Impairment of olfactory discrimination in Tbr1
+/− mice
To investigate whether Tbr1 heterozygosity has any impact on olfaction, we evaluated olfactory sensation and discrimination in Tbr1+/− mice. To set up the assay system, we first tested preferences for two distinct non-social odorants, limonene and 2-heptanol. After 2-day habituation to the presence of filter papers in their home cages, we separately spotted limonene and 2-heptanol onto two filter papers placed at two ends of the home cage (Fig. 1a, upper, preference test). The time spent sniffing limonene and 2-hepatonal was then measured. We found that both wild-type (WT) littermates and Tbr1+/− mice spent similar amounts of time sniffing these two odorants (Fig. 1b; odorant effect: F(1,9) = 2.437, p = 0.153, two-way RM ANOVA), suggesting that both WT and Tbr1+/− mice can sense both limonene and 2-hepatonal and have no preference for either of them.
We then used these two odorants in six consecutive trials to examine the olfactory sensation and discrimination abilities of mice. Limonene and mineral oil (a control) were presented to mice in the first five trials with 15-min intervals (Fig. 1a, lower panel). The time spent sniffing limonene in the first trial indicated the olfactory sensation of mice. WT littermates and Tbr1+/− mice spent comparable amounts of time sniffing limonene in trial 1 (Fig. 1c; t(20) = 1.23, p = 0.2331, unpaired t test). Compared with mineral oil, both Tbr1+/− mice and WT littermates spent significantly longer time sniffing limonene in trial 1 (Fig. 1d, trial 1; WT, t(10) = 6.559, p < 0.0001; Tbr1+/−, t(10) = 5.147, p = 0.0004, paired t test). The results suggested Tbr1+/− mice exhibit normal olfactory sensation. The repetitive exposure to limonene in consecutive trials 2 to 5 habituated mice to limonene and adapted their olfactory responses to this odor (Fig. 1d). We found that time spent sniffing limonene rapidly decreased to levels comparable to those recorded for responses to mineral oil in both Tbr1+/− mice and WT littermates during subsequent trials 2–5 (Fig. 1d; trial 2: WT, t(10) = 1.762, p = 0.1085; Tbr1+/−, t(10) = 0.5437, p = 0.5986, paired t test), indicating that habituation, i.e., olfactory adaptation, is also normal in Tbr1+/− mice.
The olfactory discrimination ability of Tbr1+/− mice was then investigated in trial 6, representing the dishabituation test. Limonene (the familiar odorant) and 2-heptanol (a novel odorant) were simultaneously presented in the home cages of mice during trial 6 (Fig. 1a, lower panel). All WT littermates spent significantly more time sniffing 2-heptanol (Fig. 1e; WT, t(10) = 6.981, p = 0.001, paired t test), suggesting that WT mice were able to distinguish 2-heptanol from limonene. However, of the 11 Tbr1+/− mice we assayed, only five animals spent more time sniffing 2-heptanol (Fig. 1e; Tbr1+/−, t(10) = 2.109, p = 0.0611, paired t test). We then calculated an odor preference index by comparing the limonene and mineral oil data from trial 1 and the 2-heptanol and limonene data from trial 6 (see “Methods” section and Fig. 1f). We found that the preference indices for trial 1 were comparable between WT and Tbr1+/− mice (Fig. 1f; t(20) = 0.4123, p = 0.6845, unpaired t test), further supporting the conclusion that Tbr1+/− mice have no defect in olfactory sensation. However, for trial 6, the preference indices of Tbr1+/− mice were significantly lower than those of WT littermates (Fig. 1f; t(20) = 2.981, p = 0.0074, unpaired t test). These results suggest that deletion of one allele of the Tbr1 gene impairs olfactory discrimination but not olfactory sensation or adaptation.
Tbr1 expression in the olfactory system of mouse brains
To investigate how Tbr1 haploinsufficiency regulates olfaction, we examined Tbr1 expression in the olfactory system of WT mouse brains (Fig. 2a). Consistent with previous findings that TBR1 is expressed in mitral cells, tufted cells, and juxtaglomerular excitatory neurons of the olfactory bulb [26, 32, 61, 62], we also found that TBR1 was mainly expressed in the mitral cell layer and glomerular layer of the olfactory bulb in adult WT mice (Fig. 2b). In addition to the olfactory bulb, immunostaining also detected TBR1 expression in the piriform cortex (PC), mainly at the layer II projection neurons and in the perirhinal cortex (PrC), enriched at layer VI (Fig. 2c). However, there was no TBR1 signal in the olfactory tubercle (OT) (Fig. 2c). These immunostaining results indicate that TBR1 is expressed in several regions of the olfactory system of mouse brains.
