Different levels of [3H]MK-801 binding in long-sleep and short-sleep lines of mice

Alcohol,Vol. 13, No. 3,315-320, 1996 Copyright©1996ElsevierScienceInc. Printedin the USA.All rightsreserved 0741-8329/96$15.00 + .00 ELSEVIER 0741-8329(95)02113-2

Different Levels of [3H]MK-801 Binding in Long-Sleep and Short-Sleep Lines of Mice W A L T E R R. W I L S O N A N D A L L A N C. C O L L I N S 1

Institute for Behavioral Genetics and the School o f Pharmacy, University of Colorado, Boulder, CO 80309 Received 31 July 1995; Accepted 7 December 1995 WILSON, W. R. AND A. C. COLLINS. Different levels of [3H]MK-801 binding in long-sleep and short-sleep lines of mice. ALCOHOL 13(3) 315-320, 1996.-The long-sleep (LS) and short-sleep (SS) lines of mice were selectively bred for differential sensitivity to the hypnotic effects of ethanol. Several studies suggest that excitatory amino acid receptor systems are involved in these genetically determined differences in sensitivity to ethanol. The experiments described in this article examine further the potential role of NMDA excitatory amino acid receptors in genetically determined differences in hypnotic sensitivityto ethanol by measuring [3H]MK-801binding in eight brain regions of LS and SS lines of mice. Significantly greater levels of binding were found in SS hippocampus and striatum. Binding levels in the remaining brain regions revealed no significant between-line differences. Affinity differences between regions were seen but no between-line differences in affinity were found in any brain region. These findings lend support to the hypothesis that differences in NMDA receptor systems are part of the genetically determined biochemistry that produces differential hypnotic sensitivity to ethanol in LS and SS mice. Ethanol

NMDA receptors

Glutamate

Genetics

take up more [3H]glutamate compared to LS vesicles (2), and that slices of cortex and striatum from SS mice, stimulated with potassium, release more endogenous glutamate, in a calcium-dependent manner, than do the same regions from LS mice (the opposite result is seen in hippocampus) (4). Furthermore, NMDA agonists, administered centrally, decrease sensitivity to ethanol (measured by the blood ethanol concentration taken at loss of righting response) whereas NMDA antagonists increase sensitivity to ethanol (18). The various experimental findings suggest further studies that would determine 1) how ethanol interacts with the NMDA excitatory amino acid receptor subtype, and 2) whether any differences exist between LS and SS NMDA receptor systems that may contribute to the demonstrated line differences in hypnotic sensitivity to ethanol. Accordingly, possible differences in NMDA receptors were examined by measuring binding of [3H]MK-801 to LS and SS brain membranes from eight brain regions. Statistical analysis revealed significant between-line differences in maximal binding levels in hippocampus and striatum. Receptor affinities did not differ between the two lines within a region. These findings lend further support to the hypothesis that the NMDA receptor system is involved in LS-SS genetically determined differences in hypnotic sensitivity to ethanol.

THE LS and SS lines of mice were selectively bred for differential hypnotic sensitivity following intraperitoneal administration of ethanol (12,13). The two lines differ only slightly in rate of ethanol elimination (15) but differ markedly in CNS sensitivity to the depressant effects of ethanol. Success in breeding for differential sensitivity confirms a role for genetic regulation of the response to ethanol and suggests that gene expression produces differences in LS and SS neurochemistry through which ethanol acts to produce its differential hypnotic effects. Several studies reveal that ethanol has inhibitory actions at the NMDA excitatory amino acid receptor subtype (2,6,8). Physiologically relevant ethanol concentrations of less than 50 mM inhibit NMDA-stimulated current measured electrophysiologically (8) and calcium measured using fluorescent imaging (2). Ethanol also inhibits NMDA-stimulated production of cGMP (6). These examples demonstrate that ethanol has specific effects on the NMDA receptor subtype and suggest that the actions of ethanol are partially mediated by interacting with NMDA receptors. Other reports suggest that excitatory amino acid systems are involved in the differential acute hypnotic response to ethanol seen in the LS and SS lines of mice (3,4,18). These studies demonstrate that whole-brain vesicles from SS mice

