Nurl. Med. Bid. Vol. 19, No. 8, pp. 841-849, 1992 ht. .I. Rodiat. A&. Instrum. Part B Printed in Great Britain. All rights reserved

0883-2897/92 $5.00 + 0.00 Copyright 6 1992 Pergamon Press Ltd

Quantification of h Vivo Binding of [3H]RX 821002 in Rat Brain: Evaluation as a Radioligand for Central a,-Adrenoceptors

S. P. HUME’*, A. A. LAMMERTSMA’, J. OPACKA-JUFFRY’, R. G. AHIER’, R. MYERS’, J. E. CREMER’, A. L. HUDSON’,

D. J. NUTT* and V. W. PIKE’

‘MRC Cyclotron Unit, Hammersmith Hospital, Ducane Road, London W12 OHS and ‘Reckitt & Colman Psychopharmacology Unit, School of Medical Sciences, University Walk,

Bristol BS8 ITD, England

(Received 23 December 1991; in revised form 1 April 1992)

On the basis of its established in vitro characteristics, [3H]RX 821002 was evaluated in rats as an in vivo radioligand for central a,-adrenoceptors. Estimates for in vivo binding potential, obtained by compartmen- tal analyses of time-radioactivity data, ranged between 1.9 for hypothalamus and 0.2 for cerebellum, with a regional distribution in brain which was similar to that observed in vitro. Selectivity and specificity of the signal were checked by predosing with either the a,-antagonists, idazoxan or yohimbine, the a,-agonist, clonidine, or the q-antagonist, prazosin. Pretreatment of the rats with the selective neurotoxin, DSP4, had no significant effect on rH]RX 821002 binding, suggesting that the majority of labelled sites were situated post-junctionally. The studies indicate that [‘H]RX 821002 can be used experimentally as an in vivo marker for central a,-adrenoceptors. The size and rate of expression of the specific signal encourage the development and assessment of [“C]RX 821002 for clinical PET studies.

Introduction

The 2-methoxy analogue of [3H]idazoxan, rH]RX 821002, has been demonstrated in vitro to be a potent and selective radioligand for a,-adrenoceptors in several tissues, including human frontal cortex (Vauquelin et al., 1990) and rat brain (Hudson et al., 1992) and in an established human cell-line express- ing aM receptors (Langin et al., 1989). Although idazoxan itself labels an additional non-a2 site, the non-stereoselective “idazoxan site” (Convents et al., 1989), the introduction of the alkoxy substituent on the 2-position of the benzodioxan ring (Stillings et al., 1985) increases the selectivity of the antagonist for the a,-site versus the non-a* site approx. 400-fold without reducing the affinity of the ligand for the a,-adreno- ceptor (Vauquelin et al., 1990; De Vos et al., 1991).

On the basis of its established in vitro character- istics, RX 821002 was identified as a suitable candi- date for assessment as an in vivo ligand for central a,-adrenoceptors, as part of an ongoing programme to prepare prospective ligands for positron emission tomography (PET). The present study is an investi- gation of the biodistribution of [‘H]RX 821002 in rat brain (i.e. the time-course of its in vivo uptake and retention) as an initial step in the evaluation of RX 821002 as a PET radioligand, when labelled

*Author for correspondence.

with carbon- 11. Regional specificity and selectivity of binding were assessed by predosing the rats with either the a,-antagonists, idazoxan or yohimbine, the al-agonist, clonidine, or the a,-antagonist, prazosin, and by pretreatment of the rats with the selective neurotoxin, DSP4.

Materials and Methods

Ex vivo biodistribution

Approximately 2 h prior to radioligand injection, rats (male SpragueDawley weighing 240-280 g) were anaesthetized with Isoflurane in N,O/O, and the tail vein and artery catheterized. After recovery, the animals were lightly restrained. [‘H]RX 821002 (26 MBq, 0.15 nmol per animal) was given i.v. in 0.20mL saline. Blood samples were withdrawn at designated times (6 per rat) and a composite curve for plasma radioactivity as a function of time was obtained by pooling data from all animals. The radioactive contents of various brain regions were obtained post-mortem by tissue dissection followed by scintillation counting, at times ranging between 2 and 150 min post-injection. Tissue samples were dis- solved overnight in 1 mL soluene, at 37”C, and blood and plasma samples were dissolved in 200 HL 1 M NaOH and acidified with 200 PL 3 M HCl to mini- mize quenching. All samples were counted using a Beckman LS 6800, with automatic quench correction

841

842 S. P. HUME et al.

Table 1. Fitted parameters (with SE), estimated from two tissue (control) or single tissue (predosed) compartment models using the

