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Decreased 5-HT2 but not 5-HT1 receptor binding in cortex of rat after chronic administration of dothiepin
Neuropharmacology Vol. 24, No. 4, pp. 275-278, Printed in Great Britain. All rights resewed
1985 Copyright
0
0028-3908/85 %3.00 + 0.00 1985 Pergamon Press Ltd
DECREASED 5-HT,BUT NOT 5-HT, RECEPTOR BINDING IN CORTEX OF RAT AFTER CHRONIC ADMINISTRATION OF DOTHIEPIN APPLICATION
OF THE WOOLF PLOT TO ANALYSIS PARAMETERS
OF BINDING
G. L. DIGGORY and W. R. BUCKETT Research Department, The Boots Company PLC, Nottingham, NG2 3AA, England (Accepted
5 September
1984)
Summary-Prolonged administration of the antidepressant drug, dothiepin hydrochloride (30 mg/kg orally twice daily for 24 days), resulted in a significant decrease in the population of serotonin, (5-HT,) binding sites in the frontal cortex of rats whereas serotonin, (5-HT,) binding sites remained unaltered. No significant differences in affinity constants for either ligand-binding site interaction were observed. Analyses of the binding parameters was performed using linear transformation methods of the specific binding isotherms according to Scatchard (1949) [Ann. N. Y. Acad. Sci. 51: 660-6721 or Woolf (see Haldane, 1957: Nature 179: 832). The resulting parameter estimates generated in each analysis were compared. Although both methods demonstrated the decreased B,, for 5-HT, binding sites with no change in 5-HT, sites after prolonged administration of dothiepin, Woolf analyses gave reliably better estimates of the binding parameters as judged by examination of the respective correlation coefficients for best fit linear regression lines. Key words: 5-HT, receptor, 5-HT, receptor, Woolf analysis, rat cortex, dothiepin.
The mode of action of antidepressant drugs is still not completely understood although the acute effects of these compounds on uptake systems and turnover of neurotransmitters are well described. Increasing effort is now focussed on the biochemical consequences of extended treatment in view of the delayed clinical efficacy of the drugs (Oswald, Brezinova and Dunleavy, 1972). Tricyclic antidepressant drugs, including dothiepin, induce decreases in the number of /J-receptors (Banerjee, Kung, Riggi and Chanda, 1977; ClementsJewery, 1978; Buckett and Thomas, 1982) and reduce the sensitivity of the adenylate cyclase linked to noradrenaline (NA) in limbic forebrain of the rat (Mobley and Sulser, 1981; Buckett and Diggory, 1982). Subsensitivity of 5-HT, but not 5-HT, receptors has been shown to occur only after prolonged treatment with the tricyclic antidepressants (Maggi, U’Prichard and Enna, 1980; Peroutka and Snyder, 1980a, b) and with the atypical antidepressants, iprindole (Peroutka and Snyder, 1980a) and mianserin (Blackshear and Sanders-Bush, 1982). The present studies were carried out to determine whether dothiepin affects subpopulations of 5-HT receptors after prolonged administration and to investigate the value of the Woolf plot in the analysis of binding parameters for neurotransmitter systems, as has already been described for steroid hormone systems (Keightley and Cressie, 1980). A preliminary account of this work has already been presented (Buckett and Diggory, 1984).
