LOCALIZATION OF BINDING SITES FOR CALCITONIN GENE-RELATED PEPTIDE IN RAT BRAIN BY IN C’!TRO

AUTORADIOGRAPHY

P. M. SEXTON. J. S. McK~szre. * R. T. M-\soh.+ J. M. Most;!.l:y. T. J. MARTIS and F. ,4. 0. MEst)I,IsoHN

University of Melbourne. Departments of Medicine and Surgeryt. Austin and Repatrlatlon General Hospitals Medical School. Heidelberg. Victoria 3084 and *Department of Ph>siolog). Ilni\erslt\ of

Melbourne. Parkville. Victoria 3057. Australia

Abstract-The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat hraln w~crc studied using in r>irro autoradiography.

In a radioreceptor assay using [‘Z51]human calcitomn gene-related pcptlde as the radiollgand. \hlth cerebellar cortical membranes, rat calcitonin gene-related peptlde had a hinding affinity constant 01 1.16+0.23x 10’“M ’ and a site concentration of 43.4 i 3 4 fmol mg protein. In this syhtcm. human calcitinin gene-related peptide had a bmdmg affinity constant of 3.9 + 0.7 Y IO M ’ whereas salmon calcitonin was very weak with a binding affinity constant of only 6.X I 4.0 x IO‘ M ’

CGRP binding localized by in vim autoradiography. using [“‘l]rat calcitonin gene-related peptidc. had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina tcrmlnalis, ventral caudatc putamen. median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus. the superior and inferior colliculi. pontine nuclei. molecular and PurklnJe ccl1 layers of the cerehcllar cortex. the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the \estibular dnd cochlear nuclei.

The distribution of these binding sites suggests multiple roles for CGRP in the central nerv!)us \!\tcm including auditory. visual. gustatory and somatosensory proce\slng. and in neuroendocrlnc contrail

Calcitonin gene-related peptide (CGRP) is a novel

neuropeptide which arises from alternative pro- cessing of the primary RNA transcript of the gene encoding calcitonin.‘~‘J.J7 Central administration o!

this peptide decreases appetiteS and gastric acid secretior?’ and increases sympathetic noradrenergic outflow with accompanying hypertension and tachy- cardia I3 CGRP immunoreactivity has been localized within the central nervous system -’ I’ IS.?5.J~.J9.~?,CJ.~‘.CX

and In eye.” pituitary”‘.“” and peripheral tis- sUeS,7.1u2H.18.44.5H In addition. release of CGRP has been demonstrated from cultured rat trigeminal ganglion cells.‘”

In rirro CGRP binding, using membrane prepara-

tions. has revealed binding sites in the rat and human CNS.‘“,“-” Autoradiographic analysis of binding to human brain localized sites to the molecular and Purkinje cell layers of the cerebellar cortex. and to the posterior substantia gelatinosa of the spinal cord.‘-

To elucidate the central role of CGRP we have undertaken a detailed anatomical localization of

Address correspondence to: P. M. Sexton. Universit) of Melbourne, Department of Medicine. Austin Hospital. Heidelberg 3084. Victoria. Australia.

.4hhrr~~ia/ions: BSA. bovine serum albumin; CGRP. calcitonin gene-related peptide: CGRP-I. CGRP- Immunoreactive: CT, calcitonin: GH. growth hormone; GHRH. growth hormone releasing hormone: IC\‘. intracerebroventricular: Ka. binding affinity constant; O\‘LT. organum vasulosum of the lamina terminalis.

CGRP binding sites in the rat CNS h! i/f I i/r.o autoradiograph!.

EXPERI’MENTAI. PRO(‘EI)l RE:h

Radlolabelled human CGRP ([“‘l]lodohlstldiLI’“-h CGRP) and rat CGRP ([“‘l]iodohlstid)I”‘-rC(;RP) UC’IC prepared using the chloramme T method.:’ and purified hb reverse phase high pressure liquid chromatograph) on ‘I Waters Cl8 /t Bondapak column. eluted In i?“. acctomtrilc in ().50/o trifluoroacctlc acid The following synthetic CGRP peptides: human CGRP (I 37). (II-CGRP). rat (‘GRP (I 37). (r-CGRP): [Tyr”] rat CGRP (23 37): and [Tyr”] human CGRP (23 37) were kindly made a\alluble by Dr J. Rivlcr (The Sdlk Institute. San Diego. CA). Salmon calcitonin (sCT) ,tnd de\ Ser’ [Gly’] aCT v.erc gcncr~,ualy supplied b> Dr R. C. Orlowskl (Armour Pharmaceutical Cornpan!. K,lnknkce. IL. L1.S.A )_

