Response profiles to amino acid odorants of ... - Wiley Online Library

3 downloads 11 Views 732KB Size Report
We describe the general features of AA-responsive glomeruli and ... Email: [email protected] ... creating a functional olfactory map (Hálasz & Greer,. 1993 ...

567

J Physiol 581.2 (2007) pp 567–579

Response profiles to amino acid odorants of olfactory glomeruli in larval Xenopus laevis Ivan Manzini1,2 , Christoph Brase1 , Tsai-Wen Chen1 and Detlev Schild1,2 1

Department of Neurophysiology and Cellular Biophysics, and 2 DFG Research Center for Molecular Physiology of the Brain (CMPB), University of G¨ottingen, Humboldtallee 23, 37073 G¨ottingen, Germany

Glomeruli in the vertebrate olfactory bulb (OB) appear as anatomically discrete modules receiving direct input from the olfactory epithelium (OE) via axons of olfactory receptor neurons (ORNs). The response profiles with respect to amino acids (AAs) of a large number of ORNs in larval Xenopus laevis have been recently determined and analysed. Here we report on Ca2 + imaging experiments in a nose–brain preparation of the same species at the same developmental stages. We recorded responses to AAs of glomeruli in the OB and determined the response profiles to AAs of individual glomeruli. We describe the general features of AA-responsive glomeruli and compare their response profiles to AAs with those of ORNs obtained in our previous study. A large number of past studies have focused either on odorant responses in the OE or on odorant-induced responses in the OB. However, a thorough comparison of odorant-induced responses of both stages, ORNs and glomeruli of the same species is as yet lacking. The glomerular response profiles reported herein markedly differ from the previously obtained response profiles of ORNs in that glomeruli clearly have narrower selectivity profiles than ORNs. We discuss possible explanations for the different selectivity profiles of glomeruli and ORNs in the context of the development of the olfactory map. (Resubmitted 15 February 2007; accepted after revision 5 March 2007; first published online 8 March 2007) Corresponding author I. Manzini: Department of Neurophysiology and Cellular Biophysics, University of G¨ottingen, Humboldtallee 23, 37073 G¨ottingen, Germany. Email: [email protected]

In the vertebrate olfactory system, odorants are detected by olfactory receptor neurons (ORNs) embedded in the olfactory epithelia of the olfactory organ. Individual adult ORNs express only one type of olfactory receptor (OR) from a repertoire of ∼1000 in rodents and ∼100 in fish (Mombaerts, 1999). In rodents, ORNs expressing a given OR are widely distributed within the olfactory epithelium (OE) (Ressler et al. 1993; Vassar et al. 1993; Strotmann et al. 1994), yet their axons converge onto few specific glomeruli in the olfactory bulb (OB), thereby creating a functional olfactory map (H´alasz & Greer, 1993; Ressler et al. 1994; Vassar et al. 1994; Mombaerts et al. 1996; Wang et al. 1998; Strotmann et al. 2000; Zheng et al. 2000; Potter et al. 2001). Glomeruli are spheroidal neuropil structures comprising ORN axon terminals, which convey the afferent inputs to glomeruli, as well as dendritic tufts of OB projection neurons and interneurons. They are thought to be functional units of olfactory information processing because they gather and integrate specific afferent inputs from ORNs (Lancet et al. 1982; Hildebrand & Shepherd, 1997; Shepherd &

I. Manzini and C. Brase contributed equally to this work.  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

Greer, 1998; Kratskin & Belluzzi, 2003; Lledo et al. 2005). However, the molecular processes that determine how the olfactory map is set up are poorly understood. Axon guidance and sorting (R¨ossler et al. 1999; St John et al. 2003; Feinstein & Mombaerts, 2004; Feinstein et al. 2004; Schwarzenbacher et al. 2006) as well as synapse formation over development (Graziadei et al. 1978; Valverde et al. 1992; Treolar et al. 1999) certainly play important roles here. In mice, ‘ectopic’ ORs appear to be involved in glomerular targeting, but several other factors have also been implicated in this complex assembly (Mombaerts et al. 1996; Wang et al. 1998; Feinstein & Mombaerts, 2004; Feinstein et al. 2004; Strotmann et al. 2004; Miyasaka et al. 2005; Schwarzenbacher et al. 2006; Serizawa et al. 2006; Imai et al. 2006). To understand the development of the olfactory map, aquatic vertebrates are particularly suited. The fertilized eggs of most aquatic species develop into free-swimming larvae before metamorphosing into juvenile animals. Ontogenetic stages of some amphibians are well characterized and easy to handle. Carrying out corresponding experiments in prenatal mammals is considerably more difficult. Another reason to study the setup of the olfactory map in aquatic species is that DOI: 10.1113/jphysiol.2007.130518

568

I. Manzini and others

a number of behaviourally relevant odorants (Sorensen & Caprio, 1998) are well known, e.g. prostaglandins (Sorensen et al. 1988; Kitamura et al. 1994), nucleotides (Kang & Caprio, 1995), bile acids (Kang & Caprio, 1995; Sato & Suzuki, 2001), and amino acids (AAs) (Caprio & Byrd, 1984; Kang & Caprio, 1995; Vogler & Schild, 1999; Sato & Suzuki, 2001; Manzini et al. 2002a,b; Manzini & Schild, 2003a, 2004; Czesnik et al. 2006). The crucial signal processing step lying between ORNs and the relay neurons of the OB are the olfactory glomeruli. Functional Ca2+ imaging of glomerular activities can tell precisely which stimulus qualities are processed in every individual glomerulus. Given that each glomerulus is innervated only by ORNs of a specific class, one would assume that the response profiles of ORN classes and of glomeruli are identical. As preliminary evidence of ours indicated, however, that this is not the case, we set out to systematically record stimulus responses of individual glomeruli using a nose–brain preparation of larval Xenopus laevis. We describe the general features and selectivity profiles of glomerular responses and compare them with ORN response profiles. The obvious divergence between both is discussed. Methods Nose–brain preparation for Ca2 + imaging

