The refinement of mitral and tufted cell apical dendrite is not affected by massive cell ablation at perinatal stages.

A. Schematic representation of the transgenic strategy to ablate M/T cells at perinatal stages. The Tbx21 promoter is specific for M/T cells. The Tbx::cre transgene controls the expression of the Cre recombinase in M/T cells, thus regulating the selective expression in M/T cells of the subunit A of the diphtheria toxin (DTA), from a CAG::loxp stop loxP-DTA reporter mouse.

B. Schematic representation of the two possible scenarios expected after M/T cell ablation. (top) Reduction of M/T cells density during perinatal maturation does not affect the apical dendrite refinement. (bottom) The lack of peer M/T cells interferes with the normal refinement of the apical dendrite, thus retaining an immature morphology with multiple apical dendrites.

C. Quantification of the number of remaining M/T cells (tdT positive cells) in the OB at different times after perinatal cell ablation in Tbx::DTA mice crossed with the Ai9 reporter mouse. In Tbx::DTA::Ai9 mice, approximately 25% of the M/T cells remained at P10 (9,605.7 ± 1,229.6; n=4), ∼3% at P21 (1,024.7 ± 282.2; n=9), and ∼1% at P120 (437.6 ± 119.2; n=8) compared with wild type (Tbx) mice at P120 (38,546.8 ± 2,912.2; n=3). Data are shown as average ± standard deviation.

D. Confocal images of individually labeled M/T cells in the OB of a P10 Tbx::DTA::Confetti mouse after perinatal cell ablation. Note that M/T cells present a single apical dendrite innervating a glomerulus. Scale bar is 100 µm.

Massive ablation of M/T cells in adult mice induces plasticity of apical dendrite.

A. Schematic representation of the transgenic strategy to ablate M/T cells in adult animals. The Tbx21 promoter is specific for M/T cells. The Tbx-cre transgene controls the expression of the Cre recombinase in M/T cells, enzyme, thus regulating the selective expression in M/T cells of the receptor (iDTR) for diphtheria toxin (DT), from a CAG:loxp stop loxP-iDTR reporter mouse. Upon systemic injection of DT into the bloodstream, DT gets transported into M/T cells expressing iDTR, and selectively kills them.

B. Two possible scenarios are expected after ablating most of the M/T cells in adult animals. (top) M/T cells density reduction does not induce changes in the apical dendrite structure. (bottom) The absence of peer M/T cells induces structural plasticity in remaining M/T cells, whose apical dendrites extend into nearby glomeruli.

C. Quantification of the number of remaining M/T cells (tdTomato (tdT) positive cells) in the OB of adult mice (P120) in different cell ablation experiments. 437.6 ± 119.2 M/T cells remained in Tbx::DTA::Ai9 mice (∼1% of the full set of M/T counted in wt mice; n=8; light red). 1,158 ± 99 M/T cells remained in Tbx::iDTR::Ai9 mice (∼3% compared to wt mice; n=4; light green). 9,819 ± 1544 M/T cells remained in Tbx::iDTR::Ai9 mice when half of the DT concentration (10 μg/Kg of body weight) was injected (∼25% compared to wt; n=3; dark green). Wild type (Tbx::Ai9) mice had 38,546.8 ± 2,912.2; n=3; gray). Data are shown as average ± standard deviation.

D-H. Confocal images of M/T cells after sparse labeling using AAV-DiO-GFP. (C) Tbx (wild type) mice at P120. (D) Tbx::DTA mice at P120. (E) Tbx::iDTR mice at P120 (cell ablation induced at P60). (F) Tbx::iDTR mice at P240 (cell ablation induced at P180). (G) Tbx::iDTR mice at P120 (cell ablation induced at P60 using half of the DT dose). Blue = DAPI staining. Scale bar in H is 50 µm and applies to D-H.

I. Percentage of M/T cells with an apical dendrite innervating one, two, three or more glomeruli for experimental conditions in D-H. In Tbx::DTA ∼35% of M/T cells had an apical dendrite innervating more than one glomerulus (n=149; p-value<0.0001; χ2 test). In Tbx:iDTR mice, when ablation was induced at P60, 48% of M/T cells had an apical dendrite innervating more than one glomerulus (n=188; p-value<0.0001). No significant differences were observed in Tbx:iDTR mice when comparing cell ablation performed at P60 or P180 (n=216; p=0.15) or using half of the DT dose (n=236; p=0.05).

M/T cell apical dendrite plasticity caused by reduction in the number of neurons is independent of neuronal activity.

A. (left) Schematic representation of the strategy to perform sensory deprivation (naris occlusion) in Tbx::iDTR prior to cell ablation induced at P60. (right) Two possible scenarios are expected in the remaining M/T cells of sensory deprived OBs. (left cartoon) Sensory deprivation prevents the structural changes of M/T caused by ablation of M/T peers and retain a single apical dendrite, or (right cartoon) M/T cells undergo structural plasticity in sensory deprived OB, as shown in Figure 2F.

