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Regional changes in [3H]diazepam binding in the brains of mice after removal of the olfactory bulbs
EXPERIMENTAL
NEUROLOGY
72, 91-98 (1981)
Regional Changes in [3H]Diazepam Binding in the Brains of Mice after Removal of the Olfactory Bulbs JAMES
D. HIRSCH’
Department of Biological Research, G. D. Searle & Company, P.O. Box 5110, Chicago, Illinois 60680 Received May I, 1980; revision received September 18, 1980 [sH]Diazepam binding was assayed 3 weeks after surgery in 13 brain regions derived from sham-operated and bilaterally olfactory bulbectomized mice. In bulbectomized mice, [Wjdiazepam binding was elevated 30% in the frontal cortex and 24% in the thalamus but decreased 26% in the striatum and 28% in the midbrain. These changes represented alterations in the number of binding sites. No binding changes were observed in the olfactory tubercle, amygdala, piriform cortex, olfactory peduncle, hypothalamus, hippocampus, coiliculi, cerebellum, or pons-medulla of bulbectomized animals. These results are compared with the effects of other types of brain lesions on the brain benzodiazepine receptor, and are possibly related to the altered sensitivity of bulbectomized mice to benzodiazepines and convulsants.
INTRODUCTION There is strong evidence to suggest that benzodiazepine tranquilizers act in the brain via membrane-bound receptors (4,6,19,22,23,26). Part of this evidence was obtained by measuring the effects of various brain lesions or neuropathologic conditions on the binding of radiolabeled benzodiazepines in vitro. Braestrup et al. (5) reported 40 to 65% decreases in [3H]flunitrazepam binding in rat cerebella 9 to 10 days after local injections of the neurotoxin, kainic acid. Similarly, Braestrup and Squires (6) demonstrated decreases of 30 to 40% in [3H]diazepam and [3H]fluniAbbreviation: BBX-bilaterally olfactory bulbectomized. 1 The author is indebted to Dr. Frank L. Margolis for his help and encouragement. I would also like to gratefully acknowledge postdoctoral fellowship support from the Roche Institute of Molecular Biology where this work was performed. Jill Cluesmann at Roche and Carole Ryan at Searle provided excellent secretarial assistance. 91 OOW4886/81/040891-08$02.00/O Copyri&t AU rights
0 1961 by Academic Press, Inc. of reproduction in my form reserved.
92
JAMES D. HIRSCH
trazepam binding in kainic acid-injected rat substantia nigra 8 to 50 days after the lesion. In addition, injection of kainic acid into the rat caudate nucleus resulted in about a threefold decrease in the affinity of [3H]diazepam binding in the ipsilateral substantia nigra (3) 10 days after the lesion with no change in the number of ligand receptors. Mohler and Okada (24) reported decreases in [3H]diazepam binding in the putamen and caudate nucleus of brains obtained from victims of Huntington’s chorea, a neurodegenerative disorder, and benzodiazepine receptor binding was shown to decrease in the cerebella of the “nervous” mutant mouse where F’urkinje cells degenerate during development (5, 2 1). In contrast, Lippa and co-workers reported an increase of about 28% in [3H]diazepam binding after 3 weeks in the rat frontal cortex ipsilateral to a coronal knife cut made on the anterior border of the caudate nucleus (20). Also, Hirsch and Margolis (18,19) reported a 20% increase in r3H]diazepam binding in the remaining mouse olfactory bulb 6 months after a unilateral olfactory bulbectomy. Several studies demonstrated no effects of various treatments or conditions on the benzodiazepine receptor. Benzodiazepine receptor binding was not altered in the rat occipital cortex by injection of 6-hydroxydopamine into ascending noradrenergic neurons, nor was binding affected in the rat striatum and cerebellum after parenteral administration of 3-acetylpyridine. X irradiation of the rat hippocampus also had no effect on benzodiazepine binding (6). In addition, binding was unaltered in cerebella of “weaver” mutant mice where granule cells degenerate (5). When these biochemical studies were compared with reports showing altered behavioral responses to benzodiazepines and convulsant treatments (1, 28) in mice subjected to bilateral olfactory bulbectomy, it was postulated that this brain lesion might also result in changes in [3H]benzodiazepine binding in various regions of the brain. Thus, we investigated the binding of [3H]diazepam to membranes prepared from 13 brain regions from bilaterally olfactory bulbectomized (BBX) mice 3 weeks after the lesion. The results indicate that BBX-induced changes in [3H]diazepam binding were quantitatively similar to those reported by other workers using other lesioning techniques and may be related to the alterations in response of BBX mice to benzodiazepines and convulsants. METHODS Bilateral olfactory bulbectomy on anesthetized IO- to 12-month-old female CD-l mice (40 to 45 g; Charles River, Wilmington, Mass.) was accomplished by aspiration as previously described (30). Sham surgery consisted of all surgical steps but the animals received a partial craniotomy
[3H]DIAZEPAM
BINDING
AFTER
BULBECTOMY
93
