Quantification of presynaptic α2-adrenoceptors in rat brain after short-term DSP-4 lesioning

European Journal of Pharmacology, 249 (1993) 37-41 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00

37

EJP 53352

Quantification of presynaptic a2-adrenoceptors in rat brain after short-term DSP-4 lesioning D a v i d J. H e a l *, S a r a h A. B u t l e r , M i c h a e l R. P r o w a n d W. R o g e r B u c k e t t Boots Pharmaceuticals ResearchDepartment, Nottingham NG2 3AA, UK Received 10 June 1993, revised MS received 6 August 1993, accepted 13 August 1993

The relative numbers of pre- and postsynaptic az-adrenoceptors were determined in various rat brain regions after short-term DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) lesioning. For these studies, rats pretreated with zimeldine (10 mg/kg i.p.) were injected with DSP-4 (100 mg/kg i.p.) and were killed either 3 or 15 days later. At the 3 day time-point, DSP-4 treatment produced marked reductions in the noradrenaline content of the cortex (93%), hippocampus (89%), hypothalamus (83%) and cerebellum (92%) with no change in the levels of dopamine or 5-HT. This treatment also decreased the number of az-adrenoceptors labelled with [3H]idazoxan in the cortex (20%), hippocampus (18%), cerebellum (24%) and hypothalamus (39%). Fifteen days after DSP-4 lesioning, the marked reductions of noradrenaline were sustained in the cortex, hippocampus and cerebellum, but there was a considerable reversal of the effect of DSP-4 in the hypothalamus. At this time-point, the decrease in az-adrenoceptors was attenuated in cortex (4%) and cerebellum (0%) and their number was increased in hippocampus (8%) and hypothalamus (7%). Together, the data argue that presynaptic az-adrenoceptors comprise approximately 20% of the total az-adrenoceptor population in the cortex, hippocampus and cerebellum, but about 40% of it in the hypothalamus. Furthermore, they also demonstrate that although the number of presynaptic a2-adrenoceptors in rat brain can be determined by the reduction of radioligand-receptor binding shortly after DSP-4 lesioning, this effect is rapidly masked by receptor proliferation in response to noradrenergic denervation. DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine); a2-Adrenoceptor; Brain; (Presynaptic a2-adrenoceptors); (Postsynaptic a2-adrenoceptors)

1. Introduction

Although it is possible to demonstrate functionally the presence of az-adrenoceptors on noradrenergic nerve terminals in the central nervous system (CNS) via their modulatory effect on noradrenaline release in vitro (Starke and Montel, 1973; Mulder et al., 1978) and in vivo (Dennis et al., 1987; Itoh et al., 1990), attempts to quantify their n u m b e r by radioligand-receptor binding have generally proved unsuccessful. Thus, it has been shown that the number of az-adrenoceptors present in most brain regions is either unaltered or even increased following selective destruction of noradrenergic neurones with 6-hydroxydopamine (U'Prichard et al., 1977; 1980) or DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine) (Dooley et al., 1983). As rats were left to recover for periods of 10-42 days in these studies, the failure consistently to detect decreases in a2-adrenoceptor binding after neuronal lesioning could have been due to receptor proliferation

* Corresponding author. Tel. 0602 492149, fax 0602 492330.

on postsynaptic sites in response to the marked reduction in noradrenergic tone. We have tested this hypothesis by determining the effects on a2-adrenoceptor binding 3 and 15 days after noradrenergic denervation; the former time-point having been selected to permit m e a s u r e m e n t of the proportion of presynaptic oL2adrenoceptors in relation to the total population before their contribution was masked by denervation-induced receptor proliferation. In these experiments, we have used DSP-4 to lesion selectively noradrenergic neurones. This neurotoxin covalently binds to electrophilic centres (Dudley et al., 1990) and inactivates these neurones after being transported into them via the high affinity noradrenaline reuptake system (Cho et al., 1980; Hallman and Jonsson, 1984).