METHODS Male Sprague-Dawley
rats (175-225 g) were given
30 mg/kg dothiepin hydrochloride (Prothiaden, Boots Co. PLC, Nottingham) orally twice daily for 24 days and subsequent doses and results are expressed in terms of this salt. Control animals were treated with 0.9% saline for 24 days. Twenty-four hours after the final treatment, the animals were killed and the brains rapidly removed, washed in ice-cold saline followed by dissection of the frontal cortices on an ice-cold porcelain plate. Cortices were then weighed and homogenised (4°C) in 40 vol of 50 mM Tri-HCl buffer (pH 7.7, 25°C) using a Polytron (setting 6, 15 set). The homogenate was centrifuged (4O,OOOg,15 min) and the washing procedure repeated. The washed membrane preparation was resuspended and preincubated for 15 min at 37°C. After recentrifugation (4O,OOOg, 10 min), the final membrane pellet was resuspended in 100 vol Tri-HCI buffer, as described above, but containing 4 mM CaCl,, 10 PM pargyline and 0.1% (w/v) ascorbic acid. Tissue aliquots equivalent to 18 mg original wet weight of cortex (1.8 ml) were added to polypropylene incubation tubes (84 x 13 mm) followed by ligands (100 ~1) and drugs (100 ~1) as appropriate. For 5-HT, binding sites, l&20.0 nM [3H]5-HT (100~1) was used with lO/*M unlabelled 5-HT (100 ~1) to determine non-specific binding. Binding sites for 5-HT, were measured using 0.25-5.0 nM [‘Hlspiperone (100 ~1) together with 275
276
G. L. DIGG~KY and W. R. BUCUTT 2.5
2.0
\ \
1.5 4 Q 1.0
0.5
8
8
Fig. 1. Effect of chronic administration of dothiepin on the binding of [‘HIS-HT and [3H]spiroperidol to 5-HT, and 5-HT, binding sites in the frontal cortex of the rat. Rats were treated twice daily with 30 mg/kg (p.0.) dothiepin for 24 days and killed 24 hr later. In the illustrated Scatchard analyses the individual points plotted represents the means obtained from several binding experiments performed on individual animals (for n values, see Table 1). The B,,, and K, values obtained in the conventional manner were
as follows for 5-HT, binding: controls: B,,, = 13.49, K, = 6.71, I = 0.91; dothipein: B,,,,, = 13.12, K, = 5.85, r = 0.94. For 5-HT, binding: controls: B,,,,, = 12.78, K, = 1.36, Y = 0.95; dothiepin: Bmax= 8.59, K, = 1.18, I = 0.97. 1.O p M d-lysergic acid diethylamide (d-LSD; 100~1). Tubes were gently mixed by vortexing and incubated at 37°C for 15 min; after incubation, tube contents were rapidly filtered under vacuum using Whatman GF/B filters, washed with 3 x 4ml aliquots of icecold buffer and the filters counted for radioactivity in lOm1 of scintillant (Packard Emulsifier, 299 TM) using a Packard Tri-Carb 4530 at a counting efficiency of 55-60$//,. ANALYSIS
OF BINDING PARAMETERS
Estimates of the parameters of specific saturation binding isotherms (K, and I?,,,) were generated by
F
linear transformation methods chard (1949) and Woolf (see outlined by Keightley and Michaelis-Menten relationship perbola of the form: B
=
as described by ScatHaldane, 1957). As Cressie (1980), the is described by a hy-
vL,JF K,+F
and linearisation can be achieved in several ways and as described by Scatchard (1949) thus: B &a, _=F K,
(B,,, = B at B/F = 0, l/K,
= slope of plotted
F
Fig. 2. Effect of chronic administration of dothiepin on the binding of [3H]5-HT and [3H]spiroperidol to 5-HT, and 5-HT, binding sites in the frontal cortex of the rat. Rats were treated twice daily with 30 mg/kg (p.0.) dotl,iepin for 24 days and killed 24 hr later. In the illustrated Woolf analyses, the individual points plotted represent the means obtained from several binding experiments performed on individual animals (for n values, see Table 1). The B,,,,, and K, values obtained as described in the Methods section were as follows for 5-HT, binding: controls: B,, = 12.14, K, = 6.0, r = 0.99; dothiepin: B,,,,, = 14.46, K, = 7.9, Y = 0.98. For 5-HT, binding : controls: B,,,,, = 11.89, Ko = 1.26, r = 0.99; dothiepin: B,,, = 8.22, K, = 1.25, r = 0.99.
line)
Decreased Table 1. The elkts of extended 5-HT7 binding.
