Effects of JO 1784, A selective sigma ligand, on the autoradiographic localization of M2 and M2 muscarinic receptor subtypes in trimethyltin treated rats

~

Pergamon

0197-0186(95)00004-6

Neurochem. Int. Vol. 26, No. 6, pp. 559-570, 1995 Copyright © 1995 ElsevierScienceLtd Printed in Great Britain. All rights reserved 01974)186/95 $9.50+0.00

EFFECTS OF JO 1784, A SELECTIVE SIGMA LIGAND, ON THE AUTORADIOGRAPHIC LOCALIZATION OF M1 A N D M2 MUSCARINIC RECEPTOR SUBTYPES IN TRIMETHYLTIN TREATED RATS B E R N A D E T T E E A R L E Y 1, M A R Y G L E N N O N , B R I A N E. L E O N A R D I* and JEAN-LOUIS JUNIEN 2 ~Department of Pharmacology, University College, Galway, Ireland 2Institut de Recherche Jouveinal, 94265 Fresnes, Paris, France (Received 22 August 1994 ; accepted 12 January 1995)

Abstract--The distribution patterns of Mt and M2 muscarinic receptor subtypes following TMT and JO 1784 administration in the male Sprague-Dawley rat were investigated. In the present study, JO 1784 was injected in doses of 1, 4 and 16 mg/kg i.p. for one week prior to the single injection of TMT (8 mg/kg i.p.) and subsequently for 33 days. The effects of JO 1784 on the density of muscarinic receptor sub-types (M~ and M2) in the control and trimethyltin (TMT) treated rats were then evaluated. The topographic distribution and changes in muscarinic (ML and M2) receptor densities were determined by means of autoradiography using [3H]quinuclidinylbenzilate (QNB). Both sub-types of muscarinic receptors contributed to the observed decrease in total muscarinic receptor binding in TMT-treated rats. In control rats, JO 1784 alone decreased M~ receptor density in the amygdaloid nuclei, basal ganglia, cortex and hippocampus and decreased M2 receptor density in the amygdaloid nuclei, basal ganglia, cortex, hippocampus, hypothalamus and septal regions. In TMT treated rats, chronic JO 1784 administration has a "neuroprotective effect" on both M~ and M 2 receptors subtypes. Thus, following chronic administration of JO 1784 to TMT treated rats, both increases and decreases in M~ receptor density were observed relative to TMT animals. A significant increase in Mt receptor density was found in the cortex, olfactory regions, septum, thalamus and basal forebrain nuclei. In the hippocampus (CA2 and CA3), a significant decrease in M~ receptor density was observed. In TMT-treated rats, JO 1784 produced a significant increase in M2 receptor density in several brain regions with the most marked effects occurring in the amygdaloid nuclei, basal ganglia, cortex, hippocampus and hypothalamus. The ability of the selective sigma ligand, JO 1784, to attenuate the loss of muscarinic receptors in TMT treated rats could be of importance in the development of novel neuroprotective drugs.

Chang, 1986) making it possible to examine the effects of lesions of varying severity on neurochemical parameters. The primary neuropathological effects of T M T appear in the hippocampal pyramidal cells in regions CA~, CA3 and CA4 (Bouldin et al., 1981 ; Dyer et al., 1982, 1983 ; Earley et al., 1992). Following T M T administration, maximal hippocampal damage occurs 21 days after exposure and the clearance of T M T from the brain has a calculated t m of approximately 16 days (Dyer et al., 1982; Brown et al., 1979). It has previously been established that there is a slow increase in tin concentrations in the brain (Cook et al., 1984). The slow increase in the brain concentrations may be related to the high affinity of T M T for haemoglobin (Brown et al., 1979 ; Aldridge et al., 1981). Haemoglobin may therefore act as a store * Author to whom all corespondence should be addressed. 559

Trimethyltin (TMT) a neurotoxic organotin compound, is a c o m m o n byproduct in the manufacture of dimethyltin chloride, a stabilizing agent for certain plastics. H u m a n exposure to T M T results in epileptic seizures, rage reactions, anorexia, mental confusion and m e m o r y impairment (Fortemps et al., 1978; Fuller and Olney, 1981; Ross et al., 1981). In rats, T M T produces a characteristic behavioural syndrome consisting of learning deficits as measured by appetitive learning tasks, impairments in spatial navigation and deficits in passive avoidance behaviour (Earley et al., 1992; O'Connell et al., 1994a). Neuropathological changes induced by T M T are dose related (Dyer et al., 1982; Chang and Dyer, 1983;

Bernadette Earley el a/.

560

resulting in the slow release of T M T into the plasma from which it enters the brain. This may account for the fact that m a x i m u m d a m a g e occurs in the rat brains 21 days after a single a d m i n i s t r a t i o n of T M T (Dyer et al., 1982; C h a n g and Dyer, 1983). N a a l s u n d et al. (1985) reported a maximal reduction in the high affinity uptake o f glutamate on postinjection day 21 with an 8 mg/kg single injection of T M T . Muscarinic cholinergic receptors in the brain are directly involved in m e m o r y processing a n d cognition and it is generally accepted that the h i p p o c a m p u s is pivotally involved in learning and m e m o r y processes (Bronzetti et al., 1993). The h i p p o c a m p u s is an area of high cholinergic activity with 10% of its n e u r o n a l input estimated to be cholinergic. Thus, destruction of muscarinic receptors can lead to p r o f o u n d memory deficits (Earley et al., 1992; O ' C o n n e l l et al., 1994a, 1994b). We have previously d e m o n s t r a t e d that T M T is a potent n e u r o t o x i n which can contribute to the loss of muscarinic receptors (Earley et al., 1989, 1992: O ' C o n n e l l et al., 1994a, 1994b). Rats of the same strain and weight which were previously injected with a single injection of T M T (6, 7 and 8 mg/kg) were shown to be deficient in the acquisition of a water maze task, as well as in acetylcholinesterase activity a n d total muscarinic binding (Earley et al., 1989). The purpose of the present study was to quantify the density of M t and M2 receptors by means of autoradiographic procedures in several brain regions 34 days after a single injection of T M T a n d to determine if JO 1784 ( ( + ) - N - c y c l o p r o p y l - m e t h y l - N - m e t h y l - 1,4diphenyl- I-yl-but-3-en- l-ylamine, hydrochloride), had a " n e u r o p r o t e c t i v e effect" in preventing the loss of cholinergic receptors induced by T M T . We have previously d e m o n s t r a t e d that JO 1784, a selective sigma ligand, is effective in reversing scopolamine induced amnesia in the rat (Earley et al., 1991).

