Isolation of mouse vomeronasal receptor genes and their co-localization with specific G-protein messenger RNAs1

Molecular Brain Research 60 Ž1998. 215–227

Research report

Isolation of mouse vomeronasal receptor genes and their co-localization with specific G-protein messenger RNAs 1 H. Saito b

a,b,2

, M.L. Mimmack b, E.B. Keverne a , J. Kishimoto

a,3

, P.C. Emson

b,)

a Sub-Department of Animal BehaÕior, Department of Zoology, UniÕersity of Cambridge, Madingley, Cambridge CB3 8AA, UK Laboratory of Molecular and CognitiÕe Neuroscience, Department of Neurobiology, The Babraham Institute, Cambridge CB2 4AT, UK

Accepted 21 July 1998

Abstract Four mouse vomeronasal receptors ŽmV1Rs. have been isolated by similarity to rat vomeronasal receptor ŽV1R. motifs. The four mV1Rs identified in this study are members of two distinct subfamilies. Specific in situ hybridization probes ŽISH. derived from the 3X non-coding regions of the mV1R genes, were used to detect expression of a single receptor and probes from the homologous coding regions were used to detect expression of subfamily members. The ISH results showed that the mV1Rs expressing neurons were scattered in the middlerupper layer of the vomeronasal organ ŽVNO. sensory epithelium in serial VNO sections but were excluded from the deeper layers of the VNO sensory epithelium and these neurons were found to co-express the mRNA for the G-protein Ga i2, and were distinct from the deeper layers of the VNO sensory epithelium where the mRNA for Ga o positive neurons was located. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Ga i2-protein; Ga o-protein; In situ hybridization; Mouse; Vomeronasal receptor

1. Introduction In the mammalian olfactory system, odors are detected by sensory neurons mainly at two locations: the main olfactory epithelium ŽMOE. and vomeronasal organ ŽVNO.. Although the MOE and the VNO are derived from the same olfactory placode w14,20,25,26,28x and have similar morphological structures and lifespan w3,16,48,62,63x, they are functionally distinct. The olfactory neurons in the MOE detect volatile odors w44x while the vomeronasal sensory neurons respond to non-volatile pheromones and play an important role in sexual behavior w24,31,51x and neuroendocrine responses. They have their own individual receptors and mechanisms of signal transduction w8,9,23,38,66x.

)

Corresponding author. Fax: q44-1223-836614; E-mail: [email protected] 1 Sequence data from this article have been deposited with the EMBLrGenbank Data Libraries under Accession numbers Y12724 and Y12725. 2 Current address: Riken, Wako, Saitama, 351-0198 Japan. 3 Current address: Shiseido Cutaneous Biology Research, MGH East Bldg., 149 13th Street, Charlestown, MA 02129, USA.

In the mammalian MOE, volatile odors bind to olfactory receptors which are members of the seven transmembrane domain G-protein coupled receptor superfamily and consist of some 1000 different genes w15x. In higher vertebrates, the neurons expressing distinct subfamilies of receptors are localized in topographically restricted zones within the MOE. Within a given zone, however, the distribution of receptor types is random w40,49,52,61x. The axons of one receptor subfamily project to the same glomerulus in spatially defined regions of the main olfactory bulb ŽMOB. w47,52,53x. The VNO is an anatomically distinct part of the olfactory system with direct ‘hardwired’ connections through the accessory olfactory bulb to the amygdala and hypothalamus w21,30,58x. This connectivity underlies the involvement of the vomeronasal system in the detection of chemical signals of a social nature which have consequences for reproduction and endocrine state w19,31,39,55,65x. The VNO detects chemical signals Žpheromones. produced and received across both sexes. Pheromones can alter the course of pregnancy Žfemales. w11x or can induce early puberty Žfemales. w41,42x. Detection also encourages reproductive behavior and aggression Žfemales and males. w29,31x, and influences female cyclicity and ovulation w5–7,12x. Receptors on vomeronasal

0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 1 8 3 - 1

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X

Fig. 1. Nucleotide alignment showing the similarity of the mV1R 1 and mV1R 2 sequences. 5 non-coding regions of mV1R 1 and mV1R 2 are shown in ŽA. X and 3 non-coding regions are shown in ŽB.. The N-terminal and the C-terminal amino acid sequences are shown alongside the nucleotide sequences. The putative polyadenylation signals are boxed. The ATTT and TATT motifs are underlined. The sequences relating to specific probes expressing single receptors are shown with grey boxes. Accession numbers of the mV1R 1 and mV1R 2 cDNAs are Y12724 and Y12725, respectively.