2. Materials and methods 2.1. Animals, drugs and administration protocols

Adult male CD rats (Charles River, Margate) weighing 180-200 g at the start of treatment were used. They

38 were housed two or three per cage on a 12 h light/dark cycle (lights on 07:00 h) at 21°C and 55% humidity. Rats were allowed free access to tap water and, as animals consumed less food after DSP-4 lesioning, bowls of wet-mash were provided in each cage. Drugs were obtained from the following sources: zimeldine, DSP-4 (RBI, Natick USA). Rats were pretreated with zimeldine (10 m g / k g i.p.) to protect 5-hydroxytryptamine (5-HT)-containing neurones as described by Jonsson et al. (1981) with DSP-4 (100 m g / k g i.p.) being injected 30 min later. Control animals received zimeldine (10 m g / k g i.p.) followed by saline (2 m l / k g i.p.). Animals were killed 72 h or 15 days later.

2.2. Tissue preparation Rats were killed by cervical dislocation and their brains were rapidly removed. The right halves of the cerebral cortex, cerebellum and hippocampus were used for monoamine determinations and the left halves were taken for a2-adrenoceptor binding. Hypothalami were dissected from groups of four rats, three of which were pooled for receptor binding and the fourth was taken for monoamine measurement. All brain samples were snap frozen in liquid nitrogen. Monoamine concentrations were measured immediately, whereas samples for az-adrenoceptor binding were stored at - 20°C until assayed.

2.3. a2-Adrenoceptor binding assay Brain tissue was homogenised in ice-cold 0.25 M sucrose (1:30 w / v ) using a motor driven homogeniser with teflon pestle (12 strokes, 800 rpm) and centrifuged at 1000 x g for 10 min. The supernatant was stored on ice and the pellet was rehomogenised (1 : 15 w / v ) and centrifuged at 850 x g for 10 min. Combined supernatants were diluted (1:80 w / v ) with Tris-HC1 containing 0.5 mM E D T A (pH 7.5 at 25°C) and centrifuged at 39000 x g for 12 min. The resulting pellet was again resuspended (1:80 w / v ) in this buffer and recentrifuged at 39 000 X g for 12 min. The final pellet was suspended (1 : 80 w / v ) in 50 mM Tris-HCl (pH 7.5, 25°C) containing 5.68 mM L-ascorbic acid and 5 mM E D T A (equivalent to 12.5 mg wet weight tissue/ml) and used immediately in the binding assay. All centrifugations were performed at 4°C. Eight point saturation binding analyses were performed in a total volume of 500 ~,1, using 400 ~1 of membrane suspension and 50 ~1 [3H]idazoxan (0.1-8.0 riM; 42 C i / m m o l , Amersham International, Amersham). Specific binding was defined using 50 t*1 of 5 b~M phentolamine or 100 /~M ( - ) - a d r e n a l i n e . Incubations were performed at 0°C for 75 min and were terminated by rapid filtration through Skatron 11734 filter mats followed by washing with ice-cold 50 mM

Tris-HCl (pH 7.7 at 25°C) using a Skatron cell harvester. Radioactivity was counted in 4 ml Packard 299 scintillant at an efficiency of approximately 45%. Protein determinations were performed by the method of Lowry et al. (1951) modified by the use of bovine serum albumin as the reference standard. Receptor number (Bma X) and affinity constant ( K d) values were calculated using the L I G A N D iterative non-linear curve fitting programme (McPherson, 1985).

