Recording of odor-evoked responses in a genetically defined glomerulus.

A) Schematic diagram illustrating the experimental preparation. A puff of 200 µM methionine was applied for 100 ms to the left olfactory epithelium of Dre.mxn1:GFP Xenopus tropicalis tadpoles. Changes in the Local Field Potential (LFP) were measured ipsilaterally with an electrode targeted to the lateral glomerulus formed by axon terminals of olfactory sensory neurons (OSNs) expressing GFP. The inset shows a confocal projection illustrating labelled OSNs (s: soma, a:axon) and the bilateral formation of glomeruli (g). The Dre.mxn1 promoter also drives GFP expression in the endothelial cells of some blood vessels (e). B) Representative glomerular odor-evoked response (black) obtained by averaging individual responses (gray) of the LFP following stimulation (arrow). The asterisk shows a positivity that was evident in 37% of the recordings and preceded the characteristic negativity associated with glomerular activation. C) Negative deflections of LFP (individual responses, gray; average trace, black) were observed when the recording electrode was placed in the GFP labelled glomerulus but disappeared in the mitral cell layer (ML, red traces; GL, glomerular layer). D) Experiment showing LFP recordings performed in four different locations of the glomerular layer spaced by 50 μm. The characteristic odor-evoked response was observed in only one of the positions tested. The representative glomerular odor-evoked response (black) was obtained by averaging individual responses (gray) following stimulation with methionine (arrow). E) Hyperstack projections of the left olfactory bulb of three different Dre.mxn1:GFP X. tropicalis tadpoles. The lateral glomerular cluster (L) is always evident, and some medial projections (M) are apparent in two of the illustrated examples. The color scale indicates dorsoventral disposition.

Odor-evoked responses are mediated by glutamatergic neurotransmission.

A) The representative glomerular odor-evoked response (black) was obtained by averaging individual responses (gray) of the Local Field Potential (LFP) following ipsilateral stimulation of the olfactory epithelium using 200 μM methionine (arrow). In this example, the pipette solution contained 100 µM CNQX and upon local injection of 1 μL, there was a reduction in the amplitude of LFP negativities. B) Mean LFP changes obtained under control conditions (n=21) were reduced after the application of 100 µM CNQX (n=8), 100 µM AP5 (n=5), or both 100 µM CNQX and 100 µM AP5 (n=8). C) Box plot illustrating how the initially recorded peak negativities were affected by the application of 100 µM CNQX, or, 100 µM AP5 together with 100 µM CNQX. Boxes represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups. Statistical differences were evaluated using paired t-test. D) Rubi-glutamate was injected into the GFP labelled glomerulus and locally uncaged with a 500 ms pulse of blue light (circle). The example shows the change in LFP induced by two flashes (squares) delivered at an interval of 9 seconds. E) Application of picrotoxin, a GABAA antagonist, did not modify odor-evoked changes. Recordings show average responses (n=9). Statistical differences were evaluated using paired t-test. F) Odor-evoked LFP changes were exclusively triggered by ipsilateral stimulation. Individual responses are indicated in gray, with the representative average response in black. Contralateral stimuli (yellow) did not modify the LFP, as shown in the average representative response (brown).

Potentiation of odor-evoked responses by transection of the contralateral olfactory nerve.

A) The number of olfactory sensory neurons (OSNs) and the amplitude of odor-evoked negative deflections of the Local Field Potential (LFP) were related to olfactory nerve width according to linear and exponential functions, respectively. Individual data points are represented by circles (n=48). Each bin indicates the mean ± s.e.m. of n=6 tadpoles. The dotted line indicates the steady-state LFP amplitude reached during development. B) Representative odor-evoked responses obtained in a control tadpole (black) and in a different animal (red), 24 h after contralateral nerve transection. Gray traces indicate individual responses to the application of 200 μM methionine solution (arrow). C) Odor-evoked LFP changes exhibit amplitudes above the expected values (dotted line as in A) after contralateral olfactory nerve transection at the indicated time points. The dots represent the mean ± s.e.m. obtained 2 to 7 hours (n=10), 1 to 2 days (n=14), and 10 to 11 days (n=11) post-injury. There was a 75% increase in animals recorded 1 to 2 days after transection of the contralateral olfactory nerve (red arrow) compared to control tadpoles (dotted line). D) Dots (mean ± s.e.m.) connected by a line illustrate odor-evoked glomerular responses at the indicated times after injury. The superimposed violin plot displays individual data. Most LFPpeak values are above the level expected for the developmental period studied (dotted line as in A).

