Adrenergic receptor and catecholamine distribution in rat cerebral cortex: binding studies with [3H]prazosin, [3H]idazoxan and [3H]dihydroalprenolol

Brain Research, 402 (1987) 403-408

41)3

Elsevier BRE 22016

Adrenergic receptor and catecholamine distribution in rat cerebral cortex: binding studies with [3H]prazosin, [3H]idazoxan and [3H]dihydroalprenolol Laurent Diop, Richard Bri6re, Louise Grondin and Tomfis A. Reader Centre de Recherche en Sciences Neurologiques, Ddpartement de Physiologie, Facultd de Mddecine, Universitd de Montrdal, Montrdal, QuO. (Canada)

(Accepted 23 September 1986) Key words: al-Adrenoceptor; a2-Adrenoceptor; fl-Adrenoceptor; Distribution; [3H]Prazosin; [3H]Idazoxan:

[3H]Dihydroalprenolol; Catecholamine

The tritiated adrenergic antagonists [3H]dihydroalprenolol ([3H]DHA; fl-receptors), [3H]prazosin ([~H]PRZ; cq-rcceptors), and [3HJidazoxan ([3H]IDA; a2-receptors ) were used to determine the distribution of these sites in 5 defined areas of the adult rat cerebral cortex. The highest density of [3H]PRZ binding was found in the prefrontal cortex, with a lower and homogeneous distribution for the frontal, parietal, occipital and temporal areas. The [3H]IDA binding sites were fairly uniform for all areas, except for the temporal cortex where it was very dense. In contrast, fl-adrenoceptors labelled by [3H]DHA were very homogeneous for all the regions cxamined. The functional significance of the distribution of al, a 2 and fl-adrenoceptors is discussed in relation to the catecholamine innervation and monoamine contents measured by high performance liquid chromatography.

The aim of the present study was to determine biochemically the distribution (receptor number or density) and apparent affinities (dissociation constants) of adrenergic receptors using 3 noradrenergic antagonists, i.e. [3H]prazosin ([3H]PRZ) for the al-adrenoceptors 6'11'j7, [3H]idazoxan ([3H]IDA, RX781094) for the a2-adrenoceptors 15'is and [3H]dihydroalprenolol ([3H]DHA) for total fl-adrenergic 1"2 binding sites, in 5 well-defined areas of the rat cerebral cortex. In order to relate the biochemical mapping of receptor sites to the distribution of the endogenous catecholamine (CA) levels, the monoamine contents in the different cortical regions were determined by high performance liquid chromatography (HPLC) coupled to electrochemical detection. The 5 cortical areas in this study represent distinct functional regions, of relative ease of dissection and for which the tissular C A contents have been documented in previous biochemical studies 7,14.16,24.

Adult male S p r a g u e - D a w l e y rats (250-300 g) were decapitated and their brains quickly removed and placed on crushed ice. The neocortex was dissected under a binocular stereomicroscope as previously described 7'16, with a plastic chamber ~ to perform the coronal sections. Briefly, samples of prefrontal cortex (PF) included the most rostral part of the cerebral cortex, from anterior (A) 10,000 ~tm as the posterior limit to the poles of the hemispheres s. The frontal cortex (FR) was obtained between A 10,000 u m and A 7000 # m and included all the grey matter dorsal to the horizontal plane 0.0. The parietal (PAl area (A 7000#m to A 4000]Lm), was the dorsolateral cortex above horizontal (H) 0.0, limited internally by the corpus callosum. The occipital cortex (OCC) was obtained from A 4000g~m to posterior (P) 500~tm. The temporal cortex (TE, or region R in ref. 7) was dissected from the slices used for the PA and OCC areas, and comprised tissue (entorhinal and py-

Correspondence: T.A. Reader, Centre de Recherche en Sciences Neurologiques, Departement de Physiologie. Facult6 de M6dc-

cine, Universit~ de Montreal. Q@. H3C 3J7, Canada. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

404 r i f o r m cortices} b e t w e e n A 5 0 0 0 # m to P 5 0 0 ~ m : lat-

at 25 °C fl)r 45 m i n . F o r t h e i # u d r c n o c e p t o r

eral ( L ) 2 0 0 0 / ~ m to 6000 u m a n d f r o m H

t h e m e m b r a n e s w e r e h o m o g e n i z e d )n Tris-! tCI buf-

2600/~m to

-4500/ml.

