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Effect of prior nicotine treatment on drug induced changes in serum LH concentrations in rats
Life Sciences, Vol. 60, No. 25, pp. 23032308, 1997 Copright0 1997Ekwier Science Inc. Printed in the USA. All rights merved 0024-3205/97 s17.w t .Gu
PI1 s0024-3205(97)00285-3
ELSEVIER
EFFECT OF PRIOR NICOTINE TREATMENT ON DRUG INDUCED CHANGES IN SERUM LH CONCENTRATIONS IN RATS
C. A. Hodson lBt2,M. C. Davis’ and H. W. Burden3
’ Department of Obstetrics and Gynecology and 3 Department of Anatomy and Cell Biology, School of Medicine, East Carolina University, Greenville, NC 27858 (Received in final form March 17,1997)
Summary The effect of daily injections of nicotine on drug induced changes in LH secretion was investigated in male rats. Daily administration of nicotine for 7 days resulted in decreased basal serum LH concentrations. Nicotine treatment blocked naloxone induced LH release and reduced LHRH induced increases in serum LH. Clonidine induced increases in serum LH were not altered by nicotine treatment and haloperidol treatment did not alter nicotine induced decreases in serum LH. In an acute study nicotine blocked LH secretion induced by the long acting opioid antagonist naltrexone. Collectively these results indicate that opioidergic neurons are involved in the reduction in serum LH that occurs following nicotine. They also indicate that chronic nicotine treatment can reduce the pituitary gland response to LHRH. Key Word: nicotine, luteinizing hormone, clonidine, pituitary gland, haloperidol, luteinking hormone, naltrexone, naloxone
hormone releasing
Acute administration of nicotine has an inhibitory effect on luteinizing hormone secretion in rats (1). This effect of nicotine has a major dopaminergic component but other nerurotransmitters may be involved (2). We have reported that nicotine treatment can prevent naloxone induced LH secretion, and these results suggest that opiodergic neurons may be involved in the suppressive effect of nicotine on LH (3). In the central nervous system nicotine alters neural responsiveness to other neurotransmitters such as glutamate and acetylcholine (4). Nicotine may cause a similar change in responsiveness to opioids within the central nervous system. Tolerance develops rapidly to many effects of nicotine (5) and it is not clear that the acute decrease in LH following nicotine treatment is biologically significant. Nicotine treatment also causes acute increases in ACTH and corticoid release in rats (6,7) and tolerance to this effect develops rapidly. Thereafter ACTH and corticoids are not altered by nicotine treatment (8,9).
* Corresponding author
2304
Nicotine and Luteinizing Hormone
Vol. 60, No. 25,1997
Because previous reports (8,9) showing that tolerance to nicotine develops rapidly used similar nicotine doses used in our previous study (l), we reasoned that a similar attenuation of the LH response to nicotine might develop. In this study, we determined whether chronic treatment with nicotine affected naloxone, clonidine and luteinizing hormone releasing hormone induced changes in LH secretion. We were also interested in determining if other neurotransmitters were involved in the nicotine induced inhibition of LH and whether these responses were attenuated after repetitive injection of nicotine.
aM teri Animals: Male Sprague-Dawley rats weighing 500 grams, obtained from Charles River, Inc. were used for these studies. They were housed in a 12-12 hour light/dark cycle, and food and water were provided ad libitum. Exneriment 1: Rats were randomly divided into 4 treatment groups. Two groups were given daily subcutaneous injections of 0.5 mg nicotine, and two groups received saline injections. On day 7, the rats were given their final injections of nicotine or saline. Fifteen minutes after these injections were given, the time 0 blood sample was collected. One of the following drugs: A. 5 mg naloxone HCWkg bw, B. 0.3 mg clonidine HCL/kg bw, C. 1 mg haloperidol/kg bw, D. 500 ng luteinizing hormone releasing hormone or E. saline was injected immediately after the time 0 bleeding. Additional blood samples were collected from the retro-orbital sinus at 15, 30, and 60 minutes after time 0 using ether anesthesia. Exueriment 2: Rats were randomly assigned to one of four treatment groups. Two groups were injected subcutaneously with .5 mg of nicotine, and the other two with an equivalent volume of saline. Fifteen minutes later, at time 0, an orbital sinus blood sample was collected. The rats were immediately injected subcutaneously with naltrexone 5 mg/kg bw or saline. Additional blood samples were collected at 15, 30, and 60 minutes. The four treatment combinations were: A. saline followed by saline, B. nicotine then saline injection, C. saline then naltrexone, and D. nicotine followed by naltrexone. -methods: Serum LH concentrations were measured by radioimmunoassay using reagents provided by the Hormone Distribution Program of the National Institutes of Health. LH was iodinated using the method of Greenwood, Hunter, and Glover (10). Serum LH was measured according to the instructions provided with the assay kits. Serum LH concentrations are expressed in ng/ml in terms of NIH rLH RP 1 (3). Statistical comparisons were made using analysis of variance and the Student Newman-Keuls procedures (11).