Cerehcllar cortical membranes were prepared h\ differcntlal ccntrlfupation.“’ ” Rat ccrchcllar cortex \\;I; placed In 20 LOI. of ice-cold 0.035 M Trls H<‘l hutl’er. pli 7.4, homogenized for 30 35 s using a polytron (Luzcrn Kinematlsches. Hochfrequenr-Gerat) and centrifuged at 48.000~ ‘it 4 C for 20 min. The pcllct\ were then rc- suspended in the lnltial \olumc of hulTer. reh~~mogenl.vd. and again ccntrlfuged for 2Om1n. This procedure wa\ repeated once more and the pellets were then \ucpendicd In 50 mM Trls HCI. 100 mM NaC‘I. pH 7.4 and \tored at -20 C until used. Protein concentratlom wcrc dctermlned hq a modllied Lowry method.“’

The membranes (250 llg protein) were lncuhated at 20 (‘ for I h In 1).5 ml of 5OmM Trls HCI. IO0 mM NaC‘l contatnlnp 0 2% ho\~nc 5erum alhumm (BSA). I) 4 mM

12.35

1236 P.M. SEXTON et al.

bacitracin, [ml]hCGRP (56nCi/ml, ~85pM) and in- creasing concentrations of unlabelled competing ligand. After incubation, the mixtures were centrifuged at 12,000 g for 3 min at 4°C in an Eppendorf (Brinkmann) micro- centrifuge. The supernatant was discarded and the pellet washed with 1 ml of cold 0.9% NaCI, recentrifuged and the supernatant aspirated. The radioactivity present in the pellets was measured in a Crystal Multi Detector Gamma System (United Technologies Packard).

Results are means of triplicate determinations. Binding isotherms were analysed by a non-linear, iterative, model fitting computer programme. 39

A utoradiography For autoradiography, male Sprague-Dawley rats

(250-300g) were decapitated, their brains were rapidly removed and frozen for 20 s by immersion in isopentane at -40°C. Twenty /~m sections were cut in a cryostat at - 2 0 ° C and thaw-mounted onto gelatin-coated slides, dehy- drated at reduced pressure in a dessicator at 4°C .for 2-4 h and stored at - 80°C in sealed boxes containing silica gel for 2-7 days. 2: Alternate sections were air-dried and stained with thionin for anatomical localization. The sections were preincubated for 15 min in 50 mM Tris-HC1 buffer, pH 7.4, containing 100mM NaC1, 0.2% BSA, and 0.4mM baci- tracin at 20°C and then incubated for 1 h in 10 ml of the same buffer containing either [mI]hCGRP or [mI]rCGRP (113 nCi/ml ~ 164 pM). Non-specific binding was measured in the presence of either 1/~M unlabelled hCGRP or rCGRP. The slide-mounted sections were then rapidly washed using four l min transfers through 50mM Tris-HCl, pH 7.4, at 0°C and dried in a stream of cold air. The dried sections were fixed for 2 h in paraformaldehyde vapour at 80°C and then exposed to Agfascopix CR3 (Agfa Gevaert, Belgium) X-ray film for 7-21 days in X-ray cassettes. A set of radioactivity standards was included in each cassette. The standards were prepared as follows: 20/~m sections were cut from a 5 mm diameter brain core that had been snap frozen in isopentane at -40°C. The sections were then thaw mounted onto gelatin-coated slides and air-dried. A range of known [mI]radioactivity standards was applied to brain discs in a volume of 5 ktl and dried. Mean protein content of adjacent brain sections was measured after digestion in 1.0 M NaOH. 2° The area of these sections was measured using the image analyser and the value of mean protein content per unit area was calculated. The radioactivity standards were corrected for decay using the exponential equation, the decay constant and the elapsed time interval to the middle of the exposure period. This data enabled computer calibration of the optical density of the autoradiographs in terms of dpm or fmol/mg protein. The X-ray films were processed in a Kodak RP X-omat automatic developer.

Autoradiographs were analysed using an EyeCom Model 850 image processor (Log E/Spatial .Data Systems, Springfield, VA) coupled to a DEC 11/23 LSI computer to obtain colour-coded images calibrated in terms of radio- ligand density.

Following autoradiography, the sections were stained with thionin to enable anatomical identification of binding sites. Reference was made to rat brain atlases of Albe- Fesard et al) and Paxinos and Watson, 4t and to detailed descriptions of the amygdala, 26 hypothalamus, 4 and thalamus 5.

RESULTS

Characterization of binding sites for ['25I]hCGRP

In the radioreceptor assay using membranes from rat cerebellar cortex, [mI]hCGRP bound to a single class of high affinity sites (Fig. I). In this system, r C G R P (1-37) showed a binding affinity constant,

1:] ~ 601

20

0 i / - -

0 -11 -10 -9 -8 -7 -6 -5 Log10 [pepfide] M

Fig. 1. Specificity of binding displacement of [m[]hCGRP to cerebellar cortical membranes by rCGRP (1-37), &; hCGRP (1-37), x ; [Tyr 23] hCGRP (23-37), Q; [Tyr 23] rCGRP (23-37), ~ ; des Ser 2 [Gly 8] SCT, I-I; SCT

(1-32), O.