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sensory neurons detect non-volatile molecules Žpheromones. w24,45x. In 1995, Dulac and Axel w23x isolated seven vomeronasal receptors ŽV1Rs. in the rat using a single cell approach. These receptors were also members of the seven transmembrane G-protein coupled receptor superfamily of proteins with a coding region composed of only one exon, similar to olfactory receptors. However, their sequences do not show any clear homology to olfactory receptors, or indeed to any other members of the family of seven transmembrane domain receptors w50x. In situ hybridization ŽISH. used to examine the pattern of V1R expression in the rat for seven different receptor

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probes, showed they were expressed only in vomeronasal sensory neurons. Neurons expressing V1R subfamilies did not appear to be topographically organized but were randomly distributed in the anterior–posterior axis of the VNO sensory epithelium in several sections. The rat V1R neurons were localized in the apical two-thirds of the vomeronasal sensory epithelium. Recently, another family of vomeronasal receptors ŽV2Rs. related to the Ca2q sensing receptor and metabotropic glutamate receptors were identified w33,46,56x. ISH showed that expression of some members of V2Rs appears concentrated in the vomeronasal sensory neurons located in the deep layer of the VNO, and

Fig. 2. Alignment of the deduced amino acid sequences of four mV1Rs. The predicted positions of the seven transmembrane domains are indicated ŽTM I–TM VII. and black boxes indicate the positions of conserved amino acid residues.

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a few members of V2Rs were expressed in the middle or upper layer of the VNO w33,56x. Recently, several articles have suggested that the G-proteins, Ga i2 and Ga o play important roles in sensory transduction in the vomeronasal sensory neurons w43x. Moreover, Ga i2 and Ga o are expressed in different regions of the VNO w9x. The neurons expressing Ga i2 are located in the apical layer of the rat VNO sensory epithelium, whereas Ga o neurons are located in the deep layers. Each group of neurons projects to the anterior or posterior part of the accessory olfactory bulb ŽAOB., respectively w36x. It is likely that these two topographically distinct neural layers that are characterized by Ga i2 and Ga o have distinct functions in the context of vomeronasal recognition. In this study, we have cloned four mouse vomeronasal receptors ŽmV1Rs. by the similarity to rat V1R motifs and have performed ISH to look at their detailed localization. To complement these studies ‘double’ ISH using radioactive 35 S or 33 P-Ga i2 and Ga o probes, and digoxigenin ŽDIG.-labelled mV1R probes was carried out. Additionally a series of thin sections throughout the extent of the VNO were hybridized with DIG-Ga o, Ga i2 and DIG-mV1R probes, analysed and serially reconstructed. 2. Materials and methods 2.1. Cloning of the mouse Õomeronasal receptors PolyŽA.q RNA was isolated from mouse vomeronasal tissue using the acidic guanidinium thiocyanate–phenol–

chloroform method. cDNAs were produced from DNAse I treated Ž0.1 Urmg RNA for 15 min at 378C. polyŽA.q RNAs. First-strand cDNA synthesis was performed with vomeronasal RNA Ž0.5 mg. by random hexamer priming using a first-strand cDNA synthesis kit ŽClontech.. cDNAs prepared from 0.1 mg RNA were used in subsequent PCR reactions. PCR amplification was performed under the following conditions: 10 cycles at 958C, 45 s; 55–648C, 60 s; 728C, 60 s and then continued for a further 20 cycles at 958C, 45 s; 648C, 60 s; 728C, 60 s. Degenerate PCR primers were designed from the conserved regions of the cloned rat V1Rs expressed in the VNO. Primer nucleotide sequences are 5X TGCrGrTTTArGGCIAArGArGTTCr TAAArGCAAr GCAArC AA, GrTCrGrTGrTIGCCrTCTGTGCrTTCrGIGG3X which spans transmembrane regions TM 3 to TM 6, and 5X CATACAATGAATAAGArGACAArGC, GCrGTCATArGAGCAGr TCAGGATGG, GGGCTGrTACrTIGTGGCATAG3X which spans the whole coding region of the V1R gene. PCR products were subcloned into pBluescript ŽStratagene. and analysed by cycle sequencing using an ABI 373A DNA sequencer. 2.2. Library screening A mouse vomeronasal lZAP II cDNA library Ž10 6 recombinant phages, Stratagene. was screened Ž; 10 5 plaques. with random hexamer primed 32 P-labelled DNA probes derived from the PCR products. Primary screening of filters was performed by hybridizing overnight at 588C in Church buffer Ž0.5 M NaH 2 PO4 pH 7.2r7% SDSr1

