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Autoradiographic studies on the effect of ethanol on muscarinic receptors in rat brain
Neurochem. Int. Vol. 15, No. I, pp. 91-95, 1989 Printed in Great Britain
0197-0186/89 $3.00 + 0.00 Pergamon Press plc
AUTORADIOGRAPHIC STUDIES ON THE EFFECT OF ETHANOL ON MUSCARINIC RECEPTORS IN RAT BRAIN G. F. DUNGLISON,P. A. WILCE* and B. C. SHANLEY Alcohol Research Unit, Department of Biochemistry, University of Queensland, St Lucia 4067, Queensland, Australia (Received 5 September 1988; accepted 30 January 1989)
Abstract--The effect of ethanol vapour inhalation on the binding characteristics of muscarinic receptors in rat brain has been studied using quantitative autoradiography. After 14 days in ethanol vapour there was increased binding of [3H]QNB to the cortex of 4- and 6-week-old animals, but not 1l-week-old animals, and to the hippocampus of the 6-week-old animals. Displacement studies indicated that the effect of ethanol was due to an increase in the density of low-affinity carbachol binding sites. There was no effect on the binding of [3H]QNB in the presence of pirenzipine. The data strongly suggest that the increase in binding after ethanol exposure is due to an increase in M~ sites in the younger animals possibly linked to age-related plasticity of the nervous system.
There has been considerable interest in the effect of ethanol on cholinergic neurotransmission since this mechanism appears to play a major role in m e m o r y and learning (Deutsch, 1971; D r a c h m a n and Leavitt, 1974). O f particular importance is the effect of ethanol on muscarinic receptor density. We have previously shown that administration of ethanol (15% v/v) in the drinking water of juvenile animals for 3 months resulted in an increase in muscarinic receptor density particularly in the cerebral cortex, while the affinity of the receptors appeared to be unaffected (Pietrzak et al., 1988). In other studies on the effect of ethanol on the G A B A - r e c e p t o r complex we have utilised an ethanolinhalation regime to produce symptoms of tolerance in a relatively short time (10-14 days). Marked ethanol-induced changes in the coupling of the various receptor sites in this complex were noted (de Vries et al., 1987; Hillmann et al., 1988). We have now applied this technique to determine effect of shortterm ethanol vapour treatment on cholinergic receptors in young animals during rapid growth and development. Significant advances in the understanding of central cholinergic activity have come from studies of receptors by in vitro autoradiography. These studies have confirmed the distribution of brain muscarinic receptors as deduced from homogenates in vitro and
have extended the number of regions known to be cholinoceptive. Further, using this technique, as in homogenate assays, it has been possible to identify muscarinic receptor subtypes using the M~-selective antagonist, pirenzepine, or the agonist carbachol (Mash and Potter, 1986). Particular advantages of in vitro autoradiography are the enhancement of the specific to nonspecific binding ratio, the efficient use of radioligands and quantitation of receptor populations in small brain areas.
EXPERIMENTAL PROCEDURES
L-Quinuclidinyl [phenyl-4-3H]benzilate (specific activity 39 Ci/mmol) and autoradiographic [3H] microscales were supplied by Amersham, Australia. Carbamylcholine chloride, alcohol dehydrogenase and fl-nicotinamide adenine dinucleotide (fl-NAD) were supplied by Sigma Chemical Co., St Louis. Unlabeled L-quinuclidinyl benzilate and pirenzepine were supplied by Hoffman La Roche and Boehringer Ingelheim, respectively. All other chemicals were analytical grade as required. The ethanol inhalation method used was as described by Hillmann et al. (1988). Three separate experiments were performed using groups of 5 male Wistar rats weighing on average 390 g (11-week-old), 200 g (6-week-old) and 57 g (4-week-old). Age-matched controls not exposed to ethanol vapour received similar care and handling as the experimental rats. Blood alcohol levels were determined using the method of Lundquist (1959). For autoradiographic analysis, animals were decapitated and brains quickly removed. The brains were sliced to the desired level before being snap frozen on a metal plate immersed in liquid nitrogen. Brains were stored at -70°C
*To whom reprint requests should be addressed. 91
92
G.F. DUNGLISONet
until required. Ten micron sections were thaw-mounted onto microscope slides which had been previously immersed in a 1% (w/v) gelatin solution containing 1% (w/v) formalin and oven dried at 70°C. Slide mounted sections were dried at room temperature and stored at -20°C. The standard assay procedure was incubation at 25°C min, followed by two 5-min washes in ice-cold Na+-K +-phosphate buffer (pH 7.4). A further l-min wash in cold distilled water removed excess salts from tissues. Slides were dried with cold air immediately. In all assays non-specific binding was determind by the inclusion of 1 mM QNB. Brain sections (triplicate per slide) were incubated in 10mM Na + K+-phosphate buffer containing 1 nM [3H]QNB. Displacement assays were performed according to Mash and Potter (1986). Sections were preincubated in 10mM Na+-K+-phosphate buffer containing 10mM EDTA and then labelled with l nM [3H]QNB in the presence of pirenzepine (3 mM) or carbachol (0.1 mM). Radiolabelled slidemounted tissue sections were then opposed to sheets of LKB Ultrofilm in X-ray cassettes for approx. 3-4 weeks at - 2 0 ° C . Films were then removed and developed by standard procedures. A set of commercial and brain-paste standards was included in each X-ray cassette. The images on the [3H]sensitivefilm were analysed using a computer image analysis system based on the AVID software system (Biomedical Computer Systems, Melbourne, Australia). This allows selection of specific areas of the image for comparison against a stored calibration curve and calculation of specific radioactivity expressed as fmoles of bound ligand per mm2 of tissue. This information can be fed directly into progams for analysis of binding parameters. Analysis of drug-receptor interactions was performed using the computer program EBDA (McPherson, 1983). Binding parameters determined are those given by subsequent analysis of the Scatchard data with the iterative, nonlinear, curve-fitting program of Munson and Rodbard (1986).