Tadpoles of Xenopus laevis (stages 51–56; staged after Nieuwkoop & Faber, 1994) were cooled to produce complete immobility, and then killed by transection of the brain at its transition to the spinal cord. All procedures for animal handling and tissue dissections were carried out according to the guidelines of the G¨ottingen University Committee for Ethics in Animal Experimentation. A block of tissue containing the olfactory epithelia, the olfactory nerves and the anterior two-thirds of the brain was cut out and kept in bath solution (see below). The tissue was glued onto the stage of a vibroslicer (VT 1000S; Leica, Bensheim, Germany) and only the dorsal surface of the OBs was sliced off. Thereby the cutting angle was chosen in a way to enter the OB straight above the olfactory nerve entrance. The olfactory epithelia were left intact. For a more detailed description of this preparation see the work of Czesnik et al. (2003). As well-developed glomeruli have been found only in the more ventral OB (Nezlin & Schild, 2000, 2005; Nezlin et al. 2003) the described slicing technique ensures an ideal access to the glomerular layer. The more dorsal part of the OB consists of an apparently structureless fibre meshwork without any clear discernible glomeruli (Nezlin & Schild, 2000). For Ca2+ imaging experiments the tissue slices were transferred to a recording chamber, and 200 μl of bath solution (see below) containing 50 μm Fluo-4/AM (Molecular Probes, Leiden, The Netherlands) was added. The fluorescence of Fluo-4

J Physiol 581.2

increases with increasing intracellular Ca2+ concentration. Fluo-4/AM was dissolved in DMSO (Sigma, Deisenhofen, Germany) and Pluronic F-127 (Molecular Probes). To avoid transporter-mediated destaining of neuropil of the OB (Manzini & Schild, 2003b), MK571 (Alexis Biochemicals, Gr¨unberg, Germany), a specific inhibitor of the multidrug-resistance-associated proteins (Gekeler et al. 1995; Abrahamse & Rechkemmer, 2001) was added to the incubation solution. After incubation on a shaker at room temperature for 1 h, the nose–brain preparations were placed under a grid in a recording chamber (Edwards et al. 1989) and placed on the microscope stage of an Axiovert 100M (Zeiss, Jena, Germany) to which a laser scanning unit (LSM 510; Zeiss) was attached. Before starting the Ca2+ -imaging experiments, the slices were rinsed with bath solution for at least 20 min. Ca2 + imaging of odour responses, and data analysis

Intracellular Ca2+ concentrations were monitored using a laser-scanning confocal microscope (Zeiss LSM 510/Axiovert 100M). Fluorescence images (excitation at 488 nm; emission >505 nm) of the OB were acquired at 5 Hz and 200 ms exposure time per image, with a number of images taken as control images before the onset of odour delivery. The fluorescence changes (F/F) were calculated for individual glomeruli as F/F = (F 1 − F 2 )/F 2 , where F 1 is the fluorescence averaged over the pixels of a glomerulus (glomerular borders were outlined manually), while F 2 is the average fluorescence of that glomerulus prior to stimulus application. A response was assumed if the following two criteria were met: (a) the first two glomerular intensity values F/F (t 1 ) and F/F (t 2 ) after stimulus arrival at the OE had to be larger than the maximum of the prestimulus intensities; and (b) F/F (t 2 ) > F/F (t 1 ), with t 2 > t 1 . To obtain high-quality maps of the pixels in the recorded image sequences that showed odorant-induced responses (see Figs 2 and 3), we analysed the data using a custom-written program in MATLAB (Mathworks, Natick, USA). A ‘pixel correlation map’ was obtained by calculating the cross-correlation between the fluorescence signals of a pixel to that of its immediate neighbours and then displaying the resulting value as a greyscale map. As physiological responses often give similar signals in adjacent pixels, this method specifically highlights those pixels. In contrast, pixels that contain only noise show uncorrelated traces and thus appear dark in the cross-correlation map. Solutions and stimulus application

The composition of the bath solution was (mm): 98 NaCl, 2 KCl, 1 CaCl2 , 2 MgCl2 , 5 glucose, 5 sodium pyruvate,  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

Response profiles of olfactory glomeruli

J Physiol 581.2

Table 1. Water-soluble mixtures of L-amino acids Mixture LCN SCN BAS AROM AA

Amino acids Proline, valine, leucine, isoleucine, methionine Glycine, alanine, serine, threonine, cysteine Arginine, lysine, histidine Tryptophan, phenylalanine LCN, SCN, BAS and AROM

Mixtures of L-amino acids were prepared as described by Caprio & Byrd (1984). LCN, long-chain neutral; SCN, short-chain neutral; BAS, basic; AROM, aromatic; and AA, mixture of all 15 amino acids.

10 Hepes. The pH of the bath solution was adjusted to 7.8, which is the physiological pH in this poikilothermal species (Howell et al. 1970). The osmolarity of the bath solution was 230 mosmol l−1 . As odorants, we used a mixture of 15 AAs (listed in Table 1) applied as a mixture of all 15 AAs, as submixtures, or as single AAs. All AAs were purchased from Sigma. The AAs were dissolved in bath solution (10 mm stock, each) and used at a final concentration of 200 μm in all of the experiments. CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) was dissolved in bath solution (stock of 10 mm) and used at a final concentration of 50 μm. d-APV (d-2-amino-5-phosphonovaleric acid) was dissolved in bath solution (stock of 10 mm) and used at a final concentration of 200 μm. The solutions were prepared immediately before use by dissolving the respective stock solution in bath solution. The bath solution was applied by gravity feed from a storage syringe through a funnel drug applicator (Schild, 1985) to the recording chamber. The flow was 350 μl min−1 . Odorants were pipetted directly into the funnel without stopping the flow of the bath solution. The tip of the applicator was placed close to the ipsilateral OE. The dilution of the odorants within the funnel was less than 1%, the delay between the odorants leaving the funnel outlet and reaching the mucosal surface was less than 1 s, and after the end of stimulation, odorants were completely rinsed from the mucosa within 15 s (for details see Manzini et al. 2002b). Outflow was through a syringe needle placed close to the OE to ensure that odorant molecules were removed rapidly. Direct effects of the AA stimuli on the OB were excluded by a series of control experiments. After stimulation with the mix of AAs, we cut the olfactory nerves and repeated the stimulation. We did not observe any responses to AA after transection of the olfactory nerves, and we found no differences from control conditions. However, to further exclude any direct effects on OB neurons we renounced to apply critical AAs (l-glutamate, l-aspartate, l-glutamine and l-asparagine) known to have direct effects on the OB. Therefore, the AA mixture used in the experiments described in this paper contained four AAs less than the  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

569

mixture used in our previous work where we determined response profiles to AAs of individual ORNs (see Manzini & Schild, 2004). The minimum interstimulus interval between odorant applications was at least 2 min.

