Canine narcolepsy is associated with an elevated number of α2-receptors in the locus coeruleus

Brain Research, 500 (1989) 209-214

209

Elsevier BRES 14890

Canine narcolepsy is associated with an elevated number of az-receptors in the locus coeruleus Beate Fruhstorfer, Emmanuel Mignot, Scott Bowersox, Seiji Nishino, William C. Dement and Christian Guilleminault Sleep Research Center, Stanford School of Medicine, Palo Alto, CA 94304 ( U. S. A. )

(Accepted 21 March 1989) Key words: a2-Receptor; [3H]Yohimbine binding: Narcolepsy: Dog: Locus ceruleus; Noradrenaline: Autoinhibition

a2-Receptors in the canine brain were pharmacologically characterized using [~H]yohimbine binding. Competition studies revealed a single class of binding sites in frontal cortex but two distinct subtypes in nucleus caudatus. The role of central ~2-receptors in narcolepsy was investigated in 5 normal and 5 narcoleptic Doberman pinschers. Scatchard analysis of [3H]yohimbine binding in different brain areas revealed an increase in the number of a2-binding sites limited to the locus coeruleus. This suggests that altered autoinhibition of norepinephrine release may be associated with the narcoleptic symptomatology. INTRODUCTION Narcolepsy is a disorder characterized by excessive daytime sleepiness, a disturbed sleep pattern and, in many cases, by sudden attacks of partial or complete loss of muscle tone due to emotional stimulation (cataplexy). A n abnormal pattern of R E M sleep during the attacks of sleepiness, as well as a similarity between cataplexy and the muscular paralysis found during regular R E M sleep, have led to the assumption that narcolepsy is a R E M sleep disorder ~°. Canine narcolepsy is a spontaneous animal model of the human syndrome with essential homologies, that allow the investigation of brain neurotransmitter abnormalities potentially involved in this disorder 3. It has generally been accepted that brainstem cholinergic mechanisms play a major role in R E M sleep and that cholinergic neurons in the pontine reticular formation regulate muscle atonia during R E M sleep '2~3. However, also a monoamine involvement in R E M sleep regulation was proposed 14 and the role of norepinephrine in the overall regulation of sleep and wakefulness is now well documented 9"22 yet the exact contribution of norepiCorrespondence:

nephrine in the executive mechanisms of R E M sleep remains still unclear 7. Norepinephrine might also be involved in the expression of cataplexy in narcoleptic dogs, as a pharmacologically induced deficit of norepinephrine in the synaptic cleft increased cataplexy 21. It was concluded that the cholinergic neurons involved in the regulation of muscle tone during R E M sleep were controlled by adrenergic mechanisms. A subclassification of ct-adrenergic receptors has been invented according to their postsynaptic (al) or presynaptic location (a2) (see ref. 26). This anatomic subdivision has been proved to be not entirely correct, insofar as some postsynaptic receptors also have a 2 properties, but other attempts at definition on the basis of functional or biochemical approaches also lack a general applicability (see ref. 5). Now there is increasing evidence that both, cq- and a2-receptors provide a further receptor heterogeneity and thus additional subdivision is necessary (a~A and ct~BI°, a2A and a2~6). The function of the 'classical' postsynaptically located a-receptors can be described as mediators of adrenergic actions while the presynaptically localized alpha receptors act as autoinhibitors of norepinephrine release 27.

C. Guilleminault, Stanford Sleep Research Center, 701 Welch Road, Suite 2226, Palo Alto, CA 94304, U.S.A.

210 Both a-adrenergic receptor types, (h- and ~z,receptors, are distributed throughout the whole central nervous system, but certain areas show marked differences, in autoradiographic studies, especially high densities of a2-receptors were observed in the locus coeruleus 2~, the area with the largest number of norepinephrine-containing cell bodies in the brain. Furthermore, evidence was provided that it is in the locus coeruleus where substances acting on a2-adrenoceptors affect sleep and arousal mechanisms s. It was shown that microinfusion of clonidine - - an a2-receptor stimulating substance - - into the locus coeruleus of rats induced sleep, while microinfusion of yohimbine - - an a2-receptor blocking agent - - caused arousal. Canine narcolepsy is associated with an altered number of several receptor types, e.g., muscarinic cholinergic receptor levels have been shown to be higher in the nucleus reticularis gigantocellularis of the brainstem ~s, brain dopamine receptors were shown to be increased in the amygdala 4 and recently also an increased number of am-receptors in the amygdala was reported 2t. The aim of the present study was to characterize a2-adrenergic receptor binding in the normal dog brain by means of [3H]yohimbine binding studies and to investigate possible a2-receptor abnormalities in the brain of narcoleptic dogs. MATERIALS AND METHODS Doberman pinscher dogs from the Stanford Sleep Disorders Research Center breeding colony were killed at 100-150 days of age by intravenous infusion of thiopental sodium. The brains were removed immediately postmortem, frozen by immersion in cold 2-methylisobutane (-40°C) and stored at -80 °C. Brains from 5 affected bomozygous (3 separate litters, 2 males and 3 females) and 5 normal (2 separate litters, 3 males and 2 females) Dobermans of either sex were used. Dissections were made or described in Mefford et al.19. Sections were taken and photographic plates were made of successive sections (1 mm in thickness) of the brainstem of a control Doberman and were used as templates for further dog dissections. The locus coeruleus was identified histologically in one 16-month-old narcoleptic Doberman (Fig. 1) and its position superim-

