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. 2018 Sep 4;12:295.
doi: 10.3389/fncel.2018.00295. eCollection 2018.

P/Q Type Calcium Channel Cav2.1 Defines a Unique Subset of Glomeruli in the Mouse Olfactory Bulb

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Free PMC article

P/Q Type Calcium Channel Cav2.1 Defines a Unique Subset of Glomeruli in the Mouse Olfactory Bulb

Martina Pyrski et al. Front Cell Neurosci. .
Free PMC article

Abstract

Voltage-gated calcium (Cav) channels are a prerequisite for signal transmission at the first olfactory sensory neuron (OSN) synapse within the glomeruli of the main olfactory bulb (MOB). We showed previously that the N-type Cav channel subunit Cav2.2 is present in the vast majority of glomeruli and plays a central role in presynaptic transmitter release. Here, we identify a distinct subset of glomeruli in the MOB of adult mice that is characterized by expression of the P/Q-type channel subunit Cav2.1. Immunolocalization shows that Cav2.1+ glomeruli reside predominantly in the medial and dorsal MOB, and in the vicinity of the necklace glomerular region close to the accessory olfactory bulb. Few glomeruli are detected on the ventral and lateral MOB. Cav2.1 labeling in glomeruli colocalizes with the presynaptic marker vGlut2 in the axon terminals of OSNs. Electron microscopy shows that Cav2.1+ presynaptic boutons establish characteristic asymmetrical synapses with the dendrites of second-order neurons in the glomerular neuropil. Cav2.1+ glomeruli receive axonal input from OSNs that express molecules of canonical OSNs: olfactory marker protein, the ion channel Cnga2, and the phosphodiesterase Pde4a. In the main olfactory epithelium, Cav2.1 labels a distinct subpopulation of OSNs whose distribution mirrors the topography of the MOB glomeruli, that shows the same molecular signature, and is already present at birth. Together, these experiments identify a unique Cav2.1+ multiglomerular domain in the MOB that may form a previously unrecognized olfactory subsystem distinct from other groups of necklace glomeruli that rely on cGMP signaling mechanisms.

Keywords: Cacna1a; Cav2.1α-1a subunit; olfactory bulb; olfactory epithelium; olfactory glomerulus; olfactory subsystem; synaptic localization; voltage-gated calcium channel.

