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. 2018 Dec 5;38(49):10424-10437.
doi: 10.1523/JNEUROSCI.0740-18.2018. Epub 2018 Oct 19.

Congenital Cytomegalovirus Infection Alters Olfaction Before Hearing Deterioration In Mice

Françoise Lazarini  1   2 , Lida Katsimpardi  3   2 , Sarah Levivien  3   2   4 , Sébastien Wagner  3   2 , Pierre Gressens  4   5   6 , Natacha Teissier  4   5   7 , Pierre-Marie Lledo  1   2
Affiliations

Congenital Cytomegalovirus Infection Alters Olfaction Before Hearing Deterioration In Mice

Françoise Lazarini et al. J Neurosci. .

Abstract

In developed countries, cytomegalovirus (CMV)-infected newborns are at high risk of developing sensorineural handicaps such as hearing loss, requiring extensive follow-up. However, early prognostic tools for auditory damage in children are not yet available. In the fetus, CMV infection leads to early olfactory bulb (OB) damage, suggesting that olfaction might represent a valuable prognosis for neurological outcome of this viral infection. Here, we demonstrate that in utero CMV inoculation causes fetal infection and growth retardation in mice of both sexes. It disrupts OB normal development, leading to disproportionate OB cell layers and rapid major olfactory deficits. Olfaction is impaired as early as day 6 after birth in both sexes, long before the emergence of auditory deficits. Olfactometry in males reveals a long-lasting alteration in olfactory perception and discrimination, particularly in binary mixtures of monomolecular odorants. Although sensory inputs to the OB remain unchanged, hallmarks of autophagy are increased in the OB of 3-postnatal week-old mice, leading to local neuroinflammation and loss of neurons expressing tyrosine hydroxylase and calbindin. At the cellular level, we found CMV-infected cells and an increased number of apoptotic cells scattered throughout the OB layers, whereas cell proliferation in the neurogenic subventricular zone was decreased. These cellular observations were long-lasting, persisting up to 16 weeks after birth in both males and females and thus providing a mechanism supporting olfactory loss. Despite obvious differences in neurogenesis between human and mouse, these findings offer new strategies aimed at early detection of neurological dysfunctions caused by congenital infections.SIGNIFICANCE STATEMENT In developed countries, congenital cytomegalovirus (CMV)-infected newborns are at high risk of developing sensory handicaps such as hearing loss, thus requiring prolonged follow-up. In this study, we describe for the first time the functional impact of congenital CMV infection on the olfactory system and its associated sense of smell. We demonstrate that a mouse model of congenital CMV infection shows defects in olfactory bulb (OB) normal development and pronounced olfactory deficits affecting acuity and discrimination of odorants. These major olfactory deficits occur long before the emergence of auditory deficits through the upregulation of OB autophagy inducing local neuroinflammation and altered neuron content. Our findings provide new opportunities for designing olfactory means to monitor the possible neurological outcome during congenital CMV infection.

Keywords: congenital; cytomegalovirus; deafness; hyposmia; interneuron; olfactory bulb.

