letters to nature
the number, order and duration of copulations, and determination ofoffspring paternity. Further studies of sperm competition involving
taxa at different stages of divergence are needed to determine thefrequency of conspecific sperm preference and the rapidity withwhich it arises. Drosophila is an ideal genus in which to pursue genetic
and anatomic studies of conspecific sperm preference because there
are mutant markers in many species, the fate of sperm can befollowed easily within a female, and there are tools to produce malesMlacking sperm or various components of the seminal fluid20.
.........................................................................................................................

Methods

All stocks were reared at 24 8C with a 12-h light–dark cycle. Stock information
is available from the author on request. Males and females were collected as
virgins under CO2 anaesthesia and stored in 8-dram food vials. Each female’s
first mating took place on the fourth day after eclosion, and her second mating(if any) occurred two days later. All males were four days old at the time ofmating. Flies were transferred without anaesthesia into a food vial forobservation, and 10–60 vials were observed simultaneously. All mating
observations began within 1 h of lights coming on in the incubator, andlasted from 45 min to 10 h depending on the ease with which mating occurred.
All copulations were observed and timed (unless otherwise noted), and males
were removed from the observation vial within 5 min of copulation ending.
Females failing to mate on the first day were discarded. Of the females that did
mate successfully, a random subset was never given the opportunity to remate.
Females that refused to remate were discarded. Females were stored individually in food vials for the two days between the first and second mating. They
were transferred to a fresh vial for the second mating, and thereafter transferred
to fresh vials every three days until they stopped laying fertile eggs. All offspringfrom each of these vials were reared to adulthood and scored for paternity.
Paternity was determined by the presence of a mutant marker, except in the caseof hybrids between D. simulans and D. mauritiana, which were distinguished by
the shape of the male genital arch. F1 females were obtained by crossing D.
simulans ebony females with D. mauritiana singed males. XO males were
produced by crossing virgin D. simulans females to males from a D. simulans
attached-X, attached-XY stock. All male offspring from this cross lack only a Y
chromosome, and so should produce normal seminal fluids21. All matings were
performed as described above with the following exceptions. Copulations
between D. simulans females and D. mauritiana males are often abnormallybrief, resulting in interruptions of sperm transfer22. To control for this, allmatings known not to result in sperm transfer were excluded from the study.
Insemination was determined directly for single matings and first matings by
the presence of larvae in the female’s food vial. Insemination could not be
assessed directly for second matings, but all matings shorter than thoseresulting in insemination after a single mating (,8 min) were excluded fromthe study. Matings of D. mauritiana females with D. simulans males could not
be observed owing to the extreme discriminatory behaviour of the females.
Instead, 10 virgin males and 10 virgin females were crowded together on thethird day after eclosion, and left for 24 h at 24 8C. Females were transferredunder CO2 anaesthesia to individual vials on the morning of the fourth day
after eclosion. Insemination was determined by the presence of larvae in thevials. Inseminated females were aged and re-mated to D. mauritiana males as
described above.
Received 25 February; accepted 19 May 1997.
1. Fisher, R. A. The Genetical Theory of Natural Selection (Clarendon, Oxford, 1930).
2. Rice, W. R. Sexually antagonistic male adaptation triggered by experimental arrest of female
evolution. Nature 381, 232–234 (1996).
3. Palumbi, S. R. & Metz, E. C. Strong reproductive isolation between closely related tropical sea urchins(genus Echinometra). Mol. Biol. Evol. 8, 227–239 (1991).
4. Lee, Y. H. & Vacquier, V. D. The divergence of species-specific abalone sperm lysins is promoted bypositive Darwinian selection. Biol. Bull. 182, 97–104 (1992).
5. Rice, W. R. & Hostert, E. E. Laboratory experiments on speciation: what have we learned in 40 years?
Evolution 47, 1637–1653 (1993).
6. Parker, G. A. Sperm competition and its evolutionary consequences in the insects. Biol. Rev. 45, 525–
567 (1970).
7. Pyle, D. W. & Gromko, M. H. Repeated mating by female Drosophila melanogaster: The adaptive
importance. Experientia 34, 449–450 (1978).
8. Chapman, T., Liddle, L. R., Kalb, M. J., Wolfner, M. F. & Partridge, L. Cost of mating in Drosophilamelanogaster females is mediated by male accessory gland products. Nature 373 241–244 (1995).
9. Hewitt, G. M., Mason, P. & Nichols, R. A. Sperm precedence and homogamy across a hybrid zone in
the alpine grasshopper Podisma pedestris. Heredity 62, 343–353 (1989).
10. Bella, J. L., Butlin, R. K., Ferris, C. & Hewitt, G. M. Asymmetrical homogamy and unequal sex ratio

