2. MorleyJE. Extrahypothalamic thyrotropin releasing hormone (TRH) - its distribution and its functions. LifeSci 1979; 25: 1539-1550. 3. Winokur A, Utiger R D. Thyrotropin releasing hormone: regional distribution in rat brain. Science 1974; 185: 265-266.
4. Sharp T, Bennett G W, Marsden C A. Thyrotropin-releasing hormone analogues increase dopamine release from slices of rat brain. J Neurochem 1982; 39:1763-1766. 5. KerwinR W, Pycock C J. Thyrotropin releasing hormone stimulates release of [3H]-dopaminefrom slices of rat nucleus accumbens in vitro. BrJ Pharmacol 1979; 67: 323-326. 6. Narumi S, Nagai Y, Saji Y, Nagawa Y.A possible mechanism of action of thyrotropin-releasing hormone (TRH)and its analog DN-1417 on the release of dopamine from the nucleus accumbens and striatum in rats. JpnJ Pharmacol 1985; 39: 425-435. Z KalivasP W, Stanley D, Prange A J Jr. Interaction between thyrotropin-releasing hormone and the mesolimbic dopamine system. Neuropharmacology 1987; 26: 33-38. 8. PlotnikoffN P, Breese G R, Prange AJ Jr. Thyrotropin-releasing hormone (TRH):DOPApotentiation and biogenic amine studies. Pharmacol BiochemBehav 1975; 3: 665-670. 9. Manberg P J, NemeroffC B, Prange AJ Jr. Thyrotropin-releasing hormone and amphetamine: a comparison of pharmacological profiles in animals. Prog Neuropsychopharmacol 1979; 3: 303-314.
10. Jaworska-Feil L, BudziszewskaB, LasofiW. The effects of repeated amphetamine administration on the thyrotropinreleasing hormone level. Its release and receptors in the rat brain. Neuropeptides 1995; 29:171-176. 11. SevarinoK A, Primus RJ. Cocaine regulation of brain preprothyrotropin-releasing hormone mRNA.J Neurochem 1993; 60: 1151-1154. Neuropeptides (1997) 31(3), 253-258
258
Jaworska-Feil et al
12. Spangler R, Unterwald E M, Kreek M J. "Binge" cocaine administration induces a sustained increase of prodynorphin mRNA in rat caudate-putamen. Mol Brain Res 1993; 19: 323-32Z 13. Przegaliflski E, Jaworska L. The effect of repeated administration of antidepressant drugs on the thyrotropin-releasing hormone (TRH) content of rat brain structures. Psychoneuroendocrinology 1990; 15: 147-153. 14. PrzegalifisM E, Jaworska L, Budziszewska B. The role of dopamine receptors in the release of thyrotropin-releasing hormone from the rat striatum and nucleus accumbens: an in vitro study. Neuropeptides 1993; 25: 277-282. 15. Sharif N A, Burt D R. Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects. Regul Pept 1983 7:399-411. 16. Przegalifiski E, Jaworska L, Konarska R. Repeated treatment with amitriptyline or electroconvulsise shock does not affect thyrotropin-releasing hormone (TRIg) receptors in discrete rat brain structures. Neurosci Lett 1990; 115:86-91. 1Z Lowry O H, Rosenbrough N J, Farr A L, Randall R J. Protein measurement with the Folin reagent. J Biol Chem. 1951; 193: 265-275.
Neuropeptides (1997) 31(3), 253-258
18. Trujillo K A, Akil H. Inhibition of opiate tolerance by noncompetitive N-methyl-D-aspartate receptor antagonists. Brain Res 1994; 633: 178-188. 19. Canonico P L, Bruno V, Fiore L, Nicoletti F, Scapagnini U. Thyrotropin-releasing hormone (TRH): a positive modulation of excitatory amino acid transmission in neuronal cultures. Pharmacol Res Commun 1988; 20 Suppl. II: 66. 20. Kasparov S A, Chizh B A. The NMDA-receptor antagonist dizocflpine (MK-801) suppresses the memory facflatory action of thyrotropin-releasing hormone. Neuropeptides 1992; 23: 87-92. 21. Jaworska-FeiI J, Budziszewska B, Lasofl W. The effects of single and repeated morphine administration on the level of thyrotropin-releasing hormone and its receptors in the rat brain. Neuropeptides 1995; 29: 343-349. 22. Bhargava H N. The effects of thyrotropin releasing hormone on the central nervous system responses to chronic morphine administration. Psychopharmacology 1980; 68: 185-189. 23. Bhargava H N. Modification of development of physical dependence on morphine by thyrotropin releasing hormone. In: Way EL, ed. Endogenous and Exogenous Opiate Agonists and Antagonists. New York: Pergamon. 1980: 549.
© Pearson Professional Ltd 1997