Ka, of 1.16 _ 0.23 × 10 '° M - ' and had a site concen- tration of 43.4 -I- 3.4 fmol /mg protein. Human C G R P was less potent with a Ka of 3 . 9 + 0 . 7 × 109M -l .

The C-terminal fragments of these peptides re- tained little affinity for the receptor, showing Ka 's for [Tyr23]-rat C G R P (23-37) and [Tyr23]-human C G R P (23-37) of only 6 .8 -1 -0 .5x10SM - ' and 1.4 -t- 0.1 x 10 6 M - l , respectively.

Salmon calcitonin and its des Ser 2 [Gly s] analogue also had very low activity in this system with Ka 's of 6 . 8 _ 4 . 0 0 × 1 0 5 M - l and 8 .3 - t -0 .6x 105M -I, respectively.

Binding of p25I]hCGRP to tissue sections was also assessed. This was evaluated both by counting the sections in a gamma counter and by computer analysis of the autoradiographs.

Equilibrium binding of [125I]hCGRP to rat hind- brain at the level of the inferior olivary nuclei at 20°C was achieved by 1 h. Under these conditions total binding was approximately 3100dpm/sect ion and non-specific binding was <250 dpm/section. Com- puter analysis of binding isotherms obtained by displacement of [mI ]hCGRP to the inferior olivary nuclei by unlabelled h C G R P revealed a single class of sites with a binding affinity of 1.0-I-0.4 x l0 9 M -1.

Distribution of binding sites for [mI]rCGRP revealed by in vitro autoradiography

[l:5I]Rat C G R P showed a characteristic distribu- tion of binding sites within the rat brain. For descrip- tive purposes, these sites have been assigned to the following classes of relative densities based upon quantitative computerized densitometry:

CGRP binding sites in rat brain

Table I. Relative densities for (“‘I]rat CGRP binding obtained by quantltati\e UI r/rro autoradioeranhk

Structure

Tclcncephalon Nucleus accumbens

roitral caudal

Basolatcral nucleus of amygdala Central nucleus of amygdala Lateral nucleus of amygdala Mcdlal nucleus of amygdala Postcromedial cortical nucleus of amygdala (‘audate putamen (caudoventral) Lateral prefrontal cortex Medial prefrontal cortex External plexiform layer, of olfactory bulb Olfactory ventricle Ventral nucleus of diagonal band Primary olfactor) cortex

Diencephalon Amygdala~hlppocampal cortex Antcrlor hypothalamus Anterodorsal thalamic nucleus Arcuate nucleus. hypothalamus (rostral) CA, (Ammons horn) CA, (Ammons horn) Dorsomedlal hypothalamic nucleus Habenular nuclei (lateral and medial) Lateral geniculate nucleus Lateral mammillary body Rest of mammillary body Medial preoptic area Median eminence OVLT Red nucleus Reuniens nucleus Suhthalamic nucleus Entorhlnal cortex Vcntromedial posterior nucleus (thalamus) Prcmammillary area

Mescnceph,ilon Darkschewitsch nucleus Inferior colliculus Oculnmotor and trochlearis nucleus Superior colliculus Sustantia nigra pars reticulata

Rhomhencephalon Facial nucleus Hypoglossal nucleus Inferior olivar) nuclei Medial wstibular nucleus Molecular layer (cerebellar cortex) Motor trlgeminal nucleus Nuclcw of lateral lemniscus Nucleus of the solitary tract Pontine nuclei Prlnctpal sensor> trigeminal nucleus SpInal trlgeminal tract Superior oli\ary nuclei

I1

Mean + SEM (number ol (fmol mg protein) sectlons)

2 I .7 i 0.4 33.8 i_ 1.4 13.x 2 I .7 24.3 i 3.’ 30.5 i_ 1.6 X.8 + 6 2 I? 6 i 6.2 27 3 * 2.2

6.5 + I.0 15.3 i_ 0.4 IO.1 * 0.4

3.6 14.4 T 1.6 14.3 * I.0

?<?+‘4 __ ._ _ _. 14.7 * 0.4 1.1 i0.z

25 7.3 & 0.8

14.5 * 0.7 28.0

20.3 * I.7 18.1 io.3 57.6 + 1.6 314+19

17.6 36.7 * 5.7 45.5 * 5.2 13.8 * I.1

17.4 70.3 * 0.7 18.1 iO.6 15.x +0.7 39.3 + 0.6

17.9 f 0.3 2x.5 * 0.4 I6 2 * 0.2

26.8 * 2.4 19.1 & I.5

75.2 f I.5 40.4 * 0.6 42.5 * I.1 41.7 * 3.6 16.X * 0.h 16.8 * 0.5 21.9 * 1.7 32.0 * 0.7 24.1 + 2.4 19.0 k 0.9

4.2 16.9 + 1.x

low = I@ 19: moderate = X-28: high = 29-33; bery Tehcephulor~

high > 33 fmol,mg protein (Table 1). Between animal variation over 3 hindhrain nuclei was approximately There was a low binding site density in the granular.

