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Brain Research, 418 (1987) 174-177 174 Elsevier BRE 22430 Central adrenergic receptor changes in the inherited noradrenergic hyperinnervated mutant mouse tottering Pat Levitt, Christopher Lau*, Arlene Pylypiw and Leonard L. Ross Department of Anatomy, The Medical College of Pennsylvania, Philadelphia, PA 19129 (U.S.A.) (Accepted 5 May 1987) Key words: Mutant mouse; Adrenergic receptor; Locus coeruleus; Hyperinnervatlon Adrenergic receptor binding characteristics were analyzed in the mutant mouse tottering (tg/tg), a single gene locus autosomal recessive mutation causing hyperinnervation by locus coeruleus neurons of their target regions, which results in epilepsy. Instead of the expected down-regulation of receptors due to the hyperinnervation, both [3H]prazosin (al-receptor) and [125I]iodopindolol(fl-receptor) binding were normal in the tg/tg hippocampus, spinal cord and slightly increased in the cerebellum. This lack of postsynaptic receptor modulation in the target ceils, combined with increased levels of norepinephrine due to the aberrant axon growth, may be critical factors in the expression of the abnormal spike-wave absence seizures in the tg/tg mouse. Tottering (tg/tg) is an autosomal recessive gene mutation in the C57B1/6 J mouse that results in stereotyped focal motor and spike-wave absence seizures 14. While it is characteristic of many neurological mutant mice to have severe brain malformations, the tg/tg mouse appears to express a single anatomical alteration. The pontine noradrenergic nucleus locus coeruleus (LC) exhibits an abnormal overgrowth of its terminal axons in specific targets, resulting in a 30-150% increase in norepinephrine (NE) in each area x°. This hyperinnervation is involved in the genesis of the epilepsy, because deletion of the ascending LC fibers prior to the onset of the epilepsy results in normal E E G patterns in tg/tg mice 13. While the presynaptic changes have been well characterized in this mouse, direct tg gene effects on adrenergic receptors or potential postsynaptic responses of the adrenergic receptors to the hyperinnervation have not been defined. A decrease in cholinergic muscarinic receptor number was described recently, but it does not appear until after the onset of the cortical spike-wave absence seizures 4 weeks postnatally ~1, suggesting that the increase in spontaneous membrane excitabil- ity and not the tg gene causes the receptor alteration. In contrast, the N E hyperinnervation is present at birth and perhaps even prenatally 15. Because of the altered presynaptic noradrenergic component in the tg/tg mouse, it might be expected that substantial receptor changes would also occur. In the CNS, different mechanical and chemical lesion paradigms affecting adrenergic neurons have been used to create either denervated or hyperinnervated target areas. In many instances, removal of a defined adrenergic afferent system results in classic receptor supersensitivity responses of the postsynaptic cells 3A6'18. Conversely, induction of sprouting of NE fibers can result in a down-regulation of the fl-receptor ~'5. Adrenergic receptor subtypes in all brain regions, however, do not necessarily respond to altered afferent input in a similar manner. In several exampies, no permanent change occurred following target denervation 6,19. The tg/tg mouse provides a hyperinnervation model that does not require the use of mechanical or chemical intervention and is amenable to examination of receptor changes due to a developmental in- * Present address: Mail Drop 67, U.S. EPA, Research Triangle Park, NC 27711, U.S.A. Correspondence: P. Levitt, Department of Anatomy, Medical College of Pennsylvania, 3200 Henry Avenue, Philadelphia, PA 19129, U,S.A. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division) 175 crease in afferent input. In the present study, we have examined both a t- and fl-receptors in regions of the tg/tg mouse that receive an abnormal (hippocampus and cerebellum) or normal (spinal cord) complement of noradrenergic fibers arising from the LC and compared them to their + / + counterparts. The results indicate a remarkable conservation of receptor number in the hippocampus and only minor changes in the cerebellum in spite of extensive, permanent hyperinnervation in the tg/tg mouse. Colonies of tg/tg mutant and C57B1/6 J wild type ( + / + ) mice were maintained by generating offspring from mating pairs obtained from Jackson laboratories. Age and sex-matched mature tg/tg and + / + mice were used for the receptor binding studies. Animals were sacrificed by cervical dislocation and the hippocampus, cerebellum and spinal cord were rapidly dissected on ice, weighed and stored frozen for subsequent analysis for NE content and receptor binding assays. The levels of NE were determined by high-pressure liquid chromatography using electrochemical detection as described by Felice et al. 4. flAdrenergic receptors were measured by the specific binding of [125I]iodopindolol essentially as described by McMillian et al. 12. Crude membrane fractions were prepared by the method of Witkin and Harden 2° and protein analysis by the method of Bradford e. Triplicates of 0.1 ml of the crude membrane fraction were incubated in 61 pM [125I]iodopindolol with or without 100 p M D,L-isoproterenol to displace specific binding. Scatchard analysis 17 was performed on membrane preparations pooled from each brain area for all the mice (n = 14-16) of each genotype. [125I]iodopindolol concentrations ranged from 12 to 178 pM. al-Adrenergic receptors were assessed by the specific binding of [3H]prazosin by the modifications of Hornung et al. 7 as described previously 8. Triplicates of the crude membrane fraction were incubated in 0.7 nM [3H]prazosin with or without 1 p M phentolamine to displace specific binding. Results are reported as means ___S.E.M. with levels of significance calculated by the AVOVA test. Straight lines in the Scatchard analysis were fitted by linear regression. [125I]Iodopindolol (2200 Ci/mmol) was obtained from New England Nuclear; [3H]prazosin (23 Ci/mmol) was obtained from Amersham; D,L-isoproterenol hydrochloride and phentolamine hydrochloride were obtained from Sigma. In the present study, preferential elevations of NE observed in the hippocampus and cerebellum (but not spinal cord) of the tg/tg mouse (Table I) further confirm and support our original findings l°. fl-Adrenergic receptors in the hippocampus, cerebellum and spinal cord of the tg/tg mouse were evaluated by [125I]iodopindolol binding and compared to those of the + / + mouse. As shown in Fig. 1, there was little or no difference in fl-adrenergic receptor binding between tg/tg and + / + mice in the hippocampus and spinal cord. Scatchard analysis further confirmed that the binding kinetics between these two genotypes are indistinguishable from each other (Fig. 1 and Table I). (For hippocampus, the small but statistically significant 5% elevation of Bmax in the tg/tg mouse more than likely reflects the fit of the data to the linear regression rather than meaningful physiologic alterations.) In the cerebellum, Scatchard analysis (Fig. 1 and Table I) reveals a statistically significant decrease in Kd in the tg/tg mouse, representing a slight increase in ligand affinity for fl-receptors. Determination of Bmax, however, demonstrates an opposite change for the apparent receptor number, decreasing by approximately 10%. These changes may account for the rather minor increase (12%) in the TABLE I NE content, [~251]iodopindolol binding affinity (Ka) and capacity (BmaQ in various brain regions of tg/tg and +/+ mice NE content is expressed as mean + S.E.M. of duplicate determinations of 6 or more animals of each genotype. Kinetic data of receptor binding are derived from Scatchard analysis performed in each tissue. Each analysis was composed of 8-9 concentrations of radiolabeled ligand ranging from 12 to 178 pM. Straight lines were fitted by linear regression with a minimum correlation coefficient of 0.97. Each point of the Scatchard plot represents triplicate binding determinations. Ko and Bmax values are expressed as means + S.E.M. Hippocampus +/+ tg/tg Cerebellum +/+ tg/tg Spinal cord +/+ tg/tg *P < 0.05 vs +/+ NE (pg/mg tissue) K d (pM) Bma. (fmoles/ mg protein) 446 + 23 614 + 18" 147 + 3 142 + 3 74.8 _+0.6 77.3 + 0.6* 422 + 17 706 + 45* 72.0 + 2.9 57.8 + 1.8" 37.4 + 0.6 33.7+ 0.4* 516 + 23 455 + 41 90.6 + 1.9 101 + 2* 20.5 _+0.2 20.3 ___0.2 176 - Receptors #- Receptors A B 30 I "1" I-~ tg/tg 25 0 2.0 !. 