Results The day seven nicotine injection resulted in a significant decrease in LH with respect to saline treatment at 0 and 15 minutes @< 0.05) Figure 1A. The opioid antagonist, naloxone, did not stimulate increased LH secretion in rats treated with nicotine, while in saline treated rats, naloxone treatment caused a significant increase in serum LH concentrations (P < 0.01). The effect of clonidine on serum LH concentrations in nicotine and saline pre-treated rats is shown in Figure 1B. This alpha adrenergic agonist increased serum LH concentrations in both nicotine and saline treated rats (P < 0.05).
Vol. 60, No.
25,1!297
Nicotine and Luteinizing Hormone
2305
The effect of the dopamine agonist haloperidol on serum LH concentrations is shown in Figure 1C. Haloperidol treatment did not affect serum LH concentrations in either nicotine or saline pretreated rats. 60
1
SAL
60
A
SAL-
SAL NAL &------a NIC SAL -
SO --__ ii 2 E
_*
NIC NALC-----. --__ --__
,I
40.
P
/I
OJ
15
4s
I
0
60
SAL
400
60
SAL
D
M
SALLHRH b----a Nlc ML NIC LHRH e-----.
NIC HAL*-----.
z
E 3
ML
SAL HALO-------o NIC SAL’
SO
45
MINUTES AFTER CLONIDINE
C
SAL_
30
15
MINUTES AFTER NALOXONE
60
o---o o-------o M
OJ,
-I
30
0
SAL SAL S*L CL0 NIC SAL
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‘0 P
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0
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15
30
15
.
< 0
60
15
;0
is
60
MINUTES AFTER LHRH
MINUTES AFTER HALOPERIDOL
FIG. 1 Serum LH concentrations in ng/ml in male rats treated for 7 days with saline (open circles) or nicotine (closed circles). On day 7 the rats were injected immediately after collection of the time 0 blood sample (15 minutes after nicotine treatment) with: A. naloxone HCl (NAL), B. clonidine HCl (CLO), C. haloperidol (HAL) or D. luteinizing hormone releasing hormone (LHRH). *Significant difference from control @ < 0.05), ** significant difference from control (p < O.Ol), *** significant difference from nicotine treated rats (p
2336
Nicotine and Luteinking Hormone
Vol. 60, No. 25,1997
stimulatory effect of naltrexone on LH was blocked in the nicotine pre-treated rats. Nicotine treatment caused a modest decrease in basal LH concentrations in intact male rats, but the effect was not statistically significant.
OJ ,
0
15
SAL
SAL
o-----o
SAL
N*L
o------o
NIC
SAL
NC
NAL c-----4
-
30
MINUTES AFTER NALTREXONE
t 45
60
TREATMENT
FIG. 2 Serum LH concentrations in ng/ml in male rats given a single injection of nicotine (solid circles) or a single injection of saline (open circles) and then injected with saline (SAL) solid line or naltrexone (NAL) dashed line. ** Denotes significance from saline control (p
Vol. 60, No. 25,199l
Nicotine and Luteinking Hormone
2307
In mice, tolerance to the ACTH releasing effect of nicotine injections can be induced by administration of 2mg nicotine/kg bw 3 times a day for 2 weeks (13). Studies in rodents on the development of tolerance to nicotine indicate that it develops over a time span similar to our study. Since we observed lower basal LH levels and naloxone induced LH secretion were blocked in rats given nicotine for 7 days, we think that tolerance to the LH inhibiting effects of nicotine develops at a slower rate than ACTH tolerance. In addition we did not observe LH secretion when the long lasting opioid agonist naltrexone was given to rats treated acutely with nicotine. This observation suggests that we were using large enough doses of opioid antagonists to stimulate LH secretion in our study. Collectively our data suggest that endogenous opioids mediate some of the inhibitory effects of nicotine on LH secretion and tolerance is slow in developing. LH secretion one hour after the administration of a pharmacological dose of LHRH was reduced in rats that received nicotine daily for 7 days. Nicotine did not alter LHRH induced LH secretion at earlier times. These results suggest that pituitary capacity for LH secretion is reduced by repetitive nicotine treatment. We have found that