Fig 1.
Zebrafish OBs are connected by two interhemispheric pathways.
(A) Two-photon microscopy images showing unilateral dye labeling of OB cells in an adult zebrafish forebrain. This image is reconstructed by juxtaposing several partially overlapping images. Labeled mitral cell axons cross the midline at the level of the anterior commissure and terminate in the contralateral OB. Dense ipsilateral axonal projections to Dp, the fish homolog of the olfactory cortex, course through the lateral olfactory tract. Red asterisks indicate dye electroporation sites in the OB, and red arrowheads point at axonal projections in the contralateral OB. (B) Schematic of dye labeling in the OB. The filled red ellipse indicates the labeling site. The gray dashed square indicates the imaging zone in (C). (C) Horizontal, sagittal, and coronal views of mitral cell axons projecting to the contralateral OB in a representative brain (n = 12 fish). (D) Schematic of dye labeling of Dp. The gray dashed square indicates the imaging zone in (E). (E) Horizontal, sagittal, and coronal views of Dp projections to the contralateral OB in a representative brain (n = 5 fish). Scale bars represent 100 μm. Dp, dorsal part of the dorsolateral pallium; OB, olfactory bulb; OE, olfactory epithelium.
Fig 2.
The direct interbulbar connections are topographically organized and glomeruli specific.
(A) Schematic of the dye labeling in the medial OB. The filled red ellipse indicates the labeling site. The gray dashed squares indicate the two imaging fields. (B) Two-photon microscopy images showing the labeled neurons in the medial OB (bottom) and their reconstructed axonal projections (red) to the contralateral medial OB (top, n = 3 fish). The yellow dashed line separates each OB into a medial and a lateral half. (C) Schematic of the dye labeling in the lateral OB. (D) Two-photon microscopy images showing the labeled neurons in the lateral OB (bottom) and their reconstructed axonal projections (red) to the contralateral lateral OB (top) in a representative brain (n = 7 fish). (E) Schematic of the dye labeling of the lVPG. (F and G) Two-photon microscopy images showing the labeled neurons innervating the lVPG (bottom) and their reconstructed axonal projections (red) to the contralateral OB (top, n = 2 fish). (H). Schematic of the dye labeling of the mVPG. (I and J) Two-photon microscopy images showing the labeled neurons innervating the mVPG (bottom) and their reconstructed axonal projections (red) to the contralateral OB (top, n = 2 fish). The lVPG is indicated by a yellow asterisk, and the mVPG is indicated by a yellow cross. Scale bar represents 100 μm for (B–J). (K) Two-photon microscopy image of an adult zebrafish forebrain showing the reconstructed projection of a single mitral cell to the contralateral OB (n = 4 cells projecting to the contralateral OB, see Materials and methods). The gray background is elavl3:GCaMP6s labeling. The axonal reconstruction is color-coded for depth. The plain gray line highlights the contour of the zebrafish forebrain explant. The gray dashed square indicates the imaging zone in (B). The scale bar represents 50 μm. (L) Close-up image of the labeled mitral cell’s axonal terminals at the contralateral OB. Note the close apposition of the labeled axon with contralateral mitral cells’ dendritic tuft. The dashed gray line highlights the contour of the brain explant. The scale bar represents 25 μm. elavl3, ELAV-like RNA binding protein 3; GCaMP6, genetically encoded calcium sensor, circular permutated green florescent protein-Calmodulin-M13 peptide 6; lVPG, lateral ventroposterior glomerulus; mVPG, medial ventroposterior glomerulus; OB, olfactory bulb; TMR, tetramethylrhodamine; Vv, ventral nucleus of the ventral telencephalon.
Fig 3.
Interhemispheric connections modulate odor-evoked responses in the contralateral OB.
(A) Illustration of the experimental setup depicting an adult zebrafish brain explant expressing GCaMP6s in mitral cells. A tube delivers food odor (od) selectively to the ipsilateral nostril. Contralateral stimulations (cs) of varying intensities (low, medium, and strong, see Materials and methods) are delivered using a glass microstimulation electrode inserted in the contralateral olfactory nerve. (B) The contralateral olfactory tract, which enables interhemispheric olfactory connections, is sectioned in control conditions. (C–E and O) Mean odor response time course of all mitral cells recorded in the ipsilateral OB following odor presentation (od, gray bar) or odor combined with simultaneous contralateral microelectrode stimulation (od + cs; cs indicated by a red bar) for low, medium, and strong stimulation intensities and the control condition, respectively. For each condition, mitral cells from multiple animals are pooled and sorted by the amplitude of contralateral modulation ([od + cs] − od). (F–H and P) Average mitral cells’ responses to odor (red) are plotted against their response to odor combined with cs for low, medium, and strong cs (in red) and the control condition (in black), respectively. Mitral cells above the unity line are positively modulated by contralateral inputs. (I–K and Q) The contralateral modulation ([od + cs] − od) is displayed for all mitral cells for low, medium, and strong cs and the control condition, respectively. (L–N and R) The cumulative frequency distribution of the contralateral modulation is displayed for all mitral cells for low, medium, and strong cs (in red) and the control condition (in black), respectively. The numbers of mitral cells are as follows: low cs, 335 cells in three fish; medium cs, 335 cells in three fish; strong cs, 857 cells in six fish; control cs, 477 cells in two fish (*p < 0.05; ***p < 0.001; two-sample, two-tailed Kolmogorov–Smirnov test). Numerical data used to generate this figure can be found in S1 Data and S2 Data. cs, contralateral microstimulation; GCaMP6, genetically encoded calcium sensor, circular permutated green florescent protein-Calmodulin-M13 peptide 6; OB, olfactory bulb; od, food odor extract.
