Brain Research, 177 (1979) 241-252 © Elsevier/North-Holland Biomedical Press

241

THE LOCALIZATION OF RECEPTOR BINDING SITES IN THE SUBSTANTIA NIGRA AND STRIATUM OF THE RAT

T. D. REISINE, J. I. N A G Y , K. B E A U M O N T , H. C. FIBIGER and H. I. YAMAMURA

Department of Pharmacology, College of Medicine, University of Arizona Health Sciences Center, Tucson, Ariz. 85724 (U.S.A.) and (J.I.N., H.C.F.) Division of Neurological Sciences, University of British Columbia, Vancouver, B.C. V6T 114/5 (Canada) (Accepted February 22nd, 1979)

SUMMARY

Neurotransmitter receptor binding of 5 ligands was examined in the striatum, substantia nigra (SN) and frontal cortex of rats which had received either unilateral 6hydroxydopamine (6-OHDA) lesions of the nigrostriatal pathway (NSP) or unilateral kainic acid lesions of the striatum. 6-OHDA lesions of the NSP significantly reduced [aH]dihydroalprenolol ([aH]DHA) and [3H]naloxone ([aH]Nal) binding by 31 ~ and 28 ~, respectively, in the denervated striatum compared to the contralateral side. Scatchard analysis revealed that the alteration in [aH]DHA binding was not due to a change in the affinity of the fl-adrenergic receptor for [aH]DHA. In marked contrast to these changes in the striatum, destruction of the NSP resulted in a significant increase in [3H]DHA and [aH]Nal binding by 44 7ooand 26 ~, respectively, in the frontal cortex of the lesioned compared to the control side. 6OHDA lesions in the NSP did not alter striatal receptor binding for [aH]quinuclidinyl benzilate ([aH]QNB), [8H]muscimol ([aH]Mus) or [3H]flunitrazepam ([SH]FIu). Similarly, intrastriatal kainic acid injections did not alter striatal receptor binding for [3H]Nal, [all]Flu or [SH]Mus. Of the various receptor densities measured in the SN after the above lesions the only alteration observed was a 43 70 increase in [all]Flu binding following 6-OHDA lesions of the NSP. Scatchard analysis indicated no change in the affinity of the benzodiazepine receptor for [SH]FIu. 6-OHDA lesions of the NSP did not alter [aH]QNB or [3H]Nal binding in the SN. Striatal kainic acid lesions did not alter nigral [SH]QNB or [all]Flu binding. The results are discussed in terms of neurotransmitter localization and plasticity within the striatum, SN and frontal cortex.

242 INTRODUCTION With the advent of receptor binding methodology it has become possible to quantitate the receptor densities of various neurotransmitters in the central nervous system. The basal ganglia, particularly the substantia nigra (SN) and striatum (caudate-putamen), lend themselves well to receptor measurements since these brain areas have been the subject of intense neuroanatomical and neurochemical investigations. From the anatomical studies, it is known that the SN projects dopamine (DA)containing efferents to the striatum t and to frontal cortical areas ~7. Reciprocally, the SN receives gamma-aminobutyric acid (GABA)-containing 7 and substance P-containing 7 afferents from the striatum. In addition there is evidence for the existence in the striatum of cholinergic 19 and GABAergic z9 neurons, as well as neurons containing enkephalins 21. In the striatum there is evidence that the release of DA from DA-containing terminals may be regulated through the action of acetylcholine on muscarinic or nicotinic receptors located on DA terminals 20. In addition, opiate receptors have been demonstrated to be located on DA-containing terminals in the striatumZ7, 38, and various studies have indicated that opiates may regulate the release of DA from these terminalslL GABA receptors have been demonstrated in the striatum. However, there are conflicting reports regarding the changes in these receptors after lesionsg,sL Attempts have also been made to characterize and localize the benzodiazepine (BDZ) binding sites in the CNS. The density and affinity of these binding sites have been shown to decrease in H D striatum 41 where there is marked neuronal loss. However, it has been suggested that the BDZ receptor is localized to glia ~2. Recently, a direct effect of BDZ has been shown on DA synthesis in DA terminals in the striatum 5 suggesting the presence of BDZ binding sites on these structures. In the present report an attempt was made to further clarify the nature of the various neurochemical interactions of the above systems. To this end we have lesioned the nigrostriatal DA-containing pathway (NSP) with 6-hydroxydopamine (6-OHDA) which has been shown to selectively destroy catecholamine containing neurons 47. In addition, we have lesioned the striatum with kainic acid which is believed to destroy neuronal cell bodies near the site of injection while sparing adjacent axons or terminals14,30. Following these lesions the densities of fl-adrenergic, opiate, muscarinic cholinergic, benzodiazepine and GABAergic receptors in the striatum, SN and frontal cortex were measured. METHODS A total of 65 male Wistar rats weighing about 300 g were used. All surgery was conducted in a stereotaxic apparatus while the animals were under Nembutal anesthesia. Unilateral 6-OHDA lesions of the NSP were achieved by injections of 2.0 #1 of a physiological saline solution containing 4 #g/2/zl 6-OHDA and 0.3 mg/ml ascorbic acid at the co-ordinates AP + 4.4, ML + 1.8 and DV --2.5, according to the atlas of Kfnig and Klippel z4. Animals received desipramine hydrochloride (25 mg/kg) intraperitoneally, 30 min before 6-OHDA injections to prevent damage to noradrener-