only. After surgery they were housed 10 to 13 per cage and experiments began 3 weeks later. The mice were killed by CO2 asphyxiation and the brains were rapidly removed. Some brains were dissected on ice into eight major regions using the boundries described by Cicero et al. (10). We separated the midbrain from the pons, which was left attached to the medulla. In other brains, the limbic forebrain was removed by transecting the brain coronally at the level of the mammillary bodies. This region was then dissected at -20°C as described by Hirsch (19) into the amygdalae, olfactory tubercles, olfactory peduncles, piriform cortices, and hypothalami. Brains with damage to the frontal cortex, olfactory peduncles, olfactory tubercles, or any other region were discarded. After dissection the tissues were stored at -70°C. Freezing to 1 month had no effect on [3H]diazepam binding. [3H]Diazepam binding to membrane preparations from each region was assayed as previously described (20). Briefly, twice-washed membrane fractions prepared by Polytron (Brinkmann Instruments, Westbury, N.Y.) homogenization of brain regions in 50 ~0120 mM Tris-HCl buffer (PH 7.4) followed by centrifugation were incubated 20 min at 0°C with 1.9 nM [3H]diazepam (39.1 Ci/mmol, New England Nuclear, Boston, Mass.) in the presence or absence of 10 PM unlabeled diazepam (Hoffman-LaRoche, Nutley, N.J.) for determination of nonspecific binding. Each 200~~1 assay contained 200 to 300 pg membrane protein. Reactions were terminated by dilution with 2 ml ice-cold buffer followed by a rapid vacuum filtration through Whatman GF/B glass fiber filters. The filters were then washed twice more with 2 ml buffer and dried 20 min at 60°C. Bound [3H]diazepam was determined by liquid scintillation spectrometry at 33% efficiency. In each experiment, three to six samples of each brain region derived from more than one sham-operated or BBX animal were pooled. Effects of BBX on the ligand binding affinity or on the number of ligand receptors in the brain regions were determined by measuring specific binding for the range of 0.08 to 13.5 nM [3H]diazepam in the presence or absence of 10 PM unlabeled diazepam and constructing double reciprocal plots of the saturation isotherms obtained. RESULTS There were no significant differences in [3H]diazepam binding between sham-operated and BBX mice in the amygdalae, olfactory tubercles, piriform cortices, hypothalami, or olfactory peduncles in the limbic system 3 weeks after the lesion (Table 1). These results were in sharp contrast to the effects of BBX on opiate and muscarinic cholinergic binding in these regions (18, 19). In addition, there were no changes in r3H]diazepam
94
JAMES D. HIRSCH TABLE
1
Regional Changes in [3H]Diazepam Binding in Sham-Operated and Bilaterally Bulbectomized Mice” fmol/mg protein Brain region Striatum Olfactory tubercle Hippocampus Amygdala Piriform cortex Colliculi Frontal cortex Olfactory peduncle Midbrain Hypothalamus Cerebellum Thalamus Pons-medulla
Sham (n = 4)
BBX (n = 4)
386 k 377 2 345 2 292 2 290 2 282 + 265 + 254 + 239 + 205 + 183 ? 174+ 135 +
285 t 367? 381 + 284 ? 306 + 314r 346 + 250 + 173 -c 212 k 188 * 216 f 121 k
30 13 24 10 11 21 8 13 14 5 12 5 6
15 6 20 11 11 2 16 10 18 11 11 13 7
Percentage change
P
-26 -3 +10 -3 +5 +11 +30 -2 -28 +3 +3 +24 -6
co.025 NS NS NS NS NS 10.005 NS co.05 NS NS CO.025 NS
n Specific binding of [3H]diazepam at a concentration of 1.9 nM was determined + 10 pM unlabeled diazepam in membrane preparations of the various brain regions. Incubations were at 0°C for 20 min, and assays were terminated by vacuum filtration through Whatman GF/B filters followed by washing. Experiments were carried out 3 weeks after surgery. Values in fmol/mg protein are means f SE from four separate experiments. In each experiment, three to six pieces of each region from two or more animals were pooled. Statistical significance was determined by Student’s t test. NS-not significant.
binding in the hippocampus, colliculi, cerebellum, or pons-medulla. However, binding of [3H]diazepam increased 30% in the frontal cortex (P < 0.005) and 24% in the thalamus (P < 0.025) and decreased 26 (P < 0.025) and 28% (P < 0.05), respectively, in the striatum and midbrain. Saturation isotherms followed by double-reciprocal plot analysis of the binding data indicated that primarily the number of [3H]diazepam receptors (B,,,) was altered in these four brain regions rather than the affinity for the ligand (K,). These plots are single, straight lines indicating a single [3H]diazepam binding site in each brain region (Fig. 1). In sham-operated striata, a B,,, of 1670 fmol/mg protein was obtained and 1250 fmoYmg protein was obtained in BBX striata. The sham and BBX KD was 6.3 nM. In frontal cortex, where [3H]diazepam binding increased after BBX (Table 1). the shamB,ax was 2000 fmoYmg protein, and the BBX value was 2857 fmol/mg protein. In this region the apparent KD for the sham-operated animals was 7.1 nM and 7.4 nM for the BBX animals. Similar
[3HJDIAZEPAM
c
2I
4I
6I s”
0I
BINDING
lo
I2
95
AFTER BULBECTOMY
14 r
x10%
2I
41
6
8
10I
12I
S~x108M
AG. 1. Double reciprocal plots of saturable [sH]diazqam bii in brain regions from bilaterally olfactory bulbectomized (BBX) and sham-operated mice. Specific [SH]diazepam binding was measured for the range ofO.08 to 13.5 nt+t[Wldiazepam in the presence or absence of 10 PM unlabeled diazepam. Binding assay conditions are described in detail under Methods. Membranes were prepared from frozen brain regions obtained from BBX and sham-operated animals as described under Methods. Each experiment was replicated twice with diierent membrane preparations and results varied by about 10%.