EXPERIMENTAL PROCEDURES

Animals

Male Sprague Dawley rats with a mean body weight of 28G320 g (Harlan Olac, UK.) were used. They were housed 4 per cage and maintained on a 12-h (8.00 a.m. 8.00 p.m.) light/12-h (dark 8.00 p.m.-8.00 a.m.) dark cycle, food and water were available ad libitum. A single dose of TMT chloride (Aldrich, U.K.) was dissolved in 0.89% physiological saline and administered by i.p. injection. Groups of rats (n = 8) were treated with a single injection of TMT (8 mg/kg, expressed as total salt). Control animals received an equivalent volume of physiological saline. JO 1784 ((+)-Ncyclopropyl-methy[-N-methyl-1,4-diphenyl- l-yl-but-3-en- I ylamine, hydrochloride), was dissolved in physiological saline and administered by the i.p. route in doses of l, 4 and 16 mg/kg (expressed as free base). The experimental animals

were administered JO 1784 or saline for 7 days before the single injection of TMT and for 33 days following the TMT injection. Twenty four hours alter the last administration of JO 1784 (i.e. 34 days post TMT) the animals were sacrificed by decapitation, their brains rapidly removed, frozen on dry ice powder and stored at - 7 0 ' C until use. The frozen brains were cut into 20/~M thick coronal sections at - 15 C, then thaw mounted on chrome alum/gelatin coated slides. Autoradiography of cholinergic muscarinic receptors was performed by the method described by Kuhar and Yamamura 11976) and Rainbow et al. (1982). Frozen 20 jzm thick brain sections were labeled in t'itro with I nM [~H]quinuclidinyl benzilate (QNB)(41.5 Ci/mmol, New England Nuclear) or with 1 nM [~H]QNB in the presence of 1 /~M atropine to determine non-specific binding. The sections were incubated with the ligand for 2 h at 25 C in 0.9% saline containing 15 mM Na2PO4, pH 7.0, washed twice lbr 5 min in 4 C buffer, dried briefly on a 6 0 C slide warmer and apposed against Amersham [3Hi sensitive Hyperfilm (U.K.) for 7 days to generate autoradiograms. Alternate sections of a brain sample were assigned to the M~ and M2 assays. 100 ,uM carbachol was added for M~ receptor determination, while 100 nM pirenzepine was included for M 2 analysis. Pirenzepine, an Mrselective antagonist, was used in the assays as a counter ligand to occlude M] sites, allowing the primary ligand, [3H]QNB to label the remaining M 2 muscarinic receptors. The brain sections were co-exposed with brain mash standards and Amersham commercially available radioactive standards (microscales 1 630 nCi/mg, Amersham. U.K.) against Amersham [3Hi sensitive Hyperfilm (U.K.) for 7 days at 2YC in cardboard X-ray film cassettes (Earley et al., 1989). Films were developed and fixed with Kodak developer (DI9) and fixer (Unifix). Non-specific binding was not visually apparent. Tritium brain-mash standards

Tritium brain-mash standards were made by thoroughly mixing one half of a rat forebrain with 0.5M8 #Ci of [)Hi leucine (53 Ci/mmol, New England Nuclear) in 0.01 N HCt (1 liCi/#l). Brain tissue was mixed with [3H]leucine in a polyethylene tube with a teflon-coated spatula to minimize tissue adherence to glass or metal. The brain mash was transferred to conical shaped aluminium-foil molds, approximately I cm in height and 0.5 cm in base diameter, and tYozen in powdered dry ice. After removal of the aluminiumli~il, the frozen brain-mash standards were mounted onto crystat chucks and cut into 20 #m thick sections at - 15C. At intervals through the frozen brain-mash, representative sections were collected with forceps and were homogenized in 200 id of double distilled water. Triplicate 20/~1 aliquots of the homogenized sections were used either for scintillation counting or for protein measurements according to the method of Bradford (1976). The remaining sections were thaw-mounted onto chrome alum/gelatin subbed slides, dried briefly on a 60'C slide warmer and apposed against Amersham [3Hi sensitive Hyperfilm (U.K.) for 7 days at 23:C in cardboard X-ray film cassettes as described previously (Rainbow et al., 1982). Individual films containing the brain sections were coexposed to commercially available (Amersham) tritiated standards and brainmash standards as described. Generation q[tritium standard curz,e

After development and fixation, the optical densities of the standard autoradiograms were plotted against their tritium

Effects of JO 1784 on M~ and M: receptors concentration per mg protein. The optical density (O.D.) was defined as the log incident light/transmitted light, where incident and transmitted light were expressed as digital voltage. The voltage of the incident light typically measured 14.3 V. This procedure reduced variability that resulted from using different batches of film and corrected the low density standards for the spurious contribution made by film background to their readings (Ehn and Larsson, 1979). Both the R.D. and the O.D. of standard autoradiograms were plotted against their tritium concentrations. A minimum of 3 separate densities was averaged for each standard and used for analysis.

Analysis of [ 3H] QNB autoradioyrams The tritium standard curve was used to convert densitometric measurements into molar quantities of bound ligand. The tritium concentrations of grey matter in muscarinic autoradiograms were converted into picomoles of bound ligand/mg protein by dividing by the specific radioactivity of the [3H]QNB and by cross reference to brain-mash standards and commercially available standards containing known amounts of radioactivity and protein per section. Quantitative analysis of the autoradiograms was performed on a video camera based computerized image analysis system. This system utilizes an IBM/PC/CAT compatible computer, a frame grabber loaded from Imaging Technology International and a software package especially written for this application (Biegon and Rainbow, 1982).

561

between the pyramidal and molecular layers of the C A fields of the hippocampus in the M~ and M2 receptor distribution, these layers were analysed together as a single structure. Digital analysis of the autoradiograms revealed significant decreases in both M~ (Table 1) and M2 (Table 2) receptor density in T M T treated rats (Fig. 1).

EJ]bcts of T M T on M ~ receptors M~ receptor density was highest in the CA~ pyramidal cell layer of the hippocampus, the olfactory tubercle, the basal ganglia (nucleus accumbens and caudate putamen), and the amygdaloid nuclei with moderately low densities occurring in the thalamus, septum, and ventral forebrain nuclei (Table 1). T M T administration produced a marked reduction in M~ receptor density in several brain regions: CA~ pyramidal cell layer (38%), nucleus accumbens (22%), septal nuclei (35-42%), olfactory tubercle (41%). Regions containing both cholinergic terminals and intrinsic cholinergic neurons also exhibit significant reductions (5-40%) in M~ binding (Table 1 and Fig. 1).