243 gic neurons. Unilateral kainic acid lesions consisted of injecting 10 nmol kainic acid (Sigma) in 1.0 #1 of buffered saline into the striatum at the co-ordinates AP + 8.4, ML + 2.8, and DV + 4.5. One month after surgery the animals were killed by cervical fracture and the brains removed. The SN was dissected from coronal sections obtained on a freezing microtome while the striatum and frontal cortex were dissected freehand. The brain tissue was immediately frozen (--60 °C) until the time of assay. For the determination of enzyme activities, tissues were homogenized in 20-30 vols of 50 mM Tris-acetate buffer (pH 6.4) containing 0.2 ~ Triton X-100. Glutamic acid decarboxylase (GAD) 11 and tyrosine hydroxylase (TH) 2s activities were assayed as previously described except that saturating concentrations of both cofactor and substrate were employed. Choline acetyltransferase (CAT) activity was assayed according to the method of Fonnum is. The ligands employed to measure the various receptor levels were as follows: cholinergic muscarinic receptor, [3H]QNB; opiate receptor, [ZH]Nal; fl-adrenergic receptor, [3H]DHA; benzodiazepine receptor, [SH]Flu; g a m m a - a m i n o b u t y r i c acid ( G A B A ) receptor, [3H]Mus. The interaction of these ligands with their respective receptors has been previously characterized a n d the conditions of the assays used in the present study have been described elsewherea,a,aa,42,46, 50. Some of the above receptor levels were n o t measured in the b r a i n areas u n d e r investigation due to limited supplies of tissue. Statistics were performed by using the Students t-test. Protein was determined by the m e t h o d of Lowry et al.~L TABLE I The effect o f 6-OHDA lesions o f the N S P and kainic acid lesions o f the striatum on TH, GAD and C A T activities in the striatum and S N

Values represent the mean ± S.E.M. of 7 or 8 determinations of the lesioned and contralateral control side. ND, not detectable. Lesion

6-OHDA lesion of the NSP

Kainic acid lesion of the striatum

* P < 0.001. ** P < 0.05. *** P < 0.01.

Area measured

Striatum control lesioned SN control lesioned Striatum control lesioned SN control lesioned

Enzyme activities (nmol/mg prot./h) TH

GAD

CAT

5.03 :~ 0.19 ND

242 £ 12 288 i 14"**

65.6 ± 3.0 69.5 ± 2.5

3.05 i 0.25 0.26 ± 0.09***

930 ± 43 912 4- 62

5.38 -¢- 0.54 4.37 + 0.30**

242 4- 9.1 107 ~ 9.9*

2.86 ± 0.50 3.41 ~- 0.93

862 ~ 58 501 ~ 23*

76.1 ± 4.4 23.0 ± 2.9*

w

244 TABLE II Brain neurotransmitter receptor densities after 6-OHDA lesions of the nigrostriatal path way Values are the means d- S.E.M. of 5 animals. Final concentration of aH-ligands used was 0.1 nM, [3H]QNB; 0.25 nM, [aH]DHA; and 5 nM, [aH]Nal. Tissue concentrations of 50, 250 and 300/~g protein per assay tube were used for the QNB, DHA and Nal binding assays, respectively. Area studied