I?“;, (Table 2). ependymal and external plexiform layers of the olfac-

Non-specific binding measured in the presence of tory bulb. The glomeruli. and the olfactory nerves

I i(M CGRP M;IS al\\a>s <5”b of the total binding. were free of binding. and did not produce u visible image. Over most of the cerebral cortex. binding was very

Table 7. Between animal variation of [“sl]rCGRP binding to four hindbrain nuclei (fmol,‘mg protein)

Rat *NTS nl2 MeVe IO

I 32.0 + 0.7 40.4+0.5 41.2 & 3.6 42.5 I I.1 2 25.7i2.7 33.2kl.5 35.6,tJ.i 31.6f2.1 3 32.9 + 0.8 44.2 & I.0 34.x i_ 0.9 30.3 + 1.Y 4 33.3 7 2. I 43.9 _c 2 I 40.x * 1.6 35.7 i 1.9

Mean 31.0+1.8 40.4k2.6 38.lF I.7 37.312.4

Measurements for each nuclei of each rat are mean + SEM of 5 sections. *Abbreviations as per Table I.

low or undetectable. A low binding site density occurred in medial prefrontal cortex (Fig. 2a). How- ever, moderate binding site densities were seen in the visual association cortex (area 18) and auditory cor- tex in the middle layers, becoming more intense in the

perirhinal region (gustatory and post-gustatory area 13) (Figs 2f-3d). Moderate binding site densities

occurred in primary olfactory and entorhinal cortex.

In the hippocampus, the subiculum showed low

binding ventrally, somewhat higher in the dorsal

region. Low density binding occurred in fields CA3 and CA4 of Ammon’s horn. particularly ventrally (Figs 2h-3b).

anterior commissure nucleus accumbens anterodorsal thalamic nucleus anterior hypothalamic nucleus nucleus ambiguus arcuate hypothalamic nucleus area postrema basolateral amygdaloid nucleus field CA3 of Ammon’s horn field CA4 of Ammon’s horn corpus callosum central amygdaloid nucleus central gray centrolateral thalamic nucleus central medial thalamic nucleus caudate putamen cuneate nucleus dorsal cochlear nucleus nucleus of Darkschewitsch dorsal lateral geniculate nucleus dorsal raphe nucleus dorsal tegmental nucleus fundus striati gracile nucleus inferior colliculus inferior olivary nuclei lateral amygdaloid nucleus lateral vestibular nucleus laterodorsal thalamic nucleus lateral habenular nucleus nucleus of the lateral lemniscus lateral mammillary nucleus lateral reticular nucleus medial amygdaloid nucleus medial vestibular nucleus medial geniculate nucleus medial habenular nucleus medial mammillary nucleus

:cb AD

AHY Amb Arc AP BL CA3 CA4 cc Ce CG CL CM CPU cu DCo Dk DLG DR

DTg FStr Gr IC IO La LaVe LD LHb LL LM LRt Me MeVe MG MHb MM

The ventral striatum. includmg the nucleus accum- bens and ventral lateral regions of the caudarc pnt‘:i- men showed moderate to vcr! high blndtnp \rtc densities urth patches where mceptor btndlng \%a> undetectable (Fig. 2b--d). Rostrally. the b~ndmg was over a medial band of small. densely packco cells. The anterior commissurc uas unmarked (t:g. 2b cj. The rostra1 part of the strta terminalis nucleus and olfactory tubercle showed a continuous regron ot moderate to very high density of bmding srtc> con-

tinuous with the nucleus accumbens. The ventral putamen caudally, above the lateral

amygdala, showed moderate to high binding (Fig 2g.h). No binding occurred in the rest of 111s neo-

striatum. nor in globus pallidus. ventral pallidurn, or entopeduncular nucleus. The islands of Calicja were free of binding.