Fig. t. Histologic slide obtained on narcoleptic Doberman No, 79R9 Luxol fast blue-Cresyl violet (magnification × 4.6). Circles indicate locus coeruleus. Photograph du~' to the courtesy of Dr. Lysia Forno, M.D. Stanford University, Neuro Pathology Laboratory, who performed histologic investigations on narcoleptic dog brains.

posed onto atlas drawings of Lim et al. ~7 and Adrianov and Merring ~. Dog atlases were used as templates and the locus coeruleus region was dissected free-hand from coronal sections of the brainstem. Dog brains were dissected in pairs (one narcoleptic with one control). Radioligand assay conditions were determined with tissue samples from the frontal cortex of normal and narcoleptic dogs. The frozen brain samples were homogenized in 20 vols./weight tissue of ice-cold 51) mM Tris-HCl buffer (pH 8.0) and centrifuged at 15,000 x g for 10 min at 4 °C. The pellets were twice resuspended in the same buffer and centrifuged as before. For final tissue preparation, the pellets were suspended in 30 vols. of distilled water and diluted in 300 vols. of 25 mM

211 glycylglycine buffer (pH 7.6) (final concentration 22.7 mM). All binding experiments were performed with [3H]yohimbine (New England Nuclear; spec. act. 72.5 Ci/mmol). Tissue was always added in 90(1 ul aliquots to tubes containing ligands disolved in 100ul distilled water. Non-specific binding was defined by {I.1 mM (-)-norepinephrine. After incubation at 30 °C for 30 rain binding was terminated by rapid vacuum filtration through Whatman GF/C glassfiber filters that were subsequently rinsed twice with ice-cold 50 mM Tris-HC1 (pH 8.0). Filters had been pretreated with 0.1% polyethylenimine solution to reduce non-specific binding. Radioactivity retained on the filters was measured in 4 ml of Cytoscint (Westchem) by liquid scintillation spectrometry (Beckman LS3801). Counting efficiencies typically exceeded 50%. Protein concentrations were determined with the Bio-Rad protein assay using bovine serum albumin as standard.

Kinetic studies The dissociation rate constant (K 1 (min i) was determined by preincubation of a tissue suspension from frontal cortex and [3H]yohimbine at prior determined K d concentration for 30 min; then 0.1 mM norepinephrine was added and incubation was continued for various times from 30 s to 60 min. The observed association rate constant Ko~,~(min 1) was determined by incubation of the same tissue suspension with the same fixed radioligand concentration, with or without norepinephrine, for various times from 10 s to 6(1 rain. The association rate constant K+l (M -I rain 1) was determined from K+l = (K,,~.,~-K ~/L, where L is the radioligand concentration. The equilibrium dissociation constant K d equals K ]K+~. The data were analyzed using the program K I N E T I C Is.

with only one tube at each point. In competition studies the tissue suspension of frontal cortex or nucleus caudatus was incubated with 8-14 concentrations of the competing drug and [3H]yohimbine and K d concentration, ldazoxan, prazosin and yohimbine were used as antagonists and clonidine, norepinephrine and oxymetazoline as agonists. All drugs were purchased from Sigma, with the exception of idazoxan, a gift from Necker Medical School (Paris). Assuming one or two independent binding site models, the data were analyzed using the programs E B D A ~s and L I G A N D 24. For the statistical analysis of differences between the normal and the narcoleptic brain tissue, the M a n n - W h i t n e y U-test statistic was used. RESULTS Specific binding of [)Hlyohimbine to frontal cortex increased linearly as a function of protein content over the range of 2.0-4.5 mg tissue/ml. All assays were done within this range. Time course measurements of [3H]yohimbine binding to dog frontal cortex at K d concentrations indicated a rapid association with a steady state reached after 10 min and maintained for at least 60 rain (Fig. 2). The association rate constant K+l calculated from such experiments was found to be 0.54 _+ 0.03 nM l min ~ (n = 3). Dissociation