Striatum control lesion SN control lesion Frontal cortex control lesion

Receptor binding (fmol/mg protein) f3H]QNB % control [3H/DHA

% control

/3H TNal

% control

554 ± 11 528 ± 9

95

69*

192 ± 11 138 ± 11

72*

147 ± 8 162 zk 6

110

576 ± 17 560 ± 14

97

35 ± 2 24 ± 2

m

25 i 3.6 35 4- 0.8

144"

85±7 81 ± II

95

119 ~ 10 150 z[__ 10

126"

* P < 0.05. RESULTS The effects of 6 - O H D A lesions of the N S P a n d kainic acid lesions of the striatum on tyrosine hydroxylase (TH), glutamic acid decarboxylase ( G A D ) , a n d choline acetyltransferase (CAT) activities in the striatum a n d SN, shown in Table I, are similar to those reported previously 40. 6 - H y d r o x y d o p a m i n e lesions of the N S P resulted in over a 90 ~ decrease in b o t h striatal a n d nigral T H activity, a significant increase of 19 ~ in striatal G A D activity a n d no change in nigral G A D or striatal C A T activity. Striatal kainic acid lesions resulted i n a significant decrease of 19 ~ in striatal T H activity while nigral T H activity remained unaltered. These lesions decreased striatal

3. I00

E ~

50

al

.05

BIF

(frn°i

• mgprotein

.I x ~--,-I pM"

Fig. 1. Scatchard plot of [aH]dihydroalprenolol binding to unlesioned contralateral (@) and ipsilatera~ NSP lesioned ( . ) corpus striatum. The apparent KD values (from the slope of the line) are 0.4 nM for the unlesioned and 0.35 nM for lesioned striatum. Maximum specific binding (from y-intercept) was 75 fmol/mg protein for unlesioned and 52 fmol/mg protein for the lesioned striatum.

245 and nigral G A D activity by 56 70 and 42 70 respectively. Striatal CAT activity was reduced by 70 % on the lesioned compared to the contralateral side. Interestingly, however, CAT activity contralateral to the kainic acid lesioned striatum increased significantly by about 18 70 when compared to the activity in saline injected striatum (data not shown). There was no alteration in any other enzymes on the side contralateral to the lesions relative to saline injected controls. The receptor densities for [aH]QNB, [3H]DHA and [3H]Nal in the striatum, SN and frontal cortex after 6 - O H D A lesions of the NSP are shown in Table II. The binding of [SH]QNB was not altered in any of these brain areas, nor was the binding of [3H]Nal altered in the SN. The binding of [SH]Nal was significantly decreased in the striatum by 28 % and increased in the frontal cortex by 26 70. Similarly, the binding of [aH]DHA was significantly decreased by 31 70 in the striatum and increased by 44 700in the frontal cortex. Scatchard analysis (Fig. 1) of saturation isotherms of [aH]DHA binding in tissue homogenates from control and NSP lesioned striatum revealed no change in the affinity of [3H]DHA for its receptor. The receptor binding of [aH]Mus and [3H]Flu in the striatum and SN after NSP lesions is shown in Table III. Also shown is the binding of these ligands in striatal and nigral tissue obtained from animals which had received unilateral saline injections into the NSP. There was no difference between ipsilateral and contralateral binding of [aH]Mus and [3H]FIu in the striatum of animals which had received unilateral saline NSP injections. N o r was there any difference in these receptor levels in striatal or nigral tissues derived from saline injected animals compared to the receptor levels in the striatum contralateral to the 6 - O H D A NSP lesioned side. NSP lesions did not alter TABLE III Neurotransmitter receptor densities after 6-OHDA of nigrostriatal pathway

Receptor binding of [SH]muscimoland [3H]flunitrazepamwas measured in the ipsilateral and contralateral striatum and SN after unilateral saline injections into the NSP, and in the ipsilateral and eontralateral striatum and SN after unilateral 6-OHDA lesions of the NSP. Values are the means 4- S.E.M. of 5 animals. Final concentration of [3H]Musand laH]Flu was 0.25 and 5 nM, respectively. Tissue concentrations of 250 and 50/~g protein per assay tube were used in the Mus and Flu binding assays, respectively. Area studied

Treatment

Side

Receptor binding (fmol/mg protein) [SH]Mus

Striatum

SN

* P < 0.05.