High binding site densities were found m parts ot

the amygdala. particularly the caudal part of the central nucleus (Fig. 2g,h), and medial ventral regions of the lateral nucleus (Fig. 2hj. Most of the medial and basolateral nuclei (Fig. ?g,h) had moderate bind-

ing site densities, as did the cortical-amygdaloid nucleus. cortico-amygdala transition zone (F;ig. 2g,h)

Mo5 Mol MPO n3 n7 n12 NTS ox OVLT PB PCL PFr PH PM Pn PO Pr5 PrH PT PVA Re RtTg SC SHy SNR so spsc STh SuVe TeAud Tu VDB VLG VHM VP VPM vs

motor trigeminal nucleus molecular layer, cerebellum medial preoptic area principal oculomotor nucleus facial nuclei hypoglossal nucleus nucleus of the solitary tract optic chiasm organum vasculosum of the lamina terminalis parabrachial nucleus Purkinje cell layer, cerebellum prefrontal cortex posterior hypothalamic nucleus premammillary area pontine nuclei primary olfactory cortex principle sensory trigeminal nucleus prepositus hypoglossal nucleus pretectal area paraventricular thalamic nucleus anterior reuniens thalamic nucleus reticulotegmental nucleus. pons superior colliculus septohypothalamic nucleus substantia nigra pars reticulata superior olivary nuclei nucleus of the spinal trigeminal nerve, caudal subthalamic nucleus superior vestibulal nucleus temporal cortex, auditory area olfactory tubercle nucleus of vertical limb of diagonal band vaentral lateral geniculate nucleus ventrornedial hypothalamic nucleus ventral pallidurn ventroposterior thalamic nucleus, medial ventral striatum

Figs 2 and 3. Autoradiographic localization of [‘z51]rCGRP binding m rat brain. I6 days exposure

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CGRP bindmp sites in rat hraln 1211

and the amygdaloid-hippocampal area (Fig. 3a).

Binding was largely absent from the basomcdial

nucleus and adjacent interstitial zones.

Spotty binding site densities of moderate intensity

\vcrc \cen in the ventral nucleus of diagonal band

( Ficr _. .Ic). which were related to the scatter of large

neurons. Elsewhere in the septal complex, bindlng

site densities wcrc low or absent.

Lou binding was observed in the internal capsule

(Fi?. 2g.h) and optic chiasm (Fig. 2d.e). In the

thalamus. low binding densities were seen in a variety

of nuclei anterodorsal (Fig.Zf). anteroventral, lat-

crodor\al. midline nuclei (but not nucleus rhom-

hoidcus). parts of the rostra1 intralaminar group.

lateral posterior. pretcctal suprageniculate areas,

medial geniculatc (Fig. 3a). parafascicular and medial

vcntr;ll posterior nuclei (Fig. 2g,h). A moderate den-

sit! 01‘ binding sites occurred in the medial and lateral

hahcnula (Fig. 3a). Low binding site densities

occurred over the caudal. central lateral. lateral

dol-somedial. dorsal and ventral lateral geniculate

nuclei (Fig. 3~). Binding was low or absent in

the \cntrolateral complex, lateral ventroposterior.

vcntr~m~cdial. reticular and gelatinous nuclei.

In ~hc subthalamic area. the zona incerta including

par\ occllular nucleus had low binding site densities

rostrall!. but binding was undetectable caudallq or

later-all!. The subthalamic nucleus of Luys had a

moderate density of binding sites (Fig. 2h).

Thi: hypothalamus showed moderate to very high

binding site densities distributed from the prcoptic to

mammillar) regions (Figs 2d-3a). In the medial prc-

optic (Fig. ?d) and anterior hypothalamic areas (Fig.

1f ). bInding site densities were moderate whereas the

jupraoptic and paraventricular nuclei were un-

lahcll~d. In the tuberal region. the external margin 01

the vc,ntromcdial nucleus had a moderate binding site

denGty. with ;I lighter centre. The arcuate nucleus

(FIN. 2g). median eminence (Fig. Zg). and lateral

tubcr,il hypothalamus (Fig. Zh) had moderate to \‘erq

high rcccptor densities. The dorsal anterior and dor-

somcdial hypothalamic areas had low binding densi-

ties. the circumscribed dorsal nucleus was fret of

b~nd~np (Fig. 2g.h). Posteriorly. the premammilary

complex had high density binding sites (Fig. 3a) and

the c audal magnoccllular and lateral mammillarq

nuclc~ a very high binding density (Fig. 3a). Modcrate

handing occurred in the posterior hypothalamic area.

stlpr;lmammlll~~ry nucleus. and medial mammillary

complex (Figs ?h 3a). A very high density of binding

\ltcs occurred o\cr the organum vasulosum of the

lamin, terminalis (OVLT) (Fig. le). but little in the

suhfornical organ.

A modcrate binding site density was seen in the

dor-s.11 (visual) layers of the superior colliculus (Fig.