“metabohte” corrected plasma input

Control IDA-predosed

Region BP (W,) f’, K/M f’, (4 I&)

Olfactory tubercles 1.43 f. 0.03 4.2 f 0.3 4.3 *0.1 Frontopolar cortex 0.80 + 0.12 5.3 f 0.6 5.6 k 0.1 Anterior cingulate 0.65 -& 0.21 5.3 f 0.7 5.3 *0.1 Striatum 0.44 + 0.17 4.5 f 0.6 4.5 kO.1 Parietal cortex 0.84 &- 0.20 5.3 k 0.6 5.5 + 0.2 Thalamus I.18 + 0.21 4.6 f 0.4 4.7*0.1 Hypothalamus 1.89 + 0.34 4.3 rf. 0.5 4.2 +O.l Hippocampus 0.74 k 0.14 4.2 + 0.3 4.6 * 0.1 Occipital cortex 0.80 f 0.32 4.8 f 0.9 5.2 *0.1 Superior colliculi 0.78 k 0.20 4.6 + 0.5 4.OkO.l Inferior colliculi 1.14+0.32 5.0 k 0.8 4.2 + 0.1 Entorhinal cortex 1.70 + 0.25 5.0 f 0.5 5.2 -+ 0. I Rostra1 medulla 0.93 + 0.18 3.8 rtr 0.4 3.1 + 0.1 Caudal medulla 0.90 + 0.19 3.7 * 0.4 3.7 fO.l Cerebellar lobules 0.43 IO.09 3.6 2 0.2 3.8;O.l Cerebellar vermis 0.24 2 0.04 4.0 f 0.1 3.7 *0.1

measured using an internal 13’Cs standard. Further details of the method have been reported previously (Hume et a!., 1991). The dissection was based on the method originally described by Glowinski and Iversen (1966). The tissues (listed in Table 1) were identified on brain slices made according to Palkovits and Brownstein (1988). The locus coeruleus was included in the “rostra1 medulla” sample. Both blood and tissue data were normalized for injected radio- activity and body weight, giving “uptake units” defined as (Bq g-’ tissue)/(injected Bq g-’ body wt), as described previously (Cremer et al., 1992).

Animals were given [3 H]RX 82 1002 either alone or Smin after predosing with either an a,-antagonist, idazoxan (IDA) or yohimbine hydrochloride (YOH), the a,-agonist, clonidine hydrochloride (CLO), or the cc,-antagonist, prazosin hydrochloride (PRZ). The drugs were given i.v. at the following doses: IDA, 2 mg kg-‘, YOH, 2 mg kg-‘; CLO, 1 mg kg-‘; PRZ, 2 mg kg-‘. In addition, two groups of rats received chronic pretreatment. One group was given the selective noradrenergic neurotoxin, DSP-4 [N- (chloroethyl)-N-ethyl-2-bromobenzylamine] i.p., at a dose of 50 mg kg-’ in 0.5 mL saline, 3 days before

C plasma = cp+c m

radioligand injection. The other group was given twice-daily i.p. injections of the cholinesterase inhibi- tor, neostigmine methyl sulphate (NEO), at a dose of 0.1 mg kg-‘, for 2 weeks prior to radioligand injection.

The radioligand, racemic [3H]RK 821002 (2- methoxy-1,4-[6,7-3H]benzodioxan-2-yl-2-imidazo- lidine HCI), specific activity 47.5 Ci mmol-‘, was prepared by Amersham International plc for Reckitt & Colman Ltd, Kingston upon Hull, England. Ida- zoxan (RX 781094) was kindly supplied by the Reckitt & Colman Psychopharmacology Unit, Bristol. All other chemicals were purchased from Sigma, England. The studies were carried out by licensed investigators in accordance with the British

Council’s Guidelines on the Use of Living Animals in ScientijSc Research, 2nd Edn.

Analyses

Initially, single-tissue or two-tissue compartment models with a plasma input function (e.g. Lammertsma et al., 1991) were used in an attempt to describe the time-activity data from IDA-predosed and control groups, respectively. However, using standard non-linear regression techniques, neither set of data could be satisfactorily fitted because of a gradual recuperation of plasma counts with time, presumably due to build up of labelled metabolites. To circumvent the need for metabolite measurement in individual animals, a “metabolite compartment” was added to the models, with the basic assumption that the metabolites did not cross the blood-brain barrier.