Saline

contralateral ipsilateral 6-OHDA lesions contralateral ipsilateral Saline contralateral ipsilateral 6-OHDAlesions contralateral ipsilateral

247 203 230 210 -----

4± 44-

26 8 19 10

%

82 91

[all]Flu

47 45 48 43 72 76 70 100

44444444-

2 3 2 2 3 7 4 2

%

96 90 105 143"

246 35C

25O W

E

150

50

.I

B/F

.2

( fmol x l ~ ~rngprotein pM/

Fig. 2. Scatchard plot o f [ZH]flunitrazepam binding to unlesioned contralateral (O) a n d ipsilateral N S P lesioned (Ill) s u b s t a n t i a nigra. The a p p a r e n t KD (from the slope o f the line) are 1.1 n M for u n lesioned a n d 0.9 n M for lesioned substantia nigra. M a x i m u m specific binding (from the y-intercept) was 432 f m o l / m g protein for unlesioned a n d 578 f m o l / m g protein for the lesioned substantia nigra.

[ZH]Mus or [3H]FIu binding in the striatum. These lesions, however, significantly increased [ZH]Flu binding in the SN by 43 ~. Scatchard analysis (Fig. 2) of saturation isotherms of [3H]FIu binding of control and NSP lesioned tissue showed that the increase was not due to a change in the affinity of [3H]Flu for its receptor. The effect of kainic acid lesions of the striatum on various receptor levels in the striatum and SN is shown in Table IV. In the striatum these lesions did not alter [ZH]Nal, [ZH]Mus or [all]Flu binding. In the SN, striatal kainic acid injections had no significant effect on [3H]QNB or [3H]FIu binding.

T A B L E IV

Neurotransmitter receptor densities in the striatum and S N after injection o f kainic acid into the striatum Values are the m e a n s ± S.E.M. of 5 determinations.

Ligand

Receptor binding (fmoles/mg protein) Striatum

[ZH]QNB [ZH]Nal [aH]Mus [all]Flu

SN

Control

Lesioned

%

Control

Lesioned

%

-185 ± 10 184 ± 20 44 ± 5

-194 ± 18 249 ± 31 41 ± 3

169 z~ 6 --68 ± 7

198 ± 7 --76 ± 8

112

105 130 93

112

247 DISCUSSION The measurement of neurotransmitter receptor levels is a valuable addition to the arsenal of techniques available to the investigator of brain function since it is an excellent complement to neuroanatomical and neurochemical studies. Thus, by producing appropriate lesions, receptors of specific neurotransmitters can be localized to distinct neuron types and to neuronal elements. These studies aid in the determination of (1) connections at the synaptic level, (2) the direction of transmitter release and (3) the potential site of action of drugs. Caution, however, should be emphasized. It is becoming apparent that the CNS is a highly adaptive structure and therefore certain lesions may produce changes secondary to the initial lesion such as diaschisis, transneuronal degeneration, supersensitivity or axon sprouting44. These phenomena may lead to misinterpretation of results in receptor localization studies particularly since they occur over widely varying periods of time. The present results should be viewed with this in mind.

Muscarinic cholinergic receptor Pharmacological studies have suggested that acetylcholine enhances the release of striatal DA through actions on presynaptic muscarinic and nicotinic receptors located on striatal DA terminals2°. Recently, it has been demonstrated that 40 days after 6-OHDA lesions of the NSP, striatal [all]atropine binding was unaltered23. Our results with [aH]QNB binding confirm these reports that there are no measurable presynaptic muscarinic cholinergic receptors, as detected by [3H]QNB binding, on striatal DA terminals. Thus, acetylcholine probably exerts its effects on striatal DA release either through nicotinic cholinergic presynaptic receptors on DA terminals or by an indirect mechanism. A point of controversy for some time has been the significance of the presence of acetylcholinesterase (ACHE) on non-cholinergic neurons such as the DA-containing cells of the SN26,a~,a3. It has been suggested by some that the presence of AChE in neurons indicates a cholinergic input and is thus a marker for cholinoception. The present observations that DA neuron loss in the SN does not alter nigral [aH]QNB receptor binding provide evidence that these dopaminergic cells do not receive cholinergic (muscarinic) afferents. The findings that striatal kainic acid lesion did not alter [aH]QNB binding in the SN further suggests that striatonigral terminals do not contain [aH]QNB binding sites. It cannot be excluded, however, that these neuronal elements in the nigra may contain cholinergic receptors of the nicotinic type.