5-HT,
but not 5-HT,
receptor
administration of dothiepin to rats on the parameters as assessed by both Scatchard and Woolf analysis
KO Dosing
details
217
binding of 5-HT, and
Bmiix (pmol g-‘)
Data analysis
n
Dothiepin 0.9% Saline Dothiepin 0.9% Saline
Scatchard
I2 5 12 5
8.0 10.8 8.55 9.07
1.35 2.24 1.46 1.99
14.35 16.93 14.86 15.46
f k k +
1.22 1.46 1.43 2.21
0.89 0.87 0.97 0.95
f + * +
0.02 0.03 0.01 0.02
Dothiepin 0.9% Saline Dothiepin 0.9% Saline
Scatchard
16 8 16 8
1.54*0.17 1.64 If: 0.14 1.58 +O.lS 1.61 + 0.20
9.69 14.24 8.51 14.01
f 0.74’ & 0.90 + l.OO* * 1.12
0.89 0.93 0.98 0.98
+ * + f
0.02 0.01 0.01 0.01
(nm)
r
5-HT, 24-day 24-day 24-day 24-day 5-HT, 24-day 24-day 24-day 24-day
Woolf
Woolf
k i ir i
*P < 0.01 compared to 0.9% saline controls. Values are expressed as the mean f SE (n as indicated) of estimates obtained in the individual linear analyses, which were then pooled and treated statistically using the Student’s f-test (see Methods).
or by Woolf
(1957) thus: F
Kn
1
z=K+$F nlax[ Inix (Kn = F at F/B = 0,l/B,,, = slope of plotted line). In the present study single estimates of K, and B,,,
Methods section. Whilst no change in the parameters of 5-HT, binding sites was apparent, the decrease in the population of 5-HT, binding sites is clearly demonstrated, with no change in affinity constant. Again, the superiority of Woolf analysis in reliably providing estimates of binding parameters is clearly illustrated.
for both 5-HT, and S-HT, binding sites obtained from individual animals generated using either linear analysis were pooled and treated statistically DISCUSSION (Student’s t-test). Table 1 presents the results of these The 5-HT, and 5-HT, binding sites were measured analyses for both control and animals treated with dothiepin. using procedures previously reported (Peroutka and As an alternative method of analysis of the data, Snyder, 1980a, b) and estimates of the binding estimates of the K, and B,,,values were generated parameters (Kn and B,,,,,) have been generated using linear transformations of the binding isotherms. The by Scatchard and Woolf analyses using data points commonly used Scatchard and the comparatively representing the mean of several separate experiments unknown Woolf plots were compared and the Woolf performed on individual animals. The graphical representations of such studies are illustrated in Fig. 1 analysis found to produce more reliable estimates of the binding parameters. This finding extends to a (Scatchard) and Fig. 2 (Woolf); the K, and B,,,,, neurotransmitter system, for the first time, the imvalues obtained together with the correlation coefficients for the linear regression lines are also provements in data analysis previously reported for presented. a steroid hormone receptor binding assay using oestradiol-17/? (Keightley and Cressie, 1980). Prolonged administration of dothiepin to rats reRESULTS sulted in a decrease in the number of 5-HT, but not of 5-HT, sites in the cortex and was demonstrated The results presented in Table 1 demonstrate that using either linear analysis method. This extends to prolonged administration of dothiepin to rats did not dothiepin the finding that antidepressant drugs affect the parameters of the S-HT, binding site. Thus, “down-regulate” 5-HT2 binding sites in the cortex of the mean values for K, and B,,, were not the rat (inter alia, Peroutka and Snyder, 1980a, b; significantly different in the drug-treated group comBlackshear and Sanders-Bush, 1982). It is of interest pared to saline-treated controls, using either Scatto note that previous studies have demonstrated that, chard or Woolf analysis. However, examination of consistent with these biochemical findings, prolonged correlation coefficients demonstrated the better administration of dothiepin to rats attenuated reliability of such parameters using Woolf as compared to Scatchard analysis. In contrast to the results behavioural paradigms induced by 5-methoxyN’,N’-dimethyltryptamine and 5-hydroxytryptophan of 5-HT, binding sites, prolonged administration of (Buckett and Luscombe, 1983), facets of which are dothiepin significantly decreased the population of thought to be mediated by the 5-HT, receptor S-HT, binding sites (decreased B,,,) with no corresponding alteration in K, values as assessed by both (Green, 1984). Scatchard and Woolf analysis. Again, better correlation coefficients were obtained after Woolf REFERENCES analysis, thus providing more reliable estimates of Banerjee S. P., King L. S., Riggi S. J. and Chanda S. K. generated parameter values. (1977) Development of /?-adrenergic receptor subFigures 1 and 2 present graphically, the Scatchard sensitivity by antidepressants. Nature 268: 455-456. and Woolf plots obtained as described in the Blackshear M. A. and Sanders-Bush E. (1982) Serotonin