Assuming no specific binding of radioligand in the brains of the IDA-predosed group, time-radio- activity data for each region were then satisfactorily fitted using a single tissue compartment model with the additional plasma “metabolite” compartment and two further rate constants (km, and k,), for transfer into and out of the metabolite pool. Since the values for k,, and kti were found to he independent of the brain region used in their estimation, mean

values were used to define the additional plasma

I

I 5 ,,

k *_m2 Gkrnl

I

I *, k2 +I

metaboliie I parent tracer free +

I nowqecific

I spbe~~311y

I

Fig. 1. Schematic representation of the model(s) used. Measured quantities are the total concentrations of radioactivity in plasma (parent tracer and metabolite) = (Cp + C,,,) and tissue = (C, + C,,,). For the single tissue model, C belld = zero. K, is the rate constant for transfer from tissue to plasma (mL g-’ min-I) and k2, k3, k,, km, and kti (min-‘) are rate constants for transfer between the compartments illustrated.

In uiuo binding of [‘H]RX 821002 in rat brain 843

metabolite compartment in the single or two tissue compartment models, schematically illustrated in Fig. 1. The compartment models were then used to obtain best fits to the brain region data: for the control group, the two-tissue compartment model was used, estimating both binding potential (BP = k, /k4) and volume of distribution (V,, = k, /k2). For the IDA-predosed group, a single tissue compart- ment approach was used, estimating V, only (i.e. no specific binding).

Results

Following iv. injection, the initial clearance of [3H]RX 821002 from plasma was very rapid, followed by a gradual recuperation of counts. Composite data are shown in Fig. 2, together with an exponential interpolation of the points. This curve was used as the input function in the non-linear regressions of IDA-predosed regional time-radioactivity data to a single tissue compartment model, allowing for a metabolite compartment in the plasma (Fig.1). The average values for k,, and k,, were used to generate the metabolite corrected plasma curve, shown as the dashed line in Fig. 2, which was then used for all subsequent fits of regional time-activity data.

Figure 3 illustrates time-radioactivity data from three of the dissected regions: entorhinal cortex, hippocampus and cerebellum vermis (representing a range of radioactivity retentions) from control rats and rats predosed with the cc,-antagonist, IDA (Chapleo et al., 1981; Pimoule et al., 1983). In the control brain, the initial uptake of radioligand was similar in all regions but was retained differentially. In the IDA-predosed brain, although the initial up- take was high, especially in cortex, radioactivity was

Q i 3

B .3 P z!!

3.5 plasma

3.0

2J

2.0

1.5 I

1.0

0.5

0.0 0 100 ntzier injection(mln)

150

Fig. 2. Radioactive content of plasma (0) as a function of time after i.v. injection of [‘H]RX 821002, together with the best interpolation of the data points (-), obtained as the sum of three exponentials. The dashed line represents the plasma curve corrected for metabolites, as predicted from single tissue compartment fits of IDA-predosed time-radio- activity curves using the model shown in Fig. 1 (excluding k, and k,; no specific binding). The ordinate axis has been limited to enable the data to be seen more clearly. At the minimum time of assay (10 s after injection), the mean f SD

radioactivity level was 6.2 f 0.9 uptake units (n = 5).

(a) Entorhinal cortex

0 50 100 150 Time after injection (min)

(b) Hippocampus 2 3.5 - .- 2 1 3.0-

i 2.5-Q,

‘= B 2.0-

s 1.5- : .& l.O- P . Q 0.5 - -“.o._..~.._.......e__.--_o

& cz o.oI 0 50 100 150

Time after injection (mm)

(c) Cerebellar vetmis

z 3.5 -

L! 3.0-

“.” r

0 ;o ic’ GO Time after injection (mitt)

Fig. 3. Time-activity data for three of the dissected regions: (a) entorhinal cortex, (b) hippocampus and (c) cerebellar vermis following i.v. injection of -26 MBq [‘H]RX 821002, given to control rats (0) or rats predosed with IDA (0) at a dose of 2 mg kg-’ iv. 5 min prior to radioligand injection. Each time point represents data from an individual animal. Solid and dashed lines are the best fits to control and predosed data, respectively, using the compartmental

models described in Methods.

lost rapidly from all regions and approached plasma levels approx. 60min after injection of radioligand. The solid lines represent best fits to the control (tracer alone) data obtained using the two-tissue compart-

S. P. HUME et al.

0.0 ! I I I 1

0 50 100 150 200 In vitro autoradiography (fmol/mg wet tissue)

Fig. 4. Correlation of individual regional values for BP with in vitro binding distribution obtained using conventional autoradiographic techniques (Hudson et al., 1992). The correlation coefficient was 0.898 (P ~0.001). The in vitro data (from 2 nM [‘H]RX 821002) are averages of the published values for subregions or individual nuclei within the ex viuo dissected regions listed in Table 1. Note, not all of the dissected regions had representative in vitro data.

ment model shown in Fig. 1, and the dashed lines represent fits to the data from the IDA-predosed rats, obtained using the single-tissue compartment model.