Results We recorded odorant-induced responses of individual olfactory glomeruli of X. laevis tadpoles by Ca2+ imaging using Fluo-4 as Ca2+ -indicator dye and AAs as olfactory stimuli. Figure 1A shows the OB of a nose–brain preparation stained with Fluo-4 (image acquired at rest). Application of the mixture of all AAs to the intact ipsilateral OE induced changes of the Ca2+ -dependent fluorescence in the neuropil of the glomerular layer of the OB (Fig. 1B). Figure 1C shows the time courses of the [Ca2+ ]i transients of the four regions marked in Fig. 1B (nos 1–4). To ascertain that the responding structures were individual glomeruli and not simply unstructured fibre meshwork, it was necessary to focus into the responding neuropil and repeat the odorant application. The sequence of three images shown in Fig. 1D–F gives the details of one individual AA-sensitive glomerulus identified using this approach. Mucosal application of AAs transiently increased the Ca2+ -dependent glomerular fluorescence in a spatially inhomogeneous way. The corresponding time course of the [Ca2+ ]i transient of the whole glomerulus is shown in Fig. 1G (black trace). Application of the bath solution as a negative control stimulus did not evoke any comparable change in the [Ca2+ ]i , either in this (Fig. 1G, red trace) or in any other preparation tested. In the 93 nose–brain preparations tested with the mixture of all AAs we clearly identified 181 individual glomeruli that were activated upon mucosal AA stimulation. They were all situated in the lateral half of the OB and had an oval or rounded shape with diameters ranging from 10 to 34 μm. The average diameter was 21.01 ± 4.58 μm (mean ± s.d.). To differentiate between the pre- and postsynaptic origin of the Ca2+ responses obtained from individual glomeruli, we carried out a number of experiments (n = 10) where we added d-APV (200 μm) and CNQX (50 μm) to the bath solution. These NMDA and non-NMDA glutamate receptor antagonists are known to block the synaptic transmission between ORNs and mitral cells (Berkowicz et al. 1994; Ennis et al. 1996; Shipley & Ennis, 1996). Figure 2A displays the intraglomerular structure of an individual glomerulus activated upon mucosal application of the mixture of all AAs (a1 , before; a2 , during; a3 , after application of both antagonists). Note the patchy response of the glomerulus under influence of the antagonists (see a2 ). Figure 2B exemplarily shows that the AA-induced [Ca2+ ]i response of an individual glomerulus is attenuated but not completely blocked by addition of both antagonists. The reduction of the

570

I. Manzini and others

[Ca2+ ]i responses in the 14 responsive glomeruli tested (10 nose–brain preparations) was 46 ± 17 and 49 ± 13% (mean ± s.d.) after 10 and 15 min drug incubation, respectively. After 20 min wash-out, the Ca2+ responses recovered. To determine the response profiles to the 15 AAs of individual glomeruli we first searched for individual glomeruli that responded to the mixture of all 15 AAs using the above described procedure and subsequently applied the four submixtures of AAs (LCN, SCN, BAS and AROM, see Table 1). Finally we tested the 15 single AAs, one after another. Figure 3A shows an individual AA-responsive glomerulus. Mucosal application of the mixture of all AAs elicited a transient Ca2+ response as seen in the series of three pseudocoloured images (a1 –a3 ). The intraglomerular finestructure of the glomerulus

J Physiol 581.2

is shown in a4 . Its temporal response traces (the whole glomerulus taken as one region of interest) are shown below the images. This glomerulus was monoresponsive, i.e. it responded only to l-arginine and, accordingly, to the basic (BAS) AAs and the mixture of all AAs. Figure 3B–D shows three more mono- or biresponsive individual glomeruli taken from different nose–brain preparations and their temporal response traces (for a more detailed description see the figure legend). Figure 3E shows a multiresponsive glomerulus that responded to l-glycine, l-alanine, l-serine, l-threonine, l-cysteine, l-valine, l-leucine, l-isoleucine, l-methionine, l-arginine and l-lysine and, accordingly, to the short-chain neutral (SCN), long-chain neutral (LCN) and BAS AAs, and to the mixture of all AAs. The five images shown in the right panel of Fig. 3E

Figure 1. Nose–brain preparation and amino-acid-induced Ca2 + signals in individual glomeruli in the olfactory bulb of a Xenopus laevis tadpole A, the imaged region of the olfactory bulb (OB) of a nose–brain preparation (stage 52) has been superimposed to a sketch of an OB (ON, olfactory nerve; GL, glomerular layer; MCL, mitral cell layer; GCL, granule cell layer; MOB, main olfactory bulb; AOB, accessory olfactory bulb; and V, ventricle). The nose–brain preparation was stained with the Ca2+ -indicator dye Fluo-4 (green fluorescence, image acquired at rest). B, the image shows areas of the neuropil in the glomerular layer activated by mucosal application of the mixture of all amino acids (AAs) (see encircled areas indicated by the white arrows 1–4). C, time courses of the [Ca2+ ]i transients of the four regions (1–4). D–F, sequence of three images acquired at a higher magnification clearly showing that mucosal application of a mixture of all AAs (200 μM, each) transiently increases Ca2+ -dependent fluorescence in an individual glomerulus (stage 52). D, image taken before the application of the amino acid mixture. E, image taken at the peak of the response; F, image taken after return to the base line fluorescence. G, time course of [Ca2+ ]i transients of the glomerulus shown in D–F evoked by the mucosal application of the mixture of all AAs (black line). Application of bath solution as a control stimulus showed no comparable response (red line).  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