2.4. Measurement of brain monoamine concentrations Measurement of monoamine (noradrenaline, 5-HT and dopamine) concentrations in various regions of rat brain was performed by HPLC with electrochemical detection (HPLC-ECD) to confirm the selectivity and extent of the DSP-4 lesioning procedure. Tissues were homogenised in 5 volumes (w/v) of 0.1 M perchloric acid containing 0.4 mM sodium metabisulphite (antioxidant) and 0.8 g M isoprenaline (internal standard) using a Polytron PT 10-35 disruptor (setting 5-6). After centrifugation at 1100 x g for 15 min at 4°C and 15000 × g for 5 min at 4°C, 30 ~1 of the resulting supernatant was injected onto the HPLC system for the determination of monoamine concentrations. This comprised a Spectra Physics 8810 HPLC pump (flow rate 1 m l / m i n ) connected via a WISP 712 refrigerated autoinjector to a 5 p~m Hypersil ODS 1 reversed-phase analytical column (length 250 × 4.6 mm i.d.) maintained at 45°C and protected by a Brownlee Aquapore RP-300 pre column (length 30 x 4.6 mm i.d.). The H P L C mobile phase was 0.1 M sodium dihydrogen orthophosphate-orthophosphoric acid buffer (pH 3.20) containing 16% v / v methanol, 2.8 mM 1-octane sulphonic acid sodium salt and 0.7 mM EDTA. Noradrenaline, 5-HT and dopamine were detected by use of a BAS LC-4A amperometric detector connected to a TL-5 flow-cell set at a potential of + 0.75 V versus an Ag/AgC1 reference electrode.

2.5. Statistical analysis All results were analysed using Student's unpaired t-test.

3. Results

3.1. The effects of DSP-4 on ol2-adrenoceptor number in various regions of rat brain Three days following DSP-4 (100 m g / k g i.p.) administration, az-adrenoceptor number was decreased by approximately 20% in the cortex, hippocampus and cerebellum and by about 40% in the hypothalamus of lesioned rats (fig. 1A). However 15 days after DSP-4

39

Bmax ± S.E. A

400

20%$

18%;

39%~

800

r--I

,--1

e

:×:

600 300 2O0

24%,~

i

[

I

400

.×.

IO0

200

[]

0

o

B

400

4% ,~

I

I

7% t

8% [--1

I--]

800

300 200

0%

r - - i

100

I ~

400 200

0

0 HIPPOCAMPUS CORTEX

) M

I--'1

HYPOTHALAMUS

CEREBELLUM

Fig. 1. Effects of DSP-4 treatment on ae-adrenoceptor number in four rat brain regions. Rats were given an i.p. injection of zimeldine (10 m g / k g ) 30 min prior to DSP-4 (100 m g / k g ) or saline. (A) Three or (B) 15 days later, rats were killed and binding assays to determine a2-adrenoceptor number were performed as described in Methods. Results are Bmax _+S.E. ( f m o l / m g protein). Hatched columns represent DSP-4 treatment and open columns saline controls. The number of determinations is shown in each column. Significantly different from saline control * * P < 0.01, * * * P < 0.001.

treatment, these deficits had disappeared and there were no differences in ch-adrenoceptor number in any of the brain regions examined (fig. 1B). 3.2. The effects of DSP-4 on monoamine concentrations in various regions of rat brain Three days after DSP-4 (100 m g / k g i.p.) injection, there were very marked depletions of noradrenaline in

cortex, hippocampus, cerebellum and hypothalamus of the lesioned rats with no significant changes in 5-HT or dopamine concentrations (table 1). In the cortex, hippocampus and cerebellum similar results were obtained 15 days after DSP-4 treatment (88-94% depletions of noradrenaline). However in the case of the hypothalamus, there was a partial reversal of the effect of DSP-4 with only a 45% reduction in noradrenaline concentrations at 15 days. Once again, there was no effect of DSP-4 on 5-HT and dopamine concentrations in these four brain regions.

4. Discussion DSP-4 and its 2-methyl analogue, xylamine, are alkylating agents which covalently bind to electrophilic centres at or near their site of action (Dudley et al., 1990). In order to exert their effects, these compounds have to be transported into noradrenergic neurones (Cho et al., 1980; Hallman and Jonsson, 1984). In zimeldine-pretreated rats, DSP-4 was found to produce a profound decrease in the concentrations of noradrenaline in the cortex, hippocampus, cerebellum and hypothalamus and this effect was sustained over the 15 day period in all regions, except the hypothalamus; this finding agrees with earlier reports that DSP-4 selectively lesions noradrenergic neurones originating in the locus ceruleus (Jonsson et al., 1981; Dooley et al., 1983). In zimeldine-pretreated rats, concentrations of the other monoamine neurotransmitters, 5-HT and dopamine, were unaltered by DSP-4 treatment demonstrating a specific action of the neurotoxin on noradrenergic neurones under these experimental conditions. When a2-adrenoceptors were quantified 3 days after DSP-4 administration, the number of these recep-