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Figures

Figure 1.
Figure 1.
Impact of CMV congenital infection on hearing. A, Timetable of the experiments. Mice were infected with CMV or received PBS at E13. They were analyzed using olfactometers and with buried food-finding tests. Their ABRs were recorded twice, at W4 and W16. B, Animal model of CMV infection in pregnancy. Murine CMV or PBS was intraplacentally inoculated in each embryo of pregnant mice under deep anesthesia. C, Body weight of the 8-postnatal day-old mice after infection with murine CMV at day 13 of gestation (n = 21 CTL, n = 35 CMV). CTL mice were injected with saline only. Outliers were identified by ROUT. Variances between the CTL and CMV groups without outliers are different (F(33,19) = 2.433, *p < 0.05), underlying the growth retardation of 9/34 CMV pups. D, Click-evoked ABR waveforms at different sound intensities. E, ABR hearing thresholds for a click. n = 8 male mice per group. p-values were calculated by Mann–Whitney test (C, D: comparison between CTL and CMV groups) or Wilcoxon matched-pairs signed–rank test (D: comparison between W4 and W16 for each mouse group). *p < 0.05; **p < 0.01, ***p < 0.001; mean ± SEM in bar graphs.
Figure 2.
Figure 2.
Impact of CMV congenital infection on neonate olfaction. A, Emission and quantitation of ultrasonic vocalizations. The recording of ultrasonic calls began 30 s after placing the pups in the test chamber of the olfactometer. Ultrasonic vocalizations were detected using an ultrasonic microphone connected to a bat detector that converts ultrasonic sounds to the audible frequency range. B, Timetable of the experiments. Mice were infected in utero with CMV or received PBS at E13. They were analyzed using olfactometers at 6 and 8 d after birth. C, Typical wave traces of spontaneous call series from preweaning 6-day-old pups after congenital CMV infection. CTL mice were inoculated with PBS only. D, Experimental paradigm. Ultrasonic emission responses were recorded during the first period without odorant (1 min), followed by a period of odorant exposure (1 min) and finally the last period of exhaust odorant (1 min and 30 s). E–G, Emission of ultrasonic calls for citral odorant on day 6 after birth (n = 18 CTL, n = 19 CMV). H, I, Emission of ultrasonic calls for male scent odorant on day 8 after birth (n = 8 CTL, n = 11 CMV). p-values were calculated by Mann–Whitney test (E) or Wilcoxon matched-pairs signed–rank test (H, I). **p < 0.01, ***p < 0.001, ****p < 0.0001; mean ± SEM in E.
Figure 3.
Figure 3.
Impact of CMV congenital infection on olfactory perception in adult male mice. A, B. Latency to find the buried (A) or visible (B) food reward in the buried food-finding test. Results are percentage of mice that did not find the food over a 15 min period. C, Go–no go procedure. The olfactometer isolator comprises an odorant sampling port and a water delivery tube to reward mice. Animals are trained to distinguish between 2 odorants: a positive stimulus (S+) and a negative stimulus (S−). A licking response following an S+ trial and no licking following an S− trial were scored as correct. D, percentage of mice that did not reach the criterion performance (75% correct responses in the block) on a discrimination task using n-butanol (10−3 dilution) and its solvent. A block is a series of 20 trials with random 10 S+ and 10 S−. E, Effects of congenital CMV infection on olfactory detection. Results are expressed as detection thresholds (−log10 of odorant dilution; mean ± SEM) for n-butanol (left) or the percentage of mice for the last dilution performance criterion (right). Results in A, B, and F are expressed as the mean ± SEM of correct responses. n = 8 CMV, n = 11 CTL. p-values were calculated by Gehan–Breslow–Wilcoxon test (A, B, D) and Mann–Whitney test (E). *p < 0.05; **p < 0.01.
Figure 4.
Figure 4.
Impact of CMV congenital infection on olfactory discrimination in adult male mice. A, B, Graph depicting the percentage of correct responses in each block of the easy (A) discrimination task between isoamylacetate (S+) and anethol (S−) or the difficult (B) discrimination tasks between their binary mixtures. The mixture ratio of isoamylacetate and anethol is indicated on the graph. C–E, Percentage of mice that did not reach the performance criterion for the discrimination task between isoamylacetate and anethol (C), d-limonene and citronellal (D), and d-limonene and anethol (E). F, Long-term memory test. To assess olfactory memory, mice were trained during 5 consecutive days to recall distinguishing d-limonene and anethol. Mice were then retested at W16 following the end of the training session (W12). In A, B, and F, a score of 50% corresponds to the success rate expected on the basis of chance alone (dashed line). Results in A, B, and F are expressed as the mean ± SEM of correct response. n = 8 CMV, n = 11 CTL. p-values were calculated by ANOVA with repeated-measures (A, B), Gehan–Breslow–Wilcoxon test (C–E), or Mann–Whitney test (F). *p < 0.05, **p < 0.01.
Figure 5.
Figure 5.
Impact of CMV congenital infection on the postnatal olfactory system at W3. A, Sagittal section of a murine brain showing the neurogenic dentate gyrus (DG) of the hippocampus, the neurogenic SVZ, the RMS, and the OB. Neuroblasts born in the neonatal and postnatal SVZ migrate via the RMS until the OB, where they differentiate into GCL or GL interneurons. B, D–F, Representative staining of coronal SVZ (D, bottom) and OB slices with DAPI (B, D, E) murine CMV IE1 (B, D, bottom), cleaved caspase 3 (Casp3, D, top), OMP expressed by OSN (D, middle), Iba (D, middle; E), CD68 expressed by activated microglia and macrophages (E), Ki67 (D), TH (F), CB (F), and CR (F), antibodies showing CMV+, apoptotic Casp3+, OSN, macrophages, microglia, Ki67+ neural progenitor cells, TH+, CB+ and CR+ cells in CTL and congenital CMV-infected mice at W3 after birth. C, G–I, Screening of the OB proteins from congenital CMV-infected mice at W3 for autophagy induction (C, I), cell apoptosis (C, G), and microglial reaction (C, G). Lysates were extracted from the OB of CTL and congenital CMV-infected mice at W3 and analyzed by immunoblot (C) using antibodies to detect Casp3, Iba1, CD68, Beclin1, phospho-AMPK, Atg5, p62, LC3 I/II, LAMP, phospho-mTOR, and actin (three mice each). The levels of Casp3, Iba, CD68, Beclin1, pAMPK, Atg5, p62, LC3 II/ LC3 I, LAMP, pmTOR, and actin were quantified (G,I) by band intensity with Fiji software. H, J, Ki67+, TH+, CB+, and CR+ cell densities in the SVZ, GL, glomerulus (glom), and GCL at W3 following congenital CMV inoculation. All mice are W3-old males injected at E13 with PBS (CTL) or CMV. For immunoblot analysis (G, I), n = 4 mice per group. For cell density analysis (H) and area ratio (J), n = 4–6 mice per group. For glom size (J), n = 409 glom from 4 CTL, n = 352 glom from 4 CMV. Results are shown as mean ± SEM. p-values were calculated by Mann–Whitney test. *p < 0.05, **p < 0.01. Scale bars: 100 μm in D, bottom; 50 μm in B, D, middle, and F; 25 μm in D, bottom; and 5 μm in D, top, and E.
Figure 6.
Figure 6.
Long-lasting impact of CMV congenital infection on the adult OB. A, Representative staining of coronal SVZ (top) and OB (middle, bottom) slices with Ki67 (top), TH (middle) and murine CMV IE1 (bottom). B–D, Ki67+ cell densities in the SVZ (B), TH+ cell densities in the GL (C), and glomerulus size (D) at W16 after birth following congenital CMV inoculation. For cell density analysis, n = 5 male CTL, n = 2 female CTL, n = 4 male CMV, n = 4 female CMV. For glomerulus size, n = 212 glomeruli from 2 CTL females, n = 409 glomeruli from 4 CMV females, n = 499 glomeruli from 4 CTL males, n = 262 glomeruli from 4 CMV males. Results in D are shown as mean ± SEM. p-values were calculated by Mann–Whitney test. *p < 0.05, ***p < 0.001. Cc, Corpus callosum; LV, lateral ventricle; str, striatum. Scale bars: 100 μm in A, top; 50 μm in A, middle and bottom).
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