Requests for reprints should be addressed to A. C. Collins, Ph.D., Institute for Behavioral Genetics, Campus Box 447, Boulder, CO 80309. 315

316

WILSON AND COLLINS METHOD

Animals Naive male and female LS/Ibg and SS/Ibg mice were used. The animals were housed five per cage; temperature (27°C), humidity (28070), and a 12-h light cycle were maintained (lights on at 0700 h). Food and water were provided ad lib. All procedures involving animal subjects conform to guidelines established by the University of Colorado at Boulder Institutional Animal Care and Use Committee.

Membrane Preparation The whole brain, including cerebellum, was removed and dissected into regions on ice. Hypothalamic, cerebellar, hindbrain, collicular, hippocampal, midbrain, striatal, and cortical regions were isolated and each region from one animal was placed into 1 ml of ice-cold modified Krebs-Ringer-HEPES buffer (I x KRH) consisting of NaC1 121 mM, KC1 3 mM, CaC12 2.5 mM, and HEPES hemisodium 20 mM. Each region was homogenized with a Teflon pestle in a 10 x 75 mm polypropylene tube. The homogenate was then centrifuged at 20,000 x g for 20 min. The supernatant was discarded, the pellet resuspended in 1 ml of deionized distilled water, and the volume doubled by addition of more water. The resuspended tissue was incubated at 37 °C for 30 min and again centrifuged at 20,000 x g for 20 min. The resulting pellet was resuspended in fresh water (no further incubation) and centrifuged again as described above. The supernatant was discarded and replaced with 0.1 x KRH and the pellet was then frozen at - 7 0 ° C overnight, or until prepared for binding. Upon thawing, the membrane pellet was washed seven more times by resuspending in room temperature 0.1 x KRH buffer followed by centrifugation. The seven washes in 0.1 x KRH were followed by two final washes using 1 x KRH buffer. The final pellet was resuspended in distilled water at a protein concentration of 1-3 #g/#l (10) for use in binding experiments. The 12 wash cycles attempted to remove endogenous glutamate (GLU) and glycine (GLY). The efficiency in removing GLU and GLY was estimated by experiments in which cortical tissue was dissected and homogenized and spiked with approximately 2 x 106 cpm of [14C]GLU or [14C]GLY. Aliquots of the spiked homogenate, each wash supernatant, and the final pellet were counted by liquid scintillation spectroscopy. The removal of radioactive counts proceeded in an apparent firstorder manner (data not shown) and after 12 washes the membrane pellet contained approximately 0.05°70 of the initial concentration of radioactivity. Other evidence that the concentrations of endogenous GLU and GLY were effectively reduced comes from experiments in which the binding of [~H]MK-801 increased in a dose-dependent manner in the presence of increasing concentrations of GLU or 100 nM GLU plus increasing concentrations of GLY (data not shown).

f H]MK-801 Binding Binding was performed in 96-well plates (Sumilon) in a volume of 100/zl. The solutions were combined in three steps. First, 25 #1 of a concentrated solution of binding modulators (GLU, GLY, spermidine (SPMD), Mg ÷ +, and unlabelled MK801 for nonspecific binding) in KRH buffer was added so that final concentrations were 0.1 x KRH buffer, G L U / G L Y concentrations from 10-100 #M, SPMD 100 #M, and Mg 0-15 mM. Next, 25 #1 of concentrated [3H]MK-801 (15-30 Ci/mmol, Dupont NEN, Boston, MA) in distilled water was