RESULTS
The ethanol inhalation program involved a slow induction phase. The concentration of ethanol in the chamber was increased slowly from day 1 to 6. Blood alcohol concentration remained below 1 mg/ml during this induction phase. Ethanol vapour concentration as then further increased and the blood alcohol concentration rose steadily to the final level of 3 mg/ml by day 12. Four-week-old and 6-week-old rats gained 9 6 . 4 + 0 . 3 g and 3 0 . 4 + 3 . 4 g (mean+_SD) body weight, respectively, during the period of ethanol exposure, while the 11-week-old rats maintained their intial weight (data not shown). Preliminary experiments indicated that saturation of muscarinic receptors with [3H]QNB occurred at 1 nM with half-maximal saturation at 0.3 nM. At the concentration of [3H]QNB used routinely, nonspecific binding in the presence of 1 mM QNB did not generate a detectable image on the film.
al.
Autoradiograms of ethanol-treated and control animals showed a wide distribution of muscarinic binding sites (Fig. 1). In particular, there was a high density of [3H]QNB-binding sites in the cerebral cortex, hippocampus and striatum and a low density in the thalamus and cerebellum. Quantitation revealed that rats treated with ethanol vapour showed distinct changes in muscarinic receptor density in the cerebral cortex and hippocampus (Table l). In the cerebral cortex there was an 18% increase in muscarinic receptor density in 4-week-old rats, while in the 6-week-old rats the increase was 25%. There were no changes in muscarinic receptor density in the oldest animals. In the hippocampus a significant increase in muscarinic receptor density was observed only in 6-week-old rats. Using data from the 6-week-old ethanol-treated and control rats, saturation curves were generated for [~H]QNB binding in the cerebral cortex and hippocampus. Scatchard analysis of the saturation data is shown in Fig. 2. As indicated above, ethanol treatment resulted in an increase in Bmaxin both brain areas. However, in neither area was there a change in K0. At the concentration used (3 mM), pirenzepine selectively occupies approx. 90% of the M~ sites allowing [3H]QNB to interact with approx. 60% of the remaining M 2 sites. Similarly 1 mM carbachol occupies 95% of the high-affinity agonist binding sites revealing 80% of the low-affinity carbachol receptors. Pirenzepine reduced binding in forebrain structures such as the cerebral cortex, hippocampus and corpus striatum [Fig. l, PZ]. Quantitation of the remaining radioactivity indicated that there was no change in the remaining M 2 receptors in either brain region studied in any age group following ethanol treatment (Table 2). Carbachol (0.1mM) produced a general reduction of [3H]QNB binding in the thalamus and hypothalamus, but showed weaker effects in the hippocampus, striatum and cerebral cortex [Fig. 1, CARB]. There was a significant increase in low-affinity carbachol receptors in the cortex of the younger animals (approx. 15% in each case, Table 3) but not in the ll-week-old animals. DISCUSSION
The development of autoradiographic techniques has increased the anatomical resolution of receptor distribution to the micron range and increased the sensitivity of the ligand-binding measurement when
Effect of ethanol on muscarinic receptors
93
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fmol/mm a
8.5
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Fig. 1. Pseudo-colour image of autoradiographs of cortical slices from control and ethanol-treated animals. Slices from ethanol-treated animals (ETH) or control animals (CON) were incubated with [aH]QNB. To illustrate the areas displaced by 3#M pirenzipine (PZ) or 0.1mM carbachol (CARB) + representative sections from control animals are shown in the lower panels. compared with assays of tissue homogenates (Clarke and Hall, 1981). Further, the possibility of crosscontamination of brain areas such as that which occurs during dissection for homogenate assays is greatly reduced and, since the tissue remains un-