Fig. 3. Southern blot hybridized with mV1R 1 ŽA., mV1R 2 ŽB., mV1R 1C ŽC., and mV1R 4C ŽD. probes. Mouse genomic DNA was digested with HindIII ŽH., EcoRI ŽE., or Pst I ŽP.. The scale Žin kb. is indicated on the right handside of the blot.

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mM EDTA.. Filters were washed twice at high stringency in Church buffer at 588C for 15 min. Fifty positive plaques were detected and replated for secondary screening. Secondary and tertiary screens were performed in duplicates at high stringency Ž658C. and low stringency Ž588C. resulting in the identification of two positive plaques mV1R 1 and mV1R 2 , respectively. Isolation of pBluescript clones mV1R 1 and mV1R 2 was performed using the ‘in vivo’ excision procedure ŽStratagene.. A series of deletions were generated for mV1R 1 and mV1R 2 using the ExoIIIrMung

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Bean Nuclease deletion kit ŽStratagene. and subsequently analysed by cycle sequencing using an ABI 373A DNA sequencer. After base calling with the ABI analysis software ŽVersion 2.1., the analysed data were transferred to the software package DNASIS for assembly of overlapping sequence and contigs to generate the complete nucleotide sequence of mV1R 1 and mV1R 2 . In order to compare the sequences of these genes, the GAP program of the GCG package w22x was used for alignment of the receptors.

Fig. 4. ISH of coronal VNO sections. Sections were hybridized with DIG-labelled mV1R 1 ŽA., mV1R 2 ŽB., mV1R 1C ŽC., or mV1R 4C ŽD. riboprobes. ŽE. and ŽF. show the higher magnifications of the regions boxed in ŽC. and ŽD., respectively. The ventral side of mouse nasal cavity is to the left of each photograph. The cells indicated by arrows show strong signal and arrow heads show weak signal. Scale barss 100 mm.

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2.3. Genomic analysis Genomic DNA was isolated from liver samples dissected from adult Balbrc mice. Initially, the samples were resuspended in TNE Ž50 mM Tris–HCl pH 8.0, 100 mM NaCl, 5 mM EDTA pH 8.0. and homogenized. They were then digested by addition of 20 mgrml proteinase K and 0.5% SDS, overnight at 558C. The following day, the digested liver tissue was extracted with phenolrchloroform 1:1. Genomic DNA was then precipitated with ethanol and resuspended in 1 = TE, digested with restriction endonucleases for 4–16 h. Following electrophoresis on a 0.8% agarose gel in the presence of 1 = TAE ŽTris– Acetate, EDTA., the DNAs were then transferred to nylon membrane ŽHybond-Nq Amersham. in 0.4 M NaOH, and cross-linked under UV light. Blots were prehybrized for 20 min at 688C in Church buffer and hybridized with a 32 P-labelled DNA probe Ž5 = 10 5 cpmrml. at 688C overnight. DNA probes were obtained from the coding region of mV1R 1 Ž500 bp., mV1R 3 Ž743 bp., mV1R 4 Ž858 bp., the non-coding region of mV1R 1 Ž410 bp., and mV1R 2 Ž864 bp. by PCR or subcloning. The following day, the blots were washed twice with Church buffer for 15 min at 688C. 2.4. Paraffin-embedded and frozen tissue preparation Paraffin sections were produced for ISH experiments using DIG-labelled riboprobes ŽBoehringer Mannheim.. Twenty-day old Balbrc mice were perfused transcardially with PBS ŽpH 7.2., followed by fixation with 4% PFA in PBS for 1 h. The VNO was dissected from the head and post-fixed in the same fixative overnight. The following day, the VNO was processed through a standard paraffin embedding protocol and 5 mm serial sections were prepared using a microtome. Double-ISH was performed using frozen sections as follows: the VNO was dissected, fixed, resuspended in 30% sucrose for 2 h and stored at y708C until further use. Serial 15-mm sections were prepared with a cryostat.