added; when diluted, final concentrations of [3H]MK-801 ranged from 0.3 to 100 nM. In the last step, binding was initiated by adding 50/~1 of membrane suspension that contained 50-100/zg of protein in distilled water. A one-tenth concentration of KRH buffer was used for binding because initial experiments attempted in a final concentration of 1 x KRH gave very little binding (data not shown). This result is consistent with other reports in which high concentrations of ions inhibit binding (14,19). Further evidence that the concentration of ions in 0.1 x KRH do not significantly alter binding comes from experiments in which membranes were prepared using a Tris buffer (Tris acetate 20 mM, pH 7.4). Scatchard plots for tissue in Tris vs. KRH buffer resulted in the same maximal levels of binding; however, Tris buffer resulted in slightly higher affinities (data not shown). After initiating binding the plates were tightly covered with Parafilm and placed in a closed humidified chamber at room temperature. In association experiments, binding was allowed to proceed for times of 1-30 rain. Initial equilibrium studies showed slightly increasing binding even at 48 h (data not shown). Accordingly, equilibrium binding experiments were allowed to proceed for 48 h. Six concentrations of [3H]MK801 were used; the total amount bound was < 15°70 at the lowest concentration of [3H]MK-801. Nonspecific binding was determined by inclusion of 10 ~tM unlabelled MK-801 (final concentration). Binding was terminated and free [3H]MK-801 was separated from bound by dilution with ice-cold HEPES (20 mM)EDTA (1 mM) buffer (HE) followed by filtration using a cell harvester apparatus (Inotech). Two filters (Gelman Type A/E, Ann Arbor, MI; Micro Filtration Systems GB100RN, Dublin, CA) were used and were soaked in 0.5°70 polyethylenimine prior to filtration. Following the initial filtration, the filters were washed three times with approximately 100 ml (about I ml per filter) of HE buffer. Filters containing membrane-bound [3H]MK-801 were placed in vials containing 1.5 ml of Budget-Solve scintillation cocktail (Research Products International Corp., Mount Prospect, IL) and shaken for 20 min. The radioactivity was then determined by liquid scintillation spectroscopy (Packard TR 1600) at an efficiency of at least 50070. [3H]MK-801 maximal binding (Bma0 and affinity (Kd) were obtained by Scatchard analysis.

Heat Denaturation of Binding Heat stability of [3H]MK-801 binding was determined by the same procedures described above. However, prior to initiating the binding reaction the membrane suspension was incubated at 50°C for times of 1-30 min. The decrease in binding was then plotted as log (070 control binding) vs. time of incubation.

Ethanol Inhibition of Binding Ethanol (0-500 mM) was included in some experiments because ethanol has been shown to inhibit various NMDAinduced responses. Given the problems of loss of ethanol with long incubation times, short association times of 5 and 15 min were used. These points reflect an early and later point on the ascending limb of the association curve. GLU and GLY (10 #M) were included to stimulate the receptors and allow any potential competitive interaction to be seen.

Statistical Analysis Statistical comparisons between LS and SS lines of mice for maximal binding, binding affinity, and the effect of etha-