fer (5 m M al p H 7.4) c o n t a i n i n g 250 m M s u c r o s e ,

F o r m o n o a m i n e assays, t h e s a m p l e s w e r e h o m o g e n i z e d in 0.1 Na,EGTA.

assays

w a s h e d twice in this b u f f e r , a n d finally" r e s u s p e n d e d

N cold H C I O a c o n t a i n i n g 4 m M o f

in T r i s - H C I 511 m M . p H 7.4. T h e s e m e m b r a n e p r e p a -

c e n t r i f u g e d ( 2 0 , 0 0 0 g f o r 45 m i n at 4 °C)

r a t i o n s w e r e a d d e d in 1511ul aliquot.~ to t u b e s a l r e a d y

a n d t h e s u p e r n a t a n t s u s e d f o r assays by h i g h p e r -

c o n t a i n i n g 75 ul o f c o l d b u f f e r , with o r w i t h o u t 10 u M

f o r m a n c e liquid c h r o m a t o g r a p h y

w i t h ion-

p r o p r a n o l o l for n o n - s p e c i f i c b i n d i n g . T h e ~,aturanon

p a i r i n g a n d e l e c t r o c h e m i c a l d e t e c t i o n , f o l l o w i n g es-

c u r v e s w e r e p e r f o r m e d at e q u i l i b r i u m w i t h lit dilu-

t a b l i s h e d p r o c e d u r e s m')9. T h e p e l l e t s w e r e d i s s o l v e d

t i o n s o f [ 3 H ] D H A (82 C i / m m o l _ A m e r s h a ) n : 0.2 --l(l

o v e r n i g h t in 1 N N a O H

(HPLC)

for p r o t e i n a s s a y u, u s i n g b o -

n M ) , t h e l i g a n d a d d e d in 75 ul a l i q u o t s (final w)ls. -;011 u l ) , a n d t h e t u b e s i n c u b a t e d at 25 :C" t()) 30 thin. A t

v i n e s e r u m a l b u m i n as s t a n d a r d . F o r b i n d i n g assays o f c ~ - a d r e n o c e p t o r s t h e c o r t i c a l

the end of the incubations, the mixtures were diluted

s a m p l e s w e r e h o m o g e n i z e d in 40 vols. (w/v) o f c o l d

in 2 ml of cold b u f f e r f o l l o w e d by r a p i d f i l t r a t i o n ( < 5

s o d i u m / p o t a s s i u m p h o s p h a t e Is')s b u f f e r (50 m M . p H

s) u n d e r v a c u u m t h r o u g h W h a t m ~ m G F / C glasb f i b r e

7.4) u s i n g a P o l y t r o n 115 st. T h e s e h o m o g e n a t e s

filters, a n d t h e filters w a s h e d twu'e ( 5 ) n i l with cold

w e r e c e n t r i f u g e d 149,0(/0 g, 15 rain at 4 ° C L t h e pel-

b u f f e r . R a d i o a c t i v i t y was c o u n t e d by h q u i d scintilta-

lets w a s h e d t w i c e a n d a f t e r a final c e n t r i f u g a t i o n , re-

(ion ( E c o n o f l u o r . N e w E n g l a n d N u c l e a r ) i n Rackbetta

11 c o u n t e r

s u s p e n d e d in t h e s a m e c o l d buffer. T h e s e m e m b r a n e

1215

p r e p a r a t i o n s w e r e a d d e d in 60 ltl a l i q u o t s to t u b e s al-

Non-specific

r e a d y c o n t a i n i n g 15 u l o f cold b u f f e r , w i t h o r w i t h o u t

b i n d i n g ( T B ) for [ 3 H ] P R Z . < 2 5 % of T B for [ s H ] t D A

10 I(M p h e n t o l a m i n e

for non-specific binding. The

a n d <21F~, of T B for [ 3 H ] D H A . P r o t e i n c o n c e n t r a -

s a t u r a t i o n e x p e r i m e n t s w e r e p e r f o r m e d at e q u i l i b r i -