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formed as described by Kishimoto et al. w40x and Smith et al. w60x. 2.6. Digoxigenin in situ hybridization Sections were digested with 0.02% pepsin in 0.2 N HCl for 15 min at 378C. The sections were hybridized at 508C Žlow stringency. or 578C Žhigh stringency. for 16–20 h Ž0.1 mg riboproberml of hybridization buffer; 50% formamide, 1 = Denhardt’s, 10% dextran sulfate, 4 = SSC.. The sections were washed twice in 2 = SSCr0.1% SDS at 558C Žlow stringency. or 628C Žhigh stringency. for 30 min and treated with RNase A Ž10 mgrml. in 2 = SSC. VNO sections treated with RNase required increased stringency washes enabling detection of single receptor genes. RNAs were detected with an alkaline phosphatase conjugated anti-DIG monoclonal antibody ŽBoehringer. and signals were visualized using Nitro blue tetrazolium ŽNBT, Boehringer. and 5-bromo-4-chloro-3-indolyl-phosphate ŽBCIP, Boehringer. as substrate. 2.7. Double in situ hybridization Sections were digested with 0.02% pepsin in 0.2 N HCl for 5 min at 378C. The sections were hybridized at 578C for 16–20 h Ž0.1 mg DIG-labelled probes, 10 7 cpm radiolabelled proberml hybridization buffer plus 0.01% b-mercaptoethanol.. The sections were washed twice in 2 = SSCr50% formamide containing 0.001% b-mercaptoethanol at 628C for 30 min. The remaining steps were identical to the DIG-ISH protocol described above. After visualization of positive signals using NBTrBCIP as substrate, the slides were washed with water five times and dried at room temperature overnight. The following day, the slides were dipped in Ilford K5D emulsion ŽIlford, UK. and after approximately 2 weeks exposure, developed in D19 developer.

3. Results

2.5. Riboprobe preparation

3.1. Cloning of mouse Õomeronasal receptors

mV1R riboprobes were prepared from the same templates used for the genomic Southern blots. The Ga o Ž1000 bp. and Ga i2 Ž400 bp. templates were obtained by RT-PCR from the coding region of each gene. DIG, 35 S or 33 P-labelled RNA probes were synthesized using an RNA labelling kit ŽBoehringer.. DIG and double-ISH were per-

Only one full length receptor gene was obtained from 10 positive clones by high stringency screening. To obtain additional V1R cDNAs, the library was re-screened at a lower stringency and a further receptor cDNA obtained. The size of two vomeronasal receptors ŽmV1R 1 and the mV1R 2 . obtained from the library were 2674 and 3403 bp,

Note to Table 1: Analysis of the spatial distribution of mV1R 1 , mV1R 2 , mV1R 1C , and mV1R 4C positive cells along the rostral–caudal axis in the adult mouse VNO. The left vertical axis shows the number of positive cells per slide and the right vertical axis shows the size of the VNO ŽA.. The population of the positive cells expressing mV1R 1,2 and mV1R 1C,4C in each position through the rostro-caudal extent of the VNO is shown in ŽB. and ŽC., respectively. The number of positive cells is the average of eight sections from two animals.

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respectively. The homology between mV1R 1 and mV1R 2 in the coding regions was 90%, but for the whole sequence only 77% due to the less-conserved 5X and 3X non-coding regions. Alignment of sequences from the 5X and 3X noncoding regions of mV1R 1 and mV1R 2 are shown in Fig. 1. The 5X non-coding regions of both receptors are of similar length. The first 100 bp of mV1R 1 and mV1R 2 are not conserved but the rest of these regions are well conserved with aspect to each other Ž80%. ŽFig. 1A.. The 3X non-coding regions of mV1R 1 and mV1R 2 shown in Fig. 1B were 900 and 957 bp, respectively. A total of 34 repeats of ATTT or TATT motifs were identified in the 3X non-coding regions of mV1R 1 and 27 repeats of ATTT or TATT motifs were identified in 3X non-coding regions of mV1R 2 . The homology of the 3X non-coding regions of mV1R 1 and mV1R 2 was relatively high in the initial 500 bp, but not in the rest of the 3X non-coding regions. The predicted proteins encoded by mV1R 1 and mV1R 2 were

