Influence of unilateral olfactory bulbectomy on opiate and other binding sites in the contralateral bulb

Brain Research, 199 (1980) 39-47

39

© Elsevier/North-HollandBiomedicalPress

I N F L U E N C E OF U N I L A T E R A L OLFACTORY BULBECTOMY ON OPIATE AND OTHER BINDING SITES IN THE CONTRALATERAL BULB

JAMES D. HIRSCH* and FRANK L. MARGOLIS** Department of Physiological Chemistry and Pharmacology, Roche Institute of Molecular Biology, Nutley, N.J. 07110 (U.S.A.)

(Accepted April 4th, 1980) Key words: olfactory bulbectomy -- opiate binding -- glutamic acid decarboxylase -- naloxone

SUMMARY The neurochemical consequences of unilateral olfactory bulbectomy (UBX) in mice were determined in the remaining olfactory bulb at various times after surgery. The most significant finding was a progressive decline in opiate ligand (dihydromorphine and naloxone) binding that appeared within 11 days after surgery and persisted throughout the study. Statistically significant declines in spiroperidol (--67 ~), clonidine ( 4 8 ~ ) and muscimol ( - - 1 6 ~ ) binding were also observed 90 days after surgery, At 180 days postsurgery we observed a 20 ~o increase in diazepam binding. No effect of UBX on dihydroalprenolol, quinucludinylbenzilate or serotonin ligand binding was observed. Bulbectomy resulted in a moderate decrease (--28 ~ ) in DOPA decarboxylase activity 14 days after surgery, which returned to normal by 30 days. Glutamic acid decarboxylase activity decreased by 37 ~ 7 days after UBX, returned to normal by 14 days after surgery and then increased by 25 ~ 90 days after UBX. Unilateral bulbectomy had no effect on cholineacetyltransferase activity in the remaining bulb. Thus, following a unilateral procedure, one bulb cannot necessarily serve as a valid control for the other. Possible explanations for the neurochemical changes observed are discussed.

INTRODUC]?ION The neurotransmitters utilized within the olfactory bulb have been intensively studied in recent years. The granule cells and some periglomerular cells are thought to * Present address: Department of BiologicalResearch, G.D. Searle and Company, P.O. Box 5110, Chicago, Ill. 60680, U.S.A. ** To whom all correspondence should be addressed.

40 use GABA31,34,z8 as transmitter, and there is also evidence that some of the periglomerular and tufted cells are dopaminergic22, 23. The mitral cells may use glutamate or aspartate21, ~4, and there is ample evidence to suggest that the dipeptide carnosine is the neuroaffector utilized by afferent olfactory axons in the glomerular layer of the bulb20,z6,27,30,33. In addition, centrifugal pathways contribute at least cholinergic, dopaminergic, noradrenergic and serotonergic fibers to various neuronal layers of the bulb6,as,2z,z3. Recent findings have also shown that opiate binding sites and enkephalins (refs. 1, 26, 27 and 50 and R.V. Lewis and F.L. Margolis, unpublished observations), as well as other peptides with suspected neuroaffector activityV,S,2Z,2s, 37, are found in the bulb. A large number of benzodiazepine binding sites are also found in the bulb27,33. These observations, coupled with the distinct and well-studied laminar arrangement of bulb neurons, ligand binding sites and centrifugal input in the bulb6,1s,22,23,33,4°, stimulated us to investigate the effects of unilateral bulbectomy [UBX] on various neurochemical parameters in the remaining olfactory bulb. Even though there is no evidence at present that direct neuronal connections exist from bulb to bulb4, 5, it was anticipated that information about the transmitters involved in bulbto-bulb communication could be obtained with this approach, especially since the anterior olfactory nucleus does exhibit projections to the contralateral bulbS,15,TM. MATERIALSAND METHODS Methods Unilateral olfactory bulbectomy (UBX) on retired adult female CD-1 exbreeder mice (Charles River Breeding Laboratories, Wilmington, Mass.) under Chloropent anesthesia was performed by aspiration as previously described a6. The mice were 10-12 months old at the beginning of these experiments and were used within 14-30 days of their arrival in our colony. After surgery animals were housed in groups of 10-13 on a 24 h clock (12 h light, 12 h dark) at 75 °F and 5 0 ~ relative humidity. Animals were killed by COz asphyxiation, and the remaining bulbs were rapidly removed, frozen on dry ice and stored at --70 °C. Any animals showing damage to the remaining bulb, the frontal cortex, or any areas more caudal to either bulb were discarded. Sham operations consisted of all surgical steps, stopping at a partial craniotomy. Pilot experiments revealed that neither ligand binding nor enzyme activity varied significantly in sham animals over the time course of these experiments. Thus, for experiments up to and including 30 days post-UBX, age-matched shams were not used and the shams were killed all at once. However, as a precaution, age-matched shams were used for the 90- and 180-day points and were killed along with their respective lesioned counterparts. Specific binding of [3H]spiroperidol (SPI), [3H]dihydroalprenolol (DHA), [3H]clonidine (CL), [3H]quinuclidinylbenzilate (QNB), [3H]diazepam, (DZ), [3H]muscimol (MU), [aH]dihydromorphine (DHM), [3H]naloxone (NAL), [3H]serotonin and [3H]lysergic acid diethylamide (LSD) were determined in bulb membranes prepared by homogenization in 20 mM Tris.HC1 buffer (pH 7.4) or 50 mM Tris.citrate buffer (pH 7.1) (MU binding) as previously described 3,9,1°,13,17,~6,z7,33,41-43,47,49. Specific SPI