J Physiol 581.2

Response profiles of olfactory glomeruli

show the intraglomerular response patterns elicited by the applications of the mixture of all AAs and of the different submixtures of AAs. Using the same approach we were able to determine the response profiles to 15 AAs (see Table 1) from a total of 67 individual glomeruli (41 nose–brain preparations). In some cases the attempt to record the entire response profile of a glomerulus did not succeed because experimental conditions differed or otherwise changed during the experiment and we were unable to take a complete response profile. Such incomplete response profiles were not considered for further evaluation. Figure 4 shows the resulting 67 × 15 response matrix. A response of a glomerulus to a particular AA is indicated by a ‘square’ in the response matrix, the colour standing for a specific AA subgroup. For the sake of clarity, the time courses and response amplitudes are neglected in this representation and the 67 response profiles are ordered by the number of effective stimuli (S1, S2, etc.). Thirty out of the 67 glomerular response profiles differed from each other (A, B, C, etc.). While 23 of them occurred once, 7 of them occurred more than once (I, II, III, IV, V, VI and VII), the number of occurrences being between 2 and 12. Thirty glomeruli responded exclusively to one AA (S1 group), and these AAs were l-phenylalanine (9 glomeruli), l-arginine (6 glomeruli), l-histidine (12 glomeruli), l-methionine (2 glomeruli) and l-glycine (1 glomerulus). Interestingly, four of these five AAs were also involved in the responses profiles of the 17 glomeruli activated by two AAs (S2 group). The remaining 20 glomeruli were activated by 3–14 AAs. At this point it may be important to mention that also the glomeruli whose response profiles were determined

only partially (see above) had a tendency to respond to few AAs. The frequency of glomerular responses to the 15 different AAs used varied considerably from AA to AA (see Figs 4 and 5). The response frequency to the BAS AAs l-arginine and l-histidine, the aromatic (AROM) AA l-phenylalanine, and the LCN AA l-methionine, was markedly higher than that to the other AAs. While l-arginine, l-histidine, l-phenylalanine and l-methionine were the most effective stimuli, respectively, activating 33 (approx. 50%), 33 (approx. 50%), 21 (approx. 32%) and 16 (approx. 24%) of the 67 glomeruli tested, l-valine (6 responsive glomeruli), l-threonine (6 responsive glomeruli) and l-proline (3 responsive glomeruli) were the least effective stimuli. The histogram of effective AAs per glomerulus shown in Fig. 6 clearly suggests that glomeruli tend to respond to few AAs rather than too many. Seventy per cent of the recorded glomeruli (47 out of the 67) were activated by one (45%) or two AAs (25%). Only nine glomeruli (approx. 13%) responded to more than five AAs, and no glomerulus responded to all 15 AAs. In our previous study with ORNs (Manzini & Schild, 2004) we observed a narrowing of ORN selectivity over developmental stages. To check whether glomeruli, too, gain specificity over developmental stages we split the dataset of Fig. 4 in two subsets, i.e. glomeruli recorded from lower larval stages (51, 52, 53) and glomeruli recorded from higher larval stages (54, 55, 56). The frequency histograms of the number of effective AAs in each subset of glomeruli are plotted in Fig. 7. The histograms of the number of effective AAs per glomerulus of the lower

Figure 2. Effects of non-NMDA and NMDA glutamate receptor antagonists on AA-induced Ca2 + responses of an individual glomerulus Intraglomerular structures of an individual responding glomerulus (stage 51, greyscale map, see Methods for details) activated by mucosal application of the mixture of all AAs (200 μM each). a1 , before; a2 , during; and a3 , after application of non-NMDA and NMDA glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 μM) and D-2-amino-5-phosphonovaleric acid (D-APV; 200 μM). The arrows in a2 indicate intraglomerular spots that remained almost unaffected upon application of the antagonists. B, time courses of the AA-induced [Ca2+ ]i transient (whole glomerulus taken as region of interest) evoked by the mucosal application of the mixture of all amino acids (black line, control). Application of the antagonists attenuated, but did not completely block, the AA-induced [Ca2+ ]i transient (red lines, AA applications after 10 and 15 min antagonist application, respectively). After a wash-out time of 20 min, the AA-induced [Ca2+ ]i transient recovered almost completely (black line, wash-out).  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

571

572

I. Manzini and others

J Physiol 581.2

 C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

Response profiles of olfactory glomeruli

J Physiol 581.2

stages (Fig. 7A) and the higher stages (Fig. 7B) do not differ at p = 0.05 (Mann–Whitney U test), indicating that the specificity to AAs in glomeruli remains constant over the developmental stages examined. Discussion General features of glomerular responses

We have recorded responses of individual glomeruli in the OB of X. laevis tadpoles upon application of AA to the intact ipsilateral OE. All AAs were applied at a concentration of 200 μm. This concentration lies in the dynamic range of the dose–response curve for AAs and is sufficiently high to activate ORs with high as well as those with low affinity for AAs (I. Manzini, unpublished). All of the 181 individual glomeruli that were identified to respond to AA were situated in the lateral half of the OB. This result is in line with previous studies in larval X. laevis showing that only cells in the lateral half of the OB respond to mucosal AA application (Manzini et al. 2002b; Czesnik et al. 2003), and also with evidence from other aquatic species in which AAs have been shown to be mapped in the lateral OB (Friedrich & Korsching, 1997, 1998; Hara & Zhang, 1998; Nikonov & Caprio, 2001; Hansen et al. 2003). As expected, and confirmed by our experiments using CNQX/APV (see Fig. 2), the responding intraglomerular fibre meshwork consisted of both ORN axon terminals and mitral cell dendrites. Blockage of the

postsynaptic non-NMDA and NMDA receptors attenuated the odorant-induced Ca2+ signal but never completely blocked it, indicating that the overall [Ca2+ ]i signal measured reflected Ca2+ influx in both ORN axon terminals and mitral cell dendrites. It has been shown that mammalian glomeruli are made up of interdigitating subcompartments predominantly composed of either axons or dendrites (Kosaka et al. 1997; Kasowski et al. 1999; Potter et al. 2001; Wachowiak et al. 2004). Our results seem to be in line with these studies (see arrows in a2 of Fig. 2A), indicating that the glomeruli in X. laevis show a similar subcompartmentalization as seen in mammalian glomeruli. Glomerular specificity profiles

The main focus of this study was the recording of glomerular response profiles to AAs. We succeeded in recording the exact response profiles to 15 AAs (see Table 1) in 67 individual glomeruli (Fig. 4). It stands out that individual glomeruli tend to respond to few AAs rather than too many. Most of them are mono- or biresponsive. If a glomerulus was activated by more than one AA, in most cases these AAs were members of one or two subgroups of AAs (see Table 1). Response profiles to AAs of glomerular modules have also been determined in zebrafish (Friedrich & Korsching, 1997). In contrast to larval X. laevis, individual glomerular modules in zebrafish typically have broad and complex response profiles to AAs, though some glomerular modules preferentially respond