Opiate receptor Several studies have suggested that opiates interact with striatal DA-containing terminals 15. Recently, a 30-40 ~o reduction in striatal opiate receptor density 6-22 days after either intranigral or intraventricular injections of 6-OHDA has been demonstratedlO,a6,sa,4L It has also been found that there is a 30-45 ~o reduction in striatal 3Hopiate binding 3-5 days after striatal kainic acid lesions10,18,aa,4L The present study is in agreement with the decrease in opiate receptor levels in the striatum after the

248 degeneration of DA terminals. However, after kainic acid lesions of the striatum we found no significant alteration in [3H]Nal binding in this structure. Several workers have interpreted the results of their lesioning studies as indicating the presence of at least two populations of opiate receptors in the striatum, one population presynaptic on DA terminals and another on striatal perikarya or dendrites10,38, 45. Childers et al. lz have also suggested that a third poPUlation of striatal opiate receptors exists on the terminals of corticostriatal neurons. The presence of opiate receptors on axon terminals in other neuronal systems such as the sensory afferents to the dorsal horn of the spinal cord e5 and on trigeminal afferents to the brain stem 2z provide a precedent for the suggestion of the presence of opiate receptors on DA terminals in the striatum. However, the absence in the present study of a decrease in striatal [ZH]Nal binding 30 days following kainic acid lesions of the striatum could be the result of various transynaptic processes that have resulted from the destruction of striatal cell bodies. If a substantial amount of striatal opiate receptors are located on either DA or corticostriatal terminals, then sprouting of these neurons after degeneration of striatal perkarya could increase striatal opiate receptor densities to control levels. Thus, in the present study, the depletion of striatal opiate receptors, as previously reportedX3,38,45, following intrastriatal kainic acid lesion may have been masked by the sprouting of opiate receptor containing striatal nerve terminals. Interestingly, Pollard et al. 3s reported a 33 ~ decrease in [3H]Nal binding in the SN 10-15 days after lesion of the NSP with 6-OHDA. Recently, however, Pert et al. 34 have demonstrated by electrophysiological studies that there is no direct interaction of opiates with nigral DA cell bodies. Our results reveal that opiate receptors are not located on nigral DA cell bodies but may be on GABAergic or substance P afferents, from the striatum (work in progress). The results of Pollard et al. as may have been due to non-specific damage in the SN by intranigral injections of 6-OHDA. The present method of eliminating DA cell bodies in the SN by intra-NSP injections of 6-OHDA avoids this problem. Recently, a reduction has been reported in [3H]naloxone binding in the nucleus accumbens and septum after DA deafferentation of these mesolimbic areas 36. Interestingly, we obtained an increase in [3H]Nal binding in the frontal cortex after 6O H D A lesions of the NSP. Nigral DA neurons have been shown to project to frontal cortical regions and these fibers follow, in part, the course of the NSP (ref. 17). Therefore, the present 6-OHDA lesions of the NSP most likely interrupt some of the nigral DA projection to the frontal cortex. However, in the absence of knowledge of the degree of effect of those lesions on frontal cortical DA terminals (work currently in progress) it is difficult to interpret the increase in [3H]Nal binding in this area.

fl Adrenergic receptor In spite of the low levels of noradrenaline (NA) (ref. 48) and dopamine-fl-hydroxylase (D/3H) (ref. 43) in the striatum, this structure appears to receive a significant amount of noradrenergic afferents from the locus coeruleus 31. The presence of [3H]DHA receptors in this structure supports this contention. In fact, of the brain areas measured, the striatum is among the areas containing the highest densities of

249 [aH]DHA binding a. The present lesions of the NSP with 6-OHDA do not affect the noradrenergic fiber systems as all animals receiving these lesions were pretreated with desipramine, a drug which has been shown to prevent the uptake of 6-OHDA into noradrenergic axons and terminals 16. The noradrenergic system in the striatum and frontal cortex, however, may have undergone secondary changes leading to the decrease and increase in [3H]DHA binding in the striatum and frontal cortex, respectively. Alternatively, in the striatum at least, [3H]DHA receptors may be present on DA terminals. The exact changes in NA levels after 6-OHDA NSP lesions and the nature of the changes in [SH]DHA binding in the striatum and frontal cortex are currently being investigated.