intermittent treatment with nicotine reduces anterior pituitary gland LH content in adult rats (unpublished observation), and we believe that the reduction in pituitary gland LH content may explain the reduction in the response to LHRH. The reduced LHRH induced LH release in the nicotine treated rats support our conclusion that tolerance to the inhibiting effect of nicotine on LH secretion did not develop in the 7 day time frame of this study. Clonidine induced increases in LH were not prevented by nicotine treatment. Clonidine treatment resulted an acute increase in serum LH concentrations in both the nicotine treated and control rats. Clonidine acts to stimulate acute increases in LHRH. In the time frame of clonidine stimulation, 15 minutes post injection, we found a normal LH response to LHRH. Differences in LH secretion in response to clonidine might appear in a long term clonidine administration study. Our results with clonidine indicate that nicotine does not alter the activity of noradrenergic neurons which stimulate LH secretion. Andersson & al (14) and Fuxe et al (15) have indicated that hypothalamic dopaminergic neurons mediate the suppressive effect of nicotine on luteinizing hormone secretion. In our study we administered the dopamine receptor blocker haloperidol after nicotine, to see if dopaminergic blockade would increase LH in nicotine treated rats. We noted a modest attenuation of the nicotine effect after haloperidol, but it was not statistically significant. Likewise the effects of haloperidol on LH secretion in control rats were not significant. Administering haloperidol before nicotine treatment may make the inhibitor effect of dopamine more apparent. Our results though negative do not exclude dopaminergic mediation of the inhibitory effect of nicotine on LH secretion (14,15). Collectively these data indicate that repetitive administration of a pharmacologic dose of nicotine over a 7 day period can effect decreases in basal LH secretion (14). The repetitive administration reduces the capacity of the pituitary gland for LH release in response to a high dose challenge with LHRH. Repetitive nicotine administration prevented opioid induced LH secretion but it did not affect LH secretion induced by clonidine. These results suggest that the neural mechanisms controlling gonadotrophin secretion are not immediately desensitized by repetitive administration of nicotine and suggest an opioidergic component mediates the suppressive effects of nicotine on LH secretion.
Nicotine and Luteinking Hormone
1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Vol. 60, No. 25,lW
C.A. BLAKE, R.J. SCARAMUZZI, R.L. NORMAN, S. KANEMATSU and C.H. SAWYER, Endocrinology 91 1253-1258 (1972). K. ANDERSSON, K. FUXE, P. ENEROTH, A. HARFSTRAND and L.F. AGNATI, Neuroendocrinology $8 188-200 (1988). C.A. HODSON, A. DAVENPORT, G. PRICE and H.W. BURDEN, Life Sci. a 839-846 (1993). D.S. MCGEHEE, M.J.S. HEATH, S. GELBER, P. DEVAY and L.W. ROLE, Science m 1692-1696 (1995). R.T. JONES, T.R. FARRELL III and R.I. HERNING, NIDA Res. Monogr. 211202-208 (1978). C.J. MELISKA and D.G. GILBERT, Pharmacol. B&hem. Behav. @ 229-235 (1991). G.R. CAM, J.R. BASSETT and K.D. CARINCROSS, Arch. Int. Pharmacodyn. Ther. a 49-66 (1979). G.R. CAM and J.R. BASSETT, Pharmacol. B&hem. Behav. 24 221-226 (1984). B.M. SHARP and S. BEYER, J. Pharmacol. Exp. Ther. m 486-491 (1986). F.C. GREENWOOD, W.M. HUNTER and J.S. GLOVER, B&hem. J. @ 114-123 (1963). G.W. SNEDECOR and W.G. COCHRAN, Statistical Methods, 6th ed, Iowa State University Press, Ames, Iowa (1968). A.R. CAGGIULA, L.H. EPSTEIN, S.M. ANTLEMAN, S. SAYLOR, S. KNOPF, K.A. PERKINS and R. STILLER, Psychopharmacology 111499-507 (1993). J.R. PAULY, E.U. GRUN and A.C. COLLINS, Psychopharmacology mS 33-39 (1992). K. ANDERSSON, P. ENEROTH, K. FUXE, F. MASCAGNI and L.F. AGNATI, Neuroendocrinology 41462-466 (1985). K. FUXE, L. AGNATI, P. ENEROTH, J.A. GUSTAFSSON, T. HOKFELT, A. LOFSTROM, B. SKE’IT and P. SKETI’, Med. Biol. 55 148-157 (1977).