666

from reciprocal mating-order crosses between Chorthippus parallelus subspecies. Heredity 68, 345–
352 (1992).
11. Gregory, P. G. & Howard, D. J. A postinsemination barrier to fertilization isolates two closely related
ground crickets. Evolution 48, 705–710 (1997).
12. Wade, M. J., Patterson, H., Chang, N. W. & Johnson, N. A. Postcopulatory, prezygotic isolation inflour beetles. Heredity 72, 163–167 (1994).
13. Smith, R. L. (ed.) Sperm Competition and the Evolution of Animal Mating Systems (Academic, New
York, 1984).
14. Gromko, M. H., Gilbert, D. G. & Richmond, R. C. in Sperm Competition and the Evolution of Animal
Mating Systems (ed. Smith, R. L.) 371–426 (Academic, New York, 1984).
15. Harshman, L. G. & Prout, T. Sperm displacement without sperm transfer in Drosophila melanogaster.
Evolution 48, 758–766 (1994).
16. Clark, A. G., Aguade, M., Prout, T., Harshman, L. G. & Langley, C. H. Variation in sperm displacement
and its association with accessory gland protein loci in Drosophila melanogaster. Genetics 139, 189–
201 (1995).
17. Hughes, K. A. Quantitative genetics of sperm competition in Drosophila melanogaster. Genetics 145,
139–151 (1997).
18. David, J., Lemeunier, F., Tsacas, L. & Bocquet, C. Hybridation d’une nouvelle espèce, Drosophila
mauritiana avec D. melanogaster et D. simulans. Ann. Génét. 17, 235–241 (1974).
19. Eberhard, W. G. Female Control: Sexual Selection by Cryptic Female Choice (Princeton Univ. Press, New
Jersey, 1996).
20. Herndon, L. A. & Wolfner, M. F. A Drosophila seminal fluid protein, Acp26Aa, stimulates egg laying infemales for 1 day after mating. Proc. Natl Acad. Sci. USA 92, 10114–10118 (1995).
21. Ingman-Baker, J. & Candido, E. P. Proteins of the Drosophila melanogaster male reproductive system:
two-dimensional gel patterns of proteins synthesized in the XO, XY, and XYY testis and paragonial
gland and evidence that the Y chromosome does not code for structural sperm proteins. Biochem.
Genet. 18, 809–828 (1980).
22. Coyne, J. A. The genetics of an isolating mechanism between two sibling species of Drosophila.
Evolution 47, 778–788 (1993).
Acknowledgements. I thank K. C. Price, P. Rooney, K. Kyle, C. Kim and K. Dyer for technical assistance; J.
Coyne for inspiration and help; and H. A. Orr, M. Turelli, M. Noor, N. Johnson and M. Wade forcomments. This work was supported by an NSF predoctoral fellowship to the author, and by an NIH grant
to J. Coyne.
Correspondence and requests for materials should be addressed to the author (e-mail: csprice@midway.
uchicago.edu).

Molecular evidence fromretroposons that whales
form a clade withineven-toed ungulates
Mitsuru Shimamura*, Hiroshi Yasue†,
Kazuhiko Ohshima*, Hideaki Abe*, Hidehiro Kato‡,
Toshiya Kishiro‡, Mutsuo Goto§, Isao Munechikak& Norihiro Okada** Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Yokohama, Kanagawa 226, Japan† Animal Genome Research Group, National Institute of Animal Industry,2 Ikenodai, Kukisaki-machi, Inashiki-gun, Ibaraki 305, Japan‡ Large Cetacean Section, National Research Institute of Far Seas Fisheries,
5-7-1 Orido, Shimizu, Shizuoka 424, Japan§ Genetic Ecology Section, The Institute of Cetacean Research, 4-18 Toyomi-cho,
Chuo-ku, Tokyo 104, Japank Chiba Zoological Park, 280 Minamoto-cho, Wakaba-ku, Chiba 264, Japan.........................................................................................................................

