Developmental Bram Research, 54 (1990) 295-298 295 Elsevier

BRESD 611367

Calcitonin gene-related peptide in the developing mouse olfactory system

Harriet Baker

Cornell Untversuv Medtcal College, The Burke Rehabdttatlon Center, Whzte Plams, NY 10605 (U S A )

(Accepted 27 March 1990)

Key words Olfactory marker protein, Tyrosme hydroxylase, Phenotypic expression, Rodent, Olfactory epithelium

The development of calcltonm gene-related pepttde (CGRP) was investigated m the mouse olfactory system The peptlde was not found in olfactory receptor neurons in embryos examined from gestatlonal day 13 (El3) to El8 but was present in other brain regions and in peripheral tissues At El8, CGRP-immunoreactive fibers, presumably of tngeminal, not olfactory receptor cell, origin were observed within the lamina proprla These data suggest that m wvo CGRP does not act through olfactory receptor neurons to regulate phenotypic expression m the olfactory bulb

A number of studies have now established that

expression of the dopamine phenotype in olfactory bulb

(OB) juxtaglomerular neurons is under transneuronal regulation from peripheral olfactory afferent receptor cell lnnervatton (see ref. 3 for review). Both chemical and

surgical deafferentatlon result in decreased levels of dopamine In addition, activity, immunoreactlvity, and

mRNA levels are reduced for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamme biosynthesm. Re-

cent experiments, employing cultured embryonic mouse

olfactory bulb, supported a role for calcitonin gene related peptide (CGRP), a 37-amino acid neuropeptide,

in the induction of the OB dopamme phenotype during

development, and by extension in the control of TH expression in the adult 5

CGRP is derived by alternative RNA splicing of the same gene which encodes calcitonln 1'13. The peptide is

highly expressed m discrete regions of the neural axis including the olfactory system 9"13 In order to be imph-

cated in the regulation of TH enzyme expression In the adult, CGRP would have to be localized within olfactory afferent receptor cells and their axons, since lesion of the

receptor neurons profoundly alters the dopamine pheno-

type in the OB Several recent communications indicated that CGRP lmmunoreactwity in adults is found in fibers within the olfactory mucosa and is colocalized with substance p6 7.14 yet there has been no demonstration of

CGRP in olfactory receptor cells or their processes. Alternatively, if CGRP is important only to the

induction of the dopamine phenotype during develop- ment, then it m~ght be expressed transiently in receptor

cells exclusively during gestation. The current studies

investtgated the prenatal distribution of CGRP in the

mouse olfactory system in order to ascertain if the

peptlde can be implicated in the reduction of the dopamine phenotype in vivo, as in vitro.

Embryos were obtained by caesarian section from timed pregnant CD-1 mice (Charles River Breeding Laboratories, Kingston, NY) which were anesthetized

with Nembutal (30 mg/kg, i p.). Gestational day 13 embryos (El3) were immersed in fixative (4% buffered,

0.1 M phosphate buffer, formaldehyde generated from

paraformaldehyde) for 6-12 h Embryos from El5 to E18

were perfused with saline followed by the above fixative, and then postfixed for several hours. All tissue was

cryoprotected in 30% sucrose, 30/~m cryostat sections were collected on subbed slides, and tissue was immu- noreacted as previously described z using specific antlsera to CGRP (provided by Susan Amara and Randy Blakely,

Yale University, New Haven, CT) or olfactory marker protein (OMP, provided by Frank L Margolis, Roche Institute, Nutley, N J) OMP was utilized to demonstrate the developing receptor cell neurons and their processes

in the olfactory epithelium and bulb as this protein previously was localized to these cells during embryo- genesis 8

OMP was demonstrable in olfactory receptor neurons

and processes from about El3 (Fig 1A,C). On the other hand, CGRP-hke immunoreactivlty was not found in the receptor cells or their processes at El3 (Fig. 1B,D) or any gestational age (Fig 2B,D). However, CGRP immuno- reactwe cells and fibers were observed in the spinal cord

Correspondence H Baker, Cornell Umverslty Medical College, The Burke Rehablhtatlon Center, 785 Mamaroneck Avenue, White Plains, NY 10605, U S A