3c.d in the inferior colliculus (Fig. 3c~~). and in the

small interfascicular nucleus (not shown). LOU tnnd-

ing densities were seen in the dorsal central gay

(Fig. 3b). the nucleus of Darkschcwitsch (Fig. XI).

rostra1 raphe linearis, occulomotor and trochlear

nuclei (Fig. 3b). dorsal raphe. and reticulotcgmcnt~ll

nucleus. The substantia nigra had ;I modcrate dcnsitq

of binding sites over the pars rcticulata. but not over

the pars compacta or ventral tcgmcntal arc;1 (Fig. ia ).

The red nucleus had a low densit! of bIndIng sites

(not shown). The interpeduncular nucleus N;IS I’rec of

binding except for a lateral horder. the pc)\tcrlor

subnucleus.

Very high densities of binding L;lte:, ucrc found 111

the inferior ollvary complex. the lateral rctlcular

nucleus (parkoccllular) (Fig. 3g J ). the h~poplossal

nuclear complex (Fig. 3h j ). in the dorsal cc>chlcal-

nucleus and dorsal region of icntral nuclcu~ (Fig.

3e.f). High binding densities were found 111 the WII-

tary tract and dorsal vagal nuclei (Fig. 3h.l 1.

A very high density of binding sites ~a:, found in

the medial vestibular nucleus. but was modcrate in

the superior, lateral and inferior nuclcl 01‘ the WS-

tibular complex (Fig. 3f.g).

Moderate binding site denslticj hcrc obzcrvcd 111

the cercbellar-iclated pontinc nuclcl in ;I patchy dls-

tribution (Fig. 3b). in the molecular and Pwkinyz cell

layers. but none in the granular layer of rhc cerchellar

cortex (Fig. 3d-g).

Moderate receptor densities \+crc lound In the

facial nuclei (Fig. 3e.f). in the dorsal and Icntral

nuclei of lateral lemniscus (Fig. 3~). nuclcua ;imhl-

guus (Fig. 3h). area postrema. and in the prlnclplc

sensor? nucleus of the trigetninal nerve (Fig. 3d.e). In

the dorsal column nuclei binding densltlc\ wcrc mod-

erate to high (Fig. 31). Moderate le\cls 01‘ handing

sites appeared in the pontinc and mcdullar! rcucular

formation (Fig. 3g j ). the gigantoccllular rcgiun. the

dorsal tegmental nucleus. parabrachlal (particularly

caudovcntral). and mesenccphallc trigcmlnal nuclei

(Fig. 3g j ). Low binding densities were found in the trlgcmlnal

motor nucleus (Fig. 3f.g). dorsal raphc n~~clcu~. the

superior olivary complex (Fig. 3f.p) and the deep

cerehellar nuclei (not shown). IAM bIndIng densltlc\

also occurred in the mtddle and infcrlor cerehcllar

peduncles and cercbellar white ma1tcl-. -2 high dcnslt)

of binding was also found over the pa mater I Fig5 2

and 3). This binding appeared to bc hpecltic 111 that

it was totally displaced by IO ” M cold r(‘GRP

The pattern of distribution ol‘[“‘l]hC‘GRP hlndlng

was very Gmilar to that of [““l]rC‘GRP. However

there wcrc some minor diffcrenccs in that the mod-

crate to \crq high binding dcnsltics seen Mlth r(‘GRP

radioligand o\cr: the prcoptic to mammilla~-1 region\

of the hypothalamus: the subthalamic nuclcu~: the

OVLT and the medial amygdalold nucleus Mere

not detected in the studies using [“‘l]hC‘<jRP.

[‘2’I]hCGRP had hlpher rclatl\c binding dens~t~c~

than [“‘l]rCGRP over the optic nerves and chiasm. internal capsule and stria terminalis.

In this study we have demonstrated high affinity binding sites with both rat and human CGRP in rat

cerebellar cortex, with the rat CGRP having slightly higher affinity than the human CGRP. The C- terminal fragments had very low potency, as has been found previously with rat and human nervous

tissue”“.” and rat pancreatic tissue.5’ Salmon calcitonin and its des Se? [Gly”] analogue

were both very weak in displacing [“51]hCGRP.

demonstrating that the CGRP binding site labelled in these studies is distinct from the calcitonin binding

site.