Alteration of the olfactory system in Tbr1
+/− mouse brains
We then investigated whether the deletion of an allele of the Tbr1 gene alters anatomic or histological features of the olfactory system in mouse brains. We performed MRI to compare the size of the olfactory system of Tbr1+/− mice and WT littermates. Consistent with previous histological analysis [23], our MRI results showed that the posterior part of the anterior commissure is the most sensitive region to Tbr1 haploinsufficiency, even without normalization against whole-brain size (Fig. 3a and b). After normalizing against whole-brain size, the anterior part of the anterior commissure and the olfactory bulb (including the glomerular, external plexiform, mitral cell, internal plexiform, and granule cell layers) were smaller in Tbr1+/− mice (Fig. 3a and b). However, the olfactory tubercle, piriform cortex, or perirhinal cortex were not affected by Tbr1 haploinsufficiency (Fig. 3b). We then conducted Nissl staining to investigate whether histological features of the olfactory system were altered by Tbr1 haploinsufficiency. We found that cellular organization and the laminar structure of the olfactory bulb, olfactory tubercle, piriform cortex, and perirhinal cortex were all normal in Tbr1+/− mice (Fig. 3c). Thus, our MRI analysis and Nissl staining suggest that the size, but not structure, of the anterior commissure and olfactory bulb are particularly sensitive to Tbr1 haploinsufficiency.
Characterization of Tbr1
+/− olfactory bulbs using various markers
We performed immunofluorescence staining with various markers to further characterize Tbr1+/− olfactory bulbs. The first set of markers we used comprised members of the TBR1 subfamily of T-box transcription factors, including TBR1, TBR2 (T-brain-2, also known as Eomesodermin or EOMES), and TBX21 (also known as T-bet). Although TBR1 subfamily members are all expressed in mitral cells, tufted cells, and juxtaglomerular excitatory neurons of the olfactory bulb, only some of those cells express all three of these transcription factors [32]. Thus, differential expression of TBR1 subfamily members defines subpopulations of excitatory neurons in olfactory bulbs, though the biological functions of these different subpopulations are still unknown. In Tbr2−/− neurons, TBR1 expression is upregulated, whereas TBX21 protein levels are reduced [32]. We wondered whether Tbr1 haploinsufficiency also influences expression of other members of the TBR1 subfamily. We carried out triple immunostaining using TBR1, TBR2, and TBX21 antibodies to analyze adult olfactory bulbs. Our results revealed that TBR2:TBR1:TBX21 triple-positive mitral cells accounted for a sizeable proportion (~ one third) of all mitral cells in WT mice (Fig. 4, white nuclei in WT). Double-positive cells were also frequently found in WT olfactory bulbs (Fig. 4, yellow or purple nuclei in WT). In Tbr1+/− olfactory bulbs, the general patterns of TBR1 subfamily-positive cells were similar to those of WT mice, but TBR2 seemed to be dominant and the number of triple-positive cells was reduced (Fig. 4, Tbr1+/−). Thus, the properties of projection neurons in olfactory bulbs are likely altered by Tbr1 deficiency.
A previous study indicated that Tbr2 deletion alters expression of vesicular glutamate transporters (VGLUTs) in mitral and tufted cells, and influences dendrodendritic synapses in the external plexiform layer of olfactory bulbs [32]. To investigate whether Tbr1 haploinsufficiency influences the expression of VGLUTs, we performed immunostaining using antibodies against VGLUT1 and VGLUT2. We found that ratios of VGLUT1 and VGLUT2 signals in the glomerular to external plexiform layers were not altered in Tbr1+/− olfactory bulbs compared to those of WT littermates (Fig. 5a and b, Additional file 1: Figure S1). Thus, unlike Tbr2 deficiency [32], Tbr1 haploinsufficiency does not result in a shift from VGLUT1 to VGLUT2. Based on immunostaining with neurofilament antibody, Tbr2−/− mitral cells exhibit thinner and more disorganized dendrites compared with those of WT cells [32]. In contrast to the outcome of Tbr2 deletion, the dendrites of Tbr1+/− mitral cells became thicker (Fig. 5c, Additional file 1: Figure S1). Thus, TBR1 and TBR2 play differential roles in controlling synapse transmission and dendritic organization of mitral cells.
We then used calretinin, parvalbumin, and calbindin antibodies as markers to monitor interneurons in olfactory bulbs. Calretinin+ neurons were widely distributed at different layers, including the glomerular layer (GL), external plexiform layer (EPL), mitral cell layer (MCL), and granular cell layer (GCL) (Fig. 5d, Additional file 1: Figure S1). All these layers showed fewer calretinin+ neurons in Tbr1+/− olfactory bulbs compared to WT littermates, though only the differences for EPL, MCL, and GCL were significant (Fig. 5g; GL, t(5) = 2.429, p = 0.0595; EPL, t(5) = 11.55, p = < 0.0001; MCL, t(5) = 4.065, p = 0.0097; GCL, t(5) = 2.898, p = 0.0339, unpaired t test). Parvalbumin+ interneurons were enriched at the EPL (Fig. 5e, Additional file 1: Figure S1) and had a lower cell density in Tbr1+/− olfactory bulbs relative to WT littermates (Fig. 5h; t(5) = 4.88, p = 0.0046, unpaired t test). Calbindin+ interneurons were present in the GL (Fig. 5f, Additional file 1: Figure S1) but there was no significant difference between Tbr1+/− mice and WT littermates (Fig. 5i; t(4) = 0.3014, p = 0.7782, unpaired t test). Since TBR1 is not expressed in interneurons, the reduction of calretinin+ and parvalbumin+ interneurons is a non-cell autonomous effect. Only parvalbumin signals were obviously altered in Tbr2−/− mice [32]. Therefore, Tbr1 and Tbr2 exhibit differing non-cell-autonomous effects on interneurons.