The origin of whales and their transition from terrestrial life to a
fully aquatic existence has been studied in depth. Palaeontological1,2, morphological3 and molecular studies4–7 suggest thatthe order Cetacea (whales, dolphins and porpoises) is more closely
related to the order Artiodactyla (even-toed ungulates, including
cows, camels and pigs) than to other ungulate orders. The
traditional view that the order Artiodactyla is monophyletic has
been challenged by molecular analyses of variations in mitochondrial and nuclear DNA5–7. We have characterized two families ofshort interspersed elements (SINEs) that were present exclusively
in the genomes of whales, ruminants and hippopotamuses, but
not in those of camels and pigs. We made an extensive survey ofretropositional events that might have occurred during the divergence of whales and even-toed ungulates. We have characterizednine retropositional events of a SINE unit, each of which provides
phylogenetic resolution of the relationships among whales, ruminants,

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letters to nature
hippopotamuses and pigs. Our data provide evidence that whales,
ruminants and hippopotamuses form a monophyletic group.
We attempted to resolve the issue of whether the order Artiodactyla is monophyletic or paraphyletic by basing our analysis on
the presence or absence of SINEs at particular orthologous loci of
certain groups of species. SINEs are retroposons that have been
amplified and integrated into genomes by retroposition8–11, that is,by the integration of a reverse-transcribed copy of RNA. As aconsequence of the nature of retroposons, SINEs can be foundspecifically within members of a particular clade10–13. It is generally
believed that SINEs are not excised precisely and, moreover, that
SINEs have not been inserted independently at orthologous lociwithin different evolutionary lineages. These features mean that
SINEs are very useful for the reconstruction of phylogenetic
relationships among closely related species12,13.
We have characterized two new and different families of SINEs,
designated the CHR-1 (for Cetacea, hippopotamus and Ruminantia) and CHR-2 family of repeats, from the genomes of several
species of whales. The consensus sequences of these two families of
SINEs are shown in Fig. 1a. The order Artiodactyla is traditionallydivided into three suborders: Ruminantia (chevrotains, deer, cows,
sheep), Tylopoda (camels) and Suiformes (pigs, peccaries andhippopotamuses). Dot-hybridization studies showed that these
two families of SINEs are distributed extensively in the genomesof Cetacea, Ruminantia and hippopotamus, but were not detectedin those of Tylopoda or of Suiformes other than the hippopotamus(Fig. 1b). These results suggest that whales, ruminants and hippopotamuses form a monophyletic group. This possibility prompted
us to isolate specific genomic loci at which SINEs had been inserted.
The first approach to this involved random screening, to identify
loci that contained a CHR-1 or CHR-2 SINE unit, followed bycloning and sequencing. Polymerase chain reactions (PCRs) were
performed with genomic DNA from various cetacean and artiodactyl species to determine whether or not the locus might be
informative from a phylogenetic perspective. Second, we performed
a comprehensive survey of the protein-coding genes, in standard
databases, in which an intron contained one unit of CHR-1 or CHR2. When the length of the intron was short enough for generation ofa PCR product from the entire intron, we designed one set ofprimers by reference to the sequences of exons. We used these two
approaches to characterize seven different loci with a CHR-1 orCHR-2 SINE unit, as described below.