[3H]MK-801 BINDING IN LS AND SS MICE

317

nol on [3H]MK-801 binding were made using the multiple ANOVA routine in SPSS. Significant effects revealed by analysis of variance (ANOVA) were further tested using the Dunnett post hoc test. RESULTS Initial experiments using cortex, hippocampus and striatum demonstrated that GLU increased the binding of [3H]MK-801 in a dose-dependent manner with an ECs0 of approximately 1 /zM, and that GLY, in the presence of 0.1 /~M GLU, further increased [3H]MK-801 binding with an ECs0 of approximately 0.3 #M (data not shown). In experiments using cortical tissue the competitive antagonist 2-aminophosphonovaleric acid (APV) was added in the presence and absence of binding stimulators (GLU, GLY) and binding allowed to proceed for 1-60 min. APV only inhibited binding at concentrations of 10 #M or greater in the absence of GLU and GLY and had no effect on binding of [3H]MK-801 when GLU and GLY were included in the incubation (data not shown). These results also are evidence that the multiple wash steps were effective in removing endogenous GLU and GLY. Also, in cortex, the noncompetitive antagonist magnesium (Mg) added to either association or equilibrium binding experiments dose-dependently inhibited [3H]MK-801 binding (data not shown). No significant differences in response between LS and SS lines of mice were detected in these experiments. No difference in the rate of association of [aH]MK-801 binding in cortex between LS and SS was seen at times of 130 min (data not shown) in the presence of GLU and GLY. Likewise, in determining time points for use in equilibrium binding conditions, LS and SS mice showed similar time courses in reaching apparent equilibrium; in the presence of high concentrations of binding stimulators (100 #M GLU, GLY, and SPMD) binding increased rapidly for 4-10 h and also showed a slight, nonsignificant increase still seen at 48 h (data not shown). Experiments using apparent equilibrium conditions (48-h incubation at room temperature in the presence of 100 #M GLU, GLY, and SPMD) to estimate Bm~ and Kd values in eight brain regions are shown in Figs. 1-3 and in Table 1. Binding in all regions was saturable and resulted in apparent linear relationships; single and pooled experiments analyzed with the program LIGAND never resulted in a two-site model fitting the data better than a single-site model. However, six point curves are not reliably analyzed using the LIGAND program, so other experiments in cortex (also in striatum, hippocampus, colliculi, midbrain, and hindbrain) using 12 concentrations of [3H]MK-801 were done; these experiments also failed to give a two-site fit when analyzed with the program LIGAND (data not shown). Scatchard plots are shown for cortex (Fig. 1), hippocampus (Fig. 2), and striatum (Fig. 3). Bma~ values (Table 1) for SS mice were significantly greater in the hippocampus and striatum whereas LS mice had greater levels in the cerebellum. Although a LS-SS difference was found in the cerebellum, day-to-day variation in this region was greater, and any attempt to increase the amount of protein slowed filtration, which would also increase variability by increasing the time for separating free from bound ligand. Therefore, cerebellar data are reported in Table 1 but will not be discussed further. In the cortex the Bm~ was consistently higher in SS mice but did not quite achieve statistical significance (p < 0.07). No Bmax differences between LS and SS mice were found in the remaining brain regions.

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FIG. 1. Scatchard plot of [3H]MK-801 binding (0.3-100 nM in half-log increments) to well-washed LS (O) and SS (©) cortical membranes. B ~ and Kd values estimated from the line-of-best-fitare found in the respective equations. Data points represent the mean ± SEM from 6-10 experiments.

Binding affinities determined by Scatchard analysis did not differ between LS and SS mice within a brain region (Table 1). However, comparing the K s of a brain region to all the other brain regions within and across lines did reveal significant K s differences between regions. Differences in binding parameters could indicate that the receptor proteins differ in LS and SS mice. If this is the case, different levels of binding may reflect different rates of receptor protein degradation instead of true differences in levels of binding. To initially examine this possibility, binding of [3H]MK-801 to heat-exposed (50°C) membranes was done. Experiments using membranes from the cortex, hippocampus, and striatum demonstrated no difference in the rate of loss of binding between LS and SS lines of mice; log (070 control binding) vs. time plots were linear for both LS and SS mice (data not shown). The final set of experiments examined the effects of ethanol (0-500 mM) on [aH]MK-801 binding at 5 min (data not shown) and 15 min in the presence of l0 #M GLU and GLY. The effects of ethanol in the brain regions that demonstrated

400 ~

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FIG. 2. Scatchard plot of [3H]MK-801 binding to well-washed LS ( e ) and SS (©) hippocampal membranes. See Fig. l legend for further details.

318

WILSON AND COLLINS 300

900 f(y) =-5.97(y) + 1015.4110 LS I

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FIG. 3. Scatchard plot of [3H]MK-801 binding to well-washed LS ( e ) and SS (©) striatal membranes. See Fig. 1 legend for further details.

30O 200 0

1O0

200

300

400

500

[ E t h a n o l ] (mM)

significant LS-SS Bmax differences are shown in Fig. 4. Because other studies have reported that ethanol inhibits NMDAinduced responses, 10 #M GLU and GLY were included to ensure that the receptors were significantly activated so that the binding could be inhibited. Ethanol significantly inhibited binding only at high nonphysiological concentrations of 250 mM or greater in the cortex, hippocampus, and striatum. In the cerebellum, significant inhibition of binding was also seen at lower ethanol concentrations. ANOVA revealed no effects of line or a line-by-dose interaction in any brain region. DISCUSSION