B. Confocal images of M/T cells labeled using AAV-DiO-GFP showing that remaining M/T cells in sensory deprived OBs still remodel their apical dendrites, such that they branch toward multiple nearby glomeruli, as the open OB. Blue = DAPI staining. Scale bar in B is 50 µm.

C. Schematic representation of the strategy to selectively reduce neuronal activity in M/T cells in Tbx::iDTR by expressing the Kir2.1 channel before inducing cell ablation at P60. Two possible scenarios are expected in the remaining M/T cells. Neuronal activity reduction prevents the structural changes of M/T caused by ablation of M/T peers and retain a single apical dendrite, or M/T cells with reduced neuronal activity undergo structural plasticity in sensory deprived OB, as shown in Figure 2F.

D. Confocal images of M/T cells labeled using AAV-DiO-GFP show that M/T cells expressing the Kir2.1 channel in the remaining cells when cell ablation was performed in Tbx::iDTR mice, showed structural changes in their apical dendrite. Blue = DAPI staining. Scale bar in D is 50 µm.

E. Percentage of M/T cells with apical dendrites innervating one, two, three or more glomeruli for experimental conditions shown B and D. Similar results were obtained in M/T cells from both OBs, closed (39%; n=231) or open (44%; n=228) nostril. No significant differences were observed between Tbx::iDTR mice with open or closed nostrils (p-value=0.17; χ2 test). Similarly, expression of Kir2.1 starting before cell ablation did not cause significant differences in Tbx::iDTR mice (p-value=0.05).

F.

Odor map maintenance is disrupted by the drastic reduction in the number of M/T cells.

A. Schematic representation of the transgenic strategy to perform cell ablation in perinatal (Tbx::DTA) and adult (Tbx::iDTR) mice to analyze the axon projection of the M72 olfactory sensory neurons into the OB.

B. Two possible scenarios are expected for the OSN axon projection to the OB after M/T cells ablation. (left) M72 OSN axon project to a single glomerulus per hemibulb as in wt animals, or (right) M72 OSN axons project to more than one glomeruli per hemibulb, perturbing the odor map in the OB surface.

C. Confocal images of M72 OSN axons (green) in a wild type mouse (Tbx, top row) and Tbx::DTA mice (bottom row). Each column represents the analysis of the OSN axon at two different time points (P10 and P21). Immunostaining against olfactory marker protein (OMP, red) labels all OSN axons. Blue= DAPI staining.

D. Confocal images of M72 OSN axons (green) in Tbx, Tbx::DTA and Tbx::iDTR mice at P120. The glomerular projection was disrupted in adult mice, both in animals in which ablation occurred perinatally (Tbx::DTA) or as adults (Tbx::iDTR injected at P60 with DT). Immunostaining against OMP (red) labels all OSN axons. Blue = DAPI staining. Scale bar in D is 50 µm and applies to A-D.

E. Percentage of M72 OSN axons projecting to one, two, three or more glomeruli per hemibulb in Tbx, Tbx::DTA and Tbx::iDTR mice. No differences were observed at P10 and P21 between the M72 OSN axon in normal conditions or with reduced M/T cells (Tbx: P10 (n=12), P21 (n=20); Tbx::iDTR: P10 (n=12), P21 (n=12). At P10 (p-value=0.59; χ2 test), P21 (p-value=0.23). However, significant differences in M72 OSN axons were observed in Tbx::DTA (n=64; p-value<0.001) and Tbx::iDTR (n=52; p-value<0.001) mice at P120, projecting to multiple glomeruli instead of a single glomerulus as in Tbx mouse (n=24).

Mouse behavior after massive M/T cells ablation.

A. Olfactory performance of Tbx (n=12), Tbx::DTA (n=13), and Tbx::iDTR (n=11) mice in a go/no go paradigm to investigate their odor detection ability. Decreasing cineole concentrations were presented each experimental day. Dots represent the mean percentage of accuracy responses in a single day. Accuracy lower than 80% was considered as a deficit in olfactory detection. Tbx::DTA and Tbx::iDTR mice successfully performed odor detection with concentrations of cineole as low as 0.05%, but failed when the concentration of odorant was further reduced. Control mice (Tbx) successfully detected cineole at all concentrations tested (up to 0.001%).

B. Odor discrimination experiment exposing mice to increasing binary mixtures of cineole and eugenol (left) and (+)-carvone and (−)-carvone odors (right). Both experimental mice (Tbx::DTA and Tbx::iDTR) could successfully detect cineole and eugenol when they were presented as individual odors, or with 80/20 mixtures. However, they failed when mixtures of cineole::eugenol were 55/45. However, Tbx::iDTR mice discriminated between (+)-carvone and (−)-carvone odors presented individually, but not when odor mixtures were presented. Tbx::DTA could not discriminate between (+)-carvone and (−)-carvone even when they were presented as individual odors.