data were obtained in the thalamus and midbrain. In the thalamus, the sham and BBX B,,, values were 1000 and 1667 fmoYmg protein, respectively. The sham K, was 6.3 nM and the BBX KD was 7.7 nM. In the midbrain, the sham B,,, was 2000 fmoYmg protein and the BBX B,,, was 1724 fmol/mg protein. The KD values for this region were 14.3 nM for BBX animals and 16.6 nM for the controls. These results confirm the data in Table 1 that were obtained at a single concentration of [SH]diazepam. DISCUSSION Perhaps the most striking finding of this study was that there was no effect of BBX on [3H]diazepam binding in regions of the limbic system. Olfactory bulbectomy results in numerous neuroanatomic and neurochemical changes in denervated limbic areas (8, 12, 15, 16, 18, 29) and other receptors in these regions are markedly affected by BBX (18, 19). The changes in [3H]diazepam binding reported here occurred in regions not known to be directly associated with bulb efferent fibers and may represent
14 ’
%
JAMES
D. HIRSCH
indirect effects of denervation. This hypothesis is supported by neurophysiologic, neuroanatomic, and neurochemical evidence for an association of cortical regions, the thalamus, the striatum, and the midbrain with olfactory pathways. Giachetti and MacLeod (11) reported that cells in the ventroposteromedial nucleus of the rat thalamus responded to electrical stimulation of the lateral olfactory tract and Tanabe et al. reported (27) an area in the monkey prefrontal cortex that responded electrically to delivery of odorants in the nostrils. Similarly, Heimer (16) and Heimer and Wilson (17) demonstrated a projection from olfactory cortical areas to the ventral part of the head of the caudate nucleus in the striatum where this structure overlays the olfactory tubercle. Neuroanatomic results reveal that serotonergic afferent fibers from the midbrain travel to the olfactory bulb in rodents (7) and bulbectomy may result in disruption of this pathway as well. There is also neurochemical evidence to support this serotonergic pathway (18). Thus, in view of the apparent neuronal localization of benzodiazepine receptors (5,6, 21), the present results may provide biochemical support for the apparent pathways described above. At present it is not known why receptor number increases in the frontal cortex and thalamus or decreases in the striatum and midbrain. Increases may represent denervation supersensitivity and decreases may be due to loss of neurons or terminals bearing benzodiazepine receptors, but proof of this is not yet available. The present results may also relate to observations made of BBX animals administered benzodiazepines or subjected to convulsant drugs or treatments. In 1972 Araki and Ueki (1) reported that BBX mice had higher shock thresholds for development of convulsions and were less sensitive to several convulsant drugs when tested 2 10 days after surgery. Later, it was reported (28) that BBX mice tested 214 days after surgery were more sensitive to the anticonvulsant effects of the benzodiazepines, chlordiazepoxide, diazepam, and nitrazepam, against both electrically and pentylenetetrazol-induced convulsions. It was suggested that alterations in neuronal function in the cerebral cortex, midbrain, thalamus, ponsmedulla, and possibly other brain regions were responsible for the altered responses shown by mice with lesions (1, 28). It was also reported that benzodiazepines prevented spreading of seizures from the thalamus, cortex, and other regions (2,9, 13, 14). Therefore, although the changes in [3H]diazepam binding reported here were small, their location suggests that they could be related to the behavioral alterations observed in BBX mice. It should be noted that only 10 to 20% of brain benzodiazepine receptors need to be occupied by diazepam in order for the drug to prevent pentylenetetrazol-induced convulsions or exhibit anticonflict activity (20, 25). Thus, a small change in diazepam binding in a specific brain region could have a marked effect on the action of the drug in that region.
[WjDIAZEPAM
BINDING
AFTER
BULBECTOMY
!27
In conclusion, these data suggest that BBX in mice results in changes in E3H]diazepam binding in the frontal cortex, thalamus, striatum, and midbrain 3 weeks after the lesion. The magnitude of these changes is similar to that reported by other workers using a variety of other lesioning techniques. In addition, it can be hypothesized that these changes are related to the altered responses of BBX mice to benzodiazepines and possibly to convulsants (1, 28). Further work will be necessary to determine if BBX-induced alterations in benzodiazepine receptors really reflect neurodegenerative or plasticity changes in the affected brain regions. Also, it remains to be determined whether the physiological responses of affected cells and regions to benzodiazepines and convulsants are attenuated or enhanced after BBX. Additional experiments may also reveal the effects of BBX on the y-aminobutyric acid receptor in mouse brain and the regulation of the benzodiazepine receptor by this neurotransmitter amino acid. REFERENCES 1. ARAKI, Y., AND S. UEKI. 1972. Changes in sensitivity to convulsion in mice with olfactory bulb ablation. Jap. J. Pharmacol. 22: 447-452. 2. BEN-Am, Y., E. TREMBLAY, 0. P. OTTERSEN, AND R. NAQUET. 1979. Evidence suggesting secondary epileptogenic lesions after kainic acid: pretreatment with diazepam reduced distant but not local brain damage. Brain Res. 165: 362-365. 3. BIGGIO, G., M. G. CORDA, C. A. LAMBERTI, AND G. L. GESSA. 1979. Changes in benzodiazepine receptors following gabaergic denervation of substantia nigra. Eur. J. Pharmacol. 58: 215-216. 4. BOSMANN, H. B., K. R. CASE, AND P. DISTEFANO. 1977. Diazepam receptor characterization: specific binding of a benzodiazepine to macromolecules in various areas of rat brain. FEES Lert. 82: 368-372. 5. BRAESTRUP, C., M. NIELSON, G. BIGGIO, AND R. F. SQUIRES. 1979. Neuronal localisation of benzodiazepine receptors in cerebellum. Neurosci. Left. 13: 219-224. 6. BRAESTRUP, C., AND R. F. SQUIRES. 1978. Brain specific benzodiazepine receptors. Br. .I. Psychiatr. 133: 249-260. 7. BROADWELL, R. D., AND D. M. JACOBOWITZ. 1976. Olfactory relationships of the telencephalon and diencephalon in the rabbit. III. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations. J. Comp. Neurol. 170: 321-346. 8. CAIN, D. P. 1974. The role of the olfactory bulb in limbic mechanisms. Psychol. Bull. 81: 654-671. 9. CHUSID, J. G., AND L. M. KOPELOFF. 1%2. Chlordiazepoxide as an anticonvulsant in monkeys. Exp. Biol. Med. 109: 546-548. 10. CICERO, T. J., L. G. SHARPE, E. ROBINS, AND S. S. GROTE. 1972. Regionaldistributionof tyrosine hydroxylase in rat brain. J. Neurochem. 19: 2241-2243. 11. GIACHETTI, I., AND P. MACLEOD. 1977. Olfactory input to the thalamus: evidence for a ventroposteromedial projection. Brain Res. 125: 166- 169. 12. GRAZIADEI, P. P. C., R. R., LEVINE, AND G. A. MONTI GRAZIADEI. 1978. Regeneration of olfactory axons and synapse formation in the forebrain after bulbectomy in neonatal
mice. Proc. Natl. Acad. Sci. U.S.A. 75: 5230-5234. 13. GUERRERO-FIGUEROA,
R.,
M.