Significant between-line differences in maximal [3H]MK801 binding were observed in the hippocampus and striatum; SS mice display greater levels of binding than LS mice. A trend towards a higher Bm~x in SS cortex was seen but the apparent difference was not statistically significant. There were no apparent between-line differences 1) in affinity of [3H]MK-801, 2) in stimulation or inhibition of binding by vari-

TABLE 1 MAXIMAL BINDING (Bm~) AND AFFINITY (Kd) OF [JHIMK-801 BINDING TO LS AND SS BRAIN MEMBRANES Bm~ LS Hypothalamus Cerebellum Hindbr ain CoUiculi Hippoeampus Midbrain Striatum Cortex

426 499 211 382 2779 771 1028 2124

+ ± 4± ± ± ± ±

ga SS

20 30* 22 73 300* 30 198" 120

359 389 188 424 3576 733 1560 2319

4± ± ± ± ± ± ±

LS 29 34* 75 28 168" 92 61" 189

5.7 16.5 6.6 5.6 6.4 6.2 5.6 8.9

4± ± ± ± ± 4±

SS 0.3 1.2 1.1 0.6 0.9 0.8 0.8 1.2

7.0 16.0 5.8 7.1 7.0 6.9 6.1 7.9

4± ± ± ± ± ± 4-

0.6 1.9 0.4 1.2 0.4 0.8 0.8 0.9

Bmax units are f m o l / m g protein. Kd units are riM. Kds did not vary between lines within a brain region. *LS vs. SS significantly different: p < 0.05 Student's t-test.

FIG. 4. Effect of ethanol (0, 25, 50, 100, 250, or 500 mM) on [3H]MK-801 binding (5 nM) at 15 min in the presence of 10 #M GLU and GLY. D a t a points represent the mean ± SEM from six experiments. *p < 0.05, **p < 0.01 Dunnett's test.

ous pharmacological agents, or 3) in heat inactivation of binding. These results suggest that LS and SS lines of mice differ in the number of NMDA receptors but apparently do not differ in the type of receptor. Binding of [3H]MK-801 to the NMDA receptor ion channel is often biphasic; a fast component reflects binding to the activated open channel and a slower component reflects binding by hydrophobic diffusion into the unactivated closed ion channel (7). In the present experiments biphasic binding was rarely seen. This may be due to factors mentioned by Javitt and Zukin (7), including long incubation times and saturating levels of GLU, GLY, and SPMD that would not allow one to differentiate the fast component from the slow. In any case, if both fast and slow components do reflect binding to the same population of receptors, long incubation times should be used to more accurately determine receptor density. Biphasic binding kinetics could also indicate the existence of NMDA receptor subtypes. This possibility was not addressed in the present study; therefore, more complicated differences in LS and SS [3H]MK-801 binding may exist that could only be determined by more rigorous analysis. Further studies using different assay conditions may be needed to resolve these issues. The experiments comparing binding in KRH vs. binding in Tris demonstrate that the binding density is unaffected by the use of these two buffers. Therefore, the Bm~x values obtained are reliable in spite of the presence of minimal concentrations of ions in the buffer. The present study did not find any LS-SS differences in apparent affinity of [3H]MK-801 binding within a brain region. As discussed above, the conditions used undoubtedly measured binding to both open and closed states of the ion channel. Therefore, the Kd obtained probably lies somewhere between the true Kd values for the fast and slow components. Recently reported findings (1) give further support to the