Our analysis indicates that a CHR-2 SINE had been integrated at
the locus Pm52 in a common ancestor of cetaceans (Fig. 2A). The
patterns of PCR products are shown in Fig. 2A, a. We performed
hybridization experiments with the SINE sequence to confirm that
the SINE unit had been integrated in a common ancestor of all
cetaceans (Fig. 2A, b), and with the flanking sequence to confirm
that the orthologous locus of each species had been amplified
accurately (Fig. 2A, c). The presence of the SINE unit in longer
fragments (about 620 base pairs in length) in cetaceans (lanes 1–7)
and the absence of the SINE unit in shorter fragments (about
230 base pairs (bp) in length) in artiodactyls (lanes 8–15) wereconfirmed by sequencing. The small fluctuations in fragment length
were due to insertions and deletions of several nucleotides (data notshown). The Pm72 locus yielded similar results (Fig. 2B). The
presence of these two loci indicates that the order Cetacea forms amonophyletic group.
The locus pgha3, at which a CHR-1 SINE was integrated in intron
C of the gene for the a-subunit of a pituitary glycoprotein hormone(Fig. 2C), and locus c21-352, at which a CHR-1 SINE was integrated
in intron C of the gene for steroid 21-hydroxylase (Fig. 2D),
demonstrate the monophyly of ruminants.
Locus Gm5 was isolated by random screening by using CHR-1
SINE as probe. The SINE unit seems to have been integrated in acommon ancestor of cetaceans, ruminants and hippopotamuses,suggesting that these three evolutionary lineages are monophyletic(Fig. 2E). The sequences of the fragments from the short-finned
pilot whale, cow, hippopotamus and Bactrian camel confirmed thepresence of the SINE unit in the longer fragment, and its absence inthe shorter fragment, respectively. In lane 15 for the pig, a longer
band was detected, but sequencing showed that this was due toinsertion of another SINE unit, PRE-1 (ref. 14) in another site of thislocus (data not shown).
The loci aaa228 and aaa792 are both derived from the gene for thea-subunit of the F(0)F(1) ATP synthase (designated the atpA1 gene)in the bovine genome. At locus 228, a CHR-1 SINE is present in the
intron between exons 2 and 3, suggesting monophyly of cetaceans,ruminants and hippopotamuses (Fig. 2F, a). Hybridization experiments using two different kinds of probe (Fig. 2F, b and c)
confirmed this conclusion.
Locus aaa792, between exons 10 and 11, is more complex. Threedifferent families of SINEs (CHR-1, CHR-3 and Bov-tA15) became
associated independently with this locus during the evolution of

Figure 1 The two newly isolated families of SINEs, CHR-1 and CHR-2, were
present exclusively in the genomes of whales, ruminants and hippopotamuses.
a, The consensus sequences of CHR-1 and CHR-2. The tRNA-derived region isunderlined in each case. These sequences will appear in the DDBJ, EMBL and
GenBank databases under the accession numbers: AB005033 and AB005034. b,
Dot hybridization experiment.

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letters to nature
Figure 2 Analysis of the seven loci at which a
SINE unit(s) was inserted during the evolution of
cetaceans, ruminants and hippopotamuses: A,
Pm52; B, Pm72; C, pgha3; D, c21-352; E, Gm5; F,
aaa228; G, aaa792. a, Products of PCR; b, c,
results of hybridization experiments with different kinds of probe, namely, a unit sequence of
the SINE (b) and the flanking sequence (c),respectively. In G, d and e show results ofhybridization experiments with two different
SINE probes, the CHR-2 SINE and the Bov-tA
SINE, respectively.

cetaceans and even-toed ungulates. The first integration event,involving a CHR-1 SINE, occurred in a common ancestor of
cetaceans, ruminants and hippopotamuses (Fig. 2G, a–c). The
pattern of PCR products is shown in Fig. 2G, a. Hybridization
experiments with the CHR-1 sequence (Fig. 2G, b) and the flanking
sequence (Fig. 2G, c) as probe, respectively, showed that the SINE
unit was integrated at the orthologous loci of the species designatedabove lanes 1–13, but not at those of the camel (lane 14) or the pig(lane 15). These results confirm the monophyly of cetaceans,ruminants and hippopotamuses, excluding camels and pigs. However, the lengths of fragments generated by PCR (Fig. 2G, a) variedamong species (lanes 1–13), but we deduced from the sequences ofthe main fragments that the other two different kinds of SINE wereinvolved in this locus. Experiments using new probes confirmedthat a CHR-2 SINE was integrated in the lineage of the minke andhumpback whales (Fig. 2G, d, lanes 1 and 2), and that a Bov-tA
SINE was integrated in the lineage of the pecora (cows, sheep, deerand giraffes), indicating that the lineage forms a monophyletic
668