Figures

FIGURE 1
FIGURE 1
Cav2.1 staining reveals a unique subset of glomeruli in the MOB. (A) Cav2.1 immunoreactivity (red) exemplified in a whole-mount preparation of the left and right MOB (dorso-caudal view) of an adult B6 mouse. Cav2.1+ axon bundles (arrows) terminate into several individual glomeruli (arrowheads). Large Cav2.1+ glomeruli reside in the caudal aspect of the MOB, close to the accessory olfactory bulb (AOB). Frequently, two glomeruli reside next to each other (brackets). Staining is absent in the AOB. (B) Negative control reaction using peptide blocking is devoid of Cav2.1 staining (dorso-caudal view and right bulb). (C) Sagittal section (14 μm) at about the MOB midline depicting four individual Cav2.1+ glomeruli (arrows 1–4) and their afferent axonal fibers (arrowheads). The inset (bottom) shows a higher magnification of two glomeruli (2, 3) and axonal projections from the nerve layer of the MOB. Nuclei staining with Hoechst dye (blue) verifies glomerular boundaries. (D) Coronal section (14 μm) of the posterior MOB showing two large Cav2.1+ glomeruli medial and lateral to the AOB. The inset at the bottom depicts a higher magnification of the medial glomerulus. (E) Cav2.1+ glomeruli (arrows) are already evident at postnatal day 7 (P7) including afferent axons (arrowheads, inset, and bottom). (A–E) Orientations are as depicted by arrows indicating a, anterior; p, posterior; d, dorsal; v, ventral; l, lateral; m, medial; AON, anterior olfactory nucleus. Scale bars (A–E) overviews, 500 μm; (C,D) insets, 100 μm; (E) inset, 50 μm.
FIGURE 2
FIGURE 2
Distribution and position of Cav2.1+ glomeruli in the MOB. (A) Representative bilateral 3D reconstruction of the Cav2.1+ glomeruli in the MOB of an adult mouse (4 month, caudal view). Both left and right olfactory bulbs were collected as serial 50 μm sections from the same mouse. Closely adjacent glomeruli are differently colored (yellow, red, and white) to facilitate identification. The majority of Cav2.1+ glomeruli is located on the dorsal and medial MOB surface. The location of the AOB is as indicated in blue. (B) Dorsal view of the two MOBs shown in (A) illustrating the Cartesian localization of Cav2.1+ glomeruli in reference to their position along the medial-to-lateral (ML) axis and their relative position along the anterior-to-posterior (AP) extent of MOBs. (C) 3D reconstruction of the left and right MOB hemispheres of two additional mice (8 weeks) further illustrates the distribution of Cav2.1+ glomeruli in the dorsal and medial MOB.
FIGURE 3
FIGURE 3
Cav2.1 is localized to presynaptic OSN axon terminals. Confocal images showing immunoreactivity (red) for (A) OMP, (B) vGlut2, (C) Map2, and (D) vGlut1 in combination with Cav2.1 (green) in coronal MOB sections of adult B6 mice. (A,B) The merged images illustrate that the presynaptic markers OMP and vGlut2 colocalize with Cav2.1 yielding the yellow fluorescence signal. (C,D) In contrast, the post-synaptic dendritic markers Map2 and vGlut1 do not colocalize with Cav2.1 in the glomerular neuropil. This is illustrated in the magnified merge at the bottom (boxed area) showing separate red and green fluorescence signals. Scale bars (A–D) 40 μm, inset magnifications, 20 μm.
FIGURE 4
FIGURE 4
Cav2.1+ OSN axons form typical asymmetrical synapses with post-synaptic dendrites in glomeruli. (A–E) Examples of Cav2.1+ OSN terminals (ONT) establishing asymmetric synapses with unstained and electron lucent dendrites (D) are shown. Arrows (red) indicate the polarity of the synapses. Labeled axonal terminals were evident from the accumulation of the DAB precipitate among the presynaptic vesicles and membrane specializations. There was also a typical darkening of mitochondrial membranes in labeled terminals. In Cav2.1+ glomeruli, only ONT are visible suggesting that Cav2.1 is homogeneously expressed by the axons innervating glomeruli. Scale bars: 500 nm.
FIGURE 5
FIGURE 5
Cav2.1+ glomeruli and expression of neural adhesion molecules. (A,B) Confocal images of the dorsal-caudal MOB (sagittal plane) stained with Cav2.1 (red) and neural cell adhesion molecule Ncam2 (green). (A) The single Cav2.1+ glomerulus in the central-dorsal MOB exhibiting immunoreactivity for Ncam2 (arrow) represents a rare observation. Other Ncam2+ glomeruli in this area are devoid of Cav2.1 immunoreactivity (arrowheads). (B) Cav2.1+ glomeruli in the dorso-caudal MOB are Ncam2– (arrow) as depicted by the large glomerulus. (C,D) Double-labeling immunohistochemistry for Cav2.1 (red) and Taar (green) in the dorsal center (C) and dorso-caudal border (D) of the MOB. Cav2.1+ glomeruli are not labeled by the panTaar antibody used. Images are representatives of (N = 3) adult B6 mice, with N representing every second section per mouse. Scale bars: 100 μm.
FIGURE 6
FIGURE 6