8 !.@, 1.4 C ~2o o ~'~ 7.2 0 ~ °'6 ~E ~o 1.0 0.6 0.4 C.2 0 - - Hippocampus Spinal Cord Hippocampus Cerebellum 125-I-Pindolol binding to hippocampus ~ound/Free 125-1 - Pindolol binding to cerebellum Bound/Free Spinal Cord 125-1-Pindolol binding to spinal cord Bound / Free •.~ t g l t 9 ~o- 0.56 0.48 0.40 U.32 0.24 0.1 0.08[ 0.00 - - 20 40 60 80 Bound(f moles/mg protein) 0 I 10 20 30 40 50 Bound (f moles/rag protein) O 5 10 15 20 ÷/* 25 Bound (tmoles/mg protein) Fig. 1. A: determination of fl-adrenergic receptor binding with [125I]iodopindolol(61 pM) in the brain regions of wildtype (+/+) and mutant tottering (tg/tg) mice. Bars represent means -+ S.E.M. of determination from 14-16 mice of each genotype. Asterisk denotes statistically significant difference (P < 0.01) between tg/tg and +/+ mice. B: determination of al-adrenergic receptor binding with [3H|prazosin (0.7 nM) in brain regions of +/+ and tg/tg mice. Bars represent means + S.E.M. of determinations from 6-8 mice of each genotype. C: Scatchard analysis of [125I]iodopindololbinding to the brain regions in +/+ and tg/tg mice. Data of binding kinetics are summarized in Table I. overall radioligand binding in the cerebellum (Fig. 1). Nevertheless, this small alteration is opposite from the decrease observed when a comparable but selective adrenergic hyperinnervation was produced by chemical lesion 5'19. The physiological significance of the slight changes in cerebellar/3-adrenergic receptors in the tg/tg mouse is probably minimal, particularly in light of previous results where central administration of 6-hydroxydopamine in neonates did not eliminate the LC-cerebellar projection, yet the spike-wave absence seizures were prevented 13. Similarly, when a-adrenergic receptors were assessed by [3H]prazosin binding in the hippocampus and spinal cord, no differences between tg/tg and + / + mice were measured (Fig. 1). Furthermore, in each of the brain regions selected for analysis, no significant change in size was observed between brain regions of the two genotypes 9, thus ruling out the possibility that concomitant changes of LC target volumes might obscure detection of receptor alterations in the mutant brain. Results from the current study therefore suggest that in spite of the abnormal presynaptic innervation pattern in the tg/tg mouse, there is little or no corresponding modulation of the postsynaptic receptors on the target cells. One possibility which may account for this observation is that despite the increased number of NE axons in the hippocampus and cerebellum, the neuronal activity of the LC neurons projecting to these regions might be less than expected. This possibility, however, is tinlikely, because our recent study on neurotransmitter turnover indicates similar metabolism and release rates of NE per terminal axon between tg/tg and + / + mice 9. Therefore, the absence of postsynaptic receptor modulation (down-regulation) in the hyperinnervated brain regions of the tg/tg mouse might be, in part, responsible for the abnormal physiological ac- 177 tivity in the CNS that is characteristic of this genotype. A n additional possibility that merits consideration is that the adrenergic receptor profiles in the mature animal determined in the present study might reflect an 'adaptive' or 'recovered' state of the postsynaptic components on the target cells after an initial and perhaps transient period of receptor modulation in the developing tg/tg mouse. A recent study showed that the N E hyperinnervation in the hippocampus and cerebellum in the tg/tg mouse (by the first postnatal week) 15 well preceded the onset of epilepsy (one month postnatally) 14. Thus, further study to examine the ontogeny of postsynaptic adrenergic receptors during the period when hyperinnervation is initially expressed will be required to resolve this possibility. The results from the current study demonstrate that the permanent N E hyperinnervation is not accompanied by a corresponding, compensatory per1 Beaulieu, M. and Coyle, J.T., Fetally-induced noradrenergic hyperinnervation of cerebral cortex results in persistent down-regulation of beta-receptors, Dev. Brain Res., 4 (1982) 491-494. 