0165-3806/90/$03 50 © 1990 Elsevter Sctence Pubhshers B V (Blomedtcal Dwlslon)

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Fig 2 Dark-field (A ,B) and bright-field (C,D) photomicrographs of OMP- and CGRP-lmmunoreact ive elements in adjacent sections from an E l 8 mouse A robust OMP-lmmunostamed cells and fibers are observed in the olfactory mucosa, in nerve fascicles in the lamina proprla (lp) which innervate the OB, and within the olfactory nerve layer of the OB (ob). Some fibers also exist in deeper layers of the OB Large arrow indicates a region illustrated at higher magnification in C B in a section adjacent to that in A , CGRP-immunostalned fibers are observed m the lamina propria (lp) and in some fibers which approach the OB (small arrows) At this age stained centrifugal afferents are not yet present in the OB Neither receptor cells nor their processes appear to contain C G R P lmmunoreactivity Data from other laboratories 6'7't°'12'i4 suggest that the stained processes in the lamina propria are of trigemmal origin Large arrow indicates region illustrated at higher magnification in D C large numbers of OMP immunoreactlve receptor cells are found in the eplthehal layer (oe) and large bundles of fibers are observed m the lamina propria D' a few varicose fibers lmmunostamed with C G R P are observed in the lamina propna, but no receptor cell staining is observed m the epithelial layer Bar = 260 pm for A and B and 40/~m for C and D

Fig. 1 0 M P and C G R P immunoreactivitles in the mouse OB. A OMP lmmunoreactlvity In the embryonic day 13 (E l3 ) mouse occurs in many receptor cells m the olfactory mucosa (small arrows) and in stamed fibers (arrowheads) which reach the region of the presumptive OB. As it was not possible to perfuse the E13 mouse, darkly stained blood cells are apparent outside the neuroeplthehum, but are distinct from the OMP-lmmunostalned receptor cells and fibers Large arrow indicates region illustrated at higher magnification in C B in a section adjacent to that in A , CGRP-lmmunoreactlvity is not observed in olfactory receptor cells in the E13 mouse or at any gestational age Large arrow indicates regions illustrated at higher magnification in D. C' numerous OMP-immunostamed olfactory receptor cells, some with apical dendrites, are apparent at this magnification D no CGRP-contalnlng cells or processes are observed even at higher magmficatlon E. at E l 3 , CGRP-contalnlng neurons (arrows) are observed in the ventral horn of the spinal cord F in the adult mouse , CGRP-immunosta lned fibers are observed m the olfactory nerve layer (onl, curved arrows) and in centrifugal fibers (straight arrow) of unknown origin. The CGRP-lmmunoreact lve fibers surround but do not enter the glomerulus (gl). a, anterior, cv, cerebral vesicle, nc, nasal cavity, v, ventral A ,B , bar = 1 7 0 # m C - E , bar = 4 0 / ~ m , E bar = 20/~m