group (Fig. 2G, e, lanes 8–11). The sequences of major fragments
are shown in Fig. 3.
All results for the seven loci are congruent (Fig. 4), and provide
conclusive evidence for the paraphyly of the order Artiodactyla,
which should include the order Cetacea, and for the paraphyly of thesuborder Suiformes, from which hippopotamuses should be
excluded. Hippopotamuses form a monophyletic group with cetaceans and ruminants.
The inclusion of cetaceans within the order Artiodactyla has been
proposed previously5, as the Ruminantia/Cetacea clade with the
Suiformes (pigs and peccaries) as an outgroup. The possibility ofclustering the hippopotamus with the Cetacea has also beensuggested6,7, even though hippopotamuses have traditionally beengrouped with pigs and peccaries on morphological grounds16.
However, careful reanalyses of available molecular data17–19 indicatethat the hypothesis of artiodactyl paraphyly was not supported
convincingly from a statistical point of view. However, recent
analyses of genes for milk casein7 provided new, convincing support

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Figure 3 An alignment of sequencesof the aaa792 locus in the cow (BT,Bos taurus), minke whale (BA,
Balaenoptera acutorostrata), hippopotamus(HA,

Hippopotamus

amphibius) and bactrian camel(CB, Camelus bactrianus). Boxed
sequences indicate direct repeatsarising from duplication upon retroinsertion. The underlined sequences
show the sequences used for primers.

Bars

indicatedeletions.

Nucleotides identical to those in
the cow are indicated by dots.

Figure 4 Phylogenetic relationships among cetaceans and artiodactyls, as
deduced from the sites of insertion of SINEs. Arrows indicate the timing ofinsertion of SINEs. Types of SINE are shown in parentheses.

for the ((cetaceans, hippopotamuses), ruminants) tree, supportinga previous hypothesis5. Our analysis of SINE retrotranspositions
seems to provide unambiguous support for that hypothesis.
The conclusions from our retropositional analysis are inconsistent with earlier morphologically based hypotheses16,20,21. Paleontological and morphological data suggest that modern whales
originated from the Archaeocetes (primitive aquatic cetaceans),
which first appeared in the early Eocene epoch22. The Archaeocetes
are believed to have originated from mesonychians, which appeared
before the Eocene20. However, the most primitive artiodactyls(Dichobunids) first appeared in the early Eocene, and the originof nearly all the families of artiodactyls can only be traced back to
the middle or the late Eocene23,24. Such a sequence of appearance of
these animals is inconsistent with our molecular data. However, a
recent calibration of molecular clocks suggests that divergencesamong orders of eutherian mammals can be traced back more than
100 Myr. Hence, diversification of avian and mammalian orders
might not have been an adaptive radiation after the Cretaceous/
Tertiary extinction event (65 Myr ago), but might have beencorrelated with the fragmentation of emergent land areas during
the Cretaceous25. We believe that recent molecular data will lead tothe reinterpretation by palaeontologists of many fossil records of
Artiodactyla to match our conclusions. Extensive morphologicalreversals and convergences, as well as large gaps in the fossil record,Mwill then have to be acknowledged.
.........................................................................................................................

Methods

Polymerase chain reaction. PCR was performed in a 50-ml reaction mixture

containing 0.2 mM dNTP, 200 ng of primer, Tth buffer (final Mg2+ concentration, 1.5 mM) and 1 unit of Tth DNA polymerase (Toyobo, Osaka). Annealing
temperature was chosen from 49 8C to 58 8C. A portion of the PCR products
was analysed by electroporesis in an agarose gel containing 2% (w/v) Nusieve
GTG and 1% (w/v) Seakem GTG (FMC BioProducts, Rockland, ME). Hybridization and washing were performed as described13.
Sequences of primers for PCR. Pm52 locus, 59 primer, 59-TCCTGATTCC(C/T)CTGAACAAA-39, and 39 primer, 59-GGG(G/A)AAGACT(C/T)CCA(G/A)
NATURE | VOL 388 | 14 AUGUST 1997