Figure 3. Response profiles of five individual glomeruli upon mucosal application of AAs A, sequence of three pseudocoloured images representing the imaged OB part of a nose–brain preparation (stage 53) showing that mucosal application of a mixture of all AAs (200 μM, each) transiently increases Ca2+ -dependent fluorescence in an individual glomerulus. a1 , Image taken before the application of the AA mixture; a2, image taken at the peak of the response; a3 , image taken after return to the base line fluorescence; a4 , image showing the fine intraglomerular structures of the activated glomerulus (greyscale map, see Methods for details). The time courses of the [Ca2+ ]i transients of the glomerulus shown in A, evoked by the application of AAs, are given below the images. The traces show the responses to the mixture of all AAs, to the mixture of basic (BAS) and to L-arginine. There was no response to the long-chain neutral (LCN), the short-chain neutral (SCN) and or the aromatic (AROM) AAs. There was no response to the remaining single AAs of the BAS mixture. B, images b1 –b4 (same explanation as A) of another AA-activated glomerulus (stage 52) and time courses of [Ca2+ ]i transients evoked by the application of AAs. This glomerulus responded to the mixture of all AAs, to the mixture BAS, and to L-arginine and L-histidine. There was no response to LCN, SCN, AROM or to L-lysine, the third AA of the BAS mixture. C, images c1 –c4 (same explanation as A) of a third AA-activated glomerulus (stage 55) and time courses of [Ca2+ ]i transients evoked by the application of AAs. The traces show the responses to the mixture of all AAs, to the mixture BAS and to L-histidine. There was no response to the LCN, the SCN and or AROM AAs. There was no response to the remaining single AAs of the BAS mixture. D, images d1 –d4 (same explanation as A) of a fourth AA-activated glomerulus (stage 56) and time courses of [Ca2+ ]i transients evoked by the application of AAs. This glomerulus responded to the mixture of all AAs, to the mixture BAS, to L-arginine and L-histidine. There was no response to LCN, SCN, AROM or to L-lysine, the third AA of the BAS mixture. E, images e1 –e4 (same explanation as A) of a fifth AA-activated glomerulus (stage 52). The time courses of [Ca2+ ]i transients of this glomerulus evoked by the application of AAs are given on the right panel. The glomerulus responded to the mixture of all AAs, the mixture of LCN, to L-isoleucine, L-leucine, L-methionine and L-valine, the mixture of SCN, to L-cysteine, L-glycine, L-alanine, L-serine and L-threonine, and to the mixture BAS, and to L-arginine and to L-lysine. There was no response to the mixtures AROM, or to the remaining single AAs of the LCN or BAS mixtures. The images above the respective response traces show the fine intraglomerular structures of the responding glomerulus upon application of the mixture of all AAs, LCN, SCN, BAS and AROM.  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

573

574

I. Manzini and others

J Physiol 581.2

Figure 4. Response profiles of 67 individual glomeruli to 15 AAs A 67 × 15 matrix representing the responses of 67 individual glomeruli each tested for 15 AAs as abbreviated by the common one-letter code for AAs (first line). A ‘rectangle’ or a ‘free space’ in the matrix indicates whether or not a particular glomerulus responded to a specific AA (rectangle, ‘response’; free space, ‘no response’). The response profiles are ordered by the number (S1, S2, etc.) of effective AAs. The different colours indicate the subdivision of the 15 AAs in four subgroups (red, SCN; blue, LCN; green, BAS; and black, AROM). The 30 response patterns that differed from each other are marked with the letters A–AD. The seven response patterns occurring more than once are marked with roman numbers (I–VII).  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

J Physiol 581.2

Response profiles of olfactory glomeruli

575

responses of the receptor cell and glomerular stages of the same species has been lacking so far. Here, we compared the response profiles to AAs of individual glomeruli recorded in the present study with response profiles of ORNs of the same species of identical developmental stages (stages 51–56) obtained in a previous study (Manzini & Schild, 2004). The most conspicuous finding of this study is that 47 out of the 67 glomerular response profiles recorded (i.e. 70%) responded to one or two stimuli while only nine glomeruli (13%) responded to more than five AAs (see Figs 4 and 6). These response profiles clearly diverge from the response profiles of ORNs. In contrast to the glomeruli recorded in the present study, only 76 out of 283 ORNs (27%) responded to one or two AAs and almost half of them responded to more than five AAs. These numbers only marginally change if the four AAs that were not used in Figure 5. Response incidences of the 15 different AAs The histogram shows for each of the 15 AAs used in how many glomeruli (out of 67) a response was observed. Results are plotted as relative numbers, i.e. normalized to 67. As a number of individual glomeruli (n = 67) responded to more than one AA, the sum of the response frequencies is much higher than 100%.

to subgroups of AAs with similar chemical properties (Friedrich & Korsching, 1997). Comparison of glomerular and ORN response profiles

A number of past studies conducted on various species have focused on responses and response profiles of either ORNs (Sicard & Holley, 1984; Duchamp-Viret et al. 1999; Ma & Shepherd, 2000; Sato & Suzuki, 2001) or cells and glomeruli in the OB (Johnson et al. 1999; Rubin & Katz, 1999; Uchida et al. 2000; Meister & Bonhoeffer, 2001; Fried et al. 2002). However, a comparison of odorant-induced

Figure 6. Number of effective AAs per individual glomerulus Frequencies of glomeruli (n = 67, 41 nose–brain preparations) that responded to a certain number n of AAs (n out of 15 AAs, Table 1).  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

Figure 7. Lacking stage dependence of glomerular response profiles A, frequency distribution of glomeruli (n = 39, 24 nose–brain preparations) responding to n stimuli evaluated for earlier stages (stages 51, 52 and 53). B, frequency distribution of glomeruli of later stages (n = 28, 17 nose–brain preparations, stages 54, 55 and 56).