M.
RYE,
AND
R. G.
HEATH.
1969.
Effects
of two
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benzodiazepine derivatives on cortical and subcortical epileptogenic tissues in the cat and monkey. I. Limbic system structures. Curr. Ther. Res. Clin. Exp. 11: 27-39. 14. GUERRERO-FIGUEROA, R., M. M. RYE, AND R. G. HEATH. 1%9. Effects of two benzodiazepine derivatives on cortical and subcortical epileptogenic tissues in the cat and monkey. II. Cortical and centrencephalic structures. Curr. Ther. Res. C/in. Exp. 11: 40-50.
15. HEIMER, L. 1972. The olfactory connections of the diencephalon of the rat. An experimental light and electron microscopic study with special emphasis on the problem of terminal degeneration. Brain Behav. Evol. 6: 484-523. 16. HEIMER, L., AND R. J. KALIL. 1978. Rapid transneuronal degeneration and death of cortical neurons following removal of the olfactory bulb in adult rats. J. Comp. Neural. 178: 559-610. 17. HEIMER, L., AND R. D. WILSON. 1975. The subcortical projections of the allocortex: similarities in the neuronal associations of the hippocampus, the piriform cortex, and the neocortex. In M. SANTINI, Ed., “Golgi Centennial Symposium,” Raven Press, New York. pp. 177-193. 18. HIRSCH, J. D. 1980. The neurochemical sequelae of olfactory bulbectomy. Life Sci. 26: 1551-1559. 19. HIRSCH, J. D. 1980. Opiate and muscarinic ligand binding in five limbic areas after bilateral olfactory bulbectomy. Bruin Res. 198: 271-283. 20. LIPPA, A. S., D. CRITCHETT, M. C. SANO, C. A. KLEPNER, J. COUPET, AND B. A. BEER. 1979. Benzodiazepine receptors: cellular and behavioral characteristics. Pharmacol. Biochem. Behav. 10: 831-842. 21. LIPPA, A. S., M. C., SANO, J. COUPET, AND B. A. BEER. 1978. Evidence that benzodiazepine receptors reside on cerebellar Purkinje cells: studies with “nervous” mutant mice. Life Sci. 23: 2213-2218. 22. MACKERER, C. R., R. L. KOCHMAN, B. A. BIERSCHENK, AND S. S. BREMNER. 1978. The binding of [sH]diazepam to rat brain homogenates. J. Pharmacol. Exp. Ther. 206: 405-413. binding to 23. MOHLER, H., AND T. OKADA. 1977. Properties of [3H]diazepam benzodiazepine receptors in rat cerebral cortex. Life Sci. 20: 2101-2110. 24. MOHLER, H., AND T. OKADA. 1978. The benzodiazepine receptor in normal and pathological human brain. Br. J. Psychiutr. 133: 261-268. 25. PAUL, S. M., P. J. SYAPIN, B. A. PALJGH, V. MONCADA, AND P. SKOLNICK. 1979. Correlation between benzodiazepine receptor occupation and anticonvulsant effects of diazepam. Nature (London) 281: 688-689. 26. SPETH, R. C., G. J. WASTEK, P. G. JOHNSON, AND H. I. YAMAMURA. 1978. Benzodiazepine binding in human brain. Characterization using [3H]flunitrazepam. Life
Sci. 22: 859-866.
27. TANABE, T., M. IINO, Y. OOSHIMA, AND S. TAKAGI. 1974. An olfactory area in the prefrontal lobe. Bruin Res. 80: 127-130. 28. UEKI, S., Y. ARAKI, AND S. WATANABE. 1977. Changes in sensitivity of mice to anticonvulsant drugs following bilateral olfactory bulb ablations. Jup. J. Pharmucol. 27: 183-192. 29. WENZEL, B. M. 1974. The olfactory system and behavior. Pages l-40 in L. V. DICARA, Ed., “Limbic and Autonomic Nervous Systems Research.” Plenum, New York. 30. WHITTEN, W. K. 1956. The effect of removal of the olfactory bulbs on the gonads of mice. J. Endocrinol. 14: 160-163.