The potentiation of odor-evoked responses is not mediated by injury derived cues.

A) Odor-evoked Local Field Potential (LFP) changes were recorded by an electrode targeted to the GFP-positive glomerulus of Dre.mxn1:GFP tadpoles one to two days after bilateral transection of optic nerves. B) Peak LFP negativities recorded in tadpoles with sectioned optic nerves (n=18) did not exhibit the characteristic potentiation observed after transection of the contralateral olfactory nerve (n=14), as they remained within the range of values observed during normal development (solid line, as in Fig. 3A). Bins indicate mean±s.e.m., circles show individual values. C) Imaging of reactive oxygen species (ROS) two hours after transecting one olfactory nerve (arrow). The ratio between the fluorescence emitted by HyPer-YFP when excited at 488 nm and 405 nm is indicated in pseudocolor. Notice that ROS were increased at the injury site but remained at basal levels in both olfactory bulbs as indicated by the box plot. Each circle shows values collected in a single tadpole. D) Block of ROS production by incubating tadpoles with 200 μM apocynin (n=10) or 2 μM diphenyleneiodonium (DPI, n=5) did not modify the amplitude of odor-evoked LFP responses recorded 24 h after contralateral olfactory nerve transection (n=10). Boxes represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups.

The presynaptic component of glomerular activation is affected by damage to contralateral olfactory sensory neurons.

A) Application of a puff of 200 μM methionine to the olfactory epithelium activates a set of glomeruli in the ipsilateral olfactory bulb (arrows) of tubb2b:GCaMP6s tadpoles. Images show the relative changes in GCaMP6s fluorescence (ΔF/F) obtained after two sequential stimulations carried out in a single tadpole. B) Time course of the responses detected in the glomeruli indicated in A). C) An example showing the simultaneous recording of Local Field Potential (LFP) and changes in GCaMP6s fluorescence in the region targeted by the electrode. Colored traces and gray traces show the change in GCaMP6s fluorescence (ΔF/F) and LFP respectively observed after three sequential applications of 200 μM methionine. Black traces show the average ΔF/F and LFP responses. D) Kinetics of the change in LFP observed in tadpoles with the contralateral olfactory nerve transected between 2 h and 48h prior to recording. The differences are illustrated by representative recordings obtained in two different tadpoles. E) Intracellular calcium increases detected in glomeruli of control tadpoles with intact olfactory pathways (35 glomeruli, 10 tadpoles, black), and, in tadpoles subjected to the transection of the contralateral olfactory nerve (10 glomeruli, 3 tadpoles, red). Each trace indicates the response of a glomerulus to a single stimulus. Solid lines and error bars indicate mean ± s.e.m. F) Calcium transients detected in tadpoles with an olfactory nerve transected showed a larger amplitude and a rising phase with a shorter time constant (τ). Boxes in D) and F) represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups. Statistical differences in D) and F) were evaluated using paired and unpaired t-tests, respectively. Circles in D) indicate tadpoles and in F) refer to glomeruli.

Tyrosine hydroxylase positive neurons project to the glomerular layer of the olfactory bulb.

A) Cell bodies of neurons expressing tyrosine hydroxylase (TH+, magenta) were sparsely distributed at the border of the glomerular (GL) and mitral cell (ML) layers of the olfactory bulb and sent their neuronal processes (arrows) to innervate the glomerular layer (asterisks). B) Projections of TH+ neurons (arrows) contacted axon terminals of olfactory sensory neurons labelled with DiI (cyan, asterisks). C) The lateral glomerular cluster (L) labeled in Dre.mxn1:GFP Xenopus tropicalis tadpoles was contacted by projections (asterisks) of processes emerging from TH+ neurons (arrows).

Contralateral input modulates presynaptic inhibition mediated by dopamine D2 receptors and is involved in the potentiation of glomerular responses.