t i o n s w e r e a s s a y e d " in I00 tt[ aliqu()ts of m e m b r a n e

u m w i t h 111-12 d i l u t i o n s of e i t h e r [ 3 H ] P R Z ( 19.8 o r

p r e p a r a t i o n s , a n d w e r e usuall~ b e t w e e n 1 5 a n d ?',.~

binding

(etficienc~

a LKB

represented

<20%

55-~151;-) ()f t o t a l

22.6 C i / m m o l . N e w E n g l a n d N u c l e a r 0 . 0 0 5 - 4 n M fi-

m g ml. T h e a n a l y s e s o f t h e b i n d i n g e x p e r i m e n t s w e r e

nal

p e r f o r m e d u s i n g S C A T . i.e. ~t Sc~ktchard p l o t 2'. b u t

concentration)

or

[3H]IDA

156.1

Ci/mmol

A m e r s h a m : 0 . 0 5 - 4 0 n M ) , t h e t i g a n d s a d d e d in 75 ttl

with a n o n - l i n e a r , l e a s t - s q u a r e s c a r v e - f i t t i n g ((era-

a l i q u o t s (final vols. 150 u l ) , a n d t h e t u b e s i n c u b a t e d

rive p r o c e d u r e b a s e d o n F e l d m a n .~ p a r a m e t e r

fit-

"FABLE I Endogenous m o n a m m e contents in th'e regtons o f the cerebral (ortex

Results are expressed as the mean contents m ngtmg protein ~_ standard error of the means sample. The less s~gmficant difference t~c* tween the Temporal and the other cortical regions was determined by unpaired Student's t-test: ~P < 0.115. ' P < 11.()1 and " ~ ' P < 0.001; n is given in parentheses. The compounds are: NA. noradrenaline: MHPG. 4-hydroxy-3-methoxyphen ylgtycol: NMN. normctanephrine: AD. adrenaline: MTN. metanephrine: DA. dopamine: DOPAC. 3.4-dihydroxyphenylacetic acid: HVA, homovanillic acid: 3-MT. 3-methoxytyramine: 5-HT. serotonin: HIAA. 5-h~ droxvindole-3-acetic acid: 5-HTP. 5-hydroxytrvptophan.

NA MHPG NMN AD MTN DA DOPAC HVA 3-MT 5-lIT HIAA 5-HTP

E141 1121 81 141 n) 141 141 14) 141 141 141 81

Pre@ontal

Frontal

Parietal

Occtpital

2.58 + 0.33 2.43 +_ 0.58 (I.211 _+ 0.(17 0.05 +_0.01 U. lO z 0.02 0.23 z 0.08 0.50 m 0.08 0.81 _~ I).15 S.3t) --~ 0.42 1.41) *_0. I() 5.84 z 0.75 0.14 z 0.03

2.58 z 0.34 2.18 +_ 0.48 0.33 z 1/.117 0.06 z 0.01 0.15 +- 0.04 11.21 z 0.05 0.40 _+ 0.05 0.78 ~_ 0.14 8.52 --~0.47 1.30±0. I0 3.97 ± 0.48 ~).15 _*:o.03

2.24 ± 0.28 2.49 _+ tl.611 ().lg ± 0.(13 0.07 _+ (t.O[ 0.19 z 0.03 ")'3 0.__ ~_ 0.06 0.28 _+ 0.06 0.45 ~_0. II 8.(.)8 ~ 0.4g 1.05 7 0 ( 7 3.12 ~_ 0.30 i).()~ ~ 0.02

2.61 z 0.35 2.60-2 ~).~ 0.23 -+ 11.03 0.()5 _+ O.Ol ().11) _+ ().02 0.21 .+. .0. (I~, (). I~ ± 0.03 11.55 _~ 0.12 7.74 --+,).41 1.17 ~().211 3,49 = ().3') 0.22 z ().()I

Temporal

~>.4~,)± 1.17" 4.53 z 1.33 IL3t ± I.( 4 ~1.14 2 (.).I13 t).13 4 0.03 .52 _~ () 29 .... t 78 z ().22 ~ "" 1.41 ~ ~).lh~* ().i)g ± U.34' 2.(~t,)-'~ 2 0 " 7.85 ~ 1.07 ~).211z. ~).04

405 ting at-'.