576

I. Manzini and others

the experiments with glomeruli (see Methods) are left out in the evaluation of the ORN response profiles of our earlier paper (Manzini & Schild, 2004). This comparison shows that individual glomeruli tend to have much narrower specificity profiles than ORNs. How can these obvious disparities be explained? Or, asked differently, where do the axons of the high number of ORNs with broad sensitivity terminate? One explanation to this question could be that ORNs with broad sensitivity are immature ORNs not yet having their axon properly and functionally connected with their target glomerulus in the OB. In this context the experiments carried out by Gesteland et al. (1982) in rat embryos should be remembered. They suggested that ORNs which are not functionally connected to the OB are less selective in their responses to odorants and that development of selective responsiveness occurs when they mature morphologically just before birth. Gesteland et al. (1982) interpreted their data the following way. ORNs could first have ‘receptor sites’, i.e. ORs, with broad specificity that are subsequently replaced by more specific sites, or alternatively, immature ORNs could have a variety of specific ‘receptor sites’, some of which are lost during maturation. These data and their interpretation match well with the results of the present work, as well as with results of previous studies (Manzini & Schild, 2004; Schild & Manzini, 2004) where we have shown a narrowing selectivity of ORNs over stages and the appearance of pattern cascades consistent with the gradual removal of individual ORs over stages. Furthermore, a narrowing of the selectivity over developmental stages as observed in ORNs (Manzini & Schild, 2004) does not occur in glomeruli in the OB (see Fig. 7). This result is also in line with the above explanation. A second explanation of the above discrepancies could be the extrabulbar olfactory system consisting of ORNs, the axons of which bypass the OB without synapsing on mitral cells in olfactory glomeruli and directly terminate in higher brain areas. This system has been reported in various fish species (Bazer et al. 1987; Honkanen & Ekstrom, 1990; Szabo et al. 1991; Hofmann & Meyer, 1992, 1995; Becerra et al. 1994), and also in larval X. laevis (Pinelli et al. 2004). The possibility that at least some of the ORN axons with broad sensitivity are part of the extrabulbar system cannot be ruled out. As a third explanation, there is the possibility of a sharpening of response profiles in glomeruli by means of lateral inhibition within the neuronal network of the OB (Aungst et al. 2003; Cleland & Linster, 2005). This can generally not be excluded. It would rather be expected. However, in the tadpole’s glomeruli we have so far no indication for inhibitory interneuronal connections, so that it is very unlikely that lateral inhibition is responsible for the narrower response profiles observed in glomeruli. Specifically, the OB of larval X. laevis is relatively sparsely

J Physiol 581.2

populated with periglomerular cells, which are interneurons thought to be involved in mechanisms of lateral inhibition (Cleland & Linster, 2005), and the few periglomerular cells that exist in premetamorphic stages do not yet form tufts within glomeruli (Nezlin et al. 2003). A fourth explanation that could be considered to explain the narrower response profiles of glomeruli is the possibility that small Ca2+ responses in ORNs may not be detectable in glomeruli and therefore may create the impression of narrower response profiles in glomeruli. This is also highly improbable. First, we employed the same experimental approach with the same Ca2+ -sensitive dye in our experiments in the OE and the OB. Methodical differences in detection sensitivity can thus be excluded. Second, a large number of ORNs converge onto every glomerulus (Mombaerts, 1996), thus increasing the stimulus sensitivity of the olfactory system in general (Duchamp-Viret et al. 1989) and of glomeruli in particular. It would therefore be more plausible to assume that hardly detectable ORN responses are easier to detect in glomeruli. This, in turn, would rather broaden than narrow the glomerular response profiles. The above explanations are of course not mutually exclusive. Specifically, axons of individual ORNs need to be traced after having established their response profiles by calcium imaging. Also, in future experiments, glomeruli in the adult frog should be imaged using two-photon absorption microscopy and checked for the presence or lack of multiresponsive specificity profiles. Two-photon absorption microscopy will most probably also permit to acquire a glomerular odour map in the OB, which in turn will reveal whether the position of glomeruli with the same response profile is conserved across different animals. References Abrahamse SL & Rechkemmer G (2001). Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A. Pflugers Arch 441, 529–537. Aungst JL, Heyward PM, Puche AC, Karnup SV, Hayar A, Szabo G & Shipley MT (2003). Centre-surround inhibition among olfactory bulb glomeruli. Nature 426, 623–629. Bazer GT, Ebbesson SOE, Reynolds JB & Bailey RP (1987). A cobalt-lysine study of primary olfactory projections in king salmon fry (Oncorhynchus tshawytscha Walbaum). Cell Tissue Res 248, 499–503. Becerra M, Manso M, Rodriguez-Moldes I & Anadon R (1994). Primary olfactory fibres project to the ventral telencephalon and preoptic region in trout (Salmo trutta): a developmental immunocytochemical study. J Comp Neurol 342, 131–143. Berkowicz DA, Trombley PQ & Shepherd GM (1994). Evidence for glutamate as the olfactory receptor cell neurotransmitter. J Neurophysiol 71, 2557–2561. Caprio J & Byrd RP (1984). Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory sites in the catfish. J Gen Physiol 84, 403–422.  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

J Physiol 581.2

Response profiles of olfactory glomeruli

Cleland TA & Linster C (2005). Computation in the olfactory system. Chem Senses 30, 801–813. Czesnik D, Kuduz J, Schild D & Manzini I (2006). ATP activates both receptor and sustentacular supporting cells in the olfactory epithelium of Xenopus laevis tadpoles. Eur J Neurosci 23, 119–128. Czesnik D, R¨ossler W, Kirchner F, Gennerich A & Schild D (2003). Neuronal representation of odourants in the olfactory bulb of Xenopus laevis tadpoles. Eur J Neurosci 17, 113–118. Duchamp-Viret P, Chaput MA & Duchamp A (1999). Odor response properties of rat olfactory receptor neurons. Science 284, 2171–2179. Duchamp-Viret P, Duchamp A & Vigouroux M (1989). Amplifying role of convergence in olfactory system. A comparative study of receptor cell and second-order neuron sensitivities. J Neurophysiol 61, 1085–1094. Edwards FA, Konnerth A, Sakmann B & Takahashi T (1989). A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system. Pflugers Arch 414, 600–612. Ennis M, Zimmer LA & Shipley MT (1996). Olfactory nerve stimulation activates rat mitral cells via NMDA and non-NMDA receptors in vitro. Neuroreport 7, 989–992. Feinstein P, Bozza T, Rodriguez I, Vassalli A & Mombaerts P (2004). Axon guidance of mouse olfactory sensory neurons by odorant receptors and the β2 adrenergic receptor. Cell 117, 833–846. Feinstein P & Mombaerts P (2004). A contextual model for axonal sorting into glomeruli in the mouse olfactory system. Cell 117, 817–831. Fried HU, Fuss SH & Korsching SI (2002). Selective imaging of presynaptic activity in the mouse olfactory bulb shows concentration and structure dependence of odor responses in identified glomeruli. Pro Natl Acad Sci U S A 99, 3222–3227. Friedrich RW & Korsching SI (1997). Combinatorial and chemotopic odorant coding in the zebrafish olfactory bulb visualized by optical imaging. Neuron 18, 737–752. Friedrich RW & Korsching SI (1998). Chemotopic, combinatorial, and noncombinatorial odorant representations in the olfactory bulb revealed using a voltage-sensitive axon tracer. J Neurosci 18, 9977–9988. Gekeler V, Ise W, Sanders KH, Ulrich WR & Beck J (1995). The leukotriene LTD4 antagonist MK571 specifically modulates MRP associated multidrug resistance. Biochem Biophys Res Commun 208, 345–352. Gesteland RC, Yancey RA & Farbman AI (1982). Development of olfactory receptor neuron selectivity in the rat fetus. Neuroscience 7, 3127–3136. Graziadei PP, Levine RP & Graziadei GA (1978). Regeneration of olfactory axons and synapse formation after bulbectomy in neonatal mice. Proc Natl Acad Sci U S A 75, 5230–5234. H´alasz N & Greer CA (1993). Terminal arborizations of olfactory nerve fibers in the glomerula of the olfactory bulb. J Comp Neurol 337, 307–316. Hansen A, Rolen SH, Anderson K, Morita Y, Caprio J & Finger TE (2003). Correlation between olfactory receptor cell type and function in the channel catfish. J Neurosci 23, 9328–9339.  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