Fig 4.
Natural odors elicit odor-selective and chemotopically organized responses in the contralateral OB.
(A) Illustration of the experimental setup depicting an adult zebrafish brain explant expressing GCaMP6s in mitral cells. A tube delivers the olfactory cues to the nostrils. The stimuli used are AAs, bile acids, and pgf2α. The olfactory nerve is sectioned (red mark) on the contralateral OB. Mitral cell odor responses are recorded in the OB with an intact olfactory nerve using two-photon calcium imaging. (B) Illustration of the experimental setup depicting an adult zebrafish brain explant. Mitral cell odor responses are recorded in the deafferented OB using two-photon calcium imaging. (C) Odor response time course of all ipsilateral mitral cells pooled and sorted by their response amplitude (760 cells in six fish). (D) Odor response time course of three representative odor-selective ipsilateral mitral cells. Arrowheads indicate odor onset. (E) Odor response time course of all contralateral mitral cells pooled and sorted by decreasing responsiveness (715 cells in six fish). (F) Odor response time course of three representative odor-selective contralateral mitral cells. Arrowheads indicate odor onset. (G) The percentage of mitral cells responding to at least one olfactory cue is larger in the ipsilateral OB (black) than in the deafferented contralateral OB (red) (**p < 0.01; Student t test). (H) The percentage of mitral cells responding to only one of the presented odors was similar in the ipsilateral (black) and contralateral OB (red) (Student t test). (I) The cumulative frequency of lifetime sparseness for all mitral cells was similar in both OBs. ns; two-sample, two-tailed Kolmogorov–Smirnov test. (J) Ipsilateral (left) and contralateral (right) OB responses to pgf2α in the same fish (representative planes located 50 μm deep from the ventral OB surface). (K) Ipsilateral (left) and contralateral (right) OB responses to AA mix in the same animal than in (J) (representative planes located 90 μm deep from the ventral OB surface). (L) Spatial distribution of mitral cells responding selectively to pgf2α, along the rostrocaudal axis in both OBs for all fish. Values close to 1 indicate caudal locations, whereas values close to 0 indicate rostral locations. Note the caudal location of pgf2α-selective ipsilateral cells compared with the random distribution indicated by the gray dashed line (*p < 0.05; Mann–Whitney U test). The caudality index of contralateral pgf2α-selective cells is similar to that of ipsilateral pgf2α-selective cells (ipsi- versus contralateral, ns; Mann–Whitney U test). (M) Location of mitral cells responding selectively to AAs along the mediolateral axis in both OBs for all fish. Values close to 1 indicate lateral locations, whereas values close to 0 indicate medial locations. Note the lateral location of ipsilateral AA-selective cells compared with the random distribution indicated by the gray dashed line (***p < 0.001; one-sample Student t test). The laterality index of contralateral AA-selective cells is similar to that of ipsilateral AA-selective cells (ipsi- versus contralateral, ns; Student t test). (N) The pairwise similarity in mitral cell responses as a function of the distance between them (ipsilateral, n = 6 fish; contralateral, n = 6 fish; ns; Student t test). Dashed lines indicate the average correlation after shuffling the spatial locations of mitral cells. Numerical data used to generate this figure can be found in S1 Data and S2 Data. AA, amino acids; contra, contralateral; GCaMP6, genetically encoded calcium sensor, circular permutated green florescent protein-Calmodulin-M13 peptide 6; ipsi, ipsilateral; ns, nonsignificant; OB, olfactory bulb; pgf2α, prostaglandin 2α.
Fig 5.
OB interneurons are diffusely activated by the contralateral olfactory inputs.
