Biochemical characterization and autoradiographic localization of central substance P receptors using [125I]physalaemin

Brain Research, 332 (1985) 299-307 Elsevier

299

BRE 10680

Biochemical Characterization and Autoradiographic Localization of Central Substance P Receptors Using [125I]Physalaemin STEVENS WOLF 1, TERRY W MOODY l, REMI QUIRION 2 * and THOMAS L O'DONOHUE 2

~Departrnent of BlochernlstO', The George Washington University School of Medicine and Health Scwnces, Washington, DC 20037 and 2Experimental Therapeutics Branch, NINCDS, Bethesda, MD 20205 (U S A ) (Accepted July 23rd, 1983)

Ke) words peptlde receptors - - autoradlographlc locahzatlon - - physalaemm binding sites - - substance P - - eledolsln

The binding of [125I]physalaemln to rat brain slices was investigated Radlolabeled physalaemln bound ~lth high affinity (Kl = 0 3 nM) to a single class of s i t e s (Bma x = 22 fmol/mg protein) Kinetic studies indicated that binding was time-dependent and all specific binding was reversible Pharmacology studies indicated that specific [i2SI]physalaemm binding was inhibited by structurally related peptldes such as substance P and eledolsxn Biochemical studies indicated that specific binding of radlolaheled physalaemln was greatly reduced if the brain shces were pretreated with heat, trypsin or N-ethyl malelmlde Autoradlographlc studies indicated that the [125I]physalaemln binding sites were dxscretely distributed throughout the brain Highest gram densities were present in the olfactory bulb, dentate gyrus, amygdala, superficial layers of the superior colhculus, sublculum, dorsal parabrachial nucleus, locus coeruleus. nucleus tractus sohtarn and dorsal horn of the spinal cord Moderate grain densities were present m the nucleus accumbens, olfactor) tubercle, pyriform cortex, strlatum, hlppocampus, inferior colhculus and central gray of the mldbraln Low grain densmes were present m most thalamlc nuclei, the substantla nlgra and cerebellum The corpus callosum and controls treated with 1 ~M unlabeled physalaemln had negligible levels of binding The unique pharmacological and regional &strlbUtlOn data obtained suggest that [lzSI]ph)salaemm may serve as a valuable probe to study central substance P receptors INTRODUCTION

Subsequently, substance P receptors usmg rat parotld gland m e m b r a n e s 20 and mouse mesencephalic

Substance P, an un d e c a p e p t id e lnitxally isolated

cellsi were characterized using [t25I]Bolton H u n t e r

from b o v m e hypothalamus6, represents one class of

substance P. Recently, the discrete regional distribu-

peptldes biologically active in the CNS. E n d o g e n o u s

tion of substance P receptors was described using

substance P has been d e t e c t e d in high density m dis-

[125I]Bolton H u n t e r substance p30 and [3H]substance

crete brain areas such as the substantta gelatmosa

p2v Previously, we described the regional distribu-

and substantla nlgra by radioimmunoassay 3 and us-

tion o f ' b m d i n g of [t25I]physalaemln, a potent sub-

mg i m m u n o c y t o c h e m l c a l techniques7,21

stance P analogue, to rat brain 3a H e r e we describe

Also, sub-

stance P is present in m o d e r a t e density in the rat hy-

the

pothalamus and is released from synaptosomes by

[1251]physalaemm binding to rat brain slices and the

depolarizing stlmuh m a Ca2+-dependent m a n n e r 29.

discrete regional distribution of these blndmg sites

When released from neurons, substance P may activate specific receptors

using in vitro autoradlographlc techniques.