A ~

3o,, FFR! F R t n N ' :',[

© m

The HPLC assays of endogenous m o n o a m i n e s showed the highest levels in the TE region, in agree-

• T[ MF {'q,',

Z 0

ment with previous fluorometric 7, and radioenzymatic 14>-'4 studies. The noradrenaline (NA) concentra-

o

To

2(;O-

I(:,~

E

tion was highest in the TE region, and homogeneously m

lower for the other cortical areas (Table 1). The two metabolites of NA, i.e. 4-hydroxy-3-methoxy-phenylglycol ( M H P G ) and n o r m e t a n e p h r i n e (NMN) were equally distributed, but for a slightly higher al-

r

I H!

B

c~-

g

~ q-'

,

1

~, F,

'#,'

-;~':-

1

ffJl_

though not significantly different level of M H P G for the TE region. The dopamine (DA) content was also

ii .....

highest m the TE region (P < ().001), as was that of its 3 metabolites, i.e. homovanillic acid (HVA), 3,4-

i ~, ~ :,,,

dihydroxyphenyhtcetic acid ( D O P A C ) and 3-methoxytyramine (3-MT). Traces of adrenaline (AD)

/

J

CL

~'~, 7

and metanephrine (MTN) were detected, but since '"

the existence of a defined adrenergic pathway to the cortcx has not been established we cannot warrant further specuhttion, although we could exclude contamination of other peaks by these compounds. The indoleamine serotonin (5-HT) was more a b u n d a n t in the -I'E region (f' < 0.001) as compared to the other areas. The 5-HT metabolite 5-hydroxyindole-3-acetic acid (H1AA) was slightly higher in TE and PF regions, but 'a'c could not document significant differcnces in this study. There were also traces of 5-hydroxytryptophan (5-HTP) in all the cortical areas. The saturation curves obtained with the tritiated adrenergic antagonists in m e m b r a n e preparations from total cortex and from the 5 cortical regions. showed in every case that specific binding was saturable and of high affinity (Fig. 1). For homogenates of total cortex, the density of binding sites (Bm~x) and the equilibrium dissociation constants (Kd) were of the same order as those earlier reported with [-~H]PRZ for ezI- (refs. 6, 11, 17), [ ; H ] I D A for a,- (refs. 15, 18) and [ ; H I D H A for/%adrenoceptors I.-' The distribution of the a-adrenergic receptors in the 5 cortical areas examined was not homogeneous, and two different patterns could be determined for the two types of ~z-adrenergic binding sites (Table II). The highest concentration of a~-adrenoceptors was located in the prefrontal cortex (PF). where it accounted for more than 60Cb of the total a-adrenergic binding, giving an ~zl,"~z2 ratio of 1.4. The a I sites labelled by' [;H]PRZ are postsynaptic in the cerebral cortex ~-. so that this ratio indicates a predominance

" ,.

I ~:"J

'.1

>

-L

Fig. 1. Representative binding experiments performed with [Hlprazosm, [H]ldazoxan ~ ' ~ " and [H]d~hydroalprenolol in membrane preparations from total cortex and the prefrontal (PF) and temporal (TE) areas. The saturation curves are shown on the left while the right panels illustrate the Scatchardz{~plots determined by iteration, according to Feldman's procedure4"12. A: [~H]PRZ labelling of %-adrenoceptors: Total cortex: Bm,,,= 203 fmot/mg protein, K i = 0.24 nM; PF: Bm~,~ = 26(I l'mol,'mgprotein. Kd = 0.20 nM, and TE: BIll,IX = 204 fmol,'mgprotein. Kd = (t.26 nM. B: [~H]IDA labelling of a,-adrenoceptors: Total: Bma ` = 159 fmol,'mg protein. Kd = 2.01[) nM; PF: Bin,,,= 186 fmol/mg protein. Kd = 1.995 nM. and TE: Bm~,~= 369 fmol/mg protein, Kd = 1. 114 nM. (': the/~'-adrenoceptors labelled with [3H]DHA were homogeneously distributed: Torah Bm~' = 114 fmol/mg protein,/x~ = 1.486 nM: PF: Bm~,, = 100 I'mol,,'mgprotein, Kd = 1.015 nM and TE = 91 fmol/mg protein, K~ = 0.823 riM. of postsynaptic a r a d r e n o c e p t o r s over a 2 binding sites. The anatomical localization of a~-adrenoceptors at presynaptic locations has not been proven. However, an up-regulation of a , sites after surgical or chemical N A denervation of cortical neurones has been reported, and this speaks in favour of their existence at postsynaptic locations 23. Indeed, (~-adrenoceptors may not be entirely situated on NA termi-