PI1 s0024-3205(97)00285-3
ELSEVIER
EFFECT OF PRIOR NICOTINE TREATMENT ON DRUG INDUCED CHANGES IN SERUM LH CONCENTRATIONS IN RATS
C. A. Hodson lBt2,M. C. Davis’ and H. W. Burden3
’ Department of Obstetrics and Gynecology and 3 Department of Anatomy and Cell Biology, School of Medicine, East Carolina University, Greenville, NC 27858 (Received in final form March 17,1997)
Summary The effect of daily injections of nicotine on drug induced changes in LH secretion was investigated in male rats. Daily administration of nicotine for 7 days resulted in decreased basal serum LH concentrations. Nicotine treatment blocked naloxone induced LH release and reduced LHRH induced increases in serum LH. Clonidine induced increases in serum LH were not altered by nicotine treatment and haloperidol treatment did not alter nicotine induced decreases in serum LH. In an acute study nicotine blocked LH secretion induced by the long acting opioid antagonist naltrexone. Collectively these results indicate that opioidergic neurons are involved in the reduction in serum LH that occurs following nicotine. They also indicate that chronic nicotine treatment can reduce the pituitary gland response to LHRH. Key Word: nicotine, luteinizing hormone, clonidine, pituitary gland, haloperidol, luteinking hormone, naltrexone, naloxone
hormone releasing
Acute administration of nicotine has an inhibitory effect on luteinizing hormone secretion in rats (1). This effect of nicotine has a major dopaminergic component but other nerurotransmitters may be involved (2). We have reported that nicotine treatment can prevent naloxone induced LH secretion, and these results suggest that opiodergic neurons may be involved in the suppressive effect of nicotine on LH (3). In the central nervous system nicotine alters neural responsiveness to other neurotransmitters such as glutamate and acetylcholine (4). Nicotine may cause a similar change in responsiveness to opioids within the central nervous system. Tolerance develops rapidly to many effects of nicotine (5) and it is not clear that the acute decrease in LH following nicotine treatment is biologically significant. Nicotine treatment also causes acute increases in ACTH and corticoid release in rats (6,7) and tolerance to this effect develops rapidly. Thereafter ACTH and corticoids are not altered by nicotine treatment (8,9).
* Corresponding author
2304
Nicotine and Luteinizing Hormone
Vol. 60, No. 25,1997
Because previous reports (8,9) showing that tolerance to nicotine develops rapidly used similar nicotine doses used in our previous study (l), we reasoned that a similar attenuation of the LH response to nicotine might develop. In this study, we determined whether chronic treatment with nicotine affected naloxone, clonidine and luteinizing hormone releasing hormone induced changes in LH secretion. We were also interested in determining if other neurotransmitters were involved in the nicotine induced inhibition of LH and whether these responses were attenuated after repetitive injection of nicotine.
aM teri Animals: Male Sprague-Dawley rats weighing 500 grams, obtained from Charles River, Inc. were used for these studies. They were housed in a 12-12 hour light/dark cycle, and food and water were provided ad libitum. Exneriment 1: Rats were randomly divided into 4 treatment groups. Two groups were given daily subcutaneous injections of 0.5 mg nicotine, and two groups received saline injections. On day 7, the rats were given their final injections of nicotine or saline. Fifteen minutes after these injections were given, the time 0 blood sample was collected. One of the following drugs: A. 5 mg naloxone HCWkg bw, B. 0.3 mg clonidine HCL/kg bw, C. 1 mg haloperidol/kg bw, D. 500 ng luteinizing hormone releasing hormone or E. saline was injected immediately after the time 0 bleeding. Additional blood samples were collected from the retro-orbital sinus at 15, 30, and 60 minutes after time 0 using ether anesthesia. Exueriment 2: Rats were randomly assigned to one of four treatment groups. Two groups were injected subcutaneously with .5 mg of nicotine, and the other two with an equivalent volume of saline. Fifteen minutes later, at time 0, an orbital sinus blood sample was collected. The rats were immediately injected subcutaneously with naltrexone 5 mg/kg bw or saline. Additional blood samples were collected at 15, 30, and 60 minutes. The four treatment combinations were: A. saline followed by saline, B. nicotine then saline injection, C. saline then naltrexone, and D. nicotine followed by naltrexone. -methods: Serum LH concentrations were measured by radioimmunoassay using reagents provided by the Hormone Distribution Program of the National Institutes of Health. LH was iodinated using the method of Greenwood, Hunter, and Glover (10). Serum LH was measured according to the instructions provided with the assay kits. Serum LH concentrations are expressed in ng/ml in terms of NIH rLH RP 1 (3). Statistical comparisons were made using analysis of variance and the Student Newman-Keuls procedures (11).