Substance P receptors have been characterized using rat brain h o m o g e n a t e and [3H]substance plo

MATERIALS AND METHODS

These studies indicated that substance P receptors

Preparation o f radtolabeled pepttde All pepttdes used were purchased from Bachem

were enriched m the strlatum but not cerebellum

kinetics,

equilibrium

and

pharmacology

of

* Present address Douglas Hospital Research Center, 6875 Boulevard LaSalle Verdun, Que , Canada H4H 1R3 Correspondence T W Moody, Biochemistry Department, The George Washington Umverslty Me&cal School, 23t)0 Eye St , N W , Washington, DC 20037, U S A 0006-8993/85/$03 30© 1985 Elsevier Science Publishers B V (Biomedical Division)

~l)ll

q ABLE I

Laboratortes (Torrance, CA) o r Peninsula Laboratortes (San Carlos, CA) Physalaemm, ~ 4/gg, was radlolabeled wnh 2 mCl of Na~2Sl (Amersham, Chicago, IL) using 0 4/zg chloramme T (Stgma Chemical Co , St. Louts, MO) After 30 sec the reaction was quenched by the addmon of 1 ug ot sodmm metab~sulfite (Sigma) Then [12~l]physalaemm was purified usmg the gel filtratton techmques described previously ~ The specific act~vtty of ou) tracer preparattons ranged from 400-6(/0 Ct/mmol

Binding assays were perfolmed m tnphcatc using l t) nM [~251]physalaemm The densJt) ol binding sites ~as expressed a~ fmol of radlolabeled peptlde bound/rag protein and the K was calculated based on the ability of unlabeled physalaemm to displace specifically bound [12Sl]physalaemm

Regton

('oordmate~

Demtty (Jmol/ rngprotelt;

K

A 12,130/~m A 79801~m A 4110/m) A 1800rum P 3 0 mm

14 19 17 7 1

() '~

Ph vsalaemm bmdmg aasay

Oltactory bulb Stnatum Hlppocampub Mldbram Pons/Cerebellum

Twelve-!tm thick coronal secuons of rat brain were cut using an IEC mmotome and thaw mounted onto cover slips or shdes Secttons were air drted and incubated in assay buffer composed of 130 mM NaC1, 5 mM MgC1z, 5 mM MnCI2, 5 mM KCI, 1 mM E G T A , 100ktg/ml bacltracln and 0 1% bovine serum albumm (BSA) m 10 mM Hepes-NaOH, pH 7.4, plus rad~olabeled peptlde at 22 °C in the presence or absence ot unlabeled pepttde After incubation, free radlolabeled pept~de was removed by two consecutive 4-mln washes m buffer at 4 °C Then sections, which contamed bound pept~de, on cover slips were crushed and assayed for radloacttvtty using a LKB gamma counter or sections on shdes were exposed to [~H]ultrofilm (LKB) as described -'4 Protein concentration was determmed b2¢ the Lowry method 22 When biochemical experiments were conducted, rat brain shces were treated with the chemtcal or enzymatic reagents as indicated, then extenswely washed m buffer to remove excess reagent Equlhbrmm bmdlng studtes were conducted by incubating the treated rat bram shces with [12~llphysalaemm for 30

Regional dtsotbutton of [1251]ph~sa/aemm bma,n~ t(, tea/ram, ~ltce~

04 (I ,~ n d n d

n d indicates not determined

quently, bindmg studtes were conducted using telencephalon slices derived from Konig and Klippe! coordinates A7980-892016.

Kmettc bmdmg studws The kinetics of [125I]physalaemm binding were investigated using rat brain shces which contained the strlatum Fig. 1A shows that [125I]physalaemm rapidly associated to the rat brain shces. Total binding increased raptdly the first 15 mln, then slowly for the next 75 mm. In contrast, non-specific binding, in the presence ot 1/~M unlabeled physalaemln, mcreased slowly throughout the time-course of the experiment The differences between the two represents specific

B

2

rain

)

I

RESULTS

v

5

[ i

buttal bmdlng studtes

[125I]Physalaemm bound to coronal rat brain shces with high afflmty. Table I shows that [t25I]physalaemm bmdmg was greatest m slices which contained the strtatum, htppocampus and olfactory bulb, mtermedmte m the mtdbraln and lowest m the pons and cerebellum Because pharmacology studies indicated that physalaemm bound with s~mdar affimty m each of these regions, these differences may reflect different receptor densities rather than afflntttes. Subse-