homogenised, the interaction of receptor with lipids and other membrane modulators in the native state is maintained. Obviously, autoradiography may reveal small but potentially significant changes that could not be detected by conventional means. In this
Table I. The effect of ethanol vapour inhalation on [3H]QNB binding in vitro to muscarinic receptors in rat brain section Cerebral cortex Age 4 weeks (n = 5) 6 weeks (n = 5) I 1 weeks (n = 3)
Hippocampus
Control
Ethanol
Control
Ethanol
4.95 _+0.2 4.4 + 0.7 4.95 +_0.10
5.85 _+0.5* 5.5 + O.1* 5.15 + 0.35
5.10 _+0.6 6.5:1:0.41 5.15 _+0.10
5.8 _+0.75 7.60 + 0.42* 5.75 + 0.75
Animals were exposed to ethanol vapour for 10-14 days. The density of rcmeptors is expresscxi as fmol/mm2 tissue. Values (mean _+ SD) represent measurements from individual animals. These were obtained by duplicate measurements of triplicate sections from each animal. In all cases there was minimum non-specific binding. *Value is significantly different from control (P < 0.01).
94
G.F. DUNGLISONet al. .20 .16
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Fig. 2. Scatchard analysis of [3H])QNB binding to cortex and hippocampus measured by quantitative autoradiography. Left panel: (O) control, (O) ethanol-treated. Right panel: hippocampus (l-q) control, (11) ethanol-treated. initial study we have examined the possibility of using this technique to investigate the effects o f ethanol on brain muscarinic receptors. Significant increases in total muscarinic receptor a n d in low-affinity agonistbinding sites particularly in the cerebral cortex o f young animals have been revealed. Scatchard analysis of s a t u r a t i o n binding d a t a for the intact cerebral cortex a n d h i p p o c a m p u s of ethanol-treated a n d control animals showed a single, u n i f o r m set o f binding-sites for [3H]QNB. We have also confirmed the d a t a o b t a i n e d from assays o f b r a i n h o m o g e n a t e t h a t e t h a n o l t r e a t m e n t does n o t alter the K~ ( T a b a k o f f et al., 1979; R a b i n et al., 1980; Pietrzak et al., 1988). This finding indicates t h a t any changes in [3H]QNB binding reflect changes in receptor number. The increase in total muscarinic receptors revealed by a u t o r a d i o g r a p h y is consistent with o t h e r work using b r a i n h o m o g e n a t e u n d e r different experimental
conditions. After ethanol a d m i n i s t r a t i o n to mice ( T a b a k o f f et al., 1979; R a b i n et al., 1980), or to rats (Pietrzak et al., 1988) a n increase in total muscarinic receptor density in the range o f 1 4 - 5 0 % has been observed. In the latter study, the change was solely due to an increase in M j receptor density. A similar increase in the density of cortical high-affinity [3H]pirenzepine binding sites was also seen in mice chronically treated with ethanol by the oral route ( H o f f m a n et al., 1986). A u t o r a d i o g r a p h i c studies have indicated t h a t brain regions rich in M 1 receptors contain low-affinity agonist sites, while regions in M2 receptors have an a b u n d a n c e of high-affinity agonist sites (Potter et al., 1984; Cortes a n d Palacois, 1986). T h e displacement of [3H]QNB by c a r b a c h o l in the present study from high-affinity binding sites revealed increased binding to low-affinity c a r b a c h o l binding sites. This g r o u p of binding-sites p r o b a b l y includes the M~ sites described by classic pirenzepine binding studies. There was no increase in the binding remaining in the presence o f pirenzepine (M 2 receptors). Both of these results strongly suggest t h a t the increase in muscarinic receptors seen after ethanol v a p o u r exposure is due to an increase in M t receptors. The increase in muscarinic receptor density observed in 4- a n d 6-week-old rats, but not 1 l-weekold rats, m a y be an adaptive response by the young animals to c o m p e n s a t e for this drug-induced insult. Certainly, a loss of plasticity in muscarinic receptors following long-term cholinergic drug a d m i n i s t r a t i o n has been observed in animals over 3 m o n t h s o f age (Pedigo a n d Polk, 1985). The results reported here
