The role of calretinin-expressing granule cells in olfactory bulb functions and odor behavior

Abstract

The adult mouse olfactory bulb is continuously supplied with new neurons that mostly differentiate into granule cells (GCs). Different subtypes of adult-born GCs have been identified, but their maturational profiles and their roles in bulbar network functioning and odor behavior remain elusive. It is also not known whether the same subpopulations of GCs born during early postnatal life (early-born) or during adulthood (adult-born) differ in their morpho-functional properties. Here, we show that adult-born calretinin-expressing (CR⁺) and non-expressing (CR^-) GCs, as well as early-born CR⁺ GCs, display distinct inhibitory inputs but indistinguishable excitatory inputs and similar morphological characteristics. The frequencies of inhibitory post-synaptic currents were lower in early-born and adult-born CR⁺ GCs than in adult-born CR^- neurons. These findings were corroborated by the reduced density of gephyrin⁺ puncta on CR⁺ GCs. CR⁺ GCs displayed a higher level of activation following olfactory tasks based on odor discrimination, as determined by an immediate early gene expression analysis. Pharmacogenetic inhibition of CR⁺ GCs diminished the ability of the mice to discriminate complex odor mixtures. Altogether, our results indicate that distinct inhibitory inputs are received by adult-born CR⁺ and CR^- GCs, that early- and adult-born CR⁺ neurons have similar morpho-functional properties, and that CR⁺ GCs are involved in complex odor discrimination tasks.

Figure 1

Morphological characterization of early-born and adult-born CR⁺ GCs and adult-born CR⁻ GCs (a) Schematic diagram showing the injection of GFP-encoding lentivirus into the RMS of adult mice and the morphometric parameters used to analyze the structural properties of CR⁺ and CR⁻ GCs. (b) Representative images of CR⁺ and CR⁻ GCs at three wpi. Insets show CR immunolabeling (white). Scale bars: 20 µm (left image), 4 µm (right image), and 10 µm (inset). (c–f) The mean lengths of the primary dendrite (c), secondary dendrites (d), and basal dendrites (e), and the spine densities (f) of adult-born CR⁺ and CR⁻ GCs at three and five wpi. (g) Schematic diagram showing the co-injection of the Flex-GFP and Tdtomato AAVs into the RMS of adult and P12 CR-Cre mice. (h) Example of adult-born CR⁺ and CR⁻ GCs at one wpi. (i) Examples of early-born and adult-born CR⁺ GCs and adult-born CR⁻ GCs at five wpi. Scale bars: 20 µm (left image) and 2 µm (right image). (j–m) The mean lengths of the primary dendrites (j), secondary dendrites (k), basal dendrites (l), and spine densities (m) of early-born and adult-born CR⁺ and adult-born CR⁻ GCs at one, three and five wpi. See Table 1.

Figure 2

The excitatory postsynaptic inputs of CR⁺ and CR⁻ GCs are indistinguishable (a). Labeling of early-born CR⁺ GCs in CR-Cre::CAG-CAT-GFP mice. The inset shows a higher magnification of GFP⁺ GCs in the GCL. Scale bars: 500 µm (left image) and 10 µm (right image). (b) Examples of sEPSC and mEPSC events recorded from early-born CR⁺ GCs, adult-born CR⁺ GCs, and CR⁻ GCs. (c,d) The mean amplitudes (c) and frequencies (d) of sEPSCs and mEPSCs recorded from early and adult-born CR⁺ GCs and adult-born CR⁻ GCs are indistinguishable. See Table 3.

Figure 3

CR⁺ GCs receive weaker inhibitory inputs than CR⁻ GCs and have fewer gephyrin⁺ puncta on their primary dendrites (a). Examples of sIPSCs and mIPSCs recorded from early-born CR⁺ GCs, and adult-born CR⁺ and CR⁻ CGs. (b,c). Mean amplitudes (b) and frequencies (c) of sIPSCs and mIPSCs recorded from early and adult-born CR⁺ GCs and adult-born CR⁻ GCs. See Table 4. (d). Representative image of virally labeled GCs (green) that have also been labeled for gephyrin (white). Arrowheads indicate gephyrin⁺ puncta. Scale bars: 10 µm. (e). Mean densities of gephyrin⁺ puncta on the primary and basal dendrites and cell soma of CR⁺ and CR⁻ adult-born GCs at three wpi. See Table 4. *p < 0.05 Mann-Whitney U-test for IPSCs frequencies and paired t-test for gephyrin analysis.

