Download Central adrenergic receptor changes in the

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.