41 binding was determined 4- 0.1 mM dopamine. Specific DHA binding was determined 4- 10 #M (--)-propranolol and specific CL binding was performed 4- 10 /~M phentolamine. Specific binding of QNB was assayed 4- 1 /~M atropine, while DZ specific binding was determined 4- 10 #M diazepam. Specific binding of MU was assayed 4- 1 mM GABA. Specific binding of DMH and NAL was determined 4- 10 /~M levallorphan, specific serotonin binding was determined ± 0.1 mM serotonin and specific LSD binding was determined 4- 10/~M serotonin. Cholineacetyltransferase (CAT) activity (CoA:choline O-acetyltransferase, EC 2.3.1.6) was assayed by the method of Wilson et al. 4s. Glutamic acid decarboxylase (GAD) (L-glutamic 1-carboxylase, EC 4.1.1.14) activity was determined by the method described by Baxter 2. DOPA decarboxylase (DDC) (3,4-dihydroxy-L-phenylalanine carboxylase, EC 4.1.1.26) was assayed by the method of Christenson et al. 11 with minor modifications. In each binding experiment at each time point, at least 3 and usually 6 bulbs were pooled in order to obtain enough tissue. For each of the enzyme experiments, at least two bulbs were pooled at each time point. Protein was measured by the method of Lowry et al. 29 using bovine serum albumin as the standard. Materials

The following 8H-labeled materials were obtained from New England Nuclear (Boston, Mass.): [3H-methyl]diazepam (39.1 Ci/mmol), [3H(N)methylene]muscimol (12.1 Ci/mmol), [4-3H]clonidine (26.7 Ci/mmol), levo[propyl-2,3-~H]dihydroalprenolol (48.6 Ci/mmol), [1-phenyl-4-3H]spiroperidol (26.4 Ci/mmol), [1,2-ZH(N)]sero tonin binoxalate (24.9 Ci/mmol), [N-methyl-SH]lysergic acid diethylamide (32.3 Ci/mmol) and [1-14C]acetyl Coenzyme A (52.8 mCi/mmol). Amsersham Corporation (Arlington Heights, Ill.) was the source of [1,7,8(N)-3H]dihydromorphine (81.0 Ci/mmol), [3-~H]quinuclidinylbenzilate (16.0 Ci/mmol), L-3[1-14C]3,4-dihydroxyphenylalanine (9.1 mCi/mmol)and DE-[1-14C]glutamicacid (23.0 mCi/mmol). Chloropent for animal anesthesia was purchased from Fort Dodge Laboratories (Fort Dodge, Iowa). RESULTS The influence of unilateral bulbectomy (UBX) on binding of 9 [aH]ligands to membranes prepared from the remaining bulb at various times after surgery is shown in Table I. It should be noted that bulbs from several mice were pooled for each of the experiments presented. The most striking effect was the progressive decrease in DHM and NAL binding in the remaining bulb. Within 11 days after UBX, NAL binding decreased 30~o in the remaining bulb, and within 14 days DHM binding declined 73 ~. Naloxone binding declined more slowly than DHM binding, and was 50 ~o lower than shams 30 days after surgery. At 240 days postsurgery DHM binding was still less than 5 0 ~ of the sham levels (not shown). Using Scatchard analysis of DHM saturation curves in bulbs obtained 14 days after UBX, it was determined that the number of DHM binding sites declined rather than there being a change in the affinity

*** P < 0.005.

* * P < 0.001.

* P < 0.01.