J

05

5 I

I

I

30

60 Time (mln)

90

10

15

qqme (mm)

Fig I Association of [125I]physalaemln to rat forebram shces A radlolabeled physalaemln (1 nM) was incubated with 12,urn coronal shces of rat brain as described m Methods Total ( , ) and non-specific ([]) binding were determined in tnphcate as a function of time The difference between the two represents specxfic binding (O) B replot of the specific [125I]physalaemm binding data where Be represents the amount bound at eqmhbrmm and B represents the amount bound al t~me t

301 TABLE II

A

Effects of chemical and enzymauc reagents on [leSl]phy~alaemm bmdmg

B

~o5 k

~.....

5

2

""2",

g 20

40

20

Tirne (mm)

40

Twelve-/xm thick secnons were incubated under the conditions described, rinsed briefly in buffer and incubated with [IzSI]physalaemm as mdmated m Methods Binding assays were performed m tnphcate and the mean value _+ S E is indicated In the absence of NEM at 37 °C, specific binding of radlolabeled physalaemm was not slgmficantly altered

Treatment

c,~ SpectJzcbinding

None Heat, 65 °C, 10 mm 2 mM NEM, 37 °C, 30 mm 100/~g/ml trypsin, 22 °C, 10 mm

100 k 4 3± 1 37 _+5 10 ± 4

Time (rmn)

F~g 2 Dlssocmtmn of bound [12SI]physalaemm A radlolabeled pepude was incubated wnh brain shces for 30 mm Then, the amount of radlolabeled physalaemm bound spemfically was determined as a funcnon of rime after a 1000-fold dflunon m buffer (O) or after addmon of 1 /xM unlabeled physalaemm (O) B replot of the specific [lZSI]physalaemln bmdmg data where Bo represents the amount bound at the zero nine point and B represents the amount bound at the lndmated time pomts

b i n d i n g which e q u l h b r a t e d after a p p r o x i m a t e l y 30 min Fig. 2 A shows that if [125I]physalaemln was inc u b a t e d with rat b r a i n slices for 30 rain, specifically b o u n d r a d l o l a b e l e d p e p t i d e was reversed by the addition of 1 u M u n l a b e l e d p h y s a l a e m i n or by d i l u t i o n in buffer The half time of dissociation was approximately 10 rain a n d after 40 rain almost all [|25I]physalaemln b o u n d specifically was r e v e r s e d A replot of the dissociation b i n d i n g data was l i n e a r (Fig 2B) a n d the dissociation c o n s t a n t (k_x = 5.7 × 10 -e rain) was calculated as described by Kltabgl et al 15 A l s o a re-

plot of the specific association rate data was linear (Fig. 1B) a n d an association rate c o n s t a n t (k I = 5 5 x 10 7 rain ~ M l) was calculated Based on these kinetic data the dissociation c o n s t a n t (Ka = k_l/kl = 1 n M ) was calculated

Equthbrtum bmdmg studtes The c o n c e n t r a t i o n d e p e n d e n c e of [leSl]physalaerain b i n d i n g to rat b r a i n shces was investigated Fig. 3 A shows that total b i n d i n g increased greatly at low a n d slightly at high c o n c e n t r a t i o n s of radlolabeled peptlde. In contrast, non-specific b i n d i n g Increased slightly as a f u n c t i o n of [l:SI]physalaemln c o n c e n t r a t i o n . The difference b e t w e e n the two represents specific b i n d i n g which was s a t u r a b l e Fig 3B shows a replot of the specific b i n d i n g data which was linear28 Based o n this e q u i l i b r i u m b i n d i n g data ra-

20

A

1 5

~

/~

lC

B

~

u_ 5C m

10

2

05

I

25 ~5 ]7 physalaemln

I

'\

50

215

(r~M)

B(fmol]