Statistics Results were analysed by one way analysis of variance using SPSS routines on measurements obtained from n = 8 animals per group. Each animal had 15 sections taken (6 in the anterior portion of the brain and 9 in the posterior portion of the brain). Each individual section was analysed in an anterior to posterior direction per individual rat brain. A maximum of 20-30 measurements were taken for each rat and included measurements for both the left and right hemispheres. A mean value for receptor density, including left and right hemisphere, was then obtained for each animal. The data from individual animals were then grouped. The values are expressed as mean pmols [3H]QNB specifically bound per mg of protein _+SD (Tables 1~4). To determine overall treatment effects, grouped data were subjected to an analysis of variance parametric procedure (Winer, 1962). If a significant overall effect was observed, Duncan's test was applied to determine statistical difference between groups. A value of P < 0.01 was considered as statistically significant (Earley et al., 1992; O'Connell et al., 1994a, 1994b). RESULTS

Quantitative autoradioyraphy of MI and M2 receptor bindin9 The specific binding of [3H]QNB was determined in 37 brain regions 34 days following T M T (8 mg/kg i.p.) and after chronic JO 1784 (40 days) (1, 4 and 16 mg/kg i.p.) treatment. Muscarinic receptor subtypes were distinguished by their differential affinities for pirenzepine and carbachol in competition with [3H]QNB. Since there was no visible distinction

Effects of T M T on M : receptors Quantitative analysis of the M 2 receptors autoradiograms revealed large reductions in receptor binding in the cholinergic nuclei such as the medial septum (18%) and the diagonal band (16%) in T M T treated rats. A 22% reduction in M2 receptor binding was observed in the CA~ pyramidal cell layer of the hippocampus. Within the parietal-motor cortex, the decrease in M2 binding was more pronounced in the deeper cortical layers, especially layers I I I - I V (36%) and layers V - V I (31%) (Table 2 and Fig. 1). As compared to the M~ receptors, the density of M2 receptors in T M T treated rats was lower overall, with moderate binding seen in the cortex, hippocampus, striatum and amygdala. However, M2 binding in the ventral forebrain cholinergic nuclei (VDB and H D B ) and thalamic regions was greater than M~ binding.

Ef[ects of JO 1784 alone on MI receptor density Following chronic administration of JO 1784, a large decrease in M~ receptor density was found in several brain regions relative to controls (Table 3). This decrease in MI receptor density was most pronounced in the amygdaloid nuclei, the basal ganglia, cortex and hippocampus with the greatest decrease occurring in the CA~ field (33%), the hippocampus at the lower dose of 1 mg/kg and in the nucleus accum-

562

B e r n a d e t t e Earley et al.

Table I. Autoradiography of M~ muscarinic receptor density in control and TMT rats, treated chronically with JO 1784. The results are expressed as mean pmol [3H]QN B specifically bound per mg of protein_+ SD

Brain region

Abbreviation

Amygdala

BE BM CE LA ME

Basolateral amygdaloid nucleus Basomedial amygdaloid nucleus Central amygdaloid nucleus Lateral amygdaloid nucleus Medial amygdaloid nucleus

Basal ganglia

Acb CPu lop ss

Accumhens Caudate putamen Lateral caudate putamen Striatal streak

Cortex

Acg Fr PAM PAM 1 PAM2 PAM3 PAS-P

Anterior cingulate gyrus Frontal cortex Frontoparietal motor Layers i-ii Layers iii-iv Layers v-vi Parietal motor cortex posterior part Posterior cingulate gyrus

PCG Hippocampus CAI-P CA2-P CA3-P CA4 Dgg (U)

Treatment groups TMT TMT+ saline JO 1784 (1)

Control saline

TMT+ JO 1784 (4)

TMT+ JO 1784 (16)

7.323 +0.25 6.583-+0.38 6.258+0.32 6.980-+0.23 6.337-+0.5(I

6.945-+0.28 6.345_+0.46 6.181+0.38 6.570+0.40 6.367+0.62

6.786_+0.38 6.190_+0.50 5.597 +0.56t 6.337_+0.47 6.023_+0.355"

7.154_+0.30 6.380_+0.34 5.883-+0.58 6.589_+0.36 6.263+0.45

6.957_+0.38 6.418-+0.26 6.041_+0.26 6.683_+0.26 6.206_+0.18

9.501 ±3.1)8 7.230 -+ I].43 11).145 _+3.1)4 7.891 + 1.03

7.387+0.49* 6.751 _+0.36* 7.421 _+0.36* 7.589+ 1.41

8.333_+ 1.48 6.837_+0.24 7.412_+0.29 7.368 + 0AI

8.337+ 1.44 6.987_+0.425" 8.184_+ 1.465" 7.575_+ 1.08

7.437+0.30 6.751 _+0.38 7.391 _+0.29 7.439_+0.58

6.294 _+(I.34 6.370 _+0.42 5.858_+ 0.58 6.644 + 0.40 5.285+0.87 5.488 _+0.66

5.675_+0.73* 5.389_+0.83* 5.571 +0.46 6.239_+0.33* 4.073 _+ 1.27" 4.406_+ 1.02"

5.674_+0.84 5.859_+0.455. 5.345_+0.586 6.365+0.32 4.837-+ 1.15t 4.987-+0.895"

6.020+0.73 5.886_+0.81 5.571 +0.64 6.390_+0.48 4.631 _+ 1.425. 4.837+ 1.265"

5.869_+0.66 5.762_+0.74 5.656_+0.52 6.217_+ 1.49 4.643_+ 1.32]" 4.903_+ 1.011-

5.611 _+ 1.31 4.520 + 1.47

5.426_+0.62 4.262-+0.99

4.956_+ 1.19 3.568_+ 1.39

5.423_+0.73 3.677_+ 1.40

5.439_+0.52 4.267+0.80

I 1.580_+3.23 6.468 -+ 0.33 6.536 + 0.32 7.171 _4-(I. 19

7.145_+0.33' 6.153+0.23" 6.134-+0.14" 6.213+0.57"