(C/T)TTTGAAAT-39; Pm72 locus, 59 primer, 59-TTTAAAGCATGGCAGTTGGATTT(G/A)T-39, and 39 primer, 59-GGATCTGTTTTTACTTTGACC-39;
pgha3 locus, 59 primer, 59-TCGGTGTGGTTCTC(G/C)AC(C/T)CT-39, and 39
primer, 59-TGC(C/T)CCAATCTATCA(G/A)TG(C/T)ATG-39; c21-352 locus, 59
primer, 59-GAGAATTCCTTCTG(G/A)AT(G/A)GT(G/C)AC-39, and 39 primer,
59-(G/A)(C/T)CCGCAGCTCCATGGA(G/A)CC-39; Gm5 locus, 59 primer, 59GTAATGTGATTTGGCTTAGTGC-39, and 39 primer, 59-TCAGCTCCTGGTGGCAGTCT-39; aaa228 locus, 59 primer, 59-GCTTGATACCTACCACTATGAA-39, and 39 primer, 59-CCTGG(A/C)(A/T)GTCT(G/C)AATTTGCAC-39;
and aaa792 locus, 59 primer, 59-TGTGGA(A/T)(G/T)NTG(G/C)CAGATTT(A/T)AAAG-39, and 39 primer, 59-CAGCCACTTGCTCTTCAATAGC-39.
Locus. Of the seven loci described, Pm52 and Pm72 were newly isolated bycloning and sequencing from a genomic library of sperm whales (Physeter
macrocephalus), and Gm5 was isolated from that of short-finned pilot whale(Globicephala macrorhynchus). The other four loci were found in bovine
genomic sequences in the EMBL database, as follows (accession numbers, locus
name, SINE family): (X00004, pgha3, CHR1); (M11267 and M13545, c21-352,
CHR-1); (X64565 and S48112, aaa228, CHR-1); (X64565 and S48112; aaa792;
CHR-1, CHR-2 and Bov-tA).
Received 31 January; accepted 28 May 1997.
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Acknowledgements. We thank the Zoological Society of San Diego’s Center and Y. Mukai in the Meat
Hygenic Inspection Office in Ueda, Nagano prefecture for providing a sample of DNA from the lesser
Malayan chevrotain and samples of DNA from calf, pig and sheep, respectively. This work was supported
by a Grant-in-Aid for Specially Promoted Research from the Ministry of Education, Science, Sports andCulture of Japan.
Correspondence and requests for materials should be addressed to N.O. (e-mail: nokada@bio.titech.ac.jp).

Formation of olfactory
memories mediated bynitric oxide
K. M. Kendrick, R. Guevara-Guzman, J. Zorrilla,
M. R. Hinton, K. D. Broad, M. Mimmack & S. Ohkura
Department of Neurobiology, The Babraham Institute, Babraham,
Cambridge CB2 4AT, UK.........................................................................................................................

Sheep learn to recognize the odours of their lambs within twohours of giving birth, and this learning involves synaptic changes
within the olfactory bulb1,2. Specifically, mitral cells become
increasingly responsive to the learned odour, which stimulates
release of both glutamate and GABA (g-aminobutyric acid)neurotransmitters from the reciprocal synapses between the
excitatory mitral cells and inhibitory granule cells1. Nitric oxide(NO) has been implicated in synaptic plasticity in other regions of
the brain as a result of its modulation of cyclic GMP levels3–7. Here
we investigate the possible role of NO in olfactory learning. We
find that the neuronal enzyme nitric oxide synthase (nNOS) is
expressed in both mitral and granule cells, whereas the guanylyl
cyclase subunits that are required for NO stimulation of cGMP
formation8 are expressed only in mitral cells. Immediately after
birth, glutamate levels rise, inducing formation of NO and cGMP,
which potentiate glutamate release at the mitral-to-granule cell
synapses. Inhibition of nNOS or guanylyl cyclase activity prevents
both the potentiation of glutamate release and formation of theolfactory memory. The effects of nNOS inhibition can be reversed
by infusion of NO into the olfactory bulb. Once memory has
formed, however, inhibition of nNOS or guanylyl cyclase activity
cannot impair either its recall or the neurochemical releaseevoked by the learned lamb odour. Nitric oxide therefore seems
to act as a retrograde and/or intracellular messenger, being
released from both mitral and granule cells to potentiate glutamate release from mitral cells by modulating cGMP contentra670