50

Fig 3 Binding of [125I]physalaemm as a funcnon ol radlolabeled pepnde concentranon A total (m) and non-specific (IE) binding were determined m tnphcate at eqmhbrmm The difference between the two represent specific binding (©) B Scatchard plot of the specffm [12SI]physalaemm binding data The hne drawn represents the best fit assuming a single class of sites

diolabeled p h y s a l a e m l n b i n d s with high affinity ( K j = 0 3 n M ) to a single class of sites (Brad x = 22 f m o l / m g protein) Also, the effect of v a r i o u s chemical a n d e n z y m a t i c reagents u p o n [125I]physalaemin b i n d i n g was investigated T a b l e II shows that almost all specific b i n d i n g was r e d u c e d by use of heat or the e n z y m a t i c r e a g e n t trypsin Also, the chemical r e a g e n t N-ethyl malelmlde ( N E M ) greatly r e d u c e d r a d l o l a b e l e d p e p t i d e b i n d i n g These data suggest that [125I]physalaemin may brad to a p r o t e i n which has an essential sulfhvdryl group

Pharrnacology of bmdmg The ability of various p e p t l d e s to inhibit [125I]phys a l a e m l n b i n d i n g was investigated Fig 4 shows that p h y s a l a e m l n , s u b s t a n c e P a n d various s u b s t a n c e P

31t2 h e p t a ( 5 - l l ) s u b s t a n c e P were ! 3 and 1~ ~ ~, a~ pt~t:~ ,~s physalaemln, Pyr-rlexa(6-11 t ~,~lt hc'~,,(6-111substance P were It ¢7 and 0 2 ' , 4, potent as physalaemln Peptides structurally' related to substance P, such as eledolsm, did compete lot the radiolabeled physalaemln binding s~tes whereas p e p n d e , structurally unrelated to substance P did nt,r

~oo

c

z5

0

25

i,,

t

;10

....

~

I

-9 -8 -7 -6 -5 Unlabeled pept~de concentration (log M)

I

-4

Fig 4 Pharmacology of [l-~I]physalaemm binding The percentage of [~25I]physalaemln bound specifically is plotted as a

function of unlabeled pept~de concentration for physalaemm (0), substance P (©), deca(2-11)substance P (11), bepta(5-11)substance P (/7), penta(7-11)substance P (A) and m(9-11)substance P ( ~ ) The hnes were drawn point to point fragments inhibited r a d i o l a b e l e d physalaemln binding in a d o s e - d e p e n d e n t m a n n e r P h y s a l a e m m and substance P were the most p o t e n t m that 1 nM of unlabeled p e p t l d e inhibited 50% of the specific [125I]physalaemin binding (1Cs0). Because K~ = ICsd(1 + [L]/Kd), the calculated inhibition constants for physalaemin and substance P are 0 4 nM and 0 5 nM, respectlvelyiL A l m o s t all specific [125I]physalaemln binding was inhibited using 100 nM physalaemin or substance P. In comparison, the ICs0 values for d e c a ( 2 - 1 1 ) s u b s t a n c e P and hepta( 5 - l l ) s u b s t a n c e P are 10 and 180 nM, respectively. Thus, substance P fragments are not as p o t e n t in inh~bltmg r a d l o l a b e l e d p h y s a l a e m m binding as substance P. Also, the fragment p e n t a ( 7 - 1 1 ) s u b s t a n c e P weakly mhibited [125I]physalaemm b m d m g and was m o r e p o t e n t than t r i ( 9 - 1 1 ) s u b s t a n c e P, both peptides had ICs0 values greater than 1/~M These pharmacology data indmate that physalaemln may brad w~th high affinity to substance P receptors and that n u m e r o u s amino acid residues t h r o u g h o u t the substance P sequence are r e q m r e d for high affinity binding activity Also, the potency of other pepUdes was investigated. Table I I I shows that substance P was almost as potent as physalaemln In comparison, dec a ( 2 - 1 1 ) and o c t a ( 4 - 1 1 ) s u b s t a n c e P were 10 and 1 3% as potent as physalaemm. P y r - h e p t a ( 5 - 1 1 ) and