The fitted kinetic parameters from these and the other regions dissected are listed rostrocaudally in Table 1. The values for Vd (quantifying the free pool together with the non-specifically bound radio- activity) varied between different brain regions (range 3.6-5.6) but were not significantly different between control and IDA-predosed groups (linear correlation, r* = 0.717, slope = 0.94). The binding potential (quantifying specific binding) was found to be non- homogeneous throughout the brain, varying between 1.9 in hypothalamus to 0.2 in cerebellar vermis. Figure 4 illustrates the close correlation between the estimated regional in vivo BP values and the pre- viously established in vitro distribution of rH]RX 821002 in rat brain measured autoradiographically (Hudson et al., 1992).

The selectivity of the in vivo signal was further tested by acutely predosing the rats with either the a,-antagonist, PRZ (Manvaha and Aghajanian, 1982), the cc,-agonist, CL0 (U’Prichard et al., 1977) or the cr,-antagonist, YOH (Cheung et al., 1982). In these studies, only the times 90-l 50 min after injec- tion of radioligand were used, in order to minimize the numbers of animals needed. Over this period, the ratios of counts in brain regions from control (CON) rats compared with those from IDA-predosed rats were constant. Having previously established that V, was not affected by predosing, this equilibrium ratio could be taken to reflect the size of the specifically bound compartment. Figure 5(a) illustrates the highly significant correlation between the final CON/IDA ratio and the control BP in each of the dissected regions. There was no correlation between the equi- librium ratio and published values of cerebral blood flow from similarly dissected regions (Cremer and Seville, 1983) as illustrated in Fig. S(b),

31@) 0

1-l I 1 I I 1 0.8 1.0

Blob62flow (n&mln) 1.6 1.8

Fig. 5. Correlations of the ratio between tissue counts from control and IDA-predosed rats with (a) BP, for each of the regions listed in Table 1 (r* =0.924, P < 0.001) and (b) published values for blood flow in 11 of the regions, estimated using 4-iodo-[N-methyl-r4C]antipyrine (Cremer and Seville, 1983). The ratios are means of data from rats assayed at 90, 120 or 150 min after injection of -26 MBq rH]RX 821002. The size of the error on the estimates for BP are given in Table 1 and typical SD values for the ratios

CON/IDA are given in Fig. 6.

The bar diagrams in Fig. 6 illustrate final ratios of regional counts relative to IDA-predosed counts in the representative regions illustrated in Fig. 3 (i.e. entorhinal cortex, hippocampus and cerebellar vermis) in control and predosed rats. Compared with the standard of CON/IDA, the specific signal was reduced by 80-90% after YOH-predosing and by 70-80% after CLO-predosing but was not signifi- cantly affected by PRZ. The effects of the acute predosing are summarized in Fig. 7, for all of the regions dissected. The reduction in signal due to YOH or CL0 was significant at P < 0.001 or

P < 0.01 (Student’s t-test). Figure 6 also illustrates the percentage change in

regional specific binding following pretreatment with either the noradrenergic neurotoxin, DSP-4 (Jonsson et al., 1981) or the cholinesterase inhibitor, neostig- mine (Hollingsworth, 1988). The effects are summar- ized in Fig. 8. DSP-4 had no significant effect on specific binding of rH]RX 821002 in any of the regions dissected. Although NE0 caused a consistent (2060%) increase in binding, the effect was not statistically significant. This was partly due to the

In viva binding of [‘HJRX 821002 in rat brain 845

(a) Enmrhinal cortex

control

PRZ

a0

YOH

DSP-4

NE0

1.0 1.5 2.0 2.5 Counts/IDA ratio (90% mi;

4.0

(b) Hippocampus

control

PRZ

a0

YOH

DSP-4

NE0

4.0

NE0

1.0 1.5 2.0 2.5 3.0 3.5 4.0 Counts/IDA ratio (90 - 150 mitt)