7.395_+1.03 5.839-+0.575" 5.565+0.705. 6.162_+0.76

7.591_+1.07 5.909_+0.675" 5.771 -+0.845" 5.451+1.655"

7.136-+0.34 5.949_+0.311" 5.760_+0.39"}" 5.442_+1.331-

7.556 + 0.19 9.507-+ 1.91

7.378-+0.33 9.339+2.58

7.169-+0.35 8.124_+ 1.93

7.248+0.52 8.571 -+2.32

7.232+0.24 7.534+0.68

5.085+0.71 5.421 _+0.65

5.190_+0.68 5.358_+0.56

Dgm (L)

CA1 field, pyramidal CA2 field, pyramidal CA3 field, pyramidal CA4 field Dentate gyrus (upper) grim. layer Dentate gyrus (lower) tool. layer

Hypothalamus

ARC VMH

Arcuate nucleus Ventromedial

5.622 -+ 0.70 5.437-+080

5.316_+0.47 5.458+0.44

4.713_+0.885" 5.002_+0.73

Septum

lsd lsv MS

Lateral n. dorsal part Lateral n. ventral part Median n.

4.505 -+ 0.96 4.500 -+ 0.8 I 3.856_+ 1.82

2.812+1.23" 2.921_+1.72" 2.220_+ 1.63'

3.848_+1.59"t" 4.461_+0.915" 3.411+1.76 4.332_+1.41t- 4.318_+1.10t" 4.267-+0.98"i" 3.664_+ 1.175- 3.710-+ 1.445- 2.709_+ 1.36

Thalamus

LD MD VL

Laterodorsal Mediodorsal Ventrolateral

3.202 + 1.66 4.255 _+ 1.3 I 4.566 + 1.47

1.971 -+_1.21' 3.151+1.52 3.362_+1.26"

2.854+ 1.13 3.757_+1.29 4.118+1.16

2.957_+ 1.435- 2.830_+ 1.04 3.310_+1.98 3.794_+1.57 4.232_+1.31t" 4.502+0.751"

Ventral forebrain nuclei

HDB SI VDB VP

N. of horizontal limb diagonal band Substantia innominata N. of ventral limb diagonal band Ventral pallidum

5.075 _+0.97 4.999 _+0.89 4.521 _+ 1.24 4.869 4__0.86

4.388-+0.94* 3.769_+ 1.25" 3.924_+ 1.14" 3.688_+1.18"

4.748-+0.75 4.370_+ 1.32 4.341 +0.72 4.477_+1.04"i-

4.800-+0.58 4.697_+0.489 4.536_+0.815" 4.528_+0.74I" 4.593 + 1.03t 4.254_+0.84 4.373+0.735" 4.409_+0.691"

LOT TU

Lateral olfactory tract Olfactory tubercle

1.992 _+ 171 14.712 _+4.07

1.390±1.39 8.675+2.79*

2.211_+1.43 8.150_+1.30

1.655+1.28 2.389±1.38 + 12.4014-4.29I" 8.969-+2.38

Olfactory

Doses of JO 1784 are in parentheses as mg/kg i.p. *P < 0.01 versus control saline, f P < 0.01 versus TMT saline.

bens, t h ~ e a t e s t reduction occurred at the lower dose of 1 mgNg (17%) (Table 3 and Fig. 2).

hippocampus, hypothalamus and septum regions (Table 4 and Fig. 3).

Effects of JO 1784 alone on M : receptor density

L'[]ects q l J O 1784 in T M T treated rats on M i receptor density

A significant decrease in Mz receptor density was found in several brain regions relative to control values following JO 1784 administration. The regions showing the greatest decrease in M2 receptor density, were the amygdaloid nuclei, basal ganglia, cortex,

Following chronic administration of JO 1784 to TMT treated rats, both increases and decreases in M L receptor density were observed relative to those animals treated with TMT alone. A significant increase in M~ receptor density was found in the

Effects of JO 1784 on M~ and M2 receptors

563

Table 2, Autoradiography of M2 muscarinic receptor density in control and TMT rats, treated chronically with JO 1784. The results are expressed as mean pmol [3HIQNB specifically bound per mg of protein _+SD

Brain region

Abbreviation

Control saline

saline

TMT

Treatment groups TMT + TMT + JO 1784 (I) JO 1784 (4)

TMT + JO 1784 (16}

Amygdala "

BL BM CE LA ME

Basolateralamygdaloidnucleus Basomedial amygdaloid nucleus Central amygdaloid nucleus Lateral amygdaloid nucleus Medial amygdaloid nucleus

7.219_+0.20 6.159+0.83 5.539_+0.24 6.416_+0.52 5.776_+0.43

6.278-+0.86* 5.522_+0.87* 4.518_+0.98 5.413-+0.80 4.521-+0.85*

6.713_+0.44t" 5.588+_0.54 5.087-+0.4856.023+0.6855.125_0.585-

6.995_+0.2455.717_+0.69 5.008-+0.5256.263_+0.5255.275_+0.475-

6.239_+1.29 5.914-+0.83 4.882-+0.79 5.924-+1.08 4.821+__0.76

Basal ganglia

Acb CPu lcp ss

Accumbens Caudate putamen Lateral caudate putamen Striatal streak

7.815-+1.05 7.236_+0.29 8.551 -+ 1.67 7.596_+0.69

6,944___0,40* 6,078-+0.77' 7,003-+0.81" 6.575-+0.74

7.240___0.41 6.609_+0.6757.206_+0.34 6.987_+0.54~"

7.101-+0.37 6.181_+0.64 7.180+0.57 6.832-+0,22

7.107-+0.33 6.538_+0.69t" 7.399_+0.52 7.099_+0.305-

Cortex

Acg Fr PAM PAM1 PAM2 PAM3 PAS-P

Anterior cingulate gyrus Frontal cortex Frontoparietal motor Layers i-ii Layers iii-iv Layersv-vi Parietalmotor cortex posterior part Posterior cingulate gyrus

5.838_+0.78 5.842-+0.82 6.331_+0.65 6.757_+0.64 5.314_+0.95 5.909_+0.85

4.394_+1.01" 4.183_+1.13" 4.744_+1.14" 5.199_+1.10* 3.349+__1.30" 4.054_+1.25"

5.093_+0.7554.917_+0.58~f 5.026_+0.78 5.635_+0.8154.265_+0.8054.655+0.951"