298

at E l 3 (Fig. 1E). In the lamina propria, CGRP- immu-

noreactive fibers, presumably of tr igemmal origin, were

observed first at E15. At this age, other regions of the

central and peripheral nervous systems, including the

spinal cord, medulla oblongata and dorsal root ganglia,

exhibited C G R P immunoreactivity. The number and

lmmunos tamlng intensity of these fibers increased with

advancmg gestational age (Fig. 2B,D). In the adult OB,

fibers containing C G R P were seen within the olfactory

nerve layer (Fig. lF) . The origin of these fibers has not

been determined, but substance Pqmmunoreact lve fibers

in the nerve layer previously were ascribed a trtgeminal

origin 6 10,i2. Centrifugal afferent fibers lmmunosta ined

with C G R P also were observed surrounding the olfactory

glomeruh in adult mice (Fig. 1F). These processes were

not observed even in E18 embryos and may develop

postnatally, as was observed for serotonm immunoreac- tlvity in the rat li

These data demonstrate, that in contrast to the

locahzatton of C G R P to receptor cells m vitro 5, the

peptlde could not be demonstrated within neurons of the

olfactory epithelium m vivo, either during embryogenesls

or in the adult. The reason for this difference m

expression ts not clear. The same antiserum was used m

both studies and, as reported for the adult, the antiserum

stained fibers within the developing olfactory mucosa and

other brain regions. Given the absence of an appropriate

spatial-temporal expression of C G R P lmmunoreactlv~ D

within receptor cells in vlvo, the reported presence of

C G R P in vitro Is either an artifact of the culture

condittons or a misldentfflcation of the C G R P stained

cells as receptor neurons

Thus, while C G R P may influence the expression of

TH in cultures of mouse OB, the pept~de does not appear

to play a role in the development or maintenance of the

dopamine phenotype in VlVO since ~t Is not present in the

olfactory afferent neurons or their processes either at

appropriate gestatlonal ages when TH Is first observed

(E18), or in the adult mouse.

The author would hke to thank Mr Jorge Lugo for his technical assistance, Mr Jordan Pontell for photographic assistance, and Drs. Donna Stone and Charles Greer for comment~ on the manuscript Supported by DC00336 and MH44043

1 Amara, S G , Jonas, V, Rosenfeld, M G, Ong, E S and Evans, R M, Alternative RNA processing in calotonm gene expression generates mRNAs encoding &fferent polypeptlde products, Nature, 298 (1982) 240-244

2 Baker, H., Species differences in the distribution ot substance P and tyroslne hydroxylase lmmunoreactlvity m the olfactory bulb, J. Comp Neurol, 252 (1986) 206-226

3 Baker, H , Neurotransmltter plasticity m the luxtaglomerular cells of the olfactory bulb In EL Margohs and TV Getchell (Eds), Molecular Neurobtology of the Olfactory S wtem, Ple- num, New York, 1988, pp 185-216

4 Baker, H , Grillo, M and Margolls, F L, Biochemical and ~mmunocytochem~cal characterization of olfactory marker pro- tern m the rodent central nervous system J Comp Neurol, 285 (1989) 246-261

5 Dems-Domnl, S , Expression of dopammergic phenotypes in the mouse olfactory bulb reduced by the calcltonm gene-related peptlde, Nature, 339 (1989) 701-703

6 Finger, T E , Getchell, M.L., Getchell, T V. and Kmnamon, J C , Affector and effector functions of peptlderglc mnervation of the nasal cavity In B G Green, J R. Mason and M R Kare (Eds), Chemwal Irntatlon m the Nose and Mouth, Dekker, New York, in press

7 Finger, TE , Womble, M and Silver, W.L, Innervatton of the vomeronasal organ of rodents by peptlderglc nerve fibers

Jmmunoreactive for substance P and calotonln gene-related peptlde (CGRP), Chem. Senses, 14 (1989) 698

8 Farbman, A I , Prenatal development of mammalian olfactory receptor cells, Chem. Sense3, 11 (1986) 3-18

9 Gibson, S J , Polak, J.M, Bloom, S R . Sabate, I .M. Mul- derry, P M , Ghatel, M.A, McGregor, G P., Mornson, J EB., Kelly. J S. Evans, R M and Rosenfeld, M G , Calcitonm gene-retated peptlde lmmunoreactlvity m the spinal cord of man and of eight other speoes, J Neuroscl, 4 (1984) 3101-3111

10 Hokfelt, T, Ketlerth, J O, Nllsson, G and Pernow, B, Substance P locahzat~on m the central nervous system and in some primary sensory neurons, Sctence, 190 (1975) 889-890

ll McLean, J H and Shlpley, M T, Serotonerglc afferents to the rat olfactory bulb II Changes in fiber distribution during development, J Neurosct, 7 (1987) 3029-3039

12 Papka, R E and Matuhnois, D H , Association of substance Pqmmunoreact~ve nerves with the murme olfactory mucosa, Cell Tissue Res, 230 (1983) 517-525

13 Rosenfeld, M G , Mermod, J J , Amara, S G , Swanson, L.W, Sawchenko, P E , Rivier, J , Vale, WW and Evans, R.M., Production of a novel neuropeptlde encoded by the CT gene via tissue-specific RNA processing, Nature, 304 (1983) 129-135

14 Silver, W L , Farley, L G , Womble, M and Finger, T E , The etfect of neonatal capsmcm admlmstratlon on tngermnal nerve fibers m the nasal cavity, ('hem Senses. 14 (1989) 748.