Fig. 6. Ratios of counts in (a) entorhinal cortex, (b) hippocampus and (c) cerebellar vermis relative to counts in the same regions from animals predosed with IDA. For acute predosing, the drugs were given i.v. Smin prior to iniection of [‘HIRX 821002: IDA, 2mg kg-‘; PRZ, Zmg kg-‘; CLO, i mg kg-‘; YOH, 2mg kgmi. DSP-4 was given in. at a dose of SO ma ka-’ in 0.5 mL saline, 3 days nrior to injection of [rH]Rs( ~21002. NE0 (0.1 mg kg”)- was given i.p. twice-daily for 2 weeks prior to injection. Values are means (*SD) for 3 time points after nuliohgand injection, as described in Fig. 5. Signiiiamce levels (Student’s r-test) were P <O.OOl (***), P <O.Ol (**) compared with control, or P < 0.01 (++), P <0.05 (‘)

compared with CON + PRZ.

small numbers of animals in each group and the increased inter-rat variation associated with chronic rather than acute treatment. By combining the CON/IDA and PRZ/iDA data to obtain a more secure baseline, the increase in [‘H]RX 821002 bind- ing following neostigmine pretreatment could be shown to be significant at P < 0.01 or P < 0.05, for the majority of regions dissected.

Discussion

The in vitro binding of [‘HJRX 821002 to rat brain has been well characterized and quantified using membrane-binding studies in combination with auto- radiography (Hudson et al., 1992) and a hetero- geneous distribution, reflecting the noradrenergic innervation, described. Using a 2 nM concentration of RX 821002, the range of [‘H]RX 821002 binding varied SO-fold between the highest recorded level, in the nucleus tractus solitarii, and white matter. There was, however, only an &fold variation between the binding in this nucleus and that in cerebellum.

The in uiuo biodistribution, as assessed in the present study using ex oiuo dissection followed by scintillation counting, showed a differential localiz- ation of specific binding of rH]RX 821002 which was very similar to that seen in vitro (Hudson et al., 1992), with an approx. 9-fold variation in the regional estimates for binding potential. The pattern of in uiuo binding of [3H]RX 821002 was also qualitatively similar to the in vitro binding of [jH]idazoxan (Boyajian et al., 1987; Diop et al., 1987) and [‘Hip- aminoclonidine (Unnerstall et al., 1984) in rat brain.

Using a high dose of IDA as a competitive inhibi- tor, the specific in uiuo signal obtained with trace amounts of [‘H]RX 821002 was eliminated. By using a compartmental model to quantify the non-specific signal, any effects of regional variations or changes in blood flow were accounted for and values of Vd obtained. Since V, was not affected by IDA- predosing, the ratio of regional counts in control animals compared with counts from IDA-predosed animals at an “equilibrium” time (90-150 min after injection of radioactivity) was taken to represent the specific signal. At this time, the non-specific radio- activity, i.e. that in the free pool together with the non-specifically bound ligand, accounted for approx. 30% of the total radioactivity in high binding regions such aa entorhinal cortex.

Predosing with the a,-antagonist PRZ had no significant effect on the specific binding of [3H]RX 821002, consistent with the a,/a, selectivity ratio of -300 reported for RX 821002 for peripheral adrenoreceptors (Stillings et al., 1985). Both the a*-agonist clonidine and the al-antagonist yohimbine, given at pharmacologically active doses (e.g. Mogilmcka et al., 1985), reduced the specific signal but neither was as effective as IDA. The reasons for the incomplete blocking remain unclear although it may be related partly to the early suggestion that

846 S. P. HUME et al.

(a) Prazosin

olfactory tuberc1e.s hwitopolarcortex antenurcmgulate

smatum parietal cortex

tkhllUS hyporhakmus

amygdala/plntolm car hlppocampus

occipital cortex supenor colhcul1 mrenor collicul~ encodnal cortex

roshai meuullii cadal medulla

cerebelllun celebellarvelmls

-100 -80 -60 -40 -20 0 20 40 60 80 100

Per cent change in signal

(b) Clonidine

olfactofy tuhelcles frotltopolar amex antenor clngulate

smatum

@eiEE%i hypotluuatnus

=nYgdalatplntam ca WI=--Ps

occqutalcortex supenor colllcull intenor colhcuh entommalcortex

rostral medulla calxlalmeuulla

ce!rebellum ce&!ellarvermlS

-100 -80 -60 -40 -2f~ 0 20 40 60 80 100

Per cent change in signal

(c) Yohimbine

olfactoly llhexcles rrontopularcortex

““““m parktalcortex

ulalamus hypothalamus

amygdala@ultoml clx

oizl- superior colllcull intenor colllcul~ entorhlnaluMtex

KBtral meuulla caudalmeuulla

cTJ%%ellum cexmlarvermls

-100 -80 -60 -40 -20 0 20 40 60 80 loo

Per cent change in signal

Fig. 7. Percentage change in specific signal following acute predosing with (a) PRZ (2 mg kg-‘), (b) CL0 (1 mg kg-‘) or (c) YOH (2 mg kg-‘), given i.v. 5 min prior to injection of [3H]RX 821002. Using the ratios of counts relative to counts in IDA-predosed animals 90-150 min after injection of radioligand, the values

shown were calculated from [(PRZ/IDA - CON/IDA)/(CON/IDA - I)] x lOO%, etc.