90% identical to each other. Two different putative polyadenylation signals were identified in the 3X non-coding regions of both receptors. We searched the library for other members of the subfamilies of mV1Rs using additional primers to cover the whole coding region of V1R receptors. By this approach we identified mV1R 3 Ž726 bp. and mV1R 4 Ž858 bp. which had some 90% sequence homology. The homology of the predicted protein sequence of these genes to mV1R 1 – 2 was around 50%. Amino acid sequences alignments of the four identified mV1Rs are shown in Fig. 2. 3.2. Size of the receptor probes and subfamily To estimate the number of related mV1Rs from the subcloned mV1Rs probes, genomic Southern blots, using a mouse genomic DNA, were performed ŽFig. 3.. Three probes ŽmV1R 1C , mV1R 3C , and mV1R 4C . used were based

Fig. 5. ISH of coronal sections of VNO. Sequential sections were hybridized with DIG-Ga i2 ŽA. or DIG-Ga o ŽB.. Double-ISH was performed with 33 P-Ga i2 and mixed DIG-V1R probes shown with lightfield ŽC, DIG positive cells. and with darkfield ŽD, 33 P-labelled Ga i2.. The area where the signal for Ga i2 ŽD. and DIG receptor mRNA-labelled cells coincide is outlined. Scale bars s 100 mm.

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on the coding sequences of mV1R 1 , mV1R 3 , and mV1R 4 and two other probes ŽmV1R 1 and mV1R 2 . were based on the 3X non-coding regions. Southern blots with the mV1R 1C

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probe showed two intense bands and three weak bands ŽFig. 3C.. Three intense bands and three weak bands were obtained with the mV1R 4C probe ŽFig. 3D. and these

Fig. 6. Stereoimages of adjacent coronal VNO sections hybridized with a mix of DIG-labelled mV1Rs and Ga i2 riboprobes. Each pair of sections were taken at an interval of 200 mm through the rostro-caudal extent of the VNO. The vomeronasal sensory epithelium shaded in pale grey is that area which contained Ga i2 positive neurons. mV1Rs positive neurons are shown as black dots. Note the coincidence of mV1R neurons and Ga i2 staining. Scale bar s 200 mm.

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bands were different in size and distinct from the mV1R 1C boundary pattern. The pattern of bands obtained with the mV1R 3C probe was the same as that of mV1R 4C suggesting that both receptors belong to the same subfamily Ždata not shown.. These results suggest that the mV1R 1C and the mV1R 3 Ø 4C subfamilies contain at least five and six genes, respectively. The blot for the mV1R 1 probe showed only one single intense band ŽFig. 3A.. Although the mV1R 2 probe was designed from 3X non-coding region, the blot showed two distinct bands ŽFig. 3B. which also appeared to be recognized by the mV1R 1C probe suggesting that mV1R 1 and mV1R 2 belong to same subfamily.

showed that the distribution of Ga i2 and mV1Rs completely overlapped demonstrating that the DIG-labelled mV1Rs positive cells expressed 33 P-Ga i2, and that the two signals were co-localized in the same cell ŽFig. 5C,D.. The results of adjacent sections hybridized with mixed probes of all DIG-mV1Rs and the DIG-Ga i2 probe were analysed in eight regions of the VNO. All mV1R positive neurons were found within the region of Ga i2 positive neurons ŽFig. 6..

3.3. mV1Rs expression

4.1. Mouse VNO receptors

The mV1R riboprobes for ISH were synthesized from the subclones used in genomic Southern blots. All probes selectively detected neurons in the middlerupper layer of VNO using high stringency hybridization, and no probe detected any signal in the MOE of the mouse nasal cavity where odorant receptors are localized. ISH using low stringency conditions gave the same signal pattern as with high stringency. mV1R 1 and mV1R 2 signals were detected only in 0.3–0.5% of vomeronasal sensory neurons ŽFig. 4A,B.. More than 3% of neurons hybridized with the mV1R 1C as well as the mV1R 4C ŽFig. 4C–F.. ISH on serial sections was used to investigate the organization of receptors along the rostro-caudal axis of the VNO ŽTable 1.. As shown in Table 1A, the number of positive neurons increased as the size of the VNO increased in the caudal region. While there was a general rostro-caudal increase in the number of positive cells in the VNO there was no area or region selectively expressing only one single receptor or a receptor subfamily ŽTable 1B,C..