Receptor distribution

These studies have revealed a set of anatomically discrete binding sites for CGRP in the rat CNS. These receptors have a characteristic distribution

which is distinct from those previously reported for other neuropeptide receptors.2’~‘0.40~4’X43~4X

The current results are mainly consistent with the

preliminary localization of binding sites for CGRP in the rat brain by others. 9 “J Also, during the prepara-

tion of this manuscript localization of CGRP binding sites in rat brain by in vitro autoradiography was reported;‘7”.50 these reports confirm our localization

of receptors in the nucleus accumbens, the amygdala, ventral caudate, putamen, habenular, inferior olivary and vestibular nuclei, the superior and inferior col-

liculis, the cerebellum, pons, reticular formation and parts of the hypothalamus and mammillary bodies. The current study greatly extends these observations by reporting further structural detail in these areas as well as localizing receptor binding in many other regions of the brain. Furthermore, the localization of

binding sites to the molecular and Purkinje cell layers in the rat brain parallels the localization of binding sites in these layers of human cerebellar cortex.“

In the present data, CGRP binding is associated with successive levels in several functional systems. This is especially noteworthy for the auditory system, where receptors localized in the cochlear nuclei.

superior olivary nuclei, nuclei of the lateral lemniscus. central nucleus of the inferior colliculus. medial ge- niculate nucleus, and the middle or receptive layers of the auditory cortex. Along the gustatory pathways’ receptors were found in association with the nucleus of the solitary tract, the parabrachial nuclei, posterior ventromedial thalamic nuclei, central amygdaloid nucleus and in gustatory cortex. A less consistent system association was found for the visual system with high binding densities in the optic layers of the superior colliculus and a moderate binding density in the lateral geniculate nuclei and visual cortices. In the olfactory system, there was a low binding density over the mitral cell dendrites of the olfactory bulb, a

moderate binding density m prfmar> dnd .+icd?lid,:i 1. (entorhinal) olfactory cortex. and moderate 12.1 hf$ binding densities in parts of the ;mygdal.,

The neuronal sources of input ;Lxon>. II- oh< c‘t’i-~: bellar cortex included some I)/ 1 hc m0~1 10 ia!)for. dense structures in the rat brain: mfcrlol .i/i\,it.! complex, lateral reticular nucleus. pontfne nucici, ,IIU.~

intermediate spinal gray. The molcculnr I,[! ~‘i’ .)i 1 tic ccrebellar cortex. together Mlth the Purklnc <Y_+I ra)cr- showed a moderate intensttl of binding. SlnLc the granular layer was virtually dc\oid of l:ib~bi. Iiic mossy-fibre terminals of reticula-. ponto-. ,~ntl qrw- cerebellar neurons probably do not possess t’GRP receptors. Terminals of climbfng tibrch !r111n lili’ inferior olive might be responsible for bin&n)! ww over the Purkinje cell soma and dendritic (molecular 1 layers; it is equally possible that the posth>napt~c Purkinje ccl1 membranes contam CGRP hindmg sites. The binding in the deep cerebellar nuclei :.ould be attributed to climbing-fibrc collaterai. (11 li) Purkinje axon terminals.

High densities of binding sites in the vcntml drl-

atum: nucleus accumbens, olfactory tuber&. and ventral putamen, although patchy. differcntiatcd this region of the neostriatum markedly from the dorsal and lateral areas of the caudate putamen complex.

Several other comparable neurochemical distlnctions have been reported: CCK-containing axon term+

nals,‘,‘* somatostatin,’ and a number of other neuro-

peptides (Table 2 of Graybiel and Ragsdale”, have been observed to be more densely concentrated rn rhc ventral striatum-accumbens region than in rhr dorsai

striatum. The sharp demarcation of the icntral CGRP binding zone provides yet a further distinction

from the dorsal striatum. In other component!. of‘ the basal ganglia, binding was absent from the pallidal

complex, including ventral pallidum ,md i’nt(o-

peduncular nucleus, but present over the huh,tantta nigra pars reticulata regarded as part of rhc same functional output group as entopeduncular ilucleus (see Ref. 14).

Localization of CGRP binding generaIl> main- tained a fair correlation with the localiration of CGRP-immunoreactivc (C’GRP-I) fibrcs and celIs’~.17,47.49.‘1.i4 and although receptor densltq &d not always parallel fibre population. the extent of C‘GRP innervation was often similar to the dcnsit) 111’

binding sites. For example. a moderate number of CGRP-l tibres

occurred in the caudal nucleus accumbenx which decreased rostrally*’ and we found a high density (rl’ binding in caudal nucleus accumbens with httlc or nc: binding rostrally. Also, ventral caudate putamcn had high levels of both CGRP-I fibres.” and ri high density of binding site in the present study. Howw-.

in the amygdala. CGRP-I tibres were found ln most nuclei. with highest levels in the central nucleus.- although binding density was highest in the lateral nucleus with lower levels in the other nuclei (Table 1 ). In the inferior colliculus. where binding sites wcrc

CGRP binding sites in rat bran

I‘sund o\er most of the structure. CGRP-I fibres were (ion.” The vcntromedial hypothalamus. lateral hypo-

reported only in the superficial regions.“ There arc thalamus and central nucleus of the amygdala ha\c

many known cases where peptidc distribution and all been shown to modulate gastric acid secretion.‘-

hindlng site localization are not parallel. A number ol and the high densities of CGRP binding associated hypothesis have been advanced to explain this and with these nuclei may therefore mediate this actIon ol thc\c trc re\icwcd by Kuhar.” CGRP.