278
G. L. DIGGORY and W. R. BUCKETS
recentor sensitivitv after acute and chronic treatment with mianserin. J. Pharmac. exp. Ther. 221: 303-308. Buckett W. R. and Dieerorv G. L. (1982) Subsensitivitv of the noradrenaline receptor-coupled adenylate cyclase system in rat limbic forebrain induced by dothiepin. Br. J. Pharmac. 77: 5 I4P. Buckett W. R. and Thomas P. C. (1982) The antidepressant dothiepin reduces cortical p-adrenoceptor binding after subchronic oral administration to rats. Br. J. Pharmac. 75: 97P. Buckett W. R. and Luscombe G. P. (1983) Dothiepin and desipramine induce different changes in noradrenergic and serotoninergic behaviour after subchronic treatment. Br. J. Pharmac. 80: 638P. Buckett W. R. and Diggory G. L. (1984) The antidepressant dothiepin induces down-regulation of cortical 5-HTz receptors after subchronic oral treatment in the rat. Br. J. Pharmac. 82: 2lOP. Clements-Jewery S. (1978) The development of cortical B-adrenoreceptor subsensitivity in the rat by chronic treatment with trazodone, doxepin and mianserine. Neuropharmacology 17: 179-18 1. Green A. R. (1984) 5-HT and behaviour-animal studies. Neuropharnkol;gy 23: 1521-l 528. Haldane J. B. S. (1957) Graphical methods in enzyme chemistry. Nature 179: 832. II
_
.
,
Keightley D. D. and Cressie N. A. C. (I 980) The Woolf plot is more reliable than the Scatchard plot in analysing data from hormone receptor assays. J. steroid Biochem. 13: 1317-1323. Maggi A., UPrichard D. C. and Enna S. J. (1980) Differential effects of antidepressant treatment on rat brain monoaminergic receptors. Eur. J. Pharmac. 61: 91-98. Mobley P. L. and Sulser F. (1981) Down-regulation of the central noradrenergic receptor system by antidepressant therapies: biochemical and chemical aspects. In: Antidepressants: Neurochemicul, Behavioural and Clinical Perspectives (Enna S. J., Malick J. B. and Richelson E.. Eds), pp. 31-51. Raven Press, New York. Oswald I., Brezinova V. and Dunleavy D. L. F. (1972) On the slowness of action of tricyclic antidepressant drugs. Br. J. Psychiat. 120: 673-677. Peroutka S. J. and Snyder S. H. (1980a) Long-term antidepressant treatment decreases spiroperidol-labeled serotonin receptor binding. Science 210: 88-90. Peroutka S. J. and Snyder S. H. (1980b) Regulation of serotonin (5-HT,) receptors labeled with )H-spiroperidol by chronic treatment with the antidepressant amitriptyline. J. Pharmac. exp. Ther. 215: 582-587. Scatdhard G. (1949) The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51: 66&672.
1985 Copyright
0
0028-3908/85 %3.00 + 0.00 1985 Pergamon Press Ltd
DECREASED 5-HT,BUT NOT 5-HT, RECEPTOR BINDING IN CORTEX OF RAT AFTER CHRONIC ADMINISTRATION OF DOTHIEPIN APPLICATION
OF THE WOOLF PLOT TO ANALYSIS PARAMETERS
OF BINDING
G. L. DIGGORY and W. R. BUCKETT Research Department, The Boots Company PLC, Nottingham, NG2 3AA, England (Accepted
5 September
1984)
Summary-Prolonged administration of the antidepressant drug, dothiepin hydrochloride (30 mg/kg orally twice daily for 24 days), resulted in a significant decrease in the population of serotonin, (5-HT,) binding sites in the frontal cortex of rats whereas serotonin, (5-HT,) binding sites remained unaltered. No significant differences in affinity constants for either ligand-binding site interaction were observed. Analyses of the binding parameters was performed using linear transformation methods of the specific binding isotherms according to Scatchard (1949) [Ann. N. Y. Acad. Sci. 51: 660-6721 or Woolf (see Haldane, 1957: Nature 179: 832). The resulting parameter estimates generated in each analysis were compared. Although both methods demonstrated the decreased B,, for 5-HT, binding sites with no change in 5-HT, sites after prolonged administration of dothiepin, Woolf analyses gave reliably better estimates of the binding parameters as judged by examination of the respective correlation coefficients for best fit linear regression lines. Key words: 5-HT, receptor, 5-HT, receptor, Woolf analysis, rat cortex, dothiepin.