4,391 +0.52 4.501-+0.66 5.379_+0.8855.778_+0.5553.806-+0.4854.744_+0.535-

5.107+0.6254.891-+0.74~5.348_+1.2055.691+_1.017 3.951_+1.161" 4.890_+0.91t"

6.434+_0,96 5.296-+1.17 5,280___0.59 3.631_+1.19

5.113+_0.80 5.860-+0.98 4.164+0.485- 3.829+ 1.06

5.354-+1.3514.237-+1.13t"

7.257_+0.18 6.405__+0.66 6.571 _+0.46 6.083 _+1.17

5.651_+0.60" 4.673_+0.17" 4.691_+0.82* 3.738___1.05*

6.200_+0.671" 5.316_+0,72~5,225_+0.7354,865 _+0.825-

6.268+0.1255.440__+1.42I" 5.581_+1.8153.698-+ 1.74

Dgm (L)

CAI field, pyramidal CA2 field, pyramidal CA3 field, pyramidal CA4 field Dentate gyrus (upper) gram layer Dentate gyrus (lower) mol. layer

6.662_+0.45 6.888 -+0.30

5.265+0.63* 5.396-+0.99*

5.531+0.53 5.376-+0.70 5.927 -+0.685- 5.862-+0.60

Hypothalamus

ARC VMH

Arcuate nucleus Ventromedial

5.812_+0.72 5.684_+0.36

3.682_+0.76* 4.496-+.0.415- 4.607_+0.535- 4.838-+0.92 4.237_+0.88* 5.044-+0.545- 5.202-+0.61~" 5.053+_0.911"

Septum

lsd lsv MS

Lateral n. dorsalpart Lateraln. ventral part Median n.

5.479_+ 1.14 5.182-+ 1.25 5.525-+ 1.40

4.382_+0.94* 4.992+_0.69t" 4.477_+0.59 4.587+_0.72 4.370-+1.33" 4.650_+0.59"1" 3.807_+0.59t 4.350_+0.64 4.487-+0.96* 5,279_+ 0,73~" 4,362_+0,855- 4,424_+1,24

Thalamus

LD MD VL

Laterodorsal Mediodorsal Ventrolateral

4.493-+0.73 5.356_+0.80 4.737_+0.75

3,271-+0.94 4.217_+0.94 3.274_+0.71

3.980-+0.62 4.642+0.63 4.241_+0.45

3.927-+0.88 3.910_+1.03 4.282_+0,61

3.724-+1.00 5.051_+1.00 4.441_+0.85

Ventral forebrain nuclei

HDB SI VDB VP

N. of horizontal limb diagonal band Substantia innominata N. of ventral limb diagonal band Ventral pallidum

5.602 -+0.53 4.746_+0.95 5.332_+1.00 4.961+_0.81

4.694+ 0.89 4.017_+1.29 4.573___0.82 3.124_+1.61

5.034_+0.66 4.434_+0.55 4.322_+1.09 4.062_+0.94 5.182_+0.96 4.363+_0.94 3.808-+1.07 3.613-+1.07

4.842_+0.49 4.246_+0.93 4.302-+0.89 4.490-+0.80

LOT TU

Lateral olfactory tract Olfactory tubercle

1.895_+ 1.02 9.474_+3.00

0.498_+0.75 6.693_+0,81

1.436_+0.86 1.030_+0,67 7.162+0,86 7,519-+0.76

1.001 _+0,87 8,894_+0.36

PCG Hippocampus CA1-P CA2-P CA3-P CA4 Dgg (U)

Olfactory

6.307_+0.54t" 5.480-+0.8055.177_+0.8153.982-+ 1.24

5.266±1,47 5,373_+1.60

Doses of JO 1784 are in parentheses as mg/kg i.p. *P < 0.01 versus control saline, t P < 0.01 versus TMT saline.

cortex, olfactory regions, septum, thalamus and basal forebrain nuclei. This effect was observed at all doses of JO 1784 but was not dose related. In the hippocampus a significant decrease in M~ receptor density was observed (Table 1 and Fig. 4). This reduction in M~ receptor density was small, 1-3%. Chronic JO 1784 administration prevented the TMT induced reduction of M, receptors in the septum, with the greatest protection observed at the lower doses of 1 and 4 mg/kg (65 and 67%) and moderate protection

(22%) at the higher dose of 16 mg/kg. In the substantia innominata and the ventral pallidum significant protection was observed at all doses of JO 1784 but the changes in receptor density were not dose-related. Effects o f JO 1784 in T M T treated rats on M2 receptor density In TMT treated rats, chronic JO 1784 treatment attenuated the receptor loss and produced a significant

564

B e r n a d e t t e Earley et al.

Table 3. Autoradiography of M, muscarinic receptor density in comrol rats, treated chronically with JO 1784. The results are expressed as mean pmol [3HJQNB specifically bound per mg of p r o t e i n i S D

Brain region

Abbreviation

Amygdala

BL BM (E LA ME

Basolateral amygdaloid nucleu~ Basomedial amygdaloid nucleus Central amygdaloid nucleus Lateral amygdaloid nucleus Medial amygdaloid nucleus

Basal ganglia

Acb CPu lop ss

Accumbens Caudate putamen Lateral caudata putamen Striatal streak

Cortex

Acg Fr PAM PAM1 PAM2 PAM3 PAS-P

Anterior cmgulate gyrus Frontal cortex Frontoparietal motor Layers i-i± Layers ill-iv Layers v-vi Parietal motor" cortex posterior part Posterior cingulate gyrus

PCG Hippocampus CAI-P CA2-P CA3-P CA4 Dgg (U)

Treatment groups Control Control JO 1784 (I) JO 1784 (4)

(ontrol + JO 1784 (16)