TABLE 1 Concentrations of noradrenaline, 5-HT and dopamine in various rat brain regions 3 or 15 days after DSP-4 injection. Rats were given an i.p. injection of zimeldine (10 m g / k g ) 30 min prior to DSP-4 (100 m g / k g ) or saline. Three or 15 days later rats were killed and noradrenaline, 5-HT and dopamine concentrations were determined by HPLC-ECD as detailed in Meihods. Results are mean monoamine concentration ( n g / g tissue wet weight)_+ S.E. for groups of 7-10 rats. Brain region

Noradrenaline Control

5-HT DSP-4

Dopamine

Control

DSP-4

Control

DSP-4

354_+14 318 _+25 66_+ 5 802 _+49

336_+16 323 _+ 10 66_+ 6 730 _+ 19

61+14 < 2 <2 300 _+29

64_+15 < 2 <2 264 _+13

385_+16 406_+ 7 74_+ 7 912 _+35

430_+24 372_+17 70_+ 9 892 + 36

36_+ 4 30_+ 2 <2 449 + 49

45_+ 9 23_+ 6 <2 417 + 36

DSP-4 (3 days) Cortex Hippocampus Cerebellum Hypothalamus

210_+ 7 219 _+ 11 177_+11 1774 + 67

17_+ 7a 23 + 13 a 14_+ 8 a 301 _+63 a

DSP-4 (15 days) Cortex Hippocampus Cerebellum Hypothalamus a

234_+11 287_+14 212_+ 8 1951 _+82

29_+ 8 12_+ 7 13_+ 4 1071 _+83

a a ~ ~

Significantly different from appropriate saline control P < 0.001.

40

tors in the brains of the lesioned rats was decreased by approximately 20% in the cortex, hippocampus and cerebellum, and by almost 40% in the hypothalamus. Thus, these results closely agree and extend our earlier preliminary finding that short-term DSP-4 lesioning decreased rat cortical c~2-adrenoceptors by approximately 20% (Payvandi et al., 1990). In view of the findings that DSP-4 selectively inactivates noradrenergic neurones (Jonsson et al., 1981; Hallman and Jonsson, 1984) and this neurotoxin needs to be transported into them in order to exert its effects (Hallman and Jonsson, 1984), the most probable explanation for the observed reduction in cr2-adrenoceptor number is the inactivation of the autoreceptors located on the nerve terminals. These results, therefore, indicate that presynaptic az-adrenoceptors comprise approximately 20% of the total a2-adrenoceptor population in rat cortex, hippocampus and cerebellum and approximately 40% of the total in the hypothalamus. It should be emphasised that these figures may be underestimates of the presynaptic c~-adrenoceptor population for two reasons. First, the depletion of noradrenaline by DSP-4 was not complete indicating that all the noradrenergic neurones and, therefore, presynaptic a2-adrenoceptors had not been totally inactivated and, second, some postsynaptic a2-adrenoceptor proliferation could have occurred in the 3 days between DSP-4 lesioning and measurement of ch-adrenoceptor binding. In earlier investigations where noradrenergic neurones were destroyed using DSP-4 (Dooley et al., 1983) or 6-hydroxydopamine (U'Prichard et al., 1977; 1980), these treatments failed to produce a generalised decrease in a2-adrenoceptor number leading these authors to the conclusion that the overwhelming majority of az-adrenoceptors in the CNS are located postsynaptically to noradrenaline-containing neurones. Since Dooley et al. (1983) and U'Prichard et al. (1977; 1980) left the rats to recover for periods of 10-42 days before measuring a2-adrenoceptors, their findings are consistent with those obtained in the present study and by Payvandi et al. (1990) which demonstrate that marked cr2-adrenoceptor proliferation occurs in the rat brain within 2 weeks after noradrenergic lesioning. DSP-4 binds covalently and irreversibly to electrophilic centres on proteins (Dudley et al., 1990), and in brain regions innervated by the locus ceruleus this neurotoxin is reported to produce a prolonged inactivation of these noradrenergic neurones (Jonsson et al., 1981; Dooley et al., 1983) and this is consistent with our finding that noradrenaline concentrations in the cortex, hippocampus and cerebellum showed no signs of recovery 15 days after DSP-4 lesioning. Moreover, as DSP-4 almost certainly impedes the transport of newly synthesised proteins along the nerve axon (Jonsson et al., 1981; Booze eta[., 1988), the increase of a2-adren-