The mode of action of antidepressant drugs is still not completely understood although the acute effects of these compounds on uptake systems and turnover of neurotransmitters are well described. Increasing effort is now focussed on the biochemical consequences of extended treatment in view of the delayed clinical efficacy of the drugs (Oswald, Brezinova and Dunleavy, 1972). Tricyclic antidepressant drugs, including dothiepin, induce decreases in the number of /J-receptors (Banerjee, Kung, Riggi and Chanda, 1977; ClementsJewery, 1978; Buckett and Thomas, 1982) and reduce the sensitivity of the adenylate cyclase linked to noradrenaline (NA) in limbic forebrain of the rat (Mobley and Sulser, 1981; Buckett and Diggory, 1982). Subsensitivity of 5-HT, but not 5-HT, receptors has been shown to occur only after prolonged treatment with the tricyclic antidepressants (Maggi, U’Prichard and Enna, 1980; Peroutka and Snyder, 1980a, b) and with the atypical antidepressants, iprindole (Peroutka and Snyder, 1980a) and mianserin (Blackshear and Sanders-Bush, 1982). The present studies were carried out to determine whether dothiepin affects subpopulations of 5-HT receptors after prolonged administration and to investigate the value of the Woolf plot in the analysis of binding parameters for neurotransmitter systems, as has already been described for steroid hormone systems (Keightley and Cressie, 1980). A preliminary account of this work has already been presented (Buckett and Diggory, 1984).
METHODS Male Sprague-Dawley
rats (175-225 g) were given
30 mg/kg dothiepin hydrochloride (Prothiaden, Boots Co. PLC, Nottingham) orally twice daily for 24 days and subsequent doses and results are expressed in terms of this salt. Control animals were treated with 0.9% saline for 24 days. Twenty-four hours after the final treatment, the animals were killed and the brains rapidly removed, washed in ice-cold saline followed by dissection of the frontal cortices on an ice-cold porcelain plate. Cortices were then weighed and homogenised (4°C) in 40 vol of 50 mM Tri-HCl buffer (pH 7.7, 25°C) using a Polytron (setting 6, 15 set). The homogenate was centrifuged (4O,OOOg,15 min) and the washing procedure repeated. The washed membrane preparation was resuspended and preincubated for 15 min at 37°C. After recentrifugation (4O,OOOg, 10 min), the final membrane pellet was resuspended in 100 vol Tri-HCI buffer, as described above, but containing 4 mM CaCl,, 10 PM pargyline and 0.1% (w/v) ascorbic acid. Tissue aliquots equivalent to 18 mg original wet weight of cortex (1.8 ml) were added to polypropylene incubation tubes (84 x 13 mm) followed by ligands (100 ~1) and drugs (100 ~1) as appropriate. For 5-HT, binding sites, l&20.0 nM [3H]5-HT (100~1) was used with lO/*M unlabelled 5-HT (100 ~1) to determine non-specific binding. Binding sites for 5-HT, were measured using 0.25-5.0 nM [‘Hlspiperone (100 ~1) together with 275
276
G. L. DIGG~KY and W. R. BUCUTT 2.5
2.0
\ \
1.5 4 Q 1.0
0.5
8
8
Fig. 1. Effect of chronic administration of dothiepin on the binding of [‘HIS-HT and [3H]spiroperidol to 5-HT, and 5-HT, binding sites in the frontal cortex of the rat. Rats were treated twice daily with 30 mg/kg (p.0.) dothiepin for 24 days and killed 24 hr later. In the illustrated Scatchard analyses the individual points plotted represents the means obtained from several binding experiments performed on individual animals (for n values, see Table 1). The B,,, and K, values obtained in the conventional manner were