7323±0.25 6.583±0.38 6.258+0.32 6.980±0.23 6.337+-0.50

7.101 +0.28* 6.487±0,24 6.218 ±0.22 6.776_+0.25 6.437±0.37

7.086±0.17" 6.465±0.34 6.093-+0.59 6.539_+0.35* 6.411 ±0.41

7.259+-0.11 6.498-+0.42 6.108±0.33 6.788±0.29 6.203_+0.35

9.501 +_3.08 7.230_+i).43 10.145± 3.04 7891 _+ 1.03

7.871 +0.84* 7.145_+0.36" 7.927_+0,88* 7.543+0.28

7.804±0.78* 6.933+0.43* 8.292± 1.50" 7.445±0.29

8.553± 1.29 7,235±0.44 8,977± 1.80" 8.038±0.96

6294 ± 0.34 6.370±0.42 5.858±0.58 6.644+_0.40 5.285+_0.87 5.448±0.66

5.908 +_ 1.26" 6.007+_0.47* 5.822_+0.35 6.510+0.33 5.054± 1.03 5.134+_0.86

6.492 _+2.04 6.001 ±0.36" 5.731 +0.42 6.571 ±0.25 4.936_+0.91 5.348±0.57

6.256 ±0.50 6.367±0.33 6,125-+0.28" 6.809 ± 0.32 5.378±0.77 5.670_+0.60

5.611 +1.~1 4.520± 1.47

5.778 ± 0.65 3.881 ± 1.43

5.665±0.36 4.230±0.79

5.693+_0.42 4.815+_0.56

11.580!3.23 6.468±0.33 6.536±0.32 7.171±0.19

7.689± 1 . 0 1 6.121 +0.71" 6.008±0.72* 6.702±0.28

8.895± 1.98" 6.294±0.26* 6.301 +0.28" 6.933+0.19

10.174±2.37" 6.430±0.22* 6.526+-0.30 7.158_+.0.19

7.556~0.19 9.507± 1 . 9 1

7.275+0.24* 7.739±0.58

7.473 ±0.32 8.556-+ 1 . 7 1

7.564+0.17 9.517+ 1.81

Connol saline

l)gm (L)

(_AI tield, pyramidal CA2 field, pyramidal CA3 field, pyramidal CA4 field Dentate gyrus 1upper) Bran. layer Dentate gyrus (lower) tool. layer

Hypothalamus

AR(' VMH

Arcuale nucleus Ventromedial

5.622 ± 0.70 5.437±0.80

5.410±0.37" 5.423+0.55

5,540 + 0.41 5.579+0.61

5.290+0.45 5.363_+0.53

Septum

Isd Isv MS

Lateral n. dorsal part Lateral n. ventral part Median n.

4.505_+0.96 4.500-+0.81 3.856-+ 1.82

4.643±0.90 4.762±0.82 3.843± 1.16

4.693_+0.49 4.648+0.88 3.970±0.96

4.303+ 1.55 4.785± 1.19 3.981 + 1.31

Thalamus

LD MD VL

Laterodorsal Mediodorsal Ventrolateral

3.202 ± 1.66 4.255± 1 . 3 1 4.566± 1.47

3.304± 1.49 3.489±1.74 4.126± 1.77

3.386+- 1.53 4.117±0,79 4.582-+0.79

3.786-+0.61 4~831 ±0.34 4.691 +11.57

Ventral I%rebrain nuclei

HDB SI VDB VP

N. of horizontal limb diagonal band Substantia innominata N. of ventral limb diagonal band Ventral pall±alum

.~.075 ± 0.97 4.999±0.89 4.521 ± 1.24 4.869±0.86

4.818±0.53 4.775_+0.62 4.598_+0.75 4.704±0.58

4.823!0.39 4.724±0.48 4.710±0.75 4.748±0.37

5.1159+11.65 5.115 ± 0.64 4.489±0.93 5.006_+0.65

LOT TU

Lateral ollactory tract Olfactory tubercle

1.992~ 1 . 7 1 14.712±4.07

2.604+_ 1.63 I 1.048 ±3.75"

1.902 ± 1.77 14.193± 5.27

2.118 k 128 14.876 +-4.48

Ollactor}

Doses of JO 1784 are in parentheses as mg/kg i.p. *P < 0.01 versus control saline.

protection in Me receptor density in several brain regions, relative to TMT-treated rats. The most significant effects of JO 1784 were found in the amygdaloid nuclei (12-6%), the basal ganglia (8-5%), PAM 2 cortical layers (27-17%), hippocampus [(CA~, 9-10%), CA> 13-16%)] and CA~ (11-12%) and hypothalamus (22 31%) (Table 2 and Fig. 5).

DISCUSSION

In the present study, both subtypes of muscarinic receptor were decreased in TMT treated rats, but the extent of the decrease in muscarinic receptor density was region dependent. Results revealed a heterogeneous distribution of M~ and M2 receptors (Mash and Potter, 1986; Spencer et al., 1986). In a previous

Effects o f J O 1784 o n M~ a n d M2 r e c e p t o r s

565

Table 4. Autoradiography of M2 muscarinic receptor density in control rats treated chronically with JO 1784. The results are expressed as mean pmol [3H]QNB specifically bound per mg of protein_+ SD

Control saline

Treatment groups Control Control JO 1784 (1) JO 1784 (4)

Control + JO 1784 (16)

Brain region

Abbreviation

Amygdala

BL BM CE LA ME

Basolateral amygdaloid nucleus Basomedial amygdaloid nucleus Central amygdaloid nucleus Lateral amygdaloid nucleus Medial amygdaloid nucleus

7.219_+0.20 6.159_+0.83 5.539_+0.24 6.416_+0.52 5.776-+0.43

7.187_+0.36 6.174_+0.91 5.716_+0.86 6.649_+0.22 5.764-+0.78

7.048+0.31 6.047_+0.54 5.786_+0.82 6.283_+0.54 5.635_+0.62

7.314+0.25 6.159+0.85 5.753_+0.72 6.647+0.44 5.920+0.64

Basal ganglia

Acb CPu Icp ss

Accumbens Caudate putamen Lateral caudate putamen Striatal streak

7.815_+ 1.05 7.236_+ 0.29 8.551 _+ 1.67 7.596_+0.69

7.704_+ 0.63 7.293_+ 0.49 8.808 _+2.05 7.466_+0.15

7.488_+ 0.30 6.741 _+0.73 7.589 _+0.65* 7.121 _+0.33*

7.484_+ 0.18" 7.142_+ 0.25 7.677 _+0.45* 7.371 ±0.29

Cortex

Acg Fr PAM PAM 1 PAM2 PAM3 PAS-P

Anterior cingulate gyrus Fronta| cortex Frontoparietal motor Layers i-ii Layers iii-iv Layers v-vi Parietal motor cortex posterior part Posterior cingulate gyrus

5.838_+0.78 5.842_+0.82 6.331 _+0.65 6.757_+ 0.64 5.314_+0.95 5.909_+0.85

5.509_+0.68 5.530_+0.76 5.920_+0.81" 6.432_+ 0.70 4.595+0.80* 5.315_+0.71"

5.181_+0.80" 5.083_+1.10" 5.583 _+ 1.12" 6.083 + 1.03* 4.184+ 1.21" 5.063±1.21"