(A) Illustration of the experimental setup depicting an adult zebrafish brain explant injected with a synthetic calcium indicator (Rhod-2 AM) labeling inhibitory interneurons. A tube delivers odors to the nostrils. The stimuli used are amino acids, bile acids, and food odor extract. The olfactory nerve is sectioned on the contralateral OB. (B) Ipsilateral interneurons’ response to bile acid. (C) The same setup as in (A), except that the deafferented OB contralateral to odor stimulation is imaged. (D) Contralateral interneurons’ response to bile acid. (E) Odor response time course of all ipsilateral interneurons (1,302 cells in four fish). For each odor, interneurons are sorted by decreasing responsiveness. (F) Odor response time course of all contralateral interneurons (1,566 cells in five fish). (G) Cumulative frequency of lifetime sparseness for all contralateral and ipsilateral interneurons (ns; two-sample, two-tailed Kolmogorov–Smirnov test). (H) The pairwise similarity in interneuron response as a function of the distance separating them (ipsilateral, n = 4 fish; contralateral, n = 5 fish, *p < 0.05; Student t test). Dashed lines indicate the average correlation after shuffling the spatial locations of interneurons. (I) Spatial distribution of ipsilateral and contralateral interneurons responding selectively to bile acids along the mediolateral axis for all fish. Values close to 1 indicate medial locations, whereas values close to 0 indicate lateral locations. Ipsilateral bile acid–selective interneurons are located medially (*p < 0.05; one-sample Student t test for comparison, with random distribution indicated by the gray dashed line). Contralateral bile-selective interneurons are located less medially than ipsilateral ones (p = 0.054, two-sample, two-tailed Student t test with ipsilateral interneurons). Numerical data used to generate this figure can be found in S1 Data and S2 Data. contra, contralateral; ipsi, ipsilateral; ns, nonsignificant; OB, olfactory bulb; Rhod-2 AM, acetoxymethyl ester of rhodamine-2.
Fig 6.
Contralateral olfactory inputs preserve reproductive pheromone detection in food odor background.
(A) Illustration of the experimental setup depicting an adult zebrafish brain explant expressing GCaMP6s in excitatory neurons, and the tube for odor delivery to the intact nose. Three odors were used: pgf2α, food odor extract (food), and an odor mixture of pgf2α and food odor extract. Food odor was delivered at different concentrations (control [−]: 0 μg/mL; low [+]: 25 μg/mL; medium [++]: 100 μg/mL; high [+++]: 400 μg/mL). The dashed square indicates the imaged olfactory bulb. (B) Ventral fish brain dissection with the contralateral olfactory nerve sectioned, preventing contralateral inputs to modulate the odor responses measured. (C) Odor response time course of three representative pgf2α-selective MCs in the presence of contralateral olfactory inputs. Black arrowheads indicate odor onset. (D) Odor response time course of three representative pgf2α-selective MCs without contralateral olfactory inputs. (E) Odor response maps to pgf2α, food odor, and the odor mixture in the olfactory bulb receiving contralateral inputs. Black asterisks indicate pgf2α-selective glomeruli. (F) Odor response maps for the same fish and imaging plane than in (E), without contralateral inputs. (G) Relative response of pgf2α-selective cells to the odor mixture in all conditions. Each circle represents a pgf2α-selective MC with (black) or without (red) contralateral inputs. The response amplitude of each pgf2α-selective MC to the odor mixture is normalized to its response to pgf2α alone. Values close and superior to 1 represent conserved or enhanced responses to pgf2α in presence of food odor, respectively. Values close to 0 indicate suppression of the pgf2α response in the presence of food odor (**p < 0.01, Mann–Whitney U test). The number of pgf2α-selective MCs is as follows (with contralateral inputs): control (−), 57 MCs in seven fish; low (+), 61 MCs in six fish; medium (++), 83 MCs in eight fish; and high (+++), 25 MCs in five fish. The number of pgf2α-selective MCs is as follows (without contralateral input): control (−), 72 MCs in five fish; low (+), 53 MCs in six fish; medium (++), 77 MCs in six fish; and high (+++), 56 MCs in five fish. The response of the cell placed in a dashed square is out of range (3.3). (H) Correlation between MCs’ odor responses to pgf2α and odor mixtures for all conditions (**p < 0.01, Student t test). High and low pattern correlation values indicate high and low similarities with the pgf2α response pattern, respectively. Each circle represents correlation within a single fish, with contralateral input (in black) or without contralateral input (in red). (I) Correlation between MCs’ odor responses to food odor and odor mixtures for all conditions (**p < 0.01, Student t test). High and low pattern correlation values indicate high and low similarities with the food odor response pattern, respectively. Numerical data used to generate this figure can be found in S1 Data. GCaMP6, genetically encoded calcium sensor, circular permutated green florescent protein-Calmodulin-M13 peptide 6; MC, mitral cell; pgf2α, prostaglandin 2α.