Regional distribution o/ b m d m g The discrete regional distribution of [~'~l]physalaemm binding sites was investigated by him autora& o g r a p h y High gram densmes were present in certain gray matter nuclm In contrast, the grain densJtins were negligible in white matter and controls treated with 1/aM unlabeled physalaemln In the rhmencephalon, high grain densmes were observed m the molecular layer of the oltactory bulb (Fig 5A) In the telencephalon, high gram densmes were present m the frontal pole (Fig. 5A). M o d e r a t c to high gram densmes were observed in the nucleus

TABLE 1II Pharmacology ot [12-~l]physalaemm bmdmg At equihbrlum, the amount of [l:5I]physalaemln bound was determined as a function of unlabeled peptide cDncentratlon "Ihe IC50 was determined for the peptides shown abo,m Peptldes whmh did not compete for the radlolabeled physalaemln binding sites include bombesin, cholecystoklnln enkephahn, a-melanocyte stimulating hormone, neurotensln and vasoactive Intestinal polypeptlde The structures of eledo~sm, physalaemln and substance P are shown below Sequence homologies are underlined Physalaemln Pyr-Ala-Asp-Pro--Asn-Lys-Phe-Tyr-Gly-Leu-Mct-NH: Substance P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH 2 Eledotsln Pyr-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH: Pepttde

l( ,,, cnM)

% Potem v

Physalaemin Substance P (SPy deca(2-11)SP Eledolsln Octa (4-11)SP Pyr-hepta(5-11)SP Hepta(5-11 )SP Pyr-hexa(6-11)SP Hexa(6-ll)SP SP-COOH (i>Pro 2, D-Phe7, D-Trpg)SP Penta(7-I 1)SP Trl(9-11 )SP

1 I I 10 1[)0 75 75 180 180 450 2tL000 10,000 20,000 > 100,00(I

11Jl~ ~) ]l~ 1 i) !3 i ~6 06 II 2 0 005 ~) 01 0 005 <0 001

303

®

®

® ,C ©

@

®

A

I

Fig 5 DlstnbuUon of [125I]physalaemm binding s,tes m rat brain Bright-field photomicrographs of the brain autoradlograms were cut out, mounted and are shown The dark areas indicate high gram density Coronal sect,ons were derived from Komg and Khppel ~6 coordinates (A) A 12,130/~m. (B) A 9820gm, (C) A 9650/~m, (D) A 8920/~m, (E) A 8380~m, (F) A 7470/~m, (G) A 6570~m. (H) A 5660,um. and (I) A 4890~tm

31)4

® ~k C ,#

Fig 6 Distribution of radlolabeled physalaemm binding sites Coronal sections were derived from Komg and Khppel coordinates (A) A 3750,um, (B) A 2180,urn, (C) A 1610~m, (D) Palkowts and Jacobowltz25 P 0 1 mm, (E) P 1 0 mm and (F) P 2 0 mm

accumbens, striatum, lateral septal nucleus and olfactory tubercle but not the corpus callosum (Fig. 5 B - E ) The gram densities were moderate to high m the bed nucleus of the stria terminahs (Fig. 5F), anterior cortical amygdala and globus palhdus (Fig. 5G). The density of binding sttes was moderate m certain regions of the cortex, such as the pyriform cortex (Fig. 5I) and layer III of the neocortex and parietal cortex (Fig. 5C-I) Also, moderate gram densmes were observed In the hlppocampus and dentate gyrus (Figs. 5I and 6A) In the dlencephalon, certain hypothalam~c regions had moderate gram densities including the preoptlc

area (Fig 5G), suprachlasmaUc nucleus (Fig 5H), paraventricular hypothalamlc nucleus, ventromedml hypothalamic nucleus and lateral hypothalamlc area (Fig. 5I). Also, moderate gram densmes were observed in the medial habenula and medial thalamic nuclei, while the lateral thalamus had a low density of binding sites (Fig. 6A) In the mesencephalon, high grain densities were present m the superficial layers of the superior colliculus but not the medial and lateral gemculates (Fig 6B-E). Moderate gram denslues were present m the central gray, dorsal raphe nucleus and entorhinal cortex (Fig. 6E) In the myelencephalon high gram densities were