oceptors in these brain regions is much more likely to derive from receptor proliferation on postsynaptic sites rather than the regeneration of prejunctional autoreceptors. If this hypothesis is correct, it indicates that postsynaptic a2-adrenoceptors in these brain regions show a degree of plasticity as they increase by 11-16% in response to noradrenergic denervation. However, the hypothalamus is the exception because the depletion of noradrenaline produced by DSP-4 at 3 days was partially reversed at 15 days suggesting that this neurotoxin only temporarily inactivates these neurones. This suggestion is supported by the findings of Fety et al. (1986) which demonstrated that unlike noradrenergic neurones originating from the locus ceruleus, the cell bodies and nerve terminals of those innervating the hypothalamus are functionally resistant to the neurotoxic effects of DSP-4. Thus, in the case of the hypothalamus, the increase in a2-adrenoceptors observed at the latter time-point could have resulted from receptor regeneration at pre- and postsynaptic sites. It has been reported that non-adrenergic idazoxan binding sites (NAIBS) are present in rat CNS (Michel and Insel, 1989; Convents et al., 1989; Brown et al., 1990) and these sites are labelled in addition to a 2adrenoceptors when the specific binding of [3H]idazoxan is defined using high concentrations of phentolamine (Convents et al., 1989). However, as shown in table 2, the Bma× and K d values for [3H]idazoxan in the four brain regions were identical irrespective of whether specific binding was defined using 5 p.M phentolamine or 100 p~M ( - ) - a d r e n a l i n e clearly showing that under the conditions used in this study [3H]idazoxan labelled only az-adrenoceptors. As these experiments were only conducted using the brains of untreated rats, it is possible that [3H]idazoxan could label a significant proportion of NAIBS in DSP-4-1esioned brain tissue; however, this is extremely unlikely. In conclusion, the noradrenergic neurotoxin, DSP-4, can be used to selectively inactivate noradrenergic neurones in the CNS and, by measuring az-adrenoceptors shortly after lesioning, the number of these receptors present on presynaptic sites can be determined.

TABLE 2 Comparison of specific [3H]idazoxan binding defined by 5 ~zM phentolamine or 100/zM (-)-adrenaline in four rat brain regions. Results are means_+ S.E. for three determinations. Bm~,x= fmol/mg protein, K d =nM. Brain region