NEUROLOGY
72, 91-98 (1981)
Regional Changes in [3H]Diazepam Binding in the Brains of Mice after Removal of the Olfactory Bulbs JAMES
D. HIRSCH’
Department of Biological Research, G. D. Searle & Company, P.O. Box 5110, Chicago, Illinois 60680 Received May I, 1980; revision received September 18, 1980 [sH]Diazepam binding was assayed 3 weeks after surgery in 13 brain regions derived from sham-operated and bilaterally olfactory bulbectomized mice. In bulbectomized mice, [Wjdiazepam binding was elevated 30% in the frontal cortex and 24% in the thalamus but decreased 26% in the striatum and 28% in the midbrain. These changes represented alterations in the number of binding sites. No binding changes were observed in the olfactory tubercle, amygdala, piriform cortex, olfactory peduncle, hypothalamus, hippocampus, coiliculi, cerebellum, or pons-medulla of bulbectomized animals. These results are compared with the effects of other types of brain lesions on the brain benzodiazepine receptor, and are possibly related to the altered sensitivity of bulbectomized mice to benzodiazepines and convulsants.
INTRODUCTION There is strong evidence to suggest that benzodiazepine tranquilizers act in the brain via membrane-bound receptors (4,6,19,22,23,26). Part of this evidence was obtained by measuring the effects of various brain lesions or neuropathologic conditions on the binding of radiolabeled benzodiazepines in vitro. Braestrup et al. (5) reported 40 to 65% decreases in [3H]flunitrazepam binding in rat cerebella 9 to 10 days after local injections of the neurotoxin, kainic acid. Similarly, Braestrup and Squires (6) demonstrated decreases of 30 to 40% in [3H]diazepam and [3H]fluniAbbreviation: BBX-bilaterally olfactory bulbectomized. 1 The author is indebted to Dr. Frank L. Margolis for his help and encouragement. I would also like to gratefully acknowledge postdoctoral fellowship support from the Roche Institute of Molecular Biology where this work was performed. Jill Cluesmann at Roche and Carole Ryan at Searle provided excellent secretarial assistance. 91 OOW4886/81/040891-08$02.00/O Copyri&t AU rights
0 1961 by Academic Press, Inc. of reproduction in my form reserved.
92
JAMES D. HIRSCH
trazepam binding in kainic acid-injected rat substantia nigra 8 to 50 days after the lesion. In addition, injection of kainic acid into the rat caudate nucleus resulted in about a threefold decrease in the affinity of [3H]diazepam binding in the ipsilateral substantia nigra (3) 10 days after the lesion with no change in the number of ligand receptors. Mohler and Okada (24) reported decreases in [3H]diazepam binding in the putamen and caudate nucleus of brains obtained from victims of Huntington’s chorea, a neurodegenerative disorder, and benzodiazepine receptor binding was shown to decrease in the cerebella of the “nervous” mutant mouse where F’urkinje cells degenerate during development (5, 2 1). In contrast, Lippa and co-workers reported an increase of about 28% in [3H]diazepam binding after 3 weeks in the rat frontal cortex ipsilateral to a coronal knife cut made on the anterior border of the caudate nucleus (20). Also, Hirsch and Margolis (18,19) reported a 20% increase in r3H]diazepam binding in the remaining mouse olfactory bulb 6 months after a unilateral olfactory bulbectomy. Several studies demonstrated no effects of various treatments or conditions on the benzodiazepine receptor. Benzodiazepine receptor binding was not altered in the rat occipital cortex by injection of 6-hydroxydopamine into ascending noradrenergic neurons, nor was binding affected in the rat striatum and cerebellum after parenteral administration of 3-acetylpyridine. X irradiation of the rat hippocampus also had no effect on benzodiazepine binding (6). In addition, binding was unaltered in cerebella of “weaver” mutant mice where granule cells degenerate (5). When these biochemical studies were compared with reports showing altered behavioral responses to benzodiazepines and convulsant treatments (1, 28) in mice subjected to bilateral olfactory bulbectomy, it was postulated that this brain lesion might also result in changes in [3H]benzodiazepine binding in various regions of the brain. Thus, we investigated the binding of [3H]diazepam to membranes prepared from 13 brain regions from bilaterally olfactory bulbectomized (BBX) mice 3 weeks after the lesion. The results indicate that BBX-induced changes in [3H]diazepam binding were quantitatively similar to those reported by other workers using other lesioning techniques and may be related to the alterations in response of BBX mice to benzodiazepines and convulsants. METHODS Bilateral olfactory bulbectomy on anesthetized IO- to 12-month-old female CD-l mice (40 to 45 g; Charles River, Wilmington, Mass.) was accomplished by aspiration as previously described (30). Sham surgery consisted of all surgical steps but the animals received a partial craniotomy
[3H]DIAZEPAM
BINDING
AFTER
BULBECTOMY
93