305

Fig 7 Dlstrlbutmn of [125I]physalaemmbinding s,tes m rat brain Coronal secnons were derived from Palkowts and Jacobowxtzcoordinates (A) P 2 8 mm, (B) P 3 9 ram, (C) P 6 0 ram, (D) P 8 0 mm and (E) cerwcal spmal cord

present In the locus coeruleus and dorsal parabrachlal nuclei (Fig 6F). In the cerebellum, a moderate density of binding sites was observed in patches of vermion lobules 9 and 10 (Fig. 7 A - C ) . In the metencephalon, high grain densities were observed In the nucleus tractus sohtaril, nucleus ambiguus and the inferior olivary nucleus (Fig. 7 A - D ) . In the cervical spinal cord, a high density of binding sites was present in the dorsal but not ventral horn (Fig. 7E) DISCUSSION The characterization of substance P receptors has been limited due to the inavailability of adequate receptor probes. Here we utilized [12SI]physalaemin, which had a specific activity of approximately 400 Ci/mmol, as a central substance P receptor probe Previously, radlolabeled physalaemln was used to characterize substance P receptors in the exocrIne pancreas 13 Using acini dispersed from the guinea pig pancreas, radiolabeled physalaemin bound with high affinity (K a = 0.5 nM) to a single class of sites (500/cell). [125I]Physalaemin binding was competitively Inhibited with high affinity by substance P and moderate affinity by eledoisin and kassinm. Also, substance P and physalaemln stimulated Ca2+ flux and cGMP production Thus, physalaemtn serves as a valuable probe in the exocrine pancreas In the present study, [125I]physalaemin bound in a

time-dependent manner to rat brain slices and all specific binding was reversible. In particular, all specific binding was reversed with similar kinetics either by addition of excess competitor or dilution (Fig. 2A) suggesting the absence of negative cooperativity. This was verified by a Scatchard plot which was linear (Fig. 3B). The dlssoclaton constant calculated based on the kinetic, equilibrium and pharmacology data (1.0, 0 3 and 0.4 nM, respectively) were in good agreement These data indicate that [125I]physalaemln binds with high affinity to rat brain sections Further, the biochemical experiments shown in Table I indicate that [125I]physalaemin may bind to a protein which has an essential cysteine amino acid residue. The pharmacology of binding was also Investigated. Fig. 4 shows that physalaemln, substance P and substance P fragments competed for specific [125I]physalaemin binding sites. Table Ill shows that physalaemin and substance P were approximately 2 and 4 orders of magnitude more potent than eledolsin and the antagonist (D-Pro 2, D-Phe 7, D-Trpg)sub stance P, respectively. Also, as the size of the substance P fragment decreased, so did ~ts potency. Surprisingly, some investigators reported initially that hexa(6-11)substance P was more potent than substance p10.23, however, this has not been verified in recent reportsS, I8 27,31 These discrepancies in peptide potency may result from the different assay conditions used, which may lead to altered receptor binding affinities and/or peptide proteolytlc degradation rates [125I]Physalaemln is not degraded under the assay conditions used here This may result because physalaemln has a blocked N-terminal and hence IS resistant to degradation by amlnopeptidases 2. Also, the data presented in Table III indicate that ff the N-terminal m hepta(5-11) or hexa(6-11)substance P is blocked with a pyroglutamate, the potency is increased, presumably due to decreased peptide proteolysis and/or increased binding affinity A critical question is whether or not there are subclasses of central substance P receptors. Lee et al proposed that in the periphery there are two classes of substance P receptors, one class which prefers eledoisin (SP-E) and one class which prefers physalaemln (SP-P) I7. Because our pharmacology data indicate that [I25I]physalaemln binding is inhibited with 'at least two orders of magnitude greater affinity by