Figure 4

Spontaneous odor discrimination induces the activation of CR⁺ GCs (a). Spontaneous odor discrimination task based on odor habituation/dishabituation (hab/dishab). C57Bl/6 mice were habituated to the presence of a first odor (the habituation odor ((+)-carvone)), which was presented four times. Their ability to discriminate between two similar odors was then investigated by the presentation of a second odor (the dishabituation odor ((−)-carvone)), which was chemically similar to the habituation odor. As expected, mice correctly discriminated between the two odors as shown by the increase in exploration time when the dishabituation odor was presented compared to the fourth presentation of the habituation odor (a, right panel). We used mice that were not dishabituated and that received the habituation odor ((+)-carvone) during the last presentation (hab/hab) as a control (a, left panel). (b) Examples of CR and cFos immunolabeling in mice from the control and odor discrimination groups. Arrowheads indicate cFos⁺/CR⁺ GCs. Scale bar: 10 µm. (c–e) Quantification of the density of cFos⁺ and Zif268⁺ GCs (c), the percentages of cFos⁺ and Zif268⁺ GCs among CR⁺ GCs (d), and the percentage of CR⁺ (left panel) and CR⁻ (right panel) GCs among cFos⁺ and Zif268⁺ GCs (e). *p < 0.05, ***p < 0.001 paired t-test for behavioral task and unpaired t-test for IEG analysis.

Figure 5

Odor discrimination learning activates CR⁺ GCs (a). Water-restricted C57Bl/6 male mice were tested using the go/no-go odor discrimination task. Mice were randomly exposed to reward-associated and non-reward-associated odors (S+ and S−, respectively), and the percentage of correct responses (hits + correct rejections) was calculated for every 20 trials. The mice were considered to have successfully discriminated between the two odors if they reached the 85% criterion of correct responses. The odors used were 0.1% octanal and 0.1% decanal (odor pair 1). Number of blocks needed in each group to reach the 85% criterion (right panel). For mice in the control group, the water was given independently of the odor. (b). Examples of immunolabeling for CR and cFos on OB slices after the go/no-go odor discrimination task. Arrowheads indicate cFos⁺/CR⁺ GCs. Scale bar: 10 µm. (c-e) Quantification of the densities of cFos⁺ and Zif268⁺ GCs (c), the percentages of cFos⁺ and Zif268⁺ GCs among CR⁺ GCs (d), and the percentages of CR⁺ (left panel) and CR⁻ (right panel) GCs among cFos⁺ and Zif268⁺ GCs (e). Note the higher percentages of CR⁺/cFos⁺ and CR⁺/Zif268⁺ GCs, indicating that CR⁺ GCs are specifically involved in odor discrimination. *p < 0.05 unpaired t-test.

Figure 6

The pharmacogenetic inhibition of CR⁺ GCs reduces the olfactory discriminatory ability of mice (a). The EF1α-DIO-hM4D(G_i)-mCherry AAV was injected into the OB of CR-Cre mice, inducing the expression of the G_i-coupled DREADDs receptor by CR⁺ GCs. Control mice were injected with a Cre-independent GFP-encoding AAV, which labeled all GCs. The behavior experiments started at two wpi, and the mice in the two groups were given an intraperitoneal injection of 2 mg/kg of CNO 30 min prior undertaking the behavior task. (b) Example of the injection of the AAV in the OB. Scale bar: 50 µm. The inset shows a higher magnification image of DREADD-G_i infected cells. Scale bar: 10 µm. (c) Quantification of the percentages of reporter⁺ (GFP or mCherry) and cFos⁺ GCs following the CNO injections in mice previously injected with the control (EF1α-DIO-EYFP) or the DREADDs (EF1a-DIO-hM4D(G_i)-mCherry) AAV. Note the effective pharmacogenetic inhibition of CR⁺ GCs (*p < 0.05 paired t-test). (d–f) The mean scores in percentages for each block of 20 trials obtained from the control and DREADDs AAV-injected mice for the go/no-go odor discrimination task using odor pairs of different complexity. Odor pair 1: 0.1% octanal (S+) vs. 0.1% decanal (S−), n = 10 and nine mice for the control and DREADDs groups, respectively (d); Odor pair 2: 0.6% (+)- + 0.4% (−)-limonene (S+) vs. 0.4% (+)- + 0.6% (−)-limonene (S−), n = nine and 10 mice for the control and DREADDs groups, respectively (e); Odor pair 3, 0.48% (+)- + 0.52% (−)-limonene (S+) vs. 0.52% (+)- + 0.48% (−)-limonene (S−), n = 10 mice for the control and DREADDs groups, respectively (f). The dashed lines represent the 85% criterion score. Insets show the mean values for each group to reach the criterion of 85% correct responses (*p < 0.05; **p < 0.01 unpaired t-test).