[3H]MK-801 BINDING IN LS AND SS MICE

319

hypothesis that NMDA receptors have a role in the LS-SS differential sensitivity to ethanol. NMDA-stimulated elevations in intracellular calcium in microsacs from LS hippocampus were more sensitive to ethanol inhibition than were microsacs from SS hippocampus. Between-line differences were not seen in cortical microsacs. These results correlate well with the results of the current study showing LS-SS differences in hippocampal [3H]MK-801 binding but not in cortical binding. Daniell and Phillips (1) also show LS-SS between-line differences in sensitivity to MK-801 (a low concentration had greater effect in LS hippocampal microsacs than in SS microsacs), which may be expected given our data that LS and SS mice differ in number of hippocampal NMDA receptors. These results from the two studies seem to conflict because SS mice have more hippocampal NMDA receptors but LS hippocampal microsacs are more sensitive to MK-801 and ethanol. The current study has not determined if the receptors measured are all functional (e.g., some could be spare receptors), so further study will be needed to reconcile these findings. Speculating that even if all the receptors are functional, greater LS sensitivity to ethanol in the hippocampus could present if SS mice had more NMDA receptors to recruit and overcome ethanol inhibition, or LS mice with less NMDA receptors simply have a greater percentage of receptors affected by ethanol due to lower receptor levels. Furthermore, NMDA receptor subunits have been cloned and characterized, and different heteromeric subunit combinations display differential sensitivity to ethanol inhibition (11), so it would be interesting to map the distribution of the different subunits in LS and SS mice to see if the differences in amount of NMDA receptors reported here are due to differences in subunit composition. Such differences in subunit composition may also determine the differential sensitivity to ethanol seen in LS and SS hippocampal microsacs (1). Ethanol inhibited in vitro [3HIMK-801 binding only at high nonphysiological concentrations and the effect of ethanol was similar in LS and SS mice. This result is somewhat unexpected because ethanol inhibits NMDA-stimulated responses at physiologically relevant concentrations (8,9,16,17). The aforementioned experiments used living cells whereas the binding reported herein used a membrane preparation and differences between cell preparations and membrane preparations are substantial. The data presented here suggest that in vitro mem-

brane preparations require higher concentrations of ethanol to inhibit binding than is necessary to inhibit in vivo NMDA receptor function. Although the discussion above concerns NMDA differences between LS and SS lines of mice, the findings of other studies argue against such differences. For example, LS and SS whole brain mRNA injected into Xenopus oocytes results in expression of NMDA receptors that are inhibited by ethanol to a similar degree (16). Furthermore, NMDA agonists and antagonists administered prior to equally effective doses of ethanol altered blood and brain ethanol concentrations at loss of righting response to a similar degree in the two lines (18). In the present study, binding of [aH]MK-801 to LS and SS brain membranes was pharmacologically and thermally modulated in a similar manner. These findings suggest that differences in NMDA neurotransmission between LS and SS lines of mice are not due to differences in function of the NMDA receptor but may be due to differences in quantities of receptor. With the knowledge of different NMDA receptor subunit proteins and brain regional differences in NMDA sensitivity to ethanol, further studies should reconcile these apparent incongruous findings. In addition, the interaction of LS-SS differences in number of NMDA receptors with presynaptic LS-SS differences in GLU content and release (3,4) should be determined. In summary, LS and SS mice display genetically determined differences in maximal [~H]MK-801 binding in hippocampus and striatum (and possibly cerebellum). Furthermore, LS-SS genetically determined differences in brain vesicular GLU uptake and stimulated release have also been reported (3,4). Separately, and together, these findings suggest that excitatory amino acid receptor systems play a role in the differential hypnotic sensitivity to ethanol seen in LS and SS mice. Of course, further studies are needed to determine if the NMDA excitatory amino acid receptor system is one of the 711 genes estimated to be involved in the LS-SS differential hypnotic response to ethanol (5). ACKNOWLEDGEMENTS This work was supported by a grant (AA 06391) from the National Institute on Alcoholism and Alcohol Abuse. A. C. Collins is supported, in part, by a Research Scientist Award from the National Insititute on Drug Abuse (DA 00197).

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WILSON AND COLLINS 17. White, G.; Lovinger, D. M.; Weight, F. F. Ethanol inhibits NMDA-activated current but does not alter GABA-activated current in an isolated adult mammalian neuron. Brain Res. 507:332336; 1990. 18. Wilson, W. R.; Bosy, T. Z.; Ruth, J. A. NMDA agonists and antagonists alter the hypnotic response to ethanol in LS and SS mice. Alcohol 7(5):389-395; 1990. 19. Young, A. B.; Fagg, G. E. Excitatory amino acid receptors in the brain: Membrane binding and receptor autoradiographic approaches. Trends Pharmacol. Sci. 11 : 126-133; 1990.