In uioo binding of rH]RX 821002 in rat brain 847

olfactory tllbercles trontopolar cortex anbxlor cmgulatc

paliz= thalamUS

h-us amygdala@ritorln ctx

tuppocampls occlp1tal catex

supenor CmlCUll mtermr colt~cul~ entorhmaI cortex

mtral medulla cauwme!uta

cerebelhlm cerebellar VermlS

(a) DSP-4

-100 -80 -60 40 -20 0 20 40 60 80 loo Per cent change in signal

(b) Neostigmine

olfactory tubexles tronwpolar CoRex antermr cmguiak

wt+_y

hypottlalam~ amygda4pulmlln ctx

h’ppocampls occtpltalcortex

SUpeJlor co1llcu11

intenor coli~culi entorhmalcortex

rostml meuulla calNialmed~

txxt%euum cerebellar vermls

-100 -80 -60 40 -20 0 20 40 60 80 100 Per cent change in signal

Fig. 8. Percentage change in specific signal following chronic pretreatment with (a) DSP4 or (b) NEO, as described in Fig. 6. Values calculated as for Fig. 7.

there are distinct agonist and antagonist states of the a-receptor (e.g. Greenberg et al., 1976). The possi- bility that [3 H]RX 821002 labels non-adrenergic IDA sites in vivo is unlikely, based on in vitro findings (Vauqelin et al., 1990; De Vos et al., 1991; Hudson et al., 1992) and although recent in vitro studies have shown that [‘H]RX 821002 has some affinity for the 5-HT,, receptor (K, = 30 nM for 5-HT,, compared with 4.1 nM for a,) (Vauqelin et al., 1990), this is a common feature of many a,-adrenoceptor ligands, including yohimbine (Leysen et al., 1988). It is feasible, however, that any small component of binding could be identified with the 5-HT,, selective antagonist 8-OH-DPAT.

The lack of any significant effect of the selective neurotoxin DSP-4, at a dose previously shown to cause both a 95% reduction in binding to noradren- ergic uptake sites (Tejani-Butt et al., 1990) and a profound reduction in endogenous noradrenaline (Pimoule et al., 1983), is in agreement with other

work demonstrating that the majority of a,-sites in the CNS are post-synaptic (U’Prichard et al., 1977; Pimoule et al., 1983; Dennis et al., 1987). An alterna- tive explanation for the lack of effect of DSP4 is that [3 H]RX 821002 has a greater selectivity for post-junctional over presynaptic a,-adrenoceptors. This seems unlikely since there is little evidence for a subclassification of a,-adrenoceptors based on their pre- or post-junctional location (Wilson et al., 1991) and, in addition, Dennis et al. (1987) have demonstrated that the effects of idazoxan on cortical noradrenaline efflux are mediated by presynaptic receptors.

Hollingsworth (1988) has previously shown a 2- fold increase in B,,, for [3H]clonidine binding in rat brain membranes following twice-daily injections of neostigmine (0.1 mg kg-‘) for 14 days. With the implication that changes in a,-adrenoceptor number were secondary to sustained changes in synaptic noradrenaline concentration, her findings were

discussed in the context of an association between Cremer J. E. and Seville M. P. (1983) Regional brain blood

cholinergic neuronal dysfunction and the patho- flow, blood volume and haematocrit values in the adult

genesis of depressive disorders. More recently, rat. J. Cereb. Bld Flow Metab. 3, 2.54-256.

Hollingsworth and Smith (1991) have proposed that Cremer J. E., Hume S. P., Cullen B. M., Myers R.,

Manjil L. G., Turton D. R., Luthra SK., Bateman D. M. drugs which cause a sustained change in synaptic and Pike V. W. (1992) The distribution of radioactivity in

noradrenaline content do not necessarily alter the brains of rats given [N-methyl-“C]PK 11195 in vivo after

presynaptic receptor sensitivity and that changes in induction of a cortical ischaemic lesion. Nucl. Med. Biol.

receptor number measured by binding studies may 19, 159-166.

