LRP4 is required for the olfactory association task in the piriform cortex

Abstract

Background: Low-density lipoprotein receptor-related protein 4 (LRP4) plays a critical role in the central nervous system (CNS), including hippocampal synaptic plasticity, maintenance of excitatory synaptic transmission, fear regulation, as well as long-term potentiation (LTP).

Results: In this study, we found that Lrp4 was highly expressed in layer II of the piriform cortex. Both body weight and brain weight decreased in Lrp4^ECD/ECD mice without TMD (Transmembrane domain) and ICD (intracellular domain) of LRP4. However, in the piriform cortical neurons of Lrp4^ECD/ECD mice, the spine density increased, and the frequency of both mEPSC (miniature excitatory postsynaptic current) and sEPSC (spontaneous excitatory postsynaptic current) was enhanced. Intriguingly, finding food in the buried food-seeking test was prolonged in both Lrp4^ECD/ECD mice and Lrp4 cKO (conditional knockout of Lrp4 in the piriform cortex) mice.

Conclusions: This study indicated that the full length of LRP4 in the piriform cortex was necessary for maintaining synaptic plasticity and the integrity of olfactory function.

Keywords: Golgi staining; LRP4; Olfactory function; Piriform cortex; Spine density.

Fig. 1

Highly expression of Lrp4 in piriform cortex. A X-gal staining of Lrp4^+/Lac mice brain slice. PIR, piriform cortex; CTX, cerebral cortex; HIP, hippocampus; TH, thalamus; Lrp4^+/Lac mice, n = 5. B Lrp4 expression in wild-type adult mice brain was confirmed by Western blotting (Wild type mice, n = 6). C Quantification of LRP4 protein level relative to α-tubulin from Western blotting. LRP4 relative level in the HIP was normalized to a value of 1. D mRNA expression of Lrp4 in piriform cortex in postnatal wild-type mice. Lrp4 expression values were calculated relative to Gapdh by using the 2^−ΔCT methods, and the P18 group was set to a value of 1. (Wild type mice per group, n ≥ 5; RNA pool was made for qPCR in each group; Values were means ± SEM, one-way ANOVA with multiple comparisons, compared other group with P18 group. n.s. no significant, * P < 0.05, *** P < 0.001)

Fig. 2

Lrp4 was expressed in the layer II of the piriform cortex. A Lrp4^+/Lac mice brain slice was co-stained with X-gal and anti-GFAP. GFAP positive cells were mainly distributed in the second superficial area of the piriform cortex, and X-gal staining positive cells were primarily located in layer IIa of the piriform cortex; nuclei were counterstained fast red. B Layer II of the piriform cortex. X-gal co-stained with GFAP⁺ cells (red arrow) and GFAP⁻ cells (white arrow; Lrp4^+/Lac mice, n = 5). C Percent of GFAP⁺ and GFAP⁻ cells in LacZ⁺ cell population in Layer IIa of the piriform cortex (Lrp4^+/Lac mice n = 3). D Representative images of neuron staining (anti-NeuN) of the two types of mice brain. E The thickness of the piriform cortex in layers I, II, and III did not change in Lrp4^ECD/ECD mice. F The neuronal density was not different in the three layers (Mice per type, n = 7. Scale bar = 50 μm. Values were means ± SEM. **P < 0.01)

Fig. 3

Increased spine density of superficial pyramidal neuron in Lrp4^ECD/ECD mice. A Representative Golgi staining of piriform cortical neurons in mice. B The representative spine on mice’s superficial pyramidal neuron (SP, arrowhead in A) and semilunar neuron (SL, arrow in A). C Quantification of spine density on SP neurons. The mature and total spine density of SP neurons in Lrp4^ECD/ECD mice were more than in the control mice. D Quantification of spine density on SL neurons. Lrp4^ECD/ECD mice exhibited a similar spine density of SL neurons than the control mice, except for a little increase in the thin type of spine. (Control mice, n = 6; Lrp4^ECD/ECD mice, n = 6. Values were means ± SEM. * P < 0.05, **P < 0.01)

Fig. 4

Enhanced excitatory synaptic transmission of the piriform cortical neurons in Lrp4^ECD/ECD mice. A Representative recording trace of sEPSC and mEPSC in the piriform cortex. B Frequency of spontaneous excitatory postsynaptic current (sEPSC) of the piriform cortical neurons in Lrp4^ECD/ECD mice increased. C The amplitude of the piriform cortical neuronals EPSC in Lrp4^ECD/ECD mice was similar to that in the control mice (control mice, n = 4, neurons, n = 18; Lrp4^ECD/ECD mice, n = 3; neurons, n = 15). D Frequency of miniature excitatory postsynaptic current (mEPSC) of the piriform cortical neurons in Lrp4^ECD/ECD mice elevated. E The amplitude of the piriform cortical neuronal mEPSC in Lrp4^ECD/ECD mice was no different from the control mice (control mice, n = 5; neurons, n = 16; Lrp4^ECD/ECD mice, n = 6; neurons, n = 18). (Values were means ± SEM. n.s., no significant; **P < 0.01)

Fig. 5

The impaired olfactory function of Lrp4^ECD/ECD mice. A Representative trace of mice in the open-field test. B, C There was no difference between Lrp4^ECD/ECD mice and the control mice in the total travel distance (B) and average speed (C) (control mice, n = 10; Lrp4^ECD/ECD mice, n = 10). D Schematic diagram of buried food-seeking test. Food deprived for 2 days before the training, training 2 days, and testing 3 days; mice were free to access enough water all the time; food was visible in training trials and buried in testing trials. E Lrp4^ECD/ECD mice spent more time finding the buried pellet chow than control mice. F The latency to find pellet chow in the testing days. (Control mice, n = 6; Lrp4^ECD/ECD mice, n = 5. Values were means ± SEM. n.s., no significant; *P < 0.05, **P < 0.01)

Fig. 6

The impaired olfactory function of Lrp4 cKO mice in the piriform cortex. A Representative injection trace of AAV into CAG-Cas9 mice in the piriform cortex. B The representative brain of AAV infection, 21d after stereotactic injecting AAV into the piriform cortex of CAG-Cas9 mice. C–F Immunofluorescence staining showed AAV virus infection with layer II cells in the piriform cortex. G mCherry positive cell percent in the piriform cortex (AAV injection CAG-Cas9 mice n = 4). H, I Lrp4 was knockout in the piriform cortex of Lrp4 cKO mice. Piriform cortex was used for western blotting (I) and quantification of LRP4 level relative to GAPDH (H). LRP4 relative level in control mice was normalized to a value of 1. J, K Lrp4 cKO mice spent more time finding the buried food than control mice. (Lrp4 cKO mice, CAG-Cas9 male mice injected with AAV-Lrp4 gRNA, n = 9; control mice, CAG-Cas9 male mice injected with AAV vector, n = 8. Scar bar = 15 μm. Values were means ± SEM. n.s., no significant; *P < 0.05, **P < 0.01, ***P < 0.001)