C. Tbx::DTA and Tbx::iDTR males mated with wt females with a much lower frequency (Tbx::DTA=16.67%; n=12; and Tbx::iDTR=63.63%; n=11) compared to control males (90.91%; n=11).

D. In a resident/intruder paradigm, then the resident and intruder were both wt male mice, the resident started a fight in >90% of the tests. When the resident was a wild type male they fought in 33.33% of the cases if the intruder was a Tbx::DTA male (n=9) or 28,57% if the intruder was a Tbx::iDTR male (n=7). When the residents were Tbx::DTA (perinatal cell ablation) or Tbx::iDTR (cell ablation at P60) males, they did not fight when the intruder was a male with the same genotype or a wild type male mouse.

E. Quantification of M/T cells in the accessory olfactory bulb (AOB) in Tbx, Tbx::DTA, and Tbx::iDTR showed that only 1,6 % of M/T cells remained (30 ± 7, average ± S.E.M.; n=5) in Tbx::DTA mice and 16,6% in Tbx::iDTR (315 ± 35; n=3) compared to wt Tbx mice (1,900 ± 119; n=3).

M/T cells labeled at early postnatal stages in Tbx::Ai9 mice.

A. Coronal section to the OB in a P0 Tbx::Ai9 mouse. There is a high number of M/T cells that express tdT, indicating that Cre enzyme is active at embryonic stages in a significant fraction of M/T cells.

B. Coronal section to the OB in a P3 Tbx::Ai9 where most, if not all, the M/T cells are expressing tdT protein. DAPI staining (blue) labels cell nuclei. Scale bar in B is 200 µm and applies to A-B.

Histological analyses of olfactory bulbs after M/T cells ablation.

A. (left) Weight of Tbx::DTA (red bar) and Tbx::iDTR (blue bar) males mice compared with their wild type siblings (grey bar). Significant differences were observed in their weights. Tbx::DTA (27.7 ± 2.8 gr; n=19) vs Tbx (30.1 ± 3.4 gr; n=19), p-value=0.02. Tbx::iDTR (26.5 ± 1.5 gr; n=12) vs Tbx (28.2 ± 2.2 gr; n=11), p-value=0.02. (right) Weight of Tbx::DTA (red bar) and Tbx::iDTR (blue bar) females mice compared with their wild type siblings (grey bar). No significant differences were observed in their weights. Tbx::DTA (24 ± 2.8 gr; n=19) vs Tbx (25.4 ± 3.9 gr; n=19), p-value=0.22. Tbx::iDTR (22.3 ± 1.7 gr; n=22) vs Tbx (22.7 ± 2.2 gr; n=11), p-value=0.19.

B. Dorsal view of Tbx, Tbx::DTA, and Tbx::iDTR brains. Note that the OBs of the Tbx::DTA and Tbx::iDTR brains are significantly smaller than in Tbx brains (wild type).

C. Coronal sections through the Tbx::Ai9, Tbx::DTA::Ai9, and Tbx::iDTR::Ai9 OBs. M/T cells expressing the tdT fluorescent protein are labeled in red. Note that in the OB of Tbx::DTA::Ai9 and Tbx::iDTR::Ai9 mice there are much fewer M/T cells labeled compared with the OB in the Tbx::Ai9 mouse (left). blue=DAPI staining. Scale bar is 300 µm.

D. Quantification of the OB volume in the wild type (Tbx; n=3) and the two experimental mice (Tbx::DTA (n=3), and Tbx::iDTR (n=3)), including the volume for each layer in the OB. The OB volume in Tbx::DTA and Tbx::iDTR mice is reduced to half of wild type OB.

E. Confocal images of coronal sections to the OB after immunostaining with Tbr2 (red; a marker for OB excitatory neurons), TH (green; a marker for a subpopulation of PGCs), and DAPI (blue; a nuclear stain). Note that the number of Tbr2+ cells is strongly reduced in Tbx::DTA and Tbx::iDTR mice compared to Tbx mice. Scale bar is 100 µm.

M/T cells refine their apical dendrite in the absence of peer M/T cells.

A. Confocal images of M/T cells in P10 Tbx::DTA::Confetti mice after perinatal cell ablation. Most M/T cells showed a normal morphology, with a single apical dendrite innervating a single glomerulus. Scale bar is 100 µm.

Plasticity of M/T cells apical dendrite is induced by the reduction of peer cells.