A) Recordings obtained in a control tadpole showing how the amplitude of Local Field Potential (LFP) responses (gray traces) obtained during a baseline period of 8 minutes increased in a time-dependent manner after local application of 300 nM raclopride, a D2 receptor antagonist. B) Box plot showing the effect of 300 nM raclopride (blue) on the amplitude of LFP responses recorded in tadpoles with full capacity to process odors (control, n=8, black) and tadpoles subjected to the transection of the contralateral olfactory nerve (n=11, red). Boxes represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups. The effect of raclopride was evaluated using the paired t-test and the comparison between control and transected groups was performed using the unpaired t-test. C) Relative change in LFP responses induced by 300 nM raclopride in control tadpoles and in tadpoles subjected to the transection of the contralateral olfactory nerve. Dots represent mean ± s.e.m. The solid black line illustrates the fit to a Hill equation, defining a T50 at 20 minutes. D) Simultaneous recording of LFP and changes in GCaMP6s fluorescence in a tubb2b:GCaMP6s tadpole. Gray traces and light blue traces show individual responses to sequential stimulations before and after application of 300 nM raclopride, respectively. Average responses are shown in black and dark blue. E) Application of CGP-36742, a GABAB receptor antagonist, did not modify LFP responses. The box plot compares the amplitude of LFP changes recorded before (gray) and 20 min after local application of 300 μM CGP-36742 (green). Statistical differences were evaluated using paired t-test. F) Time course of relative LFP changes induced by 300 μM CGP-36742. Dots represent mean ± s.e.m (n=10). The inset shows recordings obtained in a tadpole in baseline conditions (gray) and after injection of CGP-36742 (green).

Effect on odor-evoked responses of selective photoablation of olfactory sensory neurons innervating the homologous contralateral glomerulus.

A) Odor-evoked Local Field Potential (LFP) changes were recorded one day after the selective elimination of olfactory sensory neurons (OSNs) located in the right nasal cavity. After the identification of GFP-positive OSNs in the epithelium labeled with the nuclear marker Hoechst 33342, regions containing ≥2 fluorescent neurons were identified and photobleached. Cell targeting was confirmed by the suppression of the nuclear label. Only cells found within the photobleached areas exhibited fragmented nuclei (arrows) 24 hours after photobleaching. B) Examples showing odor-evoked responses recorded in a tadpole incubated with Hoechst 33342 (black, average), and in a different tadpole 24 hours after the photobleaching of selected regions in the contralateral olfactory epithelium (violet, average). C) Photoablation of GFP positive neurons did not modify the amplitude or variance of contralateral odor-evoked responses recorded in the glomerulus innervated by cognate neurons. Boxes represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups. Statistical differences were evaluated using ANOVA followed by Tukey’s test. Circles indicate values obtained from single tadpoles.

Pallial neurons are involved in the potentiation of glomerular responses driven by contralateral injury.

A) Rhythmic calcium transients were detected in six different regions of interest (ROIs) located in the dorsolateral pallium of a X. tropicalis tubb2b:GCaMP6s tadpole. Three consecutive stimulations carried out by applying of 200 μM methionine to the ipsilateral olfactory epithelium (dotted line) evoked a synchronous response in the ROIs investigated. B,C) Odor-evoked responses were recorded 24-48 h after making a tangential injury in the contralateral dorsolateral pallium. The amplitude of LFP changes significantly increased in injured tadpoles. Boxes represent the median (horizontal line), 25th to 75th quartiles, and ranges (whiskers) of the indicated experimental groups. Statistical differences were evaluated using unpaired t-test. D) Model proposed for the bilateral modulation of glomerular output in the olfactory bulb of Xenopus tadpoles. A population of juxtaglomerular neurons releases dopamine to inhibit glomerular output by activating presynaptic D2 receptors present in OSNs (dotted box). The constant presence of dopamine within glomeruli is favored by the activity of the contralateral olfactory bulb. When the contribution of the contralateral pathway is suppressed, dopamine release diminishes, and glomerular responses become potentiated. The contralateral modulation of the tonic activity of dopaminergic juxtaglomerular neurons corrects for input differences and equalizes the synaptic output of olfactory glomeruli to achieve a bilaterally balanced transfer of information. The activity of dopaminergic interneurons is likely controlled by pallial neurons through a yet undetermined connectivity, taking advantage of their participation in the processing of olfactory information.