Our immunostaining results using various markers suggest that Tbr1 heterozygosity likely influences projection neurons and alters inhibitory interneurons. These defects are specific for Tbr1 haploinsufficiency and cannot be compensated for by the presence of Tbr2.
Reduced neuronal activation in Tbr1
+/− mouse brains
We then investigated whether neuronal activation in the olfactory system is altered by Tbr1 haploinsufficiency, resulting in impairment of olfactory responses. Two hours after exposure to limonene for 15 min, we examined C-FOS expression by immunostaining to monitor neuronal activation (Fig. 6a, b, c, and d). Compared with a mineral oil control, limonene stimulation resulted in more C-FOS-positive cells in the GL of WT mice (Fig. 6e and f; GL, WT, t(10) = 2.863, p = 0.0169, unpaired t test) but not in Tbr1+/− mice (Fig. 6e and f; GL, Tbr1+/−, t(9) = 0.09979, p = 0.9227, unpaired t test). In both the EPL and the MCL, we did not observe any change in C-FOS cell number in WT littermates or Tbr1+/− mice (Fig. 6e and f; EPL: WT, t(10) = 0.911, p = 0.3838; Tbr1+/−, t(9) = 0.6923, p = 0.5062; MCL: WT, t(10) = 1.061, p = 0.3138; Tbr1+/−, t(9) = 0.6838, p = 0.511, unpaired t test). Thus, only the GL exhibits lower neuronal activation upon odor stimulation in Tbr1+/− olfactory bulbs.
In the upper olfactory system of WT mice, the numbers of C-FOS-positive cells in both the anterior piriform and perirhinal cortices, but not olfactory tubercles, was increased upon limonene stimulation compared with a mineral oil control (Fig. 7d and e; WT: anterior piriform, t(12) = 4.486, p = 0.0007; perirhinal, t(12) = 3.17, p = 0.0081; olfactory tubercle, t(12) = 1.172, p = 0.2641, unpaired t test). In Tbr1+/− mice, neither the piriform and perirhinal cortices nor olfactory tubercles exhibited increases of C-FOS-positive cell numbers upon comparing results for limonene with mineral oil control (Fig. 7e; Tbr1+/−: anterior piriform, t(12) = 1.236, p = 0.2401; perirhinal, t(12) = 1.148, p = 0.2734; olfactory tubercles, t(12) = 0.7735, p = 0.4542, unpaired t test). Thus, the defects of neuronal activation in response to odor stimulation primarily lie in the glomerular layer of olfactory bulbs and the piriform and perirhinal cortices of Tbr1+/− mice.
D-cycloserine has a beneficial effect on olfactory discrimination of Tbr1
+/− mice
Our previous study indicated that Tbr1 haploinsufficiency impairs axonal connectivity and neuronal activation of amygdalar neurons [23]. Systemic administration or local infusion of D-cycloserine into amygdalae effectively ameliorates impaired neuronal activation of amygdalae and associated behavioral deficits in social interaction, cognitive flexibility, and memory [23]. Since neuronal activation in the glomerular layer of the olfactory bulb and its piriform and perirhinal cortices was impaired in Tbr1+/− mice upon odor stimulation (Fig. 7), we wondered if the olfactory defects of Tbr1+/− mice could be improved by D-cycloserine treatment. To test this possibility, we intraperitoneally injected D-cycloserine into both WT and Tbr1+/− mice 30 min before undergoing an olfactory discrimination test. Similar to the results without D-cycloserine treatment (Fig. 1), both WT and Tbr1+/− mice behaved comparably in terms of olfactory sensation (Fig. 8a; t(18) = 0.3053, p = 0.7636, unpaired t test) and adaptation to limonene (Fig. 8b). Importantly, in the discrimination test (trial 6), both Tbr1+/− and WT mice spent significantly more time sniffing 2-heptanol, i.e., the novel odorant (Fig. 8c; WT, t(9) = 5.479, p = 0.0004; Tbr1+/−, t(9) = 3.517, p = 0.0065; paired t test). Odor preferences of Tbr1+/− mice in both trials 1 and 6 were also comparable to those of WT mice (Fig. 8d; trial 1, t(18) = 1.601, p = 0.1269; trial 6, t(18) = 1.074, p = 0.2970; unpaired t test). These results suggest that, similar to its effect on amygdalar deficits caused by Tbr1 haploinsufficiency, increased neuronal activation by D-cycloserine ameliorates impaired olfactory discrimination in Tbr1 mutant mice.