as follows for 5-HT, binding: controls: B,,, = 13.49, K, = 6.71, I = 0.91; dothipein: B,,,,, = 13.12, K, = 5.85, r = 0.94. For 5-HT, binding: controls: B,,,,, = 12.78, K, = 1.36, Y = 0.95; dothiepin: Bmax= 8.59, K, = 1.18, I = 0.97. 1.O p M d-lysergic acid diethylamide (d-LSD; 100~1). Tubes were gently mixed by vortexing and incubated at 37°C for 15 min; after incubation, tube contents were rapidly filtered under vacuum using Whatman GF/B filters, washed with 3 x 4ml aliquots of icecold buffer and the filters counted for radioactivity in lOm1 of scintillant (Packard Emulsifier, 299 TM) using a Packard Tri-Carb 4530 at a counting efficiency of 55-60$//,. ANALYSIS
OF BINDING PARAMETERS
Estimates of the parameters of specific saturation binding isotherms (K, and I?,,,) were generated by
F
linear transformation methods chard (1949) and Woolf (see outlined by Keightley and Michaelis-Menten relationship perbola of the form: B
=
as described by ScatHaldane, 1957). As Cressie (1980), the is described by a hy-
vL,JF K,+F
and linearisation can be achieved in several ways and as described by Scatchard (1949) thus: B &a, _=F K,
(B,,, = B at B/F = 0, l/K,
= slope of plotted
F
Fig. 2. Effect of chronic administration of dothiepin on the binding of [3H]5-HT and [3H]spiroperidol to 5-HT, and 5-HT, binding sites in the frontal cortex of the rat. Rats were treated twice daily with 30 mg/kg (p.0.) dotl,iepin for 24 days and killed 24 hr later. In the illustrated Woolf analyses, the individual points plotted represent the means obtained from several binding experiments performed on individual animals (for n values, see Table 1). The B,,,,, and K, values obtained as described in the Methods section were as follows for 5-HT, binding: controls: B,, = 12.14, K, = 6.0, r = 0.99; dothiepin: B,,,,, = 14.46, K, = 7.9, Y = 0.98. For 5-HT, binding : controls: B,,,,, = 11.89, Ko = 1.26, r = 0.99; dothiepin: B,,, = 8.22, K, = 1.25, r = 0.99.
line)
Decreased Table 1. The elkts of extended 5-HT7 binding.
5-HT,
but not 5-HT,
receptor
administration of dothiepin to rats on the parameters as assessed by both Scatchard and Woolf analysis
KO Dosing
details
217
binding of 5-HT, and
Bmiix (pmol g-‘)
Data analysis
n
Dothiepin 0.9% Saline Dothiepin 0.9% Saline
Scatchard
I2 5 12 5
8.0 10.8 8.55 9.07
1.35 2.24 1.46 1.99
14.35 16.93 14.86 15.46
f k k +
1.22 1.46 1.43 2.21
0.89 0.87 0.97 0.95
f + * +
0.02 0.03 0.01 0.02
Dothiepin 0.9% Saline Dothiepin 0.9% Saline
Scatchard
16 8 16 8
1.54*0.17 1.64 If: 0.14 1.58 +O.lS 1.61 + 0.20
9.69 14.24 8.51 14.01
f 0.74’ & 0.90 + l.OO* * 1.12
0.89 0.93 0.98 0.98
+ * + f
0.02 0.01 0.01 0.01
(nm)
r
5-HT, 24-day 24-day 24-day 24-day 5-HT, 24-day 24-day 24-day 24-day
Woolf
Woolf
k i ir i
*P < 0.01 compared to 0.9% saline controls. Values are expressed as the mean f SE (n as indicated) of estimates obtained in the individual linear analyses, which were then pooled and treated statistically using the Student’s f-test (see Methods).
or by Woolf
(1957) thus: F
Kn
1
z=K+$F nlax[ Inix (Kn = F at F/B = 0,l/B,,, = slope of plotted line). In the present study single estimates of K, and B,,,
Methods section. Whilst no change in the parameters of 5-HT, binding sites was apparent, the decrease in the population of 5-HT, binding sites is clearly demonstrated, with no change in affinity constant. Again, the superiority of Woolf analysis in reliably providing estimates of binding parameters is clearly illustrated.