6000

Nompm~phrii~ 0 1 m M

1

m

Z r-

4000

Z

2000 I

Equilibrium binding studies Saturation studies were performed in frontal cortex, hippocampus, locus coeruleus, nucleus caudatus, putamen, and thalamus. [3H]yohimbine binding was determined in 6 (locus coeruleus, mean tissue weight 49.3 mg) to 12 (large size brain structures) concentrations ranging from 0.005 to 10 nM. Total binding was determined in triplicate or duplicate and non-specific binding in duplicate or

i

t

20

40

60

80

Q

100

TIME (MIN)

Fig, 2. Canine frontal cortex [3H]yohimbine association kinetics at 30 °C. Methods are described in detail in the text. Data shown are the result of a representative experiment with respective values of K i, K,l and kinetically derived Kd of 0.0617 mn i, 0.4228 nmol ~ rain i and 0.145 nM.

212 TABLE I Tissue variations for [3H]yohimbine binding in normal and narcoleptic dog brain, means and standard errors of 5 dogs For the analysis of differences between normal and narcoleptic tissue, the Mann-Whitney U-test statistic was used. Protein concentrations were determined with the Bio-Rad protein assay using bovine serum albumin as standard. Tissue

Hippocampus Locus coeruleus Nucleus caudatus Putamen Thalamus

A: normal

B: narcoleptic

K~ (nM)

Bm,~,, (fmol/mgprot)

Kj (nM)

B,~ax (fmol/mgprot)

1.~7 + 0.18 2.31 -+0.42 0.93 + 0.23 0.83 + 0.11 0.98 -+ 0.22

246.5 + 22.9 112.5 + 10.2 191.7 _+32.9 151.5 + 14.8 112.2_+21.2

0.70 _+0.ll* 1.46 + 0.11 1.07 + 0.15 0.99 + 0.07 1.00_+ 0.11

260.8 + 36.6 185.2 + 10.2"* 180.8 + 34.8 134.2 + 15.5 75.4+ 16.2

*P < 0.05, **P < 0.01. experiments lead to an exponential decay of the signal with a dissociation rate constant K_ 1 of 0.09 + 0.02 min -1 (n = 3). The kinetically derived K d is 0.17 nM, a value within the ( K 1 / K ÷ I ) of the deduced Ko. Saturation experiments of [3H]yohimbine binding to dog frontal cortex indicated a single class of

two-site model failed. The agonists clonidine, norepinephrine and oxymetazoline were also best described by a one-site binding model, although, especially for n o r e p i n e p h r i n e , a rather low slope factor was found. A n t a g o n i s t rank order affinities for the [3H]yohimbine binding site were yohimbine

binding sites over radioligand concentrations ranging from 0.005 to 10 nM. The m a x i m u m n u m b e r of binding sites, Bmax, was found to be 309.4 _+ 20.5 fmol/mg protein (n = 5), the K d was 1.35 _+ 0.08 nM. Saturation experiments over a smaller concentration range in hippocampus, locus coeruleus (see Fig. 3), nucleus caudatus, p u t a m e n and thalamus were also best described by a one site model equation. The derived values for Bin, x are given in Table I. Competition experiments for [3H]yohimbine binding sites in the frontal cortex revealed that the a-antagonists idazoxan, prazosin and yohimbine competed for specific [3H]yohimbine binding in a monophasic way, and attempts to fit the curves to a

IO0 0

E Q)

• •

80,

•

narcoleptic

0

normal

P LL

60 e~

40 0

"0 c-

20

O

m

TABLE II Inhibition of [3H]yohimbine binding to canine frontal cortex by adrenergic receptor ligands, means and standard errors of 3 experiments Slope factor

K i (nM)

Yohimbine ldazoxan Prazosin

0.82 + 0.02 0.80 + 0.03 1.04 + 0.09

3.11 + 1.06 21.17 + 7.61 594.10 _+79.0

Oxymetazoline Clonidine Norepinephrine

0.95 + 0.04 0.77 + 0.02 0.66 + 0.09

6.05 + 1.86 10.02 + 1.27 312.60 + 110.00

o

50

1 oo

15o

200

250

Bound (fmol/mg prot) Fig. 3. Scatchard plots from [SH]yohimbine binding in locus coeruleus of one normal and one narcoleptic dog. Saturation studies were conducted using [3H]yohimbine over a concentration range of 0.31 to 10 nM. The radioligand binding data were analyzed using a weighted non-linear, least-squares curve fitting program. Regression coefficients ranged from 0.90 tO 0.99. A higher number of [3H]yohimbine binding sites was found in the narcoleptic dog. The small size of the locus coeruleus in dog brain did not allow us to obtain more than 6 data points.