~(~ physalaemm or substance P than by eledoasln (]'able Ill), we may be characterizing central receptors which are m o r e similar to SP-P than SP-E receptors The a u t o r a d l o g r a p h l c studies mdlcate that the [l:51]physalaemln binding sites are discretely distributed throughout the rat brain Similar autoradaograms were g e n e r a t e d using [3H] substance W-,~ or [I-~Sl]Bolton H u n t e r substance p3~J as substance P receptor probes In particular, m o d e r a t e to high grain densities were present in the nucleus accumbens and caudate p u t a m e n Because substance P elevates homovanllhc acid and 3,4-dlhydroxyphenylacetJc acid levels in the strlatum, these substance P receptors may be present on d o p a m l n e containing neurons 32 M o d e r a t e grain densities were present in the cortex Because substance P is an excitatory agent in the cortex 26 and the strlatum 19 these substance P receptors may alter neural activity M o d e r a t e to high gram densities were present in the olfactory bulb and tubercle as well as the d e n t a t e gyrus, h l p p o c a m p u s and subiculum. The role of substance P m these brain regions remains unknown M o d e r a t e grams were present in certain hypothalamic regions such as the suprachiasmatlc nucleus as well as the central gray of the mldbraIn. Because m o d e r a t e levels of substance P are also present in these regions, these substance P receptors may play a role in the regulation of circadian cycles and pain threshold, respectively A l s o , m o d e r a t e to high grain densities were present in the superior colllculus and inferior colliculus and these areas are involved in visual and auditory information processing, respectively. High grain densities were o b s e r v e d in the amygdala Because substance P is an excitatory agent in the amygdala 19 these substance P receptors may alter neural activity High grain densities were present in the locus coeruleus and dorsal parabrachlal nuclei but not the mesencephahc tract of the trlgemmal nerve Because substance P is excitatory in the f o r m e r two regions but not the latter region 9, substance P receptors may alter neural activity in the locus coeruleus and dorsal parabrachlal nuclei. Also, high grain densities were observed in the nucleus tractus sohtarn Because substance P is a p o t e n t hypotensive peptidea~, substance P receptors in the nucleus tractus solltarll may play a role in the central regulation of blood pressure High

grain densities were present in the dorsal but not ventral horn ot the spinal cord The substatlce P receptors in the dorsal horn may mediate pare transmission via primary sensory afferent spinal cord neurons ~ It is of interest to note that the distribution of substance P receptors does not always palallel that ol substance P In some brain regions such as the nucleus accumbens, strlatum, lateral septal nucleus, bed nucleus of the stria termmahs, various hypothalamlc regions, central gray, superior colhculu,, nucleus tractus sohtarn and substantla gelatlnosa there ~s a m o d e r a t e to high content of endogenous substance P and its receptors These regions may reprt~sent areas where substance P nerve terminals come m contact with adjacent neurons which contain substance P rcceptors Surprisingly, there are some areas such as the lnterpeduncular nucleus and substantm nlgra where there is an enrichment ot endogenous ~ubstance P relative to its receptors In t h e w reglon~, substance P may bind to a different type ,~.f receptor. such as the SP-E receptor, which is not detected here. Also, there are some regions where there is an enrichment of receptors relatl'~e to substance P such as the olfactory bulb, olfactory tubercle, cortex, dentate gyrus, hlppocampus, amygdala and mterlor colhculus. In these regions, receptors may interact with endogenous tachyklnlns other than substance P, such as the recently discovered n e u r o m e d l n K ~a The discrete regional distribution of b i d i n g suggests that substance P may function as an Important regulatory peptlde in certain brain loci In particular, in Huntington's disease the level of endogenous substance P in certain regions such as the strlatum dechnes significantlya,* It would be of interest to determine if there is a concommlttant change tn central substance P receptors in the caudate p u t a m e n In summary, because [12SI]physalaemin binds with high affimty and discretely to the m a m m a l i a n brain, it may serve as a valuable probe l o t central substance P receptors in various physiological and pathological states ACKNOWLEDGEMENTS The authors wish to thank Drs R. Jensen, M Kuhar and C Shults for helpful discussions

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