Dennis T., L’Heureux R., Carter C. and Scatton B. (1987) reflect changes in post-junctional a,-adrenoceptors. Presynaptic alpha-2 adrenoceptors play a major role in

The present study demonstrated an increase in in vivo the effects of idazoxan on cortical noradrenaline release

binding of [3H]RX 821002 following long-term ad- (as measured by in vioo dialysis) in the rat. J. Pharmacol.

ministration of neostigmine, consistent with a change Exp. Ther. 241, 642649.

in post-junctional receptor function. De Vos H., DeBacker J-P. and Vauquelin G. (1991)

Efaroxan (RX 821037) is a potent and selective a,-adreno- With the proviso that a small proportion of binding ceptor antagonist in human frontal cortex membranes.

of [3 H]RX 821002 is to sites other than the q-adreno- Neurochem. Int. 18, 137-140.

ceptor, the present studies indicate that this radioli- Dickinson K. E. J., McKernan R. M., Miles C. M. M., Leys

gand can be used as an in vivo marker for the K. S. and Sever P. S. (1986) Heterogeneity of mammalian a,-adrenoceptors delineated by [3H]yohimbine binding.

a*-adrenoceptor and has a potential use in the exper- Eur. J. Pharmacol. 120, 285-293.

imental evaluation of both long-term and acute effects Diop L., BriCre R., Grondin L. and Reader T. A. (1987)

of drugs expected to affect noradrenaline neurotrans- Adrenergic receptor and catecholamine distribution in

mission, In man, a,-adrenoceptor abnormalities in rat cerebral cortex: binding studies with [‘Hlprazosin,

several psychiatric disorders and noradrenergic func- [3 Hlidazoxan and [) Hldihydroalprenolol. Brain Rex 402, 403408.

tion responses to psychotropic medication have been Glowinski J. and Iversen L. L. (1966) Regional studies of

reviewed by Glue and Nutt (1988). catecholamines in the rat brain. 1. The disposition of

Although caution has been advised in the extrapol- 3Hnorepinephrine, 3H-dopamine and 3H-dopa in the

ation of the relative potencies of a,-adrenoceptor various regions of the brain. J. Neurochem. 13, 655669.

Glue P. and Nutt D. (1988) Clonidine challenge testing antagonists in rodents to those in man (Dickinson of alpha-2-adrenoceptor function in man: the effects

et al., 1986), Convents et al. (1989) reported no major of mental illness and psychotropic medication. J. Psycho-

pharmacological differences between a,-receptors pharmacol. 2(3&4), 119-l 37.

in human and rabbit cortex using [3H]rauwolscine Greenberg D., U’Prichard D. C. and Snyder S. H. (1976)

and [‘Hlidazoxan as in vitro radioligands. The dem- Alpha-noradrenergic receptor binding in mammalian brain: differential labeling of agonist and antagonist

onstration of a significant specific signal within states. Life Sci. 19, 69-76.

60min after the i.v. injection of rH]RX 821002, Hollingsworth P. J. (1988) Chronic treatment with cholin-

supports the notion that the isotopic labelling of RX esterase inhibitors increases a,-adrenoceptors in rat brain.

821002 with carbon-l 1 at high specific activity could Eur. J. Pharmacol. 153, 167-173.

Hollingsworth P. J. and Smith C. B. (1991) Factors influenc- provide a selective and effective radioligand for the ing the function of presynaptic a,-adrenoceptors in rat

study of CNS x,-receptors in living man, using PET. brain. In Advances in the Biosciences, Vol. 82, pp. 3942.

Preliminary investigations have established the Pergamon Press, Oxford.

feasibility of labelling RX 821002 in the 2-methoxy Hudson A. L., Mallard N. J., Tyacke R. and Nutt D. J.

group with no-carrier-added carbon-11 (Pike et al., (1992) [’ HI-RX82 1002: a highly selective ligand for the identification of a,-adrenoceptors in the rat brain. Molec.

1992). Neuropharmacol. In press. Hume S. P., Pascali C., Pike V. W., Turton D. R., Ahier

R. G., Myers R., Bateman D. M., Cremer J. E., Manjil References L. G. and Dolan R. (1991) Citalopram: labelling with

carbon-l 1 and evaluation in rat as a potential radioligand Boyajian C. L., Laughlin S. E. and Leslie F. M. (1987) for in vivo PET studies of 5-HT re-uptake sites. Nucl.

Anatomical evidence for alpha-2 adrenoceptor hetero- Med. Biol. 18, 339-351. geneity-differential autoradiographic distributions of Jonsson G., Hallman H., Ponzio F. and Ross S. [3H]rauwolscine and rH]idazoxan in rat brain. .I. Phar- macol. Exp. 7’her. 24, 1079-1091.