213 > idazoxan > prazosin; agonist rank order affinities were oxymetazoline > clonidine > norepinephrine. A summary of the slope factors and K~ values is given in Table II. Competition experiments in nucleus caudatus for [3H]yohimbine binding were performed with two antagonists, prazosin and yohimbine. For yohimbine, the data were again compatible with a single class of binding sites (slope factor 0.98 + 0.05, K i 1.58 + 0.15 nM). For prazosin, two different binding sites were significantly distinguished (P < 0.01, slope factor 0.55 + 0.07), a high affinity site with K~ 11.7 + 0.91 nM and a low affinity site with K~ 924.7 + 56.9 nM. A comparison of [3H]yohimbine binding in normal and narcoleptic tissue of different brain areas revealed a significant difference in the number of binding sites in locus coeruleus: narcoleptic dogs had a higher number of a2-receptors in this region (P < 0.01; see Table I and Fig. 3); the other brain areas investigated revealed no such differences. Furthermore, a slight tendency towards a higher affinity of these binding sites for [3H]yohimbine was found in the hippocampus; the K d values of this brain area were lower in narcoleptic dogs than in normal dogs (t' < 0.05). DISCUSSION The results of this study characterize the [3H]yohimbine binding site in dog frontal cortex with the affinity profile yohimbine > idazoxan > prazosin for antagonists and oxymetazoline > clonidine > norepinephrine for agonists as a typical a2-receptor 2s. Furthermore, the data suggest a crucial involvement of the locus coeruleus in canine narcolepsy. In dog frontal cortex agonist and antagonist drugs inhibited [3H]yohimbine binding in a way consistent with a one-site binding model, although displacement curves with slope factors clearly lower than 1 were found with agonists. This finding is probably due to the fact that a2-receptors may operate intracellularly through a dual effector mechanism, direct transducer coupling and second messenger coupling 2'~4. In nucleus caudatus, however, the present data were consistent with a model of two significantly different classes of [3H]yohimbine binding sites. This result is in agreement with previous

findings for human brain 25, where one binding site for [3H]yohimbine in frontal cortex and two binding sites in nucleus caudatus were described. The second site, called a2B, found in human caudatus is closely related to the one obtained in this study for canine caudatus (affinity for prazosin 10.7 nM in humans versus 11.7 nM in dogs). In both, humans and dogs, yohimbine did not differentiate between the two subtypes suggesting a similar affinity for both binding sites. Finally, it should be emphasized that this high affinity %B site for prazosin in nucleus caudatus was not an am-adrenergic receptor > because the 0~2B affinity for yohimbine was about 4000-fold higher and the one for clonidine about 5000-fold lower than the one for %B (data from dog brain>). Recently more evidence for a2-adrenergic receptor heterogeneity has been summarized for several different tissues 6 indicating that the subtype classification may be generally applicable to at least all mammalian species. A gene for the human platelet a2-adrenergic receptor has already been cloned and identified as (/2A (low affinity for %-prazosin) 16. Comparing normal versus narcoleptic dog brain tissue, the most interesting result was an increase in the number of a2-adrenergic receptors in the locus coeruleus of narcoleptic dogs. The locus coeruleus is the brain area with the largest number of norepinephrine-containing cell bodies. These neurons possess a2-autoreceptors located either in the somadendritic area or presynaptically. They are generally accepted to act as autoinhibitors for norepinephrinergic actions by modulating either the biosynthesis of neurotransmitter or the generation of action potentials or the release of neurotransmitter by action potentials 2v. An increase in the number of receptors in the locus coeruleus of narcoleptic dogs could thus result in impaired norepinephrine release and contribute to the narcoleptic symptomatology, This finding is in accordance with earlier results showing that a deficit of norepinephrine within the central nervous system is involved in the expression of canine cataplexy 21. Previous data about the treatment of narcoleptic dogs with a 2 blocking agents like yohimbine and rauwolscine, which should increase norepinephrine release by antagonizing autoinhibition, showed indeed an improvement of the symptomatology 22 and are thus consistent with this theory.

214 ACKNOWLEDGEMENTS D i s c u s s i o n s with J. Miller, R. D e a n and the entire sleep l a b o r a t o r y w e r e helpful for the d e v e l o p m e n t of this work.

Thanks

to Dr.

J.L.

Elghozi, Necker

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