Table 2. [3H]QNB binding in vitro to muscarinic receptors in rat brain sections in the presence of pirenzepine Cerebral cortex Hippocampus Age Control Ethanol Control Ethanol 4 weeks (n = 5) 1.95 _+0.15 1.20 _+0.25 1.55 _+0.25 1.45 _+0.15 6 weeks (n = 5) 1.8 + 0.4 t.5 _+0.25 1.75 _+0.2 1.55 _+0.25 11 weeks (n = 3) 1.95 _+0.25 1.55 _+0.20 2.15 ± 0.4 1.60 ± 0.2 Animals were exposed to ethanol vapour for 10-14 days. The density of receptors is expressed as fmol/mm2 tissue. Values (mean ± SD) represent measurements from individual animals. These were obtained by duplicate measurements of triplicate sections from each animal. In all cases there was minimum non-specific binding. Table 3. [3H]QNB binding in vitro to muscarinic receptors in rat brain sections in the presence of carbachol Cerebral cortex Hippocampus Age Control Ethanol Control Ethanol 4 weeks (n = 5) 2.95 ± 0.18 3.43 ± 0.25* 4.21 _+0.39 4.22 ± 0.425 6 weeks (n = 5) 3.2 _+0.16 3.8 ± 0.31" 4.05 ± 0.33 4.90 + 0.46* II wecks (n =3) 2.97_+0.31 3.14+0.27 3.82_+0.460 4.21 ±0.22 Animals were exposed to ethanol vapour for 10-14 days. The density of receptors is expressed as fmol/mm2 tissue. Values (mean + SD) represent measurements from individual animals. These were obtained by duplicate measurement of triplicate sections from each animal. *Value is significantly different from control (P < 0.01).
Effect of ethanol on muscarinic receptors indicate that in the case of ethanol, at least, the adaptive response may be lost at a much younger age. Recently several reports of differences in muscarinic receptor ontogeny have appeared. Evans et al. (1985) showed a rapid increase in total [3H]QNB binding in mouse cerebral cortex during the first 21 days post-natally. This was shown to be due to the appearance of M1 receptors and not M E subtype. In addition, Balduini et aL (1987) have demonstrated that the appearance of MI receptor-stimulated inositol lipid turnover follows a similar time-course. It may be that this period of rapid development, reflected in the appearance of muscarinic receptors, is particularly susceptible to perturbation by ethanol exposure. It is also possible if the [3H]QNB binding sites which are discriminated by pirenzepine do represent distinct molecular entities as has been suggested, that these subtypes might exhibit differential responses following chronic ethanol-treatment. In summary, this preliminary study has demonstrated the applicability of quantitative autoradiographic techniques to the study of the effect of ethanol treatment on brain receptors. We have demonstrated using the inhalation procedure, an increase in muscarinic receptors after 14 days of exposure. Further, the data strongly suggest that this increase was due to an increment in M~ receptors, possibly linked to age-related plasticity of the nervous system. This demonstration opens the way for further studies and detailed analysis of the effects of ethanol on brain microstructures in the future.
REFERENCES Balduini W., Murphy S. D. and Costa L. A. (1987) Development changes in muscarinic receptor-stimulated phosphoinositide metabolism in rat brain. J. Pharmac. exp. Ther. 241, 421-427. Clarke C. R. and Hall M. D. (1986) Hormone receptor autoradiography: recent developments. Trends biochem. Sci. 11, 195-199. Cortes R. and Palacios J. M. (1986) Muscarinic cholinergic receptor subtypes in the rat brain. 1. Quantitative autoradiographic studues. Brain Res. 362, 227-238. Deutsh A. J. (1971) The cholinergic synapse and the site of memory. Science 174, 788-794.