577

Hara TJ & Zhang C (1998). Topographic bulbar projections and dual neural pathways of the primary olfactory neurons in salmonid fishes. Neuroscience 82, 301–313. Hildebrand JG & Shepherd GM (1997). Mechanisms of olfactory discrimination: converging evidence for common principles across phyla. Annu Rev Neurosci 20, 595–631. Hofmann MH & Meyer DL (1992). The extrabulbar olfactory pathway: primary olfactory fibers bypassing the olfactory bulb in bony fishes? Brain Behav Evol 46, 378–388. Hofmann MH & Meyer DL (1995). Aeripheral origin of olfactory nerve fibers by-passing the olfactory bulb in Xenopus laevis. Brain Res 589, 161–163. Honkanen T & Ekstrom P (1990). An immunocytochemical study of the olfactory projections in the three-spined stickleback, Gasterosteus aculeatus, L. J Comp Neurol 292, 65–72. Howell BJ, Baumgardner FW, Bondi K & Rahn H (1970). Acid–base balance in cold-blooded vertebrates as a function of body temperature. Am J Physiol 218, 600–606. Imai T, Suzuki M & Sakano H (2006). Odorant receptorderived cAMP signals direct axonal targeting. Science 314, 657–661. Johnson BA, Woo CC, Hingco EE, Pham KL & Leon M (1999). Multidimensional chemotopic responses to n-aliphatic acid odorants in the rat olfactory bulb. J Comp Neurol 409, 529–548. Kang J & Caprio J (1995). In vivo responses of single olfactory receptor neurons in the channel catfish, Ictalurus punctatus. J Neurophysiol 73, 172–177. Kasowski HJ, Kim H & Greer CA (1999). Compartmental organization of the olfactory bulb glomerulus. J Comp Neurol 407, 261–274. Kitamura S, Ogata H & Takashima F (1994). Olfactory responses of several species of teleost to F-prostaglandins. Comp Biochem Physiol Comp Physiol 107, 463–467. Kosaka K, Toida K, Margolis FL & Kosaka T (1997). Chemically defined neuron groups and their subpopulations in the glomerular layer of the rat main olfactory bulb. II. Prominent differences in the intraglomerular dendritic arborization and their relationship to olfactory nerve terminals. Neuroscience 76, 775–786. Kratskin I & Belluzzi O (2003). Anatomy and neurochemistry of the olfactory bulb. In Handbook of Olfaction and Gustation, ed. Doty RL, pp. 139–164. Dekker, New York. Lancet D, Greer CA, Kauer JS & Shepherd GM (1982). Mapping of odor-related neuronal activity in the olfactory bulb by high-resolution 2-deoxyglucose autoradiography. Proc Natl Acad Sci U S A 79, 670–674. Lledo PM, Gheusi G & Vincent JD (2005). Information processing in the mammalian olfactory system. Physiol Rev 85, 281–317. Ma M & Shepherd GM (2000). Functional mosaic organization of mouse olfactory receptor neurons. Proc Natl Acad Sci U S A 97, 12869–12874. Manzini I, Peters F & Schild D (2002a). Odorant responses of Xenopus laevis tadpole olfactory neurons: a comparison between preparations. J Neurosci Methods 121, 159–167. Manzini I, R¨ossler W & Schild D (2002b). cAMP-independent responses of olfactory neurons in Xenopus laevis tadpoles and their projection onto olfactory bulb neurons. J Physiol 545, 475–484.