Cav2.1+ glomeruli receive afferent input from a specific subset of OSNs. (A) Cav2.1 immunoreactivity (red) and endogenous GFP fluorescence in a whole-mount MOB preparation (dorsal-caudal view) of an adult OMP-GFP mouse. Cav2.1 and GFP colocalize in individual glomeruli (arrows). Coronal MOB section (14 μm) showing that Cav2.1+ glomeruli (red) colocalize with Pde4a (green) (B) and Cnga2 (green) (C). (D,E) Non-canonical markers are absent in Cav2.1+ glomeruli. (D) Whole-mount view (top) showing that Cav2.1+ glomeruli (arrows) reside anterior to the Pde2a+ glomeruli (green) and the AOB. Pde2a and Cav2.1 label separate sets of glomeruli (bottom, 14 μm coronal section). (E) Whole-mount view of the MOB (top) of a GCG-GFP mouse. Cav2.1+ glomeruli (red, arrows) reside anterior to the GFP+ glomeruli (green) that receive axonal input from GGNs. GCG-GFP+ and Cav2.1+ glomeruli are separate sets of glomeruli (bottom, 14 μm coronal section). (F) Coronal MOB section (14 μm) from a Trpc2-IRES-taulacZ mouse stained for Cav2.1 (red) and β-galactosidase (green). Both the AOB and the vomeronasal nerve (VN) but not the Cav2.1+ glomeruli (arrowheads) are positive for β-galactosidase. (G) Sagittal section (14 μm) of the caudal-ventral MOB from a Gucy1b2-IRES-tauGFP mouse. The single GFP+ (green) glomerulus shown is devoid of Cav2.1 staining (red). Hoechst nuclear dye (blue) defines glomerular boundaries in the merged images. Images are representatives of N ≥ 2 mice with N = every second section per mouse. Scale bars (A) 500 μm, (B–E) 100 μm, (F) 200 μm, (G) 50 μm.
FIGURE 7
FIGURE 7
Identification of Cav2.1+ OSNs in the MOE. (A) Coronal MOE sections (14 μm) showing the left nasal cavity of an adult B6 mouse stained with Cav2.1. Cav2.1+ OSNs reside mainly in the MOE lining the dorsal roof (dr), nasal septum (ns), the dorsal-medial tip of endoturbinate II, and the tip of endoturbinate III (dashed lines). Few labeled OSNs are detectable at the tips of ectoturbinates 2 and 3. (B) Cav2.1 staining is present in OSN knob (k), dendrite (d), soma (s), and axon (arrowheads). To delineate axonal Cav2.1 staining, contrast and brightness was increased by 50%. (C) The peptide control reaction is devoid of Cav2.1 staining. (D) RNAscope fluorescence in situ hybridization for Cav2.1 mRNA (Cacna1a, green) in the MOE of an adult B6 mouse (coronal view, left nasal cavity) shows that the distribution of labeled OSNs is closely similar to that obtained by immunohistochemistry (A). The dorsal roof, endoturbinate I, nasal septum, and the dorsal-medial tips of endoturbinates IIa and IIb (dashed lines) show labeled OSNs. Few labeled OSNs reside at the medial tip of ectoturbinate 2 (arrowheads). (E) Higher magnification shows hybridized OSNs at all depths of the epithelial layer. (F) The negative control reaction is devoid of labeling. (G) Agarose gel electrophoresis of the products obtained by RT-PCR using Cacna1a-specific primers and total RNA of adult mouse olfactory tissue. RT-PCR resulted in products (+RT) of the expected sizes using primers amplifying exons 24–38 (1,731 bp, arrowhead) and exons 37–43 (607 bp, arrowhead). Control reactions omitting reverse transcriptase (-RT) yielded no products. M, DNA size marker in base pairs as indicated at the left. (H) Magnifications of the dorsal MOE derived at different mouse ages stained with Cav2.1. Immunoreactivity for Cav2.1 (red) is absent at embryonic day 18 (E18). Cav2.1+ OSNs become visible at about postnatal day 1 (P1), and expression continues at P7, P14, P21 toward adulthood. Images are representatives of N ≥ 2 mice per age with N ≥ 10 sections per mouse. Scale bars (A,C,D) 200 μm, (E); 50 μm; (F,H) 20 μm; (B) 10 μm.
FIGURE 8
FIGURE 8
Cav2.1+ OSNs express the canonical markers OMP, Pde4a, and Cnga2. Coronal MOE sections (14 μm) of adult mice labeled for Cav2.1 and different canonical (A) and non-canonical (B) signal transduction molecules. (A) As shown at the left, Cav2.1 (red) and GFP (green) colocalize in mature OSNs (arrows) of OMP-GFP mice. Cav2.1+ OSNs (red) express Pde4a (green, middle, arrows) and Cnga2 (green, right, arrow). (B) Cav2.1 immunoreactivity (arrows) is absent in non-canonical OSNs such as those expressing Trpc2 (red, left, arrowheads), Taar (green, middle, arrowhead) or GC-D (green, right, arrowhead). Images are representatives of (N ≥ 2) mice with N = every 10th section along the anterior-to-posterior extent of the MOE in each mouse. Scale bars: 20 μm.
FIGURE 9
FIGURE 9
Summary scheme showing the organization of the major olfactory subsystems at the level of a glomerular map. (A) Dorso-caudal view of the olfactory bulb (left side) depicting the different glomerular subsystems as indicated by the color-coded legend. Cav2.1 glomeruli in the caudal MOB reside in juxtaposition to the GC-D necklace glomeruli innervated by GC-D+ OSNs of the MOE and the GC-G glomeruli innervated by GGNs of the Grueneberg ganglion (GGN). Cav2.1 glomeruli in the center of the dorsal MOB intermingle with Taar glomeruli, and are also present in the medial MOB. (B) Medial view of the right olfactory bulb visualizing the necklace glomerular tracks for GC-D and GC-G, the dorsal position of Cav2.1 and Taar glomeruli, the medial Cav2.1 glomeruli, and the rostral and ventral position of Trpc2+ glomeruli in the MOB. Arrows denote orientations a (anterior), p (posterior), d (dorsal), v (ventral), l (lateral), and m (medial); dashed lines indicate dorsal zones DI and DII, and the ventral zone V. Adapted and extended from Bear et al. (2016).

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