(1981) DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzy-

Chapleo C. B., Doxey J. C., Myers P. L. and Roach A. G. lamine)-a useful denervation tool for central and periph- eral noradrenaline neurons. Eur. J. Pharmacol. 72,

(1981) RX 781094, a new potent, selective antagonist of 173-188. cr,-adrenoceptors. Proc. E.P.S. Sept. 1981. Lammertsma A. A., Bench C. J., Price G. W., Cremer J. E.,

Cheung Y. D., Bamett D. B. and Nahorski S. R. (1982) [3H]Rauwolscine and [‘HJyohimbine binding to rat

Luthra S. K., Turton D., Wood N. D. and Frackowiak

cerebral and human platelet membranes: possible hetero- R. S. J. (1991) Measurement of cerebral monoamine

geneity of a,-adrenoceptors. Eur. J. Pharmacol. 84, oxidase B activity using L-[“Cldeprenyl and positron

79-85. emission tomography. J. Cereb. B/d Flow Metab. 11, 545-556.

Convents A., Convents D., De Backer J-P., De Keyser J. and Vauquelin G. (1989) High affinity binding of

Langin D., Lafontan M., Stillings M. R. and Paris H.

‘H rauwolscine and ‘H RX781094 to a, adreneraic (1989) [3H]RX821002: a new tool for the identification of

receptors and non-stereoselective sites in human aid a,,-adrenoceptors. Eur. J. Pharmacol. 167, 95-104.

rabbit brain cortex membranes. Biochem. PharmacoI. 38. Leysen J. E, Van Gompel P. and Gommeren W. (1988)

455-463. Distinction between adrenergic and serotonergic receptor .^. _. subtypes: specttinty of drugs and absence of cooperative

848 S. P. HUME et al.

In uivo binding of [‘H]RX 821002 in rat brain 849

interactions between adrenergic and serotonergic receptor binding sites. 1. Cardiouasc. Pharmacol. 11, (Suppl. 1) Sl8-S81.

Marwaha J. and Aghajanian G. K. (1982) Relative poten- cies of alpha-l and alpha-2 antagonists in the locus ceruleus, dorsal raphe and dorsal lateral geniculate nuclei: an electrophysiological study. J. Pharmacol. Exp. Ther. 222, 281-293.

Mogilnicka E., Klimek V., Nowak G. and Czyrak A. (1985) Clonidine and p-agonists induce hyperthermia in rats at high ambient temperature. J. Neural Trans. 63, 223-235.

Palkovits M. and Brownstein M. J. (1988) Maps and Guide to Microdissection of the Rat Brain. Elsevier, New York.

Pike V. W., Hume S. P., Aigbirhio F., Turton D. R. and Nutt D. J. (1992) [“C]RX 821002 as a potential PET radioligand for central az adrenoceptors. J. Lubelled Cmpd. Radiopharm. In press.

Pimoule C., Scatton B. and Langer S. Z. (1983) [)H]RX 781094: a new antagonist ligand labels a,-adrenoceptors in the rat brain cortex. Eur. J. Pharmacol. 95, 79-85.

Stillings M. R., Chapleo C. B., Butler R. C. M., Davies J. A., England C. D., Myers M., Myers P. L., Tweddle N., Welboum A. P., Doxey J. C. and Smith C. F. C. (1985)

a-Adrenoceptors reagents. 3. Synthesis of some 2-substi- tuted 1,Cbenzodioxans as selective presynaptic a,- adrenoceptor antagonists. J. Med. Chem. 28, 1054-1062.

Tejani-Butt S. M., Brunswick D. J. and Frazer A. (1990) rH]Nisoxetine: a new radioligand for norepinephrine uptake sites in brain. Eur. J. Pharmacol. 191, 239-243.

Unnerstall J. R., Kopajtic T. A. and Kuhar M. J. (1984) Distribution of a2 agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents. Brain Res. Rev. I, 69-101.

UPrichard D. C., Greenberg D. A. and Snyder S. H. (1977) Binding characteristics of a radiolabeled agonist and antagonist at central nervous system alpha noradrenergic receptors. Molec. Pharmacol. 13, 454-473.

Vauquelin G., De Vos H., De Backer J-P. and Ebinger G. (1990) Identification of a2 adrenergic receptors in human frontal cortex membranes by binding of t3H]RX 821002, the 2-methoxy analog of [-‘H]idazoxan. Neurochem. In!. 17, 537-546.

Wilson V. G., Brown C. M. and McGrath .I. C. (1991) Are there more than two types of a-adrenoceptors involved in physiological responses? Exp. Phwiol. 76, 3 17-346.