3.2 10.2 9.5 5.1 2.9 68.2 36.7 2.2 51.8 3.9

D i h y d r o m o r p h i n e (4.3) N a l o x o n e (6.1) S p i r o p e r i d o l (5.3) (3.8) C l o n i d i n e (3.7) Muscimol(8.3) D i a z e p a m (5.0) D i h y d r o a l p r e n o l o l (5.0) Q u i n u c l i d i n y l b e n z i l a t e (6.3) L y s e r g i c a c i d d i e t h y l a m i d e (3.1)

± ± ± 5± ± 5± 55-

Sham

Ligand (nM)

1.1 (6) 0.6 (6) 1.5 (3) 0.7 (3) 0.1 (3) 1.7(8) 2.2 (3) 0.5 (5) 8.7 (7) 0.2 (4) 3.6 £ 0.5 (4)

-7.2 5- 0.3 ( 6 ) * * * ----. . --

+ 11 days 0.91 4- 0.2 (3)* -11.9, 9.7 ---. . 2.2 ± 0.3 (3) 52.8, 59.0 . .

5- 14 days

Time after bulbectomy

2.8 62.0 5- 9.2 (3)

1.9 61.6, 46.8 .

.

0.38 --1.7 5- 0.3 (3)* 1.5 5- 0.5 (3)* 60.3 5- 3 . 7 ( 6 ) * *

-~- 90 days

1.7, 0.9 5.6 4- 0.6 ( 3 ) * * * 6.8 ----

-" 30 days

-----57.1 • 1 . 4 ( 3 ) * * 45.7 5- 2.3 (3)* ---

+ 180 days

B i n d i n g a s s a y s w e r e p e r f o r m e d as d i s c u s s e d i n ' M a t e r i a l s a n d M e t h o d s ' a n d a s p r e v i o u s l y d e s c r i b e d 3,9,t°A3,17,26,27,33,41-43,47,49. U n i l a t e r a l b u l b e c t o m y w a s p e r f o r m e d as p r e v i o u s l y d e s c r i b e d 4 L V a l u e s are f m o l [ 3 H ] l i g a n d b o u n d / r a g t i s s u e w e t w e i g h t a n d a r e m e a n s -- S.D. o r i n d i v i d u a l v a l u e s w h e r e ind i c a t e d o f t h e n u m b e r o f e x p e r i m e n t s in p a r e n t h e s e s . I n e a c h e x p e r i m e n t , a t l e a s t 3 a n d u s u a l l y 6 b u l b s w e r e p o o l e d , a n d d e t e r m i n a t i o n s w e r e p e r f o r m e d in d u p l i c a t e o r triplicate. S i g n i f i c a n c e o f differences c o m p a r e d t o s h a m s w e r e d e t e r m i n e d by S t u d e n t ' s t-test. A b l a n k in the t a b l e r e p r e s e n t s ' n o t d o n e ' .

Effect of unilateral bulbeetomy on receptor binding in the remaining bulb

TABLE I

bo

30.8 4- 3.4 (6) 24.1 ± 2.8 (9) 9.6 4- 1.0 (9)

DOPA decarboxylase Glutamic acid decarboxylase Cholineactyltransferase

* P < 0.01. ** P < 0.02.

Sham

Enzyme

29.0 ± 2.0 (3) 17.6 + 0.8 (3)* 11.8 4- 1.3 (3)

4- 1 week

Time after bulbectomy

22.1 4- 1.4 (3)** 25.3 4- 2.9 (3) 9.1 4- 0.8 (3)

+ 2 weeks

29.2 4- 0.8 (3) 24.9 -4- 1.9 (3)

+ 4 weeks

34.9 + 3.0 (3) 30.1 4- 2.5 (3)* 10.2 4- 0.9 (3)

+ 12 weeks

Enzyme activity was determined as described in 'Materials and Methods'. Values represent nmoles of 14CO~ or [laC]acetylcholine formed/mg protein/h and are means 4- S.D. of the number of separate experiments in parentheses. In each experiment, at least two bulbs were pooled, and each determination was in duplicate. Significance of differences compared to shams were determined by Student's t-test. A blank in the table represents 'not done'.