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Drachman D. A. and Leavitt J. (1974) Human memory and the cholinergic system: a relationship to aging. Arch. Neurol. 30, 113-121. Evans R. A., Watson M., Yamamura I. and Roeske W. R. (1985) Differential ontogeny of putative Mt and M 2 muscarinic receptor binding sites in the murine cerebral cortex and heart. J. Pharmac. exp. Ther. 235, 612-618. Hillmann M., Wilce P. A. and Shanley B. C. (1988) Effects of chronic ethanol exposure on the GABA-bcnzodiazepine receptor complex in rat brain. Neurochem. Int. 13, 69-73. Hoffman P. L., Moses F., Luthin G. R. and Tabakoff B. (1986) Acute and chronic effects of ethanol on receptor-mediated phosphatidylinositol-4,5-bisphosphate breakdown in mouse brain. Molec. Pharmac. 30, 13-18. Lundquist F. (1959) The determination of ethyl alcohol in blood and tissues Meth. biochem. Analysis 7, 215~251. Mash D. C. and Potter L. T. (1986) Autoradiographic localization of M~ and M2 muscarinic receptors in rat brain. Neuroscience 19, 551-564. McPherson G. A. (1983) A practical computer-based approach to the analysis of radio-ligand binding experiments. Comput. Prog. Biomed. 17, 107-114. Munson P. J. and Rodbard D. (1986) LIGAND: a versatile computerized approach for characterization of ligandbinding systems. Analyt. Biochem. 107, 220-239. Pedigo N. W. and Polk D. M. (1985) Reduced muscarinic receptor plasticity in frontal cortex of aged rats after chronic administration of cholinergic drugs. Life Sci. 37, 1443-1449. Pietrzak E. R., Wilce P. A. and Shanley B. C. (1988) The effect of chronic ethanol consumption on muscarinic receptors in rat brain. Neurochem. Int. 12, 447 452. Potter L. T., Flynn D. D., Hanchett H. E., Kalinoski D. K., Luber-Narod J. and Marsh D. C. (1984) Independent Mt and M 2 receptors: ligands, autoradiography and functions. Trends pharmac. Sci. Suppl., Jan. 1984, 22-31. Rabin R. A., Wolfe B. B., Dibner M. D., Zahniser N. R., Melchior C. and Molinoff P. B. (1980) Effects of ethanol administration and withdrawal on neurotransmitter receptor systems in C57 mice. J. Pharmac. exp. Ther. 213, 491 - 496. Tabakoff B., Munoz-Marcus M. and Fields J. Z. (1979) Chronic ethanol feeding produces an increase in muscarinic cholinergic receptors in mouse brain. Life Sci. 25, 2173-2180. de Vries D. J., Johnston G. A. R., Ward L. C., Wilce P. A. and Shanley B. C. (1987) Effects of chronic alcohol inhalation on the enhancement of benzodiazepine binding of mouse brain membranes by GABA. Neurochem. Int. 10, 231-235.
0197-0186/89 $3.00 + 0.00 Pergamon Press plc
AUTORADIOGRAPHIC STUDIES ON THE EFFECT OF ETHANOL ON MUSCARINIC RECEPTORS IN RAT BRAIN G. F. DUNGLISON,P. A. WILCE* and B. C. SHANLEY Alcohol Research Unit, Department of Biochemistry, University of Queensland, St Lucia 4067, Queensland, Australia (Received 5 September 1988; accepted 30 January 1989)
Abstract--The effect of ethanol vapour inhalation on the binding characteristics of muscarinic receptors in rat brain has been studied using quantitative autoradiography. After 14 days in ethanol vapour there was increased binding of [3H]QNB to the cortex of 4- and 6-week-old animals, but not 1l-week-old animals, and to the hippocampus of the 6-week-old animals. Displacement studies indicated that the effect of ethanol was due to an increase in the density of low-affinity carbachol binding sites. There was no effect on the binding of [3H]QNB in the presence of pirenzipine. The data strongly suggest that the increase in binding after ethanol exposure is due to an increase in M~ sites in the younger animals possibly linked to age-related plasticity of the nervous system.
There has been considerable interest in the effect of ethanol on cholinergic neurotransmission since this mechanism appears to play a major role in m e m o r y and learning (Deutsch, 1971; D r a c h m a n and Leavitt, 1974). O f particular importance is the effect of ethanol on muscarinic receptor density. We have previously shown that administration of ethanol (15% v/v) in the drinking water of juvenile animals for 3 months resulted in an increase in muscarinic receptor density particularly in the cerebral cortex, while the affinity of the receptors appeared to be unaffected (Pietrzak et al., 1988). In other studies on the effect of ethanol on the G A B A - r e c e p t o r complex we have utilised an ethanolinhalation regime to produce symptoms of tolerance in a relatively short time (10-14 days). Marked ethanol-induced changes in the coupling of the various receptor sites in this complex were noted (de Vries et al., 1987; Hillmann et al., 1988). We have now applied this technique to determine effect of shortterm ethanol vapour treatment on cholinergic receptors in young animals during rapid growth and development. Significant advances in the understanding of central cholinergic activity have come from studies of receptors by in vitro autoradiography. These studies have confirmed the distribution of brain muscarinic receptors as deduced from homogenates in vitro and
have extended the number of regions known to be cholinoceptive. Further, using this technique, as in homogenate assays, it has been possible to identify muscarinic receptor subtypes using the M~-selective antagonist, pirenzepine, or the agonist carbachol (Mash and Potter, 1986). Particular advantages of in vitro autoradiography are the enhancement of the specific to nonspecific binding ratio, the efficient use of radioligands and quantitation of receptor populations in small brain areas.