Results The day seven nicotine injection resulted in a significant decrease in LH with respect to saline treatment at 0 and 15 minutes @< 0.05) Figure 1A. The opioid antagonist, naloxone, did not stimulate increased LH secretion in rats treated with nicotine, while in saline treated rats, naloxone treatment caused a significant increase in serum LH concentrations (P < 0.01). The effect of clonidine on serum LH concentrations in nicotine and saline pre-treated rats is shown in Figure 1B. This alpha adrenergic agonist increased serum LH concentrations in both nicotine and saline treated rats (P < 0.05).
Vol. 60, No.
25,1!297
Nicotine and Luteinizing Hormone
2305
The effect of the dopamine agonist haloperidol on serum LH concentrations is shown in Figure 1C. Haloperidol treatment did not affect serum LH concentrations in either nicotine or saline pretreated rats. 60
1
SAL
60
A
SAL-
SAL NAL &------a NIC SAL -
SO --__ ii 2 E
_*
NIC NALC-----. --__ --__
,I
40.
P
/I
OJ
15
4s
I
0
60
SAL
400
60
SAL
D
M
SALLHRH b----a Nlc ML NIC LHRH e-----.
NIC HAL*-----.
z
E 3
ML
SAL HALO-------o NIC SAL’
SO
45
MINUTES AFTER CLONIDINE
C
SAL_
30
15
MINUTES AFTER NALOXONE
60
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SAL SAL S*L CL0 NIC SAL
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300
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0
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15
30
15
.
< 0
60
15
;0
is
60
MINUTES AFTER LHRH
MINUTES AFTER HALOPERIDOL
FIG. 1 Serum LH concentrations in ng/ml in male rats treated for 7 days with saline (open circles) or nicotine (closed circles). On day 7 the rats were injected immediately after collection of the time 0 blood sample (15 minutes after nicotine treatment) with: A. naloxone HCl (NAL), B. clonidine HCl (CLO), C. haloperidol (HAL) or D. luteinizing hormone releasing hormone (LHRH). *Significant difference from control @ < 0.05), ** significant difference from control (p < O.Ol), *** significant difference from nicotine treated rats (p
2336
Nicotine and Luteinking Hormone
Vol. 60, No. 25,1997
stimulatory effect of naltrexone on LH was blocked in the nicotine pre-treated rats. Nicotine treatment caused a modest decrease in basal LH concentrations in intact male rats, but the effect was not statistically significant.
OJ ,
0
15
SAL
SAL
o-----o
SAL
N*L
o------o
NIC
SAL
NC
NAL c-----4
-
30
MINUTES AFTER NALTREXONE
t 45
60
TREATMENT
FIG. 2 Serum LH concentrations in ng/ml in male rats given a single injection of nicotine (solid circles) or a single injection of saline (open circles) and then injected with saline (SAL) solid line or naltrexone (NAL) dashed line. ** Denotes significance from saline control (p
Vol. 60, No. 25,199l
Nicotine and Luteinking Hormone
2307
In mice, tolerance to the ACTH releasing effect of nicotine injections can be induced by administration of 2mg nicotine/kg bw 3 times a day for 2 weeks (13). Studies in rodents on the development of tolerance to nicotine indicate that it develops over a time span similar to our study. Since we observed lower basal LH levels and naloxone induced LH secretion were blocked in rats given nicotine for 7 days, we think that tolerance to the LH inhibiting effects of nicotine develops at a slower rate than ACTH tolerance. In addition we did not observe LH secretion when the long lasting opioid agonist naltrexone was given to rats treated acutely with nicotine. This observation suggests that we were using large enough doses of opioid antagonists to stimulate LH secretion in our study. Collectively our data suggest that endogenous opioids mediate some of the inhibitory effects of nicotine on LH secretion and tolerance is slow in developing. LH secretion one hour after the administration of a pharmacological dose of LHRH was reduced in rats that received nicotine daily for 7 days. Nicotine did not alter LHRH induced LH secretion at earlier times. These results suggest that pituitary capacity for LH secretion is reduced by repetitive nicotine treatment. We have