The VNO is a self-contained chemosensory system that is functionally committed to responding to pheromones which influence reproductive endocrine and behavioral states w13,19,24x. Our initial attempt to obtain mouse cDNA clones for VNO receptors, using degenerate primers from identified olfactory receptors, met with little success. These efforts were based on the assumption that the MOE and VNO receptors share a common developmental origin in the olfactory placode and both areas express proteins such as olfactory marker protein ŽOMP. w14,20,25,26,28x. However, even for OMP we found a differential expression pattern of OMP mRNA in the MOE and VNO w66x. Furthermore we were unable to detect, using ISH, the G-protein ŽGolf. which is believed to serve the main signal transduction pathway in olfactory receptor neurons, further supporting the view that the V1R are quite distinct from olfactory receptors. We were, however, able to detect two distinct G-proteins in the vomeronasal neurons ŽGa i2 and Ga o.. Ga i2 mRNA signal was found not to be expressed in olfactory neurons. Ga o mRNA was expressed in olfactory neurons, but only in developing neurons locating in the basal region of the MOE. In the present study, we isolated four mouse V1Rs, two ŽmV1R 1 and mV1R 2 . being full length. There are conserved sequences in the 5X and 3X non-coding regions and two different possible polyadenylation signals in the 3X non-coding regions of these two receptor genes. These elements may be stereotyped in the mV1R gene family. The significance of the high degree of similarity in the 5X non-coding regions of these two receptor genes is unclear, but may relate to transcription factor binding sites regulating individual gene expression by exclusion mechanisms w17x. Two different polyadenylation signals in the 3X noncoding region suggest that there might be two termination sites for transcription. Many repeats of ATTT or TATT motifs were identified in the mV1R 1 and the mV1R 2 3X non-coding regions. RNAs for transiently expressed genes, such as growth factors ŽGM-CSF., oncogenes, and cytokines Žinterferons and thrombospondin, have a short life w2,35x and clusters of ATTT and TATT sequences in the 3X non-coding region of these RNAs has been implicated in

3.4. Receptor oÕerlap with expression of Ga i2 The results of ISH using G-protein specific probes in adjacent coronal sections of the mouse VNO are shown in Fig. 5. A distinct population of vomeronasal sensory neurons were detected by the Ga i2 probe, that was located mainly in the middlerupper layers of the VNO sensory epithelium ŽFig. 5A.. The Ga o positive sensory neurons are located in the basal layers ŽFig. 5B.. The boundary of these two layers was waveshaped, and the signal pattern of the two G-proteins was the same throughout the rostrocaudal extent of the epithelium. Computer analysis of two adjacent sections with each G-protein showed only little overlap at the boundary of the G-protein layers Ždata not shown.. The mV1R positive neurons were located in the middlerupper layers of the VNO, so it seems likely that this family of V1R coincides with the Ga i2 positive neurons. In order to demonstrate this, double ISH and a detailed analysis of a series of adjacent sections throughout the rostro-caudal extent of the VNO were carried out ŽFig. 5C,D, Fig. 6.. All double ISH results on serial sections