EXPERIMENTAL PROCEDURES
L-Quinuclidinyl [phenyl-4-3H]benzilate (specific activity 39 Ci/mmol) and autoradiographic [3H] microscales were supplied by Amersham, Australia. Carbamylcholine chloride, alcohol dehydrogenase and fl-nicotinamide adenine dinucleotide (fl-NAD) were supplied by Sigma Chemical Co., St Louis. Unlabeled L-quinuclidinyl benzilate and pirenzepine were supplied by Hoffman La Roche and Boehringer Ingelheim, respectively. All other chemicals were analytical grade as required. The ethanol inhalation method used was as described by Hillmann et al. (1988). Three separate experiments were performed using groups of 5 male Wistar rats weighing on average 390 g (11-week-old), 200 g (6-week-old) and 57 g (4-week-old). Age-matched controls not exposed to ethanol vapour received similar care and handling as the experimental rats. Blood alcohol levels were determined using the method of Lundquist (1959). For autoradiographic analysis, animals were decapitated and brains quickly removed. The brains were sliced to the desired level before being snap frozen on a metal plate immersed in liquid nitrogen. Brains were stored at -70°C
*To whom reprint requests should be addressed. 91
92
G.F. DUNGLISONet
until required. Ten micron sections were thaw-mounted onto microscope slides which had been previously immersed in a 1% (w/v) gelatin solution containing 1% (w/v) formalin and oven dried at 70°C. Slide mounted sections were dried at room temperature and stored at -20°C. The standard assay procedure was incubation at 25°C min, followed by two 5-min washes in ice-cold Na+-K +-phosphate buffer (pH 7.4). A further l-min wash in cold distilled water removed excess salts from tissues. Slides were dried with cold air immediately. In all assays non-specific binding was determind by the inclusion of 1 mM QNB. Brain sections (triplicate per slide) were incubated in 10mM Na + K+-phosphate buffer containing 1 nM [3H]QNB. Displacement assays were performed according to Mash and Potter (1986). Sections were preincubated in 10mM Na+-K+-phosphate buffer containing 10mM EDTA and then labelled with l nM [3H]QNB in the presence of pirenzepine (3 mM) or carbachol (0.1 mM). Radiolabelled slidemounted tissue sections were then opposed to sheets of LKB Ultrofilm in X-ray cassettes for approx. 3-4 weeks at - 2 0 ° C . Films were then removed and developed by standard procedures. A set of commercial and brain-paste standards was included in each X-ray cassette. The images on the [3H]sensitivefilm were analysed using a computer image analysis system based on the AVID software system (Biomedical Computer Systems, Melbourne, Australia). This allows selection of specific areas of the image for comparison against a stored calibration curve and calculation of specific radioactivity expressed as fmoles of bound ligand per mm2 of tissue. This information can be fed directly into progams for analysis of binding parameters. Analysis of drug-receptor interactions was performed using the computer program EBDA (McPherson, 1983). Binding parameters determined are those given by subsequent analysis of the Scatchard data with the iterative, nonlinear, curve-fitting program of Munson and Rodbard (1986).
RESULTS
The ethanol inhalation program involved a slow induction phase. The concentration of ethanol in the chamber was increased slowly from day 1 to 6. Blood alcohol concentration remained below 1 mg/ml during this induction phase. Ethanol vapour concentration as then further increased and the blood alcohol concentration rose steadily to the final level of 3 mg/ml by day 12. Four-week-old and 6-week-old rats gained 9 6 . 4 + 0 . 3 g and 3 0 . 4 + 3 . 4 g (mean+_SD) body weight, respectively, during the period of ethanol exposure, while the 11-week-old rats maintained their intial weight (data not shown). Preliminary experiments indicated that saturation of muscarinic receptors with [3H]QNB occurred at 1 nM with half-maximal saturation at 0.3 nM. At the concentration of [3H]QNB used routinely, nonspecific binding in the presence of 1 mM QNB did not generate a detectable image on the film.
al.