tions. We propose that the resulting changes in the functional
circuitry of the olfactory bulb underlie the formation of olfactory
memories.
NO may mediate synaptic plasticity changes in brain regions suchas the hippocampus in order to influence both long-term potentiation (LTP) and memory formation3–7, but this idea iscontroversial9,10. In rodents, the inhibitory GABAergic granuleand periglomerular cells of the primary sensory processing region
for airborne odours, the olfactory bulb, contain large amounts ofnNOS11, which converts arginine to citrulline and NO3,4. We
investigated whether this could also be the case in sheep, andwhether the increased sensitivity of mitral-to-granule cell synapses
that results from learning the odours of their lambs might involve
potentiation of glutamate release by NO through modulation ofcGMP.
We cloned and sequenced the ovine nNOS gene and found that it
was 88% homologous with human12, rat13 and mouse14 sequences(EMBL accession number, X99042). In situ hybridization andimmunocytochemistry indicated that mitral as well as periglomerular and granule cells contained nNOS messenger RNA
and protein (Fig. 1a). The dendrodendritic connections between
the granule and mitral cells in particular contained large amounts ofnNOS immunoreactivity. Only mitral cells expressed the mRNAs
for the aa and b1 subunits of soluble guanylyl cyclase (Fig. 1a),which are both required for NO to cause cGMP formation8. The
granule and periglomerular cells expressed low levels of only the b1
subunit. Thus NO probably only stimulates cGMP formation in
mitral cells.
In brain, NO is released by glutamate acting on both NMDA (Nmethyl-D-aspartate) and AMPA ((2-aminomethyl)phenylaceticacid) receptors3,4,15. In the mouse accessory olfactory bulb, activation of both receptor types is required for the formation ofpheromonal olfactory memory16 and both are present on themain olfactory bulb mitral and granule cells17. We used in vivo
microdialysis to determine whether glutamate acts on both types ofreceptors to evoke NO and cGMP release in the olfactory bulb. Local
retrodialysis infusions of NMDA or AMPA dose-dependently
increased glutamate, GABA, NO and cGMP, although NMDA was
more potent. These actions were blocked by specific receptor
antagonists (Fig. 1b) and the NOS inhibitor L-nitroarginine (LNARG). A selective inhibitor of NO-induced guanylyl cyclase, 1H[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ)18 also prevented
stimulation of cGMP, but not NO release (Fig. 1b). Both L-NARG
and ODQ significantly reduced agonist-induced increases in glutamate and GABA, showing that agonist effects are partly mediated by
NO and cGMP. Infusions of an NO donor, S-nitrosoacetylpenicillamine (SNAP), dose-dependently increased cGMP, glutamate and
GABA; these effects were blocked by ODQ (Fig. 1b). Thus NO can
act as a retrograde or intracellular messenger in the olfactory bulb bystimulating glutamate release through modulation of cGMP.
The importance of NO release in the olfactory bulb for plasticity
changes underlying olfactory memory was investigated in post
partum animals given bilateral local infusions by microdialysis
probes of drugs targeting the NO signalling pathway. In control
animals receiving either no treatment or an inactive enantiomer of
L-NARG (D-NARG), which did not inhibit NOS, it was found thatglutamate, GABA, noradrenaline, NO and cGMP all increased
during the first 30 min after birth (Figs 2 and 3). Behavioural tests
showed that all these animals formed an olfactory memory thatallowed selective recognition of lambs (Fig. 4a). However, allanimals treated with the ionotropic glutamate receptor antagonist
g-D-glutamylglycine (DGG), and NOS inhibitor L-NARG, or theguanyl cyclase inhibitor ODQ, accepted their own and strange
lambs equally, indicating that these agents completely prevented
olfactory memory formation (Fig. 4a). The quality of maternal careshown by ewes towards their lambs during the two hours immediately post partum was unaffected by the drugs, confirming that

Nature © Macmillan Publishers Ltd 1997

NATURE | VOL 388 | 14 AUGUST 1997