4. Discussion

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accelerating mRNA degradation w2,57x. V1R mRNA stability may also be controlled by ATTT or TATT motifs. 4.2. Organization of receptor expression The four mouse VNO receptors identified in this study are probably members of two distinct subfamilies since the predicted proteins for receptors mV1R 1 and mV1R 2 showed 90% homology while those of mV1R 3 and mV1R 4 also had 90% homology, but between the two groups there was only 50% homology. We designed specific probes from unique non-coding regions of mV1R genes to detect expression of a single receptor type and from the coding regions to detect expression of receptor family members. The ISH results showed that all of the receptor probes detected neurons specifically in the vomeronasal sensory epithelium and not in the nasal cavity MOE. Localization of expression for the four receptor genes revealed no obvious organization throughout the VNO structure, but the greatest number of labelled neurons were found most caudally. The number of cells hybridized with each probe was almost identical in the right and left VNO. Approximately 4.5 = 10y4 cellsrmm2 were labelled for the mV1R 1 receptor and 2.8 = 10y4 cellsrmm2 for the mV1R 2 receptor out of a total population of 1 = 10y1 cellsrmm2 of vomeronasal sensory neurons, which represents between 0.3% and 0.5% of VNO sensory neurons. The mixed probes, mV1R 1 and mV1R 4 , which are subfamily specific, detected some 11 genes, and labelled around 3% of neurons in the middlerupper layers. Given that this would represent 3%, of 50% of all VNO neurons, i.e., the ‘middlerupper layers’ Ga i2 neurons, these results suggest that the general mV1R gene family may consist of 150–200 genes. Both type of receptors were randomly distributed along the dorsal–ventral axis of the VNO. This lack of distinct topographical symmetry along the dorsal–ventral axis seen in the VNO contrasts strikingly with the symmetry of the MOE olfactory receptors, and is almost certainly related to the differences in the sites of origin and the way receptor neurons are placed in the two structures w3,16,48,63,64x. Whereas olfactory receptor neuron replacement occurs by vertical migration of progenitor cells from the basal layer, regeneration in the VNO occurs by migration of progenitor cells from the dorsal and ventral aspects of the organ in a medial direction, which has more in common with the replacement of taste neurons in the gustatory papillae w27x. Thus each labelled neuron is gradually migrating medially and the VNO replacement pattern will depend on the rate of migration and life span of the neurons.

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with the neurons expressing Ga i2 protein in the adult mouse VNO. In the AOB, axons of the Ga i2 neurons project to the rostral part of AOB, while those VNO neurons in the deep layers of the sensory epithelium express Ga o, and project to the caudal part of AOB w9,32,43,59x. Our observations suggest that a further set of mVRs exists distinct from those we have cloned which employ the alternative Ga o signalling pathway. Indeed, the recently discovered V2Rs are found in the deep layer of the VNO, probably in Ga o neurons w33,56x. The dendrites of mitralrtufted cells in the AOB terminate in more than one glomerulus. A recent study demonstrated that there are two populations of these cells: one in the rostral and the other in the caudal regions of the AOB, and their dendrites only map to glomeruli within the same region w37x. Electophysiological mapping of field potentials suggested that these two subdivisions are functionally different w62x. These results suggest that the stereotyped organization of the VNO is maintained in the secondary neurons of the AOB. The functions of these two vomeronasal receptor families and are not clear and a particularly important question is how they share physiological functions in the vomeronasal sensory system. One possibility is that they detect the same pheromones but with distinct activity patterns. In this context it may be relevant to note that glutamate mediates its actions through two distinct classes of seven transmembrane receptor one termed ionotropic and the other metabotropic. Ionotropic receptors mediate ‘fast’ excitatory actions of glutamate w18x. The metabotropic receptors like the V2Rs have an extensive N-terminus and are coupled to a ‘slow’ intracellular transduction via G-proteins w4x. However, the two VNO receptor families have substantial structural difference and mediate their effects by different signal transduction cascades, and may therefore each respond to distinct types of ligand Žpheromones.. V1Rs and V2Rs possibly couple to different types of G-protein: V1Rs to the Ga i2 and V2Rs to the Ga o. The ability of Ga i2, but not Ga o, to inhibit adenylyl cyclases suggest that sensory ligands might have opposite effects on cAMP content w43x. These two distinct signalling pathways also might have differential effects through interactions with separate ion channels w34x. In the mammalian MOE, different odorants or different concentrations of the same odorant differentially increase IP3 vs. cAMP w10,54x. Whereas the cAMP increase opens cyclic nucleotide-gated channels and leads to depolarization w1x, the role of IP3 is not clear.

4.3. Co-localization of Õomeronasal receptors and Ga i2

Acknowledgements

The double-ISH and analyses of adjacent sections carried out in this study showed strikingly that the two different subfamilies of mV1R positive neurons co-existed

The authors wish to thank Dr. G. Glassmith for the G-protein clones, Mr. M. Hinton and W.J. Coadwell for computer analysis of the ISH data and the sequence align-

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