5.676±0.59 5.699_+0.73 6.421 +0.44 6.786 + 0.54 5.029_+0.73 5.724_+0.69

6.434_+0.96 5.280_+0.59

6.708_+0.51 4.665_+0.53*

5.926+ t.24" 4.521 +0.81"

6.897_+0.32 4.681 _+0.62

7.257_+0.18 6.405_+0.66 6.571 _+0.46 6.083_+1.17

7.184_+0.22 6.299_+0.52 6.509 _+0.58 6.223_+0.73

6.934_+0.57* 5.99l +0.99* 6.114 _+ 1.09* 6.162±0.76

7.006+0.39 6.174_+0.66 6.443 + 0.72 6.222+0.73

Dgm (L)

CAI field, pyramidal CA2 field, pyramidal CA3 field, pyramidal CA4 field Dentate gyrus (upper) gran. layer Dentate gyrus (lower) mol. layer

6.662 _+0.45 6.888_+0.30

6.248 _+0.59 6.671 _+0.56

6.081 _+0.67* 6.502+0.64

6.360 _+0.42 6.704+0.41

Hypothalamus

ARC VMH

Arcuate nucleus Ventromedial

5.812_+0.72 5.684_+0.36

5.518_+0.57" 5.601 _+0.50

4.964+0.55* 5.133 +0.73*

5.254+0.41" 5.383 +0.54

Septum

Isd Isv MS

Lateral n. dorsal part Lateral n. ventral part Median n.

5.479_+ 1.14 5.182+1.25 5.525_+ 1.40

5.437_+0.44 5.189_+0.50 5.336_+0.57

5.036_+0.79 4.728+0.65 5.161 +0.81

5.520_+0.81 5.108_+0.73 5.260_+0.66

Thalamus

LD MD VL

Laterodorsal Mediodorsal Ventrolateral

4.493 _+0.73 5.356_+0.80 4.737+0.75

4.787 _+0.46 5.337_+0.98 4.760+0.31

4.481 + I. 16 4.640_+0.72* 4.634_+0.94

4.984 + 0.67 5.013+0.68 4.507_+0.66

Ventral forebrain nuclei

HDB SI VDB VP

N. of horizontal limb diagonal band Substantia innominata N. of ventral limb diagonal band Ventral pallidum

5.602+0.53 4.746_+0.95 5.332_+ 1.00 4.961_+0.81

5.526_+0.44 5.088_+0.81 5.583-+0.63 4.546_+1.01

4.950_+0.91" 4.492+0.72 4.830_+0.58 3.815_+0.90"

5.317+0.23 4.755_+0.85 5.437_+0.56 4.130_+0.96"

LOT TU

Lateral olfactory tract Olfactory tubercle

1.895_+ 1.02 9.474 + 3.00

1.488 + 0.62 8.550 + 2.00

1.482 + 0.83 8.888 _+2.39

1.304 + 0.98* 8.658 + 2.27

PCG Hippocampus CAI-P CA2-P CA3-P CA4 Dgg (U)

Olfactory

Doses of JO 1784 are in parentheses as mg/kg i.p. *P < 0.01 versus control saline.

study (Earley et al., 1989) we reported a 60-70% depletion in total muscarinic receptor binding in TMT treated rats. This compares to a 25-30% reduction (MI/M2) in the present study suggesting that despite the large reductions in M, and M2 receptor density, the effects of TMT are not confined to the Mj and M2 receptor subtypes. When observing the effects of TMT on M~ and M 2 receptor subtypes it was necessary to sub-divide brain regions into cholinergic and noncholinergic regions. Among the cholinergic nuclei, a

large decrease was found in M, and M2 receptor density in the substantia innominata, medial septal nuclei, ventral pallidum and basal nucleus magnocellularis, diagonal band nuclei, nucleus of the horizontal and vertical limb of the diagonal band. A moderate decrease was found among cholinergic terminal areas in several of the cortical areas : these include the cingulate cortex (anterior and posterior), the 6 layers of the frontal parietal cortex, and somato-sensory cortex. Significant decreases in M, and M2 receptor density

(1)

(2)

(3}

Figs 1 3

leyendsopposite.

(4)

(5)

Figs 4 and 5. Fig. 1. Pseudocoloured images of M 1 and M2 receptor subtypes in control and TMT (8 mg/kg i.p.) treated rats at the level of the anterior hippocampus. The top left and top right sections are taken from control saline rats and represent the M ~and M2 receptor subtypes. Fig. 2. Pseudocoloured images of the M~ receptor subtype in control and JO 1784 treated rats at the level of the anterior hippocampus. Rats were injected with saline or JO 1784 (1, 4 and 16 mg/kg i.p.) for 40 days. Top left--control saline, top right--control and JO 1784 (1 mg/kg i.p.), bottom left--control and JO 1784 (4 mg/kg i.p.), bottom r i g h t ~ o n t r o l and JO 1784 (16 mg/kg i.p.). The colours represent the density of binding using the rainbow spectrum (from purple, low, to red, high). Fig. 3. Pseudocoloured images of the M2 receptor subtype in control and JO 1784 treated rats at the level of the anterior hippocampus. Rats were injected with saline or JO 1784 (1, 4 and 16 mg/kg i.p.) for 40 days. Top left Control saline, top r i g h t ~ o n t r o l and JO 1784 (1 mg/kg i.p.), bottom left--control and JO 1784 (4 mg/kg i.p.), bottom right---control and JO 1784 (16 mg/kg i.p.). The colours represent the density of binding using the rainbow spectrum (from purple, low, to red, high). Fig. 4. Pseudocoloured images of the M~ receptor subtype in TMT control and JO 1784 treated TMT rats at the level of the anterior hippocampus. Rats were injected with saline or JO 1784 (1, 4 and 16 mg/kg i.p.) for 40 days. TMT (8 mg/kg i.p.) was administered as a single injection 34 days prior to sacrifice. Top left--TMT control, top right--TMT and JO 1784 (1 mg/kg i.p.), bottom left--TMT and JO 1784 (4 mg/kg i.p.), bottom right--TMT and JO 1784 (16 mg/kg i.p.). The colours represent the density of binding using the rainbow spectrum (from purple, low, to red, high). Fig. 5. Pseudocoloured images of the M2 receptor subtype in TMT control and JO 1784 treated TMT rats at the level of the anterior hippocampus. Rats were injected with saline or JO 1784 (1, 4 and 16 mg/kg i.p.) for 40 days. TMT (8 mg/kg i.p.) was administered as a single injection 34 days prior to sacrifice. Top left--TMT saline, top right--TMT and JO 1784 (1 mg/kg i.p.), bottom left--TMT and JO 1784 (4 mg/kg i.p.), bottom right--TMT and JO 1784 (16 mg/kg i.p.). The colours represent the density of binding using the rainbow spectrum (from purple, low, to red, high).