only. After surgery they were housed 10 to 13 per cage and experiments began 3 weeks later. The mice were killed by CO2 asphyxiation and the brains were rapidly removed. Some brains were dissected on ice into eight major regions using the boundries described by Cicero et al. (10). We separated the midbrain from the pons, which was left attached to the medulla. In other brains, the limbic forebrain was removed by transecting the brain coronally at the level of the mammillary bodies. This region was then dissected at -20°C as described by Hirsch (19) into the amygdalae, olfactory tubercles, olfactory peduncles, piriform cortices, and hypothalami. Brains with damage to the frontal cortex, olfactory peduncles, olfactory tubercles, or any other region were discarded. After dissection the tissues were stored at -70°C. Freezing to 1 month had no effect on [3H]diazepam binding. [3H]Diazepam binding to membrane preparations from each region was assayed as previously described (20). Briefly, twice-washed membrane fractions prepared by Polytron (Brinkmann Instruments, Westbury, N.Y.) homogenization of brain regions in 50 ~0120 mM Tris-HCl buffer (PH 7.4) followed by centrifugation were incubated 20 min at 0°C with 1.9 nM [3H]diazepam (39.1 Ci/mmol, New England Nuclear, Boston, Mass.) in the presence or absence of 10 PM unlabeled diazepam (Hoffman-LaRoche, Nutley, N.J.) for determination of nonspecific binding. Each 200~~1 assay contained 200 to 300 pg membrane protein. Reactions were terminated by dilution with 2 ml ice-cold buffer followed by a rapid vacuum filtration through Whatman GF/B glass fiber filters. The filters were then washed twice more with 2 ml buffer and dried 20 min at 60°C. Bound [3H]diazepam was determined by liquid scintillation spectrometry at 33% efficiency. In each experiment, three to six samples of each brain region derived from more than one sham-operated or BBX animal were pooled. Effects of BBX on the ligand binding affinity or on the number of ligand receptors in the brain regions were determined by measuring specific binding for the range of 0.08 to 13.5 nM [3H]diazepam in the presence or absence of 10 PM unlabeled diazepam and constructing double reciprocal plots of the saturation isotherms obtained. RESULTS There were no significant differences in [3H]diazepam binding between sham-operated and BBX mice in the amygdalae, olfactory tubercles, piriform cortices, hypothalami, or olfactory peduncles in the limbic system 3 weeks after the lesion (Table 1). These results were in sharp contrast to the effects of BBX on opiate and muscarinic cholinergic binding in these regions (18, 19). In addition, there were no changes in r3H]diazepam
94
JAMES D. HIRSCH TABLE
1
Regional Changes in [3H]Diazepam Binding in Sham-Operated and Bilaterally Bulbectomized Mice” fmol/mg protein Brain region Striatum Olfactory tubercle Hippocampus Amygdala Piriform cortex Colliculi Frontal cortex Olfactory peduncle Midbrain Hypothalamus Cerebellum Thalamus Pons-medulla
Sham (n = 4)
BBX (n = 4)
386 k 377 2 345 2 292 2 290 2 282 + 265 + 254 + 239 + 205 + 183 ? 174+ 135 +
285 t 367? 381 + 284 ? 306 + 314r 346 + 250 + 173 -c 212 k 188 * 216 f 121 k
30 13 24 10 11 21 8 13 14 5 12 5 6
15 6 20 11 11 2 16 10 18 11 11 13 7
Percentage change
P
-26 -3 +10 -3 +5 +11 +30 -2 -28 +3 +3 +24 -6
co.025 NS NS NS NS NS 10.005 NS co.05 NS NS CO.025 NS
n Specific binding of [3H]diazepam at a concentration of 1.9 nM was determined + 10 pM unlabeled diazepam in membrane preparations of the various brain regions. Incubations were at 0°C for 20 min, and assays were terminated by vacuum filtration through Whatman GF/B filters followed by washing. Experiments were carried out 3 weeks after surgery. Values in fmol/mg protein are means f SE from four separate experiments. In each experiment, three to six pieces of each region from two or more animals were pooled. Statistical significance was determined by Student’s t test. NS-not significant.
binding in the hippocampus, colliculi, cerebellum, or pons-medulla. However, binding of [3H]diazepam increased 30% in the frontal cortex (P < 0.005) and 24% in the thalamus (P < 0.025) and decreased 26 (P < 0.025) and 28% (P < 0.05), respectively, in the striatum and midbrain. Saturation isotherms followed by double-reciprocal plot analysis of the binding data indicated that primarily the number of [3H]diazepam receptors (B,,,) was altered in these four brain regions rather than the affinity for the ligand (K,). These plots are single, straight lines indicating a single [3H]diazepam binding site in each brain region (Fig. 1). In sham-operated striata, a B,,, of 1670 fmol/mg protein was obtained and 1250 fmoYmg protein was obtained in BBX striata. The sham and BBX KD was 6.3 nM. In frontal cortex, where [3H]diazepam binding increased after BBX (Table 1). the shamB,ax was 2000 fmoYmg protein, and the BBX value was 2857 fmol/mg protein. In this region the apparent KD for the sham-operated animals was 7.1 nM and 7.4 nM for the BBX animals. Similar
[3HJDIAZEPAM
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2I
4I
6I s”
0I
BINDING
lo
I2
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AFTER BULBECTOMY
14 r
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41
6
8
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AG. 1. Double reciprocal plots of saturable [sH]diazqam bii in brain regions from bilaterally olfactory bulbectomized (BBX) and sham-operated mice. Specific [SH]diazepam binding was measured for the range ofO.08 to 13.5 nt+t[Wldiazepam in the presence or absence of 10 PM unlabeled diazepam. Binding assay conditions are described in detail under Methods. Membranes were prepared from frozen brain regions obtained from BBX and sham-operated animals as described under Methods. Each experiment was replicated twice with diierent membrane preparations and results varied by about 10%.