found that intermittent treatment with nicotine reduces anterior pituitary gland LH content in adult rats (unpublished observation), and we believe that the reduction in pituitary gland LH content may explain the reduction in the response to LHRH. The reduced LHRH induced LH release in the nicotine treated rats support our conclusion that tolerance to the inhibiting effect of nicotine on LH secretion did not develop in the 7 day time frame of this study. Clonidine induced increases in LH were not prevented by nicotine treatment. Clonidine treatment resulted an acute increase in serum LH concentrations in both the nicotine treated and control rats. Clonidine acts to stimulate acute increases in LHRH. In the time frame of clonidine stimulation, 15 minutes post injection, we found a normal LH response to LHRH. Differences in LH secretion in response to clonidine might appear in a long term clonidine administration study. Our results with clonidine indicate that nicotine does not alter the activity of noradrenergic neurons which stimulate LH secretion. Andersson & al (14) and Fuxe et al (15) have indicated that hypothalamic dopaminergic neurons mediate the suppressive effect of nicotine on luteinizing hormone secretion. In our study we administered the dopamine receptor blocker haloperidol after nicotine, to see if dopaminergic blockade would increase LH in nicotine treated rats. We noted a modest attenuation of the nicotine effect after haloperidol, but it was not statistically significant. Likewise the effects of haloperidol on LH secretion in control rats were not significant. Administering haloperidol before nicotine treatment may make the inhibitor effect of dopamine more apparent. Our results though negative do not exclude dopaminergic mediation of the inhibitory effect of nicotine on LH secretion (14,15). Collectively these data indicate that repetitive administration of a pharmacologic dose of nicotine over a 7 day period can effect decreases in basal LH secretion (14). The repetitive administration reduces the capacity of the pituitary gland for LH release in response to a high dose challenge with LHRH. Repetitive nicotine administration prevented opioid induced LH secretion but it did not affect LH secretion induced by clonidine. These results suggest that the neural mechanisms controlling gonadotrophin secretion are not immediately desensitized by repetitive administration of nicotine and suggest an opioidergic component mediates the suppressive effects of nicotine on LH secretion.
Nicotine and Luteinking Hormone
1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
Vol. 60, No. 25,lW
C.A. BLAKE, R.J. SCARAMUZZI, R.L. NORMAN, S. KANEMATSU and C.H. SAWYER, Endocrinology 91 1253-1258 (1972). K. ANDERSSON, K. FUXE, P. ENEROTH, A. HARFSTRAND and L.F. AGNATI, Neuroendocrinology $8 188-200 (1988). C.A. HODSON, A. DAVENPORT, G. PRICE and H.W. BURDEN, Life Sci. a 839-846 (1993). D.S. MCGEHEE, M.J.S. HEATH, S. GELBER, P. DEVAY and L.W. ROLE, Science m 1692-1696 (1995). R.T. JONES, T.R. FARRELL III and R.I. HERNING, NIDA Res. Monogr. 211202-208 (1978). C.J. MELISKA and D.G. GILBERT, Pharmacol. B&hem. Behav. @ 229-235 (1991). G.R. CAM, J.R. BASSETT and K.D. CARINCROSS, Arch. Int. Pharmacodyn. Ther. a 49-66 (1979). G.R. CAM and J.R. BASSETT, Pharmacol. B&hem. Behav. 24 221-226 (1984). B.M. SHARP and S. BEYER, J. Pharmacol. Exp. Ther. m 486-491 (1986). F.C. GREENWOOD, W.M. HUNTER and J.S. GLOVER, B&hem. J. @ 114-123 (1963). G.W. SNEDECOR and W.G. COCHRAN, Statistical Methods, 6th ed, Iowa State University Press, Ames, Iowa (1968). A.R. CAGGIULA, L.H. EPSTEIN, S.M. ANTLEMAN, S. SAYLOR, S. KNOPF, K.A. PERKINS and R. STILLER, Psychopharmacology 111499-507 (1993). J.R. PAULY, E.U. GRUN and A.C. COLLINS, Psychopharmacology mS 33-39 (1992). K. ANDERSSON, P. ENEROTH, K. FUXE, F. MASCAGNI and L.F. AGNATI, Neuroendocrinology 41462-466 (1985). K. FUXE, L. AGNATI, P. ENEROTH, J.A. GUSTAFSSON, T. HOKFELT, A. LOFSTROM, B. SKE’IT and P. SKETI’, Med. Biol. 55 148-157 (1977).