568

Bernadette Earley et al.

were also found in the caudate putamen (CPu), its lateral ventral part (1CP) and striatal streak areas. In non-cholinergic neurons such as the bed nucleus of the stria terminalis, moderate decreases in receptor density were found. In the present study, chronic administration of the sigma ligand JO 1784 attenuated the TMT-induced decrease in the density of M~ and M2 receptors in most brain areas. However most of these changes were not dose related. JO 1784 alone induced a decrease in M~ and M2 receptor density in the amygdaloid cortex, basal ganglia, cortex and hippocampus. There is preliminary evidence to suggest that JO 1784 modulates acetylcholine (ACh) release in vitro (Roman and Junien, unpublished observations). It would seem reasonable to suggests that JO 1784 produces a down regulation in M~ and M2 receptor sub-types when administered to control animals. The ability of JO 1784 to attenuate the actions of TMT suggest that JO 1784 has "neuroprotective" effects. In control treated rats, chronic JO 1784 produced a decrease in post-synaptic M l receptor density in the amygdaloid cortex, basal ganglia, cortex and hippocampus and a decrease in M2 receptor density in the same regions. These results, together with the previously reported involvement of the N M D A receptor in TMT toxicity (Aschner and Aschner, 1992; O'Connell et al., 1994), suggest a possible explanation for the neuroprotective effects of JO 1784 in the TMT treated rat. From these studies it would appear that the regional expression of a particular muscarinic receptor subtype may be dependent upon the source of the cholinergic innervation and the degree to which TMT produces damage to the innervation of that particular region. The primary neuropathological effects of TMT appear in the hippocampal pyramidal cells in regions CA~, CA3 and CA4 (Chang, 1986; Bouldin et al.. 1981 ; Dyer et al., 1982, 1983 ; Earley et al., 1992). The functions of the various muscarinic receptor subtypes in the brain still remain to be fully established. However, Mash and Potter (1986) have suggested that M~ receptors may mediate excitatory events while M2 sites may induce inhibition by regulating release. Muscarinic regulation of ACh release is mediated by the M2 receptor subtype which has a pre-synaptic location on cholinergic terminals (Araujo et al., 1990). On the basis of molecular biological studies, 5 different muscarinic receptor subtypes have been identified (Bonner et al., 1987, 1988 ; Peralta et al., 1987). While a large number of M~ selective compounds have been identified, only 2 classes of M2 selective compounds have been found.

These include the polymethylene tetramines including methoctramine and mefurtamine and the tricyclics compounds including A F - D X 116, A F - D X 384 and A Q - R A 741 (Gitler et al., 1992). In this study, carbachol was used to occlude the M 2 receptor allowing the M~ receptor to be unmasked and pirenzepine was used to occlude the M~ site allowing the M2 site to be unmasked. In view of the cross-reactivity of carbachol and pirenzepine with other muscarinic receptor subtypes it would be of interest to examine the effects of more selective M, ([3H]pinenzepine) and M 2 receptor ligands ([3H]AFX-DX 384 or [3H]AF-DX 116) when examining the effects of TMT on the distribution of the M~ and M2 receptor subtypes. One possible explanation for the loss of M 2 receptors in TMT animals may be related to a selective loss of cholinergic neurons, resulting in an accompanying loss of post-synaptic M, receptors. If the cellular location of M2 receptors in the brain is largely confined to cholinergic neurons and their terminals (Raiteri et al., 1984), then it may be proposed that TMT mediates its effect by producing a selective destruction of pre-synaptic M2 autoreceptors. TMT is a potent centrally acting agent which is capable of producing a long-lasting amnesia in animals. Intoxication in man produces confusion, delirium, hallucinations and memory loss (Ross, 1981). Based on these observations and studies demonstrating memory deficits after the administration of TMT, it appears that central cholinergic pathways are important for memory and cognitive function (Earley et al., 1992). We have previously reported that phencyclidine (PCP) improves TMT-induced spatial navigation deficits and TMT-induced hyperactivity and proposed that the neuroprotective effect of PCP may be primarily due to blockade of the N M D A receptor thereby preventing glutamate mediated neurotoxicity (Earley et al., 1990). A potentiation by JO 1784 of NMDA-induced excitation of CA3 pyramidal neurons in the rat dorsal hippocampus has been reported in both in vivo (Debonnel et al., 1990; Monnet et al., 1990, 1992) and in vitro release experiments from hippocampal slices (Roman et al., 1989). Furthermore, Roman et al. (1991a,b) reported that, at high concentrations, JO 1784 reduced NMDA-evoked release of [3H]noradrenaline (NA). Monney et al. (1992) confirmed the bimodal effect of JO 1784 under similar experimental conditions. In view of the selective modulation of N M D A induced neuronal activation in the CA3 hippocampal region by sigma ligands, it is proposed that JO 1784 acts as a modulator of the N M D A receptor response in the hip-

Effects of JO 1784 on M~ and M2 receptors p o c a m p u s a n d thereby blocks the excitotoxic effects of T M T o n the cholinergic system. This possibility is further strengthened by the positive effects observed with J O 1784, ( + ) 3 - [ - h y d r o x y p h e n y l ] - N - ( 1 - p r o p y l ) piperidine h y d r o c h l o r i d e ( ( + ) - 3 P P P ) a n d d i ( o r t h o tolyl) guanidine ( D T G ) in reversing scopolamine induced amnesia in the rat a n d the p r o p o s a l t h a t sigma ligands m a y interact in a positive fashion with neural systems involved in m e m o r y processing (Earley et al., 1991). The ability of J O 1784, a selective sigma ligand, to a t t e n u a t e the loss in muscarinic receptor subtypes in T M T rats could be o f i m p o r t a n c e in the develo p m e n t of novel neuroprotective agents. Acknowledgement--The authors would like to thank Dr

Jean-Louis Junien, Institut de Recherche Jouveinal, 94265 Fresnes, Paris, France for financial support towards the cost of the study.

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