data were obtained in the thalamus and midbrain. In the thalamus, the sham and BBX B,,, values were 1000 and 1667 fmoYmg protein, respectively. The sham K, was 6.3 nM and the BBX KD was 7.7 nM. In the midbrain, the sham B,,, was 2000 fmoYmg protein and the BBX B,,, was 1724 fmol/mg protein. The KD values for this region were 14.3 nM for BBX animals and 16.6 nM for the controls. These results confirm the data in Table 1 that were obtained at a single concentration of [SH]diazepam. DISCUSSION Perhaps the most striking finding of this study was that there was no effect of BBX on [3H]diazepam binding in regions of the limbic system. Olfactory bulbectomy results in numerous neuroanatomic and neurochemical changes in denervated limbic areas (8, 12, 15, 16, 18, 29) and other receptors in these regions are markedly affected by BBX (18, 19). The changes in [3H]diazepam binding reported here occurred in regions not known to be directly associated with bulb efferent fibers and may represent
14 ’
%
JAMES
D. HIRSCH
indirect effects of denervation. This hypothesis is supported by neurophysiologic, neuroanatomic, and neurochemical evidence for an association of cortical regions, the thalamus, the striatum, and the midbrain with olfactory pathways. Giachetti and MacLeod (11) reported that cells in the ventroposteromedial nucleus of the rat thalamus responded to electrical stimulation of the lateral olfactory tract and Tanabe et al. reported (27) an area in the monkey prefrontal cortex that responded electrically to delivery of odorants in the nostrils. Similarly, Heimer (16) and Heimer and Wilson (17) demonstrated a projection from olfactory cortical areas to the ventral part of the head of the caudate nucleus in the striatum where this structure overlays the olfactory tubercle. Neuroanatomic results reveal that serotonergic afferent fibers from the midbrain travel to the olfactory bulb in rodents (7) and bulbectomy may result in disruption of this pathway as well. There is also neurochemical evidence to support this serotonergic pathway (18). Thus, in view of the apparent neuronal localization of benzodiazepine receptors (5,6, 21), the present results may provide biochemical support for the apparent pathways described above. At present it is not known why receptor number increases in the frontal cortex and thalamus or decreases in the striatum and midbrain. Increases may represent denervation supersensitivity and decreases may be due to loss of neurons or terminals bearing benzodiazepine receptors, but proof of this is not yet available. The present results may also relate to observations made of BBX animals administered benzodiazepines or subjected to convulsant drugs or treatments. In 1972 Araki and Ueki (1) reported that BBX mice had higher shock thresholds for development of convulsions and were less sensitive to several convulsant drugs when tested 2 10 days after surgery. Later, it was reported (28) that BBX mice tested 214 days after surgery were more sensitive to the anticonvulsant effects of the benzodiazepines, chlordiazepoxide, diazepam, and nitrazepam, against both electrically and pentylenetetrazol-induced convulsions. It was suggested that alterations in neuronal function in the cerebral cortex, midbrain, thalamus, ponsmedulla, and possibly other brain regions were responsible for the altered responses shown by mice with lesions (1, 28). It was also reported that benzodiazepines prevented spreading of seizures from the thalamus, cortex, and other regions (2,9, 13, 14). Therefore, although the changes in [3H]diazepam binding reported here were small, their location suggests that they could be related to the behavioral alterations observed in BBX mice. It should be noted that only 10 to 20% of brain benzodiazepine receptors need to be occupied by diazepam in order for the drug to prevent pentylenetetrazol-induced convulsions or exhibit anticonflict activity (20, 25). Thus, a small change in diazepam binding in a specific brain region could have a marked effect on the action of the drug in that region.
[WjDIAZEPAM
BINDING
AFTER
BULBECTOMY
!27
In conclusion, these data suggest that BBX in mice results in changes in E3H]diazepam binding in the frontal cortex, thalamus, striatum, and midbrain 3 weeks after the lesion. The magnitude of these changes is similar to that reported by other workers using a variety of other lesioning techniques. In addition, it can be hypothesized that these changes are related to the altered responses of BBX mice to benzodiazepines and possibly to convulsants (1, 28). Further work will be necessary to determine if BBX-induced alterations in benzodiazepine receptors really reflect neurodegenerative or plasticity changes in the affected brain regions. Also, it remains to be determined whether the physiological responses of affected cells and regions to benzodiazepines and convulsants are attenuated or enhanced after BBX. Additional experiments may also reveal the effects of BBX on the y-aminobutyric acid receptor in mouse brain and the regulation of the benzodiazepine receptor by this neurotransmitter amino acid. REFERENCES 1. ARAKI, Y., AND S. UEKI. 1972. Changes in sensitivity to convulsion in mice with olfactory bulb ablation. Jap. J. Pharmacol. 22: 447-452. 2. BEN-Am, Y., E. TREMBLAY, 0. P. OTTERSEN, AND R. NAQUET. 1979. Evidence suggesting secondary epileptogenic lesions after kainic acid: pretreatment with diazepam reduced distant but not local brain damage. Brain Res. 165: 362-365. 3. BIGGIO, G., M. G. CORDA, C. A. LAMBERTI, AND G. L. GESSA. 1979. Changes in benzodiazepine receptors following gabaergic denervation of substantia nigra. Eur. J. Pharmacol. 58: 215-216. 4. BOSMANN, H. B., K. R. CASE, AND P. DISTEFANO. 1977. Diazepam receptor characterization: specific binding of a benzodiazepine to macromolecules in various areas of rat brain. FEES Lert. 82: 368-372. 5. BRAESTRUP, C., M. NIELSON, G. BIGGIO, AND R. F. SQUIRES. 1979. Neuronal localisation of benzodiazepine receptors in cerebellum. Neurosci. Left. 13: 219-224. 6. BRAESTRUP, C., AND R. F. SQUIRES. 1978. Brain specific benzodiazepine receptors. Br. .I. Psychiatr. 133: 249-260. 7. BROADWELL, R. D., AND D. M. JACOBOWITZ. 1976. Olfactory relationships of the telencephalon and diencephalon in the rabbit. III. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations. J. Comp. Neurol. 170: 321-346. 8. CAIN, D. P. 1974. The role of the olfactory bulb in limbic mechanisms. Psychol. Bull. 81: 654-671. 9. CHUSID, J. G., AND L. M. KOPELOFF. 1%2. Chlordiazepoxide as an anticonvulsant in monkeys. Exp. Biol. Med. 109: 546-548. 10. CICERO, T. J., L. G. SHARPE, E. ROBINS, AND S. S. GROTE. 1972. Regionaldistributionof tyrosine hydroxylase in rat brain. J. Neurochem. 19: 2241-2243. 11. GIACHETTI, I., AND P. MACLEOD. 1977. Olfactory input to the thalamus: evidence for a ventroposteromedial projection. Brain Res. 125: 166- 169. 12. GRAZIADEI, P. P. C., R. R., LEVINE, AND G. A. MONTI GRAZIADEI. 1978. Regeneration of olfactory axons and synapse formation in the forebrain after bulbectomy in neonatal
mice. Proc. Natl. Acad. Sci. U.S.A. 75: 5230-5234. 13. GUERRERO-FIGUEROA,
R.,
M.