for both 5-HT, and S-HT, binding sites obtained from individual animals generated using either linear analysis were pooled and treated statistically DISCUSSION (Student’s t-test). Table 1 presents the results of these The 5-HT, and 5-HT, binding sites were measured analyses for both control and animals treated with dothiepin. using procedures previously reported (Peroutka and As an alternative method of analysis of the data, Snyder, 1980a, b) and estimates of the binding estimates of the K, and B,,,values were generated parameters (Kn and B,,,,,) have been generated using linear transformations of the binding isotherms. The by Scatchard and Woolf analyses using data points commonly used Scatchard and the comparatively representing the mean of several separate experiments unknown Woolf plots were compared and the Woolf performed on individual animals. The graphical representations of such studies are illustrated in Fig. 1 analysis found to produce more reliable estimates of the binding parameters. This finding extends to a (Scatchard) and Fig. 2 (Woolf); the K, and B,,,,, neurotransmitter system, for the first time, the imvalues obtained together with the correlation coefficients for the linear regression lines are also provements in data analysis previously reported for presented. a steroid hormone receptor binding assay using oestradiol-17/? (Keightley and Cressie, 1980). Prolonged administration of dothiepin to rats reRESULTS sulted in a decrease in the number of 5-HT, but not of 5-HT, sites in the cortex and was demonstrated The results presented in Table 1 demonstrate that using either linear analysis method. This extends to prolonged administration of dothiepin to rats did not dothiepin the finding that antidepressant drugs affect the parameters of the S-HT, binding site. Thus, “down-regulate” 5-HT2 binding sites in the cortex of the mean values for K, and B,,, were not the rat (inter alia, Peroutka and Snyder, 1980a, b; significantly different in the drug-treated group comBlackshear and Sanders-Bush, 1982). It is of interest pared to saline-treated controls, using either Scatto note that previous studies have demonstrated that, chard or Woolf analysis. However, examination of consistent with these biochemical findings, prolonged correlation coefficients demonstrated the better administration of dothiepin to rats attenuated reliability of such parameters using Woolf as compared to Scatchard analysis. In contrast to the results behavioural paradigms induced by 5-methoxyN’,N’-dimethyltryptamine and 5-hydroxytryptophan of 5-HT, binding sites, prolonged administration of (Buckett and Luscombe, 1983), facets of which are dothiepin significantly decreased the population of thought to be mediated by the 5-HT, receptor S-HT, binding sites (decreased B,,,) with no corresponding alteration in K, values as assessed by both (Green, 1984). Scatchard and Woolf analysis. Again, better correlation coefficients were obtained after Woolf REFERENCES analysis, thus providing more reliable estimates of Banerjee S. P., King L. S., Riggi S. J. and Chanda S. K. generated parameter values. (1977) Development of /?-adrenergic receptor subFigures 1 and 2 present graphically, the Scatchard sensitivity by antidepressants. Nature 268: 455-456. and Woolf plots obtained as described in the Blackshear M. A. and Sanders-Bush E. (1982) Serotonin
278
G. L. DIGGORY and W. R. BUCKETS
recentor sensitivitv after acute and chronic treatment with mianserin. J. Pharmac. exp. Ther. 221: 303-308. Buckett W. R. and Dieerorv G. L. (1982) Subsensitivitv of the noradrenaline receptor-coupled adenylate cyclase system in rat limbic forebrain induced by dothiepin. Br. J. Pharmac. 77: 5 I4P. Buckett W. R. and Thomas P. C. (1982) The antidepressant dothiepin reduces cortical p-adrenoceptor binding after subchronic oral administration to rats. Br. J. Pharmac. 75: 97P. Buckett W. R. and Luscombe G. P. (1983) Dothiepin and desipramine induce different changes in noradrenergic and serotoninergic behaviour after subchronic treatment. Br. J. Pharmac. 80: 638P. Buckett W. R. and Diggory G. L. (1984) The antidepressant dothiepin induces down-regulation of cortical 5-HTz receptors after subchronic oral treatment in the rat. Br. J. Pharmac. 82: 2lOP. Clements-Jewery S. (1978) The development of cortical B-adrenoreceptor subsensitivity in the rat by chronic treatment with trazodone, doxepin and mianserine. Neuropharmacology 17: 179-18 1. Green A. R. (1984) 5-HT and behaviour-animal studies. Neuropharnkol;gy 23: 1521-l 528. Haldane J. B. S. (1957) Graphical methods in enzyme chemistry. Nature 179: 832. II
_
.
,
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