Cortex Hippocampus Cerebellum Hypothalamus

5 p.M phentolamine

100 ~.M ( - )-adrenaline

Bmax

Kd

Bmax

410+15 296_+18 123_+ 4 709-+27

0.95_+0.09 410+ 7 0.99+0.11 294_+17 0.46+_0.02 124_+ 5 0.71-+0.10 739-+36

Kd 0.85_+0.21 0.96_+0.10 0.45+_0.04 0.74-+0.14

41

References Booze, R.M., J.A. Hall, N.M. Cress, S,D. Miller and J.N. Davis, 1988, DSP-4 treatment produces abnormal tyrosine hydroxylase immunoreactivity in rat hippocampus, Exp. Neurol. 101, 75. Brown, C.M., A.C. MacKinnon, J.C. McGrath, M. Spedding and A.T. Kilpatrick, 1990, az-Adrenoceptor subtypes and imidazoline-like binding sites in the rat brain, Br. J. Pharmacol. 99, 803. Cho, A.K., R.W. Ransom, J.B. Fischer and R.C. Kammerer, 1980, The effects of xylamine, a nitrogen mustard on [3H]norepinephrine accumulation in rabbit aorta, J. Pharmacol. Exp. Ther. 214, 324. Convents, A., D. Convents, J.-P. De Backer, J. De Keyser and G. Vauquelin, 1989, High affinity binding of [3H]-rauwolscine and [3H]-RX781094 to a2-adrenergic receptors and non-stereoselective sites in human and rabbit brain cortex membranes, Biochem. Pharmacol. 38, 455. Dennis, T., R. L'Heureux, C. Carter and B. Scatton, 1987, Presynaptic alpha-2 adrenoceptors play a major role in the effects of idazoxan on cortical noradrenaline release (as measured by in vivo dialysis) in the rat, J. Pharmacol. Exp. Ther. 241, 642. Dooley, D.J., H. Bittiger, K.L. Hauser, S.F. Bischoff and P.C. Waldmeier, 1983, Alteration of central alpha 2- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergic neurotoxin, Neuroscience 9, 889. Dudley, M.W., B.D. Howard and A.K. Cho, 1990, The interaction of the beta-haloethyl benzylamines, xylamine, and DSP-4 with catecholaminergic neurons, Annu. Rev. Pharmacol. Toxicol. 30, 387. Fety, R., V. Mis~re, L. Lambas-Senas and B. Renaud, 1986, Central and peripheral changes in catecholamine-synthesizing enzyme activities after systemic administration of the neurotoxin DSP-4, Eur. J. Pharmacol. 124, 197. Hallman, H. and G. Jonsson, 1984, Pharmacological modifications of the neurotoxic action of the noradrenaline neurotoxin DSP4 on central noradrenaline neurons, Eur. J. Pharmacol. 103, 269.

Itoh, Y., R. Oishi, M. Nishibori and K. Saeki, 1990, In vivo measurement of noradrenaline and 3,4-dihydroxyphenylethyleneglycol in the rat hypothalamus by microdialysis: effects of various drugs affecting noradrenaline metabolism, J. Pharmacol. Exp. Ther. 255, 1090. Jonsson, G., H. Hallman, F. Ponzio and S. Ross, 1981, DSP4 (N-(2chloroethyl)-N-ethyl-2-bromobenzylamine) - a useful denervation tool for central and peripheral noradrenaline neurons, Eur. J. Pharmacol. 72, 173. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. McPherson, G.A., 1985, Analysis of radioligand binding experiments: a collection of computer programs for the IBM PC, J. Pharmacol. Meth. 14, 213. Michel, M.C. and P.A. Insel, 1989, Are there multiple imidazoline binding sites?, Trends Pharmacol. Sci. 10, 342. Mulder, A.H., C.DJ. de Langen, V. de Regt and F. Hogenboom, 1978, Alpha-receptor-mediated modulation of 3H-noradrenaline release from rat brain cortex synaptosomes, Naunyn-Schmied. Arch. Pharmacol. 303, 193. Payvandi, N., J.M. Elliott and D.J. Heal, 1990, Radioligand binding to presynaptic a2-adrenoceptors in rat cortex revealed by shortterm DSP-4 lesioning, Br. J. Pharmacol. 100, Proc. Suppl. 346P. Starke, K. and H. Montel, 1973, Involvement of a-receptors in clonidine-induced inhibition of transmitter release from central monoamine neurones, Neuropharmacology 12, 1073. U'Prichard, D.C., D.A. Greenberg and S.H. Snyder, 1977, Binding characteristics of a radiolabelled agonist and antagonist at central nervous system alpha noradrenergic receptors, Mol. Pharmacol. 13, 454. U'Prichard, D.C., T.D. Reisine, S.T. Mason, H.C. FiNger and H.I. Yamamura, 1980, Modulation of rat brain a- and fl-adrenergic receptor populations by lesion of the dorsal noradrenergic bundle, Brain Res. 187, 143.