M.
RYE,
AND
R. G.
HEATH.
1969.
Effects
of two
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benzodiazepine derivatives on cortical and subcortical epileptogenic tissues in the cat and monkey. I. Limbic system structures. Curr. Ther. Res. Clin. Exp. 11: 27-39. 14. GUERRERO-FIGUEROA, R., M. M. RYE, AND R. G. HEATH. 1%9. Effects of two benzodiazepine derivatives on cortical and subcortical epileptogenic tissues in the cat and monkey. II. Cortical and centrencephalic structures. Curr. Ther. Res. C/in. Exp. 11: 40-50.
15. HEIMER, L. 1972. The olfactory connections of the diencephalon of the rat. An experimental light and electron microscopic study with special emphasis on the problem of terminal degeneration. Brain Behav. Evol. 6: 484-523. 16. HEIMER, L., AND R. J. KALIL. 1978. Rapid transneuronal degeneration and death of cortical neurons following removal of the olfactory bulb in adult rats. J. Comp. Neural. 178: 559-610. 17. HEIMER, L., AND R. D. WILSON. 1975. The subcortical projections of the allocortex: similarities in the neuronal associations of the hippocampus, the piriform cortex, and the neocortex. In M. SANTINI, Ed., “Golgi Centennial Symposium,” Raven Press, New York. pp. 177-193. 18. HIRSCH, J. D. 1980. The neurochemical sequelae of olfactory bulbectomy. Life Sci. 26: 1551-1559. 19. HIRSCH, J. D. 1980. Opiate and muscarinic ligand binding in five limbic areas after bilateral olfactory bulbectomy. Bruin Res. 198: 271-283. 20. LIPPA, A. S., D. CRITCHETT, M. C. SANO, C. A. KLEPNER, J. COUPET, AND B. A. BEER. 1979. Benzodiazepine receptors: cellular and behavioral characteristics. Pharmacol. Biochem. Behav. 10: 831-842. 21. LIPPA, A. S., M. C., SANO, J. COUPET, AND B. A. BEER. 1978. Evidence that benzodiazepine receptors reside on cerebellar Purkinje cells: studies with “nervous” mutant mice. Life Sci. 23: 2213-2218. 22. MACKERER, C. R., R. L. KOCHMAN, B. A. BIERSCHENK, AND S. S. BREMNER. 1978. The binding of [sH]diazepam to rat brain homogenates. J. Pharmacol. Exp. Ther. 206: 405-413. binding to 23. MOHLER, H., AND T. OKADA. 1977. Properties of [3H]diazepam benzodiazepine receptors in rat cerebral cortex. Life Sci. 20: 2101-2110. 24. MOHLER, H., AND T. OKADA. 1978. The benzodiazepine receptor in normal and pathological human brain. Br. J. Psychiutr. 133: 261-268. 25. PAUL, S. M., P. J. SYAPIN, B. A. PALJGH, V. MONCADA, AND P. SKOLNICK. 1979. Correlation between benzodiazepine receptor occupation and anticonvulsant effects of diazepam. Nature (London) 281: 688-689. 26. SPETH, R. C., G. J. WASTEK, P. G. JOHNSON, AND H. I. YAMAMURA. 1978. Benzodiazepine binding in human brain. Characterization using [3H]flunitrazepam. Life
Sci. 22: 859-866.
27. TANABE, T., M. IINO, Y. OOSHIMA, AND S. TAKAGI. 1974. An olfactory area in the prefrontal lobe. Bruin Res. 80: 127-130. 28. UEKI, S., Y. ARAKI, AND S. WATANABE. 1977. Changes in sensitivity of mice to anticonvulsant drugs following bilateral olfactory bulb ablations. Jup. J. Pharmucol. 27: 183-192. 29. WENZEL, B. M. 1974. The olfactory system and behavior. Pages l-40 in L. V. DICARA, Ed., “Limbic and Autonomic Nervous Systems Research.” Plenum, New York. 30. WHITTEN, W. K. 1956. The effect of removal of the olfactory bulbs on the gonads of mice. J. Endocrinol. 14: 160-163.