406 FABLE 11

Specific [3H]prazosin, [3H]idazoxan and [3H]dihydroalprenolol binding to membrane preparations from rat cerebral cortex The values represent for each ligand the mean _+ S.E.M. of 6 separate experiments, performed with 6 membrane preparations and in duplicate. The densities of binding sites IBmax) are in fentomol/mg protein and the dissociation constants cKd) in nanomol. The regions are: PF. prefrontal: FR. frontal: PA. parietal: OCC, occipital: TE. temporal. Statistical significance was detcrmined using the unpaired Student's t-test to compare the homogenates from each region with those from total cerebral cortex: **P < 0.01. "~ "P < (t.001.

B,,,~ ((moiling protein ~

K, (nM)

('oetticient or Hill

[3H]Prazosin Total PF FR PA OCC TE

t73.6 + 10.3 253.0 ± 12.1"" 191.2 _ 16.4 177.1 _ 21.1 158.8 + 13.6 143.9 _+ 12.6

u. t34 0.175 0.181 0.198 0.178 0.201

z 0.030 z 0.047 z 0.050 *__0.051 z 0.055 x 0.08tl

1 307 z 0.041 1.22l ± 11075 1.222 ± 0.1t79 1.288 z. !1.1164 1.099 L (1.(t65 (i.876 z. t1.07-I

[3H]Idazoxan Total PF FR PA OCC TE

168.4 _+ 12.2 181.0 + 11.9 167.6 _+ 16.2 158.5 +_ 19.3 153.2 + 16.9 376.1 z 37.6 "~"

1.777 _ (I.173 1.214 ~- 0. i77 1.975 __*0.225 1.819 __+0.246 2 198 + 0.433 [.196 z 0.161

1.018 ~ 0.055 0.784 z. 0.073 0.889 +_ (/A)4b 1,).748 ± 0.025 1,,t.999 ± 0.1,)46 0.873 +_0.040

[-~H]Dihydroalprenolol Total PF FR PA OCC TE

102.0 + 7.6 96.5 _+6.9 107.1 ~- 11.3 106.3 + 13.2 102.8 _+ 5.8 98.6 + 11.6

1.316 _~ 0.187 1.060 _~ 0.192 0.984 ~- 0.109 0.985 _ 0.065 1.363 z (I.229 1.397 z 0.165

0.967 ± (I.875 ± 0.921,1 ± 0.813 z 0.931 ± 0.9[8 +

I.t.031 (,t.(180 0.057 1,L0211 ) 037 0.t041

[3H]-

s h o w e d the highest density o f a 2 - a d r e n o c e p t o r s with

of a ,

an aFa2 ratio of 0.41. This r e g i o n had the highest

sites 3. and the exact pre- o r p o s t s y n a p t i c n a t u r e of the

N A . D A and 5 - H T levels. Since ~t2-adrenoceptors

a 2 - a d r e n o c e p t o r s r e c o g n i z e d by [ 3 H ] I D A m a y be deb a t a b l e 15"22"23. it m a y be s u r m i s e d that t h e s e sites

are p r o p o s e d to c o n t r o l N A r e l e a s e , a significant pro-

h a v e b o t h pre- and p o s t s y n a p n c localizations. T h e e n d o g e n o u s N A c o n t e n t is m o d e r a t e in the P F r e g i o n

calization in this region. Interestingly, high levels of [ 3 H l p - a m i n o - c t o n i d i n e

(2.6 n g / m g p r o t e i n ) , so that it c o u l d be c o n s i d e r e d an

binding sites h a v e b e e n d o c u m e n t e d by r a d i o a u t o -

a r e a of r e l a t i v e l y i m p o r t a n t N A input, but with a pre-

graphy in the p i r i f o r m c o r t e x 25. which is part of t h e

d o m i n a n c e o f target r e c e p t o r s of the a r a d r e n e r g i c

T E region. W i t h the r e s e r v a t i o n s due to the use o f a

nals. A l t h o u g h the a 2 - a d r e n e r g i c a n t a g o n i s t rauwolscine

discriminates

two

populations

p o r t i o n of these r e c e p t o r s m a y h a v e a p r e s y n a p t i c lo-