Autoradiograms of ethanol-treated and control animals showed a wide distribution of muscarinic binding sites (Fig. 1). In particular, there was a high density of [3H]QNB-binding sites in the cerebral cortex, hippocampus and striatum and a low density in the thalamus and cerebellum. Quantitation revealed that rats treated with ethanol vapour showed distinct changes in muscarinic receptor density in the cerebral cortex and hippocampus (Table l). In the cerebral cortex there was an 18% increase in muscarinic receptor density in 4-week-old rats, while in the 6-week-old rats the increase was 25%. There were no changes in muscarinic receptor density in the oldest animals. In the hippocampus a significant increase in muscarinic receptor density was observed only in 6-week-old rats. Using data from the 6-week-old ethanol-treated and control rats, saturation curves were generated for [~H]QNB binding in the cerebral cortex and hippocampus. Scatchard analysis of the saturation data is shown in Fig. 2. As indicated above, ethanol treatment resulted in an increase in Bmaxin both brain areas. However, in neither area was there a change in K0. At the concentration used (3 mM), pirenzepine selectively occupies approx. 90% of the M~ sites allowing [3H]QNB to interact with approx. 60% of the remaining M 2 sites. Similarly 1 mM carbachol occupies 95% of the high-affinity agonist binding sites revealing 80% of the low-affinity carbachol receptors. Pirenzepine reduced binding in forebrain structures such as the cerebral cortex, hippocampus and corpus striatum [Fig. l, PZ]. Quantitation of the remaining radioactivity indicated that there was no change in the remaining M 2 receptors in either brain region studied in any age group following ethanol treatment (Table 2). Carbachol (0.1mM) produced a general reduction of [3H]QNB binding in the thalamus and hypothalamus, but showed weaker effects in the hippocampus, striatum and cerebral cortex [Fig. 1, CARB]. There was a significant increase in low-affinity carbachol receptors in the cortex of the younger animals (approx. 15% in each case, Table 3) but not in the ll-week-old animals. DISCUSSION
The development of autoradiographic techniques has increased the anatomical resolution of receptor distribution to the micron range and increased the sensitivity of the ligand-binding measurement when
Effect of ethanol on muscarinic receptors
93
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II 1l I 1 1 | l / COLOUR CODE 1 I n m .
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fmol/mm a
8.5
7.5
6.5
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II
I
Fig. 1. Pseudo-colour image of autoradiographs of cortical slices from control and ethanol-treated animals. Slices from ethanol-treated animals (ETH) or control animals (CON) were incubated with [aH]QNB. To illustrate the areas displaced by 3#M pirenzipine (PZ) or 0.1mM carbachol (CARB) + representative sections from control animals are shown in the lower panels. compared with assays of tissue homogenates (Clarke and Hall, 1981). Further, the possibility of crosscontamination of brain areas such as that which occurs during dissection for homogenate assays is greatly reduced and, since the tissue remains un-
homogenised, the interaction of receptor with lipids and other membrane modulators in the native state is maintained. Obviously, autoradiography may reveal small but potentially significant changes that could not be detected by conventional means. In this
Table I. The effect of ethanol vapour inhalation on [3H]QNB binding in vitro to muscarinic receptors in rat brain section Cerebral cortex Age 4 weeks (n = 5) 6 weeks (n = 5) I 1 weeks (n = 3)
Hippocampus
Control
Ethanol
Control
Ethanol
4.95 _+0.2 4.4 + 0.7 4.95 +_0.10
5.85 _+0.5* 5.5 + O.1* 5.15 + 0.35
5.10 _+0.6 6.5:1:0.41 5.15 _+0.10
5.8 _+0.75 7.60 + 0.42* 5.75 + 0.75
Animals were exposed to ethanol vapour for 10-14 days. The density of rcmeptors is expresscxi as fmol/mm2 tissue. Values (mean _+ SD) represent measurements from individual animals. These were obtained by duplicate measurements of triplicate sections from each animal. In all cases there was minimum non-specific binding. *Value is significantly different from control (P < 0.01).
94
G.F. DUNGLISONet al. .20 .16
.t2 k .08 o •
.04 0
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t]0~D (fm0l/m2)
k I
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Fig. 2. Scatchard analysis of [3H])QNB binding to cortex and hippocampus measured by quantitative autoradiography. Left panel: (O) control, (O) ethanol-treated. Right panel: hippocampus (l-q) control, (11) ethanol-treated. initial study we have examined the possibility of using this technique to investigate the effects o f ethanol on brain muscarinic receptors. Significant increases in total muscarinic receptor a n d in low-affinity agonistbinding sites particularly in the cerebral cortex o f young animals have been revealed. Scatchard analysis of s a t u r a t i o n binding d a t a for the intact cerebral cortex a n d h i p p o c a m p u s of ethanol-treated a n d control animals showed a single, u n i f o r m set o f binding-sites for [3H]QNB. We have also confirmed the d a t a o b t a i n e d from assays o f b r a i n h o m o g e n a t e t h a t e t h a n o l t r e a t m e n t does n o t alter the K~ ( T a b a k o f f et al., 1979; R a b i n et al., 1980; Pietrzak et al., 1988). This finding indicates t h a t any changes in [3H]QNB binding reflect changes in receptor number. The increase in total muscarinic receptors revealed by a u t o r a d i o g r a p h y is consistent with o t h e r work using b r a i n h o m o g e n a t e u n d e r different experimental