578

I. Manzini and others

Manzini I & Schild D (2003a). cAMP-independent olfactory transduction of amino acids in Xenopus laevis tadpoles. J Physiol 551, 115–123. Manzini I & Schild D (2003b). Multidrug resistance transporters in the olfactory receptor neurons of Xenopus laevis tadpoles. J Physiol 546, 375–385. Manzini I & Schild D (2004). Classes and narrowing selectivity of olfactory receptor neurons of Xenopus laevis tadpoles. J Gen Physiol 123, 99–107. Meister M & Bonhoeffer T (2001). Tuning and topography in an odor map on the rat olfactory bulb. J Neurosci 21, 1351–1360. Miyasaka N, Sato Y, Yeo SY, Hutson LD, Chien CB, Okamoto H & Yoshihara Y (2005). Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system. Development 132, 1283–1293. Mombaerts P (1996). Targeting olfaction. Curr Opin Neurobiol 6, 481–486. Mombaerts P (1999). Seven-transmembrane proteins as odorant and chemosensory receptors. Science 286, 707–711. Mombaerts P, Wang F, Dulac C, Chao SK, Nemes A, Mendelsohn M, Edmondson J & Axel R (1996). Visualizing an olfactory sensory map. Cell 87, 675–686. Nezlin LP, Heermann S, Schild D & R¨ossler W (2003). Organization of glomerula in the main olfactory bulb of Xenopus laevis tadpoles. J Comp Neurol 464, 257–268. Nezlin LP & Schild D (2000). Structure of the olfactory bulb in tadpoles of Xenopus laevis. Cell Tissue Res 302, 21–29. Nezlin LP & Schild D (2005). Individual olfactory sensory neurons project into more than one glomerulum in Xenopus laevis tadpole olfactory bulb. J Comp Neurol 481, 233–239. Nieuwkoop PD & Faber J (1994). Normal Table of Xenopus Laevis (Daudin). Garland Publishing, New York and London. Nikonov AA & Caprio J (2001). Electrophysiological evidence for a chemotopy of biologically relevant odors in the olfactory bulb of the channel catfish. J Neurophysiol 86, 1869–1876. Pinelli C, D’Aniello B, Polese G & Rastogi RK (2004). Extrabulbar olfactory system and nervus terminalis FMRFamide immunoreactive components in Xenopus laevis ontogenesis. J Chem Neuroanat 28, 37–46. Potter SM, Zheng C, Koos DS, Feinstein P, Fraser SE & Mombaerts P (2001). Structure and emergence of specific olfactory glomeruli in the mouse. J Neurosci 21, 9713–9723. Ressler KJ, Sullivan SL & Buck LB (1993). A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73, 597–609. Ressler KJ, Sullivan SL & Buck LB (1994). Information coding in the olfactory system: Evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell 79, 1245–1255. R¨ossler W, Oland LA, Higgins MR, Hildebrand JG & Tolbert LP (1999). Development of a glia-rich axon-sorting in the olfactory pathway of the moth Manduca sexta. J Neurosci 19, 9865–9877. Rubin BD & Katz LC (1999). Optical imaging of odorant representations in the mammalian olfactory bulb. Neuron 23, 499–511.

J Physiol 581.2

Sato K & Suzuki N (2001). Whole-cell response characteristics of ciliated and microvillous olfactory receptor neurons to amino acids, pheromone candidates and urine in rainbow trout. Chem Senses 26, 1145–1156. Schild D (1985). A computer-controlled device for the application of odours to aquatic animals. J Electrophysiol Techn 12, 71–79. Schild D & Manzini I (2004). Cascades of response vectors of olfactory receptor neurons in Xenopus laevis tadpoles. Eur J Neurosci 20, 2111–2123. Schwarzenbacher K, Fleischer J & Breer H (2006). Odorant receptor proteins in olfactory axons and in cells of the cribriform mesenchyme may contribute to fasciculation and sorting of nerve fibers. Cell Tissue Res 323, 211–219. Serizawa S, Miyamichi K, Takeuchi H, Yamagishi Y, Suzuki M & Sakano H (2006). A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting. Cell 127, 1057–1069. Shepherd GM & Greer CA (1998). Olfactory bulb. In The Synaptic Organization of the Brain, ed. Shepherd GM, pp. 159–204. Oxford University Press, New York. Shipley MT & Ennis M (1996). Functional organization of olfactory system. J Neurobiol 30, 123–176. Sicard G & Holley A (1984). Receptor cell responses to odorants: Similarities and differences among odorants. Brain Res 292, 283–296. Sorensen PW & Caprio J (1998). Chemoreception. In The Physiology of Fishes, ed. Evans DH, pp. 251–261. CRC, Boca Raton. Sorensen PW, Hara TJ, Stacey NE & Goetz FW (1988). F prostaglandins function as potent olfactory stimulants that comprise the postovulatory female sex pheromone in goldfish. Biol Reprod 39, 1039–1050. St John JA, Clarris HJ, McKeown S, Royal S & Key B (2003). Sorting and convergence of primary olfactory axons are independent of the olfactory bulb. J Comp Neurol 464, 131–140. Strotmann J, Conzelmann S, Beck A, Feinstein P, Breer H & Mombaerts P (2000). Local permutations in the glomerular array of the mouse olfactory bulb. J Neurosci 20, 6927–6938. Strotmann J, Levai O, Fleischer J, Schwarzenbacher K & Breer H (2004). Olfactory receptor proteins in axonal processes of chemosensory neurons. J Neurosci 24, 7754–7761. Strotmann J, Wanner I, Helfrich T, Beck A & Breer H (1994). Rostro-caudal patterning of receptor-expressing olfactory neurones in the rat nasal cavity. Cell Tissue Res 278, 11–20. Szabo T, Blahser S, Denizot JP & Ravaille-Veron M (1991). Extrabulbar primary olfactory projection in teleost fish. C R Acad Sci III 312, 555–560. Treolar HB, Purcell AL & Greer CA (1999). Glomerular formation in the developing rat olfactory bulb. J Comp Neurol 413, 289–304. Uchida N, Takahashi YK, Tanifuji M & Mori K (2000). Odor maps in the mammalian olfactory bulb: Domain organization and odorant structural features. Nat Neurosci 3, 1035–1043. Valverde F, Santacana M & Heredia M (1992). Formation of an olfactory glomerulus: morphological aspects of development and organization. Neuroscience 49, 255–275.  C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

J Physiol 581.2

Response profiles of olfactory glomeruli

Vassar R, Chao SK, Sitcheran R, Nunez JM, Vosshall LB & Axel R (1994). Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981–991. Vassar R, Ngai J & Axel R (1993). Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 74, 309–318. Vogler C & Schild D (1999). Inhibitory and excitatory responses of olfactory receptor neurons of Xenopus laevis tadpoles to stimulation with amino acids. J Exp Biol 202, 997–1003. Wachowiak M, Denk W & Friedrich RW (2004). Functional organization of sensory input to the olfactory bulb glomerulus analyzed by two-photon calcium imaging. Proc Natl Acad Sci U S A 101, 9097–9102.

 C 2007 The Authors. Journal compilation  C 2007 The Physiological Society

579

Wang F, Nemes A, Mendelsohn M & Axel R (1998). Odorant receptors govern the formation of a precise topographic map. Cell 93, 47–60. Zheng C, Feinstein P, Bozza T, Rodriguez I & Mombaerts P (2000). Peripheral olfactory projections are differentially affected in mice deficient in a cyclic nucleotide-gated channel subunit. Neuron 26, 81–91.

Acknowledgements This work was supported by grants of the DFG Research Center Molecular Physiology of the Brain (CMPB) to D.S. and I.M.

Suggest Documents