subtype. H o w e v e r , e n d o g e n o u s c o n t e n t m a y not re-

d i f f e r e n t r a d i o l i g a n d to label p r e s u m p t i v e cq sites, it

flect the f u n c t i o n a l i t y of the N A system and t u r n o v e r d e t e r m i n a t i o n s m a y be m o r e a d e q u a t e to assess such

has b e e n s h o w n m in vitro b i n d i n g assays 21 with

an activity. F o r F R and P A areas, the density of ch -

c e p t o r s is in the m o t o r c o r t e x , which c o r r e s p o n d s in

a d r e n o c e p t o r s was similar to that of a 2 sites T h e N A

o u r study to the P F region, in which we also m e a -

levels in F R , P A and O C C c o r t e x are a b o u t the s a m e

sured the highest density of [ 3 H ] P R Z binding sites

as in the P F a r e a : t h e s e cortices a p p e a r to h a v e a

T h e lowest c o n c e n t r a t i o n of ~ q - a d r e n o c e p t o r s l a b e l l e d with [125t}BE-2254 was f o u n d in visual, with

m o d e r a t e e n d o g e n o u s N A c o n t e n t . T h e densities of a t and a2 a d r e n o c e p t o r s in the O C C c o r t e x w e r e lower, with a r e l a t i v e p r e d o m i n a n c e of a : a d r e n o c e p t o r s (aFa2 ratio o f 1.251. In c o n t r a s t , the T E c o r t e x

[~25I]BE-2254. that the highest d e n s i t y of a v a d r e n o -

s o m e w h a t h i g h e r v a l u e s for sensory and a u d i t o r y cortical areas 21. in a g r e e m e n t with the Bma x values m e a sured with [ 3 H ] P R Z h e r e [see T a b l e II), since t h e s e

407 areas c o r r e s p o n d to the O C C , F R a n d P A regions of

renoceptors are h o m o g e n e o u s for the regions exam-

the p r e s e n t study.

ined. The distribution of adrenergic receptors is only

The d i s t r i b u t i o n of total f l - a d r e n o c e p t o r s labelled by [ 3 H ] D H A in the cortical regions here e x a m i n e d was h o m o g e n e o u s (Table 11), a n d in this respect

one of the indexes of N A i n n e r v a t i o n , but w h e n this i n f o r m a t i o n is correlated with the e n d o g e n o u s C A

m a r k e d l y differed from that of N A c o n t e n t and a~a d r e n o c e p t o r s , as shown with in vitro assays using either [3H]alprenololl or 1251-iodocyanopindolo121, as

the m a m m a l i a n cerebral cortex is hierarchically or-

well as by r a d i o a u t o g r a p h i c m a p p i n g of [ ~ H ] D H A b i n d i n g sites ~3. In the p r e s e n t study there was n o attempt to discriminate b e t w e e n fll and f12 subtypes. It is possible that some of the f l - a d r e n o c e p t o r s in cerebral cortex m a y be associated to n o n - n e u r o n a l structures such as blood vessels, pial m e m b r a n e s or glial cells, but a differential regional distribution of one or a n o t h e r subtype c a n n o t be excluded. In conclusion, this biochemical survey of a d r e n e r gic receptors clearly d e m o n s t r a t e s a differential and

contents, it clearly stems out that this i n n e r v a t i o n of ganized, and this is p r o b a b l y so for the specialized and functionally different cortical areas. This work was s u p p o r t e d by the Medical Research Council of C a n a d a (MT-6967). Personal support was provided by the F o n d s de la R e c h e r c h e en Sant6 du Q u e b e c to T . A . R . (Senior Scholar), L.D. (Postdoctoral Fellow, E q u i p e de recherche sur les n e u r o t r a n s m e t t e u r s du c e r v e a u ) , a n d R.B. (Studentship). P r o p r a n o l o l HCI and p h e n t o l a m i n e HCI were gifts from Ayerst and Ciba P h a r m a c e u t i c a l s , respectively. The aid of Miss H. Dussault in typing the m a n u s c r i p t

n o n - h o m o g e n e o u s d i s t r i b u t i o n of a I a n d a 2 b i n d i n g sites for the rat cerebral cortex. In contrast, the fl-ad-

is greatly appreciated. Credit for the graphic work goes to Mrs. G . B . Filosi and D. Cyr.

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