The major differences between the reported local- ~zatlon of CGRP”,“J”“’ and the binding site mapping are the presence of high densities of binding \itc\ l’ound in association with both the inferior oli\arv and pontine nuclei. No CGRP-I cells or fibres ha\c been rcportcd in these regions although our lindmgs indicate that a detailed study of these regions should he undcrtakcn.

In ?he brainstem. CGRP binding was observed in cranial nerve nuclei 3. 4. 5. 7. 10. 12 and nucleus of the solitaq tract. These nuclei contain many cells unmunorcactive for C‘GRP” “.” and in areas inner- vated bq thcsc nuclei: the tongue.“J palate.” eye”’ and

oc\ophapus.“J both sensory and motor CGRP-I axon

terminals have been reported. Thus. some of the C‘<iRP receptors in these nuclei may be involved in

the rccc~pt of scnhory Information and in modulating

\uhscquent ncuronal output. The presence of CGRP mRh A”’ in thcsc nuclei also suggests the possibility 01 ;Il~t~~rcgtllation of CGRP levels and output from ths CCll\.

CGRP has been colocalired to neurons containing acetylchohnestorase in some peripheral motor neur- ons associated with striated muscle.“’ Therefore It is

interesting to note that the CGRP binding OWL- sionally closely corresponds to arcah of acet>l- cholinesterase staining. ” An example of this IS the

zone of strong binding m the central nuclcuz of the inferior colliculus which closely corresponds to the arca of heavy acctylcholincsterase stalnlng.” Binding in the ventral diagonal band nucleus also corresponds with acetylcholinesterase histochemihtr> and with the presence of cholinergic ncurons.i’ Phcsc

findings suggest the possibility that acetylcholinc and

CGRP may also be coloculized in mnc ncul-on> within the CNS. However. In other chollnerglc

regions (e.g. dorsal striatum) there wcrc no (‘GRP binding sites.

Inlraccrebroventrlcular (ICV) administration 01 r( (iliP Inhibits the spontaneous secretory episodes

of growth hormone (GH) in the rat.” The majorit) 01 lihrcs contaming growth hormone releasing hor-

mont’ (GHRH) arise from cell bodies in the medlal

pcrltornical region of the lateral hypothalamus and in the :ircuatc nuclei of the hypothalamus and form a fan-llkc projection to the median eminence where a

dcnsc accumulation of GHRH-containing processes and terminals are found.” Thus. the high levels of

CXiKP binding over the arcuate nucleus and median CIIIIIICIICC suggest that the observed action of CGRP on <if-l secretion could be mediated by an inhibitory ~IYclt on GHRH release at this site.

Stimulation of the ventromedial hypothalamus de- ~I-LYI\C’$ appetite. while stimulation of the lateral hy- p~~thalamus induces feeding.” lhJh Therefore the high h~nti~np densities surrounding the ventromedial hypo- thal mus met parts of the lateral hypothalamus &nay mcci~ate the observed decrease in appetite following I(.‘\ (‘GRP injection. However. the association of ( (;RP binding sites with successive levels of gusta-

tar’! atfercnt path\vays suggest the possibility that (‘GRP ma! also ha\e an Indirect effect on appetite through taste.

Rat CGRP differs from the human by four amino acid substitutions. Three of there reprcscnt charge changes to the molecule at physiological pH The

other substitution (human Ala,. rat Scr’ ) rcpresentj the substitution of a polar residue for :I nonpolar residue. The availability of both a human and rat labelled peplide enabled the parallel investigation 01‘

binding site localization with both pcptldes. The overall pattern of binding of both radioligands wa\

very similar and they also shoued cross dlsplacemcnt

in membrane radioreceptor assays. However. thcrc

were some minor differences between the locahzation of binding using the two probes. In particular high

binding densities were seen oicr the hypothalamus. OVLT. and the subthalamic nuclcuh. u\inp [“‘I]rCGRP but not [“‘l]hCGRP. It 15 not yet clear

if this difference represents the ability of the higher- affinity radioligand ([“‘I]rCGRP) to ktbcl these sites

or a possible subclass of CGRP site of dif?crent ligand specificit); this is currently bcmp in\cstigated

Roth lateral hypothalamic and ICV administration of 1 ‘<;RP induced a decrease in gastric acid secre-

Binding sites of high affinity. and high spcciticlt>

for CGRP have been demonstrated for CGRP in rat. These sites have a characterl\tic and anatomically distinct pattern of distribution which suggest multiple

roles for the pcptide including modulation of audi- tory. visual. gustatory and somatosen<or> proce<slnp and in neuroendocrinc control.

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