conditions. After ethanol a d m i n i s t r a t i o n to mice ( T a b a k o f f et al., 1979; R a b i n et al., 1980), or to rats (Pietrzak et al., 1988) a n increase in total muscarinic receptor density in the range o f 1 4 - 5 0 % has been observed. In the latter study, the change was solely due to an increase in M j receptor density. A similar increase in the density of cortical high-affinity [3H]pirenzepine binding sites was also seen in mice chronically treated with ethanol by the oral route ( H o f f m a n et al., 1986). A u t o r a d i o g r a p h i c studies have indicated t h a t brain regions rich in M 1 receptors contain low-affinity agonist sites, while regions in M2 receptors have an a b u n d a n c e of high-affinity agonist sites (Potter et al., 1984; Cortes a n d Palacois, 1986). T h e displacement of [3H]QNB by c a r b a c h o l in the present study from high-affinity binding sites revealed increased binding to low-affinity c a r b a c h o l binding sites. This g r o u p of binding-sites p r o b a b l y includes the M~ sites described by classic pirenzepine binding studies. There was no increase in the binding remaining in the presence o f pirenzepine (M 2 receptors). Both of these results strongly suggest t h a t the increase in muscarinic receptors seen after ethanol v a p o u r exposure is due to an increase in M t receptors. The increase in muscarinic receptor density observed in 4- a n d 6-week-old rats, but not 1 l-weekold rats, m a y be an adaptive response by the young animals to c o m p e n s a t e for this drug-induced insult. Certainly, a loss of plasticity in muscarinic receptors following long-term cholinergic drug a d m i n i s t r a t i o n has been observed in animals over 3 m o n t h s o f age (Pedigo a n d Polk, 1985). The results reported here
Table 2. [3H]QNB binding in vitro to muscarinic receptors in rat brain sections in the presence of pirenzepine Cerebral cortex Hippocampus Age Control Ethanol Control Ethanol 4 weeks (n = 5) 1.95 _+0.15 1.20 _+0.25 1.55 _+0.25 1.45 _+0.15 6 weeks (n = 5) 1.8 + 0.4 t.5 _+0.25 1.75 _+0.2 1.55 _+0.25 11 weeks (n = 3) 1.95 _+0.25 1.55 _+0.20 2.15 ± 0.4 1.60 ± 0.2 Animals were exposed to ethanol vapour for 10-14 days. The density of receptors is expressed as fmol/mm2 tissue. Values (mean ± SD) represent measurements from individual animals. These were obtained by duplicate measurements of triplicate sections from each animal. In all cases there was minimum non-specific binding. Table 3. [3H]QNB binding in vitro to muscarinic receptors in rat brain sections in the presence of carbachol Cerebral cortex Hippocampus Age Control Ethanol Control Ethanol 4 weeks (n = 5) 2.95 ± 0.18 3.43 ± 0.25* 4.21 _+0.39 4.22 ± 0.425 6 weeks (n = 5) 3.2 _+0.16 3.8 ± 0.31" 4.05 ± 0.33 4.90 + 0.46* II wecks (n =3) 2.97_+0.31 3.14+0.27 3.82_+0.460 4.21 ±0.22 Animals were exposed to ethanol vapour for 10-14 days. The density of receptors is expressed as fmol/mm2 tissue. Values (mean + SD) represent measurements from individual animals. These were obtained by duplicate measurement of triplicate sections from each animal. *Value is significantly different from control (P < 0.01).
Effect of ethanol on muscarinic receptors indicate that in the case of ethanol, at least, the adaptive response may be lost at a much younger age. Recently several reports of differences in muscarinic receptor ontogeny have appeared. Evans et al. (1985) showed a rapid increase in total [3H]QNB binding in mouse cerebral cortex during the first 21 days post-natally. This was shown to be due to the appearance of M1 receptors and not M E subtype. In addition, Balduini et aL (1987) have demonstrated that the appearance of MI receptor-stimulated inositol lipid turnover follows a similar time-course. It may be that this period of rapid development, reflected in the appearance of muscarinic receptors, is particularly susceptible to perturbation by ethanol exposure. It is also possible if the [3H]QNB binding sites which are discriminated by pirenzepine do represent distinct molecular entities as has been suggested, that these subtypes might exhibit differential responses following chronic ethanol-treatment. In summary, this preliminary study has demonstrated the applicability of quantitative autoradiographic techniques to the study of the effect of ethanol treatment on brain receptors. We have demonstrated using the inhalation procedure, an increase in muscarinic receptors after 14 days of exposure. Further, the data strongly suggest that this increase was due to an increment in M~ receptors, possibly linked to age-related plasticity of the nervous system. This demonstration opens the way for further studies and detailed analysis of the effects of ethanol on brain microstructures in the future.
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