2 Bradford, M., A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1979) 248-254. 3 Dausse, J.-P., Hanh Le Quan-Bui, K. and Meyer, P., A 1and a2-adrenoceptors in rat cerebral cortex: effects of neonatal treatment with 6-hydroxydopamine, Eur. J. Pharmacol., 78 (1982) 15-20. 4 Felice, L.J., Felice, J.D. and Kissinger, P.T., Determination of catecholamines in rat brain parts by reverse-phase ion-pair liquid chromatography, J. Neurochem., 31 1461-1465. 5 Harden, T.K., Mailman, R.B., Mueller, R.A. and Breese, G.R., Noradrenergic hyperinnervation reduces the density of fl-adrenergic receptors in rat cerebellum, Brain Research, 166 (1979) 194-198. 6 Harik, S.I., Duckrow, R.B., LaManna, J.C., Rosenthal, M., Sharma, V.K. and Banerjee, S.P., Central compensation for chronic noradrenergic denervation induced by locus coeruleus lesion: recovery of receptor binding, isoproterenol-induced adenylate cyclase activity, and oxidative metabolism, J. Neurosci., 1 (1981) 641-649. 7 Hornung, R., Presek, P. and Glossman, H., Alpha adrenoreceptors in rat brain: direct identification with prazosin, Naunyn-Schmiedeberg's Arch. Pharmacol., 308 (1979) 223-230. 8 Lau, C., Pylypiw, A. and Ross, L.L., Development of serotonergic and adrenergic recptors in the rat spinal cord: effects of neonatal chemical lesions and hyperthyroidism, Dev. Brain Res., 19 (1985) 57-66. 9 Levitt, P., Normal pharmacological and morphometric parameters in the noradrenergic hyperinnervated mutant mouse tottering, Cell Tiss. Res., in press. manent postsynaptic adrenergic receptor down-regulation. This is in contrast to cerebral muscarinic cholinergic receptors, which decrease significantly in response to the increased membrane excitability caused by the abnormal synchronous physiological activity in the mature tg/tg mouse u. These data, therefore, further support our view that the tg gene selectively alters the presynaptic component of the noradrenergic system arising from the LC, producing a developmental anomaly that results in a profound pathophysiological state. This study was supported by N I H Grant NS20196 and a Fellowship from the Klingenstein Foundation (P.L.), a Pharmacology-Morphology Fellowship Award from the Pharmaceutical Manufacturers Association Foundation and Research Grant E P A 6802-4032 (C.L.) and by the Office of Mental Health of the Commonwealth of Pennsylvania. 10 Levitt, P. and Noebels, J.L., Mutant mouse tottering: selective increase of locus coeruleus axons in a defined singlelocus mutation, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 4630-4634. 11 Liles, W.C., Taylor, S., Finnell, R., Lai, H. and Nathanson, N.M., Decreased muscarinic acetylcholine receptor number in the central nervous system of the tottering (tg/tg) mouse, J. Neurochem., 46 (1986) 977-982. 12 McMillian, M.K., Schanberg, S.M. and Kuhn, C.M., Ontogeny of rat hepatic adrenoceptors, J, Pharmacol. Exp. Ther., 227 (1983) 181-186. 13 Noebels, J.L., A single gene error of noradrenergic axon growth synchronizes central neurones, Nature (London), 310 (1984) 409-411. 14 Noebels, J.L. and Sidman, R.L., Inherited epilepsy: spikewave and focal motor seizures in the mutant mouse tottering, Science, 204 (1979) 1334-1336. 15 Phillips, E. and Levitt, P., Developmental expression of the hypertrophied locus coeruleus terminal arbor in the mutant mouse tottering, Soc. Neurosci. Abstr., 12 (1986) 1361. 16 Reader, T.A. and Briere, R., Long-term unilateral noradrenergic denervation: monoamine content and 3H-prazosin binding sites in rat neocortex, Brain Res. Bull., 11 (1983) 687-692. 17 Scatchard, G., The attraction of proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660-672. 18 Sporn, J.R., Wolfe, B.B., Harden, T.K. and Molinoff, P.B., Supersensitivity in rat cerebral cortex: pre- and postsynaptic effects of 6-hydroxydopamine at noradrenergic synapses, Mol. PharmacoL, 13 (1977) 1170-1180. 19 Sutin, J. and Minneman, K.P., A- and b-adrenergic receptors are co-regulated during both noradrenergic denervation and hyperinnervation, Neuroscience, 14 (1985) 973-980. 20 Witkin, R.M. and Harden, T.K., A sensitive equilibrium binding assay for soluble B-adrenergic receptors, J. Cyclic Nucleotide Res., 7 (1981) 235-246.