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Eur J Neurosci
2007 Aug 01;264:925-34. doi: 10.1111/j.1460-9568.2007.05731.x.
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Presynaptic protein distribution and odour mapping in glomeruli of the olfactory bulb of Xenopus laevis tadpoles.
Manzini I
,
Heermann S
,
Czesnik D
,
Brase C
,
Schild D
,
Rössler W
.
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The sensory input layer in the olfactory bulb (OB) is typically organized into spheroidal aggregates of dense neuropil called glomeruli. This characteristic compartmentalization of the synaptic neuropil is a typical feature of primary olfactory centres in vertebrates and most advanced invertebrates. In the present work we mapped the location of presynaptic sites in glomeruli across the OB using antibodies to presynaptic vesicle proteins and presynaptic membrane proteins in combination with confocal microscopy. In addition the responses of glomeruli upon mucosal application of amino acid-odorants and forskolin were monitored using functional calcium imaging. We first describe the spatial distribution of glomeruli across the main olfactory bulb (MOB) in premetamorphic Xenopus laevis. Second, we show that the heterogeneous organization of glomeruli along the dorsoventral and mediolateral axes of the MOB is associated with a differential distribution of synaptic vesicle proteins. While antibodies to synaptophysin, syntaxin and SNAP-25 uniformly labelled glomeruli in the whole MOB, intense synaptotagmin staining was present only in glomeruli in the lateral, and to a lesser extent in the intermediate, part of the OB. Interestingly, amino acid-responsive glomeruli were always located in the lateral part of the OB, and glomeruli activated by mucosal forskolin application were exclusively located in the medial part of the OB. This correlation between odour mapping and presynaptic protein distribution is an additional hint on the existence of different subsystems within the main olfactory system in larval Xenopus laevis.
Fig. 1. Glomerular distribution in the MOB of larval Xenopus laevis. (A) Sagittal section of the MOB double-labelled with synaptophysin immunostaining (red) and anterograde labelling with biocytin through the olfactory nerve (green; on, olfactory nerve). The axes indicate: A, anterior; P, posterior; D, dorsal; V, ventral. (B) Schematic representation of the glomerular distribution in the MOB. The axes indicate: A, anterior; P, posterior; L, lateral; M, medial. Glomerular clusters: int, intermediate glomerular cluster; lat, lateral glomerular cluster; med, medial glomerular cluster; sc, small glomerular cluster. (C) Horizontal view of synaptophysin immunolabelled sections of the anterior part of the MOB at three different heights (C1, dorsal OB; C2, intermediate OB; C3, ventral OB). Scale bars: 100 lm.
Fig. 2. Differential distribution of presynaptic proteins in the glomerular layer of the MOB. Horizontal sections of the MOB labelled with antibodies to synaptophysin (A), syntaxin (B) and SNAP-25
(C). All three antibodies label glomeruli in all glomerular clusters (int, intermediate glomerular cluster; lat, lateral glomerular cluster; med, medial glomerular cluster). The axes in (A) indicate: A, anterior; P,
posterior; L, lateral; M, medial. (D) Horizontal section of the MOB anterogradely labelled with biocytin through the olfactory nerve. Double-staining of the olfactory receptor axons with biocytin in (D) clearly
visualizes three glomerular clusters. (E) Synaptotagmin immunostaining of the same slice shown in (D). Synaptotagmin-immunoreactivity (IR) was limited to the lateral and partly to the intermediate glomerular
cluster. (F) An overlay of the two channels (D and E) accents the unequal distribution of synaptotagmin-IR in the different clusters. Scale bars: 100 lm.
Fig. 3. Nose–brain preparation and amino acid-induced calcium signals in individual glomeruli in the lateral glomerular cluster of the MOB. (A) Fluo-4-stained OB of a nose–brain preparation. The dotted lines indicate the approximate borders of the accessory OB and the mitral and granule cell layer in the MOB and
the position of the lateral glomerular cluster. The image was acquired at rest (AOB, accessory olfactory bulb; GCL, granule cell layer; GL, glomerular layer; lat,
lateral glomerular cluster; MCL, mitral cell layer). (B) image of the same OB showing various glomeruli, mitral cells and granule cells activated by mucosal
application of amino acids (200 lm). (C) higher magnification of the lateral cluster of another nose–brain preparation showing a number of amino acid-responsive
glomeruli (see areas marked by numbers). The arrows indicate individual responsive fibres. (D) Time courses of the [Ca2+]i transients of the five glomeruli indicated
by numbers in (C). Scale bars: 50 lm (A); 20 lm (C).
Fig. 4. Differential activation of glomerular clusters by amino acids and
forskolin. (A) Fluo-4-stained OB of a noseâbrain preparation (image acquired
at rest). The dotted lines indicate the approximate borders of the three main
glomerular clusters of the MOB (int, intermediate glomerular cluster; lat, lateral
glomerular cluster; med, medial glomerular cluster). (B) Time courses of the
[Ca2+]i transients of the three glomerular clusters upon mucosal application of
100 lm forskolin (fsk; medial cluster: blue line; intermediate cluster: green
line; lateral cluster: red line). (C) Time courses of the [Ca2+]i transients of the
three glomerular clusters upon mucosal application of 200 lm amino acids
(same colours as in B). The entire clusters were taken as regions of interest.
Scale bar: 100 lm.