GABA receptor It has been suggested that GABA interacts with DA terminals in the striatum 2. In a previous study, we demonstrated that in Parkinson's disease, in which there is a large loss of nigrostriatal DA neurons, there is no alteration in striatal [aH]GABA binding ag. Thirty days after lesions of the NSP with 6-OHDA we found no alteration of striatal [aH]Mus binding. This confirms our previous suggestion that there are no measurable presynaptic GABA receptors on striatal DA terminals ag. Thirty days after intrastriatal injection of KA, there was no alteration in striatal [3H]Mus binding. These results alone would suggest that GABA receptors are not located on striatal cell bodies. However, Campochiaro et al. have reported that 10 days after intrastriatal KA lesions there was an increase in striatal [aH]GABA binding 9. It has also been found that 9 months after intrastriatal KA lesions there is an 80 ~ decrease in aH-GABA binding in the striatum 51. Thus, it appears that striatal GABA receptor alterations induced by intrastriatal KA lesions are dependent on the length of time after the KA lesion. Alternatively, if the striatum contralateral to the lesion is used as a control any effect of the lesion may tend to be obscured by changes in the control side. However (compared with saline injected striatum) no changes in striatal [aH]Mus binding in striatum contralateral to kainic acid injected side were observed (Table III).

Benzodiazepine receptor Recently, it has been suggested that there is an intimate relationship between GABA and the pharmacological actions of benzodiazepines 49. It is possible that this interaction results from the close association of benzodiazepine and GABA receptors. In the present study, it was demonstrated that 30 days after either intrastriatal injection of KA or destruction of the NSP with 6-OHDA neither GABA nor benzodiazepine receptor density in striatum was altered. However, there were alterations in the other neurotransmitter receptors such as muscarinic cholinergic, opiate, fl-adrenergic and dopaminergic 4o receptors after either striatal KA lesions or nigral 6-OHDA lesions. We 41 have previously demonstrated a 35~o decrease in benzodiazepine receptor density in the striatum of Huntington's diseased (HD) brains, in which there are extensive losses of striatal neurons. In view of the other biochemical similarities between H D and striatal KA lesions, long survival times (greater than 30

250 days) m a y p r o d u c e decreases in striatal [3H]FIu b i n d i n g paralleling the findings in H D brains. Such a finding would s u p p o r t the previously p r o p o s e d m o d e l 49 o f an interaction between G A B A a n d b e n z o d i a z e p i n e receptors, p e r h a p s located o n the same striatal neurons, since the G A B A r e c e p t o r density in K A injected s t r i a t u m has a l r e a d y been shown to decrease 9 m o n t h s after i n i t i a t i o n o f the lesion 51. Braestrup et al. 7 have shown t h a t [aH]diazepam b i n d i n g decreases in the SN after i n t r a n i g r a l kainic acid injections suggesting t h a t [aH]diazepam receptors are located on neurons. In the present study, the increase in [ZH]Flu b i n d i n g t h a t was o b t a i n e d in the S N after 6 - O H D A lesions o f the N S P (Table II]) suggests t h a t these receptors are n o t located o n nigral D A - c o n t a i n i n g neurons. N o r are these b i n d i n g sites located on striatonigral terminals since no c h a n g e was f o u n d in nigral [ZH]Flu b i n d i n g after striatal kainic acid lesions. These results are n o t due to artifacts o f using tissue controls c o n t r a l a t e r a l to the lesion because [3H]Flu b i n d i n g in the s t r i a t u m a n d S N on the side c o n t r a l a t e r a l to the lesions was u n c h a n g e d c o m p a r e d to saline injected controls.

ACKNOWLEDGEMENTS S u p p o r t e d by grants f r o m the M e d i c a l R e s e a r c h C o u n c i l a n d H u n t i n g t o n ' s C h o r e a F o u n d a t i o n to H . C . F . ; U.S.P.H.S. G r a n t s , H u n t i n g t o n ' s C h o r e a F o u n d a t i o n a n d H e r e d i t a r y Disease F o u n d a t i o n grants to H.I.Y. W e wish to t h a n k S. A t m a d j a a n d A. Chen for their excellent technical assistance a n d C. K o u s e n for the t y p i n g o f this manuscript. J. I. N a g y is a fellow o f the H u n t i n g t o n ' s Society for C a n a d a . H.I.Y. is a recipient o f a Research Scientist D e v e l o p m e n t A w a r d ( R S D A ) f r o m the N a t i o n a l Institute o f M e n t a l H e a l t h (MH-00095).

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