A. Confocal images of M/T cells at P120 labeled with AAV-DiO-GFP in Tbx::DTA and (B) Tbx::iDTR mice (cell ablation performed at P60). (C) M/T cells in Tbx::iDTR mice at P240 (cell ablation at P180). (D) M/T cells in Tbx::iDTR at P120 (cell ablation at P60 using half the DT dose). Blue = DAPI staining. Scale bar in D is 50 µm and applies to A-D.

Tuft length of remaining M/T cell apical dendrites.

A. Quantification of the apical dendrite tuft length of remaining M/T cells after cell ablation in experimental conditions as in Figure 2 D-H. Significant differences were observed when the tuft length of M/T cells in Tbx mice (6,234.2 ± 554.8 µm, n=22) was compared to M/T cells in Tbx::DTA (12,889.5 ± 1,378.1 µm; n=24; p<0.0001), and Tbx::iDTR mice (cell ablation at P60; 14,211.5 ± 1,527.3 µm; n=24; p<0.0001)(Mann Whitney U test). No significant differences were observed when comparing ablation performed at P60 versus P180 in Tbx::iDTR mice (cell ablation at P180; 11,583.4 ± 1,005 µm; n=23; p=0.72) or using half of the DT doses in P60 Tbx::iDTR mice (13,122.8 ± 1,005 µm; n=30; p=0.29). Data are shown as average ± S.E.M.

Confirmation of sensory deprivation by naris occlusion.

A-D. Confocal images of coronal sections through the OB of a Tbx (A and B) and Tbx::iDTR (C and D) mice labeled with tyrosine hydroxylase antibody (TH, red) which labels a subset of periglomerular cells input. Upon sensory deprivation, the levels of TH are strongly reduced in PGs (A, C) Sensory deprived OBs in wild type (A) and Tbx::iDTR (C) mice. (B, D) Open OBs in wild type (B) and Tbx::iDTR

(D) mice. Blue = DAPI staining. Scale bar in D is 50 µm and applies to A-D.

M/T cell apical dendrite plasticity caused by reduction in the number of neurons is independent of neuronal activity.

A-B. Confocal images of remaining M/T cells labeled with AAV-DiO-GFP in the closed (A) and open (B) nostril in P120 Tbx::iDTR mice where sensory deprivation was performed before cell ablation at P60.

C-D. Remaining M/T cells in Tbx (C) and Tbx::iDTR (D) mice at P120 expressing the Kir2.1 channel before cell ablation at P60. Blue= DAPI staining. Scale bar in D is 50 µm and applies to A-D.

M/T cells activity reduction by overexpressing the Kir2.1 channel.

A. Example of membrane potential traces in response to current steps of various amplitudes. Recorded from one M/T cell in a control (Tbx::Ai9) mice.

B. Example of membrane potential traces recorded from one M/T cell expressing Kir, with the same stimuli as in A.

C-D. Resting membrane potential (C) and input resistance (D) of M/Tss in control (Tbx::Ai9) mice (n=14 in C and n=19 in D), Kir-negative cells MCs in Tbx mice injected with Kir virus (Kir- control; n=8 in C and n=8 in D), or Kirexpressing MCs (Kir+; n=21 in C and n=17 in D).

E. F-I (firing frequency/injected current) curve of control and Kir+ MCs.

Apical dendrite tuft length of remaining M/T cell with reduced activity.

A. Quantification of the apical dendrite tuft length of remaining M/T cells after cell ablation in experimental conditions as in Figure 3B and D. Significant differences were observed when comparing the tuft length of M/T cells in the sensory deprived OB (closed nostril; 9,176.9 ± 618 µm; n=30) to those in the contralateral open OB (open nostril; 12,276.9 ± 1,272.2 µm; n=23; p=0.045), and control mice (Tbx::iDTR; 14,211.5 ± 1,527.3 µm; n=24; p=0.025) (Mann Whitney U test). Significant differences were observed when comparing the tuft length of remaining M/T cells expressing the Kir2.1 channel in Tbx::iDTR injected with DT(9,642.7 ± 618 µm; n=19) with M/T cells expressing the Kir2.1 channel in control Tbx::iDTR mice that did not receive any DT injection(Tbx::iDTR; 6,417.9 ± 546.7 µm; n=20; p=0.019). Data are shown as average ± S.E.M.

Odor detection accuracy after reduction of M/T cells in adult mice.

A. The Tbx::iDTR mice (n=14) could be grouped in three different groups depending on the type of behavior that they exhibited in the go/no go paradigm for olfactory detection. Group 1: 50% of the mice (n=7) performed the test accurately from the first day [black line]; Group 2: 29% (n=4) were able to perform the task after one week of training [dark grey line], and Group3: 21% of the mice (n=3) were unable to do the test even after several weeks of training [stippled gray line]. Dots represent the mean percentage of accuracy responses in a single day. Accuracy lower than 80% was considered as a deficit in odor detection.