Effect o f unilateral bulbectomy on enzyme activity in the remaining bulb

TABLE 1I

4~ t.~

44 of DHM for the binding sites (J.D. Hirsch and F.L. Margolis, unpublished observations). A possible decrease in SPI binding was seen at 30 days, and a large decrease in SPI binding (--67 ~o) was observed 90 days after U BX. At 180 days a a~ o/decrease in CL binding was found as well. Small but statistically significant decreases in M U binding were found 90 and 180 days after surgery, and 180 days after UBX there was an apparent increase in DZ binding in the remaining bulb, which was no longer observed at 240 days postsurgery (not shown). At no time after UBX could changes in DHA or QNB binding be detected in the remaining bulb. At 11 days post-UBX there was no change in LSD binding, and in a single experiment using [3H]serotonin as the ligand we saw no changes in binding at 90 days postsurgery (not shown). There were also effects of U BX on the activity of neurotransmitter synthesizing enzymes in the remaining bulb (Table ll). There was a moderate decrease in DDC activity (--28 ~o) 14 days after surgery that disappeared by 30 days. Paradoxically, GAD activity decreased 7 days after surgery, returned to normal levels at 14 and 30 days post-UBX and then increased by 25 ~ at 90 days after UBX. There was no effect of UBX on CAT activity in the remaining bulb. DISCUSSION The major finding of this study is that when monitored with either naloxone or dihydromorphine, opiate ligand binding declined significantly and progressively in the contralateral bulb after UBX. Although the cause of this decline is unknown, a probable explanation for these results is that the decrease in opiate binding sites in the remaining bulb is due to their presence on fibers or terminals of some pathway that degenerates extensively and fairly rapidly after UBX. Thus, any fibers projecting from one bulb to the other via the anterior olfactory nucleus and anterior commissure could be candidates for this roleS,15,TM. For example, if there is rapid transneuronal degeneration of fibers that enter the contralateral bulb via the anterior olfactory nuclei and anterior commissure, then opiate sites located presynaptically on these fibers and terminals would disappear rapidly as well. Since DDC activity declined at 14 days (Table II), we might expect that evidence for presynaptic degeneration would be observed before a decline in presumed postsynaptic receptors (ref. 39 and Table I). A similar interpretation would apply to noradrenergic fibers entering the bulbs from the locus coeruleus6,18 that synapse on cells in the bulbs bearing a-adrenergic binding sites. As shown in Table I, a-ligand binding declined markedly 90 days after UBX. However, the laminar distribution of itand fl-adrenergic binding sites in the bulbs is differenta3, the laminar content of noradrenaline in the bulb corresponds best with the location of fl-sites32 and fl-ligand binding is unaffected by UBX (Table I). Thus, an alternative explanation for the longterm loss of a-sites in the bulb may relate to their possible role as presynaptic autoregulatory sites on slowly degenerating neurons. In contrast to the opiate binding sites, there is only a small loss of GABA binding sites in the bulb at long times after UBX, accompanied by a small loss of GAD that precedes the loss of binding sites (Tables I and II). This implies that GABA

45 binding sites may also respond to changing levels of their transmitter, as dopamine and norepinephrine binding sites do 39. The loss of GABA binding sites in the bulb would require that there be small degenerative changes in mitral-cell dendrites (the probable location of the GABA binding sites 4°) after UBX. Unfortunately, there is as yet no morphological data to support this view. Current data suggest that essentially all of the cholinergic parameters in the bulbs result from centrifugal input19,44, 51. Since this input is apparently entirely ipsilateral6, 51, it is not surprising that neither QNB binding nor CAT activity were affected by UBX (Tables I and II). As reported by others, CAT activity in the bulb is relatively low 19,44. The neurochemical evidence presented in this paper suggests that opiate systems may play a significant and hitherto unsuspected role in olfactory bulb function. The opiate binding sites in the bulb, as elsewhere in the brain 14, may be located presynaptically to regulate the release of several important transmitters 12. Further, if enkephalins inhibit firing of neurons in the bulb as they do in other brains areas86, 4~, opiate systems could have a more direct effect on neural activity in the bulbs. These considerations may be related to the role of the olfactory bulbs in modulating limbic function35, 45, and thus modulating emotional behaviors. The present data also serve to emphasize the need for a thorough investigation of the neuroanatomical consequences of UBX on the remaining bulb. This is especially true since our results suggest that the olfactory bulbs communicate with each other in a complex way, presumably via the anterior olfactory nuclei and anterior commissure4-6,15,16. In view of the importance of the olfactory bulbs not only in olfaction itself but in modulating many behaviors 45, more research on this brain area is necessary. Finally, it is important to note, in view of these data, that the use of one olfactory bulb as a control for testing the effect of unilateral surgery needs to be validated for each experimental situation. ACKNOWLEDGEMENTS We would like to thank Drs. Jean Rossier and Elinor Cantor for reviewing the manuscript, and Jill M. Cluesmann for her excellent secretarial assistance.

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